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Poly(L-lysine) (PLL)-3-hydroxyphenylboronic acid (3-PBA) composite was prepared onto an Au electrode via cyclic voltammetry (CV), which was characterized by electrochemical and quartz crystal microbalance (QCM) techniques, and used to promote adhesion of plant cells. 3-PBA can interact with glycosyl compounds on the cell wall to form negatively charged covalent compounds while PLL increases the number of positively charged sites, which enhances the electrostatic interactions with the negatively charged cell wall, both promoting cell adhesion. The PLL-poly(3-PBA) composite was successfully modified on the Au electrode surface, as demonstrated by QCM and Fourier transform infrared spectrophotometry (FTIR). Moreover, we confirmed the synergistic effect of PLL and 3-PBA to adhere rice suspension cells using CV, electrochemical impedance spectroscopy (EIS), QCM and optical microscope observations. Finally, the dynamic process of rice cells’ adhesion to PLL-poly(3-PBA)/Au followed by salt stress treatment of NaCl was monitored in real-time by QCM. The cells softened at first then hardened under the action of 40 mM NaCl. This work presents a novel and conventional way to immobilize plant cells onto sensors, and has importance in the study of the structure, function and behavior of plants under various stresses at the cellular level.

• PDA/PLL hybrid coatings were developed to functionalize hydroxyapatite scaffolds. • The hybrid coatings realize tunable and sustained release of BMP2. • The hybrid coatings promote BMSC adhesion and differentiation. • The coating-functionalized scaffolds possess excellent osteoinductive properties. The scaffold for bone tissue engineering should possess proper porosity, adequate mechanical properties, cell affinity for cell attachment, and the capability to bind bioactive agents to induce cell differentiation. In this study, we successfully prepared a porous hydroxyapatite (HA) scaffold that is functionalized by poly(L-lysine)/polydopamine (PLL/PDA) hybrid coating. The PLL/PDA coating takes advantages of the high protein and cell affinity of PDA, as well as the biodegradability of PLL. Therefore, the coating can anchor bone morphogenic protein-2 (BMP2) to the HA scaffold via catechol chemistry under a mild condition so as to protect the bioactivity of BMP2. Meanwhile, the coating can also release BMP2 in a tunable and sustainable manner as the PLL degrades in the physiological environment. The BMP2-entrapped PLL/PDA coating on the HA scaffold can more efficiently promote osteogenic differentiation of bone marrow stromal cells (BMSCs) in vitro and induce ectopic bone formation to a much greater level in vivo compared with a bare HA scaffold that delivers BMP2 in a burst manner. All of these results suggest that the PDA-mediated catechol modification of the HA scaffold can be an effective strategy to develop sustainable protein delivery system, and that the PLL/PDA-coated HA scaffold could be a promising candidate for bone tissue engineering applications. [ABSTRACT FROM AUTHOR]

Background: Cell tracking is a powerful tool to understand cellular migration, dynamics, homing and function of stem cell transplants. Nanoparticles represent possible stem cell tracers, but they differ in cellular uptake and side effects. Their properties can be modified by coating with different biocompatible polymers. To test if a coating polymer, poly(L-lysine), can improve the biocompatibility of nanoparticles applied to neural stem cells, poly(L-lysine)-coated maghemite nanoparticles were prepared and characterized. We evaluated their cellular uptake, the mechanism of internalization, cytotoxicity, viability and proliferation of neural stem cells, and compared them to the commercially available dextran-coated nanomag®-D-spio nanoparticles.Results: Light microscopy of Prussian blue staining revealed a concentration-dependent intracellular uptake of iron oxide in neural stem cells. The methyl thiazolyl tetrazolium assay and the calcein acetoxymethyl ester/propidium iodide assay demonstrated that poly(L-lysine)-coated maghemite nanoparticles scored better than nanomag®-D-spio in cell labeling efficiency, viability and proliferation of neural stem cells. Cytochalasine D blocked the cellular uptake of nanoparticles indicating an actin-dependent process, such as macropinocytosis, to be the internalization mechanism for both nanoparticle types. Finally, immunocytochemistry analysis of neural stem cells after treatment with poly(L-lysine)-coated maghemite and nanomag®-D-spio nanoparticles showed that they preserve their identity as neural stem cells and their potential to differentiate into all three major neural cell types (neurons, astrocytes and oligodendrocytes).Conclusion: Improved biocompatibility and efficient cell labeling makes poly(L-lysine)-coated maghemite nanoparticles appropriate candidates for future neural stem cell in vivo tracking studies.

The deposition of polyelectrolyte (PEL) multilayers (PEMs) of poly(l-lysine)/cellulose sulfate (PLL/CS) onto germanium (Ge) substrates depending on salt concentration (cS) and deposition step z at constant PEL concentration cPEL = 0.01 M and pH = 7.0 was studied. In situ ATR-FTIR spectroscopy was used for the quantitative determination of alternate PLL/CS deposition profiles (adsorbed amount versus z) and total deposited PEM amount. By varying cS from 0 M to 1.0 M, a maximum of deposited amount was obtained at 0.1 M, so that both no salinity (0 M) and high salinity (1.0 M) revealed deposited amounts that were far lower than for mean salinity (0.1 M). Furthermore, in situ ATR-FTIR allowed to determine the detailed modulation of the PEL composition during the consecutive PEM deposition, which was interpreted as being due to both diffusion of given PEL from the PEM interior towards the outermost region and release of the PEM upon contact with the bulk oppositely charged PEL solution. Finally, ex situ ATR-FTIR measurements on the PEL solutions after deposition of PEM-20 revealed the distinct release of PEL from the PEM solely for cS = 1.0 M, due to the highest mobility of PEL under high salt conditions. These studies help to prepare functional PEM coatings with defined thicknesses and morphologies for the passivation and activation of material surfaces in the biomedical and food field.

The polyplexes formed by nucleic acids and polycations have received a great attention owing to their potential application in gene therapy. In our study, we report experimental results and modeling outcomes regarding the optimization of polyplex formation between the double-stranded DNA (dsDNA) and poly(L-Lysine) (PLL). The quantification of the binding efficiency during polyplex formation was performed by processing of the images captured from the gel electrophoresis assays. The design of experiments (DoE) and response surface methodology (RSM) were employed to investigate the coupling effect of key factors (pH and N/P ratio) affecting the binding efficiency. According to the experimental observations and response surface analysis, the N/P ratio showed a major influence on binding efficiency compared to pH. Model-based optimization calculations along with the experimental confirmation runs unveiled the maximal binding efficiency (99.4%) achieved at pH 5.4 and N/P ratio 125. To support the experimental data and reveal insights of molecular mechanism responsible for the polyplex formation between dsDNA and PLL, molecular dynamics simulations were performed at pH 5.4 and 7.4. [ABSTRACT FROM AUTHOR]

Angiogenesis plays an important role in both soft and hard tissue regeneration, which can be modulated by therapeutic drugs. If nanoparticles (NP) are used as vectors for drug delivery, they have to encounter endothelial cells (EC) lining the vascular lumen, if applied intravenously. Herein the interaction of unloaded polyelectrolyte complex nanoparticles (PECNP) composed of cationic poly(l-lysine) (PLL) and various anionic polysaccharides with human vascular endothelial cells (HUVEC) was analyzed. In particular PECNP were tested for their cell adhesive properties, their cellular uptake and intracellular localization considering composition and net charge. PECNP may form a platform for both cell coating and drug delivery. PECNP, composed of PLL in combination with the polysaccharides dextran sulfate (DS), cellulose sulfate (CS) or heparin (HEP), either unlabeled or labeled with fluorescein isothiocyanate (FITC) and either with positive or negative net charge were prepared. PECNP were applied to human umbilical cord vein endothelial cells (HUVEC) in both, the volume phase and immobilized phase at model substrates like tissue culture dishes. The attachment of PECNP to the cell surface, their intracellular uptake, and effects on cell proliferation and growth behavior were determined. Immobilized PECNP reduced attachment of HUVEC, most prominently the systems PLL/HEP and PLL/DS. A small percentage of immobilized PECNP was taken up by cells during adhesion. PECNP in the volume phase showed no effect of the net charge sign and only minor effects of the composition on the binding and uptake of PECNP at HUVEC. PECNP were stored in endosomal vesicles in a cumulative manner without apparent further processing. During mitosis, internalized PECNP were almost equally distributed among the dividing cells. Both, in the volume phase and immobilized at the surface, PECNP composed of PLL/HEP and PLL/DS clearly reduced cell proliferation of HUVEC, however without an apparent cytotoxic effect, while PLL/CS composition showed minor impairment. PECNP have an anti-adhesive effect on HUVEC and are taken up by endothelial cells which may negatively influence the proliferation rate of HUVEC. The negative effects were less obvious with the composition PLL/CS. Since uptake and binding for PLL/HEP was more efficient than for PLL/DS, PECNP of PLL/HEP may be used to deliver growth factors to endothelial cells during vascularization of bone reconstitution material, whereas those of PLL/CS may have an advantage for substituting biomimetic bone scaffold material.

Narrowly distributed poly(l-lysine-b-N-isopropylacrylamide) (PLL-b-PNIPAM) was prepared through ring-opening polymerization of ε-benzyloxycarbonyl-l-lysine N-carboxy-α-amino anhydride and atom transfer radical polymerization of NIPAM, followed with the removal of ε-benzyloxycarbonyl group. Then gold nanoparticles (AuNPs) grafted with PLL-b-PNIPAM (PNIPAM-PLL-AuNPs) were obtained by the reduction of chloroauric acid with sodium citrate in the presence of PLL-b-PNIPAM. PNIPAM-PLL-AuNPs and its precursors were thoroughly characterized by proton magnetic resonance spectroscope, Fourier transform infrared spectroscope, UV-vis spectroscope, transmission electron microscopy, dynamic light scattering, thermogravimetric analysis, and circular dichroism. The obtained PNIPAM-PLL-AuNPs exhibited high colloid stability even at strong alkaline (pH = 12) and acidic (pH = 2) conditions. The thermal and pH dual-responsive behaviors of the grafting PLL-b-PNIPAM chains was observed to be affected by AuNPs, while not for the secondary structure of PLL chains. Correspondingly, the surface plasmon resonance (SPR) of AuNPs was found to be sensitive to both pH value and temperature. A blue shift in the SPR happened both with increasing pH value and increasing temperature. The stimuli-response was reversible in heating-cooling cycles. The gold nanoparticles with both pH and temperature response may have potential applications in biomedical areas and biosensors.

A novel, simple and selective electrochemical method was investigated for the simultaneous detection of dopamine (DA) and uric acid (UA) on a poly(L-lysine)/graphene oxide (GO) modified glassy carbon electrode (PLL/GO/GCE) by differential pulse voltammetry (DPV). The electrochemically prepared PLL/GO sensory platform toward the oxidation of UA and DA exhibited several advantages, including high effective surface area, more active sites and enhanced electrochemical activity. Compared to the PLL-modified GCE (PLL/GCE), GO-modified GCE and bare GCE, the PLL/GO/GCE exhibited an increase in the anodic potential difference and a remarkable enhancement in the current responses for both UA and DA. For the simultaneous detection of DA and UA, the detection limits of 0.021 and 0.074 μM were obtained, while 0.031 and 0.018 μM obtained as the detection limits for the selective detection of UA and DA, using DPV in the linear concentration ranges of 0.5 to 20.0 and 0.5 to 35 μM, respectively. In addition, the PLL/GO/GCE demonstrated good reproducibility, long-term stability, excellent selectivity and negligible interference of ascorbic acid (AA). The proposed modified electrode was successfully implemented in the simultaneous detection of DA and UA in human blood serum, urine and dopamine hydrochloride injection with satisfactory results. [ABSTRACT FROM AUTHOR]

Abstract: A hydrophilic star block co-polymer was synthesized, characterized, and evaluated as a protein nanocarrier. The star block co-polymer was composed of a hyperbranched polyethylenimine (PEI) core, a poly(l-lysine) (PLL) inner shell, and a poly(ethylene glycol) (PEG) outer shell. The model protein insulin can be rapidly and efficiently encapsulated by the synthesized polymer in aqueous phosphate buffer at physiological pH. Complexation between PEI–PLL–b-PEG and insulin was investigated using native polyacrylamide gel electrophoresis. The uptake of enhanced green fluorescent protein into Ad293 cells mediated by PEI–PLL–b-PEG was also investigated. The encapsulated insulin demonstrated sustained release at physiological pH and showed accelerated release when the pH was decreased. The insulin released from the star block co-polymer retained its chemical integrity and immunogenicity. [Copyright &y& Elsevier]

Abstract: Star-block copolymers PEI-g-(PLL-b-PEG) with a branched polyethylenimine (PEI) core, a poly(l-lysine) (PLL) inner shell, and a poly(ethylene glycol) (PEG) outer shell have been synthesised and evaluated as potential nanocarriers for anionic drugs. The star-block copolymers were synthesised by a ring-opening polymerisation of ɛ-benzyloxycarbonyl-l-lysine N-carboxyanhydride initiated by the peripheral primary amino groups of PEI, surface modification with activated PEG 4-nitrophenyl carbonate, and subsequent deprotection of benzyl groups on the side chains of the PLL inner shell. The synthesised star-block copolymers were characterised by 1H NMR, gel permeation chromatography (GPC), and dynamic light scattering (DLS). The encapsulation properties of these star-block copolymers were characterised by spectrophotometric titration and dialysis. These techniques demonstrated that anionic model dyes, such as methyl orange and rose Bengal, and the model drug diclofenac sodium can be encapsulated efficiently by PEI-g-(PLL-b-PEG) at physiological pH. The entrapped model compounds demonstrated sustained release at physiological pH and accelerated release when the pH was either increased to 10.0–11.0 or decreased to 2.0–3.0. The efficient encapsulation as well as the pH-responsive releasing properties of these star-block copolymers could be potentially used in the controlled release of anionic drugs. [Copyright &y& Elsevier]

Abstract: Although polymers, polyplexes, and cells are exposed to various extracellular and intracellular pH environments during polyplex preparation and polymeric transfection, the impact of environmental pH on polymeric transfection has not yet been investigated. This study aims to understand the influence of environmental pH on polymeric transfection by modulating the pH of the transfection medium or the culture medium. Changes in the extracellular pH affected polymeric transfection by way of complex factors such as pH-induced changes in polymer characteristics (e.g., proton buffering capacity and ionization), polyplex characteristics (e.g., size, surface charge, and decomplexation), and cellular characteristics (e.g., cellular uptake, cell cycle phases, and intracellular pH environment). Notably, acidic medium delayed endocytosis, endosomal acidification, cytosolic release, and decomplexation of polyplexes, thereby negatively affecting gene expression. However, acidic medium inhibited mitosis and reduced dilution of gene expression, resulting in increased transfection efficiency. Compared to pH 7.4 medium, acidic transfection medium reduced gene expression 1.6–7.7-fold whereas acidic culture medium enhanced transfection efficiency 2.1–2.6-fold. Polymeric transfection was affected more by the culture medium than by the transfection medium. Understanding the effects of extracellular pH during polymeric transfection may stimulate new strategies for determining effective and safe polymeric gene carriers. [Copyright &y& Elsevier]

Among the well-studied polypeptide-type gene carriers, transfection efficiency is empirically known to be higher for poly(L-arginine) (PR) than poly(L-lysine) (PK). The big difference between PR and PK should be determined at one of the intracellular trafficking steps based on the different charge densities, structures or PKa values. However, the endosomal escape and the intranuclear transcription efficiency in living cells have not been clarified yet. In this study, a novel method for quantifying the intranuclear transcription efficiency and the nuclear transport of the polyplex is established based on the nuclear and the cytosolic microinjection technique, and the results for PK and PR with different molecular weights (MWs) are compared in living cells. The intranuclear transcription efficiency is the same in PR and PK and it decreases rapidly with increasing MW, in spite of the commonly measured transfection efficiency. The transcription efficiency is strongly suppressed at high MW and strongly correlates with the polyplex forming ability expressed as a critical ratio of the number of polypeptide cationic groups to the number of pDNA anionic groups. When considered with the results of the cellular uptake and in vitro transfection with or without chloroquine, the rate-limiting step for their gene transfer is the buffering effect-independent endosomal escape. [ABSTRACT FROM AUTHOR]

Abstract: The adsorption behavior of poly(ethylene oxide)-b-poly(l-lysine) (PEO113-b-PLL10) copolymer onto silica nanoparticles was investigated in phosphate buffer at pH 7.4 by means of dynamic light scattering, zeta potential, adsorption isotherms and microcalorimetry measurements. Both blocks have an affinity for the silica surface through hydrogen bonding (PEO and PLL) or electrostatic interactions (PLL). Competitive adsorption experiments from a mixture of PEO and PLL homopolymers evidenced greater interactions of PLL with silica while displacement experiments even revealed that free PLL chains could desorb PEO chains from the particle surface. This allowed us to better understand the adsorption mechanism of PEO-b-PLL copolymer at the silica surface. At low surface coverage, both blocks adsorbed in flat conformation leading to the flocculation of the particles as neither steric nor electrostatic forces could take place at the silica surface. The addition of a large excess of copolymer favoured the dispersion of flocs according to a presumed mechanism where PLL blocks of incoming copolymer chains preferentially adsorbed to the surface by displacing already adsorbed PEO blocks. The gradual addition of silica particles to an excess of PEO-b-PLL copolymer solution was the preferred method for particle coating as it favoured equilibrium conditions where the copolymer formed an anchor-buoy (PLL-PEO) structure with stabilizing properties at the silica-water interface. [Copyright &y& Elsevier]

Abstract: A major challenge in gene therapy is the development of effective gene delivery vectors with low toxicity. In the present study, linear poly(ethylenimine) (lPEI) with low molecular weight was grafted onto the block copolymer (PPL) of poly(l-lysine) (PLL) and poly(ethylene glycol)(PEG), yielding a ternary copolymer PEG-b-PLL-g-lPEI (PPI) for gene delivery. In such molecular design, PLL, lPEI and PEG blocks were expected to render the vector biodegradability, proton buffering capacity, low cationic toxicity and potentially long circulation in vivo, respectively. Given proper control of molecular composition, the copolymers demonstrated lower cytotoxicity, proton buffering capacity, ability to condense pDNA and mediate effective gene transfection in various cell lines. With folate as an exemplary targeting ligand, the FA-PPI/pDNA complex showed much higher transgene activity than its nontargeting counterpart for both reporter and therapeutic genes in folate receptor(FR)-positive cells. FA-PPI mediated effective transfection of the TNF-related apoptosis-inducing ligand gene (TRAIL) in human hepatoma Bel 7402 cells, leading to cell apoptosis and great suppression of cell viability. Our results indicate that the copolymers might be a promising vector combining low cytotoxicity, biodegradability, and high gene transfection efficiency. [Copyright &y& Elsevier]

Abstract: Central to gene therapy technology has been the use of cationic polymers as vectors for DNA and RNA (polyfectins). These have been presumed to be safer than viral systems which, for example, have been found to switch on oncogenes. Two key polycations that have been intensively researched for use as synthetic vectors are poly(ethylenimine) and poly(l-lysine). A frequent stumbling block with these polyfectins is that long-term gene expression in cell lines has not been achieved. Recently it has transpired that both of these polycations can induce mitochondrially mediated apoptosis. It is the aim of this review to discuss the mechanisms behind the observed polycation toxicity including roles for little studied cellular organelles in the process such as the lysosome and endoplasmic reticulum. [Copyright &y& Elsevier]

Abstract: Optical reflectometry was used to study adsorption behavior of poly(l-lysine) (PLL) on the water–silica interface for different pH and concentrations of the monovalent salt NaBr. At low salt level and low pH, the adsorbed amount increases with increasing salt concentration and pH. This adsorption behavior can be qualitatively explained by electrostatic interactions between the negatively charged substrate and the positively charged polyelectrolyte. At higher salt levels and pH, the adsorbed amount passes through a maximum. This maximum is probably related to the competition between adsorbing monovalent cations and the polyelectrolyte. The initial adsorption kinetics is mainly controlled by the mass transport to the surface. [Copyright &y& Elsevier]

Abstract: While multidrug resistance (MDR) has been a significant issue in cancer chemotherapy, delivery resistance to various anti-cancer biotherapeutics, including genes, has not been widely recognized as a property of MDR. This study aims to provide a better understanding of the transfection characteristics of drug-sensitive and drug-resistant cells by tracing microenvironmental pHs of two representative polymer vectors: poly(l-lysine) and polyethyleneimine. Drug-sensitive breast MCF7 cells had four- to seven-times higher polymeric transfection efficiencies than their counterpart drug-resistant MCF7/ADR-RES cells. Polyplexes in MCF7/ADR-RES cells after endocytosis were exposed to a more acidic microenvironment than those in MCF7 cells; the MDR cells show faster acidification rates in endosomes/lysosomes than the drug-sensitive cells after endocytosis (in the case of PLL/pDNA complexes, ∼ pH 5.1 for MCF7/ADR-RES cells vs. ∼ pH 6.8 for MCF7 cells at 0.5 h post-transfection). More polyplexes were identified trapped in acidic subcellular compartments of MCF7/ADR-RES cells than in MCF7 cells, suggesting that they lack endosomal escaping activity. These findings demonstrate that the design of polymer-based gene delivery therapeutics should take into account the pH of subcellular compartments. [Copyright &y& Elsevier]

Polylysine is a biocompatible, biodegradable, water soluble polypeptide. Thanks to the pendant primary amines it bears, it is susceptible to modification reactions. In this work Poly(L-lysine) (PLL) was partially modified via the effortless free-catalysed aza-Michael addition reaction at room temperature by grafting N-isopropylacrylamide (NIPAm) moieties onto the amines. The resulting PLL-g-NIPAm exhibited LCST-type thermosensitivity. The LCST can be tuned by the NIPAm content incorporated in the macromolecules. Importantly, depending on the NIPAm content, LCST is highly dependent on pH and ionic strength due to ionization capability of the remaining free lysine residues. PLL-g-NIPAm constitutes a novel biodegradable LCST polymer that could be used as "smart" block in block copolymers and/or terpolymers, of any macromolecular architecture, to design pH/Temperature-responsive self-assemblies (nanocarriers and/or networks) for potential bio-applications. [ABSTRACT FROM AUTHOR]

Abstract: Hyperbranched poly(amido amine)s containing vinyl and hydroxyl groups were successfully synthesized via Michael addition polymerization of triacrylamide (TT) and 3-amino-1,2-propanediol (APD) with equal molar ratio in feed. 1H, 13C and HSQC NMR techniques were used to clarify the structure of hyperbranched polymers and polymerization mechanism. The Michael addition reaction of hyperbranched poly(1TT-1APD)s with primary amine-terminated poly(ε-benzyloxycarbonyl-l-lysine)s [PLys(Z)] yielded a star-like hyperbranched polymers with poly(1TT-1APD) core and Plys(Z) shell. The Z groups in PLys(Z) were removed under acidolysis, and thus star-like hyperbranched polymers with hydroxyl groups inside and primary amine groups outside were obtained successfully. [Copyright &y& Elsevier]

Abstract: A promising application of the multivariate curve resolution based on alternating least squares (MCR-ALS) method is reported for the analysis of the temperature-dependent conformational changes in poly(l-lysine) (PLL). The MCR-ALS approach has enabled detection of three structural components developed during the melting experiment. Most important is an infrared signature of the intermediate component described as a mixture of native α-helical conformation and its extended forms. [Copyright &y& Elsevier]

The potential of novel amphiphilic water soluble and degradable poly(e-caprolactone)-g-poly(L-lysine) as carriers for DNA transfection has been investigated. Two graft copolymers having the same proportion of lysine units but different structures have been synthesized following two grafting techniques. The chemical composition of these copolymers, their expected architectures and their behaviour in aqueous solutions have been studied. The benefits resulting from the use of these degradable polycationic structures as well as their ability to form polyplexes are discussed. Finally, preliminary transfection assays of MCF-7 cells by pRL-TK plasmid using poly(ϵ-caprolactone)-g-poly(L-lysine) copolymers as carriers are reported. [ABSTRACT FROM AUTHOR]

Abstract: The current study compared the effectiveness of an amphiphilic biomaterial poly(l-lysine)-palmitic acid (PLL–PA), and the lipid-based transfection agent Lipofectamine™ 2000 for plasmid delivery to bone marrow stromal cells (BMSC). We investigated the utility of the carriers to deliver a plasmid containing enhanced green fluorescent protein (pEGFP) to BMSC in vitro. Confocal microscopy was used to investigate the intracellular trafficking of pEGFP/carrier complexes. pEGFP delivery and EGFP expression were assessed by flow cytometry. PLL–PA formed condensed structures with pEGFP and successfully delivered the plasmid into the nucleus within 5h of incubation with the cells. PLL–PA delivered the pEGFP to ∼80% of the cells, achieving a maximum transfection efficiency of ∼22%. This was significantly higher than Lipofectamine™ 2000-mediated transfection, which was 11% under most optimal conditions. Dosing the BMSC two or three times during the 24h period increased the transfection efficiency by 2–3 folds, without compromising cell viability. When chloroquine was employed as an ensomolytic agent, 100μm of the drug increased the transfection efficiency while reducing cell viability, but lower concentrations (1–10μm) were not beneficial for transfection. Combining PLL–PA with Lipofectamine™ 2000 created an additive effect, increasing the transfection efficiency of PLL–PA. Long-term evaluation of gene expression with pEGFP/PLL–PA yielded ∼17% transfection on day 1, which gradually decreased over a 12-day period. We conclude that PLL–PA is an effective biomaterial carrier and a promising candidate for non-viral gene delivery to BMSC. [Copyright &y& Elsevier]

Abstract: Electrostatically driven layer-by-layer (LbL) assembly is a simple and robust method for producing structurally tailored thin film biomaterials, of thickness ca. 10nm, containing biofunctional ligands. We investigate the LbL formation of multilayer films composed of polymers of biological origin (poly(l-lysine) (PLL) and dextran sulfate (DS)), the adsorption of fibronectin (Fn)—a matrix protein known to promote cell adhesion—onto these films, and the subsequent spreading behavior of human umbilical vein endothelial cells (HUVEC). We employ optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microgravimetry with dissipation (QCMD) to characterize multilayer assembly in situ, and find adsorbed Fn mass on PLL-terminated films to exceed that on DS terminated films by 40%, correlating with the positive charge and lower degree of hydration of PLL terminated films. The extent and initial rate of Fn adsorption to both PLL and DS-terminated films exceed those onto the bare substrate, indicating the important role of electrostatic complexation between negatively charged protein and positively charged PLL at or near the film surface. We use phase-contrast optical microscopy to investigate the time-dependent morphological changes of HUVEC as a function of layer number, charge of terminal layer, and the presence of Fn. We observe HUVEC to attach, spread, and lose circularity on all surfaces. Positively charged PLL-terminated films exhibit a greater extent of cell spreading than do (negatively charged) DS-terminated films, and spreading is enhanced while circularity loss is suppressed by the presence of adsorbed Fn. The number of layers plays a significant role only for DS-terminated films with Fn, where spreading on a bilayer greatly exceeds that on a multilayer, and PLL-terminated films without Fn, where initial spreading is significantly higher on a monolayer. We observe initial cell spreading to be followed by retraction (i.e. decreased cell area and circularity with time) for films without Fn, and for DS-terminated films with Fn. Overall, the Fn-coated PLL monolayer and the Fn-coated PLL-terminated multilayer are the best performing films in promoting cell spreading. We conclude the presence of Fn to be an important factor (more so than film charge or layer number) in controlling the interaction between multilayer films and living cells, and thus to represent a promising strategy toward in vivo applications such as tissue engineering. [Copyright &y& Elsevier]

Interactions between charged macromolecules (e.g., proteins, nucleic acids, polyelectrolytes) and charged surfaces govern many natural and industrial processes. We investigate here the influence of an applied electric potential on the adsorption of charged polymers, and report the following significant result: the adsorption of certain amine side chain-containing polycations may become continuous, i.e., asymptotically linear (or nearly linear) in time over hours, upon the application of a modest anodic potential. Employing optical waveguide lightmode spectroscopy (OWLS) and an indium tin oxide (ITO) substrate, we show that asymptotic kinetics, and the adsorbed mass at the onset of the asymptotic regime, depend sensitively on polymer chemistry (in particular, side chain volume and charge location), increase with applied potential and ionic strength (conditions favoring a thicker initial layer), and are independent of bulk polymer concentration (suggesting postadsorption events to be rate limiting). X-ray photoelectron spectra reveal a suppressed polymer charge within layers formed via continuous adsorption, but no evidence of electrochemical reactions. We propose a mechanism based on polymer-polymer binding within the adsorbed layer, enabled by suppressed electrostatic repulsion and/or enhanced ionic correlations near the conducting surface, and stabilized by short-range attractive interactions. Continuous adsorption under an applied electric potential offers the possibility of nanoscale films of tailored polymer content realized in a single step. [ABSTRACT FROM AUTHOR]

Abstract: Poly(l-lysine)s, PLLs, are commonly used for DNA compaction and cell transfection. We report that, although PLLs of low (2.9kDa), L-PLL, and high (27.4kDa), H-PLL, Mw in free form and DNA-complexed cannot only cause rapid plasma membrane damage in human cell lines, phosphatidylserine “scrambling” and loss of membrane integrity, but later (24h) initiate stress-induced cell death via mitochondrial permeabilization without the involvement of processed caspase-2. Mitochondrially mediated apoptosis was confirmed by detection of cytochrome c (Cyt c) release, activation of caspases-9 and -3, and subsequent changes in mitochondrial membrane potential. Plasma membrane damage and apoptosis were most prominent with H-PLL. Cytoplasmic level of Cyt c was more elevated following H-PLL treatment, but unlike L-PLL case, inhibition of Bax channel-forming activity reduced the extent of Cyt c release from mitochondria by half. Inhibition of Bax channel-forming activity had no modulatory effect on L-PLL-mediated Cyt c release. Further, functional studies of isolated mitochondria indicate that H-PLL, but not L-PLL, can directly induce Cyt c release, membrane depolarization, and a progressive decline in the rate of uncoupled respiration. Combined, our data suggest that H-PLL and L-PLL are capable of initiating mitochondrially mediated apoptosis differently. The observed PLL-mediated late-phase apoptosis may provide an explanation for previously reported transient gene expression associated with PLL-based transfection vectors. The importance of our data in relation to design of novel and safer cationic non-viral vectors for human gene therapy is discussed. [Copyright &y& Elsevier]

Abstract: Titanium has been a successful implant material owing to its excellent strength to weight ratio, toughness, and bioinert oxide surface. Significant progress has been made on the improvement of titanium''s bioactivity by coating its oxide surface with calcium phosphates and bioactive molecules. Here, we report on the coating of titanium with a poly(l-lysine)-calcium phosphate hybrid material with a nanoscale texture. This hybrid coating was grown by first nucleating seed crystals of calcium phosphate, directly on the Ti surface and then exposing this surface to solutions containing Ca2+, PO4 3−, and poly(l-lysine). The resultant hybrid coating was characterized by electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, thermogravimetric analysis, X-ray photoelectron spectroscopy, and elemental analysis. This material contained 14% by weight poly(l-lysine), and this organic component decreased greatly the dimensions of the surface features, thus enhancing surface area relative to the inorganic control. The highly textured hybrid material was more susceptible than the control to acidic and enzymatic degradation. The amino acid cysteine was covalently linked to the hybrid material, demonstrating the potential of this coating for further functionalization. These hybrid coatings may prove useful in enhancing the bioactivity of titanium. [Copyright &y& Elsevier]

• A conventional interface for plant cell adhesion was created by CV polymerization. • Co-polymerization of 3-hydroxyphenylboric acid with PLL has a synergistic effect. • The dynamic adhesion of rice suspension cells to the interface was monitored by QCM. • Rice cells softened at first and then hardened under the salt stress of 40 mM NaCl. Poly(L-lysine) (PLL)-3-hydroxyphenylboronic acid (3-PBA) composite was prepared onto an Au electrode via cyclic voltammetry (CV), which was characterized by electrochemical and quartz crystal microbalance (QCM) techniques, and used to promote adhesion of plant cells. 3-PBA can interact with glycosyl compounds on the cell wall to form negatively charged covalent compounds while PLL increases the number of positively charged sites, which enhances the electrostatic interactions with the negatively charged cell wall, both promoting cell adhesion. The PLL-poly(3-PBA) composite was successfully modified on the Au electrode surface, as demonstrated by QCM and Fourier transform infrared spectrophotometry (FTIR). Moreover, we confirmed the synergistic effect of PLL and 3-PBA to adhere rice suspension cells using CV, electrochemical impedance spectroscopy (EIS), QCM and optical microscope observations. Finally, the dynamic process of rice cells' adhesion to PLL-poly(3-PBA)/Au followed by salt stress treatment of NaCl was monitored in real-time by QCM. The cells softened at first then hardened under the action of 40 mM NaCl. This work presents a novel and conventional way to immobilize plant cells onto sensors, and has importance in the study of the structure, function and behavior of plants under various stresses at the cellular level. [ABSTRACT FROM AUTHOR]

The deposition of polyelectrolyte (PEL) multilayers (PEMs) of poly(l-lysine)/cellulose sulfate (PLL/CS) onto germanium (Ge) substrates depending on salt concentration (c S ) and deposition step z at constant PEL concentration c PEL = 0.01 M and pH = 7.0 was studied. In situ ATR-FTIR spectroscopy was used for the quantitative determination of alternate PLL/CS deposition profiles (adsorbed amount versus z) and total deposited PEM amount. By varying c S from 0 M to 1.0 M, a maximum of deposited amount was obtained at 0.1 M, so that both no salinity (0 M) and high salinity (1.0 M) revealed deposited amounts that were far lower than for mean salinity (0.1 M). Furthermore, in situ ATR-FTIR allowed to determine the detailed modulation of the PEL composition during the consecutive PEM deposition, which was interpreted as being due to both diffusion of given PEL from the PEM interior towards the outermost region and release of the PEM upon contact with the bulk oppositely charged PEL solution. Finally, ex situ ATR-FTIR measurements on the PEL solutions after deposition of PEM-20 revealed the distinct release of PEL from the PEM solely for c S = 1.0 M, due to the highest mobility of PEL under high salt conditions. These studies help to prepare functional PEM coatings with defined thicknesses and morphologies for the passivation and activation of material surfaces in the biomedical and food field.

• Gene complexation affinity of polycation controls tightness and size of polyplex. • Smaller, tighter polyplex delivered more pDNA into nucleus than larger, looser one. • Larger polyplex had slower cytosolic and nuclear transport of pDNA than smaller one. • Too tighter polyplex made poor gene expression due to poor pDNA release in nucleus. • Tightness and size of polyplex strongly influence polymeric gene transfection. The delivery of plasmid DNA (pDNA) using polycations has been investigated for several decades; however, obstacles that limit efficient gene delivery still hinder the clinical application of gene therapy. One of the major limiting factors is controlling pDNA binding affinity with polymers to control the complexation and decomplexation of polyplexes. To address this challenge, polycations of α-poly(L -lysine) (APL) and ε-poly(L -lysine) (EPL) were used to prepare variable complexation/decomplexation polyplexes with binding affinities ranging from too tight to too loose and sizes ranging from small to large. APL-EPL/ATP-pDNA polyplexes were also prepared to compare the effects of endosomolytic ATP on complexation/decomplexation and the sizes of polyplexes. The results showed that smaller and tighter polyplexes delivered more pDNA into the cells and into the nucleus than the larger and looser polyplexes. Larger polyplexes exhibited slower cytosolic transport and consequently less nuclear delivery of pDNA than smaller polyplexes. Tighter polyplexes exhibited poor pDNA release in the nucleus, leading to no improvement in transfection efficiency. Thus, polyplexes should maintain a balance between complexation and decomplexation and should have optimal sizes for effective cellular uptake, cytosolic transport, nuclear import, and gene expression. Understanding the effects of complexation/decomplexation and size is important when designing effective polymer-based electrostatic gene carriers. [ABSTRACT FROM AUTHOR]

Angiogenesis plays an important role in both soft and hard tissue regeneration, which can be modulated by therapeutic drugs. If nanoparticles (NP) are used as vectors for drug delivery, they have to encounter endothelial cells (EC) lining the vascular lumen, if applied intravenously. Herein the interaction of unloaded polyelectrolyte complex nanoparticles (PECNP) composed of cationic poly(l-lysine) (PLL) and various anionic polysaccharides with human vascular endothelial cells (HUVEC) was analyzed. In particular PECNP were tested for their cell adhesive properties, their cellular uptake and intracellular localization considering composition and net charge. PECNP may form a platform for both cell coating and drug delivery. PECNP, composed of PLL in combination with the polysaccharides dextran sulfate (DS), cellulose sulfate (CS) or heparin (HEP), either unlabeled or labeled with fluorescein isothiocyanate (FITC) and either with positive or negative net charge were prepared. PECNP were applied to human umbilical cord vein endothelial cells (HUVEC) in both, the volume phase and immobilized phase at model substrates like tissue culture dishes. The attachment of PECNP to the cell surface, their intracellular uptake, and effects on cell proliferation and growth behavior were determined. Immobilized PECNP reduced attachment of HUVEC, most prominently the systems PLL/HEP and PLL/DS. A small percentage of immobilized PECNP was taken up by cells during adhesion. PECNP in the volume phase showed no effect of the net charge sign and only minor effects of the composition on the binding and uptake of PECNP at HUVEC. PECNP were stored in endosomal vesicles in a cumulative manner without apparent further processing. During mitosis, internalized PECNP were almost equally distributed among the dividing cells. Both, in the volume phase and immobilized at the surface, PECNP composed of PLL/HEP and PLL/DS clearly reduced cell proliferation of HUVEC, however without an apparent cytotoxic effect, while PLL/CS composition showed minor impairment. PECNP have an anti-adhesive effect on HUVEC and are taken up by endothelial cells which may negatively influence the proliferation rate of HUVEC. The negative effects were less obvious with the composition PLL/CS. Since uptake and binding for PLL/HEP was more efficient than for PLL/DS, PECNP of PLL/HEP may be used to deliver growth factors to endothelial cells during vascularization of bone reconstitution material, whereas those of PLL/CS may have an advantage for substituting biomimetic bone scaffold material.

Narrowly distributed poly(l-lysine- b - N -isopropylacrylamide) (PLL- b -PNIPAM) was prepared through ring-opening polymerization of ε-benzyloxycarbonyl-l-lysine N -carboxy-α-amino anhydride and atom transfer radical polymerization of NIPAM, followed with the removal of ε-benzyloxycarbonyl group. Then gold nanoparticles (AuNPs) grafted with PLL- b -PNIPAM (PNIPAM-PLL-AuNPs) were obtained by the reduction of chloroauric acid with sodium citrate in the presence of PLL- b -PNIPAM. PNIPAM-PLL-AuNPs and its precursors were thoroughly characterized by proton magnetic resonance spectroscope, Fourier transform infrared spectroscope, UV-vis spectroscope, transmission electron microscopy, dynamic light scattering, thermogravimetric analysis, and circular dichroism. The obtained PNIPAM-PLL-AuNPs exhibited high colloid stability even at strong alkaline (pH = 12) and acidic (pH = 2) conditions. The thermal and pH dual-responsive behaviors of the grafting PLL- b -PNIPAM chains was observed to be affected by AuNPs, while not for the secondary structure of PLL chains. Correspondingly, the surface plasmon resonance (SPR) of AuNPs was found to be sensitive to both pH value and temperature. A blue shift in the SPR happened both with increasing pH value and increasing temperature. The stimuli-response was reversible in heating-cooling cycles. The gold nanoparticles with both pH and temperature response may have potential applications in biomedical areas and biosensors.