Elsevier

Microbial Pathogenesis

Volume 99, October 2016, Pages 271-281
Microbial Pathogenesis

Amphotericin B-conjugated biogenic silver nanoparticles as an innovative strategy for fungal infections

https://doi.org/10.1016/j.micpath.2016.08.031Get rights and content

Highlights

  • Preparation of biogenic silver nanoparticles.

  • Covalent conjugation of amphotericin B to biogenic nanoparticles.

  • Enhanced antifungal activity.

  • A novel green approach to treat fungal infections.

Abstract

New strategies are required to improve the efficacy of drugs and to treat the emerging microbial resistance. An effective strategy is to combine drugs with metal nanoparticles for the control of microbial infections and resistance. Keeping in view this fact, we developed a facile and eco-friendly protocol for the synthesis of amphotericin B-conjugated silver nanoparticles and their assessment as an antifungal agent. Phytochemicals from the aqueous extract of Maytenus royleanus and amphotericin B were used as capping agents to prepare two types of silver nanoparticles i.e. (i) biogenic silver nanoparticles (b-AgNPs) and (ii) amphotericin B-conjugated biogenic silver nanoparticles (Amp-bAgNPs). UV-Vis spectroscopy was used to detect the characteristic surface Plasmon resonance peaks (SPR) for the prepared nanoparticles (424–433 nm). High-resolution transmission electron microscopy (HRTEM) study revealed the formation of well dispersed and spherical silver nanoparticles and Amp-bAgNPs with an average particles size of 10 and 15 nm. EDX and FTIR studies confirmed the elemental composition and surface adhered biomolecules in the prepared nanoparticles respectively. Biogenic silver nanoparticles revealed low to moderate antifungal activity (4–8 mm ± 0.2), however, the amphotericin B conjugated silver nanoparticles exhibited significant activity against Candida albicans (16 mm ± 1.4) and Candida tropicalis (18 mm ± 1.5). In conclusion, the enhanced antifungal activity of the Amp-AgNPs conjugate system is due to the synergy between the antifungal activity of amphotericin B and the antimicrobial property of silver. The findings of this study suggest that the conjugated nanoparticles could be used as efficient antifungal agents and drug delivery vehicles. Furthermore, this is the first report describing the synthesis of silver nanoparticles using the aqueous extract of Maytenus royleanus and the conjugation of amphotericin B, an antifungal drug, to the phytosynthesized silver nanoparticles.

Introduction

The ‘greener’ and eco-friendly procedures in chemical science and technology are becoming increasingly popular and are much needed because of global problems related with environmental contamination. The chemists and technologists around the globe follow the basic principles of green chemistry to develop less hazardous synthesis procedures. Among the modern line of technological advancements, nano-biotechnology occupies an important position to create and enhance the utility of nano-size materials in advanced biotechnology [1]. Nanoscale materials and devices have unique physiochemical characteristics that make them applicable in fields ranging from electronic to medicine [2], [3]. Among the metal nanoparticles, nano silver is widely used in a wide array of consumer products and clinical care, which possess strong toxicity against a broad spectrum of pathogenic microbes [4], [5], [6], [7].

The pathogenesis caused by fungal species has a significant influence on animal and plant life. The most common fungal pathogenesis that affects approximately 1.7 billion people worldwide is the superficial skin and nail infections in humans [8]. Oral infections in babies and vulvovaginal candidiasis in women are also common fungal pathogenesis that affects up to 75% of the women in the childbearing age [9]. Most of these superficial complications are caused by various species of Candida. Candida albicans, an opportunistic pathogen, is a normal commensal of the gastrointestinal microflora in healthy individuals. Candida albicans is the common agent of candidiasis and is considered as the fourth most frequent infection in the united states hospitals [10], mainly due to the increasing number of susceptible compromised individuals that at risk of this systemic infection [11], [12]. Several Candida species have been reported that cause invasive Candidiasis in human. Invasive fungal infection is a major cause of mortality that kills approximately 1.5 million people every year. More than 17 different species of Candida have been reported to be etiologic agents of invasive candidiasis in humans [13], [14].

Amphotericin B is the most common antifungal agent used in clinical practice; however, fungal resistance has been reported to this drug [15], [16]. Furthermore, amphotericin B is believed to self-assembled into higher-order molecular forms that result in its toxic property. The immobilization of this drug on a metal substrate would minimize its chance of self-assembly and would enhance its efficacy towards target binding (ergosterol), thus diminishing its toxicity. Hence, new strategies are needed to conjugate amphotericin B with metal nanoparticles in order to increase its efficacy, reduce its toxicity and to minimize the chance of fungal resistance to amphotericin B.

Nanoscale materials have emerged as the new class of promising antimicrobial agents due to their unique physiochemical characteristics and a large surface to volume ratio [17]. Among the metal structure, silver nanoparticles are well known for their antimicrobial activities, which exert a complex mechanism of action by interfering DNA replication, respiratory chain mechanism, and other cellular proteins [18], [19], [20]. In addition, nano-silver also promotes the generation of several reactive oxygen species (ROS), which destroy pathogenic microbes by a process called respiratory burst mechanism [21]. With nanotechnology, the availability of AgNPs has enabled the use of pure silver to accomplish a rapid growth in medical practice.

Both physical and chemical methods have been successfully used to synthesize metal nanoparticles. However, all those procedures generally rely on the use of expensive and hazardous chemicals and are therefore not eco-friendly. The synthesis of metal nanoparticles using eco-friendly and biocompatible reagents could minimize the toxicity of the resulting nanomaterials and the environmental impact of the byproducts [22]. Silver nanoparticles with desirable features can be prepared by the green approach. Plants represent a renewable source of bioactive molecules that can be effectively used as reducing and capping agents for the synthesis of large-scale metal nanoparticles [23]. Maytenus royleanus belongs to the family Celastraceae, widely distributed in the Northern region of Pakistan and is commonly known as “sur azghee. Maytenus has been used in folk medicine for the treatment of a variety of ailments including chest pains, rheumatism, diarrhea, eye infection, dyspepsia and wounds healing [24], [25]. The biological actions of Maytenus species are due to the presence of various bioactive secondary metabolites such as flavonoids, phenolic glucosides, and triterpenes [26].

In the present contribution, an eco-friendly and facile method was developed to synthesize silver nanoparticles. The aqueous extract of Maytenus royleanus was used to reduce and stabilize silver ions into silver nanoparticles without any supportive chemicals. The antifungal drug, amphotericin B was conjugated with biogenic silver nanoparticles via EDC and NHS coupling reaction. The antifungal effect of biogenic and amphotericin B- conjugated silver nanoparticles was tested against Candida albicans and Candida tropicalis.

Section snippets

Materials

Maytenus royleanus was collected from the Northern area (Swat) of Pakistan. Amphotericin B, 1-(3-(dimethylamino) propyl) 3-ethylcarbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich while silver nitrate was purchased from Beijing Chemical Works China respectively. All the chemicals used in this study were of analytical grade.

Preparation of plant extract

Dried and powdered plant material was washed several times with de-ionized distilled water to remove any dust particles. The

UV-visible spectroscopy

The progress of nanoparticles synthesis was monitored by UV-visible spectroscopy. Silver nanoparticles exhibit a characteristic surface plasmon resonance phenomenon (SPR) which can be used to detect the synthesis of metal nanoparticles. Aliquots of the reaction mixture were taken at regular intervals of time and analyzed for the evolution of specific SPR peaks for silver nanoparticles. Fig. 1 presents the time-dependent evolution of UV-vis spectra for silver nanoparticles synthesized from

Conclusion

The aqueous extract of Maytenus royleanus was successfully used to reduce and stabilize silver ions into silver nanoparticles. The developed protocol was purely green as no toxic reagents were used to prepare metal nanoparticles. The phytosynthesized silver nanoparticles exhibited low to moderate activity against Candida albicans and Candida tropicalis respectively. However, the antifungal activity of these nanoparticles was enhanced by conjugating with amphotericin B. The antifungal drug

Acknowledgments

The authors are highly grateful to Chinese Scholarship Council (CSC No. 2014DFH974) and International Science & Technology Cooperation Program of China (Grant No. 2013DFR90290) for supporting the current project.

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