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Publication numberCN1245156 C
Publication typeGrant
Application numberCN 94191236
PCT numberPCT/US1994/001985
Publication date15 Mar 2006
Filing date22 Feb 1994
Priority date22 Feb 1993
Also published asCA2155947A1, CA2155947C, CN1118136A, DE69433723D1, DE69433723T2, DE69433723T3, EP0693924A1, EP0693924A4, EP0693924B1, EP0693924B2, US5498421, US5635207, US5639473, US20090048331, WO1994018954A1
Publication number94191236.1, CN 1245156 C, CN 1245156C, CN 94191236, CN-C-1245156, CN1245156 C, CN1245156C, CN94191236, CN94191236.1, PCT/1994/1985, PCT/US/1994/001985, PCT/US/1994/01985, PCT/US/94/001985, PCT/US/94/01985, PCT/US1994/001985, PCT/US1994/01985, PCT/US1994001985, PCT/US199401985, PCT/US94/001985, PCT/US94/01985, PCT/US94001985, PCT/US9401985
Inventors马克W格瑞恩斯达夫, 帕特里克树恩雄, 迈克尔王, 保罗A桑福德, 肯尼思S沙斯利克, 内尔P迪塞
Applicant美国生物科学有限公司
Export CitationBiBTeX, EndNote, RefMan
External Links: SIPO, Espacenet
Methods for in vivo delivery of biologics and compositions therefor
CN 1245156 C
Abstract  translated from Chinese
本发明提供了用于体内传送生物制品的组合物,其中生物制品与由生物相容材料制备的聚合物外壳相结合。 The present invention provides a composition for the in vivo transfer of biological products, biological products and wherein the polymer shell made of a biocompatible material prepared by combining. 该生物制品可以与聚合物外壳本身相结合,和/或生物制品选择性地悬浮/分散于生物相容的分散剂中,被聚合物外壳所包裹。 The biological products may be combined with the polymer shell itself, and / or biological products selectively suspended / dispersed in a biocompatible dispersing agent, the polymer shell is wrapped. 另一方面,把与聚合物外壳相结合的生物制品给某主体给药,可以是分散在一种合适的生物相容液体中给药。 On the other hand, the combination of the polymer shell of biological products to be administered in a body, may be dispersed in a suitable biocompatible liquid administered.
Claims(28)  translated from Chinese
1.一种用于体内传送生物制品的组合物,其中所述的生物制品选自:一种固体,任意分散于一种生物相容分散剂中,它基本上完全被包裹在一种聚合物外壳中,一种液体,任意分散于一种生物相容分散剂中,它基本上完全被包裹在聚合物外壳中,一种气体,任意分散于一种生物相容分散剂中,它基本上完全被包裹在聚合物外壳中,一种与聚合物外壳结合的气体,或它们任何两种或多种的组合,其中所述外壳的最大截面直径不大于10微米,其中所述的聚合物外壳由一种实质上是通过二硫键方式交联的生物相容材料制备,并且其中所述的聚合物外壳任意被一种适当的试剂修饰,该试剂通过一种任意的共价健连接所述的聚合物外壳。 1. A composition for in vivo transfer of biological products, wherein said biological product is selected from: a solid, optionally dispersed in a biocompatible dispersing agent, substantially completely wrapped in a polymer housing, a liquid, optionally dispersed in a biocompatible dispersing agent, substantially completely encased in the polymer housing, a gas, optionally dispersed in a biocompatible dispersing agent, substantially is completely wrapped in a polymer shell, a combination of gas and polymer shell, or any combination of two or more thereof, wherein the maximum cross-sectional diameter of the housing is not more than 10 microns, wherein said polymeric shell by way of a substantially crosslinked by disulfide bonds prepared biocompatible material, and optionally wherein said polymer shell is a suitable modifying agent, the agent through a covalent bond to connect to any of the The polymer shell.
2.根据权利要求1的组合物,其中所述的生物制品是选自药物活性试剂、诊断试剂或营养剂。 2. The composition of claim 1, wherein said biological product is selected from a pharmaceutically active agent, a diagnostic agent or a nutritional agent.
3.根据权利要求2的组合物,其中所述的药物活性试剂是选自止痛剂,麻醉剂,止喘药,抗菌素、抗抑郁剂、抗糖尿病药,抗真菌剂、抗高血压药、抗炎剂、抗肿瘤剂、抗焦虑剂、酶活性剂、核酸结构,免疫促进剂,免疫抑制剂,生理活性气体或疫苗。 3. The composition of claim 2, wherein said pharmaceutically active agent is selected from analgesic agents, anesthetic agents, antiasthmatics, antibiotics, antidepressants, antidiabetics, antifungal agents, antihypertensive agents, anti-inflammatory agents, anti-tumor agents, anti-anxiety agents, activator of the enzyme, nucleic acid structure, immune enhancers, immunosuppressants, physiologically active gases or vaccine.
4.根据权利要求3的组合物,其中所述的药物活性试剂是核酸结构。 4. The composition of claim 3, wherein said pharmaceutically active agent is a nucleic acid structure.
5.根据权利要求2的组合物,其中所述的诊断试剂选自超声对比剂,放射对比剂,或磁对比剂。 5. The composition of claim 2, wherein said diagnostic agent is selected from ultrasound contrast agents, radiocontrast agents, or magnetic contrast agents.
6.根据权利要求5的组合物,其中所述的磁对比剂是一种含氟的磁共振成像剂。 6. The composition of claim 5, wherein said magnetic contrast agent is a fluorine-containing magnetic resonance imaging agent.
7.根据权利要求6的组合物,其中所述的含氟的磁共振成像剂选自:(a)CXF2X+Y-ZAZ,其中X=1-30,Y=2;或当X≥2时Y是0或-2;或当X≥4时Y是-4,Z=0至(2X+Y-1)的任何整数,和A是选自氢,除氟外的卤素,-CN,-OR,其中R是氢,烷基、氟烷基,链烯基,氟链烯基,炔基,氟炔基,芳基,氟芳基,链烷酰基,氟链烷酰基,链烯酰基,氟链烯酰基,炔酰基,氟炔酰基,(b)[CXF2X+Y′-ZAZ]aJRb-a,其中:X,Z,A和R如上所定义,Y′=+1;或当X≥2时Y′是-1或-3;或当X≥4时Y′是-5,J=O,S,N,P,Al或Si,a=1,2,3,或4和b=对2价的J是2,或对3价的J是3,或对4价的J是4,(c)A′-[(CF2)XO]cA″,其中:X如上所定义,A′是选自氢,卤素,-CN,-OR,其中R是H,烷基,氟烷基,链烯基,氟链烯基,炔基,氟炔基,芳基,氟芳基,链烷酰基,氟链烷酰基,链烯酰基,氟链烯酰基,炔酰基,氟炔酰基,A″是选自H或R,其中R同上所定义,C=1-300,或 The composition according to claim 6, wherein said fluorine-containing magnetic resonance imaging agent is selected from: (a) CXF2X + Y-ZAZ, where X = 1-30, Y = 2; or when when X≥2 Y is 0 or -2; or when X≥4 when Y is -4, Z = 0 to (2X + Y-1) is any integer, and A is selected from hydrogen, halogen fluoride outside, -CN, - OR, wherein R is hydrogen, alkyl, fluoroalkyl, alkenyl, fluoro alkenyl, alkynyl, fluoro alkynyl, aryl, fluoroaryl, alkanoyl, fluoro alkanoyl, alkenoyl, fluoro alkenoyl, alkynoyl, fluoro alkynoyl, (b) [CXF2X + Y'-ZAZ] aJRb-a, wherein: X, Z, A and R are as defined above, Y '= + 1; or when X≥ 2 Y 'is -1 or -3; or when X≥4 when Y' is -5, J = O, S, N, P, Al or Si, a = 1,2,3, or 4 and b = of divalent J is 2, or 3-valent J is 3, or tetravalent J is 4, (c) A '- [(CF2) XO] cA ", wherein: X is as defined above, A' is selected from hydrogen, halogen, -CN, -OR, wherein R is H, alkyl, fluoroalkyl, alkenyl, fluoro alkenyl, alkynyl, fluoro alkynyl, aryl, fluoroaryl, alkanoyl acyl, fluoro alkanoyl, alkenoyl, fluoro alkenoyl group, an alkynyl group, an alkynyl group fluorine, A "is selected from H or R, wherein R is as defined above, C = 1-300, or 其中:X同上定义,和c′=2-20,以及它们任何两者或更多的混合物。 Wherein: X is defined above, and c '= 2-20, and their mixtures of any two or more.
8.根据权利要求2的组合物,其中所述的营养剂是选自氨基酸,蛋白质,核酸,糖,碳水化合物,脂溶性维生素,脂肪,或它们任何两者或更多的组合。 The composition according to claim 2, wherein said nutritional agent is selected from amino acids, proteins, nucleic acids, sugars, carbohydrates, fat-soluble vitamins, fat, or any two or more combination thereof.
9.根据权利要求2的组合物,其中所述的在所述外壳内的药物活性试剂溶解于或悬浮于一种生物相容分散剂中。 The composition according to claim 2, wherein said housing within said pharmaceutically active agent is dissolved or suspended in a biocompatible dispersing agent.
10.根据权利要求9的组合物,其中所述的生物相容分散剂是选自豆油,椰子油,橄榄油,红花油,棉籽油,具有4-30个碳原子的脂肪族、环脂族或芳香族的烃,具有1-30个碳原子的脂族或芳族的醇,具有2-30个碳原子的脂族或芳族的酯,具有2-30个碳原子的烷基、芳基或环醚,具有1-30个碳原子并且还可以有一个以上卤素取代基的烷基或芳基卤化物,具有3-30个碳原子的酮,聚二醇,或它们任意两者或更多的组合。 10. The composition of claim 9, wherein said biocompatible dispersing agent is selected from soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, an aliphatic 4 to 30 carbon atoms, cycloaliphatic aliphatic or aromatic hydrocarbons, aliphatic or aromatic alcohols of 1-30 carbon atoms, aliphatic or aromatic esters of 2-30 carbon atoms, an alkyl group having 2-30 carbon atoms, an aryl group or a cyclic ether having 1 to 30 carbon atoms and may also have more than one halogen substituent alkyl or aryl halide, a ketone having 3 to 30 carbon atoms, polyalkylene glycol, or any two of them or more combinations.
11.根据权利要求2的组合物,其中所述的药物活性试剂是完整地包裹在所述外壳之内。 11. The composition of claim 2, wherein said pharmaceutically active agent is completely encased within the housing of the.
12.根据权利要求1的组合物,其中所述的交联聚合物是一种天然存在的聚合物,一种合成聚合物,或它们的组合,其中所述的聚合物,在交联之前,已共价连接上了巯基或二硫键。 12. The composition of claim 1, wherein said crosslinked polymer is a naturally occurring polymer, a synthetic polymer, or combinations thereof, wherein said polymer, prior to cross-linking, is covalently linked to a thiol group or the disulfide bonds.
13.根据权利要求12的组合物,其中所述的天然存在的聚合物是选自含有巯基和/或二硫基的蛋白质,含有巯基和/或二硫基的多肽,含有巯基和/或二硫基的脂,含有巯基和/或二硫基的多核酸,或含有巯基和/或二硫基的多糖。 13. The composition of claim 12, wherein said naturally occurring polymer is selected from the group containing a mercapto group and / or disulfide group of the protein, a mercapto group and / or a polypeptide containing a disulfide group, a mercapto group-containing and / or di- aliphatic group, a mercapto group-containing and / or polynucleic acid disulfide group, or a mercapto group and / or disulfide group-containing polysaccharide.
14.根据权利要求13的组合物,其中所述的蛋白质是选自血红蛋白、肌红蛋白、白蛋白,胰岛素、溶菌酶、免疫球蛋白、α-2-巨球蛋白、纤连蛋白、玻连蛋白、纤维蛋白原、或它们任意两者或更多的组合。 14. The composition of claim 13, wherein said protein is selected from hemoglobin, myoglobin, albumin, insulin, lysozyme, immunoglobulins, α-2- macroglobulin, fibronectin, vitronectin protein, fibrinogen, or a combination of any two or more of them.
15.根据权利要求14的组合物,其中所述的蛋白质是白蛋白。 15. The composition of claim 14, wherein said protein is albumin.
16.根据权利要求14的组合物,其中所述的蛋白质是血红蛋白。 16. The composition of claim 14, wherein said protein is hemoglobin.
17.根据权利要求14的组合物,其中所述的蛋白是白蛋白与血红蛋白的组合物。 17. The composition of claim 14, wherein said protein is albumin and hemoglobin of the composition.
18.根据权利要求13的组合物,其中所述的多糖是选自藻酸盐或酯、多聚甘露糖醛酸、多聚甘露糖醛酸盐、透明质酸、透明质酸盐、肝素、葡聚糖、脱乙酰壳多糖、几丁质、纤维素、淀粉、糖原、瓜耳胶、刺槐豆胶、果聚糖、旋复花粉、环葡聚糖、琼脂糖、黄原胶、卡拉吉兰、果胶、盖兰胶、硬葡聚糖、或它们任意两者或更多组合。 18. The composition of claim 13, wherein said polysaccharide is selected from alginate or esters, poly-mannuronic acid, poly-mannuronic acid, hyaluronic acid, hyaluronate, heparin, glucan, chitosan, chitin, cellulose, starch, glycogen, guar gum, locust bean gum, fructans, inulin, cyclodextrin, agarose, xanthan gum, Kara Guillen, pectin, blue plastic cover, scleroglucan, or a combination of any two or more of them.
19.根据权利要求12的组合物,其中所述的合成聚合物是选自合成的含有半胱氨酸残基和/或二硫基的多氨基酸,含有巯基和/或二硫基的合成多肽,含有游离巯基和/或二硫基的改性的聚乙烯醇,经改性含有游离巯基和/或二硫基的甲基丙烯酸多羟基乙基酯,经改性含有游离巯基和/或二硫基的聚丙烯酸,经改性含有游离巯基和/或二硫基的聚乙烯基噁唑啉,经改性含有游离巯基和/或二硫基的聚乙烯吡咯烷酮,经改性含有游离巯基和/或二硫基的聚丙烯酰胺,经改性含有游离巯基和/或二硫基的聚二醇,以及它们任意两者或多者的混合物。 19. The composition of claim 12, wherein said synthetic polymer is chosen from synthetic containing a cysteine residue and / or multiple amino acid disulfide group, a mercapto group, and a synthetic polypeptide / or disulfide group-containing containing free sulfhydryl groups and / or disulfide group-modified polyvinyl alcohol, the modified containing free mercapto group and / or disulfide group of polyhydroxy ethyl methacrylate esters, containing a free sulfhydryl group of the modified and / or two thio polyacrylic acid, modified by containing free sulfhydryl groups and / or disulfide group polyvinyl oxazoline, containing a free sulfhydryl group of the modified and / or disulfide group of polyvinylpyrrolidone, the modified contain free sulfhydryl groups and / polyacrylamide or disulfide group, the modified contain free sulfhydryl groups and / or disulfide group of polyalkylene glycol, and mixtures of two or more of any of them.
20.根据权利要求1的组合物,其中所述的气体是选自空气、氧气、氩气、氮气、一氧化碳、二氧化碳、氦气、氙气、一氧化二氮、一氧化一氮、或它们任意二者或多个的组合。 20. The composition of claim 1, wherein said gas is selected from air, oxygen, argon, nitrogen, carbon monoxide, carbon dioxide, helium, xenon, nitrous oxide, nitric oxide, or any two or a combination of a plurality of.
21.根据权利要求1的组合物,其中交联聚合物上的二硫键是由超声辐射形成的。 21. The composition of claim 1, wherein the disulfide bonds on the crosslinked polymer are formed by ultrasonic irradiation.
22.根据权利要求1的组合物,其中所述的含有生物制品的聚合物外壳是悬浮在一种生物相容介质中,并且其中所述的生物相容介质是选自水、缓冲水溶液介质、生理盐水、缓冲的生理盐水、氨基酸溶液、蛋白质溶液、糖溶液、维生素溶液、碳水化合物溶液、合成的聚合物溶液、含有脂类的乳化液、或它们任意两者或多个的组合。 22. The composition of claim 1, wherein said polymeric shell containing biologic is suspended in a biocompatible medium, and wherein said compatible biological medium is selected from water, buffered aqueous medium, physiological saline, buffered physiological saline, amino acid solution, a protein solution, sugar solution, vitamin solution, a carbohydrate solution, synthetic polymer solution, containing a lipid emulsion, or a combination of any two or more of them.
23.根据权利要求1的组合物,其中所述的聚合物外壳是被一种合适的试剂改性的,其中所述的合适的试剂是选自合成的聚合物、磷脂、蛋白质、多糖、表面活性剂、化学改性剂、或它们的组合,其中所述的试剂与所述的聚合物外壳通过任意的共价键相结合。 23. The composition of claim 1, wherein said polymer shell is a suitable reagent-modified, wherein said suitable agent is selected from synthetic polymers, phospholipids, proteins, polysaccharides, surface active agents, chemical modifiers, or combinations thereof, wherein said reagent and said polymeric shell through any covalent bond combination.
24.一种制备用于体内传送的生物制品的方法,所述方法包括将含有能够被二硫键交联的生物相容材料的水性介质与生物制品经过高强度超声条件处理一段时间,足以促使所述生物相容材料被二硫键交联,其中所述生物制品基本上完全被包裹在聚合物外壳中,所述的聚合物外壳由一种实质上是通过二硫键方式交联的生物相容材料制备,并且其中所述外壳的最大截面直径不超过10微米。 24. A method for the preparation of biological products in vivo delivery, said method comprising the biocompatible material containing disulfide bonds can be crosslinked in aqueous medium and biological products through the high intensity ultrasound conditions for processing a time sufficient to induce The biocompatible material is disulfide cross-linking, wherein the biological products are substantially completely encased in the polymer shell, the polymer shell is formed of a substantially cross-linked by disulfide bonds biological way compatible material preparation, and the maximum cross-sectional diameter of said enclosure is no more than 10 microns.
25.有效量的权利要求1的组合物在制备给患者体内传送生物制品的药物中的应用。 25. The composition of an effective amount of a claim in the manufacture of biological products to patients in vivo transfer of medicament.
26.有效量的权利要求1的组合物在制备给患者输送血液代用品的药物中的应用。 26. The composition of claim effective amount to a patient in the manufacture of a medicament for delivery of a blood substitute applications.
27.有效量的权利要求22的组合物在制备给患者体内传送生物制品的药物中的应用。 22 27. The composition of claim effective amount to a patient in the manufacture of a medicament for the transfer of biological products applied.
28.权利要求7的组合物在制备获得体内磁共振影像的制剂中的应用。 28. The composition of claim 7 obtained in vivo magnetic resonance imaging in preparation for the preparation.
Description  translated from Chinese
用于体内传送生物制品的方法及用于该方法的组合物 Transfer of biological products for the in vivo method and composition for this method

本发明涉及生物制品的体内传送。 The present invention relates to in vivo transfer of biological products. 一方面,是将生物制品与生物相容材料制成的聚合物外壳相结合。 On the one hand, is a biological product and a biocompatible polymer shell made of a material combination. 该生物制品可以聚合物外壳本身结合在一起,和/或将生物制品(或者是悬浮于/分散于生物相容分散剂中的生物制品)包含在聚合物外壳之内。 The biological products may be combined with a polymer shell itself, and / or biological products (or suspended / dispersed in a biocompatible dispersing agent in biological products) contained within the polymer shell. 另一方面,将与聚合物外壳相结合的生物制品给一个体给药,或者是分散于一种合适的生物相容液体中给药。 On the other hand, the polymer shell combination with biologics administration to a body, or dispersed in a suitable biocompatible liquid administered.

存在于血液中的微粒体和外来小体一般是由“血液过滤器官”,即脾脏,肺脏和肺脏,从血液循环中清除的。 Present in the blood of microsomes and foreign bodies is normally provided by the "blood filtering organs", namely the spleen, lungs and lung, cleared from the blood circulation. 正常全血中所含有颗粒物质包括红细胞(一般直径8微米),白细胞(一般直径6-8微米)和血小板(一般直径1-3微米)。 Normal whole blood contained in particulate matter, including red blood cells (typically 8 microns in diameter), white blood cells (typically 6-8 microns in diameter), and platelets (typically 1-3 microns in diameter). 大多数器官和组织中的微循环可以使这些血液细胞自由通过。 Most organs and tissues microcirculation of blood cells can make these freely. 当血液循环中存在直径大于10-15微米的小血栓(血块)时,就会有毛细管梗塞或阻塞的危险,导致局部缺血或氧缺乏以及可能出现组织坏死。 When there are more than 10-15 microns in diameter small thrombus (blood clot) in the blood circulation, or there will be a risk of blocking the capillary infarction, leading to ischemia or oxygen deficiency and possible tissue necrosis. 因此,必须避免向血液循环中注射直径大于10-15微米的颗粒。 Therefore, must be avoided to the blood circulation is greater than 10-15 microns in diameter injected particles. 尽管如此,小于7-8微米的颗粒混悬液还是相对安全的,曾经用它传送过脂质体和乳剂形式的药物活性成份,营养试剂,和成像用的对比剂。 Nevertheless, less than 7-8 microns suspension is relatively safe, it was transferred through the use of liposomes and emulsions in the form of pharmaceutically active ingredients contrast agents, nutritional agents, and for imaging.

颗粒的大小及其传送方式决定了它们的生物功能。 Particle size and transmission scheme determines their biological functions. Strand等人[inMicrosphere-Biomedical Applications,ed.A.Rembaum,PP193-227,CRC Press(1988)]曾报道过颗粒的传送状况取决于它们的大小。 Strand et al. [InMicrosphere-Biomedical Applications, ed.A.Rembaum, PP193-227, CRC Press (1988)] have reported the transfer status particles depends on their size. 在组织间质注射之后,大小在几毫微米至100毫微米之间的颗粒进入毛细淋巴管,在淋巴结中就会出现吞噬作用。 After between interstitial injection, the particle size of between a few nanometers to 100 nanometers into the lymphatic capillaries, phagocytosis occurs in the lymph nodes. 静脉/动脉注射之后,小于大约2微米的颗粒会很快被血液中的网状内皮系统(RES),也称之为单核吞噬细胞系统(MPS),所清除。 After intravenous / intraarterial injection, particles less than about 2 microns will soon be the blood by the reticuloendothelial system (RES), also known as the mononuclear phagocyte system (MPS), are cleared. 大于约7微米的颗粒在静脉注射之后,会被肺毛细管所捕获。 Greater than about 7 microns, after intravenous injection, be trapped lung capillaries. 动脉注射之后,在到达的第一个毛细血管床把颗粒捕获。 After intra-arterial injection, reaching the first capillary bed of the particle capture. 小泡状的巨噬细胞捕获吸入的颗粒。 Vesicle-like macrophages capture particles inhaled.

对于不溶于水或难溶于水对胃酸环境敏感的药物来说,不能按常规方式给药(如静脉注射或口服给药)。 For insoluble or poorly soluble in water sensitive drugs on gastric acid environment, it can not be administered in a conventional manner (such as intravenous or oral administration). 通过在表面活性剂或乳化稳定剂存在下将油稳定的药物与水溶液(如生理盐水)一起乳化制成稳定的微粒乳剂可以实现这些药物的非肠道给药。 By a surfactant or emulsifying stabilizer stable pharmaceutical oil with an aqueous solution (such as physiological saline) emulsified together into a stable emulsion can be achieved microparticles for parenteral administration of these drugs. 只要乳化剂的成份在药理学是隋性的,即可以将这些乳化剂静脉注射。 As long as the emulsifier in the composition is pharmacologically inert, i.e. These emulsifiers may be intravenous. 例如,美国专利NO.4,073,943描述了在表面活性剂如卵磷脂,普卢兰尼克(聚丙二醇与聚乙二醇的共聚物),油酸聚甘油酯等存在下将溶于油的非水溶性药理活性成份与水乳化后的给药。 For example, U.S. Patent NO.4,073,943 describes a surfactant such as lecithin, Pluronic (polypropylene glycol and polyethylene glycol copolymer), polyglycerol ester of oleic acid, etc. The presence of oil-soluble and water-insoluble administration of a pharmacologically active ingredient with water by emulsifying. PCT国际专利公开号NO,WO85/00011描述了具有适合于皮内或静脉注射大小范围的,用磷脂,如二肉豆蔻酰卵磷脂,包裹的麻醉药物微小滴剂。 PCT International Patent Publication No. NO, WO85 / 00011 describes a suitable for intradermal or intravenous injection size range, with a phospholipid, such as dimyristoyl phosphatidylcholine, anesthetics tiny drops wrapped.

有文献报道用蛋白微粒作用药理或诊断试剂的载体。 It has been reported with the carrier protein particle effects of pharmacological or diagnostic reagents. 通过热变性或化学交联已制备出了白蛋白微球体。 Heat denaturation or chemical crosslinking has been prepared by the albumin microspheres. 热变性微球体是在100℃至150℃的温度之间由一种乳化混合物(如,白蛋白,所要加入的试剂,和一种合适的油)制得。 Thermal denaturation temperature of the microspheres is between 100 ℃ to 150 ℃ from an emulsified mixture (e.g., albumin, the agent to be added, and a suitable oil) was prepared. 然后用一种合适的溶剂洗涤微球体,并贮存。 Then washed with a suitable solvent microspheres, and stored. Leucuta等人(Internation-al Journal of Pharmaceutics vol 41:213-217(1988))描述热变性微球体的制备方法。 Leucuta et al. (Internation-al Journal of Pharmaceutics vol 41: 213-217 (1988)) describe the thermal denaturation microsphere preparation.

制备化学交联微球体的方法包括用戊二醛处理乳化液以使蛋白交联,然后洗涤和贮存。 Preparation of chemically crosslinked microspheres comprising emulsion with glutaraldehyde to crosslink the protein so that, then washed and stored. Lee等人[Science Vol.213:233-235(1981)]和美国专利No.4,671,954报导了这种制备方法。 Lee et al. [Science Vol.213: 233-235 (1981)] and U.S. Patent No.4,671,954 reported this preparation.

上述用于制备作为药理活性成份载体的蛋白微球体的方法,虽然适用传送水溶性试剂,但不能携带不溶于水的试剂。 Said protein microsphere method for the preparation of a carrier as a pharmacologically active ingredient, although suitable water-soluble transfer agent, but can not carry water-insoluble reagent. 这种限制是基于使油包水乳剂的水相中蛋白成份交联或热变性的制剂技术所固有的。 This limitation is based on the oil-in-water emulsion composition of the aqueous phase protein crosslinking or heat denaturation of the inherent formulation techniques. 任何水溶性试剂溶于含有蛋白的水相中可以被这种所产生的交联或热变性蛋白基质所包裹携带,但是难溶于水或溶于油的试剂不能结合到由这些方法形成的蛋白基质中。 Any water-soluble reagent comprising a water-soluble protein phase may be crosslinked or heat-denatured protein matrix generated such wrapped carry, but insoluble in water or oil-soluble agent can not bind to proteins formed by these methods matrix.

因此,许多难溶于水的生物制品给人体给药带来了问题。 Thus, many of sparingly water-soluble biologics presents a problem for human administration. 的确,如果口服给药传送药物是无效的,这种本来不溶于或难溶于水介质的药理活性成份的传送会受到严重削弱。 Indeed, if the oral administration of drugs is ineffective transmission, which originally insoluble or poorly soluble in aqueous medium of a pharmacologically active ingredient of the transfer will be seriously weakened. 因此,目前用于传送本来不溶于或难溶于水介质的药理活性成份的制剂中需要加入试剂以稳定药理活性成份。 Accordingly, currently used for transmitting the original formulation is insoluble or poorly soluble in aqueous medium in the pharmacological active ingredient necessary to add a reagent to stabilize the pharmacologically active ingredient. 尽管如此,用于稳定药理活性成分的试剂(如乳化剂)还是会经常引起严重的过敏反应。 Nevertheless, stabilizing agents pharmacologically active ingredients (such as emulsifiers) is often used to cause severe allergic reactions. 因此,一般的给药方法是在注射药理活性成份之前先用抗组织胺药和类固醇治疗病人以减少药物传送所加入试剂的过敏作用。 Thus, the general method of administration before injection pharmacologically active ingredients first with antihistamines and steroids to treat patients in order to reduce drug delivery added reagents allergic effects.

在努力改进本来不溶于或难溶于水介质的药物水溶性过程中,一些研究者曾用能带来强水溶性的官能团对药物的结构进行公学改性。 In efforts to improve the originally insoluble or poorly soluble in aqueous medium-soluble drug process, some researchers have used to bring a strong water-soluble functional group structure modified drugs were public school. 现有技术中所述的化学改性是制备磺化的衍生物[Kingston et al.,美国专利5,059,699(1991)],和氨基酸酯[Mathew et al.,J.Med.Chem.Vol.35:145-151(1992)],它表现出明显的生物活性。 Chemical modification according to the prior art is the preparation of sulfonated derivatives [Kingston et al, U.S. Patent No. 5,059,699 (1991).], And amino acid esters [Mathew et al, J.Med.Chem.Vol.35.: 145-151 (1992)], which showed significant biological activity. 这种改性产生的水溶性衍生物有助于本来不溶于或难溶于水的药物在水介质(溶于无害的载体如生理盐水)中的静脉内传送。 The modified water-soluble derivatives have been generated helps water-insoluble or poorly soluble drugs in aqueous medium (dissolved in innocuous carrier such as physiological saline) transmission within a vein. 尽管如此,但这种改性给药物制备增加了成本,还会引起不希望发生的副作用和/或过敏反应,和/或降低药物的效果。 Nevertheless, such a modification adds cost to the preparation of a pharmaceutical, can also cause undesirable side effects and / or allergic reactions, and / or reduced drug effect.

常常难以传送的生物制品是氧。 Biological products are often difficult to transfer oxygen. 的确,不能过分强调用作红细胞代用品(“血液代用品”或“人造血”)的临床安全性和有效的氧携带介质的要求。 Indeed, can not be overemphasized used as a substitute for red blood cell ("blood substitutes" "human blood" or) clinical safety and efficacy requirements for oxygen carrying media. 这些介质的一些可能的用途包括(A)一般的输液应用,包括常规和紧急情况下替代急性失血,(B)在器官移植前体外支持器官或体内手术中支持器官,(C)向体内局部缺血的组织和器官加强氧传送,(D)向血管供应不足的肿瘤加强氧传送以增加放疗或化疗的效果,(E)在实验研究中支持器官或动物,和(F)向培养基中的活细胞增加氧输送。 Some possible uses of these media include (A) the general infusion applications, including the normal and emergency substitution acute blood loss, (B) in an organ transplant prior to in vitro or in vivo support support organ surgery, (C) to the partial lack of in vivo blood, tissues and organs to strengthen the oxygen transmission, lack of (D) to strengthen the oxygen supply to the tumor blood vessels transferred to increase the effectiveness of chemotherapy or radiotherapy, (E) support organ or animal experimental studies, and (F) to medium living cells increase oxygen delivery.

输血可以用来补充患有各种疾病的病人的血液动力系统,这些疾病包括血溶量的减少,(如由于失血引起),红细胞数目的减少(如,由于骨髓破坏引起的)或血细胞受损或破坏(如由于溶血性贫血引起)。 Blood transfusion may be used to supplement a patient suffering from various diseases of the hemodynamic system, these diseases include reducing the amount of blood solution, (e.g., caused due to loss of blood), decrease in the number of red blood cells (e.g., due to damage caused by bone marrow) or blood cell damage or damage (e.g., hemolytic anemia due to induced). 输血不仅可以增加血管内的容量,而且还可以提供携带有溶解氧的红细胞有助于向组织供氧。 Transfusion can not only increase the capacity of intravascular, but can also provide red blood cells that carries oxygen to the tissue oxygen helps.

在向已经过大量失血的病人的输血过程中,对于供血者和接受输血者的血型要小心匹配以使之相容。 In the transfusion process has been a lot of blood loss to the patient, for blood donors and blood transfusion recipients who have to be careful to make it compatible match. 这样常使病人经历一段有害的缺氧时间。 This often makes the patient experience a harmful hypoxia. 而且,即使是有通过预先采血和贮存的病人自已提供的自体红细胞,由于存放时间的原因这些自体细胞氧携带能力和安全性都会降低。 Moreover, even if there is blood through the patient in advance and stored autologous red blood cells to provide their own, due to storage time of these from the cell body of oxygen carrying capacity and safety will be reduced. 因此,在输液后24小时的时间内,病人会处于非最佳氧传送状态。 Accordingly, within 24 hours after the infusion time, the patient will be in a non-optimal oxygen delivery status. 最后,在所有的全血或从中得到的红细胞输血过程中,一直存在着病人受病毒和/或细菌感染的危险。 Finally, in all of the whole blood or red blood cell transfusion from obtained, there has been a patient for viruses and dangerous / or bacterial infection.

因此,大家公认需要有一种在正常环境状况下用于氧运输和传送的替代物质,它应具有如下特性。 Therefore, there is a recognized need in normal environmental conditions for oxygen transport and delivery of alternative substances, and it should have the following characteristics. 理想的是,用于氧运输和传送的物质应当能够携带和传送氧到达装置、器官和组织以致于使这些环境保持正常的氧压。 Ideally, the material used for oxygen transport and delivery should be able to carry and transfer the oxygen to reach the apparatus, organs and tissues so that these environments to maintain normal oxygen pressure. 理想的这种物质应当是安全和无毒,无细菌和/或病毒污染,和无抗原性和无热原性(也即小于0.25EU/ml)。 This material should preferably be safe and non-toxic, non-bacterial and / or viral contamination, and non-antigenic and non-pyrogenic (i.e. less than 0.25EU / ml). 另外,用于氧运输和传送的物质应当具有与血液相匹配的粘度、胶态和渗透特性。 In addition, the material used for the transport and delivery of oxygen should have a viscosity, colloidal and permeability characteristics of the blood matches. 还希望这种物质能够长时间地保持在病人的血液系统内,从而使得病人自已的红细胞再生和成熟。 This substance is also desirable to be able to maintain for a long time in the patient's blood system, so that the patient's own red blood cell regeneration and maturation. 而且,还希望使用的这种物质不影响或阻碍红细胞的生成。 And, also hope that this matter does not affect or hinder the use of erythropoiesis.

目前,已有一些静脉液体可用于治疗急性血容量减少,包括类晶体的,如乳酸盐林格溶液或生理盐水,和胶体溶液,如正常人血清白蛋白。 Currently, there are a number of intravenous fluid can be used to treat acute hypovolemia, including crystalloid, such as Ringer's lactate solution or normal saline, and colloidal solution, such as normal human serum albumin. 类晶体和胶体的溶液可以暂时地纠正血容量的不足,但是不能直接补充给组织的氧传送。 Class crystalloid and colloid solutions may temporarily correct hypovolemia, but can not be directly transferred to the organization of supplementary oxygen. 尽管输血是优选的治疗方式,但是是否能够有足够量的安全的血液来源是一个永久性的问题。 Although blood transfusion is the preferred treatment modality, but whether there is a sufficient amount of blood is a source of secure permanent problem.

其他一些经常是不溶于或难溶于水介质,并且希望是溶于一种无毒载体如生理盐水中给药的,且有利于尽可能减少不希望发生的副作用和/或过敏反应的生物制品是一些诊断试剂,如对比试剂。 Others are often insoluble or poorly soluble in aqueous medium, and desirably dissolved in a non-toxic carrier such as physiological saline administration, and is advantageous to minimize the undesirable side effects and / or allergic reactions occurring biological products are diagnostic agents such as contrast agents.

对比剂在放射学成像过程中是需要的,因为它能够有利于观察器官(也即,它们的位置,大小和形态)和其它的细胞结构与周围组织的区分。 In contrast radiology imaging process is needed, because it can contribute to observe organs (i.e., their location, size and shape) to differentiate and other cellular structures with the surrounding tissue. 例如,即使软组织具有显著不同的生物学功能(如,肝和脾),但它们具有类似的细胞组成(即,它们基本由水组成)。 For example, even if the soft tissue have significantly different biological functions (e.g., liver and spleen), but they have similar cell composition (i.e., they are composed substantially of water).

磁共振成像(MRI)或核磁共振成像技术是应用射频辐射检查应用磁场强度下的特定原子核。 Magnetic resonance imaging (MRI) or magnetic resonance imaging techniques are specific nuclei radiofrequency radiation checks under the applied magnetic field strength. 在某些方面它类似于X-线计算机体层成像(CT),它能提供(在某些情况下)器官的断层成像,并且有可能具有明显的软组织分辨率。 In some aspects it is similar to X- ray computed tomography (CT), it can provide (in some cases) tomographic imaging of organs, and may have significant soft tissue resolution. 目前应用当中,它的影像是器官和组织中质子的分散图。 Among the current application, and its image is decentralized organs and tissues chart protons. 但是,与X-线计算机体层成像不同的是,MRI不使用离子化辐射。 However, with the X- ray computed tomography is different, MRI does not use ionizing radiation. 因此,MRI是一种安全无害的医学成像技术。 Therefore, MRI is a safe and harmless medical imaging techniques.

尽管NMR现象早在1954年就被发现了,但是将其作为描述内部结构的工具用于医学诊断只是近些年的事情。 Despite early NMR phenomenon was discovered in 1954, but as a description of the internal structure of the tool for medical diagnosis only in recent thing. 该技术是由Lauterbur[Nature242:190-191(1973)]首次建立的。 The technology was developed by Lauterbur [Nature242: 190-191 (1973)] for the first time established.

大家熟知具有合适的核旋转的原子核在应用磁场的方向上排列成直线。 Nuclear familiar with a suitable core rotation arranged in a line in the direction of the applied magnetic field. 核旋转可以是两种方式排列成直线:与外部磁场方向相同或相反。 Nuclear rotation can be arranged in a straight line in two ways: with the same or opposite direction of the external magnetic field. 沿磁场方向排列更为稳定;而在不稳定状态中排列(即,逆磁声方向)必须吸收能量。 Aligned along the magnetic field is more stable; are arranged (i.e., the sound diamagnetic direction) in an unstable state must absorb energy. 对于质子来说,在1特斯拉(1特斯拉=104高斯)磁场存在下这些核以42.6MHZ的频率旋进或共振,在此频率下,辐射的射率脉冲(RF)会激发原子核,并改变它们的旋转方向以逆磁场方向排列成直线。 For protons, in 1 tesla (1 tesla = 104 gauss) magnetic field in the presence of the nuclear precession frequency 42.6MHZ or resonance, at this frequency, the emissivity of radiation pulse (RF) will excite nuclei and change their direction of rotation in the reverse direction of the magnetic field are arranged in a straight line. RF脉冲之后,被激发的原子核“松驰”或反回到平衡状态或沿磁场方向排列成行。 After the RF pulse, the excited nuclei "relax" or return to equilibrium, or Anti-direction are arranged in rows along the magnetic field. 可以用两种松驰术语描述松驰信号减弱。 You can describe the relaxation signal weakened by two relaxation terms. T1,旋转—点阵松驰时间或纵向松驰时间,是原子核返回到沿外部应用磁场方向的平衡状态所需要的时间。 T1, rotating - lattice relaxation time or longitudinal relaxation time, the nuclei return to equilibrium along the time direction of the external magnetic field application required. 第二,T2,或旋转———旋转松驰时间,是与单个质子旋转的起始连贯旋进时间间隔有关。 Second, T2, or rotating --- rotation relaxation time is consistent with the starting time of a single proton precession rotation interval related. 不同种类的哺乳动物中各种体液、器官和组织的松驰时间都有完整的记录。 Different species of mammals in various body fluids, organs and tissues of the relaxation time has a complete record.

MRI的优点之一是可以选择不同的扫描平面和切面厚度而不会失去分辨率。 One of the advantages is that you can choose different MRI scanning plane and slice thickness without losing resolution. 这使得能够直接获得高质量的横向、冠状和矢状的影像。 This makes it possible to obtain transverse, coronal and sagittal image quality directly. MRI设备中没有任何移动部件保证了高度的可靠性。 MRI device has no moving parts ensure a high degree of reliability. 一般认为在选择性地检查组织方面MRI比X-线计算机体层成像(CT)具有更大的潜能。 Generally believed that the selective inspection organization MRI than X- ray computed tomography (CT) has greater potential. 在CT中,X-线衰减系数单独确定影像对比,而至少有三种分离的变量(T1,T2和原子核旋转密度)决定了磁共振影像。 In the CT, X- ray attenuation coefficients alone determine image contrast, whereas at least three separate variables (T1, T2 and nuclei spin density) determines the magnetic resonance image.

由于器官和组织中的细微生理—化学差异,MRI能够区别组织的类型并检查出X-线或CT所不可能检查出的疾病。 Because organs and tissues subtle physiological - chemical differences, MRI can distinguish the type of organization and check out the X- ray or CT of the disease can not be checked out. 比较而言,CT和X-线只能对组织和器官中电子密度的差异敏感。 In comparison, CT and X- line only tissues and organs of the difference electron density sensitive. 由MRI技术能够得到的影像,由于具有更好的立体分辨率,还能使医生检查出比CT能够检查出的更小的结构。 By MRI imaging techniques can be due to better resolution, three-dimensional, so that doctors can check out the CT ratio can check out the smaller structures. 另外,使用MRI技术可以容易地获得任何影像扫描平面,包括横向,冠状和矢状的。 Further, using MRI techniques can be easily obtained for any image scanning plane, including transverse, coronal and sagittal.

目前,MRI被广泛用于许多医学疾病的辅助诊断。 Currently, MRI is widely used to assist in the diagnosis of many medical conditions. 例如,关节损伤,骨髓疾病,软组织肿瘤,纵隔浸润,淋巴结病,海绵状血管瘤,血友病,肝硬变,肾细胞癌,子宫平滑肌瘤,子宫内膜异位,乳腺癌,狭窄性病性,冠状动脉疾病,分割性动脉瘤,脂肪瘤肥大,房间隔病,缩窄性心包炎,等等。 For example, joint damage, bone marrow disease, soft tissue tumors, mediastinal invasion, lymphadenopathy, cavernous hemangioma, hemophilia, liver cirrhosis, renal cell carcinoma, uterine fibroids, endometriosis, breast cancer, sexually transmitted diseases stenosis sex, coronary artery disease, aneurysm segmentation, lipoma hypertrophy, atrial septal disease, constrictive pericarditis, and so on. [见,例如,Edelman & Warach,Medical Progress 328:708-716(1993);Edelman & Warach,New England J.of Medicine328:785-791(1993)]。 [See, e.g., Edelman & amp; Warach, Medical Progress 328: 708-716 (1993); Edelman & amp; Warach, New England J.of Medicine328: 785-791 (1993)].

常规使用的磁共振成像目前是基于来自细胞内水分子的质子信号。 Conventional magnetic resonance imaging is currently used is based on a signal from the protons of water molecules within cells. 因此,经常难以解释影像和区分单独器官及细胞结构。 Therefore, it is often difficult to explain and distinguish images of organs and cell structure alone. 有两种可能的方法来更好地区分质子信号。 There are two possible ways to better differentiate proton signals. 第一种是使用对比剂来改变一个区域内水分子的T1或T2以与其他区域比较。 The first is to change the use of contrast agents within a region T1 or T2 of the water molecules in comparison with other regions. 例如,亚乙基三胺五乙酸钆(Gd-DTPA)可以缩短它附近水分子的质子T1松驰时间,从增强了所获得的影像。 For example, diethylenetriamine pentaacetic acid gadolinium (Gd-DTPA) shortens the proton it can be close to the T1 relaxation time of water molecules, from the enhanced image obtained.

如上所述,顺磁阳离子,例如,Gd,Mn,和Fe是良好的MRI对比剂。 As mentioned above, the paramagnetic cations, for example, Gd, Mn, and Fe are good MRI contrast agents. 它们能够缩短周围水的质子T1松驰时间,以增强所获得的MRI影像,否则这种影像是无法阅读的。 They can shorten the surrounding water proton T1 relaxation time, in order to enhance MRI images obtained, otherwise the image is unreadable.

第二种区分单独器官和细胞结构的方法是导入另一种能够成像的原子核(即,成像剂)。 A second distinction between individual organs and cellular structures is to import another method capable of imaging nuclei (i.e., an imaging agent). 使用第二种方法,只能在对比剂到达的地方成像。 With the second method, only imaging contrast agent to reach places. 这种方法的优点是排除周围水的干扰而获得影像。 The advantage of this method is to exclude interference of the surrounding water is obtained images. 合适的对比剂必须是生物相容的(即,无毒,化学稳定性,不与组织发生反应)并能在有限的时间内从体内消除。 Suitable contrast agents must be bio-compatible (i.e., non-toxic, chemical stability, does not react with the tissue) and can be eliminated from the body within a limited time.

虽然氢一直被选为MRI扫描的典型基础(因为体内有大量的氢),但是由于缺乏对比,产生的是非常差的影像区域。 Although hydrogen has been chosen as the basis of a typical MRI scan (because the body has a lot of hydrogen), but due to lack of contrast, the resulting image is very poor area. 因此,使用其他的活性MRI原子核(如氟)具有优越性。 Therefore, the use of other active MRI nuclei (such as fluorine) having a superiority. Mattery曾在文章中[见SPIE,626,XIV/PACS IV,18-23(1986)]描述了在不同诊断成像技术如超声波,磁共振,放射照相和计算机体层照相中使用特定的全氟化碳。 Mattery once in the article [see SPIE, 626, XIV / PACS IV, 18-23 (1986)] describes the use of various diagnostic imaging technologies such as ultrasound, magnetic resonance, radiography and computer tomography in particular perfluorinated Carbon. 使用氟是有益的,因为在体内天然不存在氟。 Fluorine is advantageous since fluorine is not present naturally in the body.

现有技术提到的可用于医学诊断目的的磁共振的含氟化合物限于从一组水溶性或能够形成乳剂的含氟分子中选择。 Fluorine-containing compounds may be used for medical diagnostic purposes mentioned prior art magnetic resonance limited or selected from the group of water-soluble fluorine-containing molecules capable of forming an emulsion of. 因此,现有技术中使用水溶性氟化碳的氟化碳乳剂具有行多缺点,例如,1)使用了不稳定的乳剂,2)缺乏器官特异性和目标性,3)由于使用了乳化剂和表面活性剂(如卵磷脂和卵黄卵磷脂)有可能诱发过敏反应,4)传送能力有限,和5)静泳注射后这种水溶性氟化碳很快被血液稀释。 Thus, the prior art use a water-soluble carbon fluorinated fluorocarbon emulsions having rows and drawbacks, e.g., 1) the use of unstable emulsions, 2) the lack of organ-specific and targeted, 3) the use of emulsifiers and a surfactant (such as lecithin and egg yolk lecithin) may induce allergic reactions, 4) a limited transmission capacity, and 5) water soluble fluorocarbons such static swimming after injection blood was quickly diluted.

根据本发明,提供了用于体内传送生物制品的,适合于以水悬浮液非肠道给药的微颗粒形式的组合物。 According to the present invention, provides a method for in vivo transfer of biological products, adapted to form an aqueous suspension of microparticles for parenteral administration of the compositions. 本发明组合物含有与聚合物外壳相结合的生物制品(固体、液体或气体的)。 The compositions of the invention contain a polymer shell combination biological products (solids, liquids or gases). 该聚合物外壳是一种有二硫键存在下交联的生物相容材料。 The polymer shell has a disulfide bond in the presence of a crosslinked biocompatible material. 与生物制品相结合的聚合物外壳可以悬浮于一种生物相容基质中给药。 And a combination of biological products can be suspended in a polymer shell one biocompatible matrix is administered. 用本发明组合物传送生物制品排除了以含有,例如,乙醇和多乙氧基蓖麻油,并用生理盐水稀释的乳剂形式给药生物制品的必要性(见,例如,Norton et al.,inAbstracts of the 2nd National Cancer Institute Workshopon Taxol & Taxus,September 23-24,1992)。 Transfer Biologicals composition of the present invention excludes containing, for example, ethanol and polyethoxylated castor oil, diluted with normal saline and the necessity of administration in the form of an emulsion with biological products (see, for example, Norton et al., InAbstracts of the 2nd National Cancer Institute Workshopon Taxol & amp; Taxus, September 23-24,1992). 这些已知组合物的缺点是它们有可能产生过敏副作用。 The disadvantage of these known compositions is that they may produce allergic side effects.

根据本发明的另一方面,惊奇和未预料地发现根据本发明制备的不溶结构的血红蛋白(Hb)可以可逆性地结合氧。 According to another aspect of the invention, surprising and unexpected to find that can be reversibly bound oxygen prepared in accordance with the present invention insoluble hemoglobin structure (Hb). 本发明的不溶性血红蛋白结构(IHC)结合氧的氧亲合性与用红细胞中可溶性血红蛋白分子或现有技术中曾描述过的用作可能的血液代用品的可溶性改性的血红蛋白分子获得的氧亲合性相似。 Oxygen affinity hemoglobin molecule insoluble hemoglobin structure (IHC) of the present invention binds oxygen with an oxygen affinity and hemoglobin molecules in red blood cells or soluble prior art has described the use as a blood substitute soluble possible to obtain the modified adhesion similar.

根据本发明的另一方面,还提供了将生物制品包裹在聚合物外壳中的方法。 According to another aspect of the present invention, there is also provided a method of biological products wrapped in a polymer housing. 本发明的另一方面,还提供了获得局部氧和温度数据的方以及获得体内器官和组织的氟磁共振影像的方法。 Another aspect of the present invention also provides a method for obtaining local oxygen and temperature data side and obtaining fluorine magnetic resonance images of body organs and tissue.

以微颗粒混悬液的形式传送生物制品,通过使不同大小的颗粒和通过不同途径给药,可以在某种程度上到达靶器官如肝脏,肺脏,脾脏,淋巴循环系统,等。 To form a suspension of microparticles transfer biological products, by making different sizes of particles and administered by different routes, could somehow reach the target organ such as the liver, lungs, spleen, lymphatic circulation, and the like. 本发明的传送方法还使得生物制品给药,例如基本上水不溶性药理活性成份的给药,以比现有技术的传送系统所需的给药体积和时间(即:传送一典型人体剂量的200-400mg的紫杉醇需要在24小时的时间内静脉输液大约1至2升的液体)更小的液体量和大大减少的给药时间来完成。 Transmission method of the present invention also enables administration of biological products, such as administration of substantially water insoluble pharmacologically active ingredient, the administration volume and the time to transfer than the prior art systems required (ie: transmitting a typical human dose of 200 -400mg paclitaxel required within 24 hours of intravenous infusion of approximately one to two liters of liquid) and a smaller amount of the liquid greatly reduced administration time to complete.

例如,本发明聚合物外壳的混合液可以静脉内给药,使得产生血管化器官(如肝脏,脾脏,淋巴和肺)和骨髓的影像成为可能。 For example, the present invention is a mixture of polymer shell may be administered intravenously, such that a vascularized organs (e.g. liver, spleen, lymph and lung) and bone marrow images becomes possible. 由于网状内皮系统(RES)(也称之为单核吞噬细胞(MNP)系统)能够摄取微米大小的含有机氟的聚合物外壳,因此可以获得器官靶特异性。 Since the reticuloendothelial system (RES) (also known as the mononuclear phagocyte (MNP) system) can image micron-sized polymer shell containing fluorine, organ target specificity can be obtained. 象肝脏和脾脏这样的器官在从血液中清除外来品种物质(如,颗粒物质)过程中起着重要作用,因此经常被称之为“血液过滤器官”。 Organs such as liver and spleen remove foreign matter from the blood species (e.g., particulate matter) plays an important role in, it is often referred to as "blood filtering organs."

这些器官组成了大部分的网状内皮系统。 These organs make up most of the reticuloendothelial system. 此外,淋巴循环中的淋巴结合有网状内皮系统的细胞。 In addition, lymph circulation lymphoid reticuloendothelial system incorporating cells. 所以,使用本发明的微米大小的含有机氟聚合物外壳使淋巴系统的成像成为可能。 Therefore, the present invention is the use of micron size of the fluorine-containing polymer shell of the imaging of the lymphatic system is possible. 口服给药或作成一种栓剂,也能进行胃和胃肠道的成像。 For oral administration or suppositories made one kind, can be carried out in the stomach and gastrointestinal tract imaging. 这种悬浮液也能被注入非血管部位,如脑脊髓腔使这些部位成像。 This suspension can also be injected into the non-vascular sites, such as the chamber so that these parts of the brain and spinal cord imaging.

作为本发明的又一实施例,顺磁阳离子如Gd.Mn,Fe等能与多聚阴离子如藻酸盐连结,并用作一种有效的MRI对比试剂。 As a further embodiment of the present invention, paramagnetic cations with polyanions such as alginates Quick e.g. Gd.Mn, Fe, etc., and used as an effective MRI contrast agent.

本发明通过下述方法克服了现有技术中的缺陷,即提供了1)含生物制品的聚合物外壳的可注射用悬浮液,2)与简单的乳化液相比具有增强了的稳定性形式的生物制品,3)由于RES或MNP系统摄取本发明的聚合物外壳,而具有靶器官的特异性(例如,肝,脾,肺等),4)无乳化剂体系,由此避免了可能潜在引发过敏反应的试剂,以及5)由于本发明含有生物制品的聚合物外壳能够把一个特定器官作为靶目标,从而具有注射相对少剂量的生物制品并仍能获得较好反应的性能。 By a method of the present invention overcomes the deficiencies of the prior art, i.e., provided a) biological products containing polymer shell injectable suspension, 2) as compared with a simple emulsion form having enhanced stability biological products, 3) due to the RES or MNP system uptake polymer shell of the invention, having target organ specificity (e.g., liver, spleen, lung, etc.), 4) emulsifier-free system, thereby avoiding the potentially Reagents trigger allergic reactions, and 5) Since the present invention is a polymer shell containing biological products able to target a particular organ, as a target, so as to have a relatively small doses of injection of biological products and still obtain a better performance of the reaction.

图1是按照本发明所制备的一种聚合物外壳的示意图。 Figure 1 is a schematic diagram in accordance with the present invention is prepared in a polymer shell. 图中,A指不溶性二硫化物交联的聚合物外壳,B指该聚合物外壳的内部,它含有氧或其它气体,含有溶有氧的氟代烃,溶有生物制品的一种生物相容油,含水基质中溶有生物制品的油包水型乳化液,液体中扩散有固体颗粒的一种混悬液等等,C表示该聚合物外壳的厚度,一般大约为5-50毫微米,而D是表示该聚合物外壳的直径,一般大约在0.1至20微米的范围内。 Figure, A refers to the insoluble disulfide polymer shell crosslinked, B refers to the interior of the polymeric shell, which contains oxygen or other gases, including a dissolved oxygen fluorocarbon, a solution of a biological phase of biological products Yung oil, an aqueous solution of the matrix of biological products in-oil type emulsion, the liquid diffusion of a suspension of solid particles, etc., C represents the thickness of the polymeric shell, typically about 5-50 nm and D is the diameter of the polymer shell, generally approximately in the range of 0.1 to 20 microns.

图2表示无基质的血红蛋白溶液的氧结合曲线(虚线曲线)和含有本发明不溶性血红蛋白结构的溶液的氧结合曲线(实线曲线)(即,以希耳氏系数与氧分压的函数表示的曲线)。 Figure 2 shows oxygen stroma-free hemoglobin solution binding curve (dashed curve) and an oxygen-containing solution of the present invention insoluble hemoglobin structure binding curve (solid curve) (i.e., to the Greek ear's coefficient expressed as a function of oxygen partial pressure curve). 本发明不溶性血红蛋白结构的实际数值点用实心框表示。 The actual value of the present invention insoluble hemoglobin dot structure is represented by a solid box.

图3表示无基质的血红蛋白溶液的氧结合曲线(虚线曲线)和含有用1.7mM变构效应物2,3-双磷酸甘油酯(2,3-BPG)处理后的本发明不溶性血红蛋白结构的溶液的氧结合曲线(实线曲线)。 Figure 3 shows a solution of the present invention, stroma-free hemoglobin solution after the oxygen binding curve (dashed curve), and contains from 1.7mM allosteric effector 2,3-bis-phosphoglycerate (2,3-BPG) treated insoluble hemoglobin structure oxygen binding curve (solid curve). 本发明不溶性血红蛋白结构的实际数值点用实心框表示。 The actual value of the present invention insoluble hemoglobin dot structure is represented by a solid box.

按照本发明,提供一种体内传送生物制品的组合物,其中所述的生物制品选自于:基本上完全包含在一种聚合物外壳中的一种固体,它可以是分散在一种生物可适应的扩散剂中,基本上完全包含在一种聚合物外壳中的一种液体,它可以是分散在一种生物可适应的扩散剂中,基本上完全包含在一种聚合物外壳中的一种气体,它可以是分散在一种生物可适应的扩散剂中,与聚合物外壳相结合的一种气体,或者是上述任意两种或多种的混合物,其中该壳最大横切面直径不大于10微米左右,其中该聚合物外壳含有一种基本上以二硫键交联的生物可接受的物质,以及其中该聚合物外壳的外部可以用一种适当试剂任意改性,其中该试剂是通过共价键与该聚合物外壳连接的。 According to the present invention, there is provided a composition for in vivo transfer of biological products, wherein said biological product is selected from: substantially completely solid in a polymer comprising a housing, which can be dispersed in a biologically Adaptation of the diffusing agent, substantially completely contained in a liquid in a polymer shell, it can be dispersed in a biological adaptable diffusing agent, substantially completely contained within a polymeric shell of a gases, which may be dispersed in a biological adaptable diffusing agent, a combination of gas and the polymer shell, or to any two or more thereof, wherein the shell diameter is not greater than the maximum cross-section about 10 microns, wherein the polymer housing comprises a substantially disulfide crosslinked biologically acceptable material, and wherein the exterior of the polymer shell can be arbitrarily modified with a suitable reagent, wherein the reagent by covalently attached to the polymer shell.

这里所用的术语“体内传送”是指通过口服、静脉、皮下、腹膜内、鞘内、肌肉内、颅内、吸入、局部、经皮、栓剂(直肠)、阴道栓(阻道)等等给药途径来输送一种生物制品。 The term "in vivo transfer" as used herein refers to oral, intravenous, subcutaneous, intraperitoneal, intrathecal, intramuscular, intracranial, inhalation, topical, transdermal, suppository (rectal), pessary (channel resistance) and the like to ways to convey a biological drug products.

这里所用的术语“生物制品”是指药物活性剂(如止痛剂,麻醉剂,止喘药,抗菌素,抗抑郁药,抗糖尿病药,抗真菌剂,抗高血压药,抗炎剂,抗肿瘤剂,抗焦虑剂(anxiolytic a-gents),酶活性剂,核酸结构,免疫促进剂,免疫抑制剂,生理活性气体,疫苗等),诊断试剂(如超声对比剂,放射对比剂,或磁对比剂),有营养价值的试剂等等。 As used herein, the term "biological products" refers to pharmaceutically active agents (such as analgesic agents, anesthetic agents, antiasthmatics, antibiotics, antidepressants, antidiabetics, antifungal agents, antihypertensive agents, antiinflammatory agents, antitumor agents , anxiolytics (anxiolytic a-gents), activator of the enzyme, nucleic acid structure, immune enhancers, immunosuppressants, physiologically active gases, vaccines, etc.), diagnostic reagents (such as ultrasound contrast agents, radiocontrast agents, or magnetic contrast agents ), nutritional value reagents and so on.

这里所用的术语“微米”是指毫米的千分之一的测量单位。 As used herein, the term "micron" refers to millimeters of thousandth of the unit of measurement.

许多生物相容物质可以被用于实施本发明形成一种聚合物外壳。 Many biocompatible materials may be used in the practice of the present invention to form a polymer shell. 这里所用的术语“生物相容”是指不会以任何有害方式明显改变或影响它所被导入的生物系统的物质。 As used herein, the term "biocompatible" means not significantly changed in any way harmful or Substances which it is introduced into the biological system. 基本上任何天然或合成的结构中含有巯基或二硫键的物质可用来制备二硫键交联的外壳。 Essentially any natural or synthetic structures containing a mercapto group or a disulfide linkage of substance used to prepare disulfide crosslinked shell. 巯基或二硫键可预先存在于生物相容物质结构中,或者通过一种适宜的化学改性将它们导入。 Mercapto group or a disulfide linkage may be pre-existing in the structure of the biocompatible material, or by a suitable chemical modification import them. 例如,对这种改性来说天然产生的生物相容物质如蛋白质,多肽,多核苷酸,多糖(如藻酸酯或盐、高甘露糖醛酸含量的藻酸酯或盐、多聚甘露糖醛酸、多聚甘露糖醛酸盐,透明质酸,透明质酸盐,脱乙酰壳多糖、几丁质、纤维素、淀粉、糖原、瓜耳胶、刺槐豆胶、葡聚糖、果聚糖、旋复花粉、环葡聚糖、琼脂糖、黄原胶、卡拉吉兰(carageenan)、肝素、果胶、盖兰(gellan)胶、硬葡聚糖、或它们任意两者或更多组合),脂类,等等都是选择的物质。 For example, such modified organisms is naturally occurring compatible substances such as proteins, polypeptides, polynucleotides, polysaccharides (e.g., alginic acid esters or salts, high mannuronic acid content of the alginic acid esters or salts thereof, poly-mannose uronic acid, poly mannuronate, hyaluronic acid, hyaluronate, chitosan, chitin, cellulose, starch, glycogen, guar gum, locust bean gum, dextran, fructan, inulin, cyclodextrin, agarose, xanthan gum, Kara selegiline (carageenan), heparin, pectin, Gerland (gellan) gum, scleroglucan, or any two of them, or more combinations), lipids, and the like substances are selected. 其它连接键如酯键,酰胺键,醚键等也能在超声照射步骤中形成(只要在起始物中含有必要的官能团)。 Other linkages such as an ester bond, an amide bond, an ether bond or the like can be formed (as long as the necessary starting materials containing functional groups) in the ultrasonic irradiation step.

作为生物相容物质的适当例子,天然产生的或合成的蛋白只要具有足够的巯基或二硫键以形成交联(例如,在超声照射中由于氧化作用通过二硫键形成),这些蛋白质都可以使用。 The suitable examples of biocompatible materials, naturally occurring or synthetic protein, as long as having a mercapto group or a disulfide bond sufficient to form a crosslinked (e.g., in the ultrasonic irradiation is formed by a disulfide bond by oxidation), these proteins can be use. 蛋白质的适当例子包括白蛋白(含有35个半胱氨酸残基)、胰岛素(含有6个半胱氨酸残基)、血红蛋白(每个α2β2单位含有6个半胱氨酸残基)、溶菌酶(含有8个半胱氨酸残基)、免疫球蛋白、α-2-巨球蛋白、纤连蛋白(fibronectin)、玻连蛋白(vitronectin)、纤维蛋白原等以及它们中任意两种或多种的组合物。 Examples of suitable proteins include albumin (containing 35 cysteine residues), insulin (containing six cysteine residues), hemoglobin (α2β2 each unit containing six cysteine residues), lysozyme and any two or enzymes (containing 8 cysteine residues), immunoglobulins, α-2- macroglobulin, fibronectin (fibronectin), vitronectin (vitronectin), fibrinogen them a variety of compositions.

目前用于形成聚合物外壳的优选蛋白质是白蛋白。 Preferably the protein is currently used to form the polymer shell is albumin. 用于形成聚合物外壳的其它优选蛋白质是血红蛋白。 Other preferred proteins used to form the polymer shell is hemoglobin. 还有用于形成聚合物外壳的其它优选蛋白质是白蛋白和血红蛋白的混合物。 Other preferred proteins as well as for forming a polymer shell is a mixture of albumin and hemoglobin. 或者,象α-2-巨球蛋白(一种已知的调理素)这样的蛋白质可以用来促进巨噬细胞样细胞对包裹有生物制品颗粒的外壳的摄取,或者促进肝脏、脾脏对外壳包裹颗粒的摄取。 Or, like the α-2- macroglobulin (a known opsonin) Such proteins can be used to promote macrophage-like cells of biological products wrapped shell particle uptake, or promote liver, spleen parcel of the housing ingest particles. 在形成聚合物外壳过程中,也可以使用其他的功能性蛋白质,如抗体或酶,它们能够促进传送生物制品到达所希望的靶位点。 In the process of formation of the polymer shell, it is also possible to use other functional proteins, such as antibodies or enzymes, which can promote the transfer of biological products to reach the desired target site.

类似地,含有巯基或二硫键的合成多肽对形成具有一种聚合物外壳的颗粒来说也是良好的可选择物质。 Similarly, synthetic polypeptides containing a mercapto group or a disulfide linkage to the formation of particles having a polymer shell material is also a good alternative. 此外,聚二醇(如,直链或支链的),聚乙烯醇,甲基丙烯酸多羟基乙酯,聚丙烯酸,聚乙基噁唑啉,聚丙烯酰胺,聚乙烯吡咯烷酮等等对化学改性(导入巯基和/或二硫键以及壳体形成(通过引起它们的交联)来说都是良好的可选择物。 In addition, polyalkylene glycols (e.g., straight chain or branched chain), polyvinyl alcohol, polyhydroxy ethyl methacrylate, polyacrylic acid, polyethyloxazoline, polyacrylamide, polyvinyl pyrrolidone and the like on the chemically modified of (introducing a mercapto group and / or a disulfide linkage and the housing is formed (by causing the crosslinking thereof) selectable for both a good thing.

在制备本发明组合物中,人们能够任意使用一种分散剂来悬浮或溶解生物制品。 In preparing the compositions of the present invention, one can optionally use one dispersing agent to suspend or dissolve the biological products. 能够用于实施本发明的分散剂包括能悬浮或溶解生物制品但不与形成壳体所用的聚合物或者生物制品本身产生化学反应的任何液体。 Can be used in the practice of this invention include dispersing agents can be suspended or dissolved in any liquid biological products but not used in forming the shell polymer or a bio-chemical reaction of the article itself. 例如水,植物油(如,大豆油,矿物油,玉米油,菜子油,椰子油,橄榄油,红花油,棉花籽油等),具有4-30个碳原子的脂族、环脂族、或芳香烃(如正-十二烷,正-癸烷,正-己烷,环己烷,甲苯,苯,等),具有1-30个碳原子的脂肪醇或芳香醇(如,辛醇等),具有2-30个碳原子的脂肪酸酯或芳香酯(如辛酸乙酯等),具有2-30个碳原子的烷基,芳基、环醚(如,二乙醚,四氢呋喃等),具有1-30个碳原子的烷基卤化物或芳基卤化物(和可以是一个以上的卤素取代基,如,CH3Cl,CH2Cl2,CH2-Cl-CH2Cl等),具有3-30个碳原子的酮类(如丙酮,丁酮等),聚二醇(如,聚乙二醇等),或者它们中任意两种或多种的混合物。 For example, water, vegetable oils (e.g., soybean oil, mineral oil, corn oil, rapeseed oil, coconut oil, olive oil, safflower oil, cotton seed oil, etc.), aliphatic 4 to 30 carbon atoms, cycloaliphatic, or an aromatic hydrocarbon (such as n - dodecane, n - decane, n - hexane, cyclohexane, toluene, benzene, etc.), fatty alcohols or aromatic alcohols having 1-30 carbon atoms (e.g., octanol etc.), having 2 to 30 carbon atoms, a fatty acid ester or aromatic esters (e.g., ethyl caprylate, etc.), an alkyl group having 2-30 carbon atoms, an aryl group, a cyclic ether (e.g., diethyl ether, tetrahydrofuran, etc.) , having 1-30 carbon atoms, an alkyl halide or aryl halide (and may be more than one halogen substituent, e.g., CH3Cl, CH2Cl2, CH2-Cl-CH2Cl, etc.), having 3-30 carbon atoms ketones (such as acetone, methyl ethyl ketone, etc.), polyglycols (e.g., polyethylene glycol, etc.), or of any two or more thereof.

特别优选的分散剂组合物包括挥发性液体如二氯甲烷,乙酸乙酯,苯等(即,对药物活性剂有高级溶解性并能溶于所用的其它分散剂的溶剂)和一种挥发性较低的分散剂。 Particularly preferred dispersant composition comprising a volatile liquid such as dichloromethane, ethyl acetate, benzene and the like (i.e., have a high level of solubility of the pharmaceutically active agent is soluble in the solvent used and other dispersant) and a volatile lower dispersant. 当加入到其它分散剂时,这些挥发性添加剂有助于促进药物活性成份在该分散剂中的溶解。 When added to the other dispersing agent, these Volatile additives help to promote the pharmaceutically active ingredient is dissolved in the dispersant. 由于该步骤常耗费时间所以这种混合物是最理想的。 Since this step is often time consuming so this mixture is ideal. 溶解完成后,可以蒸发除去这些挥发性组分(任选在真空下)。 After complete dissolution, the volatile components can be removed by evaporation (optionally under vacuum).

按本发明所述制备的,基本上完全包含在聚合物外壳中或与之结合的生物制品颗粒,可以是只以颗粒形式给药,或者是以一种溶于生物相容基质中的混悬液给药。 Prepared according to the present invention, substantially completely contained in the polymer particles of biological products or in connection with the housing, and may be administered in the form of particles only, or based on a biocompatible matrix suspended dissolved fluid administration. 这种基质可以选自于水,缓冲的含水基质,生理盐水,缓冲的生理盐水,任选缓冲的氨基酸溶液,任选缓冲的蛋白溶液,任选缓冲的糖溶液,任选缓冲的碳水化合物溶液,任选缓冲的维生素溶液,任选缓冲的合成聚合物溶液,含脂类的乳化液,或它们任意两者或多者的组合。 The matrix can be a carbohydrate solution selected from water, buffered aqueous medium, physiological saline, buffered saline, optionally buffered solutions of amino acids, optionally buffered protein solution, optionally buffered solutions of sugars, optionally buffered , optionally buffered solutions of vitamins, optionally buffered solutions of synthetic polymers, lipid-containing emulsions, or any combination of two or more of them.

按照本发明的其它实施方案,还提供了用于体内传送的生物制品制剂的制备方法,该方法包括把含有能够被二硫键交联的生物相容物质的基质和生物制品经过高强度超声条件处理一段时间,足以促使该生物相容物质被二硫键所交联;其中所述的生物制品基本上完全包含在聚合物外壳中,并且其中该壳的最大横切直径不大于10微米。 According to other embodiments of the present invention, there is provided a method for the preparation of biological products for the in vivo transfer agents, the method comprising containing disulfide bonds can be crosslinked by a biocompatible matrix material and biological products through the high intensity ultrasound conditions processing a time, sufficient to cause the biocompatible material being cross-linked disulfide bonds; wherein said biological product is substantially completely contained in the polymer shell, wherein the shell and the maximum transverse diameter of not more than 10 microns.

因此,按照本发明,包含在聚合物外壳中的生物制品是用高强度超声合成的。 Thus, according to the present invention, comprising in the polymer shell of the biological products are synthesized using high intensity ultrasound. 在形成稳定的聚合物外壳中包括两个非线性声学处理步骤(即,声波乳化和空化)首先,声波乳化使该生物制品分散在蛋白水溶液中。 Two non-linear acoustic processing includes the step of forming a stable polymeric shell (i.e., acoustic emulsification and cavitation) First, acoustic emulsification so that the biological products dispersed in an aqueous solution of the protein. 然后通过形成二硫键来化学交联和稳定所形成的分散体。 Then by a disulfide bond to form a dispersion and stable chemical crosslinking formed. 用经过声波空化产生的过氧化物氧化半胱氨酸的残基(这里的聚合物是一种蛋白质如白蛋白)来形成二硫键。 After oxidation with peroxide generated acoustic cavitation cysteine residue (where polymer is a protein such as albumin) to form a disulfide bond.

可选用离心过滤器(100KDa截获)过滤所得到的悬浮液并把滤得的结构或微囊悬浮于生理盐水或适当的缓冲液中。 Optional centrifugal filter (100KDa intercepted) filtering the resulting suspension was filtered and the resulting structure or microcapsules suspended in a physiological saline or a suitable buffer. 图1显示了这样一种结构的示意图。 Figure 1 shows a schematic of such a structure. 这些结构的平均直径为2微米左右。 The average diameter of these structures is about 2 microns. 用一台Elzone颗粒计数器所测定的颗粒大小分范围是相当窄的(一般观察到平均直径在3微米左右的高斯分布)。 Particle size range using a Elzone divided particle counter measured is relatively narrow (typically on the average, about 3 microns in diameter Gaussian distribution). 用这种技术得到的颗粒大小范围是在0.1至20微米之间。 Particle size range obtained by this technique is between 0.1 to 20 microns. 优选颗粒大小范围在0.5到10微米之间,最优选范围是1到5微米。 Preferably a particle size range between 0.5 to 10 microns, most preferably in the range of 1 to 5 microns. 这种颗粒大小特别适合于医学应用,这是因为能够进行静脉内或动脉内注射,而没有小血管堵塞和继发组织(因氧气缺乏而局部缺血)坏死的危险。 Such particle size is particularly suitable for medical applications, this is because it can be intravenous or intra-arterial injection, a small blood vessel blockage and without secondary tissue (ischemia due to oxygen deficiency) dangerous necrosis. 作为对照,正常红细胞直径大约是8微米。 As a control, normal red blood cell diameter of about 8 microns.

上述步骤的非显而易见特征是分散剂的选择,物别涉及到分散剂的极性选择。 Non-obvious features of the above step is to select a dispersant, was not related to the polarity select dispersant. 有关生物制品颗粒的壳体的形成过程包括,在水相和非水相之间的交界处,使生物相容物质重新取向,从而使生物相容物质中的亲水区暴露于水相,而生物相容物质中的疏水区定向于非水相。 For the formation of particles of biological products include a housing, at the junction between the aqueous phase and the non-aqueous phase, biocompatible material reoriented so that the biocompatible material of the hydrophilic region is exposed to the aqueous phase, and biocompatible substances in hydrophobic regions oriented in a non-aqueous phase. 在该生物相容物质是一种蛋白质的情况下,为了完成其构象的有效展开或改变,必须给这种聚合物提供能量。 In the case where the biocompatible material is a protein, in order to complete its conformation effective to expand or change, energy must be supplied to such a polymer. 在两种液相(即水相和非水相)间的界面自由能(界面张力)使界面处的蛋白构象发生改变。 Interface in two liquid phases (i.e., aqueous and non-aqueous phase) between the free energy (interfacial tension) so that the conformation of the protein at the interface change. 热能也是蛋白构象展开和/或改变所需的能量库。 Thermal energy is protein conformation expand and / or change the energy required libraries.

热能输入是下列变量的函数,即:高强度超声波辐射过程中所用的声音功率,高强度超声波辐射的时间,经受高超率超声波辐射的物质性能,经受高强度超声波辐射的物质性质等。 Heat input is a function of the following variables, namely: high intensity ultrasound radiation used during the sound-power, high intensity ultrasonic irradiation time, undergo substance superb performance rate of ultrasonic radiation, undergo physical properties such as high-intensity ultrasonic radiation. 高强度超声波辐射过程中的声音功率变化范围很大,一般在1到1000特/厘米2的范围内;50到200瓦特/厘米2是声音功率的优选范围。 Sound power range of high intensity ultrasonic irradiation process is large, generally in the range from 1 to 1000 dtex / cm 2; 50 to 200 watts / cm 2 is preferred sound power range. 类似地,暴露于高强度超声波辐射的时间变化范围也很大,一般在几秒钟到大约5分钟的范围内。 Similarly, exposure time to high intensity ultrasonic irradiation range of variation is also large, typically in the range of a few seconds to about 5 minutes. 在高强度超声波辐射中的暴露时间最好在大约15到60秒钟范围内。 In the high intensity ultrasonic irradiation in the exposure time is preferably in the range of about 15 to 60 seconds. 本领域熟练技术人员公知所用的声音功率越高,需要在高强度超声波辐射的暴露时间就越短,反之亦然。 The known to a person skilled sound used for higher power, the shorter the required high-intensity ultrasonic irradiation exposure time and vice versa.

界面自由能与两种液体间极性差值成正比。 Interfacial free energy is proportional to the polarity difference between the two liquids. 所以在指定操作温度下两种液体间界面处的最小自由能是形成所需聚合物外壳的基本能量。 Therefore, at the minimum specified operating temperature at the interface between the two liquids free energy is the energy required to form the basic polymer shell. 因此,如果使用同源系列的分散剂而逐渐改变其极性,如,烷基酸乙酯,那么同源性越大就越没有极性,即,这些分散剂和水间的界面张力是随着酯类中碳原子数目的增大而增加的。 Thus, if a homologous series of dispersing agents gradually changes its polarity, e.g., ethyl group, then the greater the more homology no polarity, i.e., the interfacial tension between these dispersing agents and water is with the esters increased the number of carbon atoms increases. 所以,我们发现,尽管乙酸乙酯是与水不溶混的(即,一种两个碳原子酸的酯),但是在室温下(~20℃),这种分散剂单独不会产生较大产率的聚合物外壳包裹的颗粒。 Therefore, we found that, although ethyl acetate is water-immiscible (i.e., one in which two carbon atoms, esters of the acids), but at room temperature (~ 20 ℃), this dispersing agent alone will not produce a greater yield polymer shell wrapped particle rate. 相反,一种高级酯如辛酸乙酯(一种8个碳原子酸的酯)却产生较高产率的聚合物外壳包裹的颗粒。 In contrast, a high ester such as ethyl octanoate (8 carbon atoms, one kind of acid ester) has produced a higher yield of polymer shell coated particles. 实际上,庚酸乙酯(一种7个碳原子酸的酯)产生中等产率的颗粒而较低级的酯(3,4,5或6个碳原子酸的酯类)得到更低的产率。 In fact, ethyl heptanoate (7 carbon atoms, one kind of acid ester) generation of particles moderate yields and lower level of ester (4, 5 or 6 carbon atoms, an acid ester) to obtain a lower Yield. 因而,在指定温度下,人们能够设定一个形成高产率聚合物外壳包裹的颗粒所需的最小含水分散剂界面张力的条件。 Thus, at a given temperature, it is possible to set a minimum interfacial tension of aqueous dispersions of the conditions required for a high yield of polymer shell coated particle formation.

温度是可以被控制影响聚合物外壳包裹颗粒产率的另一个变量。 Temperature is another variable that can be controlled polymer shell wrapped particles affect yield. 通常,液体表面张力随着温度的增长而降低。 Typically, the surface tension of the liquid with the growth temperature decreases. 温度对表面张力的变化率经常因不同液体而不同。 Rate of change of temperature on the surface tension of the liquid often vary different. 所以,例如,两种液体之间的界面张力(Δγ)可以是T1温度下的Δγ1和T2温度的Δγ2。 Therefore, for example, the interfacial tension between the two liquids (Δγ) may be Δγ1 temperature T1 and temperature T2 of the Δγ2. 如果T1下的Δγ1与形成本发明聚合物外壳所需的最小值接近,并且,如果Δγ2(T2温度下)大于Δγ1,那么,从T1到T2的温度改变将增加聚合物外壳的产率。 If Δγ1 T1 under the minimum required polymer formed shell close to the present invention, and, if Δγ2 (at temperature T2) is greater than Δγ1, then the temperature change from T1 to T2 will increase the yield of polymer shell. 实际上,这种变化在使用庚酸乙酯的情况下可以观察到,在20℃庚酸乙酯得到中等产率而在10℃得到较高产率。 In fact, this change in the case of ethyl heptanoate can be observed, at 20 ℃ heptanoate to give moderate yields obtained in high yields at 10 ℃.

温度也影响所用液体的蒸气压。 Temperature also affects the vapor pressure of the liquid. 温度越低,总蒸气压就越低。 The lower the temperature, the lower the total vapor pressure. 总蒸气压越低,空泡的破裂就越有效。 The total vapor pressure is lower, the more effective the vacuole rupture. 超声波辐射空泡的崩破率增加与过氧化物(HO2-)形成率的增加相关。 The collapse of cavitation ultrasonic irradiation breaking rate increases with peroxide (HO2-) associated with an increased rate of formation. 过氧化物生成率增加可以使较低温上聚合物外壳的产率增加。 Peroxide generation rate can increase the yield of polymer shell on the lower temperature increase. 但是,尽管如此,与此相反,用过氧化物离子氧化巯基(即,形成二硫键)的反应率是随着温度的增长而增加的。 But, nevertheless, on the contrary, the reaction rate of oxidation of a mercapto group with a peroxide (i.e., disulfide bond formation) is that as the growth temperature increases. 所以,对一种经过超声波辐射条件处理的指定液体来说,存在一个能够获得高产率聚合物外壳的相当窄的最佳温度操作范围。 Therefore, for a specified liquid through ultrasonic irradiation process, the existence of a shell polymer can be obtained in a high yield of a relatively narrow range of optimum operating temperature.

因而两种作用的结合,即,表面张力随温度的变化(它直接影响到该聚合物的展开和/或构象的改变)和反应产率随温度的变化(该反应是指通过形成二硫键而发生的聚合物交联)这两者的结合表示了聚合物外壳包裹颗粒的总体变化和产率。 Thus combining the two effects, i.e., change in surface tension with temperature (which directly affects the polymer to expand and / or conformational change), and the reaction yield change with temperature (the reaction is through the formation of disulfide bonds The cross-linking of the polymer) represents a combination of the two yields the overall change and coated particles of polymer shell. 适合于制备本发明聚合物外壳的温度在大约0-80℃的范围内。 Temperature suitable for the preparation of the polymers of the present invention, the housing is in the range of about 0-80 ℃.

可对上述超声波辐射步骤进行操作来生产含有具有一定大小范围的生物制品的聚合物外壳包裹的颗粒。 The ultrasonic irradiation step may be operated to produce a package containing a polymer shell particles having a certain size range of biological products. 这里优选颗粒半径在大约0.1至5微米的范围内。 Here radius of the particles is preferably in the range of about 0.1 to 5 microns. 在这个范围内较窄的大小分布非常适合于生物制品的静脉内给药。 Within this range is very narrow size distribution within the biological products suitable for intravenous administration. 最好把聚合物外壳包裹的颗粒悬浮于生物相容的基质中(如本文所描述的),再用适当方式给药。 Preferably the polymer shell coated particles are suspended in a biocompatible matrix (as described herein), then the appropriate mode of administration.

此外,可以用一种适当的试剂对该聚合物外壳进行改性,其中该试剂是通过一种任意共价键与该聚合物外壳结合的。 In addition, with a suitable reagent to the modified polymer shell, wherein the agent is an optionally through covalent bonding with the polymer shell. 能够用于这种结合的共价键有酯键,醚键,氨基甲酸乙酯键,二酯键,酰胺键,仲胺或叔胺键,磷酸酯键,硫酸酯键以及其它类似的键。 This can be used for covalent binding of the ester bond, ether bond, urethane bond, ester bond, an amide bond, a secondary or tertiary amine bond, phosphorothioate bond, ester bond sulfate, and other similar keys. 适合于这种任意改性聚合物外壳的适当试剂有合成聚合物(聚二醇(如直链或支链的聚乙二醇)),聚乙烯醇,甲基丙烯酸多羟基乙酯,聚丙烯酸,聚乙基恶唑啉,聚丙烯酰胺,聚乙烯吡咯烷酮等),磷脂(如磷脂酰胆碱(PC),磷酸酰乙醇胺(PE),磷脂酰肌醇(PI),神经鞘磷脂等),蛋白质(如酶,抗体等),多糖(如淀粉,纤维素,葡聚糖,藻酸盐,脱乙酰壳多糖,果胶,透明质酸等),化学改性剂(如5′-磷酸吡哆醛,吡哆醛的衍生物,二醛,琥珀酰水杨酸酯等),或者它们中任何两种或多种的混合物。 Any appropriate reagent suitable for such a modified polymer shell synthetic polymers (polyalkylene glycols (e.g. linear or branched chain polyethylene glycol)), polyvinyl alcohol, polyhydroxy ethyl methacrylate, polyacrylic acid , polyethyloxazoline, polyacrylamide, polyvinylpyrrolidone, etc.), phospholipids (such as phosphatidylcholine (PC), phosphatidylinositol-ethanolamine (PE), phosphatidylinositol (PI), sphingomyelin, etc.), proteins (such as enzymes, antibodies, etc.), polysaccharides (such as starch, cellulose, dextran, alginate, chitosan, pectin, hyaluronic acid, etc.), chemical modifiers (e.g. 5'-phosphate-pyrazol , or a mixture of any two or more of pyridoxal, pyridoxal derivatives, dialdehydes, succinyl salicylate) them.

用一种聚合物外壳包裹的溶解生物制品的一般情况是可能发生变化的。 Biologics generally dissolved in a polymer shell wrapped possible changes. 可以使用生物制品颗粒在生物相容分散剂中的混悬液(替代含有溶解的生物制品的生物相容分散剂)来生产一种含有分散剂悬浮的生物制品颗粒的的聚合物外壳。 Biologicals may be used in a biocompatible dispersing agent particles in suspension (instead of biocompatible biological products containing dissolved dispersing agents) to produce a polymer shell containing a particle dispersant Biologicals suspension of the. 换句话说,这种聚合物壳体能含有一种生物制品在分散剂中的饱和溶液。 In other words, the polymeric shell could contain a saturated solution of biological products in a dispersing agent. 另一种变化是含有一种生物制品固体核的聚合物壳体,它是先把生物制品溶于一种挥发性有机溶剂(如苯)中,形成聚合物外壳,再在真空下,如在旋转蒸发器中,蒸发挥发性溶剂或者将全部悬浮液冷冻干燥来制成的。 Another variation is a biological product comprising a solid core of a polymeric shell, it is first dissolved in a volatile organic Biologicals solvent (such as benzene), forming a polymer shell, and then under vacuum, such as in rotary evaporator, the evaporation of the volatile solvent or by freeze-drying the entire suspension was made. 这样就形成了一种用聚合物包衣包裹生物制品团体核的结构。 Thus forming a polymer-coated bodies wrapped biologics nuclear structure. 后一种方法对传送相对小体积大剂量的生物制品是特别有益的。 The latter method of transferring a relatively small volume of high doses of biological products is particularly advantageous. 在有些情况下,形成该核外壳的生物相容物质本身可以是一种治疗剂或诊断剂,例如,对于胰岛素来说,它可以作为上述超声波辐射步骤中所形成的一种聚合物壳体的一部分来传送。 In some cases, forming the core housing biocompatible substance itself may be a therapeutic or diagnostic agent, e.g., insulin, it can be said as a polymeric shell formed in the ultrasonic irradiation step of part of the transmission. 在其它情况下,形成外壳的聚合物也能参与生物制品的传送,例如,当生物制品是胰岛素时,它可以作为上述超声波辐射步骤中所形成的聚合物壳体的一部分来传送,由此来提供一种具有高度氧结合力的血液代用品。 In other cases, the polymer forming the shell could participate in the transfer of biological products, for example, when the biological products are insulin, it can be used as a part of said polymeric shell formed in the ultrasonic irradiation step to transmit, thereby providing a blood substitute having a high degree of oxygen-binding capacity.

聚合物壳体的变化也是有可能的。 Changes polymeric shell are also possible. 例如,含有巯基的少量PEG能包括在该聚合物中。 For example, a small amount of PEG containing sulfhydryl groups could be included in the polymer. 一旦暴露于超声波辐射,PEG就与该聚合物交联并形成该聚合物外的一部分。 Upon exposure to ultrasonic irradiation, PEG is cross-linked with the polymer and the outer portion of the polymer formed. 另一方面,可以在制备该壳后将PEG连接在该聚合物外壳上(而不是作为制成该外壳的基质一部分)。 On the other hand, the preparation will be in the PEG polymer shell attached to the housing (rather than as part of a matrix formed of the housing).

PEG以其非粘合特性为人们所熟知,并且已经把它附着于蛋白质和酶上来增加它们在体内的循环时间(Abu-chowski等,J.Biol.Chem.Vol.252:578(1977))。 PEG in its non-adhesive properties are well known, and it has been attached to proteins and enzymes in the body up to increase their circulation time (Abu-chowski etc., J.Biol.Chem.Vol.252: 578 (1977)) . PEG还被附着于脂质体中形成脂质双层的磷脂上来降低它们在体内的摄取量,并延长它们在体内的停留时间(Klibanov等,FEBS Letters Vol.268:235(1990))。 PEG has also been attached to phospholipids in the lipid bilayer of liposomes formed up to reduce their intake in the body, and to prolong their in vivo residence time (Klibanov, etc., FEBS Letters Vol.268: 235 (1990)). 因此把PEG掺合进交联蛋白外壳的壁上能够改变它们的血液循环时间。 Thus the PEG incorporated into the crosslinked protein shell wall can change their blood circulation time. 这种性能能被用来维持生物制品的较高血液浓度,并能延长该生物制品的释放时间。 This capability can be used to maintain a high blood concentration of biological products, and can extend the release time of the biological products.

适用于改性聚合物外壳的是亲电子PEG衍生物,它包括PEG-咪唑,琥珀酸琥珀酰亚胺酯,碳酸硝基苯酯,tresy-lates,等;亲核的PEG衍生物,它包括PEG-胺,氨基酸酯,酰肼,硫醇等。 Applies to modified polymer shell is an electrophilic PEG derivatives including PEG- imidazole, succinimidyl succinate carbonate, p-nitrophenyl ester, tresy-lates, etc; PEG derivative nucleophilic comprising PEG- amine, amino acid esters, hydrazides, thiols and the like. PEG改性的聚合物壳体比未改性的壳体能够在循环中持续更长的时间。 PEG-modified polymeric shell can last longer in the circulation than the unmodified housing. 用PEG改性聚合物的外壳可以在形成外壳之前或形成外壳之后进行。 Were followed by PEG-modified polymer shell may be formed prior to forming the housing or shell. 现在优选的技术是在形成聚合物外壳后对其进行改性。 Presently preferred technique is to form a polymer shell after its modification. 其它聚合物包括葡聚糖,藻酸盐,羟乙基淀粉等可以用于该聚合物外壳的改性。 Other polymers including dextran, alginates, hydroxyethyl starch and the like may be used for modifying the polymer shell.

本领域熟练技术人员可预想到在本发明范围和构思之内可以有一些改变。 Those skilled in the art can envision within the scope and spirit of the present invention may have some changes. 例如,在形成聚合物外壳壁过程中,可以改变该聚合物外壳中的分散剂,可以使用许多不同的生物制品,以及可以使用大范围的蛋白质和其它天然和合成的聚合物。 For example, in the process of formation of the polymer shell wall, the housing can be changed in the polymer dispersant, can be used a number of different biological products, and can use a wide range of proteins and other natural and synthetic polymers. 它的应用范围也是相当广的。 It is quite a wide range of applications. 除了生物医学应用,如传送药物、诊断剂(在成像中使用)、人造血液(声化学交联的血红蛋白)和胃肠外营养剂,本发明的聚合物壳体结构还可以掺入化妆品中使用,如在皮肤乳剂或护发产品、香水中使用,在压敏墨水中使用,在农药中使用。 In addition to biomedical applications, such as the transfer of drugs, diagnostic agents (used in imaging), artificial blood (hemoglobin sonochemical crosslinked) and parenteral nutritional agents, the polymeric shell structures of the present invention may also be incorporated into cosmetic use , such as skin creams or hair care products, perfume use, use in pressure sensitive inks, pesticides used.

按照本发明的一个实施方案,如上所述制成的聚合物壳体可用于体内传送生物制品,如药物活性剂,诊断剂或有营养价值的试剂。 According to one embodiment of the present invention, the polymer housing manufactured as described above can be used for in vivo transfer of biological products, such as pharmaceutically active agents, diagnostic agents or agents of nutritional value. 可用于实施本发明的药物活性剂的例子有止痛剂(如,醋酸米诺芬(acetominophen),阿斯匹林,布洛芬,吗啡及其衍生物等),麻醉气体(如,环丙烷,安氟醚,氟烷,异氟烷,甲氧氟烷,氧化亚氮等),平喘药(如,苄酞嗪,甲哌噻庚酮,traxanox等),抗生素(如,新霉素,链霉素,氯霉素,头孢菌素,氨苄青霉素,青霉素,四环素等),抗抑郁药(如,甲苯噁唑辛,氧苯哌吲哚,丙咪嗪,氯哌三唑酮(trazadone)等),抗糖尿病药(如,双胍,激素,磺尿类衍生物等),抗真菌剂(如,两性霉素B,制霉菌素,杀念珠菌素等),抗高血压药(如,心得安,苯丙酰苯心安,心得平,心痛定,利血平等),甾体抗炎药(如,可的松,氢化可的松,地塞米松,强的松龙,强的松,氟恶米松等),非甾体抗炎药(如,消炎痛,布洛芬,拉米芬酮(ramifenizone),吡氧噻嗪等),抗肿瘤剂(如阿霉素,环磷酰胺,放线菌素,博来霉素,段诺霉素(duanorubicin),阿霉素,艾波霉素(epirubicin),自力霉素,甲氨蝶呤,氟尿嘧啶,碳铂(Carboplatin),卡氮芥(BCNU),顺氯氨铂,鬼臼乙叉甙,干扰素,苯芥胆甾醇,紫杉醇(这里所用的术语“紫杉醇”是指包括紫杉醇类似物及其前体药物,紫杉烷以及其它类似于紫杉醇的药物,如taxotere等),喜树碱及其衍生物(该化合物对治疗结肠癌具有较可靠的疗效),长春花碱,长春新碱,以及激素类抗肿瘤药,如雌激素,孕激素类,三苯氧胺等),抗焦虑药(如,硝苯呋海因,安定等),酶活性剂(如,去氧核糖核酸酶,核蛋白体酶(ribozyme)等),核酸结构(如,IGF-1编码序列,VIII因子编码序列,IX因子编码序列,反义核苷酸序列等),免疫刺激剂(如,白芥素,干扰素,疫苗等),免疫抑制剂(如,环孢菌素(C,A),硫唑嘌呤,布雷青霉素(mizorobi-ne,FK506,强的松等),生理活性气体(如,空气,氧气,氩气,氨气,一氧化碳,二氧化碳,氦气,氙气,一氧化二氮,一氧化氮,二氧化氮等,以及它们中任意两种两种或多种的混合物),还有其它药物活性剂,如甲氰咪胍,邻氯苯对氯苯二氯乙烷,维沙定(visadine),卤代亚硝基脲类(halonitrosoureas),蒽环类(anthracycline),椭圆玫瑰树碱,苯佐卡因,巴比妥类等。 Examples of pharmaceutically active may be used in the practice of the present invention include analgesic agents (e.g., acetic acid Mi Nuofen (acetominophen), aspirin, ibuprofen, morphine and derivatives thereof, etc.), anesthetic gas (e.g., cyclopropane, enflurane, halothane, isoflurane, methoxyflurane, nitrous oxide, etc.), asthma drugs (eg,  benzyl phthalazine A ketotifen, traxanox etc.), antibiotics (eg, neomycin , streptomycin, chloramphenicol, cephalosporin, ampicillin, penicillin, tetracycline, etc.), antidepressants (e.g., toluene oxazole-octyl, oxybenzyl piperazine-indole, imipramine, chloro piperazine triadimefon (trazadone ), etc.), antidiabetics (e.g., biguanides, hormones, sulfo urine derivatives, etc.), antifungal agents (e.g., amphotericin B, nystatin, kill Candida factors, etc.), antihypertensives (e.g. , propranolol, metoprolol benzene styrene-acrylic acid, experience level, nifedipine, reserpine), NSAIDs (eg, cortisone, hydrocortisone, dexamethasone, prednisolone, prednisone fluorine evil betamethasone, etc.), non-steroidal anti-inflammatory drugs (eg, indomethacin, ibuprofen, La Mifen ketone (ramifenizone), piroxicam, etc.), anti-tumor agents (such as doxorubicin, cyclophosphamide actinomycin, bleomycin, daunorubicin paragraph (duanorubicin), doxorubicin, doxorubicin Appleby (epirubicin), self-adriamycin, methotrexate, fluorouracil, carboplatin (Carboplatin), card nitrogen mustard (BCNU), cisplatin, etoposide, interferons, benzene mustard cholesterol, paclitaxel (as used herein the term "paclitaxel" is intended to include paclitaxel analogs and prodrugs, taxanes, and other drugs like taxol, taxotere and other such), camptothecin and its derivatives (the compound for the treatment of colon cancer has a more reliable effect), vinblastine, vincristine, and hormonal antineoplastic agents, such as estrogen , progesterone, tamoxifen, etc.), anti-anxiety drugs (eg, dantrolene result, stability, etc.), activity of agents (eg, Deoxyribonuclease, ribosomal enzyme (ribozyme), etc.), nucleic acid structure ( e.g., IGF-1 coding sequence, VIII factor coding sequence, IX factor encoding sequence, antisense nucleotide sequence, etc.), immunostimulating agents (e.g., Sinapis hormone, interferons, vaccines, etc.), immunosuppressants (e.g., cyclosporin (C, A), azathioprine, Bray penicillin (mizorobi-ne, FK506, prednisone, etc.), physiologically active gases (e.g., air, oxygen, argon, ammonia, carbon monoxide, carbon dioxide, helium , xenon, nitrous oxide, nitric oxide, nitrogen dioxide, and any two of them two or more thereof), as well as other pharmaceutically active agents, such as cimetidine, o chlorobenzene for dichloroethane, chlorobenzene, vysha set (visadine), halogenated nitrosoureas (halonitrosoureas), anthracyclines (anthracycline), ellipticine, benzocaine, such as barbiturates.

可用于实施本发明的诊断剂的例子有超声对比剂,放射对比剂(如碘辛烷类,卤化碳类,肾造影剂等),磁对比剂(例如碳氟化合物,脂溶性顺磁化合物,GdDTPA,顺磁化合物水溶液等),以及其它试剂(如气体,氩气,氮气,一氧化碳,二氧化碳,氦气,氙气,一氧化二氮,一氧化一氮,二氧化氮等以及它们中任意两种或多种的混合物)。 Examples of diagnostic agents useful in the practice of the present invention include ultrasound contrast agents, radiocontrast agents (such as iodine octanes, halocarbons, renal angiography and the like), magnetic contrast agents (fluorocarbons, lipid soluble paramagnetic compounds, for example, GdDTPA, aqueous paramagnetic compounds, etc.), as well as other agents (such as a gas, argon, nitrogen, carbon monoxide, carbon dioxide, helium, xenon, nitrous oxide, nitric oxide, and any two of them is a nitrogen, nitrogen dioxide or more thereof).

可用于实施本发明的有营养价值的试剂例子包括氨基酸、糖、蛋白质、核酸、碳水化合物、脂溶性维生素(如维生素A,D,E,K等)或者脂肪,或者它们中任意两种或多种的混合物。 Examples of reagents useful in the practice of this invention include nutritionally valuable amino acids, sugars, proteins, nucleic acids, carbohydrates, fat-soluble vitamins (such as vitamins A, D, E, K, etc.) or fat, or any two or more of them mixture thereof.

本发明含生物制品的聚合物壳体与现有技术中的蛋白微球之间的主要区别在于构成的性质和形成该聚合物外壳后蛋白质的最终状态,以及运送难溶于水或基本不溶于水的试剂的能力。 The present invention is a polymeric shell containing biological products and the main difference between the prior art in that the protein between the microspheres and the properties constituting the final state of the protein after formation of the polymer shell, and transporting water-insoluble or substantially insoluble in capacity of the water reagent. 按照本发明,该聚合物(如蛋白质)是通过与许多蛋白质的天然结构中存在的氨基酸。 According to the present invention, the polymer (such as proteins) is the natural structure of many proteins by amino acids present. 例如半胱氨酸形成二硫键来进行选择性化学交联的。 E.g., cysteine to form a disulfide bond selective chemical crosslinking. 使用超声波辐射方法将含有溶解的或悬浮的生物制品的分散剂分散到一种具有巯基或二硫化物基团(如白蛋白)的生物相容物质的水溶液中,由此在非水溶性基小滴的周围,形成一种交联聚合物外壳。 Ultrasonic irradiation method using an aqueous solution containing dissolved or suspended biologic dispersed in a dispersing agent having a mercapto group or disulfide groups (e.g., albumin) biocompatible substance, whereby the small water-insoluble base around the droplets to form a crosslinked polymer shell. 超声波辐射步骤在液体中产生空穴作用,引起局部大量产热,导致过氧化物离子的形成,该离子通过氧化巯基残基(和/或断裂已存在的二硫键)交联聚合物而形成新的交联的二硫键。 Ultrasonic irradiation step of generating cavitation in the liquid, causing local heat production lot, resulting in the formation of peroxide ions, the ions by oxidation of sulfhydryl residues (and / or breaking existing disulfide bonds) a crosslinked polymer and to form a new disulfide bond crosslinked.

本发明步骤相反,现有技术中戊二醛交联的方法是非特异性的,基本上与蛋白质结构中存在的任意的亲核基团(如,胺,巯基和羟基)反应。 Step of the present invention contrary, the prior art method of glutaraldehyde crosslinking is nonspecific and essentially any protein present in the structure of a nucleophilic group (e.g., an amine, a mercapto group and a hydroxyl group) reaction. 如现有技术中所述的加热变性作用可明显而不可逆地改变蛋白质的结构。 The heating denaturation according to the prior art can be significantly and irreversibly alter the protein structure. 相比而言,本发明所形成的二硫化物特异性非常强,并且基本上没有使蛋白质变性。 In contrast, the present invention is formed by a disulfide specificity is very strong, and substantially no protein denaturation. 此外,因为本发明方法制备的聚合物外壳比包裹颗粒的直径相对更薄,所以聚合物外壳中含有的生物制品颗粒或液滴不同于现有技术中交联或加热变性了的蛋白微球。 In addition, since the method of the invention is relatively thinner than the diameter of the shell polymer coated particles, so the particles or droplets of biological products contained in the polymer shell is different from the prior art crosslinked or heat denatured protein microspheres. (用透射电子显微镜检查法)已经测得聚合物包衣的“外壳厚度”大约为25微米,而被包裹的颗粒直径为1微米(1000毫微米)。 (Transmission electron microscopy) a polymer coating has been measured "shell thickness" of approximately 25 microns, and a particle diameter wrapped 1 micron (1000 nm). 比较起来,现有技术的微球没有蛋白外壳,但是,却有分散于整个微球体积中的蛋白质。 By comparison, the prior art does not microsphere protein coat, however, there is dispersed throughout the volume of the protein microspheres.

含有固体、液体或气体的生物制品核的聚合物外壳可以用相对小的体积来传送大剂量的生物制品。 Biologicals core polymer shell containing solid, liquid or gas may be used to transmit a relatively small volume of a large dose of biological products. 这将减少在接受大量液体时患者的不适,并将减少住院时间。 This will reduce patient discomfort in accepting large amounts of liquid and reduce hospitalization time. 此外,聚合物外壳的壁一般可被蛋白水解酶在体内全部降解(如,当聚合物是一种蛋白质时),使得传送系统没有副作用,对现在的制剂来说这是常有的情况。 Further, the polymer wall of the housing may be generally all proteolytic enzymes in vivo degradation (e.g., when the polymer is a protein), the transmission system such that no adverse effect on the present formulation, this is often the case.

按照本发明这种实施方案,生物制品的液滴或颗粒被包含在截面直径不大于10微米左右的外壳中。 According to this embodiment of the invention, droplets or particles of biologic are contained in the cross-sectional diameter of no greater than about 10 microns housing. 截面直径小于5微米是较优选的,而对静脉内途径给药来说,2微米左右截面直径是最优选的。 Cross-sectional diameter of less than 5 microns is more preferred, while for intravenous routes, the cross-sectional diameter of about 2 microns are most preferred.

按照本发明的另一个实施方案,人们已经发现这里所述的聚合物外壳,当由血红蛋白制备时,具有令人惊奇的较高的氧结合能力,因此可被用作血液代用品。 According to another embodiment of the present invention, it has been found that the polymer housing described herein, when prepared from hemoglobin, have high oxygen binding capacity Surprisingly, it can be used as a blood substitute. 血红蛋白(Lehninger,in Biochemistry,Worth Publishers,Inc.New York,PP.145-149,1975)是由四聚体(两个α和两个β链)组成的64,500MW的蛋白质。 Hemoglobin (Lehninger, in Biochemistry, Worth Publishers, Inc.New York, PP.145-149,1975) is 64,500MW protein by tetramer (two α and two β chains) thereof. 每条α和β链以一个非共价键连接一个血红素残基。 Each α and β chains of a non-covalently bonded a heme residue. α和β键也由氢结合和范德伏尔斯力形成的非共价键结合在一起。 α and β are combined by a hydrogen bond and non-covalent Van der Volscian force formed together. 这四个血红素基团,每个亚单位中有一个能够结合四分子的氧。 The four heme groups, one in each subunit capable of binding four molecules of oxygen. 这些扁平的血红素基团在正方平面的配位处含有一个铁离子。 These flat heme group contains an iron ion in the coordination of the square plane. 在完整的分子中,这四个血红素位于相对远离于另外一个的位置。 In the intact molecule, which is located relatively far from the four heme to another location.

在四聚物的血红蛋白分子中,血红素单位间在结合氧方面的相互作用或协同作用大大增加了每个血红素单位的氧结合能力。 In tetrameric hemoglobin molecule, the interaction or synergy between oxygen binding heme units in terms of greatly increased oxygen binding capacity of each heme unit. 一般来说,一个单独的血红素单位应当能结合一个单一分子的氧。 In general, a single hemoglobin unit should be able to combine in a single molecule of oxygen. 然而,在血红蛋白分子中相邻血红素单位的协同作用增加了每个血红素单位的氧结合。 However, neighboring heme unit in hemoglobin molecule synergy increases oxygen binding heme units each. 这种协同作用用术语“希尔系数”来描述,它的数值反应了相互作用的氧结合位点的数目。 This synergy term "Hill Coefficient" to describe the value of which reflects the number of interacting oxygen binding sites. 对天然血红蛋白来说,希尔系数大约为2.8。 Natural hemoglobin, the Hill coefficient of approximately 2.8.

在红细胞中可溶性血红蛋白占总蛋白的90%左右。 Soluble hemoglobin in red blood cells is about 90% of the total protein. 由于血红蛋白的结合力,100ml的全血能够吸收大约21ml的氧气。 Since the binding force of hemoglobin, 100ml of whole blood is capable of absorbing oxygen of about 21ml. 与结合氧同样重要的是,血红蛋白也能有效地把结合的氧释放给组织。 And equally important is bound oxygen, hemoglobin can be effectively bound to the tissue oxygen release. 血红蛋白结合和释放氧的这种能力经常被定量表达为P50(或 Hemoglobin oxygen binding and release of this capability is often quantitatively expressed as P50 (or )。 ). 例如,整个血液的P50,即,导致血红蛋白50%饱和的氧分压,大约是28mmHg。 For example, whole blood P50, i.e., leading to a 50% saturation of hemoglobin oxygen partial pressure, about 28mmHg.

氧分压和血红蛋白饱和百分比之间的关系可用一个S形曲线来表示,曲线的位置受pH值的影响(波耳氏效应)。 Oxygen partial pressure and the relationship between the percentage of saturated hemoglobin available an S-shaped curve is represented, the curve position influenced by pH (the Bohr's effect) by. 在确定的氧分压下血红蛋白溶液的pH值越高,氧饱和的百分数就越大,而P50越低;氧饱和曲线在横坐标上向左移动。 Determining the oxygen partial pressure at the pH of the hemoglobin solution is higher, the greater the percentage of oxygen saturation, and the lower the P50; oxygen saturation curve moved to the left on the abscissa. 相反,血红蛋白溶液的pH值越低,氧饱和的百分数也越小,而P50就越高;氧饱和曲线在横坐标上向右移动。 Conversely, the lower the pH of the hemoglobin solution, the smaller the percentage of oxygen saturation, and the higher P50; oxygen saturation curve to the right on the abscissa. 因此,当血红蛋白从相对碱性pH值的肺部移向相对酸性pH值的缺氧组织(由压氧呼吸产生乳酸)蛋白时,血红蛋白将有一个释放其运载的氧的趋向。 Thus, when the hemoglobin from the lungs is relatively alkaline pH toward hypoxic tissue relative to the acidic pH (lactic acid produced by the pressure of oxygen breathing) protein, the hemoglobin would have an oxygen carrying release its tendency. 所以,血红蛋白对氧的亲和力是与血红蛋白的P50成反方向变化的。 Therefore, the oxygen affinity of hemoglobin is hemoglobin P50 into the opposite direction changes.

血红蛋白分子或其构象的改变是与氧结合亲合力的变化有关的。 Changing the hemoglobin molecule or its conformation is combined with the oxygen affinity of the changes associated. 例如,与2,3一二磷酸甘油酯的结合(2,3-DPG,存在于红细胞中)放松了氧与血红蛋白的结合,从而促进了氧向组织的释放;例如在高海拔和怀孕期,这些需要增加氧气输送的生理条件下,血浆中的2,3-DPG含量上升。 For example, glycerol and 2,3 bisphosphate binding (2,3-DPG, present in red blood cells) and hemoglobin oxygen binding to relax, thereby promoting the release of oxygen to the tissues; for example, at high altitude and pregnancy, these need to increase oxygen delivery physiological conditions, plasma 2,3-DPG content increased. 在血红素取代基中由Fe(II)到Fe(III)的铁离子的氧化导致了高铁血红蛋白(met-Hb)的形成,这种高铁血红蛋白和水结合得很紧以致于妨碍了氧的运送。 In heme substituents from Fe (II) oxidation to Fe (III) of the ferric ions leads to methemoglobin (met-Hb) is formed, this combination of methemoglobin and water tight so prevents oxygen transport . 这种氧化或者“自身氧化”是在体内进行的一个过程,红细胞中的氧化还原酶系统一直监视着这种氧化过程。 This oxidation or 'self-oxidation "is a process carried out in vivo, erythrocytes oxidoreductase system has been monitoring this oxidation process.

血红蛋白,这种运输和传递氧的蛋白质,能从红细胞壁的膜上或基质中(基质中含有决定血型的特殊抗原)和从其它细胞和血浆组分中分离。 Hemoglobin, such transport and transfer oxygen proteins from red cell wall or membrane matrix (matrix containing specific antigen blood group decision) and isolated from other cells and plasma components. 如果这种分离和离析是有效的话,所得到无介质的血红蛋白不含抗原物质;因此,不必要再进行按血型配血。 If such separation and isolation is effective, the resulting hemoglobin-free medium does not contain antigenic material; therefore, unnecessary then press blood with blood.

从红细胞微环境中提取出的无基质的血红蛋白(SFH)已被发现具有特别紧密地结合氧的特性(低的P50),并且输血后还具有一个较短的循环半衰期。 Extracted from erythrocytes microenvironment stroma-free hemoglobin (SFH) has been found tightly bound oxygen having particular characteristics (low P50), and also has a short circulating half-life after transfusion. 较低的P50,是血红蛋白氧结合曲线左移的反应,部分原因是无介质血红蛋白暴露于比红细胞内的pH值(7.2)更高的血浆pH值(7.4)的结果;而且,当血红蛋白从红细胞中除去时,血红蛋白和2,3一二磷酸甘油酯之间的自然关系被破坏,从而更加降低了P50。 Lower P50, is the hemoglobin oxygen binding curve to the left of the reaction, in part because no medium is exposed to hemoglobin within red blood cells than the pH (7.2) higher plasma pH (7.4) results; and, when the hemoglobin from the red blood cells When removed, the natural relationship between hemoglobin and 2,3 bisphosphate glyceride is disrupted, thus further reducing the P50. 就从循环中清除而论,我们观察到无介质血红蛋白可由肾迅速清除,输血半衰期 Cleared from the circulation is concerned, we observed no media hemoglobin rapidly cleared by the kidney, blood half-life 仅100分钟左右,SFH的希尔系数在2.3-2.8范围中。 Only 100 minutes, SFH Hill coefficient in the range 2.3-2.8.

我们已经测定出化学改性的血红蛋白克服了无介质血红蛋白的一些缺点。 We have determined that the chemical modification of hemoglobin to overcome some of the shortcomings of hemoglobin-free medium. 在现有技术中所描述的改良方法包括无介质血红蛋白的各种分子内交联方法;无介质血红蛋白与低分子量试剂的分子内和分子间的交联的方法;以及无介质血红蛋白与其它聚合物配合的方法。 Improved methods described in the prior art include the various molecules of hemoglobin-free medium crosslinking method; method of the non-crosslinked hemoglobin with low molecular weight reagents medium molecular and intermolecular; and hemoglobin-free medium with other polymers fit method.

无介质血红蛋白分子内交联的方法在现技术中是已知的。 No media methods within crosslinked hemoglobin molecules in the current technology is known. 例如,见美国专利4,584,130,4,598,064和4,600,531。 For example, see US Patent 4,584,130,4,598,064 and 4,600,531. 这种方法是通过一个反丁烯二酸根桥在蛋白质的α链上共价连接赖氨酸99残基的方法来改性无介质血红蛋白。 This method is a method by means of a fumaric acid radical in the α-chain protein bridge covalently attached to a lysine residue 99 No media modified hemoglobin. 这种分子内交联的结果是,双阿斯匹林交联的血红蛋白具有等同于血液的氧亲和力。 Results The cross-linking of this molecule is double-aspirin crosslinked hemoglobin has an oxygen affinity equivalent to blood. 而且,双阿斯匹林交联的血红蛋白(分子量为64,500)不能够再分解为二聚物(分子量为32,250)。 Furthermore, double-aspirin crosslinked hemoglobin (molecular weight 64,500) can no longer be broken down into dimers (molecular weight 32,250). 所以,双阿斯匹林α-α交联的血红蛋白的保留时间为4到8小时(这是无介质血红蛋白保留时间的两到四倍)。 Therefore, the retention time of hemoglobin α-α double aspirin crosslinked for 4-8 hours (this is no media hemoglobin retention time of two to four times). 尽管如此,当患者失去大量血液时,由于需要一种能在几天内运送氧气的氧载体,对治疗急性出血来说这种血红蛋白还是不能够提供足够长的维持时间。 Nonetheless, when patients lose a lot of blood, due to a need for a delivery of oxygen to the oxygen carrier in a few days, for the treatment of acute bleeding for this hemoglobin or not able to provide sufficient long duration. 双阿斯匹林交联的血红蛋白的P50在生物范围内(24-28mmHg)希尔系数也是这样(2.5-2.8)。 Hemoglobin P50 double aspirin crosslinked within the biological range (24-28mmHg) Hill coefficient, too (2.5-2.8).

使用低分子量的交联剂也能使血红蛋白分子进行分子内相互交联。 Low molecular weight crosslinker also make hemoglobin molecules cross-linked to the molecule. 例如,在美国专利4,336,248中描述了优选加入磷酸吡哆醛之后,使用双醛使血红蛋白分子互相之间和/或血浆蛋白以及明胶衍生物进行配合。 After e.g., in U.S. Patent No. 4,336,248 describes a preferred pyridoxal phosphate was added, using dialdehyde between hemoglobin molecules to one another and / or plasma proteins and gelatin derivatives were blended. 美国专利NOS.4,001,401,4,001,200,4,05,590和4,061,736中描述了与双官能团或多官能团的低分子量交联剂进行的交联。 U.S. Patent 4,061,736 describes NOS.4,001,401,4,001,200,4,05,590 and crosslinking with bifunctional or polyfunctional crosslinking agent of low molecular weight. 分子间血红蛋白交联产物常常不是单一可溶性四聚物,而是多个血红蛋白四聚物通过共价键连接形成的可溶性低聚物。 Intermolecular hemoglobin crosslinking are often not single soluble product tetramers, but multiple tetramers of hemoglobin soluble oligomer by the formation of a covalent bond. 典型的是,这类分子间交联的产物通常具有不等同于血液的氧携带和输送性能(与全血的P50值28相比戊二醛聚合的血红蛋白P50为18-23)并且它的希尔系数在1.8-2.8范围内。 Typically, the product of such inter-molecular cross-linking generally have not equivalent to blood oxygen carrying and conveying performance (compared to P50 values of whole blood of 28 glutaraldehyde polymerized hemoglobin P50 of 18-23) and its Greek Dole coefficient in the 1.8-2.8 range. 而且,现有技术中已知通过戊二醛进行分子间交联的产物是具有抗原性的[见DH Marks等,Militry Med.152:473(1987)]。 Furthermore, known in the art by glutaraldehyde intermolecular crosslinked product having antigenic [see DH Marks, etc., Militry Med.152: 473 (1987)].

一般来说,血红蛋白分子内和分子间的交联降低一些由未改性的血红蛋白离解为αβ-二聚物所产生的肾的毒性问题。 In general, the crosslinked hemoglobin molecule and reduces some of the intermolecular unmodified hemoglobin from the dissociation of dimers αβ- renal toxicity problems arising. 然而,由可溶性血红蛋白产生的胶体渗透压(COP)却并没有因分子内交联而显著降低。 However, colloid osmotic pressure (COP) of soluble hemoglobin produced by has not been affected by intramolecular crosslinking significantly reduced. 所以,这就限制了可溶性血红蛋白血液代用品适于给药的剂量。 Therefore, this limits the soluble hemoglobin blood substitutes suitable for administration of the dose. 一般来说,COP的增加会引起流体静压力的降低和肾小球过滤率的相应降低,引起了少尿,严重情况下无尿。 Generally, COP increase will cause a corresponding decrease hydrostatic pressure and glomerular filtration rate is reduced, causing oliguria, anuria severe cases. 现有技术中所述的可溶性血红蛋白的给药已经引起了心动过缓,血压升高和肌酐清除下降。 The administration of soluble hemoglobin of the prior art has caused bradycardia, decrease blood pressure and creatinine clearance. 已发现血管收缩和血管梗阻是肾脏效应的病因,这些都是与使用可溶性血红蛋白血液代用品有关的。 It has been found vasoconstriction and vascular obstruction is the cause kidney effects, these are the use of soluble hemoglobin blood substitutes concerned. 使用如本文所述制备的一种高度聚合形式的血红蛋白作为血液代用品以可缓解这些问题。 Using a highly polymerized form prepared as described herein as hemoglobin blood substitute in order to alleviate these problems.

高度氟化的化合物,特别是全氟化碳化合物,由于其对氧的高溶解性,也已经被当作是红细胞代用品,适合于这种用途的高氟化化合物有全氟化碳类如,全氟萘烷,全氟二氢化茚,全氟甲基金刚烷,全氟三丙基胺,全氟三丁胺,全氟辛溴等。 Highly fluorinated compound, especially a perfluorocarbon compound, because of its high solubility for oxygen, has been used as a red blood cell substitutes, highly fluorinated compounds suitable for this purpose are hydrocarbons such as perfluorocarbons , perfluorodecalin, perfluoro indane, perfluoromethyl adamantane, perfluoro tripropylamine, perfluorotributylamine, perfluorooctyl bromide and the like. 在静脉内使用时,与水不混溶的这些全氟化碳类必须分散成注射乳剂。 When used intravenously, and water immiscible these perfluorocarbons must be dispersed into injectable emulsion. 在这些应用中常用的乳化剂是蛋黄卵磷脂和卵磷脂,二者都具有沉淀变态反应物的能力。 Commonly used in these applications are egg yolk lecithin and lecithin emulsifier, both of which have the ability to precipitate allergy thereof. 例如参见,PCT 92/06517,它描述了含有氟化物和磷脂,如溶血磷脂酰胆碱和溶血磷脂酰胆胺,作为表面活性剂的一种乳剂,或者PCT 93/11868描述了用蛋黄卵磷脂作乳化剂的乳剂,它含有高氟化的,氯取代的,非环状有机化合物作为氧载体。 See, for example, PCT 92/06517, which describes a fluoride and phospholipids, such as lysophosphatidylcholine and lysophosphatidic acid, cholestyramine, as an emulsion surfactant, or PCT 93/11868 describes the use of egg yolk lecithin as emulsifiers in emulsion, which contains high fluoride and chlorine-substituted acyclic compounds as an oxygen carrier.

Fluosol-DA(α治疗剂),是一种全氟萘烷和全氟三丙胺的乳浊液,它是FDA唯一批准用于预防球形冠状血管成形术中暂时局部缺血的药品。 Fluosol-DA (α therapeutic agent), is a perfluorodecalin and perfluoro tripropylamine emulsion, it is the only FDA approved for the prevention of coronary angioplasty spherical temporary ischemic drugs. 另一个氟化碳产品,含氧剂(Alliance Pharmaceuticals)或全氟辛溴也已被批准作为口服造剂。 Another fluorocarbon product, oxygen-containing agent (Alliance Pharmaceuticals), or perfluorooctyl bromine has also been approved as an oral agent made. 全氟化合物用作血液代用品的综述参见Riess等,Angew Chem.Int.Ed.Engl.17.621-634(1978). Perfluoro compound is used as a blood substitute for review see Riess, etc., Angew Chem.Int.Ed.Engl.17.621-634 (1978).

现有技术中所述的血液代用品仅使用溶解性血红蛋白作氧载体。 According to the prior art soluble hemoglobin blood substitutes used only as an oxygen carrier. 的确,一般传统所接受的是,一种不溶性血红蛋白分子(例如,它是与其它血红蛋白分子过量聚合或交联到不溶的程度,或者它是由过度变性引起不溶的,等等)由于很有可能分子中氧结合位置而不适合用作可逆性结合氧。 Indeed, generally accepted tradition is an insoluble hemoglobin molecule (for example, it is associated with other hemoglobin molecules excessive degree of polymerization or crosslinking to insoluble, or it is caused by excessive degeneration insoluble, etc.) is likely due to molecular oxygen binding site is not suitable for use as reversibly bind oxygen. 此外,现有技术的可溶性血红蛋白的希尔系数并不大于未改性的天然血红蛋白的希尔系数。 In addition, the prior art soluble hemoglobin Hill coefficient is not greater than the unmodified natural hemoglobin Hill coefficient.

比较起来,本文所述的由血红蛋白制得的聚合物壳体是“巨大”的肉眼可见的分子(由于它大量聚合或交联了许多血红蛋白的四聚物分子),因为它有较大的体积,所以不溶于含水介质。 By comparison, as described herein produced polymer by hemoglobin shell is "great" macroscopic molecules (due to its large number of polymerization or crosslinking of a number of hemoglobin tetramer molecules), because it has a larger volume , which is not soluble in an aqueous medium. 在超声波辐射步骤中由于蛋白质半胱氨酸残基上的巯基的交联而发生聚合。 In ultrasonic irradiation step crosslinking cysteine residues on the protein mercapto group and the polymerization occurs. 按照本发明所制备的聚合物外壳,一般含有至少104个交联的聚合物分子,并且有1012个血红蛋白四聚物交联成一个肉眼可见的血红蛋白“大聚合物”(megamer)。 According to the present invention is prepared polymer shell, generally at least 104 containing polymer molecules cross-linked, and there are 1012 hemoglobin tetramers crosslinked into a visible hemoglobin "big polymer" (megamer). 出人意料地发现氧能与这些具有亲和力的不溶性结构可逆性地结合,对一种红细胞(RBC)代用品来说它的亲和力在可用的范围中,即P50在约10mmHg到约50mmHg之间。 Surprisingly found that oxygen can bind reversibly to these insoluble structures with affinity for a red blood cell (RBC) substitutes for its affinity for the available range, i.e., P50 between about 10mmHg to about 50mmHg.

有关本发明不溶性血红蛋白结构(IHC)的另一个令人惊奇并出人意料的发现是它有极高的希尔系数(n)。 Another thing about the structure of the present invention is insoluble hemoglobin (IHC) surprises and unexpected discovery is that it has a high Hill coefficient (n). 希尔系数是测定血红蛋白四聚物分子中氧结合位置(血红素单位)之间协同作用程度的一个指标。 Hill coefficient is an indicator measuring the degree of synergy between the hemoglobin tetramer molecular oxygen binding site (heme units). 天然血红蛋白最大的希尔系数大约是2.8,而现有技术中改性的血红蛋白一般报道的希尔系数少于2.8。 Largest natural hemoglobin Hill coefficient is about 2.8, and the art of modified hemoglobin generally reported a Hill coefficient of less than 2.8. 对本发明不溶性血红蛋白结构所测得的希尔系数特别大,一般在约5到约25范围内。 The present invention insoluble hemoglobin structure measured Hill coefficient is particularly large, typically in the range of about 5 to about 25. 不需要由任何作用理论来限制,这些极高的数值是由邻近交联四聚物血红蛋白单位的氧结合位置之间相互作用或相互连接所产生的。 Do not need to be limited by any theory of action, these values are extremely high oxygen binding between a position adjacent the crosslinked tetrameric hemoglobin units interconnected by the interaction or generated. 基本上,人们相信希尔系数增大,表明在结合氧时不溶结构中多个四聚物在由脱氧-T(紧张的)状态转化为氧-R(松驰)状态过程中起着协同作用。 Basically, it is believed Hill coefficient increases, indicate when a plurality of bound oxygen-insoluble tetramer structure by deoxygenation -T (tense) state is converted to oxygen -R (relaxed) state plays a synergistic effect .

在本发明血红蛋白结构中所观察到的出人意料的大的希尔系数具有的优点在于每个血红蛋白四聚物单位所运载氧的量远远超出了天然血红蛋白或现有技术中修饰的血红蛋白所能达到的运载氧的量。 Hill coefficient surprisingly large advantage in having a structure of the present invention, hemoglobin observed that the amount of each hemoglobin tetramer units for carrying oxygen far beyond the prior art natural hemoglobin or modified hemoglobin that can be achieved The amount of the carrier of oxygen. 这种增长了的氧运载能力大大有益于本发明用作RBC代用品。 This increased oxygen carrying capacity greatly benefit the present invention is used RBC substitutes.

在氧分压约为4-100mmHg范围内本发明血红蛋白结构具有最大的希尔系数。 The oxygen partial pressure in the range of about 4-100mmHg hemoglobin structure of the present invention has the greatest Hill coefficient. 换句话说,在这个氧分压范围内能达到最大的协同作用。 In other words, in this range of oxygen partial pressure to achieve maximum synergy. 因为典型的肺泡pO2在这个范围之内,当用本发明结构作为血液代用品时,血红蛋白结构在肺中可以最大量的摄取氧。 Because the typical alveolar pO2 within this range, the structure of the present invention when used as a blood substitute, the maximum amount of hemoglobin structure may oxygen uptake in the lungs.

另一方面,本发明结构向组织释放氧与生理血红蛋白非常相似,即,在典型的组织pO2(<40mmHg)时,大部分结合在不溶性血红蛋白结构上的氧被释放给了组织充氧。 On the other hand, the structure of the present invention with a physiologically oxygen to the tissues is very similar to hemoglobin, i.e., in a typical tissue pO2 (<40mmHg), the majority of the insoluble hemoglobin bound oxygen is released to the structure of the tissue oxygenation. 所以,本发明的交联的不溶性血红蛋白在典型负载压下(如肺中)具有比现有技术中的血红蛋白更高的不寻常的氧结合能力(由于希尔系数较大),并且,在组织中所遇到的,典型压力下保持有效的氧释放能力。 Therefore, the insoluble crosslinked hemoglobin of the present invention in a typical load pressure (e.g. lung) higher than in the prior art unusual hemoglobin oxygen binding capacity (due to the Hill coefficient is larger), and, in the tissue encountered, the ability to maintain an effective oxygen release under typical pressure.

由于本发明不溶性血红蛋白结构的交联性质和其体积,它可能比现有技术的红细胞(RBC)代用品更具有相当长的体内循环时间。 Since the structure of the present invention insoluble hemoglobin crosslinked nature and its volume, it may be more in vivo circulation time considerably longer than the prior art erythrocytes (RBC) substitutes. 而且,由于它们的较大分子(肉眼可见的)体积,它们不可能诱发肾毒性问题,而这正是现有技术中所述的一般性四聚物或寡聚物可溶解形式的血红蛋白所存在的问题。 Moreover, due to their large molecular (macroscopic) size, they are unlikely to induce renal toxicity problems, and this is generally a tetramer or oligomer soluble forms of hemoglobin described in the prior art present problem.

本发明的空的(“泡状”或微泡)不溶性血红蛋白结构可以在血红蛋白外壳或膜内装载一种适当的气体。 Empty the present invention ("bubble" or microbubbles) insoluble hemoglobin structure may be loaded in a suitable gas or hemoglobin shell membrane. 所以,当血红蛋白“微泡”例如在一个外界设备或在肺中用氧平衡时,该结构或泡的中心核被未结合的或游离氧所饱和,这些氧通过分子扩散进入核中。 So, when hemoglobin "microbubbles" For example, in an external device or when the lungs with oxygen balance, the central core of the structure or the bubble is unbound or free oxygen saturated with oxygen by these molecules diffuse into the nucleus. 所以这种结构除了与形成微囊外壳或膜的血红蛋白结合氧外,还在其空核的小空间内携带未结合的分子氧。 So in addition to the structure of hemoglobin to form microcapsules with shell or membrane bound oxygen, but still within its hollow core of a small space unbound oxygen carrying molecule. 这种系统运送未结合(但被包裹了的)氧的能力大大增加了核系统的氧运载容量,超过了单独由血红蛋白所运载的氧的量。 This system is shipped unbound (but was wrapped in) ability of oxygen greatly increases the oxygen carrying capacity of the nuclear system, exceeds the amount separately carried by hemoglobin oxygen. 现有技术中没有记载这种在氧与血红蛋白结合的同时还通过小空泡携带未结合氧的能力。 This prior art is not described in the binding of oxygen to hemoglobin through small vacuoles unbound oxygen carrying capacity.

在血管内给药前也能用氧气预先装载或饱和不溶性血红蛋白结构。 Before administration of oxygen in the blood vessels can also pre-loaded or saturated insoluble hemoglobin structure. 这样可以象冠状血管成形术或肿瘤治疗的短期使用中最大量的送氧。 This tumor as coronary angioplasty or short-term use in the treatment of forming the maximum amount of the oxygen.

本发明不溶性血红蛋白结构的疏散“细胞”性能使得它们能够以一种生理学方式运送氧,而与体内的红细胞没有什么不同。 Evacuation "Cell" performance of the present invention insoluble hemoglobin structure such that they can in a physiologically transported oxygen, but no different from red blood cells in vivo. 由于本发明不溶性血红蛋白结构的“大聚合物”特性,它们产生的胶体渗透压或膨胀压与现有技术中任何等量的(就氧运载量而言)可溶性血红蛋白相比是微不足道的。 As the "big polymer" feature of the present invention insoluble hemoglobin structure, colloid osmotic pressure or swelling pressure of the prior art in that they produce the same amount of any of (in terms of oxygen carrying capacity) of soluble hemoglobin is negligible compared. 这就可以把高浓度的本发明血红蛋白结构进行静脉输液,而对现有技术的可溶性血红蛋白来说,由于担心渗透梯度引起血管周围组织严重的脱水,只能以最大浓度6-8g/dl来输液。 This can put a high concentration of hemoglobin structure of the invention for intravenous infusion, while soluble hemoglobin of the prior art, because of fear perivascular tissue osmotic gradient caused severe dehydration, only with the greatest concentration of 6-8g / dl for infusion .

本发明还可以扩展到使用其它氧结合蛋白用作RBC代用品。 The present invention can also be extended to other oxygen-binding proteins used as RBC substitutes. 作为一个例子,肌红蛋白具有一个单独的氧结合血红素基团(但没有可交联的半胱氨酸残基),应当有与本发明相同方式的作用。 As an example, myoglobin has a separate oxygen binding heme group (but no cross-linkable cysteine residues), should have the same effect of the present invention. 具有至少两个可交联的半胱氨酸残基的一种遗传工程产生的肌红蛋白可被用来生成一种不溶性肌红蛋白结构。 A genetic engineering has at least two crosslinkable cysteine residues produced myoglobin can be used to generate an insoluble myoglobin structure. 氧结合蛋白与对氧没有亲和力的蛋白的组合物可以用于形成本发明的不溶性结构,例如可以使用血红蛋白和肌红蛋白。 Oxygen binding protein with no affinity for oxygen composition protein can be used to form an insoluble structure of the present invention, for example, using hemoglobin and myoglobin.

本发明组合物比现有技术的微囊包裹血红蛋白组合物具有更显著的优点。 Compositions of the present invention has significant advantages over microencapsulated hemoglobin compositions of the prior art. 现有技术的血红蛋白的脂质体制剂在脂质体外壳含有可溶性血红蛋白。 Hemoglobin liposomal formulation in liposomes prior art housing contains soluble hemoglobin. 现有技术的脂质体包裹血红蛋白组合物具有的一些缺点已被本发明所克服。 Some disadvantages of the prior art liposome-wrapped hemoglobin compositions have been overcome by the present invention. 从脂质体组合物中裂解出的可溶性血红蛋白有可能会引起肾中毒。 Cleaved out from the liposomal composition soluble hemoglobin may cause renal toxicity. 本发明不溶性结构由于它大范围地交联性质将不会裂解出可溶性血红蛋白。 Structure of the present invention is insoluble due to its wide range of cross-linking properties will not be cleaved soluble hemoglobin. 脂质体的聚集已知能激活补体蛋白C3α。 Aggregation of liposomes is known to activate the complement protein C3α. 如果是不溶性结构,由于它的体积远远大于脂质体的体积范围,这种聚集将是不可能的。 If insoluble structure, because of its volume is much greater than the volume range of liposomes, this concentration would be impossible.

本发明不溶性交联的血红蛋白组合物避免了与现有技术的可溶性血红蛋白组合物有关的毒性。 Hemoglobin compositions of the present invention is insoluble crosslinked hemoglobin avoids soluble compositions of the prior art related toxicity. 血红蛋白的肾中毒或肾毒性主要是与从循环中清除可溶性二聚物,四聚物,或寡聚物血红蛋白有关。 Nephrotoxicity or renal toxicity of hemoglobin is mainly related to remove soluble dimer, tetramer or oligomer related hemoglobin from the circulation. 已广泛交联的或“大聚合物”的本发明血红蛋白不能由肾脏清除,因而是不可能有肾毒性的。 Has been widely crosslinked or "large polymer" of the present invention can not be cleared by the kidneys hemoglobin, hence there can be no renal toxicity. 本发明的不溶性结构不能被肾脏清除,因而解决了这个问题。 Insoluble structure of the present invention is not cleared by the kidneys, thus solving this problem. 本发明广泛交联的血红蛋白结构比现有技术具有的另一个优点是由于其不溶的形式,增加了在血管内的保留特性。 Another advantage of the present invention broadly cross-linked hemoglobin structure than in the prior art is due to its having the insoluble form, increasing the retention characteristics in the blood vessel.

用透射电子显微测定法(TEM)测得不溶性血红蛋白(IHC)的形态。 By transmission electron microscopy assay (TEM) measurements could not be soluble hemoglobin (IHC) form. 为获得牛IHC的横切片的TEM显微照片,把IHC用戊二醛固定,用四氧化锇和亚铁氰化钾染色(为提供高蛋白浓度区的对照),用一种低粘度树脂包埋并进行超薄切片(片厚为~75nm)。 In order to obtain a cross-section of bovine IHC TEM photomicrograph, the IHC were fixed with glutaraldehyde, osmium tetroxide and potassium ferrocyanide dyeing (high protein concentration region for providing a control), with a low viscosity of the resin package buried and thin slices (slice thickness of ~ 75nm). 由于在这些步骤中可能会有整个直径的一些缩小和IHC的一些形状扭曲,所以IHC的真实直径最好用溶液颗粒大小的分布(3微米;std.dev.1)来表示,而不是直接测量TEM显微照片。 Since some of these steps may be reduced and the entire diameter of the IHC some distorted shape, so the real diameter of the IHC is best to use the solution particle size distribution (3 microns; std.dev.1) expressed, rather than direct measurement TEM micrographs. 仔细观察TEM显微照片发现三个明确的区域:一个清晰的中心区;一个暗的颗粒薄层;和一个与颗粒外表面相关的松散附着的,分散的,有斑点的灰色区。 Careful observation TEM micrograph found three distinct regions: a clear central region; a dark layer of particles; and an outer surface of a loosely associated with the particle attached, dispersed, speckled gray region. 黑色薄层就是IHC壳。 IHC is black thin shell. 它含有一种高密度蛋白,在染色步骤中,显色最明显。 It contains a high density protein, in the dyeing step, the most obvious color. 松散附着的灰色物似乎是在样片制备的固定步骤中粘在IHC壳上的天然蛋白。 Loosely attached to the gray matter seems to be stuck in a fixed sample preparation step in the IHC shell of natural proteins. 对这个照片和许多其它显微照片的初步测量表明牛血红蛋白的IHC的壳厚约为25-35nm。 Initial measurements of the photos and many other micrographs showed that bovine hemoglobin IHC shell thickness of 25-35nm. 血红蛋白是一种直径为5.5mn的粗糙的球体蛋白(L.Stryer,Biochemistry,WHFree-man,New York,1988)。 Hemoglobin is a diameter of 5.5mn rough sphere protein (L.Stryer, Biochemistry, WHFree-man, New York, 1988). 所以,IHC的蛋白外壳大约是血红蛋白分子(四聚物)的4到20倍厚。 Therefore, IHC shell around the protein hemoglobin molecules (tetramers) is 4-20 times thicker. 因此,一个直径为3.0μm的泡状物应当能含有大约104到1012个血红蛋白分子。 Therefore, a diameter of 3.0μm foamy substance should be able to contain from about 104 to 1012 hemoglobin molecules.

用圆二色性检测本发明的不溶性血红蛋白结构(IHC)(微泡或微球)发现IHC中α-螺旋和β-折叠的含量与纯化的无介质血红蛋白(SFH)并没有明显差别。 IHC found in α- and β- folded helix content of insoluble hemoglobin purified by circular dichroism detector structure of the present invention (IHC) (microbubbles or microspheres) No Media hemoglobin (SFH) and no significant difference. 这种发现十分重要,因为它表明不溶性血红蛋白的交联步骤和形成没有导致蛋白变性(即,三级和四级结构的改变)。 This finding is important because it indicates that the crosslinking step and the formation of insoluble hemoglobin does not result in protein denaturation (i.e., change the tertiary and quaternary structure). 当然用代表合成步骤后保留了可逆性氧结合和氧结合血红素单位之间的协同作用的功能性数据也证实了这种发现。 After the course, with representatives of synthetic steps to retain the reversible oxygen binding heme and oxygen combine synergies between the functional units of the data also confirmed this finding.

现已测出了IHC的氧结合性能。 Now measured the oxygen binding properties of the IHC. 由于met-Fe(III)形式的血红蛋白不能结合氧,可使用Hyashi等人的还原体系(A.Hyashi,T.suzuki,M.shin.Biochim.Biophys.Acta310,309,1973)来把Fe(III)还原为Fe(II)。 Since the met-Fe (III) in the form of hemoglobin can not bind oxygen, may be used Hyashi et al reduction system (A.Hyashi, T.suzuki, M.shin.Biochim.Biophys.Acta310,309,1973) to the Fe (III ) reduced to Fe (II). 该还原体系是由不同浓度的葡糖-6-磷酸,葡糖-6磷酸脱氢酶,NADP,铁氧化还原蛋白,铁氧化还原蛋白还原酶和过氧化氢酶组成。 The reduction system is composed of different concentrations of glucose-6-phosphate, glucose 6-phosphate dehydrogenase, NADP, ferredoxin, ferredoxin reductase and catalase components. 在每次氧结合实验前,向IHC中加入该还原体系并在4℃保持24-36小时。 Before each binding experiment oxygen, IHC was added to the reduction system and kept at 4 ℃ 24-36 hours.

按照实施例14所述可以合成牛和人的血红蛋白IHC。 As described in Example 14 can be synthesized bovine and human hemoglobin IHC. 象本领域熟练技术人员所知道的,所用的血红蛋白可来源于任何脊推动物,非脊椎动物或真核源,或者脊椎动物,非脊推动物或真核细胞的基因操作产物。 Like skilled personnel know, the use of hemoglobin can be derived from any vertebrate material, non-vertebrate or eukaryotic sources, or vertebrates, genetic manipulation of non-vertebrate product or eukaryotic cells. 表1提供了本文结果的总结。 Table 1 provides a summary of the results of this paper.

表1声处理过的Hb微泡和未经声处理的BHb在不同浓度的磷酸酯中n最大和P50的总结表 Table 1 sound-treated Hb microbubbles and unsonicated's largest and P50 BHb n summary tables in different concentrations of phosphate in

注:BHb微泡的希尔系数(n)是按公式计算的:n=&Delta;log(Y/1-Y)&Delta;logPo2,]]>其中Y是氧合部分而Po2是氧压,对微泡而言,每项Δlog(Y/1-Y)都是五个连续点的平均值全部结合实验都是在25℃三羟甲基氨基甲烷缓冲液(pH 7.4)中进行的。 NOTE: BHb microbubbles Hill coefficient (n) is calculated according to the formula: n = & Delta; log (Y / 1-Y) & Delta; logPo2,]]> where Y is the oxygenation portion Po2 is oxygen pressure, for microbubbles, the each Δlog (Y / 1-Y) are the average of five consecutive points all binding experiments were carried out at 25 ℃ Tris buffer (pH 7.4) in. 如IHC紫外可见光谱所证实的,IHC保留了它的可逆性结合氧的性能,这表明有met-Fe(III),氧-Fe(II)和脱氧-Fe(II)形式存在。 IHC as evidenced by UV-visible spectroscopy, IHC retained its reversible oxygen binding properties, which indicates a met-Fe (III), oxygen -Fe (II) and deoxy -Fe (II) form. IHC能够在脱氧状态和有氧状态之间循环,十次以上循环也基本不会被降解。 IHC can deoxy state between the state and the aerobic cycle, more than ten cycles are not substantially degraded. 这一点是很重要的,因为它表明在制备IHC红细胞代用品的方法中不会明显改变活性血红素部位周围的环境。 This is very important because it indicates that in the preparation of the IHC red blood cell substitute process does not significantly alter the activity of heme site surroundings.

这些氧结合数据表明IHC基本上不含有变性血红蛋白。 These oxygen binding data suggest that IHC substantially denatured hemoglobin. 如果它被变性,就不会(或很少)观察到生理反应性。 If it is modified, it will not (or rarely) observed physiological reactivity.

在无磷酸酯存在下还原的血红蛋白IHC和天然无介质血红蛋白的氧结合曲线在形状上都是S形,这种曲线表明了氧结合的作用性。 Phosphate in the presence of oxygen binding curve without reducing hemoglobin IHC and natural hemoglobin-free media are S-shaped in shape, this curve shows the effect of the oxygen-bound. 在两种曲线中P50值(血红蛋白上存在的氧结合位置有一半结合氧所需的压力)相近(21托与22托)。 In both curves P50 values (oxygen binding sites present on hemoglobin half required for binding oxygen pressure) similar to (21 Torr and 22 Torr). 这种结果表明IHC是在与天然血红蛋白相似的氧气压力下结合并释放氧的。 Such results show binding in the IHC is similar to natural hemoglobin oxygen pressure and release of oxygen. 令人吃惊的是,IHC的最大希尔系数,n最大值(代表氧结合位置间协同作用的程度)明显高于无介质血红蛋白溶液(9.5对2.6;见图2)。 Surprisingly, IHC's largest Hill coefficient, n the maximum value (the degree of oxygen binding sites between representatives of synergy) was significantly higher than those without media hemoglobin solution (9.5 pairs of 2.6; see Figure 2). 用下列公式来计算希尔系数(n):&Delta;log(Y/1-Y)&Delta;logPO2]]>其中:Y=氧合部分,和PO2=氧分压用5个连续点的平均值获得每个(Δ)log(Y/1-Y)值,然后作一些光滑曲线。 Using the following formula to calculate the Hill coefficient (n): & Delta; log (Y / 1-Y) & Delta; logPO2]]> where: Y = oxygenation portion, and the oxygen partial pressure PO2 = the average of five consecutive points with obtained for each (Δ) log (Y / 1-Y) value, and then make some smooth curve.

已经证明天然血红蛋白的变构效应物如肌醇六磷酸酯(IHP)和2,3-双磷酸甘油酯(2,3-BPG)能够增加P50(即,较低氧亲和力)和提高协同性。 Has proven natural allosteric hemoglobin effector, such as inositol hexaphosphate (IHP) and 2,3-bis-phosphoglycerate (2,3-BPG) can increase the P50 (i.e. lower oxygen affinity) and to improve interoperability. IHC中也可以见到相同的作用。 IHC may be seen in the same effect. 即使用P50值的增加相同,也能看到更为引入注目的作用IHC的协同性。 That is the same as using the P50 value increases, we could see a more compelling role IHC synergy. 在1.7mMIHP(17.6对2.8)和2,3-BPG(14对2.8)存在下n最大值比天然血红蛋白有更显著的增加(见图3和表1)。 In 1.7mMIHP (17.6 对 2.8) and 2,3-BPG (14 对 2.8) in the presence of n more significant increase in the maximum value (see Fig. 3 and Table 1) than native hemoglobin.

协同性上出人意料的较大增强的明显是因为IHC壳中血红蛋白四聚物间的共价连接。 The unexpected synergistic enhancement is apparently due to the larger covalent IHC shell connection between hemoglobin tetramers. 希尔系数不可能大于相互作用位点数目。 Hill coefficient can not be greater than the number of points of interaction. 天然血红蛋白中大约2.8的数值反应了一个四聚物的协同性。 Natural hemoglobin value of approximately 2.8 reflects a tetramer of interoperability. 尽管如此,在IHC壳中,一旦结合氧后就有几个交联的四聚物之间有连接(通过形成二硫键)。 Nevertheless, in the IHC shell, there is a connection between tetramer bound oxygen once there are several cross-linked (via disulfide bond formation). 最近四聚物之间的相互作用可能是最强的;然而相距更远的四聚物之间也存在较弱的相互作用。 Recently the interactions between tetramers may be strongest; there are also weak interactions between tetramers further away, however. n最大值基本上表示结合氧后IHC外壳中由脱氧-T转化到氧-R状态过程中多个四聚物的协同作用。 n represents the maximum Basically IHC shell upon binding oxygen converted to oxygen by deoxy -T -R status process synergies multiple tetramers. 血红蛋白IHC的TEM显微照片也反应了大约6个血红蛋白聚合物的壳厚度。 TEM micrographs of hemoglobin IHC also reflects the shell thickness of about six hemoglobin polymers. 直径20μm的泡状物应当能包含大约104到1012个血红蛋白分子。 20μm in diameter vesicles should be able to contain approximately 104-1012 hemoglobin molecules.

用制备后不同时期的颗粒数目来测定IHC贮存的稳定性。 The number of particles at different times after preparation determined using IHC storage stability. IHC可在和生理盐水中4℃下存放最多6个月。 IHC can be stored for up to six months in saline at 4 ℃ and in. 在第3个月,IHC的浓度已经下降大约10%,而在第6个月,浓度已下降约25-30%。 At 3 months, the concentration of the IHC have fallen by around 10%, while in the first six months, the concentration has dropped by about 25-30%.

分别在37℃,25℃和4℃下测出了IHC的自身氧化速率(从Fe(II)向met-Fe(III)转化)是大于60小时,96小时和25天。 Respectively at 37 ℃, 25 ℃ and 4 ℃ measured the self-oxidation rate of the IHC (conversion to met-Fe (III) from Fe (II)) is greater than 60 hours, 96 hours and 25 days. 保持在惰性气体中,获得这些结果就不需采用特殊的预防保护措施。 Maintained in an inert gas, to obtain these results without the use of special preventive and protective measures. 现有技术清楚地证明保存在象氮气这样的惰性气体中对降低血红蛋白的自身氧化率是有益的。 Clearly demonstrates that the prior art stored in an inert gas such as nitrogen to reduce the rate of autoxidation of hemoglobin is advantageous. 在这种条件下贮放一个较长时期所保存的Fe(II)血红蛋白部分将有极大的增长。 In this condition storage is stored for a longer period of Fe (II) hemoglobin section will have tremendous growth.

此外,用如上所述的Hyashi等的还原系统贮存IHC悬浮液可以防止自身氧化。 In addition, as described above with a reducing system of Hyashi like storage IHC suspensions may prevent their oxidation.

我们已研究了用巴氏灭菌作为IHC悬浮液最后阶段灭菌的一种方法。 We have studied a method pasteurized IHC suspension as the final stage of sterilization. 使用几种不同的巴氏灭菌条件。 Using several different pasteurization conditions. 用在每种条件下灭菌后的颗粒数目来衡量温度对IHC的作用。 With the number of particles in each condition after sterilization to measure the effect of temperature on the IHC.

条件1:IHC悬浮液的温度在8分钟内从25℃上升到62.8℃,并在这个温度下保持30分钟。 Condition 1: Temperature of the IHC suspension in 8 minutes increased from 25 ℃ to 62.8 ℃, and maintained at this temperature for 30 minutes. 颗粒数目表明降解低于20%。 The number of particles showed less than 20% degradation.

条件2:IHC悬浮液的温度在10分钟内从25℃上升到71.7℃并在该温度下保持15秒。 Condition 2: Temperature of the IHC suspension was increased over 10 minutes from 25 ℃ to 71.7 ℃ and held at this temperature for 15 seconds. 颗粒数目表明有低于20%的降解。 There are number of particles showed less than 20% degradation.

条件3:IHC悬浮液的温度在12分钟内从25℃上升到89.5℃并在该温度下保持2秒钟。 Condition 3: Temperature of the IHC suspension was increased in 12 minutes from 25 ℃ to 89.5 ℃ and held at this temperature for 2 seconds. 颗粒数目表明有高于70%的严重降解。 The number of particles greater than 70% indicates a serious degradation.

所以,发现条件1和2适合作巴氏灭菌方法。 So, to discover the conditions 1 and 2 are suitable for the pasteurization methods. γ射线照射也适合作最后阶段的灭菌方法。 γ-ray irradiation sterilization method is also suitable for the final stage.

用已知的变构效应物对血红蛋白的化学改性可以改变IHC的氧亲和性(或P50)。 Using known chemical modification allosteric effector of hemoglobin oxygen affinity IHC can change (or P50). 对血红蛋白的改性一般都限制了有氧和脱氧这两种构象之间的转换,因此氧合功能几乎总是以某种方式发生改变。 Modified hemoglobin generally limits the aerobic and deoxy conformational conversion between the two, so the oxygenation function is almost always in some way changed. 例如,在有氧形式下改性血红蛋白,通常对高氧亲和力有好处,而在脱氧条件下进行改性则作用是相反的。 For example, in the form of modified hemoglobin oxygen, high oxygen affinity is usually good, but under deoxidation conditions modification is the opposite effect. 由于吡哆醛衍生物分子很象天然变构效应物2,3一二磷酸甘油吡哆酯(DPG),所以可用吡哆醛衍生物作改性剂。 Because of pyridoxal derivative molecules, much like the natural allosteric effector 2,3 twelve pyridoxal phosphate, glycerol ester (DPG), so it can be pyridoxal derivatives as modifiers. 它们能结合血红蛋白末端的氨基。 They can bind hemoglobin terminal amino group. 例如通过类似于2,3-DPG的自然相互作用的5/-磷酸吡哆醛(PLP)与血红蛋白反应来增加P50。 For example, by a method similar natural interaction of 2,3-DPG 5 / - pyridoxal phosphate (PLP) to increase the reaction with the hemoglobin P50. 可用作改性剂的还有其它吡哆醛衍生物,如2-去甲-2-甲酰PLP(一种能连接血红蛋白β链的双官能团试剂)或者四磷酸双吡哆醛。 There may be used as modifiers of other derivatives of pyridoxal such as 2-nor-2-formylpropyl PLP (a bifunctional reagent capable of connecting the hemoglobin β chains) or bis pyridoxal tetraphosphate. 也可以用其它交联剂如酰基三甲基磷酸钠)来交联β链。 Can also be cross-linked with other cross-linking agents such as β-chain acyl-trimethyl phosphate).

还可以使用醛类改性剂。 You can also use aldehydes modifier. 例如用戊二醛聚合血红蛋白和用戊醛连接PLP。 For example with glutaraldehyde polymerized hemoglobin and connect with pentanal PLP.

双阿司区林酯例如3,5-双(二溴水杨基),富马酸和相应的单阿斯匹林可以用作变构改性剂。 Double A Division District Forest esters such as 3,5-bis (dibromo salicyl), fumaric acid and the corresponding single aspirin can be used as an allosteric modifier. 阿斯匹林在血红蛋白的a链和单官能团试剂之间结合到一个内部赖氨酸上。 Aspirin between hemoglobin chains and a monofunctional reagent to an internal lysine binding. 这二者都能增加血红蛋白的P50。 Both of which can increase the hemoglobin P50.

所以,可以制备“低亲和力”或“高亲和力”结构以用于除外伤和急性失血之外的情况,例如在需要局部传送氧并且是有益的情况下使用。 Therefore, can be prepared, "low affinity" or "high affinity" structure, except for the case of trauma and acute blood loss than, for example of the partial transfer of oxygen and is used under advantageous circumstances.

以上技术生产的一种“低亲和力”结构,即具有较高P50(>28mmHg)的结构,已在放射或化学方法治疗肿瘤中使用氧时作为一种辅剂使用。 Structure more than one technology to produce "low affinity" structure, that has a higher P50 (> 28mmHg) has been the treatment of tumors in radiation or chemical methods are used as an adjuvant when using oxygen. 这种结构在体外装载最大容量的氧,然后给药到肿瘤循环中。 In this configuration the maximum capacity of the oxygen loading in vitro, and then administered to the tumor circulation. 它使大量氧释放在肿瘤部位。 It makes a lot of oxygen release at the tumor site. 在放射或化学疗法存在下产生的活性氧使肿瘤部位产生较大的细胞毒素活性。 Reactive oxygen species in the presence of radiation or chemotherapy produced a greater portion of the tumor cytotoxic activity.

“高亲和力”结构(P50<28mmHg)适用于“局部缺血的氧输送”。 "High affinity" structure (P50 <28mmHg) applies to "ischemia oxygen delivery." 局部缺血或组织缺氧会在许多病理状态中出现,如中风,心肌梗塞等。 Ischemic or hypoxic tissue may occur in many pathological states, such as stroke, myocardial infarction and the like. 在这些部位优先释放氧将有助于尽可能减少组织的持续受损。 In these areas a priority will help to minimize the release of oxygen sustained damaged tissue. 具有与全血相似氧亲和力的氧载体或RBC代用品在这一类部位不会优先释放氧。 Whole blood has a similar affinity for oxygen or oxygen carrier RBC substitutes will not release oxygen priority sites in this category. 然而,具有高氧亲和力(即与全血相比P50较低)并且因时在正常遇到的氧梯度条件下能保留其大部分氧的结构,由于血和组织间氧梯度较大的缘故交替地在这类局部缺血部位优先释放氧。 However, with high oxygen affinity (ie, lower compared with whole blood P50) and when due under normal oxygen gradient conditions encountered in most of the oxygen can retain its structure, because the gradient between blood and tissue oxygen larger reason alternately Such ischemic sites in priority to release oxygen. 通过改变交联性质,用一种具有所需亲和力的合适天然血红蛋白,或用基因工程方法获得的具有合适亲合力的血红蛋白可以很容易地操作本发明的不溶性血红蛋白结构的亲和力,以获得一种适合这种使用的数值(P50)。 By changing the nature of crosslinking, using a suitable natural hemoglobin with the desired affinity, or by genetic engineering methods obtained having a suitable affinity hemoglobin can easily operate affinity insoluble hemoglobin structure of the present invention, to obtain a suitable Such use values (P50).

本发明不溶性血红蛋白结构能制成胶囊,并由此作为药中氧载体(如氟化碳类),药物,诊断剂等的有效载体。 Insoluble hemoglobin structure of the invention can be made into capsules, and thus as a drug carrier effective oxygen carrier (such as fluorinated carbon-based), drugs, diagnostic agents of. 被胶囊包裹的氟化碳类(FC)是有效的氧载体,它与氧分压是以线性关系运送的释放溶解的氧,而IHC的血红蛋白外壳与氧压却是以S形曲线关系运送和释放结合的氧。 By encapsulating a carbon fluoride type (FC) are effective oxygen carriers, which release oxygen and the oxygen partial pressure is based on a linear relationship transported dissolved, and hemoglobin IHC shell and the oxygen pressure is in the S-shaped curve, but the relationship between the delivery and the release of bound oxygen. 在同一制剂中血红蛋白和氟化碳的这种特殊结合使体内能够最大地运送和释放氧。 In the same formulation of this particular combination of hemoglobin and fluorocarbon maximum so that the body can be transported and releasing oxygen.

现有技术中都没有公开同时运送血红蛋白(Hb)和氟化碳(FC)能力的技术。 Are not disclosed in the prior art while transporting hemoglobin (Hb) and fluorocarbon (FC) technology capabilities. 在血红蛋白外壳的核内包裹的氟化碳能作为一个氧的贮存库。 In hemoglobin housing nuclear carbon fluoride can be wrapped as a reservoir for oxygen. 这种结合使载体结合的氧的运送对压力呈S形关系(即对血红蛋白)以及线性关系(即对氟化碳)。 This combination enables the delivery of oxygen bound to a carrier pressure was S-shaped relationship (ie hemoglobin) and linear relationship (ie, carbon fluoride). 此结合也使其对组织pO2为线性的“背景”释放氧(从氟化碳)以及S型的“团块”(bolus)的氧释放(从血红蛋白)。 This combination also makes the tissue pO2 linear "background" release of oxygen (from the carbon fluoride), and S-type "lumps" (bolus) of oxygen release (from hemoglobin). 特别是例如组织局部贫血或肿瘤治疗过程中,短时间内需要大量氧时,它可更有效地进行氧的输送。 Especially e.g. tumor tissue of ischemia or the course of treatment, a short period of time requires a lot of oxygen, it can be more effective oxygen delivery.

Hb/FC结合可外部监测血管内运送剂量的位置而显示其优点。 Hb / FC combination can position the external monitoring intravascular delivery of doses and show its advantages. 因为通过MRI很容易使19F核成像,因此很可能觉察血管和组织内运送悬浮液的累积。 By MRI because it is easy to 19F nuclear imaging, it is likely to accumulate in the blood vessels and tissues perceive transport suspension. 这在肿瘤治疗中具有很大优点,因在此过程中,氧被同来作为放射或化疗剂的辅助剂,所以它可精确地监视载氧血红蛋白/FC悬浮液输送到预想的位点。 This has the great advantage in cancer therapy, because in this process, oxygen is the same as radiation or chemotherapeutic agents adjuvants, it can accurately monitor the oxygen-carrying hemoglobin / FC suspension to the desired site of delivery.

本发明实践中,有许多氟化碳(FCs)适合使用,详述如下。 Practice of the invention, there are many carbon fluoride (FCs) suitable for use, as detailed below.

此外,无氧的结合力但可交联半胱氨酸残基或巯基(天然或人工引入)的蛋白质可包裹合适氧亲合性的生物相容氟化碳用作血液替代物。 In addition, oxygen-free binding force but crosslinkable cysteine residues or sulfhydryl groups (native or artificially introduced) may be wrapped in a protein affinity for oxygen suitable biocompatible carbon fluoride used as a blood substitute. 作为其例,血蛋白包裹全氟萘烷或全氟三丙胺用作血液替代品。 As examples thereof, blood proteins wrapped perfluorodecalin or perfluoro tripropylamine is used as a blood substitute.

有一些药物适合于包裹在本发明血红蛋白微胶囊中。 There are a number of drugs suitable for hemoglobin encapsulated in microcapsules of the present invention. 一些化学治疗剂需要有氧存在才可获得最大肿瘤细胞毒性。 Some chemotherapeutic agents require the presence of oxygen in order to obtain the maximum tumor cytotoxic. 在一种氧载体如血红蛋白的结构中传送这类药物可以有效地把细胞毒素的基本成分结合进一种单一包装中。 The transmission of such drugs in an oxygen carrier such as hemoglobin structure of the basic components can be effectively incorporated into the cell toxin in a single package. 许多适用的细胞毒素药物是油溶性的。 Many suitable cytotoxic drug is oil soluble. 这些药物可溶于一种氟化碳或者其它生物相容的油中,如大豆油,红花油,椰子油,橄榄油,棉花籽油等。 These drugs can be dissolved in a fluorocarbon or other biocompatible oil such as soybean oil, safflower oil, coconut oil, olive oil, cotton seed oil and the like. 将油/药溶液和一种血红蛋白溶液一起经超声波辐射来生产在一种包裹在交联的不溶性血红蛋白外壳中的油/药微球。 The oil / drug solution and a hemoglobin solution to produce together by ultrasonic irradiation in a package housing oil-insoluble crosslinked hemoglobin in / microspheres. 在血管内给药前把该悬浮液用氧饱和。 In a blood vessel prior to administration to the suspension was saturated with oxygen. 油溶性细胞毒性药物包括环磷酰胺,GCNU,左旋溶肉瘤素,自力霉素,紫杉醇及其衍生物,taxotere及其衍生物,喜树碱,阿霉素,鬼臼乙叉甙,阿莫西芬,长春花碱,新长春碱等;非甾体类抗炎剂如:布洛芬、阿斯匹林、吡氧噻嗪、甲氰咪胍等;甾体类如雌激素,氢化泼尼松,可的松,氢化可的松,二氟松等,一些药物如胆甾醇对苯乙酸氮芥,邻氯苯对氯苯二氯乙烷,维沙定(Visadine),卤代亚硝基脲类(helonitrosoureas),蒽环类(anthrocydine),椭圆玫瑰树碱,安定,等;免疫抑制剂如环孢菌素,硫唑嘌呤,FK506等。 Oil-soluble cytotoxic drugs include cyclophosphamide, GCNU, L dissolved sarcoma, self-adriamycin, paclitaxel and its derivatives, taxotere and derivatives, camptothecin, doxorubicin, etoposide, amoxicillin Finland, vinblastine, vinblastine and other new; non-steroidal anti-inflammatory agents such as: ibuprofen, aspirin, piroxicam, cimetidine, etc; steroid hormones such as estrogen, hydrogenated prednisone pine, cortisone, hydrocortisone, fluoride pine, some drugs such as cholesterol of phenylacetic acid mustard, dichloroethane, chlorobenzene o-dichlorobenzene, vysha set (Visadine), halo-nitroso Urea (helonitrosoureas), anthracyclines (anthrocydine), ellipticine, stability, etc; immunosuppressants such as cyclosporine, azathioprine, FK506, etc.

用双乳化的方法也可以以胶囊的形式把水溶性药物包在IHC壳内。 Using double emulsion method may also be in the form of capsules in the water-soluble drug package IHC shell. 首先,用一种生物相容油乳化药物水溶液得到一种油包水(w/o)乳化液。 First, a biocompatible oil-emulsifying drug solution to give a water-in-oil (w / o) emulsion. 把w/o乳化液当作油相,并与上述血红蛋白水溶液一起经受超声波辐射处理,生成壳中含有IHC的所需水溶性药物的微浮化液,适用于本发明这个实施方案的乳化剂包括普卢兰尼克斯(Pluronics)(聚环氧乙烷和聚环氧丙烷的嵌段共聚物),来源蛋黄的磷脂(如卵磷脂,蛋黄卵磷脂等);脂肪酸酯(如,单和双硬脂酸甘油酯,单和双棕榈酸甘油酯等)。 The w / o emulsion as the oil phase, and is subjected to ultrasonic irradiation treatment together with the above aqueous solution of hemoglobin, to generate micro-emulsified liquid IHC shell containing the desired water-soluble drug, suitable for this embodiment of the present invention include emulsifiers Pulu Raney Alex (Pluronics) (polyethylene oxide and polypropylene oxide block copolymers), sources of egg yolk phospholipids (such as lecithin, egg yolk lecithin and the like); fatty acid esters (e.g., mono- and di glyceryl monostearate, glyceryl mono- and di-palmitate, etc.). 用于本发明这个实施方案中的水溶性药物包括抗肿瘤药,如放线菌素,博来霉素,环磷酰胺,段诺霉素(duanorubicin),阿霉素,艾波霉素(epirubicin),氟尿嘧啶,碳铂(carboplatin),顺氯氨铂,干扰素,白芥素,氨甲叶酸,自力霉素,它莫西芬,雌激素,孕激素等。 Used in this embodiment, the present invention is a water-soluble drugs, including anticancer drugs such as actinomycin, bleomycin, cyclophosphamide, daunorubicin segment (duanorubicin), doxorubicin, adriamycin Appleby (epirubicin ), fluorouracil, carboplatin (carboplatin), cisplatin, interferon, plain white mustard, methotrexate, self-adriamycin, tamoxifen, estrogen, progesterone and other.

双乳化方法也适合于传送其它水溶性治疗剂,诊断剂或有营养价值的物质。 Double emulsion method is also adapted to transmit other water soluble therapeutic agent, diagnostic agent or nutritional value of the substance. 例如,以胶囊形式把血红蛋白微乳化液包裹于IHC中可以增加IHC中的血红蛋白的含量。 For example, in capsule form the hemoglobin microemulsion wrapped in IHC can increase the content of hemoglobin IHC.

为了使IHC更象红细胞,可在交联的血红蛋白微泡周围形成磷脂双层。 In order to make the IHC more like red blood cells, can be crosslinked hemoglobin microbubbles formed around the phospholipid bilayer. 这类双层形成了一种真正的“红细胞类似物”,并可在两步骤工艺中生成用于形成这种双层结构的带电荷磷脂或脂类包括磷脂酰胆碱,磷脂酰乙醇胺,磷脂酰丝氨酸,磷脂酰肌醇,磷脂酰甘油,神经鞘髓磷脂,双十四烷基磷酸,双十六烷基磷酸,肌氨酸酯(肌氨酰胺),甜菜碱,单体和双体醇酸等。 Such bilayer forming a true 'red cell analog' and may be used to generate such a two-layer structure formed of charged phospholipids or lipids include phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl in a two step process serine, phosphatidyl inositol, phosphatidyl glycerol, nerve myelin sheath, bis-tetradecyl phosphate, dicetyl phosphate, sarcosinate (sarcosinate amide), betaines, monomeric and catamaran alcohol acid and the like. 在本发明中也可以使用非离子脂类,它包括聚乙烯脂肪酸酯,聚乙烯脂肪酸醚,二乙醇酰胺,长链酰基己糖酰胺,长链酰基氨基酸酰胺,长链氨基酸胺,聚氧乙烯山梨糖醇酯,聚氧单甘油酯和双甘油酯,单和双硬脂酸酯甘油酯,单和双油酸甘油脂,单和双棕榈酸甘油酯该技术上的另外一个变化是使用可光聚合的脂类或经化学反应容易被交联的脂类来生产一种更加稳定的脂类“膜”包衣。 In the present invention, non-ionic lipids may also be used, including polyethylene fatty acid esters, polyethylene fatty acid ethers, diethanolamides, long chain acyl amides hexose, long chain acyl amino acid amides, long chain amino acid amines, polyoxyethylene sorbitan esters, polyoxyethylene mono- and diglycerides, mono- and di-stearates, glyceryl mono- and di-glycerides of oleic acid, palmitic acid mono- and di-glycerides of the technically Another variation is to use photopolymerizable lipids or by chemical reactions are readily crosslinked lipids to produce a more stable lipid 'membrane' coat. 可用于本发明中的可光聚合的脂类有丙烯酸酯或甲基丙烯酸酯取代的脂类(如,磷脂酰胆碱,磷脂酰乙醇胺,磷脂酰丝氨酸,磷脂酰甘油,双十四烷基磷酸,双十六烷基磷酸等),具有天然可聚合的不饱和的脂类(如具有丙二醛基或可连接的双烯基的不饱和的磷脂酰胆碱等),等等。 Useful in the present invention are photopolymerizable lipids substituted acrylate or methacrylate lipids (e.g., phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl glycerol, bis-tetradecyl phosphate , dicetyl phosphate, etc.), having a polymerizable unsaturated natural lipids (such as those having aldehyde or bis alkenyl malonic connectable unsaturated phosphatidylcholine, etc.), and the like. 经硫代-二硫化物交换容易进行交联的脂类也适合于形成IHC的稳定脂类包衣。 After thio - disulfide exchange readily crosslinked lipids are also suitable for the formation of stable lipid coated IHC. 这种脂类的例子有用硫辛酸酯化的磷脂酰胆碱的衍生物,等等。 Examples of such useful lipoic acid esterified lipid phosphatidylcholine derivatives, and the like.

以超声波辐射合成的IHC类可以按如上所述以在生物相容介质中的混悬液形成给药,也可以与其他营养剂一起给药。 Ultrasonic radiation can be synthesized as described above in class IHC suspension in a biocompatible medium is formed of administration, may be administered together with other nutrients.

体内给药的优选途径是静脉内,动脉内,肌肉内,皮下,腹膜内,口腔内,吸入,局部,透皮,栓剂,阴道栓等给药途径。 Preferred route of administration is in vivo intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, oral, inhalation, topical, transdermal, suppository, vaginal suppository, etc. administration route.

总之,本发明不溶性血红蛋白结构比现有技术的可溶性血红蛋白,用胶囊包裹的可溶性血红蛋白,氟化碳血液代用品或氧载体有许多优点。 In summary, the insoluble hemoglobin structure of the present invention over prior art soluble hemoglobin, soluble hemoglobin encapsulating, fluorocarbon blood substitutes or oxygen carriers have many advantages. 这些优点包括:—较高的氧容量;—可变的氧亲和力;—不溶性“大聚合物”血红蛋白,它比现有技术中的四聚物或寡聚物的可溶性血红蛋白可在循环中保留较长时间;—由于分子体积较大而具有较低可能性的肾细胞毒性;—血红蛋白渗漏的可能性比脂质体包裹的血红蛋白更小;—由于它的体积比脂质体更大,所以不可能有激发补体蛋白的聚集物形成;—与现有技术的可溶性血红蛋白相比,由于其独立的“细胞”性,其作用更象RBC;—在运载与血红蛋白结合的氧的同时还能运送一个未结合氧的贮存库;—能作为不含有潜伏变态反应性和毒性乳化剂的一种氟化碳(FC)载体;—Hb/FC结构中的交联血红蛋白能提供比现有技术中使用卵磷脂和/或其它合成表面活性剂的乳化系统更强的稳定性;—从Hb/FC的氧释放曲线是与组织pO2有关的一种S形和线形结合的曲线;—可用19FMRI体内测定和监控Hb/FC结构;—除了运送氧外,血红蛋白或Hb/FC结构也可用作药物载体;—可以将脂类双层膜用于血红蛋白结构,使其更象生理结构;—用聚合物如PEG改性血红蛋白结构来进一步增加血管内的保留时间。 These advantages include: - higher oxygen capacity; - variable oxygen affinity; - insoluble "Great polymer" hemoglobin, it may retain comparing the prior art soluble hemoglobin tetramer or oligomer in a loop long; - due to larger molecules having a low possibility of renal cell toxicity; - the possibility of leakage of hemoglobin liposome-encapsulated hemoglobin ratio less; - due to its larger volume than the liposomes, so there can stimulate complement proteins aggregate formation; - compared to soluble hemoglobin of the prior art, because of their separate "cells", and its effect is more like RBC; - in carrying and hemoglobin oxygen delivery while an unbound oxygen depot; - can be used as a fluorocarbon (FC) does not contain a latent allergic and toxic emulsifiers carrier; -Hb / FC structure crosslinked hemoglobin can be used than in the prior art to provide lecithin and / or greater stability of other synthetic surfactants emulsifying systems; - from Hb / FC oxygen release profile is associated with tissue pO2 and a linear combination of S-shaped curve; - and in vivo assays are available 19FMRI Monitoring Hb / FC structure; - besides transporting oxygen, the hemoglobin or Hb / FC structure can be used as drug carriers; - can be used for hemoglobin lipid bilayer structure, making it more like the physical structure; - with polymers such as PEG-modified hemoglobin structure to further increase intravascular retention time.

本发明的另一方面,可以将一般是疏水的,与水不混容的这种难以给药的含有机氟化合物包裹在聚合物外壳中(如上所述),使其容易地传送。 Another aspect of the present invention can be generally hydrophobic, water immiscible and difficult to administer such a fluorine-containing compound in the polymer shell wrap (as described above), making it easy to transfer. 用聚合物外壳包裹的含有机氟化合物是容易使用的,并且是生物相容的。 A fluorine-containing compound with a polymer shell is wrapped easy to use, and is biocompatible. 按照本发明所生产的聚合物壳体颗粒大小有大约2微米的平均直径,因为它用于静脉内或动脉内注射没有小血管堵塞并随后引起组织损伤(例如由于局部失血而引起氧丢失)的危险,所以医学应用是最理想的,相比较起来,红细胞直径大约是8微米(所以为防止血管堵塞注射性生物材料的直径应当小于8-10微米。 According to the present invention is produced by the particle size of the polymer shell have an average diameter of about 2 microns, as it for intravenous or intraarterial injection No small blood vessel blockage and subsequent tissue damage (e.g., due to partial loss of oxygen caused by loss of blood) of dangerous, so is ideal for medical applications, phase comparison, red blood cell is about 8 microns in diameter (so as to prevent blood vessel blockage injectable biomaterial should be smaller than the diameter of 8-10 microns.

天然产生的氟原子(19F)能给出一个清晰的核磁共振信号,所以在MRI中能作为对比剂或“探针”使用19F的特殊优点包括:1)在体内的天然浓度相当低(一般在体内不存在氟),2)核磁共振的高敏感性,3)与1H相近的磁旋比,所以仅对现有的MRI装置作微小改动就能用来使19F磁共振成像,以及4)多数含有机氟化合物毒性低。 Naturally occurring fluorine atoms (19F) can give a clear nuclear magnetic resonance signal, so can be used as a contrast agent in MRI or "probe" used 19F Special advantages include: 1) natural in vivo relatively low concentration (generally in absence of in vivo fluoro), 2) a high nuclear magnetic resonance sensitivity, 3) and the 1H gyromagnetic ratio similar, so only the conventional MRI apparatus can be used to make a small change 19F magnetic resonance imaging, and 4) the majority of fluorine-containing compounds of low toxicity.

氟化碳一般无毒,并且是生物相容的。 Carbon fluoride is generally non-toxic and biocompatible. 由于氟化碳的碳-氟键较强(约130kcal/摩尔),所以它是稳定的,并不具有反应性的,固此,也不可能被代谢。 Since the carbon fluoride carbon - fluorine bonds stronger (about 130kcal / mol), so that it is stable, not reactive with the solid of this, can not be metabolized. 相比而言,CH键(约100千卡/摩尔)较弱并有反应性较强,FDA已批准了两种氟化碳,全氟三丙胺和全氟萘烷,以商品名Fluosol DA作为血液代用品在医学上使用。 In contrast, CH bond (approximately 100 kcal / mole) is weak and there is a stronger reactivity, FDA has approved two kinds of carbon fluoride, perfluoro tripropylamine and perfluorodecalin, as the trade name Fluosol DA blood substitutes used in medicine.

在实施本发明中能够使用许多不同的氟化碳。 In the embodiment of the present invention can be used in many different carbon fluoride. 例如,可把符合下列通式的化合物混入本文所述的本发明方法所用的聚合物外壳中:(a)Cx F2x+y-zAz,其中x=1-30,优选5-15, For example, the compound may be incorporated in line with the formula described herein a method used in the present invention the polymer shell: (a) Cx F2x + y-zAz, where x = 1-30, preferably 5-15,

y=2;或当X≥2时,y=0或-2;或者当x≥4时,y=-4,z=0到(2x+y-1)的任何整数;和A选自于H,除F外的卤素,-CN,-OR,其中R是H,烷基,氟烷基,链烯基,氟代链烯基,炔基,氟代炔基,芳香基,氟代芳香基,链烷酰基,氟链烷酰基,链烯酰基,氟链烯酰基,炔酰基,氟炔酰基,(b)[CxF2x+y′-zAz]aJRb-a,其中X,Z,A和R如上所定义,Y′=+1;当X≥2时Y′=-1或-3;当X≥4时Y′=-5,J=O,S,NP,Al或Si,a=1,2,3或4,和b=对二价J是2,或对三价J是3,对四价J是4,(c)A′-[(CF2)xO]cA″,其中:X如上所定义,A′选自于H,卤原子,-CN,-OR,其中R是H,烷基,氟烷基,链烯基,氟代链烯基,炔基,氟代炔基,芳香基,氟代芳香基,链烷酰基,氟链烷酰基,链烯酰基,氟链烯酰基,炔酰基,氟炔酰基,A″选自H或R,其中R如上所定义,C=1-300,优选2-50,或 y = 2; or when X≥2 time, y = 0 or -2; or when x≥4 time, y = -4, z = 0 to (2x + y-1) is any integer; and A is selected from H, halogen except F, -CN, -OR, wherein R is H, alkyl, fluoroalkyl, alkenyl, fluoroalkenyl, alkynyl, fluorinated alkynyl group, an aromatic group, an aromatic fluoro group, alkanoyl, fluoro alkanoyl, alkenoyl, fluoro alkenoyl group, an alkynyl group, an alkynyl group fluorine, (b) [CxF2x + y'-zAz] aJRb-a, wherein X, Z, A and R as defined above, Y '= + 1; when X≥2 when Y' = - 1 or -3; when X≥4 when Y '= - 5, J = O, S, NP, Al or Si, a = 1 , 2, 3 or 4, and b = 2 for a divalent J, or 3 for a trivalent J, for a tetravalent J is 4, (c) A '- [(CF2) xO] cA ", wherein: X as defined above, A 'is selected from H, a halogen atom, -CN, -OR, wherein R is H, alkyl, fluoroalkyl, alkenyl, fluoroalkenyl, alkynyl, fluoro alkynyl, aryl, fluorinated aryl, alkanoyl, fluoro alkanoyl, alkenoyl, fluoro alkenoyl group, an alkynyl group, an alkynyl group fluorine, A "is selected from H or R, wherein R is as defined above, C = 1 -300, preferably 2 to 50, or

(d) (D) 其中:X如上所定义,而c′=2-20,优选2-8,以及它们中任意两种或多种混合物。 Wherein: X is as defined above, and c '= 2-20, preferably 2-8, and any mixture of two or more of them.

上述通式中所包括的是具有如下通式的化合物:CxF2x,例如,全氟-1-己烯(C6F12),全氟-正己烯(C6F12),全氟-3-己烯(C6F12)等;环状-CxF2x,例如,全氟环己烷(C6F12),全氟环辛烷(C8F16)等,CxF2x-2,例如,全氟-1-己炔(C6F10),全氟-2-己炔(C6F10),全氟-3-己炔(C6F10)等,双环-CxF2x-2,例如全氟萘烷(C10F18)等,CxF2x+2,例如,全氟己烷(C6F14),全氟辛烷(C8F18),全氟壬烷(C9F20),全氟癸烷(C10F22),全氟十二烷(C12F26)等,CxF2x-4例如,全氟-2,4-己二烯等,CxF2x+1A,例如,全氟三丙胺[(C4F9)3N],全氟三丁胺,全氟三叔丁胺,等,CxF2x-2A2,例如,C10F18H2,等,以及这类高度氟化的化合物如全氟-1,2-二氢化茚,全氟甲基金刚烷,全氟辛基溴,全氟二甲基环辛烷,全氟环辛溴,全氟冠醚等。 In the above general formula include compounds having the general formula is: CxF2x, e.g., perfluoro-1-hexene (C6F12), perfluoro - n-hexene (C6F12), perfluoro-3-hexene (C6F12), etc. ; cyclic -CxF2x, for example, perfluorocyclohexane (C6F12), perfluoro cyclooctane (C8F16), etc., CxF2x-2, e.g., perfluoro-1-hexyne (C6F10), perfluoro-2-hexyl alkyne (C6F10), perfluoro-3-hexyne (C6F10), etc., bicyclic -CxF2x-2, e.g., perfluorodecalin (C10F18), etc., CxF2x + 2, for example, perfluorohexane (C6F14), perfluoro-octyl alkyl (C8F18), perfluoro nonane (C9F20), perfluoro decane (C10F22), perfluoro dodecane (C12F26), etc., CxF2x-4, for example, perfluoro-2,4-hexadiene, CxF2x + 1A, e.g., perfluoro tripropylamine [(C4F9) 3N], perfluorotributylamine, perfluoro-tri-t-butylamine, etc., CxF2x-2A2, e.g., C10F18H2, etc., as well as such highly fluorinated compounds such as perfluoro - indan, perfluoromethyl adamantane, perfluorooctyl bromide, perfluoro dimethyl cyclooctane, perfluoro cyclooctadiene bromo, perfluoro crown ether.

除了如上所述的直链,支链和环状含氟化合物外,在实施本发明中还可以使用氟化冠醚(如,全氟-12-冠-4,全氟15-冠-5,全氟18-冠-6等)。 In addition to the straight chain, branched chain and cyclic fluorine-containing compounds as described above, in the embodiment of the present invention may also be used fluorinated crown ethers (e.g., perfluoro -12- crown-4, perfluoro 15-Crown-5, perfluoro-crown-6, etc.).

为了获得具有较高信号噪声比的好的磁共振图像,使用大量等价氟是有益的,这里所用的术语“等价氟”是指那些含氟化合物的存在于基本上类似的微环境(即;基本上相似的磁环境)中的氟取代基,等价氟将会产生一个显像信号。 In order to obtain good magnetic resonance images with high signal to noise ratio, the use of a large number of equivalent fluorine is advantageous, as used herein the term "fluorine equivalence" refers to the presence of those fluorine-containing compound in a substantially similar micro-environment (i.e. ; substantially similar magnetic environment) of fluorine substituents, fluorine will produce an equivalent imaging signal. 大量等价氟将会产生一个不被“非等价”氟的对抗信号冲淡的强信号。 Will generate a large number of equivalent fluorine signal is not strong "non-equivalent" dilute fluorine against signal.

这里所用术语“非等价氟”是指那些与相同含氟化合物上的其他氟取代基相比存在于基本上不相似的微环境(即基本上不相似的磁环境)中的含氟化合物的氟取代基。 As used herein the term "non-equivalent fluorine" refers to the group as compared with the present in a substantially non-similar micro-environment (i.e., do not substantially similar magnetic environment) in the fluorine-containing compound are identical to those of other fluorine-containing compound with fluorine substituents on the fluoro substituents. 因此,与等价氟相比,非等价氟由于其不同的化学位移会产生多个信号。 Thus, as compared with equivalent fluorine, non-equivalent fluorine due to their different chemical shifts will produce a plurality of signals. 所以,尽管具有大量非等价氟的化合物可以满足MRI的应用,但要获得最佳的图像这些化合物是不理想的。 So, even though a large number of non-equivalent fluorine compounds can meet MRI applications, but to get the best picture of these compounds are not ideal.

对于血管显像的应用特别有意义的是具有延长了循环时间的含氟化碳聚合物外壳。 Vascular imaging of particular interest is the application of a fluorinated carbon having a prolonged cycle time of polymer shell. 目前所用的血管照像技术使用X-射线对比剂,这是一种损伤性的方法。 The presently used photographic techniques using vascular X- ray contrast agent, which is an invasive method. 近来已证实1H-MRI能够用于血管照像术[Edelman和Warach,New Eng-land J.of Medicine328:785-791(1993)]。 1H-MRI has recently been confirmed by photography can be used in vascular surgery [Edelman and Warach, New Eng-land J.of Medicine328: 785-791 (1993)]. 同样地,19F-MRI适用于血管照像术,并具有许多优点,例如能获得与周围组织(它不含任何天然氟)参照的较强烈的对比。 Similarly, 19F-MRI is suitable for photographic vascular surgery, and has many advantages, such as can be obtained with the surrounding tissue (which does not contain any native fluorine) with reference to the relatively strong contrast. 可应用这种方法的例子有诊断和鉴别颅内动脉瘤,动静脉畸形,上腔静脉闭塞,下腔静脉闭塞,门静脉闭塞,盆腔静脉闭塞,肾静脉闭塞,肾肠系膜动脉闭塞,外周肠系膜动脉闭塞等。 Examples of this approach can be applied to diagnostic and identification of intracranial aneurysms, arteriovenous malformation, occlusion of the superior vena cava, inferior vena cava occlusion of the portal vein occlusion, pelvic vein occlusion, renal vein occlusion, renal mesenteric artery occlusion, peripheral arterial occlusive mesenteric and so on.

按照本发明用聚合物外壳包裹的含氟化合物能用于各种目的,如,获得各种器官和/或组织的磁共振图像,获得器官和/或组织的氟分布情况,以及测量局部温度。 The fluorine-containing compound according to the present invention is wrapped with a polymer shell can be used for various purposes, e.g., to obtain various organs and / or magnetic resonance image of the tissue, organ distribution obtained fluorine and / or tissue, and measuring the local temperature. 本发明对比剂并不仅局限在MRI的使用,而且也能用于超声波扫描术和放射学的使用中。 The present invention is not limited to the contrast agents in MRI use, but can also be used using ultrasonography and radiology in. 氟的其它同位素18F也能用作正电子放射X线体层照相中(PET)的对比剂。 Other isotopes 18F fluoride can also be used as a positron emission tomography in the X-ray (PET) contrast agent. 所以,使用一种含氟对比剂,能够进行PET和MRI两种诊断。 Therefore, use of a fluorine-containing contrast agent, capable of two diagnostic PET and MRI. 也能捕获用于放射对比剂中的其它显影剂如锝和铊化合物。 Radiation can also be used to capture the contrast agent other developers such as technetium and thallium compounds. 这类对比剂的两个例子是神经松驰剂(Neurolyte)和心松解剂(Cardiolyte)。 Two examples of this type of contrast agent is nerve relaxant (Neurolyte) and cardiac release agent (Cardiolyte).

使用本发明组合物测定氧是基于在顺磁物质如氧的存在下19F的NMR松驰率的显著变化。 Using the composition of the present invention is based on the determination of oxygen in the presence of paramagnetic species such as oxygen at a significant change in the NMR relaxation rate of 19F. 由于氧是顺磁性的,它将与氟核相互作用,增加19F从激发态到正常态的松驰率。 Because oxygen is paramagnetic, it will work with fluorine nuclear interaction, increased 19F from an excited state to the normal state of relaxation rate. 用监控这种松驰率变化的方法,能够测定局部区域的氧浓度(把MRI信号校准到一个已知氧浓度)。 By monitoring the rate of change of this relaxation method, the oxygen concentration can be measured local area (the MRI signal is calibrated to a known concentration of oxygen).

例如,这种体系的新颖性在于1)使用MRI能获得氧的信息;2)利用氧的顺磁性影响19FMRI(NMR)信号;3)使用聚合物外壳提供一个对氧也具有通透性的恒定的保护性环境等等。 For example, the novelty of this system is that a) the use of MRI to obtain oxygen information; 2) the use of oxygen paramagnetic influence 19FMRI (NMR) signal; 3) the use of a polymer shell also has a pair of oxygen permeability constant protection of the environment and so on.

通过使用经过生理温度范围内的相变的固体含氟化合物(如,高分子量的化合物,或含氟化合物的混合物),也能使用MRI测量局部温度。 By using the phase change through the physiological temperature range of solid fluorine-containing compound (e.g., high molecular weight compound, or a mixture of fluorine-containing compounds), MRI can also be used measure the local temperature. 固体比液体的松驰时间更长。 Longer than the relaxation time of solid liquid. 所以当达到相变转化(即,从固体到液体)时,松驰时间将显著降低。 So when it reaches the phase change transition (i.e., from solid to liquid), the relaxation time will be significantly reduced. 在固体到液体的相变中可观察到NMR谱的显著变化。 In the solid to liquid phase transition can be observed a significant change in NMR spectrum. 把指定含氟化合物的MRI信号形状可以校正到一个已知温度。 The MRI signal shape specified fluorine-containing compound can be calibrated to a known temperature. 通过使用聚合物外壳内的高分子量含氟化合物(即,具有≥15℃熔点的含氟化合物),或者通过使用一种聚合物外壳内的含氟化合物与非氟化合物的混合物,来选择聚合物外壳的内含物,以提供一种发生相变所需的温度范围(一般在约22-55℃范围内)。 By using a high molecular weight fluorine-containing compound within the polymeric shell (i.e., a fluorine-containing compound having a melting point of ≥15 ℃), or by using a mixture of a fluorine-containing compound and a non-fluorinated compound within the polymeric shell, the polymer is selected The contents of the housing, to provide a desired phase transition temperature range (typically in the range of about 22-55 ℃). 该壳内的氟化碳将在指定温度范围内经过从固体到液体的相变,随之也改变所观察到的松驰率,这样就可以测定体内温度。 The carbon fluoride shell will pass within the specified temperature range the phase change from solid to liquid, and with it changes observed relaxation rate, so the temperature can be measured in vivo. 局部温度数据将是特别有用的,例如在癌症的高体温治疗中监控患者或者在测定癌细胞中(癌细胞比正常细胞更冷)使用。 Local temperature data would be particularly useful, for example, to monitor patients at high temperature treatment of cancer or cancer cells was measured (colder cancer cells than in normal cells) in use.

所使用的含氟组合物将决定相变的温度范围。 The fluorine-containing composition employed will determine the phase transition temperature range. 所以,只是简单地通过改变含氟组合物的组分即可在较宽的温度范围内使用这种技术。 Therefore, simply to use this technology in a wide range of temperatures by varying the component of the fluorine-containing composition. 例如,在一种聚合物外壳中包裹的纯全氟十二烷(C12F26)将在氟化碳的熔点(75℃)进行固体到液体的相变。 For example, in a polymer shell wrapped pure perfluoro dodecane (C12F26) will be a solid to liquid phase transition at the melting point of the carbon fluoride (75 ℃). 然而,这种转变是急速的,只能得到少量的温度数据。 However, this change is rapid, and can only get a small amount of temperature data. 为得到大量的数据,可以把含氟组合物的熔点扩大到较宽范围内,例如,只是简单地向纯的含氟组合物中加入其它组分。 To obtain large amounts of data, the melting point of the fluorine-containing composition can be expanded to a wide range, for example, simply to the pure fluorine-containing composition added to the other components. 现有技术中众所周知混合物比相应纯的组分有较低的和较宽的熔点范围。 The mixture was well known in the prior art has a lower melting point and a wider range than the corresponding pure components. 所以,例如,用一种较低分子量的氟化碳配制成的全氟十二烷将会加宽用胶囊形式包裹的组合物的熔点范围。 Therefore, for example, with a lower molecular weight carbon fluoride formulated as perfluoro dodecane capsule form will be widened wrapped composition melting point range. 相似地,用一种烷烃(如戊烷)和含氟化合物(如,全氟十二烷)的混合物将会加宽包裹组合物的熔点范围。 Similarly, with an alkane (e.g., pentane), and fluorine-containing compounds (e.g., perfluoro dodecane) was will widen the range of the melting point of the coating composition.

此外,在实施本发明中也能使用化学加成氟的化学改性长链脂肪酸(如,正十七烷酸[C17H34O2],十九烷酸[C19H38O2]等),醇类(如十九烷醇[C19H40O],二十二烷醇[C22H46O]等)。 Further, in the embodiment of the present invention can also be used fluorine chemical addition chemically modified long chain fatty acids (e.g., n-heptadecanoic acid [C17H34O2], nonadecanoic acid [C19H38O2], etc.), alcohols (e.g. nonadecanoic alcohol [C19H40O], Docosanol [C22H46O], etc.). 例如,全氟-叔-丁醇(t-[C4F9-OH;PCR CHEMI-CALS)]与上述任意反应性含氧化合物之间的脱水偶联反应将会产生经过固体到液体相变的一个分子,和一个具有9个等价氟的分子。 For example, perfluoro - tert - butoxide (t- [C4F9-OH; PCR CHEMI-CALS)] dehydration coupling reaction between any of the above-described reactive oxygen-containing compounds will produce a molecule through a solid to liquid phase transition and one has nine equivalent fluorine molecules. 同样,例如,一种氟化脂肪酸加宽熔点范围,由此可用于进行局部温度的测定。 Similarly, e.g., fluorinated fatty acids, melting point range is widened, thereby can be used for measuring the local temperature.

这种温度测定体系的新颖性在于,例如1)可使用MRI获得空间分解的温度数据,2)利用温度与MRI(NMR)信号的依赖关系,3)使用了在指定温度范围经过固体到液体相变的一种含氟化碳组合物,4)使用了聚合物外壳对介质提供恒定和保护性的环境,和4)在得到形态数据的同时可得到温度数据。 Novelty is that such a temperature measurement system, e.g., 1) may be used to obtain spatially resolved temperature MRI data, 2) the use of temperature and MRI (NMR) signal dependency, 3) used in the specified temperature range after the solid to liquid phase A fluorine-containing carbon variable composition, 4) a polymer shell on the medium used to provide a constant and protective environment, and 4) to obtain temperature data in the form data obtained simultaneously.

按照本发明,在一个截面直径不大于约10微米(这里使用的术语“微米”是指毫米的千分之一测量单位)的壳内包裹含氟组合物的颗粒。 According to the present invention, in a cross-sectional diameter no greater than about 10 microns (as used herein the term "micron" refers to the thousandth of a millimeter unit of measurement) particles of the fluorine-containing composition of the housing package. 较优选截面直径小于5微米,而对静脉内途径给药来说目前最优选的是小于1微米的截面直径。 More preferably a cross-sectional diameter of less than 5 microns, while intravenous routes is currently most preferred is a cross-sectional diameter of less than 1 micron.

根据显像的需要可用各种方式把本发明的对比剂引入体内部位。 The contrast agent can be used in various ways into the body portion of the present invention according to need imaging. 例如,由口腔摄取或栓剂形式把悬浮水溶液导入胃肠道(如,以获得到胃和胃肠道的显像),注射到非血管部位如脑脊髓腔,或者注射到血管系统或特定器官的脉管如冠状动脉中。 For example, by oral ingestion or suppository form of an aqueous suspension of the import of the gastrointestinal tract (eg, to get to the stomach and gastrointestinal tract imaging), injected into non-vascular sites such as cerebral spinal cavity, or injected into the vascular system or specific organs vessels such as coronary arteries. 此外,也能把本发明对比剂注射到其它身体部位,如前,后眼腔,耳朵,膀胱(如,通过尿道),腹膜腔,输尿管,尿道肾盂,骨关节腔,淋巴管,蛛网膜下腔,脑室腔,心窒腔等。 In addition, the present invention can also contrast agent injected into other body parts, such as front, rear eye cavity, ear, bladder (e.g., through the urethra), the peritoneal cavities, ureter, urethra, renal pelvis, bone articular cavity, lymphatic vessels, the subarachnoid chamber ventricular cavity, the heart chambers and so stifled.

含有氟组合物的固体或液体核的聚合物外壳能以相对小的体积直接传送高剂量的含氟组合物试剂。 Solid or liquid core shell polymer compositions containing fluorine can be relatively small volume directly deliver high doses of the fluorine-containing composition agent. 这就减少了患者在接受大量液体时的不适。 This reduces patient discomfort when accepting a lot of liquid.

按照本发明的其它实施方案,提供了一种对现有文献中没有描述过的基本水不溶性药物如紫杉醇给药问题的解决方法。 According to other embodiments of the invention, there is provided a method of solving the existing literature is not described as substantially water-insoluble drug paclitaxel administration problems. 所以,我们已经发现用微米大小颗粒的水悬浮液,或含有这种药物颗粒或溶于生物相容非水液体中的药物的水悬浮液,能够完成这类药物的传送。 Therefore, we have found that the aqueous suspension with the micron-sized particles, or drug-containing particles or dissolved in a biocompatible non-aqueous liquid of an aqueous suspension of the drug, it is possible to complete the transfer of such drugs. 这种方法有利于传送高浓度的这类药物,并由此避免了乳化剂的使用和其相关的毒副作用。 This approach allows delivery of high concentrations of these drugs, and thereby avoiding the use of emulsifiers and their associated toxic side effects.

按照本发明的又一个实施方案,通过一种新的含药组合物可以改进上述的给药方式,其中把基本上不溶于水的药物如紫杉醇悬浮在一种生物相容液体中,并且所得到的悬浮液含有截面尺寸不大于10微米左右的这类药物(如紫杉醇)的颗粒,用不同方法如研磨,喷雾干燥,沉淀,超声波辐射等能获得小于10微米的所需大小颗粒。 According to yet another embodiment of the present invention, by a novel drug-containing compositions can be administered to improve the above-described manner, wherein the substantially water-insoluble drugs such as paclitaxel is suspended in a biocompatible liquid, and the resulting The suspension containing the cross-sectional dimension no greater than about 10 microns such drugs (such as paclitaxel) particles, using different methods such as grinding, spray drying, precipitation, ultrasonic irradiation, etc. to obtain the desired particle size less than 10 microns.

由于常规方法得到的不溶于水的药物如紫杉醇的晶体大小大于20微米,所以这种药物(如紫杉醇)的固体颗粒尚没有以生理盐水这类载体的悬浮液形式传送。 Because the crystal size of the water-insoluble drug paclitaxel conventional means such as greater than 20 microns, so that the drug (e.g., paclitaxel) have not yet transferred the solid particles in suspension in physiological saline such vectors. 然而,本发明却公开了把基本不溶于水的药物(如紫杉醇)研成大小低于10微米左右,优选低于5微米左右,最优选低于1微米左右的颗粒,并以该颗粒的悬浮液的传送给药,其中所述的颗粒大小能以悬浮液形式进行静脉内传送而没有器官和组织微循环堵塞的危险。 However, the present invention has disclosed a substantially water insoluble drugs (such as paclitaxel) and pulverized into a size of less than about 10 microns, preferably less than about 5 microns, and most preferably less than about 1 micron particles, and the particles suspended administration of liquid transfer, wherein the particle size can be performed without the microcirculation of organs and tissues clogging risk intravenously transmitted in the form of a suspension.

由于被传送药物的微粒特性,具有网状内皮组织系统的器官如脾、肝、和肺能够从循环中将多数药物清除。 Because drug particle characteristics are transmitted, having a reticuloendothelial system organ such as the spleen, liver, and lungs can remove it from the circulation most drugs. 这就使得颗粒形式的生理活性剂能够达到体内的这些部位。 This makes the form of particles of physiologically active agent can be achieved in vivo these parts.

在这个实施方案中所用的生物相容液体与上述那些相同。 In this embodiment, the biocompatible liquid used the same as those described above. 此外,非肠道营养剂如Intralipid(用作非肠道营养剂的商业上可买到的脂肪乳化液的商品名;可从Kabi vitrum,Inc.,Clayton,Ncrth(Carolina买到),Nutralipid(用作非肠道营养剂的商业上可买到的脂肪乳化液的商品名;可从McGaw,Irvine,alifornia买到),LiposynIII(用作非肠道营养剂的可从商业上买到的脂肪乳化液的商品名(含有20%大豆油,1.2%卵磷脂和2.5%甘油);可从Abbott Labora-tories,North Chicago,Illinois买到),等可用作该药物颗粒的载体。或者,如果生物相容液体含有象大豆油这样的溶解药物的物质(如,象Intralipid中),药物可部分或全部溶于载体液体中,有助于它的传送。这种情况的例子是用Intralipid作载体传送紫杉醇。在这里用于该实施方案的优选生物相容液体是非肠道营养剂,如上所描述的那些。 In addition, parenteral nutritional agents such as Intralipid (used as a parenteral nutrition agent commercially available fat emulsion trade name; available from Kabi vitrum, Inc., Clayton, Ncrth (Carolina available), Nutralipid ( used as a parenteral nutrition agent commercially available fat emulsion trade name; available from McGaw, Irvine, alifornia buy), LiposynIII (used as a parenteral nutrition agent commercially available from fat tradename emulsion (containing 20% soybean oil, 1.2% lecithin and 2.5% glycerin); available from Abbott Labora-tories, North Chicago, Illinois available), etc. can be used as a carrier of the drug particles or, if. biocompatible liquid containing substances such as soybean oil was dissolved drug such (e.g., as Intralipid), the drug may be partially or fully dissolved in a liquid carrier, contributes its transport. Examples of this are used as a carrier Intralipid transfer paclitaxel. Here the preferred embodiment the biological compatible liquid parenteral nutritional agents, those as described above.

根据本发明的另一个实施方案,还提供了一种用于体内传送紫杉醇的组合物,其中将紫杉醇溶于一种非肠道营养剂中。 According to another embodiment of the present invention also provides a composition for the in vivo transfer of paclitaxel, wherein the paclitaxel is dissolved in a parenteral nutrition agent.

根据本发明的一个实施方案,本发明提供了一种用于体内传送生物制品的组合物,其中所述的生物制品选自:一种固体,任意分散于一种生物相容分散剂中,它基本上完全被包裹在一种聚合物外壳中,一种液体,任意分散于一种生物相容分散剂中,它基本上完全被包裹在聚合物外壳中,一种气体,任意分散于一种生物相容分散剂中,它基本上完全被包裹在聚合物外壳中,一种与聚合物外壳结合的气体,或它们任何两种或多种的组合,其中所述外壳的最大截面直径不大于10微米,其中所述的聚合物外壳由一种实质上是通过二硫键方式交联的生物相容材料制备,并且其中所述的聚合物外壳任意被一种适当的试剂修饰,该试剂通过一种任意的共价键连接所述的聚合物外壳。 According to one embodiment of the present invention, the present invention provides a composition for in vivo transfer of biological products, biological products, wherein said is selected from: a solid, optionally dispersed in a biocompatible dispersing agent, it substantially completely encased in a polymeric shell, a liquid, optionally dispersed in a biocompatible dispersing agent, substantially completely wrapped in a polymer shell, a gas, optionally dispersed in a biocompatible dispersing agent, substantially completely encased in the polymer shell, a polymer shell with a gas combination, or any combination of two or more thereof, wherein the maximum cross-sectional diameter of the housing is not more than 10 microns, wherein said polymeric shell is prepared from a substantially biocompatible material cross-linked by disulfide bonds manner, and wherein any of said polymer shell is a suitable reagent modified by the agent an arbitrary covalently linked to the polymer shell.

进一步地,其中所述的合适的试剂是选自合成的聚合物、磷脂、蛋白质、多糖、表面活性剂、化学改性剂、或它们的组合,其中所述的试剂与所述的聚合物外壳通过任意的共价键相结合。 Further, wherein the suitable reagent is selected from synthetic polymers, phospholipids, proteins, polysaccharides, surface active agents, chemical modifiers, or combinations thereof, wherein said agent to said polymer shell by any combination of covalent bonds.

根据本发明的再一个实施方案,本发明提供了一种用于体内传送生物制品的组合物,其中所述的生物制品选自:一种固体,任意分散于一种生物相容分散剂中,它基本上完全被包裹在一种聚合物外壳中,一种液体,任意分散于一种生物相容分散剂中,它基本上完全被包裹在聚合物外壳中,一种气体,任意分散于一种生物相容分散剂中,它基本上完全被包裹在聚合物外壳中, According to the present invention, a further embodiment, the present invention provides a composition for the in vivo transfer of biological products, biological products, which is selected from: a solid, optionally dispersed in a biocompatible dispersing agent, It is substantially completely enclosed in a polymer shell, a liquid, optionally dispersed in a biocompatible dispersing agent, substantially completely wrapped in a polymer shell, a gas, optionally dispersed in a kind of a biocompatible dispersing agent, substantially completely encased in the polymer shell,

一种与聚合物外壳结合的气体,或它们任何两种或多种的组合,其中所述外壳的最大截面直径不大于10微米,其中所述的聚合物外壳由一种实质上是通过二硫键方式交联的生物相容材料制备,并且其中所述的聚合物外壳任意被一种适当的试剂修饰,该试剂通过一种任意的共价键连接所述的聚合物外壳。 A combination of gas and polymer shell, or any combination of two or more thereof, wherein the maximum cross-sectional diameter of the housing is not more than 10 microns, wherein said polymer shell is made of a substantially by disulfide Preparation of bio-compatible material key mode crosslinked, and optionally wherein said polymer shell is a suitable modifying agent, the agent through an optional covalent linkage of the polymer shell.

进一步地,所述的生物制品是选自药物活性试剂、诊断试剂或营养剂。 Further, the biological product is selected from a pharmaceutically active agent, a diagnostic agent or a nutritional agent. 进一步地,所述的诊断试剂选自超声对比剂,放射对比剂,或磁对比剂。 Further, the diagnostic agent is selected from ultrasound contrast agents, radiocontrast agents, or magnetic contrast agents. 进一步地,所述的磁对比剂是一种含氟的磁共振成像剂。 Further, the magnetic contrast agent is a fluorine-containing magnetic resonance imaging agent.

根据本发明的又一个实施方案,本发明提供了一种用于体内传送生物制品的组合物,其中所述的生物制品选自:一种固体,任意分散于一种生物相容分散剂中,它基本上完全被包裹在一种聚合物外壳中,一种液体,任意分散于一种生物相容分散剂中,它基本上完全被包裹在聚合物外壳中,一种气体,任意分散于一种生物相容分散剂中,它基本上完全被包裹在聚合物外壳中,一种与聚合物外壳结合的气体,或它们任何两种或多种的组合,其中所述外壳的最大截面直径不大于10微米,其中所述的聚合物外壳由一种实质上是通过二硫键方式交联的生物相容材料制备,并且其中所述的聚合物外壳任意被一种适当的试剂修饰,该试剂通过一种任意的共价键连接所述的聚合物外壳。 According to yet another embodiment of the present invention, the present invention provides a composition for the in vivo transfer of biological products, biological products, wherein said is selected from: a solid, optionally dispersed in a biocompatible dispersing agent, It is substantially completely enclosed in a polymer shell, a liquid, optionally dispersed in a biocompatible dispersing agent, substantially completely wrapped in a polymer shell, a gas, optionally dispersed in a species biocompatible dispersing agent, substantially completely encased in the polymer shell, a polymer shell with a gas combination, or any combination of two or more of them, wherein the maximum cross-sectional diameter of the housing is not greater than 10 microns, wherein said polymer shell made of a biocompatible material was prepared essentially by way of disulfide cross-linked, and optionally wherein said polymer shell is a suitable modifying agent, the agent connecting said polymeric shell through an optional covalent bond.

进一步地,其中所述的交联聚合物是一种天然存在的聚合物,一种合成聚合物,或它们的组合,其中所述的聚合物,在交联之前,已共价连接上了巯基或二硫键。 Further, wherein said crosslinked polymer is a naturally occurring polymer, a synthetic polymer, or combinations thereof, wherein said polymer, prior to crosslinking, has covalently attached the sulfhydryl or disulfide bonds.

进一步地,所述的天然存在的聚合物是选自含有巯基和/或二硫基的蛋白质,含有巯基和/或二硫基的多肽,含有巯基和/或二硫基的脂,含有巯基和/或二硫基的多核酸,或含有巯基和/或二硫基的多糖。 Further, the naturally occurring polymer is selected from the group containing a mercapto group and / or disulfide group of the protein, a mercapto group and / or a polypeptide containing a disulfide group, a mercapto group-containing and / or disulfide group of fat, containing a mercapto group and / polynucleic acid or disulfide group, or a mercapto group and / or disulfide group-containing polysaccharide.

进一步地,其中所述的蛋白质是选自血红蛋白、肌红蛋白、白蛋白,胰岛素、溶菌酶、免疫球蛋白、α-2-巨球蛋白、纤连蛋白、玻连蛋白、纤维蛋白原、或它们任意两者或更多的组合。 Further, wherein said protein is selected from hemoglobin, myoglobin, albumin, insulin, lysozyme, immunoglobulins, α-2- macroglobulin, fibronectin, vitronectin, fibrinogen, or any two or more of them in combination.

本发明将通过下列非限定性实施例来更详细地说明。 The present invention will be implemented by the following non-limiting Examples will be described in more detail.

实施例1含油的蛋白外壳的制备将3ml USP(美国药典)5%人血清白蛋白(Alpha治疗公司)溶液置入放有声波探子(Heat System XL2020型)的圆柱形试管。 Preparation Example 1 of the protein coat of oil will 3ml USP (United States Pharmacopoeia) 5% human serum albumin (Alpha Therapeutics, Inc.) was placed into sonic probe (Heat System XL2020 type) of a cylindrical tube. 在白蛋白溶液上覆盖6.5ml的USP等级的豆油(大豆油)。 Covering 6.5ml albumin solution in USP grade soybean oil (soybean oil). 将声波探子的尖端放于两溶液的界面,并把此组合装置保持在20℃的冷浴中。 Sonic probe tip put on the interface of the two solutions, and the combination of this device in a cold bath of 20 ℃. 使此系统平衡后,声波装置开通30秒。 After equilibration to make this system, the acoustic wave device opening 30 seconds. 强烈混合得到一种白色乳状悬浮液。 Strong mixing to obtain a white milky suspension. 将悬浮液用生理盐水稀释成1∶5。 The suspension was diluted with saline to 5. 利用颗粒计数器(颗粒数据系统Elzone,280PC型)测定含油蛋白壳的大小分布和浓度。 Determination of the size distribution and concentration of oil-containing protein shell using a particle counter (Particle Data Systems Elzone, 280PC type). 测定后的蛋白壳被确定具有约1.350.73微米的最大横径,其在原悬浮液的浓度为~109个/ml外壳。 After determination of the protein shell is determined to have the maximum diameter of about 1.35 0.73 microns, its concentration in the original suspension of ~ 109 / ml housing.

作为对照,使上述组合物中不含蛋白,当受到超声辐射时则不能形成稳定的微乳化液。 As a control, the above-mentioned composition does not contain proteins, when subjected to ultrasonic irradiation can not form a stable microemulsion. 此结果表明,对于微球体的形成,蛋白是必不可少的。 These results indicate that, for the formation of microspheres, the protein is essential. 此将通过如下所述的电子显微照片观察和透射式电子显徽镜研究得到证实。 This will be described below by electron micrograph observation and transmission electron microscopy studies confirmed emblem.

实施例2影响聚合物外壳形成的参数对几种变量,如蛋白浓度、温度、声波处理时间、管理活性剂的浓度和声波强度进行测试,以便获得聚合物外壳的最佳制备方式。 Parameter Example 2 Effect of forming the polymer shell on several variables such as protein concentration, temperature, sonication time, acoustic intensity and concentration of test agent administration, so as to obtain the best way of preparing the polymer shell. 这些参数通过含有甲苯的交联牛血清白蛋白外壳进行确定。 These parameters of bovine serum albumin by cross-linking the shell containing toluene were determined.

用颗粒计数器测定由蛋白浓度为1%、2.5%、5%和10%的溶液制备的聚合物外壳的大小和数量的变化,发现聚合物外壳的大小不随蛋白浓度的变化而大幅度地改变,但是每ml由“乳状悬浮液制备的聚合物外壳的数量,在5%浓度以下随蛋白浓度的增加而增加。未发现在此蛋白浓度以上,聚合物外壳的数量产生显著的变化。 Measured by the particle counter by protein concentration of 1%, 2.5%, the size and number of changes in a 5% solution was prepared and 10% of the polymer shell, the size of the polymer shell is not found to change with protein concentration greatly changed, However, by the number of "milky suspension prepared polymer shell, in a concentration of 5% or less with increasing protein concentration increases per ml. are not found in the protein concentration or more, the amount of polymer shell have a significant change.

对于聚合物外壳的最佳制备,发现试管最初温度是重要的。 For optimum preparation of the polymers of the housing, the first temperature is an important discovery of the tube. 典型地,最初试管温度保持在0~45℃之间。 Typically, the first test tube maintained at a temperature between 0 ~ 45 ℃. 用于聚合物外壳形成的油的水油界面张力是一个重要参数,它也以温度的函数形式而变化。 Water-oil interfacial tension of the polymer shell for the formation of oil is an important parameter, it is also in the form of a function of temperature change. 药理活性剂对蛋白外壳的产生没有重要的影响。 Pharmacologically active agent is no significant effect on the protein coat of the produce. 不管药理活性剂是以溶解状态加入,还是悬浮于分散介质中,它是相对不重要的。 Regardless of pharmacologically active agent is added to the dissolved state, or suspended in a dispersion medium, it is relatively unimportant.

声波处理时间是决定每ml所产生的聚合物外壳数量的重要因素。 Sonication time is an important factor determining the number of polymer shell produced per ml. 发现声波处理时间大于3分钟,聚合物外壳总量则减少,说明过度的声波处理可能导致聚合物外壳的破坏,声波处理时间少于3分钟,则产生足够数量的聚合物外壳。 Found sonication time greater than three minutes, the total amount of the polymer shell is decreased, indicating excessive sonication may result in the destruction of the polymer shell, sonication time is less than 3 minutes, then a sufficient quantity of polymer shell.

按照发声器生产厂家提供的列线图,此外使用的发声器超声功率大约为150w/cm2。 According to nomogram manufacturers to provide a sounder, sounder uses ultrasonic power addition of about 150w / cm2. 应用三种逐渐增高的功率设定,发现最高功率的功率设定产生的聚合物外壳数量最多。 Application of three progressively higher power settings, find the largest number of the highest power setting power generation polymer shell.

实施例3含溶解紫杉醇的聚合物外壳的制备将紫杉醇以2mg/ml的浓度溶解在USP等级豆油中。 Preparation of the polymer containing the dissolved paclitaxel housing Example 3 Paclitaxel 2mg / ml concentration was dissolved in USP grade soybean oil. 将3ml USP 5%人血清白蛋白溶液置于连接有声波探子的圆柱形试管中。 The 3ml USP 5% human serum albumin solution was placed in the acoustic wave probe is connected to a cylindrical tube. 在白蛋白溶液上覆盖6.5ml豆油/紫杉醇溶液,声波探子尖端置于两溶液的界面上。 Covering 6.5ml soybean oil / taxol solution, sonic probe tip is placed on the interface of the two solutions in the albumin solution. 使此组合进行平衡,发声器开通30秒。 This combination makes the balance, sounder opening 30 seconds. 强烈混合,得到一种稳定的白色乳状悬浮液,其中含有包裹了/油紫杉醇溶液的蛋白壁聚合物外壳。 Intensive mixing, to obtain a stable white milky suspension containing wrapped / oil solution of paclitaxel protein polymer shell walls.

为获得此交联蛋白外壳的较高载荷量,将一种油和药物的互溶剂(药物在其中有相当大的溶解度)与油混合在一起。 To obtain this high crosslinked protein shell of the amount of load, and the drug will be an oil mutual solvent (in which the drug has a relatively large solubility) were mixed together with the oil. 只要此互溶剂相对无毒(例如乙酸乙酯),它便可以同原始载体一起注射,另一方面,可以在聚合物外壳制备之后,通过真空下液体的蒸发而被去除。 As long as the mutual solvent is relatively non-toxic (e.g., ethyl acetate), which can be injected along with the original carrier, on the other hand, after the polymer shell can be prepared, by evaporation under vacuum of the liquid is removed.

实施例4聚合物外壳的稳定性在3种不同温度下(即4℃、25℃和38℃),分析已知浓度的聚合物外壳悬浮液的稳定性。 Example 4 Stability of the polymer shell in three different temperatures (i.e., 4 ℃, 25 ℃ and 38 ℃), analyze the stability of a known concentration of polymer shell suspension. 通过特定时间内颗粒数的改变来测定其稳定性。 Stability was determined by varying the number of particles within a specific time. 如上所述(参照实施例1)制备出含有豆油的交联蛋白(白蛋白)外壳,在盐水中稀释,使最后的油浓度达到20%,并在上述温度下保存。 As described above (see Example 1) was prepared containing soybean oil crosslinked protein (albumin) housing, diluted in saline to make a final oil concentration of 20%, and stored at that temperature. 所获得的每个样品的颗粒数(Elzone)与时间的函数总结于表2中。 A function of particle number (Elzone) obtained for each sample and the time are summarized in Table 2.

表2 Table 2

上面的数据说明:经过实验时间,所计数的颗粒(即聚合物外壳)的浓度仍保持相当恒定。 The above data show that: After the experiment time, the count of particles (ie polymer shell) concentrations remained fairly constant. 相当恒定的范围在7-91010/ml之间。 Fairly constant in the range of 7-9 1010 / ml. 它表明,在经过几乎四周的时间,在不同的温度下,聚合物外壳具有良好的稳定性。 It shows that, after almost four weeks, at different temperatures, a polymer shell with good stability.

实施例5活体内生物分布——含有荧光团的交联蛋白外壳为确定静脉内注射后,包裹在蛋白聚合物外壳体内的液体的摄取及生物分布,将一种荧光染料(红荧烯,从Aldrich中得到)包裹在人血清白蛋白(HSA)聚合物外壳体内,用作一种标记物。 Example 5 In vivo biodistribution embodiment - crosslinked protein shell containing a fluorophore is determined after intravenous injection, wrapped in a protein polymeric shell body of liquid uptake and biodistribution will be a fluorescent dye (rubrene, from obtained Aldrich) wrapped in human serum albumin (HSA) polymer shell body, used as a marker. 这样,将红荧烯溶解于甲苯中,通过如上所述的超声辐射制备出含有甲苯/红荧烯的交联白蛋白外壳。 Thus, the rubrene was dissolved in toluene, as described above by ultrasonic irradiation to prepare a crosslinked albumin housing containing toluene / rubrene. 所得的乳状悬浮液在生理盐水中稀释5倍。 The resulting milky suspension was diluted five times in normal saline. 然后将2ml稀释后的悬浮液以10分钟的速率注射入小鼠的尾静脉。 Then 2ml diluted suspension at 10 minutes were injected into the tail vein of mice. 在注射后1小时处死一只动物,另一个在注射24小时后处死。 1 hours after injection the animals were sacrificed one, the other sacrificed at 24 hours after injection. 在荧光显微镜下检查肺、肝、胃、脾、骨髓的100微米冷冻切片,以确定包裹了荧光染料或释放了染料的聚合物外壳的存在。 Under a fluorescence microscope examination of lung, liver, stomach, spleen, bone marrow 100 micron frozen sections, to determine the release of a fluorescent dye or wrapped presence dye polymer shell. 在1小时的时候,大多数聚合物外壳是完整的(即呈现明亮的荧光颗粒,直径大约1微米),并且位于肺和肝内。 In one hour's time, most of the polymer shell is intact (i.e. showing a bright fluorescent particle diameter of about 1 micron), and in the lung and liver. 24小时的切片,染料在肺、肝、脾、骨髓中都能观察列。 24 hours slices dye in the lung, liver, spleen, bone marrow can be observed columns. 对普通的组织染色进行了观察,显示聚合物外壳的壳壁已经被水解(消化),染料从中释放出来。 Staining of normal tissue was observed, showing the shell wall has been hydrolyzed polymer shell (digestion), the dye is released therefrom. 此与预期的结果相一致,说明本发明组合物具有延缓和控制内容药物活性剂(如紫杉醇)释放的潜在用途。 This is consistent with the expected results, indicating the potential use of the composition of the present invention having a delay and control the content of pharmaceutically active agents (such as paclitaxel) release.

实施例6含有豆油(SBO)的聚合物外壳的毒性如实施例1所述制备含豆油的聚合物外壳,在生理盐水中稀释为两种不同的溶液,一种含20%豆油,另一种含30%豆油。 Example 6 Toxicity polymer shell containing soybean oil (SBO) is as described in the preparation of a polymer shell containing soybean oil described in Example 1, was diluted in physiological saline for two different solutions, one containing 20% soybean oil and another containing 30% soybean oil.

内脂,一种商业上可得到的TPN剂,含有20%豆油(SBO)。 Lactone, a commercially available TPN agent, contains 20% soybean oil (SBO). 当以1毫升/分钟的速度注射内脂时,小鼠的半数致死量是120ml/kg,或者说对于1个30g的小鼠大约为4ml。 When 1 ml / min rate of injection of grease, the median lethal dose in mice was 120ml / kg, or for a 30g mouse approximately 4ml.

用含有豆油的本发明组合物对两组小鼠(3个为1组,每个重约30g)作如下处理。 Composition of the present invention comprises soybean oil to two groups of mice (three per group, each weighing about 30g) treated as follows. 给每个小鼠注射4ml制备的含豆油聚合物外壳的悬浮液。 Polymer shell containing soybean oil to a suspension of each mouse injected 4ml prepared. 一组中的每个小鼠接受含20%SBO的悬浮液,另一组的每个小鼠接受含30%SBO的悬浮液。 A group of mice received each containing 20% SBO suspension, each of the other group of mice receiving the suspension containing 30% SBO of.

如此处理后,接受含20%SBO悬浮液组中的3个小鼠全部存活,并且在SBO处理后一周观察,所有组织或器官均未显示肉眼所见毒性。 After this treatment, accept suspension containing 20% SBO group of three mice all survived, and one week after SBO treatment was observed in all tissues or organs showed no toxicity to the naked eye can see. 接受含30%SBO悬浮液的一组三个小鼠中仅有1个在注射后死亡。 Accept a group of three mice containing 30% SBO suspension only one death after injection. 此结果清楚地表明,按本发明聚合物外壳内所含的油,与商业上可得到的SBO制剂(内脂)相比,在它的半数致死量时是无毒的。 These results clearly indicate that the oil within the polymer according to the present invention is contained in the housing, compared with a commercially available SBO formulation (lactone), the median lethal dose when it is non-toxic. 此作用可能是由于聚合物外壳内的油的缓慢释放(即控制的生物利用率)的结果。 This effect may be due to the slow release of the results (i.e., control of bioavailability) of the polymer within the housing of the oil. 与商业上可得到的高油量的乳剂相比,如此的缓慢释放阻止了油的致死量的出现。 Compared with the commercially available high-oil emulsions, such slow release prevents the lethal dose of oil occurs.

实施例7从聚合物外壳中释放的豆油的活体生物利用率在向鼠血流中注射聚合物外壳悬浮液后,进行一项试验以确定被聚合物外壳包裹的物质的缓慢或持续释放。 Vivo bioavailability of soybean oil in Example 7 released from the polymer to the housing in the blood stream after injection mice polymer shell suspensions, conducted an experiment to determine the polymer shell material wrapped slow or sustained release. 通过如上所述的声波处理,制备含有豆油(SBO)的交联蛋白(白蛋白)壁的聚合物外壳。 By sonication as described above, is prepared containing soybean oil (SBO) crosslinked protein (albumin) walled polymeric shell. 用生理盐水将所得到的含油的聚合物外壳混悬液稀释成最终含20%油的混悬液。 With saline the resulting oily suspension was diluted to a final shell polymer containing 20% oil suspension. 用10多分钟将5ml此种悬浮液注入有插管的小鼠颈外静脉中。 10 minutes with 5ml of such suspension is injected into the external jugular vein cannula in mice. 在注射后的几个时间点,从这些小鼠采血,并通过常规分析确定血中甘油三酯的含量(豆油主要是甘油三酯)。 At several time points after injection, blood was collected from these mice, and to determine the content of triglyceride in the blood by conventional analysis (mainly soybean oil triglycerides).

用5ml商业上可得到的脂肪乳剂(内脂,一种含水非肠道营养剂—含有20%豆油,1.2%蛋黄卵磷酯和2.25%甘油)作对照物。 With 5ml commercially available fat emulsion (lactone, an aqueous parenteral nutrition agent - containing 20% soybean oil, 1.2% egg yolk lecithin and 2.25% glycerin) was used as a control. 此对照物利用卵磷酯作为乳化剂,以稳定此乳剂。 This controls the use of lecithin as an emulsifier to stabilize the emulsion. 这两种情况下的甘油三酯的血清水平的比较将得出作为时间函数的油的生物利用率的直接比较。 Serum levels of triglyceride in both cases the comparison will yield a function of time as the bioavailability of the oil direct comparison. 除含油20%的聚合物外壳悬浮液以外,还注射5ml生理盐水稀释的最终浓度为含油30%的聚合物外壳悬浮液样品。 In addition to a 20% oil suspension of polymer shell, further diluted with normal saline injection 5ml final concentration of 30% oil suspension of polymer shell samples. 三个组中,每个组使用2个小鼠。 Three groups, each group use two mice. 将每种情况下血中甘油三酯水平列于表3中,其单位为mg/dl。 In each case the blood triglyceride levels are shown in Table 3, in units of mg / dl.

表3 Table 3

标有“注射前”的一栏中表示注射前血中甘油三酯水平。 Column labeled "before injection" indicates the level of blood triglycerides prior to injection. 很明显,对于内脂对照物,可以看到注射后甘油三酯水平很高,大约需要24小时的时间甘油三酯开始降到注射前水平。 Clearly, for the lipid controls, can see a high triglyceride levels after injection, approximately 24 hours prior to injection triglyceride levels are starting to fall. 可见,这种油在注射后能被立即利用于代谢中。 Seen, this oil can be immediately after injection use in metabolism.

与内脂含有相同油总量(20%)的含油聚合物外壳悬浮液,其血清中可测得的甘油三酯则显示出一种很不同的利用率。 Oily suspension polymer shell and the inner lipid containing the same total amount of oil (20%), which can be measured in serum triglycerides are showing a very different utilization. 其含量升高到其正常值的2倍左右,并且在此水平上维持好几小时。 The content thereof is increased to about twice its normal value, and maintained at this level for hours. 这表明甘油三酯以一个相当接近于正常值的水平缓慢或持续地释放入血。 This shows a fairly close to normal triglyceride level or a slow continuously released into the blood. 接受含油30%的聚合物外壳组显示较高甘油三酯水平(伴随较高的给药量),并在48小时内降至正常。 Accepted 30% of the polymer shell oil group showed higher triglyceride levels (with a higher dose of), and returned to normal within 48 hours. 重复一次,与接受内脂的对照组相比,此组中血甘油三酯水平没有极大的升高。 Repeated once, compared with the control group received an aliphatic, serum triglyceride levels in this group did not greatly increased. 这又一次表明了本发明组合物中油的缓慢和持续释放,其优点是避免了聚合物外壳内物质的危险性高血浓度,并且以可接受的水平在较长时间内被利用。 This again shows that the compositions of the invention are slow and sustained release of oil, the advantage of avoiding the risk of high blood concentrations of the polymer shell material, and to an acceptable level to be used in a long time.

很明显,本发明的聚合物外壳内所传送的药物也将具有同样的优越性。 Obviously, the drug within the polymer shell of the present invention will also have transmitted the same advantages.

这种含豆油的聚合物外壳系统可悬浮于一种由氨基酸、基本电解质、维生素和糖组成的水溶液中,以构成一种全胃肠外营养剂(TPN)。 This polymer shell system containing soybean oil may be suspended in an aqueous solution of amino acids, essential electrolytes, vitamins and sugar composition, to form a total parenteral nutrition agent (TPN). 这种TPN不能用目前常用的脂肪乳(如内脂)来制成,因为有电解质存在的乳剂是不稳定的。 This TPN can not be currently used fat emulsion (e.g. a lactone) to be made, because the presence of electrolyte emulsion is unstable.

实施例8含有药物活性剂固体核心的交联蛋白壁聚合外壳的制备在聚合物外壳内传送水难溶性药物(如紫杉醇)的另一种方法是制备一种包裹有固体药物核心的聚合物外壳。 Another method of preparing a crosslinked protein wall 8 comprising a pharmaceutically active agent in a solid core shell polymerization Example transferred water insoluble drug in the polymer shell (such as paclitaxel) is wrapped with the preparation of a solid drug core shell polymer . 这种“蛋白包衣”药物颗粒可按下述方法获得。 This "protein coated 'drug particle may be obtained by the following method. 重复实施例3中所描述的步骤,使用有机溶剂溶解紫杉醇达到较高浓度。 Step 3 as described in Example II was repeated, using an organic solvent to dissolve taxol to achieve a higher concentration. 通常使用的溶剂是有机溶剂,如苯、甲苯、己烷、乙醚等。 The solvent is generally used an organic solvent, such as benzene, toluene, hexane, diethyl ether and the like. 聚合物外壳的制备如实施例3。 Preparation of polymer shell as in Example 3. 将5ml含溶解紫杉醇的聚合物外壳乳状悬浮液用生理盐水稀释至10ml。 5ml of a polymer shell containing the dissolved taxol milky suspension was diluted to 10ml with saline. 将此悬浮液放入室温的旋转式蒸发器中,挥发性的有机物通过真空而被去除。 The suspension was placed in a rotary evaporator at room temperature, the volatile organics were removed by vacuum. 在旋转式蒸发器中2小时后,在显微镜下观察,这些聚合物外壳暴露出模糊的核心,这表明基本上所有有机溶剂确实已被除去而固体紫杉醇存在于蛋白壳内。 After 2 hours, observed under a microscope in a rotary evaporator, these polymeric shell exposed fuzzy core, indicating that substantially all of the organic solvent has been removed and the solid was indeed present in the protein shell of paclitaxel.

还可以将带有有机溶剂溶解的药物核心的聚合物外壳冷冻干燥,以获得一种不干碎粉沫,它能在使用时再次悬浮于盐水中(或其它适宜的液体中)。 Polymer shell with the drug core can also be dissolved in an organic solvent, freeze-dried, crushed to obtain a powder quit, it can be used again when suspended in saline (or other suitable liquid). 如果是在室温下不能保持固态的药物,可获得液体核心聚合物外壳。 If it is not the drug remains solid at room temperature, the liquid obtained core shell polymer. 这种方法可以用来制备含非稀释药物的交联蛋白壁壳体。 This method can be used to prepare cross-linked protein walls of the housing containing a non-diluted drugs. 颗粒大小分析显示这些聚合物外壳比含油的聚合物外壳小。 The particle size analysis shows these polymeric shell is smaller than the oil-polymer shell. 尽管目前用于制备聚合物外壳的优选蛋白是白蛋白,其实其它蛋白如α-2-巨球蛋白,一种已知的调理素,也可用来增加巨噬细胞对聚合物外壳的摄取量。 Although the presently preferred protein for preparing a polymeric shell is albumin, other proteins such as actually α-2- macroglobulin, a known opsonin, also be used to increase the intake of macrophages on the polymer shell. 还可以,在形成聚合物外壳过程中将PEG-巯其加入其中,以产生一种可延长体内循环时间的聚合物外壳。 May also be, during the formation of the polymer shell will PEG- thiol added therein to produce a circulation time in vivo extended polymer shell.

实施例9聚合物外壳在体内的循环及释放动力学按上述(见,实施例3)制备含紫杉醇的固体核心聚合物外壳,并悬浮于生理盐水中。 Example 9 In vivo kinetics of polymer shell embodiment of circulation and release according to the above (see, Example 3) A solid core shell polymer prepared containing paclitaxel, and suspended in physiological saline. 用下述的HPLC来测定悬浮液中紫杉醇的浓度。 By the following HPLC to determine the concentration of the suspension of paclitaxel. 首先,聚合物外壳内的紫杉醇被加入的0.1M巯基乙醇游离出来(引起蛋白二硫交联键的互换,并破坏聚合物外壳的交联);然后,用乙腈将游离的紫杉醇从悬浮液中提取出来。 First, the taxol within the polymeric shell was added 0.1M mercaptoethanol was freed (to cause protein disulfide crosslinks interchangeable, and destruction of crosslinked polymer shell); then with acetonitrile to free paclitaxel from the suspension are extracted. 离心所得的混合液,并将其上清液冻干。 The resulting mixture was centrifuged, and the supernatant was lyophilized. 将冻干物溶解于甲醇中,并注射于HPLC上,以确定悬浮液中紫杉醇的浓度,大约为1.6mg/ml。 The lyophilized material was dissolved in methanol and injected on the HPLC, to determine the concentration of paclitaxel in the suspension, is about 1.6mg / ml.

通过颈静脉导管给小鼠注射此悬浮液2ml,2小时时处死此动物,并通过HPLC测定存在于肝脏中的紫杉醇量。 Injection to the mice via the jugular vein catheter suspension was 2ml, the animals were sacrificed 2 hours this, and in the liver was measured in the amount of paclitaxel by HPLC. 此需要对肝脏进行均浆,然后用乙腈提取,在离心后冻干上清液。 The need for liver homogenates, and then extracted with acetonitrile and lyophilized supernatant after centrifugation. 将冻干物溶解于甲醇中,并注射于HPLC上。 The lyophilized material was dissolved in methanol and injected onto HPLC. 从2小时的肝脏中回收了约给药量15%的紫杉醇。 Recovery of about 15% of paclitaxel administered in an amount from 2 hours liver. 显示出肝脏的有效剂量。 Shows effective dose liver. 此结果与已知的肝脏网状内皮系统从血中清除小颗粒的功能相一致。 This result with known liver reticuloendothelial systems of small particles from the blood is consistent functionality.

实施例10交联PEG-壁聚合物外壳的制备制备含巯基的聚乙二醇,它是制备本发明聚合物外壳的含巯基蛋白的替代物,或是制备本发明聚合物外壳的添加剂。 Example 10 A crosslinked polymer shell wall PEG- Preparation of mercapto group-containing polyethylene glycol, which is the present invention for preparing a polymer shell substitute mercapto group-containing protein, or an additive for preparing a polymer shell of the invention. 已知PEG对细胞无毒性、无炎性、无粘连性,并且通常无生物活性。 PEG is known not toxic to cells, non-inflammatory, non-blocking, and usually no biological activity. 曾将它连接于蛋白上以减少其抗原性,连接于构成脂类的脂质体上可以延长它们在体内的循环时间。 Has connected to it to reduce its antigenicity, they are connected to the cycle time can be extended in vivo lipid constituting the liposome protein. 因此,将PEG结合于基本蛋白壳,可以预期地延长聚合物外壳的循环时间和提高其稳定性。 Therefore, the PEG bound to the basic protein shell, the shell polymer can be expected to prolong the cycle time and improve its stability. 通过改变加入到5%白蛋白溶液中的巯基聚乙二醇的浓度,有可能获得具有不同体内稳定性的聚合物外壳。 By changing the 5% albumin solution was added to a concentration of mercapto group in the polyethylene glycol, it is possible to obtain a polymer shell having a different in vivo stability. 可以通过文献中可得到的技术制备巯基聚乙二醇(如Harris和Herati的技术,公开于PolymerPreprints Vol.32:154-155(1991))。 Can be obtained by the literature in the art for preparing mercapto-polyethylene glycol (such as Harris and Herati technique disclosed in PolymerPreprints Vol.32: 154-155 (1991)).

将分子量2000g/mol的巯基聚乙二醇以1%(0.1g加入10ml中)的浓度溶解于5%的白蛋白溶液中。 The molecular weight of 2000g / mol of polyethylene glycol with mercapto-1% (0.1g in 10ml was added) was dissolved in a concentration of 5% albumin solution. 在此蛋白/PEG溶液上覆盖如实施例1所述的油,并于声处理后产生带有由交联蛋白和PEG构成的壳壁的含油聚合物外壳。 Covering the oil as described in Example 1 on the protein / PEG solution, and produce oil polymer shell having a shell wall of crosslinked protein and PEG constituted after sonication. 按实施例4所述检验这些聚合物外壳的稳定性。 The test described in Example 4 the stability of these polymer shell.

可被巯基修饰并用来替代PEG的其它水溶性合成聚合物包括:聚乙烯醇、异丁烯酸聚羟乙酯、聚丙烯酸、聚乙烯恶唑啉、聚丙烯酰胺、聚乙烯吡咯烷酮、多糖等(如:聚氨基葡糖、藻酸盐、透明质酸、右旋糖酐、淀粉、果胶等)。 A mercapto group which may be modified and used to replace PEG of other water-soluble synthetic polymers include: polyvinyl alcohol, poly hydroxy ethyl methacrylate, polyacrylic acid, polyvinyl oxazoline, polyacrylamide, polyvinyl pyrrolidone, polysaccharides (such as: chitosan, alginate, hyaluronic acid, dextran, starch, pectin, etc.).

例如,可延长体内循环时间的含碳氟化物的蛋白壳,对血管系统成像特别有益。 For example, to extend the circulation time in vivo protein shell carbon fluoride, particularly useful for imaging the vascular system. 与壳壁中不含PEG的外壳相比,这些壳体在延长期中仍保留在循环中。 Compared with the PEG-free shell wall in a housing, which housing is extended period remain in the circulation. 例如,它可使心脏循环直观化,并提供一种非损伤性的评价冠状循环的方法,代替了如血管造影术这样的传统损伤性技术。 For example, it allows the visualization of the cardiac cycle, and provides a non-invasive method for evaluation of the coronary circulation, instead of angiography such as traditional invasive techniques.

实施例11通过含免疫抑制剂聚合物壳体的静脉给药将免疫抑制剂传送到移植器官免疫抑制剂广泛应用于如下的器官移植中以防止排斥反应的发生。 11 by intravenous administration of immunosuppressive agents containing polymeric shell of the immunosuppressant to the transplanted organ transferred embodiment is widely used as an immunosuppressant in organ transplants to prevent rejection reactions. 特别是,环孢菌素(Cyclosporine),一种有效的免疫抑制剂,可延长同种异体移植的存活时间,包括:皮肤、心脏、肾脏、胰腺、骨髓、小肠和肺的移植。 In particular, cyclosporine (Cyclosporine), a potent immunosuppressant, prolong allograft survival time, comprising: a skin, heart, kidney, pancreas, bone marrow, small intestine and lung transplantation. 已证明环孢菌素可抑制某些体液免疫和在更大程度上抑制细胞调节的反应如同种移植排斥反应、迟发型过敏反应、试验性过敏性脑脊髓炎、布氏佐剂关节炎和许多动物品种中不同器官的移植物对抗宿主的移植性疾病。 Cyclosporin has been shown to inhibit the suppression of some humoral and cell-mediated responses to a greater extent as allograft rejection, delayed hypersensitivity, experimental allergic encephalomyelitis, arthritis, and many Brandt adjuvant animal species in different organs transplanted graft against host disease. 使用环孢菌素已在人体中成功地进行了肾、肝和心脏的同种异体移植。 Cyclosporin has been successfully carried out using the kidney, liver and heart allogeneic transplants in humans.

环孢菌素目前以口服给药,可以是以含环孢菌素溶液的胶囊形式给药,该环孢菌素溶液可以是它的乙醇溶液和油溶液(如玉米油、聚氧乙烯化的甘油酯等),也可以环孢菌素的橄榄油、聚氧乙烯化的甘油酯等的溶液形式给药。 Cyclosporin present in oral administration, may be in the form of capsules containing a solution of cyclosporin administration, the cyclosporin solution may be a solution of ethanol and its oil solution (such as corn oil, polyoxyethylated of in the form of glycerides, etc.), may be cyclosporin olive oil, polyoxyethylated glycerides and the like solution dose. 它也可以通过静脉注射给药,这种情况下它被溶解于乙醇(约30%)和Cremaphor(聚氧乙烯化的蓖麻油)溶液中,注射前必须在生理盐水或5%葡萄糖中稀释为1∶20至1∶100。 It can also be administered by intravenous injection, in which case it is dissolved in ethanol (about 30%) and Cremaphor (polyoxyethylated castor oil), a solution must be diluted prior to injection in saline or 5% glucose 1:20 to 100. 与静脉输注相比,口服溶液的绝对生物利用率大约是3%(SandozPharmaceutica Corporation,PublicationSDI-Z10(A4),1990)。 Compared to intravenous infusion, the absolute bioavailability of an oral solution is approximately 3% (SandozPharmaceutica Corporation, PublicationSDI-Z10 (A4), 1990). 通常,环孢菌素的静脉给药与目前使用的紫杉醇静脉给药存在着相似的问题,即被认为是由静脉给药载体Cremaphor引起的过敏和变态反应。 In general, intravenous administration of cyclosporin and paclitaxel currently used intravenous administration exist similar problems, and is considered to be allergic allergy caused by the intravenous administration of the carrier Cremaphor. 另外,如此所述用胶囊包裹药物(如环孢菌素)的静脉给药,可避免给药后立即出现危险的血液峰值。 Further, so the encapsulating drugs (such as cyclosporin) intravenous administration, a dangerous peak blood immediately after administration can be avoided. 例如,目前所用的环孢菌素制剂与上述胶囊包裹形式的环孢菌素制剂的比较,显示出后者的注射后立即出现的血液峰值水平有5倍的下降。 E.g., cyclosporin formulations currently used in the above-described encapsulated form of comparative formulations with cyclosporin, showing the blood level peak appears immediately after the injection of the latter is 5-fold decrease.

为了避免与Cremaphor相关的问题,包裹于如上所述的聚合物外壳内的环孢菌素可通过静注给药。 In order to avoid the problems associated with the Cremaphor, cyclosporine encapsulated in the polymer within the housing as described above may be administered by intravenous injection. 可以将它溶解于一种生物适合性油类或许多其它溶剂。 It can be dissolved in a biocompatible oil or a number of other solvents. 然后通过如上所述的声波处理,将它分散入聚合物外壳内。 Then by sonication as described above, it will be dispersed into the polymer shell. 另外,聚合物外壳包裹的环孢菌素(或其它免疫抑制剂)给药的重要优点是由于肝内网状内皮系统对注射物质的摄取而具有局部定位特点。 Further, the polymer shell wrapped cyclosporin (or other immunosuppressants) administration important advantage is due to the reticuloendothelial system of the liver uptake of injected material having local localization characteristics. 在一定程度上,由于局部定位特点可避免系统毒性作用并减少有效剂量。 To some extent, due to the characteristics of the local positioning system can be avoided and to reduce the toxicity of the effective dose. 在实施例9中描述了静脉给药之后,可将聚合物外壳包裹的紫杉醇有效地输送到肝脏。 Following Example 9 describes the intravenous administration, the paclitaxel-polymer shell wrapped efficiently transported to the liver. 根据本发明输送环孢菌素(或其他可能的免疫抑制剂)会得到类似的结果。 Conveying cyclosporin (or possibly other immunosuppressants) will get a similar result according to the present invention.

实施例12聚合物外壳的抗体靶作用本发明的聚合物外壳的特征是可以把单克隆或多克隆抗体附着在聚合物外壳上,或者把抗体结合到聚合物外壳内。 Antibodies are target Example 12 polymer shell of shell polymer of the present invention is characterized in that a monoclonal or polyclonal antibody can be attached to the polymer shell, or the antibody bound to the polymer shell. 当制备聚合物微型胶囊壳时,可以把抗体结合到聚合物外壳内,或者在制备之后将抗体附着在聚合物微型胶囊壳上。 When preparing the polymer microcapsule shell, the antibody can be incorporated into the polymer shell, or after the preparation of the antibody attached to the polymeric microcapsule shell. 可用标准蛋白固定技术来实现这一目的。 Standard protein immobilization techniques available to achieve this goal. 例如,蛋白质如白蛋白制备的蛋白微型胶囊,白蛋白赖氨酸残基上的大量的氨基基团可以用来附着适宜的改性抗体。 For example, proteins such as albumin protein microcapsules prepared, a large number of amino groups on the albumin lysine residues may be modified for attachment of suitable antibodies. 例如,制备聚合物外壳时,将抗肿瘤抗体结合到聚合物外壳内;或者在制备之后将抗肿瘤抗体附着在聚合物微型胶囊壳上,从而将抗肿瘤剂输送到肿瘤部位。 For example, when preparing a polymer shell, the anti-tumor antibody binding to the polymer shell; or after the preparation of the anti-tumor antibodies attached to the polymeric microcapsule shell, so that the anti-tumor agent is delivered to the tumor site. 再如,在制备聚合物外壳时,将抗靶细胞上受体的抗体结合到聚合物外壳内,者在制备之后,将抗靶细胞上受体的抗体附着在聚合物微型胶囊壳上。 Again, in the preparation of the polymer shell, the anti-receptor antibody on the target cell binding to the polymer shell, were after preparation, the anti-receptor antibody on the target cells attached to the polymeric microcapsule shell. 使基因产品被传送到特定细胞上(如肝细胞上或骨髓中的某些干细胞上);另外,可用抗核心受体的单克隆抗体将包裹的产品作用于特定细胞类型的细胞核。 So that the gene product is transferred to the specific cell (e.g. liver cells or bone marrow stem cells in some); In addition, the core of the available anti-receptor monoclonal antibody coated product will effect the nucleus in a particular cell type.

实施例13聚合物外壳作为多核苷酸结构、酶和疫苗的载体基因疗法作为一个可行性的选择性疗法,越来越被广泛接受(目前,40多项人基因转移的方案已经被NIH和/或FDA检查委员会认可)。 Example 13 polymer shell structure implemented as polynucleotides, enzymes and vaccines as a gene therapy vector feasibility of selective therapy, more and more widely accepted (at present, more than 40 human gene transfer program has been NIH and / or FDA inspection committee approval). 但实行这种疗法的障碍之一就是人们不愿意使用病毒载体将基因物质导入到人细胞基因组中。 But one obstacle to the implementation of this therapy is that people are reluctant to use viral vectors to import genetic material into human cell genome. 病毒本身是具有毒性的,因此,在基因疗法中冒险使用病毒载体,特别是对非致死性、非遗传性疾病的治疗来说是不被接受的。 The virus itself is toxic, so in gene therapy using viral vectors adventure, especially for the treatment of non-lethal, non-hereditary disease is not acceptable. 可惜的是,没有利用病毒载体而转移的质粒通常不被导入到靶细胞的基因组中。 Unfortunately, there is no use of viral vectors and plasmid transfer usually not be imported into the genome of the target cells. 另外,如同常规药物一样,这种质粒在体内的半衰期是有限的。 Further, as conventional drugs, such plasmids in vivo half-life is limited. 因此,使用基因疗法(以及反义疗法,它是一种相反形式的基因疗法。其中将核酸或寡聚核苷酸导入来抑制基因表达)的一般限制不能有效地向体内传送核酸或寡聚核苷酸,因为它们太大不能透过细胞膜。 Therefore, the use of gene therapy (as well as antisense therapy, which is a form of gene therapy opposite. Wherein introducing the nucleic acid or oligonucleotide to inhibit gene expression) generally limits can not be efficiently transmitted to the body of the nucleic acid or oligonucleosomes nucleotide, through the cell membrane because they are too large.

将DNA、RNA、质粒、寡聚核苷酸、酶等包裹在本发明所述的蛋白微胶囊壳中,可以促进它们向肝、肺、脾、淋巴和骨髓的靶传送。 The DNA, RNA, plasmids, oligonucleotides, enzymes wrapped in protein microcapsule shells according to the present invention, it is possible to facilitate their liver, lung, spleen, lymph and bone marrow transfer target. 因此,按照本发明,可以将这些生物制品传送到细胞内部而不会伴随有与使用病毒(载体相关的危险性)。 Thus, according to the present invention, these biological products can be transferred to the internal cell and not accompanied by (risk associated carrier) with the use of the virus. 这种类型的制剂可以促进从血流中直接对聚合物外壳的非特异性摄入或者RES细胞的摄粒作用。 This type of formulation can promote granulation taken directly from the bloodstream polymer shell nonspecific RES uptake or cell. 或者通过肌肉注射进入肌肉细胞中,或者通过直接注射进入肿瘤中。 Or by intramuscular injection into the muscle cells, or by direct injection into the tumor. 另外,可以用抗核心受体的单克隆抗体将包裹产物靶传送到某种细胞类型的细胞核。 Further, the target product can be wrapped receptor with anti-core monoclonal antibodies are transferred to the nucleus of certain cell types.

用这种结构可以治疗的疾病包括糖尿病、肝炎、血友病、膀胱纤维变性、多发性硬化症、肿瘤、流感、艾滋病等疾病。 This structure can be treated with diseases including diabetes, hepatitis, hemophilia, cystic fibrosis, multiple sclerosis, cancer, influenza, AIDS, and other diseases. 例如,可以将类胰岛素生长因子基因(IGF-1)包裹在蛋白微囊外壳中给药来治疗糖尿病性外周神经病和恶病质。 For example, insulin-like growth factor gene may be (IGF-1) for the treatment of diabetic peripheral neuropathy and cachexia wrapped administered protein microcapsule shell. 可以将编码IX因了和VIII因了(用于治疗血友病)的基因包裹在本发明的蛋白微囊外壳中,以特异性地传送肝脏。 Can be encoded by the IX and VIII by the gene parcel (for the treatment of hemophilia) protein microcapsule shell of the present invention, to specifically transfer the liver. 同样,可以将低密度脂蛋白(LDL)受体基因包裹在本发明的蛋白微囊壳体中,特异性地传送到肝脏以治疗动脉粥样硬化。 Similarly, the low-density lipoprotein (LDL) receptor gene wrapped in protein microcapsule shell of the present invention, specifically delivered to the liver for treatment of atherosclerosis.

适用于实施本发明的其它基因是那些重新激活机体对癌细胞的免疫反应的基因。 Applicable to other embodiments of the invention the gene is a gene that re activate the body's immune response against cancer cells. 例如,由质粒中所含DNA编码的抗原,如HLA-B7,可以加入到本发明的蛋白微囊壳体中,直接注射入癌灶(例如皮肤癌)。 For example, the plasmid DNA encoding the antigen contained, such as HLA-B7, may be added to a protein microcapsule shell of the present invention, injected directly into the foci (e.g., skin cancer). 一旦进入癌灶,抗原将激发癌特异性细胞,使细胞激活素(如,IL-2)水平升高,它能使肿瘤具有免疫系统所攻击的靶目标。 Once foci, cancer-specific antigens will stimulate cells, cytokines (e.g., IL-2) elevated levels, which enables the target for tumors of the immune system attack.

再如,也可以将含有部分腺相关病毒基因组的质粒包裹到本发明的蛋白微胶囊壳体中。 Again, it may be part of a plasmid containing the AAV genome to protein microcapsule shell wrapped present invention. 另外,本发明的蛋白微囊壳体可以用于传送治疗基因到达CD8+T细胞,作为对各种癌症和感染性疾病的可接受性免疫治疗。 In addition, protein microcapsule shell of the present invention can be used to transfer the therapeutic gene to reach the CD8 + T cells, as an acceptable immunotherapy of various cancers and infectious diseases.

本发明的蛋白微囊壳体还可用作抵抗感染性疾病的给药系统,来靶传送如抗肝炎B病毒的反义核苷酸。 Protein shell microcapsules of the present invention can also be used to resist infectious diseases, drug delivery systems to target the transfer of anti-hepatitis B virus as antisense nucleotides. 这种反义寡聚核苷酸的一例子是一种抗肝炎B病毒的聚腺苷酸信号的21聚体含磷硫的核苷酸(phosphoro thioate)。 One example of such an antisense oligonucleotide 21-mer nucleotide phosphorus-sulfur (phosphoro thioate) An anti-hepatitis B virus polyadenylation signal.

本发明的蛋白微囊壳体还可用来传送囊性纤维变性透膜调节基因(CFTR)。 Protein microcapsule shell of the present invention may also be used to transfer the cystic fibrosis gene regulation permeable membrane (CFTR). 缺乏这种基固的人会发生囊性纤维变性,可以通过喷雾含有CFTR基因的本发明的蛋白微囊壳体,并直接吸入肺中来治疗这种疾病。 This lack of a solid base of cystic fibrosis will occur, by a spray containing the CFTR gene protein microcapsule shell of the present invention, and directly inhaled into the lungs to treat this disease.

用本发明的微囊壳体也可以传送酶。 Housing with microcapsules of the invention may also be transferred enzyme. 例如,可以将酶(DNAse)包裹,并通过在聚合物壳体外部连接上合适的抗体而将它靶传送到病毒包裹蛋白或病毒感染的细胞。 For example, the enzyme (DNAse) package, and the external connection through the polymer shell on a suitable antibodies it is transferred to the target viral envelope proteins or virus infected cells. 也可以将疫苗包裹在本发明的聚合物微囊中,并用于皮下,肌肉内或静脉内给药。 Vaccines can also be encapsulated in polymeric microcapsules of the present invention, and for subcutaneous, intramuscular or intravenous administration.

实施例14用作血红细胞代用品的不溶性血红蛋白结构(HIC)的制备合成前用乙醇和无菌生理盐水冲洗一个20ml玻璃反应皿、钛发生器和垫圈、以及所用的全部仪器。 Example 14 Insoluble Hemoglobin structure used as blood substitutes embodiment erythrocytes (HIC) was prepared prior to the synthesis of ethanol and 20ml sterile saline wash a glass reaction capsule, titanium generator and gaskets, as well as all the instruments used. 在典型反应中,将3.5ml 5%w/v血红蛋白(人或牛的)加入反应皿,连接上超声波发声器(Heat Systems XL2020,20KHz,最大功率400W)。 In a typical reaction, 3.5ml 5% w / v hemoglobin (human or bovine) was added to the reaction cuvette, the ultrasonic horn connected (Heat Systems XL2020,20KHz, maximum power 400W). 然后将此发声器和反应皿浸入55℃的控温槽中,虽然产物可以在较宽的温度范围内(0~80℃)合成,但最佳温度为55℃,pH值为6.8。 This sound is then immersed in the dish and the reaction tank temperature of 55 ℃, although the product may be (0 ~ 80 ℃) synthesized over a wide temperature range, but the optimum temperature is 55 ℃, pH value of 6.8. 对于物质的高收率,温度控制是关键,并且最佳温度依赖于特殊的实验设计。 For high yield material, temperature control is critical, and the optimal temperature depends on the particular experimental design. 超声波源在功率7位置上开通。 Ultrasonic power source to open 7 position. 利用厂家的列线图推测输出功率大约为150W/cm2。 Use factory nomogram speculated output power of about 150W / cm2. 在30秒内完成反应。 The reaction is completed within 30 seconds. 较长或较短的反应时间,其产量都比较少。 Longer or shorter reaction time, which is less than the yield. 对于牛血红蛋白来说,使2.5%w/v溶液通过一种Sephadex G-25凝胶渗透柱以除去所有阴离子,如磷酸根。 For bovine hemoglobin, the make 2.5% w / v solution by means of a Sephadex G-25 gel permeation column to remove any anions, such as phosphate. 在典型的人血红蛋白IHC的合成中,将超声发生器置于气和水的交界面上。 In a typical synthesis of human hemoglobin IHC, the ultrasonic generator placed in the interface between the gas and water. 产生的均匀悬浮液含有蛋白质类血红细胞。 The homogeneous suspension produced contains proteinaceous red blood cells. 将水悬浮液贮藏于4℃的无菌容器内。 The aqueous suspension was stored in a sterile container at 4 ℃.

典型的反应产生每ml含有大约3108IHC壳体的溶液,其壳体平均直径3微米,标准误差为1微米。 Typical reaction per ml of solution containing about 3 108IHC housing, which housing 3 micron average diameter, standard deviation of 1 micron. 此合成过程产生了高浓度的微米大小的生物物质,其大小分布范围很窄。 This synthesis process produces high concentrations of micron-sized biological material, the size distribution is narrow.

合成以后,IHC仍保持为天然蛋白溶液中的悬浮体。 After synthesis, IHC remain as a suspension in the native protein solution thereof. 为了从未反应的蛋白中分离出IHC,可用如下几种方法:过滤、离心和透析。 To unreacted protein isolate IHC, several methods are available as follows: filtration, centrifugation and dialysis. 第1种方法包括过滤混合物,通过一种Anotop注射过滤器,其孔径为0.2μm(Whatman,Inc.),用几体积的水冲洗过滤器,直到滤液含有很少或不含蛋白(通过UV-可见光谱测定)。 The first method comprises filtering the mixture by means of a Anotop syringe filter, a pore size of 0.2μm (Whatman, Inc.), Washed with several volumes of water will filter until the filtrate containing little or no protein (by UV- Visible spectroscopy). IHC被回冲(backwashed)出过滤器,重新悬浮于相同容积的盐水中。 IHC is back to red (backwashed) the filter, resuspended in the same volume of saline. 第二种纯化过程包括向心或离心过滤器的应用,其截止分子量为100KD。 The second purification process involves the heart or the application of centrifugal filter with a cutoff molecular weight 100KD. 离心过滤是一种被滤过膜从中间隔开的离心管。 Centrifugal filtration is a filtration membrane is separated from the middle of the tube. 在1000G将IHC溶液离心5分钟,可使大多数未反应的血红蛋白(64.5KD)通过滤过膜。 The solution was centrifuged at 1000G The IHC five minutes, make most of the unreacted hemoglobin (64.5KD) through a filtration membrane. 最后,用大分子量(300KD)的滤过膜透析以纯化IHC。 Finally, with a large molecular weight (300KD) dialysis membrane filtration to purify the IHC. 然而,此方法需要透析2天左右。 However, this method requires dialysis two days or so. 纯化IHC的优选方法是应用向心离心。 Purification IHC preferred method is the application of centrifugal centripetal.

实施例15用作红细胞替代物的不溶性血32蛋白/白蛋白结构(IHAC)的制备合成前用乙醇和无菌生理盐水冲洗一个20ml玻璃反应皿、钛发声器和垫圈以及所用的全部仪器。 Insoluble in Example 15 is used as the blood erythrocyte substitutes 32 protein / albumin structure (IHAC) is prepared before synthesis of ethanol and 20ml sterile saline wash a glass reaction capsule, titanium sounder and washer and the entire instrument used. 在典型反应中,将3.5ml 5%m/v血红蛋白和白蛋白(人或牛的,血红蛋白/白蛋白比值在0.5~2之间)加入反应皿,连接超声发生器(Heat Systems,XL2020,20kHz,最大功率400W),然后将此发声器和反应皿浸入55℃的控温槽中。 In a typical reaction, 3.5ml 5% m / v hemoglobin and albumin (human or bovine hemoglobin / albumin ratio of between 0.5 to 2) was added to the reaction cuvette, the connection of ultrasonic generator (Heat Systems, XL2020,20kHz The maximum power 400W), then this dish sounder and reaction immersion bath temperature of 55 ℃. 虽然产物可在较宽的温度范围内(0~80)合成,但在55℃下反应进行最佳。 Although the product can be synthesized over a wide temperature range (0 to 80), but the reaction was carried out at 55 ℃ best. pH值为6.8。 pH value of 6.8. 对于产物的高收率,温度的控制是关键,并且最佳温度依赖于特殊的实验设计。 For high yield of the product, temperature control is critical, and the optimal temperature depends on the particular experimental design. 超声波源在功率7位置上开通,利用厂家的列线图推测其输出功率大约为150W/cm2。 Ultrasonic power source 7 in the opening position, the use of the manufacturer's nomograph presumed output power of about 150W / cm2. 在30秒内完成反应。 The reaction is completed within 30 seconds. 较长或较短的反应时间,其产量都比较少。 Longer or shorter reaction time, which is less than the yield. 所产生的均匀悬浮液中含有蛋白类血红细胞的替代物。 The homogeneous suspension produced contains proteinaceous red blood cell substitute of. 含水悬浮液经过过滤、冲洗,重新悬浮于无菌缓冲盐水中,并在4℃的无菌容器内贮存。 The aqueous suspension was filtered, rinsed, resuspended in sterile buffered saline and stored in sterile containers at 4 ℃.

如上所述,典型的反应产生每ml含有大约108个壳体的溶液,其壳体平均直径为3微米,标准误差为1微米。 As described above, typical reaction produces approximately 108 per ml of solution containing the housing, the average diameter of the casing 3 microns, standard deviation of 1 micron. 此合成过程产生高浓度、大小分布狭窄的微米大小的生物物质。 This synthesis process produces a high concentration, size distribution narrow biomass micron size.

还可以利用一种流水线系统,使IHC的生成反应连续进行。 Can also take advantage of a pipelined system, the resulting reaction IHC continuously performed. 这种系统由蠕动泵组成,它可持续地将血红蛋白流以及任意一种的生物可溶性油或碳氟化物泵入带有声波发声探针的反应皿中。 This system is composed by a peristaltic pump, which sustainably hemoglobin flow and any oil or bio-soluble fluoride carbon pumped into the reactor dish with acoustic sound of the probe. 在反应皿中停留一段合适的时间,IHC通过从反应皿中溢出进入回收罐而得到回收。 Stay for a suitable period of time in the reaction capsule, IHC from the reaction capsule by overflow into the recovery tank and recovered. 未反应血红蛋白溶液重新循环进入反应皿中。 The unreacted hemoglobin solution is recycled to the reaction vessel.

实施例16含有包裹碳氟化物的不溶性血红蛋白结构的制备合成前用乙醇和无菌生理盐水冲洗一个20ml玻璃反应皿、钛发生器和垫圈,以及所用的全部仪器。 Preparation 16 Synthesis of ethanol before use package containing fluorocarbon insoluble hemoglobin configuration of an embodiment and a 20ml sterile saline flush glass reaction capsule, titanium generator and gaskets, as well as all the instruments used. 在典型的反应中,将3.5ml 5%m/v血红蛋白(人或牛的)加入反应皿,连接超声发声器(Heat Systems XL2020,20KHz,最大功率400W)。 In a typical reaction, 3.5ml 5% m / v hemoglobin (human or bovine) was added to the reaction cuvette, connector ultrasonic sounder (Heat Systems XL2020,20KHz, maximum power 400W). 将一种碳氟化物,全氟萘烷3.5ml,加入反应皿中。 The A carbon fluoride, perfluorodecalin 3.5ml, added to the reaction vessel. 然后将此超声发声器和反应皿浸入20℃的控温槽内。 Then this ultrasonic immersion dish Sounders and reaction temperature of 20 ℃ tank. 此水相pH为6.8。 This aqueous phase pH of 6.8. 超声波源在功率7位置上开通。 Ultrasonic power source to open 7 position. 利用厂家的列线图推测出输出功率大约为150W/cm2。 Use factory nomogram inferred output power of about 150W / cm2. 在30秒内完成反应。 The reaction is completed within 30 seconds. 所得的均匀悬浮液中含有在内部包裹有全氟萘烷的交联不溶性血红蛋白壳体的微囊或微球体。 The resulting homogeneous suspension is contained within microcapsules or microspheres wrapped perfluorodecalin crosslinked insoluble hemoglobin shell. 此乳状悬浮液经过过滤、冲洗,重新悬浮于上述的无菌缓冲生理盐水中,并在4℃的无菌容器内贮存。 This milky suspension was filtered, rinsed, resuspended in sterile buffered saline mentioned above, and stored in a sterile container at 4 ℃.

如上所述,典型的反应产生每ml含有大约108个壳体的溶液,其壳体平均直径为3微米,标准误差是1微米。 As described above, typical reaction produces approximately 108 per ml of solution containing the casing, the casing of which an average diameter of 3 microns with a standard error of 1 m. 此合成过程产生了高浓度、大小分布狭窄的微米大小的生物物质。 This synthesis process produces a high biomass concentration, size distribution narrow micron size.

实施例17含有包裹碳氟化物的不溶性白蛋白结构的制备合成前用乙醇和无菌生理盐水冲洗一个20ml玻璃反应皿、钛发声器和垫圈,以及所有的全部仪器。 Preparation 17 Synthesis of ethanol before use package containing fluorocarbon insoluble albumin structure of an embodiment of sterile saline flush and a 20ml glass reaction capsule, titanium sounder and washer, and all all instruments. 在典型反应中,将3.5ml 5%w/v白蛋白(人或牛的)加入反应皿中,连接超声发声器(Heat Systems XL2020,20KHz,最大功率400W)。 In a typical reaction, 3.5ml 5% w / v albumin (human or bovine) was added to the reaction cuvette, the connector ultrasonic sounder (Heat Systems XL2020,20KHz, maximum power 400W). 将一种碳氟化物,全氟萘烷(或全氟三丙胺)3.5ml加入反应皿中。 The A carbon fluoride, perfluorodecalin (or perfluoro tripropylamine) 3.5ml added to the reaction vessel. 然后将超声发声器和反应皿浸入20℃的控温槽内。 Then the ultrasonic sound and response dish immersion bath temperature of 20 ℃. 此水相pH为6.8。 This aqueous phase pH of 6.8. 超声波源在功率7位置上开通。 Ultrasonic power source to open 7 position. 利用厂家的列线图推测出输出功率约为150W/cm2。 Use factory nomogram inferred output power of about 150W / cm2. 在30秒内完成反应。 The reaction is completed within 30 seconds. 所得的均匀悬浮液中含有在内部包裹有全氟萘烷(或全氟三丙胺)的交联不溶性白蛋白壳体的微囊或微球体。 Homogeneous suspension obtained contained inside wrapped with perfluorodecalin (or perfluoro tripropylamine) crosslinked insoluble Albumin microspheres or microcapsules housing. 此乳状悬浮液经过过滤、冲洗,重新悬浮于上述无菌缓冲盐水中,并在4℃的无菌容器中贮存。 The milky suspension is filtered, washed and resuspended in sterile buffered saline above, and stored in sterile containers at 4 ℃ in.

如上所述,典型的反应产生每ml含有大约108个壳体的溶液,其壳体平均直径为3微米,标准误差为1微米。 As described above, typical reaction produces approximately 108 per ml of solution containing the housing, the average diameter of the casing 3 microns, standard deviation of 1 micron. 此合成过程产生了高浓度、大小分布狭窄的微米大小的生物物质。 This synthesis process produces a high biomass concentration, size distribution narrow micron size.

实施例18用变构改良剂如5′-磷酸吡哆醛(PLP)进一步改良的不溶性血红蛋白结构为获得对氧具有不同亲合力(即不同的P50的血红蛋白结构,用IHC与PLP(一种已知的变构调节剂)作进一步反应。将三羟甲基氨基甲烷缓冲液中的IHC(按实施例14获得)悬浮液,在10℃氮气下脱氧。将10ml脱氧后的IHC悬浮液分别放入6个独立的反应皿中。每个反应皿中加入的PLP/Hb摩尔比不同,它们是:0.1/3.0,0.75/3.0,1.5/3.0,3.0/3.0,4.2/3.0,6.0/3.0。30分钟后,加入超过10倍量的硼氢化钠,持续30分钟以减少席夫碱。然后通过离心过滤此悬浮液,用缓冲盐水回冲3次,重新悬浮于缓冲盐水中,并在4℃下贮存。这种修饰作用于脱氧血红蛋白中6-珠蛋白链上的氨基末端基因。从这种意义上讲,在稳定脱氧构象方面,此种改性与2,3-DPG它连接于赖氨酸EF6(82)b上的作用非常相似。 Example 18 allosteric modifiers such as pyridoxal 5'-phosphate (PLP) further modified embodiment of the structure of the insoluble hemoglobin obtained having different oxygen affinity (i.e., P50 of different hemoglobin structures, using IHC with PLP (one has known allosteric modulator) for further reaction. The Tris buffer of IHC (obtained as described in Example 14) was, at 10 ℃ deoxygenated nitrogen. After 10ml of deoxygenated IHC suspension were placed . into six separate reactions each dish was added the reaction capsule PLP / Hb molar ratio different and are: 0.1 / 3.0,0.75 / 3.0,1.5 / 3.0, 3.0 / 3.0,4.2 / 3.0,6.0 / 3.0. After 30 minutes, added more than 10 times the amount of sodium borohydride for 30 minutes to reduce the Schiff base. The suspension is then filtered by centrifugation with buffered saline backflush 3 times, resuspended in buffered saline, and 4 ℃ Under storage. This modification in deoxygenated hemoglobin in the 6-amino-terminal globin chain gene. In this sense, in terms of stability deoxy conformation, such modified with 2,3-DPG which is connected to the lysyl acid EF6 (82) b on the role very similar.

这六种不同程度改性的结果是:IHC的P50随着PLP取代的增多而递增(P50的递增,也就是氧亲合力的下降)。 The results of the six different degrees of modification are: IHC PLP replaced with the increase of the P50 is incremented (P50 increment, which is decreased oxygen affinity).

实施例19带有血红蛋白和聚乙二醇交联壳体的不溶性结构物已知聚乙二醇(PEG)对细胞无毒性、无炎症性、无粘连性,通常无生物活性。 Example 19 and polyethylene glycol with a hemoglobin crosslinked insoluble casing structure known as polyethylene glycol (PEG) not toxic to cells, non-inflammatory, non-adhesive, usually no biological activity. 附着在PEG上的蛋白很少发现有抗原性。 PEG attached to the protein found to have little antigenicity. 发现脂质体的循环会固PEG的联接或结合而增加。 Circulating liposomes will find a solid connection or a combination of PEG increases. 因此,这种将PEG结合入RBC中,将会预期地延长循环时间。 Therefore, this will be incorporated into PEG RBC, it will be expected to prolong the cycle time. 通过改变加入蛋白(如血红蛋白)的巯基PEG的浓度,可制备出具有不同稳定性的PEG血红蛋白RBC。 By varying the concentration of added protein (e.g. hemoglobin) mercapto group of PEG, PEG can be prepared with different RBC hemoglobin stability. 巯基PEG可通现有技术制备(如:Harris和Heart Polymer Preprints 32:154(1991))。 Mercapto PEG may be prepared through the prior art (such as: Harris and Heart Polymer Preprints 32: 154 (1991)).

将分子量为2000g/mol的巯基PEG以1%的浓度(即0.1g加入10ml)溶解于5%血红蛋白溶液中。 The molecular weight of 2000g / mol mercapto PEG concentration of 1% (i.e., added 0.1g 10ml) was dissolved in a 5% hemoglobin solution. 用声波处理这种蛋白-Peg溶液以生成如实施例14中描述的蛋白类红细胞替代物。 By sonication this protein -Peg solution as in Example 14 to produce the proteins described in erythrocyte substitutes.

实施例20带有以共价键附着于壳体外部的聚乙二醇的不溶性血红蛋白结构按实施例14所述制备IHC。 Example 20 Insoluble Hemoglobin structure with covalently attached to the exterior of the housing of the embodiment of polyethylene glycol prepared as described in Example 14 IHC. 采用文献中(Beauchamp etal.Analytical Biochemistry 131:25-33,1983)的技术,将分子量为10,000的聚乙二醇(PEG 10K)与1,1′一羰基二咪唑(CDI)反应。 Using the literature (Beauchamp etal.Analytical Biochemistry 131: 25-33,1983) technique, the molecular weight of 10,000 of polyethylene glycol (PEG 10K) and 1,1 'a carbonyl diimidazole (CDI) reaction. 将IHC悬浮于50mM pH为8.0的硼酸缓冲液中,并且加入PEG-CDI(超过血红蛋白赖氨酸总量的2倍摩尔量),在室温下搅拌反应混合物6小时,所得的PEG-IHC通过过滤分离,用盐水冲洗,重新悬浮于无菌缓冲盐水中。 The IHC were suspended in 50mM pH 8.0 borate buffer, and added to PEG-CDI (2 fold molar amount of hemoglobin over total lysine), the reaction mixture was stirred at room temperature for 6 hours, the resulting PEG-IHC by filtration separated, washed with brine, resuspended in sterile buffered saline.

实施例21 Example 21

影响不溶性血红蛋白结构形成的参数检验几个变量,如蛋白浓度,温度,声处理时间,声强度,pH值,以获得IHC的最佳生成方式。 Parameters on structure formation of insoluble hemoglobin test several variables, such as protein concentration, temperature, sonication time, acoustic intensity, pH value, in order to obtain the best IHC generation scheme.

由1%,2.5%,5%和10%的血红蛋白溶液制备这些物质,也可由混和蛋白溶液制备,如血红蛋白和人、牛的白蛋白,其浓度变化为1-10%。 From 1%, 2.5%, 5% and 10% of the preparation of hemoglobin solutions of these substances, can also be prepared mixed protein solution such as hemoglobin and human, bovine albumin, which change in concentration of 1-10%. 用粒子计数器测定其大小和浓度。 Determination of particle size and concentration of the counter. 发现其大小不随蛋白起始浓度出现有意义的变化。 Found that size does not appear significant changes with the initial concentration of the protein. 在蛋白自起始浓度至5%之间时,所得数量随浓度增加而增加。 In proteins from the time between an initial concentration of 5%, the resulting amount increases with the concentration. 在此浓度以上,其数量没有发现有明显变化。 In this concentration more than the number found no significant change.

发现初始试管温度对IHC的最佳制备有重要意义。 The initial temperature of the tube was found to have the best preparation IHC important. 典型初始温度保持在0-80℃,最佳初始温度为70℃左右。 Typical initial temperature was maintained at 0-80 ℃, the optimum initial temperature is about 70 ℃.

超声处理时间也是决定每ml产生IHC数量的重要因素。 Sonication time is also an important factor per ml determine the number of IHC. 超声处理约30秒左右,有利于合成高浓度的IHC。 Sonication for about 30 seconds, is conducive to the synthesis of a high concentration of IHC. 更长或更短的超声处理时间产生足量但较前为少的IHC。 Longer or shorter time to produce a sufficient amount of ultrasonic treatment, but less than before the IHC.

按照超声发生器厂家提供的列线图,用于此实验的超声发生器的声音功率约为150W/cm2。 Nomogram according to the ultrasonic generator provided by the manufacturer, for this experiment ultrasonic sound generator power is about 150W / cm2. 其它设定功率也可产生大量的IHC。 Other settings can produce a lot of power IHC.

实施例22作为油溶性药物的载体的不溶性血红蛋白结构几种抗肿瘤药物的细胞毒作用在有氧情况下大大提高。 22 as a carrier oil-soluble drug cytotoxicity insoluble hemoglobin structures of several antineoplastic drugs embodiment greatly increased under aerobic conditions. 因此,在将药物输送到肿瘤灶的同时,需要提高那个部位的氧浓度。 Thus, in the drug delivery to the tumor at the same time, the need to improve the oxygen concentration in that part of the. 本发明的血红蛋白微球体具有此作用。 Hemoglobin microspheres of the present invention have this effect. 上面实施例16描述了不溶性血红蛋白壳体对碳氟化物液体的包裹。 16 The above embodiments describe the case of parcels insoluble hemoglobin fluorocarbon liquid. 细胞毒药物,例如环磷酰胺、BCNU、左旋苯丙氨酸氮芥、紫杉醇、喜树碱、阿霉素、鬼臼乙叉戒等等,可被溶解在碳氟化物或其它适宜的油中,例如豆油,并且被包裹于血红蛋白结构内。 Cytotoxic drugs, such as cyclophosphamide, BCNU, melphalan, paclitaxel, camptothecin, doxorubicin, etoposide ring, etc., can be dissolved in a fluorocarbon or other suitable oil , such as soybean oil, and wrapped in the hemoglobin structure.

以5mg/ml的浓度将紫杉醇溶解于豆油(SBO)中,将3.5ml 5%的血红蛋白溶液加入反应中,然后再加入3.5ml的SBO/紫杉醇。 At a concentration of 5mg / ml of paclitaxel was dissolved in soybean oil (SBO), the 3.5ml 5% hemoglobin solution is added to the reaction, and then adding 3.5ml of SBO / Taxol. 对两相混合物进行如实施例16中的声波处理,得到含有SBO/紫杉醇的交联不溶性血红蛋白壳体。 The two-phase mixture was subjected as in Example 16 was sonicated to give a crosslinked containing SBO / Taxol insoluble hemoglobin shell.

实施例23作为水溶性药物的载体的聚合物壳体几种水溶性药物被选用包裹于聚合物壳体内。 Example 23 A water-soluble drug as a carrier of a polymeric shell is chosen wrapped several water-soluble drug in a polymeric shell. 例如,将氨基甲叶酸以5mg/ml的浓度溶于水中。 For example, the methotrexate concentration of 5mg / ml dissolved in water. 使用普卢兰尼克-65(聚环氧乙烷和聚环氧丙烷的嵌段共聚物)将每ml这种水溶液和4ml的豆油一起乳化,形成一种稳定的油包水(w/o)微乳液。 -65 Using pluronic (polyethylene oxide and polypropylene oxide block copolymers) to each ml of this aqueous solution and 4ml of soybean oil are emulsified together to form a stable water in oil (w / o) microemulsion. 在3.5ml 5%血红蛋白溶液上覆盖3.5ml这种w/o微乳化液。 Such covering 3.5ml w / o microemulsion at 3.5ml 5% hemoglobin solution. 声处理30秒后得到含有包裹的氨基甲叶酸微乳化液的不溶性血红蛋白结构。 After sonicated for 30 seconds to give methotrexate microemulsion containing encapsulated insoluble hemoglobin structure.

实施例24作为蛋白载体的聚合壳体几种蛋白被选用包裹于聚合物外壳内,如血红蛋白,白蛋白等等。 Example 24 Polymerization housing as a protein carrier is selected wrapped in several proteins within the polymer shell, such as hemoglobin, albumin and the like. 例如,作为一种增加IHC的血红蛋白负荷量的方法,把血红蛋白包裹入IHC中,取代实施例23中的水溶性药物。 For example, as a method of increasing the hemoglobin loading of the IHC method, wrapped into the hemoglobin IHC, the substituted water-soluble drug in Example 23 embodiment. 将血红蛋白以10%的浓度溶于水中。 The hemoglobin concentration of 10% was dissolved in water. 使用普卢兰尼克(Pluronic)-65(聚环氧乙烷和聚环氧丙烷的嵌段共聚物)将1ml的这种水溶液与4ml的豆油一起乳化,以形成一种稳定的油包水(w/o)微乳化液。 Using pluronic (Pluronic) -65 (polyethylene oxide and polypropylene oxide block copolymers) to 1ml of this aqueous solution together with 4ml of soybean oil emulsion to form a stable water in oil ( w / o) microemulsion. 在3.5ml 5%的血红蛋白溶液上覆盖3.5ml这种含有血红蛋白的油包水微乳化液。 On 3.5ml 5% hemoglobin solution covering 3.5ml hemoglobin containing such a water-oil microemulsion. 这两相混合物经声波处理30秒后得到不溶性血红蛋白结构,这种结构含有本身也含血红蛋白的包裹的微乳化液。 The two phase mixture was sonicated for 30 s after obtain insoluble hemoglobin structure, which itself contains encapsulated hemoglobin-containing microemulsion. 这种方法可提高每个IHC微球体的血红蛋白总量,因而为结合氧提高了携氧能力。 This method can improve the total amount of hemoglobin per microsphere of the IHC and therefore increased the oxygen is combined with the oxygen carrying capacity.

实施例25白蛋白/碳氟化物结构的体内给药——磁共振成像(19F-MRI)检测生物分布按实施例17制备含全氟壬烷的白蛋白结构。 Example 25 Albumin / vivo administration fluorocarbon structure - Magnetic Resonance Imaging (19F-MRI) detecting the biodistribution of Example 17 was prepared containing perfluorononane albumin structure. 最后的悬浮液由含20%体积的碳氟化物的无菌盐水构成。 The final suspension is composed of sterile saline containing 20% by volume of the carbon fluoride. 将2ml此悬浮液经尾静脉注入氯胺酮麻醉的Sprague Dawley小鼠。 2ml of this suspension was injected into the tail vein of mice ketamine anesthetized Sprague Dawley. 通过使用Bruker 500MHz NMR仪器以19F-MRT检测碳氟化物的活体分布。 By using a Bruker 500MHz NMR instrument to detect carbon fluoride 19F-MRT vivo distribution. 将小鼠放入一个10cm19F线圈内使用T1加权序列以TR=1秒,TE=20毫秒和256128数据矩阵而获得影像。 The mice were placed in a 10cm19F coil using T1-weighted sequences with TR = 1 秒, TE = 20 ms and 256 128 data matrix to obtain images.

给药后1小时发现大多数FC蓄积在肝、肺、脾中。 One hour after the administration found that most FC accumulate in the liver, lung and spleen. 一些FC也能在骨髓中检测到。 Some FC can also be detected in the bone marrow. 在组织定位和蓄积方面,血红蛋白结构有预期的完全相同的作用。 In terms of tissue localization and accumulation of hemoglobin structure identical to the expected effect. 这些观察结果对肝、肺肿瘤的治疗,以及在用高剂量氧配合细胞毒药物的定位给药或作为放射治疗的辅助药物治疗骨髓肿瘤细胞方面具有重要的启示。 These observations on the liver, lung cancer treatment, as well as in terms of positioning with high doses of oxygen administered with cytotoxic drugs or radiation therapy as adjuvant therapy of tumor cells in bone marrow has important implications.

实施例26带有药物的结构的活体给药按实施例22制备含包裹的紫杉醇(在SBO中)的不溶性血红蛋白结构,所得的悬浮液由含20%体积的SBO的无菌盐水构成。 Example 26 in vivo administration of the drug with the structure of embodiment in Example 22 was prepared containing encapsulated Taxol (in SBO are) insoluble hemoglobin structure, the resulting suspension containing 20% by volume of the SBO in sterile saline composition. 将2ml此悬浮液经尾静脉注入氯胺酮麻醉的Sprague Dawley小鼠中。 2ml of this suspension was injected into the tail vein of ketamine anesthesia Sprague Dawley mice.

注射2小时后,处死小鼠,取出肝脏。 2 hours after injection, mice were sacrificed, livers. 用少量的盐水使肝脏匀浆,并用乙酸乙酯提取。 Saline with a small amount of the liver homogenate, and extracted with ethyl acetate. 将提取物冻干,溶于甲醇中并注入到一种HPLC柱。 The extract was lyophilized, dissolved in methanol and injected into one kind of the HPLC column. 从肝中回收了未代谢的起始剂量大约15%的紫杉醇。 Recovering the initial dose of unmetabolized taxol from about 15% of the liver. 这说明,在向这些部位传送氧的同时将抗肿瘤药物特异性地输送到肝脏是可行的。 This shows that, in the transfer of oxygen to these sites while the antitumor agent specifically transported to the liver is feasible.

实施例27作为不溶性血红蛋白血液代用品的稳性血液置换模型通过颈外静脉,给麻醉的Sprague-Dawley小鼠插管。 Example 27 as an insoluble hemoglobin blood substitutes stability blood replacement model through external jugular vein to anesthetized Sprague-Dawley mice intubation. 在10分钟时间取出其血量的70%左右。 In 10 minutes about 70% of the time taken its blood volume. 小鼠保持此状态再经过10分钟。 Mice remain in this state and then after 10 minutes. 然后重新注入一种P50为28mmHg的充氧的IHC等渗悬浮液。 And then re-injecting a P50 to 28mmHg of oxygenated IHC isotonic suspension. 连续监测小鼠的平均动脉压,心率和呼吸频率。 Continuous monitoring of mean arterial blood pressure in mice, heart rate and respiratory rate. 随着时间的推移这些小鼠存活下来。 Over time, these mice survive.

实施例28难溶性血红蛋白对组织局部缺血逆转的作用对难溶性血红蛋白(IHC)选择性地将氧气传输到局部缺血部位的功能进行研究。 Example 28 Insoluble Hemoglobin implement role reversal of ischemic tissue of poorly soluble hemoglobin (IHC) to selectively transfer oxygen to the ischemic site features research. 具有“高亲合力”,即P50<28mmHg的IHC适用于这一目的,因为它们仅在此循环系统中正常碰到氧梯度大的部位,也就是所说的在局部缺血部位释放氧气。 Having a "high affinity", i.e., P50 <28mmHg of IHC suitable for this purpose, since they are only in this circulation system normally encountered oxygen gradients large parts, also known as the release of oxygen in the ischemic area. 将具有P50为20mmHg的IHC用于这目的。 P50 will have to 20mmHg of IHC for this purpose.

用大鼠双侧颈动脉闭塞模型作为“中风”或大脑局部缺血模型。 Bilateral carotid artery occlusion rat model as a "stroke" or cerebral ischemia model. 通过临时结扎使氯胺酮麻醉的Sprague-Dawley小鼠的两侧动脉闭塞,在对照组中,15分钟后移去结扎而恢复正常的血液流动。 By making the temporary ligation of ketamine sides Sprague-Dawley mice artery occlusion in the control group, 15 minutes after ligation and removed to restore normal blood flow. 在实验鼠中,用加氧设备使IHC混悬液在外部充氧后,直接将1ml高亲合力的IHC盐水混悬液注入每一侧的颈动脉。 In mice, the equipment used to make oxygenated IHC suspension in the external oxygenation, directly to a high affinity IHC 1ml saline was injected into the carotid artery on each side. 治疗24小时后,处死小鼠,取出它们的大脑,固定,切片并用氮蓝四唑(NBT)或锥虫蓝染色来测定细胞死亡的程度。 After 24 hours of treatment, the mice were sacrificed, their brains removed, fixed, sectioned and stained with nitro blue tetrazolium (NBT) or trypan blue dye to determine the extent of cell death. 通过锥虫蓝染色的测定得知在接受了本发明IHC的实验小鼠中细胞死亡程度较低。 By measuring trypan blue staining accepted that in the present invention, a lower degree of cell death IHC experiments mice.

实施例29不溶血红蛋白结构在活体循环系统中半衰期的估价通过外颈静脉向麻醉的Sprague-Dawley小鼠(350-400g)插入导管。 Example 29 Insoluble Hemoglobin structure in vivo half-life in the circulation system to the valuation embodiment catheterized mice anesthetized Sprague-Dawley (350-400g) via external jugular vein. 将相当于20%动物血容量的IHC等渗混悬浓注射液通过导管给药。 Will be equivalent to 20% of blood volume IHC animal isotonic suspension concentrated injection administered through the catheter. 在0.25到92小时之间的采样时间范围内采血。 Blood sampling time within the range of between 0.25 to 92 hours. 离心血样并且观察血浆的溶血迹迹象或可溶血红蛋白的出现。 Blood samples were centrifuged and plasma was observed dissolved or soluble hemoglobin blood signs appear. 由于IHC中的“微泡”具有气态的空间(并且因此比水的密度低),它们在离心后上升到血浆的表面。 Since the IHC in the "microbubbles" having gaseous space (and thus lower than the density of water), they rise to the surface after centrifugation plasma. 滤除这些微泡,在盐水中重新悬浮,并且用粒子计数器计数。 These microbubbles filtered, re-suspended in saline and counted using a particle counter. 循环系统中IHC半衰期就测定出来了。 Circulating half-life is determined IHC system out. 与现有技术的源于血红蛋白的人工血浆代用品相比,发现本IHC显示出增加了的循环半衰期。 Compared with the prior art plasma substitute from artificial hemoglobin, found this IHC exhibit increased circulating half-life.

实施例30IHC的器官保存作用—对大鼠心脏的保藏作用用外科手术摘除麻醉Sprague-Dawley鼠的心脏并利用室内空气对其进行人工呼吸。 Organ Preservation embodiment 30IHC - the preservation effect on the heart in rats with surgical removal of the Sprague-Dawley rat cardiac anesthesia and the use of indoor air its artificial respiration. 将心脏浸入具有同IHC(或IHC/FC,或白蛋白/FC)保藏基质有相同组分而没有血红蛋白组分的晶体基质(“心脏麻痹基质”——CM)中。 Will have the same heart immersed IHC (or IHC / FC, or Albumin / FC) preservation matrix have the same composition but no crystal matrix component of hemoglobin (the "heart paralysis matrix" --CM) in. 心脏用CM浸泡几分钟,将其冷却到10℃。 Heart with CM soak a few minutes, it was cooled to 10 ℃. 然后用140mlIHC保藏基质在12℃保藏心脏12小时。 Then 140mlIHC deposited matrix stored at 12 ℃ heart 12 hours. 连续以低压(18mmHg)使IHC基质通过心脏,并且以95%O2/5%CO2使其持续地平衡。 Continuous low pressure (18mmHg) so that the matrix by IHC heart, and with 95% O2 / 5% CO2 to the continuance of balance. 保藏12小时后,通过使用离体的工作大鼠心脏检测设备检测心脏的收缩、舒张和功能活动实施例31利用IHC基质在心脏外科手术中进行心脏麻痹进行心肺分流术,并且在恰当的主动脉交叉关闭和开口之后,将含有IHC(或IHC/FC,或白蛋白/FC)的等渗混悬液作为氧载体,在4℃经团块注射把500至100ml的团块输送到主动脉根部。 12 hours after accession, from the contraction of the heart to work through the use of rat cardiac testing equipment to detect body relaxation and functional activities embodiment 31 of a heart attack the use of IHC matrix were performed cardiopulmonary bypass in cardiac surgery, and in the right aortic After closing and opening cross isotonic, containing IHC (or IHC / FC, or Albumin / FC) as an oxygen carrier suspension, injection at 4 ℃ by mass to 500 to 100ml of mass delivery to the aortic root . 向左侧、右侧冠状动脉口输送另外剂量的冷基质,在外科分流术的情况下,在最终的吻合术之前也将介质传输到移植物的末端。 To the left and right coronary ostia conveying additional dose of cold substrate, in the case of bypass surgery, and before the final anastomosis will also be a transmission medium to the end of the graft. 每15到20分钟以足以保持较低的心肌温度的量输送该基质。 Every 15 to 20 minutes in an amount sufficient to maintain a low temperature conveyance of the myocardial matrix. 完成整个操作过程后,移去主动脉夹,使心脏开始恢复温暖。 After the completion of the entire operation, aortic clamp is removed, the heart began to restore warmth.

实施例32IHC基质在血管成形术或动脉疏通术(Atherectomy)中的应用在用来恢复向器官的闭塞或灌注不足区域灌注的介入手术过程中给药IHC(或IHC/F,或白蛋白/FC)介质。 EXAMPLE 32IHC stromal artery angioplasty or surgery to clear (Atherectomy) application in to restore the organ perfusion occlusion or hypoperfusion area of intervention is administered during surgery IHC (or IHC / F, or albumin / FC ) media. 这种手术的例子有血管成形术或动脉通术。 Examples of such surgery have angioplasty or artery through surgery. 在经皮透腔状血管成形术方法的膨胀充气过程中通过膨胀充气导管的中心腔以大约60ml/分钟的速度给药充氧的IHC基质可以缓解局部的缺血情况。 In percutaneous transluminal angioplasty shaped cavity forming method can ease the process of expansion of the inflatable local ischaemia inflatable catheter through the expansion of the central chamber at a rate of about 60ml / min administered oxygenated IHC matrix. 以体温的温度将这种基质给药,它含有例如生理相容的Ringer氏电解质和底物。 In this matrix body temperature will be administered, for example, it contains a physiologically compatible Ringer's electrolytes and substrates. 在每一膨胀充气期间注入一定剂量的氧气平衡的IHC基质。 Inflatable injected during each dose of the oxygen balance IHC matrix. 还可以在动脉疏通术的充气膨用户阶段使用类似的方法,该手术可用来以刀或激光的形式物理清除血管中的障碍。 Can also clear the artery surgery in stage pneumatic expansion users use a similar approach, the procedure can be used in the form of physical knife or laser removal of blood vessel disorders. 在酶促溶血过程中把基质直接注入被堵塞的血管能够在堵塞物溶解时为堵塞物提供氧。 In the course of the enzymatic hemolytic matrix directly into the blood vessels can be blocked when the blockage is dissolved to provide oxygen for the blockage. 目前在一些血管成形术过程中使用Fluosol-DA,本发明的IHC(或IHC/FC,或白蛋白/FC)基质能够取代Fluosol-DA。 Currently Fluosol-DA used in some angioplasty process, IHC present invention (or IHC / FC, or Albumin / FC) matrix can replace Fluosol-DA.

实施例33合成由聚合物外壳包裹的十二氟壬烷(C9F20)必须在合成反应进行之前将一个20ml的玻璃反应槽、钛制的发声器、套管以及使用的全部设备用乙醇和无菌盐水洗涤干净。 Synthesis reaction must be carried out prior to the reaction vessel a 20ml glass, the titanium sounder Example 33 Synthesis of polymer shell wrapped by the twelve-fluoro nonane (C9F20) embodiment, the sleeve and the entire device for use with ethanol and sterile washed clean brine. 在这一典型反应中,将3.5ml无菌5%W/v USP(美国药典标准)人体血清白蛋白(Alpha医疗公司提供)加入到反应槽中,反应槽连接于超声波发声器(Heat Systems,XL2020,20KHz,最大功率400W)。 In this typical reaction, 3.5ml of sterile 5% W / v USP (United States Pharmacopoeia standards) human serum albumin (Alpha Medical Corp.) was added to the reaction vessel, the reaction vessel is connected to the ultrasonic horn (Heat Systems, XL2020,20KHz, maximum power of 400W). 然后将发声器与槽一起浸入温度恒定在22℃的水浴中。 Then, together with the slot sounder immersed in a water bath at a constant temperature of 22 ℃. 反应在22℃进行为最适宜条件,而产品能在更宽的温度范围内(0℃到约40℃)合成。 Reaction was carried out at 22 ℃ for optimum conditions, and the product can be over a wider temperature range (0 ℃ to about 40 ℃) synthesis. 温度控制是高产量的关键,而最适宜温度条件则依赖于特定的实验条件。 Temperature control is the key to high yield, and the optimum temperature conditions are dependent on the particular experimental conditions.

接着加入6ml十二氟壬烷(C9F20),超声波旋钮旋至第七挡功率处。 Then join 6ml twelve perfluorononane (C9F20), ultrasonic power knob to place seventh gear. 加入的碳氟化合物的量可在小于1ml到约13ml之间变化,这时的蛋白质聚合物外壳产率良好。 The amount of fluorocarbon added can be varied between less than about 1ml to 13ml, then the protein polymer shell high yield. 反应在约30秒内完成。 The reaction is completed in about 30 seconds. 反应时间过短和过长产量都会减少。 The reaction time is too short and long yields will be reduced. 所生产的均匀的混悬液包含由蛋白质聚合物外壳包裹的十二氟壬烷,按体积计算为大致60%体积的全氟壬烷。 The homogeneous suspension produced contains the protein polymer shell wrapped by the twelve-fluoro-nonane, by volume of approximately 60% by volume of the perfluoro nonane. 在4℃的无菌容器内保存这一含水混悬液。 Save this aqueous suspension in sterile containers at 4 ℃.

典型的反应生产的溶液每ml含大致1109个外壳。 A typical reaction for producing a solution containing per ml of approximately 1 109 个 housing. 平均外壳直径为2微米,标准差为1微米。 The average shell diameter of 2 m, a standard deviation of 1 micron. 观察到这一合成过程生产出高浓度的、大小分布范围较窄的微米大小的生物制品。 The synthesis process was observed to produce high concentration, size distribution narrower micron sized biological products.

实施例34合成由聚合物外壳包裹的全氟三丁基胺(C12F27N)或全氟三丙基胺(C9F21N)将5%w/v USP的人体血清白蛋白(3.5ml)和氟胺化合物(6ml)加入到玻璃反应槽中并用高强度的超声波辐射,反应条件是功率定在第7档,水浴温度为22℃,反应时间大致在30秒。 Example 34 Synthesis of a polymeric shell wrapped perfluoro tributylamine (C12F27N) or perfluoro tripropylamine (C9F21N) embodiment the 5% w / v USP human serum albumin (3.5ml) and fluorine amine compound ( 6ml) was added to a glass reaction vessel and radiation with high intensity ultrasound, the reaction conditions are given in the first seven-speed power, water bath temperature of 22 ℃, the reaction time is approximately 30 seconds. 又一次合成了高浓度的由蛋白质聚合物外壳包裹的高浓度的全氟三丙基胺[(C3F7)3N]和全氟三丁基胺[(C4F9)3N](1109个外壳/ml),外壳平均直径为2微米。 Again a high concentration of synthetic protein polymer shell wrapped by the high concentration of perfluoro tripropylamine [(C3F7) 3N] and perfluoro tributylamine [(C4F9) 3N] (1 109 个 housing / ml ), an average shell diameter of 2 microns.

实施例35合成由聚合物外壳包裹的全氟萘烷(C10F18) Example 35 Synthesis of polymer shell wrapped by perfluorodecalin (C10F18)

将5%w/v USP的人体血清白蛋白(3.5ml)和全氟萘烷(C10F18;6ml)加入到玻璃反应槽中,并用高强度的超声波辐射。 The 5% w / v USP human serum albumin (3.5ml) and perfluorodecalin (C10F18; 6ml) was added to a glass reaction vessel, and the high intensity ultrasonic irradiation. 反应条件为功率定在第七挡,水浴温度为22℃,反应时间大致在30秒。 The reaction conditions for power set in the seventh block, water bath temperature of 22 ℃, the reaction time is approximately 30 seconds. 合成了由蛋白质聚合物外壳包裹的高浓度、大小分布范围较窄的全氟萘烷。 Synthesis of a high concentration of polymer shell wrapped by the protein, a narrower size distribution perfluorodecalin. 此外,由于全氟萘烷和全氟三丙基胺都是FDA批准的碳氟化合物,Fluosol DA的主要成分,因此将这些化合物用于医学成像很容易被管理机构所认可。 Further, since the main component of the fluorocarbon perfluorodecalin and perfluoro tripropylamine are FDA-approved, Fluosol DA, and therefore these compounds in medical imaging can easily be recognized by regulatory agencies.

实施例36合成由聚合物外壳包裹的全氟15-冠-5(C10F20O5)将5%w/v USP的人体血清白蛋白(3.5ml)和氟冠醚(C10F20O5;6ml)加入到玻璃反应槽中,并且用高强度的超声波辐射。 Example 36 Synthesis of a polymeric shell wrapped perfluoro 15-crown -5 (C10F20O5) embodiment the 5% w / v USP human serum albumin (3.5ml) and fluorine crown ethers (C10F20O5; 6ml) was added to a glass reaction vessel , and radiation with high intensity ultrasound. 反应条件定在功率的第七挡,水浴温度为22℃,反应时间大致在30秒。 The reaction conditions are given in the seventh power block, water bath temperature of 22 ℃, the reaction time is approximately 30 seconds. 同前,合成了高浓度的由蛋白质聚合物外壳包裹的氟冠醚,其颗粒大小分布较窄。 With the former, a high concentration of synthetic protein polymer shell wrapped by fluorine crown ether, a narrow particle size distribution. 事实上这一合成由聚合物外壳包裹的碳氟化合物的实验过程是典型的所有碳氟化合物的研究方法。 In fact this experimental procedure synthetic polymer shell wrapped by the fluorocarbons are typical methods all fluorocarbon.

实施例37合成由聚合物外壳包裹的全氟-叔丁基丁烯(C10F18H2) Example 37 Synthesis of a polymer shell wrapped perfluoro - tert-butyl-butene (C10F18H2)

将5%w/v USP的人体血清白蛋白(3.5ml)和C10F18H2(6ml)加入到玻璃反应槽中,并用高强度的超声波辐射,功率定在第七挡,水浴温度为22℃和反应时间大致在30秒。 The 5% w / v USP human serum albumin (3.5ml) and C10F18H2 (6ml) was added to a glass reaction vessel, and use of high intensity ultrasonic irradiation, the power given in the seventh block, water bath temperature is 22 ℃ and reaction time approximately 30 seconds. 通过这一过程,能够合成由蛋白质聚合物包裹的高浓度的全氟-叔丁基丁烯。 Through this process, the protein can be synthesized by a high concentration of polymer-wrapped perfluoro - tert-butyl-butene.

实施例38由聚合物外壳包裹的碳氟化合物的毒性在10分钟内给五只大鼠通过外插的静脉导管注射5ml 2%v/v碳氟化合物混悬液(由HSA蛋白质聚合物外壳包裹的全氟壬烷)。 Toxicity fluorocarbon polymer shell wrapped by Example 38 in 10 minutes to five rats by extrapolation of intravenous catheter injection of 5ml 2% v / v fluorocarbon suspension (HSA protein polymeric shell wrapped by perfluoro nonane). 一般的碳氟化合物由于其牢固的氟-碳键而没有毒性;的确,碳氟化合物已经被成功地用作FDA批准的人工血浆代用品(Fluosol DA)。 Usually fluorocarbons due to their solid-fluoro - carbon bond without toxicity; indeed, fluorocarbons have been successfully used as FDA approved artificial blood plasma substitutes (Fluosol DA). 在特定的时间收集大鼠并且解剖。 Rats were collected and dissected at a specific time. 除了观察大鼠的一般性健康之外,要仔细检查肝、脾、肺和肾。 In addition to the general health of rats were observed outside, to carefully check the liver, spleen, lungs and kidneys. 在0.5,2,8和24小时检查时大鼠都是健康的,没有发炎的组织或器官。 In 0.5, 2 and 24 hours to check the health of rats are not inflamed tissues or organs. 90天后第五只大鼠仍然存活,而且仍很健康。 90 days after the fifth rats were still alive, but still healthy. 作为对照组,这一FDA批准的大鼠体内豆油的用量为半数致死量,进一步证实了碳氟化合物是无毒的和安全的。 As a control group, soybean oil rats in an amount of the FDA approved the median lethal dose, further confirmed the fluorocarbons are nontoxic and safe.

实施例39纯碳氟化合物和由聚合物外壳包裹的碳氟化合物的19F Example 39 pure fluorocarbon and a polymer shell wrapped fluorocarbons 19F

核磁共振光谱由蛋白质聚合物外壳包裹的碳氟化合物和纯碳氟化合物的NMR光谱可以在Bruker 500MHzNMR光谱仪上获得。 Fluorocarbon polymer shell proteins by nuclear magnetic resonance spectroscopy and NMR spectra of pure wrapped fluorocarbon may be obtained on Bruker 500MHzNMR spectrometer. 仪器调到19F,它的共振频率为470.56NHz。 Instrument transferred 19F, its resonance frequency 470.56NHz. 用氘溶剂作为阻断剂,并且所有光谱都以在0ppm的Freon(CCl3F)为客观的参考标准。 With solvent deuterium as blockers, and all spectra are in Freon (CCl3F) at 0ppm for the objective reference standard. 将全氟壬烷和CDCl3放入5mm NMR试管中。 The perfluorononane and CDCl3 into 5mm NMR tubes. 纯全氟壬烷的光谱得到两组尖峰,一组在-87ppm,第二组在-127,-128和-133ppm。 Pure perfluorononane the spectrum of the resulting two sets of spikes, a group -87ppm, a second group in -127, -128 and -133ppm.

由HSA蛋白质聚合物外壳包裹的全氟壬烷混悬液在D2O中重新混悬,并且获得了类似的NMR光谱。 HSA protein polymeric shell wrapped by the perfluorononane suspension was resuspended in D2O, and a similar NMR spectrum obtained. 从20%v/v碳氟化合物混悬液得到强的信号,其峰值或共振频率是在-81,-121,-122和-126ppm处。 To give a strong signal from 20% v / v fluorocarbon suspension, the peak or resonant frequency is -81, -121, -122 and -126ppm place. 由聚合物外壳包裹的碳氟化合物在超声波辐射期间没有引起全氟壬烷化学或结构的改变。 Fluorocarbon polymer shell by the wrapped during ultrasonic irradiation did not cause the chemical or structural changes nonane perfluoro. 例如,对于C9F20分别观察到两组共振频率:一组相应的是在大约-80ppm,第二组共振频率大致在-125ppm,与CF2基团相符合。 For example, the two groups were observed for C9F20 resonance frequency: a set of corresponding at about -80ppm, a second set of resonance frequency is substantially in -125ppm, consistent with the CF2 group.

实施例40利用碳氟化合物的19F核磁共振光谱测定局部温度在Bruker 500MHzNMR光谱仪上测得碳氟化合物的可变温度NMR光谱。 Example 40 19F NMR spectroscopy utilizing fluorocarbon local temperature was measured on Bruker 500MHzNMR spectrometer fluorocarbon measured variable temperature NMR spectroscopy. 仪器调整至19F,它的共振频率为470.50Hz。 Instruments adjusted to 19F, it's resonance frequency 470.50Hz. 用氘溶剂(d6-二甲基亚砜[d6-DMSO)])作为阻断剂,并且所有光谱以在0ppm的Freon(CCl3F)为客观的参考标准。 With deuterium solvent (d6- DMSO [d6-DMSO)]) as a blocking agent, and all spectra with Freon (CCl3F) at 0ppm for the objective reference standard. 全氟十二烷的熔点为77℃,将它与d6-MDSO一同在室温下放入5mm NMR试管。 Perfluoro dodecane a melting point of 77 ℃, with d6-MDSO it together into 5mm NMR tube at room temperature. 在不同的温度下检测到氟的光谱,并测量其谱线宽度,将-81ppm处的谱线宽度以温度的函数形式表述如下:-81ppm(Hz)的谱线宽度温度(℃)51.1 10257.0 8264.65 60随着温度上升,低温时的宽谱开始变尖锐,这是由于全氟十二烷正处于从固态到液态的相变阶段。 At different temperatures detected fluorine spectrum, and measure the line width, the line width at -81ppm as a function of temperature expressed as follows: -81ppm (Hz) of the spectral line width of the temperature (℃) 51.1 10257.0 8264.65 60 as the temperature rises, low temperature begins to sharply broad spectrum, which is due to perfluoro dodecane is changing from solid to liquid phase transformation. 这种变化随着温度变化而尖锐、迅速变化,如同纯物质所测得的结果。 This sharp change with temperature change, rapid change, as a result of pure material measured.

为了减低溶点温度、加宽溶点温度范围,在全氟十二烷中加入(约2%v/v)的戊烷。 In order to reduce the melting point temperature, widen the melting point temperature range, was added (approximately 2% v / v) in pentane perfluoro dodecane. 如上所示,在全氟十二烷从固态向液态相变阶段,低温时的宽峰变尖锐了。 As indicated above, perfluoro dodecane phase change from a solid to a liquid phase, a broad peak at low temperature becomes acute. 全氟十二烷/戊烷混合物的谱线宽度作为温度的函数表示如下:谱线宽度(Hz)-82ppm-123.3ppm温度(℃)21.26 87.17 77165.89 280.50 67216.6 341.2 57290.77 436.15 47578.27 451.33 37577.62 525.11 27 Perfluoro dodecane / pentane mixture of linewidth as a function of temperature as follows: line width (Hz) -82ppm-123.3ppm temperature (℃) 21.26 87.17 77165.89 280.50 67216.6 341.2 57290.77 436.15 47578.27 451.33 37577.62 525.11 27

全氟十二烷/戊烷混合物具有所要求的有较宽范围的低熔点。 Perfluoro dodecane / pentane mixture having the desired low melting point has a wide range. 使用这一系统,可在27℃至77℃范围内进行温度测定。 Using this system, the temperature can be measured in the range of 27 ℃ to 77 ℃. 因此,只要给出一个谱线宽度值,就能测定当时的温度。 Therefore, as long as a given line width value, can be measured at the time of temperature.

举例来说,利用这种技术测量活体的局部温度,包括注射含有碳氟化合物混合物(如上所述)的蛋白质外壳,该混合物的宽的熔化相变过程具有温度——谱线宽度的相互关系(能从实验中获得)。 For example, the use of such techniques to measure the local temperature of the living body, including injection containing fluorocarbon mixtures (as described above) of the protein shell, the width of the molten mixture having a phase transition temperature of - line width correlation ( obtained from the experiment). 这种制剂除了可以作为19FMRI的对比剂之外,它可以作用于肝脏和脾脏,因此同时可用测定器官内的局部变化温度(以阐明组织内明显异常的病理状态)。 In addition to this formulation can be used as contrast agents 19FMRI outside, it can act on the liver and spleen, and therefore can be measured at the same time the local changes in temperature within the organ (to clarify the apparent anomaly in the pathological state).

实施例4119F核磁共振模型图象将两种类型的聚合物外壳包裹的碳氟化合物用于这种模拟成像的研究。 Example 4119F NMR model images embodiments two types of fluorocarbon polymer shell encapsulated compounds for imaging studies such simulations. 按照如实施例33和34所述合成由HSA蛋白质聚合物外壳包裹的全氟壬烷和全氟三丁基胺。 Following the procedure as tributylamine Examples 33 and 34 Synthesis of the HSA protein polymeric shell wrapped by perfluoro nonane and perfluoro. 用盐水稀释合成的每个体积含60%碳氟化合物的混悬液,然后取2ml放入聚苯乙烯试管。 Each volume of diluted suspension containing 60% synthetic fluorocarbon with brine, and then placed in a polystyrene test tubes of 2ml. 将这些聚苯乙烯管放入商业上可得到的在1.5特斯拉下工作的Siemens 2T MRI仪器(10cm19F线圈)。 These polystyrene tubes placed in a commercially available Siemens 2T MRI instrument working at 1.5 Tesla (10cm19F coil). 在5分钟时间内以10毫秒的回声时间(TE)和300秒的重复时间(TR)(256256矩阵)获得试管的19F核磁共振图象。 Over a 5 minute period to 10 ms echo time (TE) and 300 seconds of the repetition time (TR) (256 256 matrix) tubes 19F NMR image obtained.

聚合物外壳包裹的全氟壬烷稀释[浓度],M成象清晰度 Polymer shell wrapped perfluorononane diluted [concentration], M image sharpness

1 1.8 极佳1/2 0.9 极佳1/4 0.45 较好1/10 0.18 较好1/50 0.09 较好1/100 0.02 勉强良好的MR模拟成象甚至在低浓度的由聚合物外壳包裹的全氟壬烷情况下就可观测到。 1 1.8 0.9 Excellent Excellent 1/2 1/4 1/10 0.18 0.45 better better better 1/100 1/50 0.09 0.02 reluctantly good MR imaging even in low concentrations simulated by a polymer shell wrapped Under perfluorononane situation can be observed. 也可以在由聚合物外壳包裹的全氟三丁基胺中观测到非常接近的数值。 May be very close to the value observed in the polymer shell wrapped perfluoro tributyl amine. 仅在高稀释度(1/100;0.02M)时成象质量和分辨率较差。 Only at high dilution; time (1/100 0.02M) image quality and poor resolution.

实施例42离体肝脾的19F核磁共振成象向300g大鼠体内注射2ml 20%v/v由HSA蛋白质聚合物外壳包裹的全氟壬烷混悬液。 Example 42 In Vitro 19F nuclear magnetic resonance imaging of the liver and spleen to 300g rats injected 2ml 20% v / v HSA protein polymeric shell wrapped by the perfluorononane suspension. 分别在2小时后和5天后处死大鼠,摘除肝、脾、肾和肺。 After 2 hours, respectively, and five days later the rats were killed, removed liver, spleen, kidney and lung. 例如,将整个儿肝脏立即放入一个4特斯拉的MRI仪器,使用一只10cm的19F线圈工作。 For example, the entire liver children immediately into a 4 tesla MRI instrument, use a 10cm of 19F coil work. 使用T1加权序列且TR=1秒,TE=20毫秒,数据矩阵为256128(即,128个相编码步骤,16个信号平均值)获得肝、脾和肾的19F磁共振图像。 Using T1-weighted sequence and TR = 1 秒, TE = 20 ms, the data matrix is 256 128 (i.e., 128 phase encoding steps, 16 signal averages) obtained 19F magnetic resonance images in liver, spleen and kidney.

肝脏的19F MRI图象表现出与肝脏吸收聚合物外壳的可变度有关的局部可变强度。 19F MRI images showed the liver and liver-absorbing polymer shell variable degree of local variable intensity related. 例如,可以观察到与门静脉相应的暗区,在该区域看不到聚合物外壳包裹的全氟壬烷出现,因为多数壳体都集中在肝的RES细胞内。 For example, the portal vein can be observed with the corresponding dark area, in the region do not see the polymer shell wrapped perfluorononane appear, because most of the housing are concentrated in the liver cells of the RES.

在注射两小时后扫描的肝脏平均成象强度大约比注射五天后扫描记录的成象强度高20-30%。 Two hours after the injection liver average intensity imaging scans approximately 20-30% higher than five days after the injection intensity imaging scan records. 这表明可能由于聚合物外壳的破裂而使部分全氟壬烷弥散了。 This indicates that the polymer shell may be partially broken to diffuse the perfluorononane. 总之,获得了表现肝脏形态的高质量的成象。 In short, get the performance of high-quality imaging liver morphology. 表明这一技术在诊断以及定位肝脏内部异常病理改变方面的可行性。 Suggests that this technique in the diagnosis of abnormal liver pathology and internal orientation change aspects of feasibility.

实施例43体内肝、脾的19F核磁共振成象在10分钟内给150g的大鼠注射2ml 20%v/v由HSA聚合物外壳包裹的全氟壬烷(c9F20)。 Example 43 in vivo liver embodiment, spleen 19F NMR imaging in rats within 10 minutes of injection of 150g 2ml 20% v / v HSA polymer shell wrapped by perfluoro nonane (c9F20). 然后将大鼠置于4特斯拉MRI仪器内,使用10cm19F线圈工作。 Then rats were placed in 4 Tesla MRI instrument, using 10cm19F coil work. 在收集图象前用氯胺酮麻醉大鼠。 Before collecting the rats were anesthetized with ketamine image. 用T1加权序列和TR=1秒,TE=20毫秒,以及数据距阵256128(即128个相编码步骤,16个信号平均数)来获得整个大鼠和单个器官如肝、脾和肾的19F磁共振图像。 T1-weighted sequence with TR = 1 and 秒, TE = 20 msec, and a data Inmerse Matrix 256 128 (i.e., 128 phase encoding steps, 16 signal averages) to obtain the whole rat and a single organ, such as liver, spleen and kidney The 19F magnetic resonance images.

分别在注射由HSA蛋白质聚合物外壳包裹的全氟壬烷15分钟,2小时和24小时后使大鼠成象。 Respectively injected by HSA protein polymeric shell wrapped perfluorononane 15 minutes, 2 hours and 24 hours after the rats were imaged. 总之,获得了表现肝和脾形态的高质量成象,表明了这一技术在诊断以及定位含肝脏RES的器官内异常病理改变可行性。 In short, access to the performance of the liver and spleen morphology of high-quality imaging, showed abnormal pathological changes in the organs of this technology in the diagnosis and localization of liver RES containing feasibility.

实施例44利用体内19F核磁共振成象测定局部温度在10分钟内给300g.的大鼠注射5ml 20%v/v由HSA聚合物外壳包裹的全氟壬烷/2%戊烷(或全氟十九酸和1%胆固醇)。 Example 44 rats were injected with the use of magnetic resonance imaging in vivo 19F localized temperature measured within 10 minutes to 300g. Of 5ml 20% v / v HSA polymer shell wrapped by the perfluorononane / 2% pentane (or perfluoro nineteen acid and 1% cholesterol). 然后将该鼠放入15cm线圈内(Siemens 1.5特斯拉MRI磁共振机)。 Then put the mouse within 15cm coil (Siemens 1.5 Tesla MRI magnetic resonance machine). 以TE为10毫秒和TR为300秒来收集成象(256256矩阵)。 With TE 10 ms and TR for 300 seconds to collect imaging (256 256 matrix). 在采集数据前用氯胺酮麻醉大鼠。 Before the rats were anesthetized with ketamine data collection. 在15分钟时间内以5毫米的片厚使肝、脾成象。 In the period of 15 minutes to 5 mm thick sheet liver, spleen imaging. 用加热垫包住温和的大鼠,分别在室温和约37℃时采集数据。 With mild heating pad wrapped rats, data were collected at room temperature and about 37 ℃.

实施例45利用19F核磁共振成象进行体内氧测定在10分钟内给300g的大鼠注射5ml 20%v/v由HSA聚合物外壳包裹的全氟壬烷。 Example 45 utilizing 19F nuclear magnetic resonance imaging oxygen in vivo assays in rats were injected within 10 minutes to 300g of 5ml 20% v / v HSA polymer shell wrapped by the perfluorononane. 然后将大鼠放入16cm线圈中(Sienens 1.5特斯拉MRI磁共振机)。 Then the rats were placed in 16cm coil (Sienens 1.5 Tesla MRI MRI machine). 以TE为70毫秒和TR为3秒来采集成象(256256矩阵)。 With TE 70 ms and TR of 3 seconds to capture an image forming (256 256 matrix). 在采集数据前将大鼠放入一可控制装置中。 Before the rats were placed in a data collection device can be controlled. 首先将大鼠放入氧气室以增加氧气的新陈代谢,然后收集谱线宽度和成象。 The rat is placed in an oxygen chamber to increase oxygen metabolism, and the linewidth and image collection. 下一步给大鼠注射氯胺酮以减少氧气的消耗量,并再一次收集谱线宽度和成象,观察到谱线宽度和成象强度发生改变,这与大鼠体内溶解氧的含量一致。 Next to the injection of ketamine in rats to reduce consumption of oxygen, and once again collected and imaged line width, and line width was observed imaging intensity changes, which is consistent with the dissolved oxygen content of the rats. 在较高的氧浓度下测到最大的谱线宽度。 At higher oxygen concentration maximum measured linewidth. 用5毫米厚的片使肝脾在15分钟后成象。 With a 5 mm thick slices so that the liver and spleen at 15 points Zhong Houcheng like. 用加热垫包住麻醉鼠,分别在室温和37℃时采集到两组数据。 Anesthetized rats using a heating pad wrapped, the two sets of data were collected at room temperature and at 37 ℃.

实施例46制备紫杉醇颗粒用球磨机研磨紫杉醇晶体(Sigma Chemical)直到获得低于10微米粒度的紫杉醇固体颗粒。 Example 46 Preparation of particles with a ball mill paclitaxel Paclitaxel crystals (Sigma Chemical) until a particle size less than 10 microns paclitaxel solid particles. 将颗粒悬浮于等渗盐水中,并借助粒子计数器(Elzone,Particle Data)计数来测定其颗粒大小。 The pellet was suspended in isotonic saline, and by means of a particle counter (Elzone, Particle Data) counting to determine their particle size. 继续研磨直到颗粒的粒度100%均低于5微米。 Continue grinding until the particle size of 100% are less than 5 microns. 用于静脉输液的优选粒度是低于5微米,最理想的是低于1微米。 Preferred particle size for intravenous infusion is less than 5 microns, and most preferably less than 1 micron.

另外还可通过在水中对紫杉醇混悬液进行声处理直到所有颗粒粒度低于10微米而获得紫杉醇颗粒。 It may also be in the water of taxol by suspension was sonicated until all particles less than 10 microns to obtain paclitaxel particles.

通过向紫杉醇乙醇溶液中加水直至得到混浊液而使紫杉醇从其乙醇液中沉淀出来,也能获得低于10微米的紫杉醇颗粒,也可以在加水过程中对紫杉醇溶液进行声处理,直到获得混浊液。 Paclitaxel in ethanol by adding water to the solution until a cloudy solution leaving the Taxol precipitated from ethanol solution, can be obtained Paclitaxel particles less than 10 microns, and to be in the process of adding water to the paclitaxel solution was sonicated until a turbid liquid . 对最终的混合液进行过滤和干燥,就获得了理想粒度的纯紫杉醇颗粒。 The final mixture was filtered and dried, to obtain the desired particle size of the pure paclitaxel particles.

通过对溶于挥发性有机溶剂如乙醇的紫杉醇溶液进行喷雾干燥可制备优质的紫杉醇颗粒。 By paclitaxel dissolved in a volatile organic solvent such as ethanol may be prepared by spray drying high quality paclitaxel particles. 使溶液流过超声喷嘴形成含乙醇的紫杉醇液滴。 The solution flowing through the ultrasonic nozzle forming droplets containing paclitaxel ethanol. 随着乙醇在喷雾干燥器中蒸发,得到优质紫杉醇颗粒。 As the ethanol was evaporated in a spray dryer, to obtain high quality paclitaxel particles. 可通过改变紫杉醇乙醇液的浓度、调节流过喷嘴的液体流速和声处理强度而改变粒度。 By changing the concentration of paclitaxel in ethanol solution, adjusting the flow rate of the liquid flowing through the nozzle is changed the particle size and sonication intensity.

实施例47合成附着于聚阴离子的顺磁性阳离子举例来说,经过把藻酸盐分散到二氯化钆(GdCl3)溶液中,可实现藻酸钆的合成,例如,可以通过对含钆离子(如,GdCl3)的溶液进行超声波辐射和加入少量藻酸钠溶液来合成适于细胞内注射的小球型藻酸钆。 Paramagnetic cation Synthesis Example 47 attached to the polyanion, for example, after the alginate dispersed dichloride gadolinium (GdCl3) solution can be achieved gadolinium alginic acid synthesis, for example, by containing gadolinium ions ( e.g., GdCl3) was subjected to ultrasonic radiation and a small amount of sodium alginate solution is adapted to synthesize intracellular injection of small spherical alginic acid gadolinium. 将藻酸盐分散到钆离子溶液中,经超声波辐射和多价钆离子的交联。 The gadolinium ion alginate dispersed in the solution, the ultrasonic horn and the crosslinked polyvalent gadolinium ions. 产生微米粒度的藻酸钆颗粒。 Alginic acid gadolinium produce micron size of the particles. 除了使用超声波辐射以外,也能应用低或高速混合法。 In addition to the use of ultrasonic radiation, can also be applied low or high speed mixing.

或者,将藻酸钠溶液覆盖在不相混溶的有机溶剂或油(例如:豆油、葵花油、甲苯,二氯甲烷,氯仿以及类似物)上或被其覆盖,使液体接受超声波辐射,并由此使含藻酸盐水相分散到有机相中,然后加入多价离子溶液(例如:GdCl3,Mn-Cl3,FeCl3以及类似物)。 Alternatively, the sodium alginate solution covering immiscible organic solvent or oil (e.g.: soybean oil, sunflower oil, toluene, methylene chloride, chloroform and the like) or on its cover, so that the liquid receiving ultrasonic radiation, and whereby the alginate-containing water phase dispersed into the organic phase, and then adding a solution of multivalent ions (eg: GdCl3, Mn-Cl3, FeCl3 and the like). 因此藻酸钠被交联,产生出适合于在血管内注射后作MRI对比剂使用的极小的藻酸钆球型粒子。 Thus, sodium alginate crosslinked, produce suitable for injection was made after the MRI contrast agent in the blood vessels of very small spherical particles of alginic acid gadolinium. 可使用任何基本的合成技术用藻酸盐和多价阳离子用来制造球体,纤维,平片等等。 May be used with any basic synthetic techniques alginate and multivalent cations used to make spheres, fiber, flat sheet and the like.

本发明以其特定优选的实施例进行了具体介绍,应当指出,任何修改和变化都在所描述的和权利要求的范围之内。 The present invention, in its preferred specific embodiments have been specifically described, it should be noted that any modifications and variations are within the scope of the claimed and described in claims.

Classifications
International ClassificationA23L33/00, A61K9/51, A61K9/50, A61K9/48, A61K47/30, A61K51/00, A61K47/48, A61K49/00, A61K49/22, A61K49/04, A61K49/18, A61K9/00
Cooperative ClassificationA61K47/6927, A61K47/6925, A61K49/1863, A61K9/5146, A61K9/5169, A61K9/5138, A61K9/0026, A61K49/126, A61K49/18, A61K9/5161, A61K49/1818, A61K9/5052, A23L33/40, A61K49/222, B82Y5/00, A61K49/226, A61K49/223
European ClassificationB82Y5/00, A61K49/12P, A61K49/18R, A61K47/48W8E, A61K49/18R2N2K8, A61K49/22P, A61K9/00M5F, A61K9/50H6H, A61K49/22P12, A23L1/29F, A61K47/48W8D, A61K49/18, A61K49/22P4, A61K9/51H6B, A61K9/51H6D, A61K9/51H6F, A61K9/51H6H
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