WO2020235697A1

WO2020235697A1 - Improved method for manufacturing microneedle-based diagnostic skin patch coated with aptamer, and patch

Info

Publication number: WO2020235697A1
Application number: PCT/KR2019/005921
Authority: WO
Inventors: 손인식
Original assignee: 주식회사 넥스모스
Priority date: 2019-05-17
Filing date: 2019-05-17
Publication date: 2020-11-26

Abstract

The present invention relates to a method for manufacturing a microneedle-based diagnostic skin patch coated with an aptamer, the method comprising a step for treating the surface of a microneedle with a base to modify the surface with a carboxyl group, and binding an aptamer having an amine group. A patch according to the present invention is advantageous in that a large number of aptamers, which are much smaller in size than antibody materials, can be attached to a relatively large number of microneedle tip surfaces. In addition, since aptamers for various kinds of biomarkers can be attached at the same time, various kinds of materials can be detected simultaneously (multiplexing), and thus even a microneedle tip-based skin patch can be used as a protein chip using aptamers.

Description

Improved aptamer-coated microneedle-based diagnostic skin patch manufacturing method and its patch

The present invention relates to an improved aptamer-coated microneedle-based diagnostic skin patch manufacturing method and a patch manufactured by the method.

Skin diseases represent a major health care challenge in today's world. With over a million new skin cancer diagnoses each year in the United States (National Cancer Institute, www.cancer.gov), predicting and diagnosing skin disorders is important in terms of its management. Current diagnostic methods rely primarily on visual observation and biopsy. However, a detection method that relies on visual observation is not necessarily effective in diagnosing a skin condition or disease, and does not detect a 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 carried out by a physician in order to be safely practiced, and an abundant sample of cells on the skin surface, which are cells involved in the reaction in general, is not usually provided.

Thus, non-invasive methods of diagnosing and monitoring skin conditions and diseases represent an important means for the management 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 not only about the genetic predisposition of the test subject to the occurrence of skin diseases, but also the 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 prophylactic measures and determining whether a person actually responds to a particular therapeutic, skin care product or regimen. In addition, detection and diagnostic methods are important in evaluating the safety of such treatments, products and measures.

In addition, it has been reported that the composition of skin substances changes 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 are detected in the skin in various ways depending on the disease state. In addition to atopic dermatitis, melanoma, and bacterial inflammation of the skin, biomarker analysis of the skin is being performed in 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, etc. are used as non-invasive methods, but their 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).

[Prior patent literature]

Korean Patent Publication No. 1020120006945

The present invention solves the above problems and is conceived by necessity, and an object of the present invention is to provide an improved patch manufacturing method for use in diagnosis of various diseases.

Another object of the present invention is to provide a patch for use in diagnosis of various diseases.

In order to achieve the above object, the present invention is a microneedle-based skin diagnostic patch coated with an aptamer comprising the step of treating the surface of the microneedle with a base, modifying the surface with a carboxyl group, and binding an aptamer having an amine group. Provides a manufacturing method.

In one embodiment of the present invention, the microneedles are preferably made of polycarbonate, but are not limited thereto.

In another embodiment of the present invention, the base is preferably 2N sodium hydroxide, but is not limited thereto.

In another embodiment of the present invention, the bonding step is preferably to use NHS (N-Hydroxysuccinimide), and EDC[1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide] catalyst, but is not limited thereto.

In the present invention, a polycarbonate microneedle is sequentially coated with titanium and gold to prepare a coated polycarbonate microneedle, and an aptamer bonded to polyethylene glycol (PEG) having a thiol group A microneedle-based aptamer coated comprising the step of combining the titanium-and-gold-coated polycarbonate microneedle and the aptamer bonded to the polyethylene glycol (PEG) having a thiol group It provides a method of manufacturing a skin diagnostic patch.

In one embodiment of the present invention, the thickness of the coated gold is preferably 10 to 40 nm, but is not limited thereto.

In addition, the present invention provides a step of bonding an amine group to an aptamer; b) attaching silanol to the surface of the microneedle tip by plasma oxidation, and then 3-glycidoxypropyltrimethoxysilane (3) to the hydroxyl group. -GPTMS) after silane treatment (silanizing), aptamer-coated microneedle-based skin diagnostic patch manufacturing method comprising the step of bonding an amine group bound to the aptamer to an epoxy group included in the 3-GPTMS Provides.

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

Further, the present invention provides a microneedle-based skin diagnostic patch coated with an aptamer prepared by the method of the present invention.

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

The present invention will be described below.

The present invention is a diagnostic skin capable of effectively detecting various biomarkers present in the intercellular interstitial in the skin by coating the surface of a microneedle of about 200 um in length that can penetrate the stratum corneum of the skin and lead to epidermis with an aptamer. It's a patch.

The types of biomarkers that can be collected by being included in the intracellular interstitial in the skin are substances secreted by the infectious bacteria themselves or the infectious bacteria in various infectious diseases. For example, in the case of malaria, the malaria bacteria themselves or proteins released by the bacteria are representative proteins. PfHRP2 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, Tau protein is increased in Parkinson's or Alzheimer's disease and can be used as a marker, and Alpha- Synuclein can also be used as an important diagnostic marker for Parkinson's disease, Alymoid beta can also be used as a diagnostic marker for Alzheimer's disease, and various cancer markers can also be used as a biomarker capable of capturing.

Regarding the fabrication of the aptamer-coated microneedle of the present invention, various types of microneedles have been developed so far for effective drug delivery through brain-machine interfaces (BMIs) or through the skin, and tested for measurement of brain waves through the actual scalp.

For example, Ami, Y et al, J of Micro/Nanolithography, MEMS, and MOEMS published in 2011. 10 (1), 011503, March 2011 Referring to the paper, it can be seen that a microneedle for skin penetration with a sharp tip of 50 um in thickness x 200 um in length was successfully developed using polydimethylsiloxane (PDMS) (Fig. 1). .

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

Aptamer is a method of detecting a specific substance using the three-dimensional structure of single strand DNA or RNA. It is similar to the antigen-antibody reaction, but the size of the substance is much smaller so that a large number of aptamers can be bound to the end or inner hole of the microneedle. It has the advantage of being able to.

In addition, since aptamers for several types of biomarkers can be attached together (similar concept to a gene chip), several types of substances can be detected at the same time (Multiplexing). In the case of a gene chip, a genome capable of detecting more than 10,000 kinds of DNA or RNA can be combined on a nail-sized chip, so in theory, a skin patch can also be used as a biomarker chip by activating the microneedle as an aptamer for various substances.

In the case of the development of a skin diagnostic patch using an aptamer, there is a problem in that it must pass through the stratum corneum of about 20 μm since it is necessary to detect biomarkers present in the interstitial cells of the epidermal layer. To make a diagnostic skin patch that solved this problem through a microneedle, an aptamer can be attached to the microneedle as follows.

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

In addition, since aptamers for various types of biomarkers can be attached together, multiple types of substances can be detected at the same time (multiplexing), so a microneedle tip-based skin patch can also be used as a protein chip using an aptamer.

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

2 is an example of a microneedle and a patch using the same,

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

4 is an aptamer linked to PEG to which an amine (NH2-) group is bonded,

Figure 5 is a method of coating a polycarbonate microneedle surface with an aptamer bonded to one end of PEG. (A) After treatment with HNO ₃ on the PC microneedle surface, it is reduced with NaBH ₄ to finally bind the amine group. Afterwards, a carboxyl group at one end and PEG bound to an aptamer at the other end are treated with PC-NH2 together with EDC/NHS to complete a PEG-aptamer functionalized PC microneedle. (B) NH2-conjugated PC through ATR-FTIR. (C) Fluorescence tagged anti-sense oligonucleotide was used to analyze the amount of aptamer attached to the PC surface.

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

Figure 7 relates to an example using a gold coated polycarbonate microneedle, a PC microneedle is sequentially coated with 10 nm-thick titanium and 40 nm-thick gold through sputtering, and then PEG-aptamer is bonded to the microneedle surface through a Thiol-gold reaction. Let it.

FIG. 8 shows the results of testing the number of molecules of PEG-aptamer coated through thiol-gold bonding to a PC microneedle and stability at room temperature for about 40 days, showing that the number of PEG-aptamer-binding molecules increased in proportion to the gold coating thickness. It was confirmed, and it was confirmed that the binding was stably maintained at room temperature for a long period of time.

9 is a diagram showing a method of modifying the surface of a polycarbonate microneedle,

10 is a diagram showing a method of manufacturing an aptamer coated microneedle patch,

11 is a diagram showing a method of attaching an aptamer to silica nanoparticles including a dye,

12 is a diagram showing the fluorescence spectrum result,

13 is a diagram showing the TEM results of SiNP after attaching PEG-SiNP and aptamer,

14 is an example of confirming an analysis method through an in vitro experiment.

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

Example 1:Microneedle tip on the surface Aptamer Attached Reaction example

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

A. PDMS microneedle Yes:

Silanol (SiOH) is formed through plasma oxidation on the surface of PDMS constituting the microneedle. After silanizing this hydroxy group with 3-glycidoxypropyltrimethoxysilane (3-GPTMS) having an epoxy group, aptamer-PEG can be formed on the surface of the microneedle by binding the amine group of PEG (Fig. 4) bound to the aptamer to this expoxy group. (Fig. 3).

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

PC-NH ₂ is prepared by attaching a primary amine group by reducing nitro groups after electrophilic substitution on the surface of the PC microneedle by nitric acid treatment. Here, the aptamer bonded to PEG having a carboxyl group is bonded using N-Hydroxysuccinimide (NHS) and 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (EDC) as a catalyst (Fig. 5A). That is, after HNO3 is treated on the PC microneedle surface, it is reduced to NaBH4 and finally amine group is bound. Afterwards, a carboxyl group at one end and PEG bound to an aptamer at the other end are treated with PC-NH2 together with EDC/NHS to complete a PEG-aptamer functionalized PC microneedle.

The binding of the amine group to the polycarbonate was confirmed by the ATR-FTIR method (FIG. 5B), and the binding of the aptamer bonded to the PEG by the second reaction was verified by the method using a fluorescence tagged oligonucleotide (FIG. 5C).

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

C. Example using gold coated polycarbonate microneedle:

After sputtering the surface of the PC microneedle with titanium and gold in turn, the aptamer bound to the PEG having a thiol group was reacted with TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) for 1 hour at room temperature, and then aptamer-PEG By combining -TCEP with the prepared gold-coated PC microneedle at room temperature for 24 hours, it is possible to implement a diagnostic microneedle whose surface is functionalized with DNA aptamer (FIG. 7). The coating state of the surface was confirmed through a scanning electron microscope (SEM).

As a result of testing the number of PEG-aptamers bound to the PC microneedle surface and binding stability at room temperature, it was found that about 2 x 10 ¹⁰ aptamers were stably bound to the microneedle surface for more than 40 days (FIG. 8).

D. Improved Polycarbonate microneedle surface After modification With aptamer How to coat:

1) Examples of microneedle surface modification reaction.

Polycarbonate (PC) microneedles are treated with 2N sodium hydroxide (NaOH) aqueous solution, hydrolyzed at 70 °C for 30 minutes, washed with distilled water, treated with acetic acid, and reacted at room temperature for 15 minutes (Fig. 9 ).

Regarding this reaction condition, 2N, 5N, and 10N NaOH treatment resulted in no significant difference for each concentration condition, but at high concentration, surface damage appeared, so it was confirmed that 2N is the optimal concentration, and in the case of reaction time in 2N NaOH In the case of 30 minutes, the concentration of the attached aptamer was 21.61±1.18 nM, and in the case of 2 hours, the reaction efficiency decreased as the time increased to 15.92±1.06 nM, so the reaction was fixed for 30 minutes.

2) An example of an aptamer attachment method.

The aptamer is attached by treating the aptamer with amine groups using NHS (N-Hydroxysuccinimide) and EDC[1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide] catalyst to the PC microneedle whose surface is modified with COOH. Complete the PC microneedle patch.

The attached aptamer was verified through a method using fluorescence tagged oligonucleotide. It was confirmed through the above two-step reaction.

The aptamer sequence attached to the microneedle is 5'-C6 Amine-ACC GAC AGG GGA GCG CCT CGC ACT GAC TCG-3' (SEQ ID NO: 1).

Example 2: Microneedle By patch Capture Disease Biomarker Examples of analysis methods

In order to analyze the biomarkers collected by the microneedle patch, a method of using silica nanoparticles into which a dye is introduced, a method of developing color through gold nanoparticles, and a method of using quantum dots as a chromophore may be used, but the present invention is not limited thereto.

As an example of the analysis method, a method of preparing silica nanoparticles into which a dye is introduced includes the steps of: i) preparing silica nanoparticles including a dye ii) attaching an aptamer to the dye silica nanoparticles.

i) Silica nanoparticles (Dye-SiNP) containing dyes are prepared through reverse phase microemulsion method. That is, Ru(bpy) ₃ as a dye is mixed with Tetraethyl orthosilicate (TEOS), distilled water, cyclohexane, 1-hexanol, and Triton X-100 for 1 hour at room temperature, and then ammonia water is added and reacted at room temperature for 24 hours. After that, PEGylation is performed by adding TEOS and Tetraethyl ortho-silicate-Polyethylene glycol (PEG-TEOS). Then, avidin is bound using cyanobromide. After that, the aptamer to which biotin is attached is combined using avidin-biotin reaction. (Fig. 11)

ii) The aptamer-attached Dye-SiNP (Apt-SiNP) confirmed that the spectroscopic properties of the dye were preserved through fluorescence spectroscopy (FIG. 12), and the size of the particles after attachment of the aptamer using Transmission electron microscopy It was confirmed that the aptamer was attached through the increase. (Fig. 13)

The aptamer sequence attached to the dye silica nanoparticles of the present invention is 5'-Biotin-GCA CAC GCA GGC AGG CCG AGG GGG GAG CTG CT-3' (SEQ ID NO: 2).

Example 3: Example of confirmation of analysis method through in vitro experiment

Amyloid-β that was not collected after 2 hours after treatment with 100 ng of Amyloid-β (1-42) as a disease biomarker on a PC microneedle patch with aptamers that were surface-modified using different concentrations of NaOH. After washing (1-42), Apt-SiNP was treated at a concentration of 100 μg/mL, and the result was confirmed by fluorescent spectroscopy after 2 hours. As a result, it was confirmed that the most Amyloid-β (1-42) was collected in the PC microneedle patch subjected to surface modification by treatment with 2N NaOH (FIG. 14).

Claims

A method of manufacturing a microneedle-based skin diagnostic patch coated with an aptamer comprising the step of treating the microneedle surface with a base, modifying the surface with a carboxyl group, and binding an aptamer having an amine group.
The method of claim 1, wherein the microneedles are made of polycarbonate.
The method of claim 1, wherein the base is 2N sodium hydroxide.
The microneedle-based aptamer-coated microneedle according to claim 1, wherein the bonding is performed using a catalyst of NHS (N-Hydroxysuccinimide) and EDC[1-ethyl-3-(3′-dimethylaminopropyl) carbodiimide]. Skin diagnostic patch manufacturing method.
A microneedle-based skin diagnostic patch coated with an aptamer prepared by the method of any one of claims 1 to 4.
The microneedle-based skin diagnostic patch of claim 5, wherein the skin diagnostic patch has an aptamer attached to a surface of the microneedle tip, and a diagnostic component can be attached to an end of the aptamer.
The microneedle-based skin diagnostic patch of claim 5, wherein the diagnostic component is protein, peptide, DNA or RNA.