JP2011219427A - Process for producing nanoparticles of water soluble elastin and skin permeable carrier including the nanoparticles - Google Patents

Abstract

PROBLEM TO BE SOLVED: To further develop a technique of coacervate droplets of water soluble elastin to provide a skin permeable carrier which can be used as a drug delivery system (DDS) for percutaneous administration.SOLUTION: The skin permeable carrier includes nanoparticles of water soluble elastin which can carry a medicine thereon, the nanoparticles preferably having a particle size ranging from 40 to 400 nm, more preferably, from 80 to 220 nm. The carrier is produced by a method of irradiating coacervate droplets of the water soluble elastin with radiation of, for example, 5-60 kGy, to obtain particles having a particle size of nano-order.

Description

The present invention relates to a method for producing water-soluble elastin nanoparticles and a skin-permeable carrier comprising the obtained nanoparticles and capable of carrying a drug or the like.

Drug delivery systems (DDS), which are systems that efficiently act on a target site, are classified into oral administration, pulmonary administration, transdermal administration, intravenous injection, and the like according to the administration method. Oral administration is simple among the administration methods, but the barrier of the gastrointestinal mucosa is a bottleneck, and studies focusing on the permeation mechanism and absorption methods mediated by receptors have been studied. Transpulmonary administration is said to be the most non-invasive, and more efficient nebulizers are being developed along with research on liquid and spray-dried powder inhalants. Although transdermal administration has already been put into practical use, since protein pharmaceuticals hardly penetrate the skin, they are often used in combination with ultrasound, iontophoresis or the like that promotes permeation. In the intravenous injection, a protein pharmaceutical is modified with polyethylene glycol (PEG) and used for the purpose of eliminating antigenicity or increasing blood retention. In addition, as these DDS carriers, nanoparticles made of living body-derived liposomes and PLGA (lactic acid / glycolic acid polymer) have been studied.

Transdermal administration has many advantages because it can avoid hepatic metabolism compared to oral administration and is easier to supply to patients than intravenous injection, but it has many advantages, such as stratum corneum and skin barrier. Due to its intrinsic mechanism, high molecular weight substances are said to be more difficult to penetrate into the skin than low molecular weight substances. However, the EPR effect that the administered substance is selectively supplemented to the lesion site such as a tumor and stays for a long time can be expected from a polymer substance rather than a low-molecular substance. Permeation is a great advantage.

Other factors that cause substances to penetrate the skin include physical properties and particle size. Fat-soluble substances are more likely to show penetration than water-soluble substances, and particles with a particle size of 200 nm or less penetrate into the skin and can be expected to be applied to transdermal preparations and functional cosmetics. Since the particles are predated by macrophages and the like due to the phagocytic (RES) effect by the reticuloendothelial tissue, the retention in blood is low.

On the other hand, the present inventors have focused on elastin, a biopolymer, and have studied its application as a drug carrier. Elastin is a major constituent of animals such as the aorta, ligaments and skin of animals. It is also a major component of fish, such as arterial spheres. Elastin is insoluble but can be solubilized by acid or alkali treatment. The solubilized elastin causes a phenomenon called coacervation in an aqueous solution. This is a reversible series of solutions in which an elastin aqueous solution becomes cloudy when heated to near body temperature, and when left as it is, it is separated into two layers of a transparent equilibrium solution and a pale yellow highly viscous coacervate, and when cooled, it returns to the original homogeneous solution. This refers to a phenomenon (see Patent Documents 1 and 2). The present inventors paid attention to the coacervate droplets formed during the coacervation, and succeeded in producing more stable nanoparticles by irradiating them with γ rays. The inventors of the present invention have paid attention to the use of such nanoparticles as a DDS carrier.

JP 2007-45722 A JP 2009-219422 A

An object of the present invention is to further develop the technique of coacervate droplets of water-soluble elastin and provide a skin-permeable carrier that can be used as a DDS for transdermal administration.

The inventors of the present invention prepared nanoparticles having different particle diameters by irradiating water-soluble elastin coacervate droplets under various conditions to examine the effects of differences in γ-ray irradiation conditions. The present invention was completed by finding that nanoparticles in the range of 1000 nm can be obtained.

That is, the present invention is a skin-permeable carrier comprising water-soluble elastin nanoparticles capable of carrying a drug. The water-soluble elastin nanoparticles preferably have a particle diameter in the range of 40 nm to 400 nm, particularly in the range of 80 nm to 220 nm.

Another aspect of the present invention relates to a method for producing such a carrier, characterized in that water-soluble elastin coacervate droplets are irradiated with radiation to obtain particles having a nano-order particle size. It is a manufacturing method of the nanoparticle of sex elastin. As a radiation dose to irradiate, the range of 5-60 kGy is preferable.

Transdermal administration has many advantages because it can avoid hepatic metabolism compared to oral administration and is easier to supply to the patient, but it has intrinsic benefits such as stratum corneum and skin barrier. Due to the mechanism, it is considered that high molecular weight substances are more difficult to penetrate into the skin than low molecular weight substances. However, when the water-soluble elastin nanoparticles of the present invention are used, the administered substance is selectively supplemented to a lesion such as a tumor, and the EPR effect, which tends to stay for a long time, is higher than that of a low-molecular substance. Since the substance can be expected, it can be used as a DDS preparation by transdermal administration.

The figure which dye | stained hematoxylin eosin (HE) the mini-pig skin tissue specimen. The figure which dye | stained the elastic pig | wing Wang Gieson (EVG) the mini-pig skin tissue specimen. The figure which dye | stained Masson trichrome (MT) dyeing | staining of the mini-pig skin tissue specimen.

Various methods and means for obtaining water-soluble elastin have been proposed. Preferable is the following method proposed by the present inventor (see Japanese Patent Application Laid-Open No. 2007-45722 (Patent Document 1)).

In the first method, insoluble elastin is obtained by removing collagen and other unnecessary proteins from animal biological tissue, and then this insoluble elastin is converted into an acidic solubilizing solution such as oxalic acid or an alkaline solution such as sodium hydroxide. It is immersed and dissolved in a solubilizing solution to produce water-soluble elastin. Collagen and other unwanted protein removal treatment is an alkaline solution containing at least one of sodium hydroxide, potassium hydroxide, calcium hydroxide, and barium hydroxide, and sodium hydroxide added to the alkaline solution. It is carried out by immersing the animal living tissue in an alkaline solution in which the total amount of potassium hydroxide, calcium hydroxide and barium hydroxide is 0.05 to 0.5 mol per liter at 90 to 105 ° C. for 5 to 60 minutes. Is preferred. In addition, when removing collagen and other unnecessary proteins, prior to treatment with an alkaline solution, animal biological tissue is placed in a salt solution containing at least one of sodium chloride, potassium chloride, calcium chloride, and barium chloride. It is also preferable to perform an immersion treatment (pretreatment) for immersion.

The animal living tissue is not particularly limited, but it is preferable to use a ligament or aortic blood vessel obtained from mammals such as pigs, horses, cows, sheep, etc. in terms of a high content of elastin. Also, arterial spheres of fish with a high elastin content may be used. Animal biological tissue is preferably homogenized first using a homogenizer. For homogenization, any animal tissue such as a mixer or meat chopper can be shredded, and preferably a tool capable of shredding into 3 mm square or less, more preferably paste-like. It is preferable that the size of the shredded animal biological tissue is smaller because the efficiency of removing collagen and other unnecessary proteins can be increased. The homogenized animal biological tissue may be degreased by boiling it with hot water or a hot dilute alkaline aqueous solution, or treating it with an organic solvent, for example.

Examples of the solubilized solution include oxalic acid, formic acid, acetic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, betaine, difluoroacetic acid, trifluoroacetic acid, phosphoric acid, sulfamic acid, perchloric acid, and trichloroacetic acid. An acidic solution containing at least one of them is used. The total amount of acid in this acidic solution is preferably 0.05 to 5 mol per liter, and the liquid temperature is preferably 90 to 105 ° C.

The solubilizing solution may also be an alkaline solution containing at least one of sodium hydroxide, potassium hydroxide, calcium hydroxide, and barium hydroxide. The total amount of sodium hydroxide, potassium hydroxide, calcium hydroxide and barium hydroxide added to this alkaline solution is 0.05 to 5 mol per liter, and the solution temperature is 90 to 105 ° C. Is preferred.

Before the second method includes at least one of removal processing of unnecessary parts of animal living tissue, degreasing treatment of animal living tissue, shredding processing of animal living tissue, and removal processing of unnecessary protein with salt. Predetermined treatment process, alkaline extraction process in which pre-treated animal living tissue is immersed in an alkaline solution to filter out collagen and other unwanted proteins, and an alkaline dissolution process in which the residue after the alkaline extraction process is dissolved with alkali It is a method for producing water-soluble elastin by sequentially performing a filtrate recovery step for obtaining a filtrate containing water-soluble elastin by repeated filtration and a water-soluble elastin production step for producing water-soluble elastin from the filtrate. The alkali used in the alkali dissolution step is preferably any one of sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide, or a mixture.

This operation is different from the first method in which collagen and other unwanted proteins are removed from the tissue to obtain insoluble elastin, and then the insoluble elastin is solubilized to obtain water-soluble elastin. This is a method for directly obtaining water-soluble elastin without obtaining insoluble elastin. That is, degreased, shredded, and salt-treated animal living tissue is immersed in an alkaline solution of 0.05 to 0.5 mol per liter at 90 to 105 ° C. for 5 to 60 minutes, and collagen and unnecessary proteins other than elastin Then, the treated tissue is obtained, and then the treated tissue is 0.05 to 5 mol per liter (the concentration of the alkaline solution is higher) in an alkaline solution at 90 to 105 ° C. for 5 to 240 minutes (longer time). This is a method of obtaining water-soluble elastin by immersing and dissolving.

The water-soluble elastin obtained by the first or second method as described above is then removed from the low-molecular-weight one by, for example, dialysis treatment, so that the relatively high molecular weight preferably used in the present invention is used. Water-soluble elastin is obtained.

The obtained water-soluble elastin was adjusted with a solvent of a wide-range buffer solution (pH 1.0 to 11.0) in a concentration range of 0.1 to 60 mg / ml, and the temperature rising rate was 0.1 to 40 ° C./min. By heating, coacervate droplets can be formed. And the production | generation of a coacervate droplet can be confirmed by measuring the turbidity of the aqueous solution of water-soluble elastin in the temperature range of 5-80 degreeC.

The coacervate droplets obtained as described above may be irradiated with radiation, for example, γ rays by a Co-60 γ ray irradiation apparatus. As irradiation conditions, an irradiation temperature of 20 to 80 ° C. and an irradiation amount of about 5 to 60 kGy are appropriate. The particle size of the obtained nanoparticles is preferably in the range of 40 nm to 400 nm, particularly in the range of 80 nm to 220 nm.

In this example, nanoparticles of water-soluble elastin derived from porcine aorta were produced by γ-irradiation, and by conducting a penetration experiment into the skin of minipigs, high molecular weight proteins that did not originally penetrate into the skin were absorbed into the skin. Shown to penetrate.

[Preparation of water-soluble elastin from pig]
1) Isolation of insoluble elastin derived from pigs In accordance with the following procedure, NaCl-soluble and NaOH-soluble unwanted proteins were extracted from porcine aortic defatted tissue.

Using porcine aortic defatted tissue (living tissue), remove the unnecessary part by scraping off the low elastin content such as fat and muscle attached as pretreatment with a blade etc., and then acetone treatment And a degreasing process such as hot water or hot dilute alkali treatment. Next, the defatted biological tissue was subjected to shredding by homogenizing using a homogenizer. The homogenized biological tissue was added with 1M sodium chloride having a volume of about 10 times the weight, and stirred at room temperature for 1 hour to remove NaCl-soluble unnecessary protein. Then, the biological tissue and the NaCl solution were separated. For the separated NaCl solution, for example, the total protein is quantified by the Burette method, and this operation is repeated 5 times until the total protein content in the NaCl solution is 0.1 mg / mL or less. And then drained by centrifugation (3000 rpm, 5 minutes).

Next, about 10 times the volume of the defatted biological tissue (10 ml per 1 g of weight) of 0.1N sodium hydroxide aqueous solution is added and stirred at 100 ° C. for 15 minutes to remove collagen and alkali-soluble unnecessary proteins. The process to do was performed. Then, the biological tissue and the alkaline solution were separated. For the separated alkaline solution, the total protein was quantified by, for example, the burette method, and this operation was repeated 5 times until the total protein amount contained in the alkaline solution was 0.1 mg / mL or less. Thereafter, acetic acid was added for neutralization, washing was performed by centrifugation (5000 rpm, 20 minutes), and the residue was dried to obtain insoluble elastin.

2) Preparation of porcine-derived water-soluble elastin 0.5N sodium hydroxide having a volume 10 times the dry weight of porcine-derived insoluble elastin was added and stirred at 100 ° C for 30 minutes. After the reaction, the solution was quickly cooled with ice and neutralized with acetic acid. Then, it dialyzed for 1 week using the dialysis membrane which fractionates molecular weight 6,000-8,000 or more. Thereafter, it was freeze-dried to obtain a relatively high molecular weight porcine-derived water-soluble elastin.

[Molecular weight and turbidity measurement of water-soluble elastin]
The obtained water-soluble elastin was subjected to SDS-PAGE under non-reducing conditions, and the molecular weight distribution was examined. The gel concentration was 15.0%, the staining method was CBB, and the molecular weight marker was protein molecular weight marker “Daiichi” II. As a result, since a dialysis membrane having a molecular weight fraction of 6,000 to 8,000 cut was used, a relatively high molecular weight water-soluble elastin having a molecular weight of 8,000 or more was obtained. The average molecular weight was 252 kDa.

After adjusting water-soluble elastin to a concentration of 2.2 mg / ml, turbidity was measured using a spectrophotometer with a Peltier temperature controller (JASCO: V-560) under a nitrogen stream. The measurement conditions were a wavelength of 400 nm, a temperature range of 5 to 65 ° C., and a temperature increase rate of 0.5 ° C./min. As a result, turbidity increased with increasing temperature. Also, a sharp rise was observed from around 25 ° C to around 40 ° C. Thereafter, the turbidity intensity became constant from around 40 ° C.

[Preparation of nanoparticles by gamma irradiation]
First, in order to examine the solution concentration at the time of γ-irradiation, which is optimal for preparing nanoparticles of around 150 nm, water-soluble elastin is added to each concentration (2.0 mg / ml, 5.0 mg / ml, The temperature was increased at a rate of 20 ° C./min, the irradiation temperature was 60 ° C., and the irradiation amount was 10 kGy.

[Particle size measurement]
NICOMP (Imaging Technology Group, 380ZLS) was used to measure the particle size of the nanoparticles crosslinked by γ-ray irradiation, and was performed by dynamic light scattering (DLS). Each sample was placed in a 3.0 ml cell, and the temperature in the cell was set to 4 ° C., 37 ° C., and 60 ° C., and measurement was performed for 8 minutes each. Table 1 shows the particle diameters at 4 ° C., 37 ° C., and 60 ° C. of each concentration of water-soluble elastin irradiated with γ rays. The irradiation conditions were an irradiation amount of 10 kGy, a temperature increase rate of 20 ° C./min, and an irradiation temperature of 60 ° C.

From Table 1, the change in the particle size with temperature is large at concentrations of 2.0 mg / ml and 10 mg / ml, and at a concentration of 5.0 mg / ml, a particle size of about 100 to 150 nm is maintained at any temperature. It was found that the optimum concentration for producing nanoparticles having a particle size of around 150 nm, which is the purpose of this time, is 5.0 mg / ml.

[Yield / Yield of produced nanoparticles]
The yield of nanoparticles obtained by γ-irradiation of 150 ml of water-soluble elastin adjusted to 5.0 mg / ml was 0.345 g, and the yield was 46.0% (water-soluble elastin 0.750 g was 100% Yield and yield).

^{[3 H} labeling of porcine water-soluble elastin and Nanoparticles
³ H labeling of nanoparticles prepared from water-soluble elastin and water-soluble elastin was performed by the following method.

[Production of ³ H-labeled water-soluble elastin]
200 μg of water-soluble elastin was weighed into a siliconized eppen, 60 μl of TFE was added thereto, 250 μl of pyridine was added, and further, [ ³ H] acetic anhydride was mixed with 250 μl of toluene to a radioactivity of 5.0 mCi. It was. The mixture was allowed to stand at room temperature for 4 hours, then precipitated by centrifugation, and the solvent was blown off under a nitrogen stream. The residue was dissolved in TFE, 1.0 ml of PBS was added, and the mixture was stirred with a sonicator and vortex to obtain the above ³ H-labeled reaction solution of water-soluble elastin. To a Sephadex G-25 gel filtration chromatography column pretreated with PBS 10 ml, 2.0 mg / 10 ml unlabeled elastin 500 μl, PBS 10 ml in order, and then pretreated by flowing 4 ° C. PBS until the column carrier was sufficiently cooled, The above ³ H-labeled reaction solution of water-soluble elastin was poured. Next, 1.0 ml, 1.0 ml, and 3.0 ml of PBS were flowed sequentially. ^A fraction of 3.0 ml (3 fractions x 1.0 ml) of 3H-labeled acetyl-water soluble elastin was collected. The obtained ³ H-labeled acetyl-water-soluble elastin had a radioactivity of 141 μCi / mg.

[Production of ³ H-labeled nanoparticles]
200 μg of nanoparticles were weighed in a siliconized eppen, to which 60 μl of TFE was added, 250 μl of pyridine was added, and [ ³ H] acetic anhydride was further added to 250 μl of toluene mixed to a radioactivity of 5.0 mCi. The mixture was allowed to stand at room temperature for 4 hours, precipitated by centrifugation, and the solvent was blown off under a nitrogen stream. Further, it was washed by centrifugation (10,000 rpm, 4 ° C., 10 min × 3 times), 500 ml of PBS was added to the residue, and washed by centrifugation (10,000 rpm, 4 ° C., 10 min × 3 times). 500 μl was added to obtain 500 μl of ³ H-labeled acetyl-nanoparticles. The radioactivity of the obtained ³ H-labeled acetyl-nanoparticles was 9.30 μCi / mg.

[Radioactivity of ³ H-labeled water-soluble elastin and ³ H-labeled nanoparticles]
The radioactivity of ³ H-labeled acetyl-water-soluble elastin and ³ H-labeled acetyl-nanoparticles is shown in Table 2. When compared with the radioactivity per coating amount, ^{3 H-labeled} acetyl - was found to be approximately 0.4 times the water-soluble elastin - radioactivity nanoparticles ^{3 H-labeled} acetyl.

^{[3 H-labeled} water-soluble elastin into minipigs skin tissue, and, ^{3 H-labeled} nanoparticle penetration Experiment
The mini-pig skin tissue was thawed at room temperature, cut into 5 cm ² squares, lightly wiped with a cotton wool soaked with 70% ethanol, and floated on a dish containing 10 ml of the culture solution. After applying 50 μl (20 μg, 3.3 μg) of ³ H-labeled acetyl-water-soluble elastin and ³ H-labeled acetyl-nanoparticles on each skin floated on a dish, it was placed in a 37 ° C. carbon dioxide incubator. Left to stand. At 0h, 9h, and 24h after application, the application site was punched and collected using a 7 mm punch and hammer. Under the same conditions, a sample that was not coated with any sample was collected as a control.

[Preparation of miniature pig skin tissue by microtome]
The collected skin sample was placed in 10% formalin overnight and washed with running water for 5 hours. Thereafter, dehydration was performed by the following method.

Samples are sequentially put in 70% ethanol (90 min, 37 ° C.), 80% ethanol (90 min, 37 ° C.), 90% ethanol (90 min, 37 ° C.), and 100% ethanol (90 min × 4, 37 ° C.), dehydrated in 100% xylene (30 min × 3, 37 ° C.), then dehydrated in paraffin (45 min × 3, 63 ° C.) and embedded in paraffin bath (PB-150) .

For the untreated skin sample, the same operation was performed fully automatically by SAKURA ETP.
In addition, the dehydrated sample was embedded in paraffin using Tissue-TekIII (Dispensing Console, Cryo Console). After smoothing and thinly cooling the sliced surface of the embedded sample, the sample is sliced into 8μm thicknesses by 3 each time using a microtome (Reichert-Jung, 2030 BIOCUT) and floated in water. Then, the glass was scooped into a slide glass and the paraffin was stretched with an extender. After fully stretching and drying, it was allowed to stand at 37 ° C. overnight.

[Confirmation of ^{the 3 H-labeled} samples by autoradiography]
The sample which was allowed to stand at 37 ° C. overnight was immersed in 100% xylene prepared in three layers for 5 minutes each time, and then sequentially immersed in 100%, 99%, 95%, 85% and 75% ethanol for deparaffinization. Immerse the deparaffinized sample (except for the untreated sample) in a 45 ° C emulsion (KODAK Autoradiography Emulsion Type, NTB: distilled water = 1: 1) in a dark room. The case was placed side by side, the lid was closed, light-shielded with vinyl tape and a black vinyl bag, and left at 4 ° C. for 3 weeks. After standing for 3 weeks, the sample is again in a dark room for 2 min in room temperature developer (KODAK DEKTOL Developer, stock solution: distilled water = 1: 1 (mixed at 32-38 ° C.)), and 10 min in distilled water for fixation.
, Fixer (KODAK
Fixer) was immersed for 5 minutes with stirring, and then washed with running water for 20 minutes.

[Staining of miniature pig skin tissue specimens]
The sample after the deparaffinization or autoradiography was stained by the following method. Untreated skin samples after deparaffinization are stained with hematoxylin and eosin (HE) staining for cell nuclei, elastica Wangyson (EVG) staining for elastin, and Masson trichrome (MT) staining for collagen. The skin samples treated by the method and subjected to autoradiography were treated with only eosin stained with hematoxylin and eosin (HE).

[Hematoxylin and eosin (HE) staining]
The deparaffinized sample was washed with running water and then immersed in distilled water. The Mayer's hematoxylin solution was lightly applied and immersed in the same solution for 5 min. Washing with running water for coloration was performed for 10 minutes, soaking in distilled water, and soaking for 2 minutes in eosin diluted 5-fold with 95% ethanol, followed by washing with running water. Thereafter, it was dehydrated with ethanol and xylene in the same manner as described above and sealed in paraffin. An optical microscope (Olympus BX 50) photograph of the HE-stained skin tissue section is shown on the left side of FIG.

[Elastica Wangyson (EVG) staining]
The deparaffinized sample is sequentially washed with running water, then immersed in distilled water, soaked in 70% ethanol, soaked in resorcin fuchsin for 2 to 3 hours, soaked in 100% ethanol, soaked in distilled water after running in running water, and iron hematoxylin solution ( Immerse in iron hematoxylin solution I: II = 1: 1) for 10 minutes, wash with running water for 10 minutes for coloration, soak in distilled water, soak in one-Geeson solution (One-Geeson solution A: B = 20: 3) for 3 minutes, 70% Put in ethanol. Thereafter, dehydration treatment was performed in the same manner as described above, and sealed in paraffin. An optical microscope (Olympus BX 50) photograph of a skin tissue section stained with EVG is shown in the center of FIG.

[Masson trichrome (MT) staining]
The deparaffinized sample was sequentially washed with running water, then immersed in distilled water, immersed in mordant for 20 min, washed with running water (3 min), then immersed in distilled water, soaked in caratchi hematoxylin for 1 h, and washed with running water (10 min). Then, it was soaked in Orange G for 1 min, sequentially soaked in 1% acetic acid prepared in 2 bats, soaked in fuchsin ponceau solution for 10 min, and soaked in 1% acetic acid prepared in 2 bats. Further, soak in phosphotungstic acid for 10 min, sequentially soak in 2% 1% acetic acid, soak in aniline blue for 3 min, soak in 2 bats in 1% acetic acid, and soak in 2% in 100% propanol. It was. Thereafter, dehydration treatment was performed in the same manner as described above, and sealed in paraffin. A TM stained optical microscope (Olympus BX 50) photograph is shown on the right side of FIG.

[Penetration confirmation of ^{the 3 H-labeled} samples by autoradiography]
After application of ³ H-labeled acetyl-water-soluble elastin and ³ H-labeled acetyl-nanoparticles, an optical microscope (Olympus BX 50) photograph of an eosin-stained skin tissue section was taken and the results were taken. These are shown in FIGS. 2 and 3, respectively.

[Analysis of results]
From the photographs of HE staining, EVG staining, and MT staining (FIG. 1), cells constituting the epidermis (HE staining), elastin (EVG staining), and collagen (TM staining) were observed. In addition, the following was found from autoradiography after application of ³ H-labeled acetyl-water-soluble elastin (FIG. 2) and autoradiography after application of ³ H-labeled acetyl-nanoparticles (FIG. 3).

^As shown by the arrow in FIG. 2, in the mini-pig skin tissue to which ³ H-labeled acetyl-water-soluble elastin was applied, it started to deposit in the stratum corneum at 0 h after application and almost deposited at 9 h, but 24 hours passed However, almost no penetration into the skin was observed. On the other hand, ^{3 H-labeled} acetyl - In miniature swine skin tissue nanoparticles coated (FIG. 3), as indicated by the arrow in FIG. 3, ^{3 H-labeled} acetyl in after 0h coating - as with a water-soluble elastin, the stratum corneum Although 9 hours after application, penetration into the epidermis was observed as the number of samples on the stratum corneum decreased, confirming the difference from ³ H-labeled acetyl-water-soluble elastin. Further, 24 hours after application, the whole epidermis and the upper layer of the dermis penetrated. Moreover, elastin content in per coating weight 50μl is, ^{the 3 H-labeled} acetyl - despite the water-soluble elastin is large, radioactivity ^{3 H-labeled} acetyl - less about 0.4 times the radioactivity of the water-soluble elastin This suggests that the nanoparticles may penetrate the skin more than the water-soluble elastin.

From these results, it was confirmed that water-soluble elastin, which does not normally penetrate into the skin, could penetrate into the skin by γ-irradiation to form nanoparticles. This suggests that elastin permeates into the skin as nanoparticles and is absorbed inside the skin to become an important component of the skin, and at the same time elastin may be a new DDS carrier for transdermal administration. It was.

Claims (4)

A skin-permeable carrier comprising water-soluble elastin nanoparticles capable of carrying a drug. The skin-permeable carrier according to claim 1, wherein the particle diameter of the water-soluble elastin nanoparticles is in the range of 40 nm to 400 nm. A method for producing a water-soluble elastin nanoparticle comprising irradiating a water-soluble elastin coacervate droplet with radiation to obtain a particle having a nano-order particle size. The method for producing nanoparticles of water-soluble elastin according to claim 3, wherein the radiation applied to the water-soluble elastin coacervate droplets is γ rays with an irradiation amount of 5 to 60 kGy.

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