Reversible exposure of hydrophobic residues on albumin as a novel strategy for formulation of nanodelivery vehicles for taxanes

AG Garro1, DM Beltramo1,2,3, RV Alasino1, V Leonhard1,2, V Heredia1, ID Bianco1,2,41Center of Excellence in Products and Processes of Córdoba; 2National Research Council of Argentina (CONICET); 3School of Chemistry, Catholic University of Córdoba; 4Department of Exact, Physical and Natural Sciences, National University of La Rioja, ArgentinaBackground: We report herein a novel strategy for the preparation of protein-based nanodelivery vehicles for hydrophobic active pharmaceutical ingredients.Methods: The procedure consisted of three steps, ie, exposure of hydrophobic residues of a protein to a pH-induced partial unfolding: interaction between hydrophobic residues on the protein and the hydrophobic active pharmaceutical ingredient, and a final step where the structure of the protein was reversed to a native-like state by returning to neutral pH. As proof of concept, the interaction of paclitaxel with partially unfolded states of human serum albumin was evaluated as a potential method for the preparation of water-soluble complexes of the taxane with albumin.Results: We found that paclitaxel readily binds to pH-induced partially unfolded albumin, leading to the formation of optically clear water-soluble complexes. The complexes thus formed were more stable in solution when the albumin native state was at least partially restored by neutralization of the solution to a pH around 7. It was also observed that the hydrodynamic radius of human serum albumin was only slightly increased after the cycle of pH changes, remaining in a monomeric state with a size according to paclitaxel binding. Furthermore, paclitaxel binding did not affect the overall exposure of charged groups of human serum albumin, as evaluated by its interaction with an ionic exchange resin.Conclusion: The in vitro biological activity of the complexes formed was qualitatively equivalent to that of a Cremophor®-based formulation.Keywords: human serum albumin, paclitaxel, unfolded states, solubilit

Biochemical characterization of the interactions between doxorubicin and lipidic GM1 micelles with or without paclitaxel loading

Victoria Leonhard,1,2 Roxana V Alasino,1,2 Ismael D Bianco,1–3 Ariel G Garro,1 Valeria Heredia,1 Dante M Beltramo1,2,4 1Centro de Excelencia en Productos y Procesos de Córdoba (CEPROCOR), Córdoba, Argentina; 2Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina; 3Departamento de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de La Rioja, La Rioja, Argentina; 4Laboratorio de Biotecnología, Facultad de Ciencias Químicas, Universidad Católica de Córdoba, Córdoba, Argentina Abstract: Doxorubicin (Dox) is an anthracycline anticancer drug with high water solubility, whose use is limited primarily due to significant side effects. In this study it is shown that Dox interacts with monosialoglycosphingolipid (GM1) ganglioside micelles primarily through hydrophobic interactions independent of pH and ionic strength. In addition, Dox can be incorporated even into GM1 micelles already containing highly hydrophobic paclitaxel (Ptx). However, it was not possible to incorporate Ptx into Dox-containing GM1 micelles, suggesting that Dox could be occupying a more external position in the micelles. This result is in agreement with a higher hydrolysis of Dox than of Ptx when micelles were incubated at alkaline pH. The loading of Dox into GM1 micelles was observed over a broad range of temperature (4°C–55°C). Furthermore, Dox-loaded micelles were stable in aqueous solutions exhibiting no aggregation or precipitation for up to 2 months when kept at 4°C–25°C and even after freeze–thawing cycles. Upon exposure to blood components, Dox-containing micelles were observed to interact with human serum albumin. However, the amount of human serum albumin that ended up being associated to the micelles was inversely related to the amount of Dox, suggesting that both could share their binding sites. In vitro studies on Hep2 cells showed that the cellular uptake and cytotoxic activity of Dox and Ptx from the micellar complexes were similar to those of the free form of these drugs, even when the micelle was covered with albumin. These results support the idea of the existence of different nano-domains in a single micelle and the fact that this micellar model could be used as a platform for loading and delivering hydrophobic and hydrophilic active pharmaceutical ingredients. Keywords: cancer drugs, nano-domains, drug delivery, hydrophobic interaction