Biomedical Engineering Reference
In-Depth Information
Biodegradable NMs, ~200 nm in diameter biocompatible polymer, PLGA
have the potential for sustained gene delivery have been reported.
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The wild-
type (wt) p53 gene (DNA) was loaded in NMs (PLGA~DNA) and used in a breast
cancer cell line to determine its antiproliferative activity.
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The PLGA~DNA
were formulated using a multiple-emulsion-solvent evaporation technique before
these were used for cell transfection. Cells transfected with wt-p53 DNA that
were loaded in the PLGA NMs demonstrated higher sustained antiproliferative
effect than those with naked wt-p53 DNA or wt-p53 DNA complexed with a com-
mercially available transfecting agent (Lipofectamine).
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The breast cancer cells
that were transfected with wt-p53 DNA-loaded PLGA NMs showed sustained
levels of p53 mRNA compared with cells which were transfected with naked
wt-p53 DNA or the wt-p53 DNA-Lipofectamine complex. Further studies using
fluorescent labeled DNA under a confocal microscopy and quantitative analyses
with a microplate reader also indicated sustained intracellular localization of
PLGA~DNA which suggested the slow release of DNA from the NMs that were
localized inside the cells. The loading of the DNA in PLGA is described in the
protocol below.
7.3.2.1 Protocol for Gene Delivery with Biodegradable
Polymers PLGA
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(1)
Dissolve 1 mg DNA and 2 mg of nuclease free Bovine serum albumin
(BSA) in 200 uL of Tris-EDTA, pH 8.
(2)
Prepare the primary emulsion by sonicating 30 mg of PLGA 50:50 (intrin-
sic viscosity of 1.32 g/dL, Birmingham Polymers) in 1 mL of chloroform
with the above DNA solution for 2 min over an ice bath using a probe
sonicator set at 55 W of energy output.
(3)
Emulsify the result further into a 2% w/v polyvinyl alcohol (PVA, 30-
70 kDa, Sigma) solution for 5 min to form a multiple (water-in-oil-in-
water) emulsion.
(4)
Stir this emulsion overnight on a magnetic stir plate that is kept in a
vacuum desiccator for 1 h to evaporate chloroform.
(5)
Separate the NMs using ultracentrifugation at 35,000 rpm for 20 min at
4 °C.
(6)
Wash twice to remove PVA and unencapsulated DNA.
(7)
Resuspend the pellet in 5 mL of sterile water by sonication for 30 s.
(8)
Lyophilize the suspension (−80 °C and <10 umHg) for 48 h.
(9)
Collect all washing of the PLGA~DNA.
(10)
Measure the OD at 260 nm.
(11)
Compare with original DNA that is not exposed to PLGA and calculate
the amount of DNA loaded on the PLGA.
(12)
Take a 0.1 mg/mL suspension of the PLGA~DNA and sonicate in ice bath
for 30 s.
(13)
Measure the size of the NMs in a particle size analyzer.
(14)
Establish the zeta potential before and after the DNA loading.
