Non-viral Gene Therapy - Intracellular Delivery: Fundamentals and Applications - page 606

transfection activity. For polymer vectors, their molecular weight and molecular
weight distribution also affect the physicochemical characteristics of polyplexes.
The structure and morphology of polyplexes are thought to be kinetically controlled
(Lai and van Zanten 2001 ), which is often related to the mixing order (for example,
adding polycation to DNA solution or DNA to polycation solution). Although the
presence of cationic moieties is necessary for complexation, the positive charges of
lipoplexes or polyplexes are responsible for their cytotoxicity, at least to some
degree (Lv et al. 2006 ). Furthermore, the electrostatic forces should be carefully
modulated so that the payload genes can be disassociated at the site of action.
Alternatively, therapeutic genes can be incorporated into nano- or micro-particles
based on biodegradable polymers like polyesters, polyanhydrides or poly(ortho
esters). DNA-loaded microparticles can be produced by methods frequently adopted
to fabricate microparticles for sustained drug delivery (Jilek et al. 2005 ). Their size
can vary from nanometer to micrometer length scale, depending on preparation
techniques and process parameters. Potential DNA degradation during fabrication
and release, and low DNA bioavailability are the main issues for these sustained
delivery vectors. These problems can be partly addressed by adsorbing DNA onto
microparticles that have been peripherally decorated with cationic moieties (Singh
et al. 2000 ; Kasturi et al. 2005 ). In addition, a recent study by Discher's group has
demonstrated that siRNA and oligonucleotides (ODNs) can also be incorporated
into polymersomes assembled from neutral amphiphiles (Kim et al. 2009c ).

2.2 Cellular Entry

2.2.1 Non-specific Uptake

Sulfated proteoglycans are among the most negatively charged components of the
cell, which are considered to be the targets of nonviral complexes carrying positive
charges. For cellular uptake, the cationic moieties of lipoplexes or polyplexes first
bind to anionic proteoglycans by electrostatic interactions. Study by Mounkes et al.
demonstrated that lipoplexes could not transfer the payload DNA in Raji cells that
do not express proteoglycans (Mounkes et al. 1998 ). Mislick and Baldeschwieler
have showed optimal gene transfer and expression in HeLa cells by polylysine/
polyplexes when the N/P ratio was above one, while a N/P ratio <1 led to the low
level of gene expression (Mislick and Baldeschwieler 1996 ). Significant reduction
in luciferase expression was observed after the treatment of HeLa cells with
sodium, a potent inhibitor of proteoglycan sulfation. Additionally, transfection was
also dramatically inhibited by heparin and heparan sulfate as well as chondroitin
sulfate B. Other unidentified cell surface components may also mediate the binding
and subsequent uptake of nonviral vectors.
The cellular uptake of nonviral vectors may be accomplished mainly through
endocytic pathways such as clathrin-mediated, caveolae-mediated, or macropino-
cytic routes. Whereas drugs that can inhibit both clathrin- and caveolae-mediated