A hydrogel microneedle and a preparation method thereof

Abstract

The application belongs to the technical field of hydrogel microneedles, and relates to a hydrogel microneedle and a preparation method thereof, and the preparation method comprises the following steps: placing a methacrylated chitosan solution in a centrifugal tube, adding a photoinitiator, vortexing and ultrasonicating until the photoinitiator is completely dissolved to obtain a hydrogel microneedle filling solution; and taking the hydrogel microneedle filling solution and injecting the hydrogel microneedle filling solution into a PDMS microneedle mold, centrifuging, irradiating with an ultraviolet lamp and drying.The hydrogel microneedle prepared from methacrylated chitosan has superior performance in wound closure, re-epithelialization and hair follicle formation, and the hydrogel microneedle is combined with a rhodioside drug depot to construct a transdermal drug delivery system, the drug delivery system has high drug loading and no polymer deposition, can efficiently transdermally deliver rhodioside, promotes wound closure and tissue remodeling, and provides a potential strategy for wound treatment.

Description

Technical Field

[0001] This invention belongs to the field of hydrogel microneedle technology, and particularly relates to a hydrogel microneedle and its preparation method. Background Technology

[0002] Currently, the most common treatment for acute and chronic wounds is to select different types of wound dressings for different wounds. Traditional wound dressings, such as sterile gauze and bandages, can only cover the wound surface and absorb exudate, playing no positive role in wound healing and skin regeneration. They may also adhere to newly formed tissue, causing pain upon removal. Modern wound dressings, including hydrocolloids, hydrogels, alginates, foams, and films, still have various limitations in their application. Furthermore, necrotic tissue or bacterial biofilms at the wound site significantly restrict the penetration of drugs through these barriers to exert their therapeutic effects.

[0003] The needle-like structure of microneedles can overcome these barriers, effectively delivering drugs and increasing effective drug concentrations at the wound site. However, some challenges remain in the preparation and application of microneedles. For example, microneedle patches are arrays of micron-sized needles, resulting in low drug loading within the needles. Furthermore, continuous use of soluble microneedles may lead to significant polymer deposition in the skin. Therefore, improving the drug efficacy of microneedles without leaving polymer residues in the skin remains a challenge in microneedle development. Summary of the Invention

[0004] To address the shortcomings of the existing technology, this invention provides a hydrogel microneedle and its preparation method, the specific technical solution of which is as follows:

[0005] The first objective of this invention is to provide a method for preparing hydrogel microneedles, comprising the following steps:

[0006] Methacrylamide chitosan water (CSMA) was placed in a centrifuge tube, a photoinitiator was added, and the mixture was vortexed and sonicated until the photoinitiator was completely dissolved to obtain a hydrogel microneedle filling solution. The hydrogel microneedle filling solution was injected into a PDMS microneedle mold and centrifuged at 4000-6000 rpm for 5-20 min. Finally, the mold was irradiated with a UV lamp with a wavelength of 300-380 nm for 0.25-3 min and dried at 20-40℃. The hydrogel microneedles (HFMN) were obtained after demolding.

[0007] The structural formula of the methacrylamide chitosan is shown in formula (1):

[0008]

[0009] Under the action of a photoinitiator and after ultraviolet light irradiation, the photosensitive group amide double bond can undergo a free radical polymerization reaction, covalently cross-linking to form a hydrogel. Hydrogel microneedles can be prepared in this way, based on the principle as follows: Figure 1As shown. The hydrogel microneedles prepared by this invention have a complete appearance, beautiful needle shape, sharp needle tip, and a four-sided pyramidal needle body with high mechanical strength, which can overcome the barrier of the stratum corneum and meet the requirements for skin penetration. The needle-like structure of the hydrogel microneedles penetrates the stratum corneum in a minimally invasive and painless manner, and rapidly absorbs tissue fluid when inserted into the skin. At the same time, chitosan has a number of valuable properties, such as hemostasis, antibacterial properties, and biocompatibility, and can be used to treat various wounds, especially chronic wounds, infections, or ulcers. Therefore, the hydrogel microneedles of this invention have antibacterial properties, can break through necrotic tissue and bacterial biofilms, and maintain a moist environment in the wound, which is conducive to promoting the generation of epithelial cells and skin tissue remodeling. Therefore, the hydrogel microneedles have superior performance in wound closure, re-epithelialization, and hair follicle formation, and have potential application prospects in wound healing.

[0010] Furthermore, the degree of substitution of the methacrylamide chitosan is 10-50%, the concentration of the methacrylamide chitosan solution is 3-5 wt%, and the concentration of the photoinitiator is 0-0.15 wt%.

[0011] Furthermore, the photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (Irgacure2959).

[0012] Furthermore, the vortex duration is 1-5 minutes, the ultrasonic power is 100-300W, and the ultrasonic time is 10-20 minutes.

[0013] Furthermore, the method for preparing the methacrylamide chitosan includes the following steps:

[0014] (1) Preparation of chitosan solution: Add 1-3wt% acetic acid solution to chitosan, stir at room temperature and speed of 400-800 rpm for 0.5-2h until chitosan is completely dissolved, to prepare a chitosan solution with a concentration of 2-4wt%.

[0015] (2) Preparation of methacrylated chitosan: Take a chitosan solution and place it in a reaction vessel. Protect it from light and slowly add methacrylic anhydride while stirring. The molar ratio of chitosan to methacrylic anhydride is (1-4):1. Continue stirring at 25-60℃ for 6-24 hours. Then slowly add NaHCO3 solution to the reaction solution. Adjust the pH of the reaction solution to neutral and dialyze. After dialyzing, pre-freeze the solution at -70 to -100℃ for 1-3 hours and then transfer it to -50 to -70℃ for freeze-drying for 3-4 days. After completion, store the freeze-dried CSMA at -20℃ for later use.

[0016] The preparation of methacrylated chitosan in this invention utilizes the reaction between the amino group (-NH2) of chitosan and the double bond (C=C) of methacrylic anhydride to introduce a photoresponsive group, namely a methacryloyl group, thus preparing a photoresponsive chitosan. The acylation reaction equation of chitosan is as follows:

[0017]

[0018] Furthermore, the NaHCO3 solution has a mass fraction of 10%.

[0019] Furthermore, the dialysis method involves pouring the neutral reaction solution into a dialysis bag with a molecular weight cutoff of 3500, placing the dialysis bag in a 5L beaker, and dialyzing for 5 days, changing the water 3 times a day.

[0020] The second objective of this invention is to provide a hydrogel microneedle prepared by the above-described method for preparing hydrogel microneedles.

[0021] Furthermore, the hydrogel microneedles also include a rhodioloside drug reservoir, comprising, by weight, 5-30 parts rhodioloside, 5-25 parts excipients, and 45-90 parts water; the excipients are at least one of gelatin, methylcellulose, carboxymethylcellulose, carboxyethylcellulose, sodium carboxymethyl starch, polyvinylpyrrolidone K30 (PVP K30), sorbitol, and glycerin.

[0022] In use, the rhodioloside drug reservoir is attached to the backing of the hydrogel microneedle, and the rhodioloside drug reservoir can be placed on the hydrogel microneedle and fixed with medical tape.

[0023] A transdermal drug delivery system was constructed by combining hydrogel microneedles prepared from methacrylamide chitosan with a rhodioloside drug reservoir. Upon insertion into the skin, the hydrogel microneedles rapidly absorb tissue fluid. A continuous pathway is formed between the rhodioloside drug reservoir attached to the backing of the hydrogel microneedles and the dermal microcirculation, allowing the drug to enter the dermis through this pathway. This effectively promotes drug penetration and significantly improves the release and permeation behavior of rhodioloside. Therefore, the drug delivery system combines the anti-inflammatory and antioxidant properties of rhodioloside with the antibacterial properties of chitosan hydrogel microneedles, their ability to penetrate necrotic tissue and bacterial biofilms, and their ability to maintain a moist wound environment, which is beneficial for promoting epithelial cell regeneration and skin tissue remodeling.

[0024] Furthermore, the rhodioloside storage is a rhodioloside freeze-dried storage, prepared as follows: Rhodioloside, excipients, and water are added to a centrifuge tube and mixed evenly; then, at 20-30℃, ultrasonic treatment is performed for 10-30 minutes at an ultrasonic power of 60-80Hz to obtain a formulation solution; the formulation solution is poured into a mold and pre-frozen at -70 to -100℃ for 1-3 hours, then transferred to a cold environment of -50 to -70℃ and a vacuum degree of 10-20 Pa for freeze-drying for 20-30 hours; after drying, the freeze-dried storage is peeled off from the mold to obtain the rhodioloside freeze-dried storage.

[0025] Furthermore, the rhodioloside reservoir is a rhodioloside hydrogel reservoir, which is prepared as follows: rhodioloside, excipients and water are added to a centrifuge tube and mixed evenly; then, at 20-30℃, ultrasonic treatment is performed for 10-30 minutes with an ultrasonic power of 100-300W to obtain a formulation solution; the formulation solution is poured into a mold, and after solidification at room temperature, the hydrogel reservoir is peeled off from the mold to obtain the rhodioloside hydrogel reservoir.

[0026] Furthermore, the rhodioloside freeze-dried storage container, by weight, comprises 10-30 parts of rhodioloside, 2-5 parts of sodium carboxymethyl starch, 5-10 parts of PVP K30, 4-6 parts of sorbitol, and 50-90 parts of water; preferably, it comprises 10 parts of rhodioloside, 3 parts of sodium carboxymethyl starch, 10 parts of PVP K30, 5 parts of sorbitol, and 72 parts of water.

[0027] Furthermore, the rhodioloside hydrogel reservoir, by weight, comprises 10-30 parts rhodioloside, 10-20 parts gelatin, 1-3 parts glycerin, 4-6 parts sorbitol, and 50-70 parts water; preferably, it comprises 10 parts rhodioloside, 15 parts gelatin, 2 parts glycerin, 5 parts sorbitol, and 68 parts water.

[0028] The beneficial effects of this invention are as follows:

[0029] The hydrogel microneedles prepared from methacryloylchitosan of this invention possess antibacterial properties, can penetrate necrotic tissue and bacterial biofilms, and maintain a moist environment in wounds, which is beneficial for promoting the generation of epithelial cells and skin tissue remodeling. They also exhibit superior performance in wound closure, re-epithelialization, and hair follicle formation. Furthermore, when combined with a rhodioloside drug reservoir, a transdermal drug delivery system is constructed. This system has a high drug loading capacity and no polymer deposition, enabling efficient transdermal delivery of rhodioloside to promote wound closure and tissue remodeling, providing a promising strategy for wound treatment. Attached Figure Description

[0030] Figure 1 A schematic diagram illustrating the principle of photocrosslinking of methacrylamide chitosan;

[0031] Figure 2 This is an image of the freeze-dried CSMA of the present invention.

[0032] Figure 3 CSMA obtained from reactions with different molar ratios 1 H-NMR spectrum

[0033] (A1: 1; B2: 1; C3: 1; D4: 1);

[0034] Figure 4 The effect of molar ratio on the degree of substitution of CSMA;

[0035] Figure 5 CSMA obtained at different reaction times according to the present invention 1 H-NMR spectrum

[0036] (A6h; B12h; C24h);

[0037] Figure 6 This invention illustrates the effect of reaction time on the degree of substitution of CSMA.

[0038] Figure 7 CSMA obtained at different reaction temperatures according to the present invention 1 H-NMR spectrum

[0039] (A 25℃; B 40℃; C 60℃);

[0040] Figure 8 This invention illustrates the effect of reaction temperature on the degree of substitution of CSMA.

[0041] Figure 9 The DSC spectra of CS and CSMA in this invention;

[0042] Figure 10 The PXRD patterns of CS and CSMA in this invention;

[0043] Figure 11 The IR spectra of CS and CSMA in this invention;

[0044] Figure 12 For the present invention CS and CSMA 1 H-NMR spectrum;

[0045] Figure 13 This is a schematic diagram of the hydrogel microneedle preparation process of the present invention;

[0046] Figure 14 The images (A) and (B) under an upright microscope show the appearance of the HFMN under different ultraviolet light irradiation times according to the present invention.

[0047] Figure 15 This invention relates to the effect of ultraviolet irradiation time on the swelling rate of HFMN.

[0048] Figure 16 The ultraviolet light irradiation time of this invention affects HFMN puncture The effect of sealing film capability;

[0049] Figure 17 This invention relates to the effect of ultraviolet light irradiation time on the sensory score of HFMN.

[0050] Figure 18 The effect of drying temperature on the morphology of HFMN in this invention

[0051] (A 25℃; B 40℃; C 60℃);

[0052] Figure 19 The images (A) and (B) under an upright microscope show the appearance of HFMN with different degrees of substitution according to the present invention.

[0053] Figure 20 The swelling diagram of HFMN with different degrees of substitution in PBS buffer at 0.5 h is shown.

[0054] Figure 21 The degree of substitution of CSMA in this invention for HFMN puncture parafilm The sealing film's ability to seal is affected;

[0055] Figure 22 The effect of the degree of substitution on the sensory score of HFMN according to the present invention;

[0056] Figure 23 The images (A) and (B) under an upright microscope show the appearance of HFMN with different concentrations of CSMA according to the present invention.

[0057] Figure 24 This invention relates to the effect of CSMA concentration on the swelling rate of HFMN.

[0058] Figure 25 Parafilm of CSMA concentration versus HFMN in this invention The effect of sealing film capability;

[0059] Figure 26 The effect of CSMA concentration on the sensory score of HFMN in this invention;

[0060] Figure 27 The images (A) and (B) under an upright microscope show the appearance of HFMN with different concentrations of photoinitiator of the present invention.

[0061] Figure 28 The effect of photoinitiator concentration on the swelling rate of HFMN is presented in this invention.

[0062] Figure 29 The photoinitiator concentration of this invention affects the HFMN puncture parafilm. The sealing film's ability to seal is affected;

[0063] Figure 30 The effect of photoinitiator concentration on the sensory score of HFMN in this invention;

[0064] Figure 31 The contour plot and response surface plot show the effect of the interaction between the degree of CSMA substitution and the concentration of CSMA solution on the sensory score in this invention.

[0065] Figure 32 The contour plot and response surface plot show the effect of the interaction between the degree of CSMA substitution and the concentration of photoinitiator on the sensory score in this invention.

[0066] Figure 33 The contour plot and response surface plot show the effect of the interaction between CSMA solution concentration and photoinitiator concentration on sensory scores in this invention.

[0067] Figure 34 Here are the external images (A) and stereomicroscopic images (B) of the HFMN of the present invention;

[0068] Figure 35 This is a mechanical strength diagram of the hydrogel microneedles of the present invention;

[0069] Figure 36 This is a staining diagram of isolated pig skin according to the present invention;

[0070] A. Exterior image of isolated pig skin after HFMN puncture; B. Image of skin after puncture stained with pansambal blue; C. Image of skin after puncture stained with H&E (400×);

[0071] Figure 37 Macroscopic photographs of the freeze-dried storage tank (A) and the hydrogel storage tank (B) of the present invention;

[0072] Figure 38 Physical characteristic diagrams of different storage tanks and different formulations of the present invention.

[0073] A. Weight of the Sal lyophilized reservoir after demolding; B. Dissolution time of the Sal lyophilized reservoir in PBS buffer; C. Drug content of the Sal lyophilized reservoir; D. Weight of the Sal hydrogel reservoir after demolding; E. Dissolution time of the Sal hydrogel reservoir in PBS buffer; F. Drug content of the Sal hydrogel reservoir (n=3; * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001);

[0074] Figure 39 The in vitro permeation amount of rhodioloside in this invention

[0075] (A. Total transdermal drug release over 48 hours, including drug amount in pigskin and receiving pool; B. Drug amount extracted from hydrogel microneedles over 48 hours; C. Drug amount retained in pigskin over 48 hours; D. Release amount in receiving pool at each sampling point) (n = 3; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001);

[0076] Figure 40 The wound closure rate of each group of mice in this invention was measured at 0, 3, 7, 10, and 14 days.

[0077] Figure 41 Macroscopic images (A) and magnified views (B) of the wound repair in mice of each group at 0, 3, 7, 10 and 14 days.

[0078] Figure 42 H&E staining of tissue sections from wounds at 7 days and 14 days for this invention;

[0079] Figure 43 Masson staining of tissue sections from wounds at 7 days and 14 days for this invention;

[0080] Figure 44 The collagen volume fraction of each group of wounds in this invention at 7 and 14 days.

[0081] (n = 5; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001). Detailed Implementation

[0082] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0083] Example:

[0084] 1. Preparation of methacrylamide chitosan, the steps of which include:

[0085] (1) Preparation of acetic acid solution: Measure 2 mL of acetic acid solution, slowly pour it into 98 mL of distilled water, shake well, and you will get a 2% acetic acid solution.

[0086] (2) Preparation of chitosan solution: Weigh about 1.5g of chitosan accurately, add 50mL of 2% acetic acid solution, stir magnetically for 1h at room temperature and 600rpm until the chitosan is completely dissolved, and a 3% chitosan solution is obtained.

[0087] (3) Preparation of methacrylated chitosan: Measure 50 mL of 3% chitosan solution and place it in a 250 mL beaker. Protect from light. Slowly add an appropriate amount of methacrylic anhydride to the beaker while stirring. Continue stirring at 25–60 °C for 6–24 hours. After the reaction time is reached, measure an appropriate amount of 10% NaHCO3 solution and slowly add it dropwise to the reaction solution to adjust the pH to neutral. Pour the neutral reaction solution into a dialysis bag with a molecular weight cutoff of 3500. Place the dialysis bag in a 5 L beaker and dialyze for 5 days, changing the water 3 times a day. After dialysis, remove the solution and place it in a 50 mL centrifuge tube. Pre-freeze at -80 °C for 2 hours, then transfer it to a freeze dryer and freeze-dry at -60 °C for 3 days. After completion, store the freeze-dried CSMA at -20 °C for later use. After freeze-drying, if… Figure 2 .

[0088] The effects of different molar ratios of methacrylic anhydride (MA) and chitosan (CS), reaction time, and reaction temperature on the properties of the CSMA product were investigated using three factors: the molar ratio of MA to CS (4:1, 3:1, 2:1, and 1:1), reaction time (6, 12, and 24 h), and reaction temperature (25, 40, and 60 °C). The degree of substitution (DS) was calculated using 1H NMR spectroscopy. The different reaction parameters used in the CSMA preparation process are shown in Table 1.

[0089]

[0090] Table 1 Different reaction parameters in the study of CSMA preparation process

[0091]

[0092] 1.1.1 Molar ratio of MA to CS

[0093] Different molar ratios of MA and CS (1:1, 2:1, 3:1 and 4:1) were selected, and the reaction was carried out at 40℃ for 12 h with stirring. 1 H-NMR spectrum as follows Figure 3 As shown, the degree of substitution results are as follows: Figure 4 As shown in the figure, the degree of substitution was 9.60 ± 0.00% when the molar ratio of MA to CS was 1:1. The degree of substitution gradually increased with the increase of the methacrylic anhydride ratio. At a molar ratio of 2:1, the degree of substitution was 16.00 ± 1.38%; at a molar ratio of 3:1, the degree of substitution was 23.20 ± 1.38%; and at a molar ratio of 4:1, the degree of substitution was 28.80 ± 0.00%. The degree of substitution of the product was positively correlated with the molar ratio of C=C and NH2.

[0094] 1.1.2 Reaction Time

[0095] To investigate the effect of reaction time on the degree of substitution, the reaction was carried out for 6, 12, and 24 hours, with a molar ratio of MA to CS of 4:1 and a temperature of 40℃. 1 H-NMR spectrum as follows Figure 5 As shown, the degree of substitution results are as follows: Figure 6 As shown in the figure. The results showed that the degree of substitution of CSMA was 29.60±1.38% when the reaction time was 6 h; 30.40±1.38% when the reaction time was 12 h; and 28.00±0.00% when the reaction time was 24 h. According to statistical analysis, there was no significant difference in the degree of substitution of the product at reaction times of 6, 12, and 24 h (p>0.05).

[0096] 1.1.3 Reaction Temperature

[0097] To investigate the effect of reaction temperature on the degree of substitution, the reaction was carried out at 25, 40 and 60 °C, with a molar ratio of MA to CS of 4:1 and a reaction time of 6 h. 1 H-NMR spectrum as follows Figure 7 As shown, the degree of substitution results are as follows: Figure 8 As shown in the figure. The results show that the degree of substitution is 20.00±1.39% at a reaction temperature of 25℃; 29.60±0.00% at a reaction temperature of 40℃; and 46.40±2.77% at a reaction temperature of 60℃. The degree of substitution of the product CSMA increases with increasing reaction temperature.

[0098] 1.2 Structural characterization of methacrylated chitosan

[0099] 1.2.1 Differential Calorimetry (DSC)

[0100] like Figure 9 As shown, both CS and CSMA DSC curves exhibit significant endothermic peaks, corresponding to the detachment of water molecules from the CS molecular chain within the 25-150℃ range. During dehydration, the maximum temperatures of both are similar, indicating that the content of bound water in their molecular chains is similar. Furthermore, both CS and CSMA curves show exothermic peaks, with onset points of 280℃ and 256℃ respectively, corresponding to the thermal degradation of glucosamine units on the CS molecular chain. In contrast, the thermal degradation temperature of CSMA is lower, possibly due to weakened intermolecular hydrogen bonding. This suggests that MA is grafted onto the -NH2 group of the CS molecular chain, as the -NH2 group on the CS molecular chain readily forms hydrogen bonds.

[0101] 1.2.2 Powder X-ray Diffraction Analysis (PXRD)

[0102] To investigate the crystal structures of CS and CSMA, powder analysis was performed using X-ray diffraction. Figure 10As shown, CS exhibits two distinct crystallization peaks at 2θ of 11° and 20°, respectively. The strong diffraction peak at 20° is a typical characteristic pattern of CS. This is due to the strong hydrogen bonding within and between CS molecules, which is also the reason for CS's poor water solubility. In the CSMA spectrum, the crystallization peak at 20° is significantly reduced, showing a blunt and broad peak. This proves that the introduction of methacrylic anhydride at the NH position disrupts the hydrogen bonding between molecular chains to some extent, thus reducing crystallinity and forming a hypocrystalline or amorphous state. PXRD experimental results indicate a decrease in the crystallinity of CSMA, preliminarily proving that MA has been successfully grafted onto the CS molecular chain.

[0103] 1.2.3 Fourier Transform Infrared Spectroscopy (FTIR)

[0104] FTIR spectra of CS and CSMA are as follows Figure 11 As shown in the figure, both CS and CSMA exhibited NH stretching vibration absorption peaks in the 3400 cm⁻¹ region. In the 2900 cm⁻¹ region, the absorption peak corresponded to the CH₂ asymmetric stretching vibration peak, while in the 2800 cm⁻¹ region, the absorption peak was due to the saturated CH stretching vibration. The absorption at 1157 cm⁻¹ was caused by the asymmetric stretching vibration of COC, while the absorption peaks at 1072 cm⁻¹ and 1032 cm⁻¹ corresponded to the stretching vibration of the CO skeleton. In the FTIR spectrum of CSMA, new absorption peaks appeared at 1711 cm⁻¹, 1658 cm⁻¹, and 1452 cm⁻¹, which are characteristic vibration peaks of amides, corresponding to amide I, amide II, and amide III bands (Table 1-4). The appearance of these new absorption peaks confirms that methacrylic anhydride (MA) successfully underwent an acylation reaction with CS. Simultaneously, no significant absorption peaks were observed in the characteristic wavelength range of the ester group (1730-1740 cm⁻¹), thus concluding that no reaction occurred between the hydroxyl group of CS and MA. The results of FTIR spectroscopy further confirmed that MA was successfully grafted onto the CS molecular chain.

[0105] 1.2.4 Nuclear magnetic resonance hydrogen spectroscopy analysis ( 1 H-NMR)

[0106] pass 1 ¹H-NMR spectra can confirm the chemical structure of CSMA. CSMA is obtained by acylation of MA with the amino group in CS. Figure 12 As shown, cyclic proton peaks (b1, b2, c, d, e, f) of the monosaccharides glucosamine and acetylglucosamine appeared in the range of 2.5–4.0 ppm. Specifically, the Hb2 chemical shift of the monosaccharide glucosamine was at 3.0 ppm, and the Hb1 chemical shift of the monosaccharide acetylglucosamine was at 3.5–4.0 ppm, consistent with the CS structure. In CSMA… 1The 1H-NMR spectrum showed two distinct signal peaks with chemical shifts of 5.6 and 5.8 ppm, which are vinyl proton peaks (g) introduced by methacrylic anhydride. The structural formula of this 1H-NMR spectrum is consistent with the molecular structure of CSMA. 1 Compared to H-NMR spectra, CSMA 1 The H-NMR spectrum showed a vinyl proton peak (g), which confirmed that the methacrylamide group was grafted onto the chitosan molecular chain, and CSMA was successfully prepared.

[0107] 2. Screening of preparation process for hydrogel microneedles

[0108] The preparation of hydrogel microneedles includes the following steps:

[0109] Accurately weigh a methacrylamide chitosan solution (CSMA) with a substitution degree of 10-50% and a concentration of 3-5 wt%, and place it in a centrifuge tube. Accurately weigh a photoinitiator, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (Irgacure 2959), and add it to the centrifuge tube. Vortex for 2 min, then sonicate for 15 min (ultrasonic power 150W) until Irgacure 2959 is completely dissolved to obtain an HFMN-filled solution. The photoinitiator concentration is 0-0.15 wt%. Inject an appropriate amount of the HFMN-filled solution into a PDMS mold, centrifuge at 5000 rpm for 10 min. Finally, irradiate with a UV lamp (wavelength 365 nm) for 30 s, dry at 25°C, and demold to obtain HFMN. The microneedle preparation process is as follows: Figure 13 .

[0110] HFMN was prepared according to the above method, and the effects of different UV irradiation times (0, 0.5, 1, 2, 3, and 5 min) and drying temperatures (25, 40, and 60 °C) on HFMN were investigated. Appearance, swelling ratio, and mechanical strength were used as evaluation indicators.

[0111] Sensory score = Appearance inspection score + Swelling rate score + Puncture capability inspection score

[0112] 2.1 Screening of UV Irradiation Time

[0113] This experiment investigated the effect of UV irradiation time (0, 0.5, 1, 2, and 5 min) on HFMN. Preparation conditions: CSMA substitution degree of 30%, 4 wt% CSMA, 0.05 wt% photoinitiator Irgacure 2959, dried at 25℃.

[0114] Depend on Figure 14It is evident that the duration of ultraviolet (UV) irradiation has little effect on the external morphology of HFMN. HFMN prepared under UV irradiation conditions of 0, 0.5, 1, and 2 min exhibits intact appearance and good needle morphology. HFMN prepared under UV irradiation conditions of 3 and 5 min has a relatively intact appearance, but the edges of the microneedle backing are slightly curved, and the needle body is slightly bent.

[0115] like Figure 15 As shown, HFMN prepared after 30 seconds of UV irradiation exhibited the highest swelling rate, reaching 629.43 ± 43.22% after 24 hours. HFMN without UV irradiation completely dissolved in water within 0.5 hours, likely due to the absence of covalent cross-linking and covalent bond formation within its molecular structure. No significant differences in swelling rate were observed between HFMN prepared under UV irradiation for 1, 2, 3, and 5 minutes (p > 0.05).

[0116] Figure 16 This study reflects the skin penetration of HFMN in simulated eight layers of sealing film. Results show that HFMN at various light exposure times can penetrate two layers of sealing film and one layer of parafilm. The sealing film thickness was approximately 127 μm, and the HFMN insertion depth was approximately 254 μm. The puncture rates were 68.52±11.56%, 99.07±1.61%, 94.44±7.35%, 98.15±1.61%, 98.15±3.21%, and 88.89±11.11%, respectively. The HFMN without UV irradiation had the lowest puncture rate. The HFMN irradiated for 0.5, 1, 2, and 3 minutes had similar puncture rates. The HFMN irradiated for 5 minutes had a lower puncture rate, possibly because the needle became thinner and more brittle due to prolonged irradiation.

[0117] Calculate sensory scores, specifically as follows: Figure 17 As shown in the figure. The results indicate that the sensory score was highest when the illumination time was 30 seconds, indicating that its overall performance was the best. Therefore, the ultraviolet light illumination time was 30 seconds in subsequent experiments.

[0118] 2.2 Drying Temperature Screening

[0119] This experiment investigated the morphology of HFMN under three different temperature conditions: 25℃, 40℃, and 60℃. Preparation conditions: CSMA substitution degree 30%, 4wt% CSMA, 0.05wt% photoinitiator Irgacure 2959, UV irradiation for 30s. HFMN was prepared according to the method for preparing hydrogel microneedles, and its morphology was observed and recorded.

[0120] At 25℃, HFMN requires 36 hours to dry and form; at 40℃ and 60℃, the drying times are 10 hours and 5 hours, respectively. The morphological results are as follows: Figure 18As shown, the morphology of HFMN deteriorates with increasing temperature. Therefore, to ensure a smooth morphology of HFMN, a drying temperature of 25℃ was selected for subsequent experiments.

[0121] 3Box-Behnken response surface methodology for optimizing hydrogel microneedle formulations

[0122] The effects of different CSMA substitution degrees (10%, 30%, and 50%), CSMA solution concentrations (3 wt%, 4 wt%, and 5 wt%), and photoinitiator concentrations (0, 0.03 wt%, 0.05 wt%, 0.1 wt%, and 0.15 wt%) on the preparation of HFMN were investigated, with sensory scores as the indicator.

[0123] 3.1 Substitution degree of CSMA

[0124] The effects of CSMA solutions with DS values ​​of 10%, 30%, and 50% on HFMN were investigated. Preparation conditions: CSMA solution concentration of 3 wt%, Irgacure 2595 concentration of 0.05 wt%, UV irradiation for 30 s, and drying at 25 °C.

[0125] Depend on Figure 19 It is evident that the DS (dissolved matter) of CSMA affects the appearance of HFMN. HFMN prepared using a CSMA solution with a DS of 10% has an overall intact appearance and good needle morphology; HFMN prepared using a CSMA solution with a DS of 30% has a relatively intact appearance but the backing edge is distorted, the needle morphology is good, and the needle tip is slightly bent; HFMN prepared using a CSMA solution with a DS of 50% has a relatively intact appearance, but the backing is severely distorted, the needle morphology is inconsistent in thickness, and the needle tip is noticeably bent. Therefore, it is not recommended to use a CSMA solution with a DS of 50% to prepare HFMN.

[0126] Depend on Figure 20 It is evident that HFMN with a DS of 10% was completely dissolved at 0.5 h; HFMN with DS of 30% and 50% swelled in PBS buffer, but were easily broken upon removal for weighing, making weighing impossible. The swelling property of HFMN increased with increasing degree of substitution. The breakage may be due to insufficient cross-linking or lack of cross-linking of HFMN.

[0127] Figure 21The results show the skin penetration performance of HFMN prepared with different degrees of substitution under simulated eight layers of sealing film. The results indicate that HFMN prepared with CSMA at a DS of 10% can penetrate up to three layers of sealing film, while HFMN prepared with DS of 30% and 50% can only penetrate two layers. This suggests that HFMN prepared with CSMA at a DS of 10% has slightly stronger mechanical strength than the other two. The HFMN prepared with CSMA at a DS of 50% may have had poor needle morphology due to excessive cross-linking, thus affecting its penetration ability.

[0128] Calculate sensory scores, such as Figure 22 As shown, the sensory score first increases and then decreases. When the DS of the CSMA increases from 10% to 30%, the sensory score gradually increases. With increased substitution, the content of carbon-carbon double bonds in the photosensitive group also increases, which can increase the chemical crosslinking strength of the hydrogel. However, when the substitution is too high, the crosslinking is too tight, resulting in a finer HFMN morphology after demolding, thus affecting mechanical strength. Therefore, the optimal point for response surface methodology optimization is determined to be a CSMA with a DS of 30%.

[0129] 3.2 Concentration of CSMA solution

[0130] This experiment investigated the effects of 3 wt%, 4 wt%, and 5 wt% CSMA solutions on the preparation of HFMN. Preparation conditions: CSMA substitution degree 30%, 0.05 wt% Irgacure 2595, UV irradiation for 30 s, and drying at 25 °C.

[0131] Depend on Figure 23 It is evident that the concentration of CSMA affects the morphology of HFMN. HFMN prepared using a 3wt% CSMA solution has a relatively intact appearance, with a distorted backing edge, good needle morphology, and slightly bent needle tips. HFMN prepared using a 4wt% CSMA solution also has a relatively intact appearance, with slightly distorted edges, good needle morphology, and slightly bent needle tips. HFMN prepared using a 5wt% CSMA solution exhibits severe distortion, with thinner and slightly tilted needles and noticeably bent needle tips. The 3wt% CSMA solution is too dilute, resulting in significant evaporation at the needle tips after drying, leading to poor needle tip morphology. The 5wt% CSMA solution is too viscous, resulting in high cross-linking levels and distorted HFMN patches after cross-linking. Therefore, HFMN prepared using 3wt% or 4wt% CSMA solutions exhibits better morphology.

[0132] At 0.5 h, HFMN prepared using 3 wt% CSMA solution swelled in PBS buffer, but broke upon removal and could not be weighed, possibly due to insufficient cross-linking of HFMN. Figure 24As shown, the swelling rate of HFMN prepared with 4 wt% CSMA solution increased slowly, reaching 629.43 ± 43.22% at 4 h, with slight fluctuations at 24 h. At 0.5 h, the swelling rate of HFMN prepared with 5 wt% CSMA solution reached 540.00 ± 41.58%, with only slight fluctuations at 24 h. This indicates that with increasing CSMA concentration, the degree of cross-linking of HFMN increases, and the swelling rate decreases. The swelling rate of HFMN decreases with increasing CSMA concentration. Furthermore, statistical analysis showed a significant difference in the swelling rates between 4 wt% and 5 wt% HFMN (p < 0.05).

[0133] like Figure 25 The results show that HFMN prepared with different concentrations of CSMA can penetrate two layers of sealing film in simulated skin penetration. The results indicate that HFMN prepared with different concentrations of CSMA can penetrate two layers of sealing film, with penetration rates of 65.74±9.75%, 91.67±0.00%, and 85.18±8.49%, respectively, indicating that HFMN prepared with 4wt% CSMA has slightly stronger mechanical strength than the other two.

[0134] Calculate sensory scores, such as Figure 26 As shown, the HFMN prepared with 4 wt% CSMA achieved the highest sensory score. Therefore, the central point for optimizing the response surface methodology was determined to be 4 wt% CSMA.

[0135] 3.3 Concentration of photoinitiator

[0136] This experiment investigated the effects of 0, 0.03, 0.05, and 0.1 wt% photoinitiator Irgacure 2959 on HFMN. Preparation conditions: CSMA substitution degree of 30%, 4 wt% CSMA, UV irradiation for 30 s, drying at 25 °C.

[0137] Depend on Figure 27 It is evident that the HFMN without the added photoinitiator Irgacure 2959 has a more intact appearance and better needle morphology. The HFMN with added 0.03%, 0.05%, and 0.1% wt% Irgacure 2959 has slightly twisted edges, but still has a better needle morphology.

[0138] The swelling properties of HFMN were examined. The results are as follows: Figure 28As shown, HFMN without Irgacure 2959 dissolved completely at 0.5 h without a swelling curve; HFMN with 0.03 wt% Irgacure 2959 had the highest swelling rate, rapidly swelling at 0.5 h to 598.64 ± 97.34%, reaching 900.98 ± 227.08% at 4 h, with only slight fluctuations at 24 h; HFMN with 0.05 wt% and 0.10 wt% Irgacure 2959 had swelling rates of 629.43 ± 43.22% and 625.75 ± 78.31% at 24 h, respectively. Statistical analysis showed no significant difference in swelling rates between the two groups (p > 0.05).

[0139] Figure 29 This study reflects the penetration ability of HFMN with different concentrations of photoinitiator into eight layers of sealing film. The results show that HFMN with added 0, 0.03, 0.05, and 0.1 wt% Irgacure 2959 can all puncture two layers of sealing film, with penetration rates of 68.00±11.56%, 91.67±2.78%, 99.07±1.60%, and 98.15±3.21%, respectively. HFMN without a photoinitiator exhibits the worst penetration ability, while HFMN with 0.05 wt% photoinitiator shows the best penetration ability.

[0140] like Figure 30 As shown, 0.03 wt% Irgacure had the highest sensory score for HFMN. However, its puncture ability was poor, so the center point for response surface methodology optimization was chosen as 0.05 wt% Irgacure.

[0141] 3.4 Box-Behnken Response Surface Experiment

[0142] 3.4.1 Regression Modeling and Analysis of Variance

[0143] The results of the response surface optimization experiment are shown in Table 2. Using Design-Expert 12 software, the experimental results were fitted with sensory scores as the response values ​​to obtain the regression equation: Y = 24.20 - 1.12A + 1.88B - 1.00C + 1.50AB + 0.75AC - 0.75BC - 1.85A² - 5.35B² - 4.60C².

[0144] Table 2. Response Surface Experiment Design and Results

[0145]

[0146] The results of the analysis of variance for the fitted equation are shown in Table 3. The p-value of the fitted model is <0.0001, indicating that the regression model is highly significant and statistically significant. The lack-of-fit term p = 0.1639 > 0.05, indicating that the lack-of-fit term is not significant, suggesting that the established model has a good fit, high reliability, and no lack of fit factors, making the experimental data meaningful. Furthermore, the adjusted R² of the regression equation is 0.9811, and the predicted R² is 0.9050, with a difference of less than 0.2. The signal-to-noise ratio is 26.0850 > 4, indicating that the model established in this experiment can accurately predict the actual situation and can be used for screening optimal prescriptions.

[0147] Significance tests were performed on the individual factors A, B, and C in the regression equation. The results showed that each factor had a highly significant effect on the sensory score (P < 0.01). The F-value reflects the degree of influence of each factor on the sensory score; the larger the F-value, the greater the influence. Therefore, based on the F-value, the order of influence of the three factors on the sensory score is: B (solution concentration) > C (photoinitiator concentration) > A (degree of substitution).

[0148] Table 3. Response Surface Experiment Design and Results

[0149]

[0150] Note: * indicates p < 0.05, the difference is significant; ** indicates p < 0.01, the difference is highly significant; *** indicates p < 0.001, the difference is extremely significant.

[0151] 3.4.2 Response Surface Analysis

[0152] like Figure 31 As shown, the response surface has a large curvature, and the contour plot is elliptical. Combined with the significance test of AB in Table 3 (p = 0.0016, < 0.01), it can be concluded that the interaction between the degree of CSMA substitution and the concentration of CSMA solution has a very significant impact on the sensory score. Furthermore, the response surface curves show that as the degree of CSMA substitution and the concentration of CSMA solution increase, the sensory score increases, reaches a maximum, and then begins to decline.

[0153] like Figure 32 As shown, the response surface is curved, and the contour plot is elliptical. Combined with the significance test of AC in Table 3 (p = 0.0419, < 0.05), this indicates that the interaction between the degree of CSMA substitution and the photoinitiator concentration has a significant impact on sensory scores.

[0154] like Figure 33 As shown, the response surface is curved, and the contour plot is elliptical. Combined with the significance test of BC in Table 3 (p = 0.0419, < 0.05), this indicates that the interaction between CSMA solution concentration and photoinitiator concentration has a significant impact on sensory scores.

[0155] Response surface methodology (RSM) experiments can clearly understand the impact of individual factors and their interactions on sensory scores. Through experimental design and results analysis, a formulation model for HFMN was established, capable of predicting the optimal combination of the three factors. Analysis of variance and 3D response surface methodology were performed on the experimental results, concluding that the degree of CSMA substitution, CSMA solution concentration, and photoinitiator concentration all significantly affect the formation of HFMN. Among the interactions among the three, the degree of CSMA substitution and CSMA solution concentration have the most significant impact on sensory scores.

[0156] The optimal formulation for HFMN was predicted using a model built with Design Expert 12, resulting in the following optimal formulation: CSMA substitution degree of 24.51%, CSMA solution concentration of 4.15 wt%, and photoinitiator concentration of 0.043 wt%. Under these conditions, the theoretical sensory evaluation score was 26.56. Based on practical considerations, the experimental conditions were changed to: CSMA substitution degree of 23%, CSMA solution concentration of 4.15 wt%, and photoinitiator concentration of 0.04 wt%.

[0157] 4. Basic performance evaluation of hydrogel microneedles

[0158] 4.1 Morphological Characterization

[0159] like Figure 34 As shown, HFMN has a complete appearance, a beautiful needle shape, a sharp needle tip, and a four-sided pyramidal needle body.

[0160] 4.1 Mechanical strength test

[0161] The required penetration force into the skin should exceed 0.1 N / needle, meaning the breaking force of the microneedle must be greater than 0.1 N to overcome the barrier of the stratum corneum. The pressure-deformation diagram of HFMN is shown below. Figure 35 As shown, no significant breakage occurred during the test, indicating high mechanical strength. Maximum pressure was generated at a deformation of 500 μm, with each HFMN strand bearing an average pressure of approximately 1.02 N, sufficient for skin penetration.

[0162] 4.2 Assessment of the ability to perform ex vivo skin puncture

[0163] To verify whether HFMN can overcome the barrier of the stratum corneum, this section uses ex vivo porcine skin for puncture experiments. The results are as follows... Figure 36 As shown, trypan blue staining results indicate that HFMN forms distinct microporous channels on the skin surface. H&E staining sections show that HFMN successfully penetrates the stratum corneum, further confirming that HFMN has high mechanical strength and can overcome the stratum corneum barrier, demonstrating its potential for transdermal drug delivery.

[0164] 5. Rhodioloside Reservoir Prescription Screening

[0165] The optimal formulation should possess the following characteristics: high drug loading capacity, successful detachment from the mold, uniform appearance, high mechanical strength, and rapid dissolution in PBS buffer. The faster the drug reservoir dissolves, the faster the drug is released from the reservoir and delivered transdermally via the HFMN hydrogel network. Therefore, to screen for the optimal formulation, this section characterizes the morphology and physical properties of the drug reservoir. Formulation screening results are shown in Tables 4 and 5.

[0166] The preparation of the rhodioloside freeze-dried storage cell includes the following steps: As shown in Table 4 below, take appropriate amounts of rhodioloside, various excipients (such as gelatin, methylcellulose, carboxymethylcellulose, carboxyethylcellulose, sodium carboxymethyl starch, PVP K30, sorbitol), and water, add them to a centrifuge tube, and mix thoroughly; then sonicate at 25℃ for 15 min. Take about 0.5 g of this formulation solution, accurately weigh it, and cast it into a cryopreservation embedding box with a length of 7 mm, a width of 7 mm, and a depth of 5 mm; pre-freeze it at -80℃ for 2 h, place it in a freeze dryer (temperature -60℃, vacuum degree 10 Pa), and freeze-dry for 24 h; after drying, peel the freeze-dried storage cell from the mold to obtain the rhodioloside freeze-dried storage cell.

[0167] The preparation of the rhodioloside hydrogel reservoir includes the following steps: as shown in Table 5 below, take appropriate amounts of rhodioloside, gelatin, glycerol, sorbitol and water, accurately weigh them, add them to a centrifuge tube, mix them evenly, and then sonicate them at 25℃ for 15 min; take about 0.5 g of the formula solution, accurately weigh it, and cast it into a cryopreservation embedding box with a length of 7 mm, a width of 7 mm and a depth of 5 mm; after solidification at room temperature, peel the hydrogel reservoir from the mold to obtain the rhodioloside hydrogel reservoir.

[0168] Table 4. Formula for Rhodioloside Freeze-Dried Storage

[0169]

[0170] Table 5. Formulation of Rhodiola Rosea Glycoside Hydrogel Storage Facility

[0171]

[0172] 5.1 Physical Appearance

[0173] Freeze-dried storage facilities F1-F12 are too brittle or too sticky to meet drug storage requirements. F13-F15 have the same excipients in their formulations, but the drug quality gradually increases. For example... Figure 37 As shown, F13 is easy to demold, has a uniform and complete shape, and meets the requirements of drug storage. However, F14 and F15 overflow the mold after freeze-drying, are larger in size, cannot be completely peeled out of the mold, have visible fragments, suffer greater losses, have loose holes and are fragile, and do not meet the requirements of drug storage.

[0174] Among the various hydrogel reservoir formulations, F1 is easy to demold and has good toughness, with visually uniform drug distribution; F2 is easy to demold, slightly sticky, with slight sedimentation at the four corners; F3 is more sticky, making it difficult to demold, with slight overall deformation and obvious sedimentation.

[0175] 5.2 Physical property characterization

[0176] Figure 38 A, B, and C reflect the physical characteristics of the freeze-drying storage. Freeze-drying storage F15 has the shortest dissolution time and shows a significant difference compared to F13 and F14. Figure 38 (B) This may be due to the presence of many loose pores, allowing moisture to easily penetrate. However, its hardness is insufficient, making it prone to breakage during demolding. Therefore, the actual measured content is 105.89±6.77 mg, which differs greatly from the theoretical value of 150 mg, resulting in a recovery rate of only 70.59% (Table 6). In contrast, F13 dissolves in less than 5 minutes, with an actual content of 49.91±6.90 mg, close to the theoretical content of 50 mg, and a recovery rate of 99.82%.

[0177] Figure 38 D, E, and F reflect the physical properties of the hydrogel reservoir. The weights of F1-F3 remained stable after demolding, and statistical analysis showed no significant differences (p > 0.05). Figure 38 D). There was no significant difference in dissolution time between F1 and F2, while F3 showed significant differences from both F1 and F2 (e.g., Figure 38 E). This may be due to the excessive viscosity of F3, making it difficult to dissolve. The actual drug contents of F1-F3 were 48.00±1.92 mg, 77.71±23.56 mg, and 134.12±37.93 mg, respectively, with recovery rates of 96.00%, 77.71%, and 89.41%. The high recovery rate of F1 and the low recovery rates of F2 and F3 may be due to uneven distribution of F2 and F3 deposition, or loss during demolding caused by excessive viscosity.

[0178] Compared to hydrogel reservoirs, freeze-dried reservoirs have a shorter dissolution time. Figure 38 (B and E). This is likely because sodium carboxymethyl starch was added to freeze-dried storage tank F15. Sodium carboxymethyl starch is a commonly used, highly effective disintegrant and excipient in tablets, possessing strong water absorption and swelling properties. Therefore, adding 3% sodium carboxymethyl starch can significantly shorten the dissolution time of the freeze-dried storage tank.

[0179] In summary, freeze-dried reservoir F13 and hydrogel reservoir F1 were selected as rhodioloside reservoirs for in vitro permeation studies.

[0180] Table 6. Characteristics of different storage formulations (X±SD, n=3)

[0181]

[0182] 6. In vitro permeation study of hydrogel microneedles

[0183] Two reservoir formulations were selected: lyophilized reservoirs and hydrogel reservoirs. Both exhibited high recovery rates after demolding and rapid dissolution. The rapid dissolution of the drug reservoirs in PBS buffer demonstrated that rhodioloside can be readily released from the reservoirs and rapidly permeate through the hydrogel network.

[0184] The transdermal experiment was divided into three groups: the lyophilized reservoir-HFMN group, the hydrogel reservoir-HFMN group, and the topical formulation group.

[0185] like Figure 39 As shown in Figure A, at 48 h, the total cumulative skin penetration (including skin and receiver pool) in the hydrogel reservoir group and the lyophilized reservoir group was significantly higher than that in the topical formulation group. Statistically, there was no significant difference in the total cumulative penetration between the two drug reservoirs (p > 0.05).

[0186] Figure 39 B shows the retention of rhodioloside (Sal) in HFMN at 48 h. The Sal released from the lyophilized reservoir group and the hydrogel reservoir group were retained in HFMN at levels of 1468.32±782.36 μg and 2829.22±224.31 μg, respectively. The retention of Sal in HFMN in the hydrogel reservoir group was significantly higher than that in the lyophilized reservoir group.

[0187] Figure 39 Figure C shows the amount of Sal retained in the skin at 48h. At 48h, the amount of Sal retained in the skin in the lyophilized reservoir group and the hydrogel reservoir group was greater than that in the topical formulation group, and the retention amount in the lyophilized reservoir group was significantly higher than that in the topical formulation group.

[0188] like Figure 39As shown in Figure D, the cumulative release of Sal through the skin increased over time at each sampling point. From 0-4 h, the cumulative release of Sal in the lyophilized reservoir group was higher than that in the hydrogel reservoir group. This is likely due to the high solids content and disintegrant in the lyophilized reservoir, which utilizes the microporous pathways formed by HFMN to absorb water from the interstitial fluid of the skin. Therefore, the bottom of the lyophilized reservoir dissolves rapidly, subsequently increasing the diffusion effect of Sal. In contrast, the diffusion of Sal in the hydrogel reservoir was caused by the active diffusion of the drug itself. Over time, the cumulative permeation of Sal in the hydrogel reservoir group gradually increased and was higher than that in the lyophilized reservoir group. This is because the concentration of Sal diffusing into HFMN in the hydrogel group continuously increased, and compared to the lyophilized reservoir group, there was no process of water absorption, disintegration, and drug release, resulting in a faster release rate. At 48 h, although the cumulative permeation of Sal in the hydrogel reservoir group was slightly higher than that in the lyophilized reservoir, statistically, there was no significant difference between the cumulative permeation of the two drug reservoirs (p > 0.05).

[0189] Compared with the topical formulation, the binding of HFMN to the rhodioloside reservoir significantly enhanced the cumulative release of Sal. Figure 39 (D) At 12, 24, and 48 h, the lyophilized reservoir group released 677.88±79.31 μg, 1504.21±183.75 μg, and 3386.32±413.84 μg Sal, respectively; the hydrogel reservoir group released 794.12±119.51 μg, 1769.30±272.60 μg, and 3988.87±593.97 μg Sal, respectively; while the topical formulation group released only 152.66±8.72 μg, 326.38±19.56 μg, and 738.82±45.75 μg Sal. At 48 h, the cumulative permeation of Sal in the lyophilized and hydrogel reservoir groups was 4.59 times and 5.40 times that of the topical formulation group, respectively. The hydrogel and lyophilized reservoir groups significantly increased the transdermal delivery of rhodioloside. This indicates that the binding of hydrogel and lyophilized reservoirs to HFMN can both promote the transdermal permeability of rhodioloside, and both types of reservoirs can be used as options to increase the rhodioloside reservoir.

[0190] 7. Pharmacodynamic Study of Hydrogel Microneedles

[0191] 7.1 Wound Model Establishment

[0192] Establishment of the wound model: After 3 days of environmental acclimatization, 4% (w / v) chloral hydrate was prepared and administered intraperitoneally at a dose of 0.1 mL / 10 g. Once the mice were completely anesthetized and their limbs were weak, 44 cm² of hair was removed from their backs using a razor. One day after hair removal, 18 mice with intact skin were selected. The skin was wiped with 75% ethanol, and a circular full-thickness skin wound (epidermis, dermis, subcutaneous tissue, and muscle layer) with a diameter of 5 mm was created on the back of each mouse using a skin biopsy device. Hemostasis was achieved with cotton.

[0193] 7.2 Experimental grouping and dosing regimen

[0194] Mice were randomly divided into three groups of nine each: a model control group, an HFMN group, and a Sal-HFMN group. In the model control group, the wound was covered with sterile gauze and secured with medical tape. In the HFMN group, one HFMN tablet was inserted vertically into the wound daily after moistening the skin with saline solution, and then secured with medical tape. In the Sal-HFMN group, one Sal-HFMN tablet was inserted vertically into the wound daily after moistening the skin with saline solution, then a Sal drug reservoir was placed on top of the HFMN tablet, and finally secured with medical tape.

[0195] 7.3 Wound observation and wound closure rate

[0196] The gross condition of all rat wounds was recorded daily by photograph. ImageJ software was used to process the photos, measure the size of the wounds, and calculate the wound closure rate.

[0197] 7.4 Biological Sample Collection and Evaluation

[0198] At day 7, three mice from each group were removed, and the skin around the wound was cut off, the hair was trimmed, and excess adipose tissue was removed. The cut pieces were then cut into 1x1cm pieces and placed on aluminum foil with the epidermis facing up to prevent the skin from curling during fixation. They were then fixed in a 4wt% paraformaldehyde solution for 3 days.

[0199] On day 14, the skin around the wound was cut, hair was removed, and excess fatty tissue was taken out. The cut piece was then cut into 1x1cm pieces and placed on aluminum foil with the epidermis facing up to prevent the skin from curling during fixation. It was then placed in a 4wt% paraformaldehyde solution for fixation for 3 days.

[0200] H&E staining: The fixed 7-day and 14-day mouse skin was dehydrated, embedded, sectioned, dewaxed, stained with hematoxylin and eosin (H&E), and dehydrated and mounted. The re-epithelialization of the wound surface was observed under an optical microscope.

[0201] Masson staining: Fixed 7-day and 14-day mouse skin was dehydrated, embedded, sectioned, dewaxed, stained with iron hematoxylin-pounose red-aniline blue (Masson stain), and then dehydrated and mounted. Collagen deposition in the wound was observed under an optical microscope, and the collagen volume fraction (CVF) was calculated using ImageJ software. Five fields of view were taken from each sample under a 400x optical microscope, and the results were averaged.

[0202] 7.5 Discussion of Results

[0203] 7.5.1 Wound Closure Rate

[0204] Figure 41 The closure status of wounds in each group at days 0, 3, 7, 10, and 14 is shown. Results indicate that the wound closure rate was significantly faster in the HFMN and Sal-HFMN groups, with wounds essentially closed within 14 days. On day 3, the wound size in both the HFMN and Sal-HFMN groups showed significant changes compared to the model control group. (Based on Table 7 and...) Figure 40 Quantitative results show that the wound closure rate of the HFMN group and the Sal-HFMN group exceeded 20%.

[0205] In the medical field, achieving a 50% wound closure rate in a shorter time is crucial for reducing the risk of infection. On day 7, thick scabs were observed in the model control group, while significant changes in wound size were observed in the HFMN and Sal-HFMN groups. The wound closure rate in the model control group was only 11.24±7.81%, while the wound closure rates in the HFMN and Sal-HFMN groups exceeded 50% (67.83±10.59% and 77.57±5.04%, respectively). Therefore, it is speculated that the use of HFMN and Sal-HFMN may achieve better medical outcomes.

[0206] On day 10, the wound closure rate in the model control group was still less than 50%, at only 27.65±6.89%, while the rates in the other two groups were 85.71±3.37% and 89.14±6.54%, respectively. On day 14, the eschar on the surface of the model control group fell off, and it could be observed that the wound surface was basically closed, but the skin was pink, suggesting that the skin remodeling was incomplete.

[0207] The wound closure rate results showed that HFMN and Sal-HFMN had an advantage in promoting wound closure compared to the model control group.

[0208] Table 7. Wound closure rates of mice in each group at 3, 7, 10, and 14 days.

[0209]

[0210] 7.5.2 Skin histological analysis

[0211] 7.5.2.1 HE staining

[0212] like Figure 42 As shown, on day 7, the wound epidermis in the HFMN and Sal-HFMN groups showed significant hyperplasia and rapid re-epithelialization; in contrast, the model control group was covered with a large amount of inflammatory necrotic tissue (eschar), and re-epithelialization was slow. Furthermore, thicker granulation tissue was clearly visible in the wounds of the HFMN and Sal-HFMN groups, while only a very small amount of newly formed tissue was observed in the wounds of the control group.

[0213] Perfect wound healing typically involves the regeneration of some dermal appendages during the remodeling phase, and hair follicle regeneration is also an indicator of wound repair. For example... Figure 42 On day 14, although the model control group showed complete re-epithelialization, very few hair follicles were observed. In contrast, the HFMN and Sal-HFMN groups had more hair follicles in the dermis. This demonstrates that HFMN exhibits superior performance in wound closure, re-epithelialization, and hair follicle formation.

[0214] 7.5.2.2 Masson staining

[0215] In order to assess the level of collagen deposition in each group of this experiment, Masson staining was performed on the skin of each group, and the collagen volume fraction was calculated according to the formula.

[0216]

[0217] like Figure 43 As shown, collagen deposited at the wound site stained blue. On day 7, the wounds of mice in the model control group produced only a small amount of collagen fibers; the collagen fibers in the HFMN group were loosely structured and disordered, with low maturity; while in the Sal-HFMN group, collagen deposition was rapid, with increased deposition and a more compact arrangement. The experimental results showed that collagen deposition was most significant in the Sal-HFMN group. On day 14, the collagen fibers at the center of the wound in the model control group were loosely structured, disordered, and had low maturity, while the surrounding collagen fibers were denser; the entire wound surface of mice in the HFMN group showed blue, densely structured, neatly arranged, and highly mature collagen fibers; while the wound surface of mice in the Sal-HFMN group showed a large number of dark blue, densely structured, neatly arranged, and highly mature collagen fibers.

[0218] Figure 44Table 8 shows the CVF of each group on days 7 and 14. On day 7, the CVF of the skin at the wound site in the model control group was 11.45±4.47%, significantly lower than that in the HFMN group (66.98±13.45%) and the Sal-HFMN group (68.90±11.23%). On day 14, the CVF of both the HFMN and Sal-HFMN groups was greater than that in the model control group. Quantitative analysis of the mean collagen density of Masson stained sections further confirmed that HFMN and Sal-HFMN can promote collagen deposition and maturation in the later stages of healing, which is beneficial to tissue regeneration and improves healing quality. The excellent re-epithelialization effect and significant collagen deposition indicate that HFMN and Sal-HFMN have potential application prospects in wound healing.

[0219] Table 8. Collagen volume fraction (X±SD, n=5) of mice in each group at 7 and 14 days.

[0220]

[0221] Therefore, the hydrogel microneedles provided by this invention combine a rhodioloside drug reservoir and hydrogel microneedles prepared from methacryloxychitosan to construct a transdermal drug delivery system. This system has a high drug loading capacity and no polymer deposition, and can efficiently deliver rhodioloside transdermally, promoting wound closure and tissue remodeling, and has a significant effect on wound treatment.

[0222] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A transdermal drug delivery system, characterized in that, include: (a) a hydrogel microneedle prepared by photocrosslinking of methacrylamide chitosan; and (b) a rhodioloside drug reservoir attached to the backing of the hydrogel microneedle; wherein the rhodioloside drug reservoir is a lyophilized reservoir made of the following components in parts by weight: 10 parts rhodioloside, 3 parts sodium carboxymethyl starch, 10 parts polyvinylpyrrolidone K30, 5 parts sorbitol, and 72 parts water.

2. The transdermal drug delivery system according to claim 1, characterized in that, The preparation method of the rhodioloside freeze-dried storage is as follows: Rhodioloside, excipients and water are added to a centrifuge tube and mixed evenly; then, at 20-30℃, ultrasonic treatment is carried out for 10-30 minutes with an ultrasonic power of 100-300W to obtain the formulation solution. The formulation solution is poured into a mold and pre-frozen at -70~-100℃ for 1-3 hours, then transferred to -50~-70℃ and freeze-dried under a vacuum of 10 Pa for 20-30 hours; after drying, the freeze-dried storage is peeled off from the mold to obtain the product.

3. A transdermal drug delivery system, characterized in that, include: (a) a hydrogel microneedle prepared by photocrosslinking of methacrylamide chitosan; and (b) a rhodioloside drug reservoir attached to the backing of the hydrogel microneedle; wherein the rhodioloside drug reservoir is a hydrogel reservoir made of the following components in parts by weight: 10 parts rhodioloside, 15 parts gelatin, 2 parts glycerol, 5 parts sorbitol, and 68 parts water.

4. The transdermal drug delivery system according to claim 3, characterized in that, The preparation method of the rhodioloside hydrogel reservoir is as follows: Rhodioloside, excipients and water are added to a centrifuge tube and mixed evenly; then, at 20-30℃, ultrasonic treatment is performed for 10-30 minutes with an ultrasonic power of 100-300W to obtain the formulation solution; the formulation solution is poured into a mold, solidified at room temperature and then peeled off to obtain the product.

5. Use of the transdermal drug delivery system according to any one of claims 1-4 in the preparation of a medicament for promoting wound healing.

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