Nano-formulation of a drug for skin cancer penetrates the stratum corneum and induces high rate apoptosis in tumoral cell line and alterations in in vivo zebrafish model

PRIETO MJ; TEMPRANA, F.; MONTANARI, JORGE; CALIENNI, M. N.; ALONSO, S. DEL VALLE

Basilea

Congreso; CLINAM 10th European and Global Conference and Exhibition for Clinical Nanomedicine and Targeted Medicine: Witnessing the Solutions and Tackilng the Hurdles; 2017

CLINAM European Foundation for Clinical Nanomedicine

Abstract Ultradeformable liposomes (UL) are a drug delivery nanosystem with an elastic modulus lower than conventional liposomes. This feature makes UL capable to penetrate the stratum corneum (SC) of the skin and release their content into the viable epidermis, where neoplastic events occur in skin cancer. 5-Fluorouracil (5FU) is a classic antineoplastic drug, administered parenterally, with severe side effects. Therefore, the incorporation of 5FU in UL could improve specific-site delivery and aims to reduce side effects. In this work, a UL 5FU-loaded formulation (UL5FU) of soy phosphatidylcholine and sodium cholate, as border activator, was obtained as a future topical treatment for skin cancer. The nanosystem was biophysically characterized in size, encapsulation efficiency, stability in time, lamellarity, drug release, ultradeformability, drug to lipid ratio and drug-membrane interaction. Penetration properties were studied on a Saarbrücken Penetration Model device with human skin explants. The skin penetration was determined by tape stripping technique (Figure 1), removing each layer of SC and analyzing the presence of the drug in the upper SC (layers 1-10), bottom SC (layers 11-20) and viable epidermis plus dermis (VE). Also, skin was incubated with UL5FU performed with two fluorescent labels, Rhodamine-DPPE for the tracking of membrane lipids and FITC for the tracking of aqueous content. Intact skin and transversal sections of 20 µm in thickness (Figure 2) were studied by confocal laser scanning microscopy. Penetration profile of 5FU was quantified in each layer of the SC and in the VE after tape stripping by fluorescence. In vitro studies were carried out in two human cell lines: HaCaT (non tumoral) and SK-Mel-28 (tumoral). Cytotoxicity was studied by MTT, Crystal Violet and Neutral Red at 4 and 24 hs. Uptake of UL5FU with Rhodamine-DPPE and FITC was analyzed at 4 and 37 ºC in both lines. Induction of apoptosis after 6 hs of incubation was assessed by flow cytometry, it was detected with annexin V conjugated with FITC (Figure 3). In vivo studies in zebrafish (Danio rerio) larvae (4-7 days post-fecundation) were performed to determine toxicologic and teratogenic effects of 5FU and UL5FU. Zebrafish is an increasingly accepted animal model for nanotoxicological studies because it high correlation effects and other advantages if compare to other animals. Effects were assessed by determination of swimming activity, with an automated system with infrared microbeam arrangement that detects interruption by the larva body in real time; alterations in the heart rate; morphological changes (changes in eye area, rostrocaudal length and spinal cord length, absence of swim bladder, arched body, tissue ulceration and pericardial edema); and histological analysis of brain -particularly the raphe populations-, spinal cord and liver in parasagittal serial sections of 10 µm thickness staining with hematoxylin-eosin (Figure 4). The UL5FU formulation was stable over time and incorporation of 5FU did not alter significantly its deformability properties, although it interacts with the liposomal membrane. It was capable to penetrate the SC and deliver 5FU in the VE of intact skin, at similar conditions of temperature and water gradient of in vivo context. Even though, UL5FU affected both cell lines, the formulation increased strongly the cytotoxic effect of 5FU in the tumoral line after 24 hs of treatment. Furthermore, SK-Mel-28 shown a higher uptake than HaCaT and apoptosis studies shown a differential effect between both lines. After 6 hs of incubation there was significant induction of apoptosis only in the tumor line by UL5FU and higher than the free drug. From in vivo studies, valuable toxicological and teratogenic information was obtained. UL5FU and the free drug produced alterations in the swimming activity -but a very different range of doses-, which could be related to neurological damage, and induced morphological changes in larvae. Cardiological effects of 5FU observed correspond to a secondary effect of the drug, because the heart is a target organ of it. The research continues to develop a novel formulation of UL as delivery system of Vismodegib for topical treatment of basal cell carcinoma. Vismodegib is a recently approved by FDA drug for oral administration. References -Betancourt, T., Doiron, A., Homan, K. A., & Brannon-Peppas, L. (2009). Controlled release and nanotechnology. In Nanotechnology in drug delivery (pp. 283-312). Springer New York. -Cevc, G., & Blume, G. (1992). Lipid vesicles penetrate into intact skin owing to the transdermal osmotic gradients and hydration force. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1104(1), 226-232. -Montanari, J., Maidana, C., Esteva, M. I., Salomon, C., Morilla, M. J., & Romero, E. L. (2010). Sunlight triggered photodynamic ultradeformable liposomes against Leishmania braziliensis are also leishmanicidal in the dark. Journal of Controlled Release, 147(3), 368-376. -Prieto, M. J., Gutierrez, H. C., Arévalo, R. A., Chiaramoni, N. S., & del Valle Alonso, S. (2012). Effect of risperidone and fluoxetine on the movement and neurochemical changes of zebrafish. Open Journal of Medicinal Chemistry, 129-138. -Schaefer, U., & Loth, H. (1996). An ex-vivo model for the study of drug penetration into human skin. Pharm. Res, 13(366), b24. -Wagner, H., Kostka, K. H., Lehr, C. M., & Schaefer, U. F. (2000). Drug distribution in human skin using two different in vitro test systems: comparison with in vivo data. Pharmaceutical research, 17(12), 1475-1481.