Gene Delivery Using Chemical Methods - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 146

and Nanoparticles
DNA-loaded PLGA is generally formulated using the water-oil-water double-emulsion
solvent-evaporation technique [115-117] . However, the spray-drying technique was
also reported in some studies [113,116] . Initially, poor loading of DNA was observed
with the double-emulsion technique until Tinsley Bown et al. succeeded in optimizing
the double-emulsion process by changing the organic solvent from dichloromethane to
ethyl acetate and optimizing various process parameters [118] . Optimized techniques
of water-oil-water emulsion/solvent evaporation as well as a water-oil emulsion/
solvent diffusion to produce PEGylated PLGA nanoparticles with high DNA loading
(up to 10-12 g pDNA/mg polymer) and high encapsulation efficiency were reported
(60-90%) [119] .
During preparation of DNA-loaded particles, shear force generated should be min-
imum to retain DNA supercoiling [120] . However, it must be balanced for obtaining
the desired droplet size. Condensation of DNA with PLL reduces its susceptibility to
shear forces [121,122] . Also, creation of a more viscous solution by reducing temper-
ature minimizes the effects of shear forces during creation of the DNA-PLG emul-
sion. Ando et al. proposed a cryopreparation technique to prevent the exposure of the
pDNA to shear forces [123] . Positively surface charged microparticle using cationic
surfactants 1,2-dioleoyloxy-3-(trimethylammonio) propane chloride (DOTAP) were
prepared, and DNA adsorbed on positively charged surface efficiently to give high
drug lading. However, surface adsorbed DNA may result in a burst effect and degrade
easily [124] .
Attempts to enhance the loading of PLGA as the encapsulation of pDNA in PLGA
have been challenging. It was demonstrated that hydrophilic PLGA shows higher
encapsulation efficiency. The molecular weight of the PLGA also affects encapsu-
lation efficiency significantly. Preparations of negatively charged PLL grafts onto
PLGA facilitate the adsorption of the negatively charged DNA, thereby improving
drug loading [125] . Another approach was to reduce the negative charge of pDNA
by condensing it with poly(amino acids) (like PLL) before encapsulation in PLGA
particles. This approach demonstrated 75-85% loading [122,126] . A similar strategy
using poly(ethyleneimine), pDMAEMA, and chitosan has been reported to enhance
loading PLGA particles with DNA [127-130] .
PLGA particles exhibit an initial burst release of pDNA, followed by slow release
for several days/weeks [113,114,118] . Release of pDNA is controlled by degradation
of the PLGA particles through the hydrolytic process. Increase in hydrophilicity of
PLGA resulted in a faster release of pDNA. In addition, release patterns also depend
on the method of manufacturing; nanoparticles prepared by the water-oil emulsion-
diffusion technique released DNA rapidly, whereas those obtained by the water-oil-
water emulsion method showed an initial burst followed by a slow release for at least
28 days [119] . Surface adsorption of pDNA, rather than entrapping the pDNA in the
PLGA particles, resulted in a substantially faster release in vitro [114] .
The process of internalizing PLGA particles into cells is a concentration and time
dependent endocytic process. Uptake of particulate systems could occur through var-
ious processes such as by phagocytosis, fluid phase pinocytosis, or receptor-mediated
endocytosis [131,132] . DNA-loaded micro- and nanoparticles are internalized by

4.2.2.2.1 PLGA Microparticles and Nanoparticles particles and Nanoparticles