Nano preparation based on redox reactivity drug delivery and preparation method thereof

Abstract

The invention discloses a drug delivery nano preparation based on redox reactivity and a preparation method, the drug delivery nano preparation comprises a conjugate as a carrier, the conjugate is a natural polymer prodrug prepared by coupling carboxymethyl chitosan (CMC) with doxorubicin (DOX) through a redox reactivity joint; 3-(5 '-hydroxymethyl-2-furyl)-1-benzyl indazole (YC-1) is encapsulated in a micelle formed by self-assembly of the conjugate, so that the double-drug nano platform can sequentially release two drugs, namely YC-1 and DOX. And the redox reactive joint is 3-(2-pyridine dimercapto) propionic acid N-hydroxysuccinimide ester (SPDP). The single drug components can show a synergistic effect after being sequentially released, DOX and YC-1 show a synergistic effect under a low-oxygen condition within a set concentration range, the synergistic effect is stronger under a relatively high concentration, DOX promotes nuclear DNA damage and mitochondrial damage and induces tumor cell apoptosis, and the effect of inhibiting tumor apoptosis is achieved. Load of YC-1 can effectively reverse chemotherapy drug resistance of hypoxia tumor cells.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceutical preparations, and in particular to a redox-reactive drug delivery nanoformulation and a preparation method thereof. Background Art

[0002] Chemotherapy remains crucial in clinical oncology and enhances therapeutic efficacy by inducing tumors to release damage-associated pattern molecules (DAMPs) that trigger immunogenic cell death (ICD), thereby activating anti-tumor immunity.

[0003] For example, a Chinese invention patent application (application publication number: CN116688140A, application publication date: 2023.09.05) discloses a near-infrared light-responsive drug delivery system and its preparation method and application, which includes upconversion nanoparticles, a mesoporous silica shell coated on the surface of the upconversion nanoparticles, drug molecules adsorbed in the pores of the mesoporous silica shell, a near-infrared dye coupled to the surface of the mesoporous silica shell, adamantane coupled to the near-infrared dye, and a β-cyclodextrin complexed with adamantane. The nano drug delivery system of the above scheme can stably encapsulate drug molecules in the mesopores, and respond quickly to release based on the self-sensitive photooxidation bond breaking of the near-infrared dye in the system under the irradiation of near-infrared light (700-900nm) with a lower power density, and control the illumination time to meet the quantitative drug delivery requirements. In addition, the upconversion luminescence of the nano drug delivery system of the above scheme under 980nm laser excitation does not affect the release of drug molecules, and can realize the drug positioning and bioimaging functions of the nano drug delivery system.

[0004] As can be seen from the above scheme, in recent years, drug delivery systems with time-controlled release function have developed rapidly, and the ideal sustained-release function has given it excellent transformation potential. Among them, near-infrared light-responsive drug delivery systems mainly use the time-controlled quantitative drug delivery requirements of near-infrared light excitation; however, the thermal effect generated by near-infrared has become one of the key factors limiting the further development and application of such drug delivery systems.

[0005] For example: China's invention patent application (application publication number: CN113713120AA, application publication date: 2021.11.30) discloses a carboxymethyl chitosan nanogel for delivering anti-tumor drugs, in which CMCS is modified with glycidyl methacrylate (GMA), and the GCMCS grafted with double bonds is cross-linked with N,N-bis(acryloyl)cysteamine (BAC) containing disulfide bonds as a cross-linking agent, and then modified with the tumor active targeting molecule folic acid (FA) to prepare a targeted nanogel for the delivery of the anti-tumor drug doxorubicin (DOX); after the nanogel is loaded with DOX, under the stimulation of high concentrations of GSH in the tumor environment, the disulfide bonds in its structure are cleaved, thereby releasing DOX to play a role in killing tumor cells; the modification with folic acid adds an active targeting effect to the nanogel, and the carrier remains stable during the circulation in the body. After reaching the tumor site, it can release the loaded drug at a fixed point through stimulus response, thereby increasing the drug concentration in tumor tissues and cells, thereby improving the efficacy.

[0006] However, the presence of inhibitory DAMPs often disrupts the process of anti-tumor immune activation and easily leads to an immunosuppressive tumor microenvironment (TEM), thereby reducing the therapeutic effect. Summary of the invention

[0007] The purpose of the present invention is to provide a redox-reactive drug delivery nanoformulation and a preparation method thereof to solve the problems raised in the above-mentioned background technology.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] A redox-responsive drug delivery nanoformulation, comprising a conjugate as a carrier, wherein the conjugate is a natural polymer prodrug prepared by coupling carboxymethyl chitosan (CMC) with doxorubicin (DOX) via a redox-responsive linker;

[0010] The micelles formed by the self-assembly of the conjugate encapsulate 3-(5'-hydroxymethyl-2-furyl)-1-benzylindazole (YC-1).

[0011] As a further embodiment of the present invention, the redox-reactive linker is 3-(2-pyridyldithiol) propionic acid N-hydroxysuccinimide ester (SPDP).

[0012] The conjugate CMC-DOX of the present invention is an amphiphilic macromolecule, which is composed of a hydrophilic polysaccharide skeleton and a hydrophobic DOX part with an anthracycline drug group. In an aqueous solution, the hydrophobic chain segments with anthraquinone groups self-aggregate to form multiple hydrophobic microdomains, while the hydrophilic segments of CMC show high affinity for water molecules, curling on the surface of the hydrophobic microdomain to form a hydrophilic shell, thereby stabilizing the structure in the lowest energy state; at the same time, the hydrophobic YC-1 can aggregate into the core of the hydrophobic microdomain through hydrophobic interactions, thereby forming NP-DY. The encapsulated YC-1 shows the characteristics of rapid release in the presence of GSH, which can inhibit the HIF-1α / P-gp axis, reduce drug efflux, and restore the balance between immune stimulation and DAMPs inhibition to promote ICD. After YC-1 is released, the coupled DOX can achieve continuous redox-dependent release, can be highly accumulated in cancer cells, thereby playing a dual role of chemotherapy and triggering anti-tumor immune response. NP-DY shifts the balance of DAMPs in the tumor microenvironment toward immunostimulatory properties, ultimately activating a robust antitumor immune response in hypoxia-induced chemoresistant breast cancer.

[0013] A method for preparing a redox-reactive drug delivery nanoformulation comprises the following steps:

[0014] S1, combining carboxymethyl chitosan and a redox-reactive linker to obtain carboxymethyl chitosan having a redox-reactive linker;

[0015] S2, combining carboxymethyl chitosan having a redox linker and doxorubicin to prepare a conjugate as a carrier;

[0016] S3. Encapsulating 3-(5'-hydroxymethyl 1-2-furyl)-1-benzylindazole (YC-1) into micelles formed by self-assembly of the conjugate can obtain a drug delivery nanoformulation.

[0017] As a further solution of the present invention, the preparation method of the carboxymethyl chitosan having a redox joint in step S1 is:

[0018] 60 mg of 3-(2-pyridyldithiol) propionic acid N-hydroxysuccinimide ester (SPDP) dissolved in 6 mL of dimethyl sulfoxide (DMSO) was added dropwise to 40 mg of carboxymethyl chitosan (CMC) dissolved in 40 mL of phosphate buffered saline (PBS, pH 8.0), and the mixture was allowed to react at 30 °C for 24 h;

[0019] Then, the reaction product was dialyzed in ultrapure water for 72 h using a dialysis bag (MWCO 3500 Da), and after freeze-drying, carboxymethyl chitosan with a redox linker (CMC-SPDP powder) was obtained.

[0020] As a further embodiment of the present invention, the method of using the conjugate as a carrier in step S2 is:

[0021] 25 mg of carboxymethyl chitosan with redox linker (CMC-SPDP) was dissolved in 10 mL of PBS (pH 8.0), and 12.5 mg of doxorubicin (DOX) (dissolved in 1 mL of DMSO) was added under argon atmosphere, and the reaction was stirred at 30 °C for 24 h;

[0022] Then, the reaction product was dialyzed in ultrapure water for 48 h using a dialysis bag (MWCO 3500 Da) to produce a conjugate (CMC-DOX).

[0023] As a further solution of the present invention, the specific steps of encapsulating 3-(5'-hydroxymethyl 1-2-furyl)-1-benzylindazole (YC-1) into the conjugate (CMC-DOX) micelle in step S3 are:

[0024] The conjugate (CMC-DOX) with a doxorubicin (DOX) content of 2 mg (DOX content was measured by UV spectrophotometry (TECAN, Spark)) was dissolved in 10 mL PBS (pH 8.0);

[0025] Then, 4 mg of 3-(5′-hydroxymethyl 1-2-furyl)-1-benzylindazole (YC-1) dissolved in 20 μL of dimethyl sulfoxide (DMSO) was added to the solution, and sonicated at 300 W for 30 min using a non-contact ultrasonic instrument (Diaggenode, Bioruptor Plus).

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The drug delivery nanoformulation of the present invention can respond to the reductive tumor environment, promote the sequential release of YC-1 and DOX, achieve more drug accumulation and sustained retention in tumor cells, thereby enhancing the therapeutic effect and reducing systemic toxicity;

[0028] 2. The single drug components of the redox-responsive drug delivery nanoformulation of the present invention exhibit synergistic effects. DOX and YC-1 exhibit synergistic effects under hypoxic conditions within a set concentration range, and the synergistic effects are stronger at higher concentrations. DOX promotes nuclear DNA damage and mitochondrial damage, and induces tumor cell apoptosis. The loading of YC-1 can effectively reverse the chemotherapy resistance of hypoxic tumor cells.

[0029] 3. The drug delivery nanoformulation of the present invention can inhibit the growth rate of tumors. Compared with single therapy, combined drug therapy can enhance the recruitment and activation of immunosuppressive cells. It first inhibits the HIF-1α / p-gp signaling axis through the release of YC-1, overcomes the chemotherapy resistance of tumors under hypoxic environment, and thus enhances the anti-tumor effect of the subsequently released DOX, which is beneficial to promote the release of immunogenic DAMPs in vitro and in vivo, overcomes the obstacles of hypoxic tumor environment, has excellent tumor ablation effect, and can effectively induce local ICD in tumors;

[0030] In summary, the redox-responsive drug delivery nanoformulation of the present invention can overcome the problem of hypoxia in the tumor immune microenvironment, achieve synergistic effects, and further improve the tumor ablation effect; redox-responsive combined drug therapy shows more effective biological effects, providing broad application prospects for the treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A schematic diagram of the preparation process of a redox-responsive drug delivery nanoformulation;

[0032] Figure 2 It is a transmission electron microscopy, particle size, potential and UV absorption spectrum of various drugs of a redox-responsive drug delivery nanoformulation;

[0033] Figure 3 This is a sequential drug release result diagram of a redox-responsive drug delivery nanoformulation;

[0034] Figure 4 Cellular endocytosis of a redox-responsive drug delivery nanoformulation

[0035] Figure 5 The results of CCK-8 testing for a redox-responsive drug delivery nanoformulation;

[0036] Figure 6 This is a graph showing the in vitro tumor inhibition results of a redox-responsive drug delivery nanoformulation;

[0037] Figure 7 The in vivo tumor-suppressing effect of a redox-responsive drug delivery nanoformulation;

[0038] Figure 8 This is the WB test result of the in vivo tumor-suppressing protein expression associated with a redox-responsive drug delivery nanoformulation. DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] See also Figures 1 to 8 , the present invention provides a technical solution: a redox-responsive drug delivery nanoformulation, comprising a conjugate as a carrier, wherein the conjugate is prepared by coupling carboxymethyl chitosan (CMC) with doxorubicin (DOX) via a redox-responsive linker;

[0041] The micelles formed by the self-assembly of the conjugate encapsulate 3-(5'-hydroxymethyl-2-furyl)-1-benzylindazole (YC-1).

[0042] Specifically, the redox-reactive linker is 3-(2-pyridyldithiol) propionic acid N-hydroxysuccinimide ester (SPDP), and carboxymethyl chitosan (CMC) is combined with SPDP and then combined with the chemotherapy drug doxorubicin to obtain a conjugate with drug-carrying capacity.

[0043] Among them, 3-(5'-hydroxymethyl-2-furyl)-1-benzylindazole (YC-1), as a hypoxia-inducible factor 1-α (HIF-1α) inhibitor, can overcome the chemoresistance of hypoxic tumors by inhibiting the HIF-1α / p-gp signaling axis, thereby enhancing the antitumor effect of doxorubicin (DOX) and promoting the release of immunogenic DAMPs in vitro and in vivo.

[0044] In the present invention, 3-(2-pyridyldithiol) propionic acid N-hydroxysuccinimide ester (SPDP) is used as a bifunctional cross-linking agent to conjugate the amino group of carboxymethyl chitosan (CMC) with the thiol group of doxorubicin (DOX) to form a conjugate containing a disulfide bond (CMC-DOX), and then the conjugate is self-assembled with 3-(5'-hydroxymethyl-2-furyl)-1-benzylindazole (YC-1) to obtain the drug delivery nanoformulation (NP-DY) of the present invention.

[0045] In the nanoformulation (NP-DY) of the present invention, CMC-DOX is an amphiphilic macromolecule consisting of a hydrophilic polysaccharide backbone and a hydrophobic DOX portion with an anthracycline drug group;

[0046] In aqueous solution, the hydrophobic chains with anthraquinone groups spontaneously aggregate to form multiple hydrophobic microdomains, while the hydrophilic domains of CMC show high affinity for water molecules and curl up to form a hydrophilic shell on the surface of the hydrophobic domains, thereby stabilizing the structure of the preparation in the lowest energy state; at the same time, the hydrophobic YC-1 aggregates into the core of the hydrophobic domain through hydrophobic interactions to form NP-DY.

[0047] The encapsulated YC-1 in the present invention exhibits the characteristics of rapid release in the presence of GSH, which can inhibit the HIF-1α / P-gp axis, reduce drug efflux, and restore the balance between immune stimulation and DAMPs inhibition to promote ICD; at the same time, YC-1 also cooperates with DOX coupled to NP-DY to achieve sustained and redox-dependent release, and can be highly accumulated in cancer cells, thereby exerting a dual effect of chemotherapy and triggering an anti-tumor immune response.

[0048] A method for preparing a redox-responsive drug delivery nanoformulation (i.e., a dual-drug nanoplatform) comprises the following steps:

[0049] S1. Preparation of carboxymethyl chitosan with redox linker:

[0050] 60 mg of 3-(2-pyridyldithiol) propionic acid N-hydroxysuccinimide ester (SPDP) dissolved in 6 mL of dimethyl sulfoxide (DMSO) was added dropwise to 40 mg of carboxymethyl chitosan (CMC) dissolved in 40 mL of phosphate buffered saline (PBS, pH 8.0), and the mixture was allowed to react at 30 °C for 24 h;

[0051] Then, the reaction product was dialyzed in ultrapure water for 72 h using a dialysis bag (MWCO 3500 Da), and after freeze-drying, CMC-SPDP powder, i.e., carboxymethyl chitosan with a redox linker, was obtained.

[0052] S2. Construction of conjugate (CMC-DOX):

[0053] 25 mg of carboxymethyl chitosan with redox linker (CMC-SPDP) was dissolved in 10 mL of PBS (pH 8.0), and 12.5 mg of doxorubicin (DOX) (dissolved in 1 mL of DMSO) was added under argon atmosphere, and the reaction was stirred at 30 °C for 24 h;

[0054] Then, the reaction product was dialyzed in ultrapure water for 48 h using a dialysis bag (MWCO 3500 Da) to produce a conjugate (CMC-DOX).

[0055] Preparation of dual-drug nanoplatform (NP-DY) co-loaded with S3, YC-1 and DOX:

[0056] The conjugate (CMC-DOX) with a doxorubicin (DOX) content of 2 mg was dissolved in 10 mL PBS (pH 8.0), wherein the DOX content was measured by UV spectrophotometry (TECAN, Spark);

[0057] Then, 4 mg of 3-(5′-hydroxymethyl 1-2-furyl)-1-benzylindazole (YC-1) dissolved in 20 μL of dimethyl sulfoxide (DMSO) was added to the solution, and sonicated at 300 W for 30 min using a non-contact ultrasonic instrument (Diaggenode, Bioruptor Plus).

[0058] To obtain the DOX-loaded nanoplatform (NP-D), the reaction mixture of CMC-DOX was dialyzed in ultrapure water for 48 h and then sonicated at 300 W for 30 min.

[0059] Similarly, to prepare the YC-1-loaded nanoplatform (NP-Y), 1 mg of CMC-SPDP in 10 mL of PBS (pH 8.0) was mixed with 4 mg of YC-1 (dissolved in 20 μL of DMSO) and sonicated for 30 min at 300 W using the same non-contact ultrasonic instrument. The dual-drug nanoplatform was then dispersed in ultrapure water for characterization.

[0060] The morphology of the dual-drug nanoplatform (NC-PY) was observed by transmission electron microscopy using phosphotungstic acid negative staining. Figure 2 A shows a transmission electron microscope image of the dual-drug nanoplatform (NC-PY); the hydrodynamic size and zeta potential of the dual-drug nanoplatform (NC-PY) were measured using a dynamic light scattering instrument. Figure 2 B shows the hydrated particle size of the dual-drug nanoplatform. Figure 2 C shows the Zeta potential and dispersibility index of the dual-drug nanoplatform in ultrapure water. Figure 2 D shows the UV absorption peaks of each drug and each nanoplatform analyzed by UV spectrophotometry.

[0061] 1. Detection of sequential drug release of dual-drug nanoplatform (NC-PY):

[0062] The drug release profiles of NP-DY under different conditions were evaluated using a multifunctional microplate reader. 1 mL of NP-DY dispersion was added to a dialysis bag (MWCO 3500 Da) and placed in 4 mL of PBS (pH 7.4, with / without GSH) containing 0.2% (w / v) Tween 80. The assay was performed in triplicate.

[0063] The samples were incubated in a shaking water bath at 37 °C with an agitation speed of 150 rpm, and at designated time points, 1 mL of the release medium was collected and replaced with an equal volume of fresh medium. Figure 3 A shows the concentration of DOX in the release medium measured by fluorescence (excitation wavelength: 479 nm; emission wavelength: 587 nm); Figure 3 B shows the concentration of YC-1 in the release medium measured by UV spectrophotometry.

[0064] 2. Study on in vitro cell uptake of dual-drug nanoplatform:

[0065] 4T1 cells were suspended in medium containing 10% fetal bovine serum and then seeded into 35 mm glass-bottomed culture dishes (Biosharp). Incubate at 37°C for 24 hours to allow cells to adhere. Subsequently, the cells were transferred to a normoxic incubator or hypoxic chamber for 6 hours, and the medium was replaced with 1 mL of fresh medium containing PBS, NP-Y, NP-D or NP-DY, respectively, and incubated at 37°C for different time intervals (12 hours and 16 hours). After incubation, the cells were washed three times with 1 mL of PBS and the nuclei were stained with Hoechst 33342. Cell imaging was performed using CLSM (Zeiss LSM880). Figure 4 A shows the condition of cell hypoxia. Figure 4 B shows the process of endocytosis mechanism. Figure 4 C shows the cell endocytosis effect determined by immunofluorescence technique.

[0066] 3. Detection of cytotoxicity of dual-drug nanoplatform (NC-PY):

[0067] 4T1 cells (1×10 4 ) were seeded in 96-well plates and incubated at 37°C for 24 hours to maintain adhesion; the cells were then transferred to a normoxic incubator or an anoxic chamber for 6 hours; thereafter, the cells were incubated at 37°C for 22 hours with 1 mL of fresh medium containing PBS, NP-Y, NP-D, or NP-DY, respectively, where DOX concentrations were 2.4, 4, 8, 12, and 16 μg / mL, and YC-1 concentrations were 4.8, 8, 16, 24, and 32 μg / mL; after treatment, 10 μL of CCK-8 was added to each well, and the plates were incubated at 37°C for another hour; the absorbance at 450 nm was measured using a microplate reader (TECAN Spark), Figure 5 a and Figure 5 b shows the cell viability reflected by the ratio of absorbance values ​​between the experimental group and the control group.

[0068] To verify the cell-killing ability of the dual-drug nanoplatform, 4T1 cells (1×105 per well) were seeded in 12-well plates and incubated at 37°C for 24 hours to maintain adhesion; then transferred to a normoxic incubator or a hypoxic chamber for 6 hours; then, the cells were incubated at 37°C for 22 hours with 1 mL of fresh culture medium containing DOX or YC-1, where the DOX concentration was 12.5 μg / mL and the YC-1 concentration was 25 μg / mL; after incubation, the culture medium was replaced with freshly prepared calcein AM / PI solution, the cells were incubated for 30 minutes, and then observed under an inverted fluorescence microscope (Mshot, MF52-N); Figure 6 Shown are the results of evaluating cytotoxicity using the Annexin V-FITC / SYTOX-APC double staining apoptosis detection kit.

[0069] 4. Detection of the anti-tumor efficacy of dual-drug nanoplatform cells in vivo:

[0070] Female BALB / c mice were injected subcutaneously on the right flank with 4T1 tumor cells (3 × 10 per mouse). 6 4T1 tumor-bearing mice were randomly divided into 4 groups (n=5) one week later, and then received the following treatments: intravenous injection of PBS, NP-Y, NP-D or NP-DY (8 mg / kg DOX or 16 mg / kg YC-1 per dose) for 6 times; during the treatment, the tumor volume of the mice was monitored every two days, and the tumor volume was calculated as follows: Tumor volume (mm 3 ) = Width 2 (mm 2 )×length (mm)×0.5.

[0071] Figure 7 A shows the changes in tumor volume in different groups during treatment; Figure 7 B shows the actual status of mouse tumors at the end of the detection on day 14.

[0072] 5. Expression of specific proteins acted by the dual-drug nanoplatform:

[0073] At the end of the 14th day of monitoring, mice were killed and tumors were collected. Tissue samples were taken and crushed, and total tumor cell protein was extracted. The total protein extract was analyzed by SDS-PAGE, electrophoresed at 100V, and then transferred to the membrane at 100V for 40min. In order to block nonspecific binding sites, 5% skim milk was prepared with PBST to block the membrane for 1.5h. After blocking, the primary antibody was incubated at 4℃ overnight, washed 3 times with PBST, and then incubated with goat anti-rabbit IgG antibody (1 / 10000 dilution) for 1.5h. Finally, the expression of protein bands was observed by development (such as Figure 8 shown).

[0074] Figure 8 Western blotting was used to analyze the expression of apoptosis-related proteins in tumor cells after different treatments. The P-gp and HIF-1α protein bands in the "NP-DY" group were significantly reduced compared with those in other groups, inhibiting the HIF-1α / p-gp signaling axis and overcoming the chemotherapy resistance of hypoxic tumors.

[0075] From the above preparation process and experimental data, it can be seen that:

[0076] 1. The dual-drug nanoplatform of the present invention does not require complicated design and preparation processes and will not cause functional biological toxicity;

[0077] 2. The drug loading rate of the dual-drug nano-platform of the present invention can respond to the reductive tumor environment, promote the continuous release of the dual-drug, and have good biocompatibility and a long blood circulation time;

[0078] 3. The therapeutic agent (YC-1) overcomes the chemoresistance of hypoxic tumors by inhibiting the HIF-1α / p-gp signaling axis, thereby enhancing the anti-tumor effect of DOX and promoting the release of immunogenic DAMPs in vitro and in vivo, enhancing the inhibitory effect on tumors.

[0079] The use of the natural polymer carboxymethyl chitosan of the present invention to co-load the chemotherapeutic agent DOX and the HIF-1 inhibitor YC-1 can shift the DAMP balance to an immunostimulatory state and enhance the systemic anti-tumor immune response by inhibiting the HIF-1α signaling axis. The natural polymer prodrug loading YC-1 leads to the sequential release of the two drugs, allowing tumor cells to preferentially take up YC-1 and promote the effective accumulation of DOX in hypoxic tumor cells. The dual-drug nanoplatform of the present invention exhibits an enhanced inhibitory effect on primary tumors. These encouraging results guarantee the translational potential of the combined treatment strategy in cancer treatment. The dual-drug nanoplatform developed in this study provides a potential strategy to enhance the systemic tumor treatment effect through chemotherapy and provides a theoretical basis for the development of clinical combined treatment strategies.

[0080] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A redox-responsive drug delivery nanoformulation, characterized in that: The invention comprises a conjugate as a carrier, wherein the conjugate is prepared by coupling carboxymethyl chitosan with doxorubicin via a redox reactive linker; The micelles formed by the self-assembly of the conjugate encapsulate 3-(5'-hydroxymethyl-2-furyl)-1-benzylindazole.

2. A redox-responsive drug delivery nanoformulation according to claim 1, characterized in that: The redox-reactive linker is 3-(2-pyridyldithiol) propionic acid N-hydroxysuccinimide ester.

3. A method for preparing a redox-reactive drug delivery nanoformulation, characterized in that: The steps include: S1, combining carboxymethyl chitosan and a redox-reactive linker to obtain carboxymethyl chitosan having a redox-reactive linker; S2, combining carboxymethyl chitosan having a redox linker and doxorubicin to prepare a conjugate as a carrier; S3. Encapsulating 3-(5'-hydroxymethyl 1-2-furyl)-1-benzyl indazole into micelles formed by self-assembly of the conjugate can obtain a drug delivery nanoformulation.

4. The method for preparing a redox-responsive drug delivery nanoformulation according to claim 3, characterized in that: The preparation method of the carboxymethyl chitosan having a redox joint in step S1 is: 60 mg of 3-(2-pyridyldithiol)propionic acid N-hydroxysuccinimide ester dissolved in 6 mL of dimethyl sulfoxide was added dropwise to 40 mg of carboxymethyl chitosan dissolved in 40 mL of phosphate buffered saline, and the mixture was allowed to react at 30°C for 24 h; Then, the reaction product was dialyzed in ultrapure water for 72 h using a dialysis bag and freeze-dried to obtain carboxymethyl chitosan with a redox linker.

5. The method for preparing a redox-responsive drug delivery nanoformulation according to claim 3, characterized in that: The method of using the conjugate as a carrier in step S2 is: 25 mg of carboxymethyl chitosan with redox linkers was dissolved in 10 mL of PBS, and 12.5 mg of doxorubicin was added under argon atmosphere and stirred at 30 °C for 24 h; Then, the reaction product was dialyzed in ultrapure water for 48 h using a dialysis bag to produce a conjugate.

6. The method for preparing a redox-responsive drug delivery nanoformulation according to claim 3, characterized in that: The specific steps of encapsulating 3-(5'-hydroxymethyl 1-2-furyl)-1-benzylindazole into the conjugate micelle in step S3 are: Dissolve 2 mg of the conjugate containing doxorubicin in 10 mL of PBS; Then, 4 mg of 3-(5′-hydroxymethyl 1-2-furyl)-1-benzylindazole dissolved in 20 μL of dimethyl sulfoxide was added to the solution, and sonicated at 300 W for 30 min using a non-contact ultrasonic instrument.

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