Folate Conjugated Carfilzomib Loaded Lactoferrin Nanoparticles as Biotherapeutics for Cancer

Carfilzomib(CFZ) is a second generation irreversible proteosome inhibitor that have shown strong clinical efficacy in relapsed cancer management.Despite of having high potency in cancer treatment, poor solubility, short half-life, and poor in vivo stability hinders its therapeutic application. Proteins like lactoferrin with structural tunability and iron homeostasis resembling multifunctional roles in cancer research, that could enhance the accumulation of drug in vicinity of tumor tissues. Exploring the surface amenities of proteins by conjugating tumor associated antigens(TAAs) can be an emerging approach as cancer theragnostic. Hence, the study aimed to fabricate a formulation of folate functionalized carfilzomib loaded lactoferrin nanoparticles for the treatment of ovarian cancer. Lactoferrin nanoparticles were fabricated using desolvation technique, which is based on nanoprecipitation principle. Quality-by-design paradigms were used to obtain optimal composition of nanoformulation. The carfilzomib loaded lactoferrin nanoparticles(CFZ-LF-NPs) were found to be uniformly distributed in range of 100- 150nm. Moreover, crosslinking of protein nanoparticles resulted in high drug entrapment of CFZ-LF-NPs showing 91.21 ± 0.209% entrapment efficiency. Owing to unique architectures of protein, its multi-functional surface ammenties can be conjugated with overexpressed biomarkers such as folate to employ a target specific delivery of hydrophobic cargo like carfilzomib. CFZ-LF-NPs were conjugated with folic acid using EDC-NHS based carbodiimide chemistry. The developed formulation was evaluated for physicochemical characterisation interpreted via NMR, FTIR, DSC, and in vitro release studies .FA conjugated nanoparticles resulted in high %drug release of 91.932 ± 0.549% at tumor pH 5.5, while controlled drug release at pH 7.4 till 6 days, thereby indicating pH triggered drug release. Further, FITC was attached to folic acid conjugated carfilzomib loaded lactoferrin nanoparticles( FA-CFZ-LF-NPs) as a bioimaging tag for monitoring the cellular uptake using Flow cytometry and Confocal microscopy. Folate conjugated nanoparticles showed high cellular uptake and more than 60% of apoptosis was observed in Flow cytometry analysis. Moreover, MTT assay of FA-CFZ-LF-NPs delineated 89% cell cytotoxicity till 72 hours in SKOV-3 cells as compared to non-conjugated nanoparticles showing less than 50% cell inhibition. As high iron storage in folic acid and lactoferrin played a cornerstone in cell uptake of nanoparticles. Additionally, overexpression of folate receptor leveraged receptor mediated uptake, that synergistically enhanced the drug retention and cytotoxicity in cancer cells. Therefore, the MTT assay, apoptosis, and cellular uptake assay unraveled that nanoconjugates showed high cytotoxicity in SKOV-3 cells as compared to non-conjugated NPs and plain drug in SKOV-3 cells. Hence, the potential approach may emerge in future as a molecular-docks comprising nanomedicine with enormous applications such as biosensing, molecular labelling, bio-imaging as well as immunotherapy-based theragnostic. This delineates a precise targeting of nanoconjugates with prolonged retention that sets forth motif towards advancement in cancer theragnostic.
Therefore, the attendees would be able to understand the urge to explore theragnostic approach in cancer management and importance of molecular tagging in order to leverage the precise treatment. The study would garner the attention of research community in developing the scale up of protein based biotherapeutics, that can be a potential future of cancer treatment.