Purpose: The purpose of the work is to improve tumor regression and survival of animals by effective formulation of the natural anti-oxidant compound
Methods: Two batches of chitosan nanosuspension was prepared using 3:1 ratio of chitosan and TPP using ionotropic gelation method. One batch was coated with polysorbate 80 while the other was kept uncoated. The formulations so developed was analyzed using Differential scanning calorimetry (DSC) and Fourier transform infra-red spectroscopy (FTIR) by adsorbing them on mannitol followed by lyophillization. Also, their particle size, zeta potential, polydispersity index, drug entrapment, morphology and in vitro drug release was also evaluated using suitable instruments. All the reagents were suitable analytical grade and were used as received.
For in vitro cellular cytotoxicity study, MCF-7 was chosen as a breast cancer cell line and HEK293T cells were taken to study any effect on normal cells. 3000 cells/well of both MCF-7 and HEK293T seeded in 96 well plate in 200µl DMEM media supplemented with 10% FBS. Cells were treated with different concentrations of A, B and cultured for 48hrs. After that 10% MTT (prepared in PBS) was added and incubated for 3hrs followed by solubilization of formazan crystals by DMSO and read at 570nm in ELISA reader. Next, for in vivo studies, 1x106 sarcoma-180 mouse cancer cells were injected in female Swiss albino mice to form solid tumor in left flank of hind leg. After 2 weeks of palpable tumor formation, treatment started with parent drug and different nanoformulations in various concentrations to find effective dose and toxicity. The formulation showing best tumor regression and survival of mice at minimum dose with least toxicity were compared to vehicle treated control , and selected as most effective drug candidate. Further, normalization of cellular architecture was evaluated in tumor tissues by histological analysis and different parameters like angiogenesis,proliferation, apoptosis were checked by immunohistochemical analysis as mechanisms of tumor regression.
Results: The size of the formulation was found to be in nanorange and that was reduced for the batch in which surfactant was used. The range of zeta potential revealed stable nanosuspension. No polymorphic changes/physical incompatibility has been observed among the drug, different excipients and the final formulation during DSC and FTIR study. The TEM image revealed nanorange, smooth surface and surfactant coating on the surface of formulations.In both coated and uncoated formulation, 45-55% drug release was observed after 24 hr offering sustained release of the drug.
MTT assay revealed coated nanodrug delivery as more effective on cancer cell lines without significant effect on normal Human kidney cell line. Coated encapsulated drug showed higher cytotoxicity than uncoated Nanoformulation and parent compound. In tumor bearing mice both nanoformulation treatments showed better tumor regression followed by ellagic acid itself compared to vehicle treated control, without any significant changes in vital organs like liver, kidney, spleen and heart. Coated nanoformulation showed highest efficacy at 20 mg/Kg body weight dose followed by uncoated nanoformulation and Ellagic acid at at 30mg/Kg body weight and 50 mg/Kg body weight respectively. Coated nanoformulation also significantly improved survival of tumor bearing mouse.The mechanism of tumor regression was attributed to reduction of proliferation and angiogenesis and increase in apoptosis of tumor cells.
Kalpana Nagpal– Noida, Uttar Pradesh, India
Sandip Ghosh– Kolkata, West Bengal, India
Biswarup Basu– Kolkata, West Bengal, India
Kalpana Nagpal– Assistant Professor-III, Amity Institute of Pharmacy, Amity University, Noida, Noida, India, India