Nano-drug with hypoxia responsiveness and photoisomerization function as well as preparation and application of nano-drug

Abstract

The invention discloses a nano-drug with hypoxia responsiveness and a photoisomerization function as well as preparation and application thereof, and belongs to the technical field of biological medicines. The 4, 4 '-diacyl chloride azobenzene is used as a low-oxygen response unit and is covalently linked with the 10-hydroxycamptothecine and the drug carrier mPEG to form the size-controllable nano-drug, and the nano-drug has a proper particle size, is more beneficial to enrichment and curative effect exertion of the drug at a tumor site through blood circulation, and has a good application prospect. When the nano-drug reaches a hypoxia tumor site, the azo group in the nano-drug is reduced by the special azo reductase in a hypoxia environment, so that the structure of the nano-drug is disintegrated, the 10-hydroxycamptothecine is released to play a role in chemotherapy, and the toxic and side effects of the drug on normal tissues are reduced; besides, the nano-drug is high in utilization rate and has better biocompatibility, so that the nano-drug has a better application prospect in preparation of antitumor drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to nanomedicine with hypoxia responsiveness and photoisomerization function and its preparation and application. Background Art

[0002] According to statistics from cancer research institutions, the number of cancer deaths in China reached 3 million in 2020, ranking first in the world. It is estimated that by 2030, there will be 27 million new cancer patients worldwide. Traditional tumor treatments, 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 timeliness of treatment of tumor tissues, as well as people's 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, angiogenesis, invasiveness, metastasis, resistance to cell death, metabolic changes and genomic instability. Overexpressed hypoxia-inducible factors (HIFs) exist in tumors, which can induce the expression of vascular endothelial growth factor, promote angiogenesis in malignant tumors, increase vascular permeability, and promote immune escape and multidrug resistance of tumor cells by regulating immune cells. The presence of hypoxia is a major dilemma and difficulty in cancer treatment today.

[0003] Camptothecin (CPT) and its derivative 10-hydroxycamptothecin (HCPT) are natural alkaloids extracted from the Chinese plant Camptotheca acuminata, which have good anti-tumor effects. However, they have problems in clinical applications, such as poor water solubility, small drug molecules that are easily eliminated by the metabolic system, poor stability, lack of targeting, and easy to develop drug resistance.

[0004] Therefore, it is necessary to provide a nanomedicine having hypoxia responsiveness and photoisomerization function and loaded with 10-hydroxycamptothecin to solve the deficiencies in the prior art. Summary of the invention

[0005] The purpose of the present invention is to provide a nano drug with hypoxia responsiveness and photoisomerization function and its preparation and application. In the present invention, a hypoxia responsive unit 4,4'-diacyl chloride azobenzene is used as a hypoxia responsive unit, which is covalently linked with 10-hydroxycamptothecin and a drug carrier mPEG to form a size-controlled nano drug, and the drug release rate is controlled by adjusting the size of the nano drug and the drug concentration is maintained within a 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 a high utilization rate and good biocompatibility.

[0006] In the first aspect, the present invention provides a nano drug with hypoxia responsiveness and photoisomerization function, wherein the nano drug 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.

[0007] In the present invention, the inventors have found that the hypoxia response unit 4,4'-diazobenzene chloride is used as the hypoxia response unit, which is covalently linked with 10-hydroxycamptothecin and the drug carrier mPEG to form a size-controlled nanodrug. The nanodrug has a high utilization rate and can target the lesion site to reduce the side effects of the drug. In addition, in vitro and in vivo experiments have confirmed that the nanodrug has good biocompatibility and can be reduced by specific reductases in hypoxic tumor cells. The structure disintegrates to release the anticancer drug 10-hydroxycamptothecin, thereby effectively exerting a chemotherapeutic effect and inhibiting tumor growth and killing cancer cells.

[0008] In a second aspect, the present invention provides a method for preparing a nano drug having hypoxia responsiveness and photoisomerization function as described above, comprising the following steps: S1, using 4,4'-diazobenzene chloride (AZO), 10-hydroxycamptothecin (HCPT) 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 PHA through self-assembly, and then obtaining a nano drug PHAU having hypoxia responsiveness and photoisomerization function through light treatment.

[0009] In the present invention, the preparation process of the nano drug with hypoxia responsiveness and photoisomerization function is shown in the following formula (1): .

[0010] In the preparation method provided by the present invention, AZO is used as a hypoxia response unit, and is covalently linked with the anticancer drug HCPT and the drug carrier mPEG to form an amphiphilic block copolymer, and the amphiphilic block copolymer is self-assembled into a polymer micelle PHA in water, and then the PHA is irradiated with ultraviolet light and visible light alternately to prepare a size-controlled nano drug PHAU. Generally, when a drug circulates in the blood of a subject, if the drug size is small (less than 20nm), it will be cleared by the reticuloendothelial system within a few hours; if the drug size is large, it will stay in the liver and spleen for 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 of HCPT and poor solubility, in the present invention, the prepared nano drug PHAU has a size-controlled particle size, which can deliver HCPT to the tumor site and accelerate the release rate of HCPT; in addition, the molecular structure of the nano drug PHAU contains two configurations, cis and trans, and compared with PHA, the particle size is smaller and the structure is more compact, which is more conducive to improving the stability in the body, targeting the tumor site and prolonging the blood circulation time. In addition, the azo group in PHAU is used to controllably release HCPT, which has high stability and can effectively reduce the leakage of drugs outside the tumor tissue and reduce the toxic side effects on healthy cells.

[0011] In some embodiments, in step S1, the molar ratio of 4,4'-diazobenzene chloride, 10-hydroxycamptothecin and mPEG is (0.12-0.2): (0.4-0.6): (0.005-0.015).

[0012] In some embodiments, in step S1, the molar ratio of the catalyst to 4,4'-diacyl chloride azobenzene is (1.64-1.66):(0.12-0.2), and the catalyst comprises 4-dimethylaminopyridine.

[0013] In some embodiments, the reaction specifically comprises: stirring the reaction at room temperature for 8-20 hours.

[0014] It is understandable that the reaction is carried out in an organic solvent, and the organic solvent can be selected from conventional organic solvents. In the present invention, the organic solvent is preferably dichloromethane.

[0015] In some embodiments, the molecular weight of mPEG is 4000-6000.

[0016] In some preferred embodiments, the molecular weight of mPEG is 5,000.

[0017] In some embodiments, in step S1, separation and purification specifically comprises: dialyzing the reaction product for 24-72 hours using a dialysis bag with a molecular weight cutoff of 4000-6000.

[0018] In some preferred embodiments, in step S1, separation and purification specifically comprises: dialyzing the reaction product for 48 hours using a dialysis bag with a molecular weight cutoff of 5000.

[0019] In some embodiments, in step S3, the light treatment specifically includes: 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 3-5 times.

[0020] In some preferred embodiments, in step S3, the light treatment specifically includes: first irradiating with ultraviolet light for 1 minute, then irradiating with visible light for 1 minute; repeating the irradiation 3 times.

[0021] In some embodiments, the ultraviolet light has a wavelength of 365 nm.

[0022] In some embodiments, in step S3, the average particle size of the nanomedicine having hypoxia responsiveness and photoisomerization function is 40-60 nm.

[0023] In some preferred embodiments, in step S3, the average particle size of the nanomedicine having hypoxia responsiveness and photoisomerization function is 50 nm.

[0024] In a third aspect, the present invention provides the use of the above-mentioned nanomedicine with hypoxia responsiveness and photoisomerization function or the nanomedicine with hypoxia responsiveness and photoisomerization function prepared according to any of the above-mentioned preparation methods in the preparation of anti-tumor drugs.

[0025] The beneficial effects of the present invention are as follows: different from the prior art, the present invention uses 4,4'-diacyl chloride azobenzene as a hypoxia response unit, which is covalently linked with 10-hydroxycamptothecin and a drug carrier mPEG to form a size-controllable nano drug, the nano drug has a suitable particle size, and is more conducive to the enrichment of the drug at the tumor site through blood circulation and exerting the therapeutic effect, when the nano drug reaches the hypoxic tumor site, the unique azoreductase in the hypoxic environment reduces the azo group inside the nano drug, resulting in the disintegration of the nano drug structure, thereby releasing 10-hydroxycamptothecin to exert the effect of chemotherapy, and reducing the toxic side effects of the drug on normal tissues; in addition, the nano drug has a high utilization rate and good biocompatibility, and therefore has a good application prospect in the preparation of anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The nuclear magnetic resonance hydrogen spectrum (a) of the PHA synthesized in Example 1 of the present invention and the nuclear magnetic resonance hydrogen spectrum (b) of the PHB synthesized in Comparative Example 1; Figure 2TEM and DLS images of the nanodrugs synthesized in Example 1 of the present invention, wherein (a) is a TEM image of PHA, (b) is a TEM image of PHA irradiated with 365 nm ultraviolet light for 2 min, (c) is a TEM image of PHAU, and (d) is a DLS image of PHA and PHAU and their 500-fold dilutions; Figure 3 is a fluorescence emission spectrum of PHAU synthesized in Example 1 of the present invention at an excitation wavelength of 380 nm; Figure 4 The ultraviolet spectrum of the PHA synthesized in Example 1 of the present invention after ultraviolet irradiation at 365 nm (a), the ultraviolet spectrum of the PHB synthesized in Comparative Example 1 after ultraviolet irradiation at 365 nm (b), and the schematic diagram of the photoisomerization of the PHA synthesized in Example 1 and the formation process diagram of PHAU (c); Figure 5 TEM image (a), DLS image (b) of the PHAU structure after disintegration in Example 2 of the present invention, HPLC chromatogram (c) of PHAU before and after disintegration, and DLS image (d) and TEM image (e) of PHB before and after treatment with a reducing agent; Figure 6 The graph (a) and the graph (b) of the release of HCPT by the nano-drugs PHAU and PHA under the simulated hypoxic environment (with or without Na2S2O4) in vitro in Example 3 of the present invention are as follows; Figure 7 The cell survival rate results of Hela cells treated with different drugs (nano drug PHAU, polymer micelle PHA, PHB and single drug HCPT) in Example 4 of the present invention are shown in Figure 4, wherein (a) is treated under normoxic conditions, and (b) is treated under hypoxic conditions; Figure 8 These are laser confocal images of Hela cells incubated with different drugs (nano-drug PHAU, polymer micelle PHA, PHB) in Example 5 of the present invention, wherein (a) is treated under hypoxic conditions, (b) is treated under normoxic conditions, (c) is the HCPT fluorescence intensity distribution at the point 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, with a scale of 20 µm; Fig. 9 The graphs are blood circulation curves in different drug treatment groups in Example 6 of the present invention (a) and the graph of the content of anticancer drug HCPT in tumors of tumor-bearing mice (b); Fig.10 The graphs (a) and (b) show the changes in tumor volume and weight in different drug treatment groups in Example 6 of the present invention, as well as the H&E staining and TUNEL immunofluorescence staining results of tumor tissue sections (c), with a scale of 100 μm; Fig.11 This is a graph showing the body weight of tumor-bearing mice in different drug treatment groups in Example 6 of the present invention; Fig.12 This is a diagram showing the H&E staining results of the main organs (heart, liver, spleen, lung, and kidney) of mice in different drug treatment groups in Example 6 of the present invention. DETAILED DESCRIPTION

[0027] The following will be combined with 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.

[0028] The experimental methods without specific conditions in the examples are usually carried out according to conventional conditions and conditions described in the manual, or according to conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all available from commercial channels unless otherwise specified.

[0029] Example 1 Synthesis of Nanomedicine PHAU with Hypoxia Responsiveness and Photoisomerization Function First, PHA was synthesized: 0.5 mmol of 10-hydroxycamptothecin (HCPT) and 1.65 mmol of 4-dimethylaminopyridine (DMAP) were added to 50 mL of dichloromethane to fully dissolve, and then 0.16 mmol of 4,4'-diazobenzene chloride (AZO) was added to fully dissolve, and then 0.01 mmol of mPEG (molecular weight of 5000) was added. The mixture was stirred at room temperature for 12 h. 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 h (ultrapure water was replaced every 6 h), PHA powder was obtained by freeze-drying.

[0030] Then, the nano-drug PHAU was synthesized: 5 mg of the above PHA powder was dissolved in 100 µL of dimethyl sulfoxide (DMSO), and then dripped dropwise into 5 mL of ultrapure water. After being fully stirred for 30 minutes, the polymer micelle PHA was obtained. The polymer micelle PHA was irradiated under 365 nm ultraviolet light for 1 minute and then under visible light for 1 minute. The operation was repeated three times to obtain the nano-drug PHAU.

[0031] Comparative Example 1 Synthesis of drug PHB The preparation process of the drug PHB is shown in the following formula (2): .

[0032] 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, and then 0.5 mmol of 4,4'-biphenyldiacetyl chloride was added and fully dissolved, and then 0.01 mmol of mPEG (molecular weight of 5000) was added, and the reaction was 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 replaced every 6 hours), PHB powder was obtained by freeze-drying.

[0033] Performance Characterization The PHA powder synthesized in Example 1 and the PHB powder synthesized in Comparative Example 1 were tested by hydrogen nuclear magnetic resonance spectroscopy.

[0034] 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.

[0035] 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) characteristic peaks belong 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 benzene ring of AZO, and the 5.43ppm characteristic peak belongs to the benzyl group in AZO; proton peaks e, f, g, h, j belong to the hydrogen on the benzene ring of HCPT, 0.83ppm (p) and 1.91ppm (l) characteristic peaks belong to the terminal methyl and methylene of HCPT, and i and k belong to the methylene group in the HCPT ring; the NMR spectrum of PHB ( Figure 1 (b)) There is only the peak of the methylene group in the HCPT ring between 5-6ppm. The integrated area of ​​the proton peaks c, d, e, g, and f of PHA was further analyzed using the MestReNove software, and the area ratio was 4:4:1:1:1, confirming that the PHA sample was successfully prepared; the integrated analysis of the proton peaks c, d, e, g, and f of PHB showed an area ratio of 4:4:1:1:1, and the NMR results showed that the PHB sample was successfully prepared.

[0036] The PHA powder and PHAU powder synthesized in Example 1 were subjected to transmission electron microscopy (TEM) morphology observation and dynamic light scattering particle size analyzer (DLS) test.

[0037] Specifically, PHA powder, PHA powder irradiated with 365 nm ultraviolet light for 2 min, and PHAU powder were dissolved in water to obtain PHA solution, PHA solution irradiated with 365 nm ultraviolet light for 2 min, and PHAU solution. A drop of the above aqueous solution was dropped on a copper mesh, and after the solution was naturally air-dried, a drop of uranyl acetate was dropped to dye the material on the copper mesh. After natural drying, TEM morphology was observed. At the same time, 2 mL of PHA and PHAU solutions (and 500 times dilutions thereof) were taken for DLS testing. The results are as follows: Figure 2 shown.

[0038] from Figure 2 As can be seen, the polymer micelle PHA is spherical with an average diameter of about 80nm ( Figure 2 (a)); the morphology begins to change after 365nm UV irradiation ( Figure 2 (b)), and finally formed a nanoparticle drug PHAU with a spherical particle structure with an average diameter of about 50 nm ( Figure 2 (c)), and PHAU has a more compact internal structure than PHA; by DLS ( Figure 2 (d) The hydrated particle sizes of PHA and PHAU were determined to be 89.53±4.02nm and 55.64±3.75nm, respectively. PHAU (PDI=0.133) had a narrower particle size distribution and better dispersibility than PHA (PDI=0.205). When the aqueous solutions of PHA and PHAU were diluted 500 times at the same time, it was found that the size of PHA particles changed significantly, while the particle size distribution of PHAU remained basically unchanged, indicating that the stability of polymer micelle PHA was poor, while the nanodrug PHAU had better particle stability. The above results confirmed that the structure of PHAU was more compact than that of PHA. The reason may be that after alternating irradiation of ultraviolet light and visible light, the molecules of PHA were arranged more neatly, resulting in a smaller particle size of PHAU and better stability.

[0039] The drug loading capacity of the PHAU powder synthesized in Example 1 was tested.

[0040] Specifically, HCPT with different concentration gradients was first prepared, and a standard curve model of HCPT fluorescence spectrum was established according to the emission peak intensity of the fluorescence spectrum (the fluorescence spectrum of HCPT has an emission spectrum peak intensity located at 540nm when the excitation light is 380nm, which has a consistent emission spectrum with PHAU ( Figure 3 Then, 1 mg of the lyophilized sample of nanomedicine PHAU was weighed and dissolved in ultrapure water to prepare a 10 mL solution; then, a fluorescence spectrometer was used to quantitatively analyze the content of HCPT in the nanomedicine, wherein the drug loading calculation formula of PHAU is shown in the following formula (3): .

[0041] The drug loading of nanodrug PHAU was calculated to be 10.3%.

[0042] The optical properties of the PHA powder synthesized in Example 1 and the PHB powder synthesized in Comparative Example 1 were tested.

[0043] Specifically, appropriate amounts of PHA and PHB were dissolved in 2 mL of ultrapure water, and UV-Vis was used to measure the ultraviolet absorption peak in a visible light environment. Then, the solution was placed in a dark environment and irradiated with ultraviolet light of a wavelength of 365 nm for 2 minutes, and then UV-Vis detection was performed quickly. Finally, the solution after ultraviolet light irradiation was placed in visible light for 2 minutes for UV-Vis detection. The results are as follows: Figure 4 shown.

[0044] from Figure 4 It can be seen that the polymer micelle PHA underwent photoisomerization under 365nm ultraviolet light irradiation. Before ultraviolet light irradiation, PHA had a strong absorption band near 340nm, which may be attributed to the π→π* transition in the trans structure of PHA; 2 minutes after ultraviolet light irradiation, the absorption peak at 340nm decreased significantly, which may be due to the gradual transformation of the trans structure into the cis structure; the reduction of the trans structure caused the intensity of the π→π* transition band to decrease with the increase of ultraviolet light irradiation time, while the intensity of the n→π* transition band corresponding to the cis isomer near 450nm increased with the increase of ultraviolet light irradiation time. The larger the value, the more molecules with cis configuration are. The intensity changes of π→π* and n→π* transitions indicate that PHA undergoes trans-cis isomerization under UV light. Moreover, the absorption intensities of PHAU formed by UV light irradiation and then visible light irradiation at around 365nm and around 450nm are both between the absorbance of PHA and the absorbance of PHA after UV light irradiation. This indicates that the process of trans-to-cis conversion is reversible, and the final PHAU structure is the most stable state of the molecule, with two configurations, trans and cis ( Figure 4 (a)). When PHB is irradiated with UV light, the UV absorption peak of the PHB molecule does not change ( Figure 4 (b)), this is because there is no azo group in the PHB molecule. The schematic diagram of PHAU formation is shown in Figure 4 (c) As shown. The UV-Vis characterization results of PHA and PHAU successfully confirmed that the PHAU molecule was successfully loaded with a low oxygen response unit (AZO, azo group) and that PHAU had photoisomerization properties.

[0045] Example 2 Disintegration test of nano drug PHAU in hypoxic environment In general, the hypoxic environment of tumors promotes the overexpression of many reductases, including azoreductase. Azoreductase can oxidize and reduce the N=N bond in azobenzene derivatives to break the chemical bond. Therefore, in in vitro experiments, the inorganic reducing agent sodium dithionite (Na2S2O4) can be used as a chemical mimetic of azoreductase at a concentration of 10mM in phosphate buffer solution (PBS, pH=7.4) to simulate the hypoxic environment of tumors and mediate the reduction of the azo group of PHAU. PHAU (1mg / mL) was fully dispersed in a PBS mixed solution containing Na2S2O4 and placed in a constant temperature shaker at 37°C for 60min (100r / min). TEM images before and after PHAU reduction and particle size changes characterized by DLS were recorded.

[0046] At the same time, the fluorescence detector of high performance liquid chromatography (HPLC) was used to quantitatively analyze the HCPT released after the disintegration of PHAU. The fluorescence detector parameters were set as follows: the excitation wavelength was 380nm, the emission wavelength was 540nm, the chromatographic column was a C18 reverse phase column, and the mobile phase was a methanol: water system with a volume ratio of 40:60. The results are shown in Figure 2. Figure 5 shown.

[0047] from Figure 5 It can be seen that after the PHAU aqueous solution was treated with 10 mM Na2S2O4, the spherical nanoparticles disappeared and the morphology was dissolved, and many smaller particles were piled together ( Figure 5 (a)). At the same time, DLS data also show that the particle size distribution of PHAU is no longer uniform, and large-sized substances appear ( Figure 5 (b)), which corresponds to the TEM image, indicating that the structure of PHAU may be disintegrated and small particles may aggregate. In order to further prove that PHAU disintegration can release HCPT, the experiment used HPLC fluorescence detector to monitor the peak of PHAU and Na2S2O4 mixture at different time points (2h, 4h, 8h, 12h); the peak time of PHAU alone is about 31min, and 2h after adding Na2S2O4, a new peak appears at 8min in the HPLC spectrum, which is the characteristic peak of HCPT; and as time goes by, the peak area at 31min gradually decreases, and the peak area at 8min gradually increases, which shows that PHAU is gradually disintegrating and continuously releasing free HCPT ( Figure 5 (c)). At the same time, the control group PHB was also treated with Na2S2O4 to exclude the influence of other factors on the experiment. DLS data showed that the hydrated particle size of PHB did not change ( Figure 5 (d)); and there is no significant difference in the TEM images of PHB before and after Na2S2O4 treatment ( Figure 5(e)). The above results all confirm that PHAU molecules can be reduced by reducing agents, undergo continuous elimination and decarboxylation reactions, undergo structural disintegration, and release free HCPT molecules, which are expected to respond to hypoxia stimulation in a hypoxic tumor environment.

[0048] Example 3 In vitro release test of nano drug PHAU Overexpression of azoreductase in hypoxic tumor tissue can reduce the azo groups in the compound. In order to simulate the drug release of polymer micelles in a hypoxic tumor environment, the inorganic reducing agent Na2S2O4 was used instead of azoreductase for the experiment. Specifically, two 1mL and 1mg / mL PHAU sample solutions were transferred to dialysis bags (molecular weight cutoff: 5kDa), and then immersed in a test tube containing 30mL PBS (pH 7.4) containing different concentrations of Na2S2O4 (0mM and 10mM), and released by shaking at 100r / min and 37℃ in a constant temperature shaker. At specific time points (30min, 1h, 2h, 4h, 6h, 9h, 12h, 24h, 36h, 48h), 3mL of the release medium outside the dialysis bag was taken out and 3mL of PBS solution with the corresponding concentration of Na2S2O4 was added to keep the volume of PBS solution constant. At the same time, PHB was used as a control for the experiment. The peak intensity of the emission spectrum of the release medium at each time point at 540nm was measured using a fluorescence spectrometer when the excitation light was 380nm. The HCPT concentration released by PHAU at each time point was calculated using the standard concentration curve of HCPT, thereby obtaining the cumulative release of the drug. The results are shown in Figure 6 The calculation formula of HCPT cumulative release rate is shown in the following formula (4): ; in, E r is the cumulative release of the drug (%), V e is the volume of release medium taken out at each fixed time point, n represents the number of sampling times, C i is the HCPT concentration in the release medium at the i-th sampling, V 0 is the total volume of the release medium, m is the total mass of the nanodrug loaded.

[0049] from Figure 6It can be seen that the release rate of the nano drug PHAU, which did not add Na2S2O4, slowed down significantly when the drug release reached 15%, and finally reached nearly 20% after 48 hours; the drug release rate of PHA was slightly faster than that of PHAU, but only about 30% of HCPT was released after 48 hours. This result also further confirmed that the structure of PHAU is more stable than that of PHA, and HCPT is safer in the structure formed by PHAU; after adding Na2S2O4, the release rate of PHA and PHAU accelerated sharply, and finally the amount of HCPT released in 48 hours was as high as 90% ( Figure 6 (a)). However, there is no significant difference in the drug release rate of PHB with or without the presence of Na2S2O4 ( Figure 6 (b)). This is because PHAU and PHA molecules contain azo groups, which can be reduced by Na2S2O4 to break the valence, which is conducive to the release of HCPT from micelles. This experimental result shows that PHAU has a small premature drug release rate in the blood circulation, and its structure is stable, which can maintain a long blood circulation time. When it reaches the hypoxic tumor tissue, it can quickly release the chemotherapy drug HCPT under the stimulation of hypoxia.

[0050] Example 4 Cytotoxicity test of nano drug PHAU The CCK-8 method was used to detect the toxicity of different concentrations of nanodrug PHAU, polymer micelle PHA, PHB and single drug HCPT to Hela cells in normoxic and hypoxic environments. Specifically, Hela cells in the logarithmic growth phase were first taken, the culture medium in the culture dish was discarded, 1mL PBS was added to rinse twice, and then 1mL of trypsin was added and immediately placed in the incubator for 3 minutes to digest the Hela cells that grew on the wall. After the cells were completely detached from the wall, 3mL of complete culture medium (1% double antibody, 10% fetal bovine serum, 89% DMEM) was added and mixed to terminate the digestion. After being transferred to a centrifuge tube, it was centrifuged at 1000rpm in a centrifuge for 3min, the supernatant was discarded and an appropriate amount of complete culture medium was added, and the cell suspension was gently blown with a pipette. The cell suspension was inoculated in a 96-well plate (100µL / well), and the number of cells in each well was about 5000. In order to prevent the outermost solution of the 96-well plate from evaporating, PBS (200µL / well) was added to seal the water. The experiment was divided into two groups. One group placed the culture plate in a conventional incubator at 37°C, 5% CO2, and 21% O2 for pre-culture for 24 hours; the other group placed the culture plate in a hypoxic cell culture incubator at 37°C, 2% O2, and nitrogen balance for pre-culture for 24 hours. Use complete culture medium to prepare PHAU, PHA, PHB, and HCPT solutions with different concentration gradients. Then use a spray gun to gently aspirate the culture medium in the 96-well plate, add the prepared sample solution (100µL / well), set six replicate wells for each concentration, and continue to incubate in the incubator for 24 hours. Finally, add the CCK-8 reagent to the 96-well plate (10µL / well), place it in the incubator and culture for 2 hours, and then use a microplate reader to measure the absorbance (OD) at 450nm. The toxicity of CCK-8 reagent to cells is very low and can be ignored. Since CCK-8 can continuously react with dehydrogenases in living cells, the OD value will continue to increase, so the cell survival rate can be calculated by the OD value. The results are as follows Figure 7 The cell survival rate calculation formula is shown in the following formula (5): ; in, A s The experimental wells contain culture medium, CCK-8, and drugs for cells; A c The control wells contained cell culture medium, CCK-8, and no drugs; A b The blank wells contain no cell culture medium, CCK-8, or drugs.

[0051] from Figure 7 It can be seen that with the increase of drug concentration, the cell survival rate gradually decreased ( Figure 7(a), 7(b)). When the concentration of PHAU reaches 25µg / mL, the survival rate of Hela cells in a normoxic environment is only about 38%, but it is still greater than the survival rate of about 21% in a hypoxic environment, indicating that the higher the concentration of the drug, the greater the toxicity to the cells. Among them, the single drug HCPT has similar cytotoxicity at the same drug concentration regardless of whether it is in a normoxic or hypoxic environment, indicating that the presence of oxygen has no effect on the therapeutic effect of HCPT. Moreover, when the toxicity of PHAU, PHA, and PHB at the same concentration to Hela cells in hypoxic and normoxic environments is compared, PHAU is more toxic. This shows that PHAU releases HCPT faster in Hela. Moreover, the killing effect of PHAU on Hela cells in a hypoxic environment is similar to that of HCPT at the same concentration, indicating that PHAU can release all HCPT more quickly in hypoxic cancer cells, achieving the effect of efficiently killing cancer cells.

[0052] Example 5 In vitro cellular uptake test of nano drug PHAU The uptake of nanodrug PHAU in Hela cells was analyzed by confocal laser microscopy (CLSM). Specifically, the nanodrugs PHAU, PHA, and PHB were first prepared into solutions. 1 mg of each was weighed and then added to PBS 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 inoculated in a confocal dish (1×10 5 / well), incubate for 24 hours to allow it to adhere to the wall and grow, then aspirate the culture medium. Subsequently, add 1mL of the pre-prepared PHAU, PHA and PHB sample solutions respectively, incubate with Hela cells for 4 hours, then discard the solutions, 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 1mL 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 380nm to obtain the fluorescence signal (green fluorescence) of HCPT, so as to observe the drug uptake of the cells. The results are shown in Figure 8 shown.

[0053] 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 in the cells ( Figure 8(b)). After incubation of PHA and PHB with cells, the fluorescence of HCPT was mainly distributed in the cytoplasm, while after co-incubation of PHAU with cells, HCPT was able to diffuse rapidly after entering the cells and was enriched in the nucleus. This phenomenon indicates that the nanodrug PHAU can deliver HCPT into the nucleus more effectively than PHA in a hypoxic environment. Moreover, under normoxic conditions, after co-incubation of PHAU, PHA, and PHB polymer micelles with cells, the total amount of HCPT entering the nucleus was not much different; however, in a hypoxic environment, the effect of PHAU in delivering HCPT to the nucleus was significantly improved ( Figure 8 (d)). This indicates that after PHAU is taken up by cells, the overexpressed reductase in the hypoxic environment promotes the disintegration of the nanodrug PHAU, which is conducive to the release of free HCPT, allowing more HCPT to enter the cell nucleus to exert a chemotherapeutic effect. This experimental result is consistent with the above-mentioned cytotoxicity experimental results.

[0054] Example 6 In vivo biological evaluation test of nanomedicine PHAU All animal experiments were conducted in accordance with the National Laboratory Animal Management Regulations. First, a mouse cervical cancer model was constructed using Hela cells to evaluate the anti-tumor effect of the nanodrug PHAU in vivo. Approximately 2×10 6 Hela cells in the logarithmic growth phase were selected. Four days later, 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 for parallel control. The nanodrug dispersion was injected by tail vein injection. The groups were PBS, HCPT, PHB, PHA, and PHAU, respectively. The PBS group was used as a blank control, and the calculated equivalent of HCPT in the other groups was 5 mg / kg. The experiment lasted for two weeks, with nanodrugs injected once every other day, and 50 µL was injected each time.

[0055] First, the half-life of the above drugs in the blood circulation was determined. Specifically, a fluorescence spectrum standard curve of HCPT alone was constructed, and then different time points (10min, 30min, 1h, 2h, 3h, 4h, 6h, 8h) were selected to collect blood from the tail vein of mice, and the HCPT in the blood was quantitatively analyzed by fluorescence spectroscopy. After the experiment, the tumor tissues and major organs (heart, liver, spleen, lungs, and kidneys) of the mice were removed for use. After weighing part of the tumor tissue, normal saline was added in proportion and centrifuged at 3000rpm for 10 minutes to prepare a tissue homogenate. The supernatant was taken and extracted with a mixed solution of methanol and water. After the extract was dried with nitrogen, it was dissolved in methanol for fluorescence spectroscopy detection to obtain the percentage of HCPT drug uptake per gram of tumor tissue. The results are as follows. Fig. 9 shown.

[0056] from Fig. 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 higher 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-life of the other two drugs ( Fig. 9 (a)). This shows that the blood circulation time of small molecule HCPT alone is short and it is easily metabolized by the body; while the structure of nano drug PHAU is more stable, which increases the blood circulation time of single drug HCPT from 20min to 2h. This may be related to the size of the micelles we designed, which reduces the risk of the drug being engulfed and cleared out of the body by macrophages, thereby increasing the chance of HCPT reaching the tumor site and improving the drug delivery rate. The uptake of PHAU in tumor tissue is the highest, reaching 7%ID / g, which is nearly 6 times that of single drug HCPT ( Fig. 9 (b)). The experimental results show that the efficiency of single-drug HCPT entering tumor tissue is low, it is not easy to accumulate near the tumor, and it cannot exert a chemotherapy effect; while the nano-drug PHAU is conducive to increasing the tumor targeting of the drug and greatly improving the concentration of HCPT in the tumor site.

[0057] Then, throughout the experiment, the mouse tumor volume and mouse body weight were monitored every other day using a vernier caliper and a balance. The mouse volume calculation formula is shown in the following formula (6): ; 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 Fig.10 shown.

[0058] from Fig.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 ( Fig.10(a), 10(b)); Among them, the tumor inhibition effect of single-agent HCPT was the worst, followed by PHB and PHA, and PHAU had the best effect. The experimental results show that compared with single-agent HCPT, other nanodrug carriers have better delivery effects on drugs to tumor sites. In addition, the therapeutic effect of PHA is higher than that of PHB, indicating that the hypoxia response unit in the PHA structure can undergo specific stimulation response at the tumor site and achieve targeted drug release; and compared with PHA, PHAU has a smaller particle size, which is conducive to the uptake of tumor tissue and enrichment at the tumor site, and has excellent tumor inhibition effects. In addition, the results of H&E and TUNEL staining confirmed that PHAU treatment had a more obvious effect of inducing tumor necrosis and apoptosis compared with the other four control groups ( Fig.10 (c)). According to the results of TUNEL staining, the number of live cells (green) in the tumor tissue sections after PHAU treatment was significantly reduced, and the number of dead cancer cells (blue) increased, indicating that PHAU has a good anti-tumor effect in vivo.

[0059] Next, throughout the experiment, the body weight of the tumor-bearing mice was recorded regularly. Fig.11 shown.

[0060] from Fig.11 It can be seen that the mice did not show loss of appetite or abnormal weight within two weeks after drug treatment. Different drug preparations PBS, HCPT, PHB, PHA, and PHAU did not have much effect on the weight of mice, and the weight differences of the five groups of mice were all within a reasonable range.

[0061] Finally, in order to evaluate the biosafety of the drug and detect the distribution of the drug in mice, the main organs of the mice (heart, liver, spleen, lung, and kidney) were fixed, frozen, and sectioned, and stained with H&E. The results are as follows: Fig.12 shown.

[0062] from Fig.12 It can be seen that after drug treatment, no obvious damage was found in the heart, liver, spleen, lungs, and kidneys. This is because the release effect of HCPT is poor under normoxic conditions, and the redox environment in normoxic tissues reaches a balance, which cannot provide PHAU with excessive azoreductase for structural disintegration, and is metabolized by the body after blood circulation. Therefore, the nanomedicine PHAU has good biocompatibility and tumor tissue specificity, and can significantly reduce the toxic side effects of drugs on normal tissues.

[0063] In summary, the present invention uses the hypoxia response unit 4,4'-diacyl chloride azobenzene as the hypoxia response unit, which is covalently linked with 10-hydroxycamptothecin and the drug carrier mPEG to form a size-controlled nanodrug. The drug release rate is controlled by adjusting the size of the nanodrug and the drug concentration is maintained within the required range. At the same time, the nanodrug can target the lesion site to reduce the side effects of the drug. In addition, the nanodrug has a high utilization rate and good biocompatibility.

[0064] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0065] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A nanomedicine with hypoxia responsiveness and photoisomerization function, characterized in that: The nano drug 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 a nano drug having hypoxia responsiveness and photoisomerization function as claimed in claim 1, characterized in that: The steps include: S1, using 4,4'-diacyl chloride azobenzene, 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 by self-assembly, and then treating 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'-diacidyl chloride azobenzene 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 cutoff 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, and then irradiating with visible light for 0.5-1.5 minutes; 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 nanodrug with hypoxia responsiveness and photoisomerization function as claimed in claim 1 or the nanodrug 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.

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