A thermo-sensitive NIPA-based co-polymer and monosize polycationic nanoparticle for non-viral gene transfer to smooth muscle cells

Primary smooth muscle cells (SMC) isolated from the aorta of fetal calf were transfected with a green fluorescent protein (GFP)-encoding plasmid DNA, which was carried by a water-soluble and temperature-sensitive N-isopropylacrylamide-based (NIPAAm-based)-co-polymer, either poly(N-isopropylacrylamide-co-2-methacryloamidohistidine) (poly(NIPAAm-co-MAH)) or monosized PEGylated nanoparticle poly(styrene/poly(ethylene glycol) ethyl ether methacrylate/N-(3-(dimethylamino)propyl) methacrylamide) (poly(St/PEG-EEM/DMAPM)). Poly(NIPAAm-co-MAH) co-polymer was synthesized by solution polymerization of n-isopropylacrylamide (NIPAAm) and 2-methacrylamidohistidine (MAH). Monosized cationic nanoparticles were produced by emulsifier-free emulsion polymerization of styrene, PEG ethyl ether methacrylate and N-[3-(dimethyl-amino) propyl] methacrylamide, in the presence of a cationic initiator, 2,2-azobis (2-methylpropionamidine) dihydrochloride. The structure of poly(St/PEG-EEM/DMAPM) and poly(NIPAAm-co-MAH) was confirmed by(1) H-NMR and FT-IR spectroscopy. Particle size/size distribution and surface charges of both carriers were measured by Zeta Sizer. The LCST behavior of poly(NIPAAm-co-MAH) co-polymer was followed spectrophotometrically. Poly(St/PEG-EEM/DMAPM) nanoparticles, with an average size of 78 nm and zeta potential of 54.4 mV, and an average size of 200 nm with a zeta potential of 54.2 mV, and poly(NIPAAm-co-MAH) were used in the transfection studies. The cytotoxicity of the vectors was tested using the MTT method. According to conditions for the transfection study (polymer/cell ratio and polymer-cell incubation period), cell loss was only 4 and 15% with poly(St/PEG-EEM/DMAPM) sized 78 and 200 nm, respectively. Poly(NIPAAm-co-MAH) cytotoxicity was insignificant. Poly(NIPAAm-co-MAH) uptake efficiency in SMCs was around 85%, but gene expression efficiency were low compared to poly(St/PEG-EEM/DMAPM)/pEGFP-N2 conjugates because of the low zeta potential of the co-polymer. Polymer uptake efficiencies of the nanoparticles were 90-95%. GFP expression efficiency was 68 and 64% after transfection with pEGFP-N2 conjugate with 78 and 200 nm sized poly(St/PEG-EEM/DMAPM) nanoparticles.     

Effect of ionic strength on the loading efficiency of model polypeptide/protein drugs in pH-/temperature-sensitive polymers

In this report, the effect of ionic strength on the loading efficiency of three model polypeptide/protein drugs, namely angiotensin II, insulin, and cytochrome c, in pH- and temperature-sensitive terpolymers of poly(NIPAAm-co-butylmethacrylate-co-acrylic acid) (poly(NIPAAm-co-BMA-co-AA)) has been investigated. Loading efficiency of polypeptides in pH-/temperature-sensitive beads composed of poly(NIPAAm-co-BMA-co-AA) terpolymer is predominantly governed by hydrophobic interactions, both nonspecific surface interactions and/or specific interactions (binding pockets) between the protein and the polymer molecules. Thus, loading efficiency increases with ionic strength. However, as ionic strength increases further, polymer deswelling (collapse), which is also controlled by hydrophobic forces, becomes more pronounced, and consequently, a higher fraction of water is squeezed out during bead formation and the loading efficiency starts to decrease.     

Synthesis,Characterization and Properties of a Physically and Chemically Gelling Polymer System Using Poly(NIPAAm-co-HEMA-acrylate) and Poly(NIPAAm-co-cysteamine)

The aim of this work was to develop a simultaneous physically and chemically gelling system using NIPAAm co-polymers. The in situ polymer gel was obtained by synthesizing poly(NIPAAm-co-HEMAacrylate) and poly(NIPAAm-co-cysteamine) through free radical polymerization and further nucleophilic substitution. The purpose of the dual gelation is that physical gelation would take place at higher temperatures as the NIPAAm chains associate, while chemical gelation would occur through a Michael-type addition reaction, resulting in a cross-link forming through a nucleophilic attack of the thiolate on the acrylate. The structure of each co-polymer was then verified using ¹H-NMR and FT-IR spectroscopy. The corresponding lower critical solution temperature and phase transition behavior of each co-polymer was analyzed through cloud point and DSC, while mechanical properties were investigated through rheology. Swelling behavior was also monitored at different temperatures. The resulting polymer system demonstrated properties compatible with physiological conditions, forming a gel at pH 7.4 and at temperatures near body temperature. The hydrogel also showed reduced viscoelastic flow at low frequency stress, and increased strength than purely physical or chemical gels. Swelling behavior was determined to be temperature-dependent; however, no difference was observed in swelling percent beyond 48 h. Having the ability to alter these co-polymers through various synthesis parameters and techniques, this hydrogel can potentially be used as an injectable, waterborne gelling material for biomedical applications such as endovascular embolization.     

Furan-functionalized co-polymers for targeted drug delivery: caracterization,self-assembly and drug encapsulation

We have previously reported furan-maleimide Diels–Alder chemistry as a new methodology to couple maleimide-modified antibodies on furan-functionalized polymeric carriers in the preparation of immuno-nanoparticles for targeted drug delivery. In this report, we focus on the characterization, self-assembly behavior and drug encapsulation of two types of furan-functionalized co-polymers: poly(2-methyl, 2-carboxytrimethylene carbonate-co-D,L-lactide)-furan (poly(TMCC-co-LA)-furan) and poly(2-methyl, 2-carboxytrimethylene carbonate-co-D,L-lactide)-graft-poly(ethylene glycol)-furan (poly(TMCC-co-LA)-g-PEG-furan). The co-polymers were synthesized by modifying the carboxylic acid groups on the poly(TMCC-co-LA) backbone by either furfurylamine or PEG-furan to generate either linear co-polymers of poly(TMCC-co-LA)-furan with furan pendant groups or graft co-polymers of poly(TMCC-co-LA)-g-PEG-furan with furan-terminated PEG grafts, respectively. Using a membrane dialysis method, both of the co-polymers were self-assembled into nanoparticles in aqueous environments driven by the hydrophobic association among polymer chains. The hydrophobic domains in the nanoparticles were confirmed by the incorporation of pyrene molecules and the critical aggregation concentrations were determined to be approximately 5 × 10^?5 mM for poly(TMCC-co-LA)-furan and 2 × 10^?4 mM for poly(TMCC-co-LA)-g-PEG-furan. By the addition of borate buffer in the organic solvent used to dissolve the co-polymers in the dialysis procedure, we were able to control the size of the nanoparticles: 54–169 nm for poly(TMCC-co-LA)-furan and 28–283 nm for poly(TMCC-co-LA)-g-PEG-furan. This unique feature can be explained by the ionization of carboxylic acid groups along the co-polymer backbone. A hydrophobic anticancer drug, doxorubicin (DOX), was encapsulated within the nanoparticles, with the larger size nanoparticles incorporating greater amounts of DOX. Combining the strategy of antibody-mediated targeting, these self-assembled nanoparticles have potential as efficient anti-cancer drug carriers.     

Preparation of poly(methyl methacrylate) microspheres modified with amino acid moieties

Poly(methyl methacrylate) microspheres [poly(SerMA-co-MMA), poly(AlaMAm-co-MMA), poly(AlaEMA-co-MMA), and poly(AlaMAm/AlaEMA-co-MMA)] modified with O-methacryloyl-L -serine (SerMA), N-methacryloyl-L -alanine (AlaMAm) and L -alanine 2-methacryloyloxyethyl ester (AlaEMA) were prepared by the emulsifier-free emulsion copolymerization of methyl methacrylate (MMA) with SerMA, AlaMAm and AlaEMA, respectively, initiated with 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (ABIP). The series of polymer microspheres showed unimodal distribution of the particle size (290–800 nm) in water. From X-ray photoelectron spectroscopy it was concluded that the amino acid moieties are located on the surface of the particles in all cases. Poly(SerMA-co-MMA) showed the most effective suppression of adsorption of proteins such as albumin (Alb), γ-globulin (Glo) and fibrinogen (Fib) among the examined poly(methylmethacrylate) microspheres.     

Synthesis of Poly(ethylene glycol)-g-Chitosan-g-Poly(ethylene imine) Co-polymer and In Vitro Study of Its Suitability as a Gene-Delivery Vector

There are two main hindrances for the application of chitosan (CS) as a gene-delivery vector: poor water solubility and low transfection efficiency. To address these problems, we modified chitosan with poly(ethylene glycol) (PEG) and poly(ethylene imine) (PEI). As previously described, PEG was grafted onto CS by a reaction between the activated PEG and CS amine. This increased the solubility of CS in neutral or basic solution. Then, monomers of PEI (i.e., aziridine) were polymerized on the CS chain of the PEG(40k)-CS(50k) co-polymer obtained in the previous step. The resulting PEG-CS-PEI (PCP) co-polymer was characterized by ¹H-NMR, ¹³C-NMR and gel-permeation chromatography (GPC). It was found in the preliminary experiments that, amongst the series of PEG-CS-PEI co-polymers with various PEI molecular weights, PEG(40k)-CS(50k)-PEI(20k) was the most efficient one; therefore, it was chosen for the study. The PCP co-polymer showed lower cytotoxicity compared to PEI (25k) by MTT assay. Particle size and zeta potential of PCP/DNA complexes were measured by dynamic light scattering (DLS) and were shown to be predominantly affected by N/P ratios. PCP/DNA complexes at N/P ratio 20 were observed under a transmission electron microscope (TEM) as spherical particles with a mean diameter of about 50 nm. Plasmid DNA could be efficiently protected by PCP co-polymer from DNase I. The in vitro gene-transfection efficiency of PCP/pEGFP was higher than that of PEI(25k)/pEGFP and was markedly facilitated by serum.     

Preparation of poly(D,L-lactide) nanoparticles assisted by amphiphilic poly(methyl methacrylate-co-methacrylic acid) copolymers

When co-precipitated with amphiphilic copolymers from DMSO, poly(D,L-lactide)(PLA) can be readily converted into stable sub-200 nm nanoparticles by addition of an aqueous phase, free of any polymeric stabilizers such as poly(vinyl alcohol) or Poloxamer. In this work, the ability of random poly(methyl methacrylate-co-methacrylic acid) copolymers (PMMA-co-MA) to stabilize PLA nanoparticles was demonstrated, and the properties of PLA/PMMA-co-MA nanoparticles were investigated. When co-precipitated with PMMA-co-MA, PLA was totally converted into nanoparticles using a polymer concentration in DMSO (C_P) below 17.6 mg ml^-1, and a PMMA-co-MA proportion above a critical value depending on the content of MA repeating units (X). For instance, the lowest PMMA-co-MA proportion required was 0.9 mg mg^-1 PLA for X = 12%, and 0.5 mg mg^-1 PLA for X = 25% (for C_PLA = 16 mg ml^-1 DMSO). The nanoparticle diameter was essentially independent of X, the proportion of PMMA-co-MA, and the PLA molecular weight, except for oligomers for which the nanoparticle diameter was smaller. It decreased when the organic phase was diluted (126 ± 13 nm for C_P = 17.6 mg ml^-1, and 81 ± 5 nm for C_P = 5.6 mg ml^-1). The timedependence of the stability and the degradation of PLA/PMMA-co-MA nanoparticles was discussed. One of the main advantages of this technique is the ability to control surface properties and to bring functional groups to otherwise non-functionalized PLA nanoparticles. To illustrate this, a conjugate of PMMA-co-MA25 and biotin was synthesized, and used to prepare biotinylated nanoparticles that could be detected by fluorescence and transmission electron microscopy after infiltration into ligatured rat small intestine.     

Hydrogel Nanocomposites: A Potential UV/Blue Light Filtering Material for Ophthalmic Lenses

Poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) (poly(HEMA-co-MMA)) and ZnS hydrogel nanocomposites were prepared and characterized. The chemical composition of the inorganic nanoparticles was confirmed by X-ray diffraction, and the homogeneity of their distribution within the hydrogel was assessed by transmission electron microscopy. The influence of the content of ZnS nanoparticles on the optical performances of the nanocomposites was investigated by UV-Vis spectroscopy. The ability of the hydrogel nanocomposites to filter the hazardous UV light and part of the blue light was reported, which makes them valuable candidates for ophthalmic lens application. In contrast to the optical properties, the thermo-mechanical properties of neat poly(HEMA-co-MMA) hydrogels were found to be largely independent of filling by ZnS nanoparticles (≤2 mg/ml co-monomer mixture). Finally, in vitro cell adhesion test with lens epithelial cells (LECs), extracted from porcine lens crystalline capsule, showed that ZnS had no deleterious effect on the biocompatibility of neat hydrogels, at least at low content.     

Magnetic Poly(PEGMA–MAA) Nanoparticles: Photochemical Preparation and Potential Application in Drug Delivery

Poly(PEGMA–MAA)-coated superparamagnetic nanoparticles were synthesized by in situ photochemical polymerization in magnetite aqueous suspension under UV irradiation. The magnetic poly(PEGMA–MAA) nanoparticles were characterized by Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), photo correlation spectroscopy (PCS) and vibration sample magnetometry (VSM), respectively. The results indicated that the magnetic poly(PEGMA–MAA) nanoparticles were of regularly spherical shape and remained monodisperse. The average size measured in aqueous media was 96.4 nm, which was much bigger than that in dry state, the nanoparticles behaved superparamagnetic with saturated magnetization of 64.8 emu/g, the zeta potential was ?18.3 mV at physiological pH 7.2, and the magnetic poly(PEGMA–MAA) nanoparticles had a high stability in vitro. A typical anti-inflammatory drug, ibuprofen, was used for drug loading, and the release behavior of ibuprofen in a simulated body fluid (SBF, pH 7.4) was studied. The results indicated that these novel magnetic nanoparticles had a high drug-loading capacity and favorable release properties for ibuprofen. The magnetic poly(PEGMA–MAA) nanoparticles are very promising for application in drug delivery.     

Thermo-Responsive Mn–Zn Ferrite/Poly(N,N′-Isopropyl Acrylamide-co-N-Hydroxymethylacrylamide) Core/Shell Nanocomposites for Drug-Delivery Systems

A kind of thermo-sensitive core/shell nanoparticles (Mn–Zn ferrite/poly(N,N′-isopropylacrylamide-co-N-hydroxymethylacrylamide)) has been designed in this work. By using the surface initiated method, a thermoresponsive co-polymer composed of N,N′-isopropylacrylamide (NIPAAm) and N-hydroxymethylacrylamide (HMAAm) was grafted from ferrite core. The nanoparticles obtained were characterized by FT-IR, XRD, TGA, HRTEM and DLS. The HRTEM observation showed a clear contrast between an ordered crystalline core and a light amorphous polymeric coating. DLS results confirmed the temperature sensitivity of the nanoparticles. In vitro hyperthermia showed well-controlled self-heating properties, indicating that the nanoparticles could potentially be used as an anticancer drug carrier in the biomedical field.     

Gene delivery of PEI incorporating with functional block copolymer via non-covalent assembly strategy

A novel functional diblock polymer P(PEGMA-b-MAH) is prepared and incorporated to improve the gene delivery efficiency of poly(ethyleneimine) PEI via non-covalent assembly strategy. First, P(PEGMA-b-MAH) is prepared from l-methacrylamidohistidine methyl ester (MAH) by reversible addition fragmentation chain transfer polymerization, with poly[poly(ethylene glycol) methyl ether methacrylate] (P(PEGMA)) as the macroinitiator. Then P(PEGMA-b-MAH) is assembled with plasmid DNA (pDNA) and PEI (M_w = 10 kDa) to form PEI/P(PEGMA-b-MAH)/pDNA ternary complexes. The agarose gel retardation assay shows that the presence of P(PEGMA-b-MAH) does not interfere with DNA condensation by the PEI. Dynamic light scattering tests show that PEI/P(PEGMA-b-MAH)/pDNA ternary complexes have excellent serum stability. In vitro transfection indicates that, compared to the P(PEGMA-b-MAH) free PEI-25k/pDNA binary complexes, PEI-10k/P(PEGMA-b-MAH)/pDNA ternary complexes have lower cytotoxicity and higher gene transfection efficiency, especially under serum conditions. The ternary complexes proposed here can inspire a new strategy for the development of gene and drug delivery vectors.     

Poly(ethylenimine)-grafted-poly[(aspartic acid)-co-lysine], a potential non-viral vector for DNA delivery

A potential non-viral gene-transfer vector, poly(ethylenimine)-grafted-poly[(aspartic acid)-co-lysine] (PSL), has been developed by thermal polycondensation of aspartic acid and lysine under reduced pressure. Low-molecular-mass branch poly(ethylenimine) (PEI600) was conjugated to the backbone. The chemical structure of the resulting co-polymer was identified by H-NMR, FT-IR, TGA and X-ray diffraction. The results of the MTT assay showed that at concentration up to 4000 nmol/l of the vector cell viability was over 80% and showed low toxicity. Electrophoretic retardation and ethidum bromide assay showed that at N/P ratios 12–15 (w/w) the DNA could be condensed and neutralized. Using the zeta potential assay we discovered that it had a high positive charge on its surface of the particle (over 30 mV). The particle sizes of the co-polymer/DNA complexes were 150–170 nm, as measured by DLS and AFM. Compared with PEI600, co-polymer/DNA complexes showed a significant enhancement of transfection activity in the absence and presence of serum in NT2 and COS7 cell lines. This means that the PEI600-PSL co-polymer is a promising candidate for gene delivery.     

Poly (ethylenimine)-grafted-poly [(aspartic acid)-co-lysine], a potential non-viral vector for DNA delivery

A potential non-viral gene-transfer vector, poly(ethylenimine)-grafted-poly[(aspartic acid)-co-lysine] (PSL), has been developed by thermal polycondensation of aspartic acid and lysine under reduced pressure. Low-molecular-mass branch poly(ethylenimine) (PEI600) was conjugated to the backbone. The chemical structure of the resulting co-polymer was identified by 1H-NMR, FT-IR, TGA and X-ray diffraction. The results of the MTT assay showed that at concentration up to 4000 nmol/l of the vector cell viability was over 80% and showed low toxicity. Electrophoretic retardation and ethidum bromide assay showed that at N/P ratios 12-15 (w/w) the DNA could be condensed and neutralized. Using the zeta potential assay we discovered that it had a high positive charge on its surface of the particle (over 30 mV). The particle sizes of the co-polymer/DNA complexes were 150-170 nm, as measured by DLS and AFM. Compared with PEI600, co-polymer/DNA complexes showed a significant enhancement of transfection activity in the absence and presence of serum in NT2 and COS7 cell lines. This means that the PEI600-PSL co-polymer is a promising candidate for gene delivery.     

A nanosized,thermo-sensitive drug carrier: self-assembled Fe3O4-OA-g-P(OA-co-NIPAAm) magnetomicelles

Novel thermo-sensitive magnetomicelles that consist of a magnetic core, Fe₃O₄-oleic acid (Fe₃O₄-OA), and an amphiphilic surface layer of thermo-sensitive poly(oleic acid-co-N-isopropylacrylamide) P(OA-co-NIPAAm) co-polymer were prepared. Fe₃O₄ magnetic cores functionalized with double bonds of oleic acid were prepared by suspension–oxidation reaction. Subsequently, by the co-polymerization of hydrophilic NIPAAm, hydrophobic OA and the Fe₃O₄-OA magnetic core, the thermo-sensitive amphiphilic polymers (P(OA-co-NIPAAm)) were grafted to obtain Fe₃O₄-OA-g-P(OA-co-NIPAAm) nanoparticles. The surface polymeric layer of Fe₃O₄-OA-g-P(OA-co-NIPAAm) nanoparticles would self-assemble in aqueous media to form a micellar structure attributed to the amphiphilic P(OA-co-NIPAAm) polymers. The model drug, prednisone acetate, was loaded in the magnetomicelles and in vitro release behavior of loaded drug in magnetomicelles was further investigated, which showed a thermo-sensitive release behavior due to the thermo-sensitive structural changes of the micellar surface layer.     

Functional Poly(Dimethyl Aminoethyl Methacrylate) by Combination of Radical Ring‐Opening Polymerization and Click Chemistry for Biomedical Applications

In this work, the macromolecular design and modular synthesis of degradable and biocompatible copolymers via radical polymerization and click chemistry is highlighted and the resulting systems are evaluated as gene delivery carriers. Poly(ethylene glycol) (PEG) grafted poly[2‐methylene‐1,3‐dioxepane (MDO)‐co‐propargyl acrylate (PA)‐co‐2‐(dimethyl aminoethyl methacrylate (DMAEMA)] (MPD) is synthesized using radical polymerization and azide‐alkyne click chemistry. The polymers are less cytotoxic and are able to condense plasmid DNA into nanosized particles. The low transfection efficiency of polyplexes in HepG2 cells is significantly improved by mixing Tat peptide with polyplexes.     

Hydrolyzed Poly(2‐plenyl‐2‐oxazoline)s in Aqueous Media and Biological Fluids

Hydrolyzed poly(2‐phenyl‐2‐oxazoline)s (PPhOx) are synthesized by partial hydrolysis of PPhOx in order to produce self‐assembling copolymers with chargeable and hydrophobic units. The resulting poly(ethylene imine‐co‐2‐phenyl‐2‐oxazoline) [P(EI‐co‐PhOx)] amphiphilic copolymers contain phenyl‐oxazoline and ethylene imine segments in a random sequence and their chemical structure is confirmed by ¹H NMR and attenuated total reflection‐Fourier transform infrared spectroscopy. Static and dynamic light scattering experiments show that in aqueous solutions the random copolymers associate into aggregates of sizes in the range between 50 and 200 nm depending on the solution conditions and hydrophobic content. The positive charge of the nanoaggregates that is caused by protonation of the amine nitrogen is confirmed by zeta potential measurements. Self‐assembly in phosphate buffered saline results in large aggregates. The aggregates are proved to interact with fetal bovine serum proteins. This investigation shows that hydrolyzed phenyl oxazoline‐based copolymers provide stable amphiphilic nanoparticles able to interact with biological macromolecules for biotechnological and pharmaceutical applications.     

Facile Surface Modification and Application of Temperature Responsive Poly(N‐isopropylacrylamide‐co‐dopamine methacrylamide)

The temperature‐responsive poly(N‐isopropylacrylamide) [PNIPAAm] has been exploited for various biomedical applications. In this work, poly(N‐isopropylacrylamide‐co‐dopamine methacrylamide) [P(NIPAAm‐co‐DMAAm)] was synthesized for facile surface modification and application to cell sheets. ¹H NMR, FT‐IR, and GPC confirmed the successful synthesis of P(NIPAAm‐co‐DMAAm). The lower critical solution temperature was measured to be ca. 29.2 °C by UV–Vis spectroscopy. AFM imaging clearly visualized the transient phase transition of the temperature‐responsive polymer bound on silicon substrate by coordination bond formation. Furthermore, the adhesive and temperature responsive P(NIPAAm‐co‐DMAAm) could be successfully applied to the facile preparation of NIH‐3T3 fibroblast cell sheets.     

PEG- and PDMAEG-Graft-Modified Branched PEI as Novel Gene Vector: Synthesis,Characterization and Gene Transfection

The cytotoxicity of polyethylenimine (PEI) was a dominating obstacle to its application. Introduction of poly(ethylene glycol) (PEG) blocks to PEI is one of the strategies to alleviate the cytotoxocity of PEI. However, it is well known that the transfection efficiency of PEGylated PEI is decreased to some extent compared to the corresponding PEI. Thus, the aim of our study was to enhance the transfection efficiency of PEGylated PEI. A series of tri-block co-polymers, PEG-g-PEI-g-poly(dimethylaminoethyl L-glutamine) (PEG-g-PEI-g-PDMAEG), as novel vectors for gene therapy was synthesized and evaluated. PEG-g-PEI was first obtained by linking PEG and PEI using isophorone diisocyanate (IPDI) as coupling reagent. The anionic co-polymerization of γ-benzyl-L-glutamate N-carboxyanhydride (BLG-NCA) using PEG-g-PEI as a macro-initiator was carried out, followed by aminolysis with 2-dimethylaminoethylamine to obtain the target water-soluble tri-block co-polymer. The structures of the polymers were confirmed by FT-IR and ¹H-NMR. The influence of the molecular weight of PEI and the length of the PDMAEG chain on the physicochemical properties and transfection activity of polymer/DNA was evaluated. All PEI derivates were revealed to compact plasmid DNA effectively to give polyplexes with suitable size (approx. 100 nm) and moderate zeta potentials (10–15 mV) at N/P ratios over 10. The PEG-g-PEI-g-PDMAEG tri-block co-polymers displayed particularly low cytotoxicity, even at high concentration, reflecting an improved safety profile compared to PEI 25k. Gene transfection efficiency of PEG-g-PEI-g-PDMAEG on HeLa in the presence and absence of serum was determined. Remarkably, the transfection activity of PEG-g-PEI (10k)-g-PDMAEG (PPP-4)/DNA polyplex formulations was nearly twofold higher than PEI 25k/DNA formulations in vitro, and the transfection efficiency was less affected by the presence of serum. These results indicated that the synthesized PEG-g-PEI-g-PDMAEG tri-block co-polymers are promising candidates as carriers for gene delivery.     

Emulsion copolymerization of 2-(acryloyloxy)ethylphosphoryl-choline with vinyl monomers and protein adsorption at resultant copolymer microspheres

A series of copolymer microspheres of 2-(acryloyloxy)ethylphosphorylcholine (APC) with comonomers (M) such as methyl (MMA), ethyl (EMA), butyl (BMA), hexyl methacrylate (HMA), and styrene (St), i. e. poly(APC-co-M) microspheres, were prepared by emulsifier-free emulsion copolymerization. From the kinetics of the copolymerization, it was found that the initial rate of polymerization of St increases in the presence of small amounts of APC. The diameters of poly-(APC-co-M) microspheres were much smaller than those of the corresponding homopolymer, poly(M). It was confirmed by X-ray photoelectron spectroscopy measurements that the APC moiety is concentrated on the surface of the particles. A series of poly(APC-co-M) microspheres was found to adsorb both bovine serum albumin (Alb) and human serum γ-globulin (Glo) less that the corresponding poly(M) microspheres as a control.     

Cationic shell-crosslinked knedel-like nanoparticles for highly efficient gene and oligonucleotide transfection of mammalian cells

In this work, a robust synthetic nanostructure was designed for the effective packaging of DNA and it was shown to be an efficient agent for cell transfection. An amphiphilic block copolymer, poly(acrylamidoethylamine)₁₂₈-b-polystyrene₄₀ (PAEA₁₂₈-b-PS₄₀), was synthesized, micellized in water and shell-crosslinked using a diacid-derivatized crosslinker, to give cationic shell-crosslinked nanoparticles (cSCKs) with a mean hydrodynamic diameter of 14 ± 2 nm. A series of discrete complexes of the cSCKs with plasmid DNA (pDNA) was able to be formed over a broad range of polymer amine:pDNA phosphate ratios (N/P ratio), 2:1–20:1. The sizes of the complexes and their ability to fully bind the pDNA were dependent upon the N/P ratio, as characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM) and gel retardation assay. A luciferase activity assay and EGFP expression were used to evaluate intracellular delivery of a splice-correcting phosphorothioate and genetic material, respectively, by the cSCKs, which indicated that an N/P ratio of 6:1 gave the highest transfection. It was shown by both luciferase activity assay (48 h) and EGFP transfection data that high transfection efficiencies were achieved for HeLa cells transfected by cSCK/CCUCUUACCUCAGUUACA and cSCK/pEGFP-N1 plasmid, respectively. The cSCK/pEGFP-N1 plasmid transfection efficiency of 27% far exceeded the performance of Polyfect^® (PAMAM dendrimers), which achieved only 12% transfection efficiency, under the same conditions. Cytotoxicities for the cSCKs were evaluated for HeLa and CHO cells.