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Biomaterials

Biomaterials - Poster Session D

Poster C3 - Polymer coated gelatin nanoparticles as an antibiotic-free treatment for Staphylococcus epidermidis infections

Friday, October 25, 2024
3:30 PM - 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  More than 2.8 million antimicrobial-resistant infections occur each year in the United States.1 Among these, healthcare-associated infections pose a significant challenge. Staphylococcus epidermidis, a major healthcare-associated bacterium, commonly causes infections related to implanted medical devices like catheters and prostheses. Consequently, physicians must remove and replace the infected device, which increases both the cost and morbidity for patients.2 Furthermore, the formation of biofilms by S. epidermidis on these devices complicates treatment, as biofilms exhibit both resistance and tolerance to antibiotics, making them difficult to eradicate. This growing concern of antibiotic resistance and tolerance necessitates the development of new approaches to combat these infections. One promising strategy is the use of nanotechnology, which shows potential in treating these recalcitrant bacterial infections. Nanoparticles (NPs) can act as carriers for the delivery of antimicrobial agents due to their distinct size and physical characteristics, which enable them to target microbes and treat resistant pathogens. Here, we designed polymer-decorated gelatin NPs (P-GNPs) to treat S. epidermidis infections. We hypothesized that P-GNPs would exhibit enhanced bacterial inhibition compared to the free polymer or GNPs alone and serve as an antibiotic-free approach to eradicate the infections (Figure 1a).

Materials and

Methods:  A pH-responsive copolymer, methoxy PEG-benzoic-imine-branched polyethyleneimine (PEG-b-i-bPEI), was first synthesized and characterized as reported (Figure 1b).3 The P-GNPs were synthesized through a two-step process (Figure 1c). First, the gelatin nanoparticles (GNPs) were synthesized via a desolvation method and crosslinked with glutaraldehyde.4 Secondly, the PEG-b-i-PEI copolymer was conjugated to the GNPs via a coupling reaction using 1-ethyl-3-(3-dimethylamino)propyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), forming the P-GNPs. The success of copolymer conjugation was validated via Fourier transform infrared spectroscopy (FTIR) measurements. The amount of copolymer conjugated to the GNPs was quantified via optical density measurement using a standard curve. The morphology and size of the NPs at each step were assessed using transmission electron microscopy (TEM) and dynamic light scattering (DLS), respectively. We also tested the antimicrobial efficacy and cytotoxicity in vitro. The minimum inhibitory concentration (MIC) was examined using microdilution assays against S. epidermidis 12228, and the cytotoxicity of the NPs was monitored via cell count kit-8 (CCK-8) using fibroblast cells (NIH 3T3).

Results, Conclusions, and Discussions: In this work, we engineered antibiotic-free NPs that exhibit antibacterial properties against S. epidermidis. TEM images indicate uniform, spherical shapes (Figures 1d and e). DLS analysis showed a significant increase in the NP size and polydispersity index (PDI), indicating successful copolymer conjugation (Figure 1f and g). FTIR analysis showed a characteristic peak corresponding to the C-O-C stretching vibration at 1100 cm-1, which appears in the copolymer and P-GNP spectrum but is absent in the GNP spectrum, further confirming the conjugation of the copolymer to the GNPs (Figure 1h). Additionally, both the P-GNP and GNP formulations exhibited a net positive zeta potential when suspended in water (Figure 1i). The antibacterial activity of P-GNP was assessed in vitro and compared to that of bPEI, PEG, PEG-b-i-PEI, and GNP (Figure 1j and k). At 25.6 μg/mL, PEG-b-i-PEI exhibited a 40% inhibitory effect, whereas P-GNP showed improved antimicrobial efficacy with an 80% inhibitory effect. bPEI, PEG, and GNP did not show bacterial inhibitory effects at any tested concentrations. We further investigated the synergistic effect of the formulation. Interestingly, significant antibacterial activity was observed only with the P-GNP formulation, whereas a mixture of blank GNPs and free polymers (i.e., GNP+PEG+bPEI) or free copolymers (i.e., GNP+Copolymer) did not show bacterial inhibitory effects within the tested concentrations (Figure 1k). This indicates that the conjugation of the copolymer to the GNP is essential for antibacterial efficacy. These results suggest synergistic effects within the P-GNP system, highlighting the necessity of the formulation design. Furthermore, all tested formulations demonstrated minimal cytotoxicity in interactions with fibroblast cells (Figure 1l). In this research, we synthesized an antibiotic-free NP formulation and demonstrated its ability to inhibit the growth of S. epidermidis. Our NP formulation serves as a promising alternative approach for combating antibiotic-resistant infections.

Acknowledgements (Optional):  We would like to acknowledge the financial support from the donors of Brown University’s Undergraduate Teaching and Research Awards and Professor Robert Hurt at Brown for the use of his DLS. We thank Anthony McCormick from the Brown University Electron Microscopy Core Facility for his assistance with TEM.