Evaluation of the potential of transferrin-functionalized liposomes for docetaxel delivery in the treatment of prostate cancer.

Author(s)

Mariza Aires Fernandes

• Document type: Master's Dissertation
• Press: Araraquara. 2020-01-27.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Ciências Farmacêuticas. Araraquara
• Defense date: 2019-12-10
• Advisor: Marlus Chorilli

Abstract

Prostate cancer (PC) is the most common urological malignancy and the second leading cause of cancer-related mortality in men worldwide. Treatment is initiated through surgical interventions that include radical prostatectomy, radiotherapy treatments, androgen deprivation therapies and immunotherapies. Chemotherapy is also widely used and recent studies have shown remarkable control of CP growth using docetaxel (DTX). However, DTX has side effects associated with the toxicity of both the drug and the formulation Taxotere®. As a result, the development of nanotechnological drug delivery systems, such as liposomes, has been promising for the treatment of CP, due to the multiple favorable characteristics of these nanosystems, such as biocompatibility and biodegradability. An attractive approach is the superficial modification of liposomes with receptor-specific ligands regulated on tumor cell surfaces, such as transferrin receptors, which can be overexpressed in prostate tumor cells. The present work aimed to develop, characterize and evaluate the in vitro cytotoxicity of transferrinfunctionalized liposomes for DTX delivery in the treatment of CP. The liposomes were obtained by the classical technique of hydration of the lipid film and consisted of soybean phosphatidylcholine (SPC): cholesterol (Col): DSPE-PEG (2000) -Maleimide and SPC: Col: DSPE-PEG (2000) mixture. ) -Maleimide: DTX, in the molar ratios 10: 2: 0.78 and 10: 2: 0.78: 0.52, respectively. The characterizations were performed from the empty formulations and with DTX before and after the functionalization process. Particle average diameter, polydispersity index (IPD) and zeta potential (PZ) analyzes were performed using the Dinamic Light Scattering (DLS) and morphological analysis by transmission electron microscopy (MET). The average diameter values obtained remained below 100nm, the IPD at 0.400 and the PZ between -23.06 to -21.2mV. Through MET it was possible to prove the spherical morphology of the gall bladder, corroborating the size obtained by the DLS technique. Concentration analyzes were performed by Nanoparticle Tracking Analysis (NTA). The functionalization process was successfully obtained and the primary, secondary and tertiary transferrin structures evaluated by SDS-PAGE, circular dichroism and fluorescence spectroscopy confirmed that the process did not alter its integrity. Functionalization efficiency was 31% evaluated by the BCA assay. To evaluate the encapsulation efficiency and DTX release the analytical methodology was validated by high performance liquid chromatography. 69 and 37% of DTX were found to be encapsulated in liposomes without and with functionalization, respectively. The release assay was performed in PBS pH 7.4 with 1.25% sodium lauryl sulfate using 12-14kDa molecular weight cellulose dialysis membranes for free drug, functionalized (B) and non-functionalized (D) systems. containing docetaxel. Free drug diffusion across the membrane was greater than 60% after 72 hours of study, demonstrating that DTX release was not limited by the dialysis membrane. In the case of liposomes, DTX release was 51.70% and 31.97% for nonfunctionalized and functionalized liposomes, respectively. In vitro cytotoxicity and cell uptake assays in PC-3 and PNT2 strains suggest that empty and functionalized empty liposomes are not toxic to the strains tested; however, DTX-loaded liposomes showed high toxicity in both strains. Through the confocal microscopy and flow cytometry assay it was possible to prove that the functionalized liposomes presented pronounced uptake in PC3 strain. The interaction of these systems with the overexpressed TfRs on the cell surface through QCM-D can also be proven. The results obtained show that transferrin-functionalized liposomes are a promising strategy for the delivery of docetaxel, however additional studies in three-dimensional and in vivo cell models are necessary. (AU)

• FAPESP's process: 17/25190-8 - Evaluation of the potential of transferrin-functionalized liposomes for docetaxel delivery in the treatment of prostate cancer
• Grantee: Mariza Aires Fernandes
• Support Opportunities: Scholarships in Brazil - Master