Small nanosized poly(vinyl benzyl trimethylammonium chloride) based polyplexes for siRNA delivery.

The success of siRNA gene therapy requires the availability of safe and efficient delivery systems. In the present study, we investigated poly(vinyl benzyl trimethylammonium chloride) (PVTC) and its block copolymer with poly(oligo(ethyleneglycol) methacrylate) (POEGMA) as delivery vector for siRNA. Small polyplexes ranging from 8 to 25nm in diameter were formed in aqueous solution by spontaneous self-assembly of both the homopolymer and block copolymer with siRNA and the formed particles were stable at physiological ionic strength. It was shown that when human ovarian adenocarcinoma cells were transfected, siRNA polyplexes based on PVTC (40kDa) and PVTC-POEGMA-4 (PP4, 34kDa) efficiently induced luciferase gene silencing to the same extent as the formulation based on a commercial lipid (Lipofectamine®) (∼80%), and showed higher gene silencing than the linear polyethylenimine formulation linear polyethylenimine (∼35%). Importantly, the POEGMA block polymers displayed a significantly lower cytotoxicity as compared to L-pEI. siRNA polyplexes based on the block polymers displayed high cellular uptake resulting in ∼50% silencing of luciferase expression also in the presence of serum. These results demonstrate that PVTC-based polymers are promising siRNA delivery vectors.

Poly(vinyl benzyl trimethylammonium chloride) Homo and Block Copolymers Complexation with DNA.

In this work we focus on the use of novel homo and block copolymers based on poly(vinyl benzyl trimethylammonium chloride) as gene delivery vectors. The homopolymers and block copolymers were synthesized by RAFT polymerization schemes and molecularly characterized. DNA/polymer complexes (polyplexes) in a wide range of N/P (amino-to-phosphate groups) ratios were prepared. The ability of the novel polymers to form complexes with linear DNA was investigated by light scattering, zeta potential, and ethidium bromide fluorescence quenching measurements. The resulting polyplexes were in the size range of 80-300 nm and their surface potential changed from negative to positive depending on the N/P ratio. The stability of polyplexes was monitored by changes in their hydrodynamic parameters in the presence of salt. The novel vector systems were visualized by transmission electron microscopy. The influence of factors such as molar mass, content, and chemical structure of the polycationic moieties as well as presence of a hydrophilic poly[oligo(ethylene glycol) methacrylate] block on the structure and stability of the polyplexes, kinetics of their formation, and effectiveness of the (co)polymers to shrink and pack DNA was discussed.

Quaternized poly[3,5-bis(dimethylaminomethylene)hydroxystyrene]/DNA complexes: structure formation as a function of solution ionic strength.

The formation of complexes between the cationic polyelectrolyte poly[3,5-bis(dimethylaminomethylene)hydroxystyrene] (Q-N-PHOS), bearing two cationic sites per repeating unit, and DNA molecules in aqueous solutions is investigated at pH 7 and at various salt (NaCl) concentrations and DNA/polymer ratios. The structural characteristics of the polyplexes at different DNA/polymer ratios were characterized in terms of mass, size, and charge using static, dynamic, and electrophoretic light scattering and fluorescence spectroscopy. The results indicate that the complexes have a loose spherical morphology with size in the range of 45-100 nm depending on polymer to DNA ratio and ionic strength of the solution. Most interestingly, it is found that the polyplexes' response to changes in the ionic strength of the surrounding solution after complexation depends strongly on the initial solution ionic strength during complex formation. This also points to differences in the nanostructures of polyplexes formed at different ionic strengths.

Polyelectrolyte-surfactant complexes of poly[3,5-bis(dimethylaminomethyl)-4-hydroxystyrene]-block-poly(ethylene oxide) and sodium dodecyl sulfate: anomalous self-assembly behavior.

Polyelectrolyte-surfactant complexes (PE-S) formed by double hydrophilic cationic polyelectrolyte poly[3,5-bis(dimethylaminomethyl)-4-hydroxystyrene]-block-poly(ethylene oxide) (NPHOS-PEO) and anionic surfactant sodium dodecyl sulfate (SDS) in acidic aqueous solutions were studied by light scattering, SAXS, and scanning transmission electron microcopy in the environmental mode (wet-STEM) for various stoichiometric ratios between the numbers of SDS anions and dimethylaminomethyl groups of NPHOS in the complex. The obtained results show that the NPHOS-PEO/SDS system behaves differently from other systems of double hydrophilic block polyelectrolyte and oppositely charged ionic surfactant because it forms water-insoluble PE-S for compositions close to the zero net charge of the complex. This phase separation occurs, instead of the PE-S rearrangement to core-shell particles, which is hindered due to conformational rigidity of the NPHOS blocks. For the surfactant amounts below and above the precipitation region, large spherical aggregates and their clusters are present in the solution. SAXS measurements indicate that although the NPHOS-PEO/SDS system does not form the core-shell particles with the NPHOS/SDS core and the PEO shell as other PE-S of double hydrophilic polyelectrolytes, the aggregates contain domains of closely packed surfactant micelles which bind to both NPHOS polyelectrolyte blocks and PEO blocks.

Polyplexes based on cationic polymers with strong nucleic acid binding properties.

Cationic polymers have been studied for nucleic acid delivery both in vitro and in vivo. However, many polymer-based formulations suffer from lack of stability in biologic fluids due to interactions with anionic biomacromolecules such as proteins and polysaccharides. Likely, the stronger the electrostatic interactions between a cationic polymer and nucleic acids, the higher the stability of the polyplexes in biologic fluids will be. To get evidence for this hypothesis, quaternized poly[3,5-bis(dimethylaminomethylene)-p-hydroxyl styrene] (QNPHOS) with two permanently charged cationic sites per monomer unit as well as its block copolymer with PEG were synthesized and compared with the standard transfectant pDMAEMA, in terms of nucleic acid binding strength, gene silencing and transfection activities of the complexes which these polymers form with siRNA and plasmid DNA, respectively. It was shown that siRNA complexes based on QNPHOS and QNPHOS-PEG dissociate in the presence of a fourfold higher heparin concentration than necessary to destabilize pDMAEMA complexes. Under the same conditions, complexes of DNA and QNPHOS or QNPHOS-PEG did not show any dissociation, in contrast to pDMAEMA polyplexes. The DNA polyplexes based on QNPHOS or QNPHOS-PEG did not show transfection activity, which might be ascribed to their high physicochemical stability. On the other hand, siRNA complexes based on QNPHOS and QNPHOS-PEG showed a low cytotoxicity and an improved siRNA delivery and high gene silencing activity, even higher than those based on pDMAEMA. This might be due to the excellent binding characteristics of QNPHOS and QNPHOS-PEG to siRNA which in turn is ascribed to the presence of two permanently charged cationic groups per monomer unit. Based on the results of this study, it is concluded that formation of strong siRNA complexes with polymers containing double charges per monomer is advantageous.

Association of poly(4-hydroxystyrene)-block-poly(ethylene oxide) in aqueous solutions: block copolymer nanoparticles with intermixed blocks.

Association behavior of diblock copolymer poly(4-hydroxystyrene)-block-poly(ethylene oxide) (PHOS-PEO) in aqueous solutions and solutions in water/tetrahydrofuran mixtures was studied by static, dynamic, and electrophoretic light scattering, (1)H NMR spectroscopy, transmission electron microscopy, and cryogenic field-emission scanning electron microscopy. It was found that, in alkaline aqueous solutions, PHOS-PEO can form compact spherical nanoparticles whose size depends on the preparation protocol. Instead of a core/shell structure with segregated blocks, the PHOS-PEO nanoparticles have intermixed PHOS and PEO blocks due to hydrogen bond interaction between -OH groups of PHOS and oxygen atoms of PEO and are stabilized electrostatically by a fraction of ionized PHOS units on the surface.

Polyelectrolyte-surfactant complexes formed by poly[3,5-bis(trimethylammoniummethyl)4-hydroxystyrene iodide]-block-poly(ethylene oxide) and sodium dodecyl sulfate in aqueous solutions.

Formation of polyelectrolyte-surfactant (PE-S) complexes of poly[3,5-bis(trimethylammoniummethyl)-4-hydroxystyrene iodide]-block-poly(ethylene oxide) (QNPHOS-PEO) and sodium dodecyl sulfate (SDS) in aqueous solution was studied by dynamic and electrophoretic light scattering, small-angle X-ray scattering (SAXS), atomic force microscopy, and fluorometry, using pyrene as a fluorescent probe. SAXS data from the QNPHOS-PEO/SDS solutions were fitted assuming contributions from free copolymer, PE-S aggregates described by a mass fractal model, and densely packed surfactant micelles inside the aggregates. It was found that, unlike other systems of a double hydrophilic block polyelectrolyte and an oppositely charged surfactant, PE-S aggregates of the QNPHOS-PEO/SDS system do not form core-shell particles and the PE-S complex precipitates before reaching the charge equivalence between dodecyl sulfate anions and QNPHOS polycationic blocks, most likely because of conformational rigidity of the QNPHOS blocks, which prevents the system from the corresponding rearrangement.

Solution behavior of poly(sodium(sulfamate-carboxylate)isoprene), a pH sensitive and intrinsically hydrophobic polyelectrolyte.

The solution properties of a novel pH sensitive and intrinsically hydrophobic polyelectrolyte poly(sodium(sulfamate-carboxylate)isoprene) (SCPI) were investigated by means of dynamic, static, and electrophoretic light-scattering and fluorescence spectroscopy techniques. Because of the pH dependent charge density of the polyelectrolyte chain, the dynamics and structure of the solution were studied at both pH 7 (high charge density) and pH 3 (low charge density) and at low ionic strength conditions for two samples of different molecular weights. In all cases, a fast and a slow diffusive mode were observed. The fast mode is attributed to the diffusion of isolated polyelectrolyte chains, while the slow mode denotes the presence of multichain domains in solution, formed by electrostatic and/or hydrophobic interactions. The size and density of the multichain domains were reduced with decreasing charge density and molecular weight. Effective charge of the particles does not depend appreciably on solution pH, while fluorescence spectroscopy revealed the presence of hydrophobic domains in the studied systems. It was found that increasing temperature resulted in the compaction of both isolated chains and multichain domains due to the hydrophobic effect. Furthermore the increase of the ionic strength of the solution led to a partial dissociation of the multichain domains at short times and at pH 7, while it led to increased aggregation and precipitation at pH 3 and long times. The performed experiments allowed for a separation of the electrostatic and hydrophobic contributions to the self-assembly of the particular polyelectrolyte systems.

Self-assembled nanoparticles from a block polyelectrolyte in aqueous media: structural characterization by SANS.

We present a small angle neutron scattering (SANS) study of polystyrene-b-sodium (sulfamate/carboxylate) isoprene (PS-PSCI) nanoparticles in aqueous media. The SANS experiments are complemented by static and dynamic light scattering measurements. A detailed analysis of the scattering form factor obtained by SANS for the self-assembled block polyelectrolyte spherical nanoparticles implies a two-region power-law model for the radial volume fraction profiles. The theoretically predicted scaling of the osmotic brush regime phi(r) approximately r(-2) for the inner region and the osmotic annealing brush regime phi(r) approximately r(-8/3) for the outer region are in agreement with our experimental findings. A concentrated shell of PSCI polyelectrolyte chains collapsed on the polystyrene core is needed in the form factor analysis so that the aggregation number of the nanoparticles is self-consistent. The self-assembled nanoparticles are found to be kinetically frozen i.e. their aggregation number is not sensitive to the solution conditions and is defined by the preparation protocol. The size of the spherical nanoparticles tends to decrease upon the addition of salt and the drop of pH.

Polymer covalent functionalization of carbon nanohorns using bulk free radical polymerization.

Herein, we report on a facile approach for the covalent functionalization of carbon nanohorns, using in situ bulk free radical polymerization of methacrylic acid. The obtained material is soluble in aqueous media, facilitating its processability and has been fully characterized by means of complementary spectroscopic techniques, electron microscopy, thermogravimetric analysis, and light scattering. Simultaneously, the material has been used as a template for the synthesis of gold nanoparticles on the surface of the polymer-decorated carbon nanohorns.

Functionalization of carbon nanohorns with polyethylene oxide: synthesis and incorporation in a polymer matrix.

In the present work we describe, for the first time, the chemical functionalization of oxidized CNHs with a polymer, namely polyethylene oxide, in order to make them soluble in a variety of solvents. The success of the covalent functionalization of CNHs has been confirmed by a variety of complementary techniques, including thermogravimetric analysis, infrared and UV-vis spectroscopy. The solution behavior of the polymer functionalized CNHs has been studied in aqueous solutions at different temperatures by means of light scattering. Finally, the incorporation of the functionalized carbon nanostructure in poly(hydroxyl styrene) (PHOS) matrix is discussed.

pH-dependent self-assembly of polystyrene-block-poly((sulfamate-carboxylate)isoprene) copolymer in aqueous media.

The amphiphilic polystyrene- block-poly((sulfamate-carboxylate)isoprene) (PS-PISC) diblock copolymer was synthesized from the precursor diblock copolymer polystyrene- block-isoprene by reaction with chlorosulfonyl isocyanate. The structure and behavior of self-assembled PS-PISC nanoparticles was studied in alkaline and acidic aqueous solutions by a combination of static and dynamic light scattering, analytical ultracentrifugation, atomic force and cryogenic transmission electron microscopies, NMR spectroscopy, potentiometric titration, and fluorometry using pyrene as a polarity-sensitive fluorescent probe. It was found that PS-PISC exists in aqueous solutions in the form of micellar aggregates. The aggregation tendency increases with decreasing effective charge density in the shell, that is, with decreasing pH of the solution, and aggregates found in alkaline aqueous media have much smaller molar masses than those formed in acidic media. The latter are dense, collapsed structures with immobile PISC domains in which most of the COOH and NH 2 (+)SO 3 (-) groups are buried inside of the nanoparticles. The swelling of PISC domains and disentanglement of PISC chains after addition of a base are slow processes occurring on the time scale of days.

Grafting living polymers onto carbon nanohorns.

In the present paper we describe a new way for the covalent functionalization of carbon nanohorns by means of anionic polymerization with the "grafting to" approach. Polyisoprene homopolymers, as well as polyisoprene-b-polystyrene block copolymers have been attached on the surface of carbon nanohorns. The functionalized carbon nanostructures have been fully characterized by means of complementary spectroscopic techniques, electron microscopy, thermogravimetric analysis, and light scattering.

Synthesis and solution behavior of carbon nanotubes decorated with amphiphilic block polyelectrolytes.

The aqueous solubilization of carbon nanotubes (CNTs) with the aid of a block copolymer possessing one polyelectrolyte block (namely polystyrene-b-sodium (sulfamate/carboxylate polyisoprene)) is reported. The solubilization protocol, based on the co-dissolution of the copolymer and the CNTs, leads to the formation of supramolecular assemblies on the side walls of the tubes. Electron microscopy as well as infrared spectroscopy and thermogravimetric analysis were employed as meaningful probes to identify the adsorption of the polymer onto the surface of CNTs and the composition of the final hybrid material. Viscosity measurements on solutions of the copolymer decorated CNTs indicate that the polyelectrolyte effect, which is observed in the case of net polymers, is preserved in a lesser extent in the case of the copolymer/CNTs dispersions.