Study On Co-delivery Of Pemetrexed Disodium And Bcl-2 SiRNA By Cationic Liposomes For The Inhibition Of NSCLC

The incidence and mortality of lung cancer are increasing gradually,and 85%of all lung cancers are non-small cell lung cancer.In recent years,the combined application of anticancer drugs and genes has attracted widespread attention.The combination of anti-cancer drugs and siRNA into the same carrier can achieve combined delivery and improve the therapeutic effect.Liposome is a closed phospholipid bilayer vesicle system,which can encapsulate hydrophilic and hydrophobic drugs with good biodegradability and biocompatibility.It has great advantages as a co-carrier of anticancer drugs and siRNA.A positively charged cationic lipid carrier can statically compress the negatively charged siRNA.Meanwhile,in order to better.realize tumor targeted delivery,poly-γ-glutamate(γ-PGA),which can specifically bind to tumor-associated γ-glutamyl transpeptidase on tumor cells,was loaded on the surface of liposomes by electrostatic action to prepare actively targeted coloaded liposomes for drug and gene delivery.The main research methods and results were as follows.1 Preparation and physicochemical characterization of y-PGA-PMX/siRNA-CL complexFirsty,PMX-CL which satisfied follow-up test requirements was prepared by homogenization based on the preliminary study of the research group to prepare PMX cationic liposome(PMX-CL).Then siRNA was compressed by electrostatic incubation to obtain PMX and siRNA co-loaded cationic liposomes(PMX/siRNA-CL).Next,γ-PGA was modified onto the surface of the liposome by electrostatic action to obtain γ-PGA modified PMX and siRNA co-loaded cationic liposome(y-PGA-PMX/siRNA-CL).Lastly,the preliminary stability and in vitro release behavior of the complex were investigated.When the mass ratio of CL/siRNA was 234:1 and the γ-PGA/siRNA mass ratio was 3:1,the particle size of y-PGA-PMX/siRNA-CL complex was(222.07±1.23)run.In this particle size range,the nanocomposites could promote the accumulation of tumor sites by high retention and permeability effect of tumors.At the meantime,it could specifically bind to tumor-associated y-glutamyl transpeptidase on tumor cells to achieve tumor targeting.Then the y-PGA-PMX/siRNA-CL complex could protect siRNA from the degradation of serum,RNA enzyme A and BALF at 24 h to increase stability of siRNA.In vitro release results showed that the cumulative release rate of the γ-PGA-PMX/siRNA-CL in 48 h was(71.49 ±0.78)%.2 In vitro cell evaluation of y-PGA-PMX/siRNA-CL complexHuman A549 lung cancer cells were regarded as research subjects.Firstly,the uptake of Cy3-siRNA in A549 cells was investigated qualitatively and quantitatively by inverted fluorescence microscopy and flow cytometry.Then MTT method was used to investigate the safety of blank CL and γ-PGA-CL carriers.Next,the effects of γ-PGA-PMX/siRNA-CL complex on cell apoptosis and cycle of A549 cells were investigated by flow cytometry.Lastly,real-time quantitative qPCR and Western-Blot assay were used to investigate the effect of complex on Bcl-2 gene and protein expression level in A549 cells.The results of cellular uptake and cytotoxicity assay showed that γ-PGA-CL carrier had better uptake effect and better safety than CL.The results of cellular apoptosis assay and cycle experiment exhibited that the S phase of cells treated with γ-PGA-PMX/siRNA-CL increased significantly to inhibit DNA’s synthesis.And it promoted the apoptosis of A549 cells and inhibited the growth of tumor with better effect.The results of Western-Blot and qPCR exhibited the vector prepared in this study could deliver PMX and siRNA into A549 cells to play obvious gene and protein silencing effect.3 In vivo pharmacodynamics of y-PGA-PMX/siRNA-CL complexThe therapeutic effect of γ-PGA-PMX/siRNA-CL complex was investigated by in vivo pharmacodynamics experiments in BABL/c mice loaded with LLC.Firstly,the distribution of Cy5-siRNA in tumor tissues was investigated by small animal imaging system and frozen section of tumor tissues.Then mice were divided into eight groups randomly with five mice in each group:(1)Normal saline,(2)Free PMX,(3)γ-PGA-CL,(4)Free siRNA,(5)γ-PGANCsiRNA-CL,(6)γ-PGA-PMX-CL,(7)γ-PGA-siRNA-CL,(8)γ-PGA-PMX/siRNA-CL with five injections in two weeks.At a specific time point,weigh mice weight,tumor voulme and tumor weight to investigate the tumor inhibition effect.Finally,HE staining and the organ index of heart,liver,spleen,lung and kidney were used to evaluate the tissue safety of the complex.The tumor accumulation ability investigation proved that the γ-PGA-PMX/siRNA-CL complex could deliver siRNA to the tumor area to play a role and improve the stability of siRNA.Compared with γ-PGA-PMX-CL group and γ-PGA-siRNA-CL group,the tumor volume and tumor weight of mice in γ-PGA-PMX/siRNA-CL group were significantly lower.It could be seen that the combination of drugs and genes could enhance the tumor inhibition and improve the therapeutic effect.And in the process of experiment,the body weight tended to be stable and the results of organ index and HE staining showed no obvious toxicity compared with control,which showed that the complex and carrier had good safety.In summary,this project successfully prepared actively targeted γ-PGA-PMX/siRNA-CL complex.In vivo anti-tumor ability experiments have verified that the complex had stronger tumor inhibition than the single administration group.And the carrier had no obvious toxicity to the organ in vivo,which provides certain ideas for the clinical treatment of NSCLC and has great economic development value and clinical application prospect.