Construction And Performance Of Pirarubicin Targeted Delivery System For Anti-Lymphoma Activity In Vitro

Objective:Lymphoma is a group of hematopoietic system malignancies originating from lymph nodes and lymphoid tissue.With the deepening of human understanding of the nature of lymphoma,the incidence of lymphoma gradually increases with the increase of age,many new research results have appeared in the treatment of lymphoma.Although traditional chemotherapy,targeted therapy,chimeric antigen receptor T cell immunotherapy（CAR-T）and hematopoietic stem cell transplantation have achieved certain efficacy,some lymphoma patients will face disease recurrence and refractory,such patients have poor prognosis and high mortality.Therefore,it is of great significance to carry out relevant studies on relapsed and refractory lymphoma.With the rapid development of medical nanotechnology,there are more and more researches on the organic combination of traditional chemical drugs and nano delivery system.The research on the surface of nano delivery system can be modified with small molecules,and the research on the ligand-receptor binding on the surface of tumor cell membrane,all of them provide a new idea for the treatment of lymphoma.Studies have shown that folic acid receptors are expressed on the cell membrane surface of lymphoma Raji.There are few studies on targeting folic acid receptors on the cell membrane of lymphoma Raji.In addition,in tumor cells,if the nano delivery system cannot slow and control the release of the loaded chemotherapy drugs,the drugs cannot inhibit the proliferation of tumor cells more effectively.Therefore,in this study,lymphoma Raji cells are taken as the research object,and the Pirarubicin（THP）nano delivery system with both targeting and controlled release functions is planned to be constructed.The physical and chemical characterization was investigated to complete the study of anti-lymphoma Raji cell activity in vitro,explore the specific mechanism of action of pirarubicin nano delivery system,providing basic theoretical basis for the treatment of lymphoma by THP-NP-5%FA.Method:（1）The pirarubicin delivery system（THP-NP）was prepared by thin film method and modified by DSPE-PEG_{2 0 0 0}-FA（FA）to obtain the pirarubicin targeted delivery system（THP-NP-5%FA）.THP-NP-5%FA was successfully constructed,and its appearance properties were observed in different segments,the particle size and Zeta potential were inspected by dynamic light scattering（DLS）.（2）The encapsulation efficiency（EE）and drug loading of THP-NP and THP-NP-5%FA were determined by ultraviolet spectrophotometry.（3）Dialysis was used to simulate and evaluate the release behavior of different drugs under different p H conditions in vitro.（4）Flow cytometry was used to detect the uptake rate of THP-NP-5%FA in vitro.（5）The inhibitory effect of THP-NP-5%FA on proliferation of lymphoma Raji cells was tested by CCK-8 method.（6）Cell apoptosis was detected by Annexin V-FITC/PI double staining.All data were verified by three repeated experiments.test was used to analyze the differences between groups,and p<0.05 was considered statistically significant.Results:（1）THP-NP and THP-NP-5%FA were prepared successfully,and their appearance characters were stable.（2）The size of THPNP,THP-NP-5%FA and Zeta potential were in the ideal range.（3）The encapsulation rate of THP-NP was about（92.47±0.04）%,and the drug loading was about（30.82±0.01）%.The encapsulation rate of THP-NP-5%FA was about（91.14±0.02）%,and the drug loading was about（27.39±0.01）%.（4）To simulate drug release under different p H conditions in vitro,the results showed that:THP-NP-5%FA has p H response to drug release.The release of THP-NP and THP-NP-5%FA in the p H 7.4（simulated blood p H）group is slow,while the release of THP-NP and Thp-NP-5%FA in the p H 5.5（simulated endosome p H）group is significantly accelerated.（5）The cell intake rate was higher after the modification of pirarubicin nano-liposomes with molar ratio of 5%FA.（6）Cell proliferation inhibition experiment results show that the drug concentration of 0.5μg/m L can significantly inhibit cell proliferation,and THP-NP-5%FA group was obviously higher than that of control group.（7）Apoptosis was detected by Annexin V-FITC/PI double staining.The apoptosis of THP-NP-5%FA cells was significantly higher than that of the control group.All data were verified by three repeated experiments.Conclusions:（1）THP-NP-5%FA with high EE,high cell intake rate,strong targeting and p H sensitive was developed.（2）THP-NP-5%FA can effectively inhibit the proliferation of lymphoma Raji cells and promote the apoptosis of Raji cells.