(225d) Antibiofilm Activity of Enzyme and pH Responsive Layered Gelatin Nanoparticles

Biofilm-associated bacterial infections are one of the greatest threats to health worldwide. Biofilms exhibit a variety of mechanisms that contribute to antimicrobial resistance. Responsive nanoparticles (NPs) that target specific microbial and biofilm features show great potential to prevent and eradicate biofilms. However, developing smart drug delivery systems which respond to multiple bacteria stimuli, thereby increasing specificity to biofilms, remains a challenge. Here, we have developed a hyaluronic acid (HA) and chitosan (CS) coated doxycycline (Doxy) loaded gelatin nanoparticle (GNP) that responds to both bacterial enzymes and changes in pH. Under neutral physiological conditions, the negative charge of the HA coated NPs is expected to enhance blood circulation times when introduced systemically. At biofilm infection sites, bacterial hyaluronidases will degrade the outermost HA layer exposing the underlying CS layer. The NPs will then become positively charged and have greater ability to attach to biofilm bacteria. The acidic biofilm microenvironment will trigger the CS layer to swell providing bacterial gelatinases greater access to the GNP core. Degradation of the GNP will increase Doxy release from the NP, leading to efficient bacteria death and biofilm eradication. Compared to traditional antibiotics, these biopolymer-coated GNPs have the potential to specifically accumulate and deeply penetrate biofilm, which could eradicate bacterial biofilms after controlled release of loaded antibiotics.

By optimizing fabrication conditions (e.g., polymer concentration, assembly time, and pH), GNP, Doxy-GNP, CS-Doxy-GNP and HA-CS-Doxy-GNP were successfully prepared. The average hydrodynamic diameter increased with additional polymer coating layers, from ~215 nm for Doxy-GNP to ~243 nm and ~292 nm for CS-Doxy-GNP and HA-CS-Doxy-GNP, respectively. Additionally, zeta-potential confirmed successful coating, showing charge reversal between uncoated NPs and CS versus HA and CS coated NPs. The encapsulation efficiency and drug loading capacity of HA-CS-Doxy-GNPs was ~25.8% and 6%, respectively. Hyaluronidase treatment led to the most rapid Doxy release kinetics from HA-CS-Doxy-GNP at the conditions tested (150 U/mL hyaluronidase at pH 5), releasing ~40 % of the encapsulated Doxy in 48 hours, compared to only ~18.5 % for NPs in pH 7.4 buffer. These release kinetics were comparable to what was observed when these NPs were incubated in media from a culture of Vibrio vulnificus, a highly virulent gram-negative bacterial species. This media was expected to contain hyaluronidases, gelatinases from V. vulnificus, with a pH of ~5.5. The biofilm penetration ability of HA-CS-GNP was evaluated against pre-formed V. vulnificus biofilms (matured for 48 hours). The eradication ability of HA-CS-Doxy-GNP against V. vulnificus biofilm was demonstrated using crystal violet staining, scanning electron microscopy, live/dead staining, and colony counting. Results indicated that HA-CS-Doxy-GNP effectively disperse and eliminate V. vulnificus biofilm (nearly 100% elimination of viable bacteria). The biocompatibility of NPs was explored using a cell counting kit-8 assay (CCK-8) assay for human umbilical vein endothelial cells (HUVECs) and NIH 3T3 murine fibroblasts. We found that cells exposed to Doxy loaded NPs for 24 hours displayed lower cytotoxicity than the equivalent amount of free Doxy.

In summary, we have developed a triple stimuli-responsive HA-CS-Doxy-GNP and demonstrated its antibacterial and antibiofilm properties in vitro. This bacteria-responsive drug delivery system has the potential to be used to deliver multiple drugs including those aimed specifically at biofilms (e.g., antibiofilm peptides) or signaling molecules for infection detection (e.g., fluorescent dyes) for the effective broad-spectrum treatment and detection of infections.