Preparation method of tumor microenvironment regulation type nano-drug delivery system as well as product and application of tumor microenvironment regulation type nano-drug delivery system

Abstract

The invention discloses a preparation method of a tumor microenvironment regulation type nano-drug delivery system. The preparation method comprises the following steps: S1, enabling DSPE-PEG-COOH to react with CAI, and performing dialysis purification to obtain DSPE-PEG-CAI; s2, dissolving and mixing lecithin, cholesterol and the DSPE-PEG-CAI in the step S1, performing rotary evaporation to form a lipid film, and hydrating to obtain a liposome suspension; s3, a tumor cell membrane and the liposome suspension in the step S2 are mixed, and a tumor microenvironment regulation type biomimetic membrane is prepared through probe ultrasonic treatment; and S4, adding a chemotherapeutic drug into the biomimetic membrane in the step S3 to obtain the tumor microenvironment regulation type nano-drug delivery system. The invention further discloses the tumor microenvironment regulation type nano-drug delivery system prepared by the preparation method and application of the tumor microenvironment regulation type nano-drug delivery system in tumor treatment drugs. The tumor microenvironment regulation and control type nano-drug delivery system provided by the invention can regulate the tumor acidic microenvironment, the delivery efficiency of chemotherapeutic drugs is improved through dual tumor targeting of the biomimetic membrane, and the therapeutic effect of the chemotherapeutic drugs is effectively enhanced.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine and cell bionic drug delivery system construction, and in particular to a preparation method of a tumor microenvironment regulated nano drug delivery system, and its products and applications. Background Art

[0002] Malignant tumors seriously endanger human health. At present, the main clinical treatments for malignant tumors include surgical treatment, chemotherapy and radiotherapy. Among them, chemotherapy, as the most common and widely used treatment method, can interfere with or prevent the proliferation and metabolism of tumor cells through various mechanisms, thereby inhibiting tumor growth. However, traditional chemotherapy drugs often cause serious toxic side effects to the body. Chemotherapeutic drugs themselves have certain cytotoxicity and can cause indiscriminate damage to tumor cells and normal cells. Due to the lack of targeting of traditional chemotherapy drugs, chemotherapy drugs are difficult to effectively enrich in the tumor site, and their distribution in normal tissues makes chemotherapy also have obvious toxicity to normal organs of the human body, causing serious complications. Therefore, many patients in clinical practice often face serious physical burdens when receiving chemotherapy. In order to improve the efficacy of chemotherapy and reduce its toxic side effects on the body, in recent years, tumor targeted delivery systems have gradually become one of the hot spots in tumor treatment research.

[0003] The advantage of tumor-targeted delivery systems is that they can specifically deliver chemotherapy drugs to tumor sites, reduce the accumulation of chemotherapy drugs in normal tissues, and thus reduce the toxic side effects of chemotherapy drugs. This type of system usually encapsulates chemotherapy drugs by constructing carriers such as inorganic nanoparticles, liposomes, polymer carriers, and biomimetic membranes, and achieves drug accumulation in tumor tissues through active or passive targeting mechanisms. Compared with traditional chemotherapy modes, targeted delivery systems significantly increase the drug concentration of chemotherapy drugs in tumor tissues, effectively prolong the drug's action time, enhance the tumor treatment effect, and reduce the damage of chemotherapy drugs to normal tissues. However, although targeted delivery systems can improve the enrichment of chemotherapy drugs in tumors and enhance the efficacy of chemotherapy to a certain extent, studies have reported that the complex tumor microenvironment is also one of the main factors affecting the efficacy of chemotherapy.

[0004] The complex tumor microenvironment includes dense extracellular matrix (ECM), enriched cancer-associated fibroblasts (CAFs), angiogenesis, hypoxia, acidic microenvironment, etc. These factors constitute a natural barrier for tumor cells and severely weaken the therapeutic effect of chemotherapeutic drugs. Among them, the acidic tumor microenvironment significantly affects the permeability, stability and activity of chemotherapeutic drugs, especially for drugs with optimal activity under neutral or alkaline conditions. Under acidic conditions, the structure and activity of some chemotherapeutic drug molecules may be changed, resulting in reduced tumor treatment effects or even failure; in addition, the reduced permeability of chemotherapeutic drugs makes it difficult for them to effectively penetrate into deep areas of the tumor, which also limits the efficacy of tumor treatment. In recent years, with the increasingly profound understanding of the acidic tumor microenvironment, the acidic tumor microenvironment is considered to be an important factor in inducing tumor occurrence, metastasis and drug resistance.

[0005] Therefore, in view of the low efficiency of current chemotherapy drug delivery and the resistance of the tumor microenvironment to chemotherapy drugs, it is of great significance to develop a drug delivery system that can improve drug delivery efficiency and improve the acidic microenvironment of the tumor. Summary of the invention

[0006] In view of the insufficient tumor targeting of existing chemotherapy drugs and the acidic microenvironment of tumors, the present invention provides a method for preparing a tumor microenvironment regulated nano drug delivery system; the present invention significantly improves the accumulation of chemotherapy drugs at the tumor site through the "double" tumor targeting of the bionic membrane, and inhibits the activity of the transmembrane protein carbonic anhydrase in 4T1 breast cancer cells through the carbonic anhydrase inhibitor modified on the bionic membrane, thereby improving the acidic microenvironment of the tumor and enhancing the anti-tumor effect of the chemotherapy drugs.

[0007] The technical solution provided by the present invention is as follows:

[0008] The present invention provides a method for preparing a tumor microenvironment-regulated nano drug delivery system, the method comprising the following steps:

[0009] S1, reacting distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl (DSPE-PEG-COOH) with 4-(2-aminoethyl)benzenesulfonamide (CAI) to prepare DSPE-PEG-CAI, and collecting it by dialysis purification;

[0010] S2, dissolving lecithin, cholesterol and the DSPE-PEG-CAI in step S1 in chloroform, rotary evaporating to form a lipid film, and hydrating to obtain a liposome suspension;

[0011] S3, mixing the tumor cell membrane with the liposome suspension in step S2, and preparing a tumor microenvironment regulating bionic membrane by probe ultrasonic treatment;

[0012] S4, loading the chemotherapy drugs into the bionic membrane in step S3 to obtain a tumor microenvironment regulating nano drug delivery system.

[0013] The "dual targeting" tumor microenvironment regulating bionic membrane provided by the present invention is endowed with good tumor targeting, tumor acidic microenvironment regulating ability and biocompatibility through the combination of liposomes and tumor cell membranes, and the bionic membrane carrier has good stability and drug loading capacity. Among them, the "dual targeting" property lies in the "homologous" targeting of 4T1 tumor cells given by the tumor cell membrane in the bionic membrane, and the active targeting and binding ability of the carbonic anhydrase IX inhibitor (CAI) on the surface of the carrier to the carbonic anhydrase IX (CA IX) overexpressed in 4T1 tumor cells; its ability to regulate the tumor microenvironment lies in the inhibitory effect of CAI on CA IX on 4T1 cells, which can reduce the extracellular H + The formation of a tumor microenvironment, thereby reducing the acidity of the tumor microenvironment. In the preparation process of the "dual-targeted" tumor microenvironment regulating bionic membrane, the tumor cell membrane is extracted by repeated freezing and thawing and gradient centrifugation purification to ensure the integrity of the homologous targeting protein on the membrane, so that it can effectively target the tumor site. Chemotherapeutic drugs (such as doxorubicin hydrochloride) are successfully encapsulated in the bionic membrane through the ammonium sulfate gradient method, so that the chemotherapeutic drugs are specifically delivered to the tumor site and then achieve specific killing of the tumor. The "dual-targeted" tumor microenvironment regulating nanodrug delivery system can further amplify the anti-tumor treatment effect in the tumor microenvironment by enhancing the delivery efficiency of chemotherapeutic drugs and regulating the acidic microenvironment of the tumor.

[0014] The distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl described in the present invention is first mixed with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and sulfo-(N-hydroxysuccinimide), and stirred at room temperature for 1 to 12 hours to activate the carboxyl group, preferably for 4 to 8 hours; after the carboxyl group is activated, 4-(2-aminoethyl)benzenesulfonamide is added to the above system and stirred for 8 to 24 hours, preferably for 12 to 14 hours, to prepare DSPE-PEG-CAI.

[0015] In step S1, the feed ratio of distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, sulfo-(N-hydroxysuccinimide) and 4-(2-aminoethyl)benzenesulfonamide in the present invention is 10-30 mg: 10-60 mg: 5-30 mg: 10-50 mg.

[0016] Preferably, in step S1, the feed ratio of distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, sulfo-(N-hydroxysuccinimide) and 4-(2-aminoethyl)benzenesulfonamide is 15-25 mg: 20-40 mg: 10-20 mg: 20-50 mg.

[0017] In step S1, the molecular weight of polyethylene glycol in the distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl group may be 1K to 5K, preferably 2K.

[0018] In step S2, the feed ratio of lecithin, cholesterol and DSPE-PEG-CAI is 1-4 mg: 0.5-2 mg: 2-6 mg.

[0019] Preferably, in step S2, the feed ratio of lecithin, cholesterol and DSPE-PEG-CAI is 2-4 mg: 0.5-1 mg: 2-4 mg.

[0020] In step S2, the liposome membrane of the present invention is prepared by a thin film dispersion method, the solvent is chloroform, the rotary evaporation temperature is 20-60°C, and the rotary evaporation time is 0.5-4h. Preferably, the rotary evaporation temperature is 40-50°C, and the rotary evaporation time is 1-2h.

[0021] In step S3, the tumor cell membrane is the cell membrane of mouse breast cancer cell 4T1. 4T1 cells are cultured in a cell culture dish and scraped with a cell scraper to collect 4T1 cells. The tumor cell membrane is obtained by repeatedly freezing and thawing three times to break the cells and collect them by centrifugation.

[0022] In step S3, the feed ratio of the tumor cell membrane and liposome in the present invention is 0.5-2 mg:0.5-6 mg. That is, the feed amount of the tumor cell membrane is determined according to the amount of its membrane protein, and the mass ratio of the amount of the tumor cell membrane membrane protein to the liposome.

[0023] Preferably, the feed ratio of the tumor cell membrane to the liposome is 1-2 mg:2-4 mg.

[0024] In step S3, the ultrasonic processing power of the probe in the present invention is 5-20%, preferably working for 3s and resting for 2s, preferably the ultrasonic power of the probe is 10-15%; the ultrasonic time is 2-10min, and the preferred processing time is 3-6min.

[0025] In step S4, the concentration of the chemotherapy drug in the present invention is 1-3 mg / mL, and the chemotherapy drug is loaded by the ammonium sulfate gradient method: the incubation temperature is 20-60° C., and the incubation time is 20-60 min.

[0026] Preferably, the concentration of the chemotherapy drug is 2-2.5 mg / mL, and the chemotherapy drug is loaded by an ammonium sulfate gradient method. Preferably, the incubation temperature is 30-40° C., and the incubation time is 30-60 min.

[0027] Preferably, the chemotherapy drug can be the chemotherapy drug doxorubicin hydrochloride.

[0028] The present invention also provides a tumor microenvironment regulating nano drug delivery system capable of "dual targeting" prepared by the above-mentioned preparation method.

[0029] The present invention also provides the use of the above-mentioned "dual-targeted" tumor microenvironment-regulated nano drug delivery system in the preparation of chemotherapeutic drugs. Through the "dual" tumor targeting of the biomimetic membrane, the accumulation of chemotherapeutic drugs at the tumor site is improved (the delivery efficiency of chemotherapeutic drugs is improved), and the CAI in the biomimetic membrane is used to inhibit the activity of the transmembrane protein carbonic anhydrase IX of 4T1 tumor cells, adjust the acidity of the tumor (regulate the acidic microenvironment of the tumor), and enhance the anti-tumor effect of chemotherapeutic drugs.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The preparation conditions of the "dual-targeted" tumor microenvironment regulating bionic membrane and the "dual-targeted" tumor microenvironment regulating nanodrug delivery system of the present invention are mild and controllable, highly reproducible, and low in cost, and have good potential for industrial production and clinical transformation.

[0032] (2) The "dual-targeted" tumor microenvironment-regulated nano-drug delivery system of the present invention has excellent tumor targeting, which can effectively increase the enrichment of chemotherapy drugs in the tumor site while significantly reducing the toxic side effects on normal tissues.

[0033] (3) Through the specific inhibition of CA IX in 4T1 cells, the "dual-targeted" tumor microenvironment-regulated nanodrug delivery system can improve the acidic microenvironment of the tumor, increase the extracellular pH of the tumor cells, and further enhance the anti-tumor effect of chemotherapeutic drugs.

[0034] (4) The good biocompatibility, efficient tumor targeting ability, and ability to regulate the tumor microenvironment of the "dual-targeted" tumor microenvironment-regulated nanodrug delivery system provide new ideas for anti-tumor treatment and have broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 DSPE-PEG prepared in Example 1 2K - FT-IR image of CAI;

[0036] Figure 2This is a qualitative test result of the total protein content of the “dual-targeted” tumor microenvironment regulating bionic membrane prepared in Example 1.

[0037] Figure 3 TEM image of the "dual-targeted" tumor microenvironment-regulated nano-drug delivery system prepared in Example 1;

[0038] Figure 4 This is a detection diagram of the effect of the "dual-targeted" tumor microenvironment regulating bionic membrane prepared in Example 1 on the extracellular pH value of tumor cells;

[0039] Figure 5 This is a laser confocal imaging diagram of the effect of the "dual-targeted" tumor microenvironment regulating bionic membrane prepared in Example 1 on the acidity in tumor cells;

[0040] Figure 6 The drug release curve of the "dual-targeted" tumor microenvironment-regulated nano drug delivery system prepared in Example 1 under pH 7.4;

[0041] Figure 7 The drug release curve of the "dual-targeted" tumor microenvironment-regulated nano drug delivery system prepared in Example 1 under pH 6.5;

[0042] Figure 8 This is a diagram showing the tumor cell uptake of the "dual-targeted" tumor microenvironment-regulated nano-drug delivery system prepared in Example 1;

[0043] Fig. 9 This is a diagram showing the penetration of tumor spheres in the "dual-targeted" tumor microenvironment-regulated nano-drug delivery system prepared in Example 1;

[0044] Fig.10 This is a diagram for evaluating the cytotoxicity of the tumor microenvironment-regulated nano-drug delivery system capable of "dual targeting" in Example 1 to tumor cells;

[0045] Fig.11 This is a diagram evaluating the tumor-suppressing effect of the “dual-targeted” tumor microenvironment-regulated nanodrug delivery system in Example 1 on primary tumors. DETAILED DESCRIPTION

[0046] The present invention is described in detail below with reference to the embodiments.

[0047] Example 1

[0048] (1) 15 mg of distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl (DSPE-PEG 2K-COOH) was dissolved in 4.0 mL of deionized water, and 30 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 15 mg of sulfo-(N-hydroxysuccinimide) were added under stirring, and the stirring was continued for 4 h to activate the carboxyl group. Subsequently, 30 mg of 4-(2-aminoethyl)benzenesulfonamide (CAI) was added, and the reaction was stirred at room temperature for 24 h. The synthesized DSPE-PEG2K-CAI was purified by dialysis, freeze-dried, and stored at -20°C for later use.

[0049] (2) The mouse breast cancer cells (4T1 cells) cultured in the cell culture dish were gently scraped off with a cell scraper, washed three times with pre-cooled phosphate buffered saline (PBS), and then the obtained 4T1 cells were repeatedly frozen and thawed three times from -80°C to 37°C for cell lysis. Subsequently, the cell contents were removed by centrifugation at 4°C, 600 rpm for 5 min, and the supernatant was collected; further, the collected supernatant was centrifuged at 4°C, 14000 rpm for 20 min, and the precipitate was collected, and the collected precipitate was resuspended with PBS to obtain a tumor cell membrane suspension, which was stored at -20°C for later use.

[0050] (3) Phosphatidylcholine, cholesterol and DSPE-PEG 2K -CAI (prepared according to step (1)) was dissolved in chloroform at a mass ratio of 4:1:4 and rotary evaporated at 40°C for 1 h to form a uniform lipid film, which was then hydrated by adding PBS to obtain a liposome suspension for later use.

[0051] (4) The tumor cell membrane and liposome suspension obtained in steps (2) and (3) are mixed at a mass ratio of 0.5:1, and the mixture is then placed in an ice bath and subjected to probe ultrasonic treatment (ultrasonic power 10%, working 3s, resting 2s, a total of 5min) to obtain a "dual-targeted" tumor microenvironment regulating bionic membrane.

[0052] (5) The "dual-targeted" tumor microenvironment-regulating biomimetic membrane prepared in step (4) was resuspended in an ammonium sulfate solution (300 mM), and then transferred to a dialysis bag (MWCO: 8-14 kDa), stirred for 2 h, and the ammonium sulfate in the external phase was removed to form an ammonium sulfate gradient. Subsequently, 1 mL of doxorubicin hydrochloride (2 mg / mL) was added, and stirring was continued at 40°C for 30 min, and then ultracentrifuged at 4°C and 14,000 rpm for 20 min to remove free doxorubicin hydrochloride. The collected precipitate was resuspended in PBS to obtain a "dual-targeted" tumor microenvironment-regulating nano drug delivery system.

[0053] DSPE-PEG 2K -COOH was characterized by FT-IR. The FT-IR results are as follows Figure 1 As shown, at 1680cm -1 The obvious characteristic peaks appeared at the position of -CONH-, indicating the successful coupling of aminocarboxyl groups. The tumor microenvironment regulating biomimetic membrane capable of "dual targeting" was dissolved with sodium dodecyl sulfate, and the integrity of the membrane protein was characterized by Coomassie brilliant blue staining after PAGE gel electrophoresis. Figure 2 As shown in the figure, the "dual-targeted" tumor microenvironment-regulated bionic membrane can retain the membrane protein components of the tumor cell membrane, and thus can inherit the bionic properties related to the tumor cell membrane. The "dual-targeted" tumor microenvironment-regulated nanodrug delivery system was characterized by TEM. TEM morphology characterization is shown in the figure. Figure 3 As shown, the synthesized "dual-targeted" tumor microenvironment-regulated nanodrug delivery system has a typical "cup-shaped" structure with a diameter of about 150 nm.

[0054] Example 2

[0055] (1) 30 mg of distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl (DSPE-PEG 2K -COOH) was dissolved in 4.0 mL of deionized water, and 30 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 15 mg of sulfo-(N-hydroxysuccinimide) were added under stirring, and the stirring was continued for 4 h to activate the carboxyl group. Subsequently, 30 mg of CAI was added and stirred at room temperature for 24 h to react. The synthesized DSPE-PEG2K-CAI was purified by dialysis, freeze-dried, and stored at -20°C for later use.

[0056] (2) The 4T1 cells cultured in the cell culture dish were gently scraped off with a cell scraper, washed three times with pre-cooled PBS, and then the obtained 4T1 cells were repeatedly frozen and thawed three times from -80°C to 37°C for cell lysis. Subsequently, the cell contents were removed by centrifugation at 4°C, 600 rpm for 5 min, and the supernatant was collected; further, the collected supernatant was centrifuged at 4°C, 14000 rpm for 20 min, and the precipitate was collected. The collected precipitate was resuspended with PBS to obtain a tumor cell membrane suspension, which was stored at -20°C for later use.

[0057] (3) Phosphatidylcholine, cholesterol and DSPE-PEG 2K -CAI (prepared according to step (1)) was dissolved in chloroform at a mass ratio of 4:1:4 and rotary evaporated at 40°C for 1 h to form a uniform lipid film, which was then hydrated by adding PBS to obtain a liposome suspension for later use.

[0058] (4) The tumor cell membrane and liposome suspension obtained in steps (2) and (3) are mixed at a mass ratio of 0.5:1, and the mixture is then placed in an ice bath and subjected to probe ultrasonic treatment (ultrasonic power 10%, working 3s, resting 2s, a total of 5min) to obtain a "dual-targeted" tumor microenvironment regulating bionic membrane.

[0059] (5) The "dual-targeted" tumor microenvironment-regulating biomimetic membrane prepared in step (4) was resuspended in an ammonium sulfate solution (300 mM), and then transferred to a dialysis bag (MWCO: 8-14 kDa), stirred for 2 h, and the ammonium sulfate in the external phase was removed to form an ammonium sulfate gradient. Subsequently, 1 mL of doxorubicin hydrochloride (2 mg / mL) was added, and stirring was continued at 40°C for 30 min, and then ultracentrifuged at 4°C and 14,000 rpm for 20 min to remove free doxorubicin hydrochloride. The collected precipitate was resuspended in PBS to obtain a "dual-targeted" tumor microenvironment-regulating nano drug delivery system.

[0060] DSPE-PEG 2K -COOH was characterized by FT-IR. FT-IR results showed that at 1680 cm -1 An obvious characteristic peak appears at the position, indicating the presence of -CONH-, i.e., the successful coupling of the aminocarboxyl group. The "dual-targeted" tumor microenvironment-regulated bionic membrane was dissolved with sodium dodecyl sulfate, and the integrity of the membrane protein was characterized by Coomassie brilliant blue staining after PAGE gel electrophoresis. The "dual-targeted" tumor microenvironment-regulated bionic membrane can retain the membrane protein components of the tumor cell membrane, and thus can inherit the bionic properties related to the tumor cell membrane. The "dual-targeted" tumor microenvironment-regulated nanodrug delivery system was characterized by TEM. The synthesized "dual-targeted" tumor microenvironment-regulated nanodrug delivery system was characterized by TEM morphology, showing a typical "cup-shaped" structure.

[0061] Example 3

[0062] (1) 15 mg of distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl (DSPE-PEG 2K -COOH) was dissolved in 4.0 mL of deionized water, and 30 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 15 mg of sulfo-(N-hydroxysuccinimide) were added under stirring, and the stirring was continued for 4 h to activate the carboxyl group. Subsequently, 30 mg of CAI was added and stirred at room temperature for 24 h to react. The synthesized DSPE-PEG2K-CAI was purified by dialysis, freeze-dried, and stored at -20°C for later use.

[0063] (2) The 4T1 cells cultured in the cell culture dish were gently scraped off with a cell scraper, washed three times with pre-cooled PBS, and then the obtained 4T1 cells were repeatedly frozen and thawed three times from -80°C to 37°C for cell lysis. Subsequently, the cell contents were removed by centrifugation at 4°C, 600 rpm for 5 min, and the supernatant was collected; further, the collected supernatant was centrifuged at 4°C, 14000 rpm for 20 min, and the precipitate was collected. The collected precipitate was resuspended with PBS to obtain a tumor cell membrane suspension, which was stored at -20°C for later use.

[0064] (3) Phosphatidylcholine, cholesterol and DSPE-PEG 2K -CAI (prepared according to step (1)) was dissolved in chloroform at a mass ratio of 2:0.5:4 and rotary evaporated at 40°C for 1 h to form a uniform lipid film, which was then hydrated by adding PBS to obtain a liposome suspension for later use.

[0065] (4) The tumor cell membrane and liposome suspension obtained in steps (2) and (3) are mixed at a mass ratio of 0.5:1, and the mixture is then placed in an ice bath and subjected to probe ultrasonic treatment (ultrasonic power 10%, working 3s, resting 2s, a total of 5min) to obtain a "dual-targeted" tumor microenvironment regulating bionic membrane.

[0066] (5) The "dual-targeted" tumor microenvironment-regulating biomimetic membrane prepared in step (4) was resuspended in an ammonium sulfate solution (300 mM), and then transferred to a dialysis bag (MWCO: 8-14 kDa), stirred for 2 h, and the ammonium sulfate in the external phase was removed to form an ammonium sulfate gradient. Subsequently, 1 mL of doxorubicin hydrochloride (2 mg / mL) was added, and stirring was continued at 40°C for 30 min, and then ultracentrifuged at 4°C and 14,000 rpm for 20 min to remove free doxorubicin hydrochloride. The collected precipitate was resuspended in PBS to obtain a "dual-targeted" tumor microenvironment-regulating nano drug delivery system.

[0067] DSPE-PEG 2K -COOH was characterized by FT-IR. FT-IR results showed that at 1680 cm -1An obvious characteristic peak appears at the position, indicating the presence of -CONH-, i.e., the successful coupling of the aminocarboxyl group. The "dual-targeted" tumor microenvironment-regulated bionic membrane was dissolved with sodium dodecyl sulfate, and the integrity of the membrane protein was characterized by Coomassie brilliant blue staining after PAGE gel electrophoresis. The "dual-targeted" tumor microenvironment-regulated bionic membrane can retain the membrane protein components of the tumor cell membrane, and thus can inherit the bionic properties related to the tumor cell membrane. The "dual-targeted" tumor microenvironment-regulated nanodrug delivery system was characterized by TEM. TEM morphology characterization showed that the synthesized "dual-targeted" tumor microenvironment-regulated nanodrug delivery system had a typical "cup-shaped" structure.

[0068] Example 4

[0069] (1) 15 mg of distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl (DSPE-PEG 2K -COOH) was dissolved in 4.0 mL of deionized water, and 30 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 15 mg of sulfo-(N-hydroxysuccinimide) were added under stirring, and the stirring was continued for 4 h to activate the carboxyl group. Subsequently, 30 mg of CAI was added and stirred at room temperature for 24 h to react. The synthesized DSPE-PEG2K-CAI was purified by dialysis, freeze-dried, and stored at -20°C for later use.

[0070] (2) The 4T1 cells cultured in the cell culture dish were gently scraped off with a cell scraper, washed three times with pre-cooled PBS, and then the obtained 4T1 cells were repeatedly frozen and thawed three times from -80°C to 37°C for cell lysis. Subsequently, the cell contents were removed by centrifugation at 4°C, 600 rpm for 5 min, and the supernatant was collected; further, the collected supernatant was centrifuged at 4°C, 14000 rpm for 20 min, and the precipitate was collected. The collected precipitate was resuspended with PBS to obtain a tumor cell membrane suspension, which was stored at -20°C for later use.

[0071] (3) Phosphatidylcholine, cholesterol and DSPE-PEG 2K -CAI (prepared according to step (1)) was dissolved in chloroform at a mass ratio of 4:1:4 and rotary evaporated at 40°C for 1 h to form a uniform lipid film, which was then hydrated by adding PBS to obtain a liposome suspension for later use.

[0072] (4) The tumor cell membrane and liposome suspension obtained in steps (2) and (3) are mixed at a mass ratio of 0.5:1, and the mixture is then placed in an ice bath and subjected to probe ultrasonic treatment (ultrasonic power 15%, working 2s, resting 3s, a total of 4min) to obtain a "dual-targeted" tumor microenvironment regulating bionic membrane.

[0073] (5) The "dual-targeted" tumor microenvironment-regulating biomimetic membrane prepared in step (4) was resuspended in an ammonium sulfate solution (300 mM), and then transferred to a dialysis bag (MWCO: 8-14 kDa), stirred for 2 h, and the ammonium sulfate in the external phase was removed to form an ammonium sulfate gradient. Subsequently, 1 mL of doxorubicin hydrochloride (2 mg / mL) was added, and stirring was continued at 40°C for 30 min, and then ultracentrifuged at 4°C and 14,000 rpm for 20 min to remove free doxorubicin hydrochloride. The collected precipitate was resuspended in PBS to obtain a "dual-targeted" tumor microenvironment-regulating nano drug delivery system.

[0074] DSPE-PEG 2K -COOH was characterized by FT-IR. FT-IR results showed that at 1680 cm -1 An obvious characteristic peak appears at the position, indicating the presence of -CONH-, i.e., the successful coupling of the aminocarboxyl group. The "dual-targeted" tumor microenvironment-regulated bionic membrane was dissolved with sodium dodecyl sulfate, and the integrity of the membrane protein was characterized by Coomassie brilliant blue staining after PAGE gel electrophoresis. The "dual-targeted" tumor microenvironment-regulated bionic membrane can retain the membrane protein components of the tumor cell membrane, and thus can inherit the bionic properties related to the tumor cell membrane. The "dual-targeted" tumor microenvironment-regulated nanodrug delivery system was characterized by TEM. TEM morphology characterization showed that the synthesized "dual-targeted" tumor microenvironment-regulated nanodrug delivery system had a typical "cup-shaped" structure.

[0075] Performance test

[0076] (1) Extracellular pH detection

[0077] 4T1 cells in the logarithmic growth phase were taken and plated at 1×10 4 The cells were inoculated into a 96-well plate (200 μL / well) with RPMI 1640 culture medium containing 10% fetal bovine serum at a cell density of 10 cells, and cultured in an incubator at 37°C and 5% CO2. After the cells were cultured for 12 hours, the culture medium was replaced with a culture medium containing PBS buffer or a "dual-targeted" tumor microenvironment-regulated biomimetic membrane (TLC) (prepared according to the method of Example 1). After continuing to culture for 0 hours, 6 hours, 12 hours, 24 hours and 48 hours, the supernatant culture medium was collected and the extracellular pH value was measured with a pH meter. The results are shown in Figure 2. Figure 4 As shown, the "dual targeting" tumor microenvironment regulating bionic membrane can significantly alleviate the acidification of tumor extracellular fluid, laying the foundation for the use of "dual targeting" tumor microenvironment regulating nanodrug delivery system to improve the tumor acidic microenvironment.

[0078] (2) Intracellular acidity detection

[0079] 4T1 cells in the logarithmic growth phase were taken and plated at 1×10 5 The cells were inoculated into a confocal dish with a cell density of 10 cells using RPMI 1640 culture medium containing 10% fetal bovine serum, and cultured in an incubator at 37°C and 5% CO2. After the cells were cultured for 12 hours, the culture medium was replaced with a culture medium containing PBS buffer or a "dual-targeted" tumor microenvironment regulating biomimetic membrane (TLC) (prepared according to the method of Example 1). The culture was continued for 24 hours. The cells were washed with PBS, and the BCECF-AM working solution was added and incubated at 37°C for 30 minutes. The cells were then fixed with 4% paraformaldehyde, and the cell nuclei were stained with DAPI. A confocal laser scanning microscope (CLSM) was used to observe changes in intracellular pH. The results are as follows. Figure 5 As shown, the "dual-targeted" tumor microenvironment regulating bionic membrane can significantly increase the pH value in tumor cells and destroy the pH homeostasis in tumor cells, thus it is expected to increase the sensitivity of tumor cells to chemotherapeutic drugs.

[0080] (3) Drug release evaluation

[0081] The concentration of the chemotherapy drug doxorubicin in the "dual-targeted" tumor microenvironment-regulated nanodrug delivery system (DOX@TLC) was set to 100 μg / mL. 1 mL of the sample was added to a dialysis bag (MWCO 3500Da) and placed in PBS buffer of different pH values ​​(pH 7.4 or 6.5), and incubated with shaking at 37°C and 100 rpm. PBS solutions were collected at different time periods, and the doxorubicin concentration in the PBS solution was detected by a Multiskan MK3 microplate reader (Thermo): excitation wavelength 488 nm; emission wavelength 590 nm. The results are shown in Figure 2. Figure 6 and Figure 7 As shown, the drug release in the "dual-targeted" tumor microenvironment-regulated nanodrug delivery system under pH 6.5 conditions is significantly higher than that under pH 7.4 conditions. Therefore, the drug delivery system itself can achieve acid-responsive drug release.

[0082] (4) Evaluation of tumor cell uptake

[0083] The "dual-targeted" tumor microenvironment-regulated nano drug delivery system (DOX@TLC) prepared in Example 1 was selected and dispersed in the cell culture medium to obtain a sample of a certain concentration. 4T1 cells in the logarithmic growth phase were inoculated in a confocal dish, RPMI 1640 culture medium containing 10% fetal bovine serum was added, and cultured at 37°C in an incubator containing 5% CO2. After the cells adhered to the wall for 12 hours, the "dual-targeted" tumor microenvironment-regulated nano drug delivery system was incubated with the cells for 12 hours. Subsequently, the cell nuclei were stained with DAPI, washed with PBS, and observed under a microscope. The results are as follows: Figure 8 As shown, the "dual-targeted" tumor microenvironment-regulated nanodrug delivery system can be effectively taken up by breast cancer cells.

[0084] (5) Evaluation of tumor sphere penetration ability

[0085] The "dual-targeted" tumor microenvironment-regulated nano drug delivery system (DOX@TLC) prepared in Example 1 was selected and dispersed in a cell culture medium to obtain a sample of a certain concentration. 4T1 cells in the logarithmic growth phase were taken, and the cells were inoculated at a cell density of 8,000 cells per well in a round-bottom 96-well plate with 2% mass agarose gel on the bottom using RPMI 1640 culture medium containing 10% fetal bovine serum, and cultured at 37°C in an incubator containing 5% CO2. After the cells formed tumor spheres, they were incubated with the tumor spheres using the "dual-targeted" tumor microenvironment-regulated nano drug delivery system for 12 hours. After washing with PBS, they were observed under a microscope. The results are as follows. Fig. 9 As shown, the "dual-targeted" tumor microenvironment-regulated nanodrug delivery system can effectively penetrate tumor spheres and achieve deep drug delivery to tumors.

[0086] (6) Cytotoxicity evaluation on breast cancer cell lines

[0087] 4T1 cells in logarithmic growth phase were taken and 1×10 4 The cell density of each cell was RPMI 1640 culture medium containing 10% fetal bovine serum. The cells were inoculated in a 96-well plate and cultured in an incubator at 37°C and 5% CO2. After the cells were cultured for 12 hours, the culture medium was replaced with a culture medium containing a tumor microenvironment-regulated nano-drug delivery system (prepared according to the method of Example 1) that can be "dual targeted", wherein the dosing concentrations were 0, 1, 2, 4 and 8 μg / mL of doxorubicin hydrochloride, respectively. After continuing to culture for 24 hours, the old culture medium was aspirated and washed once with PBS buffer. MTT solution (100 μL / well) was then added to the wells, incubated in an incubator at 37°C and 5% CO2 for 1 to 4 hours, then the MTT solution was aspirated, 100 μL DMSO solution was added, and the system was shaken and shaken, and its optical density OD value was measured on an ELISA instrument.

[0088] Data processing: Use the corresponding software of the microplate reader to process the data, calculate the average OD value of the four wells of each sample, and use the average value to calculate the cell viability (Cell Viability %) according to the following formula.

[0089] Cell Viability % = average OD value of sample group / average OD value of blank control group × 100% (Cell Viability % = OD sample / OD control ×100%)

[0090] The results are as follows Fig.10 As shown. Fig.10 The analysis showed that the "dual-targeted" tumor microenvironment-regulated nano-drug delivery system exhibited obvious concentration-dependent cytotoxicity. Therefore, the "dual-targeted" tumor microenvironment-regulated nano-drug delivery system can produce obvious cytotoxicity after being taken up by tumor cells.

[0091] (7) Evaluation of tumor inhibition effect

[0092] Fifteen BALB / c mice were subcutaneously implanted with 4T1 cells on their backs to construct a subcutaneous tumor model. The mice were randomly divided into three groups, each with five mice, namely, a PBS group, a free chemotherapy drug group (DOX), and a "dual-targeted" tumor microenvironment-regulated nanodrug delivery system group (DOX@TLC) (prepared according to the method of Example 1). The drugs were administered through the tail vein for a total of three times. At the same time, the tumor volume (V) of the primary tumor was measured every other day, and the calculation formula was: V = L × W 2 / 2, W and L are the widest and longest diameters of the tumor, respectively. The tumor volume was calculated to monitor the tumor inhibition effect.

[0093] The results are as follows Fig.11 The "dual targeting" tumor microenvironment regulating nanodrug delivery system group can significantly inhibit tumor growth and has a good tumor suppression effect.

[0094] In summary, the "dual-targeted" tumor microenvironment-regulated nanodrug delivery system provided by the present invention can regulate the acidic microenvironment of the tumor, and improve the delivery efficiency of chemotherapeutic drugs through the "dual" tumor targeting of the bionic membrane, thereby effectively enhancing its therapeutic effect.

[0095] The above-described embodiments provide a detailed description of the technical solutions and beneficial effects of the present invention. It should be understood that the above descriptions are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements and equivalent substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a tumor microenvironment-regulated nano drug delivery system, characterized in that: The preparation method comprises the following steps: S1, reacting distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl with 4-(2-aminoethyl)benzenesulfonamide, and purifying by dialysis to obtain DSPE-PEG-CAI; S2, dissolving lecithin, cholesterol and the DSPE-PEG-CAI in step S1 in an organic solvent, rotary evaporating to form a lipid film, and obtaining a liposome suspension after hydration; S3, mixing the tumor cell membrane with the liposome suspension in step S2, and preparing a tumor microenvironment regulating bionic membrane by probe ultrasonic treatment; S4. Loading the chemotherapy drugs into the bionic membrane in step S3 to obtain a tumor microenvironment-regulated nano drug delivery system.

2. The method for preparing the tumor microenvironment-regulated nano drug delivery system according to claim 1, characterized in that: In step S1, the distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl is first mixed with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and sulfo-(N-hydroxysuccinimide), stirred at room temperature for 1 to 12 hours to activate the carboxyl group, and then 4-(2-aminoethyl)benzenesulfonamide is added, and the stirring is continued for 8 to 24 hours to react. The feed ratio of the distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, sulfo-(N-hydroxysuccinimide) and 4-(2-aminoethyl)benzenesulfonamide is 10 to 30 mg: 10 to 60 mg: 5 to 30 mg: 10 to 50 mg; the molecular weight of polyethylene glycol in the distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl is 1K to 5K.

3. The method for preparing the tumor microenvironment regulating nano drug delivery system according to claim 1, characterized in that: In step S2, the feed ratio of lecithin, cholesterol and DSPE-PEG-CAI is 1-4 mg: 0.5-2 mg: 2-6 mg.

4. The method for preparing the tumor microenvironment-regulated nano drug delivery system according to claim 1, characterized in that: In step S2, the organic solvent is chloroform, the rotary evaporation temperature is 20-60°C, and the rotary evaporation time is 0.5-4h.

5. The method for preparing the tumor microenvironment-regulated nano drug delivery system according to claim 1, characterized in that: In step S3, the mass ratio of the membrane protein of the tumor cell membrane to the liposome is 0.5-2 mg:0.5-6 mg.

6. The method for preparing the biomimetic membrane-loaded chemotherapeutic drug preparation according to claim 1, characterized in that: In step S3, the ultrasonic processing power of the probe is 5-20%, and the processing time is 2-10 minutes.

7. The method for preparing the tumor microenvironment regulating nano drug delivery system according to claim 1, characterized in that: In step S4, the concentration of the chemotherapy drug is 1-3 mg / mL, and the chemotherapy drug is loaded by the ammonium sulfate gradient method: the incubation temperature is 20-60° C., and the incubation time is 20-60 min.

8. A tumor microenvironment regulating nano drug delivery system obtained according to the preparation method according to any one of claims 1 to 7.

9. The tumor microenvironment regulating nano drug delivery system according to claim 8, characterized in that: It has a typical "cup-shaped" structure with a diameter of 130 to 180 nm.

10. Use of the nano drug delivery system according to claim 8 in preparing drugs for treating tumors.

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