Oral in-situ assembled gel drug delivery system and application thereof

Abstract

The invention discloses an oral in-situ assembled drug loading system and application thereof. The oral in-situ assembled drug loading system comprises a component A and a component B which can be separately administrated, the component A comprises hyaluronic acid which is modified by protocatechuic acid and coats a platelet membrane; the component B comprises hyaluronic acid which is modified by a thiourea group and is loaded with an STING agonist and traditional Chinese medicine polysaccharide. During administration, the component A is orally taken first, then the component B is orally taken after 2-10 minutes, and the component A and the component B are subjected to in-situ cross-linking gel formation through a catechol-thiourea specific coupling reaction in a high reactive oxygen inflammation environment of a postoperative wound surface of a digestive tract tumor. Wherein the STING agonist MSA-2 and the traditional Chinese medicine polysaccharide lentinan (LNT) form a supramolecular compound; the LNT can also prolong an STING signal activated by MSA-2 by depolymerizing microtubulin to generate a synergistic immune effect. The oral gel preparation has the advantages of being convenient to take, accurate in targeting, controllable in gelling, synergistic and the like, and shows a relatively high clinical transformation value in the fields of digestive tract tumor immune regulation, combined treatment and the like.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to an orally oriented in-situ assembled gel drug delivery system and its application. Background Technology

[0002] In the clinical treatment of gastrointestinal tumors, surgical resection is the primary treatment for solid tumors, but its effectiveness is severely limited by the high recurrence rate after surgery. Numerous clinical research data indicate that due to the invasive growth characteristics of tumor tissue and the limitations of the surgical field, traditional surgical resection struggles to completely eliminate tumor cells. Residual tumor cells gain a proliferative advantage in the unique immunosuppressive microenvironment after surgery, ultimately leading to tumor recurrence. This pathological process conforms to the classic "seed-soil" theory in oncology, where residual tumor cells constitute the "seed" of recurrence, while the immunosuppressive microenvironment formed after surgery provides the necessary "soil" support. Taking gastric cancer treatment as an example, clinical statistics show that even patients who undergo radical surgical resection still have a five-year recurrence rate as high as 40%–60%. In-depth research indicates that the characteristics of the postoperative microenvironment in gastric cancer mainly include: excessive secretion of pro-inflammatory factors (such as IL-6 and TNF-α) due to persistent inflammatory responses; abnormal recruitment and activation of immune cells (such as tumor-infiltrating lymphocytes and macrophages); and the resulting accelerated angiogenesis and abnormal tissue remodeling. These pathological changes collectively constitute the microenvironmental basis for promoting tumor recurrence. Therefore, developing therapeutic strategies that can effectively regulate the postoperative immune microenvironment is of great clinical significance.

[0003] In recent years, the cGAS-STING signaling pathway, as a key regulatory pathway of the innate immune system, has received widespread attention in the field of tumor immunotherapy. This pathway activates downstream signaling cascades by recognizing cytoplasmic DNA, promoting the production of pro-inflammatory factors such as type I interferon, thereby enhancing the activation of antigen-presenting cells and the anti-tumor immune response of T cells. The emergence of non-nucleotide oral small molecule STING agonists (such as MSA-2) has overcome the low oral bioavailability of traditional nucleotide agonists and shown promising application prospects. However, due to the body's self-regulation mechanisms, after STING receptor activation, it is transported to the degradation zone by the tubulin system, causing its agonistic effect to weaken over time, making it difficult to achieve sustained and stable immune activation, thus limiting its long-term immunomodulatory efficacy at the tumor site.

[0004] Meanwhile, traditional Chinese medicine polysaccharides with multiple immunomodulatory functions (such as Lentinan (LNT)) have attracted attention. LNT and other traditional Chinese medicine polysaccharides can activate immunity, promote antigen presentation, and regulate macrophage polarization through signaling pathways such as TLR4 / NF-κB. More importantly, studies have found that LNT can bind to and depolymerize microtubules, preventing the transport and degradation of activated STING protein, thus theoretically prolonging the duration of STING signaling. Therefore, combining STING agonists with traditional Chinese medicine polysaccharides to construct a "moderate activation and long-term maintenance" model represents a highly promising therapeutic approach for achieving localized, sustained, and mild immune activation while avoiding the risks of excessive signal accumulation (such as cytokine storms).

[0005] However, translating the above theories into effective clinical treatment plans faces significant challenges. The core issue lies in the lack of a drug delivery system capable of precisely and synchronously delivering the two active ingredients to the postoperative lesion and achieving controlled release. First, STING agonists and polysaccharide drugs have significantly different physicochemical properties at the molecular level. Current technologies lack an effective mechanism to form a stable complex, leading to asynchronous absorption, distribution, metabolism, and release in vivo, making it difficult to achieve optimal synergistic effects. Second, when administered orally, the two active ingredients are easily degraded and inactivated by the complex gastrointestinal environment (such as digestive enzymes and pH changes). Furthermore, conventional oral or injectable formulations are difficult to retain effectively in the dynamic wounds of the stomach or digestive tract after surgery; the drug is rapidly emptied or diluted, failing to maintain sufficient therapeutic concentrations at the lesion site. In addition, excessively rapid drug release may cause significant gastrointestinal irritation or systemic toxicity.

[0006] At the dosage form technology level, existing instant gelation technology based on the "catechol-thiourea" chemical cross-linking principle provides a solution for local drug delivery. This type of technology typically uses dopamine hydrochloride as a catechol donor, whose reaction kinetics with the thiourea group are extremely fast (usually less than 5 seconds), making it suitable for simultaneous spraying and instant curing under endoscopy. However, it is precisely this ultra-fast reaction characteristic that makes it completely unsuitable for oral drug delivery. In oral drug delivery design, if two rapidly reacting components are introduced simultaneously or sequentially into the dynamic gastrointestinal environment, the excessively rapid cross-linking reaction can cause the drug delivery system to undergo non-specific and uncontrolled gelation before reaching the target lesion (e.g., in non-lesion areas of the esophagus or stomach). This non-targeted premature gelation not only consumes a large amount of the effective drug component, significantly reducing the drug accumulation at the lesion site, but the resulting gel clumps may also affect its normal passage through the gastrointestinal tract, even causing discomfort, ultimately leading to loss of therapeutic targeting and decreased efficacy. Therefore, developing an oral in situ gelation system whose gelation rate can match the physiological transport process of the gastrointestinal tract after oral administration, and which has both excellent lesion targeting and in vivo stability, has become a technical bottleneck that must be overcome to achieve precision immunotherapy for gastrointestinal tumors.

[0007] To address the aforementioned challenges, there is an urgent need to develop an orally administered, in-situ assembled hydrogel drug delivery system. This system should allow for convenient oral administration, protect drug activity in the complex gastrointestinal environment, and intelligently identify the specific microenvironment of the postoperative wound (such as high levels of reactive oxygen species), precisely triggering in-situ gelation at that site. The resulting gel should adhere firmly to the wound, providing a physical protective barrier, and simultaneously release the STING agonist and traditional Chinese medicine polysaccharides in a controllable manner. Through the theoretically validated synergistic mechanism of these two components, it should provide long-term regulation of the local immune microenvironment, thereby fundamentally inhibiting postoperative tumor recurrence. This constitutes the research starting point and the core technical problem to be solved in this invention. Summary of the Invention

[0008] This invention aims to address the unique inflammatory and high-oxidative stress microenvironment of post-tumor resection wounds by proposing an orally administered, in-situ drug delivery system and its applications. Based on in-depth analysis of the pathological characteristics of post-operative wounds, this system, through responsive construction, is designed to achieve three main functions: First, it utilizes platelet membrane-functionalized catechol-modified HA as a drug carrier and hydrogel matrix. Leveraging the natural biocompatibility of catechol and the dual effects of platelet-specific adhesion molecules, it forms high-strength bioadhesion with wound tissue, providing an "anchor" for subsequent in-situ gelation. Second, by introducing thiourea-functionalized HA, it enables a specific "catechol-thiourea" coupling chemical reaction with the platelet membrane-HA complex in the high-ROS microenvironment of the wound, achieving in-situ cross-linking and gelation. Finally, the LNT loaded in this system and the novel STING agonist MSA-2 can be continuously and simultaneously released under the dual control of in vivo carboxylesterase and the autonomous degradation of the hydrogel network. MSA-2 and LNT synergistically form a multi-layered immune regulatory network, thereby regulating the local and systemic immune microenvironment.

[0009] To achieve the above objectives, the present invention provides an orally oriented in-situ assembled gel drug delivery system, the system comprising separately administerable component A and component B; Component A comprises a first hyaluronic acid derivative modified with a catechin group; Component B contains a second hyaluronic acid derivative modified with a thiourea group; The components A and B are configured to be administered by first orally taking component A, followed by orally taking component B at a predetermined time interval, so that in situ cross-linking and gelation can be achieved at the target inflammatory site in the digestive tract through a catechol-thiourea coupling reaction.

[0010] Furthermore, in component A, the first hyaluronic acid derivative is hyaluronic acid modified with protocatechuic acid; component A also includes a platelet membrane modified on the surface of the first hyaluronic acid derivative by a post-insertion technique.

[0011] Furthermore, the STING agonist is MSA-2, SNX281, SR-717, or CRD3874-SI; the traditional Chinese medicine polysaccharide is lentinan, tremella polysaccharide, astragalus polysaccharide, poria cocos polysaccharide, or bletilla striata polysaccharide.

[0012] Furthermore, the STING agonist is MSA-2, and the traditional Chinese medicine polysaccharide is lentinan (LNT); and the MSA-2 and the lentinan (LNT) form a supramolecular complex through non-covalent interactions, wherein the molar ratio of MSA-2 to LNT repeating units in the supramolecular complex is 1:1 to 1:2.

[0013] Furthermore, in component B, MSA-2 is covalently linked to the second hyaluronic acid derivative via an enzyme-sensitive bond, and LNT is loaded into the three-dimensional network of the second hyaluronic acid derivative through physical mixing and the supramolecular complex formed with MSA-2.

[0014] Furthermore, the predetermined time interval between the first oral component A and the subsequent oral component B is 2 to 10 minutes.

[0015] A key improvement of this invention lies in the selection of the catechol donor. Unlike the prior art, which commonly uses dopamine hydrochloride, this invention preferentially uses protocatechuic acid (3,4-dihydroxybenzoic acid) as the catechol modifying group, and finds that this substitution can solve the targeting problem of oral administration. The core mechanism is that protocatechuic acid and dopamine hydrochloride have fundamental differences in chemical structure (protocatechuic acid has a carboxyl group, while dopamine has an aminoethyl chain), which leads to differences in their electronic effects and steric hindrance, thus affecting their oxidation rate and reactivity with the thiourea group. Compared to the uncontrollable ultrafast gelation (<5 s) of the dopamine system, the reaction kinetics of protocatechuic acid-modified HA-CAT and HA-NCSN are more moderate and controllable, effectively regulating the gelation time within the range of 2–10 min. This optimized time window aligns with the environmental characteristics of the digestive tract and the stepwise oral administration strategy of this invention. This ensures that component A has sufficient time (orally administered first) to preferentially reach and anchor at the gastric wound, followed by component B (orally administered later) to effectively cross-link with the anchored component A. This greatly avoids ineffective gelation at non-target sites, significantly improving drug accumulation efficiency and targeting rate at the lesion site. Existing dopamine-based ultrafast gelation systems, due to their rapid reaction, are difficult to implement effectively in a stepwise oral administration manner. Meanwhile, the hydrogel network constructed based on protocatechuic acid exhibits higher mechanical strength and in vivo stability, providing a more durable physical barrier for drug release and wound protection.

[0016] This design fully utilizes the chemical property that catechol structures can be converted into quinone structures under oxidative conditions (inflammatory environment), and then specifically coupled with thiourea, endowing the A+B system with excellent oxidative-responsive gelling properties. The HA matrix not only provides structural support and enhances the mechanical properties of the gel system, but also, through its specific binding ability to CD44 receptors on the surface of tumor cells, enables the gel system to actively target tumor sites. To further enhance the biocompatibility and tissue adhesion of the gel system, platelet membrane bio-components are inserted into the HA molecular chain of component A through surface modification. Finally, the STING agonist and traditional Chinese medicine polysaccharides are co-loaded into the three-dimensional network structure of the hydrogel. Through the synergistic effect of the two active ingredients, the tumor immune microenvironment is effectively activated, achieving therapeutic effects of inhibiting tumor growth and preventing recurrence.

[0017] Regarding the selection of STING agonists, considering the need for oral administration and the ability to effectively activate the cGAS-STING signaling pathway to regulate the immune microenvironment, selectable drugs include, but are not limited to, MSA-2, SNX281, SR-717, CRD3874-SI, and other STING pathway agonists that can be administered orally or are suitable for gastrointestinal administration. This invention preferably uses a non-nucleotide STING agonist with good oral bioavailability as the active ingredient. In particular, MSA-2, an oral small molecule STING agonist, is preferred. As an embodiment, this compound exhibits excellent intestinal absorption properties and stable metabolic characteristics.

[0018] Regarding the selection of drug components, based on the dual considerations of meeting the requirements for immune activation while also possessing appropriate viscosity and gelling properties to facilitate hydrogel formation, this invention particularly favors polysaccharides from traditional Chinese medicines with multiple biological activities as the active ingredients of the hydrogel. Representative polysaccharides that can be selected include, but are not limited to, LNT, Tremella fuciformis polysaccharide, Bletilla striata polysaccharide, Polyporus umbellatus polysaccharide, Astragalus membranaceus polysaccharide, and other naturally derived active polysaccharides from traditional Chinese medicines. Through in-depth research on the immune activation mechanism of the STING pathway and the interaction of drug combinations, LNT is particularly preferred as an implementation scheme. This traditional Chinese medicine polysaccharide can form a complex with MSA-2 through hydrogen bonds, co-loaded into the hydrogel network, and simultaneously released through an enzyme response mechanism. LNT can prolong the STING signal by promoting microtubule depolymerization, thus synergistically working with MSA-2.

[0019] Experimental verification revealed that the supramolecular complex of MSA-2 and LNT possesses the following technical characteristics: It spontaneously forms at physiological pH through hydrogen bonding and hydrophobic interactions. The hydroxyl group (-OH) of LNT acts as a hydrogen bond donor, and the carbonyl oxygen (C=O) of MSA-2 acts as a hydrogen bond acceptor. Due to steric hindrance, the molar ratio of hydrogen bonds formed between a repeating unit of LNT and MSA-2 is approximately 1:1~2. The two self-assemble into a complex at this ratio. This complex slows down the enzymatic degradation rate of MSA-2. When carboxylesterase (CES) cleaves the ester bonds of MSA-2, the complex releases LNT synchronously with gel degradation, thus maintaining a consistent therapeutic ratio in vivo. This complex can co-load, co-transport, and synchronously release drugs, ensuring that the two drugs exert their effects at the lesion site in a fixed ratio. Furthermore, LNT, through its unique tubulin depolymerization mechanism, ensures a gentle yet sustained enhancement of the STING pathway activation effect induced by MSA-2, thereby achieving a synergistic immunotherapeutic effect.

[0020] This invention discovers that various polysaccharides from traditional Chinese medicines with multi-hydroxyl structures (such as lentinan, tremella polysaccharide, and astragalus polysaccharide) and various small-molecule STING agonists (such as MSA-2 and ADU-S100) can form supramolecular complexes with a defined molar ratio through non-covalent interactions such as hydrogen bonding and hydrophobicity. These complexes can be co-loaded in a hydrogel network and simultaneously released in vivo through mechanisms such as enzyme responses. This "co-loading and co-release" characteristic solves the core problem of pharmacokinetic mismatch in traditional combined drug administration, ensuring that the two active ingredients exert their effects simultaneously at the lesion site in a fixed ratio, thereby generating a synergistic immune activation effect. Therefore, this synergistic delivery approach based on supramolecular complexes is a universal mechanism for the "traditional Chinese medicine polysaccharide-STING agonist" combination strategy.

[0021] Building upon the general mechanism described above, this invention delves into the synergistic mechanism between LNT and MSA-2, discovering that LNT not only forms a supramolecular complex with MSA-2, but also uniquely binds to and promotes the depolymerization of tubulin. This action blocks the transport of activated STING protein to the degradation zone via the microtubule system, thereby prolonging the duration of the MSA-2-activated STING signaling pathway. This unique "activation (MSA-2) + maintenance (LNT)" model does not simply enhance the intensity of STING signaling, but aims to achieve a stable and durable state of immune activation. This model, while ensuring an effective anti-tumor immune response, significantly reduces the risk of cytokine storms caused by excessive activation of the STING pathway, constituting the core mechanism of their synergistic anti-tumor effect with added safety.

[0022] The orally edible, in-situ assembled gel system achieves autonomous cross-linking and gel formation through a specific response to the high oxidative stress microenvironment at the inflammatory site. Its mechanism of action is based on the chemical reaction characteristic of catechol structures transforming into quinone structures under oxidative conditions, utilizing the specific coupling between the generated quinone structure and thiourea groups to achieve hydrogel cross-linking. To optimize the gel system's response to the inflammatory environment, this invention preferably uses organic compounds containing catechol structures as functional modifying groups, including but not limited to: protocatechuic acid (3,4-dihydroxybenzoic acid), gallic acid, caffeic acid, catechins, dopamine and its hydrochloride salts, and other compounds with catechol structures; preferably, organic compounds containing thiol structures provide conditions for coupling with catechols, including but not limited to: aminothiourea, methyl isothiocyanate, ethyl isothiocyanate, phenyl isothiocyanate, cysteine, dithiothreitol, and other compounds.

[0023] The responsive crosslinking hydrogel system exhibits excellent gel-forming properties. Its significant technical feature is that the gel-forming process is not affected by external conditions such as ambient temperature and pH. By selecting specific catechol donors, the gel-forming process is controlled within an optimized time window of 2 to 10 minutes. This moderate gel-forming rate is consistent with the stepwise oral administration strategy, ensuring that component A is preferentially anchored to the lesion before component B can efficiently crosslink with it. Thus, it has controllable responsive gel-forming characteristics, demonstrating excellent rapid gel-forming ability while avoiding non-targeted gel-forming caused by excessively fast rates.

[0024] The present invention also provides a pharmaceutical composition comprising a traditional Chinese medicine polysaccharide and a STING agonist. The two can form a stable physical complex through hydrogen bonding and hydrophobic interactions, and synergistically exert an immune-activating effect. Furthermore, the combination of MSA-2 and LNT exhibits superior therapeutic effects due to the unique microtubule depolymerization-STING signaling prolongation mechanism of LNT.

[0025] The present invention also provides a method for preparing an orally oriented, in-situ assembled gel drug delivery system as described above, comprising the following steps: (1) Synthesize the first hyaluronic acid derivative modified with catechol groups as the precursor of component A; (2) A second hyaluronic acid derivative modified with a thiourea group was synthesized as the matrix of component B; (3) The STING agonist and traditional Chinese medicine polysaccharide were loaded onto the second hyaluronic acid derivative to obtain component B; (4) Platelet membrane is modified onto the surface of the first hyaluronic acid derivative by post-insertion technique to obtain component A.

[0026] The present invention also provides the application of the oral in situ assembled gel drug delivery system as described above in the preparation of a drug for the prevention or treatment of postoperative recurrence of gastrointestinal tumors.

[0027] The present invention also provides a method of administration for the treatment of gastrointestinal tumors, the method comprising: first orally administering the above-mentioned component A to a subject in need, and then, after an interval of 2 to 10 minutes, orally administering the above-mentioned component B to the subject.

[0028] This invention systematically evaluated the in vitro performance of the responsive in situ assembled gel (MSA-2&LNT@G), specifically including the following aspects: 1) Responsive gelling performance test: MSA-2 & LNT-NCSN and PLT / HA-CAT components were physically mixed at a predetermined ratio, and a 0.1% NaIO4 solution was used to simulate the high oxidative stress state of the in vivo inflammatory environment. The dynamic rheological parameters of the gel system under different ratios were measured using a rotational rheometer, including the changes in storage modulus (G') and loss modulus (G"), to quantitatively characterize its oxidative responsive gelling behavior. Based on the rheological test results, the hydrogel matrix ratio scheme with the best inflammatory response characteristics was selected.

[0029] 2) Environmental stability evaluation: An in vitro dissolution experiment was designed to simulate the stability of MSA-2&LNT@G under different physiological environments: simulated gastric acid environment: hydrochloric acid solution at pH 1.2; simulated tumor microenvironment: phosphate buffer at pH 6.5; physiological neutral environment: PBS buffer at pH 7.4; the structural stability of the gel under various environmental conditions was evaluated by measuring the mass loss rate of the gel at different time points.

[0030] 3) Microstructure characterization: The microstructure of MSA-2&LNT@G was observed using scanning electron microscopy (SEM): After the samples were freeze-dried, the three-dimensional porous network structure of the gel was observed at different magnifications, the pore size distribution was measured and the surface morphology characteristics were analyzed.

[0031] 4) Drug release behavior study: Establish a high performance liquid chromatography (HPLC) method to systematically investigate the drug release characteristics of MSA-2 & LNT@G: optimize chromatographic conditions, establish standard curves and validate the methodology, determine drug release kinetic curves under different simulated physiological conditions, and analyze the release mechanism.

[0032] 5) Characterization and release verification of the drug complex: Differential scanning calorimetry (DSC) was used to detect the blend of MSA-2 and LNT, and a shift in the endothermic peak was found (ΔT = 12.3℃), confirming the intermolecular interaction; Fourier transform infrared spectroscopy (FTIR) was used to observe the C=O bond peak shift of MSA-2 (1720 cm⁻¹). -1 →1705 cm -1 This indicates the formation of hydrogen bonds.

[0033] This invention conducts an in-depth study on the in vitro pharmacodynamic evaluation and molecular mechanism of MSA-2 & LNT@G. Taking a gastric cancer model as an example, the experimental design is as follows: 1) In vitro cytotoxicity evaluation: Cell models used included normal gastric mucosal epithelial cells (Ges-1), mouse gastric cancer cells (MFC), dendritic cells (DC2.4), and macrophages (RAW264.7). The following groups were established: LNT monotherapy group, MSA-2 monotherapy group, MSA-2 + LNT combination group, MSA-2 & LNT-NCSN group, PLT / HA-CAT group, hydrogel final formulation group, and blank hydrogel excipient group. The MTT assay was used to determine the toxic effects of each drug group on normal cells at different concentration gradients, and to determine the safe dosage concentration range for each formulation.

[0034] 2) Transwell assay: A DC2.4 / MFC and RAW264.7 / MFC Transwell co-culture model was established. Effector cells (DC2.4 or RAW264.7) were seeded in the upper chamber, and target cells (MFC) were cultured in the lower chamber. The treatment preparations of each group were used to determine the safe concentration for 24 hours. The survival rate of MFC cells in the lower chamber was determined by the MTT assay.

[0035] 3) Cellular uptake mechanism study: Using MFC, DC2.4, and RAW264.7 cell lines as models, each drug-treated group was labeled with C6 fluorescent dye. Intracellular fluorescence distribution was observed using laser confocal microscopy; fluorescence intensity was quantitatively analyzed by flow cytometry; sucrose (clathrin inhibition), genistein (cryptin inhibition), amiloride (macropinoplasm inhibition), and low temperature (energy inhibition) treatment groups were set up, and different drugs were co-incubated with cells to study the cellular drug uptake mechanism.

[0036] 4) Dendritic cell maturation induction: Using DC2.4 dendritic cells as a model, the above-mentioned drug groups were treated with a certain concentration. The expression of CD45-BV421, MHC-II-PE / Cy7, CD86-PE, CD11b-FITC and CD11c-APC were detected by flow cytometry to assess the maturation level of DC2.4.

[0037] 5) Macrophage polarization regulation study: Using RAW264.7 as a cell model, cells were stimulated with the classic M1 polarization inducer lipopolysaccharide (LPS, 100 ng / mL) and the M2 polarization inducer interleukin-4 (IL-4, 50 ng / mL), and intervention was performed by administering the above-mentioned drugs at specific concentrations. The expression of surface markers CD86 (M1) and CD206 (M2) was detected by flow cytometry; the cytokine secretion profile was detected by ELISA; and the expression of iNOS (M1) and Arg-1 (M2) was detected by immunofluorescence.

[0038] 6) Signaling pathway mechanism study: A DC2.4 / MFC and RAW264.7 / MFC Transwell co-culture model was established. DC2.4 and RAW264.7 cells were incubated with different concentrations of each treatment group formulation for 24 h. Western blot analysis was used to analyze the expression of key proteins in NF-κB and STING-related pathways.

[0039] This invention comprehensively evaluates the in vivo delivery and tumor recurrence prevention efficacy of the MSA-2 & LNT@G composite hydrogel formulation. Taking a gastric cancer model as an example, the experimental design is as follows: 1) In vivo delivery and distribution studies: PLT / HA-CAT was labeled with a Cy5.5 fluorescent probe, and MSA-2 & LNT-NCSN were labeled with a Cy7 fluorescent probe. Free probes served as controls. Healthy C57BL / 6 mice (20 ± 2 g) were used as models, and the above preparations were administered sequentially by gavage. The distribution and overlap of the two fluorescence methods were dynamically monitored at different time points (0.5 h, 2 h, 6 h, 12 h, 24 h) after administration using an in vivo imaging system. After euthanizing the mice, gastric tissue was harvested for in vitro imaging. Frozen sections of gastric tissue were prepared, and the fluorescence distribution of each layer of the gastric structure was observed using a confocal laser microscope (CLSM).

[0040] 2) Establishment of an orthotopic gastric cancer model: First, MFC cells were subcutaneously inoculated into 615 mice to establish a xenograft tumor model. After subcutaneous tumor formation, the mice were sacrificed, the tumor masses were removed, and 1 mm thick tumor tissue was selected from well-grown tumor tissue. 3 Tissue block. A tumor block was surgically implanted in the gastric parenchyma of C57BL / 6 mice, and the tumor block was fixed and the wound was sealed with a matrix adhesive. Pathological confirmation was performed 7 days postoperatively.

[0041] 3) Pharmacodynamic evaluation of relapse prevention: C57BL / 6 mice with tumors removed after tumor modeling were divided into groups and administered drugs for 14 days (n=10): saline control group, LNT monotherapy group, MSA-2 monotherapy group, MSA-2+LNT combination group, MSA-2&LNT-NCSN group, PLT / HA-CAT group, hydrogel final preparation group, and blank hydrogel excipient group. Body weight, survival status, and survival time were recorded daily. After 14 days, the animals were euthanized by cervical dislocation, and blood routine tests, liver function tests, TNF-α, and IL-6 levels were examined. Spleen and liver weights were measured, and tumor inhibition rate, liver and spleen indices were calculated: Tumor inhibition rate = (1 - average tumor volume in the treatment group / average tumor volume in the control group) × 100%; Liver index = liver weight (mg) / mouse body weight (g); Spleen index = spleen weight (mg) / mouse body weight (g). Routine paraffin embedding, sectioning, HE staining, Ki-67, and TUNEL histopathological examinations were performed.

[0042] 4) Immune cell subset analysis: Recurrent gastric cancer tissue, spleen, and lymph nodes of mice were dissected, and the abundance of dendritic cells (CD86+MHC-II+), T cell subsets (CD4+CD45+, CD3+CD8+), regulatory T cells (CD4+FoxP3+), and tumor-associated macrophages (M1 type: F4 / 80+CD11b+CD86+; M2 type: F4 / 80+CD11b+CD206+) was detected by flow cytometry.

[0043] 5) Cytokine profile analysis: Recurrent gastric cancer tissue, spleen, and lymph nodes of mice were dissected, pulverized, sonicated, and further centrifuged to obtain homogenate supernatants of gastric cancer tissue, spleen, and lymph nodes. The expression of relevant cytokines in the samples was detected by ELISA kit: pro-inflammatory factors (IL-6, TNF-α, IL-12), anti-inflammatory factors (IL-10), and interferon family (IFN-β, IFN-γ).

[0044] 6) Immune memory function assay: On day 15 after the first administration, draining lymph nodes, spleen cells, and peripheral blood were collected. The relative abundance of central memory T cells (Tcm, CD3+CD44+CD62L+), effector memory T cells (Tem, CD3+CD44+CD62L-), and tissue-resident memory T cells (Trm, CD3+CD44+CD62L-CD69+) was determined by flow cytometry.

[0045] 7) Signaling pathway mechanism study: Cancerous tissue from mice was dissected and processed through methods such as fragmentation and sonication to extract tumor tissue proteins. Western blot experiments were used to detect the expression of NF-κB and STING signaling pathway-related proteins in cells, as well as the level of phosphorylated proteins, to verify the molecular mechanism related to its treatment.

[0046] 8) Detection of biomarkers related to gastric cancer recurrence: Detection indicators: DAMPs-related proteins (HMGB1, HSP70, calreticulin), serum biomarkers of gastric cancer recurrence (PGⅠ, PGⅡ, AFP), tumor biomarkers (CEA, CA19-9, CA72-4), etc.

[0047] 9) In vivo safety evaluation: Healthy C57BL / 6 mice (20 ± 2 g) were used as a model, and the above preparation was administered for 14 days. Blood samples were collected. Complete blood count (red blood cells, white blood cells, platelets, etc.); major organ coefficients (heart, liver, spleen, lung, kidney); histopathological evaluation of each organ tissue (HE staining to assess organ toxicity); liver function indicators (ALT, AST, etc.).

[0048] Compared with the prior art, the present invention has the following advantages and technical effects: This invention provides an intelligent hydrogel drug delivery system based on inflammatory-responsive in-situ assembly. Its key technical feature is that, after oral administration into the digestive tract, it can specifically identify the high-oxidative-stress microenvironment of postoperative wounds from digestive tract tumors (such as oral cancer, esophageal cancer, gastric cancer, and colorectal cancer) and achieve precise in-situ gelation. The core design principle of this system is as follows: First, platelet membrane-functionalized HA forms a specific bioadhesive "anchor" with wound tissue; then, under the high ROS conditions of the wound's inflammatory microenvironment, the ortho-dihydroxyl structure in the catechol-functionalized HA is oxidized to a quinone structure, undergoing a specific "catechol-thiourea" coupling chemical reaction with the thiourea group modified on another HA molecule, achieving precise cross-linking and in-situ construction of a three-dimensional network structure at the wound site. This dual-targeting design not only ensures efficient enrichment and retention of the hydrogel at the lesion site but also significantly enhances the gel's wound retention performance through a biomimetic adhesion mechanism.

[0049] This system employs a dual-mechanism drug delivery strategy combining chemical synthesis and physical methods. The STING agonist MSA-2 is covalently coupled to the hydrogel component via enzyme-sensitive ester bonds. The traditional Chinese medicine polysaccharide LNT forms a complex with MSA-2 through hydrogen bonds, and both are co-loaded within the three-dimensional network of the hydrogel. Regarding the drug release mechanism, the system utilizes an enzyme-responsive release strategy. Once the hydrogel forms at the site of inflammation, the ester bonds break under the catalysis of carboxylesterase (CES), thereby achieving the controlled simultaneous release of the MSA-2 and LNT complex. After the ester bonds break, the hydrogel structure is affected, and it begins to slowly degrade. This release method has the following technical advantages: First, it avoids rapid drug release and maintains a stable blood drug concentration; second, the release rate is positively correlated with the degree of inflammation, and the release of LNT is positively correlated with the release of MSA-2, achieving intelligent responsive drug delivery; third, MSA-2 and LNT can be released simultaneously and produce synergistic immunomodulatory effects, achieving systematic regulation of the local and systemic immune microenvironment by activating the cGAS-STING pathway and regulating the NF-κB signaling network.

[0050] This invention is the first to discover that the combination of traditional Chinese medicine polysaccharides and STING agonists can form a stable physical complex through hydrogen bonding and hydrophobic interactions. In the representative polysaccharides MSA-2 and LNT selected in this invention, this complex can be embedded in a hydrogel network and released synchronously through ester bond cleavage and gel degradation, ensuring consistent control of the two drugs' therapeutic ratio in vivo. Although other polysaccharides and STING agonists also exhibit this self-assembly mechanism, they cannot achieve the unique tubulin depolymerization-STING signaling prolongation mechanism specific to LNT. This unique synergistic mechanism makes the combination of MSA-2 and LNT exhibit significantly superior therapeutic effects compared to other combinations.

[0051] Regarding the optimization of the drug delivery route, this invention designs an oral delivery regimen by deeply analyzing the environment and characteristics of gastrointestinal tumors. This design fully utilizes the natural affinity of HA (hyaluronic acid) for CD44 receptors on tumor cell surfaces, achieving the following technical effects: First, it significantly improves the bioavailability of the drug in the stomach and throughout the digestive tract, overcoming the poor absorption of traditional oral formulations in the gastrointestinal tract; second, the in-situ formed hydrogel layer effectively isolates the wound from direct contact with the contents of the digestive tract, reducing postoperative discomfort for patients; finally, biomimetic modification of the platelet membrane enhances the retention performance of the formulation in the digestive tract, prolonging the duration of drug action. This integrated design strategy of "inflammatory response-in-situ gelation-long-acting release" provides an innovative solution for postoperative anti-recurrence treatment of gastrointestinal tumors, such as gastric cancer.

[0052] The stepwise oral administration regimen designed in this invention is innovative and irreplaceable: First, component A is orally administered, preferentially adhering to the wound surface; then, component B is orally administered, reacting with component A to form cross-links, avoiding gelation at non-target sites. Through the biocompatibility of platelet membranes and catechol structures, and the affinity of HA for CD44 receptors overexpressed in the wound, lesion enrichment of component A is achieved. After component A is anchored, the thiourea group of component B reacts only with oxidized catechol, avoiding ineffective cross-linking under gastric acid. Based on the reaction mechanism of "catechol-thiourea" and the degree of substitution in the synthesis of each component, the molar ratio of orally administered component A to component B is approximately 1:1. Based on the "anchoring" time of component A and the rate of gastric emptying, the oral interval between the two should be controlled between 2 and 10 minutes: <2 minutes is insufficient for component A to preferentially reach and adhere to the wound surface, resulting in non-target gelation, consuming some components A and B, thus resulting in insufficient effective ingredients reaching the wound surface; >10 minutes is insufficient for component A to gel in time, which may have been emptied into the small intestine by the stomach, resulting in a reduction in the total amount of component A available for cross-linking at the wound surface, and insufficient component A to react after component B enters, resulting in poor gelation effect.

[0053] In terms of formulation design, this invention functionalizes the hydrogel matrix through chemical modification, successfully constructing a smart, responsive, in-situ gel drug delivery system. The core innovation of this system lies in utilizing the inflammatory microenvironment to specifically trigger a catechol-thiourea coupling reaction, achieving in-situ cross-linking at the wound site and forming a hydrogel with a three-dimensional network structure. This inflammatory-responsive gelation mechanism not only ensures efficient enrichment and long-term retention of the formulation at the lesion site but also enables controlled drug release through CES-sensitive ester bonds, thus overcoming the technical bottlenecks of traditional drug delivery systems, such as burst release and short retention time.

[0054] In terms of treatment strategy, this invention, based on a deep understanding of the postoperative recurrence mechanisms of various tumors (taking gastric cancer as an example), considers residual tumor cells as the "seeds" of recurrence, while the immunosuppressive microenvironment serves as the "soil" supporting their growth. Targeting this pathological characteristic, a triple mechanism of action is designed: Firstly, the cGAS-STING signaling pathway is specifically activated by the STING agonist MSA-2, promoting type I interferon secretion and antigen-presenting cell activation; secondly, LNT regulates the NF-κB signaling network, enhancing IFN-β production and promoting dendritic cell (DC) maturation and tumor-associated macrophage (TAM) polarization towards the anti-tumor M1 type; thirdly, LNT binds to tubulin, causing it to depolymerize and blocking the transport of STING to the endoplasmic reticulum for degradation, thereby prolonging the STING signal and continuously activating immunity. This synergistic effect of the three pathways not only effectively improves the immunosuppressive microenvironment ("soil") but also effectively inhibits the survival and proliferation of residual tumor cells ("seeds"), thus fundamentally blocking the postoperative recurrence pathway of tumors.

[0055] The innovation of this invention is mainly reflected in the following aspects: 1. For the first time, the "catechol-thiourea" coupling chemical reaction is used to construct gastric in-situ hydrogels. The reactants selected in this patent that provide target functional groups are all used in hydrogel synthesis for the first time; 2. The "catechol-thiourea" responsive combination constructed in this invention controls the gelation rate to 2-10 min, which is more in line with the time window for oral administration; 3. For the first time, a general strategy of "traditional Chinese medicine polysaccharide-STING agonist" is proposed to achieve simultaneous release and synergistic immunity by forming supramolecular complexes. Taking LNT and MSA-2 as examples, it is discovered for the first time that LNT and MSA-2 can form complexes through hydrogen bonds in a 1:1-2 ratio, thereby releasing both simultaneously in vivo; 4. For the first time, it is discovered and verified that the specific combination of LNT and MSA-2 can enhance the anti-tumor effect through a unique "microtubule depolymerization-STING signal prolongation" mechanism; 5. For the first time, in-situ gelation at the target site is achieved through an oral administration of component A followed by oral administration of component B. This in-situ hydrogel within the digestive tract provides both a physical barrier to protect the wound and allows for long-term controlled drug release in vivo. Furthermore, this invention is the first to propose the formation of an in-situ hydrogel in the digestive tract through oral administration for postoperative recurrence prevention and immunomodulatory intervention in digestive tract tumors. These technological innovations collectively constitute the core competitive advantage of this invention, providing a novel technical approach for the prevention and treatment of postoperative recurrence of gastric cancer. Attached Figure Description

[0056] Figure 1 This is a schematic diagram illustrating the mechanism of action of the combined MSA-2 and LNT drug combination.

[0057] Figure 2This is a schematic diagram illustrating the in vivo response of hydrogel components A (PLT / HA-CAT) and B (MSA-2 & LNT-NCSN) after stepwise oral administration to gel and enzyme decomposition of the drug.

[0058] Figure 3 The synthetic route and 1H NMR spectrum of HA-ADH ( 1 H NMR spectrum; where Figure 3 A is the synthesis roadmap. Figure 3 B is 1 H NMR spectrum.

[0059] Figure 4 The synthetic route and 1H NMR spectrum of HA-CAT ( 1 H NMR spectrum; where Figure 4 A is the synthesis roadmap. Figure 4 B is 1 H NMR spectrum.

[0060] Figure 5 The synthetic route and 1H NMR spectrum of HA-NCSN ( 1 H NMR spectrum; where Figure 5 A is the synthesis roadmap. Figure 5 B is 1 H NMR spectrum.

[0061] Figure 6 This is a diagram of the gelation reaction mechanism of "catechol-thiourea".

[0062] Figure 7 This is an in vitro pharmacodynamic evaluation diagram of the effects of MSA-2 and LNT monotherapy on the proliferation of various cell types; among them, Figure 7 A and 7B represent the effects on MFC cells. Figure 7 C and 7D represent the effects on RAW264.7 cells. Figure 7 E is the effect on DC2.4 cells. Figure 7 F represents the effect of MSA-2 regulated RAW264.7 cells on MFC cell proliferation.

[0063] Figure 8 This is a comparison of the UV-Vis absorption spectra of LNT, MSA-2 monopharmaceuticals and their supramolecular complex (LNT / MSA-2).

[0064] Figure 9 This is a particle size distribution diagram of the LNT / MSA-2 supramolecular complex.

[0065] Figure 10 This is a flow cytometry analysis of the expression of antigen-presenting cell maturation markers in tumor tissue of a 615 mouse orthotopic gastric cancer model after different treatment regimens.

[0066] Figure 11 This is a histopathological verification image of a mouse orthotopic gastric cancer model; among them, Figure 11 Image A shows H&E staining and Ki-67 immunohistochemical staining of gastric tumors in C57BL / 6 mice after orthotopic modeling. Figure 11 B shows H&E staining and Ki-67 immunohistochemical staining of gastric tumors in 615 mice after in situ modeling. Detailed Implementation

[0067] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0068] All raw materials used in this invention are not particularly restricted in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0069] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0070] Example 1 This embodiment details a method for preparing catecholized hyaluronic acid (HA-CAT) using protocatechuic acid (PCA) as a catechol donor, and then coating platelet membranes with a post-insertion technique to finally obtain hydrogel component A (PLT / HA-CAT).

[0071] 1.1 Synthesis of intermediate product HA-ADH Reference Figure 3 The synthetic route shown in A involves dissolving hyaluronic acid (HA) in deionized water to prepare a 10 mg / mL solution. Under ice-bath stirring, two molar amounts of 1-hydroxybenzotriazole (HOBt) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) are slowly added to this solution to activate the carboxyl group for 30 minutes. The ice bath is then removed, and 15 molar amounts of adipic acid dihydrazide (ADH) are added to the activated solution. The reaction is carried out under constant temperature stirring at 30°C for 6 hours. After the reaction, the reaction solution is purified by dialysis with deionized water for 24 hours to remove unreacted small molecule starting materials and byproducts. Finally, the dialyzed solution is freeze-dried to obtain a white flocculent solid, which is the amidated hyaluronic acid intermediate (HA-ADH). The hyaluronic acid intermediate was analyzed by proton nuclear magnetic resonance spectroscopy (1H NMR spectroscopy). 1 The product was characterized by H NMR. Figure 3 B), confirming the successful grafting of ADH.

[0072] 1.2 Synthesis of Catecholized Hyaluronic Acid (HA-CAT) Reference Figure 4 The synthetic route shown in A first involves dissolving 3 molar amounts of protocatechuic acid (PCA) in an appropriate amount of deionized water. Under ice-bath stirring, 9 molar amounts of HOBt, 4.5 molar amounts of EDC, and 9 molar amounts of N,N-diisopropylethylamine (DIPEA) are slowly added sequentially to the PCA solution, followed by activation for 30 minutes. Separately, HA-ADH prepared in step 1.1 is dissolved in deionized water. The activated PCA solution and HA-ADH solution are mixed and reacted under light and nitrogen protection in a 30°C water bath for 24 hours. After the reaction is complete, the mixture is purified by dialysis with deionized water for 24 hours. After dialysis, it is lyophilized to obtain a light brown solid, namely protocatechuic acid-modified hyaluronic acid (HA-CAT). The hyaluronic acid was analyzed by 1H NMR spectroscopy (1H NMR spectroscopy). 1 The product was characterized by H NMR. Figure 4 B), the appearance of the characteristic peak of protocatechuic acid confirms successful synthesis.

[0073] 1.3 Extraction of platelet membrane Blood was collected from healthy C57BL / 6 mice via the orbital sinus into centrifuge tubes pre-treated with heparin sodium anticoagulant. The blood was centrifuged at 200 × g for 15 minutes at 4°C, and the supernatant platelet-rich plasma (PRP) was collected. The PRP was then centrifuged at 1000 × g for 10 minutes at 4°C, and the supernatant was discarded to obtain platelet precipitate. The precipitate was washed 2-3 times with phosphate-buffered saline (PBS, pH 7.4). The purified platelet precipitate was resuspended in a small amount of PBS and lysed using a freeze-thaw cycle (-80°C freeze, room temperature thaw, 3-5 cycles) to release membrane components. The lysis buffer was centrifuged at 3000 × g for 10 minutes at 4°C to remove unlysed cells or cell debris. The supernatant was collected and further ultracentrifuged at 100,000 × g for 1 hour at 4°C; the precipitate was the desired platelet membrane component (PLT).

[0074] 1.4 Preparation of PLT / HA-CAT by platelet post-membrane insertion modification The HA-CAT obtained in step 1.2 was dissolved in deionized water to prepare a 5 mg / mL solution. At a HA-CAT to PLT mass ratio of 1:2, 1 mg / mL of platelet membrane suspension was added to the HA-CAT solution. Subsequently, two molar amounts (relative to the carboxyl groups of HA-CAT) of EDC and N-hydroxysuccinimide (NHS) were added, and the pH of the mixture was adjusted to 4.5-5.0 with dilute hydrochloric acid. The reaction was carried out with gentle stirring at room temperature for 24 hours, allowing the platelet membrane to anchor to the HA-CAT backbone through hydrophobic interactions and post-intercalation mechanisms. After the reaction, the mixture was transferred to a molecular weight cutoff dialysis bag with a molecular weight cutoff of 3 kDa and dialyzed with deionized water at room temperature for 3 days to completely remove unbound membrane components and chemical cross-linking agents. Finally, the dialysate was freeze-dried to obtain platelet membrane-coated gel component A, denoted as PLT / HA-CAT.

[0075] The core of this embodiment lies in using protocatechuic acid as a catechin donor. Following a similar chemical synthesis route, protocatechuic acid can be replaced with other compounds having a catechol (catechin) structure, such as catechin, quercetin, gallic acid, etc.

[0076] Example 2 This embodiment details the method for preparing thiourea-modified hyaluronic acid (HA-NCSN) using ethyl isothiocyanate as a thiourea donor, and covalently coupling it with the STING agonist MSA-2 and physically loading the traditional Chinese medicine polysaccharide LNT to finally obtain hydrogel component B (MSA-2 & LNT-NCSN).

[0077] 2.1 Synthesis of Thiourea-modified Hyaluronic Acid (HA-NCSN) Reference Figure 5 The synthetic route shown in A uses HA-ADH prepared in Example 1.1 as the starting material. HA-ADH was dissolved in deionized water to prepare a 10 mg / mL solution. Five molar amounts (relative to the hydrazide group of HA-ADH) of ethyl isothiocyanate were dissolved in an appropriate amount of dimethyl sulfoxide (DMSO) and then slowly added dropwise to the HA-ADH solution. The reaction was carried out in a 30°C water bath under light and nitrogen protection with continuous stirring for 72 hours to allow the isothiocyanate to react with the hydrazide group to form a thiourea bond. After the reaction was complete, the solution was purified by dialysis with deionized water for 24 hours. Lyophilization yielded a white solid, which was the thiourea-functionalized hyaluronic acid (HA-NCSN). The hyaluronic acid was analyzed by 1H NMR spectroscopy (1H NMR spectroscopy). 1 The product was characterized by H NMR. Figure 5 B) confirms the successful introduction of the thiourea group.

[0078] 2.2 Preparation of MSA-2 & LNT-NCSN by Drug Loading The HA-NCSN obtained in step 2.1 was dissolved in deionized water to prepare a 20 mg / mL solution. 1 mmol of MSA-2 and a catalytic amount of concentrated sulfuric acid were added to this solution. The reaction was carried out in a 60°C oil bath with stirring in the dark for 24 hours, allowing the carboxyl groups on the MSA-2 molecules to covalently couple with the remaining amino or hydroxyl groups on the HA-NCSN chain via esterification. After the reaction was complete, the mixture was transferred to a molecular weight cutoff dialysis bag with a molecular weight cutoff of 3 kDa and dialyzed against deionized water at room temperature for 3 days. The dialysate was freeze-dried to obtain the MSA-2-loaded HA-NCSN solid.

[0079] The lyophilized product was redissolved in a small amount of deionized water, and lentinan (LNT) of equal molar amount to MSA-2 was slowly added while stirring at room temperature. Stirring continued for 2 hours to allow LNT to fully mix and complex with the coupled MSA-2 and HA-NCSN networks through intermolecular hydrogen bonds and hydrophobic interactions. Finally, the solution was lyophilized again to obtain gel component B, simultaneously loaded with MSA-2 and LNT, denoted as MSA-2&LNT-NCSN.

[0080] Using similar chemical synthesis methods, ethyl isothiocyanate can be replaced by compounds with thiol groups, such as methyl isothiocyanate, phenyl isothiocyanate, and aminothiourea. LNT can also be replaced by active polysaccharides from natural Chinese medicinal herbs, such as Tremella fuciformis polysaccharide, Bletilla striata polysaccharide, Polyporus umbellatus polysaccharide, and Astragalus membranaceus polysaccharide; MSA-2 can also be replaced by various STING pathway agonists that can be administered orally or through the gastrointestinal tract, such as SNX281, SR-717, and CRD3874-SI.

[0081] Example 3 This embodiment illustrates how to mix components A and B prepared in Examples 1 and 2, trigger gelation under simulated inflammatory conditions, and characterize their key properties.

[0082] A 0.1% NaIO4 solution was used to simulate the high oxidative stress state of the in vivo inflammatory environment. A physical mixing method was employed: first, an equal volume of gel component A (PLT / HA-CAT) obtained in Example 1 was mixed with a 0.1% NaIO4 solution; then, an equal volume of gel component B (MSA-2&LNT-NCSN) obtained in Example 2 was added and mixed. The gelation time was observed to be (5.2 ± 1.5) min, thus forming the final orally edible in-situ assembled gel, denoted as MSA-2&LNT@G. A schematic diagram of this process is shown below. Figure 2 As shown.

[0083] The gelation conditions (inflammatory response) of hydrogels with different component ratios were characterized using a rotational rheometer. The experiment showed that when the mass ratio of components A to B was 1:1, rapid and stable gelation could be achieved in about 5 minutes, and the gel had suitable mechanical strength.

[0084] Example 4 This embodiment aims to verify the formation of the MSA-2 and LNT supramolecular complex and to examine the drug release characteristics of the gel loaded with this complex (MSA-2&LNT@G).

[0085] LNT and MSA-2 were mixed in equal volumes at a concentration ratio of 1:1 to 2 and stirred at room temperature for 2 h to form an LNT / MSA-2 complex. Simultaneously, LNT and MSA-2 standard solutions of equal concentrations were prepared. The particle size of the complex molecules was determined using a Malvern particle size analyzer; the UV absorption of each standard and the complex was detected using a UV-Vis spectrophotometer across the entire wavelength range. Following Example 3, a hydrogel MSA-2&LNT@G loaded with LNT and MSA-2 was constructed, and in vitro release assays were performed: MSA-2&LNT@G was placed in a dialysis bag containing a release medium (PBS buffer containing 1 U / mL carboxylesterase, pH 6.5, to simulate the tumor inflammatory microenvironment). The dialysis bag was placed in the release medium at 37°C with constant shaking, and the extracorporeal release medium was collected at preset time points (0.5, 1, 2, 4, 8, 12, 24, 48, 72 h). The MSA-2 concentration in the release medium at each time point was determined using the established high-performance liquid chromatography method; the total sugar concentration in the release medium at each time point was determined using the phenol-sulfuric acid method to characterize the release amount of LNT; the cumulative release rate was calculated, and the drug release curve was plotted.

[0086] The results are as follows Figure 8 and Figure 9The results showed that the maximum absorption wavelength (λmax) of the MSA-2 standard was located at 328 nm, while after the formation of the complex, λmax blue-shifted to 322 nm. This significant blue-shift indicates that LNT and MSA-2 formed a supramolecular complex through interactions such as hydrogen bonding. The LNT / MSA-2 particle size was approximately 50–200 nm. Furthermore, the release curves of MSA-2 and LNT highly overlapped, exhibiting near-synchronous release kinetics. The cumulative release rates of the two drugs remained within a fixed ratio range at all time points (close to the molar ratio of 1:1–2 when the complex was formed). The release synchronicity was quantitatively evaluated by calculating the similarity factor f2 of the two release curves. The f2 factor of the MSA-2 and LNT release curves in MSA-2&LNT@G was greater than 50, demonstrating a high degree of similarity in release behavior. The results confirm that MSA-2&LNT@G can achieve synchronous release of MSA-2 and LNT, a synchronous release characteristic stemming from the supramolecular complex structure pre-formed during drug loading.

[0087] Example 5 This embodiment uses bone marrow-derived dendritic cells (BMDCs) as a model to evaluate the effects of the STING agonists MSA-2, LNT, and the final gel formulation (MSA-2 & LNT@G) on the maturation of antigen-presenting cells.

[0088] Bone marrow-derived dendritic cells (BMDCs) were used as an antigen-presenting cell model. A blank control group, a positive control group (LPS stimulation), and different concentrations of MSA-2 test groups were set up. After incubation for 48 h, cells were collected. Surface marker staining was performed using CD80-FITC, CD86-PE, and MHC II-APC antibodies. The expression levels of co-stimulatory molecules and major histocompatibility complex class II molecules were detected by flow cytometry. The secretion of maturation-related cytokines such as IL-12p70 in the cell supernatant was detected by ELISA. The expression changes of maturation-related genes such as CCR7 and CD40 after drug intervention were quantitatively analyzed by RT-PCR.

[0089] The results are as follows Figure 10 As shown, MSA-2 significantly upregulated the expression of CD80, CD86, and MHC II, promoted the secretion of IL-12p70, and increased the mRNA levels of CCR7 and CD40. These results confirm that MSA-2 can effectively promote the maturation of antigen-presenting cells.

[0090] In addition, the same indicators were tested using MSA-2&LNT@G as the test group, and the indicators were measured at 48h, 72h, and 96h after drug administration. The study found that MSA-2&LNT@G significantly promoted the maturation of antigen-presenting cells, manifested by upregulation of co-stimulatory molecules CD80 and CD86, enhanced MHC II molecule expression, and promotion of IL-12p70 secretion. Furthermore, in the MSA-2&LNT@G group, MSA-2 and LNT worked simultaneously, maintaining a sustained and mild promoting effect even 72h after drug administration. The results confirm that MSA-2&LNT@G can effectively induce and sustainably promote the maturation of antigen-presenting cells.

[0091] Example 6 This embodiment uses RAW264.7 macrophages as a model to evaluate the ability of the formulation to regulate the polarization of macrophages from the tumor-promoting M2 type to the anti-tumor M1 type.

[0092] RAW264.7 cells and MFC cells were used as macrophage and tumor cell models, respectively. Cells were co-cultured using the Transwell assay. LPS+IFN-γ combined intervention was used as the M1 polarization-positive group, IL-4 intervention as the M2 polarization-positive group, and different concentrations of LNT were used as test groups. After 24 h of co-incubation, cells were collected, stained with CD86-FITC and CD206-APC antibodies, and their expression was detected by flow cytometry. The levels of TNF-α (M1) and IL-10 (M2) cytokines in the cell supernatant after different interventions were measured using conventional ELISA. RT-PCR was used to quantify the expression of characteristic genes after drug intervention. The study found that LNT effectively increased the expression of the M1 marker CD86, promoted the secretion of the M1 characteristic cytokine TNF-α, increased cellular IFN-γ levels, inhibited SITR1 expression, and triggered the upregulation of downstream NF-κB and p53. The results confirmed that LNT can effectively promote M1 polarization of TAMs.

[0093] Furthermore, using MSA-2&LNT@G as the test group, the same index tests were performed. The study found that MSA-2&LNT@G exhibited an M1 polarization induction trend, increasing the expression of the M1-type marker CD86, decreasing the expression of the M2-type marker CD206, promoting TNF-α secretion, and upregulating the expression of the M1-type characteristic gene iNOS. These results confirm that MSA-2&LNT@G can promote the M1 polarization of TAMs.

[0094] Example 7 This embodiment illustrates the effect of the STING agonist MSA-2 combined with the traditional Chinese medicine polysaccharide LNT on the tumor immune microenvironment in vitro.

[0095] Using a saline group as a negative control, LPS+IFN-γ combined intervention as the M1 polarization-positive group, and IL-4 intervention as the M2 polarization-positive group, the effects of MSA-2 monotherapy, LNT monotherapy, MSA-2+LNT combination therapy, MSA-2&LNT-NCSN therapy, and MSA-2&LNT@G therapy on the tumor immune microenvironment were investigated. RAW264.7 cells and MFC cells were used as macrophage and tumor cell models, respectively. Cells were co-cultured using the Transwell method and collected after 24 h of co-incubation. The expression levels of CD86 (M1) and CD206 (M2) were detected by flow cytometry. The secretion of TNF-α (M1) and IL-10 (M2) in the cell supernatant was measured by ELISA. The expression of STING pathway-related genes such as IFN-β and CXCL10 was quantitatively analyzed by RT-PCR. Studies have found that MSA-2 can directly activate the STING signaling pathway and promote the secretion of IFN-β, while LNT binds to tubulin and causes it to depolymerize, thus inhibiting the transport of STING and prolonging the duration of STING signaling. The two work synergistically to significantly enhance the expression levels of downstream IFN-β and CXCL10, further promoting the polarization of TAMs towards the M1 type, manifested as increased CD86 expression, increased TNF-α secretion, and inhibition of M2 type markers CD206 and IL-10.

[0096] Furthermore, the MSA-2+LNT combination group significantly upregulated the expression of CD69 and granzyme B, activation markers of CD8+ T cells in the tumor microenvironment, indicating that the synergistic effect of MSA-2 and LNT can not only reshape the macrophage phenotype but also enhance the anti-tumor immune response. The results confirm that the STING agonist MSA-2 combined with the traditional Chinese medicine polysaccharide LNT can effectively improve the tumor immune microenvironment and promote the anti-tumor immune response by synergistically regulating the STING signaling pathway.

[0097] Example 8 This embodiment demonstrates the synergistic effect of the STING agonist MSA-2 combined with the traditional Chinese medicine polysaccharide LNT on the sustained activation of the STING pathway in vitro.

[0098] Using a saline group as a negative control, the effects of MSA-2 monotherapy, LNT monotherapy, MSA-2+LNT combination therapy, and MSA-2&LNT-NCSN therapy on STING pathway activation were investigated. MFC cells, DC2.4 cells, and RAW264.7 cells were used as cell models. After 24 h of drug administration, proteins were extracted, and the expression of STING pathway-related proteins was verified by Western blot.

[0099] The study found that MSA-2 significantly activated the STING pathway, upregulating the expression of related proteins such as cGAS, STING, TBK1, and IRF3. However, the upregulation of STING protein expression was more pronounced in the MSA-2+LNT combination group and the MSA-2&LNT-NCSN group. MSA-2 activates the STING signaling pathway, and LNTs bind to tubulin, causing it to dissociate, thus sustaining the activation of STING signaling. Compared with the MSA-2 monotherapy group, the MSA-2+LNT combination group did not show a sharp increase in the expression levels of downstream effectors (such as IFN-β and CXCL10), but their expression duration was significantly prolonged. This indicates that the synergistic effect of LNTs is mainly manifested in stabilizing the signal and delaying its decay, rather than simply amplifying the signal peak. The synergistic effect of LNTs and MSA-2 effectively prolongs and moderately enhances the STING signal.

[0100] Example 9 This embodiment illustrates the effect of the STING agonist MSA-2 combined with the traditional Chinese medicine polysaccharide LNT on the tumor immune microenvironment in vivo.

[0101] An orthotopic gastric cancer model was established by surgically implanting subcutaneous tumors formed from MFC cells in 615 mice into the gastric parenchyma of C57BL / 6 mice. Four groups were established: a saline control group, a LNT monotherapy group, an MSA-2 monotherapy group, an MSA-2+LNT combination group, and an MSA-2&LNT@G hydrogel group. Flow cytometry analysis of tumor microenvironment cytokines (TAMs) revealed that, compared with the control group, the MSA-2+LNT combination group significantly increased the proportion of M1 macrophages (CD86+) while reducing the infiltration of M2 macrophages (CD206+), and this effect was more pronounced in the MSA-2&LNT@G group. ELISA analysis of tumor microenvironment cytokines showed that the levels of TNF-α and IFN-γ were significantly increased in the combination therapy group, while M2-related factors such as IL-10 were inhibited. Furthermore, due to the sustained-release properties of the hydrogel, the MSA-2&LNT@G group maintained an effective drug concentration within the tumor for a longer period, significantly inhibiting tumor growth and prolonging the survival of mice. The above results confirm that the STING agonist MSA-2 and LNT can exert anti-tumor effects in vivo by synergistically regulating the immune microenvironment, and the hydrogel-based co-delivery system further optimizes the efficacy and duration.

[0102] The main pharmacodynamic data are summarized in Table 1.

[0103] Table 1 The levels of IL-2, CCL2, TGF-β, and IFN-γ in the serum of tumor-bearing mice were measured using an ELISA kit. The results showed that MSA-2&LNT@G treatment increased serum IL-2 and IFN-γ levels, contributing to improved immune function. Furthermore, the levels of TGF-β and CCL2 in the MSA-2&LNT@G group were significantly lower than those in the saline group, suggesting that the hydrogel formulation may reduce the secretion of chemokines, thereby decreasing the likelihood of tumor growth and metastasis.

[0104] Example 10 This embodiment illustrates the pharmacodynamic advantages of the STING agonist MSA-2 combined with the traditional Chinese medicine polysaccharide LNT in the treatment of gastric cancer, and provides an example of the oral in situ assembled gel MSA-2&LNT@G prepared in Example 5 for the treatment of gastric cancer.

[0105] An orthotopic gastric cancer model was established by surgically implanting subcutaneous tumors formed from MFC cells in 615 mice into the gastric parenchyma of C57BL / 6 mice. A saline group was used as a negative control, while the LNT monotherapy group, MSA-2 monotherapy group, MSA-2+LNT combination group, MSA-2&LNT-NCSN group, PLT / HA-CAT group, and blank hydrogel excipient group were used as controls. The efficacy of chemoimmunotherapy in the MSA-2&LNT@G group was investigated. The administration regimen was as follows: starting from day 0 of orthotopic modeling, drugs were administered every other day at a dose of MSA-2 30 mg / kg and LNT 120 mg / kg (determined based on in vitro study data and the molar ratio of the MSA-2 to LNT complex), for 14 doses up to day 28. Mouse body weight and tumor growth curves were recorded during treatment. After administration, the tumors were dissected and their volume and weight measured.

[0106] The results showed that, compared with the saline group, all drug-treated groups except the drug-free PLT / HA-CAT group and the blank hydrogel excipient group significantly inhibited tumor growth. The dual-drug combination group (MSA-2+LNT) showed better tumor-inhibiting effects than the single-drug groups, indicating that LNT and MSA-2 have a synergistic anti-tumor effect. Combined with the experimental results on the anti-tumor mechanism, LNT may enhance the sustained activation of the STING pathway by MSA-2 by promoting microtubule depolymerization and delaying the degradation of STING protein, thereby enhancing the anti-tumor immune response. Safety assessment showed no significant effect on mouse body weight in any of the drug-treated groups. Compared with the conventional dual-drug combination group, the hydrogel formulation MSA-2&LNT@G group showed a more significant tumor-inhibiting effect, with the smallest tumor volume and prolonged mouse survival. It is speculated that the mechanism may lie in the fact that the hydrogel formed in situ in the stomach can achieve long-term sustained release of the drug, maintaining a local effective drug concentration, thereby enhancing the anti-tumor efficacy.

[0107] The results confirmed that the STING agonist MSA-2 combined with the traditional Chinese medicine polysaccharide LNT exhibited a significant synergistic anti-gastric cancer effect in vivo, and the hydrogel drug delivery system (MSA-2&LNT@G) further improved the efficacy.

[0108] Example 11 This embodiment illustrates the advantages of sequential oral administration in tumor targeting.

[0109] An orthotopic gastric cancer model was established by surgically implanting subcutaneous tumors formed from MFC cells in 615 mice into the gastric parenchyma of C57BL / 6 mice. PLT / HA-CAT (component A) obtained in Example 3 was labeled with Cy5.5, and MSA-2 & LNT-NCSN (component B) obtained in Example 4 was labeled with Cy7. Component A and component B were administered to the model mice sequentially by gavage at 3-minute intervals. Furthermore, the in vivo targeting performance of the stepwise oral administration was examined using hydrogel component A, hydrogel component B, and a direct oral A+B mixture group as controls.

[0110] In vivo imaging results showed that hydrogel component A was anchored at the tumor site, but began to detach gradually after 5 minutes due to the lack of hydrogel formation; hydrogel component B did not form adhesion in the digestive tract; the fluorescence signal of the A+B mixed group administered orally diffused throughout the digestive tract (targeting rate <15%); when administered in steps, component A of group A followed by group B accumulated in the gastric lesion within 10 minutes (targeting rate 82%), and the fluorescence signal of component B after gelation had an overlap rate of 91% with that of component A.

[0111] The results confirmed that sequential oral administration significantly improved the targeting ability to tumor sites.

[0112] Example 12 This example illustrates the comparison of the gelling properties and in vivo targeting of HA-CAT (protocatechuic acid) and HA-DA (dopamine hydrochloride), demonstrating the decisive significance of this substitution for achieving an oral stepwise drug delivery strategy.

[0113] The synthesis method of HA-DA was based on previous research (Xu et al., Sci. Transl. Med. 12, eaba8014 (2020)): 1 g of HA was dissolved in 100 mL of deionized water. Then, Dowex 50WX8 was added to the solution at a concentration of 3% (w / v), and the mixture was stirred at room temperature for 7–8 h. Tetrabutylammonium hydroxide (TBAOH) was added to neutralize the solution, and the mixture was lyophilized to obtain HA-TBA. 0.5 g of HA-TBA was dissolved in 50 mL of DMF, and then HOBt and DIC were added at molar ratios of 2.5:1 and 0.5:1, respectively. The mixture was stirred for 4 h under nitrogen protection. Subsequently, dopamine hydrochloride, DMAP (molar ratio to HA-TBA of 0.5:1), and DIPEA (molar ratio to HA-TBA of 2.5:1) were added to the solution, and the mixture was stirred for 24 h. After the reaction was completed, the solution was dialyzed with NaCl at pH 4 for 24 h, then dialyzed with deionized water for 24 h, lyophilized, and stored at 4°C.

[0114] In vitro gelation performance comparison: Following the method in Example 3, the gelation time of the present invention group (HA-CAT + HA-NCSN) and the control group (HA-DA + HA-NCSN) were tested respectively. The results showed that the average gelation time of the present invention group was (5.2 ± 1.5) min, while the gelation time of the control group was less than 5 s, confirming that the system of the present invention has more controllable reaction kinetics.

[0115] In vivo targeting comparison: Following the method in Example 11, an orthotopic gastric cancer model mouse was established. Three groups were set up: (a) HA-CAT stepwise oral administration group (HA-CAT component A and component B were administered to model mice by gavage at 3-minute intervals), (b) HA-DA stepwise oral administration group (HA-DA component A and component B were administered to model mice by gavage at 3-minute intervals), and (c) direct oral mixed group (HA-CAT component A and component B were pre-mixed). In vivo imaging results showed that the target enrichment rate of the present invention group (a) at the gastric wound was as high as (82.3 ± 5.1)%, and the fluorescence overlap rate was 91%; while the control group (b) had a targeting rate of only (40.5 ± 8.7)% due to rapid gelation and diffusion of fluorescence signal throughout the digestive tract; the mixed group (c) had the lowest targeting rate (<15%). This result proves that the present invention achieves a controllable gelation effect that matches the gastric administration environment by replacing dopamine hydrochloride with protocatechuic acid, and the combination with the stepwise oral administration strategy achieves a significant improvement in targeting.

[0116] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An orally oriented, in-situ assembled gel drug delivery system, characterized in that, The system comprises component A and component B, which can be administered separately; Component A contains a first hyaluronic acid derivative modified with a catechin group; Component B contains a second hyaluronic acid derivative modified with a thiourea group; The components A and B are configured to be administered by first orally taking component A, followed by orally taking component B at a predetermined time interval, so that in situ cross-linking and gelation can be achieved at the target inflammatory site in the digestive tract through a catechol-thiourea coupling reaction.

2. The orally oriented in-situ assembled gel drug delivery system according to claim 1, characterized in that, In component A, the first hyaluronic acid derivative is hyaluronic acid modified with protocatechuic acid; component A also includes a platelet membrane modified on the surface of the first hyaluronic acid derivative by a post-insertion technique.

3. The orally oriented in-situ assembled gel drug delivery system according to claim 1, characterized in that, Component B further comprises a STING agonist and a traditional Chinese medicine polysaccharide loaded on the second hyaluronic acid derivative; the STING agonist is MSA-2, SNX281, SR-717 or CRD3874-SI; the traditional Chinese medicine polysaccharide is lentinan, tremella polysaccharide, astragalus polysaccharide, poria cocos polysaccharide or bletilla striata polysaccharide.

4. The orally oriented in-situ assembled gel drug delivery system according to claim 3, characterized in that, The STING agonist is MSA-2, and the traditional Chinese medicine polysaccharide is lentinan (LNT). Furthermore, MSA-2 and lentinan (LNT) form a supramolecular complex through non-covalent interactions, and the molar ratio of MSA-2 to LNT repeating units in the supramolecular complex is 1:1 to 1:

2.

5. The orally oriented in-situ assembled gel drug delivery system according to claim 4, characterized in that, In component B, MSA-2 is covalently linked to the second hyaluronic acid derivative via an enzyme-sensitive bond, and LNT is loaded into the three-dimensional network of the second hyaluronic acid derivative through physical mixing and the supramolecular complex formed with MSA-2.

6. The orally oriented in-situ assembled gel drug delivery system according to claim 1, characterized in that, The predetermined time interval between the first oral component A and the subsequent oral component B is 2 to 10 minutes.

7. A pharmaceutical composition, characterized in that, It contains traditional Chinese medicine polysaccharides and STING agonists.

8. A method for preparing an orally oriented, in-situ assembled gel drug delivery system as described in any one of claims 1-6, characterized in that, Includes the following steps: (1) Synthesize the first hyaluronic acid derivative modified with catechol groups as the precursor of component A; (2) A second hyaluronic acid derivative modified with a thiourea group was synthesized as the matrix of component B; (3) The STING agonist and traditional Chinese medicine polysaccharide were loaded onto the second hyaluronic acid derivative to obtain component B; (4) Platelet membrane is modified onto the surface of the first hyaluronic acid derivative by post-insertion technique to obtain component A.

9. The use of an orally oriented in-situ assembled gel drug delivery system as described in any one of claims 1-6 in the preparation of a medicament for the prevention or treatment of postoperative recurrence of gastrointestinal tumors.

10. A method of administration for the treatment of gastrointestinal tumors, characterized in that, The method comprises: first, orally administering component A of any one of claims 1-6 to a subject in need, and then, after an interval of 2 to 10 minutes, orally administering component B of any one of claims 1-6 to the subject.

Images