Polymer, drug-loaded nanogel, method for adoptive immune cell surface modification of drug-loaded nanogel and application of polymer and drug-loaded nanogel

Abstract

The invention discloses a polymer, a drug-loaded nanogel, a method for adoptive immune cell surface modification of the drug-loaded nanogel and application of the drug-loaded nanogel, and belongs to the technical field of biological medicine. The structure of the polymer is shown as a formula 1. Wherein R is selected from # imgabs0 # x, y and z, k is the degree of polymerization, x + y + z + k is more than or equal to 10 and less than or equal to 4000, and xgt is more than or equal to 10 and less than or equal to 4000; 0, ygt; 0, zgt; 0, k > 0; n is the degree of polymerization and is not less than 10 and not more than 400. The drug-loaded nanogel prepared from the polymer is used for modifying the surface of adoptive immune cells, so that the adoptive immune cells have an excellent tumor treatment effect. The method for adoptive immune cell surface modification of the drug-loaded nanogel is stable in mounting, simple and high in universality, the administration dosage can be intelligently controlled, and the killing capacity or in-vivo tracing capacity of solid tumors is remarkably enhanced. # imgabs1 #

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a polymer and a drug-loaded nanogel, a method for adoptively modifying the surface of the drug-loaded nanogel with immune cells, and applications thereof. Background Art

[0002] Adoptive Cell Transfer Therapy (ACT) refers to the separation of immune-active cells from tumor patients, amplification and functional identification in vitro, and then back-transfusion to patients, so as to achieve the purpose of directly killing tumors or stimulating the body's immune response to kill tumor cells. Adoptive immune cell therapy mainly includes several categories such as TIL, LAK, CIK, DC, NK, TCR-T, and CAR-T. Among them, chimeric antigen receptor T cell therapy (CAR-T) therapy is regarded as a revolutionary breakthrough in tumor immunotherapy, and its research progress has become one of the most concerned areas. However, despite its magical effect in anti-hematoma, the treatment of solid tumors remains a daunting challenge for CAR-T therapy. Although CAR-T therapy is effective in treating hematological tumors, it faces four major challenges in treating solid tumors. The first is the problem of tumor infiltration. There are matrix barriers (such as hyaluronic acid) in solid tumors, so T cells must overcome these barriers to reach the tumor site; the second is the problem of immunosuppression. The tumor microenvironment (TME) of solid tumors has an immunosuppressive effect on T cells, which greatly weakens the anti-tumor ability of T cells; the third is the problem of CAR targets. For most solid tumors, their tumor-associated antigens (TAA) are enriched in tumors and expressed at low levels in normal tissues, making it difficult to find suitable specific recognition antigens for CAR design; the fourth is the problem of CAR-T cell tracing. It is difficult for people to grasp the proliferation and distribution of CAR-T cells after they are reinfused into the human body, so it is impossible to detect whether the CAR-T cell response is sufficient or to make early warnings for potential toxicity progression.

[0003] In order to overcome the above difficulties, people usually use genetic engineering, that is, after cell transfer, the therapeutic cells usually rely on adjuvant drugs delivered at the same time for combined therapy. These drugs are designed to maximize the efficacy and in vivo persistence of the reinfused adoptive immune cells. However, these drugs often require high concentrations and continuous delivery, resulting in dose-limiting toxicity, which limits their clinical application. One way to concentrate the effects of adjuvant drugs on transferred cells is to genetically engineer donor cells so that they secrete adjuvant drugs themselves. However, gene transfection methods have significant complexity and uncertainty, and often cannot expand too many functions; in addition, cells that have undergone genetic engineering will continue to secrete adjuvant drugs, which will also bring safety risks. At the same time, some emerging adjuvant therapies are mostly based on small molecule drugs, which cannot be encoded by genes.

[0004] Researchers have tried to develop cell surface modification technology to overcome the problem of co-delivery of adoptive cells and adjuvant drugs, providing another feasible solution to the above problems. For example, Irvine et al. used the reaction of maleimide and thiol to bind drug-loaded liposome nanoparticles to the surface of CAR-T cells. The nanoparticles release protein drugs over time. However, the number of thiol groups on the cell surface is uncertain, which makes the degree of cell surface modification uncontrollable and the delivered drug dose cannot be controlled. In addition, the newly generated CS bond in the coupling of maleimide groups and thiol groups on T cells is not stable enough under physiological conditions, and reverse Michael addition will occur, which is more unstable in the presence of competitive thiol compounds (such as cysteine). Can Zhang et al. attached liposomes loaded with cholesterol drug Avasimibe to the surface of T cells by inserting lipids into the tetrazine group of the cell membrane. However, the lipid insertion of tetrazine groups on the cell membrane is unstable and may fall off in the complex in vivo environment. Public document CN 112980786 reports a method for connecting T cells and nanoparticles based on click chemistry, which uses a metabolic sugar method to modify and label azide groups on the surface of T cells, and then uses a copper-free click chemistry reaction to modify poly(lactide-glycolide) nanoparticles on the surface of T cells; however, due to the characteristics of poly(lactide-glycolide) nanoparticles, they lack intelligent design and cannot achieve specific drug release, so the types of drugs they can carry are limited. The public document only shows a model reaction when carrying indocyanine green, so they lack universality and have a single function.

[0005] Therefore, it is of great significance to research and develop a new, simple, efficient and universal method for enhancing and tracing adoptive immune cell therapy for tumors. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a polymer and a drug-loaded nanogel, a method for modifying the surface of adoptive immune cells with the drug-loaded nanogel and its application. The drug-loaded nanogel prepared by the polymer is used to modify the surface of adoptive immune cells, thereby making the adoptive immune cells have a better anti-tumor effect.

[0007] In order to achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides a polymer, the structure of which is shown in Formula 1:

[0009]

[0010] Among them, R is selected from x, y, z, k are the degree of polymerization, 10≤x+y+z+k≤4000, x>0, y>0, z>0, k>0; n is the degree of polymerization, 10≤n≤400.

[0011] Preferably, the R is selected from Preferably, 10≤x+y+z+k≤500;

[0012] Preferably, 10≤n≤100.

[0013] The present invention also provides a drug-loaded nanogel, which is prepared by mixing an aqueous solution of a structural polymer shown in Formula 1-1, an aqueous solution of a structural polymer shown in Formula 1-2, and an aqueous solution of an auxiliary drug, and then assembling the mixture through supramolecular host-guest assembly.

[0014] The drug-loaded nanogel is prepared by cross-linking reaction under mild conditions through supramolecular host-guest interaction of the structural polymer shown in Formula 1-1 and the structural polymer shown in Formula 1-2. A large amount of auxiliary drugs are coated while forming the nanogel, so that the drug-loaded nanogel can be specifically disintegrated under the tumor microenvironment through intelligent design, and the drug loading of the drug-loaded nanogel is higher than that of general drug delivery carriers, so that local high-concentration release of drugs can be achieved, thereby significantly improving the therapeutic effect of tumors. The structural polymer shown in Formula 1-1, the structural polymer shown in Formula 1-2 and the auxiliary drugs are dissolved in aqueous solution and then mixed, so as to make the nanoparticles of the formed drug-loaded nanogel more regular and stable.

[0015] The drug-loaded nanogel of the present invention can carry many types of auxiliary drugs, including but not limited to protein drugs or small molecule drugs, etc. Preferably, the auxiliary drug is selected from one or more of hyaluronidase, sialidase, interleukin 15, cisplatin, and IR-808.

[0016]

[0017] Wherein, x, y, z, k are the degree of polymerization, 10≤x+y+z+k≤4000, x>0, y>0, z>0, k>0;

[0018] n is the degree of polymerization, 10≤n≤400.

[0019] Preferably, the concentration of the auxiliary drug is 0.1-10 mg / mL; more preferably 2 mg / mL.

[0020] Preferably, the concentration of the structural polymers represented by Formula 1-1 and Formula 1-2 is 0.1-10 mg / mL, respectively; more preferably 1 mg / mL.

[0021] Preferably, the mass ratio of the aqueous solution of the structural polymers represented by Formula 1-1 and Formula 1-2 to the aqueous solution of the auxiliary drug is (0.1-10):1:(0.1-10); more preferably (0.5-2):1:(0.1-1). In some specific embodiments of the present invention, it is preferably 1:1:0.4.

[0022] Preferably, the aqueous solution is selected from phosphate buffer solution, tris(hydroxymethylaminomethane) hydrochloride buffer solution, sodium citrate buffer solution or 4-hydroxyethylpiperazineethanesulfonic acid buffer solution.

[0023] The preparation method of the drug-loaded nanogel comprises the following steps:

[0024] (1) Dissolving the polymer of formula 1-1, the polymer of formula 1-2, and the auxiliary drug in a phosphate buffered saline solution to obtain an aqueous solution of the polymer of formula 1-1, an aqueous solution of the polymer of formula 1-2, and an aqueous solution of the auxiliary drug, respectively.

[0025] (2) mixing an aqueous solution of a polymer having a structure represented by Formula 1-1 with an aqueous solution of an auxiliary drug, and after fully mixing, adding an aqueous solution of a polymer having a structure represented by Formula 1-2 and mixing them uniformly to obtain a mixed system S;

[0026] (3) The mixed system S is dialyzed to remove the free auxiliary drug, thereby obtaining the drug-loaded nanogel. The preparation method of the drug-loaded nanogel is simple and the conditions are mild.

[0027] The drug-loaded nanogel has various functions depending on the auxiliary drugs it carries. In addition to enhancing the synergy of adoptive immune cells, it can also trace cells in the human body.

[0028] The present invention also provides a method for adoptive immune cell surface modification of drug-loaded nanogel, comprising the following steps:

[0029] The adoptive immune cells with azide groups labeled on the surface are co-incubated with the above-mentioned drug-loaded nanogel, so that the adoptive immune cells and the drug-loaded nanogel are connected through a copper-free catalytic click chemistry reaction to obtain the adoptive immune cells with surface modified drug-loaded nanogel.

[0030] The method for modifying the surface of adoptive immune cells with drug-loaded nanogels described in the present invention is based on a copper-free catalytic click chemistry reaction, which makes the connection between the drug-loaded nanogels and the adoptive immune cells very stable. At the same time, the number of azide groups expressed on the surface of the adoptive immune cells can be artificially controlled by changing the concentration of azidated mannose, thereby controlling the number of nanogels mounted on the surface of the adoptive immune cells, and realizing intelligent controllable drug dosage.

[0031] Preferably, the adoptive immune cells are selected from T cells, NK cells or macrophages isolated from human peripheral blood monocytes, or CAR-T cells, TCR-T cells, CAR-NK cells that have been genetically engineered.

[0032] Preferably, the adoptive immune cells are labeled with azide groups by adding 1,3,4,6-tetra-O-acetyl-N-azidoacetylaminomannose or 1,3,4,6-tetra-O-acetyl-N-azidoacetylaminosialic acid to the culture medium of the adoptive immune cells and incubating the cells.

[0033] In the method, the preparation method of adoptive immune cells with surface labeled azide groups comprises the following steps:

[0034] The adoptive immune cells were incubated in a culture medium containing 50 μM 1,3,4,6-tetra-O-acetyl-N-azidoacetylaminomannose (Ac4ManAz) for 48 h to label the surface of the adoptive immune cells with azide groups.

[0035] The present invention also provides the use of adoptive immune cells of the surface-modified drug-loaded nanogel prepared by the above method in the preparation of anti-tumor drugs.

[0036] The adoptive immune cells modified by the drug-loaded nanogel of the present invention significantly enhance the ability to kill solid tumors or the ability of solid tumors to be traced in the human body.

[0037] Compared with the prior art, the polymer structure provided by the present invention is shown in Formula 1. Wherein, R is selected from

[0038] x, y, z, k are the degree of polymerization, 10≤x+y+z+k≤4000, x>0, y>0, z>0, k>0; n is the degree of polymerization, 10≤n≤400. The drug-loaded nanogel prepared by the polymer is used to modify the surface of adoptive immune cells, thereby making the adoptive immune cells have a better tumor treatment effect. The method of modifying the surface of adoptive immune cells with drug-loaded nanogel has stable mounting, simple method, strong universality, can realize intelligent controllable dosage, and significantly enhances the killing ability or in vivo tracing ability of solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The poly L-asparagine (PHEA) prepared in Example 1 1 HNMR spectra;

[0040] Figure 2 The poly (L-asparagine) grafted functionalized polyethylene glycol grafted thioketal adamantane (P-Ad) prepared in Example 4 1 HNMR spectra;

[0041] Figure 3 The poly (L-asparagine) grafted polyethylene glycol grafted amino β-cyclodextrin (P-CD) prepared in Example 5 1 HNMR spectra;

[0042] Figure 4 The dynamic light scattering results of the nanogel loaded with hyaluronidase and IR-808 prepared in Examples 6 and 9 at a concentration of 0.2 mg / mL in water;

[0043] Figure 5 The experimental results of the cell surface modification of the nanogel loaded with the protein drug hyaluronidase prepared in Example 7, wherein (A) is a flow cytometry histogram of the blank group, the unfunctionalized nanogel group, and the functionalized nanogel group, and (B) is a fluorescence expression intensity diagram of the blank group, the unfunctionalized nanogel group, and the functionalized nanogel group;

[0044] Figure 6 The adoptive transfer OT1 T cells loaded with nanogel prepared in Example 8 are used for the treatment of B16OVA tumor model, wherein (A) is a tumor inhibition curve of PBS group, T cells group, T cells&NGs group, and T cells / NGs group, and (B) is a weight change diagram of the tested mice in PBS group, T cells group, T cells&NGs group, and T cells / NGs group;

[0045] Figure 7 This is a diagram showing the experimental results of cell surface modification of the nanogel loaded with near-infrared dye IR-808 prepared in Example 10. DETAILED DESCRIPTION

[0046] To further illustrate the present invention, the polymer and drug-loaded nanogel provided by the present invention, the method for modifying the surface of drug-loaded nanogel by adoptive immune cells and their applications are described in detail below in conjunction with the examples.

[0047] Example 1

[0048] 39.9g (160.0mmol) of γ-benzyl-L-aspartic acid ester-N-internal carboxylic anhydride monomer (BLA-NCA) was dissolved in 270mL of anhydrous N,N-dimethylformamide (DMF), and 1.0mL (1.0mmol / L DMF solution) of n-hexylamine (n-HA) was added after stirring and dissolving, and the mixture was sealed and stirred at 25°C for 72h. After that, 2.0g (20.0mmol) of acetic anhydride was added to the above reaction system and the reaction was continued for 12h. After the reaction was completed, the obtained reaction solution was precipitated into 2.0L of ether, filtered and washed with ether in turn, and dried under vacuum at room temperature for 24h to obtain the intermediate product poly (γ-benzyl-L-aspartic acid ester) (PBLA).

[0049] 10.0 g of the poly(γ-benzyl-L-aspartic acid ester) prepared above was dissolved in 100 mL of anhydrous N,N-dimethylformamide (DMF), and 8 mL of ethanolamine was added under stirring, and the reaction was stirred at 30° C. for 12 h. Afterwards, the obtained reaction solution was precipitated into 1.0 L of ether and centrifuged, and the obtained precipitate was re-dissolved in DMF, dialyzed with deionized water, and freeze-dried to obtain poly(L-asparagine) homopolymer (PHEA).

[0050]

[0051] The prepared poly(L-asparagine) homopolymer was subjected to nuclear magnetic resonance analysis, and the results are shown in Figure 1 , using deuterated trifluoroacetic acid as the deuterated reagent, the results showed that the chemical shift of 5.06ppm was the signal peak of the methine on the main chain, the chemical shifts of 4.50ppm and 3.92ppm were the signal peaks of the methylene connected to the secondary amine on the side group, 3.69ppm and 3.56ppm were the signal peaks of the methylene connected to the hydroxyl group on the side group, and the chemical shift of 3.06ppm was the signal peak of the methylene connected to the main chain on the side group. According to NMR calculation, the degree of polymerization of the obtained poly (L-aspartic acid) was 160, and the comprehensive yield was 80.8%.

[0052] Example 2

[0053] In a dry reaction bottle, 260 mg (1.6 mmol) of N, N'-carbonyl diimidazole, 3.5 g (70.9 mmol ethylene glycol unit) of dibenzocyclooctyne-aminopolyethylene glycol (DBCO-PEG-NH2) (5000 Da) were added, and 150 mL of DMF was added to dissolve. The reaction was sealed at a temperature of 50 ° C. After 12 hours, poly (L-aspartic acid) (PHEA) (2.0 g, 12.6 mmol aspartic acid unit) prepared in Example 1 and 196 mg (1.6 mmol) of 4-dimethylaminopyridine (DMAP) were added, and the reaction was sealed at a temperature of 50 ° C. After 12 hours, the obtained reaction solution was precipitated with 1.0 L of ether, and the obtained solid was redissolved with DMF, and then dialyzed with deionized water for 3 days, and lyophilized to obtain PHEA-g-PEG-DBCO.

[0054]

[0055] Using deuterated trifluoroacetic acid as solvent, the obtained poly (L-aspartic acid) PHEA-g-PEG-DBCO grafted with functionalized polyethylene glycol was subjected to nuclear magnetic resonance analysis, and obvious characteristic peaks of polyethylene glycol (3.65ppm) and dibenzocyclooctyne characteristic peaks (7.69ppm, 7.40ppm) were found, indicating that dibenzocyclooctyne-aminopolyethylene glycol was successfully bonded to the polymer.

[0056] Example 3

[0057] Functionalized polyethylene glycol grafted onto polyaspartic acid grafted onto phenylboronic acid pinacol ester

[0058] 234 mg (1.0 mmol) of p-hydroxymethylphenylboronic acid pinacol ester and 325 mg (2.0 mmol) of N, N'-carbonyldiimidazole (CDI) were added to the dried reaction bottle, and then 150 mL of DMF was added to dissolve. The reaction was sealed at 50 ° C. After 12 hours, polyaspartic acid grafted functionalized polyethylene glycol (PHEA-g-PEG-DBCO) (4 g, 12.3 mmol aspartic acid unit) prepared in Example 2 and 196 mg (1.6 mmol) of 4-dimethylaminopyridine (DMAP) were added, and the reaction was sealed at 50 ° C. After 12 hours, the obtained reaction solution was precipitated with 1.0 L of ether, the obtained solid was redissolved with DMF, and then dialyzed with deionized water for 3 days, and freeze-dried to obtain polyaspartic acid grafted functionalized polyethylene glycol grafted phenylboronic acid pinacol ester PHEA-g-PEG-DBCO / PBA, which was weighed and the yield was 72.5%.

[0059]

[0060] The obtained polymer was subjected to nuclear magnetic resonance analysis using trifluoroacetic acid as a deuterated reagent. The results showed that there were obvious characteristic peaks of phenylboronic acid (7.81 ppm, 7.78 ppm, 7.36 ppm, 7.33 ppm) in the spectrum, indicating that phenylboronic acid pinacol ester was successfully bonded to the polymer.

[0061] Example 4

[0062] Preparation of functionalized polyethylene glycol grafted with polyaspartic acid grafted with phenylboronic acid pinacol ester grafted with thioketal adamantane (P-Ad)

[0063] Into a dry reaction bottle, add the polyaspartic acid grafted functionalized polyethylene glycol grafted phenylboronic acid pinacol ester (1000 mg) prepared in Example 3, then add 385 mg (1.0 mmol) thioketal adamantane, and then add 150 mL of DMF to dissolve. Then, add 178 mg (1.4 mmol) of N, N-diisopropylcarbodiimide (DIC) and 196 mg (1.6 mmol) of 4-dimethylaminopyridine (DMAP), seal and react at a temperature of 25 ° C. After 48 hours, the obtained reaction solution is precipitated with 1.0 L of ether, and the obtained solid is redissolved with DMF, dialyzed with dilute hydrochloric acid aqueous solution (pH = 6.8) for 2 days to remove the pinacol ester, and then dialyzed with deionized water for 3 days to remove the hydrochloric acid. After freeze-drying, polyaspartic acid grafted polyethylene glycol grafted phenylboronic acid pinacol ester grafted thioketal adamantane is obtained, and the yield is 84.4% by weighing.

[0064]

[0065] The obtained polymer was subjected to NMR analysis using trifluoroacetic acid as a deuterated reagent. The results are shown in Figure 2 . Figure 2 This is the H NMR spectrum of polyaspartic acid grafted polyethylene glycol grafted thioketal adamantane (P-Ad) prepared in Example 4. Obvious characteristic peaks of adamantane (1.99ppm, 1.60ppm, 1.52ppm) and thioketal characteristic peak (1.53ppm) can be found, indicating that thioketal adamantane is successfully bonded to the polymer. The characteristic peak of pinacol ester (1.36ppm) disappears, indicating that pinacol ester is successfully removed from the polymer.

[0066] Example 5

[0067] Preparation of functionalized polyethylene glycol grafted with polyaspartic acid grafted with phenylboronic acid pinacol ester grafted with amino β-cyclodextrin (P-CD)

[0068] 1776 mg (1.5 mmol) of aminocyclodextrin and 486 mg (3.0 mmol) of N, N'-carbonyldiimidazole were added to the dried reaction bottle respectively, and then 150 mL of DMF was added to dissolve. The reaction was sealed at a temperature of 50 ° C. After 12 hours, the polyaspartic acid grafted functionalized polyethylene glycol (1.3 g, 4.0 mmol aspartic acid unit) prepared in Example 2 and 196 mg (1.6 mmol) of 4-dimethylaminopyridine (DMAP) were added, and the reaction was sealed at a temperature of 50 ° C. After 12 hours, the obtained reaction solution was precipitated with 1.0 L of ether, and the obtained solid was redissolved with DMF, dialyzed with dilute hydrochloric acid aqueous solution (pH = 6.8) for 2 days to remove the pinacol ester, and then dialyzed with deionized water for 3 days to remove hydrochloric acid. After freeze-drying, polyaspartic acid grafted functionalized polyethylene glycol grafted amino beta cyclodextrin was obtained, and the yield was 79.1% by weighing.

[0069]

[0070] The obtained polymer was subjected to NMR analysis using deuterated trifluoroacetic acid as the deuterated reagent. The results are shown in Figure 3 . Figure 3 This is the H NMR spectrum of the polyaspartic acid grafted functionalized polyethylene glycol grafted aminocyclodextrin (P-CD) prepared in Example 5. Figure 1 By comparison, obvious cyclodextrin characteristic peaks (3.39ppm, 3.72ppm, 4.86ppm) can be found, indicating that aminocyclodextrin is successfully bonded to the polymer. The characteristic peak of pinacol ester (1.36ppm) disappears, indicating that pinacol ester is successfully removed from the polymer.

[0071] Example 6

[0072] Loading of protein drug hyaluronidase

[0073] Add the polyaspartic acid grafted functionalized polyethylene glycol grafted phenylboronic acid pinacol ester grafted thioketal adamantane (P-Ad) (25 mg) prepared in Example 4 and the aspartic acid grafted functionalized polyethylene glycol grafted phenylboronic acid pinacol ester grafted aminocyclodextrin (P-CD) (25 mg) prepared in Example 5 to two dry bottles, and dissolve them with 2 ml of PB (phosphate buffer solution) (pH = 7.4) solution. Take the fluorescein-labeled protein drug hyaluronidase (20 mg), dissolve it with 0.4 ml of PB (pH = 7.4) solution, mix the three and vortex for 20 seconds, and stir at room temperature for 24 hours. Remove the free hyaluronidase by dialysis.

[0074] Dynamic light scattering analysis was performed on the obtained high molecular weight hyaluronidase loaded nanogel to determine the hydrodynamic radius of the nanoparticles self-assembled in water. Figure 4 The dynamic light scattering results of the hyaluronidase-loaded nanogel prepared in Example 6 at a concentration of 0.2 mg / mL in water. It can be seen from the figure that the hydrodynamic radius of the self-assembled micelles is between 100-120 nm, and the particle size distribution is uniform.

[0075] Example 7

[0076] Cell surface modification with hyaluronidase-loaded nanogels

[0077] Flow cytometry was used to detect the ability of hyaluronidase-loaded nanogels and free hyaluronidase (blank group) to modify the cell surface. 200,000 Jurkat T cells in logarithmic growth phase were plated in each well of a 12-well plate and incubated with a medium containing azido-modified mannose (Ac4ManNAz, 1,3,4,6-tetra-O-acetyl-N-azidoacetylaminomannose). After 48 hours, the original medium was replaced with a solution containing biotin-labeled DBCO-functionalized (dibenzocyclooctyne group functionalized) nanogels (functionalized nanogel group) or biotin-labeled non-DBCO-functionalized nanogels (unfunctionalized nanogel group). After incubation for 4 hours, the cells were collected and centrifuged at 1200r for 5 minutes, the supernatant was discarded and the cell pellet was retained. After repeated washing three times with 1mL of PBS (phosphate buffer solution), avidin fluorescent dye was added and incubated with the cells. After 30 minutes, 1 mL of PBS solution was added to terminate the staining. The cells were collected and centrifuged at 1200 r for 5 minutes. The supernatant was discarded and the cell pellet was retained. After repeated washing three times with 1 mL of PBS solution, the cells were resuspended with 0.3 mL of PBS. The samples were detected by BD FACSVerse flow cytometer. Figure 5 The flow cytometry results are obtained, where (A) is the flow cytometry histogram of the blank group, the unfunctionalized nanogel group, and the functionalized nanogel group, and (B) is the fluorescence expression intensity diagram of the blank group, the unfunctionalized nanogel group, and the functionalized nanogel group. It can be seen from the figure that the nanogel was successfully labeled on the cells through the bioorthogonal reaction.

[0078] Example 8

[0079] Modified drug-loaded nanogels enhance the anti-tumor effect of adoptively transferred OT1 T cells

[0080] Twenty C57BL / 6 mice (weighing about 16-18 g) bearing B16-OVA tumors were randomly divided into four groups. PBS was injected intravenously through the tail vein (PBS group), OT1 T cells were adoptively transferred (T cells group), OT1 T cells and free drug-loaded gel (T cells&NGs group) and OT1 T cells modified with drug-loaded gel (T cells / NGs group) (the concentration of hyaluronidase was 5 mg kg -1 At pre-designed time points (0, 2, 4, 6, 8, 10, 12, 14, 16 days), tumor size and mouse weight were measured. On day 18, mice were euthanized and tumors were taken for flow cytometry analysis and tissue section observation. Figure 6 The measured tumor inhibition curves are shown in Figure (A), and the tumor inhibition curves of the PBS group, T cells group, T cells&NGs group, and T cells / NGs group are shown in Figure (B). The weight changes of the tested mice in the PBS group, T cells group, T cells&NGs group, and T cells / NGs group are shown in Figure. It can be seen from the figure that the nanogel greatly enhances the anti-tumor therapeutic ability of adoptively transferred OT1 T cells. This result shows that the nanogel provided by the present invention greatly improves the anti-tumor ability of adoptively transferred cells and has great potential in the field of synergistic adoptive transfer cell therapy.

[0081] Example 9

[0082] Loading of tracer IR-808

[0083] Add polyaspartic acid grafted functionalized polyethylene glycol grafted phenylboronic acid pinacol ester grafted thioketal adamantane (P-Ad) (25 mg) prepared in Example 4 and aspartic acid grafted functionalized polyethylene glycol grafted phenylboronic acid pinacol ester grafted aminocyclodextrin (P-CD) (25 mg) prepared in Example 5 to two dry bottles, and dissolve them with 2 mL of PB (pH = 7.4) solution. Take IR-808 (20 mg), dissolve it with 0.4 ml of PB (pH = 7.4) solution, mix the three and vortex for 20 seconds, and stir at room temperature for 24 hours. Remove free IR-808 by dialysis.

[0084] The obtained tracer IR-808 loaded nanogel was subjected to dynamic light scattering analysis to determine the hydrodynamic radius of the nanoparticles self-assembled in water. The hydrodynamic radius of the self-assembled micelles was between 100-120 nm, and the particle size distribution was uniform.

[0085] Example 10

[0086] Cell surface modification with IR-808 loaded nanogels

[0087] 200,000 Jurkat T cells in logarithmic growth phase were plated in each well of a 12-well plate and incubated with a medium containing azide-modified mannose (Ac4ManNAz). After 48 hours, the original medium was replaced with a solution of DBCO-functionalized nanogels or non-DBCO-functionalized nanogels loaded with IR-808. Continue to incubate for 4 hours, collect the cells and centrifuge at 1200r for 5 minutes, discard the supernatant and retain the cell pellet, wash three times with 1mL of PBS solution, and resuspend the cells with 0.3mL of PBS. The NIRF intensity of the wells containing CAR T cells and supernatant was measured using the Odyssey NIRF imaging system. Figure 7 is the measured NIRF intensity result, from Figure 7 As can be seen, the nanogel loaded with near-infrared dyes was successfully labeled on cells through bioorthogonal reactions.

[0088] The above embodiments are only used to help understand the method and core idea of ​​the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A polymer, characterized in that The structure is shown in formula 1: Among them, R is selected from x, y, z, k are the degree of polymerization, 10≤x+y+z+k≤4000, x>0, y>0, z>0, k>0; n is the degree of polymerization, 10≤n≤400.

2. The polymer according to claim 1, characterized in that The R is selected from The 10≤x+y+z+k≤500; Said 10≤n≤100.

3. A drug-loaded nanogel, characterized in that: The solution is prepared by mixing an aqueous solution of a polymer having a structure represented by Formula 1-1, an aqueous solution of a polymer having a structure represented by Formula 1-2, and an aqueous solution of an auxiliary drug, and performing supramolecular host-guest assembly; The auxiliary drug is selected from one or more of hyaluronidase, sialidase, interleukin 15, cisplatin, and IR-808; Wherein, x, y, z, k are the degree of polymerization, 10≤x+y+z+k≤4000, x>0, y>0, z>0, k>0; n is the degree of polymerization, 10≤n≤400.

4. The drug-loaded nanogel according to claim 3, characterized in that: The concentration of the auxiliary drug is 0.1-10 mg / mL; The concentrations of the structural polymers represented by Formula 1-1 and Formula 1-2 are 0.1-10 mg / mL respectively.

5. The drug-loaded nanogel according to claim 3, characterized in that: The mass ratio of the aqueous solution of the structural polymers represented by Formula 1-1 and Formula 1-2 to the aqueous solution of the auxiliary drug is (0.1-10):1:(0.1-10).

6. The drug-loaded nanogel according to claim 3, characterized in that: The aqueous solution is selected from phosphate buffer solution, tris(hydroxymethylaminomethane) hydrochloride buffer solution, sodium citrate buffer solution or 4-hydroxyethylpiperazineethanesulfonic acid buffer solution.

7. A method for adoptive immune cell surface modification of drug-loaded nanogels, characterized in that: The following steps are involved: The adoptive immune cells with azide groups labeled on the surface are co-incubated with the drug-loaded nanogel according to any one of claims 3 to 6, so that the adoptive immune cells and the drug-loaded nanogel are connected through a copper-free click chemistry reaction to obtain the adoptive immune cells with surface-modified drug-loaded nanogel.

8. The method according to claim 7, characterized in that The adoptive immune cells are selected from T cells, NK cells or macrophages isolated from human peripheral blood monocytes, or CAR-T cells, TCR-T cells, and CAR-NK cells that have been genetically engineered.

9. The method according to claim 7 or 8, characterized in that: The adoptive immune cells are labeled with an azide group by adding 1,3,4,6-tetra-O-acetyl-N-azidoacetylaminomannose or 1,3,4,6-tetra-O-acetyl-N-azidoacetylaminosialic acid to the culture medium of the adoptive immune cells and incubating the cells.

10. Use of adoptive immune cells of the surface-modified drug-loaded nanogel prepared by the method according to any one of claims 7 to 9 in the preparation of anti-tumor drugs.

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