Preparation And In Vitro Evaluation Of Paclitaxel-loaded K237-long-circulating Targeted Liposome

Paclitaxel is a kind of diterpenes compound isolated from the bark of thePacific yew tree. PTX is a wide spectrum anti-cancer drug against cancerssuch as breast, ovarian, non-small cell lung cancer, melanoma, head and neckcancer. It is known that dehydrated ethanol and polyethoxylated castor oil candissolve PTX (Cremophor EL), TaxoL, which is widely used in clinical.However, TaxoL may result in severe side effects generally because oflacking in targeting. As a new anti-cancer drug delivery system, liposomesdemonstrates a lot of properties, such as targeting, low toxicity and controlledrelease. Liposomes pegylated can hinder the absorption of the surface ofliposomes with plasma, which could reduce the uptake of liposomes byreticulpendotheial system and prolong the cycletime, through the surface ofliposomes modified with ligand can cpecifically target to cells and reduce thedamage to normal cells.Modified with ligand modified, liposomes can specifically target to cellsand reduce side effects.Angiogenesis plays a critical role in tumor progress, besides tumorgrowth and metastasis. VEGF (vascular endothelial growth factors) is acritical factor for angiogenesis, and plays an essential role in bothphysiological and pathological angiogenesis. The VEGFR-2or KDR canspecially mediate various biological activities of VEGF-2related toproliferation of vascular endothelial cells and angiogenesis. Taken the high expression of KDR on the tumor cells surface into consideration, the bindingof K237peptide (HTMYYHHYQHHL) to KDR with highly affinity andspeciality may functionally disrupt the interaction between VEGF and theKDR. In this study, K237peptide is used as the ligand of modified liposomes,which target to tumor cells and vascular endothelial cells.In the study, K237-PEG-DSPE synthesized by the N-terminalPEGylation technique, soybean lecithin and cholesterol were used asliposomes materials to prepare paclitaxel-loaded K237-long-circulatingtargeted liposomes (K237-PTX-Lip). K237-PTX-Lip was prepared bythin-film dispersion method. Encapsulation efficiency and particle size wasregarded as detecting indexes to optimize the formulation and preparationprocess of K237-PTX-Lip. Optimized formulation was displayed as follows:the ratio of cholesterol to soybean lecithin1:10, the ratio of paclitaxel to lipidmaterial1:50, lipid material concentration of4.0%, DSPE-PEG-K2375mol%.Encapsulation efficiency of K237-PTX-Lipand PTX-Lip was93.01±0.89%and97.95±1.32%.The average size of K237-PTX-Lip and PTX-Lip was78.2±6.1nm and54.6±4.7nm, zate potential was-38.7mv and-40.9mvrespectively.The targeting effects of K237-PTX-Lip and fluorescein liposomes toSKOV-3and HUVEC were evaluated by MTT, confocal microscopy imagingsystem and flow cytometry respectively. It was shown that K237-PTX-Lipstarted to display cytotoxicity at1.0nmol L-1with HUVEC and SKOV-3cellsincubated with different concentration of PTX preparation for72h, whileTaxoL and PTX-Lip did not show cytotoxicity in SKOV-3. Further more,K237-PTX-Lip displayed significantly higher cytotoxicity than TaxoL andPTX-Lip in SKOV-3with the increase of PTX concentration. K237-PTX-Lipdisplay dramatically higher cytotoxicity on HUVEC than TaxoL andPTX-Lip at PTX concentration above3nmol L-1. The inhibition rate on HUVEC was30%after incubated72h with5nmol L-1of PTX while it didnot shown significant cytotoxicity on SKOV-3. It was concluded thatK237-PTX-Lip showed proliferation-resistant ability on HUVEC belownon-cytotoxicity concentration. The half maximal inhibiting concentration(IC50) of K237-PTX-Lip on SKOV-3and HUVEC cells was25.06nmol L-1and8.81nmol L-1, which decreased to0.336fold on SKOV-3and0.695foldon HUVEC. The results showed that K237-PTX-Lip had greater cytotoxicitycompared with common liposomes.According to Confocal microscopy imaging system study, SKOV-3andHUVEC nuclear chromatin were highly condensed and edged after incubatedwith PTX-Lip for24h, while SKOV-3and HUVEC nuclear chromatindegraded into pieces and many apoptotic bodies observed after incubated withK237-PTX-Lip for24h. The results indicated that K237-PTX-Lip uptaken bySKOV-3and HUVEC was enhanced significantly compared with commonliposomes and this uptaken enhancement could be blocked by free K237. Theresults of flow cytometry experiments also showed that the uptake level of theK237-targeted liposomes waw higher than that of the common liposomes, andthe uptake could be blocked by an excess amount of free K237.In vitro cytological evaluation results showed that liposomes modified withK237peptide had a good cellular targeting, and the uptaking of thoseliposomes by SKOV-3and HUVEC cells could be enhanced.