A blood gel sustained-release carrier for in vivo in situ engineering of immune cells and application thereof

Abstract

The application provides a blood gel sustained-release carrier for in vivo in situ engineering of immune cells and application thereof. The composition provided by the application comprises a blood gel formed by blood and a gene delivery carrier loaded therein. The blood gel is formed by fresh peripheral blood through a natural blood clotting process, has a three-dimensional porous fibrin network structure, can adsorb and slowly release the gene delivery carrier, prolongs local retention time, and reduces systemic exposure. Meanwhile, the blood gel actively recruits immune cells such as macrophages through its natural bioactive components, so that the recruited cells are placed in a local high-concentration gene carrier microenvironment, and the transfection efficiency is improved. In-vitro and in-vivo experiments prove that the carrier can significantly prolong the retention time of the viral vector in the abdominal cavity for more than 14 days; effectively recruit macrophages and B cells; successfully prepare functional CAR-M in situ in the abdominal cavity; and the macrophages after transduction present a pro-inflammatory polarization phenotype.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology, biomaterials, and immunotherapy. Specifically, it relates to an innovative platform technology for the in situ recruitment and genetic modification of immune cells in vivo. This platform is particularly suitable for developing novel tumor immunotherapies, targeting solid tumors (such as abdominal tumors like colon cancer and gastric cancer) and metastatic lesions. Through local injection, it achieves rapid recruitment, efficient transfection, and functional activation of immune cells, bypassing the complex in vitro manufacturing processes of traditional cell therapies. This platform also has the potential to be extended to other diseases requiring local immune modulation (such as infections and tissue repair). Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Chimeric antigen receptor T-cell (CAR-T) therapy has revolutionized cancer treatment, but it has yet to achieve significant breakthroughs in solid tumor therapy. Furthermore, traditional CAR cell manufacturing involves a cumbersome in vitro process, including cell isolation, activation, transduction, expansion, and reinfusion, resulting in high costs, lengthy cycles, and significant batch-to-batch variations, severely limiting its accessibility and scalability. Macrophages, due to their ability to overcome the physical barriers of solid tumors, present antigens, and possess strong phagocytic capabilities, are considered a highly promising CAR cell type, facilitating the in vivo production of chimeric antigen receptor macrophages (CAR-M). The peritoneal cavity is naturally rich in macrophages, and stimulation by foreign substances (including serum or blood clots) induces the production of even more macrophages, forming an in situ macrophage reservoir. A suitable gene delivery vector is crucial for the efficient in vivo production of chimeric antigen receptor macrophages (CAR-M). Currently, viral vectors are undoubtedly the most efficient transfection vectors. However, direct intraperitoneal injection of the virus can easily lead to rapid spread into the systemic circulation, resulting in low effective local concentrations and poor transfection efficiency, thus bringing systemic toxicity risks and drug waste. Specifically, this is due to: 1. lack of targeting and retention; 2. lack of cell recruitment ability: the vector passively waits for and encounters target cells by chance, and cannot actively create a local high-density target cell environment; 3. limited vector selection: most studies are limited to specific vectors (such as lentiviruses) and specific cells (such as T cells), lacking a universal in vivo delivery and transformation platform that can adapt to multiple vectors and cell types.

[0004] Developing an integrated solution that simultaneously satisfies the requirements of "efficient local sustained release," "active cell recruitment," and "broad compatibility" is extremely challenging. Currently, common methods include constructing dual- or multi-target CAR cells, optimizing CAR structure, transducing immunomodulatory genes into CAR cells, and enabling tumors to widely express synthetic antigens. These strategies indicate that technologies for efficiently transfecting macrophages in vivo and achieving sustained local release of viral vectors are not yet mature, and integrated systems capable of simultaneously achieving immune cell recruitment, viral sustained release, and antigen-wide amplification are also lacking. These strategies require additional engineering of CAR cells, which further complicates the problem, including complex design and manipulation, potential DNA mutation risks, and poor CAR cytotoxicity. Summary of the Invention

[0005] The purpose of this invention is to provide an integrated, in-situ, sustained-release blood gel viral vector system to achieve the following objectives: Utilizing blood to create a blood gel and specifically recruiting macrophages and sustaining viral release within the peritoneal cavity, this achieves efficient and safe in-situ preparation of CAR-M, filling the technological gap in large-scale in vivo CAR-M preparation and avoiding the complex process of in vitro CAR-M manufacturing; Utilizing the natural fibrin network of this blood gel, it enables the local retention and sustained release of various therapeutic agents (viral vectors, mRNA, protein drugs, etc.), creating a local high-concentration transfection / action microenvironment; Using this vector, it enables the local retention and sustained release of various gene therapy vectors (viral / non-viral) at target tissue sites, maximizing local transfection efficiency and minimizing systemic exposure; Establishing a universal methodology for this vector, enabling it to serve as a foundational technology that can be flexibly combined with different gene vectors (lentiviruses, adenoviruses, AAVs, etc.) and different therapeutic targets (CAR expression, cytokines, etc.).

[0006] The blood gel described in this invention not only exhibits effective recruitment activity for macrophages, but also shows significant chemotactic and enrichment effects on other immune cell subsets, including T lymphocytes, natural killer cells (NK cells), B lymphocytes, and dendritic cells (DCs). Based on this broad-spectrum immune cell recruitment characteristic, the blood gel can serve as a universal platform matrix for cell modification, suitable for in situ regulation, phenotypic remodeling, or functional editing of the aforementioned various immune cells.

[0007] The technical solution adopted in this invention is as follows: In a first aspect, the present invention provides a composition for in situ engineered immune cells in vivo, comprising a blood gel formed from blood and a gene delivery vector loaded therein.

[0008] In a preferred embodiment, the blood gel is formed from fresh peripheral blood through a natural coagulation process. Its core structure is an interwoven three-dimensional fibrin network, rich in natural bioactive components such as platelets and cytokines. The components of the blood gel itself can effectively recruit macrophages and other immune cells, facilitating in situ cell transfection.

[0009] In a preferred embodiment, the gene delivery vector is selected from lentiviruses, adenoviruses, adeno-associated viruses, poxviruses, herpesviruses, or non-viral gene delivery systems (such as mRNA or pDNA encapsulated in lipid nanoparticles).

[0010] In a preferred embodiment, the gene comprises any nucleic acid capable of encoding a chimeric antigen receptor (CAR) protein, wherein the nucleic acid is selected from plasmid DNA, messenger RNA (mRNA), self-replicating RNA (saRNA), or in vitro transcribed complementary RNA (cRNA), etc.

[0011] In a preferred embodiment, the immune cells are selected from macrophages, dendritic cells, monocytes, T cells, NK cells, or neutrophils.

[0012] It should be emphasized that the blood gel platform described in this invention is not limited to the modification of macrophages. By selecting promoters or ligands with cell type specificity, this platform can be used for in situ modification of various immune cells in vivo: Dendritic cells (DCs): CAR-DCs can be generated by using the CD11c promoter or the ZBTB46 promoter to drive the specific expression of the CAR gene in DCs, thereby enhancing antigen presentation and T cell activation. T cells: T cells can be targeted using CD3-related promoters, CD4 or CD8 promoters to generate CAR-T cells in situ near lymphoid organs to enhance tumor immune killing. NK cells: NK cells can be targeted using the NKp46 or CD244 promoter to generate CAR-NK; Neutrophils: The MRP8 promoter can be used to target neutrophils for the acute inflammatory microenvironment.

[0013] The core value of this platform lies in providing a microenvironment for "cell recruitment" and "local high-concentration transfection." The specific immune cell type ultimately modified depends on the promoter design in the viral vector, not the platform itself. Therefore, any technical solution based on the blood gel platform described in this invention, which achieves in-situ modification of different immune cell types by replacing cell-specific promoters or ligands, falls within the scope of protection of this invention.

[0014] In a particularly preferred embodiment, the gene delivery vector is a lentivirus (CV) carrying a chimeric antigen receptor gene targeting CEA, and the immune cells are macrophages, used for in situ preparation of CAR-M in the peritoneal cavity.

[0015] A second aspect of the present invention provides a method for preparing the composition described in the first aspect of the present invention, comprising the steps of: mixing the gene delivery vector with fresh blood; inducing the mixture to coagulate naturally to form a blood gel loaded with the gene delivery vector.

[0016] In a preferred embodiment, the mixing is carried out in a sterile mold and allowed to solidify at room temperature for 10-20 minutes.

[0017] A third aspect of the invention provides the use of the composition described in the first aspect of the invention in the preparation of a medicament for treating tumors, wherein the treatment is achieved by in situ modifying immune cells in vivo.

[0018] In a preferred embodiment, the tumor is a solid tumor, including abdominal cancers or metastatic abdominal cancers such as colon cancer, stomach cancer, bladder cancer, pancreatic cancer, kidney cancer, and liver cancer.

[0019] In a preferred embodiment, the immune cells are macrophages, and the gene delivery vector is a lentivirus carrying the CAR gene, used for in situ preparation of CAR-M in the peritoneal cavity.

[0020] A fourth aspect of the invention provides the use of the composition described in the first aspect of the invention in the preparation of products for in situ modification of immune cells in vivo.

[0021] The blood gel (BG) described in this invention is formed from fresh peripheral blood through a natural coagulation process. Its core structure is an interwoven three-dimensional fibrin network, rich in natural bioactive components such as platelets and cytokines. The three-dimensional network structure of BG can adsorb and slowly release viral vectors (CVs), thereby prolonging the maintenance time of local viral concentration and reducing the risk of systemic exposure. Furthermore, as a foreign substance, BG induces macrophage aggregation by activating a monocyte recruitment cascade, placing the recruited immune cells in a localized high-concentration gene vector microenvironment formed by the sustained release of the blood gel, thus improving the efficiency of viral vector uptake by cells.

[0022] The carrier preparation process of the present invention is as follows: approximately 200 microliters of peripheral blood are collected from the orbital venous plexus of mice and gently injected into a sterile mold pre-loaded with CV. The mixture is left to stand at room temperature for 15 minutes to initiate spontaneous coagulation. Subsequently, the resulting semi-solid blood gel is mechanically cut into small pieces to obtain an injectable carrier formulation.

[0023] This invention systematically proposes and validates a viral vector sustained-release system using natural blood gel (BG) formed from blood coagulation to manufacture CAR-M cells in situ within the peritoneal cavity. This differs from studies focusing solely on the delivery of a specific drug; instead, it constructs an injectable gel that is "ready to use" and integrates recruitment, sustained release, and transfection functions to modify the microenvironment. The blood gel viral sustained-release system circumvents the complex in vitro manufacturing process of CAR-M cells, utilizing the physiological characteristics of the inherent immune microenvironment within the peritoneal cavity to synergistically achieve gene transfection and macrophage recruitment. This innovative method provides a new paradigm of "targeted, efficient, and safe" treatment for peritoneal tumors. This invention does not rely on a specific virus or cell but provides a fundamental technological framework. The types of viral vectors are scalable (lentivirus, adenovirus, AAV, etc.), and the types of targeted immune cells are scalable (macrophages, dendritic cells, T cells, etc.), providing a powerful, flexible, and safe toolkit for next-generation personalized, in situ immunotherapy.

[0024] Although this invention uses lentiviruses as a vector example, further application of other alternative viruses may lead to higher transfection efficiency, lower toxicity, or stronger targeting capabilities, thereby achieving better results. This invention demonstrates the feasibility of blood gel as a viral vector sustained-release system, providing new insights into the in vivo fabrication of CAR-M.

[0025] Specifically, compared with the prior art, the present invention has the following beneficial effects: (1) Blood gel has excellent protective and sustained-release capabilities against viral vectors. In vitro experiments confirmed that the blood gel could continuously degrade for more than 15 days in PBS at 37°C. In vivo degradation and release were assessed by intraperitoneal injection of DiD-labeled virus encapsulated in either artificial hydrogel or blood gel. In the control group (free virus), the fluorescence signal rapidly decreased, while sustained fluorescence was observed in both the artificial hydrogel and blood gel groups. These results indicate that the sustained-release performance of the blood gel in vivo is comparable to that of conventional hydrogels.

[0026] Major organs and peritoneal lavage fluid were obtained from mice that received either free virus or virus-loaded blood gel. Results showed that in the control group, the virus rapidly spread from the peritoneum to the liver and other organs; fluorescence in the peritoneum no longer constituted the maximum proportion from day 2, and no fluorescence was detected in the control group from day 5. In the virus-loaded blood gel group, the virus persisted in the peritoneum and maintained its maximum proportion for over 14 days. These results indicate that blood gel prolonged the viral retention time in the peritoneum, maintained the maximum proportion of virus in the peritoneum, and reduced the systemic distribution of the virus.

[0027] (2) Blood gel has a strong ability to recruit immune cells. Flow cytometry analysis of peritoneal cells revealed increased macrophage recruitment in the virus-loaded blood gel group compared to the artificial hydrogel group, resulting in an increased number of peritoneal macrophages successfully transduced into CAR-M antibodies. Furthermore, enhanced B cell recruitment was observed in the virus-loaded blood gel group, demonstrating that blood gel has an active recruitment effect on various immune cells.

[0028] (3) Blood gel can be used to prepare functional CAR-M in situ in vivo. Peritoneal macrophages were treated with free virus, virus-loaded artificial hydrogels, or virus-loaded blood gels. The proportion of EGFP⁺ cells in the artificial hydrogel and blood gel groups was significantly higher than that in the control group, indicating successful CAR-M preparation. The proportion of EGFP⁺ cells in the artificial hydrogel and blood gel groups was slightly lower than that in the free virus group, which may be attributed to the sustained-release effect. Compared with the control group and empty vector cells, virus-transduced peritoneal macrophages also showed increased expression of pro-inflammatory markers TNF-α and IL-1β and decreased levels of anti-inflammatory IL-10, indicating that CAR-M exhibits pro-inflammatory polarization.

[0029] (4) High security The blood gel is a natural bio-derived material with no significant systemic toxicity. Preliminary safety assessments of the carrier were conducted, including changes in body weight and H&E staining of major organs. No significant differences in body weight were observed among the groups, indicating that the blood gel did not exhibit systemic toxicity. H&E staining showed no significant damage to the cellular structure, tissue structure, and morphological characteristics of major organs. Attached Figure Description

[0030] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0031] Figure 1 This is a schematic diagram illustrating the preparation and operation process of the blood gel carrier of the present invention.

[0032] Figure 2 This is a scanning electron microscope (SEM) image of the blood gel carrier. Scale bar: 10 micrometers.

[0033] Figure 3 A shows the results of in vitro degradation experiments; A represents representative photographs showing the degradation of BG over time under in vitro conditions; B shows the quantitative analysis of weight loss during the in vitro BG degradation process.

[0034] Figure 4 This is a quantitative fluorescence detection image of DiD in vivo over a period of time after blood gel injection.

[0035] Figure 5This study analyzed the in vivo distribution of blood gels; A shows the fluorescence signal distribution (CV) in major organs and peritoneal lavage fluid of different groups; B shows the average fluorescence intensity in different organs; C shows the percentage of average fluorescence intensity in different organs. Each group had n=3, representing biological replicates. All data were analyzed using GraphPad Prism. Data are presented as mean ± standard deviation and analyzed using one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0036] Figure 6 Analysis of the immune cell recruitment capacity of blood gel; A represents the number of peritoneal macrophages; B represents the number of CAR-M cells; C represents the number of B cells.

[0037] Figure 7 Efficiency and functional validation of in situ CAR-M preparation from blood gel; (A and C) EGFP was analyzed by flow cytometry and confocal microscopy after treatment with CV, HG@CV or BG@CV. + PMφ were analyzed (scale bar 1287, 300 μm); B represents the cytokine secretion profile of PMφ treated with control, pCAR, or CV. Levels of TNF-α, IL-1β, and IL-10 in the culture supernatant were detected by ELISA.

[0038] Figure 8 The results of H&E staining of major organs (heart, liver, spleen, lungs, and kidneys).

[0039] Figure 9 The expression efficiency of the EGFP reporter gene mediated by different delivery systems in cells was detected. A shows the EGFP positivity rate detected by flow cytometry after transfection of T cells with lentivirus loaded on blood gel; B shows the EGFP positivity rate detected by flow cytometry after transfection of NK cells with lentivirus loaded on blood gel; C shows the EGFP positivity rate detected by flow cytometry after transfection of B cells with lentivirus loaded on blood gel; D shows the EGFP positivity rate detected by flow cytometry after transfection of DC cells with lentivirus loaded on blood gel; E shows the EGFP positivity rate detected by flow cytometry after transfection of M cells with adenovirus loaded on blood gel; and F shows the EGFP positivity rate detected by flow cytometry after transfection of M cells with mRNA-LNP loaded on blood gel. Detailed Implementation

[0040] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, 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 invention pertains.

[0041] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0042] A universal in situ engineered carrier for immune cells based on blood gel (BG) was constructed and applied. Formed from blood coagulation, it possesses a porous structure. The choice of blood as the core substrate was not accidental, but rather based on the following three-pronged inventive concept: First, blood is widely available and carries no risk of rejection, providing fundamental feasibility for immediate clinical translation of the technology; second, endogenous materials possess inherent and excellent biocompatibility, minimizing immunogenic reactions and serving as a cornerstone for ensuring therapeutic safety; finally, and most ingeniously, the blood gel itself is not an inert carrier. The fibrin network formed during coagulation and its contained natural bioactive components (such as platelet-derived factors and fibrin degradation products) make it a powerful "immune cell recruiter," efficiently attracting the aggregation of target cells such as macrophages and monocytes, creating the necessary local cell-enriched microenvironment for subsequent in situ engineering.

[0043] The three-dimensional network structure of blood gel (BG) can adsorb and slowly release viral vectors (CVs), thereby prolonging the maintenance time of local viral concentration and reducing the risk of systemic exposure. Furthermore, as a foreign substance, BG induces macrophage aggregation or other recruited immune cells (including but not limited to macrophages and dendritic cell precursors) by activating a monocyte recruitment cascade, placing them in a microenvironment of locally high concentrations of gene vectors formed by the slow release of blood gel. This significantly improves the efficiency of cellular uptake of gene tools such as viral vectors, forming a local functional network of "viral vector-blood gel-macrophage (immune cells)," enabling them to be in situ transformed in vivo into engineered immune cells with novel functions (e.g., CAR-macrophages expressing chimeric antigen receptors).

[0044] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0045] Example 1: Preparation of CAR Lentiviral Virus (CV) A second-generation CAR lentiviral vector (named CV) targeting human CEA (CEACAM5) was constructed. The CAR structure, from N-terminus to C-terminus, consists of: a macrophage-specific promoter CD68, a CD8 leader sequence, a Myc tag, an anti-human CEA single-chain antibody (antiCEA scFv), a CD8 hinge region, a CD8 transmembrane region, a 4-1BB co-stimulatory signaling domain, a CD3ζ signaling domain, a P2A peptide, and enhanced green fluorescent protein (EGFP). The nucleotide sequence of the recombinant expression plasmid containing the coding sequence of the CAR is shown in SEQ ID NO:1. Simultaneously, an enhanced green fluorescent protein (EGFP) reporter gene is linked downstream of the CAR sequence via the P2A peptide for subsequent detection.

[0046] The CAR expression cassette was cloned into a lentiviral backbone plasmid and co-transfected into 293T cells using three plasmids (expression plasmid, psPAX2, and pMD2.G). Cell supernatants were collected at 48 and 72 hours post-transfection and concentrated by ultracentrifugation to obtain high-titer CAR lentivirus (CV). Free CAR plasmid was used as the control group (pCAR).

[0047] The CV prepared in this embodiment was used for all subsequent blood gel loading experiments.

[0048] Example 2: Preparation and Characterization of Blood Gel Carrier Preparation process: The preparation and application process of the blood gel carrier are as follows: Figure 1 As shown. First, approximately 200 μL of peripheral blood was collected from the orbital venous plexus of mice and gently injected into a sterile mold pre-loaded with a CV (chimeric antigen receptor gene vector). The mixture was allowed to stand at room temperature for 15 minutes to initiate spontaneous coagulation. The resulting semi-solid blood gel was then mechanically cut into small pieces to obtain an injectable blood gel carrier formulation. (See figure) Figure 2 Scanning electron microscopy (SEM) revealed that the gel has a porous structure, enabling efficient loading of CV.

[0049] The blood gel carrier described in this invention achieves in situ engineered immune cells and anti-tumor therapy in vivo through the following steps: After intraperitoneal injection of the blood gel carrier formulation, the blood gel plays a recruiting and activating role, attracting macrophages and promoting their uptake of CVs, thereby completing the in situ construction of CAR-M (chimeric antigen receptor macrophages) in the peritoneal cavity. The modified CAR-M can specifically recognize and bind to target antigens (such as CEA) expressed on the surface of tumor cells. Subsequently, the CAR-M infiltrates tumor tissue and directly kills tumor cells through powerful phagocytosis, achieving the treatment of colon cancer and other abdominal tumors.

[0050] Example 3: Verification of the sustained-release ability of blood gel for viral vectors As a control, this invention uses a synthetically produced hydrogel (HG), a conventional thermosensitive hydrogel made of sodium alginate and calcium chloride, to compare its sustained-release performance and cell recruitment ability with that of blood gel. HG@CV was prepared by loading viruses onto the hydrogel, and BG@CV was prepared by loading viruses onto the blood gel.

[0051] The virus-loaded blood gel carrier prepared in Example 1 was incubated in PBS at 37°C with gentle shaking to simulate the peritoneal environment. The results are as follows... Figure 3 The figures show that Figure A is a physical image of the degraded blood gel, showing the morphological changes of the blood gel on days 0, 1, 2, 4, 6, and 8; Figure B is the weight curve of the blood gel carrier degradation, showing that it is almost completely degraded by day 15. In vitro experiments confirm that the blood gel can continue to degrade for more than 15 days.

[0052] In vivo degradation and release were assessed by intraperitoneal injection of DiD-labeled CVs encapsulated in hydrogels (HG@CV) or BG (BG@CV). Figure 4 As shown, the fluorescence signal rapidly decreased in the control group (free CV), while continuous fluorescence was observed in both the HG@CV group and the BG@CV group.

[0053] Major organs and peritoneal lavage fluid were obtained from mice that received free CV (control group) or BG@CV. Results are as follows: Figure 5 The results showed that CVs in the control group rapidly diffused from the peritoneal cavity to the liver and other organs. From day 2, fluorescence in the peritoneal cavity no longer accounted for the largest proportion (the liver had the largest proportion), and from day 5, fluorescence was not detected in the control group. In the BG@CV group, CVs persisted in the peritoneal cavity and maintained the largest proportion for more than 14 days. These results indicate that BG prolonged the residence time of CVs in the peritoneal cavity, maintained the largest proportion of CVs in the peritoneal cavity, reduced the systemic distribution of CVs, and reduced the risk of systemic exposure.

[0054] Example 4: Verification of the immune cell recruitment ability of blood gel Flow cytometry analysis of peritoneal cells revealed that, for example Figure 6 A and Figure 6 As shown in Figure B, compared to the HG@CV group, the BG@CV group showed increased macrophage recruitment, resulting in an increased number of peritoneal macrophages successfully transduced into CAR-M. Furthermore, as... Figure 6 As shown in Figure C, enhanced B cell recruitment was observed in the BG@CV group, demonstrating that BG has an active recruitment effect on a variety of immune cells, laying the foundation for its use as a multi-cell type modification platform.

[0055] Example 5: Efficiency and functional verification of in situ preparation of engineered immune cells from blood gel (taking CAR-M as an example) Peritoneal macrophages (PMφ) were isolated from the peritoneal cavity of BALB / c mice via peritoneal lavage. PMφ cells were divided into four treatment groups: Control group (untreated), CV group (directly inoculated with CAR lentivirus), HG@CV group (CAR lentivirus encapsulated in hydrogel), and BG@CV group (CAR lentivirus encapsulated in blood gel). The proportion of EGFP-positive cells was detected by flow cytometry 48-72 hours after infection.

[0056] The preparation of HG@CV involved mixing CAR lentivirus with a thermosensitive hydrogel precursor solution of calcium chloride and sodium alginate at a 1:1 volume ratio and gently blowing the mixture until homogeneous. The mixture was then added to the cell culture system and incubated at 37°C for 5 minutes to form a gel, thus achieving sustained-release delivery of the virus.

[0057] Preparation of BG@CV: Approximately 200 μL of peripheral blood was collected from the orbital venous plexus of BALB / c mice and immediately injected with lentivirus (CV, 1×10⁻⁶) pre-loaded with CAR. 8 In a sterile mold containing TU / mL, gently pipette to mix. Let the mixture stand at room temperature for 15 minutes to allow it to solidify naturally and form a blood gel loaded with CV (BG@CV). Cut the formed semi-solid gel into approximately 1mm pieces using a sterile scalpel. 3 Small pieces are used to obtain an injectable carrier formulation. This formulation can be used directly in cell treatment experiments.

[0058] The results are as follows Figure 7 A showed that the proportion of EGFP⁺ cells in the HG@CV and BG@CV groups was significantly higher than that in the control group, indicating successful CAR-M preparation. Furthermore, the proportion of EGFP⁺ cells in the HG@CV and BG@CV groups was slightly lower than that in the CV group, which may be attributed to the sustained-release effect of BG or HG. Additionally, as... Figure 7 B showed that, compared with the control group and pCAR cells, CV-transduced PMφ cells also showed increased expression of pro-inflammatory markers TNF-α and IL-1β and decreased level of anti-inflammatory IL-10, indicating that CAR-M exhibits pro-inflammatory polarization.

[0059] To further verify CAR expression morphologically, the cells in each group were fixed, and the nuclei were stained with DAPI. The localization of EGFP (CAR) was then observed using a confocal microscope. The results are as follows: Figure 7 As shown in Figure C, the Control group showed only blue cell nuclei with no green fluorescence signal. The CV, BG@CV, and HG@CV groups all showed significant green fluorescence around the cell nuclei, and the Merge image showed that the green signal was mainly distributed in the cytoplasm and cell membrane region, demonstrating that all three carrier systems could successfully prepare CAR-M.

[0060] Example 6: Safety assessment of blood gel carriers A preliminary safety assessment of the carrier was conducted, such as... Figure 8 As shown, H&E staining revealed no significant damage to the cellular structure, tissue structure, and morphological characteristics of the major organs.

[0061] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0062] Example 7: Detection of EGFP reporter gene expression efficiency mediated by different delivery systems in cells In this embodiment, M cells refer to peritoneal macrophages; T cells, NK cells, B cells, and DC cells were all isolated from the spleen of BALB / c mice.

[0063] In this embodiment, T cells, NK cells, B cells, DC cells, and M cells were used as target cells. Different gene vectors were encapsulated in blood gel for transfection experiments. Transfection of T cells with lentivirus in blood gel, NK cells with lentivirus in blood gel, B cells with lentivirus in blood gel, DC cells with lentivirus in blood gel, M cells with adenovirus in blood gel, and M cells with mRNA-LNP in blood gel were performed. The proportion of EGFP-positive cells was detected by flow cytometry 48-72 hours after transfection. Figure 9 AD showed that the transfection efficiencies of lentivirus loaded on blood gel to T cells, NK cells, B cells and DC cells were 30.4%, 35.5%, 34.3% and 21.3% respectively, which were significantly higher than the expression efficiency of the control group. Figure 9 E showed that after treatment with adenovirus on blood gel, the EGFP positivity rate of M cells was as high as 37.8%, while that of the control group was only 1.96%, indicating that blood gel can significantly enhance the transduction efficiency of adenovirus. Figure 9 F indicates that in the blood gel-loaded mRNA-LNP group, only 1.16% of EGFP-positive cells were detected in the control group M cells, while the positive rate of blood gel-loaded mRNA-LNP increased to 27.6%, a significant improvement. These results demonstrate that blood gel can serve as a universal carrier platform, effectively delivering lentiviruses, adenoviruses, and mRNA-LNPs to different types of immune cells.

Claims

1. A composition for in-situ engineered immune cells, characterized in that, It contains a blood gel formed from blood and a gene delivery vector loaded therein.

2. The composition according to claim 1, characterized in that, The gene delivery vector is selected from lentiviruses, adenoviruses, adeno-associated viruses, poxviruses, herpesviruses, or non-viral gene delivery systems.

3. The composition according to claim 1, characterized in that, The immune cells are selected from macrophages, dendritic cells, monocytes, T cells, NK cells, or neutrophils.

4. The composition according to claim 1, characterized in that, The gene includes a nucleic acid that encodes a chimeric antigen receptor (CAR) protein, the nucleic acid being selected from plasmid DNA, messenger RNA (mRNA), self-replicating RNA (saRNA), or in vitro transcribed complementary RNA (cRNA).

5. The composition according to claim 1, characterized in that, The blood gel is formed from fresh peripheral blood through a natural clotting process; the blood gel has a three-dimensional fibrin network.

6. A method for preparing the composition according to any one of claims 1-5, characterized in that, Includes the following steps: The gene delivery vector is mixed with fresh blood; the mixture is induced to coagulate naturally, forming a blood gel loaded with the gene delivery vector.

7. Use of the composition according to any one of claims 1-5 in the preparation of a medicament for treating tumors, wherein the treatment is achieved by in situ modification of immune cells in vivo.

8. The application as described in claim 7, characterized in that, The tumor is a solid tumor, including colon cancer, stomach cancer, bladder cancer, pancreatic cancer, kidney cancer, liver cancer, or peritoneal metastatic cancer.

9. The application as described in claim 7, characterized in that, The immune cells are macrophages, and the gene delivery vector is a lentivirus carrying the CAR gene, used for in situ preparation of CAR-M in the peritoneal cavity.

10. Use of the composition according to any one of claims 1-5 in the preparation of a product for in situ modification of immune cells in vivo.

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