Preparation method and application of methacrylated inulin hydrogel microneedle patch

Abstract

The invention relates to a preparation method and application of a methacrylated inulin hydrogel microneedle patch, which can effectively solve the problems of complex microneedle preparation process, low transmission efficiency and poor biocompatibility in the prior art. The preparation method comprises the following steps: mixing methacrylic anhydride, inulin polysaccharide and a photoinitiator, irradiating the mixture under a UV lamp to form a semi-gel state, placing the semi-gel state in a microneedle mold, and drying the semi-gel state to prepare the methylacryloylated inulin hydrogel microneedle patch. The preparation process is simple, the cost is low, the appearance arrangement is neat, the structure is complete, and the material has relatively strong mechanical properties and relatively good biological safety.

Description

I. Technical Field

[0001] The present invention relates to the technical fields of hydrogel materials and pharmaceutical preparations, in particular to the chemical synthesis of methacrylated inulin hydrogel, and a method and application for preparing a microneedle patch using the same. II. Background Art

[0002] As an emerging transdermal drug delivery system, the microneedle patch penetrates the skin stratum corneum and directly delivers drugs into the skin, avoiding the first-pass effect of the liver and gastrointestinal side effects. The length of microneedles is usually several hundred micrometers, which can penetrate the skin stratum corneum but do not touch the nerve endings, so painless or minimally painful drug delivery can be achieved. Compared with traditional subcutaneous injection or intravenous injection, the microneedle patch greatly reduces the pain of patients and improves the acceptance of patients. It improves the bioavailability and therapeutic effect of drugs. Its characteristics such as low invasiveness, painlessness, convenient operation, and controllable drug delivery make it have unique advantages in disease treatment.

[0003] There are many types of microneedles. Currently, the microneedles visible on the market include solid microneedles, hollow microneedles, coated microneedles, hydrogel microneedles, etc. Solid microneedles are usually made of materials such as metals (such as stainless steel, titanium alloy), silicon, or non-degradable polymers. They do not carry drugs themselves and are mainly used to penetrate the skin stratum corneum to form microchannels for drug delivery. However, the drug loading capacity is limited, and an additional drug dressing step is required, making it impossible to achieve precise drug delivery. Hollow microneedles are similar to micrometer-scale syringes and have a hollow structure. Drugs can be directly loaded inside the needle body and released after the needle tip pierces the skin. However, the preparation process is complex, the cost is high, the needle body is relatively fragile, and it is easy to clog or break. Coated microneedles attach drugs to the surface of microneedles through infiltration, coating, or spraying techniques. After the microneedles penetrate the skin, the drugs dissolve and are released in the skin. The drug loading capacity is limited, and the coating on the needle body may affect the sharpness of the microneedles, and some drugs may remain in the stratum corneum. Soluble microneedles are made of biodegradable or soluble polymers. After the microneedles penetrate the skin, they gradually dissolve and release drugs. However, a high mechanical strength of the material is required, otherwise the microneedles may not be able to effectively penetrate the skin. Hydrogel microneedles are made of cross-linked polymer materials. After the microneedles penetrate the skin, they absorb interstitial fluid and swell to form micropores to release drugs. They have the advantages of high drug loading capacity, the ability to achieve continuous drug release, and the drug release rate can be controlled by adjusting the cross-linking density. They are suitable for scenarios where long-term slow-release drugs are required, such as the treatment of chronic diseases.

[0004] Hydrogel is a three-dimensional network structure material formed by hydrophilic polymer chains through chemical or physical cross-linking. It has unique physical and chemical properties, which endow it with broad application potential in multiple fields. The main properties of hydrogels include: (1) High water absorbency and swelling ability. Hydrogels can absorb a large amount of water or biological liquids and swell, but do not dissolve, while maintaining their three-dimensional network structure. This property makes it perform excellently in fields such as drug delivery and wound dressings. (2) Biocompatibility and biodegradability. Hydrogels have good biocompatibility, can reduce friction and mechanical effects on surrounding tissues, and are suitable for applications such as tissue engineering and drug release carriers. (3) Mechanical properties. Traditional hydrogels have weak mechanical properties due to uneven network and cross-linking distribution. However, in recent years, through the introduction of new cross-linking mechanisms and material designs, the mechanical properties of hydrogels have been significantly improved. Hydrogel microneedles are usually made of non-toxic and biodegradable polymer materials and have good biocompatibility. Hydrogel microneedles have sufficient hardness to penetrate the skin in a wet state, and at the same time can maintain a certain mechanical strength after insertion into the skin and can be completely removed after use, without leaving polymers in the body, reducing the risks caused by residues in the body.

[0005] Prebiotic inulin polysaccharide is a natural fructan, belonging to non-digestible carbohydrates. It is a linear straight-chain polysaccharide formed by D-fructofuranose linked by β-2,1 glycosidic bonds, and often has a glucose residue at the end, belonging to a polyhydroxy structure. After reacting with methacrylic anhydride (MA), a C=C double bond is introduced. This double bond is derived from the methacrylate group (CH2=C(CH3)-COO-), and has a photocuring effect after adding a photoinitiator.

[0006] Transdermal drug delivery is a drug delivery method that can avoid the first-pass effect and gastrointestinal reactions of oral drugs and relieve the pain of patients. However, due to the barrier of the skin stratum corneum, the efficiency of traditional transdermal delivery is relatively low. Microneedle technology can deliver drugs into the skin by penetrating the stratum corneum to form microchannels, and is highly ingeniously designed to avoid contacting blood vessels and nerve fibers under the dermis, achieving the effects of minimally invasive, painless, and bloodless. In recent years, methacrylated materials have been widely used in the biomedical field, such as injectable hydrogels and microsphere materials. However, there are currently no reports on the application of methacrylated inulin in the preparation of hydrogel microneedles and drug delivery. III. Summary of the Invention

[0007] In view of the above situation, to solve the defects of the existing technology, the purpose of the present invention is to provide a preparation method and application of a methacrylated inulin hydrogel microneedle patch, which can effectively solve the problems of complex microneedle preparation process, low transmission efficiency, and poor biocompatibility in the existing technology.

[0008] One of the technical solutions provided by the present invention is a methacrylated inulin hydrogel microneedle patch, which is prepared by mixing methacrylic anhydride with inulin polysaccharide and a photoinitiator, irradiating under a UV lamp to form a semi-gel state, and then drying in a microneedle mold.

[0009] The inulin polysaccharide described is prebiotic inulin polysaccharide.

[0010] Another technical solution provided by the present invention is a preparation method of a methacrylated inulin hydrogel microneedle patch, comprising the following steps:

[0011] 1) Add 15 mL of ultrapure water to a conical flask, add a rotor and stir. During stirring, add 10 g of inulin to dissolve.

[0012] 2) Weigh 200 - 600 mg of succinic anhydride SA and dissolve it in a certain volume of N,N-dimethylformamide (DMF), and then add it to the system of step 1).

[0013] 3) Adjust the pH to 8.5 - 9 with NaOH solution and stir at room temperature for 6 - 10 h.

[0014] 4) Then weigh 800 - 1400 mg of methacrylic anhydride MA and drop it into the reaction system of step (3) (rinse the MA on the wall of the tube with the reaction solution), adjust the pH to 8.5 - 9 with sodium hydroxide solution, and continue to stir and react at room temperature in the dark for 6 - 12 h.

[0015] 5) Collect the reaction solution and dialyze it in a dialysis bag for 48 h. Dialyze at room temperature for the first 12 h and in an ice bath for the next 36 h. Change the water every 12 h during dialysis.

[0016] 6) After dialysis, freeze at -80 °C and lyophilize with a freeze dryer to obtain a methacrylated inulin hydrogel material. Dissolve the prepared material with a photoinitiator and irradiate it under a UV lamp for 3 - 5 s to form a semi-gel state, and then place it in a microneedle mold and dry at room temperature to demold, thus obtaining a methacrylated inulin hydrogel microneedle patch.

[0017] Preferably, the inulin concentration in step 1) > 76 mM.

[0018] Preferably, the concentration of succinic anhydride SA in step 2) is 80 - 240 mM to increase the water solubility of the system.

[0019] Preferably, the concentration of methacrylic anhydride MA in step 4) is 207.8 - 363.6 mM.

[0020] Preferably, the molecular weight of the dialysis bag in step 5) is 500 D.

[0021] Preferably, the photoinitiator used in step 6) is LAP, and the concentration is 3 mg / mL.

[0022] Preferably, in step 6), the LAP initiator is dissolved in ultrapure water in the dark, pipetted and mixed well to prepare 3 mg / mL, then 600 mg of methacrylated inulin hydrogel material is weighed and added with 1 mL of initiator, and dissolved fully in the dark to obtain a methacrylated inulin hydrogel microneedle matrix. Then, the methacrylated inulin hydrogel microneedle matrix is irradiated with ultraviolet light for 4 s to form a semi-gel state, added into a TM63 type microneedle mold, centrifuged at 3500 rpm for 3 min, the backing layer is scraped off, dried at 40 °C for 30 min, then InuMA hydrogel material is added, centrifuged at 3500 rpm for 3 min, then 40% PVA is added, centrifuged at 3500 rpm for 3 min, and then PVA is added again. After drying at room temperature, the mold is removed to obtain a methacrylated inulin hydrogel microneedle patch.

[0023] The third technical solution provided by the present invention is the application of the methacrylated inulin hydrogel microneedle patch of the present invention in the preparation of a drug delivery system.

[0024] The beneficial effects of the present invention are as follows:

[0025] 1) The methacrylated inulin hydrogel microneedle patch prepared by the present invention has a simple preparation process, low cost, short time consumption, simple operation, and good biocompatibility.

[0026] 2) The inulin prebiotic polysaccharide used in the present invention has the functions of inhibiting the growth of saprophytic bacteria, reducing the production of toxic products, and improving the body's immunity, and has the advantages of good biological safety and low price.

[0027] 3) The method for preparing the microneedles of the present invention is simple in operation, only simple centrifugal drying is required, and the external morphology, stability, and mechanical properties of the microneedles are all good. IV. DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the infrared characterization diagram of Inulin, InuSA obtained by combining SA, and InuMA-L with low substitution degree and InuMA-H with high substitution degree obtained by combining different ratios of MA in the present invention.

[0029] Figure 2 It is the comparison diagram of 1H NMR spectra of Inulin, InuSA, InuMA-L, and InuMA-H in the present invention.

[0030] Figure 3 It is the SEM diagram of InuMA-L and InuMA-H in the present invention.

[0031] Figure 4This is the optical microscope image of the InuMA microneedles of the present invention.

[0032] Figure 5 This is the SEM image of the InuMA microneedles of the present invention.

[0033] Figure 6 This is the force-displacement curve of the microneedles of the present invention.

[0034] Figure 7 This is the skin insertion image of the InuMA microneedles of the present invention.

[0035] Figure 8 This is the cytotoxicity test result image of Inulin and InuMA of the present invention.

[0036] Figure 9 This is the hemolysis rate test result image of Inulin and InuMA of the present invention. V. Specific Embodiments

[0037] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments and the drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0038] Example 1

[0039] A preparation method of a methacrylated inulin hydrogel microneedle patch, comprising the following steps:

[0040] 1) Add 15 mL of ultrapure water to a conical flask, add a rotor and stir. During the stirring process, add 10 g of inulin (Inulin) with a concentration of 20 mM for dissolution;

[0041] 2) Weigh 600 mg of succinic anhydride SA and dissolve it in 10 mL of N,N-dimethylformamide (DMF), and then add it to the system in step 1);

[0042] 3) Adjust the pH to 8.5 - 9 with NaOH solution and stir at room temperature for 10 h;

[0043] 4) Then weigh 1386 mg of methacrylic anhydride MA and drop it into the reaction system in step 3), adjust the pH to 8.5 - 9 with sodium hydroxide solution, and continue to stir and react at room temperature in the dark for 12 h;

[0044] 5) Collect the reaction solution and dialyze it in a 500D dialysis bag for 48 h. Dialyze at room temperature for the first 12 h and in an ice bath for the next 36 h. Change the water every 12 h during the dialysis process;

[0045] 6) After dialysis, it is frozen at -80°C and freeze-dried by a freeze dryer to obtain methacrylated inulin hydrogel material (InuMA). The prepared material is dissolved with 3 mg / mL initiator LAP and irradiated under a UV lamp for 3 - 5 s to form a semi-gel state, and then placed in a microneedle mold and dried at room temperature to demold, thus obtaining a methacrylated inulin hydrogel microneedle patch.

[0046] The prebiotic inulin polysaccharide of the present invention has the effects of inhibiting the growth of saprophytic bacteria, reducing the production of toxic products and enhancing immunity. Succinic anhydride can increase the water solubility of the system. The used succinic anhydride SA is 6 mmol to increase the water solubility of the system. Adjusting the pH of the NaOH solution to between 8.5 and 9 can promote the formation of the esterification reaction. The photocuring time of the prepared microneedles only needs 4 s. The drying at room temperature is simple in operation and short in time-consuming. The preparation process is simple, low in cost, neat in appearance arrangement, complete in structure, with strong mechanical properties and good biosafety. The relevant test data are as follows:

[0047] Experiment 1 Preparation of Methacrylated Inulin Hydrogel Microneedles

[0048] Materials: Inulin polysaccharide (from Beijing Innochem Science & Technology Co., Ltd.)

[0049] Succinic anhydride (from Shanghai Macklin Biochemical Co., Ltd.)

[0050] N,N-Dimethylformamide (from Tianjin Damao Chemical Reagent Factory)

[0051] Methacrylic anhydride (from Shanghai Aladdin Biochemical Technology Co., Ltd.)

[0052] (1) Preparation of Methacrylated Inulin Hydrogel Material

[0053] (a) After dissolving inulin in water, succinic anhydride (SA) and N,N-dimethylformamide (DMF) are added, the pH is adjusted to between 8.5 and 9.0 and stirred for 10 h, and then methacrylic anhydride (MA) is added and the pH is adjusted to between 8.5 and 9.0, and stirred in the dark for 12 h.

[0054] (b) The reaction solution is collected and dialyzed in a 500D dialysis bag to finally obtain a dialysate; and the dialysate is frozen at -80°C and then freeze-dried by a freeze dryer to obtain methacrylated inulin hydrogel material InuMA.

[0055] From Figure 1It can be seen from the infrared spectrum that in the infrared spectrum of pure Inulin, there is an -OH absorption peak of polysaccharide near 3480 nm; after the reaction with succinic anhydride, the infrared spectrum changes significantly, and there is a carbonyl C=O absorption peak near 1732 nm; after the reaction with methacrylic anhydride MA, a C=C absorption peak appears near 1641 nm, and the peak of InuMA-H is more obvious.

[0056] From Figure 2 It can be seen from the 1H NMR spectrum that InuMA-L and InuMA-H show a C=C characteristic peak at 6.11 ppm.

[0057] Combined with Figure 1 and Figure 2 It can be proved that the preparation method of the present invention successfully synthesizes the methacrylated inulin hydrogel material.

[0058] (2) Preparation of methacrylated inulin hydrogel microneedle matrix.

[0059] Under room temperature conditions, weigh the LAP initiator, dissolve it in ultrapure water in the dark and mix well by pipetting to obtain 3 mg / mL. Then weigh 600 mg of the methacrylated inulin hydrogel material InuMA and add 1 mL of the initiator LAP, and dissolve it fully in the dark to obtain the methacrylated inulin hydrogel microneedle matrix.

[0060] Figure 3 It can be seen that the pore channels of InuMA-H are larger. In actual operation, it is found that the photocuring effect of InuMA-H is better and the time consumption is shorter.

[0061] (3) Preparation of methacrylated inulin hydrogel microneedle patch

[0062] The prepared methacrylated inulin hydrogel microneedle matrix is irradiated with ultraviolet light for 4 s to form a semi-gel state. Then add it to the A158 microneedle mold, centrifuge at 3500 rpm for 3 min, scrape off the backing layer, dry at 40 °C for 30 min, then add the InuMA hydrogel material, centrifuge at 3500 rpm for 3 min, then add 40% PVA, centrifuge at 3500 rpm for 3 min, and then add PVA again. After drying at room temperature, demold to obtain the methacrylated inulin hydrogel microneedles.

[0063] From Figure 4 and Figure 5 It can be seen that the microneedles prepared from InuMA have good appearance morphology, are arranged neatly, and the needle tips are complete.

[0064] Experiment 2 is the skin puncture test of the InuMA microneedle patch

[0065] After depilating the back of the mouse, cut the skin and fix it on a relatively hard foam board. Place the InuMA microneedles on the skin surface and press with the thumb for about 2 minutes. Then remove the microneedle backing layer, and the marks left by the microneedles piercing the skin can be seen.

[0066] From Figure 6 The test results show that the InuMA microneedles have good mechanical properties, about 0.2 N / needle.

[0067] Figure 7 The marks left by the InuMA microneedles after piercing the skin can be seen, indicating that it has the ability to pierce the skin.

[0068] Experiment 3 is the cytotoxicity test of Inulin and InuMA

[0069] The test cells include Huvec, 3T3, and SCC-7 cells. They are seeded in 96-well plates and cultured in an incubator at 37°C with 5% CO2. Huvec and 3T3 are cultured in DMEM medium containing 10% serum, and SCC-7 cells are cultured in 1640 medium containing 10% serum. After 24 hours, different concentrations of Inulin and InuMA with the same volume are added and cultured for another 24 hours. Then the supernatant is discarded, and MTT medium is added for incubation for 2.5 hours. Then the supernatant is discarded again, and dimethyl sulfoxide is added to measure the absorbance at a wavelength of 490 nm and calculate the cell survival rate. The cell survival rate = (OD of the experimental group - OD of the blank group) / (OD of the microneedle group - OD of the blank group) * 100%. Based on the fact that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-violet crystalline formazan and deposit it in the cells, this process only occurs in living cells. Since dead cells do not have this function, then DMSO is used to dissolve the formazan in the cells, and the number of living cells is indirectly reflected through enzyme-linked immunosorbent assay.

[0070] From Figure 8 The test results show that the cell survival rates of Inulin and InuMA for Huvec cells, 3T3 cells, and SCC-7 cells are all > 80%, indicating that Inulin and InuMA have no obvious toxicity to cells and have good biosafety.

[0071] Experiment 4 is the hemolysis test of Inulin and InuMA

[0072] Take about 1 mL of anticoagulant in a 10 mL centrifuge tube. Take 1 mL of mouse eyeball blood and dilute it with normal saline. Centrifuge at 3000 rpm for 5 min and repeat this operation 2 - 3 times until the supernatant is clear. Then discard the supernatant and dilute the red blood cells with normal saline to a certain concentration. Take 400 μL of the red blood cell suspension and incubate it in 600 μL of Inulin and InuMA solutions with different concentrations for 4 h. Then centrifuge at 10000×g for 5 min. Take 100 μL of the supernatant and place it in a 96 - well plate. Measure the absorbance at a wavelength of 577 nm and calculate the hemolysis rate. At the same time, set normal saline as the negative control group and DDW as the positive control group. Hemolysis rate = (OD of experimental group - OD of negative control group) / (OD of positive control group - OD of negative control group) * 100%.

[0073] Figure 9 The hemolysis test results show that neither Inulin nor InuMA has obvious hemolysis phenomenon, which fully indicates that both Inulin and InuMA have high biosafety.

[0074] The present invention has carried out a number of performance tests on the methacrylated inulin hydrogel material and InuMA microneedle patch prepared in the above examples. The cytotoxicity test and hemolysis test prove that InuMA has high biosafety. The appearance and mechanical property detection of the microneedles show that the InuMA microneedle patch not only has a complete appearance but also has strong mechanical properties.

[0075] In summary, although the present invention has encountered many difficulties in the above - mentioned preparation process, such as precipitation occurring during the cross - linking process of using methacrylic anhydride and inulin alone, resulting in an unclear photocuring effect of the obtained material. However, after introducing succinic anhydride SA, the problem of poor water solubility is significantly solved, and the methacrylated inulin hydrogel material InuMA is successfully prepared. Then, by adjusting the drug ratio, a hydrogel material that can produce a photocuring effect in only 4 s is successfully obtained. Finally, the methacrylated inulin hydrogel microneedle patch prepared by the present invention has a simple preparation process, low cost, neat appearance arrangement, complete structure, strong mechanical properties and good biosafety.

[0076] It should be noted that the above is only a preferred embodiment of the present invention, and it does not impose any form of limitation on the present invention. Any person skilled in the relevant art, without departing from the scope of the technical solution of the present invention, can make changes or modifications to equivalent embodiments by using the disclosed technical content, and all fall within the protection scope of the present invention.

Claims

1. A methacrylated inulin hydrogel microneedle patch, characterized in that, It is prepared by mixing methacrylic anhydride with inulin polysaccharide and a photoinitiator, irradiating under a UV lamp to form a semi-gel state, and then drying in a microneedle mold.

2. The methacrylated inulin hydrogel microneedle patch according to claim 1, wherein The inulin polysaccharide described is prebiotic inulin polysaccharide.

3. The preparation method of the methacrylated inulin hydrogel microneedle patch according to claim 1, characterized in that, It includes the following steps: 1) Add 15 mL of ultrapure water to a conical flask, add a rotor and stir. During the stirring process, add 10 g of inulin to dissolve. 2) Weigh 200 - 600 mg of succinic anhydride SA and dissolve it in 10 mL of N,N-dimethylformamide, and then add it to the system in step 1). 3) Adjust the pH to 8.5 - 9 with NaOH solution and stir at room temperature for 6 - 10 h. 4) Then weigh 800 - 1400 mg of methacrylic anhydride and dropwise add it to the reaction system in step 3). Adjust the pH to 8.5 - 9 with sodium hydroxide solution and continue to stir and react in the dark at room temperature for 6 - 12 h. 5) Collect the reaction solution and dialyze it in a dialysis bag for 48 h. Dialyze at room temperature for the first 12 h and in an ice bath for the next 36 h. Change the water every 12 h during the dialysis process. 6) After dialysis, freeze at -80 °C and lyophilize with a freeze dryer to obtain the methacrylated inulin hydrogel material. Dissolve the prepared material with a photoinitiator and irradiate it under a UV lamp for 3 - 5 s to form a semi-gel state. Then place it in a microneedle mold and dry at room temperature to demold, and the methacrylated inulin hydrogel microneedle patch can be obtained.

4. The preparation method of the methacrylated inulin hydrogel microneedle patch according to claim 3, characterized in that, The inulin concentration in step 1) is > 76 mM.

5. The preparation method of the methacrylated inulin hydrogel microneedle patch according to claim 3, wherein, The concentration of succinic anhydride SA in step 2) is 80 - 240 mM, which increases the water solubility of the system.

6. The preparation method of the methacrylated inulin hydrogel microneedle patch according to claim 3, wherein, The concentration of methacrylic anhydride MA in step 4) is 207.8 - 363.6 mM.

7. The preparation method of the methacrylated inulin hydrogel microneedle patch according to claim 3, characterized in that, The molecular weight of the dialysis bag in step 5) is 500 Da.

8. The preparation method of the methacrylated inulin hydrogel microneedle patch according to claim 3, characterized in that The photoinitiator used in step 6) is LAP, and the concentration is 3 mg / mL.

9. The preparation method of the methacrylated inulin hydrogel microneedle patch according to claim 3, characterized in that In step 6), the LAP initiator is dissolved in ultrapure water in the dark, blown and mixed evenly to obtain 3 mg / mL. Then weigh 600 mg of the methacrylated inulin hydrogel material, add 1 mL of the initiator and dissolve it fully in the dark to obtain the methacrylated inulin hydrogel microneedle matrix. Then irradiate the methacrylated inulin hydrogel microneedle matrix with ultraviolet light for 4 s to form a semi-gel state, add it to a TM63 type microneedle mold, centrifuge at 3500 rpm for 3 min, scrape off the backing layer, dry at 40 °C for 30 min, then add the InuMA hydrogel material, centrifuge at 3500 rpm for 3 min, then add 40% PVA, centrifuge at 3500 rpm for 3 min, and then add PVA again. After drying at room temperature and demolding, the methacrylated inulin hydrogel microneedles can be obtained.

10. Use of the methacrylated inulin hydrogel microneedle patch according to any one of claims 1 - 9 in the preparation of a drug delivery system.

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