Multimodal therapeutic protein nanocapsules for cancer therapy and imaging

Natural product derived nanotherapeutics are currently in the lime light of cancer research, due to their enhanced multifunctional therapeutic abilities, in terms of target specific drug delivery by utilization of aptamers, locked nucleic acid conjugates and also imaging strategies such as magnetic resonance imaging, positron emission tomography. Published findings from our laboratory demonstrated that iron saturated bovine lactoferrin (FebLf) and polymeric based calcium phosphate nanocarriers encapsulated with FebLf showed significant in vitro and in vivo anti tumour activity in colon and breast cancer models. The present study aimed at development of multimodal iron saturated lactoferrin (FebLf) Nanocapsules (FebLf NCs) and evaluation of anti cancer abilities in an in vitro (breast cancer MDA-MB-231) and in vivo (colon cancer Caco2 and breast cancer MDA-MB-231) human xenograft cancer models. Physical characterisation of nanocapsules: scanning electron microscopic observations confirmed a consistent size range of 60 to 80 nm with spherical morphology. Fourier transform infrared spectrum and x-ray diffraction studies indicated characteristic peaks for lactoferrin (NH3 and OH bonding patterns) in FebLf NCs. FebLf NCs magnetometric analysis demonstrated superparamagnetic behaviour in the range of 60 emu/g saturation magnetization. In vitro studies: Immunocytochemistry observations confirmed successful cellular localisation and 80 90% of cellular internalisation was observed within one hour duration as revealed in confocal microscopic observations(p<0.005). Cellular internalisation of FebLf NCs was observed in a time dependent manner at 12, 24 and 48 hours was receptor mediated via lactoferrin (Lf), transferrin (TFR, TFR 1, TFR 2), low density lipoprotein receptor-related protein (LRP 1 and LRP 2), divalent metal transporter 1 (DMT 1), ferroportin, ferritin receptors (p<0.005) as observed in quantitative real time polymerase chain reaction (qRT PCR) analysis. Apoptotic mechanism of action of FebLf NCs involved down regulation of anti apoptotic signalling molecules survivin, livin, phosphatidylinositol 3-kinase (PI3K)) pathway (p<0.005). Furthermore, a decrease in mitochondrial potential (p<0.005) was noted and caused cytochrome C (p<0.005) release. In vivo studies: In vivo nude mice (Balb C nu/nu) breast and colon cancer human xenograft model studies revealed excellent biocompatibility of FebLf NCs, when administered via oral route and tumour reduction was observed over a time period of 90 days. Haematoxylin and eosin staining showed no toxicity of FebLf NCs in the mice tissues. In vivo internalisation of FebLf NCs was mediated by following receptors lactoferrin (Lf), transferrin (TFR, TFR 1, TFR 2), low density lipoprotein receptor-related protein (LRP 1 and LRP 2), divalent metal transporter 1 (DMT 1), ferroportin, ferritin receptors (p<0.005). Further, Near infrared fluorescence live mice imaging studies showed selective localisation of FebLf NCs at tumour site and reduction in tumour was estimated in terms of radiant efficiency and area of the fluorescent signal obtained. In the light of current observations, future directions of the study includes uncovering multimodal imaging abilities by employing imaging techniques such as magnetic resonance and fluorescence based multimodal imaging strategies to uncover multifunctional ability of FebLf NCs nanoformulation. List of publications: 1. Kanwar Jagat R, Sishir K Kamalapuram, Kanwar Rupinder K. clinic 33: 765 789. (Impact Factor: 9.583 : ERA ranking A* ). Citations: 23. 2. Superparamagnetic iron oxide nanoparticles (SPIONS): The king in the cosmos of multimodal onco n Journal of Nano Studies & Technology (IJNST): 2013, Volume II Issue No.1. Citations: Yet to be cited. 3. Survivin in cancer: a spider in the w Journal of Nanomedicine & Nanotechnology (2012): Volume 3 Issue 8. Index copernicus value: 4.22. Citations: Yet to be cited. 4. cancer: the Drug Discovery Today, June (2011), Vol. 16, No. 11-12, pp. 485-94. (Impact Factor: 6.89 : ERA ranking A* ). Citations: 61.