Optical Projection Tomography Technique for Image Texture and Mass Transport Studies in Hydrogels Based on Gellan Gum.

The microstructure and permeability are crucial factors for the development of hydrogels for tissue engineering, since they influence cell nutrition, penetration, and proliferation. The currently available imaging methods able to characterize hydrogels have many limitations. They often require sample drying and other destructive processing, which can change hydrogel structure, or they have limited imaging penetration depth. In this work, we show for the first time an alternative nondestructive method, based on optical projection tomography (OPT) imaging, to characterize hydrated hydrogels without the need of sample processing. As proof of concept, we used gellan gum (GG) hydrogels obtained by several cross-linking methods. Transmission mode OPT was used to analyze image microtextures, and emission mode OPT to study mass transport. Differences in hydrogel structure related to different types of cross-linking and between modified and native GG were found through the acquired Haralick's image texture features followed by multiple discriminant analysis (MDA). In mass transport studies, the mobility of FITC-dextran (MW 20, 150, 2000 kDa) was analyzed through the macroscopic hydrogel. The FITC-dextran velocities were found to be inversely proportional to the size of the dextran as expected. Furthermore, the threshold size in which the transport is affected by the hydrogel mesh was found to be 150 kDa (Stokes' radii between 69 and 95 Å). On the other hand, the mass transport study allowed us to define an index of homogeneity to assess the cross-linking distribution, structure inside the hydrogel, and repeatability of hydrogel production. As a conclusion, we showed that the set of OPT imaging based material characterization methods presented here are useful for screening many characteristics of hydrogel compositions in relatively short time in an inexpensive manner, providing tools for improving the process of designing hydrogels for tissue engineering and drugs/cells delivery applications.

Intracellular trafficking of hyaluronic acid-chitosan oligomer-based nanoparticles in cultured human ocular surface cells.

PURPOSE: Nanoparticles are a promising alternative for ocular drug delivery, and our group has proposed that they are especially suited for ocular mucosal disorders. The goal of the present study was to determine which internalization pathway is used by cornea-derived and conjunctiva-derived cell lines to take up hyaluronic acid (HA)-chitosan oligomer (CSO)-based nanoparticles (HA-CSO NPs). We also determined if plasmids loaded onto the NPs reached the cell nucleus. METHODS: HA-CSO NPs were made of fluoresceinamine labeled HA and CSO by ionotropic gelation and were conjugated with a model plasmid DNA for secreted alkaline phosphatase. Human epithelial cell lines derived from the conjunctiva and the cornea were exposed to HA-CSO NPs for 1 h and the uptake was investigated in living cells by fluorescence microscopy. The influence of temperature and metabolic inhibition, the effect of blocking hyaluronan receptors, and the inhibition of main endocytic pathways were studied by fluorometry. Additionally, the metabolic pathways implicated in the degradation of HA-CSO NPs were evaluated by lysosome identification. RESULTS: There was intracellular localization of plasmid-loaded HACSO NPs in both corneal and conjunctival cells. The intracellular presence of NPs diminished with time. HA-CSO NP uptake was significantly reduced by inhibition of active transport at 4 °C and by sodium azide. Uptake was also inhibited by blocking hyaluronan receptors with anti-CD44 Hermes-1 antibody, by excess HA, and by filipin, an inhibitor of caveolin-dependent endocytosis. HA-CSO NPs had no effect on cell viability. The transfection efficiency of the model plasmid was significantly higher in NP treated cells than in controls. CONCLUSIONS: HA-CSO NPs were internalized by two different ocular surface cell lines by an active transport mechanism. The uptake was mediated by hyaluronan receptors through a caveolin-dependent endocytic pathway, yielding remarkable transfection efficiency. Most of HA-CSO NPs were metabolized within 48 h. This uptake did not compromise cell viability. These findings further support the potential use of HA-CSO NPs to deliver genetic material to the ocular surface.

Bioamine-crosslinked gellan gum hydrogel for neural tissue engineering.

Neural tissue engineering and three-dimensional in vitro tissue modeling require the development of biomaterials that take into account the specified requirements of human neural cells and tissue. In this study, an alternative method of producing biomimetic hydrogels based on gellan gum (GG) was developed by replacing traditional crosslinking methods with the bioamines spermidine and spermine. These bioamines were proven to function as crosslinkers for GG hydrogel at +37 °C, allowing for the encapsulation of human neurons. We studied the mechanical and rheological properties of the formed hydrogels, which showed biomimicking properties comparable to naïve rabbit brain tissue under physiologically relevant stress and strain. Human pluripotent stem cell-derived neuronal cells demonstrated good cytocompatibility in the GG-based hydrogels. Moreover, functionalization of GG hydrogels with laminin resulted in cell type-specific behavior: neuronal cell maturation and neurite migration.

Comparison of different cationized proteins as biomaterials for nanoparticle-based ocular gene delivery.

Cationized polymers have been proposed as transfection agents for gene therapy. The present work aims to improve the understanding of the potential use of different cationized proteins (atelocollagen, albumin and gelatin) as nanoparticle components and to investigate the possibility of modulating the physicochemical properties of the resulting nanoparticle carriers by selecting specific protein characteristics in an attempt to improve current ocular gene-delivery approaches. The toxicity profiles, as well as internalization and transfection efficiency, of the developed nanoparticles can be modulated by modifying the molecular weight of the selected protein and the amine used for cationization. The most promising systems are nanoparticles based on intermediate molecular weight gelatin cationized with the endogenous amine spermine, which exhibit an adequate toxicological profile, as well as effective association and protection of pDNA or siRNA molecules, thereby resulting in higher transfection efficiency and gene silencing than the other studied formulations.

Nanoparticles based on naturally-occurring biopolymers as versatile delivery platforms for delicate bioactive molecules: an application for ocular gene silencing.

Nanoparticles based on naturally-occurring biopolymers, most of them endogenous macromolecules, were designed as a versatile generation of delivery platforms for delicate bioactive molecules. The design of these nanosystems was specifically based on our recent finding about the ability of endogenous polyamine spermine (SPM) to interact with anionic biopolymers (ABs) generating ionically cross-linked nanosystems. The initial first generation of these delivery platforms, based on glycosaminoglycans and other polysaccharides, showed a very high association capacity for some delicate bioactive proteins such as growth factors, but a limited capacity to associate negatively charged molecules, such as pDNA and siRNA. However, the versatility of these nanosystems in terms of composition allowed us to customise the association of active ingredients and their physicochemical characteristics. Concretely, we prepared and incorporated gelatine cationized with spermine (CGsp) to their composition. The resulting modified formulations were characterised by a nanometric size (150-340 nm) and offer the possibility to modulate their zeta potential (from -35 to 28 mV), providing an efficient association of nucleic acids. The biological evaluation of these optimised nanosystems revealed that they are able to be internalised in vivo into corneal and conjunctival tissues and also to provide a significant siRNA gene silencing effect.