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Bioinspired synthesis of pure massicot phase lead oxide nanoparticles and assessment of their biocompatibility, cytotoxicity and in-vitro biological properties
⁎Corresponding author at: Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan. talhakhalil.qau@gmail.com (Ali Talha Khalil)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Abstract
Green and ecofriendly route for biosynthesis of lead oxide nanoparticles has been successfully demonstrated using aqueous leaf extracts of Sageretia thea (Osbeck.). Biosynthesized PbO (∼27 nm) nanoparticles were extensively characterized using XRD, FTIR, Raman, EDS etc. Morphology was studied through HR-TEM/SEM. As synthesized nanoparticles were investigated for their iv-vitro biological properties. Antibacterial activities revealed enhancement upon modulation by UV in a concentration dependent manner. Pseudomonas aeruginosa was found to be the most resistant strain (MIC = 250 µg/mL and MICuv = 31.25 µg/ml). MTT cytotoxicity on leishmania promastigotes and amastigotes revealed significant inhibition as indicated by their IC50 values of 14.7 µg/mL and 11.95 µg/m respectively. Cytotoxicity was also confirmed using brine shrimp lethality (IC50 = 27.7 µg/mL). Bio-compatibility evaluation indicated cytotoxicity to freshly isolated human macrophages (IC50 = 57.1 µg/mL). Insignificant alpha-amylase inhibition and moderate protein kinase inhibition was revealed. Antioxidant activities indicated free radical scavenging activity (58 ± 2.45) at 200 µg/mL. Moderate total reducing power and total antioxidant activity was also indicated. Overall, we conclude lead oxide as a potential candidate for biological applications, however further studies are recommended on their in vitro and in vivo cytotoxicity.
Keywords
Lead oxide
Nanoparticles
Bioinspired
Antileishmanial
Antimicrobial
1 Introduction
Biosynthesis of nanoscale metals and metal oxides has gained tremendous importance over the last few years because of their unique and interesting properties (Thema et al., 2015a,b, 2016; Thovhogi et al., 2015; Diallo et al., 2015b,a; Sone et al., 2015; Nyangiwe et al., 2015; Ezhilarasi et al., 2016; Sone et al., 2020; Fuku et al., 2016; Ismail et al., 2016). Recently, porous materials have also been used for variety of biomedical applications (Vimala et al., 2010; Lian et al., 2012; Zakaria et al., 2015; Wu et al., 2011). The interface of metal oxide nanoparticles and bio-nanotechnology provides exciting avenues for fabricating nano-scaled matter with interesting properties (Ovais et al., 2016, 2017). Applications of lead oxide has been well documented in lead acid batteries (Ghasemi et al., 2006). Lead oxide are also used in the transparent conducting films, X-ray imaging detectors and as lead graphite composite electrodes (Perry, 2010; Šljukić et al., 2007). Lead oxide is a photosensitive material and therefore can be used in optical sensors. Some of the other applications of lead oxide include their use in solar energy conversion, X-ray sensitive photoconductor and other opto-electronic applications (Perry, 2010). Because of their electronic properties and ease in fabrication, there is a great potential for their use in conjunction with other materials for a vast array of applications. Lead oxides basically exists in 2 forms. The alpha-form generally referred as litharge (red-tetragonal) and beta-form which is commonly known as massicot (yellow-orthorhombic).
Synthesis of lead oxide has primarily been done through chemical or physical routes. Thermal decomposition of lead precursors, spray pyrolysis, sono-chemical, electrochemical methods have already been applied for synthesis of PbO (Sobanska et al., 1999; Lyons et al., 1992; Ghasemi et al., 2008, 2005). However, these processes are accompanied by various disadvantages. Physical synthesis means requires high energy and high vacuum while the chemical means are associated with the generation of toxic and hazardous waste lines making them environmental un-friendly (Diallo et al., 2015b). To reduce the problem of energy consumption and hazardous waste, alternative green and eco-friendly methods have been proposed. The interface of medicinal plants and metal oxide nanoparticles has been considered as a bright area of research for synthesizing multifunctional metal and metal oxide nanoparticles. Although, chemical synthesis of lead oxide has been successfully reported, however there are no reports available in the literature about biosynthesis of lead oxide nanoparticles.
Complementing the vacuum in literature on the biosynthesis of lead oxide nanoparticles, a comprehensive research was undertaken to biosynthesize lead oxide nanoparticles using aqueous leaf extracts of medicinal plant Sageretia thea (Osbeck.). Sageretia thea is locally referred as “Momanra”/Pashto and “Bird plum”/English has well documented uses in hepatitis, jaundice, circulatory and cardio-vascular diseases. In this manuscript, biogenic synthesis of multifunctional lead oxide has been successfully reported for the first time. Furthermore, in addition to antileishmanial, antimicrobial, antioxidant and enzyme inhibition assays, their cytotoxicity was also assessed against normal human RBC’s and macrophages.
2 Materials and methods
2.1 Plant material processing
The collection of Sageretia thea was done from Islamabad, Pakistan followed by it taxonomic identification from Department of Plant Sciences, Quaid-i-Azam University (QAU), Islamabad. The herbarium specimen with voucher number MOSEL-343 was deposited at Molecular Systematics and Applied Ethnobotany Lab (MoSAEL), Department of Biotechnology, QAU. After excision, the fresh and healthy leaves were dried and grounded into fine powder via Willy mill. Aqueous extraction was performed on the plant powder, while remaining was stored for further use. In Fig. 1 the overall outline of the study has been summarized.
2.2 Biosynthesis of PbO nanoparticles
The biosynthesis of PbO nanoparticles was performed as described by Thema et al., 2015a,b with minor modifications (Thema et al., 2015b). The aqueous extract from the plant powder was obtained by mixing 30 g of plant powder into 200 mL of deionized water under continuous heat of ∼80 °C on a magnetic stirring hot plate (Snijders) for 1 h. Solid residual plant material was removed by filtering the aqueous solution 3 times with Whatman filter paper. Biosynthesis of PbO nanoparticles was performed by adding 6.0 g of the precursor salt lead acetate (Alfa Aesar) into 100 mL of aqueous plant extracts solution (pH 5.7), followed by 2 h of gentle heating and stirring at ∼60 °C. After the addition of lead acetate, the pH of the solution was recorded as 4.6 at room temperature. As the solution cooled down to room temperature, centrifugation was performed at 10,000 rpm/10 min for the collection of precipitate. Obtained pellet was washed and centrifuged (10,000 rpm/10 min) with distilled water for 3 times. For obtaining highly crystalline PbO nanoparticles the precipitate was further annealed in open air in furnace 500 °C respectively. The biosynthesized PbO nanoparticles were extensively characterized for confirmation of pure phase massicot PbO nanoparticles. The proposed mechanism of biosynthesis has been indicated in Fig. 2.
2.3 Characterization of bioinspired PbO nanoparticles
XRD analysis using X-ray diffractometer (model Bruker AXS D8 Advance) with irradiation line Kα of copper (λ = 1.5406 Å) was performed for all the samples to check the crystalline structure of PbO nanoparticles. The corresponding size of all the thermally annealed PbO nanoparticles was calculated via Scherer equation {〈Øsize〉 = K λ/Δθ1/2 cosθ}. For the study of vibrational properties Raman spectroscopy was carried out. To record the Raman spectrum (0–500 cm−1) a laser line of 473 nm with average excitation power of 2.48 mW was used. ATR-FTIR for biogenic PbO nanoparticles was carried in the spectral range from 400 to 4000 cm−1. The morphological investigation was carried out through HR-SEM and HR-TEM. Particle size distribution was calculated using image J software after digitizing the various HR-TEM images, while elemental composition was determined by Energy Dispersive X-ray spectroscopy “EDS”. Selected Area Electron Diffraction (SAED) study was also performed.
2.4 Antibacterial activity
Agar disc diffusion method (Fatima et al., 2015; Thatoi et al., 2016) was adopted for the determination of antibacterial efficacy of PbO nanoparticles while broth dilution method (Wiegand et al., 2008) was followed for the calculation of corresponding MIC’s. The microbial strains already available at Department of Biotechnology, QAU, Islamabad were refreshed on Nutrient Agar media (Oxoid-CM0003). Prior to assay, the bacterial cultures were shifted to nutrient broth and kept for 24 h in shaking incubator (Temp:37 °C; RPM:200). The bacterial broth cultures were standardized to 1 × 108 CFU/mL by adjusting the optical density to 0.5 at with sterilized nutrient broth. On the nutrient agar plate 100 µL of broth cultures were dispensed homogeneously. Filter discs (6 mm in diameter) were loaded with 10 µL of sample dilutions, dried and placed accordingly on the bacterial lawn. As a positive control, Gentamycin (10 µg) discs were used, while the bacterial plates were incubated for 24 h at 37 °C. For the measurement of zone of inhibitions vernier caliper was used. Antibacterial activity was performed over the test concentrations of 1000–31.25 µg/mL. Minimum inhibitory concentration of test samples were investigated for the samples effective at <31.25 µg/mL using broth dilution assay. MIC was defined as the minimum concentration of nanoparticle suspension that inhibit the bacterial growth.
In addition, the effect of UV-illumination was also assessed. Nanoparticles suspensions were kept in a UV-illuminator for 20 min under. The source of UV radiation was, germicidal 6 Watt UV Lamp 6GT5 (Sankyo denki- Japan).
2.5 Antileishmanial activity (Promastigotes and Amastigotes)
Promastigote cultures of Leishmania tropica KWH23 strain was used for the assessment of biogenic nanoparticles cytotoxicity via MTT cell viability assay, as described previously (Ali et al., 2017). For culturing the strain, M199 media supplemented with 10% fetal bovine serum (FBS) was used, while the density of 1 × 106 cells/mL were maintained for the assay. The assay was performed in 96 well microplate with dilutions of final concentrations ranging from 200 to 1 µg/mL. As positive and negative controls Amphotericin B and DMSO were used in the assay respectively. After incubation of seeded 96 well microplate for 72 h at 24 °C, readings were taken at 540 nm by BIOTEK microplate reader. Under the inverted microscope survived promastigotes were counted, followed by determination of LC50 values using Table curve software. Percent inhibition was calculated via following equation;
Similar procedure was applied on the amastigote cultures of Leishmania tropica KWH23.
2.6 Brine shrimp cytotoxicity
Cytotoxic potential of bioinspired PbO nanoparticles were investigated against Artemia salina larvae following already established protocol in a 96 well plate (Ali et al., 2017; Khan et al., 2015). After incubation for 24 h with the test concentrations of biogenic lead oxide nanoparticles and percent inhibition of brine shrimps were calculated by counting the dead shrimps in each well. IC50 values were calculated using table curve software.
2.7 Biocompatibility of PbO nanoparticles
2.7.1 Biocompatibility with erythrocytes
To determine the biocompatible nature of biogenic PbO nanoparticles with red blood cells of human, hemolytic assay was carried out (Malagoli, 2007). From a healthy individual fresh blood was collected and dispensed in a sterile EDTA tube. Centrifugation (14,000 rpm for 5 min) of 1 mL blood was performed for the separation of RBCs. For preparing an erythrocyte suspension in PBS, 200 µL of the pelleted erythrocyte were added to 9.8 mL of phosphate buffer saline followed by gentle shaking (pH: 7.2). In a test tube both the erythrocyte suspension (100 µL) and test nanoparticle solution (100 µL) was gently mixed, and incubated for one hour at 35 °C. The solution was further centrifuged at 10,000 rpm for 10 min and supernatant was collected. The hemoglobin release was monitored using BIOTEK microplate reader at 540 nm after dispensing the supernatant in the 96 well plate. Triton X-100 and DMSO were used as positive and negative controls respectively. Results were calculated as percentage hemolysis induced by the nanoparticle dilution calculated through the formula;
2.7.2 Biocompatibility human macrophages
For further assessment of PbO nanoparticles biocompatibility, Ficoll–Gastrografin (sodium diatrizoate) method was adopted. The cytotoxicity was inspected against human macrophages isolated from peripheral human blood. This isolation protocol is based on ficoll-gastrografin density gradient (density = 1.070 g/mL) (de Almeida et al., 2000). In 95 mL of deionized water combined with 5 mL of gastrografin, 5.7 g of ficoll was slowly dissolved. Hank’s buffer salt solution (HBSS) was used for the dilution of blood which was layered gently on the ficoll-gastrografin. The solution was further centrifuged for 30 min at 400g followed by purification with percoll gradient (density 1.064 g/mL) adjusted with sterilized deionized water. The suspension of isolated cells was made in RPMI medium supplemented with fetal bovine serum (10%), Hepes (25 mM), and antibiotics (penicillin:100 U/mL; Streptomycin: 0.1 mg/mL). In humidified incubator with 5% CO2 the isolated macrophages were cultured to the density of 1 × 105 cells/well. Percentage inhibition was calculated using formula;
2.8 Antioxidant activities
2.8.1 Free radical scavenging
By using DPPH (2,2-diphenyl 1-picrylhydrazyl) as a stable free radical, spectrophotometric method (Fatima et al., 2015; Ali et al., 2017) was employed to investigate the radical quenching ability of green synthesized PbO nanoparticles. For determination of free radical scavenging concentrations of PbO nanoparticles starting from 200 µg/mL to 1 µg/mL were tested. As a positive and negative control, ascorbic acid and DMSO were used respectively. After keeping the 200 µL reaction mixture in dark for 20 min optical densities were measured at 517 nm by BIOTEK microplate reader. Percent free radical scavenging can be found using the formula;
2.8.2 Total reducing power
For the determination of reducing power potential of biogenic PbO nanoparticles Potassium ferricyanide [K3Fe (CN)6] based method was used (Javed et al., 2016). DMSO was used as negative control while ascorbic acid as positive control. At 630 nm, the absorbance intensity was measured using BIOTEK microplate reader. Ascorbic acid equivalents per mg was regarded as reducing power.
2.8.3 Total antioxidant capacity
Previously described phosphomolybdenum based method was used to determine total antioxidant capacity (Jafri et al., 2017). The absorbance was measured at 695 nm while results are expressed as number of ascorbic acid equivalents in µg per mg of the sample i.e. µg AAE/mg.
2.9 Enzyme inhibition assays
2.9.1 Alpha amylase inhibition
In vitro alpha amylase inhibition assay was performed in a 96 well plate as described previously (Javed et al., 2016). Test samples (10 µL) and starch solution (40 µL) were added stepwise in the reaction mixture (15 µL PBS/25 µL α-amylase enzyme), followed by incubation at 50 °C for 30 min. After 30 min, 20 µl (1 M HCl) and 90 µL of iodine solution were added into reaction mixture accordingly. Blank solution contained deionized water, starch and PBS, while positive and negative controls comprised of acarbose and deionized water respectively. Enzyme inhibition was calculated by following equation; where “ODS”, “ODN” and “ODB” corresponds to the optical densities of sample, negative control and blank respectively
2.9.2 Protein kinase inhibition
Streptomyces 85E strain was used for Protein kinase inhibition assay as described previously (Fatima et al., 2015). On ISP4 minimal media a uniform lawn of the respective strain was prepared, followed by placement of 6 mm filter discs steeped with 10 µL of the test PbO nanoparticle dilution. As a positive and negative controls surfactin and DMSO were used respectively. Readings were taken after 72 h of incubation at 30 °C.
3 Results and discussion
3.1 Physical characterizations
Chemical and physical means of synthesis of nanoparticles bears certain disadvantages like their cost and hazardous waste generation. Even sometimes, noxious chemicals can remain adhered to the surface nanoparticles which limit their use especially in biological applications (Zak et al., 2011; Darroudi et al., 2014). On the contrary, biological synthesis through medicinal plants is overcome such disadvantages. Therefore green synthesis of nanoparticles is considered more acceptable route for synthesis of multifunctional nanomaterials (Ovais et al., 2016). Biosynthesis of lead oxide has never been reported previously through medicinal plants. Herein, aqueous extracts of Sageretia thea was effectively used as chelation agent for the biosynthesis of lead oxide nanoparticles. Sageretia thea has been used in making tea in parts of China and Korea. Certain phenolic and flavonoid compounds (Syringic acid, Quercetin, Myricetrin, Kaempferol, Daucosterol, Taraxerol) are already been reported from Sageretia thea (Shen et al., 2009; Chung et al., 2004; Xu et al., 1994). Such bioactive components can play a critical role in capping and stabilizing of nanoparticles (Park et al., 2011).
3.2 Characterizations
X-ray diffraction spectra of bioinspired lead oxide nanoparticles has been indicated in Fig. 3(A), while their average size was determined using Debye-Scherer approximation as indicated in Fig. 3(B). The observed Bragg peaks were the crystallographic reflections of orthorhombic pure massicot phase lead monoxide (PbO) having standard lattice parameters of 〈a〉 = 0.549 nm, 〈b〉 = 0.589 nm, 〈c〉 = 0.475 nm, 〈a/b〉 = 0.931 nm and 〈c/b〉 = 0.806 nm, which are consistent with the JCPDS pattern no. 00-038-1477. The face centered cubic lattice belonged to the space group Pcam (57). Average size was found to be 27 nm as calculated from the Debye-Scherrer formula. No other peaks were indicated by the XRD analysis which suggest the single and pure phase of bioinspired PbO nanoparticles. Morphology and particle size distribution was also studied through HR-TEM as indicated in inset of Fig. 4. From the HR-TEM results, the shape of the particles can be deduced as quasi-spherical. After digitization of the various HR-TEM images, the particle distribution was calculated and the results were found in agreement with the XRD study. Fig. 5(A)–(C) shows the HR-SEM images of PbO nanoparticles that indicate a certain degree of agglomeration. Fig. 5(D) shows a spotty ring pattern for the biosynthesized lead oxide nanoparticles which refers to their crystalline nature. To further study the elemental phase of the nanoparticles, energy dispersive spectroscopy was carried out as shown in Fig. 6. The results indicated the presence of lead (Pb) and oxygen (O) in the sample while the peak corresponding to Carbon (C) is due to the grid support.
The vibrational properties for biogenically synthesized massicot phase lead oxide nanoparticles were studied using Raman spectroscopy and ATR-FTIR as indicated in Figs. 7and 8 respectively. Raman spectra recorded over the spectral range (0–500 cm−1) indicated characteristic peaks of massicot at ∼84 cm−1, ∼162 cm−1 and ∼284 cm−1. These Raman bands are in good agreement with the previously reported studies (Burgio et al., 2001) however, relative positioning of the peaks can change with the difference in synthesis method and distribution vacancies in the unit cell (De Faria et al., 1997). In addition, the FTIR spectra was recorded over the spectral range from 0 cm−1 to 4000 cm−1. Characteristics IR peak centered at ∼500 cm−1 can be attributed to the Pb-O stretching (Arulmozhi and Mythili, 2013). The crystallinity of the PbO nanoparticles can be pre-concluded because of the intense peak. IR peak centered at ∼3400 cm−1 represents —OH stretching vibrations. FTIR spectra also show bands at centered at ∼2200 cm−1 and 1200 cm−1 which indicate the possible attached functional groups.
3.3 Antibacterial activities
Bioinspired lead oxide nanoparticles were screened for their antimicrobial potential against 3 gram positive (Staphylococcus aureus, Staphylococcus epidermis and Bacillus subtilis) and 3 gram negative bacterial strains (Klebsiella pneumonia, Pseudomonas aeruginosa and Escherichia coli) across different concentration i.e. 1000–31.25 µg/mL. In addition, the effect of UV illumination was studied in the enhancement of the antimicrobial potential. Fig. 9(A) shows the antibacterial effect of PbO nanoparticles without UV illumination. Klebsiella pneumonia was found as the most susceptible bacterial strain (MIC = 31.25 µg/mL) while Pseudomonas aeruginosa (MIC = 250 µg/mL) was the least susceptible. Upon modulation of UV-illumination, enhancement in the antibacterial potential was observed as indicated in Fig. 9(B). With UV modulation, Pseudomonas aeruginosa was inhibited at lower concentrations then 250 µg/mL, and their MIC was investigated as 31.25 µg/mL. Klebsiella pneumonia was inhibited at lower concentrations then 31.25 µg/mL. The MIC was for the samples effective up to the concentration of 31.25 µg/mL, was investigated by broth dilution assay. The tube dilution assay revealed MIC of 15.6 µg/ml for the lead oxide nanoparticles produced via aqueous root extracts of Sageretia thea. MIC values are summarized in Table 1. In general, we indicate moderate antimicrobial nature for lead oxide nanoparticles relative to the pure gentamycin disc (10 µg) which is used as a positive control. We further conclude the antibacterial response as concentration or dose dependent.
| Without UV illumination | With UV illumination | ||
|---|---|---|---|
| Gram positive | Gram positive | ||
| Bacterial strain | MIC (µg/ml) | Bacterial strain | MIC (µg/ml) |
| Staphylococcus aureus | 62.5 | Staphylococcus aureus | 31.25 |
| Staphylococcus epidermis | 62.5 | Staphylococcus epidermis | 31.25 |
| Bacillus subtilis | 125 | Bacillus subtilis | 31.25 |
| Gram negative | Gram negative | ||
| Klebsiella pneumonia | 31.25 | Klebsiella pneumonia | 15.6 |
| Pseudomonas aeruginosa | 250 | Pseudomonas aeruginosa | 31.25 |
| Escherichia coli | 125 | Escherichia coli | 31.25 |
3.4 Antileishmanial activities
Leishmaniasis is as a neglected tropical disease which is endemic to 98 countries and with ∼350 million people living under immediate threat. Until now, there are no effective treatments for its cure while the medications been used for its treatment are not only costly but also accompanied with side effects like prolonged duration for therapy and elevated toxicity (Abamor, 2017; Légaré and Ouellette, 2017). Antimonials have been widely used for their treatment which however has lost their efficiency due to drug resistance. Leishmania exist in promastigote (motile) form outside the body while transform into amastigote (non-motile) form inside the body. Antileishmanial activities of biogenically synthesized lead oxide are reported against the axenic promastigote and amastigote leishmania as indicated in Fig. 10(A). MTT cytotoxicity suggested potential lethal nature of lead oxide nanoparticles against leishmania. Percent inhibition was observed across all the tested concentrations while the IC50 values were calculated as 14.7 and 11.95 µg/mL for the promastigote and amastigote cultures of leishmania. Novel strategies involving nanoparticles are already proposed for the treatment of leishmania. Recent research has indicated a significant potential of metal oxides to be used as antileishmanial agents (Ali et al., 2017; Nadhman et al., 2016). From our results, we can conclude dose dependent cytotoxicity against leishmania, while amastigotes were found more susceptible to promastigotes.
3.5 Brine shrimp cytotoxicity
The cytotoxic potential of the lead oxide nanoparticles was also studied through brine shrimp cytotoxic activity. Brine shrimps (Artemia salina) are widely used to screen chemical entities for their cytotoxicity (Ali et al., 2017). The obtained results are summarized in Fig. 10(A). Percent inhibition was calculated by counting the succumbed shrimps relative to the dead ones after applying the test dilution. Cytotoxicity of the lead oxide nanoparticles was confirmed and the median lethal concentration was calculated as 27.74 µg/mL. The dose dependent response was also confirmed.
3.6 Bio-compatibility potential
Keeping in view the adverse effects of lead/lead oxide nanoparticles to the human exposure (de Almeida Lopes et al., 2015), their biocompatibility was assessed. Herein, it is noteworthy to mention that the bio-compatibility of the bioinspired lead oxide has never been reported. Freshly isolated human RBC’s and macrophages were used to study the bio-compatibility of the as synthesized lead oxide nanoparticles. The results are indicated in Fig. 10(B). Hemolytic activity revealed cytotoxicity of biogenic lead oxide at higher concentrations i.e. 25% at 200 µg/mL while lowering their concentrations significantly decreases percent hemolysis. On the contrary, the freshly isolated macrophage cells were inhibited significantly as compared to the RBC’s. The MTT cytotoxic assay revealed the median lethal concentration of 57.1 µg/mL against human macrophage cells. These results against human macrophage cells are in good agreement with some of the previous reports where the median lethal concentration against human cells are found to be around 50 µg/mL (Alarifi et al., 2017). These results are indicated in Fig. 10(A) and (B) while the calculated medial lethal concentration (IC50) are summarized in Table 2. Our results conclude relatively less cytotoxicity to RBC’s as compared to macrophages.
| Assay type | IC50 |
|---|---|
| Antileishmanial promastigotes | 14.7 µg/ml |
| Antileishmanial amastigotes | 11.95 µg/ml |
| Brine shrimp cytotoxicity | 27.74 µg/ml |
| Human RBC’s | >200 µg/ml |
| Human macrophages | >57.1 µg/ml |
| Alpha amylase inhibition | >200 µg/ml |
3.7 Antioxidant assays
Antioxidant potential of the bioinspired lead oxide nanoparticles was assessed as indicated in Fig. 11. Free radical scavenging activity using DPPH suggested radical scavenging potential for the biosynthesized lead oxide nanoparticles. 58% DPPH radical scavenging was observed at 200 µg/mL. Scavenging potential was directly related to the concentration of the nanoparticles. Radical scavenging activity decreased to 6% at 2 µg/mL while no activity was observed at concentration <2 µg/mL. Total reducing power and total antioxidant capacity was expressed as micrograms of ascorbic acid/mg of the test samples. Moderate reducing power i.e. 22 µg AAE/mg and moderate antioxidant capacity i.e. 19.6 µg AAE/mg were reported at 200 µg/mL. Results are summarized in Fig. 11.
3.8 Enzyme inhibition assays
Fig. 12(A) reports the alpha amylase enzyme inhibition potential of the biogenically synthesized lead oxide nanoparticles. Insignificant inhibition of alpha amylase enzyme is investigated. Enzyme inhibition was not recorded at <10 µg/mL. In addition, no protein kinase inhibition was reported across any of the tested concentration of the biogenically synthesized lead oxide nanoparticles as indicated in Fig. 12(B).
3.9 General comments on the mechanism of cytotoxicity
Taken together, the biogenically synthesized lead oxide nanoparticles showed antimicrobial and cytotoxic potential against bacteria, leishmania and also human macrophages. Generation of reactive oxygen species, mitochondrial dysfunction, DNA fragmentation and interference with the cellular proteins are considered to be the chief cause of lead oxide mediated cytotoxicity (Alarifi et al., 2017; Amiri et al., 2016). Lead oxide nanoparticles can induce cytotoxicity by oxidative stress by the reduction of GSH, increase in lipid peroxides and SOD concentrations in a dose and time dependent manner. A detailed schematic is presented in Fig. 13 which indicates the lead oxide mediated cytotoxicity.
4 Conclusion
Lead oxide nanoparticles were biogenically synthesized via complete green process without the addition of any material except for the precursor. Biological properties were assessed, and the results indicated their effectiveness against various organisms especially leishmania. UV modulation was reported to enhance the antibacterial potential. Bioinspired lead oxide nanoparticles were investigated as relatively less toxic to RBC’s as compared to macrophages. Moderate antioxidant potential while insignificant alpha amylase and protein kinase enzyme inhibition potential is reported for the bioinspired lead oxide nanoparticles.
Lead oxide has a number of industrial applications. Lead monoxide has been used in lead acid batteries, paints, lead glasses, lead glazes, lead crystals and other decorative potteries etc. Chemically synthesized lead oxide can have adverse effects when consumed at higher level. On the contrary, biogenically synthesized lead oxide presents a more viable option for industrial uses where their use cannot be avoided. In addition, biogenically synthesized lead oxide may be used for making antimicrobial lead glasses, antimicrobial paints and can be used in MRI etc. We have made a first attempt to highlight the potential biological uses of lead oxide nanoparticles, which are never said in the literature before. However, their use in nanomedicine, will require further and extensive research.
Acknowledgements
Authors are thankful to the fellows at Molecular systematics and applied ethnobotany lab, Department of Biotechnology, QAU, Islamabad, Pakistan and fellows of Materials Research Department, iThemba labs, Cape town for their encouragement, support and assistance. Authors are also indebted to Prof. Noor Muhammad Butt, for his assistance.
Conflict of interest
The authors declare no conflict of interest.
Author contributions
ATK, ZKS, MM for conceiving the idea. ATK, MO, IU and SAJ for performing the experimental. ATK, MO, MA prepared the draft manuscript. MA, ZKS and MM reviewed and improved the manuscript.
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