- Email: [email protected]
Green fabrication, characterization and antibacterial potential of zinc oxide nanoparticles using Aloe socotrina leaf extract: A novel drug delivery approach
Journal of Drug Delivery Science and Technology 55 (2020) 101465
Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Green fabrication, characterization and antibacterial potential of zinc oxide nanoparticles using Aloe socotrina leaf extract: A novel drug delivery approach
T
Basheer Ahmed Fahimmunishaa, Ramachandran Ishwaryaa, Mohamad S. AlSalhib,∗∗, Sandhanasamy Devanesanb, Marimuthu Govindarajanc,d, Baskaralingam Vaseeharana,∗ a
Biomaterials and Biotechnology in Animal Health Lab, Department of Animal Health and Management, Alagappa University, Karaikudi, 630004, Tamil Nadu, India Department of Physics and Astronomy, College of Science, King Saud University, P.O. Box -2455, Riyadh, 11451, Saudi Arabia Unit of Vector Control, Phytochemistry and Nanotechnology, Department of Zoology, Annamalai University, Annamalainagar, 608 002, Tamil Nadu, India d Department of Zoology, Government College for Women (Autonomous), Kumbakonam, 612 001, Tamil Nadu, India b c
ARTICLE INFO
ABSTRACT
Keywords: Nanotechnology Confocal laser scanning microscope XRD SEM TEM Drug delivery Antibiofilm
Currently, nanotechnology is a prolific area of scientific research. As they can be synthesized in an eco-friendly manner, zinc oxide nanoparticles (ZnO NPs) provide an attractive solution to combat bacterial infection. In this study, synthesis of ZnO NPs using Aloe socotrina (As) leaf extract and characterized by UV, FTIR, XRD, SEM, and TEM. UV spectroscopy analysis showed that the maximum absorption was at about 315 nm. TEM images of the ZnO NPs definite the structure of ZnO NPs ranging from 15 to 50 nm in size while XRD spectra revealed their crystallinity. To understand the antibacterial activity of NPs, extensive experiments were performed using several assays such as agar well diffusion, minimum inhibitory concentration, growth kinetics, intracellular uptake, reactive oxygen species (ROS) generation, and antibiofilm activity. The A. socotrina capped ZnO NPs (As–ZnO NPs) revealed significant activity against biofilms formed by four bacterial pathogens, which can interfere with the management of drug resistant bacterial diseases caused by these biofilms. In summary, this novel biosynthesis technique of As–ZnO NPs with potent bactericidal activity offers an effective solution for the management of UTI pathogens.
1. Introduction In recent years, urinary tract infection (UTI) has emerged as one of the most widespread diseases in the world. This condition is characteristically aggressive and has contributed to the rise in nosocomial bacterial infection in ambulatory and hospitalized patients as the UTI can spread from the urethra to the bladder [1]. The annual incidence of UTI is approximately between 150 and 250 million cases globally and accounts for about 8 million doctor visits. Moreover, approximately 10% of all hospitalized cases contract these infections despite the sterile hospital conditions [2]. Prolonged use of urinary catheters increases the risk of infection and directly contributes to the rise in UTI [3]. Development of biofilms on the exterior of the catheter is one of the main problems associated with the management of UTI [4,5] as it can prolong the presence of the microorganisms resulting in persistent infections that tend to be resistant to antimicrobial therapy leading to
∗
chronic disease. Some of the most common microbes that infect the urinary catheter and produce biofilms are Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, Enterococcus, Staphylococcus aureus, Proteus mirabilis, Klebsiella pneumoniae and Morganella morganii [6]. In recent times, antibiotic resistance is an important global public health issue due to the uncontrolled use of antibiotics resulting in the development of resistance in bacteria including uropathogens [7]. Therefore, the antibiotics that currently used have become ineffective and there is an increasing need to develop more efficient antibiotics against specific microbial populations. Since the early years, microbial pathogens have posed a huge challenge to the scientific community with microbial contamination being one of the major concerns [8]. Most antimicrobial agents have frequent issues such as instability at high temperature or pressure and are often toxic and cause irritation. Moreover, development of antibiotic resistant pathogens has turned into a severe health problem and
Corresponding author. Corresponding author. E-mail addresses: [email protected] (M.S. AlSalhi), [email protected] (B. Vaseeharan).
∗∗
https://doi.org/10.1016/j.jddst.2019.101465 Received 19 November 2019; Received in revised form 10 December 2019; Accepted 11 December 2019 Available online 19 December 2019 1773-2247/ © 2019 Elsevier B.V. All rights reserved.
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
hence, many researchers have attempted to come up with alternate antimicrobial remedies [9–11]. Hence, development of novel and efficient antimicrobial agents is of utmost importance and the production of small and inexpensive inorganic antimicrobial agents such as metal and metal oxide nanoparticles in place of conventional antibiotics is a promising approach in pharmaceutics and drug research [12]. Nanoparticles made with metals such as silver, zinc, titanium, silicon, copper, gold, calcium, and magnesium, which all possess different properties, capability and range of activity, have been well-established [13–19]. Of all the metal oxides used for making NPs, ZnONPs have been the center of attention due to their attractive properties and widespread applications as well as their eco-friendly nature [20,21]. Zinc oxide (ZnO) is a type of semiconducting metal oxide that has gained attention in the past 2–3 years due to its tremendous scope for application in the field of microelectronics, biomolecular, optics, biomedical systems and production of antimicrobial agents [22,23]. These NPs can attach onto and disrupt the structure of bacterial cell membranes [24]. Antibacterial agents produced using novel chemical or physical methods lead to direct absorption of several toxic chemical species onto the surface and might have poor consequences for therapeutic purpose as the chemical procedures involved in the synthesis of nanomaterials create a huge amount of harmful byproducts [25,26]. Therefore, there is a need for developing green synthesis methods rather than chemical methods as they are cost effective and eco-friendly, and there is no requirement to use elevated pressure, power, temperature and toxic chemicals. A number of studies have been performed on plants to use them as antimicrobial agents and to understand the principles behind their antimicrobial property [27]. Recent research has been focused on plants-based drugs including antimicrobials as these drugs are safer and more cost effective [28,29]. Aloe is a member of the Liliaceae family and it is well-known as ‘Socotra’ as this plant grows close to the coast of the Indian Ocean and the Island of Socotra. For a long time, Aloe socotrina has been used in herbal formulations and has been described in books related to the therapeutic use of plants. Due to its therapeutic properties, it is used to treat irritable bowel disease, ulcerative colitis, or portal congestion. Incontinence or involuntary bowel movements are common among the aged resulting in simultaneous excretion of urine and stool, and this causes UTIs that is associated with a bearing-down sensation and enlarged prostate. To our knowledge, this study describes for the first time a biological approach using the Aloe socotrina leaf extract as a reducing agent for the synthesis of ZnO NPs. The aim of this study is to synthesize ZnO NPs using the A. socotrina extract and utilize its antibacterial activity against pathogens causing UTI. 2. Materials and methods
Fig. 1. (a) UV–vis spectrum analysis (Inset showing the color of the reaction mixture (I) Aloe socotrina extract, (II) zinc acetate and (III) As–ZnO NPs; (b) and (c) XRD and FTIR analysis of As–ZnO NPs. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
2.1. Preparation of plant extract Fresh Aloe socotrina leaves were collected from Karaikudi, Tamilnadu, India and rinsed with double distilled water to eliminate
2
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
Fig. 2. SEM images of As– ZnO NPs at different magnification ranges (a) 1 μm (b) 500 nm and (c) 400 nm. Panel (d) shows the EDAX spectrum of As– ZnO NPs.
the impurities adhering to the surface of the leaves. Ten grams of finely chopped fresh leaves were soaked in Milli-Q water (100 mL), the mixture was boiled at 70 °C for 20 min and filtered through a Whatman no. 1 filter paper to obtain the extract. The filtrate was stored in a refrigerator at −4 °C till future use.
precipitate was collected and rinsed with distilled water. The pellet was then dried in a hot air oven at 80 °C for 3 h, the dried powder was calcined at 350 °C for 3 h and preserved for future studies.
2.2. Biological synthesis of ZnO NPs
The optical property of the As–ZnO NPs was characterized based on the UV-absorption spectra using a wavelength between 300 and 400 nm. The crystal density, purity, and nature of the As–ZnO NPs was determined using X-ray crystallography (Ultima IV, Rigaku, Japan). The functional groups were analyzed using FTIR spectra in the range of 4000–500 cm−1. Scanning electron microscope was used to explore the morphology of As–ZnO NPs while the particle size and size distribution
2.3. Characterization of As–ZnO NPs
ZnO NPs were effectively synthesized using the A. socotrina leaf extracts by following a previously described method with minor modifications [30]. The aqueous leaf extract of A. socotrina (10 mL) was added to 1 mM of aqueous zinc acetate and the pH was adjusted to 12. The resultant solution was pale white in color. After centrifugation, the
3
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
Fig. 3. TEM images of As-ZnO NPs at magnifications of (a) 100 nm and (b) 10 nm. (c) SAED and (d) EDAX spectrum of As– ZnO NPs. Table 1 Antibacterial activity of the As extract and As–ZnO NPs on gram-negative bacteria. In each column, different letters indicate significant differences (ANOVA, Tukey's HSD test, P < 0.05). Bacteria
Zone of inhibition (mm) As extract
E. coli K. pneumonia Proteus vulgaris P. aeruginosa
18.5 17.4 16.0 16.2
± ± ± ±
4
As– ZnO NPs b
0.7 0.4 ab 1.0 ab 0.3a
25.3 19.2 25.0 19.4
± ± ± ±
1.7b 0.8a 0.9 ab 0.8a
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
Fig. 4. MIC of the As extract and As– ZnO NPs against multidrug resistant Gram-negative pathogens.
of the sample was evaluated using transmission electron micrograph. The elemental composition of As–ZnO NPs were distinguished using Energy Dispersive X-ray analysis (EDAX) analysis.
microorganisms were analyzed by quantifying the released cytoplasmic nucleic acids and proteins [34–36]. The intracellular ROS generation in the bacterial cells treated with NPs was determined using the fluorescent dye, dichlorodihydrofluorescein diacetate (H2DCFDA or DCFHDA), which is an intracellular ROS indicator [37]. We obtained the fluorescence micrographs using confocal laser scanning microscopy. The reduction in the biofilm formation via the pathogenic bacterial strains used in this study was determined using a polystyrene microtiter plate assay [38]. The Live/Dead assay was used to analyze the live and adhered bacterial cells using the Backlight L-7012 kit (Invitrogen, Carlsbad, CA, USA) [38].
2.4. Antibacterial assays We evaluated the antibacterial activity of the As extract and As-ZnO NPs against UTI pathogens such as Escherichia coli (ATCC), Klebsiella pneumonia (MTCC), Proteus vulgaris (HQ640434), Pseudomonas aeruginosa (ATCC) and the mode of action of ZnO NPs. We tested the susceptibility of the bacteria to the As extract as well as the synthesized AsZnONPs using agar well diffusion assay [30]. The minimum inhibitory concentration (MIC) of the As extract and As–ZnO NPs was determined individually and in a mixture using the microtiter plate method [31,32]. Time-kill synergy assay [33] was performed to understand the improvement in the antibacterial effect of the As extract and As–ZnO NPs against the test bacterial pathogens that were grown at a concentration of 108 cells/mL in 10 mL of nutrient broth. The As extract and As–ZnO NPs on the damaged membranes of the
3. Results and discussion 3.1. Synthesis and characterization of As-ZnO NPs The Aloe socotrina extract was used for synthesizing the ZnO NPs. When the As extract was incubated with zinc acetate, the color of the solution changed from pale green to milky white after 1 h (Fig. 1a),
5
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
Fig. 5. Growth curve of different bacterial strains in nutrient broth supplemented with different concentrations (0, 50 and 100 μg/ml) of As extract (a) E. coli, (b) K. pneumoniae, (c) and P. vulgaris (d) P. aeruginosa.
indicating the formation of zinc nanoparticles. UV spectroscopy analysis showed maximum absorption at approximately 315 nm. (Fig. 1a). The UV–visible spectra of the freshly prepared ZnO NPs and the acquired peaks clearly revealed the occurrence of ZnO NPs in the reaction mixture. Analysis of the XRD spectra gives information regarding the crystallinity of the NPs (Fig. 1b) and the X-ray diffraction peaks acquired at 31.92°, 34.56°, 36.40°, 47.77°, 56.76°, 63.03°, 66.61° and 68.16° correlated to the lattice planes of (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 0 3), (2 0 0) and (2 0 1). JCPDS was used as a reference (75–0576) to allocate the lattice planes consistent with the peaks acquired. In the FTIR spectra, a broad peak was obtained at 3442.51 corresponding to the N–H amines, the peaks in the range of 2340.87 corresponded to the O–H stretches and the peaks observed at 1519.25,
1384.21 corresponded to the flexible N–H bonds and rigid N]O bonds, respectively (Fig. 1c). The morphology of the ZnO NPs was observed by SEM, as shown in Fig. 2. These pictures verify the formation of spherical ZnO NPs. Fig. 2a–c shows the SEM pictures of ZnO NPs at several magnifications. EDX characterization suggested that the ZnO powder has good purity and it had high zinc content (78.75%) along with oxygen (21.25%) and carbon (2.25%), as shown in Fig. 2d. Carbon was found in trace amounts, which indicates the involvement of plant phytochemical groups in the reduction and covering of the synthesized ZnO NPs [39]. Fig. 3 shows TEM images of the sample and NPs confirming the formation of ZnO NPs in the size range of 15–50 nm (Fig. 3a and b). The SAED pattern and TEM-EDS analysis are shown in Fig. 3c and d.
6
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
Fig. 6. Growth curve of different bacterial strains in nutrient broth supplemented with different concentrations (0, 50 and 100 μg/ml) of As– ZnO NPs, (e) E. coli, (f) K. pneumoniae, (g) and P. vulgaris (h) P. aeruginosa.
3.2. Antibacterial activity of As–ZnO NPs
bactericidal activity of the As extract and As–ZnO NPs for all of the tested bacterial pathogens is shown in Fig. 4a and b. Time-kill kinetic assay was performed using bacterial suspensions in nutrient broth with the addition of different concentrations of As extract and As–ZnO NPs (0, 50 and 100 μg/ml), for 24 h. The measurements were taken at 600 nm and the time-kill curve obtained is shown in Figs. 5 and 6. The bactericidal activity was seen to steadily improve and the bacteria remained vulnerable up to 8 h of incubation in the As extract and As–ZnO NPs at their individual MIC for all pathogens and whole bacterial cells were found to be destroyed within this duration. As–ZnO NPs showed time-dependent and rapid bactericidal activity against all of the tested bacterial pathogen strains, and directly acted on the bacterial cells before they entered stationary phase, as displayed in the
Agar well diffusion method was implemented to evaluate the antibacterial activity of the biosynthesized As–ZnO NPs at various concentrations against UTI pathogens. The results showed that As–ZnO NPs had dynamic dose-dependent activity against the all of the tested bacterial pathogens (Table 1) and the maximum zone of inhibition was shown against E. coli and P. vulgaris. ZnO NPs have been known to inhibit in-vitro bacterial growth [40,41]. The As extract and As–ZnO NPs showed bacteriostatic activity against Proteus vulgaris and Pseudomonas aeruginosa at low concentrations (75 μg/mL and 50 μg/mL, respectively). Similarly, Escherichia coli and Klebsiella pneumonia were inhibited at concentrations of 100 μg/mL and 75 μg/mL, respectively. The
7
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
Fig. 7. Images showing intracellular uptake of the As extract and As-ZnONPs into Gram-negative bacterial pathogens examined using a confocal laser scanning microscope.
time-kill curve. The growth curves of bacteria exposed to As extract and As–ZnO NPs reveal that they can reduce the growth as well as reproduction of bacteria. Based on these preliminary results, we propose that ZnO NPs can be used to superficially manage the spread of bacterial infections. Using MIC tests and standard growth curves, we have shown that increasing concentrations of ZnO NPs can reduce the growth of Gram-negative bacteria. It is known that Gram-negative bacteria have higher amount of negative charge on the cell wall than Grampositive bacteria [42]. Owing to the elevated negative charge on the cell surface, there is active electrostatic interaction between the ZnO
NPs and the Gram-negative bacteria compared to the Gram-positive bacteria, which aids in reducing the development of Gram-negative bacteria [43]. ZnO NPs were shown to block or slow down the development of all the tested bacterial pathogens in the lag phase itself. During the lag phase, there is a rapid acceleration of cellular metabolism resulting in the active biosynthesis of cellular macromolecules, mainly enzymes [44]. These results indicate that ZnO NPs are considerably effective against all of the tested bacterial pathogens in the lag phase. Owing to the interaction of the NPs with the cells, the cell
8
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
Fig. 8. Micrographic images showing the intracellular ROS generation in Gram-negative bacterial pathogens after treatment with As extract and As– ZnO NPs.
membrane loses its integrity initially leading to the release of its intracellular contents such as nucleic acids and proteins due to disruption of the membrane. Thus, the presence of these components in the medium indicates disruption of the membrane. We evaluated the uptake of ZnO NPs into all the tested bacterial pathogens by analyzing the fluorescence images of the As extract- and As–ZnO NPs-treated bacterial strains (Fig. 7). As described previously, the efflux of cytoplasmic content indicates bacterial cytoplasmic membrane damage [34]. We observed that with increase in the concentration of the nanoparticles, the amount of nucleic acids and proteins released from the cells also increased in a dose-dependent manner. This trend showed the quick response kinetics of the NPs by the bacterial cells, which is consistent with previous studies performed on E. coli DH5a and B. subtilis strains [36].
The generation of ROS has been shown to play a role in ZnO NPmediated cytotoxicity in bacteria. In this study, we established that the As extracts and the As–ZnO NP-treated bacterial cells from the tested strains were dichlorofluorescein positive (DCF+), which shows NPmediated cell death (Fig. 8). On the contrary, in the control cells that were not treated with As extract and As–ZnO NPs, no fluorescence was seen indicating lack of ROS generation. ROS generation can be caused by the NPs owing to obstruction of the electron transport chain in the respiratory cycle of the injured plasma membrane [45]. A similar observation was made by Das et al. [37]. We will attempt to elucidate the mechanisms responsible for ROS production in ZnO NPs treated bacteria in our future studies. Development of biofilm occurs due to long-term attachment of planktonic bacteria on several surfaces followed by formation of a
9
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
Fig. 9. Light microscopic images of Gram-negative bacterial biofilms treated with As extract and As– ZnO NPs.
bacterial community, which adheres to the surface, produces an extracellular matrix, proliferates and disintegrate around the place. Based on this, we exploited the antimicrobial activity of ZnO NPs to reduce biofilm formation and observed extensive growth reduction in the populations of all of the tested bacterial pathogens (Figs. 9 and 10). However, the inhibitory effect of ZnO NPs diminished with an increase in the cell counts of the pathogens indicating that the positively charged ZnO NPs might be electro-statically saturated due to interaction with
the negatively charged surfaces of bacterial cells, which is consistent with previous reports [30]. This suggests that the uptake of the ZnO NPs can get saturated due to the high expansion rate of biofilms thus reducing the aggregation and inhibitory capacity of ZnO NPs. We further established the antibacterial efficacy of the As extract and As–ZnO NPs using live and dead assay (Fig. 11). The presence of green fluorescence reveals the live bacterial cells, while red fluorescence represents dead bacterial cells. Consequently, we observed that
10
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
Fig. 10. CLSM image of Gram-negative bacterial biofilms treated with As extract and As– ZnO NPs.
the As–ZnO NPs treated bacterial samples exhibited higher amount of dead bacteria, which shows the proficient bactericidal property of the ZnO NPs.
strong antibacterial agent against UTI. The promising antibacterial activity enables these nanoparticles as potential bactericidal material for clinical pathogens. Conversely, further studies are required to find out the actual intracellular and cell surface target molecules in bacteria.
4. Conclusion Recently, development of the nanodrugs from greener sources has potential agents for the biological applications. We have established a simple method to synthesize zinc nanoparticles using A. socotrina extract to provide a more effective antibacterial agent based on the MIC, which has not been shown earlier. The potent antibacterial activity of these NPs enables them to be used as potential bactericidal substances for a variety of environmental and biomedical applications. In addition, the bactericidal effects of As-ZnO NPs ensure that they can act as a
Author statement Basheer Ahmed Fahimmunisha: Conceptualization, Methodology, Software; Ramachandran Ishwarya: Investigation, Methodology, Software; Mohamad S. AlSalhi: Resources, Validation; Sandhanasamy Devanesan: Formal analysis; Marimuthu Govindarajan: Reviewing and editing; Baskaralingam Vaseeharan: Supervision, Writing-reviewing and editing.
11
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
Fig. 11. Confocal images of live (green) and dead (red) gram-negative bacteria before and after treatment with As extract and As– ZnO NPs. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Declaration of competing interest
References
The Authors stated that they have no conflicts of interest.
[1] A.E. Barber, J.P. Norton, A.M. Spivak, M.A. Mulvey, Urinary tract infections: current and emerging management strategies, Clin. Infect. Dis. 57 (2013) 719–724. [2] D.D. Rahn, Urinary tract infections: contemporary management, Urol. Nurs. 28 (2008) 333–341. [3] S.M. Jacobson, D.J. Stickler, H.L.T. Mobley, M.E. Shirtliff, Complicated catheterassociated urinary tract infections due to Escherichia coli and Proteus mirabilis, Clin. Microbiol. Rev. 21 (2008) 26–59. [4] B.W. Trautner, R.O. Darouiche, Role of biofilm in catheter-associated urinary tract infection, Am. J infect. cont. 32 (2004) 177–183. [5] P. Singha, J. Locklin, H. Handa, A review of the recent advances in antimicrobial coatings for urinary catheters, Acta Biomater. 50 (2017) 20–40. [6] A.L. Flores-Mireles, J.N. Walker, M. Caparon, S.J. Hultgren, Switching Rho GTPase
Acknowledgement The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group project number RGP-023. The authors would also like to thank the Council of Scientific and Industrial Research, India, New Delhi, India [ref: 9/688 (0030)/18 EMR - I].
12
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
[7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]
[18]
[19]
[20] [21] [22] [23] [24] [25] [26] [27]
385. [28] S.Y. Pan, S.F. Zhou, S.H. Gao, Z.L. Yu, S.F. Zhang, M.K. Tang, J.N. Sun, D.L. Ma, Y.F. Han, W.F. Fong, K.M. Ko, New perspectives on how to discover drugs from herbal medicines: CAM's outstanding contribution to modern therapeutics, Evid. Based. Complement. Alternat. Med. 2013 (2013). [29] A.G. Atanasov, B. Waltenberger, E.M. Pferschy-Wenzig, T. Linder, C. Wawrosch, P. Uhrin, V. Temml, L. Wang, S. Schwaiger, E.H. Heiss, J.M. Rollinger, Potential antiosteoporotic natural product lead compounds that inhibit 17β-hydroxysteroid dehydrogenase type 2, J. Nat. Prod. 80 (2017) 965–974. [30] R. Ishwarya, B. Vaseeharan, S. Kalyani, B. Banumathi, M. Govindarajan, N.S. Alharbi, S. Kadaikunnan, M.N. Al-anbr, J.M. Khaled, G. Benelli, Facile green synthesis of zinc oxide nanoparticles using Ulva lactuca seaweed extract and evaluation of their photocatalytic, antibiofilm and insecticidal activity, J. Photochem. Photobiol., A 178 (2018) 249–258. [31] J.F. Hernandez-Sierra, F. Ruiz, D.C. Pena, F. Martinez-Gutierrez, A.E. Martinez, J. Guillen Ade, H. Tapia-Perez, G.M. Castanon, The antimicrobial sensitivity of Streptococcus mutans to nanoparticles of silver, zinc oxide, and gold, Nanomedicine 4 (2008) 237–240. [32] G.A. Martınez-Castanon, N. Nino-Martınez, F. Martınez-Gutierrez, J.R. MartınezMendoza, F. Ruiz, The antimicrobial sensitivity of Streptococcus mutans to nanoparticles of silver, zinc oxide, and gold, J. Nanoparticle Res. 10 (2008) 1343–1348. [33] R.M. Bhande, C.N. Khobragade, R.S. Mane, S. Bhande, Enhanced synergism of antibiotics with zinc oxide nanoparticles against extended spectrum β-lactamase producers implicated in urinary tract infections, J. Nanoparticle Res. 15 (2013) 1413. [34] C.Z. Chen, S.L. Cooper, Interactions between dendrimer biocides and bacterial membranes, Biomaterials 23 (2002) 3359–3368. [35] H. Liu, Y. Du, X. Wang, L. Sun, Chitosan kills bacteria through cell membrane damage, Int. J. Food Microbiol. 95 (2004) 147–155. [36] D.K. Tiwari, J. Behari, P. Sen, Time and dose-dependent antimicrobial potential of Ag nanoparticles synthesized by top-down approach, Curr. Sci. 95 (2008) 647–655. [37] B. Das, S.K. Dash, D. Mandal, T. Ghosh, S. Chattopadhyay, S. Tripathy, S. Das, S.K. Dey, D. Das, S. Roy, Green synthesized silver nanoparticles destroy multidrug resistant bacteria via reactive oxygen species mediated membrane damage, Arab. J. Chem. 10 (2017) 862–876. [38] R. Ishwarya, B. Vaseeharan, S. Suganya, A.K. Nazar, M. Govindarajan, N.S. Alharbi, S. Kadaikunnan, M.N. Al-anbr, J.M. Khaled, G. Benelli, Sargassum wightii-synthesized ZnO nanoparticles–from antibacterial and insecticidal activity to immunostimulatory effects on the green tiger shrimp Penaeus semisulcatus, J. Photochem. Photobiol., A 183 (2018) 318–330. [39] N. Bala, S. Saha, M. Chakraborty, M. Maiti, S. Das, R. Basu, P. Nandy, Green synthesis of zinc oxide nanoparticles using Hibiscus subdariffa leaf extract: effect of temperature on synthesis, anti-bacterial activity and anti-diabetic activity, RSC Adv. 5 (2015) 4993–5003. [40] Y. Xie, Y. He, P.L. Irwin, T. Jin, X. Shi, Antibacterial activity and mechanism of action of zinc oxide nanoparticles against Campylobacter Jejuni, Appl. Environ. Microbiol. 77 (2011) 2325–2331. [41] N. Padmavathy, R. Vijayaraghavan, Enhanced bioactivity of ZnO nanoparticles—an antimicrobial study, Sci. Technol. Adv. Mat. 9 (2008) 035004. [42] Y.C. Chung, Y.P. Su, C.C. Chen, G. Jia, H.L. Wang, J.G. Wu, J.G. Lin, Relationship between antibacterial activity of chitosan and surface characteristics of cell wall, Acta Pharmacol. Sin. 25 (2004) 932–936. [43] P.K. Stoimenov, R.L. Klinger, G.L. Marchin, K.J. Klabunde, Metal oxide nanoparticles as bactericidal agents, Langmuir 18 (2002) 6679–6686. [44] M.D. Rolfe, C.J. Rice, S. Lucchini, C. Pin, A. Thompson, A.D.S. Cameron, Lag phase is a distinct growth phase that prepares bacteria for exponential growth and involves transient metal accumulation, J. Bacteriol. 194 (2012) 686–701. [45] H. Su, C. Chou, D. Hung, S. Lin, I. Pao, J. Lin, F.L. Huang, R.X. Dong, J.J. Lin, The disruption of bacterial membrane integrity through ROS generation induced by nanohybrids of silver and clay, Biomaterials 30 (2009) 5979–5987.
activation into effective antibacterial defenses requires the caspase-1/IL-1beta signaling axis, Nat. rev. microbial. 13 (2015) 269–284. C.L. Ventola, The antibiotic resistance crisis: part 1: causes and threats, Pharmacol. Ther. 40 (2015) 277. F. Patrignani, L. Siroli, F. Gardini, R. Lanciotti, Determination of antibacterial and technological properties of vaginal lactobacilli for their potential application in dairy products, Front. Microbial. 17 (2016) 938. B. Li, T.J. Webster, Bacteria antibiotic resistance: New challenges and opportunities for implant‐associated orthopedic infections, J. Orthop. Res. 36 (2018) 22–32. S.B. Zaman, M.A. Hussain, R. Nye, V. Mehta, K.T. Mamun, N. Hossain, A review on antibiotic resistance: alarm bells are ringing, Cureus 9 (2017) 1403. F. Prestinaci, P. Pezzotti, A. Pantosti, Antimicrobial resistance: a global multifaceted phenomenon, Pathog. Glob. Health 109 (2015) 309–318. N. Beyth, Y. Houri-Haddad, A. Domb, W. Khan, R. Hazan, Alternative antimicrobial approach: nano-antimicrobial materials, Evid. Based. Complement. Alternat. Med. 2015 (2015) 16. R.K. Das, V.L. Pachapur, L. Lonappan, M. Naghdi, R. Pulicharla, S. Maiti, M. Cledon, L.M. Dalila, S.J. Sarma, S.K. Brar, Nanoparticle–plant interactions: two‐way traffic, nanotechnol, Environ. Eng. 2 (2017) 18. K. Pramila, Y. Ravikumar, S. Deepak Kumar, L. Kanwar, S. Sushil Kumar, Biosynthesis of iron and silver nanoparticles at room temperature using aqueous sorghum bran extracts, J. Nanostr. Chem. 8 (2018) 217–254. G. Benelli, M. Govindarajan, Green-synthesized mosquito oviposition attractants and ovicides: towards a nanoparticle-based “Lure and kill” approach? J. Clust. Sci. 28 (2017) 287–308. M. Govindarajan, G. Benelli, One-pot green synthesis of silver nanocrystals using Hymenodictyon orixense: a cheap and effective tool against malaria, chikungunya and Japanese encephalitis mosquito vectors? RSC Adv. 6 (2016) 59021–59029. A.A. Alfuraydi, S. Devanesan, M. Al-Ansari, M.S. AlSalhi, A.J. Ranjitsingh, Ecofriendly green synthesis of silver nanoparticles from the sesame oil cake and its potential anticancer and antimicrobial activities, J. Photochem. Photobiol., B 192 (2019) 83–89. M.S. AlSalhi, K. Elangovan, A.J.A. Ranjitsingh, P. Murali, S. Devanesan, Synthesis of silver nanoparticles using plant derived 4-N-methyl benzoic acid and evaluation of antimicrobial, antioxidant and antitumor activity, Saudi J. Biol. Sci. 26 (2019) 970–978. C. Vijilvani, M.R. Bindhu, F.C. Frincy, M.S. AlSalhi, S. Sabitha, K. Saravanakumar, S. Devanesan, M. Umadevi, M.J. Aljaafreh, M. Atif, Antimicrobial and catalytic activities of biosynthesized gold, silver and palladium nanoparticles from Solanum nigurum leaves, J. Photochem. Photobiol., B 192 (202) (2020) 111713. J. Jiang, J. Pi, J. Cai, The advancing of zinc oxide nanoparticles for biomedical applications, Bioinorgan. Chem. Appl. 2018 (2018) 18. V.N. Kalpana, V. Devi Rajeswari, A review on green synthesis, biomedical applications, and toxicity studies of ZnO NPs, Bioinorgan. Chem. Appl. 2018 (2018) 12. J.V. Meshram, V.B. Koli, S.G. Kumbhar, L.C. Borde, M.R. Phadatare, S.H. Pawar, Structural, spectroscopic and anti-microbial inspection of PEG capped ZnO nanoparticles for biomedical applications, Mater. Res. Express 5 (2018) 045016. I. Khan, K. Saeed, I. Khan, Nanoparticles: properties, applications and toxicities, Arab, J. Chem. 12 (2017) 908–931. A. Ali, A.R. Phull, M. Zia, Performance of silver, zinc, and iron nanoparticles-doped cotton filters against airborne E. coli to minimize bioaerosol exposure, Nanotechnol. Rev. 11 (2018) 1233–1242. A. Sharma, S.V. Madhunapantula, G.P. Robertson, Toxicological considerations when creating nanoparticle-based drugs and drug delivery systems, Expert Opin. Drug Metabol. Toxicol. 8 (2012) 47–69. J. Jeevanandam, A. Barhoum, Y.S. Chan, A. Dufresne, M.K. Danquah, Review on nanoparticles and nanostructured materials: history, sources, toxicity and regulations, Beilstein. J. Nanotech. 3 (2018) 1050–1074. S. Iravani, H. Korbekandi, S.V. Mirmohammadi, B. Zolfaghari, Synthesis of silver nanoparticles: chemical, physical and biological methods, Res. Pharm. Sci. 9 (2014)
13
Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Green fabrication, characterization and antibacterial potential of zinc oxide nanoparticles using Aloe socotrina leaf extract: A novel drug delivery approach
T
Basheer Ahmed Fahimmunishaa, Ramachandran Ishwaryaa, Mohamad S. AlSalhib,∗∗, Sandhanasamy Devanesanb, Marimuthu Govindarajanc,d, Baskaralingam Vaseeharana,∗ a
Biomaterials and Biotechnology in Animal Health Lab, Department of Animal Health and Management, Alagappa University, Karaikudi, 630004, Tamil Nadu, India Department of Physics and Astronomy, College of Science, King Saud University, P.O. Box -2455, Riyadh, 11451, Saudi Arabia Unit of Vector Control, Phytochemistry and Nanotechnology, Department of Zoology, Annamalai University, Annamalainagar, 608 002, Tamil Nadu, India d Department of Zoology, Government College for Women (Autonomous), Kumbakonam, 612 001, Tamil Nadu, India b c
ARTICLE INFO
ABSTRACT
Keywords: Nanotechnology Confocal laser scanning microscope XRD SEM TEM Drug delivery Antibiofilm
Currently, nanotechnology is a prolific area of scientific research. As they can be synthesized in an eco-friendly manner, zinc oxide nanoparticles (ZnO NPs) provide an attractive solution to combat bacterial infection. In this study, synthesis of ZnO NPs using Aloe socotrina (As) leaf extract and characterized by UV, FTIR, XRD, SEM, and TEM. UV spectroscopy analysis showed that the maximum absorption was at about 315 nm. TEM images of the ZnO NPs definite the structure of ZnO NPs ranging from 15 to 50 nm in size while XRD spectra revealed their crystallinity. To understand the antibacterial activity of NPs, extensive experiments were performed using several assays such as agar well diffusion, minimum inhibitory concentration, growth kinetics, intracellular uptake, reactive oxygen species (ROS) generation, and antibiofilm activity. The A. socotrina capped ZnO NPs (As–ZnO NPs) revealed significant activity against biofilms formed by four bacterial pathogens, which can interfere with the management of drug resistant bacterial diseases caused by these biofilms. In summary, this novel biosynthesis technique of As–ZnO NPs with potent bactericidal activity offers an effective solution for the management of UTI pathogens.
1. Introduction In recent years, urinary tract infection (UTI) has emerged as one of the most widespread diseases in the world. This condition is characteristically aggressive and has contributed to the rise in nosocomial bacterial infection in ambulatory and hospitalized patients as the UTI can spread from the urethra to the bladder [1]. The annual incidence of UTI is approximately between 150 and 250 million cases globally and accounts for about 8 million doctor visits. Moreover, approximately 10% of all hospitalized cases contract these infections despite the sterile hospital conditions [2]. Prolonged use of urinary catheters increases the risk of infection and directly contributes to the rise in UTI [3]. Development of biofilms on the exterior of the catheter is one of the main problems associated with the management of UTI [4,5] as it can prolong the presence of the microorganisms resulting in persistent infections that tend to be resistant to antimicrobial therapy leading to
∗
chronic disease. Some of the most common microbes that infect the urinary catheter and produce biofilms are Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, Enterococcus, Staphylococcus aureus, Proteus mirabilis, Klebsiella pneumoniae and Morganella morganii [6]. In recent times, antibiotic resistance is an important global public health issue due to the uncontrolled use of antibiotics resulting in the development of resistance in bacteria including uropathogens [7]. Therefore, the antibiotics that currently used have become ineffective and there is an increasing need to develop more efficient antibiotics against specific microbial populations. Since the early years, microbial pathogens have posed a huge challenge to the scientific community with microbial contamination being one of the major concerns [8]. Most antimicrobial agents have frequent issues such as instability at high temperature or pressure and are often toxic and cause irritation. Moreover, development of antibiotic resistant pathogens has turned into a severe health problem and
Corresponding author. Corresponding author. E-mail addresses: [email protected] (M.S. AlSalhi), [email protected] (B. Vaseeharan).
∗∗
https://doi.org/10.1016/j.jddst.2019.101465 Received 19 November 2019; Received in revised form 10 December 2019; Accepted 11 December 2019 Available online 19 December 2019 1773-2247/ © 2019 Elsevier B.V. All rights reserved.
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
hence, many researchers have attempted to come up with alternate antimicrobial remedies [9–11]. Hence, development of novel and efficient antimicrobial agents is of utmost importance and the production of small and inexpensive inorganic antimicrobial agents such as metal and metal oxide nanoparticles in place of conventional antibiotics is a promising approach in pharmaceutics and drug research [12]. Nanoparticles made with metals such as silver, zinc, titanium, silicon, copper, gold, calcium, and magnesium, which all possess different properties, capability and range of activity, have been well-established [13–19]. Of all the metal oxides used for making NPs, ZnONPs have been the center of attention due to their attractive properties and widespread applications as well as their eco-friendly nature [20,21]. Zinc oxide (ZnO) is a type of semiconducting metal oxide that has gained attention in the past 2–3 years due to its tremendous scope for application in the field of microelectronics, biomolecular, optics, biomedical systems and production of antimicrobial agents [22,23]. These NPs can attach onto and disrupt the structure of bacterial cell membranes [24]. Antibacterial agents produced using novel chemical or physical methods lead to direct absorption of several toxic chemical species onto the surface and might have poor consequences for therapeutic purpose as the chemical procedures involved in the synthesis of nanomaterials create a huge amount of harmful byproducts [25,26]. Therefore, there is a need for developing green synthesis methods rather than chemical methods as they are cost effective and eco-friendly, and there is no requirement to use elevated pressure, power, temperature and toxic chemicals. A number of studies have been performed on plants to use them as antimicrobial agents and to understand the principles behind their antimicrobial property [27]. Recent research has been focused on plants-based drugs including antimicrobials as these drugs are safer and more cost effective [28,29]. Aloe is a member of the Liliaceae family and it is well-known as ‘Socotra’ as this plant grows close to the coast of the Indian Ocean and the Island of Socotra. For a long time, Aloe socotrina has been used in herbal formulations and has been described in books related to the therapeutic use of plants. Due to its therapeutic properties, it is used to treat irritable bowel disease, ulcerative colitis, or portal congestion. Incontinence or involuntary bowel movements are common among the aged resulting in simultaneous excretion of urine and stool, and this causes UTIs that is associated with a bearing-down sensation and enlarged prostate. To our knowledge, this study describes for the first time a biological approach using the Aloe socotrina leaf extract as a reducing agent for the synthesis of ZnO NPs. The aim of this study is to synthesize ZnO NPs using the A. socotrina extract and utilize its antibacterial activity against pathogens causing UTI. 2. Materials and methods
Fig. 1. (a) UV–vis spectrum analysis (Inset showing the color of the reaction mixture (I) Aloe socotrina extract, (II) zinc acetate and (III) As–ZnO NPs; (b) and (c) XRD and FTIR analysis of As–ZnO NPs. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
2.1. Preparation of plant extract Fresh Aloe socotrina leaves were collected from Karaikudi, Tamilnadu, India and rinsed with double distilled water to eliminate
2
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
Fig. 2. SEM images of As– ZnO NPs at different magnification ranges (a) 1 μm (b) 500 nm and (c) 400 nm. Panel (d) shows the EDAX spectrum of As– ZnO NPs.
the impurities adhering to the surface of the leaves. Ten grams of finely chopped fresh leaves were soaked in Milli-Q water (100 mL), the mixture was boiled at 70 °C for 20 min and filtered through a Whatman no. 1 filter paper to obtain the extract. The filtrate was stored in a refrigerator at −4 °C till future use.
precipitate was collected and rinsed with distilled water. The pellet was then dried in a hot air oven at 80 °C for 3 h, the dried powder was calcined at 350 °C for 3 h and preserved for future studies.
2.2. Biological synthesis of ZnO NPs
The optical property of the As–ZnO NPs was characterized based on the UV-absorption spectra using a wavelength between 300 and 400 nm. The crystal density, purity, and nature of the As–ZnO NPs was determined using X-ray crystallography (Ultima IV, Rigaku, Japan). The functional groups were analyzed using FTIR spectra in the range of 4000–500 cm−1. Scanning electron microscope was used to explore the morphology of As–ZnO NPs while the particle size and size distribution
2.3. Characterization of As–ZnO NPs
ZnO NPs were effectively synthesized using the A. socotrina leaf extracts by following a previously described method with minor modifications [30]. The aqueous leaf extract of A. socotrina (10 mL) was added to 1 mM of aqueous zinc acetate and the pH was adjusted to 12. The resultant solution was pale white in color. After centrifugation, the
3
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
Fig. 3. TEM images of As-ZnO NPs at magnifications of (a) 100 nm and (b) 10 nm. (c) SAED and (d) EDAX spectrum of As– ZnO NPs. Table 1 Antibacterial activity of the As extract and As–ZnO NPs on gram-negative bacteria. In each column, different letters indicate significant differences (ANOVA, Tukey's HSD test, P < 0.05). Bacteria
Zone of inhibition (mm) As extract
E. coli K. pneumonia Proteus vulgaris P. aeruginosa
18.5 17.4 16.0 16.2
± ± ± ±
4
As– ZnO NPs b
0.7 0.4 ab 1.0 ab 0.3a
25.3 19.2 25.0 19.4
± ± ± ±
1.7b 0.8a 0.9 ab 0.8a
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
Fig. 4. MIC of the As extract and As– ZnO NPs against multidrug resistant Gram-negative pathogens.
of the sample was evaluated using transmission electron micrograph. The elemental composition of As–ZnO NPs were distinguished using Energy Dispersive X-ray analysis (EDAX) analysis.
microorganisms were analyzed by quantifying the released cytoplasmic nucleic acids and proteins [34–36]. The intracellular ROS generation in the bacterial cells treated with NPs was determined using the fluorescent dye, dichlorodihydrofluorescein diacetate (H2DCFDA or DCFHDA), which is an intracellular ROS indicator [37]. We obtained the fluorescence micrographs using confocal laser scanning microscopy. The reduction in the biofilm formation via the pathogenic bacterial strains used in this study was determined using a polystyrene microtiter plate assay [38]. The Live/Dead assay was used to analyze the live and adhered bacterial cells using the Backlight L-7012 kit (Invitrogen, Carlsbad, CA, USA) [38].
2.4. Antibacterial assays We evaluated the antibacterial activity of the As extract and As-ZnO NPs against UTI pathogens such as Escherichia coli (ATCC), Klebsiella pneumonia (MTCC), Proteus vulgaris (HQ640434), Pseudomonas aeruginosa (ATCC) and the mode of action of ZnO NPs. We tested the susceptibility of the bacteria to the As extract as well as the synthesized AsZnONPs using agar well diffusion assay [30]. The minimum inhibitory concentration (MIC) of the As extract and As–ZnO NPs was determined individually and in a mixture using the microtiter plate method [31,32]. Time-kill synergy assay [33] was performed to understand the improvement in the antibacterial effect of the As extract and As–ZnO NPs against the test bacterial pathogens that were grown at a concentration of 108 cells/mL in 10 mL of nutrient broth. The As extract and As–ZnO NPs on the damaged membranes of the
3. Results and discussion 3.1. Synthesis and characterization of As-ZnO NPs The Aloe socotrina extract was used for synthesizing the ZnO NPs. When the As extract was incubated with zinc acetate, the color of the solution changed from pale green to milky white after 1 h (Fig. 1a),
5
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
Fig. 5. Growth curve of different bacterial strains in nutrient broth supplemented with different concentrations (0, 50 and 100 μg/ml) of As extract (a) E. coli, (b) K. pneumoniae, (c) and P. vulgaris (d) P. aeruginosa.
indicating the formation of zinc nanoparticles. UV spectroscopy analysis showed maximum absorption at approximately 315 nm. (Fig. 1a). The UV–visible spectra of the freshly prepared ZnO NPs and the acquired peaks clearly revealed the occurrence of ZnO NPs in the reaction mixture. Analysis of the XRD spectra gives information regarding the crystallinity of the NPs (Fig. 1b) and the X-ray diffraction peaks acquired at 31.92°, 34.56°, 36.40°, 47.77°, 56.76°, 63.03°, 66.61° and 68.16° correlated to the lattice planes of (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 0 3), (2 0 0) and (2 0 1). JCPDS was used as a reference (75–0576) to allocate the lattice planes consistent with the peaks acquired. In the FTIR spectra, a broad peak was obtained at 3442.51 corresponding to the N–H amines, the peaks in the range of 2340.87 corresponded to the O–H stretches and the peaks observed at 1519.25,
1384.21 corresponded to the flexible N–H bonds and rigid N]O bonds, respectively (Fig. 1c). The morphology of the ZnO NPs was observed by SEM, as shown in Fig. 2. These pictures verify the formation of spherical ZnO NPs. Fig. 2a–c shows the SEM pictures of ZnO NPs at several magnifications. EDX characterization suggested that the ZnO powder has good purity and it had high zinc content (78.75%) along with oxygen (21.25%) and carbon (2.25%), as shown in Fig. 2d. Carbon was found in trace amounts, which indicates the involvement of plant phytochemical groups in the reduction and covering of the synthesized ZnO NPs [39]. Fig. 3 shows TEM images of the sample and NPs confirming the formation of ZnO NPs in the size range of 15–50 nm (Fig. 3a and b). The SAED pattern and TEM-EDS analysis are shown in Fig. 3c and d.
6
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
Fig. 6. Growth curve of different bacterial strains in nutrient broth supplemented with different concentrations (0, 50 and 100 μg/ml) of As– ZnO NPs, (e) E. coli, (f) K. pneumoniae, (g) and P. vulgaris (h) P. aeruginosa.
3.2. Antibacterial activity of As–ZnO NPs
bactericidal activity of the As extract and As–ZnO NPs for all of the tested bacterial pathogens is shown in Fig. 4a and b. Time-kill kinetic assay was performed using bacterial suspensions in nutrient broth with the addition of different concentrations of As extract and As–ZnO NPs (0, 50 and 100 μg/ml), for 24 h. The measurements were taken at 600 nm and the time-kill curve obtained is shown in Figs. 5 and 6. The bactericidal activity was seen to steadily improve and the bacteria remained vulnerable up to 8 h of incubation in the As extract and As–ZnO NPs at their individual MIC for all pathogens and whole bacterial cells were found to be destroyed within this duration. As–ZnO NPs showed time-dependent and rapid bactericidal activity against all of the tested bacterial pathogen strains, and directly acted on the bacterial cells before they entered stationary phase, as displayed in the
Agar well diffusion method was implemented to evaluate the antibacterial activity of the biosynthesized As–ZnO NPs at various concentrations against UTI pathogens. The results showed that As–ZnO NPs had dynamic dose-dependent activity against the all of the tested bacterial pathogens (Table 1) and the maximum zone of inhibition was shown against E. coli and P. vulgaris. ZnO NPs have been known to inhibit in-vitro bacterial growth [40,41]. The As extract and As–ZnO NPs showed bacteriostatic activity against Proteus vulgaris and Pseudomonas aeruginosa at low concentrations (75 μg/mL and 50 μg/mL, respectively). Similarly, Escherichia coli and Klebsiella pneumonia were inhibited at concentrations of 100 μg/mL and 75 μg/mL, respectively. The
7
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
Fig. 7. Images showing intracellular uptake of the As extract and As-ZnONPs into Gram-negative bacterial pathogens examined using a confocal laser scanning microscope.
time-kill curve. The growth curves of bacteria exposed to As extract and As–ZnO NPs reveal that they can reduce the growth as well as reproduction of bacteria. Based on these preliminary results, we propose that ZnO NPs can be used to superficially manage the spread of bacterial infections. Using MIC tests and standard growth curves, we have shown that increasing concentrations of ZnO NPs can reduce the growth of Gram-negative bacteria. It is known that Gram-negative bacteria have higher amount of negative charge on the cell wall than Grampositive bacteria [42]. Owing to the elevated negative charge on the cell surface, there is active electrostatic interaction between the ZnO
NPs and the Gram-negative bacteria compared to the Gram-positive bacteria, which aids in reducing the development of Gram-negative bacteria [43]. ZnO NPs were shown to block or slow down the development of all the tested bacterial pathogens in the lag phase itself. During the lag phase, there is a rapid acceleration of cellular metabolism resulting in the active biosynthesis of cellular macromolecules, mainly enzymes [44]. These results indicate that ZnO NPs are considerably effective against all of the tested bacterial pathogens in the lag phase. Owing to the interaction of the NPs with the cells, the cell
8
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
Fig. 8. Micrographic images showing the intracellular ROS generation in Gram-negative bacterial pathogens after treatment with As extract and As– ZnO NPs.
membrane loses its integrity initially leading to the release of its intracellular contents such as nucleic acids and proteins due to disruption of the membrane. Thus, the presence of these components in the medium indicates disruption of the membrane. We evaluated the uptake of ZnO NPs into all the tested bacterial pathogens by analyzing the fluorescence images of the As extract- and As–ZnO NPs-treated bacterial strains (Fig. 7). As described previously, the efflux of cytoplasmic content indicates bacterial cytoplasmic membrane damage [34]. We observed that with increase in the concentration of the nanoparticles, the amount of nucleic acids and proteins released from the cells also increased in a dose-dependent manner. This trend showed the quick response kinetics of the NPs by the bacterial cells, which is consistent with previous studies performed on E. coli DH5a and B. subtilis strains [36].
The generation of ROS has been shown to play a role in ZnO NPmediated cytotoxicity in bacteria. In this study, we established that the As extracts and the As–ZnO NP-treated bacterial cells from the tested strains were dichlorofluorescein positive (DCF+), which shows NPmediated cell death (Fig. 8). On the contrary, in the control cells that were not treated with As extract and As–ZnO NPs, no fluorescence was seen indicating lack of ROS generation. ROS generation can be caused by the NPs owing to obstruction of the electron transport chain in the respiratory cycle of the injured plasma membrane [45]. A similar observation was made by Das et al. [37]. We will attempt to elucidate the mechanisms responsible for ROS production in ZnO NPs treated bacteria in our future studies. Development of biofilm occurs due to long-term attachment of planktonic bacteria on several surfaces followed by formation of a
9
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
Fig. 9. Light microscopic images of Gram-negative bacterial biofilms treated with As extract and As– ZnO NPs.
bacterial community, which adheres to the surface, produces an extracellular matrix, proliferates and disintegrate around the place. Based on this, we exploited the antimicrobial activity of ZnO NPs to reduce biofilm formation and observed extensive growth reduction in the populations of all of the tested bacterial pathogens (Figs. 9 and 10). However, the inhibitory effect of ZnO NPs diminished with an increase in the cell counts of the pathogens indicating that the positively charged ZnO NPs might be electro-statically saturated due to interaction with
the negatively charged surfaces of bacterial cells, which is consistent with previous reports [30]. This suggests that the uptake of the ZnO NPs can get saturated due to the high expansion rate of biofilms thus reducing the aggregation and inhibitory capacity of ZnO NPs. We further established the antibacterial efficacy of the As extract and As–ZnO NPs using live and dead assay (Fig. 11). The presence of green fluorescence reveals the live bacterial cells, while red fluorescence represents dead bacterial cells. Consequently, we observed that
10
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
Fig. 10. CLSM image of Gram-negative bacterial biofilms treated with As extract and As– ZnO NPs.
the As–ZnO NPs treated bacterial samples exhibited higher amount of dead bacteria, which shows the proficient bactericidal property of the ZnO NPs.
strong antibacterial agent against UTI. The promising antibacterial activity enables these nanoparticles as potential bactericidal material for clinical pathogens. Conversely, further studies are required to find out the actual intracellular and cell surface target molecules in bacteria.
4. Conclusion Recently, development of the nanodrugs from greener sources has potential agents for the biological applications. We have established a simple method to synthesize zinc nanoparticles using A. socotrina extract to provide a more effective antibacterial agent based on the MIC, which has not been shown earlier. The potent antibacterial activity of these NPs enables them to be used as potential bactericidal substances for a variety of environmental and biomedical applications. In addition, the bactericidal effects of As-ZnO NPs ensure that they can act as a
Author statement Basheer Ahmed Fahimmunisha: Conceptualization, Methodology, Software; Ramachandran Ishwarya: Investigation, Methodology, Software; Mohamad S. AlSalhi: Resources, Validation; Sandhanasamy Devanesan: Formal analysis; Marimuthu Govindarajan: Reviewing and editing; Baskaralingam Vaseeharan: Supervision, Writing-reviewing and editing.
11
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
Fig. 11. Confocal images of live (green) and dead (red) gram-negative bacteria before and after treatment with As extract and As– ZnO NPs. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Declaration of competing interest
References
The Authors stated that they have no conflicts of interest.
[1] A.E. Barber, J.P. Norton, A.M. Spivak, M.A. Mulvey, Urinary tract infections: current and emerging management strategies, Clin. Infect. Dis. 57 (2013) 719–724. [2] D.D. Rahn, Urinary tract infections: contemporary management, Urol. Nurs. 28 (2008) 333–341. [3] S.M. Jacobson, D.J. Stickler, H.L.T. Mobley, M.E. Shirtliff, Complicated catheterassociated urinary tract infections due to Escherichia coli and Proteus mirabilis, Clin. Microbiol. Rev. 21 (2008) 26–59. [4] B.W. Trautner, R.O. Darouiche, Role of biofilm in catheter-associated urinary tract infection, Am. J infect. cont. 32 (2004) 177–183. [5] P. Singha, J. Locklin, H. Handa, A review of the recent advances in antimicrobial coatings for urinary catheters, Acta Biomater. 50 (2017) 20–40. [6] A.L. Flores-Mireles, J.N. Walker, M. Caparon, S.J. Hultgren, Switching Rho GTPase
Acknowledgement The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group project number RGP-023. The authors would also like to thank the Council of Scientific and Industrial Research, India, New Delhi, India [ref: 9/688 (0030)/18 EMR - I].
12
Journal of Drug Delivery Science and Technology 55 (2020) 101465
B.A. Fahimmunisha, et al.
[7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]
[18]
[19]
[20] [21] [22] [23] [24] [25] [26] [27]
385. [28] S.Y. Pan, S.F. Zhou, S.H. Gao, Z.L. Yu, S.F. Zhang, M.K. Tang, J.N. Sun, D.L. Ma, Y.F. Han, W.F. Fong, K.M. Ko, New perspectives on how to discover drugs from herbal medicines: CAM's outstanding contribution to modern therapeutics, Evid. Based. Complement. Alternat. Med. 2013 (2013). [29] A.G. Atanasov, B. Waltenberger, E.M. Pferschy-Wenzig, T. Linder, C. Wawrosch, P. Uhrin, V. Temml, L. Wang, S. Schwaiger, E.H. Heiss, J.M. Rollinger, Potential antiosteoporotic natural product lead compounds that inhibit 17β-hydroxysteroid dehydrogenase type 2, J. Nat. Prod. 80 (2017) 965–974. [30] R. Ishwarya, B. Vaseeharan, S. Kalyani, B. Banumathi, M. Govindarajan, N.S. Alharbi, S. Kadaikunnan, M.N. Al-anbr, J.M. Khaled, G. Benelli, Facile green synthesis of zinc oxide nanoparticles using Ulva lactuca seaweed extract and evaluation of their photocatalytic, antibiofilm and insecticidal activity, J. Photochem. Photobiol., A 178 (2018) 249–258. [31] J.F. Hernandez-Sierra, F. Ruiz, D.C. Pena, F. Martinez-Gutierrez, A.E. Martinez, J. Guillen Ade, H. Tapia-Perez, G.M. Castanon, The antimicrobial sensitivity of Streptococcus mutans to nanoparticles of silver, zinc oxide, and gold, Nanomedicine 4 (2008) 237–240. [32] G.A. Martınez-Castanon, N. Nino-Martınez, F. Martınez-Gutierrez, J.R. MartınezMendoza, F. Ruiz, The antimicrobial sensitivity of Streptococcus mutans to nanoparticles of silver, zinc oxide, and gold, J. Nanoparticle Res. 10 (2008) 1343–1348. [33] R.M. Bhande, C.N. Khobragade, R.S. Mane, S. Bhande, Enhanced synergism of antibiotics with zinc oxide nanoparticles against extended spectrum β-lactamase producers implicated in urinary tract infections, J. Nanoparticle Res. 15 (2013) 1413. [34] C.Z. Chen, S.L. Cooper, Interactions between dendrimer biocides and bacterial membranes, Biomaterials 23 (2002) 3359–3368. [35] H. Liu, Y. Du, X. Wang, L. Sun, Chitosan kills bacteria through cell membrane damage, Int. J. Food Microbiol. 95 (2004) 147–155. [36] D.K. Tiwari, J. Behari, P. Sen, Time and dose-dependent antimicrobial potential of Ag nanoparticles synthesized by top-down approach, Curr. Sci. 95 (2008) 647–655. [37] B. Das, S.K. Dash, D. Mandal, T. Ghosh, S. Chattopadhyay, S. Tripathy, S. Das, S.K. Dey, D. Das, S. Roy, Green synthesized silver nanoparticles destroy multidrug resistant bacteria via reactive oxygen species mediated membrane damage, Arab. J. Chem. 10 (2017) 862–876. [38] R. Ishwarya, B. Vaseeharan, S. Suganya, A.K. Nazar, M. Govindarajan, N.S. Alharbi, S. Kadaikunnan, M.N. Al-anbr, J.M. Khaled, G. Benelli, Sargassum wightii-synthesized ZnO nanoparticles–from antibacterial and insecticidal activity to immunostimulatory effects on the green tiger shrimp Penaeus semisulcatus, J. Photochem. Photobiol., A 183 (2018) 318–330. [39] N. Bala, S. Saha, M. Chakraborty, M. Maiti, S. Das, R. Basu, P. Nandy, Green synthesis of zinc oxide nanoparticles using Hibiscus subdariffa leaf extract: effect of temperature on synthesis, anti-bacterial activity and anti-diabetic activity, RSC Adv. 5 (2015) 4993–5003. [40] Y. Xie, Y. He, P.L. Irwin, T. Jin, X. Shi, Antibacterial activity and mechanism of action of zinc oxide nanoparticles against Campylobacter Jejuni, Appl. Environ. Microbiol. 77 (2011) 2325–2331. [41] N. Padmavathy, R. Vijayaraghavan, Enhanced bioactivity of ZnO nanoparticles—an antimicrobial study, Sci. Technol. Adv. Mat. 9 (2008) 035004. [42] Y.C. Chung, Y.P. Su, C.C. Chen, G. Jia, H.L. Wang, J.G. Wu, J.G. Lin, Relationship between antibacterial activity of chitosan and surface characteristics of cell wall, Acta Pharmacol. Sin. 25 (2004) 932–936. [43] P.K. Stoimenov, R.L. Klinger, G.L. Marchin, K.J. Klabunde, Metal oxide nanoparticles as bactericidal agents, Langmuir 18 (2002) 6679–6686. [44] M.D. Rolfe, C.J. Rice, S. Lucchini, C. Pin, A. Thompson, A.D.S. Cameron, Lag phase is a distinct growth phase that prepares bacteria for exponential growth and involves transient metal accumulation, J. Bacteriol. 194 (2012) 686–701. [45] H. Su, C. Chou, D. Hung, S. Lin, I. Pao, J. Lin, F.L. Huang, R.X. Dong, J.J. Lin, The disruption of bacterial membrane integrity through ROS generation induced by nanohybrids of silver and clay, Biomaterials 30 (2009) 5979–5987.
activation into effective antibacterial defenses requires the caspase-1/IL-1beta signaling axis, Nat. rev. microbial. 13 (2015) 269–284. C.L. Ventola, The antibiotic resistance crisis: part 1: causes and threats, Pharmacol. Ther. 40 (2015) 277. F. Patrignani, L. Siroli, F. Gardini, R. Lanciotti, Determination of antibacterial and technological properties of vaginal lactobacilli for their potential application in dairy products, Front. Microbial. 17 (2016) 938. B. Li, T.J. Webster, Bacteria antibiotic resistance: New challenges and opportunities for implant‐associated orthopedic infections, J. Orthop. Res. 36 (2018) 22–32. S.B. Zaman, M.A. Hussain, R. Nye, V. Mehta, K.T. Mamun, N. Hossain, A review on antibiotic resistance: alarm bells are ringing, Cureus 9 (2017) 1403. F. Prestinaci, P. Pezzotti, A. Pantosti, Antimicrobial resistance: a global multifaceted phenomenon, Pathog. Glob. Health 109 (2015) 309–318. N. Beyth, Y. Houri-Haddad, A. Domb, W. Khan, R. Hazan, Alternative antimicrobial approach: nano-antimicrobial materials, Evid. Based. Complement. Alternat. Med. 2015 (2015) 16. R.K. Das, V.L. Pachapur, L. Lonappan, M. Naghdi, R. Pulicharla, S. Maiti, M. Cledon, L.M. Dalila, S.J. Sarma, S.K. Brar, Nanoparticle–plant interactions: two‐way traffic, nanotechnol, Environ. Eng. 2 (2017) 18. K. Pramila, Y. Ravikumar, S. Deepak Kumar, L. Kanwar, S. Sushil Kumar, Biosynthesis of iron and silver nanoparticles at room temperature using aqueous sorghum bran extracts, J. Nanostr. Chem. 8 (2018) 217–254. G. Benelli, M. Govindarajan, Green-synthesized mosquito oviposition attractants and ovicides: towards a nanoparticle-based “Lure and kill” approach? J. Clust. Sci. 28 (2017) 287–308. M. Govindarajan, G. Benelli, One-pot green synthesis of silver nanocrystals using Hymenodictyon orixense: a cheap and effective tool against malaria, chikungunya and Japanese encephalitis mosquito vectors? RSC Adv. 6 (2016) 59021–59029. A.A. Alfuraydi, S. Devanesan, M. Al-Ansari, M.S. AlSalhi, A.J. Ranjitsingh, Ecofriendly green synthesis of silver nanoparticles from the sesame oil cake and its potential anticancer and antimicrobial activities, J. Photochem. Photobiol., B 192 (2019) 83–89. M.S. AlSalhi, K. Elangovan, A.J.A. Ranjitsingh, P. Murali, S. Devanesan, Synthesis of silver nanoparticles using plant derived 4-N-methyl benzoic acid and evaluation of antimicrobial, antioxidant and antitumor activity, Saudi J. Biol. Sci. 26 (2019) 970–978. C. Vijilvani, M.R. Bindhu, F.C. Frincy, M.S. AlSalhi, S. Sabitha, K. Saravanakumar, S. Devanesan, M. Umadevi, M.J. Aljaafreh, M. Atif, Antimicrobial and catalytic activities of biosynthesized gold, silver and palladium nanoparticles from Solanum nigurum leaves, J. Photochem. Photobiol., B 192 (202) (2020) 111713. J. Jiang, J. Pi, J. Cai, The advancing of zinc oxide nanoparticles for biomedical applications, Bioinorgan. Chem. Appl. 2018 (2018) 18. V.N. Kalpana, V. Devi Rajeswari, A review on green synthesis, biomedical applications, and toxicity studies of ZnO NPs, Bioinorgan. Chem. Appl. 2018 (2018) 12. J.V. Meshram, V.B. Koli, S.G. Kumbhar, L.C. Borde, M.R. Phadatare, S.H. Pawar, Structural, spectroscopic and anti-microbial inspection of PEG capped ZnO nanoparticles for biomedical applications, Mater. Res. Express 5 (2018) 045016. I. Khan, K. Saeed, I. Khan, Nanoparticles: properties, applications and toxicities, Arab, J. Chem. 12 (2017) 908–931. A. Ali, A.R. Phull, M. Zia, Performance of silver, zinc, and iron nanoparticles-doped cotton filters against airborne E. coli to minimize bioaerosol exposure, Nanotechnol. Rev. 11 (2018) 1233–1242. A. Sharma, S.V. Madhunapantula, G.P. Robertson, Toxicological considerations when creating nanoparticle-based drugs and drug delivery systems, Expert Opin. Drug Metabol. Toxicol. 8 (2012) 47–69. J. Jeevanandam, A. Barhoum, Y.S. Chan, A. Dufresne, M.K. Danquah, Review on nanoparticles and nanostructured materials: history, sources, toxicity and regulations, Beilstein. J. Nanotech. 3 (2018) 1050–1074. S. Iravani, H. Korbekandi, S.V. Mirmohammadi, B. Zolfaghari, Synthesis of silver nanoparticles: chemical, physical and biological methods, Res. Pharm. Sci. 9 (2014)
13