Drug-loaded nanoparticle microneedle patch with core-shell structure, preparation method and application thereof

Abstract

This invention belongs to the field of biomedicine, specifically relating to a core-shell structured drug-loaded nanoparticle microneedle patch, its preparation method, and its application. The microneedle patch consists of drug-loaded nanoparticles and a polymer matrix. The interaction between polymers with different charges can create a strong matrix polymer network, allowing antimicrobial peptides to be captured within this network. AMP-Cy3 nanoparticles (CGA-NPs) were prepared and further integrated with the microneedle patch to form a drug-loaded nanoparticle delivery microneedle patch (MN / CGA-NPs). When applied to Staphylococcus aureus infection sites, the microneedles penetrate the extracellular polymer and physically disrupt the structure of the Staphylococcus aureus biofilm. After the polymer microneedles rapidly dissolve, the drug-loaded nanoparticles are released from the microneedle matrix into the bacterial biofilm matrix, thereby killing the bacteria. This drug-loaded nanoparticle delivery microneedle patch provides an effective potential treatment for chronic wound infections.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a drug-loaded nanoparticle microneedle patch with a core-shell structure and a preparation method and application thereof. BACKGROUND

[0002] In the wound healing process, the cascade processes of hemostasis, inflammation, proliferation and remodeling occur in an orderly manner, and bacterial infection disrupts these steps of skin repair, and delayed wound healing in turn leads to persistent infection at the wound site, causing repeated infection and possibly leading to further deterioration. Bacterial biofilm has been identified as the main cause of chronic wounds. Biofilm is a collection of bacteria attached to the surface of the injured tissue, which is difficult to be cleared by the body's immune mechanism and is not sensitive to antibiotic treatment. Therefore, in the case of the increasingly declining effect of antibiotic treatment, the unique bactericidal mechanism of antimicrobial peptides allows antimicrobial peptides to have high efficiency and broad-spectrum antibacterial effect, and also makes it difficult to induce bacterial resistance. However, the non-target toxicity of antimicrobial peptides is one of the reasons why antimicrobial peptides are not widely used. In addition, in the face of a complex infection environment, especially when a biofilm is present, a single antimicrobial peptide cannot achieve the desired therapeutic effect.

[0003] Chitosan is a biodegradable biocompatible polymer, and due to its cationic nature, it has good adhesion and membrane permeability enhancement performance. The positive charge property of chitosan participates in the formation of ionic bonds with the negative charge functional groups of biological polymers, enhancing the drug loading capacity and sustained drug release characteristics of nanoparticles or composite films; gum arabic is a complex polysaccharide, which is a hydrophilic, non-toxic, phytochemical glycoprotein, biocompatible and biodegradable polymer.

[0004] The microneedle patch (MN) can penetrate the extracellular polymeric substance (EPS) of the biofilm, but the traditional microneedle is a non-soluble microneedle, which itself cannot carry drugs and is only a skin piercing tool; has poor biocompatibility; is prone to breakage when acting on the wound, leaving the skin and causing inflammatory reactions. SUMMARY

[0005] The purpose of the present application is to provide a drug-loaded nanoparticle microneedle patch capable of effectively resisting bacteria and promoting wound healing, and a preparation method and application thereof. Based on the contact between positively charged chitosan and negatively charged gum arabic, a gel is formed through intramolecular cross-linking and intermolecular cross-linking mediated by gum arabic. The interaction between the two polymers with different charges can obtain a strong matrix polymer network, so that AMP-Cy3 is captured in the matrix network, thereby preparing AMP-Cy3-loaded nanoparticles. Then, the functional nanomaterial is combined with MN to prepare a drug-loaded nanoparticle microneedle patch with a core-shell structure.

[0006] The application is made of a physical inert polymer PVP K-30 and a recombinant human collagen type III, and can deliver drug-loaded nanoparticles to a lesion site of a chronic wound.

[0007] In order to achieve the above-mentioned purpose, the application adopts the following technical solutions:

[0008] The microneedle patch provided by the application is composed of drug-loaded nanoparticles CGA-NPs and a polymer matrix.

[0009] The drug-loaded nanoparticles CGA-NPs are prepared by forming a strong matrix polymer network through intramolecular cross-linking and intermolecular cross-linking of chitosan (CS) and gum arabic (GA) mediated by GA, so that the antibacterial peptide (AMP-Cy3) is captured in the matrix network, thereby preparing the AMP-Cy3-loaded nanoparticles CGA-NPs, and the specific steps are as follows:

[0010] First, weigh CS and GA, respectively, and dissolve them in 0.1% acetic acid and deionized water, and ultrasonically dissolve them completely; and adjust the pH of the CS and GA solutions to 3 and 7, respectively. Then, dissolve AMP-Cy3 in the 0.1% acetic acid solution of CS, and stir it at room temperature with a magnetic stirrer at 200 rpm. Next, the GA solution is added dropwise to the 0.1% acetic acid solution of CS containing AMP-Cy3, and continues to be stirred at the same speed for 30 minutes. Finally, the sample is dialyzed for 2 hours to remove unreacted materials, and is freeze-dried to obtain CGA-NPs.

[0011] The preparation method of the antibacterial peptide AMP-Cy3 is as follows: the antibacterial peptide sequence KKLRLKIAFK is synthesized by using Fmoc solid-phase synthesis method, and the fluorescent dye Cy3 is coupled to the drug by dehydration condensation reaction to synthesize the final sequence KKLRLKIAFK-Cy3, which is abbreviated as AMP-Cy3.

[0012] The polymer matrix is composed of a physical inert polymer PVP K-30 and a recombinant human collagen type III (Col III) which has good biocompatibility, and the preparation method is as follows: Col III is dissolved in deionized water, and then PVP is added to prepare a mixed solution, which is stored at 4 DEG C for standby use.

[0013] Among them, the mass ratio of deionized water, PVP and Col III is 1:1.5:0.3.

[0014] The obtained drug-loaded nanoparticles have a particle size of 106 nm and a potential of +33.9 mV.

[0015] The application also provides a preparation method of the microneedle patch.

[0016] (1) Dilute the dialyzed CGA-NPs solution with deionized water. Based on its absorbance at 550 nm and the standard curve of AMP-Cy3, adjust the CGA-NPs concentration to 10 μM, and then add Col III and PVP sequentially. Pour the solution onto a mold containing microneedle patches, and centrifuge the covered mold at 2200 g for 20 minutes to fill the pores in the mold.

[0017] (2) 1 mL of polymer matrix solution was poured onto a microneedle mold, and the mold was centrifuged at 2200 g for 15 minutes. Finally, after drying in an oven at 37 °C for 36 hours, the microneedle patch was carefully peeled off and the resulting microneedle patch was stored in a sealed well plate for further study.

[0018] This invention relates to a drug-loaded nanoparticle microneedle patch composed of drug-loaded nanoparticles and a polymer matrix. Based on the interaction between positively charged CS and negatively charged GA, a gel is formed through GA-mediated intramolecular and intermolecular crosslinking. The interaction between the two polymers with different charges creates a strong matrix polymer network, allowing AMP to be captured within this network, thus preparing AMP-loaded nanoparticles CGA-NPs. These are further integrated with a microneedle patch to form a drug-loaded nanoparticle microneedle patch MN / CGA-NPs. When applied to Staphylococcus aureus infection sites, the microneedles penetrate the extracellular polymeric material (EPS) and physically disrupt the structure of the Staphylococcus aureus biofilm. After the polymer microneedles rapidly dissolve, the drug-loaded nanoparticles are released from the microneedle matrix into the bacterial biofilm matrix. This drug-loaded nanoparticle microneedle patch provides an effective potential treatment for chronic wound infections.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The process of preparing nanoparticles in this invention is an extremely mild and simple process. The nanoparticles produced have a reproducible size. In addition, the preparation of microneedle patches is also very simple and inexpensive, laying the foundation for large-scale preparation.

[0021] (2) The microneedle material contains antibacterial nanoparticles and collagen, which can easily pass through the stratum corneum and penetrate the bacterial biofilm to transfer the nanoparticles into it. It has strong invasiveness and solves the problem of easy removal or adverse reactions that exist in the local application of traditional AMP.

[0022] (3) Due to the presence of bacterial biofilms, antibiotics can effectively kill surface bacteria in the short term, but this may also lead to the emergence of drug-resistant bacteria. During the chronic healing process of wounds, symptoms of bacterial infection may recur. The unique antibacterial mechanism of AMP in the CGA-NPs prepared in this invention can effectively kill bacteria without producing drug-resistant bacteria. At the same time, AMP is encapsulated by CS and GA, which slows down the release of AMP to a certain extent, which is sufficient to cope with the recurrence of bacterial infection. Attached Figure Description

[0023] Figure 1 (A) and (B) are HPLC chromatograms of the antimicrobial peptide and the Cy3-labeled antimicrobial peptide, respectively.

[0024] Figure 2 This is the mass spectrum of the antimicrobial peptide;

[0025] Figure 3 Transmission electron microscope (TEM) image of drug-loaded nanoparticles;

[0026] Figure 4 (A) and (B) are particle size distribution diagrams of CG-NPs and CGA-NPs, respectively;

[0027] Figure 5 (A) and (B) are the particle size change diagram of CGA-NPs over seven days and the potential diagram of CG-NPs and CGA-NPs, respectively.

[0028] Figure 6 The UV-Vis absorption spectra of AMP-Cy3, CG-NPs, and CGA-NPs are shown.

[0029] Figure 7 This is a microscopic structure diagram of the microneedles;

[0030] Figure 8 The dissolution of microneedles on the back skin of mice within 30 minutes;

[0031] Figure 9 (A) and (B) are colony plots and statistical graphs of E. coli after co-incubation of different concentrations of AMP-Cy3 and E. coli for one hour, respectively.

[0032] Figure 10 (A) and (B) are colony diagrams and statistical graphs of different concentrations of AMP-Cy3 and S. aureus after co-incubation for one hour;

[0033] Figure 11 (A) and (B) are colony plots and statistical graphs of CG-NPs and E. coli after co-incubation for one hour at different dilution ratios;

[0034] Figure 12 (A) and (B) are colony plots and statistical graphs of CG-NPs and S. aureus after co-incubation for one hour at different dilution ratios;

[0035] Figure 13 (A) and (B) are colony plots and statistical graphs of different concentrations of CGA-NPs and E. coli after co-incubation for one hour;

[0036] Figure 14 (A) and (B) are colony diagrams and statistical graphs of different concentrations of CGA-NPs and S. aureus after co-incubation for one hour;

[0037] Figure 15 (A) and (B) are respectively Ic 90 Live / dead staining assay of CGA-NPs concentrations in E. coli and S. aureus;

[0038] Figure 16 (A) and (B) are respectively Ic 50 and Ic 90 Effect of CGA-NP concentration on the growth curves of E. coli and S. aureus;

[0039] Figure 17 The results show the biocompatibility of CGA-NPs with L929 cells;

[0040] Figure 18 This represents the results of in vitro drug release;

[0041] Figure 19 Photographs of wound healing within 10 days of treating Staphylococcus aureus infection wounds on the backs of mice with different material groups in vivo;

[0042] Figure 20 A quantitative graph showing the area of ​​wound healing within 10 days of treating Staphylococcus aureus-infected wounds on the backs of mice with different material groups in vivo. The wound healing rate (percentage of the original wound) represents the average from three independent experiments;

[0043] Figure 21 Image of wound tissue smear experiment in the CGA-NPs treatment group;

[0044] Figure 22 This image illustrates the preparation method and application of drug-loaded nanoparticle microneedle patches. Detailed Implementation

[0045] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0046] Example 1: Preparation of drug-loaded nanoparticles CGA-NPs and blank nanoparticles CG-NPs

[0047] First, using 5 equivalents of amino acids in MBHA resin, HBTU and HOBt were added and activated in DMF for 45 minutes each time. Second, the Fmoc protecting groups on the amino acids were removed using piperidine / DMF (20%, v / v) for 30 minutes each time. The above steps were repeated until the sequence KKLRLKIAFK was synthesized. HPLC purification is shown in the appendix. Figure 1 (A). The molecular weight of AMP was confirmed by LC-MS: m / z was calculated as [M+2H]. 3+ 489.95, measured value 489.6; [M+2H] 2+ 734.425, actual measured value is 733.8, see attached. Figure 2 ;

[0048] The Fmoc group at the N-terminus of the sequence was then cleaved using TFA / TIS / DCM (1:5:94). The exposed N-terminal amino group was coupled with 5 times excess free Cy3 in the presence of DIPEA for 12 hours to form the sequence AMP-Cy3. The peptide chain AMP-Cy3 was cleaved from the resin using a cleavage mixture of TFA, EDT, deionized water, and TIS (94:2.5:2.5:1, v / v / v / v), and purified by high-performance liquid chromatography (HPLC). HPLC purification details are attached. Figure 1 (B)

[0049] Finally, 3.75 mg of CS and 17.5 mg of GA were weighed and dissolved in 5 mL of 0.1% acetic acid and 3.5 mL of deionized water, respectively, to achieve concentrations of 750 μg / mL and 5 mg / mL. The solutions were then sonicated until completely dissolved, and the pH of the CS and GA solutions was adjusted to 3 and 7, respectively. Subsequently, 5 mg of AMP-Cy3 was dissolved in 5 mL of CS solution and stirred at 200 rpm with a magnetic stirrer at room temperature. Immediately afterwards, 3.5 mL of GA solution was added dropwise to the 0.1% acetic acid solution containing AMP-Cy3 dissolved in CS, and stirring continued at the same speed for 30 minutes. Finally, the sample was dialyzed for 2 hours to remove unreacted material, yielding CGA-NPs with an encapsulation efficiency of 78.4% and a drug loading of 22%. Transmission electron microscopy images are attached. Figure 3 .

[0050] The preparation of blank CG-NPs is relatively simple. It involves adding 3.5 mL of GA solution at pH 7 dropwise to 5 mL of GA solution at pH 3, and stirring at 200 rpm for 30 minutes at room temperature. Finally, the sample is dialyzed for 2 hours to remove unreacted material, yielding CG-NPs.

[0051] Determination of particle size and potential of drug-loaded nanoparticles CGA-NPs and blank nanoparticles CG-NPs

[0052] The dialyzed CGA-NPs solution was diluted with deionized water, and the concentration was adjusted to 10 μM based on its absorbance at 550 nm and the AMP-Cy3 standard curve. The particle size and potential were determined using a Malvern ZSE dynamic light scattering particle size analyzer (UK), and the particle size was 106 nm (see attached figure). Figure 4 (B), the potential is +33.9mV (see appendix). Figure 5 (B). The blank CG-NPs nanoparticles have a particle size of 68.1 nm (see attached image). Figure 4 (A), potential is +37.4mV (see appendix) Figure 5 (B) The particle size of the nanoparticles increased and the potential decreased before and after drug loading. The particle size was measured daily, and the particle size of the drug-loaded nanoparticles did not change significantly over seven days, reflecting the stability of the drug-loaded nanoparticles to some extent. The results are shown in the appendix. Figure 5 (A)

[0053] Experiment on the determination of ultraviolet absorption of drug-loaded nanoparticles

[0054] Take 200 μL of AMP-Cy3, CG-NPs, and CGA-NPs solutions of the same concentration in a 96-well plate, and scan the absorbance across the entire wavelength range of 300 nm to 800 nm using a microplate reader. The UV absorption spectrum is shown in the attached figure. Figure 6 As shown in the figure, standalone CG-NPs have no characteristic absorption in the 450nm–600nm range, while CGA-NPs have a characteristic absorption peak of Cy3 in the 450nm–600nm range, and this peak shows a trend of decreasing and broadening, indicating that AMP-Cy3 has been successfully encapsulated in the nanoparticles.

[0055] AMP-Cy3 in vitro antibacterial activity assay

[0056] 125 μL of AMP-Cy3 at concentrations of 0 μM, 2.5 μM, 5 μM, 7.5 μM, 10 μM, 12.5 μM, 15 μM, 17.5 μM, and 20 μM were respectively mixed with 250 μL of Escherichia coli and Staphylococcus aureus cultures (10 μL / mL). 8The mixture (CFU / mL) was mixed and incubated for 1 hour. Then, the antibacterial properties were evaluated using the agar plate counting method, which involved diluting the mixture of bacteria and AMP-Cy3 by 20,000 times, spreading it on a plate, and incubating it in a biochemical incubator. The colonies on the agar plate were then counted (n≥3) to evaluate the antibacterial properties of AMP-Cy3.

[0057] The antibacterial effect against E. coli is shown in the appendix. Figure 9 (A) and (B) are colony plots and statistical graphs of E. coli after co-incubating different concentrations of AMP-Cy3 with E. coli for one hour, respectively.

[0058] As shown in the figure, AMP-Cy3 has a good killing effect on E. coli, achieving a killing effect of over 90% at 12.5 μM.

[0059] The antibacterial effect against S. aureus is shown in the appendix. Figure 10 (A) and (B) are colony diagrams and statistical graphs of different concentrations of AMP-Cy3 and S. aureus after co-incubation for one hour.

[0060] As shown in the figure, AMP-Cy3 has a better killing effect on S. aureus, achieving a killing effect of over 90% at 10μM.

[0061] In vitro antibacterial activity assay of blank nanoparticles

[0062] 1. Dilute the dialyzed CG-NPs to concentrations of 3%, 6%, 9%, 12%, 15%, 18%, 21%, and 24% with sterile water, and then take 125 μL and 250 μL of E. coli culture (10 8 The mixture of E. coli and CG-NPs was mixed (CFU / mL) and incubated for 1 hour. Then, the mixture of E. coli and CG-NPs was diluted 20,000 times, plated, and incubated in a biochemical incubator. The colonies on the agar plates were counted (n≥3) to evaluate the antibacterial properties of CGA-NPs.

[0063] The antibacterial effect against E. coli is shown in the appendix. Figure 11 (A) and (B) are colony plots and statistical graphs of E. coli after co-incubating different concentrations of CG-NPs with E. coli for one hour, respectively.

[0064] As shown in the figure, the blank nanoparticles have a good killing effect on E. coli, and the killing effect can reach more than 90% when the dilution concentration is 21%.

[0065] 2. Dilute the dialyzed CG-NPs to concentrations of 16%, 32%, 48%, 64%, 80%, 96%, and 100% with sterile water, and then take 125 μL and 250 μL of S. aureus culture (108 The mixture of S. aureus and CG-NPs was mixed (CFU / mL) and incubated for 1 hour. Then, the mixture of S. aureus and CG-NPs was diluted 20,000 times, plated, and incubated in a biochemical incubator. The colonies on the agar plates were counted (n≥3) to evaluate the antibacterial properties of CGA-NPs.

[0066] The antibacterial effect against S. aureus is shown in the appendix. Figure 12 (A) and (B) are colony diagrams and statistical graphs of different concentrations of CG-NPs and S. aureus after co-incubation for one hour.

[0067] As shown in the figure, the blank nanoparticles have virtually no killing effect on S. aureus.

[0068] In vitro antibacterial activity assay of drug-loaded nanoparticles

[0069] 1. 125 μL of CGA-NPs at concentrations of 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, and 3.5 μM were respectively mixed with 250 μL of E. coli culture (10... 8 The mixture of CFU / mL was mixed and incubated for 1 hour. Then, the mixture of E. coli and CGA-NPs was diluted 20,000 times, plated, and incubated in a biochemical incubator. The colonies on the agar plates were counted (n≥3) to evaluate the antibacterial properties of CGA-NPs.

[0070] The antibacterial effect against E. coli is shown in the appendix. Figure 13 (A) and (B) are colony plots and statistical graphs of different concentrations of CGA-NPs and E. coli after co-incubation for one hour.

[0071] As shown in the figure, the drug-loaded nanoparticles have a good killing effect on E. coli, achieving a killing effect of over 90% at 3 μM, indicating the excellent bactericidal effect of the drug-loaded nanoparticles.

[0072] 2. 125 μL of CGA-NPs at concentrations of 2.5 μM, 5 μM, 7.5 μM, 10 μM, 12.5 μM, 15 μM, and 17.5 μM were respectively mixed with 250 μL of S. aureus culture (10... 8 The mixture of S. aureus and CGA-NPs was mixed (CFU / mL) and incubated for 1 hour. Then, the mixture of S. aureus and CGA-NPs was diluted 20,000 times, plated, and incubated in a biochemical incubator. The colonies on the agar plates were counted (n≥3) to evaluate the antibacterial properties of CGA-NPs.

[0073] The antibacterial effect against S. aureus is shown in the appendix. Figure 14(A) and (B) are colony diagrams and statistical graphs of different concentrations of CGA-NPs and S. aureus after co-incubation for one hour.

[0074] As shown in the figure, the nanoparticles have a good killing effect on S. aureus, and the killing effect can reach more than 90% at 10 μM, indicating the excellent bactericidal effect of drug-loaded nanoparticles.

[0075] Bacterial Live / Dead Assay

[0076] The bacterial death of *E. coli* and *S. aureus* before and after treatment with CGA-NPs was verified by a live / dead bacterial staining experiment. 1 mL of *E. coli* and *S. aureus* bacterial suspensions were centrifuged at 5000 rpm, 4°C, for 5 min, and the supernatant was discarded. The suspensions were then resuspended in 250 μL of sterile PBS, followed by 125 μL of CGA-NPs. The suspensions were incubated at 37°C for 30 min. The control group consisted of PBS. After incubation, the suspensions were centrifuged at 37°C. All experimental groups were then mixed with a dye solution containing 3 μM SYTO 9 and 3 μM propidium iodide (PI) at room temperature for 20 min, and finally imaged using a confocal microscope. According to the manufacturer's instructions, live bacterial cells were stained with SYTO 9 dye (green), while dead bacterial cells were labeled with propidium iodide dye (red) due to damage to the cell wall and cell membrane. Results for *E. coli* and *S. aureus* are shown in the appendix. Figure 15 (A) and (B) indicate that CGA-NPs have a good bactericidal effect on both E. coli and S. aureus, and also suggest that its bactericidal mechanism is related to cell membrane disruption.

[0077] Effect of drug-loaded nanoparticles on the growth curves of E. coli and S. aureus

[0078] To investigate the effect of drug-loaded nanoparticles on bacterial growth, 50 μL of Ic solution was added to a 96-well plate. 50 Ic 90 The CGA-NPs and 50 μL of PBS were added, followed by 150 μL of bacterial culture (bacterial concentration of 10). 5 CFU / mL), sequentially set to Ic 50 Group, Ic 90 The study included a control group and three replicates for each group, with E. coli and S. aureus grown in separate 96-well plates. The absorbance of each well at 600 nm was measured continuously for 12 hours using a microplate reader to evaluate the effect of the drug-loaded nanoparticles on bacterial growth. Results for E. coli and S. aureus are shown in the appendix. Figure 16(A) and (B), as shown in the figures, the effects of CGA-NPs on the growth curves of E. coli and S. aureus are not significantly different. The OD600 value of the PBS group increases rapidly in the first 6 hours, reaching its peak around 10 hours; Ic 50 The OD600 value of the group increased rapidly in the first 4 hours, reaching a peak around 10 hours, at which point the peak value was approximately half that of the PBS group; Ic 90 The OD600 values ​​of the group did not change significantly, and did not exhibit the growth and reproduction state of normal bacterial cells; cell proliferation was almost completely inhibited. This result indicates the excellent bactericidal effect of the drug-loaded nanoparticles.

[0079] Biocompatibility assay

[0080] The MTT assay was used to investigate the toxicity of nanomaterials to L929 cells, thereby evaluating the biocompatibility of drug-loaded nanoparticles. First, cells were seeded in 96-well microplates (10 cells per well). 4 Cells were incubated overnight on DMEM culture medium. Drug-loaded nanoparticles (92 μM) were diluted to 2 μM, 4 μM, 8 μM, and 16 μM. Different concentrations (0 μM, 2 μM, 4 μM, 8 μM, and 16 μM) of CGA-NPs solutions were then co-incubated with the overnight-cultured cells. After 24 h and 48 h of incubation, cell viability was assessed by measuring absorbance at 490 nm using a microplate reader to evaluate the biocompatibility of CGA-NPs. Results are attached. Figure 17 The results showed that the cell survival rate was greater than 90% at a drug-loaded nanoparticle concentration of 12 μM. Combined with the minimum inhibitory concentration of the nanoparticles, this indicates that CGA-NPs can effectively inhibit bacterial growth without causing cytotoxicity.

[0081] In vitro drug release

[0082] An appropriate amount of CGA-NPs was completely dissolved in 3 mL of deionized water and placed in a sealed dialysis membrane (molecular weight cutoff 3.5 KD). The dialysis membrane was then immersed in a 50 mL centrifuge tube containing 40 mL of release medium. The centrifuge tube was placed vertically in a 37°C constant temperature shaking incubator and shaken (180 rpm). Every 30 min, 0.8 mL of release medium was taken for concentration determination, and the same volume, temperature, and pH of release medium were added to ensure that the total volume of release medium remained constant. The OD450 of the release medium was measured using a UV spectrometer to determine the AMP concentration. Skin pH plays an important role in maintaining normal skin flora and preventing pathogen invasion. Normal skin is slightly acidic, with an average pH of 5–6. When a skin wound is infected by S. aureus, the skin pH changes from acidic to slightly alkaline. Therefore, we measured the cumulative release of AMP from CGA-NPs over 4 h and the release of AMP-Cy3 alone at pH 5.5 and 7.2. The results are shown in the attached figure. Figure 18As shown.

[0083] This AMP exhibits excellent hydrophilicity, with a maximum release rate of 90% within the first hour. If directly loaded into microneedles, the AMP would be completely released in a short time as the needle tip dissolves, insufficient to handle the repeated exposure to biofilms. Encapsulating it in nanoparticles not only protects the AMP from degradation but also provides a sustained-release effect. A pH of 7.2 simulates infection by *S. aureus*. In this environment, GA absorbs water and swells, creating larger gaps in the CS-GA chains, from which AMP-Cy3 is released. Release continues for the first 4 hours, reaching a maximum of 59% and showing a continued release trend. A pH of 5.5 simulates normal skin, which is slightly acidic. CS dissolves under acidic conditions, leading to an explosive release of AMP-Cy3 in the first 0.5 hours, with the maximum release rate reaching approximately 80% after 3 hours.

[0084] Comparative Example 1

[0085] 1.25 mg of CS and 17.5 mg of GA were weighed and dissolved in 5 mL of 0.1% acetic acid and 3.5 mL of deionized water, respectively, to achieve concentrations of 250 μg / mL and 5 mg / mL. The solutions were sonicated until completely dissolved, and the pH of the CS and GA solutions was adjusted to 3 and 7, respectively. Subsequently, 5 mg of AMP-Cy3 was dissolved in 5 mL of CS solution and stirred at 200 rpm with a magnetic stirrer at room temperature. Immediately afterwards, 3.5 mL of GA solution was added dropwise to the 0.1% acetic acid solution containing AMP-Cy3 dissolved in CS, and stirring continued at the same speed for 30 minutes. Finally, the sample was dialyzed for 2 hours to remove unreacted material, yielding CGA-NPs. At this point, the nanoparticle size was 58.2 nm, and the PDI was 0.289; however, due to the low CS content, the Zeta potential was only +29.1 mV.

[0086] Comparative Example 2

[0087] 5 mg of CS and 17.5 mg of GA were weighed and dissolved in 5 mL of 0.1% acetic acid and 3.5 mL of deionized water, respectively, to achieve concentrations of 1 mg / mL and 5 mg / mL. The solutions were sonicated until completely dissolved, and the pH of the CS and GA solutions was adjusted to 3 and 7, respectively. Subsequently, 5 mg of AMP-Cy3 was dissolved in 5 mL of CS solution and stirred at 200 rpm with a magnetic stirrer at room temperature. Immediately afterwards, 3.5 mL of GA solution was added dropwise to the 0.1% acetic acid solution containing AMP-Cy3 dissolved in CS, and stirring continued at the same speed for 30 minutes. Finally, the sample was dialyzed for 2 hours to remove unreacted material, yielding CGA-NPs. At this point, due to the high CS content, the nanoparticle size was relatively large at 184.2 nm, the PDI was 0.312, and the Zeta potential was +36.3 mV.

[0088] Comparative Example 3

[0089] 3.75 mg of CS and 3.5 mg of GA were weighed and dissolved in 5 mL of 0.1% acetic acid and 3.5 mL of deionized water, respectively, to achieve concentrations of 250 μg / mL and 1 mg / mL. The solutions were sonicated until completely dissolved, and the pH of the CS and GA solutions was adjusted to 3 and 7, respectively. Subsequently, 5 mg of AMP-Cy3 was dissolved in 5 mL of CS solution and stirred at 200 rpm with a magnetic stirrer at room temperature. Immediately afterwards, 3.5 mL of GA solution was added dropwise to the 0.1% acetic acid solution containing AMP-Cy3 dissolved in CS, and stirring continued at the same speed for 30 minutes. Finally, the sample was dialyzed for 2 hours to remove unreacted material, yielding CGA-NPs. At this point, the nanoparticle size was 58.2 nm. Due to the low CS and GA content, the PDI was high at 0.415, and the Zeta potential was low at +28.8 mV.

[0090] Comparative Example 4

[0091] 3.75 mg of CS and 35 mg of GA were weighed and dissolved in 5 mL of 0.1% acetic acid and 3.5 mL of deionized water, respectively, to achieve concentrations of 1 mg / mL and 10 mg / mL. The solutions were then sonicated until completely dissolved, and the pH of the CS and GA solutions was adjusted to 3 and 7, respectively. Subsequently, 5 mg of AMP-Cy3 was dissolved in 5 mL of CS solution and stirred at 200 rpm with a magnetic stirrer at room temperature. Immediately afterwards, 3.5 mL of GA solution was added dropwise to the 0.1% acetic acid solution containing AMP-Cy3, and stirring continued at the same speed for 30 minutes. During stirring, the solution exhibited severe emulsification and the Tyndall effect disappeared, possibly due to excessive aggregation of nanoparticles, resulting in a non-uniform particle size distribution and the appearance of peaks above 1000 nm.

[0092] Comparative Example 5

[0093] 3.75 mg of CS and 17.5 mg of GA were weighed and dissolved in 5 mL of 0.1% acetic acid and 3.5 mL of deionized water, respectively, to achieve concentrations of 750 μg / mL and 5 mg / mL. The solutions were sonicated until completely dissolved, and the pH of the CS and GA solutions was adjusted to 3 and 7, respectively. Subsequently, 1 mg of AMP-Cy3 was dissolved in 5 mL of CS solution and stirred at 200 rpm with a magnetic stirrer at room temperature. Immediately afterwards, 3.5 mL of GA solution was added dropwise to the 0.1% acetic acid solution containing AMP-Cy3 dissolved in CS, and stirring continued at the same speed for 30 minutes. Finally, the sample was dialyzed for 2 hours to remove unreacted material, yielding CGA-NPs. At this point, the nanoparticle size was 98 nm, the PDI was 0.268, the Zeta potential was +36.3 mV, and the encapsulation efficiency was as high as 89%, but the drug loading was low, only 18%.

[0094] Comparative Example 6

[0095] 3.75 mg of CS and 17.5 mg of GA were weighed and dissolved in 5 mL of 0.1% acetic acid and 3.5 mL of deionized water, respectively, to achieve concentrations of 750 μg / mL and 5 mg / mL. The solutions were sonicated until completely dissolved, and the pH of the CS and GA solutions was adjusted to 3 and 7, respectively. Subsequently, 10 mg of AMP-Cy3 was dissolved in 5 mL of CS solution and stirred at 200 rpm with a magnetic stirrer at room temperature. Immediately afterwards, 3.5 mL of GA solution was added dropwise to the 0.1% acetic acid solution containing AMP-Cy3 dissolved in CS, and stirring continued at the same speed for 30 minutes. Finally, the sample was dialyzed for 2 hours to remove unreacted material, yielding CGA-NPs. At this point, the nanoparticle size was 106 nm, the PDI was 0.270, and the Zeta potential was +34.1 mV. Under these synthesis conditions, the drug loading rate decreased slightly to 21%. 5 mg of AMP-Cy3 was sufficient to achieve the maximum drug loading rate. Adding too much AMP-Cy3 actually caused the encapsulation efficiency to decrease significantly to only 43%.

[0096] Comparative Example 7: Preparation of Unloaded Blank Microneedle Patches

[0097] The blank microneedles consist of a polymer matrix composed of physically inert polymer PVP K-30 and recombinant human type III collagen (Col III). The preparation method is as follows: First, 0.20 g of Col III is dissolved in 1 mL of deionized water, and then 1.5 g of PVP is added to prepare a mixed solution, i.e., the polymer matrix solution. Second, 1 mL of the polymer matrix solution is poured onto a microneedle mold, and the mold is centrifuged at 2200 g for 15 minutes. Finally, after drying in an oven at 37°C for 36 hours, the microneedle patch is carefully peeled off. The resulting microneedle patch (e.g., ...) is then... Figure 7 (As shown), stored in a sealed orifice plate for further study.

[0098] Example 2: Preparation of drug-loaded nanoparticle microneedle patches

[0099] When preparing microneedle patches, the mass ratio of deionized water:PVP:Col III should be strictly followed as 1:1.5:0.3. (When the ratio of deionized water to PVP is greater than 1:1.5, the mechanical strength of the microneedles formed after drying is insufficient, making it impossible to penetrate the skin; when the ratio of deionized water to PVP is less than 1:1.5, the microneedles are too brittle and difficult to peel off from the mold; when Col III reaches its maximum solubility, the ratio of deionized water to Col III should be 1:0.3.)

[0100] The drug-loaded nanoparticle microneedle patch consists of drug-loaded nanoparticles and a polymer matrix. The specific preparation method is as follows:

[0101] Dissolve 0.30g Col III in 1mL of deionized water, then add 1.5g PVP to prepare a mixed solution, i.e., a polymer matrix solution, and store at 4℃ for later use.

[0102] First, the dialyzed CGA-NPs solution was diluted with deionized water. Based on its absorbance at 550 nm and the AMP-Cy3 standard curve, the CGA-NPs concentration was adjusted to 10 μM. 1 mL of this solution was then added sequentially with 0.3 g of Col III and 1.5 g of PVP. This solution was poured onto a mold containing microneedle patches, and the covered mold was centrifuged at 2200 g for 20 minutes to fill the pores.

[0103] Next, 1 mL of polymer matrix solution was poured onto a microneedle mold, and the mold was centrifuged at 2200 g for 15 minutes. Finally, after drying in an oven at 37 °C for 36 hours, the microneedle patch was carefully peeled off and the resulting microneedle patch was stored in a sealed well plate for further study.

[0104] Microneedle dissolution experiment in mouse skin

[0105] The prepared microneedles were inserted into the skin on the backs of three mice, and left in place for 10 min, 20 min, and 30 min, respectively, before being removed. The dissolution of the microneedle tips was observed under a microscope, and the results are shown in the attached figure. Figure 8 As shown, the microneedles dissolved completely after 30 minutes on the skin of a mouse's back, releasing the drug-loaded nanoparticles.

[0106] Wound Repair Experiment

[0107] The specific experimental steps are as follows:

[0108] (1) Bacterial infection trauma model of back wound in BALB / c mice

[0109] To establish an infected mouse wound model, an elliptical full-thickness wound (8 mm × 6 mm) penetrating the fascia was created on the back of mice using a disposable biopsy puncturator. Bacteria (10 μL) were then inoculated at the wound site. 7 S. aureus (CFU / mL) was infected for 72 hours. During the modeling period, mice were housed individually with free access to water and food. After the modeling was completed, each mouse's wound was infected with bacteria and appeared yellow.

[0110] (2) Grouping of animals

[0111] Eighteen mice were randomly divided into six groups:

[0112] Group A was the PBS control group, and the dosage of the drug was 200 μL / wound, applied externally.

[0113] Group B received PVP+Col III, with a dosage of 200 μL / wound, applied topically.

[0114] Group C consisted of PVP+CoI III+CG-NPs, with a dosage of 200 μL / wound, applied topically.

[0115] Group D received PVP+CoI III+AMP-Cy3, with a dosage of 200 μL / wound, applied topically.

[0116] Group E consisted of PVP+CoI III+CGA-NPs, with a dosage of 200 μL / wound, applied topically.

[0117] Group F consists of drug-loaded microneedle patches (MN / PVP+CoI III+CGA-NPs), applied topically;

[0118] (3) Apply each group of materials to the wound on the back of the mouse, treat once every two days, stop treatment after three treatments, and euthanize the mouse after 10 days of feeding and observation.

[0119] Experimental observations showed that by day ten, the wounds on the backs of mice in all experimental groups had healed to varying degrees. Wound images, quantitative data on wound area, and results of wound tissue smears are attached. Figure 19 , 20 As shown in Figure 21, it can be seen that group F (MN / PVP+CoI III+CGA-NPs) showed significant improvement in wound healing after Staphylococcus aureus infection, exhibiting the best wound healing effect and minimal scarring. The wound tissue smear results also indicated that Staphylococcus aureus at the wound site was completely eradicated, verifying the excellent antibacterial effect of CGA-NPs. Furthermore, compared to group E, group F utilized a microneedle delivery method to puncture the biofilm and directly deliver CGA-NPs to the lesion site, fundamentally eliminating Staphylococcus aureus and preventing recurrent biofilm infection.

[0120] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A core-shell structured drug-loaded nanoparticle microneedle patch, characterized in that, The microneedle patch is composed of drug-loaded nanoparticles and a polymer matrix; Drug-loaded nanoparticles CGA-NPs are prepared by chitosan CS and gum arabic GA forming a strong matrix polymer network through GA-mediated intramolecular and intermolecular crosslinking, which allows the antimicrobial peptide AMP-Cy3 to be captured in the matrix polymer network, thus preparing AMP-Cy3-loaded nanoparticles CGA-NPs. The method for preparing the antimicrobial peptide AMP-Cy3 is as follows: the antimicrobial peptide sequence KKLRLKIAFK is synthesized using the Fmoc solid-phase synthesis method, and then the fluorescent dye Cy3 is coupled to the antimicrobial peptide using a dehydration condensation reaction to synthesize the final sequence KKLRLKIAFK-Cy3, abbreviated as AMP-Cy3. The polymer matrix is ​​composed of physically inert polymer PVP K-30 and recombinant human type III collagen Col III.

2. The core-shell structured drug-loaded nanoparticle microneedle patch as described in claim 1, characterized in that, The preparation steps of the AMP-Cy3 nanoparticles CGA-NPs are as follows: First, weigh CS and GA, dissolve them in 0.1% acetic acid and deionized water respectively, sonicate them until completely dissolved, and adjust the pH of the CS and GA solutions to 3 and 7 respectively. Next, AMP-Cy3 was dissolved in a 0.1% acetic acid solution of CS and stirred with a magnetic stirrer at room temperature. Then, GA solution was added dropwise to the acetic acid solution of CS containing AMP-Cy3 and stirred at the same speed for 30 minutes. Finally, the sample was dialyzed for 2 hours to remove unreacted material and lyophilized to obtain CGA-NPs.

3. The core-shell structured drug-loaded nanoparticle microneedle patch as described in claim 2, characterized in that, The concentration of CS was 0.75 mg / mL; the concentration of GA was 5 mg / mL; and the amount of AMP-Cy3 added was 5 mg.

4. The core-shell structured drug-loaded nanoparticle microneedle patch as described in claim 1, characterized in that, The polymer matrix is ​​prepared by dissolving Col III in deionized water, then adding PVP to mix and prepare a mixed solution, which is then stored at 4 ℃ for later use.

5. The core-shell structured drug-loaded nanoparticle microneedle patch as described in claim 4, characterized in that, The mass ratio of deionized water, PVP and Col III is 1:1.5:0.

3.

6. The core-shell structured drug-loaded nanoparticle microneedle patch as described in claim 1, characterized in that, The drug-loaded nanoparticles have a particle size of 106 nm and a potential of +33.9 mV.

7. A method for preparing a core-shell structured drug-loaded nanoparticle microneedle patch as described in claim 1, characterized in that, The microneedle patch is prepared by solvent casting, and the preparation method steps are as follows: First, the dialyzed CGA-NPs solution was diluted with deionized water to adjust the CGA-NPs concentration to 10 μM, and then Col III and PVP were added sequentially. The solution was poured onto a mold containing microneedle patches, and the covered mold was centrifuged at 2200 g for 20 minutes to fill the pores in the mold. Next, the polymer matrix solution was cast onto the microneedle mold, and the mold was centrifuged at 2200 g for 15 minutes. Finally, after drying in an oven at 37°C for 36 hours, the microneedle patch was peeled off.

8. The application of a core-shell structured drug-loaded nanoparticle microneedle patch as described in claim 1 in the preparation of wound dressings.

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