Graphene oxide with acid-activated bacterial membrane anchoring for improving synergistic antibacterial performances

Highlights

• Azithromycin (AZI) was loaded on the graphene oxide (GO) surface.

• Polyethyleneimine (PEI) increased the anchoring of AZI@GO to bacteria.

• Physical film breaking of GO synergistic azithromycin sterilization.

Abstract

Bacterial resistance toward antibiotics has become a major threat to current anti-infective therapy, and now it's very urgent to develop new therapeutic drugs and coping strategies for overcoming bacterial resistance. Although graphene oxide (GO)-based nanocomposite has been widely used as antibacterial material, its antibacterial activity is still low and needs to be improved. Herein, we have presented a synergistic antibacterial agent, polyethyleneimine-citraconic anhydride modified and azithromycin-loaded GO nanosheet (AZI@GO-PEI-CA), which could selectively anchor bacterial membrane and improve antibacterial activity. The primary amine groups of polyethyleneimine (PEI) react with citraconic anhydride (CA), but could be recovered at acidic condition, which not only could increase its biocompatibility at physiological condition due to almost neutral charge of AZI@GO-PEI-CA after the introduction of CA, but also can enhance the anchoring of bacteria because AZI@GO-PEI-CA become highly positive after removal of CA under the acidic inflammatory microenvironment. Azithromycin (AZI) was conjugated onto GO due to that GO can not only physically insert the membrane of bacteria to kill bacteria but also help AZI enter the bacteria (such as Gram-positive S. aureus and Gram-negative E. coli), which could further inhibit ribosome biogenesis and protein synthesis. PEI-CA could increase anchor bacteria, GO and AZI could kill bacteria via different mechanism, therefore, the synergistic effect of PEI-CA, AZI, and GO in AZI@GO-PEI-CA nanoparticles could effectively kill bacteria. In vivo skin wound healing experiments also confirmed AZI@GO-PEI-CA could highly reduce the risk of S. aureus infection and accelerate wound healing. Therefore, this multicomponent antibacterial agent with synergistic antibacterial mechanism is very promising in the treatment of bacterial infection.

Introduction

Bacterial infections have emerged as a worldwide public health concern [1]. In recent years, many bacteria associated with human diseases have evolved into multidrug-resistant microbial pathogens due to multiple mutations and antibiotic abuse, leading to higher morbidity and mortality [2], [3], [4], [5]. It has been reported that these pathogen infections caused millions of deaths annually with a huge burden on global medical resources and socioeconomic development [6], [7]. Consequently, new and effective antibacterial materials need to be developed urgently. In fact, there is a growing interest in developing nonspecific and broad-spectrum antimicrobial agents such as metallic materials, antimicrobial peptides (AMPs), cationic polymers, and metal-based nanoparticles, etc [8], [9], [10], [11], [12], [13], [14], [15]. Recently, two-dimensional nanomaterials represented by graphene oxide (GO), have been widely used in biomedical applications due to its excellent physical and chemical properties [15], [16], [17], [18], [19], [20]. It was reported that the easy formation of hydrogen bonds (HBs) between the oxygen-containing groups of GO and the lipid bilayer on the membrane surface could significantly promote penetration and insertion into the bacterial membrane [21]. Moreover, some graphene-based nanomaterials, such as reduced graphene oxide (rGO) and graphene quantum dots (GQD), have the activity of causing membrane perturbation, increasing intracellular reactive oxygen species (ROS) and killing bacteria [22], [23]. It was found that the physicochemical properties of GO could be used to express a certain antibacterial ability against many bacterial pathogens, such as E. coli, S. aureus, Bacillus subtilis, etc [24], [25], [26]. However, the working mechanism of GO is still unitary, which is unable to effectively exert high antibacterial activity within a safe dose. A promising approach is to use the combination of antibiotics with GO-based nanosheet, which could further increase bacterial-killing and overcome bacterial resistance due to the rapid entry of antibiotics into bacterial cells after membrane fragmentation by GO-based nanosheet [27]. For examples, Li and co-workers reported that a glycopeptide antibiotic vancomycin was used to synergize GO at a weak alkaline condition to treat Gram-positive bacterial infections [28], and Mariana et al. presented a new tuberculostatic agents, isoniazid and pyrazine-2-carbohydrazide covalently linked to GO, which enhanced respective antibacterial activity and extended antimicrobial spectrum towards other microbial strains [29]. Therefore, we envisage that the antimicrobial efficacy of GO nanosheet can be further improved through surface modification and loading antibiotics or other fungicides, especially for resistant bacteria [30], [31], [32].

Cationic polymers are the most popular macromolecules which have been applied in destroying the outer and cytoplasm membranes of bacteria to promote the antimicrobial activity of nanoparticles since the negative charge on the surface of microbial cell membrane [33], [34], [35], [36], [37], [38]. However, the direct immobilization of cationic polymers on graphene oxide may cause changes in stability and biological toxicity, because cationic components could be absorbed by opposite charges in body fluids, resulting in inevitable systemic cytotoxicity. To solve this concern, we have presented a synergistic antimicrobial agent AZI@GO-PEI-CA, which can self-remove CA structure, increase the positive charge density, and selectively anchor bacterial surface via electrostatic adsorption. As previously reported, an acid-responsive GO-based nanocarrier coated with polymers, poly(allylamine hydrochloride) (PAH) modified with 2,3-dimethylmaleic anhydride, exhibited low cytotoxicity, and anchored bacteria via acid-triggered charge reversal [39]. Also, it was reported that citraconic anhydride reacted with the primary amine groups of polyethyleneimine could increase biocompatibility of PEI and promote cytotoxic efficacy to cancer cells [40], [41]. Herein, we conjugated azithromycin (AZI) onto GO via the reaction of carboxyl group with the hydroxyl group of azithromycin. Subsequently, in order to selectively anchor the bacterial surface and minimize cytotoxicity, CA was linked onto PEI, and then PEI-CA was further adsorbed on GO surface, forming AZI@GO-PEI-CA, as shown in Scheme 1. After CA was removed via the amide bond hydrolysis, the surface charge of AZI@GO-PEI-CA could change from almost neutral to highly positive, thereby increasing the anchoring ability to bacteria [42], [43], [44], [45], [46], [47], [48]. After AZI@GO-PEI-CA reached the bacterial surface, GO nanosheet could easily insert the bacterial membrane, destroyed the membrane integrity [49], facilitated antibacterial agent AZI to enter bacteria, which could further inhibit protein synthesis, thereby enhancing the bactericidal efficacy and overcoming the problem of bacterial resistance (Scheme 1).