Nano-drug with low oxygen response and photoisomerization function, preparation and application thereof

Abstract

The application discloses a kind of nano-drugs with low oxygen response and photoisomerization function and preparation and application, belong to the field of biomedicine technology.The present application uses 4,4'-dichloro azobenzene as low oxygen response unit, which is covalently linked with 10-hydroxy camptothecin and drug carrier mPEG to form size-controllable nano-drugs, which have suitable particle size, more conducive to the enrichment of drug circulation in tumor site and play curative effect, when the nano-drugs reach the low-oxygen tumor site, the azo reductase unique to low-oxygen environment will reduce the azo group inside the nano-drugs, resulting in the disintegration of the nano-drug structure, thereby releasing 10-hydroxy camptothecin to play the efficacy of chemotherapy, and reduce the toxic and side effects of drug on normal tissues;In addition, the nano-drug has high utilization rate and good biocompatibility, so it has good application prospect in the preparation of antitumor drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a nano-drug with hypoxia responsiveness and photoisomerization function and preparation and application. BACKGROUND

[0002] According to the statistical data of the Cancer Research Institute, the number of cancer deaths in China reached 3 million in 2020, ranking first in the world. It is estimated that there will be 27 million new cancer patients in the world by 2030. Traditional tumor treatment methods, such as chemotherapy, radiotherapy and surgical resection, have many problems in clinical application. These methods often cannot accurately distinguish between tumor tissue and normal tissue, resulting in serious side effects on normal tissue while killing cancer cells. In addition, due to the individual differences, tissue heterogeneity and treatment timeliness of tumor tissue, and the incomplete understanding of the pathogenesis, the actual effect of traditional treatment methods is not ideal. The tumor microenvironment builds a natural barrier for cancer cells to escape the clearance of the body's immune system. Hypoxia, as one of the characteristics of most solid tumors, plays a key role in chemotherapy resistance, radiation resistance, angiogenesis, vascular permeability, invasiveness, metastasis, resistance to cell death, metabolic changes and genomic instability. There is overexpression of hypoxia-inducible factors (HIFs) in the tumor site, which can induce the expression of vascular endothelial growth factor to promote malignant tumor angiogenesis and increase vascular permeability, and promote the immune escape and multidrug resistance of tumor cells by regulating immune cells. The presence of hypoxia is a big dilemma and difficulty in current cancer treatment.

[0003] Camptothecin (CPT) and its derivative 10-hydroxycamptothecin (HCPT) are natural alkaloids extracted from the unique plant Camptotheca acuminata in China, which have good antitumor effect. However, they have problems such as poor water solubility, small drug molecules easy to be cleared by the metabolic system, poor stability, lack of targeting and easy to produce drug resistance in clinical application.

[0004] Therefore, it is necessary to provide a nano-drug with hypoxia responsiveness and photoisomerization function and loaded with 10-hydroxycamptothecin to solve the problems existing in the prior art. SUMMARY

[0005] The purpose of the present application is to provide a nano-drug with hypoxia responsiveness and photoisomerization function and preparation and application. In the present application, the hypoxia-responsive unit 4,4'-dichloroazobenzene is used as a hypoxia-responsive unit, which is covalently connected with 10-hydroxycamptothecin and the drug carrier mPEG to form a size-controllable nano-drug. The size of the nano-drug is adjusted to control the drug release rate and maintain the drug concentration in the required range. At the same time, the nano-drug can target the lesion site to reduce the side effects of the drug. In addition, the nano-drug has high utilization rate and good biocompatibility.

[0006] In a first aspect, the present application provides a nano-drug with low oxygen response and photoisomerization functions, which is formed by covalently connecting 4,4'-dichloroazobenzene, 10-hydroxycamptothecin and mPEG, and has the structure shown in the following formula (I):

[0007] ;

[0008] wherein m and n are both positive integers greater than or equal to 1.

[0009] In the present application, the inventors have found that using 4,4'-dichloroazobenzene as a low oxygen response unit, which is covalently connected with 10-hydroxycamptothecin and drug carrier mPEG to form a nano-drug with controllable size, the nano-drug has high utilization rate and can target the lesion site to reduce the side effects of the drug. In addition, in vivo and in vitro experiments have confirmed that the nano-drug has good biocompatibility, can be reduced by specific reductase in low oxygen tumor cells, and the structure is disintegrated to release the anticancer drug 10-hydroxycamptothecin, thereby efficiently exerting the effect of chemotherapy and inhibiting tumor growth and killing cancer cells.

[0010] In a second aspect, the present application provides a preparation method of the nano-drug with low oxygen response and photoisomerization functions as described above, which comprises the following steps: S1, reacting 4,4'-dichloroazobenzene (AZO), 10-hydroxycamptothecin (HCPT) and mPEG in the presence of a catalyst to obtain an amphiphilic block copolymer after separation and purification; S2, dissolving the amphiphilic block copolymer in water to obtain polymer micelles PHA by self-assembly, and then treating by light to obtain the nano-drug PHAU with low oxygen response and photoisomerization functions.

[0011] In the present application, the preparation process of the nano-drug with low oxygen response and photoisomerization functions is shown in the following formula (1):

[0012] .

[0013] The preparation method provided by the application is characterized in that AZO is used as a low-oxygen response unit, and is covalently connected with an anticancer drug HCPT and a drug carrier mPEG to form an amphiphilic block copolymer, the amphiphilic block copolymer is self-assembled into a polymer micelle PHA in water, and then the PHA is irradiated alternately under ultraviolet light and visible light to prepare a size-controllable nano drug PHAU. Generally, when a drug circulates in a body of a subject, if the size of the drug is small (less than 20 nm), the drug is removed by a reticuloendothelial system in a few hours; if the size of the drug is large, the drug stays in the liver and spleen in a short time; and the blood circulation time of the drug in the body is related to the particle size of the nano drug. Compared with the small molecule and poor solubility of HCPT, the nano drug PHAU prepared in the application has a size-controllable particle size, which can deliver HCPT to a tumor site and accelerate the release speed of HCPT. In addition, the molecular structure of the nano drug PHAU contains cis and trans configurations, which are smaller in size and more compact in structure, and are more beneficial to improve the in-vivo stability, target the tumor site and prolong the blood circulation time. In addition, the azo group in the PHAU is used to controllably release HCPT, has high stability, can effectively reduce the leakage of the drug outside the tumor tissue, and reduce the toxic side effects on healthy cells.

[0014] In some embodiments, in step S1, the molar ratio of 4,4'-dichloroazobenzene, 10-hydroxy camptothecin and mPEG is (0.12-0.2):(0.4-0.6):(0.005-0.015).

[0015] In some embodiments, in step S1, the molar ratio of the catalyst to 4,4'-dichloroazobenzene is (1.64-1.66):(0.12-0.2), and the catalyst comprises 4-dimethylaminopyridine.

[0016] In some embodiments, the reaction specifically comprises: stirring the reaction at room temperature for 8-20 h.

[0017] It can be understood that the reaction is carried out in an organic solvent, and the organic solvent can be selected from conventional organic solvents, and in the application, the organic solvent is preferably dichloromethane.

[0018] In some embodiments, the molecular weight of mPEG is 4000-6000.

[0019] In some preferred embodiments, the molecular weight of mPEG is 5000.

[0020] In some embodiments, in step S1, the separation and purification specifically comprises: dialyzing the reaction product in a dialysis bag with a molecular weight cut-off of 4000-6000 for 24-72 h.

[0021] In some preferred embodiments, in step S1, the separation and purification specifically comprises: dialyzing the reaction product using a dialysis bag with a molecular weight cut-off of 5000 for 48 h.

[0022] In some embodiments, in step S3, the light irradiation treatment specifically comprises: first irradiating with ultraviolet light for 0.5-1.5 min, and then irradiating with visible light for 0.5-1.5 min; repeating the irradiation for 3-5 times.

[0023] In some preferred embodiments, in step S3, the light irradiation treatment specifically comprises: first irradiating with ultraviolet light for 1 min, and then irradiating with visible light for 1 min; repeating the irradiation for 3 times.

[0024] In some embodiments, the wavelength of the ultraviolet light is 365 nm.

[0025] In some embodiments, in step S3, the average particle size of the nano-drug with the functions of hypoxia-responsiveness and photo-isomerization is 40-60 nm.

[0026] In some preferred embodiments, in step S3, the average particle size of the nano-drug with the functions of hypoxia-responsiveness and photo-isomerization is 50 nm.

[0027] In a third aspect, the present application provides use of the nano-drug with the functions of hypoxia-responsiveness and photo-isomerization as described above or prepared according to any of the preparation methods described above in the preparation of an anti-tumor drug.

[0028] The present application has the following beneficial effects: different from the prior art, the present application uses 4,4'-dichloroazobenzene as a hypoxia-responsive unit, which is covalently connected with 10-hydroxy camptothecin and a drug carrier mPEG to form a nano-drug with controllable size, the nano-drug has a suitable particle size, which is more conducive to the enrichment of the drug in the tumor site through blood circulation and the exertion of the therapeutic effect, when the nano-drug reaches the hypoxic tumor site, the azo reductase unique to the hypoxic environment reduces the azo group inside the nano-drug, resulting in the disintegration of the nano-drug structure, thereby releasing 10-hydroxy camptothecin to exert the chemotherapeutic effect, and reducing the toxic and side effects of the drug on normal tissues; in addition, the nano-drug has high utilization rate and good biocompatibility, and therefore has good application prospect in the preparation of an anti-tumor drug. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 NMR hydrogen spectrum of PHA synthesized in Example 1 of the present application (a) and NMR hydrogen spectrum of PHB synthesized in Comparative Example 1 (b);

[0030] Figure 2TEM and DLS graphs of the nanodrug synthesized in Example 1 of the present application, wherein (a) is a TEM graph of PHA, (b) is a TEM graph of PHA irradiated by 365 nm ultraviolet light for 2 min, (c) is a TEM graph of PHA U, and (d) is a DLS graph of PHA and PHA U and after dilution of 500 times;

[0031] Figure 3 Fluorescence emission spectrum graph of PHA U synthesized in Example 1 of the present application at an excitation wavelength of 380 nm;

[0032] Figure 4 Ultraviolet spectrum graph of PHA synthesized in Example 1 of the present application after 365 nm ultraviolet irradiation (a) and ultraviolet spectrum graph of PHB synthesized in Comparative Example 1 after 365 nm ultraviolet irradiation (b) as well as the photoisomerization schematic diagram of PHA synthesized in Example 1 and the formation process graph of PHA U (c);

[0033] Figure 5 TEM graph (a), DLS graph (b) and HPLC chromatogram (c) of PHA U after the structure of PHA U synthesized in Example 2 of the present application disintegrates, and DLS graph (d) and TEM graph (e) of PHB before and after treatment with a reducing agent;

[0034] Figure 6 Result graph of the release of HCPT by nanodrug PHA U and PHA in vitro under simulated low-oxygen environment (with or without Na2S2O4) (a) and result graph of the release of HCPT by PHB (b) in Example 3 of the present application;

[0035] Figure 7 Cell survival rate result graph of Hela cells treated with different drugs (nanodrug PHA U, polymeric micelles PHA, PHB and single drug HCPT) in Example 4 of the present application, wherein (a) is treated under normoxic conditions, and (b) is treated under hypoxic conditions;

[0036] Figure 8 Laser confocal graph of Hela cells incubated with different drugs (nanodrug PHA U, polymeric micelles PHA, PHB) in Example 5 of the present application, wherein (a) is treated under hypoxic conditions, (b) is treated under normoxic conditions, (c) is the HCPT fluorescence intensity distribution at the position indicated by the arrow in (a), and (d) is the fluorescence intensity of HCPT in the nucleus of Hela cells under hypoxic and normoxic conditions, and the scale is 20 µm;

[0037] Figure 9 Blood circulation curve graph (a) and content graph of anti-cancer drug HCPT in tumors of tumor-bearing mice (b) in different drug treatment groups in Example 6 of the present application;

[0038] Figure 10 Figures (a) and (b) are graphs showing the tumor volume and weight changes in different drug treatment groups in Example 6 of the present application, and Figure (c) is a graph showing the H&E staining and TUNEL immunofluorescence staining results of tumor tissue sections, with a scale of 100 µm;

[0039] Figure 11 Figure is a graph showing the body weight of tumor-bearing mice in different drug treatment groups in Example 6 of the present application;

[0040] Figure 12 Figure is a graph showing the H&E staining results of the main organs (heart, liver, spleen, lung, kidney) of mice in different drug treatment groups in Example 6 of the present application. DETAILED DESCRIPTION

[0041] The technical solutions in the embodiments of the present application will be described below in detail with reference to the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of the present application.

[0042] The experimental methods in the embodiments not specified with specific conditions are generally performed according to the conventional conditions and the conditions described in the manuals, or according to the conditions suggested by the manufacturers. The general equipment, materials, reagents, etc. used, if not specifically stated, can be obtained from commercial channels.

[0043] Example 1 Synthesis of nano-drug PHAU with hypoxia responsiveness and photoisomerization function

[0044] First, synthesize PHA: add 0.5 mmol of 10-hydroxycamptothecin (HCPT) and 1.65 mmol of 4-dimethylaminopyridine (DMAP) into 50 mL of dichloromethane to dissolve thoroughly, then add 0.16 mmol of 4,4'-dichloroazobenzene (AZO) to dissolve thoroughly, and then add 0.01 mmol of mPEG (molecular weight 5000), stir at room temperature for 12 h, after the reaction is completed, transfer the mixed solution to a dialysis bag with a molecular weight cut-off of 5000, seal, and dialyze in ultrapure water with uniform stirring, dialyze for 48 h (replace the ultrapure water every 6 h), and then freeze-dry to obtain PHA powder.

[0045] Then synthesize nano-drug PHAU: take 5 mg of the above PHA powder, dissolve in 100 µL of dimethyl sulfoxide (DMSO), and then drop into 5 mL of ultrapure water, stir thoroughly for 30 min, obtain polymer micelles PHA, irradiate the polymer micelles PHA under 365 nm ultraviolet light for 1 min, then irradiate under visible light for 1 min, repeat the operation three times, and obtain nano-drug PHAU.

[0046] Comparative Example 1 Synthesis of drug PHB

[0047] The preparation process of the drug PHB is shown in the following formula (2):

[0048] .

[0049] Specifically, 0.5 mmol of 10-hydroxycamptothecin (HCPT) and 1.65 mmol of 4-dimethylaminopyridine (DMAP) were added to 20 mL of N,N-dimethylformamide and fully dissolved. Then, 0.5 mmol of 4,4'-biphenyldiacetyl chloride was added and fully dissolved. Then, 0.01 mmol of mPEG (molecular weight 5000) was added and stirred at room temperature for 12 hours. After the reaction was completed, the mixed solution was transferred to a dialysis bag with a molecular weight cutoff of 5000, sealed, and dialyzed in ultrapure water under uniform stirring. After dialysis for 48 hours (ultrapure water was changed every 6 hours), PHB powder was obtained by freeze-drying.

[0050] Performance Characterization

[0051] The PHA powder synthesized in Example 1 and the PHB powder synthesized in Comparative Example 1 were subjected to hydrogen nuclear magnetic resonance spectroscopy.

[0052] Specifically, the PHA powder and PHB powder were vacuum dried and dissolved in deuterated chloroform, and then subjected to H NMR spectroscopy ( 1 HNMR) characterization, the results are as follows Figure 1 shown.

[0053] from Figure 1 As can be seen from the NMR spectrum of PHA ( Figure 1 (a)), 3.5ppm is the peak of the deuterated reagent DMSO-d6, 3.27ppm (a) and 3.55ppm (b) are characteristic peaks belonging to the methoxy group of mPEG and the methylene group in the repeating unit; proton peaks c and d come from the hydrogen on the AZO benzene ring, and the 5.43ppm characteristic peak is attributed to the benzyl group in AZO; proton peaks e, f, g, h, and j are attributed to the hydrogen on the HCPT benzene ring, 0.83ppm (p) and 1.91ppm (l) are characteristic peaks belonging to the terminal methyl and methylene of HCPT, and i and k are attributed to the methylene group within the HCPT ring; the NMR spectrum of PHB ( Figure 1 In (b), only the HCPT intramethylene peaks are present between 5 and 6 ppm. Further analysis of the integrated areas of the proton peaks c, d, e, g, and f of PHA using MestReNove software revealed an area ratio of 4:4:1:1:1, confirming the successful preparation of the PHA sample. Furthermore, integrated analysis of the proton peaks c, d, e, g, and f of PHB revealed an area ratio of 4:4:1:1:1, confirming the successful preparation of the PHB sample using NMR results.

[0054] Transmission electron microscope (TEM) morphology observation and dynamic light scattering particle size instrument (DLS) test were performed on the PHA powder and PHAU powder synthesized in Example 1.

[0055] Specifically, the PHA powder, the PHA powder irradiated by 365 nm ultraviolet light for 2 min and the PHAU powder were respectively dissolved in water to obtain a PHA solution, a PHA solution irradiated by 365 nm ultraviolet light for 2 min and a PHAU solution, one drop of the above aqueous solution was dropped on a copper mesh, and after the solution was naturally air-dried, one drop of uranyl acetate was dropped on the material on the copper mesh for staining, and after natural drying, TEM morphology observation was performed, at the same time, 2 mL of the PHA and PHAU solutions (and 500 times dilutions thereof) were respectively taken for DLS test, and the results are shown in Figure 2

[0056] As can be seen from Figure 2 , the polymer micelles PHA are spherical, with an average diameter of about 80 nm Figure 2 (a); the morphology starts to change Figure 2 (b) after 365 nm ultraviolet irradiation, and finally forms a nano-drug PHAU with a spherical particle structure, with an average diameter of about 50 nm Figure 2 (c), and the PHAU has a more compact internal structure than the PHA; the hydration particle sizes of the PHA and PHAU are determined by DLS Figure 2 (d) to be 89.53±4.02 nm and 55.64±3.75 nm, respectively, and the PHAU (PDI=0.133) has a narrower particle size distribution and better dispersibility than the PHA (PDI=0.205); and when the aqueous solutions of the PHA and PHAU are simultaneously diluted 500 times, it is found that the size of the PHA particles changes greatly, while the particle size distribution of the PHAU remains basically unchanged, which indicates that the stability of the polymer micelles PHA is poor, and the nano-drug PHAU has good particle stability; the above results confirm that the structure of the PHAU is more compact than that of the PHA, and the reason may be that after the molecules of the PHA are alternately irradiated by ultraviolet light and visible light, the molecules are arranged more regularly, resulting in a decrease in the particle size of the PHAU and better stability.

[0057] The drug loading capacity of the PHAU powder synthesized in Example 1 was tested.

[0058] Specifically, first, HCPT of different concentration gradients was prepared, and a standard curve model of the HCPT fluorescence spectrum was established according to the emission peak intensity of the fluorescence spectrum (the fluorescence spectrum of HCPT has a peak intensity of the emission spectrum at 540 nm when the excitation light is 380 nm, which has the same emission spectrum as the PHAU Figure 3 ​The fluorescence spectrometer was used to quantitatively analyze the content of HCPT in the nano-drug, and the drug loading amount of PHAU was calculated according to the following formula (3):

[0059] .

[0060] The drug loading amount of the nano-drug PHAU was calculated to be 10.3%.

[0061] The optical properties of the PHA powder synthesized in Example 1 and the PHB powder synthesized in Comparative Example 1 were tested.

[0062] Specifically, an appropriate amount of PHA and PHB was respectively dissolved in 2 mL of ultrapure water, and UV-Vis was used to determine the ultraviolet absorption peak in the visible light environment. Then, after being irradiated with ultraviolet light of a wavelength of 365 nm for 2 min, UV-Vis was quickly detected. Finally, the solution after ultraviolet irradiation was placed in visible light for 2 min to detect UV-Vis, and the results are shown in Figure 4 .

[0063] As can be seen from Figure 4 , the polymer micelles PHA undergoes photoisomerization under the irradiation of ultraviolet light of 365 nm. Before ultraviolet irradiation, PHA has a strong absorption band near 340 nm, which may be due to the π→π* transition in the trans structure of PHA. After 2 min of ultraviolet irradiation, the absorption peak at 340 nm is obviously reduced, which may be due to the gradual conversion of the trans structure to the cis structure. The decrease in the trans structure leads to a decrease in the intensity of the π→π* transition band with the increase of ultraviolet irradiation time, while the intensity of the n→π* transition band corresponding to the cis isomer near 450 nm increases with the increase of ultraviolet irradiation time, corresponding to the increase of cis configuration molecules. The change in the intensity of π→π* and n→π* transition indicates that the anti-cis isomerization of PHA occurs under ultraviolet irradiation. Moreover, the absorption intensity of PHAU formed after ultraviolet irradiation and visible light irradiation is between the absorbance of PHA and the absorbance of PHA after ultraviolet irradiation, which indicates that the process of trans to cis is reversible, and the structure of the finally formed PHAU is the most stable state of the molecule, which exists in the form of trans and cis configurations Figure 4 (a). When PHB is irradiated with ultraviolet light, the ultraviolet absorption peak of PHB molecules does not change Figure 4 (b), because there is no azo group in the PHB molecule. The schematic diagram of the formation of PHAU is as follows Figure 4(c) shown. The UV-Vis characterization results of PHA and PHAU successfully proved that the low oxygen response unit (AZO, azo group) was successfully carried in the PHAU molecule and the PHAU had photoisomerization characteristics.

[0064] Example 2 Disintegration test of nano-drug PHAU in low oxygen environment

[0065] Generally, the low oxygen environment of tumor promotes the overexpression of many reductases, including azo reductase. Azo reductase can oxidize and reduce the N=N bond in azobenzene derivatives, causing the chemical bond to break. Therefore, in vitro experiments can use inorganic reducing agent sodium dithionite (Na2S2O4) as a chemical mimic of azo reductase, with a concentration of 10 mM in phosphate buffer solution (PBS, pH = 7.4) to simulate the low oxygen environment of tumors and mediate the reduction of the azo group of PHAU. PHAU (1 mg / mL) was fully dispersed in a PBS mixed solution containing Na2S2O4 and placed in a constant temperature shaker at 37°C for 60 min (100 r / min). The TEM images and DLS characterization of the particle size change before and after PHAU reduction were recorded.

[0066] At the same time, high performance liquid chromatography (HPLC) with a fluorescence detector was used to quantitatively analyze the HCPT released after PHAU disintegration. The fluorescence detector parameters were set as follows: excitation wavelength 380 nm, emission wavelength 540 nm, C18 reverse phase column, and mobile phase of methanol: water system with a volume ratio of 40:60. The results are shown in Figure 5 .

[0067] As can be seen from Figure 5 , after the PHAU aqueous solution was treated with 10 mM Na2S2O4, the spherical nanoparticles disappeared, the morphology disintegrated, and a lot of small particles accumulated together (Fig. 8 Figure 5 (a)). At the same time, the DLS data also showed that the particle size distribution of PHAU was no longer uniform, and large-sized substances appeared (Fig. 8 Figure 5 (b)), which corresponded to the TEM images, indicating that the structure of PHAU may have disintegrated and small particles may have aggregated. In order to further prove that PHAU disintegration can release HCPT, the HPLC fluorescence detector was used to monitor the peak of PHAU and Na2S2O4 mixture at different time points (2 h, 4 h, 8 h, 12 h). The peak of PHAU alone appeared at about 31 min, and after adding Na2S2O4, a new peak appeared at 8 min after 2 h, which was the characteristic peak of HCPT. With the increase of time, the peak area at 31 min gradually decreased, and the peak area at 8 min gradually increased, which indicated that PHAU was gradually disintegrating and continuously releasing free HCPT.Figure 5 (c) ). Meanwhile, the control group PHB was also tested, and PHB was also treated with Na2S2O4 to exclude the influence of other factors. DLS data showed that the hydrated particle size of PHB did not change Figure 5 (d) ); and the TEM images of PHB before and after Na2S2O4 treatment also had little difference Figure 5 (e) ). The above results all confirmed that PHAU molecules can be reduced by reducing agents, undergo continuous elimination and decarboxylation, can undergo structural disintegration, and release free HCPT molecules, which are expected to occur in a hypoxic tumor environment.

[0068] Example 3 In vitro release test of nano-drug PHAU

[0069] Hypoxic tumor tissues overexpress azo reductase, which can reduce the azo group in the compound. In order to simulate the drug release of polymer micelles in the hypoxic tumor environment, inorganic reducing agent Na2S2O4 was used instead of azo reductase for the experiment. Specifically, two 1 mL, 1 mg / mL PHAU sample solutions were transferred into dialysis bags (molecular weight cut-off: 5 kDa), and then immersed in test tubes containing 30 mL of PBS (pH 7.4) containing different concentrations of Na2S2O4 (0 mM and 10 mM). The release was carried out in a 100 r / min, 37°C constant temperature shaker. At specific time points (30 min, 1 h, 2 h, 4 h, 6 h, 9 h, 12 h, 24 h, 36 h, 48 h), 3 mL of release medium outside the dialysis bag was taken out and 3 mL of PBS solution of the corresponding concentration of Na2S2O4 was added to maintain the constant volume of the PBS solution. At the same time, PHB was used as a control for the experiment. The peak intensity of the emission spectrum at 540 nm when the excitation light was 380 nm was measured at each time point using a fluorescence spectrometer. The HCPT concentration released by PHAU at each time point was calculated by the standard concentration curve of HCPT, and the cumulative drug release amount was obtained, as shown in Figure 6 The formula for calculating the cumulative release rate of HCPT is shown in the following formula (4):

[0070] ;

[0071] wherein, E r is the cumulative drug release amount (%), V e is the volume of the release medium taken out at each fixed time point, and n represents the number of samples taken, C i is the HCPT concentration in the release medium at the i-th sampling time, V 0 is the total volume of the release medium, mThe total mass of the drug loaded in the nanodrug.

[0072] It can be seen from Figure 6 The release rate of the nanodrug PHAU without Na2S2O4 is significantly slowed down when the drug release amount reaches 15%, and the final release amount is only about 20% after 48 h. The drug release rate of PHA is slightly faster than that of PHAU, but only about 30% of HCPT is released after 48 h. This result further confirms that the structure of PHAU is more stable than that of PHA, and HCPT is safer in the structure formed by PHAU. After the addition of Na2S2O4, the release rates of PHA and PHAU are dramatically accelerated, and the final release amount of HCPT is as high as 90% after 48 h Figure 6 (a). However, there is no significant difference in the drug release rate of PHB with or without Na2S2O4 Figure 6 (b). This is because the intramolecular azo group of PHAU and PHA can be reduced by Na2S2O4 to break the valence, which is beneficial to the release of HCPT from the micelles. This experimental result shows that PHAU has a small rate of premature drug release in the blood circulation, is stable in structure, and can maintain a long blood circulation time. When it reaches the hypoxic tumor tissue, it can quickly release the chemotherapeutic drug HCPT under the stimulation of hypoxia.

[0073] Example 4 Cell toxicity test of nanodrug PHAU

[0074] The toxicity of different concentrations of nano-drug PHAU, polymeric micelles PHA, PHB and single drug HCPT on Hela cells in normoxia and hypoxia environment was detected by CCK-8 method. Specifically, first, the Hela cells in logarithmic growth phase were taken, the culture solution in the culture dish was discarded, 1 mL PBS was added for two times of rinsing, 1 mL trypsin was added immediately and then placed in the incubator for 3 minutes, the purpose was to digest the Hela cells growing adherently. After the cells were completely detached, 3 mL of complete culture medium (1% double antibody, 10% fetal bovine serum, 89% DMEM) was added to mix and terminate digestion, then moved to the centrifuge tube and centrifuged at 1000 rpm for 3 min, the supernatant was discarded and an appropriate amount of complete culture medium was added, and the cell suspension was prepared by gently blowing with a pipette gun. The cell suspension (100 µL / well) was inoculated in a 96-well plate, and the number of cells in each well was about 5000. In order to prevent the solution in the outermost circle of the 96-well plate from volatilizing, PBS (200 µL / well) was added for water sealing. The experiment was divided into two groups, one group was pre-cultured in a conventional incubator at 37°C, 5% CO2 and 21% O2 for 24 hours; the other group was pre-cultured in a hypoxic cell culture box at 37°C, 2% O2 and nitrogen balance for 24 hours. The complete culture medium was used to prepare different concentration gradients of PHAU, PHA, PHB and HCPT solutions. Then the culture medium in the 96-well plate was gently sucked out with a pipette, and the prepared sample solution (100 µL / well) was added respectively, six replicate wells were set for each concentration, and the incubation was continued in the incubator for 24 hours. Finally, CCK-8 reagent was added to the 96-well plate (10 µL / well), and after incubation in the incubator for 2 hours, the absorbance (OD) at 450 nm was measured by a microplate reader. The toxicity of CCK-8 reagent to cells was very low and could be ignored. Since CCK-8 can continuously react with dehydrogenase in living cells, the OD value will continue to rise, so the cell survival rate can be calculated by the OD value, and the results are shown in Figure 7 As shown in the following formula (5):

[0075] ;

[0076] Wherein, A s The experimental hole, that is, the culture medium containing cells, CCK-8, and drugs; A c The control hole, that is, the culture medium containing cells, CCK-8, and no drugs; A b The blank hole, that is, the culture medium without cells, CCK-8, and no drugs.

[0077] As can be seen from Figure 7 , with the increase of the concentration of each drug, the cell survival rate is gradually reduced Figure 7(a), 7(b)). When the concentration of PHAU reached 25µg / mL, the survival rate of Hela cells in a normoxic environment was only approximately 38%, but this was still greater than the approximately 21% survival rate in a hypoxic environment, indicating that higher drug concentrations increase cellular toxicity. HCPT alone exhibited similar cytotoxicity at the same drug concentration in both normoxic and hypoxic environments, indicating that the presence of oxygen has no effect on the therapeutic efficacy of HCPT. Furthermore, a comparison of the toxicity of PHAU, PHA, and PHB to Hela cells at the same concentrations in hypoxic and normoxic environments revealed that PHAU was more toxic. This suggests that PHAU releases HCPT more rapidly in Hela cells. Furthermore, the cytotoxic effect of PHAU on Hela cells in a hypoxic environment was similar to that of HCPT at the same concentration, indicating that PHAU can more rapidly release all of its HCPT in hypoxic cancer cells, resulting in a highly effective killing effect on cancer cells.

[0078] Example 5 In vitro cellular uptake test of nanodrug PHAU

[0079] The uptake of the nanodrug PHAU in Hela cells was analyzed using confocal laser scanning microscopy (CLSM). Specifically, 1 mg of each nanodrug PHAU, PHA, and PHB was prepared into a solution. PBS was added to make a 4 mM stock solution, which was then diluted with culture medium to a 40 µM sample solution for later use. The Hela cell suspension was then seeded in a confocal dish (1 × 10 5 / well), incubate for 24 hours until it adheres to the wall and grows, then aspirate the culture medium. Then add 1 mL of the pre-prepared PHAU, PHA and PHB sample solutions respectively, incubate with Hela cells for 4 hours, discard the solution, and then add PBS to gently wash three times, mainly to remove materials that are not bound to the cells. After gently aspirating the PBS, add 1 mL of 4% paraformaldehyde tissue fixative to fix the cells. After fixation for 30 minutes, discard the fixative, wash three times with PBS, and finally use CLSM to excite at a wavelength of 380 nm to obtain the fluorescence signal of HCPT (green fluorescence), so as to observe the drug uptake of the cells. The results are shown in the figure. Figure 8 shown.

[0080] from Figure 8 It can be seen that after hypoxia treatment ( Figure 8 (a), 8(c)), compared with PHB, HeLa cells co-incubated with PHAU and PHA showed significantly enhanced fluorescence, while under normoxic conditions, after the three drugs were incubated with cells for 4 hours, there was little difference in the fluorescence intensity of the cells ( Figure 8(b) ). After incubation with PHA and PHB, the fluorescence of HCPT mainly distributed in the cytoplasm, while after incubation with PHAU, HCPT could quickly diffuse into the nucleus after entering the cell, which indicated that PHAU could deliver HCPT into the nucleus more effectively than PHA in the hypoxic environment. Moreover, under normoxic conditions, the total amount of HCPT entering the nucleus was not significantly different after incubation with PHAU, PHA and PHB respectively; but under hypoxic conditions, the effect of PHAU on delivering HCPT into the nucleus was significantly improved Figure 8 (d) ). This indicates that after being taken up by cells, the overexpressed reductive enzymes in the hypoxic environment promote the disintegration of the nanodrug PHAU, which is beneficial to the release of free HCPT, so that more HCPT enters the nucleus to play a chemotherapeutic role. This experimental result is consistent with the above cytotoxicity experimental results.

[0081] Example 6 In vivo biological evaluation test of nanodrug PHAU

[0082] All animal experiments were carried out in accordance with the provisions of the National Experimental Animal Management Regulations. First, a mouse cervical cancer model was constructed using Hela cells to evaluate the in vivo anti-tumor effect of nanodrug PHAU. About 2 x 10 6 logarithmic growth phase of Hela cells were injected subcutaneously into the abdomen of BALB / C mice (6-week-old female mice, body weight 20-25 g), and after 4 days, mice with similar tumor sizes were selected to establish a tumor-bearing mouse model. The tumor-bearing mice were divided into 5 groups, with 3 mice in each group. The nanodrug dispersion was injected into the tail vein, and each group was injected with PBS, HCPT, PHB, PHA and PHAU respectively. The PBS group was used as a blank control, and the calculated equivalent of HCPT in each group was 5 mg / kg. The experiment lasted for two weeks, and the nanodrug was injected every other day, with each injection being 50 µL.

[0083] First, the half-life of the above drugs in the blood circulation was determined. Specifically, a fluorescence spectrum standard curve of HCPT single drug was first constructed, and then different time points (10 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h) were selected, and the mouse tail vein blood was collected for quantitative analysis of HCPT in the blood by fluorescence spectrum. After the experiment, the tumor tissues and main organs (heart, liver, spleen, lung, kidney) of the mice were taken out for standby. Part of the tumor tissue was weighed, then physiological saline was added in proportion, and then centrifuged at 3000 rpm for 10 minutes to prepare tissue homogenate. The supernatant was taken and extracted with a mixed solution of methanol and water. The extract was blown dry with nitrogen, and then dissolved in methanol for fluorescence spectrum detection to obtain the HCPT drug uptake percentage per gram of tumor tissue, as shown in Figure 9 the results.

[0084] from Figure 9 It can be seen that the concentration of HCPT alone in the blood decreases rapidly, and its blood half-life is only about 20 minutes. The circulation time of PHAU, PHA and PHB in the blood is significantly longer than that of HCPT alone; the blood half-life of PHA and PHB is about 40 minutes, while the blood half-life of PHAU can be as long as 2 hours, which is longer than the half-lives of the other two drugs ( Figure 9 (a)). This suggests that the small molecule HCPT alone has a short blood circulation time and is easily metabolized by the body. The nanopharmaceutical PHAU, on the other hand, has a more stable structure, increasing the blood circulation time of single-drug HCPT from 20 minutes to 2 hours. This may be related to the micelle size we designed, which reduces the risk of drug engulfment and clearance by macrophages, thereby increasing the chance of HCPT reaching the tumor site and improving the drug delivery rate. PHAU has the highest uptake in tumor tissue, reaching 7% ID / g, nearly six times that of single-drug HCPT. ( Figure 9 (b) Experimental results show that single-agent HCPT has low efficiency in entering tumor tissue and is not easily accumulated near the tumor, thus failing to exert its chemotherapy effect. However, the nanopharmaceutical PHAU enhances the drug's tumor targeting and significantly increases the concentration of HCPT at the tumor site.

[0085] Then, throughout the experiment, the tumor volume and mouse body weight were monitored every other day using a vernier caliper and a balance. The mouse volume was calculated using the following formula (6):

[0086] ;

[0087] At the same time, the tumor tissues after drug treatment were fixed and frozen, then sectioned and stained with hematoxylin-eosin (H&E) and TUNEL, and observed under CLSM. The results are as follows Figure 10 shown.

[0088] from Figure 10 It can be seen that the tumors of mice treated with PBS grew faster, while the other four groups showed different tumor inhibition effects ( Figure 10(a), 10(b); wherein the tumor inhibition effect of HCPT alone is the worst, followed by PHB and PHA, and the effect of PHAU is the best. The experimental results show that, compared with HCPT alone, other nano-drug carriers have better effect of delivering drugs to tumor sites. Moreover, the treatment effect of PHA is higher than that of PHB, indicating that the low-oxygen response unit in the structure of PHA can specifically stimulate response at the tumor site to achieve site-specific drug release; and compared with PHA, PHAU has smaller particle size, which is beneficial to the uptake of tumor tissue and the enrichment of tumor site, and has excellent tumor inhibition effect. In addition, the H&E and TUNEL staining results all confirm that PHAU treatment has a more obvious effect of inducing tumor necrosis and apoptosis compared with the other four control groups Figure 10 (c). According to the TUNEL staining results, the number of living cells (green) in the tumor tissue section after PHAU treatment is significantly reduced, and the number of dead cancer cells (blue) is increased, indicating that PHAU has good in vivo anti-tumor effect.

[0089] Then, during the entire experiment, the body weight of the tumor-bearing mice was recorded regularly, and the results are shown in Figure 11 .

[0090] As can be seen from Figure 11 , within two weeks after drug treatment, the mice did not show loss of appetite and abnormal body weight. Different drug preparations PBS, HCPT, PHB, PHA and PHAU had little effect on the body weight of mice, and the body weight differences of the five groups of mice were within a reasonable range.

[0091] Finally, in order to evaluate the biological safety of the drug, the distribution of the drug in the mouse body was detected, and the main organs (heart, liver, spleen, lung, kidney) of the mouse were fixed and frozen for sectioning, and H&E staining was used, and the results are shown in Figure 12 .

[0092] As can be seen from Figure 12 , after drug treatment, the heart, liver, spleen, lung and kidney showed no obvious damage. This is because the release effect of HCPT under normoxic conditions is poor, and the redox environment in the normoxic tissue reaches equilibrium, which cannot provide excess azo reductase for the disintegration of PHAU structure, and after blood circulation, it is metabolized by the body. Therefore, the nano-drug PHAU has good biocompatibility and tumor tissue specificity, which can significantly reduce the toxic side effects of drugs on normal tissues.

[0093] To sum up, the low oxygen response unit 4,4'-dichloro azobenzene is used as the low oxygen response unit in the application, which is covalently connected with 10-hydroxy camptothecin and the drug carrier mPEG to form the size-controllable nano drug, the drug release rate is controlled by adjusting the size of the nano drug, the drug concentration is maintained in the required range, meanwhile, the nano drug can target the lesion site to reduce the side effects of the drug, in addition, the nano drug has high utilization rate and good biocompatibility.

[0094] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own emphasis, and the description not fully described in individual embodiments can be referred to the description in other embodiments.

[0095] The above-described embodiments only express the implementation of the application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the application, a number of modifications and improvements can be made, which are all within the protection scope of the application. Therefore, the protection scope of the patent of the application should be subject to the appended claims.

Claims

1. A nanomedicine with hypoxia responsiveness and photoisomerization function, characterized in that: The nanomedicine is formed by covalently linking 4,4'-diacyl chloride azobenzene, 10-hydroxycamptothecin and mPEG, and has a structure shown in the following formula (I): ； Wherein, m and n are both positive integers ≥1.

2. A method for preparing the nanomedicine having hypoxia responsiveness and photoisomerization function as claimed in claim 1, characterized in that: The steps include: S1, using 4,4'-diazobenzene dichloride, 10-hydroxycamptothecin and mPEG as raw materials, reacting in the presence of a catalyst, and obtaining an amphiphilic block copolymer through separation and purification; S2. Dissolving the amphiphilic block copolymer in water, obtaining polymer micelles through self-assembly, and then treating the micelles with light to obtain the nanomedicine with hypoxia responsiveness and photoisomerization function.

3. The method for preparing the nanomedicine having hypoxia responsiveness and photoisomerization function according to claim 2, characterized in that: In step S1, the molar ratio of the 4,4'-diazobenzene chloride, the 10-hydroxycamptothecin, and the mPEG is (0.12-0.2): (0.4-0.6): (0.005-0.015).

4. The method for preparing the nanomedicine having hypoxia responsiveness and photoisomerization function according to claim 2, characterized in that: In step S1, the molar ratio of the catalyst to the 4,4'-diazolyl chloride is (1.64-1.66): (0.12-0.2), and the catalyst includes 4-dimethylaminopyridine.

5. The method for preparing the nanomedicine having hypoxia responsiveness and photoisomerization function according to claim 2, characterized in that: In step S1, the reaction specifically includes: stirring the reaction at room temperature for 8-20 hours.

6. The method for preparing the nanomedicine having hypoxia responsiveness and photoisomerization function according to claim 2, characterized in that: The molecular weight of the mPEG is 4000-6000.

7. The method for preparing the nanomedicine having hypoxia responsiveness and photoisomerization function according to claim 2, characterized in that: In step S1, the separation and purification specifically includes: dialyzing the reaction product for 24-72 hours using a dialysis bag with a molecular weight cut-off of 4000-6000.

8. The method for preparing the nanomedicine having hypoxia responsiveness and photoisomerization function according to claim 2, characterized in that: In step S2, the light treatment specifically includes: first irradiating with ultraviolet light for 0.5-1.5 minutes, then irradiating with visible light for 0.5-1.5 minutes; and repeating the irradiation 3-5 times.

9. The method for preparing the nanomedicine having hypoxia responsiveness and photoisomerization function according to claim 2, characterized in that: In step S2, the average particle size of the nanomedicine having hypoxia responsiveness and photoisomerization function is 40-60 nm.

10. Use of the nanomedicine with hypoxia responsiveness and photoisomerization function according to claim 1 or the nanomedicine with hypoxia responsiveness and photoisomerization function prepared by the preparation method according to any one of claims 2 to 9 in the preparation of anti-tumor drugs.

Images