Elsevier

Biomaterials

Volume 34, Issue 38, December 2013, Pages 10328-10337
Biomaterials

Bioconjugated nanoparticles for attachment and penetration into pathogenic bacteria

https://doi.org/10.1016/j.biomaterials.2013.09.045Get rights and content

Abstract

As an antimicrobial agent, silver nanoparticles functionalized with both bacitracin A and polymyxin E (AgNPs-BA&PE) were designed and synthesized with complementary antibacterial functions to act against gram-positive and gram-negative bacteria. AgNPs-BA&PE could easily get attached and penetrate into the bacterial cell membrane through surface-immobilized BA and PE with a membrane target, resulting in up to 10-fold increase in the antibacterial activity, without the emergence of bacterial resistance. Analysis of the antimicrobial mechanism confirmed that the synthesized nanoparticles caused disorganization of the bacterial cytomembrane and leakage of cytoplasmic contents. This antimicrobial agent with better biocompatibility can promote healing of infected wounds, and has promising and useful applications in biomedical devices and antibacterial control systems.

Introduction

Infectious diseases induced by the bacteria continue to be one of the greatest health challenges worldwide. The introduction of antibiotics has made a striking impact on the treatment of infectious diseases and has dramatically decreased mortality. However, emergence of multiple antibiotics-resistant bacteria poses a new threat to human health [1], [2]. Therefore, the design and development of new antimicrobial agents that have high antibacterial activity and low propensity to induce resistance are crucial. Nanomaterials, as new antimicrobial agents, which are capable of getting attached to the bacterial membrane and disrupting its integrity, have attracted increasing attention in the field of biomedicine [3]. To date, many kinds of antibacterial nanoparticles, such as carbon nanotubes [4], fullerene [5], metal oxide nanoparticles [6], and metallic nanoparticles [7] have been examined. Among them, carbon nanotubes and fullerene C60 are found to exhibit strong antibacterial properties. However, tedious synthesis procedure, high cost, and use of large amounts of organic solvents have hampered their further applications. Although metal oxide nanoparticles obtained by aqueous synthesis also exhibit antimicrobial activities, problems such as induction of bacterial resistance (e.g. TiO2 and ZnO nanoparticles) and requirement of UV illumination for antibacterial actions (e.g. TiO2 nanoparticles) are still not resolved [6].

When compared with the above-mentioned nanomaterials, silver nanoparticles (AgNPs) are the most exceptional antimicrobial agents among metallic nanoparticles used against different bacteria, viruses, and fungi, with minimal perturbation to human cells and low propensity to induce bacterial resistance [3], [8]. AgNPs can increase the permeability of bacterial cell membrane, penetrate into the cytoplasm [9], and inactivate essential respiratory enzymes and proteins responsible for RNA and DNA replication, leading to bacterial death [7]. Unfortunately, these antibacterial actions of AgNPs are often dependent on high concentration because of the random physical collision of AgNPs with the bacterial surface, leading to penetration of AgNPs into the cytoplasm [10]. Recent studies have indicated that cationic polymers-stabilized AgNPs with positively charged surface can easily bind to the negatively charged bacterial surface through non-specific electrostatic interaction [11]. However, toxicity is still an obstacle to the biomedical applications of cationic polymers [12], [13].

It is generally accepted that antimicrobial agents have single or multiple target sites within the microbial cell, and that damage to these target sites results in the antibacterial effect [14]. The major target sites for antimicrobial agents are often present at the outermost layers of the bacterial cell. In order to be effective, an antimicrobial agent should reach and interact with its microbial target site(s). In recent years, antimicrobial peptides (AMPs) have been demonstrated to be excellent antimicrobial agents for the treatment of multidrug-resistant infections [15], [16]. The macrocyclic amido groups of some AMPs chelate Mg2+ and Ca2+ of the bacterial cell surface to easily fuse into the bacteria and disrupt membrane organization, eventually leading to bacterial death [17], [18]. AMPs, acting as “a molecular knife” by inserting into and damaging the bacterial cell membrane, mediate the penetration of AgNPs into the bacteria [19]. Moreover, AMPs serve as stabilizers protecting AgNPs against agglomeration [20] and achieve polyvalent effects by getting concentrated on the surface of AgNPs [21]. In addition, the concurrent emergence of multiple antimicrobial agents in a single antimicrobial agent does not induce bacterial resistance [22]. To the best of our knowledge, the use of AMPs-functionalized AgNPs as antimicrobial agents against gram-positive and gram-negative bacteria has not yet been reported.

In this study, we designed and synthesized AMPs-functionalized AgNPs as an antimicrobial nanomaterial. We chose bacitracin A (BA) and polymyxin E (PE) with macrocyclic amido groups as the target molecules from commercial AMPs. BA and PE have potent bactericidal activity directed primarily against gram-positive and gram-negative bacteria, respectively. Both gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and gram-positive bacteria (Staphylococcus aureus and Bacillus amyloliquefaciens) were used as bacterial models. To achieve highly efficient antimicrobial activity, the synthesis conditions of AgNPs functionalized with both BA and PE (AgNPs-BA&PE) were optimized comprehensively. The minimum inhibitory concentration (MIC), fractional inhibitory concentration (FIC), zone of inhibition test, and LIVE/DEAD bacterial viability assay were used to characterize the antibacterial activity of AgNPs-BA&PE and the synergistic effects between AgNPs and AMPs. Furthermore, the mechanism of antibacterial action of AgNPs-BA&PE was also systematically studied. Finally, we further explored the biocompatibility of AgNPs-BA&PE using mouse fibroblast cells (NIH3T3 cells) and determined the effect of this antimicrobial agent on the treatment of bacteria-induced wound infection.

Section snippets

Materials

Silver nitrate (AgNO3, 99.995%, metals basis, Ag 63% min) and sodium borohydride (NaBH4, 98% min) were purchased from Alfa Aesar (Ward Hill, MA, US). BA (from Bacillus licheniformis, ≥50,000 U/g) and PE (Colistin sulfate salt, ≥15,000 U/mg) were from Sigma–Aldrich (St. Louis, MO, US). E. coli ATCC 8739, P. aeruginosa ATCC 9027, S. aureus ATCC 6538 and B. amyloliquefaciens ATCC 23842 strains were provided by Department of Microbiology of Nankai University (Tianjin, China). The ultrapure water

Results and discussion

AgNPs-BA&PE were synthesized via reduction of AgNO3 by NaBH4 in the presence of BA and PE, respectively, in water. The amine groups of BA and PE were observed to assemble silver cations and then cap the growing AgNPs surface following reduction of the cations. As a result, co-localized BA and PE exhibited complementary antimicrobial functions toward gram-positive and gram-negative bacteria.

Conclusions

An antimicrobial agent with high efficient antibacterial effect was designed and synthesized by simply conjugating AgNPs with AMPs. BA and PE of the AgNPs-BA&PE surface were found to have the ability to attack biological targets on the bacterial cell membranes and mediate AgNPs to get attached and penetrate into the bacteria. AgNPs-BA&PE efficiently inhibited the growth of gram-positive and gram-negative bacteria, and did not induce resistance in bacteria. These nanoparticles were observed to

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant No. 21174071 and 81170773) and the Program for Changjiang Scholars and Innovative Research Team in University (IRT 1257).

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