Anti-tumor Study Of Palliative Intratumoral Injection Therapy Through Lecithin-Chitosan Nanoparticles Loading Paclitaxel-Cholesterol Complex And SiRNA

Palliative intratumoral injection is a treatment for patients inoperable or who cannot tolerate systemic chemotherapy for poor general condition,tumor recurrence,etc.Palliative intratumoral injection therapy can control or reduce tumor volume,alleviate complications caused by tumor occupying,improve patient quality of life,prolong patient survival,and thus apllied to treat head and neck squamous cell carcinoma,lung cancer,esophageal cancer,and digestive tract tumors.However,current clinical chemotherapeutic drug dosage form is not suitable for intratumoral injection.The retention time of common chemotherapeutic injection is relatively short with poor cell uptake.The formulations of intratumoral injection were mostly thermosensitive or pH sensitive gels.They would turn into solid once injected into tumor so that the drug distributed inside the solid tumor unevenly and pressure problems followed.It is a hot topic in current research that the synergistic effect of small molecule chemical drugs combined with gene drugs in cancer treatment.Nanosystems loaded with chemical drugs and gene drugs can improve drug resistance channels and increase cellular drug intake to increase chemotherapy efficacy.Gene drugs,such as siRNA,can inhibit the proliferation of tumor cells by silencing specific genes of tumor cells,and play a synergistic anti-tumor effect.On one hand,the dual-loaded nanoparticles can increase the solubility of the small molecule drug and reduce its toxic side effects.On the other hand,siRNA is protected not be degraded by nuclease in plasma,increasing its cellular uptake.Based on the above purposes,this study designed a lecithin-chitosan nanoparticle?LCS_NPs?loaded with paclitaxel cholesterol complex?PTX-CH?and siRNA^VEGF.On the one hand PTX-CH has the anti-tumor effect,on the other hand siRNA^VEGF inhibits the expression of VEGF protein in tumor cells which can reduce the formation of new blood vessels in squamous cell carcinoma tumor tissues.When the siRNA^VEGF/PTX-CH-loaded LCS_NPs used for palliative intratumoral injection of squamous cell carcinoma of the head and neck can play a role in inhibiting tumors synergistically.First,we prepared paclitaxel cholesterol complex?PTX-CH?with PTX and cholesterol?CHO?by solvent evaporation method in order to increase the lipophilicity of PTX and to increase the drug loading of LCS_NPs.PTX-CH was tested by DSC,FTIR,X-ray powder diffraction to make sure the preparation was successful.Secondly,paclitaxel–cholesterol complex loaded lecithin-chitosan nanoparticles?PTX-CH-loaded LCS_NPs?were prepared by solvent-injection method.The particle size of the nanoparticles was 142.83±0.25 nm,the Zeta potential was 13.5±0.2 mV,and the encapsulation efficiency was 92.83±0.29%with the optimal formulation and preparation conditions.The stability experiment proved that the nanoparticles had good stability within 3 days and met the requirements of in vitro and in vivo experiments.Release behavior of PTX from PTX-CH-loaded LCS_NPs showed a pH-sensitive pattern.The microenvironment of tumor tissue is acidic.The drug released from the nanoparticles in tumor sites could be faster and targeted.4T1 cells were used in vitro anti-tumor evaluation of PTX-CH-loaded LCS_NPs.The results of laser confocal and flow cytometry showed that the cell uptake of nanoparticles was significantly higher than that of free drugs.The mechanism of cell uptake of nanoparticles was the energy-dependent caveolae-mediated endocytosis and macropinocytosis in company with the Golgi apparatus.Apoptosis experiments showed that PTX-CH-loaded LCS_NPs could induce more tumor cell apoptosis.In vivo experiments with tumor-bearing mice showed that intratumoral injection of PTX-CH-loaded LCS_NPs was more effective than PTX solution in inhibiting tumor growth and prolonging the survival cycle of mice,and inhibited the distant metastasis of tumor cells in some degree.Based on the research of PTX-CH-loaded LCS_NPs,we selected VEGF siRNA as gene drug to prepare siRNA^VEGF/PTX-CH-loaded LCS_NPs.The average particle size of siRNA^VEGF/PTX-CH-loaded LCS_NPs was 149.63±2.05nm,the Zeta potential was10.70±0.30 mV.The results of gel retardation experiments indicated that PTX-CH-loaded LCS_NPs could immobilize siRNA^VEGF and protect siRNA^VEGF from nuclease degradation in plasma within 12hs.Human squamous cell carcinoma cell line CAL-27 was selected in vitro anti-tumor evaluation of siRNA^VEGF/PTX-CH-loaded LCS_NPs.Laser confocal and flow cytometry experiments showed that LCS_NPs could be a carrier to deliver siRNA to the interior of cells efficiently.The results of Western Blot experiment showed that siRNA^VEGF/PTX-CH-loaded LCS_NPs could effectively silence the expression of VEGF gene in CAL-27 cells and decrease the expression of VEGF protein in cells.SiRNA^VEGF/PTX-CH-LCS_NPs could inhibit CAL-27 more effectively and promote more CAL-27 cells apoptotic.A nude mouse model of Cal-27squamous cell carcinoma was used to investigate the anti-tumor effect of intratumoral injection of siRNA^VEGF/PTX-CH-loaded LCS_NPs in vivo.Intratumoral injection of siRNA^VEGF/PTX-CH-loaded LCS_NPs was more effective in inhibiting of tumor.The results of rtPCR and Western Blot showed that the expression of siRNA^VEGF mRNA and protein in siRNA^VEGF/Blank LCS_NPs group and siRNA^VEGF/PTX-CH-loaded LCS_NPs group were significantly lower than the control group.More apoptotic tumor cells could be observed in TUNEL staining sections of the siRNA^VEGF/PTX-CH-loaded LCS_NPs group.It showed that the anti-tumor effect of siRNA^VEGF/PTX-CH-loaded LCS_NPs is stronger than that of PTX injection.The results of this study indicated that LCS_NPs could be a carrier for both PTX and siRNA.SiRNA^VEGF/PTX-CH-loaded LCS_NPs could inhibit tumor cell growth more effectively in the intratumoral injection treatment of head and neck squamous cell carcinoma tumors and reduce the expression of VEGF protein in tumor cells.