Puerarin hydrogel for enhancing solid tumor removal capability of CAR-NK therapy and application thereof

Abstract

The invention discloses puerarin hydrogel for enhancing solid tumor removal capacity of CAR-NK (Chimeric Antigen Receptor-Natural Killer) therapy and application of the puerarin hydrogel. The injectable puerarin hydrogel is based on hydrogen peroxide reaction hydrogel formed by mixing (polyethylene glycol dimethyl) acrylate, ferrous chloride and puerarin. After the injectable puerarin hydrogel is fixed in a tumor, endothelial cells can be activated to generate nitric oxide, so that tumor blood vessels are expanded, tumor hypoxia is relieved, tumor immunosuppression is reversed, intratumor infiltration, survival and targeted killing functions of CAR-NK cells are promoted, growth of breast tumors and lung tumors is finally inhibited, and obvious toxic and side effects are not generated. The invention provides an effective strategy for reprogramming tumor immunosuppression to activate CAR-NK cells through injectable puerarin hydrogel so as to enhance solid tumor treatment, and application of the puerarin hydrogel or combined CAR-NK cells in preparation of a medicine for removing solid tumors through an effective CAR-NK therapy.

Description

Technical Field

[0001] The present invention belongs to the research fields of purified traditional Chinese medicine, injectable hydrogel, and CAR-NK cell therapy for solid tumors, and specifically relates to the application of injectable puerarin hydrogel in reprogramming immunosuppression to enhance the targeted killing of solid tumors by chimeric antigen receptor NK cells. Background Art

[0002] Natural killer (NK) cells are a major subtype of innate immune effector cells. NK cells can distinguish pathological tissues from normal tissues based on stimulatory and inhibitory receptors on the tissue surface. Therefore, they can rapidly kill various abnormal cells, including cancer cells, without the time-consuming activation process of antigen-specific adaptive immune responses and without secreting various cytokines and chemokines, as the latter may cause unnecessary cytokine storms. Thus, they are considered a powerful method for effectively eliminating tumor cells with significantly reduced or even no expression of major histocompatibility complex (MHC) class I. Inspired by the success of chimeric antigen receptor T (CAR-T) cell therapy, chimeric antigen receptor NK (CAR-NK) cell therapy has been widely carried out in the research of eradicating solid tumors (such as NCT03940820, NCT02839954). Compared with CAR-T cell therapy, CAR-NK cell therapy also has better safety (such as no or minimal neurotoxicity, cytokine release syndrome, or graft-versus-host disease), less interference from tumor heterogeneity, and a larger cell source.

[0003] However, similar to CAR-T cell therapy, the therapeutic effect of CAR-NK cell therapy on the corresponding solid tumors is still significantly restricted by the harsh tumor microenvironment. The irregular and dysfunctional blood vessels of most solid tumors not only significantly inhibit the tumor infiltration of CAR-NK cells, but also seriously affect their survival and effector ability by promoting the formation of tumor acidity and hypoxia. In addition, abnormal expression of cytokines (such as transforming growth factor-β (TGF-β)) and soluble ligands (such as soluble NKG2D-L (sNKG2D-L), proline-rich large protein BAG6 (BAG6)) can also affect the recruitment and activation of NK cells in solid tumors. Therefore, in preclinical and clinical studies, researchers are committed to developing a new generation of CAR or rationally combining it with other cancer treatment methods to enhance the efficacy of CAR-NK cell therapy. For example, overexpression of interleukin 15 (IL-15) can make CAR-NK cells have better tumor infiltration or proliferation efficacy, thereby significantly improving anti-tumor activity. Platelet-based nanomedicines anchoring specific immunoglobulin G (IgG) can achieve IgG implantation into tumor cell membranes through membrane fusion, thereby more effectively improving the efficacy of CAR-NK cell therapy through antibody-dependent cellular cytotoxicity. However, few studies have reported that the efficacy of CAR-NK cell therapy can be enhanced by directly normalizing tumor vasculature, even though tumor vasculature is closely related to tumor oxygenation, tumor immune microenvironment, and tumor infiltration of CAR-NK cells. Summary of the invention

[0004] Purpose of the invention: In view of the problem that the current CAR-NK therapy for solid tumors has poor effects, the present invention proposes a puerarin hydrogel that reprograms immunosuppression to enhance CAR-NK cells to inhibit solid tumors, which can induce endothelial cells to produce nitric oxide, dilate tumor blood vessels, relieve hypoxia, reverse tumor immunosuppression, enhance CAR-NK infiltration, survival and function, and eliminate solid tumors. It can be effectively used in the preparation of anti-tumor or tumor-suppressing drugs, improve the efficacy of CAR-NK, and effectively solve the existing solid tumor vascular abnormalities, hypoxia, immunosuppression, etc. that limit the function of CAR-NK.

[0005] The present invention also provides the use of the puerarin hydrogel in enhancing CAR-NK in clearing solid tumors such as triple-negative breast cancer and lung cancer.

[0006] Technical solution: To achieve the above-mentioned purpose of the invention, the present invention provides a puerarin hydrogel for enhancing the ability of CAR-NK therapy to eliminate solid tumors. The puerarin hydrogel is formed by mixing puerarin with a hydrogen peroxide-reactive hydrogel composed of (polyethylene glycol dimethyl) acrylate and ferrous chloride.

[0007] The puerarin hydrogel is prepared by mixing (polyethylene glycol dimethyl)acrylate, ferrous chloride, hydrogen peroxide and puerarin, and then incubating at room temperature to form a gel.

[0008] Preferably, the puerarin hydrogel is prepared by mixing (polyethylene glycol dimeth)acrylate, ferrous chloride, hydrogen peroxide and puerarin, and then incubating at 25° C. to form a gel.

[0009] The puerarin hydrogel of the present invention is used in the preparation of anti-tumor or tumor-inhibiting drugs.

[0010] The puerarin hydrogel promotes endothelial cells to produce nitric oxide, thereby dilating tumor blood vessels, alleviating hypoxia, and reversing tumor immunosuppression.

[0011] The puerarin hydrogel of the present invention is used in the preparation of drugs for promoting CAR-NK infiltration, survival and function and clearing solid tumors.

[0012] The puerarin hydrogel promotes endothelial cells to produce nitric oxide, dilates tumor blood vessels, relieves hypoxia, reverses tumor immunosuppression, promotes CAR-NK infiltration, survival and function, and clears solid tumors.

[0013] The puerarin hydrogel of the present invention is used in combination with CAR-NK cells in the preparation of drugs or reagents for clearing solid tumors.

[0014] The puerarin hydrogel of the present invention is used in the preparation of a drug for enhancing CAR-NK therapy to eliminate solid tumors.

[0015] Wherein, the tumors include breast tumors and lung tumors.

[0016] The pharmaceutical composition for enhancing CAR-NK therapy to eliminate solid tumors of the present invention comprises the puerarin hydrogel as an active ingredient and a pharmaceutically acceptable excipient.

[0017] Preferably, the pharmaceutical composition is an injection.

[0018] The present invention proposes for the first time the use of a traditional Chinese medicine extract, puerarin hydrogel, in the treatment of solid tumors by reprogramming immunosuppression to enhance CAR-NK cells, wherein the reprogramming immunosuppression is to relieve tumor immunosuppression.

[0019] The present invention combines puerarin with (polyethylene glycol dimethyl) acrylate (PEGDMA) and ferrous chloride (FeCl 2 ) composed of hydrogen peroxide (H 2 O 2)After the reactive hydrogels are mixed and the obtained puerarin-loaded PEGDMA hydrogel (Puerarin@PEGel) is injected into solid tumors, it can prompt endothelial cells to produce nitric oxide, dilate tumor blood vessels, relieve hypoxia, and reverse tumor immunosuppression. At the same time, Puerarin@PEGel can enhance the infiltration of CAR-NK cells injected via the tail vein into tumors, promote their survival and function in solid tumors, and Puerarin@PEGel combined with CAR-NK therapy can significantly eliminate solid tumors. In summary, the injectable puerarin hydrogel can reprogram immunosuppression to enhance the effect of chimeric antigen receptor NK cells in targeting and killing solid tumors, and can be used in the preparation of anti-tumor or tumor-inhibiting drugs; or the application of puerarin hydrogel combined with CAR-NK in the preparation of drugs for eliminating solid tumors.

[0020] The present invention proposes an injectable hydrogel composed of (polyethylene glycol dimethyl) acrylate (PEGDMA) and ferrous chloride (FeCl 2 ), which is reactive to hydrogen peroxide (H 2 O 2 ). After the Chinese medicine puerarin extract puerarin (Puerarin) is encapsulated and injected into solid tumors, Puerarin@PEGel can continuously release puerarin to long-acting prompt endothelial cells to produce nitric oxide, dilate tumor blood vessels, relieve hypoxia, reverse tumor immunosuppression, while enhancing the infiltration of CAR-NK cells injected via the tail vein into tumors, promote their survival and function in solid tumors, and finally eliminate solid tumors. In summary, the injectable puerarin hydrogel can reprogram immunosuppression to enhance the effect of chimeric antigen receptor NK cells in targeting and killing solid tumors. The present invention first proposes a simple and efficient method or combination, that is, to enhance the effect of chimeric antigen receptor NK cells in targeting and killing solid tumors by reprogramming immunosuppression with puerarin hydrogel.

[0021] Due to the abnormal vascular network in solid tumors, the infiltration of CAR-NK cells is hindered. At the same time, the immunosuppressive tumor microenvironment also restricts the function of CAR-NK cells infiltrating into the tumor. The present invention uses PEGel hydrogel to slowly release Puerarin for a long time, which can improve the tumor vascular network and the tumor immunosuppressive microenvironment for a long time, and enhance the anti-tumor ability of CAR-NK. For the first time, the present invention combines puerarin hydrogel to enhance the clearance of solid tumors by CAR-NK. Compared with the injection of Puerarin alone, Puerarin@PEGel can reshape the tumor vascular network for a longer time and improve the tumor immunosuppressive microenvironment. Compared with the injection of CAR-NK alone, when Puearin@PEGel is combined with CAR-NK, the proportion of CAR-NK infiltrating into the solid tumor is higher, the survival time is longer, the release amounts of telomerase and perforin are higher, and the ability to clear solid tumors is better. The present invention discovers for the first time that Puerarin@PEGel has a significant effect in synergistically clearing solid tumors with CAR-NK. In addition, when Puerarin is directly injected into the tumor, the drug effect cannot be maintained for a long time, and the anti-tumor ability in synergy with CAR-NK is weak.

[0022] The present invention mainly utilizes for the first time that Puerarin can dilate tumor blood vessels. After combining with hydrogel, it can dilate tumor blood vessels for a long time, improve the tumor immune microenvironment, enhance the survival and effector functions of CAR-NK cells, and ultimately enhance the ability of CAR-NK cells to target and kill solid tumors. After the injectable puerarin hydrogel is fixed in the tumor, it can activate endothelial cells to produce nitric oxide, thereby dilating tumor blood vessels, relieving tumor hypoxia, reversing tumor immunosuppression, promoting the infiltration, survival and targeted killing functions of CAR-NK cells in the tumor, and ultimately inhibiting the growth of breast tumors and lung tumors without obvious toxic side effects.

[0023] Although CAR-NK has achieved great success in the treatment of hematological tumors, its effect in treating solid tumors is not good, mainly because the complex microenvironment of solid tumors restricts the function of CAR-NK. The puerarin hydrogel of the present invention can effectively reshape the tumor immune microenvironment and enhance the ability of CAR-NK to target and kill solid tumors.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0025] The present invention prepares puerarin hydrogel (Puerarin@PEGel) for the first time, and discovers for the first time that Puerarin@PEGel can continuously release Puerarin to promote endothelial cells to produce nitric oxide for a long time, dilate tumor blood vessels, relieve hypoxia, reverse tumor immunosuppression, and its application in the preparation of anti-tumor or tumor-inhibiting drugs.

[0026] Through experimental research, the present invention discovers that after Puerarin@PEGel is injected into solid tumors, it enhances the infiltration of CAR-NK cells injected via the tail vein, and enhances the survival and function of CAR-NK in solid tumors of triple-negative breast cancer and lung cancer. Further, Puerarin@PEGel in combination with CAR-NK cells can efficiently eliminate solid tumors; therefore, the puerarin hydrogel of the present invention can be used in the application of preparing drugs for enhancing the elimination of solid tumors by CAR-NK. Brief Description of the Drawings

[0027] Figure 1 It is for the preparation, characterization and biological function evaluation of Puerarin@PEGel. (A) Photos of the mixed solution of PEGDMA, puerarin and ferric chloride without (left) and with (right) the addition of H 2 O 2 (left) and with the addition of H 2 O 2 (right). (B) Representative scanning microscope images of the prepared PEGel (left) and Puerarin@PEGel (right). (C-E) Rheological properties of the unprepared PEGel when the shear frequency (C), strain (D) and temperature (E) change. (F) Cumulative release curve of puerarin in Puerarin@PEGel. (G) Expression of P-eNOS and eNOS in HUVECs determined by Western blotting. (H) Relative production amount of NO after treating HUVECs with puerarin for 24 hours. (I-M) Relative cell viability after treating HUVEC (I), con-CAR-NK (J), HER1-CAR-NK (K), MDA-MB-468 (L) and NCI-H23 (M) cells with puerarin.

[0028] Figure 2Construction of HER1-CAR-NK cells and its inhibition of effector functions under hypoxia mediation. (A) Schematic diagram of gene construction of con-CAR and HER1-CAR. (B) Electrophoretogram of vector or enzyme cleavage products. Lane 1, con-CAR vector; Lane 2, con-CAR DNA product treated with EcoRI / XbaI; Lane 3, HER1-CAR vector; Lane 4, HER1-CAR DNA product treated with EcoRI / XbaI. (C-E) Detection of the expression level of CD3ζ in NK, con-CAR-NK, and HER1-CAR-NK cells by real-time PCR (C), Western blot (D), and flow cytometry (E). (F) Relative cell viability of NK, con-CAR-NK, and HER1-CAR-NK cells cultured under hypoxic or normoxic conditions. (G&H) As shown in the figure, the levels of granzyme B and perforin secreted by con-CAR-NK or HER1-CAR-NK cells under hypoxic and normoxic conditions when the ratio of NK cells / tumor cells (MDA-MD-468 or NCI-H23) is 2.5:1. (I&J) Lysis rates (detected by LDH) of con-CAR-NK or HER1-CAR-NK co-cultured with MDA-MB-468 (I) and NCI-H23 (J) at different ratios under hypoxic and normoxic conditions. (K) Expression levels of p-SHP-1, SHP-1, p-ERK, and ERK in NK, con-CAR-NK, and HER1-CAR-NK cells under hypoxic or normoxic conditions. (L) Schematic diagram of the mechanism by which hypoxia inhibits the effector functions of CAR-NK cells.

[0029] Figure 3 In vivo retention curve of Puerarin@PEGel and its ability to activate the eNOS / NO / cGMP axis. (A) Schematic diagram of the experiment. (B-C) Intratumoral retention of fluorescein@PEGel over time in MDA-MB-468 and NCI-H23 tumor-bearing mice 2 days or 9 days after injection. (D-I) Intratumoral profiles of p-eNOS and eNOS expression (D, E), NO production (F, G), and cGMP production (H, I) in MDA-MB-468 and NCI-H23 tumor-bearing mice 2 days or 9 days after injection of fluorescein@PEGel. (J) Schematic diagram of the mechanism by which Puerarin@PEGel treatment activates the eNOS / NO / cGMP axis.

[0030] Figure 4Puerarin@PEGel-mediated tumor microenvironment remodeling. (A) Schematic diagram of the experiment. (B-D) Representative CD31 and NG2 immunofluorescence staining images (B) of tumor sections collected from MDA-MB-468 tumor-bearing mice after treatment and the corresponding semi-quantitative analysis results (C&D) are shown as follows. (E-G) Representative CD31 and NG2 immunofluorescence staining images (CD31 and NG2) and the corresponding semi-quantitative analysis results (F&G) are shown as follows. (H-K) Representative immunofluorescence staining images of tumor sections collected from MDA-MB-468 and NCI-H23 tumors, anti-pimonidazole (H&I) and anti-HIF-1α staining (J&K). Relative contents of lactates (L&M) and TGF-β (N&O) in MDA-MB-468 or NCI-H23 tumors.

[0031] Figure 5 Puerarin@PEGel can improve the infiltration, survival time and effector function of CAR-NK cells in vivo. (A) Schematic diagram of the experimental procedure. (B&C) Flow cytometry analysis of the intratumoral percentages of con-CAR-NK or HER1-CAR-NK cells treated differently in MDA-MB-468 (B) and NCI-H23 (C) tumors. (D&E) Real-time quantitative PCR detection of the relative intratumoral infiltration rates of con-CAR-NK or HER1-CAR-NK cells in MDA-MB-468 (D) and NCI-H23 (E) tumors under different treatment methods. (F-I) Immunofluorescence staining and semi-quantitative analysis of the infiltration rates of con-CAR-NK and HER1-CAR-NK cells in MDA-MB-468 (F&G) and NCI-H23 (H&I) tumors under different treatment methods. (J-M) Relative intratumoral expression levels of granzyme B (J&K) and perforin (L&M) in MDA-MB-468 and NCI-H23 tumors under different treatment methods.

[0032] Figure 6 Puerarin@PEGel enhanced the therapeutic effect of HER1-CAR-NK cells on solid tumors. (A) Flow chart of the experiment. (B-D) Average tumor volume (B), individual tumor volume (C) and median survival time (D) of NCI-H23 tumors after different treatments as shown. (E-G) Average tumor volume (E), individual tumor volume (F) and median survival time (G) of MDA-MB-468 tumors after different treatments. Detailed implementation manners

[0033] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0034] Materials, reagents, etc. used in the examples can be obtained from commercial sources unless otherwise specified.

[0035] Biomaterials, reagents, and kits used in the present invention can all be obtained by conventional commercial purchase, and the experimental techniques used are all conventional operations in the art or are carried out with reference to the instructions of the corresponding products.

[0036] The sources or models of the biomaterials used in the present invention are as follows:

[0037] Lung cancer cell NCI-H23( CRL-5800 TM );

[0038] Breast cancer cell MDA-MB-468( HTB-132 TM );

[0039] PBMCs cells were purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd.

[0040] HUVECs cells were purchased from Shanghai Bihe Biochemistry Technology Co., Ltd.;

[0041] NCI-H23 cells were purchased from Shanghai Chuanqiu Biotechnology Co., Ltd.;

[0042] con-CAR-NK cells were constructed in Example 2;

[0043] HER1-CAR-NK cells were constructed as in Example 2.

[0044] The sources of the biological and chemical reagents used in the examples of the present invention are shown in Table 1:

[0045] Table 1 Sources of biological and chemical reagents

[0046]

[0047]

[0048] Example 1

[0049] (Preparation and characterization of (polyethylene glycol dimethyl) acrylate gel:

[0050] Mix the substances in a pure water system at the final concentrations, and mix PEGDMA (10 wt%) with FeCl 2 (1.6 mM), H 2 O 2(1 mM) and puerarin (10 μM) were mixed and then incubated at room temperature for 1 min until gel formation. The microstructure of the prepared hydrogel was observed using a scanning electron microscope (SEM, ZEISS Supra55). The rheological properties of the prepared hydrogel at different shear rates, strains, and temperatures were evaluated using a rotational rheometer (HAAKE MARS 40). The release of puerarin was evaluated in PBS (pH 7.4) at 37 °C. Absorbance was recorded at a wavelength of 306 nm using a multimode reader (Thermo Variskan) (mass extinction coefficient is 13.4 mL mg -1 cm -1 ), and the content of released puerarin was determined.

[0051] It was found that, according to the above method, when an equimolar concentration of PEGDMA monomer and H 2 O 2 and FeCl 2 were mixed in the system, a hydrogel (PEGel) could be formed within one minute at room temperature ( Figure 1 A). Scanning electron microscopy showed that the obtained plain PEGel or puerarin-loaded PEGel (Puerarin@PEGel) exhibited a similar micro-porous network structure ( Figure 1 B). Using a rotational rheometer, it was found that at different angular frequencies and strain conditions, the storage modulus (G') value was higher than the loss modulus (G”) value ( Figure 1 C-D), indicating that PEGel has good mechanical strength. In addition, the G' and G” values of PEGel measured under the conditions of 25 - 60 °C fluctuated very little, further indicating its excellent stability ( Figure 1 E), which also proved from the side that the puerarin hydrogel Puerarin@PEGel also has good stability and mechanical properties. By recording the typical absorbance of puerarin in Puerarin@PEGel in PBS at 37 °C at a wavelength of 306 nm, it can be seen that after Puerarin@PEGel was soaked in PBS for 48 hours, about 60% of the puerarin was released from PEGel, indicating that Puerarin@PEGel can achieve the sustained release of puerarin ( Figure 1 F).

[0052] Example 2

[0053] Cell experiment:

[0054] All cell lines were purchased from ATCC. MDA-MB-468 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. HUVECs and NCI-H23 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. All cells were cultured in a humidified incubator at 37°C with 5% CO 2 2. NK cells were isolated from peripheral blood mononuclear cells of healthy human whole blood and cultured in NK cell-specific medium supplemented with 10% human serum at 37°C.

[0055] To evaluate the ability of puerarin treatment to activate the eNOS / NO axis in HUVECs, HUVECs were pre-cultured in 6-well plates (75% cell density per well), treated with puerarin (0.01 μM - 10 μM) for 24 hours, then HUVECs were collected, and the expression levels of p-eNOS and eNOS were detected by Western blotting, and the production of NO was detected by a commercial NO detection kit. Western blotting results showed that incubation with puerarin (10 μM, 24 hours) led to a significant increase in the intracellular p-eNOS level in HUVECs, while the eNOS level remained unchanged ( Figure 1 G). Detection by the NO detection kit found that the NO production in HUVECs treated with puerarin was significantly increased compared with HUVECs not treated with puerarin ( Figure 1 H). In summary, puerarin can activate the eNOS / NO axis in endothelial cells.

[0056] To evaluate the cytotoxicity of puerarin, HUVECs, con-CAR-NK cells, HER1-CAR-NK cells (the construction of the two types of cells is as follows), MDA-MB-468 cells and NCI-H23 cells were pre-seeded in 96-well plates at a density of 1×10 4 cells per well, treated with different concentrations of puerarin for 24 hours, and then the viability of these cells was detected using the CCK-8 or MTT method according to the standard procedure. The study showed that after incubation with puerarin for 24 hours at a series of concentrations from 0.01 μM to 10 μM, the cell viability of HUVECs, con-CAR-NK cells, HER1-CAR-NK cells, MDA-MB-468 cells and NCI-H23 cells remained almost unchanged ( Figure 1 I-M), demonstrating its safety.

[0057] HER1-CAR-NK cells were generated by transducing NK cells with a lentiviral vector encoding the HER1-specific third-generation CAR sequence. Con-CAR-NK was generated by transducing with a non-targeting third-generation CAR lentiviral vector. The successful construction of HER1-CAR-NK cells and con-CAR-NK cells was determined by detecting the expression of CD3ζ by Western blot, real-time PCR, and flow cytometry.

[0058] The specific construction of con-CAR-NK cells and HER1-CAR-NK cells is as follows: First, the third-generation CAR plasmid containing the CD8 signal, CD8 Hinge, CD28, 4-1BB, and CD3ζ sequences (Yan Liu et al. Cell Prolif. 2020 Aug; 53(8): e12858.) was fused with the sequence encoding the HER1-specific single-chain fragment variable (scFv) (Lin Xia et al. Clin Transl Immunol. 2020 May 3; 9(5): e01135.) to construct the HER1-CAR vector. The non-specific third-generation CAR plasmid was used as con-CAR( Figure 2 A). The successful construction of the con-CAR and HER1-CAR vectors was verified by EcoRI / XbaI restriction digestion assay( Figure 2 B). Subsequently, NK cells obtained from human peripheral blood mononuclear cells (PBMCs) were transduced with the lentivirus expressing HER1-CAR to generate HER1-CAR-NK cells, and NK cells were transduced with the lentivirus containing the con-CAR expression plasmid to generate Con-CAR-NK cells. Specifically: CAR (HER1-CAR / con-CAR), pMD2.G, and psPAX2 lentiviral plasmids were mixed with lipo2000 at a ratio of 4:1:3 and transfected into 293T / 17 cells. The fresh complete medium was replaced 8 hours after transfection. The culture medium was collected three days later, filtered through a 0.45 μm filter, and the virus was concentrated by ultracentrifugation to collect the high-titer lentiviral solution. The lentivirus was added to NK cells to obtain the relevant con-CAR-NK and HER1-CAR-NK cells. Then, the expression of CD3ζ was detected by real-time PCR, Western blot, and flow cytometry analysis, which also confirmed the successful construction of con-CAR-NK and HER1-CAR-NK cells( Figure 2 C-E).

[0059] To evaluate the effect of hypoxic culture on the effector function of CAR-NK cells, at normoxia and hypoxia, at 1×10 per well 4The density of the cells was pre-seeded with MDA-MB-468 and NCI-H23 cells in 96-well plates and co-cultured with HER1-CAR-NK cells at different ratios for 24 hours (at 1:1, 2.5:1, 5:1). Then, ELISA kits or LDH kits were used to detect granzyme B, perforin, and lactate dehydrogenase (LDH) secreted in the supernatant. It was found by MTT assay that under hypoxic culture (<2% O 2 , 24 h) conditions, the cell viabilities of NK, con-CAR-NK, and HER1-CAR-NK cells were only 67%, 62%, and 72% respectively compared to the viabilities of the corresponding cells under normoxic culture conditions ( Figure 2 F). And it was detected by ELISA that after incubation of HER1-CAR-NK cells with MDA-MB-468 and NCI-H23 cells, the secretion amounts of granzyme B and perforin were significantly higher than those of con-CAR-NK cells incubated with cancer cells under the same conditions ( Figure 2 G-H). In sharp contrast, hypoxic incubation significantly restricted the secretion of cytotoxic granzyme B and perforin by HER1-CAR-NK cells incubated with targeted cancer cells. It was found by the standard LDH release assay that compared with con-CAR-NK cells, the cell killing ability of HER1-CAR-NK cells against targeted MDA-MB-468 and NCI-H23 cells was significantly enhanced. However, hypoxic culture conditions significantly restricted the cell killing ability of HER1-CAR-NK cells against the corresponding targeted cancer cells ( Figure 2 I-J). This experiment effectively demonstrated that hypoxic culture conditions significantly restricted the cell killing ability of HER1-CAR-NK cells against the corresponding targeted cancer cells.

[0060] To evaluate the effect of hypoxia on CAR-NK cells, NK cells, con-CAR-NK cells, and HER1-CAR-NK cells were cultured under normoxic or hypoxic (<2% O 2 ) conditions for 24 hours, and then the survival rates of all treated cells were evaluated by MTT assay, and the expression levels of intracellular p-SHP-1, SHP-1, p-ERK, and ERK were detected by Western blot. Western blot detection showed that hypoxic incubation led to an increase in the phosphorylation amount of SHP-1, but had little effect on the total SHP-1 expression. This hypoxic incubation promoted the phosphorylation of SHP-1, further indicating that this led to a decrease in the phosphorylation level of ERK compared with the corresponding cells under normoxic incubation ( Figure 2 K). Therefore, these results together indicate that the introduction of HER1-CAR will significantly enhance the targeted killing activity of NK cells, while under hypoxic culture conditions, the killing activity of NK cells will be significantly inhibited, which may be achieved through the attenuation effect of activating the SHP-1-mediated ERK axis.Figure 2 L).

[0061] In vivo animal experiments:

[0062] NSG mice were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. All animal experiments were approved by the Experimental Animal Center of Nanjing Normal University. An NCI-H23 tumor model was established by subcutaneous injection, and an MDA-MB-468 tumor model was established by injection into the mammary fat pad. Two weeks after injecting the tumor cells, the drug was injected.

[0063] To evaluate the sustained release effect of PEGel in vivo, Fluorescein@PEGel and free Fluorescein solution were injected into the tumors of MDA-MB-468 or NCI-H23 tumor NSG mice (maintaining Fluorescein = 1 mg / kg for the above two, and the method of loading the fluorescent agent Fluorescein onto PEGel refers to Example 1). At 10 minutes, 2 days, and 9 days after injection, the tumors of each tumor NSG mouse were collected, and Fluorescein fluorescence was detected by immunofluorescence. The results of the confocal microscope showed that the tumor sections of MDA-MB-468 and NCI-H23 tumor mice collected 10 minutes and 2 days after intratumoral injection of free Fluorescein and Fluorescein@PEGel showed similar fluorescence signals ( Figure 3 A-C). In sharp contrast, obvious fluorescence signals were only observed in the tumor sections collected 9 days after intratumoral injection of Fluorescein@PEGel in mice, while free Fluorescein only showed obvious fluorescence signals in the tumor sections at 10 min and 2 days after injection, and no fluorescence signals were detected in the tumor sections at 9 days. These results indicate that PEGel can retain the loaded small molecules in the tumor for a long time, proving that PEGel has good sustained release ability, suggesting that PEGel drug loading can have good effects. In this experiment, since puerarin has no detectable label, Fluorescein was used instead of puerarin to prove that the PEGel system can sustainably release drugs for a long time.

[0064] To evaluate the effect of Puerarin@PEGel treatment on remodeling tumor blood vessels, four groups of MDA-MB-468 or NCI-H23 tumor-bearing NSG mice were intratumorally injected with PBS, PEGel, Puerarin, and Puerarin@PEGel (maintaining Puerarin = 30 mg / kg and PEGgel = 50 μL as above). On the 2nd and 9th days after injection, tumors from each mouse were collected, and the expression levels of p-eNOS and eNOS, as well as the production of NO and cGMP, were detected by Western blot, NO detection kit, and cGMP detection kit. In addition, the expression levels of CD31 and NG2, as well as pimonidazole and HIF-1α, were detected by immunofluorescence staining. The contents of lactates and TGF-β in the tumors were detected by lactate kit and ELISA kit. Western blot results showed that in tumors treated with Puerarin@PEGel for 2 days and 9 days, the expression of p-eNOS increased significantly, but the change in the expression of total eNOS was very small ( Figure 3 D-E). In sharp contrast, ordinary Puerarin only significantly increased the expression of p-eNOS at 2 days after treatment, while the effect of ordinary PEGel on p-eNOS in tumors was negligible. In addition, through the above commercial NO detection kit, it was found that the amount of NO in MDA-MB-468 and NCI-H23 tumors increased significantly at 2 days and 9 days after Puerarin@PEGel treatment. At the same time, compared with mice injected with PEGel or the control group, ordinary Puerarin treatment could promote NO production in these tumors at 2 days after treatment ( Figure 3 F-G). Compared with the control group and the PEGel injection group, when injected with Puerarin and Puerarin@PEGel for two days, both could promote NO production in tumors, and only the Puerarin@PEGel injection group could promote NO production until the ninth day with a significant effect. In addition, through ELISA experiments, it was found that Puerarin@PEGel treatment could increase the production of cGMP in these two types of tumors for up to 9 days ( Figure 2 H-I). Compared with PEGel and the control group, ordinary Puerarin treatment only led to a short-term increase in cGMP production. Compared with the control group and the PEGel injection group, when injected with Puerarin and Puerarin@PEGel for two days, both could promote cGMP production in tumors, and only the Puerarin@PEGel injection group could promote cGMP production until the ninth day with a significant effect. These results together indicate that puerarin@PEGel (Puerarin@PEGel) can activate the eNOS / NO / cGMP axis in endothelial cells and adjacent vascular smooth muscle cells (Figure 3 J)

[0065] NO can dilate blood vessels and promote blood vessel maturation. Therefore, in this example, the regulatory effect of Puerarin@PEGel treatment on the normalization of blood vessels in MDA-MB-468 and NCI-H23 mouse models was studied. The method was the same as the effect of Puerarin@PEGel treatment on remodeling tumor blood vessels. Control, PEGel, Puerarin, and Puerarin@PEGel were injected into the tumors, and blood vessel normalization indexes were detected. The results of immunofluorescence staining showed that there was no significant difference in the expression level of CD31 among the groups ( Figure 4 A-G), indicating that the effect of Puerarin@PEGel treatment on intratumoral blood vessel density was minimal. In sharp contrast, the detection of the expression of NG2 at 2 days and 9 days after treatment showed that Puerarin@PEGel treatment significantly increased the maturity of tumor blood vessels ( Figure 3 B-G). At the same time, using the same method, ordinary Puerarin treatment could only promote the maturation of tumor blood vessels at 2 days after treatment. Compared with the control group and the PEGel injection group, there was no significant change in the CD31 index after injecting Puerarin and Puerarin@PEGel for two or nine days, indicating that there was no obvious promotion of angiogenesis. However, NG2 / CD31 was significantly increased on the second day of Puerarin treatment, indicating that Puerarin could promote blood vessel maturation, but the effect did not last until the ninth day. In fact, Puerarin is water-soluble, and water-soluble drugs are effective within 1-2 days and are metabolized by the kidneys after three days. The increase in NG2 / CD31 could be observed on the second day and the ninth day after Puerarin@PEGel injection, indicating that Puerarin@PEGel could promote blood vessel maturation for a long time. In addition, pimonidazole and HIF-1α were detected by immunofluorescence, and the results showed that the area of hypoxic tumors in the tumor and the expression of HIF-1α were significantly reduced ( Figure 4 H-K). Consistently, ordinary Puerarin treatment only led to a short-term 2-day tumor oxygenation effect, and puerarin@PEGel could long-term promote the tumor oxygenation effect up to 9 days. These results together indicate that Puerarin@PEGel can reduce tumor hypoxia by promoting tumor blood vessel normalization. Consistently, ordinary Puerarin treatment only led to a short-term tumor oxygenation effect. These results together indicate that Puerarin@PEGel can reduce tumor hypoxia by promoting tumor blood vessel normalization.

[0066] Tumor hypoxia can promote the accumulation of immunosuppressive lactate in tumors by promoting glycolysis. Therefore, using a commercial lactate content detection kit, it was found that Puerarin@PEGel treatment could inhibit lactate production at 2 days and 9 days after treatment ( Figure 4 L,M). Ordinary Puerarin treatment only had a short-term inhibitory effect on the production of intratumoral lactate. Compared with the control group and the PEGel injection group, the content of lactate in the tumor decreased on the second day after Puerarin injection, but the content of lactate rebounded on the ninth day, indicating that Puerarin could inhibit lactate in the tumor, but the effect did not last until the ninth day. The content of lactate in the tumor decreased on the second and ninth days after Puerarin@PEGel injection, indicating that Puerarin@PEGel could significantly inhibit lactate for a long time. In addition, through the use of the corresponding ELISA detection, it was found that the expression of TGF-β could also be effectively downregulated at 2 days and 9 days after Puerarin@PEGel treatment ( Figure 4 N,O). Similarly, ordinary Puerarin treatment only had a short-term inhibitory effect on intratumoral TGF-β. Compared with the control group and the PEGel injection group, the content of TGF-β in the tumor decreased on the second day after Puerarin injection, but the content of TGF-β rebounded on the ninth day, indicating that Puerarin could inhibit TGF-β in the tumor, but the effect did not last until the ninth day. The content of TGF-β in the tumor decreased on the second and ninth days after Puerarin@PEGel injection, indicating that Puerarin@PEGel could inhibit TGF-β for a long time. These results indicate that Puerarin@PEGel treatment can inhibit the secretion of TGF-β.

[0067] To evaluate the effectiveness of Puerarin@PEGel treatment in promoting the infiltration, function, and survival time of CAR-NK cells in vivo, five groups of NSG mice carrying MDA-MB-468 or NCI-H23 tumors were treated as follows: Group 1, Con-CAR-NK injection; Group 2, HER1-CAR-NK injection; Group 3, PEGel fixation + HER1-CAR-NK injection; Group 4, Puerarin injection + HER1-CAR-NK injection; Group 5, Puerarin@PEGel fixation + HER1-CAR-NK injection, where the injection of con-CAR-NK cells or HER1-CAR-NK cells was 5×10 6, Puerarin@PEGel, and Puerarin (Puerarin = 30 mg / kg), with PEGgel and PBS having the same volume as Puerarin@PEGel. On day 0, Puerarin@PEGel was injected intratumorally (puerarin 30 mg / kg). On day 1, 5×10 6 con-CAR-NK cells or HER1-CAR-NK cells were injected via the tail vein. At 2 days and 9 days after treatment, tumors were collected, and the levels of RFP labeling were detected by flow cytometry, real-time quantitative PCR, and immunofluorescence. The expression levels of telomerase B and perforin were detected using a telomerase detection kit and a perforin detection kit.

[0068] By flow cytometry, it was found that Puerarin@PEGel treatment could effectively promote the infiltration and survival of such HER1-CAR-NK cells in MDA-MB-468 and NCI-H23 tumors for up to 9 days ( Figure 5 A-C). In contrast, ordinary Puerarin only significantly infiltrated tumors with HER1-CAR-NK cells within 2 days after injection. Recording the intratumoral RFP signal by real-time PCR and immunofluorescence staining further confirmed the high efficiency of Puerarin@PEGel treatment in promoting the intratumoral infiltration and survival of HER1-CAR-NK cells ( Figure 5 D-I). In addition, the expressions of granzyme B and perforin were significantly increased in the MDA-MB-468 and NCI-H23 tumors of mice at 2 days and 9 days after continuous treatment with Puerarin@PEGel and injection of HER1-CAR-NK cells. In sharp contrast, continuous use of ordinary Puerarin and injection of HER1-CAR-NK cells only led to significant intratumoral expression of granzyme B and perforin within 2 days after treatment, while other treatments had little effect on the intratumoral expression of granzyme B and perforin ( Figure 5 J-M). In summary, these results indicate that Puerarin@PEGel treatment can significantly enhance the infiltration, survival, and cytolytic activity of HER1-CAR-NK cells in solid tumors.

[0069] To evaluate the tumor inhibitory effect of Puerarin@PEGel in combination with CAR-NK cells, another batch of NSG mice bearing MDA-MB-468 and NCI-H23 tumors were injected with con CAR-NK cells or HER1 CAR-NK cells (5×10 6) Except as described above, other treatment methods are as described above. The tumor length (L) and width (W) of each mouse were measured using a digital caliper, and the tumor volume (V) was calculated according to the following formula: V = L * W * W / 2. During the entire monitoring process, the body weight of each mouse was measured using a digital balance.

[0070] By evaluating the effect of Puerarin@PEGel treatment on the inhibitory ability of HER1-CAR-NK cells against NCI-H23 tumors in NSG mice ( Figure 6 A-E), it was found that Puerarin@PEGel combined with HER1-CAR-NK treatment completely inhibited the growth of NCI-H23 tumors ( Figure 6 B-C). In sharp contrast, intratumoral injection of ordinary Puerarin combined with HER1-CAR-NK treatment only showed a slightly increased tumor inhibitory effect compared with single HER1-CAR-NK treatment or treatment with ordinary PEGel plus HER1-CAR-NK. The median survival time of mice receiving ordinary Puerarin-assisted HER1-CAR-NK treatment was determined to be 46 days, while the median survival times of tumor-bearing mice receiving PEGel plus HER1-CAR-NK cells, single HER1-CAR-NK cells, and non-HER1-CAR-NK cell treatment (Con-CAR-NK) were only 33 days, 38 days, and 25 days, respectively ( Figure 6 D). In addition, the synergistic effect of Puerarin@PEGel therapy on HER1-CAR-NK therapy was further evaluated. For this purpose, five groups of mice received the same treatment as described above. Consistently, Puerarin@PEGel combined with HER1-CAR-NK treatment completely inhibited the growth of MDA-MB-468 tumors ( Figure 6 E-F). In sharp contrast, ordinary Puerarin combined with HER1-CAR-NK treatment only moderately inhibited the growth of MDA-MB-468 tumors, and the median survival time of these mice was determined to be 55 days. The median survival times of mice receiving PEGel plus HER1-CAR-NK cells, single HER1-CAR-NK cells, and non-HER1-CAR-NK cell treatment were 40 days, 41 days, and 26 days, respectively ( Figure 6 G). In summary, compared with HER1-CAR-NK, Puerarin-assisted HER1-CAR-NK, and PEGel-assisted HER1-CAR-NK treatments, Puerarin@PEGel combined with HER1-CAR-NK can more effectively inhibit solid tumors, has a significant inhibitory effect on tumors, is significantly superior to other groups, and ultimately comprehensively inhibits solid tumors.

Claims

1. A puerarin hydrogel for enhancing the ability of CAR-NK therapy to eliminate solid tumors, characterized in that, the puerarin hydrogel is formed by mixing a puerarin with a hydrogen peroxide-reactive hydrogel composed of (polyethylene glycol dimethyl) acrylate and ferrous chloride.

2. The puerarin hydrogel for enhancing the ability of CAR-NK therapy to eliminate solid tumors according to claim 1, characterized in that, the puerarin hydrogel is prepared by mixing (polyethylene glycol dimethyl) acrylate, ferrous chloride, hydrogen peroxide, and puerarin, and then incubating at room temperature to form a gel.

3. Use of the puerarin hydrogel according to claim 1 in the preparation of an anti-tumor or tumor-inhibiting drug.

4. The use according to claim 3, characterized in that, the puerarin hydrogel preferably promotes the production of nitric oxide by endothelial cells, dilates tumor blood vessels, relieves hypoxia, and reverses tumor immunosuppression.

5. Use of the puerarin hydrogel according to claim 1 in the preparation of a drug for promoting CAR-NK infiltration, survival and function and eliminating solid tumors.

6. The use according to claim 5, characterized in that, the puerarin hydrogel promotes the production of nitric oxide by endothelial cells, dilates tumor blood vessels, relieves hypoxia, reverses tumor immunosuppression, promotes CAR-NK infiltration, survival and function, and eliminates solid tumors.

7. Use of the puerarin hydrogel according to claim 1 in combination with CAR-NK cells in the preparation of a drug or reagent for eliminating solid tumors.

8. Use of the puerarin hydrogel according to claim 1 in the preparation of a drug for enhancing the ability of CAR-NK therapy to eliminate solid tumors.

9. The use according to claim 3 or 5 or 7, characterized in that, the tumors include breast tumors and lung tumors.

10. A pharmaceutical composition for enhancing the ability of CAR-NK therapy to eliminate solid tumors, characterized in that, it contains the puerarin hydrogel according to claim 1 as an active ingredient and a pharmaceutically acceptable excipient.

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