Preparation And Evaluation Of Paclitaxel-loaded PEGylated Immunoliposome

Despite its strong anti-tumor activity against ovarian cancer, breast cancer, non-small cell lung cancer and so on, paclitaxel (Taxol^?) has limited clinical applications due to its low aqueous solubility and hypersensitivity caused by Cremophor^? EL and ethanol which is the vehicle used in the current commercial product. In an attempt to develop a pharmaceutically acceptable formulation that could replace Taxol^?, a paclitaxel incorporated liposome has been constructed to improve solubility and physicochemical stability. The effect of various components of the liposome, including cholesterol and lipid, on the solubility and entrapment efficiency (EE) of paclitaxel was systematically investigated. The results showed that 3% (v/v) of Tween 80 in the hydration medium of liposome significantly increased the solubility (up to 3.39 mg/mL) in the liposome formulation composed of 10% (w/v) of S₁₀₀PC with cholesterol (cholesterol-to-lipid molar ratio=10:90). The addition of 5% (v/v) of PEG 400 can further increase the entrapment efficiency of liposome to 89.26%, as well as the paclitaxel content in the liposome. When sucrose (sugar-to-lipid molar ratio=2.3) was added as a lyoprotectant during the freeze-drying of the liposome, physicochemical stability of liposome was significantly improved. Moreover, the cytotoxicity of the liposome formulation against MDA-MB-231 human breast cancer cell line was not significantly different from that of Taxol^?. The enhanced aqueous solubility as well as the physicochemical stability of paclitaxel in the liposome formulation developed in this study could be a safer and effective alternative to the Cremophor^? EL and ethanol formulation.On the base of developed plain liposome, PEGylated liposomal formulation of paclitaxel has been developed with the purpose of improving the circulating time of paclitaxel in vivo. The use of 3% (v/v) Tween 80 in the hydration media was able to increase the solubility of drug to 3.28 mg/mL, which is enough for the clinical application. The addition of sucrose as a lyoprotectant in the freeze-drying process increased the stability of the liposome particles. From the in vitro release studies, only 33% of paclitaxel was released from PEGylated liposome within 24 h, which is much lower than those of plain liposome and Taxol^?. Cytotoxicity in human breast cancer cell lines (MDA-MB-231 and SK-BR-3) of our paclitaxel formulation was less potent compared to Taxol^? after 24 h incubation, but was equipotent after 72 h.Then, a sterically stabilized paclitaxel-loaded liposome tailored to target human epidermal cell growth factor receptor 2 (HER2) over-expressing human breast cancer cells was developed by post-insertion method. Results indicated that the targeting moiety (thiolated Herceptin) was successfully coupled to the distal reactive maleimide terminus of the poly (ethylene glycol)-phospholipid conjugate as well as being incorporated in the liposomal bilayers. The structure of Herceptin in the whole preparation procedure of PEGylated immunoliposome can maintain intact according to SDS-PAGE analysis. The immunological activity of Herceptin in different conjunctions (thiolated Herceptin and liposome-attached Herceptin) was evaluated in over-expressing HER2 human breast cancer cells, SK-BR-3 and BT-474, compared with free Herceptin, which showed that the activity of Herceptin conjugates was comparable to that of free Herceptin. In vitro release studies showed that only 36% of paclitaxel was released from PEGylated immunoliposome within 24 h, which had no difference with that from PEGylated liposome. Confocal laser scanning microscopy studies showed that the PEGylated immunoliposome was uptaken into the interior of the tumor cell through the receptor-mediated endocytosis pathway. The result from the combined effect analysis proved that the cytotoxicity of Herceptin and free paclitaxel or liposomal paclitaxel against BT-474 and SK-BR-3 cells had additive effect. Moreover, the PEGylated immunoliposome is more cytotoxic than the formulation including equal amount of Herceptin and PEGylated liposomal paclitaxel. The PEGylated immunoliposome showed substantially higher cellular uptake than the PEGylated liposome in cancer cells (BT-474 and SK-BR-3) over-expressing HER2 at 37℃, while no difference was found in low HER2 expressing cells (MDA-MB-231) or at low temperature (4℃), which is consistent with the results from confocal laser scanning microscopy.Pharmacokinetics of paclitaxel in the PEGylated immunoliposome was compared with that in Taxol^?, plain liposome and PEGylated liposome in rats. The PEGylated liposome showed the longest circulating time in vivo, following PEGylated immunoliposome, plain liposome and Taxol^?. The relative bioavailability of plain liposome was 160% relative to Taxol^?, while the values of PEGylated liposome and PEGylated immunoliposome were 712.75% and 390.83%, respectively. Biodistribution studies in breast cancer xenografted nude mouse model (MDA-MB-231) showed that PEGylated liposome had the highest cumulative amount of paclitaxel in tumor, following PEGylated immunoliposome, plain liposome and Taxol^?; While in the case of BT-474 nude mouse model, the cumulative amount of PEGylated immunoliposome had no difference with PEGylated liposome, following plain liposome and Taxol^?.In the end, the pharmacodynamics of Taxol^?, plain liposome, PEGylated liposome and PEGylated immunoliposome were evaluated in two different kinds of nude mouse models and saline as control. PEGylated liposome and PEGylated immunoliposome showed similar antitumor effect in MDA-MB-231 nude mouse models, following plain liposome and Taxol^?. As to BT-474 nude mouse model, PEGylated immunoliposome showed the best antitumor effect, following PEGylated liposome, plain liposome and Taxol^?. The results confirmed that paclitaxel loaded liposome can improve its anticancer effect and decrease its adverse effect and it can serve as a promising model for future breast cancer therapy.Pharmacodynamic results confirmed PEGylated immunoliposome had higher therapeutic effect in nude mice-bearing BT-474 breast cancer models than PEGylated liposome. However, PEGylated immunoliposome showed similar anticancer effect to PEGylated liposome in nude mice-bearing MDA-MB-231 breast cancer models. In all, paclitaxel-loaded PEGylated immunoliposome was developed successfully to increase the antitumor effect in over-expressing HER2 human breast cancer models.In all, the solubility of paclitaxel can be increased greatly (＞3 mg/mL) by the use of Tween 80 in the hydration medium and the versatile liposome technique. Freeze drying procedure was used to increase the stability of liposome. Due to the rapid elimination of plain liposome, PEGylated lipid was incorporated into plain liposome and the mean residence time can be increased from 3.15 h for plain liposome to 18.75 h for PEGylated liposome. In order to further increase the anticancer effect of paclitaxel, Herceptin was connected with PEGylated liposome to attain antibody mediated active targeting. Pharmacodynamics confirmed that PEGylated immunoliposome can significantly inhibit the tumor growth in HER2 over-expressing nude mouse models. Therefore, the liposomal formulation of paclitaxel could serve as a better alternative for the targeting of human breast tumors.