Multifunctional nano-carrier with drug resistance, biotin receptor targeting and hypoxia / low pH dual responsiveness as well as preparation method and application of multifunctional nano-carrier

Abstract

The invention discloses a multifunctional nano-carrier with drug resistance, biotin receptor targeting and hypoxia / low pH dual responsiveness and a preparation method and application thereof, and belongs to the technical field of biomedical polymer materials. The AGT inhibitor conjugate with biotin receptor targeting and low oxygen and low pH dual response provided by the invention comprises a hydrophobic 2-amino-O4-benzyl pteridine group (BD), a low oxygen response group azobenzene (AZO) and a hydrophilic high polymer material modified by biotin (Bio). According to the system, hydrophobic chemotherapeutic drugs can be loaded in a drug carrying system through intermolecular acting force, target biotin receptors on tumor cells and are enriched in tumor tissues by utilizing high permeability and long-term effectiveness of the hydrophobic chemotherapeutic drugs; the tumor microenvironment characterized by low oxygen and low pH can induce azo bond breakage and pH response on the nano-carrier to release BD with AGT activity inhibition and an alkylating agent with anti-tumor activity, so that the drug resistance and anti-tumor activity of the drug are improved, and the targeting property is increased.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical polymer materials, and in particular to the preparation and application of a multifunctional nanocarrier having anti-drug resistance, biotin receptor targeting and dual responsiveness to low oxygen / low pH. Background Art

[0002] DNA alkylating agents are among the earliest chemotherapy drugs used in tumor treatment, but their low stability and low targeting ability greatly limit their clinical application. In addition, the resistance of tumor cells to chemotherapy drugs is also an important factor affecting their efficacy. Various proteins involved in the DNA repair mechanism can effectively repair DNA damage, reduce the cytotoxicity of anticancer alkylating agents, and make tumor cells resistant to alkylating agents, which seriously reduces the chemotherapy effect of this type of drug. 6 -Alkylguanine-DNA alkyltransferase (AGT) is a class of DNA damage repair proteins present in cells. 6 The alkylation damage at position 145 is transferred to the cysteine ​​residue at position 145 to repair the damaged DNA. In order to overcome the drug resistance caused by AGT, researchers have used AGT inhibitors in combination with alkylating agents such as semustine, fotemustine, and dacarbazine to increase the sensitivity of anticancer alkylating agents to tumor cells and improve the effect of tumor treatment. 4 -Benzylpteridine (BD) is an AGT inhibitor that can be used to overcome AGT-induced drug resistance.

[0003] However, due to the lack of targeting and functionality, as well as the problems of poor solubility and stability of the drugs, the drug distribution is not coordinated after the co-administration of AGT inhibitors and anticancer alkylating agents, and the dose of the drug to exert anticancer activity is limited. In addition, in addition to inhibiting the activity of AGT in tumor tissues, AGT inhibitors also inhibit the activity of AGT in normal tissues, resulting in serious side effects and reducing the therapeutic effect. Therefore, it is necessary to develop a new tumor-targeted drug delivery system that can target the delivery of alkylating agents and AGT inhibitors to the tumor site, so that they can work together to make them both anti-tumor and anti-drug resistance, thereby achieving efficient and low-toxic chemotherapy.

[0004] Utilizing the unique microenvironment of tumors to construct a responsive drug delivery system has broad application prospects in the field of targeted tumor therapy. Tumor tissue is highly heterogeneous, and the internal area of ​​solid tumors is in a hypoxic microenvironment with significantly lower oxygen content than normal tissues. At the same time, tumor tissues usually have an acidic microenvironment with a pH between 6.0 and 6.5, which is significantly lower than that of normal tissues. In addition, biotin receptors are overexpressed in various types of cancer cells and play an important role in tumor metabolism, growth, and metastasis. Targeting biotin receptors on tumor cells can improve the efficiency of chemotherapy and reduce the side effects of chemotherapy. Therefore, by constructing a new nano-drug delivery system that is both anti-resistance, tumor-targeted and stimuli-responsive, it is possible to achieve targeted release of anticancer alkylating agents in tumor tissues, thereby achieving precise treatment of tumors. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a multifunctional nanocarrier having anti-drug resistance, biotin receptor targeting and dual response to hypoxia / low pH.

[0006] The biotin receptor targeting and hypoxia and low pH dual-responsive AGT inhibitor conjugate provided by the present invention comprises a hydrophobic 2-amino-O 4 -Hydrophilic polymer material modified with benzylpteridine group (BD), low oxygen responsive group azobenzene (AZO) and biotin (Bio).

[0007] This system can load hydrophobic chemotherapy drugs into the drug delivery system through intermolecular forces, target biotin receptors on tumor cells, and enrich them in tumor tissues by virtue of their high permeability and long-lasting effect; the tumor microenvironment characterized by hypoxia and low pH can induce the rupture of azo bonds and pH response on the nanocarriers, releasing BD with AGT inhibitory activity and alkylating agents with anti-tumor activity, thereby improving the drug's resistance to drug resistance and anti-tumor activity, and increasing its targeting.

[0008] The hypoxic group is azobenzene, which can break the azo bond in the hypoxic area of ​​the tumor.

[0009] The AGT inhibitor is BD.

[0010] The hydrophilic polymer material is carboxymethyl chitosan (CMCS), which can be protonated in the low pH microenvironment of the tumor.

[0011] The hydrophilic polymer material is modified with biotin, which can specifically bind to the biotin receptor overexpressed on the surface of tumor cells and has good biocompatibility, thereby further improving the accuracy of drug delivery and reducing toxic side effects.

[0012] The present invention provides a preparation method of "a multifunctional nanocarrier having both anti-drug resistance, biotin receptor targeting and dual responsiveness to hypoxia / low pH" comprising the following steps:

[0013] 1) Synthesis of Bio-CMCS-AZO-BD (BCAB), a biotin receptor-targeted and hypoxia / low pH dual-responsive AGT inhibitor conjugate:

[0014] The present invention provides a biotin receptor targeting and hypoxia / low pH dual responsive AGT inhibitor conjugate BCAB with a structural formula of:

[0015]

[0016] CMCS was dissolved in water and stirred thoroughly to obtain a CMCS solution with a concentration of 1-50 mg / mL, dimethylaminopyridine (DMAP) and N,N′-dicyclohexylcarbodiimide (DCC) were added thereto, wherein the mass ratio of CMCS, DMAP and DCC was 1:(0.1-2):(0.1-2), and stirring was continued. 2-amino-6-((3-((4-aminophenyl)diazenyl)benzyl)oxy)-9H-pteridine (BDAZO) was weighed and dissolved in dimethyl sulfoxide (DMSO) to obtain a BDAZO solution with a concentration of 1-20 mg / mL, which was added to the above reaction solution, and the reaction time was 10-36 h in the dark at 25-55° C. under nitrogen protection. After the reaction is completed, the product is placed in a dialysis bag for dialysis under the conditions of deionized water / DMSO (V / V = 1:3 to 1:1) for 24-96 hours, and then dialyzed with pure water for 24-96 hours. The liquid in the dialysis bag is collected and passed through a water membrane to remove impurities, and CMCS-AZO-BD is obtained by freeze drying.

[0017] Dissolve biotin in N-hydroxysuccinimide (NHS) to obtain a biotin solution with a concentration of 1-30 mg / mL, then add DCC, wherein the mass ratio of biotin to DCC is 1:(0.1-10), add it to the CMCS-AZO-BD aqueous solution after magnetic stirring, the mass ratio of biotin solution to CMCS-AZO-BD is 1:(0.05-2), and incubate the obtained solution under magnetic stirring at room temperature for 48-98 hours. The obtained mixture is dialyzed with deionized water for 24-98 hours, and collected after freeze drying to obtain the conjugate BCAB.

[0018] 2) Preparation of a multifunctional nanocarrier BCAB NPs with anti-drug resistance, biotin receptor targeting and dual responsiveness to hypoxia / low pH:

[0019] The lyophilized powder of the conjugate BCAB was weighed and dissolved in distilled water to obtain a 10-150 mg / mL aqueous solution of the conjugate, and ultrasonic treatment was performed in an ice bath for 1-50 min, and the ultrasonic power was set to 100-800 W. Then, the reaction solution was filtered through a mixed cellulose filter membrane and vacuum freeze-dried for 12-48 h to obtain the nanocarrier BCAB NPs.

[0020] 3) Preparation of BCAB / Drug NPs, a multifunctional anti-tumor nanodrug loaded with chemotherapeutic drugs with anti-drug resistance, biotin receptor targeting and dual response to hypoxia / low pH:

[0021] The hydrophobic chemotherapy drug is dissolved in an organic solvent to obtain a drug solution with a concentration of 1-120 mg / mL, the lyophilized powder of the conjugate BCAB is dissolved in distilled water to obtain a conjugate solution with a concentration of 1-150 mg / mL, and the drug solution is evenly added to the conjugate solution with a syringe, and the stirring reaction is continued for 1-60 min. Then the reaction solution is ultrasonically treated for 1-60 min under ice bath conditions, and the ultrasonic power is set to 100-800 W. Next, the above-mentioned ultrasonic reaction solution is added to a dialysis bag, the molecular weight cutoff of the dialysis bag is 1000-10000 Da, and the dialysate is a phosphate buffered saline (PBS) with a pH of 7.4, and the dialysate is dialyzed for 1-18 hours to remove the unencapsulated drug. Finally, the liquid in the dialysis bag is freeze-dried to obtain the multifunctional anti-tumor nanomedicine BCAB / Drug NPs with dual response to anti-drug resistance and hypoxia / low pH of the chemotherapeutic drug.

[0022] Preferably, in step 1), the concentration of CMCS solution is preferably 1-25 mg / mL, the mass ratio of CMCS, DMAP and DCC is 1:(0.5-1):(0.5-1), the concentration of BDAZO solution is preferably 2-15 mg / mL, the reaction temperature is 25-45°C, the reaction time is 10-24 h, and the dialysis conditions are deionized water / DMSO dialysis for 24-72 h (deionized water / DMSO V / V=1:3), and pure water dialysis for 24-72 h; the concentration of biotin solution is preferably 1-20 mg / mL, the mass ratio of biotin and DCC is 1:(0.5-8), the mass ratio of biotin solution and CMCS-AZO-BD is 1:(0.05-1), the reaction time is 48-96 h, and the deionized water dialysis is 24-96 h.

[0023] Preferably, in step 2), the concentration of the BCAB aqueous solution is preferably 10-100 mg / mL; the ultrasonic treatment time is preferably 5-40 min; the ultrasonic power is preferably 100-600 W; and the vacuum drying time is preferably 18-48 h.

[0024] Preferably, the hydrophobic chemotherapy drug described in step 3) is a DNA alkylating agent, preferably a chloroethyl nitrosourea anticancer alkylating agent and dacarbazine (DITC); further preferably semustine (Me-CCNU), fotemustine (FTMS) and DITC. The organic solvent is one or more of DMSO, ethanol, methanol, and DMAC, preferably DMAC or DMSO; the concentration of the drug solution is preferably 1-100 mg / mL; the concentration of the BCAB aqueous solution is preferably 1-120 mg / mL. The reaction time of the drug solution and the conjugate solution is preferably 1-50 min; the ultrasonic treatment time is preferably 1-40 min, the dialysis bag cutoff molecular weight is preferably 1000-6000 Da, the ultrasonic power is preferably 100-600 W; the dialysis time is preferably 1-15 h.

[0025] The tumor described in the present invention is one or more of colorectal cancer, lymphoma, brain tumor, myeloma, liver cancer, glioma, prostate cancer, leukemia, brain glioma, lymphoma, malignant melanoma, and breast cancer; preferably one or more of colorectal cancer, brain tumor, glioma, liver cancer, breast cancer, and prostate cancer.

[0026] The present invention has the following essential features:

[0027] 1) The functional nanocarrier prepared by the present invention has anti-drug resistance, biotin receptor targeting and dual responsiveness to hypoxia / low pH. Biotin targets the biotin receptor overexpressed on the tumor surface. In the tumor microenvironment of hypoxia and low pH, the azo bond on the carrier is reduced to release the AGT inhibitor 2-amino-O 4 -benzylpteridine, plays an anti-drug resistance role. At the same time, chitosan is protonated under acidic conditions, the polymer network expands, and the shell permeability increases, further releasing the anti-tumor drugs encapsulated in the nanomicelle core, thereby achieving targeted delivery and controlled release of anti-tumor drugs, and playing a role in enhancing efficacy and reducing toxicity.

[0028] 2) The amphiphilic block copolymer material used in the present invention has good biocompatibility and biodegradability, and can self-assemble in aqueous solution to form spherical nanocarriers with uniform particle size and good dispersion.

[0029] 3) The functional nanocarrier designed in the present invention can encapsulate hydrophobic chemotherapy drugs, thus overcoming the shortcomings of chemotherapy drugs such as instability and easy clearance in the body and low bioavailability caused by the poor solubility of hydrophobic drugs.

[0030] 4) The functional nanocarrier prepared by the present invention is a spherical nanoparticle with a shell-core structure, and its average particle size is 100-200nm. Under the enhanced permeability retention effect (EPR) of tumor tissue, it is effectively enriched in the tumor site and enters the cell through endocytosis.

[0031] 5) The preparation method of the present invention is simple, economical and low in cost, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings are used to further illustrate the present invention and are used together with the following specific embodiments to explain the present invention, but do not constitute a limitation of the present invention. In the accompanying drawings:

[0033] Figure 1 The preparation route of the present invention's "a multifunctional nanocarrier with anti-drug resistance, biotin receptor targeting and dual responsiveness to hypoxia / low pH, and its preparation method and application" and a schematic diagram of drug delivery in tumor tissue;

[0034] Figure 2 The particle size distribution and transmission electron microscopy morphology of BCAB NPs in Example 1 of the present invention;

[0035] Figure 3 The particle size distribution and transmission electron microscopy morphology of BCAB / Me-CCNU NPs in Example 3 of the present invention;

[0036] Figure 4 The particle size distribution and transmission electron microscopy morphology of BCAB / FTMS NPs in Example 5 of the present invention;

[0037] Figure 5 The particle size distribution and transmission electron microscopy morphology of BCAB / DITC NPs in Example 7 of the present invention;

[0038] Figure 6 The stability of each functional drug carrier in Experimental Example 2 of the present invention in H2O, PBS and DMEM medium containing 10% serum (FBS);

[0039] Figure 7 The hypoxic reduction sensitivity of each functional drug carrier in Experimental Example 3 of the present invention under neutral pH conditions (Figures A, C, and E are the results of measurement under normoxic conditions, and Figures B, D, and F are the results of measurement under hypoxic conditions) (normoxic conditions: air environment; hypoxic conditions: 90% nitrogen, 5% oxygen, and 5% carbon dioxide);

[0040] Figure 8 The pH responsiveness of each functional drug carrier in Experimental Example 4 of the present invention under normoxic conditions (Figures A, C, and E are the results of the measurement under neutral pH conditions, and Figures B, D, and F are the results of the measurement under low pH conditions) (neutral pH conditions: pH = 7; low pH conditions: pH = 5.3);

[0041] Fig. 9 is the cumulative drug release curve of each free drug and the functional nanocarrier in the normoxic reaction system under the condition of neutral pH in Experimental Example 5;

[0042] Fig.10 is the cumulative drug release curve of each free drug and the functional nanocarrier in the hypoxic reaction system under the condition of neutral pH in Experimental Example 5;

[0043] Fig.11 The cumulative drug release curves of free drugs and functional nanocarriers in the normoxic reaction system under low pH conditions in Experimental Example 5;

[0044] Fig.12 The cumulative drug release curves of free drugs and functional nanocarriers in the hypoxic reaction system under low pH conditions in Experimental Example 5; DETAILED DESCRIPTION

[0045] In order to explain the present invention more clearly, the applicant will further explain the present invention in detail according to the embodiments of the technical solution of the present invention.

[0046] Example 1 Preparation of a multifunctional nanocarrier (BCAB NPs) with anti-drug resistance, targeting and dual response to hypoxia / low pH:

[0047] (1) Synthesis of BCAB

[0048] Dissolve 200 mg CMCS in 20 mL water and stir thoroughly to obtain a CMCS solution with a concentration of 10 mg / mL, add 100 mg DMAP and 150 mg DCC, and continue stirring. Weigh 50 mg of BDAZO and dissolve it in 10 mL DMSO to obtain a BDAZO solution with a concentration of 5 mg / mL, add it to the above reaction solution, and react in the dark at 35 ° C under nitrogen protection for 12 hours. After the reaction is completed, the product is placed in a dialysis bag for dialysis. The dialysis conditions are deionized water / DMSO dialysis for 24 hours (deionized water / DMSO V / V=1:3), and then dialyzed with pure water for 48 hours. Collect the liquid in the dialysis bag and pass it through a water membrane to remove impurities, and obtain CMCS-AZO-BD by freeze drying.

[0049] 24 mg of biotin was dissolved in 4 ml of NHS to obtain a biotin solution with a concentration of 6 mg / mL, and then 104 mg of DCC was added, and after magnetic stirring, it was added to 20 mL of deionized water solution containing 6 mg of CMCS-AZO-BD, and the resulting solution was incubated at room temperature under magnetic stirring for 72 hours. The resulting mixture was dialyzed against deionized water for 96 hours, freeze-dried and collected to obtain the conjugate BCAB.

[0050] IR (KBr tablet)υ / cm -1:3432.6(NH),2938.9(CH),1687.8(C=O),1685.6(C=N),1683.5(C=C),1248.4(COC),1178.2(CO);

[0052] 1 H NMR (400MHz, DMSO) δ: 1.25-1.58 (m, 6H, CH2); 1.7 (s, 2H, NH2); 2.05 (m, 2H, CH2); 2.93 (m, 1H, CH); 3.10 (m, 2H, CH 2);3.27-3.36(m,3H,CH);3.55-3.60(m,6H,CH2);3.85-4.00(m,4H,CH);4.31(d,2H,CH2);4.37(d,1H,OH);4.50 -4.60(m,3H,CH);5.16(s,2H,CH2);5.35-6.02(d,2H,CH);6.33(s,2H,NH2);7.58(m,1H,CH);7.67-7.88(d,4H,C H);7.96(s,1H,CH);8.14(s,1H,NH);8.41(d,2H,CH);8.60(s,2H,CH);9.96-10.86(s,3H,NH);12.80(s,1H,OH);

[0053] (2) Preparation of BCAB NPs

[0054] Weigh 250 mg of the BCAB freeze-dried powder prepared in step (1) into a round-bottom flask, add 10 mL of distilled water to fully dissolve, and obtain a 25 mg / mL conjugate aqueous solution. Ultrasonic treatment was performed for 15 min under ice bath conditions, and the ultrasonic power was set to 400 W. Then, the above reaction solution was filtered through a 0.45 μm mixed cellulose filter membrane, and the filtrate was vacuum freeze-dried for 48 hours to obtain BCAB NPs.

[0055] Example 2 Preparation of a multifunctional nanocarrier (BCAB NPs) with anti-drug resistance, targeting and dual response to hypoxia / low pH:

[0056] (1) Synthesis of BCAB

[0057] 630 mg CMCS was dissolved in 35 mL water and stirred thoroughly to obtain a CMCS solution with a concentration of 18 mg / mL, to which 500 mg DMAP and 350 mg DCC were added and stirred continuously. 80 mg BDAZO was weighed and dissolved in 18 mL DMSO to obtain a BDAZO solution with a concentration of 4.4 mg / mL, which was added to the above reaction solution and reacted in the dark at 35 ° C under nitrogen protection for 15 h. After the reaction, the product was placed in a dialysis bag for dialysis under the dialysis conditions of deionized water / DMSO dialysis for 24 h (deionized water / DMSO V / V = 1:3) and pure water dialysis for 48 h. The liquid in the dialysis bag was collected and passed through a water membrane to remove impurities, and CMCS-AZO-BD was obtained by freeze drying.

[0058] 36 mg of biotin was dissolved in 6 ml of NHS to obtain a biotin solution with a concentration of 6 mg / mL, and then 106 mg of DCC was added, and after magnetic stirring, it was added to a solution of 5.4 mg of CMCS-AZO-BD dissolved in 15 mL of deionized water, and the resulting solution was incubated at room temperature under magnetic stirring for 72 hours. The resulting mixture was dialyzed against deionized water for 96 hours, freeze-dried, and collected to obtain the conjugate BCAB.

[0059] IR (KBr tablet)υ / cm -1 :3422.8(NH),2932.6(CH),1681.7(C=O),1688.8(C=N);1681.2(C=C),1246.6(COC),1172.1(CO);

[0061] 1 HNMR (400MHz, DMSO) δ: 1.25-1.58 (m, 6H, CH2); 1.69 (s, 2H, NH2); 2.05 (m, 2H, CH2); 2.92 (m, 1H, CH); 3.10 (m, 2H, C H2);3.27-3.36(m,3H,CH);3.55-3.60(m,6H,CH2);3.85-4.00(m,4H,CH);4.33(d,2H,CH2);4.37(d,1H,OH);4.5 0-4.60(m,3H,CH);5.16(s,2H,CH2);5.35-6.02(d,2H,CH);6.33(s,2H,NH2);7.58(m,1H,CH);7.67-7.88(d,4H, CH);7.96(s,1H,CH);8.14(s,1H,NH);8.42(d,2H,CH);8.62(s,2H,CH);9.96-10.86(s,3H,NH);12.82(s,1H,OH);

[0062] (2) Preparation of BCAB NPs

[0063] Weigh 200 mg of the BCAB freeze-dried powder prepared in step (1) into a round-bottom flask, add 10 mL of distilled water to fully dissolve, and obtain a 20 mg / mL conjugate aqueous solution. Ultrasonic treatment was performed for 20 min under ice bath conditions, and the ultrasonic power was set to 250 W. After filtering the above reaction solution through a 0.45 μm mixed cellulose filter membrane, the filtrate was vacuum freeze-dried for 24 h to obtain BCABNPs.

[0064] Example 3 Preparation of a multifunctional nanocarrier (BCAB / Me-CCNU NPs) carrying Me-CCNU with anti-drug resistance, targeting and dual response to hypoxia / low pH

[0065] (1) Synthesis of BCAB

[0066] Dissolve 450 mg CMCS in 30 mL water and stir thoroughly to obtain a CMCS solution with a concentration of 15 mg / mL, add 300 mg DMAP and 250 mg DCC, and continue stirring. Weigh 50 mg of BDAZO and dissolve it in 15 ml DMSO to obtain a BDAZO solution with a concentration of 3.3 mg / mL, add it to the above reaction solution, and react in the dark at 35 ° C under nitrogen protection for 14 hours. After the reaction is completed, the product is placed in a dialysis bag for dialysis. The dialysis conditions are deionized water / DMSO dialysis for 24 hours (deionized water / DMSO V / V = 1:3) and pure water dialysis for 48 hours. Collect the liquid in the dialysis bag and pass it through a water membrane to remove impurities, and obtain CMCS-AZO-BD by freeze drying.

[0067] 25 mg of biotin was dissolved in 5 mL of NHS to obtain a biotin solution with a concentration of 5 mg / mL, and then 105 mg of DCC was added, and after magnetic stirring, it was added to a solution of 5.3 mg of CMCS-AZO-BD dissolved in 15 mL of deionized water, and the resulting solution was incubated at room temperature under magnetic stirring for 72 hours. The resulting mixture was dialyzed against deionized water for 96 hours, freeze-dried, and collected to obtain the conjugate BCAB.

[0068] IR (KBr tablet)υ / cm -1 :3428.7(NH),2942.8(CH),1679.2(C=O),1692.6(C=N),1678.2(C=C),1235.6(COC),1159.6(CO);

[0070] 1H NMR (400MHz, DMSO) δ: 1.25-1.58 (m, 6H, CH2); 1.66 (s, 2H, NH2); 2.05 (m, 2H, CH2) 2.93 (m, 1H, CH); 3.10 (m, 2H, CH) 2);3.27-3.36(m,3H,CH);3.55-3.60(m,6H,CH2);3.85-4.00(m,4H,CH);4.33(d,2H,CH2);4.38(d,1H,OH);4.50 -4.60(m,3H,CH);5.18(s,2H,CH2);5.35-6.02(d,2H,CH);6.33(s,2H,NH2);7.58(m,1H,CH);7.67-7.88(d,4H,C H);7.96(s,1H,CH);8.16(s,1H,NH);8.43(d,2H,CH);8.64(s,2H,CH);9.96-10.86(s,3H,NH);12.84(s,1H,OH);

[0071] (2) Preparation of BCAB / Me-CCNU NPs

[0072] Weigh 3 mg Me-CCNU and dissolve it in 100 μL anhydrous ethanol to obtain a Me-CCNU ethanol solution with a concentration of 30 mg / mL. Weigh 40 mg BCAB and dissolve it in 1 mL deionized water to obtain a conjugate aqueous solution with a concentration of 40 mg / mL. The above Me-CCNU solution is evenly added to the conjugate aqueous solution with a syringe, and the reaction is continued to stir for 20 minutes. Then the reaction solution is ultrasonically treated for 10 minutes under ice bath conditions, and the ultrasonic power is set to 400 W. Next, the above ultrasonic reaction solution is added to a dialysis bag with a molecular weight cutoff of 3500 Da, and the dialysate is PBS with a pH of 7.4. The dialysis time is 8 hours to remove unencapsulated drugs. Finally, the liquid in the dialysis bag is freeze-dried to obtain BCAB / Me-CCNU NPs.

[0073] Example 4 A multifunctional nanocarrier carrying Me-CCNU with anti-drug resistance, targeting and dual response to hypoxia / low pH

[0074] (1) Synthesis of BCAB

[0075] 900 mg CMCS was dissolved in 45 mL of water and stirred thoroughly to obtain a CMCS solution with a concentration of 20 mg / mL, to which 900 mg DMAP and 500 mg DCC were added and stirred continuously. 100 mg of BDAZO was weighed and dissolved in 20 mL of DMSO to obtain a BDAZO solution with a concentration of 5 mg / mL, which was added to the above reaction solution and reacted in the dark at 35 ° C under nitrogen protection for 17 h. After the reaction was completed, the product was placed in a dialysis bag for dialysis under the dialysis conditions of deionized water / DMSO dialysis for 24 h (deionized water / DMSO V / V=1:3) and pure water dialysis for 48 h. The liquid in the dialysis bag was collected and passed through a water membrane to remove impurities, and CMCS-AZO-BD was obtained by freeze drying.

[0076] 81 mg of biotin was dissolved in 9 mL of NHS to obtain a biotin solution with a concentration of 9 mg / mL, and then 108 mg of DCC was added, and after magnetic stirring, it was added to a solution of 7.41 mg of CMCS-AZO-BD dissolved in 19 mL of deionized water, and the resulting solution was incubated at room temperature under magnetic stirring for 72 hours. The resulting mixture was dialyzed against deionized water for 96 hours, freeze-dried, and collected to obtain the conjugate BCAB.

[0077] IR (KBr tablet)υ / cm -1 :3438.2(NH),2946.7(CH),1686.5(C=O),1698.4(C=N),1671.3(C=C),1242.7(COC),1163.9(CO);

[0079] 1 H NMR (400MHz, DMSO) δ: 1.25-1.58 (m, 6H, CH2); 1.65 (s, 2H, NH2); 2.06 (m, 2H, CH2); 2.94 (m, 1H, CH); 3.11 (m, 2H, CH 2);3.27-3.36(m,3H,CH);3.55-3.60(m,6H,CH2);3.85-4.00(m,4H,CH);4.33(d,2H,CH2);4.38(d,1H,OH);4.50 -4.60(m,3H,CH);5.18(s,2H,CH2);5.35-6.02(d,2H,CH);6.35(s,2H,NH2);7.58(m,1H,CH);7.67-7.88(d,4H,C H);7.96(s,1H,CH);8.16(s,1H,NH);8.47(d,2H,CH);8.65(s,2H,CH);9.96-10.86(s,3H,NH);12.85(s,1H,OH);

[0080] (2) Preparation of BCAB / Me-CCNU NPs

[0081] Weigh 2 mg Me-CCNU and dissolve it in 100 μL DMSO to obtain a Me-CCNU DMSO solution with a concentration of 20 mg / mL. Weigh 30 mg BCAB and dissolve it in 1 mL deionized water to obtain a conjugate aqueous solution with a concentration of 30 mg / mL. The above Me-CCNU solution is evenly added to the conjugate aqueous solution with a syringe, and the reaction is continued to stir for 15 minutes. Then the reaction solution is ultrasonically treated for 5 minutes under ice bath conditions, and the ultrasonic power is set to 350 W. Next, the above ultrasonic reaction solution is added to a dialysis bag with a molecular weight cutoff of 3000 Da, and the dialysate is PBS with a pH of 7.4. The dialysis time is 8 hours to remove unencapsulated drugs. Finally, the liquid in the dialysis bag is freeze-dried to obtain BCAB / Me-CCNU NPs.

[0082] Example 5 Preparation of a multifunctional nanocarrier (BCAB / FTMS NPs) carrying FTMS with anti-drug resistance, targeting and dual response to hypoxia / low pH

[0083] (1) Synthesis of BCAB

[0084] Dissolve 760 mg CMCS in 40 mL water and stir thoroughly to obtain a CMCS solution with a concentration of 19 mg / mL, add 700 mg DMAP and 400 mg DCC, and continue stirring. Weigh 90 mg BDAZO and dissolve it in 19 mL DMSO to obtain a BDAZO solution with a concentration of 4.7 mg / mL, add it to the above reaction solution, and react in the dark at 35 ° C under nitrogen protection for 16 hours. After the reaction is completed, the product is placed in a dialysis bag for dialysis. The dialysis conditions are deionized water / DMSO dialysis for 24 hours (deionized water / DMSO V / V = 1:3) and pure water dialysis for 48 hours. Collect the liquid in the dialysis bag and pass it through a water membrane to remove impurities, and obtain CMCS-AZO-BD by freeze drying.

[0085] 64 mg of biotin was dissolved in 8 mL of NHS to obtain a biotin solution with a concentration of 8 mg / mL, and then 107 mg of DCC was added, and after magnetic stirring, it was added to a solution of 6.84 mg of CMCS-AZO-BD dissolved in 18 mL of deionized water, and the resulting solution was incubated at room temperature under magnetic stirring for 72 hours. The resulting mixture was dialyzed against deionized water for 96 hours, freeze-dried, and collected to obtain the conjugate BCAB.

[0086] IR (KBr tablet)υ / cm -1:3439.3(NH),2941.6(CH),1687.2(C=O),1699.8(C=N),1676.2(C=C),1241.9(COC),1166.1(CO);

[0088] 1 H NMR (400MHz, DMSO) δ: 1.25-1.58 (m, 6H, CH2); 1.66 (s, 2H, NH2); 2.08 (m, 2H, CH2); 2.94 (m, 1H, CH); 3.11 (m, 2H, CH 2);3.27-3.36(m,3H,CH);3.55-3.60(m,6H,CH2);3.85-4.00(m,4H,CH);4.33(d,2H,CH2);4.38(d,1H,OH);4.50 -4.60(m,3H,CH);5.18(s,2H,CH2);5.35-6.02(d,2H,CH);6.36(s,2H,NH2);7.59(m,1H,CH);7.67-7.88(d,4H,C H);7.98(s,1H,CH);8.18(s,1H,NH);8.48(d,2H,CH);8.66(s,2H,CH);9.96-10.86(s,3H,NH);12.86(s,1H,OH);

[0089] (2) Preparation of BCAB / FTMS NPs

[0090] Weigh 6 mg FTMS and dissolve it in 100 μL DMSO to obtain a 60 mg / mL FTMSDMSO solution. Weigh 70 mg BCAB and dissolve it in 1 mL deionized water to obtain a 70 mg / mL conjugate aqueous solution. Add the above FTMS solution evenly to the conjugate aqueous solution with a syringe and continue to stir and react for 40 minutes. Then, ultrasonically treat the reaction solution for 30 minutes under ice bath conditions, and the ultrasonic power is set to 550 W. Next, add the above ultrasonic reaction solution to a dialysis bag with a molecular weight cutoff of 5000 Da, a dialysate of PBS with a pH of 7.4, and a dialysis time of 11 hours to remove unencapsulated drugs. Finally, freeze-dry the liquid in the dialysis bag to obtain BCAB / FTMS NPs.

[0091] Example 6 Preparation of a multifunctional nanocarrier (BCAB / FTMS NPs) carrying FTMS with anti-drug resistance, targeting and dual response to hypoxia / low pH

[0092] (1) Synthesis of BCAB

[0093] 1380 mg CMCS was dissolved in 60 mL water and stirred thoroughly to obtain a CMCS solution with a concentration of 23 mg / mL, to which 1100 mg DMAP and 800 mg DCC were added and stirred continuously. 400 mg BDAZO was weighed and dissolved in 35 mL DMSO to obtain a BDAZO solution with a concentration of 11.4 mg / mL, which was added to the above reaction solution and reacted in the dark at 35 ° C under nitrogen protection for 20 h. After the reaction was completed, the product was placed in a dialysis bag for dialysis under the dialysis conditions of deionized water / DMSO dialysis for 24 h (deionized water / DMSO V / V=1:3) and pure water dialysis for 48 h. The liquid in the dialysis bag was collected and passed through a water membrane to remove impurities, and CMCS-AZO-BD was obtained by freeze drying.

[0094] 130 mg of biotin was dissolved in 13 mL of NHS to obtain a biotin solution with a concentration of 10 mg / mL, and then 111 mg of DCC was added, and after magnetic stirring, it was added to a solution of 10 mg of CMCS-AZO-BD dissolved in 23 mL of deionized water, and the resulting solution was incubated at room temperature under magnetic stirring for 72 hours. The resulting mixture was dialyzed against deionized water for 96 hours, freeze-dried, and collected to obtain the conjugate BCAB.

[0095] IR (KBr tablet)υ / cm -1 :3442.3(NH),2943.7(CH),1689.3(C=O),1692.6(C=N),1678.5(C=C),1249.2(COC),1168.8(CO);

[0097] 1H NMR (400MHz, DMSO) δ: 1.25-1.58 (m, 6H, CH2); 1.67 (s, 2H, NH2); 2.09 (m, 2H, CH2); 2.95 (m, 1H, CH); 3.12 (m, 2H, CH 2);3.27-3.36(m,3H,CH);3.55-3.60(m,6H,CH2);3.85-4.00(m,4H,CH);4.34(d,2H,CH2);4.38(d,1H,OH);4.50 -4.60(m,3H,CH);5.19(s,2H,CH2);5.35-6.02(d,2H,CH);6.36(s,2H,NH2);7.59(m,1H,CH);7.67-7.88(d,4H,C H);7.98(s,1H,CH);8.18(s,1H,NH);8.49(d,2H,CH);8.67(s,2H,CH);9.96-10.86(s,3H,NH);12.87(s,1H,OH);

[0098] (2) Preparation of BCAB / FTMS NPs

[0099] Weigh 7 mg FTMS and dissolve it in 100 μL DMSO to obtain a 70 mg / mL FTMSDMSO solution. Weigh 80 mg BCAB and dissolve it in 1 mL deionized water to obtain a 80 mg / mL conjugate aqueous solution. Add the above FTMS solution evenly to the conjugate aqueous solution with a syringe and continue to stir and react for 45 minutes. Then, ultrasonically treat the reaction solution for 35 minutes under ice bath conditions, and the ultrasonic power is set to 600 W. Next, add the above ultrasonic reaction solution to a dialysis bag with a molecular weight cutoff of 5500 Da, a dialysate of PBS with a pH of 7.4, and a dialysis time of 12 hours to remove unencapsulated drugs. Finally, freeze-dry the liquid in the dialysis bag to obtain BCAB / FTMS NPs.

[0100] Example 7 Preparation of a multifunctional nanocarrier (BCAB / DITC NPs) carrying DITC with anti-drug resistance, targeting and dual response to hypoxia / low pH

[0101] (1) Synthesis of BCAB

[0102] 1210 mg CMCS was dissolved in 55 mL water and stirred thoroughly to obtain a CMCS solution with a concentration of 22 mg / mL, to which 850 mg DMAP and 700 mg DCC were added and stirred continuously. 300 mg BDAZO was weighed and dissolved in 30 mL DMSO to obtain a BDAZO solution with a concentration of 10 mg / mL, which was added to the above reaction solution and reacted in the dark at 35 ° C under nitrogen protection for 19 h. After the reaction was completed, the product was placed in a dialysis bag for dialysis under the dialysis conditions of deionized water / DMSO dialysis for 24 h (deionized water / DMSO V / V = 1:3) and pure water dialysis for 48 h. The liquid in the dialysis bag was collected and passed through a water membrane to remove impurities, and CMCS-AZO-BD was obtained by freeze drying.

[0103] 110 mg of biotin was dissolved in 12 mL of NHS to obtain a biotin solution with a concentration of 9.2 mg / mL, and then 110 mg of DCC was added, and after magnetic stirring, it was added to a solution of 9 mg of CMCS-AZO-BD dissolved in 22 mL of deionized water, and the resulting solution was incubated at room temperature under magnetic stirring for 72 hours. The resulting mixture was dialyzed against deionized water for 96 hours, freeze-dried, and collected to obtain the conjugate BCAB.

[0104] IR (KBr tablet)υ / cm -1 :3434.6(NH),2948.2(CH),1682.9(C=O),1691.3(C=N),1672.1(C=C),1243.5(COC),1163.4(CO);

[0106] 1 H NMR (400MHz, DMSO) δ: 1.25-1.58 (m, 6H, CH2); 1.68 (s, 2H, NH2); 2.09 (m, 2H, CH2); 2.96 (m, 1H, CH); 3.13 (m, 2H, CH 2);3.27-3.36(m,3H,CH);3.55-3.60(m,6H,CH2);3.85-4.00(m,4H,CH);4.35(d,2H,CH2);4.39(d,1H,OH);4.50 -4.60(m,3H,CH);5.19(s,2H,CH2);5.35-6.02(d,2H,CH);6.36(s,2H,NH2);7.59(m,1H,CH);7.67-7.88(d,4H,C H);7.98(s,1H,CH);8.19(s,1H,NH);8.48(d,2H,CH);8.68(s,2H,CH);9.96-10.86(s,3H,NH);12.88(s,1H,OH);

[0107] (2) Preparation of BCAB / DITC NPs

[0108] Weigh 4 mg DITC and dissolve it in 100 μL anhydrous ethanol to obtain a DITC ethanol solution with a concentration of 40 mg / mL. Weigh 50 mg BCAB and dissolve it in 1 mL deionized water to obtain a conjugate aqueous solution with a concentration of 50 mg / mL. Add the above DITC solution evenly to the conjugate aqueous solution with a syringe and continue to stir and react for 30 minutes. Then, ultrasonically treat the reaction solution for 20 minutes under ice bath conditions, and the ultrasonic power is set to 450 W. Next, add the above ultrasonic reaction solution to a dialysis bag with a molecular weight cutoff of 4000 Da, a dialysate of PBS with a pH of 7.4, and a dialysis time of 9 hours to remove unencapsulated drugs. Finally, freeze-dry the liquid in the dialysis bag to obtain BCAB / DITC NPs.

[0109] Example 8 Preparation of a multifunctional nanocarrier (BCAB / DITC NPs) carrying DITC with anti-drug resistance, targeting and dual response to hypoxia / low pH

[0110] (1) Synthesis of BCAB

[0111] Dissolve 1050 mg CMCS in 50 mL water and stir thoroughly to obtain a CMCS solution with a concentration of 21 mg / mL, add 1000 mg DMAP and 600 mg DCC, and continue stirring. Weigh 200 mg of BDAZO and dissolve it in 25 mL DMSO to obtain a BDAZO solution with a concentration of 8 mg / mL, add it to the above reaction solution, and react in the dark at 35 ° C under nitrogen protection for 18 hours. After the reaction is completed, the product is placed in a dialysis bag for dialysis. The dialysis conditions are deionized water / DMSO dialysis for 24 hours (deionized water / DMSO V / V = 1:3) and pure water dialysis for 48 hours. Collect the liquid in the dialysis bag and pass it through a water membrane to remove impurities, and obtain CMCS-AZO-BD by freeze drying.

[0112] 100 mg of biotin was dissolved in 10 mL of NHS to obtain a biotin solution with a concentration of 10 mg / mL, and then 109 mg of DCC was added, and after magnetic stirring, it was added to a solution of 8 mg of CMCS-AZO-BD dissolved in 20 mL of deionized water, and the resulting solution was incubated at room temperature under magnetic stirring for 72 hours. The resulting mixture was dialyzed against deionized water for 96 hours, freeze-dried, and collected to obtain the conjugate BCAB.

[0113] IR (KBr tablet)υ / cm -1:3438.9(NH),2942.2(CH),1676.5(C=O),1698.4(C=N),1665.5(C=C),1241.7(COC),1159.2(CO);

[0115] 1 H NMR (400MHz, DMSO) δ: 1.25-1.58 (m, 6H, CH2); 1.69 (s, 2H, NH2); 2.08 (m, 2H, CH2); 2.97 (m, 1H, CH); 3.15 (m, 2H, CH 2);3.27-3.36(m,3H,CH);3.55-3.60(m,6H,CH2);3.85-4.00(m,4H,CH);4.37(d,2H,CH2);4.39(d,1H,OH);4.50 -4.60(m,3H,CH);5.19(s,2H,CH2);5.35-6.02(d,2H,CH);6.39(s,2H,NH2);7.57(m,1H,CH);7.67-7.88(d,4H,C H);7.99(s,1H,CH);8.17(s,1H,NH);8.49(d,2H,CH);8.69(s,2H,CH);9.96-10.86(s,3H,NH);12.89(s,1H,OH);

[0116] (2) Preparation of BCAB / DITC NPs

[0117] Weigh 5 mg DITC and dissolve it in 100 μL anhydrous ethanol to obtain a DITC ethanol solution with a concentration of 50 mg / mL. Weigh 60 mg BCAB and dissolve it in 1 mL deionized water to obtain a conjugate aqueous solution with a concentration of 60 mg / mL. The above DITC solution is evenly added to the conjugate aqueous solution with a syringe, and the stirring reaction is continued for 35 minutes. Then the reaction solution is ultrasonically treated for 25 minutes under ice bath conditions, and the ultrasonic power is set to 500 W. Next, the above ultrasonic reaction solution is added to a dialysis bag with a molecular weight cutoff of 4500 Da, and the dialysate is PBS with a pH of 7.4. The dialysis time is 10 hours to remove unencapsulated drugs. Finally, the liquid in the dialysis bag is freeze-dried to obtain BCAB / DITC NPs.

[0118] Experimental Example 1: Testing of BCAB NPs encapsulation efficiency and drug loading

[0119] 1. Experimental Materials

[0120] The reaction solution of each drug-loaded functional nanocarrier before dialysis was placed in an ultrafiltration tube (3KD) and centrifuged at 1000 rpm for 10 min. The filtrate was collected and an appropriate amount of methanol was dripped into the reaction solution using an organic solvent destruction method. The solution was filtered through a 0.22 μm water membrane and the drug content was determined by high performance liquid chromatography (HPLC) to calculate the encapsulation efficiency and drug loading.

[0121]

[0122] 2. Experimental results: see Table 1.

[0123] Table 1 Encapsulation efficiency and drug loading of various drug-loaded functional nanocarriers

[0124]

[0125] From the data in Table 1, it can be seen that the prepared multifunctional nanocarrier with anti-drug resistance, targeting and dual responsiveness to hypoxia / low pH has an encapsulation rate of more than 75% for each hydrophobic drug and a drug loading capacity of more than 8%, indicating that the carrier can efficiently encapsulate various hydrophobic drugs and has a high drug loading capacity.

[0126] Experimental Example 2: Stability test of functional nanocarriers loaded with drugs

[0127] Take an appropriate amount of the freeze-dried powder of the drug-loaded functional nanocarrier prepared in Examples 3-8, dissolve it in distilled water, PBS, or DMEM medium containing 10% serum (FBS), and measure the particle size changes of the three drug-loaded functional nanocarriers at 0, 12, 24, 48, 72, and 96 hours. The results are as follows: Figure 6 As shown. Figure 6 It can be seen that the average particle size of the three drug-loaded functional nanoparticles in each solvent has no obvious change, indicating that the drug-loaded nanoparticles have good stability.

[0128] Experimental Example 3: Hypoxia reduction sensitivity test of functional nanocarriers loaded with drugs

[0129] The Na2S2O4 method was used to simulate hypoxic reducing conditions. 6 mg of the drug-loaded functional nanocarrier freeze-dried powder prepared in Example 3-8 was weighed into a centrifuge tube and dissolved in 3 mL of distilled water. 2 mL of 2 μM Na2S2O4 solution was added to the system. The EP tube was sealed and incubated in a 37°C water bath for 30 min. The average particle size change was detected using a Malvern particle size analyzer. The results are as follows: Figure 7After adding Na2S2O4, the average particle size of each drug-loaded functional nanocarrier changed significantly, and the average particle size distribution was wide, indicating that the azobenzene in each nanodrug carrier was reduced under hypoxic conditions, resulting in the cracking of the nanodrug carrier and the fragmentation of the particles; while under normoxic conditions, the average particle size of the three drug-loaded functional nanocarriers did not change significantly, indicating that azobenzene would not be reduced under normoxic conditions, each nanodrug carrier could not be cracked, the particles remained stable, and had good hypoxic selectivity.

[0130] Experimental Example 4: pH responsiveness test of functional nanocarriers loaded with drugs

[0131] 10 mg of the drug-loaded functional nanocarrier freeze-dried powder prepared in Example 3-8 was weighed into a centrifuge tube, dissolved in 10 mL of PBS, and 0.1 mL of 5 mol / mL hydrochloric acid solution was added. The mixture was shaken and incubated in a 37°C water bath for 10 min. The average particle size change was detected using a Malvern particle size analyzer. The results are as follows: Figure 8 As shown. In the reaction system of low pH simulating the acidic microenvironment of tumors, the average particle size of each nanodrug changed significantly. Compared with the neutral pH group, the average particle size distribution of the nanodrug in the low pH group became wider, and the particle size distribution in the solution was uneven. Under neutral pH conditions, the average particle size distribution of the nanodrug was more concentrated, indicating that the nanodrug has good low pH responsiveness.

[0132] Experimental Example 5: Drug release behavior test in various drug-loaded functional nanocarriers

[0133] The drug-loaded functional nanocarriers prepared in Examples 3-8 and free chemotherapeutic drugs (Me-CCNU, FTMS and DITC) were placed in regenerated fiber dialysis bags (dialysis bags with a molecular weight cutoff of 1000-2000Da), and the dialysis bags were placed in a PBS working solution containing rat liver microsomes and nicotinamide adenine dinucleotide phosphate (NADPH). Nitrogen was filled into the system and 0.1 mL of 5 mol / mL hydrochloric acid was added to simulate the hypoxic and low pH tumor microenvironments, respectively. Each group of samples was dialyzed under the above conditions. 0.5 mL of PBS outside the dialysis bag was taken at 10, 20, 40, 60, 90, 120, 150, 180, and 240 min, and the contents of Me-CCNU, FTMS and DITC in the solution were determined by high performance liquid chromatography to obtain the cumulative release of the three drugs. Under neutral pH conditions, the cumulative release of each free drug and functional nanocarrier in the normoxic and hypoxic systems is as follows: Fig. 9 and 10 As shown, the three drug-loaded functional nanocarrier groups showed different drug release abilities under normoxic and hypoxic conditions: under normoxic conditions (e.g. Fig. 9), the cumulative drug release of each group was less than 20%, indicating that under normoxic conditions, the nanoparticles remained intact and did not break, so the encapsulated drugs could not be released; while under hypoxic conditions (such as Fig.10 ), the cumulative drug release of each nanogroup increased significantly, and reached more than 40% at 60 minutes of treatment. This was because the hypoxic conditions triggered the reduction of the azobenzene groups in the BCAB carrier, which in turn caused the nanocarrier to disintegrate, allowing the drug in the nanocore to be effectively released.

[0134] Under low pH conditions, the cumulative release of free drugs and functional nanocarriers in normoxic and hypoxic systems is shown in Figure 2. Fig.11 and 12 As shown in the figure, the three free drug groups released drugs rapidly under normoxic and hypoxic conditions, and the final cumulative drug release was higher than 90%. The three drug-loaded functional nanocarrier groups showed different drug release abilities under normoxic and hypoxic conditions, and showed obvious differences from the neutral pH nanocarrier group: under normoxic conditions (such as Fig.11 ), the cumulative drug release of each nano group was significantly higher than that of the nanocarrier group at neutral pH, which indicates that the low pH environment can promote the release of drugs from the nano core; moreover, under low oxygen and low pH conditions (such as Fig.12 ), the cumulative drug release of each nano-drug group reached the highest level, which was not only higher than the normoxic group at neutral pH, but also higher than the hypoxic group at neutral pH and the normoxic group at low pH. This indicates that under the synergistic effect of hypoxia and low pH, while the azo bonds on the nanocarriers were reduced, chitosan was also protonated, resulting in the expansion of the polymer network and the fragmentation of the shell, which promoted the full release of the drugs in the nano-core. The experimental results show that BCAB NPs can maintain good stability in normal tissues, which is conducive to prolonging the blood circulation time of drugs and preventing premature drug release; and in the tumor microenvironment with hypoxia and low pH, BCAB NPs can quickly disintegrate and efficiently release drugs, thereby exerting a good targeted anti-tumor effect.

[0135] Experimental Example 6: Cytotoxicity test of functional nanocarriers loaded with drugs

[0136] 1. Experimental Materials

[0137] Test compounds: free Me-CCNU, FTMS, DITC, BCAB / Me-CCNU NPs, BCAB / FTMS NPs and BCAB / DITC NPs prepared in Examples 3-8.

[0138] Cell lines: human glioma SF763, human brain glioma cells T98G, human liver cancer HepG2 cells, human breast cancer MCF-7 cells, and human colorectal adenocarcinoma HT29 cells.

[0139] 2. Experimental methods

[0140] All five types of cells were seeded in 96-well plates at 1000 cells / well. After culturing at 37°C and 5% CO2 for 24 hours, they were treated with functional nanocarriers and free drugs loaded with drugs at concentrations of 20μM, 50μM, 100μM, 200μM, 500μM, 800μM, 1000μM and 2000μM, with 6 replicates in each group, and a control group was set up. Normoxia / neutral pH, hypoxia / neutral pH, normoxia / low pH and hypoxia / low pH groups were set up; nitrogen was filled into the system and hydrochloric acid was added to simulate the tumor microenvironment expressed by hypoxia / low pH. Then 10μL of CCK-8 solution was added to each well for 4 hours. Finally, the absorbance value was detected at a wavelength of 450nm, and the cell survival rate was calculated according to the following formula, and the half-inhibition rate IC of the drug was calculated 50 .

[0141]

[0142] A 药物处理组 is the absorbance value of the sample group with culture medium, tumor cells, drug solution, and CCK-8 solution;

[0143] A 空白组 is the absorbance value of the sample group with culture medium and CCK-8 solution but without tumor cells and drugs;

[0144] A 对照组 The absorbance values ​​of the sample group containing culture medium, tumor cells, and CCK-8 solution but no drug solution.

[0145] 3. Experimental results: see Table 2

[0146] Table 2 The half inhibitory concentration (IC) of drugs on tumor cells under normoxic and hypoxic conditions 50 ,μM)

[0147]

[0148] From the data in Table 2, it can be seen that under normoxic / neutral pH conditions, the IC 50 The values ​​are significantly higher than those of free chemotherapy drugs. This is because the nanocarrier BCAB NPs effectively encapsulates the chemotherapy drugs and cannot be cleaved under normoxic / neutral pH conditions, so the drugs cannot be released and therefore cannot inhibit tumor cells. This shows that BCAB NPs remain stable in normal tissues, cannot break up, and cannot release the drugs in the nanocore, thus effectively reducing the toxic side effects of drugs on normal tissues.

[0149] IC of each chemotherapeutic drug encapsulated by each functional nanocarrier under hypoxia / neutral pH 50 The value is significantly lower than that of free chemotherapy drugs. This indicates that BCAB NPs have good hypoxia responsiveness, and the azobenzene in them can be effectively reduced under hypoxic conditions, thereby fragmenting the nanocarriers and releasing the drugs and AGT inhibitor BD in the nanocore.

[0150] IC of each chemotherapeutic drug encapsulated by functional nanocarriers under normoxic / low pH conditions 50 IC value of free chemotherapeutic drugs 50 The value decreased. This indicates that the synthesized BCAB NPs have good pH responsiveness. In a low pH environment, the nanocarrier polymer network expands, the shell permeability increases, and the encapsulated drug is released. However, since the azo bond failed to break, the AGT inhibitor BD was not released, so the tumor cells still showed a certain resistance to anticancer drugs.

[0151] Under low oxygen and low pH conditions, the IC 50 The value is significantly lower than that of free chemotherapy drugs and nanogroups under various other conditions. The reason for this result is that low pH conditions cause chitosan to be protonated, and the low oxygen environment causes the azo bond to break, which in turn increases the permeability of the shell and further fragments it. This allows the encapsulated anti-tumor drug to be fully released specifically under low oxygen and low pH conditions, and releases the AGT inhibitor BD, thereby exerting a synergistic effect of targeted anti-cancer and anti-resistance.

[0152] The above results show that the functional nanocarrier synthesized by the present invention can exert good anti-drug resistance and dual responsiveness to hypoxia / low pH. In the tumor microenvironment of hypoxia and low pH, the hypoxia targeting group azobenzene on the carrier can be specifically reduced, and the permeability of chitosan is enhanced in a low pH environment, which promotes the fragmentation of the nanocarrier and releases the anticancer drugs encapsulated in the core and the AGT inhibitor in the shell structure. While the present invention targets the delivery of anti-tumor drugs, it also exerts an anti-drug resistance effect, improves the bioavailability of chemotherapeutic drugs, and reduces the systemic toxicity of free drugs. In addition, the functional nanocarrier BCAB NPs encapsulates anticancer drugs, significantly improving the stability of the drugs and the blood circulation time, which is conducive to the drugs to better exert their anti-tumor effects.

[0153] Finally, the embodiments of the present invention are only relatively simple implementation schemes and are not used to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A multifunctional nanocarrier with anti-drug resistance, biotin receptor targeting and dual responsiveness to hypoxia / low pH, characterized in that The structural formula is as follows:

2. A method for preparing a "multifunctional nanocarrier having both anti-drug resistance, biotin receptor targeting and dual responsiveness to hypoxia / low pH", characterized in that The following steps are involved: 1) Synthesis of the biotin receptor-targeted and hypoxia / low pH dual-responsive AGT inhibitor conjugate Bio-CMCS-AZO-BD, i.e. BCAB: The structural formula of BCAB is: CMCS was dissolved in water and stirred thoroughly to obtain a CMCS solution with a concentration of 1-50 mg / mL, dimethylaminopyridine (DMAP) and N,N′-dicyclohexylcarbodiimide (DCC) were added thereto, wherein the mass ratio of CMCS, DMAP and DCC was 1:(0.1-2):(0.1-2), and stirring was continued to obtain a reaction solution; 2-amino-6-((3-((4-aminophenyl)diazenyl)benzyl)oxy)-9H-pteridine (BDAZO) was weighed and dissolved in dimethyl DMSO (dimethyl sulfoxide) is added to obtain a BDAZO solution with a concentration of 1-20 mg / mL, and the solution is added to the above reaction solution, and the reaction time is 10-36 h in the dark at 25-55°C under nitrogen protection; after the reaction, the product is placed in a dialysis bag for dialysis, and the dialysis conditions are deionized water / DMSO volume ratio of 1:3 to 1:1, the dialysis is performed for 24-96 h, and then the dialysis is performed with pure water for 24-96 h; the liquid in the dialysis bag is collected and passed through a water membrane to remove impurities, and CMCS-AZO-BD is obtained by freeze drying; Dissolve biotin in N-hydroxysuccinimide (NHS) to obtain a biotin solution with a concentration of 1-30 mg / mL, then add DCC, wherein the mass ratio of biotin to DCC is 1:(0.1-10), add to the CMCS-AZO-BD aqueous solution after magnetic stirring, the mass ratio of biotin solution to CMCS-AZO-BD is 1:(0.05-2), and incubate the obtained solution under magnetic stirring at room temperature for 48-98 hours; dialyze the obtained mixture with deionized water for 24-98 hours, collect it after freeze drying, and obtain the conjugate BCAB; 2) Preparation of a multifunctional nanocarrier BCAB NPs with anti-drug resistance, biotin receptor targeting and dual responsiveness to hypoxia / low pH: The lyophilized powder of the conjugate BCAB was weighed and dissolved in distilled water to obtain a conjugate aqueous solution with a concentration of 10-150 mg / mL, and ultrasonic treatment was performed in an ice bath for 1-50 min, and the ultrasonic power was set to 100-800 W; then, the above reaction solution was filtered through a mixed cellulose filter membrane and vacuum freeze-dried for 12-48 h to obtain the nanocarrier BCAB NPs.

3. The method according to claim 2, characterized in that: In step 1), the concentration of CMCS solution is 1-25 mg / mL, the mass ratio of CMCS, DMAP and DCC is 1:(0.5-1):(0.5-1), the concentration of BDAZO solution is 2-15 mg / mL, the reaction temperature is 25-45°C, the reaction time is 10-24 h, the dialysis conditions are deionized water / DMSO dialysis for 24-72 h (deionized water / DMSO V / V=1:3), and pure water dialysis for 24-72 h; the concentration of biotin solution is 1-20 mg / mL, the mass ratio of biotin and DCC is 1:(0.5-8), the mass ratio of biotin solution and CMCS-AZO-BD is 1:(0.05-1), the reaction time is 48-96 h, and the deionized water dialysis is 24-96 h.

4. The method according to claim 2, characterized in that: In step 2), the concentration of the BCAB aqueous solution is 10-100 mg / mL; the ultrasonic treatment time is 5-40 min; the ultrasonic power is 100-600 W; and the vacuum drying time is 18-48 h.

5. A method for preparing a multifunctional anti-tumor nanodrug BCAB / Drug NPs having anti-drug resistance, biotin receptor targeting and dual response to hypoxia / low pH, characterized in that: The preparation of the chemotherapeutic drug is as follows: A hydrophobic chemotherapy drug is dissolved in an organic solvent to obtain a drug solution with a concentration of 1-120 mg / mL, a conjugate BCAB lyophilized powder or a nanocarrier BCAB NPs is dissolved in distilled water to obtain a conjugate solution with a concentration of 1-150 mg / mL, the drug solution is evenly added to the conjugate solution with a syringe, and the reaction is continued to be stirred for 1-60 minutes; then the reaction solution is ultrasonically treated for 1-60 minutes under ice bath conditions, and the ultrasonic power is set to 100-800W; next, the above-mentioned ultrasonic reaction solution is added to a dialysis bag, the dialysis bag has a molecular weight cutoff of 1000-10000Da, and the dialysate is a phosphate buffered saline (PBS) with a pH of 7.4, and dialyzed for 1-18 hours to remove unencapsulated drugs; finally, the liquid in the dialysis bag is freeze-dried to obtain a multifunctional anti-tumor nanodrug BCAB / Drug NPs with anti-drug resistance and hypoxia / low pH dual response that encapsulates chemotherapy drugs.

6. The method according to claim 5, characterized in that: The hydrophobic chemotherapy drug is a DNA alkylating agent, or a chloroethylnitrosourea anticancer alkylating agent and dacarbazine DITC; the organic solvent is one or more of DMSO, ethanol, methanol, and DMAC, and is DMAC or DMSO; the concentration of the drug solution is 1-100 mg / mL; the concentration of the BCAB aqueous solution is 1-120 mg / mL; the reaction time of the drug solution and the conjugate solution is 1-50 min; the ultrasonic treatment time is 1-40 min, the dialysis bag molecular weight cutoff is 1000-6000 Da, the ultrasonic power is 100-600 W; and the dialysis time is 1-15 h.

7. The method according to claim 5, characterized in that: The tumor is colorectal cancer, lymphoma, brain tumor, myeloma, liver cancer, glioma, prostate cancer, leukemia, brain glioma, lymphoma, malignant melanoma or breast cancer.

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