WO2017007271A1 - Aptamer-coated microneedle-based diagnostic skin patch - Google Patents

Abstract

The present invention relates to an aptamer-coated microneedle-based diagnostic skin patch, and the patch of the present invention has advantages in that the size of a material is greatly smaller than an antibody material and a large number of aptamers can be bound to tip surfaces of a relatively large number of microneedles. In addition, the patch of the present invention allows the attachment of aptamers for several kinds of biomarkers at the same time, and thus can detect several kinds of materials at the same time (multiplexing), so that a microneedle tip-based skin path can also be used as a protein chip using an aptamer.

Description

Aptamer-Coated Microneedle Based Diagnostic Skin Patches

The present invention relates to aptamer coated microneedle based diagnostic skin patches.

Skin diseases represent a major healthcare challenge in the world today. With over one million skin cancers diagnosed each year in the United States (National Cancer Research Institute, www.cancer.gov), predicting and diagnosing skin diseases is important in terms of their management. Current diagnostic methods rely primarily on visual observation and biopsy. However, detection methods that rely on visual observation are not necessarily effective in diagnosing a skin condition or disease and do not detect risk or disease until clinical signs appear. In addition, invasive methods such as biopsies increase the likelihood of infection as well as trauma to the test subject. In addition, the method must be performed by a physician in order to be safe and usually does not usually provide abundant cell samples on the skin surface, the cells involved in the reaction.

Thus, noninvasive methods of diagnosing and monitoring skin conditions and diseases represent an important means for the care of patients and for evaluating the efficacy of existing and new therapeutics, skin care products and skin care regimens. In addition, the method can provide important information regarding the genetic predisposition of the test subject to the occurrence of skin disease, as well as specific genetic changes based on the skin condition of the test subject. Identifying these genetic changes can be important in identifying potential drug targets and preventive measures and in determining whether a person actually responds to a particular therapeutic agent, skin care product or regimen. In addition, detection and diagnostic methods are important for evaluating the safety of such treatments, products, and measures.

In addition, it has been reported that the composition of the substance of the skin is changed in various disease states as well as local skin diseases. It is known that various substances such as lipids, structural proteins, inflammatory substances, nucleic acids, and metabolites can be detected in the skin according to the disease state. In addition to atopic dermatitis, melanoma, and bacterial inflammation of the skin, biomarkers are being analyzed for various diseases such as Alzheimer's disease, Parkinson's disease, breast cancer, cardiovascular disease, diabetes, and drug addiction. However, in most cases very invasive skin biopsy is used. Iontophoresis, Microdialysis, Tape stripping, Ultrasound, Microneedle are used as non-invasive methods, but the efficiency is low (Paliwal et al., 2013 Diagnostic opportunities based on skin biomarkers.European journal of pharmaceutical sciences: official journal of the European Federation for Pharmaceutical Sciences 50: 546-556.

[Previous Patent Document]

Republic of Korea Patent Publication No. 1020120006945

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems and needs, and an object of the present invention is to provide a patch for use in the diagnosis of various diseases.

In order to achieve the above object, the present invention comprises the steps of: a) binding an amine group to an aptamer; b) attaching a silanol to the surface of the microneedle tip by plasma oxidation, and then adding a hydroxyl; Apylmer-coated microneedle comprising the step of silanizing with 3-glycidoxypropyltrimethoxysilane (3-GPTMS) to a group, and then bonding an amine group bonded to the aptamer to an epoxy group included in the 3-GPTMS. It provides a method for manufacturing a base skin diagnostic patch.

In addition, the present invention is treated with HNO ₃ on the surface of the polycarbonate microneedle, and then reduced to NaBH ₄ to finally combine the amine group, polyethylene glycol (PEG) is bonded to the carboxyl group at one end and the aptamer at the other end Provides a method for preparing aptamer-coated microneedle-based skin diagnostic patch comprising the step of combining using NHS (N-Hydroxysuccinimide), and EDC [1-ethyl-3- (3'-dimethylaminopropyl) carbodiimide] as a catalyst .

The present invention also provides an aptamer-coated microneedle-based skin diagnostic patch prepared by the method of the present invention.

In one embodiment of the present invention, the skin diagnostic patch is characterized in that the aptamer is attached to the microneedle tip surface and the diagnostic component can be attached to the end of the aptamer, but is not limited thereto. The diagnostic component is preferably a protein, peptide, DNA or RNA, but is not limited thereto.

Hereinafter, the present invention will be described.

The present invention provides a diagnostic skin that effectively detects various biomarkers present in intracellular interstitial cells by coating a microneedle surface of about 200 um in length that can penetrate the stratum corneum of the skin and lead to epidermis. It's a patch.

The types of biomarkers that can be collected in the intracellular interstitial tissue are secreted by the infecting bacteria or infectious organisms in various infectious diseases. For example, in case of malaria, malaria bacteria or proteins released by the bacteria are representative proteins. This is PfHRP2, and in the case of various viruses, the virus itself can be detected, and in the case of various degenerative brain diseases, for example, the Tau protein is increased in Parkinson's or Alzheimer's disease and can be used as a marker. Synuclein can also be used as an important diagnostic marker for Parkinson's disease, Alymoid beta can be used as a diagnostic marker for Alzheimer's disease, and various cancer markers can be used as a biomarker that can be collected.

In connection with the production of aptamer-coated microneedle of the present invention, various types of microneedles have been developed for effective delivery of drugs through brain-machine interfaces (BMIs) or skin and have been tested for measurement of EEG through actual scalp.

For example, Ami, Y et al, J of Micro / Nanolithography, MEMS, and MOEMS, published in 2011. 10 (1), 011503, March 2011 It can be seen that the successful development of the skin penetrating microneedle with a pointed tip with a thickness of 50 um x 200 um in length using polydimethylsiloxane (PDMS) (FIG. 1). .

In addition, microneedles or microprojection arrays of various materials and lengths are being manufactured. The types of chemicals used in the microneedle are as follows. Polyurethane (PU), Polypropylene (PP), Polyethylene (PE), Polystyrene (PS), Poly (methyl methacrylate) (PMMA), Polydimethylsiloxane (PDMS), Polycarbonate (PS), Liquid crystal polymer (LCP).

An aptamer is a method that detects a specific substance by using a three-dimensional structure of single strand DNA or RNA, which is similar to an antigen-antibody reaction, but because the substance is much smaller, aptamers can be attached to the ends or holes of microneedles. There is an advantage that it can.

In addition, aptamers for various biomarkers can be attached at once (similar to gene chips) to detect multiple kinds of materials at the same time (Multiplexing). In the case of a gene chip, a genome capable of detecting more than 10,000 DNA or RNA can be combined on a nail size chip, so the skin patch can also be used as a biomarker chip by activating a microneedle as an aptamer for various substances.

In the development of a skin diagnostic patch using aptamers, biomarkers present in the epilepsy of the epidermal layer need to be detected. To create a diagnostic skin patch that solves this problem with a microneedle, you can attach an aptamer to the microneedle as follows:

As can be seen through the present invention, in the present invention, a method for detecting a specific substance by using a three-dimensional structure of single strand DNA or RNA called aptamer is similar to an antigen-antibody reaction, but the size of the substance is much smaller and relatively large. The advantage is that a large number of aptamers can be bonded to the microneedle tip surface.

In addition, since aptamers for various biomarkers can be attached at one time, multiple substances can be detected at the same time (Multiplexing), and microneedle tip-based skin patches can be used as protein chips using aptamers.

1 is a structure according to the fabrication of the microneedle and the skin layer using PDMS,

2 is a microneedle and an example patch using the same;

3 is a method of attaching silanol to the surface of the microneedle tip by plasma oxidation, silanizing the hydroxyl group with 3-GPTMS, and then combining the amine group of PEG coupled with the aptamer to the epoxy group included in 3-GPTMS.

4 shows aptamers linked to PEG with Amine (NH2-) groups bound,

Figure 5 is a method of coating a polycarbonate microneedle surface with an aptamer bonded to one end of the PEG. (A) After treating HNO3 on the surface of PC microneedle, it is reduced to NaBH4 to finally bind the amine group. Thereafter, PEG, which combines a carboxyl group at one end with an aptamer at the other end, is treated with PC-NH2 along with EDC / NHS to complete the PEG-aptamer functionalized PC microneedle. (B) Identification of NH2-coupled PC via ATR-FTIR. (C) Analysis of the amount of aptamer attached to the surface of the PC using fluorescence tagged anti-sense oligonucleotide.

6 is a PC microneedle surface coated with PEG-aptamer.

   Hereinafter, the present invention will be described in more detail with reference to non-limiting examples. However, the following examples are intended to illustrate the present invention and the scope of the present invention is not to be construed as limited by the following examples.

Example 1: Microneedle  on the tip surface Aptamer  Attached Example of reaction

In order to avoid inaccuracy of diagnosis due to proteins that bind nonspecifically to the microneedle, the microneedle surface is treated with polyethylene glycol (PEG), and then aptamer is attached to the PEG end to construct the device.

A. PDMS microneedle Yes:

Silanol (SiOH) is formed through plasma oxidation of the surface of PDMS constituting the microneedle. By silanizing this Hydroxyl group with 3-glycidoxypropyltrimethoxysilane (3-GPTMS) having an epoxy group, aptamer-PEG can be formed on the surface of the microneedle by combining the amine group (Fig. 4) of PEG with an aptamer combined with this expoxy group. (FIG. 3).

B. Polycarbonate microneedle surface  Bound to one end of the PEG With aptamer  How to coat:

The surface of the PC microneedle was treated with nitric acid, followed by electrophilic substitution, and reduced nitro groups to prepare PC-NH ₂ with primary amine groups. The aptamer bound to PEG having a carboxyl group is bound thereto using N-Hydroxysuccinimide (NHS) and 1-ethyl-3- (3′-dimethylaminopropyl) carbodiimide (EDC) as a catalyst (FIG. 5A). That is, HNO3 is treated on the surface of the PC microneedle and then reduced to NaBH4 to finally bind the amine group. Thereafter, PEG, which combines a carboxyl group at one end with an aptamer at the other end, is treated with PC-NH2 along with EDC / NHS to complete the PEG-aptamer functionalized PC microneedle.

The binding of amine groups to polycarbonate was confirmed by the ATR-FTIR method (FIG. 5B), and the binding of aptamers bound to PEG by the second reaction was demonstrated by the method using fluorescence tagged oligonucleotide (FIG. 5C).

Through the two-step reaction described above, a skin patch for diagnosing a biomarker of the skin coated with aptamer attached to PEG was coated on the surface of the polycarbonate microneedle as shown in FIG. 6.

Claims (5)

1. a) binding an amine group to an aptamer; b) attaching a silanol to the surface of the microneedle tip with plasma oxidation, followed by 3-glycidoxypropyltrimethoxysilane (3-GPTMS) to the hydroxyl group. After silane treatment (silanizing), a method for producing a microneedle-based skin diagnostic patch coated with an aptamer comprising the step of bonding an amine group bonded to the aptamer to an epoxy group contained in the 3-GPTMS.
2. After treating HNO ₃ on the surface of polycarbonate microneedle, reducing it to NaBH ₄ to finally combine the amine group, polyethylene glycol (PEG), which combines a carboxyl group with an aptamer at one end, is NHS (N- Hydroxysuccinimide), and a method for producing aptamer-coated microneedle-based skin diagnostic patch comprising the step of binding using EDC [1-ethyl-3- (3'-dimethylaminopropyl) carbodiimide] as a catalyst.
3. An aptamer-coated microneedle based skin diagnostic patch prepared by the method of claim 1.
4. 4. The microneedle-based skin diagnostic patch of claim 3, wherein the skin diagnostic patch has an aptamer attached to the surface of the microneedle tip and a diagnostic component can be attached to the end of the aptamer.
5. 5. The microneedle based skin diagnostic patch of claim 4, wherein the diagnosable component is a protein, peptide, DNA or RNA.

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