Symmetric nucleoside microneedle patch loaded with polymer vesicles as well as preparation method and application of symmetric nucleoside microneedle patch

Abstract

The invention discloses a symmetric nucleoside microneedle patch loaded with polymer vesicles as well as a preparation method and application of the symmetric nucleoside microneedle patch. The symmetric nucleoside microneedle patch loaded with the polymer vesicles comprises a guanosine-quadruplex hydrogel microneedle tip doped with drug-loading polymer vesicles, the raw material of the drug-loaded polymer vesicle comprises a polycaprolactone-polyglutamic acid copolymer; the guanosine-quadruplex hydrogel is prepared from the following raw materials: guanosine and KB (OH) 4. The microneedle patch is constructed on the basis of dynamic covalent bond and non-covalent interaction, has a sharp tip array capable of penetrating the epithelium and the lower layer of a mucous membrane, can efficiently deliver a drug to a canker lesion part by bypassing a mucous membrane barrier, can be controllably broken under a high ROS condition of an inflammation microenvironment, has the characteristics of high drug delivery efficiency and high accuracy, and can be used for preparing a medicine for treating the canker lesion part. The effect of effectively improving chemotherapy-induced oral ulceration can be achieved by reducing inflammatory response, relieving pain feeling and inhibiting pain sensitization.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a symmetrical nucleoside microneedle patch loaded with polymer vesicles, and a preparation method and application thereof. Background Art

[0002] Oral ulcers are a common mucosal disease, often associated with significant pain and severely impacting patients' quality of life. Currently, the clinical treatment of oral ulcers faces three major challenges: 1) Due to the moist nature of the oral environment, drugs have difficulty remaining at the lesion site for extended periods, resulting in limited efficacy; 2) the pseudomembrane on the ulcer surface forms a physical barrier, hindering effective drug penetration into deeper tissues; and 3) commonly used drugs, such as dexamethasone, have limitations such as poor solubility and inhibition of cell migration, which in turn impair the healing process. These challenges collectively hinder therapeutic efficacy, necessitating new drug delivery strategies. Numerous topical formulations, such as hydrogels, films, and patches, have been developed to enhance drug adhesion. However, these formulations lack sufficient mucosal adhesion and are unable to maintain prolonged adhesion in the dynamic, moist environment of the oral cavity. This limitation not only impairs therapeutic efficacy but also causes an uncomfortable foreign body sensation. Furthermore, the pseudomembrane formed on the ulcer surface acts as a physical barrier, further hindering the penetration of topical drugs and reducing their effective retention time at the lesion site, thus impacting clinical efficacy.

[0003] Guanosine-quadruplex hydrogel, as a self-assembled supramolecular hydrogel based on guanine derivatives, has been shown to have significant potential in drug delivery. Its unique structure can effectively physically encapsulate drugs or combine with drugs through chemical modification, thereby improving the stability and sustained release performance of drugs. In addition, the nucleoside component can effectively remove excess reactive oxygen species (ROS) at the ulcer site, providing a highly efficient carrier with great application potential for drug delivery. Although a series of drug delivery systems based on guanosine-quadruplex hydrogels have been developed, in the process of realizing the present invention, the inventors found that there are at least the following problems in the prior art:

[0004] Existing guanosine-quadruplex hydrogel drug delivery systems have relatively weak mechanical properties and unstable drug release properties, which hinder their clinical application. In addition, the formation of pseudomembranes on the ulcer surface can hinder drug penetration, reduce the drug's residence time at the lesion site, and affect the therapeutic effect. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to address the deficiencies of the above-mentioned prior art and provide a symmetrical nucleoside microneedle patch loaded with polymer vesicles, a preparation method and an application thereof. The symmetrical nucleoside microneedle patch loaded with polymer vesicles has a neat needle tip array, a complete needle body, a clear boundary between the needle tip component and the backing component, and the maximum force that a single needle tip can withstand exceeds the mechanical strength required for the microneedle to pierce the skin, and has a significant therapeutic effect on chemotherapy-induced oral ulcers in mice.

[0006] Compared with the prior art, the present invention has the following advantages:

[0007] The symmetrical nucleoside microneedle patch loaded with polymer vesicles of the present invention is constructed based on dynamic covalent bonds and non-covalent interactions. It has a sharp tip array that can penetrate the mucosal epithelium and the underlying layer, and can bypass the mucosal barrier to efficiently deliver drugs to the ulcer lesion site. It can be controllably broken under high ROS conditions in the inflammatory microenvironment, and has the characteristics of high drug delivery efficiency and high precision. It can effectively improve chemotherapy-induced oral ulcers by reducing inflammatory reactions, alleviating pain perception and inhibiting pain sensitization.

[0008] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 PCL in Example 1 48 -b-PGA 21 H NMR spectrum of the copolymer;

[0010] Figure 2 TEM image (left) and SEM image (right) of the polymer vesicles in Example 2;

[0011] Figure 3 Schematic diagram of the 90°C stability test results of the polymer vesicles in Example 2;

[0012] Figure 4 Schematic diagram of the preparation process of the symmetrical nucleoside microneedle patch and the therapeutic mechanism of the microneedle patch in Example 4;

[0013] Figure 5 This is a schematic diagram of the hydration driving process of Example 4;

[0014] Figure 6 is the Fourier transform infrared spectrum of guanosine-quadruplex hydrogel;

[0015] Figure 7 Guanosine-quadruplex hydrogel 11 B NMR spectrum;

[0016] Figure 8 Guanosine-quadruplex hydrogel 1 H NMR spectrum;

[0017] Figure 9 XRD pattern of guanosine-quadruplex hydrogel;

[0018] Figure 10 SEM image of guanosine-quadruplex hydrogel;

[0019] Figure 11 Schematic diagram of surface morphology of symmetrical nucleoside microneedle patch;

[0020] Figure 12 Morphology diagram of symmetrical nucleoside microneedle patch with fluorescent label;

[0021] Figure 13 Schematic diagram of mechanical property test results of symmetrical nucleoside microneedle patch;

[0022] Figure 14 Schematic diagram of dissolution performance test results of symmetrical nucleoside microneedle patch;

[0023] Figure 15 Schematic diagram of guanosine-quadruplex hydrogel scavenging efficiency test results on different reactive oxygen species (ROS);

[0024] Figure 16 Schematic diagram of guanosine-quadruplex hydrogel in vitro antioxidant capacity test results;

[0025] Figure 17 Schematic diagram of guanosine-quadruplex hydrogel in vitro anti-inflammatory capacity test results;

[0026] Figure 18 Schematic diagram of guanosine-quadruplex hydrogel in vitro anti-inflammatory test expression level test results of pro-inflammatory factor (TNF-α) and anti-inflammatory factor (IL-10);

[0027] Figure 19 Schematic diagram of fluorescence microscopic image of macrophage phenotype in guanosine-quadruplex hydrogel in vitro anti-inflammatory capacity test and corresponding macrophage expression analysis results;

[0028] Figure 20 Schematic diagram of guanosine-quadruplex hydrogel cell migration and tube formation experiment results;

[0029] Figure 21 Schematic diagram of symmetrical nucleoside microneedle patch pain behavior evaluation test results;

[0030] Figure 22 Schematic diagram of symmetrical nucleoside microneedle patch treatment effect evaluation results on 5-fluorouracil induced chemotherapy induced oral ulcer model.

[0031] BRIEF DESCRIPTION OF DRAWINGS

[0032] 1—needle cavity; 2—groove area. DETAILED DESCRIPTION

[0033] The following will be combined with the embodiments of this application to clearly and completely describe the technical solution. Obviously, the embodiments described are part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0034] In the following description, the term "and / or" is used to describe the association relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can represent: A exists alone, B exists alone, and A and B exist at the same time. A and B can be singular or plural.

[0035] In the following description, the terms "include", "comprising", "having" and "containing" are open-ended terms, meaning including but not limited to.

[0036] Those skilled in the art should understand that in the following description of the embodiments of the present application, the order of serial numbers does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0037] It will be understood by those skilled in the art that the numerical ranges in the examples of the present application are to be understood as also specifically disclosing each intermediate value between the upper and lower limits of the ranges. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also included in the present application. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

[0038] Unless otherwise indicated, the technical / scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this application belongs. Although this application describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of this application. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail.

[0039] On the one hand, a symmetrical nucleoside microneedle patch loaded with polymer vesicles is provided, comprising a guanosine-quadruplex hydrogel microneedle tip incorporated with drug-loaded polymer vesicles; the raw material of the drug-loaded polymer vesicles comprises a polycaprolactone-polyglutamic acid copolymer.

[0040] A guanosine-quadruplex hydrogel microneedle tip incorporating drug-loaded polymer vesicles was constructed through dynamic covalent bonds and non-covalent interactions, wherein the dynamic covalent bond is a borate bond, and the non-covalent interactions are hydrogen bonds, ionic crosslinking, and π-π stacking. The dynamic nature of the borate bond can be fully utilized to achieve controllable breakage under high ROS conditions in the inflammatory microenvironment.

[0041] In some specific embodiments, the drug in the drug-loaded polymer vesicles is dexamethasone. Polymer vesicles assembled with polycaprolactone-polyglutamic acid copolymer can efficiently encapsulate poorly soluble drugs and achieve sustained release.

[0042] In some embodiments, the guanosine-quadruplex hydrogel raw materials include guanosine and KB(OH)4. In some specific embodiments, the molar ratio of guanosine to KB(OH)4 is 2:1. In the present invention, a 2:1 molar ratio of guanosine to H3BO3 or KB(OH)4 facilitates the formation of a stable guanosine solution. As the amount of H3BO3 increases, the structural stability decreases. The dissolution temperature is ≥90°C to ensure complete dissolution of guanosine.

[0043] In some embodiments, the guanosine-quadruplex hydrogel material further comprises adenosine.

[0044] Furthermore, the guanosine-quadruplex hydrogel raw material also includes adenosine. The adenosine component in the microneedle synergizes with dexamethasone and has a significant anti-inflammatory and analgesic effect. This synergistic enhancement is more significant in promoting angiogenesis. The adenosine-containing hydrogel of the present invention can promote the expression of CD31 and Ki67, reverse the inhibitory effect of glucocorticoids on cell migration, and promote epithelial repair.

[0045] In another aspect, a method for preparing the aforementioned symmetrical nucleoside microneedle patch loaded with polymer vesicles is provided, comprising:

[0046] Provided is a gel preparation solution for guanosine-quadruplex hydrogel incorporating drug-loaded polymer vesicles; the raw materials of the preparation solution include polymer vesicles, guanosine, H3BO3 aqueous solution and K-containing + alkaline aqueous solution;

[0047] The gel preparation liquid of guanosine-quadruplex hydrogel doped with drug-loaded polymer vesicles is placed in the needle cavity of the microneedle mold to obtain the hydrogel, and the backing solution is added to obtain a symmetrical nucleoside microneedle patch loaded with polymer vesicles.

[0048] Taking advantage of the property of guanosine-quadruplex hydrogel that it can quickly gel after cooling, based on the ultrasound-assisted hydration needle casting method, it specifically solves the defect that the current vacuum casting technology is difficult to prepare microneedle patches with clear structure.

[0049] In some embodiments, the volume of the backing solution is 1 to 2 times the volume of the preparation solution.

[0050] In some embodiments, placing a gel preparation solution of a guanosine-quadruplex hydrogel incorporating drug-loaded polymer vesicles into the microneedle mold cavity comprises: filling the microneedle mold cavity with ultrapure water, injecting the gel preparation solution of the guanosine-quadruplex hydrogel incorporating drug-loaded polymer vesicles, allowing the surface of the gel preparation solution of the guanosine-quadruplex hydrogel incorporating drug-loaded polymer vesicles to contact the surface of the ultrapure water in the cavity, and performing air drying to evaporate the ultrapure water, thereby allowing the gel preparation solution of the guanosine-quadruplex hydrogel incorporating drug-loaded polymer vesicles to enter the cavity. In some specific embodiments, the air drying temperature for evaporating the ultrapure water is 90° C. and the time is 10 minutes.

[0051] During the research process, the inventors found that the guanosine-quadruplex hydrogel could not be directly injected into the needle cavity. The method of loading the needle tip matrix solution into the microneedle mold by vacuum or centrifugation and then adding the backing layer matrix solution to obtain the microneedle after drying and demolding is difficult to solve the bubble problem in the microneedle preparation process. The bubbles that are not completely eliminated have a significant impact on the appearance of the microneedle, the integrity of the microneedle array and the mechanical strength. The present invention uses the hydration effect of ultrapure water to drive the gel preparation solution into the needle cavity, which can effectively avoid the generation of bubbles during the pipetting process, and has the characteristics of high microneedle insertion rate, sharp microneedle tip morphology and complete array.

[0052] In some embodiments, the method for preparing the polymer vesicles comprises:

[0053] Step 1, providing deprotected polycaprolactone and γ-benzyloxycarbonyl-L-glutamic acid anhydride;

[0054] Step 2: dissolving the deprotected polycaprolactone and γ-benzyloxycarbonyl-L-glutamic acid anhydride in a first organic solvent, and performing a ring-opening polymerization reaction under an inert atmosphere to obtain a polycaprolactone-polypolypeptide copolymer; the first organic solvent is dimethylformamide;

[0055] Step 3: dissolving the polycaprolactone-polypolypeptide copolymer in a second organic solvent and performing a deprotection reaction to obtain a polycaprolactone-polyglutamic acid copolymer; the second organic solvent is a HBr / CH3COOH solution;

[0056] Step 4: dissolving the polycaprolactone-polyglutamic acid copolymer in a third organic solvent, adding physiological saline dropwise, stirring to assemble, and dialyzing to obtain polymer vesicles; the third organic solvent is dimethyl sulfoxide; in some specific embodiments, the concentration of the polycaprolactone-polyglutamic acid copolymer in the system after dissolving the polycaprolactone-polyglutamic acid copolymer in the third organic solvent is 0.5-2 mg / mL; and the volume of physiological saline is 2-5 times the volume of the third solvent.

[0057] The process involves dripping physiological saline for assembly. The charge shielding effect of salt ions weakens the electrostatic repulsion of the PGA segments, enhancing the aggregation driving force of the PCL hydrophobic chains. Under the hydrophilic-hydrophobic interaction, the copolymer spontaneously assembles into vesicles. The research found that inducing copolymer self-assembly in physiological saline resulted in vesicles with more uniform particle size and more stable structure, especially when the volume of physiological saline is 2-5 times that of dimethyl sulfoxide, which ensures full and thorough assembly.

[0058] The concentration of polycaprolactone-polyglutamic acid copolymer is 0.5-2 mg / mL, which can form suitable drug-loaded vesicles. When the copolymer concentration is too high, it is difficult to achieve sufficient assembly of the vesicles and precipitation is likely to occur. When the concentration is too low, subsequent drug loading is affected.

[0059] Some embodiments further provide a method for preparing drug-loaded polymer vesicles, comprising:

[0060] Step 1, providing deprotected polycaprolactone and γ-benzyloxycarbonyl-L-glutamic acid anhydride;

[0061] Step 2: dissolving the deprotected polycaprolactone and γ-benzyloxycarbonyl-L-glutamic acid anhydride in a first organic solvent, and performing a ring-opening polymerization reaction under an inert atmosphere to obtain a polycaprolactone-polypolypeptide copolymer; the first organic solvent is dimethylformamide;

[0062] Step 3: dissolving the polycaprolactone-polypolypeptide copolymer in a second organic solvent and performing a deprotection reaction to obtain a polycaprolactone-polyglutamic acid copolymer; the second organic solvent is a HBr / CH3COOH solution;

[0063] Step 4: dissolving the polycaprolactone-polyglutamic acid copolymer and the drug in a third organic solvent, dropping physiological saline into the solvent, stirring to assemble, and dialyzing to obtain drug-loaded polymer vesicles; the third organic solvent is dimethyl sulfoxide.

[0064] In some embodiments, a gel preparation solution for guanosine-quadruplex hydrogel incorporating drug-loaded polymer vesicles is provided, comprising: using a solution containing drug-loaded polymer vesicles as a solvent, adding guanosine and H3BO3 aqueous solution respectively, and adding K-containing solution after ultrasonication. + In some specific embodiments, the K-containing + The alkaline solution is KOH aqueous solution;

[0065] The process involves placing guanosine in an H3BO3 aqueous solution, followed by the addition of a KOH aqueous solution. The boronic acid group specifically coordinates with the guanosine cis-diol, inducing the formation of a stable boronate dimer. Subsequently, after ultrasound treatment, the molecules stack in an orderly manner, providing a template for the hierarchical assembly of the G-quadruplex. The addition of KOH +In an alkaline solution, potassium ions chelate and stabilize the Hoogsteen hydrogen bonds in the G4 plane, triggering cross-linking of the gel network to obtain a guanosine hydrogel precursor with shear thinning and self-healing properties.

[0066] In some embodiments, when the raw material of the preparation solution includes adenosine, the preparation method includes: using a solution containing drug-loaded polymer vesicles as a solvent, adding guanosine, adenosine and H3BO3 aqueous solution respectively, and adding K-containing solution after ultrasonication. + of alkaline solution to obtain a preparation solution.

[0067] In another aspect, the present invention provides a use of the above-mentioned symmetrical nucleoside microneedle patch loaded with polymer vesicles in the preparation of drugs for sustained analgesia and high-efficiency anti-inflammatory.

[0068] On the other hand, the present invention provides a use of a symmetrical nucleoside microneedle patch loaded with polymer vesicles in the preparation of a drug for repairing oral ulcers.

[0069] The present invention has been subjected to a series of experiments before the application is filed. Some of the experimental results are listed below to further describe the invention in detail, and the following embodiments are used to describe the invention in detail.

[0070] Example 1

[0071] This embodiment provides a polycaprolactone-polyglutamic acid copolymer (PCL 48 -b-PGA 21 ), comprising:

[0072] Provide deprotected polycaprolactone (PCL 48 -NH2) and γ-benzyloxycarbonyl-L-glutamic acid cyclic anhydride (Bz-Glu-NCA);

[0073] The deprotected polycaprolactone (PCL 48 -NH2) and γ-benzyloxycarbonyl-L-glutamic acid cyclic anhydride (Bz-Glu-NCA) are dissolved in a first organic solvent and reacted under the protection of an inert atmosphere to obtain a polycaprolactone-polypeptide copolymer (PCL 48 -b-PBLG 21 ); the first organic solvent is anhydrous dimethylformamide;

[0074] The polycaprolactone-polypeptide copolymer (PCL 48 -b-PBLG 21 ) is dissolved in a third organic solvent and subjected to a deprotection reaction to obtain a polycaprolactone-polyglutamic acid copolymer (PCL 48 -b-PGA 21 ); the third organic solvent is HBr / CH3COOH;

[0075] The specific operations are as follows:

[0076] Step 1: Prepare the polymer PCL-NH-Boc by ring-opening polymerization, specifically comprising:

[0077] Step 101: 30.00 g of caprolactone monomer (262.8 mmol) and 350 mL of toluene were added to a 500 mL round-bottom flask, and the mixture was azeotroped in an oil bath at 145° C., and about 330 mL of toluene solution containing water vapor was evaporated;

[0078] Step 102: The system was cooled to room temperature, purged with argon for 30 minutes to remove oxygen, and 0.777 g of initiator tert-butyloxycarbonyl (Boc)-protected ethanolamine (4.800 mmol) and 0.004 g of catalyst Sn(Oct)2 (0.01 mmol) were added. Under argon protection, the reaction was carried out at 110° C. for 48 hours, and then cooled to room temperature. The tert-butyloxycarbonyl-protected ethanolamine was N-(tert-butyloxycarbonyl)ethanolamine, purchased from Merck Chemicals (Shanghai) Co., Ltd.

[0079] Step 103: Remove impurities from the system cooled to room temperature in step 102 to obtain the final product PCL. 48 -NH-Boc; Specifically comprising: dissolving the system after cooling to room temperature in step 102 in 10 mL of dichloromethane (DCM), adding icy methanol dropwise to precipitate three times, with the volume of icy methanol used for each precipitation being 200 mL, filtering to obtain the product, and drying it in a vacuum oven at 40°C for 2 days to obtain a white powdery solid PCL 48 -NH-Boc, the yield is about 85%.

[0080] Step 2: PCL 48 -NH-Boc was used as raw material to prepare deprotected polycaprolactone (PCL 48 -NH2), specifically including:

[0081] Step 201: 4.395 g PCL 48 -NH-Boc was dissolved in 5 mL of anhydrous DCM, and 5 mL of anhydrous trifluoroacetic acid (TFA) was added. The mixture was stirred at room temperature for 4 h, and the solvent was removed by rotary evaporation to obtain a polymer;

[0082] Step 202: The polymer was redissolved in 30 mL of DMF, transferred to a 3.5 kDa dialysis bag, and dialyzed with a 5% by mass aqueous solution of NaHCO3 and deionized water to remove the organic solvent, and lyophilized to obtain a polymer with -NH2 as the terminal group, i.e., deprotected polycaprolactone (PCL). 48 -NH2), with a yield of about 81.4%;

[0083] Step 3: providing a γ-benzyloxycarbonyl-L-glutamic acid cyclic anhydride monomer (Bz-Glu-NCA), specifically comprising:

[0084] Step 301: In a 250 mL round-bottom flask, 10 g of the small molecule amino acid γ-benzyl ester-L-glutamic acid (42.10 mmol) and 29.25 g of α-pinene (210.7 mmol) were added, followed by the addition of 100 mL of anhydrous THF. The mixture was heated to reflux at 55° C. When the reflux temperature was reached, 50 mL of a THF solution containing triphosgene was added, and the mixture was refluxed for approximately 5 h until the solution became clear. The mass of triphosgene in the THF solution containing triphosgene was 9.476 g (31.6 mmol).

[0085] Step 302: Cool the clarified system to room temperature and precipitate with n-hexane three times to obtain a white Bz-Glu-NCA monomer with a yield of about 88.3%;

[0086] Step 4: Deprotected polycaprolactone (PCL 48 -NH2) as the initiator, γ-benzyloxycarbonyl-L-glutamic acid anhydride (Bz-Glu-NCA) as the main raw material, and the polycaprolactone-polypeptide copolymer (PCL) was prepared by ring-opening polymerization. 48 -b-PBLG 21 ), specifically including:

[0087] Step 401: 2.3 g freshly prepared Bz-Glu-NCA monomer (8.76 mmol) and 2 g PCL 48 -NH2 (0.53 mmol) was dissolved in 10 mL of anhydrous dimethylformamide (DMF) and reacted at room temperature for 48 h under argon protection;

[0088] Step 402: The reaction system was precipitated with ether, centrifuged, and the precipitate was washed with ether and dried in a vacuum oven at 40° C. for 48 h to obtain a diblock polymer, namely, polycaprolactone-polypeptide copolymer (PCL 48 -b-PBLG 21 ), with a yield of about 65%;

[0089] Step 5: using the polycaprolactone-polypeptide copolymer (PCL 48 -b-PBLG 21 ) as raw materials to prepare polycaprolactone-polyglutamic acid copolymer (PCL 48 -b-PGA 21 ), specifically including:

[0090] Step 501: 2g PCL 48 -b-PBLG 21Dissolved in 5 mL of 33 wt% concentration

[0091] Stirred at room temperature in fume hood for 4 h in HBr / CH3COOH solution;

[0092] Step 502, the post-reaction system was repeatedly precipitated three times with ether for purification;

[0093] Step 503, for further purification, the purified material was dissolved in DMF and placed in a dialysis bag, dialyzed in deionized water for 3 days to remove HBr / CH3COOH, and freeze-dried to obtain a white powder solid, i.e., polycaprolactone-polyglutamic acid copolymer (PCL 48 -b-PGA 21 ) with a yield of 73%.

[0094] The test results of the polycaprolactone-polyglutamic acid copolymer of the example by nuclear magnetic resonance hydrogen spectrum are shown in Table 1. Figure 1 As can be seen from Table 1, Figure 1 it can be seen that the polycaprolactone-polyglutamic acid copolymer of the example is successfully prepared.

[0095] Example 2

[0096] The example provides a preparation method of a polymer vesicle, comprising:

[0097] Step one, dissolve the polycaprolactone-polyglutamic acid copolymer (PCL 48 -b-PGA 21 ) described in Example 1 in dimethyl sulfoxide (DMSO) to obtain a copolymer solution; the PCL 48 -b-PGA 21 concentration in the copolymer solution is 2.0 mg / mL;

[0098] Step two, under the condition of 25℃ and 380 rpm stirring, add physiological saline to the copolymer solution at a speed of 3 drops per minute until the volume of the physiological saline is 2 times the volume of dimethyl sulfoxide; the physiological saline is added by a peristaltic pump;

[0099] Step three, continue to stir at 25℃ and 380 rpm for 30 minutes, transfer to regenerated cellulose dialysis membrane, and dialyze with deionized water for 2 days; the water is changed every 6 hours during the dialysis process to completely remove the organic solvent, and a polymer vesicle aqueous solution (PVs aqueous solution) is obtained; the molecular weight cut-off value MWCO of the regenerated cellulose dialysis membrane is 14.0 kDa; the amount of deionized water used each time during the dialysis process is 2 L; the concentration of the polymer vesicle (PVs) in the polymer vesicle aqueous solution is 330 μg / mL.

[0100] This embodiment also provides a method for preparing dexamethasone polymer vesicles (Dex-PVs), which is basically the same as the method for preparing polymer vesicles (PVs) described above, except that:

[0101] Step 1 is to prepare the polycaprolactone-polyglutamic acid copolymer (PCL 48 -b-PGA 21 ) and dexamethasone were dissolved in dimethyl sulfoxide (DMSO) to obtain a copolymer solution; PCL in the copolymer solution 48 -b-PGA 21 The concentration was 2.0 mg / mL, and the dexamethasone concentration was 1.0 mg / mL;

[0102] In step three, the dialysis time with deionized water is 3 hours, and the water is changed every 30 minutes to ensure the encapsulation of the drug dexamethasone and remove the organic solvent; the result of step three is a dexamethasone polymer vesicle aqueous solution (Dex-PVs aqueous solution); the concentration of Dex-PVs vesicles in the dexamethasone polymer vesicle aqueous solution is 330 μg / mL.

[0103] The morphology of polymer vesicles was observed using electron microscopy. Figure 2 As shown, Figure 2 The left picture is a TEM picture. Figure 2 The right picture is the SEM picture. Figure 2 It can be seen that the size is about 220nm, the edges are clear, and the morphology is stable and uniform.

[0104] Figure 3 The stability test results of the polymer vesicles at 90°C were determined by DLS analysis of particle size changes. The results showed that the vesicle size and dispersion did not change significantly over a test period of more than 50 hours, indicating that the vesicles can be stably and uniformly maintained under the preparation conditions (90°C) and are suitable for use as drug carriers.

[0105] Example 3

[0106] This embodiment provides a method for preparing a guanosine-quadruplex hydrogel (GBG hydrogel), which is prepared by a one-pot process, comprising:

[0107] Step 1: Prepare 0.29M H3BO3 aqueous solution and 0.29M KOH aqueous solution in advance;

[0108] Step 2: 42 mg of guanosine (G, 0.145 mmol) and 0.25 mL of H3BO3 aqueous solution (0.0725 mmol) were added to 4.5 mL of ultrapure (DI) water as the solvent, and the mixture was ultrasonicated for 30 s. The ultrasonicated system was heated in a 90°C forced air drying oven until the guanosine was completely dissolved to obtain a mixed system;

[0109] Step 3: Add 0.25 mL of KOH aqueous solution (0.0725 mmol) to the above mixture, and continue stirring in a 90° C. air drying oven for 20 minutes to obtain a gel preparation solution;

[0110] Step 4: Cool the gel preparation solution to room temperature to obtain a stable GBG hydrogel (1 wt%); in this embodiment, the mass of G / KB(OH)4 is 50 mg, and the concentration of GBG hydrogel is 1 wt%.

[0111] This example also provides a method for preparing a guanosine-quadruplex hydrogel (GBA hydrogel) to which adenosine is added. The method is the same as the above-mentioned method for preparing the guanosine-quadruplex hydrogel (GBG hydrogel), except that 12 mg of adenosine (0.0435 mmol) is added at the same time as guanosine in step 2.

[0112] This example also provides a method for preparing a guanosine-quadruplex hydrogel (Dex-PVs@GBG hydrogel) incorporating dexamethasone polymer vesicles (Dex-PVs), which is the same as the method for preparing the GBG hydrogel described above, except that in step 2, 4.5 mL of Dex-PVs aqueous solution is used as the solvent.

[0113] This example also provides a method for preparing a guanosine-quadruplex hydrogel (Dex-PVs@GBA hydrogel) doped with dexamethasone polymer vesicles (Dex-PVs). The method is the same as the above-mentioned method for preparing the GBA hydrogel, except that in step 2, 4.5 mL of Dex-PVs aqueous solution is used as the solvent.

[0114] Example 4

[0115] This embodiment provides a method for preparing a symmetrical nucleoside microneedle patch, the process and treatment mechanism are as follows Figure 4 Shown, including:

[0116] Step 1: inject ultrapure water into the PDMS mold, ultrasonicate for 10 minutes, ensure that the ultrapure water completely fills each needle cavity, and then place the mold filled with ultrapure water in a 90°C blast drying oven for preheating for 5 minutes, remove the ultrapure water remaining in the groove, and obtain a mold with ultrapure water in the needle cavity; the PDMS mold includes a needle cavity area arranged in an array, and a groove area (2) located on the needle cavity area and connected to the needle cavity area, the needle cavity (1) array of the needle cavity area is 20*20, the needle cavity (1) has a tetrahedral morphology, the vertical height from the needle cavity vertex to the bottom of the tetrahedron is 370μm, the bottom surface size of the tetrahedron is 390*390μm, the groove size is 19.2*19.2mm, and the depth is 2mm;

[0117] Step two, keep the temperature of the blast drying oven unchanged, inject 0.8 mL of the gel preparation solution of Example 3 into the mold filled with ultrapure water, the gel preparation solution fills the upper area of the needle cavity (1) and the groove area (2), keep the temperature of the blast drying oven unchanged, stand for 10 min, the ultrapure water evaporates, the gel preparation solution enters the needle cavity; the gel preparation solution enters the needle cavity driven by hydration, the ultrapure water contacts with the gel preparation solution, the ultrapure water gradually evaporates under the condition of 90℃ blast drying, accompanied by the change of water molecules from liquid to gaseous state with volume contraction, surface tension is generated, the gel preparation solution is sucked into the needle cavity through capillary force, the hydration driven process is as shown in Figure 5 The gel preparation solution is the gel preparation solution of Dex-PVs@GBG hydrogel of Example 3.

[0118] Step three, then, transfer the mold filled with the gel preparation solution to a 25℃ blast drying oven to dry for 1 hour, form an incompletely dried hydrogel, use a spatula to scrape off the hydrogel outside the needle cavity, use a pipette to apply 0.8 mL of the backing layer solution to the surface of the hydrogel, the backing layer solution fills the groove area (2); the backing layer solution is a PVA solution, the PVA solution is a mixed solution of PVA, water and glycerol; in the PVA solution, the mass percentage content of PVA is 5%, the mass percentage content of glycerol is 0.5%; the PVA is PVA-203, the molecular weight is 31000.

[0119] Step four, continue to dry in the 25℃ blast drying oven for about 7 h until completely dried, remove the mold, obtain the nucleoside microneedle patch.

[0120] The present embodiment also provides a preparation method of Dex-PVs@GBA microneedle patch, which is the same as the above method for preparing the nucleoside microneedle patch, except that the gel preparation solution is the gel preparation solution obtained by taking the Dex-PVs aqueous solution as the solvent in Example 3.

[0121] Performance evaluation

[0122] 1. To verify the structure of the guanosine-quadruplex hydrogel, Fourier transform infrared spectroscopy test, nuclear magnetic resonance, X-ray diffraction measurement and SEM image analysis were performed.

[0123] 1.1 Fourier transform infrared spectroscopy: lyophilize the guanosine-quadruplex hydrogel to be tested to obtain a lyophilized powder, mix the lyophilized powder with KBr according to a mass ratio of 1:100, and place it in a Fourier transform infrared spectrometer (INVENIO) to observe the spectrum, the scanning frequency is 7.5 kHz, and the result is as shown in Figure 6 .

[0124] According to Figure 6 It can be seen that the infrared spectra of the GBA hydrogel and the GBG hydrogel are 1086 cm -1A characteristic peak obviously attributed to the asymmetric stretching vibration peak of the borate bond BOC appears nearby, indicating that the borate bond exists in the GBA hydrogel and GBG hydrogel of the present invention.

[0125] 1.2 Nuclear Magnetic Resonance ( 11 B NMR, 1 H NMR) was recorded using a Bruker AV 400 MHz spectrometer with DMSO-d6 as solvent, TMS as standard, and the guanosine-quadruplex hydrogel to be tested as the sample at room temperature. 11 B NMR ( Figure 7 ), 1 H NMR ( Figure 8 ) spectrum.

[0126] The boron spectrum showed that in the boron spectrum of the guanosine-quadruplex hydrogels GBA and GBG, the signal peak at 20 ppm disappeared, and a broad absorption band was presented, indicating that the boron atom underwent chemical conversion to form a boronate ester bond. In the hydrogen spectrum, the -OH signal peak of the guanosine-quadruplex hydrogels GBA and GBG disappeared, and the peak attributable to the diester appeared, proving the formation of the diester and the consumption of -OH. The hydrogen spectrum and the boron spectrum jointly proved the formation of boronate ester bonds in the guanosine-quadruplex hydrogels GBA and GBG of the present invention.

[0127] 1.3 X-ray diffraction (XRD) measurements: X-ray diffractometer (Bruker D8 ADVANCE X-ray diffractometer) was used to measure the XRD spectra of GBG freeze-dried powder at 25°C in an angle range of 1° to 40°. Figure 9 ).

[0128] according to Figure 9 It can be seen that a characteristic Bragg diffraction peak appears at 26.5°. According to the WLBragg equation, the interplanar spacing d is This is the same as the spacing between two adjacent G-quartets. The results are basically consistent, which shows that there are G-quartet planes in the hydrogel system of the present invention, and the planes are stacked to form a higher-order G-quartet structure.

[0129] 1.4 SEM images of guanosine-quadruplex hydrogel (GBG) (left) and guanosine-quadruplex hydrogel incorporated with dexamethasone vesicles (Dex-PVs@GBG) (right) Figure 10 As shown, according to Figure 10 It can be seen that the SEM image of GBG hydrogel shows an entangled nanofiber structure with an average nanofiber width of 40 nm. The Dex-PVs@GBG hydrogel presents a structure in which vesicles and fibers coexist, and the average nanofiber width is 30 nm, indicating that the vesicles are successfully loaded into the hydrogel.

[0130] 2. To verify the structure and performance of the symmetrical nucleoside microneedle patch, scanning electron microscopy, double-layer structure confirmation, mechanical property testing, and solubility testing were performed.

[0131] 2.1 SEM images: The specific morphological details of the surface morphology of the symmetrical nucleoside microneedle patch of Example 4 were observed using a scanning electron microscope. Figure 11 As shown, according to Figure 11 The microneedle tips are neatly arranged in a 20 × 20 mm array, demonstrating a complete morphology. Each microneedle has a quadrangular pyramid base diameter of 390 μm and a tip height of 365 μm. Compared to the mold, the microneedles are slightly smaller, primarily due to volume shrinkage of the PDMS mold during the curing process, and further shrinkage of the microneedles during drying and dehydration.

[0132] 2.2 Double-layer structure: Using the gel preparation solution and PVA solution with dye added as raw materials, a symmetrical nucleoside microneedle patch was prepared by the method described in Example 4 to obtain a microneedle patch with fluorescent markers, wherein the dye in the gel preparation solution is rhodamine dye and the dye in the PVA solution is fluorescein isothiocyanate (FITC). The microneedle patch with fluorescent markers was placed under an inverted fluorescence microscope to observe the color development. The results are as follows: Figure 12 As shown, according to Figure 12 It can be seen that the needle tip is red and the back is green, clearly showing the layered structure, indicating that the needle tip of the nucleoside microneedle patch of the present invention is completely formed by the nucleoside solution.

[0133] 2.3 Mechanical Properties: The test method includes: using a universal testing machine, fixing the symmetrical nucleoside microneedle patch of Example 4 on the base of the force gauge, with the needle tip facing upward, and moving the sensor vertically downward at a rate of 0.5 mm / min. When contacting the needle tip, an interactive force is generated, which is recorded by the pressure sensor to obtain an axial pressure-displacement image. When the preset maximum pressure (20 N) is reached, the measurement is automatically terminated.

[0134] The tip morphology of the symmetrical nucleoside microneedle patch was recorded before and after mechanical testing and examined using an optical microscope to assess potential bending or fracture under the applied axial force. Figure 13 As shown, according to the force-compression displacement curve of the microneedle, the maximum force that a single needle tip can withstand is about 0.19N, which meets the mechanical strength required for the microneedle to pierce the skin (0.058N). Figure 13 The right image is an image of the microneedle after compression, indicating that the microneedle tip breaks under stress.

[0135] 2.4 Solubility: The symmetrical nucleoside microneedle patch of Example 4 was immersed in PBS solution for a predetermined time, and the state of the symmetrical nucleoside microneedle patch was observed using an optical microscope to confirm the detachment of the needle tip. The results were as follows: Figure 14 As shown, according to Figure 14 It can be seen that after contacting with PBS solution, the background PVA is quickly dissolved and the needle tip is independent, and the monitoring shows that complete separation is completed within 90 seconds.

[0136] 3. To detect the scavenging efficiency of guanosine-quadruplex hydrogel on different reactive oxygen species (ROS), total antioxidant capacity experiment, hydrogen peroxide detection kit, superoxide anion scavenging experiment, hydroxyl radical scavenging experiment and in vitro antioxidant capacity test experiment were used for testing.

[0137] 3.1 The total antioxidant capacity experiment was carried out by ABTS method, which specifically included: taking a 96-well plate, adding 10 μL PBS buffer in the well as control group 1, labeled as "blank control 1", adding 100 μL of ABTS working solution in the well as sample well, adding 10 μL gel preparation liquid to the sample well, taking the well with 100 μL PBS and 10 μL gel preparation liquid as control group 2, labeled as "blank control 2", to evaluate the effect of the hydrogel itself. Mix the solution in each well gently to ensure uniform distribution, then incubate at 37°C for 5 minutes, after incubation, measure the absorbance value A of each group at 734 nm, and calculate the corresponding inhibition percentage, i.e. clearance rate, the results are shown in the upper left graph of Figure 15 .

[0138]

[0139] 3.2 The determination of hydrogen peroxide scavenging efficiency was carried out by H2O2 scavenger detection kit, which was purchased from Biyun Tian Biological Technology Co., Ltd. The test method included: diluting the H2O2 standard solution provided in the kit by 1:10, mixing the diluted H2O2 solution with the gel preparation liquid in a mass ratio of 4:1, incubating at 37°C for 12h, then adding the color developing agent as the experimental sample group, mixing the diluted H2O2 solution with PBS buffer in a mass ratio of 1:4, incubating at 37°C for 12h, adding the color developing agent, and labeling as "blank control 1". Another blank control was set, i.e. mixing the diluted H2O2 solution with the gel preparation liquid in a mass ratio of 4:1, incubating at 37°C for 12h without adding the color developing agent, as "blank control 2". Incubate each group with color developing agent at room temperature for 5 minutes, then measure the absorbance A at 405 nm, and calculate the corresponding inhibition percentage according to the following formula, the results are shown in the lower left graph of Figure 15 .

[0140]

[0141] 3.3 The superoxide anion scavenging efficiency was determined by superoxide anion detection (O2 ·- ) kit, wherein the superoxide anion detection (O2 ·-The kit was purchased from Suzhou Keming Biotechnology Co., Ltd. The test method includes: according to the kit instructions, reagent 1, reagent 2 and water were mixed, incubated at 25°C for 1 minute, then, gel preparation solution and reagent 3 were added, and the mixture was gently pipetted to ensure thorough mixing, and incubated at 37°C for 30 minutes. Reagents 4 and 5 were added and ensured to be completely mixed to obtain a mixed solution, and the mixed solution was incubated at room temperature for 20 minutes. 200 μL of the mixed solution was transferred to a 96-well plate, and the absorbance A at 530 nm was measured. The corresponding inhibition percentage was calculated according to the following formula. The results are shown in FIG. Figure 15 As shown in the upper right picture:

[0142]

[0143] 3.4 The hydroxyl radical scavenging efficiency was determined using a ·OH scavenger detection kit, which was purchased from Suzhou Keming Biotechnology Co., Ltd. The test method included: according to the kit instructions, the reagent was mixed with the gel preparation solution, mixed thoroughly, and subjected to colorimetric reaction at 37°C for 20 minutes. 200 μL was transferred to a 96-well plate, and the absorbance A was measured at 510 nm. The corresponding inhibition percentage was calculated according to the following formula. The results are shown in the figure. Figure 15 As shown in the lower right figure:

[0144]

[0145] according to Figure 15 It can be seen that the scavenging rates of the four hydrogels on ABTS free radicals are all over 90%, showing good overall antioxidant effect. ·- The scavenging rates of the four hydrogels were all about 60%, while the scavenging rates of ·OH and H2O2 were close to 70%, indicating that the hydrogels of the present invention have good scavenging ability for reactive oxygen species. The antioxidant capacity of the four hydrogels was not much different, which may be because the four hydrogels all rely on the "self-consumption" reducing ability of the nucleoside material to scavenge reactive oxygen species.

[0146] 3.5 The in vitro antioxidant capacity test of guanosine-quadruplex hydrogel was performed using a reactive oxygen species (ROS) detection kit, including: human oral mucosal epithelial cells (Leuk-1 cells) were seeded in a 96-well plate at a density of 8000 cells per well, incubated in a CO2 incubator for 12 hours to allow cell attachment, and after the incubation, 100 μL of a mixture of preheated Leuk-1 cell complete culture medium and Rosup solution was added to each well. After a further incubation for 2 hours, the solution in the well was aspirated, and then 100 μL of gel preparation solution was added. An equal amount of PBS buffer was added to the control group, and then the incubation was continued for 1 hour. 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was added, and the cells were incubated at 37°C for 30 minutes. The cells were washed with PBS to remove free fluorescent dyes, and the intracellular fluorescence intensity was observed under an inverted fluorescence microscope to evaluate the level of reactive oxygen species. The results are shown in FIG. Figure 16 As shown; wherein, in the mixed solution of Leuk-1 cell complete culture medium and Rosup solution, the Rosup concentration is 1 μg / mL; the guanosine-quadruplex hydrogel solution is a mixed solution of guanosine-quadruplex hydrogel and Leuk-1 cell complete culture medium, wherein the guanosine-quadruplex hydrogel concentration is 500 μg / mL. Figure 16 It can be seen that compared with the control group, the green fluorescence of the PBS group after 24 hours of Rosup stimulation was significantly enhanced, indicating that Rosup stimulation produces reactive oxygen species and induces oxidative stress. Compared with cells stimulated only by Rosup, the fluorescence intensity of cells co-incubated with hydrogels was significantly lower, close to the fluorescence level of normal cells, indicating that hydrogels can effectively reduce reactive oxygen species and bring the reactive oxygen level close to a healthy state. At the same time, when co-incubated with vesicles (Dex-PVs) or adenosine (A) alone, the fluorescence intensity of cells was reduced compared to the PBS group, but was still significantly higher than the cell group co-incubated with hydrogels, indicating that the hydrogel of the present invention has a synergistic in vitro antioxidant capacity.

[0147] 4. In vitro experiments on guanosine-quadruplex hydrogels, including in vitro anti-inflammatory ability tests, in vitro migration and tube formation promotion tests, and in vitro analgesic ability tests.

[0148] 4.1 The in vitro anti-inflammatory ability was verified using RAW 264.7 cells, including: RAW 264.7 cells were divided into 6 groups and treated according to the following experimental groups. The initial RAW 264.7 cell concentration in each group was 5×10 5 / mL:

[0149] 1) Control group: only 1 mL of PBS was added;

[0150] 2) PBS group: pretreated with 1 mL of 1 μg / mL LPS for 24 h and then added with 1 mL of PBS;

[0151] 3) GBG group: GBG wet granules were added after pretreatment with 1 mL of 1 μg / mL LPS for 24 hours; the concentration of GBG wet granules in the system was 500 μg / mL. The GBG wet granules were prepared by placing the GBG hydrogel in complete culture medium at a volume ratio of 1:9, thoroughly crushing the GBG hydrogel, and filtering to obtain GBG wet granules;

[0152] 4) GBA group: First, treat with 1 mL of 1 μg / mL LPS for 24 h, then add GBA wet granules; the concentration of GBA wet granules in the system is 500 μg / mL; the preparation method of GBA wet granules is basically the same as that of GBG wet granules;

[0153] 5) Dex-PVs@GBG group: First, treat with 1 mL of 1 μg / mL LPS for 24 h, then add Dex-PVs@GBG wet granules; the concentration of Dex-PVs@GBG wet granules in the system is 500 μg / mL; the preparation method of Dex-PVs@GBG wet granules is basically the same as that of GBG wet granules;

[0154] 6) Dex-PVs@GBA group: First treated with 1 mL of 1 μg / mL LPS for 24 h, then added with Dex-PVs@GBA wet granules; the concentration of Dex-PVs@GBA wet granules in the system was 500 μg / mL; the preparation method of Dex-PVs@GBA wet granules was basically the same as that of GBG wet granules;

[0155] The corresponding basal culture medium was used as a control.

[0156] The cells in each group were cultured in 90 mL of complete culture medium for 24 h.

[0157] After the culture, RAW 264.7 cells were collected and stained with PE-labeled CD206 and APC-labeled CD86 antibodies. The cell phenotype was analyzed by flow cytometry. Figure 17 As shown, according to Figure 17 It can be seen that compared with the control group, after LPS treatment, the transformation of M0 macrophages to M1 macrophages was significant, and the change of M2 phenotype was almost negligible, indicating that LPS effectively induced inflammatory response. Hydrogel treatment can enhance the anti-inflammatory phenotype M2. However, this improvement in anti-inflammatory performance was not obvious in the GBG hydrogel treatment group, nor was it significant in the GBA hydrogel treatment group. However, in the Dex-PVs@GBG, especially the Dex-PVs@GBA treatment group, this anti-inflammatory performance increased significantly. Specifically, the proportion of M2 macrophages in all cells increased from 1.61% to 19%, while the proportion of M1 macrophages decreased from 36.6% to 11.8%. Especially in the Dex-PVs@GBA treatment group, compared with the PBS group, the M2 / M1 ratio increased by 36.6 times, compared with the GBG treatment group, the M2 / M1 ratio increased by 25.52 times, compared with the GBA treatment group, the M2 / M1 ratio increased by 71 times, and compared with the Dex-PVs@GBG treatment group, the M2 / M1 ratio increased by 1.25 times, which has a significant immunomodulatory effect.

[0158] After the culture was completed, the contents of TNF-α and IL-10 in the cell culture supernatant were detected by ELISA. Figure 18 As shown, according to Figure 18It can be seen that the PBS group treated with LPS can significantly induce macrophages to secrete the proinflammatory cytokine TNF-α. Compared with the PBS group, the TNF-α content in the hydrogel-treated group was significantly decreased, while the IL-10 content was significantly increased, indicating that the hydrogel of the present invention can effectively inhibit the expression of proinflammatory factors and promote the release of anti-inflammatory factors.

[0159] After the culture, immunofluorescence staining was performed using rabbit anti-CD86 polyclonal antibody (FITC-labeled goat anti-rabbit secondary antibody) and rabbit anti-CD206 polyclonal antibody (Alexa Fluor 594-labeled goat anti-rabbit secondary antibody), and then the polarization state of macrophages was observed under a confocal microscope. The results are shown in Figure 2. Figure 19 As shown, according to Figure 19 It can be seen that the PBS group treated with LPS showed significant green fluorescence expressing CD86, indicating that LPS can induce the expression of anti-inflammatory factors. The hydrogel-treated group showed reduced green fluorescence intensity. The green fluorescence in the GBA, Dex-PVs@GBG and Dex-PVs@GBA groups decreased significantly, indicating a significant decrease in CD86 expression. In particular, the green fluorescence intensity in the Dex-PVs@GBA group decreased most significantly, and the red fluorescence increased most significantly, indicating that the Dex-PVs@GBA hydrogel can significantly inhibit CD86 polarization and upregulate the expression of CD206.

[0160] 4.2 In vitro migration and tube formation promotion tests were verified by scratch test, including: human microvascular endothelial cells (HMEC-1) were cultured at 1×10 6 The cells were seeded at a density of 100 μg / mL in a 12-well plate and cultured overnight in a basal medium at 37°C. After serum starvation, a linear scratch with a width of 500 μm was formed on the cell monolayer using a 200 μL pipette tip. The cells were washed with PBS to remove cell debris. Subsequently, 1 mL of low-serum medium was added to the wells, and 1 mL of a hydrogel extract with a concentration of 1000 μg / mL was added to obtain a treatment solution. The treatment solution was cultured for a preset time, and the scratch healing was observed under an optical microscope. The scratch area and width were quantitatively analyzed using ImageJ software, and the migration rate was calculated based on the ratio of the gap area at the preset time to the gap area at 0 h. The concentration of the guanosine-quadruplex hydrogel in the treatment solution was 500 μg / mL. 1 mL of basal medium was added to the control group. The results are shown in FIG. Figure 20 As shown in A to D, according to Figure 20As can be seen from A to D, the unhealed area of ​​each group of materials was significantly reduced after being co-incubated with HMEC-1 cells for 24 hours. The migration distances of each group were: 376.53 μm in the control group, 410.65 μm in the GBG group, 462.38 μm in the GBA group, 300.35 μm in the Dex-PVS@GBG group, and 502.89 μm in the Dex-PVS@GBA group. The migration rate of HMEC-1 cells in the Dex-PVS@GBA group reached 69.64%. Compared with the control group, this performance of promoting endothelial cell migration was not significant in the GBG group, especially Dex-PVS@GBG. The area of ​​the unhealed area in the Dex-PVS@GBA group was significantly reduced, indicating that the Dex-PVS@GBA hydrogel of the present invention can effectively promote endothelial cell migration.

[0161] 4.3 The testing method for the in vitro pro-angiogenesis experiment includes: pre-cooling the Matrigel at 4°C overnight, diluting it with HMEC-1-specific basal culture medium to a final concentration of 50% to obtain diluted Matrigel, pre-cooling all experimental materials at -20°C overnight, adding 50 μL of the diluted Matrigel to each well of a 96-well plate, and incubating the plate at 37°C for 30 minutes to allow the Matrigel to solidify, seeding HMEC-1 cells that have been well passaged after digestion on the solidified Matrigel at a density of 20,000 cells per well, adding guanosine-quadruplex hydrogel to a final concentration of 500 μg / ml of guanosine-quadruplex hydrogel in the system, and using a group to which an equal amount of basal culture medium was added as a control. The cell sediment attachment was defined as 0 h, and images were captured using an optical microscope to evaluate the cell status at 0 h. The cells were then incubated in a 37°C cell culture incubator for 4 h. Angiogenesis was observed and photographed under a bright field using an inverted phase-contrast fluorescence microscope. The vascular nodes and junctions in each group were counted and statistically analyzed using ImageJ software. The results are shown in Figure 2. Figure 20 As shown in B, E and F, Figure 20 Figures B, E, and F are schematic diagrams of tube formation after 4 hours of co-incubation. Compared to the control and other experimental groups, the Dex-PVS@GBA group exhibited distinct tubule structures, while only sparse cell connections were observed in the other groups. Quantification of branch number and intersection points revealed a significant increase in the Dex-PVS@GBA group, demonstrating that the Dex-PVS@GBA method can significantly ameliorate the inhibitory effect of dexamethasone on tube formation and effectively promote angiogenesis.

[0162] 4.4 A mouse model of chemotherapy-induced oral ulcers was established to evaluate the analgesic ability of the symmetrical nucleoside microneedle patch in vitro, including: male C57BL / 6 mice (6 weeks old, weighing 15-25 g) were acclimated to the environment in plastic cages under standard laboratory conditions for 5 days and randomly divided into 7 groups (n=8). Each mouse was intraperitoneally injected with 200 μL of 5-fluorouracil at a concentration of 5 mg / mL every other day for a total of 3 times. After the third injection, a small cotton swab soaked in 99% by mass acetic acid aqueous solution was applied to the tongue of each mouse for 35 seconds to induce oral ulcers. On the first day after induction, a single 3×3 array symmetrical nucleoside microneedle patch was applied to the ulcerated area, and pain was assessed 24 hours after treatment. The pain behavior experiment was conducted in a 30×30×20 cm monitoring room with a grid floor. No food or water was provided. Before the experiment, the mice were allowed to relax in the monitoring room for 15 minutes to reduce stress. After that, the experiment was carried out. The pain response of the mice was observed and recorded based on the camera installed in the front of the monitoring room. The inflammatory pain of the mandible and face was assessed by the frequency of wiping (wiping the face with unilateral or bilateral forelimbs) and the duration of scratching (scratching the lips with bilateral forelimbs). Each observation period was 3 minutes, and there was an interval of 3 minutes between two adjacent observation periods. The number of wiping times and the duration of scratching were recorded in each observation period. The degree of pain of oral ulcers in each group of mice was compared. The pain assessment was conducted in a double-blind manner.

[0163] Electrophysiological testing was performed on mice 24 hours after treatment, including applying 1V electrical stimulation to the tip of the tongue bilaterally under gas anesthesia, recording the compound muscle action potential (CMAP amplitude) using a pair of electrodes, and quantitatively analyzing the CMAP amplitude to assess the severity of oral pain in mice. The results are as follows: Figure 21 As shown, according to Figure 21 It can be seen that compared with mice without model establishment (baseline group), the number of wiping times and scratching duration of the control group mice with chemotherapy-induced oral ulcers were significantly increased. After treatment with the patch, except for the GBG microneedle group, the number of wiping times and scratching duration of the remaining groups were significantly reduced, indicating that the patch of the present invention can significantly reduce inflammatory pain in the mandible and face of mice. Compared with mice without model establishment (baseline group), the myoelectric amplitude generated by electrical stimulation was about 5mV, showing a complete and clear waveform, while the control group with chemotherapy-induced oral ulcers showed a significantly enhanced myoelectric amplitude of about 20mV, with a stronger waveform. The possible reason is that long-term inflammation leads to pain sensitization, a lower pain threshold of the nervous system, and a more acute pain response. After treatment with different patches, except for the GBG microneedle group, all treatment groups showed obvious electrical signal attenuation, indicating that the microneedles of the present invention have a more significant analgesic effect, especially the Dex-PVs@GBA microneedle patch group, which showed the most significant electrical signal attenuation, indicating that it has excellent effect in relieving inflammatory pain. The Dex-PVs@GBA microneedle patch of the present invention can effectively improve chemotherapy-induced oral ulcers by reducing inflammatory response, alleviating pain perception and inhibiting pain sensitization.

[0164] 5. A mouse model of chemotherapy-induced oral ulcers was established to evaluate the in vivo healing effect of the symmetrical nucleoside microneedle patch. Male C57BL / 6 mice (6 weeks old, weighing 15-25 g) were acclimated in plastic cages under standard laboratory conditions for 5 days and randomly divided into 7 groups (n=8). Each mouse was intraperitoneally injected with 200 μL of 5-fluorouracil at a concentration of 5 mg / mL every other day for a total of 3 times. After the third injection, a small cotton swab soaked in 99% acetic acid was applied to the tongue of each mouse for 35 seconds to induce oral ulcers. On the first day after induction, a single 3×3 array symmetrical nucleoside microneedle patch was applied to the ulcer area. The ulcers were photographed on days 1, 3, 5, 7, and 9, and the ulcer area was quantified using ImageJ software. The blank group consisted of mice that did not receive any treatment after induction. In addition, the clinical severity of tongue ulcers in mice was assessed according to the following scoring criteria:

[0165] 0. Normal mucosa, no bleeding, swelling, or ulcers;

[0166] 1. Mild redness or bruising, without bleeding, swelling or ulceration;

[0167] 2. Severe redness and congestion, possibly with bleeding, small ulcers, or scar tissue, but no swelling;

[0168] 3. Severe redness and congestion, with visible bleeding, large ulcers, and swelling.

[0169] Mice were euthanized on days 5 and 9, and tissue and blood samples were collected for subsequent characterization. The reduction in ulcer area, re-epithelialization, and tissue regeneration were quantified compared with the untreated control group to determine the healing effect. HE staining was used to observe tissue structure and inflammatory cell infiltration. Immunofluorescence staining was used to evaluate markers related to healing and inflammation. Figure 22 As shown, according to Figure 22As can be seen, mice in the control group still showed obvious ulcers after 9 days, and wound healing was slow. After 9 days of treatment, the relative wound areas of the gel and microneedle patch groups were: 30.49% for the commercial patch group, 29.86% for the GBG microneedle group, 22.73% for the GBA microneedle group, 26.17% for the Dex-PVs@GBG gel group, 31.31% for the Dex-PVs@GBG microneedle group, and 6.27% for the Dex-PVs@GBA microneedle group. The Dex-PVs@GBA microneedle group significantly accelerated wound healing and displayed the lowest clinical severity score, indicating that the Dex-PVs@GBA microneedle group exhibited the most significant efficacy in promoting wound healing. We also monitored the weight changes of mice in each group. The weight of mice in the control group continued to decrease throughout the experiment and showed no significant recovery by the end of the experiment. The mice in the patch or gel treatment group also showed varying degrees of weight loss in the early stages of the experiment (the first three days), which may be due to the pain caused by the ulcer, which led to a decrease in the mice's appetite. After receiving two treatments, the weight of the mice in the patch or gel treatment group gradually recovered, indicating that the patch or gel treatment of the present invention can effectively relieve the pain of mice and improve their appetite.

[0170] HE staining revealed minimal inflammatory cell infiltration in the Dex-PVS@GBA microneedle group, and relatively complete tissue recovery in the wound area, indicating that Dex-PVS@GBA microneedle treatment significantly reduced local inflammatory responses and accelerated tissue healing. In particular, at the junction of the tongue mucosa and underlying muscle layer, the tissue structure was compact and showed no significant structural deformation, demonstrating excellent tissue repair.

[0171] Masson trichrome staining further assessed the progress of collagen deposition and tissue remodeling. Results showed that in the Dex-PVS@GBA microneedle group, collagen fiber deposition increased significantly, with orderly and dense fiber arrangement, indicating a significant acceleration of collagen deposition and tissue remodeling. In particular, in the transition zone between the tongue mucosa and muscle layer, collagen deposition was abundant, with parallel and uniform fiber arrangement, demonstrating mature tissue repair characteristics. These results demonstrate that Dex-PVS@GBA microneedles not only promote collagen deposition but also accelerate the formation of functional tissue, enhancing the mechanical strength and structural integrity of local tissues. In contrast, the other treatment groups exhibited more dispersed and irregular collagen deposition, with loose and disordered fiber arrangement, reflecting a lag in collagen synthesis and tissue repair, and failed to effectively suppress local inflammatory responses. This demonstrates that the Dex-PVS@GBA microneedles of the present invention significantly improve the quality and structure of collagen deposition, effectively promoting wound healing and accelerating tissue remodeling.

Claims

1. A symmetrical nucleoside microneedle patch loaded with polymer vesicles, characterized in that: The invention comprises a guanosine-quadruplex hydrogel microneedle tip doped with drug-loaded polymer vesicles; the raw materials of the drug-loaded polymer vesicles include polycaprolactone-polyglutamic acid copolymer; the raw materials of the guanosine-quadruplex hydrogel include guanosine and KB(OH)4.

2. The symmetrical nucleoside microneedle patch loaded with polymer vesicles according to claim 1, characterized in that: The guanosine-quadruplex hydrogel raw material also includes adenosine.

3. A method for preparing a symmetrical nucleoside microneedle patch loaded with polymer vesicles according to any one of claims 1 to 2, characterized in that: include: providing a gel preparation solution for a guanosine-quadruplex hydrogel incorporating drug-loaded polymer vesicles; The gel preparation liquid of guanosine-quadruplex hydrogel doped with drug-loaded polymer vesicles is placed in the needle cavity of the microneedle mold to obtain the hydrogel, and the backing solution is added to obtain a symmetrical nucleoside microneedle patch loaded with polymer vesicles.

4. The method according to claim 3, characterized in that The volume of the backing solution is 1 to 2 times the volume of the gel preparation solution of the guanosine-quadruplex hydrogel incorporated with the drug-loaded polymer vesicles.

5. The method according to claim 3, characterized in that Placing a gel preparation liquid of guanosine-quadruplex hydrogel doped with drug-loaded polymer vesicles into the needle cavity of the microneedle mold comprises: filling ultrapure water into the needle cavity of the microneedle mold, injecting the gel preparation liquid of guanosine-quadruplex hydrogel doped with drug-loaded polymer vesicles, making the liquid surface of the gel preparation liquid of guanosine-quadruplex hydrogel doped with drug-loaded polymer vesicles contact the liquid surface of ultrapure water in the needle cavity, and performing forced drying to evaporate the ultrapure water, so that the gel preparation liquid of guanosine-quadruplex hydrogel doped with drug-loaded polymer vesicles enters the needle cavity.

6. The method according to claim 3, characterized in that The preparation method of the drug-loaded polymer vesicles comprises: Step 1, providing deprotected polycaprolactone and γ-benzyloxycarbonyl-L-glutamic acid anhydride; Step 2: dissolving the deprotected polycaprolactone and γ-benzyloxycarbonyl-L-glutamic acid anhydride in a first organic solvent, and performing a ring-opening polymerization reaction under an inert atmosphere to obtain a polycaprolactone-polypeptide copolymer; Step 3, dissolving the polycaprolactone-polypolypeptide copolymer in a second organic solvent and performing a deprotection reaction to obtain a polycaprolactone-polyglutamic acid copolymer; Step 4: dissolving the polycaprolactone-polyglutamic acid copolymer and the drug in a third organic solvent, dropping physiological saline into the solvent, stirring to assemble the solvent, and dialyzing the solvent to obtain drug-loaded polymer vesicles.

7. The method according to claim 6, characterized in that The first organic solvent is dimethylformamide; and / or the second organic solvent is HBr / CH3COOH solution; and / or the third organic solvent is dimethyl sulfoxide.

8. The method according to claim 6, characterized in that In step 4, the polycaprolactone-polyglutamic acid copolymer and the drug are dissolved in a third organic solvent to obtain a system in which the concentration of the polycaprolactone-polyglutamic acid copolymer is 0.5-2 mg / mL; and / or, in step 4, the volume of the physiological saline is 2-5 times the volume of the third organic solvent.

9. The method according to claim 3, characterized in that The preparation method of the gel preparation solution of the guanosine-quadruplex hydrogel doped with drug-loaded polymer vesicles comprises: using a solution containing drug-loaded polymer vesicles as a solvent, adding guanosine and H3BO3 aqueous solution respectively, and adding K-containing + to obtain the preparation solution.

10. An application of the symmetrical nucleoside microneedle patch loaded with polymer vesicles as claimed in claim 1, characterized in that: The application includes application in preparing drugs for sustained analgesia and high-efficiency anti-inflammatory, or application in preparing drugs for repairing oral ulcers.

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