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Development of solid lipid nanoparticles in loading alcaloídico extract of Solanum Lycocarpum and biological evaluation in vitro in bladder cancer cells

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Ivana Pereira Santos Carvalho
Total Authors: 1
Document type: Master's Dissertation
Press: Ribeirão Preto.
Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
Defense date:
Examining board members:
Priscyla Daniely Marcato Gaspari
Advisor: Priscyla Daniely Marcato Gaspari

Solanum lycocarpum is a native species of the Brazilian savannah known for its diversity in glycoalkaloids (GA). The GA solasonine (SS) and solamargine (SM), extracted from the fruits of their plant, have antioxidant and antitumoral activity; acting on different molecular mechanisms interesting for cancer therapy. However, the low water solubility of these molecules and their toxicity limits their application. Thus, the development of a nanostructured formulation containing these glycoalkaloids may allow their clinical application in bladder cancer therapy (BC). Thus, this project aims at the development and characterization of nanostructured solid lipid (NLC) as carrier systems of alkaloidic extract of S. lycocarpum (AE), rich in GA, and their cytotoxic evaluation in order to obtain a nanostructured system for future intravesical therapy of bladder cancer. The NLC were prepared by the emulsion and sonication method and optimized using a factorial design 23. The influence of three independent variables was evaluated as type and amount of lipid and type of surfactant in response factors diameter and polydispersity index (PdI). NLCs were characterized according to average diameter, size distribution and zeta potential by dynamic light scattering (DLS), recrystallization index (RI) by differential scanning calorimetry (DSC), percentage of encapsulation efficiency (EE%) by LC MS/MS, morphological analysis by atomic force microscopy (AFM), distribution and concentration of nanoparticles by nanoparticle tracking analysis (NTA), the in vitro release profile of GA from NLC was done by Franz\'s cells diffusion, and stability over time by DLS. Cytotoxicity assay was evaluated by neutral red uptake assay in bladder cancer cells (RT4). Cells were treated with different concentrations of GA free and encapsulated in NLC (5-40 ?g/mL) and empty NLC for 24h, 48h and 72h. The NLC developed showed diameter smaller than 80 nm and zeta potential of -9.26 mV. The PdI was less than 0.2 indicating dispersion with low polydispersity. The NLC was stable for 60 days regarding to size, PdI and zeta potential. Recrystallization index (RI) of NLC-AE was 47.23%, indicating a less crystalline structure of the lipid in the particle, which provides high encapsulation efficiency of SS (86%) and SM (89.1%). This low RI prevents the expulsion of GA from NLC during the storage. NLCs exhibited semi-spherical form as observed by AFM. The particles concentration, measured by NTA, was 9.74 x 1011 particles/mL and the diameter was about 125 nm. The GA present in NLC-AE showed an in vitro sustained release profile after 36 h of release in a sink condition. The release mechanisms of SS and SM were different. The release mechanism of SS was non-Fickan while SM was Fickan. The AE free and NLC-AE was cytotoxic to RT4 cells, and the NLC-AE was more cytotoxic than the AE free after 48 hours of treatment. The IC50 of NLC-AE, after 24 hours of treatment, was 15.94 ?g.mL-1 and 12.35 ?g.mL-1 after 48 h of treatment. This reduction in IC50 value can be related to the release profile of the GA (SS and SM) of the nanoparticles. The results demonstrate that NLC developed is an interesting system to encapsulate AE with potential for future therapy of bladder cancer (AU)

FAPESP's process: 14/16727-0 - Development of solid lipid nanoparticles as carrier system of glycoalkaloids of Solanum lycocarpum for bladder cancer therapy
Grantee:Ivana Pereira Santos Carvalho
Support Opportunities: Scholarships in Brazil - Master