Novel oncolytic virus loaded hydrogel and application of novel oncolytic virus loaded hydrogel to preparation of medicine or preparation for controlling tumor postoperative recurrence

Abstract

The invention relates to the field of oncolytic virus tumor immunotherapy, in particular to application of oncolytic virus loaded hydrogel to preparation of drugs or preparations for controlling tumor postoperative recurrence. The invention discloses a preparation prepared by wrapping an oncolytic virus with hydrogel prepared from self-assembled polypeptide, which can induce an I-type interferon pathway, activate innate immunity and adaptive immunity and generate anti-tumor immune memory during in-situ continuous release in an operation so as to control postoperative recurrence of tumors and prolong survival. In addition, the invention also discovers that different types of oncolytic viruses wrapped by hydrogel have the same effect of controlling postoperative recurrence of tumors. Therefore, the invention provides a new normal form for continuous anti-tumor immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of oncolytic virus tumor immunotherapy, especially the effect of preparing hydrogel-encapsulated oncolytic virus for controlling tumor recurrence after surgery. Specifically, it relates to a preparation that starts with the in-situ placement of a hydrogel-loaded oncolytic virus preparation during surgery to induce anti-tumor immune activation, inhibit tumor recurrence after surgery, and prolong survival. Background Art

[0002] Tumor is a major disease threatening human health and life. For patients with the opportunity for surgery, surgical resection of the tumor mass is often the first choice; radiotherapy, chemotherapy, and immunotherapy are also treatment options. However, for patients who have undergone surgery, postoperative recurrence (in-situ and metastatic recurrence) is one of the greatest challenges to be addressed. The recurrence time is also uncertain, and some metastatic recurrence cases may occur several years or even decades after surgery. In addition, the wound healing process and related inflammation lead to postoperative tumor recurrence. Currently, chemotherapy and radiotherapy are commonly used clinically to control postoperative recurrence. However, these methods often have strong toxic and side effects in some cases. Therefore, there is an urgent need for safe and effective treatment methods to control postoperative tumor recurrence.

[0003] Regarding the reasons for postoperative tumor recurrence, due to the complexity of the tumor itself, local tumor micro-infiltration may occur in the tumor, and there may be circulating tumor cells; these factors can induce future tumor recurrence, including in-situ recurrence and distant metastasis. Specifically, local tumor micro-infiltration and circulating tumor cells not only inhibit the innate immune response in various ways, but also mainly promote the escape of tumor cells from infiltrating immune effector cells by activating immune checkpoints. In addition, the wound healing process and related inflammation can lead to an immunosuppressive microenvironment at the surgical site, and then enter a systemic immunosuppressive state, helping tumor cells achieve immune escape and wait for recurrence. With the continuous emergence of exciting clinical research results and the continuous listing of anti-tumor immunotherapy drugs, anti-tumor immunotherapy has brought hope to tumor patients. Among these methods or drugs, some can eliminate the immune escape of tumor cells and directly induce anti-tumor immunity; some of these methods have potential value in controlling postoperative tumor recurrence and metastasis. Therefore, tumor immunotherapy has great potential application value in preventing postoperative recurrence.

[0004] For the administration route, systemic administration of immunotherapy is mainly used to achieve long-term effective systemic immune surveillance, such as immune checkpoint inhibitors, CAR-T cells or CAR-NK cells. Studies have shown that intratumoral immunotherapy is safer and sometimes more effective than systemic therapy. Localized treatment at the tumor site can disrupt local immune tolerance, induce systemic anti-tumor immunity, and avoid serious side effects. Therefore, in-situ immune activation is a promising strategy for tumor immunotherapy and prevention of postoperative tumor recurrence. Another important point is that wound healing and inflammation begin at the moment the tumor is removed; therefore, to effectively control postoperative recurrence, immune activation should be induced as early as possible (i.e., immediately after tumor resection). In addition, postoperative wound healing is not a short-term process; therefore, in-situ immune regulation immediately after surgery is preferably carried out once and maintained for a period of time, because repeated induction may cause new damage.

[0005] As an important branch of tumor immunotherapy, oncolytic virus (OVs) therapy has received increasing attention in recent years. OVs are a type of virus with anti-tumor effects that utilize the ability of some viruses to selectively replicate and destroy cancerous tissues without damaging normal tissues. In addition to having a direct oncolytic effect on tumor cells, OVs can also induce a systemic anti-tumor immune response, converting "cold" tumors into "hot" tumors. This result increases the susceptibility of tumors to other treatment modalities, thus providing a practical opportunity for synergistic anti-cancer strategies. Among all types of exogenous viruses, adenovirus, herpes simplex virus (HSV), and vaccinia virus (VV) are the three exogenous viruses that have been commonly studied in clinical trials in the past decade, indicating their potential for future applications. However, some challenges related to efficacy and safety still hinder the use of immunotherapy. Promisingly, biomaterials with novel drug delivery properties have promoted significant progress in cancer immunotherapy. In recent years, hydrogels, as a versatile biomaterial, have been widely used in drug release, including local focused drug release, continuous drug release, and responsive drug release. Due to their biodegradability and the ability to stably exist in the body for a period of time, biomaterial scaffolds such as hydrogels are particularly suitable for immune regulation. Some hydrogels have been used as carriers for immune regulatory drugs targeting tumor cells to prevent recurrence and metastasis. In summary, these pioneering works on hydrogels have given us a lot of inspiration for tumor prevention and treatment.

[0006] Therefore, the present invention constructs a newly designed supramolecular hydrogel (adv@Nap hydrogel) to achieve the sustained release of oncolytic adenovirus type V (adv). The adv@Nap hydrogel is placed in situ during surgery to treat an orthotopic breast cancer mouse model, to evaluate its inhibitory effect on tumor recurrence, and to analyze its anti-tumor immune response. In addition, the present invention also expands the scope of application to include other types of OVs and verifies it in a humanized mouse tumor model. These findings provide new treatment strategies and theoretical bases for the development of methods for controlling tumor recurrence after surgery; importantly, the method developed by the present invention has strong clinical application prospects. Summary of the Invention

[0007] In the present invention, we demonstrate the preparation of a hydrogel formulation loaded with oncolytic virus, and find through animal models that such an oncolytic virus formulation can effectively control tumor recurrence after surgery when placed in situ starting from during the operation.

[0008] The oncolytic virus formulation disclosed by the present invention that can control tumor recurrence after surgery is an oncolytic virus formulation encapsulated by self-assembling polypeptide.

[0009] In a mouse orthotopic breast cancer model constructed with 4T1 mouse breast cancer cells or MDA-MB-231 human breast cancer cell line, a hydrogel loaded with an oncolytic virus formulation prepared by encapsulating the oncolytic virus with polypeptide NapGFFYK can effectively activate the host's anti-tumor immune response, control tumor recurrence after surgery, and prolong the survival period of mice when released in situ starting from during the operation.

[0010] Therefore, the specific content of the present invention is as follows:

[0011] A hydrogel-encapsulated oncolytic virus formulation is prepared by encapsulating an oncolytic virus with a self-assembling polypeptide, and the self-assembling polypeptide is NapGFFYK.

[0012] A hydrogel-encapsulated oncolytic virus formulation is prepared by encapsulating an oncolytic virus with a self-assembling polypeptide, and the oncolytic virus is human adenovirus type V.

[0013] A hydrogel-encapsulated oncolytic virus formulation is prepared by encapsulating an oncolytic virus with a self-assembling polypeptide, and the oncolytic virus is herpes simplex virus.

[0014] A hydrogel-encapsulated oncolytic virus formulation is prepared by encapsulating an oncolytic virus with a self-assembling polypeptide, and the oncolytic virus is vaccinia virus.

[0015] A hydrogel-encapsulated oncolytic virus formulation is prepared by encapsulating an oncolytic virus with a self-assembling polypeptide, and the formulation can stably exist in vivo for about 10 days.

[0016] A hydrogel-encapsulated oncolytic virus formulation is prepared by encapsulating an oncolytic virus with a self-assembling polypeptide, and the formulation can stably exist in an in vitro simulated body fluid environment for about 15 days.

[0017] The oncolytic virus preparation encapsulated in hydrogel prepared by self-assembling polypeptide to wrap the oncolytic virus is used to control the recurrence after tumor resection, and the tumor model is a mouse orthotopic breast cancer model constructed by 4T1 cells.

[0018] The oncolytic virus preparation encapsulated in hydrogel prepared by self-assembling polypeptide to wrap the oncolytic virus is used to control the recurrence after tumor resection, and the tumor model is a mouse orthotopic breast cancer model constructed by MDA-MB-231 cells.

[0019] The oncolytic virus preparation encapsulated in hydrogel is used to control the recurrence after tumor resection, and the administration method of the preparation is in-situ administration.

[0020] The oncolytic virus preparation encapsulated in hydrogel is used to control the recurrence after tumor resection, and the administration timing of the preparation is to administer immediately after tumor resection.

[0021] The oncolytic virus preparation encapsulated in hydrogel is used to control the recurrence after tumor resection, and the action mode of the preparation is to activate the type I interferon pathway, induce innate and adaptive immune responses, and generate immune memory.

[0022] The oncolytic virus preparation encapsulated in hydrogel is used to control the recurrence after tumor resection, and the action result of the preparation is to inhibit the recurrence and distant metastasis after tumor resection and extend the survival period of mice. Description of the Drawings

[0023] Figure 1 Antitumor immune regulation effect of oncolytic adenovirus in orthotopic breast cancer model. a Schematic diagram of activating immune response by adenovirus (adv) (in orthotopic breast cancer mouse model). Female BALB / c mice were inoculated with 4T1 cells (1×10 6 cells per mouse) in the fourth mammary fat pad. When the tumor volume reached about 80 mm 3 (starting from 7 days after tumor inoculation), mice were intraperitoneally injected with adv (1×10 8Three times with pfu or phosphate-buffered saline (PBS). Two days after the last adv injection, the spleens and tumors of the mice were collected, and single-cell suspensions were prepared for staining and analysis by flow cytometry. b Flow cytometry analysis of natural killer cells (NKp46+), activated natural killer cells (CD69+NKp46+), highly activated natural killer cells (CD11b+CD27+NKp46+), M1 cells (CD86+F4 / 80+CD11b+), M2 cells (CD206+F4 / 80+CD11b+), activated dendritic cells (MHCⅡ+CD11c+CD11b+), myeloid-derived suppressor cells (Gr-1+CD11b+), CD4+ T cells (CD4+), regulatory T cells (CD25+CD4+), and cytotoxic CD8+ T cells (GZMB+CD8a+) in the spleens of mice. c Representative flow cytometry analysis of CD8+ T cells (CD8a+), cytotoxic CD8+ T cells I (FN-γ+CD8a+), central memory (CD44+CD62L+), and effector memory (CD44+CD62L-) CD8+ T cells in the spleens of mice. Flow cytometry analysis of natural killer cells (NKp46+), activated natural killer cells (CD69+NKp46+), highly activated natural killer cells (CD11b+CD27+NKp46+), M1 cells (CD86+F4 / 80+CD11b+), M2 cells (CD206+F4 / 80+CD11b+), activated dendritic cells (MHCⅡ+CD11c+CD11b+), myeloid-derived suppressor cells (Gr-1+CD11b+), CD4+ T cells (CD4+), regulatory T cells (CD25+CD4+), and cytotoxic CD8+ T cells (GZMB+CD8a+) in the tumor tissues of mice. f Representative flow cytometry analysis of CD8+ T cells (CD8a+), cytotoxic CD8+ T cells I (FN-γ+CD8a+), central memory (CD44+CD62L+), and effector memory (CD44+CD62L-) CD8+ T cells in the tumor tissues of mice.

[0024] Figure 2The release of oncolytic virus adv from the hydrogel in vitro and in vivo is a stable and slow process. a Design and preparation of a supramolecular hydrogel NapGFFYK micelle loaded with the anti-tumor drug adv. b Representative scanning electron microscope image of adv loaded in the NapGFFYK colloid. c Solids content of adv@Nap after soaking in vitro for several days. (D-E) Rheological property analysis of adv@Nap after soaking in vitro for the corresponding number of days, with frequency (d) and strain (e) as functions. The inset is an image of adv@Nap hanging at the bottom of the centrifuge tube without falling off after inverting the centrifuge tube for 14 days. f Representative images of PBS@Nap and adv@Nap at the surgical site on the 3rd and 7th days. g Real-time PCR detection of adv hexamers in adv@Nap after soaking in vitro for several days or after placement in vivo. h Representative fluorescence images of 293T cells infected with adv@Nap after soaking in vitro for several days or after placement in vivo.

[0025] Figure 3 Analyze the relationship between the rheological properties of the PBS@Nap gel and frequency and strain at corresponding exposure days in vitro. The inserted picture is of the PBS@Nap gel hanging at the bottom of the centrifuge tube without falling off after inverting the centrifuge tube for 14 days.

[0026] Figure 4 Analyze the relationship between the rheological properties of the PBS@Nap gel and the adv@Nap gel and frequency and strain at corresponding exposure days in vivo.

[0027] Figure 5 Starting from the in-situ placement of the hydrogel adv@Nap during the operation to control tumor recurrence and metastasis and activate the anti-tumor immune response. a Schematic diagram of the ability of the adv@Nap gel to control tumor recurrence in a mouse model of breast cancer postoperative recurrence. Female BALB / c mice were inoculated with 4T1-Luc cells (1×10 6 / mouse) in the fourth mammary fat pad. On the 10th day after inoculation, when the tumor grew to approximately 100 mm 3At that time, the tumor was surgically resected (day 0), and the corresponding surgeries were performed on each group. On the 3rd and 7th days after surgery, the spleens, tdLNs, and lungs of the mice were collected, and single-cell suspensions were prepared for flow cytometry staining analysis. b IVIS imaging of 4T1-Luc cells at different time points in each group. c Survival curves of mice in each group, and the significance of the differences was analyzed. The spleen samples of mice in each group were analyzed by flow cytometry to show the proportions of various immune cells. d Activated NK cells (CD69+NKp46+). e Highly efficient NK cells (CD11b+CD27+NKp46+). f DCs that cross-present antigens and generate anti-tumor immune responses (CD103+CD11c+CD11b+). g Activated DCs (CD86+CD11c+CD11b+). h Plasmacytoid DCs (B220+CD11c+CD11b+). i Activated CD4+ T cells (CD69+CD4+). j Activated CD8+ T cells (CD69+CD8a+). k Central memory CD8+ T cells (Ly6C+CD62L+CD8a+). l Representative images of immunohistochemical staining of CD4+, CD8+, and CD11c+ in splenocytes of each group 7 days after different treatments. (m-n) CD8+ T cells were extracted from the spleens of mice in each group and stimulated with the peptide SPSYVYHQF to detect the activation of immune cells, including IFN-γ+CD8a+ T cells (m) and GZMB+CD8a+ T cells (n).

[0028] Figure 6 Flow cytometry analysis was performed on the immune cells shown in the mouse spleen. Activated dendritic cells (CD8a+CD11c+CD11b+, CD40+CD11c+CD11b+, MHC II+CD11c+CD11b+).

[0029] Figure 7 Flow cytometry analysis of the indicated immune cell clusters in the mouse tdLNs and lungs. Activated NK cells (CD69+NKp46+), M1 cells (CD86+F4 / 80+CD11b+), M2 cells (CD206+F4 / 80+CD11b+), DC cells (CD86+CD11c+), CD8+ T cells (CD8a+), cytotoxic CD8+ T cells (IFN-γ+CD8a+), central memory cells (CD44+CD62L+, Ly6C+CD62L+) CD8+ T cells in the mouse spleen.

[0030] Figure 8 Tumor growth curve after CT-26 rechallenge. Mice that were rechallenged with 4T1 cells in the left axilla of the mouse and did not develop tumor masses were inoculated with the CT-26 colorectal cancer cell line (1×10 5 / mouse) in the right axilla. When the tumor volume exceeded 1500 mm 3When the time comes, the mice are euthanized in accordance with animal ethics.

[0031] Figure 9 Adv@Nap gel was placed in situ to induce the type I interferon pathway and immune chemotaxis. a ELISA was used to detect the concentration of IFN-α in the serum of mice on the 3rd day after surgery. b ELISA was used to detect the concentration of IFN-β in the serum of mice on the 3rd day after surgery. c ELISA was used to detect the concentration of IFN-α in the serum of mice on the 14th day after surgery. d ELISA was used to detect the level of IFN-β in the serum of mice on the 14th day after surgery. e ELISA was used to detect the concentration of IL-15 in the serum of mice on the 3rd day after surgery. f ELISA was used to detect the concentration of CXCL-9 in the serum of mice on the 3rd day after surgery. g ELISA was used to detect the level of CXCL-10 in the serum of mice on the 3rd day after surgery. h ELISA was used to detect the concentration of IL-15 in the serum of mice on the 14th day after surgery. i ELISA was used to detect the concentration of CXCL-9 in the serum of mice on the 14th day after surgery. j ELISA was used to detect the concentration of CXCL-10 in the serum of mice on the 14th day after surgery. k Schematic diagram of the mouse antibody depletion experiment. A mouse model was constructed as described above, and adv@Nap gel was administered in situ immediately after tumor resection. Antibodies against different immune cells (NK cells, CD8+ T cells, or CD4+ T cells) were used to deplete immune cells, or IFNAR1, an inhibitor of the type I interferon pathway, was used. In the antibody depletion experiment, the postoperative survival curves of each group of mice were monitored, and the significance of the differences between the adv@Nap group and other groups was analyzed. The mouse model was constructed as described above, and the tumor was treated with adv@Nap immediately after surgery or after storing adv@Nap at 4 °C for one week. The postoperative survival curves of each group of mice were monitored, and the significance of the differences between the adv@Nap group and other groups was analyzed.

[0032] Figure 10Necessity of intraoperative and in-situ administration for adv@Nap gel-induced immune activation to control postoperative tumor recurrence. a Flow chart of the experiment on the effect of different administration methods on controlling postoperative tumor recurrence. A mouse model of postoperative breast cancer recurrence was constructed as previously described. At the time of tumor resection, adv@Nap colloid was placed in-situ or subcutaneously, or intravenously injected in the form of adv solution. b Survival curves of mice in each group were shown, and the significance of differences was analyzed. (c-h) On the 3rd and 7th days after surgery, mouse spleens were harvested, single-cell suspensions were prepared, stained, and analyzed by flow cytometry to show the proportions of various immune cells. i ELISA was used to detect the concentration of IFN-α in the sera of mice on the 7th day after surgery. j ELISA was used to detect the level of IFN-β in the sera of mice on the 7th day after surgery. k Flow chart of the effect of different administration durations of adv@Nap on controlling postoperative tumor recurrence. A mouse model of postoperative breast cancer recurrence was constructed as described above, and adv@Nap colloid was placed immediately or 7 days after surgery. Survival curves of mice in each group were shown, and the significance of differences was analyzed. (m-t) On the 3rd and 7th days after surgery, mouse spleens were harvested, single-cell suspensions were prepared, stained, and analyzed by flow cytometry to show the proportions of various immune cells. u ELISA was used to detect the concentration of IFN-α in the sera of mice on the 7th day after surgery. v ELISA was used to detect the level of IFN-β in the sera of mice on the 7th day after surgery.

[0033] Figure 11 Flow cytometry analysis of immune cells involved in mouse spleens. Activated dendritic cells (CD86+CD11c+CD11b+, MHCⅡ+CD11c+CD11b+, CD8a+CD11c+CD11b+). Activated CD4+ T cells (CD69+CD4+).

[0034] Figure 12 Flow cytometry analysis of the indicated immune cells in mouse spleens. Activated dendritic cells (CD86+CD11c+CD11b+, MHCⅡ+CD11c+CD11b+, CD8a+CD11c+CD11b+).

[0035] Figure 13Initiating the in-situ placement of HSV@Nap or VV@Nap during the operation can effectively control tumor recurrence and metastasis. a Schematic illustration of the ability of HSV@Nap to control tumor recurrence in a postoperative breast cancer recurrence mouse model. On the 3rd and 7th days after the operation, the spleens of the mice were harvested, single-cell suspensions were prepared, stained, and analyzed by flow cytometry. b IVIS imaging of 4T1-Luc cells in each group treated with HSV at different time points after the operation. c Postoperative survival curves of each group of mice were shown, and the significance of the differences was analyzed. The spleens of each group of mice were analyzed by flow cytometry, and the proportions of various immune cells were shown. d Activated NK cells (CD69+). e DC cells (CD103+) that cross-present antigens and generate anti-tumor immune responses. f Activated CD4+ T cells (CD69+CD4+). g Activated CD8+ T cells (CD69+CD8a+). h Cytotoxic CD8+ T cells (IFN-γ+CD8a+). i Central memory CD8+ T cells (Ly6C+CD62L+). j ELISA detection of the level of serum IFN-α on the 7th day after the operation. k ELISA detection of the level of serum IFN-β on the 7th day after the operation. l IVIS imaging of 4T1-Luc cells in all groups at different time points after the operation. m Postoperative survival curves of each group of mice were shown, and the significance of the differences was analyzed. The spleens of each group of mice treated with VV were analyzed by flow cytometry, and the proportions of various immune cells were shown. n Activated NK cells (CD69+). o Highly effector NK cells (CD11b+CD27+). p Activated DC cells (CD86+). q Activated CD8+ T cells (CD69+CD8a+). r Cytotoxic CD8+ T cells (IFN-γ+CD8a+). s Central memory CD8+ T cells (Ly6C+CD62L+). t ELISA detection of the concentration of serum IFN-α on the 7th day after the operation. u ELISA detection of the level of serum IFN-β on the 7th day after the operation.

[0036] Figure 14 Flow cytometry analysis of specified immune cells in the spleens of mice treated with HSV@Nap. Effector natural killer cells (CD11b+CD27+NKp46+) and activated dendritic cells (CD86+CD11c+CD11b+, MHCⅡ+CD11c+CD11b+, CD8a+CD11c+CD11b+).

[0037] Figure 15Flow cytometry was used to analyze the specified immune cells in the spleens of VV@Nap-treated mice. Effector NK cells (CD11b+CD27+NKp46+), M1 cells (CD86+F4 / 80+CD11b+), M2 cells (CD206+F4 / 80+CD11b+), activated DC cells (MHCⅡ+CD11c+CD11b+), activated CD4+ T cells (CD69+CD4+), and cytotoxic CD+ T cells (IFNγ+CD4+).

[0038] Figure 16 In a mouse model with humanized immune system, the in-situ placement of hydrogel adv@Nap during surgery could control tumor recurrence. a Schematic diagram of the ability of adv@Nap gel to control tumor recurrence in a mouse model of breast cancer recurrence after surgery with human immune system. Female NCG mice were inoculated with MDA-MB-231 cells (1×10 5 ) in the fourth mammary fat pad. Two days before tumor resection surgery, hPBMCs were extracted from human blood and injected intraperitoneally into the mice at a density of 5×10 6 / mouse. On the 10th day after inoculation, when the tumor grew to approximately 100 mm 3 , the tumor was resected surgically (day 0), and the corresponding treatments were given to each group. On the 3rd and 7th days after surgery, the spleens of the mice were taken to prepare single-cell suspensions, and flow cytometry staining analysis was performed. b The survival curves of the mice in each group were given, and the significance of the differences was analyzed. c Representative flow cytometry analysis of the hCD45+ cell population on the 3rd day after surgery. d Representative immunohistochemical staining images of hCD45+ in the spleens of the mice in each group 7 days after different treatments. e Representative flow cytometry data of the proportion of hCD45+hCD11b+ cells in the spleens of the mice in each group on the 3rd day after surgery. f Representative flow cytometry data of the proportion of hCD45+hCD8a+ cells in the spleens of the mice in each group on the 7th day after surgery. g Representative flow cytometry data of the proportion of hCD8a+GZMB+ cells in the spleens of the mice in each group on the 7th day after surgery. h Representative flow cytometry data of the proportion of hCD8a+IFNγ+ cells in the spleens of the mice in each group on the 7th day after surgery. i ELISA was used to detect the level of serum hIFN-α on the 7th day after surgery. j ELISA was used to detect the level of serum hIFN-β on the 7th day after surgery.

[0039] Figure 17The in - vivo placement of adv@Nap in situ at the beginning of the operation is safe in the body and has no obvious toxic effects. Composition of whole blood of mice on the 7th day after treatment with an adv@Nap gel. WBC: white blood cell; Lymph: lymphocyte; Mon: monocyte; Gran: neutrophil; RBC: red blood cell; HGB: hemoglobin concentration; HCT: hematocrit; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; RDW: red blood cell distribution width; PLT: platelet count; MPV: mean platelet volume; PDW: platelet distribution width; PCT: plateletcrit. The dotted line represents the determined normal range. b Concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) in the serum of mice on the 7th day after treatment with an adv@Nap gel. The dotted line represents the determined normal range. c Representative hematoxylin and eosin (H&E) staining images of major organs such as the heart, liver, spleen, lung, and kidney of mice on the 7th day after treatment with an adv@Nap gel. d Body weight of mice after the operation. Detailed implementation mode

[0040] All experimental materials and methods involved in the example part

[0041] 1. Cell line

[0042] The cell lines used in this project mainly include: 4T1 / 4T1-luciferase (mouse breast cancer cells), H22 (hepatoma cells derived from Balb / C mice), CT-26 (colorectal cancer cell line derived from Balb / C mice), B16F10 (melanoma cell line derived from C57BL / 6 mice), LLC (Lewis lung cancer cell line derived from Balb / C mice), PANC-02 (pancreatic cancer cell line derived from C57BL / 6 mice), GL261 (glioma cell line derived from C57BL / 6 mice), MB49 (bladder cancer cell line derived from C57BL / 6 mice), (renal cell carcinoma cell line), MDA-MB-231 (human triple-negative breast cancer cell line), HEK293 (human embryonic kidney cells), Hela-S3 (human cervical cancer cell line). All of the above cell lines are stored in the laboratory. 4T1, B16F10, LLC, GC261, HEK293, CT-26 and Hela-S3 cells were purchased from ATCC. H22, PANC-02, MB49, MDA-MB-231 cells were purchased from the China Center for Type Culture Collection (CCTCC; China). 4T1-luciferase cells were purchased from Ubigene Biosciences. 4T1, B16F10, LLC, GC261, HEK293, CT-26, PANC-02, MB49, MDA-MB-231 cells were cultured in DMEM medium (Dulbecco’s modified Eagle’s medium, DMEM; Cat#11965092, Gibco-Thermo Fisher Scientific) containing 10% fetal bovine serum (Fetal bovine serum, FBS; Cat#10099158, Gibco-Thermo Fisher Scientific) and 1% penicillin-streptomycin solution (Penicillin-Streptomycin, PS; Cat#450-201-EL, WISENT). H22, Hela-S3 cells were suspended in serum-free medium (Cat#H740KJ, Basalmedia, Shanghai) and cultured by rotation in a spinner flask (Cat#TCB002002, Jetbiofil, Guangzhou, China). All cells were cultured at 37°C under 5% CO2 conditions.

[0043] 2. Virus

[0044] Oncolytic adenovirus (Type V adenovirus), oncolytic reovirus (Oncolytic orthoreovirus), oncolytic Coxsackievirus (Coxsackievirus A21), oncolytic Newcastle disease virus (NDV Herts / 33 strain), oncolytic measles virus (Oncolytic measles virus Edmonston strain), oncolytic vesicular stomatitis virus (Mudd-Summer strain, Indiana serotype), oncolytic poliovirus (Poliovirus type 1, sabin strain) are from virus species stored in the laboratory. Oncolytic herpes simplex virus (Herpes simplex virus type 1, strain F), oncolytic vaccinia virus (Elstree strain) were purchased from Wuhan Bin Hui Biotechnology Co., Ltd.

[0045] 2. Mice

[0046] All animal experiments were conducted in accordance with the guidelines approved by the Ethics Committee of Nanjing Drum Tower Hospital, School of Medicine, Nanjing University. Six- to eight-week-old mice were used in these experiments. Female wild-type BALB / c mice were purchased from GemPharmatech Co., Ltd (Nanjing). Female NOD-Prkdc-scid IL2rgem1 / Cyagen (C-NKG) mice were purchased from Cyagen Biosciences Inc. (Shanghai, China). The mice were placed under specific pathogen-free (SPF) conditions at a temperature of 18°C - 24°C, with access to water and food, and maintained on a 12-h light / dark cycle.

[0047] 3. Preparation of hydrogel

[0048] 1) Using polypeptide as the matrix: A DMSO solution of NapGFFYK (concentration 80 mg / ml) and an equal volume of a virus mixture (adenovirus at 2×10^9 pfu / ml, herpes simplex virus at 1×10^8 pfu / ml, vaccinia virus at 1×10^7 pfu / ml, reovirus at 1×10^7 pfu / ml, Coxsackievirus at 1×10^7 pfu / ml, Newcastle disease virus at 5×10^8 pfu / ml, measles virus at 5×10^6 pfu / ml, vesicular stomatitis virus at 5×10^7 pfu / ml, poliovirus at 5×10^8 pfu / ml) were rapidly mixed. The mixture was oscillated on a vortex mixer (product of Scientific Instruments, USA) for 20 seconds, and a translucent hydrogel was instantly formed. [1,2] .

[0049] 2) Using collagen as a matrix: Mix collagen (2 mg / ml) with virus solutions (adenovirus at 2×10^9 pfu / ml, herpes simplex virus at 1×10^8 pfu / ml, vaccinia virus at 1×10^7 pfu / ml, reovirus at 1×10^7 pfu / ml, coxsackievirus at 1×10^7 pfu / ml, Newcastle disease virus at 5×10^8 pfu / ml, measles virus at 5×10^6 pfu / ml, vesicular stomatitis virus at 5×10^7 pfu / ml, poliovirus at 5×10^8 pfu / ml) at a ratio of 1:3 at 4°C, and then warm up to 37°C to form oncolytic viruses encapsulated by collagen matrix hydrogel. [3,4] 。

[0050] 3) Using gelatin as a matrix: Mix 20% (w / v) gelatin methyl acrylate with virus solutions (adenovirus at 2×10^9 pfu / ml, herpes simplex virus at 1×10^8 pfu / ml, vaccinia virus at 1×10^7 pfu / ml, reovirus at 1×10^7 pfu / ml, coxsackievirus at 1×10^7 pfu / ml, Newcastle disease virus at 5×10^8 pfu / ml, measles virus at 5×10^6 pfu / ml, vesicular stomatitis virus at 5×10^7 pfu / ml, poliovirus at 5×10^8 pfu / ml) at a ratio of 1:1 at room temperature, then add 0.5% (w / v) photoinitiator and gel at 80°C to prepare oncolytic viruses encapsulated by gelatin matrix hydrogel. [2,5] 。

[0051] 4) Using hyaluronic acid as a matrix: First, modify hyaluronic acid with 3,3'-dithiobis(propionic acid hydrazide) to form DTPH-HA. Then mix DTPH-HA (2% w / v) with virus solutions (adenovirus at 2×10^9 pfu / ml, herpes simplex virus at 1×10^8 pfu / ml, vaccinia virus at 1×10^7 pfu / ml, reovirus at 1×10^7 pfu / ml, coxsackievirus at 1×10^7 pfu / ml, Newcastle disease virus at 5×10^8 pfu / ml, measles virus at 5×10^6 pfu / ml, vesicular stomatitis virus at 5×10^7 pfu / ml, poliovirus at 5×10^8 pfu / ml) at a ratio of 1:4 at room temperature, and add the crosslinking agent 2,5-hexanedione. The mixture is rotated vigorously and then defoamed to form oncolytic viruses encapsulated by light yellow hyaluronic acid matrix hydrogel. [6,7] 。

[0052] 5) Using alginate as the matrix: Mix the sodium alginate solution (4% w / v) with the virus solution (adenovirus at 2×10^9 pfu / ml, herpes simplex virus at 1×10^8 pfu / ml, vaccinia virus at 1×10^7 pfu / ml, reovirus at 1×10^7 pfu / ml, coxsackievirus at 1×10^7 pfu / ml, Newcastle disease virus at 5×10^8 pfu / ml, measles virus at 5×10^6 pfu / ml, vesicular stomatitis virus at 5×10^7 pfu / ml, poliovirus at 5×10^8 pfu / ml) at a ratio of 1:2 at 4°C. Subsequently, add the cross-linking agent calcium carbonate solution and glucono-δ-lactone, and slowly mix to form a hydrogel of alginate matrix encapsulating oncolytic virus [8] 。

[0053] 6) Using fibrin as the matrix: Mix fibrinogen (160 mg / ml) with the virus solution (adenovirus at 2×10^9 pfu / ml, herpes simplex virus at 1×10^8 pfu / ml, vaccinia virus at 1×10^7 pfu / ml, reovirus at 1×10^7 pfu / ml, coxsackievirus at 1×10^7 pfu / ml, Newcastle disease virus at 5×10^8 pfu / ml, measles virus at 5×10^6 pfu / ml, vesicular stomatitis virus at 5×10^7 pfu / ml, poliovirus at 5×10^8 pfu / ml) evenly at a ratio of 1:1. Subsequently, simultaneously extrude the two solutions through a double-barrel syringe with a buffer containing thrombin (100 - 500 IU / mL) and CaCl2 (10 - 40 mM), and let it stand for 1 - 5 minutes after mixing to complete cross-linking, thus preparing a hydrogel of fibrin matrix encapsulating oncolytic virus [9,10] 。

[0054] 7) Using chitosan as the matrix: Dissolve chitosan in acetic acid to form a solution with a concentration of 20% (w / v). Subsequently, add the virus solution (adenovirus at 2×10^9 pfu / ml, herpes simplex virus at 1×10^8 pfu / ml, vaccinia virus at 1×10^7 pfu / ml, reovirus at 1×10^7 pfu / ml, coxsackievirus at 1×10^7 pfu / ml, Newcastle disease virus at 5×10^8 pfu / ml, measles virus at 5×10^6 pfu / ml, vesicular stomatitis virus at 5×10^7 pfu / ml, poliovirus at 5×10^8 pfu / ml) at a ratio of 1:10. Then, add a chemical cross-linking agent such as glutaraldehyde to initiate an amino cross-linking reaction, thus preparing a hydrogel of chitosan matrix encapsulating oncolytic virus [11,12] 。

[0055] 8) Using agarose as the matrix: After mixing low melting point agarose powder with buffer solution, heat it to completely dissolve the agarose powder to form a 0.5% (w / v) solution. Then, after lowering the temperature by a certain degree, add the virus solution (adenovirus at 2×10^9 pfu / ml, herpes simplex virus at 1×10^8 pfu / ml, vaccinia virus at 1×10^7 pfu / ml, reovirus at 1×10^7 pfu / ml, coxsackievirus at 1×10^7 pfu / ml, Newcastle disease virus at 5×10^8 pfu / ml, measles virus at 5×10^6 pfu / ml, vesicular stomatitis virus at 5×10^7 pfu / ml, poliovirus at 5×10^8 pfu / ml), mix well. When the temperature drops to room temperature, a hydrogel with agarose matrix encapsulating oncolytic virus is formed. [13,14] 。

[0056] 9) Using polyethylene glycol or polycaprolactone as the matrix: Copolymerize hydrophilic polyethylene glycol (PEG) with hydrophobic PCL to form an amphiphilic block structure. Then, in a 60% (w / v) copolymer, add the virus solution (adenovirus at 2×10^9 pfu / ml, herpes simplex virus at 1×10^8 pfu / ml, vaccinia virus at 1×10^7 pfu / ml, reovirus at 1×10^7 pfu / ml, coxsackievirus at 1×10^7 pfu / ml, Newcastle disease virus at 5×10^8 pfu / ml, measles virus at 5×10^6 pfu / ml, vesicular stomatitis virus at 5×10^7 pfu / ml, poliovirus at 5×10^8 pfu / ml) at a volume ratio of 1:2, and then self-assemble to form micelles or gel networks to prepare a hydrogel with polycaprolactone and polyethylene glycol as the matrix encapsulating oncolytic virus. [15 , 16] 。

[0057] 10) Using polyvinyl alcohol as the matrix: Mix a 30% polyvinyl alcohol solution with the virus solution (adenovirus at 2×10^9 pfu / ml, herpes simplex virus at 1×10^8 pfu / ml, vaccinia virus at 1×10^7 pfu / ml, reovirus at 1×10^7 pfu / ml, coxsackievirus at 1×10^7 pfu / ml, Newcastle disease virus at 5×10^8 pfu / ml, measles virus at 5×10^6 pfu / ml, vesicular stomatitis virus at 5×10^7 pfu / ml, poliovirus at 5×10^8 pfu / ml) at a ratio of 1:4, then freeze at -20°C for 24 hours and thaw at room temperature for 1 hour, and repeat the cycle multiple times to form a hydrogen bond and microcrystalline region cross-linked network to prepare a hydrogel with polyvinyl alcohol as the matrix encapsulating oncolytic virus. [17,18] 。

[0058] 11) Using sodium carboxymethyl cellulose as the matrix: After restoring the virus solutions (adenovirus at 2×10^9 pfu / ml, herpes simplex virus at 1×10^8 pfu / ml, vaccinia virus at 1×10^7 pfu / ml, reovirus at 1×10^7 pfu / ml, coxsackievirus at 1×10^7 pfu / ml, Newcastle disease virus at 5×10^8 pfu / ml, measles virus at 5×10^6 pfu / ml, vesicular stomatitis virus at 5×10^7 pfu / ml, poliovirus at 5×10^8 pfu / ml) to room temperature, add 10% (w / v) sodium carboxymethyl cellulose in a 1:1 ratio. A physical gel network is spontaneously formed through hydrogen bonds and entanglements between molecular chains to prepare a hydrogel encapsulating oncolytic viruses using sodium carboxymethyl cellulose as the matrix [19,20] 。

[0059] 12) Using polyacrylamide or poly(N - isopropylacrylamide) as the matrix: Mix 30% (w / w) acrylamide or N - isopropylacrylamide monomer with the cross - linker bisacrylamide solution and the virus solutions (adenovirus at 2×10^9 pfu / ml, herpes simplex virus at 1×10^8 pfu / ml, vaccinia virus at 1×10^7 pfu / ml, reovirus at 1×10^7 pfu / ml, coxsackievirus at 1×10^7 pfu / ml, Newcastle disease virus at 5×

[0060] 10^8 pfu / ml, measles virus at 5×10^6 pfu / ml, vesicular stomatitis virus at 5×10^7 pfu / ml, poliovirus at 5×10^8 pfu / ml) in a 1:1 ratio to form a homogeneous solution. Then add 0.5% (w / v) initiator ammonium persulfate. Free radicals are generated through redox reactions to trigger monomer polymerization, preparing a hydrogel encapsulating oncolytic viruses using polyacrylamide as the matrix [21,22] 。

[0061] 13) Based on polyurethane: Mix diisocyanate and polyethylene glycol in a ratio of 1:3. Subsequently, add 30% of the mass of the chain extender dimethylolpropionic acid based on the mass of polyethylene glycol and 0.2% of the total reaction mass of the catalyst dibutyltin dilaurate, and react at 60 - 80 °C to form a prepolymer. Disperse the prepolymer in 4 times the volume of the virus solution (adenovirus at 2×10^9 pfu / ml, herpes simplex virus at 1×10^8 pfu / ml, vaccinia virus at 1×10^7 pfu / ml, reovirus at 1×10^7 pfu / ml, coxsackievirus at 1×10^7 pfu / ml, Newcastle disease virus at 5×10^8 pfu / ml, measles virus at 5×10^6 pfu / ml, vesicular stomatitis virus at 5×10^7 pfu / ml, poliomyelitis virus at 5×10^8 pfu / ml), and finally solidify it into a hydrogel to prepare a hydrogel based on polyurethane encapsulating oncolytic virus [23,24] 。

[0062] 4. Determination of the solid content of the adv@Nap hydrogel

[0063] Weigh the hydrogel and record its wet weight as W1. Then, subject the hydrogel sample to freeze-drying treatment. Record its dry weight as W2 again. The formula for calculating the solid content is: (W2 / W1)×100%.

[0064] 5. Rheological measurement and scanning electron microscope observation of the adv@Nap hydrogel

[0065] Before measurement, carefully transfer the hydrogel to the rheometer plate (Thermo Scientific Haake RheoStress 6000) with a spatula. Subsequently, conduct a rheological experiment at a frequency of 1 Hz in the strain sweep mode with a strain amplitude range of 0.1% - 10%, and conduct an experiment in the frequency sweep mode with a strain of 1% in the frequency range (gap: 1.5 mm; temperature: 20 °C). Take scanning electron microscope images with a Gemini500 scanning electron microscope (acceleration voltage: 10 kV).

[0066] 6. qRT-PCR

[0067] To detect the viral copy number after adenovirus (adv) infects cells, HEK293T cell samples at different time points need to be collected. Each sample is treated with 500 μL of proteinase K lysis buffer (containing 100 μg / mL proteinase K, 50 mmol / L potassium salt, 10 mmol / L tris(hydroxymethyl)aminomethane, 0.5% Tween) to lyse the cells by repeatedly pipetting. The lysate is incubated at 56 °C for 45 minutes to completely lyse 293T cells and release viral genomic DNA. Subsequently, the lysate is heated to 100 °C and maintained for 10 minutes to inactivate proteinase K. The prepared viral genomic DNA template is used for subsequent qPCR amplification. The primers used for amplification are as follows: GAPDH forward primer (5'-GGACCTGACCTGCCGTCTAG) and reverse primer (5'-GTAGCCCAGGATGCCCTTGA); and hexamer forward primer (5'-TGGGCATCCTACACCAACAC) and reverse primer (5'-AGTGCGCCCATGGACATAAA).

[0068] 7. Establishment and treatment of tumor models

[0069] For the orthotopic breast tumor model, 4T1 cells (1×10 6 per mouse) are injected into the fourth mammary fat pad of female BALB / C mice. When the tumor volume reaches approximately 80 - 100 mm 3 , the mice are randomly divided into a control group and an adv group, and 100 μL of PBS or 2×10 9 cfu / mL of adv solution are injected intraperitoneally into the mice on days 0, 2, and 4, respectively.

[0070] For the orthotopic postoperative recurrent breast tumor model, 4T1 or 4T1-luciferase cells (1×10 6 per mouse) are injected into the fourth mammary fat pad of female BALB / C mice. Ten days later, the mice are randomly divided into different groups. The tumors are surgically resected and treated differently according to the above method. The wound is closed with a medical clip.

[0071] For the tumor rechallenge experiment, 5×10 5 4T1 or 1×10 5 CT-26 cells are injected into the axilla or contralateral axilla of each mouse. The body weight of the mice is monitored. The tumor length (L) and width (W) are measured every two days with a digital caliper, and the tumor size (V) is calculated as V = (L×W2) / 2. If the tumor volume exceeds 1500 mm 3 , the mice are sacrificed. For the humanized breast tumor recurrence model, MDA-MB-231 cells (5×10 5were injected into the fourth mammary fat pad of female C-NKG mice. Eight days later, human peripheral blood mononuclear cells (hPBMCs) were intraperitoneally injected (5×10 6 per mouse). Two days after tumor inoculation, the mice were randomly divided into different groups and treated as described above.

[0072] 8. In vivo bioluminescence and imaging

[0073] One day before surgery and weekly after surgery, the local tumor recurrence and metastasis of the mice were examined using an IVIS imaging system. Before each imaging, the mice were intraperitoneally injected with potassium D-luciferin (150 mg / kg), and then anesthetized with 2% isoflurane. Then, bioluminescence images were captured using an IVIS Spectrum in vivo imaging system (Perkin Elmer).

[0074] 9. Biochemical tests of blood components

[0075] To examine the safety of the treatment measures, whole blood samples were collected from the mice 7 days after surgery. For alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN), the blood samples were first allowed to stand at room temperature for 2 hours, and then centrifuged at 3000 revolutions per minute for 15 minutes, and the supernatant was collected for immediate detection. For other components in the blood, plasma samples were stabilized using EDTA or heparin as anticoagulants. Subsequently, blood components were detected using an automatic biochemical analyzer (Chemray 800, Rayto), and the test results were exported after the automatic biochemical analyzer test was completed.

[0076] 10. Depletion of immune cells and neutralization of IFNAR1

[0077] To confirm the necessity of specific immune subsets and type I IFN for the treatment effect, starting from 1 day before surgery, antibodies against NK cells (anti-asialo GM1, FUJIFILM Wako, Japan), CD4+ T cells (anti-mouse CD4, Bioxcell, USA), CD8+ T cells (anti-mouse CD8a, Bioxcell, USA), or IFNAR1 (anti-mouse IFNAR-1, Bioxcell, USA) were intraperitoneally injected every 3 days. All antibodies were used in a timely manner at a dose of 200 μg per mouse.

[0078] 11. Flow cytometry

[0079] All samples were detected on a BD FACSAria III analyzer and analyzed using FlowJo 10 software. All antibodies were purchased from BioLegend or eBioscience. For immune cells in the tumor microenvironment, spleens, thymic lymph nodes, or lung tissues were collected and digested with collagenase IV (50 mg / ml) at 37 °C for 2 hours, and then filtered through a 70-μm cell strainer to generate single-cell suspensions. The obtained single-cell suspensions were stained with different antibodies. Dead cells were excluded using 4′,6-diamidino-2-phenylindole (DAPI) before analysis. Fluorescent antibodies included APC-CD45, APC / Cyanine7-CD3, FITC-NKp46, FITC-CD4, PerCP / Cy5.5-CD8a, PE-CD11b, PE / Cyanine7-CD11c, PE-GZMB, PE / Cyanine7-IFNγ, APC / Cyanine7-CD25, PE / Cyanine7-CD69, APC / Cyanine7-CD27, APC / Cyanine7-CD3, PE / Cyanine7-F4 / 80, PE-CD86, PerCP / Cy5.5-CD206, APC / Cyanine7-MHCⅡ, PE-Gr1, APC-CD62L, PE-CD44, and PE-Ly6C. Cells were grouped as follows: lymphocytes (FSC-H, SSC-H), single cells (FSC-H, FSC-A), CD4+ T cells (CD4+ gated CD3+ cells), CD8+ T cells (CD8a+ gated CD3+ cells), activated CD4+ T cells (CD69+ gated CD4+ cells), activated CD8+ T cells (CD69+ or CD11b+CD27+ gated NKp46+ cells), cytotoxic CD8+ T cells (IFNγ+ or GZMB+ gated CD8a+ cells), regulatory T cells (CD25+ gated CD4+ T cells), natural killer cells (NKp46+ gated CD45+ cells), activated natural killer cells (CD69+ or CD11b+CD27+ gated NKp46+ cells), dendritic cells (CD11c+MHCⅡ+ gated CD11b+CD45+ cells), activated dendritic cells (CD8a+, CD103+, or CD86+ gated cells), plasmacytoid dendritic cells (B220+ gated cells), macrophages (CD11b+

[0080] F4 / 80+ gated CD45+ cells), "M1-type" macrophages (CD86+ gated cells), "M2-type" macrophages (CD206+ gated cells), and

[0081] To verify tumor-specific antigen recognition, experiments were conducted using the antigen peptide SPSYVYHQF (GenScript) at a concentration of 10 μg / mL. Spleen cells were extracted from mice in the corresponding groups (Mouse CD8+ T Cell Isolation Kit, Selleck) and cultured with the antigen peptide for 6 hours. Cytotoxic CD8+ T cells (IFNγ+ or GZMB+ gated CD8a+ cells) were analyzed by flow cytometry.

[0082] 12. Enzyme-linked immunosorbent assay (ELISA)

[0083] To test cytokine levels in mice, whole blood was collected from the eyes of mice. After incubation at room temperature for 2 h, serum was extracted by centrifugation at 3000 rpm / min for 15 min, and the supernatant was collected. An ELISA kit (Shanghai Enzyme-linked Biotechnology Co., Ltd.) was used to detect the expression levels of cytokines IFNα, IFNβ, IL-15, CXCL-9, and CXCL-10.

[0084] 13. Immunohistochemistry (IHC)

[0085] Mouse tissues were fixed in 4% neutral buffered formalin solution, paraffin-embedded, cut into 5-μm sections, and subjected to hematoxylin-eosin (H&E) staining and immunohistochemical staining. First, the paraffin sections were dewaxed with warm water: the sections were dewaxed with environmental protection solution I for 10 minutes, environmental protection dewaxing solution II for 10 minutes, environmental protection dewaxing solution III for 10 minutes, dehydrated with absolute ethanol I for 5 minutes, absolute ethanol II for 5 minutes, absolute ethanol III for 5 minutes, and rinsed with distilled water. For H&E staining, the sections were stained with hematoxylin solution for 1-2 minutes or eosin solution for 0.5 minutes. Then, the sections were rinsed with water for 2 minutes, dehydrated with 95% ethanol (2 changes), treated with absolute alcohol (2 changes), and cleared with xylene (3 changes) for 3 minutes. Finally, the sections were covered with cover slips. For immunohistochemistry (IHC), the dewaxed sections were subjected to antigen retrieval (the retrieval solution and conditions were determined according to the tissue). Then, the endogenous peroxidase activity was blocked, and the cells were incubated with serum. After that, each component was incubated with primary antibodies (including rabbit anti-mouse CD4 antibody, rabbit anti-mouse CD8a antibody, rabbit anti-mouse CD11c antibody, and goat anti-human CD45 antibody). Then, secondary antibody was added, and the samples were developed with 3,3'-diaminobenzidine (DAB). The development results were brown and yellow. After that, the cell nuclei were stained with hematoxylin. Finally, the sections were taken out of xylene, slightly air-dried, and then sealed with glue.

[0086] 14. Quantification and statistical analysis

[0087] The survival rate of mice was analyzed using the Kaplan-Meier method and log rank test. All data were expressed as mean ± sem. Two-group comparisons were performed using two-tailed Student's t-test, and multiple-group comparisons were performed using ordinary one-way ANOVA. NS, no significant difference; *p≤0.05, **p≤0.01, ***p≤0.001.

[0088] Example 1 Oncolytic adenovirus adv can significantly activate the anti-tumor immune response

[0089] To evaluate the potential of oncolytic adenovirus (adv) to prevent postoperative tumor recurrence, we evaluated the ability of oncolytic adenovirus to induce immune activation in solid tumors. An orthotopic breast cancer model was constructed using the murine breast cancer 4T1 cell line. Adenovirus was administered to mice by intratumoral injection ( Figure 1 A). Two days after the last adenovirus administration, tumor tissues were excised, and the activation of immune cells in the tumor microenvironment and spleen of mice was detected by flow cytometry. The results showed that adenovirus had no significant effect on innate immune cells in the tumor microenvironment or spleen, including natural killer cells, activated natural killer cells (CD69+, CD11b+CD27+), activated dendritic cells (MHCⅡ+), M1 (CD86+), M2 (CD206+), and other immune clusters ( Figure 1 B and 1D). However, during the adaptive immune response, adenovirus treatment significantly increased the proportions of CD8+ T cells and cytotoxic CD8+ T cells (IFN-γ+ and GZMB+) ( Figure 1 C and 1E). Notably, adenovirus treatment significantly induced the infiltration of central memory CD8+ T cells (CD44+CD62L+) into the tumor microenvironment and spleen, as well as the infiltration of effector memory CD8+ T cells (CD44+CD62L-) in the spleen ( Figure 1 C and 1E). These results indicate that oncolytic adenovirus can induce a systemic anti-tumor immune response by inducing the activation of anti-tumor memory T cells, suggesting that oncolytic adenovirus has potential value in controlling postoperative tumor recurrence.

[0090] Example 2 Design and characterization of a supramolecular hydrogel loaded with oncolytic virus adv

[0091] Activation of the immune memory response by adenovirus (Adv) is a prerequisite for its inhibition of postoperative tumor recurrence. To fully exert the ability of Adv to activate anti-tumor immunity, a strategy for continuous immune activation is required. In addition, to address the problem that oncolytic viruses are not feasible for in-situ treatment after tumor resection, we adopted a hydrogel system capable of achieving local drug delivery in vivo, which can continuously release oncolytic viruses in-situ as the hydrogel matrix gradually degrades. We selected a short peptide, namely 2-naphthyl-glycine-phenylalanine-phenylalanine (NapGFF), as the motif of the hydrogelator to enable it to self-assemble into a tangled fiber network structure of supramolecular hydrogel. This motif has been widely studied by our team and other teams for constructing peptide hydrogels. In this study, tyrosine and lysine were added to the C-terminus to improve the self-assembly efficiency, so the complete peptide sequence is NapGFFYK, as Figure 2 shown in A. The π-π stacking and hydrophobic interactions of NapGFFYK result in efficient self-assembly of the peptide to form a hydrogel. Adv is dispersed in the hydrogel supported by peptide fibers in the form of nanoparticles ( Figure 2 B).

[0092] The mechanical stability of the Adv@Nap gel is crucial for its application as a virus vector. The prepared hydrogel was immersed in simulated body fluid (containing 10% fetal bovine serum) for 14 days, and its solid content was evaluated at different time points. After 14 days of immersion, the solid content of the Adv@Nap gel decreased from 8.5% to 4% and gradually tended to be stable ( Figure 2 C), indicating that it will not completely disintegrate within 14 days in vivo. The rheological properties of the hydrogel changing with time are one aspect of its stability. Rheological property analysis shows that the storage modulus (G') of the hydrogel on day 0 is about 40 - 60 kPa, more than 3 times that of the loss modulus (G"), while it drops to 200 Pa on day 14, about 3 times that of the loss modulus, indicating that the hydrogel exhibits a solid rather than a viscous response ( Figure 2 D). In addition, the hydrogel has mechanical stability within a strain range of about 6% ( Figure 2 E). We also took images of the Adv@Nap gel. This hydrogel can still stably adhere to the bottom of the centrifuge tube without falling off when the centrifuge tube is inverted for 14 days, indicating that the hydrogel has good physical stability. The same stability test was carried out on the hydrogel without virus (PBS@Nap gel) as a control group, and the results show that the introduction of the virus did not affect the mechanical stability of the hydrogel ( Figure 3 ).

[0093] In addition, we also studied the mechanical stability of the Adv@Nap gel and the PBS@Nap gel in vivo. On the 3rd and 7th days after implanting the hydrogel into mice, visible hydrogel fragments were observed when the surgical suture site was opened (Figure 2 F). Analysis of its rheological properties showed that the storage modulus (4 kPa) and loss modulus (2 kPa) of the hydrogel were similar to those in vitro, indicating that the hydrogel had sustained mechanical stability ( Figure 4 ). Notably, we evaluated the presence of viral particles after the hydrogel was soaked in liquid in vitro for several days or placed in vivo for several days. Real-time PCR was used to detect the hexon protein of adv, and the results showed that the viral particles in vitro were maintained for about 14 days and the number of particles gradually decreased. At the surgical site in vivo, although the number of particles decreased faster, the viral particles in the hydrogel still remained for more than 10 days, ( Figure 2 G). In addition to confirming the presence of the virus, we also confirmed the infectivity of the virus loaded in the hydrogel after long-term exposure by fluorescence imaging ( Figure 2 H). In summary, these results indicate that the supramolecular hydrogel loaded with oncolytic adenovirus has sufficient stability and degradability to maintain the continuous release and infectivity of the virus in vitro and in vivo.

[0094] Example 3 Placing adv@Nap in situ immediately after tumor resection can control postoperative tumor recurrence

[0095] Since adv can be continuously released from the adv@Nap gel, combined with the ability of adv to induce adaptive anti-tumor immune responses in vitro and in vivo, especially cytotoxicity and central memory CD8+ T cells, we next evaluated the ability of the hydrogel loaded with adenovirus to prevent postoperative tumor recurrence. For this purpose, we constructed a mouse model of in situ breast cancer. On the 10th day after tumor inoculation, the tumor was surgically resected. Then, the adv@Nap gel or PBS@Nap gel was placed at the original tumor site, and the incision was sutured. The control group of mice was directly sutured after tumor resection. The adv group of mice was injected with the same amount of adv solution at the tumor site. To prove that adv needs to be loaded into the hydrogel to exert its effect, we implemented the adv&Nap gel group, in which the mice were treated with PBS@Nap gel and adv solution was injected at the resected tumor site. After all these operations were completed, each wound was sutured, and the mice were returned to the cage to continue feeding after regaining consciousness ( Figure 5 A).

[0096] To evaluate the effect of the adv@Nap gel on postoperative tumor recurrence, the tumor burden of the mice was monitored weekly using the IVIS imaging system after surgery. The results showed that there was no obvious tumor recurrence or metastasis in the adv@Nap gel group, while there were varying degrees of tumor recurrence and metastasis in the other groups ( Figure 5B). The results shown by the survival curve were consistent with those of IVIS imaging, indicating that continuous release of adv encapsulated in the hydrogel at the tumor site immediately after tumor resection was more effective than single - use adv in controlling tumor recurrence and prolonging the survival of mice. Figure 5 C).

[0097] To explore how the adv@Nap gel controls postoperative tumor recurrence and prolongs the survival of mice, we evaluated the immune responses of mice in each group. Spleen samples were collected from mice on the 3rd and 7th days after surgery, and the activation ratios of immune cells were analyzed by flow cytometry. The results showed that on the 3rd day after surgery, the ratios of activated (CD69+) and highly effector NK cells (CD11b+CD27+) in the adv@Nap group were significantly higher than those in the adv group. Figure 5 D and 5E). For antigen - presenting cells, the ratios of CD103+, CD86+CD8a+CD40+ and MHCⅡ+ dendritic cells (DCs) capable of cross - presenting antigens were significantly increased in the adv@Nap gel group. Figure 5 F, 5G and 6), indicating a significant enhancement of the innate immune response in mice treated with adv@Nap. The ratio of plasmacytoid DC cells (B220+) that can secrete type I interferon was also significantly increased. Figure 5 H), indicating that adv@Nap treatment may induce the activation of the type I interferon pathway in mice.

[0098] Since the activation of the innate immune response, especially the change in the ratio of antigen - presenting cells, usually induces the activation of adaptive immunity, we further evaluated these adaptive - immunity - related cell populations in mice on the 7th day after surgery. These results were consistent with those of the innate immune analysis, because the ratios of CD4+ T cells (CD69+) and CD8+ T cells (CD69+) in the spleens of mice in the adv@Nap group were significantly higher than those in the adv group. Figure 5 I and 5J). Notably, the significantly increased central memory T cells (Ly6C+CD62L+) in the adv@Nap group are crucial for long - term suppression of tumor recurrence. Figure 5 K). The results of immunohistochemical staining also confirmed these phenomena. Figure 5 L). In addition, we also detected the ratios of immune cells in the tumor - draining lymph nodes (tdLNs) and lungs of mice, which were similar to those in the spleen in terms of innate immunity, adaptive immunity and central memory T cells. Figure 7)。 These findings indicate that adv@Nap treatment can induce the activation of the systemic immune response, thereby inhibiting in situ recurrence and distant metastasis of postoperative tumors. To investigate whether the ability of adv@Nap to control postoperative tumor recurrence is related to the activation of antigen-specific CD8+ T cells against mouse tumor cells, we isolated CD8+ T cells from the corresponding groups and restimulated them with the specific antigen peptide SPSYVYHQF expressed by 4T1 cells. The results showed that the proportions of cytotoxic IFNγ+CD8+ T cells and GZMB+CD8+ T cells in the adv@Nap group were significantly higher than those in the adv group, indicating that adv@Nap controls postoperative tumor recurrence through CD8+ T cell-mediated antigen-specific immunity( Figure 5 M and 5N). The tumor rechallenge experiment verified the specificity of the anti-tumor effect. The same tumor cells (4T1) were implanted into the left axilla of mice that had survived for more than 70 days after treatment with adv@Nap, and no tumor masses formed. Subsequently, different tumor cells (CT-26) were implanted into the contralateral axilla of the mice. Obvious tumor masses formed 6 days after tumor implantation( Figure 8 ).

[0099] Collectively, these results indicate that placing adv@Nap in situ immediately after tumor resection can induce tumor antigen-specific immune responses by activating innate and adaptive immunity, while inducing persistent immune memory and ultimately inhibiting postoperative tumor recurrence and metastasis.

[0100] Example 4 The in situ continuous release of adv@Nap starting from intraoperative can cause significant systemic activation of the type I interferon pathway and immune chemotaxis

[0101] Recombinant adenovirus vectors can stimulate plasmacytoid dendritic cells to secrete IFN-α and IFN-β in vitro and in vivo, thereby inducing the activation of the type I interferon pathway. We also found that the release of adv from adv@Nap can stimulate the activation of plasmacytoid dendritic cells( Figure 5 H). Therefore, we hypothesized that the in situ continuous release of adv from adv@Nap starting from intraoperative controls postoperative tumor recurrence by activating the type I interferon pathway. To this end, we detected the levels of type I interferon effector molecules in the sera of mice in all groups. The results showed that the contents of IFN-α and IFN-β in the adv@Nap group were significantly higher than those in the adv group on the 3rd and 14th days after surgery, indicating that the type I interferon pathway in the adv@Nap group was continuously activated( Figure 9A-9D). In addition, we also detected a variety of chemokines that recruit immune cells. IL-15 plays an important role in maintaining the function and development of NK cells and memory CD8+ T cells. Both CXCL-9 and CXCL-10 can bind to CXCR3, thereby recruiting and regulating immune cells, including effector T cells, NK cells, dendritic cells (DCs), and macrophages, etc. Enzyme-linked immunosorbent assays (ELISAs) showed that, compared with the adv group, when used on the 3rd and 14th days after surgery, the adv@Nap group significantly promoted the release of IL-15, CXCL-9, and CXCL-10( Figure 9 E-9J). To further verify the necessity of the type I interferon pathway for adv@Nap to control tumor recurrence, an IFNAR inhibitory antibody was used to block this pathway. The results showed that the protective effect of adv@Nap against tumor recurrence was weakened( Figure 9 K). In addition, we used blocking antibodies against NK cells, CD4+ T cells, and CD8+ T cells to confirm that these immune cells are essential for the hydrogel adv@Nap to successfully control tumor recurrence( Figure 9 L). To illustrate the clinical translation potential of the hydrogel adv@Nap, we demonstrated that adv@Nap stored at 4°C for one week had the same efficacy as newly prepared adv@Nap in controlling postoperative tumor recurrence( Figure 9 M). Taken together, these results indicate that starting with the in-situ placement of the adv@Nap hydrogel during surgery can induce systemic activation and chemotaxis of the type I interferon pathway, and the type I interferon pathway and its downstream immune cells are indispensable for inhibiting tumor recurrence.

[0102] Example 5 The hydrogel adv@Nap for controlling postoperative tumor recurrence requires in-situ placement starting from surgery

[0103] To verify that in-situ placement of adv@Nap during surgery is necessary for controlling recurrence and activating the immune response, we used two other common administration methods: subcutaneous placement of the adv@Nap gel and intravenous injection of three equal doses of the adv solution( Figure 10 A). The survival curves of the mice clearly showed that placing the adv@Nap gel at the resection site had the greatest effect on prolonging the survival of the mice compared with other treatment methods( Figure 10 B). Flow cytometry analysis showed that the number of innate immune cells in the in-situ administration group was significantly greater than that in other groups( Figure 10C-10E and 11). We also evaluated the proportions of cells related to adaptive immunity. The results showed that, compared with the other two groups, activated CD8+ T (CD69+) cells, cytotoxic CD8+ T (IFNγ+) cells, and central memory CD8+ T cells (Ly6C+CD62L+) were significantly enriched in the in-situ treatment group, while there was no differential enrichment of activated CD4+ T cells (CD69+) ( Figure 5 F-5H and S6). The concentrations of IFN-α and IFN-β in the sera of mice in the in-situ treatment group were also higher ( Figure 10 I and 10J). These results indicate that the adv@Nap gel requires in-situ operation to induce the type I interferon pathway, activate innate and adaptive immune responses, form immune memory, and ultimately control tumor recurrence.

[0104] Notably, we also demonstrated the necessity of placing the adv@Nap gel immediately after surgical resection of the tumor ( Figure 10 K). The survival time of mice with the adv@Nap gel placed in-situ on the 7th day after tumor resection was significantly shorter than that of mice with the adv@Nap gel placed immediately after surgery ( Figure 10 L). In terms of immune system activation, as expected, on the 7th day after surgery, the levels of innate immunity, adaptive immunity, and immune memory were significantly reduced after the adv@Nap gel was placed in-situ ( Figure 10 M-10T and 12). The cytokine levels of the type I interferon pathway also decreased significantly ( Figure 10 U and 10V). These results suggest that timely placement of the adv@Nap gel during surgery is crucial for its efficacy.

[0105] Example 6 Hydrogels loaded with oncolytic herpes simplex virus (HSV) or oncolytic vaccinia virus (VV) can also control tumor recurrence after surgery

[0106] In addition to adenovirus adv, HSV and VV are the other two oncolytic viruses most studied in clinical trials. In preclinical and clinical studies, they have been shown to induce immune activation. To demonstrate that the ability of hydrogels loaded with oncolytic viruses to control tumor recurrence after surgery is not limited to adv, we performed the same treatment on a mouse model of breast cancer recurrence after surgery using a hydrogel loaded with HSV at the tumor site ( Figure 13 A). The IVIS imaging results showed that, similar to the adv@Nap gel, in-situ treatment with the HSV@Nap gel immediately after tumor resection could effectively control postoperative tumor recurrence ( Figure 13 B). The survival curves also supported these results ( Figure 13C). As before, we also measured the proportions of innate and adaptive immune cells in the spleens of mice on days 3 and 7. The results were also similar to those reported previously. On day 3 after surgery, the proportion of innate immune cells (including NK cells and DC cells) in the HSV@Nap hydrogel group was significantly higher than that in the HSV or HSV&Nap hydrogel groups when compared with the HSV or HSV&Nap hydrogel groups ( Figure 13 D, 13E, and 14). On day 7, we also tested the adaptive immunity of the mice. The proportions of activated CD4+ T cells (CD69+) and activated CD8+ T cells (CD69+) in the HSV@Nap hydrogel group were significantly increased when compared with the HSV or HSV&nap hydrogel groups ( Figure 13 F and 13G), and the proportion of cytotoxic CD8+ T cells (IFN-γ+) was also significantly increased ( Figure 13 H). Similarly, the concentrations of IFN-α and IFN-β were significantly increased under the treatment of HSV@Nap hydrogel ( Figure 13 J and 13K). These results indicate that the in-situ placement of the hydrogel HSV@Nap during surgery can effectively induce an anti-tumor immune response and control tumor recurrence. Notably, compared with the HSV&Nap group, HSV@Nap did not significantly increase the proportion of central memory CD8+ T cells, although the proportion in the HSV@Nap hydrogel group was significantly higher than that in the HSV group ( Figure 13 I). This finding was confirmed by IVIS imaging because tumor recurrence occurred in the mice treated with HSV@Nap hydrogel 4 weeks after surgery ( Figure 13 B).

[0107] In another validation test using VV instead of adv, similar to the results of adv and HSV hydrogels, the hydrogel VV@Nap loaded with VV could effectively control tumor recurrence and metastasis, significantly prolong the survival of mice, and induce the activation of innate and adaptive anti-tumor immune responses, as well as the type I interferon pathway ( Figure 13 L-13U and 15). Specifically, VV@Nap significantly increased the proportion of central memory CD8+ T cells (Ly6C+CD62L+) in mice ( Figure 13 S), indicating that VV@Nap is more effective than HSV@Nap in promoting immune memory and controlling postoperative tumor recurrence and metastasis. These results suggest that the in-situ placement of the hydrogel loaded with oncolytic virus during surgery can induce immune activation and effectively control tumor recurrence and metastasis. These results greatly expand the application prospects of the hydrogel loaded with oncolytic virus in anti-tumor immunotherapy.

[0108] Example 7 Humanized mouse model demonstrated the clinical translation potential of the hydrogel adv@Nap in controlling tumor recurrence

[0109] Oncolytic virus drugs with good therapeutic effects need to be experimented on humanized models before they can be applied to clinical research. For this purpose, we inoculated severe immunodeficient NCG mice with the human triple-negative breast cancer cell line MDA-MB-231 and transplanted human peripheral blood mononuclear cells (hPBMCs) to construct a tumor model of humanized immune system mice ( Figure 16 A). The survival curve of the mice showed that the in situ placement of adv@Nap hydrogel at the beginning of the operation could significantly extend the survival time of the mice ( Figure 16 B). To confirm the successful construction of the humanized immune system in immunodeficient mice, we extracted spleen cells on the 3rd day after the operation for flow cytometry analysis. The results showed an obvious hCD45 cluster ( Figure 16 C). Immunohistochemical results also showed that hCD45+ cells were present in the spleens of mice in each group ( Figure 16 D), indicating that the humanized immune system mouse model was successfully established. Then, we analyzed the innate immune cells and adaptive immune cells of mice in each group, and the results were similar to those of previous mouse models; among them, the proportion of CD11b+CD45+ cells in the adv@Nap group was significantly higher than that in other groups, indicating that the proportion of monocytes and macrophages in the adv@Nap group was higher, that is, the adv@Nap group had a richer type of innate immune cells ( Figure 16 E). In addition, the analysis of the adaptive immune cell group of mice on the 7th day after the operation showed that the proportions of CD8+ T cells and cytotoxic CD8+ T cells (GZMB+ or IFNγ+) in the adv@Nap group were significantly higher than those in the adv or adv&Nap groups ( Figure 16 F-16H). As expected, the type I interferon pathway in the adv@Nap group was also significantly activated, as confirmed by the cytokines IFN-α and IFN-β ( Figure 16 I and 16J). These results indicate that in the mouse tumor model with humanized immune system, the in situ placement of adv@Nap hydrogel at the beginning of the operation can induce anti-tumor immune response and the activation of type I interferon pathway, and control tumor recurrence after the operation. These findings demonstrate the potential of the hydrogel OVs@Nap loaded with oncolytic virus in clinical applications and provide a basis for further clinical translational research.

[0110] Example 8 Preliminary safety evaluation of the hydrogel adv@Nap

[0111] To confirm the clinical translation ability, we evaluated the in vivo safety of the hydrogel adv@Nap. Hematological examinations were performed on the whole blood of mice, and all parameters were within the normal range ( Figure 17 A). In addition, we also evaluated its systemic toxicity, including liver toxicity and kidney toxicity. There were no significant differences in the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) among the groups (Figure 17 B). Additionally, histological examinations of major organs by hematoxylin and eosin (H&E) staining revealed no obvious toxicity among groups. Figure 17 C). Monitoring of body weight confirmed that the mice maintained homeostasis throughout the experiment. Figure 17 D). These results indicate that the in situ placement of adv@Nap hydrogel during surgery can induce in situ prolonged immune activation, control postoperative tumor recurrence, and have no obvious toxic effects in mice, confirming its in vivo safety and laying a foundation for further clinical translation.

[0112] Example 9 Effects of various hydrogels loaded with different oncolytic viruses on controlling tumor recurrence after surgery Various hydrogels loaded with different oncolytic viruses and based on polypeptides: hydrogels based on polypeptides loaded with oncolytic adenovirus (Type V adenovirus), hydrogels based on polypeptides loaded with oncolytic orthoreovirus, hydrogels based on polypeptides loaded with coxsackievirus A21, hydrogels based on polypeptides loaded with Newcastle disease virus (NDV Herts / 33 strain), hydrogels based on polypeptides loaded with oncolytic measles virus (Edmonston strain), hydrogels based on polypeptides loaded with vesicular stomatitis virus (Mudd-Summer strain, Indian serotype), hydrogels based on polypeptides loaded with poliovirus type 1 (sabin strain), hydrogels based on polypeptides loaded with herpes simplex virus type 1 (strain F), hydrogels based on polypeptides loaded with vaccinia virus (Elstree strain). All of these various hydrogels loaded with different oncolytic viruses can significantly control tumor recurrence after surgery and inhibit distant tumor metastasis.

[0113] Collagen-based hydrogels loaded with different oncolytic viruses: Collagen-based hydrogels loaded with oncolytic adenovirus (Type V adenovirus), collagen-based hydrogels loaded with oncolytic orthoreovirus, collagen-based hydrogels loaded with coxsackievirus A21, collagen-based hydrogels loaded with NDV Herts / 33 strain, collagen-based hydrogels loaded with oncolytic measles virus Edmonston strain, collagen-based hydrogels loaded with oncolytic vesicular stomatitis virus (Mudd-Summer strain, Indiana serotype), collagen-based hydrogels loaded with poliovirus type 1, sabin strain, collagen-based hydrogels loaded with herpes simplex virus type 1, strain F, collagen-based hydrogels loaded with vaccinia virus (Elstree strain). These various hydrogels loaded with different oncolytic viruses can significantly control tumor recurrence after surgery and inhibit distant tumor metastasis.

[0114] Gelatin-based hydrogels loaded with different oncolytic viruses: Gelatin-based hydrogels loaded with oncolytic adenovirus (Type V adenovirus), gelatin-based hydrogels loaded with oncolytic orthoreovirus, gelatin-based hydrogels loaded with coxsackievirus A21, gelatin-based hydrogels loaded with Newcastle disease virus (NDV Herts / 33 strain), gelatin-based hydrogels loaded with oncolytic measles virus (Edmonston strain), gelatin-based hydrogels loaded with vesicular stomatitis virus (Mudd-Summer strain, Indiana serotype), gelatin-based hydrogels loaded with poliovirus type 1 (sabin strain), gelatin-based hydrogels loaded with herpes simplex virus type 1 (strain F), gelatin-based hydrogels loaded with vaccinia virus (Elstree strain). These various hydrogels loaded with different oncolytic viruses can significantly control tumor recurrence after surgery and inhibit distant tumor metastasis.

[0115] Hyaluronic acid-based hydrogels loaded with different oncolytic viruses: hyaluronic acid-based hydrogels loaded with oncolytic adenovirus (Type V adenovirus), hyaluronic acid-based hydrogels loaded with oncolytic orthoreovirus, hyaluronic acid-based hydrogels loaded with coxsackievirus A21, hyaluronic acid-based hydrogels loaded with Newcastle disease virus (NDV Herts / 33 strain), hyaluronic acid-based hydrogels loaded with oncolytic measles virus (Edmonston strain), hyaluronic acid-based hydrogels loaded with vesicular stomatitis virus (Mudd-Summer strain, Indiana serotype), hyaluronic acid-based hydrogels loaded with poliovirus type 1 (sabin strain), hyaluronic acid-based hydrogels loaded with herpes simplex virus type 1 (strain F), hyaluronic acid-based hydrogels loaded with vaccinia virus (Elstree strain). These various hydrogels loaded with different oncolytic viruses can significantly control tumor recurrence after surgery and inhibit distant tumor metastasis.

[0116] Alginate-based hydrogels loaded with different oncolytic viruses: Alginate-based hydrogels loaded with oncolytic adenovirus (Type V adenovirus), alginate-based hydrogels loaded with oncolytic orthoreovirus, alginate-based hydrogels loaded with coxsackievirus A21, alginate-based hydrogels loaded with Newcastle disease virus (NDV Herts / 33 strain), alginate-based hydrogels loaded with oncolytic measles virus (Edmonston strain), alginate-based hydrogels loaded with oncolytic vesicular stomatitis virus (Mudd-Summer strain, Indiana serotype), alginate-based hydrogels loaded with poliovirus type 1 (sabin strain), alginate-based hydrogels loaded with herpes simplex virus type 1 (strain F), alginate-based hydrogels loaded with vaccinia virus (Elstree strain). These various hydrogels loaded with different oncolytic viruses can significantly control tumor recurrence after surgery and inhibit distant tumor metastasis.

[0117] Fibrin-based hydrogels loaded with different oncolytic viruses: fibrin-based hydrogels loaded with oncolytic adenovirus (Type V adenovirus), fibrin-based hydrogels loaded with oncolytic orthoreovirus, fibrin-based hydrogels loaded with Coxsackievirus A21, fibrin-based hydrogels loaded with NDV Herts / 33 strain, fibrin-based hydrogels loaded with Oncolytic measles virus Edmonston strain, fibrin-based hydrogels loaded with Vesicular stomatitis virus (Mudd-Summer strain, Indiana serotype), fibrin-based hydrogels loaded with Poliovirus type 1, sabin strain, fibrin-based hydrogels loaded with Herpes simplex virus type 1, strain F, fibrin-based hydrogels loaded with Vaccinia virus (Elstree strain). These various hydrogels loaded with different oncolytic viruses can significantly control tumor recurrence after surgery and inhibit distant tumor metastasis.

[0118] Chitosan-based hydrogels loaded with different oncolytic viruses: chitosan-based hydrogels loaded with oncolytic adenovirus (Type V adenovirus), chitosan-based hydrogels loaded with oncolytic orthoreovirus, chitosan-based hydrogels loaded with Coxsackievirus A21, chitosan-based hydrogels loaded with NDV Herts / 33 strain, chitosan-based hydrogels loaded with Oncolytic measles virus Edmonston strain, chitosan-based hydrogels loaded with Mudd-Summer strain, Indiana serotype of oncolytic vesicular stomatitis virus, chitosan-based hydrogels loaded with Poliovirus type 1, sabin strain, chitosan-based hydrogels loaded with Herpes simplex virus type 1, strain F, chitosan-based hydrogels loaded with Elstree strain of oncolytic vaccinia virus. These various hydrogels loaded with different oncolytic viruses can significantly control tumor recurrence after surgery and inhibit distant tumor metastasis.

[0119] Agarose-based hydrogels loaded with different oncolytic viruses: agarose-based hydrogels loaded with oncolytic adenovirus (Type V adenovirus), agarose-based hydrogels loaded with oncolytic orthoreovirus, agarose-based hydrogels loaded with coxsackievirus A21, agarose-based hydrogels loaded with Newcastle disease virus (NDV Herts / 33 strain), agarose-based hydrogels loaded with oncolytic measles virus (Edmonston strain), agarose-based hydrogels loaded with oncolytic vesicular stomatitis virus (Mudd-Summer strain, Indiana serotype), agarose-based hydrogels loaded with poliovirus type 1 (sabin strain), agarose-based hydrogels loaded with herpes simplex virus type 1 (strain F), agarose-based hydrogels loaded with vaccinia virus (Elstree strain). These various hydrogels loaded with different oncolytic viruses can significantly control tumor recurrence after surgery and inhibit distant tumor metastasis.

[0120] Hydrogels based on polyethylene glycol or polycaprolactone loaded with different oncolytic viruses: hydrogels based on polyethylene glycol or polycaprolactone loaded with oncolytic adenovirus (Type V adenovirus), hydrogels based on polyethylene glycol or polycaprolactone loaded with oncolytic orthoreovirus, hydrogels based on polyethylene glycol or polycaprolactone loaded with coxsackievirus A21, hydrogels based on polyethylene glycol or polycaprolactone loaded with NDV Herts / 33 strain, hydrogels based on polyethylene glycol or polycaprolactone loaded with oncolytic measles virus Edmonston strain, hydrogels based on polyethylene glycol or polycaprolactone loaded with oncolytic vesicular stomatitis virus (Mudd-Summer strain, Indiana serotype), hydrogels based on polyethylene glycol or polycaprolactone loaded with poliovirus type 1, sabin strain, hydrogels based on polyethylene glycol or polycaprolactone loaded with herpes simplex virus type 1, strain F, hydrogels based on polyethylene glycol or polycaprolactone loaded with vaccinia virus (Elstree strain). These various hydrogels loaded with different oncolytic viruses can significantly control tumor recurrence after surgery and inhibit distant tumor metastasis.

[0121] Hydrogels based on polyvinyl alcohol loaded with different oncolytic viruses: hydrogels based on polyvinyl alcohol loaded with oncolytic adenovirus (Type V adenovirus), hydrogels based on polyvinyl alcohol loaded with oncolytic orthoreovirus, hydrogels based on polyvinyl alcohol loaded with coxsackievirus A21, hydrogels based on polyvinyl alcohol loaded with Newcastle disease virus (NDV Herts / 33 strain), hydrogels based on polyvinyl alcohol loaded with oncolytic measles virus (Edmonston strain), hydrogels based on polyvinyl alcohol loaded with oncolytic vesicular stomatitis virus (Mudd-Summer strain, Indiana serotype), hydrogels based on polyvinyl alcohol loaded with poliovirus type 1 (sabin strain), hydrogels based on polyvinyl alcohol loaded with herpes simplex virus type 1 (strain F), hydrogels based on polyvinyl alcohol loaded with vaccinia virus (Elstree strain). These various hydrogels loaded with different oncolytic viruses can significantly control tumor recurrence after surgery and inhibit distant tumor metastasis.

[0122] Hydrogels based on sodium carboxymethyl cellulose loaded with different oncolytic viruses: hydrogels based on sodium carboxymethyl cellulose loaded with oncolytic adenovirus (Type V adenovirus), hydrogels based on sodium carboxymethyl cellulose loaded with oncolytic orthoreovirus, hydrogels based on sodium carboxymethyl cellulose loaded with Coxsackievirus A21, hydrogels based on sodium carboxymethyl cellulose loaded with NDV Herts / 33 strain, hydrogels based on sodium carboxymethyl cellulose loaded with oncolytic measles virus Edmonston strain, hydrogels based on sodium carboxymethyl cellulose loaded with oncolytic vesicular stomatitis virus (Mudd-Summer strain, Indiana serotype), hydrogels based on sodium carboxymethyl cellulose loaded with Poliovirus type 1, sabin strain, hydrogels based on sodium carboxymethyl cellulose loaded with Herpes simplex virus type 1, strain F, hydrogels based on sodium carboxymethyl cellulose loaded with Vaccinia virus Elstree strain. These various hydrogels loaded with different oncolytic viruses can significantly control tumor recurrence after surgery and inhibit distant tumor metastasis.

[0123] Hydrogels based on polyacrylamide or poly(N-isopropylacrylamide) loaded with different oncolytic viruses: hydrogels based on polyacrylamide or poly(N-isopropylacrylamide) loaded with oncolytic adenovirus (Type V adenovirus), hydrogels based on polyacrylamide or poly(N-isopropylacrylamide) loaded with oncolytic orthoreovirus, hydrogels based on polyacrylamide or poly(N-isopropylacrylamide) loaded with coxsackievirus A21, hydrogels based on polyacrylamide or poly(N-isopropylacrylamide) loaded with Newcastle disease virus (NDV Herts / 33 strain), hydrogels based on polyacrylamide or poly(N-isopropylacrylamide) loaded with oncolytic measles virus (Edmonston strain), hydrogels based on polyacrylamide or poly(N-isopropylacrylamide) loaded with oncolytic vesicular stomatitis virus (Mudd-Summer strain, Indianas erotype), hydrogels based on polyacrylamide or poly(N-isopropylacrylamide) loaded with poliovirus type 1 (sabin strain), hydrogels based on polyacrylamide or poly(N-isopropylacrylamide) loaded with herpes simplex virus type 1 (strain F), hydrogels based on polyacrylamide or poly(N-isopropylacrylamide) loaded with vaccinia virus (Elstree strain). These various hydrogels loaded with different oncolytic viruses can significantly control tumor recurrence after surgery and inhibit distant tumor metastasis.

[0124] Polyurethane-based hydrogels loaded with different oncolytic viruses: polyurethane-based hydrogels loaded with oncolytic adenovirus (Type V adenovirus), polyurethane-based hydrogels loaded with oncolytic orthoreovirus, polyurethane-based hydrogels loaded with coxsackievirus A21, polyurethane-based hydrogels loaded with Newcastle disease virus (NDV Herts / 33 strain), polyurethane-based hydrogels loaded with oncolytic measles virus (Edmonston strain), polyurethane-based hydrogels loaded with oncolytic vesicular stomatitis virus (Mudd-Summer strain, Indiana serotype), polyurethane-based hydrogels loaded with poliovirus type 1 (sabin strain), polyurethane-based hydrogels loaded with herpes simplex virus type 1 (strain F), polyurethane-based hydrogels loaded with vaccinia virus (Elstree strain). These various hydrogels loaded with different oncolytic viruses can significantly control postoperative tumor recurrence and inhibit distant tumor metastasis.

[0125] [1] Hu C, Zhang M, Wu J, et al. Bisphosphonate-Modified Functional Supramolecular Hydrogel Promotes Periodontal Bone Regeneration by Osteoclast Inhibition[J]. ACS Appl Mater Interfaces, 2023.

[0126] [2] Wu J, Lu A D, Zhang L P, et al. [Study of clinical outcome and prognosis in pediatric core binding factor-acute myeloid leukemia][J]. Zhonghua Xue Ye Xue Za Zhi, 2019, 40(1): 52-7.

[0127] [3]Davidenko N, Schuster C F, Bax D V, et al. Evaluation of cell binding to collagen and gelatin: a study of the effect of 2D and 3D architecture and surface chemistry[J]. J Mater Sci Mater Med, 2016, 27(10): 148.

[0128] [4]Ding L, QiQ, Zhang S, et al. Hydroxypropyl methylcellulose reinforced collagen / PVA composite hydrogel wound dressing with self-adaptive, hemostasis and antibacterial ability for wound

[0129] healing[J]. Int J Biol Macromol, 2025, 304(Pt 2): 140811.

[0130] [5]Nichol J W, Koshy S T, Bae H, et al. Cell-laden microengineered gelatin methacrylate hydrogels

[0131] [J]. Biomaterials, 2010, 31(21): 5536 - 44.

[0132] [6]Griesser J, Hetényi G, Bernkop-Schnürch A. Thiolated Hyaluronic Acid as Versatile

[0133] Mucoadhesive Polymer: From the Chemistry Behind to Product Developments - What Are the Capabilities?[J]. Polymers(Basel), 2018, 10(3).

[0134] [7]Yang X,Wang B,Peng D D,et al.Hyaluronic Acid-Based InjectableHydrogels for Wound

[0135] Dressing and Localized Tumor Therapy:A Review[J].ADVANCED NANOBIOMEDRESEARCH,

[0136] 2022,2(12).

[0137] [8]Lee K Y,Mooney D J.Alginate:properties and biomedicalapplications[J].Prog Polym Sci,2012,

[0138] 37(1):106-26.

[0139] [9]LiY,Meng H,Liu Y,et al.Fibrin gel as an injectable biodegradablescaffold and cellcarrier for tissue engineering[J].ScientificWorldJournal,2015,2015:685690.

[0140]

[10] Lai J H,Kong D X,Liu Y T,et al.A Flexible Fibrin-Based Platformfor Driving One-Day Self-

[0141] Assembly of Millimeter-Sized Cell-Rich Spheroids and TheirApplications:An Old Materialwith

[0142] New Tricks[J].ADVANCED FUNCTIONAL MATERIALS,2024,34(39).

[0143]

[11] Fiamingo A, Montembault A, Boitard SE, et al. Chitosan Hydrogels for the Regeneration of Infarcted Myocardium: Preparation, Physicochemical Characterization, and Biological

[0144] Evaluation[J]. Biomacromolecules, 2016, 17(5): 1662 - 72.

[0145]

[12] Zhang Q, Duan SY, Huo ZY, et al. New Dressings Based on Chitosan and Its Application[J].

[0146] PROGRESS IN CHEMISTRY, 2023, 35(10): 1450 - 60.

[0147]

[13] Zareei A, Jiang H, Chittiboyina S, et al. A lab - on - chip ultrasonic platform for real - time and

[0148] nondestructive assessment of extracellular matrix stiffness[J]. LabChip, 2020, 20(4): 778 - 88.

[14] Alegre - Sánchez A, Bernárdez C. A new nonhydrophilic agarose gel as subdermal filler for facial

[0149] rejuvenation: Aesthetic results and patient satisfaction[J]. J Cosmet Dermatol, 2020, 19(8): 1900 - 6.

[0151]

[15] Della Sala F, Biondi M, Guarnieri D, et al. Mechanical behavior of bioactive poly(ethylene glycol)

[0152] diacrylate matrices for biomedical application[J].J Mech Behav BiomedMater,2020,110:103885.

[0153]

[16] Zhang S, Zhao X, Yang W.[Bacterial Blocking and Repair ofIntestinalDefects With Well-Alighed

[0154] Lamellar MXene / Polyvinyl Alcohol Hydrogels Prepared by BidirectionalFreezing Method][J].

[0155] Sichuan da xue xue bao Yi xue ban=Journal of Sichuan University Medical science edition,

[0156] 2024,55(4):838-44.

[0157]

[17] Dong L, Wang SJ, Zhao XR, et al. 3D-Printed Poly(ε-caprolactone)Scaffold Integrated with Cell-laden Chitosan Hydrogels for Bone TissueEngineering[J]. Sci Rep, 2017, 7(1):13412.

[0158]

[18] Wu Jing, Yang Kuan, Yan Xiaoyan, et al. Application of natural polysaccharide-based self-healing hydrogels in the treatment of diabetic wounds[J]. Polymer Bulletin: 1-20.

[0159]

[19] Bao D, Chen M, Wang H, et al. Preparation and characterization of double crosslinked hydrogel films from carboxymethylchitosan and carboxymethylcellulose[J]. Carbohydr Polym, 2014, 110: 113-20.

[0160]

[20] Tian ZH, Zhao WJ, Du QY, et al. Construction of collagen / sodium carboxymethyl cellulose oxide / zinc oxide nanocomposite hydrogels[J]. China Leather, 2022, 51(05): 9-17+21.

[0161]

[21] Trappmann B, Gautrot JE, Connelly JT, et al. Extracellular-matrix tethering regulates stem-cell fate[J]. Nat Mater, 2012, 11(7): 642-9.

[22] Bei Z, Ye L, Tong Q, et al. Thermostimulated shrinking and adhesive hydrogel dressing for treating chronic diabetic wounds[J]. Cell Reports Physical Science, 2024, 5(11).

[0162]

[23] Farrukh A, Zhao SF, Del Campo A. Microenvironments Designed to Support Growth and Function of Neuronal Cells[J]. FRONTIERS IN MATERIALS, 2018, 5.

[0163]

[24] Han L, Yan HH, Mei GH, et al. Preparation and application of a hydrophilic traditional Chinese medicine polyurethane dressing for general surgery[J]. Adhesion, 2025, 52(01): 94-7.

Claims

1. A novel hydrogel loaded with oncolytic virus.

2. The novel hydrogel loaded with oncolytic virus according to claim 1, characterized in that: The oncolytic virus is one or a combination of several of oncolytic adenovirus, oncolytic herpes simplex virus, oncolytic vaccinia virus, oncolytic reovirus, oncolytic coxsackievirus, oncolytic Newcastle disease virus, oncolytic measles virus, oncolytic vesicular stomatitis virus, or oncolytic poliovirus.

3. The novel hydrogel loaded with oncolytic virus according to claim 1, characterized in that: The hydrogel is one or a combination of several of collagen hydrogel, gelatin hydrogel, hyaluronic acid hydrogel, alginate hydrogel, fibrin hydrogel, chitosan hydrogel, agarose hydrogel, polypeptide hydrogel, polyethylene glycol, polyvinyl alcohol, polycaprolactone, sodium carboxymethyl cellulose, polyacrylamide, poly(N-isopropylacrylamide), polyurethane.

4. The novel hydrogel loaded with oncolytic virus according to claim 2, wherein: The hydrogel is one or a combination of several of collagen hydrogel, gelatin hydrogel, hyaluronic acid hydrogel, alginate hydrogel, fibrin hydrogel, chitosan hydrogel, agarose hydrogel, polypeptide hydrogel, polyethylene glycol, polyvinyl alcohol, polycaprolactone, sodium carboxymethyl cellulose, polyacrylamide, poly(N-isopropylacrylamide), polyurethane.

5. A novel hydrogel loaded with oncolytic virus according to any one of claims 1-4, characterized in that: The hydrogel is a self-assembling polypeptide hydrogel.

6. The novel hydrogel loaded with oncolytic virus according to claim 5, characterized in that: The self-assembling polypeptide is NapGFFYK.

7. Use of a novel hydrogel loaded with oncolytic virus according to any one of claims 1-4 in the preparation of a drug or preparation for controlling postoperative recurrence of tumors or inhibiting distant metastasis of tumors.

8. Use of a novel hydrogel loaded with oncolytic virus according to claim 5 in the preparation of a drug or preparation for controlling postoperative recurrence of tumors or inhibiting distant metastasis of tumors.

9. Use of a novel hydrogel loaded with oncolytic virus according to claim 6 in the preparation of a drug or preparation for controlling postoperative recurrence of tumors or inhibiting distant metastasis of tumors.

10. Use of a novel hydrogel loaded with oncolytic virus in the preparation of a drug or preparation for controlling postoperative recurrence of tumors or inhibiting distal metastasis of tumors, characterized in that: The tumor is breast cancer, liver cancer, colorectal cancer, lung cancer, melanoma, pancreatic cancer, glioma, or bladder cancer.

11. Use of a novel hydrogel loaded with oncolytic virus in the preparation of a drug or preparation for controlling postoperative recurrence of tumors or inhibiting distant metastasis of tumors, characterized in that: The tumor is breast cancer, liver cancer, colorectal cancer, lung cancer, melanoma, pancreatic cancer, glioma, or bladder cancer.

12. Use of a novel hydrogel loaded with oncolytic virus in the preparation of a drug or preparation for controlling postoperative recurrence of tumors or inhibiting distal metastasis of tumors, characterized in that: The tumor is breast cancer, liver cancer, colorectal cancer, lung cancer, melanoma, pancreatic cancer, glioma, or bladder cancer.

13. Use of a novel hydrogel loaded with oncolytic virus in the preparation of a drug or preparation for controlling postoperative recurrence of tumors or inhibiting distal metastasis of tumors, characterized in that: The administration mode of the drug or preparation is in-situ administration during surgery.

14. Use of a novel hydrogel loaded with oncolytic virus in the preparation of a drug or preparation for controlling postoperative recurrence of tumors or inhibiting distal metastasis of tumors, characterized in that: The administration mode of the drug or preparation is in-situ administration during surgery.

15. Use of a novel hydrogel loaded with oncolytic virus in the preparation of a drug or preparation for controlling postoperative recurrence of tumors or inhibiting distal metastasis of tumors, characterized in that: The administration mode of the drug or preparation is in-situ administration during surgery.

16. Use of a novel hydrogel loaded with oncolytic virus in the preparation of a drug or preparation for controlling postoperative recurrence of tumors or inhibiting distal metastasis of tumors, characterized in that: The administration timing of the drug or preparation is immediate administration after tumor resection.

17. Use of a novel hydrogel loaded with oncolytic virus in the preparation of a drug or preparation for controlling postoperative recurrence of tumors or inhibiting distant metastasis of tumors, characterized in that: The administration timing of the drug or preparation is immediate administration after tumor resection.

18. Use of a novel hydrogel loaded with oncolytic virus in the preparation of a drug or preparation for controlling postoperative recurrence of tumors or inhibiting distal metastasis of tumors, characterized in that: The administration timing of the drug or preparation is immediate administration after tumor resection.

19. Use of a novel hydrogel loaded with oncolytic virus in the preparation of a drug or preparation for controlling postoperative recurrence of tumors or inhibiting distal metastasis of tumors, characterized in that: The novel hydrogel loaded with oncolytic virus activates the type I interferon pathway, induces innate and adaptive immune responses, and generates immune memory.

20. Use of a novel hydrogel loaded with oncolytic virus in the preparation of a drug or preparation for controlling postoperative recurrence of tumors or inhibiting distal metastasis of tumors, characterized in that: The novel hydrogel loaded with oncolytic virus activates the type I interferon pathway, induces innate and adaptive immune responses, and generates immune memory.

21. Use of a novel hydrogel loaded with oncolytic virus in the preparation of a drug or preparation for controlling postoperative recurrence of tumors or inhibiting distal metastasis of tumors, characterized in that: The novel hydrogel loaded with oncolytic virus activates the type I interferon pathway, induces innate and adaptive immune responses, and generates immune memory.

22. Use of a novel hydrogel loaded with oncolytic virus in the preparation of a drug or preparation for controlling postoperative recurrence of tumors or inhibiting distal metastasis of tumors, characterized in that: The novel hydrogel loaded with oncolytic virus activates the type I interferon pathway, induces innate and adaptive immune responses, and generates immune memory.

23. Use of a novel hydrogel loaded with oncolytic virus in the preparation of a drug or preparation for controlling postoperative recurrence of tumors or inhibiting distant metastasis of tumors, characterized in that: The novel hydrogel loaded with oncolytic virus activates the type I interferon pathway, induces innate and adaptive immune responses, and generates immune memory.

24. Use of a novel hydrogel loaded with oncolytic virus in the preparation of a drug or preparation for controlling postoperative recurrence of tumors or inhibiting distal metastasis of tumors, characterized in that: The novel hydrogel loaded with oncolytic virus activates the type I interferon pathway, induces innate and adaptive immune responses, and generates immune memory.

Images