Biomedical Engineering Reference
In-Depth Information
system. In their study, the L-DNA-AuNP was used for improved transfection
efficiency and reduced cytotoxicity. DNA strands were loaded onto the AuNPs
to increase the payload per delivery event that enhances transfection efficiency.
But, negatively charged DNA strands that are exposed in this system deterio-
rates during contact between DNA-AuNPs and cells because the cell membrane
itself carries a weak negative charge. This phenomenon is overcome by attach-
ing a cationic lipid layer to DNA-modified AuNP that neutralizes the nega-
tive charge thereby, facilitating cell membrane penetration of DNA-modified
AuNPs as well as protecting the modified DNA.
7.3.1.1 Protocol for Loading DNA in AuNP and Encapsulating
with Liposome
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The modification of the AuNP is based on electrostatic interaction with the DNA
at a controlled pH. The process involved adjusting the pH of the citrate AuNPs to
9 and exposure to the pDNA followed by subsequent modification with the lipids.
(1)
Centrifuge the citrate-stabilized AuNPs (15, 30,50, and 80 nm) and collect
the pellet.
(2)
Dilute the pDNA to 20 ng/uL (in 10 mM phosphate buffer at pH 9).
(3)
Measure the concentration of the pDNA using a UV/Vis absorbance at
260 nm.
(4)
Add the pDNA to the AuNPs and mix vigorously. The negatively charged
phosphates backbone of the pDNA chain facilitates the exchange with the
citrate to form pDNA~AuNP which leads to the formation of the higher
bonding energy between Au and phosphates compared with that between
Au and carboxylates in citrates.
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(5)
Incubate this mixture in an orbital shaker at 300 rpm at 37 °C overnight.
(6)
Centrifuge the pDNA-loaded AuNPs and separate the supernatant from
the residue.
(7)
Measure the amount of unreacted pDNA from the supernatant by taking
the absorbance at 260 nm.
(8)
Calculate the amount of pDNA loaded per AuNP particle by subtracting
the amount that was unreacted from the original concentration. The reacted
pDNA is divided by the number of AuNPs used during the pDNA loading.
(9)
Check the stability of the pDNA-AuNPs by adding 200 nM NaCl salt
concentration to 50 mM AuNP. During this saline addition, color change
from reddish wine to purple means aggregation of AuNPs due to unstable
or incomplete binding of the pDNA to the AuNPs.
(10)
Mix the pDNA-AuNP with 1.5 uL or 3 uL liposomes and incubate at
25 °C for 20 min with vigorous shaking.
Results from the studies of Rhim and collaborators that published this proto-
col
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showed that the AuNPs had 10-100 pDNA strands per particle depending
on the size. Zeta potential measurements indicated that the AuNPs contained
