Strategies for antimicrobial peptide coatings on medical devices: a review and regulatory science perspective.

Indwelling and implanted medical devices are subject to contamination by microbial pathogens during surgery, insertion or injection, and ongoing use, often resulting in severe nosocomial infections. Antimicrobial peptides (AMPs) offer a promising alternative to conventional antibiotics to reduce the incidence of such infections, as they exhibit broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria, microbial biofilms, fungi, and viruses. In this review-perspective, we first provide an overview of the progress made in this field over the past decade with an emphasis on the local release of AMPs from implant surfaces and immobilization strategies for incorporating these agents into a wide range of medical device materials. We then provide a regulatory science perspective addressing the characterization and testing of AMP coatings based on the type of immobilization strategy used with a focus on the US market regulatory niche. Our goal is to help narrow the gulf between academic studies and preclinical testing, as well as to support a future literature base in order to develop the regulatory science of antimicrobial coatings.

Bioprinted Osteogenic and Vasculogenic Patterns for Engineering 3D Bone Tissue.

Fabricating 3D large-scale bone tissue constructs with functional vasculature has been a particular challenge in engineering tissues suitable for repairing large bone defects. To address this challenge, an extrusion-based direct-writing bioprinting strategy is utilized to fabricate microstructured bone-like tissue constructs containing a perfusable vascular lumen. The bioprinted constructs are used as biomimetic in vitro matrices to co-culture human umbilical vein endothelial cells and bone marrow derived human mesenchymal stem cells in a naturally derived hydrogel. To form the perfusable blood vessel inside the bioprinted construct, a central cylinder with 5% gelatin methacryloyl (GelMA) hydrogel at low methacryloyl substitution (GelMALOW ) was printed. We also develop cell-laden cylinder elements made of GelMA hydrogel loaded with silicate nanoplatelets to induce osteogenesis, and synthesized hydrogel formulations with chemically conjugated vascular endothelial growth factor to promote vascular spreading. It was found that the engineered construct is able to support cell survival and proliferation during maturation in vitro. Additionally, the whole construct demonstrates high structural stability during the in vitro culture for 21 days. This method enables the local control of physical and chemical microniches and the establishment of gradients in the bioprinted constructs.

Mussel-Inspired Multifunctional Hydrogel Coating for Prevention of Infections and Enhanced Osteogenesis.

Prevention of postsurgery infection and promotion of biointegration are the key factors to achieve long-term success in orthopedic implants. Localized delivery of antibiotics and bioactive molecules by the implant surface serves as a promising approach toward these goals. However, previously reported methods for surface functionalization of the titanium alloy implants to load bioactive ingredients suffer from time-consuming complex processes and lack of long-term stability. Here, we present the design and characterization of an adhesive, osteoconductive, and antimicrobial hydrogel coating for Ti implants. To form this multifunctional hydrogel, a photo-cross-linkable gelatin-based hydrogel was modified with catechol motifs to enhance adhesion to Ti surfaces and thus promote coating stability. To induce antimicrobial and osteoconductive properties, a short cationic antimicrobial peptide (AMP) and synthetic silicate nanoparticles (SNs) were introduced into the hydrogel formulation. The controlled release of AMP loaded in the hydrogel demonstrated excellent antimicrobial activity to prevent biofilm formation. Moreover, the addition of SNs to the hydrogel formulation enhanced osteogenesis when cultured with human mesenchymal stem cells, suggesting the potential to promote new bone formation in the surrounding tissues. Considering the unique features of our implant hydrogel coating, including high adhesion, antimicrobial capability, and the ability to induce osteogenesis, it is believed that our design provides a useful alternative method for bone implant surface modification and functionalization.

Biomarkers and diagnostic tools for detection of Helicobacter pylori.

Helicobacter pylori is responsible for worldwide chronic bacterial infection in humans affecting approximately half of the world's population. H. pylori is associated with significant morbidity and mortality including gastric cancer. The infection has both direct and indirect impacts on economic and overall well-being of patients; hence, there is a great need for diagnostic markers that could be used in the development of diagnostic kits. Here, we briefly review general aspects of H. pylori infection and the diagnostic biomarkers used in laboratory tests today with a focus on the potential role of microfluidic systems in future immunodiagnosis platforms.

Adenosine-associated delivery systems.

Adenosine is a naturally occurring purine nucleoside in every cell. Many critical treatments such as modulating irregular heartbeat (arrhythmias), regulation of central nervous system (CNS) activity and inhibiting seizural episodes can be carried out using adenosine. Despite the significant potential therapeutic impact of adenosine and its derivatives, the severe side effects caused by their systemic administration have significantly limited their clinical use. In addition, due to adenosine's extremely short half-life in human blood (<10 s), there is an unmet need for sustained delivery systems to enhance efficacy and reduce side effects. In this article, various adenosine delivery techniques, including encapsulation into biodegradable polymers, cell-based delivery, implantable biomaterials and mechanical-based delivery systems, are critically reviewed and the existing challenges are highlighted.

Chitosan nanoparticles as adenosine carriers.

The objective of this research project was to evaluate the potential use of chitosan (CS) nanoparticles (NPs) as a drug delivery system for the molecule adenosine. Adenosine is an essential drug used for treating several health issues especially irregular heart rhythm. However, due to its extremely short half-life in vivo (<10 s), the effective delivery of adenosine in clinical applications is a significant challenge. In this research, adenosine was encapsulated into NPs formed by ionic gelation of CS. The encapsulation efficiency and loading capacity of 20% and 3% were obtained, respectively, by forming a complex between CS NPs and adenosine. The obtained CS NPs had a spherical shape in the size range of 260.6 ± 20.1 nm. Spectrophotometry analysis of the adenosine released in vitro showed an initial burst release phase, a plateau phase, followed by a steady release over a week.

Multilayered coating on titanium for controlled release of antimicrobial peptides for the prevention of implant-associated infections.

Prevention of bacterial colonization and formation of a bacterial biofilm on implant surfaces has been a challenge in orthopaedic surgery. The treatment of implant-associated infections with conventional antibiotics has become more complicated by the emergence of multi-drug resistant bacteria. Antimicrobial eluting coatings on implants is one of the most promising strategies that have been attempted. This study reports a controlled release of an antimicrobial peptide (AMP) from titanium surface through a non-cytotoxic multilayered coating. Three layers of vertically oriented TiO2 nanotubes, a thin layer of calcium phosphate coating and a phospholipid (POPC) film were impregnated with a potent broad-spectrum AMP (HHC-36). The coating with controlled and sustained release of AMP was highly effective against both Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria. No cytotoxicity to osteoblast-like cells (MG-63) was observed. Moderate platelet activation and adhesion on the implant surface with no observable activation in solution, and very low red blood cell lysis was observed on the implant. This multi-layer assembly can be a potential approach to locally deliver AMPs to prevent peri-implant infection in orthopaedics without being toxic to host cells.

Drug release and bone growth studies of antimicrobial peptide-loaded calcium phosphate coating on titanium.

Preventing infection is one of the major challenges in total hip and joint arthroplasty. The main concerns of local drug delivery as a solution have been the evolution of antibiotic-resistant bacteria and the potential inhibition of osseointegration caused by the delivery systems. This work investigated the in vitro drug release, antimicrobial performance, and cytotoxicity, as well as the in vivo bone growth of an antimicrobial peptide loaded into calcium phosphate coated Ti implants in a rabbit model. Two potent AMP candidates (HHC36: KRWWKWWRR, Tet213: KRWWKWWRRC) were first investigated through an in vitro cytotoxicity assay. MTT absorbance values revealed that HHC36 showed much lower cytotoxicity (minimal cytotoxic concentration 200 µg/mL) than Tet 213 (50 µg/mL). The AMP HHC36 loaded onto CaP (34.7 ± 4.2 µg/cm(2)) had a burst release during the first few hours followed by a slow and steady release for 7 days as measured spectrophotometrically. The CaP-AMP coatings were antimicrobial against Staphylococcus aureus and Pseudomonas aeruginosa strains in colony-forming units (CFU) in vitro assays. No cytotoxicity was observed on CaP-AMP samples against MG-63 osteoblast-like cells after 5 days in vitro. In a trabecular bone growth in vivo study using cylindrical implants, loading of AMP HHC36 did not impair bone growth onto the implants. Significant bone on-growth was observed on CaP-coated Ti with or without HHC36 loading, as compared with Ti alone. The current AMP-CaP coating thus offers in vivo osteoconductivity to orthopedic implants. It also offers in vitro antimicrobial property, with its in vivo performance to be confirmed in future animal infection models.

The biocompatibility and biofilm resistance of implant coatings based on hydrophilic polymer brushes conjugated with antimicrobial peptides.

Bacterial colonization on implant surfaces and subsequent infections are one of the most common reasons for the failure of many indwelling devices. Several approaches including antimicrobial and antibiotic-eluting coatings on implants have been attempted; however, none of these approaches succeed in vivo. Here we report a polymer brush based implant coating that is non-toxic, antimicrobial and biofilm resistant. These coating consists of covalently grafted hydrophilic polymer chains conjugated with an optimized series of antimicrobial peptides (AMPs). These tethered AMPs maintained excellent broad spectrum antimicrobial activity in vitro and in vivo. We found that this specially structured robust coating was extremely effective in resisting biofilm formation, and that the biofilm resistance depended on the nature of conjugated peptides. The coating had no toxicity to osteoblast-like cells and showed insignificant platelet activation and adhesion, and complement activation in human blood. Since such coatings can be applied to most currently used implant surfaces, our approach has significant potential for the development of infection-resistant implants.

Antimicrobial peptides on calcium phosphate-coated titanium for the prevention of implant-associated infections.

Prevention of implant-associated infections has been one of the main challenges in orthopaedic surgery. This challenge is further complicated by the concern over the development of antibiotic resistance as a result of using traditional antibiotics for infection prophylaxis. The objective of this study was to develop a technique that enables the loading and local delivery of a unique group of cationic antimicrobial peptides (AMP) through implant surfaces. A thin layer of micro-porous calcium phosphate (CaP) coating was processed by electrolytic deposition onto the surface of titanium as the drug carrier. The broad spectrum AMP Tet213 (KRWWKWWRRC) was selected and loaded onto the CaP coating. SEM, XRD and FTIR analyses confirmed the CaP coating to be micro-porous octacalcium phosphate. By using a luminescence spectrometer technique, it was demonstrated that a 7 µm thick porous CaP coating could load up to 9 µg of AMP/cm² using a simple soaking technique. The drug-loaded CaP coating (CaP-Tet213) was not cytotoxic for MG-63 osteoblast-like cells. The CaP-Tet213 implants had antimicrobial activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria with 106-fold reductions of both bacterial strains within 30 min as assessed by measuring colony-forming units (CFU). Repeated CFU assays on the same CaP-Tet213 specimen demonstrated retention of antimicrobial activity by the CaP-Tet213 surfaces through four test cycles. The susceptibility of bacteria to the CaP-Tet213 surfaces was also evaluated by assessing the inhibition of luminescence of P. aeruginosa containing a luxCDABE cassette at 4 h and 24 h with ∼92% and ∼77% inhibition of luminescence, respectively. It was demonstrated that CaP-Tet213 was a more efficient antimicrobial coating than CaP-MX226, CaP-hLF1-11 or CaP-tobramycin following incubation of CaP implants with equimolar concentrations of Tet213, the commercially developed antimicrobial peptide MX-226, hLF1-11 or tobramycin. A device coated with CaP-Tet213 could be a potential solution for the prevention of the peri-implant infection in orthopaedics.