Novel drug-loaded microneedle applied to construction of oral leukoplakia animal model

Abstract

The invention discloses a method for constructing a 4-nitroquinoline-N-oxide (4NQO) supported soluble microneedle patch (4NQO-PVA / CS-MNs). The design of the 4-nitroquinoline-N-oxide (4NQO) supported soluble microneedle patch (4NQO-PVA / CS-MNs) has both scientific perspectiveness and clinical transformation potential. The needle body is made of a polyvinyl alcohol / chitosan (PVA / CS) composite material with excellent biocompatibility, and the structural stability of the backing layer is enhanced through polyvinyl alcohol / polyvinylpyrrolidone (PVA / PVP) crosslinking. According to the delivery system, the effective concentration of the drug in the local mucous membrane is maintained through a slow release mechanism, typical oral leukoplakia (OLK) can be induced in a four-week modeling period, and the research period is remarkably shortened. Meanwhile, a micro-channel formed by microneedle puncture can excite a tissue repair cascade reaction, and the dual effects of drug delivery and microenvironment regulation and control are synchronously achieved. The research provides a precise and controllable novel tool for OLK pathological mechanism analysis and drug screening, and is expected to promote the innovation of oral mucosa precancerous lesion research paradigm.

Description

Technical Field

[0001] The present invention relates to the technical field of constructing an oral leukoplakia animal model with soluble novel drug-loaded microneedles, specifically to the preparation of soluble microneedles loaded with 4-nitroquinoline-N-oxide (4NQO) and their application to the dorsal tongue mucosa of rats to construct oral leukoplakia. Background Art

[0002] Oral leukoplakia (OLK), as one of the typical potentially malignant disorders of oral mucosa, its occurrence and development involve the interaction of multiple factors such as smoking, Candida infection, human papillomavirus infection, immune disorder and microenvironmental disturbance, and has a significant potential for malignancy. At present, the 4NQO induction method has become the mainstream strategy for constructing an OLK animal model. This water-soluble carcinogen can simulate the oral carcinogenesis process by drinking water or local coating, but the traditional drug delivery mode has significant limitations: the modeling period is as long as 12-16 weeks, and systemic drug delivery causes non-specific toxicity in multiple organs (such as esophageal cancer), resulting in a high mortality rate of experimental animals. It is difficult to form an effective concentration gradient in the oral mucosa after drug metabolism and diffusion. Moreover, the oral or coating method is easily affected by the first-pass effect and drug loss, resulting in low bioavailability and unstable lesion induction efficiency, seriously restricting the research efficiency.

[0003] Microneedles (MN) technology provides an innovative solution to break through the above bottlenecks. This technology painlessly penetrates the mucosal barrier through an array of micron-scale needles (height 10-2000 μm, width 10-50 μm), and realizes deep drug delivery on the premise of avoiding nerve stimulation. Summary of the Invention

[0004] This study innovatively constructs a 4NQO-loaded soluble novel drug-loaded microneedle patch (4NQO-PVA / CS-MNs), whose design has both scientific foresight and clinical transformation potential. The needle body uses a polyvinyl alcohol / chitosan (PVA / CS) composite material with excellent biocompatibility, and the backing layer enhances the structural stability through cross-linking of polyvinyl alcohol / polyvinylpyrrolidone (PVA / PVP). This delivery system maintains the effective concentration of the drug locally in the mucosa through a sustained-release mechanism, and can induce typical OLK within a 4-week modeling period, significantly shortening the research period. At the same time, the microchannels formed by microneedle puncture can stimulate the tissue repair cascade reaction, and simultaneously achieve the dual effects of drug delivery and microenvironment regulation. This study provides a precise and controllable new tool for the analysis of the pathological mechanism of OLK and drug screening, and is expected to promote the innovation of the research paradigm for oral mucosal precancerous lesions.

[0005] To solve the above problems, the technical solution of the present invention is as follows: Preparation of 4NQO-PVA / CS-MNs: It includes the synthesis of PVA / CS solution, the preparation of the backing layer PVA / PVP, and the preparation of 4NQO-PVA / CS-MNs;

[0006] Synthesis of PVA / CS solution: Slowly add 2 g of CS powder (degree of deacetylation ≥ 85%) to 100 mL of pre-cooled 1% acetic acid solution, and stir magnetically (at room temperature, 500 - 800 rpm) until completely dissolved. Transfer the solution to a dialysis bag with a molecular weight cut-off of 10 kDa, and dialyze with deionized water until the pH of the dialysis solution is close to 6.0. After dialysis, the solution is filtered through a 0.45 μm filter membrane, and the nearly neutral CS solution is transferred to a constant temperature drying oven at 37°C to remove the moisture therein, thereby obtaining a CS solution with a concentration of 10%. The solution needs to be stored refrigerated (at 4°C).

[0007] Weigh 10 g of PVA (PVA 1788, degree of alcoholysis 88%) and add it to 100 mL of deionized water, stir at 90°C until completely dissolved, cool to room temperature, and then mix it with the 10% CS solution in a volume ratio of 7:3, stir magnetically for 30 min, and store it in a refrigerator at 4°C for later use.

[0008] Preparation of PVA / PVP solution: Prepare a 1:1 (v / v) mixture of 10% PVA and 10% PVP aqueous solutions.

[0009] Preparation of PVA / CS-MNs: Drop 200 μL of the PVA / CS mixed solution onto the surface of a PDMS microneedle mold (needle length 650 μm, bottom diameter 280 μm, 10×10 array), centrifuge at 3500×g for 10 min, and repeat 3 times to ensure complete filling of the mold cavity. Use a ruler to scrape off the excess solution above the mold, and place it in a vacuum drying oven at 40°C for 30 min to form the tip layer. Mix 10% PVA and 10% PVP aqueous solutions in a 1:1 (v / v) ratio, and evenly coat the back of the tip layer, dry at 40°C for 12 h to form the base layer. Peel the microneedle patch from the mold and store it in a desiccator (relative humidity ≤ 20%) in the dark.

[0010] Preparation of 4NQO-PVA / CS-MNs:

[0011] (1) Dissolve 4NQO in 1,2-propanediol to prepare a 30 mg / mL solution. Vortex-mix the 4NQO solution and the PVA / CS mixture in proportion (avoid introducing air bubbles), let it stand at 4°C for 12 h, and then sterilize it through a 0.22 μm filter membrane. Store it in the dark at 4°C.

[0012] (2) After evacuating the PDMS microneedle mold, 200 μL of the 4NQO-PVA / CS mixed solution was spread on the surface of the mold, placed in a 50 mL centrifuge tube, and centrifuged at 3500×g for 10 min. This was repeated 3 times to ensure complete filling of the mold cavities. After scraping off the excess solution, the mold was transferred to a vacuum drying oven and dried at 40 °C for 20 min to form the tip layer. A solution of 10% PVA and 10% PVP in a 1:1 (v / v) ratio was mixed and coated on the back of the tip layer, and then vacuum dried at 30 °C for 24 h to form the base layer. After peeling off the microneedle patch, it was stored in a nitrogen-filled desiccator (4 °C, RH≤10%) for use in the dark.

[0013] Furthermore, the morphological characteristics of the 4NQO-PVA / CS-MNs were observed by scanning electron microscopy (SEM), and at the same time, elemental distribution analysis of the samples was carried out. The acceleration voltage was set at 10 kV, and quantitative analysis of the microneedle size was performed using Image J software; the Fourier transform infrared spectrum (FTIR) of 4NQO-PVA / CS was obtained by Fourier transform infrared spectrometer to determine the relevant characteristic groups.

[0014] Furthermore, the mechanical properties of the 4NQO-PVA / CS-MNs include mechanical property testing, in vitro drug release, adhesion testing, puncture performance testing, and irritation to the skin.

[0015] Mechanical property testing: The compressive strength of the prepared PVA / CS-MNs and 4NQO-PVA / CS-MNs was tested using a universal mechanical testing machine. The microneedle base was fixed flat on the lower fixture base, and an upper fixture probe with a diameter of 10 mm was selected. It moved downward from the contact with the tip at a rate of 0.1 mm / min, and the force values between 0 - 600 μm of displacement were recorded. The obtained data were recorded and the stress-strain curve was plotted.

[0016] To evaluate the effect of 4NQO-PVA / CS-MNs on the proliferation of HOK cells, the specific method was as follows: 4NQO-PVA / CS-MNs were added to the complete medium (containing 10% fetal bovine serum and 1% double antibody) at a concentration of 1.6 mg / mL, and incubated at 37 °C and 5% CO2 for 24 hours to simulate the cell culture environment to release the soluble components in the material; then centrifuged at 3000 rpm for 10 minutes, the supernatant was taken to remove insoluble particles, and extracts with concentrations of 0.2, 0.4, 0.8, and 1.6 mg / mL were obtained by gradient dilution, labeled and stored at 4 °C for use (it is recommended to use within 24 hours).

[0017] In the experimental procedure, HOK cells were seeded into a 96-well plate at a density of 3000 cells / well, and 100 μL of cell suspension was added to each well. The cells were pre-cultured in an incubator at 37 °C and 5% CO2 for 24 hours to allow them to adhere. Subsequently, the original medium was replaced with 4NQO-PVA / CS-MNs extracts at different concentrations (experimental group) or normal complete medium (blank control group). Each group was set with 4 replicates, and the detection was carried out after co-incubation for 1, 2, and 3 days. When detecting, the original medium should be aspirated first to avoid interference from residual materials. 100 μL of fresh medium containing 10% CCK-8 reagent (90 μL medium + 10 μL CCK-8) was added to each well, and it was incubated in the dark for 1-4 hours (the optimal color development time was determined by preliminary experiments, usually 2 hours). Subsequently, the absorbance (OD value) was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm.

[0018] The experimental results were analyzed for statistical differences by ANOVA. Technical replicates (3 wells / concentration) and biological replicates (3 independent experiments) were set for each group to improve reliability.

[0019] Adhesion test: Fresh excised pig skin was taken, the hair on the surface was cleaned, and the excess grease was scraped off. 4NQO-PVA / CS-MNs were punctured into the inner side of the pig skin, and then it was continuously rinsed with running water for 1 min to observe the adhesion of the microneedle patch.

[0020] Puncture performance test: The microneedle patch was pressed onto the excised pig skin (2 kg / cm 2 , 30 s), and after removal, 0.1% methylene blue solution (5 min) was added dropwise. The rhodamine B-labeled microneedles were pressed onto the flat excised pig skin and placed under a confocal laser scanning microscope (CLSM) for layer-by-layer scanning at an excitation wavelength of 562 nm. CLSM images were captured every 50 μm in depth. The distribution of rhodamine B at different skin depths was observed to evaluate the puncture of the microneedles in the tissue.

[0021] Stimulation to the skin: Rats were intraperitoneally anesthetized with 2% pentobarbital sodium, and the back skin was gently depilated. The microneedle patch was placed on the back skin and continuously pressed for 5 min and then removed. The recovery of the back skin was observed at 10 min, 20 min, and 30 min after removal, respectively.

[0022] Furthermore, the in vitro biocompatibility of 4NQO-PVA / CS-MNs was investigated. The CCK-8 method was used to evaluate the effect of 4NQO-PVA / CS-MNs on the proliferation of HOK cells. The experiment was divided into a blank control group, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, 1.6 mg / mL 4NQO-PVA / CS-MNs groups, and each group had 4 replicate wells. The HOK cell suspension was inoculated into a 96-well plate at a cell density of 3000 cells / well. After culturing at 37 °C and 5% CO2 for 24 h, the cell culture medium of 4NQO-PVA / CS-MNs was replaced with the complete medium of 4NQO-PVA / CS-MNs, and the blank control group still used the cell culture medium. After co-incubation for 1, 2, and 3 days, the medium was replaced with a medium containing 10% CCK-8 and incubated for 1 - 4 h. The optical density (OD) value of the medium was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0023] Furthermore, the experiment explored the appropriate concentration of 4NQO in the microneedle patch. To explore the appropriate concentration of 4NQO for the suitable microneedles, the literature showed that the applied concentration of 4NQO was 0.5 - 2 mg / mL, 2 - 3 times a week [24 - 27]. Based on this, the 4NQO concentrations set in this experiment were 0.4 mg / mL, 0.8 mg / mL, and 1.6 mg / mL, respectively. They were prepared into 4NQO-PVA / CS-MNs, and then a pre-experiment on rats was carried out to prove their effects.

[0024] Furthermore, 4NQO-PVA / CS-MNs were used to establish OLK formation in Wistar rats.

[0025] Sixty 8-week-old male Wistar rats were purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd. (Shandong, China). All animals were bred under a 12 h light-dark cycle for one week, and the experiment was carried out after the rats adapted to the environment. All experimental protocols were approved by the Animal Protection and Use Committee of Qingdao University. The experimental animals were strictly raised according to the Chinese national standard requirements for laboratory animal environment and facilities (GB 14925 - 2010 / xg1 - 2011), fed with standard feed, and the breeding conditions were 12 h light - 12 h dark, humidity 50 ± 15%, and temperature 22 ± 2 °C. The experimental operations followed the eighth edition of the "Guide for the Care and Use of Laboratory Animals", ISBN-10: 0 - 309 - 15394 - 4 and the relevant regulations of the Laboratory Animal Management Measures of the Medical College of Qingdao University. The animals were adaptively raised for one week before the experiment, and they had free access to food and water during this period.

[0026] Experimental grouping: The sixty male Wistar rats were randomly divided into a blank control group, a drug-drinking group, a PVA / CS-MNs group, and a 4NQO-PVA / CS-MNs group, with 20 rats in each group (n = 20).

[0027] Blank control group: Distilled water was provided daily.

[0028] Drug administration group: The 4NQO was prepared into a stock solution with a concentration of 0.1% (0.004 g made up to 40 mL) with distilled water and stored in the refrigerator at 4 °C in the dark.

[0029] PVA / CS-MNs: The prepared PVA / CS-MNs were applied to the anterior 1 / 2 of the dorsal tongue of the rats and pressed for 20 s.

[0030] 4NQO-PVA / CS-MNs: The 4NQO-PVA / CS-MNs with a concentration of 0.8 mg / mL were applied to the anterior 1 / 2 of the dorsal tongue of the rats and pressed for 20 s.

[0031] Experimental procedure

[0032] (1) Preparation of anesthetic: Weigh 1.5 g of pentobarbital sodium powder, dissolve it in 100 mL of ultrapure water, disperse it thoroughly with an ultrasonic oscillator, aliquot and seal it in 20-mL centrifuge tubes, and store it in the refrigerator at 4 °C. Freshly prepare it each time after use.

[0033] (2) Two trained experimenters administered drugs to the Wistar rats in each experimental group at a fixed time every week for 4 consecutive weeks.

[0034] In this experiment, the operations on the PVA / CS-MNs and 4NQO-PVA / CS-MNs groups of rats were performed under general anesthesia. The single anesthesia time was maintained at 1.5 h - 2 h. Anesthesia method: Intraperitoneal injection of 1.5% pentobarbital sodium at a dose of 1.0 mL / 400 g, combined with inhaled isoflurane anesthesia. Precautions during peritoneal anesthesia: The head of the rat was placed in a lower position to move the internal organs upward. Insert a needle subcutaneously in the right lower abdomen, move forward 0.5 cm, then pass through the abdominal muscles at a 45° angle. Aspirate to check for no blood, and then inject the drug. After anesthesia, place the rat on a heating blanket to keep warm. If there are signs of awakening, give inhaled isoflurane anesthesia for 10 s each time. During the operation, place the rat on a fixing plate. One person uses a non-toothed forceps to pull out the rat's tongue flat, and the other person applies the microneedle patch to the anterior 1 / 2 of the ventral tongue of the rat and presses for 20 s.

[0035] Drug administration group: Before each use, mix the 0.1% 4NQO stock solution evenly with an ultrasonic oscillator, dilute the stock solution with drinking water to a concentration of 0.02%, place it in a light-proof drinking water bottle for free drinking, and change it 2 to 4 times a week.

[0036] Blank control group: Provide drinking water daily.

[0037] During the experiment, observe the changes in the color and shape of the white lesion area on the tongue mucosa of the rats every day, and take pictures before treatment, at the 1st, 2nd, 3rd week of treatment, and at the 4th week of treatment.

[0038] After the treatment cycle ended, the rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital. After being sacrificed by an overdose of anesthetic, tongue specimens were taken and fixed in 10% neutral buffered formalin solution at 5 times the volume, and then conventional paraffin sections of the specimens were made.

[0039] Observation indicators

[0040] The water intake, body weight, changes in the tongue mucosa, and mental state were recorded weekly; gross photographs were taken before sampling at the 4th week. The rats were sacrificed by an overdose of sodium pentobarbital anesthesia, and the tongue tissues were fixed in 10% formalin.

[0041] Histopathological examination and immunohistochemical staining analysis. At 4 weeks of the experiment, the rats were sacrificed and the OLK tissues of each group were excised. The specimens were immediately washed clean on the surface with normal saline and placed in 10% neutral buffered formalin at 5 times the volume for 48 h. The specimens were placed in embedding cassettes and rinsed with running tap water overnight. Then dehydration, clearing in paraffin, embedding, and sectioning were performed, with a thickness of 5 μm. HE staining and immunohistochemical staining (Ki-67) were performed. The hearts, livers, spleens, lungs, and kidneys of the rats in each group were dissected, and HE-stained histopathological sections were made in the same way. The images were taken and observed using a microscope and a polarizer device.

[0042] Statistical analysis

[0043] Graphpad Prism 10 software was used for statistical processing. All experimental data were expressed as mean ± standard deviation (X±SD). One-way analysis of variance was used, and pairwise comparisons were performed using the t-test. For those with unequal variances, an approximate F-test was used. *p<0.05 indicated statistical significance, **p<0.01, and ***p<0.001 indicated significant statistical significance.

[0044] The experimental instruments included an autoclave, a constant temperature water bath, a precision electronic balance, an oven, an enzyme-linked immunosorbent assay (ELISA) reader, a Fourier transform infrared spectrometer, a pure water instrument, a vacuum drying oven, a shaking incubator, a sterile operation bench, pipettes, a centrifuge, an anesthesia machine, a scanning electron microscope, an inverted fluorescence microscope, a laser confocal microscope, a Nicolet iN10 FTIR spectrometer, a refrigerator, and a universal testing machine.

[0045] The advantages of the present invention compared with the existing technologies are as follows: In this study, a 4NQO-PVA / CS-MNs delivery system with sustained-release function was successfully constructed. Through the synergistic effect of the physical puncture of the microneedles and the controlled release of the polymer, precise targeted release of the drug in the sublingual mucosa was achieved. This system not only overcomes the limitations of traditional modeling techniques, but also provides a standardized animal model for the study of the pathogenesis of oral leukoplakia, drug screening and nanomedicine applications, showing good application prospects. The microneedle patch quickly established an OLK animal model in 4 weeks, significantly shortening the modeling cycle and effectively avoiding systemic exposure, providing an efficient tool for subsequent related research on pathological mechanisms and materials science on the OLK animal model. Therefore, the soluble novel microneedle patch of 4NQO developed in this project has good application prospects in oral leukoplakia modeling. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 FIG. is the preparation flow chart of 4NQO-PVA / CS-MNs for constructing OLK of the present invention (A) and a schematic diagram of its use for establishing an oral leukoplakia animal model (B).

[0047] Figure 2 FIG. is a schematic diagram of the animal experiment flow chart of the present invention.

[0048] Figure 3 FIG. is the topographic maps of (A) PVA / CS-MN under optical microscope and scanning electron microscope, (B) 4NQO-PVA / CS-MNs under optical microscope and scanning electron microscope, (C) the topographic map of microneedles under fluorescence inverted microscope with rhodamine B-labeled 4NQO, (D) the FT-IR spectrogram of the components of 4NQO-PVA / CS-MNs, and (E) the pressure-strain curve of the microneedle patch.

[0049] Figure 4 FIG. is the standard curve graph of drug release (A) and the drug release curve graph of the microneedle patch (B) of the present invention.

[0050] Figure 5 FIG. shows (A) the photo of the microneedle patch attached to pigskin (a), the photo of the microneedle patch attached to pigskin under running water flushing (b), and the photo of the microneedle patch attached to pigskin after running water flushing (c). B. (a) Uniform dot distribution left by the punctured ex vivo pigskin can be seen, (b) after methylene blue staining; C. The confocal laser scanning microscope image shows the depth map of rhodamine-labeled MNs (red fluorescence) piercing into pigskin. D. The microneedles piercing into the back of rats at (a) 0 min, (b) 10 min, (c) 20 min, and (d) 60 min.

[0051] Figure 6 FIG. is the CCK-8 experimental result graph of 4NQO-PVA / CS-MNs of the present invention.

[0052] Figure 7 These are the morphological and pathological result diagrams of the tongues of Wistar rats after being treated with 4NQO-PVA / CS-MNs at different concentrations for 4 weeks.

[0053] Figure 8 These are the morphological change diagrams under different modeling methods during the establishment of the OLK animal model in Wistar rats.

[0054] Figure 9 These are the (A) tongue morphology and (B) white lesion area diagrams of each group in Wistar rats during the establishment of the OLK animal model under different modeling methods.

[0055] Figure 10 These are the (A) body weight change, (B) daily water intake change, and (C) survival analysis table diagrams of each group.

[0056] Figure 11 (A) These are the histopathological result diagrams of each group after 4 weeks and (B) these are the epithelial thickness statistical diagrams of each group.

[0057] Figure 12 (A) These are the Ki-67 immunohistochemical staining result diagrams of each group and (B) these are the quantitative analysis diagrams of the number of positive cells. All data are expressed as mean ± standard deviation (n = 3), p < 0.0001.

[0058] Figure 13 (A) These are the H&E result diagrams of the heart, liver, spleen, lungs, and kidneys of rats at 24 hours and (B) these are the H&E result diagrams at 4 weeks. Detailed implementation manners

[0059] To make the content of the present invention easier to be clearly understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0060] Preparation of 4NQO-PVA / CS-MNs: It includes the synthesis of PVA / CS solution, the preparation of the backing layer PVA / PVP, and the preparation of 4NQO-PVA / CS-MNs;

[0061] Synthesis of PVA / CS solution: Slowly add 2 g of CS powder (degree of deacetylation ≥ 85%) to 100 mL of pre-cooled 1% acetic acid solution, and stir magnetically (at room temperature, 500 - 800 rpm) until completely dissolved. Transfer the solution to a dialysis bag with a molecular weight cut-off of 10 kDa, and dialyze with deionized water until the pH of the dialysis solution is close to 6.0. After dialysis, the solution is filtered through a 0.45 μm filter membrane, and the nearly neutral CS solution is transferred to a constant temperature drying oven at 37°C to remove the moisture therein, thereby obtaining a CS solution with a concentration of 10%. The solution needs to be stored refrigerated (at 4°C).

[0062] Weigh 10 g of PVA (PVA 1788, degree of alcoholysis 88%) and add it to 100 mL of deionized water. Stir at 90 °C until completely dissolved. After cooling to room temperature, mix it with 10% CS solution in a volume ratio of 7:3, stir magnetically for 30 min, and store it in a refrigerator at 4 °C for later use.

[0063] Preparation of PVA / PVP solution: Prepare an aqueous solution of 10% PVA and 10% PVP and mix them in a ratio of 1:1 (v / v).

[0064] Preparation of PVA / CS-MNs: Drop 200 μL of PVA / CS mixed solution onto the surface of a PDMS microneedle mold (needle length 650 μm, bottom diameter 280 μm, 10×10 array), centrifuge at 3500×g for 10 min, and repeat 3 times to ensure complete filling of the mold cavity. Use a ruler to scrape off the excess solution above the mold, and place it in a vacuum drying oven at 40 °C for 30 min to form the tip layer. Mix an aqueous solution of 10% PVA and 10% PVP in a ratio of 1:1 (v / v), evenly coat it on the back of the tip layer, and dry it at 40 °C for 12 h to form the base layer. Peel the microneedle patch from the mold and store it in a desiccator (relative humidity ≤20%) in the dark.

[0065] Preparation of 4NQO-PVA / CS-MNs:

[0066] (1) Dissolve 4NQO in 1,2-propanediol to prepare a 30 mg / mL solution. Vortex-mix the 4NQO solution and the PVA / CS mixture in proportion (avoid introducing air bubbles), let it stand at 4 °C for 12 h, and then sterilize it through a 0.22 μm filter membrane. Store it in the dark at 4 °C.

[0067] (2) After evacuating the PDMS microneedle mold, spread 200 μL of 4NQO-PVA / CS mixed solution on the surface of the mold, place it in a 50 mL centrifuge tube, centrifuge at 3500×g for 10 min, and repeat 3 times to ensure complete filling of the mold cavity. After scraping off the excess solution, transfer the mold to a vacuum drying oven and dry it at 40 °C for 20 min to form the tip layer. Mix a 10% PVA solution and a 10% PVP solution in a ratio of 1:1 (v / v), coat it on the back of the tip layer, and dry it in a vacuum at 30 °C for 24 h to form the base layer. After peeling the microneedle patch, store it in a nitrogen-filled desiccator (4 °C, RH≤10%) in the dark for later use.

[0068] The morphological characteristics of 4NQO-PVA / CS-MNs were observed by scanning electron microscopy (SEM), and at the same time, elemental distribution analysis of the samples was carried out. The acceleration voltage was set at 10 kV, and quantitative analysis of the microneedle size was performed using Image J software; the Fourier transform infrared spectrum (FTIR) of 4NQO-PVA / CS was obtained by a Fourier transform infrared spectrometer to determine the relevant characteristic groups.

[0069] The mechanical properties of 4NQO-PVA / CS-MNs include mechanical property tests, in vitro drug release, adhesion tests, puncture performance tests, and skin irritation.

[0070] Mechanical property test: Use a universal mechanical testing machine to conduct a compressive strength test on the prepared PVA / CS-MNs and 4NQO-PVA / CS-MNs. The flat base of the microneedles is fixed on the lower fixture base. Select an upper fixture probe with a diameter of 10 mm and move downward from the contact tip of the needle at a rate of 0.1 mm / min. Record the force values between 0 - 600 μm of displacement, record the obtained data, and plot the stress-strain curve.

[0071] In vitro drug release: Use ultraviolet-visible spectrophotometry to analyze 4NQO at different concentrations (n = 3) immersed in 10 mL of artificial saliva, make a standard curve of the 4NQO solution, and obtain its correlation coefficient and regression equation. Place 4NQO-PVA / CS-MNs (n = 3) in artificial saliva (pH 6.8), shake in a constant temperature shaker at 37 °C (rpm), and use an ultraviolet-visible spectrophotometer to detect the absorbance value at 260 nm of the solution at 10 min, 20 min, 30 min, 40 min, 50 min, 1 h, 2 h, 3 h......12 h, 24 h, 36 h, 72 h respectively to make an in vitro drug release curve.

[0072] Adhesion test: Take fresh excised pig skin, clean the hair on the surface, scrape off the excess grease, puncture 4NQO-PVA / CS-MNs into the inner side of the pig skin, and then continuously flush with running water for 1 min to observe the adhesion of the microneedle patch.

[0073] Puncture performance test: Press the microneedle patch on the excised pig skin (2 kg / cm 2 , 30 s), and after removal, drop 0.1% methylene blue solution (5 min). Press the rhodamine B-labeled microneedles onto the flat excised pig skin and place them under a laser confocal scanning microscope for layer-by-layer scanning at an excitation wavelength of 562 nm. The CLSM images are captured at a depth of every 50 μm. Observe the distribution of rhodamine B at different skin depths to evaluate the puncture situation of the microneedles in the tissue.

[0074] Skin irritation: Anesthetize rats intraperitoneally with 2% pentobarbital sodium, gently depilate the back skin, place the microneedle patch on the back skin and press continuously for 5 min and then remove it. Observe the recovery of the back skin at 10 min, 20 min, and 30 min after removal respectively.

[0075] To evaluate the effect of 4NQO-PVA / CS-MNs on the proliferation of HOK cells, the specific method is as follows: Add 4NQO-PVA / CS-MNs at a concentration of 1.6 mg / mL to the complete medium (containing 10% fetal bovine serum and 1% double antibody), incubate at 37 °C and 5% CO2 for 24 hours to simulate the cell culture environment and release the soluble components in the material; then centrifuge at 3000 rpm for 10 minutes, take the supernatant to remove insoluble particles, and obtain extracts with concentrations of 0.2, 0.4, 0.8, and 1.6 mg / mL by gradient dilution. After labeling, store at 4 °C for later use (it is recommended to use within 24 hours).

[0076] In the experimental procedure, inoculate HOK cells into a 96-well plate at a density of 3000 cells / well, add 100 μL of cell suspension to each well, and pre-culture in a 37 °C and 5% CO2 incubator for 24 hours to allow them to adhere. Subsequently, replace the original medium with extracts of 4NQO-PVA / CS-MNs at different concentrations (experimental group) or ordinary complete medium (blank control group). Set 4 replicates for each group and perform detection after co-incubation for 1, 2, and 3 days. When detecting, first aspirate the original medium to avoid interference from residual materials. Add 100 μL of fresh medium containing 10% CCK-8 reagent (90 μL of medium + 10 μL of CCK-8) to each well, incubate in the dark for 1 - 4 hours (determine the optimal color development time through preliminary experiments, usually 2 hours), and then measure the absorbance (OD value) at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0077] The experimental results need to be analyzed for statistical differences by ANOVA. Set technical replicates (3 wells / concentration) and biological replicates (3 independent experiments) for each group to improve reliability.

[0078] Furthermore, the experiment explores the appropriate concentration of 4NQO in the microneedle patch. To explore the appropriate concentration of 4NQO for the microneedles, the literature shows that the applied concentration of 4NQO is 0.5 - 2 mg / mL, 2 - 3 times a week [24 - 27]. Based on this, the 4NQO concentrations set in this experiment are 0.4 mg / mL, 0.8 mg / mL, and 1.6 mg / mL, respectively. They are respectively prepared into 4NQO-PVA / CS-MNs, and then a pre-experiment on rats is carried out to prove their effects.

[0079] Sixty 8-week-old male Wistar rats were purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd. (Shandong, China). All animals were bred for one week under a 12-h light / dark cycle and the experiments were conducted after the rats had adapted to the environment. All experimental protocols were approved by the Animal Protection and Use Committee of Qingdao University. The experimental animals were strictly raised in accordance with the Chinese national standards for laboratory animal environment and facilities (GB 14925-2010 / xg1-2011), fed with standard feed, and the breeding conditions were 12-h light - 12-h darkness, humidity 50 ± 15%, and temperature 22 ± 2°C. The experimental operations followed the eighth edition of the "Guidelines for the Care and Use of Laboratory Animals", ISBN-10: 0-309-15394-4 and the relevant regulations of the Laboratory Animal Management Measures of the Medical College of Qingdao University. The rats were adaptively raised for one week before the experiment, during which they had free access to food and water.

[0080] Experimental grouping: Sixty male Wistar rats were randomly divided into three experimental groups: blank control group, drug-administered group, PVA / CS-MNs group, and 4NQO-PVA / CS-MNs group (n = 20).

[0081] Blank control group: Distilled water was provided daily.

[0082] Drug-administered group: 4NQO was prepared into a stock solution with a concentration of 0.1% (0.004 g made up to 40 mL) with distilled water and stored in the dark in a 4°C refrigerator.

[0083] PVA / CS-MNs: The prepared PVA / CS-MNs were applied to the anterior 1 / 2 of the dorsal tongue of the rats and pressed for 20 s.

[0084] 4NQO-PVA / CS-MNs: 0.8 mg / mL of 4NQO-PVA / CS-MNs was applied to the anterior 1 / 2 of the dorsal tongue of the rats and pressed for 20 s.

[0085] Experimental procedure

[0086] (1) Preparation of anesthetic: Weigh 1.5 g of pentobarbital sodium powder, dissolve it in 100 mL of ultrapure water, disperse it thoroughly with an ultrasonic oscillator, aliquot and seal it in 20-mL centrifuge tubes, and store it in a 4°C refrigerator. Freshly prepare it each time after use.

[0087] (2) Every week, two uniformly trained experimental personnel administered drugs to the Wistar rats in each experimental group at a fixed time for 4 consecutive weeks.

[0088] In this experiment, rats in the PVA / CS-MNs and 4NQO-PVA / CS-MNs groups were operated under general anesthesia. The single anesthesia time was maintained at 1.5h-2h. The anesthesia method was intraperitoneal injection of 1.5% sodium pentobarbital at a dose of 1.0mL / 400g, combined with isoflurane inhalation anesthesia. Precautions for intraperitoneal anesthesia: The rat's head was low, the internal organs were moved to the upper abdomen, and the subcutaneous puncture was made in the right lower abdomen, 0.5cm forward, and then through the abdominal muscle at a 45° angle. No blood was aspirated and the drug was injected. After anesthesia, it was placed on a heating blanket to keep warm. If there were signs of awakening, isoflurane inhalation anesthesia was given for 10s each time. During the operation, the rat was placed on a fixed board, one person pulled out the rat's tongue flat with toothless forceps, and the other person attached the microneedle patch to the front 1 / 2 of the rat's tongue abdomen and pressed for 20S.

[0089] Drug oral group: Before each use, 0.1% 4NQO stock solution was mixed evenly with an ultrasonic oscillator, diluted with drinking water to a concentration of 0.02%, placed in a light-proof drinking bottle for free drinking, and replaced 2 to 4 times a week.

[0090] Blank control group: drinking water was provided daily.

[0091] During the experiment, the changes in color and shape of the white lesions on the tongue mucosa of the rats were observed every day, and photos were taken before treatment, at the 1st, 2nd, 3rd and 4th week of treatment.

[0092] After the treatment cycle, rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital and killed by overdose of anesthetic. Tongue specimens were fixed in 5 volumes of 10% neutral buffered formalin solution and then routine paraffin sections were prepared.

[0093] Observation indicators

[0094] Water intake, body weight, changes in tongue mucosa, and mental state were recorded weekly; gross photographs were taken before sampling at week 4. Rats were sacrificed by anesthesia with an overdose of sodium pentobarbital, and tongue tissues were fixed in 10% formalin.

[0095] Histopathological examination and immunohistochemical staining analysis. At 4 weeks of the experiment, the rats were killed and the OLK tissues of each group were cut. The surface of the specimens was immediately cleaned with physiological saline and placed in 5 times the volume of 10% neutral buffered formalin for 48 hours. The specimens were placed in an embedding box and rinsed with tap water overnight. Then they were dehydrated, paraffin-permeated, embedded, and sliced ​​to a thickness of 5μm. H&E staining and immunohistochemical staining (Ki-67) were performed. The hearts, livers, spleens, lungs, and kidneys of the rats in each group were dissected and H&E-stained histopathological sections were prepared in the same way. Images were taken and observed using a microscope and a polarizer device.

[0096] Statistical analysis

[0097] Statistical analysis was performed using GraphPad Prism 10 software. All experimental data were expressed as mean ± standard deviation (X±SD). One-way analysis of variance was used, and pairwise comparisons were made using the t-test. For data with unequal variances, an approximate F-test was used. *p<0.05 indicates statistical significance, **p<0.01, ***p<0.001 indicates significant statistical significance.

[0098] Results

[0099] 1 Microneedle patch morphology characterization

[0100] 1.1 Morphological characteristics of PVA / CS-MNs

[0101] First, PVA / CS-MNs were prepared, and the prepared microneedle patch was observed under an optical microscope and a scanning electron microscope to observe its appearance morphology at different angles. As Figure 3 shown in A, the base of the microneedle patch was flat, and 100 microneedles (10×10) were arranged neatly and uniformly on it. The needle body morphology was complete and uniform, with the same size, no fractures or cracks. Each needle body was conical, with a sharp tip, which was beneficial for penetrating the skin and stratum corneum. The surface of the microneedle body was smooth, which helped to reduce the pain during insertion and tissue damage. The length of the microneedle body was about 600 μm, the width of the bottom was about 280 μm, and the center spacing of the needle bodies was about 600 μm. Compared with the PDMS mold, the height was reduced by about 7.6%, probably due to water evaporation during the drying process.

[0102] 1.2 Morphological characteristics of 4NQO-PVA / CS-MNs

[0103] The prepared microneedle patch was observed under an optical microscope and a scanning electron microscope to observe its appearance morphology at different angles.

[0104] Figure 3 As shown in B, the base of the microneedle patch was flat, and 100 pale yellow microneedles (10×10) were arranged neatly and uniformly on it. The needle body morphology and size were the same as those of PVA / CS-MNs.

[0105] The tips and backings of the microneedles were labeled with rhodamine B, Figure 3 and C shows the fluorescence image of the rhodamine B-labeled microneedle patch. Under the excitation of green fluorescence, the red fluorescent dye rhodamine B was evenly and highly concentrated in the microneedle body part. This indicates that the microneedle body part was filled with 4NQO, which ensured the good mechanical strength of the microneedles while increasing the drug penetration amount and effectively improving the drug delivery efficiency.

[0106] 2 Physicochemical property tests

[0107] 2.1 FTIR spectral test

[0108] FTIR spectroscopy was used to determine the types of functional groups of the microneedle materials. As Figure 3 shown in -1 D, for the characteristic peak analysis of CS: stretching vibration of O-H would appear near 3650 cm -1 , absorption peak of C=O would appear at 1714 cm -1 , and stretching vibration of C-O in the sugar ring would appear at 1090 cm -1 . For the characteristic peak analysis of PVA: stretching vibration peak of O-H at 3200–3500 cm -1 . The –CH2 in the main chain of PVA molecule produced the peak at 2947 cm -1 , and the absorption peak of stretching vibration of C-O was at 1094 cm -1 . For the characteristic peak analysis of PVA / CS: in the PVA / CS precursor, the characteristic peaks of PVA and CS were clearly visible. The O-H of PVA overlapped with the O-H and -NH2 absorption peaks of CS, forming a strong hydrogen bond interaction, and a particularly wide and strong peak was formed at 3200~3500 cm -1 . The vibration peak of N-H in CS also appeared at 3200–3300 cm -1 . The vibration peak near 1542 cm -1 corresponded to the vibration peak of amide bond (II) in CS, indicating that CS had successfully loaded PVA. For the characteristic peak analysis of 4NQO-PVA / CS: since both CS, PVA and 4NQO contained hydroxyl groups, the vibration of O-H appeared at 3200–3500 cm -1 . In the spectrum, the stretching vibration peak of C=C on the aromatic ring was near 1504 cm-1, and the absorption peak of C=O was near 1650 cm

[0109] 2.2 Mechanical property test

[0110] As Figure 3 .E shown: with the increase of the compression distance, the required compression stress gradually increased, and the microneedle patch showed an obvious non-linear strain response. The compression stress required for the non-drug-loaded microneedle patch was always greater than that of the drug-loaded microneedle patch, indicating that the non-drug-loaded microneedle patch had a higher compression strength. Adding 4NQO to the microneedles reduced their mechanical strength. The tip 1 / 3 of the microneedle was thinner and required less force to produce deformation. With the increase of the cross-sectional area of the microneedle body, its mechanical strength gradually increased, and it could withstand a pressure of about 224 N at most. Previous studies have shown that the force required for each needle to penetrate the skin is less than 0.1 N, indicating that the MNs prepared in this study have sufficient mechanical strength to penetrate the epithelial barrier without being broken.

[0111] 2.3 In vitro drug release

[0112] As a transdermal drug delivery system, the in vitro drug release experiment of the microneedle patch aims to evaluate the efficacy of continuous drug release from the microneedle patch under preset conditions. As Figure 4 .A shows that the 4NQO release standard curve is: Y = 0.01530*X + 0.01439, R2 = 0.9998, which conforms to a linear relationship. As Figure 4 .B shows that within the 5 days of detection, the microneedle patch exhibited stable drug release behavior, and the drug concentration showed a biphasic release pattern over time. In the initial stage of drug release, 4NQO was rapidly released from the microneedles, reaching 12% at 3 hours, 20% at 6 hours, 50% at 13 hours, and 80% at 24 hours, forming an initial concentration peak, which may be due to the fact that part of the 4NQO on the surface or near the surface of the microneedles can diffuse out quickly. As time goes by, it enters the slow release stage, and the 4NQO in the microneedles is gradually released at a slower rate and is almost completely released in 5 days, thus maintaining the drug concentration at a relatively stable level. The slow release helps to extend the action time of the drug, can meet the need for continuous drug delivery to the oral mucosa, reduce the frequency of drug administration, and improve patient compliance.

[0113] 2.4 Adhesion test

[0114] The mucosal adhesion experiment is a key step in evaluating the ability of the microneedle patch to maintain its position and effectively deliver drugs on the target mucosal tissue. The microneedle patch was placed on pig skin to simulate the moist mucosa, and running water was used to wash it, and the adhesion of the microneedles was observed. As Figure 5 As shown in A, after being washed by running water, the microneedles still adhered firmly to the pig skin and were consistent with the surface morphology of the pig skin, indicating that it has good adhesion performance.

[0115] 2.5 Penetration performance test

[0116] The performance test of the microneedle patch on pig skin provides an evaluation of the actual penetration effect in vivo and helps to understand the performance of the microneedles on living skin. Figure 5 As shown in B(a), the ex vivo pig skin was punctured, Figure 5 and the dot-like distribution consistent with the microneedle array after methylene blue staining in B(b) indicates that the microneedles have good mechanical strength and can puncture the skin smoothly; in order to evaluate the penetration depth, the depth of rhodamine b-labeled MNs penetrating the skin was recorded by CLSM layer by layer. It is reported that the thickness of the rat oral mucosa epithelium is about 100 μm, which is also consistent with the results of the pathological sections of the rat tongue mucosa. Figure 5 .C shows that the insertion depth of the MN patch can reach 280 μm, which indicates that the MN patch can easily penetrate the rat oral mucosa epithelial layer to reach the lamina propria.

[0117] 2.6 Stimulation of the skin by microneedle patch puncture

[0118] The microneedles were placed on the shaved back skin of rats and pressed for 5 min and then removed, and the skin recovery was observed. As Figure 5 . As shown in D, the skin pinhole array was clearly visible immediately after removal, and there was no redness or inflammation. At about 1 hour, the skin gaps formed by microneedle puncture were basically restored, and the skin was not red and swollen, indicating the minimally invasive nature of microneedle puncture.

[0119] CCK-8 cell compatibility characterization of 34NQO-PVA / CS-MNs

[0120] The cell compatibility of biomaterials is a key test for evaluating whether the materials are suitable for biomedical applications. HOK cells were cultured in conditioned media of 4NQO-PVA / CS-MNs at different concentrations. At the 1st, 2nd, and 3rd days, cell viability was evaluated by the CCK-8 method. When HOK cells were cultured in the conditioned media of 4NQO-PVA / CS-MNs for the 1st day and 2nd day, the cell proliferation in all microneedle groups was lower than that of the control group ( Figure 6 ). At the 3rd day, there was no difference between the 4NQO-PVA / CS at 0.2 mg / mL, 0.4 mg / mL, and 0.8 mg / mL and the control group, and they were non-toxic to cells, indicating good biocompatibility.

[0121] The experimental results showed that after HOK cells were cultured for 3 days under the conditions of low-concentration (0.2, 0.4, 0.8 mg / mL) 4NQO-PVA / CS-MNs, there was no significant difference in cell proliferation compared with the control group (p>0.05), indicating its good biocompatibility. This was mainly attributed to the sustained-release characteristics of the microneedle matrix and the protective effect of the PVA / CS material: 4NQO was slowly released locally through the gradual degradation of the soluble microneedles, avoiding direct damage to cells caused by short-term high-concentration drug exposure; at the same time, PVA and CS maintained intracellular homeostasis by adsorbing some free drugs and activating the cell antioxidant defense system (such as SOD, GSH). However, in the 1.6 mg / mL group, excessive drug release led to the accumulation of reactive oxygen species (ROS) and increased DNA damage, significantly inhibiting cell viability. The above concentration-dependent differences suggested that 0.8 mg / mL was the safe drug-loading threshold of 4NQO-PVA / CS-MNs, which could not only effectively induce lesions but also avoid toxicity risks, providing a key dosage basis for the optimization of the oral leukoplakia animal model.

[0122] 4 Experiment to explore the 4NQO concentration

[0123] This study innovatively adopted a microneedle drug delivery mode of single administration once a week for 4 weeks. By systematically observing the lesion process of the rat tongue mucosa and combining in vitro toxicity verification, the optimal working concentration was determined to be 0.8 mg / mL. Specifically, the 0.4 mg / mL group did not cause pathological changes in the tongue; the 0.8 mg / mL group successfully induced typical OLK, and the pathological diagnosis was moderate epithelial dysplasia (Figure 7 );However, in the 1.6 mg / mL group, tongue mucosal ulceration and bleeding occurred due to drug overdose, and the CCK-8 assay showed significant cytotoxicity. The results of this concentration screening provided a key parameter basis for subsequent modeling studies.

[0124] The realization of this concentration effect can be explained by the following mechanism: The microneedle array achieves programmed drug sustained release through the controllable degradation of the PVA / CS matrix. 80% of the drug is released rapidly within the initial 24 hours to form an effective concentration, and continuous release is maintained at a sub-toxic level for the subsequent 5 days. This spatio-temporal distribution characteristic not only ensures the continuous stimulation required for DNA damage accumulation but also avoids the oxidative stress burst triggered by the instantaneous 1.6 mg / mL group. The 0.8 mg / mL drug delivery system is exactly in the critical range that induces epithelial dysplasia without triggering the apoptosis threshold. The positive charge property of the CS matrix enhances the drug-DNA binding efficiency, significantly reducing the requirement for the effective concentration.

[0125] Through spatio-temporal pharmacokinetic regulation, this drug delivery system shortens the modeling period to 4 weeks at a dose of 0.8 mg / mL. The study confirmed that the local sustained release strategy mediated by microneedles not only breaks through the dose limitation of traditional drug delivery methods but also provides a technological innovation path for the standardized establishment of oral precancerous lesion models, demonstrating the potential for translational application in drug screening and mechanism research.

[0126] Study on the Use of 4NQO-PVA / CS-MNs Microneedle Patches for Establishing an OLK Animal Model

[0127] 5.1 Morphological Changes and Mental States of Wistar Rats during the Experiment

[0128] During the experiment, the rats in each group were monitored for morphology and mental state. The results showed that neither the carrier material itself nor the microneedle drug delivery system caused significant systemic toxic reactions ( Figure 8 ). Rats in the blank microneedle group and the PVA / CS-MNs group maintained normal physiological states throughout the experimental period (1 - 4 weeks), showing good hair gloss, agile and coordinated movement, active exploratory behavior, and no abnormal weight fluctuations or listlessness.

[0129] Rats in the traditional drug administration group showed drug-related side effects at the initial stage of drug administration: within 30 minutes after drug administration, there was a significant increase in saliva secretion (the hair in the submandibular area remained wet continuously), accompanied by a decrease in activity. By the 3rd week, the skin in the submandibular area showed signs of hyperkeratosis (dry desquamation of the epidermis and loss of normal luster) due to long-term drug stimulation, but no severe skin toxicity such as hair loss was observed. It is worth noting that the diarrhea symptoms often reported in the literature after long-term oral administration of 4NQO did not occur in this study, presumably related to the effective avoidance of direct contact with the gastrointestinal tract by the microneedle drug delivery. By the 4th week, the saliva secretion and mental state of this group of rats gradually recovered.

[0130] In the rats of the 4NQO-PVA / CS-MNs group, only a transient increase in salivary secretion occurred within 1 hour after drug administration, and no persistent moistening in the submandibular area or skin pathological changes were observed. There was no significant difference in the animal behavior pattern throughout the experiment compared with the blank control group. This significant reduction in toxicity confirmed the technical advantage of the microneedle drug delivery system in effectively reducing the systemic exposure of drugs through local targeted delivery, providing a safer modeling scheme for subsequent long-term carcinogenicity studies.

[0131] 5.2 Condition of the lingual mucosa of Wistar rats

[0132] Dynamic observation of the lingual mucosa of rats in each group showed ( Figure 9 ), and there was a significant time-effect correlation in the modeling system. The blank control group and the PVA / CS-MNs group maintained the normal physiological state of the lingual mucosa throughout the experimental period (1 - 4 weeks), showing uniform glossiness on the mucosal surface, no congestion, erosion or abnormal keratinization, which confirmed the biosafety of the microneedle carrier material itself. No immediate injury was observed in the lingual mucosa of the traditional drug-drinking group during the experimental observation period (1 - 4 weeks), but its long-term carcinogenic effect appeared at 12 weeks as reported in the literature, manifested as the formation of multifocal keratotic leukoplakia on the dorsal lingual mucosa, showing granular protrusions, which was in line with the pathological characteristics of typical OLK.

[0133] The 4NQO-PVA / CS-MNs group showed a unique pattern of lesion progression: the first week was the induction latency period, and the lingual mucosa maintained a normal morphology; the second week entered the initial lesion stage, with a local congestion focus appearing in the center of the dorsal tongue, indicating microcirculation changes and inflammatory reactions; the third week formed a definite pathological focus, with 2 - 3 irregular white keratinized plaques appearing on the dorsal tongue of each rat, with a size range of 0.3×0.3 mm to 0.8×0.8 mm, and pathological sections showed thickening of the epithelial spinous layer with cellular atypia; by the fourth week, the lesion entered the exponential growth phase, the number of white lesions increased to 4 - 6, with an average diameter of 0.8×0.8 mm, and some lesions fused to form a patchy hyperkeratotic area, and histopathology confirmed moderate dysplasia with dyskeratosis, belonging to pre-cancerous lesions. The lesion area ( Figure 9 B) was significantly different from that of other groups.

[0134] Compared with the traditional drug-drinking model, the microneedle drug delivery system can complete the induction of typical lesions of OLK within 4 weeks, shortening the modeling period by 67%, and the lesion characteristics are highly uniform. Before the formation of cancer, the oral mucosal tissue of the animal model will show lesions similar to human OLK. This significant compression of the time window not only improves the research efficiency, but also realizes the dynamic balance of the mucosal injury threshold and repair mechanism through precise spatiotemporal pharmacokinetic regulation, providing a more stable model basis for the mechanism research and drug intervention of oral pre-cancerous lesions.

[0135] 5.3 Body weight, water intake, and survival analysis

[0136] Dynamic monitoring of body weight during the experimental period showed that ( Figure 10 A), the average body weight of rats in the traditional drug - drinking group was significantly lower than that of the blank control group, PVA / CS - MNs group, and 4NQO - PVA / CS - MNs group at 4 weeks (p < 0.01).

[0137] Analysis of drinking behavior found that ( Figure 10 B), there was a significant difference between the MNs group and the blank control group, which might be because the insertion of MNs affected the water intake of rats due to discomfort. There was a significant difference between the drug - drinking group and the other three groups (p < 0.01).

[0138] Kaplan - Meier survival curve analysis showed that ( Figure 10 C), the 4 - week survival rate of the traditional drug - drinking group was significantly lower than that of the other three groups (p < 0.05). The micro - needle drug delivery system precisely controls the spatio - temporal distribution of drugs in the mucosal layer, which not only ensures the local effective concentration but also controls the plasma drug concentration at a sub - toxic level, significantly reducing the risk of liver and kidney toxicity.

[0139] 5.4 Histopathological analysis of the OLK animal model

[0140] The pathological H&E staining results of each group ( Figure 11 A). Control group: The stratum corneum is composed of multiple layers of tightly arranged squamous keratinized cells with large cell volume and homogeneous cytoplasm; the granular layer is composed of 2 - 3 layers of fusiform cells with keratohyalin granules in the cytoplasm; the stratum spinosum is the thickest area of the epithelium, composed of 4 - 8 layers of polygonal cells with distinct intercellular bridges; the basal cells are cuboidal, connected to the basement membrane through hemidesmosomes, and the nuclear - cytoplasmic ratio is moderate.

[0141] The mucosa of the PVA / CS - MNs group maintained the normal keratinization process, the cells in the granular layer and stratum spinosum were regularly arranged, and there were no atypia changes in the basal cells, confirming that the micro - needle carrier itself did not induce pathological changes.

[0142] The traditional drug - drinking group showed the characteristics of simple epithelial hyperplasia, with uniform thickening of the stratum corneum but good cell differentiation, increased number of cells in the stratum spinosum but maintaining normal polarity, and the basement membrane was intact. The 4NQO - PVA / CS - MNs group showed typical moderate dysplasia: the stratum corneum showed parakeratosis (residual keratinized cell nuclei), the granular layer was thickened with disordered cell arrangement, the cell density in the stratum spinosum was significantly increased (the number of cell layers reached 12 - 15 layers), the epithelial rete pegs extended finger - like into the deep lamina propria. The basal cells showed pleomorphic changes, with an increased nuclear - cytoplasmic ratio, deep - stained nuclei with prominent nucleoli, and mitotic figures were seen in some areas, which was in line with the cytological characteristics of precancerous lesions.

[0143] Quantitative analysis of epithelial thickness ( Figure 11B) Results showed that there were no significant differences in mucosal thickness among all groups at 2 weeks (p > 0.05), indicating that the microneedle drug delivery system mainly induced cell function changes rather than structural remodeling in the early stage. By 4 weeks, the epithelial thickness of the drug-loaded microneedle group was significantly different from that of the other three groups (p < 0.05). This thickening effect was due to the disordered layers caused by abnormal hyperplasia rather than simple proliferative changes, which was highly consistent with the pathological evolution pattern of clinical OLK. The data indicated that the microneedle drug delivery system could complete the pathological evolution from molecular damage to tissue remodeling within 4 weeks through programmed drug release, which was significantly superior to the 12-week cycle of the traditional model.

[0144] Ki-67 is a cell proliferation marker, and its function is closely related to mitosis. The increase in the number of its positive cells usually means the enhancement of cell proliferation activity. The Ki-67 positive cells in human and canine mucosa are mainly located in the upper basal layer, while in other species, most proliferating cells are located in the basal layer. Only immunohistochemical quantitative analysis showed ( Figure 12 ), the Ki-67 positive cells of 4NQO-PVA / CS-MNs were significantly higher than those of the blank control group, the traditional drug-drinking group, and PVA / CS-MNs (p < 0.01). There was no statistical difference in proliferation activity among the latter three groups (p > 0.05), indicating that the microneedle carrier itself did not activate the cell cycle, while traditional drug-drinking only caused a moderate proliferative response.

[0145] In this experiment, the number of positive cells in the drug-loaded microneedle group was significantly higher than that in other groups, indicating that its cell proliferation ability was stronger. This might be related to the microneedle promoting drug penetration and enhancing local drug concentration, thus accelerating the cell renewal and repair process in the leukoplakia area. Due to different drug delivery methods or drug carriers in other groups, the same proliferative effect was not achieved. Therefore, the Ki-67 results supported that the drug-loaded microneedle group successfully induced the formation of leukoplakia within 4 weeks, and its cell proliferation activity was significantly higher than that in other groups.

[0146] Systemic toxicity assessment was performed by histological staining to analyze the potential damage of the drug to major organs ( Figure 13 ). Studies have shown that topical application of 4-NQO on the rat tongue for 16 weeks caused liver, kidney, and spleen toxicity. The acute toxicity experiment in this study showed ( Figure 13 A), 24 hours after a single dose, no abnormal tissue structures were observed in the organs such as the heart, liver, spleen, lung, and kidney of rats. The myocardial cells were arranged neatly, the hepatic cell cords were clear, the alveolar structure was intact, and there were no necrotic foci in the renal tubules, indicating that short-term drug exposure did not cause obvious organ toxicity. The sub-chronic toxicity experiment further verified ( Figure 13B) After 4 consecutive weeks of drug administration, the main organs of the animals in each experimental group still maintained normal morphology, without myocardial hypertrophy or interstitial fibrosis, no lipid droplet accumulation or necrosis in hepatocytes, the splenic lymph follicle structure was intact, the alveolar wall was not thickened, and the glomerular filtration barrier was normal. These results confirmed that neither the traditional oral drug administration nor the microneedle drug delivery method caused systemic pathological damage to the experimental animals during the four-week observation period. In particular, the microneedle drug delivery system significantly reduced the systemic exposure risk while maintaining the local effective drug concentration, providing an important guarantee for the safe intervention of the oral premalignant lesion model.

[0147] In summary, this study successfully developed a soluble microneedle patch loaded with 4NQO (4NQO-PVA / CS-MNs).

[0148] Using physicochemical characterization techniques, it was verified that 4NQO-PVA / CS-MNs had excellent morphological characteristics and mechanical properties, could effectively penetrate the oral mucosa and achieve controlled drug release. And an OLK animal model was successfully established using 4NQO-PVA / CS-MNs, demonstrating that it could rapidly and stably induce OLK lesions, with controllable lesion locations, and significantly shortened the model establishment time. In animal experiments, 4NQO-PVA / CS-MNs showed good induction effects, could simulate the pathological process of OLK, providing an efficient and controllable tool for related research. And it provided a new technical means for the research of the pathological mechanism of OLK and drug screening, with important scientific research value and clinical application potential.

[0149] The above describes the present invention and its implementation manners. Generally speaking, if those of ordinary skill in the art are inspired by it and, without departing from the gist of the present invention, design similar structural modes and embodiments to this technical solution without creative efforts, they shall fall within the protection scope of the present invention.

Claims

1. Preparation of soluble microneedles based on the payload 4-nitroquinoline-N-oxide (4NQO) and their application in constructing oral leukoplakia on the dorsal tongue mucosa of rats. It includes the synthesis of polyvinyl alcohol / chitosan (PVA / CS) solution, the preparation of the backing layer of polyvinyl alcohol / polyvinylpyrrolidone (PVA / PVP), and the preparation of 4NQO-PVA / CS-MNs; Synthesis of PVA / CS solution: Slowly add 2 g of CS powder (degree of deacetylation ≥ 85%) to 100 mL of pre-cooled 1% acetic acid solution, and stir magnetically (at room temperature, 500 - 800 rpm) until completely dissolved. Transfer the solution to a dialysis bag with a molecular weight cut-off of 10 kDa, and dialyze with deionized water until the pH of the dialysis fluid is close to 6.

0. After dialysis, the solution is filtered through a 0.45 μm filter membrane, and the nearly neutral CS solution is transferred to a constant temperature drying oven at 37 °C to remove the moisture therein, thereby obtaining a CS solution with a concentration of 10%. The solution needs to be stored refrigerated (at 4 °C). Weigh 10 g of PVA (PVA 1788, degree of alcoholysis 88%) and add it to 100 mL of deionized water. Stir at 90 °C until completely dissolved. After cooling to room temperature, mix it with the 10% CS solution in a volume ratio of 7:3, stir magnetically for 30 min, and store it in a refrigerator at 4 °C for later use. Preparation of PVA / PVP solution: Prepare a 1:1 (v / v) mixture of 10% PVA aqueous solution and 10% PVP aqueous solution. Preparation of PVA / CS-MNs: 200 μL of PVA / CS mixed solution was dropped onto the surface of a PDMS microneedle mold (needle length 650 μm, bottom diameter 280 μm, 10×10 array), and centrifuged at 3500×g for 10 min. This was repeated 3 times to ensure complete filling of the mold cavities. The excess solution above the mold was scraped off using a ruler and placed in a vacuum drying oven at 40 °C for 30 min to form the tip layer. A 1:1 (v / v) mixture of 10% PVA and 10% PVP aqueous solution was uniformly coated on the back of the tip layer and dried at 40 °C for 12 h to form the base layer. Peel the microneedle patch from the mold and store it in a desiccator (relative humidity ≤ 20%) in the dark. Preparation of 4NQO-PVA / CS-MNs: (1) Dissolve 4NQO in 1,2-propanediol to prepare a 30 mg / mL solution. Vortex-mix the 4NQO solution and the PVA / CS mixture in proportion (avoid introducing air bubbles), let it stand at 4 °C for 12 h, and then sterilize it through a 0.22 μm filter membrane. Store it in the dark at 4 °C. (2) After evacuating the PDMS microneedle mold, spread 200 μL of the 4NQO-PVA / CS mixed solution on the surface of the mold, place it in a 50 mL centrifuge tube, centrifuge at 3500 × g for 10 min, and repeat 3 times to ensure complete filling of the mold cavity. After scraping off the excess solution, transfer the mold to a vacuum drying oven and dry it at 40 °C for 20 min to form the tip layer. Mix 10% PVA and 10% PVP solution in a 1:1 (v / v) ratio and coat it on the back of the tip layer, and dry it in a vacuum at 30 °C for 24 h to form the base layer. After peeling off the microneedle patch, store it in a desiccator protected by nitrogen (at 4 °C, RH ≤ 10%) in the dark for later use.

2. Preparation of the soluble microneedles based on 4NQO as the payload, characterized in that The morphological characteristics of the 4NQO-PVA / CS-MNs were observed by scanning electron microscopy (SEM), and at the same time, the elemental distribution analysis of the samples was carried out. The acceleration voltage was set at 10 kV, and the quantitative analysis of the microneedle size was carried out through Image J software; the Fourier transform infrared spectrum (FTIR) of 4NQO-PVA / CS was obtained by a Fourier transform infrared spectrometer to determine the relevant characteristic groups.

3. Preparation of soluble microneedles based on 4NQO load according to claim 1, characterized in that The mechanical properties of the 4NQO-PVA / CS-MNs include mechanical property testing, in vitro drug release, adhesion testing, puncture performance testing, and skin irritation. Mechanical property testing: The compressive strength of the prepared PVA / CS-MNs and 4NQO-PVA / CS-MNs was tested using a universal mechanical testing machine. The micro-needle base was flatly fixed on the lower fixture base, and an upper fixture probe with a diameter of 10 mm was selected. It moved downward from the contact tip at a rate of 0.1 mm / min, and the force values between 0 - 600 μm of displacement were recorded. The obtained data were recorded and the stress-strain curve was plotted. In vitro drug release: 4NQO-PVA / CS-MNs (n = 3) were immersed in 10 mL of artificial saliva (pH 6.8, containing 0.5% SDS to maintain sink conditions), and oscillated at a constant temperature of 37°C (100 rpm). At preset time points, 1 mL of the supernatant (while adding an equal volume of pre-warmed medium) was taken, filtered through a 0.22 μm filter membrane, and the absorbance was measured at λ = 260 nm using a UV spectrophotometer (Shimadzu UV-2600). The cumulative release rate was calculated based on the standard curve, and the in vitro drug release curve was made. Adhesion testing: The hair on the surface of freshly excised pig skin was removed, and the excess grease was scraped off. The 4NQO-PVA / CS-MNs were punctured into the inner side of the pig skin, and then continuously rinsed with running water for 1 min to observe the adhesion of the micro-needle patch. Puncture performance test: Press the microneedle patch onto ex vivo porcine skin (2 kg / cm 2 , 30 s), and after removal, add 0.1% methylene blue solution (5 min). Press the rhodamine B-labeled microneedles into the flat ex vivo porcine skin, place it under a confocal laser scanning microscope (CLSM), and perform layered scanning at an excitation wavelength of 562 nm. CLSM images are captured at every 50-μm depth. Observe the distribution of rhodamine B at different skin depths to evaluate the puncture situation of the microneedles in the tissue. Skin irritation: Rats were intraperitoneally anesthetized with 2% sodium pentobarbital, and the back skin was gently depilated. The micro-needle patch was placed on the back skin and continuously pressed for 5 min and then removed. The recovery of the back skin was observed at 10 min, 20 min, and 30 min after removal respectively.

4. Preparation of soluble microneedles based on 4NQO load according to claim 1, characterized in that In vitro biocompatibility of 4NQO-PVA / CS-MNs. To evaluate the effect of 4NQO-PVA / CS-MNs on the proliferation of HOK cells, the specific method was as follows: 4NQO-PVA / CS-MNs were added to the complete medium (containing 10% fetal bovine serum and 1% double antibody) at a concentration of 1.6 mg / mL, and incubated at 37°C and 5% CO2 for 24 hours to simulate the cell culture environment to release the soluble components in the material; then centrifuged at 3000 rpm for 10 minutes, the supernatant was taken to remove insoluble particles, and extracts with concentrations of 0.2, 0.4, 0.8, and 1.6 mg / mL were obtained by gradient dilution, labeled and stored at 4°C for later use (it is recommended to use within 24 hours). In the experimental procedure, HOK cells were seeded into a 96-well plate at a density of 3000 cells / well, and 100 μL of cell suspension was added to each well. The cells were pre-cultured in an incubator at 37 °C and 5% CO2 for 24 hours to allow them to adhere. Subsequently, the original medium was replaced with 4NQO-PVA / CS-MNs extracts at different concentrations (experimental group) or normal complete medium (blank control group). Four replicate wells were set up for each group, and the detection was carried out after co-incubation for 1, 2, and 3 days. When detecting, the original medium should be aspirated first to avoid interference from residual materials. Then, 100 μL of fresh medium containing 10% CCK-8 reagent (90 μL of medium + 10 μL of CCK-8) was added to each well, and the mixture was incubated in the dark for 1-4 hours (the optimal color development time was determined by preliminary experiments, usually 2 hours). Subsequently, the absorbance (OD value) was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm. The experimental results were analyzed for statistical differences by ANOVA. Technical replicates (3 wells / concentration) and biological replicates (3 independent experiments) were set up for each group to improve reliability.

5. The preparation of the soluble microneedles based on the payload 4NQO according to claim 1, wherein The experiment explored the appropriate concentration of 4NQO in the microneedle patch. To explore the appropriate concentration of 4NQO for the suitable microneedles, the literature showed that the concentration of 4NQO applied was 0.5-2 mg / mL, 2-3 times a week. Based on this, the 4NQO concentrations set in this experiment were 0.4 mg / mL, 0.8 mg / mL, and 1.6 mg / mL, respectively. 4NQO-PVA / CS-MNs were prepared respectively, and then a preliminary experiment on rats was carried out to prove its effect.

6. The preparation of the soluble microneedles based on the payload 4NQO according to claim 1, wherein: The 4NQO-PVA / CS-MNs were used for the establishment of OLK. Sixty 8-week-old male Wistar rats were purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd. (Shandong, China). All animals were bred under a 12-hour light-dark cycle for one week, and the experiment was carried out after the rats adapted to the environment. All experimental protocols were approved by the Animal Protection and Use Committee of Qingdao University. The experimental animals were strictly raised according to the Chinese national standards for experimental animal environment and facilities (GB 14925-2010 / xg1-2011), fed with standard feed, and the breeding conditions were 12-hour light - 12-hour darkness, humidity 50±15%, and temperature 22±2 °C. The experimental operations followed the eighth edition of the "Guidelines for the Protection and Use of Laboratory Animals", ISBN-10: 0-309-15394-4 and the relevant regulations of the Laboratory Animal Management Measures of the Medical Faculty of Qingdao University. The animals were adaptively raised for one week before the experiment, and they had free access to food and water during this period. Experimental grouping: Sixty male Wistar rats were randomly divided into a blank control group and three experimental groups: the drug-drinking group, PVA / CS-MNs group, and 4NQO-PVA / CS-MNs group (n = 20). Blank control group: Distilled water was provided daily. Drug-drinking group: 4NQO was prepared into a stock solution with a concentration of 0.1% (0.004 g was made into 40 mL) with distilled water and stored in the refrigerator at 4 °C in the dark. PVA / CS-MNs: The prepared PVA / CS-MNs were pasted on the anterior 1 / 2 of the dorsal tongue of the rats and pressed for 20 s. 4NQO-PVA / CS-MNs: 0.8 mg / mL of 4NQO-PVA / CS-MNs was applied to the anterior 1 / 2 of the dorsum of the rat tongue and pressed for 20 seconds. Experimental Procedure (1) Preparation of anesthetics: Weigh 1.5 g of sodium pentobarbital powder, dissolve it in 100 mL of ultrapure water, disperse it thoroughly using an ultrasonic oscillator, divide it into 20 mL centrifuge tubes, and store it in a refrigerator at 4°C. Prepare freshly after each use. (2) Two uniformly trained experimenters conducted drug administration experiments on Wistar rats in each experimental group at a fixed time every week for 4 consecutive weeks. In this experiment, rats in the PVA / CS-MNs and 4NQO-PVA / CS-MNs groups were operated under general anesthesia. The single anesthesia time was maintained at 1.5h-2h. The anesthesia method was intraperitoneal injection of 1.5% sodium pentobarbital at a dose of 1.0mL / 400g, combined with isoflurane inhalation anesthesia. Precautions for intraperitoneal anesthesia: The rat's head was low, the internal organs were moved to the upper abdomen, and the subcutaneous puncture was made in the right lower abdomen, 0.5cm forward, and then through the abdominal muscle at a 45° angle. No blood was aspirated and the drug was injected. After anesthesia, it was placed on a heating blanket to keep warm. If there were signs of awakening, isoflurane inhalation anesthesia was given for 10s each time. During the operation, the rat was placed on a fixed board, one person pulled out the rat's tongue flat with toothless forceps, and the other person attached the microneedle patch to the front 1 / 2 of the rat's tongue abdomen and pressed for 20S. Drug oral group: Before each use, 0.1% 4NQO stock solution was mixed evenly with an ultrasonic oscillator, diluted with drinking water to a concentration of 0.02%, placed in a light-proof drinking bottle for free drinking, and replaced 2 to 4 times a week. Blank control group: drinking water was provided daily. During the experiment, the changes in color and shape of the white lesions on the tongue mucosa of the rats were observed every day, and photos were taken before treatment, at the 1st, 2nd, 3rd and 4th week of treatment. After the treatment cycle, rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital and killed by overdose of anesthetic. Tongue specimens were fixed in 5 volumes of 10% neutral buffered formalin solution and then routine paraffin sections were prepared. Observation indicators Water intake, body weight, changes in tongue mucosa, and mental state were recorded weekly; gross photographs were taken before sampling at week 4. Rats were sacrificed by anesthesia with an overdose of sodium pentobarbital, and tongue tissues were fixed in 10% formalin. Histopathological examination and immunohistochemical staining analysis. At 4 weeks of the experiment, the rats were killed and the OLK tissues of each group were cut. The surface of the specimens was immediately cleaned with physiological saline and placed in 5 times the volume of 10% neutral buffered formalin for 48 hours. The specimens were placed in an embedding box and rinsed with tap water overnight. Then they were dehydrated, paraffin-permeated, embedded, and sliced ​​to a thickness of 5μm. H&E staining and immunohistochemical staining (Ki-67) were performed. The hearts, livers, spleens, lungs, and kidneys of the rats in each group were dissected and H&E-stained histopathological sections were prepared in the same way. Images were taken and observed using a microscope and a polarizer device. The preparation and application of 4NQO-PVA / CS-MNs according to claims 1-6 are characterized in that: All data were presented as mean ± standard deviation (SD). Statistical analysis was performed using Grapgpad Prism 10 software. All experimental data were expressed as mean ± standard deviation (X±SD). One-way analysis of variance was used, and pairwise comparisons were made using the t-test. For data with unequal variances, an approximate F-test was used. *p<0.05 indicated statistical significance, **p<0.01, and ***p<0.001 indicated significant statistical significance. The experimental instruments included an autoclave, a constant temperature water bath, a precision electronic balance, an oven, an enzyme-labeled instrument, a Fourier transform infrared spectrometer, a pure water instrument, a vacuum drying oven, a shaking incubator, aseptic operation equipment, pipettes, a centrifuge, an anesthesia machine, a scanning electron microscope, an inverted fluorescence microscope, a laser confocal microscope, a Nicolet iN10 FTIR spectrometer, a refrigerator, and a universal mechanical testing machine.

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