Virus vector polymer matrix hydrogel lyophilized preparation as well as preparation method and application thereof

Abstract

The invention provides a viral vector polymer matrix hydrogel freeze-drying preparation as well as a preparation method and application thereof, and relates to the technical field of biological medicines. The viral vector polymer matrix hydrogel freeze-drying preparation provided by the embodiment of the invention comprises polymer matrix hydrogel, the virus vector is loaded in a three-dimensional network of the crosslinked polymer matrix hydrogel. The viral vector polymer matrix hydrogel lyophilized preparation enables the viral vector to be stably stored at 4 DEG C for at least 12 weeks. By utilizing the local retention and sustained release characteristics of the hydrogel, the viral vector polymer matrix hydrogel can realize high drug concentration in a lesion area, and meanwhile, systemic toxicity and antibody neutralization reaction of an antiviral vector are effectively avoided. In a mouse melanoma and cancer postoperative resection model, the combined treatment plays a powerful anti-tumor role, and tumor recurrence is remarkably inhibited.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of biomedical technology, and in particular to viral vector polymer matrix hydrogel freeze-dried formulations, their preparation methods and applications. Background Technology

[0002] Postoperative tumor recurrence remains a major challenge in clinical cancer treatment because surgery alone cannot eliminate residual shadows and drug-resistant cell populations. Although routine postoperative adjuvant radiotherapy and chemotherapy are widely used in clinical practice, their efficacy is often limited by two key factors: significant systemic toxicity and the frequent emergence of drug resistance when controlling microlesions.

[0003] Localized hydrogel delivery systems possess excellent biocompatibility, a biodegradable three-dimensional network structure, and controllable drug release characteristics, making them suitable as drug depots at surgical sites. Hydrogel delivery systems increase drug concentrations in the tumor bed while significantly reducing systemic toxicity. However, despite the advantages of hydrogel-based delivery in reducing toxicity, there remains an urgent need to develop novel synergistic therapies to overcome drug resistance issues associated with conventional treatments. Summary of the Invention

[0004] The embodiments of the present invention provide a viral vector polymer matrix hydrogel freeze-dried formulation, its preparation method and application, so as to at least partially solve the problems existing in the prior art.

[0005] The first aspect of this invention provides a lyophilized formulation of a viral vector polymer matrix hydrogel, the lyophilized formulation comprising: a polymer matrix hydrogel, wherein the viral vector is loaded in a three-dimensional network of a cross-linked polymer matrix hydrogel.

[0006] Optionally, the viral vector includes adeno-associated virus, lentivirus, adenovirus, and herpes simplex virus.

[0007] Optionally, the lyophilized formulation further includes: protein drugs, small molecule drugs, and / or peptide drugs.

[0008] A second aspect of this invention provides a method for preparing the lyophilized formulation of the viral vector polymer matrix hydrogel described in the first aspect, the method comprising: Methacrylated hyaluronic acid was prepared based on methacrylic anhydride and hyaluronic acid; Preparation of polymer matrix hydrogels based on methacrylated hyaluronic acid; After cross-linking the polymer matrix hydrogel, it was freeze-dried and then dissolved in a viral vector solution to obtain a hydrogel containing a viral vector. The hydrogel containing the viral vector was freeze-dried to obtain a freeze-dried formulation of a viral vector polymer matrix hydrogel.

[0009] Optionally, methacrylated hyaluronic acid is prepared based on methacrylic anhydride and hyaluronic acid, including: Methacrylic anhydride was added to a hyaluronic acid solution, and the pH value of the reaction was maintained between 8 and 9 by adding NaOH while continuously stirring at 4 degrees Celsius. Methacrylamide-modified hyaluronic acid was precipitated using acetone, and the precipitate was washed with ethanol and then dissolved in deionized water. After dialysis to remove impurities, the hyaluronic acid was freeze-dried to obtain purified methacrylamide.

[0010] Optionally, a polymer matrix hydrogel is prepared based on methacrylated hyaluronic acid, comprising: Methacrylamide-modified hyaluronic acid, a crosslinking agent, and the photoinitiator Lrgacure2959 were added to the albumin solution; Free polymerization is carried out through ultraviolet radiation to form a polymer matrix hydrogel loaded with albumin nanoparticles.

[0011] Optionally, the cross-linking methods for cross-linking the polymer matrix hydrogel include: physical cross-linking, chemical cross-linking, enzyme-catalyzed cross-linking, or radiation cross-linking.

[0012] The third aspect of this invention provides the application of the viral vector polymer matrix hydrogel lyophilized formulation described in the first aspect, for the preparation of tumor therapeutic agents, wherein the viral vector in the therapeutic agent is AAV, and the viral vector delivers the p53 gene.

[0013] Optionally, the therapeutic agent further includes: a ferroptosis inducer.

[0014] Optionally, the tumor includes melanoma and bladder cancer.

[0015] The polymer matrix hydrogel provided in this embodiment of the invention has a three-dimensional porous network structure, which can be used as a local drug library to stably fix viral vectors to postoperative tumor lesions.

[0016] In vivo experiments have demonstrated that hydrogel delivery significantly increases the amount of AAV retained at the injection site compared to administration of free AAV. At the same time, through its sustained release properties, it avoids rapid drug diffusion and ensures that an effective therapeutic concentration is maintained in the tumor microenvironment for a long time.

[0017] The local delivery method of hydrogels avoids the liver isolation problem of systemic drug administration, avoids the problem of free AAV being easily taken up by the liver, reduces drug distribution in off-target tissues (liver, kidney, lung), and reduces ineffective dose consumption.

[0018] The hydrogel can respond to hyaluronidase in the tumor microenvironment to achieve on-demand release. In the presence of 1 mg / mL hyaluronidase, 80% of AAV can be rapidly released within 24 hours, while the release is slow in an enzyme-free environment (normal tissue), further improving the targeting and controllability of delivery.

[0019] Local hydrogel delivery avoids systemic exposure of the viral vector. No abnormalities were found in hematological analysis (white blood cells, red blood cells, liver and kidney function indicators, etc.) and H&E staining of major organs (heart, liver, spleen, lungs, and kidneys). The mouse body weight did not change significantly, demonstrating its excellent biocompatibility.

[0020] Hydrogels can encapsulate viral vectors, reducing their contact with the systemic immune system, significantly decreasing the production of antiviral vector neutralizing antibodies, avoiding treatment failure caused by antibody neutralization, and reducing the risk of immunogenicity.

[0021] In this invention, the viral vector-loaded hydrogel can be freeze-dried into a powder and stored stably at 4°C for at least 12 weeks, maintaining high transduction activity. The stability of the freeze-dried formulation stems from the fixation of viral vector particles by the porous network of the hydrogel, reducing viral aggregation and physical degradation. Simultaneously, the freeze-drying process reduces hydrolysis, addressing the industry pain point of difficult storage and transportation of viral vectors. Furthermore, the freeze-dried hydrogel powder can be rapidly rehydrated, restoring its three-dimensional network structure, making it suitable for local injection into the postoperative tumor bed without requiring complex equipment, thus meeting the operational needs of clinical adjuvant therapy.

[0022] In summary, the lyophilized formulation of the viral vector polymer matrix hydrogel provided in this invention enables the viral vector to be stably stored at 4°C for at least 12 weeks. By utilizing the local retention and sustained release properties of the hydrogel, the viral vector polymer matrix hydrogel can achieve high drug concentrations in the lesion area, while effectively avoiding systemic toxicity and neutralizing antibody reactions against the antiviral vector. In mouse models of melanoma and postoperative resection of cancer, the combined treatment exhibited a strong antitumor effect and significantly inhibited tumor recurrence. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Confocal microscopy images of B16F10 cells infected with lyophilized powder loaded with AAV-GFP after storage at 4 degrees Celsius for different times are shown. Figure 2The flow cytometry results of B16F10 cells infected with lyophilized powder loaded with AAV-GFP after storage at 4 degrees Celsius for different times are shown. Figure 3 A schematic diagram is shown illustrating the screening of AAV serotypes to efficiently deliver the p53 gene to tumor cells; Figure 4 A schematic diagram illustrating the characterization of a hydrogel loaded with adeno-associated virus (AAV) and albumin nanoparticles and the evaluation of its in vivo and in vitro drug release curves is shown. Figure 5 A schematic diagram illustrating the antitumor effects of hydrogel co-delivery of AAV-p53 and ferroptosis inducer in mouse melanoma and bladder cancer models is shown. Figure 6 A schematic diagram illustrating the mechanism of action of combined AAV-p53 and iron sag inducer in the in vivo antitumor efficacy is shown. Detailed Implementation

[0025] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0026] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents and other instruments whose manufacturers are not specified are all commercially available products.

[0027] Recombinant adeno-associated virus (rAAV) is widely used in clinical gene therapy for diseases such as Leber congenital amaurosis (Luxturna®) and hemophilia due to its low immunogenicity, high gene delivery efficiency, and inability to replicate. These advantages make rAAV an ideal vector for delivering the p53 gene to tumor cells. However, systemic administration of AAV is often limited by the presence of neutralizing antibodies, which reduces its infectivity at the target site. Furthermore, after intravenous injection, a large proportion of viral particles are isolated in the liver, requiring high doses to achieve therapeutic effects. This not only increases systemic toxicity but also increases production costs. In contrast, local delivery of AAV using hydrogels offers significant advantages. By releasing the viral vector directly into the target tissue, this method improves transduction efficiency, minimizes systemic exposure, reduces immune recognition, and mitigates the effects of pre-existing neutralizing antibodies.

[0028] To improve expression efficiency, this invention incorporates a spider silk-derived domain (NT*) into the N-terminus of p53, thereby enhancing the stability and expression level of the p53 protein. Furthermore, to increase the water solubility and tumor uptake of the ferroptosis inducer, this invention encapsulates it in human serum albumin nanoparticles (HSA-NP). Finally, to optimize local treatment, this invention co-loads the optimized AAV-p53 and the nanoparticle-encapsulated ferroptosis activator into a hyaluronic acid-based hydrogel, constructing a local treatment system for precise application to postoperative tumor lesions.

[0029] The viral vector polymer matrix hydrogel provided in this invention can be freeze-dried, allowing the viral vector to be stably stored at 4°C for at least 12 weeks. By utilizing the local retention and sustained release properties of the hydrogel, the viral vector polymer matrix hydrogel achieves high drug concentrations in the lesion area, while effectively avoiding systemic toxicity and neutralizing antibody reactions against the antiviral vector. In mouse models of melanoma and postoperative resection of cancer, the combined treatment exhibited a strong antitumor effect and significantly inhibited tumor recurrence.

[0030] Specifically, this invention provides a viral vector polymer matrix hydrogel lyophilized formulation, the lyophilized formulation comprising: a polymer matrix hydrogel, wherein the viral vector is loaded into a three-dimensional network of the cross-linked polymer matrix hydrogel.

[0031] Specifically, the lyophilized formulation includes a polymer matrix hydrogel, wherein the viral vector is loaded into a three-dimensional network of the cross-linked polymer matrix hydrogel.

[0032] Specifically, the lyophilized formulation also includes: protein drugs, small molecule drugs and / or peptide drugs.

[0033] Specifically, the polymer matrix includes: hyaluronic acid, polyvinylpyrrolidone, collagen, silk fibroin, gelatin, hydroxypropyl methylcellulose, chondroitin sulfate, dextrin, polyvinyl alcohol, carboxymethyl cellulose, carboxymethyl chitosan, dextran sulfate, or glycogen.

[0034] In this embodiment of the invention, a method for preparing a viral vector polymer matrix hydrogel lyophilized formulation is also provided, the method comprising: S1, Methacrylated hyaluronic acid is prepared based on methacrylic anhydride and hyaluronic acid.

[0035] S2, a polymer matrix hydrogel was prepared based on methacrylated hyaluronic acid.

[0036] S3, after cross-linking the polymer matrix hydrogel, freeze-drying it, and then adding the viral vector solution to dissolve it, a hydrogel containing the viral vector is obtained.

[0037] In this embodiment of the invention, in order to avoid the influence of ultraviolet radiation on the activity of viral vectors (common gene therapy vectors such as adeno-associated virus, lentivirus, adenovirus, and herpes simplex virus), the polymer matrix hydrogel is lyophilized after ultraviolet cross-linking and then dissolved in a viral vector solution to form a hydrogel containing the viral vector.

[0038] S4. The hydrogel containing the viral vector is freeze-dried to obtain a freeze-dried formulation of the viral vector polymer matrix hydrogel.

[0039] In this embodiment of the invention, taking hyaluronic acid as the polymer matrix as an example, a method for preparing a viral vector hyaluronic acid hydrogel freeze-dried formulation is provided.

[0040] Specifically, step S1 includes: Methacrylic anhydride was added to a hyaluronic acid solution, and the pH value of the reaction was maintained between 8 and 9 by adding NaOH while continuously stirring at 4 degrees Celsius.

[0041] In this embodiment of the invention, the stirring can be carried out continuously for 24 hours.

[0042] Methacrylamide-modified hyaluronic acid was precipitated using acetone, and the precipitate was washed with ethanol and then dissolved in deionized water.

[0043] After dialysis to remove impurities, the hyaluronic acid was freeze-dried to obtain purified methacrylamide.

[0044] Specifically, step S2 includes: Methacrylamide-modified hyaluronic acid, a crosslinking agent, and the photoinitiator Lrgacure2959 were added to the albumin solution.

[0045] In this embodiment of the invention, the crosslinking agent can be N,N-methylenebisacrylamide (MBA).

[0046] Free polymerization is carried out through ultraviolet radiation to form a polymer matrix hydrogel loaded with albumin nanoparticles.

[0047] In this embodiment of the invention, the crosslinking methods for crosslinking the polymer matrix hydrogel in step S3 include: physical crosslinking, chemical crosslinking, enzyme-catalyzed crosslinking, or radiation crosslinking.

[0048] This invention also provides an application of a viral vector polymer matrix hydrogel lyophilized formulation for the preparation of tumor therapeutic agents, wherein the viral vector in the therapeutic agent is AAV, and the viral vector delivers the p53 gene.

[0049] Specifically, the therapeutic agent also includes: ferroptosis inducers.

[0050] Specifically, the tumors include melanoma and bladder cancer.

[0051] To verify and illustrate the viral vector polymer matrix hydrogel lyophilized formulation provided in the embodiments of the present invention, specific examples are provided below: Example 1: Preparation and stability study of lyophilized formulation of viral vector hyaluronic acid hydrogel This invention investigates the stability of adeno-associated virus (AAV)-loaded hydrogel lyophilized powder at 4 degrees Celsius. The AAV-loaded hydrogel lyophilized powder was prepared using steps S1-S4 described above.

[0052] Infection experiments with B16F10 cells showed (e.g.) Figure 1 and Figure 2 As shown, Figure 1 This image shows confocal microscopy images of B16F10 cells infected with lyophilized powder loaded with AAV-GFP after storage at 4°C for different times. Figure 2 The results of flow cytometry analysis of B16F10 cells infected with AAV-GFP-loaded lyophilized powder after storage at 4°C for different times are shown. The AAV-loaded hydrogel lyophilized powder remained stable at 4°C for at least 12 weeks. This is likely due to the three-dimensional mesh structure of the hydrogel, which effectively immobilizes AAV virus particles within the pores, preventing aggregation and collision-induced inactivation. Simultaneously, lyophilization of the hydrogel removes most of the water, effectively reducing degradation.

[0053] Example 2: AAV6-NT*p53 effectively restores the expression of functional p53 in tumor cells. Given that different AAV serotypes exhibit varying transduction efficiencies in tumor cells, this invention first compared several common serotypes (AAV1, 2, 5, 6, 8, 9) carrying the GFP reporter gene in vitro (e.g., Figure 3 As shown in Part A of the diagram. Figure 3A schematic diagram is shown for screening AAV serotypes to effectively deliver the p53 gene to tumor cells. (A) shows a schematic diagram of an in vitro AAV serotype screening strategy. (B) shows a confocal microscopy image of green fluorescent protein expression after transducing B16F10 cells with different AAVGFP serotypes, scale bar, 20 μm. (C) shows a flow cytometry analysis of green fluorescent protein expression after transducing B16F10 cells with different AAV-GFP serotypes (n=4). (D) shows a confocal microscopy image of green fluorescent protein expression after transducing MB49 cells with different AAV-GFP serotypes, scale bar, 20 μm. (E) shows a flow cytometry analysis of green fluorescent protein expression after transducing MB49 cells with different AAV-GFP serotypes (n=4). (F) shows a schematic diagram of AAV6-p53 and AAV6-NT*p53 expression cassettes (top) and a transmission electron microscopy image of purified AAV6 particles (bottom), scale bar, 20 nm. (G) indicates Western blot analysis of p53 expression after transduction of B16F10 cells with AAV6-p53 or AAV6-NT*p53.

[0054] Using flow cytometry and confocal microscopy, this embodiment of the invention determined that AAV6 is a transducer of B16F10 (e.g., ...). Figure 3 (as described in (B), (C)) and MB49 (as...) Figure 3 The cell lines shown in (D) and (E) were the most effective serotypes, with a transduction rate exceeding 95%, significantly higher than other tested serotypes. Based on this high efficiency, AAV6 was selected as the vector for delivering p53 in subsequent experiments. To address the issues of low expression levels and poor conformational stability of wild-type p53 protein, this embodiment of the invention fuses a highly soluble spider silk-derived domain (NT*) to its N-terminus, a strategy previously shown to enhance protein stability and bioactivity. This embodiment of the invention constructed two plasmid variants, pCAG-p53 and pCAG-NT*p53, driving high-level expression in mammalian cells using the CAG promoter. Next, this embodiment of the invention used AAV6 as a capsid to package and purify AAV6-p53 and AAV6-NT*p53, respectively. Transmission electron microscopy confirmed that the purified AAV particles exhibited the expected ~20 nm icosahedral morphology (e.g., ...). Figure 3 (As shown in Figure (F)). Next, in this embodiment of the invention, Western blot analysis was used to evaluate the expression of p53 protein in B16F10 cells after transduction with AAV6-p53 or AAV6-NT*p53. The results showed that both vectors effectively restored p53 expression, and the protein level produced by AAV6-NT*p53 was significantly higher than that produced by AAV6-p53 (as shown in Figure (F)). Figure 3(as shown in G). These findings confirm that AAV6-NT*p53 effectively transduced mouse tumor cells and powerfully restored the expression of functional p53.

[0055] Example 3: Hyaluronic acid hydrogel loaded with AAV and albumin nanoparticles can enhance tumor accumulation and sustained release of therapeutic drugs. The therapeutic effect of AAV in vivo is often limited by off-target effects and pre-existing antibody neutralization, typically requiring high doses to achieve a therapeutic effect. To minimize off-target effects and reduce the required dose, embodiments of this invention encapsulate AAV in a hyaluronic acid-based hydrogel for localized treatment. (e.g.) Figure 4 As shown in Part (A), Figure 4The following diagram illustrates the characterization of hydrogels loaded with adeno-associated virus (AAV) and albumin nanoparticles, and the evaluation of their in vitro and in vivo drug release profiles. (A) shows a schematic diagram of the preparation process of hydrogels co-loaded with AAV and HSA nanoparticles. (B) shows confocal microscopy images of hydrogels loaded with Cy5-labeled AAV in lyophilized and hydrated states, scale bar 50 μm. (C) shows the hydrodynamic size distribution of empty HSA nanoparticles as determined by dynamic light scattering (DLS). (D) shows transmission electron microscopy (TEM) images of Erastin nanoparticles and RSL3 nanoparticles, scale bar 200 nm. (E) shows a scanning electron microscopy (SEM) image of lyophilized hydrogel powder loaded with HSA nanoparticles. Scale bar, 3 micrometers. (F) shows the in vitro release curve of AAV from hyaluronic acid hydrogel in PBS with or without hyaluronidase, quantified by qPCR. (G) shows the in vivo release kinetics of DiD-labeled HSA nanoparticles in the hydrogel after subcutaneous injection in mice, monitored by fluorescence imaging. (H) shows the in vivo distribution of free AAV6-Luc and hydrogel-encapsulated AAV6-Luc after subcutaneous injection (n=4). (I) shows the viral genome quantification at the injection site after subcutaneous injection of free or hydrogel-encapsulated AAV6-Luc (n=4). (J) shows a schematic diagram of the experimental procedure for detecting AAV6-specific antibody levels in mouse blood. (K) shows the AAV6-specific antibody levels in mouse serum after subcutaneous injection of free or hydrogel-encapsulated AAV6-Luc (n=3). The embodiments of this invention first characterized the AAV-loaded hydrogel system. To visualize the distribution of AAV in the hydrogel matrix, the virus was labeled with Cy5 (Cy5-AAV). Subsequently, the Cy5-AAV-hydrogel composite material was lyophilized and cryosectioned. Confocal imaging of the sections confirmed that AAV was successfully encapsulated in the hydrogel, with the virus distributed throughout the porous network structure. Furthermore, the lyophilized powder could be easily rehydrated to regenerate the hydrogel, during which time the AAV particles remained uniformly dispersed in the modified matrix (e.g., Figure 4 (As shown in Part B).

[0056] Ferroplasm inducers such as Erastin and RSL3 exhibit poor solubility and lack of targeting, resulting in a narrow therapeutic window and frequent overlap between effective and toxic doses. As relevant studies have shown, conjugating AAV-p53 with ferroplasm inducers can sensitize tumor cells, potentially reducing the required dose. To further improve water solubility and targeting efficiency, embodiments of this invention encapsulate ferroplasm inducers in human serum albumin nanoparticles (hereinafter referred to as NP), subsequently integrating them into HA hydrogels to promote local accumulation and reduce systemic distribution (e.g., Figure 4(As shown in (A)). Characterization of the NPs revealed that the hydrodynamic diameter of the empty nanoparticles was approximately 161.4 nm (PDI = 0.157). Drug loading increased the size of Erastin NPs to 164.2 nm (PDI = 0.165) and the size of RSL3 NPs to 172.6 nm (PDI = 0.229) (as shown in (A)). Figure 4 As shown in (C). Transmission electron microscopy (TEM) confirmed the spherical morphology and uniform distribution (as shown in the image). Figure 4 (As shown in D). Scanning electron microscopy (SEM) of the lyophilized hydrogel containing NPs shows that surface protrusions indicate the embedding of nanoparticles, confirming their uniform dispersion in the hydrogel matrix (as shown in D). Figure 4 (As shown in (E)).

[0057] This invention evaluated the drug release behavior of hydrogels in vitro and in vivo. For AAV release, hydrogel samples were incubated in PBS with or without hyaluronidase. qPCR analysis showed that AAV release peaked at 144 hours in plain PBS, while in the presence of 1 mg / mL hyaluronidase, over 80% of AAV was released within 24 hours, indicating enzyme-triggered release (e.g., ...). Figure 4 (See Figure (F)). To evaluate in vivo release kinetics, DiD-labeled NPs loaded in the hydrogel were subcutaneously injected into mice. Compared to free DiD injection, the hydrogel formulation significantly prolonged dye retention and release, supporting its potential for sustained single-dose administration (e.g., ...). Figure 4 (as shown in G). Next, embodiments of the invention evaluated how hydrogel encapsulation affects the in vivo distribution of AAV. Quantitative analysis of viral genomes in different organs revealed that, compared with free AAV administration, hydrogel-based delivery increased AAV retention at the injection site by approximately 46.1-fold, while significantly reducing off-target transduction in the liver, kidneys, and lungs (e.g., Figure 4 (As shown in (H)(I)). This targeted distribution improves therapeutic efficacy by enhancing local transduction while minimizing potential toxicity to non-target tissues. Furthermore, embodiments of the invention evaluated whether local hydrogel delivery could mitigate the production of neutralizing antibodies, a common limitation of systemic AAV administration. Measurements of serum anti-AAV6 capsid antibodies indicated that while subcutaneous injection of free AAV6-Luc induced a significant antibody response compared to the blank control, hydrogel-encapsulated AAV6-Luck did not cause a significantly elevated antibody level (e.g., Figure 4 (As shown in (J)(K)). These results demonstrate that hydrogel-based AAV delivery avoids the immunogenicity associated with conventional drug administration, thus preserving the therapeutic potential of AAV-p53.

[0058] Example 4: Co-delivery of AAV-p53 and ferroptosis inducer hydrogels in vivo exhibits potent antitumor efficacy. To evaluate the potential of AAV-p53 combined with ferroptosis inducers for in vivo therapy, this invention established a mouse melanoma model by subcutaneous injection of luciferase-expressing B16F10 cells (B16F10-Luc) from C57BL / 6 mice. Tumor growth was monitored using bioluminescence imaging to measure mean irradiance. When the tumor reached approximately 100 mm³, mice were randomly assigned to groups, and approximately 90% of the tumor was surgically removed. Subsequently, mice received peritumoral injections of PBS, free AAV-p53+Erastin NP, gel (AAV-p53), gel (Erastin NPs), or a combination of gels (AAV-p53+Erastin NPs). Bioluminescence imaging (e.g., D-luciferin) was performed on days 0, 4, 8, and 13 after D-luciferin injection. Figure 5 As shown in (A), Figure 5 The diagram illustrates the antitumor effects of hydrogel co-delivery of AAV-p53 and ferroptosis inducers in mouse melanoma and bladder cancer models. (A) shows the timeline of B16F10-Luc tumor inoculation and treatment experiments. (B) shows the monitoring of B16F10-Luc tumor growth kinetics over time by bioluminescence intensity changes (n=6). (C) shows the quantification of B16F10-Luc tumor bioluminescence intensity on day 13 after treatment (n=6). (D) shows the survival curves of B16F10-Luc tumor-bearing mice under different treatment regimens. (E) shows representative in vivo bioluminescence images of B16F10-Luc tumor-bearing mice in different treatment groups. (F) shows a schematic diagram of the tumor re-attack experiment (top) and corresponding bioluminescence images of mice with completely eliminated tumors after treatment with Gel (AAV-p53+Erastin NPs) and the untreated control group (bottom). (G) shows MB49-Luc... A schematic diagram of the timeline of the bladder tumor inoculation and treatment intervention experiment. (H) represents the monitoring of MB49-Luc tumor growth kinetics in different treatment groups (n=6) by bioluminescence intensity; (I) represents the survival curves of MB49-Luc tumor-bearing mice under different treatment regimens; (J) represents representative in vivo bioluminescence images of B16F10-Luc tumor-bearing mice under different treatment conditions. Figure 5 As shown in Figure (E), compared with the PBS and free combination groups, both the gel (AAVp53) and gel (Erastin NPs) monotherapy groups effectively inhibited tumor growth, benefiting from the local retention of therapeutic drugs mediated by the hydrogel. The gel (AAV-p53 + Erastin NP) group exhibited the strongest antitumor effect, indicating that AAV-p53 and Erastin have a synergistic effect in vivo (e.g., Figure 5(As shown in (B) and (C)). Notably, 70% of the mice in the combined gel group achieved complete tumor eradication, resulting in a significant survival benefit (e.g., Figure 5 (D) As shown in the middle. To investigate whether this treatment could induce immune memory against melanoma, tumor-free survivors in the gel (AAV-p53+ErastinNPs) group received a second subcutaneous injection of 4×10 5 B16F10-Luc cells were administered to juvenile mice on day 60. The mice received the same injections as the control group. Tumor growth was monitored by bioluminescence starting on day 67 (e.g., ...). Figure 5 (As shown in F). Although the tumors grew rapidly in juvenile mice, leading to complete death by day 95, the treated mice exhibited complete rejection of the re-attacked tumor cells. Interestingly, two treated mice showed detectable subcutaneous tumors on day 67, but these tumors were subsequently cleared by day 95, indicating the establishment of adaptive immune memory (e.g., ...). Figure 5 (as shown in (F)).

[0059] To comprehensively evaluate the synergistic effect between AAV-p53 and different classes of ferroptosis inducers, this embodiment of the invention further evaluated its combination with the GPX4 inhibitor RSL3 in the same B16F10-Luc tumor model. After surgical resection of approximately 90% of the tumor, mice were treated with PBS, free AAV-p53+RSL3-NP, gel (AAV-p53), gel (RSL3-NPs), or gel (AAV-p53+RSL3NPs). Bioluminescence imaging showed that the gel (AAV-p53+RSL3-NP) group again produced the most effective tumor suppression and significantly prolonged survival, confirming the synergistic effect of AAV-p53 with two major classes of ferroptosis inducers.

[0060] Finally, this invention validates the broad applicability of this strategy in MB49-Luc bladder cancer cells, a model known for its poor response to deferroinducers due to altered lipid metabolism and reduced ROS production. This invention establishes a mouse bladder cancer model by subcutaneously injecting luciferase-expressing MB49 cells (MB49-Luc) into C57BL / 6 mice and monitoring tumor growth using bioluminescence imaging. When the tumor reached approximately 100 mm³, approximately 90% of the MB49 tumor mass was surgically removed. Mice were then subcutaneously injected at the resection site with PBS, AAV-p53+Erastin NP, gel (AAV-p53), gel (Erastin NPs), or gel (AAV-p53+Erastin NPs), followed by analyses as described above (e.g.). Figure 5(As shown in G). The results showed that Erastin (gel (Erastin NP)) alone had limited effect in inhibiting tumor growth. Notably, the combination therapy (gel (AAV-p53 + Erastin NPs)) achieved the most profound inhibition of tumors and significantly prolonged animal survival (e.g., Figure 5 (As shown in (H)(I)(J)). These results indicate that AAV-p53 can even sensitize anti-ferrous bladder cancer to ferrous inducers, thereby producing a potent synergistic antitumor activity.

[0061] Example 5: AAV-p53 works synergistically with ferroptosis inducers by activating anti-tumor immunity and enhancing susceptibility to ferroptosis. The powerful synergistic effect of AAV-p53 and ferroptosis inducers in inhibiting tumor recurrence prompted this invention to investigate the underlying cellular mechanisms. This invention first analyzed the infiltration of immune cells in tumor tissue after treatment (e.g., Figure 6 As shown in (A), Figure 6 A schematic diagram illustrating the mechanism of action of the combination therapy of AAV-p53 and iron sag inducer in vivo is shown, wherein (A) represents a schematic diagram illustrating the proposed mechanism of action of the combination therapy. (BE) represents the flow cytometry analysis of immune cell infiltration in tumor tissue after treatment: the proportion of macrophages (B), CD4+ T cells (C), CD8+ T cells (D), NK cells (E) and mature dendritic cells (DC) (F) (n=5), (G) represents the percentage of mature DCs in tumor draining lymph nodes (TDLN) (n=5), (H) represents the cytokines (IFN-γ, TNF-α, IL-1β) secreted by spleen cells after in vitro restimulation with tumor antigen (n=5), (I) represents a representative image of tumor tissue sections stained with TUNEL (apoptosis) and Ki-67 (proliferation), (J) represents the concentration of cysteine ​​in tumor tissue, and the mRNA expression levels of iron sag-related genes (KO) in tumor tissue: CHAC1 (K), HMOX1 (L), SLC7A11 (M), TFRC (N) and PTGS2 (O) (N=5)). Flow cytometry showed that, compared with the blank control group, the proportions of macrophages, CD4+ T cells, CD8+ T cells, NK cells, and mature dendritic cells (DCs) in the gel (AAVp53+Erastin NP) group were significantly increased (e.g., Figure 6(As shown in (B)(C)(D)(E)(F)). Notably, Erastin monotherapy failed to induce this change, suggesting that combination therapy with AAV-p53 is crucial for reversing the immunosuppressive tumor microenvironment (TME). Upon acquiring antigens in the tumor, dendritic cells (DCs) migrate to the tumor draining lymph nodes (TDLN) to activate T cells. Consistent with intratumoral data, combination therapy significantly increased the proportion of mature DCs in the TDLN compared to the PBS group, an effect not observed with Erastin alone (e.g., ...). Figure 6 (As shown in middle (F)). Furthermore, analysis of tumor-specific immune responses in the spleen showed that, in the combined group, spleen cells secreted the highest levels of IFN-γ and TNF-α and the lowest levels of IL-1β after restimulation with tumor antigens (as shown in middle (F)). Figure 6 (H) These results indicate that the combination therapy effectively initiated a systemic antitumor immune response. In this embodiment of the invention, Ki-67 immunofluorescence and TUNEL staining were also used to assess in situ proliferation and death of tumor cells. The gel (AAV-p53+Erastin NP) group showed the strongest TUNEL signal (indicating apoptosis) and the weakest Ki-67 signal (indicating inhibition of proliferation), confirming the superior efficacy of the combination therapy (e.g., Figure 6 As shown in (I).

[0062] Because cysteine ​​is crucial for cellular defense against ferroptosis, this invention measured its concentration in tumors. Cysteine ​​levels were significantly reduced in tumors treated alone or in combination with the gel (AAV-p53) (e.g., Figure 6 As shown in Figure (J), AAV-p53-mediated p53 reactivation impairs the tumor's antioxidant capacity. Furthermore, qPCR analysis of ferroptosis-related genes revealed that combination therapy resulted in the highest mRNA expression of pre-ferroptosis genes (CHAC1, HMOX1, TFRC, PTGS2). Compared with monotherapy, the expression of the antiferritin gene SLC7A11 was the lowest (e.g. Figure 6 (As shown in (K)(L)(M)(N)(O)). This gene expression profile confirms that AAV-p53 sensitizes tumors to ferroptosis, and that this combination induces the strongest ferroptotic response.

[0063] Example 6: The hydrogel-based delivery system for AAV-p53 and ferroptosis activator showed good safety in vivo.

[0064] To assess the in vivo safety of AAV-p53 alone and in combination with a ferroptosis activator, mouse body weight was monitored throughout the study, and blood and major organs (e.g., heart, liver, spleen, lung, and kidney) were collected at the endpoint. No significant changes in body weight were observed in any treatment group. Hematological analyses were performed based on complete blood counts and blood biochemistry tests, assessing a set of parameters including white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), lymphocytes (Lymph), platelets (PLT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea (urea), uric acid (UA), and creatine kinase MB isoenzyme (CKMB). No significant changes in hematological parameters were detected in any group, indicating that the side effects of AAV-p53 and its combination with a ferroptosis activator are negligible. Furthermore, major organs were examined by H&E staining. Histological analysis showed no significant abnormalities or identifiable differences between treatment groups, further supporting the in vivo safety of the combination therapy.

[0065] As a key tumor suppressor, p53 has been shown to establish defense mechanisms by regulating multiple classical tumor suppressor pathways, including cell cycle arrest and apoptosis. Reactivation of p53 further enhances tumor cell susceptibility to ferroptosis through transcriptional repression of the cysteine ​​transporter SLC7A11 and upregulation of the lipoxygenase ALOX12, thereby reducing the required dose of ferroptosis inducers and broadening the therapeutic window. Furthermore, p53 reactivation counteracts the immunosuppressive effects of ferroptosis by activating the immune response. Therefore, the combined use of p53 restoration and ferroptosis inducers is a promising synergistic anti-tumor strategy, which can not only effectively activate ferroptosis to eliminate residual drug-resistant cells but also reshape the immune microenvironment and establish long-term immune surveillance, thereby significantly inhibiting postoperative tumor recurrence.

[0066] Ferroprelation can combat postoperative tumor recurrence by bypassing apoptosis resistance to conventional therapies. However, its clinical potential is limited by a narrow therapeutic window and lack of immunogenicity. Embodiments of this invention have found that AAV-mediated p53 reactivation directly offsets these limitations. Therefore, embodiments of this invention propose a therapeutic agent that combines AAV-p53 with ferroptosis induction.

[0067] This invention first demonstrates that the AAV6 serotype exhibits high transduction efficiency in mouse melanoma B16F10 and bladder cancer MB49 cell lines. Compared to conventional adenovirus-mediated p53 delivery, the AAV vector exhibits lower immunogenicity and improved safety. By fusing a soluble spider silk domain (NT*) to the N-terminus of p53, this invention successfully constructed AAV-p53, which makes p53 protein expression more efficient and stable in tumor cells, thus overcoming a key limitation of conventional p53 gene expression—poor protein stability. In vitro transcriptomic analysis further reveals that AAV-p53 extensively reprograms the gene expression network of tumor cells, not only activating canonical p53 pathways (e.g., inducing cell cycle arrest and apoptosis) but also significantly regulating genes involved in immune regulation and metabolic reprogramming. Notably, this invention revealed that AAV-p53 downregulated the key cysteine ​​transporter SLC7A11 and upregulated the lipoxygenase ALOX12, confirming the role of p53 in promoting ferroptosis through transcriptional repression of SLC7A11. Mechanistically, this demonstrates its potential to sensitize tumor cells to ferroptosis. AAV-p53 alone effectively inhibited tumor cell proliferation and induced cell cycle arrest and apoptosis. More importantly, it exhibited a strong synergistic effect with two classes of ferroptosis inducers acting through different mechanisms, namely Xc... - The systemic inhibitor Erastin and the GPX4 inhibitor RSL3 / ML162 significantly increased intracellular lipid peroxidation and enhanced cytotoxicity. This finding highlights the potent antitumor potential of the therapeutic formulations provided in the embodiments of the present invention.

[0068] This invention also found that AAV-p53 effectively counteracts the negative immunomodulatory effects of ferroptosis. Mechanistic studies showed that tumor cells treated with AAV-p53 more effectively promoted dendritic cell maturation. Furthermore, when combined with Erastin to induce ferroptosis, this treatment strongly activated T-cell responses. This suggests that p53 may reverse the immunosuppressive tendency associated with ferroptosis by upregulating antigen presentation and related chemokines. This highlights the significant advantage of p53 gene therapy compared to strategies that merely enhance ferroptosis intensity without altering its immunogenicity, such as the combination of cucurbitacin B and Erastin34.

[0069] To achieve localized, efficient drug delivery while minimizing systemic toxicity, this invention presents a hyaluronic acid-based hydrogel delivery system. Unlike systemic administration of small molecules, local gene therapy avoids off-target toxicity and the problems of frequent dosing. The advantages of the hydrogel delivery system of this invention are clearly demonstrated: it can sustainably release albumin nanoparticle-encapsulated drugs at the tumor site; local administration effectively circumvents the challenges associated with systemic AAV delivery, such as liver isolation and antibody neutralization; and the lyophilized hydrogel formulation significantly improves the storage stability of AAV. In vivo efficacy evaluation in mouse models of melanoma and cancerous bladder yielded encouraging results.

[0070] Although preferred embodiments of the present invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0071] The present invention provides a detailed description of a viral vector polymer matrix hydrogel freeze-dried formulation, its preparation method, and its application. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A viral vector polymeric matrix hydrogel lyophilized formulation, characterized in that, The freeze-dried preparation comprises: a high-molecular-matrix hydrogel, and the viral vector is loaded in the three-dimensional network of the cross-linked high-molecular-matrix hydrogel.

2. The viral vector polymer matrix hydrogel lyophilizate according to claim 1, characterized in that The viral vector comprises: an adeno-associated virus, a lentivirus, an adenovirus, and a herpes simplex virus.

3. The viral vector polymer matrix hydrogel lyophilizate according to claim 1, characterized in that, The freeze-dried preparation further comprises: a protein drug, a small-molecule drug, and / or a polypeptide drug.

4. The viral vector polymer matrix hydrogel lyophilizate according to claim 1, characterized in that The high-molecular-matrix comprises: hyaluronic acid, polyvinylpyrrolidone, collagen, silk fibroin, gelatin, hydroxypropyl methylcellulose, chondroitin sulfate, dextrin, polyvinyl alcohol, carboxymethyl cellulose, carboxymethyl chitosan, dextran sulfate, or glycogen.

5. A method of preparing a lyophilized preparation of a viral vector macromolecular matrix hydrogel according to any one of claims 1 to 4, characterized in that, The method comprises: Preparation of methacrylated hyaluronic acid based on methacrylic anhydride and hyaluronic acid; Preparation of a high-molecular-matrix hydrogel based on methacrylated hyaluronic acid; After cross-linking the high-molecular-matrix hydrogel, freeze-drying is performed, and then a viral vector solution is added for dissolution, to obtain a hydrogel embedded with a viral vector; Freeze-drying of the viral vector-embedded hydrogel to obtain a viral vector high-molecular-matrix hydrogel freeze-dried preparation.

6. The method of claim 5, wherein the viral vector polymer matrix hydrogel lyophilizate is prepared by, Preparation of methacrylated hyaluronic acid based on methacrylic anhydride and hyaluronic acid, comprising: Methacrylic anhydride is added to a hyaluronic acid solution, the pH value of the reaction is maintained at 8-9 by adding NaOH, and continuous stirring is performed at 4 degrees Celsius; Methacrylated hyaluronic acid is precipitated using acetone, the precipitate is washed with ethanol, and then dissolved in deionized water; After dialysis to remove impurities, freeze-drying is performed to obtain purified methacrylated hyaluronic acid.

7. The method of claim 4, wherein the viral vector polymer matrix hydrogel lyophilizate is prepared by, Preparation of a high-molecular-matrix hydrogel based on methacrylated hyaluronic acid, comprising: Methacrylated hyaluronic acid, a cross-linking agent, and a photo initiator Lrgacure2959 are added to an albumin solution; Free radical polymerization is performed by ultraviolet radiation to form a high-molecular-matrix hydrogel loaded with albumin nanoparticles.

8. Use of a viral vector macromolecular matrix hydrogel lyophilisate according to any one of claims 1 to 4, characterized in that, It is applied to the preparation of a tumor treatment preparation, the viral vector in the treatment preparation is AAV, and the viral vector delivers a p53 gene.

9. Use of a viral vector macromolecular matrix hydrogel lyophilisate according to claim 8, characterized in that, The treatment preparation further comprises: an iron death inducer.

10. Use of a viral vector macromolecular matrix hydrogel lyophilizate according to claim 8, characterized in that, The tumor comprises melanoma and bladder cancer.

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