Silver/gold nanoalloy implant coatings with antibiofilm activity via pH-triggered silver ion release.

Implant infections are a major challenge for the healthcare system. Biofilm formation and increasing antibiotic resistance of common bacteria cause implant infections, leading to an urgent need for alternative antibacterial agents. In this study, the antibiofilm behaviour of a coating consisting of a silver (Ag)/gold (Au) nanoalloy is investigated. This alloy is crucial to reduce uncontrolled potentially toxic Ag+ ion release. In neutral pH environments this release is minimal, but the Ag+ ion release increases in acidic microenvironments caused by bacterial biofilms. We perform a detailed physicochemical characterization of the nanoalloys and compare their Ag+ ion release with that of pure Ag nanoparticles. Despite a lower released Ag+ ion concentration at pH 7.4, the antibiofilm activity against Escherichia coli (a bacterium known to produce acidic pH environments) is comparable to a pure nanosilver sample with a similar Ag-content. Finally, biocompatibility studies with mouse pre-osteoblasts reveal a decreased cytotoxicity for the alloy coatings and nanoparticles.

Antimicrobial Potency of Fmoc-Phe-Phe Dipeptide Hydrogels with Encapsulated Porphyrin Chromophores Is a Promising Alternative in Antimicrobial Resistance.

Antimicrobial resistance (AMR) poses a significant global health risk as a consequence of misuse of antibiotics. Owing to the increasing antimicrobial resistance, it became imperative to develop novel molecules and materials with antimicrobial properties. Porphyrins and metalloporphyrins are compounds which present antimicrobial properties especially after irradiation. As a consequence, porphyrinoids have recently been utilized as antimicrobial agents in antimicrobial photodynamic inactivation in bacteria and other microorganisms. Herein, we report the encapsulation of porphyrins into peptide hydrogels which serve as delivery vehicles. We selected the self-assembling Fmoc-Phe-Phe dipeptide, a potent gelator, as a scaffold due to its previously reported biocompatibility and three different water-soluble porphyrins as photosensitizers. We evaluated the structural, mechanical and in vitro degradation properties of these hydrogels, their interaction with NIH3T3 mouse skin fibroblasts, and we assessed their antimicrobial efficacy against Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Escherichia coli (E. coli) bacteria. We found out that the hydrogels are cytocompatible and display antimicrobial efficiency against both strains with the zinc porphyrins being more efficient. Therefore, these hydrogels present a promising alternative for combating bacterial infections in the face of growing AMR concerns.

The Enrichment of Whey Protein Isolate Hydrogels with Poly-γ-Glutamic Acid Promotes the Proliferation and Osteogenic Differentiation of Preosteoblasts.

Osseous disease accounts for over half of chronic pathologies, but there is a limited supply of autografts, the gold standard; hence, there is a demand for new synthetic biomaterials. Herein, we present the use of a promising, new dairy-derived biomaterial: whey protein isolate (WPI) in the form of hydrogels, modified with the addition of different concentrations of the biotechnologically produced protein-like polymeric substance poly-γ-glutamic acid (γ-PGA) as a potential scaffold for tissue regeneration. Raman spectroscopic analysis demonstrated the successful creation of WPI-γ-PGA hydrogels. A cytotoxicity assessment using preosteoblastic cells demonstrated that the hydrogels were noncytotoxic and supported cell proliferation from day 3 to 14. All γ-PGA-containing scaffold compositions strongly promoted cell attachment and the formation of dense interconnected cell layers. Cell viability was significantly increased on γ-PGA-containing scaffolds on day 14 compared to WPI control scaffolds. Significantly, the cells showed markers of osteogenic differentiation; they synthesised increasing amounts of collagen over time, and cells showed significantly enhanced alkaline phosphatase activity at day 7 and higher levels of calcium for matrix mineralization at days 14 and 21 on the γ-PGA-containing scaffolds. These results demonstrated the potential of WPI-γ-PGA hydrogels as scaffolds for bone regeneration.

A Doubly Fmoc-Protected Aspartic Acid Self-Assembles into Hydrogels Suitable for Bone Tissue Engineering.

Hydrogels have been used as scaffolds for biomineralization in tissue engineering and regenerative medicine for the repair and treatment of many tissue types. In the present work, we studied an amino acid-based material that is attached to protecting groups and self-assembles into biocompatible and stable nanostructures that are suitable for tissue engineering applications. Specifically, the doubly protected aspartic residue (Asp) with fluorenyl methoxycarbonyl (Fmoc) protecting groups have been shown to lead to the formation of well-ordered fibrous structures. Many amino acids and small peptides which are modified with protecting groups display relatively fast self-assembly and exhibit remarkable physicochemical properties leading to three-dimensional (3D) networks, the trapping of solvent molecules, and forming hydrogels. In this study, the self-assembling fibrous structures are targeted toward calcium binding and act as nucleation points for the binding of the available phosphate groups. The cell viability, proliferation, and osteogenic differentiation of pre-osteoblastic cells cultured on the formed hydrogel under various conditions demonstrate that hydrogel formation in CaCl2 and CaCl2-Na2HPO4 solutions lead to calcium ion binding onto the hydrogels and enrichment with phosphate groups, respectively, rendering these mechanically stable hydrogels osteoinductive scaffolds for bone tissue engineering.

Antibiofilm activity of nanosilver coatings against Staphylococcus aureus.

Implant infections due to bacterial biofilms constitute a major healthcare challenge today. One way to address this clinical need is to modify the implant surface with an antimicrobial nanomaterial. Among such nanomaterials, nanosilver is arguably the most powerful one, due to its strong and broad antimicrobial activity. However, there is still a lack of understanding on how physicochemical characteristics of nanosilver coatings affect their antibiofilm activity. More specifically, the contributions of silver (Ag)+ ion-mediated vs. contact-based mechanisms to the observed antimicrobial activity are yet to be elucidated. To address this knowledge gap, we produce here nanosilver coatings on substrates by flame aerosol direct deposition that allows for facile control of the coating composition and Ag particle size. We systematically study the effect of (i) nanosilver content in composite Ag silica (SiO2) coatings from 0 (pure SiO2) up to 50 wt%, (ii) the Ag particle size and (iii) the coating thickness on the antibiofilm activity against Staphylococcus aureus (S. aureus), a clinically-relevant pathogen often present on the surface of surgically-installed implants. We show that the Ag+ ion concentration in solution largely drives the observed antibiofilm effect independently of Ag size and coating thickness. Furthermore, co-incubation of both pure SiO2 and nanosilver coatings in the same well also reveals that the antibiofilm effect stems predominantly from the released Ag+ ions, which is especially pronounced for coatings featuring the smallest Ag particle sizes, rather than direct bacterial contact inhibition. We also examine the biocompatibility of the developed nanosilver coatings in terms of pre-osteoblastic cell viability and proliferation, comparing it to that of pure SiO2. This study lays the foundation for the rational design of nanosilver-based antibiofilm implant coatings.

Phloroglucinol-enhanced whey protein isolate hydrogels with antimicrobial activity for tissue engineering.

Aging populations in developed countries will increase the demand for implantable materials to support tissue regeneration. Whey Protein Isolate (WPI), derived from dairy industry by-products, can be processed into hydrogels with the following desirable properties for applications in tissue engineering: (i) ability to support adhesion and growth of cells; (ii) ease of sterilization by autoclaving and (iii) ease of incorporation of poorly water-soluble drugs with antimicrobial activity, such as phloroglucinol (PG), the fundamental phenolic subunit of marine polyphenols. In this study, WPI hydrogels were enriched with PG at concentrations between 0 and 20% w/v. PG solubilization in WPI hydrogels is far higher than in water. Enrichment with PG did not adversely affect mechanical properties, and endowed antimicrobial activity against a range of bacteria which occur in healthcare-associated infections (HAI). WPI-PG hydrogels supported the growth of, and collagen production by human dental pulp stem cells and - to a lesser extent - of osteosarcoma-derived MG-63 cells. In summary, enrichment of WPI with PG may be a promising strategy to prevent microbial contamination while still promoting stem cell attachment and growth.

Responsive Quaternized PDMAEMA Copolymers with Antimicrobial Action.

In this work, the antimicrobial action of partially quaternized poly(2-(dimethylamino)ethyl methacrylate) (PQDMAEMA) copolymers using different alkyl halides is presented. The poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) homopolymer was synthesized by group transfer polymerization, followed by the modification of its tertiary amine groups, using bromoethane, iodoethane, bromohexane and bromoethanol, to introduce permanent cationic, quaternary ammonium salt moieties, randomly distributed along the polymer chains. In all cases, the degree of quaternization was low, at ~10 mol%, as verified by proton nuclear magnetic resonance spectroscopy to preserve the thermo-responsive character of the PDMAEMA precursor polymer. The biocidal activity of the lightly quaternized PQDMAEMA copolymers against Escherichia coli and Staphylococcus aureus was evaluated by calculating the minimum inhibitory concentration (MIC) as well as the minimum bactericidal concentration (MBC) of the polymers and by comparing them to the respective values of the precursor non-quaternized PDMAEMA homopolymer. The antibacterial mechanism of action in the solution was studied by zeta potential measurements, scanning electron microscopy and protein leakage tests signifying the disruption of the outer membrane of the bacterial cells to release their periplasmic proteins.

In Vitro Biocompatibility Assessment of Nano-Hydroxyapatite.

Hydroxyapatite (HA) is an important component of the bone mineral phase. It has been used in several applications, such as bone regenerative medicine, tooth implants, drug delivery and oral care cosmetics. In the present study, three different batches of a commercial nanohydroxyapatite (nHA) material were physicochemically-characterized and biologically-evaluated by means of cytotoxicity and genotoxicity using appropriate cell lines based on well-established guidelines (ISO10993-5 and OECD 487). The nHAs were characterized for their size and morphology by dynamic light scattering (DLS) and transmission electron microscopy (TEM) and were found to have a rod-like shape with an average length of approximately 20 to 40 nm. The nanoparticles were cytocompatible according to ISO 10993-5, and the in vitro micronucleus assay showed no genotoxicity to cells. Internalization by MC3T3-E1 cells was observed by TEM images, with nHA identified only in the cytoplasm and extracellular space. This result also validates the genotoxicity since nHA was not observed in the nucleus. The internalization of nHA by the cells did not seem to affect normal cell behavior, since the results showed good biocompatibility of these nHA nanoparticles. Therefore, this work is a relevant contribution for the safety assessment of this nHA material.

Antibacterial Effect of Colloidal Suspensions Varying in Silver Nanoparticles and Ions Concentrations.

A lot of effort has been dedicated recently to provide a better insight into the mechanism of the antibacterial activity of silver nanoparticles (AgNPs) colloidal suspensions and their released silver ionic counterparts. However, there is no consistency regarding whether the antibacterial effect displayed at cellular level originates from the AgNPs or their ionic constitutes. To address this issue, three colloidal suspensions exhibiting different ratios of AgNPs/silver ions were synthesized by a wet chemistry method in conjunction with tangential flow filtration, and were characterized and evaluated for their antimicrobial properties against two gram-negative, Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), and two gram-positive, Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis), bacterial strains. The produced samples contained 25% AgNPs and 75% Ag ions (AgNP_25), 50% AgNPs and 50% Ag ions (AgNP_50), and 100% AgNPs (AgNP_100). The sample AgNP_100 demonstrated the lowest minimum inhibitory concentration values ranging from 4.6 to 15.6 ppm for all four bacterial strains, while all three samples indicated minimum bactericidal concentration (MBC) values ranging from 16.6 ppm to 62.5 ppm against all strains. An increase in silver ions content results in higher bactericidal activity. All three samples were found to lead to a significant morphological damage by disruption of the bacterial cell membranes as analyzed by means of scanning electron microscopy (SEM). The growth kinetics demonstrated that all three samples were able to reduce the bacterial population at a concentration of 3.1 ppm. SEM and growth kinetic data underline that S. epidermidis is the most sensitive among all strains against the investigated samples. Our results showed that all three AgNPs colloidal suspensions exhibited strong antibacterial properties and, thus, they can be applied in medical devices and antimicrobial control systems.