Complex coacervation of Mg(ii) phospho-polymethacrylate, a synthetic analog of sandcastle worm adhesive phosphoproteins.

The highly phosphorylated Pc3 proteins, major components of the sandcastle worm adhesive, are sequestered with Mg as spherical sub-granules within heterogeneous secretory granules in adhesive gland cells. The phase behavior of a synthetic phospho-polymethacrylate analog of the Pc3 phosphoproteins, in the presense of Mg(ii), was characterized to determine whether it is chemically possible for the natural adhesive components to be packaged and stored as liquid complex coacervates. Of several multivalent metal salts tested, only MgCl induced complex coacervation of the phospho-copolymer. Complex coacervates formed at Mg/P ratios from 0.5-8, and in [NaCl]s from 0-3 M. At low temperature and pH, the complex coacervates were clear and homogeneous. At higher temperatures and pH, the coacervate phases were translucent. The elastic and viscous moduli initially decreased as temperature increased, but then increased significantly near the temperature boundary between clear and translucent forms. A mechanism is proposed in which relatively weak, ionic strength-independent, outer shell crossbridging of -PO32- sidechains by Mg[H2O]62+ complex ions is responsible for the clear homogeneous lower viscosity coacervate form. At higher temperature and pH, displacement of inner shell H2O molecules by phosphate O- ligands creates stronger crossbridges, additional dehydration, and more viscous coacervates. The results demonstrate that Pc3 phosphoproteins can exist as condensed phospho/Mg(ii) complex coacervates under conditions expected in the adhesive glands of sandcastle worms in their natural environment. Considering the common regulatory role of phosphorylation and the intracellular abundance of Mg2+ it is possible that soft bridging of phosphate groups by Mg[H2O]n2+ may promote other regulated cellular liquid liquid phase separation phenomena.

The role of coacervation and phase transitions in the sandcastle worm adhesive system.

Sandcastle worms, Phragmatopoma californica (Fewkes), live along the western coast of North America. Individual worms build tubular shells under seawater by gluing together sandgrains and biomineral particles with a multipart, rapid-set, self-initiating adhesive. The glue comprises distinct sets of condensed, oppositely charged polyelectrolytic components-polyphosphates, polysulfates, and polyamines-that are separately granulated and stored at high concentration in distinct cell types. The pre-organized adhesive modules are secreted separately and intact, but rapidly fuse with minimal mixing and expand into a crack-penetrating complex fluid. Within 30s of secretion into seawater, the fluid adhesive transitions (sets) into a porous solid adhesive joint. The nano- and microporous structure of the foamy solid adhesive contributes to the strength and toughness of the adhesive joint through several mechanisms. A curing agent (catechol oxidase), co-packaged into both types of adhesive granules, covalently cross-links the adhesive and becomes a structural component of the final adhesive joint. The overall effectiveness of the granulated sandcastle glue is more a product of the cellular sorting and packaging mechanisms, the transition from fluid to solid following secretion, and its final biphasic porous structure as it is of its composition or any particular amino acid modification.

PEGylation and HAylation via catechol: α-Amine-specific reaction at N-terminus of peptides and proteins.

UNLABELLED: The development of chemoselective, site-specific chemistries for proteins/peptides is essential for biochemistry, pharmaceutical chemistry, and other fields. In this work, we found that catechol, which has been extensively utilized as an adhesive molecule for material-independent surface chemistry and as a crosslinker in hydrogel preparation, specifically reacts with N-terminal α-amines, avoiding the ε-amine group in lysine. A conjugate of methoxy-poly(ethylene glycol)-catechol called mPEG-cat chemoselectively reacts with N-terminal amine groups at neutral pH resulting in site-specific PEGylation. To demonstrate the versatility of this catechol chemoselective reaction, we used four proteins (lysozyme, basic-fibroblast growth factor (bFGF), granulocyte-colony stimulating factor (G-CSF), insulin, and erythropoietin (EPO)) as well as two peptides (hinge-3 and laminin-derived peptide (LDP)). All the tested macromolecules showed N-terminal site-specific modifications. Furthermore, we prepared another catechol grafted conjugate called hyaluronic acid-catechol (HA-cat) to demonstrate that this catechol-involved chemoselective chemistry is not specific for PEG conjugates. This new catechol chemoselective chemistry could be a new platform for the functionalization of proteins and peptides for a variety of purposes. STATEMENT OF SIGNIFICANCE: Considering the fact that biological activities of proteins or peptides depend largely on their 3-dimensional conformation, the orientation-controllable reaction is very important for preserving the intrinsic functionality of them. In addition to PEG, many other bio-polymers such as oligonucleotides, antibodies, and oligosaccharides have been conjugated with proteins or peptides for various biomedical applications. Although several chemoselective conjugation chemistries have been reported, conjugation efficiencies are different depending on types of proteins or polymers, and thus there've been strong needs for the development of alternative strategy of chemoselective conjugation that can be applied for a variety of therapeutic proteins towards high biological activities. We are certain this new catechol chemoselective chemistry could be a new platform for the functionalization of proteins and peptides for various purposes.

Therapeutic Effect of Akt1 siRNA Nanoparticle Eluting Coronary Stent on Suppression of Post-Angioplasty Restenosis.

For effective treatment of restenosis, therapeutic genes are delivered locally from a coated stent at the site of injury, leading to inhibition of smooth muscle proliferation and neo-intimal hyperplasia while promoting re-endothelialization. In a previous study, we delivered Akt1 siRNA nanoparticles (ASNs) from a hyaluronic acid (HA)-coated stent surface to specifically suppress the pro-proliferative Akt1 protein in smooth muscle cells (SMCs). In the present study, therapeutic efficacy was investigated in a rabbit restenosis model after percutaneous implantation of an ASN-immobilized stent in a rabbit iliac artery. Quantitative and qualitative analyses of in-stent restenosis were investigated in an in vivo animal model by micro-CT imaging and SEM observation, respectively. Proliferation status and neo-intima formation of the vascular tissues located near ASN-immobilized stents were analyzed by immunohistochemical staining using anti-Akt1 and anti-Ki67 antibodies and histological analyses, such as hematoxylin and eosin staining and Verhoeff's elastic stain. Re-endothelialization after implantation of an ASN-immobilized stent was also analyzed via immunohistochemistry using an anti-CD31 antibody. To elucidate the molecular mechanism related to reducing SMC proliferation and subsequent inhibition of in-stent restenosis in vivo, protein and mRNA expression of Akt1 and downstream signaling proteins were analyzed after isolating SMC-rich samples from the treated vasculature. The implanted Akt1 siRNA-eluting stent efficiently mitigated in-stent restenosis without any side effects and can be considered a successful substitute to current drug-eluting stents.

Novel Fabrication of MicroRNA Nanoparticle-Coated Coronary Stent for Prevention of Post-Angioplasty Restenosis.

BACKGROUND AND OBJECTIVES: MicroRNA 145 is known to be responsible for cellular proliferation, and its enhanced expression reportedly inhibits the retardation of vascular smooth muscle cell growth specifically. In this study, we developed a microRNA 145 nanoparticle immobilized, hyaluronic acid (HA)-coated stent. MATERIALS AND METHODS: For the gene therapy, we used disulfide cross-linked low molecular polyethylenimine as the carrier. The microRNA 145 was labeled with YOYO-1 and the fluorescent microscopy images were obtained. The release of microRNA 145 from the stent was measured with an ultra violet spectrophotometer. The downstream targeting of the c-Myc protein and green fluorescent protein was determined by Western blotting. Finally, we deployed microRNA 145/ssPEI nanoparticles immobilized on HA-coated stents in the balloon-injured external iliac artery in a rabbit restenosis model. RESULTS: Cellular viability of the nanoparticle-immobilized surface tested using A10 vascular smooth muscle cells showed that MSN exhibited negligible cytotoxicity. In addition, microRNA 145 and downstream signaling proteins were identified by western blots with smooth muscle cell (SMC) lysates from the transfected A10 cell, as the molecular mechanism for decreased SMC proliferation that results in the inhibition of in-stent restenosis. MicroRNA 145 released from the stent suppressed the growth of the smooth muscle at the peri-stent implantation area, resulting in the prevention of restenosis at the post-implantation. We investigated the qualitative analyses of in-stent restenosis in the rabbit model using micro-computed tomography imaging and histological staining. CONCLUSION: MicroRNA 145-eluting stent mitigated in-stent restenosis efficiently with no side effects and can be considered a successful substitute to the current drug-eluting stent.

New antifouling platform characterized by single-molecule imaging.

Antifouling surfaces have been widely studied for their importance in medical devices and industry. Antifouling surfaces mostly achieved by methoxy-poly(ethylene glycol) (mPEG) have shown biomolecular adsorption less than 1 ng/cm(2) which was measured by surface analytical tools such as surface plasmon resonance (SPR) spectroscopy, quartz crystal microbalance (QCM), or optical waveguide lightmode (OWL) spectroscopy. Herein, we utilize a single-molecule imaging technique (i.e., an ultimate resolution) to study antifouling properties of functionalized surfaces. We found that about 600 immunoglobulin G (IgG) molecules are adsorbed. This result corresponds to ∼5 pg/cm(2) adsorption, which is far below amount for the detection limit of the conventional tools. Furthermore, we developed a new antifouling platform that exhibits improved antifouling performance that shows only 78 IgG molecules adsorbed (∼0.5 pg/cm(2)). The antifouling platform consists of forming 1 nm TiO2 thin layer, on which peptidomimetic antifouling polymer (PMAP) is robustly anchored. The unprecedented antifouling performance can potentially revolutionize a variety of research fields such as single-molecule imaging, medical devices, biosensors, and others.

Suppression of post-angioplasty restenosis with an Akt1 siRNA-embedded coronary stent in a rabbit model.

Restenosis is the formation of blockages occurring at the site of angioplasty or stent placement. In order to avoid such blockages, the suppression of smooth muscle cells near the implanted stent is required. The Akt1 protein is known to be responsible for cellular proliferation, and specific inhibition of Akt1 gene expression results in the retardation of cell growth. To take advantage of these benefits, we developed a new delivery technique for Akt1 siRNA nanoparticles from a hyaluronic acid (HA)-coated stent surface. For this purpose, the disulfide cross-linked low molecular polyethyleneimine (PEI) (ssPEI) was used as a gene delivery carrier because disulfide bonds are stable in an oxidative extracellular environment but degrade rapidly in reductive intracellular environments. In this study, Akt1 siRNA showed efficient ionic interaction with the ssPEI carrier, which was confirmed by polyacrylamide gel electrophoresis. Akt1 siRNA/ssPEI nanoparticles (ASNs) were immobilized on the HA-coated stent surface and exhibited stable binding and localization, followed by time-dependent sustained release for intracellular uptake. Cellular viability on the nanoparticle-immobilized surface was assessed using A10 vascular smooth muscle cells, and the results revealed that immobilized ASNs exhibited negligible cytotoxicity against the adhering A10 cells. Transfection efficiency was quantified using a luciferase assay; the transgene expression of Akt1 suppression through the delivered Akt1 siRNA was measured using RT-PCR and western blot, demonstrating higher gene silencing efficiency when compared to other carriers. ASN coated on HA stents were deployed in the balloon-injured external iliac artery in rabbits in vivo. It was shown that the Akt1 released from the stent suppressed the growth of the smooth muscle at the peri-stent implantation area, resulting in the prevention of restenosis in the post-implantation phase.