6 research outputs found
Polyplex Micelles with Double-Protective Compartments of Hydrophilic Shell and Thermoswitchable Palisade of Poly(oxazoline)-Based Block Copolymers for Promoted Gene Transfection
Improving the stability of polyplex
micelles under physiological
conditions is a critical issue for promoting gene transfection efficiencies.
To this end, hydrophobic palisade was installed between the inner
core of packaged plasmid DNA (pDNA) and the hydrophilic shell of polyplex
micelles using a triblock copolymer consisting of hydrophilic polyÂ(2-ethyl-2-oxazoline),
thermoswitchable amphiphilic polyÂ(2-<i>n</i>-propyl-2-oxazoline)
(PnPrOx) and cationic polyÂ(l-lysine). The two-step preparation
procedure, mixing the triblock copolymer with pDNA below the lower
critical solution temperature (LCST) of PnPrOx, followed by incubation
above the LCST to form a hydrophobic palisade of the collapsed PnPrOx
segment, induced the formation of spatially aligned hydrophilic–hydrophobic
double-protected polyplex micelles. The prepared polyplex micelles
exhibited significant tolerance against attacks from nuclease and
polyanions compared to those without hydrophobic palisades, thereby
promoting gene transfection. These results corroborated the utility
of amphiphilic polyÂ(oxazoline) as a molecular thermal switch to improve
the stability of polyplex gene carriers relevant for physiological
applications
Poly(ethylene glycol) Crowding as Critical Factor To Determine pDNA Packaging Scheme into Polyplex Micelles for Enhanced Gene Expression
A critical
role of polyethylene glycol (PEG) crowding in the packaging
of plasmid DNA (pDNA) into polyplex micelles (PMs) was investigated
using a series of PEG-<i>b</i>-polyÂ(l-lysine) (PEG–PLys)
block copolymers with varying molecular weights of both PEG and PLys
segments. Rod-shaped PMs preferentially formed when the tethered PEG
chains covering pDNA in a precondensed state were dense enough to
overlap one another (reduced tethering density (RTD) > 1), whereas
globular PMs were obtained when they were not overlapped (RTD <
1). These results submitted a scheme that steric repulsive effect
of PEG regulated packaging pathways of pDNA either through folding
into rod-shape or collapsing into globular depending on whether the
PEG chains are overlapped or not. The rod-shaped PMs gave significantly
higher gene expression efficacies in a cell-free system compared to
the globular PMs, demonstrating the practical relevance of regulating
packaging structure of pDNA for developing efficient gene delivery
systems
Toroidal Packaging of pDNA into Block Ionomer Micelles Exerting Promoted <i>in Vivo</i> Gene Expression
Selectively spooling single plasmid
DNA (pDNA), as a giant polyelectrolyte,
into a nanosized toroidal structure or folding it into a rod-like
structure has been accomplished by polyion complexation with block
catiomers to form polymeric micelles in varying NaCl concentrations.
The interactive potency between the pDNA and block catiomers was determined
to play a critical role in defining the ultimate structure of the
pDNA; the formation of toroidal or rod-like structures was achieved
by complexation in 600 or 0 mM NaCl solutions, respectively. Compared
with the rod-like structure, the toroidal structure possessed superior
biological functions capable not only of elevating <i>in vitro</i> transcription but also of elevating <i>in vivo</i> gene
transduction efficiency. This demonstrated the great utility of the
toroidal pDNA packaging as a distinct structured gene carrier. Furthermore,
the fact that the NaCl concentration at which the toroidal structure
was specifically formed corresponds to seawater stimulates interest
in this ordered nanostructure as a possible inherent structure for
DNA
Acidic pH-Triggered Release of Doxorubicin from Ligand-Decorated Polymeric Micelles Potentiates Efficacy against Cancer Cells
Current chemotherapeutic strategies against various intractable
cancers are futile due to inefficient delivery, poor bioavailability,
and inadequate accumulation of anticancer drugs in the diseased site
with toxicity caused to the healthy neighboring cells. Drug delivery
systems aiming to deliver effective therapeutic concentrations to
the site of action have emerged as a promising approach to address
the above-mentioned issues. Thus, as several receptors have been identified
as being overexpressed on cancer cells including folate receptor (FR),
where up to 100–300 times higher overexpression is shown in
cancer cells compared to healthy cells, approximately 1–10
million receptor copies per cancer cell can be targeted by a folic
acid (FA) ligand. Herein, we developed FA-decorated and doxorubicin-conjugated
polymeric micelles of 30 nm size. The hydrophilic block comprises
poly(ethylene glycol) units, and the hydrophobic block contains aspartic
acid. Decoration of FA on the micelle surface induces ligand–receptor
interaction, resulting in enhanced internalization into the cancer
cell and inside the endolysosomal compartment. Under acidic pH, the
micelle structure is disrupted and the hydrazone bond is cleaved,
which covalently binds the doxorubicin with the hydrophobic backbone
of the polymer and release the drug. We observed that the cellular
uptake and nuclear colocalization of the targeted micelle are 2–4
fold higher than the control micelle at various incubation times in
FR-overexpressed various cancer cell lines (KB, HeLa, and C6). These
results indicate significant prospects for anticancer therapy as an
effective and translational treatment strategy
Tethered PEG Crowdedness Determining Shape and Blood Circulation Profile of Polyplex Micelle Gene Carriers
Surface modification by polyÂ(ethylene
glycol) (PEG) onto gene carrier
prepared through the electrostatic assembly of pDNA and polycation
(polyplex) is a widely acknowledged strategy to advance their systemic
application. In this regard, PEG crowdedness on the polyplex surface
should give important contribution in determining blood circulation
property; however its accurate quantification has never been demonstrated.
We report here the first successful determination of PEG crowdedness
for PEGylated polyplexes (polyplex micelle) formed from PEG–polyÂ(l-lysine) block copolymers (PEG–PLys) and plasmid DNA
(pDNA). Tethered PEG chains were found to adopt mushroom and even
squeezed conformation by modulating PEG crowdedness through PLys segment
length. Energetic analysis was conducted on the polyplex micelle to
elucidate effect of PEG crowdedness on shape and clarify its essential
role in regulating packaging structure of pDNA within the polyplex
micelle. Furthermore, the PEG crowdedness significantly correlated
to blood retention profile, approving its critical role on both shape
and systemic circulation property
A Nanoparticle Platform To Evaluate Bioconjugation and Receptor-Mediated Cell Uptake Using Cross-Linked Polyion Complex Micelles Bearing Antibody Fragments
Targeted nanomedicines are a promising
technology for treatment
of disease; however, preparation and characterization of well-defined
protein-nanoparticle systems remain challenging. Here, we describe
a platform technology to prepare antibody binding fragment (Fab)-bearing
nanoparticles and an accompanying real-time cell-based assay to determine
their cellular uptake compared to monoclonal antibodies (mAbs) and
Fabs. The nanoparticle platform was composed of core-cross-linked
polyion complex (PIC) micelles prepared from azide-functionalized
PEG<i>-<i>b</i>-</i>polyÂ(amino acids), that is,
azido-PEG<i>-<i>b</i>-</i>polyÂ(l-lysine)
[N<sub>3</sub>–PEG<i>-<i>b</i>-</i>PLL]
and azido-PEG<i>-<i>b</i>-</i>polyÂ(aspartic acid)
[N<sub>3</sub>–PEG<i>-<i>b</i>-</i>PAsp].
These PIC micelles were 30 nm in size and contained approximately
10 polymers per construct. Fabs were derived from an antibody binding
the EphA2 receptor expressed on cancer cells and further engineered
to contain a reactive cysteine for site-specific attachment and a
cleavable His tag for purification from cell culture expression systems.
Azide-functionalized micelles and thiol-containing Fab were linked
using a heterobifunctional cross-linker (FPM-PEG<sub>4</sub>-DBCO)
that contained a fluorophenyl-maleimide for stable conjugation to
Fabs thiols and a strained alkyne (DBCO) group for coupling to micelle
azide groups. Analysis of Fab–PIC micelle conjugates by fluorescence
correlation spectroscopy, size exclusion chromatography, and UV–vis
absorbance determined that each nanoparticle contained 2–3
Fabs. Evaluation of cellular uptake in receptor positive cancer cells
by real-time fluorescence microscopy revealed that targeted Fab–PIC
micelles achieved higher cell uptake than mAbs and Fabs, demonstrating
the utility of this approach to identify targeted nanoparticle constructs
with unique cellular internalization properties