Evaluation of the potential of nanostructured lipid carriers functionalized with folic acid for incorporation of rapamycin in the treatment of breast cancer

Author(s)

Camila Fernanda Rodero

• Document type: Doctoral Thesis
• Press: Araraquara. 2022-01-17.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Ciências Farmacêuticas. Araraquara
• Defense date: 2021-12-10
• Advisor: Marlus Chorilli

Abstract

Breast cancer is the second most common type of cancer in the world. Among the candidate drugs for use in the treatment of this type of cancer is rapamycin, which is capable of inhibiting the mTOR protein, controlling its growth and progression in the cell cycle. However, it is a lipophilic drug, undergoes sequestration by erythrocytes and has chemical instability, factors that compromise its bioavailability. Thus, its incorporation in nanostructured lipid carriers functionalized with folic acid can be an interesting strategy, as this system can protect the drug from possible degradation, in addition to providing slower and more sustained action. Functionalization of carriers with folic acid enables drug release at the target site by binding to folate receptors that are expressed in large amounts by a variety of tumor cells, including breast cells. Based on the above, the objective was to develop and characterize carriers functionalized with folic acid for incorporation of rapamycin and to evaluate its potential in the treatment of breast cancer. Six different carriers were developed, which presented mean hydrodynamic diameter values from 97.29±0.79 to 119.05±0.23 nm respectively, and zeta potential values from -12.45±0.48 to -28, 75±0.16 mV, respectively. All formulations showed spherical and nanoscale morphology, according to transmission electron microscopy analyses. Thermal analysis shows a fusion event at 50°C, which is endothermic, showing a new compound. The functionalization of the lipid with folic acid was confirmed by spectrophotometric analysis in the visible ultraviolet, by nuclear magnetic resonance and vibrational spectroscopy in the infrared region. Quantification of rapamycin was performed by high-performance liquid chromatography, showing that it was within the parameters required and recommended by ANVISA and ICH, with linearity of r2=0.99, precision and robustness. Encapsulation efficiency tests showed that the carrier can encapsulate about 95% of the drug. The release assay showed for the functionalized carriers the release of 82% of rapamycin after 16 hours, while the non-functionalized carrier released about 45% of the drug in the same period. In vitro cytotoxicity assays showed that carriers did not show toxicity against LEWIS, MCF-7 and MCF10-A cell lines. For the system, it was observed IC50 of 84, 176 and 110nM, respectively, and for free rapamycin the IC50 against the same cell lines were 81.8, 141 and 95nM, respectively. Confocal microscopy evidenced the cellular internalization of the system incorporated with DiO in MCF-7 and LEWIS lines. Flow cytometry data corroborate those from confocal microscopy, suggesting the internalization of the formulation. The alternative model of Caenorhabditis elegans demonstrated that the formulation does not present toxicity. The in vivo xenographic model of breast cancer showed that the system, both administered intravenously and intraperitoneally, within 18 days of treatment, led to tumor regression, showing a statistical difference in relation to controls. The results suggest that the carrier containing rapamycin and functionalized with folic acid has promising potential for the treatment of breast cancer. (AU)

• FAPESP's process: 17/22758-3 - Evaluation of the nanostructured lipid carriers potential functionalized with folic acid incorporated with rapamycin for breast cancer treatment
• Grantee: Camila Fernanda Rodero
• Support Opportunities: Scholarships in Brazil - Doctorate