A multi-responsive degradable nanodrug delivery system and a preparation method and application thereof

Abstract

This invention relates to a multi-responsive biodegradable nanomedicine delivery system, its preparation method, and its applications, belonging to the field of biomedical materials and nanomedicine technology. The invention discloses a biodegradable hydrogen-bonded supramolecular polymer, a triple-responsive nanomedicine based on this polymer, its microneedle patch, its preparation method, and its uses. The nanomedicine is formed by self-assembly of a biodegradable amphiphilic polymer carrier containing disulfide bonds and multiple hydrogen bond recognition units, and a multifunctional prodrug linked by chemical bonds to a chemotherapeutic drug and a near-infrared photothermal molecule. The resulting nanomedicine exhibits triple-responsive properties of pH, redox (GSH), and photothermal properties, enabling precise and controllable drug release at tumor sites and depletion of intracellular GSH. Furthermore, by loading this nanomedicine into a matrix such as hyaluronic acid to form a microneedle patch, it can penetrate the stratum corneum for painless transdermal drug delivery.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and nanomedicine technology, and more specifically, relates to a multi-responsive degradable nanomedicine delivery system, its preparation method and application. Background Technology

[0002] The tumor microenvironment (TME) is characterized by weak acidity and high glutathione (GSH) concentrations, while traditional chemotherapy drugs suffer from significant systemic toxicity, poor targeting, and a high risk of developing drug resistance. Existing nanomedicine carriers often lack biodegradability, leading to long-term toxicity due to accumulation in the body; or they lack multiple stimulus-response mechanisms, hindering precise and controllable drug release. Furthermore, single treatment modalities (such as chemotherapy alone or photothermal therapy alone) are often insufficient to completely eliminate tumors, and recurrence is common after drug discontinuation.

[0003] While some responsive nanocarriers have been reported, most suffer from drawbacks such as complex synthesis, low drug loading capacity, single response mechanism, or lack of effective transdermal delivery methods. Particularly for superficial or dermal tumors like melanoma, traditional intravenous administration methods have low bioavailability. While microneedle patch technology can achieve transdermal drug delivery, there is a lack of intelligent microneedle systems that combine high-efficiency drug loading, multi-stimulus release, and synergistic therapeutic functions. Therefore, developing a nanomedicine system that is biodegradable, multi-stimulus responsive, and capable of efficient combined therapy, and suitable for transdermal delivery, has significant clinical implications. Summary of the Invention

[0004] This invention aims to provide a biodegradable hydrogen-bonded supramolecular polymer and multi-responsive nanomedicines and microneedle patches prepared based on this polymer. The invention utilizes RAFT polymerization to prepare a biodegradable polymer containing disulfide bonds, and loads multifunctional prodrugs through hydrogen bonding, achieving synergistic drug release in the tumor microenvironment through GSH consumption, acidic pH response, and photothermal triggering, significantly improving antitumor efficacy and reducing side effects. This solves the technical problems of complex nanocarrier synthesis, low drug loading capacity, and single response mechanism in existing technologies.

[0005] According to a first aspect of the present invention, a biodegradable amphiphilic hydrogen-bonded supramolecular polymer is provided, having the structural formula shown in Formula I:

[0006] Formula I Where m and n are both positive integers, and the values ​​of m and n range from 2 to 1000; R and Z represent the leaving group and stabilizing group of the reversible addition-fragmentation chain transfer reagent, respectively; X is a hydroxyl group, an amino group, a functional group connected by an ester bond or an amide bond, or X is an OM structure, where M is a metal ion; Y is an acrylate monomer or an acrylamide monomer, and Y contains a structure with a DAD-type hydrogen bond array.

[0007] Preferably, the structure having a DAD-type hydrogen bond array is melamine, 2,6-diaminopyridine, or 2,4-diaminopyrimidine; Preferably, the acrylate monomer is The acrylamide monomer is or , where y is , or .

[0008] Preferably, the leaving group R is: cyanoisopropyl, benzyl, tertiary carbon group, primary / secondary alkyl or ester-substituted alkyl; The stabilizing group Z is phenyl, thiophenyl, alkylthio, or alkoxy.

[0009] According to another aspect of the present invention, a method for preparing the aforementioned degradable amphiphilic hydrogen-bonded supramolecular polymer is provided, comprising the following steps: (1) A monomer containing disulfide bonds, monomer Y, a reversible addition-fragmentation chain transfer reagent and an initiator are dissolved in an organic solvent. The monomer Y is an acrylate monomer or an acrylamide monomer containing a DAD-type hydrogen bond array. After heating and polymerization purification, a biodegradable hydrogen-bonded supramolecular polymer is obtained. (2) The biodegradable hydrogen-bonded supramolecular polymer obtained in step (1) is mixed with an alkaline solution to deprotonate the polymer, thus obtaining a biodegradable amphiphilic hydrogen-bonded supramolecular polymer.

[0010] Preferably, the monomer containing disulfide bonds is such as lipoic acid or a lipoic acid derivative; the structure having a DAD-type hydrogen bond array is melamine, 2,6-diaminopyridine, or 2,4-diaminopyrimidine. Preferably, the acrylate monomer is The acrylamide monomer is or , where y is , or .

[0011] According to another aspect of the present invention, a multi-responsive nanomedicine is provided, using the aforementioned degradable amphiphilic hydrogen-bonded supramolecular polymer as a carrier, wherein the carrier loads a prodrug via hydrogen bonding, and the prodrug is formed by linking a near-infrared photothermal conversion molecule and a chemotherapeutic drug through chemical bonds. Preferably, the nanomedicine has a particle size of 10-500 nm and a zeta potential of -100 to 100 mV.

[0012] According to another aspect of the present invention, a method for preparing the aforementioned multi-responsive nanomedicine is provided, wherein the biodegradable amphiphilic hydrogen-bonded supramolecular polymer and the prodrug are mixed in a solvent and self-assembled to form nanoparticles by means of nanoprecipitation, emulsion solvent evaporation or self-assembly.

[0013] According to another aspect of the present invention, a microneedle patch for nanomedicine is provided, wherein the multi-responsive nanomedicine is loaded onto the tip portion of the microneedle using a biocompatible polymer material as a matrix; the matrix material is selected from hyaluronic acid, polyvinylpyrrolidone, polyvinyl alcohol, chitosan, or silk fibroin.

[0014] According to another aspect of the present invention, a method for preparing the microneedle patch of the nanomedicine is provided, wherein a solution of the multi-responsive nanomedicine is mixed with a matrix material solution, cast into a microneedle mold, and then degassed, dried, and demolded to obtain the microneedle patch of the nanomedicine.

[0015] The application of the aforementioned multi-responsive nanomedicine, or the microneedle patch of the aforementioned nanomedicine, in the preparation of antitumor drugs; Preferably, the tumor is melanoma, squamous cell carcinoma of the skin, basal cell carcinoma, malignant fibrous histiocytoma, or liposarcoma.

[0016] In summary, compared with the prior art, the above-described technical solutions conceived by this invention mainly possess the following technical advantages: (1) The biodegradable supramolecular polymer of the present invention is the first to realize the RAFT copolymerization of thioctic acid monomers with acrylic acid or acrylamide monomers, and has degradation characteristics under specific conditions.

[0017] (2) The biodegradable amphiphilic hydrogen-bonded supramolecular polymer in this invention has a dual mechanism of biodegradability and GSH consumption: the carrier backbone contains disulfide bonds, which undergo reductive breakage and degradation in the high GSH environment of tumors, while consuming intracellular GSH, disrupting the redox balance of tumor cells, and enhancing chemotherapy sensitivity.

[0018] (3) The nanomedicine in this invention has triple response characteristics of redox, pH and photothermal: on the one hand, after the nanomedicine enters the tumor cells, the polymer carrier can respond to the degradation and release of the drug in response to the high GSH environment; on the other hand, the prodrug carried by the triple hydrogen bond can respond to the low pH value of the tumor microenvironment and release the prodrug; furthermore, after being irradiated by 808 nm laser, the temperature rise will also cause the hydrogen bond to break in response, promoting the release of the prodrug; under the synergistic effect, the triple response characteristics of the nanomedicine in this invention realize the precise burst release of the drug at the tumor site, and the release rate can reach 92%, which is much higher than that of single / dual response systems.

[0019] (4) The nanomedicines in this invention have a highly efficient synergistic therapeutic effect: for example, when combined with the chemotherapy effect of FUA780 and the photothermal therapy (PTT) effect of IR780, the photothermal conversion efficiency reaches 26.9%, and it shows a significant synergistic anti-tumor effect (tumor inhibition rate of 93%) in in vitro and in vivo experiments, overcoming the limitations of single therapy.

[0020] (5) The microneedle patch in this invention has the advantage of transdermal drug delivery: the microneedle patch can penetrate the stratum corneum to achieve painless and efficient local drug delivery, avoiding the toxic side effects of systemic drug delivery, and is particularly suitable for the treatment of skin and superficial tumors.

[0021] (6) This invention is applicable to different thioctic acid comonomers and prodrugs (different near-infrared photothermal conversion molecules and chemotherapeutic drugs): the comonomer can be a monomer containing melamine, 2,6-diaminopyridine, 2,4-diaminopyrimidine, etc., with a DAD-type hydrogen bond array structure; the chemotherapeutic drug can be a drug or prodrug with an ADA-type hydrogen bond array, such as 5-fluorouracil and its derivatives, carmofluorine and its derivatives, deoxyfluorouridine and its derivatives; and the photothermal conversion molecule can be IR780, IR808, ICG, Cy series dyes, etc. The chemotherapeutic drug and the photothermal conversion molecule are linked by responsively breaking or hydrolyzable chemical bonds, and the comonomer and the chemotherapeutic drug are linked by hydrogen bonds.

[0022] (7) Excellent biocompatibility of the present invention: The carrier material and nanomedicine have good blood compatibility and cell compatibility, no obvious systemic toxicity, and high safety. Attached Figure Description

[0023] Figure 1 (a) Synthetic steps of RAFT reagent BnMAT; (b) 1 H NMR spectrum; (c) 13 C10 NMR spectrum, solvent is DMSO- d 6.

[0024] Figure 2 :(a)P(DAP- what-LA) 1 1H NMR spectrum, solvent is DMSO- d 6; (b) P(DAP- what -LANa) 1 HNMR spectrum, solvent is DMSO- d 6; (c) P(DAP- what -LANa) 1 1H NMR spectrum, solvent is DMSO- d 6.

[0025] Figure 3 (a) The synthetic steps of the prodrug FUA780; (b) 1 1H NMR spectrum, solvent is DMSO- d 6.

[0026] Figure 4 (a) Schematic diagram of hydrogen bonding between DAP and FUA780; (b) 1 H NMR titration showed that -N in FUA780 H - Proton displacement as DAP content increases.

[0027] Figure 5 (a) Nanomedicine FUA780@P(DAP- what (a) UV-vis spectrum of LANa; (b) TEM image of nanomedicine (inset) and DLS hydrated particle size distribution.

[0028] Figure 6 (a) Photothermal images of nanomedicine solutions under different laser power densities; (b) Illumination heating and natural cooling curves; (c) Photothermal conversion efficiency based on cooling curves. η =26.9%) Calculation graph; (d) In vitro release curves of FUA780 under different stimulation conditions (pH, GSH, laser).

[0029] Figure 7 (a) Vector P(DAP- what (b) toxicity of LANa) to 3T3 normal cells and B16-F10 tumor cells; (c) vector P(DAP- what (c) GSH depletion of B16-F10 tumor cells by LANa; (d) Toxicity of different nano-formulations to B16-F10 cells (± laser); (e) Live / Dead staining to visually demonstrate the cell-killing effects of different treatment groups.

[0030] Figure 8 (a) Equipped with FUA780@P(DAP- what(a) Flowchart of nanomedicine preparation using microneedles; (b) SEM image of microneedles; (c) CLSM 3D reconstruction showing the distribution of drug at the needle tip; (d) Mechanical strength (fracture force) analysis of microneedles before and after drug loading.

[0031] Figure 9 Statistical chart of isolated melanoma weight and tumor inhibition rate. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0033] In this invention, the RAFT reagent is a chain transfer agent used in reversible addition-fragmentation chain transfer polymerization (RAFT Polymerization).

[0034] This invention provides a RAFT reagent and its synthesis method. The RAFT reagent is 2-[benzylthio(thiocarbonyl)thio]-2-methylpropionic acid (BnMAT), with the following general structural formula: [Chemical structural description: S=C(S-CH2-Ph)(SC(CH3)2-COOH)]. The synthesis method includes: using benzyl thiol, carbon disulfide, and 2-bromo-2-methylpropionic acid as raw materials, a nucleophilic substitution reaction is carried out in an organic solvent in the presence of a basic catalyst. Preferably, the basic catalyst is at least one of anhydrous potassium phosphate, sodium hydroxide, or potassium carbonate; the organic solvent is at least one of tetrahydrofuran, dichloromethane, or acetonitrile. Preferably, the reaction temperature is 0-80°C, and the reaction time is 6-24 hours. More preferably, the synthesis steps include: dissolving benzyl thiol in an anhydrous organic solvent containing a basic catalyst, stirring at room temperature, adding carbon disulfide, continuing to stir, then adding 2-bromo-2-methylpropionic acid, stirring overnight; filtering after the reaction is complete, washing, drying, concentrating the organic phase, and purifying by recrystallization to obtain the RAFT reagent.

[0035] This invention provides a biodegradable amphiphilic polymer and its preparation method. The biodegradable amphiphilic polymer has a main chain containing disulfide bonds and side chains containing recognition units capable of forming multiple hydrogen bonds, as shown in Formula I:

[0036] Formula I Where m and n are both positive integers, and the range of m and n is 2-999; Wherein R and Z represent the leaving group and stabilizing group of the reversible addition-fragmentation chain transfer (RAFT) reagent, respectively; the leaving group R is: cyanoisopropyl, benzyl, tertiary carbon group, primary / secondary alkyl or ester-substituted alkyl, etc.; wherein X is a hydroxyl, amino, functional group connected by ester bond or amide bond, or X is an OM structure, wherein M is a metal ion; wherein Y is an acrylate or acrylamide monomer; and Y contains a structure with a DAD type hydrogen bond array.

[0037] Preferably, y represents the presence of melamine, 2,6-diaminopyridine, or 2,4-diaminopyrimidine.

[0038] Preferably, the polymer is prepared by RAFT polymerization of an acrylamide monomer (DAP) containing 2,6-diaminopyridine and a monomer containing a disulfide bond (such as a thioctic acid derivative). More preferably, the preparation method includes: dissolving the above-mentioned RAFT reagent, initiator, monomer DAP, and monomer containing a disulfide bond in an organic solvent, removing oxygen, heating to carry out a polymerization reaction, and precipitating and purifying after the reaction to obtain copolymer P(DAP- what -LA); further, P(DAP- what -LA) reacts with a base to convert the carboxyl group into a carboxylate, yielding the more water-soluble amphiphilic polymer P(DAP- what -LANa).

[0039] This invention provides a multifunctional prodrug and its synthesis method. The multifunctional prodrug is formed by linking a near-infrared photothermal conversion molecule and a chemotherapeutic drug through a responsively breaking or hydrolyzable chemical bond, with the following general structural formula: Drug-Linker-PTT. Wherein, Drug is a chemotherapeutic drug selected from at least one of drugs or prodrugs possessing an ADA-type hydrogen bond array, such as 5-fluorouracil and its derivatives, carmofluoride and its derivatives, and deoxyfluorouridine and its derivatives; PTT is a near-infrared photothermal conversion molecule selected from at least one of IR780, IR808, ICG, and Cy series dyes; and Linker is an amide bond, ester bond, disulfide bond, or hydrazone bond. Preferably, the multifunctional prodrug is FUA780, formed by linking 5-fluorouracil acetic acid (FUA) and an IR780 derivative (IR780-NH2) through an amide bond. Preferably, the synthesis method includes: activating the chemotherapeutic drug derivative, then performing a condensation reaction with a near-infrared dye derivative containing an amino group, and purifying to obtain the multifunctional prodrug.

[0040] This invention provides a method for preparing a triple-responsive nanomedicine. The nanomedicine comprises the aforementioned degradable amphiphilic polymer as a carrier, and the aforementioned multifunctional prodrug is loaded via hydrogen bonding. The nanomedicine exhibits pH-responsive, redox-responsive, and photothermal-responsive properties. Preferably, the nanomedicine is prepared by nanoprecipitation, emulsion solvent evaporation, or self-assembly. Preferably, the nanomedicine has a particle size of 50-300 nm, a zeta potential of -60 to -10 mV, and a drug loading (DLC) of 5%-30%. Preferably, the cumulative drug release rate of the nanomedicine under tumor microenvironment conditions (pH 5.5, 10 mM GSH, 808 nm laser irradiation) can reach over 80%.

[0041] This invention provides a microneedle patch loaded with the aforementioned nanomedicine. The microneedle patch uses a biocompatible polymer material as a matrix, with the nanomedicine loaded onto the tip of the microneedle. Preferably, the matrix material is selected from at least one of hyaluronic acid, polyvinylpyrrolidone, polyvinyl alcohol, chitosan, and silk fibroin. Preferably, the microneedle patch has a needle height of 200-1000 μm, a needle base width of 100-500 μm, and mechanical strength sufficient to penetrate the stratum corneum of the skin (breaking force > 0.05 N / needle). Preferably, the preparation method of the microneedle patch includes: mixing a nanomedicine solution with a matrix material solution, casting the mixture into a microneedle mold, degassing, drying, and demolding to obtain the drug-loaded microneedle patch.

[0042] This invention provides the application of the aforementioned nanomedicine or microneedle patch in the preparation of antitumor drugs. Preferably, the tumor is melanoma, squamous cell carcinoma of the skin, basal cell carcinoma, malignant fibrous histiocytoma, or liposarcoma. More preferably, the application is a combination of chemotherapy and photothermal therapy. The system provided by this invention significantly improves the antitumor efficacy and reduces systemic toxicity, and is particularly suitable for the combination of chemotherapy and photothermal therapy for solid skin tumors or subcutaneous tumors such as melanoma.

[0043] The following are specific examples.

[0044] Example 1: Synthesis method of the RAFT reagent BnMAT of the present invention.

[0045] Under argon protection, benzyl mercaptan (3.52 mL, 30 mmol, 1.5 eq) was dissolved in a suspension of anhydrous THF (30 mL) containing K3PO4 (7.42 g, 35 mmol, 1.75 eq). After stirring at room temperature for 20 minutes, CS2 (5.43 mL, 90 mmol, 4.5 eq) was added, and stirring was continued for another 20 minutes. Subsequently, 2-bromo-2-methylpropionic acid (3.34 g, 20 mmol, 1.0 eq) was added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was filtered, and the organic phase was washed successively with 1M HCl (3 times), saturated NaCl solution (2 times), and deionized water (3 times), dried, and concentrated. The crude product was recrystallized from n-hexane to give a pale yellow flocculent solid BnMAT (4.12 g, 14.4 mmol), with a yield of 72%. Its structure was determined by... 1 H NMR, 13 Characterization by C NMR and ESI-MS confirmed the results.

[0046] Figure 1 The synthesis steps and related spectra of this embodiment are shown in (a) the synthesis steps of the RAFT reagent BnMAT; (b) 1 HNMR spectrum; (c) 13 C10 NMR spectrum, solvent is DMSO- d 6.

[0047] BnMAT (C 12 H 14 O2S3), yield 72%. 1 H NMR (600 MHz, DMSO- d 6) δ 12.97 (s, 1H), 7.40 – 7.27 (m, 5H), 4.60 (s, 2H), 1.64 (s, 5H). 13 C NMR (150 MHz, DMSO- d 6) δ221.08, 173.57, 135.43, 129.73, 129.11, 128.18, 56.80, 40.83, 25.49. Example 2: The biodegradable amphiphilic polymer carrier P(DAP-) of the present invention what Synthesis and characterization of LANa.

[0048] The RAFT reagent BnMAT (10.3 mg, 0.036 mmol) obtained in Example 1, the initiator azobisisobutyronitrile (AIBN) (0.59 mg, 0.0036 mmol), the monomer DAP containing 2,6-diaminopyridine (251.6 mg, 1.08 mmol), and lipoic acid LA (890.3 mg, 4.32 mmol) were dissolved in tetrahydrofuran and placed in a reaction tube sealed with a rubber stopper. Argon gas was bubbled through the solution for deoxygenation at 0°C for 30 minutes, followed by heating in an oil bath at 65°C for 6 hours. After the reaction was complete, the reaction solution was precipitated twice in isopropyl ether to remove excess monomer. After vacuum drying, the target polymer P(DAP-) was obtained. what -LA). Through 1 Characterization by 1H NMR, SEC and FT-IR confirmed the successful synthesis of its structure, with a number-average molecular weight (NMR) of 1H NMR, SEC and FT-IR. M n The value is 13.4 kDa, and the dispersion is ( The value is 1.38.

[0049] P(DAP- what P(DAP-) (210 mg, 0.2 mmol) was dissolved in THF (5 mL), and an aqueous solution of NaOH (8 mg, 0.2 mmol) (5 mL) was slowly added dropwise while stirring for 24 hours. The solvent was removed by rotary evaporation, and then freeze-dried to obtain a white powdery solid P(DAP-) what -LANa). Through 1 H NMR and FT-IR (at 1545 cm⁻¹) -1 The appearance of a new characteristic peak of carboxylate group confirmed that the carboxyl group was successfully converted into sodium carboxylate.

[0050] Figure 2 For the relevant spectra in this embodiment, (a) P(DAP- what -LA) 1 1H NMR spectrum, solvent is DMSO- d 6; (b) P(DAP- what -LANa) 1 1H NMR spectrum, solvent is DMSO- d 6; (c) P(DAP- what -LANa) 1 1H NMR spectrum, solvent is DMSO- d 6.

[0051] The degree of polymerization of lipoic acid (LA) is 65, while the degree of polymerization of the monomer DAP containing 2,6-diaminopyridine is 16. LA is characterized by -S(C H 2)- and -S(C H)-and the characteristics of DAP-N H - and the pyridine ring H It can be proven that the P(DAP- what -LA) and P(DAP- what The successful synthesis of -LANa).

[0052] Example 3: Synthesis and characterization method of the multifunctional prodrug FUA780 of the present invention.

[0053] First, IR780 (0.667 g, 1.00 mmol), 4-aminothiophenol (0.25 g, 2.00 mmol) and triethylamine (7 μL) were reacted overnight in anhydrous DMF (15 mL), and then post-processed to give IR780-NH2 (0.657 g, 87% yield).

[0054] Then, at 0°C, FUA (206.9 mg, 1.10 mmol) and HOBT (178.4 mg, 1.32 mmol) were dissolved in anhydrous DMF (10 mL), and a DMF solution of EDCI (253 mg, 1.32 mmol) (7.5 mL) was slowly added dropwise, with stirring for 30 minutes. Subsequently, an anhydrous DMF solution of IR780-NH2 (453 mg, 0.6 mmol) (7 mL) was added dropwise to the reaction system, and the mixture was stirred overnight at room temperature. After the reaction was completed, the mixture was post-processed and purified by column chromatography (eluent: CH2Cl2 / CH3OH = 10 / 1). in / out The experiment yielded dark green crystals FUA780 (254.3 mg), with a yield of 45.8%. Its structure was determined by... 1 H NMR, 13 Confirmed by C NMR, ESI-MS and UV-vis spectroscopy (maximum absorption peak redshifted to 793 nm).

[0055] Figure 3 (b) The synthesis steps of the prodrug FUA780 obtained in this embodiment; 1 1H NMR spectrum, solvent is DMSO- d 6.

[0056] FUA780 (C 48 H 53 FIN5O3S), yield 45.8%, 1 H NMR (400 MHz, DMSO- d6) δ 11.89 (d, J = 5.1 Hz, 1H), 10.30 (s, 1H), 8.63 (d, J = 14.1 Hz, 2H), 8.05 (d, J = 6.7 Hz, 1H), 7.54 (dd, J = 8.1, 6.0 Hz, 4H), 7.46 – 7.35 (m, 4H), 7.28 – 7.18 (m,4H), 6.35 (d, J = 14.2 Hz, 2H), 4.44 (s, 2H), 4.16 (t, J = 7.2 Hz, 4H), 2.76(t, J = 6.2 Hz, 4H), 1.93 (t, J = 6.1 Hz, 2H), 1.75 (h, J = 7.4 Hz, 4H), 1.46(s, 12H), 0.95 (t, J = 7.3 Hz, 6H). Example 4: The nanomedicine FUA780@P(DAP- of the present invention what Methods for constructing and characterizing LANa.

[0057] Prepared using a nanoprecipitation method. The P(DAP-) obtained in Example 2 was used... what -LANa) (10 mg) was dissolved in a water / ethanol mixture (1 mL water + 1 mL ethanol). After complete dissolution, an ethanol solution (1 mL) of FUA780 (5 mg) obtained in Example 3 was added. Under vigorous stirring, 12 mL of water was added dropwise to the mixture using a syringe pump. After stirring for another hour, the solution was transferred to a dialysis bag (MWCO 3500 Da) and dialyzed against water for 12 hours to remove free prodrug and organic solvent, yielding the nanomedicine FUA780@P(DAP- what -LANa). The drug loading was characterized by UV-Vis and fluorescence spectroscopy. TEM (average particle size 148 nm), DLS (hydrodynamic diameter 164 nm), and Zeta potential (from -45.9 mV to -40.6 mV after loading) were also used for characterization. The drug loading capacity (DLC) was measured to be 17.3 ± 0.8%, and the drug loading efficiency (DLE) was 41.8 ± 2.3%.

[0058] Figure 4 Characterization of hydrogen bonding interactions of the nanomedicine prepared in this embodiment: (a) Schematic diagram of hydrogen bonding interactions between DAP and FUA780; (b) 1 H NMR titration showed that -N in FUA780 H - Proton displacement as DAP content increases.

[0059] A mixed solvent system (CDCl3 / DMSO-) was used. d 6, in / out ,19 / 1), optimized to ensure FUA780 and P(DAP- what The solubility of -LA) is used to trace the -N of FUA780. H -proton chemical shift ( Figure 4 (The blue spots in a). When the molar ratio of DAP increased from 1 to 5, the unique -N of FUA780 (final concentration 1.05 mM) H - The proton signal exhibited a low-field shift, decreasing from 10.96 ppm to 11.12 ppm. Figure 4 (b) This change is attributed to the formation of hydrogen bonds, which deshield -N by reducing the surrounding electron density. H -Protons, thereby shifting the resonance frequency to a lower field (higher ppm).

[0060] Figure 5 Particle size-related characterization of the nanomedicine prepared in this embodiment: (a) Nanomedicine FUA780@P(DAP- what (a) UV-vis spectrum of LANa; (b) TEM image of nanomedicine (inset) and DLS hydrated particle size distribution.

[0061] In the UV-vis spectrum, FUA780 exhibits a characteristic absorption peak at 793 nm. Figure 5 The absorption peaks of a) and P(DAP-co-LANa) at 296 nm are simultaneously observed at FUA780@P(DAP- what The presence of -LANa indicates the successful loading of the prodrug FUA780. TEM images and dynamic light scattering (DLS) results further demonstrate that the nanomedicine possesses a uniform size ( Figure 5 (b) in the middle.

[0062] Example 5: Experimental method for the photothermal properties and in vitro release behavior of the nanomedicine of the present invention.

[0063] Under 808 nm NIR laser irradiation, the nanomedicine solution obtained in Example 4 exhibited a concentration- and power-dependent photothermal effect at 2.0 W / cm². 2 Under irradiation, the temperature of the 36 μg / mL solution rose to a maximum of 52.3℃. After four laser-switched cycles, it exhibited excellent photothermal stability. Its photothermal conversion efficiency was calculated (…). η The figure was 26.9%.

[0064] Drug release under different stimulation conditions was studied using dialysis. Under physiological conditions (pH 7.4, 20 μM GSH), only 18% was released within 48 h. The release rate reached 69% under dual stimulation (pH 5.5 + 10 mM GSH), and the cumulative release rate reached as high as 92% after 48 h under triple stimulation combined with laser (pH 5.5, 10 mM GSH, L+).

[0065] Figure 6 For the photothermal performance and drug release characterization of this embodiment: (a) photothermal images of the nano-drug solution under different laser power densities; (b) illumination heating and natural cooling curves; (c) photothermal conversion efficiency based on the cooling curves. η =26.9%) Calculation graph; (d) In vitro release curves of FUA780 under different stimulation conditions (pH, GSH, laser).

[0066] The above results indicate that nanomedicines have excellent photothermal conversion efficiency and can release more drugs in response to three stimuli.

[0067] Example 6: Experimental method for the in vitro antitumor effect of the nanomedicine of the present invention.

[0068] CCK-8 experiments showed that the vector P(DAP-) obtained in Example 2 what -LANa) showed negligible cytotoxicity to normal 3T3 cells. Example 4 yielded drug-loaded nanoparticles FUA780@P(DAP- what -LANa) exhibited concentration-dependent cytotoxicity, and its efficacy was superior to the control group (vector non-degradable) FUA780@P(DAP- what -OEGA). At the highest tested concentration (4.8 μg / mL FUA780), cell viability decreased to 51%; after combined with 808 nm laser irradiation, the viability further decreased sharply to 15%, showing a significant synergistic therapeutic effect. Live / Dead staining visually confirmed that the degradable nanocarrier combined with laser irradiation had the strongest tumor cell killing effect.

[0069] Figure 7 For the characterization of the nanomedicine for cell compatibility in this embodiment: (a) Carrier P(DAP- what (b) toxicity of LANa) to 3T3 normal cells and B16-F10 tumor cells; (c) vector P(DAP- what (c) GSH depletion of B16-F10 tumor cells by LANa; (d) Toxicity of different nano-formulations to B16-F10 cells (± laser); (e) Live / Dead staining to visually demonstrate the cell-killing effects of different treatment groups.

[0070] The above results indicate that the nanocarrier exhibits low toxicity to normal cells but some toxicity to B16-F10 tumor cells, and it can deplete GSH within tumor cells. Compared with non-degradable nanomedicines, the FUA780@P(DAP- what -LANa) exhibited higher cytotoxicity.

[0071] Example 7: Preparation and characterization method of microneedle patch loaded with nanomedicine according to the present invention.

[0072] Using hyaluronic acid / polyvinylpyrrolidone (2 / 1) as the matrix material, the reconstituted FUA780@P(DAP-) obtained in Example 4 was... what -LANa) nanomedicine lyophilized powder and matrix solution (250 mg / mL) were mixed at a volume ratio of 1:1. This mixture was poured into a PDMS negative mold, vacuum-sealed to remove air bubbles, and excess solution in the backing layer was scraped off. Then, pure matrix solution was backfilled to form the base layer. After drying and demolding, a microneedle patch loaded with the nanomedicine was obtained. The microneedles exhibited a complete tetrahedral pyramidal structure with a regular array (10 × 10) and uniform needle size (base width 310 μm, height 750 μm). CLSM 3D imaging confirmed that the drug fluorescence signal was concentrated at the needle tip. Mechanical testing showed that the breaking force of the drug-loaded microneedles was still much higher than the minimum force required for skin puncture (0.058 N / needle).

[0073] Figure 8 Characterization of the microneedle patch in this embodiment: (a) Equipped with FUA780@P(DAP- what (a) Flowchart of nanomedicine preparation using microneedles; (b) SEM image of microneedles; (c) CLSM 3D reconstruction showing the distribution of drug at the needle tip; (d) Mechanical strength (fracture force) analysis of microneedles before and after drug loading.

[0074] The above results indicate that the load FUA780@P(DAP- what The microneedles for LANa nanomedicine have been successfully prepared and have good mechanical strength, enabling them to penetrate the stratum corneum and deliver nanomedicine subcutaneously.

[0075] Example 8: Evaluation method of in vivo anti-melanoma efficacy of microneedle patch.

[0076] Treatment experiments were conducted on a B16-F10 tumor-bearing C57BL / 6 mouse model. Infrared thermography showed that the temperature at the tumor site in the drug-loaded microneedle patch treatment group prepared in Example 7 could rapidly rise to approximately 50°C under laser irradiation. Tumor growth curves showed that FUA780@P(DAP- whatThe LANa microneedling combined with laser irradiation group (L+) showed the best effect, with a tumor inhibition rate of 93%, significantly superior to other groups. The body weight of mice in all groups remained stable during treatment. H&E, TUNEL, and Ki67 staining analyses of tumor tissues consistently indicated that FUA780@P(DAP- what The LANa) MNs L+ treatment group showed the highest proportion of tumor cell necrosis / apoptosis and the lowest proportion of proliferating cells. No significant toxicity was found in the pathological analysis of major organ tissues and hematological parameters, confirming the good biocompatibility of this treatment system.

[0077] Figure 9 This is a statistical chart showing the effect of microneedle patches on the weight and tumor inhibition rate of isolated melanomas in this embodiment.

[0078] Tumor weight and tumor suppression rate results indicate that FUA780@P(DAP- what The LANa microneedle combined with laser irradiation group (L+) showed the best effect, with a tumor inhibition rate of 93%, which was significantly better than other groups, further proving the beneficial effects of the present invention.

[0079] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A biodegradable amphiphilic hydrogen-bonded supramolecular polymer, characterized in that, The structural formula is shown in Formula I: Formula I Where m and n are both positive integers, and the values ​​of m and n range from 2 to 1000; R and Z represent the leaving group and stabilizing group of the reversible addition-fragmentation chain transfer reagent, respectively; X is a hydroxyl group, an amino group, a functional group connected by an ester bond or an amide bond, or X is an OM structure, where M is a metal ion; Y is an acrylate monomer or an acrylamide monomer, and Y contains a structure with a DAD-type hydrogen bond array.

2. The biodegradable amphiphilic hydrogen-bonded supramolecular polymer as described in claim 1, characterized in that, The structure with the DAD-type hydrogen bond array is melamine, 2,6-diaminopyridine, or 2,4-diaminopyrimidine; Preferably, the acrylate monomer is The acrylamide monomer is or , where y is , or .

3. The biodegradable amphiphilic hydrogen-bonded supramolecular polymer as described in claim 1 or 2, characterized in that, The leaving group R is cyanoisopropyl, benzyl, tertiary carbon group, primary alkyl, secondary alkyl or ester-substituted alkyl; The stabilizing group Z is phenyl, thiophenyl, alkylthio, or alkoxy.

4. The method for preparing the biodegradable amphiphilic hydrogen-bonded supramolecular polymer according to any one of claims 1-3, characterized in that, Includes the following steps: (1) A monomer containing disulfide bonds, monomer Y, a reversible addition-fragmentation chain transfer reagent and an initiator are dissolved in an organic solvent. The monomer Y is an acrylate monomer or an acrylamide monomer containing a DAD-type hydrogen bond array. After heating and polymerization purification, a biodegradable hydrogen-bonded supramolecular polymer is obtained. (2) The biodegradable hydrogen-bonded supramolecular polymer obtained in step (1) is mixed with an alkaline solution to deprotonate the polymer, thus obtaining a biodegradable amphiphilic hydrogen-bonded supramolecular polymer.

5. The method for preparing the biodegradable amphiphilic hydrogen-bonded supramolecular polymer as described in claim 4, characterized in that, The monomer containing disulfide bonds is such as lipoic acid or lipoic acid derivatives; the structure with a DAD-type hydrogen bond array is melamine, 2,6-diaminopyridine or 2,4-diaminopyrimidine. Preferably, the acrylate monomer is The acrylamide monomer is or , where y is , or .

6. A multi-responsive nanomedicine, characterized in that, Using the biodegradable amphiphilic hydrogen-bonded supramolecular polymer as described in any one of claims 1-3 as a carrier, the carrier loads a prodrug through hydrogen bonding, and the prodrug is formed by linking a near-infrared photothermal conversion molecule and a chemotherapeutic drug through chemical bonds; Preferably, the nanomedicine has a particle size of 10-500 nm and a zeta potential of -100 to 100 mV.

7. The method for preparing multi-responsive nanomedicine as described in claim 6, characterized in that, The biodegradable amphiphilic hydrogen-bonded supramolecular polymer and the prodrug are mixed in a solvent using a nanoprecipitation method, an emulsion solvent evaporation method, or a self-assembly method, and then self-assembled to form nanoparticles.

8. A microneedle patch for nanomedicine, characterized in that, Using a biocompatible polymer material as the matrix material, the multi-responsive nanomedicine described in claim 6 is loaded onto the tip of a microneedle; the matrix material is selected from hyaluronic acid, polyvinylpyrrolidone, polyvinyl alcohol, chitosan, or silk fibroin.

9. The method for preparing the microneedle patch of nanomedicine as described in claim 8, characterized in that, The solution of the multi-responsive nanomedicine described in claim 6 is mixed with the matrix material solution, cast into a microneedle mold, and then degassed, dried, and demolded to obtain a microneedle patch of the nanomedicine.

10. The application of the multi-responsive nanomedicine as described in claim 6, or the microneedle patch of the nanomedicine as described in claim 8, in the preparation of antitumor drugs; Preferably, the tumor is melanoma, squamous cell carcinoma of the skin, basal cell carcinoma, malignant fibrous histiocytoma, or liposarcoma.

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