Silver-enriched microdomain patterns as advanced bactericidal coatings for polymer-based medical devices.

Today, it would be difficult for us to live a full life without polymers, especially in medicine, where its applicability is constantly expanding, giving satisfactory results without any harm effects on health. This study focused on the formation of hexagonal domains doped with AgNPs using a KrF excimer laser (λ=248 nm) on the polyetheretherketone (PEEK) surface that acts as an unfailing source of the antibacterial agent - silver. The hexagonal structure was formed with a grid placed in front of the incident laser beam. Surfaces with immobilized silver nanoparticles (AgNPs) were observed by AFM and SEM. Changes in surface chemistry were studied by XPS. To determine the concentration of released Ag+ ions, ICP-MS analysis was used. The antibacterial tests proved the antibacterial efficacy of Ag-doped PEEK composites against Escherichia coli and Staphylococcus aureus as the most common pathogens. Because AgNPs are also known for their strong toxicity, we also included cytotoxicity tests in this study. The findings presented here contribute to the advancement of materials design in the biomedical field, offering a novel starting point for combating bacterial infections through the innovative integration of AgNPs into inert synthetic polymers.

A New Promising Material for Biological Applications: Multilevel Physical Modification of AgNP-Decorated PEEK.

In the case of polymer medical devices, the surface design plays a crucial role in the contact with human tissue. The use of AgNPs as antibacterial agents is well known; however, there is still more to be investigated about their anchoring into the polymer surface. This study describes the changes in the surface morphology and behaviour in the biological environment of polyetheretherketone (PEEK) with immobilised AgNPs after different surface modifications. The initial composites were prepared by immobilising silver nanoparticles from a colloid solution in the upper surface layers of polyetheretherketone (PEEK). The prepared samples (Ag/PEEK) had a planar morphology and were further modified with a KrF laser, a GaN laser, and an Ar plasma. The samples were studied using the AFM method to visualise changes in surface morphology and obtain information on the height of the structures and other surface parameters. A comparative analysis of the nanoparticles and polymers was performed using FEG-SEM. The chemical composition of the surface of the samples and optical activity were studied using XPS and UV-Vis spectroscopy. Finally, drop plate antibacterial and cytotoxicity tests were performed to determine the role of Ag nanoparticles after modification and suitability of the surface, which are important for the use of the resulting composite in biomedical applications.

Polymer-Antimicrobial Peptide Constructs with Tailored Drug-Release Behavior.

Microbial resistance is one of the main problems of modern medicine. Recently, antimicrobial peptides have been recognized as a novel approach to overcome the microbial resistance issue, nevertheless, their low stability, toxicity, and potential immunogenic response in biological systems have limited their clinical application. Herein, we present the design, synthesis, and preliminary biological evaluation of polymer-antibacterial peptide constructs. The antimicrobial GKWMKLLKKILK-NH2 oligopeptide (PEP) derived from halictine, honey bee venom, was bound to a polymer carrier via various biodegradable spacers employing the pH-sensitive or enzymatically-driven release and reactivation of the PEP's antimicrobial activity. The antibacterial properties of the polymer-PEP constructs were assessed by a determination of the minimum inhibitory concentrations, followed by fluorescence and transmission electron microscopy. The PEP exerted antibacterial activity against both, gram-positive and negative bacteria, via disruption of the bacterial cell wall mechanism. Importantly, PEP partly retained its antibacterial efficacy against Staphylococcus epidermidis, Escherichia coli, and Acinetobacter baumanii even though it was bound to the polymer carrier. Indeed, to observe antibacterial activity similar to the free PEP, the peptide has to be released from the polymer carrier in response to a pH decrease. Enzymatically-driven release and reactivation of the PEP antimicrobial activity were recognized as less effective when compared to the pH-sensitive release of PEP.

Laser-Processed PEN with Au Nanowires Array: A Biocompatibility Assessment.

Although many noble metals are known for their antibacterial properties against the most common pathogens, such as Escherichia coli and Staphylococcus epidermidis, their effect on healthy cells can be toxic. For this reason, the choice of metals that preserve the antibacterial effect while being biocompatible with health cells is very important. This work aims to validate the effect of gold on the biocompatibility of Au/Ag nanowires, as assessed in our previous study. Polyethylene naphthalate (PEN) was treated with a KrF excimer laser to provide specific laser-induced periodic structures. Then, Au was deposited onto the modified PEN via a vacuum evaporation method. Atomic force microscopy and scanning electron microscopy revealed the dependence of the surface morphology on the incidence angle of the laser beam. A resazurin assay cytotoxicity test confirmed safety against healthy human cells and even cell proliferation was observed after 72 h of incubation. We have obtained satisfactory results, demonstrating that monometallic Au nanowires can be applied in biomedical applications and provide the biocompatibility of bimetallic Au/AgNWs.

Targeting the Virus Capsid as a Tool to Fight RNA Viruses.

Several strategies have been developed to fight viral infections, not only in humans but also in animals and plants. Some of them are based on the development of efficient vaccines, to target the virus by developed antibodies, others focus on finding antiviral compounds with activities that inhibit selected virus replication steps. Currently, there is an increasing number of antiviral drugs on the market; however, some have unpleasant side effects, are toxic to cells, or the viruses quickly develop resistance to them. As the current situation shows, the combination of multiple antiviral strategies or the combination of the use of various compounds within one strategy is very important. The most desirable are combinations of drugs that inhibit different steps in the virus life cycle. This is an important issue especially for RNA viruses, which replicate their genomes using error-prone RNA polymerases and rapidly develop mutants resistant to applied antiviral compounds. Here, we focus on compounds targeting viral structural capsid proteins, thereby inhibiting virus assembly or disassembly, virus binding to cellular receptors, or acting by inhibiting other virus replication mechanisms. This review is an update of existing papers on a similar topic, by focusing on the most recent advances in the rapidly evolving research of compounds targeting capsid proteins of RNA viruses.

SERS and advanced chemometrics - Utilization of Siamese neural network for picomolar identification of beta-lactam antibiotics resistance gene fragment.

The enormous development and expansion of antibiotic-resistant bacterial strains impel the intensive search for new methods for fast and reliable detection of antibiotic susceptibility markers. Here, we combined DNA-targeted surface functionalization, surface-enhanced Raman spectroscopy (SERS) measurements, and subsequent spectra processing by decision system (DS) for detection of a specific oligonucleotide (ODN) sequence identical to a fragment of blaNDM-1 gene, responsible for ß-lactam antibiotic resistance. The SERS signal was measured on plasmonic gold grating, functionalized with capture ODN, ensuring the binding of corresponded ODNs. Designed DS consists of a Siamese neural network (SNN) coupled with robust statistics and Bayes decision theory. The proposed approach allows manipulation with complex multicomponent samples and predefine the desired detection level of confidence and errors, automatically determining the number of required spectra and samples. In constant to commonly used classification-type SNN, our method was applied to analyze samples with compositions previously "unknown" to DS. The detection of targeted ODN was performed with ≥99% level of confidence up to 3 × 10-12 M limit on the background of 10-10 M concentration of similar but not targeted ODNs.

Physically Switchable Antimicrobial Surfaces and Coatings: General Concept and Recent Achievements.

Bacterial environmental colonization and subsequent biofilm formation on surfaces represents a significant and alarming problem in various fields, ranging from contamination of medical devices up to safe food packaging. Therefore, the development of surfaces resistant to bacterial colonization is a challenging and actively solved task. In this field, the current promising direction is the design and creation of nanostructured smart surfaces with on-demand activated amicrobial protection. Various surface activation methods have been described recently. In this review article, we focused on the "physical" activation of nanostructured surfaces. In the first part of the review, we briefly describe the basic principles and common approaches of external stimulus application and surface activation, including the temperature-, light-, electric- or magnetic-field-based surface triggering, as well as mechanically induced surface antimicrobial protection. In the latter part, the recent achievements in the field of smart antimicrobial surfaces with physical activation are discussed, with special attention on multiresponsive or multifunctional physically activated coatings. In particular, we mainly discussed the multistimuli surface triggering, which ensures a better degree of surface properties control, as well as simultaneous utilization of several strategies for surface protection, based on a principally different mechanism of antimicrobial action. We also mentioned several recent trends, including the development of the to-detect and to-kill hybrid approach, which ensures the surface activation in a right place at a right time.

Printable Resin Modified by Grafted Silver Nanoparticles for Preparation of Antifouling Microstructures with Antibacterial Effect.

The usage of three-dimensional (3D) printed materials in many bioapplications has been one of the fastest-growing sectors in the nanobiomaterial industry in the last couple of years. In this work, we present a chemical approach for grafting silver nanoparticles (AgNPs) into a resin matrix, which is convenient for 3D printing. In this way, the samples can be prepared and are able to release silver ions (Ag+) with excellent antibacterial effect against bacterial strains of E. coli and S. epidermidis. By the proposed process, the AgNPs are perfectly mixed and involved in the polymerization process and their distribution in the matrix is homogenous. It was also demonstrated that this approach does not affect the printing resolution and the resin is therefore suitable for the construction of microstructures enabling controlled silver ion release and antifouling properties. At the same time the physical properties of the material, such as viscosity and elasticity modulus are preserved. The described approach can be used for the fabrication of facile, low-cost 3D printed resin with antifouling-antibacterial properties with the possibility to control the release of Ag+ through microstructuring.

Bimetallic Nanowires on Laser-Patterned PEN as Promising Biomaterials.

As inflammation frequently occurs after the implantation of a medical device, biocompatible, antibacterial materials must be used. Polymer-metal nanocomposites are promising materials. Here we prepared enhanced polyethylene naphthalate (PEN) using surface modification techniques and investigated its suitability for biomedical applications. The PEN was modified by a KrF laser forming periodic ripple patterns with specific surface characteristics. Next, Au/Ag nanowires were deposited onto the patterned PEN using vacuum evaporation. Atomic force microscopy confirmed that the surface morphology of the modified PEN changed accordingly with the incidence angle of the laser beam. Energy-dispersive X-ray spectroscopy showed that the distribution of the selected metals was dependent on the evaporation technique. Our bimetallic nanowires appear to be promising antibacterial agents due to the presence of antibacterial noble metals. The antibacterial effect of the prepared Au/Ag nanowires against E. coli and S. epidermidis was demonstrated using 24 h incubation with a drop plate test. Moreover, a WST-1 cytotoxicity test that was performed to determine the toxicity of the nanowires showed that the materials could be considered non-toxic. Collectively, these results suggest that prepared Au/Ag nanostructures are effective, biocompatible surface coatings for use in medical devices.

Influence of Drying Method and Argon Plasma Modification of Bacterial Nanocellulose on Keratinocyte Adhesion and Growth.

Due to its nanostructure, bacterial nanocellulose (BC) has several advantages over plant cellulose, but it exhibits weak cell adhesion. To overcome this drawback, we studied the drying method of BC and subsequent argon plasma modification (PM). BC hydrogels were prepared using the Komagataeibacter sucrofermentans (ATCC 700178) bacteria strain. The hydrogels were transformed into solid samples via air-drying (BC-AD) or lyophilization (BC-L). The sample surfaces were then modified by argon plasma. SEM revealed that compared to BC-AD, the BC-L samples maintained their nanostructure and had higher porosity. After PM, the contact angle decreased while the porosity increased. XPS showed that the O/C ratio was higher after PM. The cell culture experiments revealed that the initial adhesion of human keratinocytes (HaCaT) was supported better on BC-L, while the subsequent growth of these cells and final cell population density were higher on BC-AD. The PM improved the final colonization of both BC-L and BC-AD with HaCaT, leading to formation of continuous cell layers. Our work indicates that the surface modification of BC renders this material highly promising for skin tissue engineering and wound healing.

Antibacterial Properties of a Honeycomb-like Pattern with Cellulose Acetate and Silver Nanoparticles.

This study involved the preparation and characterization of structures with a honeycomb-like pattern (HCP) formed using the phase separation method using a solution mixture of chloroform and methanol together with cellulose acetate. Fluorinated ethylene propylene modified by plasma treatment was used as a suitable substrate for the formation of the HCP structures. Further, we modified the HCP structures using silver sputtering (discontinuous Ag nanoparticles) or by adding Ag nanoparticles in PEG into the cellulose acetate solution. The material morphology was then determined using atomic force microscopy (AFM) and scanning electron microscopy (SEM), while the material surface chemistry was studied using energy dispersive spectroscopy (EDS) and wettability was analyzed with goniometry. The AFM and SEM results revealed that the surface morphology of pristine HCP with hexagonal pores changed after additional sample modification with Ag, both via the addition of nanoparticles and sputtering, accompanied with an increase in the roughness of the PEG-doped samples, which was caused by the high molecular weight of PEG and its gel-like structure. The highest amount (approx. 25 at %) of fluorine was detected using the EDS method on the sample with an HCP-like structure, while the lowest amount (0.08%) was measured on the PEG + Ag sample, which revealed the covering of the substrate with biopolymer (the greater fluorine extent means more of the fluorinated substrate is exposed). As expected, the thickness of the Ag layer on the HCP surface depended on the length of sputtering (either 150 s or 500 s). The sputtering times for Ag (150 s and 500 s) corresponded to layers with heights of about 8 nm (3.9 at % of Ag) and 22 nm (10.8 at % of Ag), respectively. In addition, we evaluated the antibacterial potential of the prepared substrate using two bacterial strains, one Gram-positive of S. epidermidis and one Gram-negative of E. coli. The most effective method for the construction of antibacterial surfaces was determined to be sputtering (150 s) of a silver nanolayer onto a HCP-like cellulose structure, which proved to have excellent antibacterial properties against both G+ and G- bacterial strains.

Effect of Small Polyanions on In Vitro Assembly of Selected Members of Alpha-, Beta- and Gammaretroviruses.

The assembly of a hexameric lattice of retroviral immature particles requires the involvement of cell factors such as proteins and small molecules. A small, negatively charged polyanionic molecule, myo-inositol hexaphosphate (IP6), was identified to stimulate the assembly of immature particles of HIV-1 and other lentiviruses. Interestingly, cryo-electron tomography analysis of the immature particles of two lentiviruses, HIV-1 and equine infectious anemia virus (EIAV), revealed that the IP6 binding site is similar. Based on this amino acid conservation of the IP6 interacting site, it is presumed that the assembly of immature particles of all lentiviruses is stimulated by IP6. Although this specific region for IP6 binding may be unique for lentiviruses, it is plausible that other retroviral species also recruit some small polyanion to facilitate the assembly of their immature particles. To study whether the assembly of retroviruses other than lentiviruses can be stimulated by polyanionic molecules, we measured the effect of various polyanions on the assembly of immature virus-like particles of Rous sarcoma virus (RSV), a member of alpharetroviruses, Mason-Pfizer monkey virus (M-PMV) representative of betaretroviruses, and murine leukemia virus (MLV), a member of gammaretroviruses. RSV, M-PMV and MLV immature virus-like particles were assembled in vitro from truncated Gag molecules and the effect of selected polyanions, myo-inostol hexaphosphate, myo-inositol, glucose-1,6-bisphosphate, myo-inositol hexasulphate, and mellitic acid, on the particles assembly was quantified. Our results suggest that the assembly of immature particles of RSV and MLV was indeed stimulated by the presence of myo-inostol hexaphosphate and myo-inositol, respectively. In contrast, no effect on the assembly of M-PMV as a betaretrovirus member was observed.

A new way to prepare gold nanoparticles by sputtering - Sterilization, stability and other properties.

We have developed a novel simple method for effective preparing gold nanoparticles (AuNPs) intended for utilization in biomedicine. The method is based on gold sputtering into liquid poly(ethylene glycol) (PEG). The PEG was used as a basic biocompatible stabilizer of the AuNP colloid. In addition, two naturally occurring polysaccharides - Chitosan (Ch) and Methylcellulose (MC) - were separately diluted into the PEG base with the aims to enhance the yield of the sputtering without changing the sputtering parameters, and to further improve the stability and the biocompatibility of the colloid. The colloids were sterilized by steam, and their stability was measured before and after the sterilization process by dynamic light scattering and UV-Vis spectrophotometry. The results indicated a higher sputtering yield in the colloids containing the polysaccharides. The colloids were also characterized by atomic absorption spectroscopy (AAS) to reveal the composition of the prepared nanoparticles by transmission electron microscopy (TEM) to visualize the nanoparticles and to evaluate their size and clustering, and by rheometry to estimate the viscosity of the colloids. The zeta-potential of the AuNPs was also determined as an important parameter indicating the stability and the biocompatibility of the colloid. In addition, in vitro tests of antimicrobial activity and cytotoxicity were carried out to estimate the biological activity and the biocompatibility of the colloids. Antimicrobial tests were performed by a drip test on two bacterial strains - Gram-positive Staphylococcus epidermidis and Gram-negative Escherichia coli. AuNP with chitosan proved to possess the highest antibacterial activity, especially towards the Gram-positive S. epidermidis. In vitro tests on eukaryotic cells, i.e. human osteoblastic cell line SAOS-2 and primary normal human dermal fibroblasts (NHDF), were performed after a 7-day cultivation to determine the effect and the toxic dose of the colloids on human cells. The studied colloid concentrations were in the range from 0.6 µg/ml to 6 µg/ml. Toxicity of the colloids started to reappear at a concentration of 4.5 µg/ml, especially with chitosan in the colloid, where the colloid with a concentration of 6 µg/ml proved to be the most toxic, especially towards the SAOS-2 cell line. However, the PEG and PEG-MC containing colloids proved to be relatively non-toxic, even at the highest concentration, but with a slowly decreasing tendency of the cell metabolic activity.

In Vitro Quantification of the Effects of IP6 and Other Small Polyanions on Immature HIV-1 Particle Assembly and Core Stability.

Proper assembly and disassembly of both immature and mature HIV-1 hexameric lattices are critical for successful viral replication. These processes are facilitated by several host-cell factors, one of which is myo-inositol hexaphosphate (IP6). IP6 participates in the proper assembly of Gag into immature hexameric lattices and is incorporated into HIV-1 particles. Following maturation, IP6 is also likely to participate in stabilizing capsid protein-mediated mature hexameric lattices. Although a structural-functional analysis of the importance of IP6 in the HIV-1 life cycle has been reported, the effect of IP6 has not yet been quantified. Using two in vitro methods, we quantified the effect of IP6 on the assembly of immature-like HIV-1 particles, as well as its stabilizing effect during disassembly of mature-like particles connected with uncoating. We analyzed a broad range of molar ratios of protein hexamers to IP6 molecules during assembly and disassembly. The specificity of the IP6-facilitated effect on HIV-1 particle assembly and stability was verified by K290A, K359A, and R18A mutants. In addition to IP6, we also tested other polyanions as potential assembly cofactors or stabilizers of viral particles.IMPORTANCE Various host cell factors facilitate critical steps in the HIV-1 replication cycle. One of these factors is myo-inositol hexaphosphate (IP6), which contributes to assembly of HIV-1 immature particles and helps maintain the well-balanced metastability of the core in the mature infectious virus. Using a combination of two in vitro methods to monitor assembly of immature HIV-1 particles and disassembly of the mature core-like structure, we quantified the contribution of IP6 and other small polyanion molecules to these essential steps in the viral life cycle. Our data showed that IP6 contributes substantially to increasing the assembly of HIV-1 immature particles. Additionally, our analysis confirmed the important role of two HIV-1 capsid lysine residues involved in interactions with IP6. We found that myo-inositol hexasulphate also stabilized the HIV-1 mature particles in a concentration-dependent manner, indicating that targeting this group of small molecules may have therapeutic potential.

Optomechanical Processing of Silver Colloids: New Generation of Nanoparticle-Polymer Composites with Bactericidal Effect.

The properties of materials at the nanoscale open up new methodologies for engineering prospective materials usable in high-end applications. The preparation of composite materials with a high content of an active component on their surface is one of the current challenges of materials engineering. This concept significantly increases the efficiency of heterogeneous processes moderated by the active component, typically in biological applications, catalysis, or drug delivery. Here we introduce a general approach, based on laser-induced optomechanical processing of silver colloids, for the preparation of polymer surfaces highly enriched with silver nanoparticles (AgNPs). As a result, the AgNPs are firmly immobilized in a thin surface layer without the use of any other chemical mediators. We have shown that our approach is applicable to a broad spectrum of polymer foils, regardless of whether they absorb laser light or not. However, if the laser radiation is absorbed, it is possible to transform smooth surface morphology of the polymer into a roughened one with a higher specific surface area. Analyses of the release of silver from the polymer surface together with antibacterial tests suggested that these materials could be suitable candidates in the fight against nosocomial infections and could inhibit the formation of biofilms with a long-term effect.

Conserved cysteines in Mason-Pfizer monkey virus capsid protein are essential for infectious mature particle formation.

Retrovirus assembly is driven mostly by Gag polyprotein oligomerization, which is mediated by inter and intra protein-protein interactions among its capsid (CA) domains. Mason-Pfizer monkey virus (M-PMV) CA contains three cysteines (C82, C193 and C213), where the latter two are highly conserved among most retroviruses. To determine the importance of these cysteines, we introduced mutations of these residues in both bacterial and proviral vectors and studied their impact on the M-PMV life cycle. These studies revealed that the presence of both conserved cysteines of M-PMV CA is necessary for both proper assembly and virus infectivity. Our findings suggest a crucial role of these cysteines in the formation of infectious mature particles.