Active oxygen / hypoxia dual-response drug delivery carrier as well as preparation method and application thereof

Abstract

The invention provides an active oxygen / hypoxia dual-response drug delivery carrier as well as a preparation method and application thereof. The method comprises the following steps: S1, synthesizing a methoxy polyethylene glycol-b-polylysine block copolymer; s2, synthesizing a block copolymer PEG (Polyethylene Glycol)-b-PLL (Poly L-Lactic Acid)-g-NI modified by nitroimidazole; s3, reacting cinnamyl aldehyde with thiohydracrylic acid to generate an active oxygen responsive cinnamyl aldehyde prodrug TCA; s4, dissolving PEG (Polyethylene Glycol)-b-PLL-g-NI, TCA (Trichloroacetic Acid), FeCl3. 6H2O and a medicine into DMSO (Dimethylsulfoxide); s5, carrying out self-assembly through a nano-precipitation method to form drug-loaded micelles; and S6, dialyzing the synthesized drug-loaded micelle with deionized water to obtain the drug-loaded micelle. The dual-response drug delivery carrier prepared according to the invention can realize responsive controlled release of drugs under in-vitro hydrogen peroxide stimulation and sodium hydrosulfite simulated hypoxia environment stimulation, reduce leakage of drugs at non-focus parts, and realize maximum utilization of drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanotechnology and biomedical science, and particularly relates to an active oxygen / hypoxia dual-responsive drug delivery carrier, a preparation method and application thereof. BACKGROUND

[0002] Active oxygen and hypoxic microenvironment are the core regulatory factors of various pathological processes. Abnormal increase of active oxygen and hypoxic microenvironment often accompanies metabolic imbalance and treatment resistance, which can directly cause mitochondrial dysfunction, immune escape and drug resistance, and become a key problem in disease treatment such as wound repair and cancer. Studies have shown that dynamic regulation targeting pathological microenvironment heterogeneity is the core path to cope with disease evolution. Precise identification of microenvironment characteristics and realization of time and space controllable release of therapeutic drugs are the key to break through the pathological barrier. However, the traditional drug delivery system is difficult to match the dynamic evolution law of lesion microenvironment due to the single recognition dimension of stimulation signal and the rigid response logic.

[0003] The existing single-stimulus-responsive carriers such as pH-sensitive and enzyme-cleavage drug carriers can achieve basic stimulus-responsive targeted release, but still have two core defects. First, the recognition signal of the carrier is relatively single, which cannot distinguish the microenvironment gradient difference in the pathological area, and there may be a certain non-specific drug leakage. Second, the release kinetics of the single-stimulus-responsive drug carrier is simple. When the stimulus reaches the threshold, the drug is completely released, which is difficult to adapt to the multi-stage treatment needs.

[0004] The multi-responsive carrier developed in recent years solves the defects of traditional single-stimulus-responsive carriers by combining multiple pathological microenvironment signals such as oxidation-reduction, temperature, pH, enzyme, etc., and partially improves the release accuracy. However, it still faces great challenges to match the stimulus response logic with the drug action period. Therefore, developing an intelligent drug carrier system with logic-gated stimulus response, self-feedback hierarchical release and ultra-low drug non-target leakage has become a technical challenge that needs to be overcome in the field of targeted microenvironment regulation. SUMMARY

[0005] The purpose of the present application is to provide an active oxygen / hypoxia dual-responsive drug delivery carrier, a preparation method and application thereof, so as to solve the problems of single signal recognition logic, mismatched release kinetics and insufficient structural stability of the existing stimulus-responsive carriers.

[0006] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0007] According to a first aspect of the present application, a preparation method of an active oxygen / hypoxia dual-responsive drug delivery carrier is provided, comprising the following steps:

[0008] S1, Lys-NCA was induced to open ring polymerization by mPEG-NH2 as initiator to obtain PEG-b-PLL(Z); then the benzyloxy carbonyl protection was removed by HBr / acetic acid solution in trifluoroacetic acid, and PEG-b-PLL was obtained by precipitation with ether;

[0009] S2, after NI-COOH was activated by EDCI / HOBt in DMF, it was coupled with mPEG-b-PLL and DIPEA under nitrogen; the reaction solution was added dropwise into pre-cooled ether to precipitate the crude product, which was filtered under reduced pressure and washed with ether to obtain nitroimidazole modified PEG-b-PLL-g-NI;

[0010] S3, CA and 3-mercaptopropionic acid were dissolved in ethyl acetate, and trifluoroacetic acid was added as catalyst under dark condition, and the active oxygen responsive cinnamaldehyde prodrug TCA was generated by stirring in the dark through the sulfur acetal reaction between cinnamaldehyde and mercaptopropionic acid;

[0011] S4, PEG-b-PLL-g-NI, TCA, FeCl3·6H2O and model drug were weighed and dissolved in anhydrous DMSO, and a uniform solution was formed by ultrasonic treatment;

[0012] S5, the above solution was added dropwise into the vigorously stirred deionized water to induce the formation of drug-loaded micelles;

[0013] S6, the synthesized drug-loaded micelles were dialyzed against deionized water to remove the unloaded components and DMSO, and the active oxygen / hypoxia responsive drug carrier was obtained.

[0014] Preferably, in step S1, the volume ratio of n-hexane / tetrahydrofuran mixed solvent used for recrystallization of NCA monomer is (2-5) ︰ 1.

[0015] Preferably, in step S1, the molecular weight of mPEG-NH2 is in the range of 2000-10000 Da.

[0016] Preferably, in step S1, the molar ratio of Lys-NCA to mPEG-NH2 is (20-60) ︰ 1, which is selected according to the desired lysine polymerization degree, and it mainly provides reaction and coordination sites for the subsequent, and also affects the surface charge of the final assembly.

[0017] Preferably, in step S1, the volume ratio of trifluoroacetic acid and HBr / acetic acid solution used for deprotection is (2-4) ︰ 1.

[0018] Preferably, in step S1, all reagents used for synthesis are super dry reagents to prevent unnecessary side reactions.

[0019] Preferably, in step S2, the amount of EDCI and HOBt used for activating the carboxyl group is 1-1.5 equivalents of the carboxyl group in NI-COOH, and the activation time is 0.5-1.5 h.

[0020] Preferably, in step S3, the molar ratio of CA to 3-mercaptopropionic acid is 1:(2-3), and the amount of trifluoroacetic acid used is a catalytic amount.

[0021] Preferably, in step S3, the developing agent for monitoring the progress of the reaction is n-hexane and ethyl acetate in a volume ratio of (3-4):1.

[0022] Preferably, in step S4, the mass ratio of PEG-b-PLL-g-NI, TCA, FeCl3·6H2O and the drug is 5:(1-4):(0.5-3):(0.5-3).

[0023] Preferably, in step S4, the volume of DMSO used is 100-1000 μL.

[0024] Preferably, in step S5, the volume of deionized water is 4-10 times the volume of DMSO to facilitate the formation of the assembly.

[0025] Preferably, in step S6, the dialysis medium is replaced every 2 h, and the dialysis is performed for a total of 24 h to remove the unloaded chemotherapeutic drugs and organic reagents.

[0026] It should be understood that, in the preparation method provided by the present application, the molecular weight of the PEG segment in step S1 will affect the drug release behavior, and preferably the molecular weight of mPEG-NH2 is in the range of 2000-10000 Da; if the molecular weight is too large, the steric hindrance will hinder drug release, and if the molecular weight is too small, a stable assembly cannot be formed. The feed ratio in step S4 will also have some effect on the drug release behavior of the product and the stability of the nanoparticles. Preferably, the mass ratio of PEG-b-PLL-g-NI, TCA, FeCl3·6H2O and the drug is 5:(1-4):(0.5-3):(0.5-3).

[0027] According to one preferred embodiment of the present application, a preparation method of an active oxygen / hypoxia dual-responsive drug delivery carrier is provided, comprising the following steps:

[0028] S1, using benzyloxycarbonyl-protected lysine (H-Lys(Z)-OH) as a raw material, reacted with triphosgene in tetrahydrofuran (THF), and high-purity lysine-N-carboxylic anhydride (Lys-NCA) was obtained by vacuum distillation and recrystallization in a hexane / THF mixed solvent; then, using monomethoxy polyethylene glycolamine (mPEG-NH2) as an initiator, the ring-opening polymerization of Lys-NCA was initiated in anhydrous N,N-dimethylformamide (DMF), and purified by precipitation with ice-cold diethyl ether to obtain polyethylene glycol-b-polybenzyloxycarbonyl lysine block copolymer (PEG-b-PLL(Z)); further, PEG-b-PLL(Z) was dissolved in trifluoroacetic acid, and the benzyloxycarbonyl(Z) protecting group was removed by adding HBr / acetic acid solution, and finally purified by precipitation with ice-cold diethyl ether to obtain methoxy polyethylene glycol-b-polylysine block copolymer (PEG-b-PLL);

[0029] In step S2, NI-COOH was dissolved in anhydrous DMF, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBt) were added sequentially. The mixture was stirred at room temperature to activate the carboxylic acid groups. Subsequently, a DMF mixture of mPEG-b-PLL and N,N-diisopropylethylamine (DIPEA) from step S1 was added dropwise to the above activation system, and the reaction was continued under nitrogen protection. After the reaction was completed, the mixture was added dropwise to pre-cooled diethyl ether to precipitate the crude product. The solid was collected by vacuum filtration and washed three times with diethyl ether. The final product, PEG-b-PLL-g-NI, was dried under vacuum and stored under nitrogen protection.

[0030] S3, cinnamaldehyde (CA) and 3-mercaptopropionic acid were dissolved in ethyl acetate, and trifluoroacetic acid was added as a catalyst under light-protected conditions; the mixture was stirred in the dark and monitored by thin-layer chromatography until the starting material spot disappeared. After the reaction was complete, the mixture was filtered through a Buchner funnel and washed successively with pre-cooled deionized water and hexane to remove unreacted 3-mercaptopropionic acid and byproducts, and then dried under vacuum to obtain TCA;

[0031] S4, weigh out PEG-b-PLL-g-NI, TCA, FeCl3·6H2O and model drug, dissolve them in anhydrous DMSO, and sonicate until completely dissolved;

[0032] S5. The above solution is added dropwise to deionized water at room temperature with vigorous stirring at 400-700 rpm to induce the formation of nano-assemblies.

[0033] S6. Subsequently, the mixture is transferred to a dialysis bag and dialyzed with deionized water to remove free drugs and uncoordinated metal ions, ultimately obtaining nanoassemblies.

[0034] According to a second aspect of the present application, there is provided an active oxygen / hypoxia dual-responsive drug delivery carrier prepared by the above method, wherein the core of the active oxygen / hypoxia dual-responsive drug delivery carrier is a hypoxia-responsive nitroimidazole unit, the shell layer is an electrostatic coordination network of TCA, iron ions and amino groups, the core hypoxia-responsive nitroimidazole unit endows the polymer with hydrophobicity, and the active oxygen / hypoxia dual-responsive drug delivery carrier releases model drugs only when treated with hydrogen peroxide and hypoxia, and the drug release reaches a peak when treated with hydrogen peroxide and hypoxia.

[0035] According to the present application, when the dual-responsive drug delivery carrier is treated with hydrogen peroxide, the sulfur acetal bond in the cinnamaldehyde prodrug is attacked and cleaved to release cinnamaldehyde and iron ions.

[0036] According to the present application, when the dual-responsive drug delivery carrier is treated with simulated hypoxia, the nitroimidazole groups in the micellar core hydrophobe react to form aminoimidazole, realizing a change in hydrophobicity and thus inducing drug release.

[0037] The active oxygen / hypoxia dual-responsive drug delivery carrier prepared according to the present application has the following specific dissociation process under active oxygen / hypoxia stimulation: when the carrier is in an active oxygen environment, the sulfur acetal bond in the TCA is oxidized and broken, releasing cinnamaldehyde and dissociating part of Fe 3+ ; when in a hypoxic environment, the core nitroimidazole unit is converted into aminoimidazole, resulting in a change in hydrophobicity; only when the dual stimuli coexist, the coordination network of the shell layer is completely destroyed and the core structure is disintegrated, realizing maximum drug release.

[0038] Preferably, in a microenvironment of a disease overexpressing ROS and hypoxia, the drug carrier can respond to cleavage, realizing drug release. It should be understood that in tumor treatment, the higher the degree of hypoxia and the content of ROS in the tumor, the better the effect of the drug carrier. In a preferred embodiment, 803 gastric cancer cells are incubated in a hypoxic environment for verification.

[0039] According to a third aspect of the present application, there is provided a use of the ROS / hypoxia dual-responsive drug carrier in the preparation of a medicament for treating tumors. According to an embodiment of the present application, it is verified that the drug carrier has a drug delivery therapeutic effect in tumor cells 803.

[0040] The key point of the present application is that a dual-signal gating unit is constructed by exploratory combination of the chemical response characteristics of sulfur ketone and nitroimidazole, which can effectively improve the recognition accuracy of the lesion site compared to a single responsive carrier. On this basis, the dynamic coordination network of Fe 3+ -carboxyl is further optimized, i.e., the polymer PEG-b-PLL-g-NI, TCA and Fe 3+Fe 3+ The Fenton reaction can be catalyzed by the released Fe to generate hydroxyl radicals from H2O2, thereby increasing the local reactive oxygen species level and further accelerating the cleavage of the thioacetal bond of TCA to form an "active oxygen -> TCA dissociation -> Fe 3+ release -> active oxygen enhancement -> accelerated dissociation" self-feedback cycle, thereby improving the drug release efficiency. 3+ After the dissociation of the surface TCA-Fe

[0041] Compared with the prior art, the active oxygen / hypoxia dual-responsive drug delivery carrier provided by the application, and the preparation method and application thereof, have the following remarkable advantages:

[0042] 1) The responsive unit used in the application can release cinnamaldehyde synchronously and consume NADPH at the lesion site in addition to its own response characteristics, thereby increasing the sensitivity of tumors to ferroptosis and cooperating with cinnamaldehyde to stimulate mitochondria and Fe 3+ Fenton to perform tumor ferroptosis treatment.

[0043] 2) The structure is stable and controllable, and the surface Fe 3+ and the dynamic coordination network of the TCA carboxyl group can be stable for up to seven days under physiological conditions.

[0044] 3) The shell is formed by one-step self-assembly of an amphiphilic block copolymer and ionic coordination, and the assembly method is simple and has high batch repeatability.

[0045] 4) The molecular logic "AND" gate design of the thioacetal (ROS response) and nitroimidazole (hypoxia response) reduces non-specific drug release and realizes cascade responsive drug controlled release. Only when both "active oxygen (ROS) + hypoxia" characteristics of the tumor microenvironment exist, the double-sensitive bonds on the micelles will be cleaved in turn to trigger drug release; if either condition is not met, the drug will be "locked" in the carrier, thereby reducing the damage to healthy tissues.

[0046] In summary, the responsive drug-loaded micelles prepared according to the present application can realize responsive controlled release of drugs under the stimulation of hydrogen peroxide in vitro and sodium dithionite simulated hypoxic environment, reduce drug leakage in non-lesion sites, and realize maximum utilization of drugs. The active oxygen / hypoxia dual-responsive drug carrier provided by the present application realizes precise activation and self-enhanced release through pathological microenvironment characteristics, has high drug loading rate, low systemic toxicity and synergistic treatment advantages. The innovative design integrates microenvironment response, microenvironment regulation and synergistic treatment together, and shows broad application prospects in the treatment of diseases such as tumor treatment and infected wound repair, in which oxidative stress / hypoxia coexist. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 is a preparation method flow chart of an active oxygen / hypoxia dual-responsive drug carrier according to the present application;

[0048] Figure 2 is a nuclear magnetic resonance hydrogen spectrum diagram of nitroimidazole modified PEG-b-PLL-g-NI prepared in example 1 of the present application;

[0049] Figure 3 is a nuclear magnetic resonance hydrogen spectrum diagram of active oxygen-responsive cinnamaldehyde prodrug TCA prepared in example 1 of the present application;

[0050] Figure 4 is a mass spectrum diagram of active oxygen-responsive cinnamaldehyde prodrug TCA prepared in example 1 of the present application;

[0051] Figure 5 is a pure PEG-b-PLL-g-NI polymer micelle PM prepared in example 1 of the present application, a sorafenib model drug-loaded micelle sora@PM, a TCA-Fe 3+ complexed sorafenib drug-loaded micelle sora@FTPM and TCA-Fe 3+ complex;

[0052] Figure 6 is a PDI diagram of PM, sora@PM, sora@FTPM and FT prepared in example 1 of the present application;

[0053] Figure 7 is a TEM diagram of PM, sora@PM, sora@FTPM and FT prepared in example 1 of the present application;

[0054] Figure 8 is a Zeta potential diagram of PM, sora@PM, sora@FTPM and FT prepared in example 1 of the present application;

[0055] Figure 9 is the nuclear magnetic resonance hydrogen spectrum of TCA prepared according to the embodiment 1 of the present application before and after hydrogen peroxide treatment;

[0056] Figure 10 is the ultraviolet-visible spectrum of PEG-b-PLL-g-NI prepared according to the embodiment 1 of the present application before and after sodium hydrosulfite treatment;

[0057] Figure 11 is the fluorescence spectrum of PEG-b-PLL-g-NI prepared according to the embodiment 1 of the present application before and after sodium hydrosulfite treatment with fluorescence amine as amino indicator;

[0058] Figure 12 is the Ce6 drug release curve of Ce6@FTPMs prepared according to the embodiment 1 of the present application before and after different treatments;

[0059] Figure 13 is the CA drug release curve of Ce6@FTPMs prepared according to the embodiment 1 of the present application before and after different treatments;

[0060] Figure 14 is the Fe drug release curve of Ce6@FTPMs prepared according to the embodiment 1 of the present application before and after different treatments; 3+

[0061] Figure 15 is the treatment effect of sora@FTPMs prepared according to the embodiment 1 of the present application on 803 tumor cells with CCK-8 as indicator. DETAILED DESCRIPTION

[0062] The present application will be further described in conjunction with specific examples. It should be understood that the following examples are intended to be illustrative only and are not intended to limit the scope of the present application. Unless otherwise specified, the technical means used in the examples are routine operations in the art, or follow the experimental methods suggested by the manufacturers of the instruments and equipment. The reagents and materials used in the examples are commercially available unless otherwise specified.

[0063] According to the present application, a preparation method of an active oxygen / hypoxia dual-responsive drug carrier is provided, which comprises the following steps: Figure 1 ​The synthesis process is shown, including steps: 1) inducing ring-opening polymerization of lysine-NCA to synthesize PEG-b-PLL with methoxyl polyethylene glycol amine as an initiator; 2) modifying PEG-b-PLL through amide reaction to obtain PEG-b-PLL-g-NI; 3) generating active oxygen responsive cinnamaldehyde prodrug TCA (3,3-bis-(2-carboxy-ethyl sulfanyl)-1T phenylpropylene-(1)) through the sulfur acetal reaction between cinnamaldehyde and mercaptopropionic acid; 4) dissolving PEG-b-PLL-g-NI, TCA, FeCl3·6H2O and drugs in DMSO in a certain proportion to form a uniform solution; 5) self-assembling to form drug-loaded micelles through the nano-precipitation method; 6) dialyzing the synthesized drug-loaded micelles against deionized water to remove unloaded components and DMSO, thereby obtaining an active oxygen / hypoxia-responsive drug carrier.

[0064] Example 1

[0065] Lys(Z)-NCA (0.38 g, 1.2 mmol) and PEG-NH2 (Mn = 5000, 1 g, 0.10 mmol) were dissolved in anhydrous N,N-dimethylformamide (DMF, 30 mL) under an argon atmosphere, and the reaction was continued at 35°C for 3 days to obtain PEG-b-PLL(Z). Under argon protection, PEG-b-PLL(Z) (0.72 g, 0.06 mmol) was dissolved in trifluoroacetic acid, followed by the addition of a 33% (w / v) HBr / acetic acid solution, and a deprotection reaction was carried out at room temperature for 2 h to synthesize PEG-b-PLL.

[0066] NI-COOH (338.572 mg, 1.7 mmol) was dissolved in anhydrous DMF, and EDCI (0.33 g, 1.7 mmol) and 1-hydroxybenzotriazole (HOBt, 0.23 g, 1.7 mmol) were added in sequence, and the carboxylic acid was stirred at room temperature for 1 h. Subsequently, a DMF mixed solution of PEG-b-PLL (0.2 g, 0.017 mmol) and N,N-diisopropylethylamine (DIPEA, 0.3 mL, 5.1 mmol) was injected into the reaction system, and the reaction was continued at 35°C under nitrogen protection for 24 h. After the reaction was completed, the crude product was precipitated with ice ether (3×50 mL), and the nitroimidazole-modified PEG-b-PLL-g-NI was obtained by reduced pressure filtration, Figure 2 The presence of nitroimidazole characteristic peaks 13 and 14 proves the successful grafting.

[0067] CA (3.25 g, 24.5 mmol) and 3-mercaptopropionic acid (5.72 g, 54.01 mmol) were dissolved in 10 mL of ethyl acetate, and a catalytic amount of trifluoroacetic acid was added to the reaction system in the dark. The reaction mixture was continuously stirred at room temperature for 24 h, and the reaction progress was monitored by thin layer chromatography (n-hexane / ethyl acetate = 3:1, v / v). After the reaction was completed, the mixture was filtered through a Buchner funnel, washed with pre-cooled deionized water and hexane in sequence to remove unreacted raw materials and by-products, and dried at 40 °C under vacuum for 12 h to obtain white crystalline TCA. The product was confirmed by proton nuclear magnetic resonance spectroscopy (1H NMR) ( Figure 3 ), and its mass spectrum is shown in Figure 4 .

[0068] PEG-b-PLL-g-NI (5.0 mg), TCA (3.0 mg), FeCl3·6H2O (1.656 mg, 6 μmol), and sorafenib (1.0 mg) were weighed and dissolved in 200 μL of DMSO, and ultrasonically treated for 15 min until completely dissolved. The above solution was added dropwise to 5 mL of vigorously stirred deionized water to induce nanoscale assembly formation. Subsequently, the micelle solution was dialyzed using a dialysis bag with a molecular weight cutoff of 14 kDa to remove free drugs and uncoordinated metal ions, and finally obtained nanoscale assembly sora@FTPMs. The remaining synthesis method and feed ratio were consistent with Ce6@FTPMs, PMs, FT, etc., only the feed material was changed, and the particle size and PDI of the obtained product were characterized by transmission electron microscopy and DLS. TEM images ( Figure 7 ) confirmed that the drug-loaded composite micelles had uniform and stable spherical morphology, and DLS results ( Figure 5 ) showed that the particle size of the drug-loaded composite micelles was about 100 nm. The PDI results ( Figure 6 ) confirmed that the uniformity of the drug-loaded composite micelles formed by coordination was significantly improved. Zeta potential images ( Figure 8 ) proved that the drug was successfully modified to the micelle surface.

[0069] To verify the responsiveness of different components, 10 mg of TCA was treated with 1 mM of hydrogen peroxide and detected by NMR ( Figure 9 ). The cleavage of TCA and the release of cinnamaldehyde were confirmed by the addition of characteristic peaks. The characteristic absorption peak change of PEG-b-PLL-g-NI aqueous solution treated with 5 mM sodium dithionite was verified by UV-visible spectroscopy ( Figure 10 ) to determine its responsiveness. At the same time, fluorescent amine was used as an indicator to characterize the change in the number of free primary amines in the solution ( Figure 11 ). The increase in fluorescence intensity in the fluorescence spectrum can confirm that the nitroimidazole is changed to aminoimidazole after hypoxic treatment.

[0070] To verify the drug stimulation response, the model drug was replaced by Ce6, and its release in different media was detected by dialysis diffusion. Specifically, the oxidative stress microenvironment was simulated by 1 mM H2O2, and the hypoxic microenvironment was simulated by 1-5 mM sodium dithionite solution. 3 mL of drug solution was placed in a dialysis bag and dialyzed in 30 mL of release medium. At a given time point, 3 mL of dialysis medium was taken for determination. The concentration of Ce6 and cinnamaldehyde was confirmed by UV-visible spectroscopy, and the release concentration of Fe was confirmed by ICP. Ce6 drug release curve of Ce6@FTPMs before and after different treatments, CA drug release curve, Fe 3+ release curve are shown in Figure 12 、 Figure 13 、 Figure 14

[0071] Finally, the effectiveness of sora@FTPMs was verified in 803 tumor cell lines. After 803 cells were plated in a 96-well plate for adherent culture, the culture medium was replaced with fresh culture medium containing different concentrations of materials. After 24 h of continuous culture, cell activity was detected using a CCK-8 kit. The results are shown in Figure 15 The composite drug-loaded micelles produced the most excellent tumor inhibition effect in tumor cells endowed with hypoxia.

[0072] The above is only a preferred embodiment of the present application, not to limit the scope of the present application, the above embodiment of the present application can also be made various changes. Any simple, equivalent changes and modifications made according to the content of the claims and the specification of the present application, all fall within the scope of the present application. The present application is not described in detail, all are conventional technical content.​

Claims

1. A method for preparing a reactive oxygen species / hypoxia dual-response drug delivery carrier, characterized in that, Includes the following steps: S1, using mPEG-NH2 as an initiator to induce the ring-opening polymerization of Lys-NCA to obtain PEG-b-PLL(Z); then, the benzyloxycarbonyl group protection is removed in trifluoroacetic acid with HBr / acetic acid solution, and PEG-b-PLL is obtained by precipitating with diethyl ether. S2, after activating NI-COOH in DMF with EDCI / HOBt, is coupled with mPEG-b-PLL and DIPEA under nitrogen; the reaction solution is added dropwise to pre-cooled diethyl ether to precipitate the crude product, which is then filtered under reduced pressure and washed with diethyl ether to obtain nitroimidazole modified PEG-b-PLL-g-NI; S3, cinnamaldehyde and 3-mercaptopropionic acid are dissolved in ethyl acetate, and trifluoroacetic acid is added as a catalyst under light-protected conditions. The mixture is stirred in the dark to generate the reactive oxygen species-responsive cinnamaldehyde prodrug TCA by the thioacetal reaction between cinnamaldehyde and mercaptopropionic acid. S4, weigh PEG-b-PLL-g-NI, TCA, FeCl3·6H2O and the drug and dissolve them in anhydrous DMSO, and sonicate to form a homogeneous solution; S5. The above solution is added dropwise to vigorously stirred deionized water to induce the formation of drug-loaded micelles from the nano-assemblies. S6. Dialyze the synthesized drug-loaded micelles with deionized water to remove unloaded components and DMSO, thereby obtaining a reactive oxygen species / hypoxia responsive drug carrier.

2. The preparation method according to claim 1, characterized in that, Step S1 includes: using H-Lys(Z)-OH as a raw material, reacting it with triphosgene in THF, and then recrystallizing it under reduced pressure and in a mixed solvent of hexane / tetrahydrofuran to obtain high-purity Lys-NCA; subsequently, using mPEG-NH2 as an initiator, initiating the ring-opening polymerization of Lys-NCA in anhydrous DMF, and purifying it by precipitation with ice-cold diethyl ether to obtain PEG-b-PLL(Z); further dissolving PEG-b-PLL(Z) in trifluoroacetic acid, adding HBr / acetic acid solution to remove the benzyloxycarbonyl protecting group, and finally obtaining PEG-b-PLL after precipitation with ice-cold diethyl ether.

3. The preparation method according to claim 2, characterized in that, In step S1, the molecular weight of mPEG-NH2 is in the range of 2000-10000 Da, and the molar ratio of Lys-NCA to mPEG-NH2 is (20-60):

1.

4. The preparation method according to claim 2, characterized in that, In step S1, the volume ratio of the hexane / tetrahydrofuran mixed solvent used for NCA single recrystallization is (2-5):1; the volume ratio of the trifluoroacetic acid and HBr / acetic acid solution used for deprotection is (2-4):

1.

5. The preparation method according to claim 1, characterized in that, In step S2, the amount of EDCI and HOBt used to activate the carboxyl group is 1-1.5 equivalents of the carboxyl group in NI-COOH, and the activation time is 0.5-1.5h.

6. The preparation method according to claim 1, characterized in that, In step S3, the molar ratio of cinnamaldehyde to 3-mercaptopropionic acid is 1:(2-3), and the amount of trifluoroacetic acid used is the catalytic amount.

7. The preparation method according to claim 1, characterized in that, In step S4, the mass ratio of PEG-b-PLL-g-NI, TCA, FeCl3·6H2O and the drug is 5:(1-4):(0.5-3):(0.5-3).

8. The preparation method according to claim 1, characterized in that, In step S5, the volume of deionized water is 4-10 times the volume of DMSO; in step S6, the dialysis medium is deionized water, which is replaced every 2 hours for a total of 24 hours of dialysis.

9. A reactive oxygen species / hypoxia dual-response drug delivery carrier prepared by the preparation method according to any one of claims 1-8, characterized in that, The core of the reactive oxygen species (ROS) / hypoxia dual-response drug delivery carrier is a hypoxia-responsive nitroimidazole unit. The shell is an electrostatic coordination network formed by the carboxyl group of TCA, iron ions, and amino groups of polylysine segments through coordination. The ROS / hypoxia dual-response drug delivery carrier only partially dissociates under single ROS or hypoxia stimulation, and only completely dissociates under dual ROS and hypoxia stimulation, thereby achieving responsive controlled drug release.

10. The use of the reactive oxygen species / hypoxia dual-response drug delivery carrier as described in claim 9 in the preparation of a medicament for treating tumors characterized by reactive oxygen species overexpression and hypoxic microenvironment.

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