Category: Formulation and Quality
Purpose: The primary purpose of this project was the development of self-assembling nanomicelles as a potential treatment for breast cancer. Here we employed polymeric nanomicelles with targeting agent hyaluronic acid (HA) covalently attached on the corona of the nanomicelles. HA binds to CD44 receptors on the cancerous cells and can selectively target the cancer cells with minimal toxicity to healthy cells. HA conjugated to the nanomicelles via a disulphide bond can be easily be cleaved by glutathione enzyme present in the cytosol of the cancer cells. These cells express gluththione 10-100 times more than the normal cells and the extracellular fluid. Here we developed HA-targeted polymeric nanomicelles encapsulated a mixture of two drugs; paclitaxel (PAC) and ritonavir (Rit). Rit being a P-gp inhibitor can reduce the efflux of chemotherapeutic agents and make the currently available therapy more potent and reduce toxicity due to dose dumping. Thus this project was aimed at design, synthesis, formulation, characterization and optimization of HA targeted nanomicelles encapsulating PTX+Rit for breast cancer.
Methods: HA targeted polymeric naomicelles encapsulating PTX+Rit were prepared by solvent evaporation film rehydration method. In the first step, HA was conjugated to PLA-PEG-PLA polymer a simple addition reaction to form a graft triblock co-polymer. Similarly a reduction sensitive graft polymer
(HA-ss-PLA-PEG-PLA) was also prepared. In the second step, an optimized ratio of the two polymers; HA-PLA-PEG-PLA triblock polymer and Vitamin E TPGS was used for encapsulating PTX+Rit in the core of the nanomicelles. The design of experiment was performed by student version of JMP software. HA-PLA-PEG-PLA graft polymer was verified by FT-IR and NMR spectroscopy. The hydrodynamic size, zeta potential and the poly dispersity index was determined by a zetasizer particle size analyzer. Entrapment efficiency and the loading efficiency of PTX+Rit was determined simultaneously by an LC-MS method. The cell cytotoxicity of the formulation was verified using breast cancer cell line; MCF-7A, triple negative breast cancer cell line; MDA-MB-231 and normal breast cell line MCF-12A. The in-vitro uptake was quantitatively determined by flow cytometry (FACS) and qualitatively by confocal laser scanning microscopy (CLSM).
Results: An aqueous PTX+Rit nanomicellar formulation targeted with hyaluronic acid was prepared and optimized using Full Factorial design of JMP software. HA conjugation to PLA-PEG-PLA was confirmed by H1NMR. PTX+Rit nanomicellar formulation had a mean diameter of 117.22 nm, a polydispersity index of 0.19 and a zeta potential of -17.22 mV. Entrapment and loading efficiencies of the formulation were 97.74% and 0.79% for PTX and 95.13 % and 0.72 for Rit respectively. In-vitro cytotoxicity studies such as MTT assay and LDH assay revealed dose reduction in the IC50 value for a combination of PTX+Rit nanomicelles as compared to PTX nanomicelles for MCF-7 and MDA-MB-231 cell lines. While the cell viability was almost 100% for the blank formulation indicating the high safety profile of the polymers HA-PLA-PEG-PLA and Vitamin E TPGS used in this nanomicellar formulation. Cellular uptake studies using FACS and CLSM indicated an increase in uptake of PTX+Rit nanomicelles targeted with HA as compared to non-targeted PTX+Rit nanomicelles in a time-dependent manner.
Conclusion: An aqueous PTX+Rit nanomicellar formulation was formulated and evaluated for its effectiveness. The potential of targeted nanomicelles to selectively target metastatic breast cancer cells coupled with co-delivery of Paclitaxal with P-gp inhibitor Ritonavir can have a strong anti-cancer effect and possible reversal of multi drug resistance in metastatic breast cancer.