Marya Ahmed

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.

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.

Well-controlled cationic water-soluble phospholipid polymer-DNA nanocomplexes for gene delivery.

The facile synthesis of biocompatible and nontoxic gene delivery vectors has been the focus of research in recent years due to the high potential in treating genetic diseases. 2-Methacryloxyethyl phosphorylcholine (MPC) copolymers were recently studied for their ability to produce nontoxic and biocompatible materials. The synthesis of well-defined and water-soluble MPC polymer based cationic vectors for gene delivery purposes was therefore attractive, due to the potential excellent biocompatibility of the resulting copolymers. Herein, cationic MPC copolymers of varying architectures (block versus random) were produced by the reversible addition--fragmentation chain transfer (RAFT) polymerization technique. The copolymers produced were evaluated for their gene delivery efficacy in the presence and absence of serum. It was found that copolymer architectures and molecular weights do affect their gene delivery efficacy. The statistical copolymers produced larger particles, and showed poor gene transfection efficiency as compared to the diblock copolymers. The diblock copolymers served as efficient gene delivery vectors, in both the presence and absence of serum in vitro. To the best of our knowledge, this is the first report where the effect of architecture of MPC based copolymer on gene delivery efficacy has been studied.

Intracellular delivery of DNA and enzyme in active form using degradable carbohydrate-based nanogels.

The facile encapsulation of biomolecules along with efficient formulation and storage makes nanogels ideal candidates for drug and gene delivery. So far, nanogels have not been used for the codelivery of plasmid DNA and proteins due to several limitations, including low encapsulation efficacy of biomolecule of similar charges and the size of cargo materials. In this study, temperature and pH sensitive carbohydrate-based nanogels are synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization technique and are studied in detail for their capacity to encapsulate and codeliver plasmid DNA and proteins. The temperature sensitive property of nanogels allows the facile encapsulation of biomaterials, while its acid-degradable profile allows the burst release of biomolecules in endosomes. Hence these materials are expected to serve as efficient vectors to deliver biomolecules of choice either alone or as codelivery system. The nanogels produced are relatively monodisperse and are around 30-40 nm in diameter at 37 °C. DNA condensation efficacy of the nanogels is dependent on the hydrophobic property of the core of the nanogels. The DNA-nanogel complexes are formed by the interaction of carbohydrate residues of nanogels with the DNA, and complexes are further stabilized with linear cationic glycopolymers. The DNA-nanogels complexes are also studied for their protein loading capacity. The degradation of the nanogels and the controlled release of DNA and proteins are then studied in vitro. Furthermore, the addition of a nontoxic, cationic glycopolymer to the nanogel-DNA complexes is found to improve the cellular uptake and hence to improve gene expression.

Study of Transfection Efficiencies of Cationic Glyconanoparticles of Different Sizes in Human Cell Line

The growing attention toward the synthesis and uses of gold nanoparticles for biomedical applications is based on their biocompatibility, ease of functionalization, and unique optical and electronic properties. Recently, the gold nanoparticles are also found to induce the size-dependent interactions with living tissues. It has been found that gold nanoparticles of different sizes are uptaken by the cells in vitro and by the organs of living specimens in vivo at different rates. Herein, we report the use of gold nanoparticles of different sizes as a gene delivery agent. The gold nanoparticles of 10, 40, and 100 nm diameter were surface functionalized with cationic glycopolymer, and their biocompatibility under physiological conditions was investigated. The stable nanoparticles were then complexed with enhanced cyanine fluorescence protein plasmid (pECFP) and their transfection efficiencies in Hela cell line were studied. It was found that gold nanoparticles of 40 nm core diameter exhibit highest transfection efficiencies compared to the other sizes of nanoparticles studied.

Rapid Synthesis of Gold Nanorods Using a One-Step Photochemical Strategy

Rapid synthesis of gold nanorods of controlled dimensions is one of the desired aspects of nanotechnology as a result of the potential of these nanomaterials for biomedical applications. The synthesis of gold nanorods has been achieved using a photoinitiator as an instant source of ketyl radicals, which allows the synthesis of gold nanorods in minutes. This is the first report providing a one-step synthesis of nanorods of controlled dimensions in 20-30 min using photoinitiator I-2959 as a source of ketyl radicals. Furthermore, the role of UV intensity, the concentration of silver ions, and the presence of cosolvents and a cosurfactant have been studied in detail in an effort to produce nanorods with controlled dimensions in higher yields. The role of acetone in nanorod synthesis has been explored in detail, and it has been demonstrated that, for the photochemical synthesis of nanorods using a photoinitiator, acetone is not a critical component and can be replaced by other water-miscible solvents, thus the successful synthesis of nanorods in tetrahydrofuran (THF) has been demonstrated. It has also been found that a cosurfactant and an organic solvent are not required for the synthesis of nanorods; however, their presence is found to improve the monodispersity of nanorod samples, in addition to providing a higher yield.