Design Of Gene Delivery Vectors Research Articles

A Kinetic Model of Oligonucleotide-Brush Interactions for the Rational Design of Gene Delivery Vectors.

Polymer brushes are attractive candidates for the design of gene delivery vectors as they allow the systematic study of the impact of structural (type, size, and shape of nanomaterials core) and physicochemical parameters (cationic monomer chemistry, brush thickness, and grafting density) on transfection efficiency. However, relatively little is known of their interactions of oligonucleotides. To study such interactions, we use surface plasmon resonance and developed a kinetic model of brush binding and infiltration. We identify the striking impact that brush grafting density and thickness have on oligonucleotide kinetics of infiltration, binding affinity, and maximum loading. Surprisingly, double-stranded RNA molecules are found to load at significantly higher levels compared to DNA molecules of identical sequence (apart from uracils/thymines). Furthermore, analysis of the kinetics of adsorption of these oligonucleotides indicates that the stoichiometry of binding (ratio of amine versus phosphate residues) is close to parity for the uptake of 20 bp double-stranded RNA. Finally, nanoparticles were designed to be used as gene transfection vectors and to quantify if the brush grafting density and thickness significantly impact transfection efficiencies in a small interfering RNA knockdown assay. Therefore, this study demonstrates the rational design of polymer brush-based nanoparticle vectors for efficient delivery of oligonucleotides. The model developed will allow to uncover how the refinement of the physicochemical and structural properties of polymer brushes enable the tuning of RNA binding and allow the systematic study of cationic vectors efficiency for RNA delivery.

Solution Conformation of Polymer Brushes Determines Their Interactions with DNA and Transfection Efficiency.

Polymer brush-functionalized nanomaterials offer interesting features for the design of gene delivery vectors as their physicochemical and structural properties can be designed independently of the chemistry, size and shape of the nanomaterial core. However, little is known of the parameters regulating the adsorption and infiltration of DNA molecules at the surface of positively charged polymer brushes, despite the importance of such processes for gene delivery. Here we investigate the role of the molecular environment (e.g., pH, type of buffer, concentration) on the interactions between plasmid DNA and positively charged poly(dimethylaminoethyl methacrylate) (PDMAEMA) brushes using a combination of light scattering, electrophoretic light scattering, in situ ellipsometry, and surface plasmon resonance. We show that the conformation of swollen PDMAEMA brushes is modulated by the surrounding buffer and that this impacts strongly on the ability of such brushes and nanomaterials based on these coatings to complex DNA molecules. In turn, the levels of transfection efficiency measured correlate with changes in brush conformation and DNA binding. Therefore, this work demonstrates the importance of molecular design of polymer brushes to control DNA complexation and release in order to optimize the performance of polymer brush-functionalized nanomaterials for gene delivery applications.

Graphene oxide–cationic polymer conjugates: Synthesis and application as gene delivery vectors

Nanomedicine as the interface between nanotechnology and medical sciences is a new area that has attracted the attention of vast groups of researchers. Carbon nanomaterials are common platform for synthesis of nanoparticles for biomedical applications due to their low cytotoxicity and feasible internalization into mammalian cell lines (Yang et al., 2007; Arora et al., 2014; Oh and Park, 2014). Synthesis of vectors based on various cationic polymers polyethylenimine (PEI), polypropylenimine (PPI) and polyamidoamine (PAMAM) and their derivatives were considered as a strategy for transferring plasmid DNA and treatment of genetic diseases. Considering the low cytotoxicity of graphene, chemical modification of its surface has led to fabrication of novel gene delivery systems based on graphene and graphene oxide. Herein we report the synthesis of three groups of vectors based on conjugation of graphene oxide (GO) with alkylated derivatives of three different cationic polymers (polyethylenimine (PEI), polypropylenimine (PPI) and polyamidoamine (PAMAM)) through different linkers including surface carboxyl group, glycine and spermidine. Two main challenges in design of gene delivery vectors is decreasing cytotoxicity while improving the transfection efficiency. All synthesized vectors showed significantly lower cellular toxicity compared to bare polymer. A plasmid encoding green fluorescent protein (GFP) was used to evaluate the transfection efficiency of nanoparticles both qualitatively using live cell fluorescent imaging and quantitatively using flow cytometry and each vector was compared to its polymer base. Most successful conjugation strategy was observed in the case of PEI conjugates among which most efficient vector was PEI–GO conjugate bearing glycine linker. This vector was 9 fold more effective in terms of the percent of EGFP transfected cells.

Evaluation of Rod‐Shaped Nanoparticles as Carriers for Gene Delivery

Fabrication of nonspherical particles for gene delivery remains a major challenge. In this study, novel rod‐like nanoparticles are prepared for efficient gene delivery by self‐assembly of α‐cyclodextrin (α‐CD) and polyethylenimine‐methoxy poly(ethylene glycol) (PEI‐mPEG). The study reveals that the rod‐like PEI‐mPEG/α‐CD particles can bind DNA effectively and the resulting PEI‐mPEG/α‐CD/DNA complexes show over four times higher gene delivery capability than their spherical counterparts and PEI(25K) due to more efficient cellular uptake. Furthermore, the cytotoxicity of rod‐like PEI‐mPEG/α‐CD is about five times lower than that of the nanospheres, and 50 times lower than that of DNA/PEI(25K). These results indicate that shape is an important parameter for the design of gene delivery vectors.

Hybrid Nanomaterial Complexes for Advanced Phage-guided Gene Delivery.

Developing nanomaterials that are effective, safe, and selective for gene transfer applications is challenging. Bacteriophages (phage), viruses that infect bacteria only, have shown promise for targeted gene transfer applications. Unfortunately, limited progress has been achieved in improving their potential to overcome mammalian cellular barriers. We hypothesized that chemical modification of the bacteriophage capsid could be applied to improve targeted gene delivery by phage vectors into mammalian cells. Here, we introduce a novel hybrid system consisting of two classes of nanomaterial systems, cationic polymers and M13 bacteriophage virus particles genetically engineered to display a tumor-targeting ligand and carry a transgene cassette. We demonstrate that the phage complex with cationic polymers generates positively charged phage and large aggregates that show enhanced cell surface attachment, buffering capacity, and improved transgene expression while retaining cell type specificity. Moreover, phage/polymer complexes carrying a therapeutic gene achieve greater cancer cell killing than phage alone. This new class of hybrid nanomaterial platform can advance targeted gene delivery applications by bacteriophage.

Pharmacodynamics of adenovector distribution within the inner ear tissues of the mouse

Recent studies have demonstrated that delivery of genes to the inner ear can achieve a variety of effects ranging from support of auditory neuron survival to protection and restoration of hair cells, demonstrating the utility of vector based gene delivery. Translation of these findings to useful experimental systems or even clinical applications requires a detailed understanding of the pharmacokinetics of gene delivery in the inner ear. Ideal gene delivery systems will employ a well tolerated vector which efficiently transduces the appropriate target cells within a tissue, but spare non-target structures. Adenovectors based on serotype 5 (Ad 5) are commonly used vectors, are easy to construct and have a long track record of efficacious gene transfer in the inner ear. In this study we demonstrate that distribution of Ad5 vector occurs in a basal to apical gradient with rapid distribution of vector to the vestibule after delivery via a round window cochleostomy. Transduction of the vector and expression of the delivered transgene occurs by 10 min post vector delivery. At 24 h post delivery only 16% of vector that was initially detectable within the inner ear by quantitative PCR remained. Perilymph sampling was used to determine that vector concentrations in perilymph peaked at 30 min post delivery and then declined rapidly. Understanding these basic distribution patterns and parameters for delivery are important for the design of gene delivery vectors and vital for modeling dose responses to achieve safe efficacious delivery of a therapeutic agent.

Influence of Physicochemical Properties on Pharmacokinetics of Non-viral Vectors for Gene Delivery

The influence of physicochemical properties on the in vivo pharmacokinetics of gene delivery vectors after systemic administration is reviewed based on our studies. We have been studying the development of DNA delivery systems, such as plasmid DNA complexed with cationic polymers (polyplexes) and cationic liposomes (lipoplexes). Even if target-recognizable ligand is incorporated into the system, the overall physicochemical properties, notably size and charge, are predominant factors influencing in vivo disposition characteristics of the vector. Based on this consideration, liver cell-specific carrier systems via receptor-mediated endocytosis were successfully developed by optimizing physicochemical characteristics. In conclusion, rational design of gene delivery vectors requires an understanding of their pharmacokinetics in relation to the physicochemical properties. Optimization of the physicochemical properties is important for successful in vivo gene delivery by non-viral vectors.