of the polymer [109, 110]. It is important to note that internalization of positively
charged polyplexes is facilitated, given that the cell surface is negatively charged
(because of the presence of proteoglycans) so, in general, nanoparticles with
smaller size and higher zeta potential are most likely to be uptaken by cells.
Once in the blood stream, hydrophobic nanospheres are rapidly opsonized and
extensively cleared by the mononuclear phagocyte system (MPS). This problem
can be prevented by surface modification, such as coating with hydrophilic
polymers, or by formulating nanospheres with biodegradable copolymers with
hydrophilic characteristics [111]. Moreover, the introduction of hydrophilic
polymers, such as oligo(ethylene glycol) or poly(ethylene glycol) (PEG, probably
the most widely used), or others such as the zwiterrionic 2-methacryloxyethyl
phosphorylcholine (MPC) or poly(hydroxyethyl methacrylate) (PHEMA) can provide steric stabilization to otherwise unstable polyplexes in water. They can also
protect DNA against protein adsorption and degradation by enzymatic nucleases.
Active targeting to certain cells or organs can be attained by the recognition at
the molecular level between a ligand and receptors overexpressed on cell
membranes through specific interactions. Once the molecules bind to the receptors,
the complex is internalized via receptor-mediated endocytosis, facilitating the
cellular uptake of the carrier of this ligand. This will not be treated in this review
because, most of the time, active targeting with nanoparticles is achieved by
conjugating an antibody or protein to a polymer, thus influencing the physicochemical properties of the polymer and polyplex formed thereof. Nevertheless, this
is an extremely important aspect of gene delivery for in vivo applications [112].
Intracellular Processes
As previously mentioned, for most cell types, the size requirement for particle
uptake via endocytosis is in the order of 200 nm or less, and a net positive charge
on the surface of the conjugate has been shown to be important for triggering
uptake. Moreover, to be effective, these polyplexes must be optimized at all stages
of the delivery process, ranging from target-cell recognition (attachment of
targeting ligands in order to be recognized and taken up by specific cells)
[113–115] to their escape from the endosome-enclosed milieu, resistance to cytoplasmic degradative enzymes such as nucleases, and release of the genetic material
at the desired site of action [116]. Thus, polymers should bind efficiently and
protect the genetic material against nonspecific interactions with proteins and cell
membranes in blood, but efficiently release it in the cytosol in order to favor gene
expression (Scheme 13) [117]. Indeed, when the polycation binds too strongly, it
results in impaired gene expression.
Concerning the intracellular trafficking of polyplexes, it begins in early
endosomal vesicles. These early endosomes subsequently fuse with sorting
endosomes, which in turn transfer their contents to the late endosomes. Late
endosomal vesicles are acidified (pH 5–6) by membrane-bound proton-pump
ATPases. The normal process is that the endosomal content is then relocated to
Polyelectrolyte Complexes of DNA and Polycations as Gene Delivery Vectors
129
charged polyplexes is facilitated, given that the cell surface is negatively charged
(because of the presence of proteoglycans) so, in general, nanoparticles with
smaller size and higher zeta potential are most likely to be uptaken by cells.
Once in the blood stream, hydrophobic nanospheres are rapidly opsonized and
extensively cleared by the mononuclear phagocyte system (MPS). This problem
can be prevented by surface modification, such as coating with hydrophilic
polymers, or by formulating nanospheres with biodegradable copolymers with
hydrophilic characteristics [111]. Moreover, the introduction of hydrophilic
polymers, such as oligo(ethylene glycol) or poly(ethylene glycol) (PEG, probably
the most widely used), or others such as the zwiterrionic 2-methacryloxyethyl
phosphorylcholine (MPC) or poly(hydroxyethyl methacrylate) (PHEMA) can provide steric stabilization to otherwise unstable polyplexes in water. They can also
protect DNA against protein adsorption and degradation by enzymatic nucleases.
Active targeting to certain cells or organs can be attained by the recognition at
the molecular level between a ligand and receptors overexpressed on cell
membranes through specific interactions. Once the molecules bind to the receptors,
the complex is internalized via receptor-mediated endocytosis, facilitating the
cellular uptake of the carrier of this ligand. This will not be treated in this review
because, most of the time, active targeting with nanoparticles is achieved by
conjugating an antibody or protein to a polymer, thus influencing the physicochemical properties of the polymer and polyplex formed thereof. Nevertheless, this
is an extremely important aspect of gene delivery for in vivo applications [112].
Intracellular Processes
As previously mentioned, for most cell types, the size requirement for particle
uptake via endocytosis is in the order of 200 nm or less, and a net positive charge
on the surface of the conjugate has been shown to be important for triggering
uptake. Moreover, to be effective, these polyplexes must be optimized at all stages
of the delivery process, ranging from target-cell recognition (attachment of
targeting ligands in order to be recognized and taken up by specific cells)
[113–115] to their escape from the endosome-enclosed milieu, resistance to cytoplasmic degradative enzymes such as nucleases, and release of the genetic material
at the desired site of action [116]. Thus, polymers should bind efficiently and
protect the genetic material against nonspecific interactions with proteins and cell
membranes in blood, but efficiently release it in the cytosol in order to favor gene
expression (Scheme 13) [117]. Indeed, when the polycation binds too strongly, it
results in impaired gene expression.
Concerning the intracellular trafficking of polyplexes, it begins in early
endosomal vesicles. These early endosomes subsequently fuse with sorting
endosomes, which in turn transfer their contents to the late endosomes. Late
endosomal vesicles are acidified (pH 5–6) by membrane-bound proton-pump
ATPases. The normal process is that the endosomal content is then relocated to
Polyelectrolyte Complexes of DNA and Polycations as Gene Delivery Vectors
129