Glyco Star Polymers as Helical Multivalent Host and Biofunctional Nano-Platform

A series of amylose-based star polymers (1, 2, 4, and 8 arms) as a new glyco biomaterial was synthesized by a click reaction and enzymatic polymerization of specific primers with phosphorylase. The molecular weights were controlled by the enzymatic reaction. Further polymerization resulted in a viscous solution and, especially, for the 8-arm primer, a hydrogel was obtained due to effective cross-linking between the multiarmed structures. The star polymers with a degree of polymerization of about 60 per arm acted as an allosteric multivalent host for hydrophobic molecules by helical formation. A cationic 8-arm star polymer catalyzed DNA strand exchange as a nucleic acid chaperone. Amylose-based star polymers are promising building blocks for producing advanced hybrid glyco biomaterials

A Stimulus-Responsive Shape-Persistent Micelle Bearing a Calix[4]arene Building Block: Reversible pH-Dependent Transition between Spherical and Cylindrical Forms

A series of cationic calix[4]­arene-based lipids with alkyl chains of varying length were newly synthesized, and the ones with propyl and hexyl tails, denoted by CaL[4]­C3 and C6, respectively, were found to form spherical micelles at low pH (protonated state of the amine headgroup). Upon deprotonation with increasing pH, CaL[4]­C3 showed a sphere-to-cylinder transition, while CaL[4]­C6 changed from sphere, to cylinder, to monolayer vesicle. Synchrotron small-angle X-ray scattering (SAXS) patterns from both spherical and cylindrical CaL[4]­C3 micelles exhibited a sharp intensity minimum, indicating shape monodispersity. The monodispersity of the CaL[4]­C3 spherical micelles was further confirmed by analytical ultracentrifugation (AUC). SAXS, AUC, and static light scattering agreeingly indicated an aggregation number of 6. In contrast, CaL[4]­C6 exhibited polydispersity with an average aggregation number of 12. When the number of carbons of the alkyl chain was increased to 9 (CaL[4]­C9), cylinder formed at low pH, while at high pH, no clear morphology could be observed. The present results indicate that a very precise combination of tail length, head volume, and rigidity of the building block is required to produce shape-persistent micelles and that the shape-persistence can be maintained upon a structural transition. An attempt to reconstruct a molecular model for the spherical CaL[4]­C3 micelle was made with an ab initio shape determining program

Long-Lasting and Efficient Tumor Imaging Using a High Relaxivity Polysaccharide Nanogel Magnetic Resonance Imaging Contrast Agent

Clinically approved small-molecule magnetic resonance imaging (MRI) contrast agents are all rapidly cleared from the body and offer weak signal enhancement. To avoid repeated administration of contrast agent and improve signal-to-noise ratios, agents with stronger signal enhancement and better retention in tumors are needed. Therefore, we focused on hydrogels because of their excellent water accessibility and biodegradability. Gadolinium (Gd)-chelating cross-linkers were incorporated into self-assembled pullulan nanogels to both impart magnetic properties and to stabilize this material that has been extensively studied for medical applications. We show that these Gd-chelating pullulan nanogels (Gd-CHPOA) have the highest reported relaxivity for any hydrogel-based particles and accumulate in the 4T1 tumors in mice at high levels 4 h after injection. This combination offers high signal enhancement and lasts up to 7 days to delineate the tumor clearly for longer imaging time scales. Importantly, this long-term accumulation does not cause any damage or toxicity in major organs up to three months after injection. Our work highlights the clinical potential of Gd-CHPOA as a tumor-imaging MRI contrast agent, permitting tumor identification and assessment with a high signal-to-background ratio

Molecular Engineering of a Fluorescent Bioprobe for Sensitive and Selective Detection of Amphibole Asbestos

<div><p>Fluorescence microscopy-based affinity assay could enable highly sensitive and selective detection of airborne asbestos, an inorganic environmental pollutant that can cause mesothelioma and lung cancer. We have selected an <i>Escherichia coli</i> histone-like nucleoid structuring protein, H-NS, as a promising candidate for an amphibole asbestos bioprobe. H-NS has high affinity to amphibole asbestos, but also binds to an increasingly common asbestos substitute, wollastonite. To develop a highly specific Bioprobe for amphibole asbestos, we first identified a specific but low-affinity amosite-binding sequence by slicing H-NS into several fragments. Second, we constructed a streptavidin tetramer complex displaying four amosite-binding fragments, resulting in the 250-fold increase in the probe affinity as compared to the single fragment. The tetramer probe had sufficient affinity and specificity for detecting all the five types of asbestos in the amphibole group, and could be used to distinguish them from wollastonite. In order to clarify the binding mechanism and identify the amino acid residues contributing to the probe’s affinity to amosite fibers, we constructed a number of shorter and substituted peptides. We found that the probable binding mechanism is electrostatic interaction, with positively charged side chains of lysine residues being primarily responsible for the probe’s affinity to asbestos.</p> </div

SDS–PAGE analysis of amosite-binding proteins isolated from <i>E. coli</i> lysate.

<p>SDS–PAGE analysis of amosite-binding proteins isolated from <i>E. coli</i> lysate.</p

Adsorption of the engineered peptide tetramers on amosite.

<p>Pep1-Pep7 were used to identify the binding sequence and optimal linker length. Pep8-Pep14 were used to determine the binding mechanism and the contribution of individual amino acids of the binding sequence. The percentage values indicate the adsorption of each peptide tetramer relative to that of pep3, which is referred to as the “original binding sequence”.</p

Scatchard analysis of H-NS_60-90 monomer and its streptavidin-based tetramer’s adsorption on amosite.

<p>Scatchard analysis of H-NS_60-90 monomer and its streptavidin-based tetramer’s adsorption on amosite.</p

Specificity of fluorescently labeled tetramers of H-NS fragments.

<p>The fibers are stained with fluorescently labeled tetramers of the indicated H-NS fragments. Each pair of phase contrast and fluorescence micrographs of amosite and wollastonite fibers shows the same field of view. Bar, 50 µm.</p

Amosite fibers stained with fluorescently labeled monomers and streptavidin-based tetramers of H-NS_60-90.

<p>Each pair of phase contrast and fluorescence micrographs shows the same field of view. Due to lower affinity of H-NS_60-90 monomer, its adsorption on amosite at the indicated concentration is minimal. Bar, 10 µm.</p

Self-Assembled Polypeptide Nanogels with Enzymatically Transformable Surface as a Small Interfering RNA Delivery Platform

Nanometer-size gel particles, or nanogels, have potential for delivering therapeutic macromolecules. A cationic surface promotes cellular internalization of nanogels, but undesired electrostatic interactions, such as with blood components, cause instability and toxicities. Poly­(ethylene glycol) coating has been used to shield charges, but this decreases delivery efficiency. Technical difficulties in synthesis and controlling molecular weights make it unfeasible to, instead, coat with biodegradable polymers. Our proposed solution is cationized nanogels enzymatically functionalized with branched polysaccharide chains, forming a shell to shield charges and increase stability. Biodegradation of the polysaccharides by an endogenous enzyme would then expose the cationic charges, allowing cellular internalization and cargo delivery. We tested this concept, preparing maltopentaose functionalized cholesteryl poly­(l-lysine) nanogel and using tandem enzymatic polymerization with glycogen phosphorylase and glycogen branching enzyme, to add branched amylose moieties, forming a CbAmyPL nanogel. We characterized CbAmyPL nanogels and investigated their suitability as small interfering RNA (siRNA) carriers in murine renal carcinoma (Renca) cells. The nanogels had neutral ζ potential values that became positive after degradation by α-amylase. Foster resonance energy transfer demonstrated that the nanogels formed stable complexes with siRNA, even in the presence of bovine serum albumin and after α-amylase exposure. The nanogels, with or without α-amylase, were not cytotoxic. Complexes of CbAmyPL with siRNA against vascular endothelial growth factor (VEGF), when incubated alone with Renca cells decreased VEGF mRNA levels by only 20%. With α-amylase added, however, VEGF mRNA knockdown by the siRNA/nanogels complexes was 50%. Our findings strongly supported the hypothesis that enzyme-responsive nanogels are promising as a therapeutic siRNA delivery platform