Elsevier

Carbohydrate Polymers

Volume 256, 15 March 2021, 117498
Carbohydrate Polymers

Boosting the antibacterial activity of chitosan–gold nanoparticles against antibiotic–resistant bacteria by Punicagranatum L. extract

https://doi.org/10.1016/j.carbpol.2020.117498Get rights and content

Highlights

Abstract

The present work describes the synthesis of a new series of chitosan–gold hybrid nanoparticles (CS–AuNPs) for the delivery of Punicagranatum L. extract (PE). It proposes CS and PE as reducing agents for gold ions in aqueous solution. The effect of PE on the physicochemical properties of the CS–AuNPs was investigated with UV spectroscopy, DLS, DSC, XRD, FTIR, SEM/EDX and TEM. Interestingly, about 50 % reduction in size was observed with using PE alone for gold reduction. The ζ–potential of CS–AuNPs was shifted from +53.1 ± 6.7 mV to 31.0 ± 6.0 mV upon conjugation of the negatively–charged PE polyphenols. The developed PE–conjugated CS–AuNPs exhibited higher stability at different pH values. About 87 % of the loaded PE was released from the NPs over 24 h. The antibacterial activity of CS–PE–AuNPs displayed a synergetic affect against methicillin–resistant S. aureus with MIC and MBC values of 15.6 and 62.5 μg/mL, respectively.

Introduction

Antimicrobial resistance is an emerging health/life and food security threat that needs concerted global action to be overcome (Wester, Gopinathan, Gjefle, Solberg, & Røttingen, 2017). When infections cannot be treated with first–line antibiotics, highly expensive medicines must be used. A longer illness and treatment duration, even in hospitals, increases health–care costs as well as the economic burden on families and societies. According to the World Health Organization (WHO), antibiotic resistance is putting the achievements of modern medicine at risk. Therefore, the WHO set a global action plan on antimicrobial resistance which has five strategic objectives one of which is to strengthen surveillance and research. Therefore, new natural antibacterial agents should be developed to combat this alarming problem.

Failure to tackle antibiotic–resistant bacteria is estimated to cost an extra 10 million deaths per year worldwide by 2050 (de Kraker, Stewardson, & Harbarth, 2016) with major global economic burden (Llp, 2014). Superbugs or supergerms is a phenomenon which refers to that bacterial strains which show resistance to the majority of antibiotics (Huang et al., 2016) such as S.aureus which has become resistant to both methicillin (Loomba, Taneja, & Mishra, 2010) and vancomycin (Thati, Shivannavar, & Gaddad, 2011).

In 1968, emergency of methicillin–resistant Staphylococcus aureus (MRSA) outbreaks in hospital has been documented (Barrett, McGehee, & Finland, 1968). MRSA has been described firstly as a hospital–acquired MRSA (HA–MRSA) or nosocomial outbreaks. After that, MRSA could emerge its virulence out of hospitals (in the community) and known as community–acquired MRSA (CA–MRSA) (Kali et al., 2013). MRSA are notorious for their ability to develop its resistance even against new antibiotics. MRSA causes mild skin infections, such as sores, boils, impertigo, or abscesses. But it can also cause more serious skin infections or infect surgical wounds, the lungs (acute pneumonia), or osteomyelitis. In severe cases, it causes meningitis, the bloodstream infection (septicaemia), or endocarditis which are considered fatal cases (Kali et al., 2013; Sharifi–Rad, Hoseini Alfatemi, Sharifi Rad, & Iriti, 2014; Ye, Li, & Fang, 2014). MRSA shows resistance also to aminoglycosides, macrolides, tetracycline, chloramphenicol, and lincosamides. Lately, transferable resistance has occurred to MRSA against vancomycin which had been used to overcome such strain. Moreover, MRSA has shown resistance to disinfectant and could cause acquired infection in hospital.

Traditional therapy using antibiotics for wound healing are faced with several challenges, due to the emergence of multidrug–resistant (MDR) organisms and a decrease in newer antibiotics (Unemo & Nicholas, 2012).

As a result of insufficiency of conventional antibiotics to treat MDR bacteria wound infection, the research applicable strategies of natural products in remedying such infections against MDR bacteria is a major challenge and in huge demand.

Gold nanoparticles (AuNPs) research is a subject of substantial interest around the world due to their wide potential applications in areas such as nanotechnology, electronics and biomedicine. In addition, AuNPs can be synthesized with a variety of sizes and shapes according to the desired application. Moreover, it is easy to modify their surfaces with several kinds of molecules including proteins, carbohydrates and lipids (Pissuwan, 2017). However, it was reported that AuNPs have weak antibacterial activity compared to other metal NPs (Mokammel, Islam, Hasanuzzaman, & Hashmi, 2019). The antibacterial properties of AuNPs can be regarded to the production of reactive oxygen species that induce oxidative stress inside bacterial cells. Therefore, composites of AuNPs are formed with other antibacterial agents in order to enhance the antimicrobial activity of the former.

Chitosan (CS), a long biopolymer chain of N–acetylglucosamine, was reported to have several advantages such as high broad–spectrum antibacterial activity and low toxicity to mammalian cells (Das & Patra, 2017). CS is a natural aminated polysaccharide obtained via the deacetylation of chitin, a homopolymer constituting the major structural component of shrimp exoskeleton, crab shells and fungi cell walls (Pokhrel & Yadav, 2019; Sánchez–Machado et al., 2019). In addition, CS is biocompatible, hydrophilic, biodegradable and susceptible to chemical modification (Agrawal, Strijkers, & Nicolay, 2010). It was reported that CS with sulfamethoxazole have the ability to treat the antibiotic–resistant P. aeruginosa (Tin, Sakharkar, Lim, & Sakharkar, 2009). Similarly, we expect that the combination of CS and PE in the shell of CS–coated AuNPs may boost their antimicrobial activity against antibiotic–resistant Staphylococcus aureus.

Punica granatum L. extract (PE) is known for its high antibacterial, antifungal and antiviral activities (Angamuthu, Purushothaman, Kothandan, & Swaminathan, 2019; Wafa et al., 2017). Several studies reported the use of P. granatum fruit extract for the synthesis of metallic NPs due to its high reducing properties (Sukri et al., 2019; Yusefi, Shameli, Ali, Pang, & Teow, 2020). Shahbazi and Shavisi used Mentha spicata essential oil and PE to enhance the antibacterial/antioxidant characteristics of nano–chitosan films (Shahbazi & Shavisi, 2018). Moreover, Patel et al. used PE to induce the synthesis of AuNPs and studied the influence of the produced AuNPs on the lifespan and fertility of C. elegans (Patel, Siddiqi, Sharma, Alhomida, & Khan, 2019). In another study reported by Gubitosa and coworkers, a one–pot synthesis method was used for the preparation of AuNPs using Punica granatum juice for dermatological and cosmetic applications (Gubitosa et al., 2018). A combination of CS with plant extract (Lignosus rhinocerotis) was used as a reducing agent for the biosynthesis of AuNPs that demonstrated good antibacterial activity against P. aeruginosa, Escherichia coli, Staphylococcus aureus and Bacillus sp. (Katas, Lim, Nor Azlan, Buang, & Mh Busra, 2019).

In one of our recent studies, the effect of different concentrations of CS (as a reducing/capping agent) on the physicochemical and antibacterial properties of CS–AuNPs was investigated (Hussein et al., 2020). The approach of hybrid nanocomposites is considered an interesting trend for obtaining new materials of advanced properties. Fabrication of polymeric nano–spheres based on metal NPs in the core, combines both the advantages of physicochemical characteristics and biological properties of nano–sized nobel metal and polymer respectively (Zienkiewicz–Strzałka et al., 2019).

With the aim of gaining more impact, better efficiency and specific targeting, the study here hypothesized the use of AuNPs as a platform incorporated by other polymeric and material components of biocompatible properties with maintaining the hybrid water solubility and the status of colloidal stability. Such features are so crucial to trigger the specific biomedical tasks on–demand and to address the basic design requirements of the service life cycle. By such complex hybrid AuNPs, it could introduce and present multiple functionalities within a single nanostructure.

The main objective of the present study is to incorporate PE into the CS–AuNPs in order to boost their antibacterial activity against antibiotic–resistant bacteria. The physicochemical properties, antibacterial activity against methicillin–resistant Staphylococcus aureus (as a model bacterial species), loading capacity (LC), encapsulation efficiency (EE) and PE–release profile were thoroughly investigated using a number of analytical tools.

Section snippets

Materials

P. granatum was obtained from the local market. CS was purchased from Sigma–Aldrich (448877–250 G). The molecular weight of CS is in the order of 45 kDa and its degree of acetylation is about 80 %, according to the manufacturer’s data. Glacial acetic acid (Panreac) and HAuCl4.3H2O (99.5 %, Sigma–Aldrich, Germany) were used throughout the experimental procedure. All chemicals were of analytical grade and were used without further purification. Methicillin–resistant Staphylococcus aureus

Results and discussion

It is well–known that AuNPs are prepared via the reduction of gold ions (Au+) to metallic gold (Au0) by various means. In the present work, we aimed at fabricating CS–AuNPs functionalized with PE. The process involves the reduction of gold ions by CS to form AuNPs in the presence of PE. A mixture of CS and PE was stirred for 4 h and then the temperature was increased to 80 °C. This step is necessary for the homogeneous mixing of PE with CS and it allowed the PE components to self–assemble into

Conclusions

We report herein a method for boosting the antibacterial properties of CS–AuNPs against antibiotic–resistant bacteria via incorporating PE into the CS coating. The physicochemical properties of the as synthesized CS–PE–AuNPs were enhanced compared to their CS–AuNPs and PE–AuNPs counterparts. The current study provides a promising antibacterial agent based on a natural/edible fruit extract and carbohydrate polymer against antibiotic–resistant bacteria such as MSRA. It was found that the proposed

CRediT authorship contribution statement

Mohamed A. Mohamady Hussein: Conceptualization, Methodology, Investigation, Visualization, Writing - original draft, Funding acquisition. Mariusz Grinholc: Resources, Methodology, Funding acquisition. Ahmed S. Abo Dena: Methodology, Writing - review & editing. Ibrahim M. El-Sherbiny: Conceptualization, Methodology, Resources, Writing - review & editing. Mosaad Megahed: Conceptualization, Methodology, Resources, Supervision, Project administration, Writing - review & editing.

Acknowledgements

The authors are grateful to the Islamic Development Bank (IDB), Jeddah, Saudi Arabia, for financially supporting this work through the IDB Merit Scholarship Program (Ref. 36/11207330, File No. 23/EGT/P34).

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