A cross-linked nanoprodrug, a nano-self-assembled hydrogel precursor sheet prepared therefrom, and its applications.

Abstract

This invention discloses a cross-linked nanoprodrug, a nano-self-assembled hydrogel precursor tablet prepared therefrom, and its applications. The nano-self-assembled hydrogel precursor tablet of this invention is prepared by direct compression of the cross-linked nanoprodrug or by compression of a cross-linked nanoprodrug mixed with a drug-loaded orthoester compound. It exhibits high nanodrug content, enhanced nanoparticle release, and high storage and transport stability, as well as good patient compliance. This nano-self-assembled hydrogel precursor tablet responds to the pH gradient environment of the gastrointestinal tract. In acidic gastric juice, through protonation of functional groups, it forms a three-dimensional network structure of nano-self-assembled hydrogel mediated by hydrogen bonds. Subsequently, it stably passes through the small intestine into the colon. In the alkaline colonic environment, its functional groups deprotonate, hydrogen bonds break, and the drug is efficiently released under the electrostatic repulsion between nanoparticles and the synergistic repulsion of the orthoester compound. While preferentially adhering to colonic lesions through electrostatic interactions, it significantly improves the targeted and synergistic therapeutic effect on CRC.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a cross-linked nanoprodrug and a nano self-assembled hydrogel precursor sheet prepared therefrom, and its applications. Background Technology

[0002] Colorectal cancer (CRC), the third most common malignant gastrointestinal tumor worldwide, is characterized by complex carcinogenic factors, high recurrence rates, and severe damage. Patients commonly experience symptoms such as rectal bleeding and loose stools, seriously threatening human health. For early-stage CRC composed of polyps and small tumors, endoscopic surgical resection is typically used clinically. Ideally, this can remove the tumor while restoring bowel function. However, for intermediate-to-late-stage CRC, where the tumor has breached the basement membrane and blood vessels proliferate extensively, surgical resection often carries serious risks such as anastomotic leakage and bleeding, reducing postoperative survival rates. Clinically, chemotherapy drugs including fluorouracil, oxaliplatin, irinotecan, and capecitabine are commonly used for systemic treatment; however, the lack of tumor targeting and severe toxic side effects limit further application. Therefore, there is an urgent need to develop a novel drug delivery system to achieve targeted therapy for CRC while improving patient prognosis.

[0003] Nanomedicines, due to their targeting and low toxicity, have been widely studied and applied in clinical practice. In particular, orally delivered nanomedicines, once they overcome gastrointestinal barriers such as the destructive effects of strong acidic gastric juice, the influence of gastrointestinal enzymes, and absorption by small intestinal villi to reach the colonic mucosal tumor microenvironment, can significantly enhance tumor tissue enrichment through dynamic changes in their physicochemical properties, such as removable PEGylation, potential reversal, and particle size transformation. Therefore, the use of nanomedicines for targeted drug delivery in CRC has enormous development potential.

[0004] Chinese patent application CN116920109A discloses a zwitterionic functionalized small-molecule oral nanoprodrug system, which is prepared by the self-assembly of a hydrophilic betaine and a hydrophobic drug-vinyl ether methacrylate complex. This system can load polyhydroxy chemotherapeutic drugs and exhibits gastrointestinal stability and good biocompatibility. However, this nanoprodrug is largely taken up by villi in the small intestine and enters the bloodstream, where it is subsequently affected by immune clearance and the first-pass effect in the liver, severely reducing the drug content at the tumor site and limiting efficacy. Chinese patent application CN116785234A discloses a drug-loaded nanocomposite hydrogel, prepared by loading manganese dioxide nanoparticles encapsulating chemotherapeutic drugs onto inulin hydrogel. This hydrogel exhibits gastrointestinal stability and colonic flora responsiveness, protecting the nanodrugs from destruction and absorption in the gastrointestinal tract and enabling specific release at colonic lesions. However, due to the limitations of inorganic nanomedicines, including structural rigidity and easy precipitation, and the limitations of microbial response, including slow response speed and insufficient specificity, this nanocomposite hydrogel suffers from insufficient particle stability and incomplete drug release due to slow degradation. Furthermore, the inherent limitations of hydrogels, including low storage and transport stability and low patient compliance due to oral stickiness, further limit its clinical application.

[0005] Therefore, there is an urgent need to develop a novel oral nanodelivery system to overcome the shortcomings of current delivery systems and further improve the accuracy of CRC targeted delivery. Summary of the Invention

[0006] To address the shortcomings of the prior art, the present invention aims to provide a cross-linked nanoproduce and a nano-self-assembled hydrogel precursor sheet prepared therefrom. This precursor sheet exhibits high nanodrug content, enhanced particle release, significantly improved accuracy of CRC targeted delivery, and high stability.

[0007] This invention is achieved through the following technical solution:

[0008] In a first aspect, the present invention provides a cross-linked nanoprodrug, which is constructed by cross-linking a macromolecular polymer with a dicarboxylic chemotherapeutic drug via amide bonds and then self-assembling, and its structural formula is shown in formula (I):

[0009]

[0010] Wherein, P1 represents a macromolecular polymer, specifically selected from carboxymethyl chitosan, collagen, silk fibroin, or whey protein; M1 represents any one of the following (i) to (x) dicarboxylic acid chemotherapeutic drugs:

[0011]

[0012]

[0013] Preferably, the molecular weight of the carboxymethyl chitosan is 10,000-1,000,000 Da, the molecular weight of the collagen is 50,000-300,000 Da, the molecular weight of the silk fibroin is 100,000-400,000 Da, and the molecular weight of the whey protein is 14,000-150,000 Da.

[0014] In this invention, the dicarboxylated chemotherapy drug is selected from methotrexate or synthesized by esterification of mitoxantrone with succinic anhydride, maleic anhydride or norcanthone, or by oxidizing cisplatin or oxaliplatin with an oxidizing agent (such as hydrogen peroxide) and then synthesizing dicarboxylated chemotherapy drugs by esterification of succinic anhydride, maleic anhydride or norcanthone.

[0015] This invention selects macromolecular polymers and dicarboxylated chemotherapy drugs, and by adjusting the types, improves the biocompatibility, stability and antitumor activity of nanoprodrugs while preparing cross-linked nanomedicines.

[0016] Secondly, the present invention provides a method for preparing the above-mentioned cross-linked nanoprodrug, wherein the cross-linked nanoprodrug is constructed by cross-linking a macromolecular polymer containing both amino and carboxyl groups with a dicarboxyl chemotherapeutic drug via amide bonds and then self-assembling, specifically including the following steps:

[0017] (1) Dissolve the dicarboxylic chemotherapeutic drug, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in water and stir at room temperature to obtain an activated dicarboxylic chemotherapeutic drug solution.

[0018] (2) Dissolve the macromolecular polymer in water and add it dropwise to the activated dicarboxylic chemotherapeutic drug solution, and stir the reaction at room temperature to obtain the cross-linked solution;

[0019] (3) The cross-linked solution was dialyzed to obtain an aqueous solution of cross-linked nano-prodrug, which was then freeze-dried after being rapidly frozen in liquid nitrogen to obtain the cross-linked nano-prodrug.

[0020] Preferably, in step (1), the molar ratio of the dicarboxylated chemotherapeutic drug, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) is 1:(2.0-5.0):(2.0-5.0); and the stirring time is 2-6 h.

[0021] Preferably, in step (2), the stirring reaction time is 12-24 hours.

[0022] Preferably, in step (3), the dialysis involves placing the cross-linked solution in a dialysis bag (1000-3500 Da) and dialyzing it in double-distilled water for 24-72 hours; the liquid nitrogen quick-freezing time is 1-5 minutes; the freeze-drying time is 24-72 hours, and the freeze-drying temperature is -50 to -40°C.

[0023] Thirdly, the present invention provides a nano-self-assembled hydrogel precursor tablet, which is obtained by pressing the above-mentioned cross-linked nano-prodrug into tablets, or by pressing the above-mentioned cross-linked nano-prodrug into tablets mixed with a drug-loaded orthoester compound.

[0024] Preferably, the mass ratio of the orthoester compound to the cross-linked nanoparticle prodrug is 1:4-4:1 (e.g., 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, or any ratio within the above range). This invention controls the mixing and tableting of the orthoester compound and the cross-linked nanoparticle drug within the above range, achieving thorough mixing of the orthoester compound while improving the storage and transportation stability of the tablets.

[0025] Preferably, the orthoester compound is selected from any one of the following compound structural formulas (II) to (IV):

[0026]

[0027] R1 is selected from any one of hydrogen, methyl, ethyl, propyl, isopropyl, butyl, or phenyl;

[0028] R2 is selected from any one of ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl;

[0029]

[0030] R1 is selected from any one of hydrogen, methyl, ethyl, propyl, isopropyl, butyl, or phenyl;

[0031] R2 is selected from any one of ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl;

[0032] R3 is selected from any one of methyl, ethyl, propyl, butyl, pentyl, or hexyl;

[0033]

[0034] R1 is selected from any one of hydrogen, methyl, ethyl, propyl, isopropyl, butyl, or phenyl;

[0035] R2 is selected from any one of ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl;

[0036] R3 is selected from any one of methyl, ethyl, butyl, or hexyl.

[0037] The preparation method of the orthoester compound as shown in structural formula (II) of the present invention includes the following steps: under nitrogen protection, diglycerides, triesters and catalysts are dissolved in a first organic solvent at a molar ratio of 1:(2.2-5.0):(0.01-0.04), and the mixture is stirred at room temperature for 12-48 hours. Then, the mixture is extracted with saturated sodium carbonate, dried with anhydrous magnesium sulfate, and the excess triester raw material is removed by vacuum distillation to obtain the orthoester compound of type I.

[0038] The preparation method of the orthoester compound as shown in structural formula (III) of the present invention includes the following steps: under nitrogen protection, a type I diol, a triester, and a catalyst are dissolved in a first organic solvent at a molar ratio of 1:(1.1-2.0):(0.01-0.02), and the mixture is stirred at room temperature for 12-48 hours. Then, the mixture is extracted with saturated sodium carbonate, dried with anhydrous magnesium sulfate, and the excess triester raw material is removed by vacuum distillation to obtain the type II orthoester compound.

[0039] The method for preparing the orthoester compound as shown in structural formula (Ⅳ) of the present invention includes the following steps: under nitrogen protection, type II diols, triesters and catalysts are dissolved in a first organic solvent at a molar ratio of 1:(1.1-2.0):(0.01-0.02), and the mixture is stirred at room temperature for 12-48 hours. Then, the mixture is extracted with saturated sodium carbonate, dried with anhydrous magnesium sulfate, and the excess triester raw materials are removed by vacuum distillation to obtain type III orthoester compounds.

[0040] Preferably, the first organic solvent includes, but is not limited to, acetonitrile, dichloromethane, tetrahydrofuran, and dioxane; the triester raw materials include, but are not limited to, triethyl orthoformate, triethyl orthoacetate, triethyl orthopropionate, triethyl orthoisopropionate, triethyl orthobutyrate, triethyl orthobenzoate, tripropyl orthoformate, tripropyl orthoacetate, tripropyl orthopropionate, tripropyl orthoisopropionate, tripropyl orthobutyrate, tripropyl orthobenzoate, triisopropyl orthoformate, triisopropyl orthoacetate, triisopropyl orthopropionate, and triisopropyl orthopropionate. Triisopropyl orthobutyrate, triisopropyl orthobenzoate, tributyl orthoformate, tributyl orthoacetate, tributyl orthopropionate, tributyl orthoisopropionate, tributyl orthobutyrate, tributyl orthobenzoate, triisobutyl orthoformate, triisobutyl orthoacetate, triisobutyl orthopropionate, triisobutyl orthoisopropionate, triisobutyl orthobutyrate, triisobutyl orthobenzoate, tri-tert-butyl orthoformate, tri-tert-butyl orthoacetate, tri-tert-butyl orthopropionate, tri-tert-butyl orthoisopropionate, tri-tert-butyl orthobutyrate, tri-tert-butyl orthobenzoate.

[0041] Preferably, the type I diols include, but are not limited to, 1,2-propanediol, 1,2-butanediol, 1,2-pentanediol, 1,2-hexanediol, 1,2-heptanediol, and 1,2-octanediol;

[0042] Preferably, the Class I diols include, but are not limited to, 1,3-propanediol, 1,3-butanediol, 1,3-hexanediol, and 1,3-octanediol; and the catalysts include, but are not limited to, p-toluenesulfonic acid and p-toluenesulfonic acid pyridinium.

[0043] This invention improves biocompatibility by adjusting the type and length of substituents in orthoester compounds without altering the properties of the orthoesters before and after the modification. The orthoester compounds of this invention contain only carbon-oxygen bonds and do not disrupt the drug structure.

[0044] Fourthly, the present invention also provides a method for preparing the above-mentioned nano-self-assembled hydrogel precursor sheet.

[0045] In one embodiment, the nano-self-assembled hydrogel precursor tablet is obtained by grinding and compressing the above-mentioned cross-linked nano-produce powder into tablets (20-200 mg / tablet).

[0046] As another embodiment, the preparation method of the nano-self-assembled hydrogel precursor sheet includes the following steps:

[0047] S1. Dissolve the drug in an orthoester compound to prepare a drug-loaded oil phase;

[0048] S2. The drug-loaded oil phase is mixed with the cross-linked nano-prodrug aqueous solution, and a homogeneous emulsion is prepared by ultrasonication. After being rapidly frozen in liquid nitrogen, it is then freeze-dried.

[0049] The nano-self-assembled hydrogel precursor tablets loaded with drug-loaded orthoester compounds were obtained by grinding and pressing.

[0050] Preferably, the ultrasound time is 1-5 min; the liquid nitrogen quick-freezing time is 1-5 min; the freeze-drying time is 24-72 h; and the freeze-drying temperature is -50 to -40 °C.

[0051] Preferably, the drug is selected from antitumor drugs used to treat CRC, including but not limited to paclitaxel, docetaxel, etoposide, teniposide, vincristine, vinorelbine, vindesine, homoharringtonine, irinotecan, topotecan, daunorubicin, doxorubicin, epirubicin, mitoxantrone, actinomycin D, mitomycin, methotrexate, carboplatin, cisplatin, and oxaliplatin; and anti-inflammatory drugs, including but not limited to aspirin, indomethacin, diclofenac, ibuprofen, naproxen, ketoprofen, and flurbiprofen. Meloxicam, piroxicam, celecoxib, etoricoxib, prednisone, prednisolone, dexamethasone, betamethasone, hydrocortisone, fluocinolone acetonide, budesonide, triamcinolone acetonide, methylprednisolone; antibacterial drugs including but not limited to oxacillin, cloxacillin, ceftriaxone, cefoperazone, ceftazidime, erythromycin, azithromycin, clarithromycin, roxithromycin, doxycycline, minocycline, tetracycline, chloramphenicol, ciprofloxacin, levofloxacin, moxifloxacin, sulfadiazine, sulfamethoxazole, metronidazole, tinidazole Ornidazole, ketoconazole, fluconazole, itraconazole, voriconazole, amphotericin B, clindamycin, lincomycin, rifampin; antiviral drugs including but not limited to acyclovir, valacyclovir, ganciclovir, famciclovir, oseltamivir, peramivir, amantadine, rimantadine, ribavirin, entecavir, tenofovir disoproxil fumarate, lamivudine, adefovir dipivoxil, efavirenz, nevirapine, ritonavir, lopinavir, atazanavir, sodium phosphonoformate; traditional Chinese medicine natural products including but not limited to matrine, small Berberine, corydaline, ephedrine, artemisinin, andrographolide, oleanolic acid, ursolic acid, paclitaxel, ginsenosides, quercetin, kaempferol, baicalin, hesperidin, soy isoflavones, bone fat, coumarin, osthol, xanthotoxin, schisandrin, magnolol, emodin, chrysophanol, shikonin, β-sitosterol, stigmasterol, curcumin, gingerol, capsaicin, oleanane-type triterpenes, glycyrrhetinic acid, menthol, eugenol, cinnamaldehyde, atractylodesone; the dosage of the drugs is the therapeutically effective amount.

[0052] The preparation method of the nano-self-assembled hydrogel precursor sheet in this invention is simple, with strong storage and transportation stability and patient compliance. Furthermore, by loading orthoester compounds, multiple drugs can be loaded while improving the release of nanomedicines, thereby further enhancing the targeted and synergistic therapeutic effect on CRC.

[0053] Fifthly, the present invention also provides the application of the above-mentioned nano-self-assembled hydrogel precursor sheet in the preparation of drugs for treating colorectal cancer (CRC).

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] The nano-self-assembled hydrogel precursor tablets provided by this invention are prepared by direct compression of cross-linked nano-prodrugs or by compression of cross-linked nano-prodrugs mixed with drug-loaded orthoester compounds. They exhibit high nano-drug content, enhanced nanoparticle release, and high storage and transportation stability, as well as improved patient compliance. Figure 13 As shown, this nano-self-assembled hydrogel precursor sheet can respond to the pH gradient environment of the gastrointestinal tract. In acidic gastric juice, through protonation of its groups, it forms a nano-self-assembled hydrogel with a three-dimensional network structure under the mediation of hydrogen bonds. Subsequently, it stably passes through the small intestine and enters the colon. In the alkaline colonic environment, its groups are deprotonated and hydrogen bonds are broken. Under the electrostatic repulsion between nanoparticles and the synergistic repulsion of orthoester compounds, the drug is released efficiently. While preferentially adhering to the colonic lesion site through electrostatic interaction, it significantly improves the targeted synergistic therapeutic effect on CRC. Attached Figure Description

[0056] Figure 1 Characterization of the cross-linked nanoprodrugs (NPs) in Example 1 (a is the particle size distribution and SEM image of NPs, b is the titration curve of NPs, c is the particle size change of NPs at different pH values, and d is the drug release curve of NPs at different pH values).

[0057] Figure 2 Characterization of cross-linked nanoprodrugs in Examples 2, 3 and 4 (a is the particle size distribution of cross-linked nanoprodrug (collagen-maleic anhydride cisplatin (IV)), b is the particle size distribution of cross-linked nanoprodrug (silk fibroin-succinic anhydride oxaliplatin (IV)), c is the particle size distribution of cross-linked nanoprodrug (whey protein-methotrexate));

[0058] Figure 3 The orthoester compound (OE) in Example 5 1 H NMR spectrum;

[0059] Figure 4 This is a schematic diagram illustrating the preparation of the nano-self-assembled hydrogel precursor sheet in Example 5;

[0060] Figure 5 Characterization of the nano-self-assembled hydrogel precursor sheet in Example 5 (a is the macroscopic morphology and Pt content of the precursor sheet, b is the particle size recovery curve of NPs in the precursor sheet, and c is the release curve of NPs in the precursor sheet).

[0061] Figure 6 The stability of the nano-self-assembled hydrogel precursor sheet in Example 6 is characterized (a is the particle size distribution of NPs released after 30 days of the precursor sheet being placed, and b is the macroscopic stability diagram of the precursor sheet after 30 days of placement).

[0062] Figure 7Characterization of the gelation properties of the nano-self-assembled hydrogel precursor sheet in Example 7 (ac is the microstructure and SEM image of the hydrogel NT-Gel, NOT-Gel and NOMT-Gel, d is the single frequency test image of the hydrogel, e is the XRD image, and f is the FT-IR image).

[0063] Figure 8 The spatiotemporal dynamic response of the nano-self-assembled hydrogel precursor sheet in Example 8 is characterized by the following: (a) gradient degradation curve of the precursor sheet, (b) gradient release curve of Ma in the precursor sheet, and (c) gradient release curve of NPs in the precursor sheet.

[0064] Figure 9 The in vitro antitumor activity of the nano-self-assembled hydrogel precursor sheet in Example 9 is characterized (a is the cytotoxicity of the precursor sheet to CT26 tumor cells, b is the cytotoxicity of the precursor sheet to H22 tumor cells, c is the cytotoxicity of the precursor sheet to HepG2 tumor cells, and d is the cytotoxicity of the precursor sheet to L929 cells).

[0065] Figure 10 The in vitro anti-inflammatory activity of the nano-self-assembled hydrogel precursor sheet in Example 10 is characterized as follows: (a) Effect of the precursor sheet on the generation of reactive oxygen species (ROS) in RAW cells after LPS induction; (b) Effect of the precursor sheet on the expression of tumor necrosis factor (TNF-α) in RAW cells after LPS induction; (c) Effect of the precursor sheet on the expression of interleukin 1β (IL-1β) in RAW cells after LPS induction; (d) Effect of the precursor sheet on the expression of interleukin 6 (IL-6) in RAW cells after LPS induction.

[0066] Figure 11 The in vivo antitumor activity of the nano-self-assembled hydrogel precursor sheet in Example 11 is characterized (a is a curve of tumor volume change, b is a curve of tumor weight comparison, c is a curve of tumor morphology comparison, and d is a curve of mouse body weight change).

[0067] Figure 12 The in vivo anti-inflammatory activity of the nano-self-assembled hydrogel precursor sheet in Example 12 is characterized (a is the curve of mouse body weight change, b is the curve of mouse disease activity index (DAI) change, c is the comparison of mouse colon length, and d is the comparison of mouse spleen tumor).

[0068] Figure 13 This is a schematic diagram illustrating the dynamic changes of the nano-self-assembled hydrogel precursor sheet in the pH gradient of the gastrointestinal tract. Detailed Implementation

[0069] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The described embodiments are only some embodiments of the present invention, and not all embodiments.

[0070] Example 1: Preparation and characterization of cross-linked nanoprodrugs NPs (carboxymethyl chitosan-demethylcanthionized cisplatin(IV)):

[0071] Cisplatin (DDP) was oxidized by hydrogen peroxide (H2O2) and then esterified with norcanthionine (NCTD) at a molar ratio of 1:3 at 65 °C for 24 h to synthesize norcanthionine-modified cisplatin Pt(IV)-1.

[0072] Pt(IV)-1 (13.4 mg, 19.94 μmol), EDC (9.6 mg, 49.08 μmol), and NHS (5.4 mg, 46.01 μmol) were dissolved in 1 mL of deionized water and activated for 4 h. Then, 5 mL of carboxymethyl chitosan (CMCS) (100,000 Da - 200,000 Da) aqueous solution (10.7 mg / mL) was added dropwise and the mixture was stirred at room temperature in the dark for 12 h.

[0073] After dialyzing with deionized water for 72 hours using a dialysis bag (3500 Da), the sample was flash-frozen in liquid nitrogen for 5 minutes and then freeze-dried at -45°C for 48 hours to obtain a white powder, which is the cross-linked nanoprodrug NPs.

[0074] The particle size and size variations of NPs in different PBSs (pH 7.4, pH 6.8, pH 5.0, and pH 5.0 / GSH) were determined by DLS (Zeta-sizer Nano-ZS90, Malvern, Britain), and their morphology was observed by SEM (Regulus 8230, HITACHI, Japan). The isoelectric point of the NPs was determined by an automated potentiometric titrator. Drug release from the NPs in various PBSs (pH 7.4, pH 6.8, pH 5.0, and pH 5.0 / GSH) was determined by dialysis.

[0075] The results are as follows Figure 1 As shown in Figure ac, NPs stably maintained a small particle size (197.3 nm) with a dispersion index of 0.189 in an environment of pH 7.4, while in an environment of pH 5.0, due to isoelectric point aggregation and precipitation, the particle size increased rapidly (>1000 nm). The dynamic change in particle size, gradually increasing from the normal physiological environment (pH 7.4), to the tumor extracellular environment (pH 6.5-7.0), to the tumor intracellular environment (pH 4.0-6.0), is beneficial for enhancing tumor tissue enrichment. Furthermore, as... Figure 1 As shown in Figure d, the NPs exhibit pH / GSH-responsive drug release. Their selective drug release in the low pH and high GSH environment within tumor cells is beneficial for specifically enhancing tumor cytotoxicity and reducing side effects in normal tissues.

[0076] Example 2: Preparation and characterization of cross-linked nanoprodrugs (collagen-maleic anhydride cisplatin(IV)):

[0077] Cisplatin (DDP) was oxidized by hydrogen peroxide (H2O2) and then reacted with maleic anhydride (MA) at a molar ratio of 1:2 at 70°C for 24 h to synthesize maleic anhydride-modified cisplatin (IV).

[0078] Maleic anhydride cisplatin (IV) (10 μmol), EDC (25 μmol) and NHS (25 μmol) were dissolved in 1 mL of deionized water and activated for 6 h. Then, 5 mL of collagen (COL) (250000 Da-300000 Da) aqueous solution (8 mg / mL) was added dropwise and the mixture was stirred at room temperature in the dark for 24 h.

[0079] After dialysis with deionized water for 72 hours using a dialysis bag (3500 Da), the sample was flash-frozen in liquid nitrogen for 5 minutes and then freeze-dried at -45°C for 48 hours to obtain a powder, which is the cross-linked nano-prodrug (collagen-maleic anhydride cisplatin (IV)).

[0080] The particle size distribution of cross-linked nanoprodrug (collagen-maleic anhydride cisplatin(IV)) in PBS (pH 7.4) was determined by DLS.

[0081] The results are as follows Figure 2 As shown in Figure a, the cross-linked nanoprodrug (collagen-maleic anhydride cisplatin (IV)) in PBS (pH 7.4) had an average particle size of 216.7 nm and a dispersion index of 0.09.

[0082] Example 3: Preparation and characterization of cross-linked nanoprodrugs (silk fibroin-succinic anhydride-substituted oxaliplatin(IV)):

[0083] Oxaliplatin (OXA) was oxidized with hydrogen peroxide (H2O2) and then esterified with succinic anhydride (SA) at a molar ratio of 1:2 at 70 °C for 24 h to synthesize succinic anhydride-modified oxaliplatin (Ⅳ).

[0084] Succinic anhydride oxaliplatin (IV) (10 μmol), EDC (25 μmol) and NHS (25 μmol) were dissolved in 1 mL of deionized water and activated for 6 h. Then, 5 mL of silk fibroin (SF) (100000 Da-150000 Da) aqueous solution (8 mg / mL) was added dropwise and the reaction was carried out at room temperature in the dark with stirring for 24 h.

[0085] After dialysis with deionized water for 72 hours using a dialysis bag (3500 Da), the product was rapidly frozen in liquid nitrogen for 5 minutes and then freeze-dried at -45°C for 48 hours to obtain a powder, which is the cross-linked nanoprodrug (silk fibroin-succinic anhydride-oxaliplatin (IV)).

[0086] The particle size distribution of cross-linked nanoprodrug (silk fibroin-succinic anhydride-oxaliplatin(IV)) in PBS (pH 7.4) was determined by DLS.

[0087] The results are as follows Figure 2 As shown in Figure b, the cross-linked nanoprodrug (silk fibroin-succinic anhydride-oxaliplatin(IV)) in PBS (pH 7.4) had an average particle size of 278.9 nm and a dispersion index of 0.113.

[0088] Example 4: Preparation and characterization of cross-linked nanoprodrugs (whey protein-methotrexate):

[0089] Methotrexate (MTX) (10 μmol), EDC (25 μmol) and NHS (25 μmol) were dissolved in 1 mL of deionized water and activated for 6 h. Then, 5 mL of whey protein (WP) (15000 Da-20000 Da) aqueous solution (8 mg / mL) was added dropwise and the mixture was stirred at room temperature in the dark for 24 h.

[0090] After dialysis with deionized water for 72 hours using a dialysis bag (3500 Da), the sample was flash-frozen in liquid nitrogen for 5 minutes and then freeze-dried at -45°C for 48 hours to obtain a powder, which is the cross-linked nanoprodrug (whey protein-methotrexate).

[0091] The particle size distribution of cross-linked nanoprodrug (whey protein-methotrexate) in PBS (pH 7.4) was determined by DLS.

[0092] The results are as follows Figure 2 As shown in Figure c, the cross-linked nanoprodrug (whey protein-methotrexate) in PBS (pH 7.4) has an average particle size of 235.5 nm and a dispersion index of 0.131.

[0093] Example 5: Preparation and characterization of nano-self-assembled hydrogel precursor sheets:

[0094] The NPs powder prepared in Example 1 was ground and compressed to obtain nano-self-assembled hydrogel precursor tablets NT (100 mg / tablet).

[0095] Diglycerides, triethyl orthoacetate, and p-toluenesulfonic acid monohydrate (PTSA) were dissolved in anhydrous dichloromethane (DCM) at a molar ratio of 1:3:0.0025, and the mixture was stirred at room temperature under nitrogen purging for 12 h. The reaction was terminated by adding triethylamine (TEA), and triethyl orthoacetate and DCM were removed by rotary evaporation at 40 °C. The mixture was dissolved in ethyl acetate and extracted three times sequentially with saturated sodium carbonate and saturated sodium chloride solutions, then dried over anhydrous magnesium sulfate (MgSO4) for 24 h. Next, ethyl acetate was removed by rotary evaporation at 50 °C, followed by vacuum distillation at 140 °C to obtain the orthoester compound OE.

[0096] Furthermore, matrine (Ma) was dissolved in OE to prepare a clear and transparent drug-loaded oil phase OE-Ma (Ma concentration: 1 g / mL). OE or OE-Ma was mixed with an aqueous solution of NPs (OE or OE-Ma to NPs mass ratio: 1:1), and sonicated for 5 min to prepare a homogeneous emulsion. The emulsion was then rapidly frozen in liquid nitrogen for 5 min, freeze-dried at -45℃ for 48 h, and ground and compressed into tablets to obtain nano-self-assembled hydrogel precursor tablets NOT or NOMT (100 mg / tablet) loaded with orthoester compounds.

[0097] NT, NOT, and NOMT were placed in PBS (pH 8.0), and the supernatant was collected at the corresponding time points (0h, 1h, 2h, 4h, 8h, 12h, 24h, and 48h) to determine the particle size of the released NPs by DLS and the release rate of NPs by ICP-MS.

[0098] like Figure 3 As shown, an OE consisting of two five-membered rings containing orthoester bonds was prepared, and then... 1 The accuracy of the structure was verified by H NMR, and the integral ratio of proton peak a to proton peak e was observed to be close to 3:1, proving that the OE structure is correct.

[0099] The preparation process and structural schematic diagram of the nano-self-assembled hydrogel precursor sheet are shown below. Figure 4 As shown.

[0100] The platinum contents in NT, NOT, and NOMT, determined by ICP-MS, were 20.8 μg / mg, 10.5 μg / mg, and 10.4 μg / mg, respectively. Figure 5 a). For example Figure 5 As shown in Figures b and c, compared to NT, the addition of OE significantly accelerated the particle size recovery rate and significantly increased the particle release rate of NPs in the NOT and NOMT groups, indicating that OE promoted the release and particle size recovery of NPs. Example 6: Stability Characterization of Nano-Self-Assembled Hydrogel Precursor Sheets

[0101] The nano-self-assembled hydrogel precursor sheet NOMT prepared in Example 5 was placed on oil-absorbing paper at room temperature for 30 days. The particle size distribution of released NPs was measured by DLS on day 0 and day 30, and the exudation of oil droplets was observed by pressing.

[0102] like Figure 6 As shown in Figure a, the particle size of NPs after reconstitution of NOMT on day 30 was not significantly different from that on day 0. Furthermore, no oil stains appeared on the blotting paper regardless of whether the tablet was left to stand or pressed, indicating that OE was uniformly dispersed in the tablet and could maintain stability. Figure 6 (b) Good stability is beneficial for storage, transportation and further application.

[0103] Example 7: Characterization of the gel-forming properties of nano-self-assembled hydrogel precursor sheets:

[0104] Hydrogels NT-Gel, NOT-Gel, and NOMT-Gel were prepared by placing NT, NOT, and NOMT in PBS (pH 1.5) for 1 hour. The microstructure of the hydrogels was photographed using a microscope. After lyophilization, the cross-sectional morphology of the hydrogels was photographed using SEM (S-4800, HITACHI, Japan). Single-frequency scanning rheometer was used to determine the changes in G' and G” of the three hydrogels over time. Furthermore, molecular interactions during hydrogel formation were detected by XRD and FT-IR.

[0105] like Figure 7 As shown in Figure 1, SEM scans revealed that all three groups possessed a uniform porous structure. Microscopic examination showed that NOT-Gel and NOMT-Gel contained numerous oil droplets, indicating successful loading of OE and OE-Ma. Furthermore, as... Figure 7 As shown in Figure d, the elastic modulus (G') of all three groups is greater than the viscous modulus (G”), indicating successful hydrogel formation. To investigate the gelation mechanism of the precursor sheets, XRD and FI-TR were used to further characterize the structure, such as... Figure 7 As shown in Figure e, compared to NPs, NT-Gel exhibits a different crystal structure, with the broad peak position shifting from 21.07° to 21.21°. Furthermore, as... Figure 7 As shown in f, compared with NPs, the characteristic peak of NT-Gel shifts, with υC=O changing from 1648 cm⁻¹. -1 Offset to 1604cm -1 υO-H from 3449cm -1 Offset to 3418cm -1 This indicates that the precursor sheet successfully formed a nano-self-assembled hydrogel precursor sheet in a pH 1.5 environment through protonation of groups and mediated by hydrogen bonds.

[0106] Example 8: Spatiotemporal dynamic response characterization of gastrointestinal tract to self-assembled nano-hydrogel precursor sheets

[0107] NT, NOT, and NOMT were sequentially placed in different PBS solutions at time gradients (pH 1.5, 1h; pH 5.0, 1h; pH 6.0, 2h; pH 7.4, 3h; and pH 8.0, 41h). The gels were stirred at 100 rpm at 37°C to simulate gastrointestinal motility. The wet weight of the NOMT gel was measured at each time point, and the supernatant was used to determine the release of NPs from the NT, NOT, and NOMT groups by ICP-MS. Additionally, the supernatant from the NOMT group was analyzed at each time point using a SpectraMax M2 microplate reader. eThe release of Ma was measured at 220 nm by Molecular Devices, America. The mass remainder was calculated using the following formula:

[0108]

[0109] Among them, M0 and M r These represent the initial weight of the tablet and the weight at a fixed time point, respectively.

[0110] The abundant functional groups on the nano-self-assembled hydrogel precursor sheet endow it with spatiotemporal dynamic responsiveness to gastrointestinal pH gradients. For example... Figure 8 As shown in Figure a, NOMT rapidly absorbs water to form a hydrogel, resulting in an overall increase in weight before reaching pH 8.0. After immersion in a pH 8.0 environment for 41 hours, due to deprotonation of functional groups and the breakage of hydrogen bonds between nanoparticles, the hydrogel swells and degrades under the synergistic effect of charge repulsion and OE repulsion. The degradation of NOMT-Gel further triggers the release of Ma and NPs, such as... Figure 8 As shown in Figures b and c, due to the stability of the hydrogel in the pH range of 1.5–7.4, the release rates of Ma and NPs did not exceed 40% before entering the pH 8.0 environment. After entering the pH 8.0 environment, due to the rapid disintegration of the hydrogel, Ma and NPs were released efficiently. Notably, compared with the NT group, the release rates of NPs in both the NOT and NOMT groups were significantly increased, indicating that OE can promote the release of NPs from the hydrogel.

[0111] Example 9: Characterization of the in vitro antitumor activity of nano-self-assembled hydrogel precursor sheets

[0112] The cytotoxicity of various formulations was evaluated in L929, HepG2, CT26, and H22 cells using the MTT toxicity assay. In short, cells (1 × 10⁻⁶) were subjected to MTT assays. 4 Cells were seeded in 96-well plates and cultured overnight for proliferation. Then, NT, NOT, and NOMT solutions with the same Pt ​​concentration of 2-64 μM were added and co-incubated for 48 h. Subsequently, MTT solution (20 μL, 5 mg / mL) was added to each well, and incubation continued for 4 h. The culture medium for adherent cells (L929, HepG2, CT26) was replaced with DMSO (150 μL), and the culture medium for suspension cells (H22) was replaced with 1 mL of triple lysis buffer (10% SDS, 5% isobutane, and 0.012 mol / L HCl). Cell viability was measured using a microplate reader at UV-Vis absorption wavelengths of 570 nm and 490 nm, respectively, and calculated. Cell viability was calculated using the following formula:

[0113]

[0114] Among them, OD sample OD negative and OD blank These represent the OD values ​​of the sample group, negative control group, and blank group, respectively.

[0115] like Figure 9 As shown in the diagram, based on efficient NP release, enhanced cellular uptake and intracellular retention, pH / GSH-specific drug release within tumor cells, and the synergistic effect of DDP and DMC, all three prodrug groups significantly inhibited the proliferation of tumor cells CT26, H22, and HepG2, and were safe for normal L929 cells. Notably, due to the promoting effect of OE on NP release and particle size recovery, the NOT and NOMT groups showed significantly enhanced inhibitory effects on tumor cell proliferation compared to the NT group.

[0116] Example 10: In vitro anti-inflammatory activity characterization of nano-self-assembled hydrogel precursor sheets

[0117] RAW264.7 cells (1×10⁻⁶) 4 Cells were evenly distributed in 96-well plates and cultured for 24 h. Intracellular ROS generation was stimulated by co-incubation with LPS (25 μg / mL) for 24 h. Subsequently, Ma aqueous solution (Ma-sol), NT, NOT, and NOMT (Ma concentration: 1.2 mM, Pt concentration: 64 μM) were added to each well, and the cells were co-cultured for another 48 h, with physiological saline as a control. Then, the cells were treated with a reactive oxygen species detection kit, and ROS output was quantitatively analyzed using a microplate reader (excitation wavelength: 488 nm, emission wavelength: 525 nm).

[0118] In addition, RAW264.7 cells (1×10⁻⁶) were used. 5 Cells were evenly distributed in 6-well plates and cultured for 24 h. Cells were then co-incubated with LPS (25 μg / mL) for 24 h to stimulate the expression of pro-inflammatory cytokines. Subsequently, Ma-sol, NT, NOT, and NOMT (Ma concentration: 1.2 mM, Pt concentration: 64 μM) were added to each well, and the cells were co-cultured for another 48 h. Physiological saline was used as a control. The expression levels of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), were then detected using an ELISA kit.

[0119] like Figure 10As shown in Figure a, LPS-induced ROS levels significantly increased in RAW264.7 cells, while NOMT effectively inhibited ROS production by promoting the Nrf2 signaling pathway and differentiating M1 macrophages into M2 macrophages. To investigate the anti-inflammatory mechanism of NOMT, the levels of pro-inflammatory cytokines in the cell culture supernatant were measured using ELISA. Figure 10 As shown in Figure 2bd, under LPS induction, the expression levels of pro-inflammatory cytokines, including tumor necrosis factor (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), were significantly increased in RAW264.7 cells. NOMT, through efficient release of Ma and effective uptake by M1 macrophages, effectively inhibited the expression levels of pro-inflammatory cytokines by inhibiting the NF-κB signaling pathway, thus exhibiting significant in vitro anti-inflammatory activity, which is beneficial for the synergistic treatment of CRC.

[0120] Example 11: In vivo antitumor activity characterization of nano-self-assembled hydrogel precursor sheets

[0121] Establish 150mm 3 CT26 tumor-bearing Balb / c mice were randomly divided into 4 groups (n=6), and each group was orally administered NT, NOT, or NOMT (Pt dose: 4 mg / kg) daily, with saline as a control. Tumor volume and body weight were monitored daily, and after 7 days, mice were sacrificed and tumor tissue was photographed. Tumor volume (V) was calculated using the following formula:

[0122]

[0123] Among them, D max and D min These represent the long and short diameters of the tumor, respectively.

[0124] like Figure 11 As shown in Figure a, compared to the saline control group, the tumor volume in mice rapidly increased to 1400 mmHg within 7 days. 3 In mice treated with NT, NOT, and NOMT, tumor growth was inhibited and slowed, indicating that NPs in the precursor tablets could be effectively released and, in response to the tumor cell intracellular environment, efficiently released DDP and DMC to synergistically induce tumor cell apoptosis. Notably, compared to the NT group, the NOT and NOMT groups showed significantly higher inhibition rates against CT26 solid tumors, suggesting that the OE-mediated promotion of NP release and particle size restoration further enhanced the tumor tissue targeting and enrichment capacity, thereby improving therapeutic efficacy.

[0125] In addition, such as Figure 11As shown in Figure bc, after treatment, the tumor size in the treatment group was significantly smaller than that in the control group (1.2g), and after the addition of OE, the tumor weight in the NOT group (0.3g) and NOMT group (0.3g) was significantly lower than that in the NT group (0.7g). Figure 11 As shown in Figure d, the body weight of mice in each group gradually increased during the treatment period, indicating that the prodrug tablets had low side effects.

[0126] The above results indicate that the nano-self-assembled hydrogel precursor tablets can be converted into hydrogels after oral administration, targeting and delivering NPs to colonic lesions. NPs are specifically adsorbed through electrostatic interactions and released under the mediation of OE. The NPs are further enriched in tumor tissue. Under the enhanced tumor tissue penetration, cellular uptake, intracellular retention, intracellular responsive drug release triggered by the tumor microenvironment, and the synergistic effect of DDP and DMC, significant inhibition of solid tumors and reduction of side effects on normal tissues can be achieved.

[0127] Example 12: In vivo anti-inflammatory activity characterization of nano-self-assembled hydrogel precursor sheets

[0128] C57BL / 6 mice (8 weeks old) that had been pre-acclimated for 7 days were randomly divided into 6 groups (n=6): Control, DSS, NT, NOT, Ma-sol, and NOMT. The Control group had free access to water for 10 days, during which time they were given oral saline daily. The other groups had free access to 3% DSS for 7 days, followed by free access to water for 3 days, during which time they were given oral saline, Ma-sol (Ma dose: 92 mg / kg), and NT, NOT, and NOMT (Pt dose: 4 mg / kg, Ma dose: 92 mg / kg), respectively. Mouse weight, occult blood status, and fecal consistency were recorded daily, and the Disease Activity Index (DAI) was calculated. After 10 days, the spleens of the mice were sacrificed for weight measurement, and the colons for length measurement. DAI is a comprehensive score based on the percentage of weight loss, occult blood status, and fecal consistency; the average score of these three parameters was calculated to obtain the DAI. The indicators are shown in Table 1 below.

[0129] Table 1

[0130]

[0131]

[0132] like Figure 12 As shown in Figure a, during the modeling and treatment periods, compared to the saline group where mouse weight gradually increased, all groups of mice experienced weight loss and exhibited significant colitis symptoms, including bleeding and diarrhea, after DSS modeling. Based on this, the DAI index was calculated using three indicators: weight loss, occult blood, and fecal consistency. Figure 12As shown in Figure b, the DAI index in the DSS model group increased sharply compared to the saline group. From days 7 to 10, after replacing DSS with saline, compared to the DSS group, the NT group, and the NOT group, mice continued to lose body weight, and their bleeding and diarrhea symptoms worsened, with the DAI index continuing to rise. Mice in the Ma-sol and NOMT groups showed symptom relief and a decrease in the DAI index, indicating a significant anti-inflammatory effect. Notably, compared to the Ma-sol group, because NOMT can target and deliver Ma to the colonic lesion site, the NOMT group showed a greater degree of relief of colitis symptoms than the Ma-sol group, and the DAI index decreased faster in the NOMT group. After treatment, the colon and spleen of mice in each group were sacrificed for analysis. Figure 12 As shown in the middle CD, compared with the saline group, after DSS modeling, the colon of mice was significantly shortened and the spleen was significantly enlarged. The NOMT and Ma-sol groups significantly alleviated these symptoms, and the NOMT group had a higher therapeutic effect than the Ma-sol group.

[0133] The above results demonstrate that the nano-self-assembled hydrogel precursor sheet prepared in this invention can be precisely released to target colonic lesions.

[0134] Ma is effective in treating colitis and is beneficial for the synergistic treatment of CRC.

[0135] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nano-self-assembled hydrogel precursor tablet, obtained by compression of cross-linked nano-prodrugs, or by compression of cross-linked nano-prodrugs mixed with drug-loaded orthoester compounds, wherein the cross-linked nano-prodrugs are constructed by cross-linking macromolecular polymers with dicarboxylic chemotherapeutic drugs via amide bonds and then self-assembling, and its structural formula is shown in formula (I): ； （I） in, P1 represents a macromolecular polymer, specifically selected from carboxymethyl chitosan, collagen, silk fibroin, or whey protein; M1 represents any one of the following (i) to (x) dicarboxylic acid chemotherapeutic drugs: 、 、 (i) (ii) 、 、 （ⅲ） （ⅳ） 、 、 (v) (Vi) 、 、 （ⅶ） （ⅷ） 、 ； （ⅸ） （ⅹ） 。 2. The nano-self-assembled hydrogel precursor sheet according to claim 1, characterized in that, The molecular weight of the carboxymethyl chitosan is 10,000-1,000,000 Da, the molecular weight of the collagen is 50,000-300,000 Da, the molecular weight of the silk fibroin is 100,000-400,000 Da, and the molecular weight of the whey protein is 14,000-150,000 Da.

3. The nano-self-assembled hydrogel precursor sheet according to claim 1 or 2, characterized in that, The preparation method of the cross-linked nanoprodrug includes the following steps: (1) Dissolve the dicarboxylic chemotherapeutic drug, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in water and stir at room temperature to obtain an activated dicarboxylic chemotherapeutic drug solution. (2) Dissolve the macromolecular polymer in water and add it dropwise to the activated dicarboxylic acid chemotherapy drug solution, and stir the reaction at room temperature to obtain the cross-linked solution; (3) The cross-linked solution was dialyzed to obtain an aqueous solution of cross-linked nano-prodrug, which was then freeze-dried after being rapidly frozen in liquid nitrogen to obtain the cross-linked nano-prodrug.

4. The nano-self-assembled hydrogel precursor sheet according to claim 3, characterized in that, In step (1), the molar ratio of the dicarboxylic chemotherapeutic drug, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) is 1:(2.0-5.0):(2.0-5.0); the stirring time is 2-6 h.

5. The nano-self-assembled hydrogel precursor sheet according to claim 3, characterized in that, In step (3), the liquid nitrogen quick-freezing time is 1-5 min; the freeze-drying time is 24-72 h, and the freeze-drying temperature is -50~-40℃.

6. The nano-self-assembled hydrogel precursor sheet according to claim 1, characterized in that, The orthoester compound is selected from any one of the following compound structural formulas (II): (II) R1 is selected from any one of hydrogen, methyl, ethyl, propyl, isopropyl, butyl, or phenyl; R2 is selected from any one of ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl.

7. The nano-self-assembled hydrogel precursor sheet according to claim 6, characterized in that, The mass ratio of the orthoester compound to the cross-linked nanoprodrug is 1:4-4:

1.

8. The method for preparing the nano-self-assembled hydrogel precursor sheet according to any one of claims 1 to 7, characterized in that, Including the following methods: Method 1: Grind and compress the cross-linked nanoprodrug to obtain a nano-self-assembled hydrogel precursor tablet; Method 2: Includes the following steps: S1. Dissolve the drug in an orthoester compound to prepare a drug-loaded oil phase; S2. The drug-loaded oil phase is mixed with the cross-linked nano-prodrug aqueous solution, and a uniform emulsion is prepared by ultrasonication. After being rapidly frozen in liquid nitrogen, the emulsion is freeze-dried, ground, and pressed into tablets to obtain nano-self-assembled hydrogel precursor tablets loaded with drug-loaded orthoester compounds.

9. The use of the nano-self-assembled hydrogel precursor sheet according to any one of claims 1 to 7 in the preparation of a drug for treating colorectal cancer.

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