Tunable Nanocarrier Morphologies from Glycopolypeptide-Based Amphiphilic Biocompatible Star Copolymers and Their Carbohydrate Specific Intracellular Delivery.

Nanocarriers with carbohydrates on the surface represent a very interesting class of drug-delivery vehicles because carbohydrates are involved in biomolecular recognition events in vivo. We have synthesized biocompatible miktoarm star copolymers comprising glycopolypeptide and poly(ε-caprolactone) chains using ring-opening polymerization and "click chemistry". The amphiphilic copolymers were self-assembled in water into morphologies such as nanorods, polymersomes, and micelles with carbohydrates displayed on the surface. We demonstrate that the formation of nanostructure could be tuned by chain length of the blocks and was not affected by the type of sugar residue. These nanostructures were characterized in detail using a variety of techniques such as TEM, AFM, cryogenic electron microscopy, spectrally resolved fluorescence imaging, and dye encapsulation techniques. We show that it is possible to sequester both hydrophobic as well as hydrophilic dyes within the nanostructures. Finally, we show that these noncytotoxic mannosylated rods and polymersomes were selectively and efficiently taken up by MDA-MB-231 breast cancer cells, demonstrating their potential as nanocarriers for drug delivery.

Cationic charged helical glycopolypeptide using ring opening polymerization of 6-deoxy-6-azido-glyco-N-carboxyanhydride.

Glycopolypeptides with a defined secondary structure are of significance in understanding biological phenomena. Synthetic glycopolypeptides, or polypeptides featuring pendant carbohydrate moieties, have been of particular interest in the field of tissue engineering and drug delivery. In this work, we have synthesized charged water-soluble glycopolypeptides that adopt a helical conformation in water. This was carried out by the synthesis of a glyco-N-carboxyanhydride (glyco-NCA) containing an azide group at the sixth position of the carbohydrate ring. Subsequently, the NCA was polymerized to obtain azide-containing glycopolypeptides having good control over molecular weight and polydispersity index (PDI) in high yields. We were also able to control the incorporation of the azide group by synthesizing random co-glycopolypeptide containing 6-deoxy-6-azido and regular 6-OAc functionalized glucose. This azide functionality allows for the easy attachment of a bioactive group, which could potentially enhance the biological activity of the glycopolypeptide. We were able to obtain water-soluble charged glycopolypeptides by both reducing the azide groups into amines and using CuAAC with propargylamine. These charged glycopolypeptides were shown to have a helical conformation in water. Preliminary studies showed that these charged glycopolypeptides showed good biocompatibility and were efficiently taken up by HepG2 cells.

Multiple topologies from glycopolypeptide-dendron conjugate self-assembly: nanorods, micelles, and organogels.

Glycopolypeptides (GPs) were synthesized by ring-opening polymerization of glycosylated N-carboxyanhydride monomer and attached to hydrophobic dendrons at one chain end by "click" reaction to obtain amphiphilic anisotropic macromolecules. We show that by varying polypeptide chain length and dendron generation, an organogel was obtained in dimethylsulfoxide, while nanorods and micellar aggregates were observed in aqueous solutions. Assemblies in water were characterized by electron microscopy and dye encapsulation. Secondary structure of the GP chain was shown to affect the morphology, whereas the chain length of the poly(ethylene glycol) linker between the GP and dendron did not alter rod-like assemblies. Bioactive surface chemistry of these assemblies displaying carbohydrate groups was demonstrated by interaction of mannose-functionalized nanorods with ConA.

Synthesis of silk fibroin-glycopolypeptide conjugates and their recognition with lectin.

Silk fibroin (SF), the natural fibrous protein created by the Bombyx mori silk worm, is being increasingly explored as a biomaterial for tissue engineering due to its excellent mechanical strength, high oxygen/water permeability, and biocompatibility. It is also well known that surface modification of SF with organic ligands such as the extracellular protein binding Arg-Gly-Asp (RGD) peptides help adhesion and proliferation of cells better-a key requirement for it to function as extracellular matrices. In this work, we have conjugated synthetic glycopolypeptides (GPs) that were synthesized by controlled ring-opening polymerization of α-manno-lys N-carboxyanhydrides (NCAs) onto SF by using Cu catalyzed click reaction to synthesize a new hybrid material (SF-GP), which we believe will have both the mechanical properties of native SF and the molecular recognition property of the carbohydrates in the GP. By controlling the amount of GP grafted onto SF, we have made three SF-GP conjugates that differ in their ability to assemble into films. SF-GP conjugates having a very high content of GP formed completely water-soluble brush-like polymer that displayed very high affinity toward the lectin concanavalin-A (Con-A). Films cast from SF-GP conjugates using lower amounts of grafted GP were more stable in water, and the stability can be modulated by varying the amount of GP grafted. The water-insoluble film SF-GP(25) was also found to bind to fluorescently labeled Con-A, as was seen by confocal microscopy. Such SF-GP hybrid films may be useful as mimics of extracellular matrices for tissue engineering.

Controlled synthesis of O-glycopolypeptide polymers and their molecular recognition by lectins.

The facile synthesis of high molecular weight water-soluble O-glycopolypeptide polymers by the ring-opening polymerization of their corresponding N-carboxyanhydride (NCA) in very high yield (overall yield > 70%) is reported. The per-acetylated-O-glycosylated lysine-NCA monomers, synthesized using stable glycosyl donors and a commercially available protected amino acid in very high yield, was polymerized using commercially available amine initiators. The synthesized water-soluble glycopolypeptides were found to be α-helical in aqueous solution. However, we were able to control the secondary conformation of the glycopolypeptides (α-helix vs nonhelical structures) by polymerizing racemic amino acid glyco NCAs. We have also investigated the binding of the glycopolypeptide poly(α-manno-O-lys) with the lectin Con-A using precipitation and hemagglutination assays as well as by isothermal titration calorimetry (ITC). The ITC results clearly show that the binding process is enthalpy driven for both α-helical and nonhelical structures, with negative entropic contribution. Binding stoichiometry for the glycopolypeptide poly(α-manno-O-lys) having a nonhelical structure was slightly higher as compared to the corresponding polypeptide which adopted an α-helical structure.