A polymer and drug-loaded nanogel, a method for surface modification of drug-loaded nanogel by adoptive immune cells and application thereof

Abstract

The application discloses a kind of polymer and drug-loaded nanogel, the method for surface modification of drug-loaded nanogel of adoptive immune cell and application thereof, belong to the field of biological medicine.The polymer structure is as shown in formula 1. Wherein, R is selected from x, y, z, k is 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 cell, so that the adoptive immune cell has excellent tumor treatment effect.The method for surface modification of drug-loaded nanogel of adoptive immune cell is stable, simple, and has strong universality, can realize the intelligent controllable of dosing, significantly enhances the killing ability of solid tumor or in vivo tracing ability.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a polymer and drug-loaded nanogel, a method for modifying the surface of adoptive immune cells with drug-loaded nanogels, and their applications. Background Technology

[0002] Adoptive cell transfer therapy (ACT) involves isolating immune-active cells from a cancer patient, expanding and functionally identifying them in vitro, and then reinfusing them into the patient to directly kill tumor cells or stimulate the body's immune response to kill tumor cells. Adoptive 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) is considered a revolutionary breakthrough in tumor immunotherapy, and its research progress has become one of the most closely watched areas. However, despite its remarkable efficacy in treating hematomas, treating solid tumors remains a significant challenge for CAR-T therapy. While CAR-T therapy has shown good results in treating hematologic malignancies, it faces four major challenges in treating solid tumors. First, there is the challenge of tumor invasion. Solid tumors contain stromal barriers (such as hyaluronic acid), which T cells must overcome to reach the tumor site. Second, there is the challenge of immunosuppression. The tumor microenvironment (TME) of solid tumors has an immunosuppressive effect on T cells, greatly weakening their anti-tumor ability. Third, there is the challenge of CAR target identification. For most solid tumors, tumor-associated antigens (TAAs) are enriched on the tumor but also expressed at low levels in normal tissues, making it difficult to find suitable specific recognition antigens for CAR design. Fourth, there is the challenge of CAR-T cell tracking. It is difficult to monitor the proliferation and distribution of CAR-T cells after infusion into the human body, thus making it impossible to detect whether the CAR-T cell response is adequate or to provide early warning of potential toxic progression.

[0003] To overcome these difficulties, genetic engineering is commonly used, where therapeutic cells are typically delivered in combination with adjuvant drugs after cell transfer. These drugs aim to maximize the efficacy and persistence of the reinfused adoptive immune cells. However, these drugs often require high concentrations and sustained delivery, leading to dose-limiting toxicity, which restricts their clinical application. One approach to focusing the adjuvant drug's effect on the transferred cells is to genetically engineer donor cells to secrete the adjuvant drug themselves. However, gene transfection methods are significantly complex and uncertain, often limiting the functional expansion; furthermore, the continuous secretion of adjuvant drugs by genetically engineered cells poses safety concerns. Meanwhile, many emerging adjuvant therapies are based on small-molecule drugs that are not genetically encoded.

[0004] Researchers have attempted to develop cell surface modification techniques to overcome the challenges of co-delivering adoptive cells and adjuvant drugs, offering another feasible solution to these problems. For example, Irvine et al. used the reaction of maleimide with thiol groups 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, making the degree of cell surface modification uncontrollable and the delivered drug dosage unpredictable. Furthermore, the newly formed CS bond in the coupling between the maleimide group and the thiol group on the T cell is not stable enough under physiological conditions, undergoing reverse Michael addition, and is even more unstable in the presence of competing thiol compounds (such as cysteine). Can Zhang et al. attached liposomes carrying the cholesterol drug Avasimibe to the surface of T cells by inserting the tetrazine group into the cell membrane. However, the insertion of the tetrazine group into the cell membrane is unstable and may detach in the complex in vivo environment. Publication CN 112980786 reports a method for linking T cells to nanoparticles based on click chemistry. It uses a metabolic sugar method to modify and label the T cell surface with azide groups, and then uses a copper-free click chemistry reaction to modify the T cell surface with poly(ethylene lactide) nanoparticles. However, due to the characteristics of poly(ethylene lactide) nanoparticles, they lack intelligent design and cannot achieve specific drug release, which limits the types of drugs they can carry. The publication only shows a model reaction when carrying indocyanine green, thus lacking universality and only achieving a single function.

[0005] Therefore, it is of great significance to research and develop a new, simple, efficient, and universally applicable 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 drug-loaded nanogel, a method for modifying the surface of adoptive immune cells with drug-loaded nanogel, and the application thereof. The drug-loaded nanogel prepared by the polymer is used to modify the surface of adoptive immune cells, thereby enabling the adoptive immune cells to have a better anti-tumor effect.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides a polymer with the structure shown in Formula 1:

[0009]

[0010] Formula 1;

[0011] Where R is selected from , or .

[0012] x, y, z, and k represent the degree of aggregation, where 10 ≤ x + y + z + k ≤ 4000, x > 0, y > 0, z > 0, and k > 0.

[0013] n is the degree of aggregation, 10≤n≤400.

[0014] Preferably, in this invention, R is selected from... ,or ;

[0015] Preferably, 10 ≤ x + y + z + k ≤ 500;

[0016] Preferably, 10 ≤ n ≤ 100.

[0017] The present invention also provides a drug-loaded nanogel, which is prepared by mixing an aqueous solution of a polymer with the structure shown in Formula 1-1, an aqueous solution of a polymer with the structure shown in Formula 1-2, and an aqueous solution of an auxiliary drug, and then assembling them as a supramolecular host-guest assembly.

[0018] The drug-loaded nanogel is prepared through a cross-linking reaction under mild conditions using supramolecular host-guest interactions between polymers with structures shown in Formula 1-1 and Formula 1-2. This process forms a nanogel while simultaneously encapsulating a large amount of adjuvant drug, allowing the drug-loaded nanogel to be intelligently designed and specifically disintegrate in the tumor microenvironment. Furthermore, the drug loading capacity of the drug-loaded nanogel is higher than that of typical drug delivery carriers, thus enabling localized high-concentration drug release and significantly improving the therapeutic effect on tumors. The polymers with structures shown in Formula 1-1 and Formula 1-2, along with the adjuvant drug, are dissolved in aqueous solutions and then mixed to ensure that the nanoparticles of the formed drug-loaded nanogel are more regular and stable.

[0019] The drug-loaded nanogel of the present invention can carry a variety of adjuvant drugs, including but not limited to protein drugs or small molecule drugs. Preferably, the adjuvant drug is selected from one or more of hyaluronidase, sialidase, interleukin-15, cisplatin, and IR-808.

[0020]

[0021] Equation 1-1;

[0022]

[0023] Equation 1-2;

[0024] Where x, y, z, and k are the degree of aggregation, 10≤x+y+z+k≤4000, x>0, y>0, z>0, and k>0;

[0025] n is the degree of aggregation, 10≤n≤400.

[0026] Preferably, the concentration of the adjuvant drug is 0.1-10 mg / mL; more preferably, it is 2 mg / mL.

[0027] Preferably, the concentrations of the polymers with the structures shown in Formula 1-1 and Formula 1-2 are 0.1-10 mg / mL; more preferably, they are 1 mg / mL.

[0028] Preferably, the mass ratio of the aqueous solution of the polymer with the structure shown in Formula 1-1 and Formula 1-2 to the aqueous solution of the adjuvant drug is (0.1-10):1:(0.1-10); more preferably, it is (0.5-2):1:(0.1-1). In some specific embodiments of the present invention, it is preferably 1:1:0.4.

[0029] Preferably, the aqueous solution is selected from phosphate buffer solution, tris(hydroxymethyl)aminomethane hydrochloride buffer solution, sodium citrate buffer solution, or 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution.

[0030] The preparation method of the above-mentioned drug-loaded nanogel includes the following steps:

[0031] (1) Dissolve the polymer with the structure shown in Formula 1-1, the polymer with the structure shown in Formula 1-2, and the auxiliary drug in phosphate buffer solution to obtain aqueous solutions of the polymer with the structure shown in Formula 1-1, the polymer with the structure shown in Formula 1-2, and the auxiliary drug, respectively.

[0032] (2) Mix the aqueous solution of the polymer with the structure shown in Formula 1-1 with the aqueous solution of the adjuvant drug, mix thoroughly, add the aqueous solution of the polymer with the structure shown in Formula 1-2 and mix evenly to obtain the mixed system S.

[0033] (3) Dialyze the mixed system S to remove the free auxiliary drug, and the drug-loaded nanogel can be obtained. The above preparation method of drug-loaded nanogel is simple and the conditions are mild.

[0034] The drug-loaded nanogels can achieve a variety of functions depending on the auxiliary drugs they carry. In addition to enhancing adoptive immune cells, they can also be used to trace cells in the human body.

[0035] This invention also provides a method for modifying the surface of adoptive immune cells with drug-loaded nanogels, comprising the following steps:

[0036] Adoptive immune cells with surface-labeled azide groups were co-incubated with the aforementioned drug-loaded nanogel, allowing the adoptive immune cells and the drug-loaded nanogel to connect via a copper-free click chemistry reaction, resulting in adoptive immune cells with surface-modified drug-loaded nanogel.

[0037] The method for modifying the surface of adoptive immune cells with drug-loaded nanogels described in this invention is based on copper-free catalytic click chemistry, resulting in a highly stable connection between the drug-loaded nanogels and adoptive immune cells. Furthermore, the number of azide groups expressed on the surface of adoptive immune cells can be artificially controlled by altering the concentration of azidomannose, thereby controlling the number of nanogels attached to the surface of the adoptive immune cells and achieving intelligent and controllable drug delivery.

[0038] Preferably, the adoptive immune cells of the present invention are selected from T cells, NK cells or macrophages isolated from human peripheral blood mononuclear cells, or CAR-T cells, TCR-T cells or CAR-NK cells that have been genetically engineered.

[0039] Preferably, in this invention, the adoptive immune cells are labeled with azide groups by incubating them in a culture medium containing 1,3,4,6-tetra-O-acetyl-N-azidoacetylaminomannose or 1,3,4,6-tetra-O-acetyl-N-azidoacetylaminosialic acid.

[0040] The method for preparing adoptive immune cells with surface-labeled azide groups includes the following steps:

[0041] Adoptive immune cells can be labeled with azide groups by incubating them in a medium containing 50 μM 1,3,4,6-tetra-O-acetyl-N-azidoacetylaminomannose (Ac4ManAz) for 48 h.

[0042] This invention also provides the application of adoptive immune cells of surface-modified drug-loaded nanogels prepared by the above method in the preparation of antitumor drugs.

[0043] Adoptive immune cells modified with the drug-loaded nanogel described above in this invention exhibit significantly enhanced killing ability against solid tumors, or the ability to trace solid tumors in the human body.

[0044] Compared with the prior art, the polymer structure provided by the present invention is shown in Formula 1. Wherein, R is selected from... , or x, y, z, and k represent the degree of polymerization, where 10 ≤ x + y + z + k ≤ 4000, x > 0, y > 0, z > 0, and k > 0; n represents the degree of polymerization, where 10 ≤ n ≤ 400. The drug-loaded nanogel prepared from the polymer is used to modify the surface of adoptive immune cells, thereby enhancing their therapeutic effect on tumors. The method for modifying the surface of adoptive immune cells with drug-loaded nanogels is stable, simple, and widely applicable, enabling intelligent control of the dosage and significantly enhancing the killing ability or in vivo tracking ability of solid tumors. Attached Figure Description

[0045] Figure 1 The poly-L-asparagine (PHEA) prepared in Example 1 1 HNMR spectrum;

[0046] Figure 2 The poly-L-asparagine-grafted functionalized polyethylene glycol-grafted thioketane (P-Ad) prepared in Example 4 1 HNMR spectrum;

[0047] Figure 3 The poly-L-asparagine-grafted polyethylene glycol-grafted amino-β-cyclodextrin (P-CD) prepared in Example 5 1 HNMR spectrum;

[0048] Figure 4 The dynamic light scattering results of the nanogels loaded with hyaluronidase and IR-808 prepared in Examples 6 and 9 in water at a concentration of 0.2 mg / mL are shown.

[0049] Figure 5 The figure shows the experimental results of modifying the cell surface of the nanogel carrying the protein drug hyaluronidase prepared in Example 7. Figure (A) shows the flow cytometry histograms of the blank group, the unfunctionalized nanogel group, and the functionalized nanogel group. Figure (B) shows the fluorescence expression intensity of the blank group, the unfunctionalized nanogel group, and the functionalized nanogel group.

[0050] Figure 6Figure 1 shows the therapeutic results of adoptive transfer of OT1 T cells loaded with nanogel prepared in Example 8 to the B16OVA tumor model. Figure 2 shows the tumor inhibition curves of the PBS group, T cells group, T cells & NGs group, and T cells / NGs group. Figure 3 shows the weight changes of the tested mice in the PBS group, T cells group, T cells & NGs group, and T cells / NGs group.

[0051] Figure 7 The figure shows the experimental results of modifying the cell surface with the nanogel loaded with near-infrared dye IR-808 prepared in Example 10. Detailed Implementation

[0052] To further illustrate the present invention, the following describes in detail, with reference to embodiments, the polymers and drug-loaded nanogels provided by the present invention, the method for modifying the surface of adoptive immune cells with drug-loaded nanogels, and their applications.

[0053] Example 1

[0054] 39.9 g (160.0 mmol) of γ-phenylmethyl-L-aspartic ester-N-lactalic anhydride monomer (BLA-NCA) was dissolved in 270 mL of anhydrous N,N-dimethylformamide (DMF). After stirring and dissolving, 1.0 mL (1.0 mmol / L DMF solution) of n-hexylamine (n-HA) was added, and the mixture was sealed and reacted at 25 °C for 72 h. Then, 2.0 g (20.0 mmol) of acetic anhydride was added to the above reaction system, and the reaction was continued for 12 h. After the reaction was completed, the resulting reaction solution was precipitated into 2.0 L of diethyl ether, filtered and washed with diethyl ether, and dried under vacuum at room temperature for 24 h to obtain the intermediate product poly(γ-phenylmethyl-L-aspartic ester) (PBLA).

[0055] 10.0 g of the poly(γ-phenylmethyl-L-aspartic acid ester) prepared above was dissolved in 100 mL of anhydrous N,N-dimethylformamide (DMF). 8 mL of ethanolamine was added under stirring, and the reaction was carried out at 30 °C for 12 h. The resulting reaction solution was then settled into 1.0 L of diethyl ether, centrifuged, and the precipitate was reconstituted with DMF, dialyzed against deionized water, and lyophilized to obtain the poly(L-aspartic acid) homopolymer (PHEA).

[0056]

[0057] Nuclear magnetic resonance analysis was performed on the prepared poly(L-asparagine) homopolymer, and the results are shown in the figure. Figure 1Using deuterated trifluoroacetic acid as the deuterating agent, the results showed that the chemical shift of 5.06 ppm was the signal peak of the methylene group on the main chain; chemical shifts of 4.50 ppm and 3.92 ppm were the signal peaks of the methylene group attached to the secondary amine on the side group; chemical shifts of 3.69 ppm and 3.56 ppm were the signal peaks of the methylene group attached to the hydroxyl group on the side group; and chemical shift of 3.06 ppm was the signal peak of the methylene group attached to the main chain on the side group. According to NMR calculations, the degree of polymerization of the obtained poly(L-aspartic acid) was 160, and the overall yield was 80.8%.

[0058] Example 2

[0059] To a dry reaction flask, add 260 mg (1.6 mmol) of N,N'-carbonyldiimidazole, 3.5 g (70.9 mmol ethylene glycol unit) of dibenzocyclooctyne-amino polyethylene glycol (DBCO-PEG-NH2) (5000 Da), and then add 150 mL of DMF to dissolve. The reaction was sealed and reacted at 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. The reaction was sealed and reacted at 50°C. After 12 hours, the resulting reaction solution was precipitated with 1.0 L of diethyl ether. The resulting solid was reconstituted with DMF, dialyzed against deionized water for 3 days, and then lyophilized to obtain PHEA-g-PEG-DBCO.

[0060]

[0061] Nuclear magnetic resonance analysis of the grafted functionalized polyethylene glycol poly(L-aspartic acid) PHEA-g-PEG-DBCO using deuterated trifluoroacetic acid as solvent revealed obvious polyethylene glycol characteristic peaks (3.65 ppm) and dibenzocyclooctyne characteristic peaks (7.69 ppm, 7.40 ppm), indicating that dibenzocyclooctyne-amino polyethylene glycol was successfully bonded to the polymer.

[0062] Example 3

[0063] Polyaspartic acid-grafted functionalized polyethylene glycol-grafted pinacol ester

[0064] 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 a dry reaction flask, followed by dissolution in 150 mL of DMF. The reaction was sealed and reacted at 50 °C. After 12 hours, the 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. The reaction was sealed and reacted at 50 °C. After 12 hours, the resulting reaction solution was precipitated with 1.0 L of diethyl ether. The resulting solid was reconstituted with DMF, dialyzed against deionized water for 3 days, and lyophilized to obtain polyaspartic acid-grafted functionalized polyethylene glycol-grafted phenylboronic acid pinacol ester PHEA-g-PEG-DBCO / PBA. The yield was 72.5%.

[0065]

[0066] Nuclear magnetic resonance (NMR) analysis of the obtained polymer was performed using trifluoroacetic acid as a deuteration 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 pinacol ester of phenylboronic acid was successfully bonded to the polymer.

[0067] Example 4

[0068] Preparation of polyaspartic acid-grafted functionalized polyethylene glycol-grafted pinacol ester-grafted thioketane (P-Ad)

[0069] To a dry reaction flask, 1000 mg of polyaspartic acid-grafted functionalized polyethylene glycol-grafted pinacol ester (prepared in Example 3) was added, followed by 385 mg (1.0 mmol) of thioketane, and then 150 mL of DMF was added to dissolve it. Next, 178 mg (1.4 mmol) of N,N-diisopropylcarbodiimide (DIC) and 196 mg (1.6 mmol) of 4-dimethylaminopyridine (DMAP) were added. The mixture was sealed and reacted at 25°C for 48 hours. The resulting reaction solution was precipitated with 1.0 L of diethyl ether, and the resulting solid was reconstituted with DMF. The solution was dialyzed against dilute hydrochloric acid aqueous solution (pH=6.8) for 2 days to remove pinacol ester, and then dialyzed against deionized water for 3 days to remove hydrochloric acid. After lyophilization, polyaspartic acid-grafted polyethylene glycol-grafted pinacol ester-grafted thioketane was obtained and weighed; the yield was 84.4%.

[0070]

[0071] The obtained polymer was subjected to nuclear magnetic resonance analysis using trifluoroacetic acid as the deuteration reagent. The results are shown in [reference needed]. Figure 2 . Figure 2 The 1H NMR spectrum of the polyaspartic acid-grafted polyethylene glycol-grafted thioketane (P-Ad) prepared in Example 4 is shown. Distinct characteristic peaks of adamantane (1.99 ppm, 1.60 ppm, 1.52 ppm) and thioketane (1.53 ppm) are observed, indicating that the thioketane adamantane was successfully bonded to the polymer. The disappearance of the characteristic peak of pinacol ester (1.36 ppm) indicates that the pinacol ester was successfully removed from the polymer.

[0072] Example 5

[0073] Preparation of polyaspartic acid-grafted functionalized polyethylene glycol-grafted pinacol ester-grafted amino-β-cyclodextrin (P-CD)

[0074] 1776 mg (1.5 mmol) of aminocyclodextrin and 486 mg (3.0 mmol) of N,N'-carbonyldiimidazole were added to a dry reaction flask, followed by dissolution in 150 mL of DMF. The reaction was sealed and reacted at 50 °C for 12 hours. Then, 1.3 g (4.0 mmol) of polyaspartic acid-grafted functionalized polyethylene glycol (DMAP) prepared in Example 3 and 196 mg (1.6 mmol) of 4-dimethylaminopyridine (DMAP) were added. The reaction was sealed and reacted at 50 °C for 12 hours. The resulting reaction solution was precipitated with 1.0 L of diethyl ether, and the resulting solid was reconstituted with DMF. The solution was dialyzed against dilute hydrochloric acid aqueous solution (pH=6.8) for 2 days to remove pinacol ester, and then dialyzed against deionized water for 3 days to remove hydrochloric acid. After lyophilization, polyaspartic acid-grafted functionalized polyethylene glycol-grafted aminobetacyclodextrin was obtained and weighed, with a yield of 79.1%.

[0075]

[0076] The obtained polymer was subjected to nuclear magnetic resonance analysis using deuterated trifluoroacetic acid as the deuteration reagent. The results are shown in [reference needed]. Figure 3 . Figure 3 The image shows the 1H NMR spectrum of the polyaspartic acid-grafted functionalized polyethylene glycol-grafted aminocyclodextrin (P-CD) prepared in Example 5. (The image is then compared with...) Figure 1 In comparison, distinct characteristic peaks of cyclodextrin (3.39 ppm, 3.72 ppm, 4.86 ppm) were observed, indicating that aminocyclodextrin was successfully bonded to the polymer. The characteristic peak of pinacol ester (1.36 ppm) disappeared, indicating that pinacol ester was successfully removed from the polymer.

[0077] Example 6

[0078] Loading of protein drugs with hyaluronidase

[0079] Add 25 mg of polyaspartic acid-grafted functionalized polyethylene glycol-grafted phenylboronic acid pinacol ester-grafted thioketal adamantane (P-Ad) prepared in Example 4 and 25 mg of aspartic acid-grafted functionalized polyethylene glycol-grafted phenylboronic acid pinacol ester-grafted aminocyclodextrin (P-CD) prepared in Example 5 to two drying flasks, respectively, and dissolve them in 2 ml of PB (phosphate buffer) solution (pH=7.4). Take 20 mg of fluorescein-labeled protein drug hyaluronidase and dissolve it in 0.4 ml of PB (pH=7.4) solution. Mix the three solutions, vortex for 20 s, and stir at room temperature for 24 h. Remove free hyaluronidase by dialysis.

[0080] Dynamic light scattering analysis was performed on the obtained polymeric hyaluronidase-loaded nanogel to determine the hydrodynamic radius of the nanoparticles that self-assembled in water. Figure 4 The hyaluronidase-loaded nanogel prepared in Example 6 is shown in the dynamic light scattering results in water at a concentration of 0.2 mg / mL. As can be seen from the figure, the hydrodynamic radius of the self-assembled micelles is between 100-120 nm, and the particle size distribution is uniform.

[0081] Example 7

[0082] Cell surface modified nanogels loaded with hyaluronidase

[0083] 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 seeded into each well of a 12-well plate and incubated with a medium containing azide-modified mannose (Ac4ManNAz, 1,3,4,6-tetra-O-acetyl-N-azidoacetylaminomannose). After 48 h, the original medium was replaced with a solution containing either biotin-labeled DBCO-functionalized (dibenzocyclooctynyl-functionalized) nanogels (functionalized nanogel group) or biotin-labeled non-DBCO-functionalized nanogels (unfunctionalized nanogel group). Incubation continued for another 4 h. Cells were collected, centrifuged at 1200 rpm for 5 min, the supernatant was discarded, and the cell pellet was retained. The cells were washed three times with 1 mL of PBS (phosphate-buffered saline) and then co-incubated with avidin fluorescent dye. After 30 minutes, 1 mL of PBS solution was added to stop staining. Cells were collected and centrifuged at 1200 rpm for 5 minutes. The supernatant was discarded, and the cell pellet was retained. The cells were washed three times with 1 mL of PBS solution, and then resuspended in 0.3 mL of PBS. The samples were analyzed using a BD FACSVerse flow cytometer. Figure 5The flow cytometry results are shown in Figure (A), which is the histogram of the flow cytometry for the blank group, the unfunctionalized nanogel group, and the functionalized nanogel group. Figure (B) is the fluorescence expression intensity graph for the blank group, the unfunctionalized nanogel group, and the functionalized nanogel group. As can be seen from the figures, the nanogels were successfully labeled onto the cells through a bioorthogonal reaction.

[0084] Example 8

[0085] Modified drug-loaded nanogels enhance the tumor-suppressive effect of adoptive OT1 T cells.

[0086] Twenty C57BL / 6 mice (weighing approximately 16-18g) bearing B16-OVA tumors were randomly divided into four groups. The groups received intravenous injection of PBS via tail vein (PBS group), adoptive transfer of OT1 T cells (T cells group), adoptive transfer of OT1 T cells with free drug-loaded gel (T cells & NGs group), and OT1 T cells modified with drug-loaded gel (T cells / NGs group). (The hyaluronidase concentration was 5 mg / kg in all groups.) -1 Tumor size and mouse weight were measured at pre-designed time points (days 0, 2, 4, 6, 8, 10, 12, 14, and 16). Mice were euthanized on day 18, and tumors were collected for flow cytometry analysis and histological examination. Figure 6 The tumor inhibition curves are shown in Figure (A), which depicts the tumor inhibition curves of the PBS group, T cells group, T cells & NGs group, and T cells / NGs group. Figure (B) shows the changes in body weight of the tested mice in the PBS group, T cells group, T cells & NGs group, and T cells / NGs group. The figures show that the nanogel significantly enhances the anti-tumor therapeutic ability of adoptive OT1 T cells. This result indicates that the nanogel provided by this invention greatly improves the anti-tumor ability of adoptive cells and has great potential in the field of enhancing adoptive cell therapy.

[0087] Example 9

[0088] Loading of tracer IR-808

[0089] Add 25 mg of polyaspartic acid-grafted functionalized polyethylene glycol-grafted phenylboronic acid pinacol ester-grafted thioketal adamantane (P-Ad) prepared in Example 4 and 25 mg of aspartic acid-grafted functionalized polyethylene glycol-grafted phenylboronic acid pinacol ester-grafted aminocyclodextrin (P-CD) prepared in Example 5 to two drying flasks, respectively, and dissolve them in 2 mL of PB solution (pH=7.4). Take 20 mg of IR-808, dissolve it in 0.4 mL of PB solution (pH=7.4), mix the three solutions, vortex for 20 s, and stir at room temperature for 24 h. Remove free IR-808 by dialysis.

[0090] Dynamic light scattering analysis was performed on the obtained tracer IR-808-loaded nanogels to determine the hydrodynamic radii of the nanoparticles that self-assembled in water. The hydrodynamic radii of the self-assembled micelles were between 100 and 120 nm, and the particle size distribution was uniform.

[0091] Example 10

[0092] Cell surface modified nanogels loaded with IR-808

[0093] 200,000 Jurkat T cells in logarithmic growth phase were seeded into each well of a 12-well plate and incubated in medium containing azide-modified mannose (Ac4ManNAz). After 48 hours, the original medium was replaced with a solution of DBCO-functionalized nanogel loaded with IR-808 or a non-DBCO-functionalized nanogel. Incubation continued for another 4 hours. Cells were collected, centrifuged at 1200 rpm for 5 minutes, and the supernatant was discarded, retaining the cell pellet. The pellet was washed three times with 1 mL of PBS and then resuspended in 0.3 mL of PBS. The NIRF intensity of the wells containing CAR T cells and supernatant was measured using an Odyssey NIRF imaging system. Figure 7 For the measured NIRF intensity results, from Figure 7 As can be seen, the nanogels carrying near-infrared dyes were successfully labeled onto cells via a bioorthogonal reaction.

[0094] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A polymer, characterized in that, The structure is shown in Equation 1: Formula 1; Where R is selected from , or ; x, y, z, and k represent the degree of aggregation, where 10 ≤ x + y + z + k ≤ 4000, x > 0, y > 0, z > 0, and k > 0. n is the degree of aggregation, 10≤n≤400.

2. The polymer according to claim 1, characterized in that, The R is selected from ,or ; 10≤x+y+z+k≤500; 10≤n≤100。 3. A drug-loaded nanogel, characterized in that, It is prepared by mixing an aqueous solution of a polymer with the structure shown in Formula 1-1, an aqueous solution of a polymer with the structure shown in Formula 1-2, and an aqueous solution of an auxiliary drug, and then assembling them as a supramolecular host-guest assembly. The adjuvant drug is selected from one or more of hyaluronidase, sialidase, interleukin-15, cisplatin, and IR-808; Equation 1-1; Equation 1-2; Where x, y, z, and k are the degree of aggregation, 10≤x+y+z+k≤4000, x>0, y>0, z>0, and k>0; n is the degree of aggregation, 10≤n≤400.

4. The drug-loaded nanogel according to claim 3, characterized in that, The concentration of the adjuvant drug is 0.1-10 mg / mL; The concentrations of the polymers with the structures shown in 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 polymer with the structure shown in Formula 1-1 and the aqueous solution of the polymer with the structure shown in Formula 1-2 to the aqueous solution of the adjuvant 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(hydroxymethyl)aminomethane hydrochloride buffer solution, sodium citrate buffer solution or 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution.

7. A method for modifying the surface of adoptive immune cells with drug-loaded nanogels, characterized in that, Includes the following steps: Adoptive immune cells with surface-labeled azide groups are co-incubated with the drug-loaded nanogel according to any one of claims 3-6, so that the adoptive immune cells and the drug-loaded nanogel are linked by a copper-free click chemistry reaction to obtain 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 mononuclear cells, or CAR-T cells, TCR-T cells, or CAR-NK cells that have undergone genetic engineering.

9. The method according to claim 7 or 8, characterized in that, The adoptive immune cells are labeled with azide groups by incubating them in a culture medium containing 1,3,4,6-tetra-O-acetyl-N-azidoacetylaminomannose or 1,3,4,6-tetra-O-acetyl-N-azidoacetylaminosialic acid.

10. The use of adoptive immune cells of the surface-modified drug-loaded nanogel prepared by the method of any one of claims 7-9 in the preparation of antitumor drugs.

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