Broadening the spectrum of small-molecule antibacterials by metallic nanoparticles to overcome microbial resistance

Accepted Manuscript Title: Broadening the spectrum of small-molecule antibacterials by metallic nanoparticles to overcome microbial resistance Authors: Mahendra Rai, Avinash P. Ingle, Raksha Pandit, Priti Paralikar, Indarchand Gupta, Marco V. Chaud, Carolina Alves dos Santos PII: DOI: Reference:

S0378-5173(17)30846-3 http://dx.doi.org/10.1016/j.ijpharm.2017.08.127 IJP 16980

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

15-6-2017 31-8-2017 31-8-2017

Please cite this article as: Rai, Mahendra, Ingle, Avinash P., Pandit, Raksha, Paralikar, Priti, Gupta, Indarchand, Chaud, Marco V., dos Santos, Carolina Alves, Broadening the spectrum of small-molecule antibacterials by metallic nanoparticles to overcome microbial resistance.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2017.08.127 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Broadening the spectrum of small-molecule antibacterials by metallic nanoparticles to overcome microbial resistance 1Mahendra 2Marco 1

Rai*, 1Avinash P. Ingle, 1Raksha Pandit, 1Priti Paralikar, 1Indarchand Gupta,

V. Chaud, 2Carolina Alves dos Santos Nanobiotechnology Lab., Department of Biotechnology, SGB Amravati University,

Amravati-444 602, Maharashtra 2

LaBNUS – Biomaterials and Nanotechnology Laboratory, University of Sorocaba, Sorocaba/SP, Brazil

Corresponding author: E-mail: [email protected]

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Graphical Abstract Antimicrobial resistances is responsible for the development of multi-drug resistant pathogens and it become one of the most important global public health concerns. The overuse of antibiotics leads to the microbial resistance. In this context, various nanomaterials showed strong antibacterial activity against MDR pathogens through different mechanisms and hence it is believed that nanotechnology will be the one of solution to overcome the problem of MDR infections.

Abstract Now-a-days development of microbial resistancce have become one of the most pressing global public health concerns. It is estimated that about 2 million people are infected in USA with multidrug resistant bacteria and out of these, about 23,000 die per year. In Europe, the number of deaths associated with infection caused by MDR bacteria is about 25,000 per year, However, the situation in Asia and other devloping countries is more critical. Considering the increasing rate

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of antibiotic resistance in various pathogens, it is estimated that MDR organisms can kill about 10 million people every year by 2050. The use of antibiotics in excessive and irresponsible manner is the main reason towards its ineffectiveness. However, in this context, promising application of nanotechnology in our everyday life has generated a new avenue for the development of potent antimicrobial materials and compounds (nanoantimicrobials) capable of dealing with microbial resistance. The devlopement and safe incorporation of nanoantimicrobials will bring a new revolution in health sector. In this review, we have critically focused on current worldwide situation of antibiotic resistance. In addition, the role of various nanomaterials in the management of microbial resistance and the possible mechanisms for antibacterial action of nanoparticles alone and nanoparticle-antibiotcs conjuagte are also discussed.

Keywords: Nanoparticles, Drug resistance, Nanotechnology, Antimicrobials, Nanomedicine.

1. Introduction The use of antimicrobials in therapeutics has increased considerably during the last few decades (Sandoval-Motta and Aldana, 2016). Moreover, new and emerging infections caused by microorganisms are also increased (Tanwar et al., 2014). The mechanism of action of antimicrobials is responsible for different range of action spectrum, as well as its application, from therapeutic use in humans, food additives/supplements, plants and animals (Nikaido, 2009). The antimicrobials available for the use are obtained by fermentation, synthetic and semisynthetic routes and cause the death of microorganisms, because they act on membrane of the microorganisms, affect nucleic acid synthesis and alter metabolism (Marinho et al., 2016). The use of antimicrobials has generated a positive impact on the prolongation and improvement of the quality of life of the population. However, its excessive use has given rise to the emergence of antimicrobial resistance (AMR) to the compounds traditionally used, which is becoming a grave problem of world-wide public health (Fernadez et al., 2016). The bacterial strains, which acquired frequent emergence of resistance to antibiotics are referred to as “multidrug resistant bacteria”. Holmstrup et al. (2017) reported that in USA, AMR causes 2 million infections with 23,000 deaths annually and in Europe AMR is associated with about 25,000 deaths per year. However, the situation in Asia and other devloping countries is more critical. 3

Moreover, considering the increasing rate of antibiotic resistance in various pathogens, it is estimated that MDR pathogens can kill about 10-million people every year by 2050, shockingly, this would surpass all other life threatening diseases including cancer (O’Neill, 2014). Microbial resistance has caused a negative impact under the health system, increasing hospitalization time and treatment expenses (Holmstrup et al., 2017). The mechanism of resistance is defined as the insensitivity of the microorganisms to a compound for which it was previously sensitive (Tanwar et al., 2014). Microbial resistance is classified as: (i) intrinsic resistance, (ii) incorporation of genetic material, and (iii) adaptive resistance. One of the major problems of microbial resistance is environmental dissemination and the absence of new compounds capable of reversing this scenario (Marinho et al, 2016). The most cases of increase in number of infections caused by microorganisms which are resistant to many antimicrobials (Fernandez et al., 2016; Nikaido, 2009) . Nanotechnology represents a promising alternative for the development of new materials and compounds capable of being incorporated into everyday life, with applicability in health and other areas of science. According to Director General of the World Health Organization (WHO), Dr. Margaret Chan, use of nanomaterials as potential antimicrobial agents (nanomedicine) will be a post-antibiotic era, which has potential of overcoming the problem of MDR infections (http://www.nanowerk.com/spotlight/spotid=32188.php). Studies have demonstrated the ability of some organic and inorganic nanoparticles, which act as antimicrobial agents (Rudramurthy et al., 2016). The silver and gold nanoparticles, have the property to act on different parameters and functions of the microorganisms, which causes greater difficulty for the development of microbial resistance (Santos et al., 2014). Hoseinzadeh et al., (2016) reported that some characteristics in nanomaterials are responsible for their antimicrobial effectiveness, which include nanomaterial load, type of material, shape, concentration, etc. Despite the advantages and potential applicability of nanomaterials in combating AMR, their toxicity and safety are barriers that limits their efficient and safe use (Wacker et al., 2016). The aim of this review is to focus on the current worldwide situation of AMR and role of various nanomaterials in overcoming the antibiotic resistance. In addition, possible antibacterial mechanisms of nanoparticles and nanoparticles-antibiotics conjugates against MDR bacteria and toxicity of nanomaterials is also discussed.

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2. Role of metal nanoparticles in the management of AMR The resistant bacteria include both Gram positive and Gram negative bacteria such as vancomycin-resistant Enterococcus faecium (VRE), methicillin-resistant Staphylococcus aureus (MRSA), multidrug resistant (MDR) Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. It is well known that metal nanoparticles (MNPs) exhibit antimicrobial activity, which can efficiently deal with resistant strains of microorganisms. Table 1 summarizes the antibacterial efficacy of various nanoparticles against MDR bacteria. However, the detail explanation has been given below.

2.1. Silver nanoparticles (AgNPs) From the studies performed in the past it is evident that AgNPs possesses strong antimicrobial potential including MDR pathogens (Patra and Baek, 2017; Thapa et al., 2017). Percival et al. (2007) synthesized AgNPs and evaluated its antimicrobial activity against MDR Gram positive and Gram negative bacteria such as methicillin and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA) and Enterococcus faecium. The study reported that AgNPs not only act as potential antimicrobial agent but also help in the inhibition of biofilm formation. Ingle et al. (2008) demonstrated the potential of AgNPs synthesized from Fusarium acuminatum against MDR Staphylococcus aureus. Nanda and Saravanan (2009) evaluated the antimicrobial activity of AgNPs against MRSA, methicillin-resistant Staphylococcus epidermidis (MRSE). The authors suggested that AgNPs can be used as an alternative to combat the problem of drug resistant microorganisms. The antimicrobial activity of AgNPs against MDR P. aeruginosa, ampicillin resistant Escherichia coli O157:H7 and erythromycin resistant Streptococcus pyogenes (ERSP) showed that AgNPs help in the reduction of the infections caused by MDR bacteria (Lara et al., 2010). Behera and Nayak (2013) agreed upon these findings and further presented the data supporting the use of AgNPs against MDR microbes. Rai et al. (2012) also reviewed that AgNPs can act as a powerful weapon against various multi-drug resistant bacteria. AgNPs synthesized from endophytic fungus Penicillium sp. possess antimicrobial activity against MDR S. aureus and E. coli. Both biologically and chemically synthesized AgNPs showed excellent antimicrobial activity against MDR pathogens (Singh et al., 2014). Chemically synthesized AgNPs inhibit 56% of biofilm formation by drug resistant strains of P. aeruginosa 5

(Palanisamy et al., 2014). The antimicrobial activity of AgNPs depend on the stabilizing agent, charge and size of synthesized nanoparticles (Cavassin et al., 2015). Comparative antibacterial study of commercially available AgNPs and AgNPs stabilized with different compounds such as citrate, chitosan, polyvinyl alcohol against 54 drug resistant bacteria including oxacillin resistant S. aureus, carbapenem and polymyxin resistant A. baumannii (PRAB), carbapenem-resistant Enterobacteriaceae, vancomycin-resistant Enterococcus spp. demonstrated that citrate and chitosan AgNPs showed maximum antimicrobial activity. The antimicrobial activity of AgNPs was higher against Gram negative microorganisms as compared to Gram positive MDR resistant strains (Cavassin et al., 2015). Similarly, antibacterial efficacy of AgNPs was demonstrated against MDR Enterobacter sp., P. aeruginosa, K. pneumoniae, E. coli (Gopinath et al., 2015) and against MDR -Extended Spectrum Beta Lactamase producing E. coli (Kar et al., 2016).

2.2. Gold nanoparticles (AuNPs) Compared to AgNPs, there are less reports available on the antimicrobial study of AuNPs. However, there are some reports which suggests that AuNPs do not have or possess very weak antimicrobial properties. The inconsistency in the antimicrobial activity of AuNPs may be due to improper purification of AuNPs (Shareena-Desari et al., 2015). AuNPs did not show inherent activity against MDR bacteria. On the other hand, when AuNPs were functionalized with cationic group, antimicrobial activity against MDR microorganisms was demonstrated. S. aureus is considered as most deadly due to its resistance against various antibiotics. However, Li et al. (2014) demonstrated that AuNPs functionalized with thiol-group can be used as potential agent against MDR S. aureus. Vinoj et al. (2015) synthesized AuNPs and functionalized their surface with Acyl Homoserine Lactone Lactonase protein and the surface functionalized AuNPs were evaluated against MDR Proteus spp. In vitro effect of functionalized AuNPs was tested against biofilm of Proteus spp. Biofilm produced by Proteus spp. are the major cause of catheter-related urinary tract infections and the problems associated with it. Hence, synthesized AuNPs can act as a potential nanomaterial, which can be used against urinary tract infections caused by MDR bacteria such as Proteus spp. Zhao et al. (2013) reported the synthesis of AuNPs and coated its surface with dimethyl-biguanide. In vitro antimicrobial study of AuNPs alone and the coated 6

nanoparticles against MDR E. coli, MRSA and P. aeruginosa showed that only coated AuNPs have antimicrobial activity against MDR microorganisms. Various antibiofilm agents with potential antimicrobial activity showed promising results in control of biofilm in medical sector. Combined use of antibiotics with antibiofilm agents like nanoparticles will upgrade action and prevent resistance problem (Abdel-Rahim and Mohamed, 2015).

2.3. Bismuth nanoparticles (BiNPs) Bismuth is a crystalline, brittle metal and its compounds are used to treat gastrointestinal disorders (Tillman et al., 1996; Maev et al., 2008). Bismuth derivatives have been used in medicine to treat vomiting, diarrhea, nausea and stomach pain (Figueroa-Quintanilla et al., 1993). BiNPs in combination with chlorhexidine, nystatin, and terbinafine controls the growth and biofilm formation (Hernandez-Delgadillo et al., 2012, 2013). It was also reported that zerovalent BiNPs have potential to reduce cell growth of Streptococcus mutans in biofilm formation (Hernandez-Delgadillo et al., 2012). These nanoparticles prevent biofilm formation up to 69%. Bismuth-containing nanoparticles with X-ray irradiation treatment also have potential to kill MDR bacteria (Luo et al., 2013). The authors reported that the surface modified BiNPs showed up to 90% growth inhibition against MDR P. aeruginosa. Since, X-rays can easily penetrate human tissues, this bactericidal strategy has the potential to be used effectively in killing of MDR bacteria in vivo.

2.4. Copper nanoparticles (CuNPs) Efficacy of CuNPs against MDR pathogens have been less studied. There are only a few reports which suggests the efficient activity of CuNPs against MDR bacteria. Ashajyothi et al. (2014a) demonstrated the efficacy of biogenic CuNPs synthesized from Enterococcus faecalis against various strains of MDR bacteria like E. coli, K. pneumoniae and MRSA. The characterization carried out by different analytical techniques including transmission electron microscopy (TEM) confirmed the synthesis of CuNPs in the average size range of 20-90 nm. Further, in vitro efficacy of the CuNPs showed significant antibacterial nature of these biogenic CuNPs. MRSA was found to be most sensitive followed by E. coli and K. pneumoniae.

2.5 Selenium nanoparticles (SeNPs) 7

There are reports suggesting the potential antimicrobial activity of organo-selenium. Considering these facts, researcher started exploring antimicrobial properties of selenium at nanosize. Interestingly, it was observed that SeNPs possesses strong antimicrobial and antioxidant activities suggesting its ability to be used as therapeutic agent to combat various microbial infections (Ramya et al. 2015). Tran and Webster (2011) reported that SeNPs at concentration of 7.8 µg/ml potentially inhibit the growth of S. aureus. In another study, SeNPs synthesized from bacterium Ralstonia eutropha showed excellent antimicrobial activity against P. aeruginosa, S. aureus, E. coli and Streptococcus pyogenes and showed 99% growth inhibition at concentration of 100, 100, 250 and 100 µg/ml respectively (Srivastava and Mukhopadhyay, 2015). Cremonini et al. (2016) demonstrated the efficacy of biogenic SeNPs synthesized from Stenotrophomonas maltophilia and Bacillus mycoides against clinical isolate of P. aeruginosa. Recently, it was demonstrated that SeNPs significantly checked the growth of important foodborne pathogens such as E. coli O157:H7, S. aureus, Salmonella, and Listeria monocytogenes which confirms the bacteriostatic activity of SeNPs at 10 µg/ml. Moreover, among these pathogens S. aureus found to have dose dependent sensitivity (Nguyen et al., 2017). However, there is any report available on evaluation of antibacterial activity of SeNPs against MDR bacteria, hence, further investigations are needed to explore the potency of SeNPs MDR microorganisms. Overall, it can be proposed that biogenic SeNPs appear to be reliable candidates for safe medical applications to inhibit the growth of various pathogens.

3. Role of various metal oxide nanoparticles in the management of MDR pathogens Like metal nanoparticles, oxides of some metal like zinc oxide nanoparticles (ZnONPs), copper oxide nanoparticles (CuONPs), titanium dioxide nanoparticles (TiO2NPs), magnesium oxide nanoparticles (MgONPs), aluminium oxide nanoparticles (Al2O3NPs), etc. also found to have potential efficacy against MDR pathogens (Ashajyothi et al., 2014b; Beyth et al., 2015; Ingle et al., 2009) (Table 1).

3.1 Zinc oxide nanoparticles (ZnONPs) Ashajyothi et al. (2014b) demonstrated the antibacterial efficacy of biosynthesized ZnONPs from E. faecalis and commercial antibiotics against three MDR pathogens viz. E. coli, K. pneumoniae and MRSA. It was reported that ZnONPs showed more activity as compared to commercial 8

antibiotics against all the test pathogens. Similarly, Al-Hartomy and Mujahid (2014) studied the antibacterial activity of ZnO nanosheets synthesized by hydrothermal method using zinc nitrate and urea against MDR E. coli and MRSA. The high resolution transmission scanning electron microscopy (HRTEM) analysis showed the synthesis of hexagonal structure having average thickness of 12 nm. Thus, synthesized ZnO nanosheets showed significant activity against tested MDR bacteria like E. coli and MRSA confirming that not only spherical but other structures of ZnO also showed significant antibacterial activity. ZnONPs were also found to have good antibacterial activity against MDR bacteria such as Streptococcus agalactiae and S. aureus by the mechanisms of membrane disorganization, which ultimately resulted in increased permeability leading to cell death. In addition, surface modified ZnONPs with polyvinyl alcohol also demonstrated oxidative stress along with increase in membrane permeability and the cellular internalization, which cause cell death (Huang et al., 2008). Hamed et al. (2015) demonstrated the activity of ZnONPs against various clinical isolates of MDR S. aureus and E. coli singly and in combination with N-acetyl cysteine. It was reported that, when ZnONPs tested singly it showed MIC’s of 100 and 60 μg/ml, however; when it was used in combination with N-acetyl cysteine its activity found to increase which showed lesser MIC’s of 3.9 and 3.9 μg/ml against S. aureus and E. coli respectively. Functionalization of ZnONPs with various biomolecules including antibiotics enhance their bioactivity (Patra et al., 2014). Patra et al. (2014) demonstrated the activity of microwave assisted synthesized ZnONPs in range of 18-20 nm and functionalized with ciprofloxacin (an antibiotic) against clinical isolates of MDR such as E. coli, S. aureus and Klebsiella sp. It was reported that ciprofloxacinconjugated ZnONPs exhibited excellent antibacterial activity against these bacterial strains. In another study, Jan et al. (2013) studied the doping effect of tin (Sn) on the antibacterial activity of ZnO nanoparticles. The activity of undoped and doped ZnONPs was demonstrated against MRSA. The results showed that ZnONPs doped with Sn enhanced antibacterial activity as compared to undoped ZnONPs. Therefore, authors concluded that such changed behavior of ZnONPs after doping with Sn, will be a novel approach, hence, can be used for the prevention of infections caused by S. aureus especially on skin.

3.2 Copper oxide nanoparticles (CuONPs)

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Among different metal oxides nanoparticles, researcher around the globe are focusing their research on copper based nanoparticles including CuONPs in biomedical field. It is only because of their broad-spectrum bioactivity, safe and cost effective nature (Ingle et al., 2009). Ingle et al. (2009) extensively reviewed various bioactivities of CuONPs including their antimicrobial nature against wide range of pathogens including MDR bacteria. Concerning the biofilm formation, Agarwala et al. (2014) demonstrated the comparative activity of CuO and Fe2O3 nanoparticles against biofilm forming bacteria (viz. MRSA and E. coli). Here, CuONPs showed higher activity as compared to Fe2O3 nanoparticles. Among these two bacteria, maximum activity was reported against MRSA (zone of inhibition of 22 ± 1 mm) followed by E. coli (zone of inhibition of 18 ± 1 mm) in case of CuONPs. Malka et al. (2013) carried out a similar study to Jan et al. (2013) only differing in the doping effect of zinc (Zn) on CuONPs. In the study of Malka et al. (2013), Zn-doped CuONPs were synthesized and simultaneously they are deposited on cotton fabrics in different concentration using ultrasound irradiation. Further, their activity was evaluated against MDR E. coli and MRSA. A significant enhancement (about 10,000 times more) in the antimicrobial activity of the Zn-doped CuONPs was reported as compared to CuONPs and ZnONPs alone. 3.3 Titanium dioxide nanoparticles (TiO2NPs) TiO2NPs are another type of metal oxide nanoparticles that has been extensively studied for their antimicrobial efficacy. These nanoparticles proved to have potential ability to kill both Grampositive and Gram-negative bacteria (Beyth et al., 2015). Li et al. (2013) developed conjugates of geometrical isomer ferrocene-carborane derivatives (designated as FcSB1 and FcSB2) and TiO2NPs for the management of nosocomial infection caused by MDR bacteria like A. baumannii. The different assays performed such as drug interaction assay and time-kill studies showed the considerable enhancement in the antibacterial efficacy of TiO2NPs when conjugated with FcSB1/or FcSB2 against clinically MDR-resistant A. baumannii. Similarly, Arora et al. (2015) demonstrated the effectiveness of TiO2NPs against MDR P. aeruginosa recovered from pus, sputum, endo-tracheal tract and broncho-alveolar lavage samples alone and in combination with antibiotics viz. ceftazidime and cefotaxime. The significant rise in the activity of nanoparticles was reported when used in combination with antibiotic, ceftazidime (zone of inhibition 24 mm), whereas the combination of TiO2NPs and cefotaxime did not show any change. Similarly, MDR E. coli also demonstrated susceptibility towards TiO2NPs (Haghi et al., 10

2012). In another study, Roy et al. (2010) investigated antibacterial efficacy of TiO2NPs in combination with 23 different commercial antibiotics against clinical isolates of MRSA. The results reported showed that the combination of TiO2NPs with antibiotics is more active as compared to almost all the commercial antibiotics when used alone. Out of all the tested antibiotics, the combination of TiO2NPs with Amikacin and Penicillin G showed maximum rise in the efficacy.

3.4 Magnesium oxide nanoparticles (MgONPs) Magnesium oxides are inorganic metal oxide and its nano form has a potential to reduce bacterial contamination (Tang and Lv, 2014). MgONPs inhibits the function of S. aureus and P. aeruginosa in the presence of ultrasound (Mirhosseini, 2015). Antimicrobial potential of MgONPs depend upon their size. The smaller size MgONPs have more antibacterial and antibiofilm efficacy as compared with larger particles against E. coli and S. aureus (Lellouche et al., 2012). MgONPs possess positive charge in suspension, opposite to that of bacterial cell, which leads to enhancement of bactericidal activity (Koper et al., 2002; Stoimenov et al., 2002). The authors also reported that nano form of MgO possess active halogens that enhances bactericidal potential of MgONPs. Vatsha et al. (2013) demonstrated the antibacterial activity of Mg(OH)2 and MgONPs against E. coli and S. aureus. The study revealed that MgO showed more activity as compared to Mg(OH)2 because of its nanoform, as the smaller nanoparticles are more reactive than larger ones. After interaction with bacterial cell wall they were reported to generate reactive oxygen species (ROS), leading to cell death (Makhluf et al., 2005).

3.5 Aluminium oxide nanoparticles (Al2O3NPs) and Iron oxide nanoparticles (Fe2O3NPs) Antibacterial properties of Al2O3NPs are not much studied. The treatment of Al2O3NPs cause cell wall damage and disorganization of cell membrane, which leads to bacterial cell death (Ansari et al., 2013). The study revealed that Al2O3NPs penetrate inside bacterial cell by forming pits and smalls holes on its surface. Inside the bacterial cell nanoparticles interact with cellular macromolecules and cause cell death. Ansari and coworkers (2014) studied the interaction of Al2O3NPs with E. coli and their cell envelope biomolecules. The study evidenced that Al2O3NPs after interaction with membrane biomolecules, affect membrane integrity and fluidity, which contributes to bacterial cell damage. Ansari et al. (2015) evaluated bactericidal activity of 11

Al2O3NPs against MDR P. aeruginosa. Mechanistically Al2O3NPs penetrates into bacterial cell through the cell membrane, resulting into cell growth inhibition. Iron oxide nanoparticles (Fe2O3NPs) are the least toxic among all the magnetic nanoparticles with excellent magnetic properties. It was found that Fe2O3NPs core coated with silica- functionalized group can be used as an excellent agent to combat with multi-drug resistant like E. coli and S. aureus. Hence, functionalized nanoparticles can be used in prevention of multidrug resistance (Ansari et al., 2015; Chaurasia et al., 2016). 4. Role of synergistic combination in management of MDR bacteria The poor intracellular bioavailability and non-target specific mode of action reduces efficacy of antibiotics. Moreover, its widespread use leads to development of resistance. In addition, use of some antibiotics has cytotoxicity and side effects. These problems can be solved by the development of new therapeutic strategies such as combined therapy of nanoparticles with other antimicrobial agents. Similarly, encapsulation of nanoparticles with essential oils evidenced improved antimicrobial efficacy against MDR bacteria (Rai et al., 2017). The combination efficacies of nanoparticles with antibiotics have been exploited by many researchers. Abed et al. (2015) developed nano-device coupled with antibiotics, which is capable of intracellular delivery of beta lactam antibiotics to overcome bacterial resistance. The combination of nanocarriers with drugs offer many advantages over conventional therapy which includes controlled drug release at specific site of action, which increase drug efficiency, bioavailability of drug in right proportion with prolong effect, improved solubility of hydrophobic drugs as well as multiple drugs can also be delivered at specific cells, etc. (Ranghar et al., 2014; Zhang et al., 2010). Non-antibiotic drugs and pyrimidinethiol loaded AuNPs promote broad spectrum bactericidal activity against MDR bacteria (Zhao et al., 2013). This study demonstrated that antihyperglycemic drug (metform) showed better synergy with DAPT (4,6-diamino-2pyrimidinethiol) on AuNPs against MDR Gram positive and Gram negative bacteria by penetrating bacterial cell membrane. Antibiotic loaded nanocarriers can be efficiently used for targeted delivery at infection site. In addition, vancomycin-coated AuNPs were found to be effective and inhibit the growth of VRE significantly (Gu et al., 2003). Furthermore, amoxicillin is hydrophilic whereas AgNPs are hydrophobic in nature. Thus, AgNPs can easily interact with bacterial membrane containing hydrophobic phospholipids and glycoproteins. This characteristic 12

of AgNPs facilitate the transportation of amoxicillin to the bacterial cell surface (Li et al., 2005; Allahverdiyev et al., 2011). Many nanocarriers are synthesized with an aim of improving therapeutic potential of antibiotics by increasing their spectrum of action which lowers antibiotics toxicity level. Synergistic combination helps in restoring the activity of antimicrobial drugs against resistant bacteria (Shimanovich and Gedanken, 2016). AgNPs and AuNPs functionalized with ampicillin showed potent antibacterial activity against MDR isolates of P. aeruginosa, E. aerogenes and MRSA (Brown et al., 2012). The study showed that ampicillin capped AuNPs have potent bactericidal activity against resistant isolates of test organisms, which proved it as novel therapeutics to destroy ampicillin resistant bacteria. Fayaz et al. (2010) investigated that antibacterial activity of cepoferazone was increased when used in combination with AgNPs against Gram positive and Gram negative bacteria. Moreover, application of imipenem, trimethoprime, gentamicin, vancomycin, ciprofloxacin and amoxicillin in combination with AgNPs showed significant effect against MDR bacteria (Allahverdiyev et al., 2011; Birla et al., 2009; Li et al., 2005; Naqvi et al., 2013). Activities of fluoroquinolones, cephalosporins, penicillins, lincosamides, sulfonamides, glycopeptides, azalides, macroolides and aminoglycosides antibiotics with TiO2NPs against MRSA were studied by Roy et al. (2010). The results demonstrated increase in inhibition zone area around disc. Vancomycin conjugated to the surface of Fe2O3NPs (Choi et al. 2012), porous silica nanoparticles (Qi et al., 2013) and AuNPs (Gu et al., 2003), resulted in targeted delivery of vancomycin for selected killing of pathogenic bacteria. Table 2 showed the antibacterial activity of nanoparticles in combination with different antibiotics against Gram-positive and Gram-negative bacteria.

5. Polymeric nanomaterials for targeted drug delivery Polymeric nanoparticles have been efficiently studied as drug carrier. Polymeric nanoparticles like liposomes, solid lipid nanoparticles, chitosan were successfully used for targeted delivery of antibiotics (Ibrahim et al., 2015; Ranghar et al., 2014). For example, encapsulation of vancomycin in liposome enhance killing efficacy of MRSA compared with free vancomycin (Liu and Omri, 2014; Sande et al., 2012). Jiang et al. (2016) demonstrated enhanced targeted delivery of cell wall and cell membrane active daptomycin antibiotic against Staphylococcal pneumonia infection. Here, daptomycin performed dual function as an antibiotic and as a targeting ligand for 13

MRSA. The results revealed that daptomycin-liposomal formulation acts as promising therapeutic agent against MRSA and exhibit highly specific targeting against infection. Polymeric nanoparticles in combination with vancomycin against S. aureus, Enterococcus clinical infection (Lotfipour et al., 2013; Radovic-Moreno et al., 2012), penicillin against MRSA (He et al., 2013), ampicillin against P. aeruginosa and E. aerogenes and MRSA (Balland et al., 1996; Brown et al., 2012), etc. were efficiently used as delivery system for antibiotics. Similarly, liposomes in combination with vancomycin against MRSA (Pumerantz et al., 2011; Sande et al., 2012), rifampicin against Mycobacterium spp. (Vyas et al., 2004; Zaru et al., 2009), dendrimers with gatifloxacin against MRSA (Durairaj et al., 2010), vancomycin against drug resistant S. aureus (Choi et al., 2013), solid lipid nanoparticles in combination with norfloxacin against E. coli (Wang et al., 2012 ) and gatifloxacin against MRSA (Kalam et al., 2010; 2013) found to be effective for target specific drug delivery.

6. Antibacterial mechanism of nanoparticles and nanoparticle-antibiotics conjugate Various reports are available which proposed mechanism of antimicrobial action of different nanoparticles. Nanoparticles inhibit bacterial growth by several mechanisms such as inhibition of cell wall, alternation in cell membrane and inhibition of protein synthesis, etc. (Chatterjee et al., 2014; Golinska et al., 2016). However, mechanism of combination effect of nanoparticles and antimicrobial agents is still not well known and under investigation. Among metallic nanoparticles AgNPs has been found to be more effective against bacteria, viruses and eukaryotic microorganisms. At the cellular level, AgNPs attack the respiratory chain and cell division, leading to the cell death. Pal et al. (2007) claimed that the antibacterial mechanism of AgNPs was due to release of silver ions. After interaction with AgNPs, it release silver ions which inhibits cellular and respiratory enzymes essential for ATP production, induces the production of ROS, damages the DNA so that it loses its replicative ability and inhibits the expression of ribosomal subunit proteins. It has greater tendency to react with sulfur or phosphorus containing molecules, hence proteins and DNA are the preferential targets for them. Damage to membrane structure causes the substantial rise in its permeability, causing the uncontrolled damage, leading to cell death. In another study, Bawskar et al. (2015) proposed that AgNPs have demonstrated to induce efflux of phosphate, interact with thiol-group of membrane, which alters cytoplasmic constituents and inhibit respiratory enzymes and DNA replication in 14

pathogenic bacteria (Bawskar et al., 2015). Moreover, AuNPs exert antibacterial activity through the attachment to membrane which change the membrane potential leading to decrease in ATP level and affects protein synthesis by the inhibition of tRNA binding to the ribosome (Lima et al. 2013; Tiwari et al. 2011). Similarly, Li et al. (2015) showed that catechin-CuNPs interact with bacterial cell membrane leading to membrane lipid and protein bilayer disruption in S. aureus. The leakage of intracellular materials occur due to membrane damage by synergistic antibacterial effect of catechin-CuNPs as evidenced from SEM, TEM and AFM analysis. Antimicrobial action of CuNPs was also reported due to lipid peroxidation and generation of reactive oxygen species (ROS), protein oxidation and DNA degradation in E. coli (Chatterjee et al., 2014). According to Xie et al. (2011) ZnONPs affects the permeability of membranes and damage bacterial cell membrane where nanoparticles enter and induce oxidative stress subsequently resulting in the inhibition of cell growth and eventually in cell death. Moreover, ZnONPs were reported to inhibit growth of MRSA, methicillin-sensitive S. aureus (MSSA), and MRSE strains, K. pneumoniae, E. coli, S. enteritidis, S. mutans (Beyth et al., 2015) and also against extended spectrum β-lactamase producing E. coli and K. pneumoniae (Hameed et al., 2016). Induction of oxidative stress through the generation of ROS is considered as important antibacterial mechanism for TiO2NPs. Generation of ROS commonly cause site specific DNA damage (Cioffi and Rai, 2012). In addition, Roy et al. (2010) demonstrated the remarkable antibacterial activity of TiO2NPs against MRSA which proposed that interaction of TiO2NPs with cell membrane inhibits the bacterial growth. In another study, Carre et al. (2014) claimed that the antibacterial photocatalytic activity of TiO2NPs was resulted due to lipid peroxidation which leads to enhance membrane fluidity and disrupt the cell integrity in case of E. coli. Based on these observations and hypothesis, schematic representation of various antibacterial mechanisms for different nanoparticles has been illustrated in Figure 1. Moreover, antibiotics (amoxicillin) in combination with metallic nanoparticles like AgNPs shown to possess the enhanced synergistic antimicrobial effect against both Grampositive and Gram-negative bacteria. It is possible for resistant pathogens to develop resistance against antibiotics when applied singly, but it is difficult to develop resistance against combination of antibiotics and nanoparticles (antibiotic conjugated nanoparticles). It was proposed that, in case of combination, if pathogen develop resistance against either nanoparticles 15

or antibiotics, then another antimicrobial partner have bactericidal activity. Actually, due to nanoparticle-antibiotic conjugation (AgNPs- amoxicillin), the concentration of antibiotics increases at the actual place of antibiotic-pathogen contact (cell membrane), which accelerates the binding between pathogen and antibiotics (Allahverdiyev et al., 2011; Li et al., 2005). In another study, Gu et al. (2003) proposed the possible mechanism of antibacterial action for vancomycin-AuNPs conjugates against VRE. Generally, in case of vancomycin sensitive bacteria, vancomycin binds to d-alanine repeats present on the bacterial surface. This binding cause the inhibition of peptidoglycan biosynthesis of cell wall. Whereas, in case of VRE, terminal cell-surface peptide undergo modifications and thus lowers the vancomycin activity (Taylor and Webster, 2011). However, it was reported that through gold and sulphur interactions the vancomycin activity against VRE increases (Gu et al., 2003). Vancomycin capped AuNPs possibly interact in multivalent way with amino acid residues of glycanpeptidyl precursors on glycosides present on bacterial membrane (Allahverdiyev et al., 2011). Mechanistically it has been claimed that close contacts between vancomycin molecules, approximately 31 units per nanoparticles results into the modifications in binding properties by multivalent inhibition (Allahverdiyev et al., 2011; Taylor and Webster, 2011; Xing et al., 2003). These nanoparticles also been reported to inhibit VRSA non-specifically by binding to cell surface peptides involved in their cell wall synthesis (Fayaz et al., 2011). Hence, such combination therapy plays important role in management of MDR pathogens. Figure 2 showed the schematic representation of proposed mechanisms for antibacterial activity of antibiotics-conjugates nanoparticles. However, polymeric nanoparticles mediated drug delivery have different mechanism of action as compared to the antibiotic-conjugated nanoparticles. For example, aminoglycosidesloaded liposomes interact with the outer membrane of MDR P. aeruginosa, leading to the membrane deformation (Mugabe et al., 2006). The liposome carrying the antimicrobial drugs fuses with the cell membrane and thus immediately introduce drug inside the bacteria, potentially overtaking the efflux pump and suppressing the microbial drug resistance (Huh et al., 2011; Omri et al., 2002; Zhang et al., 2010).

7. Limitations in the use of nanoparticles With any kind of applications, the safety of the nanomaterials is also important. But their novel physico-chemical properties contribute to the concerns associated with them, which can affect 16

humans and environment. Chances of direct exposure to nanoparticles may occur preferably in individuals, whose occupation is to deal with the nanoparticles. Researchers and workers manufacturing nanoparticles are at high risk of getting exposed to them. Nanoparticles present in the environment can be internalized via either airway through the respiratory system or via digestive system through ingested food material or via direct entry through damaged skin. By all of these means, nanoparticles exposure leads to systemic entry whereby they can disturb the normal functioning of the metabolism (Gupta et al., 2012). Many scientific groups have rigorously studied the in vitro and in vivo toxicity of metal and metal oxide nanoparticles. As discussed earlier, nanoparticles have many applications, and therefore, there are high chances of generation of aerosols of those nanoparticles during their manufacturing and handling. But unfortunately, scanty literature is available regarding their toxicological effects which limit their uses. After single airway exposure of ZnO, TiO2, Al2O3 and CeO2 to BALB/cJ mice, they reduced tidal volume depending on the exposure duration and dose. However, ZnO and TiO2 were reported to induce nasal irritation. Al2O3 and TiO2 were found to strongly induce the inflammation in lungs, whereas ZnO with least inflammatory inducer (Larsen et al., 2016). Intranasal exposure of TiO2 and MWCNTs, induces the proinflammatory cytokines such as tumor necrosis factor-alpha and interleukin-6 in dose dependent manner. Similar results are also being reported while assessing the probable hazardous effects of metallic nanoparticles. They were reported to inflammatory inducer, inviting neutrophil and lymphocyte. Metal oxide nanoparticles were found to cause DNA strand breaks (Larsen et al., 2016). The nanoparticles reaching to blood circulation, can induce the peripheral inflammation in lungs, liver and kidney (Sukwong et al., 2016). All of these studies point towards the fact that nanoscale size, enhanced reactivity, heightened characteristics to translocate through tissues and cell contributes the cytotoxicity of nanoparticles. Concerns about environmental safety of nanoparticles are also rising. The elevated release of nanoparticles in aquatic ecosystem is one of those environmental threats. Aquatic exposure of ZnONPs lead to the biochemical and physiological changes in goldfish. Zn was found to get accumulated in fish liver brain and muscle (Yin et al., 2017). However, evidence suggests that subchronic exposure of AgNPs to Chapalichthys pardalis caused greatest damage to liver and gills (Valerio-García et al., 2017). Uptake through roots is the easiest and faster mode of translocation of nanoparticles form soil to the plants. After absorption, nanoparticles are 17

distributed throughout the plant and found to be accumulated. The accumulation of nanoparticles thus acts as obstacle in plant growth. In such condition, nanoparticles pose hazard to the plant and also invites risk to the complete food chain (Wang et al., 2017). These studies imply the importance of studying the impact of nanoparticles on environment, which also needs great consideration in finding the threats associated with them. The role of nanoparticles aggregation and their dissolution is still the matter of debate concerning the nanoparticle toxicities. Some studies claims that aggregation of nanoparticles play the profound role in exerting the toxicity. Some other studies proved that nanoparticles get accumulated in the highly active metabolic organs such as kidney and liver. In one hand, the accumulation of nanoparticles act as the obstacle in maintaining the homeostasis of such organs, indirectly affecting the entire body function. It is also speculated that nanoparticles release ions in the medium in which they are suspended. In such cases, it is difficult to give credit to the exerted toxic effect to the nanoparticles itself or the ion generated by their dissolution. This dissolution effect is negligible for AuNP, whereas it is significantly high for less stable nanoparticles such as AgNPs and CdSe NPs (Carrillo-Carrion et al., 2014). AgNPs dissolute to generate the Ag+ ions which is responsible for damage to the cells (Navarro et al., 2008). Similarly, other past studies confirmed that toxicity of various nanoparticles such as AgNPs, CuONP and ZnONPs is partially driven by the ions released from them (Bondarenko et al. 2013; Ivask et al., 2014). Santoro et al. (2007) also claimed that toxic effects of AgNPs are contributed by the ions released by their dissolution. The AgNPs and Ag+ ions treatment caused the differential expression of genes related to oxidative stress. Those genes were induced highly in presence of Ag+ ions as compared to the AgNPs (Kim et al., 2009). Although many upcoming studies are focusing on the issue of nanotoxicity, many unknown are still remaining. Therefore, there is a strong need of investigating nanotoxicity in greater depth to explore at cellular and molecular level. These studies can act as indicators for nanoparticle driven cytotoxicity which will help in deciding the safe use of this technology. 8. Conclusions The discovery of different antibiotics has brought revolution in biomedical field, which saves human life by providing a simple cure for various infections including life threatening diseases. However, widespread production and overuse of such antibiotics have contributed to emergence 18

of antibiotic resistance and development of MDR infectious organisms which is considered as a major concern for global public health. The rapid increase in the rate of antibiotic resistance in last few decades, has generated the need to develop powerful antibiotics or potent alternatives in order to tackle MDR problem. Current situation is really worst, and hence antibiotics alone can never be a solution for MDR pathogens. Recently, nanotechnology is emerging as a new tool to fight against antibiotic resistance as well as MDR infections and prevent the world from its catastrophic consequences. The evolution of nanotechnology is occurring very fast with wide range applications of nanomaterials in therapeutic treatments as nanomedicine can be new hope for keeping us in the race against antibiotic resistance. Various nanomaterials such as metal and metal oxide nanoparticles solely or in combination can be efficiently used as nanomedicine against the infections caused by MDR bacteria. The main reason behind the potential antibacterial efficacy of nanomaterials is that they can act by various mechanisms and bacteria are not yet immune to their effects. Thus, it would take time to develop resistance against nanomaterials. Moreover, antibiotics conjugated nanoparticle are proving to be potential weapon to prevent the menace of MDR microorganism. Herein, nanoparticles help to increase the concentration of antibiotics at the bacterial surface and thus increases their effectiveness. Similarly, nano-carrier mediated antibiotic delivery also useful to bypass the drug resistance mechanisms. Although, certain toxicological issues are associated with the use of nanomaterials, it is necessary to introduce nanomedicines in health care after extensive in vivo studies using animal models. More advance studies on similar path will surely give a nanotechnology based remedy to treat dreadfulness of multiple drug resistance so as to save each and every prestigious human life.

Conflict of interest Authors do not have conflict of interest to declare

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Figure Legends

Figure 1. Schematic representation of various antibacterial mechanisms for different nanoparticles against MDR bacteria.

Figure 2. Possible mechanisms of interaction between antibiotic-conjugated nanoparticles and MDR bacteria. Where, (1) AgNPs-amoxicillin conjugation increases the concentration of amoxicillin at the bacterial surface and inhibit bacterial growth (2) Vancomycin-capped AuNPs binds to amino acid residues of glycanpeptidyl precursors on glycosides present on bacterial membrane in different ways and thus help to restore antibacterial property of vancomycin which further inhibit cell wall synthesis.

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Table 1: Antibacterial efficacy of various nanoparticles against MDR bacteria Nanoparticles

Dose/ Size of Concentration nanoparticles 66.7 mM 100 nm

Tested microorganisms ERSP

References

AgNPs 83.3 mM

100 nm

AgNPs

80 µl

25 nm

Ampicillin resistant Escherichia coli 0157:H7; P. aeruginosa E. coli, S. aureus

Citrate-AgNPs

6.7 µg/ml

40 nm

PRAB, MRSA

ChitosanAgNPs

13.5 µg/ml

25 nm

P. aeruginosa

PVP-AgNPs

3.3 µg/ml

25 nm

27 µg/ml

10 nm

PVP-AgNPs

13.5 µg/ml 54 µg/ml

10 nm 10 nm

AgNPs

3.1 µg/ml

37-168 nm

Enterobacteriaceae sp. Enterobacteriaceae sp. P. aeruginosa S. aureus, MRSA E. coli, K. pneumonia

BiNPs

1.56 µg/ml 0.5mM

37-168 nm 20 nm

P. aeruginosa S. mutans

BiNPs

30 µg/ml

30 nm

P. aeruginosa

ZnONPs

0.55 µg/ml

90 nm

K. pneumoniae, P. aeruginosa, A. baumannii

0.45 µg/ml

90 nm

0.60 µg/ml

90 nm

E. coli, Proteus mirabilis

Lara et al., 2010

Singh et al., 2014 Cavassin et al., 2015 Cavassin et al., 2015

Cavassin et al., 2015 Cavassin et al., 2015 Shaker and Shaaban, 2017 HernandezDelgadillo et al., 2012 Luo et al., 2013

Al-Hartomy and Mujahid 2014

35

Staphylococcus epidermis, Streptococcus pneumoniae 0.65 µg/ml

90 nm Salmonella typhi, Bacillus subtilis

0.75 µg/ml

90 nm S. aureus, MRSA, Micrococcus luteus

ZnONPs functionalized with N-acetyl cysteine TiO2NPs

3.9 μg/ml

50 nm

E. coli, S. aureus

Hamed et al., 2015

350 μg/ml

**

P. aeruginosa

CuONPs

500 μg/ml

**

MgONPs

500 μg/ml

**

Al2O3NPs

1700 μg/ml

10 nm

S. aureus, P. aeruginosa S. aureus, P. aeruginosa S. aureus

9-179 nm

E. coli

9-180 nm

P. aeruginosa

Arora et al. 2015 Mirhosseini, 2015 Mirhosseini, 2015 Ansari et al., 2013 Ansari et al., 2014 Ansari et al., 2015

Al2O3NPs

1600-320 μg/ml Al2O3NPs 1600-320 μg/ml ** Details are not given.

36

Table 2: Antibacterial activity of nanoparticles in combination with different antibiotics against Gram-positive and Gram-negative bacteria. Nanoparticles Size (nm)

Antibiotics

AgNPs

60-80

Kanamycin, ampicillin, gentamicin, streptomycin, vancomycin

AgNPs

4

AgNPs

Bacteria

References

Grampositive MRSA

Gramnegative **

Ampicillin

MRSA

P. aeruginosa, Enterobacter aerogenes

Brown et al., 2012

5-40

Erythromycin, kanamycin, ampicillin, chloramphenicol

S. aureus, M. luteus

S. typhi, E. coli

Fayaz et al., 2010

TiO2NPs

20

Penicillin, amikacin, ampicillin, gentamicin, oxacillin, cloxacillin

MRSA

**

Roy et al., 2010

TiO2NPs

25

Ceftazidime

**

P. aeruginosa

AuNPs

4-5

Vancomycin

**

VRE

Arora et al., 2015 Burygin et al., 2009

AuNPs

4-5

Vancomycin

VRSA

**

Mandal et al., 2006

ZnONPs

66

Proteus sp., S. aureus

E. coli, P. aeruginosa, Acinetobacter sp.

Ehsan and Sajjad, 2017

ZnONPs

**

Gentamicin, Amkacin, oxacillin, erythromycin, ceftriaxone, amoxyclav, fodfomycin, clindamycin, ciprofloxacin Ciprofloxacin

S. aureus

Fe3O4

**

Vancomycin

MRSA

E. coli, Klebsiella sp. VRE

Patra et al., 2014 Choi et al., 2012

Birla et al., 2009

** Details are not given.

37