JP6567716B1 - Biodegradable microneedle array - Google Patents

Abstract

A new transdermal delivery system is provided. A method for fabricating a microneedle-based array for transdermal delivery of therapeutic agents comprising mixing carboxymethylcellulose (CMC) and dextrin (DEX) to provide a polysaccharide-based combination And adding the therapeutic agent to the polysaccharide-based combination. [Selection figure] None

Description

  The present invention relates to a method for making a polysaccharide-based dissolvable microneedle-based array and a microneedle array produced therefrom.

  Developing a delivery system for a newly developed drug is a challenging task. Drugs are administered via various general routes such as oral, parenteral, ophthalmic, transdermal, nasal, pulmonary, and buccal routes (Langer, 2001). Of these routes, the transdermal drug delivery system (TDDS) is a promising system that can be used without the help of medical staff (Prausnitz and Langer, 2008; Wokovich et al., 2006). TDDS also has several advantages such as liver first pass metabolism and elimination of side effects of oral dosage forms in the gastrointestinal tract (Asbill and Michniak, 2000; Hadgraft et al., 1996). However, the system still has some drawbacks such as low efficiency. Efficiency can be reduced for various reasons such as skin structure and stratum corneum (SC) barrier properties (Bronaugh and Maibach, 2005). The presence of the SC barrier means that only oil-soluble molecules and low molecular weight compounds can be transported.

  In order to overcome the troublesome SC barrier, several methods have been developed to enhance skin penetration for drug delivery. One such method is the use of a microneedle array, which shows better performance in delivering therapeutic agents through the skin barrier without causing damage (Kaushik et al., 2001; McAllister et al. 2003; Mikszta et al., 2002). A microneedle array (MN) is applied to the skin surface and the epidermis is perforated without stimulating nociceptors (Martin et al., 2012). These MNs form micropores through the skin and diffuse drugs or vaccines into the dermal microcirculation (Cheung et al., 2014; Kim et al., 2012; Liu et al., 2012; Sullivan et al., 2010; Tuan-Mahmood et al. , 2013). MN arrays are made of various materials such as polymers, metals, silicone rubbers, and polysaccharides (Badran et al., 2009; Ding et al., 2009; Hafeli et al., 2009; Li et al., 2009; Lin et al., 2001; Martanto et al., 2004). Among them, polymer MN arrays are increasingly attractive because they are cheaper to mass produce than silicon or metal arrays and are safe during application. When using polymer dissolution, drugs and biomolecules can be incorporated within the MN (Chu and Prausnitz, 2011; Hirobe et al., 2015; Lee et al., 2008; Lee et al., 2011; Liu et al., 2012; Sullivan et al., 2010). After releasing the drug, the polymer dissolves in the skin, avoiding the risk of MN remaining in the body (Ke et al., 2012; Sullivan et al., 2008). The polymer used for MN application should have characteristics such as biocompatibility, strong mechanical properties, and rapid dissolution rate. Considering drug-dependent release times, polymers with different dissolution rates will be required to deliver controlled amounts of drug.

  Solid MN is cast and formed using biodegradable polymers as two types of structures (“one-piece” and “layer-by-layer”). In the “one-piece” method, the solution containing the drug and polymer is injected and cast simultaneously on the same mold; in the “multi-layer” method, the model structure is cast in combination with multiple coating steps or each slice of the model. The Organic solid MN extends the molecular weight range from hundreds to thousands of synthetic and natural materials, dextrin (DEX), carboxymethylcellulose (CMC), amylopectin (AMP), polylactide-co-glycolide, galactose, maltose, and Polyvinylpyrrolidone is included (Lee et al., 2008; Raphael et al., 2010). Many model compounds of various sizes can be added according to their different molecular compatibility; for example, β-galactosidase enzyme, sulforhodamine, sodium salicylate, calcein, and bovine serum albumin have been successfully incorporated into MN doing. Over the past decade, preclinical evaluation of MN delivery using various drugs, bioactive proteins, and vaccines has progressed (Pettis and Harvey, 2012). Animal and human studies have demonstrated the application of various drugs and vaccines. However, relatively few MN clinical trials have been published (Pettis and Harvey, 2012). Stability of drugs or biomolecules encapsulated in MN, reliable insertion of MN into the skin, assessment of skin irritation and skin infection, assessment of pharmacokinetics and pharmacodynamics, and assessment of pain and other perceptions Further research is needed (Kim et al., 2012; Pettis and Harvey, 2012).

  New transdermal delivery systems remain desirable.

SUMMARY OF THE INVENTION In the present invention, it is unexpectedly found to make a biocompatible microneedle array (MN) using a combination of CMC and dextrin DEX. Accordingly, the present invention provides a method for making polysaccharide-based dissolvable microneedle-based arrays that can be used as invasive and transdermal delivery systems. The microneedle array obtained from the method of the present invention is safe and biocompatible and can be used for transdermal administration of drugs such as insulin.

In one embodiment, the present invention is a method for fabricating a microneedle-based array for transdermal delivery of a therapeutic agent comprising:
Mixing carboxymethylcellulose (CMC) and dextrin (DEX) to provide a polysaccharide-based combination; and adding the therapeutic agent to the polysaccharide-based combination;
A method comprising:

  In one embodiment of the invention, the polysaccharide-based combination comprises 30-70% CMC and 30-70% DEX.

  In one example of the invention, the polysaccharide-based combination comprises 50% CMC and 50% DEX.

  The term “therapeutic agent” as used herein refers to a compound or substance, such as a drug, that provides a therapeutic benefit to a mammal when administered to the mammal in a therapeutically effective amount.

  In one embodiment of the invention, the therapeutic agent is a drug.

  In one embodiment of the invention, the therapeutic agent is insulin.

  In another aspect, the present invention provides a microneedle array for transdermal delivery of therapeutic agents made from a polysaccharide-based combination of carboxymethylcellulose (CMC) and dextrin (DEX). The microneedle array is manufactured by the method of the present invention.

  The foregoing summary, as well as the following detailed description of the present invention, will be better understood when read in conjunction with the appended drawings.

An image showing an embodiment of the present invention providing a microneedle array, including a polysaccharide-based microneedle array produced by a computer-aided design (CAD) system integrated with a high speed cutting machine and a casting area in the middle of a mode Where NA means needle area, BA means blank area, and RA means ridge area; CT means connecting trench. A scheme for the fabrication of microneedle arrays according to the present invention is provided. Figures provide for the analysis of different polysaccharides, including (A) thermogravimetric analysis (TGA) X-ray diffraction (XRD); and (B) analysis of the original powder form, respectively. Provide a diagram showing the results of cytotoxicity testing of individual polysaccharides of carboxymethylcellulose (CMC) and dextrin (DEX), trehalose and control; where NIH 3T3 cells are used to test polysaccharide toxicity Used in 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay. Viscosity measurements of carboxymethylcellulose (CMC) and dextrin (DEX) in (A) deionized water, (B) phosphate buffer, pH 7.4, and (C) phosphate buffered saline (pH 7.4) Where C represents a mixture containing 100% CMC (C), 1C1D represents a mixture containing 50% CMC and 50% DEX, and 1C2D represents 34% CMC and 66 2C1D represents a mixture containing 66% CMC and 34% DEX, and 3C2D represents a mixture containing 60% CMC and 40% DEX. Provided are images of the microneedle array according to the present invention, such as (A) scanning electron microscope (SEM) images and (B) optical microscope (OM) observations. Use deionized water or phosphate buffered saline (PBS) as solvent, (A) deionized water as solvent; (B) phosphate buffer, pH 7.4, as solvent; (C) phosphate buffered physiology Saline, pH 7.4, and X-ray diffraction (XRD); and (D) Thermogravimetric analysis (TGA), which provides an analysis of fabricated polysaccharide microneedles consisting of carboxymethylcellulose (CMC) and dextrin (DEX) Provide a diagram showing the results; where C indicates a mixture containing 100% CMC (C), 1C1D indicates a mixture containing 50% CMC and 50% DEX, 1C2D indicates 34% CMC and A mixture containing 66% DEX is shown, 2C1D is a mixture containing 66% CMC and 34% DEX, and 3C2D is a mixture containing 60% CMC and 40% DEX. 1 shows the cytotoxicity test of a microneedle array according to the present invention; where NIH3T3 cells are used to test the toxicity of polysaccharides (carboxymethylcellulose (CMC); mixture of CMC and dextrin (CMC + dextrin); and control). -(4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay. Shows observation of tissue sections under an optical microscope for in vivo skin penetration testing; nude mice were treated with a microneedle array according to the invention supplemented with bovine serum albumin (BSA) as the test drug: (A) and ( B) provides two representative images of hematoxylin and eosin (H & E) staining, arrows indicate microneedle penetration; (C) tracks BSA release after penetrating the skin Provide representative images of mouse tissue sections after immunohistochemistry and H & E (IHC + H) staining; (D) provides an enlarged view of IHC + H stained tissue showing BSA release (scale bar Is 50 μm for (A) and (C) and 25 μm for (B) and (D)). Shown are representative frozen sections of time-tracked methylene blue (MB) dye released from polysaccharide microneedles and diffused into the skin. Mice were treated with microneedles containing MB for (A) 2 hours, (B) 4 hours, and (C) and (D) 8 hours. Shown are representative images of microneedle arrays before (top image) and after (bottom image) before (A) 4 hour, (B) 8 hour and (C) 24 hour mouse attachment.

Description of the invention
Mold Design for MN Array As shown in Figure 1, a mold was fabricated on an aluminum (Al) plate using a high speed cutting machine and a computer aided design (CAD) system (AutoCAD 2012). The aluminum mold was redesigned based on preliminary results. One aluminum mold contains two array units for making two microneedle arrays in a casting process. There were four functional areas in the aluminum mold: (1) needle area (NA); and (2) blank area (BA); (3) ridge area (RA); and (4) connecting trench (CT). The central NA of the mold has two groups of 10 × 10 conical needle arrays. The NA was surrounded by a BA that provides thickness to the model to ensure toughness and avoid stress failure when released from the mold. RA was used to separate each single array unit, and CT helped slow the flow of the viscous solution and avoid the formation of bubbles during rapid injection.

  To analyze the composite material for MN fabrication, a procedure was designed using two test loops as shown in FIG. 2 to provide a fabrication scheme for a microneedle array according to the present invention. The initial test loop was based on moisture content and results from X-ray diffraction (XRD) and thermogravimetric analysis (TGA). Furthermore, the first checkpoint was to test whether the composite mixture could not be cut by temperatures up to 100 ° C. A second checkpoint tested whether the mixture was soluble. If the mixed solution is soluble, the next test is a biotoxicity test. In the absence of toxicity, a series of fluidized coatings were tested using checkpoints for good film formation from a 5% mixed solution. When all checkpoints were tested, MN production proceeded. All work with test films and MN fabrication were done in a single “all-in-one” mold.

Polysaccharide materials Based on their biocompatibility and water solubility, we first identified four species to analyze their physical properties to identify suitable molecules for making dissolvable MN. Different polysaccharides were selected. CMC, a cellulose derivative, is a water-soluble natural polysaccharide that is widely used in various foods (Chillo et al., 2007) and medical applications. DEX contains various sizes of water-soluble glucose polymers produced by hydrolysis of starch. DEX is used as a food additive in the food industry and in many pharmaceutical products. Hyperbranched amylopectin (AMP) is a water-soluble polysaccharide that is one of the two major components of starch. AMP is used in various applications such as food thickening. Trehalose (TRE) is a natural disaccharide containing two glucose units linked by α, α-1,1-glucoside bonds (Higashiyama, 2002). TRE is widely distributed in nature and is an attractive material for industrial use. This saccharide is used as a sweetener in the food industry (Higashiyama, 2002).

  CMC sodium salt (99.5% purity), DEX (from 99% purity potato starch), TRE dehydrate (Saccharomyces cerevisiae, suitable for cell culture, purity 99%) and AMP (from potato starch) were purchased from Sigma-Aldrich .

Physical properties of polysaccharides for MN production The moisture content of all polysaccharides was measured using an infrared moisture meter MA150, Sartorius. Approximately 0.1 g of each sugar in its powder form was heated and analyzed for the percentage of moisture. In order to obtain the heating temperature range of the polysaccharide material in the casting process, TGA was performed using a Setaram Labsys-TGDSC, DSC131 apparatus. A sample of 0.008 g of each sugar in its powder form was heated for TGA analysis from room temperature to 500 ° C. at a heating rate of 5 ° C./min. The material in powder form was also used for XRD analysis using a Rigaku (Copyright) TTRAXIII powder X-ray diffractometer to obtain information on the crystallization and molecular arrangement of the material.

  The viscosity of the polysaccharide in the aqueous solution was measured using a Haake Viscotester 550 Rotational Viscometer (Thermo Scientific). A 5% solution containing 2.5 g polysaccharide dissolved in 47.5 g solvent (deionized water, phosphate buffer, and phosphate buffered saline (PBS), respectively) was poured into the viscometer for testing. .

Microneedle (MN) casting
Vacuum deposition was performed at 65 ° C. for MN casting. Polysaccharide solutions (5% w / v) were prepared by dissolving them in phosphate buffer and sonicating for 60 minutes for complete dispersion and dissolution. To remove bubbles, the solution was centrifuged at 3500 rpm for 10 minutes at room temperature. The solution was then slowly poured along the edge of the aluminum mold. The mold containing the solution was held in vacuum for 15-20 minutes at room temperature to lift the bubbles and repeated until no visible bubbles were observed. The mold was heated from room temperature to 65 ° C and heated at a steady temperature of 65 ° C for up to 24 hours. The heating rate was 1 ° C./min.

  The finished polysaccharide film or MN product was removed vertically from the aluminum mold by manual peeling. A poorly formed product will not completely peel off. Selection criteria were set as a complete separation from the mold and an MN array without cracks.

After evaluation casting of the fabricated MN, the physical properties of the polysaccharide film or fabricated microneedle were characterized by TGA and XRD analysis (see section 2.3) and hardness test.

  After separation from the mold, the final product was tested for hardness using a GARDCO (copyright) Wolff-Wilborn hardness pencil tester (Paul N. Gardner) according to the manufacturer's protocol. This scratch hardness test determined the resistance of the polysaccharide film to the scratch effect on the surface.

  We determined the structure of MN using a scanning electron microscope (SEM) and an optical microscope (OM) on a Hitachi (copyright) H-7100 microscope.

Cytotoxicity
In vitro toxicity was performed on a normal NIH 3T3 (mouse embryonic cell) cell line using a 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay. NIH 3T3 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS). For toxicity studies, cells were seeded in 24-well plates in DMEM containing 10% FBS at a seeding density of 5,000 cells / well and 24% at 37 ° C. with 5% CO ₂ before addition of test compound. Incubated for hours. The polymer was dissolved in PBS and filtered through a 0.22 μM filter. After 24 hours, 10 μl of medium containing test compound at 0.05% (w / w) concentration was added and incubated for 48 hours at 37 ° C. with 5% CO ₂ . The experiment was performed 3 times and cells without test compound served as controls. After 48 hours, 200 μl of MTT solution (5 mg / ml) in PBS was added to each well and incubated at 37 ° C. for 4 hours. After removing the medium containing MTT, the formed formazan crystals were solubilized in 150 μL of dimethyl sulfoxide, and the absorbance at 540 nm was measured using a microplate reader. Cell inhibition% = 100-Abs (drug) / Abs (control) x 100 (where Abs means absorbance at 540 nm) to determine the percentage of cell inhibition.

The nude mouse, BALB / cAnN.Cg-Foxn1nu / Cr1Nar1, used in the in vivo skin permeability measurement study was obtained from the National Laboratory Animal Center in Taiwan. The MN was attached to the mouse using a bandage bundle. Following treatment, the skin was surgically removed and prepared for tissue sections.

  To track protein release after MN skin penetration, 1% BSA (bovine serum albumin, Sigma-Aldrich) powder was mixed with casting polysaccharide (50% CMC + 50% DEX). Skin tissue pieces were fixed with 4% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin (H & E). Immunohistochemistry (IHC) was performed using an antibody against BSA (Millipore® 07248).

  0.25% methylene blue (MB) powder was mixed with casting polysaccharide (CMC alone or 50% CMC + 50% DEX) for time tracking of pigment release from MN and diffusion to skin. Skin tissue pieces were frozen and prepared as frozen sections.

  The use of animals in this study was approved by IACUC of Taipei Medical University.

Material investigation and selection for MN fabrication This study presents a method for producing biodegradable MN for transdermal drug rally delivery. In order to select the appropriate materials, the polysaccharides CMC, DEX, TRE, and AMP were analyzed in their powder form to examine their moisture content, crystallinity and thermal stability. Polysaccharides were mixed at various ratios and tested for their solubility in aqueous buffers. Subsequently, individual or combined soluble polysaccharides were poured onto the mold surface to check their film formation. The cytotoxicity of individual polysaccharides was also tested.

  Table 1 shows the results of the moisture content test. Water molecules were present in all tested polysaccharides even after drying at 55 ° C. for 24 hours. The water capacity of the material and its affinity with water molecules prevents the material from drying out completely or moisture is lost during the casting process, and the material is compatible with the incorporated drug or protein Made sure.

データは、3回反復測定値からの平均として提供される。カルボキシメチルセルロース、CMC；デキストリン、DEX；トレハロース、TRE；アミロペクチン、AMP table 1
Water content of various polysaccharides

Data are provided as an average from 3 replicate measurements. Carboxymethylcellulose, CMC; Dextrin, DEX; Trehalose, TRE; Amylopectin, AMP

  Individual sugars in powder form were analyzed by XRD and TGA to determine crystallinity and thermal stability. Crystallization properties provided information on the molecular arrangement of the materials and their water retention capacity. This information can be useful in combining different polysaccharides. Analyzing the thermal properties using TGA ensured that the material was stable at the drying temperatures used in the casting process.

  All materials tested showed a significant signal peak due to crystallization (Figure 3A). TRE produced the most obvious signal. DEX is a group of carbohydrates produced by hydrolysis of starch and therefore showed a signal similar to that of AMP (FIG. 3A). The lower signal of CMC and the smoother curve indicated that more amorphous regions were present in CMC.

  The mass loss profile with temperature is shown in FIG. 3B and Table 2. The first mass loss (90-130 ° C) indicates the critical temperature of water loss, and the second loss (above 240 ° C) reflects molecular decomposition. CMC and TRE showed curves that decreased more significantly for water loss than AMP and DEX (FIG. 3B). This result indicated that AMP and DEX had better moisture capacity in their powder form. This was confirmed by a moisture content test (Table 1). TGA data showed that the polysaccharide is thermally stable for casting in a typical low temperature MN manufacturing process having a temperature of 55-75 ° C. When the casting temperature was lowered, the drying time was 15 hours at 75 ° C, 24 hours at 65 ° C, and 28 hours at 55 ° C. The subsequent MN casting temperature was set at 65 ° C. because the polysaccharide matrix and BSA were stable.

カルボキシメチルセルロース、CMC；デキストリン、DEX；トレハロース、TRE；アミロペクチン、AMP
T₁₀：10％質量損失についての熱分解温度
T₅₀：50％質量損失についての熱分解温度
T_d：分子分解温度
R₃₄₀：340℃における残留重量パーセント Table 2
Thermogravimetric analysis (TGA) of various polysaccharides showing thermal decomposition temperature

Carboxymethylcellulose, CMC; Dextrin, DEX; Trehalose, TRE; Amylopectin, AMP
T ₁₀ : Thermal decomposition temperature for 10% mass loss
T ₅₀ : Thermal decomposition temperature for 50% mass loss
T _d : Molecular decomposition temperature
R ₃₄₀ : Residual weight percentage at 340 ° C

  Sugar solubility and viscosity have a direct impact on the quality of the casting product. Polysaccharides were mixed at different ratios and tested for their solubility in aqueous buffers. Amylopectin (AMP) is suitable for manufacturing MN because it has low solubility in water-soluble buffer solution and requires continuous heat to dissolve it. It wasn't.

  The viscosity of the sugar solution increases at high concentrations. The viscosity of the material affects the filling effect after the sugar solution is poured into the mold and affects the final product. A highly viscous solution cannot fill the mold and generate bubbles. Low viscosity solutions do not give the final product sufficient thickness and result in a decrease in physical properties. It was therefore necessary to find a suitable viscosity to fill the mold surface. Use CMC at various concentrations (5-50% w / v) in the test tube to check the viscosity suitable for injection, and visually observe the smooth flow of liquid as the tube is rotated (Table 3). Only the 5% solution was able to completely cover the test tube, whereas the 10% and 20% solutions showed only partial coverage. The 30% and 50% solutions moved slowly due to their high viscosity and could not completely cover the tube. We selected a 5% solution as a baseline for further study.

N/A：流動不可能
AC：完全に被覆、C：部分的に被覆、LC：管を被覆することはできない、ゆっくりと流れる。 Table 3
Overview of fluid coating tests with various concentrations (5-50%) of carboxymethylcellulose (CMC) to cover test tubes

N / A: Non-flowable
AC: Completely covered, C: Partially covered, LC: Cannot cover tube, flow slowly.

  Subsequently, the blank surface on the back side of the aluminum mold was used to examine the mold film (dried at 65 ° C. for 24 hours) using a 5% solution of TRE, DEX, or CMC, respectively. TRE produced a thin, irregular, translucent film that could not be secured to the sheet structure. DEX made relatively thick blocks that could break into pieces. Therefore, TRE and DEX alone were not suitable for casting in MN arrays. They were considered for use as a mixture with CMC.

  TRE in combination with CMC was difficult to separate from the mold surface (data not shown), resulting in surface scars and repeated cleaning periods. When the concentration of TRE combined with CMC was reduced, a sufficiently thick polymer film could not be formed, it was difficult to separate by sticking to the mold, and several pieces were produced (data not shown). XRD analysis (FIG. 3A) showed a large difference in crystallinity between the two polysaccharides. TRE showed more crystallization and less amorphous regions, whereas CMC showed less crystallization and more amorphous regions (FIG. 3A). TRE exists as small molecular weight molecules in the solution and shows macromolecular cracks in the crystals during the drying process that damage the overall structure. Therefore, TRE was not suitable for combination with CMC to produce MN.

  DEX was similar to CMC in terms of crystallographic signal (FIG. 4A) and molecular size; therefore, compatibility with CMC may be improved during the casting process. CMC alone or in combination with DEX was considered for further study.

Pre-casting evaluation (viscosity test)
The viscosity test of CMC combined with DEX was performed (Fig. 6). Three different aqueous buffers (deionized water, phosphate buffer, pH 7.4 and PBS, pH 7.4) were used to dissolve CMC and DEX in various ratios (100% CMC, 50% CMC + 50 % DEX, 34% CMC + 66% DEX, 66% CMC + 34% DEX, and 60% CMC + 40% DEX). By mixing the materials, the properties of the fluid changed significantly. As the CMC ratio increased, the viscosity increased (FIG. 5). The solvent increased the viscosity slightly due to the presence of salt in the buffer. Because of the presence of salt in the buffer, the solvent slightly increased the viscosity. Even if the solvent was replaced, the viscosity could not be reduced. However, the solvent PBS significantly increased the viscosity of pure CMC. Finally, PB of MN casting was selected.

Fabrication of polysaccharide-based MN array aluminum molds offers several advantages for model casting. It is compatible with conventional MN casting methods (Lee et al., 2008; Migalska et al., 2011; Park et al., 2016). Al ASTM 6061 (FDA approved for use as a food container) is a highly corrosion resistant material for heavy structures and was used in this study to prepare reusable molds. For the production of MN, the polysaccharide mixture was poured onto an Al mold and dried at 65 ° C. for 24 hours. Low temperatures were used for casting because the final product is made of biomolecules and drugs with polysaccharides. The thermal stability of the target molecule was considered during the drying process (Figure 3B and Table 2). FIG. 5 shows SEM and OM observations of polysaccharide-based MN with a length of about 480 μm, a substrate of 370 μm, and a needle center spacing of 600 μm.

  In this study, MN was made using CMC alone and CMC / DEX mixture. CMC is a naturally soluble polymer that is miscible with bioactive compounds, is relatively easy to process at low temperatures, and is inexpensive. It is widely used in medical applications (Agarwal et al., 2015; Chen et al., 2015; Pasqui et al., 2014) such as MN production (Kommareddy et al., 2012; Lee et al., 2017; Lee et al., 2008; Park et al., 2016). ing.

A pencil hardness test was performed to ensure the hardness of the final product after separation of the produced MN characteristic mold. Table 4 shows the details of the pencil hardness test. CMC combined with DEX showed better hardness than CMC alone. The polysaccharide combination showed an increase in hardness in phosphate buffer compared to deionized water.

  TGA results showed that the thermal properties of different combinations of CMC and DEX in aqueous buffer were stable up to 200 ° C. (FIGS. 5A-5C, Table 5). The initial mass loss curve after the casting process showed a gentle and continuous CMC and DEX curve (see FIG. 7A for film, see FIG. 3B compared to powder form). However, when PB or PBS was used as the solvent, the TGA curve of the casting film showed no significant difference, indicating that no direct chemical reaction was formed between the material and the phosphate buffer (FIG. 7A-FIG. Curves CMC, 1C1D, 1C2D, 2C1D at 7C). The mass loss for casting the film was due to water loss.

カルボキシメチルセルロース、CMC；デキストリン、DEX；リン酸緩衝生理食塩水、PBS；リン酸緩衝液、PB Table 4
Pencil hardness test of various combinations of polysaccharides in aqueous buffer

Carboxymethylcellulose, CMC; dextrin, DEX; phosphate buffered saline, PBS; phosphate buffer, PB

Table 5
TGA analysis and thermal decomposition temperature of microneedle array manufactured using various polysaccharides

  The XRD results of the polysaccharide film showed a large difference before the casting treatment (FIG. 3A) and after the casting treatment (FIG. 7D). Crystallization signals appeared at 18 ° C.-21 ° C., 22 ° C.-24 ° C., 40 ° C.-42 ° C. (FIG. 7D). A comparison of signal area and spacing showed that the signal was mainly caused by CMC, not DEX or solvent. XRD showed no extra crystallization signal for the solvent product (compare 3C2D curve with PBS 3C2D and 1C2D curve with PBS 1C2D in FIG. 7D). Mixing with DEX only affected the height of the signal peak. This result supports the hypothesis that the casting process delays CMC recrystallization. Recrystallization may be due to the gradual loss of water during heating, resulting in an orderly and ordered molecular arrangement (Jeong et al., 2016). Crystallization is an important indicator of materials that retain moisture. An ordered array of molecules effectively resists the penetration of external water into the crystal structure and prevents evapotranspiration from the inside to the outside.

  A cytotoxicity test was performed to confirm the biocompatibility of the produced MN (FIG. 8). The MTT assay of the NIH-3T3 cell line confirmed that MN does not show toxicity and toxic substances accumulated in MN during the fabrication process. Cell viability exceeded 95% in both CMC alone and CMC / DEX mixtures compared to controls. We made MN for subsequent in vivo studies using CMC alone and CMC / DEX mixture (50% CMC + 50% DEX).

In vivo skin penetration test and drug release time test For in vivo skin penetration test, CMC / DEX mixture combined with BSA was used for MN fabrication. MN was allowed to attach to mice for 24 hours. Light microscopy of mouse skin sections stained with H & E clearly showed microneedle penetration on the surface (FIGS. 9A, 9B). IHC + H staining revealed release of BSA after penetrating the skin (dark brown Tracking (shown as) (FIGS. 9C, 9D). Further confirmation was performed by combining H & E and IHC + H staining to assess the integrity of the drug delivery method. The manufactured MN array could be used on the skin of nude mice, indicating that BSA penetration and release were successful. The fabricated MN array can be used on the skin of nude mice, indicating that BSA penetration and release was successful.

  For drug dissolution and diffusion time tracking, CMC alone or CMC / DEX mixture in combination with methylene blue was used for MN fabrication. After inserting MN into nude mice through a series of checkpoints, the methylene blue representing the drug was followed for release on the surface and diffusion to the skin. Frozen sections of mouse skin did not show a significant dye signal for the first 2 hours after treatment (FIG. 10A). After MN treatment, methylene blue diffusion and skin penetration were observed for 4 hours (FIGS. 10B and 10C). The CMC / DEX mixture showed a better release of methylene blue (FIG. 10D) compared to the release of pure CMC methylene blue (FIG. 10C).

Evaluation of MN dissolution
In order to evaluate the dissolution of MN, the appearance of the CMC needle was confirmed before and after insertion into the skin of nude mice (FIG. 11). No significant visual changes in needle appearance were observed after 4 hours (Figure 11A) and 8 hours (Figure 11B) treatment. After 24 hours of MN treatment, the appearance of the needles clearly changed (FIG. 11C). The needle became looser and softer as it was dissolved and absorbed by body fluids. The results also showed that MN was partially dissolved after treatment for up to 24 hours. Therefore, MN may be suitable for long-term release in drug delivery.

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Claims (4)

  A microneedle array for transdermal delivery of therapeutic agents, consisting of a polysaccharide-based combination of carboxymethylcellulose (CMC) and dextrin (DEX). The combination of polysaccharide-based, comprises 30 to 70% CMC and 30% to 70% of DEX, microneedle array according to claim 1. The microneedle array of claim 2 , wherein the polysaccharide-based combination comprises 50% CMC and 50% DEX. The microneedle array according to any one of claims 1 to 3 , wherein the therapeutic agent is insulin.

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