Hypoxic bioreductive responsive amphiphilic block polymers, methods of making and using the same

Abstract

The application belongs to the technical field of high-molecular anti-tumor drug delivery carriers, and particularly relates to an oxygen-deficient bioreduction responsive amphiphilic block polymer anti-tumor drug carrier and a preparation method and application thereof. The oxygen-deficient responsive block copolymer (POC7A-PCL) with a hydrophobic segment of PCL and a hydrophilic segment of POC7A is constructed through ring-opening polymerization and atom transfer radical polymerization. The polymer is used to load an anti-tumor drug doxorubicin to construct a responsive polymer nanoparticle, which shows stronger anti-tumor capacity. Therefore, the POC7A-PCL can be applied to the preparation of anti-tumor drugs as a drug carrier.

Description

Technical Field

[0001] The present invention belongs to the technical field of polymer anti-tumor drug delivery carriers, and particularly relates to an hypoxic bioreduction-responsive amphiphilic block polymer anti-tumor drug carrier, a preparation method thereof, and an 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, cellular 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 reaction effect (FBR). In general, the ability of the drug delivery system to be invisible 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 anti-fouling 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 for resisting 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 seriously 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 drug delivery systems using PEG as a hydrophilic shell in vivo, 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 anoxic bioreduction-responsive amphiphilic block polymer, the chemical structure of which is shown in Formula I:

[0007]

[0008] Wherein, n is 28 to 35, and m is 44 to 48.

[0009] Hypoxia is a hallmark of the tumor microenvironment. The hypoxic microenvironment within tumors 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 regions of various solid tumors.

[0010] N-oxide-based polymers (PTAO) can be used to construct efficient drug delivery vehicles. In the present invention, we prepared a block copolymer POC7A-block-poly(ε-caprolactam) (POC7A-PCL) using poly(2-(N-oxy-hexamethyleneimino)ethyl methacrylate) (POC7A) and poly(ε-caprolactone) (PCL) with a cyclic seven-membered ring as the hydrophilic and hydrophobic segments, respectively. The POC7A-PCL of the present invention has efficient bioreductive activity and tumor penetrability and is responsive to the acidic tumor microenvironment (pH 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) 2-(azepan-1-yl)ethyl methacrylate and m-chloroperbenzoic acid are reacted to obtain a zwitterionic monomer N-oxide-2-(azepan-1-yl)ethyl methacrylate, which is recorded as OC7A-MA;

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

[0014] (3) PCL-OH is reacted 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, the hypoxic bioreduction-responsive amphiphilic block polymer.

[0016] Furthermore, in step (1), the specific reaction conditions are as follows: 2-(azepan-1-yl)ethyl methacrylate is dissolved in anhydrous dichloromethane, and m-chloroperbenzoic acid is added under ice bath conditions. After reacting at room temperature for 2-3 hours, the product is purified by column chromatography to obtain 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 a freeze-pump-thaw cycle, and then reacted at 30° C. to 40° C. for 4 to 6 hours. 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 as follows: PCL-OH and triethylamine are dissolved in anhydrous dichloromethane, and 2-bromoisobutyryl bromide dissolved in dichloromethane is added dropwise in an ice bath. The reaction is carried out at room temperature for 12 to 16 hours. After completion of the reaction, the solvent is removed using a rotary evaporator, and PCL-Br is precipitated with methanol and diethyl ether.

[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 as follows: PCL-Br, OC7A-MA, CuBr and PMDETA are placed in DMF, degassed by freeze-pump-thaw cycles, 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 hypoxia 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 a delivery vehicle for 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, enhancing cellular uptake and tumor penetration by binding to negatively charged cell membranes. Finally, POC7A-PCL / DOX exhibited stronger anti-tumor ability, which means that POC7A has great potential as an anti-fouling 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 1 H NMR spectroscopy characterization.

[0035] Figure 6Figure 1. Schematic diagram of the preparation of POC7A and POEGMA hydrogels and incubation with BSA.

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

[0037] b is a fluorescence image of FITC-BSA absorbed by POC7A hydrogel, poly(methacrylic acid oligoethylene glycol ester) (POEGMA) hydrogel and polypropylene (PP) after 6 hours and 24 hours incubation. Scale bar is 150 pm.

[0038] c is quantification of the mean fluorescence intensity (MFI) in the fluorescence image using ImageJ.

[0039] d is the Bradford detection of adsorbed proteins on different hydrogels after 24 hours incubation.

[0040] Figure 7 Figure 2. Schematic diagram of the preparation of POC7A-PCL and POEGMA-PCL hydrogels and incubation with BSA.

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

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

[0043] Figure 8 Figure 3. Schematic diagram of the preparation of POC7A-PCL / DOX nanoparticles.

[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 change in particle size of DOX micelles over time in PBS.

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

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

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

[0050] Figure 9 Figure 4. Schematic diagram of the preparation of POC7A-PCL / DOX nanoparticles and incubation with MCF-7 cells.

[0051] a is a 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 DOX-containing micelles under hypoxic conditions.

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

[0055] Figure 10 middle:

[0056] a) Fluorescence image of C8161 cells cultured with DOX-loaded micelles at a DOX concentration of 0.5 μg / mL under normoxic conditions. Scale bar, 100 μm. Cell nuclei stained with DAPI appear blue, and DOX appears 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 DOX-containing micelles under normoxic conditions.

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

[0060] Figure 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 results of C8161 cells in the lower chamber of the transwell culture dish after being treated with FITC-labeled micelles under hypoxic conditions.

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

[0064] d shows the penetration of FITC-labeled micelles and DOX-loaded micelles into C8161 MTS under hypoxic conditions. Scale bar, 200 μm. Z-stack imaging interval, 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] Figure 12 middle:

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

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

[0070] c shows the penetration of FITC-labeled micelles and DOX-loaded micelles into C8161 MTS under normoxic conditions. Scale bar, 200 μm. Z-stack imaging interval, 15 μm.

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

[0073] Figure 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 after intravenous injection in each group.

[0076] Figure 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] Figure 15The tissue sections were stained with hematoxylin-eosin and observed under a light microscope (scale bar, 100 μm). DETAILED DESCRIPTION

[0083] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they 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] Currently, commonly used PEGylated drug delivery vehicles can increase the blood circulation time of doxorubicin. However, the PEG on the surface of these vehicles reduces cellular endocytosis, which is detrimental to the anti-tumor effect of doxorubicin. Therefore, the use of nano-micelle shell structures with stronger cellular internalization instead of PEG 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 First, PCL-OH is obtained by ring-opening polymerization of ε-caprolactone, and then the block copolymer is obtained by atom transfer radical polymerization using PCL-Br obtained after bromination as a macromolecular initiator. 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, we obtained PCL-OH through the ring-opening polymerization of ε-caprolactone, and reacted PCL-OH with 2-bromoisobutyryl bromide to obtain the macroinitiator PCL-Br. Finally, using PCL-Br as a macroinitiator, we obtained the amphiphilic block polymer poly(2-(N-oxide-hexamethyleneimino)ethyl methacrylate)-b-polycaprolactone through atom transfer radical polymerization of OC7A-MA monomer, 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 similar method was used to synthesize the block polymer poly(2-(N-oxide-N,N-dimethylamino)ethyl methacrylate)-b-poly(ε-caprolactone) (PODMA-PCL) 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-cooling conditions. After reacting at room temperature for 3 hours, the mixture was purified by column chromatography to obtain an off-white solid, N-oxide-2-(azepan-1-yl)ethyl methacrylate, designated as OC7A-MA. The mass-to-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 and degassed via freeze-pump-thaw cycles. The mixture was then reacted at 38°C for 5 hours. The reactant was quenched, precipitated, and dried to obtain PCL-OH. The mass-to-volume ratio of N-(tert-butyloxycarbonyl)ethanolamine, diphenyl phosphate, ε-caprolactone, and anhydrous toluene was 0.37 g:0.58 g:12.10 g:30.00 mL.

[0092] PCL-OH and triethylamine (CAS No. 121-44-8) were dissolved in anhydrous dichloromethane. 2-Bromoisobutyryl bromide (CAS No. 20769-85-1) dissolved in dichloromethane was added dropwise in an ice bath. The reaction was allowed to proceed at room temperature for 15 hours. After completion of the reaction, the solvent was removed using a rotary evaporator, and PCL-Br was precipitated with methanol and diethyl ether. The mass-to-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 via 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 via three freeze-pump-thaw cycles and then placed in a preheated oil bath at 40°C. After 24 hours, the reaction was quenched by exposure to air and dialyzed against methyl sulfoxide (DMSO) (500 mL x 3) and deionized water (500 mL x 3). The solution was lyophilized to afford POC7A-PCL (0.69 g, 56.1% yield) as a pale yellow solid, the structural formula of which is shown in Formula I:

[0094]

[0095] Wherein: n is 30 and m is 48.

[0096] PODMA-PCL block copolymers were synthesized using 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 using three freeze-pump-thaw cycles and then placed in a preheated oil bath at 40°C. After 24 hours, the reaction was quenched by exposure to air and dialyzed against dimethyl sulfoxide (DMSO) (500 mL x 3) and deionized water (500 mL x 3). The solution was lyophilized to yield POC7A-PCL as a pale yellow solid (0.69 g, 56.1% yield).

[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). Polymerization was initiated by the addition of diphenyl phosphate (DPP) (25.19 mg, 0.10 mmol) in 1 mL of toluene. After polymerization at 30°C for 4 hours, the reaction solution was poured into 100 mL of cold ether. The precipitate was isolated by filtration, washed with ether, and dried under vacuum to yield PEG-PCL as a white solid (0.54 g, 54.0% yield).

[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 then FITC (2 mg) and triethanolamine (0.4 mL) were added. The solution was stirred in the dark at room temperature overnight, 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, after removing the residual FITC molecules, was lyophilized 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-encapsulated 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 plated in DMEM medium containing 10% (v / v) FBS at a density of 4,000 cells per well (100 μL) in a 96-well plate 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 the 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 μg / mL in glass-bottomed culture dishes and cultured under normoxic conditions for 12 hours. 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 minutes. The culture medium was then removed, and the cells were rinsed three times with cold PBS and subjected to fluorescence imaging. The absorption of DOX-loaded micelles by the cells was further quantified by flow cytometry. C8161 cells in culture medium (1 mL) were cultured at a density of 1×10 5 Cells were seeded at a density of 100 μg / mL in 12-well plates and cultured under normoxic conditions for 12 hours. The culture medium was replaced with fresh culture medium (1 mL, pH 6.7) containing DOX, PEG-PCL / DOX, or POC7A-PCL / DOX at a DOX concentration of 1.0 μg / mL under normoxic or hypoxic conditions, respectively. After rinsing three times with PBS, the cells were harvested and analyzed by flow cytometry.

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

[0108] C8161 cells were plated at 10 per well 4 Cells were seeded at a density of 100 μL into 96-well 3D spheroid culture plates (Corning) and cultured under normoxia (20% O2) for 3 days to form multicellular tumor spheroids (MTS). For 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, followed by incubation at 37°C under normoxia (20% O2) or hypoxia (1% O2) for 4 hours. All 3D 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 hours of incubation, the MTs were rinsed with PBS, transferred to chamber coverslips, and imaged using confocal microscopy.

[0110] Example 5: Transwell assay to study transcytosis

[0111] A concentration of 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, and 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 approximately 120 mm 3 Afterwards, 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. 24 hours later, the mice were sacrificed, and the tumors and major organs (heart, liver, spleen, lungs, and kidneys) 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 hours, and the fluorescence intensity of DiR in the blood was analyzed using 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) bearing B16 tumors were randomly divided into five groups (n=5) and intravenously injected with 0.2 mL of PBS, DOX, PEG-PCL / DOX, PODMA-PCL / DOX, or POC7A-PCL / DOX solution, with a DOX equivalent dose 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 tumor inhibition rate (TIR) ​​was calculated as TIR (%) = (average tumor weight of the control group - average tumor weight of the treatment group) / average tumor weight of the control group × 100%.

[0116] To demonstrate the advantages of the block polymers of the present invention in enhancing the efficacy of doxorubicin, we first characterized the structure and properties of the self-assembled drug-loaded polymer nanoparticles. We then compared the anti-tumor effects of the drug-loaded nanoparticles of the present invention and small-molecule doxorubicin, as well as a control group of drug-loaded micelles, in vitro cell experiments and mouse tumor models. The results are described 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 showed 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 for 60 h.

[0118] Next, pyrene was used as a fluorescent probe, excited with 334 nm light and its fluorescence intensity at 373 nm and 384 nm was recorded. The ratio of the fluorescence intensity at 373 nm to the fluorescence intensity at 384 nm was then plotted against the polymer concentration. The critical micelle concentration of the polymer PEG-PCL was found to be 17.0 μg / mL ( Figure 7 a), the critical micelle concentration of the 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 approximately 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 nanoparticle accumulation in tumor tissue. Furthermore, the drug loading and encapsulation efficiencies 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, POC7A-PCL / DOX nanoparticles could release DOX more effectively ( Figure 8 e, Figure 8 f). This may be due to the stronger hydrophilicity of POC7A.

[0120] Under hypoxia, CYP450-mediated bioreduction of N-oxides and 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 culture medium (pH 6.7) simulating the slightly acidic nature of tumors, 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. Figure 9 , Figure 10 As shown in Figure 2, under hypoxia, POC7A-PCL / DOX exhibited bright red fluorescence that was much stronger than that of 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). Figure 9 However, under normoxia, there was no significant difference in fluorescence intensity among the groups (as shown in Figure 10 These results indicate that POC7A-PCL / DOX has stronger cellular uptake than DOX and PEG-PCL / DOX under hypoxia, and that 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 treatment with different drugs 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 of up to 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 than that of PEG-PCL / DOX under hypoxic conditions. Specifically, the half-maximal inhibitory concentration (IC 50) was 3.85 μg / mL, far below the half-maximal inhibitory concentration of DOX and PEG-PCL / DOX under hypoxia. POC7A-PCL / DOX exhibited a higher cellular internalization rate under hypoxia, leading to enhanced anticancer effects. However, they exhibited similar cytotoxicity under normoxic conditions.

[0122] Since the transcellular transport of nanomedicines can effectively promote their tumor penetration, it is necessary to study the transcellular transport ability of nanomedicines. We have demonstrated that POC7A-PCL micelles can be efficiently internalized by cells under hypoxia. We then established a transwell model to study exocytosis. Figure 11 、 Figure 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 the main barrier to the penetration of nanomedicines. We used POC7A-PCL micelles to co-incubate with multicellular tumor spheroids (MTS) model with rich matrix. FITC PCL and PEG- FITC PCL was cultured under hypoxia for 4 h and then fluorescence imaging was performed using CLSM. Almost no PEG- FITC FITC fluorescence (green) in the PCL-treated group ( Figure 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. In addition, 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 tumor penetration was significantly reduced. Similarly, when POC7A- FITC When PCL was co-incubated with MTS, only weak FITC green fluorescence was observed. These results indicate that hypoxia, CYP450, and the acidic pH of the tumor microenvironment 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 zwitterionic amphiphilic block polymer carrier PODMA-PCL that has been reported as a comparison. In order to study the in vivo performance of 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. Figure 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 beneficial 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 every two days with a DOX equivalent dose of 2 mg / kg in an average volume of approximately 150 mm. 3 Tumor-bearing mice were treated with four treatments in total ( Figure 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 ( Figure 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 to the PODMA-PCL / DOX group, with a TIR as high as 84.1% ( Figure 14 c).

[0126] The anti-tumor effect of POC7A-PCL / DOX was further verified by H&E, Ki 67 and TUNEL staining ( Figure 14 e). H&E staining images showed that the tumor tissue in the POC7A-PCL / DOX group underwent extensive vacuolation, 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 show that compared with traditional PEGylated micelles, POC7A-covered micelles show effective tumor accumulation, deep tumor tissue penetration, and strong cell internalization, ultimately producing better therapeutic effects. In addition, it is important to note that 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 ( Figure 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 additional 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 may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A hypoxic bioreduction-responsive amphiphilic block polymer, characterized in that: The chemical structure is shown in Formula I: Wherein, 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) 2-(azepan-1-yl)ethyl methacrylate and m-chloroperbenzoic acid are reacted to obtain a zwitterionic monomer N-oxide-2-(azepan-1-yl)ethyl methacrylate, which is recorded 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, 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 then 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 as follows: dissolving the initiator N-(tert-butyloxycarbonyl)ethanolamine, the catalyst diphenyl phosphate and the monomer ε-caprolactone in anhydrous toluene, degassing through a freeze-pump-thaw cycle, and then reacting at 30° C. to 40° C. for 4 to 6 hours. The reactants are 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 0.37-0.74 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 as follows: 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. After the reaction is complete, removing the solvent using a rotary evaporator, and precipitating with methanol and diethyl 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 as follows: PCL-Br, OC7A-MA, CuBr and PMDETA are placed in DMF, degassed by freeze-pump-thaw cycles, and then reacted at 38°C to 42°C for 22 to 24 hours. 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 hypoxia 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 hypoxic bioreduction-responsive amphiphilic block polymer according to claim 1 and doxorubicin are self-assembled in an aqueous solution.

10. The antitumor drug according to claim 9, characterized in that Pharmaceutically acceptable excipients are also included.

Images