Nanometer albumin paclitaxel particle, preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to nano albumin paclitaxel particles as well as a preparation method and application thereof. According to the invention, DCC and NHS are used as condensing agents, so that the conjugate HSA-RA with relatively high in-vivo stability can be obtained; hSA-RA and PTX are self-assembled according to a specific ratio, so that the effect of synergistically inhibiting cancer cells by PTX and RA can be improved, and the effective release rate of the medicine can also be improved. The nano albumin paclitaxel particle provided by the invention not only enhances the killing effect of paclitaxel on living tumor cells, but also has an obvious improvement effect on cervical cancer cell inflammation and oxidative damage, so that by inhibiting the expression of CYP3A4 and CYP2C8, the nano albumin paclitaxel particle has obvious and positive effects on treatment and prognosis of cervical cancer.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and specifically relates to a nano-albumin paclitaxel particle, a preparation method and application thereof. Background Art

[0002] Paclitaxel (PTX) drugs have been used clinically to treat cancer for many years. They have broad-spectrum anti-tumor activity and can be used in chemotherapy for various cancers, such as head and neck cancer, lung cancer, breast cancer, etc. However, due to the extreme hydrophobicity of paclitaxel, its water solubility is poor, which seriously limits the scope of paclitaxel in practical applications.

[0003] Although there are drugs for treating cancer by nano-encapsulating paclitaxel in the prior art, there are still some problems in the process of nano-encapsulating paclitaxel. For example, the nano-encapsulated paclitaxel drug used to treat metastatic breast cancer has better efficacy than paclitaxel alone, but due to the imperfect preparation method during the nano-encapsulation of paclitaxel, the nano-encapsulated paclitaxel drug has poor stability and low effective release rate, which is not conducive to the full release and exertion of the efficacy of the nano-encapsulated paclitaxel drug. Summary of the invention

[0004] The purpose of the present invention is to provide a nano-albumin paclitaxel particle, a preparation method and an application thereof, which not only has strong stability and high release rate, but also has a significant positive effect on the treatment and prognosis of cervical cancer by inhibiting the expression of CYP3A4 and CYP2C8.

[0005] The invention provides a method for preparing nano albumin paclitaxel particles. 1,3-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) are used as condensing agents, retinoic acid (RA) is grafted onto human serum albumin (HSA) through an amidation reaction, and a conjugate HSA-RA is obtained; paclitaxel (PTX) is dripped into the conjugate HSA-RA for mixing, and after dialysis, the retentate is dried to obtain the nano albumin paclitaxel particles; the volume ratio of the paclitaxel (PTX) to the conjugate HSA-RA is 1:10.

[0006] Preferably, the amidation reaction comprises: activating a retinoic acid solution with a mixed solution containing 1,3-dicyclohexylcarbodiimide and N-hydroxysuccinimide to obtain activated retinoic acid; mixing the activated retinoic acid with a human serum albumin solution at a volume ratio of 1:45, and after dialysis, drying the retentate to obtain a conjugate HSA-RA.

[0007] Preferably, the mixed solution uses DMSO as a solvent and includes 14.8 mg / mL of 1,3-dicyclohexylcarbodiimide and 8.28 mg / mL of N-hydroxysuccinimide.

[0008] Preferably, the volume ratio of the retinoic acid solution to the mixed solution is 1:1; the retinoic acid solution uses DMSO as a solvent and includes 180 mg / mL of retinoic acid.

[0009] Preferably, the human serum albumin solution uses PBS as a solvent, and the volume mass ratio of the solvent to human serum albumin is 4.5 mL:10 mg.

[0010] Preferably, the activation and mixing are performed separately under light protection.

[0011] Preferably, the solvent of the paclitaxel solution includes ethanol, and the concentration of paclitaxel in the paclitaxel solution is 1 mg / mL; the solution containing the conjugate HSA-RA uses PBS solution as solvent, and the concentration of HSA-RA in the solution is 1 mg / mL.

[0012] The present invention provides nano-albumin paclitaxel particles obtained by the method described in the above technical solution.

[0013] The present invention provides the use of nano-albumin paclitaxel particles obtained by the method described in the above technical scheme in the preparation of drugs for treating and / or prognosing cervical cancer.

[0014] Preferably, the drug includes a drug that inhibits the expression of CYP3A4 and / or CYP2C8.

[0015] Beneficial effects:

[0016] The present invention provides a method for preparing nano-albumin paclitaxel particles, comprising the following steps: using 1,3-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) as condensing agents, grafting retinoic acid (RA) onto human serum albumin (HSA) through an amidation reaction to obtain a conjugate HSA-RA; dropping paclitaxel (PTX) into the conjugate HSA-RA for mixing, and after dialysis, drying the retentate to obtain nano-albumin paclitaxel particles; the volume ratio of paclitaxel (PTX) to the conjugate HSA-RA is 1:10. The present invention uses DCC and NHS as condensing agents, which is conducive to obtaining a conjugate HSA-RA with high in vivo stability; by self-assembling HSA-RA and PTX in a specific ratio, it is not only conducive to enhancing the synergistic inhibition of cancer cells by PTX and RA, but also can improve the effective release rate of the drug.

[0017] Based on the above technical advantages, the present invention also provides the use of nano-albumin paclitaxel particles in the preparation of drugs for the treatment and / or prognosis of cervical cancer. Experiments have shown that the nano-albumin paclitaxel particles provided by the present invention not only enhance the killing effect of paclitaxel on living tumor cells, but also have a significant improvement effect on inflammation and oxidative damage of cervical cancer cells. Therefore, by inhibiting the expression of CYP3A4 and CYP2C8, it has a significant positive effect on the treatment and prognosis of cervical cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0019] Figure 1 To verify the characterization observation of Nano-HSA-PTX in Example 1;

[0020] Figure 2 To verify the difference between Nano-HSA-PTX and PTX in Example 2;

[0021] Figure 3 To verify the toxicity observation results of Nano-HSA-PTX in Example 3;

[0022] Figure 4 To verify the activity detection of CC cells by Nano-HSA-PTX in Example 4;

[0023] Figure 5 To verify the Transwell experiment results in Example 5;

[0024] Figure 6 To verify the number of HeLa and SiHa cells that penetrated the membrane in Example 5;

[0025] Figure 7 To verify the results of the HeLa cell scratch experiment in Example 5;

[0026] Figure 8 To verify the results of the SiHa cell scratch experiment in Example 5;

[0027] Fig. 9 To verify the cell migration rate of HeLa and SiHa in Example 5;

[0028] Fig.10 To verify the flow cytometry experimental results in Example 6;

[0029] Fig.11 To verify the apoptosis rate of HeLa and SiHa cells in Example 6;

[0030] Fig.12 To verify the expression of proteins Bax and Bcl-2 in cells detected in Example 6;

[0031] Fig.13 To verify the effect of Nano-HSA-PTX on CC cell inflammation and oxidative damage in Example 7;

[0032] Fig.14 To verify the effect of Nano-HSA-PTX on CYP3A4 and CYP2C8 in Example 8;

[0033] Fig.15 To verify the effect of Nano-HSA-PTX on living tumors in Example 9. DETAILED DESCRIPTION

[0034] The present invention provides a method for preparing nano-albumin paclitaxel particles, comprising the following steps: using 1,3-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) as condensing agents, grafting retinoic acid (RA) onto human serum albumin (HSA) through an amidation reaction to obtain a conjugate HSA-RA; dropping paclitaxel (PTX) into the conjugate HSA-RA for mixing, and after dialysis, drying the retentate to obtain nano-albumin paclitaxel particles; the volume ratio of paclitaxel (PTX) to the conjugate HSA-RA is 1:10. The nano-albumin paclitaxel particles prepared in this way are not only conducive to improving the synergistic inhibition of cancer cells by PTX and RA, but also can improve the stability and effective release rate of paclitaxel nano-drugs.

[0035] In the present invention, unless otherwise specified, all reagents used are purchased conventionally.

[0036] The present invention preferably prepares a mixed solution containing DCC and NHS. In the present invention, the mixed solution preferably uses DMSO as a solvent; in the mixed solution, the concentration of DCC is preferably 14.8 mg / mL; in the mixed solution, the concentration of NHS is preferably 8.28 mg / mL.

[0037] After obtaining the mixed solution containing DCC and NHS, the present invention preferably activates the RA solution with the mixed solution containing DCC and NHS to obtain activated RA. The RA solution of the present invention preferably uses DMSO as a solvent; the concentration of RA in the RA solution is preferably 180 mg / mL; the volume ratio of the RA solution to the mixed solution containing DCC and NHS is preferably 1:1; the activation is preferably stirred under light-proof conditions; the stirring time is preferably 24 hours. The present invention preferably prepares an RA solution.

[0038] After obtaining the activated RA, the present invention preferably mixes the HSA solution with the activated RA to obtain a reaction solution. The HSA solution of the present invention preferably uses PBS as a solvent; the concentration of the PBS is preferably 0.1M; there is no special requirement for the pH value of the PBS, and the technology well known in the art can be used; in the HSA solution, the volume mass ratio of the solvent to HSA is preferably 4.5mL:10mg; the volume ratio of the HSA solution to the activated RA is 45:1; during the mixing, the activated RA is preferably added dropwise to the HSA solution; there is no special requirement for the method of adding dropwise, and the technology well known in the art can be used; the process of adding dropwise is accompanied by stirring; the stirring time is preferably 24h. The above-mentioned amidation reaction is conducive to grafting RA to HSA.

[0039] After obtaining the reaction solution, the present invention preferably dialyzes the reaction solution and dries the retentate to obtain the conjugate HSA-RA. In the present invention, the dialysis is preferably performed in deionized water; the molecular weight cutoff (MWCO) of the dialysis bag is preferably 10000Da; the dialysis time is preferably 48h; the drying is preferably freeze drying; there is no special requirement for the freeze drying method, and the technology well known in the art can be used.

[0040] After obtaining the conjugate HSA-RA, the present invention preferably dissolves the conjugate HSA-RA in a PBS solution to obtain an HSA-RA solution. The concentration of the PBS of the present invention is preferably 0.1M; there is no special requirement for the pH value of the PBS, and the technology well known in the art can be used; in the HSA-RA solution, the concentration of HSA-RA is preferably 1 mg / mL.

[0041] After obtaining the HSA-RA solution, the present invention drips the paclitaxel solution into the HSA-RA solution to obtain a mixed solution. The paclitaxel solution of the present invention preferably uses ethanol as a solvent, and more preferably uses anhydrous ethanol as a solvent; in the paclitaxel solution, the concentration of paclitaxel is preferably 1 mg / mL; during the dripping, the volume ratio of the paclitaxel solution to the HSA-RA solution is preferably 1:10; the dripping process is accompanied by stirring; and the stirring time is preferably 10 minutes.

[0042] After obtaining the mixed solution, the present invention preferably dialyzes the mixed solution and dries the retentate to obtain nano-albumin paclitaxel particles. In the present invention, the dialysis is preferably performed in deionized water; the molecular weight cutoff of the dialysis bag is preferably 10000Da; the dialysis time is preferably 48h; the drying is preferably freeze drying; there is no special requirement for the freeze drying method, and the technology well known in the art can be used.

[0043] The present invention provides nano-albumin paclitaxel particles obtained by the method described in the above technical solution. The nano-albumin paclitaxel particles obtained by the above method have strong stability and high effective release rate.

[0044] The present invention also provides the use of the nano-albumin paclitaxel particles obtained by the method described in the above technical solution in the preparation of drugs for treating and / or prognosing cervical cancer, wherein the drugs preferably include drugs that inhibit the expression of CYP3A4 and / or CYP2C8.

[0045] Experiments have shown that the nano-albumin paclitaxel particles provided by the invention not only enhance the killing effect of paclitaxel on living tumor cells, but also have a significant improvement effect on inflammation and oxidative damage of cervical cancer cells. By inhibiting the expression of CYP3A4 and CYP2C8, it has a significant positive effect on the treatment and prognosis of cervical cancer.

[0046] In order to further illustrate the present invention, a nano-albumin paclitaxel particle, a preparation method and an application thereof provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0047] Preparation before the experiment:

[0048] Cell information: Human CC cells (HeLa), human cervical squamous cell carcinoma cells (SiHa) and human normal cervical epithelial cells (End1 / E6E7) were purchased from ATCC and cultured in a medium consisting of 10% FBS and 90% DMED (culture conditions were 37°C, 5% CO2). The medium was replaced every 2 days and the cells were passaged when the growth rate reached 70-80%.

[0049] Animal data: 20 SPF-grade C57BL / 6 female mice aged 6 to 8 weeks were purchased from Jiangsu Ruike Biotechnology Co., Ltd., weighing 20 to 25 g, with an animal use license number of SYXK (Su) 2021-0062. Five mice were kept in a cage at a temperature of 20 to 26 °C, a humidity of 40 to 70%, a 12-h day / night cycle, and free access to food and water.

[0050] Statistical methods: SPSS23.0 software was used for statistical analysis. All experiments in this study were repeated three times. The results were expressed as (±s). The independent sample t-test was used for inter-group comparison, and analysis of variance and Bonferroni test were used for comparison among multiple groups. P<0.05 was considered statistically significant.

[0051] Example 1

[0052] A method for preparing nano-albumin paclitaxel particles (Nano-HSA-PTX), comprising the following steps:

[0053] (1) Synthesis of HSA-RA: Using DCC and NHS as condensing agents, RA was grafted onto HSA through an amidation reaction. Specifically, 8.28 mg of NHS and 14.8 mg / mL of DCC were weighed and dissolved in 1 mL of DMSO to prepare a mixed solution containing NHS and DCC; RA was dissolved in the DMSO solution to prepare an RA solution with a concentration of 180 mg / mL; 100 μL of the mixed solution containing NHS and DCC was measured and mixed with 100 μL of the RA solution. To achieve activation of the RA solution, stir in the dark for 24 hours to obtain activated RA; weigh 10 mg of HSA (human serum albumin) and dissolve it in 4.5 mL of 0.1 M PBS to obtain an HSA solution; measure 100 μL of the activated RA, add it dropwise into the HSA solution, stir and react in the dark for 24 hours at room temperature to obtain a reaction solution; dialyze the reaction solution in deionized water (molecular weight cutoff is 10000 Da) for 48 hours to obtain a retentate, and freeze-dry the retentate to obtain HSA-RA.

[0054] (2) Preparation of PTX@HSA-RA (i.e., Nano-HSA-PTX): The drug-loaded nanoparticles PTX@HSA-RA were prepared by a self-assembly method, specifically:

[0055] Weigh 5 mg of the HSA-RA prepared in step (1) and dissolve it in 5 mL of 0.1 M PBS to obtain an HSA-RA solution;

[0056] Weigh 0.5 mg of PTX and dissolve it in 0.5 mL of anhydrous ethanol to obtain an anhydrous ethanol solution of PTX;

[0057] The anhydrous ethanol solution of PTX was added dropwise to the HSA-RA solution while stirring, and stirred for 10 min to obtain a reaction solution;

[0058] The reaction solution was dialyzed in deionized water (MWCO: 10000Da) for 48 h to obtain a retentate, which was freeze-dried to obtain PTX@HSA-RA.

[0059] Comparative Example 1

[0060] Paclitaxel (PTX) without any treatment.

[0061] Verification Example 1: Characterization Observation

[0062] The particle size distribution and surface Zeta potential of Nano-HSA-PTX prepared in Example 1 were measured using a nano-laser particle size analyzer, and the morphological characteristics of Nano-HSA-PTX were observed using a transmission electron microscope (TEM). Figure 1 A in

[0063] Another 1 mL of Nano-HSA-PTX was placed in DMEM complete medium (37°C) containing 10% FBS and PBS (4°C) with a pH value of 7.4 and incubated for 7 days. The particle size distribution and surface Zeta potential changes were detected every 24 hours. The results are shown in Figure 1 B~D in.

[0064] Note: Figure 1 In the figure, Figure A shows the morphological characteristics of Nano-HSA-PTX observed under TEM; Figure B shows the particle size distribution of Nano-HSA-PTX; Figure C shows the PDI of Nano-HSA-PTX; and Figure D shows the Zeta potential of Nano-HSA-PTX.

[0065] like Figure 1 As shown in A in the figure, it can be seen that Nano-HSA-PTX presents a relatively complete and uniform circular or quasi-circular state under TEM, with dispersed arrangement, large gaps, and relatively uniform particle size distribution.

[0066] Depend on Figure 1 As can be seen from Figures B to D, there was no significant difference in the particle size distribution, PDI and Zeta potential of Nano-HSA-PTX in DMEM complete medium containing 10% FBP by volume and PBS with a pH value of 7.4 (P>0.05), which indicates that Nano-HSA-PTX has high in vivo stability.

[0067] Verification Example 2: Encapsulation Efficiency (EE%) and Drug Loading Rate (DL%) Test and In Vitro Release Test

[0068] 1 mL of Nano-HSA-PTX prepared in Example 1 and 1 mL of PTX in Comparative Example 1 were respectively taken, and Nano-HSA-PTX and PTX were ultrasonically mixed with 9 mL of acetonitrile, and centrifuged (1200 rpm) for 15 min to obtain a supernatant, and the content of PTX in the supernatant (i.e., A value) was detected by high performance liquid chromatography (HPLC);

[0069] 1 mL of Nano-HSA-PTX prepared in Example 1 and 1 mL of PTX in Comparative Example 1 were freeze-dried and the total mass (i.e., B value) was measured.

[0070] Subsequently, the EE% and DL% of Nano-HSA-PTX and PTX were calculated, respectively; the results are shown in Figure 2 A and Figure 2 B in Figure 2Figure A shows the difference in encapsulation efficiency EE% between Nano-HSA-PTX and PTX; Figure B shows the difference in drug loading rate DL% between Nano-HSA-PTX and PTX); wherein, EE%=A value / PTX dosage×100%; DL%=A value / B value×100%.

[0071] In addition, 3 mL of Nano-HSA-PTX and PTX were taken and added to dialysis bags, respectively, and placed in a container containing 30% release medium at 37°C for oscillation and mixing. 3 mL of samples were taken every 2 h, and the content of PTX in the removed release medium was determined by HPLC. The cumulative drug release rates of Nano-HSA-PTX and PTX were calculated. The results are shown in Figure 2 C in Figure 2 Figure C shows the drug release rate comparison between Nano-HSA-PTX and PTX).

[0072] Depend on Figure 2 It can be seen that the EE% and DL% of Nano-HSA-PTX are (50.03±2.93)% and (9.50±0.95)%, respectively, which are significantly higher than PTX (P<0.05). Comparing the drug release rates between the two, it can be seen that normal PTX is basically completely released at 6h; while the release rate of Nano-HSA-PTX is significantly reduced, and it is completely metabolized at about 22h.

[0073] Verification Example 3: In vitro cytotoxicity observation

[0074] HeLa and SiHa were inoculated in 96-well plates and incubated overnight at 37°C and 5% CO2. The culture medium in each well was then replaced with Nano-HSA-PTX prepared in Example 1 at concentrations of 25, 50, 100, and 200 μg / mL, respectively. After 48 hours of continuous culture, 20 μL of MTT solution was added to each well. The absorbance was detected by a microplate reader (490 nm). The concentration with the most significant inhibitory effect was used as the basis for subsequent research. The specific results are shown in Figure 3 (exist Figure 3 Figure A shows the toxicity observation of Nano-HSA-PTX to HeLa; Figure B shows the toxicity observation of Nano-HSA-PTX to SiHa).

[0075] Depend on Figure 3 It can be seen that under the treatment of 25, 50, 100, and 200 μg / mL Nano-HSA-PTX, the activity of HeLa and SiHa cells gradually decreased with the increase of concentration (P<0.05), among which the cell activity was lowest under the treatment of 200 μg / mL Nano-HSA-PTX (P<0.05), so this dose was selected for subsequent studies.

[0076] CC cell grouping and intervention treatment:

[0077] According to the results of the above in vitro cytotoxicity observations, cervical cancer cells (HeLa and SiHa) were cultured in culture media containing 200 μg / mL Nano-HSA-PTX and 200 μg / mL PTX, respectively. Cells cultured with an equal amount of saline were set up as a control group. After culturing for 24 hours, Nano-HSA-PTX group (HeLa and SiHa), PTX group (HeLa and SiHa) and control group (HeLa and SiHa) cells were obtained for verification of related experiments in Examples 4 to 9:

[0078] Verification Example 4: Cell Viability Detection

[0079] The cells of the Nano-HSA-PTX group, PTX group and control group after the above intervention treatment were inoculated in 96-well plates, and 20 μL MTT solution (5 mg / mL) was added to each well after culturing for 24 h, 48 h and 72 h, respectively. The cell absorbance value was detected and the growth curve was drawn. The results are shown in Figure 4 A and Figure 4 B in Figure 4 In the figure, A shows the cell growth curve of HeLa; B shows the cell growth curve of SiHa);

[0080] Cells from the Nano-HSA-PTX group, PTX group, and control group were inoculated into 12-well plates (300 cells / well) and cultured for 14 days, with the culture medium replaced every 3 days. The cells were then stained with 2% crystal violet at 37°C for 30 minutes, and cloned cells were counted under an optical microscope to calculate the cloning rate. Figure 4 C and Figure 4 D in Figure 4 Figure C shows the results of the cell cloning experiment; Figure D shows the cell cloning rates of HeLa and SiHa).

[0081] Depend on Figure 4 As can be seen from A to D in the figure, the cell growth of the Nano-HSA-PTX group and the PTX group was significantly lower than that of the control group, and the cell absorbance value of the Nano-HSA-PTX group was lower than that of the PTX group (P<0.05). Similarly, the cell cloning experiment also showed that the cell cloning rate of the control group was the highest among the three groups, and the cell cloning rate of the Nano-HSA-PTX group was lower than that of the PTX group (P<0.05).

[0082] Verification Example 5: Cell invasion and migration ability detection

[0083] The cells in the Nano-HSA-PTX group, PTX group and control group were starved for 12 h and then resuspended in DMSO. The cell concentration was adjusted to 5×10 5 100 cells / mL were inoculated in the upper chamber of the Transwell chamber, and DMSO medium was added to the lower chamber. The cells were cultured for 24 h, fixed with formaldehyde, and stained with 4% crystal violet solution. Five fields of view were randomly selected for counting under a microscope (200×). The results are shown in the table. Figures 5-6 ( Figure 5 Indicates the results of Transwell experiment; Figure 6 represents the number of HeLa and SiHa transmembrane cells);

[0084] When the cell fusion degree of Nano-HSA-PTX group, PTX group and control group was close to 100%, a 10 μL pipette tip was used to make a straight scratch. After 24 hours, the scratch healing was observed under a 40x microscope and photographed for preservation. The results are shown in Figures 7-8 ( Figure 7 The results of the HeLa cell scratch test are shown in the first row. The three figures show the results of the 0h control group, PCX group and Nano-HSA-PTX group respectively. The three figures in the second row show the results of the 0h control group, PCX group and Nano-HSA-PTX group respectively. Figure 8 The results of the SiHa cell scratch test are shown in Figure 9. The first three figures show the results of the 0h control group, PCX group and Nano-HSA-PTX group, and the second three figures show the results of the 0h control group, PCX group and Nano-HSA-PTX group. ImageJ was used to calculate the migration area on the scratch and the healing rate of the scratch. The results are shown in Figure 9. Fig. 9 represents the cell migration rate of HeLa and SiHa).

[0085] Depend on Figures 5 to 9 It can be seen that the number of transmembrane cells in the control group was higher than that in the Nano-HSA-PTX group and the PTX group, while the number of transmembrane cells in the Nano-HSA-PTX group was the lowest among the three groups (P<0.05). The results of the cell scratch experiment were consistent with the above situation, showing that the cell migration rate in the control group was the highest among the three groups, while that in the Nano-HSA-PTX group was the lowest (P<0.05).

[0086] Verification Example 6: Cell apoptosis ability detection

[0087] The cells of Nano-HSA-PTX group, PTX group and control group were taken respectively, added to 6-well culture plates and cultured for 24 h, then the supernatant was discarded, the cells were washed 3 times with pre-cooled PBS and collected, resuspended in 100 μL binding buffer, and then Annexin V and PI were added in sequence, incubated in the dark for 15 min, and the cell apoptosis rate was detected by flow cytometry. The results are shown in Figure 10-11 ( Fig.10 represents the results of flow cytometry experiments; Fig.11 represents the apoptosis rate of HeLa and SiHa cells).

[0088] In addition, Western blot was used to detect the expression of apoptosis-related proteins Bax and Bcl-2 in cells. Each cell was lysed by adding RIPA buffer, separated by SDS-PAGE electrophoresis, and transferred to PVDF membrane. The membrane was sealed with 5% skim milk for 2 hours, washed and added with primary antibody, and incubated at 4°C overnight. The next day, the membrane was washed and added with horseradish peroxidase-labeled secondary antibody, incubated at 4°C for 2 hours, and the gray value of the target band was analyzed after development. The results are shown in Fig.12 C~ Fig.12 F in Fig.12 In the figure, Figure C shows the Western blot experimental results of HeLa; Figure D shows the expression of Bax and Bcl-2 proteins in HeLa; Figure E shows the Western blot experimental results of SiHa; Figure F shows the expression of Bax and Bcl-2 proteins in SiHa).

[0089] Depend on Fig.12 It can be seen that the apoptosis rate of the control group was higher than that of the other two groups. The apoptosis rates of the Nano-HSA-PTX group in HeLa and SiHa were (12.66±0.66)% and (13.55±0.68)%, respectively, which were lower than those of the PTX group (P<0.05). The results of apoptosis protein detection showed that the expression of Bax protein in the control group was the highest among the three groups, and Bcl-2 was the lowest, while the Nano-HSA-PTX group was the opposite, that is, the expression of Bax protein was the lowest and Bcl-2 was the highest (P<0.05).

[0090] Verification Example 7: CC Inflammatory Response and Oxidative Damage Detection

[0091] PCR was used to detect oxidative damage markers SOD, GSH-Px and inflammatory factors IL-6, IL-8, and TNF-α. The total RNA of the cells in the Nano-HSA-PTX group, PTX group, and control group was extracted by Trizol method, and then 10 μg of total RNA was reverse transcribed into cDNA, and PCR amplification reaction was performed using primers of SEQ ID NO.1 to SEQ ID NO.12. PCR amplification reaction conditions: 95℃ pre-denaturation for 30s, 95℃ denaturation for 10s, 60℃ annealing for 30s, for a total of 40 cycles. The primer sequences were designed and constructed by Invitrogen, USA (see Table 1 for details), with GAPDH as the internal reference, and 2 -△△CT The relative expressions of SOD, GSH-Px and inflammatory factors IL-6, IL-8, and TNF-α were calculated. The results are shown in Fig.13 (exist Fig.13Figure A shows the expression levels of inflammatory factors in HeLa; Figure B shows the expression levels of inflammatory factors in SiHa; Figure C shows the expression levels of oxidative damage indicators in HeLa; Figure D shows the expression levels of oxidative damage indicators in SiHa).

[0092] Table 1 Primer information

[0093]

[0094] Depend on Fig.13 It can be seen that compared with the control group, the IL-6, IL-8, and TNF-α mRNA levels in the Nano-HSA-PTX group and the PTX group were significantly lower than those in the control group, while the SOD and GSH-Px mRNA levels were increased (P<0.05); among them, the inflammatory factors and oxidative damage indicators in the Nano-HSA-PTX group were more significantly improved than those in the PTX group (P<0.05).

[0095] Verification Example 8: CYP3A4 and CYP2C8 expression detection

[0096] Western blot and PCR were used to detect the expression of CYP3A4 and CYP2C8 in the cells of Nano-HSA-PTX group, PTX group and control group, respectively. The expression of CYP3A4 and CYP2C8 in HeLa, SiHa and End1 / E6E7 was also detected to confirm the relationship between CYP3A4 and CYP2C8 and CC. The results are shown in Fig.14 (exist Fig.14 In the figure, Figure A shows the expression levels of CYP3A4 and CYP2C8 in three groups of cells in HeLa; Figure B shows the expression levels of CYP3A4 and CYP2C8 in three groups of cells in SiHa; Figure C shows the expression levels of CYP3A4 and CYP2C8 in HeLa, SiHa and End1 / E6E7, among which the left figures in A, B and C all show Western blot results, and the left figures all show PCR amplification results).

[0097] Depend on Fig.14 It can be seen that in the Nano-HSA-PTX group and the PTX group, the expression levels of CYP3A4 and CYP2C8 were significantly lower than those in the control group, and the Nano-HSA-PTX group was even lower than that in the PTX group (P<0.05). Further detection of the expression of CYP3A4 and CYP2C8 in HeLa, SiHa and End1 / E6E7 showed that the expression of CYP3A4 and CYP2C8 in HeLa and SiHa was higher than that in End1 / E6E7 (P<0.05).

[0098] Verification Example 9: Nude Mouse Tumor Formation Experiment

[0099] Nano-HSA-PTX cells (HeLa and SiHa) and normal cultured HeLa and SiHa were taken and the concentration was adjusted to 1×10 7 / mL. They were injected into the subcutaneous tissue of the armpit of mice (0.5mL / mouse) and raised for 7 days until tumor nodules appeared at the injection site, which was considered a successful modeling. Mice injected with Nano-HSA-PTX-intervention cells were used as the Nano-HSA-PTX group (n=10), and mice injected with normal cells were used as the control group (n=10). The culture was continued for 15 days, and 3 mice in each group were randomly selected every 5 days and killed by cervical dislocation under anesthesia. The complete subcutaneous tumor tissue was separated, weighed, and the volume was calculated. The results are shown in Fig.15 A in Fig.15 C in Fig.15 In the figure, A shows the growth of tumor cells in different treatments; B shows the volume change of tumor cells; C shows the weight change of tumor cells);

[0100] Hematoxylin and eosin (HE) staining: Tumor tissues from different treatments were fixed with formaldehyde, embedded in paraffin, and sliced ​​(4 μm thick); blocked with goat serum, and primary and secondary antibodies diluted 1:100 were added. DAB staining solution was used for 6 to 8 minutes, and hematoxylin counterstained for 10 seconds. The changes in tumor tissue structure were observed under a microscope (200×). The results are shown in Fig.15 D( Fig.15 Figure D shows the changes in different tumor tissue structures observed under a microscope).

[0101] Depend on Fig.15 It can be seen that at 5-15 days, the tumor volume and weight of the control group were significantly higher than those of the Nano-HSA-PTX group (P<0.05). The HE staining results of the tumor tissue showed that the tumor cells in the tumor tissue of the control group transplanted with HeLa and SiHa grew in clusters and whirlpools, the cells were oval or round, the nuclei were large, pathological nuclear division was observed, the nuclear staining was deepened, and the nuclear condensation, dissolution, and fragmentation were observed. Necrotic lesions were formed between the tumor clusters, showing a homogeneous red staining. In contrast, the tumor tissue of the Nano-HSA-PTX group showed abundant blood supply, clear boundaries, fish-like cross-section, and relatively uniform tumor cell size and lighter color.

[0102] In summary, the Nano-HSA-PTX provided by the present invention can effectively enhance the killing effect of PTX on CC cells and reduce oxidative damage in cells, and its mechanism may be related to inhibiting the expression of CYP3A4 and CYP2C8. Therefore, Nano-HSA-PTX can effectively improve the therapeutic effect of PTX on CC and provide more reliable protection for the prognosis of patients.

[0103] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing nano-albumin paclitaxel particles, characterized in that: The steps include: Using 1,3-dicyclohexylcarbodiimide and N-hydroxysuccinimide as condensation agents, retinoic acid was grafted onto human serum albumin via an amidation reaction to obtain the conjugate HSA-RA; The paclitaxel solution is added dropwise to the solution containing the conjugate HSA-RA to mix, and after dialysis, the retentate is dried to obtain nano-albumin paclitaxel particles; The volume ratio of the paclitaxel solution to the conjugate HSA-RA solution is 1:

10.

2. The method according to claim 1, characterized in that The amidation reaction comprises: Activating the retinoic acid solution with a mixed solution containing 1,3-dicyclohexylcarbodiimide and N-hydroxysuccinimide to obtain activated retinoic acid; The activated retinoic acid and human serum albumin solution are mixed at a volume ratio of 1:45, and after dialysis, the retentate is dried to obtain the conjugate HSA-RA.

3. The method according to claim 2, characterized in that The mixed solution uses DMSO as a solvent and includes 14.8 mg / mL of 1,3-dicyclohexylcarbodiimide and 8.28 mg / mL of N-hydroxysuccinimide.

4. The method according to claim 2, characterized in that: The volume ratio of the retinoic acid solution to the mixed solution is 1:1; The retinoic acid solution uses DMSO as a solvent and includes 180 mg / mL retinoic acid.

5. The method according to claim 2, characterized in that: The human serum albumin solution uses PBS as a solvent, and the volume mass ratio of the solvent to human serum albumin is 4.5 mL:10 mg.

6. The method according to claim 2, characterized in that The activation and mixing were performed separately under light protection.

7. The method according to claim 1, characterized in that The solvent of the paclitaxel solution includes ethanol, and the concentration of paclitaxel in the paclitaxel solution is 1 mg / mL; The solution containing the conjugate HSA-RA uses PBS solution as solvent, and the concentration of HSA-RA in the solution is 1 mg / mL.

8. Nano-albumin paclitaxel particles obtained by the method according to any one of claims 1 to 7.

9. Use of the nano-albumin paclitaxel particles obtained by the method according to any one of claims 1 to 7 in the preparation of drugs for the treatment and / or prognosis of cervical cancer.

10. The use according to claim 9, characterized in that: The drugs include drugs that inhibit the expression of CYP3A4 and / or CYP2C8.

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