ABSTRACT
Scaffold-based systems have become essential in biomedical research, providing the possibility of building in vitro models that can better mimic tissue/organic physiology. A relatively new family of biomimetics-pseudo-proteins (PPs)-can therefore be considered especially promising in this context. Three different artificial leucine-based LPP films were tested in vitro as potential scaffolding materials. In vitro experiments were performed using two types of cells: primary mouse skin fibroblasts and a murine monocyte/macrophages cell line, RAW264.7. Cell adhesion and cell spreading were evaluated according to morphological parameters via scanning electron microscopy (SEM), and they were assessed according to actin cytoskeleton distribution, which was studied via confocal laser microscopy. Cell proliferation was evaluated via an MTT assay. Cell migration was studied using time-lapse microscopy. SEM images for both types of cells demonstrated prominent adhesion and perfect cell spreading on all three LPPs. Analyses of actin cytoskeleton organization revealed a high number of focal adhesions and prominent motility-associated structures. A certain stimulation of cell proliferation was detected in the cases of all three LPPs, and two of them promoted macrophage migration. Overall, our data suggest that the LPPs used in the study can be considered potential cell-friendly scaffolding materials.
ABSTRACT
Purpose: Drug delivery to treat ocular diseases still is a challenge in ophthalmology. One way to achieve drug delivery that is investigated currently is topical administration of drug-loaded polymeric nanoparticles (NPs) that are able to penetrate ocular barriers. The purpose of this study was optimal preparation of NPs made from pseudo-proteins and evaluation of their ability to penetrate ocular tissues. Methods: Biodegradable NPs of various types were prepared by nanoprecipitation of pseudo-protein composed of l-leucine (L), 1,6-hexanediol (6), and sebacic acid (8) (8L6). Arginine-based cationic polyester amides 8R6 and comb-like polyester amide containing lateral PEG-2000 chains along with 8L6 anchoring fragments in the backbones were used to construct positively charged and PEGylated NPs. They were loaded with fluorescein diacetate (FDA) or rhodamine 6G (Rh6G) as fluorescent probes. Suspensions of the NPs were given to cultivated microglial cells and retinal pigment epithelial (RPE) cells as well as topically on eyes of C57BL/6 mice. Penetration of NPs into the eyes was checked by fluorescence analysis. Results: NPs were prepared, and their properties were characterized. Cultured microglial cells and RPE cells took up the NPs. After topical administration, penetration of NPs into the cornea of the eyes was clearly seen. Small amounts of fluorescent dyes were also found in the lens, the retina, and the sclera depending on the type of NPs. Conclusions: The results showed that the new NPs penetrate ocular tissues after topical administration and are internalized by the cells. This raises confidence that the NPs may be useful carriers of therapeutic agents for ocular delivery.
