Ravin Narain

The effect of polymer architecture, composition, and molecular weight on the properties of glycopolymer-based non-viral gene delivery systems

Although a variety of non-viral gene delivery vectors has been synthesized and used for gene delivery purposes, well-defined glycopolymer-based gene delivery carriers is not well explored. Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization technique allows successful and facile synthesis of cationic glycopolymers containing pendant sugar moieties in the absence of protecting group chemistry. A library of cationic glycopolymers of pre-determined molar masses and narrow polydispersities ranging from 3 to 30 kDa has been synthesized using RAFT polymerization technique. These polymers differ from each other in their architectures (block versus random), molecular weights, and monomer ratios (carbohydrate to cationic segment). It is shown that the above-mentioned parameters can largely affect the toxicity, DNA condensation ability and gene delivery efficacy of these polymers. Statistical copolymers of high degree of polymerization are found to be the ideal vector for gene delivery purposes. These statistical copolymers show lower toxicity and higher gene expression in the presence and absence of serum, as compared to the corresponding diblock copolymers. This is the first example of well-defined synthetic glycopolymers as DNA carriers that works both in the presence and absence of serum proteins. The critical composition of carbohydrate segment in copolymers for enhanced gene delivery and low toxicity was determined and an increase in carbohydrate residues in copolymers resulted in a decrease in transfection efficiencies of these polymers. The effect of serum proteins on statistical and diblock copolymer based polyplexes and hence gene delivery efficacy was studied. The results showed that the diblock copolymer-based polyplexes showed lower interactions with serum proteins, lower cellular uptake and very low gene expression in both Hep G2 and Hela cells in comparison to statistical copolymers.

Biotinylated glyco-functionalized quantum dots: synthesis, characterization, and cytotoxicity studies.

Quantum dots (QDs) containing surface carboxylic groups have been successfully modified using biotinylated glycopolymer and carbohydrate/biotin reagents via EDC coupling. The biotinylated glycopolymer was synthesized in controlled dimension via the reversible addition-fragmentation chain transfer (RAFT) polymerization of the three monomers containing biotin, sugar, and amine groups as pendent groups, respectively. The modified QDs were analyzed by dynamic light scattering and fluorescence spectrophotometry, and the data revealed no change in the physical properties of QDs after surface modification. Furthermore, the surface modified QDs showed excellent water solubility and colloidal stability. Subsequently, the availability of the biotin ligand on the surface of functionalized QDs was quantified using 4-hydroxyazobenzene 2-carboxylic acid (HABA)/avidin binding assay. Cell viability studies revealed that the cytotoxicity of QDs after surface functionalization is improved and that the biotinylated glycopolymer modified QDs showed an enhancement in biocompatibility as compared to that of the original QDs. The biotinylated glyco-functionalized quantum dots may act as new suitable fluorescent probes in biomedical applications.

Cationic glyco-functionalized single-walled carbon nanotubes as efficient gene delivery vehicles.

Carbon nanotubes are an emerging class of nanomaterials that are receiving enormous attention in the field of biomedicines due to their biocompatibility, degradability, cell penetrating abilities, and, more specifically, their remarkable ability to localize in the nucleus of the cell without the need of nuclear localizing signals. They are used as drug and gene delivery agent for in vitro studies; however, their transfection efficiencies in vitro are still questionable. We report here the surface functionalization of single-walled carbon nanotubes (SWNTs) with cationic glycopolymers and their use as an in vitro gene transfer agent. The copolymer modified SWNTs are found to be biocompatible and exhibit transfection efficiencies that are comparable to the commercially available agent lipofectamine 2000.

The effect of molecular weight, compositions and lectin type on the properties of hyperbranched glycopolymers as non-viral gene delivery systems

The architectures of gene delivery vectors, in addition to their molecular weights and compositions, can play a critical role in DNA condensation and hence on their gene expression. In general, branched polymers are superior gene delivery vectors as compared to their linear analogs. This study reports the efficacy of cationic hyperbranched glycopolymers for DNA condensation and gene expression. Hyperbranched glycopolymers of varying molecular weights and compositions are synthesized via reversible addition fragmentation chain transfer (RAFT) process and are further explored for their gene expression in vitro. Galactose-based hyperbranched polymers are compared to glucose-derived hyperbranched polymers for their cellular uptake, toxicity and gene expression. It is found that molecular weight of hyperbranched polymers, and carbohydrate content of copolymers are critical factors in determining the gene expression as well as in imparting the specificity to these novel gene delivery vectors. The galactose-based hyperbranched glycopolymer of ~30 kDa or lower show improved gene expression at varying polymer/plasmid ratios. The incubation of hyperbranched polyplexes in the presence of serum protein show the presence of stable particles and gene expression of these hyperbranched polyplexes is unaffected in the presence of serum proteins. Furthermore, the cellular uptake and gene expression are studied in two different cell lines in the presence of lectins. It is found that polyplexes-lectin conjugates show enhanced cellular uptake in vitro, however their gene expression is cell line and lectin type dependent.

Synthesis of low polydispersity, controlled-structure sugar methacrylate polymers under mild conditions without protecting group chemistryElectronic supplementary information (ESI) available: experimental protocols, spectroscopic characterization and rates of polymerization. See http://www.rsc.org/suppdata/cc/b2/b208654a/

We report the synthesis of low polydispersity, controlled-structure sugar methacrylate polymers by the ring-opening reaction of 2-aminoethyl methacrylate with D-gluconolactone, followed by the atom transfer radical polymerisation of the resulting sugar methacrylate in methanol at 20 degrees C.

Degradable Thermoresponsive Nanogels for Protein Encapsulation and Controlled Release

Reversible addition-fragmentation chain transfer (RAFT) polymerization technique was used for the fabrication of stable core cross-linked micelles (CCL) with thermoresponsive and degradable cores. Well-defined poly(2-methacryloyloxyethyl phosphorylcholine), poly(MPC) macroRAFT agent, was first synthesized with narrow molecular weight distribution via the RAFT process. These CCL micelles (termed as nanogels) with hydrophilic poly(MPC) shell and thermoresponsive core consisting of poly(methoxydiethylene glycol methacrylate) (poly(MeODEGM) and poly(2-aminoethyl methacrylamide hydrochloride) (poly(AEMA) were then obtained in a one-pot process by RAFT polymerization in the presence of an acid degradable cross-linker. These acid degradable nanogels were efficiently synthesized with tunable sizes and low polydispersities. The encapsulation efficiencies of the nanogels with different proteins such as insulin, BSA, and β-galactosidase were studied and found to be dependent of the cross-linker concentration, size of protein, and the cationic character of the nanogels imparted by the presence of AEMA in the core. The thermoresponsive nature of the synthesized nanogels plays a vital role in protein encapsulation: the hydrophilic core and shell of the nanogels at low temperature allow easy diffusion of the proteins inside out and, with an increase in temperature, the core becomes hydrophobic and the nanogels are easily separated out with entrapped protein. The release profile of insulin from nanogels at low pH was studied and results were analyzed using bicinchoninic assay (BCA). Controlled release of protein was observed over 48 h.

Degradable Thermoresponsive Core Cross-Linked Micelles: Fabrication, Surface Functionalization, and Biorecognition

We report on the fabrication of core cross-linked (CCL) micelles possessing thermoresponsive and degradable cores and biocompatible coronas cofunctionalized with carbohydrate and biotin moieties. Well-defined poly(2-aminoethylmethacrylamide) (PAEMA) homopolymer was first synthesized in a controlled fashion via the reversible addition-fragmentation chain transfer (RAFT) process. CCL micelles comprising of well-solvated PAEMA coronas and thermoresponsive cores were then obtained in a one-pot manner via RAFT copolymerization of N-isopropylacrylamide (NIPAM) and bis(2-methacryloyloxyethyl) disulfide (DSDMA) difunctional monomers by employing PAEMA as the macro-RAFT agent. In the presence of dithiothreitol (DTT), the obtained CCL micelles can be disintegrated into unimers due to the cleavage of disulfide cross-linkers, whereas deswelling of micellar cores can be achieved via heating above the phase transition temperature of PNIPAM. Thus, the release profiles of this type of nanocarriers are expected to be triggered by temperature and thiols or a combination of both. Furthermore, primary amine residues located within coronas of CCL micelles have been further exploited for surface functionalization with biotin and carbohydrate moieties, rendering them biocompatible and bioactive. The availability of biotin within the coronas of CCL micelles was confirmed by HABA/avidin binding assay and Diffractive Optics Technology (DOT) biosensing instrument. After the micelles were immobilized on the surface of avidin-sensor chip, specific biorecognition of the available biotins and carbohydrate moieties on the CCL micelles was further confirmed. We expect that this novel type of bioactive and potentially biocompatible CCL micelles can be employed as smart nanocarriers for targeted drug delivery and controlled release.

Hyperbranched Glycopolymers for Blood Biocompatibility

Carbohydrate-based drug and gene delivery carriers are becoming extremely popular for in vitro and in vivo applications. These carriers are found to be nontoxic and can play a significant role in targeted delivery. However, the interactions of these carriers with blood cells and plasma components are not well explored. To the best of our knowledge, there are currently no reports that explore the role of carbohydrate based carriers for blood biocompatibility. Hyperbranched glycopolymers of varying molecular weights are synthesized by reversible addition-fragmentation chain transfer polymerization (RAFT) and are studied in detail for their biocompatibility, including hemocompatibility and cytotoxicity against different cell lines in vitro. The hemocompatibility studies (such as hemolysis and platelet activation) indicate that hyperbranched glycopolymers of varying molecular weights produced are highly hemocompatible and do not induce clot formation, red blood cell aggregation, and immune response. Hence, it can be concluded that glycopolymers functionalized carriers can serve as an excellent candidate for various biomedical applications. In addition, cytotoxicity of these hyperbranched polymers is studied in primary and malignant cell lines at varying concentrations using cell viability assay.

Cationic Glyconanoparticles: Their Complexation with DNA, Cellular Uptake, and Transfection Efficiencies

There is a need to synthesize new gene delivery vehicles that can deal with the problems of endosomal escape and nuclear entry. We propose cationic glycopolymer-stabilized gold nanoparticles as an effective gene delivery system. The cationic glyconanoparticles synthesized were revealed to be biocompatible and are resistant to aggregation in physiological conditions. The complexation of DNA to the cationic glyconanoparticles is determined by agarose gel electrophoresis. The localization of the DNA-glyconanoparticles inside the Hela cell line and their mechanism of uptake is studied by confocal microscopy. Finally, the efficacy of the glyconanoparticles as gene delivery vehicles in vitro is studied by their complexation with cyanine fluorescence protein encoded plasmid, and the transfection efficiency is found to be comparable to the commercially available control Lipofectamine 2000.

Synthesis of Biotinylated α-D-Mannoside or N-Acetyl β-D-Glucosaminoside Decorated Gold Nanoparticles: Study of Their Biomolecular Recognition with Con A and WGA Lectins

Gold nanoparticles (NPs) functionalized with a mixed shell of well-defined biotinylated glycopolymers and polyethylene glycol (PEG) provide an effective platform for the biomolecular recognition of proteins both in solution and on surfaces. Well-defined biotinylated glycopolymers were first synthesized by the reversible addition-fragmentation chain transfer (RAFT) process. They contain two types of carbohydrate residues either N-acetyl β-D-glucosaminopyranoside (GlcNAc) or α-D-mannopyranoside (Man) as pendent groups. The biotinylated glycopolymers and polyethylene glycol were subsequently used in the in situ formation of gold glyconanoparticles via an easy photochemical process. The obtained biotinylated glyconanoparticles were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The bioavailability of the biotin and specific carbohydrate residues at the periphery of the NPs were assessed using the diffraction optic technology (DOT) system. The studies showed the accessibility of the biotin ligands for conjugation to immobilized avidin on the DOTLab biosensor. Furthermore, these avidin conjugated glyconanoparticles were found to selectively immobilize lectins. The specificity of lectin binding was dependent on the type of carbohydrate residues. As such, N-acetyl β-D-glucosaminoside decorated gold nanoparticles were found to specifically interact with wheat germ agglutinin (WGA) lectin, whereas α-D-mannoside ones were found to specifically interact with Concanavalin A (Con A) lectin.