금속 나노입자의 생체 합성과 항균적 적용

Biogenic Synthesis of Metallic Nanoparticles and Their Antibacterial Applications

Abstract

최근 생물의학 분야에서의 광범위한 응용 가능성에 의하여 식물이나 미생물을 이용한 은(Ag), 금(Au), 백금(Pt), 세륨(Ce), 아연(Zn), 구리(Cu) 등의 금속 나노물질 합성에 관한 연구가 주목받고 있다. 식물은 플라보노이드, 알칼로이드, 사포닌, 스테로이드 탄닌과 각종 영양 성분과 같은 생리 활성 물질을 풍부하게 가지고 있으며, 유사하게 미생물들도 단백질과 같은 생리활성 대사산물이나 색소, 항생제 및 산과 같은 가치가 있는 화학물질을 분비한다. 최근 보고된 바에 의하면, 나노입자의 생체 합성은 무해한 방법일 뿐만 아니라 항균, 항진균, 세포 증식 억제 및 항플라스모디아 활성과 같은 생물의학 분야로서의 적용에 주요한 후보로 여겨진다. 나노입자의 이러한 생리 활성은 농도에 의존적이며, 나노입자의 모양과 크기에도 따라 달라질 수 있다. 미생물과 식물은 나노입자의 친환경적합성에 사용되는 대사산물이나 화학물질 등의 훌륭한 공급원으로서 생물 의학 분야에서 유용하게 사용된다. 미생물 또는 식물 원료를 사용하여 합성된 나노입자는 화학적인 방법으로 합성된 나노입자보다 더 낮은 독성을 나타낸다. 본 리뷰논문에서는 미생물이나 식물과 같은 생물학적 재료를 이용한 나노입자의 합성과, 합성에 사용되는 다양한 기술의 특성 및 나노입자의 항균 분야에서의 적용에 대하여 중점적으로 서술하였다.

Recent studies on synthesis of metallic nanomaterials such as silver (Ag), gold (Au), platinum (Pt), cerium (Ce), zinc (Zn), and copper (Cu) nanoparticles (NPs) using plants and microbes are attracted researchers for their wide range of applications in the field of biomedical sciences. The plant contains abundant of bioactive contents such as flavonoids, alkaloids, saponins, steroids tannins and nutritionals components. Similarly, microbes produce bioactive metabolites, proteins and secretes valuable chemicals such as color pigments, antibiotics, and acids. Recently reported, biogenic synthesis of NPs in non-hazardous way and are promising candidates for biomedical applications such as antibacterial, antifungal, anti-cell proliferative and anti-plasmodia activity. All those activities are dose dependent, along with their shape and size also matters on potential of NPs. Microbes and plants are great source of metabolites, those useful in biomedical field, such metabolites or chemicals involved in synthesis of NPs in an ecofriendly way. NPs synthesized using microbes or plant materials are reveals more non-toxic, facile, and cost-effective compare to chemically synthesized NPs. In present review we are focusing on NPs synthesis using biological agents such as microbes (bacteria, fungi and algae) and plant, characterization using different techniques and their antibacterial applications on pathogenic Gram-positive and Gram-negative organisms.

Introduction

Nanotechnology is one of the most active research fields and deal with synthesis and usage of nano-sized materials in the field of science and technology. Research on nano-materials is rapidly growing because of their different properties at the nanoscale. The decoration of nanoscale material is the backbone of nanotechnology and their applications in the field of biology, chemistry and engineering. Nano particles (NPs) synthesis is mainly performed by two ways, top down and bottom-up. In top-down approach involves cutting or crushing of bulk materials into fine nanoscale particles. The bottom-up approaches involves the creation of materials at nanoscale by assembling them atom to atom, molecules by molecules and cluster by cluster. The different methods have been developed for the NPs synthesis including physical, chemical and biological [33]. NPs synthesis using different methods are schematically presented in the Fig. 1.

Fig. 1. Schematic presentation of nanoparticles synthesis through physical, chemical and biological method.

The physical methods for synthesis of nanomaterials utilizes high energy, pressure, and required high temperature [33]. Most commonly used physical methods include attrition and pyrolysis. Attrition involves the grinding of particles by a size-reducing mechanism and pyrolysis involves burning of precursor and passing at high pressure. Chemical methods for synthesis of NPs utilizes toxic chemicals which is harmful for human beings as well as to environment [42]. Most commonly used chemical methods are chemical reduction [105], electrochemical techniques [46], and photo-chemical reactions [78]. Nowadays researches focusing on biological methods for synthesis of NPs [57,96] which includes use of biological agents such as microorganisms [58,59], and plants [34]. The biological method for synthesis of nano materials have been utilized as an alternative method to the chemical or physical methods because of their ecofriendly approach. In biological synthesis of NPs involves use of bacteria [59,61], fungi [31], algae [22] and plants [44,57]. The NPs synthesized using biological methods are known as biogenic NPs. Such NPs are non-toxic and shows different potential because of their different shape and size [92]. Because of the optoelectronic and physicochemical properties of nanoscale materials are applied in the field of chemical catalysis, immunosensors, pharmaceutical products, anti-microbial, anti-cancer, drug delivery and medical diagnosis. This review is concerned with the ecofriendly route for the synthesis of metallic NPs using biological resources including plants and microbes, short introduction to the characterization techniques employed for metallic NPs, antibacterial applications of metallic NPs and future prospects and challenges of the metallic NPs.

Biological Method for Nanoparticles Synthesis

Traditionally NPs synthesized by physical, chemical and biological methods. Among that, physical and chemical methods need higher degree of energy / heat and hazardous chemicals, respectively. Because of these unfavorable issues and the byproducts of these methods are very harmful and hazardous to human beings and environment too. While, in the biological method of NPs synthesis, use of biological agents such as bacteria, fungi or plants has been reported with an ecofriendly approach. Its byproducts are not harmful to environment, and because of that this method has gained more attraction of researches. Here in this review we will focus more on biological method of NPs synthesis only and their antibacterial applications.

The use of biological method for NPs synthesis is facile and ecofriendly [44,57], in that the plant materials and microorganisms used as a reducing agents [58,59]. The step-wise process for NPs synthesis in biological method are presented in Fig. 2. The plant material is extracted in water or other solvents and used as a reducing agent [85]. While in case of microorganisms, the extracellular synthesis of NPs can be performed by using supernatant of microbes after microbial growth [61] and intracellular NPs formation observed inside of the microbial cells [58,59]. The biochemical reaction between metal salt and plant extracts or supernatant of microbial broth results in formation of specific color to the reaction solution because of formation of NPs by reducing metal ions [28]. Aggregation of NPs creates specific shape (spherical, oval, triangular, rod, wire, square, pentagonal, hexagonal, diamond shape, flower like shape etc.) and size of NPs [40]. The in the present review, biogenic synthesis of few metallic NPs are explained with the help of some selected research studies only.

Fig. 2. Nanoparticles synthesis by biological method are presented in stepwise order.

Use of Plant Materials

The plant crude extracts have been considered a green route and reliable method for synthesis of metallic NPs owing to its ecofriendly nature [103]. Plant extract containing different active chemicals (phytochemicals) and secondary metabolites including phenols, alkaloids, flavonoids, terpenoids and acids in which these components are involve in reduction process and formation of NPs [44]. These natural products are derived from various parts of plants such as root, stem, leaf, flower, fruits, seeds, rhizomes, tubers and gums etc. The extract of plant containing secondary metabolites act as a reducing as well as stabilizing agents in the process of metallic NPs synthesis [25,49]. When plant extracts mixed with metal salts, it results in formation of specific color to the reaction solution because of formation of NPs [57]. The characteristic color observed from yellow to brown and yellow to deep red or purple for AgNPs and AuNPs, respectively due to surface plasmon vibration [67]. The different plants reported for synthesis of metallic NPs are listed in Table 1.

Table 1. Different type of nanoparticles synthesized using plant materials and their applications

The flower extract of Madhuca longifolia mixed with silver nitrate solution and reaction performed for AgNPs synthesis. 30-35 nm, spherical and oval shape AgNPs were observed under transmission electron microscope (TEM) and NPs surface covered by phytochemicals were observed by using Fourier transform infrared spectroscopy (FT-IR). The interaction of functional groups such as alcohols, phenols, and amines were confirmed by comparing specific peaks between AgNPs and M. longifolia extracts FT-IR spectra [64]. Likewise, aqueous extract of Lonicera japonica prepared by boiling of flowers in water and obtained crude extract were used for NPs synthesis. Flower extract mixed with choloauric acid and formation of AuNPs were noticed by visible color change from yellow to ruby red and confirmed by observing Ultraviolet-visible (UV-Vis) spectroscopic absorbance peak at 530 to 580 nm [60]. It was observed that increasing of AuNPs formation when reaction temperature is increasing and also different pH studies suggested that acidic condition of reaction inhibit formation of NPs while alkaline condition enhances NPs formation [43,60]. In biogenic synthesis of NPs, different shape and sizes forms because of various phytochemicals present in extract of plant. Sequential fractions of Ocimum sanctum leaves were obtained by using different polarity solvents includes water, n-butanol, chloroform and hexane and reported for synthesis of morphologically different AuNPs; chloroform extract produced more than 200 nm circular disc shape with rough edge, hexane extract produced 100 nm spherical NPs, water extract produces anisotropic NPs, and n-butanol produced aggregation of AuNPs [45]. The leaves extract of medicinal plants (Jatropa grossypifolia and Jatropa glandulifera) reported for synthesis of PtNPs [37]. The medicinal plants leaves were extracted in water and crude extract mixed with hexachloroplatinic acid; the reaction mixture developed a dark brown color which indicated formation of PtNPs and confirmed by observing absorbance peak at 260 nm by UV-vis spectrophotometry, also proposed that -SO₂ functional group were found to be prone for reduction of platinum ions to PtNPs based on FT-IR studies. Likewise, dates extract also reported for biogenic synthesis of PtNPs, and found NPs were potential as an antibacterial activity against Gram-positive and Gram-negative bacteria and cytotoxic effect against different cancer cell lines [2]. Nanoceria synthesis reported by using leaf extract of plant Pisonia alba; produced CeO₂ NPs were reported for having antioxidant effect and antifungal activity [86]. Single step, photosynthesis of CeO₂ NPs reported recently; Gloriosa superba leaf extract mediated CeO₂ NPs were spherical shape with an average of 5 nm and showed antibacterial activity [9]. Zinc oxide (ZnO) NPs synthesis using leaf extract of Mentha pulegium; plant extract mixed with zinc nitrate hexahydrate and boiled until reaction mixture becomes paste and after that heated at 400ºC for 2 hours to obtain powder form of ZnONPs. The synthesized ZnONPs found potential as an antibacterial activity against human pathogens [69]. Similarly, other group of researchers reported ZnONPs synthesis using stem bark extract from Albizia lebbeck and reported for potential biomedical applications includes antimicrobial, antioxidant, and cytotoxic activity against human breast cancer cell lines [98]. Synthesis of Copper (Cu) NPs using Hagenia abyssinica was reported as an ecofriendly and green method, synthesized CuNPs indicates absorbance band at 403 nm due to surface plasmon resonance and has been reports as an antibacterial agent [7]. In another research, synthesis of CuNPs using Syzygium aromaticum bud extract reported, formation of CuNPs visually observed by reaction solution color change from blue to green and conformed by observing absorbance peak at about 580 nm by VU-vis spectrometry [71].

Use of Microorganisms

The biogenic synthesis of metallic NPs using living cells is a promising and novelty tool in nano-biotechnology. Intraor extracellular biosynthesis of metallic NPs can be achieved by using microorganisms including bacteria, fungi, yeast, actinomycetes and algae [58, 59, 90, 99]. Microorganisms are preferred for synthesis of metallic NPs because of required moderate conditions, easy purification and high yield. Therefore, recently microbes reported widely for NPs synthesis with title of “bacteria as nano-factory” [90]. Different metallic NPs synthesized by microorganisms are listed in Table 2. The synthesis of NPs can be carried out both intracellularly and extracellularly using microorganisms [88]. In recent years, CuNPs synthesis using bacterium Morganella morganii reported and the reduction mechanism were proposed is CuSO₄.5H₂O + bacteria = CuNPs + Byproducts [73]. For extracellular synthesis, the culture filtrate or supernatant is collected and mixed with metal salt solution. While for intracellular synthesis, cell biomass or cells inoculated with metal salt solution. Visual observation for formation of NPs can be determined by color change or reaction mixture. For example, yellow to brown color is an indication of AgNPs synthesis [4], and yellow to ruby red color formation for AuNPs formation [61]. The extracellular formation of NPs is depending on the presence of microbial proteins or enzymes secreted by microbes in the cell free supernatant. It is widely accepted that nitrate reductase enzyme involved in formation of NPs by reducing metal ions [32]. Recently reported extracellular synthesis of NPs by using cell free supernatant including AgNPs (spherical, 3-20 nm) by bacterium Bacillus subtilis [4], AuNPs (spherical, 20.93 ± 3.46) by bacterium Paracoccus haeundaensis [61], Zinc oxide NPs (hexagonal, 34-55 nm) by fungi Xylaria acuta [93]. The hypothetical intracellular NPs synthesis mechanism is carried out by trapping of metal ions (positive charge) on the surface of cell (negative charge) or negative group containing enzymes / protein in the cytoplasm; the trapped ions reduced to nuclei and subsequently for different shaped NPs [32, 58, 59]. In an intracellular synthesis of NPs, a cellular enzymes involved in bio-reduction process for formation of NPs, a research work reported intracellular synthesis of AgNPs by using Pseudomonas stutaeri AG259; a small sized AgNPs ranging from 1-200 nm were observed inside the bacterial cell. The formation of AgNPs because of NADH dependent enzymes [41]. The synthesized NPs collection or separation needs an additional step, mechanical breakdown of cell wall followed by centrifugation and purification of NPs. The mechanism for NPs synthesis is differs because of different biochemical properties of microorganisms, which is the one major reason for many mechanisms still not clear.

Table 2. Different type of nanoparticles synthesized using microorganisms and their applications

Abbreviations: NPs–nanoparticles, Ag–silver, Au–gold, Zn–zinc, ZnO–Zinc oxide, Cu–Copper, CuO–copper oxide. Ce– cerium, CeO–Cerium oxide, Pt–platinum, nm–nanometer, NA–Not applicable.

Characterizations of Nanoparticles

To date, there are many techniques employed for characterization of NPs. However, reaction color change is primary indication and NPs formation is confirmed by observation of peak at specific wavelength by UV-visible spectrophotometry [19]. The formation of specific shape, size can be determined by visible color formation of solution containing nanoparticles and confirmation can be done by electron microscopy [91]. Formation of different sizes of NPs also determined by electrophoretic light scattering spectroscopy [95], scanning electron microscopy and transmission electron microscopy [26]. The elemental metallic nature determined by energy dispersive x-ray analysis [97] and the crystalline nature of metallic nanoparticles determined by X-ray diffraction analysis [77]. The presence of positive or negative charges on surface of NPs are studied by zeta potential measurements [61]. The interaction of biomolecules from plant extract or microbial supernatant which involves in formation of NPs are analyzed by FT-IR, in which specific transmission spectrums analyzed. These are the only few techniques listed here for characterization of metallic NPs.

Antibacterial Applications of Nanoparticles

The advantages of nanotechnology are growing quickly in several fields [55, 79, 80, 103]. Metallic NPs are applicable to many emerging technologies such as catalysis, water filtration, food packaging, agricultural, cosmetics, dressing for infection or burn [44, 47, 82]. The major biomedical applications of selected metallic NPs are presented in Fig. 3. The development of novel drug is necessary to treat multi drug resistant microbes. The application of NPs in biomedical field is like a revolution because of their antibacterial activity [44,59]. There are many studies reported for biogenic synthesis of metallic NPs such as Ag, Au, Zn, ZnO, Cu, CuO, Ce, CeO and Pt using different microorganisms and plants. The antibacterial activity of metallic NPs against different pathogenic bacteria are listed in Table 3.

Fig. 3. The diagrammatic presentation of biomedical applications of metallic nanoparticles.

Table 3. Metallic nanoparticles and their antibacterial effect on pathogenic bacteria

The exact mechanism of metallic NPs action on microbes is still not known, but the possible mechanism of action of metallic NPs, have been suggested according to the injuries and changes, induced in bacterial cells death [44]. AgNPs have an extremely large surface area, which provides better contact with microorganisms. Several mechanisms have been proposed to explain the antimicrobial properties of AgNPs [79]. One of them is based on the understanding that the key element in antimicrobial action of AgNPs is high affinity of Ag toward sulfur and phosphorus. Due to the abundance of sulfur-containing structural proteins and enzymes on the bacterial cell membrane, AgNPs can interact with them and in turn reduce cell functionality and viability. Moreover, they interact with phosphorus-containing compounds like DNA. Smaller (Nano sized) AgNPs attach to the cell membrane, make pores on the cell wall, lead to greater permeability, and release cytoplasmic content, suppress enzymes, attack the respiratory chain, and cell division, which cause the death of bacteria [53,57]. The effect of AgNPs on bacterial cell morphology has been studied using TEM and SEM image analysis. It was found that similar morphological alterations detachment of cytoplasmic membrane from the cell wall occur after treatment with AgNPs in both the Gram positive and Gram-negative bacteria. Nevertheless, the Gram negative bacteria are more susceptible to this treatment in comparison with Gram-positive bacteria.

The exact molecular mechanism of action of Zn or ZnO NPs on bacteria is still unclear. Zhang et al. [104] studied the susceptibility of Gram-negative bacteria to ZnO NPs and pointed out that interaction between NPs and bacterium is caused by electrostatic forces; at pH 7 E. coli have a negative charge (-7.2 mV) because excess of carboxylic groups. Conversely, ZnO nanoparticles have a positive charge, with a zeta potential of +24 mV [104]. As a result, opposite charges between the bacterium and nanoparticles generate electrostatic interactions, leading to a strong bind between the NPs and bacterial surface. So, this interaction produces multiple cell membrane injuries, which trigger total collapse of membrane and eventually cell death. Other authors indicate that the occurrence of reactive oxygen species (ROS) is the main mechanism responsible for the killing efficiency of ZnO NPs [30]. The morphological changes in cell surface leads to formation of bleb on cell surface, which causes shrinkage of bacterial cells, leakage of intracellular components and eventually death [38].

Recently, the Nanoceria (Cecerium, CeO–cerium oxide) also reported for antibacterial activity against different pathogenic bacteria. The exact mechanism of killing microbes is yet not clearly elucidated. However, it is proposed that CeNPs attach to the cell surface due to electrostatic properties and leads to death of microbes [51]. Due to strong electrostatic potential CeO₂ NPs interact with membrane proteins thiols groups, which results in protein denaturation, membrane impermeability eventually leads to microbial death [100, ]. Other researchers proposed that CeO₂ NPs mostly kill microbes via a massive production of ROS) in cells [102]. Cu is widely used as cheap and effective material for sterilizing liquids and textile [17]. Therefore, CuNPs may be a promising antibacterial agent in the future. But the most challenging job is to get stabilized CuNPs, after synthesis of CuNPs is rapidly undergoes oxidation in water and air. The antibacterial activity of CuNPs reported against both, Gram-positive and Gram-negative bacteria as listed in Table 3. But the complete mechanism behind antibacterial activity of CuNPs is not yet reported clearly. The proposed or hypothetical mechanism are the most accepted and reported recently is (i) Changes in the bacterial cell membrane permeability due to accumulation and dissolution of NP on cell surface [6,15]. (ii) Cellular structures dis-functioning or destruction because of ROS generation from CuNPs [8]. (iii) Uptake of ions derived from CuNPs or of NPs as whole into cells leads to disruption of DNA replication due to depletion of ATP [75]. All above mentioned proposed mechanisms causes injuries / leakage of cell contents / arrest of biochemical reactions of bacterial cells are the causes for bacterial cell death.

Challenges and Future Prospects

The biological method for synthesis of NPs is inexpensive, simple, easy to scale up and ecofriendly. The biogenic NPs are more suitable for biomedical applications due to the tocchemical free nature and shape size can be manipulated or controlled in biogenic process of NPs synthesis. Despite these opportunities, the underlying challenges for biogenic NPs are includes stability of synthesized NPs, formation of nanoclusters, aggregation of nanomaterials and determination of toxicity on animals or environment is still not cleared well. Research on biogenic synthesis of NPs is still ongoing and day-by-day many updated research added in the field of biomedical science and medical science for the use of NPs in human wellness. The progress in the growth of nanotechnology and its translation to clinical practices required comprehensive characterization, regulatory guidelines and suitable methods for detection of their toxicity.