A photothermal combined drug composite soluble sugar-based microneedle and a preparation method thereof

Abstract

The present application relates to a kind of photothermal combined drug composite soluble sugar-based microneedle and its preparation method.The microneedle is soluble polymer as needle body matrix, load fucoidan-epipodophyllotoxin covalent coupling and the carbon dot with near-infrared photothermal conversion performance, by mould pouring, vacuum filling, drying and demoulding obtain square pyramidal soluble microneedle array.Fucoidan (FU) and epipodophyllotoxin (PPT) are coupled by hydrolysable connecting arm, while retaining antitumor activity, improve the dispersion and local delivery compatibility of epipodophyllotoxin;Carbon dot (CDs) under near-infrared laser irradiation makes the rapid heating of administration site, and FU-PPT generates photothermal-drug combined tumor inhibition effect.The needle body of the microneedle obtained in the present application is complete, array is regular, mixed medicine microneedle still has sufficient mechanical strength to pierce into skin, while can realize short time rapid heating, with excellent tumor inhibition effect.The present application can be used for local delivery and photothermal combined treatment of skin tumor or superficial solid tumor.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, and particularly relates to a photothermal combined drug composite soluble glycosyl microneedle and its preparation method. Background Technology

[0002] Melanoma and other superficial solid tumors of the skin are characterized by their superficial location, high local invasiveness, and high risk of recurrence. Current treatment methods mainly include surgical resection, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. For early or localized lesions, surgical resection remains an important treatment, but postoperative residual disease, recurrence of small lesions, and insufficient local adjuvant therapy remain clinical problems. For advanced lesions, although systemic drug administration can cover the entire body, it is often accompanied by problems such as wide drug exposure, significant toxic side effects, and insufficient drug concentration at local lesions. Therefore, developing a material system for skin or superficial tumors that can achieve efficient local delivery with low systemic exposure and can be combined with exogenous triggering therapy has clear application value.

[0003] Podophyllotoxin (PPT) is a class of natural products with significant anti-proliferative activity, capable of interfering with microtubule assembly and tumor cell mitosis, exhibiting strong cytotoxic activity. However, free podophyllotoxin suffers from drawbacks such as poor water solubility, poor dispersibility, narrow therapeutic window, and high systemic toxicity. Direct application to local or systemic administration can easily lead to problems such as insufficient formulation stability, local irritation, or damage to non-target tissues. Improving its hydrophilicity, formulation compatibility, and local delivery suitability while preserving its antitumor activity is a key issue that needs to be addressed for the further development of this class of drugs.

[0004] Fucoidan (FU) is a sulfated polysaccharide derived from brown algae. It possesses excellent hydrophilicity, biocompatibility, and a multi-hydroxyl structure, making it suitable as a glycosyl drug carrier or a hydrophilically modified backbone. Covalent coupling of fucoidan with podophyllotoxin via a hydrolyzable linker forms a fucoidan-podophyllotoxin covalent conjugate, FU-PPT. On one hand, the fucoidan backbone improves the dispersion and processing properties of podophyllotoxin in aqueous systems; on the other hand, the hydrolyzable linker enables the gradual release of the drug from local tissue fluid or the tumor microenvironment, thereby prolonging the local action time and reducing the risk of sudden release of free drug.

[0005] Soluble microneedles are a type of minimally invasive percutaneous drug delivery platform capable of penetrating the stratum corneum of the skin and dissolving in tissue fluid to release drugs. They offer advantages such as low pain, ease of operation, avoidance of sharps residue, and suitability for local lesion administration. For superficial skin tumors such as melanoma, soluble microneedles can deliver drugs directly to the intradermal or superficial tumor area, resulting in local drug accumulation and reduced systemic exposure. However, drug-only microneedles may still have limitations such as limited local killing intensity, insufficient penetration into larger tumor tissues, and a single drug action mechanism, making it difficult to fully meet the needs of efficient ablation of superficial tumors.

[0006] Photothermal therapy is a treatment method that utilizes photothermal materials to absorb near-infrared light and convert it into heat energy, thereby raising the temperature of local tissues and inducing thermal damage to tumor cells or enhancing drug sensitivity. Compared with traditional thermotherapy, near-infrared light irradiation can achieve exogenous triggering within a certain spatial range, making it suitable for combination with local drug delivery materials. Carbon dots have the characteristics of small particle size, good dispersibility, abundant surface functional groups, and compatibility with water-soluble polymer systems. Furthermore, their photothermal conversion properties can be controlled through precursors and preparation conditions, making them suitable as photothermal functional components in soluble microneedles.

[0007] In existing technologies, microneedle drug delivery, glycosyl drug conjugation, and photothermal material therapy are mostly relatively independent technical routes, and there is still a lack of composite systems that integrate multiple functions. In particular, how to simultaneously maintain microneedle shapeability, mechanical penetration strength, drug release capacity, and near-infrared photothermal response under high drug loading conditions is a core technical problem that needs to be solved in the preparation of such composite microneedles. Summary of the Invention

[0008] The technical problem solved by this invention is: how to stably and uniformly load drugs and photothermal materials with different physicochemical properties into the needle matrix of soluble microneedles while ensuring high drug loading; and how to maintain microneedle shapeability, mechanical penetration strength, drug release capacity and near-infrared photothermal response under high drug loading conditions, so as to improve the therapeutic effect on superficial tumors.

[0009] In view of the technical problems existing in the prior art, the present invention designs a photothermal combined drug composite soluble glycosyl microneedles and its preparation method.

[0010] It should be noted that, in this invention, unless otherwise specified, the specific meaning of "comprising" in relation to composition definition and description includes both open-ended meanings such as "comprising," "including," etc., and closed-ended meanings such as "composed of," etc., and similar meanings.

[0011] To solve the aforementioned technical problems, the present invention adopts the following solution:

[0012] [First technical solution]

[0013] A method for preparing a photothermal combined drug-derived soluble glycosyl microneedles includes the following steps:

[0014] S1: Preparation of fucoidan-podophyllotoxin covalent conjugate;

[0015] S2: Preparation of carbon dots with photothermal conversion properties;

[0016] S3: Preparation of needle precursor solution:

[0017] The fucoidan-podophyllotoxin covalently coupled compound prepared in step S1, the carbon dots prepared in step S2, polyvinyl alcohol and polyvinylpyrrolidone are dissolved or dispersed in an aqueous system to form a needle precursor solution.

[0018] S4: Preparation of photothermal combined drug-derived soluble glycosyl microneedles:

[0019] The needle precursor liquid prepared in step S3 is added to the microneedle negative mold, and the mold is filled with vacuum and centrifugation. After drying and curing, the mold is removed to obtain the photothermal combined drug composite soluble glycosyl microneedles.

[0020] Furthermore, the specific steps for preparing the fucoidan-podophyllotoxin covalent conjugate in S1 are as follows:

[0021] Podophyllotoxin was dissolved in a solvent, and succinic anhydride, a catalyst and an acid-binding agent were added to induce succinylation of the hydroxyl groups of podophyllotoxin to obtain a podophyllotoxin intermediate with a carboxyl linkage arm.

[0022] The podophyllotoxin intermediate with the carboxyl linker is activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and then reacted with an aqueous solution of fucoidan or a mixture of fucoidan and an organic solvent, so that fucoidan and podophyllotoxin are coupled through a hydrolyzable linker.

[0023] After the reaction was completed, the mixture was dialyzed and then freeze-dried to obtain the fucopolysaccharide-podophyllotoxin covalent conjugate, denoted as FU-PPT.

[0024] Furthermore, the process of preparing the needle precursor solution in step S3 is as follows:

[0025] Weigh out polyvinyl alcohol and polyvinylpyrrolidone, add deionized water, heat and stir until completely dissolved to obtain a blank needle matrix solution;

[0026] The mass ratio of polyvinyl alcohol to polyvinylpyrrolidone is (8-15):(10-25);

[0027] The FU-PPT prepared in step S1 and the CDs prepared in step S2 are added to the blank needle matrix solution to obtain the needle precursor solution.

[0028] Furthermore, the process of preparing photothermal combined drug-based soluble glycosyl microneedles in step S4 is as follows:

[0029] Add the needle precursor liquid from step S3 to the surface of the needle mold, and degas it under vacuum for 5-30 min and centrifuge it at 1000-5000 rpm for 5-30 min to ensure that the needle precursor liquid fully fills the needle cavity.

[0030] After scraping off excess liquid from the mold surface, the mold is dried and demolded to obtain photothermal combined drug soluble glycosyl microneedles.

[0031] Furthermore, in step S1, the solvent is one of anhydrous dimethyl sulfoxide, N,N-dimethylformamide, or dichloromethane;

[0032] The molar ratio of succinic anhydride to podophyllotoxin is (1-1.5):1;

[0033] The mass ratio of fucoidan to carboxylated podophyllotoxin is (2-5):1;

[0034] Furthermore, the catalyst is one of 4-dimethylaminopyridine or N-methylimidazole;

[0035] The acid-binding agent is triethylamine, and the molar ratio of the acid-binding agent to podophyllotoxin is (1-1.5):1.

[0036] In this invention, there are no specific requirements for the amount of catalyst used; generally, the amount of catalyst added is 1-2% of the mass of podophyllotoxin.

[0037] Furthermore, in step S3, the mass fraction of FU-PPT in the needle precursor solution is 30%-65%, and the concentration of CDs in the needle precursor solution is 50-500 μg / mL.

[0038] In step S1 of this invention, podophyllotoxin is dissolved using a solvent. There is no particular limitation on the amount of solvent used; generally, it is sufficient to fully dissolve the podophyllotoxin PPT. The organic solvent for dissolving fucoidan can be one of phosphate-buffered saline or 1-butyl-3-methylimidazolium hydrogen sulfate.

[0039] In the step of activating the intermediate with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, the mass of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is equal to the mass of the podophyllotoxin intermediate with a carboxyl linker arm.

[0040] In this invention, the drying conditions in step S3 can be drying at room temperature, 37°C or under vacuum for 12-48 hours, followed by demolding.

[0041] The purpose of dialysis is to remove unreacted small molecules and solvents;

[0042] In this invention, the specific operation for preparing carbon dots with photothermal conversion properties in step S2 is as follows:

[0043] Citric acid was used as the carbon source, dimethyl sulfoxide as the sulfur source, and urea as the nitrogen source. The mixture was placed in a reaction vessel and reacted at 160-220℃ for 4-12 h. After cooling to room temperature, a dark-colored reaction solution was obtained. The mass ratio of citric acid to urea was 1:(1-5), and the amount of dimethyl sulfoxide used was 10-20 ml per 1 g of citric acid.

[0044] The dark reaction solution was centrifuged at high speed, then filtered, dialyzed, and freeze-dried to obtain the carbon dots with photothermal conversion properties, denoted as CDs.

[0045] In step S2 of the present invention, the reactor is a polytetrafluoroethylene-lined reactor; the high-speed centrifugation is to remove large particles in the reaction; the filtration process is carried out by filtration through a 0.22 μm filter membrane.

[0046] In this invention, the catalyst selected is N-methylimidazolium (NMI), and its catalytic mechanism is as follows:

[0047] The nitrogen atom of NMI attacks the carbonyl group of succinic anhydride; the succinic anhydride undergoes ring opening to form a highly reactive acyl imidazolium intermediate; the PPT hydroxyl group attacks the carbonyl group of this intermediate; through proton transfer, NMI leaves and is regenerated.

[0048] In this invention, the working principle of the acid-binding agent is as follows: by using the lone pair electrons on the nitrogen of the tertiary amine to accept protons, the acid produced in the reaction is converted into triethylamine salt, thereby reducing the acidity of the system and promoting the reaction process.

[0049] In this invention, the fucoidan-podophyllotoxin covalent conjugate comprises fucoidan and podophyllotoxin linked by an ester bond linker to form a hydrolyzable linker structure. The fucoidan-podophyllotoxin covalent conjugate is obtained by a condensation reaction between the hydroxyl group of podophyllotoxin and carboxylated or activated fucoidan.

[0050] In this invention, depending on the specific application scenario requirements, the microneedles can be prepared as a square pyramid, cone, or pyramidal array with a needle height of 300-1200 μm, a needle body base width of 200-800 μm, and a distance between adjacent needle tips of 500-1500 μm.

[0051] [Second Technical Solution]

[0052] A photothermal combined drug composite soluble glycosyl microneedle prepared according to the above method, wherein the soluble glycosyl microneedle comprises a soluble polymer needle body, FU-PPT distributed within the needle body, and carbon dots distributed within the needle body.

[0053] Furthermore, after the microneedle is inserted into the skin or superficial tumor tissue, the needle matrix absorbs tissue fluid and dissolves and releases FU-PPT and carbon dots, and under near-infrared laser irradiation, the local temperature rises to 42-58 ℃.

[0054] [Third technical solution]

[0055] The application of the above-mentioned soluble glycosyl microneedles in pharmaceuticals or medical materials for the local treatment of skin tumors, melanoma, superficial solid tumors, or postoperative residual tumors.

[0056] Specific applications are as follows:

[0057] 1. For topical treatment of skin tumors, melanoma and other superficial solid tumors.

[0058] 2. Used for administration to the wound or tumor bed after tumor resection to eliminate residual tumor cells.

[0059] 3. Used for local delivery of chemotherapy drugs, inhibiting tumor growth and reducing systemic toxicity.

[0060] 4. Used to load photothermal agents to achieve photothermal therapy.

[0061] 5. Used for combined delivery of antitumor drugs and photothermal agents.

[0062] 6. Used for local sustained-release, controlled-release, or stimulus-responsive drug release in tumors.

[0063] 7. Used to inhibit postoperative tumor recurrence, local invasion and distant metastasis.

[0064] This invention first utilizes chemical coupling technology to link hydrophilic fucoidan with the hydrophobic antitumor drug podophyllotoxin, solving the problem of drug application in aqueous microneedle systems and forming a "prodrug" conjugate that combines hydrophilicity and pharmacodynamics. Second, carbon dots with photothermal effects are prepared as functional elements for physical therapy. Finally, using a soluble polymer (PVA / PVP) as a matrix, the various components are integrated into a unified microneedle array via a molding method. In application, the physical insertion of the microneedles overcomes the barrier problem of transdermal drug penetration. After dissolution, the conjugate releases the drug sustainably through hydrolyzable connecting arms, while the carbon dots generate a thermotherapy effect triggered by external near-infrared light. The synergistic effect of these two components achieves highly effective localized combined therapy.

[0065] This invention provides a photothermal combined drug-derived soluble glycosyl microneedles and its preparation method, which have the following beneficial effects:

[0066] 1. The hydroxyl group in the PPT molecule undergoes nucleophilic attack on the carbonyl group of succinic anhydride under the action of a catalyst, causing the succinic anhydride to open the ring and form carboxylated podophyllotoxin, thereby introducing a terminal carboxyl group into the PPT molecule; subsequently, the terminal carboxyl group is activated by a coupling agent and undergoes an esterification reaction with the hydroxyl group in the FU molecule, so that PPT is covalently grafted to FU through the succinic acid linker arm to obtain the FU-PPT coupling product.

[0067] 2. This invention significantly improves the water solubility and dispersibility of PPT in aqueous systems by covalently coupling PPT with FU, providing a basis for high drug loading and uniform distribution in microneedles. The prepared FU-PPT conjugate combines the hydrophilicity of FU and the antitumor activity of PPT, making it suitable for high-drug delivery in soluble microneedles.

[0068] 3. The PVA / PVP matrix used in this invention can dissolve and release drugs in tissue fluid while improving the needle's shapeability and skin penetration strength. By optimizing the ratio of PVA / PVP matrix to FU-PPT drug loading, the prepared composite microneedles are complete, have a regular array, and possess high mechanical strength sufficient to penetrate the skin.

[0069] 4. The carbon dots used in this invention possess excellent near-infrared photothermal conversion properties, enabling the microneedle delivery site to rapidly heat up under near-infrared laser irradiation, with the heating effect being concentration-dependent. The carbon dots impart a near-infrared photothermal response to the microneedles, allowing for synergistic killing with FU-PPT and enhancing the tumor-killing effect.

[0070] 5. Experimental data from this invention show that the drug-mixed microneedles still have high mechanical strength, the carbon dots have concentration-dependent heating capacity, and the combined treatment group in animal models has excellent tumor-suppressing effects. Attached Figure Description

[0071] Figure 1 NMR verification image of the FU-PPT conjugate prepared in Example 1 of this invention.

[0072] Figure 2 Transmission electron microscope (TEM) and high-resolution electron microscope (HRTEM) images of carbon dots prepared in Example 1 of this invention;

[0073] Figure 3 Infrared thermal images of carbon dots of different concentrations prepared in Example 1 of this invention under near-infrared irradiation;

[0074] Figure 4 Scanning electron microscope image of the soluble microneedle array prepared in Example 1 of this invention;

[0075] Figure 5 Mechanical performance curves and drug loading-maximum compressive force data of the microneedles prepared in Example 1 of this invention;

[0076] Figure 6 The glycosyl microneedles prepared in Example 1 of this invention were used to measure mouse tumor volume, tumor weight, and in vitro tumor images. Detailed Implementation

[0077] The present invention will be further described below with reference to specific embodiments and accompanying drawings:

[0078] Example 1

[0079] S1: Preparation of Fucoidan-Polypodophyllotoxin Covalent Conjugate

[0080] 0.1 mol of podophyllotoxin was dissolved in anhydrous dimethyl sulfoxide. Succinic anhydride, 4-dimethylaminopyridine (catalyst), and triethylamine (acid-binding agent) were added to induce succinylation of the hydroxyl group in podophyllotoxin, yielding a podophyllotoxin intermediate with a carboxyl linker. The amount of succinic anhydride added was 0.1 mol, the amount of catalyst was 3.39 mmol, and the amount of acid-binding agent was 0.12 mol.

[0081] The above intermediate was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and then reacted with an aqueous solution of fucoidan to couple FU and PPT through a hydrolyzable linker arm.

[0082] The specific amounts of activating agents were 373 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 224 mg of N-hydroxysuccinimide. The masses of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were equal to the mass of the podophyllotoxin intermediate with the carboxyl linker. The added masses of FU and carboxylated PPT were 3 g and 1 g, respectively.

[0083] After the reaction was completed, unreacted small molecules and solvents were removed using a dialysis bag, and then FU-PPT was obtained by lyophilization.

[0084] S2: Preparation of carbon dots with photothermal conversion properties

[0085] Citric acid was used as the carbon source, dimethyl sulfoxide as the sulfur source, and urea as the nitrogen source. The mixture was placed in a polytetrafluoroethylene-lined reactor and reacted at 200 °C for 8 h. After cooling to room temperature, a dark-colored reaction solution was obtained. The solution contained 15 ml of dimethyl sulfoxide, 3 g of urea, and 1 g of citric acid.

[0086] The reaction solution was centrifuged at high speed to remove large particles, then filtered through a 0.22 μm filter membrane, dialyzed, and lyophilized to obtain CDs.

[0087] S3: Preparation of needle precursor solution

[0088] Weigh 100 mg of polyvinyl alcohol (PVA) and 100 mg of polyvinylpyrrolidone (PVP), add 4 mL of deionized water, heat and stir until completely dissolved to obtain a blank needle matrix solution.

[0089] The fucose-podophyllotoxin covalent conjugate prepared in step S1, CDs, PVP and PVA prepared in step S2 are dissolved or dispersed in an aqueous system to form a needle precursor solution; the mass fraction of FU-PPT is 50% and the concentration of CDs is 300 μg / mL.

[0090] S4: Preparation of photothermal combined drug-derived soluble glycosyl microneedles:

[0091] Add the needle precursor solution prepared in step S3 to the surface of the square pyramidal microneedle negative mold, and degas under vacuum for 20 min and centrifuge at 4000 rpm for 15 min to ensure that the solution fully fills the needle cavity.

[0092] After scraping off excess liquid from the mold surface, dry at room temperature, 37 ℃ or under vacuum drying conditions for 12-48 h, and demold to obtain photothermal combined drug composite soluble glycosyl microneedles.

[0093] Example 2

[0094] S1: Preparation of Fucoidan-Polypodophyllotoxin Covalent Conjugate

[0095] 1 mol of podophyllotoxin was dissolved in anhydrous N,N-dimethylformamide. Succinic anhydride, N-methylimidazole catalyst, and triethylamine acid-binding agent were added to induce succinylation of the hydroxyl groups of podophyllotoxin, yielding a podophyllotoxin intermediate with a carboxyl linker. The amount of succinic anhydride added was 1.5 mol, the amount of catalyst was 0.101 mol, and the amount of acid-binding agent was 1 mol.

[0096] The above intermediate was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and then reacted with an aqueous solution of fucoidan to couple FU and PPT through a hydrolyzable linker arm.

[0097] The specific amounts of activating agents used were 373 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 224 mg of N-hydroxysuccinimide. The masses of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were equal to the mass of the podophyllotoxin intermediate with the carboxyl linker. The added masses of FU and carboxylated PPT were 2 g and 1 g, respectively.

[0098] After the reaction was completed, unreacted small molecules and solvents were removed using a dialysis bag, and then the FU-PPT conjugate was obtained by lyophilization.

[0099] S2: Preparation of carbon dots with photothermal conversion properties

[0100] Citric acid was used as the carbon source, dimethyl sulfoxide as the sulfur source, and urea as the nitrogen source. The mixture was placed in a polytetrafluoroethylene-lined reactor and reacted at 160 °C for 12 h. After cooling to room temperature, a dark-colored reaction solution was obtained. The reaction mixture contained 20 ml of dimethyl sulfoxide, 5 g of urea, and 1 g of citric acid.

[0101] The reaction solution was centrifuged at high speed to remove large particles, then filtered through a 0.22 μm filter membrane, dialyzed, and lyophilized to obtain CDs.

[0102] S3: Preparation of needle precursor solution

[0103] Weigh 80 mg of PVA and 250 mg of PVP, add 5 mL of deionized water, heat and stir until completely dissolved to obtain a blank needle matrix solution.

[0104] The FU-PPT prepared in step S1, CDs, PVP and PVA prepared in step S2 are dissolved or dispersed in an aqueous system to form a needle precursor solution; the mass fraction of FU-PPT is 65% and the concentration of carbon dot dispersion is 500 μg / mL.

[0105] S4: Preparation of photothermal combined drug-derived soluble glycosyl microneedles:

[0106] Add the needle precursor solution prepared in step S3 to the surface of the square pyramidal microneedle negative mold, and degas under vacuum for 5 min and centrifuge at 5000 rpm for 5 min to ensure that the solution fully fills the needle cavity.

[0107] Example 3

[0108] S1: Preparation of Fucoidan-Covalent Conjugates

[0109] 0.5 mol of podophyllotoxin was dissolved in anhydrous dichloromethane. Succinic anhydride, N-methylimidazole catalyst, and triethylamine acid-binding agent were added to induce succinylation of the hydroxyl group of podophyllotoxin, yielding a podophyllotoxin intermediate with a carboxyl linker. The amount of succinic anhydride added was 0.5 mol, the amount of catalyst added was 12.62 mmol, and the amount of acid-binding agent added was 0.75 mol.

[0110] The above intermediate was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and then reacted with fucoidan dissolved in phosphate buffered saline solution, so that FU and PPT were coupled through a hydrolyzable linker arm. The mass ratio of fucoidan to intermediate was 1:5; the molar ratio of activator to intermediate was 1:1.

[0111] The specific activation amounts were 373 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 224 mg of N-hydroxysuccinimide. The mass of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide was equal to the mass of the podophyllotoxin intermediate with the carboxyl linker arm. The added masses of FU and carboxylated PPT were 5 g and 1 g, respectively.

[0112] After the reaction was completed, unreacted small molecules and solvents were removed using a dialysis bag, and then the FU-PPT conjugate was obtained by lyophilization.

[0113] S2: Preparation of carbon dots with photothermal conversion properties

[0114] Citric acid was used as the carbon source, dimethyl sulfoxide as the sulfur source, and urea as the nitrogen source. The mixture was placed in a polytetrafluoroethylene-lined reactor and reacted at 220 °C for 4 h. After cooling to room temperature, a dark-colored reaction solution was obtained. The reaction mixture contained 10 ml of dimethyl sulfoxide, 1 g of urea, and 1 g of citric acid.

[0115] The reaction solution was centrifuged at high speed to remove large particles, then filtered through a 0.22 μm filter membrane, dialyzed, and lyophilized to obtain CDs.

[0116] S3: Preparation of needle precursor solution

[0117] Weigh 150 mg of PVA and 100 mg of PVP, add 1 mL of deionized water, heat and stir until completely dissolved to obtain a blank needle matrix solution.

[0118] The FU-PPT prepared in step S1, CDs, PVP and PVA prepared in step S2 are dissolved or dispersed in an aqueous system to form a needle precursor solution; the mass fraction of FU-PPT is 30% and the concentration of carbon dot dispersion is 50 μg / mL.

[0119] S4: Preparation of photothermal combined drug-derived soluble glycosyl microneedles:

[0120] Add the needle precursor solution prepared in step S3 to the surface of the square pyramidal microneedle negative mold, and degas under vacuum for 30 min and centrifuge at 1000 rpm for 30 min to ensure that the solution fully fills the needle cavity.

[0121] Application Example 1: Photothermal Combined Drug Microneedling Therapy

[0122] After establishing a mouse melanoma tumor model, the animals were divided into four groups: blank control, FU group, CDs + laser group, PPT group, FU-PPT group, FU-PPT + CDs without laser group, and FU-PPT + CDs + laser group. The treatment effects were as follows: Figure 6 As shown.

[0123] Each drug administration group can be treated with microneedle patch, local drug administration or equivalent dose method, and the laser group is irradiated with near-infrared after drug administration.

[0124] The tumor volume change curve over time showed that the tumors in the blank control group and the fucoidan group continued to grow, reaching approximately 1.2-1.4 cm by day 14. 3 ;

[0125] PPT, FU-PPT, CDs+Laser, and FU-PPT+CDs without laser can all inhibit tumor growth to varying degrees.

[0126] The FU-PPT+CDs+Laser group had the lowest tumor volume, reaching approximately 0-0.05 cm by day 14. 3 .

[0127] Further analysis of ex vivo tumor photographs and tumor weight statistics showed that the FU-PPT+CDs+Laser group had the lowest tumor weight, significantly lower than the blank control, FU, PPT, FU-PPT, and no-laser combination groups, indicating a synergistic therapeutic effect between the drug action of FU-PPT and the photothermal effect of carbon dots.

[0128] In this application example, FU-PPT provides local chemotherapy, while carbon dots provide thermal damage under near-infrared irradiation, together forming a photothermal-drug combined therapy.

[0129] Comparative Example 1: Preparation of FU-soluble microneedles

[0130] Weigh 100 mg of PVA and 200 mg of PVP in S3 of Example 1, add 4 mL of deionized water, heat and stir until completely dissolved to obtain a blank needle matrix solution.

[0131] FU was added to the PVA / PVP matrix solution to fully dissolve or uniformly disperse it, thus obtaining a fucoidan microneedle precursor solution. This precursor solution was added to a microneedle negative mold, and after vacuum filling, centrifugation, drying, and demolding, fucoidan-soluble microneedles were obtained.

[0132] The microneedles obtained in this comparative example did not contain PPT and CDs and were used to evaluate the effects of FU itself and the glycosyl matrix on microneedle shapeability and in vivo treatment outcomes.

[0133] Comparative Example 2: Preparation of CDs photothermal soluble microneedles

[0134] The carbon dots prepared in S2 of Example 1 were added to the PVA / PVP matrix solution prepared in S3 of Example 1 to make the final concentration of carbon dots 300 μg / mL. The solution was then stirred or sonicated to form a uniform carbon dot microneedle precursor solution.

[0135] The carbon dot microneedle precursor solution was added to the microneedle negative mold, and carbon dot photothermal soluble microneedles were obtained by vacuum filling, centrifugation, drying and demolding.

[0136] The comparative example, carbon dot photothermal soluble microneedles, exhibits localized temperature rise under near-infrared laser irradiation. It can be used as a comparative example of CDs+Laser formulation to evaluate the contribution of photothermal action alone to tumor suppression.

[0137] Comparative Example 3: Preparation of Free PPT-Soluble Microneedles

[0138] The free podophyllotoxin is first dissolved or dispersed in a small amount of dimethyl sulfoxide, ethanol, polyethylene glycol or other acceptable solubilizing system;

[0139] Then add the PVA / PVP matrix solution prepared in S3 of Example 1, and stir or ultrasonically disperse to form a free podophyllotoxin microneedle precursor solution.

[0140] The precursor solution was added to the microneedle negative mold, and after vacuuming, centrifugation filling, drying and demolding, free PPT soluble microneedles were obtained.

[0141] This comparative example of free PPT soluble microneedles was used to compare with FU-PPT microneedles to demonstrate the improvement of PPT dispersibility, formulation compatibility and local delivery by FU covalent coupling.

[0142] Comparative Example 4: Preparation of FU-PPT soluble glycosyl microneedles

[0143] Weigh 100 mg of PVA and 200 mg of PVP, add 4 mL of deionized water, heat and stir to dissolve, then add the FU-PPT covalent coupling compound prepared in Example 1 to make the FU-PPT content 50%.

[0144] After stirring and ultrasonic dispersion, the FU-PPT microneedle precursor solution was added to the microneedle negative mold, vacuum degassed and centrifuged to fill, and dried and demolded to obtain FU-PPT soluble glycosyl microneedles.

[0145] This FU-PPT soluble glycosyl group does not contain carbon dots and is used to evaluate the antitumor efficacy of FU-PPT drug conjugates when delivered alone.

[0146] Comparative Example 5: Preparation of FU-PPT+CDs Composite Soluble Glycosyl Microneedles

[0147] The CDs prepared in Example 1 were added to the FU-PPT microneedle precursor solution described in Comparative Example 4 to bring the final carbon dot concentration to 300 μg / mL.

[0148] The FU-PPT conjugate content was controlled at 50%. After stirring and ultrasonic treatment, the FU-PPT+CDs composite microneedle precursor solution was obtained.

[0149] The composite microneedle precursor solution was added to a square-pyramidal microneedle negative mold, and after vacuum degassing for 5-30 min, centrifugation at 1000-5000 rpm for 5-30 min, drying for 12-48 h, and demolding, FU-PPT+CDs composite soluble glycosyl microneedles simultaneously loaded with FU-PPT and carbon dots were obtained.

[0150] This comparative example, the FU-PPT+CDs composite soluble glycosyl laser-free group, was used to evaluate the therapeutic effect of drug conjugates co-loaded with carbon dots without photoirradiation.

[0151] Performance testing:

[0152] Firstly, regarding the screening of the mechanical properties and solution formulations of the composite soluble glycosyl microneedles prepared in the embodiments of the present invention.

[0153] The mechanical properties of the microneedles were evaluated using compression tests. In the orthogonal screening, PVA was fixed at 100 mg, PVP at 100-200 mg, and water at 1-5 mL.

[0154] The results showed that the water and PVA / PVP content significantly affected the microneedle compression curve and maximum force, with some formulations reaching approximately 130-185 N near a 1.0 mm displacement. The testing process is shown in Table 1. Table 1 indicates that the matrix exhibited optimal mechanical properties when the PVA content was 100 mg, the PVP content was 100 mg, and the water content was 4 mL.

[0155] Table 1. Formulation table for evaluating the mechanical properties of microneedles using compression testing.

[0156] Secondly, microneedle formulations with different FU-PPT conjugate content ratios.

[0157] Under matrix conditions of 100 mg PVA, 100 mg PVP, and 4 mL water, the amount of FU-PPT conjugate added was varied. (The drug content mentioned below refers to the amount of FU-PPT conjugate added.) Drug-mixed microneedles with drug contents of 30%, 35%, 40%, 45%, 50%, 55%, 60%, and 65% were prepared. The formulations are shown in Table 2 below.

[0158] As shown in Table 2, when the FU-PPT conjugate content is 30%-60%, the maximum force of the drug-mixed microneedles is approximately 255.80-323.08 N, which still has good mechanical strength; when the drug content is increased to 65%, the maximum force decreases significantly to approximately 62.13 N.

[0159] Therefore, the preferred drug content is 45%-60%, and more preferably about 50%, which can balance high drug loading and needle strength.

[0160] Table 2. Statistical Table of Microneedle Mechanics for Different Drug Contents

[0161] Further comparison can be made using the accompanying diagrams in the instruction manual:

[0162] Figure 1 NMR verification image of the FU-PPT conjugate prepared in Example 1 of this invention.

[0163] from Figure 1 As can be seen, the characteristic signal changes of fucoidan, podophyllotoxin, and the coupling product are observed. The NMR spectra show that the coupling product simultaneously possesses the characteristic signal of the aromatic region of podophyllotoxin (7.2-8.5 ppm) and the related signal of the fucoidan sugar ring (3-5.5 ppm), indicating the formation of the FU-PPT coupling product.

[0164] Figure 2 Transmission electron microscope (TEM) and high-resolution electron microscope (HRTEM) images of carbon dots prepared in Example 1 of this invention;

[0165] from Figure 2 It can be seen that the carbon dots prepared in Example 1 of the present invention are distributed in nanoscale particle-level dots; lattice fringes can be observed in the high-resolution image, indicating that it has the characteristics of carbon nanomaterials.

[0166] Figure 3 Infrared thermal images of carbon dots of different concentrations prepared in Example 1 of this invention under near-infrared irradiation;

[0167] Carbon dot dispersions were prepared at different concentrations of 50, 100, 200, 300, and 500 μg / mL, irradiated under a near-infrared laser, and the temperature changes were recorded.

[0168] Infrared thermal imaging data showed that after 10 minutes of irradiation, the blank control was approximately 30.3 °C, 50 μg / mL was approximately 34.3 °C, 100 μg / mL was approximately 37.1 °C, 200 μg / mL was approximately 42.9 °C, 300 μg / mL was approximately 47.7 °C, and 500 μg / mL was approximately 58.0 °C. Figure 3 It can be seen that the higher the carbon point concentration, the more obvious the temperature rise, indicating that carbon points have a significant concentration-dependent photothermal conversion capability.

[0169] Figure 4 Scanning electron microscope image of the soluble microneedle array prepared in Example 1 of this invention;

[0170] from Figure 4 It can be seen that the square pyramidal needles are complete and arranged in a regular manner.

[0171] Figure 5Mechanical performance curves and drug loading-maximum compressive force data of the microneedles prepared in Example 1 of this invention;

[0172] from Figure 5 It can be seen that the drug-mixed microneedles possess the mechanical strength required to penetrate the skin.

[0173] Figure 6 The glycosyl microneedles prepared in Example 1 of this invention were used to measure mouse tumor volume, tumor weight, and in vitro tumor images.

[0174] As can be seen from Figure 6, the photothermal combined drug composite soluble glycosyl microneedles FU-PPT+CDs+Laser group prepared in the embodiment of the present invention has the strongest antitumor effect.

[0175] The present invention has been described above by way of example with reference to the embodiments and accompanying drawings. Obviously, the implementation of the present invention is not limited to the above-described manner. Any improvements made by adopting the inventive concept and technical solution of the present invention, or the direct application of the inventive concept and technical solution of the present invention to other occasions without modification, are all within the protection scope of the present invention.

Claims

1. A method for preparing a photothermal combined drug-derived soluble glycosyl microneedles, characterized in that, Includes the following steps: S1: Preparation of fucoidan-podophyllotoxin covalent conjugate; S2: Preparation of carbon dots with photothermal conversion properties; S3: Preparation of needle precursor solution: The fucoidan-podophyllotoxin covalently coupled compound prepared in step S1, the carbon dots prepared in step S2, polyvinyl alcohol and polyvinylpyrrolidone are dissolved or dispersed in an aqueous system to form a needle precursor solution. S4: Preparation of photothermal combined drug-derived soluble glycosyl microneedles: The needle precursor liquid prepared in step S3 is added to the microneedle negative mold, and the mold is filled with vacuum and centrifugation. After drying and curing, the mold is removed to obtain the photothermal combined drug composite soluble glycosyl microneedles.

2. The method for preparing the photothermal combined drug-derived soluble glycosyl microneedles according to claim 1, characterized in that: The specific steps for preparing the fucoidan-podophyllotoxin covalent conjugate using S1 are as follows: Podophyllotoxin was dissolved in a solvent, and succinic anhydride, a catalyst and an acid-binding agent were added to induce succinylation of the hydroxyl groups of podophyllotoxin to obtain a podophyllotoxin intermediate with a carboxyl linkage arm. The podophyllotoxin intermediate with the carboxyl linker is activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and then reacted with an aqueous solution of fucoidan or a mixture of fucoidan and an organic solvent, so that fucoidan and podophyllotoxin are coupled through a hydrolyzable linker. After the reaction was completed, the mixture was dialyzed and then freeze-dried to obtain the fucopolysaccharide-podophyllotoxin covalent conjugate, denoted as FU-PPT.

3. The method for preparing the photothermal combined drug-derived soluble glycosyl microneedles according to claim 2, characterized in that: The process of preparing the needle precursor solution in step S3 is as follows: Weigh out polyvinyl alcohol and polyvinylpyrrolidone, add deionized water, heat and stir until completely dissolved to obtain a blank needle matrix solution; The mass ratio of polyvinyl alcohol to polyvinylpyrrolidone is (8-15):(10-25); The FU-PPT prepared in step S1 and the CDs prepared in step S2 are added to the blank needle matrix solution to obtain the needle precursor solution.

4. The method for preparing the photothermal combined drug-derived soluble glycosyl microneedles according to claim 3, characterized in that: The process of preparing photothermal combined drug-based soluble glycosyl microneedles in step S4 is as follows: Add the needle precursor liquid from step S3 to the surface of the needle mold, and degas it under vacuum for 5-30 min and centrifuge it at 1000-5000 rpm for 5-30 min to ensure that the needle precursor liquid fully fills the needle cavity. After scraping off excess liquid from the mold surface, the mold is dried and demolded to obtain photothermal combined drug soluble glycosyl microneedles.

5. The method for preparing the photothermal combined drug-derived soluble glycosyl microneedles according to claim 2, characterized in that: In step S1, the solvent is one of anhydrous dimethyl sulfoxide, N,N-dimethylformamide, or dichloromethane. The molar ratio of succinic anhydride to podophyllotoxin is (1-1.5):1; The mass ratio of fucoidan to carboxylated podophyllotoxin was (2-5):

1.

6. The method for preparing the photothermal combined drug-derived soluble glycosyl microneedles according to claim 2, characterized in that: The catalyst is one of 4-dimethylaminopyridine or N-methylimidazole; The acid-binding agent is triethylamine, and the molar ratio of the acid-binding agent to podophyllotoxin is (1-1.5):

1.

7. The method for preparing the photothermal combined drug-derived soluble glycosyl microneedles according to claim 4, characterized in that: In step S3, the mass fraction of FU-PPT in the needle precursor solution is 30%-65%, and the concentration of CDs in the needle precursor solution is 50-500 μg / mL.

8. A photothermal combined drug-soluble glycosyl microneedle prepared according to any one of claims 1-7, characterized in that, The soluble glycosyl microneedles comprise a soluble polymer needle body, a fucoidan-podophyllotoxin covalently coupled compound distributed within the needle body, and carbon dots distributed within the needle body.

9. The photothermal combined drug-soluble glycosyl microneedles according to claim 8, characterized in that, After the microneedle is inserted into the skin or superficial tumor tissue, the needle matrix absorbs tissue fluid and dissolves and releases fucopolysaccharide-podophyllotoxin covalently coupled with carbon dots, and under near-infrared laser irradiation, the local temperature rises to 42-58 ℃.

10. The use of the soluble glycosyl microneedles of claim 9 in medicaments or medical materials for the local treatment of skin tumors, melanoma, superficial solid tumors or postoperative residual tumors.

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