CN109432051B - Targeting nanoparticle with anti-ovarian cancer activity and preparation and application thereof - Google Patents
Targeting nanoparticle with anti-ovarian cancer activity and preparation and application thereof Download PDFInfo
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- CN109432051B CN109432051B CN201811591942.6A CN201811591942A CN109432051B CN 109432051 B CN109432051 B CN 109432051B CN 201811591942 A CN201811591942 A CN 201811591942A CN 109432051 B CN109432051 B CN 109432051B
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Abstract
The invention provides a targeting nanoparticle with anti-ovarian cancer activity, which consists of stearic acid, polyethyleneimine and hyaluronic acid, wherein the carried drugs are paclitaxel and curcumin, and respectively account for 0.4 percent and 0.4 percent of the total nanoparticle. And (2) taking carbodiimide as an activating agent of carboxyl, dehydrating and condensing the carboxyl of stearic acid and primary amino of polyethyleneimine, mixing with the medicament, filtering and freeze-drying to obtain the medicament-carrying targeted nanoparticle. According to the invention, through a high-molecular nano material with cell uptake and cytoplasm retention functions, the characteristic of receptor-ligand is utilized to encapsulate a drug with a target positioned in a cell, so that the drug uptake of ovarian cancer cells is increased, the distribution of the drug in normal tissues is favorably reduced, the toxic and side effects of the drug are reduced, and the combination of curcumin coordinates the toxicity of paclitaxel on the ovarian cancer cells, enhances the anti-ovarian cancer activity of the paclitaxel and reverses the drug resistance of the paclitaxel. The targeting nanoparticle can be applied to the preparation of targeting drugs loaded with anti-ovarian cancer activity.
Description
Technical Field
The invention belongs to the field of pharmacy, and relates to a targeting nanoparticle with anti-ovarian-cancer activity, and preparation and application thereof in preparation of a medicament for treating ovarian cancer diseases.
Background
Compared with other gynecological malignant tumors, the ovarian cancer is more latent in onset. More than 75% of ovarian cancer patients have been reported to have advanced stages at the time of diagnosis. In recent years, despite the continuous improvement of treatment means of ovarian cancer, the mortality rate of ovarian cancer is still the highest in female gynecological tumors, and the five-year survival rate of ovarian cancer patients is only improved from 33.7% in 1975 to 45.6% in 2011. The conventional approach to ovarian cancer treatment is to surgically remove macroscopic tumors and residual lesions and to apply chemotherapy, although ovarian cancer is sensitive to first-line chemotherapeutic drugs (such as paclitaxel), but is short-lived and is prone to developing resistance. It is statistically determined that 90% of patients with stage III-IV ovarian cancer die because of multidrug resistance. Therefore, reducing the death rate of ovarian cancer is a great clinical challenge, and finding an effective method for reversing the drug resistance of ovarian cancer-paclitaxel is of great significance.
Curcumin is a natural polyphenolic compound and can be used as functional food, pharmaceutical and nutritional supplements. It has various pharmacological properties, such as antioxidant and anti-inflammatory properties, and has been shown to inhibit the proliferation of tumor cells. In recent years, with the progress of drug resistance research, people find that curcumin can improve the sensitivity of drugs to a certain extent and reverse tumor drug resistance by regulating various signal pathways. However, the use of curcumin is limited by its chemical instability, low water solubility and poor oral bioavailability.
The nano delivery vehicle can greatly improve the treatment efficacy by improving the solubility and protecting the biological activity. In addition, nanoparticles can also enhance drug uptake by drug-resistant cells and provide precise drug therapy to tumor tissues to reduce systemic toxicity. Ovarian cancer tissues express higher levels of CD44, which is driving tumor metastasis and may be one of the reasons for tumor resistance to chemotherapy. When CD44 was knocked down, proliferation, invasive activity and spheroid formation were significantly inhibited with increased drug sensitivity. Therefore, strategies to target CD44 were developed to prevent metastasis and drug resistance of ovarian cancer.
Hyaluronic Acid (HA) is a polyanionic polysaccharide, HAs low toxicity, is biodegradable and biocompatible, and its product induces receptor-mediated intracellular signaling. The nanoparticles modified with HA have high affinity for CD44 overexpressed on cancer cell membranes, enabling their targeting into ovarian cancer cells.
Disclosure of Invention
The invention aims to provide a targeting nanoparticle with anti-ovarian cancer activity, which is loaded with paclitaxel and curcumin, wherein the nanoparticle material consists of stearic acid (molecular weight of 284.48Da), polyethyleneimine (molecular weight of 10kDa) and hyaluronic acid (molecular weight of 10KDa), the mass ratio of stearic acid to polyethyleneimine is 2:1-2:5, the hyaluronic acid accounts for 10% of the total mass of stearic acid, polyethyleneimine and hyaluronic acid, and the loaded paclitaxel and curcumin respectively account for 0.4% and 0.4% of the total nanoparticles.
The invention also aims to provide a preparation method of the targeted nanoparticle, which is to take carbodiimide (EDC) as an activating agent of carboxyl, and dehydrate and condense primary amine groups of Stearic Acid (SA) and Polyethyleneimine (PEI) to obtain PEI-SA. The method is realized by the following steps:
(1) 199mg of Stearic Acid (SA) was previously dissolved in 50ml of hot ethanol and 500mg of Polyethyleneimine (PEI) was dissolved in 40ml of distilled water, then they were mixed at 80 ℃ with stirring, 750mg of carbodiimide (EDC) was added to the mixture with stirring at 300rpm for 24 hours, the reaction solution was dialyzed against 10% ethanol solution using a dialysis membrane (MWCO: 3.5KDa, Spectrum Laboratories, Laguna Hills, Calif.) for 48 hours to remove by-products, finally, dialyzed against distilled water for 2 hours, the dialyzed product was lyophilized, PEI-SA was collected, and then the PEI-SA solution was coated with 5mg/ml of HA to obtain a lyophilized product of HA of (PEI-SA).
(2) Accurately weighing 10mg (PEI-SA) HA lyophilized product, adding appropriate amount of deionized water for dispersion to obtain 1mg/ml solution, slowly adding 0.8ml paclitaxel and curcumin (1mg/ml) ethanol solution into the nanometer solution under stirring at room temperature, and subjecting the mixture to ultrasonic treatment in ice bath for 15 min. Thereafter, the mixture was stirred for 4 hours, and then dialyzed overnight in pure water using a dialysis membrane (MWCO: 7000Da, USA) and the pure water was changed frequently. Then, the solution was centrifuged at 3500rpm for 15 minutes to remove unloaded paclitaxel and curcumin, filtered with a 0.45 μm pore size filter and lyophilized to obtain (PEI-SA) HA nanoparticles (abbreviated as (PEI-SA) HA/PC) of paclitaxel and curcumin.
The invention also aims to provide application of the targeting nanoparticle with anti-ovarian cancer activity in preparation of anti-ovarian cancer activity-loaded targeting drugs, wherein the loaded drugs are paclitaxel and curcumin. Research shows that compared with free medicines (free paclitaxel and curcumin), the drug-loaded nanoparticles can obviously improve the anti-ovarian cancer activity of the drug-loaded nanoparticles at a cellular level.
According to the invention, amphiphilic nanoparticles are formed by grafting by utilizing the lipophilicity of SA and the hydrophilicity of PEI, and ovarian cancer cells are targeted by utilizing the receptor-ligand characteristic of HA so as to improve the enrichment of a medicament in a target tissue, thereby achieving the purpose of improving the anti-ovarian cancer activity of the medicament, and simultaneously, the participation of curcumin coordinates the anti-tumor effect of paclitaxel and reverses the medicament resistance.
The invention has the advantages that: the drug uptake of ovarian cancer cells can be greatly increased by using the macromolecule nanometer material with the functions of cell uptake and cytoplasm retention and encapsulating the drug with the target positioned in the cells by utilizing the characteristic of receptor-ligand. The ingestion of ovarian cancer cells to the medicine is increased, the distribution of the medicine in normal tissues is favorably reduced, and the toxic and side effects of the medicine are reduced; the combination of curcumin coordinates the toxicity of paclitaxel to ovarian cancer cells, enhances the anti-ovarian cancer activity of paclitaxel and reverses the drug resistance.
Drawings
FIG. 1 is a confocal fluorescence microscopy (PEI-SA) observation of cellular uptake of HA/PC at SKOV 3.
Fig. 2 is a schematic representation of nanoparticles in an ovarian cancer tissue microenvironment.
Detailed Description
The invention is further explained by combining the drawings and the embodiments.
Example 1: preparation of anti-ovarian cancer active nanoparticles
(1) Preparation of anti-ovarian cancer active nanoparticles
Carbodiimide (EDC) is used as an activating agent of carboxyl, and the carboxyl (SA) of stearic acid and the primary amine group of Polyethyleneimine (PEI) are subjected to dehydration condensation to obtain PEI-SA. 199mg of SA were previously dissolved in 50ml of hot ethanol, and 100mg of PEI were dissolved in 40ml of distilled water, respectively. Then, they were mixed at 80 ℃ with stirring. 750mg of EDC were added to the mixture with stirring at 300rpm for 24 hours. The reaction solution was dialyzed against a 10% ethanol solution for 48 hours using a dialysis membrane (MWCO: 3.5kDa, Spectrum Laboratories, Laguna Hills, Calif.) to remove by-products. Finally, the mixture was dialyzed against distilled water for 2 hours, and the dialyzed product was lyophilized to collect PEI-SA. The PEI-SA solution was then coated with 5mg/ml HA to give a lyophilized product of (PEI-SA) HA.
Accurately weighing 10mg (PEI-SA) HA lyophilized product, adding appropriate amount of deionized water for dispersion to obtain 1mg/ml solution, slowly adding 0.8ml paclitaxel and curcumin (1mg/ml) ethanol solution into the nanometer solution under stirring at room temperature, and subjecting the mixture to ultrasonic treatment in ice bath for 15 min. After that, the mixture was stirred for 4 hours, and then dialyzed overnight in pure water using a dialysis membrane (MWCO: 7000Da, USA) with frequent replacement of fresh water. Then, the solution was centrifuged at 3500rpm for 15 minutes to remove unloaded paclitaxel and curcumin, filtered with a 0.45 μm pore size filter and lyophilized to obtain (PEI-SA) HA nanoparticles (abbreviated as (PEI-SA) HA/PC) of paclitaxel and curcumin.
A particle size and surface potential tester is adopted to respectively test the particle size, the surface potential, the drug encapsulation efficiency and the drug loading capacity of (PEI-SA) HA/PC nanoparticles (Table 1). Table 1 shows the particle size and distribution of (PEI-SA) HA/PC nanoparticles (mass ratio of SA to PEI 2: 1).
Table 1 particle size, surface potential and drug encapsulation efficiency of anti-ovarian cancer active nanoparticles.
Example 2: preparation of anti-ovarian cancer active nanoparticles
(1) Preparation of anti-ovarian cancer active nanoparticles
Carbodiimide (EDC) is used as an activating agent of carboxyl, and the carboxyl (SA) of stearic acid and the primary amine group of Polyethyleneimine (PEI) are subjected to dehydration condensation to obtain PEI-SA. 199mg of SA were pre-dissolved in 50ml of hot ethanol, and 300mg of PEI were dissolved in 40ml of distilled water, respectively. Then, they were mixed at 80 ℃ with stirring. 750mg of EDC were added to the mixture with stirring at 300rpm for 24 hours. The reaction solution was dialyzed against a 10% ethanol solution for 48 hours using a dialysis membrane (MWCO: 3.5kDa, Spectrum Laboratories, Laguna Hills, Calif.) to remove by-products. Finally, the mixture was dialyzed against distilled water for 2 hours, and the dialyzed product was lyophilized to collect PEI-SA. The PEI-SA solution was then coated with 5mg/ml HA to give a lyophilized product of (PEI-SA) HA.
Accurately weighing 10mg (PEI-SA) HA lyophilized product, adding appropriate amount of deionized water for dispersion to obtain 1mg/ml solution, slowly adding 0.8ml paclitaxel and curcumin (1mg/ml) ethanol solution into the nanometer solution under stirring at room temperature, and subjecting the mixture to ultrasonic treatment in ice bath for 15 min. After that, the mixture was stirred for 4 hours, and then dialyzed overnight in pure water using a dialysis membrane (MWCO: 7000Da, USA) with frequent replacement of fresh water. Then, the solution was centrifuged at 3500rpm for 15 minutes to remove unloaded paclitaxel and curcumin, filtered with a 0.45 μm pore size filter and lyophilized to obtain (PEI-SA) HA nanoparticles (abbreviated as (PEI-SA) HA/PC) of paclitaxel and curcumin.
A particle size and surface potential tester is adopted to respectively test the particle size, the surface potential, the drug encapsulation efficiency and the drug loading capacity of the (PEI-SA) HA/PC nanoparticles (Table 2). Table 2 shows the particle size and distribution of (PEI-SA) HA/PC nanoparticles (mass ratio of SA to PEI 2: 3).
Table 2 particle size, surface potential and drug encapsulation efficiency of anti-ovarian cancer active nanoparticles.
Example 3: preparation of anti-ovarian cancer active nanoparticles
1) Preparation of anti-ovarian cancer active nanoparticles
Carbodiimide (EDC) is used as an activating agent of carboxyl, and the carboxyl (SA) of stearic acid and the primary amine group of Polyethyleneimine (PEI) are subjected to dehydration condensation to obtain PEI-SA. 199mg of SA were previously dissolved in 50ml of hot ethanol, and 500mg of PEI were dissolved in 40ml of distilled water, respectively. Then, they were mixed at 80 ℃ with stirring. 750mg of EDC were added to the mixture with stirring at 300rpm for 24 hours. The reaction solution was dialyzed against a 10% ethanol solution for 48 hours using a dialysis membrane (MWCO: 3.5kDa, Spectrum Laboratories, Laguna Hills, Calif.) to remove by-products. Finally, the mixture was dialyzed against distilled water for 2 hours, and the dialyzed product was lyophilized to collect PEI-SA. The PEI-SA solution was then coated with 5mg/ml HA to give a lyophilized product of (PEI-SA) HA.
Accurately weighing 10mg (PEI-SA) HA lyophilized product, adding appropriate amount of deionized water for dispersion to obtain 1mg/ml solution, slowly adding 0.8ml paclitaxel and curcumin (1mg/ml) ethanol solution into the nanometer solution under stirring at room temperature, and subjecting the mixture to ultrasonic treatment in ice bath for 15 min. After that, the mixture was stirred for 4 hours, and then dialyzed overnight in pure water using a dialysis membrane (MWCO: 7000Da, USA) with frequent replacement of fresh water. Then, the solution was centrifuged at 3500rpm for 15 minutes to remove unloaded paclitaxel and curcumin, filtered with a 0.45 μm pore size filter and lyophilized to obtain (PEI-SA) HA nanoparticles (abbreviated as (PEI-SA) HA/PC) of paclitaxel and curcumin.
A particle size and surface potential tester is adopted to respectively test the particle size, the surface potential, the drug encapsulation efficiency and the drug loading capacity of the (PEI-SA) HA/PC nanoparticles (Table 3). Table 3 shows the particle size and distribution of (PEI-SA) HA/PC nanoparticles (mass ratio of SA to PEI 2: 5).
Table 3 particle size, surface potential and drug encapsulation efficiency of anti-ovarian cancer active nanoparticles.
Example 4: application of anti-ovarian cancer active nanoparticles
(1) Application of anti-ovarian cancer active nanoparticles
IC of (PEI-SA) HA vs SKOV350At 478. mu.g/ml, indicating that (PEI-SA) HA HAs a relatively low cytotoxicity.
IC of free PTX in SKOV3 cells501.35. mu.g/ml, whereas (PEI-SA) HA mediated post IC50It was 0.11. mu.g/ml (as shown in Table 4). IC of free curcumin502.49 μ g/mL, IC when mediated by (PEI-SA) HA vector50It was 0.68. mu.g/ml. The effect of (PEI-SA) HA/curcumin on drug-resistant cells was similar to that of sensitive cells. IC when both PTX and curcumin were loaded in (PEI-SA) HA vehicle ((PEI-SA) HA/PC)50At a minimum, it is suggested that these two drugs have a synergistic effect. IC of free PTX in SKOV3-TR30 cells509.33. mu.g/ml, while (PEI-SA) IC in HA50After loading, it was 0.13. mu.g/ml. Curcumin IC when (PEI-SA) HA vector mediated500.70 μ g/ml, IC of free curcumin50It was 3.83. mu.g/mL. In the SKOV3-TR30 cell line, (PEI-SA) HA/PC showed the best cytotoxicity. Table 4 shows the IC50 values of PTX, (PEI-SA) HA/PTX, curcumin, (PEI-SA) HA/curcumin and (PEI-SA) HA/PC on SKOV3/SKOV3-TR30 cells.
Toxicity of free PTX to drug-resistant cells is significantly reduced. PTX is effective on both sensitive and resistant cells after being mediated by (PEI-SA) HA. Curcumin has similar effects on sensitive and resistant cells after vector mediation. This indicates, on the one hand, that the efficacy of (PEI-SA) HA may be effective in improving the effect of PTX on resistant cells. On the other hand, the combination of curcumin and PTX had a stronger toxic effect on drug resistant cell lines. Third, the involvement of curcumin reversed the role of PTX in the drug-resistant cell line SKOV3-TR 30.
TABLE 4 IC50 values of PTX, (PEI-SA) HA/PTX, curcumin, (PEI-SA) HA/curcumin and (PEI-SA) HA/PC versus SKOV3/SKOV3-TR30 cells.
Example 5: application of anti-ovarian cancer active nanoparticles
(1) Cellular uptake of (PEI-SA) HA/PC
Uptake in SKOV3 cells was measured by using fluorescently labeled (PEI-SA) HA/PC nanoparticles. Figure 1 shows fluorescence microscopy images of FITC-labeled complexes and RITC-labeled nanoparticle distributions incubated with SKOV3 cells for 0,1,2,4, and 8 hours. It can be seen that the fluorescence intensity gradually increases with the incubation time. After 24 hours, significant fluorescence was detected in all cells. Notably, the fluorescence uptake in the drug-resistant cell line SKOV3-TR30 was similar to that in SKOV 3. In addition, intracellular uptake of fluorescently labeled nanoparticles was also performed in a quantitative manner. The average fluorescence intensity results of (PEI-SA) HA/PC measured by flow cytometry after 4 hours of incubation are consistent with the results of the fluorescence photographs. FIG. 1 is confocal fluorescence microscopy image observation. SKOV3 cells were incubated for 1 hour, 2 hours, 4 hours and 8 hours for fluorescent image observation of FITC-labeled (PEI-SA) HA/PC; SKOV3 cells were incubated for 1 hour, 2 hours, 4 hours, and 8 hours for fluorescence image observation of RITC-labeled (PEI-SA) HA/PC. Abbreviations: FITC, fluorescein isothiocyanate; RITC, rhodamine isothiocyanate; combinations of nuclear staining and fluorescence were combined.
Example 6
Biocompatible Stearic Acid (SA) modified Polyethyleneimine (PEI) is used as a mother nucleus of the nanoparticle and coated with HA, and then paclitaxel and curcumin are loaded (fig. 2), so that the nanoparticle targets ovarian cancer tissues by virtue of an antibody-receptor principle. The nanoparticles can be used for strongly and synergistically enhancing the cytotoxicity of the paclitaxel, and can change a paclitaxel signal conduction path to enhance the synergistic effect of drug combination and reverse the drug resistance of the paclitaxel.
The application examples show that the drug-loaded nanoparticles constructed by the research have the effect of high-efficiency anti-ovarian cancer activity.
Claims (2)
1. A targeted nanoparticle with anti-ovarian cancer activity is characterized in that the nanoparticle is composed of stearic acid with the molecular weight of 284.48Da, polyethyleneimine with the molecular weight of 10kDa, hyaluronic acid with the molecular weight of 10KDa and carried drugs, wherein the mass ratio of the stearic acid to the polyethyleneimine is 2:1-2:5, the hyaluronic acid accounts for 10% of the total mass of the stearic acid, the polyethyleneimine and the hyaluronic acid, and the carried drugs are paclitaxel and curcumin which respectively account for 0.4% and 0.4% of the total nanoparticle;
the targeted nanoparticles are prepared by the following steps:
(1) dissolving stearic acid in hot ethanol in advance, dissolving polyethyleneimine in distilled water, then mixing them at 80 ℃ under stirring, adding carbodiimide to the mixture under stirring for 24 hours, dialyzing the reaction solution against 10% ethanol solution using a dialysis membrane for 48 hours to remove by-products, finally, dialyzing against distilled water for 2 hours, lyophilizing the dialyzed product, collecting polyethyleneimine-stearic acid, and then coating the polyethyleneimine-stearic acid solution with 5mg/ml concentration of HA to obtain (polyethyleneimine-stearic acid) HA lyophilized product;
(2) weighing (polyethyleneimine-stearic acid) HA freeze-dried products, adding a proper amount of deionized water for dispersion to prepare a solution with the concentration of 1mg/ml, then slowly adding 0.8ml of ethanol solution of paclitaxel and curcumin into the nanoparticle solution under stirring at room temperature, carrying out ultrasonic treatment on the mixture in an ice bath for 15 minutes, then stirring the mixture for 4 hours, then dialyzing overnight in pure water by using a dialysis membrane and frequently replacing purified water, then centrifuging the solution to remove the unloaded paclitaxel and curcumin, filtering and freeze-drying to obtain paclitaxel and curcumin-loaded (PEI-SA) HA nanoparticles.
2. The use of the targeted nanoparticle with anti-ovarian cancer activity of claim 1 in the preparation of targeted drug loaded with anti-ovarian cancer activity, wherein the loaded drug is paclitaxel and curcumin.
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| Double-layered hyaluronic acid/stearic acidmodified polyethyleneimine nanoparticles encapsulating (-)-gossypol: a nanocarrier for chiral anticancer drugs;Hao Liu等;《Journal of Materials Chemistry B》;20141231;第2卷;第5238–5248页，尤其是第5238页右栏第2段，第5239页Scheme1，第5240页左栏第3段、右栏第1、3段，第5241页左栏第1段，第5242页右栏倒数第2段，第5244页左栏第2段 * |
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