Nanopharmacology - Nanomaterials for Medical Applications - page 300

The studies of Prabha 84 used the wild-type p53-gene loaded in PLGA NMs for
the transfection of breast cancer cell MDA MB231, a cell line that is derived from
a human ductal carcinoma from patient with metastatic disease and had not been
subjected to chemotherapy. 92 The p53 gene rearrangement in this cell line causes
a reduced level of expression of p53 making it suitable for studying the effect
of p53 gene therapy. 93 Using multiple-emulsion-solvent evaporation technique,
wild-type p53-gene was loaded in PLGA NMs to determine the gene's antipro-
liferative activity in a breast cancer cell line. The intracellular trafficking of both
the NPs and the nanoparticle-entrapped DNA were monitored through the use
of coumarin and luciferase labeled genes. The results 93 indicated that cells trans-
fected with wt-p53 DNA-loaded NPs showed significantly greater antiprolifera-
tive effect than those with naked wt-p53 DNA or wt-p53 DNA complexed with a
commercially available transfecting agent (Lipofectamine). The cells transfected
with wt-p53 DNA-loaded NPs also showed sustained p53 mRNA levels compared
to cells which were transfected with naked wt-p53 DNA exhibiting the sustained
antiproliferative activity of NPs. Microscope examination of cells transfected with
fluorescently labeled DNA indicated intracellular localization of DNA with NMs
that may be interpreted as a result of the slow release of DNA from NMs localized
inside the cells. Thus, this study showed that nanoparticle-mediated wt-p53 gene
delivery resulted in antiproliferative activity, that can be used for cancer therapy.
Chitosan nanoparticles (cNPs) have also been demonstrated as effective gene-
delivery vehicles. 73 pDNAs were transported in cNPs and expressed in the lung
epithelium of mice. The results indicated that intranasal delivery of plasmids by
cNPs resulted in sustained expression of the encoded protein in the lung which led
to an effective supply therapeutic or prophylactic levels of an immunomodulatory
molecule.
7.3.3 Electrostatic Loading of DNA in NMs
The negative nature of the DNA backbone is advantageous in linking with posi-
tively charged NMs leading to a DNA~NMs complexes that are formed through
electrostatic binding between the positive charges of the NMs and the negative
charges of the DNA ( Figure 7.2 ). This electrostatic interaction is strong enough
and is very easy to carry out in any laboratory. The 30 nm IOMNPs that are
modified with amines exhibit an positive charge at low pH <5. When exposed
to single strands of DNA (ssDNA), the IOMNPs easily form ssDNA~IOMNP
complex. When the ssDNA~IOMNP complex is exposed to the complementary
ssDNA, the double stranded DNA (dsDNA are formed on the surface of the
IOMNP resulting in dsDNA~IOMNP. The formation of the dsDNA~IOMNP
complex is verified with a dye that intercalates with dsDNA but not with a
ssDNA. Acridine orange can intercalate with the dsDNA~IOMNP complex.
The same process has also been used in liposome and other polymer-mediated
gene transfer. 55,56,94 At Ocean NanoTech, 30 nm IOMNPs were used to capture
dsDNA from lysed breast cancer cell line SK-BR3 as described below.