4 research outputs found
How Does the Spacer Length of Cationic Gemini Lipids Influence the Lipoplex Formation with Plasmid DNA? Physicochemical and Biochemical Characterizations and their Relevance in Gene Therapy
Lipoplexes formed by the pEGFP-C3 plasmid DNA (pDNA)
and lipid
mixtures containing cationic gemini surfactant of the 1,2-bis(hexadecyl
dimethyl ammonium) alkanes family referred to as C<sub>16</sub>C<sub><i>n</i></sub>C<sub>16</sub>, where <i>n</i> =
2, 3, 5, or 12, and the zwitterionic helper lipid, 1,2-dioleoyl-<i>sn</i>-glycero-3-phosphatidylethanolamine (DOPE) have been studied
from a wide variety of physical, chemical, and biological standpoints.
The study has been carried out using several experimental methods,
such as zeta potential, gel electrophoresis, small-angle X-ray scattering
(SAXS), cryo-TEM, gene transfection, cell viability/cytotoxicity,
and confocal fluorescence microscopy. As reported recently in a communication
(<i>J. Am. Chem. Soc.</i> <b>2011</b>, <i>133</i>, 18014), the detailed physicochemical and biological studies confirm
that, in the presence of the studied series lipid mixtures, plasmid
DNA is compacted with a large number of its associated Na<sup>+</sup> counterions. This in turn yields a much lower effective negative
charge, <i>q</i><sub>pDNA</sub><sup>–</sup>, a value that has been experimentally
obtained for each mixed lipid mixture. Consequently, the cationic
lipid (CL) complexes prepared with pDNA and CL/DOPE mixtures to be
used in gene transfection require significantly less amount of CL
than the one estimated assuming a value of <i>q</i><sub>DNA</sub><sup>–</sup> = −2.
This drives to a considerably lower cytotoxicity of the gene vector.
Depending on the CL molar composition, α, of the lipid mixture,
and the effective charge ratio of the lipoplex, ρ<sub>eff</sub>, the reported SAXS data indicate the presence of two or three structures
in the same lipoplex, one in the DOPE-rich region, other in the CL-rich
region, and another one present at any CL composition. Cryo-TEM and
SAXS studies with C<sub>16</sub>C<sub><i>n</i></sub>C<sub>16</sub>/DOPE-pDNA lipoplexes indicate that pDNA is localized between
the mixed lipid bilayers of lamellar structures within a monolayer
of ∼2 nm. This is consistent with a highly compacted supercoiled
pDNA conformation compared with that of linear DNA. Transfection studies
were carried out with HEK293T, HeLa, CHO, U343, and H460 cells. The
α and ρ<sub>eff</sub> values for each lipid mixture were
optimized on HEK293T cells for transfection, and using these values,
the remaining cells were also transfected in absence (-FBS-FBS) and
presence (-FBS+FBS) of serum. The transfection efficiency was higher
with the CLs of shorter gemini spacers (<i>n</i> = 2 or
3). Each formulation expressed GFP on pDNA transfection and confocal
fluorescence microscopy corroborated the results. C<sub>16</sub>C<sub>2</sub>C<sub>16</sub>/DOPE mixtures were the most efficient toward
transfection among all the lipid mixtures and, in presence of serum,
even better than the Lipofectamine2000, a commercial transfecting
agent. Each lipid combination was safe and did not show any significant
levels of toxicity. Probably, the presence of two coexisting lamellar
structures in lipoplexes synergizes the transfection efficiency of
the lipid mixtures which are plentiful in the lipoplexes formed by
CLs with short spacer (<i>n</i> = 2, 3) than those with
the long spacer (<i>n</i> = 5, 12)
Effects of a Delocalizable Cation on the Headgroup of Gemini Lipids on the Lipoplex-Type Nanoaggregates Directly Formed from Plasmid DNA
Lipoplex-type
nanoaggregates prepared from pEGFP-C3 plasmid DNA
(pDNA) and mixed liposomes, with a gemini cationic lipid (CL) [1,2-bis(hexadecyl
imidazolium) alkanes], referred as (C<sub>16</sub>Im)<sub>2</sub>C<sub><i>n</i></sub> (where C<sub><i>n</i></sub> is
the alkane spacer length, <i>n</i> = 2, 3, 5, or 12, between
the imidazolium heads) and DOPE zwitterionic lipid, have been analyzed
by zeta potential, gel electrophoresis, SAXS, cryo-TEM, fluorescence
anisotropy, transfection efficiency, fluorescence confocal microscopy,
and cell viability/cytotoxicity experiments to establish a structure–biological
activity relationship. The study, carried out at several mixed liposome
compositions, α, and effective charge ratios, ρ<sub>eff</sub>, of the lipoplex, demonstrates that the transfection of pDNA using
CLs initially requires the determination of the effective charge of
both. The electrochemical study confirms that CLs with a delocalizable
positive charge in their headgroups yield an effective positive charge
that is 90% of their expected nominal one, while pDNA is compacted
yielding an effective negative charge which is only 10–25%
than that of the linear DNA. SAXS diffractograms show that lipoplexes
formed by CLs with shorter spacer (<i>n</i> = 2, 3, or 5)
present three lamellar structures, two of them in coexistence, while
those formed by CL with longest spacer (<i>n</i> = 12) present
two additional inverted hexagonal structures. Cryo-TEM micrographs
show nanoaggregates with two multilamellar structures, a cluster-type
(at low α value) and a fingerprint-type, that coexist with the
cluster-type at moderate α composition. The optimized transfection
efficiency (TE) of pDNA, in HEK293T, HeLa, and H1299 cells was higher
using lipoplexes containing gemini CLs with shorter spacers at low
α value. Each lipid formulation did not show any significant
levels of toxicity, the reported lipoplexes being adequate DNA vectors
for gene therapy and considerably better than both Lipofectamine 2000
and CLs of the 1,2-bis(hexadecyl ammnoniun) alkane series, recently
reported
Efficient Cellular Knockdown Mediated by siRNA Nanovectors of Gemini Cationic Lipids Having Delocalizable Headgroups and Oligo-Oxyethylene Spacers
The use of small interfering RNAs
(siRNAs) to silence specific
genes is one of the most promising approaches in gene therapy, but
it requires efficient nanovectors for successful cellular delivery.
Recently, we reported liposomal gene carriers derived from a gemini
cationic lipid (GCL) of the 1,2-bis(hexadecyl dimethyl imidazolium)
oligo-oxyethylene series ((C<sub>16</sub>Im)<sub>2</sub>(C<sub>2</sub>H<sub>4</sub>O)<sub><i>n</i></sub>C<sub>2</sub>H<sub>4</sub> with <i>n</i> = 1, 2, or 3) and 1,2-dioleyol phosphatidylethanolamine
as highly efficient cytofectins for pDNA. On the basis of the satisfactory
outcomes of the previous study, the present work focuses on the utility
of coliposomes of these gemini lipids with the biocompatible neutral
lipid mono oleoyl glycerol (MOG) as highly potent vectors for siRNA
cellular transport in the presence of serum. The (C<sub>16</sub>Im)<sub>2</sub>(C<sub>2</sub>H<sub>4</sub>O)<sub><i>n</i></sub>C<sub>2</sub>H<sub>4</sub>/MOG-siRNA lipoplexes were characterized
through (i) a physicochemical study (zeta potential, cryo-transmission
electron microscopy, small-angle X-ray scattering, and fluorescence
anisotropy) to establish the relationship between size, structure,
fluidity, and the interaction between siRNA and the GCL/MOG gene vectors
and (ii) a biological analysis (flow cytometry, fluorescence microscopy,
and cell viability) to report the anti-GFP siRNA transfections in
HEK 293T, HeLa, and H1299 cancer cell lines. The in vitro biological
analysis confirms the cellular uptake and indicates that a short spacer,
a very low molar fraction of GCL in the mixed lipid, and a moderate
effective charge ratio of the lipoplex yielded maximum silencing efficacy.
At these experimental conditions, the siRNA used in this work is compacted
by the GCL/MOG nanovectors by forming two cubic structures (<i>Ia</i>3<i>d</i> and <i>Pm</i>3<i>n</i>) that are correlated with excellent silencing activity. These liposomal
nanocarriers possess high silencing activity with a negligible cytotoxicity,
which strongly supports their practical use for in vivo knockdown
studies
Magnetic Silica Nanoparticle Cellular Uptake and Cytotoxicity Regulated by Electrostatic Polyelectrolytes–DNA Loading at Their Surface
Magnetic silica nanoparticles show great promise for drug delivery. The major advantages correspond to their magnetic nature and ease of biofunctionalization, which favors their ability to interact with cells and tissues. We have prepared magnetic silica nanoparticles with DNA fragments attached on their previously polyelectrolyte-primed surface. The remarkable feature of these materials is the compromise between the positive charges of the polyelectrolytes and the negative charges of the DNA. This dual-agent formulation dramatically changes the overall cytotoxicity and chemical degradation of the nanoparticles, revealing the key role that surface functionalization plays in regulating the mechanisms involved