Abstract
Purpose: :
Our research objective is to develop a biodegradable nanoparticle capable of delivering CTGF–B antisense oligonucleotides through the corneal epithelium and stromal layers to moderate wound healing. The purpose of this study is to synthesize and characterize chitosan (CS) tripolyphosphate (TPP) nanoparticles conjugated with polyethylene glycol (PEG) for use in our corneal drug delivery system. The system is attractive due to its enhanced ability to transfect cells and degrade harmlessly in vivo.
Methods: :
Nanoparticle suspensions were prepared by adding TPP drop–wise to dilute CS solutions under gentle stirring and acidic conditions. Particle formation went to completion within 1 hour via ionic gelation. To determine the effect of CS concentration on particle size and charge, TPP was fixed at a 5:1 ratio and five CS concentrations were made ranging from .10% – .40%. To determine the effect of TPP on particle size and charge, CS was fixed at .25% and TPP was added at ratios ranging from 2:1 to 6:1. Suspensions of CS with low MW and high MW PEG were made using the same methods, but prior to TPP addition, varying concentrations of PEG 0–40% (weight/volume) were added to the CS solution. The particles were analyzed using the Coulter Particle Counter, Brookhaven ZetaPlus, and transmission electron microscopy. The particles were evaluated for mean particle size, polydispersity, morphology and surface charge.
Results: :
The increase in CS concentration resulted in an increase in particle size and a decrease in zeta potential. Particles formed, ranged from 150nm to10µm. The larger sizes appear to be the result of agglomeration, which occurred at concentrations near .40%. The TPP study also showed a trend for linear increase in particle size with an increase in the ratio of chitosan to TPP. However the zeta potential also increased linearly with the CS to TPP ratio. The morphology of the particles under microscope examination revealed particles with a profile more polygonal than spherical. The addition of PEG to the suspension showed similar shapes, except for an additional cloudy halo.
Conclusions: :
The CS–TPP system produced nanoparticles in the desired size range (less than one micron) and without the use of chemicals damaging to the oligonucleotide or toxic to the host. Adjusting the amount of TPP in the system can modify the affinity of the particle for the cell surface. These nanoparticles have the potential to be useful in treating corneal disease and injury as a sustained release system.
Keywords: gene transfer/gene therapy • wound healing • cornea: basic science