Preparation method and application of an antitumor preparation based on supramolecular hydrogel delivery of engineered NK cells

Abstract

The application relates to the field of biological medicines, and discloses a preparation method and application of an anti-tumor preparation based on a supramolecular hydrogel for delivering engineered NK cells. The anti-tumor preparation comprises IL15-loaded microcapsules, NK92 cells modified on the surface of the microcapsules, and an injectable supramolecular hydrogel embedding the modified NK92 cells; the IL15-loaded microcapsules are prepared by taking calcium carbonate microspheres as templates, depositing a multilayer film of PAH / PSS alternately, coating a PAH / alded hyaluronic acid layer, and removing the calcium carbonate cores. The application breaks through the traditional cell delivery concept, constructs a local and self-sustaining immune-regulating ecosystem at a tumor site, and provides a powerful new means for resisting solid tumors.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and more specifically, it relates to a method for preparing and applying an engineered NK cell antitumor agent based on supramolecular hydrogel delivery. Background Technology

[0002] Natural killer (NK) cell-based immunotherapy is a promising approach to cancer treatment, as it can identify and kill tumor cells without prior sensitization. Adoptive NK cell therapy has shown significant efficacy in hematologic malignancies, but its effectiveness in solid tumors remains limited. Key challenges include poor tumor invasiveness, short in vivo retention time, and rapid functional depletion within the immunosuppressive tumor microenvironment (TME). These factors collectively prevent it from exerting a potent and sustained anti-tumor effect, significantly restricting its clinical application.

[0003] Various strategies for enhancing the anti-tumor activity of NK cells have been explored, including genetic engineering, immune checkpoint blockade, and cytokine pretreatment. Among these, cytokine-based regulation, particularly interleukin-15 (IL15), has shown great potential. IL15 regulates the interaction between NK cells and CD8+. + IL-15 is a core cytokine for T cell homeostasis and activation, making it an ideal choice for enhancing cellular immunotherapy. However, IL-15 has a short plasma half-life, limiting its clinical translation and requiring high-frequency, high-dose administration, which can easily lead to severe dose-limiting toxicities such as capillary leakage syndrome and cytokine release syndrome. Against this backdrop, injectable hydrogels have attracted much attention because they can serve as local reservoirs for therapeutic cells and cytokines, enabling sustained release and significantly improving local retention and bioavailability. However, a key limitation remains: simply co-encapsulating cells and cytokines in a hydrogel cannot guarantee the continued coupling of key survival signals as cells actively infiltrate the harsh tumor microenvironment, leading to easy cell inactivation after infiltration.

[0004] Therefore, developing a novel bio-hybrid system that can simultaneously achieve long-term retention of NK cells, continuous in situ activation, and cascade activation of endogenous anti-tumor immunity has significant clinical value and application prospects. Summary of the Invention

[0005] The purpose of this invention is to provide a novel bio-hybrid system that can simultaneously achieve long-term retention of NK cells, continuous in-situ activation, and cascade activation of endogenous anti-tumor immunity.

[0006] To achieve the aforementioned objectives, this application first synthesizes IL15-loaded microcapsules, then modifies the surface of NK92 cells using these microcapsules as functional materials, and finally embeds the engineered NK92 cells in an injectable hydrogel formed from hydrophobically modified hydroxypropyl methylcellulose (HPMC-C12), polyethylene glycol-polylactic acid (PEG-PLA) nanoparticles, and α-cyclodextrin (α-CD). This application breaks through the traditional cell delivery model, successfully constructing a self-sustaining local immune ecosystem, providing a robust new strategy with clinical translational potential for solid tumor treatment.

[0007] Specifically, this application adopts the following technical solution:

[0008] In a first aspect, this application provides a supramolecular hydrogel-based delivery system for engineered NK cell antitumor agents, including IL-loaded... 15 microcapsules, NK92 cells modified on the surface of the microcapsules, and an injectable supramolecular hydrogel encapsulating the modified NK92 cells; The load IL The microcapsules of 15 were obtained by using calcium carbonate microspheres as templates, depositing multiple layers of films alternately with PAH / PSS, then coating them with a PAH / aldehyde-based hyaluronic acid layer, and finally removing the calcium carbonate core.

[0009] Secondly, the preparation method of engineered NK cell antitumor agents based on supramolecular hydrogel delivery described in this application includes the following steps: Step 1, Prepare IL-loaded 15% calcium carbonate microspheres were prepared by alternating deposition of PAH / PSS on the surface of the microspheres using a layer-by-layer self-assembly technique, followed by coating with aldehyde-modified hyaluronic acid. After removing the core, IL-loaded microspheres were obtained. 15 microcapsules; Step 2, transfer the load IL 15 microcapsules were co-incubated with NK92 cells to allow the IL-loaded cells to... 15 microcapsules are chemically bonded to the cell surface to obtain IL-loaded microcapsules. 15 microcapsule-modified engineered NK92 cells; Step 3, Preparation of HPMC C12, PEG PLA nanoparticles and α An injectable supramolecular hydrogel containing cyclodextrin was used to embed engineered NK92 cells within the hydrogel, resulting in a supramolecular hydrogel-based delivery system for engineered NK cell antitumor agents.

[0010] Further, step 1 includes: Sodium carbonate solution was added to calcium chloride solution, and the mixture was stirred vigorously at room temperature. After centrifugation, washing, and drying, calcium carbonate microspheres were obtained. Calcium carbonate microspheres were dispersed in buffer solution, and IL15 solution was added and mixed. The mixture was then shaken and incubated to obtain IL15-loaded calcium carbonate microspheres. Sodium hyaluronate was dissolved in deionized water, and sodium periodate solution was added. The mixture was stirred at room temperature in the dark, and PBS buffer was added until completely dissolved to obtain a precipitate. The precipitate was then purified to obtain aldehyde-modified hyaluronic acid. IL15-loaded calcium carbonate microspheres were incubated alternately in PAH and PSS solutions for multiple cycles. Then, they were incubated sequentially in PAH and aldehyde-modified hyaluronic acid solutions. Finally, the microspheres were incubated with EDTA to remove the calcium carbonate core, and the IL15-loaded microcapsules were collected by centrifugation.

[0011] Further, step 2 includes: NK92 cells were resuspended in serum-free medium and seeded in 12-well plates. IL15-loaded calcium carbonate microspheres (MA to cell ratio 3:1) were added to the cell culture medium. After centrifugation, the IL15-loaded calcium carbonate microspheres were brought into contact with the cells. After co-incubation, the cell suspension was placed on lymphocyte separation medium and the engineered cells were purified by density gradient centrifugation. The cells were then washed and collected.

[0012] Furthermore, the ratio of the IL15-loaded calcium carbonate microspheres to NK92 cells is 3:1.

[0013] Further, step 3 includes: HPMC was dissolved in N-methylpyrrolidone, dodecyl isocyanate was added, and then N,N-diisopropylethylamine was added as a catalyst. The reaction solution was stirred at room temperature, and the resulting solution was precipitated with acetone. The hydrophobically modified HPMC was collected by filtration and vacuum dried at room temperature to obtain a white amorphous powder HPMC-C12. A solution of PEG-b-PLA in N,N-dimethylformamide was added dropwise to ultrapure water under stirring to obtain PEG. PLA nanoparticles; Dissolve HPMC-C12 in PBS buffer to prepare a stock solution, fill a syringe with the solution, and add PEG to another syringe. PLA nanoparticle stock solution, α-CD stock solution and engineered NK92 cell suspension; The two syringes were connected by a double-port connector and repeatedly injected to mix them, thereby obtaining an engineered NK cell anti-tumor agent based on supramolecular hydrogel delivery.

[0014] Thirdly, this application provides the application of the aforementioned supramolecular hydrogel-based engineered NK cell antitumor agents in the preparation of drugs for treating solid tumors.

[0015] Furthermore, the solid tumor is breast cancer.

[0016] Fourthly, this application provides a pharmaceutical composition comprising the aforementioned supramolecular hydrogel-based engineered NK cell antitumor agent.

[0017] Fifthly, this application provides the use of the pharmaceutical composition in the preparation of a drug for treating solid tumors.

[0018] In summary, this application has the following beneficial effects: This application successfully constructs a living biohybrid platform that synergistically integrates MA (manipulated amino acids) with NK92 cells and an injectable supramolecular hydrogel, providing a robust and transformable new strategy for local immunotherapy of solid tumors. This platform simultaneously addresses a classic challenge in NK cell therapy against solid tumors—ensuring persistent tumor residency and maintaining functional durability within an immunosuppressive tumor microenvironment. The supramolecular hydrogel, acting as a dynamic protective microenvironment, enables long-term local retention and controlled release of NK92 cells. More importantly, the IL15-loaded MA serves as a cytokine reservoir, providing persistent in-situ activation signals to NK92 cells, achieving potent tumor killing. The core finding of this application is that this platform can initiate a self-propagating immune cascade: the MA, loaded onto infiltrating NK92 cells, delivers IL15 into the tumor, effectively activating dormant cytotoxic CD8. + T cells were used, and these results were validated in an in vitro model of human breast cancer. This application breaks through the traditional concept of cell delivery, constructing a local, self-sustaining immune regulatory ecosystem at the tumor site, providing a powerful new approach to combat solid tumors. Attached Figure Description

[0019] Figure 1 Preparation and characterization of MA-loaded NK92 cells; (a) AFM image of MA; scale bar = 4 μm. (b) Schematic diagram of cell modification process based on aldehyde (Ald) modification of hyaluronic acid surface. (c–d) Representative flow cytometry plots (c) and quantitative analysis (d) of FITC-labeled cell binding ratios with and without aldehyde modification, n = 4. (e–f) Three-dimensional confocal imaging (e) and two-dimensional imaging (f) of MA-loaded NK92 cells; NK92 cells were labeled with PKH26 (orange), MA was labeled with FITC (green), and cell nuclei were labeled with DAPI (blue); scale bar = 2 μm. (g) Cryo-scanning electron microscopy (cryo-SEM) image of the surface morphology of MA-loaded NK92 cells; scale bar = 1 μm. (h) Percentage of MA remaining on the surface of NK92 cells after 5 days of in vitro culture, n = 3. (i) Cumulative release curve of IL15 in MA at 37 °C, n = 3.

[0020] Figure 2 Preparation and characterization of HPMC-based supramolecular hydrogels for embedding engineered NK92 cells; (a) Schematic diagram of injectable HPMC-based supramolecular hydrogels constructed based on dual supramolecular interactions. (b) Rheological frequency scanning curves of different hydrogels at 0.5% strain. (c) Shear rates 0.1–100 s⁻¹. -1 (d) Shear viscosity scanning curves of different hydrogels within the range. (e-f) Cryo-SEM image of PNP1 / 5-5CD hydrogel (inset) and its porous structure; scale bar = 200 μm. (e-f) Degradation process of Dil-labeled PNP1 / 5-5CD hydrogel under mouse skin observed by in vivo imaging system (IVIS) (e) and quantitative results of fluorescence intensity at each time point (f), n=3. (g) Live / dead staining confocal images of MA-loaded NK92 cells cultured in PNP1 / 5-5CD hydrogel for 3 and 7 days, reflecting cell activity and proliferation. (h) In vitro cumulative release curve of engineered NK92 cells from PNP1 / 5-5CD hydrogel.

[0021] Figure 3 Durable and potent in vitro function of NK92 cells with surface-bound MA; (a) Schematic diagram of the in vitro experimental protocol for evaluating the sustained activation effect of MA modification on NK92 cells; NK92 cells were treated with PBS, free IL15, blank MA, and IL15-loaded MA for 24 h or 72 h, respectively. (b–c) Representative histogram (b) and mean fluorescence intensity (MFI) analysis of CD107a expression on the surface of NK92 cells after 24 h of treatment, n=3. (d–e) Representative histogram (d) and mean fluorescence intensity analysis of IFN-γ expression in NK92 cells after 24 h of treatment, n=3. (f–g) Representative histogram (f) and quantitative analysis of mean fluorescence intensity of CD107a expression on the surface of NK92 cells after 72 h of treatment, n=3. (h–i) Representative histogram (h) and quantitative analysis of mean fluorescence intensity of IFN-γ expression in NK92 cells after 72 h of treatment, n=3. (j) Schematic diagram of the in vitro experimental protocol for evaluating the sustained tumor-killing effect of MA modification on NK92 cells; NK92 cells were pretreated with free IL15 or MA modification for 24 h or 72 h, and then co-cultured with various breast cancer cell lines at different effector-to-target ratios (E:T). (k–l) Killing activity of NK92 cells against MDA-MB-231(k) and 4T1(l) breast cancer cells after 24 h of treatment, n=3. (m–n) Killing activity of NK92 cells against MDA-MB-231(m) and 4T1(n) breast cancer cells after 72 h of treatment, n=3. Statistical difference criteria: P<0.05 ( P<0.05, P<0.01, P<0.001, P < 0.0001; NS indicates no significant difference.

[0022] Figure 4 The hydrogel system prolonged NK92 cell retention and intratumoral IL15 delivery in a mouse subcutaneous 4T1 breast cancer model. (a) Schematic diagram of the in vivo experimental protocol for monitoring NK92 cell retention in a mouse subcutaneous 4T1 breast cancer model. (b–c) Representative bioluminescence imaging images (b) and fluorescence intensity decay curves over time (c) of NK92 cells at different time points after different treatments, n=5. (d–e) CD56 levels in tumor tissues of each group at the experimental endpoint. + Representative immunohistochemical (IHC) staining images (d) and quantitative analysis (e) of NK92 cells; scale bar = 50 μm, n = 5. (f–g) Representative immunohistochemical staining images (f) and statistical analysis (g) of IL15 levels in tumor sections of each group at the experimental endpoint; scale bar = 50 μm, n = 5. Statistical criteria for difference: P < 0.05 ( P<0.05, P<0.01, P<0.001, P < 0.0001; NS indicates no significant difference.

[0023] Figure 5 Antitumor effect of hydrogel system in mouse subcutaneous 4T1 breast cancer model; (a) Schematic diagram of the treatment regimen for a mouse subcutaneous breast cancer model. (b–d) Actual images of tumors in each group at the experimental endpoint (b), single tumor volume (c), and single tumor weight (d), n=5. (e) Representative H&E staining images of tumor tissues in each group; scale bar = 100 μm. (f) Representative TUNEL staining images (green) of tumor tissues after different treatments; cell nuclei are labeled with DAPI (blue); scale bar = 100 μm. Statistical difference criteria: P < 0.05 ( P<0.05, P<0.01, P<0.001, P < 0.0001; NS indicates no significant difference.

[0024] Figure 6 NK92 cells carrying MA mediated the reactivation of intratumoral cytotoxic T cells in human breast cancer tissue. (a) Schematic diagram of the experimental protocol for analyzing the immune response and antitumor effect of the biohybrid system in a human breast cancer ex vivo tissue model. (b–c) Representative flow cytometry histograms (b) and mean fluorescence intensity analysis (c) of CD107a expression on the surface of NK92 cells in the co-culture system after different treatments, n=10. (d–e) Representative flow cytometry histograms (d) and quantitative analysis (e) of IFN-γ expression in NK92 cells in the co-culture system after different treatments, n=10. (f–g) CD45 in tumor + CD3 in cell population + CD8 + Representative flow cytometry plots (f) and statistical analysis (g) of the proportion of cytotoxic T cells, n=10. (h–i) Intratumoral CD8 + IFN-γ + Representative flow cytometry plots (h) and quantitative analysis (i) of the proportion of cytotoxic T cells, n=10. Statistical criteria for difference: P<0.05 ( P<0.05, P<0.01, P<0.001, P < 0.0001; NS indicates no significant difference.

[0025] Figure 7 The antitumor effect of hydrogel systems in human breast cancer tissue samples; (a) Representative H&E staining images of tumor tissues from each group at the experimental endpoint; scale bar = 50 μm. (b) Representative TUNEL staining images (green) of human tumor sections after different treatments; cell nuclei are labeled with DAPI (blue); scale bar = 100 μm.

[0026] Figure 8 Synthesis of aldehyde-modified hyaluronic acid (HA-Ald); (a) Synthetic route; (b) Fourier transform infrared (FTIR) spectrum at 1741 cm⁻¹. - The characteristic peak at ¹ is indicated by a green arrow.

[0027] Figure 9 The surface Zeta potential was measured by dynamic light scattering (DLS) to verify each step of the CaCO3 core-surface layer-by-layer assembly (LbL) process (n = 3).

[0028] Figure 10 Height distribution curve of IL15-loaded microcapsules in AFM image.

[0029] Figure 11 Characterization of HPMC-C12; (a) Synthetic route; (b) ¹H-NMR spectrum of HPMC-C12, in which the dodecyl terminal methyl peak is marked by a black box, indicating that the dodecyl chain has been successfully modified onto HPMC.

[0030] Figure 12 Hydrated particle size distribution of PEG-PLA nanoparticles as measured by DLS.

[0031] Figure 13 Photograph of the injectable PNP1 / 5-5CD hydrogel.

[0032] Figure 14 FITC-labeled MA encapsulated in PNP1 / 5-5CD hydrogel and applied subcutaneously to mice for IVIS imaging observation (a) and corresponding fluorescence intensity quantitative analysis (b) (n = 3).

[0033] Figure 15 : Figure 5 Figure a shows the statistical analysis of the percentage of TUNEL-positive cells in tumor tissues after different treatments (n = 5). Statistical significance was defined as P < 0.05. p<0.05, p<0.01, p<0.001, p<0.0001; NS indicates no statistically significant difference.

[0034] Figure 16 : Figure 5 Figure a shows the changes in mouse body weight during the subcutaneous 4T1 tumor antitumor experiment (n = 5).

[0035] Figure 17 : Figure 5At the end of the treatment experiment shown in a, the serum levels of alanine aminotransferase (ALT, a), aspartate aminotransferase (AST, b), gamma-glutamyl transferase (γ-GT, c), total bilirubin (T-Bil, d), blood urea nitrogen (BUN, e), serum creatinine (SCr, f), lactate dehydrogenase (LDH-L, g), and creatine kinase isoenzyme MB (CK-MB, h) in subcutaneous 4T1 tumor-bearing mice were measured.

[0036] Figure 18 : Figure 5 Representative H&E stained sections of the heart, liver, lungs, and kidneys of subcutaneously tumor-bearing mice at the end of the treatment experiment shown in figure a. Scale bar = 100 μm.

[0037] Figure 19 : Figure 6 Figure a shows the quantitative analysis of the percentage of TUNEL-positive cells in human tumor sections after different treatments (n = 5). Statistical significance was defined as P < 0.05. p<0.05, p<0.01, p<0.001, p<0.0001; NS indicates no statistically significant difference. Detailed Implementation

[0038] The technical solutions and effects of this application will be further described in detail below with reference to the embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely for explaining the invention and are not intended to limit the invention.

[0039] Example I. Experimental Materials HPMC (number-average molecular weight ~86kDa), polyallylamine hydrochloride (PAH, molecular weight ~50kDa), sodium polystyrene sulfonate (PSS, molecular weight ~70kDa), ultrafiltration tubes (molecular weight cutoff 10kDa), PKH26 staining kit, DNase I, collagenase IV, and mitomycin C were all purchased from Sigma-Aldrich.

[0040] PEG (molecular weight 5kDa)-b-PLA (molecular weight 15kDa) was purchased from Daigang Biotechnology.

[0041] PAH-FITC (molecular weight ~50kDa) was purchased from Ruixi Biotechnology.

[0042] Dil dye and 7-aminoactinomycin D (7-AAD) were purchased from Beyotime Biotechnology.

[0043] Other chemical reagents were purchased from Aladdin Reagent Co., Ltd.

[0044] Recombinant human IL15 and recombinant human IL2 were purchased from Novizan.

[0045] The IL15 enzyme-linked immunosorbent assay (ELISA) kit was purchased from Linke Biotechnology.

[0046] The lymphocyte separation solution was purchased from GE Healthcare Life Sciences.

[0047] The anti-human CD56 antibody was purchased from Abcam, and the anti-human IL15 antibody was purchased from R&D Systems.

[0048] The live / dead cell staining kit was purchased from Yisheng Biotechnology.

[0049] Cell activation mixture, Zombie Violet live / dead cell staining kit, Fc receptor blocking solution, and fluorescently labeled antibody were all purchased from BioLegend.

[0050] The flow cytometry fixative and perforation solutions were purchased from Thermo Fisher Scientific.

[0051] II. Preparation of IL15-loaded calcium carbonate microspheres 2 mL of 0.33 M sodium carbonate solution was rapidly poured into 2 mL of 0.33 M calcium chloride solution, and the mixture was vigorously stirred at 1200 rpm for 40 s at room temperature. The resulting calcium carbonate microspheres were centrifuged (8000 rpm, 5 min), washed three times with water, and dried overnight at 50 °C for later use. 1 mg of calcium carbonate microspheres were dispersed in 1 mL of HEPES buffer (0.1 mM, pH 7.4), and 1 mL of IL15 solution (50 μg / mL, dissolved in 0.1 mM HEPES) was added and mixed. After incubation with shaking at 4 °C for 4 h, the IL15-loaded calcium carbonate microspheres were collected and washed three times with HEPES buffer (0.1 mM, pH 7.4) to remove unbound IL15. The IL15 loading efficiency was approximately 82%.

[0052] III. Preparation of aldehyde-modified hyaluronic acid (HA-Ald) Sodium hyaluronate was dissolved in 100 mL of deionized water (2.5 mg / mL), and 667 μL of sodium periodate solution (10 mg / mL, dissolved in deionized water) was added. The reaction mixture was stirred vigorously at room temperature in the dark for 2 h, and then phosphate-buffered saline (PBS) was added until completely dissolved. The product was precipitated with 1000 mL of acetone, and excess solvent was removed by gently pressing with a spatula. The precipitate was redissolved in 100 mL of PBS and precipitated again. This dissolution-precipitation cycle was repeated 3 times. The purified product was then lyophilized for later use. The successful aldehyde modification was verified by Fourier transform infrared spectroscopy (FTIR).

[0053] IV. Preparation of IL15-loaded microcapsules (MA) Calcium carbonate microspheres loaded with IL15 and coated with three layers of PAH / PSS and one layer of PAH / HA-Ald were prepared using LbL technology.

[0054] Take 1 mg of IL15-loaded calcium carbonate microspheres or blank microspheres and incubate them alternately in 3 mL of PAH solution (2 mg / mL, 0.5 M NaCl, pH 6.5) and 3 mL of PSS solution (2 mg / mL, 0.5 M NaCl, pH 6.5) for 3 cycles. Each layer preparation was incubated for 15 min with stirring, followed by washing three times with ultrapure water (pH 6.5) by centrifugation (3000 rpm, 5 min). The final PAH / HA-Ald coating on the microsphere surface was applied using the same method, incubating sequentially in 3 mL of PAH solution (2 mg / mL, 0.5 M NaCl, pH 6.5) and 3 mL of HA-Ald or HA solution (2 mg / mL, 0.5 M NaCl, pH 6.5).

[0055] The zeta potential of the microsphere samples after each step of LbL was detected by dynamic light scattering (DLS). In the fluorescence correlation experiment, PAH labeled with fluorescein isothiocyanate (FITC) was used for MA synthesis. Finally, the microspheres were incubated with 10 mL of EDTA (0.2 M, pH 8.5) for 20 min to remove the calcium carbonate core. The microcapsules loaded with IL15 (named MA) were collected by centrifugation (3000 rpm, 5 min), washed, and resuspended in PBS for later use.

[0056] The surface morphology of MA was examined using atomic force microscopy (AFM, Bruker). The release behavior of IL-15 from MA was investigated: MA samples were incubated in PBS at 37°C with shaking. The supernatant was collected by centrifugation at predetermined time points, and fresh PBS was added. The IL-15 content in the solution was quantitatively detected by ELISA to analyze the cumulative release behavior.

[0057] V. The binding of MA to NK92 cells NK92 cells were resuspended in serum-free medium and seeded in 12-well plates (1.5 × 10⁶ cells / well).6 (cells / well), centrifuge at 300g for 5 min to allow cells to settle to the bottom of the well. Add 4.5 × 10⁻⁶ cells to the cell culture medium. 6 One MA (MA to cell ratio 3:1) was centrifuged at 300g for 10 min to bring the MA into contact with the cells. After co-incubation for 2 h, the cell suspension was placed on lymphocyte separation medium, and the engineered cells were purified by density gradient centrifugation. After washing, the cells were collected.

[0058] VI. Characterization of NK92 cells equipped with MA Binding experiments were conducted using FITC-labeled amino acids (MA). NK92 cell samples were analyzed by flow cytometry to determine the binding rate of MA to NK92 cells. To further verify MA modification on the NK92 cell surface, NK92 cells were first labeled with PKH26 and co-incubated with FITC-labeled MA, then observed using a confocal microscope. To observe the surface morphology of MA-loaded NK92 cells, cell samples were treated with liquid nitrogen and observed using a cryo-scanning electron microscope (cryo-SEM, Hitachi Regulus 822). To monitor the retention of FITC-labeled MA on the NK92 cell surface, NK92 cells were first treated with 25 μg / mL mitomycin C for 30 min to inhibit cell proliferation, followed by washing three times with PBS. The pretreated cells were then bound to FITC-labeled MA, and the MA-loaded NK92 cells were cultured in complete culture medium for 5 days. Cell samples were collected at predetermined time points, purified using lymphocyte separation medium, and the proportion of NK92 cells with fluorescent signals was analyzed by flow cytometry.

[0059] VII. Synthesis of HPMC-C12 1 g of HPMC was dissolved in 45 mL of N-methylpyrrolidone (NMP) and stirred at 80 °C for 1 h. After cooling to room temperature, 0.5 mmol of dodecyl isocyanate (dissolved in 5 mL of NMP) was added, followed by 3 drops of N,N-diisopropylethylamine as a catalyst. The reaction solution was stirred at room temperature for 16 h, and the resulting solution was precipitated with acetone. The hydrophobically modified HPMC was collected by filtration and dried under vacuum at room temperature for 24 h to obtain a white amorphous powder, HPMC-C12.

[0060] VIII. Preparation of PEG-PLA Nanoparticles 1 mL of a 50 mg / mL solution of PEG (5 kDa)-b-PLA (15 kDa) in N,N-dimethylformamide (DMF) was added dropwise at 80 μL / min to 10 mL of ultrapure water under stirring (1200 rpm). The resulting nanoparticles were purified by ultrafiltration and resuspended to a concentration of 20 wt% as a stock solution. The nanoparticle size was detected by dynamic light scattering (DLS). Nanoparticle fluorescent labeling: 250 μL of Dil stock solution (2 mg / mL) was added to 750 μL of PEG-b-PLA solution, followed by the above nanoprecipitation process.

[0061] IX. Preparation of PNP-CD hydrogels and cell embedding In the 1 mL PNP1 / 5 hydrogel formulation, 1 / 5 represents 1 wt% HPMC-C12 and 5 wt% PEG-PLA nanoparticles (total solid polymer 6 wt%), with the remaining 94 wt% being buffer, α-CD solution, PBS, or cell suspension. PNP1 / 5-5CD and PNP1 / 5-10CD hydrogels contain 5% and 10% α-CD, respectively.

[0062] The preparation method for 1 mL PNP1 / 5-5CD hydrogel is as follows: Dissolve HPMC-C12 in PBS to prepare a 6 wt% stock solution, and take 167 mg and load it into a 1 mL syringe. Add 250 μL of nanoparticle stock solution (20 wt%), 250 μL of α-CD stock solution (20 wt%), and 333 μL of PBS or cell suspension to another 1 mL syringe. Connect the syringe containing the nanoparticle / α-CD / cell suspension through a double-ended connector, and slowly inject the solution until the connector is visible. Then connect the syringe containing HPMC-C12 to the other end of the connector. Repeatedly inject and mix the two syringes through the connector for 30 s to 1 min to form a homogeneous PNP-CD hydrogel.

[0063] 10. Hydrogel Characterization PNP1 / 5-5CD hydrogel samples were treated with liquid nitrogen and their morphology was observed using cryo-scanning electron microscopy (cryo-SEM, Hitachi Regulus 822). The cumulative release of engineered NK92 cells from the PNP1 / 5-5CD hydrogel was investigated: 500 μL of cell-loaded hydrogel was injected into the top cell filter (40 μm pore size) of a 6-well plate, and the number of released NK92 cells was counted using a cell counter (Thermo Fisher Scientific), and the cumulative release curve was analyzed. The rheological properties of the hydrogel were tested at room temperature using a 20 mm plate rotational rheometer (TA, HR20). Frequency sweep tests were performed in the range of 0.1–100 rad / s at a constant strain of 0.5%; shear rate sweep tests were performed in the range of 0.1–100 s⁻¹ at a fixed frequency of 1 Hz.

[0064] XI. Detection of cell viability in hydrogels Evaluation of the activity of NK92 cells carrying MA in hydrogels: Cells were embedded in PNP1 / 5-5CD hydrogels (1×10⁻⁶ cells / mL). 3 (Engineered cells / mL), 100 μL of hydrogel was injected into a 96-well glass plate. After 3 and 7 days of culture, live / dead cells were stained with calcein and propidium iodide (PI) for 15 min, and observed by Z-axis stacking three-dimensional scanning using a confocal microscope (Zeiss LSM98).

[0065] XII. In vivo degradation of PNP1 / 5-5CD hydrogel and MA PNP1 / 5-5CD hydrogels were prepared using Dil-labeled nanoparticles. 150 μL of the Dil-labeled hydrogel was subcutaneously injected into each mouse, and fluorescence monitoring was performed using an in vivo imaging system (IVIS, PerkinElmer, USA). Similarly, 150 μL containing ~2 × 10⁻⁶ nanoparticles was injected... 6 Subcutaneous injection of FITC-labeled MA PNP1 / 5-5CD hydrogel was performed, and the in vivo degradation of FITC-labeled MA was analyzed by IVIS.

[0066] XIII. Cell Lines and Culture Human NK cell line NK92 was cultured in complete medium: α-MEM medium supplemented with 12.5% ​​fetal bovine serum (FBS), 12.5% ​​horse serum, 0.2 mM inositol, 0.1 mM β-mercaptoethanol, and 0.02 mM folic acid (with 100 U / mL recombinant human IL2 added during routine passage expansion). Human breast cancer cell line MDA-MB-231 and mouse breast cancer cell line 4T1 were cultured in DMEM medium (MDA-MB-231) and RPMI 1640 medium (4T1), respectively, both supplemented with 10% FBS and 1% penicillin-streptomycin, under standard conditions (37℃, 5% CO2). When cell confluence reached 80%, cells were passaged every 2-3 days at a 1:3 ratio.

[0067] XIV. In vitro functional assay of NK92 cells equipped with MA To evaluate the durable activation effect of MA modification on NK92 cells: NK92 cells were modified with MA or blank MA (without IL15), with unmodified NK92 cells treated with PBS and free IL15 (50 ng / mL) as controls. After 24 h of culture, the expression of cell surface degranulation marker CD107a and intracellular interferon-γ (IFN-γ) was analyzed by flow cytometry. To simulate in vivo cytokine consumption, cells in each group were cultured for another 72 h without changing the culture medium, and activation markers were analyzed using the same flow cytometry method.

[0068] Cell samples from each group were first blocked with Fc receptor blocking solution at room temperature for 10 min to inhibit non-specific Fc receptor binding. Then, fluorescently labeled antibodies (1:100 diluted in staining buffer) were added, and the samples were incubated at 4°C in the dark for 30 min for surface staining. For intracellular marker detection, the cells were first fixed and perforated, then fluorescent antibodies (1:100 diluted in perforation solution) were added, and the samples were incubated at room temperature in the dark for 30 min. Finally, all samples were washed, resuspended, and analyzed.

[0069] The durable cytotoxicity conferred by MA on NK92 cells was evaluated using NK92 cells expressing green fluorescent protein (GFP) (NK92-GFP). NK92 cells loaded with MA (cultured in complete medium) and NK92 cells cultured in medium containing free IL15 (50 ng / mL) were pretreated for 24 h or 72 h, respectively (without changing the medium). Subsequently, the two groups of pretreated NK92 cells (2 × 10⁻⁶ cells / mL) were... 5 Cells were co-incubated for 16 h with mouse (4T1) or human (MDA-MB-231) breast cancer cells at different effector-to-target ratios (E:T). Cell samples from each group were collected, stained with 7-AAD, and the proportion of 7-AAD-positive dead cells in GFP-negative target cells was analyzed by flow cytometry. All flow cytometry assays were performed using a BDL SFR Ortessa flow cytometer, and data were analyzed using FlowJo software.

[0070] XV. Construction of a mouse subcutaneous breast cancer model All animal experiments were conducted in accordance with institutional ethical guidelines and approved by the Animal Care and Use Committee of the Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (Approval No.: SH9H-2025-A1579-1). All in vivo experiments followed the ARRIVE (Animal Research: In vivo experimental reporting) guidelines. 4–6-week-old BALB / c nude mice were purchased from commercial facilities and housed under specific pathogen-free (SPF) conditions. Female mice were selected because they are commonly used to establish breast cancer models. 5 × 10 5 Four T1 cells were suspended in 100 μL PBS and subcutaneously seeded into the lateral dorsal flank of mice. The inoculation continued until the tumor volume reached approximately 50 mm². 3 At that time, the mice were randomly divided into groups.

[0071] XVI. Local retention of NK92 cells in a mouse subcutaneous breast cancer model Subcutaneous breast cancer mice were randomly divided into 5 groups (n=5 per group) and NK92 cells expressing luciferase (NK92-Luc) were used. Each 1 mL hydrogel contained 1×10⁶ cells. 7 NK92 cells or NK92 cells equipped with MA were injected subcutaneously around the tumor with different hydrogels (150 μL per mouse), and divided into three groups: NK92@Gel group, NK92+IL15@Gel group, and NK92-MA@Gel group. Simultaneously, a free NK92 group (1.5 × 10⁻⁶ cells) was also included.6 Free NK92 cells were lysed in 150 μL PBS and injected into the control group (150 μL PBS injection). Each mouse in the NK92+IL15@Gel group contained approximately 6 μg of IL15 in the hydrogel, comparable to the total IL15 in the NK92-MA@Gel group. Local retention of NK92 cells in each group was monitored: D-fluorescein (150 mg / kg) was injected intraperitoneally every 3 days, followed by isoflurane anesthesia and IVIS imaging for 15 days. Bioluminescence decay curves were quantitatively analyzed using Living Image software, with luminescence intensity expressed as mean radiance (p·s⁻¹). -1 ·cm -2 ·sr -1 The endpoint of the experiment was reached by taking tumor tissues from each group and performing immunohistochemical (IHC) staining with anti-human CD56 and anti-human IL15 primary antibodies to detect the presence of NK92 cells and the distribution of IL15 within the tumor.

[0072] XVII. Treatment Experiment of Mouse Subcutaneous Breast Cancer Model After 15 days of topical application of different formulations, the therapeutic effects on a mouse subcutaneous breast cancer model were evaluated, using the same administration regimen as in 2.16. Mouse body weight was monitored throughout the experiment. At the experimental endpoint, the tumor was dissected, and its length, width, and weight were recorded. Tumor volume was calculated using the formula V = (length × width) / (length × width) * ... 2 The tumor tissue from each group was stained with H&E and tested for TUNEL to evaluate the tumor-killing effect of the biohybrid system. Peripheral blood serum was collected to detect cardiac, hepatic, and renal function indicators. Major organs (heart, liver, lung, and kidney) were stained with H&E to assess systemic biocompatibility. TUNEL results were quantitatively analyzed using ImageJ software.

[0073] 18. Construction of an in vitro model of human breast cancer tissue Fresh breast cancer tumor specimens were obtained from surgical patients at Huashan Hospital affiliated with Fudan University, with informed consent obtained from the patients and approval from the institution's ethics committee (approval number: 2025-871). The experiment strictly followed the institution's ethics guidelines, fully protecting patient privacy. Fresh tumor tissue was cut into small pieces, embedded in 4% low-melting-point agarose, and cut into 500μm thick sections using a vibratory microtome (Leica VT1200S). Patient-derived tumor sections were randomly divided into 5 groups and treated in a cell culture incubator for 40 h: PBS control group, free NK92 group, NK92@Gel group, NK92+IL15@Gel group, and NK92-MA@Gel group. An in vitro co-culture system was established using Transwell chambers (0.4μm, 6-well plates). Tumor tissue was placed at the bottom of the upper chamber, and different formulations were applied to the tissue surface. Culture medium was added to the lower chamber. The culture medium in the lower chamber was changed every 8 h to simulate in vivo IL15 clearance. For the hydrogel group, 500μL of 5×10⁻⁶ IL15-containing gels were applied to each well. 4Hydrogels containing either NK92 cells or NK92 cells loaded with MA. The NK92+IL15@Gel group contained 200 ng IL15 per well, comparable to the NK92-MA@Gel group. The free NK92 group contained 5 × 10⁶ ng IL15 per well. 4 Free NK92 cells were placed on the tumor surface.

[0074] 19. Evaluation of Immune Activation and Therapeutic Effect in Ex vivo Human Breast Cancer Tissue Model To evaluate the antitumor effects of different formulations: Tumor sections were fixed and stained with H&E and TUNEL to analyze tumor cell apoptosis / necrosis rates. Simultaneously, remaining co-culture samples from each group were collected for flow cytometry analysis of immune responses. Tissues were minced and digested with 1 mg / mL collagenase IV and 0.2 mg / mL DNase I at 37°C for 45 min. The cell suspension was filtered and stained. GFP-positive NK92 cell activation was assessed using the Zombie Violet live / dead cell staining kit, anti-human CD107a-BV510, and anti-human IFN-γ-BV605 staining. Cytotoxicity of CD8 cells was also assessed. + T lymphocytes: stained with Zombie Violet live / dead cell staining kit, anti-human CD45-BV785, anti-human CD3-APC, anti-human CD8-PE-Cy7, and anti-human IFN-γ-BV605. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) + Quantitative CD3 in a population + CD8 + T cell ratio, in CD3 + CD8 + CD8 determination in subgroups + IFN-γ + T cell ratio.

[0075] 20. Statistical Analysis Unless otherwise specified, all data are expressed as mean ± standard error (SEM). Statistical analysis was performed using GraphPad Prism 8 software. Unpaired two-tailed t-tests were used for comparisons between two groups, and one-way ANOVA combined with Tukey's post-hoc test was used for comparisons among multiple groups. P < 0.05 was considered statistically significant. P<0.05, P<0.01, P<0.001, P < 0.0001; NS indicates no significant difference.

[0076] 21. Experimental Results 1. Preparation and characterization of NK92 cells equipped with MA IL15-loaded microcapsules were modified onto the surface of NK92 cells using LbL technology to enhance cell function. Figure 1 As shown in Figure a, using calcium carbonate microspheres loaded with IL15 as a template, PAH and PSS were used for layer-by-layer assembly, and aldehyde-modified hyaluronic acid (HA-Ald) was used as the outer cell adhesion layer of MA. Figure 8 This allows microcapsules to adhere to the surface of NK92 cells. Dynamic light scattering (DLS) analysis ( Figure 9 The results showed that the change in surface Zeta potential after each step of LbL confirmed that the calcium carbonate core was successfully loaded with IL15 and completed the surface coating cycle of PAH, PSS, and HA-Ald.

[0077] Dissolving the calcium carbonate core yielded IL15-loaded microcapsules (MA). Atomic force microscopy (AFM) images showed that the MAs were approximately 1–2 μm in size. Figure 1 a), the height profile shows its thickness to be approximately 13 nm ( Figure 10 Subsequently, NK92 cells were co-incubated with MA at 37°C for 2 hours, allowing MA to adhere to the surface of NK92 cells. Figure 1 (b) This process is mediated by a Schiff base reaction between the aldehyde group (Ald) on the MA and the primary amine on the cell surface. Flow cytometry analysis ( Figure 1 c- Figure 1 d) Confirmed that the binding rate of fluorescently labeled MA to NK92 cells was significantly higher than that of the group without aldehyde modification (P<0.001), indicating that the aldehyde group on MA can achieve more efficient cell adhesion. Three-dimensional confocal imaging ( Figure 1 e) and two-dimensional view ( Figure 1 f) consistently shows that FITC-labeled MA adheres to the surface of PKH26-labeled NK92 cells. Cryo-scanning electron microscopy images further confirm the presence of microvesicle-like MA on the surface of NK92 cells. Figure 1 g). After 5 days of in vitro culture, the MA binding rate on the cell surface decreased slightly from 88.27% to 74.8%, indicating that MA binding to NK92 cells was relatively stable. Figure 1 Furthermore, MA exhibited sustained release behavior, with a cumulative IL15 release rate of approximately 88.2% after 12 days of incubation at 37°C. Figure 1 i). In summary, a simple method was used to successfully construct a live in vivo biological hybrid system of NK92 cells equipped with MA.

[0078] 2. Characterization of HPMC-based supramolecular hydrogels used to embed engineered NK92 cells Synthesis of hydrophobically modified HPMC with dodecyl chains (C12) Figure 11PEG-PLA nanoparticles were prepared via nanoprecipitation. Dynamic light scattering (DLS) detection was then performed. Figure 12 The results show that the hydrodynamic size of the obtained PEG-PLA nanoparticles is approximately 50 nm, making them suitable for constructing polymer-nanoparticle hydrogels. For example... Figure 2 As shown in Figure a, the hydrophobic interaction between the hydrophobic C12 chains and the PLA core of the PEG-PLA nanoparticles serves as a physical crosslinking point, bridging the nanoparticles into a three-dimensional network to form an injectable polymer-nanoparticle (PNP) hydrogel suitable for cell embedding. Furthermore, α-CD is added as a supramolecular reinforcing agent, strengthening the hydrogel through the formation of inclusion complexes between the host molecule and the PEG guest molecules on the nanoparticle surface. Rheological analysis of the hydrogel was performed, and the frequency sweep results are as follows (…). Figure 2 (b) shows that the storage modulus (G') of each group of hydrogels is higher than the loss modulus (G''), and both storage modulus and loss modulus increase with increasing α-CD concentration, with PNP1 / 5-10CD hydrogel exhibiting the highest G' and G'' values. The results confirm that the hydrogels exhibit typical elastic behavior, and the addition of α-CD enhances their elastic properties. The mechanism is that α-CD can aggregate around the PEG nanoparticle chains via intermolecular hydrogen bonds, forming rigid physical cross-linked clusters, thus strengthening the hydrogel network. Figure 2 a). PNP1 / 5-5CD hydrogel is injectable ( Figure 13 ), shear rate scan results ( Figure 2 c) shows that the viscosity of the hydrogel continuously decreases with increasing shear rate, further verifying its injectability. Figure 2 The PNP1 / 5-5CD sample image in d shows supramolecular hydrogel formation. Cryo-scanning electron microscopy reveals its porous three-dimensional network structure, which can provide a favorable microenvironment for the survival of embedded NK92 cells. The degradation of the fluorescently labeled PNP1 / 5-5CD hydrogel after subcutaneous injection was observed using an in vivo imaging system (IVIS). The results (…) Figure 2 (e-2f) shows that the PNP1 / 5-5CD hydrogel gradually degrades within 4 weeks, providing a long-term reservoir for engineered NK92 cells.

[0079] Engineered NK92 cells were embedded in a PNP1 / 5-5CD hydrogel and co-cultured for different time periods. Cell viability in the hydrogel was detected by live / dead cell staining. Confocal imaging ( Figure 2 g) showed that almost all NK92 cells equipped with MA survived after 3 days of culture in the hydrogel; after 7 days of culture, the number of live cells increased significantly, with only a small number of dead cells, indicating that the PNP1 / 5-5CD hydrogel has excellent biocompatibility and can support the survival and proliferation of NK92 cells. Furthermore, engineered NK92 cells were continuously released from the PNP1 / 5-5CD hydrogel into the culture medium (g) during the 16-day observation period. Figure 2h). In summary, the injectable supramolecular hydrogel hybrid system for embedding MA-equipped NK92 cells can support the survival and proliferation of engineered NK92 cells and achieve continuous cell release.

[0080] 3. NK92 cells equipped with MA exhibit sustained and potent in vitro activity. To investigate the persistent effect of MA on NK92 cells, NK92 cells were treated with PBS, free IL15, blank MA, and MA for different time periods. The activation status of NK92 cells was analyzed by flow cytometry. Figure 3 a). After 24 hours of treatment, the cell surface CD107a (a) of the free IL15 group and the MA group... Figure 3 b-3c) and intracellular IFN-γ ( Figure 3 d-3e) expression was significantly higher in both groups than in other groups, with no significant difference between the two groups. Figure 3 (b-3e) indicates that IL15 released by MA can activate NK92 cells in the same way as free IL15. However, after 72 hours of treatment, there was no significant difference in activation markers between the free IL15 group and the PBS group for NK92 cells, while NK92 cells loaded with MA maintained significantly higher levels of CD107a and IFN-γ expression. Figure 3 (f-3i). The results showed that, unlike the rapid depletion of IL15 within 72 hours in the free IL15 group, IL15 was continuously released in the MA group, ensuring long-term stimulation of NK92 cells. The persistent upregulation of CD107a triggered the cytotoxic activity of engineered NK92 cells and initiated tumor killing.

[0081] To further investigate the durable tumor-killing ability of NK92 cells equipped with MA, MA-modified cells and cells treated with free IL15 were pretreated for 24 h and 72 h, respectively, and then co-incubated with two types of breast cancer cell lines for 16 h. NK92 cytotoxicity was analyzed by flow cytometry. Figure 3 j). After 24 hours of pretreatment, the antitumor effects of free IL15 and MA-treated NK92 cells on 4T1 and MDA-MB-231 breast cancer cells were comparable. Figure 3 k-3l); however, after 72 hours of pretreatment, the free IL15 group was significantly less effective than the MA group (k-3l); Figure 3 In summary, MA modification can provide NK92 cells with a self-sustaining supply of IL15, achieving sustained and potent activation and cytotoxicity of NK92 cells in vitro.

[0082] 4. Biological hybrid systems enhance NK92 cell retention and intratumoral IL15 delivery in a mouse subcutaneous breast cancer model. To evaluate the effect of engineered cell embedding hydrogels on prolonging cell retention time in a mouse subcutaneous breast cancer model, mice were divided into a control group, a free NK92 group, an NK92@Gel group, an NK92+IL15@Gel group, and an NK92-MA@Gel group. NK92 cell bioluminescence was monitored by IVIS for 15 consecutive days. Figure 4 a). Figure 4 b shows that, compared with the free NK92 group, the NK92 cell group encapsulated in hydrogel had a slightly higher bioluminescence signal on day 3, which was maintained until day 6, indicating that the hydrogel, as a reservoir, can physically assist in the retention of NK92 cells. The cell retention time in the NK92+IL15@Gel group was further prolonged to day 9, and the bioluminescence intensity on day 6 was significantly higher than that in the NK92@Gel group (P<0.05). Figure 4 c). It is worth noting that the NK92-MA@Gel group had the longest retention time, at least 15 days, and its luminescence signal remained higher than that of other groups throughout the experiment.

[0083] The endpoint of the in vivo imaging experiment was to perform CD56 immunohistochemical staining on subcutaneous tumor tissue to analyze the presence of NK92 cells within the tumor. Figure 4 Day 4e showed that 15 days after treatment, only a small number of cells were present in the central region of solid tumor tissue in the NK92@Gel group, indicating that the hydrogel itself has limited advantage in protecting NK92 cells within the tumor. In contrast, the number of CD56+NK92 cells in the tumor was significantly higher in the NK92-MA@Gel group than in the NK92+IL15@Gel group (P<0.0001), indicating that the MA-mediated in situ persistent IL15 signaling activation can significantly support the functional survival of infiltrated NK92 cells in the harsh immunosuppressive tumor microenvironment.

[0084] In addition, IL15 immunohistochemical staining was performed on tumor tissue sections at the experimental endpoint to detect the presence of IL15 in the tumor. Figure 4 f-4g analysis showed that the distribution of IL15 in tumor tissue of the NK92-MA@Gel group was significantly higher than that of other groups (P<0.0001), indicating that IL15 can be delivered intratumorally via infiltrating MA-modified NK92 cells. Importantly, in addition to directly maintaining engineered NK92 cells, IL15 delivered to the immunosuppressive tumor microenvironment can also exert multifaceted immunostimulatory effects, bringing additional immunotherapeutic benefits. In vivo imaging results ( Figure 14 The results showed that the fluorescently labeled MA could remain around mouse breast tumors for at least 4 weeks, indicating that MA on NK92 cells can serve as a persistent intratumoral carrier for IL15, facilitating the long-term retention of IL15 within the tumor. In summary, this biohybrid system can simultaneously enhance the survival time of engineered NK92 cells and achieve intratumoral IL15 distribution in a mouse subcutaneous 4T1 breast cancer model.

[0085] 5. Antitumor effect of biological hybrid system in mouse subcutaneous breast cancer model To evaluate the in vivo therapeutic effect of the bio-hybrid system, different formulations were subcutaneously injected around the subcutaneous tumor and treated for 15 days. Figure 5 a). Figure 5 b-5d showed that the tumor inhibition rate of the NK92@Gel group was higher than that of the free NK92 group, but there was no significant difference compared with the control group; the NK92+IL15@Gel group effectively inhibited the progression of 4T1 tumors compared with the NK92@Gel group; while the NK92-MA@Gel group showed the strongest anti-tumor activity among all groups. TUNEL and H&E staining results ( Figure 5 e-5f; Figure 15 The results showed that the tumor cell apoptosis and necrosis rates induced by NK92+IL15@Gel and NK92-MA@Gel treatment groups were significantly higher than those in other groups. TUNEL quantification results showed that the proportion of apoptotic cells in the NK92-MA@Gel group was significantly higher than that in the NK92+IL15@Gel group (P<0.0001). The enhanced tumor apoptosis and necrosis in the NK92-MA@Gel group may be due to the direct cytotoxicity of NK92 cells carrying MA and their secretion of pro-inflammatory cytokines such as IFN-γ.

[0086] Physiological and biochemical parameters of 4T1 tumor-bearing mice were monitored throughout the process to assess the in vivo biosafety of the hybrid platform. No significant changes in mouse body weight were observed in any group, indicating that each formulation had good systemic tolerability. Figure 16 At the treatment endpoint, serum biochemical analysis showed that cardiac, hepatic, and renal function indicators in all groups were within the normal reference range. Figure 17 Histopathological examination of major organs (heart, liver, lungs, kidneys) using H&E staining showed no obvious tissue damage or inflammatory infiltration. Figure 18 This confirms the good biocompatibility of the treatment regimen in vivo. In summary, the results indicate that the local hydrogel and MA-loaded NK92 cells have a synergistic effect, demonstrating a potent anti-tumor effect.

[0087] 6. Engineered NK92 cell-mediated intratumoral cytotoxic T cell immune remodeling To investigate the immune response elicited by NK92 cells carrying MA in solid tumors, an in vitro model of freshly resected human breast cancer tissue was used. Figure 6 a). Fresh patient samples were obtained and co-cultured with different formulations in Transwell chambers for 40 hours, with the culture medium changed every 8 hours to simulate in vivo IL-15 clearance. Tumor tissue was then analyzed by flow cytometry. Figure 6 a). Results of NK92 cell function within the tumor ( Figure 6(b-6e) showed that CD107a expression in NK92 cells of the NK92-MA@Gel group was approximately twice that of the NK92+IL15@Gel group, and 4.2 times and 4.1 times that of the free NK92 group and the NK92@Gel group, respectively; IFN-γ expression was approximately 1.5 times that of the NK92+IL15@Gel group, and 2.4 times and 2.3 times that of the free NK92 group and the NK92@Gel group, respectively. The superior activation level of NK92 cells in the NK92-MA@Gel group stemmed from the MA backpack's on-demand delivery of IL15 signaling, constructing a durable, personalized cytokine microenvironment for each cell, ensuring a continuous supply of signaling during and after tumor invasion. In contrast, the free IL15 released by NK92+IL15@Gel formed a diffusion-limiting gradient, resulting in insufficient signaling supply to cells migrating deeper into the tissue.

[0088] Given that NK92 cells equipped with MA deliver intratumoral IL15, further analysis of cytotoxic CD8 was conducted. + T cells. The NK92-MA@Gel group elicited the strongest cytotoxic T cell immunity, CD8. + The percentage of T cells was 12.93 ± 1.01%, CD8 + IFN-γ + The proportion of T cells was 43.07±4.52%, approximately twice that of the NK92+IL15@Gel group (P<0.0001). Furthermore, the proportions of both cell types were significantly higher in the NK92+IL15@Gel group than in the NK92@Gel group (P<0.0001). Based on these results, the superior efficacy of the NK92-MA@Gel group stems from the delivery of IL15 to ex vivo tumor tissue, which is a dysfunctional CD8+ cell. + T cells provide crucial survival and activation signals. Furthermore, after NK92 cells kill tumor cells, dendritic cells cross-present tumor antigens, which can further enhance cytotoxicity through cross-sensitization. + T cell response. Activated CD8 + T cells, in turn, can promote and maintain NK92 cytotoxicity, forming a positive feedback loop that powerfully enhances the anti-tumor response. In summary, this biohybrid system not only achieves NK92 cell self-sustaining but also, through engineered NK92 cell-mediated intratumoral IL15 distribution, cascades activation of dormant cytotoxic CD8+. + T cells synergistically and potently activate innate and adaptive immunity in human breast cancer tissue, demonstrating enormous therapeutic potential.

[0089] 7. Antitumor effects of biohybrid systems in in vitro human breast cancer tissue models To further evaluate the antitumor effect of the biohybrid system, histological analysis was performed on human breast cancer tissue samples. TUNEL and H&E staining results and quantitative analysis were conducted. Figure 7 a-7b; Figure 19 The results showed that the control group exhibited only a small amount of apoptosis and necrosis; the free NK92 group and the NK92@Gel group only slightly killed tumor cells compared to the control group, with no significant difference between the two groups, indicating that the tumor-killing ability of NK92 cells is limited without IL15 signaling support. The NK92+IL15@Gel group significantly enhanced NK92-mediated tumor killing compared to the NK92@Gel group (P<0.05). Notably, the NK92-MA@Gel group exhibited the strongest anti-tumor activity, with TUNEL results showing that its tumor apoptosis rate was significantly higher than all other groups (P<0.0001). H&E staining results also showed that the NK92-MA@Gel group had the best effect in inducing tumor necrosis. The significant tumor-killing activity of the NK92-MA@Gel group is attributed to the combined effect of persistently activated NK92 cells and reactivated endogenous cytotoxic T cells. Figure 6 In summary, the results confirm that the hydrogel biohybrid system embedding NK92 cells with MA exhibits robust therapeutic efficacy in human breast cancer ex vivo tissue, revealing the clinical translational potential of this platform against solid tumors.

[0090] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A supramolecular hydrogel-based delivery system for engineered NK cell antitumor agents, characterized in that, Including load IL 15 microcapsules, NK92 cells modified on the surface of the microcapsules, and an injectable supramolecular hydrogel encapsulating the modified NK92 cells; The load IL The microcapsules of 15 were obtained by using calcium carbonate microspheres as templates, depositing multiple layers of films alternately with PAH / PSS, then coating them with a PAH / aldehyde-based hyaluronic acid layer, and finally removing the calcium carbonate core.

2. The method for preparing engineered NK cell antitumor agents based on supramolecular hydrogel delivery as described in claim 1, characterized in that, Includes the following steps: Step 1, Prepare IL-loaded 15% calcium carbonate microspheres were prepared by alternating deposition of PAH / PSS on the surface of the microspheres using a layer-by-layer self-assembly technique, followed by coating with aldehyde-modified hyaluronic acid. After removing the core, IL-loaded microspheres were obtained. 15 microcapsules; Step 2, transfer the load IL 15 microcapsules were co-incubated with NK92 cells to allow the IL-loaded cells to... 15 microcapsules are chemically bonded to the cell surface to obtain IL-loaded microcapsules. 15 microcapsule-modified engineered NK92 cells; Step 3, Preparation of HPMC C12, PEG PLA nanoparticles and α An injectable supramolecular hydrogel containing cyclodextrin was used to embed engineered NK92 cells within the hydrogel, resulting in a supramolecular hydrogel-based delivery system for engineered NK cell antitumor agents.

3. The preparation method according to claim 2, characterized in that, Step 1 includes: Sodium carbonate solution was added to calcium chloride solution, and the mixture was stirred vigorously at room temperature. After centrifugation, washing, and drying, calcium carbonate microspheres were obtained. Calcium carbonate microspheres were dispersed in buffer solution, and IL15 solution was added and mixed. The mixture was then shaken and incubated to obtain IL15-loaded calcium carbonate microspheres. Sodium hyaluronate was dissolved in deionized water, and sodium periodate solution was added. The mixture was stirred at room temperature in the dark, and PBS buffer was added until completely dissolved to obtain a precipitate. The precipitate was then purified to obtain aldehyde-modified hyaluronic acid. IL15-loaded calcium carbonate microspheres were incubated alternately in PAH and PSS solutions for multiple cycles. Then, they were incubated sequentially in PAH and aldehyde-modified hyaluronic acid solutions. Finally, the microspheres were incubated with EDTA to remove the calcium carbonate core, and the IL15-loaded microcapsules were collected by centrifugation.

4. The preparation method according to claim 2, characterized in that, Step 2 includes: NK92 cells were resuspended in serum-free medium and seeded in 12-well plates. IL15-loaded calcium carbonate microspheres (MA to cell ratio 3:1) were added to the cell culture medium. After centrifugation, the IL15-loaded calcium carbonate microspheres were brought into contact with the cells. After co-incubation, the cell suspension was placed on lymphocyte separation medium and the engineered cells were purified by density gradient centrifugation. The cells were then washed and collected.

5. The preparation method according to claim 4, characterized in that, The ratio of IL15-loaded calcium carbonate microspheres to NK92 cells was 3:

1.

6. The preparation method according to claim 2, characterized in that, Step 3 includes: HPMC was dissolved in N-methylpyrrolidone, dodecyl isocyanate was added, and then N,N-diisopropylethylamine was added as a catalyst. The reaction solution was stirred at room temperature, and the resulting solution was precipitated with acetone. The hydrophobically modified HPMC was collected by filtration and vacuum dried at room temperature to obtain a white amorphous powder HPMC-C12. A solution of PEG-b-PLA in N,N-dimethylformamide was added dropwise to ultrapure water under stirring to obtain PEG. PLA nanoparticles; Dissolve HPMC-C12 in PBS buffer to prepare a stock solution, fill a syringe with the solution, and add PEG to another syringe. PLA nanoparticle stock solution, α-CD stock solution and engineered NK92 cell suspension; The two syringes were connected by a double-port connector and repeatedly injected to mix them, thereby obtaining an engineered NK cell anti-tumor agent based on supramolecular hydrogel delivery.

7. The application of the supramolecular hydrogel-based engineered NK cell antitumor agent according to claim 1 in the preparation of drugs for treating solid tumors.

8. The application according to claim 7, characterized in that, The solid tumor is breast cancer.

9. A pharmaceutical composition, characterized in that, Includes the engineered NK cell antitumor agent based on supramolecular hydrogel delivery as described in claim 1.

10. Use of the pharmaceutical composition of claim 9 in the preparation of a medicament for treating solid tumors.

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