Self-Assembled Cationic Nanogels for Intracellular Protein Delivery

An effective intracellular protein delivery system with self-assembled cationic nanogels is reported. Interaction of proteins with self-assembled nanogels of cationic cholesteryl group-bearing pullulans (CHPNH2) was investigated by dynamic light scattering (DLS), transmission electron micrograph (TEM), fluorescence resonance energy transfer (FRET), and fluorescence correlation spectroscopy (FCS). The cationic nanogels strongly interacted with bovine serum albumin (BSA) and formed monodispersed nanoparticels (<50 nm). The complex more effectively internalized into HeLa cells than cationic liposomes and a protein transduction domain (PTD) based carrier even in the presence of serum. The higher efficiency of the nanogel carrier is probably due to the formation of colloidally stable nanoparticles with the protein. The enzymatic activity of β-galactosidase (β-Gal) was retained after internalization into cells. The nanogel carrier promoted nuclear delivery of a GFP-conjugated nuclear localization signal and Tat as a PTD (Tat-NLS-GFP). A blocking experiment with chemical inhibitors revealed the possible involvement of macropinocytosis in the uptake of the nanogel complex. After cellular uptake, the complex of the nanogel−protein was dissociated and the protein was released inside the cell. Such a self-assembled cationic nanogel system should create opportunities for novel applications of protein delivery

Functional Cycloamylose as a Polysaccharide-Based Biomaterial: Application in a Gene Delivery System

Cycloamylose (CA) exhibits differences in geometry and greater colloidal stability compared with amylose. Here we report the synthesis of a cationic CA derivative and its application for gene delivery. Cationic CA (catCA) and cationic amylose (catAmy) were synthesized by introducing spermine groups. The interactions between catCA or catAmy with plasmid DNA encoding firefly luciferase was examined by gel electrophoresis, dynamic light scattering, and transmission electron microscopy. Activity as a gene delivery system was evaluated by flow cytometry and luciferase assays. CatCA formed a condensed pDNA complex (∼250 nm in size). The catCA complex showed enhanced cellular uptake and greater transfection efficiency than the catAmy complex. Hemolysis by membrane destabilization and the effects of hydroxychloroquine on transfection ability suggest that the formation of a supramolecular complex with CA is important for high transfection activity. These results suggested that CA can be used as new polysaccharide-based biomaterials

Novel Thermoresponsive Polymers Having Biodegradable Phosphoester Backbones

Novel Thermoresponsive Polymers Having Biodegradable Phosphoester Backbone

Amphiphilic Polysaccharide Nanoballs: A New Building Block for Nanogel Biomedical Engineering and Artificial Chaperones

Enzymatically synthesized glycogen (ESG), a highly branched (1→4)(1→6)-linked α-glucan, is a new monodisperse spherical hyperbranched nanoparticle (molecular weight, 106−107; diameter, 20−30 nm), polysaccharide nanoball. Amphiphilic ESG nanoballs were synthesized by introducing a cholesterol group to enzymatically synthesized glycogen (CHESG). CHESG assembled into a structure containing a few molecules to form cluster nanogels (approximately 35 nm in diameter) in water. The cluster nanogels were dissociated by the addition of cyclodextrin (CD) to form a supramolecular CHESG−CD nanocomplex due to complexation with the cholesterol group and CD. The CHESG nanogel showed high capacity for complexation with proteins, and the CHESG−CD nanocomplex showed high chaperone-like activity for thermal stabilization of enzymes. CHESG has great potential to become a new building block for nanogel biomedical engineering and to act as an artificial chaperone for protein engineering

Nanogelation and Thermal Stabilization of Enzyme by Vitamin B₆‑Bearing Polysaccharide as Biocrosslinker

Nanogels containing a protein (carbonic anhydrase, CA) were prepared by cross-linking CA and pyridoxal (vitamin B6)-bearing pullulan (PLPP) as a biocrosslinker via Schiff base formation. UV titration and high-performance liquid chromatography confirmed that CA was quantitatively complexed with PLPP in the presence of zinc ions. Dynamic light scattering and transmission electron microscopy showed that the nanogel diameter was about 20 nm. CA retained 90% of its native activity after complexation with PLPP. Moreover, the residual enzymatic activity of CA after heating and its long-term storage stability at room temperature were improved by complexation with PLPP. Enzyme nanogelation with PLPP is an efficient method for enzyme stabilization

Additional file 3 of CD63-positive extracellular vesicles are potential diagnostic biomarkers of pancreatic ductal adenocarcinoma

Additional file 3: Table S2. Summary of Power calculation

Polysaccharide-Hair Cationic Polypeptide Nanogels: Self-Assembly and Enzymatic Polymerization of Amylose Primer-Modified Cholesteryl Poly(l‑lysine)

In this study, we prepared a new associating polymer, ChMaPLL, by the substitution of the poly­(l-lysine) moiety with oligosaccharide amylose primer and cholesterol. ChMaPLL formed positively charged polypeptide nanogels (∼50 nm) via self-assembly in water. The nanogels showed a secondary structural transition to an α-helix structure induced by poly­(l-lysine) in response to an increase in pH. Oligosaccharides of the nanogels reacted with the phosphorylase a enzyme. Amylose-conjugated nanogels were obtained by enzymatic polymerization. The elongation of the saccharide chain shielded the positive charge of the nanogels. The multiresponsive polysaccharide–polypeptide hybrid nanogels might prove to be useful in the areas of biotechnology and biomedicine

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

Enzyme-Responsive Molecular Assembly System with Amylose-Primer Surfactants

The association of amylose-primer surfactants was controlled by changing the amphiphilicity with a chain-elongation reaction triggered by the addition of phosphorylase. By using this property, the micelle-to-vesicle transition of mixed lipid/ primer systems can be controlled