Development of a nanostructured lipid carrier containing a pool of siRNAs associated with an antitumor agent as a multifunctional therapeutic approach for skin cancer

Author(s)

Juliana Santos Rosa Viegas

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2022-03-24

Examining board members

Maria Vitoria Lopes Badra Bentley; Emanuel Carrilho; Vanessa Carla Furtado Mosqueira; Laura de Oliveira Nascimento

• Advisor: Maria Vitoria Lopes Badra Bentley

Abstract

Skin cancer advances in number of cases worldwide, especially in countries where there is a predominance of Caucasians. The available treatments are less effective due to the mutations and to overcome these challenges, gene silencing therapies have been widely adopted. In the present work, a nanostructured lipid carrier (NLC) containing 5-Fluororuracil and a pool of siRNAs (siRNA Gli-1, siRNA EGFR and siRNA Bcl-2) was developed in order to act on different skin cancer pathways. The NLC was developed in high pressure homogenizer and optimized by experimental design. NLC was characterized and presented diameter between 200 - 250 nm, polydispersion index less than 0.3, zeta potential of 49 mV, and number of nanoparticles 1012/mL. Scaling up studies from 20 mL to 200 mL were performed, presenting the same physicochemical characteristics in all volumes. Complexation with the siRNAs occurred at N/P 2 ratio for control and Bcl-2 siRNAs, and N/P 5 ratio for siRNA Gli-1 and EGFR, and all decomplexed efficiently with anionic competition using heparin. The release of 5-FU occurred by Higuchi kinetics, releasing about 60 to 80% of 5-FU in 8 hours in a sustained manner. In vitro and ex vivo cutaneous studies indicated that 5-FU is mostly retained in the epidermis. In addition, it does not cause tissue damage and reduces the desquamation of the stratum corneum. Cellular studies (2D and 3D spheroids) indicated that the system itself is not toxic, but when associated with active agents it significantly reduces cell viability. The cells A431 and A375 were the most susceptible lines to the combination NLC 5-FU + Bcl-2 siRNA, the cells Sk-mel-103 and 1205Lu had their viability reduced for NLC 5-FU + pool, showing that they are more aggressive and resistant. Cell uptake occurred efficiently in the cells and the internalization pathway determined was endocytosis dependent by clathrin and a minority by caveolin. It was determined in vitro that the multifunctional NLC containing 5-FU + pool siRNA decreased the migration as well as decrease the 1205Lu invasiveness. The NLC 5-FU + siRNA Bcl-2, evaluated in vivo in a xenotransplant model of the A431, can suppress tumor growth with controlled inflammatory infiltration (low levels of myeloperoxidase, N-acetylglucosaminidase and TNF-α). Furthermore, NLC 5-FU + siRNA Bcl-2 presented effective tumor apoptosis, Bcl-2 knockdown and negativity for extracellular matrix remodeling. (AU)

• FAPESP's process: 19/04448-2 - Development of a nanostructured lipid carrier containing pool of siRNAs associated with the antitumor agent as a multifunctional therapeutic approach for skin Cancer
• Grantee: Juliana dos Santos Rosa
• Support Opportunities: Scholarships in Brazil - Doctorate