Polymer coated liposomes for use in the oral cavity - a study of the in vitro toxicity, effect on cell permeability and interaction with mucin.

In this study we investigated the in vitro toxicity, impact on cell permeability and mucoadhesive potential of polymer-coated liposomes intended for use in the oral cavity. A TR146 cell line was used as a model. The overall aim was to end up with a selection of safe polymer coated liposomes with promising mucoadhesive properties for drug delivery to the oral cavity. The following polymers were tested: chitosan, low-methoxylated pectin (LM-pectin), high-methoxylated pectin (HM-pectin), amidated pectin (AM-pectin), Eudragit, poly(N-isopropylacrylamide-co-methacrylic acid) (p(NIPAAM-co-MAA)), hydrophobically modified hydroxyethyl cellulose (HM-HEC), and hydrophobically modified ethyl hydroxyethyl cellulose (HM-EHEC). With chitosan as an exception, all the systems exhibited no significant effect on cell viability and permeability at the considered concentrations. Additionally, all the formulations showed to a varying degree an interaction with mucin (BSM type I-S); the positively charged formulations exhibited the strongest interaction, while the negatively and neutrally charged formulations displayed a moderate or low interaction. The ability to interact with mucin makes all the liposomal formulations promising for oromucosal administration. Although the chitosan-coated liposomes affected the cell viability, this formulation also influenced the cell permeability, which makes it an interesting candidate for systemic drug delivery from the oral cavity.

Characterization of temperature induced changes in liposomes coated with poly(N-isopropylacrylamide-co-methacrylic acid).

Positively charged liposomes were coated with the negatively charged and temperature sensitive poly(N-isopropylacrylamide-co-methacrylic acid) by electrostatic deposition. Too low or too high polymer concentrations lead to unstable suspensions. However, intermediate polymer concentrations (0.05-0.2 wt.%) result in relatively stable suspensions of polymer-coated liposomes. At elevated temperatures the thickness of the polymer layer around the coated liposomes increased sharply at 40 °C, due to the formation of polymer multilayers. At higher temperatures, a contraction of the adsorbed polymer layer was observed. The uncoated liposomes exhibited an interesting transition in size and intensity of the scattered light when heated, attributed to the transition from the gel to liquid crystalline phase. Rheo-SALS (small angle light scattering under shear conditions) measurements demonstrated that the polymer coating was stable under shear at physiological temperature. It also revealed an anomalous high scattered intensity of the uncoated liposomes compared to the coated liposomes. This discrepancy was diminished at higher temperatures, and can probably be attributed to the change from a non-spherical, polyhedron-like conformation of the uncoated liposomes in the gel phase to a spherical shape above the phase transition.

Studies on pectin-coated liposomes and their interaction with mucin.

Pectin is a polymer with well-known mucoadhesive properties. In this study, liposomes were coated with three different types of pectin. Their properties were characterized and their mucoadhesiveness was estimated by a novel in vitro approach. Two different types of commercially available mucin were investigated in order to choose the best candidate for the method. The effect of pH on the properties of the coated liposomes and the interaction with mucin was also studied. The pectin-coated liposomes and the complexes they formed with mucin were characterized by dynamic light scattering (DLS), zeta potential and turbidity measurements. The zeta potential of the liposomes shifted from positive to negative after coating with pectin. They also exhibited larger diameters, and the liposomes coated with HM-pectin were the largest. After the addition of mucin, the zeta potential shifted to a less negative value and the sizes of the pectin-coated liposomes increased. The complexes formed between mucin and the HM-pectin-coated liposomes were the largest, while the smallest were formed with the LM-pectin-coated liposomes. The pH was found to affect the interaction between the coated liposomes and mucin. DLS was conducted on an ALV goniometer to gain information about the diffusivity of the samples, the relative scattered intensities and to obtain an optimal characterization of the size distributions. The results correlated well with measurements from an automatized light scattering instrument (Zetasizer Nano ZS).

Effect of chitosan malate on viability and cytoskeletal structures morphology of Caco-2 cells.

The aim of this work was to evaluate the effects of the treatment with chitosan malate (CM) on viability of Caco-2 cells and on the morphology and the integrity of their cytoskeletal structures (microtubules, microfilaments). Cytotoxicity of CM, both as a solution and as microparticles obtained by spray drying, was evaluated by using the reduction of MTT reagent; microtubule and microfilaments organization of Caco-2 cells treated with CM solution was examined with immunofluorescence techniques in monolayers fixed with the glutaraldehyde-borohydride method. CM as a solution displayed a concentration-dependent cytotoxicity towards Caco-2 cells, with viability percentages of 5 ± 2%, 7 ± 3% and 31 ± 15% at 15, 10 and 5mg/mL, respectively, while at 2.5mg/mL or less cell viability was 90% or higher. CM microparticles also produced a remarkable cytotoxic effect (cell viability 84 ± 17%, 16 ± 8% and 5 ± 6% after treatment with 1, 5 and 10mg CM per well, respectively), resulting more toxic than CM solution. Microtubules pattern of Caco-2 cells, which is a network regularly arranged around the nucleus, appeared deeply modified by CM treatment in a concentration-dependent way, with progressive microtubule changes in length and spatial disposition and deposition of fluorescent aggregates at the periphery of the cells. Furthermore, after treatment with 5-15mg/mL CM, remarkable alterations of actin organization were observed, with a progressive disruption of the network of stress fibers and the appearance of actin aggregates inside the cell cytoplasm. In conclusion, viability and the cytoskeletal pattern of Caco-2 cells are modified by treatment with CM at high concentrations, which might be locally reached in vivo after application of drug-loaded chitosan microparticles onto mucosal cells.