Study On Long - Circulating Liposomes Of Folic Acid Modified By

Isoalantolactone (IAL), a sesquiterpene lactones, was one of the mainly active ingredient of the extract from Inula helenium L.. Pharmacological investigations showed IAL possessed the effects of anticancer, significant anti-inflammatory and hepatoprotective activity similar to that of silymarin, anti-dematophytic and antifungal activity.However, the clinical application was restricted by its insolubility and toxicity.As the novel drug delivery carrier, liposomes could reduce untoward effects of chemotherapy drugs, increase the therapeutic index (TI), and improve clinical effects. However, the poor stability during storage and the short half-life in the blood restricted the application and development of liposomes. The domestic and international recent research indicated that the surface-modified of liposomes could enhance the stablity on the storage and in vivo. Generally, PEG was used to modified the surface of liposomes to reduce phagocytosis, prolonged the half-life in the blood and increase the concentration of drug in tumor site. Nevertheless, recent research showed PEG-modified liposomes induced accelerated blood clearance (ABC) effect by multi-clinical application.In this paper, IAL liposomes were prepared and modified with folic acid,PEG and HSA in order to improve its half-time and targeting in vivo. The main research contents and results were as follows:1. PEG-bis-amine synthesis and characterizationPEG-bis-amine was respectively prepared by the azide reduction method and the benzene sulfonyl chloride-amine substitution method. The former yield was48.2%and the latter was54.4%. The melting ranges were respectively48.4～51.1℃and44.4～53.6℃, and the control sample melting range was48.7～50.5℃. The former product was more pure than the latter. Therefore, the synthesis of the former has higher efficiency than the latter.The FTIR spectrum shows the stretching vibration and bending vibration of PEG-bis-amine N-H bond in3426cm-1,1577cm-1. A strong absorption peak was appeared in1109cm-1for the characteristic peaks of C-O-C.’H-NMR (CDCl3) spectrum shows the peak of δ=2.86ppm was a-methylene proton peak with the amine, and the peak of δ=3.48ppm was methylene proton peaks with oxygen, and the peak of δ=3.55-3.63was ethylene oxide chain proton peak. The above data suggested that PEG-bis-amine was prepared successfully.2. Folate-PEG-DSPE synthesis and characterizationFolate-PEG-DSPE was synthesized with folic acid, PEG-bis-amine, and DSPE with three-stepped reaction. The γ-carboxyl of folic acid actived by NHS was reacted with PEG-bis-amine to synthesize Folate-PEG-NH2. Then the N-succinyl-DSPE was sythesized by the hydroxyl activation of DSPE with succinic anhydride containing imidazole groups. Finally the above two compounds were mixed to generate Folate-PEG-DSPE.The lyophilized product melting range was187.8～206.6℃. The FTIR spectrum showed there was a strong absorption peak at1675cm-1, which indicated the amide bond between PEG-bis-amine and Folate or SUC-DSPE respectively. In addition, the peaks at1608cm-1and at1574cm-1were the absorption peaks of the benzene group of the folic acid and showed that compounds contained folic acid. The peak at1721cm-1showed the carboxyl absorption peak of two fatty acid chains of DSPE. Furthermore, the NMR also showed that the peaks of δ=1.26ppm and δ=1.59ppm were-CH3(CH2)14and-CH3-(CH2)14-CH2-proton peaks, which were the fatty acid chains of DSPE respectively. The peak of δ=2.40ppm showed the beta, gamma methylene groups of the glutamate in folic acid molecules. The peak of δ=3.75ppm showed the ethylene oxide chain of PEG. Hence, this compound contained folic acid, DSPE and PEG components, which should be the Folate-PEG-DSPE. The final yield of Folate-PEG-DSPE was19.7%.3. Preparation and characterization of IAL long circulating liposomes with folic acidThe orthogonal design was used to optimized the formula and technology of preparation of IAL long circulating liposomes with folic acid. Blank traditional liposome, f-PEG-IAL liposomes and f-HSA-PEG-IAL liposome were prepared by film evaporation method and their spherical shapes were observed by the electron microscopy. The "shell" formed by PEG and HSA was observed clearly with the diameter136.27nm and the zeta potential-21.54mV, which indicated the HSA increased mutual repulsion between particles and enhanced the stability of liposomes.4. IAL long circulating liposomes with folic acid releaseing in vitro and cell uptaking testThe traditional IAL liposomes, f-PEG-IAL liposomes and f-HSA-PEG-IAL liposomes released IAL in vitro by matrix diffusion type.The cumulative leaching rates during24h were only42.35%,12.45%,14.23%and87.10%,29.38%and48.40%during120h respectively. The drug release process in vitro were accords with Higuchi equation:y=ax1/2.The f-PEG and f-HSA-PEG liposmes uptaked by MPM were slower than that of the traditional liposomes. The phagocytosis rate was IAL traditional liposome> f-PEG-IAL liposome> f-HSA-PEG-IAL liposomes successively. In addition, the blank plasma of mice could increase the phagocytosis rate of MPM for liposomes. f-HSA-PEG-IAL liposomes had higher resistance to plasma opsonic protein binding, and then lower the ability of MPM phagocytosis.5. The pharmacokinetic study of IAL long circulating liposomes with folic acidIAL traditional liposome, f-PEG-IAL liposome and f-HSA-PEG-IAL liposome were injected into body of rats by the rat tail vein. After administration of8h, IAL concentrations were347ng/mL,999ng/mL and1171ng/mL respectively. Untill12h, IAL concentration of traditional IAL liposome group below the detecting limitation and after24h, IAL concentration in blood of f-PEG-IAL and f-HSA-PEG-IAL liposomes groups were761ng/mL and851ng/mL. The concentration of f-HSA-PEG-IAL liposomes in blood decreased slower than that of f-PEG-IAL liposomes. It indicated that HSA as an opsonin proteins could further decreased the blood clearance of liposomes.5days later, ABC phenomenon was observed by a second injection of liposomes with PEG. The concentration-time curve was fitted to one compartment model with the change of its pharmacokinetic parameters. When the first injections were IAL(PBS), IAL traditional liposomes, f-PEG-IAL liposomes and f-HSA-PEG-IAL liposome, the half-life (t1/2) of f-HSA-PEG-IAL liposome were1.06,2.76,2.74and1.87fold and AUC0→12value of1.19,2.85,2.18and1.27fold than that of f-PEG-IAL liposome. These results showed that the IAL liposomes containing HSA were more stable than liposomes with PEG only by repetitious injection of liposome. Therefore, IAL liposomes modified by HSA and PEG were more difficult for the MPS phagocytosis than those only with PEG, which had higher stability and the longer retention time in vivo with high bioavailability.6. The anti-tumor ability in vitro and the inhibition rate of IAL long circulating liposomes in tumor bearing miceAccording to the results of MTT, the proliferations of HeLa, MCF-7and PANC-1tumor cells had obviously inhibitied by f-PEG-IAL and f-HSA-PEG-IAL liposomes. The concentration-time curve of IAL was fitted to logarithm regression curve. The inhibition rates for above three types of tumor cells were:MCF-7> HeLa> PANC-1. The data in the experiments showed the inhibition rate of liposomes with folic acid were higher than those without folic acid. Accordingly, folic acid could effectively target to tumors. For the f-PEG-IAL liposomes, the IC50of HeLa, MCF-7and PANC-1cells were29.41μmol/L,9.24μmol/L and21.14μmol/L; for the f-HSA-PEG-IAL liposomes was48.87μM,20.66μmol/L and29.67μmol/L respectively. These values were smaller than that of IAL dispersant(>100μ M), and also found the uptake of liposomes for tumor cells was reduced by HSA steric hindrance effect. Annex in V-FITC/PI double-labeled flow cytometry (FCM) results showed that f-HSA-PEG-IAL liposomes induced the apoptosis rates of MCF-7were39.2±2.93%,66.8±4.37%,85.8±3.32%and90.9±3.86%during the24h,36h,48h and72h cultivation time at the100μmol/L and the control group (48h) was0.21±0.03%. The tumor cell apoptosis rate increased with the prolongation of culture time and the early apoptosis gradually developed to late apoptosis and necrosis.That may be one of the anticancer mechanism of f-HSA-PEG-IAL liposomes.In this paper, the MCF-7cells in nude mice was used as the model of breast cancer by the subcutaneous implant method. The tumor tissues growth rate was reduced by the administration of IAL liposomes. The tumor inhibition rate of f-HSA-PEG-IAL liposome, f-PEG-IAL liposome and IAL traditional liposome were45.8%,71.4%and84.8%respectively.For the toxicity study, the body weight of nude mice increased after administration, which indicated the the affinity of IAL liposomes for tumor cells was higher than normal cells, and folic acid enhenced these characteristics. The morphological results showed that10mg/kg f-HSA-PEG-IAL could effectively inhibit the growth of tumor cells.