Development and Optimisation of Nanoparticulate System for Brain Delivery of L-DOPA

Abstract

Background and Aims: Parkinson’s disease (PD) is the second most common movement disorder and can cause substantial morbidity and a shortened life span. L-DOPA (LD) is the primary drug used for the treatment of PD. However, only 1% of the given dose is transported unchanged to the central nervous system (CNS) after oral administration due to its peripheral decarboxylation. As a result, LD has to be administered at high dose in combination with a peripheral decarboxylase inhibitor that causes severe side-effects. The aim of this project was to design nanoparticulate drug delivery system by using poly (D, Llactide- co-glycolide) (PLGA) as a polymeric matrix for the delivery of LD across blood-brain barrier (BBB). Nanoparticles (NPs) provide a feasible choice as drug delivery device to cross the BBB because it may overcome the biological barrier and increase the bioavailability of the drug in the brain. Methods: LD-loaded PLGA NPs were prepared using modified water-in-oil-in-water (W1/O/W2) emulsion solvent evaporation technique. The NPs were coated with polysorbate 80 (P80) to enhance their BBB permeability. Samples in in vitro drug release study and stability study were measured by a novel isocratic high performance liquid chromatography (HPLC) method. A central composite design (CCD) was applied for optimisation of the formulation parameters. The chemical and physical properties of PLGA NPs such as particles size, entrapment efficiency, zeta potential, porosity, surface charge and drug-polymer compatibility were measured. The cytotoxicity of PLGA NPs to the Caco-2 cells and the rate and extent of Caco-2 cell uptake of drug-loaded PLGA NPs were evaluated. In vitro BBB model was developed by co-culturing rat brain microvascular endothelial cells (RBMVECs) and rat astrocytes on Transwell insert. This model was morphologically and functionally characterised. Eventually, the transport of LD-loaded P80 coated PLGA NPs across BBB model was evaluated. Results and discussion: The optimum formulation was obtained at the concentration of PLGA (5%, w/v) and PVA (6%, w/v); and faster organic solvent removal rate (700 rpm). The corresponding particle size was 256 nm and the entrapment efficiency was 62%. The in vitro LD release profile showed a burst release and followed by slow and steady release over 7 days. Differential scanning calorimetry (DSC) and Fourier transform infrared (FT-IR) revealed that LD was presented in the NPs in an amorphous state and no interactions between drug and polymer were observed. The SEM images of NPs showed that all particles had spherical shape with porous outer skin. The LD-loaded PLGA NPs exhibited good stability over 3 months stored at 2-8°C. The in vitro evaluation study on Caco-2 cells showed that LD-loaded PLGA NPs were non-toxic up to 1000 μg/ml. The cellular uptake of LD-loaded PLGA NPs would be increased when decreasing the hydrophilicity of particle. In vitro BBB model (based on coculturing RBMVEC cells and astrocytes to mimic in vivo BBB) was successfully established. Results indicated that P80 coated NPs were efficiently taken up by brain endothelial cells. NPs coated with P80 successfully transported across BBB model with an apparent permeability coefficient (Papp) value of ~3.13x10-5 cm/sec. The transport process was energydependent. No opening of tight junction was observed when NPs were applied to the BBB model. This suggested that P80 coated PLGA NPs preferentially transported across BBB without disrupting the BBB integrity. Conclusion: This project has demonstrated that PLGA NPs can be utilised as controlled release drug delivery system (DDS) for the oral delivery of LD. Encapsulated LD can be protected from peripheral decarboxylation before they are released. P80 coated PLGA NPs represent a very promising preparation for the delivery of LD across the BBB without opening the tight junction. This technique provides a potential to enhance LD bioavailability by protecting LD from peripheral decarboxylation.