WO2016049791A1

WO2016049791A1 - In-situ gel used as vitreous body substitute material, and preparation method and use thereof

Info

Publication number: WO2016049791A1
Application number: PCT/CN2014/000885
Authority: WO
Inventors: 常菁; 陶勇; 王斌; 郭宝华; 姜燕荣; 黄延宾
Original assignee: 清华大学
Priority date: 2014-09-29
Filing date: 2014-09-29
Publication date: 2016-04-07

Abstract

Provided are a set for generating ophthalmic zwitter-ion in-situ gel, and application thereof. The set consists of a zwitter-ion polymer as represented by formula (I), a polymer as represented by formula (II) and a solvent, wherein, a molar ratio of sulfhydryl in formula (I) and carbon-carbon double bond functional group in formula (II) is 1:1-5:1, and the solvent is a buffered solution with a pH of 7.0-8.0. When using the set, various components in the set for generating ophthalmic zwitter-ion in-situ gel are mixed according to a proportion and under a room temperature for 5-40 minutes, and the mixed solution is injected into a vitreous body cavity, thus generating the zwitter-ion in-situ gel in a body via in-situ crosslinking. The prepared zwitter-ion in-situ gel is used as the vitreous body substitute material and compared with a traditional artificial vitreous body substitute material, the prepared zwitter-ion in-situ gel remains stable for a long period of time after being planted into an eye, and has no obvious toxic reaction or inflammatory reaction.

Description

In-situ gel for vitreous substitute material and preparation method and application thereof

Technical field

The invention belongs to the technical field of gel preparation, and particularly relates to an in situ gel for a vitreous substitute material and a preparation method and application thereof.

Background technique

In view of the fact that the zwitterionic polymer has a large number of super-hydrophilic zwitterionic groups, it can form a super-ionic ionized layer, so it has unique biocompatibility and anti-biofouling properties, and can inhibit the adsorption of non-specific proteins. Bacterial adhesion and biofilm formation. These excellent properties make these materials increasingly used in antifouling coatings, antibacterial coatings, anticoagulant materials, biomedical diagnostics, drug delivery, gene delivery vehicles, separation membranes, and hull coatings.

At present, more studies are the phosphorylcholine (PC), sulfobetaine (SB), carboxylic acid betaine (CB) zwitterionic materials used to modify the surface of biomedical materials to achieve anti-nonspecific protein adsorption. . Among them, Jiang Shaoyi's research group and others have carried out a large number of anti-non-specific protein adsorption properties, mechanism and application of poly(sulfobetaine amphoteric polymethacrylic acid) and poly(carboxylic acid betaine methyl acrylate) materials. the study. According to Jiang Shaoyi et al., the adsorption of fibrin on the surface of poly(sulfobetaine amphoteric polymethacrylic acid) and poly(carboxylic acid betaine methyl acrylate) modified materials is less than 0.3 nanograms per square centimeter; and zwitterionics The polymer-modified vascular catheter material can effectively adsorb the protein to the surface of the material in 100% plasma and serum, inhibiting the occurrence of blood coagulation. At the same time, Jiang Shaoyi's research group also prepared a zwitterionic hydrogel. The in vitro cytotoxicity test showed that the gel was not cytotoxic; the gel was implanted under the skin of the mouse and collagen fibers were not formed around the gel within 3 months. bag. Therefore, these advantages provide potential for zwitterionic materials to be used as tissue scaffold materials and artificial organs in vivo.

However, no reports of zwitterionic polymers with in situ gels have been reported so far, and no zwitterionic in situ gels have been used for the study of vitreous fillers.

Invention disclosure

It is an object of the present invention to provide a kit for forming an ophthalmic zwitterionic in situ gel.

The kit for forming an ophthalmic zwitterionic in situ gel provided by the present invention comprises a zwitterionic polymer having a polyfluorenyl functional group represented by formula (I), a polymer represented by formula (II) (α-PEG-MA), and Solvent composition;

In the kit, the zwitterionic polymer having a polyfluorenyl functional group represented by the formula (I), the polymer represented by the formula (II) (α-PEG-MA), and the solvent are separately packaged;

Wherein the molar ratio of the fluorenyl group in the zwitterionic polymer having a polyfluorenyl functional group represented by the formula (I) to the carbon-carbon double bond functional group in the polymer represented by the formula (II) is 1:1. -5:1, preferably in a molar ratio of 1:1 to 3:1, most preferably in a molar ratio of 2:1; the solvent may be a buffer having a pH of 7.0 to 8.0, such as a phosphate buffer having a pH of 7.0 to 8.0. liquid. Specifically, it may be a phosphate buffer having a pH of 7.4 and a concentration of 0.02 to 0.2 mol/L.

Wherein, a and b in the formula (I) are integers greater than 1, preferably a is an integer of 100-600, b is an integer of 8-50; m and n are each greater than or equal to 1 and less than 5, and X is O. Or an NH group, Y is SO ₃ ^- or COO ^- , and the zwitterionic polymer represented by the formula (I) has a weight average molecular weight of from 1,000 Da to 1000 KDa, preferably a weight average molecular weight of from 10 kDa to 500 kDa.

z in the formula (II) may be an integer greater than 1, and preferably z is an integer greater than 20 and less than 200, further preferably 45 to 90, and the specific z value may be 45.

The concentration of the zwitterionic polymer having a polyfluorenyl functional group represented by the formula (I) in the kit may be 0.1-500 mg/mL (preferably 10-100 mg/mL), and the polymer represented by the formula (II) (α-PEG) The concentration of -MA) may be from 0.01 to 50 mg/mL (preferably from 0.5 to 5 mg/mL).

In use, the components in the kit for forming the ophthalmic zwitterionic in situ gel are mixed at room temperature for 5-40 minutes (preferably 10-20 minutes), and then the mixed solution is injected into the vitreous cavity (eg, It can be injected in situ by gas-liquid exchange method to form a zwitterionic in situ gel in situ.

It is still another object of the present invention to provide a zwitterionic in situ gel for use as an artificial vitreous and a method of preparing the same.

The zwitterionic in situ gel provided by the present invention is obtained by in situ crosslinking of a zwitterionic polymer having a polyfluorenyl functional group represented by the above formula (I) and a polymer represented by the formula (II) (α-PEG-MA). Prepared by the combined reaction.

The in-situ crosslinking reaction can be carried out in a solvent which may be a buffer having a pH of 7.0 to 8.0, such as a phosphate buffer having a pH of 7.0 to 8.0. Specifically, it may be a phosphate buffer having a pH of 7.4 and a concentration of 0.02 to 0.2 mol/L.

The in situ crosslinking reaction is carried out at room temperature.

The molar ratio of the fluorenyl group in the zwitterionic polymer having a polyfluorenyl functional group represented by the formula (I) and the carbon-carbon double bond functional group in the polymer represented by the formula (II) initially added in the in-situ crosslinking reaction is 1 : 1-5:1, preferably a molar ratio of 1:1 to 3:1, and most preferably a molar ratio of 2:1.

The zwitterionic polymer having a polyfluorenyl functional group represented by the formula (I) is added in the form of a solution having a polyfluorenyl zwitterionic polymer having a mass concentration of 0.1 to 500 mg/mL, preferably 10 to 100 mg/mL. .

The polymer (α-PEG-MA) represented by the formula (II) is also added in the form of a solution in which the mass concentration of α-PEG-MA is from 0.01 to 50 mg/mL, preferably from 1 to 5 mg/mL.

The volume ratio of the solution of the polyfluorenyl zwitterionic polymer represented by the formula (I) to the solution of the α-PEG-MA of the formula (II) is from 1:10 to 10:1.

The invention also provides the use of the above zwitterionic in situ gel in the preparation of artificial vitreous.

The zwitterionic in situ gel prepared by the invention is used for the vitreous substitute material to maintain long-term stability after being implanted into the eye, and has no obvious toxicity reaction and inflammatory reaction (mainly as a vitreous cavity) compared with the traditional artificial vitreous substitute material. Transparent, no turbidity appears, and the time of mixing in vitro is significantly shortened (only about 15min), which is more conducive to surgical operations.

DRAWINGS

Figure 1 is a schematic view showing the structure of a zwitterionic in situ gel prepared by the present invention.

2 is a synthetic route diagram of a polyfluorenyl zwitterionic polymer prepared according to the present invention.

Figure 3 is a nuclear magnetic resonance spectrum of a polyfluorenyl zwitterionic polymer prepared according to the present invention.

Figure 4 is a nuclear magnetic resonance carbon spectrum of a polyfluorenyl zwitterionic polymer prepared according to the present invention.

Figure 5 is a graph showing the storage modulus characterization data of different formulations of zwitterionic in situ gels prepared according to the present invention.

Figure 6 is a graph showing swelling data of zwitterionic in situ gels prepared in different formulations at 37 °C.

Figure 7 is a graph showing light transmittance test data of a zwitterionic in situ gel (molar ratio of sulfhydryl groups to carbon-carbon double bonds of 1:1) prepared by the present invention.

Figure 8 is a graph showing the results of cytotoxicity test of the zwitterionic polymer, α-PEG-MA and zwitterionic in situ gel prepared by the present invention.

Fig. 9 is a graph showing the test results of the zwitterionic in situ gel prepared by the present invention after filling the vitreous cavity of the rabbit eye.

The best way to implement the invention

The invention is illustrated by the following specific examples, but the invention is not limited thereto.

The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and biological materials are commercially available unless otherwise specified.

The polyfluorenyl zwitterionic polymer (poly(MPDSA-SH)) used in the following examples was prepared as follows: [3-(methacrylamido)propyl]dimethyl (3- Thiopropyl) ammonium hydroxide inner salt 0.8g, N, N'-bis (acrylic) cystamine 0.047g, dissolved in 4mL 75% ethanol solution, adding ammonium persulfate 15.7mg, N, N, 11 μL of N',N'-tetramethylethylenediamine, reacted at room temperature for 20 h, and then immersed in H ₂ O for 3 days to remove small molecules. Then, the obtained product was added to 30 mL of DTT solution for 1 hour, and after dialysis for 3 days, The final product is obtained after lyophilization.

The structure of the obtained polymer can be confirmed from the nuclear magnetic hydrogen spectrum of FIG. 3 and the nuclear magnetic carbon spectrum of FIG.

The product prepared by the above method, in the corresponding polymer of the formula (I), a is 100-600, b is 8-50, m is 3, n is 3, X is NH, and Y is SO ^3- . The polymer produced has a molecular weight of 10 KDa to 500 KDa.

The α-PEG-MA used in the following examples was prepared as follows:

A compound of the formula (II) wherein z is 45 is prepared by using a hydroxyl terminated PEG (number average molecular weight of about 2,000, supplied by Alfa-Aesar, Cat. No. B22181) as a starting material. The specific preparation method is as follows:

(1) 9.0 g of ethyl hydroxyethyl methacrylate (TCI, product number H0916) was dissolved in 75 mL of anhydrous diethyl ether, and 2.3 mL of phosphorus tribromide was added dropwise under ice bath, and the reaction was continued at room temperature for 3 hours. After 24 hours, ethyl bromomethacrylate was obtained.

(2) 20.0 g of terminal hydroxyl group PEG was dissolved in 300 mL of dichloromethane, 2.4 g of sodium hydride was added, and the mixture was stirred for 1 hour. 8 mL of the bromoethyl acrylate prepared above was added dropwise under an ice bath, and reacted at room temperature for 24 hours to obtain a compound of the formula (II).

Example 1. Preparation of zwitterionic in situ gel

Dissolve poly(MPDSA-SH) and α-PEG-MA (z is 45) in phosphate buffer solution with a concentration of 0.02mol/L at pH=7.4, respectively, and prepare a concentration of 100mg/mL, 1.6mg/mL. The solution is mixed in a volume ratio of 1:1 (the molar ratio of sulfhydryl groups to carbon-carbon double bonds in the mixed system is 2:1), and the in-situ cross-linking reaction is carried out at room temperature for 24 hours to obtain zwitterionic in situ coagulation. gum.

The corresponding zwitterionic in situ gel product can be prepared by adjusting the molar ratio of sulfhydryl groups to carbon-carbon double bonds in the mixed system to 1:1 and 3:2, respectively, according to the above method.

Example 2: Characterization of in vitro performance of zwitterionic in situ gel of the present invention

The gel prepared in Example 1 was used for the swelling, strength, and light transmittance performance test.

The experimental results are shown in Figure 5-7.

The performance characterization data of in vitro zwitterionic in situ gels showed that the equilibrium swelling ratio of the gel was relatively small at 37 °C and remained unchanged for 60 days (see Figure 6). The strength of the gel The zwitterionic polymer changes with the ratio of α-PEG-MA, and the higher the zwitterionic polymer content, the weaker the strength (see Figure 5). The light transmittance of the gel can be maintained above 90% (see Figure 7). The above results indicate that the zwitterionic in situ gel fully meets the basic requirements for vitreous substitute materials.

Example 3 Preparation of a zwitterionic in situ gel and cell safety experiment

1. Preparation of zwitterionic in situ gel

Dissolve poly(MPDSA-SH) and α-PEG-MA (z is 45) in phosphate buffer solution with pH=7.4 concentration of 0.2mol/L, respectively, and prepare a concentration of 100mg/mL, 1.6mg/mL. The solution is mixed in a ratio of 1:1 to 2:1 by volume (ie, the molar ratio of sulfhydryl to carbon-carbon double bonds is 1:1, 2:1, respectively), and the in-situ cross-linking reaction is carried out at room temperature. At 24 h, a zwitterionic in situ gel was obtained for cell experiments.

2. Cell line (human retinal pigment epithelial cells, RPE cells), routine culture, the culture medium is DMEM/F12=1:1 (purchased in Gibco) containing 10% fetal bovine serum, and transferred to cells of 3-6 passages. Used for experiments.

3. Take a sterile 12-well plate, add 2 mL of RPE cell suspension to each well (count to 500,000/mL), and incubate for 12 h in a 37 ° C 5% CO ₂ incubator, remove the culture solution, and add 2 mL of poly ( MPDSA-SH) or α-PEG-MA or a culture solution containing an in situ gel (wherein the concentration of poly(MPDSA-SH) is 5 wt%, the concentration of α-PEG-MA is 1.6 wt%, and the concentration of the gel is 10 wt%; set 6 replicates per group). The control group was added with 2 mL of the culture solution, and the culture was continued for 24 hours.

4. The cells were collected by trypsin digestion, and apoptosis was measured by BD PE Annexin V Apoptosis Detection Kit I. The results are shown in Fig. 8.

Cellular experimental data showed that although RPE cells were co-cultured with 1.6 wt% α-PEG-MA for 24 h, the proportion of apoptotic and necrotic cells was significantly increased compared with the control group, but RPE cells were 5 wt% poly ( MPDSA-SH) solution, 5 wt% or 10 wt% in situ gel co-culture for 24 h, compared with the control group, the proportion of apoptotic and necrotic cells did not change significantly. The above results indicate that the zwitterionic in situ gel prepared by the present invention has no obvious cytotoxicity.

Example 4: Animal intraocular filling safety test of zwitterionic in situ gel of the present invention

Three cyan rabbits were selected in the experiment, and the right eye was implanted into the in situ gel.

The animal experiment steps are as follows:

1. Anesthetize blue-blue rabbits.

2. Cut the semi-lateral bulbar conjunctiva along the limbus, and use a 19G scleral puncture knife to cut the perfusion hole and the glass-cut hole.

3, insert the perfusion head, vitrectomy head, remove all vitreous, continue to inject perfusate during the resection process, the perfusate formula is as follows: 500mL lactated Ringer's solution contains glucose 2g, dexamethasone 8mg, adrenaline hydrochloride 0.5mg, Botomycin 16,000 U.

4. The formulation of the gel solution is: 0.2 mol/L phosphate buffer (pH 7.4) containing 50 mg/mL of poly(MPDSA-SH), and 0.8 mg/mL of α-PEG-MA (thiol and double bond) The ratio of functional groups is 2:1). After mixing for 20 minutes at room temperature, the mixed solution is injected into the vitreous cavity by gas-liquid exchange method, and cross-linked in situ to form a zwitterionic in situ gel.

5. Suture the wound and inject 2 mg of triamcinolone acetonide into the conjunctiva.

The zwitterionic in situ gel material prepared by the invention can form a complete transparent gel in the vitreous cavity of the rabbit eye, and maintains good stability, and remains intact when removed from the vitreous cavity after two months. Sexuality and good transparency (see Figure 9B). Through fundus photo observation and fluoroscopy (see Figure 9A, C), after 3 months of gel filling, the vitreous cavity remained clear and transparent, and the retina and choroid were normal without any bleeding or exudation. A B-ultrasound examination of the gel-filled vitreous cavity for 2 months (see Figure 9D) showed no abnormal changes compared to the control eye, so the material did not cause significant damage to the retina and other tissues. Histopathological examination was performed 2 months after the gel-filled vitreous cavity (see Figure 9E). The structure of the retina was intact, no abnormal changes were observed, and no inflammatory cell infiltration was observed, reflecting that the material was effective against postoperative Inflammation occurs and does not cause significant toxicity to other tissues such as the retina.

Example 5: Animal intraocular filling safety test of zwitterionic in situ gel of the present invention

Three cyan rabbits were selected in the experiment, and the right eye was used to implant the in situ gel.

The animal experiment steps are as follows:

1. Anesthetize blue-blue rabbits.

4. The formulation of the gel solution is: 0.2 mol/L phosphate buffer (pH 7.4) containing 50 mg/mL poly(MPDSA-SH), and 1.6 mg/mL α-PEG-MA (sulfhydryl and double bonds) The ratio of functional groups is 1:1). After mixing for 15 minutes at room temperature, the mixed solution is injected into the vitreous cavity by gas-liquid exchange method, and cross-linked in situ to form a zwitterionic in situ gel.

The experimental results were the same as those in Example 4. The injected solution formed a complete transparent gel in the vitreous cavity of the rabbit eye, and no abnormal changes were observed in the retina and other tissues, and no inflammatory cell infiltration was observed.

In summary, the zwitterionic in situ gel material can form a complete gel in the vitreous cavity of the rabbit eye, and can be stable and transparent for a long time. After filling the vitreous cavity, the gel effectively inhibits inflammatory cell infiltration, and the vitreous cavity can remain transparent for a long time without any punctate turbidity. It has not caused any toxicity to the retina and other tissue structures and has been pathologically altered. Therefore, it has been proved by animal experiments that the filling of the gel in the vitreous cavity is non-toxic and safe.

Industrial application

The zwitterionic in situ gel prepared by the invention is used for the vitreous substitute material to maintain long-term stability after implantation in the eye, and has no obvious toxicity reaction and inflammatory reaction as compared with the traditional artificial vitreous substitute material.

Claims

A kit for forming an ophthalmic zwitterionic in situ gel, comprising a zwitterionic polymer represented by formula (I), a polymer represented by formula (II), and a solvent;

In the kit, the zwitterionic polymer represented by the formula (I), the polymer represented by the formula (II) and the solvent are separately packaged;

Wherein the molar ratio of the fluorenyl group in the zwitterionic polymer represented by the formula (I) to the carbon-carbon double bond functional group in the polymer represented by the formula (II) is 1:1 to 5:1; a buffer having a pH of 7.0-8.0;

Wherein, a and b in the formula (I) are integers greater than 1, m and n are integers greater than 1 and less than 5, X is an O or NH group, and Y is SO 3 - or COO - , (I The zwitterionic polymer shown has a weight average molecular weight of from 1000 Da to 1000 KDa.

In the formula (II), z is an integer greater than 1.
The kit according to claim 1, wherein a in the formula (I) is an integer of from 100 to 600, and b is an integer of from 8 to 50; a weight average molecular weight of the zwitterionic polymer represented by the formula (I) The amount is 10KDa-500KDa;

In the formula (II), z is an integer greater than 20 and less than 200, preferably 45 to 90.
The kit according to claim 1 or 2, characterized in that the molar ratio of the thiol group in the zwitterionic polymer represented by the formula (I) to the carbon-carbon double bond functional group in the polymer represented by the formula (II) The ratio is 1:1-3:1, preferably 2:1; the solvent is a phosphate buffer having a pH of 7.4 and a concentration of 0.02-0.2 mol/L.
The kit according to any one of claims 1 to 3, wherein the concentration of the zwitterionic polymer represented by the formula (I) in the kit is 0.1-500 mg/mL, and the polymer represented by the formula (II) The concentration is 0.01-50 mg/mL.
A method for preparing a zwitterionic in situ gel, comprising the steps of: cross-linking a zwitterionic polymer represented by formula (I) according to claim 1 or 2 with a polymer represented by formula (II) in situ The reaction is obtained;

The molar ratio of the fluorenyl group in the zwitterionic polymer represented by the formula (I) to the carbon-carbon double bond functional group in the polymer represented by the formula (II) is from 1:1 to 5:1.
The preparation method according to claim 5, wherein the in-situ crosslinking reaction is carried out in a solvent which is a buffer having a pH of 7.0 to 8.0, preferably a pH of 7.4 and a concentration of 0.02 to 0.2. Mol/L phosphate buffer.
The preparation method according to claim 5 or 6, wherein the molar group of the sulfhydryl group in the zwitterionic polymer represented by the formula (I) and the carbon-carbon double bond functional group in the polymer represented by the formula (II) The ratio is 1:1 to 3:1, and the most preferable molar ratio is 2:1.
The method according to any one of claims 5 to 7, wherein the zwitterionic polymer represented by the formula (I) is added in the form of a solution, and the zwitterionic polymerization represented by the formula (I) The mass concentration of the solution is 0.1-500 mg / mL, preferably 10-100 mg / mL;

The polymer represented by the formula (II) is added in the form of a solution, the mass concentration of the polymer solution represented by the formula (II) is 0.01-50 mg / mL, preferably 1-5 mg / mL;

The volume ratio of the solution of the zwitterionic polymer represented by the formula (I) to the solution of the polymer of the formula (II) is from 1:10 to 10:1.
A zwitterionic in situ gel prepared by the method of any of claims 5-8.
Use of the kit of any of claims 1-3 or the zwitterionic in situ gel of claim 9 for the preparation of an artificial vitreous.