CUBOSOMES AS NANOCARRIERS FOR CANCER TREATMENT: SYNCHROTRON LIGHT TECHNIQUES APPLIED IN STRUCTURAL AND PERFORMANCE STUDIES OF NANOPARTICLES IN BIOLOGICAL SYSTEMS

Cancer is a disease with high mortality rate worldwide causing approximately 10 million of deaths in 2020 according to National Cancer Institute. Chemotherapy is the most used method to treat this disease, but it is extremely cytotoxic. In this regard, the use of nanoparticles (NPs) incorporating drugs is promising for therapeutic purposes as it increases the effectiveness against cancer and minimizes adverse effects. Particularly, cubosomes (lipid bicontinuous cubic phase NPs) have been emerged as a powerful platform for cancer treatment due to their (i) biocompatibility and ability to (ii) encapsulate both hydrophilic and lipophilic drugs and (iii) provide a controlled release of drugs. While these advantages are of pivotal importance toward high-performance therapeutic procedures, investigations on the structural variations of cubosomes throughout the drug encapsulation process and detailed analyses of the performance of these systems in cell cultures or complex biological environments are still lacking in the literature. Accordingly, this project seeks remarkable advances regarding the characterization of cubosomes in the absence and presence of doxorubicin (DOX) and a comprehensive understanding of their action mechanism into healthy and tumor cell lines, using synchrotron radiation as the main analysis tool.
Phytantriol (PHY) and monoolein (MO) cubosomes with approximate sizes of 200 nm were produced. The encapsulation efficiency of doxorubicin was 53% and 68% for the PHY and MO cubosomes, respectively. SAXS measurements confirmed the formation of bicontinuous cubic structures, with symmetry Pn3m for PHY cubosomes and symmetry Im3m for MO cubosomes. Colloidal stability assays of the NPs in biological medium (DMEM supplemented with fetal bovine serum) indicated that both remain relatively stable for at least 24 h, which is essential to ensure the internalization of the cubosomes in the target cells. DOX release assays showed that both NPs release 80% of the drug within approximately 200 min at pH 7.4. Cell viability tests (Alamar blue) were initially performed to ensure the maximum concentration of cubosome (without DOX) that can be used without being toxic to cells. PHY cubosomes were more toxic than MO cubosomes, with maximum working concentrations of 30 and 100 ug mL–1, respectively. Flow cytometry reinforced the results obtained using Alamar blue. According to viability tests with DOX incorporation, free DOX appeared to be more toxic than DOX-with cubosomes. Moreover, it was possible to observe that both DOX-containing NPs were less toxic for healthy cells than for tumor cells, which is a fundamental feature for a more efficient and safer use in nanomedicine. Finally, confocal microscopy images revealed that the cubosomes were internalized by the cells, being mostly located around the cell nucleus. New experiments using synchrotron radiation are in progress. The goal is determining the cytolocation of such systems based on the use of coherent diffraction imaging (CDI), named Ptychography CDI (PCDI). These experiments play a key role for the translation of increasingly efficient nanomedicines into the clinical practice.
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2022/02378-0).