16 research outputs found
Probing of the Assembly Structure and Dynamics within Nanoparticles during Interaction with Blood Proteins
Fully understanding the influence of blood proteins on the assembly structure and dynamics within nanoparticles is difficult because of the complexity of the system and the difficulty in probing the diverse elements and milieus involved. Here we show the use of site-specific labeling with spin probes and fluorophores combined with electron paramagnetic resonance (EPR) spectroscopy and fluorescence resonance energy transfer (FRET) measurements to provide insights into the molecular architecture and dynamics within nanoparticles. These tools are especially useful for determining nanoparticle stability in the context of blood proteins and lipoproteins and have allowed us to quantitatively analyze the dynamic changes in assembly structure, local stability, and cargo diffusion of a class of novel telodendrimer-based micellar nanoparticles. When combined with human plasma and individual plasma components, we find that non-cross-linked nanoparticles immediately lose their original assembly structure and release their payload upon interaction with lipoproteins. In contrast, serum albumins and immunoglobulin gamma have moderate affects on the integrity of the nanoparticles. Disulfide cross-linked nanoparticles show minimal interaction with lipoproteins and can better retain their assembly structure and payload <i>in vitro</i> and <i>in vivo</i>. We further demonstrate how the enhanced stability and release property of disulfide cross-linked nanoparticles can be reversed in reductive conditions. These findings identify factors that are crucial to the performance of nanomedicines and provide design modes to control their interplay with blood factors
Disulfide Cross-Linked Micelles for the Targeted Delivery of Vincristine to B-Cell Lymphoma
Vincristine (VCR) is a potent anticancer drug, but its
clinical efficacy is limited by neurotoxicity. The field of drug delivery
may provide an opportunity to increase the therapeutic index of VCR
by delivering the drug specifically to tumor sites while sparing normal
tissue. We have recently developed a telodendrimer (PEG<sup>5k</sup>-Cys<sub>4</sub>-L<sub>8</sub>-CA<sub>8</sub>) capable of forming
disulfide cross-linked micelles (DCMs) which can encapsulate a variety
of chemotherapeutics. In the present study, we encapsulated VCR into
these micelles (DCM-VCR) and used them to treat lymphoma bearing mice.
DCM-VCR particles have a size of 16 nm, which has been shown to be
optimal for their accumulation into tumor via the enhanced permeability
and retention (EPR) effect. Compared to our first-generation non-cross-linked
micelles (NCMs), DCM-VCR demonstrated greater stability and slower
drug release under physiological conditions. In addition, DCM-VCR
exhibited a maximum tolerated dose (MTD) of 3.5 mg/kg while the MTD
for conventional VCR was only 1.5 mg/kg. Using a near-infrared cyanine
dye (DiD) as the surrogate drug, we showed that DCM-VCR accumulated
at the tumor site starting 1 h after injection and persisted up to
72 h in lymphoma xenografted nude mice. In an <i>in vivo</i> efficacy study, high dose (2.5 mg/kg) DCM-VCR produced the greatest
reduction in tumor volume. High dose DCM-VCR was well tolerated with
no significant changes in complete blood count, serum chemistry and
histology of the sciatic nerve. Mice treated with an equivalent dose
(1 mg/kg) of conventional VCR and DCM-VCR controlled tumor growth
equally; however, in combination with on-demand addition of the reducing
agent <i>N</i>-acetylcysteine, DCM-VCR exhibited a superior
antitumor effect compared to conventional VCR
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Discovery and Characterization of a Potent and Specific Peptide Ligand Targeting Endothelial Progenitor Cells and Endothelial Cells for Tissue Regeneration
Endothelial progenitor
cells (EPCs) and endothelial cells (ECs)
play a vital role in endothelialization and vascularization for tissue
regeneration. Various EPC/EC targeting biomolecules have been investigated
to improve tissue regeneration with limited success often due to their
limited functional specificity and structural stability. One-bead
one-compound (OBOC) combinatorial technology is an ultrahigh throughput
chemical library synthesis and screening method suitable for ligand
discovery against a wide range of biological targets, such as integrins.
In this study, using primary human EPCs/ECs as living probes, we identified
an αvβ3 integrin ligand LXW7 discovered by OBOC combinatorial
technology as a potent and specific EPC/EC targeting ligand. LXW7
overcomes the major barriers of other functional biomolecules that
have previously been used to improve vascularization for tissue regeneration
and possesses optimal stability, EPC/EC specificity, and functionality.
LXW7 is a disulfide cyclic octa-peptide (cGRGDdvc) containing unnatural
amino acids flanking both sides of the main functional motif; therefore
it will be more resistant to proteolysis and more stable <i>in
vivo</i> compared to linear peptides and peptides consisting
of only natural amino acids. Compared with the conventional αvβ3
integrin ligand GRGD peptide, LXW7 showed stronger binding affinity
to primary EPCs/ECs but weaker binding to platelets and no binding
to THP-1 monocytes. In addition, ECs bound to the LXW7 treated culture
surface exhibited enhanced biological functions such as proliferation,
likely due to increased phosphorylation of VEGF receptor 2 (VEGF-R2)
and activation of mitogen-activated protein kinase (MAPK) ERK1/2.
Surface modification of electrospun microfibrous PLLA/PCL biomaterial
scaffolds with LXW7 <i>via</i> Click chemistry resulted
in significantly improved endothelial coverage. LXW7 and its derivatives
hold great promise for EPC/EC recruitment and delivery and can be
widely applied to functionalize various biological and medical materials
to improve endothelialization and vascularization for tissue regeneration
applications
CTA095, a Novel Etk and Src Dual Inhibitor, Induces Apoptosis in Prostate Cancer Cells and Overcomes Resistance to Src Inhibitors
<div><p>Etk is a non-receptor tyrosine kinase, which provides a strong survival signal in human prostate cancer cells. Src, another tyrosine kinase that cross-activates with Etk, has been shown to play an important role in prostate cancer metastasis. Herein, we discovered a new class of Etk inhibitors. Within those inhibitors, CTA095 was identified as a potent Etk and Src dual inhibitor. CTA095 was found to induce autophagy as well as apoptosis in human prostate cancer cells. In addition, CTA095 inhibited HUVEC cell tube formation and “wound healing” of human prostate cancer cells, implying its role in inhibition of angiogenesis and metastasis of human prostate cancer. More interestingly, CTA095 could overcome Src inhibitor resistance in prostate cancer cells. It induces apoptosis in Src inhibitor resistant prostate cancer cells, likely through a mechanism of down regulation of Myc and BCL2. This finding indicates that simultaneously targeting Etk and Src could be a promising approach to overcome drug resistance in prostate cancer.</p></div
CTA095 induces apoptosis in Src inhibitor resistant prostate cancer cells through Myc and BCL2 inhibition.
<p>PC3-AZD20 (PC3 cell resistant to 20 μM AZD0530) cells were seeded at 10<sup>6</sup> cells/well in 6 well plates overnight. The cells were treated with AZD0530 or CTA095 at 10 μM. Apoptosis was analyzed using Annexin-V FITC apoptosis detection kit (A). The mRNA levels of Myc and BCL2 were measured using real-time PCR (B). pEtk, Etk, pSrc, Src, pStat3, Stat3, Myc and BCL2 levels were measured using the corresponding antibodies through Western blot (C). Columns, mean; bars, standard deviation, n = 3.</p
Induction of apoptosis of PC3 cells following treatment with CTA095.
<p>PC3 cells were seeded at 10<sup>6</sup> cells/ml (2 ml) in a 6-well plate overnight and then treated with CTA095 at the indicated concentrations for 24 h. Cell cycle arrest was analyzed using PI staining (A). Apoptosis was analyzed using Annexin-V FITC apoptosis detection kit (B). Caspase 9 activation was measured using western blot (D). For caspase 3/7 activity, PC3 cells were seeded at 5000 cells/well in 96 well plate overnight and treated with CTA095 at 0–10 μM for 24 h. Caspase-3/7 activities were measured using the Apo-ONE Homogeneous Caspase-3/7 Assay kit (Promega, Madison, WI) according to the manufacturer's instruction. Columns, mean; bars, standard deviation, n = 3. 5 μM and 10 μM are significantly different from 0 μM (*, p<0.05, one-way ANOVA with Tukey test for pair wise comparison).</p
Growth Inhibition of CTA095 to LNCAP, CWR22Rv1, PC3 prostate cancer and normal prostate (RWPE1) cells.
<p>Cells were seeded at 5,000 cells/well in 96-well plate overnight and treated with CTA095 at the indicated concentrations. The cell viability was measured using MTT assay after 72 h. dots, mean; bars, standard deviation, n = 3.</p
CTA095 overcomes Src inhibitor resistance in prostate cancer cells.
<p>PC3 and PC3-AZD20 (PC3 cell resistant to 20 μM AZD0530) cells were seeded at 2000 cells/well in 96 well plates overnight. The cells were treated with AZD0530 or CTA095 at the indicated concentrations. Cell viability was measured using MTT assay after 72 h. Symbols, mean; bars, standard deviation, n = 3.</p
Inhibition of cell signaling in PC3 cells following treatment with CTA095.
<p>Cells were grown in 10 mm plate to 50% confluence and treated with CTA095. Cells were harvested after 24 h. pEtk, Etk, pSrc, Src, pStat3, Stat3, pAkt and Akt levels were measured using the corresponding antibodies by Western blot. One of three similar experiments depicted.</p
Inhibition of PC3 xenograft tumor growth by CTA095nano.
<p>(CAT095 formulated in nano-micelles.) 2×10<sup>6</sup> PC3 cells were injected subcutaneously to nude mice. The tumors were grown the indicated size and the mice were randomly divided into two groups (8 mice/group). The control group was treated with vehicle. The treatment group was treated with CTA095nano at 10 mg/kg twice a week with iv injection. The tumor size (A) and body weight (B) were measured once a week. Marks, mean; bars, sd. n = 8.</p