| Popis: |
Age-related macular degeneration (AMD) is the leading cause of vision loss and blindness in people over age 65 in industrialized nations. The exudative AMD accounts for only 10% to 20% of AMD cases, but causes 80 to 90% of cases with severe vision loss related to AMD. The current intravitreal injection of anti-VEGF (vascular endothelial growth factor) therapies using Avastin or Lucentis has several unmet clinical needs, such as to minimize the frequency of injection, to decrease the systemic side effects, and to evaluate the effect of therapies. We propose to develop a drug delivery system that could perform sustained release of anti-VEGF drug by multifunctional drug-loaded microparticles (MPs).A modified double emulsion technique has firstly been used to fabricate biodegradable poly-lactic-co-glycolic acid (PLGA) MPs. The MPs could provide fluorescence, ultrasound, and OCT contrast simultaneously. Meanwhile, linear correlations has been observed between fluorescence intensity and MPs concentration, same as ultrasound intensity and MPs concentration. One of the anti-VEGF drugs (Avastin) has been conjugated to MPs to increase binding efficiency for target delivery. However, the modified double emulsion process is not suitable for encapsulating fragile protein drugs, such as Lucentis. The electrospray process has been introduced to overcome the limitations of the double emulsion process. Both monolayer MPs and double-layer MPs with core-shell structure are fabricated by single-axial and co-axial electrospray, respectively. To optimize the fabrication process, the effects of the applied voltage and the flow rate on the resulting and co-axial jet and the MPs size has been investigated. The results indicate that both the average size of MPs and the diameter of the co-axial jet increased as the flow rate increasing and decreased as the applied voltage increasing. A theoretical model using instability analysis has been developed to understand the process physics and predict the process outcome. Meanwhile, the biodegradation time of PLGA MPs as well as the bioavailability of Lucentis under single-axial electrospray is tested by enzyme-linked immunosorbent assay (Elisa).After the MPs fabrication and characterization, the inflammation experiment on intravitreal injection of MPs has been investigated by immunocytochemical analysis on in vivo chick model. CD45, Islet1, and Draq5 are used to label microglia, horizontal cells, and the nuclear DNA, respectively. TUNEL and CC3 are selected as cell death model to further investigate the cell apoptosis after injection. The results show that MP injection at a dose of 20¿¿g and below does not cause inflammation under our experimental condition. However, significant microglia reactive as well as cell death (labeled by TUNEL) provided the evidence that MPs at a dose of 200¿¿g and above induces significant retinal inflammation and cell death. |
