PLGA-based gene delivering nanoparticle enhance suppression effect of miRNA in HePG2 cells
Nanoscale Research Letters
PLGA-based gene delivering nanoparticle enhance suppression effect of miRNA in HePG2 cells
Gao Feng Liang 0
Yan Liang Zhu 0
Bo Sun 0
Fei Hu Hu 0
Tian Tian 0
Shu Chun Li 0
Zhong Dang Xiao 0
0 State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University , Nanjing, 210096 , China
The biggest challenge in the field of gene therapy is how to effectively deliver target genes to special cells. This study aimed to develop a new type of poly(D,L-lactide-co-glycolide) (PLGA)-based nanoparticles for gene delivery, which are capable of overcoming the disadvantages of polyethylenimine (PEI)- or cationic liposomebased gene carrier, such as the cytotoxicity induced by excess positive charge, as well as the aggregation on the cell surface. The PLGA-based nanoparticles presented in this study were synthesized by emulsion evaporation method and characterized by transmission electron microscopy, dynamic light scattering, and energy dispersive spectroscopy. The size of PLGA/PEI nanoparticles in phosphate-buffered saline (PBS) was about 60 nm at the optimal charge ratio. Without observable aggregation, the nanoparticles showed a better monodispersity. The PLGA-based nanoparticles were used as vector carrier for miRNA transfection in HepG2 cells. It exhibited a higher transfection efficiency and lower cytotoxicity in HepG2 cells compared to the PEI/ DNA complex. The N/P ratio (ratio of the polymer nitrogen to the DNA phosphate) 6 of the PLGA/PEI/DNA nanocomplex displays the best property among various N/P proportions, yielding similar transfection efficiency when compared to Lipofectamine/DNA lipoplexes. Moreover, nanocomplex shows better serum compatibility than commercial liposome. PLGA nanocomplexes obviously accumulate in tumor cells after transfection, which indicate that the complexes contribute to cellular uptake of pDNA and pronouncedly enhance the treatment effect of miR-26a by inducing cell cycle arrest. Therefore, these results demonstrate that PLGA/PEI nanoparticles are promising non-viral vectors for gene delivery.
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Introduction
MicroRNAs (miRNAs) are small, highly conserved,
noncoding RNAs that regulate gene expression at the
posttranscriptional level. They involve in various cellular
mechanisms including development, differentiation,
proliferation, and apoptosis. The pivotal roles of these
miRNAs in human cancers have been discovered [1,2], and
the therapeutic applications of miRNA have been
developed using various viral vectors [3,4].
However, the disadvantages of viral vectors limited
their application in gene delivery, such as immunogenic/
inflammatory responses, low loading capacity, large scale
manufacturing, and quality control [5]. Consequently,
more attention have been paid on non-viral gene delivery
vectors in recent years, such as liposomes (lipoplexes),
polycationic polymers (polyplexes), and organic or
inorganic nanoparticles (nanoplexes) [6]. To enhance gene
delivery effect, various cationic complexes have been
developed for delivering plasmid DNA, antisense, or
siRNA into cells [7-9]. Poly(D,L-lactide-co-glycolide)
(PLGA) were extensively assessed for their ability of
delivering variety of therapeutic agents [10-12]. PLGA
nanoparticles were shown to escape from the endo-lysosomal
compartment to the cytoplasmic compartment and
release their contents over extended periods of time [13].
These features rendered PLGA nanoparticles as potential
tool for gene delivery efficiently.
Polyethylenimine (PEI) is water-soluble, linear, or
branched polymers with a protonable amino group [14,15].
Due to their high cationic charge density at physiological
pH, PEIs are able to form non-covalent complexes with
DNA, siRNA, and antisense oligodeoxynucleotide.
Therefore, PEIs hold a prominent position among the
polycationic polymers used for gene delivery [16-18]. The
intracellular release of PEI/nucleic acids complexes from
endosomes is considered as relying on the protonation of
amines in the PEI molecule, which leading to osmotic
swelling and subsequent burst of the endosomes.
Moreover, PEIs also facilitate nucleic acid entry into the nucleus
[19,20]. However, it has been reported that long PEI chains
are highly effective in gene transfection, but more cytotoxic
[14,21,22].
In order to overcome these hurdles in gene therapy
and improve gene delivery efficiency, we developed
novel non-liposome-based cationic polymers which are
composed of PLGA as the core and cationic PEI as the
shell. The biodegradable PLGA nanoparticles, modified
with a polyplexed PEI coating, were tested by loading
the expression vector (pDNA) of miR-26a, which is
capable of inducing cell cycle arrest in HepG2 cells. In this
study, nanoparticles of controlled size and persistent
shape have been obtained by an emulsion evaporation
method and characterized by transmission electron
microscopy (TEM), dynamic light scattering (DLS), and
energy dispersive spectroscopy (...truncated)