In vitro and in vivo studies of ocular topically administered NLC for the treatment of uveal melanoma

Uveal melanoma is one of the most common and aggressive intraocular malignancies, and, due to its great capability of metastasize, it constitutes the most incident intraocular tumor in adults. However, to date there is no effective treatment since achieving the inner ocular tissues still constitutes one of the greatest challenges in actual medicine, because of the complex structure and barriers. Uncoated and PEGylated nanostructured lipid carriers were developed to achieve physico-chemical properties (mean particle size, homogeneity, zeta potential, pH and osmolality) compatible for the ophthalmic administration of (S)-(–)-MRJF22, a new custom-synthetized prodrug for the potential treatment of uveal melanoma.

Highlights

• NLCs with adequate features for ocular administration were designed and prepared.
• Prodrug encapsulation into NLCs protects the cornea from irritative side effect.
• Antiangiogenic assay confirmed the antitumoral activity of the loaded NLCs.
• Loaded NLCs were able to prevent ocular inflammation in vivo.
• Preliminary fluorescence images suggested NLCs ability to reach the inner eye.

The colloidal physical stability was investigated at different temperatures by Turbiscan® Ageing Station. Morphology analysis and mucoadhesive studies highlighted the presence of small particles suitable to be topically administered on the ocular surface. In vitro release studies performed using Franz diffusion cells demonstrated that the systems were able to provide a slow and prolonged prodrug release. In vitro cytotoxicity test on Human Corneal Epithelium and Human Uveal Melanoma cell lines and Hen’s egg-chorioallantoic membrane test showed a dose-dependent cytotoxic effect of the free prodrug on corneal cells, whose cytocompatibility improved when encapsulated into nanoparticles, as also confirmed by in vivo studies on New Zealand albino rabbits.

Antiangiogenic capability and preventive anti-inflammatory properties were also investigated on embryonated eggs and rabbits, respectively. Furthermore, preliminary in vivo biodistribution images of fluorescent nanoparticles after topical instillation in rabbits’ eyes, suggested their ability to reach the posterior segment of the eye, as a promising strategy for the treatment of choroidal uveal melanoma.

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Materials

Kolliphor RH40 was provided by BASF Italia S.p.a. (Cesano Modena, Italy); Oleoyl Macrogol-6 Glycerides (Labrafil) was a gift from Gattefossé Italia s.r.L. (Milano, Italy); Hydrogenated Coco-Glycerides (Softisan 100) was bought from IOI Oleo GmbH (Oleochemicals, IOI group); Isopropyl myristate (IPM) was purchased from Farmalabor (Canosa di Puglia, Italy). Tris (hydroxymethyl)aminomethane buffer, methanol (MeOH) and ethanol (EtOH) were bought from Merck (Darmstadt, Germany). Didodecyldimethylammonium bromide (DDAB), Polyethylene Glycol 1500 (PEG 1500), Fluorescein isothiocyanate (FITC), phosphate buffer saline (PBS) components (NaCl, KCl, Na2HPO4, KH2PO4), artificial tear fluid (ATF) components (CaCl2·2H2O, NaHCO3, NaCl), mucin (mucin from porcine stomach type II), simulated tear fluid (STF) components (NaCl, NaHCO3, CaCl2·2H2O and KCl), benzalkonium chloride, trypsin-EDTA (1X), 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), DMSO, DAPI (4′,6-diamidino-2-phenylindole), sodium arachidonate, Hank’s solution components (CaCl2·2H2O, MgSO4, KCl, K2HPO4, NaHCO3, NaCl, Na2PO4, glucose), paraformaldehyde, glucose, Triton X-100, Mowiol components (Mowiol, glycerol) were purchased from Sigma Aldrich (MO, USA). OCT compound (Sakura Finetek, Torrance, CA, USA). Regenerated cellulose membranes (Spectra/Por CE; Mol. Wet. Cutoff 3500) were supplied by Spectrum (Los Angeles, CA, USA). (S)-(–)-MRJF22 was synthetized by the research group of Prof. Agostino Marrazzo, in the Medicinal Chemistry Laboratory of the Department of Drug and Health Sciences (Università di Catania) (Barbaraci et al., 2021). All solvents (LC grade) were from VWR International (Milan, Italy).

Cinzia Cimino, Elena Sánchez López, Angela Bonaccorso, Lorena Bonilla, Teresa Musumeci, Josefa Badia, Laura Baldomà, Rosario Pignatello, Agostino Marrazzo, Carla Barbaraci, María Luisa García, Claudia Carbone,
In vitro and in vivo studies of ocular topically administered NLC for the treatment of uveal melanoma,
International Journal of Pharmaceutics, 2024, 124300, ISSN 0378-5173, https://doi.org/10.1016/j.ijpharm.2024.124300.