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

Abstract

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 antiinfective coating strategies to meet a range of clinical challenges where the risks for S. aureus prosthetic infections range from transient to long-lasting.

Triggered Release of Ampicillin from Metallic Implant Coatings for Combating Periprosthetic Infections

“…Upon treating the Ti6Al4V pin surfaces with DopaMA, Oligo 5′-carboxy-mCmGTTmCmG-3′-acrydite was UV-cross-linked with PEGDMA to form a coherent and uniform hydrogel coating around the pin (Scheme B, top row). This Oligo sequence was previously validated for selective cleavage by MN at the T–T phosphodiester linkage, with its 3′-acrydite end responsible for copolymerizing with the hydrogel while the 5′-carboxylate end subsequently conjugating with the amino group of ampicillin. ,, Unincorporated Oligo was thoroughly washed out with sterile DI water, and the UV absorption at 260 nm of the aqueous wash solution after 24 h revealed that ∼90% Oligo effectively incorporated into the hydrogel. Ampicillin was then covalently coupled to the 5′ end of the tethered Oligo via amidation upon EDC/sNHS activation of the 5′-carboxyl end (Scheme B, middle row).…”

“…A rat femoral canal infection model was used to evaluate the ability of the Oligo-Amp hydrogel coated Ti6Al4V IM pins (Ti-Oligo-Amp + S. aureus) to prevent S.…”

“…Such a S. aureus inoculation dose mimics implant-related infections that occur in a controlled operating room environment instead of those arising from gross contaminations . PEGDMA control hydrogel coatings applied to the IM pins without covalently tethered Oligo and ampicillin were used in infected (Ti-Ctl + S.…”

“…The rat femoral IM pin infection model was surgically created following the procedures we previously described . Briefly, after a skin incision was made over the knee joint of an anesthetized rat, the patella and patellar tendon were dislocated, revealing the femoral intercondylar notch.…”

“…The MN-triggered vancomycin release effectively cleared S. aureus inoculated in a mouse or rat femoral canal [40–1000 colony-forming units (cfu)] in a timely manner before the bacteria had a chance to colonize on intramedullary (IM) titanium alloy pins or invade surrounding bone matrices. , Additionally, we demonstrated that the on-demand release kinetics of vancomycin could be modulated by incorporating phosphorothioate modifications into the Oligo linker …”