Background: Paclitaxel is one of the most effective and most widely-used anti-cancer agents.However, paclitaxel is difficult to formulate for parenteral administration because of its low aqueous solubility.Presently paclitaxel is formulated for clinical use in ethanol and Cremophor EL, a solvent system associated with severe adverse effects.The extensive clinical use of this drug is somewhat delayed due to the lack of appropriate delivery vehicles.To avoid these adverse effects and inconvenience of this formulation, alternative formulations have been exploited for both parenteral and oral delivery of PTX.Polymeric micro-and nanoparticles, lipid nanoparticles polymeric micelles, liposomes and emulsions have been examined, and all of these formulations achieved the improvement of PTX solubility.Lipid microspheres are heterogeneous mixtures of oil in water, where the oil droplets are confined to nanometer size(typically less than 200 nm).The advantages of lipid microspheress include an opportunity to solubilize hydrophobic compounds, such as PTX.The obvious benefits of lipid microspheres as drug delivery systems have led to the development of several systems for i.v.and oral administrations of PTX over the past many years.However, the solubilizing capacity of lipid microspheres are quite limited.An alternative approach is to use the paclitaxel-lipid complex, which is retained more stable in lipid carrier preparations.In this paper, we developed a novel Cremophor-free, autoclave stable, intravenous microspheres involving a lipid complex, paclitaxel-lipid complex, incorporated in the core of a nano-size sterically stabilized lipid microspheres(lipo-paclitaxel).The paclitaxel-lipid complex can improve the solubility of paclitaxel in the oil phase of emulsion significantly.Objective: To develope a novel Cremophor-free, autoclave stable, intravenous lipid microspheres involving a lipid complex, paclitaxel-lipid complex, incorporated in the core of a nano-size sterically stabilized lipid microspheres(lipo-paclitaxel) and evaluate antiproliferative effects of lipo-paclitaxel on MCF-7 cell line.Methods: The Paclitaxel-lipid Complex were prepared by a rotary evaporation method.Then we associated lipid complex supermolecular to lipid microspheres by High pressure homogenizer and analyzed the new formulations size, zeta potential, incorporation efficiency, chemical and physical stability as previously described.Apart from physicochemical properties mentioned above, in vitro cellular uptake, antiproliferative activity and cell-apoptosis assay of lipid-rich paclitaxel lipid microspheres on MCF-7 cell line were evaluated.Results: In vitro cytotoxicity assay Cytotoxicity of free paclitaxel, Taxol, Paclitaxel-lipid complex, and lipid-rich paclitaxel lipid microspheres was compared by conducting an CCK-8 assay.As presented in Figure 1, the survival curves of MCF-7 showed a concentration-dependent tendency in the range of 0.001-1 μg/mL.The cell-survival rate in the lipid mierospheres group was significantly lower than that in the free paclitaxel group, demonstrating that lipid microspheres showed higher anticancer efficacy than free paclitaxel solution.Compared with Taxol, the lipid microspheres exhibited as well as Taxol.In vitro cellular uptake study To investigate the causes of antineoplastic effect of lipid microspheres, an in vitro cellular uptake assay was conducted on MCF-7 cells.As shown in Figure 8A, more paclitaxel was taken up by cells in the form of lipid microspheres under different time at 10μg/mL concentration (P < 0.05) when compared with free paclitaxel solution.Cell-apoptosis assay As shown in Figure 3, the morphology of MCF-7 cells significantly changed after paclitaxel treatment.The cells became partly round and fragmentized, and this phenomenon was more obvious in lipid microspheres group.It is well known that the apoptotic cell nucleus will show many morphologic modifications, such as nuclear pyknosis and condensation, chromatin fragmentation, and formation of apoptotic bodies.In Figure 3, the morphology of the cell nucleus in the control group was maintained with homogeneous chromatin, while the significant chromatin condensation, nuclear fragmentation, or even apoptotic body formation could be found in the paclitaxel-treated groups.It was also found that the nucleus-morphology change was more severe in the lipid microspheres group.To measure the apoptotic effect of Taxol and lipid microspheres quantitatively, MCF-7 cell apoptosis was evaluated, and the percentage of cell apoptosis treated with Taxol and lipid microspheres was determined by flow cytometer.As shown in Figure 4 and Figure 5, the lipid microspheres could induce more cell apoptosis (52.18 % ±5.72 %) than Taxol (32.49% ± 7.56%) (P < 0.05).Conclusion: The lipid microspheres formulation exhibited a homogeneous round shape.The mean particle size, zeta potential, and encapsulation efficiency were 150nm,-38.3 mV and 97.3%, respectively.Compared with Taxol, the lipid microspheres exhibited a significant improvement in cellular uptake of PTX on MCF-7 at different incubation time.Moreover, the lipid microspheres exhibited the higher cytotoxicity on MCF-7 cells than Taxol and it could induce more cell apoptosis (52.18 % ±5.72 %) than Taxol (32.49% ± 7.56%) (P < 0.05).The lipid microspheres has great potential to serve as a potential system for the delivery of PTX with higher antitumor effects.Further study on in vivo anticancer effect is being explored.