Polypeptide Coatings Research Articles

Mixed-charge glycopolypeptides as antibacterial coatings with long-term activity

Planktonic bacteria adhere and subsequently form biofilms on implantable medical devices can cause severe infections that have become the major types of hospital-acquired infections. Traditional coatings for the implants are frequently lack of long-term antifouling and bactericidal activities. It is still a big challenge to simultaneously improve the antifouling and bactericidal activities of the coatings. Herein, we report that mixed-charge glycopolypeptide coatings are of long-term antibacterial activities to efficiently inhibit the biofilm growth. The glycosylation of mixed-charge polypeptides has led to a significant improvement of both antifouling and bactericidal activities. The cooperative effect of the saccharide residues and mixed-charge residues improved the resistance of the polypeptide coatings against protein adsorption. The saccharide and l-glutamic acid (E) residues collectively enhanced the bacterial membrane-disruption of cationic l-lysine (K) residues, leading to potent bactericidal activity. Meanwhile, the glycopolypeptide coatings showed superior biocompatibility, long-term antibiofilm and anti-infection properties in two types of mouse subcutaneous infection models and one type of mouse urinary tract infection model. This work provides a new strategy to achieve antibacterial coatings with long-term activities for preventing implantable medical device associated infections.

Impact of Charge Composition and Distribution on the Antibacterial Properties of Polypeptide Coatings.

Inspired by the charge composition and distribution of proteins and peptides, we designed and prepared a series of brush polypeptides with positive and negative charges separately distributed in the side chains and the backbones. The brush polypeptides can self- or co-deposit on various substrates forming ultrathin and stable coatings. They showed potent bactericidal activity and antibiofilm property, outperforming conventional linear polypeptide coatings with randomly distributed positive and negative charges. Keeping the balance of positive/negative charges and increasing the numbers of positive/negative charges can further improve the antibacterial property of brush polypeptide coatings without sacrificing their biocompatibility.

Polypeptide coatings on biominerals with superior antimicrobial and antifouling properties inspired by human salivary proteins

Inhibiting the growth of bacteria on biomedical implants is of great clinical significance due to the associated life-threatening infections. Peptide sequence DDDEEK (SAP) and NFKRKYGHHRKA (P1) are inspired from the salivary proteins statherin and histatin, respectively. SAP enables stable immobilization on the biomineral surface, and P1 has a strong antimicrobial effect against clinically pathogenic bacteria. Herein, we report the surface-immobilized multifunctional coating based on a hybrid peptide NFKRKYGHHRKAKEEDDD (P1-SAP). This coating can form on hydroxyapatite (HA)/tricalcium phosphate (TCP) surfaces by a simple dip-coating or spraying method and remain on the surface for at least 1 month. The P1-SAP coating exhibits 3-fold effective antimicrobial properties against bacteria/biofilm and shows 2-fold antifouling functions than the bare group due to the synergistic effect of superhydrophilic surface, cationic peptides and surface hydrophobic groups. Additionally, the combination of SAP with P1 can reduce the cytotoxicity of P1 to MG63 cells because the negative charges of SAP can effectively neutralize the positive charges of P1. Moreover, in vivo studies in rats show a significant decrease in infection associated with the P1-SAP-coated HA implants, indicating the efficacy of this coating against subcutaneous infections. Our findings provide valuable guidance to the design and evaluation of coatings with antimicrobial and antifouling properties for implant surfaces.

Phytic Acid-Promoted rapid fabrication of natural polypeptide coatings for multifunctional applications

Surface modification or reconstruction of biomaterials, medical devices or other materials hold great promise for improved functionality and applications. However, it is essential to develop coating layers possessing a wide range of functionalities, such as antibacterial, anticorrosive and biocompatible properties. In this work, a simplified method was investigated for the supramolecular assembly of natural bio compounds, phytic acid (PA) and antimicrobial peptide of ε-polylysine (Ply), via one-pot co-deposition. The PA and Ply formed a coating layer on the substrate surface through the inherent surface affinity of PA and ionic bond formation between the electro-positive Ply and electro-negative PA. The co-deposition of PA-Ply is a surface-independent. The physio-chemical features of PA-Ply on titanium (Ti) were characterized to reveal the polymer decoration, surface composition, and microtopography. It was noticed that the PA-Ply coating on Ti (Ti-PA-Ply) could kill greater than 99.9% of both Gram-positive and Gram-negative bacterial pathogens upon their adhesion as compared to that of the pristine Ti surface. Furthermore, the in vivo rat subcutaneous infection model demonstrated effective anti-infection and bactericidal properties of the Ti-PA-Ply surface. In addition, the PA-Ply coating exhibited anti-corrosive property for medical-grade magnesium (Mg) in an electrochemical corrosion model, and promoted the proliferation of endothelial cells. The versatile PA-Ply coating also imparted an improved anti-fogging performance. Thus, the present one-step co-depostition of PA-Ply provided a simple, envirementelly-benign and sustainable approach to the design and fabrication of multi-functional surfaces.

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.

Antibacterial Brush Polypeptide Coatings with Anionic Backbones and AMP Mimetic Side-Chains

Antibacterial Brush Polypeptide Coatings with Anionic Backbones and AMP Mimetic Side-Chains

Biomineral interface with superior cell adhesive and antibacterial properties based on enzyme-triggered digestion of saliva acquired pellicle-inspired polypeptide coatings

With the long-term clinical study of implantation materials, the problem of bioinertness on the implant surface emerged. The bioinert implant surface can cause inflammation, fibrosis, coagulation, and infections, and even threaten the life of patients. Herein, we designed and synthesized a saliva-acquired pellicle (SAP)-inspired multifunctional polypeptide DDDEEKRWRWRWGPLGVRGD (SAP-MP196-G-1) by orderly assembling the hydroxyapatite (HA)-adsorbing motif DDDEEK, antibacterial motif RWRWRW, bacterial enzyme-responding motif GPLGV, and cell adhesion motif RGD. The bioinspired polypeptide can self-assemble into nanoparticles and tightly adsorb onto the biomineral, such as the HA surface. The newly formed bio-interface exhibited two-fold higher adhesion and proliferation of MG63 cells and five-fold higher antibacterial properties against Staphylococcus aureus (S. aureus) compared to bare HA group. An in vivo study indicated that the bio-interface can promote implant wound healing and decrease infection of S. aureus on HA-coated implants. This study proposes a strategy to design a cell-adhesive and antibacterial-biomineral interface based on a dynamic enzyme-digestion reaction to effectively inhibit the side effects of implantation surface presence.

Relating polymeric microparticle formulation to prevalence or distribution of fibronectin and poly-d-lysine to support mesenchymal stem cell growth

Protein-coated polymer-based microparticles are attractive supports for cell delivery, but the interplay between microparticle properties, protein coating, and cell response is poorly understood. The interest in alternative microparticle formulations increases the need for a better understanding of how functional protein coatings form on different microparticles. In this work, microparticle formulations based on biodegradable polymers [poly (lactic-co-glycolic acid) (PLGA) and the triblock copolymer PLGA-poloxamer-PLGA] were prepared via an emulsion-based process. To explore the impact that the use of a surfactant has on the properties of the microparticles, the emulsion was stabilized by using either a surfactant, poly(vinyl alcohol), or an organic solvent, propylene glycol. Four different types of microparticles were prepared through combinations of the two types of polymers and the two types of stabilizers. The coating of microparticles with proteins/polypeptides such as fibronectin and poly-d-lysine has been demonstrated before and is an integral step for their application as microcarriers, e.g., for cell delivery; however, the impact of the microparticles' surface chemical properties on the formation (prevalence and distribution) of the mixed polypeptide coatings and the influence on subsequent cell attachment remain to be elucidated. Using a colocalization analysis approach on ToF-SIMS images of protein-coated microparticles, we show that the use of propyleneglycol over PVA as well as the substitution of PLGA by the triblock copolymer resulted in enhanced protein adsorption. Furthermore, if propyleneglycol is used, the substitution of PLGA with the triblock copolymer leads to increased stem cell adhesion.

Development of regenerated silk films coated with fluorinated polypeptides to achieve high water repellency and biodegradability in seawater

In this study, we prepared silkworm silk films with fluorinated polypeptide coatings on their surface to achieve water-repellent silk materials. The silk films were smoothly and uniformly coated with the fluorinated polypeptides to give a transparent film. The contact angles of water droplets on the polypeptide-coated silk films were measured to investigate the ability of the films to resist water. The silk films coated with the fluorinated polypeptides exhibited higher contact angles compared to those of the uncoated silk film, indicating that the fluorinated polypeptides formed a hydrophobic surface on the silk film. The structure of the fluorinated polypeptides influenced the water-repellent ability, and the polypeptide containing both perfluoroalkyl chains and biphenyl groups imparted the highest water repellency. A biochemical oxygen demand (BOD) assay for the polypeptide-coated silk films in seawater revealed that the silk films showed high biodegradability in seawater even with the fluorinated polypeptide coating. The polypeptide-coated silk films provide not only high water repellency for practical use but also high biodegradability in marine environments.

Antibiofilm elastin-like polypeptide coatings: functionality, stability, and selectivity

Antimicrobial peptides (AMPs) are currently receiving interest as an alternative to conventional antibiotics to treat biomaterial-associated infection. However, the inherent instability of such peptides often limits their efficacy in intended clinical applications. Covalent immobilization of AMPs to surfaces is one strategy to increase the long-term stability and minimize the toxicity. In this work, an antimicrobial peptide, RRPRPRPRPWWWW-NH2 (RRP9W4N), was used to modify elastin-like polypeptide (ELP) surface coatings containing cell-adhesive peptide domains (RGD) using covalent chemistry. The AMP retained its antibacterial activity against Staphylococcus epidermidis, Staphylococcus aureus, and Pseudomonas aeruginosa when covalently bonded to ELP surfaces. Simultaneously, the AMP functionalization had insignificant effect on the viability, function, and differentiation of human osteosarcoma MG63 cells and human mesenchymal stem cells (hMSCs). Furthermore, stability of the immobilized AMP in human blood serum was investigated, and the results suggested that the AMP preserved its antibacterial activity up to 24 h. Combined, the results show that covalently attached AMPs onto RGD-containing ELP are an excellent candidate as an antimicrobial coating for medical devices. Statement of SignificanceBiomaterial associated infection, caused by adherent biofilm, is usually difficult to treat. There is a high demand for new materials and treatments to decrease the infection rates, especially with increasing threats concerning resistant bacteria. Formation of biofilms on medical devices lowers the bacteria susceptibility towards traditional antibiotics and also circumvent our immune system often resulting in revisional surgery and extensive use of antibiotics. One promising strategy is to develop surfaces having low bacterial attractiveness or bacterial killing properties, but still retaining the main function of the device. In this study, we have developed an implant coating that demonstrates a high antimicrobial effect and at the same time showing no negative affect on human cells.

Tuning the Stiffness of Surfaces by Assembling Genetically Engineered Polypeptides with Tailored Amino Acid Sequence.

We introduce elastin-like recombinamers (ELRs) as polypeptides with precise amino acid positioning to generate polypeptide coatings with tunable rigidity. Two ELRs are used: V84-ELR, a hydrophobic monoblock, and EI-ELR, an amphiphilic diblock. Both were modified with the amine-reactive tetrakis (hydroxymethyl) phosphonium chloride compound. We evaluated the affinity, conformation, and dissipative behavior of ELRs assembled on alkanethiol self-assembled coatings by quartz crystal microbalance with dissipation monitoring, multiparametric surface plasmon resonance, and atomic force microscopy. The thickness of the polypeptide coatings showcases the preferential affinity of ELRs to NH2- and CH3-terminated surfaces. We demonstrate that V84-ELR strongly bonded to the substrate and reorganizes into an extended and more hydrated layer as the adsorbed amount increases, whereas EI-ELR has a less dissipative behavior. The results suggest that ELR adsorption depends on the amino acid sequence and the substrate chemistry, ultimately influencing the stiffness of the polypeptide coatings.

Effect of amine content and chemistry on long-term, three-dimensional hepatocyte spheroid culture atop aminated elastin-like polypeptide coatings.

Culture conditions that induce hepatic spheroidal aggregates sustain liver cells with metabolism that mimics in vivo hepatocytes. Here we present an array of elastin-like polypeptide conjugate coating materials (Aminated-ELPs) that are biocompatible, have spheroid-forming capacity, can be coated atop traditional culture surfaces, and maintain structural integrity while ensuring adherence of spheroids over long culture period. The Aminated-ELPs were synthesized either by direct conjugation of ELP and various polyelectrolytes or by conjugating both ELP and various small electrolytes to the reactive polymer poly(2-vinyl-4,4-dimethyl azlactone) (PVDMA). Spheroid morphology, cellular metabolic function, and liver-specific gene expression over the long-term, 20-day culture period were assessed through optical microscopy, measurement of total protein content and albumin and urea production, and quantitative real-time (qRT) PCR. We found that the amine content of the Aminated-ELP coatings dictated the initial hepatocyte attachment, but not the subsequent hepatocyte spheroid formation and their continued attachment. A lower amine content was generally found to sustain higher albumin production by the spheroids. Out of the 19 Aminated-ELP coatings tested, we found that the lysine-containing substrates comprising ELP-polylysine or ELP-PVDMA-butanediamine proved to consistently culture productive spheroidal hepatocytes. We suggest that the incorporation of lysine functional groups in Aminated-ELP rendered more biocompatible surfaces, increasing spheroid attachment and leading to increased liver-specific function. Taken together, the Aminated-ELP array presented here has the potential to create in vitro hepatocyte culture models that mimic in vivo liver functionality and thus, lead to better understanding of liver pathophysiology and superior screening methods for drug efficacy and toxicity. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 377-388, 2017.

Evaluation of the bulk platelet response and fibrinogen interaction to elastin‐like polypeptide coatings

In this work, we expand on our understanding of the thrombogenicity of coatings prepared with three different recombinant elastin-like polypeptides (ELPs). The bulk platelet response of the ELP coatings was characterized following whole blood contact under physiological shear flow (300 s(-1) ) using flow cytometry. Prolonged exposure to shear flow (1-h) indicated that materials coated with the longer ELP coatings (ELP2 and ELP4) had less bulk platelet activation and microparticle formation than materials coated with the shorter ELP1. Quartz crystal microbalance with dissipation (QCM-D) was used to monitor the binding of the platelet membrane receptor GPIIb/IIIa to ELP-adsorbed fibrinogen (Fg) surfaces. Compared to the shorter ELPs, a lower amount of Fg adsorbed to the ELP4 coated material and ELP4 appeared to form a softer, more structurally flexible coating layer. When Fg was adsorbed to the ELP coated surface it demonstrated an altered binding for GPIIb/IIIa that was inhibited in the presence of an AGDV-containing peptide but not an RGD-containing peptide. Conversely, on the shorter ELP coatings, binding of GPIIb/IIIa to an adsorbed Fg layer was partially inhibited in the presence of an RGD-containing peptide. These results indicate that both the quantity and conformational state of Fg varies when adsorbed to surfaces coated with ELPs of varying sequence length, which may be mediating their platelet response. Collectively, the findings reinforce the applicability of the ELPs as potential thromboresistant coatings, especially with the use of the longer polypeptide-ELP4.

A Simple and Versatile Synthetic Strategy to Functional Polypeptides via Vinyl Sulfone-Substituted l-Cysteine N-Carboxyanhydride

Vinyl sulfone-substituted l-cysteine N-carboxyanhydride (VSCys-NCA) monomer was designed and developed to afford a novel and versatile family of vinyl sulfone (VS)-functionalized polypeptides, which further offer a facile access to functional polypeptide-based materials including glycopolypeptides, functional polypeptide coatings, and in situ forming polypeptide hydrogels through Michael-type addition chemistry under mild conditions. VSCys-NCA was obtained in two straightforward steps with a high overall yield of 76%. The copolymerization of γ-benzyl l-glutamate NCA (BLG-NCA), N-benzyloxycarbonyl-l-lysine NCA (ZLL-NCA), or l-leucine NCA (Leu-NCA) with VSCys-NCA using 1,1,1-trimethyl-N-2-propenylsilanamine (TMPS) as an initiator proceeded smoothly in DMF at 40 °C, yielding P(BLG-co-VSCys), P(ZLL-co-VSCys), or P(Leu-co-VSCys) with defined functionalities, controlled molecular weights, and moderate polydispersities (PDI = 1.15–1.50). The acidic deprotection of P(BLG-co-VSCys) and P(ZLL-co-VSCys) furnished water-soluble VS-functionalized poly(l-glutamic acid) (P(Glu-co-VSCys)) and VS-functionalized poly(l-lysine) (P(LL-co-VSCys)), respectively. These VS-functionalized polypeptides were amenable to direct, efficient, and selective postpolymerization modification with varying thiol-containing molecules such as 2-mercaptoethanol, 2-mercaptoethylamine hydrochloride, l-cysteine, and thiolated galactose providing functional polypeptides containing pendant hydroxyl, amine, amino acid, and saccharide, respectively. The contact angle and fluorescence measurements indicated that polymer coatings based on P(Leu-co-VSCys) allowed direct functionalization with thiol-containing molecules under aqueous conditions. Moreover, hydrogels formed in situ upon mixing aqueous solutions of P(Glu-co-VSCys) and thiolated glycol chitosan at 37 °C. These vinyl sulfone-functionalized polypeptides have opened a new avenue to a broad range of advanced polypeptide-based materials.

Surface and adsorption characteristics of three elastin-like polypeptide coatings with varying sequence lengths

The surface properties of a family of elastin-like polypeptides (ELPs), differing in molecular weight and sequence length, were investigated to understand how the nature of the polypeptide film might contribute to their thrombogenic profile. Physical adsorption of the ELPs onto Mylar increased surface wettability as the sequence length decreased while X-ray spectroscopy analysis showed an increasing amide content with sequence length. Chemical force microscopy analysis revealed that the ELP-coated surfaces displayed purely hydrophilic adhesion forces that increased as the ELP sequence length decreased. Adsorption isotherms performed using the quartz crystal microbalance with dissipation, showed that the surface coverage increased with ELP sequence length. The longer polypeptides (ELP2 and ELP4) also displayed higher specific dissipation values indicating that they established films with greater structural flexibility and associated water content than the shorter polypeptide, ELP1. Additionally, the stability of the ELP coating was lower with the shorter polypeptides. This study highlights the different surface properties of the ELP coatings as well as the dynamic nature of the ELP adsorbed layer wherein the conformational state may be an important factor contributing to their blood response.

Photochemically immobilized polymer coatings: effects on protein adsorption, cell adhesion, and leukocyte activation

Amphiphilic chains of 4-benzoylbenzoic acid moieties and polymer were photochemically immobilized onto silicone rubber to ask whether the covalently coupled polymers would passivate the silicone rubber by inhibiting protein adsorption and subsequent cell adhesion and activation. Three groups of polymers were utilized: the hydrophilic synthetic polymers of polyacrylamide, polyethylene glycol, and polyvinylpyrrolidone; the glycosaminoglycan, hyaluronic acid; and poly(glycine-valine-glycine-valine-proline), a polypeptide derived from the sequence of clastin. Each coating variant decreased the adsorption of fibrinogen and immunoglobulin G compared to uncoated silicone rubber. All except the methoxy-polyethylene glycol coating nearly abolished fibroblast growth, but none of the coating variants inhibited monocyte or polymorphonuclear leukocyte adhesion. Interleukin-1β, interleukin-1 receptor antagonist, and tumor necrosis factor-α secretion by leukocytes were not statistically different between any of the coating variants and uncoated silicone rubber. However, the methoxy-polyethylene glycol and elastin-based polypeptide coatings, which supported the highest numbers of adherent monocytes, also elicited the lowest levels of pro-inflammatory cytokine secretion. When these in vitro data were collectively evaluated, the coating that most effectively passivated silicone rubber was the polypeptide derived from elastin.