Preparation,in Vitro/in Vivo Anti-tumor Activity And Preliminary Pharmacokinetics Of Gambogenic Acid-loaded Pegylated Liposomes

Gambogenic acid?GNA?,isolated from gamboge,is one of the major bioactive caged xanthones.During the past few decades,GNA were identified as bioactive compounds that possessed diverse anti-tumor activities both in vitro and in vivo.However,The excessive irritation to the blood vessel,short elimination half-life and poor aqueous solubility restricted its clinical application.Objective:In this study,Gambogenic acid-loaded PEGylated liposomes?GNA-PEG-LPs?were developed to reduce toxicity,prolong the half-life and enhance anticancer efficacy both in vitro and in vivo.Methods:?1?GNA-PEG-LPs were prepared by ethanol injection method and optimized by single factor test and orthogonal experiment.The encapsulation efficiency,drug loading,morphology,Zeta potential,DSC analysis,storage stability,in vitro release of GNA-PEG-LPs were evaluated.?2?Effects of GNA-PEG-LPs on lung?A549?,stomach?SGC-7901?and liver?HepG2?cancer cells were investigated by MTT assay,observation of apoptotic morphological changes,flow-cytometric analysis of apoptosis and Western blot analysis of the molecular mechanism.?3?The blood vessel irritation and hemolysis rate were determined to evaluate the safety of GNA-PEG-LPs.?4?LLC tumor-bearing C57BL/6 mouse model was adopted to evaluated the antitumor efficacy in vivo.Immunohistochemistry experiment was performed to examine the changes of apoptosis-related proteins.HE staining was conducted to evaluate the safety of GNA-PEG-LPs.?5?The pharmacokinetic parameters of GNA and GNA-PEG-LPs were compared after a single dose to SD rats.Results:?1?The encapsulation efficiency,drug loading and zeta potential of GNA-PEG-LPs were 88.13±1.31%,3.72±0.04%,-22.10±0.20 mV,respectively.The morphology observed by TEM revealed a uniform and spherical shape.The average particle size of GNA-PEG-LPs was 90.13±0.16 nm and their polydispersity index?PDI?was 0.092±0.003.DSC analysis suggested that GNA dispersed homogeneously into liposomes and no crystallization of GNA occurred during the process for the preparation of liposomes.GNA-PEG-LPs could maintain the integral stability within four weeks at 4?.The in vitro release study revealed that GNA-PEG-LPs exhibited a slow drug release feature.?2?Blank PEGylated liposomes demonstrated no obvious cytotoxicity,indicating good safety of blank liposomes to cells.Compared with GNA solution,GNA-PEG-LPs possessed more obvious inhibiting effect and promoting apoptosis of A549,SGC-7901 and HepG2 cells.Compared with free GNA group,there was enhanced up-regulation of Bax,cleaved caspase-3 protein and down-regulation of Bcl-2 protein in GNA-PEG-LPs,indicating that GNA-PEG-LPs is likely to induce apoptosis through the mitochondrial pathway.?3?The vascular irritation of GNA could be reduced by liposomal encapsulation.There was no hemolysis phenomenon when the total lipid concentration was less than 2.28mg/mL.?4?In vivo antitumor experiment indicated a statistically improved antitumor effect of GNA-PEG-LPs compared to the free drug group.Immunohistochemistry analysis suggested that GNA upregulated the expression of Bax and caspase-3proteins and downregulated the expression of Bcl-2 protein in tumor tissues of LLC tumor-bearing mice,and these changes were augmented by GNA-PEG-LPs.Major organs of the mice,including the heart,liver,spleen,lung,and kidney,did not show any obvious histopathological abnormities or lesions in free GNA group or GNA-PEG-LPs group,suggesting no adverse effect on these organs upon treatment.The therapy that was used in this study?2mg/kg every 2 days?displayed a lower toxicity,suggesting the safety of GNA-PEG-LPs.?5?The HPLC-UV method for the determination of GNA in SD rats was established and validated.Pharmacokinetic study showed that the t_1/2/2 and the AUC of GNA-PEG-LPs were higher than GNA solution approximately 2.84-fold and 2.97-fold,respectively.Conclusion:PEGylated liposomes could be a potential nanocarrier to prolong half-life,reduce toxicity and enhance anticancer efficacy both in vitro and in vivo,thus representing an effective and safe nanocarrier for GNA delivery in cancer treatment.