A dual response nano-carrier to hypoxia and pH, drug-loaded nanoparticles and application thereof

Abstract

The application discloses a low-oxygen and pH dual-response nano-carrier, a drug-loaded nanoparticle and application thereof, and belongs to the technical field of biological medicines. The nano-carrier with moderate particle size and low-oxygen and pH dual-response is obtained by using the reaction of aldehyde benzoic acid, nonaethylene glycol and azo-p-phenetidine, the nano-carrier is specifically targeted to a tumor site, and then specifically releases drugs, thereby reducing toxic side effects; meanwhile, after the nano-carrier responds, benzaldehyde groups are released, the benzaldehyde groups are covalently connected with proteins on a lysosome membrane, the structure and activity of the proteins are changed, the lysosome membrane is broken after the proteins are denatured, the permeability of the lysosome membrane is increased, the nano-carrier cannot be discharged to the extracellular, the drug concentration in cells is greatly improved, and the drug bioavailability of the nano-carrier is significantly improved; in addition, the drug loading capacity of the nano-carrier is high, and therefore, the nano-carrier has a good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a low-oxygen and pH dual-response nano-carrier, a drug-loaded nanoparticle and application thereof. BACKGROUND

[0002] Cancer is one of the major diseases causing human death at present, and chemotherapy is one of the most important means for treating cancer at present. However, the treatment effect is poor due to no specificity and strong drug resistance. Therefore, it is of great significance in the treatment of cancer to design a treatment strategy which can improve the specificity of drugs to target tumor sites and improve the drug utilization rate to reduce drug resistance. Supramolecular nanomaterials can be passively targeted to accumulate in tumor sites in vivo due to their suitable size through the enhanced permeability and retention (EPR) effect. Moreover, the designability of supramolecular nanomaterials provides a wide design platform for treatment strategies.

[0003] Studies have shown that tumor cells have different specific tumor microenvironments (TEM) from normal cells, which mainly manifest in low-oxygen microenvironment, acidic pH, overexpression of reactive oxygen species (ROS) and glutathione (GSH), etc. On the one hand, these specific expressions will have a negative impact on chemotherapy, but on the other hand, they also provide ideas for targeting tumors. Due to the abnormal proliferation and metabolism of tumor cells, the number of intracellular acidic organelles lysosomes will also increase. Lysosomes are mainly responsible for clearing intracellular foreign substances, including anti-tumor drugs, which is one of the reasons for drug resistance. Studies have found that tumor cells are relatively sensitive to lysosome disorder, and once the permeability of lysosome membrane changes, various hydrolytic enzymes (such as cathepsin B) and other substances (such as iron and H + ) in lysosomes enter the cytoplasm, which will induce lysosome-dependent cell death (LDCD). The commonly used lysosome membrane permeability (LMP) inducers at present mainly include lysosome-dwelling detergents (such as Siramesine), dipeptide methyl ester (such as Leu-Leu-OMe) and reactive oxygen species (ROS), etc. However, these inducers are rarely able to selectively induce LMP of cancer cells, and also cause strong toxic side effects and bring pain to patients.

[0004] At present, the existing drug delivery nano-carriers have problems of poor targeting, low drug bioavailability, poor drug loading capacity, etc. SUMMARY

[0005] The application aims to provide a low-oxygen and pH dual-response nano-carrier, drug-loaded nanoparticles and application thereof.

[0006] In the first aspect, the application provides a low-oxygen and pH dual-response nano-carrier, which has the structure shown in the following formula (I):

[0007] ;

[0008] The molecular weight of the low-oxygen and pH dual-response nano-carrier is 800-10000 g / mol.

[0009] In the application, the inventors find that by using the reaction of p-formaldehyde benzoic acid, nonaethylene glycol and azo-p-phenylamine, a nano-carrier with moderate particle size and low-oxygen and pH dual-response is obtained. The nano-carrier contains an azo group, so it has low-oxygen response, and contains an imine group, so it has pH response. The above dual response makes the nano-carrier specifically target tumor sites, and then specifically releases drugs to reduce toxic side effects. At the same time, after the nano-carrier responds, benzaldehyde groups are released, which covalently bond with proteins on the lysosome membrane, causing changes in the structure and activity of the proteins. After the protein denatures, the lysosome membrane ruptures, the permeability increases, and the nano-carrier (loaded with drugs) cannot be discharged outside the cell, greatly increasing the intracellular drug concentration, thereby significantly improving the drug bioavailability of the nano-carrier. In addition, the drug loading capacity of the nano-carrier is high, so it has good application prospects.

[0010] In the second aspect, the application provides a preparation method of the low-oxygen and pH dual-response nano-carrier, which comprises the following steps: S1, reacting p-formaldehyde benzoic acid and nonaethylene glycol in the presence of a catalyst and an activator to obtain an intermediate copolymer after separation and purification; S2, mixing the intermediate copolymer with azo-p-phenylamine and reacting to obtain the low-oxygen and pH dual-response nano-carrier after separation and purification.

[0011] In the application, the preparation process of the low-oxygen and pH dual-response nano-carrier is shown in the following formula (1) and (2):

[0012] ;

[0013] .

[0014] The preparation method is simple, the raw materials are green, non-toxic, cheap and easy to obtain, and has good biological safety, so that large-scale production and application are facilitated.

[0015] In some embodiments, in step S1, the molar ratio of aldehyde benzoic acid, nonaethylene glycol, catalyst and activator is (3-10):(3-10):(0.3-1):(3-10); and the catalyst comprises 4-dimethylaminopyridine, and the activator comprises 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride.

[0016] In some preferred embodiments, the molar ratio of aldehyde benzoic acid, nonaethylene glycol, catalyst and activator is 6:6:0.6:6.

[0017] In some embodiments, in step S1, the reaction in the presence of the catalyst and the activator specifically comprises: reacting for 18-25 h at room temperature.

[0018] In some preferred embodiments, the reaction in the presence of the catalyst and the activator specifically comprises: reacting for 21 h at room temperature.

[0019] 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 present application, the organic solvent is preferably dichloromethane; triethylamine can also be added to the reaction for better reaction, and the amount of triethylamine added is added according to the actual use requirement.

[0020] In some embodiments, in step S1, the intermediate copolymer obtained after separation and purification specifically comprises: washing the obtained product with a hydrochloric acid solution with a mass concentration of 10%, and after drying, column chromatography separation is performed to obtain the intermediate copolymer.

[0021] In some embodiments, in step S2, the molar ratio of the intermediate copolymer to azo p-aniline is (0.13-0.16):(0.13-0.16).

[0022] In some preferred embodiments, the molar ratio of the intermediate copolymer to azo p-aniline is 0.1475:0.1475.

[0023] In some embodiments, in step S2, after the intermediate copolymer and azo p-aniline are mixed, the reaction specifically comprises: reacting for 20-30 h at room temperature.

[0024] In some preferred embodiments, the reaction after mixing the intermediate copolymer with azo-p-phenylenediamine specifically comprises: reacting for 24 h at room temperature.

[0025] 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. In the present application, the organic solvent is preferably dichloromethane.

[0026] In some embodiments, when the reaction after mixing the intermediate copolymer with azo-p-phenylenediamine, further comprises the step of adding glacial acetic acid to the reaction system.

[0027] It can be understood that the addition of glacial acetic acid facilitates better reaction, and the amount of glacial acetic acid added is added according to the actual use needs.

[0028] In some embodiments, in step S2, the low-oxygen and pH dual-responsive nanocarrier obtained by separation and purification specifically comprises: re-precipitating the obtained product with diethyl ether three times to obtain the low-oxygen and pH dual-responsive nanocarrier.

[0029] In a third aspect, the present application provides a drug-loaded nanoparticle, which is obtained by loading a drug on the low-oxygen and pH dual-responsive nanocarrier prepared by the above-mentioned low-oxygen and pH dual-responsive nanocarrier or any of the above-mentioned preparation methods.

[0030] In some embodiments, the drug comprises doxorubicin.

[0031] It can be understood that the drug can be selected according to the type of target cells, and in the present application, the drug preferably comprises doxorubicin.

[0032] In a fourth aspect, the present application provides a preparation method of the drug-loaded nanoparticle as described in any of the above, comprising the following steps: mixing the low-oxygen and pH dual-responsive nanocarrier and the drug after being dissolved in an organic solvent respectively, then adding ultrapure water, and then obtaining the drug-loaded nanoparticle after dialysis, filtration and freeze-drying.

[0033] In some embodiments, the dialysis specifically comprises: using a dialysis bag with a molecular weight cut-off of 3000-4000 Da, and dialyzing for 18-32 h.

[0034] In some preferred embodiments, a dialysis bag with a molecular weight cut-off of 3500 Da is used, and dialysis is performed for 24 h.

[0035] It can be understood that the organic solvent can be selected from conventional organic solvents. In the present application, the organic solvent is preferably dimethyl sulfoxide.

[0036] In a fifth aspect, the present application provides the use of the drug-loaded nanoparticle as described in any of the above or prepared by the above-mentioned preparation method in the preparation of an antitumor drug.

[0037] The beneficial effects of the present application are: different from the prior art, the present application uses the reaction of aldehyde benzoic acid, nonaethylene glycol and azo-p-phenylamine to obtain a nano carrier with moderate particle size and low oxygen and pH dual response, the nano carrier contains an azo group, thus having low oxygen response, and contains an imine group, thus having pH response, the above-mentioned dual response makes the nano carrier specifically target the tumor site, and then specifically release the drug, reducing the toxic side effects; at the same time, after the response of the nano carrier, benzaldehyde groups are released, which covalently connect with the proteins on the lysosome membrane, causing changes in the structure and activity of the proteins, and the lysosome membrane is ruptured after the denaturation of the proteins, the permeability is increased, the nano carrier (loaded drug) cannot be discharged outside the cell, the intracellular drug concentration is greatly increased, and thus the drug bioavailability of the nano carrier is significantly improved, in addition, the nano carrier has high drug loading capacity, thus having good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 The GPC chart of the intermediate copolymer F-PEG synthesized in Example 1 of the present application is shown in Figure 1. 1 The H-NMR chart (a) of the intermediate copolymer F-PEG synthesized in Example 1 of the present application is shown in Figure 2. 1 The H-NMR chart (b) of the intermediate copolymer F-PEG synthesized in Example 1 of the present application is shown in Figure 3.

[0039] Figure 2 The GPC chart of the intermediate copolymer F-PEG synthesized in Example 1 of the present application is shown in Figure 1.

[0040] Figure 3 The UV-vis chart of the intermediate copolymer F-PEG synthesized in Example 1 of the present application is shown in Figure 4.

[0041] Figure 4 The FT-IR chart of the intermediate copolymer F-PEG synthesized in Example 1 of the present application is shown in Figure 5.

[0042] Figure 5 The DLS chart of the low oxygen and pH dual response nano carrier FPA solution prepared in Example 1 of the present application after being placed for 0, 5 days, respectively, is shown in Figure 6.

[0043] Figure 6 The TEM chart of the low oxygen and pH dual response nano carrier FPA solution prepared in Example 1 of the present application is shown in Figure 7.

[0044] Figure 7 The DLS chart of the PBS aqueous solution containing FPA, the acetic acid buffer (pH value is 5.0) containing FPA, the PBS aqueous solution containing FPA and Na2S2O4, and the acetic acid buffer (pH value is 5.0) containing FPA and Na2S2O4 prepared in Example 1 of the present application is shown in Figure 8.

[0045] Figure 8 UV-Vis spectra of acetic acid buffer (pH 5.0) containing FPA and Na2S2O4 prepared for Example 1 of the present application;

[0046] Figure 9 TEM images of acetic acid buffer (pH 5.0) containing FPA and Na2S2O4 prepared for Example 1 of the present application after 24 h;

[0047] Figure 10 Drug loading efficiency and encapsulation efficiency of drug-loaded nanoparticles FPA / DOX prepared in Example 2 of the present application;

[0048] Figure 11 In vitro release curve of drug-loaded nanoparticles FPA / DOX in Example 3 of the present application;

[0049] Figure 12 Raman spectra of BSA, F-PEG, BSA and F-PEG mixed freeze-dried powder in Example 4 of the present application;

[0050] Figure 13 Morphology of BSA, F-PEG, F-PEG and BSA co-incubated for 24 h in Example 4 of the present application;

[0051] Figure 14 FT-IR spectra of BSA, F-PEG, BSA and F-PEG mixed freeze-dried powder in Example 4 of the present application;

[0052] Figure 15 CLSM images of Hela cells incubated with amiloride, M-β-CD, Hypertonic sucrose for 30 min and untreated Hela cells (a) and average intensity of DOX fluorescence signal in each treatment group (b) in Example 5 of the present application;

[0053] Figure 16 CLSM images of Hela cells co-incubated with FPA for 0, 1, 2, 4, 6 h and then treated with AO (a) and average intensity of AO red fluorescence signal in cells treated at different times (b) in Example 6 of the present application;

[0054] Figure 17 TEM images of lysosomes in Hela cells incubated with drug-loaded nanoparticles FPA / DOX in Example 6 of the present application;

[0055] Figure 18A CLSM images of Hela cells co-incubated with FPA / DOX for 1, 2, 4, 6, 9 h and then stained with Lyso-Tracker and DAPI in Example 7 of the present application;

[0056] Figure 18B for based on Figure 18A Fig. 6 is a diagram of the red light of DOX and the blue light of DAPI, the corresponding fluorescence signal distribution and the intensity diagram of the DOX fluorescence signal in the nucleus in the embodiment 5 of the present application;

[0057] Figure 19 Fig. 7 is a diagram of the cell survival rate after the FPA treatment of Hala cells in the embodiment 8 of the present application (a), the cell survival rate of the FPA / DOX treatment of Hala cells under the condition of normal oxygen and pH value of 7.4 (b) and the cell survival rate of the FPA / DOX treatment of Hala cells under the condition of low oxygen and pH value of 5.0 (c);

[0058] Figure 20 Fig. 8 is a flow cytometry diagram of the co-incubation of the FPA / DOX, free DOX and PBS with Hela cells for 6h in the embodiment 9 of the present application;

[0059] Figure 21 Fig. 9 is a diagram of the in vivo anti-tumor efficacy of the drug-loaded nanoparticles FPA / DOX in the embodiment 10 of the present application; wherein, (a) is a diagram of the body weight change of mice, (b) is a diagram of the tumor volume change of mice, (c) is a diagram of the average weight of tumors of the treated mice at 18 days, (d) is a diagram of the tumors of mice at different time in different treatment groups; (e) is a diagram of the H&E staining and TUNEL staining of the tumor site of mice after treatment;

[0060] Figure 22 Fig. 10 is a diagram of the in vivo biological safety evaluation of the drug-loaded nanoparticles FPA / DOX in the embodiment 11 of the present application; wherein, (a) is a diagram of the serum detection results of mice treated with PBS, free DOX and drug-loaded nanoparticles FPA / DOX, (b) is a diagram of the H&E staining results of the main organs (heart, liver, spleen, lung, kidney) of mice treated with PBS, free DOX and drug-loaded nanoparticles FPA / DOX;

[0061] Figure 23 Fig. 11 is a diagram of the synthesis of the drug-loaded nanoparticles FPA / DOX and the principle of the targeting tumor cells in the present application. DETAILED DESCRIPTION

[0062] The technical solutions in the embodiments of the present application will be clearly and completely described below 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 the other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0063] For experimental methods in the examples where specific conditions are not specified, generally conventional conditions and conditions described in the manual or conditions recommended by the manufacturer were followed. The general equipment, materials, reagents, etc. used were all commercially available unless otherwise specified.

[0064] Example 1 Synthesis of Hypoxia and pH Dual-Response Nanocarrier FPA

[0065] First, the intermediate copolymer F-PEG was synthesized: 6 mmol of p-formylbenzoic acid was dissolved in 20 mL of tetrahydrofuran, and 2.18 mL of thionyl chloride was added under ice-bath stirring. After stirring for 8 h under ice-bath stirring, the solvent was removed by rotary evaporation, and 20 mL of dichloromethane was added to obtain solution a; 6 mmol of nonaethylene glycol was dissolved in 20 mL of dichloromethane, and 209 μL of triethylamine, 0.6 mmol of 4-dimethylaminopyridine (DMAP), 6 mmol of 1-( 3-Dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC·HCl) was added to obtain solution b; solution a was added dropwise into solution b, and the mixture was stirred at room temperature for 21 hours. The solvent was removed by rotary evaporation, and the mixture was washed with 10% hydrochloric acid solution and dried. The resulting product was separated by column chromatography using methanol:ethyl acetate (volume ratio) of 2:98, 4:96, 8:92, 12:88, 16:84, and 20:80 as eluent to obtain F-PEG.

[0066] Then, the hypoxia and pH dual-responsive nanocarrier FPA was synthesized: 0.1475 mmol of F-PEG was dissolved in 2 mL of dichloromethane, and then 50 μL of glacial acetic acid was added, followed by 0.1475 mmol of azobenzene, mixed and stirred, and reacted at room temperature for 24 hours. After the reaction, the mixture was precipitated three times with glacial ether to obtain the hypoxia and pH dual-responsive nanocarrier FPA.

[0067] Performance Testing

[0068] First, using H NMR spectroscopy ( 1 H-NMR) was used to detect the hypoxia and pH dual-responsive nanocarrier FPA synthesized above, and the results were as follows Figure 1 shown.

[0069] from Figure 1 As can be seen from the figure, the hypoxia and pH dual-responsive nanocarrier FPA was successfully synthesized.

[0070] Then, the hypoxia and pH dual-responsive nanocarrier FPA synthesized above was subjected to gel permeation chromatography (GPC) using tetrahydrofuran as solvent. The results are shown in Figure 2 shown.

[0071] from Figure 2As can be seen from FIG. 1 and FIG. 2, the molecular weight of the hypoxia and pH dual-responsive nanocarrier FPA is 4194 g / mol.

[0072] Next, the UV-vis spectrum of the above-synthesized hypoxia and pH dual-responsive nanocarrier FPA, F-PEG and azo-p-phenetidine was tested using a UV-visible spectrophotometer, and the FT-IR spectrum of the above-synthesized hypoxia and pH dual-responsive nanocarrier FPA was measured using a tablet pressing method, and the results are shown in FIG. 3 and FIG. 4, respectively. Figure 3 and 4

[0073] As can be seen from FIG. 5 and FIG. 6, the hypoxia and pH dual-responsive nanocarrier FPA was successfully synthesized. Figure 3 and 4

[0074] Further, an aqueous PBS solution containing 0.25 mg / mL FPA was prepared, the hydration particle size of the above-synthesized hypoxia and pH dual-responsive nanocarrier FPA was measured using a Malvern particle size tester, and then the sample was placed at room temperature for 5 days, and the hydration particle size was detected again to verify the stability of the nanoparticles; an aqueous PBS solution containing 0.25 mg / mL FPA was continuously prepared, and uranyl acetate was used as a staining agent, and the morphology of the above-synthesized hypoxia and pH dual-responsive nanocarrier FPA was observed using a transmission electron microscope (TEM), and the results are shown in FIG. 7 and FIG. 8, respectively. Figure 5 and 6

[0075] As can be seen from FIG. 9 and FIG. 10, the hypoxia and pH dual-responsive nanocarrier FPA has a suitable nanosize and can exist stably under physiological conditions. Figure 5 and 6

[0076] Further, the hypoxia and acid dual responsiveness of the nanocarrier FPA was verified.

[0077] ​​​​Specifically, an inorganic reducing agent, sodium dithionite (Na2S2O4), was used as a chemical mimic of azoreductase. 10 mM Na2S2O4 was added to an acetic acid buffer (pH 5.0) to simulate the hypoxic and acidic environment in vivo. PBS aqueous solution containing 0.05 mg / mL FPA, acetic acid buffer (pH 5.0) containing 0.05 mg / mL FPA, PBS aqueous solution containing 0.05 mg / mL FPA and 10 mM Na2S2O4, and PBS aqueous solution containing 0.05 mg / mL FPA and 10 mM The acetic acid buffer (pH 5.0) of Na2S2O4 was placed in a constant temperature shaker at 37°C and shaken (100 r / min) for 2 days. Samples were taken at 0, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, and 48 hours to test their DLS to characterize the change in the particle size of the nanoparticles, and samples were taken at 24 hours to test their TEM images. In addition, an acetic acid buffer (pH 5.0) containing 0.25 mg / mL FPA and 10 mM Na2S2O4 was prepared and placed in a constant temperature shaker at 37°C (100 r / min) for 0, 6, 9, 12, and 24 hours, and its UV-vis spectrum was measured. The results are as follows: Figures 7-9 shown.

[0078] from Figures 7-9 It can be seen that the nanocarrier FPA will disintegrate and break in a hypoxic and / or acidic environment to obtain compounds with smaller particle size. The above results show that the nanocarrier FPA has dual responsiveness to hypoxia and acid, and therefore can be used as a drug loading carrier for targeting tumors.

[0079] Example 2 Preparation of drug-loaded nanoparticles FPA / DOX

[0080] The drug-loaded nanoparticles FPA / DOX were prepared by dialysis method.

[0081] Specifically, 30 mg of FPA was weighed and dissolved in 500 μL of dimethyl sulfoxide (DMSO), and 3 mg of doxorubicin (DOX) was weighed and dissolved in 200 μL of dimethyl sulfoxide (DMSO). The two were mixed and injected into 10 mL of ultrapure water in a vortex manner. After stirring for 4 hours in the dark, the mixture was dialyzed (MWCO = 3500 Da) for 24 hours, filtered and freeze-dried to obtain drug-loaded nanoparticles FPA / DOX. The FPA / DOX (1 mg / ml) solution was measured using a microplate reader. Lambda ex =485nm, Lambda em =592nm fluorescence curve to determine the content of DOX loaded therein; and the drug loading capacity (DLC) and encapsulation efficiency (EE) were calculated using the following formulas (3) and (4). The results are shown in Figure 10 shown.

[0082] .

[0083] From Figure 10 it can be seen that the low oxygen and pH dual-responsive nanocarrier FPA has good drug loading and encapsulation efficiency.

[0084] Example 3 In vitro release of drug-loaded nanoparticles FPA / DOX

[0085] This example studies the drug release behavior of drug-loaded nanoparticles FPA / DOX at 37℃ in 10mM Na2S2O4 solution (pH value is 5.0), 10mM Na2S2O4 solution (pH value is 7.4), buffer (pH value is 5.0), and buffer (pH value is 7.4), respectively.

[0086] Specifically, first, the drug-loaded nanoparticle FPA / DOX dispersion (1mg / mL) was placed in a dialysis bag with a MWCO of 3500Da, and placed in 30mL of the corresponding release medium, and shaken in a constant temperature shaker at 37℃ and 100r / min. At 0.5, 1, 2, 4, 6, 9, 12, 24, 36, 48h, 3mL of dialysate was taken, and the corresponding fresh release medium solution was added. The cumulative DOX release was determined by fluorescence analysis (λex=485nm, λem=592nm). Each group of experiments was designed in triplicate; the drug release rate was calculated by the following formula (5), and the results are shown in Lambda ex =485nm, Lambda em =592nm). Figure 11

[0087] ;

[0088] Among them, C n : the DOX concentration measured at the nth time; C i : the DOX concentration measured at the ith time; V : the volume of the release medium; V 0: the volume of the removed external liquid; m: the total mass of DOX in the system.

[0089] From Figure 11 it can be seen that the drug-loaded nanoparticles FPA / DOX have good release rate for DOX after low oxygen and acid dual response.

[0090] Example 4 Study on the cross-linking behavior of intermediate copolymer F-PEG and lysosomal membrane proteins

[0091] ​In this embodiment, bovine serum albumin (BSA) is used as a mimic of lysosomal proteins to study the cross-linking behavior of intermediate copolymer F-PEG and bovine serum albumin.

[0092] Specifically, 10 mg of F-PEG and BSA were weighed according to a mass ratio of 1:1, dissolved in 10 mL of ultrapure water, mixed uniformly, and placed in a constant temperature shaker at 37°C and 100 r / min for 24 h. The liquid was observed, and the results are shown in Figure 13 After that, the solid sample powder was obtained by freeze-drying. A 532 nm solid laser was used to measure the Raman spectra of BSA, F-PEG, and the freeze-dried powder of BSA and F-PEG at 1500-1800 cm -1 -1. Meanwhile, the FT-IR spectra of BSA, F-PEG, and the freeze-dried powder of BSA and F-PEG were measured at 1500-1800 cm -1 -1 using a spectrum pure potassium bromide mixed and ground with the samples and a tablet press method. The results are shown in Figure 12 , 14

[0093] As can be seen from Figures 12-14 , after mixing F-PEG and BSA, a precipitate appeared in the solution Figure 13 , and it can be clearly seen from the Raman spectra Figure 12 and FT-IR spectra Figure 14 that F-PEG and BSA reacted after mixing to produce new peaks. The above results show that F-PEG can react with bovine serum albumin, causing changes in the structure of bovine serum albumin.

[0094] Example 5: Investigation of the cellular entry pathway of drug-loaded nanoparticles FPA / DOX

[0095] In this embodiment, laser confocal microscopy was used to investigate the cellular entry pathway of drug-loaded nanoparticles FPA / DOX.

[0096] ​Specifically, in order to explore the endocytosis pathway of the drug-loaded nanoparticles FPA / DOX, 2 mM amiloride endocytosis inhibitor was selected to inhibit macropinocytosis, 5 mM methyl-β-cyclodextrin (M-β-CD) was selected to inhibit clathrin-mediated endocytosis, 450 mM hypertonic sucrose was selected to inhibit clathrin-mediated endocytosis, and complete medium was set as a blank control. The HeLa cells in the logarithmic growth phase (incubated at 37°C in a 5% CO2 incubator) were plated on a confocal dish at a density of 30-40%, and the three inhibitors and the blank control were used for 30 min, respectively. The inhibitor solution was aspirated and washed with PBS three times, and then the complete medium solution of FPA / DOX (1 μg / mL, based on the DOX content) was added, and after 1-2 h of action, laser confocal microscopy was used for imaging, and the results are shown in Figure 15

[0097] As can be seen from Figure 15 , the red fluorescence of DOX can be observed in the three groups using inhibitors and the blank control group without using inhibitors, indicating that FPA / DOX can be taken up by cells; the fluorescence intensity of DOX in the hypertonic sucrose group is reduced to 66.6% relative to the blank control group, while the relative fluorescence intensity of the amiloride and M-β-CD groups can still be maintained at 90% and above, indicating that FPA / DOX enters cells through clathrin-mediated endocytosis to exert its effect.

[0098] Example 6 Study on the behavior of low-oxygen and pH dual-responsive nanocarriers FPA on lysosome membrane permeabilization

[0099] In this embodiment, the acridine orange fluorescence detection kit for lysosome membrane permeability (LMP) / integrity was used to study the effect of low-oxygen and pH dual-responsive nanocarriers FPA on the lysosome membrane permeability of HeLa cells.

[0100] ​Specifically, HeLa cells (incubated at 37°C in a 5% CO2incubator) were seeded in confocal dishes at a density of 30-40% and incubated overnight. The medium in the dishes was aspirated and washed with PBS for 2-3 times, and medium containing 53.53 μg / mL FPA was added and incubated for 1, 2, 4, and 6 hours, with a group without FPA added as a blank control for 0 hours. The cell culture solution was carefully aspirated, 500 μL of 37°C preheated cell cleaning solution was added to the dish along the wall, the cleaning solution was aspirated, and the operation was repeated twice. Then, 5 μL of acridine orange fluorescent staining solution was added to the dish along the wall, and the dish was incubated at 37°C in a cell incubator for 15 minutes in the dark. After the staining solution was carefully aspirated, 500 μL of cell cleaning solution was added and aspirated, and the operation was repeated 3 times. Finally, 500 μL of cell cleaning solution was added, and the red fluorescence intensity in the cells was observed under a laser confocal microscope at an excitation wavelength of 555 nm and an emission wavelength of 617 nm, and the green fluorescence intensity in the cells was observed at an excitation wavelength of 490 nm and an emission wavelength of 528 nm. The results are shown in Figure 16 .

[0101] As can be seen from Figure 16 , AO is a lysosome-dissolving heterochromatic fluorescent dye. In intact lysosomes, it is in the form of protonated oligomers and exhibits red fluorescence, while in the cytoplasm, it is in the form of monomeric deprotonated form and exhibits green fluorescence, which is used to analyze the permeability of lysosome membranes. When the permeability of lysosomes increases, the red fluorescence of AO entering the lysosomes gradually weakens, and the green fluorescence gradually increases. In this embodiment, after the cells were treated with FPA, the red fluorescence of AO gradually weakened and the green fluorescence gradually increased with the extension of the treatment time, indicating that after FPA treatment, the AO dye was converted from the protonated oligomer form in the lysosomes to the monomer form in the cytoplasm, indicating that the treatment of FPA increased the permeability of the lysosome membrane and destroyed the original function of the lysosome membrane.

[0102] Further, TEM was used to observe the morphology of lysosomes in cells treated with different drugs. HeLa cells were placed in a 6-well plate and incubated overnight. The medium in the plate was carefully aspirated and washed with PBS for 2-3 times, and FPA / DOX (1 μg / mL, based on the DOX content) was added, and a group without drug was set as a blank control. After 12 hours of incubation, the medium was carefully aspirated, washed with PBS for 2-3 times, and the supernatant was discarded after trypsin digestion and centrifugation. The sample was prepared according to the biological TEM sample preparation procedure and observed using biological TEM. The results are shown in Figure 17 .

[0103] As can be seen fromFigure 17 It can be seen that after treating cells with FPA / DOX, it can be observed that the lysosomes of the cells cannot maintain their normal regular spherical shape, and swelling and other phenomena will appear. The complete lysosomal membrane of normal cell lysosomes can no longer be intuitively observed. The results further show that FPA / DOX can cause the lysosomal membrane to rupture.

[0104] Example 7 Study on the behavior of drug-loaded nanoparticles FPA / DOX after entering cells

[0105] In this example, the behavior of drug-loaded nanoparticles FPA / DOX after entering cells was studied.

[0106] Specifically, HeLa cells (5 × 10 4 / well) were inoculated in 2 mL of complete culture medium in a confocal dish and incubated overnight. The culture medium in the culture dish was aspirated and rinsed with PBS 2-3 times. Culture medium containing FPA / DOX (1 μg / mL, based on DOX content) was added to the culture dish and incubated for 1, 2, 4, 6, and 9 hours respectively. Then, the medium containing the drug was aspirated and rinsed with PBS 2-3 times. Lyso-Tracker Red (lysosomal red fluorescent probe) and complete culture medium were added to the culture dish at a ratio of 1 μL:10 mL and incubated in the dark for 20 minutes. The cells were aspirated and rinsed three times with PBS. Then, 4,'6-diamidino-2-phenylindole dihydrochloride (DAPI) solution was added to stain the cell nuclei. After incubation in the dark for 10 minutes, the cells were rinsed three times with PBS. Finally, the cells were observed under a laser confocal microscope. The results were as follows: Figure 18A and 18B shown.

[0107] from Figure 18A and 18B It can be seen that after the drug-loaded nanoparticles FPA / DOX enter the cells, as the drug action time increases, the released DOX can enter the cell nucleus more, thereby improving the drug utilization rate.

[0108] Example 8 Cytotoxicity Test of Drug-Loaded Nanoparticles FPA / DOX

[0109] In this example, the MTT assay was used to determine the toxicity of FPA, drug-loaded nanoparticles FPA / DOX, and free DOX to HeLa cells under normoxia, pH 7.4, and hypoxia, pH 5.0.

[0110] Specifically, first, HeLa cells in logarithmic growth phase were seeded into 96-well plates at a density of 5000, incubated normally in an incubator for 24 h, different concentrations of FPA solution were prepared with complete medium respectively, 100 μL was added to each well, 6 duplicate wells were set in each group, and incubation was continued for 24 h. Finally, MTT solution was added to each well, incubated for 4 h, and DMSO solution was added for color development. The absorbance (OD) of each well at 570 nm was measured, and the cell survival rate was calculated according to the following formula (6):

[0111] ;

[0112] Wherein: A s The experimental group, i.e. the medium containing cells, MTT, and drugs, A c The control group, i.e. the medium containing cells, MTT, and no drugs, A b The blank group, i.e. the medium containing no cells, MTT, and no drugs.

[0113] In addition, the cytotoxicity experiments of drug-loaded nanoparticles FPA / DOX and free DOX under normoxia and pH 7.4 conditions were consistent with the above operation. In the cytotoxicity operation under hypoxia and pH 5.0 conditions, the complete medium with pH 5.0 was used to prepare FPA / DOX and free DOX solutions, and was incubated in a micro-aerobic anaerobic bag for 24 h. The rest of the operation was consistent with the above, and the results are shown in Figure 19 .

[0114] As can be seen from Figure 19 , the nanocarrier FPA itself can kill tumor cells, which is attributed to lysosome death-induced apoptosis; drug-loaded nanoparticles FPA / DOX have stronger cytotoxicity under hypoxia and acidic conditions, which is more conducive to targeted killing of tumor cells.

[0115] Example 9 Apoptosis experiment of drug-loaded nanoparticles FPA / DOX

[0116] HeLa cells (1.0-5.0 x 10 7The 12-well plates were inoculated with the cells and incubated overnight. The complete medium in each well of the 12-well plate was aspirated, and then the medium containing FPA / DOX (0.5 μg / mL, based on the content of DOX), free DOX was added, respectively, and the complete medium containing PBS without drug was set as a blank control, and three parallel groups were set in each group, and incubated in a 37°C cell incubator for 6 h. The medium was aspirated and reserved, and the cells were trypsinized for 3 min, and then the original medium was added to terminate the digestion, and then centrifuged at 1000 r / min for 3 min, and the supernatant was carefully discarded, and then the cells were washed twice with the cell staining buffer, and then resuspended in the cell staining buffer, so that the cell suspension was maintained at 0.25-1.0 x 10 7 cells / mL, 100 μL of the cell suspension was taken from each sample, 5 μL of Annexin V Alexa Fluor 647 dye was added to incubate in the dark for 10 min, 5 μL of 7-AAD dye was added to incubate in the dark for 5 min, and then 400 μL of the cell staining buffer was added to resuspend the cells, and then the cells were detected by using a flow cytometer, and the results are shown in Figure 20

[0117] As can be seen from Figure 20 , the drug-loaded nanoparticles FPA / DOX kill tumor cells in the form of apoptosis, and the drug-loaded nanoparticles FPA / DOX have strong toxicity to tumor cells.

[0118] Example 10 Anti-tumor efficacy of drug-loaded nanoparticles FPA / DOX in vivo

[0119] This example evaluates the anti-tumor efficacy of drug-loaded nanoparticles FPA / DOX in vivo.

[0120] Specifically, BALB / C mice (6 weeks old, female mice, body weight 20-25 g) were purchased, and the mice were adapted in the animal facility for 1 week before the experiment. The HeLa cells were used to construct a mouse cervical cancer model, and about 5 x 10 6 logarithmic growth phase of HeLa cells were subcutaneously injected into the abdomen of BALB / C mice, and when the tumor diameter reached about 100 mm 2 , the tumor-bearing mice were randomly divided into 5 groups (n = 6).

[0121] ​The tumor-bearing mice were injected with drugs in different treatment groups by tail vein injection, which were PBS group, drug-loaded nanoparticle FPA / DOX group, wherein PBS group was used as a blank control, and the calculated equivalent of DOX in FPA / DOX group was 1 mg / kg. The experiment was carried out for two weeks, and different drugs in different treatment groups were injected every other day, 100 μL each time. During the whole experiment, the change of tumor size and the change of mouse body weight were measured every other day, and the mouse tumor volume was calculated using the following formula (7):

[0122] .

[0123] When the treatment was 18 days, the mice were sacrificed, and the sections of the tumor site of the mice were stained with H&E and TUNEL to evaluate the killing effect of the drugs on the tumor, and the results are shown in Figure 21 .

[0124] As can be seen from Figure 21 , after the tumor-bearing mice were treated with drug-loaded nanoparticles FPA / DOX, the body weight of the mice did not change much and basically remained stable; with the increase of treatment time, the tumor volume and weight of the tumor-bearing mice decreased significantly, and the above results showed that the drug-loaded nanoparticles FPA / DOX had good antitumor effect in mice, and the final tumor inhibition rate reached 95.27%.

[0125] Example 11 In vivo biological safety evaluation of drug-loaded nanoparticles FPA / DOX

[0126] In this embodiment, the in vivo biological safety of drug-loaded nanoparticles FPA / DOX was evaluated.

[0127] Specifically, the toxicology data of healthy BALB / c female nude mice were used to evaluate the toxicity of the drug. The mice (6 weeks old, 20-25 g) were randomly divided into 3 groups (n=6), and were injected with PBS, free DOX and drug-loaded nanoparticles FPA / DOX respectively, and were sacrificed after 24 h, and the serum samples of the mice were collected for ALT, AST, ALP, BUN, TP, ALB, GLOB and CREA detection, and the main organs (including heart, liver, spleen, lung, kidney) of the mice were collected, and further histological analysis was performed on the sections of the organs using H&E staining, and the results are shown in Figure 22 .

[0128] As can be seen from Figure 22 , after the mice were injected with drug-loaded nanoparticles FPA / DOX, there were no abnormalities in blood biochemistry, blood routine and main organs, and the above results showed that the drug-loaded nanoparticles FPA / DOX had good biological safety.

[0129] In summary, please refer to Figure 23It is a schematic diagram of synthesis of the drug-loaded nanoparticles FPA / DOX and the targeted tumor cell effect in the application. Specifically, in the application, by using the reaction of aldehyde benzoic acid, nonaethylene glycol and azo p-toluidine, a nano-carrier with moderate particle size and low oxygen and pH dual response is obtained, the nano-carrier specifically targets tumor sites, then specifically releases drugs, reduces toxic side effects; at the same time, after the response of the nano-carrier, benzaldehyde groups are released, which covalently bond with proteins on the lysosome membrane, leading to changes in protein activity, thereby causing the lysosome membrane to rupture and increase in permeability, unable to expel the nano-carrier outside the cell, thereby significantly improving the drug bioavailability of the nano-carrier, and in addition, the nano-carrier has high drug loading capacity.

[0130] It should be noted that each of the above embodiments belongs to the same inventive concept, and the description of each embodiment has its own emphasis. If not fully described in an individual embodiment, the description in other embodiments can be referred to.

[0131] The above-described embodiments only express the implementation of the application, and the description is more specific and detailed, but it should not be construed as limiting the scope of the patent. It should be noted that for those skilled in the art, without departing from the inventive concept, a number of modifications and improvements can be made, which are within the scope of the application. Therefore, the protection scope of the patent of the application should be subject to the appended claims.

Claims

1. A hypoxia and pH dual-responsive nanocarrier, characterized in that: It has the structure shown in the following formula (I): ； Wherein, the molecular weight of the hypoxia and pH dual-responsive nanocarrier is 800-10000 g / mol.

2. A method for preparing a hypoxia and pH dual-responsive nanocarrier according to claim 1, characterized in that: The steps include: S1. Using p-formylbenzoic acid and nonaethylene glycol as raw materials, reacting in the presence of a catalyst and an activator, and obtaining an intermediate copolymer through separation and purification; S2. The intermediate copolymer is mixed with azo-paraaniline and reacted, and the low oxygen and pH dual-responsive nanocarrier is obtained through separation and purification.

3. The method for preparing the hypoxia and pH dual-responsive nanocarrier according to claim 2, characterized in that: In step S1, the molar ratio of the p-formylbenzoic acid, the nonaethylene glycol, the catalyst, and the activator is (3-10): (3-10): (0.3-1): (3-10); the catalyst includes 4-dimethylaminopyridine, and the activator includes 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride.

4. The method for preparing the hypoxia and pH dual-responsive nanocarrier according to claim 2, wherein: In step S1, carrying out the reaction in the presence of the catalyst and the activator specifically includes: reacting at room temperature for 18-25 hours.

5. The method for preparing the hypoxia and pH dual-responsive nanocarrier according to claim 2, characterized in that: In step S2, the molar ratio of the intermediate copolymer to the azo-paraaniline is (0.13-0.16): (0.13-0.16).

6. The method for preparing the hypoxia and pH dual-responsive nanocarrier according to claim 2, characterized in that: In step S2, after mixing the intermediate copolymer and azo-paraaniline, reacting the mixture specifically includes: reacting at room temperature for 20-30 hours.

7. A drug-loaded nanoparticle, characterized in that: The drug-loaded nanoparticles are obtained by loading drugs on the hypoxia and pH dual-responsive nanoparticles according to claim 1 or the hypoxia and pH dual-responsive nanoparticles prepared by the preparation method of any one of claims 2 to 6.

8. The drug-loaded nanoparticles according to claim 7, characterized in that: The drugs include doxorubicin.

9. A method for preparing drug-loaded nanoparticles according to any one of claims 7 to 8, characterized in that: The steps include: The hypoxia and pH dual-responsive nanocarrier and the drug are dissolved in an organic solvent respectively and then mixed, and then ultrapure water is added. After dialysis, filtration and freeze-drying, the drug-loaded nanoparticles are obtained.

10. Use of the drug-loaded nanoparticles according to any one of claims 7 to 8 or the drug-loaded nanoparticles prepared by the preparation method according to claim 9 in the preparation of anti-tumor drugs.

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