Amphiphilic block polymer with hypoxic biological reduction responsiveness as well as preparation method and application of amphiphilic block polymer

Abstract

The invention belongs to the technical field of macromolecular anti-tumor drug delivery carriers, and particularly relates to a hypoxic biological reduction responsive amphiphilic block polymer anti-tumor drug carrier as well as a preparation method and application thereof. The hypoxia responsive block copolymer (POC7A-PCL) of which the hydrophobic segment is PCL and the hydrophilic segment is POC7A is constructed through ring opening polymerization and atom transfer radical polymerization. The POC7A-PCL is used as a polymer, the polymer is used for loading an anti-tumor drug doxorubicin to construct responsive polymer nanoparticles, and the polymer nanoparticles show stronger anti-tumor capability, so that the POC7A-PCL can be used as a drug carrier to be applied to preparation of anti-tumor drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of polymer anti-tumor drug delivery carriers, and specifically relates to hypoxia bioreduction responsive amphiphilic block polymer anti-tumor drug carriers and a preparation method and application thereof. Background Art

[0002] The ultimate goal of anticancer drug delivery systems is to deliver drugs into solid tumors to achieve potent treatment with minimal side effects. Due to the complexity of the physiological environment, a series of physiological barriers, the so-called "CAPIR", must be effectively overcome, including blood circulation, tumor accumulation, tumor penetration, cell internalization, and drug release. It is worth noting that the protein corona formed by the nonspecific adsorption of proteins on the drug delivery system can significantly change its physicochemical properties and fate in the body, thereby inducing a foreign body response effect (FBR). In general, the invisibility of drug delivery systems to cells and proteins is considered to be the key to long-term circulation in the blood and accumulation in tumor tissues.

[0003] To date, a variety of hydrophilic polymer materials have been explored as antifouling shells for drug delivery systems to prolong blood circulation. Typical polymers include polyethylene glycol (PEG), polysaccharides, proteins, and protein analogs. Among them, PEG has been widely used as a "gold standard" strategy against nonspecific protein adsorption. However, although the PEG layer is conducive to long-term blood circulation, it affects cellular internalization. In addition, given the dense structure of solid tumors and extracellular matrix, PEG usually leads to poor tumor penetration, which severely limits the anti-tumor efficacy. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the defects of the drug delivery system using PEG as the hydrophilic shell in the in vivo application, thereby improving the efficacy of anti-tumor drugs.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides an anaerobic bioreduction responsive amphiphilic block polymer, the chemical structure of which is shown in Formula I:

[0007]

[0008] In the formula, n is 28 to 35, and m is 44 to 48.

[0009] Hypoxia is a characteristic of the tumor microenvironment. The hypoxic microenvironment within the tumor is closely associated with drug resistance, tumor metastasis, and poor prognosis. However, hypoxia also provides opportunities for targeted drug delivery. Hypoxia-activated prodrugs, such as the anticancer drugs aquinone (AQ4N) and telapazolidone (TPZ), are characterized by hypoxia-responsive N-oxide molecules. Under hypoxic conditions, the N-oxide group can be reduced to a tertiary amine under the catalysis of cytochrome P450 (CYP450) enzymes, which are highly expressed in hypoxic areas of various solid tumors.

[0010] N-oxide-based polymers (PTAO) can be used to construct efficient drug delivery carriers. In the present invention, we use poly (2-(N-oxide-hexamethyleneimino) ethyl methacrylate) (POC7A) and poly (ε-caprolactone) (PCL) with a cyclic seven-membered ring as the hydrophilic segment and the hydrophobic segment, respectively, to prepare a block copolymer POC7A-block-poly (ε-caprolactam) (POC7A-PCL). The POC7A-PCL of the present invention has efficient bioreduction activity and tumor penetrability, and is responsive to the acidic microenvironment of the tumor (pH value is 6.5-6.9).

[0011] In a second aspect, the present invention provides a method for preparing the hypoxic bioreduction responsive amphiphilic block polymer, comprising the following steps:

[0012] (1) reacting 2-(azepan-1-yl)ethyl methacrylate and m-chloroperbenzoic acid to obtain a zwitterionic monomer N-oxide-2-(azepan-1-yl)ethyl methacrylate, denoted as OC7A-MA;

[0013] (2) PCL-OH was obtained by ring-opening polymerization of ε-caprolactone using N-(tert-butyloxycarbonyl)ethanolamine as initiator;

[0014] (3) reacting PCL-OH with 2-bromoisobutyryl bromide to obtain the macroinitiator PCL-Br;

[0015] (4) Using PCL-Br as a macromolecular initiator, poly(2-(N-oxide-hexamethyleneimino)ethyl methacrylate)-b-polycaprolactone was obtained by atom transfer radical polymerization of OC7A-MA monomer, which was named POC7A-PCL, i.e., the hypoxic bioreduction responsive amphiphilic block polymer.

[0016] Furthermore, in step (1), the specific reaction conditions are: dissolving 2-(azepan-1-yl)ethyl methacrylate in anhydrous dichloromethane, and adding m-chloroperbenzoic acid under ice bath conditions. After reacting at room temperature for 2 to 3 hours, purifying by column chromatography, finally obtaining an off-white solid.

[0017] The mass volume ratio of the 2-(azepan-1-yl)ethyl methacrylate, m-chloroperbenzoic acid and dichloromethane is 3.14-4.71 g: 3.42-5.13 g: 100-150 mL.

[0018] Furthermore, in step (2), the specific reaction conditions are as follows: the initiator N-(tert-butyloxycarbonyl)ethanolamine, the catalyst diphenyl phosphate and the monomer ε-caprolactone are dissolved in anhydrous toluene, degassed by freeze-pump-thaw cycle, and then reacted at 30°C to 40°C for 4 to 6 hours, and the reactants are quenched, precipitated and dried to obtain the PCL-OH.

[0019] The mass volume ratio of the N-(tert-butyloxycarbonyl)ethanolamine, diphenyl phosphate, ε-caprolactone and anhydrous toluene is 0.37-0.74 g: 0.58-1.16 g: 12.10-24.20 g: 30.00-60 mL.

[0020] Furthermore, in step (3), the specific reaction conditions are: dissolving PCL-OH and triethylamine in anhydrous dichloromethane, adding 2-bromoisobutyryl bromide dissolved in dichloromethane dropwise under ice bath conditions, reacting at room temperature for 12 to 16 hours, and after the reaction is completed, removing the solvent using a rotary evaporator, and precipitating with methanol and ether to obtain PCL-Br.

[0021] The mass volume ratio of the PCL-OH, triethylamine, 2-bromoisobutyryl bromide and anhydrous dichloromethane is 11.00-16.5 g: 1.52-2.28 g: 2.30-3.45 g: 70.00-105 mL.

[0022] Furthermore, in step (4), the specific reaction conditions are: PCL-Br, OC7A-MA, CuBr and PMDETA are placed in DMF, degassed by freeze-pump-thaw cycle, and then reacted at 38°C to 42°C for 22 to 24 hours, and the reactants are quenched, dialyzed, and freeze-dried to obtain the POC7A-PCL;

[0023] The mass volume ratio of the PCL-Br, OC7A-MA, CuBr, PMDETA and DMF is 0.550-1.100 g: 0.680-1.360 g: 0.014-0.028 g: 0.017-0.034 g: 4-8 mL.

[0024] In a third aspect, the present invention provides the use of the hypoxic bioreduction responsive amphiphilic block polymer in the preparation of an anti-tumor drug carrier.

[0025] Furthermore, the anti-tumor drug includes doxorubicin.

[0026] In a fourth aspect, the present invention provides an anti-tumor drug, which is formed by self-assembly of the hypoxic bioreduction responsive amphiphilic block polymer and doxorubicin in an aqueous solution.

[0027] Furthermore, the anti-tumor drug also includes pharmaceutically acceptable excipients.

[0028] The present invention has the following beneficial effects:

[0029] The POC7A-PCL block copolymer micelles of the present invention are used as delivery carriers of doxorubicin (DOX). Since POC7A-PCL has efficient bioreduction activity and is responsive to the acidic microenvironment of the tumor (pH 6.5-6.9), the cellular uptake capacity of POC7A-PCL loaded with DOX (POC7A-PCL / DOX) is about 2.7 times higher than that of PEG-PCL loaded with DOX (PEG-PCL / DOX) under hypoxic conditions. Under hypoxic conditions, POC7A-PCL is effectively reduced to poly(tertiary amine) (PTA-PCL) by CYP450 enzymes and further protonated in the acidic microenvironment of the tumor, which enhances cellular uptake and tumor penetration by binding to negatively charged cell membranes. Finally, POC7A-PCL / DOX exhibits stronger anti-tumor ability, which means the great potential of POC7A as an antifouling and efficient drug delivery polymer. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a synthetic route for the amphiphilic block polymer poly(2-(N-oxide-hexamethyleneimino)ethyl methacrylate)-b-polycaprolactone (POC7A-PCL) in the present invention.

[0031] Figure 2 is the zwitterionic monomer OC7A-MA of the present invention. 1 H NMR spectroscopy characterization.

[0032] Figure 3 The polycaprolactone (PCL-OH) of the present invention is 1 H NMR spectroscopy characterization.

[0033] Figure 4 is the macromolecular initiator PCL-Br in the present invention 1 H NMR spectroscopy characterization.

[0034] Figure 5 The poly (2- (N-oxide-hexamethyleneimino) ethyl methacrylate) -b-polycaprolactone (POC7A-PCL) of the present invention is 1 H NMR spectroscopy characterization.

[0035] Figure 6middle:

[0036] a Schematic diagram of the preparation of POC7A and POEGMA hydrogels and their incubation with BSA.

[0037] b Fluorescence images of FITC-BSA absorption by POC7A hydrogel, poly(ethylene glycol methacrylate) (POEGMA) hydrogel, and polypropylene (PP) after 6 and 24 hours of culture. Scale bar: 150 μm.

[0038] c The mean fluorescence intensity (MFI) in the fluorescence images was quantified using ImageJ.

[0039] d Bradford assay of proteins adsorbed on different hydrogels after 24 h of culture.

[0040] Figure 7 middle:

[0041] a is a plot of the ratio of the concentration of polymer PEG-PCL to the intensity of the fluorescence emission at wavelengths of 373 nm and 384 nm using pyrene as a fluorescent probe. The concentration of pyrene in the solution is 8×10 -7 M, the excitation wavelength of the optical test is 334 nm.

[0042] b is a plot of the ratio of the intensity of the polymer POC7A-PCL concentration to the intensity of the fluorescence emission at 373 nm and 384 nm using pyrene as a fluorescent probe. The concentration of pyrene in the solution is 8×10 -7 M, the excitation wavelength of the optical test is 334 nm.

[0043] Figure 8 middle:

[0044] a is the particle size distribution of PEG-PCL / DOX nanoparticles.

[0045] b is the particle size distribution of POC7A-PCL / DOX nanoparticles.

[0046] c is the particle size change of DOX micelles in PBS over time.

[0047] d is the change in particle size of DOX micelles in DMEM over time.

[0048] e is the drug release curve of DOX-loaded micelles at pH 7.4.

[0049] f is the drug release curve of DOX-loaded micelles at pH 5.5.

[0050] Fig. 9 middle:

[0051] a is the fluorescence image of C8161 cells cultured with DOX-loaded micelles at a DOX concentration of 0.5 μg / mL under hypoxic conditions. The scale bar is 100 μm.

[0052] b The mean fluorescence intensity (MFI) in the fluorescence images was quantified using ImageJ.

[0053] c Flow cytometric analysis of C8161 cells treated with micelles containing DOX under hypoxic conditions.

[0054] d is the quantification graph of fluorescence intensity in c.

[0055] Fig.10 middle:

[0056] a is a fluorescence image of C8161 cells cultured with DOX-loaded micelles at a DOX concentration of 0.5 μg / mL under normoxic conditions. The scale bar is 100 μm. The cell nuclei stained with DAPI are shown in blue and DOX is shown in red.

[0057] b The mean fluorescence intensity (MFI) in the fluorescence images was quantified using ImageJ.

[0058] c Flow cytometric analysis of C8161 cells treated with micelles containing DOX under normoxic conditions.

[0059] d is the quantification graph of fluorescence intensity in c.

[0060] Fig.11 middle:

[0061] a shows the transport process of FITC-labeled micelles across the C8161 cell layer on the Transwell membrane.

[0062] b is the flow cytometry detection result after treating C8161 cells in the lower chamber of the transwell culture dish with FITC-labeled micelles under hypoxic conditions.

[0063] c is the quantification graph of fluorescence intensity in b.

[0064] d shows the penetration of FITC-labeled micelles and DOX-loaded micelles in C8161 MTS under hypoxia. The scale bar is 200 μm. The Z-stack tomography interval is 15 μm.

[0065] e is a randomly selected white arrow on PTAO- FITC FITC fluorescence intensity of PCL- and PTAO-PCL / DOX-treated MTs.

[0066] f is a randomly selected white arrow on PTAO- FITCDOX fluorescence intensity of PCL- and PTAO-PCL / DOX-treated MTs.

[0067] Fig.12 middle:

[0068] a is the flow cytometry detection result after treating C8161 cells in the lower chamber of the transwell culture dish with FITC-labeled micelles under normoxic conditions.

[0069] b is the quantification graph of fluorescence intensity in a.

[0070] c shows the penetration of FITC-labeled micelles and DOX-loaded micelles in C8161 MTS under normoxic conditions. The scale bar is 200 μm. The Z-stack tomography interval is 15 μm.

[0071] d is a randomly selected white arrow on the PTAO- FITC FITC fluorescence intensity on PCL- and PTAO-PCL / DOX-treated MTs.

[0072] e is a randomly selected white arrow on PTAO- FITC DOX fluorescence intensity on MTs treated with PCL and PTAO-PCL / DOX.

[0073] Fig.13 middle:

[0074] a Quantitative analysis of the fluorescence intensity of tumors and major organs after intravenous injection in each group.

[0075] b shows the blood clearance kinetics of each group after intravenous injection.

[0076] Fig.14 middle:

[0077] a: Schematic diagram of subcutaneous B16 melanoma treatment.

[0078] b Tumor volume growth curves of different treatment groups, mean ± sd (n = 5).

[0079] c is the average tumor volume of mice in different treatment groups.

[0080] d is the average weight of mice in different treatment groups.

[0081] e is a representative image of tumor tissue stained with H&E to identify tumor necrosis, Ki67 as a tumor cell proliferation marker, and TUNEL as a tumor cell apoptosis marker. Scale bar, 150 μm.

[0082] Fig.15The representative histological features of the main organs. Tissue sections were stained with hematoxylin-eosin and observed under an optical microscope (scale bar, 100 μm). DETAILED DESCRIPTION

[0083] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0084] The commonly used PEGylated drug delivery carriers can increase the blood circulation time of drugs when carrying doxorubicin, but the PEG on the surface of the carrier has the effect of reducing cell endocytosis, which is not conducive to the anti-tumor effect of doxorubicin. Therefore, the nano-micelle shell structure with stronger cell internalization is selected to replace PEG, and the drug delivery carrier can significantly increase the endocytosis of drugs by tumor cells.

[0085] The synthetic method route of the block polymer of the present invention is as follows Figure 1 As shown. First, PCL-OH is obtained by ring-opening polymerization of ε-caprolactone, and then the PCL-Br obtained after acyl bromide is used as a macromolecular initiator to obtain a block copolymer by atom transfer radical polymerization. The technical solution is described as follows:

[0086] like Figure 1 As shown, first we reacted 2-(azepan-1-yl)ethyl methacrylate (C7A-MA) with m-chloroperbenzoic acid to obtain the zwitterionic monomer N-oxide-2-(azepan-1-yl)ethyl methacrylate (OC7A-MA). Then, using N-(tert-butyloxycarbonyl)ethanolamine as an initiator, ε-caprolactone was used to obtain PCL-OH through ring-opening polymerization, and PCL-OH was reacted with 2-bromoisobutyryl bromide to obtain the macroinitiator PCL-Br. Finally, using PCL-Br as a macroinitiator, the atom transfer radical polymerization of OC7A-MA monomer was used to obtain the amphiphilic block polymer poly(2-(N-oxide-hexamethyleneimino)ethyl methacrylate)-b-polycaprolactone, named POC7A-PCL. The reactions of OC7A-MA, PCL-OH, PCL-Br and PCL-POC7A 1 H NMR characterizations are shown in Figure 2 , Figure 3 , Figure 4 and Figure 5A block polymer poly(2-(N-oxide-N,N-dimethylamino)ethyl methacrylate)-b-poly(ε-caprolactone) (PODMA-PCL) was synthesized by a similar method for comparison in in vivo experiments. A control block polymer PEG-PCL was synthesized by ring-opening polymerization.

[0087] By self-assembling the block polymer POC7A-PCL and the anti-tumor drug doxorubicin in aqueous solution, drug-loaded micelle nanoparticles with POC7A as the hydrophilic shell and polycaprolactone and doxorubicin as the hydrophobic core can be obtained.

[0088] Example 1: Preparation of polymer and its nanoparticles

[0089] (1) Atom transfer radical polymerization of N-oxide monomers

[0090] 2-(Azepan-1-yl)ethyl methacrylate (CAS No.: 20602-94-2) was dissolved in anhydrous dichloromethane, and m-chloroperbenzoic acid (CAS No.: 937-14-4) was added under ice bath conditions. After reacting at room temperature for 3 hours, the mixture was purified by column chromatography to finally obtain an off-white solid N-oxide-2-(azepan-1-yl)ethyl methacrylate, recorded as OC7A-MA, wherein the mass volume ratio of 2-(azepan-1-yl)ethyl methacrylate, m-chloroperbenzoic acid and DMF was 3.14 g:3.42 g:100 mL.

[0091] The initiator N-(tert-butyloxycarbonyl)ethanolamine (CAS No.: 26690-80-2), the catalyst diphenyl phosphate (CAS No.: 838-85-7) and the monomer ε-caprolactone (CAS No.: 502-44-3) were dissolved in anhydrous toluene, degassed by freeze-pump-thaw cycle, and then reacted at 38°C for 5 hours, and the reactants were quenched, precipitated, and dried to obtain the PCL-OH. The mass volume ratio of the N-(tert-butyloxycarbonyl)ethanolamine, diphenyl phosphate, ε-caprolactone and anhydrous toluene is 0.37g:0.58g:12.10g:30.00mL.

[0092] PCL-OH and triethylamine (CAS No.: 121-44-8) were dissolved in anhydrous dichloromethane, and 2-bromoisobutyryl bromide (CAS No.: 20769-85-1) dissolved in dichloromethane was added dropwise under ice bath conditions. The reaction was carried out at room temperature for 15 hours. After the reaction was completed, the solvent was removed by rotary evaporator, and PCL-Br was precipitated with methanol and ether. The mass volume ratio of PCL-OH, triethylamine, 2-bromoisobutyryl bromide and anhydrous dichloromethane was 11.00 g: 1.52 g: 2.30 g: 70.00 mL.

[0093] The block copolymer was synthesized by atom transfer radical polymerization (ATRP). PCL-Br (0.550 g, 0.10 mmol) and OC7A-MA (0.680 g, 3.00 mmol) were added to a mixture of CuBr (0.014 g, 0.10 mmol) and PMDETA (CAS No.: 3030-47-5, 0.017 g, 0.10 mmol) in N,N-dimethylformamide (DMF) (4 mL). The mixture was degassed by three freeze-pump-thaw cycles and then placed in a preheated oil bath at 40 °C. After 24 h, the reactants were quenched by exposure to air and dialyzed with methyl sulfoxide (DMSO) (500 mL×3) and deionized water (500 mL×3). The solution was lyophilized to obtain a light yellow solid POC7A-PCL (0.69 g, yield: 56.1%), the structural formula of which is shown in Formula I:

[0094]

[0095] In the formula: n is 30, m is 48.

[0096] PODMA-PCL block copolymer was also synthesized according to a similar method. PCL-Br (0.550 g, 0.10 mmol) and OC7A-MA (0.680 g, 3.00 mmol) were added to a mixture of CuBr (0.014 g, 0.10 mmol) and PMDETA (0.017 g, 0.10 mmol) in N,N-dimethylformamide (DMF) (4 mL). The mixture was degassed by three freeze-pump-thaw cycles and then placed in a preheated oil bath at 40 ° C. After 24 hours, the reactants were quenched by exposure to air and dialyzed with dimethyl sulfoxide (DMSO) (500 mL×3) and deionized water (500 mL×3). The solution was lyophilized to obtain a light yellow solid POC7A-PCL (0.69 g, yield: 56.1%).

[0097] (2) Ring-opening polymerization of block copolymer PEG-PCL

[0098] Under argon, ε-CL (0.50 g, 4.38 mmol) and mPEG5k-OH (CAS: 9004-74-4) (0.50 g, 0.10 mmol) were dissolved in anhydrous toluene (2 mL). Diphenyl phosphate (DPP) (25.19 mg, 0.10 mmol) was injected into 1 mL of toluene to start polymerization. After polymerization at 30 ° C for 4 hours, the reaction solution was poured into 100 mL of cold ether. The precipitate was separated by filtration, washed with ether, and vacuum dried to obtain a white solid PEG-PCL (0.54 g, yield: 54.0%).

[0099] (3) Preparation of FITC-labeled block copolymers

[0100] The specific steps are as follows: POC7A-PCL (0.2 g) was dissolved in a mixture of CH2Cl2 (4 mL) and trifluoroacetic acid TFA (2 mL). After stirring at room temperature for 3 hours, the solvent was removed by rotary evaporation. The residue was redissolved in DMF (4 mL), and FITC (2 mg) and triethanolamine (0.4 mL) were added. The solution was stirred in the dark at room temperature overnight, and then dialyzed with DMSO (500 mL×3) for 24 hours and with deionized water (2 L×3) for 24 hours. POC7A-PCL was further purified by Sephadex column. FITC PCL, remove the residual FITC molecules, and then freeze-dry to obtain an orange powder (0.14 g, yield: 69.6%). FITC-labeled PODMA-PCL and PEG-PCL were also synthesized by a similar method.

[0101] (4) Preparation of drug-loaded micelles

[0102] The specific steps are as follows: DOX·HCl (1 mg) was dissolved in DMSO (1 mL), and then TEA (10 μL) was added. After stirring in the dark for 4 hours, PEG-PCL or POC7A-PCL (10 mg) was added and stirred for another hour, and then the solution was added dropwise to deionized water (10 mL) and stirred for 30 minutes. Then, the mixture was dialyzed with PBS (pH 8.0, 1 L×3) and deionized water (1 L×2). Since the unloaded DOX formed large aggregates in the micellar solution, a membrane filter with a pore size of 220 nm was used to remove the DOX aggregates to obtain drug-loaded nanoparticles. The preparation of DiR-embedded nanoparticles and FITC-labeled nanoparticles was also achieved by similar steps.

[0103] Example 2: Cytotoxicity Experiment

[0104] The CCK-8 assay was used to evaluate the in vitro cytotoxicity of PEG-PCL / DOX and POC7A-PCL / DOX. C8161 cells were placed in DMEM medium containing 10% (v / v) FBS and cultured in 96-well plates at a density of 4000 cells (100 μL) per well, and then cultured overnight at 37°C for attachment. Fresh medium (100 μL) with a pH of 6.7 was then replaced. The cells were then exposed to different formulations for 48 hours under normoxic or hypoxic conditions. The absorbance of each well was measured at a wavelength of 450 nm using a multiwell plate reader.

[0105] Example 3: Cellular uptake experiment

[0106] C8161 cells in culture medium (1 mL) were plated at 1×105 The cells were cultured at a density of 100 cells / well in a glass-bottomed dish and cultured under normoxic conditions for 12 h. The culture medium was replaced with fresh culture medium (1 mL, pH 6.7) containing DOX, PEG-PCL / DOX, or POC7A-PCL / DOX, with a DOX dose of 1.0 μg / mL, respectively, and cultured under normoxic or hypoxic conditions. After culture, the cell nuclei were stained with DAPI (2.5 μg / mL) for 15 min. The culture medium was then removed, and the cells were rinsed three times with cold PBS for fluorescence imaging. The cell uptake of DOX-loaded micelles was further quantified by flow cytometry. C8161 cells in culture medium (1 mL) were cultured at 1×10 5 Cells were seeded at a density of 100 μg / mL in 12-well plates and cultured under normoxic conditions for 12 h. The culture medium was replaced with fresh culture medium (1 mL, pH 6.7) containing DOX, PEG-PCL / DOX, or POC7A-PCL / DOX, with a DOX concentration of 1.0 μg / mL under normoxic or hypoxic conditions, respectively. After rinsing three times with PBS, cells were harvested for flow cytometric analysis.

[0107] Example 4: MTS permeation experiment under hypoxia or normoxia

[0108] C8161 cells were plated at 10 4 The density of 100 μL was seeded into a 96-well three-dimensional cell spheroid culture plate (Corning) and cultured for 3 days under normoxic (20% O2) conditions to form multicellular tumor spheroids (MTS). When performing bioassays, FITC-labeled PEG-PCL or POC7A-PCL (150 μL, FITC concentration of 0.5 μg / mL) was added to fresh culture medium (pH 7.4 or 6.7) with or without 500 μM DPI, and then cultured for 4 hours at 37°C, normoxic (20% O2) or hypoxic (1% O2). All three-dimensional MTS were imaged using a confocal microscope (Olympus FV3000).

[0109] Mature C8161 cell MTs were incubated with DOX, PEG-PCL / DOX, or POC7A-PCL / DOX (DOX concentration was 1.0 μg / mL) in 96-well plates. After 4 h of incubation, MTs were rinsed with PBS, transferred to chamber coverslips, and imaged using confocal microscopy.

[0110] Example 5: Transwell assay to study transcytosis

[0111] The upper chamber (200 μL) and lower chamber (1 mL) of a Transwell polycarbonate cell culture vessel (Corning) were filled with 2×10 5cells / mL of C8161 cell culture medium, and the culture medium was replaced every 24 hours. After 4 days of culture, culture medium (200 μL, pH 6.7) containing FITC-labeled PEG-PCL or POC7A-PCL (FITC concentration of 0.5 μg / mL) was added to the upper chamber, and the PBS group was used as a negative control. The cells were cultured in 1% and 20% oxygen incubators, respectively. After 24 hours, the culture medium in the lower chamber was aspirated and discarded, the cells in the lower chamber were rinsed three times with PBS, trypsinized and collected, and then the cell concentration was measured by flow cytometry.

[0112] Example 6: Animal Model and Biodistribution

[0113] To establish the B16 tumor model, B16 cells (2 × 10 6 ) was injected subcutaneously into the right abdomen of female C57 BL / 6 mice. The tumor volume reached about 120 mm 3 Afterwards, the mice were randomly divided into three groups (n=3) and fasted for 12 hours before the experiment with free access to water. C57 BL / 6 mice bearing B16 tumors were injected with free DiR, PEG-PCL / DiR, PODMA-PCL / DiR or POC7A-PCL / DiR at a dose of 0.5 mg / kg. After 24 hours, the mice were killed, and the tumors and major organs (heart, liver, spleen, lung, and kidney) were removed and analyzed using the IVIS Spectrum system. To evaluate the pharmacokinetics, after injection of each formulation (100 μL) via the tail vein, blood was drawn from the mouse orbit at 0.1, 0.5, 1, 2, 6, 12, and 24 h, and the fluorescence intensity of DiR in the blood was analyzed by the IVIS Spectrum system to evaluate the pharmacokinetics of the nanoparticles.

[0114] Example 7: Evaluation of anti-tumor effects in animals

[0115] Will carry about 150mm 3 Female C57 BL / 6 mice (5-6 weeks old) with B16 tumors were randomly divided into five groups (n=5) and injected intravenously with 0.2 mL of PBS, DOX, PEG-PCL / DOX, PODMA-PCL / DOX or POC7A-PCL / DOX solution, with an equivalent dose of DOX of 2 mg / kg, once every two days for a total of four injections. Tumor volume and body weight were measured every other day. On the 14th day after treatment, the mice were euthanized, and tumors and major organs (heart, liver, spleen, lung, and kidney) were collected. The calculation formula for tumor inhibition rate (TIR) ​​is TIR (%) = (average tumor weight of the control group - average tumor weight of the treatment group) / average tumor weight of the control group × 100%.

[0116] In order to illustrate the advantages of the block polymer of the present invention in improving the efficacy of doxorubicin, we first characterized the structure and properties of the drug-loaded polymer nanoparticles obtained by self-assembly, and compared the anti-tumor effects of the drug-loaded nanoparticles of the present invention and small molecule doxorubicin and the drug-loaded micelles of the control group by in vitro cell experiments and the construction of mouse tumor models. The results we obtained are explained below.

[0117] Excellent antifouling properties facilitate drug delivery in vivo. First, we evaluated the antifouling properties of POC7A and PEG. We prepared POC7A hydrogel and POEGMA hydrogel by photoinitiated polymerization of OC7A-MA and OEGMA, respectively. After incubation with luciferase-labeled BSA (FITC-BSA) for 6 h and 24 h ( Figure 6 a) Use confocal microscopy to observe protein adhesion. Figure 6 As shown in b, POC7A had almost no protein adhesion even after 24 hours of incubation. We further verified this conclusion by Bradford test ( Figure 6 c, Figure 6 d). These results indicate that POC7A has excellent antifouling properties, which is crucial for the subsequent in vivo drug delivery. At the same time, we monitored the stability of PEG-PCL / DOX and POC7A-PCL / DOX micelles in PBS and DMEM culture media. Figure 8 c, Figure 8 As shown in (d), the sizes of the two drug-loaded nanoparticles did not change significantly in PBS and DMEM culture media after 60 h.

[0118] Next, pyrene was used as a fluorescent probe, 334 nm light was used for excitation, and its fluorescence intensity at 373 nm and 384 nm was recorded. Then, the ratio of the fluorescence intensity at 373 nm to the fluorescence intensity at 384 nm was plotted against the polymer concentration, and the critical micelle concentration of the polymer PEG-PCL was obtained to be 17.0 μg / mL ( Figure 7 a), the critical micelle concentration of polymer POC7A-PCL is 6.0 μg / mL ( Figure 7 b). Dynamic light scattering analysis revealed that the average particle size of the DOX-loaded PEG-PCL nanoparticles (PEG-PCL / DOX) was about 64.6 nm ( Figure 8 a), the average particle size of POC7A-PCL nanoparticles loaded with DOX (POC7A-PCL / DOX) is about 42.0 nm (e.g. Figure 8 b), this size facilitates the enrichment of nanoparticles in tumor tissue. Moreover, the drug loading rate and encapsulation rate of PEG-PCL / DOX and POC7A-PCL / DOX nanoparticles were similar.

[0119] In addition, we also evaluated the release behavior of DOX from drug-loaded nanoparticles. The test results showed that both PEG-PCL / DOX and POC7A-PCL / DOX could effectively release DOX drug molecules. Under acidic conditions of pH 5.5, DOX from POC7A-PCL / DOX nanoparticles could be released more effectively ( Figure 8 e, Figure 8 f). This may be due to the stronger hydrophilicity of POC7A.

[0120] CYP450-mediated bioreduction of N-oxides under hypoxia, and the subsequent protonation of tertiary amines and binding to negatively charged cell membranes may facilitate cellular uptake. Therefore, we co-cultured C8161 cells with DOX, PEG-PCL / DOX, and POC7A-PCL / DOX in a medium simulating the slightly acidic nature of tumors (pH 6.7) with equal amounts of DOX, under hypoxia and normoxia, and then performed fluorescence imaging and flow cytometry tests to characterize the cellular uptake of nanoparticles. Fig. 9 , Fig.10 As shown in Figure 2, under hypoxia, POC7A-PCL / DOX exhibited much stronger bright red fluorescence than PEG-PCL / DOX. Further flow cytometry tests showed that the fluorescence intensity of cells treated with POC7A-PCL / DOX was 2.7 times and 1.7 times that of the PEG-PCL / DOX and DOX groups, respectively (Figure 2). Fig. 9 However, under normoxia, there was no significant difference in fluorescence intensity among the groups (as shown in Fig.10 These results indicate that POC7A-PCL / DOX has stronger cellular uptake than DOX and PEG-PCL / DOX under hypoxia, and hypoxia is crucial in promoting the cellular uptake of POC7A-PCL / DOX.

[0121] Effective cellular uptake plays a key role in improving the efficacy of anticancer drugs. The cell activity of C8161 cells after different drug treatments was determined by the CCK-8 method. POC7A-PCL and PEG-PCL micelles without DOX had minimal effect on the proliferation of C8161 cells in vitro even at concentrations as high as 100 μg / mL under normoxic and hypoxic conditions, indicating that the drug carrier itself has good biocompatibility. After loading with DOX, the cytotoxicity of POC7A-PCL / DOX was significantly higher compared with PEG-PCL / DOX under hypoxic conditions. Specifically, the half maximal inhibitory concentration (IC 50) was 3.85 μg / mL, which is much lower than the half-maximal inhibitory concentration of DOX and PEG-PCL / DOX under hypoxia. POC7A-PCL / DOX had a higher cellular internalization rate under hypoxia, thus enhancing the anticancer effect. However, they also showed similar cytotoxicity under normoxic conditions.

[0122] Since the transcellular transport of nanodrugs can effectively promote their tumor penetration, it is necessary to study the transcellular transport ability of nanodrugs. We have demonstrated that POC7A-PCL micelles can be efficiently internalized by cells under hypoxia. We then established a transwell model to study exocytosis. Fig.11 , Fig.12 As shown, POC7A-PCL micelles were able to penetrate the tumor cell layer more efficiently under hypoxia compared with PEG-PCL micelles, whereas no significant difference was observed in transcytosis under normoxia.

[0123] Polysaccharide gel-filled tumor stroma is a major barrier to the penetration of nanomedicines. We used POC7A-PCL micelles to co-incubate with a multicellular tumor spheroid (MTS) model with rich matrix. FITC PCL and PEG- FITC PCL was cultured under hypoxia for 4 h and then subjected to fluorescence imaging using CLSM. Almost no PEG- FITC FITC fluorescence (green) in the PCL-treated group ( Fig.11 d). In contrast, POC7A- FITC PCL-treated MTs showed bright fluorescence both on the surface and inside the spheroids. Similarly, POC7A-PCL / DOX showed bright fluorescence under hypoxia. Furthermore, line scan analysis of fluorescence intensity as a function of distance showed that POC7A- FITC PCL and POC7A-PCL / DOX-treated MTs showed a higher FITC On the other hand, in the presence of the CYP 450 inhibitor diphenylammonium iodonium chloride (DPI), POC7A- FITC PCL significantly reduced tumor penetration. Similarly, when POC7A- FITC When PCL was co-incubated with MTS, only weak FITC green fluorescence was observed. These results suggest that hypoxia, CYP450, and the acidic pH of the tumor microenvironment together play a crucial role in enhancing the cellular uptake and tumor penetration of POC7A-PCL micelles.

[0124] Next, we conducted an in vivo efficacy study. In order to further illustrate the in vivo performance of the present invention, we also added another zwitterionized amphiphilic block polymer carrier PODMA-PCL that has been reported as a comparison. In order to study the in vivo performance of the micelles, we used DiR-loaded PEG-PCL (PEG-PCL / DiR) and DiR-loaded PTAO-PCL (PTAO-PCL / DiR) for fluorescence imaging. Mice were intravenously injected with PEG-PCL / DiR, PODMA-PCL / DiR and POC7A-PCL / DiR. After 24 hours, the mice were killed and the main organs and tumor tissues were collected. Fig.13 As shown, the fluorescence intensity of tumor tissue in the POC7A-PCL / DiR group was the strongest, even stronger than that in the PODMA-PCL / DiR group. However, the free DiR and PEG-PCL / DiR groups did not show obvious tumor tissue aggregation. All these results indicate that POC7A-PCL micelles can effectively aggregate in tumors. Finally, the pharmacokinetic behavior of POC7A-PCL was studied accordingly. After intravenous injection, mouse blood was collected through the orbital venous plexus at different time points. The blood circulation half-lives of POC7A-PCL / DiR, PODMA-PCL / DiR and PEG-PCL / DiR were 8.29 hours, 7.06 hours and 6.57 hours, respectively. The benefit to blood circulation is mainly due to the super anti-fouling properties of PTAO.

[0125] The antitumor activity of POC7A-PCL / DOX was also evaluated in B16 tumor-bearing C57 BL / 6 mice. PBS, DOX, PEG-PCL / DOX, PODMA-PCL / DOX, and POC7A-PCL / DOX were intravenously injected with a DOX equivalent dose of 2 mg / kg every two days in an average volume of about 150 mm 3 Tumor-bearing mice were treated four times in total ( Fig.14 a). In contrast to the rapid growth of the PBS group, DOX and DOX-loaded micelle groups showed different inhibitory abilities on B16 tumor growth ( Fig.14 b). The DOX and PEG-PCL / DOX groups showed lower tumor inhibition rates of 31.0% and 32.7%, respectively. In contrast, POC7A-PCL / DOX and PODMA-PCL / DOX largely inhibited tumor growth throughout the experiment, which we attribute to the stronger tumor uptake and penetration of PTAO-PCL / DOX, which greatly increased the bioavailability of DOX. In addition, POC7A-PCL / DOX exhibited stronger tumor inhibition compared with the PODMA-PCL / DOX group, with a TIR as high as 84.1% ( Fig.14 c).

[0126] The anti-tumor effect of POC7A-PCL / DOX was further verified by H&E, Ki 67 and TUNEL staining ( Fig.14 e). H&E staining images showed that tumor tissues in the POC7A-PCL / DOX group underwent extensive cavitation, severe nuclear shrinkage, and reduced cell density. In addition, there was less red fluorescence in the Ki 67 staining images and more green fluorescence in the TUNEL staining images, indicating that POC7A-PCL / DOX can effectively inhibit tumor cell proliferation and promote tumor cell apoptosis. Overall, these results indicate that compared with traditional PEGylated micelles, POC7A-covered micelles show effective tumor accumulation, deep tumor tissue penetration, and strong cell internalization, ultimately resulting in better therapeutic effects. In addition, importantly, no significant changes in the body weight of POC7A-PCL / DOX-treated mice were observed throughout the treatment process. And compared with the control group, there was no obvious damage to the major organs of POC7A-PCL / DOX-treated mice ( Fig.15 ), indicating the biosafety and biocompatibility of POC7A-PCL / DOX.

[0127] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0128] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. An anaerobic bioreduction responsive amphiphilic block polymer, characterized in that: The chemical structure is shown in Formula I: In the formula, n is 28 to 35, and m is 44 to 48.

2. The method for preparing the hypoxic bioreduction responsive amphiphilic block polymer according to claim 1, characterized in that: The following steps are involved: (1) reacting 2-(azepan-1-yl)ethyl methacrylate and m-chloroperbenzoic acid to obtain a zwitterionic monomer N-oxide-2-(azepan-1-yl)ethyl methacrylate, denoted as OC7A-MA; (2) PCL-OH was obtained by ring-opening polymerization of ε-caprolactone using N-(tert-butyloxycarbonyl)ethanolamine as an initiator; (3) reacting PCL-OH with 2-bromoisobutyryl bromide to obtain the macroinitiator PCL-Br; (4) Using PCL-Br as a macromolecular initiator, poly(2-(N-oxide-hexamethyleneimino)ethyl methacrylate)-b-polycaprolactone was obtained by atom transfer radical polymerization of OC7A-MA monomer, which was named POC7A-PCL, i.e., the hypoxic bioreduction responsive amphiphilic block polymer.

3. The method for preparing the hypoxic bioreduction responsive amphiphilic block polymer according to claim 2, characterized in that: In step (1), the specific reaction conditions are: dissolving 2-(azepan-1-yl)ethyl methacrylate in dichloromethane, adding m-chloroperbenzoic acid under ice bath conditions, reacting at room temperature for 2 to 3 hours, and purifying by column chromatography to finally obtain an off-white solid; The mass volume ratio of the 2-(azepan-1-yl)ethyl methacrylate, m-chloroperbenzoic acid and dichloromethane is 3.14-4.71 g: 3.42-5.13 g: 100-150 mL.

4. The method for preparing the hypoxic bioreduction responsive amphiphilic block polymer according to claim 3, characterized in that: In step (2), the specific reaction conditions are: dissolving the initiator N-(tert-butyloxycarbonyl)ethanolamine, the catalyst diphenyl phosphate and the monomer ε-caprolactone in anhydrous toluene, degassing by freeze-pump-thaw cycle, and then reacting at 30°C to 40°C for 4 to 6 hours, quenching the reactants, precipitating and drying to obtain the PCL-OH. The mass volume ratio of the N-(tert-butyloxycarbonyl)ethanolamine, diphenyl phosphate, ε-caprolactone and anhydrous toluene is 0.37-0.74 g: 0.58-1.16 g: 12.10-24.20 g: 30.00-60 mL.

5. The method for preparing the hypoxic bioreduction responsive amphiphilic block polymer according to claim 4, characterized in that: In step (3), the specific reaction conditions are: dissolving PCL-OH and triethylamine in anhydrous dichloromethane, adding 2-bromoisobutyryl bromide dissolved in dichloromethane dropwise under ice bath conditions, reacting at room temperature for 12 to 16 hours, and after the reaction is completed, removing the solvent using a rotary evaporator, and precipitating with methanol and ether to obtain PCL-Br. The mass volume ratio of the PCL-OH, triethylamine, 2-bromoisobutyryl bromide and anhydrous dichloromethane is 11.00-16.5 g: 1.52-2.28 g: 2.30-3.45 g: 70.00-105 mL.

6. The method for preparing the hypoxic bioreduction responsive amphiphilic block polymer according to claim 5, characterized in that: In step (4), the specific reaction conditions are: PCL-Br, OC7A-MA, CuBr and PMDETA are placed in DMF, degassed by freeze-pump-thaw cycle, and then reacted at 38°C to 42°C for 22 to 24 hours, and the reactants are quenched, dialyzed, and freeze-dried to obtain the POC7A-PCL; The mass volume ratio of the PCL-Br, OC7A-MA, CuBr, PMDETA and DMF is 0.550-1.100 g: 0.680-1.360 g: 0.014-0.028 g: 0.017-0.034 g: 4-8 mL.

7. Use of the hypoxic bioreduction responsive amphiphilic block polymer according to claim 1 in the preparation of anti-tumor drug carriers.

8. The use according to claim 7, characterized in that: The anti-tumor drug includes doxorubicin.

9. An anti-tumor drug, characterized in that: The method is formed by self-assembling the hypoxic bioreduction responsive amphiphilic block polymer according to claim 1 and doxorubicin in an aqueous solution.

10. The anti-tumor drug according to claim 9, characterized in that: Pharmaceutically acceptable excipients are also included.

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