Biomedical Engineering Reference
In-Depth Information
on the fact that divalent metal cations are easily to form ionic complexes with nucleic
acids and these complexes have excellent transportability across the cell membrane
via ion channel, the technique of CaP co-precipitation for
in vitro
transfection is
employed as a routine laboratory procedure (Graham and van der Eb
1973
).
The standard transfection method of calcium phosphate was originally estab-
lished by Graham and van der Eb in 1973 (Graham and van der Eb
1973
). For the
preparation of the CaP carrier for transfection, calcium chloride solution with DNA
and phosphate-buffered saline solution are mixed together, which results in the
formation of fine precipitates (nano- and micropaticles) of calcium phosphate with
DNA. There are several factors that are concerned with cell transfection efficiency
of the standard calcium phosphate method, including the precipitation conditions
(such as pH value, concentrations of calcium chloride and DNA, temperature, and
the time between precipitation and transfection), as well as the morphology and
size of the nanoparticles. Insufficiently protected nanocrystals will grow to micro-
crystals by Ostwald-ripening and therefore loss their transfection ability as investi-
gated by Orrantia and colleagues (Orrantia and Chang
1990
). Moreover, the
transfection efficiency also strongly depends on the cell type. Quite often, the trans-
fection reproducibility of calcium phosphate is poor.
Inspired by the observation that CaP formed nanoparticles generally possess good
biocompatibility and biodegradability as well as relatively high transfection activity,
a number of groups have concentrated on CaP nanoparticles for transfection (Zhang
and Kataoka
2009
). Jordan et al. reported the application of CaP/DNA coprecipitates
to achieve efficient transfection in cultured cell lines (Jordan et al.
1996
; Jordan and
Wurm
2004
). They found that highly efficient transient and stable transfections could
be achieved by optimizing physicochemical conditions of initiation and growth of
precipitate complexes, such as the concentrations of calcium and phosphate, tem-
perature, DNA concentration, and reaction time. One of the successful examples in
cancer gene therapy using DNA-loaded CaP nanoparticles (CPNP-DNA) of 23.5-
34.5 nm was reported by Cai et al. in 2005 (Liu et al.
2005
). The CPNP-DNA com-
plex efficiently adhered to the cell membrane and entered the cells, leading to an
increase in the amount of DNA in the cells, thus resulting in a successful enhance-
ment in transfection efficiency. It should be noted that for these CaP-based nano-
systems loaded with genetic materials, the transfection efficiency decreased with
increasing incubation time for nanoparticles formation, which was attributed to the
time-dependent particle growth in the presence of a protective outer layer (Fasbender
et al.
1998
). Furthermore, the genetic material located on the surface of the nanopar-
ticles was very susceptible to enzymatic attacks by nuclease, resulting in the desta-
bilization of nanoparticles and thus reduced transfection activity.
Apparently, CaP-based transfection systems alone do not meet the requirements
for
in vivo
applications. Since PEGylation of CaP nanoparticles helps to minimize
the nonspecific interactions between the particles and the biological species present
in the biological medium, PEGylated CaP nanoparticles are expected to yield stable
and biocompatible nanocarriers for nucleic acid delivery (Kakizawa and Kataoka
2002
; Kakizawa et al.
2004
). Kataoka and coworkers have synthesized PEG-
b
-
poly(aspartic acid) (PEG-PAA) block copolymers to prepare CaP nanoparticles
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