Tunable film degradation and sustained release of plasmid DNA from cleavable polycation/plasmid DNA multilayers under reductive conditions

The controllable and sustained release of DNA from the surfaces of biomaterials or biomedical devices represents a new method for localized gene delivery. We report the synthesis of a novel polycation containing disulfide bonds in its backbone and the fabrication of polycation/plasmid DNA multilayered thin films by layer-by-layer assembly. The films are very stable during preparation and in storage, however, they gradually degrade and release the incorporated DNA when incubated in PBS buffer containing dithiothreitol (DTT), which results from the degradation of a disulfide-contained polymer under reductive conditions. The film degradation rate and DNA release rate can be tuned by the concentration of reducing agent. This approach will be useful in gene therapy and tissue engineering by controlled administration of therapeutic DNA deposited on the surface of implantable biomedical devices or tissue engineering scaffolds.     

Stability of poly(ethylene glycol)-graft-polyethylenimine copolymer/DNA complexes: influences of PEG molecular weight and PEGylation degree

Polyethylenimine (PEI) is one of the most widely investigated cationic polymers for gene delivery. However, PEI/DNA complexes are unstable and tend to aggregate. PEGylation was used to improve the stability. The stability of polymer/DNA complexes was investigated including complexation stability, aggregation stability, sedimentation stability, and nuclease stability. PEI25K/DNA complexes were liable to aggregate to large particles (500–700 nm). The aggregation was proved to be induced by phosphate anion. In the medium without phosphate anion, aggregation was prevented by electrostatic repulsion. Owing to more efficient steric repulsion, PEG2 and PEG5K excelled PEG750 in facilitating copolymers to form stable small polyplexes (below 100 nm) without aggregation regardless of phosphate anion. The steric repulsion predominated over electrostatic repulsion in stabilization.     

O-Carboxymethyl-chitosan/organosilica hybrid nanoparticles as non-viral vectors for gene delivery

Polymeric non-viral vectors, such as chitosan nanoparticles show good biocompatibility, but low transfection efficiency. The objective of this study was to improve the transfection efficiency of chitosan based non-viral vectors by using o-carboxymethyl-chitosan which is a kind of water-soluble chitosan derivative and also has good biocompatibility. O-Carboxymethyl-chitosan-organosilica hybrid nanoparticles (CMG NPs) were synthesized through a rapid one-step aqueous synthetic approach for gene delivery. The size of nanoparticles was 276 ± 25 nm and zeta potential was 31.6 ± 0.4 mV in deionized water. Zeta potential increased with the decrease of pH, and it had been discovered that pH = 5.5 is the best point for CMG NPs to bond with plasmid DNA. DNA inclusion and integrity was evaluated by gel electrophoresis, and it is indicated that CMG NPs could protect DNA against DNase I and serum degradation. The results of MTT for cell viability and in vitro transfection also support the idea that CMG NPs could be used as efficient and safe vectors for gene delivery.     

Capture and release of genomic DNA by PEI modified Fe3O4/Au nanoparticles

Polyethylenimine (PEI) modified Fe₃O₄/Au nanoparticles were synthesized in aqueous solution and characterized by photo correlation spectroscopy (PCS) and vibrating sample magnetometer (VSM). The so-obtained Fe₃O₄/Au-PEI nanoparticles were capable of efficient electrostatic capture of DNA. The maximum amount of genomic DNA captured on 1.0 mg Fe₃O₄/Au-PEI nanoparticles was 90 μg. The DNA release behavior was studied and the DNA recovery from Fe₃O₄/Au-PEI nanoparticles approached 100% under optimal conditions. DNA extraction from mammalian cells using Fe₃O₄/Au-PEI nanoparticles was successfully performed. Up to approximately 43.1 μg of high-purity (OD₂₆₀/OD₂₈₀ ratio = 1.81) genomic DNA was extracted from 10 mg of liver tissue. The results indicated that the prepared Fe₃O₄/Au-PEI nanoparticles could be successfully used for DNA capture and release.     

PEGylated bioreducible poly(amido amine)s for non-viral gene delivery

A facile method for PEGylated bioreducible poly(amido amine)s is described by a one-pot Michael-type addition polymerization of N, N′-cystaminebisacrylamide (CBA) with a mixture of 4-amino-1-butanol (ABOL) and mono-tert-butoxycarbonyl (Boc) PEG diamine. By this approach, two Boc-amino-PEGylated p(CBA-ABOL) copolymers were obtained with the PEG/ABOL composition ratio of 1/10 (1a) and 1/6 (2a), respectively. These copolymers were characterized by ¹H NMR and gel permeation chromatography. The PEGylated copolymers 1a, and its deprotected analog 1b with a terminal amino group at the PEG chain, were further evaluated as gene delivery vectors. The copolymers 1a and 1b condense DNA into nano-scaled PEGylated polyplexes (< 250 nm) with near neutral (2–5 mV, 1a) or slightly positive (9–13 mV, 1b) surface charge which remain stable in 150 mM buffer solution over 24 h. UnPEGylated polyplexes from p(CBA-ABOL), however, are relatively less stable and increase in size to more than 1 μm. The PEGylated polyplexes showed very low cytotoxicity in MCF-7 and NIH 3T3 cells and induced appreciable transfection efficiencies in the presence of 10% serum, although that are lower than those of p(CBA-ABOL) lacking PEG. The lower transfection efficiency of the PEGylated p(CBA-ABOL) polyplexes is discussed regarding the effect of PEGylation on endosomal escape of the PEGylated polyplexes.     

pH-Triggered DNA delivery based on multilayer film of DNA polyplexes and charge-reversible poly(ethylenimine)

Development of materials with stimuli-responsive properties is of interest for biotechnical applications including gene delivery and regenerative medicine. Here, we report a multilayer film through layer-by-layer self-assembly of DNA polyplexes and charge-reversible poly(ethylenimine) (cPEI). Through functionalizing PEI with cyclohexanedicarboxylic acid, cPEI showed negatively charged and therefore was used for electrostatic self-assembly with positively charged DNA polyplexes. Side chains of cPEI can be hydrolyzed in acidic environment while it is stable in neutral condition. Such pH-triggered hydrolysis led to charge reverse of cPEI from negative to positive, which consequently led to a disassembly of multilayer film. Both UV-vis and ellipsometry spectrum measurements suggested that the multilayer film grew with a thickness of 150 nm for twelve bilayers. Under low pH condition, the multilayer film collapsed and DNA polyplexes were released. The multilayer film containing cPEI could be served as a local gene delivery system in specific low pH conditions such as extracellular acidity of solid tumor and lysosomal.     

Enhanced gene transfer with multilayered polyplexes assembled with layer-by-layer technique

Successful gene therapy asks for multifunctional vectors which can not only protect DNA from degradation but also transfer it into nuclear and subsequently express the loaded gene. Here we reported a novel multilayered delivery system constructed with DNA, protamine (Pro) and polyethylenimine (PEI) via lay-by-layer (LbL) technique, which posed multifunctions. DNA was previously condensed into a compact core with Pro which also contained nuclear localisation signals (NLS) domains for nuclear transfer. Then additional DNA was deposited as the first layer onto the cationic core via the electrostatic attraction which would increase the loading dose of DNA. At last, PEI was absorbed as the outmost layer to achieve the endosomal escape. Therefore a quaternary polyplexes which offered high loading of DNA, nuclear transfer ability and endosomal escape capability was constructed with the LbL technique. The obtained quaternary polyplexes showed positive surface charge, spherical morphology, a relatively narrow particle size distribution and strong DNA protection capability. Compared with commercially available PEI/DNA complexes, the novel multifuctional vector exhibited not only lower cytotoxicity (P<0.05) but also higher transfection efficiency in HepG2 and HeLa cells (P<0.05) in vitro test.     

Polyplexes of polyethylenimine and per-N-methylated polyethylenimine-cytotoxicity and transfection efficiency

For non-viral gene delivery, the carriers for DNA transfer into cells must be vastly improved. The branched cationic polymer polyethylenimine has been described as an efficient gene carrier. However, polyethylenimine was demonstrated to mediate substantial cytotoxicity. Therefore, this study is aimed at investigating per-N-methylated polyethylenimine, which is thought to have a much lower cytotoxicity due to its lower charge density. Results from a gel retardation assay and laser light scattering indicated that per-N-methylated polyethylenimine condenses DNA into small and compact nanoparticles with a mean diameter <150 nm. Furthermore, polyplexes of polyethylenimine and per-N-methylated polyethylenimine with DNA had a positive zeta potential and the polymers protected DNA from nuclease-mediated digestion. The transfection efficiency of polyethylenimine and per-N-methylated polyethylenimine was tested in CHO-K1 cells. Using green fluorescent protein as reporter gene and flow cytometry analysis, we demonstrated that per-N-methylated polyethylenimine has a lower cytotoxicity, but also a significantly lower transfection efficiency. Using propidium iodide staining, we could additionally distinguish between viable and dead cells. At NP > or = 12, per-N-methylated polyethylenimine showed a much higher cell viability and the ratio of viable and transfected cells to dead and transfected cells was about 1.5 to 1.7 fold higher than for polyethylenimine. The results of cell viability from flow cytometry analysis were confirmed by the MTS assay. Using luciferase reporter gene for transfection experiments, the gene expression of per-N-methylated polyethylenimine was lower at NP 6, 12 and 18 as compared to polyethylenimine, but at NP 24 it yielded similar levels.     

Novel hyperbranched polyamidoamine nanoparticles for transfecting skeletal myoblasts with vascular endothelial growth factor gene for cardiac repair

We investigated the feasibility and efficacy of hyperbranched polyamidoamine (hPAMAM) mediated human vascular endothelial growth factor-165 (hVEGF₁₆₅) gene transfer into skeletal myoblasts for cardiac repair. The hPAMAM was synthesized using a modified one-pot method. Encapsulated DNA was protected by hPAMAM from degradation for over 120 min. The transfection efficiency of hPAMAM in myoblasts was 82.6 ± 7.0% with cell viability of 94.6 ± 1.4% under optimal conditions. The hPAMAM showed much higher transfection efficiency (P < 0.05) than polyetherimide and Lipofectamine 2000 with low cytotoxicity. The transfected skeletal myoblasts gave stable hVEGF₁₆₅ expression for 18 days. After transplantation of hPAMAM–hVEGF₁₆₅ transfected cells, apoptotic myocardial cells decreased at day 1 and heart function improved at day 28, with increased neovascularization (P < 0.05). These results indicate that hPAMAM-based gene delivery into myoblasts is feasible and effective and may serve as a novel and promising non-viral DNA vehicle for gene therapy in myocardial infarction.     

Synthesis and properties of a novel methoxy poly(ethylene glycol)-modified galactosylated chitosan derivative

Chitosan and its derivatives are attractive non-viral vectors. To produce target-cell specificity and improve the solubility of chitosan, a novel chitosan derivative, modified with galactose and methoxy poly(ethylene glycol) (mPEG) was synthesized, and structure changes of chitosan and its derivatives were characterized. Compared to chitosan, the solution viscosity of the novel chitosan derivative drastically decreased. And, the degree of substitution (DS) of chitosan by galactose and mPEG were calculated as 0.09 and 0.30. The average diameter and zeta potential of mPEGylated galactosylated chitosan (GaC) nanoparticle containing VRMFat plasmid were 178 nm and +2.93 mV, suggesting suitable properties for gene delivery system. The gel electrophoresis confirmed that the plasmid DNA was remained completely by the mPEGylated GaC nanoparticle. And, the cytotoxic effect of mPEGylated GaC nanoparticles on human embryonic kidney (HEK 293) cells was negligible in comparison with that of control chitosans. Therefore, it is expected that the mPEGylated GaC will have the potential as a targeting gene delivery system for a further application. Tao Zhang and Dong Li equally contributed to this research.     

Lead-Free Cesium Thulium Halide Perovskite Microcrystals for Near Ultraviolet Luminescence

Lead halide perovskites attract tremendous research attention due to excellent optoelectronic properties. However, realizing efficient near ultraviolet (NUV) luminescence with these materials is still a big challenge. Herein, a novel rare-earth perovskite cesium thulium chloride (CsTmCl₃) with high crystallinity has been synthesized via a simple hot-injection method. The obtained CsTmCl₃ microcrystals have a size distribution of around 1–5 µm, and demonstrate a highly efficient NUV emission at 337 nm with a full width at half maximum (FWHM) of 68 nm. The determined band gap of CsTmCl₃ microcrystals is ≈3.92 eV, which is supported by theoretical calculations. Moreover, a high photoluminescence quantum yield (PLQY) of up to 12% in NUV region has been achieved in such a lead-free perovskite. The findings suggest that CsTmCl₃ perovskite microcrystal is a promising low-toxic material for applications in NUV optoelectronic devices.     

Strategic Fast Induction Heating to Combat Hysteresis Barriers in a Flexible MOF for Rapid CO2 Desorption in Biogas Upgrading

A major challenge in Cyclic Swing Separation using flexible adsorbents that have high equilibrium CO₂ adsorption capacity is their very low-pressure hysteresis that hinders efficient desorption. Mg-Gallate MOF is such a flexible adsorbent that only begins to release CO₂ at its pore closing pressure at 0.08 bar and 30 °C, showing very slow and inefficient desorption in pressure or temperature swing. Therefore, a novel strategy is presented that combines state of art technique Magnetic Induction Heating with a vacuum swing for fast and efficient CO₂ desorption from flexible adsorbents at a moderately elevated temperature (70 °C).     

Peptide‐like Polymers Exerting Effective Glioma‐Targeted siRNA Delivery and Release for Therapeutic Application

Lipopolymer 49, a solid‐phase synthesized T‐shaped peptide‐like oligoamide containing two central oleic acids, 20 aminoethane, and two terminal cysteine units, is identified as very potent and biocompatible small interfering RNA (siRNA) carrier for gene silencing in glioma cells. This carrier is combined with a novel targeting polymer 727, containing a precise sequence of Angiopep 2 targeting peptide, linked with 28 monomer units of ethylene glycol, 40 aminoethane, and two terminal cysteines in siRNA complex formation. Angiopep‐polyethylene glycol (PEG)/siRNA polyplexes exhibit good nanoparticle features, effective glioma‐targeting siRNA delivery, and intracellular siRNA release, resulting in an outstanding gene downregulation both in glioma cells and upon intravenous delivery in glioma model nude mice without significant biotoxicity. Therefore, this novel siRNA delivery system is expected to be a promising strategy for targeted and safe glioma therapy.     

Transfection efficiency of depolymerized chitosan and epidermal growth factor conjugated to chitosan–DNA polyplexes

An efficient non-viral gene delivery for varieties of cells has been considered essential for gene therapy and tissue engineering. This study evaluated transfection efficiency of chitosan (HW) with molecular weights (Mw) at 470 and degree of deacetylation (DDA) 80% and its depolymerization product (LW) with Mw at 16 kDa and DDA 54%, as well as epidermal growth factor (EGF) conjugated to chitosan–DNA microparticles of both HW and LW by using either disulfide linkage or NHS-PEO₄-Maleimide as a cross linker. The results revealed that the depolymerized LW at chitosan/DNA charge ratio 56:1 and pH 6.9 gave high transfection efficiency in both KB, a cancer cell line, and fibroblast cells at about the same level of Lipofectamine^TM, but the EGF-conjugated chitosan–DNA polyplexes from these methods did not improve transfection efficiency, which may come from the aggregation and fusing of the complexes as shown in scanning electron microscopy. However, this depolymerized LW chitosan showed the potential for further development as a safe and cost-effective non-viral gene delivery vehicle.     

Degradable poly(amino ester) based on poly(ethylene glycol) dimethacrylate and polyethylenimine as a gene carrier: molecular weight of PEI affects transfection efficiency

The aim of the research is to study the effect of polyethylenimine (PEI) molecular weight on the gene transfection efficiency of degradable poly(amino ester) based on poly(ethylene glycol) dimethacrylate (PEGDMA) and polyethylenimine (PEG-cr-PEI) as a gene carrier. Various low molecular weight (LMW) branched PEI based PEG-cr-PEI was synthesized via Michael addition. The degradation half-life of PEG-cr-PEI was longer at pH 5.6 than that at pH 7.4. The plasmid condensation and protection ability of the PEG-cr-PEI were confirmed by agarose gel electrophoresis assay. PEG-cr-PEI/DNA nanoparticles showed high positive zeta potential (>+20 mV), narrow size distribution, and spherical shapes with size below 250 nm when N/P ratios of PEG-cr-PEI to DNA were above 10, suggesting that they have endocytosis potential. The cytotoxicity of PEG-cr-PEI/DNA complexes was lower than that of PEI 25K/DNA complexes, and the transfections mediated by PEG-cr-PEI were checked in 293T, HeLa and HepG2 cell lines. The report gene expression was increased with increasing the molecular weight of LMW PEI. The “proton sponge effect” was proposed as the mechanism of PEG-cr-PEI mediated gene transfection.     

Interfacially organized DNA/polycation complexes: a route to new planar polymeric and composite nanostructures

Effects of nano-scale supramolecular organization and patterning in amphiphilic polycation monolayer formed by poly-4-vinilpyridine with 16% cetylpyridinium groups and in novel planar DNA/amphiphilic polycation complexes formed at the air-aqueous DNA solution interface and deposited on the solid substrates have been studied using AFM. Stable ordered quasi-crystalline planar polymeric monolayer structures were formed by amphiphilic polycation molecules. Extended net-like and quasi-circular toroidal condensed conformations of deposited planar DNA/amphiphilic polycation complexes were obtained in dependence on the amphiphilic polycation Langmuir monolayer state during the DNA binding. Those monolayer and multilayer DNA/polycation complex Langmuir–Blodgett films were used as templates and nanoreactors for generation of inorganic nanostructures. As a result, ultrathin polymeric nanocomposite films with integrated DNA building blocks and inorganic semiconductor (CdS) and iron oxide nanoparticle quasi-linear arrays or aggregates (nanorods) were formed successfully and characterized by TEM. The data obtained give evidence for effectiveness of the monolayer techniques for study mechanisms of DNA structural transformations caused by complexation with cationic compounds and demonstrate its perspectives for creation of new planar DNA-based self-organized stable polymeric complex nanostructures and nanocomposites with nano-scale structural ordering.     

Homogeneous chitosan/carbonate apatite/citric acid nanocomposites prepared through a novel in situ precipitation method

Homogeneous nanocomposites composed of carbonate apatite and chitosan in the presence of citric acid were synthesized by a novel in situ precipitation method. The morphological and componential properties of composites were investigated. The carbonate apatite particulates, in sizes of about 50–100 nm, were distributed within the network chitosan hydrogel homogeneously, moreover, inorganic particles could be controlled by the size of networks of organic matrix which was mediated with crosslink degree of chitosan. Through highly magnified TEM observation, it can be shown that inorganic particles were composed of more fine sub-particles whose diameters were between 2 and 5 nm in size without regular crystallographic orientation. The concept of multiple-order template mediation was brought forward for the first time. A novel hierarchical porous nanocomposite scaffold was also prepared by multilevel freeze-drying technique and its mechanical performance had obvious increase compared with neat chitosan. These results provided an efficient approach toward new biomimetic tissue scaffold for the biomedical applications with enhanced intensity/bioactivity and controlled resorption rates.     

Carboxymethyl derivative of scleroglucan: a novel thermosensitive hydrogel forming polysaccharide for drug delivery applications

A carboxymethyl derivative of scleroglucan (Scl-CM) with a derivatization degree of 65 ± 5% was synthesized. The rheological behaviour of this novel polymer was studied and compared with that of the starting polymer. We observed that the charged moieties carried on the chains could prevent the triple helix formation of Scl. Scl-CM aqueous solutions behave like true polymer solutions up to 1% w/v, whereas above this concentration a weak gel behaviour was observed. CaCl₂ addition to aqueous Scl-CM solutions led to a physical gel formation; the hydrogel strength was related to polymer and CaCl₂ concentrations. Temperature sweeps, registered at 1 Hz on hydrogels differing in CaCl₂ concentration, evidenced a gel → sol transition in the range of 30–40°C, depending on the molar ratio between carboxylic groups and Ca⁺². In order to verify a possible use of these hydrogels as drug delivery systems, acyclovir was loaded into the network. Rheological analysis evidenced that the loaded drug can affect the hydrogel elastic modulus. The release of acyclovir in phosphate buffer was evaluated at different temperatures in order to assess the suitability of this novel drug delivery system in topical applications.     

Effect of ionic strength solution on the stability of chitosan–DNA nanoparticles

Chitosan–DNA nanoparticles employed in gene therapy protocols consist of a neutralised, stoichiometric core and a shell of the excess of chitosan which stabilises the particles against further coagulation. At low ionic strength, these nanoparticles possess a high stability; however, as the ionic strength increases, it weakens the electrostatic repulsion which can play a decisive part in the formation of highly aggregated particles. In this study, new results about the effect of ionic strength on the colloidal stability of chitosan–DNA nanoparticles were obtained by studying the interaction between chitosans of increasing molecular weights (5, 10, 16, 29, 57 and 150?kDa) and calf thymus DNA. The physicochemical properties of polyplexes were investigated by means of dynamic light scattering, static fluorescence spectroscopy, optic microscopy, transmission electronic microscopy and gel electrophoresis. After subsequent addition of salt to the nanoparticles solution, secondary aggregation increased the size of the polyplexes. The nanoparticles stability decreased drastically at the ionic strengths 150 and 500?mM, which caused the corresponding decrease in the thickness of the stabilising shell. The morphologies of chitosan/DNA nanoparticles at those ionic strengths were a mixture of large spherical aggregates, toroids and rods. The results indicated that to obtain stable chitosan–DNA nanoparticles, besides molecular weight and N/P ratio, it is quite important to control the ionic strength of the solution.     

Synthesis and characterization of octahedral PbF2

The novel 3D octahedron-like PbF₂ structures with dimension of 2-4 µm have been successfully synthesized by a simple route at low temperature. The morphologies and structures of as-prepared products were characterized by X-ray powder diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The growth mechanism has been proposed for octahedral PbF₂. It was found that the aging time is important for the formation of 3D octahedron-like PbF₂ structures. The room-temperature photoluminescence measurements revealed a strong blue emission band at 485 nm. It was indicated that the as-prepared octahedral PbF₂ could have the potential application in optoelectronic devices.