Application Of Bile Acids And Phospholipids Modified With Bile Acids And Their Analogues In Development Of Secretory Phospholipase A₂-responsive Liposomes

Cancer is a serious threat to human health and quality of life.Applications of anticancer drugs often compromise in tumor therapy because of their toxic and side effects and their susceptibility to drug resistance.As a drug delivery system,liposomes can improve the targeting,effectiveness and safety of drugs,and provide a better choice for tumor treatment compared to conventional chemotherapy.However,after reaching the tumor site,liposomes are unable to actively release drugs due to their own stability,resulting in low bioavailability of drugs at tumor sites.A typical example is cisplatin-loaded liposomes（SPI-77）.In clinical phase II,the anti-tumor activity of liposomal cisplatin was much lower than that of free cisplatin,mainly because cisplatin cannot penetrate through liposomal membranes at tumor sites.Therefore,active release mechanisms are urgently needed for liposomes.Here in our study,a new class of liposomes containing bile acids and phospholipids modified by bile acids or their analogues were designed.Secretory phospholipase A₂（s PLA₂）which is overexpressed in tumor sites was used to promote drug release from those novel liposomes.Liposomes containing bile acids were constructed.Liposomes containing chenodeoxycholic acid（CDCA-Lip）,3-alpha-hydroxy-7-keto-5-beta-cholanic acid（7-k CDCA-Lip）,3,7-dketo-5-beta-cholanic acid（d-k CDCA-Lip）and liposomes without any bile acids（Normal-Lip）were prepared by thin film hydration method.The particle size,dispersion coefficient and Zeta potential of liposomes were measured by nano-particle analyzer.The encapsulation efficiency（EE）of oxaliplatin（L-OHP）in liposomes were determined by HPLC.Their responsiveness to s PLA₂from different sources was tested using 5（6）-carboxyl fluorescein（CF）as drug surrogates.The effect of CF release under s PLA₂influence was tested.The stability of the L-OHP-loaded liposomes in the presence or absence of fetal bovine serum（FBS）was investigated.The anti-proliferation activity of L-OHP-loaded liposomes,blank liposomes,and cholic acid compounds on HT-29 and Colo205 human colorectal cancer cells was evaluated by MTT assay.Flow cytometry was used to determine the liposome uptake behavior by two cell lines.The results showed that the liposomes prepared by thin film method showed similar particle sizes（70-90 nm）,good dispersion（PDI<0.1）,and a negative surface charge.The encapsulation efficiency（EE）of oxaliplatin（L-OHP）was about 3.8-4.3%,but the EE of CDCA-Lip was less than 1%.When bee-venom（Bv）s PLA₂was used to induce CF release,liposomes containing bile acids didn’t show better release profiles than Normal-Lip.However,when Colo205 s PLA₂was used,the liposomes containing bile acids released more CF than Normal-Lip.These results indicated that liposomes containing CDCA or d-k CDCA were more responsive to Colo205 s PLA₂than Normal-Lip.The results of cell uptake experiments showed that the addition of bile acids reduced the uptake of liposomes by tumor cells.Encapsulated with L-OHP,liposomes were stable in the presence of different volumes of FBS,indicating as the leakage rates of L-OHP at 48 h was less than 30%in 10%or 50%FBS.7-k CDCA-Lip and d-k CDCA-Lip showed stronger anti-proliferative activity against s PLA₂-secreting Colo205 cells than Normal-Lip,while showing similar antitumor activity to Normal-Lip against non-s PLA₂-secreting HT-29 cells.In addition,we designed and synthesized a novel phospholipid（1-stearoyl-2-（3α-deoxy-5β-cholan-24-oic acid）-sn-glycero-3-phosphocholine,d LCA-PC）modified with 3α-deoxy-lithocholic acid（d LCA）.Liposomes containing d LCA-PC（d LCA-PC-Lip）were constructed and prepared.Their responsiveness to s PLA₂from different sources was tested using CF as indicators.The results showed that the liposomes containing d LCA-PC were more responsive to Bv s PLA₂than liposome constructed with normal lipids,but were not less responsive to Colo205 s PLA₂.Liposomes drug delivery system containing cholic acids compounds or phospholipids modified by cholic acids was successfully constructed.Experiments in CF release and in vitro antitumor activity showed that 7-k CDCA and d-k CDCA improved responsiveness of liposomes to containing s PLA₂for content release.However,the liposomes containing d LCA-PC were only highly responsive to Bv s PLA₂,and could not increase the release of its contents when induced by s PLA₂secreted from tumor cells.