A sodium ion-responsive injectable self-assembling hydrogel system and preparation method and application thereof

Abstract

This invention discloses a sodium ion-responsive injectable self-assembled hydrogel system, its preparation method, and its applications. Specifically, it involves in vitro and in vivo experimental studies on the loading and delivery capacity, attenuation capacity, and enhancement of immunotherapeutic and antitumor efficacy of 1,6-DAS hydrogel for oncolytic bacteria. Through molecular biology experiments, pharmacodynamic experiments, flow cytometry, and molecular dynamics simulations, it elucidates the mechanisms by which the 1,6-DAS hydrogel achieves further attenuation of oncolytic bacteria, enhanced antitumor immunity, and improved efficacy without affecting the inherent targeted antitumor activity of oncolytic bacteria. This invention belongs to the field of biotechnology. By combining innovative materials with live bacteria, it systematically elucidates the key effects and corresponding mechanisms of 1,6-DAS hydrogel-loaded oncolytic bacteria administration on their toxicity and efficacy, providing direct practical evidence for the wider application of 1,6-DAS hydrogel in the field of in vivo microbial therapy.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a sodium ion-responsive injectable self-assembly hydrogel system, its preparation method, and its application. Background Technology

[0002] Cancer remains one of the leading causes of death today, and the global cancer burden is projected to increase by 77% by 2050, with over 35 million new cases annually. Live microbial drugs (oncolytic bacteria) represent one of the most cutting-edge strategies in anticancer drug development, offering unique advantages in treating refractory tumors. Among these, attenuated Salmonella VNP20009 (VNP) is the most widely studied and clinically promising candidate drug. In the more than 20 years since VNP was first approved for clinical trials in 1999, numerous clinical trials have been conducted on VNP and its engineered strains, but a clinical breakthrough has yet to be achieved. The fundamental reason lies in the "binding effect" between bacterial toxicity and efficacy; enhancing the antitumor activity of the strain often leads to increased toxicity, while attenuation reduces efficacy. Therefore, "unbinding" the toxicity and efficacy of oncolytic bacteria or engineered oncolytic bacteria is key to overcoming the bottleneck in live microbial drug development.

[0003] Hydrogels are multifunctional soft materials with high water content, exhibiting good compatibility and drug encapsulation capabilities. In recent years, some studies have applied hydrogels with different properties to the sustained-release delivery of microorganisms. For example, Chinese patent CN202511125038.6 discloses a probiotic polyphenol nanoparticle colon-targeted co-delivery system. However, no research or patent reports have yet applied hydrogels to engineered oncolytic bacteria with strong adverse reactions, such as attenuated Salmonella, to achieve attenuation or enhancement of the strain through sustained release or other pathways. The main reason for this is that the gelling and injectability of hydrogels have potential impacts on bacterial activity. The administration of oncolytic bacteria, such as attenuated Salmonella, is mainly through systemic or intraperitoneal administration. This means that the hydrogel must be injectable. However, the current methods for controlling injectable hydrogels are mainly ultrasound response (Chinese patent CN202511796385.1), temperature response (Chinese patent CN202110630245.2), or pH response (Chinese patent CN202511818958.6). These response methods have a high probability of destroying the activity of the bacteria themselves.

[0004] Chinese patent CN201911100567.5 discloses a gel with the potential to deliver oncolytic bacteria, such as attenuated Salmonella, namely an isosteval-based metal gel. The gel solvent mentioned in the patent is not water, and the isosteval-based metal gel reaction can most likely occur in an aqueous solvent as well.

[0005] Currently, there is a lack of sodium ion-responsive injectable self-assembly hydrogel systems, their preparation methods, and applications. Summary of the Invention

[0006] The purpose of this invention is to provide a sodium ion-responsive injectable self-assembly hydrogel system, its preparation method, and its application.

[0007] This invention elucidates the application of injectable hydrogels in oncolytic bacterial antitumor immunodelivery therapy and the corresponding mechanisms of toxicity reduction and efficacy enhancement. Through experiments, this invention evaluates the loading and delivery capacity, toxicity reduction capacity, and immuno-antitumor efficacy of 1,6-DAS hydrogels for oncolytic bacteria or engineered oncolytic bacteria, as well as their related mechanisms. The oncolytic bacteria include, but are not limited to, attenuated Salmonella Typhimurium, Escherichia coli, Bifidobacterium, BCG, Shigella, Klebsiella pneumoniae, and Pseudomonas aeruginosa, either original or mutant strains, or engineered strains expressing TNF-α nanobodies or PD-1 nanobodies.

[0008] In vivo and in vitro experiments have demonstrated that 1,6-DAS hydrogel has the ability to load and deliver oncolytic bacteria or engineered oncolytic bacteria into the peritoneum. By sustaining the release of bacteria and blocking flagellar proteins on the bacterial surface, it can significantly improve systemic toxicity caused by oncolytic bacteria or engineered oncolytic bacteria, including weight loss, organ enlargement, tissue damage and low long-term survival. Furthermore, by enhancing the proportion of activated dendritic cells and CD8+ T cells in the tumor immune microenvironment, it can further enhance the anti-tumor response induced by oncolytic bacteria or engineered oncolytic bacteria, thereby significantly improving the therapeutic effect.

[0009] This invention elucidates the feasibility of using 1,6-DAS hydrogels in the delivery of oncolytic bacteria or engineered oncolytic bacterial strains, as well as the significant toxicity reduction and efficacy enhancement effects on the strains after loading, and the related mechanisms. This is beneficial for the further application of hydrogels in clinical microbial drug therapy.

[0010] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: In a first aspect, this application provides a sodium ion-responsive injectable self-assembling hydrogel system.

[0011] Secondly, this application provides a method for preparing an injectable self-assembling hydrogel system.

[0012] Thirdly, this application provides a method for loading oncolytic bacteria onto a sodium ion-responsive injectable in vivo self-assembling hydrogel.

[0013] Fourthly, this application provides the use of a hydrogel system in the preparation of a drug for treating tumors.

[0014] Fifthly, this application provides a kit for delivering oncolytic bacteria.

[0015] Sixthly, this application provides a sodium ion-responsive injectable self-assembling hydrogel containing oncolytic bacteria.

[0016] The first aspect of this application provides a sodium ion-responsive injectable self-assembly hydrogel system, the injectable self-assembly hydrogel system comprising: A hydrogel precursor solution comprising isostevol-diester sulfonic acid derivative and oncolytic bacteria; wherein, after the hydrogel precursor solution and oncolytic bacteria are mixed, they can self-assemble into a hydrogel containing the oncolytic bacteria under conditions of contact with sodium ions.

[0017] Furthermore, the isosteviol-diester sulfonic acid derivative is selected from any one of isosteviol-1,6-diester sulfonic acid (1,6-DA), sodium isosteviol-1,6-diester sulfonate (1,6-DAS), potassium isosteviol-1,6-diester sulfonate, calcium isosteviol-1,6-diester sulfonate, isosteviol-1,4-diester sulfonic acid (1,4-DA), sodium isosteviol-1,4-diester sulfonate (1,4-DAS), potassium isosteviol-1,4-diester sulfonate, or calcium isosteviol-1,4-diester sulfonate. Oncolytic bacteria include, but are not limited to, original or mutant strains of attenuated Salmonella Typhimurium, Escherichia coli, Bifidobacterium, BCG, Shigella, Klebsiella pneumoniae, and Pseudomonas aeruginosa, or engineered attenuated Salmonella Typhimurium, Escherichia coli, Bifidobacterium, BCG, Shigella, Klebsiella pneumoniae, or Pseudomonas aeruginosa strains expressing exogenous functional proteins.

[0018] Furthermore, the engineered oncolytic bacterial strains are oncolytic bacterial strains expressing TNF-α nanobodies or PD-1 nanobodies.

[0019] A second aspect of this application provides a method for preparing an injectable self-assembled hydrogel system, comprising the following steps: (1) Prepare a solution of isosteviol-diestersulfonic acid derivative as a hydrogel precursor solution; (2) Mix the oncolytic bacteria cultured to the exponential growth phase with the hydrogel precursor solution described in step (1); (3) The mixture obtained in step (2) is incubated in an environment containing sodium ions to form a hydrogel system by self-assembly.

[0020] Furthermore, the sodium-containing environment is a phosphate buffer solution, a sodium chloride solution, or the in vivo environment of a living organism.

[0021] A third aspect of this application provides a method for loading oncolytic bacteria onto a sodium ion-responsive injectable in vivo self-assembly hydrogel, comprising the following steps: (1) Preparation of an aqueous solution of isosteviol-diester sulfonic acid derivative; (2) After centrifuging the oncolytic bacteria, collect the bacterial precipitate and resuspend the bacterial precipitate in the aqueous solution described in step (1) to obtain a bacterial solution mixture; (3) Contact the bacterial mixture obtained in step (2) with a solution containing sodium ions, or inject the bacterial mixture into a living organism to trigger self-assembly to form a hydrogel containing oncolytic bacteria.

[0022] Further, in step (1), the isosteviol-diester sulfonic acid derivative is isosteviol-1,6-diester sulfonic acid (1,6-DA) or isosteviol-1,4-diester sulfonic acid (1,4-DA); the concentration of the aqueous solution is 5-20 mg / mL; the preparation of the aqueous solution includes the steps of heating and dissolving the isosteviol-diester sulfonic acid derivative in a solvent and then cooling it. In step (2), the oncolytic bacteria are, but are not limited to, attenuated Salmonella Typhimurium, Escherichia coli, Bifidobacterium, BCG, Shigella, Klebsiella pneumoniae, and Pseudomonas aeruginosa, either original or mutant strains, or engineered strains expressing TNF-α nanobodies or PD-1 nanobodies; the amount of bacterial precipitate is not less than 1 × 10⁻⁶ per 100 μL of aqueous solution. 9 CFU; In step (3), the sodium-containing solution is a phosphate buffer or a sodium chloride solution; the biological body is subcutaneous or peritoneal.

[0023] The fourth aspect of this application provides the application of a hydrogel system in the preparation of drugs for treating tumors. In vivo and in vitro experiments were conducted to evaluate the hydrogel's loading and delivery capacity for oncolytic bacteria, its attenuation capacity, and its ability to enhance immunotherapeutic efficacy against tumors, as well as the corresponding molecular mechanisms.

[0024] Furthermore, the drug is administered via injection, and the hydrogel system self-assembles in vivo to achieve sustained-release delivery of oncolytic bacteria; the application of sodium ion-responsive injectable self-assembling hydrogels in oncolytic bacterial antitumor immunodelivery therapy, specifically low molecular weight injectable self-assembling hydrogels of isosteval-1,6-diester sulfonate sodium (1,6-DAS) derived from isosteval-1,6-diester sulfonate (1,6-DA).

[0025] The application of sodium ion-responsive injectable in vivo self-assembling hydrogel in oncolytic bacterial antitumor immunodelivery therapy is characterized by a low molecular weight injectable in vivo self-assembling hydrogel of isostevol-1,4-diester sulfonate sodium (1,4-DAS) derived from isostevol-1,4-diester sulfonic acid (1,4-DA).

[0026] The application of sodium isostevine-1,6-diester sulfonate (1,6-DAS), a low molecular weight injectable in vivo self-assembling hydrogel derived from isostevine-1,6-diester sulfonic acid (1,6-DA), in oncolytic bacterial antitumor immunodelivery therapy is characterized by its application in oncolytic bacteria, represented by wild-type attenuated Salmonella strains.

[0027] The application of sodium isostevine-1,6-diester sulfonate (1,6-DAS), a low molecular weight injectable in vivo self-assembly hydrogel derived from isostevine-1,6-diester sulfonic acid (1,6-DA), in oncolytic bacterial antitumor immunodelivery therapy is characterized by its application in engineered oncolytic bacterial strains (e.g., oncolytic bacteria such as VNP expressing TNF-α nanobody plasmid [VNP-TNF-α nanobody, VNP-TNF-α nb], VNP expressing PD-1 nanobody plasmid [VNP-PD-1 nanobody, VNP-PD-1 nb], etc.).

[0028] The application of sodium isostevine-1,4-diester sulfonate (1,4-DAS), a low molecular weight injectable in vivo self-assembling hydrogel derived from isostevine-1,4-diester sulfonic acid (1,4-DA), in oncolytic bacterial antitumor immunodelivery therapy is characterized by its application in oncolytic bacterial strains represented by VNP.

[0029] The application of sodium isostevine-1,4-diester sulfonate (1,4-DAS), a low molecular weight injectable in vivo self-assembling hydrogel derived from isostevine-1,4-diester sulfonic acid (1,4-DA), in oncolytic bacterial antitumor immunodelivery therapy is characterized by its application in engineered oncolytic bacterial strains (e.g., VNP expressing TNF-α nanobody plasmid [VNP-TNF-α nb], VNP expressing PD-1 nanobody plasmid [VNP-PD-1 nb], etc.).

[0030] Hydrogel systems are used for: (i) Reduce systemic toxicity associated with oncolytic bacterial therapy, including weight loss, organ enlargement, tissue damage, and / or reduced long-term survival; and / or (ii) Enhance the antitumor efficacy of oncolytic bacteria; The reduction in systemic toxicity is achieved by the hydrogel system through the slow release of oncolytic bacteria and / or by blocking immunogenic proteins on the surface of oncolytic bacteria.

[0031] Furthermore, the immunogenic protein is flagellin; the enhanced antitumor efficacy is achieved by enhancing the activation of dendritic cells (DCs) and / or CD8+ in the tumor immune microenvironment. +The proportion of T cells is used to achieve this; the tumor is a solid tumor; solid tumors include any one of melanoma, lung cancer, lymphoma, bladder cancer, liver cancer, colorectal cancer, or breast cancer.

[0032] A fifth aspect of this application provides a kit for delivering oncolytic bacteria, characterized in that: the kit comprises: (a) A hydrogel precursor composition containing isosteviol-diestersulfonic acid derivatives; and (b) Oncolytic bacteria, including but not limited to attenuated Salmonella Typhimurium, Escherichia coli, Bifidobacterium, BCG, Shigella, Klebsiella pneumoniae, Pseudomonas aeruginosa original strains or mutant strains or engineered bacterial strains expressing TNF-α nanobodies or PD-1 nanobodies; Among them, components (a) and (b) can form hydrogels containing oncolytic bacteria after contact with sodium ions.

[0033] Furthermore, the isosteviol-diester sulfonic acid derivative is selected from any one of isosteviol-1,6-diester sulfonic acid (1,6-DA), sodium isosteviol-1,6-diester sulfonate (1,6-DAS), potassium isosteviol-1,6-diester sulfonate, calcium isosteviol-1,6-diester sulfonate, isosteviol-1,4-diester sulfonic acid (1,4-DA), sodium isosteviol-1,4-diester sulfonate (1,4-DAS), potassium isosteviol-1,4-diester sulfonate, or calcium isosteviol-1,4-diester sulfonate.

[0034] The sixth aspect of this application provides a sodium ion-responsive injectable self-assembling hydrogel containing oncolytic bacteria, prepared by a method.

[0035] 100 μL of the hydrogel can encapsulate more than 1 × 10 9 The oncolytic bacteria in CFU are preserved without affecting their activity. Under in vitro conditions, the hydrogel can release approximately 80% of the oncolytic bacteria it encapsulates within 72 hours.

[0036] Beneficial effects: This invention is the first to construct an oncolytic bacterial delivery system based on a sodium ion-responsive injectable in vivo self-assembling hydrogel. This system achieves further attenuation and enhancement of the toxicity of oncolytic bacteria, with comprehensive patent protection, and provides experimental basis for the development of oncolytic bacterial drugs represented by attenuated Salmonella.

[0037] Compared with the prior art, the present invention has the following advantages: (1) This invention is the first to achieve in vivo loading and delivery of oncolytic bacteria without affecting their activity. Breaking away from the limitations of previous hydrogels that required changes in temperature, pH, or specific wavelengths of light to gel, this method achieves gelation and release of hydrogels only under the mild conditions of the sodium ion environment in vivo, minimizing the risk of impacting the activity of oncolytic bacteria.

[0038] (2) Breaking through the constraint of synergistic increase or decrease in the toxicity and efficacy of oncolytic bacteria for the first time The application of this hydrogel delivery platform breaks through the core problem faced in oncolytic bacterial therapy for the first time, namely: improving efficacy often means increasing toxicity, and reducing toxicity often means reducing efficacy. It perfectly achieves both toxicity reduction and efficacy enhancement of oncolytic bacteria.

[0039] (3) Elucidate the specific mechanism by which the hydrogel attenuates oncolytic bacteria, and demonstrate that the delivery platform is safe and efficient. The application of this hydrogel delivery platform achieves further systemic attenuation of oncolytic bacteria through a dual approach of sustained release and hydrogel molecule shielding of bacterial immunogenic proteins.

[0040] (4) Elucidate the specific mechanism by which the hydrogel enhances the efficacy of oncolytic bacteria, and realize the significant improvement in the therapeutic effect of oncolytic bacteria through the delivery platform. The application of this hydrogel delivery platform enhances the proportion of activated DC cells and CD8+ T cells in the tumor immune microenvironment, thereby further improving the anti-tumor response induced by oncolytic bacteria.

[0041] (5) The technology is simple to operate and has a low threshold for conversion. The process involves preparing a 10 mg / mL hydrogel, dissolving it in pure water, heating it in a pressure-resistant tube until dissolved, cooling it, and then directly injecting the encapsulated bacterial strain. Each step is convenient and quick, and the reaction is rapid. Attached Figure Description

[0042] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0043] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 For the present invention Figure 1 This is a schematic diagram of the chemical reaction that occurs during the gelation process of the 1,6-DAS hydrogel of the present invention, which is formed by the sodium ion response of the 1,6-DA precursor.

[0044] Figure 2This is a photograph of the gelation process of the 1,6-DAS hydrogel of the present invention, which is formed from 1,6-DA precursor in response to sodium ions.

[0045] Figure 3 This is a schematic diagram of the molecular dynamics simulation of the 1,6-DAS hydrogel of the present invention, which is formed by the sodium ion-responsive gelation process of the 1,6-DA precursor (0-600 ns).

[0046] Figure 4 The graph shows the molecular dynamics simulation results of the 1,6-DAS hydrogel of the present invention during the sodium ion-responsive gelation process (0-600 ns) of the 1,6-DA precursor. (Left: number of hydrogen bonds, Right: number of interactions)

[0047] Figure 5 This is a photograph of the 1,6-DAS hydrogel of the present invention, showing the instantaneous gelation of the 1,6-DA precursor in response to sodium ions.

[0048] Figure 6 This is a schematic diagram illustrating the principle of the 1,6-DAS hydrogel of the present invention, which is formed by the self-assembly of 1,6-DA precursors in response to sodium ions (left: before gelation, right: after gelation).

[0049] Figure 7 This is a scanning electron microscope image of the 1,6-DAS hydrogel after gelation according to the present invention.

[0050] Figure 8 The rheological analysis results of the 1,6-DAS hydrogel of the present invention after gelation are shown in the figure (left: 10 mg / mL 1,6-DAS, right: 20 mg / mL).

[0051] Figure 9 The image shows the gelation time of a 10 mg / mL 1,6-DAS hydrogel formed under different sodium ion concentrations according to the present invention.

[0052] Figure 10 The image shows a scanning electron microscope (SEM) image of a 10 mg / mL 1,6-DAS hydrogel formed under different sodium ion concentrations according to the present invention.

[0053] Figure 11 The figure shows the gel state (0-72 h) formed in the peritoneum of mice after intraperitoneal injection of the 1,6-DAS hydrogel of the present invention at different time points.

[0054] Figure 12 The graph shows the hemolysis rate of erythrocytes at different concentrations of the 1,6-DAS hydrogel of the present invention.

[0055] Figure 13This is an image showing the HE staining results of sections of major organs in mice after intraperitoneal injection of the 1,6-DAS hydrogel of the present invention.

[0056] Figure 14 This is a schematic diagram illustrating the sodium ion-responsive gelation process of the 1,6-DAS hydrogel of the present invention, which is formed by encapsulating VNP bacteria with 1,6-DA precursors in vivo and in vitro.

[0057] Figure 15 The figure shows the test results of the maximum drug loading of the 1,6-DAS hydrogel and its precursor 1,6-DA on VNP bacteria.

[0058] Figure 16 This is a scanning electron microscope image of the 1,6-DAS hydrogel of the present invention after successful loading of VNP bacteria (i.e., 1,6-DAS / VNP) and lyophilization.

[0059] Figure 17 The images show the release of VNP bacteria at different time points after loading the 1,6-DAS hydrogel of the present invention at a rotation speed of 100 rpm.

[0060] Figure 18 The graph shows the release curve of VNP bacteria from the 1,6-DAS hydrogel of the present invention at a rotation speed of 100 rpm after loading.

[0061] Figure 19 This is a growth curve of VNPs released at different time points (0-24 h) after the 1,6-DAS hydrogel of the present invention is loaded with VNP bacteria to form 1,6-DAS / VNP.

[0062] Figure 20 This is a graph showing the VNP bacterial titer in tumor tissue at different time points after intraperitoneal injection of the 1,6-DAS hydrogel of the present invention, after loading VNP bacteria to form 1,6-DAS / VNP.

[0063] Figure 21 This is a graph showing the VNP bacterial titer in major organs at different time points after intraperitoneal injection of the 1,6-DAS hydrogel of the present invention, after loading VNP bacteria to form 1,6-DAS / VNP.

[0064] Figure 22 This is a schematic timeline diagram of the pharmacodynamic experiments of a melanoma model after loading VNP bacteria onto the 1,6-DAS hydrogel of the present invention.

[0065] Figure 23 This is a graph showing the individual tumor growth curves of mice in each treatment group in a melanoma model pharmacodynamic experiment after loading VNP bacteria onto the 1,6-DAS hydrogel of the present invention.

[0066] Figure 24This image shows the tumor volume at the treatment endpoint in mice of each treatment group during pharmacodynamic experiments of a melanoma model after loading VNP bacteria onto the 1,6-DAS hydrogel of the present invention.

[0067] Figure 25 The graph shows the weight change curves of mice in each treatment group in the pharmacodynamic experiment of a melanoma model after loading VNP bacteria onto the 1,6-DAS hydrogel of the present invention.

[0068] Figure 26 Survival curves of mice in each treatment group in the pharmacodynamic experiment of a melanoma model after loading VNP bacteria onto 1,6-DAS hydrogel of the present invention.

[0069] Figure 27 This is a graph showing the spleen coefficient as the therapeutic endpoint in a melanoma model pharmacodynamic experiment using 1,6-DAS hydrogel loaded with VNP bacteria according to the present invention.

[0070] Figure 28 The images show the spleen photographs and HE staining results of tissue sections used in the pharmacodynamic experiment of a melanoma model after loading VNP bacteria onto the 1,6-DAS hydrogel of this invention, representing the therapeutic endpoint.

[0071] Figure 29 The liver coefficient is a therapeutic endpoint in a pharmacodynamic experiment of a melanoma model after loading VNP bacteria onto the 1,6-DAS hydrogel of the present invention.

[0072] Figure 30 The images show liver photographs and HE staining results of tissue sections used in a melanoma model pharmacodynamic experiment after loading VNP bacteria onto 1,6-DAS hydrogel according to the present invention.

[0073] Figure 31 The graph shows the alanine aminotransferase result, the therapeutic endpoint, in a pharmacodynamic experiment of a melanoma model after loading VNP bacteria onto the 1,6-DAS hydrogel of the present invention.

[0074] Figure 32 The image shows the white blood cell count, the therapeutic endpoint, in a pharmacodynamic experiment of a melanoma model after loading VNP bacteria onto the 1,6-DAS hydrogel of this invention.

[0075] Figure 33 This image shows the platelet count results, the therapeutic endpoint, in a pharmacodynamic experiment of a melanoma model after loading VNP bacteria onto the 1,6-DAS hydrogel of the present invention.

[0076] Figure 34 The images show scanning electron microscope (SEM) images of VNP bacteria before and after loading the 1,6-DAS hydrogel of the present invention.

[0077] Figure 35The image shows the zeta potential detection results of VNP bacteria before and after loading the 1,6-DAS hydrogel of the present invention.

[0078] Figure 36 This is a heatmap showing the binding affinity between the 1,6-DAS hydrogel molecules and the VNP flagellin protein domains of the present invention.

[0079] Figure 37 This is a schematic diagram of the binding affinity between the 1,6-DAS hydrogel molecule of the present invention and the D0 and D1 domains of VNP flagellin (top left is the 3D structure of the D0 domain, top right is the 2D structure of the D0 domain, bottom left is the 3D structure of the D1 domain, and bottom right is the 2D structure of the D1 domain).

[0080] Figure 38 This is a flow cytometry analysis result of various immune cells in the spleen, which is the therapeutic endpoint of a pharmacodynamic experiment in a melanoma model after loading VNP bacteria onto the 1,6-DAS hydrogel of the present invention.

[0081] Figure 39 This is a flow cytometry analysis result of blood immune cells as the therapeutic endpoint in a melanoma model pharmacodynamic experiment after loading VNP bacteria onto the 1,6-DAS hydrogel of the present invention.

[0082] Figure 40 This is a flow cytometry analysis result of various immune cells in tumor tissue as the therapeutic endpoint in a melanoma model pharmacodynamic experiment after loading VNP bacteria onto the 1,6-DAS hydrogel of the present invention.

[0083] Figure 41 This is a landscape heatmap of the Gibbs free energy of the 1,6-DAS hydrogel molecules of the present invention in a POPC lipid bilayer with the sodium ion channel protein Nav1.7 of DC cells, as simulated by a molecular dynamics simulation system.

[0084] Figure 42 This diagram shows the number of 1,6-DAS hydrogel molecules of the present invention that have a contact distance of less than 0.6 nm with the sodium ion channel protein Nav1.7.

[0085] Figure 43 This figure shows the channel opening analysis results of the sodium ion channel protein Nav1.7 in the 1,6-DAS hydrogel molecule of the present invention under the condition of sodium ion presence or absence.

[0086] Figure 44 This is a schematic timeline diagram of the pharmacodynamic experiments of a lung adenocarcinoma model after loading VNP bacteria onto the 1,6-DAS hydrogel of the present invention.

[0087] Figure 45 The graph shows the weight change curves of mice in each treatment group in the pharmacodynamic experiment of a lung adenocarcinoma model after loading VNP bacteria onto the 1,6-DAS hydrogel of the present invention.

[0088] Figure 46 This is a graph showing the individual tumor growth curves of mice in each treatment group in the pharmacodynamic experiment of a lung adenocarcinoma model after loading VNP bacteria onto the 1,6-DAS hydrogel of the present invention.

[0089] Figure 47 This image shows the tumor volume at the treatment endpoint in mice of each treatment group during the pharmacodynamic experiment of a lung adenocarcinoma model after loading VNP bacteria onto the 1,6-DAS hydrogel of the present invention.

[0090] Figure 48 The images show the spleen photographs and HE staining results of tissue sections used in the pharmacodynamic experiment of a lung adenocarcinoma model after loading VNP bacteria onto the 1,6-DAS hydrogel of this invention, representing the therapeutic endpoint.

[0091] Figure 49 This is a graph showing the spleen coefficient as the therapeutic endpoint in a pharmacodynamic experiment of a lung adenocarcinoma model after loading VNP bacteria onto the 1,6-DAS hydrogel of the present invention.

[0092] Figure 50 The images show liver photographs and HE staining results of tissue sections used in the pharmacodynamic experiment of a lung adenocarcinoma model after loading VNP bacteria onto 1,6-DAS hydrogel according to the present invention.

[0093] Figure 51 The liver coefficient is a graph representing the therapeutic endpoint in a pharmacodynamic experiment of a lung adenocarcinoma model after loading VNP bacteria onto the 1,6-DAS hydrogel of the present invention.

[0094] Figure 52 The graph shows the alanine aminotransferase result, the therapeutic endpoint, in a pharmacodynamic experiment of a lung adenocarcinoma model after loading VNP bacteria onto the 1,6-DAS hydrogel of the present invention.

[0095] Figure 53 The image shows the white blood cell count results, the therapeutic endpoint, in a pharmacodynamic experiment of a lung adenocarcinoma model after loading VNP bacteria onto the 1,6-DAS hydrogel of the present invention.

[0096] Figure 54 This is a graph showing the platelet count results, the therapeutic endpoint, in a pharmacodynamic experiment of a lung adenocarcinoma model after loading VNP bacteria onto the 1,6-DAS hydrogel of the present invention.

[0097] Figure 55 This is a schematic timeline diagram of the pharmacodynamic experiments of a melanoma model after loading VNP-TNF-α nb bacteria onto the 1,6-DAS hydrogel of the present invention.

[0098] Figure 56 This image shows the tumor volume at the treatment endpoint in mice of each treatment group during pharmacodynamic experiments of a melanoma model after loading VNP-TNF-α nb bacteria onto the 1,6-DAS hydrogel of the present invention.

[0099] Figure 57 This is a graph showing the individual tumor growth curves of mice in each treatment group in a melanoma model pharmacodynamic experiment after loading VNP-TNF-α nb bacteria onto the 1,6-DAS hydrogel of the present invention.

[0100] Figure 58 This is a graph showing the weight change of mice in each treatment group in a melanoma model pharmacodynamic experiment after loading VNP-TNF-α nb bacteria onto the 1,6-DAS hydrogel of the present invention.

[0101] Figure 59 The images show liver and spleen images of mice in each treatment group at the treatment endpoint in a pharmacodynamic experiment of a melanoma model after loading VNP-TNF-α nb bacteria onto the 1,6-DAS hydrogel of the present invention.

[0102] Figure 60 This is a graph showing the spleen coefficient of mice in each treatment group during the pharmacodynamic experiment of a melanoma model after loading VNP-TNF-α nb bacteria onto the 1,6-DAS hydrogel of the present invention.

[0103] Figure 61 The liver coefficient diagram shows the treatment endpoints of mice in each treatment group in the pharmacodynamic experiment of a melanoma model after loading VNP-TNF-α nb bacteria onto the 1,6-DAS hydrogel of the present invention.

[0104] Figure 62 This is a flow cytometry analysis result of various immune cells in the spleen of mice in each treatment group during the pharmacodynamic experiment of a melanoma model after loading VNP-TNF-α nb bacteria onto the 1,6-DAS hydrogel of the present invention.

[0105] Figure 63 This is a flow cytometry analysis result of blood immune cells in mice at the treatment endpoint of a melanoma model pharmacodynamic experiment after loading VNP-TNF-α nb bacteria onto the 1,6-DAS hydrogel of the present invention.

[0106] Figure 64 This is a flow cytometry analysis result of various immune cells in the tumor tissue of mice in each treatment group at the treatment endpoint of a melanoma model pharmacodynamic experiment after loading VNP-TNF-α nb bacteria onto the 1,6-DAS hydrogel of the present invention.

[0107] Figure 65 This is a schematic timeline diagram of the pharmacodynamic experiments of a bilateral melanoma model after loading VNP-TNF-α nb bacteria onto the 1,6-DAS hydrogel of the present invention.

[0108] Figure 66This is a growth curve of the contralateral tumor in a pharmacodynamic experiment of a bilateral melanoma model after loading VNP-TNF-α nb bacteria onto the 1,6-DAS hydrogel of the present invention. Detailed Implementation

[0109] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0110] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0111] This invention attempts to use original or mutant strains of attenuated Salmonella Typhimurium, Escherichia coli, Bifidobacterium, BCG, Shigella, Klebsiella pneumoniae, and Pseudomonas aeruginosa, or engineered strains expressing TNF-α nanobodies or PD-1 nanobodies. Because attenuated Salmonella Typhimurium, Escherichia coli, Bifidobacterium, BCG, Shigella, Klebsiella pneumoniae, and Pseudomonas aeruginosa are similar in size, growth rate, physicochemical properties, antitumor properties, and mechanisms, these oncolytic bacteria exhibit similar activities. Attenuated Salmonella is the oncolytic bacterium with the most published research papers, clinical trials, and professional R&D companies, and is the most representative bacterium among oncolytic bacteria. Therefore, this invention focuses on showcasing the research results of attenuated Salmonella. The results of other oncolytic bacteria are similar to those of attenuated Salmonella. Our research team has repeatedly demonstrated similar results among different oncolytic bacteria in previous studies (Adv Mater. 2024; 36(36):e2406140.), so this invention will not repeat them one by one. In addition, this invention has also studied different tumor models, including melanoma, lung cancer, lymphoma, bladder cancer, liver cancer, colorectal cancer, and breast cancer, and obtained similar anti-tumor results in all of them (Adv Mater. 2024; 36(36):e2406140). Our research team has repeatedly demonstrated similar biological anti-tumor efficacy among different oncolytic bacteria in previous studies, so this invention will not repeat the results in different tumor models one by one.

[0112] The first aspect of this application provides a sodium ion-responsive injectable self-assembly hydrogel system, the injectable self-assembly hydrogel system comprising: A hydrogel precursor solution comprising isostevol-diester sulfonic acid derivative and oncolytic bacteria; wherein, the hydrogel precursor solution, when mixed with original or mutant strains of oncolytic bacteria, including but not limited to attenuated Salmonella typhimurium, Escherichia coli, Bifidobacterium, BCG, Shigella, Klebsiella pneumoniae, and Pseudomonas aeruginosa, or engineered oncolytic bacterial strains expressing TNF-α nanobodies or PD-1 nanobodies, can self-assemble into a hydrogel containing the oncolytic bacteria under conditions of contact with sodium ions.

[0113] In some embodiments, the isosteviol-diester sulfonate derivative is selected from any one of isosteviol-1,6-diester sulfonate (1,6-DA), sodium isosteviol-1,6-diester sulfonate (1,6-DAS), potassium isosteviol-1,6-diester sulfonate, calcium isosteviol-1,6-diester sulfonate, 1,4-diester sulfonate (1,4-DA), sodium isosteviol-1,4-diester sulfonate (1,4-DAS), potassium isosteviol-1,4-diester sulfonate, or calcium isosteviol-1,4-diester sulfonate. Oncolytic bacteria include, but are not limited to, original or mutant strains of attenuated Salmonella typhimurium, Escherichia coli, Bifidobacterium, BCG, Shigella, Klebsiella pneumoniae, and Pseudomonas aeruginosa, or engineered oncolytic bacterial strains expressing exogenous functional proteins.

[0114] In some embodiments, the engineered oncolytic bacterial strain is an oncolytic bacterial strain, represented by VNP, that expresses TNF-α nanobodies or PD-1 nanobodies.

[0115] A second aspect of this application provides a method for preparing an injectable self-assembly hydrogel system, comprising the following steps: (1) Prepare a solution of isosteviol-diestersulfonic acid derivative as a hydrogel precursor solution; (2) Mix the oncolytic bacteria cultured to the exponential growth phase with the hydrogel precursor solution described in step (1); (3) The mixture obtained in step (2) is incubated in an environment containing sodium ions to form a hydrogel system by self-assembly.

[0116] In some embodiments, the sodium-containing environment is a phosphate buffer solution, a sodium chloride solution, or the in vivo environment of a living organism.

[0117] A third aspect of this application provides a method for loading oncolytic bacteria onto a sodium ion-responsive injectable in vivo self-assembly hydrogel, comprising the following steps: (1) Preparation of an aqueous solution of isosteviol-diester sulfonic acid derivative; (2) After centrifuging the oncolytic bacteria, collect the bacterial precipitate and resuspend the bacterial precipitate in the aqueous solution described in step (1) to obtain a bacterial solution mixture; (3) Contact the bacterial mixture obtained in step (2) with a solution containing sodium ions, or inject the bacterial mixture into a living organism to trigger self-assembly to form a hydrogel containing oncolytic bacteria.

[0118] In some embodiments, in step (1), the isosteviol-diester sulfonic acid derivative is isosteviol-1,6-diester sulfonic acid (1,6-DA) or isosteviol-1,4-diester sulfonic acid (1,4-DA); the concentration of the aqueous solution is 5-20 mg / mL; the preparation of the aqueous solution includes the steps of heating and dissolving the isosteviol-diester sulfonic acid derivative in a solvent and then cooling it. In step (2), the oncolytic bacteria refer to oncolytic bacteria represented by wild-type VNP20009 strain or engineered VNP strains expressing TNF-α nanobodies or PD-1 nanobodies, including but not limited to original or mutant strains of attenuated Salmonella Typhimurium, Escherichia coli, Bifidobacterium, BCG, Shigella, Klebsiella pneumoniae, and Pseudomonas aeruginosa, or engineered strains of the above-mentioned oncolytic bacteria expressing TNF-α nanobodies or PD-1 nanobodies; the amount of bacterial precipitate is not less than 1 × 10⁻⁶ per 100 μL of aqueous solution. 9 CFU; In step (3), the sodium-containing solution is a phosphate buffer or a sodium chloride solution; the biological body is subcutaneous or peritoneal.

[0119] The fourth aspect of this application provides the use of a hydrogel system in the preparation of a medicament for treating tumors.

[0120] In some embodiments, the drug is administered by injection, and the hydrogel system self-assembles in vivo to achieve sustained-release delivery of oncolytic bacteria. Hydrogel systems are used for: (i) Reduce systemic toxicity associated with oncolytic bacterial therapy, including weight loss, organ enlargement, tissue damage, and / or reduced long-term survival; and / or (ii) Enhance the antitumor efficacy of oncolytic bacteria; The reduction in systemic toxicity is achieved by the hydrogel system through the slow release of oncolytic bacteria and / or by shielding the immunogenic proteins on the surface of oncolytic bacteria.

[0121] In some embodiments, the immunogenic protein is flagellin; the enhancement of antitumor efficacy is achieved by enhancing the activation of dendritic cells (DCs) and / or CD8+ in the tumor immune microenvironment. +The proportion of T cells is used to achieve this; the tumor is a solid tumor; the solid tumor includes melanoma, lung cancer, lymphoma, bladder cancer, liver cancer, colon cancer, or breast cancer.

[0122] A fifth aspect of this application provides a kit for delivering oncolytic bacteria, characterized in that: the kit comprises: (a) A hydrogel precursor composition containing isosteviol-diestersulfonic acid derivatives; and (b) Oncolytic bacteria, including but not limited to the original strains or mutant strains of oncolytic bacteria such as attenuated Salmonella Typhimurium, Escherichia coli, Bifidobacterium, BCG, Shigella, Klebsiella pneumoniae, and Pseudomonas aeruginosa, or engineered strains of the above-mentioned oncolytic bacteria expressing TNF-α nanobodies or PD-1 nanobodies. Among them, components (a) and (b) can form hydrogels containing attenuated Salmonella bacteria after contact with sodium ions.

[0123] In some embodiments, the isosteviol-diester sulfonic acid derivative is selected from any one of isosteviol-1,6-diester sulfonic acid (1,6-DA), sodium isosteviol-1,6-diester sulfonate (1,6-DAS), potassium isosteviol-1,6-diester sulfonate, calcium isosteviol-1,6-diester sulfonate, isosteviol-1,4-diester sulfonic acid (1,4-DA), sodium isosteviol-1,4-diester sulfonate (1,4-DAS), potassium isosteviol-1,4-diester sulfonate, or calcium isosteviol-1,4-diester sulfonate.

[0124] The sixth aspect of this application provides a sodium ion-responsive injectable self-assembly hydrogel containing oncolytic bacteria, which is prepared by a method.

[0125] 100 μL of the hydrogel can encapsulate more than 1 × 10 9 The CFU contains attenuated Salmonella, a representative oncolytic bacteria, without affecting its activity. Under in vitro conditions, the hydrogel can release approximately 80% of the attenuated Salmonella and other oncolytic bacteria it encapsulates within 72 hours. Example 1

[0126] The present invention discloses a sodium ion-responsive injectable self-assembly hydrogel system, the injectable self-assembly hydrogel system comprising: The hydrogel precursor solution contains isosteviol-1,6-diester sulfonic acid derivative and oncolytic bacteria. Upon mixing with the oncolytic bacteria and in contact with sodium ions, the hydrogel precursor solution self-assembles to form a hydrogel containing the oncolytic bacteria. The isosteviol-1,6-diester sulfonic acid derivative is isosteviol-1,6-diester sulfonic acid (1,6-DA).

[0127] The engineered oncolytic bacteria is the wild-type attenuated Salmonella strain VNP20009 (VNP). The engineered oncolytic bacterial strains are represented by the VNP strain expressing TNF-α nanobodies, and are among the aforementioned engineered oncolytic bacterial strains expressing TNF-α nanobodies. Example 2

[0128] The difference between Example 2 and Example 1 is that the isosteviol-diester sulfonic acid derivative is isosteviol-1,6-diester sulfonate sodium (1,6-DAS). The engineered oncolytic bacteria is Escherichia coli. Example 3

[0129] The difference between Example 3 and Example 1 is that the oncolytic bacteria are engineered oncolytic bacteria expressing exogenous functional proteins, and the engineered oncolytic bacteria are Bifidobacterium. The isosteviol-diester sulfonic acid derivative is isosteviol-1,6-diester sulfonate potassium. Example 4

[0130] The difference between Example 4 and Example 1 is that the oncolytic bacteria are engineered oncolytic bacteria expressing exogenous functional proteins, and the engineered oncolytic bacteria are BCG. The isosteviol-diester sulfonic acid derivative is isosteviol-1,6-diester sulfonate calcium. Example 5

[0131] The difference between Example 5 and Example 1 is that the oncolytic bacteria are engineered oncolytic bacteria expressing exogenous functional proteins, and the engineered oncolytic bacteria are Shigella. The isosteviol-diester sulfonic acid derivative is isosteviol-1,4-diester sulfonic acid (1,4-DA). Example 6

[0132] The difference between Example 6 and Example 1 is that the oncolytic bacteria are engineered oncolytic bacteria expressing exogenous functional proteins, and the engineered oncolytic bacteria are Klebsiella pneumoniae. The isosteviol-diester sulfonic acid derivative is isosteviol-1,4-diester sulfonate sodium (1,4-DAS). Example 7

[0133] The difference between Example 7 and Example 1 is that the oncolytic bacteria used are engineered oncolytic bacteria expressing exogenous functional proteins, and the engineered oncolytic bacteria are the original strain of *Pseudomonas aeruginosa*. The isosteviol-diester sulfonic acid derivative is isosteviol-1,4-diester sulfonate potassium. Example 8

[0134] The difference between Example 8 and Example 1 is that the oncolytic bacteria are engineered oncolytic bacteria expressing exogenous functional proteins, and the engineered oncolytic bacteria are mutant strains. The isosteviol-diester sulfonic acid derivative is isosteviol-1,4-diester sulfonate calcium. Example 9

[0135] The present invention provides a method for preparing an injectable self-assembling hydrogel system, comprising the following steps: (1) Prepare a solution of isosteviol-diestersulfonic acid derivative as a hydrogel precursor solution; (2) Mix the oncolytic bacteria cultured to the exponential growth phase with the hydrogel precursor solution from step (1); (3) The mixture obtained in step (2) was incubated in an environment containing sodium ions to form a hydrogel system by self-assembly. The environment containing sodium ions was phosphate buffer. The engineered oncolytic bacteria was wild-type attenuated Salmonella strain VNP20009 (VNP). Example 10

[0136] The difference between Example 10 and Example 9 is that the sodium-containing environment is a sodium chloride solution, and the engineered oncolytic bacteria is Escherichia coli. Example 11

[0137] The difference between Example 11 and Example 9 is that the sodium-containing environment is the in vivo environment of a living organism. The engineered oncolytic bacteria is Bifidobacterium. Example 12

[0138] The difference between Example 12 and Example 9 is that the oncolytic bacteria are engineered oncolytic bacteria that express exogenous functional proteins, and the engineered oncolytic bacteria are BCG. Example 13

[0139] The difference between Example 13 and Example 9 is that the oncolytic bacteria are engineered oncolytic bacteria that express exogenous functional proteins, and the engineered oncolytic bacteria are Shigella. Example 14

[0140] The difference between Example 14 and Example 9 is that the oncolytic bacteria are engineered oncolytic bacteria that express exogenous functional proteins, and the engineered oncolytic bacteria are Klebsiella pneumoniae. Example 15

[0141] The difference between Example 15 and Example 9 is that the oncolytic bacteria are engineered oncolytic bacteria that express exogenous functional proteins, and the engineered oncolytic bacteria are the original strain of Pseudomonas aeruginosa. Example 16

[0142] The difference between Example 16 and Example 9 is that the oncolytic bacteria are engineered oncolytic bacteria that express exogenous functional proteins, and the engineered oncolytic bacteria are mutant strains. Example 17

[0143] The present invention discloses a method for loading oncolytic bacteria onto a sodium ion-responsive injectable in vivo self-assembling hydrogel, comprising the following steps: (1) Preparation of an aqueous solution of isosteviol-diester sulfonic acid derivative; the isosteviol-diester sulfonic acid derivative is isosteviol-1,4-diester sulfonic acid (1,4-DA); the concentration of the aqueous solution is 5 mg / mL; the preparation of the aqueous solution includes the steps of heating and dissolving the isosteviol-diester sulfonic acid derivative in a solvent and then cooling it. (2) After centrifuging the attenuated Salmonella as a representative oncolytic bacteria, collect the bacterial cell precipitate and resuspend the precipitate in the aqueous solution described in step (1) to obtain a bacterial solution mixture; the amount of bacterial cell precipitate is not less than 1×10⁻⁶ per 100 μL of aqueous solution. 9 CFU; (3) The bacterial mixture obtained in step (2) is contacted with a solution containing sodium ions, or the bacterial mixture is injected into an organism to trigger self-assembly to form a hydrogel containing attenuated Salmonella as a representative oncolytic bacteria. The solution containing sodium ions is phosphate buffer or sodium chloride solution; in the organism, it is subcutaneous or intraperitoneal. The oncolytic bacteria are engineered oncolytic bacteria expressing exogenous functional proteins. Example 18

[0144] The difference between Example 18 and Example 17 is that the method for loading oncolytic bacteria into a sodium ion-responsive injectable in vivo self-assembly hydrogel of the present invention includes the following steps: (1) Prepare an aqueous solution of isosteviol-diester sulfonic acid derivative; the isosteviol-diester sulfonic acid derivative is isosteviol-1,6-diester sulfonic acid (1,6-DA); the concentration of the aqueous solution is 20 mg / mL. (2) After centrifuging the oncolytic bacteria, collect the bacterial precipitate and resuspend the bacterial precipitate in the aqueous solution described in step (1) to obtain a bacterial solution mixture; the engineered oncolytic bacteria is Escherichia coli.

[0145] (3) The bacterial mixture obtained in step (2) is brought into contact with a sodium-containing solution, or the bacterial mixture is injected into an organism to trigger self-assembly to form a hydrogel containing oncolytic bacteria. The sodium-containing solution is a sodium chloride solution; the organism is the peritoneal cavity. Example 19

[0146] The difference between Example 19 and Example 17 is that the method for loading oncolytic bacteria into a sodium ion-responsive injectable in vivo self-assembly hydrogel of the present invention includes the following steps: (1) Prepare an aqueous solution of isosteviol-diester sulfonic acid derivative; the concentration of the aqueous solution is 16 mg / mL; the preparation of the aqueous solution includes the steps of heating and dissolving the isosteviol-diester sulfonic acid derivative in a solvent and then cooling it. (2) After centrifuging the oncolytic bacteria, collect the bacterial cell precipitate and resuspend the precipitate in the aqueous solution obtained in step (1) to obtain a bacterial solution mixture; the amount of bacterial cell precipitate is not less than 1×10⁻⁶ per 100 μL of aqueous solution. 9 CFU; the engineered oncolytic bacteria is Bifidobacterium.

[0147] (3) The bacterial mixture obtained in step (2) is brought into contact with a sodium-containing solution, or the bacterial mixture is injected into an organism to trigger self-assembly to form a hydrogel containing oncolytic bacteria. The sodium-containing solution is phosphate buffer; in an organism, it is subcutaneous. Example 20

[0148] The difference between Example 20 and Example 17 is that in step (2), the oncolytic bacteria are engineered oncolytic bacteria represented by engineered VNP strains of PD-1 nanobodies. Example 21

[0149] The difference between Example 21 and Example 17 is that in step (2), the engineered oncolytic bacteria is BCG. Example 22

[0150] The difference between Example 22 and Example 17 is that in step (2), the engineered oncolytic bacteria is Shigella. Example 23

[0151] The difference between Example 23 and Example 17 is that in step (2), the engineered oncolytic bacteria is Klebsiella pneumoniae. Example 24

[0152] The difference between Example 24 and Example 17 is that in step (2), the engineered oncolytic bacteria is the original strain of Pseudomonas aeruginosa. Example 25

[0153] The difference between Example 25 and Example 17 is that in step (2), the engineered oncolytic bacteria is a mutant strain. Example 26

[0154] The present invention relates to the application of a hydrogel system in the preparation of a drug for treating tumors.

[0155] The drug is administered via injection, and the hydrogel system self-assembles in the body to achieve sustained delivery of oncolytic bacteria, such as attenuated Salmonella. Hydrogel systems are used for: (i) Reduce systemic toxicity associated with oncolytic bacterial therapies, such as attenuated Salmonella, including weight loss, organ enlargement, tissue damage, and / or reduced long-term survival; and / or (ii) Enhance the antitumor efficacy of oncolytic bacteria, represented by attenuated Salmonella.

[0156] The reduction in systemic toxicity is achieved by the sustained release of attenuated Salmonella, representing oncolytic bacteria, and / or by shielding the immunogenic proteins on the surface of attenuated Salmonella, representing oncolytic bacteria, through the hydrogel system.

[0157] The immunogenic protein is flagellin; enhancing anti-tumor efficacy is achieved by enhancing the activation of dendritic cells (DCs) and / or CD8 in the tumor immune microenvironment. + The proportion of T cells is used to achieve this; the tumor is a solid tumor. Example 27

[0158] The difference between Example 27 and Example 26 is that the solid tumor is melanoma. Example 28

[0159] The difference between Example 28 and Example 26 is that the solid tumor is lung cancer. Example 29

[0160] The difference between Example 29 and Example 26 is that the solid tumor is lymphoma. Example 30

[0161] The difference between Example 30 and Example 26 is that the solid tumor is bladder cancer. Example 31

[0162] The difference between Example 31 and Example 26 is that the solid tumor is liver cancer. Example 32

[0163] The difference between Example 32 and Example 26 is that the solid tumor is colorectal cancer. Example 33

[0164] The difference between Example 33 and Example 26 is that the solid tumor is breast cancer. Example 34

[0165] The present invention provides a kit for delivering oncolytic bacteria, represented by attenuated Salmonella, the kit comprising: (a) A hydrogel precursor composition containing isosteviol-diestersulfonic acid derivatives; and (b) Oncolytic bacteria, including but not limited to the original strains or mutant strains of oncolytic bacteria such as attenuated Salmonella Typhimurium, Escherichia coli, Bifidobacterium, BCG, Shigella, Klebsiella pneumoniae, and Pseudomonas aeruginosa, or engineered strains of the above-mentioned oncolytic bacteria expressing TNF-α nanobodies or PD-1 nanobodies. Among them, components (a) and (b) can form hydrogels containing attenuated Salmonella bacteria upon contact with sodium ions. The isosteviol-diester sulfonic acid derivative is isosteviol-1,6-diester sulfonic acid (1,6-DA). Example 35

[0166] The isosteviol-diester sulfonic acid derivative is sodium isosteviol-1,6-diester sulfonate (1,6-DAS). Example 36

[0167] The difference between Example 36 and Example 34 is that the isosteviol-diester sulfonic acid derivative is isosteviol-1,6-diester sulfonic acid potassium. Example 37

[0168] The difference between Example 37 and Example 34 is that the isosteviol-diester sulfonic acid derivative is isosteviol-1,6-diester sulfonic acid calcium. Example 38

[0169] The difference between Example 38 and Example 34 is that the isosteviol-diester sulfonic acid derivative is isosteviol-1,4-diester sulfonic acid (1,4-DA). Example 39

[0170] The difference between Example 39 and Example 34 is that the isosteviol-diester sulfonic acid derivative is isosteviol-1,4-diester sulfonate sodium (1,4-DAS). Example 40

[0171] The difference between Example 40 and Example 34 is that the isosteviol-diester sulfonic acid derivative is isosteviol-1,4-diester sulfonic acid potassium. Example 41

[0172] The difference between Example 41 and Example 34 is that the isosteviol-diester sulfonic acid derivative is isosteviol-1,4-diester sulfonic acid calcium. Example 42

[0173] The present invention discloses an injectable self-assembling hydrogel containing sodium ion-responsive attenuated Salmonella as a representative oncolytic bacteria, which is prepared by the method described above. Example 43

[0174] Preparation of hydrogels The 1,6-DAS hydrogel precursor, 1,6-DA powder, was prepared in the laboratory. Figure 1This diagram illustrates the chemical reaction involved in the formation of the 1,6-DAS hydrogel of the present invention via a sodium ion-responsive gelation process from the 1,6-DA precursor. The attenuated Salmonella VNP, representing oncolytic bacteria, and its engineered strain VNP-TNF-α nb were provided by the laboratory. All strains were cultured in LB broth at 37 °C with shaking at 220 rpm.

[0175] In a pressure-resistant tube, a 10 mg / mL suspension of 1,6-DA was prepared and heated with an alcohol lamp for approximately 30 seconds until completely dissolved and clear. After cooling to room temperature, oncolytic bacteria, represented by VNP or engineered oncolytic bacteria, represented by VNP-TNF-α nb, were added to the 1,6-DA solution. Subsequently, a sodium-containing solution (such as PBS or NaCl solution) was added in vitro, or the precursor was directly injected into mice (subcutaneously or intraperitoneally) to trigger sodium-regulated transient self-assembly, forming a 1,6-DAS hydrogel loaded with VNP (1,6-DAS / VNP) or VNP-TNF-α nb (1,6-DAS / VNP-TNF-α nb).

[0176] Two µg of methylene blue was incorporated as a tracer into 800 µL of a 10 mg / mL 1,6-DA hydrogel solution. After adding 200 µL of PBS, the vial was inverted and photographed to record the gel state. Figure 2 This image shows the gelation process of the 1,6-DAS hydrogel of the present invention, which is formed from a 1,6-DA precursor in response to sodium ions. Furthermore, a 10 mg / mL hydrogel precursor solution of 1,6-DA was injected into PBS using a 1 mL syringe, and the gelation reaction that occurred instantaneously upon contact of the 1,6-DA solution with the sodium-containing solution was recorded using a Canon 200D II camera. Figure 5 This is a photograph of the 1,6-DAS hydrogel of the present invention, showing the instantaneous gelation of the 1,6-DA precursor in response to sodium ions.

[0177] The system was constructed using a 6×6×6 nm³ simulation chamber, randomly placing 1,6-DAS hydrogel molecules, and also included 10,6-DAS molecules. All molecular dynamics simulations were performed using Gromacs 2023.2 software (Stockholm, Sweden) under periodic boundary conditions, employing an AMBER94 all-atom force field combined with the TIP3P water model. Long-range electrostatic interactions were calculated using the particle grid Ewald method, with a real space cutoff of 1.0 nm, and van der Waals interactions were also cut off at 1.0 nm; all hydrogen-related bonds were constrained using the LINCS algorithm. The system temperature was maintained at 300 K using a V-rescale thermostat, and the pressure was controlled at 1 bar using a Parrinello-Rahman pressure equalizer. After energy minimization, the configuration was equilibrated at 300 K for 100 ps of NVT, followed by 200 ps of NPT equilibration at 300 K, and finally, a production simulation was run at 300 K with a time step of 2 fs for 600 ns. The trajectory is saved every 10 ps, ​​generating a total of 60,000 snapshots for subsequent analysis. Figure 3 This is a schematic diagram of the molecular dynamics simulation of the 1,6-DAS hydrogel of the present invention, showing the gelation process (0-600 ns) of the 1,6-DA precursor in response to sodium ions. Figure 4 The results of molecular dynamics simulations of the 1,6-DAS hydrogel of the present invention, which is formed by the sodium ion-responsive gelation process of 1,6-DA precursor (0-600 ns), are shown (left: number of hydrogen bonds, right: number of interactions). Figure 6 This is a schematic diagram illustrating the principle of the 1,6-DAS hydrogel of the present invention, which is formed by the self-assembly of 1,6-DA precursors in response to sodium ions (left: before gelation, right: after gelation). Figure 7 This is a scanning electron microscope image of the 1,6-DAS hydrogel after gelation according to the present invention.

[0178] Two concentrations of 1,6-DAS hydrogels, 10 mg / mL and 20 mg / mL, were prepared, and dynamic oscillation frequency scans were performed using a rotational rheometer (NETZSCH Kinexus Prime Lab+). All tests were conducted at 25°C with a parallel plate geometry and mm gaps. Amplitude strain measurements were performed prior to the frequency scan experiments to determine the linear viscoelastic region, ensuring that subsequent measurements were strain-independent in the 0.1–10 Hz frequency range. Figure 8 The rheological analysis results of the 1,6-DAS hydrogel of the present invention after gelation are shown (left: 10 mg / mL 1,6-DAS, right: 20 mg / mL). Figure 9 The gelation time of the 1,6-DAS hydrogel of the present invention, forming a 10 mg / mL 1,6-DAS hydrogel under different sodium ion concentrations, is given. Figure 10 The image shows a scanning electron microscope (SEM) image of a 10 mg / mL 1,6-DAS hydrogel formed under different sodium ion concentrations according to the present invention.

[0179] 1,6-DA was dissolved in saline (to maintain osmotic pressure) and hydrogels of different concentrations (10, 5, 2.5, 1.25, and 0.625 mg / mL) were prepared. After gelation, 0.5 mL of each hydrogel solution was mixed with 0.5 mL of 2% erythrocyte suspension. Deionized water and physiological saline were used as positive and negative controls, respectively, with 0.5 mL of each mixed with 0.5 mL of 2% RBC suspension. The mixtures were incubated at room temperature and then centrifuged at 1000 rpm for 5 minutes. Hemolysis of each sample was visually assessed and recorded by photographing the tubes against a white A4 background. Subsequently, 200 μL of the supernatant from each sample was carefully transferred to a 96-well plate, with each group repeated 5 times. The optical density (OD) at 540 nm was measured. 540 Measurements were taken using a microplate reader. The hemolysis rate was calculated using the following formula:

[0180] A hemolysis rate below 5% is considered acceptable, indicating good blood compatibility. Figure 12 The hemolysis rate of erythrocytes at different concentrations of the 1,6-DAS hydrogel of the present invention is shown.

[0181] To assess in vivo safety, 100 µL of 10 mg / mL 1,6-DA containing methylene blue was intraperitoneally injected into 6-8 week old C57BL / 6J mice. The peritoneum was dissected at 0, 24, 48, and 72 hours post-injection to observe hydrogel formation and degradation. Figure 11 The images show the gel state formed in the peritoneum of mice at different time points (0-72 h) after intraperitoneal injection of the 1,6-DAS hydrogel of the present invention. Furthermore, at 6 hours post-administration, whole blood, serum, and major organs were collected for complete blood cell counts, serum biochemical analysis, and H&E staining to assess the acute toxicity of the 1,6-DAS hydrogel in vivo. Figure 13 HE staining results of major organ sections after intraperitoneal injection of 1,6-DAS hydrogel in mice according to the present invention.

[0182] Figure 14 This is a schematic diagram illustrating the sodium ion-responsive gelation process of the 1,6-DAS hydrogel of the present invention, in vivo and in vitro, after the 1,6-DA precursor encapsulates oncolytic bacteria represented by VNP.

[0183] Transfer 200 µL of 10 mg / mL 1,6-DA solution to a centrifuge tube, then add 50 µL of PBS. Use a 200 mL pipette to repeatedly aspirate the solution up and down from the tip until it becomes clogged; record this time as the "gelation time." Then, apply the same procedure to oncolytic bacteria (1 × 10⁻⁶) represented by VNP containing bioluminescent plasmids. 6 The time obtained (CFU) was recorded as "VNP gelation time". Given the biofluorescence shielding properties of 1,6-DA, oncolytic bacteria, represented by VNP, were continuously introduced into 200 µL of 10‰ hydrogel precursor (using ddH2O or PBS as solvent) until red fluorescence was observed under a fluorescence microscope (Carl Zeiss). This was recorded as the maximum loading capacity of oncolytic bacteria represented by VNP. Figure 15 The results show the maximum drug loading capacity of the 1,6-DAS hydrogel and its precursor 1,6-DA of this invention against oncolytic bacteria, represented by VNP. Figure 16 This is a scanning electron microscope image of the 1,6-DAS hydrogel of the present invention after successful loading of oncolytic bacteria, represented by VNP (i.e., 1,6-DAS / VNP), and lyophilization. Figure 17 The images show the release of oncolytic bacteria, represented by VNP, at different time points after loading the 1,6-DAS hydrogel of the present invention at a rotation speed of 100 rpm.

[0184] This invention first prepared a 200 µL 1,6-DAS hydrogel in vitro, encapsulated oncolytic bacteria represented by VNP-RFP, and then immersed it in 800 µL PBS, stirring at 100 rpm. The degree of redness in the PBS solution was observed at different time points, and 200 µL samples were collected for OD measurement (an equal volume of PBS was added after each sampling). The OD was measured... 600 Measure the concentration of all bacteria and record the bacterial growth curves. Figure 18 This is a graph showing the release curve of oncolytic bacteria (represented by VNP) from the 1,6-DAS hydrogel of the present invention at 100 rpm after loading. Figure 19 The growth curves of oncolytic bacteria, represented by VNPs released at different time points (0-24 h) after the 1,6-DAS hydrogel of the present invention is loaded with VNP bacteria to form 1,6-DAS / VNP.

[0185] For in vivo release studies, a B16F10 melanoma model was established in 6-8 week old female C57BL / 6J mice. After successful model establishment, 1,6-DAS / VNP-RFP was injected intraperitoneally. Major organs were harvested at 6, 24, and 72 hours post-injection, homogenized, and colony counted using the plate method. Figure 20This invention relates to the bacterial titers of oncolytic bacteria (represented by VNP) in tumor tissue at different time points after intraperitoneal injection of the 1,6-DAS hydrogel containing oncolytic bacteria (represented by VNP) to form a 1,6-DAS / VNP ratio. Figure 21 The bacterial titers of oncolytic bacteria (represented by VNP) in major organs at different time points after intraperitoneal injection of the 1,6-DAS hydrogel of the present invention, after loading oncolytic bacteria (represented by VNP) to form 1,6-DAS / VNP, were obtained.

[0186] Evaluation of the antitumor efficacy and safety of hydrogel-encapsulated oncolytic bacteria, represented by VNP. Figure 22 This is a schematic timeline diagram of the pharmacodynamic experiments of a melanoma model after loading oncolytic bacteria, represented by VNP, onto the 1,6-DAS hydrogel of the present invention.

[0187] Solid tumor-bearing mice were constructed and injected intraperitoneally with a hydrogel delivery system containing oncolytic bacteria, represented by VNPs. Monitoring was conducted daily. Tumor volume and body weight were measured using calipers and a digital balance. On day 7, the mice were euthanized, and whole blood, serum, major organs, and tumor tissue were collected for further analysis, including whole blood count, serum biochemistry, organ weight assessment, and H&E staining of tissue sections.

[0188] Figure 23 The images show the individual tumor growth curves of mice in each treatment group during pharmacodynamic experiments of a melanoma model after loading oncolytic bacteria, represented by VNP, onto the 1,6-DAS hydrogel of this invention. Figure 24 This image shows the tumor volume at the treatment endpoint in mice of various treatment groups during pharmacodynamic experiments in a melanoma model after loading oncolytic bacteria, represented by VNP, onto the 1,6-DAS hydrogel of the present invention. Figure 25 The figures show the body weight changes of mice in different treatment groups in a melanoma model pharmacodynamic experiment after loading oncolytic bacteria, represented by VNP, onto the 1,6-DAS hydrogel of this invention. Figure 26 The survival curves of mice in each treatment group in the pharmacodynamic experiment of a melanoma model after loading oncolytic bacteria, represented by VNP, onto the 1,6-DAS hydrogel of the present invention are shown. Figure 27 The spleen coefficient was used as the therapeutic endpoint in a pharmacodynamic experiment of a melanoma model after loading oncolytic bacteria, represented by VNP, onto a 1,6-DAS hydrogel according to the present invention. Figure 28 The images and HE staining results of spleen photographs and tissue sections were obtained as the therapeutic endpoint in a pharmacodynamic experiment of a melanoma model after loading oncolytic bacteria, represented by VNP, onto a 1,6-DAS hydrogel according to the present invention. Figure 29 The liver coefficient, the therapeutic endpoint, was used in a pharmacodynamic experiment of a melanoma model after loading oncolytic bacteria, represented by VNP, onto a 1,6-DAS hydrogel according to the present invention. Figure 30The liver photographs and HE staining results of the melanoma model pharmacodynamic experiment, which was conducted after loading oncolytic bacteria, represented by VNP, onto the 1,6-DAS hydrogel of this invention, served as the therapeutic endpoint. Figure 31 The results of alanine aminotransferase, the therapeutic endpoint, were obtained in a pharmacodynamic experiment of a melanoma model after loading oncolytic bacteria, represented by VNP, onto a 1,6-DAS hydrogel according to the present invention. Figure 32 The white blood cell count results are the therapeutic endpoint in a pharmacodynamic experiment of a melanoma model after loading oncolytic bacteria, represented by VNP, onto a 1,6-DAS hydrogel according to the present invention. Figure 33 The platelet count results are the therapeutic endpoint in a pharmacodynamic experiment of a melanoma model after loading oncolytic bacteria, represented by VNP, onto a 1,6-DAS hydrogel according to the present invention. Figure 44 This is a schematic timeline diagram of the pharmacodynamic experiments in a lung adenocarcinoma model after loading oncolytic bacteria, represented by VNP, onto the 1,6-DAS hydrogel of the present invention. Figure 45 The figures show the body weight changes of mice in different treatment groups in a lung adenocarcinoma model pharmacodynamic experiment after loading oncolytic bacteria, represented by VNP, onto the 1,6-DAS hydrogel of the present invention. Figure 46 The images show individual tumor growth curves of mice in each treatment group during pharmacodynamic experiments of a lung adenocarcinoma model after loading oncolytic bacteria, represented by VNP, onto the 1,6-DAS hydrogel of this invention. Figure 47 This image shows the tumor volume at the treatment endpoint in mice of different treatment groups during a pharmacodynamic experiment in a lung adenocarcinoma model after loading oncolytic bacteria, represented by VNP, onto the 1,6-DAS hydrogel of the present invention. Figure 48 The images and HE staining results of spleen photographs and tissue sections were used as the therapeutic endpoint in a pharmacodynamic experiment of a lung adenocarcinoma model after loading oncolytic bacteria, represented by VNP, onto a 1,6-DAS hydrogel according to the present invention. Figure 49 The spleen coefficient was used as the therapeutic endpoint in a pharmacodynamic experiment of a lung adenocarcinoma model after loading oncolytic bacteria, represented by VNP, onto the 1,6-DAS hydrogel of this invention. Figure 50 The liver photographs and HE staining results of the lung adenocarcinoma model pharmacodynamic experiment, which was conducted after loading oncolytic bacteria, represented by VNP, onto the 1,6-DAS hydrogel of the present invention, were used as the therapeutic endpoint. Figure 51 The liver coefficient, the therapeutic endpoint, was used in a pharmacodynamic experiment of a lung adenocarcinoma model after loading oncolytic bacteria, represented by VNP, onto the 1,6-DAS hydrogel of this invention. Figure 52 The results of alanine aminotransferase, the therapeutic endpoint, were obtained in a pharmacodynamic experiment of a lung adenocarcinoma model after loading oncolytic bacteria, represented by VNP, onto a 1,6-DAS hydrogel according to the present invention. Figure 53 The white blood cell count results are the therapeutic endpoint in a pharmacodynamic experiment of a lung adenocarcinoma model after loading oncolytic bacteria, represented by VNP, onto a 1,6-DAS hydrogel according to the present invention. Figure 54The platelet count results are the therapeutic endpoint in a pharmacodynamic experiment of a lung adenocarcinoma model after loading oncolytic bacteria, represented by VNP, onto a 1,6-DAS hydrogel according to the present invention. Figure 55 This is a schematic timeline diagram of the pharmacodynamic experiments in a melanoma model after loading engineered oncolytic bacteria, represented by VNP-TNF-α nb, onto the 1,6-DAS hydrogel of the present invention. Figure 56 This image shows the tumor volume at the treatment endpoint in mice of various treatment groups during a pharmacodynamic experiment in a melanoma model after loading engineered oncolytic bacteria, represented by VNP-TNF-α nb, onto a 1,6-DAS hydrogel according to the present invention. Figure 57 The images show the individual tumor growth curves of mice in each treatment group during the pharmacodynamic experiment of a melanoma model after the 1,6-DAS hydrogel of this invention was loaded with engineered oncolytic bacteria, represented by VNP-TNF-αnb. Figure 58 The figures show the body weight changes of mice in different treatment groups in a melanoma model pharmacodynamic experiment after the 1,6-DAS hydrogel of this invention was loaded with engineered oncolytic bacteria, represented by VNP-TNF-α nb. Figure 59 The images show liver and spleen images of mice in various treatment groups at the treatment endpoint in a melanoma model pharmacodynamic experiment after the 1,6-DAS hydrogel of this invention was loaded with engineered oncolytic bacteria, represented by VNP-TNF-α nb. Figure 60 The spleen coefficients of mice in each treatment group were used as the treatment endpoint in a pharmacodynamic experiment of a melanoma model after loading engineered oncolytic bacteria, represented by VNP-TNF-α nb, onto the 1,6-DAS hydrogel of this invention. Figure 61 The liver coefficients of mice in each treatment group were used as the treatment endpoint in a pharmacodynamic experiment of a melanoma model after loading engineered oncolytic bacteria, represented by VNP-TNF-α nb, onto the 1,6-DAS hydrogel of the present invention.

[0189] Exploring the mechanism of enhanced antitumor efficacy after hydrogel encapsulation of oncolytic bacteria, represented by VNP. Figure 34 These are scanning electron microscope images of oncolytic bacteria, represented by VNP, before and after loading the 1,6-DAS hydrogel of the present invention. Figure 35The zeta potentials of oncolytic bacteria (represented by VNP) before and after loading the 1,6-DAS hydrogel of this invention are shown. Further molecular dynamics simulations were performed using the Gromacs 2023.2 software package. Nav1.7 sodium ion channel protein (PDB ID: 6N4Q) with full conformation was embedded in a pure POPC membrane, and 1-DAS hydrogel molecules were randomly inserted into a 14×14×14 nm³ cube simulation box of Charmmgui. The system was solvated using the TIP3P water model. A CHARMM36 force field was applied to the POPC membrane and the protein, while a GeneralAmber force field was applied to the 1-DAS hydrogel molecules. A Verlet cutoff scheme was used to update the neighbor list every 20 steps. Van der Waals interactions were calculated using a force-switching modulator with a switching distance of 1.0 nm and a cutoff wavelength of 1.2 nm. Electrostatic interactions were calculated using the particle mesh Ewald method with a Coulomb cutoff radius of 1.2 nm. All bonds involving hydrogen atoms were constrained using the LINCS algorithm. Prior to production simulation, the system underwent six equilibration steps to ensure structural stability. The production simulation was conducted with a time step of 2 fs and a total duration of 200 ns. Figure 36 This is a heatmap showing the binding affinity between the 1,6-DAS hydrogel molecule of the present invention and two protein domains of flagellin, a protein from oncolytic bacteria represented by VNP. Figure 37 This is a schematic diagram of the binding affinity between the 1,6-DAS hydrogel molecule of the present invention and the D0 and D1 domains of oncolytic bacterial flagellin represented by VNP (top left is the 3D structure of the D0 domain, top right is the 2D structure of the D0 domain, bottom left is the 3D structure of the D1 domain, and bottom right is the 2D structure of the D1 domain).

[0190] Following treatment with 1,6-DAS / VNP, tumor-bearing mice were euthanized, and spleens, blood, and tumor tissues were collected. Tumor tissues were enzymatically digested at 37°C and then at 45°C with 1 mg / mL type IV collagenase and 30 U / mL DNase I. The resulting suspension was filtered through a 40 μm cell filter to obtain a single-cell suspension. Red blood cells were lysed on ice using RBC lysis buffer (Servicebio, G2015-500 mL), followed by centrifugation and washing 1-2 times with PBS. Cells were then incubated at 4 °C in 1% bovine serum albumin for 15 minutes, and then labeled with a set of fluorophore-conjugated mouse antibodies: CD45-APC-EF780 (eBioscience, #47-0451-82), CD4-FITC (absin, #624A029), CD8A-PerCP-Cy5.5 (eBiosscience, #45-0081-80), CD69-PE-Cy7 (eBiosScience, #12-0691-81), CD11b FITC (BD, #561688), F4 / 80-APC (eBioscience, #17-4801-80), Ly6G-PerCP-Cy5.5 (BD, #4561103), CD86-PE Cy7 (BD, #560582), PI (Thermo, #P3566), CD11c APC (eBioscience, #17-0114-81) and CD80-PE (BD, #561955). Stained cells were incubated at 4°C in the dark for 30-45°C for 30-60 min, washed 1-2 times with buffer to remove unbound antibodies, and analyzed by flow cytometry (BD@FACSCantoⅡ) using FlowJo 10.8.1 software. Figure 38 The results of flow cytometry analysis of various immune cells in the spleen, representing the therapeutic endpoint of a pharmacodynamic experiment in a melanoma model after loading oncolytic bacteria, represented by VNP, onto a 1,6-DAS hydrogel according to the present invention. Figure 39 The results of flow cytometry analysis of blood immune cells were used as the therapeutic endpoint in a melanoma model pharmacodynamic experiment after loading oncolytic bacteria, represented by VNP, onto a 1,6-DAS hydrogel according to the present invention. Figure 40 The results of flow cytometry analysis of various immune cells in tumor tissue, representing the therapeutic endpoint, in a melanoma model pharmacodynamic experiment after loading oncolytic bacteria (represented by VNP) onto a 1,6-DAS hydrogel according to the present invention. Figure 62 The present invention relates to the flow cytometry analysis results of various immune cells in the spleen of mice in each treatment group during the pharmacodynamic experiment of a melanoma model after loading engineered oncolytic bacteria, represented by VNP-TNF-α nb, onto a 1,6-DAS hydrogel. Figure 63The present invention relates to the flow cytometry analysis results of various immune cells in the blood of mice in each treatment group during the pharmacodynamic experiment of a melanoma model after loading engineered oncolytic bacteria, represented by VNP-TNF-α nb, onto a 1,6-DAS hydrogel. Figure 64 The results of flow cytometry analysis of immune cells in tumor tissues of mice in each treatment group during the pharmacodynamic experiment of a melanoma model after loading engineered oncolytic bacteria, represented by VNP-TNF-α nb, onto a 1,6-DAS hydrogel according to the present invention.

[0191] Exploring the mechanism of drug toxicity reduction after hydrogel encapsulation of oncolytic bacteria, represented by VNP. This invention uses oncolytic bacterial flagellin (PDB ID: 8FML), represented by Salmonella-derived flagellin, as the model system. Through bioinformatics analysis, the flagellin structure was divided into four distinct domains, and domain-specific docking grids were calculated and defined for subsequent molecular docking simulations using 1,6-DAS hydrogels. For each domain-hydrogel interaction pair, molecular docking was performed using a rigorous sampling scheme, and the top ten energy-favorable binding postures were systematically selected based on a comprehensive scoring index. To visualize the spatial and energy patterns of these interactions, a multidimensional heatmap representation was constructed, where binding affinity (expressed as ΔG values) is plotted in matrix format, with the horizontal axis representing the docking posture and the vertical axis representing the corresponding domain. All molecular docking analyses and visualizations were performed using AutoDock Vina 1.2.7 software. Figure 41 This is a landscape thermogram of the Gibbs free energy of the 1,6-DAS hydrogel molecules in the POPC lipid bilayer and the sodium ion channel protein Nav1.7 in DC cells, as simulated by a molecular dynamics simulation system. Figure 42 The number of 1,6-DAS hydrogel molecules of the present invention with a contact distance of less than 0.6 nm with the sodium ion channel protein Nav1.7 is [not specified]. Figure 43 The results show the channel opening analysis of the sodium ion channel protein Nav1.7 in the 1,6-DAS hydrogel molecule of this invention under the conditions of sodium ion presence or absence.

[0192] Blood was collected from the orbital venous sinuses of mice after different treatments, and samples were collected into EDTA-coated anticoagulant tubes. For serum biochemistry analysis, samples were collected into sterile, enzyme-free EP tubes and allowed to coagulate at room temperature for 2 hours. The supernatant was then collected by centrifugation at 3000 rpm for 15 minutes at 2–8 °C. All complete blood count and blood biochemistry tests were performed by Servicebio.

[0193] At the experimental endpoint, mice were euthanized after different treatments, and major organs were collected and fixed with a universal tissue fixative. The samples were then sent to Nanjing Meng Biotechnology Co., Ltd. for paraffin embedding, sectioning, H&E staining, and bright-field full-slide scanning. Histological images were analyzed using CaseViewer software.

[0194] In vitro experiments have demonstrated that 100 mL of sodium ion-responsive injectable in vivo self-assembling hydrogel can encapsulate more than 1 × 10⁻⁶ bacteria without affecting bacterial activity. 9 CFU-based attenuated Salmonella, representing oncolytic bacteria, can be encapsulated in 1,6-DAS hydrogels with a capacity exceeding 1 × 10⁻⁶ without compromising activity. 9 CFU wild-type VNP bacteria are representative oncolytic bacterial strains. Figure 16 This is a scanning electron microscope image of the 1,6-DAS hydrogel of the present invention after successful loading of oncolytic bacteria (i.e., 1,6-DAS / VNP) represented by VNP and freeze-drying. Figure 19 The growth curves of VNPs released at different time points (0-24 h) after the 1,6-DAS hydrogel of the present invention is loaded with oncolytic bacteria, represented by VNP bacteria, to form 1,6-DAS / VNP.

[0195] 1,6-DAS hydrogels can encapsulate more than 1×10⁻⁶ cells without affecting activity. 9 CFU VNP-TNF-α nb is a representative engineered oncolytic bacterial strain.

[0196] 1,4-DAS hydrogels can encapsulate more than 1×10⁻⁶ cells without affecting activity. 9 CFU wild-type VNP is a representative oncolytic bacterial strain.

[0197] 1,4-DAS hydrogels can encapsulate more than 1×10⁻⁶ cells without affecting activity. 9 CFU VNP-TNF-α nb is a representative engineered oncolytic bacterial strain.

[0198] In vitro experiments have shown that 100 mL of sodium-responsive injectable in vivo self-assembling hydrogel containing attenuated Salmonella and other oncolytic bacteria can release approximately 80% of the bacteria within 72 hours when shaken at 100 rpm.

[0199] A 100 mL 1,6-DAS hydrogel containing oncolytic bacteria, represented by wild-type VNP, can release approximately 80% of the bacteria within 72 h when shaken at 100 rpm. Figure 18 The graph shows the release curve of oncolytic bacteria, represented by VNP bacteria, from the 1,6-DAS hydrogel of the present invention at a rotation speed of 100 rpm after loading VNP bacteria.

[0200] A 100 mL 1,6-DAS hydrogel containing engineered oncolytic bacteria, represented by VNP-TNF-α nb, can release approximately 80% of the bacteria within 72 h when shaken at 100 rpm.

[0201] A 100 mL 1,4-DAS hydrogel containing oncolytic bacteria, represented by wild-type VNP, can release approximately 80% of the bacteria within 72 h when shaken at 100 rpm.

[0202] A 100 mL 1,4-DAS hydrogel containing engineered oncolytic bacteria, represented by VNP-TNF-α nb, can release approximately 80% of the bacteria within 72 h when shaken at 100 rpm.

[0203] Animal experiments have demonstrated that loading and delivery of sodium ion-responsive injectable in vivo self-assembling hydrogels can significantly improve weight loss, organ enlargement, tissue damage, and long-term survival caused by oncolytic bacteria, such as attenuated Salmonella.

[0204] Loading and delivery of 1,6-DAS hydrogels significantly improved weight loss, organ enlargement, tissue damage, and long-term survival induced by oncolytic bacteria, represented by wild-type VNP. Figure 25 , 27 -33, 48-54).

[0205] Loading and delivery of 1,6-DAS hydrogels can significantly improve weight loss, organ enlargement, tissue damage, and long-term survival induced by engineered oncolytic bacteria, such as VNP-TNF-α nb. Figures 58-61 ).

[0206] Loading and delivery of 1,4-DAS hydrogels can significantly improve weight loss, organ enlargement, tissue damage and long-term survival caused by oncolytic bacteria, represented by wild-type VNP.

[0207] Loading and delivery of 1,4-DAS hydrogel can significantly improve weight loss, organ enlargement, tissue damage and long-term survival caused by engineered oncolytic bacteria, such as VNP-TNF-α nb.

[0208] The loading and delivery of 1,6-DAS hydrogels achieves further toxicity reduction without affecting efficacy by sustaining the release of flagellin from oncolytic bacteria, represented by wild-type VNPs. Figure 34-37 ).

[0209] The loading and delivery of 1,6-DAS hydrogels achieves further toxicity reduction without affecting efficacy by slow-release and masking flagellin of engineered oncolytic bacteria, represented by VNP-TNF-α nb.

[0210] The loading and delivery of 1,4-DAS hydrogels achieves further toxicity reduction without affecting efficacy by slow-release and masking flagellin of oncolytic bacteria, represented by wild-type VNP.

[0211] The loading and delivery of 1,4-DAS hydrogels achieves further toxicity reduction without affecting efficacy by slow-release and masking flagellin of engineered oncolytic bacteria, represented by VNP-TNF-α nb.

[0212] Loading and delivery of 1,6-DAS hydrogels can further enhance the antitumor efficacy of oncolytic bacteria, represented by wild-type VNP, in melanoma models. Loading and delivery of 1,6-DAS hydrogels can further enhance the antitumor efficacy of oncolytic bacteria, represented by wild-type VNP, in lung adenocarcinoma models.

[0213] Loading and delivery of 1,6-DAS hydrogels can further enhance the antitumor efficacy of engineered oncolytic bacteria, represented by VNP-TNF-αnb, in a melanoma model. Loading and delivery of 1,6-DAS hydrogels can further enhance the antitumor efficacy of engineered oncolytic bacteria, represented by VNP-TNF-αnb, in a lung adenocarcinoma model.

[0214] Animal experiments have shown that loading and delivery of sodium-responsive injectable in vivo self-assembling hydrogels can enhance the proportion of activated dendritic cells (DCs) and CD8+ T cells in the tumor immune microenvironment. Figures 38-40 This further enhances the anti-tumor immune response triggered by oncolytic bacteria, represented by attenuated Salmonella.

[0215] Loading and delivery of 1,6-DAS hydrogels can further enhance the anti-tumor immune response induced by oncolytic bacteria, represented by VNPs, by increasing the proportion of activated DC cells and CD8+ T cells in the tumor immune microenvironment.

[0216] Animal experiments have shown that loading and delivery of sodium-responsive injectable in vivo self-assembling hydrogels can further enhance the anti-tumor response induced by oncolytic bacteria, represented by attenuated Salmonella, by increasing the proportion of activated dendritic cells (DCs) and CD8+ T cells in the tumor immune microenvironment. Similarly, loading and delivery of 1,6-DAS hydrogels can further enhance the anti-tumor immune response induced by engineered oncolytic bacteria, represented by VNP-TNF-α nb, by increasing the proportion of activated DCs and CD8+ T cells in the tumor immune microenvironment. Figures 62-66 ).

[0217] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. A sodium ion-responsive injectable self-assembling hydrogel system, characterized in that: The injectable self-assembly hydrogel system includes: A hydrogel precursor solution comprising isostevol-diester sulfonic acid derivative and oncolytic bacteria; wherein, after mixing with the oncolytic bacteria, the hydrogel precursor solution can self-assemble into a hydrogel containing the oncolytic bacteria under contact with sodium ions.

2. The sodium ion-responsive injectable self-assembling hydrogel system according to claim 1, characterized in that: The isosteviol-diester sulfonic acid derivative is selected from any one of the following: isosteviol-1,6-diester sulfonic acid 1,6-DA, isosteviol-1,6-diester sulfonate sodium 1,6-DAS, isosteviol-1,6-diester sulfonate potassium, isosteviol-1,6-diester sulfonate calcium, isosteviol-1,4-diester sulfonic acid 1,4-DA, isosteviol-1,4-diester sulfonate sodium 1,4-DAS, isosteviol-1,4-diester sulfonate potassium, and isosteviol-1,4-diester sulfonate calcium; the oncolytic bacteria are, but are not limited to, attenuated Salmonella typhimurium, Escherichia coli, Bifidobacterium, BCG, Shigella, Klebsiella pneumoniae, and Pseudomonas aeruginosa, including original strains, mutant strains, or engineered strains.

3. The sodium ion-responsive injectable self-assembling hydrogel system according to claim 2, characterized in that: The engineered oncolytic bacteria strains are engineered oncolytic bacteria strains expressing TNF-α nanobodies or PD-1 nanobodies, that is, engineered strains of attenuated Salmonella Typhimurium, Escherichia coli, Bifidobacterium, BCG, Shigella, Klebsiella pneumoniae, and Pseudomonas aeruginosa expressing TNF-α nanobodies or PD-1 nanobodies.

4. A method for preparing a sodium ion-responsive injectable self-assembly hydrogel system according to any one of claims 1-3, characterized in that... Includes the following steps: (1) Prepare a solution of isosteviol-diestersulfonic acid derivative as a hydrogel precursor solution; (2) Mix the oncolytic bacteria cultured to the exponential growth phase with the hydrogel precursor solution described in step (1); (3) The mixture obtained in step (2) is incubated in an environment containing sodium ions to self-assemble into the sodium ion-responsive injectable self-assembly hydrogel system of claim 1.

5. The preparation method according to claim 4, characterized in that: The sodium-containing environment is a phosphate buffer solution, a sodium chloride solution, or the in vivo environment of a living organism.

6. A method for loading oncolytic bacteria into the sodium ion-responsive injectable self-assembly hydrogel system of claim 1, characterized in that... Includes the following steps: (1) Preparation of an aqueous solution of isosteviol-diester sulfonic acid derivative; (2) After centrifuging the oncolytic bacteria, collect the bacterial precipitate and resuspend the bacterial precipitate in the aqueous solution described in step (1) to obtain a bacterial solution mixture; (3) Contact the bacterial mixture obtained in step (2) with a solution containing sodium ions, or inject the bacterial mixture into a living organism to trigger self-assembly to form a hydrogel containing oncolytic bacteria.

7. The loading method according to claim 6, characterized in that: In step (1), the isosteviol-diester sulfonic acid derivative is isosteviol-1,6-diester sulfonic acid (1,6-DA) or isosteviol-1,4-diester sulfonic acid (1,4-DA); the concentration of the aqueous solution is 5-20 mg / mL; the preparation of the aqueous solution includes the steps of heating and dissolving the isosteviol-diester sulfonic acid derivative in a solvent and then cooling it. In step (2), the oncolytic bacteria are, but are not limited to, attenuated Salmonella Typhimurium, Escherichia coli, Bifidobacterium, BCG, Shigella, Klebsiella pneumoniae, and Pseudomonas aeruginosa, either original or mutant strains, or engineered strains expressing TNF-α nanobodies or PD-1 nanobodies; the amount of bacterial precipitate is not less than 1 × 10⁻⁶ per 100 μL of aqueous solution. 9 CFU; In step (3), the sodium-containing solution is a phosphate buffer or a sodium chloride solution; the biological body is subcutaneous or peritoneal.

8. Use of the hydrogel system according to any one of claims 1-4 in the preparation of a medicament for treating tumors.

9. The application according to claim 8, characterized in that: The drug is administered via injection, and the hydrogel system self-assembles in vivo to achieve sustained-release delivery of oncolytic bacteria. The hydrogel system is used for: (i) Reduce systemic toxicity associated with oncolytic bacterial therapy, including weight loss, organ enlargement, tissue damage, and / or reduced long-term survival; and / or (ii) Enhance the antitumor efficacy of oncolytic bacteria; The reduction in systemic toxicity is achieved by the hydrogel system through the slow release of oncolytic bacteria and / or by blocking immunogenic proteins on the surface of oncolytic bacteria.

10. The application according to claim 9, characterized in that: The immunogenic protein is flagellin; the enhanced anti-tumor efficacy is achieved by enhancing the activation of dendritic cells (DCs) and / or CD8+ in the tumor immune microenvironment. + The ratio of T cells is used to achieve this; the tumor is a solid tumor; the solid tumor is melanoma, lung cancer, lymphoma, bladder cancer, liver cancer, colon cancer, or breast cancer.

11. A kit for delivering oncolytic bacteria, characterized in that: The kit contains: (a) A hydrogel precursor composition containing isosteviol-diestersulfonic acid derivatives; and (b) Oncolytic bacteria, including but not limited to attenuated Salmonella Typhimurium, Escherichia coli, Bifidobacterium, BCG, Shigella, Klebsiella pneumoniae, Pseudomonas aeruginosa original strains or mutant strains or engineered bacterial strains expressing TNF-α nanobodies or PD-1 nanobodies; Among them, components (a) and (b) can form hydrogels containing oncolytic bacteria after contact with sodium ions.

12. The kit according to claim 11, characterized in that: The isosteviol-diester sulfonic acid derivative is selected from any one of isosteviol-1,6-diester sulfonic acid (1,6-DA), sodium isosteviol-1,6-diester sulfonate (1,6-DAS), potassium isosteviol-1,6-diester sulfonate, calcium isosteviol-1,6-diester sulfonate, isosteviol-1,4-diester sulfonic acid (1,4-DA), sodium isosteviol-1,4-diester sulfonate (1,4-DAS), potassium isosteviol-1,4-diester sulfonate, or calcium isosteviol-1,4-diester sulfonate.

13. A sodium-ion-responsive injectable self-assembling hydrogel containing oncolytic bacteria, characterized in that: It is prepared by the method described in claim 4.

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