Polypeptide Films Research Articles

Molecular mechanisms of pH-tunable stability and surface coverage of polypeptide films

Streaming potential and quartz crystal microbalance measurements, combined with all-atom molecular dynamics simulations, were used to study the pH dependency of the adsorption of two basic homopolypeptides, poly-L-lysine (PLL) and poly-L-arginine (PARG), on α-quartz surface. We report that the observed adsorption behavior rises from an interplay of i) the change in the number of possible peptide-surface ion pairs between the charged moieties and ii) repulsive electrostatic interactions between the polypeptide molecules. For low pH values, polypeptide adsorption was strongest and stable monolayers were formed. However, electrostatic repulsion between the polypeptides led to a relatively low maximum surface coverage. On the other hand, higher pH led to more weakly bound, but significantly denser, peptide films with limited stability. Simulations indicate that electrostatic interactions are the main driving force for adsorption, while hydrogen bonding and non-specific interactions also contribute. Additionally, the important role of the counterions of the negatively charged quartz surface that form a positively charged ion adlayer is highlighted. Ion release of the condensed sodium ions at the charged surface occurs via displacement by polypeptide adsorption. The mechanisms revealed by this work provide systematic guidelines to engineering active surfaces of charged peptides with controlled surface coverage and reversible binding.

Vernetzte Polypeptide durch RAFT‐vermittelte Polymerisation zum kontinuierlichen Aufbau von Polymerfilmen

AbstractPolypeptidbeschichtungen spielen aufgrund ihres großen biologischen Potentials eine wichtige Rolle in der Oberflächenmodifizierung von Materialien. Da ihre Eigenschaften durch ihren strukturellen Aufbau mitbestimmt werden, ist eine Kontrolle der Struktur durch entsprechende Herstellungsstrategien notwendig. In diesem Artikel berichten wir über eine einfache Methode zur präzisen Herstellung von dicht vernetzten Polypeptidfilmen mit ungewöhnlich hoher β‐Faltblatt – und Zufallsknäuel Konformation durch eine kontinuierliche Assemblierungspolymerisation (CAP, continuous assembly polymerization) mittels reversibler Addtitions‐Fragmentierungs‐Kettenübertragungspolymerisation (RAFT Polymerisation, reversible‐addition‐fragmentation chain‐transfer polymerization), kurz CAP‐RAFT. CAP‐RAFT wurde bereits grundlegend mit methacryliertem Poly‐l‐Lysin (PLLMA) und methacrylierter Poly‐l‐Glutaminsäure (PLGMA) untersucht. Eine sorgfältige Anpassung der Methode führte hier zu Filmen mit einer Schichtdicke von bis zu 36.1±1.1 nm, die durch mehrfache Anwendung dieser Strategie auf 94.9±8.2 nm erhöht werden konnte. PLLMA‐ und PLGMA‐Filme wiesen überraschenderweise bis zu 30–50 % Zufallsknäuel Konformation auf. Durch den enzymatischen Abbau mit einem Protease‐Mix, der Proteasen im Wundheilungsmileu repräsentiert, konnte das Potential diesen Strukturen für Anwendungen im Wirkstofftransport und in der Gewebezüchtung gezeigt werden.

Crosslinked Polypeptide Films via RAFT-Mediated Continuous Assembly of Polymers.

Polypeptide coatings are a cornerstone in the field of surface modification due to their widespread biological potential. As their properties are dictated by their structural features, subsequent control thereof using unique fabrication strategies is important. Herein, we report a facile method of precisely creating densely crosslinked polypeptide films with unusually high random coil content through continuous assembly polymerization via reversible addition–fragmentation chain transfer (CAP‐RAFT). CAP‐RAFT was fundamentally investigated using methacrylated poly‐l‐lysine (PLLMA) and methacrylated poly‐l‐glutamic acid (PLGMA). Careful technique refinement resulted in films up to 36.1±1.1 nm thick which could be increased to 94.9±8.2 nm after using this strategy multiple times. PLLMA and PLGMA films were found to have 30–50 % random coil conformations. Degradation by enzymes present during wound healing reveals potential for applications in drug delivery and tissue engineering.

Molecular Mechanisms of Ph-Tunable Stability and Surface Coverage of Polypeptide Films

Molecular Mechanisms of Ph-Tunable Stability and Surface Coverage of Polypeptide Films

Cross‐Linked Polypeptide Multilayer Films Facilitates the Cardiac Differentiation of Mouse iPSCs

Well‐designed biomaterials, which mimic physiological microenvironment and mechanical properties, can guide stem cell differentiation. Polyelectrolyte multilayer films allow for modulation of thickness, stiffness, and adhesiveness and is a useful method for substrate coating to modulate cellular behaviors. In this study, mouse induced pluripotent stem cells (iPSCs) were cultured on polypeptide multilayer films built up by the alternate deposition of cationic poly‐L‐lysine and anionic poly‐L‐glutamic acid, cross‐linked with EDC/sulfo‐NHS, and coated with gelatin. The characteristics of film thickness, surface roughness, and adsorbed film mass were measured by AFM and QCM. Cardiomyocyte differentiation was evaluated by RT‐PCR analysis of cardiac markers, cardiac troponin T (cTnT) and myosin light chain‐2a (MLC‐2a). After embryonic bodies of iPSCs were cultured for 3 days and then induced for cardiac differentiation for 0, 3, and 6 days, compared with iPSCs on culture dish, iPSCs on 6‐layer cross‐linked polypeptide films demonstrates significantly higher expressions of both cTnT and MLC‐2a on day 3 and day 6. Our results indicate a possible way to facilitate iPSCs toward cardiomyocyte differentiation on polypeptide multilayer films.Support or Funding InformationThis work was supported by the Ministry of Science and Technology, Taiwan. (Grant number: NSC 102‐2628‐B‐010‐010‐MY3).

Synthesis of antireflective silica coatings through the synergy of polypeptide layer-by-layer assemblies and biomineralization.

We report a versatile approach to synthesize silica coatings with antireflective (AR) characteristics through the combination of a layer-by-layer (LbL) assembly technique and biomineralization. LbL assembled decanoyl-modified poly(l-lysine)/poly(l-glutamic acid) (PLL-g-Dec/PLGA) multilayer films were used as templates for silica mineralization, followed by calcination. The specific deposition of silica onto the LbL polypeptide assemblies through amine-catalyzed polycondensation resulted in silica coatings that exhibited the transcription of the nano-/microstructured polypeptide films and their film thickness and porosity can be tuned by varying the number of bilayers, degree of substitution, and PLL molecular weight. AR silica coatings exhibiting more than 6% increase in transmittance in the near UV/visible spectral range can be obtained at an optimized refractive index, thickness, and surface roughness. The abrasion test showed that the silica coatings exhibited sufficient structural durability due to continuous silica nanostructures and low surface roughness. This study demonstrated that nanostructured thin films can be synthesized for AR coatings using the synergy between the LbL assembly technique and biomineralization.

ChemInform Abstract: Polypeptide Films via N‐Carboxyanhydride Ring‐Opening Polymerization (NCA‐ROP): Past, Present and Future

AbstractReview: 101 refs.

Microneedles from fish scale biopolymer

ABSTRACTThis article reports on microneedles produced from biopolymer films extracted from fish scales of tilapia (Oreochromiss sp.) using micromolding technique. Evaluation of the properties of polypeptide films prepared from the fish scales gave refractive index (1.34), protein concentration (78%), ash content (1.6%) at (22%) moisture content. The microneedles successfully inserted into artificial skin models and imaging using digital camera showed microneedles remained intact when inserted and when removed from the skin model. Microneedles also successfully inserted into porcine skin and were shown to dissolve gradually at 0 s, 60 s, 120 s, and 180 s after insertion. Microneedles containing methylene blue as model drug were also produced and successfully pierced porcine skin. 3D finite element (FEM) simulations were performed using the measured mechanical properties of the biopolymer films (Young's modulus 0.23 N/mm2 and tensile strength 1.8105 N/mm2) to evaluate the stress distribution on various dimensions of the fish scale derived microneedles and hence, their ability to withstand force necessary to pierce the skin without fracture. Results from mechanical analysis using FEM showed that microneedles with tip radius between 10 and 100 μm could withstand up to 0.12 N of force per microneedle without fracture, which is indicated when the stress at the tip of the microneedle exceeds the ultimate stress of the material of fabrication. Using skin insertion tests and finite element simulations, this study provides evidence that microneedles fabricated from fish scale biopolymer can effectively pierce and degrade into skin and therefore are good candidate for transdermal applications. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40377.

Polypeptide films via N-carboxyanhydride ring-opening polymerization (NCA-ROP): past, present and future

The formation of surface-grafted polypeptide films and interfaces via N-carboxyanhydride ring-opening polymerization (NCA-ROP) holds great potential for the facile preparation of next-generation multifunctional and responsive materials with excellent biocompatibility, biodegradability, tunable conformations and chemical functionalities. Therefore, the aim of this feature article is to provide a topical review of developments in the area of polypeptide films derived through NCA-ROP. It is evident that studies reported thus far have only started to unveil the full potential of peptide-based interfaces and materials, and with continued advancements it is anticipated that the strategic combination of NCA-ROP with modern synthetic chemistries will continue to yield versatile platforms for broader applications in the fields of polymer therapeutics, tissue engineering, (bio)nanocoatings, (bio)chemosensors, catalysis and separation technologies.

Polymerization: Assembly of Free‐Standing Polypeptide Films via the Synergistic Combination of Hyperbranched Macroinitiators, the Grafting‐From Approach, and Cross‐Chain Termination (Adv. Mater. 33/2013)

Polymerization: Assembly of Free‐Standing Polypeptide Films via the Synergistic Combination of Hyperbranched Macroinitiators, the Grafting‐From Approach, and Cross‐Chain Termination (Adv. Mater. 33/2013)

Morphology and properties of hydrophobic polypeptide-based film improved by hydrophilic poly(vinyl alcohol)

Poly(γ-benzyl l-glutamate)/poly(vinyl alcohol) (PVA) blend films were prepared by a solution casting method. Surface morphologies of the polymer blend films were investigated by atomic force microscopy and scanning electron microscopy. Thermal, mechanical, and physical properties of the polymer blend films were studied by differential scanning calorimetry, tensile tests, and contact angle tests. It was revealed that the introduction of PVA could exert pronounced effects on the morphologies and the properties of the polypeptide films.

Assembly of Free‐Standing Polypeptide Films via the Synergistic Combination of Hyperbranched Macroinitiators, the Grafting‐From Approach, and Cross‐Chain Termination

Cross-linked polypeptide-based films are fabricated via a novel and robust method employing surface-initiated ring opening polymerization of α-amino acid N-carboxyanhydrides (NCA-ROP). The judicious combination of amine-based hyperbranched macroinitiators and benzyl ester-protected NCA derivatives promotes network formation by cross-chain terminations, which allows the formation of stable cross-linked peptide-based capsules in a one-pot system.

Plasmodium falciparum synthetic LbL microparticle vaccine elicits protective neutralizing antibody and parasite-specific cellular immune responses

Epitopes of the circumsporozoite (CS) protein of Plasmodium falciparum, the most pathogenic species of the malaria parasite, have been shown to elicit protective immunity in experimental animals and human volunteers. The mechanisms of immunity include parasite-neutralizing antibodies that can inhibit parasite motility in the skin at the site of infection and in the bloodstream during transit to the hepatocyte host cell and also block interaction with host cell receptors on hepatocytes. In addition, specific CD4+ and CD8+ cellular mechanisms target the intracellular hepatic forms, thus preventing release of erythrocytic stage parasites from the infected hepatocyte and the ensuing blood stage cycle responsible for clinical disease. An innovative method for producing particle vaccines, layer-by-layer (LbL) fabrication of polypeptide films on solid CaCO3 cores, was used to produce synthetic malaria vaccines containing a tri-epitope CS peptide T1BT* comprising the antibody epitope of the CS repeat region (B) and two T-cell epitopes, the highly conserved T1 epitope and the universal epitope T*. Mice immunized with microparticles loaded with T1BT* peptide developed parasite-neutralizing antibodies and malaria-specific T-cell responses including cytotoxic effector T-cells. Protection from liver stage infection following challenge with live sporozoites from infected mosquitoes correlated with neutralizing antibody levels. Although some immunized mice with low or undetectable neutralizing antibodies were also protected, depletion of T-cells prior to challenge resulted in the majority of mice remaining resistant to challenge. In addition, mice immunized with microparticles bearing only T-cell epitopes were not protected, demonstrating that cellular immunity alone was not sufficient for protective immunity. Although the microparticles without adjuvant were immunogenic and protective, a simple modification with the lipopeptide TLR2 agonist Pam3Cys increased the potency and efficacy of the LbL vaccine candidate. This study demonstrates the potential of LbL particles as promising malaria vaccine candidates using the T1BT* epitopes from the P. falciparum CS protein.

Modification of hydrophobic polypeptide-based film by blending with hydrophilic poly(acrylic acid)

In this study, a series of poly(γ-benzyl L-glutamate)/poly(acrylic acid) (PBLG/PAA) polymer blend films were prepared by casting the polymer blend solution in dimethylsulfoxide (DMSO). The structure and morphology of the polymer blend film were investigated by Fourier Transform Infrared Spectroscopy (FT-IR) and Scanning Electron Microscopy (SEM). Thermal, mechanical, and chemical properties of PBLG/PAA polymer blend films were studied by Differential Scanning Calorimetry (DSC), Thermogravimetric (TG) Analysis, Tensile Tests, and measurements of Surface Contact Angles. The results revealed that the introduction of PAA could exert great effects on the structure and properties of the polypeptide films.

Effect of dioxane molecules on the hydration shell of polypeptides

Our interest in modifying influence of the low-molecular organic compounds on the hydration shell of biopolymers is due to a well-known fact that the former can act as regulators of the enzymatic activity changed the hydration shell of the latter. Dioxane was chosen because of its wide application in non-aqueous biocatalysis. In the present study we investigate the mechanisms of the dioxane influence on water of the first hydration layer of the model polypeptides by FTIR-spectroscopy during simultaneous sorption of water-dioxane vapors into the polypeptide films at low water activity. It was found that modification of the hydration shell of the studied polypeptides was mainly due to the indirect ordering ofpolypeptide secondary structure at penetration of dioxane molecules at these conditions.

Thermoresponsive multilayer films based on ionic elastin-like recombinamers

Thermoresponsive multilayer films consisting of ionic elastin-like recombinamers (ELRs) were prepared using a layer-by-layer technique on model substrates. The ELR polymer containing lysine units served as a polycation and the one with glutamic acid served as a polyanion. The build up process was followed by UV/Vis absorption spectroscopy and atomic force microscopy (AFM). The film formation was optimized by measuring the adsorption at various pH values. The application of monodisperse recombinamers of similar main chain with precisely controlled, evenly distributed and equidistanced charged moieties lead to the formation of stable films at biologically relevant pH values (very efficiently at pH = 7), in spite of the limited number of charged groups in these polypeptides. The fabricated films were found to be temperature-responsive around the physiological temperature, as shown using AFM, spectroscopic ellipsometry and contact angle measurements. This phenomenon increases the resistance of such polypeptide films to salty solutions, which is important for their potential applications as biocompatible nanocoatings.

Totally synthetic nanoparticle vaccines produced via layer-by-layer assembly of polypeptide films induce protective T-cell responses (52.14)

Abstract Nanoparticle vaccines were synthesized using layer-by-layer (LbL) deposition of oppositely-charged polypeptides to build up an artificial film on CaCO3 nanocores. Designed peptides (DP) containing antigenic epitopes of interest were incorporated into the layers of the film, yielding nanoparticles that delivered the DP payload to dendritic cells via phagocytosis. LbL nanoparticles induced DC maturation without triggering secretion of inflammatory cytokines including TNFα and IL-6. LbL nanoparticle delivery of DP to DC resulted in potent cross-presentation to CD8+ T-cells and more efficient presentation to CD4+ T-cells compared to presentation of soluble peptide. Mice immunized with a single dose of LbL nanoparticles without adjuvant mounted vigorous T-cell responses detectable by IFNγ and IL-4 ELISPOT and in vivo CTL assay. Mice immunized with nanoparticles containing ovalbumin-derived DP were protected from challenge with Listeria monocytogenes ectopically expressing ovalbumin. Similar to the in vitro DC maturation results, immunization with LbL nanoparticles did not induce inflammatory symptoms locally (injection site reaction) or systemically (serum cytokine spike). The potency and efficacy of polypeptide LbL nanoparticles administered in aqueous suspension without adjuvant or other formulation additive, and the absence of overt inflammatory responses, suggest that this strategy may be useful in producing novel vaccines against multiple diseases.

Tyrosine side chains as an electrochemical probe of stacked β-sheet protein conformations

The in vivo formation of β-pleated protein aggregates underlies a number of fatal neurodegenerative disorders, such as Alzheimer disease. Since molecular mechanisms of protein misfolding and aggregation remain poorly understood, this has been calling for many diverse biophysical tools capable of addressing different dynamic and conformational aspects of the phenomenon. The two model polypeptides used in this study are poly( l-tyrosine) and insulin. According to FT-IR spectra, poly( l-tyrosine) produced two distinct types of films with dominant either disordered or antiparallel β-sheet conformations depending on carrier solvent used for film's deposition. Electrochemical analysis of both the types of polypeptide films by the means of cyclic voltammetry and differential pulse voltammetry proved that different electrochemical behaviour of the tyrosine residues is determined by the conformation of polypeptide chains. We have rationalized this difference in terms of varying electrochemical accessibility of Tyr residues in each structure. We have also carried out spectral and electrochemical characterization of insulin β-sheet-rich amyloid fibrils. It appears that the detectable electrochemical response of the protein stems from the presence of four tyrosine residues per insulin monomer. Since hydrophobic residues, among them tyrosines play an important role in the formation of protein amyloid fibrils, but, on a molecular level, may be also critical in explaining neurotoxic properties of aggregates, their electrochemical properties may become a very valuable complementary tool in biophysical studies on protein misfolding.

Biomimetic deposition of hydroxyapatite on a synthetic polypeptide with β sheet structure in a solution mimicking body fluid

Deposition of a hydroxyapatite layer with similar structure to bone mineral is an attractive approach to the fabrication of bioactive coating layers to achieve direct bonding to living bone. To get successful coating of a hydroxyapatite layer on an organic polymer using a biomimetic solution, it is essential to find organic substrates that can effectively induce heterogeneous nucleation of hydroxyapatite after exposure to the body environment. Our previous study showed that sericin, a type of silk protein, has the ability to induce hydroxyapatite nucleation in a biomimetic solution when the sericin has a beta sheet structure. To confirm the effectiveness of the beta sheet structure in hydroxyapatite nucleation, we focused on investigating hydroxyapatite deposition on a synthetic polypeptide with a beta sheet structure in a biomimetic solution. The beta sheet forming polypeptides with and without carboxyl groups, poly(FE)(3)FG, poly(FQ)(3)FG, poly(LE)(3)LG and poly(LQ)(3)LG, were synthesized in this study. All the polypeptides had mainly beta sheet structure. After soaking the polypeptide films in 1.5SBF, which has 1.5 times the inorganic ion concentrations of human blood plasma, hydroxyapatite formed on the surfaces of the polypeptides with carboxyl groups, poly(FE)(3)FG and poly(LE)(3)LG, within 2 days, but not on those without carboxyl groups, poly(FQ)(3)FG and poly(LQ)(3)LG. We confirmed that the beta sheet structure was effective for hydroxyapatite nucleation even in the synthetic polypeptide. This finding is useful for the future design of organic polymers that can effectively induce nucleation of hydroxyapatite.

Immobilization of Enamel Matrix Derivate Protein onto Polypeptide Multilayers. Comparative in Situ Measurements Using Ellipsometry, Quartz Crystal Microbalance with Dissipation, and Dual-Polarization Interferometry

The buildup of biodegradable poly(L-glutamic acid) (PGA) and poly(L-lysine) (PLL) multilayers on silica and titanium surfaces and the immobilization of enamel matrix derivate (EMD) protein was followed by utilizing in situ ellipsometry, quartz crystal microbalance with dissipation, and dual-polarization interferometry (DPI). The use of the relatively new DPI technique validated earlier published ellipsometry measurements of the PLL-PGA polypeptide films. The hydrophobic aggregating EMD protein was successfully immobilized both on top of and within the multilayer structures at pH 5.0. DPI measurements further indicated that the immobilization of EMD is influenced by the flow pattern during adsorption. The formed polypeptide-EMD multilayer films are of interest since it is known that EMD is able to trigger cell response and induce biomineralization. The multilayer films thus have potential to be useful as bioactive and biodegradable coatings for future dental implants.