Hypoxia and pH dual-response nano-carrier, drug-loaded nanoparticles and application of hypoxia and pH dual-response nano-carrier

Abstract

The invention discloses a hypoxia and pH dual-response nano-carrier, a drug-loaded nanoparticle and application of the drug-loaded nanoparticle, and belongs to the technical field of biological medicine. According to the invention, p-formylbenzoic acid, nonaethylene glycol and azo p-aniline are subjected to a reaction to obtain the nano-carrier with moderate particle size and low oxygen and pH dual response, and the nano-carrier specifically targets a tumor site and then specifically releases drugs to reduce toxic and side effects; meanwhile, after the nano-carrier responds, benzaldehyde groups are released and are covalently connected with proteins on a lysosome membrane, so that the structure and activity of the proteins are changed, the lysosome membrane is broken and the permeability is increased after the proteins are denatured, and the nano-carrier cannot be discharged out of the cell, so that the intracellular drug concentration is greatly improved, and the drug effect is improved. In addition, the drug loading capacity of the nano-carrier is high, so that the nano-carrier has a relatively good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a hypoxia and pH dual-responsive nanocarrier, drug-loaded nanoparticles and applications thereof. Background Art

[0002] Cancer is one of the major diseases that cause human death. Chemotherapy is one of the most important means of cancer treatment, but its treatment effect is poor due to its non-specificity and strong drug resistance. Therefore, designing a treatment strategy that can improve drug specificity and target tumor sites and improve drug utilization to reduce drug resistance is of great significance in cancer treatment. Due to their suitable size, supramolecular nanomaterials can be passively targeted and aggregated in tumor sites in vivo through the enhanced permeability and retention (EPR) effect. The designability of supramolecular nanomaterials provides a broad design platform for therapeutic strategies.

[0003] Studies have shown that tumor cells have a specific tumor microenvironment (TEM) that is different from that of normal cells, which is mainly manifested by a hypoxic microenvironment, acidic pH, reactive oxygen species (ROS), and overexpression of glutathione (GSH). 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. Because tumor cells have abnormal proliferation and metabolism, the number of their intracellular acidic organelles, lysosomes, will also increase. Lysosomes are mainly responsible for clearing foreign substances from the cell, including anti-tumor drugs, which is also one of the causes of drug resistance. Studies have found that tumor cells are more sensitive to lysosomal disorders. Once the permeability of the lysosomal membrane changes, a variety of hydrolases (such as cathepsin B) and other substances (such as iron and H) in the lysosomes will be blocked. + ) enters the cytoplasm and induces lysosome-dependent cell death (LDCD). Currently, the commonly used lysosomal membrane permeabilization (LMP) inducers are mainly lysosomal detergents (such as Siramesine), dipeptide methyl esters (such as Leu-Leu-OMe) and reactive oxygen species (ROS), but these inducers rarely selectively induce cancer cell LMP, and they also cause extremely strong toxic side effects and bring pain to patients.

[0004] Currently, existing drug delivery nanocarriers have problems such as poor targeting, low drug bioavailability, and poor drug loading capacity. Summary of the invention

[0005] The object of the present invention is to provide a hypoxia and pH dual-responsive nanocarrier, drug-loaded nanoparticles and applications thereof. In the present invention, by using p-formylbenzoic acid, nonaglycone and azo-p-aniline to react, a nanocarrier with moderate particle size and dual response to hypoxia and pH is obtained, the nanocarrier specifically targets the tumor site, and then specifically releases the drug to reduce toxic side effects; at the same time, after the nanocarrier responds, the benzaldehyde group is released, which is covalently linked to the protein on the lysosomal membrane, resulting in a change in protein activity, thereby rupturing the lysosomal membrane, increasing permeability, and being unable to expel the nanocarrier out of the cell, thereby significantly improving the drug bioavailability of the nanocarrier, and in addition, the nanocarrier has a high drug loading capacity, and therefore has a good application prospect.

[0006] In the first aspect, the present invention provides a hypoxia and pH dual-responsive nanocarrier having a structure as shown in the following formula (I): ; Among them, the molecular weight of the hypoxia and pH dual-responsive nanocarrier is 800-10000 g / mol.

[0007] In the present invention, the inventors have found that by using p-formylbenzoic acid, nonaglycone and azoaniline to react, a nanocarrier with moderate particle size and dual response to hypoxia and pH is obtained. The nanocarrier contains an azo group, so it has hypoxia responsiveness, and contains an imine group, so it has pH responsiveness. The above dual responsiveness enables the nanocarrier to specifically target the tumor site and then specifically release the drug to reduce toxic side effects; at the same time, after the nanocarrier responds, the benzaldehyde group is released, which is covalently linked to the protein on the lysosomal membrane, resulting in changes in the structure and activity of the protein. After the protein is denatured, the lysosomal membrane is ruptured, the permeability is increased, and the nanocarrier (loaded drug) cannot be excreted from the cell, which greatly increases the intracellular drug concentration, thereby significantly improving the drug bioavailability of the nanocarrier. In addition, the nanocarrier has a high drug loading capacity, so it has a good application prospect.

[0008] In a second aspect, the present invention provides a method for preparing a hypoxia and pH dual-responsive nanocarrier as described above, comprising the following steps: S1, using p-formylbenzoic acid and nonaglycone as raw materials, reacting in the presence of a catalyst and an activator, and obtaining an intermediate copolymer through separation and purification; S2, mixing the intermediate copolymer with azoaniline and reacting, and obtaining a hypoxia and pH dual-responsive nanocarrier through separation and purification.

[0009] In the present invention, the preparation process of the hypoxia and pH dual-responsive nanocarrier is shown in the following formulas (1) and (2): ; .

[0010] The preparation method provided by the invention is simple, the raw materials used are green, non-toxic, cheap and easily available, and have good biological safety, and therefore, are convenient for large-scale production and application.

[0011] In some embodiments, in step S1, the molar ratio of p-formylbenzoic acid, nonaglycol, catalyst and activator is (3-10): (3-10): (0.3-1): (3-10); and the catalyst includes 4-dimethylaminopyridine, and the activator includes 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride.

[0012] In some preferred embodiments, the molar ratio of p-formylbenzoic acid, nonaethylene glycol, catalyst and activator is 6:6:0.6:6.

[0013] In some embodiments, in step S1, carrying out the reaction in the presence of a catalyst and an activator specifically comprises: reacting at room temperature for 18-25 hours.

[0014] In some preferred embodiments, carrying out the reaction in the presence of a catalyst and an activator specifically includes: reacting at room temperature for 21 hours.

[0015] 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. Triethylamine can also be added during the reaction to facilitate a better reaction, and the amount of triethylamine added is added according to actual use needs.

[0016] In some embodiments, in step S1, obtaining the intermediate copolymer by separation and purification specifically includes: washing the obtained product with a hydrochloric acid solution having a mass concentration of 10%, and after drying, performing column chromatography separation to obtain the intermediate copolymer.

[0017] In some embodiments, in step S2, the molar ratio of the intermediate copolymer to azo-paraaniline is (0.13-0.16): (0.13-0.16).

[0018] In some preferred embodiments, the molar ratio of the intermediate copolymer to azo-paraaniline is 0.1475:0.1475.

[0019] In some embodiments, in step S2, reacting the intermediate copolymer and azo-paraaniline after mixing them specifically comprises: reacting at room temperature for 20-30 hours.

[0020] In some preferred embodiments, the reaction after mixing the intermediate copolymer with azo-paraaniline specifically includes: reacting at room temperature for 24 hours.

[0021] 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.

[0022] In some embodiments, when the intermediate copolymer is mixed with azo-paraaniline and then reacted, the step of adding glacial acetic acid to the reaction system is also included.

[0023] It can be understood that the addition of glacial acetic acid facilitates a better reaction, and the amount of glacial acetic acid added is added according to actual use needs.

[0024] In some embodiments, in step S2, obtaining the hypoxia and pH dual-responsive nanocarrier by separation and purification specifically comprises: using glacial ether to precipitate the obtained product three times to obtain the hypoxia and pH dual-responsive nanocarrier.

[0025] In a third aspect, the present invention provides a drug-loaded nanoparticle, wherein the drug-loaded nanoparticle is obtained by loading the drug on the hypoxia and pH dual-responsive nanocarrier or the hypoxia and pH dual-responsive nanocarrier prepared by any of the above preparation methods.

[0026] In some embodiments, the drug comprises doxorubicin.

[0027] It is understandable that the drug can be routinely selected according to the type of target cells. In the present invention, the drug preferably includes doxorubicin.

[0028] In a fourth aspect, the present invention provides a method for preparing drug-loaded nanoparticles as described above, comprising the following steps: dissolving the hypoxia and pH dual-responsive nanocarrier and the drug in an organic solvent respectively and then mixing them, then adding ultrapure water, and obtaining the drug-loaded nanoparticles after dialysis, filtration and freeze-drying.

[0029] In some embodiments, the dialysis specifically comprises: using a dialysis bag with a molecular weight cut-off of 3000-4000 Da, and dialysis for 18-32 hours.

[0030] In some preferred embodiments, a dialysis bag with a molecular weight cut-off of 3500 Da is used and the dialysis is performed for 24 hours.

[0031] It is understandable that the organic solvent can be a conventional organic solvent. In the present invention, the organic solvent is preferably dimethyl sulfoxide.

[0032] In a fifth aspect, the present invention provides use of any of the above-mentioned drug-loaded nanoparticles or the drug-loaded nanoparticles prepared by the above-mentioned preparation method in the preparation of anti-tumor drugs.

[0033] The beneficial effects of the present invention are as follows: Different from the prior art, the present invention uses p-formylbenzoic acid, nonaglycone and azobenzene to react, thereby obtaining a nanocarrier with moderate particle size and dual response to hypoxia and pH. The nanocarrier contains an azo group, and thus has hypoxia responsiveness, and contains an imine group, and thus has pH responsiveness. The dual responsiveness enables the nanocarrier to specifically target the tumor site, and then specifically release the drug, thereby reducing toxic side effects; at the same time, after the nanocarrier responds, the benzaldehyde group is released, which is covalently linked to the protein on the lysosomal membrane, resulting in changes in the structure and activity of the protein. After the protein is denatured, the lysosomal membrane is ruptured, the permeability is increased, and the nanocarrier (loaded drug) cannot be excreted from the cell, thereby greatly increasing the intracellular drug concentration, thereby significantly increasing the drug bioavailability of the nanocarrier. In addition, the nanocarrier has a high drug loading capacity, and therefore has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The intermediate copolymer F-PEG synthesized in Example 1 of the present invention 1 H-NMR images (a) and the hypoxia and pH dual-responsive nanocarrier FPA 1 H-NMR spectrum (b); Figure 2 This is a GPC chart of the hypoxia and pH dual-responsive nanocarrier FPA synthesized in Example 1 of the present invention; Figure 3 This is a UV-vis image of the hypoxia and pH dual-responsive nanocarrier FPA synthesized in Example 1 of the present invention; Figure 4 FT-IR image of the hypoxia and pH dual-responsive nanocarrier FPA synthesized in Example 1 of the present invention; Figure 5 The DLS graphs of the hypoxia and pH dual-responsive nanocarrier FPA solution prepared in Example 1 of the present invention after being placed for 0 and 5 days respectively; Figure 6 TEM image of the hypoxia and pH dual-responsive nanocarrier FPA solution prepared in Example 1 of the present invention; Figure 7 The PBS aqueous solution containing FPA prepared in Example 1 of the present invention, the acetic acid buffer solution containing FPA (pH value is 5.0), the 2 S 2 O 4 PBS aqueous solution, containing FPA and Na 2 S 2 O 4 DLS graph of acetate buffer (pH 5.0); Figure 8The FPA and Na 2 S 2 O 4 UV-Vis image of acetate buffer (pH 5.0); Fig. 9 The FPA and Na 2 S 2 O 4 TEM image of acetic acid buffer (pH 5.0) after being placed for 24 hours; Fig.10 The drug loading rate and encapsulation rate of the drug-loaded nanoparticles FPA / DOX prepared in Example 2 of the present invention; Fig.11 This is an in vitro release curve of the drug-loaded nanoparticles FPA / DOX in Example 3 of the present invention; Fig.12 The Raman spectra of BSA, F-PEG, and a mixed freeze-dried powder of BSA and F-PEG in Example 4 of the present invention are shown; Fig.13 The morphological diagram of BSA, F-PEG, and F-PEG and BSA after co-incubation for 24 hours in Example 4 of the present invention; Fig.14 FT-IR images of BSA, F-PEG, and mixed lyophilized powder of BSA and F-PEG in Example 4 of the present invention; Fig.15 CLSM images (a) of Hela cells incubated with amiloride, M-β-CD, and Hypertonicsucrose for 30 min and untreated Hela cells, and the average intensity image of DOX fluorescence signal in cells of each treatment group (b) in Example 5 of the present invention; Fig.16 The CLSM images (a) of Hela cells treated with AO after being co-incubated with FPA for 0, 1, 2, 4, and 6 hours, respectively, and the average intensity images of AO red fluorescence signals in cells of the treatment groups at different time periods in Example 6 of the present invention are shown; Fig.17 This is a TEM image of intracellular lysosomes after Hela cells were incubated with drug-loaded nanoparticles FPA / DOX in Example 6 of the present invention; Fig.18A CLSM images of Hela cells in Example 7 of the present invention after being co-incubated with FPA / DOX for 1, 2, 4, 6, and 9 hours and then stained with Lyso-Tracker and DAPI; Fig.18B Based on Fig.18AThe red light of DOX and the blue light of DAPI are merged, the corresponding fluorescence signal distribution and the intensity diagram of the DOX fluorescence signal in the cell nucleus; Fig.19 The cell survival rate graph (a) of Hala cells treated with FPA in Example 8 of the present invention, the cell survival rate graph (b) of Hala cells treated with FPA / DOX under normoxia and pH 7.4, and the cell survival rate graph (c) of Hala cells treated with FPA / DOX under hypoxia and pH 5.0; Fig. 20 Flow cytometry images of FPA / DOX, free DOX and PBS in Example 9 of the present invention after incubation with Hela cells for 6 hours; Fig.21 The figures are the results of the in vivo anti-tumor efficacy of the drug-loaded nanoparticles FPA / DOX in Example 10 of the present invention; wherein (a) is a graph showing the changes in the weight of mice, (b) is a graph showing the changes in the tumor volume of mice, (c) is a graph showing the average tumor weight of mice after 18 days of treatment, (d) is a graph showing the tumors of mice at different times in different treatment groups; (e) is a graph showing H&E staining and TUNEL staining of the tumor sites of mice after treatment; Fig. 22 The in vivo biosafety evaluation results of the drug-loaded nanoparticles FPA / DOX in Example 11 of the present invention are shown in Figure 1, wherein (a) is a serum test result of mice treated with PBS, free DOX and drug-loaded nanoparticles FPA / DOX, and (b) is a H&E staining result of the main organs (heart, liver, spleen, lung, kidney) of mice treated with PBS, free DOX and drug-loaded nanoparticles FPA / DOX; Fig.23 This is a schematic diagram of the synthesis of the drug-loaded nanoparticles FPA / DOX and their targeting effect on tumor cells in the present invention. DETAILED DESCRIPTION

[0035] 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.

[0036] 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.

[0037] Example 1 Synthesis of hypoxia and pH dual-responsive nanocarrier FPA 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, the solvent was removed by rotary evaporation, and 20 mL of dichloromethane was added to obtain solution a; 6 mmol of nonaglycone 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 used to obtain solution b; solution a was added dropwise into solution b, and the mixture was stirred for reaction at room temperature for 21 hours. After the solvent was removed by rotary evaporation, the mixture was washed and dried with a hydrochloric acid solution having a mass concentration of 10%, and the obtained 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 eluents to obtain F-PEG.

[0038] 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, and then 0.1475 mmol of azobenzene was added, mixed and stirred, and reacted at room temperature for 24 hours; after the reaction, glacial ether was used to precipitate three times to obtain the hypoxia and pH dual-responsive nanocarrier FPA.

[0039] Performance Testing First, using proton nuclear magnetic resonance spectroscopy ( 1 H-NMR) was used to detect the hypoxia and pH dual-responsive nanocarrier FPA synthesized above, and the results are as follows Figure 1 shown.

[0040] from Figure 1 As can be seen, the hypoxia and pH dual-responsive nanocarrier FPA was successfully synthesized.

[0041] Then, the hypoxia and pH dual-responsive nanocarrier FPA synthesized above was subjected to gel permeation chromatography (GPC) detection using tetrahydrofuran as solvent. Figure 2 shown.

[0042] from Figure 2 It can be seen that the molecular weight of the hypoxia and pH dual-responsive nanocarrier FPA is 4194 g / mol.

[0043] Next, the UV-visible spectra (UV-vis) of the synthesized hypoxia and pH dual-responsive nanocarriers FPA, F-PEG and azobenzene were tested using a UV-visible spectrophotometer, and the Fourier transform infrared spectra (FT-IR) of the synthesized hypoxia and pH dual-responsive nanocarrier FPA were measured using a tableting method. The results are as follows: Figure 3 and 4 shown.

[0044] from Figure 3 and 4 As can be seen, the hypoxia and pH dual-responsive nanocarrier FPA was successfully synthesized.

[0045] Further, a PBS aqueous solution containing 0.25 mg / mL FPA was prepared, and the hydrated particle size of the hypoxia and pH dual-responsive nanocarrier FPA synthesized above was measured using a Malvern particle size tester, and then placed at room temperature for 5 days, and the hydrated particle size was tested again to verify the stability of the nanoparticles; a PBS solution containing 0.25 mg / mL FPA was further prepared, and uranyl acetate was used as a staining agent, and the morphology of the hypoxia and pH dual-responsive nanocarrier FPA synthesized above was observed using a transmission electron microscope (TEM), and the results were as follows: Figure 5 and 6 shown.

[0046] from Figure 5 and 6 It can be seen that the hypoxia and pH dual-responsive nanocarrier FPA has a suitable nanosize and can exist stably under physiological conditions.

[0047] Furthermore, the dual responsiveness of nanocarrier FPA to hypoxia and acid was verified.

[0048] Specifically, the inorganic reducing agent sodium dithionite (Na 2 S 2 O 4 ) as a chemical mimetic of azoreductase, 10 mM Na 2 S 2 O 4 To simulate the hypoxic and acidic environment in vivo, PBS aqueous solution containing 0.05 mg / mL FPA, acetate buffer (pH 5.0) containing 0.05 mg / mL FPA, and aqueous solution containing 0.05 mg / mL FPA and 10 mM Na 2 S 2 O 4 PBS aqueous solution containing 0.05 mg / mL FPA and 10 mM Na 2 S 2 O 4The acetic acid buffer (pH 5.0) was placed in a constant temperature shaker at 37°C and shaken (100 r / min) for 2 days. The DLS was sampled at 0, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, and 48 h to characterize the change in the particle size of the nanoparticles, and the TEM image was sampled at 24 h. In addition, a 50 μg / mL FPA-containing solution was prepared with 10 mM Na 2 S 2 O 4 The samples were placed in an acetic acid buffer (pH 5.0) at 37°C in a constant temperature shaker (100 r / min) for 0, 6, 9, 12, and 24 hours, and their UV-vis spectra were measured. The results were as follows: Figure 7-9 shown.

[0049] from Figure 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 sizes. 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.

[0050] Example 2 Preparation of drug-loaded nanoparticles FPA / DOX The drug-loaded nanoparticles FPA / DOX were prepared by dialysis method.

[0051] 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 h in the dark, the mixture was dialyzed (MWCO = 3500 Da) for 24 h, filtered and freeze-dried to obtain drug-loaded nanoparticles FPA / DOX. The FPA / DOX (1 mg / ml) solution was measured using an ELISA instrument. λ ex =485nm, λ em =592nm fluorescence curve to determine the content of DOX loaded; and the drug loading (DLC) and encapsulation efficiency (EE) were calculated using the following formulas (3) and (4). Fig.10 shown.

[0052] .

[0053] from Fig.10 It can be seen that the hypoxia and pH dual-responsive nanocarrier FPA has good drug loading capacity and encapsulation efficiency.

[0054] Example 3 In vitro release of drug-loaded nanoparticles FPA / DOX This example studies the drug-loaded nanoparticles FPA / DOX at 37°C in 10 mM Na 2 S 2 O 4 Solution (pH 5.0), 10 mM Na 2 S 2 O 4 Drug release behavior in solution (pH 7.4), buffer (pH 5.0), and buffer (pH 7.4).

[0055] Specifically, first, the drug-loaded nanoparticle FPA / DOX dispersion (1 mg / mL) was placed in a dialysis bag with a MWCO of 3500Da, placed in 30 mL of the corresponding release medium, and oscillated in a constant temperature shaker at 37°C and 100 r / min for release. 3 mL of dialysate was taken at 0.5, 1, 2, 4, 6, 9, 12, 24, 36, and 48 hours, and then supplemented with the corresponding fresh release medium solution. Fluorescence analysis ( λ ex =485nm, λ em =592nm) to measure the cumulative release of DOX. Three parallel groups were designed for each experiment; the drug release rate was calculated by the following formula (5). The results are shown in Fig.11 shown.

[0056] ; in, C n : DOX concentration measured at the nth moment; C i : DOX concentration measured at the i-th moment; V : volume of release medium; V 0 : the volume of the external liquid taken out; m: the total mass of DOX in the system.

[0057] from Fig.11 It can be seen that the drug-loaded nanoparticles FPA / DOX have a good release rate of DOX after dual response to hypoxia and acid.

[0058] Example 4 Study on the cross-linking behavior of the intermediate copolymer F-PEG and lysosomal membrane proteins In this example, bovine serum albumin (BSA) was used as a mimetic of lysosomal proteins to study the cross-linking behavior between the intermediate copolymer F-PEG and BSA.

[0059] Specifically, according to the mass ratio of F-PEG:BSA of 1:1, 10 mg of each was weighed and dissolved in 10 mL of ultrapure water. After mixing evenly, the mixture was placed in a constant temperature shaker at 37°C and 100 r / min for 24 h, and the liquid phenomenon was observed. The results were as follows. Fig.13 As shown; the solid sample powder was obtained by freeze drying, and the BSA, F-PEG, and BSA and F-PEG mixed freeze-dried powder samples were measured at 1500-1800cm using a 532nm solid laser. -1 The Raman spectra of BSA, F-PEG, and BSA and F-PEG mixed freeze-dried powder were measured by tableting method at 1500-1800 cm -1 The FT-IR spectra of Fig.12 , 14 shown.

[0060] from Figure 12-14 As can be seen from the figure, after F-PEG and BSA are mixed, precipitation appears in the solution ( Fig.13 ), and from Raman spectroscopy ( Fig.12 ) and FT-IR spectra ( Fig.14 ) It can be clearly seen in the figure that F-PEG and BSA react after mixing to produce a new peak; the above results show that F-PEG can react with bovine serum albumin, causing the structure of bovine serum albumin to change.

[0061] Example 5 Investigation of the pathway of drug-loaded nanoparticles FPA / DOX entering cells In this example, laser confocal microscopy was used to investigate the intracellular pathway of drug-loaded nanoparticles FPA / DOX.

[0062] Specifically, in order to explore the intracellular pathway of drug-loaded nanoparticles FPA / DOX, 2mM amiloride endocytosis inhibitor was used to inhibit macropinocytosis, 5mM methyl-β-cyclodextrin (M-β-CD) was used to inhibit caveolin-mediated endocytosis, and 450mM hypertonic sucrose was used to inhibit clathrin-mediated endocytosis, and complete culture medium was set as a blank control. HeLa cells in the logarithmic growth phase (in DMEM medium containing 1% double antibody (AA), 10% fetal bovine serum (FBS) and 89% at 37°C with 5% CO 2 The cells were incubated in an incubator at 30-40% density on a confocal dish, and three inhibitors and a blank control were used for 30 min, respectively. The inhibitor solution was aspirated and rinsed three times with PBS, and then FPA / DOX (1 μg / mL, based on DOX content) complete culture medium solution was added. After 1-2 h of action, imaging was performed using a laser confocal microscope. The results are shown in the figure. Fig.15shown.

[0063] from Fig.15 It can be seen that the red fluorescence of DOX can be observed in the three groups using inhibitors and the blank control group without inhibitors, indicating that FPA / DOX can be taken up by cells; the fluorescence intensity of DOX in the cells of the hypertonic sucrose group decreased to 66.6% relative to that of the blank control group, while the relative fluorescence intensity of the amiloride and M-β-CD groups was still maintained at 90% and above. The results showed that FPA / DOX entered the cells through clathrin-mediated endocytosis to exert its effect.

[0064] Example 6 Study on the permeabilization behavior of intracellular lysosomal membrane by hypoxia and pH dual-responsive nanocarrier FPA In this example, an in vivo lysosomal membrane permeability (LMP) / integrity acridine orange fluorescence detection kit was used to study the effect of hypoxia and pH dual-responsive nanocarrier FPA on the lysosomal membrane permeability of HeLa cells.

[0065] Specifically, HeLa cells were cultured in DMEM medium containing 1% double antibody (AA), 10% fetal bovine serum (FBS) and 89% CO at 37°C. 2 The cells were incubated in a 5% CO incubator at 4 °C for 24 hours (see Table 2). The cells were seeded at a density of 30-40% in a confocal dish and incubated overnight. The culture medium in the culture dish was aspirated and rinsed with PBS for 2-3 times, and the culture medium containing 53.53 μg / mL FPA was added, and cultured for 1, 2, 4, and 6 hours. The group without FPA was set as the blank control for 0 hours, and the cell culture medium was carefully aspirated. 500 μL of cell cleaning solution preheated at 37°C was added to the culture dish along the wall of the hole, and the cleaning solution was aspirated. After repeating the operation twice, 5 μL of acridine orange fluorescent staining solution was added to the culture dish along the wall of the dish, and it was placed in a 37°C cell culture incubator away from light for 15 minutes. After carefully aspirating the staining solution, 500 μL of cell cleaning solution was added and aspirated, and the operation was repeated 3 times. Finally, after adding 500 μL of cell cleaning solution, the red fluorescence intensity in the cells was observed under an excitation wavelength of 555 nm and an emission wavelength of 617 nm, and the green fluorescence intensity in the cells was observed under an excitation wavelength of 490 nm and an emission wavelength of 528 nm. The results are as follows: Fig.16 shown.

[0066] from Fig.16As can be seen in the figure, AO, as a lysosomal-affinity metachromatic fluorescent dye, is in the form of protonated oligomers in intact lysosomes, showing red fluorescence, and in the cytoplasm, it is in the form of monomeric deprotonated form, showing green fluorescence, which is used to analyze the permeability of the lysosomal membrane. When the lysosomal permeability increases, the red fluorescence of AO entering the lysosome gradually weakens, and the green fluorescence gradually increases. After the cells were treated with FPA in this embodiment, as the treatment time increased, the red fluorescence of AO gradually weakened, while the green fluorescence gradually increased, indicating that after FPA treatment, the AO dye will be converted from the protonated oligomer form in the lysosome to the monomer form in the cytoplasm, indicating that the treatment of FPA will increase the permeability of the lysosomal membrane, unable to maintain the original function, and the lysosomal membrane will be damaged.

[0067] Furthermore, TEM was used to observe the morphology of intracellular lysosomes after treatment with different drugs. HeLa cells were placed in a 6-well plate and incubated overnight. The culture medium in the well plate was carefully aspirated and rinsed with PBS 2-3 times. FPA / DOX (1μg / mL, based on DOX content) was added respectively, and a blank control without drug was set up, with 3 sub-wells for each group. After a total incubation of 12 hours, the culture medium was carefully aspirated, rinsed with PBS 2-3 times, digested with trypsin and centrifuged, and the supernatant was discarded. After fixation with 2.5% room temperature glutaraldehyde fixative, the sample was prepared according to the biological TEM sample preparation steps and then observed using biological TEM. The results are shown as follows: Fig.17 shown.

[0068] from Fig.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 occur. The complete lysosomal membrane of normal cell lysosomes can no longer be directly observed. The results further show that FPA / DOX can cause the lysosomal membrane to rupture.

[0069] Example 7 Study on the behavior of drug-loaded nanoparticles FPA / DOX after entering cells In this example, the behavior of drug-loaded nanoparticles FPA / DOX after entering cells was studied.

[0070] Specifically, HeLa cells (5×10 4 / well) were inoculated in 2 mL of complete medium in a confocal dish and incubated overnight. The medium in the dish was aspirated and rinsed with PBS 2-3 times. The culture medium containing FPA / DOX (1 μg / mL, based on DOX content) was added to the dish and incubated for 1, 2, 4, 6, and 9 hours, respectively. Then the medium containing the drug was aspirated, rinsed with PBS 2-3 times, and Lyso-Tracker Red (lysosomal red fluorescent probe) and complete medium were added to the dish at a ratio of 1 μL:10 mL and incubated in the dark for 20 minutes. The cells were aspirated and rinsed with PBS three times, and then 4,'6-diamidino-2-phenylindole dihydrochloride (DAPI) solution was added to stain the cell nucleus. After incubation in the dark for 10 minutes, the cells were rinsed with PBS three times. Finally, the cells were observed with a laser confocal microscope. The results were as follows: Fig.18A and 18B shown.

[0071] from Fig.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.

[0072] Example 8 Cytotoxicity test of drug-loaded nanoparticles FPA / DOX In this example, the MTT method 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.

[0073] Specifically, HeLa cells in the logarithmic growth phase were first inoculated into a 96-well plate at a density of 5000 and incubated in an incubator for 24 h. FPA solutions of different concentrations were prepared with complete culture medium and 100 μL was added to each well. Six replicate wells were set up in each group and incubated for another 24 h. Finally, MTT solution was added to each well. After incubation for 4 h, the MTT solution was aspirated 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): ; in: A s The experimental group, which contained cell culture medium, MTT, and drugs, A c The control group contained cell culture medium, MTT, and no drugs. A b The blank group consisted of a culture medium without cells, MTT, or drugs.

[0074] In addition, the cytotoxicity experiments of drug-loaded nanoparticles FPA / DOX and free DOX under normoxia and pH 7.4 were consistent with the above operation. In the cytotoxicity operation under hypoxia and pH 5.0, complete culture medium with pH 5.0 was used to prepare FPA / DOX and free DOX solutions, which were placed in microaerobic anaerobic bags and incubated for 24 h. The rest of the operations were consistent with the above. The results are shown in Figure 2. Fig.19 shown.

[0075] from Fig.19 It can be seen that the nanocarrier FPA itself can kill tumor cells, which is attributed to lysosomal death-induced cell apoptosis; the drug-loaded nanoparticles FPA / DOX have stronger cytotoxicity under hypoxic and acidic conditions, which is more conducive to targeted killing of tumor cells.

[0076] Example 9 Cell apoptosis experiment of drug-loaded nanoparticles FPA / DOX HeLa cells (1.0-5.0×10 7 / well) were inoculated into 12-well plates and incubated overnight. The complete medium in each well of the 12-well plate was aspirated, and then the medium containing FPA / DOX (0.5μg / mL, based on DOX content) and free DOX were added respectively, and the complete medium containing PBS without drugs was set as a blank control. Three parallel groups were set up for each group and incubated in a 37°C cell culture incubator for 6 hours. The medium was aspirated and retained, and trypsin was added to digest the cells for 3 minutes. After adding the original medium to terminate the digestion, centrifuged at 1000r / min for 3 minutes and the supernatant was carefully discarded. After adding the cell staining buffer overnight to wash the cells twice, the cell staining buffer was added overnight to resuspend the cells, and the cell suspension was kept at 0.25-1.0×10 7 100 μL of cell suspension was taken from each sample group, 5 μL of Annexin V Alexa Fluor 647 dye was added and incubated in dark for 10 min, then 5 μL of 7-AAD dye was added and incubated in dark for 5 min. After incubation, 400 μL of cell staining buffer was added to gently resuspend the cells, and the results were detected using a flow cytometer. Fig. 20 shown.

[0077] from Fig. 20 It can be seen that the drug-loaded nanoparticles FPA / DOX kill tumor cells in the form of cell apoptosis, and the drug-loaded nanoparticles FPA / DOX have strong toxicity to tumor cells.

[0078] Example 10 In vivo antitumor efficacy of drug-loaded nanoparticles FPA / DOX This example evaluates the in vivo anti-tumor efficacy of drug-loaded nanoparticles FPA / DOX.

[0079] Specifically, BALB / C mice (6 weeks old, female, weighing 20-25 g) were purchased and acclimatized in the animal facility for 1 week before the experiment. A mouse cervical cancer model was established using HeLa cells. Approximately 5 × 10 6 HeLa cells in the logarithmic growth phase, when the tumor diameter reaches about 100mm 2 The tumor-bearing mice were randomly divided into 5 groups (n=6).

[0080] Tumor-bearing mice were injected with drugs in different treatment groups by tail vein injection, namely PBS group and drug-loaded nanoparticle PFA / DOX group, where the PBS group was used as a blank control and the calculated equivalent of DOX in the FPA / DOX group was 1 mg / kg. The experiment was carried out for two weeks, and the drugs in different treatment groups were injected every other day, with 100 μL injected each time. During the entire experiment, the changes in tumor size and weight of each mouse were measured every other day, and the mouse tumor volume was calculated using the following formula (7): .

[0081] When the treatment lasted for 18 days, the mice were sacrificed, and the sections of the tumor sites of the mice were subjected to H&E staining and TUNEL staining to evaluate the killing effect of the drug on the tumor. The results are as follows: Fig.21 shown.

[0082] from Fig.21 It can be seen that after the tumor-bearing mice were treated with drug-loaded nanoparticles FPA / DOX, the weight of the mice did not change much and remained basically stable; as the treatment time increased, the tumor volume and weight of the tumor-bearing mice decreased significantly. The above results show that the drug-loaded nanoparticles FPA / DOX have good anti-tumor efficacy in mice, and the final tumor inhibition rate reached 95.27%.

[0083] Example 11 In vivo biosafety evaluation of drug-loaded nanoparticles FPA / DOX This example evaluates the in vivo biosafety of drug-loaded nanoparticles FPA / DOX.

[0084] Specifically, the toxicological data of healthy BALB / c female nude mice were used to evaluate the toxicity of the drug. The mice (age 6 weeks, 20-25g) were randomly divided into 3 groups (n=6) and intravenously injected with PBS, free DOX and drug-loaded nanoparticles FPA / DOX. The mice were killed 24 hours later. The serum samples of the mice were collected for ALT, AST, ALP, BUN, TP, ALB, GLOB and CREA detection. The main organs of the mice (including heart, liver, spleen, lung, and kidney) were collected, and the sections of each organ were further analyzed by H&E staining. The results are shown in Figure 2. Fig. 22 shown.

[0085] from Fig. 22 It can be seen that after the mice were injected with drug-loaded nanoparticles FPA / DOX, there were no abnormalities in their blood biochemistry, blood routine and major organs. The above results indicate that drug-loaded nanoparticles FPA / DOX have good biosafety.

[0086] In summary, please refer to Fig.23 , which is a schematic diagram of the synthesis of drug-loaded nanoparticles FPA / DOX and their targeting tumor cell effects in the present invention. Specifically, in the present invention, by using p-formylbenzoic acid, nonaglycone and azobenzene to react, a nanocarrier with moderate particle size and dual response to hypoxia and pH is obtained. The nanocarrier specifically targets the tumor site and then specifically releases the drug to reduce toxic side effects; at the same time, after the nanocarrier responds, the benzaldehyde group is released, which covalently connects with the protein on the lysosomal membrane, resulting in changes in protein activity, thereby rupturing the lysosomal membrane and increasing permeability, making it impossible to expel the nanocarrier out of the cell, thereby significantly improving the drug bioavailability of the nanocarrier. In addition, the nanocarrier has a high drug loading capacity.

[0087] 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.

[0088] 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 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 as claimed in claim 1, characterized in that: The steps include: S1, using p-formylbenzoic acid and nonaglycone as raw materials, reacting in the presence of a catalyst and an activator, and obtaining an intermediate copolymer through separation and purification; S2. Mixing the intermediate copolymer with azo-paraaniline and reacting them, and obtaining the hypoxia and pH dual-responsive nanocarrier 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 nonaglycol, the catalyst and the activator is (3-10): (3-10): (0.3-1): (3-10); and 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, characterized in that: 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-p-aniline, reacting the mixture specifically comprises: 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 2-6.

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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