Polymer-modified Liposome And Its Intracellular Transport

Modification of liposomes has received much attention for it can reduce sedimentation and drug leakage, enhances the drug absorption, changes the distribution, and so on. The liposomes modified with chitosan or polymethacrylate, Eudragit RL100, was prepared in the present paper and then the physicochemical properties and cellular uptake behavior was investigated, in order to evaluate the potency of its application in hydrophilic drugs intracellular delivery systems.The first part of the paper is investigating the preparation and characters of liposomes coated by chitosan(CSLP). CSLP was obtained by adding chitosan dropwise to liposomes under magnetic stirring. The effect of chitosan/lipids ratio on size, zeta potential, and coating efficiency was investigated, and then the optimum ratio of chitosan/lipids with 1:1 was employed, in which a Z-average diameter of 174.7±20.2nm, Zeta potential of 17.2mV and chitosan coated effeciency on lipds of 18.3% was found. The liposomes membrane was easily destroyed by Triton X100 after chitosan's coated. MTT assay showed that CLSP took on very low cytotoxicity, which was much lower than chitosan.Calcein was employed as the model of hydrophilic small molecular drug to investage the effect of CSLP on the drug release and cellular uptake behavior. It is found that CSLP only released 34% drug within 24h, which was much lower than liposomes. The cell uptake in Hep G2 showed that calcein solution poorly penetrated into the cells, while liposomes improved the intracellular uptake, and then CSLP improved further. Sodium azide and chlorpromazine could reduce the intracellular uptake, while indomethacin and cytochalasin B did not influence the uptake, which suggested that the mechanism of internalization was involved predominantly in clathrin-mediated endocytosis.The feasibility of CSLP as gene vehicles was investigated with antisense oligodeoxynucleotides(ODNs) as gene model. Agarose gel electrophoresis was employed to evaluate the loading efficiency of CSLP for ODNs, from which one could see ODNs was completely combined to CSLP when the mass ratio of total lipids:ASON was more than 50:1. ODNs alone poorly penetrate into cells, and it could not internalize efficiently too even under the help of liposomes. While chitosan could improve the intracellular uptake, and the CSLP improved further. When the ODNs was 1μg/mL, the intracellular uptake was found to increase with the increasing concentration of CSLP at first, then it took on a decreased uptake with the increasing CSLP above 100μg/mL, which showed that the uptake of complexes via a saturable mechansim. The internalization process was also studied as a function of time, and found that uptake reached the equilibration at 100min.In the second part of the paper, the physicochemical properties and feasibility as drug vehicles of liposomes modified by Eudragit RL100 (ERLP) was investigated. The size and Zeta potential of ERLP was determined by dynamic light scattering technology, and it was found that ERLP prepared by solvent(ethanol)-nonsolvent(water) method in the polyacrylate/lipids mass ratio of 1:1 took on a satisfactory properties with Z-average diameter of 189.5nm and Zeta potential of +40.1 mV. The accelerated test by centrifugalization showed the good physical stability of ERLP, while the membrane stability of ERLP determined by Triton X100 was poorer than liposomes. The cytotoxicity of ERLP was much lower than Eudragit RL100, in that the viability of COS-7, Hep G2 and L929 cells was above 75% even in the high concentration of 1mg/mL.Subsequenly, the potency of ERLP as doxorubicin hydrochloride(DOX) vehicles was investigated. DOX-ERLP was prepared by ammonium sulfate transmembrane gradients method, and the entrapment efficiency determined by ultracentrifugation was 44.34%. DOX-ERLP only released 25% loaded drug within 72h, which was much lower than liposomes. The intracellular uptake of DOX-ERLP in MCF-7 and MCF-7/adr was assayed by HPLC method, in which it is foud that free DOX solution cound penetrate into cells efficiently, while DOX encapsulated in liposomes showed lower intracellular uptake, but DOX-ERLP improved the uptake obviously and changed its intracellular fate. All of the DOX formulation took on less penetration into MCF-7/adr than MCF-7, and reached the uptake equilibration within 1h in MCF-7/adr, while it is not found the uptake equilibration within 12h in MCF-7 cells. The cytotoxicity of various DOX formulation to MCF-7 or MCF-7/adr cells was assayed by MTT method, from which one could see that DOX solution and DOX liposomes showed the similar cytotoxicity although the uptake of DOX liposomes was much lower than DOX solution in MCF-7 cells, while DOX-ERLP showed higher cytotoxicity than DOX solution and DOX liposomes in both MCF-7 and MCF-7/adr cells. Furthermore, to evaluate the antitumor effect in vivo, a solid tumor model was built via subcutaneous injection of H22 cells into ICR mice, after 4 days'normal feeding various DOX formulation was injected into tail vein every other day, and the size of tumor was determined at the same time. So it is found that the volumn of tumor from the solution and liposomes group was lower than the normal saline group, and the DOX-ERLP showed the lowest tumor volumn, which suggested ERLP could improve the antitumor activities obviously.At last, the feasibility of ERLP as the vehicles of ODNs was also investigated. Agarose gel electrophoresis showed that ODNs could be combined to ERLP completely when the mass ratio of ERLP to ODNs was above 16:1. ERLP could improve the intracellular uptake of ODNs significantly. When the ODNs was 0.5μg/mL, The intracellular ODNs increased with the increase of ERLP under 8μg/mL, and then it showed reduced uptake after the ERLP increased over 8μg/mL, suggested a saturable process was involved in the ERLP internalization. The cellular uptake experiments with endocytic inhibitors suggested that clathrin-mediated endocytosis was the major pathways, while macropinocytosis and passive diffusion after membran destabilization was also involved in the internalization. ODNs-ERLP was found in nucleus partly after 4h's incubation via a fluorescent labelling technology of marked nucleus with Hoechst 33342. But the antitumor effect in vitro assayed by MTT showed that the survivin or telomerase ODNs delviery by ERLP did not efficiently inhibit the proliferation for Hep G2 cells.