Preparation and Characterization of Doxorubicin Loaded Tri-Block Copolymer (PCL-PEG-PCL) and its Cytotoxic Effect on Breast Cancer Cell Line MCF-7

The present study aimed to prepare, characterize and explore the cytotoxicity effect of doxorubicin (Dox) loaded biodegradable amphiphilic triblock copolymer poly caprolactone (PCL) poly ethylene glycol (PEG) poly caprolactone  (PCL-PEG-PCL) micelles against breast cancer cell line MCF-7.The physicochemical properties and in vitro test including cytotoxicity of the micelles were examined. PCL-PEG-PCL (PCEC) micelles were prepared by nanoprecipitation method using acetone as the organic solvent, and the PCLPEG-PCL triblock copolymer was self-assembled into core/shell-like structured micelle nanoparticles due to the amphiphilic property of the PCLPEG-PCL.The average particle size determined by dynamic light scattering (DLS) of obtained micelle was (226 ± 5) nm, and polydisperse index was (0.26 ± 0.034) with a narrow monodispersed unimodal size distribution pattern. The TEM image revealed that the micelles prepared by nanoprecipitation were spherical in shape.In this study, Dox was encapsulated into micelles with encapsulation efficiency (EE) 99.7% and drug loading (DL) 28.69 %, then the release profile of Dox from the PCL-PEG-PCL micelles was studied using a dialysis method.
At pH 5.6, approximately 33% of Dox within 3 hr was released, while after 46 hr approximately 92% of Dox was released, but the rate of Dox that released at pH 7.4 is notably slower compared to pH 5.6. Moreover, the percentage of Dox released was approximately 43% within 46 hr.                                               
The in vitro safety evaluation of polymeric PCL-PEG-PCL micelles was performed and it was concluded that these nanoparticles did not induce hemolysis with the concentration of (100 µg/ml) on human erythrocyte comparing with the negative control (normal saline). The in vitro cytotoxicity of PCL-PEG-PCL micelles at different concentrations (0.1, 0.2, 0.4, 0.6, 0.8 and 1 µg/ml) on the MCF-7 cell line was
evaluated by the tetrazolium dye MTT method, and the analyzed results revealed that PCL-PEG-PCL micelles possessed negligible toxicity to MCF-7 cells even at higher concentrations, the viability percentage was above 80%,indicating good biocompatibility of these polymeric triblock. Additionally, the morphological observations of Dox loaded PCL-PEGPCL against MCF-7 cell line by inverted icroscope
revealed that after 24 hrs of treatment, no morphological changes were observed in the MCF7 cells.Whereas, after 48 hrs, the cells became rounded and detached. After 72 hrs, the number of treated cells was reduced, and they were shrinkage, rounded,detached and suspended in the culture media. Therefore, this study showed an effective delivery characteristic and the inhibition of cell growth of Dox that
loaded into PCL-PEG-PCL polymeric micelles. The in vitro cytotoxicity was investigated by MTT assay of (0.01, 0.05, 0.1,0.4, 0.8, 1.2 µg/ml) of free Dox and Dox loaded PCL-PEG-PCL micelles
against MCF-7 cell line after 24, 48 and 72 hrs. MCF-7 was more sensitive to free DOX as compared to PCL-PEG-PCL/Dox for 24hrs and 48hrs, whereas at 72hrs, it was observed that MCF-7 cells was more sensitive to PCL-PEGPCL/Dox than free Dox.moreover,it was found that the cell viability decreased significantly at p≤ 0.05 when the treatment time was increased within the experimental period.The inhibition concentration values (IC ) (inhibitory concentration to kill 50% cell death) of free Dox and Dox loaded PCL-PEG-PCL against MCF-7 cells after 72 hr were 0.56 and 0.5 respectively. 0 To track the Dox loaded PCL-PEG-PCL micelles and Dox molecules in MCF-7 living cells, label-free techniques such as coherent anti-stock Ramman spectroscopy (CARS) and two photon excitation (TPEF) was used.  
The visualization of the three dimensional distribution of the Dox molecules by CARS and TPEF showed that free Dox entered nucleus quickly and it’s concentration in cytoplasm dropped over time after 10 hr and 35 min.At the same time, Dox was released from micelles and could be observed in cytoplasm and nucleus. In addition, the cell number was relatively low of exposure MCF-7 to Dox-loaded micelles as compared with the Dox that was released after 5hr and 50 min.