Nanoparticles in the Treatment of Infections Caused by Multidrug-Resistant Organisms

1Multidrug-resistant organisms (MDROs) are becoming a growing public health crisis and make many healthcare-associated infections difficult to treat with current antibiotics. Globally, infections caused by MDROs are emerging causes of morbidity and mortality. The development of new antibiotics requires tremendous economic and labor investment and is time-consuming. For these MDRO infections, high doses of antibiotics will be administered and may generate intolerable toxic and adverse effects, which will prompt the development of alternative strategies.

2The application of nanoparticles (NPs) provides a potential strategy to manage infections caused by MDROs. In this respect, NPs have shown therapeutic promise owing to their unique physical and chemical attributes. NPs exhibiting antibacterial activities can target multiple biomolecules and have the potential to reduce or eliminate the evolution of MDROs. However, the translation of NPs to clinical use requires not only appropriate methods for the synthesis of NPs but also a thorough understanding of the physicochemical particularities, in vitro and in vivo effects, biodistribution, pharmacokinetics, and pharmacodynamics of NPs.

3In this review, we will present a broad classification of the NPs that show in vitro antimicrobial activity against MDROs, and the synergistic effects of NPs with current available antibiotics, pharmacokinetic and pharmacodynamic characteristics, and resistant mechanisms will also be discussed.

4NPs possess antimicrobial activity that can overcome common resistant mechanisms, including enzyme inactivation, decreased cell permeability, modification of target sites/enzymes, and increased efflux through overexpression of efflux pumps, to escape from the antibacterial activity of antimicrobial agents ( Figure 1 ). Moreover, NPs conjugated with antibiotics show synergistic effects against bacteria, prohibit biofilm formation, and have been utilized to combat MDROs.

5Several characteristics of NPs make them alternatives to traditional antibiotics. First, the large surface-area-to-volume ratio of NPs increases the contact area with target organisms. NPs can act as nanoscale molecules interacting with bacterial cells, regulating cell membrane penetration, and interfering with molecular pathways. Second, NPs may enhance the inhibitory effects of antibiotics. demonstrated that gold NPs conjugated with ampicillin, streptomycin, or kanamycin could lower the minimum inhibitory concentrations (MICs) of the antibiotic counterparts against both gram-negative and gram-positive bacteria. Likewise, demonstrated a synergistic effect of functionalized Au NPs and fluoroquinolone antibiotics for the treatment of multidrug-resistant Escherichia coli infections. However, the complexity of the physicochemical properties, including size, shape, chemical modification, solvent, and environmental factors, can affect the antibacterial properties of NPs during preparation of NPs and interact with bacteria. Finally, combinations of antibiotics and NPs provide complex antimicrobial mechanisms to overcome antibiotic resistance. demonstrated a synergistic effect using functionalized Au NPs and fluoroquinolone antibiotics for the treatment of multidrug-resistant E. coli bacterial strains.

6NPs are therefore regarded as next-generation antibiotics. In both in vitro and in vivo studies, NPs, mainly metallic, have been shown to exhibit activity against gram-positive and gram-negative bacteria. Though antimicrobial mechanisms that depend on the size, shape, ζ-potential, ligands, and material used are not well understood; currently accepted mechanisms include (1) direct interaction with the bacteria, leading to the disruption of membrane potential and integrity; (2) triggering of the host immune responses; (3) inhibition of biofilm formation; (4) generation of reactive oxygen species (ROS); and (5) inhibition of RNA and protein synthesis through the induction of intracellular effects ( Figure 1 ). NP coatings on implantable devices, wound dressings, bone cement, or dental materials can function as NP-based antibiotic delivery systems. Furthermore, NPs can be vectors to transfer drugs so that higher doses of antimicrobial agents can be delivered to infected sites. Thus, the combination of NPs and antimicrobial agents may be beneficial in fighting the ongoing crisis of antimicrobial resistance. Clinical applications of NPs have recently been evaluated to highlight the in vitro antimicrobial activities of NPs and the potential adverse effects of NPs on human health ( Table 1 ).

7NPs with antimicrobial activity that combats Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, and Enterobacter species include NPs containing Ag, Au, Zn, Cu, Ti, Mg, Ni, Ce, Se, Al, Cd, Y, Pd, or superparamagnetic Fe. The antimicrobial activities against MDROs, mechanisms of action, and characteristics of various NPs are shown in Table 1. Among various metallic NPs and their oxides already applied as active antimicrobial agents, silver or its ionic form is the most toxic to bacteria. This makes silver of particular interest. Silver NPs (Ag NPs) are used to a great extent since they have multiple mechanisms of antibacterial action, high biocompatibility, and functionalized potential and are easy to detect. Although Ag NPs are difficult to functionalize with biomolecules and antibiotics, Ag–gold (Au) alloys provide another path, since they combine the antimicrobial effects of Ag with the effectiveness of functionalization and the stability of Au in the form of bimetallic NPs. Furthermore, Ag–Au NPs functionalized with tetracycline have been shown to have a synergetic effect, which is attributed to the generation of ROS.

8Ag NPs and Au NPs may exhibit decreased antibacterial activity when their surfaces are modified, and copper (Cu) NPs with modified surfaces lose antimicrobial activity and fail to change the morphology of microbial cells. However, most metallic NPs, through the release of toxic ions, inflammatory cytokines, and the generation of ROS, may cause immunotoxicity, cytotoxicity, and genotoxicity in both healthy and infected cells.

9Au–Pt bimetallic NPs have antibacterial activity against multidrug‐resistant E. coli through the dissipation of bacterial membrane potential and the elevation of adenosine triphosphate (ATP) levels. Cu–Ni bimetallic NPs have been utilized as coating agents but have been used less in antimicrobial applications

10With biocompatibility and magnetic properties, iron oxide (FeO) is well known in the biomedical sector. Recently, the antibacterial properties of reduced iron and FeO NPs that damage bacteria cells through the disruption of the bacterial membrane and generation of oxidative stress inside the cell have been studied. The characteristic compatibility and safety of ZnO NPs on human skin make them appropriate additives for cosmetics, fabrics, and surfaces in close proximity to human skin. Copper oxide (CuO) NPs have been shown to exhibit excellent bactericidal and fungicidal activity, whereas TiO2 NPs possess spectacular antimicrobial properties, mainly related to ROS formation, particularly –OH free radicals.

11To overcome antibiotic resistance, NPs can be tailored and packaged with diverse antimicrobial agents. NPs act on bacteria through multiple targets and/or a unique mechanism; thus, antimicrobial resistance is unlikely to develop if NPs are combined with antibiotics since multiple simultaneous mutations are required in the same microorganism. The functionalization of NPs with antibiotics can be a promising regimen to combat bacterial resistance. Moreover, NPs can deliver antimicrobial agents to or target the infected sites and reduce the dosage and toxicity of antibiotics. For example, the synergistic antibacterial efficiency of Ag NPs and antibiotics against S. aureus, beta-lactamase- or carbapenemase-producing E. coli, P. aeruginosa, and A. baumannii strains at extremely low concentrations has been found, whereas synergistic antibacterial effects of Ag, Au, and ZnO NPs and antibiotics have been observed against S. aureus, E. faecium, E. coli, A. baumannii, and P. aeruginosa through the penetration of the bacterial cell membrane and the interference with important molecular pathways, formulating unique antimicrobial mechanisms. The efficacy of antibiotics combined with NPs was identical in both gram-positive and gram-negative bacteria, unlike the difficulty in killing MDROs with antibiotics alone. The combinations of antibiotics and functionalized Ag, Au, or ZnO NPs may promote the reversal of antimicrobial resistance and boost the antimicrobial effects of several antibiotics, including polymyxin B, ciprofloxacin, ceftazidime, ampicillin, clindamycin, vancomycin, or erythromycin, against MDROs, including antibiotic-resistant A. baumannii, P. aeruginosa, E. faecium; vancomycin-resistant Enterococcus (VRE); and methicillin-resistant S. aureus (MRSA).

12The pharmacokinetics of NPs depend on numerous aspects, such as the particle type, size, surface charge, surface coating, protein binding, exposure route, dose, and animal species. A comprehensive understanding of their pharmacokinetics is pivotal for risk assessment and biosafety in clinical practice. The pharmacokinetic and pharmacodynamic characteristics of NPs are summarized in Figure 2. The systemic or local activity and toxicity of NPs are dependent on the administration route and physicochemical characteristics, and chronic toxicity may be related to the complicated elimination pathway. A summary of the present knowledge of the pharmacokinetics and toxicity of metallic NPs is provided in Table 2.

13The oral, dermal, or pulmonary absorption of Au NPs, Ag NPs, or TiO2 NPs is generally low ( Table 2 ). The parenteral route is favored for targeting the liver or spleen. For muscle or skin targeting, the administrative route can be topical, intramuscular, intradermal, or subcutaneous, whereas oral or intranasal administration is used in the case of mucosal targeting. For example, the absorption of Au NPs by inhalation ranges from 0.06% to 5.5%, depending on the size of the NP. Oral absorption is approximately 0.01–5% for Au NPs, 1–4.2% for Ag NPs, and 0.01–0.05% for TiO2 NPs, depending on the size and coating.

14Regardless of the particle type, most metallic NPs are distributed mainly in the liver and spleen, but the physicochemical properties of Au NPs could be modified to increase their distribution to specific target organs. However, long-term studies regarding a full interpretation of the toxicological implications of NP absorption and penetration through tissues are lacking.

15The decision for the optimal dose is crucial for therapeutic targets and minimizing toxicity for medical translation. Thus far, the doses of nanomaterials causing cell damage in vitro are unrealistically high and are impossible to apply to humans. The data from animal studies may not be directly translated to human beings, and appropriate and realistic doses should be studied in the future. There have been few clinical studies on NP dosing. reported two oral doses (10 ppm with a size ranging from 5 to 10 nm and 32 ppm with a size ranging from 25 to 40 nm) of a commercial solution of Ag NP in healthy adult volunteers that did not prompt clinically significant changes in human metabolic and hematologic profiles, urine, physical findings, or imaging morphology based on comprehensive assays and tests. More clinical studies are warranted before the application of NPs to patients.

16The elimination of metallic NPs via urinary and biliary pathways is generally low, which leads to their long-term accumulation in the liver and spleen. In addition, NPs do not undergo biodegradation into biologically benign components and thus exhibit prolonged tissue retention, eventually leading to amplified toxic effects. A higher accumulation of 10-nm NPs was observed in the kidneys, but this could be caused by a lessened availability of the larger NPs due to their high accumulations in the liver and spleen.

17The degree of opsonization of NPs by serum proteins is determined by the charge and size of the NPs. By opsonization, the in vivo hydrodynamic diameter (HD) or the effective size of NPs can be altered. The endothelium usually has a pore size of 5 nm, and particles with an HD smaller than 5 nm can equilibrate with the extravascular extracellular space (EES). Conversely, larger particles with slow movement across the endothelium remain in circulation for extended periods. The kidney can remove molecules from vascular compartments, but the particles in the range of 10–20 nm are excluded from renal filtration and are eliminated through the hepatobiliary system. The remaining particles that escape degradation by Kupffer cells will be retained in the body for prolonged periods. More studies are vital to explore the ways to increase biliary and/or urine elimination of NPs to reduce organ accumulation and potential toxicity.

18The antimicrobial activity of NPs depends on several physicochemical properties, such as their size, shape, solubility, and ability to form free biocidal metal ions. Generally, smaller NPs show increased antibacterial activity compared to larger NPs. Gram-positive and gram-negative bacteria differ in terms of cell membrane components and structures and have different adsorption pathways for NPs. The susceptibility of bacteria to NPs depends on their biochemical composition since different NPs target different biomolecules. Moreover, rapidly growing bacteria are more susceptible to NPs or antibiotics than slow-growing bacteria. This may be due to the variable expression of stress-response genes between rapidly growing and slow-growing bacteria.

19The antibacterial effects of NPs have been noted to be more pronounced for gram-positive bacteria than for gram-negative bacteria. Such a finding may be related to the fact that the nonporous cell walls of gram-negative bacteria serve as penetration barriers for the entry of NPs. Cell walls of gram-positive bacteria with covalent links with neighboring proteins and components are relatively porous and allow the penetration of foreign molecules.

20Local and systemic toxic complications, as well as deleterious effects on beneficial bacteria in humans, are concerns for the use of NPs. Both NPs themselves and toxic degradation products of NPs can cause hemolysis and interfere with blood coagulation pathways. The exact mechanism of toxic complications is unclear, but it has been observed that the larger the size of the NP is, the greater the risk of adverse health effects. Among metal NPs, the toxicity of Ag NPs has been studied extensively, and Ag NPs were shown to be more toxic toward cell lines. However, most studies were performed in vitro. The deposition of Ag NPs in the liver, spleen, lungs, and other organs results in organ damage and dysfunction and seriously decreases their efficacy. Elevated Ag levels have been found in both blood and urine by the leaching of Ag from Acticoat®, a nanocrystalline Ag wound dressing, into the bloodstream and were confirmed in burn patients. Al2O3 NPs that interact with cellular biomolecules and cause adverse effects of neurotoxicity could serve as broad-spectrum bactericidal agents, regardless of drug resistance mechanisms. The oxidative damage of CuO NPs and DNA damage induced by ZnO NPs or TiO2 NPs limit their use.

21Intravenously administered NPs could accumulate in the colon, lung, bone marrow, liver, spleen, and lymphatic system, and inhalation might cause cytotoxicity in the lung. The generated free radical-mediated oxidative stress by CuO NP could interact with cell components and induce hepatotoxicity and nephrotoxicity. Though several in vivo studies have reported no apparent life-threatening toxicity related to NPs, chronic toxicity, such as nephrotoxicity, hepatotoxicity, or pulmonary toxicity, can result from the accumulation of metallic NPs in these tissues.

22However, the evaluation of toxicity at the cellular and systemic levels remains important for clinical translation, and several parameters, such as the administration route for a desired therapeutic effect and the nature and extent of the interactions between NPs and cells, tissues, and organs, should be considered. Detailed in vivo and clinical studies assessing the toxicity of NPs are highly desirable before the routine application of NPs in combating difficult-to-treat infections due to MDROs.

23NPs have multifunctional mechanisms to attack bacteria that are different from those of the currently available antibiotics ( Figure 1 ), and the combination of NPs and clinically available antibiotics allows for recovery of antimicrobial efficacy. Microbial cells need to acquire multiple mutations to develop resistance toward NPs. Furthermore, the synthesis of NPs that bind with proteins, polysaccharides, or small bioactive compounds would further enhance their antimicrobial activity toward MDROs. Resistance to NPs is always a clinical concern. Though rare, bacteria resistant to Ag, Au, or Cu NPs have been reported even after exposure to one dose of NPs. The resistance might be related to changes in the permeability of the outer membrane and high expression of efflux pumps.

24Another example of resistance to NPs is that after exposure to Cu++ and Cu-doped TiO2 NPs, reduced antimicrobial activity of TiO2 NPs to Shewanella oneidensis was noted. This effect is likely to be associated with decreased uptake and/or increased efflux of Cu++ and Cu-doped TiO2 NPs. Reduced toxic effects of both TiO2 and Al2O3 NPs to Cupriavidus metallidurans were possibly due to less uptake of plasma membrane or cell wall or increased efflux of NPs.

25The increasing clinical application of Ag NPs still raises the concern of bacterial resistance to Ag NPs. Resistance to Ag NPs attributed to sil genes has been reported in clinical K. pneumoniae and Enterobacter cloacae isolates from burn cases. Genetic changes in bacteria may result in the rapid evolution of resistance to Ag NPs, and Al2O3 NPs could trigger increased expression of conjugation-promoting genes and promote the horizontal transfer of antibiotic resistance genes. The phenotypic change in the production of flagellin in E. coli isolates resistant to Ag NPs was found to readily induce NP aggregation and attenuate the antimicrobial activity of Ag NPs.

26NPs have the potential to treat bacterial infections ( Table 3 ), but several challenges remain for their successful translation to the clinic, including further assessment of the interactions of NPs with cells, tissues, and organs; optimal dose; recognition of appropriate administration routes; and toxicity following acute and long-term exposure.

27The unique physical structure of NPs offers distinctive advantages over conventional antibiotics in terms of antibiotic resistance. The current state of NPs exhibits a strong potential to topically treat skin infections in the near future. Efforts have been made to apply NPs on the contact surfaces of medical devices, fibers, and textiles. However, systemic administration of NPs still requires multiple aspects to be addressed.

28Formulation of proper guidelines for the production and scaled-up manufacturing of these nanomaterials, the characterization of the physicochemical properties and their effect on biocompatibility, standardization of nanotoxicological assays, and protocols to compare data originating from in vitro and in vivo studies are urgent for clinical translation. Further preclinical studies have to consider the therapeutic efficacy parameters in clinical trials and the safety of NP systems. Finally, the economic impact of clinical translation of these NPs must be addressed with regard to their therapeutic efficacy.

29Given their therapeutic potential, it is essential to determine the mechanisms by which NP complexes inhibit or kill bacteria. However, there is limited information about the metabolism, clearance, and toxicity of NPs; the nature of optimal targets for certain infections; and the optimum dose for therapeutic activity at the pathogen target sites. Specific combinations of NPs and antibiotics can prevent the emergence of resistance or drive resistant bacteria back toward drug sensitivity, but translation into the clinic requires an in-depth perception of the pharmacokinetics/pharmacodynamics of NPs.

30NL wrote the manuscript, and WK and PH revised and approved the final version of the manuscript.

31This study was supported by the grants from National Cheng Kung University Hospital, Tainan, Taiwan (NCKUH-10802042) for publication fee.

32The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.