Modulating On-Demand Release of Vancomycin from Implant Coatings via Chemical Modification of a Micrococcal Nuclease-Sensitive Oligonucleotide Linker.

Periprosthetic infections are one of the most serious complications in orthopedic surgeries, and those caused by Staphylococcus aureus (S. aureus) are particularly hard to treat due to their tendency to form biofilms on implants and their notorious ability to invade the surrounding bones. The existing prophylactic local antibiotic deliveries involve excessive drug loading doses that could risk the development of drug resistance strains. Utilizing an oligonucleotide linker sensitive to micrococcal nuclease (MN) cleavage, we previously developed an implant coating capable of releasing covalently tethered vancomycin, triggered by S. aureus-secreted MN, to prevent periprosthetic infections in the mouse intramedullary (IM) canal. To further engineer this exciting platform to meet broader clinical needs, here, we chemically modified the oligonucleotide linker by a combination of 2'-O-methylation and phosphorothioate modification to achieve additional modulation of its stability/sensitivity to MN and the kinetics of MN-triggered on-demand release. We found that when all phosphodiester bonds within the oligonucleotide linker 5'-carboxy-mCmGTTmCmG-3-acrydite, except for the one between TT, were replaced by phosphorothioate, the oligonucleotide (6PS) stability significantly increased and enabled the most sustained release of tethered vancomycin from the coating. By contrast, when only the peripheral phosphodiester bonds at the 5'- and 3'-ends were replaced by phosphorothioate, the resulting oligonucleotide (2PS) linker was cleaved by MN more rapidly than that without any PS modifications (0PS). Using a rat femoral canal periprosthetic infection model where 1000 CFU S. aureus was inoculated at the time of IM pin insertion, we showed that the prophylactic implant coating containing either 0PS- or 2PS-modified oligonucleotide linker effectively eradicated the bacteria by enabling the rapid on-demand release of vancomycin. No bacteria were detected from the explanted pins, and no signs of cortical bone changes were detected in these treatment groups throughout the 3 month follow-ups. With an antibiotic tethering dose significantly lower than conventional antibiotic-bearing bone cements, these coatings also exhibited excellent biocompatibility. These chemically modified oligonucleotides could help tailor prophylactic anti-infective coating strategies to meet a range of clinical challenges where the risks for S. aureus prosthetic infections range from transient to long-lasting.

Anti-Periprosthetic Infection Strategies: From Implant Surface Topographical Engineering to Smart Drug-Releasing Coatings.

Despite advanced implant sterilization and aseptic surgical techniques, periprosthetic bacterial infection remains a major challenge for orthopedic and dental implants. Bacterial colonization/biofilm formation around implants and their invasion into the dense skeletal tissue matrices are difficult to treat and could lead to implant failure and osteomyelitis. These complications require major revision surgeries and extended antibiotic therapies that are associated with high treatment cost, morbidity, and even mortality. Effective preventative measures mitigating risks for implant-related infections are thus in dire need. This review focuses on recent developments of anti-periprosthetic infection strategies aimed at either reducing bacterial adhesion, colonization, and biofilm formation or killing bacteria directly in contact with and/or in the vicinity of implants. These goals are accomplished through antifouling, quorum-sensing interfering, or bactericidal implant surface topographical engineering or surface coatings through chemical modifications. Surface topographical engineering of lotus leaf mimicking super-hydrophobic antifouling features and cicada wing-mimicking, bacterium-piercing nanopillars are both presented. Conventional physical coating/passive release of bactericidal agents is contrasted with their covalent tethering to implant surfaces through either stable linkages or linkages labile to bacterial enzyme cleavage or environmental perturbations. Pros and cons of these emerging anti-periprosthetic infection approaches are discussed in terms of their safety, efficacy, and translational potentials.

Micrococcal-Nuclease-Triggered On-Demand Release of Vancomycin from Intramedullary Implant Coating Eradicates Staphylococcus aureus Infection in Mouse Femoral Canals.

Preventing orthopedic implant-associated bacterial infections remains a critical challenge. Current practices of physically blending high-dose antibiotics with bone cements is known for cytotoxicity while covalently tethering antibiotics to implant surfaces is ineffective in eradicating bacteria from the periprosthetic tissue environment due to the short-range bactericidal actions, which are limited to the implant surface. Here, we covalently functionalize poly(ethylene glycol) dimethacrylate hydrogel coatings with vancomycin via an oligonucleotide linker sensitive to Staphylococcus aureus (S. aureus) micrococcal nuclease (MN) (PEGDMA-Oligo-Vanco). This design enables the timely release of vancomycin in the presence of S. aureus to kill the bacteria both on the implant surface and within the periprosthetic tissue environment. Ti6Al4V intramedullary (IM) pins surface-tethered with dopamine methacrylamide (DopaMA) and uniformly coated with PEGDMA-Oligo-Vanco effectively prevented periprosthetic infections in mouse femoral canals inoculated with bioluminescent S. aureus. Longitudinal bioluminescence monitoring, µCT quantification of femoral bone changes, end point quantification of implant surface bacteria, and histological detection of S. aureus in the periprosthetic tissue environment confirmed rapid and sustained bacterial clearance by the PEGDMA-Oligo-Vanco coating. The observed eradication of bacteria was in stark contrast with the significant bacterial colonization on implants and osteomyelitis development found in the absence of the MN-sensitive bactericidal coating. The effective vancomycin tethering dose presented in this on-demand release strategy was >200 times lower than the typical prophylactic antibiotic contents used in bone cements and may be applied to medical implants and bone/dental cements to prevent periprosthetic infections in high-risk clinical scenarios. This study also supports the timely bactericidal action by MN-triggered release of antibiotics as an effective prophylactic method to bypass the notoriously harder to treat periprosthetic biofilms and osteomyelitis.

Three-Dimensional, Enzyme Biohydrogel Electrode for Improved Bioelectrocatalysis.

Higher loading of enzymes on electrodes and efficient electron transfer from the enzyme to the electrode are urgently needed to enhance the current density of biofuel cells. The two-dimensional nature of the electrode surface limits the enzyme loading on the surface, and unfavorable interactions with electrode surfaces cause inactivation of the enzyme. Benign biohydrogels are designed here to address enzyme degradation, and the three-dimensional nature of the biohydrogel enhanced the enzyme density per unit area. A general strategy is demonstrated here using a redox active enzyme glucose oxidase embedded in a bovine serum albumin biohydrogel on flexible carbon cloth electrodes. In the presence of ferricyanide as a mediator, this bioelectrode generated a maximum current density (jmax) of 13.2 mA·cm-2 at 0.45 V in the presence of glucose with a sensitivity of 67 µA·mol-1·cm-2 and a half-life of >2 weeks at room temperature. A strong correlation of current density with water uptake by the biohydrogel was observed. Moreover, a soluble mediator (sodium ferricyanide) in the biohydrogel enhanced the current density by ∼1000-fold, and citrate-phosphate buffer has been found to be the best to achieve the maximum current density. A record 2.2% of the loaded enzyme was electroactive, which is greater than the highest value reported (2-fold). Stabilization of the enzyme in the biohydrogel resulted in retention of the enzymatic activity over a wide range of pH (4.0-8.0). We showed here that biohydrogels are excellent media for enzymatic electron transfer reactions required for bioelectronics and biofuel cell applications.

"Stable-on-the-Table" Biosensors: Hemoglobin-Poly (Acrylic Acid) Nanogel BioElectrodes with High Thermal Stability and Enhanced Electroactivity.

In our efforts toward producing environmentally responsible but highly stable bioelectrodes with high electroactivities, we report here a simple, inexpensive, autoclavable high sensitivity biosensor based on enzyme-polymer nanogels. Met-hemoglobin (Hb) is stabilized by wrapping it in high molecular weight poly(acrylic acid) (PAA, M(W) 450k), and the resulting nanogels abbreviated as Hb-PAA-450k, withstood exposure to high temperatures for extended periods under steam sterilization conditions (122 °C, 10 min, 17-20 psi) without loss of Hb structure or its peroxidase-like activities. The bioelectrodes prepared by coating Hb-PAA-450k nanogels on glassy carbon showed well-defined quasi-reversible redox peaks at -0.279 and -0.334 V in cyclic voltammetry (CV) and retained >95% electroactivity after storing for 14 days at room temperature. Similarly, the bioelectrode showed ~90% retention in electrochemical properties after autoclaving under steam sterilization conditions. The ultra stable bioelectrode was used to detect hydrogen peroxide and demonstrated an excellent detection limit of 0.5 µM, the best among the Hb-based electrochemical biosensors. This is the first electrochemical demonstration of steam-sterilizable, storable, modular bioelectrode that undergoes reversible-thermal denaturation and retains electroactivity for protein based electrochemical applications.

Proton-coupled protein binding: controlling lysozyme/poly(acrylic acid) interactions with pH.

Rational design of protein-polymer composites and their use, under the influence of the stimulus, for numerous applications requires a clear understanding of protein-polymer interfaces. Here, using poly(acrylic acid) (PAA) and lysozyme as model systems, the binding interactions between these macromolecules were investigated by isothermal titration calorimetry. The binding is proposed to require and be governed by "charge neutralization of the protein/polymer interface" and predicted to depend on solution pH. Calorimetric data show strong exothermic binding of lysozyme to PAA with a molar ΔH and TΔS values of -107 and -95 kcal/mol, respectively, at pH 7 and room temperature. Both ΔH and TΔS decreased linearly with increasing pH from 3 to 8, and these plots had slopes of -17.7 and -17.5 kcal/mol per pH unit, respectively. The net result was that the binding propensity (ΔG) was nearly independent of pH but the binding stoichiometry, surprisingly, increased rapidly with increasing pH from 1 lysozyme binding per PAA molecule at pH 3 to 16 lysozyme molecules binding per PAA molecule at pH 8. A plot of stoichiometry vs pH was linear, and consistent with this result, a plot of ln(average size of the protein/polymer complex) vs pH was also linear. Thus, protonation-deprotonation plays a major role in the binding mechanism. "Charge neutralization" of the lysozyme/PAA interface controls the binding stoichiometry as well as the binding enthalpies/entropies in a predictable fashion, but it did not control the binding affinity (ΔG). The pH dependence of lysozyme binding to PAA, demonstrated here, provides a stimuli-responsive system for protein binding and release from the polymer surface.