| Paclitaxel is a kind of natural anti-tumor drug which is widely used in the treatment of various malignant tumors.Due to the poor solubility of paclitaxel,there are large amounts of polyoxyethylene castor oil and ethanol in the injection solution Taxol to increase the solubility,which causes serious allergic reactions in clinical use.In addition,paclitaxel is not selective.Paclitaxel may cause side effects such as bone marrow suppression,liver and kidney toxicity,and neurotoxicity after administration.In recent years,various nanocarriers have developed rapidly.Mesoporous silica nanoparticles(MSNs)have stand out from numerous inorganic nanomaterials because of great drug loading capacity,good biocompatibility,and being easy to modification.The surface modification and functionalization of mesoporous silica have made breakthrough progress with the deepening of research Intelligent drug delivery systems with stimulus response and targeting could be manufactured by surface modification to achieve the vision of "reducing toxicity and increasing efficiency" of anticancer drugs.Destination:In order to improve the effect of paclitaxel on tumor cells and reduce the side effects on normal cells,this study synthesized dual-targeted mesoporous silica nanoparticles MSNs-NH2-FA-RGD which could specifically recognize the folate receptor and integrin ?v?3 overexpressed on the surface of tumor cells.The dual-targeted mesoporous silica nanoparticles PTX@MSNs-NH2-FA-RGD containing paclitaxel was prepared by dipping method.We evaluated the targeting ability,safety and in vitro antitumor activity of nanoparticles through cell experiments in order to provide theoretical and experimental basis for the clinical applications of mesoporous silica nanoparticles in the future.Method:Preparation and characterization of dual-targeted MSNs-NH2-FA-RGD nanoparticles:We chose cetyltrimethylammonium bromide as template agent and tetraethylorthosilicate as silicon source.They were stirred and reacted after suction filtration.The solid obtained by suction filtration was calcined in a muffle furnace to obtain mesoporous silica nanoparticles(MSNs).MSNs were modified with silane coupling agent 3-aminopropyltri ethoxy silane(APTES)to introduce amino groups and obtain aminated mesoporous silica nanoparticles(MSNs-NH2).Folic acid targeting group(NHS-PEG-FA)reacted with MSN-NH2 nanoparticles to obtain folic acid targeted-nanoparticles(MSNs-NH2-FA).Similarly,MSNs-NH2-FA nanoparticles reacted with RGD-targeted groups(NHS-PEG-RGD)to obtain dual-targeted mesoporous silica nanoparticles(MSNs-NH2-FA-RGD).The prepared nanoparticles were characterized by transmission electron microscope,nitrogen adsorption,infrared spectroscopy,laser particle size analyzer and thermogravimetric analysis.Preparation of paclitaxel-loaded nanoparticles PTX@MSNs-NH2-FA-RGD:The experiment established a method for the determination of paclitaxel by high performance liquid chromatography.We prepared the paclitaxel-loaded nanoparticles(PTX@MSNs-NH2-FA-RGD)by dipping method.And we chose the best experiment conditions according to drug loading and encapsulation rate.Cell uptake experiment:We prepared rhodamine(RhB)-loaded fluorescent nanoparticles,which were denoted as RhB@MSNs-NH2,RhB@MSNs-NH2-FA and RhB@MSNs-NH2-FA-RGD.We selected human breast epithelial MCF-10A cells(folate receptor and integrin ?v?3 on cell surface were not expressed on cell surfaces),human cervical cancer HeLa cells(folate receptor highly expressed but integrin ?v?3 not expressed on cell surfaces)and human breast cancer MCF-7 cells(folate receptor and integrin ?v?3 were both highly expressed on cell surfaces)as the experimental subjects.HeLa,MCF-7 and MCF-10A cells in the logarithmic growth phase were seeded on cell slides.After cell culture,1 mL of complete medium containing 20?g of the above-mentioned fluorescent nanoparticles was added to each well.After culturing for 4 h,we aspirated the medium and rinsed with PBS phosphate buffer solution.We used 4%paraformaldehyde to fix the cell morphology.DAPI staining solution and Lysotracker fluorescent probe staining solution were used to stain the nucleus and cytoplasm.Then we used PBS phosphate buffer solution to rinse the cell slides and observed the uptake of fluorescent nanoparticles with fluorescence microscopes.Toxicity test of blank nanoparticles:We took MCF-7 cells and MCF-10A cells as model and investigated the cytotoxic effect of blank nanoparticles MSNs-NH2-FA-RGD.We collected MCF-7 cells and MCF-10A cells in logarithmic growth phase and seeded them in 96-well plates at the density of 1.5×105 and 6×104 cells/mL-1 per well.After the cells adhering to the well wall,we added 100?L serum-containing medium containing different concentrations of blank nanoparticles(0,10,20,40,80,160 ?g·mL-1).After incubating in the constant temperature incubator for 24 hours or 48 hours,we added 2 ?L of CCK-8 solution into each well and then placed the cells in the constant temperature incubator to incubate for 2 more hours.We utilized the microplate reader to measure the OD value of each well at 450 nm.Toxicity test of drug-loaded nanoparticles:To compare the inhibitory effects of free PTX and drug-loaded nanoparticles on MCF-7 cells,we collected MCF-7 cells in logarithmic growth phase and seeded them in 96-well plates at a density of 6×104 cells/mL-1 per well.After the cells adhering to the well wall,we added 100 ?L serum-containing medium containing different concentrations of free PTX and drug-loaded nanoparticles PTX@MSNs-NH2-FA-RGD to each well(converting to PTX concentrations of 0,10,30,100,300,1000 ng·mL-1).After incubating in the constant temperature incubator for 24 hours or 48 hours,we added 2 ?L of CCK-8 solution into each well and then placed the cells in the constant temperature incubator and continued to incubate for 2 hours.The microplate reader measured the OD value of each well at 450 nm.Result:Characterization results of MSNs-NH2-FA-RGD nanoparticles:Transmission electron microscopy observed that MSNs-NH2-FA-RGD nanoparticles were spherical with smooth surfaces and uniform pore channels.Pores could still be directly observed after modification with amino,folic acid targeting groups and RGD peptide targeting groups,proving that the post-modification process did not affect the porous channels.Nitrogen adsorption results showed that MSNs have had a large specific surface area(1023.41 m2·g-1),pore size(3.77 nm)and pore volume(1.13 cm3·g-1).After the process of modification,the specific surface area,pore size and pore volume of the MSNs-NH2,MSNs-NH2-FA and MSNs-NH2-FA-RGD nanoparticles decreased accordingly,reflecting the success of the modification process.After testing,the dual-targeted nanoparticles MSNs-NH2-FA-RGD had specific surface area(426.68 m2·g-1),Pore size(3.03 nm)and pore volume(0.40 cm3·g-1).The results of the laser particle size analyzer showed that the particle sizes of MSNs,MSNs-NH2,MSNs-NH2-FA and MSNs-NH2-FA-RGD gradually became larger,which were(131.1±34.64)nm,(168.6±39.44)nm,(191.3±61.52)nm and(229.0±100.90)nm,respectively;and the Zeta potentials were(-18.4±4.30)mV,(25.6±3.80)mV,(24.4±7.36)mV and(22.9±3.90)mV,respectively.In the infrared absorption spectrum,there were characteristic absorption peaks representing amino and amide I bands,respectively,indicating that the amino,folic acid targeting groups and RGD peptide targeting groups have been successfully grafted onto the surface of MSNs.Thermogravimetric analysis was used to determine the grafting rate of organic groups.The grafting rates of amino groups,folic acid targeting groups and RGD peptide targeting groups were 7.93%,3.87%and 4.39%,respectively.Preparation of paclitaxel-loaded nanoparticles PTX@MSNs-NH2-FA-RGD:The PTX solution showed a good linear relationship between 27.5?440 ?g·mL-1(R2=0.9998).The results of precision,stability and recovery experiment met the testing requirements.The drug-loaded experiment finally determined that PTX@MSNs-NH2-FA-RGD was prepared by magnetic stirring for 12 hours in absolute ethanol.PTX@MSNs-NH2-FA-RGD nanoparticles had the drug loading of 18.52%±-4.17%and encapsulation rate of 85.71%±2.91%.Cell uptake experiment:We noticed that the red fluorescence of RhB@MSNs-NH2,RhB@MSNs-NH2-FA,and RhB@MSNs-NH2-FA-RGD was not observed in MCF-10A cells,indicating that these three nanometers particles were not ingested by MCF-10A cells.It was observed in HeLa cells that the red fluorescence of RhB@MSNs-NH2-FA and RhB@MSNs-NH2-FA-RGD coincided with the green lysosome region,proving that RhB@MSNs-NH2-FA and RhB@MSNs-NH2-FA-RGD were distributed in the cytoplasm after endocytosed.However,there was no significant difference in fluorescence intensity between the two nanoparticles.No uptake of RhB@MSNs-NH2 by HeLa cells was observed.Compared with RhB@MSNs-NH2,RhB@MSNs-NH2-FA and RhB@MSNs-NH2-FA-RGD both had obvious red fluorescence in MCF-7 cells,and RhB@MSNs-NH2-FA-RGD showed the strongest fluorescence intensity.The results had shown that the dual-targeted modification significantly improved the enrichment of nanoparticles in MCF-7 tumor cells and fully highlighted the advantages of receptor-mediated targeting.Cytotoxicity of blank nanoparticles:The dual-targeted nanoparticles MSNs-NH2-FA-RGD showed great biocompatibility and had no significant effect on the activity of MCF-7 cells and MCF-10A cells in the concentration range of 0?160?g·mL-1.Toxicity test of drug-loaded nanoparticles:Free PTX and PTX@MSNs-NH2-FA-RGD showed great anti-tumor performance.After 48 hours of administration,the IC50 of free PTX and PTX@MSNs-NH2-FA-RGD were(35.25±2.57)ng·mL-1 and(22.21±3.4)ng·mL-1.PTX@MSNs-NH2-FA-RGD showd stronger inhibitory effect on MCF-7 cells.Conclusion:In this experiment,we synthesized the dual-targeted mesoporous silica nanoparticles MSNs-NH2-FA-RGD containing paclitaxel,which were with great drug loading capacity,excellent targeting ability and great anti-cancer activity.It showed application prospects in tumor treatment. |
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