PH / active oxygen double-response type hydrogel microneedle and preparation method thereof
By combining polyglutamic acid-phenylboronic acid copolymer with sodium alginate in a multi-network hydrogel microneedle, the shortcomings of existing microneedle systems in acne treatment, such as mechanical strength and intelligent drug release, have been overcome. This has enabled reliable transdermal drug delivery and dual-response drug release at the site of acne inflammation, and has excellent biocompatibility and antibacterial properties.
Patent Information
- Application Number
- CN202511492726.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-20
- Publication Date
- 2026-01-02
AI Technical Summary
Existing microneedle systems cannot simultaneously meet the comprehensive needs of reliable transdermal delivery, intelligent drug release, and synergistic therapy in acne treatment, especially in terms of mechanical strength, dual response to the inflammatory microenvironment of acne, and drug release control.
A multi-network hydrogel was prepared by combining polyglutamic acid-phenylboronic acid copolymer with sodium alginate and forming a multi-network hydrogel through phenylboronic acid ester bonds and Ca2+ chelation. The controlled release of the drug was achieved by utilizing the phenylboronic acid ester bonds and hydrogen bond crosslinking points.
It achieves reliable transdermal drug delivery at acne lesions, possesses excellent mechanical properties and intelligent drug release characteristics, can rapidly release drugs in low pH and high reactive oxygen species environments, and exhibits good biocompatibility and antibacterial properties.
Smart Images
Figure CN121243039A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to a hydrogel microneedle for transdermal drug delivery, and particularly to an intelligent drug release microneedle capable of responding to the acidity and high active oxygen level of the microenvironment of acne inflammation and a preparation method thereof. BACKGROUND
[0002] Inflammatory skin diseases, such as acne, are closely related to the deterioration of the local microenvironment. Propionibacterium acnes, as the key pathogenic bacterium of acne, not only can secrete various chemotactic factors to attract immune cell infiltration, but also can stimulate the production of pro-inflammatory cytokines, thereby exacerbating the inflammatory response. The two typical characteristics of the lesion area are the decrease of the local microenvironment pH value and the excessive accumulation of active oxygen. Excessive active oxygen will overflow from the cell to the extracellular, causing DNA damage and other pathways, and producing toxicity to the surrounding tissues. Therefore, it is of great significance to develop a delivery system that can target the inflammatory area of acne and simultaneously respond to acidic pH and high active oxygen level to achieve intelligent drug release, for improving the efficiency of acne treatment and reducing side effects.
[0003] As a painless and minimally invasive transdermal drug delivery technology, the hydrogel microneedle is an ideal carrier to achieve this goal. In order to endow the microneedle with environmental responsiveness, researchers have introduced various stimulus-responsive chemical bonds. Borate ester bond, due to its unique dynamic reversible characteristics, can respond to multiple stimuli such as pH and active oxygen at the same time, and has attracted much attention in intelligent drug release systems. For example, Lee et al. have disclosed a pH-regulated hyaluronic acid hydrogel using the borate ester bond between phenylboronic acid and dopamine. By precisely controlling the pH value of the precursor solution, they successfully realized rapid gelation near the physiological pH, and used it for local injection delivery of anticancer drugs. However, this system relies on complex cross-linking strategies such as dopamine self-polymerization, and its response characteristics are relatively single. Another study has disclosed the application of sodium alginate in the construction of intelligent hydrogels. As described by Pettignano et al., the abundant cis-diol structure on the molecular chain of sodium alginate enables it to combine with phenylboronic acid through dynamic covalent bonds, thereby spontaneously forming a hydrogel under neutral conditions. The hydrogel exhibits excellent elasticity, self-healing ability and multiple stimulus responsiveness. Although the hydrogel performs well in terms of elasticity, its mechanical strength is still a major limiting factor for its processing into practical microneedle arrays, resulting in that the technology still faces severe challenges when facing the reliable transdermal drug delivery required for acne treatment.
[0004] In addition, the prior art such as the research of Chen et al. discloses the application of polyglutamic acid in the preparation of dissolvable microneedles and proves the biocompatibility and immunomodulatory potential thereof. However, the technical scheme mainly utilizes the physical dissolution characteristics of polyglutamic acid to achieve drug delivery and fails to endow the microneedles with environmental stimulus responsiveness through chemical cross-linking strategy. Therefore, the microneedles are difficult to achieve precise and controllable release of drugs according to specific pathological signals (such as pH reduction and active oxygen level increase) of acne lesions, and the treatment targeting and intelligent degree need to be improved.
[0005] In summary, although the cross-linking system based on sodium alginate-phenylboronic acid provides a possibility for constructing stimulus-responsive gels, and polyglutamic acid has been proved to have the potential as a microneedle base material, the prior art has not successfully provided a technical scheme that integrates the advantages of both. Specifically, the field still lacks a hydrogel microneedle that can simultaneously meet the following comprehensive performance: sufficient mechanical strength to penetrate the stratum corneum of the skin; dual response to low pH and high active oxygen level of the acne inflammatory microenvironment and control of drug release; and inherent antibacterial and anti-inflammatory functions. SUMMARY
[0006] The purpose of the present application is to solve the problem that the existing microneedle system cannot simultaneously meet the comprehensive needs of reliable transdermal, intelligent drug release and synergistic therapy in acne treatment, and to provide a pH / active oxygen dual-responsive hydrogel microneedle with good mechanical properties, intelligent drug release characteristics and therapeutic synergistic effect and a preparation method thereof.
[0007] The pH / active oxygen dual-responsive hydrogel microneedle provided by the present application is prepared by the following method: Step 1: Preparation of polyglutamic acid-phenylboronic acid copolymer Dissolve the polyglutamic acid in the MES buffer solution, and then add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC•HCl), N-hydroxysuccinimide (NHS) and 3-aminophenylboronic acid in sequence. After stirring at room temperature for 20-24 hours, the mixed solution is extracted with ethyl acetate, and the extract is dialyzed in a dialysis bag with ultrapure water, concentrated by rotary evaporation, and freeze-dried to obtain the polyglutamic acid-phenylboronic acid copolymer.
[0008] Step 2: Preparation of polyglutamic acid-phenylboronic acid copolymer / sodium alginate / salicylic acid hydrogel The polyglutamic acid-phenylboronic acid copolymer is dissolved in ultrapure water to prepare a polyglutamic acid-phenylboronic acid copolymer solution with a concentration of 9.0% w / v-15.0% w / v, and the pH of the solution is adjusted to 6.0-8.0 by NaOH; the sodium alginate is dissolved in a PBS buffer solution to prepare a sodium alginate solution with a concentration of 2.0% w / v-3.0% w / v; the polyglutamic acid-phenylboronic acid copolymer solution and the sodium alginate solution are uniformly mixed, and salicylic acid is added, and the hydrogel formed after stirring is further soaked in a CaCl2 aqueous solution for chelation to obtain a polyglutamic acid-phenylboronic acid copolymer / sodium alginate / salicylic acid hydrogel.
[0009] Step 3: Preparation of the pH / active oxygen dual-responsive hydrogel microneedle The polyglutamic acid-phenylboronic acid copolymer / sodium alginate / salicylic acid hydrogel is placed on a microneedle mold, vacuum is drawn to make the hydrogel fully fill the microneedle mold, then the excess hydrogel is removed, and the mold is demolded after complete drying at room temperature to obtain the pH / active oxygen dual-responsive hydrogel microneedle.
[0010] Further, in the above step 1, preferably, the mass ratio of the polyglutamic acid to 3-aminophenylboronic acid is 1:0.3-1, and the molar ratio of the polyglutamic acid to 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide is 1:1-2:1-2.
[0011] Further, in the above step 1, the number average molecular weight of the polyglutamic acid is 800-1200 million.
[0012] Further, in the above step 1, the molecular weight cut-off of the dialysis bag is 12000-14000 Da, and the dialysis time is 5-7 days.
[0013] Further, in the above step 2, preferably, the mass ratio of the polyglutamic acid-phenylboronic acid copolymer solution to the sodium alginate solution is 1-5:3.
[0014] Further, in the above step 2, preferably, the concentration of the CaCl2 aqueous solution is 0.6% w / v-1.2% w / v, and the chelation time is 20-40 minutes.
[0015] Further, in the above step 3, preferably, the drug loading amount of salicylic acid in the pH / active oxygen dual-responsive hydrogel microneedle is 0.6-1.2 mg / g.
[0016] Further, in the above step 1, the pH of the MES buffer solution is 5.0-6.0; and in the above step 2, the pH of the PBS buffer solution is 7.4.
[0017] The beneficial effects of the present application are as follows: 1. The present application first modifies 3-aminophenylboronic acid (PBA-NH2) to the molecular chain of polyglutamic acid (PGA), synthesizes polyglutamic acid-phenylboronic acid copolymer (PGA-PBA), then regulates the pH of sodium alginate (Alg) and PGA-PBA precursor solutions, forms phenylboronic ester bonds between the adjacent diol groups of sodium alginate and the boric acid groups of 3-aminophenylboronic acid, and in-situ prepares polyglutamic acid-phenylboronic acid copolymer / sodium alginate / salicylic acid (PPAS) monolayer network hydrogel loaded with salicylic acid (SA) drugs under the chelation of Ca 2+ The monolayer network hydrogel forms a multi-network PPAS hydrogel under the chelation of Ca 2+ The multi-network PPAS hydrogel has hydrogen bonding, reversible dynamic phenylboronic ester bonds and the chelation of Ca 4 Pa, the tensile modulus is 1.1 MPa, and the elongation at break is 197%, the needle tip of the PPAS MNs does not break after applying a weight of 500 g, and good mechanical properties are exhibited.
[0018] 2. The cumulative release rate of SA in the PPAS MNs can reach 97.9% within 5 h in a release medium of pH=5.0 (0.1 M H2O2), indicating that the PPAS MNs have pH / ROS dual responsiveness. In addition, the in-vitro penetration test results show that the PPAS MNs can achieve drug release at a subcutaneous depth of 340 μm, and the needle body and the base are separated after 150 min of penetration, exhibiting excellent drug permeability and skin compatibility; the biocompatibility test results show that the hemolysis rate of the PPAS MNs is 0.4%, and the cell viability of the NIH-3T3 fibroblasts is greater than 80.0% as the concentration increases, exhibiting excellent biocompatibility; the antibacterial performance test results show that the antibacterial rates of the PPAS MNs containing 0.7 mg / g SA against S. aureus and E. coli are both >99.9%, and the antibacterial rate against P. acnes reaches 97.0%, exhibiting excellent antibacterial performance, and being expected to be used for the treatment of acne. BRIEF DESCRIPTION OF DRAWINGS
[0019] Figure 1 is the FT-IR spectrum of PGA, PBA-NH2, PGA-PBA, Alg and PPAS gel. 1 H NMR spectrum.
[0020] Figure 2 is the FT-IR spectrum of PGA, PBA-NH2, PGA-PBA, Alg and PPAS gel.
[0021] Figure 3XRD patterns of PGA, PGA-PBA, Alg, CaCl2, PPAS gel.
[0022] Figure 4 G' and G" as a function of stress of Alg gel, PGA-Alg gel and PPAS gel.
[0023] Figure 5 SEM images of PPAS MNs cross-section (scale bar: 500 μm).
[0024] Figure 6 SEM images of PPAS MNs after applying (a) 20 g, (b) 50 g, (c) 100 g, (d) 200 g and (e) 500 g weight (scale bar: 500 μm).
[0025] Figure 7 (a) Recovery macrograph of skin after PPAS MNs insertion into pig skin surface (scale bar: 4 mm); (b) to (d) are SEM images of PPAS MNs dissolution after 60 min, 120 min, 150 min insertion into skin respectively (scale bar: 400 μm).
[0026] Figure 8 CLSM images of PPAS MNs diffusion at different depths in skin after pressing for 5 min (scale bar: 120 μm).
[0027] Figure 9 SA release profiles of PPAS MNs under (a) different pH stimuli and (b) different pH stimuli containing 0.1 M H2O2.
[0028] Figure 10 Hemolysis rates of PGA, Alg, PGA-PBA, PPA MNs, PPAS MNs.
[0029] Figure 11 Cell viability of NIH-3T3 cells incubated with PPAS MNs loaded with different concentrations of SA for 24 h.
[0030] Figure 12 Bacterial colony counts of PPA gel, PPA MNs, PPAS gel, PPAS MNs after co-culturing with S. aureus and E. coli respectively.
[0031] Figure 13 Antibacterial rates of PPA gel, PPA MNs, PPAS gel, PPAS MNs against S. aureus and E. coli respectively.
[0032] Figure 14 are SEM images of E. coli and S. aureus before and after incubation with PPA MNs and PPAS MNs.
[0033] Figure 15 are stress-strain tensile curves of PPAS gels soaked in different Ca 2+ concentrations. DETAILED DESCRIPTION
[0034] The application will be further described in detail below in conjunction with the accompanying drawings and examples, but the scope of protection of the application is not limited to these examples. Example 1
[0035] Step 1: Preparation of PGA-PBA 2.0 g (14 mmol) of PGA with a number average molecular weight of 1 million was completely dissolved in 200 mL of MES buffer solution (100 mM, pH=5.5), 4.0 g (21 mmol) of EDC•HCl, 2.4 g (21 mmol) of NHS and 1.0 g (110 mmol) of PBA-NH2 were added in sequence, the reaction was stirred at room temperature for 24 h, then the mixed solution was extracted with ethyl acetate three times, and then the extract was added to a dialysis bag with a molecular weight cut-off of 14000 Da and dialyzed in ultrapure water for 7 d. Finally, the dialyzed solution was concentrated by a rotary evaporator and freeze-dried to obtain PGA-PBA. Figure 1 In the spectrum, a (4.14 ppm) is the proton peak of the tertiary carbon on the PGA backbone, b (2.33 ppm) and c (1.87 ppm) are the proton peaks of the methylene on the PGA backbone. Compared with the original spectrum of PGA, after coupling PBA-NH2, the proton peaks of the PGA backbone are retained at a', b' and c', and the proton peaks of the benzene ring of PBA-NH2 appear at d-g 7.63-7.34 ppm, indicating that PGA-PBA is successfully prepared.
[0036] Step 2: Preparation of PPAS hydrogel 4 g of PGA-PBA was dissolved in 40 mL of ultrapure water to prepare a 10.0% (w / v) PGA-PBA (PP) solution, and the pH of the solution was adjusted to 7.0 with NaOH. 0.9 g of Alg was dissolved in 30 mL of PBS buffer solution at pH 7.4 to prepare a 3.0% (w / v) Alg solution. 40 g of PP solution and 30 g of Alg solution were mixed uniformly, and 49 mg of SA was added simultaneously. The mixture was stirred for 30 s to form a monolayer network PPAS hydrogel. The monolayer network PPAS hydrogel (PPAS SN gel) was immersed in a 1.0% (w / v) CaCl2 aqueous solution for 30 min to chelate, obtaining a multi-network PPAS hydrogel (PPAS gel).
[0037] PGA, PGA-PBA, Alg, PBA-NH2, and PPAS gel samples were analyzed by FT-IR spectroscopy. Figure 2 As shown, compared with the original infrared spectra of PGA and PBA-NH2, PGA-PBA at 1545 cm⁻¹... -1 1426 cm -1 The skeletal vibration peak of the benzene ring in PBA-NH2 appears at 1341 cm⁻¹. -1 The stretching vibration peak of BO appeared at 1641 cm⁻¹. -1 The presence of an amide stretching vibration peak indicates that PBA-NH2 was successfully grafted onto the PGA molecular chain. The PPAS gel at 1594 cm⁻¹... -1 1407 cm -1 and 1026cm -1 The characteristic peak of Alg appears at 3394 cm⁻¹, and the stretching vibration peak of NH increases from 3394 cm⁻¹. -1 Blue shifted to 3272 cm -1 The weakening of the absorption peak of the in-plane deformation vibration of NH2 (amide I band) and the broadening of the absorption peak of -B(OH)2 indicate the formation of hydrogen bonding and further prove the existence of covalent interaction between NB or OB.
[0038] X-ray diffraction was used to analyze and test PGA, PGA-PBA, Alg, PPAS gel, and CaCl2, such as... Figure 3The original PGA has a wide and long reflection band at 24.0° due to its helical structure, and when PBA-NH2 is modified to the PGA molecular chain, PGA-PBA has a wide and long diffraction peak at 20.8°, which is slightly offset compared with the diffraction peak of PGA, indicating the successful modification of phenylboronic acid; Alg has crystal diffraction peaks at 13.6° and 21.0°, and when Alg and PGA-PBA interact to form a PPAS gel, the diffraction peaks at 10.0°-30.0° disappear, which may be due to the interaction of Alg and PGA-PBA and the transformation into an amorphous conformation.
[0039] The mechanical properties of the obtained PPAS gel were further tested, and Alg gel (0.9 g of Alg was dissolved in 30 mL of PBS buffer solution with pH = 7.4, and the hydrogel formed after stirring for 30 s was soaked in 1.0% (w / v) CaCl2 aqueous solution for 30 min for chelation to obtain Alg gel) and PGA-Alg gel (4 g of PGA and 0.9 g of Alg were dissolved in 30 mL of PBS buffer solution with pH = 7.4, and the hydrogel formed after stirring for 30 s was soaked in 1.0% (w / v) CaCl2 aqueous solution for 30 min for chelation to obtain PGA-Alg gel) were used as control groups. The rheometer was used for stress scanning of G' and G'', as shown in Figure 4 It can be seen that the G' of the three groups of samples is greater than the G'', showing an elastic-dominated property, indicating that the three hydrogels have certain strength and toughness. The G' of Alg gel reaches 1.0×10 4 Pa, and the G'' reaches 2.3×10 3 Pa, and the G' of PGA-Alg gel reaches 2.7×10 4 Pa, which is more than 2 times the G' of Alg gel, because the hydrogen bond force between PGA and Alg in the hydrogel is increased. The G' of PPAS gel reaches 6.3×10 4 Pa, which is more than 6 times the G' of Alg gel, and the gel strength is significantly enhanced, because the PBA group and the vicinal diol group of Alg are crosslinked to form a phenylboronic acid ester bond, so that the crosslinking points in the hydrogel system are increased, further improving the mechanical properties of the hydrogel.
[0040] Step 3: Preparation of PPAS hydrogel microneedle The PPAS gel was placed on the microneedle mold (20×20 microneedle array, needle height 600 μm, needle distance 800 μm, needle bottom size 290 μm × 290 μm), and placed for 25 oC The vacuum drying oven was repeatedly vacuumed for 30 min, so that the hydrogel filled the microneedle mold fully, and then the excess hydrogel was removed. After complete drying at room temperature, the PPAS MNs were demolded. The drug loading of SA in the obtained PPAS MNs was 0.7 mg / g.
[0041] The microstructure of the PPAS MNs was characterized by SEM. Figure 5 It can be observed that the prepared PPAS MNs have a regular array, and the spacing between each needle body is about 750 μm, the bottom size is about 230 μm × 230 μm, the height is about 550 μm, and the width of the needle tip is about 5 μm, indicating the successful construction of the PPAS MNs. However, compared with the specifications of the microneedle mold, the size is reduced, which may be due to the shrinkage of the PPAS MNs in the microneedle mold caused by the soaking of CaCl2.
[0042] The mechanical properties, pH / ROS responsive release behavior, transdermal drug delivery characteristics, biocompatibility and antibacterial properties of the PPAS MNs obtained in Example 1 were tested, and the specific experiments and experimental results are as follows: 1. Mechanical property test of PPAS hydrogel microneedles Figure 6 The deformation of the PPAS MNs after being pressed by different weights of weights. When the weight of the applied weight is 20 g (~ 19.6 × 10 -4 N / needle), the tip of the PPAS MNs hardly changes ( Figure 6 (a)), and when the weight increases to 50 g (~ 4.9 × 10 -3 N / needle) to 100 g (~ 9.8 × 10 -3 N / needle), the tip of the PPAS MNs is slightly curved, but the needle body and the needle tip remain intact ( Figure 6 (b), 6 (c)). When the applied weight reaches 200 g (~ 19.6 × 10 -3 N / needle), the tip of the PPAS MNs is curved ( Figure 6 (d)), and when the applied weight reaches 500 g (~ 4.9 × 10 -2 N / needle), the needle body of the PPAS MNs is severely curved, but the needle tip does not break eventually ( Figure 6 (e)), indicating that the prepared PPAS MNs have excellent mechanical strength and toughness, avoiding the danger of breaking in the epidermis during the penetration process.
[0043] 2. Transdermal drug delivery characteristics test of PPAS hydrogel microneedles (1) In vitro dissolution performance PPAS MNs were inserted into pig skin, and the skin compatibility of PPAS MNs was assessed by observing the skin's recovery. Figure 7 As shown in (a), when PPAS MNs are first removed from the skin, a neat basal pore imprint is visible on the surface. After 10 minutes, the pore imprint is no longer obvious, and after 30 minutes, the pore imprint has basically disappeared. This phenomenon indicates that the transdermal drug delivery technology of PPAS MNs has good skin compatibility.
[0044] The solubility of PPAS MNs was further investigated using SEM. Figure 7 (b) to Figure 7 (d) Images showing the dissolution of PPAS MNs after 60 min, 120 min, and 150 min of skin insertion. After 60 min of insertion, the needle partially dissolves. This is because the gel network of PPAS MNs begins to swell and degrade upon contact with body fluids, leading to the disintegration of the needle matrix. After 120 min of insertion, the needle is almost completely dissolved by the interstitial fluid. After 150 min, the needle is completely dissolved in the interstitial fluid, while the base remains intact. This indicates that during transdermal drug delivery, PPAS MNs not only allow for rapid detachment of the needle from the base, improving patient compliance, but also deliver the needle to the subepidermal lesion site, achieving better therapeutic effects.
[0045] (2) External insertion characteristics Rhodamine B was encapsulated in PPAS MNs as a marker, and a detailed study of the implantation depth of PPAS MNs was conducted using CLSM. Green fluorescence images of rhodamine B at different Z-axis heights were captured using CLSM, allowing observation not only of each insertion point but also of the diffusion of rhodamine B near each insertion point. The maximum diffusion depth of rhodamine B was also observed along the Z-axis. Figure 8 As shown, the penetration depth of PPAS MNs into the skin was 340 μm, less than the original height of 550 μm. This is partly due to the swelling of the gel matrix in body fluids during the insertion process, which weakens the mechanical properties. Another reason is the presence of Ca in PPAS MNs. 2+ As the gel network thins in bodily fluids, it gradually breaks down, leading to a decrease in insertion depth. However, a depth of 340 μm is sufficient to reach the lesion site, and PPAS MNs do not risk breaking off in the epidermis during insertion. This is also evidenced by the compressive strength test results of PPAS MNs. In conclusion, PPAS MNs exhibit excellent transdermal drug delivery performance.
[0046] 3. pH / ROS Dual-Response Release Behavior Test of PPAS Hydrogel Microneedles PPAS MNs were placed in 10 mL centrifuge tubes, and 20 mL of PBS with pH = 5.0, pH = 5.5, pH = 6.0, pH = 6.5, pH = 7.0, pH = 7.5, pH = 9.0 was added into the centrifuge tubes respectively. The centrifuge tubes were placed in a constant temperature oscillator at 37 °C in the dark, and at a set time interval, 2 mL of solution was taken out from the centrifuge tube and 2 mL of fresh PBS was added. The removed solution was detected by ultraviolet spectrophotometer to calculate the cumulative release rate of PPAS MNs in this period of time. Each group of experiments was repeated three times. o C constant temperature oscillator in the dark, and at a set time interval, 2 mL of solution was taken out from the centrifuge tube and 2 mL of fresh PBS was added. The removed solution was detected by ultraviolet spectrophotometer to calculate the cumulative release rate of PPAS MNs in this period of time. Each group of experiments was repeated three times. Figure 9 (a) is the cumulative release rate of PPAS MNs at different pH values. It can be seen from the figure that when pH = 5.0, SA can reach a release rate of 92.5% in 10 h. As the pH increases from 5.0 to 7.5, the release rate gradually slows down, and the cumulative release rate decreases from 92.5% to 72.0%. This is due to the acid cleavage of the amide bond in PPAS MNs and the pH response characteristics of the phenylboronic ester bond. When the pH is lower than 6.5, the amide bond and the phenylboronic ester bond are cleaved, thereby destroying the crosslinking points in the hydrogel, and the drug is released from the gel network. However, when the pH = 9.0, the cumulative release rate reaches 62.7%, because in the alkaline environment, the phenylboronic ester bond is firm and will not be destroyed, at this time the drug release mainly relies on the swelling of the gel network.
[0047] 0.1 M H2O2 aqueous solution was added to the PBS with pH = 5.0, pH = 5.5, pH = 6.0, pH = 6.5, pH = 7.0, pH = 7.5, pH = 9.0 as ROS response release medium, and the cumulative release rate of SA in PPAS MNs was determined. Each group of experiments was repeated three times. Figure 9 (b) is the ROS stimulation response characteristics of PPAS MNs. When 0.1 M H2O2 is added to the release medium at different pH values, the release rate of all the media is greatly increased, and when pH = 5.0, the cumulative release rate of SA reaches 97.9% in 5 h. As the pH of the release medium increases, the drug release rate gradually decreases, but even when the pH = 9.0, the release rate can reach 82.6%, indicating that the addition of H2O2 causes the phenylboronic ester bond to break, destroying the gel structure and allowing the drug to be released quickly. Figure 9
[0048] In summary, PPAS MNs have good pH / ROS dual response characteristics.
[0049] 4. Biocompatibility test of PPAS hydrogel microneedle (1) Blood compatibility To ensure the safety of PPAS MNs, the hemolysis of PGA, Alg, PGA-PBA, PPA MNs (preparation method same as Example 1, the difference is that no SA is added), and PPAS MNs were analyzed using fresh human blood. As shown in Figure 10 Figure 2, all test samples showed similar blood cell deposition behavior as the negative control group. Further statistics of the hemolysis rate of the samples showed that the blood compatibility of PGA and Alg was less than 0.2%, because they are both natural polymer materials with excellent biocompatibility. The hemolysis rate of PGA-PBA modified by phenylboronic acid was 2.3%, indicating that the introduction of PBA group would not affect the blood compatibility of PGA itself. In addition, the hemolysis rates of PPA MNs and PPAS MNs were 0.8% and 0.4%, respectively, which fully proved that the hydrogel microneedles formed by the dual action of phenylboronic acid ester bond and Ca 2+ The hydrogel microneedles formed by the dual action of phenylboronic acid ester bond and Ca
[0050] (2) Cell compatibility After NIH-3T3 fibroblasts were incubated with PPAS MNs (preparation method same as Example 1, only the amount of SA added was changed) loaded with different amounts of SA (0.6, 0.7, 0.9, 1.2, 1.4 mg / g) for 24 h, the cytotoxicity was quantitatively evaluated by MTT method. The results showed that when no SA was loaded, the cell survival rate of PPA was 94.5%, and with the gradual increase of drug concentration, the cell viability decreased, and when the drug loading was more than 1.2 mg / g, the cell survival rate was less than 80.0%, which had a certain cytotoxicity. In summary, PPAS MNs had excellent cell compatibility when the drug loading was less than 1.2 mg / g.
[0051] 5. Antimicrobial performance test of PPAS hydrogel microneedles (1) Antimicrobial performance against S. aureus and E. coli S. aureus and E. coli were used as model microorganisms to study the antimicrobial effect of PPA gel, PPA MNs, and PPAS gel and PPAS MNs. As shown in Figure 12 and 13As shown, the colony numbers of PPA gel and PPA MNs groups were not significantly different from the blank group. The antibacterial rates of PPA gel against S. aureus and E. coli were only 9.0% and 14.0%, respectively. Even with the use of microneedle technology for antibacterial, the antibacterial rates of PPA MNs against S. aureus and E. coli could only be increased to 21.0% and 23.3%, respectively, indicating that PPA without drug loading was not enough to play an antibacterial effect. Unlike this, after PPA SN gel was co-cultured with the two kinds of bacteria, the colony numbers on the plate were less, and the antibacterial rates of PPA SN gel against S. aureus and E. coli reached 93.6% and 97.0%, respectively, showing excellent antibacterial performance. In addition, the colony numbers of PPAS MNs group were almost 0, and the antibacterial rates could all reach > 99.9%, indicating that the microneedle drug delivery technology had more excellent antibacterial performance than the gel.
[0052] (2) Bacterial morphology analysis The morphologies of E. coli and S. aureus before and after treatment with PPA MNs and PPAS MNs were observed by FE-SEM. As shown in Figure 14 , before the bacterial solution was cultured with the samples, the cell membranes of E. coli and S. aureus were smooth and complete, and the bacterial morphology was full, indicating that the bacteria had a high activity. After the bacterial solution was cultured with PPA MNs, the bacterial morphology of E. coli was relatively dry compared with the control group, and the cell membrane profile of S. aureus was slightly wrinkled, indicating that PPA MNs had a certain influence on the bacterial morphology, which was due to the needle-like morphology of the microneedles. After the bacterial solution was cultured with PPAS MNs, obvious dead bacteria were observed, S. aureus appeared to be accumulated, the edge was irregular and unclear, the surface was collapsed, and the profile of the cell membrane was wrinkled. The E. coli cells were more obviously collapsed, leaked and wrinkled, indicating that the bacterial body was completely lysed or perforated, which indicated that the bacteria were in a dead state. From the observation of the bacterial morphology, it can be further indicated that the drug-loaded PPAS MNs have good antibacterial effect and are expected to be used for the treatment of acne sites.
[0053] In step 2 of the above Example 1, the PPAS SN gel was soaked in different concentrations of CaCl2 aqueous solution for 30 min for chelation to obtain the PPAS gel. As shown in Figure 15 , with the increase of the concentration of Ca 2+ , the tensile modulus of the hydrogel gradually increased. When the concentration of Ca 2+ was 0.6% w / v, 0.8% w / v and 1.0% w / v, the tensile modulus reached 0.7 MPa, 0.8 MPa and 1.1 MPa, respectively, and the tensile deformation reached 157%, 150% and 197%, respectively. This was because the Ca 2+The addition of α causes an "egg-box structure" to form between Alg molecules, increasing the mechanical strength of the system. However, when Ca... 2+ When the concentration of Ca increased from 1.0% w / v to 1.4% w / v, the tensile deformation decreased from 197% to 27%, indicating that the stronger the rigidity of the hydrogel, the easier it is to break during stretching. Therefore, this invention selects Ca concentrations of 0.6% w / v to 1.2% w / v. 2+ This is to give the hydrogel both rigidity and toughness.
Claims
1. A method for preparing a pH / reactive oxygen species dual-responsive hydrogel microneedle, characterized in that: The preparation method includes the following steps: Step 1: Preparation of polyglutamic acid-phenylboronic acid copolymer Polyglutamic acid was completely dissolved in MES buffer solution, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and 3-aminophenylboronic acid were added sequentially. After stirring at room temperature for 20-24 hours, the mixture was extracted with ethyl acetate. The extract was dialyzed with ultrapure water in a dialysis bag and concentrated by rotary evaporation. After freeze-drying, polyglutamic acid-phenylboronic acid copolymer was obtained. Step 2: Preparation of polyglutamic acid-phenylboronic acid copolymer / sodium alginate / salicylic acid hydrogel Polyglutamic acid-phenylboronic acid copolymer was dissolved in ultrapure water to prepare a polyglutamic acid-phenylboronic acid copolymer solution with a concentration of 9.0% w / v to 15.0% w / v, and the pH of the solution was adjusted to 6.0 to 8.0 with NaOH. Sodium alginate was dissolved in PBS buffer solution to prepare a sodium alginate solution with a concentration of 2.0% w / v to 3.0% w / v. The polyglutamic acid-phenylboronic acid copolymer solution and the sodium alginate solution were mixed evenly, and salicylic acid was added at the same time. After stirring, the hydrogel formed was further immersed in CaCl2 aqueous solution for chelation to obtain polyglutamic acid-phenylboronic acid copolymer / sodium alginate / salicylic acid hydrogel. Step 3: Preparation of pH / Reactive Oxygen Dual-Response Hydrogel Microneedles The polyglutamic acid-phenylboronic acid copolymer / sodium alginate / salicylic acid hydrogel was placed on a microneedle mold, and a vacuum was drawn to ensure that the hydrogel fully filled the microneedle mold. Then, the excess hydrogel was removed, and the microneedles were completely dried at room temperature before demolding to obtain pH / reactive oxygen species dual-responsive hydrogel microneedles.
2. The method for preparing pH / reactive oxygen species dual-responsive hydrogel microneedles according to claim 1, characterized in that: In step 1, the mass ratio of polyglutamic acid to 3-aminophenylboronic acid is 1:0.3-1, and the molar ratio of polyglutamic acid to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1:1-2:1-2.
3. The method for preparing pH / reactive oxygen species dual-responsive hydrogel microneedles according to claim 1, characterized in that: In step 1, the number-average molecular weight of the polyglutamic acid is 800,000 to 1,200,000.
4. The method for preparing pH / reactive oxygen species dual-responsive hydrogel microneedles according to claim 1, characterized in that: In step 1, the molecular weight cutoff of the dialysis bag is 12,000 to 14,000 Da, and the dialysis time is 5 to 7 days.
5. The method for preparing pH / reactive oxygen species dual-responsive hydrogel microneedles according to claim 1, characterized in that: In step 2, the mass ratio of the polyglutamic acid-phenylboronic acid copolymer solution to the sodium alginate solution is 1 to 5:
3.
6. The method for preparing pH / reactive oxygen species dual-responsive hydrogel microneedles according to claim 1, characterized in that: In step 2, the concentration of the CaCl2 aqueous solution is 0.6% w / v to 1.2% w / v, and the chelation time is 20 to 40 minutes.
7. The method for preparing pH / reactive oxygen species dual-responsive hydrogel microneedles according to claim 1, characterized in that: In step 3, the salicylic acid loading in the pH / reactive oxygen species dual-responsive hydrogel microneedles is 0.6–1.2 mg / g.
8. The method for preparing pH / reactive oxygen species dual-responsive hydrogel microneedles according to claim 1, characterized in that: In step 1, the pH of the MES buffer solution is 5.0 to 6.0; in step 2, the pH of the PBS buffer solution is 7.
4.
9. The pH / reactive oxygen species dual-responsive hydrogel microneedles prepared by any one of claims 1 to 8.