Dual stimuli-responsive silver loaded nanoparticles eliminate bacterial biofilms

Introduction:: Biofilm infections are extremely common and difficult to eradicate. Treatment with traditional antibiotic therapy is often ineffective due to rising antibiotic resistance and poor penetration into the biofilm. Nanoparticles (NPs) are promising drug-delivery vehicles for the targeted delivery of antimicrobial agents into biofilms. We have previously found that specific formulations of gelatin nanoparticles (GNPs) can effectively disrupt biofilms.¹ Due to the production of gelatinases by many common biofilm pathogens and at infection sites, GNPs are hypothesized to degrade at the site of infection to release loaded therapeutics. In this work, we developed GNP formulations for the stimuli-responsive release of antibacterial and antibiofilm agents. These GNPs contain silver nanoparticles (AgNPs) within the gelatinase-responsive core, as a broadspectrum antibiotic alternative. We conjugated deoxyribonuclease I (DNase I) to the GNPs; DNase I exhibits antibiofilm properties by degrading extracellular DNA, a major component of the biofilm EPS. To protect DNase stability until it encounters the biofilm microenvironment, we conjugated a pH-degradable poly(ethylene glycol) (PEG) derivative to the DNase-coated GNPs. We hypothesized that the low pH at the biofilm site, will cause the coating to be shed, exposing the DNase (Fig.1a). This work presents a dual-stimuli responsive (i.e., pH and enzyme-responsive) strategy to combat bacterial biofilms by locally delivering a combination treatment of antibiofilm and antibacterial agents.


Materials and Methods:: The pH-responsive copolymer, methoxy PEG-benzoic-imine-branched polyethyleneimine (mPEG-b-i-bPEI) was synthesized as previously reported.² The successful synthesis of mPEG-b-i-bPEI was verified with Fourier-transform infrared spectroscopy and nuclear magnetic resonance spectroscopy. The functionalized GNPs were synthesized through a three-step synthesis (Fig.1b). First, GNPs were prepared via a two-step desolvation method using glutaraldehyde as the crosslinker.¹ Then, AgNP-loaded GNPs (Ag-GNPs) were prepared by adding a solution of silver nitrate (AgNO₃) to the GNP suspension, followed by a sodium borohydride (NaBH₄) mediated reduction yielding AgNP formation within the GNPs. Finally, the Ag-GNPs were coated with DNase I and the pH-responsive copolymer via a two-step coupling reaction using 1-ethyl-3-(3-dimethylamino)propyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), yielding the complete NP formulation (P-D-Ag-GNP). The morphology and size of the NPs at each step were assessed using transmission electron microscopy (TEM) and dynamic light scattering (DLS), respectively. Thermogravimetric analysis (TGA) was used to quantify the amount of Ag in the GNPs, and NP composition was confirmed with energy-dispersive X-ray spectroscopy (EDX). The pH of the biofilm environment was measured using a pH probe, and the gelatinase activity of the biofilm was confirmed via a gelatin hydrolysis test, as previously reported.³ The minimal biofilm eradication concentration to eradicate 80% of biofilm biomass (MBEC₈₀) was examined with crystal violet (CV) assays against Staphylococcus aureus 25923, S. epidermidis 12228, and Pseudomonas aeruginosa PA01. Finally, the cytotoxicity of the NPs was monitored via a hemolysis assay against 5% (v/v) human red blood cells (RBCs).


Results, Conclusions, and Discussions:: In this work, we engineered NPs that exhibit pH- and bacterial enzyme-triggered drug release, resulting in improved antibiofilm efficacy. DLS showed an increase in NP hydrodynamic diameter with AgNP loading and DNase I functionalization, and further increase upon the addition of the pH-responsive polymer (Fig.1c). TEM imaging (Fig.1d) and EDX analysis (data not shown) confirmed that AgNPs were successfully loaded into the GNPs. TGA analysis suggested that AgNPs contribute to ∼30% of the total Ag-GNP weight. In investigating biofilm properties for different bacterial species, we found that S. aureus and S. epidermidis produced acidic biofilm environments, while P. aeruginosa exhibited a basic biofilm environment (Fig.1e). S. aureus and P. aeruginosa produced gelatinases, while S. epidermidis did not. CV staining showed that both S. aureus and S. epidermidis biofilm biomass decreased upon treatment with increasing concentrations of P-D-Ag-GNPs (MBEC₈₀ = 34 μg/mL for both) (Fig.1f), whereas P. aeruginosa biofilm biomass increased in response to P-D-Ag-GNP treatment. All NP formulations exhibited minimal hemolysis, and the DNase I and responsive polymer coating reduced hemolysis compared to Ag-GNP (i.e., at the highest concentration of NPs tested (500 μg/mL), we noted 25.45 ± 7.63 % and 4.45 ± 1.90 % hemolysis for Ag-GNP and P-D-AgGNP, respectively) (Fig.1g). Overall, the responsive NP formulation demonstrated significant eradication of biofilms that exhibited acidic environments (i.e., S. aureus and S. epidermidis). At low P-D-Ag-GNP concentrations, the lack of gelatinase production by S. epidermidis appeared to reduce biofilm eradication efficacy compared to what was observed for S. aureus. Additionally, no eradication of the basic P. aeruginosa biofilm was observed, despite gelatinase production, supporting the hypothesized mechanism of action of the P-D-Ag-GNPs.




Acknowledgements (Optional): : Financial support from the Office of Naval Research (N00014-22-1-2336) is acknowledged. We thank Professor Robert Hurt at Brown University for the use of his Zetasizer. We thank Anthony McCormick from the Brown University Electron Microscopy Core Facility for his assistance with TEM and EDX. We thank Dr. Indrek Külaots and Zachary Saleeba for their assistance with TGA.


References (Optional): : [1] Yingying Wang and Anita Shukla. Bacteria-responsive biopolymer-coated nanoparticles for biofilm penetration and eradication. Biomaterials Science, pages 2831–2843, 2022.
[2] Nayeleh Deirram, Changhe Zhang, Sarah S. Kermaniyan, Angus P.R. Johnston, and Georgina K. Such. pH-Responsive Polymer Nanoparticles for Drug Delivery. Macromolecular Rapid Communications, 40(10):1–23, 2019.
[3] Thomas Edison E. dela Cruz and Jeremy Martin O. Torres. Gelatin Hydrolysis Test Protocol. American Society for Microbiology, pages 1–10, 2012.