Studies On Folate Receptor-targeted Imatinib Liposomes And Doxorubicin/Imatinib Combination Liposomes

Platelet derived growth factor receptor (PDGFR), a promising therapeutic target of imatinib is frequently expressed in cervical cancer and plays an important role in tumor pathogenesis initiation and progression. In-vitro studies have demonstrated that imatinib can prevent PDGFR activation, induce cervical cancer cells apoptosis and inhibit cervical cancer cells proliferation. But when the drug was used in clinical treatment, the results were discouraging that minimal therapeutic activity has been shown. The poor therapeutic efficacy may be due to insufficient drug delivery to the tumor cells and the plasma protein binding. Liposome-based formulations with tumor-active targeted ligands could improve the tumor cell selectivity and uptake efficiency of liposomes. Therefore, the purpose of this study aimed at exploring a novel folate receptor-targeted delivery system via imatinib-loaded liposomes to enhance drug delivery to tumor cells and antitumor potency of imatinib through the combination of active targeting and macular targeting.Furthermore, it had been reported that imatinib could increase the sensitivity of tumor cells for classical chemotherapy. The combination of imatinib with traditional antitumor drugs could produce synergistic and additive effects in a wide range of tumor types. However, the antitumor activity of drug combinations can be significantly dependent on the molar ratio of the combined drugs and it is very difficult to achieve upon systemic administration, due to different pharmacokinetic profile of every drug in the combination. So we try to co-encapsulate imatinib (IM) and doxorubicin (DOX) in FR-targeted liposomes to maintain the appropriate drug ratio from the site of administration until it reaches the tumor cells and produce synergistic effect of the combined drugs.In chapter1, we evaluate physicochemical properties of IM using scifinder database. IM is a weak base with high oil-water partition coefficient and a certain degree of lipophilicity. The solubility of IM in water decreases with the increasing pH value. The methodology of determining IM in liposome by HPLC was established. IM liposomes were prepared by the ammonium sulfate gradient method and the effects of factors on encapsulation efficiency were investigated by single-factor test. Under the optimal condition:the ratio of HSPC and CHOL of55/40, the concentration of ammonium sulfate of300mM, the total lipids concentration of35mg/mL, and the drug-to-total lipids ratio of1/5, the mean encapsulation efficiency of FR-targeted imatinb liposomes (FLI) could be reached to90.7±0.91%.In chapter2, the physicochemical properties of FLI were investigated. Appropriate mean particle size (143.5nm) and zeta potential (-15.97mV) were obtained for FLI. The drug release profile showed minimal imatinib leakage (＜5%) in PBS at pH=7.4within72h incubation, while more leakage (＞25%) was observed in PBS at pH=5.5. It indicates that these liposomes have possessed certain degree of pH sensitivity. During storage at4℃for30days, no significant variation in liposome size and drug retention was detected. It appears that both liposomal formulations showed excellent stability during the storage and no drug precipitation or liposome aggregation was observed.In chapter3, the uptake of FR-targeted calcein fluorescent liposomes was studied. The results of fluorescence microscope and flow cytometry showed that FR-targeted liposomes could be more efficiently taken up by the FR-positive cells compared to the non-targeted liposomes, and the folate receptor mediated endocytosis could be blocked by free folic acid. Cytotoxicity assays demonstrated that the FR-targeted imatinib liposomes promoted a6-fold IC50reduction on the non-targeted imatinib liposomes from910to150uM. In addition, FR-targeted imatinib liposomes enhanced Hela cells apoptosis in vitro compared to the non-targeted imatinib liposomes.In chapter4, pharmacokinetic properties of imatinib-loaded liposomes were investigated. The methodology of determining IM in plasma by HPLC was established. The results showed that free imatinib showed rapid clearance from the plasm and both liposomal imatinib formulations showed much longer circulation time than the free imatinib did. Compared with free imatinib, liposomal imatinib can achieve much higher plasma concentration versus time, larger AUC, longer half time and mean retention time, and lower clearance rate from the body. Both targeted and non-targeted liposomes exhibited long circulation properties in Kunming mice.In chapter5, we investigated the effect of combination treatment with IM and DOX on Hela cells. The combination effect was quantitatively determined using the method of median effect principle and the combination index (CI). Both DOX and IM induced cell death in a dose-dependent manner. The IC50value in the Hela cells for DOX was0.55ug/mL and for IM it was14.24ug/mL. When DOX and IM were used concurrently according the constant ratio of1/15, synergistic inhibition of cell growth was achieved (CI＜1) and the IC50was0.27ug/mL.In chapter6, the preparation of liposomes co-encapsulated DOX and IM was investigated. The methodology of determining DOX in liposomes by HPLC was established. Both the FR-targeted liposomes co-encapsulated DOX and IM (FLDI) and the FR-targeted pH-sensitive liposomes co-encapsulated DOX and IM (FPLDI) were prepared by the ammonium sulfate gradient method and the effects of factors on encapsulation efficiency and the ratio of two drugs encapsulated in liposomes were investigated by single-factor test. FLDI can acquire a much better mean encapsulation efficiency and more appropriate drug ratio when the drug-to-lipid ratio was1/8and two drugs were loaded into liposomes at the same time. Under conditions:the ratio of DOPE and CHEMS of55/40, the pH value of ammonium sulfate of8.5, the total lipids concentration of55mg/mL, and the drug-to-total lipids ratio of1/8, the much better mean encapsulation efficiency and more appropriate drug ratio of FPLDI could be reached.In chapter7, the physicochemical properties of FLDI and FPLDI were investigated. The particle size and Zeta potential of FLDI were145.6±1.26nm and-16.41±1.73mv, while the particle size and Zeta potential of FPLDI were170.2±2.47nm and-36.45±2.56mv. The drug release results showed that both FLDI and FPLDI exhibited minimal IM and DOX leakage in pH7.4PBS within24h of incubation, and more than95%of the encapsulated IM and DOX were still retained in liposomes. At pH5.5in PBS, the release of IM and DOX from FLDI improved slightly to11.2%and19.3%, while the release of DOX from FLDI is less than FR-targeted DOX lipsosmes (FLD). FPLDI showed stronger pH sensitivity at acidic pH and the release of two drugs from FPLDI both significantly increased to90%and were kept synchronized. And furthermore, the mean diameter of FPLDI was significantly increased at low pH, indicating vesicle aggregation and membrane fusion. During storage at4℃for30days, there is no significant variation in liposome size and drug retention detected in both types of liposomes.In chapter8, the in vitro growth inhibition activity and cellular uptake efficiency of FLDI and FPLDI were investigated by a cervical cancer Hela cell model. The results of fluorescence microscope and flow cytometry showed that both FLDI and FPLDI could be efficiently uptake by Hela cells compared to the non-targeted liposomes, and the folate receptor mediated endocytosis could be blocked by free folic acid. From the MTT assays, the IC50of FPLDI, FPLD, FLDI, and FLD were2.89±0.34.5.22±0.56、14.8±1.53、9.38±0.83uM respectively. Compared to FPLD, FPLDI can induce tumor cell death more efficiently through the combination of DOX and IM. However, the antitumor effect of FLDI was weaker than FLD, because the precipitation of IM in the internal water phase of FLDI became an obstacle to the release of DOX.In conclusion, FLI could specifically target to Hela cells and thus induced specific and efficient death and apoptosis of Hela cells. This new therapeutic approach may be useful for treatment of cervix cancer with an enhanced therapeutic potential. And the FR-targeted pH-sensitive liposomes co-encapsulated DOX and IM is also a promising strategy in therapy of cervical carcinoma through the synergistic effect of DOX and IM.