electroporation are, therefore, commonly incorporated into naked DNA delivery
methods. These methods induce temporary holes in the plasma membrane, which
allows easy entry of the naked DNA into the cells. Among the two commonly used
methods, direct microinjection requires special equipment (cell injector) and
training but is suitable for non-dividing cells such as neurons and for large-sized
cells such as oocytes and embryos. Electroporation, on the other hand, is an efficient
method of naked DNA delivery but can result in a high rate of cellular death due to
the electrical stimulation. Unfortunately, despite efficient delivery into the cells,
gene integration into host genome and subsequent gene expression efficiency has
been very low in both the methods. This is primarily due to their degradation in
cytoplasm by nuclease enzymes, sequestration by DNA-binding proteins and
cytoskeletal elements in the cytoplasm and their inability to cross the nuclear
membrane.
2.2 Compacted DNA (DNA NPs)
DNA is negatively charged and, therefore, can be condensed by polycations that
range from inorganic polycations to organic polyamines to polypeptides such as
polylysine and protamine. Because condensed DNAs are compacted, they are less
accessible to nuclease degradation in the cytoplasm and can cross the nuclear
membrane at an increased efficiency. Studies have shown that naked DNA
(~1,200 nm [2]) can acquire toroidal (~50 nm [3]), ellipsoidal (~22 Â 50 nm [4]),
rod-shaped (~8–11 Â 200 nm [4]) and spherical (~130 nm [2]) configuration upon
compaction by polycations such as spermine, CK30-PEG trifluoroacetate, CK30PEG acetate and PEG-POD, respectively, in the nanometre size range and,
accordingly, may technically be called DNA NPs.
The shape and size of the DNA NPs and the percentage of the DNA that
condenses can be controlled by controlling the length of the polycation, the
concentration of the salt (e.g. NaCl), the molecular charge ratio of polycation:
DNA and the type of counterion [5–7]. The polycation may also impart additional
functionality such as cell-type-specific gene delivery. For example, DNA
compacted with galactosylated polylysine can specifically target hepatocytes,
which express the asialoglycoprotein receptor [7]. Similarly, polylysine can be
PEGylated to avoid the likelihood of particle aggregation [7]. When properly
compacted and processed, DNA NPs are homogeneous in size and shape, consist
only of compacted DNA, do not form aggregates, are colloidally stable in physiological salt concentrations and protect the DNA from digestion by cytoplasmic
nuclease. Nonetheless, problems associated with electroporation and microinjection methods of introducing the DNA or DNA NPs into the cells cannot be solved
by DNA compaction alone.
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P. Pushp et al.
methods. These methods induce temporary holes in the plasma membrane, which
allows easy entry of the naked DNA into the cells. Among the two commonly used
methods, direct microinjection requires special equipment (cell injector) and
training but is suitable for non-dividing cells such as neurons and for large-sized
cells such as oocytes and embryos. Electroporation, on the other hand, is an efficient
method of naked DNA delivery but can result in a high rate of cellular death due to
the electrical stimulation. Unfortunately, despite efficient delivery into the cells,
gene integration into host genome and subsequent gene expression efficiency has
been very low in both the methods. This is primarily due to their degradation in
cytoplasm by nuclease enzymes, sequestration by DNA-binding proteins and
cytoskeletal elements in the cytoplasm and their inability to cross the nuclear
membrane.
2.2 Compacted DNA (DNA NPs)
DNA is negatively charged and, therefore, can be condensed by polycations that
range from inorganic polycations to organic polyamines to polypeptides such as
polylysine and protamine. Because condensed DNAs are compacted, they are less
accessible to nuclease degradation in the cytoplasm and can cross the nuclear
membrane at an increased efficiency. Studies have shown that naked DNA
(~1,200 nm [2]) can acquire toroidal (~50 nm [3]), ellipsoidal (~22 Â 50 nm [4]),
rod-shaped (~8–11 Â 200 nm [4]) and spherical (~130 nm [2]) configuration upon
compaction by polycations such as spermine, CK30-PEG trifluoroacetate, CK30PEG acetate and PEG-POD, respectively, in the nanometre size range and,
accordingly, may technically be called DNA NPs.
The shape and size of the DNA NPs and the percentage of the DNA that
condenses can be controlled by controlling the length of the polycation, the
concentration of the salt (e.g. NaCl), the molecular charge ratio of polycation:
DNA and the type of counterion [5–7]. The polycation may also impart additional
functionality such as cell-type-specific gene delivery. For example, DNA
compacted with galactosylated polylysine can specifically target hepatocytes,
which express the asialoglycoprotein receptor [7]. Similarly, polylysine can be
PEGylated to avoid the likelihood of particle aggregation [7]. When properly
compacted and processed, DNA NPs are homogeneous in size and shape, consist
only of compacted DNA, do not form aggregates, are colloidally stable in physiological salt concentrations and protect the DNA from digestion by cytoplasmic
nuclease. Nonetheless, problems associated with electroporation and microinjection methods of introducing the DNA or DNA NPs into the cells cannot be solved
by DNA compaction alone.
54
P. Pushp et al.