Enhanced Clearing of Wound-Related Pathogenic Bacterial Biofilms Using Protease-Functionalized Antibiotic Nanocarriers

Abstract

Biofilms are prevalent in chronic wounds and once formed are very hard to remove, which is associated with poor outcomes and high mortality rates. Biofilms are comprised of surface-attached bacteria embedded in an extracellular polymeric substance (EPS) matrix, which confers increased antibiotic resistance and host immune evasion. Therefore, disruption of this matrix is essential to tackle the biofilm-embedded bacteria. Here, we propose a novel nanotechnology to do this, based on protease-functionalized nanogel carriers of antibiotics. Such active antibiotic nanocarriers, surface coated with the protease Alcalase 2.4 L FG, "digest" their way through the biofilm EPS matrix, reach the buried bacteria, and deliver a high dose of antibiotic directly on their cell walls, which overwhelms their defenses. We demonstrated their effectiveness against six wound biofilm-forming bacteria, <i>Staphylococcus aureus</i>, <i>Pseudomonas aeruginosa</i>, <i>Staphylococcus epidermidis</i>, <i>Klebsiella pneumoniae</i>, <i>Escherichia coli</i>, and <i>Enterococcus faecalis</i>. We confirmed a 6-fold decrease in the biofilm mass and a substantial reduction in bacterial cell density using fluorescence, atomic force, and scanning electron microscopy. Additionally, we showed that co-treatments of ciprofloxacin and Alcalase-coated Carbopol nanogels led to a 3-log reduction in viable biofilm-forming cells when compared to ciprofloxacin treatments alone. Encapsulating an equivalent concentration of ciprofloxacin into the Alcalase-coated nanogel particles boosted their antibacterial effect much further, reducing the bacterial cell viability to below detectable amounts after 6 h of treatment. The Alcalase-coated nanogel particles were noncytotoxic to human adult keratinocyte cells (HaCaT), inducing a very low apoptotic response in these cells. Overall, we demonstrated that the Alcalase-coated nanogels loaded with a cationic antibiotic elicit very strong biofilm-clearing effects against wound-associated biofilm-forming pathogenic bacteria. This nanotechnology approach has the potential to become a very powerful treatment of chronically infected wounds with biofilm-forming bacteria.