The purpose of this study was to produce poly(DL-lactic-co-glycolic acid) (PLGA) - based microparticles (MPs), externally decorated with hyaluronic acid (HA). The MPs are intended for intravitreal injections in the treatment of posterior eye segment and have been designed to prolong the release of growth factors into the vitreous body, therefore aiming to increase the time interval between two consecutive injections. The MPs, prepared by a modified double emulsion-solvent evaporation technique and loaded with bovine serum albumins (BSA) and ciliary neurotrophic factor (CNTF), were spherical, with a diameter around 70 mu m and a > 90% encapsulation efficiency. Energy Dispersive Spectroscopy (EDS) outcomes indicated that HA presence in the external aqueous phase of the emulsion did affect the surface properties of MPs. Moreover, poloxamers drastically slowed down MP degradation properties which are, in turn, closely related to their ability to prolong drug release. This is promising for the envisaged application of the produced MPs. Further work will be devoted to optimizing MP formulation with respect to the envisaged intravitreal route of administration.

Microvesicles (MVs) are lipid bilayered membranous vesicles containing functional lipids, proteins, RNA and DNA that are produced by most cells. The physiological significance of MVs has become evident, and increased MV counts and the contents of MVs are nowadays also associated with different pathophysiological phenomena. The goal of the field is to use MVs as diagnostic and therapeutic tools. To achieve this, the understanding of the mechanisms of the functions of MVs should be understood better and additionally, reliable methods for the quantification and characterization of MVs should be developed and standardized. The aim of the study was to determine differences in platelet-derived MVs produced by different activation mechanisms. The second aim was to set up and optimize a protocol based on the reaction of sulphur, phosphate and vanillin (SPV) for measuring lipid content of MVs. The third aim was to study the effect of thrombin and proteinase inhibitor PPACK to the vesiculation of platelets. Platelets were isolated from the whole blood of healthy volunteers and vesicles were produced by platelet agonists mediating thrombogenic activation (thrombin and collagen, TC), pathophysiological activation (lipopolysaccharide, LPS) and Ca-ionophore (A23187) as positive control for vesiculation. Quantification and size determination of produced MVs was done using Nanoparticle Tracking Analysis (NTA). MVs were characterized by protein content using bicinchonic acid assay (BCA) and by lipid content using SPV-reaction. MVs had great activation-dependent differences in the lipid and the protein content. Activation with Ca-ionophore produced the most MVs, but the lipid and protein content was only a fraction from (patho)physiologically induced MVs. Only TC increased vesiculation. Vesicle subpopulations had significant difference in lipid content. Thrombin and proteinase inhibitor PPACK mediated inhibition of platelet formation in all of the activations, but the effect was not statistically significant. The mechanism of inhibition was likely to be proteinase inhibitor mediated. The isolation of vesicle populations using differential centrifugation proved to isolate studied populations only partially and the quantification method with NTA was susceptible to concentrated samples. SPV protocol reacted with different intensity to different lipids. In the future, quantification and isolation methods for MVs and the subpopulations of MVs should be improved. Additionally, to understand the physiologically relevant mechanisms of platelet-derived vesicle formation, the inhibitor experiments with PPACK should be continued, because the number of replicates was too low to see significant effects due to a large donor-dependent deviation. Since MVs are heterogenous cellular multitools affecting varying (patho)physiological phenomena, optimization and standardization of methods should be continued in order to study MVs properly.