GA-PEI-PLCA Based Liver-targeting Nanoparticles As Co-delivery System For Chemotherapeutic Drugs And CpG ODN

In recent years, a large number of studies have reported that the cationic nano-carriers could be used as co-delivery systems for chemical drugs and genes, to show combination of chemotherapy and gene therapy. Based on our former studies, we further studied the capability of GA-PEI-PLGA nanoparticles to deliver CpG ODN and chemotherapeutic drugs stimultaneously. For this purpose, glycyrrhetinic acid modified PEI-PLGA copolymer(GA-PEI-PLGA) was successfully synthesized in the presence of EDC·HCl and NHS, in which GA acted as the liver targeting ligand. TNBS method was used to determine the substitution degrees of PLGA and GA in GA-PEI-PLGA, which were(11.48±0.48)% and(19.26±1.89)%, respectively. Being different from the obvious buffering capacity of PEI in the acid-base titration process, the protonation capabilities of PEI-PLGA and GA-PEI-PLGA were retained. The plasma levels of ALT, AST and TP were determined to reflect liver function and CRE was assessed to identify renal toxicity. Histologic analysis for liver toxicity and nephrotoxicity was performed by observing the morphological changes in the organs. The results showed that both PEI-PLGA nanoparticles and GA-PEI-PLGA nanoparticles had an impact on the liver function to a certain extent after multiple intravenous injections to rats. However, GA-PEI-PLGA nanoparticles exhibited much less toxic to liver than PEI-PLGA nanoparticles. The CRE showed no significant difference, implying that both PEI-PLGA and GA-PEI-PLGA nanoparticles had no impact on renal function.To investigate the co-delivery capability and its property of GA-PEI-PLGA nanoparticles, 10-hydroxycamptothecin(HCPT) and CpG ODN were chosen as model drugs. HCPT is a promising anti-cancer agent which suppresses the activity of topoisomeraseⅠand inhibits the relegation of the cleaved DNA strand. And CpG ODN has been used as an immune-stimulator to induce Th1 biasing cytokines and consequently suppress the growth of cancer cells. HCPT-loaded GA-PEI-PLGA nanoparticles were successfully prepared by emulsion-solvent evaporation method. The formulation and process were optimized by orthogonal design test with entrapment efficiency and drug loading efficiency as indexes. The optimal process was as follows: drug/GA-PEI-PLGA ratio of 3:10, oil phase/water phase ratio of 1:9, sonicating power of 60% and sonicating time of 15 min. The entrapment efficiency and drug loading efficiency of HCPT-loaded GA-PEI-PLGA nanoparticles were(87.52±3.91) % and(20.10±4.72) %, respectively. The in-vitro release behavior of HCPT-loaded GA-PEI-PLGA nanoparticles was investigated by dynamic dialysis method and compared with free HCPT. The results showed that HCPT-loaded GA-PEI-PLGA nanoparticles exhibited sustained release and the release profile conformed to Higuchi equation. Furthermore, the results of gel retardation assay showed that CpG ODN could be totally condensed into HCPT/GA-PEI-PLGA nanoparticles at the polymer/CpG ODN ratio of 10:1(w/w). CpG/HCPT/GPP nanoparticles were spherical with uniform size distribution. The average particle size was(145. 7±13.5) nm and the zeta potential was(23.11±2.29) mV.The inhibitory effect of HCPT and CpG ODN loaded GA-PEI-PLGA nanoparticles on the growth of HepG2 cells and BEL-7402 cells was investigated by MTT assay and compared with SPC-A-1 cells. The results showed that both HCPT/GA-PEI-PLGA nanoparticles and HCPT/PEI-PLGA nanoparticles exhibited greater inhibitory effect than free HCPT. The inhibitory effect of HCPT/GA-PEI-PLGA and CpG/HCPT/GA-PEI-PLGA nanoparticles on HepG2 cells and BEL-7402 cells were significantly greater than HCPT/PEI-PLGA and CpG/HCPT/PEI-PLGA nanoparticles respectively, which was not observed in SPC-A-1 cells, indicating the liver-targeting effect of GA-PEI-PLGA nanoparticles. Although CpG ODN had no direct inhibitory effect on these tumor cells, it could enhance the sensitivity of tumor cells to chemotherapeutic drugs. As a result, the inhibitory effect of CpG/HCPT/GA-PEI-PLGA nanoparticles and CpG/HCPT/PEI-PLGA nanoparticles were greater than HCPT/GA-PEI-PLGA and HCPT/PEI-PLGA nanoparticles, respectively. The immune activity of the drug-loading nanoparticles was investigated by RAW264.7 cells, a kind of mouse macrophage cell. Since HCPT inhibited the growth of RAW264.7 cells, the secretion of TNF-α stimulated by CpG/HCPT/GA-PEI-PLGA nanoparticles was less than that of CpG/GA-PEI-PLGA nanoparticles. However, the immune activity of CpG/HCPT/GA-PEI-PLGA nanoparticle was well retained.After HCPT/GA-PEI-PLGA nanoparticles were administered to Kunming mice by intravenous injection, the concentration of HCPT in liver was significantly higher than other tissues including plasma, heart, spleen, lung and kidney. And HCPT/GA-PEI-PLGA nanoparticles showed higher concentration of HCPT in liver than HCPT/PEI-PLGA nanoparticles and HCPT solution, validating the liver-targeting effect of GA-PEI-PLGA nanoparticles. The high concentration of HCPT/GA-PEI-PLGA nanoparticles in liver could prolong the effect of HCPT as well as reduce the side effect of HCPT on other tissues. These finding confirmed that GA-PEI-PLGA nanoparticle was an appropriate drug carrier for the targeting therapy of liver cancer.