Cell knapsack based on trans-isooleic acid as well as preparation method and application of cell knapsack

Abstract

The invention discloses a cell knapsack based on trans-isooleic acid and a preparation method of the cell knapsack. The preparation method comprises the following steps: (1) self-assembling trans-isooleic acid and human serum albumin to form TVA / HSA nanoparticles; and (2) carrying out cross-linking compounding on the TVA / HSA nanoparticles by adopting active ester disulfide bond active ester and active ester PEG (Polyethylene Glycol) diphenyl cyclooctyne, so as to obtain the cell knapsack based on trans-isooleic acid. The invention also provides a cell knapsack modified CAR-T cell, and a preparation method of the cell knapsack modified CAR-T cell comprises the following steps: incubating the CAR-T cell by using N-succinimide ester-azide, so that the surface of the CAR-T cell is modified with an azide group; and connecting the cell knapsack loaded with the DBCO group to the surface of the CAR-T cell by adopting a click chemical reaction of azide and DBCO. The cell knapsack disclosed by the invention is branched to the surface of the CAR-T cell in a covalent manner, and TVA is continuously released to enhance the metabolic activity and the anti-tumor effect of the CAR-T cell.

Description

[0001] This invention relates to the field of biomedical technology, and in particular to a cell dorsal sac based on trans isoleic acid, its preparation method, and its application. Background Art

[0002] T cells, or T lymphocytes, are the main components of lymphocytes, primarily responsible for immune responses and disease defense. They play roles such as directly killing target cells, assisting and inhibiting B cell antibody production, responding to specific antigens and mitogens, and producing cytokines. Chimeric antigen receptor T-cell (CAR-T) therapy involves genetically modifying human T cells in vitro to enable them to recognize and efficiently kill tumor cells, before reinfusing them into the patient to treat malignant tumors.

[0003] Chimeric antigen receptor T-cell (CAR-T) therapy has shown significant efficacy in hematological malignancies (such as B-cell leukemia and lymphoma), but it still faces the following challenges in the treatment of solid tumors: (1) Tumor microenvironment (TME) suppression, hypoxia, acidic environment and immunosuppressive factors (such as TGF-β, PD-L1) weaken CAR-T cell function; (2) T cell depletion and long-term antigen stimulation lead to CAR-T cell exhaustion, affecting persistence; (3) Insufficient metabolic adaptation: In the solid tumor microenvironment, the energy metabolism of CAR-T cells (such as fatty acid oxidation and glycolysis) is limited, affecting their proliferation and killing ability.

[0004] Current methods to improve the efficacy of CAR-T therapy include: optimization of co-stimulatory molecules (such as the 4-1BB / CD28 co-stimulatory domain) to enhance activation, but cannot overcome metabolic limitations; immune checkpoint blockade (such as PD-1 inhibitors), which may partially restore T cell function, but has significant systemic side effects; cytokine engineering (such as IL-12 / IL-15 modification), which may trigger cytokine release syndrome (CRS); and metabolic intervention (such as metformin and fatty acid supplementation), which lacks targeted delivery and has low efficiency.

[0005] Trans isoleic acid (TVA) is a long-chain fatty acid found in beef, lamb, and dairy products. It is a naturally occurring trans fatty acid. The human body cannot synthesize TVA on its own and must obtain it through diet (such as beef, lamb, and dairy products). Approximately 80% of the ingested TVA circulates in the bloodstream, while the remainder is metabolized into other substances. TVA can activate the downstream cAMP-PKA-CREB signaling pathway and increase CD8 levels by inhibiting the binding of the GPR43 receptor. +T cells have functions such as proliferation and degranulation, which promote T cell-dependent tumor killing. However, TVA cannot be synthesized in the human body and can only be obtained through exogenous intake. The existing delivery methods have the following problems: (1) poor solubility, TVA is highly hydrophobic and direct addition is inefficient; (2) lack of targeting, unable to act precisely on CAR-T cells; (3) insufficient stability, easily cleared in the body, making it difficult to maintain an effective concentration.

[0006] Existing liposomes and exosomes used for CAR-T modification have the following drawbacks: low loading efficiency, making it difficult to efficiently encapsulate small molecule fatty acids; poor binding stability, resulting in weak binding to the CAR-T cell surface and easy detachment; and insufficient metabolic regulation, failing to fully utilize fatty acid metabolism to enhance T cell function. Summary of the Invention

[0007] This invention provides a cell dorsal capsule based on trans isoleic acid and its preparation method. The cell dorsal capsule can be covalently branched to the surface of CAR-T cells and continuously release TVA to enhance the metabolic activity and anti-tumor effect of CAR-T cells.

[0008] The technical solution of the present invention is as follows: A method for preparing a cell dorsal sac based on trans isoleic acid includes the following steps: (1) Trans isoleic acid (TVA) and human serum albumin (HSA) are self-assembled to form TVA / HSA nanoparticles; (2) The TVA / HSA nanoparticles were cross-linked and compounded with active ester disulfide bond active ester (NHS-SS-NHS) and active ester PEG diphenylcyclooctylene (NHS-PEG-DBCO) to obtain cell dorsal sac based on trans isoleic acid.

[0009] Preferably, the mass ratio of trans isoleic acid to human serum albumin is 1:5~20.

[0010] Preferably, the average particle size of the TVA / HSA nanoparticles is 200~700 nm; more preferably, it is 500~700 nm.

[0011] Preferably, step (1) includes: adding trans isoleic acid organic solution dropwise into a buffer solution containing human serum albumin to form a self-assembly system, and performing composite self-assembly of trans isoleic acid and human serum albumin under vortex conditions to obtain TVA / HSA nanoparticles. In the self-assembled system, the concentration of human serum albumin is 0.5~5 mg / mL; the mass ratio of trans isoleic acid to human serum albumin is 1:5~20.

[0012] Preferably, the volume concentration of the organic solvent in the self-assembly system is no more than 5%, in order to avoid the organic solvent content being too high and interfering with the self-assembly process of the composite.

[0013] Preferably, the buffer solution is DPBS; and the organic solvent is dimethyl sulfoxide.

[0014] In step (2), the active ester disulfide bond active ester is N-hydroxysuccinimide ester-disulfide bond-disuccinimide ester, and the active ester PEG diphenylcyclooctylene is N-hydroxysuccinimide-polyethylene glycol-dibenzocyclooctylene.

[0015] Preferably, step (2) includes: mixing an active ester disulfide bond active ester organic solution with an active ester PEG diphenylcyclooctylene organic solution to form a mixed crosslinking agent solution; adding the mixed crosslinking agent solution dropwise into a TVA / HSA nanoparticle solution to form a mixed crosslinking system; and obtaining a cell dorsal sac loaded with DBCO groups through a crosslinking reaction.

[0016] Preferably, in the mixed crosslinking system, the molar ratio of the active ester disulfide bond active ester to the active ester PEG diphenylcyclooctylene is 1~10:1; the mass ratio of the active ester PEG diphenylcyclooctylene to TVA / HSA nanoparticles is 1:50~100.

[0017] Preferably, the average particle size of the cell dorsal sac is 1000~1500nm; more preferably, it is 1200~1400nm.

[0018] The present invention also provides a cell dorsal sac prepared by the above preparation method.

[0019] The cell dorsal capsule of the present invention can be covalently grafted to the surface of CAR-T cells to continuously release TVA, thereby enhancing the metabolic activity and anti-tumor effect of CAR-T cells.

[0020] This invention also provides CAR-T cells modified with cell dorsal sacs, the preparation method of which includes: CAR-T cells were incubated with N-succinimide ester-azide (NHS-N3) to modify the surface of CAR-T cells with azide groups. A click chemistry reaction of azide and DBCO was used to attach DBCO-loaded cell dorsal capsules to the surface of CAR-T cells.

[0021] Preferably, the CAR-T cells are CD19 CAR-T cells.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The cell dorsal capsule based on trans isoleic acid of the present invention can be covalently branched to the surface of CAR-T cells, and can continuously release TVA on the surface of CAR-T cells, enhance the metabolic function of CAR-T cells, and enhance their resistance to depletion and anti-tumor activity.

[0023] (2) The present invention is based on the covalent cross-linking of trans isoleic acid cell dorsal capsules with CAR-T cells to ensure that the cell dorsal capsules are firmly bound to CAR-T cells, avoid detachment in vivo, and have high stability.

[0024] (3) The modification technology of the present invention is simple and does not require complex genetic engineering modification. It can be combined with existing CAR-T cell preparation processes.

[0025] (4) HSA is an endogenous protein in the human body, and TVA is a lipid metabolite present in the human body. Both have high biosafety and low immunogenicity. Attached Figure Description

[0026] Figure 1 Particle size and zeta potential of TVA / HSA nanoparticles; Figure 2 Particle size and zeta potential of BP nanoparticles; Figure 3 Scanning electron microscopy images of BPs modified on the surface of CAR-T cells; Figure 4 The killing effect of TVA-sensitized CAR-T cells on Ramos cells is shown in the following figures: (a) ET=1:3, (b) ET=1:1, and (c) ET=3:1. Figure 5 The expression levels of CAR-T cell exhaustion-related markers under continuous antigen stimulation are shown in (a) for PD-1, (b) for LAG-3, and (c) for TIM-3. Figure 6 The diagram shows the inhibitory effect of TVA treatment on the expression of CAR-T cell exhaustion-related markers: (a) PD-1, (b) LAG-3, and (c) TIM-3. Figure 7 The image shows the effect of BPs on enhancing the ability of CAR-T cells to resist exhaustion in Example 1. (a) is PD-1, (b) is LAG-3, and (c) is TIM-3. Figure 8 The following are the effects of BPs on the killing ability of CAR-T cells in Example 1: (a) is the effector-to-target ratio ET=1:3, (b) is the effector-to-target ratio ET=1:1, and (c) is the effector-to-target ratio ET=3:1. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0028] This invention relates to TVA-based cell backpacks (BPs), which covalently attach to the surface of CAR-T cells, continuously releasing TVA to enhance their metabolic activity and anti-tumor effects. This technology exhibits strong targeting, good stability, good compatibility, and no toxic side effects.

[0029] Example 1 Methods for preparing TVA-based cell dorsal sacs include: (1) Preparation of TVA / HSA nanoparticles A self-assembly method was used to assemble trans-isooleic acid (TVA) and human serum albumin (HSA) into nanoparticles (200-500 nm in diameter). The mass ratio of TVA / HSA (1:5~1:20) and the proportion of organic solvent (5%~20%) were adjusted to prepare nanoparticles with uniform particle size, good dispersibility and strong stability.

[0030] The following steps are involved: (1-1) Solution preparation: TVA solution: Weigh trans isoleic acid (TVA), prepare a 100 mM stock solution using dimethyl sulfoxide (DMSO), and store at -20 ℃ protected from light for later use.

[0031] HSA solution: Weigh human serum albumin (HSA) and prepare a 20 mg / mL stock solution using Dulbecco's phosphate buffer (DPBS, pH 7.4). Prepare fresh before use.

[0032] (1-2) Design of the nanoparticle preparation system: TVA / HSA nanoparticles were prepared using a self-assembly method. The final volume percentage of DMSO in the system was controlled at 5% to avoid interference from excessive organic solvent content on the self-assembly process. Referring to the concentration of HSA under human physiological conditions, the final concentration of self-assembled HSA was set at 1 mg / mL, and the mass ratio of TVA to HSA was 1:10.

[0033] (1-3) Nanoparticle preparation steps: Take a 1.5 mL transparent EP tube and add 25 μL of HSA stock solution (20 mg / mL) and 450 μL of DPBS sequentially. Mix gently and then sonicate the tube in a 40 ℃ water bath for 10 minutes to fully disperse the HSA molecules. While sonicating, prepare the TVA working solution: mix 1.77 μL of TVA stock solution (100 mM) with 23.23 μL of DMSO and gently vortex. After sonication in the water bath, transfer the HSA solution to a 2 mL glass bottle and place the bottle on a small magnetic vortex apparatus. Place a suitable magnetic rotor in the bottle and set the vortex speed to low (500 rpm). Under continuous vortexing, slowly add the prepared TVA working solution dropwise to the HSA solution using a pipette to ensure uniform complexation and self-assembly of TVA and HSA molecules. After the addition is complete, continue vortexing at low speed for 60 minutes to fully stabilize the complex.

[0034] (1-4) DLS measurement of TVA / HSA nanoparticle size and zeta potential: After the TVA / HAS nanoparticles were prepared, their particle size and zeta potential were characterized using a potentiometric particle size analyzer. The prepared nanoparticles were diluted 10-fold with DPBS to ensure the particle concentration remained within the instrument's optimal detection range during the assay. One mL of the diluted solution was slowly transferred to a Malvern Zetasizer cuvette using a pipette. Vigorous agitation during transfer should be avoided to prevent air bubble formation and ensure accurate results. Figure 1 The experimental results showed that the average particle size of TVA / HSA nanoparticles was 576±38.76 nm, the average zeta potential was -11.8±0.458 mV, and the average dispersion index (PDI) was 0.109±0.0093.

[0035] (2) Construction of BPs The prepared TVA / HSA nanoparticles were cross-linked and composited with active ester disulfide bond active ester (NHS-SS-NHS) and active ester PEG diphenylcyclooctylene (NHS-PEG-DBCO) to prepare BPs with larger particle size and loaded with DBCO groups.

[0036] The following steps are involved: (2-1) Solution preparation: NHS-SS-NHS solution: Weigh N-hydroxysuccinimide ester-disulfide bond-disuccinimide ester (NHS-SS-NHS), prepare a 100 mM stock solution using anhydrous DMSO, and store at -20 ℃.

[0037] NHS-PEG-DBCO solution: Weigh N-hydroxysuccinimide-polyethylene glycol-dibenzocyclooctylene (NHS-PEG-DBCO) and prepare a stock solution of 250 μg / mL using sterile DPBS. Store at -20 °C.

[0038] (2-2) Preparation of cross-linked mixture system: According to the molar ratio of n(NHS-SS-NHS): n(NHS-PEG-DBCO) = 5:1, measure 7.3 μL of NHS-SS-NHS stock solution (100 mM) and 1.5 μL of NHS-PEG-DBCO stock solution (250 μg / mL) respectively, add them to sterile EP tubes, and gently pipette to mix, thus preparing a cross-linked mixture. After preparation, store the mixture on ice in the dark for later use.

[0039] (2-3) Crosslinking modification of TVA / HSA nanoparticles: Take 500 μL of the pre-prepared TVA / HSA nanoparticle solution into a 2 mL glass bottle. Place the glass bottle on a magnetic vortex apparatus, insert a suitable magnetic rotor, and set a low-speed vortex (500 rpm) to keep the nanoparticle solution in a continuous vortex state. Under vortex conditions, use a pipette to slowly add 8.8 μL of the prepared crosslinking mixture to the TVA / HSA nanoparticle solution to ensure sufficient contact and uniform reaction between the crosslinking agent and the nanoparticles (DBCO-linked nanoparticles rely on the binding of NHS esters to the amino groups on the surface of HSA proteins, so its calculation is based on the amount of HSA, and the final mass ratio of NHS-PEG-DBCO to nanoparticles is 1:66.67). After the addition is complete, continue the low-speed vortex reaction for 30 minutes to allow NHS-SS-NHS to be fully crosslinked on the nanoparticle surface, forming submicron-sized complexes (BPs) with larger particle sizes.

[0040] (2-4) DLS measurement of BP particle size and zeta potential: After the BPs were prepared, their particle size and zeta potential were characterized using a potential particle size analyzer. For example... Figure 2 The experimental results showed that the average particle size of BPs was 1269±57.66 nm, the average zeta potential was -13.83±0.404 mV, and the average dispersion index (PDI) was 0.195±0.017.

[0041] (3) BPs covalently modify the surface of CAR-T cells CAR-T cells were incubated with N-succinimide-azide (NHS-N3) for 30 min to modify the surface of CAR-T cells with azide groups. Subsequently, a click chemistry reaction of azide with DBCO was used to attach BPs to the surface of CAR-T cells.

[0042] The following steps are involved: (3-1) Preparation of NHS-N3 solution: Weigh N-hydroxysuccinimide-azide (NHS-N3) and prepare a 50 mM stock solution using DPBS. Store at -20 °C. Take 1 μL of the 50 mM NHS-N3 stock solution and add it to 2.5 mL of DPBS. Gently vortex to mix and prepare a 20 μM working solution for later use.

[0043] (3-2) Azide group modification in CAR-T cells: CD19 CAR-T cells were collected and washed twice with DPBS (1500 rpm, 5 min) to remove residual components from the culture medium. The cells were then resuspended in DPBS and the cell density was adjusted to 1 × 10⁶ cells / mL. 6 Add 20 μM NHS-N3 working solution per milliliter to allow NHS-N3 to react with the amino groups on the cell surface, modifying the azide group (-N3). Incubate the cell suspension at room temperature (25°C) with low-speed rotation (50 rpm) for 30 minutes to ensure the reaction proceeds fully. After incubation, centrifuge at 1200 rpm for 3 minutes, discard the supernatant, and wash the cells twice with DPBS to remove unreacted NHS-N3. Resuspend the modified cells (N3-CAR-T) in 500 μL of DPBS for later use.

[0044] (3-3) Click chemistry of BPs with N3-CAR-T cells: Take the pre-prepared BPs and adjust the concentration to 20 μM (based on TVA concentration).

[0045] Add the BPs solution to the N3-CAR-T cell suspension and mix gently to allow DBCO to undergo a click chemical reaction with the azide group, forming a stable covalent link.

[0046] The mixture was incubated at room temperature (25 °C) for 30 minutes at low speed (50 rpm) to ensure the reaction proceeded fully. After incubation, the cells were centrifuged at 1200 rpm for 3 minutes, the supernatant was discarded, and the cells were washed twice with DPBS to remove unbound BPs. The modified cells (BPs-CAR-T) were resuspended in an appropriate amount of DPBS or culture medium for subsequent experiments.

[0047] (3-4) Evaluation of BP modification efficiency using scanning electron microscopy Cell surface morphology was characterized using JEOL field emission scanning electron microscopy.

[0048] Cell sample preparation: Sampling, approximately 5 × 10⁶ cells in the logarithmic growth phase. 6 Collect the cells into a 1.5 mL clear centrifuge tube, centrifuge at 500 g for 3 min, discard the supernatant, wash once with 1×PBS, centrifuge at 500 g for 3 min to remove the PBS, and a pea-sized cell pellet should be visible at the bottom of the tube. Fixation (glutaraldehyde-osmium tetroxide double fixation method): Slowly add 1 mL of glutaraldehyde electron microscopy fixative along the tube wall (be careful not to blow away the cell clumps), fix at 4 ℃ for 4 hours (or overnight), after glutaraldehyde fixation, wash three times with 0.1 M phosphate buffer, 15 min each time; after washing, fix with 1% osmium tetroxide for 2–4 hours, after osmium tetroxide fixation, wash three times with 0.1 M phosphate buffer, 15 min each time.

[0049] The cell surface morphology was examined using a scanning electron microscope. For example... Figure 3 Scanning electron microscopy (SEM) images showed that blood biomarkers (BPs) were stably loaded onto the surface of CAR-T cells in a mesh-like structure, which was clearly visible in the highlighted areas. High-resolution imaging revealed the tight binding between BPs and the CAR-T cell membrane, forming a uniform and stable modification layer.

[0050] Comparative Example 1 TVA enhances CAR-T cell function validation: (1) TVA sensitizes CAR-T cell killing: In co-culture experiments, CD19 CAR-T cells and target Ramos cells were co-incubated at effector-to-target ratios of 1:3, 1:1, and 3:1. The experiments were conducted in 96-well cell culture plates, with 100 μL of cell suspension added to each well, resulting in a total cell count of 1 × 10⁶ cells. 5 During co-incubation monitoring, the cells were divided into two groups: one group without TVA and the other group treated with 20 μM TVA. Subsequently, dead cells were stained in real-time using PI dye, and the cells were continuously monitored for 48 hours using the Incucyte live cell analysis system to dynamically assess the cytotoxic ability of CAR-T cells against Ramos cells.

[0051] like Figure 4 The experimental results showed that, compared with the untreated negative control (NC) group, TVA treatment significantly enhanced the killing effect of CAR-T cells on Ramos cells.

[0052] (2) TVA resists CAR-T cell depletion: First, a CAR-T cell exhaustion model was constructed using a co-culture system of CAR-T cells and Ramos cells to establish a CAR-T cell exhaustion phenotype induced by long-term antigen exposure. CAR-T cells and Ramos cells were mixed at an effector-to-target ratio of 3:1 and seeded in RPMI-1640 complete medium containing 10% fetal bovine serum (FBS). A control group was set up: CAR-T cells were cultured alone (without antigen stimulation). The co-culture system was incubated at 37 ℃ and 5% CO2 for 10 days, with the medium replaced every 48 hours to maintain T cell viability. During the co-culture process, fresh Ramos cells (effector-to-target ratio of 3:1) were added every 48 hours to maintain a high-intensity antigen-stimulated state of CAR-T cells.

[0053] Detection of CAR-T cell exhaustion phenotype: After 5 days of co-culture, CAR-T cells were collected and washed twice with PBS to remove residual culture medium and cell debris. CAR-T cells were surface-stained with fluorescently labeled antibodies to detect the expression levels of exhaustion-related markers, including PD-1, TIM-3, and LAG-3. Isotype controls were included to exclude non-specific staining interference. Data were acquired using flow cytometry (Thermo Attune NxT), with at least 10,000 cell events collected for each sample. The proportion of positive cells for PD-1, TIM-3, and LAG-3 and the mean fluorescence intensity (MFI) were analyzed using FlowJo software (v10.10).

[0054] like Figure 5 Flow cytometry results showed that compared with unstimulated CAR-T cells, prolonged antigen stimulation significantly upregulated the expression levels of CAR-T cell surface exhaustion markers PD-1, LAG-3, and TIM-3 (p<0.01), indicating that CAR-T cells exhibited a functional exhaustion phenotype under continuous antigen exposure.

[0055] Next, the inhibitory effect of TVA on CAR-T cell exhaustion was investigated. In a co-culture system of CAR-T cells and Ramos cells, the following experimental groups were set up: Experimental group: 20 μM TVA was added (TVA stock solution was prepared with DMSO); Control group: An equal volume of DMSO was added as a negative control (NC group). After 5 days of co-culture, CAR-T cells were collected, and the expression levels of PD-1, LAG-3, and TIM-3 were detected according to the above method. Figure 6The experimental results showed that, compared with the NC group, TVA treatment significantly inhibited the upregulation of PD-1 expression on the surface of CAR-T cells (reduced positive cell ratio, p<0.01) for 5, 7, and 10 days, and also had a certain inhibitory effect on the expression levels of LAG-3 and TIM-3. TVA can partially reverse the functional exhaustion phenotype of CAR-T cells by inhibiting the upregulation of exhaustion indicators, thereby enhancing their anti-tumor activity.

[0056] Example 2 Example 1: Validation of BPs-enhanced CAR-T cell function (1) BPs enhance the ability of CAR-T cells to resist exhaustion: The exhaustion index detection method in Comparative Example 1 was used to evaluate the degree of exhaustion of CAR-T cells by BPs under continuous stimulation by tumor antigens.

[0057] like Figure 7 Under continuous antigen stimulation for 5 and 7 days, CAR-T cells modified with BPs showed lower cell exhaustion indices, with significant downregulation of PD1, LAG3, and TIM3 (p<0.01). This indicates that BP modification enhances the cell exhaustion resistance of CAR-T cells.

[0058] (2) BPs enhance the killing ability of CAR-T cells The cell killing assay method in Comparative Example 1 was used to evaluate the enhancement effect of BPs on the killing of Ramos target cells by CAR-T cells.

[0059] like Figure 8 The experimental results showed that CAR-T cells modified with BPs had a stronger killing effect on target Ramos cells, with the killing efficiency increasing to 250% at an effector-to-target ratio of 1:3, 230% at an effector-to-target ratio of 1:1, and 190% at an effector-to-target ratio of 3:1. This indicates that BP modification enhances the anti-tumor effect of CAR-T cells.

[0060] The embodiments described above provide a detailed explanation of the technical solutions and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, and equivalent substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. (1) Trans isoleic acid and human serum albumin are self-assembled to form TVA / HSA nanoparticles; (2) The TVA / HSA nanoparticles were cross-linked and composited with active ester disulfide bond active ester and active ester PEG diphenylcyclooctylene to obtain cell dorsal sac based on trans isoleic acid.

2. The method for preparing a cell dorsal sac according to claim 1, characterized in that, The mass ratio of trans isoleic acid to human serum albumin is 1:5~20.

3. The method for preparing a cell dorsal sac according to claim 1, characterized in that, The average particle size of the TVA / HSA nanoparticles is 200~700 nm.

4. The method for preparing a cell dorsal sac according to claim 1, characterized in that, The average particle size of the cell dorsal sac is 1000~1500nm.

5. The method for preparing a cell dorsal sac according to claim 1, characterized in that, Step (1) includes: adding trans isoleic acid organic solution to a buffer solution containing human serum albumin to form a self-assembly system, and then performing composite self-assembly of trans isoleic acid and human serum albumin under vortex conditions to obtain TVA / HSA nanoparticles.

6. The method for preparing a cell dorsal sac according to claim 5, characterized in that, In the self-assembled system, the concentration of human serum albumin is 0.5~5 mg / mL; the mass ratio of trans isoleic acid to human serum albumin is 1:5~20.

7. The method for preparing a cell dorsal sac according to claim 1, characterized in that, Step (2) includes: mixing an active ester disulfide bond active ester organic solution with an active ester PEG diphenylcyclooctylene organic solution to form a mixed crosslinking agent solution; adding the mixed crosslinking agent solution dropwise into a TVA / HSA nanoparticle solution to form a mixed crosslinking system; and obtaining a cell dorsal sac loaded with DBCO groups through a crosslinking reaction.

8. The method for preparing a cell dorsal sac according to claim 7, characterized in that, In the mixed crosslinking system, the molar ratio of active ester disulfide bond active ester to active ester PEG diphenylcyclooctylene is 1~10:

1.

9. A cell dorsal sac prepared by the preparation method according to any one of claims 1 to 8.

10. A CAR-T cell modified with a cell dorsal sac as described in claim 9, characterized in that, Its preparation methods include: CAR-T cells were incubated with N-succinimide ester-azide to modify the surface of CAR-T cells with azide groups. A click chemistry reaction between azide and DBCO was used to attach DBCO-loaded cell dorsal capsules to the surface of CAR-T cells.

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