Poly L-lysine Layer Research Articles

Nanofluidic diodes based on asymmetric bio-inspired surface coatings in straight glass nanochannels.

In this study, we present nanofluidic diodes fabricated from straight glass nanochannels and functionalized using bio-inspired polydopamine (PDA) and poly-L-lysine (PLL) coatings. The resulting PDA coatings are shown to be asymmetric due to a combination of transport considerations which can be leveraged to provide a measure of control over the effective channel geometry. By subsequently introducing a layer of amine-bearing PLL chains covalently bound to the PDA, we enhance heterogeneities in the charge and ion distributions within the channel and enable significant current rectification between forward-bias and reverse-bias modes; our PDA-PLL-coated channels yielded a rectification ratio greater than 1000 in a 100 nm channel filled with 0.01× phosphate-buffered saline solution (PBS). We further demonstrated that at higher ionic strength conditions, reducing the solution pH increased the number of protonated amines within the PLL layer, amplifying the charge disparities along the channel and leading to greater rectification. As nanofluidic diodes with bipolar surface charge distributions tend to provide superior performance compared to those with a single wall charge polarity, we imposed a more bipolar charge distribution in our devices by partially coating our PDA-PLL-coated channels with negatively charged polyacrylic acid (PAA). These enhanced bipolar channels exhibited greater current rectification than the PDA-PLL-coated channels, reaching rectification ratios in excess of 100 even in more physiologically-relevant 1× PBS solutions. Our fabrication approach and the results herein provide a promising platform from which the scientific community can build upon in the relentless endeavor for improved sensitivity in biosensors and other analytical devices.

Differentiation of Alginate-Encapsulated Wharton Jelly-Derived Mesenchymal Stem Cells into Insulin Producing Cells.

Objective Insulin insufficiency due to the reduced pancreatic beta cell number is the hallmark of diabetes, resulting in an intense focus on the development of beta-cell replacement options. One approach to overcome the problem is to search for alternative sources to induce insulin-producing cells (IPCs), the advent of mesenchymal stem cells (MSCs) holds great promise for producing ample IPCs. Encapsulate the MSCs with alginate improved anti-inflammatory effects of MSC treatment. This study aimed to evaluate the differentiation of wharton jelly-derived MScs into insulin producing cells using alginate encapsulation. Materials and Methods In this experimental study, we established an efficient IPCs differentiation strategy of human MSCs derived from the umbilical cord’s Wharton jelly with lentiviral transduction of Pancreas/duodenum homeobox protein 1 (PDX1) in a 21-day period using alginate encapsulation by poly-L-lysine (PLL) and poly-L-ornithine (PLO) outer layer. During differentiation, the expression level of PDX1 and secretion of insulin proteins were increased. Results Results showed that during time, the cell viability remained high at 87% at day 7. After 21 days, the differentiated beta-like cells in microcapsules were morphologically similar to primary beta cells. Evaluation of the expression of PDX1 and INS by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) on days 7, 14 and 21 of differentiation exhibited the highest expression on day 14. At the protein level, the expression of these two pancreatic markers was observed after PDX1 transduction. Results showed that the intracellular and extracellular insulin levels in the cells receiving PDX1 is higher than the control group. The current data showed that encapsulation with alginate by PLL and PLO outer layer permitted to increase the microcapsules’ beta cell differentiation. ConclusionEncapsulate the transduced-MSCs with alginate can be applied in an in vivo model in order to do the further analysis.

Ag‐Coupled Polymeric Nanohybrids with Synergistic Photodynamic and Photothermal Activities for Advanced Antibacterial Therapy

AbstractConsidering the implications of inherent resistance of pathogenic bacteria, it is important to design a new generation of nonchemotherapic nanoagents for effective antibacterial therapy. Herein, we illustrate a one‐step reprecipitation‐encapsulation strategy to incorporate hydrophobic photosensitizers (ZnPc) and photothemal agents (dopamine hydrochloride) with a cationic poly‐L‐lysine (PLL) layer. Upon light irradiation, the polymeric nanohybrid exhibits high photo‐to‐thermal conversion efficiency (30.3%) and effective 1O2 generation capability. To further enhance the antibacterial efficiency, Ag nanoparticle, a typical broad‐spectrum antibacterial agent, was decorated on the surface of PLL layer by in‐situ reduction. The as‐synthesized nanohybrids, referred to as ZnPc/DA@Ag, achieved a robust photodynamic/photothermal/Ag antibacterial capability without apparent systemic toxicity, thus holding great potential for broad‐spectrum eradication of pathogenic bacteria.

Enhancement of Probe Density in DNA Sensing by Tuning the Exponential Growth Regime of Polyelectrolyte Multilayers.

Surface-based biosensing devices benefit from a dedicated design of the probe layer present at the transducing interface. The layer architecture, its physicochemical properties, and the embedding of the receptor sites affect the probability of binding the analyte. Here, the enhancement of the probe density at the sensing interface by tuning the exponential growth regime of polyelectrolyte multilayers (PEMs) is presented. PEMs were made of poly-l-lysine (PLL), with appended clickable dibenzocyclooctyne (DBCO) groups and oligo(ethylene glycol) chains, and poly(styrene sulfonate) (PSS). The DNA probe loading and target hybridization efficiencies of the PEMs were evaluated as a function of the PLL layer number and the growth regime by a quartz crystal microbalance (QCM). An amplification factor of 25 in the target DNA detection was found for a 33-layer exponentially grown PEM compared to a monolayer. A Voigt-based model showed that DNA probe binding to the DBCO groups is more efficient in the open, exponentially grown films, while the hybridization efficiencies appeared to be high for all layer architectures. These results show the potential of such engineered gel-like structures to increase the detection of bio-relevant analytes in biosensing systems.

Proliferation and osteogenic differentiation of human mesenchymal stem cells on cross‐linked polypeptide multilayer films

Polyelectrolyte multilayer film is an emerging method for substrate coating to regulate cellular behaviors. This material allows for modulation of thickness, stiffness, and adhesiveness. In this study, human mesenchymal stem cells (hMSCs) were cultured on polypeptide multilayer films built up by the alternate deposition of cationic poly‐L‐lysine (PLL) and anionic poly‐L‐glutamic acid (PLGA), cross‐linked with EDC/sulfo‐NHS, and coated with type I collagen. The in‐vitro degradation of the cross‐linked PLL/PLGA films in the presence or absence of hMSCs were followed by fluorimetry after labeling of the outermost PLL layer with FITC. Cell proliferation was measured by CyQuant proliferation assay and osteogenic differentiation was evaluated by q‐PCR of osteogenic specific genes such as ALP, OCN, and OPN. The results showed that these cross‐linked films, with or without hMSCs on top of them, were quite stable in the culture medium and not subjected to degradation at least for a few weeks. While hMSCs maintained similar attachment and spreading behavior regardless of layer number (1–6 layers) of films, they demonstrated better proliferation rate on even number layer of films, where the top layers are PLGA films. In addition, compared with cells cultured on polystyrene culture wells and treated with osteogenic induction medium, hMSCs cultured on 6‐layer cross‐linked PLL/PLGA films showed a significant increase in the osteogenic differentiation rate. Our observations indicate a feasible way to manipulate stem cell behavior on polypeptide multilayer films by changing the number of layers, the outermost polypeptide layer, and the concentration of cross‐linkers.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Adsorption Kinetics and Reversibility of Linear Plasmid DNA on Silica Surfaces: Influence of Alkaline Earth and Transition Metal Ions

A quartz crystal microbalance with dissipation monitoring is used to study the adsorption of linear plasmid DNA on silica surfaces and silica surfaces coated with poly-L-lysine (PLL) in solutions containing either alkaline earth (calcium and magnesium) or transition (cobalt, copper, and zinc) metals. Our results show that electrostatic attraction alone does not fully explain the significantly higher adsorption rate of DNA on the positively charged PLL layer in Cu(2+) solution compared to solutions containing Ca(2+), Mg(2+), Co(2+), or Zn(2+). Diffusion coefficients measured by dynamic light scattering reveal that the compactness of plasmid DNA molecules is greater in solutions containing Cu(2+) compared to that of DNA in other divalent electrolyte solutions. When the adsorption rate of plasmid DNA on silica is normalized to the corresponding adsorption rate on PLL-coated surfaces at the same solution condition, the attachment (adsorption) efficiencies are about 0.01 for Ca(2+) or Mg(2), but close to unity for Co(2+), Cu(2+), or Zn(2+). Results from viscoelastic modeling of adsorbed DNA layers suggest that the DNA layer formed in Cu(2+) solutions is thicker and more viscous compared to that formed in Co(2+) solutions. This study demonstrates that plasmid DNA has a strong affinity to Cu(2+), which results in a more compact conformation of DNA molecules compared to the case with the other divalent cations investigated.

Non-invasive evaluation of alginate/poly- l-lysine/alginate microcapsules by magnetic resonance microscopy

In this report, we present data to demonstrate the utility of 1H MR microscopy to non-invasively examine alginate/poly- l-lysine/alginate (APA) microcapsules. Specifically, high-resolution images were used to visualize and quantify the poly- l-lysine (PLL) layer, and monitor temporal changes in the alginate gel microstructure during a month long in vitro culture. The thickness of the alginate/PLL layer was quantified to be 40.6±6.2 μm regardless of the alginate composition used to generate the beads or the time of alginate/PLL interaction (2, 6, or 20 min). However, there was a notable difference in the contrast of the PLL layer that depended upon the guluronic content of the alginate and the alginate/PLL interaction time. The T 2 relaxation time and the apparent diffusion coefficient (ADC) of the alginate matrix were measured periodically throughout the month long culture period. Alginate beads generated with a high guluronic content alginate demonstrated a temporal decrease in T 2 over the duration of the experiment, while ADC was unaffected. This decrease in T 2 is attributed to a reorganization of the alginate microstructure due to periodic media exchanges that mimicked a regular feeding regiment for cultured cells. In beads coated with a PLL layer, this temporal decrease in T 2 was less pronounced suggesting that the PLL layer helped maintain the integrity of the initial alginate microstructure. Conversely, alginate beads generated with a high mannuronic content alginate (with or without a PLL layer) did not display temporal changes in either T 2 or ADC. This observation suggests that the microstructure of high mannuronic content alginate beads is less susceptible to culture conditions.

Self-assembled monolayers of alkanethiols on gold modulate electrophysiological parameters and cellular morphology of cultured neurons

Self-assembled monolayers (SAMs) of ω-substituted alkanethiols on gold have been explored as well defined in vitro model surfaces for the investigation of neuronal growth and function. When used as cell culture substrates, surfaces with monolayers functionalized with terminal −COOH groups support neuron attachment and growth even without an intermediate protein layer. Addition of a poly- l-lysine layer (PLL) to the −COOH terminated monolayers significantly increases total neurite outgrowth. Mixed monolayers containing −COOH and −CH 3 terminal groups in 1:10 and 1:100 ratios poorly support neuron adhesion and preclude neurite extension. A layer of PLL improves the ability of mixed monolayer surfaces to support neuronal growth in culture. The morphology of cultured neurons depends on the chemical composition of SAMs on the support surface. Using glass microelectrode intracellular recording, the properties of cell culture substrates modulate the dynamic properties of action potentials of cultured neurons. These findings provide insight into the cellular responses of excitable cells to the chemical details of a surface and, thus, may help direct the rational design of biologically active materials.

Microencapsulation of Cells Producing Therapeutic Proteins: Optimizing Cell Growth and Secretion

Microencapsulation of genetically engineered cells may have important applications as delivery systems for therapeutic proteins. However, optimization of the microcapsules with regard to mechanical stability, cell growth, and secretion of proteins is necessary in order to evaluate the future use of this delivery technology. We have explored the growth, survival, and secretion of therapeutic proteins from 293-EBNA cells producing endostatin (293 endo cells) and JJN3 myeloma cells producing hepatocyte growth factor (HGF) that have been embedded in various types of alginate capsules. Parameters that affect capsule integrity such as homogenous and inhomogenous gel cores and addition of an outer poly-L-lysine (PLL)-alginate coating were evaluated in relation to cell functions. When cells were encapsulated, the PLL layer was found to be absolutely required for the capsule integrity. The JJN3 and 293 endo cells displayed completely different growth and distribution patterns of live and dead cells within the microcapsules, as shown by 3D pictures reconstructed from images taken with confocal laser scanning microscopy (CLSM). Encapsulated JJN3 cells showed a bell-shaped growth and HGF secretion curve over a time period of 5 months. The 293 endo cells reached a plateau phase in growth after 23 days postencapsulation; however, after around 30 days a fraction of the microcapsules started to disintegrate. Microcapsule disintegration occurred with time irrespective of capsule and cell type, showing that alginate microcapsules possessing relatively high gel strength are not strong enough to keep proliferating cells within the microcapsules for prolonged time periods. Although this study shows that the stability of an alginate-based cell factory can be increased by a PLL-alginate coating, further improvement is necessary with regard to capsule integrity as well as controlling the cell growth before this technology can be used for therapy.

Structural Characterization of Multilayered DNA and Polylysine Composite Films: Influence of Ionic Strength of DNA Solutions on the Extent of DNA Incorporation

Thin films of alternating DNA and poly-l-lysine (PLL) layers were fabricated onto various planar surfaces by the layer-by-layer (LbL) self-assembly technique. For both the polydeoxynucleotide (PDN, polyguanylic acid or poly[G]) and the oligodeoxynucleotide (ODN, a 30-mer) investigated, UV−visible spectrometry and contact angle measurements showed that a uniform layer of DNA can be fully adsorbed onto each alternate PLL layer. Various parameters affecting the DNA loading into the composite film were investigated with a particular emphasis placed on the effect of the ionic strength in the DNA solution used for the film preparation. For the PLL/poly[G] film, it was found that 150 μg/mL of poly[G] solution containing 0.5 M NaCl attains the maximum loading for every poly[G] layer. Atomic force microscopy was utilized to measure the DNA surface density and structure at the topmost layers of several representative composite films. While the DNA film thickness increases with the ionic strength of the DNA solution...

Poly-L-Lysine Induces Fibrosis on Alginate Microcapsules via the Induction of Cytokines.

Alginate - poly-l-lysine (PLL) microcapsules can be used for transplantation of insulin-producing cells for treatment of type I diabetes. In this work we wanted to study the inflammatory reactions against implanted microcapsules due to PLL. We have seen that by reducing the PLL layer, less overgrowth of the capsule is obtained. By incubating different cell types with PLL and afterwards measuring cell viability with MTT, we found massive cell death at concentrations of PLL higher than 10 μg/ml. Staining with annexin V and propidium iodide showed that PLL induced necrosis but not apoptosis. The proinflammatory cytokine, tumor necrosis factor (TNF), was detected in supernatants from monocytes stimulated with PLL. The TNF response was partly inhibited with antibodies against CD14, which is a well-known receptor for lipopolysaccharide (LPS). Bactericidal permeability increasing protein (BPI) and a lipid A analogue (B-975), which both inhibit LPS, did not inhibit PLL from stimulating monocytes to TNF production. This indicates that PLL and LPS bind to different sites on monocytes, but because they both are inhibited by a p38 MAP kinase inhibitor, they seem to have a common element in the signal transducing pathway. These results suggest that PLL may provoke inflammatory responses either directly or indirectly through its necrosis-inducing abilities. By combining soluble PLL and alginate both the toxic and TNF-inducing effects of PLL were reduced. The implications of these data are to use alginate microcapsules with low amounts of PLL for transplantation purposes.