TWI688405B - Biodegradable microneedle array - Google Patents

Abstract

The present invention provide a method for fabricating a polysaccharide-based, dissolvable microneedle-based array, wherein the polysaccharide-based microneedle is made of a combination of 30-70% carboxymethyl cellulose (CMC) and 30-70% dextrin (DEX).

Description

Biodegradable microneedle array

The invention relates to a method for manufacturing a polysaccharide-based soluble microneedle array and a microneedle array prepared thereby.

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

To overcome the difficult SC barrier, several methods have been developed to enhance skin penetration for drug delivery. One method is to use a microneedle array, which shows better performance in delivering therapeutic drugs through the skin barrier without causing any damage (Kaushik et al., 2001; McAllister et al., 2003; Mikszta et al., 2002). The microneedle array (MN) is applied to the skin surface and penetrates the epidermis without stimulating nociceptors (Martin et al., 2012). These MNs create tiny holes through the skin and allow drugs or vaccines to diffuse into the skin's microcirculation (Cheung et al., 2014; Kim et al., 2012; Liu et al., 2012; Sullivan et al., 2010; Tuan-Mahmood et al., 2013). The MN array is made of various materials, such as polymers, metals, silicone rubber, 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 becoming more and more attractive because they are expected to be mass-produced cheaper than silicon or metal arrays, and are safer in application. When dissolving polymers are used, drugs and biomolecules can bind inside 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 the drug is released, the polymer dissolves in the skin, which avoids the risk of MN remaining in the body (Ke et al., 2012; Sullivan et al., 2008). Polymers used in MN applications should have characteristics such as biocompatibility, strong mechanical properties, and rapid dissolution rates. When considering the drug-dependent release time, polymers with different dissolution rates will be required to deliver a controlled amount of drug.

The solid MN is cast and is formed using biodegradable polymers as two types of structures: "single piece" and "layer by layer". In the "single piece" method, the solution containing the drug and polymer is injected and simultaneously cast on the same mold; while in the "layer by layer" method, the model structure is obtained by multiple coating steps or with each of the models The combination of flakes is cast. Organic solid MN extends the molecular weight range of synthetic and natural materials from hundreds to thousands, including dextrin (DEX), carboxymethyl cellulose (CMC), amylopectin (AMP), polylactide-co-glycolide Ester, galactose, maltose and polyvinylpyrrolidone (Lee et al., 2008; Raphael et al., 2010). According to their different molecular compatibility, many model compounds of different sizes can be added; for example, β-galactosidase, sulforhodamine, sodium salicylate, calcein and bovine serum albumin have been successfully Merged into MN. Over the past decade, preclinical evaluation of MN delivery with various drugs, bioactive proteins, and vaccines has shown progress (Pettis and Harvey, 2012). Animal and human studies have proven the application of various drugs and vaccines. However, there are relatively few published MN clinical studies (Pettis and Harvey, 2012). Including the stability of drugs or biomolecules encapsulated in MN, reliable insertion of MN into the skin, evaluation of skin irritation and skin infections, evaluation of pharmacokinetics and pharmacodynamics, and evaluation of pain and other perceptions require further detailed studies (Kim et al. People, 2012; Pettis and Harvey, 2012).

New transdermal delivery systems are still needed.

It was unexpectedly discovered in the present invention that the combination of CMC and dextrin DEX is used to manufacture biocompatible microneedle arrays (MN). Accordingly, the present invention provides a method of manufacturing a polysaccharide-based soluble microneedle array, which can be used as an invasive and transdermal delivery system. The microneedle array obtained by this method is safe and biocompatible, and can be used for transdermal administration of drugs such as insulin.

In one aspect, the present invention provides a method of manufacturing a microneedle array for transdermal delivery of a therapeutic agent, comprising: mixing carboxymethyl cellulose (CMC) and dextrin (DEX) to provide a polysaccharide-based combination; and The therapeutic agent is added to the polysaccharide-based combination.

In a specific embodiment of the present invention, the polysaccharide combination comprises 30 to 70% CMC and 30 to 70% DEX.

In an example of the present invention, the polysaccharide-based combination includes 50% CMC and 50% DEX.

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

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

In a specific 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, which is made of a combination of carboxymethyl cellulose (CMC) and dextrin (DEX) polysaccharides. The microneedle array is prepared by the method of the present invention.

For MN Array mold design

As shown in Figure 1, a high-speed cutting machine and a computer-aided design (CAD) system (AutoCAD 2012) are used to manufacture molds on aluminum (Al) plates. Based on the preliminary results, the aluminum mold was modified. An aluminum mold includes two array units to create two microneedle arrays during the casting process. There are four functional areas in the aluminum mold: (1) the needle area (NA); and (2) the blank area (BA); (3) the ridge area (RA); and (4) the connection groove (CT). The NA in the center of the mold has two sets of 10×10 tapered needle arrays. NA is surrounded by BA, providing thickness to the model to ensure toughness and avoid stress cracking when released from the mold. RA is used to separate each individual array unit, while CT helps the viscous solution flow slower and avoid air bubbles during rapid injection.

In order to analyze the composite material used for MN manufacturing, as shown in FIG. 2, the program is designed to have two test cycles, providing a solution for manufacturing the microneedle array according to the present invention. The first test cycle is based on the results of water content and X-ray diffraction (XRD) and thermogravimetric analysis (TGA). In addition, the first checkpoint is to test whether the compound mixture cannot be cut at temperatures up to 100°C. The second checkpoint tests whether the mixture is soluble. If the mixed solution is soluble, the next test will be a biotoxicity test. If there is no toxicity, test the flow coverage with a good film formation checkpoint of 5% mixed solution. After all the inspection points have been tested, the manufacture of MN is started. All manufacturing work with test films and MN is performed in a single "integrated" casting mold.

Polysaccharide material

Based on their biocompatibility and water solubility, we initially selected four different polysaccharides to analyze their physical properties to identify suitable molecules to make soluble MN. CMC, a cellulose derivative, is a water-soluble natural polysaccharide that has been widely used in various foods (Chillo et al., 2007) and medical applications. DEX contains water-soluble glucose polymers of different sizes produced by the hydrolysis of starch. DEX is used as a food additive and some pharmaceutical products in the food industry. Highly branched amylopectin (AMP) is a water-soluble polysaccharide, which is one of the two main components of starch. AMP is used in different applications, including food thickening. Trehalose (TRE) is a natural disaccharide that contains two glucose units connected by α,α-1,1-glucosidic bonds (Higashiyama, 2002). TRE is widely distributed in nature and is an attractive substance for industrial applications. This sugar is used as a sweetener in the food industry (Higashiyama, 2002).

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

For MN Physical characteristics of the manufactured polysaccharide

The water content of all polysaccharides was measured using an infrared moisture analyzer MA150, Sartorius. Approximately 0.1 g of each sugar in powder form was heated and analyzed for moisture percentage. TGA was performed using the Setaram Labsys-TGDSC DSC131 instrument to obtain the heating temperature range of the polysaccharide material during casting. Each sugar sample in 0.008g powder form was heated from room temperature to 500°C for TGA analysis at a heating rate of 5°C min-1. Materials in powder form are also used for XRD analysis, using a Rigaku© TTRAXIII X-ray powder diffractometer to obtain information about the crystallization and molecular arrangement of the material.

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

Microneedles( MN ) Casting

We use vacuum deposition for MN casting at 65°C. The polysaccharide solution (5% w/v) was prepared by dissolving them in phosphate buffer and sonicating for 60 minutes to completely disperse and dissolve. To remove air bubbles, the solution was centrifuged at 3500 rpm for 10 minutes at room temperature. Then the solution was slowly injected along the edge of the aluminum mold. The mold with the solution was kept under vacuum at room temperature for 15 to 20 minutes to make the bubbles upward and repeat until no visible bubbles were observed. The mold was heated from room temperature to 65°C and the stable temperature was 65°C for 24 hours. The heating rate is set to 1°C min-1.

The finished polysaccharide film or MN product is removed vertically from the aluminum mold by manual peeling. Badly formed products will not be completely peeled off. The selection criteria was set to be completely separated from the mold and the crack-free MN array.

made MN evaluation of

After casting, the physical properties of the polysaccharide film or manufactured microneedles were characterized by TGA and XRD analysis (see Section 2.3) and hardness testing.

According to the manufacturer's procedure guidelines, after separation from the mold, use the GARDCO © Wolff-Wilborn hardness pencil tester (Paul N. Gardner) to test the hardness of the finished product. This scratch hardness test determines the resistance of the polysaccharide film to the scratch effect of the surface.

We used Scanning Electron Microscope (SEM) and Optical Microscope (OM) on Hitachi©H-7100 microscope to determine the structure of MN.

Cytotoxicity

In vitro cytotoxicity of normal NIH 3T3 (mouse embryonic cell) cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay test. NIH 3T3 cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). For toxicity studies, before adding test compounds, cells with a density of 5,000 cells/well were seeded in 24-well dishes in DMEM containing 10% FBS and incubated at 37° C., 5% CO ₂ for 24 hours. The polymer was dissolved in PBS and filtered through a 0.22-μM filter. After 24 hours, 10 μl of a medium containing the test compound at a concentration of 0.05% (w/w) was added, and cultured at 37°C and 5% CO ₂ for 48 hours. The experiment was repeated three times, and cells containing no test compound served as a control group. After 48 hours, 200 μl of MTT solution (5 mg ml-1) dissolved in PBS was added to each well and incubated at 37°C for 4 hours. After removing the MTT-containing medium, the formazan crystals formed were dissolved in 150 μL of dimethyl sulfoxide and the absorbance at 540 nm was measured using a microplate analyzer. Use the following formula to determine the percentage of cell inhibition:% cell inhibition = 100-Abs (drug)/Abs (control group) × 100, where Abs refers to the absorbance at 540 nm.

In vivo skin permeability measurement

The nude mice used in this study, BALB/cAnN.Cg-Foxn1nu/CrlNarl, were obtained from the National Laboratory Animal Center of Taiwan. Bandage bundles were used to connect the MN to the mice. After treatment, the skin is surgically peeled off and prepared for tissue sectioning.

To track the release of protein after MN skin penetration, 1% BSA (bovine serum albumin, Sigma-Aldrich) powder was mixed into polysaccharide (50% CMC + 50% DEX) for casting. The skin tissue pieces were fixed in 4% paraformaldehyde and embedded in paraffin. The sections were stained with hematoxylin and eosin (H&E). Immunohistochemistry (IHC) was performed using antibodies against BSA (Millipore ^TM 07248).

In order to track the time of dye release from MN and diffusion into the skin, 0.25% methylene blue (MB) powder was mixed with polysaccharides (CMC alone, or 50% CMC + 50% DEX) for casting. The skin tissue pieces were frozen and prepared into frozen sections.

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

Examples

Examples 1 For MN Manufacturing materials investigation and selection

In this study, we propose a method to produce biodegradable MN for transdermal drug delivery. In order to select suitable materials, the polysaccharides CMC, DEX, TRE and AMP in powder form were analyzed to check their water content, crystallinity and thermal stability. The polysaccharides were mixed in different ratios to test their solubility in aqueous buffer. Subsequently, soluble polysaccharides, alone or in combination, were poured onto the mold surface to check their film formation. The cytotoxicity of individual polysaccharides was also tested.

The results of the water content test are shown in Table 1. Even when the material has been dried at 55°C for 24 hours, water molecules are present in all polysaccharides tested. The moisture content of the material and its affinity with water molecules prevent the material from completely drying out or losing moisture during the casting process, which ensures that the material is compatible with the incorporated drug or protein. Table 1 Water content of various polysaccharides

The individual sugars in powder form were analyzed by XRD and TGA to check their crystallinity and thermal stability. Crystallization features provide information about the molecular arrangement of materials and their ability to retain water. This information may be helpful when combining different polysaccharides. Use TGA to analyze the thermal properties to ensure that the material will be stable at the drying temperature used during the casting process.

All test materials showed significant crystalline signal peaks (Figure 3A). TRE produces the most obvious signal. DEX is a group of carbohydrates produced by the hydrolysis of starch and therefore shows a signal similar to AMP (Figure 3A). The lower signal and smoother curve of CMC indicate that there are more amorphous regions in CMC.

The mass loss caused by temperature is shown in Fig. 3B and Table 2. The first mass loss (90-130°C) depicts the critical temperature for moisture loss, while the second loss (over 240°C) reflects molecular decomposition. CMC and TRE showed a more significant decrease curve than AMP and DEX in water loss (Figure 3B). This result indicates that AMP and DEX have better moisture content in their powder form. This was confirmed by the water content test (Table 1). According to TGA data, polysaccharides are thermally stable in ordinary low-temperature MN preparation procedures for castings with temperatures between 55 and 75°C. Decreasing the casting temperature and increasing the drying time are as follows: 75°C for 15 hours, 65°C for 24 hours, and 55°C for 28 hours. Because the polysaccharide matrix and BSA are stable, the temperature for subsequent MN casting is set at 65°C. Table 2 Thermogravimetric analysis (TGA) of various polysaccharides showing their pyrolysis temperature

The solubility and viscosity of sugar will directly affect the quality of the cast product. The polysaccharides were mixed in different ratios to test their solubility in aqueous buffer. Among the various polysaccharide combinations tried, amylopectin (AMP) is not suitable for the manufacture of MN because of its low solubility in aqueous buffer and the need for continuous heating to dissolve it. The viscosity of the sugar solution increases at high concentrations. After pouring the sugar solution into the mold, the viscosity of the material affects the filling effect and affects the finished product. Highly viscous solutions cannot fill the mold and generate bubbles. Solutions with low viscosity may not provide sufficient thickness to the finished product, resulting in reduced physical properties. Therefore, finding the right viscosity is necessary to fill the mold surface. To determine the appropriate viscosity for pouring, use different concentrations of CMC (5-50% w/v) in the test tube, and visually observe the smooth flow of the liquid as the test tube rotates (Table 3). Only 5% solution can completely cover the test tube, while 10% and 20% solutions show partial coverage. The 30% and 50% solutions move slowly due to their high viscosity and cannot completely cover the test tube. We chose a 5% solution as a benchmark for further research. Table 3 Overview of flow coverage test by using different concentrations (5-50%) of carboxymethyl cellulose (CMC) to cover test tubes

Subsequently, we used the blank surface on the back of the aluminum mold to inspect the cast film using 5% TRE, DEX or CMC solutions (dried at 65°C for 24 hours). TRE produces thin and irregular translucent films that cannot be fixed into a sheet structure. DEX produced a relatively thick block that shattered into pieces. Therefore, TRE and DEX are not suitable for casting into MN array. They are considered to be used as a mixture with CMC.

TRE combined with CMC is difficult to separate from the mold surface (data not shown), resulting in surface scars and repeated washing rings. The decrease in the concentration of TRE combined with CMC does not produce a sufficiently thick polymer film, adheres to the mold and is difficult to separate, and generates some debris (data not shown). XRD analysis (Figure 3A) shows a huge difference in the degree of crystallinity between the two polysaccharides. TRE shows more crystalline and less amorphous regions, while CMC shows less crystalline and more amorphous regions (Figure 3A). TRE exists as small molecular weight molecules in the solution, and it shows cracks of large molecules in the crystal during the drying process, thus destroying the entire structure. Therefore, TRE is not suitable for manufacturing MN in combination with CMC.

In terms of its crystallographic signal (Figure 4A) and molecular size, DEX is similar to CMC; therefore, it may be more compatible with CMC during the casting process. CMC alone or in combination with DEX is considered for further research.

Examples 2 Pre-cast evaluation (viscosity test)

Conduct a viscosity test for the combination of CMC and DEX (Figure 6). Use three different aqueous buffers (deionized water, phosphate buffer, pH7.4 and PBS, pH7.4) to dissolve CMC and DEX at different ratios (100% CMC, 50% CMC + 50% DEX , 34% CMC + 66% DEX, 66% CMC + 34% DEX, and 60% CMC + 40% DEX). Mixing this material significantly changes the fluid properties. When the CMC ratio is higher, the viscosity increases (Figure 5). Due to the presence of salt in the buffer, the solvent will slightly increase the viscosity. Changing the solvent cannot reduce the viscosity. However, the solvent PBS significantly increased the viscosity of pure CMC. Finally, we chose PB for MN casting.

Examples 3 Polysaccharides MN Array manufacturing

Aluminum molds provide some advantages for model casting. It is compatible with traditional MN casting methods (Lee et al., 2008; Migalska et al., 2011; Park et al., 2016). Al ASTM 6061 (FDA allows it to be used as a food container) is a highly corrosion-resistant material for heavy-duty structures, and was used in this study to prepare reusable molds. To make MN, the polysaccharide mixture was poured on an Al mold and dried at 65°C for 24 hours. We use low temperature for casting because the final product will be produced with polysaccharides along with biomolecules and drugs. During the drying process, the thermal stability of the target molecule was considered (Figure 3B and Table 2). Fig. 5 shows SEM and OM observations of the polysaccharide MN, which has a length of about 480 μm, a base width of 370 μm, and a needle-center distance of 600 μm.

In this study, we used CMC and CMC/DEX mixtures alone to make MN. CMC is a naturally soluble polymer that is compatible with biologically active compounds, is relatively easy to handle at low temperatures, and is not expensive. It has been widely used in medical applications (Agarwal et al., 2015; Chen et al., 2015; Pasqui et al., 2014), including MN manufacturing (Kommareddy et al., 2012; Lee et al., 2017; Lee et al., 2008; Park et al., 2016).

Examples 4 made MN Features

Carry out a pencil hardness test to ensure the hardness of the finished product after separation from the mold. Table 4 lists the details of the pencil hardness test. The combination of CMC and DEX shows better hardness than CMC alone. The polysaccharide combination showed increased hardness in phosphate buffer than in deionized water.

表5 以各種多醣的製造的微針陣列之TGA分析，顯示其熱解溫度

TGA results show that the thermal properties of different combinations of CMC and DEX in aqueous buffer are stable up to 200°C (Figures 5A to 5C, Table 5). The curve of the first mass loss after the casting process shows a milder and continuous CMC and DEX curve (for film, see Figure 7A, compared to powder form see Figure 3B). However, when using PB or PBS as the solvent, the TGA curves of the cast film did not show significant differences (curves CMC, 1C1D, 1C2D, 2C1D in Figures 7A to 7C), and showed that there was no formation between the material and phosphate buffer Direct chemical reaction. The quality loss of the cast film is due to the loss of moisture. Table 4 Pencil hardness test of various combinations of polysaccharides in aqueous buffer

Table 5 TGA analysis of microneedle arrays made with various polysaccharides, showing their pyrolysis temperature

The XRD results of the polysaccharide film showed a sharp difference before and after the casting procedure (Figure 3A) and after (Figure 7D). Crystallization signals appear at 18°C to 21°C, 22°C to 24°C, and 40°C to 42°C (Figure 7D). Comparing the signal area and interval shows that the signal is mainly caused by CMC rather than DEX or solvent. XRD showed no additional crystallization signal of the solvent product (compare the curves of 3C2D and PBS 3C2D, and the curves of 1C2D and PBS 1C2D in Figure 7D). Mixing with DEX only affects the height of the signal peak. The results confirmed the hypothesis that the casting step caused the CMC to recrystallize slowly. The recrystallization may originate from the gradual loss of water during heating, which leads to the orderly arrangement of molecules (Jeong et al., 2016). Crystallization is an important indicator of materials that retain moisture. The neatly arranged molecular array can effectively resist the intrusion of external moisture into the crystal structure and prevent evapotranspiration from the inside to the outside.

A cytotoxicity test was conducted to check the biocompatibility of the manufactured MN (Figure 8). MTT assay on NIH-3T3 cell line confirmed that MN showed no toxicity during the manufacturing process, and no toxic substances accumulated in MN. Compared with the control group, the cell viability of CMC alone and CMC/DEX mixture exceeded 95%. We used a separate CMC and CMC/DEX mixture (50% CMC + 50% DEX) to prepare MN for subsequent in vivo studies.

Examples 5 In vivo skin penetration test and drug release time test

For in vivo skin penetration testing, the CMC/DEX mixture combined with BSA is used for MN manufacturing. MN was attached to the mice for 24 hours. Light microscopic observation of mouse skin sections stained with H&E clearly showed the penetration of microneedles on its surface (Figures 9A, 9B). IHC + H staining showed that after penetrating the skin (Figure 9C, 9D) the release of BSA was followed (highlighted in dark brown). Combining H&E and IHC + H staining further confirmed and evaluated the integrity of the drug delivery method. The fabricated MN array can be applied to the skin of nude mice, and the results show that BSA successfully penetrated and released.

For time tracking of drug dissolution and diffusion, CMC alone or a CMC/DEX mixture combined with methylene blue was used for MN manufacturing. After inserting MN into nude mice through a series of checkpoints, the methylene blue representing the drug was tracked to be released on the surface and spread into the skin. In the first 2 hours after treatment, frozen sections of mouse skin showed no significant dye signal (Figure 10A). After 4 hours of MN treatment, the diffusion of methylene blue and penetration into the skin were noticed (Fig. 10B, 10C). Compared to pure CMC (Figure 10D), the CMC/DEX mixture showed better methylene blue release (Figure 10C).

Examples 6 Assessment MN Dissolve

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no

The foregoing overview and the following detailed description of the invention will be better understood when reading in conjunction with the drawings.

In the drawings:

FIG. 1 provides an image showing a specific embodiment of the present invention that provides a microneedle array, including a polysaccharide-based microneedle array, which is prepared by a computer-aided design (CAD) system integrated with a high-speed cutting machine, which is cast in the middle of the mode Area; where NA represents the needle area, BA represents the blank area, and RA represents the ridge area; and CT represents the connection groove.

Figure 2 provides a solution for manufacturing a microneedle array according to the present invention.

Figure 3 provides diagrams for the analysis of different polysaccharides, including (A) thermogravimetric analysis (TGA) X-ray diffraction (XRD); and (B) analysis in raw powder form.

Figure 4 provides a graph showing the cytotoxicity test results of individual polysaccharides: carboxymethyl cellulose (CMC) and dextrin (DEX), trehalose, and the control group; NIH 3T3 cells were used in 3-(4,5 -Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was tested to test the toxicity of polysaccharides.

Figure 5 provides the carboxymethyl cellulose (CMC) and dextrin (DEX) in (A) deionized water, (B) phosphate buffer, pH 7.4 and (C) phosphate buffered saline, pH 7.4. Viscosity measurement diagram; where C represents a mixture containing 100% CMC (C), 1C1D represents a mixture containing 50% CMC and 50% DEX, 1C2D represents a mixture containing 34% CMC and 66% DEX, 2C1D represents a mixture containing 66% CMC and 34% DEX, and 3C2D represents a mixture containing 60% CMC and 40% DEX.

6 provides images of the microneedle array according to the present invention, including (A) scanning electron microscope (SEM) images and (B) optical microscope (OM) observations.

Figure 7 provides a graph showing the following results: (A) deionized water as a solvent; (B) phosphate buffer, pH 7.4, as a solvent; (C) phosphate buffered saline, pH 7.4, as a solvent heat Re-analysis (TGA) and X-ray diffraction (XRD); and (D) is an analysis of polysaccharide microneedles made of carboxymethyl cellulose (CMC) and dextrin (DEX), using deionized water or Phosphate buffered saline (PBS) as a solvent; C means a mixture containing 100% CMC (C), 1C1D means a mixture containing 50% CMC and 50% DEX, 1C2D means 34% CMC and 66% DEX 2C1D represents a mixture containing 66% CMC and 34% DEX, and 3C2D represents a mixture containing 60% CMC and 40% DEX.

FIG. 8 shows the cytotoxicity test of the microneedle array according to the present invention; wherein NIH3T3 cells were subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT ) Determination to test the toxicity of polysaccharides: carboxymethyl cellulose (CMC); a mixture of CMC and dextrin (CMC + dextrin), and a control group.

Fig. 9 shows the observation of tissue sections for in vivo skin penetration test under an optical microscope; where nude mice are treated with a microneedle array according to the present invention, and bovine serum albumin (BSA) is added as a test drug: (A) And (B) provide two representative images of hematoxylin and eosin (H&E) staining, where the arrows indicate the penetration of microneedles; (C) after penetrating the skin, immunohistochemistry and H&E (IHC + H) provides a representative image of the mouse tissue section after staining to track the release of BSA; (D) provides a close-up view of the IHC + H stained tissue showing BSA release (for (A) and (C), scale bar Is 50 μm, and 25 μm for (B) and (D)).

Figure 10 shows a representative frozen section of a time-tracked release of methylene blue (MB) dye released from polysaccharide microneedles and diffused into the skin. The mice were treated with microneedles containing MB for (A) for 2 hours, (B) for 4 hours, and (C) and (D) for 8 hours.

Figure 11 shows representative images of microneedle arrays before (above) and after (A) 4 hours, (B) 8 hours, and (C) 24 hours (lower image) before and after attachment to mice.

no

Claims (4)

A microneedle array for transdermal delivery of therapeutic agents is made from a combination of carboxymethyl cellulose (CMC) and dextrin (DEX) polysaccharides. The microneedle array according to claim 1, wherein the polysaccharide combination comprises 30 to 70% CMC and 30 to 70% DEX. The microneedle array as claimed in claim 1, wherein the polysaccharide combination comprises 50% CMC and 50% DEX. The microneedle array of claim 1, wherein the therapeutic agent is insulin.

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