Temperature-sensitive hydrogel delivery drug based on ALDH1 inhibitor and preparation method and application thereof

Abstract

The invention relates to the technical field of preparation of anti-cancer drugs, in particular to a temperature-sensitive hydrogel delivery drug based on an ALDH1 inhibitor and a preparation method and application of the temperature-sensitive hydrogel delivery drug. The thermo-sensitive hydrogel delivery drug is formed by compounding an ALDH1 inhibitor entrapment core, a tumor targeting modification layer and a thermo-sensitive hydrogel matrix system, and through triple technology integration, the defects that an existing delivery system is insufficient in targeting, uncontrollable in drug release and the like are overcome. The delivered drug can significantly inhibit the activity of tumor stem cells, reduce the toxicity of organs, and provide a new scheme for precise treatment of bladder cancer.

Description

Technical Field

[0001] This invention relates to the field of anticancer drug preparation technology, and in particular to a thermosensitive hydrogel delivery method for drugs based on ALDH1 inhibitors, its preparation method, and its application. Background Technology

[0002] Bladder cancer is one of the most common malignant tumors of the urinary system. Its high recurrence rate, metastasis rate, and treatment resistance are closely related to the maintenance of stemness in cancer stem cells (CSCs). Aldehyde dehydrogenase type 1 antibody (ALDH1), as a key biomarker of CSCs, promotes stemness maintenance through metabolic regulation and signaling pathway activation, and is one of the important targets for targeted therapy of bladder cancer. However, ALDH1 inhibitors (Entinostat, molecular formula C...)... 21 H 20 The clinical application of N4O3 presents numerous challenges, such as poor selectivity, short in vivo half-life (plasma half-life < 2 hours), and high systemic toxicity (liver and kidney damage), resulting in a narrow therapeutic window and difficulty in achieving precise and efficient anti-tumor effects. As one of the core technologies for targeted therapy of bladder cancer, constructing delivery drugs that combine high targeting, controllable drug release capabilities, and biocompatibility is crucial for overcoming the tumor microenvironment barrier and achieving precise delivery of ALDH1 inhibitors.

[0003] Existing ALDH1 inhibitor delivery technologies are mainly divided into two categories: direct injection of free drug and traditional nanocarrier delivery. The first method typically involves intravenous injection of the inhibitor, which is rapidly metabolized by hepatic enzymes. Due to the lack of tumor targeting, high doses are required to maintain effective concentrations, leading to serious side effects such as bone marrow suppression and gastrointestinal reactions. On the other hand, traditional nanocarrier delivery mainly includes liposome / polylactic acid-glycolic acid copolymer nanoparticle delivery systems and thermosensitive hydrogel single-component delivery systems. Liposome / polylactic acid-glycolic acid copolymer nanoparticle delivery systems can prolong drug circulation time, but lack an active targeting mechanism, resulting in low tumor accumulation efficiency and uncontrollable release rates, easily triggering sudden releases that lead to a surge in toxicity. Although thermosensitive hydrogel single-component delivery systems (such as NIPAA m-polymers) can achieve local retention, they lack specific recognition capabilities for ALDH1-positive cancer cells (CSCs) and have low drug loading rates, making it difficult to meet clinical treatment needs. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a thermosensitive hydrogel for drug delivery based on an ALDH1 inhibitor and its application, aiming to solve the problems of insufficient targeting, high systemic toxicity, uncontrollable drug release kinetics, limited biocompatibility, complex preparation process, and high cost in the prior art.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a method for preparing a drug delivery system based on an ALDH1 inhibitor using a thermosensitive hydrogel, characterized by comprising the following steps:

[0007] S1. Preparation of ALDH1 inhibitor-encapsulated core: ALDH1 inhibitor and PLGA-PEG-NH2 were dissolved in an organic solvent to obtain an organic phase solution; polyvinyl alcohol was dissolved in water and filtered to obtain an aqueous phase solution; the organic phase solution was added to the aqueous phase solution, and after ultrasonic emulsification reaction, product I was obtained; product I was heated in a water bath to evaporate the organic solvent, and the evaporated product I was centrifuged, washed and concentrated to obtain the ALDH1 inhibitor-encapsulated core;

[0008] S2. Preparation of tumor-targeting modified layer: Targeting ligand 1 was dissolved in MES buffer, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added. After oscillation activation reaction, product II was obtained. The ALDH1 inhibitor core was added to product II, and after the final volume was calibrated with Tris-HCl buffer, a oscillation coupling reaction was performed. After filtration, centrifugation, washing and concentration, the tumor-targeting modified layer was obtained.

[0009] Alternatively, prepare a tumor-targeting modified layer: dissolve the targeting ligand 2 and NHS-biotin in PBS buffer, and obtain the tumor-targeting modified layer after a shaking coupling reaction;

[0010] S3. Prepare thermosensitive hydrogels for drug delivery using tumor-targeting modification layers.

[0011] Preferably, the mass ratio of the ALDH1 inhibitor to PLGA-PEG-NH2 is (1-1.5):1.

[0012] Preferably, the organic solvent is chloroform.

[0013] Preferably, the volume ratio of the organic phase solution to the aqueous phase solution is (1-1.5):10.

[0014] Preferably, the emulsification reaction is carried out at a temperature of 20–30°C for 5–10 minutes.

[0015] Preferably, the aqueous solution is a polyvinyl alcohol solution with a mass concentration of 1-2%.

[0016] Preferably, the targeting ligand 1 is folic acid.

[0017] Preferably, the targeting ligand 2 is an anti-ALDH1 monoclonal antibody or an RGD peptide.

[0018] More preferably, the amino acid sequence of the RGD peptide is as shown in SEQ ID NO.1:

[0019] RGD: CRGDCFC (SEQ ID NO.1).

[0020] Preferably, the mass-to-volume ratio of the targeting ligand 1, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and MES buffer is (10-15) mg:(60-70) mg:(100-110) mg:(10-15) mL.

[0021] More preferably, the activation reaction is carried out at a temperature of 35–40°C for a time of 25–35 min.

[0022] More preferably, the coupling reaction is carried out at a temperature of 35–40°C for a time of 10–15 hours.

[0023] Preferably, the mass-to-volume ratio of the targeting ligand 2, NHS-biotin, and PBS buffer is (100-110) mg: (50-60) mg: (10-15) mL.

[0024] More preferably, the coupling reaction is carried out at a temperature of 3–5°C for a time of 10–15 h.

[0025] Preferably, the preparation of the thermosensitive hydrogel for drug delivery includes the following steps: dissolving N-isopropylacrylamide, cysteine ​​hydrochloride, potassium persulfate, and N,N′-methylenebisacrylamide in water to obtain product III; purging product III with nitrogen to remove oxygen, adding tetramethylethylenediamine, and rapidly mixing to obtain a linear prepolymer solution; adding the tumor-targeting modification layer to the linear prepolymer solution, vortexing and allowing it to stand to obtain the thermosensitive hydrogel for drug delivery.

[0026] Alternatively, N-vinylcaprolactam is dissolved in water, deoxygenated with nitrogen, and then potassium persulfate and N,N′-methylenebisacrylamide are added to obtain product IV; product IV undergoes a polymerization phase transition to obtain a thermosensitive hydrogel for drug delivery.

[0027] More preferably, the mass-to-volume ratio of N-isopropylacrylamide, cysteine ​​hydrochloride, potassium persulfate, N,N′-methylenebisacrylamide, water and tetramethylethylenediamine is (1-2) g:(0.05-0.15) g:(0.25-0.35) g:(20-25) mL:(50-60) μL.

[0028] More preferably, the mass-to-volume ratio of N-vinylcaprolactam, potassium persulfate, N,N′-methylenebisacrylamide to water is (0.8–1.2) g : (0.02–0.05) g : (0.02–0.05) g : (10–15) mL.

[0029] More preferably, the polymerization reaction is carried out at a temperature of 35–40°C for 10–15 hours.

[0030] Secondly, the present invention also provides a method for preparing a thermosensitive hydrogel delivery drug of a water-soluble ALDH1 inhibitor, comprising the following steps: encapsulating a water-soluble ALDH1 inhibitor using a nanoprecipitation method to obtain a thermosensitive hydrogel delivery drug of a water-soluble ALDH1 inhibitor.

[0031] Preferably, the nanoprecipitation method includes the following steps: dissolving an ALDH1 inhibitor in water to obtain an ALDH1 inhibitor aqueous solution; dissolving a polylactic acid-glycolic acid copolymer in an organic solvent to obtain an organic phase solution; adding the ALDH1 inhibitor aqueous solution to the organic phase solution, and obtaining product V after ultrasonic treatment; adding product V dropwise to a polyvinyl alcohol solution, stirring to evaporate the organic solvent, and purifying the evaporated product V by filtration to obtain a thermosensitive hydrogel for drug delivery.

[0032] More preferably, the organic solvent is acetone.

[0033] More preferably, in the process of preparing the product V, the mass-volume ratio of ALDH1 inhibitor, polylactic acid-glycolic acid copolymer, acetone and water is (100-120) mg: (200-250) mg: (10-15) mL: (10-15) mL.

[0034] More preferably, the polyvinyl alcohol solution has a mass concentration of 5-6% and is used in an amount of 50-60 mL.

[0035] Thirdly, the present invention also provides the application of the thermosensitive hydrogel for drug delivery prepared by the above preparation method in the preparation of antitumor drugs.

[0036] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0037] This invention modifies a thermosensitive hydrogel with folic acid (FA) and utilizes the FA receptor, which is highly expressed in tumor cells, to mediate endocytosis, thereby increasing drug accumulation in ALDH1-positive cancer cells (CSCs) by more than three times, reducing exposure in normal tissues, and improving the tumor-targeting efficiency of ALDH1 inhibitors. By combining a dual controlled-release mechanism of polylactic-co-glycolic acid (PLGA) nanoparticle encapsulation with a thermosensitive hydrogel network, the ALDH1 inhibitor is continuously released over 27 days, maintaining a local tumor drug concentration >10 μM, avoiding drug resistance caused by fluctuations in blood drug concentration, and achieving controlled sustained release and long-lasting effects. A one-step emulsification-polymerization process is used to construct the thermosensitive hydrogel complex, achieving a stable encapsulation rate of over 85%, shortening the production cycle to 12 hours, and reducing costs by 40% compared to traditional liposome preparation processes. Utilizing a biocompatible matrix of biodegradable PLGA and thermosensitive hydrogel, the degradation products are lactic acid and glycolic acid, which are excreted from the body through metabolism. Animal experiments have confirmed that the cardiotoxicity, hepatic toxicity, and nephrotoxicity scores are <1 (0-4 grade scoring system), thus optimizing biocompatibility and safety. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 SEM microstructure of the thermosensitive hydrogel for drug delivery (Gel@Entinostat@PLGA@FA composite hydrogel).

[0040] Figure 2 The results of thermosensitive hydrogel drug delivery tests (Gel@Entinostat@PLGA@FA composite hydrogel) are shown in the figure. Figure A shows the effect of the composite hydrogel in liquid state at room temperature (25℃), Figure B shows the effect of the composite hydrogel in viscous solid state at 37℃, and Figure C shows the drug release curve of the composite hydrogel.

[0041] Figure 3 These are confocal micrographs of the two composite materials.

[0042] Figure 4 Images of tumors in different treatment groups of a nude mouse model.

[0043] Figure 5 This is a statistical graph showing the tumor weight of different treatment groups in a nude mouse model.

[0044] Figure 6 This is a statistical graph showing the tumor volume of different treatment groups in a nude mouse model.

[0045] Figure 7 Paraffin-embedded sections of heart, liver, lung, and kidney tissues from different treatment groups in a nude mouse model. Detailed Implementation

[0046] The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0047] It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of embodiments. Furthermore, in the description of this application, the term "comprising" means "including but not limited to". Various embodiments of the present invention may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single digits within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any referenced number (fraction or integer) within the indicated range.

[0048] This invention provides a method for preparing a drug delivery system using a thermosensitive hydrogel based on an ALDH1 inhibitor, characterized by the following steps:

[0049] S1. Preparation of ALDH1 inhibitor-encapsulated core: ALDH1 inhibitor and PLGA-PEG-NH2 were dissolved in an organic solvent to obtain an organic phase solution; polyvinyl alcohol was dissolved in water and filtered to obtain an aqueous phase solution; the organic phase solution was added to the aqueous phase solution, and after ultrasonic emulsification reaction, product I was obtained; product I was heated in a water bath to evaporate the organic solvent, and the evaporated product I was centrifuged, washed and concentrated to obtain the ALDH1 inhibitor-encapsulated core;

[0050] S2. Preparation of tumor-targeting modified layer: Targeting ligand 1 was dissolved in MES buffer, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added. After oscillation activation reaction, product II was obtained. The ALDH1 inhibitor core was added to product II, and after the final volume was calibrated with Tris-HCl buffer, a oscillation coupling reaction was performed. After filtration, centrifugation, washing and concentration, the tumor-targeting modified layer was obtained.

[0051] Alternatively, prepare a tumor-targeting modified layer: dissolve the targeting ligand 2 and NHS-biotin in PBS buffer, and obtain the tumor-targeting modified layer after a shaking coupling reaction;

[0052] S3. Prepare thermosensitive hydrogels for drug delivery using tumor-targeting modification layers.

[0053] Specifically, the mass ratio of ALDH1 inhibitor to PLGA-PEG-NH2 is (1-1.5):1.

[0054] Specifically, the organic solvent is chloroform.

[0055] Specifically, the volume ratio of the organic phase solution to the aqueous phase solution is (1-1.5):10.

[0056] Specifically, the emulsification reaction is carried out at a temperature of 20–30°C for 5–10 minutes.

[0057] Specifically, the aqueous solution is a polyvinyl alcohol solution with a mass concentration of 1-2%.

[0058] Specifically, target ligand 1 is folic acid;

[0059] Specifically, the target ligand 2 is an anti-ALDH1 monoclonal antibody or an RGD peptide.

[0060] More specifically, the amino acid sequence of the RGD peptide is shown in SEQ ID NO.1.

[0061] Specifically, the mass-to-volume ratio of the targeting ligand 1, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and MES buffer is (10-15) mg: (60-70) mg: (100-110) mg: (10-15) mL.

[0062] More specifically, the activation reaction is carried out at a temperature of 35–40°C for 25–35 minutes.

[0063] More specifically, the coupling reaction is carried out at a temperature of 35–40 °C for 10–15 h.

[0064] Specifically, the mass-to-volume ratio of targeting ligand 2, NHS-biotin, and PBS buffer is (100–110) mg: (50–60) mg: (10–15) mL.

[0065] More specifically, the coupling reaction is carried out at a temperature of 3–5 °C for 10–15 h.

[0066] Specifically, the preparation of a thermosensitive hydrogel for drug delivery includes the following steps: dissolving N-isopropylacrylamide, cysteine ​​hydrochloride, potassium persulfate, and N,N′-methylenebisacrylamide in water to obtain product III; purging product III with nitrogen to remove oxygen, adding tetramethylethylenediamine, and rapidly mixing to obtain a linear prepolymer solution; adding the tumor-targeting modification layer to the linear prepolymer solution, vortexing and allowing it to stand to obtain the thermosensitive hydrogel for drug delivery.

[0067] Alternatively, N-vinylcaprolactam is dissolved in water, deoxygenated with nitrogen, and then potassium persulfate and N,N′-methylenebisacrylamide are added to obtain product IV; product IV undergoes a polymerization phase transition to obtain a thermosensitive hydrogel for drug delivery.

[0068] More specifically, the mass-to-volume ratio of N-isopropylacrylamide, cysteine ​​hydrochloride, potassium persulfate, N,N′-methylenebisacrylamide, water, and tetramethylethylenediamine is (1–2) g : (0.05–0.15) g : (0.25–0.35) g : (20–25) mL : (50–60) μL.

[0069] More specifically, the mass-to-volume ratio of N-vinylcaprolactam, potassium persulfate, N,N′-methylenebisacrylamide to water is (0.8–1.2) g : (0.02–0.05) g : (0.02–0.05) g : (10–15) mL.

[0070] More specifically, the polymerization reaction is carried out at a temperature of 35–40°C for 10–15 hours.

[0071] This invention also provides a method for preparing a thermosensitive hydrogel delivery drug of a water-soluble ALDH1 inhibitor, comprising the following steps: encapsulating a water-soluble ALDH1 inhibitor using a nanoprecipitation method to obtain a thermosensitive hydrogel delivery drug of a water-soluble ALDH1 inhibitor.

[0072] Specifically, the nanoprecipitation method includes the following steps: dissolving the ALDH1 inhibitor in water to obtain an aqueous solution of the ALDH1 inhibitor; dissolving the polylactic acid-glycolic acid copolymer in an organic solvent to obtain an organic phase solution; adding the aqueous solution of the ALDH1 inhibitor to the organic phase solution, and obtaining product V after ultrasonic treatment; adding product V dropwise to a polyvinyl alcohol solution, stirring to evaporate the organic solvent, and purifying the evaporated product V by filtration to obtain a thermosensitive hydrogel for drug delivery.

[0073] More specifically, the organic solvent is acetone.

[0074] More specifically, in the process of preparing the above product V, the mass-volume ratio of ALDH1 inhibitor, polylactic acid-glycolic acid copolymer, acetone and water is (100-120) mg: (200-250) mg: (10-15) mL: (10-15) mL.

[0075] More specifically, the polyvinyl alcohol solution has a mass concentration of 5-6% and a usage volume of 50-60 mL.

[0076] This invention also provides an application of the thermosensitive hydrogel for drug delivery prepared by the above method in the preparation of antitumor drugs.

[0077] The following specific embodiments further illustrate a thermosensitive hydrogel delivery method for drugs based on an ALDH1 inhibitor, its preparation method, and its application. This section further illustrates the content of the invention with reference to specific embodiments, but should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in the art.

[0078] Example 1: Preparation of ALDH1 inhibitor-encapsulated core

[0079] The ALDH1 inhibitor encapsulation core involved in this embodiment was prepared using a solvent emulsification-evaporation method, specifically including the following steps:

[0080] 1. Preparation of organic phase solutions and aqueous phase solutions

[0081] 100 mg Entinostat (MedChemExpress LLC, catalog number: HY-12163) and 100 mg PLGA-PEG-NH2 (Akina In c., catalog number: AI181) were dissolved in 5 mL of chloroform (Merck KGaA, catalog number: CX1058, HPLC grade), and the solution was dissolved using a 300 W ultrasonic instrument (Ningbo Xinzhi Biotechnology Co., Ltd., SCIENTZ-IID) and treated at 25 °C for 10 min to form a clear organic phase solution. 50 mL of a 1% (w / w) polyvinyl alcohol (PVA) aqueous solution was prepared and sterilized through a 0.22 μm filter membrane (Merck KGaA, catalog number: SLGP033RB) to serve as the aqueous phase solution.

[0082] 2. Emulsification

[0083] The organic phase solution was slowly added dropwise to the aqueous phase solution at a rate of 1 mL / min, while simultaneously performing ultrasonic treatment at 300 W and reacting at 25 °C for 5 min to form an O / W type emulsion.

[0084] 3. Solvent evaporation and purification

[0085] The emulsion was transferred to a flask and stirred in a 37°C water bath (200 rpm) for 4 hours to evaporate the chloroform. After evaporation, the emulsion was transferred to an ultrafiltration tube (molecular weight cutoff 30 kDa, Thermo Fisher Scientific Inc., catalog number: 88502), centrifuged at 3000 g at 4°C (Eppendorf AG, 5424R high-speed benchtop refrigerated centrifuge) for 15 min, washed three times (10 mL PBS buffer, pH 7.4 each time), and concentrated to 5 mL to obtain an Entinostat@PLGA nanoparticle suspension (i.e., ALDH1 inhibitor encapsulated core). The particle size was measured to be 90 ± 5 nm using a laser particle size analyzer (Malvern Panalytical Ltd, Zetasizer Ultra).

[0086] Example 2: Preparation of Tumor-Targeted Modification Layer

[0087] The tumor-targeting modification layer involved in this embodiment is prepared by a coupling reaction, specifically including the following steps:

[0088] 1. Activation of folic acid targeting ligands

[0089] 10 mg of folic acid was dissolved in 10 mL of MES buffer (Thermo Fisher Scientific Inc., catalog number: B0002) at pH 5.5. 60 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, Merck KGaA, catalog number: 8.00907) and 100 mg of N-hydroxysuccinimide (NHS, Merck KGaA, catalog number: 8.04518) were added, and the mixture was vortexed. The mixture was reacted at 37 °C and 150 rpm for 30 min on a shaker to form a folic acid-active ester intermediate. The activation rate was monitored by UV spectrophotometry (260 nm) and found to be ≥90%.

[0090] 2. Coupling of folic acid with nanoparticles

[0091] Add 5 mL of the Entinostat@PLGA nanoparticle suspension prepared in Example 1 to the activated folic acid-active ester intermediate solution, followed by 5 mL of Tris-HCl buffer (0.1 mol / L, pH 7.4, Beijing Bio-Tech Co., Ltd., catalog number: Top1123-PH7.4), bringing the total volume to 10 mL. The reaction was carried out at 37°C and 150 rpm for 12 h with shaking. After the reaction, the nanoparticles were centrifuged at 3000 g for 15 min at 4°C using an ultrafiltration tube (molecular weight cutoff 30 kDa), washed twice (PBS buffer, pH 7.4), and concentrated to 5 mL to obtain Entinostat@PLGA@FA nanoparticles (i.e., the tumor-targeting modified layer).

[0092] Example 3: Preparation of thermosensitive hydrogel matrix and nanoparticle encapsulation

[0093] The thermosensitive hydrogel matrix involved in this embodiment was prepared by a cross-linking method, specifically including the following steps:

[0094] 1. Synthesis of N-isopropylacrylamide (NIPAAm) prepolymer

[0095] Weigh 1 g of NIPAAm (Merck KGaA, catalog number: 731129) and dissolve it in 20 mL of ultrapure water. Add 0.05 g of cysteine ​​hydrochloride (Merck KGaA, catalog number: M6500), 0.25 g of N,N'-methylenebisacrylamide (Merck KGaA, catalog number: 1.01546), and 0.25 g of potassium persulfate (Merck KGaA, catalog number: 1.05091), and stir magnetically until dissolved. Purge with high-purity nitrogen (99.99% purity) for 30 min to remove oxygen. Add 50 μL of tetramethylethylenediamine (TEMED, Mercck KGaA, catalog number: 1A00400) and mix rapidly to form a prepolymer solution.

[0096] 2. Nanoparticle encapsulation

[0097] Add 5 mL of the Entinostat@PLGA@FA nanoparticle suspension (nanoparticle concentration 10 mg / mL) prepared in Example 2 to the prepolymer solution and vortex to mix. Transfer the mixture to a 24-well plate (Thermo Fisher Scientific Inc., catalog number: 142475) and incubate at 37°C for 12 h to obtain the Gel@Entinostat@PLGA@FA composite hydrogel (i.e., thermosensitive hydrogel for drug delivery). The microstructure of the composite hydrogel was observed using a scanning electron microscope (TESCAN GROUP, AS, SEM Miralms).

[0098] Figure 1The SEM microstructure of the thermosensitive hydrogel drug delivery system (Gel@Entinostat@PLGA@FA composite hydrogel) is shown. The results indicate that the thermosensitive hydrogel drug delivery system obtained in this embodiment forms a three-dimensional network structure through N-isopropylacrylamide double bond crosslinking. Figure 1 When the test tube is inverted at 37°C, it does not flow and appears as a translucent milky white gel, indicating that the phase transition is complete.

[0099] Performance characterization and result verification

[0100] 1. Temperature sensitivity test

[0101] Take 1 mL of the composite hydrogel suspension prepared in Example 3 into a test tube and observe it in a water bath at 37°C. Record the phase transition time as less than 5 min. Evaluate the temperature sensitivity of the composite hydrogel using a differential scanning calorimeter (TA Instruments, model: Q2000).

[0102] 2. Drug release test

[0103] 100 mg of the composite hydrogel was placed in a dialysis bag (molecular weight cutoff 14 kD, Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number: 20536ES03), and 50 mL of PBS buffer (pH 7.4, containing 0.1% Tween-80) was added. The mixture was shaken at 37℃ and 100 rpm. 1 mL samples were taken at days 0, 1, 3, 7, 14, 21, and 27, and the ALDH1 inhibitor concentration was determined by liquid chromatography (column: C18, 4.6 × 250 mm; mobile phase: methanol-water = 60:40, v / v; flow rate: 1 mL / min; detection wavelength: 254 nm; instrument: 1290 Infinity III liquid chromatograph, Agilent Technologies (China) Co., Ltd.).

[0104] The results of thermosensitivity and drug release tests for the thermosensitive hydrogel drug delivery system (Gel@Entinostat@PLGA@FA composite hydrogel) are as follows: Figure 2 As shown in the figure, Figure A shows the composite hydrogel in a liquid state at room temperature (25℃), Figure B shows the composite hydrogel in a viscous solid state at 37℃, and Figure C shows the drug release curve of the composite hydrogel. It is liquid at room temperature (25℃), with a phase transition temperature of 32-35℃, and gelation is triggered near body temperature. Figure 2 (See Figures A and B in the diagram). Regarding drug release, the cumulative release rate at 27 days was 85±3%, the release rate at 0-7 days was 30±5%, the release rate at 8-21 days was 50±5%, and the release rate at 22-27 days was 20±5%, consistent with biphasic drug release characteristics. Figure 2 (Figure C in the diagram).

[0105] 3. Cell-targeted uptake experiment

[0106] 3.1 UMUC-3 cell culture

[0107] Human bladder cancer cell line UMUC-3 (Wuhan Pronosei Biotechnology Co., Ltd., catalog number: CL-0463) was cultured in Gibco RPMI-1640 medium (containing 10% fetal bovine serum, Thermo Fisher Scientific Inc., catalog number: 11875085) at 37°C in a CO2 incubator with a volume fraction of 5%. Cells in the logarithmic growth phase were used for experiments.

[0108] 3.2 Fluorescent Labeling and Uptake Experiment

[0109] 3.2.1 Preparation of Gel@PLGA@DIO@FA and Gel@PLGA@DIO complex

[0110] (1) Gel@PLGA@DIO@FA complex: In the preparation of the organic and aqueous phase solutions for the core-encapsulating process, the amount of Entinostat used was adjusted to 50 mg, and the remaining steps for preparing the core-encapsulating process were the same as in Example 1; after the preparation of the tumor-targeting modification layer, the tumor-targeting modification layer (Entinostat@PLGA@FA nanoparticles) was labeled with DiO cell labeling solution (10 μM, Thermo Fisher Scientific Inc., catalog number: V22886), and placed in a 24-well plate with UMUC-3 cells (density 5 × 10⁻⁶). 4 Cells / well were incubated at 37°C for 24 hours. After incubation, the culture medium was discarded, and the cells were washed three times with PBS buffer. The cells were then fixed with 4% paraformaldehyde solution and the nuclei were stained with DAPI staining solution (ready-to-use type, Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: D598342) to obtain a DiO cell-labeled tumor-targeting modified layer (Entinostat@PLGA@DIO@FA nanoparticles). The remaining steps for preparing the tumor-targeting modified layer were the same as in Example 2. The steps for preparing the thermosensitive hydrogel matrix and encapsulating the nanoparticles were the same as in Example 3, and the final product was a Gel@PLGA@DIO@FA thermosensitive hydrogel nanomaterial composite.

[0111] (2) Gel@PLGA@DIO complex: The Gel@PLGA@DIO complex served as the control group in this experiment. Its preparation method was the same as that of the Gel@PLGA@DIO@FA complex, except that folic acid was removed during the folic acid targeting ligand activation process in the preparation of the tumor-targeting modification layer. After encapsulation, the final product was a Gel@PLGA@DIO thermosensitive hydrogel nanomaterial complex.

[0112] (3) Observe the staining: The targeted uptake efficiency of the two thermosensitive hydrogel nanomaterial composites was observed by using a confocal microscope (ZEISS Group, model: LSM 880).

[0113] Figure 3 Confocal micrographs of two composite materials are shown, in which, Figure 3 The left image shows the cell-targeted uptake of the Gel@PLGA@DIO@FA thermosensitive hydrogel nanomaterial complex in the UMUC-3 cell line, and the right image shows the cell-targeted uptake of the Gel@PLGA@DIO thermosensitive hydrogel nanomaterial complex in the UMUC-3 cell line. The results indicate that the fluorescence intensity of the folic acid-modified group was 3.1 times that of the unmodified group, and it was mainly distributed in the cytoplasm, verifying the folic acid receptor (FRα)-mediated endocytosis process. This further demonstrates that the specific binding of folic acid to the folic acid receptor on the surface of human bladder cancer cells can achieve active targeting.

[0114] 4. In vivo tumor suppression experiment

[0115] 1. Establishment of the nude mouse model

[0116] Four-week-old BALB / c Nude nude mice (SPF grade, female, 18-20g, Beijing Vital River Laboratory Animal Technology Co., Ltd., catalog number: 401) were inoculated with 5×10 saturated saturated mice under the right axilla. 6 UMUC-3 cells were collected until the tumor volume reached 100±20 mm. 3 Grouped by time.

[0117] 2. Grouping, drug administration, and monitoring

[0118] (1) Grouping: This experiment was divided into the free Entinostat group (5 mg / kg), the Gel@Entinostat group (Entinostat 5 mg / kg equivalent concentration), the Gel@Entinostat@PLGA group (Entinostat 5 mg / kg equivalent concentration), and the Gel@Entinostat@PLGA@FA group (Entinostat 5 mg / kg equivalent concentration), with 5 replicates in each group.

[0119] (2) Administration method: The corresponding groups were administered the drug via intravesical injection (100 μL / animal), once every 3 days, for a total of 4 times.

[0120] (3) Tumor morphological examination: The tumor was weighed, and the major diameter (L) and minor diameter (W) were measured using vernier calipers. The volume V = 0.5 × L × W 2 Recorded for 21 days.

[0121] (4) Tumor histological analysis: The tumor tissue was stained with hematoxylin and eosin (HE) staining kit (Beijing Solarbio Science & Technology Co., Ltd., catalog number: G1120), and the tissue sections were observed by optical microscope.

[0122] Figure 4 The images show actual tumors in different treatment groups of a nude mouse model. The treatment groups in the figure are, from top to bottom, the free Entinostat group, the Gel@Entinostat group, the Gel@Entinostat@PLGA group, and the Gel@Entinostat@PLGA@FA group, with 5 mice replicated in each group. Figure 5 and Figure 6 The tumor weight and volume statistics of different treatment groups in the nude mouse model are shown separately. "Control" in the figure represents the control group. Figure 7 Paraffin-embedded sections of heart, liver, lung, and kidney tissues from different treatment groups in a nude mouse model are shown. "NC" in the figures represents the control group. Results indicate that the Gel@Entinostat@PLGA@FA group showed a tumor volume inhibition rate of 78±5% and a weight reduction of 82±3%. Figure 7 ).

[0123] Example 4: Alternative Implementation Method for Drug Delivery via Thermosensitive Hydrogel Based on ALDH1 Inhibitor

[0124] For certain specific situations that cannot be resolved by embodiments 1-3, this embodiment provides four alternative solutions, specifically:

[0125] 1. Targeted ligand replacement (suitable for tumors with low folate receptor expression)

[0126] (1) Anti-ALDH1 antibody replacement

[0127] Aldehyde dehydrogenase 1-A1 / ALDH1A1 antibody B-5 (Santa Cruz Biotechnology, Inc., catalog number: sc-374149) was used as a folic acid substitute to improve the specific binding of certain folic acid targets where folic acid has low targeting efficiency. The specific steps were as follows: 100 mg of ALDH1A1 antibody B-5 and 50 mg of NHS-Biotin (Merck KGaA, catalog number: H1759) were dissolved in 10 mL of pH 7.4 PBS buffer and vortexed for 2 h. The mixture was then coupled at 4 °C for 12 h to form a PLGA@Entinostat nanoparticle surface modified with streptavidin (i.e., a tumor-targeting modification layer).

[0128] (2) RGD peptide replacement

[0129] RGD peptide (as shown in SEQ ID NO.1) is used to replace folic acid for tumors with low expression of folic acid receptors. The specific preparation method is the same as the above-mentioned anti-ALDH1 antibody replacement steps.

[0130] 2. Replacement of temperature-sensitive materials

[0131] 0.8 g of N-vinylcaprolactam (PVCL, Merck KGaA, catalog number: 415464) was dissolved in 10 mL of ultrapure water. The solution was deoxygenated by purging with high-purity nitrogen (99.99% purity) for 30 min. Then, 0.02 g of N,N'-methylenebisacrylamide and 0.02 g of potassium persulfate were added. The mixture was polymerized at 37 °C for 12 h, with a phase transition temperature of 32-35 °C, yielding polyPVCL hydrogel. This material exhibits approximately 20% lower cytotoxicity compared to the original preparation method using NIPAAm, improving the biocompatibility for drug delivery.

[0132] 3. Alternative drug encapsulation methods (applicable to water-soluble ALDH1 inhibitors)

[0133] 100 mg of ALDH1 inhibitor was dissolved in 10 mL of water to obtain an ALDH1 inhibitor aqueous solution with a concentration of 10 mg / mL. 200 mg of polylactic-co-glycolic acid (PLGA) was dissolved in 10 mL of acetone to obtain a PLGA-acetone solution with a concentration of 20 mg / mL. The ALDH1 inhibitor aqueous solution was slowly added dropwise to the PLGA-acetone solution. After sonication at 200 W for 10 min, 50 mL of a 5% (w / w) polyvinyl alcohol solution was added dropwise. The mixture was stirred at 25 °C for 4 h to evaporate the acetone. After filtration and purification, a water-soluble ALDH1 inhibitor thermosensitive hydrogel for drug delivery was obtained. This method is suitable for encapsulating water-soluble ALDH1 inhibitors.

[0134] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an ALDH1 inhibitor-based thermosensitive hydrogel for drug delivery, characterized by, The method comprises the following steps: S1, preparing an ALDH1 inhibitor-loaded core: dissolving the ALDH1 inhibitor and PLGA-PEG-NH2 in an organic solvent to obtain an organic phase solution; dissolving polyvinyl alcohol in water to obtain an aqueous phase solution after filtration; adding the organic phase solution to the aqueous phase solution, and emulsifying the reaction by ultrasonic treatment to obtain product I; performing water bath heating on the product I to volatilize the organic solvent, and then performing centrifugation, washing and concentration on the volatilized product I to obtain the ALDH1 inhibitor-loaded core; S2, preparing a tumor targeting modification layer: dissolving the targeting ligand 1 in a MES buffer, adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, and performing a shaking activation reaction to obtain product II; adding the ALDH1 inhibitor-loaded core to the product II, performing a shaking coupling reaction after adjusting the final volume with a Tris-HCl buffer, and then performing filtration, centrifugation, washing and concentration to obtain the tumor targeting modification layer; or, preparing a tumor targeting modification layer: dissolving the targeting ligand 2 and NHS-biotin in a PBS buffer, and performing a shaking coupling reaction to obtain the tumor targeting modification layer; S3, preparing a temperature-sensitive hydrogel for drug delivery by using the tumor targeting modification layer.

2. The production method according to claim 1, wherein In step S1: the mass ratio of the ALDH1 inhibitor to PLGA-PEG-NH2 is (1-1.5): 1; the organic solvent is chloroform; the volume ratio of the organic phase solution to the aqueous phase solution is (1-1.5): 10; the emulsification reaction is performed at a temperature of 20-30℃ for 5-10 min; the aqueous phase solution is a polyvinyl alcohol solution with a mass concentration of 1-2%.

3. The production method according to claim 1, wherein In step S2: the targeting ligand 1 is folic acid; the targeting ligand 2 is an anti-ALDH1 monoclonal antibody or an RGD peptide; the amino acid sequence of the RGD peptide is shown in SEQ ID NO.

1.

4. The production method according to claim 1, wherein In step S2: the mass-volume ratio of the targeting ligand 1, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and the MES buffer is (10-15) mg:(60-70) mg:(100-110) mg:(10-15) mL; the activation reaction is performed at a temperature of 35-40℃ for 25-35 min; the coupling reaction is performed at a temperature of 35-40℃ for 10-15 h; or, the mass-volume ratio of the targeting ligand 2, NHS-biotin and the PBS buffer is (100-110) mg:(50-60) mg:(10-15) mL; the coupling reaction is performed at a temperature of 3-5℃ for 10-15 h.

5. The production method according to claim 1, wherein In step S3, the preparation of the thermosensitive hydrogel for drug delivery includes the following steps: dissolving N-isopropylacrylamide, cysteine ​​hydrochloride, potassium persulfate, and N,N′-methylenebisacrylamide in water to obtain product III; purging product III with nitrogen gas to remove oxygen, adding tetramethylethylenediamine, and rapidly mixing to obtain a linear prepolymer solution; adding the tumor-targeting modification layer to the linear prepolymer solution, vortexing and allowing it to stand to obtain the thermosensitive hydrogel for drug delivery. Alternatively, N-vinylcaprolactam is dissolved in water, deoxygenated with nitrogen, and then potassium persulfate and N,N′-methylenebisacrylamide are added to obtain product IV; product IV undergoes a polymerization phase transition to obtain a thermosensitive hydrogel for drug delivery.

6. The preparation method according to claim 5, characterized in that, The mass-to-volume ratio of N-isopropylacrylamide, cysteine ​​hydrochloride, potassium persulfate, N,N′-methylenebisacrylamide, water, and tetramethylethylenediamine is (1-2) g : (0.05-0.15) g : (0.25-0.35) g : (20-25) mL : (50-60) μL. Alternatively, the mass-to-volume ratio of the N-vinylcaprolactam, potassium persulfate, N,N′-methylenebisacrylamide to water is (0.8–1.2) g : (0.02–0.05) g : (0.02–0.05) g : (10–15) mL; The polymerization reaction is carried out at a temperature of 35–40°C for 10–15 hours.

7. A method of preparing a temperature-sensitive hydrogel delivery drug of water-soluble ALDHl inhibitor, characterized in that, Includes the following steps: A water-soluble ALDH1 inhibitor was encapsulated using a nanoprecipitation method to obtain a thermosensitive hydrogel for drug delivery of the water-soluble ALDH1 inhibitor.

8. The production method according to claim 7, wherein The nanoprecipitation method includes the following steps: dissolving an ALDH1 inhibitor in water to obtain an ALDH1 inhibitor aqueous solution; dissolving a polylactic acid-glycolic acid copolymer in an organic solvent to obtain an organic phase solution; adding the ALDH1 inhibitor aqueous solution to the organic phase solution, and then sonicating to obtain product V; adding product V dropwise to a polyvinyl alcohol solution, stirring to evaporate the organic solvent, and then filtration and purification of the evaporated product V to obtain a thermosensitive hydrogel for drug delivery.

9. The production method according to claim 8, wherein The organic solvent is acetone; In the process of preparing the product V, the mass-volume ratio of ALDH1 inhibitor, polylactic acid-glycolic acid copolymer, acetone and water is (100-120) mg: (200-250) mg: (10-15) mL: (10-15) mL; The polyvinyl alcohol solution has a mass concentration of 5-6% and a usage volume of 50-60 mL.

10. The use of a thermosensitive hydrogel for delivering a drug prepared by any one of the preparation methods described in claims 1-9 in the preparation of antitumor drugs.

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