27 research outputs found
Tailoring Polymersome Bilayer Permeability Improves Enhanced Permeability and Retention Effect for Bioimaging
Self-assembled nanoparticles conjugated
with various imaging contrast
agents have been used for the detection and imaging of pathologic
tissues. Inadvertently, these nanoparticles undergo fast, dilution-induced
disintegration in circulation and quickly lose their capability to
associate with and image the site of interest. To resolve this challenge,
we hypothesize that decreasing the bilayer permeability of polymersomes
can stabilize their structure, extend their lifetime in circulation,
and hence improve the quality of bioimaging when the polymersome is
coupled with an imaging probe. This hypothesis is examined by using
poly(2-hydroxyethyl-<i>co</i>-octadecyl aspartamide), sequentially
modified with methacrylate groups, to build model polymersomes. The
bilayer permeability of the polymersome is decreased by increasing
the packing density of the bilayer with methacrylate groups and is
further decreased by inducing chemical cross-linking reactions between
the methacrylate groups. The polymersome with decreased bilayer permeability
demonstrates greater particle stability in physiological media and
ultimately can better highlight tumors in mice over 2 days compared
to those with higher bilayer permeability after labeling with a near-infrared
(NIR) fluorescent probe. We envisage that the resulting nanoparticles
will not only improve diagnosis but also further image-guided therapies
Enhancement of the Targeting Capabilities of the Paclitaxel-Loaded Pluronic Nanoparticles with a Glycol Chitosan/Heparin Composite
An enhancement of tumor-targeting capability was demonstrated
with
paclitaxel (PTX)-loaded Pluronic nanoparticles (NPs) with immobilized
glycol chitosan and heparin. The PTX-loaded Pluronic NPs were prepared
as described in our previous report by means of a temperature-induced
phase transition in a mixture of Pluronic F-68 and liquid polyethylene
glycol (PEG; molecular weight: 400) containing PTX. The liquid PEG
is used as the solubilizer of PTX, and Pluronic F-68 is the polymer
that encapsulates the PTX. The glycol chitosan and heparin were immobilized
on the surface of the Pluronic NPs in an aqueous medium, and a powdery
form of the glycol chitosan/heparin immobilized Pluronic NPs (composite
NPs) was obtained by freeze-drying. Field emission scanning electron
microscopy and a particle size analyzer were used to observe the morphology
and size distribution of the prepared NPs. To apply the composite
NPs as a delivery system for the model anticancer drug PTX, the release
pattern and pharmacokinetic parameters were observed, and the tumor
growth was monitored by injecting the composite NPs into the tail
veins of tumor-bearing mice. An enhancement of tumor-targeting capability
of NPs was verified by using noninvasive live animal imaging technology
to observe the time-dependent excretion profile, the in vivo biodistribution,
circulation time, and the tumor-targeting capability of composite
NPs
DNA Amplification in Neutral Liposomes for Safe and Efficient Gene Delivery
In general, traditional gene carriers contain strong cationic charges to efficiently load anionic genes, but this cationic character also leads to destabilization of plasma membranes and causes severe cytotoxicity. Here, we developed a PCR-based nanofactory as a safe gene delivery system. A few template plasmid DNA can be amplified by PCR inside liposomes about 200 nm in diameter, and the quantity of loaded genes highly increased by more than 8.8-fold. The liposome membrane was composed of neutral lipids free from cationic charges. Consequently, this system is nontoxic, unlike other traditional cationic gene carriers. Intense red fluorescent protein (RFP) expression in CHO-K1 cells showed that the amplified genes could be successfully transfected to cells. Animal experiments with the luciferase gene also showed <i>in vivo</i> gene expression by our system without toxicity. We think that this PCR-based nanofactory system can overcome the toxicity problem that is the critical limitation of current gene delivery to clinical application
Design of a Multicomponent Peptide-Woven Nanocomplex for Delivery of siRNA
<div><p>We developed and tested a multicomponent peptide-woven siRNA nanocomplex (PwSN) comprising different peptides designed for efficient cellular targeting, endosomal escape, and release of siRNA. To enhance tumor-specific cellular uptake, we connected an interleukin-4 receptor-targeting peptide (I4R) to a nine-arginine peptide (9r), yielding I4R-9r. To facilitate endosomal escape, we blended endosomolytic peptides into the I4R-9r to form a multicomponent nanocomplex. Lastly, we modified 9r peptides by varying the number and positions of positive charges to obtain efficient release of siRNA from the nanocomplex in the cytosol. Using this step-wise approach for overcoming the biological challenges of siRNA delivery, we obtained an optimized PwSN with significant biological activity <i>in vitro</i> and <i>in vivo</i>. Interestingly, surface plasmon resonance analyses and three-dimensional peptide models demonstrated that our designed peptide adopted a unique structure that was correlated with faster complex disassembly and a better gene-silencing effect. These studies further elucidate the siRNA nanocomplex delivery pathway and demonstrate the applicability of our stepwise strategy to the design of siRNA carriers capable of overcoming multiple challenges and achieving efficient delivery.</p></div
Detection of Active Matrix Metalloproteinase‑3 in Serum and Fibroblast-Like Synoviocytes of Collagen-Induced Arthritis Mice
The activity of rheumatoid arthritis
(RA) correlates with the expression
of proteases. Among several proteases, matrix metalloproteinase-3
(MMP-3) is one of the biological markers used to diagnose RA. The
active form of MMP-3 is a key enzyme involved in RA-associated destruction
of cartilage and bone. Thus, detection of active MMP-3 in serum or <i>in vivo</i> is very important for early diagnosis of RA. In
this study, a soluble MMP-3 probe was prepared to monitor RA progression
by detecting expression of active MMP-3 in collagen-induced arthritis
(CIA) mice <i>in vivo</i> in both serum and fibroblast-like
synoviocytes (FLSs). The MMP-3 probe exhibited strong sensitivity
to MMP-3 and moderate sensitivity to MMP-7 at nanomolecular concentrations,
but was not sensitive to other MMPs such as MMP-2, MMP-9, and MMP-13.
In an optical imaging study, the MMP-3 probe produced early and strong
NIR fluorescence signals prior to observation of erythema and swelling
in CIA mice. The MMP-3 probe was able to rapidly and selectively detect
and monitor active MMP-3 in diluted serum from CIA mice. Furthermore,
histological data demonstrated that activated FLSs in arthritic knee
joints expressed active MMP-3. Together, our results demonstrated
that the MMP-3 probe may be useful for detecting active MMP-3 for
diagnosis of RA. More importantly, the MMP-3 probe was able to detect
active MMP-3 in diluted serum with high sensitivity. Therefore, the
MMP-3 probe developed in this study may be a very promising probe,
useful as a biomarker for early detection and diagnosis of RA
Physicochemical characterization of I4R-9r variants PwSNs.
<p>(A) siRNA encapsulations by I4R-9r variants were monitored by gel retardation assay with molar ratios of 1:5 to 1:30 (siRNA: carrier) in the presence and absence of sHGP-9r. (B) Representative TEM of an I4R-9r tandem peptide variants/siRNA nanocomplex formed in water in the presence and absence of sHGP-9r; scale bar = 50 nm. (C) Stability of I4R-9r variant/sHGP-9r/siRNA nanocomplex was examined in the presence of RNase A. Undegraded siRNA of the nanocomplex was visualized on 2% agarose gel containing EtBr. The stability of free siRNA was measured as control.</p
Structural Properties of I4R-9r/sHGP-9r PwSN variants.
<p><sup><i>a</i></sup> sHGP-9r (RGWEVLKYWWNLLQYggRRRRRRRRR) is blended with I4R-9r/sHGP-9r PwSN with 1:19 molar ratios (sHGP-9r: I4R-9r). gg; Gly-Gly linker. Substituted alanine was highlighted by red.</p><p><sup><i>b</i></sup> Mean hydrodynamic size based on dynamic light scattering measurements. Errors indicate SD from at least three separate measurements.</p><p><sup><i>c</i></sup> Zeta-potential of nanocomplexes. Errors indicate SD from at least three independent measurements.</p><p>Structural Properties of I4R-9r/sHGP-9r PwSN variants.</p
Flowchart of developing multicomponent PwSN by stepwise approach to overcome biological barriers of siRNA delivery.
<p>Flowchart of developing multicomponent PwSN by stepwise approach to overcome biological barriers of siRNA delivery.</p
Dynamic interactions and structures of I4R-9r variant/siRNA nanocomplexes.
<p>(A) The released siRNA from nanocomplexes were examined upon addition of excess amount of Heparan sulfate (w:w of siRNA to Heparin sulfate as 1:5–1:40)using gel retardation assay. (B) The kinetics of association of siRNA with I4R-9r variant/sHGP-9r using SPR analysis. Increasing concentrations of I4R-9r variant/sHGP-9r peptides were injected to associate with 5`-biotinylated siRNA on the streptavidin chip. (C) The kinetics of disassociation of siRNA with I4R-9r variant/sHGP-9r using SPR analysis. After association of 5`-biotinylated siRNA with I4R-9r variant/sHGP-9r (2.5 μM) up to saturation different concentrations of heparan sulfate were injected to quantitate siRNA dissociation. The dissociation kinetics was analyzed using the Graph Prism 5.0. (D) The structures of I4R-9r variant were analyzed and compared by 3D modeling method (PEPFOLD); I4R peptide (green), Arginine of 9r (blue), Alanine (red).</p
Addition of sHGP-9r to facilitate endosome escape of nanocomplexes.
<p>(A, Top) Confocal microscopy images of HeLa cells pretreated with the early endosome marker-RFP, and subsequently incubated with I4R-9r PwSN carrying FITC labeled siRNA. Images were pseudocolored for visualization: blue = DAPI; red = early endosome marker-RFP; green = FITC-siRNA. (A, Bottom) Confocal microscopy images of HeLa cells after treatment with FITC-siRNA encapsulated in I4R-9r PwSNs, which compose of sHGP-9r at molar ratio of 1:19 (sHGP-9r: I4R-9r). (B) The gene silencing efficacies against GAPDH protein with or without the sHGP-9r peptides was assessed by Western blot. Results are presented as mean of relative immunoblot intensities ± standard deviation (n = 6).</p