Composition and hydrogel for tumor targeted therapy as well as preparation method and application of composition and hydrogel

Abstract

The invention belongs to the technical field of biology, and particularly relates to a composition for tumor targeted therapy, hydrogel and a preparation method and application thereof. The tumor targeted therapy composition provided by the invention comprises the aging tumor cells and the celecoxib, the immune activation function of the aging tumor cells is that STCs can effectively activate the maturation of dendritic cells and the immune function of T cells and NK cells, SASP of the STCs is regulated through the celecoxib, especially generation of prostaglandin E2 is inhibited, and the tumor targeted therapy effect is improved. And the combined use further enhances the anti-tumor immune effect.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to a composition for tumor targeted therapy, a hydrogel, and a preparation method and application thereof. Background Art

[0002] Tumor vaccines primarily utilize tumor-associated and tumor-specific antigens to activate the patient's innate and adaptive immune systems to eliminate tumor cells and inhibit tumor growth. However, the overall clinical efficacy of tumor vaccines is unsatisfactory, with the expected immune response observed only in a small number of patients. Researchers have attempted different strategies for tumor vaccine development, including vaccines based on recombinant antigenic peptides, proteins, RNA / DNA, or whole tumor cells (WTC).

[0003] Among these, the WTC vaccine represents a promising research and development approach. Compared to single-epitope antigen / peptide vaccines, the WTC vaccine expresses a complete antigenic spectrum, eliminating the need to recognize dominant epitopes and reducing the risk of tumor immune escape. However, its weak immunogenicity results in a low objective response rate and limited improvement in patient survival. The therapeutic efficacy of the WTC vaccine still needs to be improved.

[0004] Therefore, the development and research of anti-tumor immunotherapy is particularly important. Summary of the Invention

[0005] Based on this, one embodiment of the present application provides a composition for tumor targeted therapy, a hydrogel, and a preparation method and application thereof.

[0006] In one aspect, the present application provides a composition for tumor targeted therapy, comprising STCs and celecoxib.

[0007] In one embodiment, the composition comprises STCs and celecoxib liposomes.

[0008] On the other hand, the present application provides a hydrogel encapsulating STCs, comprising a hydrogel matrix and STCs and celecoxib liposomes encapsulated in the hydrogel matrix.

[0009] In one embodiment, the STCs concentration is 1.8×10 6 pcs / L~2.2×10 6 Pieces / L.

[0010] In one embodiment, the concentration of the celecoxib liposomes is 0.35 mg / mL to 0.45 mg / mL based on celecoxib.

[0011] In one embodiment, the STCs include pigment tumor cells.

[0012] In one embodiment, the pigment tumor cells include mouse melanoma cells.

[0013] In one embodiment, the mouse melanoma cells include B16-F10 cells.

[0014] In one embodiment, the particle size of the celecoxib liposome is 58 nm to 62 nm.

[0015] In one embodiment, the hydrogel matrix comprises a cross-linked product of chitosan and a gelling agent.

[0016] In one embodiment, the gelling agent includes 0.04M to 0.06M sodium bicarbonate.

[0017] On the other hand, the present application provides a vaccine, which includes the above-mentioned tumor-targeted therapy composition.

[0018] In one embodiment, the vaccine is in the form of a gel.

[0019] In one embodiment, the gel is a hydrogel.

[0020] Another aspect of the present application provides a method for preparing a hydrogel containing STCs, comprising:

[0021] STCs, celecoxib liposomes and gel raw materials are mixed, and the gel raw materials are cross-linked to form a hydrogel matrix encapsulating the STCs and the celecoxib liposomes, thereby preparing a hydrogel encapsulating STCs.

[0022] In one embodiment, the method for preparing STCs includes: co-culturing tumor cells with a senescence inducer to prepare STCs.

[0023] In one embodiment, the senescence-inducing agent comprises doxorubicin.

[0024] In one embodiment, the co-culture time is 44 hours to 52 hours.

[0025] In one embodiment, the method for preparing the celecoxib liposomes comprises: preparing the celecoxib liposomes by a thin film hydration method.

[0026] In one embodiment, the raw materials of the celecoxib liposomes include celecoxib and one or more of egg yolk lecithin, cholesterol and DSPE-MPEG2000.

[0027] In one embodiment, the mass ratio of the egg yolk lecithin, the cholesterol, the DSPE-MPEG2000 and the celecoxib is (44-46):(4-6):(5-2.5):(0.5-5).

[0028] In one embodiment, the thin film hydration method comprises:

[0029] The raw materials of celecoxib liposomes are dissolved in an organic solvent, the organic solvent is evaporated to dryness by rotary evaporation, and then an aqueous phase is added and shaken to hydrate and detach the lipid film to produce a liposome dispersion; and the liposome dispersion is subjected to ultrasound and membrane extrusion to prepare celecoxib liposomes.

[0030] In one embodiment, in the thin film hydration method, the organic solvent includes chloroform and methanol in a volume ratio of (1-3):1.

[0031] In one embodiment, in the thin film hydration method, the temperature of the rotary evaporation method is 40°C to 45°C.

[0032] In one embodiment, in the thin film hydration method, the ultrasonic parameters include: power of 260W to 280W, time of 10min to 20min; and

[0033] In one embodiment, in the thin film hydration method, the membrane extrusion parameters include: using a polycarbonate membrane with a thickness of 90nm to 110nm and the number of extrusions is 10 to 12 times.

[0034] On the other hand, the present application provides the use of the above-mentioned tumor-targeted therapy composition, the above-mentioned STCs-encapsulated hydrogel or the above-mentioned vaccine in the preparation of a drug for preventing postoperative tumor recurrence and / or metastasis.

[0035] In one embodiment, in the thin film hydration method, the tumor comprises melanoma.

[0036] The details of one or more embodiments of the present application are set forth in the description below, and other features, objects, and advantages of the application will become apparent from the description and from the claims thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0038] Figure 1 is the determination result of the CLX liposome concentration standard curve; Figure 1 A in the figure is the result of CLX concentration determination; Figure 1 B in the figure is the CLX concentration standard curve;

[0039] Figure 2 Characterization of senescent cells; Figure 2 A in the figure shows the observation of cell β-galactosidase staining; Figure 2 B in the figure is the content of ATP and HMGB1 in the culture supernatant;

[0040] Figure 3 The results of hydrogel preparation and characterization are shown; Figure 3 A in the figure is the CS@Gel preparation model diagram; Figure 3 B in the figure is the particle of CLX liposome; Figure 3 C in is the potential of CLX liposomes; Figure 3 D in the figure is a TEM image of CLX liposomes; Figure 3 E in the figure is the electron microscope image of CS@Gel; Figure 3 F in the figure is the SEM image of blank hydrogel; Figure 3 G in the equation is the rheological component of CS@Gel; Figure 3 H in it is fluorescence quantitative analysis; Figure 3 I in is the survival rate of senescent B16-F10 cells encapsulated in the hydrogel;

[0041] Figure 4 for the in vivo retention and tumor prevention effect of SCs+CLX-Lipo@Gel; Figure 4 A in the figure is the time-dependent imaging of SCs and SCs@Gel; Figure 4 B in the figure is the quantitative analysis of the mean fluorescence intensity at different time points; Figure 4 C in the figure is a schematic diagram of the tumor model and treatment plan; Figure 4 D in the figure is the tumor growth ratio statistics on day 7; Figure 4 E in the figure is the tumor growth curve; Figure 4 F in the figure is the tumor growth curve of mice in each group; Figure 4 G~I in the figure are flow cytometry analysis of peripheral blood immune cells ( Figure 4 G in the figure represents the proportion of NK cells; Figure 4 H in the figure is the proportion of NKG2D+ cells; Figure 4 I in the figure represents the proportion of NKp46+ cells); Figure 4 J~K in the figure are flow cytometry analysis of immune cells in spleen tissue ( Figure 4 J in the figure represents CD4+TEM; Figure 4 K in is CD8+TEM);

[0042] Figure 5 The anti-tumor activity of SCs+CLX-Lipo@Gel in a mouse subcutaneous melanoma tumor model; Figure 5 A in the figure is a schematic diagram of the tumor model and treatment plan; Figure 5 B in the figure is the tumor growth curve; Figure 5 C in the figure is a tumor picture; Figure 5 D in it is tumor mass analysis; Figure 5 E in the figure is the percentage of mature DCs and cDC1 in peritumoral lymph nodes; Figure 5 F in the figure is the percentage of CD4 + T cells in tumor tissue; Figure 5 G in the figure represents the percentage of CD8+T cells, activated CD8+T cells, and proliferating CD8+T cells in tumor tissue; Figure 5 H in the figure represents the percentage of NK cells and activated NK cells in tumor tissue;

[0043] Figure 6 To evaluate the effect of hydrogel on prolonging the survival of a mouse melanoma brain metastasis model; Figure 6 A in the figure is a schematic diagram of the establishment and treatment of a melanoma brain metastasis model; Figure 6 B in the figure is the survival rate of mice during the treatment period, and Figure 6 C in the figure is the median survival time of mice (n=12). DETAILED DESCRIPTION

[0044] Below in conjunction with embodiment and example, the application is described in further detail.Should be understood that these embodiment and example are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description hereinafter, a large amount of specific details are given in order to provide a more complete understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0046] the term

[0047] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0048] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0049] In this application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0050] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0051] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0052] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0053] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0054] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0055] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0056] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0057] In this application, % (w / w) and wt% both refer to weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0058] All documents mentioned in this application are cited as references in this application, just as each document is cited as a reference individually. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0059] Terminology: The "@" symbol is often used to indicate the presence or position of a component within a composite material or system, similar to the meaning of "located in" or "embedded." It is used to emphasize that one component is encapsulated, embedded, or combined with another material to form a composite structure.

[0060] In this application: CS@Gel represents chitosan-gelatin composite hydrogel; SCs@Gel represents senescent cell hydrogel; SCs+CLX-Lipo@Gel represents senescent cell and celecoxib liposome composite hydrogel.

[0061] The term "senescent tumor cells" (STCs) refers to cells that enter a senescent state as a tumor progresses due to factors such as DNA damage, telomere shortening, and oxidative stress. Senescent tumor cells promote tumor growth and metastasis by secreting senescence-associated secretory phenotype (SASP) factors, exerting a pro-tumor effect.

[0062] The term "celecoxib liposomes" refers to CLX liposomes or CLX-Lipo. Celecoxib liposomes can regulate the composition of the SASP of senescent tumor cells to circumvent their immunosuppressive effects, thereby significantly enhancing the immune activation ability of the whole tumor vaccine and preventing and inhibiting the occurrence and development of tumors.

[0063] Cellular senescence is a stress response program characterized by stable cell cycle arrest and a secretory program capable of reshaping the tissue environment. Senescent cells release a variety of proinflammatory cytokines, chemokines, extracellular matrix proteins, growth factors, matrix metalloproteinases, and exosome-like small extracellular vesicles. These complex secretory components are collectively referred to as the senescence-associated secretory phenotype (SASP). Senescent tumor cells can alter the tumor microenvironment through the SASP, recruiting a variety of immune cells to participate in the clearance of senescent cells. For example, the SASP of senescent tumor cells can recruit and activate CD4+ and CD8+ T cells, triggering anti-tumor immunity. In addition, damage-associated molecular patterns (DAMPs) released by senescent tumor cells, such as ATP, calreticulin, and high-mobility group protein B1 (HMGB1), can effectively recruit DCs and promote their maturation, acting as immune adjuvants.

[0064] Tumor cells can undergo senescence in response to a variety of stressors, including oncogenic signals, replication stress, hypoxia, reactive oxygen species, nutrient deprivation, and cytokines (such as TGF-β) in the tumor microenvironment. Furthermore, anticancer therapies such as radiotherapy, chemotherapy, and immunotherapy can induce tumor cell senescence. Senescent tumor cells, lacking the ability to proliferate, do not inherently promote tumor growth; however, the proteome of senescent tumor cells undergoes significant remodeling, giving them unique advantages in activating both innate and adaptive immunity. Drug-induced senescence in tumor cells significantly upregulates ligands for natural killer (NK) cell-activating receptors on their surfaces, making them more susceptible to NK cell recognition and killing. Simultaneously, senescent tumor cells also show increased expression of MHC-I molecules on their surfaces, which effectively activates the cytotoxic effects of CD8+ T cells. Furthermore, the abundance of the interferon-gamma receptor IFNGR1 increases on the surface of senescent tumor cells, making them more sensitive to IFN-gamma in the microenvironment, thereby amplifying IFN-gamma signaling and effectively activating CD8+ T cells.

[0065] In one aspect, the present application provides a composition for tumor targeted therapy, comprising senescent tumor cells and celecoxib.

[0066] The immune activation function of senescent tumor cells provided in this application, namely, STCs can effectively activate the maturation of dendritic cells (DCs), the immune function of T cells and NK cells, and regulate the SASP of STCs by celecoxib (CLX), especially inhibiting the production of prostaglandin E2 (PGE2). The combined use further enhances the anti-tumor immune effect.

[0067] In one embodiment, the composition comprises senescent tumor cells and celecoxib liposomes.

[0068] On the one hand, the present application provides a hydrogel encapsulating STCs (SCs+CLX-Lipo@Gel), comprising a hydrogel matrix, and senescent tumor cells and celecoxib liposomes encapsulated in the hydrogel matrix.

[0069] Optionally, the STCs concentration is 1.8×10 6 pcs / L~2.2×10 6 / L, for example, the STCs concentration is 1.8×10 6 / L, 1.9×10 6 pcs / L, 2.0×10 6 / L, 2.1×10 6 pcs / L or 2.2×10 6 pcs / L and any value in between.

[0070] Optionally, the concentration of the celecoxib liposomes is 0.35 mg / mL to 0.45 mg / mL, calculated as celecoxib, for example, a concentration of 0.35 mg / mL, 0.36 mg / mL, 0.37 mg / mL, 0.38 mg / mL, 0.39 mg / mL, 0.40 mg / mL, 0.41 mg / mL, 0.42 mg / mL, 0.43 mg / mL, 0.44 mg / mL or 0.45 mg / mL and any value therebetween.

[0071] This application constructs a hydrogel that co-loads senescent cells and celecoxib liposomes for co-encapsulation of celecoxib liposomes and senescent tumor cells. The hydrogel encapsulated with STCs provided in this application can significantly prolong the retention time of senescent tumor cells in the body, with the potential to continuously stimulate immune responses.

[0072] In further in vitro and in vivo studies, a single dose of the hydrogel encapsulated with STCs promoted DC maturation and recruitment of cDC1 subsets, significantly activated CD8+ T cells and NK cells within the tumor, and promoted the proliferation of CD8+ T cells and NK cells, demonstrating ideal immune activation and anti-melanoma activity. Furthermore, a single immunization of mice with the hydrogel encapsulated with STCs activated NK cells in the peripheral blood and increased the number of TEMs in the spleen, significantly inhibiting the occurrence and development of tumors.

[0073] In some embodiments, the senescent tumor cells include mouse melanoma cells. Mouse melanoma cells are a type of malignant melanoma cell derived from mice and have important applications in oncology research. These cells are highly malignant and invasive, capable of rapid growth and tumor formation, prone to metastasis, and able to spread to other sites through the blood or lymphatic system. They are also highly reproducible, and the growth and metastasis process can be repeated multiple times with stable and reliable results.

[0074] In some embodiments, the mouse melanoma cells include B16-F10 cells. B16-F10 cells are a mouse skin melanoma cell line derived from skin melanoma tissue of C57BL / 6J mice. This cell line is a subclone of B16 cells, obtained through serial in vivo passage and possessing enhanced tumor metastasis potential. B16-F10 cells exhibit a mixed spindle-shaped and epithelial-like morphology and grow adherently.

[0075] In some embodiments, the particle size of the celecoxib liposomes is 58 nm to 62 nm, for example, 58 nm, 59 nm, 60 nm, 61 nm or 62 nm and any value therebetween.

[0076] In some embodiments, the encapsulation efficiency of the celecoxib liposomes is 70% to 90%, and the drug loading is 5% to 2.5%. For example, the encapsulation efficiency is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% and any values therebetween.

[0077] In some embodiments, the celecoxib liposomes exhibit a negative potential of -12 mV to -11 mV, such as -12.0 mV, -11.9 mV, -11.8 mV, -11.7 mV, -11.6 mV, -11.5 mV, -11.4 mV, -11.3 mV, -11.2 mV, or -11.1 mV.

[0078] The present application regulates the SASP of STCs by celecoxib (CLX) liposomes (CLX-Lipo), especially inhibiting the production of prostaglandin E2 (PGE2) to enhance the anti-tumor immune effect.

[0079] In some embodiments, the hydrogel matrix comprises a cross-linked product of chitosan and a gelling agent. Chitosan is a natural polysaccharide composed of a deacetylated derivative of chitin. It exhibits excellent biocompatibility and biodegradability, and has a wide range of applications in the biomedical field. Chitosan also exhibits immunomodulatory properties and can be used as a vaccine adjuvant. Chitosan particles are readily phagocytosed by immune cells and promote innate immune responses, a process that is crucial for promoting antigen presentation.

[0080] On the other hand, the present application provides a vaccine comprising the above composition or hydrogel.

[0081] The hydrogel carrier provided in this application is thermosensitive, easy to inject, and can encapsulate STCs and CLX-Lipo, thereby prolonging the retention time of STCs in the body and achieving continuous stimulation of the immune response.

[0082] Hydrogels offer unique advantages in tumor vaccine delivery. They possess excellent hydrophilic properties and adjustable swelling behavior, sharing certain similarities with the extracellular matrix in terms of physicochemical properties. Their 3D porous structure improves the loading efficiency of tumor antigens, allowing cells to enter and exit the hydrogel and providing a suitable microenvironment for their survival. Hydrogels can also simultaneously load a variety of immunomodulatory drugs, such as immune adjuvants, cytokines, immune checkpoint inhibitors, and nanoformulations, thereby synergistically enhancing the anti-tumor efficacy of tumor vaccines. Furthermore, hydrogels can provide protection, preventing the rapid clearance of tumor antigens from the body, exerting long-lasting effects, and reducing the entry of active ingredients into the bloodstream, thereby minimizing adverse reactions.

[0083] Optionally, the gelling agent includes 0.04M to 0.06M sodium bicarbonate, for example, the concentration of sodium bicarbonate is 0.04M, 0.05M or 0.06M and any value therebetween.

[0084] This application selects melanoma as the research subject, aiming to address the dilemma of poor immunogenicity and rapid clearance from the body of whole-tumor vaccines. To this end, senescent tumor cells, which are highly immunogenic and adjuvant, are used as tumor antigens. Drug inhibition is used to modulate the SASP of senescent tumor cells, circumventing their immunosuppressive effects and enhancing the immune-stimulating capacity of whole-tumor vaccines, thereby effectively preventing and inhibiting tumor growth.

[0085] In some embodiments, the method for preparing senescent tumor cells comprises: co-culturing tumor cells with a senescence inducer to prepare senescent tumor cells.

[0086] In some embodiments, the senescence-inducing agent comprises doxorubicin (DOX) drug.

[0087] In some embodiments, the co-culture time is 44 h to 52 h, for example, 44 h, 45 h, 46 h, 47 h, 48 h, 49 h, 50 h, 51 h or 52 h, and any value therebetween.

[0088] Another aspect of the present application provides a method for preparing a hydrogel containing STCs, comprising:

[0089] Senescent tumor cells, celecoxib liposomes and gel raw materials are mixed, and the gel raw materials are cross-linked to form a hydrogel matrix encapsulating senescent tumor cells and celecoxib liposomes, thereby preparing a hydrogel encapsulating STCs.

[0090] In some embodiments, the method for preparing celecoxib liposomes comprises preparing celecoxib-loaded liposomes by thin film hydration method.

[0091] In some embodiments, the celecoxib liposome raw material includes one or more of egg yolk lecithin, cholesterol, DSPE-MPEG2000 and celecoxib.

[0092] In some embodiments, the mass ratio of egg yolk lecithin, cholesterol, DSPE-MPEG2000 and celecoxib is (44-46):(4-6):(1.5-2.5):(0.5-1.5). For example, the mass ratio is (44, 45, 46):(4, 5, 6):(1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5):(0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5).

[0093] In some embodiments, the thin film hydration method includes: dissolving the celecoxib liposome raw material in an organic solvent, evaporating the organic solvent by rotary evaporation, adding an aqueous phase and shaking to hydrate and shed the lipid film to produce a liposome dispersion; and, ultrasonicating and extruding the liposome dispersion through a membrane to prepare celecoxib-loaded liposomes.

[0094] In some embodiments, the organic solvent includes chloroform and methanol in a volume ratio of (1-3):1. For example, the volume ratio of chloroform and methanol is 1:1, 2:1, or 3:1, and any value therebetween.

[0095] In some embodiments, the temperature of the rotary evaporation method is 40° C. to 45° C. For example, the temperature of the rotary evaporation method is 40° C., 41° C., 42° C., 43° C., 44° C., or 45° C., and any values therebetween.

[0096] In some embodiments, the ultrasound parameters include: a power of 260W to 280W, and a duration of 10 minutes to 20 minutes, for example, the power is 260W, 261W, 262W, 263W, 264W, 265W, 266W, 267W, 268W, 269W, 270W, 271W, 272W, 273W, 274W, 275W, 276W, 277W, 278W, 279W, 280W, and any value therebetween.

[0097] In some embodiments, the film extrusion parameters include: using a polycarbonate film with a thickness of 90 nm to 110 nm, and extrusion times of 10 to 12 times. For example, the thickness is 90 nm, 91 nm, 92 nm, 93 nm, 94 nm, 95 nm, 96 nm, 97 nm, 98 nm, 99 nm, 100 nm, 101 nm, 102 nm, 103 nm, 104 nm, 105 nm, 106 nm, 107 nm, 108 nm, 109 nm, or 110 nm, and any value therebetween. For example, the extrusion times are 10, 11, or 12 times.

[0098] Polycarbonate film (PC film) is a thin film material made from polycarbonate (PC). Polycarbonate is a colorless, transparent thermoplastic engineering plastic that features high transparency, high strength, impact resistance, good dimensional stability, and excellent electrical insulation. Polycarbonate film is typically produced through extrusion or track etching techniques.

[0099] In some embodiments, the gel material includes chitosan.

[0100] In some embodiments, cross-linking the gel material includes adding a gelling agent.

[0101] In some embodiments, the gelling agent comprises 0.04M to 0.06M sodium bicarbonate, for example, the concentration of sodium bicarbonate is 0.04M, 0.05M, or 0.06M, and any value therebetween.

[0102] On the other hand, the present application provides the use of the above-mentioned hydrogel encapsulating STCs in the preparation of a drug for preventing postoperative tumor recurrence and / or metastasis.

[0103] In some embodiments, the tumor comprises melanoma.

[0104] The STCs-encapsulated hydrogel provided in the present application can effectively activate multiple immune cells, inhibit the growth of melanoma tumors, prevent the occurrence of melanoma tumors, and prolong the survival of mice with melanoma brain metastasis tumor models.

[0105] On the other hand, the present application provides a treatment method, which comprises administering an effective dose of the STCs-encapsulated hydrogel or the drug to a subject.

[0106] Wherein, "administer," "give," and "treat" as applied to an animal, a human, a laboratory subject, a cell, a tissue, an organ, or a biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid. "Administer," "administer," and "treat" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "administer," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. "Treatment," when applied to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0107] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0108] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0109] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0110] Example 1

[0111] This embodiment provides a method for preparing chitosan hydrogel encapsulating senescent tumors and celecoxib liposomes.

[0112] 1. Preparation process

[0113] 1. Materials

[0114] Doxorubicin hydrochloride (DOX) (Shanghai MacLean Biochemical Technology Co., Ltd., Shanghai), DMEM high glucose medium (Dalian Meilun Biotechnology Co., Ltd., Dalian), fetal bovine serum (Dalian Meilun Biotechnology Co., Ltd., Dalian), cell culture grade dimethyl sulfoxide (Shanghai MacLean Biochemical Technology Co., Ltd., Shanghai), 100× penicillin-streptomycin double antibody solution (Dalian Meilun Biotechnology Co., Ltd., Dalian), 0.25% trypsin-0.02% EDTA digestion solution (Dalian Meilun Biotechnology Co., Ltd., Dalian), celecoxib (Dalian Meilun Biotechnology Co., Ltd., Dalian), egg yolk lecithin PC-98T (Shanghai Aiweituo Pharmaceutical Technology Co., Ltd., Shanghai), DSPE-MPEG 2000 (Shanghai Aiweituo Pharmaceutical Technology Co., Ltd., Shanghai), cholesterol (Shanghai Aiweituo Pharmaceutical Technology Co., Ltd., Shanghai), chromatography grade methanol (Beijing Bailingwei Technology Co., Ltd., Beijing), chitosan (medium molecular weight) (Sigma-Aldrich, Beijing). Aldrich, USA), sodium dihydrogen phosphate (Sinopharm Chemical Reagent Co., Ltd., Shanghai), disodium hydrogen phosphate (Sinopharm Chemical Reagent Co., Ltd., Shanghai), Calcein / PI cell viability and cytotoxicity detection kit (Shanghai Beyotime Biotechnology Co., Ltd., Shanghai).

[0115] 2. Instruments

[0116] Electronic balance (BT 25S, Sartorius, Germany), biological safety cabinet (1300SERIES A2, ThermoScientific, USA), CO2 cell culture incubator (Thermo Fisher 371, Thermo Scientific, USA), rotary evaporator (Shanghai Yarong Biochemical Instrument Factory, Shanghai), liposome extruder (Mini-Extruder, Avanti, USA), laser particle size analyzer (Zetasizer Nano ZS90, Melvern, UK), high performance liquid chromatography (1260 Infinity, Agilent, USA), ultrasonic disruptor (Ningbo Xinzhi Biotechnology Co., Ltd., Ningbo).

[0117] 3. Experimental cells

[0118] Mouse melanoma cell line (B16F10) was purchased from Dalian Meilun Biotechnology Co., Ltd.

[0119] 4. Experimental methods

[0120] (1) Induction and characterization of senescent tumor cells

[0121] B16-F10 cells were digested and collected at 1×10 5 Cells were seeded at a density of 1 / well in 12-well plates. Blank, 100 nM DOX, and 200 nM DOX groups were established. The original culture medium was aspirated and the drug-containing culture medium of each group was added, and the cells were cultured for another 48 hours. After 48 hours of co-incubation with the drug, the drug-containing culture medium was aspirated and replaced with complete DMEM medium. Culture was continued for another 5 days. The cell culture supernatant was collected and stored on ice until use. Simultaneously, the cells were stained for β-galactosidase, observed under a brightfield microscope, and photographed.

[0122] (2) Detection of ATP and HMGB1 content in cell culture supernatant

[0123] The ATP content was determined according to the instructions of the Enhanced ATP Detection Kit (Meilun Biotechnology, MA0440). The HMGB1 content was determined according to the instructions of the Mouse HMGB1 ELISA Kit.

[0124] (3) Preparation of CLX liposomes (CLX-Lipo)

[0125] CLX-loaded liposomes were prepared by a thin-film hydration method. Egg yolk phosphatidylcholine (PC-98T), cholesterol, DSPE-MPEG2000, and CLX were weighed in a 45 / 5 / 2 / 1 mass ratio and dissolved in a 2 / 1 (v / v) mixture of chloroform and methanol. After rotary evaporation at 42°C in a water bath, an appropriate amount of water was added and rotary hydration was continued until a thin film fell off. The liposome suspension was sonicated using a 270W power probe for 15 minutes (2s / 3s cycle). After sonication, the liposome suspension was repeatedly extruded through a 100nm polycarbonate membrane 11 times, and free drug was removed using a G50 dextran gel column.

[0126] (4) Characterization of CLX liposomes (CLX-Lipo)

[0127] The particle size distribution and potential of liposomes were determined using a laser particle size analyzer, and the morphology of CLX liposomes was observed using a transmission electron microscope.

[0128] (5) Determination of CLX concentration standard curve

[0129] The CLX concentration standard curve was determined by high performance liquid chromatography (HPLC). Figure 1 shown.

[0130] Chromatographic conditions: chromatographic column: Diamonsil-C18 (250×4.6 mm, 5 μm); mobile phase: 15% water + 85% methanol; flow rate: 1 mL / min; column temperature: 25°C; detection wavelength: 254 nm; injection volume: 10 μL.

[0131] (6) Determination of liposome encapsulation efficiency and drug loading

[0132] Take 100 μL of liposome suspension, add 900 μL of methanol, break the emulsion by ultrasonication, filter through a 0.22 μm filter membrane, and take the filtrate for HPLC content determination. Calculate the CLX concentration according to the standard curve and calculate the encapsulation efficiency:

[0133] Encapsulation efficiency (%) = liposome encapsulated drug mass / total drug input mass × 100%.

[0134] After freeze-drying CLX-Lipo, take a certain amount of solid, add 1 mL of methanol, and fully dissolve it by ultrasonication. After filtering through a 0.22 μm filter membrane, take the filtrate for HPLC content determination. Calculate the CLX concentration according to the standard curve and calculate the drug loading:

[0135] Drug loading (%) = liposome-encapsulated drug mass / liposome total mass × 100%.

[0136] (7) Preparation of thermosensitive hydrogel

[0137] Dissolve 500 mg of chitosan in 0.1 M hydrochloric acid and stir. Prepare gelling agent solutions of varying concentrations by mixing PB buffer and 1 M sodium bicarbonate solution. Place the chitosan solution into one syringe and the gelling agent solution into another, with a volume ratio of 3:2. Connect the two syringes via a Luer connector and repeatedly push the syringe contents from side to side 15 times. Then, connect the syringes via the Luer connector to draw 0.2 mL of the CLX-Lipo cell suspension into the syringes. Repeat mixing 15 times.

[0138] The mixed gel solution was placed at 37°C for cross-linking reaction, and the appropriate gelling agent concentration was screened based on the gel formation time and cell viability.

[0139] (8) Detection of cell viability

[0140] The gel loaded with cells was loaded at 1×10 6 Cells were seeded at a density of 1 mL / well in a 12-well plate and placed in a cell culture incubator until a gel formed. The plate was removed and the gel was covered with 1 mL of complete culture medium. Culture was continued for 14 days. After the incubation period, the medium was carefully aspirated and the cells were rinsed three times with PBS. Staining was performed according to the instructions of the Calcein / PI Cell Viability and Cytotoxicity Assay Kit. The staining results were observed under a fluorescence microscope, and photos were taken to calculate the proportion of surviving cells.

[0141] (9) Morphological and rheological characterization of hydrogels

[0142] After freeze-drying, the samples were fractured in liquid nitrogen. The cross-sectional morphology was observed by scanning electron microscopy, and the modulus changes were recorded at a fixed angular frequency of 1 rad / s and a shear strain of 1%. The characterization results of senescent cells are shown in Figure 2. Figure 2 shown.

[0143] Verification results:

[0144] During cell aging, β-galactosidase with high enzymatic activity is expressed. Figure 2 As shown in Figure A, compared with the untreated group, after treatment with 200 nM DOX, B16-F10 cells showed a significant upregulation of β-galactosidase expression and a significant increase in cell volume, indicating that DOX treatment can cause B16-F10 cells to enter a senescent state. Compared with normal B16-F10 cells, senescent B16-F10 cells can secrete more ATP and HMGB1 (typical DAMPs), as shown in Figure 4. Figure 2 As shown in Figure 3 B. In summary, the administration of 200 nM DOX can successfully induce B16F10 cell senescence.

[0145] Result analysis: Figure 3As shown in Figures B to D, the CLX-Lipo prepared by the thin film hydration method exhibits a negative potential of approximately -11.6 mV; the particle size is approximately 60 nm, the shape is nearly spherical, and it exhibits a typical bilayer structure. Figure 1 As shown in Table 1, the encapsulation efficiency of CLX-Lipo is 73.49% and the drug loading is 2.00%.

[0146] Table 1: Encapsulation efficiency and drug loading of CLX liposomes

[0147] Encapsulation efficiency (%) Drug loading (%) 73.49±1.66 2.00±0.04

[0148] The present invention uses phosphate buffer containing sodium bicarbonate as a gelling agent, which can make chitosan hydrogel have temperature-sensitive properties and gel at 37 ° C ( Figure 3 To ensure rapid gelation after injection, sodium bicarbonate solutions of varying concentrations were screened. Concentrations of 0.05M, 0.075M, and 0.1M sodium bicarbonate all gelled within 1 hour at 37°C, meeting the requirement for rapid gelation. Therefore, these three concentrations were selected for further screening.

[0149] 2. In vivo immune evaluation of hydrogel vaccines

[0150] 1. Materials

[0151] RPMI 1640 medium (Dalian Meilun Biotechnology Co., Ltd., Dalian), bovine serum albumin fraction V (BSA) (Dalian Meilun Biotechnology Co., Ltd., Dalian), mouse recombinant GM-CSF (Peprotech, USA), mouse recombinant IL-4 (Peprotech, USA), NK-92 cell culture medium (Wuhan Punosai Life Science Technology Co., Ltd., Wuhan), type IV collagenase (Shanghai Yisheng Biotechnology Co., Ltd., Shanghai), hyaluronidase (Shanghai Yisheng Biotechnology Co., Ltd., Shanghai, and the rest are the same as step 1.

[0152] 2. Instruments

[0153] A high-speed micro-refrigerated centrifuge (D1524R, Dalong Xingchuang Laboratory Instrument Co., Ltd., Beijing), a microplate reader (Multiskan FC, Thermo Scientific, USA), a flow cytometer (ACEA NovoCyte 3000, Agilent, USA), a fluorescence pathology slide scanner (NanoZoomer, Japan), a small animal living imaging system (CaliperPerkinElmer, Hopkinton, USA), a super-resolution spinning disk confocal system (Olympus SpinSR10, Japan), and a horizontal shaker (TB-100, Haimen Qilin Bell Instrument Manufacturing Co., Ltd., Haimen) were used. The rest of the procedures were the same as in step 1.

[0154] 3. Experimental animals and cells

[0155] Mouse melanoma cell line (B16F10) was purchased from Dalian Meilun Biotechnology Co., Ltd. C57 / BL6 black mice (male, 8 weeks old) were purchased from Shanghai Slake Laboratory Animal Co., Ltd. and housed in a standard SPF environment (25 ± 2°C constant temperature, 12-h light-dark cycle; Laboratory Animal Use Permit: SYXK (Shanghai) 2020-0042). All animal experiments were conducted in accordance with the requirements and standards of the Institutional Animal Care and Use Committee (IACUC) of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

[0156] 4. Experimental methods

[0157] (1) Evaluation of the in vivo retention effect of hydrogels

[0158] B16F10 cells were resuspended in DiR cell membrane fluorescent probe staining working solution to prepare a hydrogel (SCs@Gel) loaded only with senescent cells. C57 / BL6 mice were randomly divided into two groups, the SCs group and the SCs@Gel group, with 3 mice in each group. The hair on the left rib and the back of the left hind limb of the mice was removed. The fluorescence intensity of the SCs cell suspension and SCs@Gel was quantitatively measured using a small animal in vivo imaging system. By adjusting the concentrations of the two, their fluorescence intensities were made consistent, and equal amounts of the suspensions were injected subcutaneously into the mice. On days 1, 2, 3, 5, 7, and 14, the fluorescence intensity of the subcutaneous injection site of the mice was measured and statistically analyzed using a small animal in vivo imaging system.

[0159] (2) Evaluation of the effect of hydrogel in preventing tumor growth

[0160] ①Evaluation of the effect of preventing tumor growth

[0161] C57 / BL6 mice were randomly divided into 6 groups, namely PBS, CS@Gel (chitosan gelatin composite hydrogel), CLX-Lipo, SCs (2×10 5cells / ), SCs+CLX-Lipo (senescent cells and celecoxib liposomes) (2×10 5 cells / mouse, 2 mg / kg) and SCs+CLX-Lipo@Gel group (2×10 5 Each group consisted of 5 mice. Hair was removed from the left flank and back of the left hind limb. Each formulation was injected subcutaneously into the mice. Seven days after injection, subcutaneous melanoma tumors were established in the mice. The day of cell injection was designated as day 0. The major and minor diameters of the tumors were recorded every other day to calculate tumor volume. On day 14, the mice were sacrificed, and samples were collected for subsequent experimental analysis.

[0162] ② Flow cytometry analysis of peripheral blood NK cells

[0163] On day 7, approximately 200 μL of blood was collected from the retroorbital venous plexus and placed on ice. 1 mL of red blood cell lysis buffer was added to the blood sample, lysed at room temperature for 10 minutes, centrifuged at 300 × g for 5 minutes, and the supernatant discarded. Rinse once with PBS, centrifuged again at 300 × g for 5 minutes, and the supernatant discarded. Flow cytometry antibody staining was performed using the specific flow cytometry protocol shown in Table 2. After staining, immune cell subset proportions were analyzed using flow cytometry.

[0164] Table 2: Flow cytometry staining protocol

[0165]

[0166] ③Flow cytometry analysis of immune cells in the spleen

[0167] On day 14, mice were euthanized and their spleens were removed for flow cytometry antibody staining. The specific flow cytometry staining protocol is shown in Table 3. After staining, the proportions of immune cell subsets were analyzed using flow cytometry.

[0168] Table 3: Flow cytometry staining protocol

[0169]

[0170]

[0171] 3. Evaluation of the efficacy of hydrogel in treating tumors

[0172] 1. Evaluation of the effect of hydrogel in inhibiting tumor growth

[0173] The melanoma subcutaneous tumors were constructed in mice, and the day of cell injection was recorded as day 0. On day 11, C57 / BL6 mice were randomly divided into 5 groups, namely PBS, CLX-Lipo, SCs (2×10 5 cells / cell), SCs+CLX-Lipo (2×10 5cells / mouse, 2 mg / kg) and SCs+CLX-Lipo@Gel group (2×10 5 Each group of mice was subcutaneously injected with each preparation peritumoral area. The weight of the mice and the long and short diameters of the tumors were recorded every other day, and the tumor volume was calculated. On day 20, the tumor volume of the PBS group was close to 2000 mm 3 , which was the end point of the experiment, the mice were euthanized, the tumors were dissected out, washed with PBS, the residual liquid was absorbed with filter paper, the tumors were weighed, and photographed for preservation.

[0174] 2. Analysis of immune cells in tumor tissue

[0175] Tumor tissue was stained with flow cytometry antibodies. The specific flow cytometry staining protocol is shown in Table 4. After staining, the proportion of immune cell subsets was analyzed using flow cytometry.

[0176] Table 4: Flow cytometry staining protocol

[0177]

[0178]

[0179] 3. Analysis of immune cells in paratumoral lymph nodes

[0180] After the end of the experiment, the inguinal lymph nodes of the mice were removed, 1 mL of PBS was added to a 12-well plate, and the cells were ground into a single-cell suspension using a blunt syringe. The suspension was then transferred to a 1.5 mL EP tube and centrifuged at 300 × g for 5 min. The supernatant was discarded, and the cells were resuspended in an appropriate amount of PBS and analyzed by flow cytometry. The flow cytometry staining protocol is shown in Table 5.

[0181] Table 5: Flow cytometry staining protocol

[0182]

[0183] 4. Evaluation of the effect of hydrogel in prolonging survival in a mouse melanoma brain metastasis model

[0184] 5 μL of B16-F10 cell suspension (5×10 3 ) was used to establish a mouse model of melanoma brain metastasis. The day of cell injection was recorded as day 0. The mice injected with tumor cells were randomly divided into three groups: PBS, STCs+CLX-Lipo (5×10 5 cells / mouse, 2 mg / kg) and STCs+CLX-Lipo@Gel group (5×10 5Each mouse received subcutaneous immunization on days 3, 6, and 9. When the body weight of the mouse decreased by 20%, it was considered a humane endpoint, and the survival rate of each group of mice was recorded.

[0185] Result analysis:

[0186] Figure 4 The in vivo retention and tumor prevention effects of SCs+CLX-Lipo@Gel, e.g. Figure 4 As shown in Figure A, the fluorescence intensity of the SCs group gradually decreased after subcutaneous injection, reaching only one-fifth of that on day 1 by day 7. In contrast, the fluorescence intensity of the SCs@Gel group remained unchanged on day 7, and the average fluorescence intensity on day 14 was approximately twice that of the SCs group. These results suggest that SCs are readily diffused and cleared after subcutaneous injection into mice. Encapsulation into the gel solution allows for rapid gelation at the injection site and slow degradation, thereby delaying the clearance of SCs from the body.

[0187] like Figure 4 As shown in Figures B to C. After day 6, the tumors of mice in the PBS, CS@Gel, and CLX-Lipo groups grew faster, and the average tumor volume exceeded 500 mm on day 14. 3 Compared with the PBS group, the tumor growth rate in the SCs group was slower, and the tumor growth rate in the SCs+CLX-Lipo group was further slowed down. The tumor growth rate in the SCs+CLX-Lipo@Gel group was the slowest, with an average tumor volume of only 181 mm on day 14. 3 , and three mice did not show tumor growth. The above results show that SCs+CLX-Lipo@Gel has a strong ability to prevent the occurrence and development of tumors. Compared with the PBS group, there was no significant change in the proportion of NK cells in the peripheral blood of mice in the CS@Gel group and the CLX-Lipo group; there was no significant change in the proportion of NK cells in the SCs group and the SCs+CLX-Lipo group, but the proportion of activated NK cells was significantly increased, and the proportion of NK cells and the proportion of activated NK cells in the peripheral blood of mice in the SCs+CLX-Lipo@Gel group were significantly increased. This shows that the addition of CLX-Lipo can further promote the activation of NK cells (such as Figure 4 GI in Figure 1).

[0188] like Figure 4 As shown in J~K, after SCs+CLX-Lipo@Gel immunization, the proportions of CD4+TEM and CD8+TEM in the spleen of mice increased significantly, indicating that the immune memory function of mice was enhanced.

[0189] Figure 5The tumor inhibitory activity of SCs+CLX-Lipo@Gel in the mouse melanoma subcutaneous tumor model is shown in Figure 2. Figure 5 As shown in Figures B-D, the tumor inhibition rate in the CLX-Lipo group was 45.10%, while in the SCs group it was 63.65%. The introduction of CLX-Lipo increased this to 77.24%. The SCs + CLX-Lipo@Gel group performed the best, achieving a 94.34% tumor inhibition rate and an average tumor mass of only 165.4 mg. Furthermore, the tumors in the SCs + CLX-Lipo@Gel group remained virtually unchanged. These results demonstrate that the SCs + CLX-Lipo@Gel group exhibits the most significant tumor inhibition activity in the mouse subcutaneous melanoma model.

[0190] After SCs+CLX-Lipo@Gel treatment, the proportion of mature DCs in the lymph nodes was about 1.35 times that of the PBS group, and the proportion of cDC1 was about 1.65 times that of the PBS group, indicating that SCs+CLX-Lipo@Gel can promote the maturation of DCs and recruit cDC1 to the peritumoral lymph nodes (e.g. Figure 5 E in the figure).

[0191] Further analysis of immune cells in tumor tissues revealed the following experimental results: Figure 5 As shown in Figures F to H. Compared with the PBS group, the proportion of CD3+CD8+T cells in the tumor tissue of the SCs+CLX-Lipo@Gel group was significantly increased, and the proportion of CD8+Ki67+ cell subsets was significantly increased, indicating that SCs+CLX-Lipo@Gel treatment can significantly promote the proliferation of CD8+T cells in the tumor. At the same time, the proportion of cell subsets of cytotoxic effectors (Granzyme B+) also increased significantly, and the proportion of SCs+CLX-Lipo@Gel group was about 5 times that of the PBS group (as shown in Figures F to H). Figure 5 In addition, analysis of NK cells in tumor tissues showed that the proportion of NK cells increased significantly after SCs+CLX-Lipo@Gel treatment. At the same time, the number of activated NK cells (NKG2D+ and NKp46+) also increased significantly, approximately 2 times and 4 times that of the PBS group. The proportion of cell subpopulations that secrete cytotoxic effector factors (Granzyme B+) also increased significantly, approximately 2 times that of the PBS group (as shown in Figure 2). Figure 5 The above results indicate that SCs+CLX-Lipo@Gel treatment can significantly activate anti-tumor immunity in mice.

[0192] Figure 6 To evaluate the effect of hydrogel on prolonging the survival of a mouse melanoma brain metastasis model. Figure 6As shown in Figures A to C, the STCs+CLX-Lipo@Gel treatment group had the longest survival time, with a median survival time of 16 days, while the STCs+CLX-Lipo group was 11.5 days and the PBS group was only 9 days. The hydrogel vaccine can significantly prolong the survival of the mouse melanoma brain metastasis model.

[0193] The embodiments described above only express several implementation methods of the present application, which are convenient for understanding the technical solutions of the present application in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of this application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A composition for tumor targeted therapy, characterized in that: The composition includes STCs and celecoxib.

2. The composition for tumor targeted therapy according to claim 1, characterized in that: The composition includes STCs and celecoxib liposomes.

3. A hydrogel encapsulating STCs, characterized in that: The method comprises a hydrogel matrix and STCs and celecoxib liposomes contained in the hydrogel matrix; Optionally, the concentration of the STCs is 1.8×10 6 pcs / L~2.2×10 6 pcs / L; Optionally, the concentration of the celecoxib liposomes is 0.35 mg / mL to 0.45 mg / mL based on celecoxib.

4. The STCs-loaded hydrogel according to claim 3, characterized in that: The STCs include pigment tumor cells; Optionally, the pigment tumor cells include mouse melanoma cells; Optionally, the mouse melanoma cells include B16-F10 cells; Optionally, the particle size of the celecoxib liposome is 58 nm to 62 nm.

5. The STCs-loaded hydrogel according to claim 1, characterized in that: The hydrogel matrix includes a cross-linked product of chitosan and a gelling agent; Optionally, the gelling agent includes 0.04M to 0.06M sodium bicarbonate.

6. A vaccine, characterized in that The vaccine comprises the tumor-targeted therapy composition according to claim 1 or 2; Optionally, the vaccine is in the form of a gel; Optionally, the gel is a hydrogel.

7. A method for preparing a hydrogel containing STCs, characterized in that: include: mixing STCs, celecoxib liposomes and a gel material, cross-linking the gel material to form a hydrogel matrix encapsulating the STCs and the celecoxib liposomes, and preparing a hydrogel encapsulating the STCs; Optionally, the STCs preparation method comprises: co-culturing tumor cells with a senescence inducer to prepare STCs; Optionally, the senescence-inducing agent comprises doxorubicin; Optionally, the co-cultivation time is 44 h to 52 h.

8. The preparation method according to claim 7, characterized in that The preparation method of the celecoxib liposome comprises: Celecoxib liposomes were prepared by thin film hydration method; Optionally, the raw materials of the celecoxib liposome include celecoxib and one or more of egg yolk lecithin, cholesterol and DSPE-MPEG2000; Optionally, the mass ratio of the egg yolk lecithin, the cholesterol, the DSPE-MPEG2000 and the celecoxib is (44-46):(4-6):(1.5-2.5):(0.5-1.5).

9. The preparation method according to claim 8, characterized in that Thin film hydration methods include: dissolving the raw material of celecoxib liposomes in an organic solvent, evaporating the organic solvent to dryness by rotary evaporation, adding an aqueous phase and shaking to hydrate and detach the lipid film to produce a liposome dispersion; and The liposome dispersion was subjected to ultrasound and membrane extrusion to prepare celecoxib liposomes; Optionally, the thin film hydration method satisfies one or more of the following conditions: (1) The organic solvent comprises chloroform and methanol in a volume ratio of (1-3):1; (2) The temperature of the rotary evaporation method is 40℃~45℃; (3) Ultrasonic parameters include: power of 260W to 280W, duration of 10min to 20min; and, (4) The film extrusion parameters include: using a polycarbonate film with a thickness of 90nm to 110nm and the number of extrusions is 10 to 12 times.

10. Use of the composition for targeted tumor therapy according to claim 1 or 2, the hydrogel encapsulating STCs according to any one of claims 3 to 5, or the vaccine according to claim 6 in the preparation of a medicament for preventing postoperative tumor recurrence and / or metastasis; Optionally, the tumor comprises melanoma.

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