TW201714610A - Soluble microneedle for protein or peptide - Google Patents

Abstract

The present invention relates to a skin administration system capable of ensuring the stability of peptides or proteins and enhancing the delivery rate of peptides or proteins through the skin and, more particularly, to a microneedle comprising peptides or proteins.

Description

Soluble microneedles for protein or peptide delivery

The present invention relates to a soluble microneedle, a transdermal delivery system capable of stably delivering a protein or peptide to the skin, and a transdermal delivery system which improves the stability of an unstable protein or peptide.

In recent years, in order to improve the skin condition (for example, wrinkles, elasticity, etc.), growth factors (epidermal growth factor, EGF), human growth hormone (hGH), and transformation gowth factor α and β have been developed. (transforming growth factor TGF-α, -β), Fibroblast growth factor 1 and 2 (fibroblast growth factor FGF-1, -2), Keratinocyte growth factor (KGF), Hepatocyte growth factor (hepatocyte) Various proteins and peptides of growth factor, HGF), Platelet derived growth factor (PDGF), and the like, and various cosmetics containing such proteins and peptides have been developed and marketed.

For example, EGF is a wound healing substance that promotes the growth of granulation (granulation tissue) and promotes blood vessel regeneration in the process of natural healing of human wounds, and is capable of exerting effects on aging. Therefore, it has recently been carried out for the content containing EGF. The development of cosmetics.

However, in particular, such a growth factor has a short biological half life and lacks sustained stability. Therefore, it has been reported that it is modified when it is incorporated into an ordinary cosmetic dosage form, so that it is difficult to exert an effect. In order to improve the stability of such unstable proteins, an encapsulation method such as a microparticle or a liposome is sometimes used. However, in this method, since the particles and the liposome are bulky (tens to hundreds of nm, or several μm or more), it is difficult to directly absorb into the skin only by application through the skin.

Secondly, for such a protein for improving the skin condition, in most cases, it is difficult to increase the desired effect only by an increase in the content. That is, epidermal growth factor (EGF), human growth hormone, and the like are one of active protein components, and their activity is more important than the content, and even if the content is increased, it is difficult to have no activity. Get the desired results.

In addition, in order to exert its efficacy on the skin, in order to exert its efficacy through the actual product, it is necessary to pass through the stratum corneum and penetrate the epidermal layer to the dermis layer when the product is used, and it is necessary to be able to uniformly transmit to the entire body. The method of the skin. Conventionally, there has been a method of improving permeability by using a surfactant or the like. However, the effect of improving the permeability is negligible, and the skin barrier is softened.

Since the delivery of the drug through the skin is convenient to use, it is applied to various fields in various forms. Such drugs that pass through the skin are mainly transmitted to the systemic circulation through the skin, and in addition, drugs such as atopic therapeutic agents, whitening or wrinkle-improving cosmetics are delivered to the skin itself. Used for the purpose. Despite such convenience and functionality, there are various difficulties in transferring drugs through the skin due to the structure of the skin, and therefore, it is not easy to develop a drug that can pass through the skin. The stratum corneum of the skin consists of a brick structure and a mortar structure composed of keratin-rich keratinocytes; the mortar structure is composed of a neurosteroid A lipid such as ceramide, fatty acid or wax fills the structure between the keratinocytes. This structure acts as a barrier and has a very low permeability. Only low molecular structural components below 500 Da can be delivered to the skin by diffusion, and only substances with excellent lipid affinity can pass through the skin.

Due to such structural characteristics of the skin, in order to increase the lipid affinity of peptides having low lipid affinity, attempts have been made to increase absorption in the skin by introducing a chain of a certain length.

However, at this time, the molecular weight of the peptide is increased, so that there is a problem that it is substantially difficult to increase absorption in the skin.

Therefore, the inventors of the present invention have begun to study a method in which various peptides and protein components capable of improving skin effects can be delivered to the skin and the effect can be enhanced.

Problems to be solved by the invention

In view of the above circumstances, an object of the present invention is to provide a protein delivery system capable of stably transferring a plurality of protein components for improving a skin condition, particularly a growth factor, into a skin, a method of manufacturing the same, and utilization. The method by which the system delivers proteins, especially growth factors, into the skin.

Further, while improving the lipid affinity of the peptide, the problem of transdermal delivery of the peptide which is difficult to penetrate the skin due to its large molecular weight has been solved.

Further, there is provided a peptide administration system capable of delivering a plurality of peptides for improving a skin condition into the skin, a method of producing the same, and a method of transferring a peptide having low lipid affinity into the skin using the system.

Technical solution to the problem

In order to solve the above problems, the present invention provides a microneedle containing a protein or a peptide. More preferably, the substance forming the microneedle is dissolved in the skin. Therefore, when the microneedle is used on the skin, the microneedle is dissolved or collapsed. The solution enables stable delivery of the peptide to the skin.

The present inventors have studied various drug delivery systems, however, as described above, in any of the systems, it is possible to achieve a lipid affinity of a peptide having a low lipid affinity while stably passing through the skin even if the molecular weight is increased. Peptide delivery systems are not easy. The present inventors have, through various efforts, surprisingly found that by incorporating a peptide or a peptide derivative having an alkyl chain at the N-terminus into a soluble microneedle in the skin, it can be efficiently delivered into the skin, thereby completing this invention.

The polypeptide as a linker of a plurality of amino acids refers to a case where amino acids different from each other are long-joined together by a chemical bond called a peptide bond, and the above-mentioned polypeptide is also simply referred to as a peptide.

In order to achieve the above problems, the microneedles need to be soluble in the skin, and in order to form soluble microneedles, hyaluronic acid, sodium carboxymethyl cellulose (Na-CMC), vinyl pyrrolidone can be used. - water-soluble polymers such as vinyl acetate copolymer, polyvinyl alcohol, and polyvinyl pyrrolidone; Xylose, Sucrose, Maltose, Lactose, seaweed a sugar such as a sugar (Trehalose); or a mixture thereof. In particular, it is preferable to use Oligo-Hyaluronic acid or sodium carboxymethyl cellulose (Na-CMC, Sodium carboxymethyl cellulose) in consideration of the skin penetration strength of the microneedle, the dissolution rate in the skin, and the like. And a mixture of saccharide (Saccharide), more preferably trehalose, more preferably mixed with Glycerine described below. Preferably, the microneedle of the present invention may further comprise a plasticizer, a surfactant, a preservative, an anti-inflammatory agent or the like in addition to the above-mentioned components forming the microneedles.

As the plasticizer, for example, ethylene glycol (Ethylene glycol), propylene glycol (Propylene glycol), dipropylene glycol (Dipropylene glycol), butylene glycol (Butylene glycol), glycerin (Glycerine), or the like may be used alone or in combination. Polyol.

Preferably, in the microneedle of the present invention, the content of the peptide is from 0.01 to 20% by weight, more preferably from 0.1 to 5% by weight, based on the total weight of the microneedle production solution.

The peptide which can be used in the present invention may be a peptide composed of 3 to 10 amino acids, and preferably may have an alkyl group having 10 to 20 carbon atoms at the N-terminus of the above amino acid. Peptide. The molecular weight of the above peptide measured by gel permeation chromatography may be 200 to 3000 Da.

Preferably, the above peptide may be selected from the group consisting of a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, a palmitoyl tripeptide, a myristoyl tetrapeptide, a hexamethylene tetrapeptide. (Caprooyl tetrapeptide), myristoyl pentapeptide, palmitoyl pentapeptide, myristoyl hexapeptide, palmitoyl hexapeptide, palmitoyl heptapeptide or Any of a group consisting of a mixture.

For example, the above-mentioned tripeptide may preferably be a peptide of the peptide 3-(Pal-Lys-Val-Lys-OH), and the myristyl tetrapeptide may be a myridine-tetrazide-12 (Myr-Lys-Ala-Lys) -Ala-NH ₂ ), the hexyl quinone tetrapeptide may be Cap-Lys-Gly-His-Lys, and the myristyl pentapeptide may be myristyl pentapeptide-17 (Myr- Lys-Leu-Ala-Lys-Lys-NH ₂ ), the five peptides may be Pal-Lys-Thr-Thr-Lys-Ser-OH, and the nutmeg hexapeptide may be nutmeg Hexapeptide--16 (Myr-Ala-Asp-Leu-Lys-Pro-Thr), the six-peptide can be hexapeptide-12 (Pal-Val-Gly-Val-Ala-Pro-Gly), seven peptides can It is a peptide -18 (Pal-Tyr-Pro-Trp-Gln-Arg-Phe).

Further, the present invention provides a microneedle patch system for peptide administration (for delivery) to which the above microneedles are attached.

Further, the present invention provides a method for producing a microneedle comprising a peptide or a peptide derivative, the method comprising: an S1 step of producing a solution comprising the above peptide and a soluble substance in the skin; and an S2 step of injecting the solution into the microneedle mold; , S3 step of drying and separating the microneedles from the above mold.

Preferably, the above microneedles may comprise peptides or peptide derivatives having a molecular weight of from 200 to 3000 Da. Further, the present invention provides a transdermal administration method of a peptide having improved skin permeability of a peptide, which is characterized in that the microneedle of the present invention is used.

Further, the present invention provides the use of a microneedle containing a large molecular weight peptide for the improvement of wrinkles.

According to still another embodiment of the present invention, there is provided a microneedle comprising microparticles containing a protein or a peptide, and more preferably, a substance forming the microneedle is dissolved in the skin, when the microneedle is used on the skin, through the microneedle Dissolving or disintegrating, the microparticles contained inside the microneedles can rapidly diffuse into the skin.

The microparticles contained inside contain a polymer that forms a hydrophobic core, so that the protein or peptide can be stably delivered to the skin.

The "protein or peptide" used herein is not necessarily used in distinction but is used in a broad sense including an amino acid polymer.

As is well known, in general, a protein refers to an amino acid polymer having a molecular weight larger than a peptide, and when the number of polymerization of an amino acid is 50 or less, it is called a peptide. However, in this context, it is not necessary to define and explain the number of polymerizations of amino acids.

The inventors have studied various drug delivery systems and, through various efforts, have surprisingly found that by immersing microparticles containing proteins in a soluble microneedle, it is possible to efficiently deliver proteins or peptides into the skin. The invention has been made. The protein-encapsulated microparticles are immersed in the soluble microneedles, and when applied to the skin, the micro-needle can make the protein painlessly penetrate into the skin, and the micro-needle is dissolved by the moisture in the skin, and the protein-containing microparticles are encapsulated. Can be delivered to the skin.

As used herein, "protein-encapsulated microparticles" means a state in which a protein is located inside a microparticle and indicates a state in which the protein is completely surrounded by the microparticle. For example, the cross section of the microparticles encapsulated with protein may have a morphology as shown in FIG. 6 of the present specification, but it is merely an example.

"Immersion" means a state of being contained, which includes not only a state inside the microneedle but completely isolated from the external environment, but also a state in which a part of the microparticles is exposed on the surface of the microneedle. "Inclusion" and "impregnation" can be used in the same meaning herein. "Immersed in a microneedle" is not only understood to be completely contained inside the microneedle, but also understood to be included in the microneedle in the form in which the microneedles and the microparticles can be transferred together when the microneedle is used on the skin. A collection of all forms.

The release of the protein component by the microparticles delivered into the skin, in particular the release of the growth factor, can therefore be effectively delivered into the skin. The growth factors used herein may comprise growth hormone.

In order to achieve the above problems, the microneedles need to be soluble in the skin, and in order to form soluble microneedles, hyaluronic acid, sodium carboxymethyl cellulose (Na-CMC), vinyl pyrrolidone can be used. - water-soluble polymers such as vinyl acetate copolymer, polyvinyl alcohol and polyvinyl pyrrolidone; Xylose, Sucrose, Maltose, Lactose, seaweed a sugar such as a sugar (Trehalose); or a mixture thereof. In particular, it is preferable to use Oligo-Hyaluronic acid or sodium carboxymethyl cellulose (Na-CMC, Sodium carboxymethyl cellulose) in consideration of the skin penetration strength of the microneedle, the dissolution rate in the skin, and the like. And a mixture of saccharide (more preferably trehalose), more preferably mixed with glycerin (Glycerine) as described below. Preferably, the microneedle of the present invention may contain a plasticizer, a surfactant, a preservative, an anti-inflammatory agent and the like in addition to the above-mentioned components containing a protein, particularly a microparticle containing a growth factor, and a microneedle. Among them, the above-mentioned plasticizer, surfactant, preservative, anti-inflammatory agent and the like can be used not only as the components described herein, but also all components generally used in the art.

In the present invention, the substance which forms the fine particles together with the above-mentioned protein must be a substance which can stably cause the structural deformation of the protein during the production of the microneedle and which can be stably contained. In particular, the substance forming the above-mentioned fine particles needs to be capable of forming a hydrophobic core to stably supply the protein without structural deformation of the protein.

As the substance for forming such fine particles, a polymer capable of forming a hydrophobic core can be used, and as a sustained-release polymer, polylactide, polyglycolide, poly(lactide-co-glycolide) can be used singly or in combination. ), polyanhydride, polyorthoester, polyether ester, polycaprolactone, methoxy polyethylene glycol-b-polycaprolactone (MPEG-PCL), polydecylamine, Biodegradable polymers such as polybutyric acid, polyvaleric acid, polyurethane or copolymers thereof; and polyacrylate, ethylene-vinyl acetate polymer, acrylic acid substituted cellulose acetate, non-degradable polyurethane a non-biodegradable polymer such as ester, polystyrene, polyvinyl chloride, polyvinyl fluoride, polyvinylimidazole, chlorosulphonate polyolefins, polyethylene oxide or a copolymer thereof, but the present invention is not Limited by this.

When considering the stability of the protein, particularly the growth factor, and the release property in the skin, as the above-mentioned hydrophobic core-forming polymer, polylactide, polyglycolide, and poly(propylene) can be preferably used. A mixture of any one or more of ester-co-glycolide and methoxypolyethylene glycol-b-polycaprolactone (MPEG-PCL).

Such microparticles may be of a matrix type or a reservoir type within a range in which the object of the present invention can be attained.

The microparticles which can be used in the present invention can be produced by various methods known in the art to which the present invention pertains. For example, fine particles which can be used in the present invention can be produced by a Solvent exchange method, a Solvent evaporation method, a Membrane dialysis method, a Spray drying method, or the like. For example, the method described in the Journal of Controlled Release, 70, 1-20, 2001 and International Journal of PharmTech Research, 3, 1242-1254, 2011 can be utilized. It can preferably be produced by a conventional emulsification and solvent evaporation method.

Preferably, the microparticles of the present invention have a diameter of from 0.01 to 10 μm. If the size of the particles exceeds 10 μm, the strength of the needle is weakened when immersed in the microneedles, so that it is difficult to penetrate the skin. The diameter of the microparticles of the present invention is measured by a laser light scattering (LLS) method, for example, using a Zetasizer ^{2000TM from} Malvern.

Preferably, in the fine particles of the present invention, the content of the protein or peptide is from 0.01 to 20% by weight, more preferably from 0.1 to 5% by weight, based on the total weight of the fine particles. Further, in the microneedle of the present invention, the content of such fine particles is preferably 0.05 to 10% by weight, more preferably 0.1 to 5% by weight based on the total weight of the microneedles.

The protein which can be used in the present invention is particularly preferably a growth factor or a growth hormone. The above growth factor or growth hormone is a protein involved in the growth, proliferation and differentiation of cells, especially in consideration of the matching with selective tissues or organs, and the modification of protein structure induced during delivery, etc. A suitable transfer body or delivery method. The present invention has confirmed through a large number of research results that in the transfer of proteins, particularly growth factors and/or growth hormones, microparticles are used in microneedles, growth factors and growth hormones. The delivery is valid.

The growth factor may be selected from the group consisting of bone morphogenetic protein (BMP), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and nerve. Growth factor (NGF), epidermal growth factor (EGF), insulin like growth factor (IGF), transforming growth factor (trans-forming growth factor-α and -β, TGF- α,-β), brain-derived neurotrophic factor (BDNF), platelet derived growth factor (PDGF), placental growth factor (PlGF), hepatocyte growth factor ( Hepatocyte growth factor (HGF), fibroblast growth factor 1 and 2 (FGF-1,-2), keratinocyte growth factor (KGF) and its analogues Any one or more.

The analogs used herein may include a case of 80% sequence homology with the above protein, preferably a protein analog having 85% sequence homology, more preferably 90% sequence homology. Protein analogs.

Further, the present invention provides a microneedle patch system for protein administration (for delivery) to which the above-described microneedles are attached. Preferably, one embodiment of the invention provides a method of transdermal administration of a protein or peptide in cosmetic form.

The present invention provides a method for producing a microneedle containing a peptide or a protein, the method comprising: an S1 step of producing a solution containing the above peptide or protein and a soluble substance in the skin; an S2 step of injecting the solution into the microneedle mold; and drying And the step S3 of separating the microneedle from the mold, wherein the step S1 further comprises the step of encapsulating the peptide or protein into the microparticle, wherein the step of encapsulating the peptide or protein into the microparticle is utilized to form a high hydrophobic core. Molecules incorporate peptides or proteins into the interior of the microparticles, thereby improving protein structure instability or protein aggregation.

Further, the present invention provides a transdermal administration method of a protein having a high skin permeation amount and excellent stability, which is characterized in that the microneedle of the present invention is used.

Further, the present invention provides a use of a microneedle for improving wrinkles, wherein the microneedle comprises microparticles containing a protein, preferably a growth factor or a growth hormone, more preferably EGF, TGF-β or hGH.

According to an embodiment of the present invention, there is provided a method of attaching a microneedle comprising a peptide having a molecular weight of 200 to 3000 Da to the skin, thereby injecting the peptide into the skin.

According to one embodiment of the invention, there is provided the use of a microneedle comprising a peptide having a molecular weight of from 200 to 3000 Da for the improvement of skin wrinkles.

According to an embodiment of the present invention, there is provided a method of attaching a microneedle comprising microparticles encapsulated with a growth factor to a skin to inject the above growth factor into the skin.

According to one embodiment of the invention, there is provided the use of a microneedle comprising microparticles encapsulated with growth factors for the improvement of skin wrinkles.

Effect of the invention

According to the present invention, there is provided a microneedle which enhances the skin permeability of a peptide having a large molecular weight.

Further, according to the present invention, there is provided a microneedle for transdermal administration comprising a peptide having improved skin permeability. Further, the present invention provides a transdermal administration method of a peptide characterized by the use of such a microneedle.

According to the present invention, there is provided a microneedle for transdermal administration of a protein which ensures stability of a protein, particularly a growth factor, and which has an increased skin permeation amount. Further, the present invention provides a microneedle for transdermal administration which does not cause deformation of a protein structure and can be stably delivered to the skin.

According to the present invention, there is provided a protein transdermal delivery system which does not cause aggregation between proteins when incorporated into a common cosmetic material dosage form, and which can stably deliver proteins into the skin.

Further, according to the present invention, there is provided a transdermal administration method of a growth factor characterized by the use of such a microneedle.

In order to facilitate an understanding of the present invention, a detailed description of the embodiments will be given below. However, various modifications may be made to the embodiments of the invention, and the scope of the invention is not limited to the embodiments described below. The embodiments of the present invention are provided to more fully illustrate the present invention by those skilled in the art.

<Manufacture of carrier-loaded microparticles>

First, 1 g of polylactic acid-glycolic acid copolymer (PLGA) was dissolved in 10 mL of dichloromethane (Methylene Chloride). A solution of 200 mg of polypeptide (epidermal growth factor, EGF) dissolved in 2 mL of purified water was slowly added to the PLGA solution to prepare a first W/O emulsion. The W/O emulsion prepared for the first time was added to a 0.2% aqueous solution of polyvinyl alcohol (100 mL) while stirring. The W/O/W double emulsion thus produced was stirred at room temperature for 24 hours, and methylene chloride as an organic solvent was evaporated to produce fine particles containing EGF. The residual dichloromethane was completely removed by a rotary evaporator, and the water was evaporated together with an organic solvent, and concentrated to a concentration of 0.2% of the total amount of EGF. As a result of analysis by ELISA kit, the EGF content was 0.21%, and the average size of the microparticles was 350 nm as a result of analysis by a particle size analyzer.

<Manufacture EGF-loaded micro-needle>

As shown in Table 1 below, EGF (added in solution form) or soluble microneedles impregnated with EGF was produced. The contents of Table 1 below are expressed in % by weight.

Table 1

Specifically, a soluble microneedle impregnated with EGF was produced as follows. An EGF solution was prepared by dissolving Oligo-HA (oligohyaluronic acid), Na-CMC (sodium carboxymethylcellulose), and trehalose in purified water, and adding glycerin, HCO-40, and EGF. The produced EGF solution was cast into a silicone microneedle mold, centrifuged at 3000 rpm for 10 minutes, and the solution was filled with a micro mold. After filling the solution, it was dried in a drying oven (70 ° C) for 3 hours. Finally, the microneedle was separated from the silicone mold by an adhesive film.

Specifically, soluble microneedles impregnated with EGF microparticles (EGF-MP) were produced as follows. A solution was prepared by dissolving Oligo-HA (oligohyaluronic acid), Na-CMC (sodium carboxymethylcellulose), and trehalose in purified water, and adding glycerin, HCO-40, and EGF microparticles (EGF 0.2%). The produced solution was cast to a silicone microneedle mold, and centrifuged at 3000 rpm for 10 minutes to fill the micro mold with a solution. After filling the solution, it was dried in a drying oven (70 ° C) for 3 hours, and the microneedle was separated from the silicone mold by an adhesive film.

<EGF Oil-in-Water Cream>

In order to compare the skin permeation amount of EGF immersed in the microneedles, in the comparative example, EGF was immersed in a common oil-in-water emulsion type for comparison. The following contents are expressed in % by weight.

Table 2

<drug release behavior>

The EGF release of the microneedle fabricated above was evaluated using a Franz diffusion cell equipped with pig skin (see Fig. 2). As the acceptor solution, a PBS solution containing 30% by weight of DPG was used. The content of EGF in pig skin tissue and receptor solution was measured over time using a Franz diffusion cell and using an ELISA kit. EGF cream was applied to the pig skin, or a microneedle impregnated with EGF or EGF-MP was applied to compare the skin permeation of the peptide over time. After the microneedle was soaked into the skin of the pig to dissolve (attachment time: 2 hours, temperature: 32 ° C), the microneedle was removed. The porcine skin in which EGF was absorbed by the microneedle was placed in a Franz diffusion cell, and the behavior of EGF released from the pig skin to the receptor solution over time was confirmed, and the results are shown in Fig. 3. As shown in Fig. 3, EGF and EGF-MP immersed in the microneedles penetrate the skin directly through the microneedle, and the permeation amount thereof is 1 μg or more, and the skin exhibits about 500 times or more of the skin compared with the cream. Throughput.

<Wrinkle improvement effect>

EGF cream, microspheres impregnated with EGF and EGF-MP were used daily at the wrinkles of the corners of the eyes. After 12 weeks, wrinkle improvement was confirmed by silicone replica and wrinkle image analysis (N=20). ), the results are shown in Fig. 4. Compared with the EGF cream, the micro-needle impregnated with EGF showed an excellent improvement effect, and in particular, the micro-needle impregnated with EGF-MP showed an outstanding wrinkle improvement effect. This is the effect that EGF is effectively delivered to the skin through the microneedle (MN) impregnated with EGF-MP, and EGF is released from the EGF-MP delivered to the skin in a stable structure, thereby making the wrinkle improvement effect outstanding. .

<EGF Stability Analysis (SEC)>

It was confirmed by SEC (size exclusion chromatography) whether or not the structure of EGF was modified when EGF immersed in the microneedles was released.

When the EGF itself is immersed in the microneedle and delivered to the skin, the EGF structure aggregates to cause deformation, and the aggregation peak is relatively increased, and when it is stably encapsulated into the microparticles and immersed in the microneedles, it is transmitted to the skin. Aggregation does not occur afterwards, showing results similar to those of the standard substance.

From this, it can be seen that when a polypeptide or a protein such as EGF is impregnated into the microneedle and delivered to the skin, the microparticles stably encapsulated can be efficiently transdermally delivered to the skin in a stable structure. .

<Making peptide microneedles>

As shown in Table 3 below, peptides (added as a solution) or soluble microneedles impregnated with peptide were produced. The contents of Table 3 below are expressed in % by weight.

table 3

Specifically, a soluble microneedle impregnated with a peptide (myristyl tetrapeptide-6) was produced as follows.

Oligo-HA (oligohyaluronic acid), Na-CMC (sodium carboxymethylcellulose) and trehalose are dissolved in purified water, and glycerin, HCO-40 and peptide solution (peptide 10%, DPG 90%) are added. A solution in which a peptide is dispersed (DPG: dipropylene glycol). The prepared peptide dispersion solution was cast into a silicone microneedle mold, and centrifuged at 3000 rpm for 10 minutes to fill the micro mold with a solution. After filling the solution, it was dried in a drying oven (70 ° C) for 3 hours. Finally, the microneedle was separated from the silicone mold by an adhesive film.

<Peptide oil-in-water cream type>

In order to compare the skin permeation amount of the peptide impregnated in the microneedles, in the comparative example, the peptides were immersed in a common oil-in-water emulsion. The following contents are expressed in % by weight.

Table 4

<drug release behavior>

The amount of skin permeation of the peptide released by the microneedle prepared above was compared using a Franz diffusion cell containing pig skin (see Fig. 2). The acceptor solution used a PBS solution containing 30% by weight of DPG.

That is, the content of the peptide in the porcine skin tissue and the receptor solution was measured over time using a Franz diffusion cell and using liquid chromatography.

As shown in Fig. 3, EGF and EGF-MP immersed in the microneedles penetrate the skin directly through the microneedle, and the permeation amount thereof is 1 μg or more, and the skin exhibits about 500 times or more of the skin compared with the cream. Transmittance

Peptide cream was applied to the pig skin, or a microneedle impregnated with the peptide was applied to compare the skin permeation of the peptide over time. After the microneedle was soaked into the skin of the pig to dissolve (attachment time: 2 hours; temperature: 32 ° C), the microneedle was removed. The porcine skin in which the peptide was absorbed by the microneedle was placed in a Franz diffusion cell, and the behavior of the peptide released from the pig skin to the receptor solution over time was confirmed, and the results are shown in Fig. 7.

As shown in Fig. 7, the peptide-containing cream has a skin permeation amount of about 0.1 μg, which is minimal, and the peptide impregnated in the microneedle penetrates directly through the skin through the microneedle, and the permeation amount thereof is 15 μg or more, and the cream In comparison, it shows a skin permeation amount of about 100 times or more.

<Wrinkle improvement effect>

The peptide cream and the peptide-impregnated microneedles were used daily at the corner wrinkles, and after 12 weeks, the degree of wrinkle improvement (N=20) was confirmed by a silicone replica and a wrinkle image analysis method.

Compared with the peptide cream, the microneedle impregnated with the peptide showed an excellent improvement effect of more than 5 times, and it was confirmed that the peptide was effectively permeated into the skin by the microneedle, so that the wrinkle improvement effect was outstanding.

Industrial applicability

The present invention can be applied to cosmetics and pharmaceutical uses for improving skin wrinkles.

The microneedles of the present invention can provide an excellent effect of reducing skin wrinkles.

The present invention has been disclosed in the above embodiments, and it is not intended to limit the present invention. Any one of ordinary skill in the art can make some modifications and refinements without departing from the spirit and scope of the present invention. The scope of the invention is defined by the scope of the appended patent application.

no

The following drawings, which are included in the specification, are used to illustrate the preferred embodiments of the present invention, and are used together with the foregoing description to provide a more profound understanding of the technical idea of the present invention. The disclosure discloses the invention.

Fig. 1 is a view showing an example of various methods of manufacturing a microneedle of the present invention. The soluble microneedles can be produced by solution casting, the solution can be cast into a mold, and the solution can be dried by filling the solution with a vacuum and/or centrifuge to a small mold. And made. As a material for forming the microneedle structure, a general synthetic water-soluble polymer and a natural water-soluble polymer can be used.

Figure 2 is a Franz diffusion cell for evaluating the drug release behavior of the microneedles of the present invention.

Fig. 3 is a graph showing the results of evaluation of evaluation of EGF release of microneedles by a Franz diffusion cell equipped with pig skin.

4 is an experimental result showing the degree of wrinkle improvement exhibited by the microneedle (EGF microneedle) impregnated with the EGF solution of the present invention and the microneedle (EGF-MP microneedle) impregnated with EGF microparticles after long-term use in wrinkles of the corners of the eye. .

Fig. 5 is a graph showing the results of the implementation of EGF stability analysis (SEC, Size Exclusion Chromatography). 5A shows the SEC data of the EGF reference material, and FIG. 5B shows the SEC data of the EGF released by the microneedle.

Fig. 6 is a schematic view exemplarily showing the morphology of fine particles.

Fig. 7 is a graph showing the results of evaluation of evaluation of peptide release of microneedles by a Franz diffusion cell equipped with pig skin.

8 is an experimental result showing the degree of wrinkle improvement exhibited by the microneedle (peptide microneedle) impregnated with the peptide solution of the present invention and the microneedle (peptide microneedle) impregnated with the peptide for a long period of time after wrinkles around the corner of the eye.

Claims (26)

A microneedle containing a peptide or a protein. The microneedle according to claim 1, wherein the peptide is a peptide having an N-terminal alkyl group having 10 to 20 carbon atoms. The microneedle according to claim 1, wherein the peptide has a molecular weight of 200 to 3000 Da. The microneedle according to claim 1, wherein the peptide is composed of a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, a palmitoyl tripeptide, a myristoyl tetrapeptide, and Caprooyl tetrapeptide, Myristoyl pentapeptide, Palmitoyl pentapeptide, Myristoyl hexapeptide, Palmitoyl hexapeptide, Palmitoyl Any one selected from the group consisting of heptapeptide) and mixtures thereof. The microneedle according to claim 1, wherein the substance forming the microneedle is dissolved in the skin. The microneedle according to claim 5, wherein the substance forming the microneedle is hyaluronic acid, sodium carboxymethyl cellulose (Na-CMC), vinyl pyrrolidone-vinyl acetate. Copolymer, polyvinyl alcohol, polyvinyl pyrrolidone, sugars or mixtures thereof. The microneedle according to claim 5, wherein the microneedle further comprises a plasticizer in addition to the substance forming the microneedle. The microneedle according to claim 1, wherein the microneedle comprises a microparticle, and the peptide or the protein is encapsulated inside the microparticle. The microneedle according to claim 8, wherein the substance forming the microneedle is dissolved in the skin, and the microparticles comprise a polymer forming a hydrophobic core. The microneedle according to claim 9, wherein the substance forming the microneedle is hyaluronic acid, sodium carboxymethyl cellulose (Na-CMC, sodium carboxymethyl cellulose), vinyl pyrrolidone-acetic acid A vinyl ester copolymer, a polyvinyl alcohol, a polyvinyl pyrrolidone, a saccharide or a mixture thereof. The microneedle according to claim 9, wherein the hydrophobic core-forming polymer is any one selected from the group consisting of a biodegradable polymer, a non-biodegradable polymer, and a mixture thereof; the biodegradation The polymer is composed of polylactide, polyglycolide, poly(lactide-co-glycolide), polyanhydride, polyorthoester, polyether ester, polycaprolactone, methoxypolyethylene glycol. Any one selected from the group consisting of -b-polycaprolactone (MPEG-PCL), polydecylamine, polybutyric acid, polyvaleric acid, polyurethane, and copolymers thereof; and the non-biodegradable The polymer is made of polyacrylate, ethylene-vinyl acetate polymer, acrylic acid substituted cellulose acetate, non-degradable polyurethane, polystyrene, polyvinyl chloride, polyvinyl fluoride, polyvinylimidazole, chlorosulfonation polymerization. Any one or more selected from the group consisting of chlorosulphonate polyolefins, polyethylene oxide, and copolymers thereof. The microneedle according to claim 11, wherein the hydrophobic core-forming polymer is at least one of polylactide, polyglycolide, and poly(lactide-co-glycolide) and methoxy A mixture of polyethylene glycol-b-polycaprolactone (MPEG-PCL). The microneedle according to claim 8, wherein the microparticles are of a matrix type or a reservoir type. The microneedle according to claim 8, wherein the microparticles have a diameter of 0.01 to 10 μm. The microneedle of claim 8, wherein the protein contained in the microparticle is a growth factor. The microneedle according to claim 15, wherein the growth factor is a bone morphogenetic protein (BMP), a fibroblast growth factor (FGF), or a vascular endothelial growth factor (VEGF). Growth factor, VEGF), nerve growth factor (NGF), epidermal growth factor (EGF), insulin like growth factor (IGF), transforming growth factor (trans-forming growth factor- α and -β, TGF-α, -β), brain-derived neurotrophic factor (BDNF), platelet derived growth factor (PDGF), placental growth factor (PlGF) ), hepatocyte growth factor (HGF), fibroblast growth factor 1 and 2 (FGF-1,-2), keratinocyte growth factor (KGF), and mixtures thereof Any one of the selected groups. The microneedle according to claim 16, wherein the growth factor is epidermal growth factor (EGF). A method for producing a microneedle containing a peptide or a protein, comprising: an S1 step of producing a solution comprising the peptide or protein, and a soluble substance in the skin; an S2 step of injecting the solution into a microneedle mold; and drying and A microneedle is separated from the microneedle mold by the S3 step. The method according to claim 18, wherein the soluble substance in the skin is hyaluronic acid, sodium carboxymethyl cellulose (Na-CMC), vinylpyrrolidone-vinyl acetate copolymerization. , polyvinyl alcohol, polyvinyl pyrrolidone, sugars or mixtures thereof. The production method according to claim 18, wherein the peptide is a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, a palmitoyl tripeptide, a myristoyl tetrapeptide, or a Caprooyl tetrapeptide, Myristoyl pentapeptide, Palmitoyl pentapeptide, Myristoyl hexapeptide, Palmitoyl hexapeptide, Palmitoyl Any one selected from the group consisting of heptapeptide) and mixtures thereof. The manufacturing method according to claim 18, wherein the step S1 further comprises a step of encapsulating a peptide or a protein into a microparticle; and encapsulating a peptide or a protein into a microparticle in the step, using A polymer forming a hydrophobic core contains the peptide or the protein inside the microparticle. The manufacturing method according to claim 21, wherein the hydrophobic core-forming polymer is any one selected from the group consisting of a biodegradable polymer, a non-biodegradable polymer, and a mixture thereof; the biodegradation The polymer is composed of polylactide, polyglycolide, poly(lactide-co-glycolide), polyanhydride, polyorthoester, polyether ester, polycaprolactone, methoxypolyethylene glycol. Any one selected from the group consisting of -b-polycaprolactone (MPEG-PCL), polydecylamine, polybutyric acid, polyvaleric acid, polyurethane, and copolymers thereof; and the non-biodegradable The polymer is made of polyacrylate, ethylene-vinyl acetate polymer, acrylic acid substituted cellulose acetate, non-degradable polyurethane, polystyrene, polyvinyl chloride, polyvinyl fluoride, polyvinylimidazole, chlorosulfonation polymerization. Any one or more selected from the group consisting of chlorosulphonate polyolefins, polyethylene oxide, and copolymers thereof. A method for transdermal administration of a peptide, wherein a microneedle containing a peptide having a molecular weight of 200 to 3000 Da is attached to the skin. A use of a microneedle comprising a peptide having a molecular weight of 200 to 3000 Da, in improving skin wrinkles. A transdermal administration method of a growth factor, wherein a microneedle comprising a microparticle encapsulated with a growth factor is attached to the skin. A use of a microneedle for improving skin wrinkles, the microneedle comprising a microparticle encapsulated with a growth factor.