nucleus through the nuclear pore complexes (NPCs) in the nuclear envelopes [150].
Using the Xenopus nuclear envelope reassembly (XNER) assay, Symens et al. [138]
found that the nuclear enclosure of NPs was dependent on the size (with 100 and
200 nm NPs being better included than the 500 nm NPs) and charge (with positively
charged NPs being better included than negatively charged or PEGylated NPs) of
the DNA–NP complexes. Accordingly, smaller NPs (generally <25 nm) are considered to be more efficient for NP-based gene delivery systems. Interestingly,
irrespective of the size of the DNA–NP complex, combining an NP-based gene
delivery system with physical methods such as electroporation does not seem to
have any beneficial effect on the gene delivery efficiency.
6.7 NP:DNA Ratio (Nitrogen:Phosphate Ratio), Concentration
and Incubation Period
The NP:DNA ratio influences the N:P ratio, which in turn determines the shape and
size of the DNA–NP complexes and the degree of DNA compaction. Furthermore,
if the ratio of NP:DNA is suboptimal, the overall charge of the complexes might be
negative, neutral or strongly positive, which can lead to inefficient adsorption to the
negatively charged surface of mammalian cells. Thus, the NP:DNA ratio needs to
be optimized to obtain an optimal rate of gene transfection. Furthermore, the
optimal concentration of a given NP–DNA complex should be evaluated for each
cell type because they are known to be dependent on cell type and cell density.
Different cell types may also require different incubation periods with the DNA–NP
complex to achieve maximal transfection level. This should be kept in mind when
determining the length of incubation after transfection. If the time-point of maximal
expression is not known for a particular cell line, a time-course experiment may be
necessary.
6.8 Controlled Intracellular Release of DNA
Besides minimizing the cytotoxicity, surface coating and functionalization of NPs
are also carried out occasionally to allow slow or delayed release of DNA until the
cells enter mitosis and dissolve their nuclear envelope. Under such circumstances,
DNA has an increased chance of coming in contact with the host genome and,
hence, of increased transfection rate. Mahor et al. [52] observed that encapsulation
of DNA in HA increased transgene expression when it was delivered into MSCs
using branched PEI as a transfecting agent. DNA-loaded NPs can also be
immobilized on the surface-coated ECM (a process termed reverse transfection),
which not only provides a surface for cell attachment but also sustains the release of
DNA from the surface, thereby inducing transgene expression for a prolonged
76
P. Pushp et al.
Using the Xenopus nuclear envelope reassembly (XNER) assay, Symens et al. [138]
found that the nuclear enclosure of NPs was dependent on the size (with 100 and
200 nm NPs being better included than the 500 nm NPs) and charge (with positively
charged NPs being better included than negatively charged or PEGylated NPs) of
the DNA–NP complexes. Accordingly, smaller NPs (generally <25 nm) are considered to be more efficient for NP-based gene delivery systems. Interestingly,
irrespective of the size of the DNA–NP complex, combining an NP-based gene
delivery system with physical methods such as electroporation does not seem to
have any beneficial effect on the gene delivery efficiency.
6.7 NP:DNA Ratio (Nitrogen:Phosphate Ratio), Concentration
and Incubation Period
The NP:DNA ratio influences the N:P ratio, which in turn determines the shape and
size of the DNA–NP complexes and the degree of DNA compaction. Furthermore,
if the ratio of NP:DNA is suboptimal, the overall charge of the complexes might be
negative, neutral or strongly positive, which can lead to inefficient adsorption to the
negatively charged surface of mammalian cells. Thus, the NP:DNA ratio needs to
be optimized to obtain an optimal rate of gene transfection. Furthermore, the
optimal concentration of a given NP–DNA complex should be evaluated for each
cell type because they are known to be dependent on cell type and cell density.
Different cell types may also require different incubation periods with the DNA–NP
complex to achieve maximal transfection level. This should be kept in mind when
determining the length of incubation after transfection. If the time-point of maximal
expression is not known for a particular cell line, a time-course experiment may be
necessary.
6.8 Controlled Intracellular Release of DNA
Besides minimizing the cytotoxicity, surface coating and functionalization of NPs
are also carried out occasionally to allow slow or delayed release of DNA until the
cells enter mitosis and dissolve their nuclear envelope. Under such circumstances,
DNA has an increased chance of coming in contact with the host genome and,
hence, of increased transfection rate. Mahor et al. [52] observed that encapsulation
of DNA in HA increased transgene expression when it was delivered into MSCs
using branched PEI as a transfecting agent. DNA-loaded NPs can also be
immobilized on the surface-coated ECM (a process termed reverse transfection),
which not only provides a surface for cell attachment but also sustains the release of
DNA from the surface, thereby inducing transgene expression for a prolonged
76
P. Pushp et al.