Tumor microenvironment acid-responsive deep-penetration nanogel and application thereof

Abstract

The application provides a tumor microenvironment acid response deep penetration nanogel and application thereof. Specifically disclosed is an acid response nanogel carrier, wherein the nanogel carrier is obtained by polymerization of double-bonded hyaluronic acid, double-bonded cyclodextrin and 2,2-dimethyl acryloyl oxy-1-ethoxy propane under the action of a free radical polymerization initiator; the double-bonded hyaluronic acid is obtained by modification of hyaluronic acid with an acrylic anhydride compound; and the double-bonded cyclodextrin is obtained by modification of a cyclodextrin compound with an olefin acyl chloride compound. Further disclosed is an acid response nanogel loaded with an active ingredient, wherein the acid response nanogel carrier is used as an active ingredient carrier, and the active ingredient is included in the double-bonded cyclodextrin. The nanogel prepared by the application has good acid response performance and drug loading capacity, and has excellent tumor penetration capacity, which is beneficial to enhancing the anti-tumor effect of the drug or for detection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to a tumor microenvironment acid-responsive deep-penetration nanogel and application thereof. BACKGROUND

[0002] Cancer is one of the most common diseases in the world, with high metastasis rate and high mortality rate. In recent years, more and more scientists have focused on drug delivery technology based on nanotechnology, and have made exciting progress. Nanodrug delivery system can effectively improve the solubility of poorly soluble drugs in water, improve the stability of drugs, avoid the combination of drugs with various proteins in blood, thereby prolonging the blood circulation time, improving the bioavailability, and at the same time, through the enhanced permeability and retention effect (EPR) passive targeting effect, enriching in tumors to enhance the treatment effect on tumors. At present, the representative nanodrugs approved for marketing include doxorubicin liposome and paclitaxel albumin nanoparticle, etc. They are mostly loaded with drugs in phospholipid bilayer or albumin particles, which enhances the pharmacokinetic parameters of drugs, and to some extent, promotes the enrichment of drugs in tumor sites, reduces the accumulation of drugs in non-target tissues, enhances the anti-tumor effect, reduces the toxic side effects, and improves the safety.

[0003] However, a large number of studies have shown that although nanodrugs can enhance the enrichment of drugs in tumor sites, most of the drugs are still difficult to effectively diffuse or penetrate in tumor sites, as they are mainly distributed in the outer layer of tumors. Without entering the interior of tumors to reach an effective concentration, it is impossible to completely eradicate all tumor cells. This not only makes the surviving tumor cells relapse, but also easily induces the generation of drug-resistant tumor cells. Therefore, constructing a nanodrug delivery system with tumor deep penetration and promoting the penetration of drugs in the deep layer of tumors is a key problem for further improving the treatment effect on tumors. After the enrichment of nanodrugs in tumors, they need to penetrate a series of interstitial spaces in tumor tissues, so as to be taken up by deep cells and exert drug efficacy. In the penetration process, the size of nanodrugs will significantly affect their diffusion rate in tumor sites, and thus affect their penetration ability. Small-sized nanodrugs are not limited by the size of intercellular space, have a faster diffusion rate, and thus have a stronger penetration ability in tumor tissues. SUMMARY

[0004] In order to solve the problem of poor penetration ability of current nanodrugs in tumor sites, the present application provides a size-variable nanodrug delivery system, which circulates in blood with a larger particle size to ensure the stability of drugs, and after enrichment in tumor sites through the EPR effect, releases drug-loaded nanocomposites with a smaller size in response to the micro-acidic environment (pH 6.5) in tumors, so as to promote the penetration of drugs into the deep layer of tumors.

[0005] The present application relates to a tumor microenvironment acid-responsive nanogel capable of promoting deep tumor penetration of drugs, solving the problem that drugs are difficult to penetrate deep into tumor tissues.

[0006] The present application provides a tumor microenvironment acid-responsive deep penetration nanogel and its application, which can release smaller nanomedicines at the tumor site, promote drug penetration in the deep tumor, and fully exert the effect of the drug.

[0007] In one aspect, the present application provides an acid-responsive nanogel carrier, which is obtained by polymerization of double-bonded hyaluronic acid, double-bonded cyclodextrin and 2,2-dimethyl acryloyl oxy-1-ethoxy propane under the action of a free radical polymerization initiator.

[0008] The double-bonded hyaluronic acid is obtained by modifying hyaluronic acid with an acrylic anhydride compound.

[0009] The double-bonded cyclodextrin is obtained by modifying a cyclodextrin compound with an olefinic acid chloride compound.

[0010] Further, the acrylic anhydride compound is selected from one or more of methyl acrylate, ethyl acrylate, and propyl acrylate.

[0011] Further, the molecular weight of the hyaluronic acid is 5000-500000. For example, 5000, 8000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000.

[0012] Further, the molar ratio of hyaluronic acid to acrylic anhydride compound is 1:1-1:6. For example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6.

[0013] Further, the cyclodextrin compound is selected from one or more of beta cyclodextrin and hydroxypropyl beta cyclodextrin.

[0014] Further, the olefinic acid chloride compound is selected from one or more of butenyl chloride, 4-pentenyl chloride, propenyl chloride, and methyl propenyl chloride.

[0015] Further, the free radical polymerization initiator is selected from hydrogen peroxide, ammonium persulfate, potassium persulfate, benzoyl peroxide, azobisisobutyronitrile, azobisisoheptyl nitrile, and dimethyl azobisisobutyrate, 4,4'-azobis(cyanopentanoic acid), and benzoyl peroxide tert-butyl ester.

[0016] Further, the average hydrated particle size of the nanogel is 150-200 nm.

[0017] Another aspect of the present application provides an acid-responsive nanogel loaded with active ingredients, which uses the above-mentioned acid-responsive nanogel carrier as the active ingredient carrier, and the active ingredient is included in the double-bonded cyclodextrin.

[0018] Further, the active ingredient is selected from an ingredient with anti-tumor activity, or an ingredient with a detection function.

[0019] Further, the ingredient with anti-tumor activity is selected from an anti-tumor drug or an immunomodulatory drug.

[0020] Further, the ingredient with a detection function is selected from one or more of di-alkyl carbocyanine dyes (DiR), indocyanine green, safranin 6, and a CY series dye.

[0021] Another aspect of the present application provides a preparation method of the acid-responsive nanogel carrier, which comprises the following steps:

[0022] S1) reacting hyaluronic acid with an acrylic anhydride compound in an alkaline aqueous solution to obtain double-bonded hyaluronic acid;

[0023] S2) dissolving a cyclodextrin compound in an organic solvent, adjusting the pH value to be alkaline, and then reacting with an olefinic acid chloride compound to obtain double-bonded cyclodextrin;

[0024] S3) preparing or obtaining 2,2-dimethyl acryloyloxy-1-ethoxy propane;

[0025] S4) preparing a drug-loaded nanogel by a free radical polymerization method, dissolving the double-bonded hyaluronic acid, the double-bonded cyclodextrin, and the 2,2-dimethyl acryloyloxy-1-ethoxy propane in an alkaline solution, adding a free radical polymerization initiator to react, and obtaining the nanogel.

[0026] Another aspect of the present application provides a preparation method of the acid-responsive nanogel loaded with active ingredients, which comprises the following steps:

[0027] S1) reacting hyaluronic acid with an acrylic anhydride compound in an alkaline aqueous solution to obtain double-bonded hyaluronic acid;

[0028] S21) dissolving a cyclodextrin compound in an organic solvent, adjusting the pH value to be alkaline, and then reacting with an olefinic acid chloride compound to obtain double-bonded cyclodextrin;

[0029] S22) dissolving the active ingredient and incubating with the double-bonded cyclodextrin to obtain an inclusion compound of the active ingredient and the double-bonded cyclodextrin;

[0030] S3) preparing or obtaining 2,2-dimethyl acryloyloxy-1-ethoxy propane;

[0031] S4) preparing drug-loaded nanogels by radical polymerization, dissolving the double-bonded hyaluronic acid, the inclusion complex of the active ingredient and the double-bonded cyclodextrin, and 2,2-dimethyl acryloyl oxy-1-ethoxy propane in an alkaline solution, adding a radical polymerization initiator to react, and obtaining the nanogels.

[0032] Preferably, the molecular weight of the hyaluronic acid in the step S1) is 5000-500000.

[0033] Preferably, the step S1) is carried out in an aqueous solvent, and more preferably, the aqueous solution is selected from one or more of physiological saline, phosphate buffer, and deionized water.

[0034] Preferably, the pH range of the alkaline condition in the step S1) is selected from 8.0-12.0.

[0035] Preferably, after the reaction in the step S1) is completed, dialysis is used to remove small molecular substances by using a dialysis bag with a molecular weight cut-off of 1000-10000.

[0036] Preferably, the molar ratio of the hyaluronic acid and the acrylic anhydride compound in the step S1) is 1:1-1:6. For example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6.

[0037] Preferably, the acrylic anhydride compound in the step S1) is selected from one or more of methyl acrylate, acrylate, ethyl acrylate.

[0038] Preferably, the cyclodextrin compound in the step S2) or S21) is selected from one or more of β-cyclodextrin, hydroxypropyl β-cyclodextrin. Further, the cyclodextrin compound can also contain a tumor-targeting compound, such as folic acid.

[0039] Preferably, the olefinic acid chloride compound in the step S2) or S21) is selected from one or more of acetyl chloride, benzoyl chloride, acryloyl chloride, chloroacetyl chloride.

[0040] Preferably, the molar ratio of the cyclodextrin compound and the olefinic acid chloride compound in the step S2) or S21) is 1:5-1:10, for example, 1:6, 1:7, 1:8, 1:9.

[0041] Preferably, the active ingredient in the step S22) is selected from an anti-tumor active ingredient or a detection reagent, and more preferably, the anti-tumor active ingredient is selected from an anti-tumor drug, an immunomodulatory drug. For example, the active ingredient is selected from tea polyphenols.

[0042] Preferably, the method of elution purification in step S3) is elution with eluent of n-hexane, ethyl acetate and triethylamine in a volume ratio of 80-90:12-16:1.

[0043] More preferably, the method of elution purification in step S3) is elution with silica gel column chromatography.

[0044] More preferably, the method of elution purification in step S3) is elution with eluent of n-hexane, ethyl acetate and triethylamine in a volume ratio of 80-90:12-16:1.

[0045] Preferably, the initiator in step S4) is selected from one or more of hydrogen peroxide, ammonium persulfate, potassium persulfate, benzoyl peroxide, azobisisobutyronitrile, azobisisoheptyl nitrile and dimethyl azobisisobutyrate.

[0046] Preferably, the reaction temperature in step S4) is 60-80℃.

[0047] Preferably, the reaction in step S4) is carried out under inert gas conditions.

[0048] Another aspect of the present application provides use of the above-mentioned acid-responsive nanogel carrier in the preparation of a drug for preventing or treating tumors or in the preparation of a carrier for a tumor detection reagent.

[0049] Another aspect of the present application provides use of the acid-responsive nanogel loaded with active ingredients in the preparation of a drug for preventing or treating tumors or in the preparation of a tumor detection reagent.

[0050] Another aspect of the present application provides a drug for preventing or treating tumors or a tumor detection reagent, which comprises a therapeutically effective amount of the above-mentioned acid-responsive nanogel loaded with active ingredients.

[0051] Another aspect of the present application provides a method for preventing or treating tumors, which comprises the step of administering the acid-responsive nanogel loaded with active ingredients to a subject.

[0052] Another aspect of the present application provides a method for detecting tumors, which comprises the step of administering the acid-responsive nanogel loaded with active ingredients to a subject.

[0053] The beneficial effects of the present application are:

[0054] (1) The nanogel prepared by the present application has good acid-responsive performance and drug loading capacity, which is conducive to the acid-responsive release of the nanogel in the tumor microenvironment.

[0055] (2) The prepared responsive nanogel has good biocompatibility, and has wide application prospects in the field of drug release.

[0056] (3) The prepared responsive nanogel can release small-size drug compounds in response to a tumor microenvironment, has excellent tumor penetration capacity, and is beneficial to enhancing the anti-tumor effect of drugs. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 (A) HCD-A synthesis route and (B) nuclear magnetic hydrogen spectrum.

[0058] Figure 2 (A) DMAEP synthesis route and (B) nuclear magnetic hydrogen spectrum.

[0059] Figure 3 (A) DMAEP synthesis route and (B) nuclear magnetic hydrogen spectrum.

[0060] Figure 4 (A) The particle size of NG after incubation in pH 7.4 PBS for 24 h and (B) the transmission electron micrograph.

[0061] Figure 5 (A) The particle size of NG after incubation in pH 6.5 PBS for 24 h and (B) the transmission electron micrograph.

[0062] Figure 6 (A-B) The survival of cells after incubation of NG with (A) B16F10 and (B) DC2.4 cells for 24 h.

[0063] Figure 7 (A) The particle size of NG after incubation in pH 7.4 PBS for 24 h and (B) the transmission electron micrograph.

[0064] Figure 8 EGCG@NG drug release curves in pH 7.4 and pH 6.5 PBS, respectively.

[0065] Figure 9 DiR, DiR@HCD-A, DiR@NG and DiR@NGlysis (pre-incubated in pH 6.5 PBS for 24 h) were incubated with B16F10 cells for 1 h, 4 h and 24 h, and the fluorescence intensity of the cells was detected by flow cytometry (n=3, P<0.05).

[0066] Figure 10Flow cytometry analysis of PD-L1 expression on the surface of B16F10 cells stimulated by IFN-γ after incubation with EGCG (E), EGCG (E), E@HCD-A, E@NG and E@NGlysis (pre-incubated in PBS at pH 6.5 for 24 h) for 24 h, respectively (n = 3, P < 0.05).

[0067] Figure 11 Fluorescence distribution and penetration of DiR, DiR@HCD-A, DiR@NG and DiR@NGlysis after incubation with multicellular tumor spheroids for 8 h.

[0068] Figure 12 Tumor growth curve of tumor-bearing mice treated with EGCG and EGCG@NG, respectively (n = 7, P < 0.05). DETAILED DESCRIPTION

[0069] In order to make the above objectives, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application are described in detail below, but it should not be understood as limiting the scope of the present application.

[0070] The term "therapeutically effective amount" is an amount effective to treat, alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and / or reduce incidence of one or more symptoms or features of cancer.

[0071] The term "anti-tumor drug" refers to a drug that kills tumor cells or induces tumor cell apoptosis by physical or chemical means, and the anti-tumor drug can be included in a cyclodextrin.

[0072] The term "immunomodulatory drug" refers to a class of drugs that act on the immune system and are effective through the immune system, which can activate the immune system, trigger an immune response, and then inhibit tumors. It does not directly kill tumor cells by chemical or physical means, or does not directly cause tumor cell apoptosis.

[0073] The term "acrylic anhydride compound" refers to a compound having an acrylic anhydride structure in the compound.

[0074] The term "cyclodextrin compound" refers to a compound having a cyclodextrin nucleus in the compound.

[0075] The term "olefinic acid chloride compound" refers to an acid chloride compound having a double bond group.

[0076] Example 1

[0077] Preparation of methacrylate hyaluronic acid:

[0078] Hyaluronic acid (HA, 0.5 g) was dissolved in 50 mL of deionized water and 0.59 mL of methacrylic anhydride was added dropwise under ice bath conditions. The pH was then adjusted to 8.0 using 1 M sodium hydroxide. After 24 h of reaction, the reaction solution was dialyzed against deionized water using a dialysis bag with a molecular weight cut-off (MWCO) of 10,000 for 72 h, changing the dialysis medium every 8 h, and then the liquid in the dialysis bag was collected and freeze-dried to obtain methacrylate hyaluronic acid (HAm). The reaction formula is as follows Figure 1 A.

[0079] Experimental results: The proton nuclear magnetic resonance spectrum of HAm prepared in this example is shown in Figure 1 B.

[0080] The data are as follows: (400 MHz, D2O-d2, δ), 1.81 (3H, H-2, C=C-CH3), 1.88 (3H, H-g), 3.21-3.79 (10H, H-b, c, d, e, f, b’, c’, d’, e’), 4.32-4.42 (2H, H-a, a’), 5.61-6.05 (2H, H-1, CH2=C).

[0081] Example 2

[0082] Preparation of acrylate hydroxypropyl β-cyclodextrin:

[0083] Hydroxypropyl β-cyclodextrin (HP-β-CD, 1 g, 0.65 mmol) and triethylamine (665 μL) were mixed in 10 mL of ice-bath DMF, and then acryloyl chloride (412 mg, 4.55 mmol) was added dropwise to the solution. After stirring for 24 h under nitrogen protection, the reaction solution was filtered and precipitated with ether. The precipitate was collected and washed three times, and vacuum dried for 24 h to obtain acrylate hydroxypropyl β-cyclodextrin (HCD-A). The reaction formula is as follows Figure 2 A.

[0084] Experimental results: The proton nuclear magnetic resonance spectrum of HCD-A prepared in this example is shown in Figure 2 B.

[0085] The data are as follows: (400 MHz, D2O-d2, δ), 1.02 (3H, H-i), 3.37-4.16 (9H, H-b, c, d, e, f, g, h), 4.95 (1H, H-a), 5.92-6.34 (3H, H-1, CH=CH2).

[0086] Example 3

[0087] Preparation of acid-responsive cross-linking molecule 2,2-dimethylacryloyloxy-1-ethoxypropane:

[0088] Hydroxyethyl methacrylate (HEMA, 2 g), 2,2-dimethoxypropane (DMOP, 5 g) and p-toluenesulfonic acid (pTSA, 60 mg) were added into 15 mL dichloromethane. After reaction at room temperature for 24 h under nitrogen protection, the dichloromethane was removed by rotary evaporation to obtain the crude product. The crude product was further purified by column chromatography with 85:14:1 n-hexane / ethyl acetate / triethylamine as eluent to obtain the final product 2,2-dimethacryloyloxy-1-ethoxypropane (DMAEP). The reaction formula is as follows Figure 3 A.

[0089] Experimental results: The proton nuclear magnetic resonance spectrum of DMAEP prepared in this example is as shown in Figure 3 B.

[0090] Data as follows: (400 MHz, CDC13-d, δ), 1.40 (6H, H-5, C(CH3)2), 1.95 (6H, H-1, C=C-CH3), 3.72 (4H, H-4, O-CH2-C), 4.28 (4H, H-3, C-CH2-O), 5.58-6.12 (4H, H-2, CH2=C).

[0091] Example 4

[0092] Preparation of tea polyphenol-loaded acid-responsive nanogel for deep penetration into tumor microenvironment:

[0093] (1) Tea polyphenol (EGCG, 2.4 mg) was dissolved in 2 mL ethanol, and then added into 2 mL deionized water containing 20 mg HCD-A. The mixture was stirred at room temperature for 1 h, and then vacuum dried to obtain a tea polyphenol-acrylate hydroxypropyl β-cyclodextrin inclusion complex (EGCG@HCD-A).

[0094] (2) The above EGCG@HCD-A was mixed with 40 mg HAm and 80 mg DMAEP in 50 mL deionized water, and 40 mg potassium persulfate, 160 mg sodium bicarbonate and 24 mL isopropyl alcohol were added. The mixture was stirred at 70°C for 4 h under nitrogen protection to obtain a nanogel.

[0095] (3) The nanogel was placed in a dialysis bag (molecular weight cut-off MWCO = 10000) and dialyzed in PBS for 12 h to collect pure tea polyphenol-loaded nanogel (EGCG@NG) in the dialysis bag. The hydrated particle size of the nanogel was detected by a dynamic light scattering instrument, and the morphology of the nanogel was observed by transmission electron microscopy.

[0096] Experimental results: As shown in Figure 4 A, the average hydrated particle size of the nanogel was about 170.99 nm. As shown in Figure 4The transmission electron micrograph shown in B shows that the nanogel presents a regular spherical shape.

[0097] Example 5

[0098] Detection of the acid-responsive size change of the tea polyphenol-loaded acid-responsive deep-penetration nanogel in a tumor microenvironment:

[0099] The EGCG@NG was dispersed in a PBS solution at pH 6.5 for 24 h to obtain the acid-cleaved EGCG-loaded nanogel (EGCG@NGlysis). The hydrated particle size of the nanogel was detected using a dynamic light scattering instrument, and the morphology of the nanogel was observed using a transmission electron microscope.

[0100] Experimental results: As shown in A, the average hydrated particle size of the nanogel increased to about 220.20 nm, and the particle size peak changed from a single peak to a double peak, with small-size particles of about 24.36 nm appearing. Figure 5 B, the transmission electron micrograph shows that the nanogel presents an irregular swollen state, and the small-size particles appearing around the nanogel prove that the nanogel can release small-size drug-loaded cyclodextrins in a slightly acidic environment. Figure 5

[0101] Example 6

[0102] Evaluation of the biocompatibility of the acid-responsive deep-penetration nanogel in a tumor microenvironment:

[0103] (1) 20 mg of HCD-A, 40 mg of HAm, and 80 mg of DMAEP were mixed in 50 mL of deionized water, and 40 mg of potassium persulfate, 160 mg of sodium bicarbonate, and 24 mL of isopropyl alcohol were added. The mixture was stirred at 70°C for 4 h under nitrogen protection, and the nanogel was collected.

[0104] (2) The nanogel was then placed in a dialysis bag (molecular weight cut-off MWCO = 10000) and dialyzed in PBS for 12 h, and the pure nanogel (NG) in the dialysis bag was collected.

[0105] (3) The in vitro cytotoxicity of the blank nanogel was determined using B16F10 and DC2.4 cells as the research objects. 5000 cells / well were seeded in a 96-well plate and cultured for 24 h, and then fresh culture medium containing different concentrations of NG (0.1, 1, 10, 50, and 500 μg / mL) was used to replace the culture medium. After 24 h, CCK-8 (10 μL) reagent was added for further incubation for 2 h. The cell activity was evaluated by reading the absorbance at 450 nm using an enzyme marker.

[0106] Experimental results: As shown in A, the average hydrated particle size of the nanogel increased to about 220.20 nm, and the particle size peak changed from a single peak to a double peak, with small-size particles of about 24.36 nm appearing. Figure 6 ​As shown in FIGs. 6A and 6B, the cell survival rate was higher than 90% under different concentrations of NG (0.1, 1, 10, 50, 500 μg / mL), indicating that the prepared blank nanogel had low cytotoxicity and good biocompatibility.

[0107] Example 7

[0108] Detection of the hemolysis effect of the tumor microenvironment acid-responsive deep-penetration nanogel on red blood cells:

[0109] (1) C57BL / 6J mouse red blood cells were taken, centrifuged at 4°C and 3500 rpm for 5 min, and then washed with cold normal saline for 3 times.

[0110] (2) The red blood cells were added to cold normal saline containing different concentrations of NG (0.001, 0.01, 0.1, 1 and 10 mg / mL) in Example 6, and the final concentration of red blood cells was adjusted to 2% (w / v).

[0111] (3) After incubation at 37°C for 4 h, the absorbance value of the supernatant at 540 nm was measured by a microplate reader.

[0112] The hemolysis level was calculated by the following formula:

[0113]

[0114] wherein A saline and A H2O are the absorbance values of red blood cells treated with normal saline and pure water at 0% (negative) and 100% (positive) hemolysis, respectively.

[0115] The experimental results are shown in FIGs. 8A and 8B. Figure 7 As shown, pure water can cause 100% hemolysis of red blood cells to release hemoglobin, and the hemolysis level of the blank nanogel is comparable to that of normal saline, which can be ignored (<1%), indicating that the blank nanogel is basically harmless to red blood cells during blood circulation and has good biocompatibility.

[0116] Example 8

[0117] Detection of the acid-responsive drug release characteristics of the tumor microenvironment acid-responsive tea polyphenol-loaded deep-penetration nanogel:

[0118] (1) EGCG@NG in Example 4 was dispersed in 1 mL of PBS at pH 7.4 or 6.5, and placed in a dialysis bag with a molecular weight of 3500.

[0119] (2) The dialysis bag was immersed in 50 mL of PBS at pH 7.4 or 6.5, and placed in a 37 °C shaker. At the predetermined time points (0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96 and 120 h), the PBS was collected and replaced with fresh dialysis medium PBS.

[0120] (3) The concentration of EGCG was determined by high performance liquid chromatography. The gradient elution mobile phase was acetonitrile-0.1% trifluoroacetic acid in water at a flow rate of 0.5 mL / min. The detection wavelength was 210 nm.

[0121] Experimental results: As shown in Figure 8 pH 6.5, the rate and concentration of EGCG release of the nanogel were significantly accelerated, and it had a pH-responsive release characteristic. At 24 h, the release rate of EGCG in pH 7.4 was 23.16 ± 3.89%, and in pH 6.5 was 61.62 ± 9.56%, with a difference of 2.66 times. At 120 h, the release rate of EGCG was 75.19 ± 5.94%, which was 2.38 times of that in pH 7.4 (31.64 ± 4.90%).

[0122] Example 9

[0123] Detection of the effect of tumor cell uptake of tumor microenvironment acid-responsive deep penetration nanogel:

[0124] (1) Dissolve di-alkyl carbocyanine dye (DiR, 240 μg) in 2 mL of ethanol, and then add to 2 mL of deionized water containing 20 mg of HCD-A. Stir the mixture at room temperature for 1 h, and then vacuum dry to obtain DiR-acrylate hydroxypropyl β-cyclodextrin complex (DiR@HCD-A).

[0125] (2) Mix 20 mg of DiR-HCD-A with 40 mg of HAm and 80 mg of DMAEP in 50 mL of deionized water, and add 40 mg of potassium persulfate, 160 mg of sodium bicarbonate and 24 mL of isopropyl alcohol, and stir the reaction under nitrogen protection at 70 °C for 4 h to obtain the nanogel.

[0126] (3) Then place the nanogel in a dialysis bag (molecular weight cut-off MWCO = 10000) for dialysis in PBS for 12 h, and collect the pure DiR-loaded nanogel (DiR@NG) in the dialysis bag.

[0127] (4) Incubate the DiR@NG in PBS at pH 6.5 for 24 h to obtain the acid-cleaved DiR-loaded nanogel (DiR@NGlysis).

[0128] (5) Seed B16F10 cells at a density of 10 5The cells were treated with DiR, DiR@HCD-A, DiR@NG and DiR@NGlysis containing 5 μg / mL DiR for 1 h, 4 h and 24 h respectively in 6-well plates with 5 μg / mL DiR per well, and the fluorescence intensity in the cells was detected by flow cytometry.

[0129] The experimental results are shown in Table 1. Figure 9 As shown in Table 1, the average fluorescence intensity (MFI) of B16F10 cells treated with DiR@NGlysis was 26.90 times, 2.49 times and 6.75 times that of DiR, DiR@HCD-A and DiR@NG respectively at 24 h, indicating that the small size DiR@HCD-A complex released after acid treatment is more conducive to cell uptake. In addition, this experiment also shows that the carrier of the application can encapsulate any kind of active ingredient or detection reagent.

[0130] Example 10

[0131] Detection of the regulation effect of tumor microenvironment acid-responsive tea polyphenol-loaded deep-penetration nanogel on PD-L1 of B16F10 tumor cells:

[0132] (1) Tea polyphenol (EGCG, 2.4 mg) was dissolved in 2 mL of ethanol, and then added to 2 mL of deionized water containing 20 mg of HCD-A. The mixture was stirred at room temperature for 1 h, and then vacuum dried to obtain a tea polyphenol-acrylate hydroxypropyl β-cyclodextrin inclusion complex (EGCG@HCD-A).

[0133] (2) The above EGCG@HCD-A was mixed with 40 mg of HAm and 80 mg of DMAEP in 50 mL of deionized water, and 40 mg of potassium persulfate, 160 mg of sodium bicarbonate and 24 mL of isopropyl alcohol were added. The reaction was stirred at 70°C for 4 h under nitrogen protection, and the nanogel was collected.

[0134] (3) Then the nanogel was placed in a dialysis bag (molecular weight cut-off MWCO = 10000) and dialyzed in PBS for 12 h, and the pure tea polyphenol-loaded nanogel (EGCG@NG) in the dialysis bag was collected.

[0135] (4) The EGCG@NG was dispersed in a PBS solution at pH 6.5 for 24 h to obtain the acid-cleaved EGCG-loaded nanogel (EGCG@NGlysis).

[0136] (5) B16F10 cells were seeded in a 6-well plate at a density of 10 5 cells per well, and after 2 h of treatment with interferon-γ (5 ng / mL), EGCG, EGCG@HCD-A, EGCG@NG and EGCG@NGlysis were added respectively, and the concentration of EGCG was 20 μg / mL.

[0137] (6) Collect the cells, stain the cells with fluorescently labeled anti-PD-L1 antibody, and detect the fluorescence intensity with flow cytometry to analyze the expression of PD-L1.

[0138] Experimental results: As shown in Figure 10 , interferon-γ induced the expression of PD-L1 in tumor cells to rise by 3.1 times. EGCG can reduce the expression of PD-L1 in tumor cells induced by interferon-γ, and compared with natural EGCG, EGCG@HCD-A and EGCG@NG, EGCG@NGlysis has stronger inhibitory effect on PD-L1, which can be reduced to 0.58 times of the interferon-γ group.

[0139] Example 11

[0140] Detection of the penetration effect of tumor microenvironment acid-responsive deep-penetration nanogel on tumor spheroids:

[0141] (1) 80 μL of 1M phosphate buffer containing 1% (w / v) sterile agarose was pre-coated in a 48-well plate.

[0142] (2) 500 μL of B16F10 cell suspension (5 x 10 4 cells / well) was inoculated and cultured for 6-8 days.

[0143] (3) When the diameter of the multicellular spheroids reached 200 μm, uniform and dense cell spheroids were selected, and DiR, DiR@HCD-A, DiR@NG and DiR@NGlysis in Example 9 were used to incubate the cells for 8 h, with a concentration of 5 μg DiR / mL.

[0144] (4) The multicellular spheroids were fixed with 4% paraformaldehyde for 30 min, and then the fluorescence distribution and penetration were observed by laser confocal scanning microscopy.

[0145] Experimental results: As shown in Figure 11 , a small amount of fluorescence signal of free DiR was observed to be aggregated at the edge of the multicellular spheroids, while the fluorescence in the DiR@HCD-A group was strong and could diffuse throughout the spheroids, indicating that HCD-A has good penetration effect. At the same time, compared with free DiR, the fluorescence intensity of DiR@NG was improved, but the fluorescence was also aggregated in the outer layer of the cell spheroids. Notably, after treatment with DiR@NGlysis, significantly enhanced fluorescence was observed throughout the cell spheroids, indicating that small-sized HCD-A inclusion complexes released from the nanogel under acid response can further effectively penetrate into the deep tumor.

[0146] Example 12

[0147] Detection of the effect of tumor microenvironment acid-responsive tea polyphenol-loaded deep-penetration nanogel on the treatment of mouse tumors:

[0148] (1) 6-week-old female C57BL / 6J mice were subcutaneously injected with B16F10 cells (1 x 10 5 per mouse) on the flank.

[0149] (2) When the tumor volume reached about 50mm 3 , the mice were randomly divided into 7 groups.

[0150] (3) Intravenous injection of EGCG solution containing 6 mg EGCG / kg or EGCG@NG nanogel was performed once every 3 days for a total of 4 times. The tumor length (L) and width (W) of the mice were recorded every other day.

[0151] (4) The tumor volume was calculated as L x W 2 / 2, and death or a tumor volume exceeding 2000mm 3 was considered as death.

[0152] Experimental results: As shown in Figure 12 , the tumor of the mouse treated with EGCG alone was only inhibited to 67.01% of the control group on day 22, while EGCG@NG could enhance the anti-tumor effect of EGCG and inhibit the tumor volume to 37.58% of the control group, indicating that the tumor microenvironment acid-responsive tea polyphenol-loaded nanogel can improve the inhibition of tumors by enhancing the penetration of drugs in tumors.

Claims

1. An acid-responsive nanogel carrier, characterized in that, The nanogel carrier is obtained by polymerization of double-bonded hyaluronic acid, double-bonded cyclodextrin and 2,2-dimethyl acryloyloxy-1-ethoxy propane under the action of a free radical polymerization initiator; The double-bonded hyaluronic acid is obtained by modification of hyaluronic acid with an acrylic anhydride compound; The double-bonded cyclodextrin is obtained by modification of a cyclodextrin compound with an olefinic acid chloride compound; The acrylic anhydride compound is selected from one or more of methyl acrylate, ethyl acrylate and propyl acrylate; The cyclodextrin compound is selected from one or more of β-cyclodextrin and hydroxypropyl β-cyclodextrin; The olefinic acid chloride compound is selected from one or more of butenyl chloride, 4-pentenyl chloride, propenyl chloride and methyl propenyl chloride; The free radical polymerization initiator is selected from one or more of hydrogen peroxide, ammonium persulfate, potassium persulfate, benzoyl peroxide, azobisisobutyronitrile, azobisisoheptyl nitrile, dimethyl azobisisobutyrate, 4,4'-azobis(cyanovaleric acid) and benzoyl peroxide tert-butyl ester; The molar ratio of hyaluronic acid to acrylic anhydride compound is 1:1-1:6; The molar ratio of the cyclodextrin compound to the olefinic acid chloride compound is 1:5-1:

10.

2. An active ingredient-loaded acid-responsive nanogel, characterized in that, The acid-responsive nanogel carrier of claim 1 is used as a carrier for an active ingredient, and the active ingredient is included in the double-bonded cyclodextrin.

3. The acid-responsive nanogel loaded with an active ingredient according to claim 2, wherein, The active ingredient is selected from an ingredient with anti-tumor activity or an ingredient with a detection function.

4. The acid-responsive nanogel loaded with an active ingredient according to claim 3, wherein The ingredient with anti-tumor activity is selected from an anti-tumor drug or an immunomodulatory drug.

5. The acid-responsive nanogel loaded with an active ingredient according to claim 4, wherein The ingredient with a detection function is selected from one or more of a dialkyl carbocyanine dye, indocyanine green, coumarin 6 and a CY series dye.

6. The method of claim 1, wherein the acid-responsive nanogel carrier is prepared by the steps of: The method comprises the following steps: S1) reacting hyaluronic acid with an acrylic anhydride compound in an alkaline aqueous solution to obtain double-bonded hyaluronic acid; S2) dissolving a cyclodextrin compound in an organic solvent, adjusting the pH value to alkaline and then reacting with an olefinic acid chloride compound to obtain double-bonded cyclodextrin; S3) preparing or obtaining 2,2-dimethyl acryloyloxy-1-ethoxy propane; S4) preparing a drug-loaded nanogel by a free radical polymerization method, dissolving double-bonded hyaluronic acid, double-bonded cyclodextrin and 2,2-dimethyl acryloyloxy-1-ethoxy propane in an alkaline solution, adding a free radical polymerization initiator and reacting to obtain a nanogel.

7. A method of preparing an acid-responsive nanogel loaded with an active ingredient according to any one of claims 2-5, characterized in that, The method comprises the following steps: S1) reacting hyaluronic acid with an acrylic anhydride compound in an alkaline aqueous solution to obtain double-bonded hyaluronic acid; S21) dissolving a cyclodextrin compound in an organic solvent, adjusting the pH value to alkaline and then reacting with an olefinic acid chloride compound to obtain double-bonded cyclodextrin; S22) dissolving an active ingredient and incubating with double-bonded cyclodextrin to obtain an inclusion compound of the active ingredient and double-bonded cyclodextrin; S3) preparing or obtaining 2,2-dimethyl acryloyloxy-1-ethoxy propane; S4) preparing a drug-loaded nanogel by a free radical polymerization method, dissolving double-bonded hyaluronic acid, an inclusion compound of the active ingredient and double-bonded cyclodextrin and 2,2-dimethyl acryloyloxy-1-ethoxy propane in an alkaline solution, adding a free radical polymerization initiator and reacting to obtain a nanogel.

8. Use of the acid-responsive nanogel carrier of claim 1 in the preparation of a carrier for a drug for preventing or treating a tumor or in the preparation of a carrier for a tumor detection reagent.

9. Use of the acid-responsive nanogel loaded with an active ingredient of any one of claims 2-5 in the preparation of a drug for preventing or treating a tumor or in the preparation of a tumor detection reagent.

10. A medicament for preventing or treating a tumor, characterized by, which comprises a therapeutically effective amount of the acid-responsive nanogel loaded with an active ingredient of any one of claims 2-5.

11. An agent for tumor detection, characterized by, which comprises a therapeutically effective amount of the acid-responsive nanogel loaded with an active ingredient of any one of claims 2-5.

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