Physicochemical and Biological Evaluation of siRNA Polyplexes Based on PEGylated Poly(amido amine)s

PURPOSE: Use of RNA interference as novel therapeutic strategy is hampered by inefficient delivery of its mediator, siRNA, to target cells. Cationic polymers have been thoroughly investigated for this purpose but often display unfavorable characteristics for systemic administration, such as interactions with serum and/or toxicity. METHODS: We report the synthesis of a new PEGylated polymer based on biodegradable poly(amido amine)s with disulfide linkages in the backbone. Various amounts of PEGylated polymers were mixed with their unPEGylated counterparts prior to polyplex formation to alter PEG content in the final complex. RESULTS: PEGylation effectively decreased polyplex surface charge, salt- or serum-induced aggregation and interaction with erythrocytes. Increasing amount of PEG in formulation also reduced its stability against heparin displacement, cellular uptake and subsequent silencing efficiency. Yet, for polyplexes with high PEG content, significant gene silencing efficacy was found, which was combined with almost no toxicity. CONCLUSIONS: PEGylated poly(amido amine)s are promising carriers for systemic siRNA delivery in vivo

Biocleavable Polycationic Micelles as Highly Efficient Gene Delivery Vectors

An amphiphilic disulfide-containing polyamidoamine was synthesized by Michael-type polyaddition reaction of piperazine to equimolar N, N′-bis(acryloyl)cystamine with 90% yield. The polycationic micelles (198 nm, 32.5 mV), prepared from the amphiphilic polyamidoamine by dialysis method, can condense foreign plasmid DNA to form nanosized polycationic micelles/DNA polyelectrolyte complexes with positive charges, which transfected 293T cells with high efficiency. Under optimized conditions, the transfection efficiencies of polycationic micelles/DNA complexes are comparable to, or even higher than that of commercially available branched PEI (Mw 25 kDa)

Therapeutic efficacy in a hemophilia B model using a biosynthetic mRNA liver depot system

DNA-based gene therapy has considerable therapeutic potential, but the challenges associated with delivery continue to limit progress. Messenger RNA (mRNA) has the potential to provide for transient production of therapeutic proteins, without the need for nuclear delivery and without the risk of insertional mutagenesis. Here we describe the sustained delivery of therapeutic proteins in vivo in both rodents and non-human primates via nanoparticle-formulated mRNA. Nanoparticles formulated with lipids and lipid-like materials were developed for delivery of two separate mRNA transcripts encoding either human erythropoietin (hEPO) or factor IX (hFIX) protein. Dose-dependent protein production was observed for each mRNA construct. Upon delivery of hEPO mRNA in mice, serum EPO protein levels reached several orders of magnitude (>125 000-fold) over normal physiological values. Further, an increase in hematocrit (Hct) was established, demonstrating that the exogenous mRNA-derived protein maintained normal activity. The capacity of producing EPO in non-human primates via delivery of formulated mRNA was also demonstrated as elevated EPO protein levels were observed over a 72-h time course. Exemplifying the possible broad utility of mRNA drugs, therapeutically relevant amounts of human FIX (hFIX) protein were achieved upon a single intravenous dose of hFIX mRNA-loaded lipid nanoparticles in mice. In addition, therapeutic value was established within a hemophilia B (FIX knockout (KO)) mouse model by demonstrating a marked reduction in Hct loss following injury (incision) to FIX KO mice

Laterally stabilized complexes of DNA with linear reducible polycations: strategy for triggered intracellular activation of DNA delivery vectors

Target-specific DNA delivery requires vectors that combine stability in the biological milieu, receptor-mediated uptake into target cells, and intracellular activation to mediate transgene expression. This is achieved here using polymer-coated vectors based on plasmid DNA complexed with a reductively degradable polycation (RPC), designed for intercellular degradation. The RPC were prepared by oxidation of the terminal cysteinyl thiol groups of Cys(Lys)<sub>10</sub>Cys. The complexes were coated and surface-cross-linked using multivalent reactive copolymers of N-(2-hydroxypropyl)methacrylamide (PHPMA), providing a unique combination of steric and reversible lateral stabilization, known to promote extended circulation in the bloodstream. Coated complexes containing RPC exhibited lateral stabilization that was reversible by treatment with 2.5 mM dithiothreitol, releasing free DNA after incubation with a polyanion. In contrast, coated complexes containing nonreducible poly(l-lysine) (PLL) were not destabilized by reduction. The biological usefulness of this trigger mechanism was examined by measuring transfection activity in human retinoblast 911 cells of coated complexes, based on PLL or RPC, targeted to cell surface receptors by covalent linkage of basic fibroblast growth factor. The levels of transgene expression observed for RPC-based targeted vectors indicated efficient intracellular activation, authenticating the concept that lateral stabilization introduced by surface coating with PHPMA can be reversed by intracellular reduction

DNA release dynamics from bioreducible layer-by-layer films

DNA release dynamics from layer-by-layer (LbL) films is an important aspect to consider with regards to localized gene delivery systems. The rate of DNA release and the condensation state of DNA during release are of particular interest in the field of gene delivery. A hyperbranched poly(amido amine) (RHB) containing bioreducible disulfide bonds is used to form interpolyelectrolyte complexes with DNA during LbL film assembly. During films disassembly, DNA is released in physiologic conditions due to the reducing nature of the RHB. Uncondensed DNA deposited on the surface was compared to DNA condensed by RHB in polyplex form by using two types of LbL films, RHB/DNA/RHB and polyplex terminated films, RHB/DNA/polyplex. LbL films with up to three layers are used in order to facilitate high-resolution AFM imaging. X-ray reflectivity, ellipsometry, and Fourier transform infrared spectroscopy are also used. The film disassembly, rearrangement and release of molecules from the surface due to thiol-disulfide exchange is conducted in reducing dithiothreitol (DTT) solutions. Salt is found to accelerate the overall rate of film disassembly. Additionally, it was found that the polyplex layer disassembles faster than the DNA layer. The predominant intermediate structure is the toroid structure for the polyplex layer and the fiber bundle structure for the DNA layer during film disassembly. This study offers a simple means to modulate DNA release from LbL films by utilizing both condensed and uncondensed DNA in different layers. The study highlights nanostructures, toroids and bundles, as dominant intermediate DNA structures during the DNA release from LbL films

Methodologies for monitoring nanoparticle formation by self-assembly of DNA with poly(l-lysine)

DNA self-assembly with polycations produces nanoparticles suitable for gene delivery, although there is no standard methodology to measure particle formation and stability. Here we have compared three commonly used assays, namely, light scattering, inhibition of ethidium bromide fluorescence, and modified electrophoretic mobility of DNA. Analysis by light scattering and loss of ethidium bromide fluorescence both showed poly(l-lysine) (pLL)/DNA nanoparticles form over the lysine/phosphate ratio range 0.6–1.0, although retardation of DNA electrophoretic mobility commenced at lower lysine/phosphate ratios. This probably indicates that the first two assays monitor DNA collapse into particles, while the electrophoresis assay measures neutralization of the charge on DNA. Gel analysis of the complexes showed disproportionation during nanoparticle formation, probably reflecting cooperative binding of the polycation. The assays were used to examine stability of complexes to dilution in water and physiological salts. Whereas all pLL/DNA nanoparticles were stable to dilution in water, the presence of physiological salts provoked selective disruption of complexes based on low-molecular-weight pLL. Polyelectrolyte complexes for targeted application in vivo should therefore be based on high-molecular-weight polycations, or should be stabilized to prevent their dissociation under physiological salt conditions

Methodologies for monitoring nanoparticle formation by self-assembly of DNA with poly(L-lysine)

DNA self-assembly with polycations produces nanoparticles suitable for gene delivery, although there is no standard methodology to measure particle formation and stability. Here we have compared three commonly used assays, namely, light scattering, inhibition of ethidium bromide fluorescence, and modified electrophoretic mobility of DNA. Analysis by light scattering and loss of ethidium bromide fluorescence both showed poly(l-lysine) (pLL)/DNA nanoparticles form over the lysine/phosphate ratio range 0.6–1.0, although retardation of DNA electrophoretic mobility commenced at lower lysine/phosphate ratios. This probably indicates that the first two assays monitor DNA collapse into particles, while the electrophoresis assay measures neutralization of the charge on DNA. Gel analysis of the complexes showed disproportionation during nanoparticle formation, probably reflecting cooperative binding of the polycation. The assays were used to examine stability of complexes to dilution in water and physiological salts. Whereas all pLL/DNA nanoparticles were stable to dilution in water, the presence of physiological salts provoked selective disruption of complexes based on low-molecular-weight pLL. Polyelectrolyte complexes for targeted application in vivo should therefore be based on high-molecular-weight polycations, or should be stabilized to prevent their dissociation under physiological salt conditions

Tuning the mechanical properties of bioreducible multilayer films for improved cell adhesion and transfection activity

A simple approach to the mechanical modulation of layer-by-layer (LbL) films is through manipulation of the film assembly. Here, we report results based on altering the salt concentration during film assembly and its effect on film rigidity. Based on changes in film rigidity, cell adhesion characteristics and transfection activity were investigated in vitro. LbL films consisting of reducible hyperbranched poly(amide amine) (RHB) have been implemented along with DNA for investigating fibroblast adhesion on [RHB/DNA](n/2) films with varying rigidities. The rigidity was varied by changing the ionic concentration of the deposition solution between 0.01 M NaCl and 1.0 M NaCl. Molecular force probe (MFP) measurements were performed to measure the apparent Young's modulus, E-APP; of the films in situ. Cell adhesion and stress-fiber characteristics were investigated using total internal reflectance microscopy (TIRF-M). The average cell peripheral area, fiber density and average fiber length during 5 days of cell growth on films with either low (below 2.0 MPa) or high (above 2.0 MPa) film elastic modulus were investigated. Transfection studies were performed using gfpDNA and SEAP-DNA to investigate if changes in cell adhesion affect transfection activity. Furthermore, cell proliferation and cytotoxicity studies were used to investigate cellular viability over a week. The results have shown that surface modification of bio-reducible LbL films of controlled thickness and roughness promotes cellular adhesion, stress-fiber growth and increased transfection activity without the need for an additional adhesive protein pre-coating of the surface or chemical cross-linking of the film