5.1. Characterization analysis
Ultraviolet-Visible (UV/Vis) spectroscopy is a technique that is used for the quantification of light that is absorbed and scattered by a sample. A sample is placed between a light source and a photo detector, and then the intensity of a beam of light is measured before and after passing through the sample. Comparison of these measurements at each wavelength was done to quantify the wavelength of the sample dependent extinction spectrum. It reveals the formation of Mn/ZnO-NCs with surface Plasmon absorption peaks. For identifying, characterizing, and studying these materials, NPs have optical properties that are sensitive to size, shape, concentration, agglomeration state, and refractive index near the NPs surface, which makes UV/Vis/IR spectroscopy a valuable tool. NPs that were made from certain metals, strongly interact with specific wavelengths of light and this is the unique optical property of these materials and that is the foundation for the field of plasmonics. For pure NPs, the spectra shows maximum absorption at 272 and 394 nm. As depicted in these values are closely related to the values in the literature reported. Figure 1.(a), shows a bathchromic shift in maximum wavelength with increase in the composition. The confirmation evidences of NPs formation was also supported as illustrated in Fig. 1.(b).shows concurrent presence of zinc oxide, Carbon and Oxygen, Manganese elements which were analysed by the energy-dispersive X-ray (EDS) within the heterojunction composite. Notably, uniform spatial distribution of these elements is observed.
The scanning electron microscopy has been used to characterize the shape and morphologies of NPs. In Fig. 1.(c).the SEM image of the sample was shown where it has got very big particles with thick texture and few pores of varied size were seen. TEM analysis revealed that NPs obtained were spherical in shape. There are dark and thick particles observed in the image which measures around 25.75nm, 20.36nm, and 21.13 nm. TEM image analysis in Fig. 1.(d) showed the average size of NPs to be around 20nm.
5.2. Antibacterial activity:
The study of the activity of manganese-zinc oxide NCs (Mn/ZnO-NCs) against four bacterial strains: Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae, and Pseudomonas aeruginosa was evaluated by the antibacterial activity Fig. 2. showed that the NCs exhibited a concentration-dependent antibacterial effect, with no inhibition at 25µg, but increasing zones of inhibition at 50, 75, and 100 µg. At 100 µg, the zones of inhibition were found to be 13 ± 0.11 mm for S. aureus, 15 ± 0.11 mm for E. coli, 15 ± 0.34 mm for S. pneumoniae, and 14 ± 0.28 mm for P. aeruginosa. These findings as shown in Table.1.highlights, the significant antibacterial potential of Mn/ZnO-NCs, suggesting their viability as effective antimicrobial agents. Enhanced efficacy is attributed to increased NPs interaction, ROS generation, and ion release.
Table.1. Zone of inhibition Mn/ZnO-NCs at various concentrations
|
|
Concentration (µg)/ Zone of Inhibition (mm)
|
S.No
|
Strain
|
25
|
50
|
75
|
100
|
1
|
Staphylococcus aureus
|
0
|
9 ± 0.34
|
11 ± 0.31
|
13 ± 0.11
|
2
|
E. coli
|
0
|
10 ± 0.14
|
13 ± 04
|
15 ± 0.11
|
3
|
Streptococcus pneumonia
|
0
|
9 ± 0.22
|
12 ± 0.11
|
15 ± 0.34
|
4
|
Pseudomonas aeruginosa
|
0
|
10 ± 0.12
|
13 ± 0.43
|
14 ± 0.28
|
5
|
Ampicilin
|
0
|
11 ± 0.34
|
14 ± 0.54
|
19 ± 0.23
|
5.3. Antitumor Activity:
The experimental protocol delineated here presents a meticulous methodology for evaluating the cytotoxic efficacy of novel NCs (NCs) across a spectrum of cancer cell lines. Initially, cryopreserved cells were thawed and resuscitated, then cultured in media to promote proliferation. Post-incubation, cells were aliquoted into vials and subjected to centrifugation to separate cellular components from nutrient media, yielding a pellet enriched with cancer cell lines. Cell quantification was performed using a Neubauer chamber to ensure precise cell density measurement and uniform distribution. A 96-well plate was meticulously prepared, with 98µL of media supplemented with 2µL of cell suspension in each well. NCs were introduced in triplicate at varying concentrations (10, 20, and 30 µL), facilitating a comprehensive dose-response assessment. Following a 48hrs incubation period under controlled conditions (37°C, CO2), cell viability was quantified via optical density (O.D) measurements, reflecting cytotoxicity levels induced by the NCs. Control wells lacking treatment and those treated with Cisplatinum served as reference points for comparative analysis. Cytotoxicity was quantified as the percentage decrease in viability compared to controls, providing a precise measure of NC-induced cell death. Additionally, IC50 values, that represents the concentration at which 50% cell viability is inhibited and were derived from dose-response curves. This rigorous methodology, combining meticulous cell culture techniques with robust cytotoxicity assays, furnishes quantitative insights into the therapeutic potential of the investigated NCs across diverse cancer cell types. In Table.2.The concentration-dependent cytotoxicity against BXPC-3 cells andOVCAR-3 cells was assessed, revealing a potent inhibitory effect at higher concentrations, with 92.2% inhibition observed at 3000nM for BXPC-3 cells. As concentration decreased, inhibition gradually diminished, though even at the lowest tested concentration (1nM), a residual cytotoxic effect of 1.0% was evident. This comprehensive evaluation highlights the dose-dependent efficacy against BXPC-3 cells of NCs, suggesting potential therapeutic relevance in pancreatic cancer treatment. The cytotoxicity against OVCAR-3 cells was investigated across a range of concentrations, revealing a concentration-dependent response. At higher concentrations (3000nM and 1000nM), significant inhibitory activity was observed, with 91.5% and 84.0% inhibition, respectively. However, inhibition gradually decreased as the concentration decreased, reaching 0.0% inhibition at the lowest concentration tested (1nM). These findings as depicted in Fig. 3. highlight the NCs potential as a cytotoxic agent against OVCAR-3 cells, emphasizing the importance of dosage in eliciting therapeutic effects. The NCs impact on MDA-MB231cells was evaluated across varying concentrations, revealing a concentration-dependent response as shown in Table.2. Significant inhibition was observed at higher concentrations (3000nM and 1000N), with 92.2% and 88.5% inhibition, respectively. As concentrations decreased, inhibitory effects gradually diminished, reaching 0.0% inhibitions at 1nM. Standard deviation values remained consistent, indicating reliable data. These findings highlight NCs potential cytotoxicity against MDA-MB231cells, emphasizing the importance of dosage in eliciting therapeutic effects. Cytotoxicity of NCs against SKOV-3 cells displayed a concentration-dependent trend. It induced significant inhibitory effects at higher concentrations (3000nM and 1000nM), yielding 94.3% and 93.7% inhibition, respectively. As concentration decreased, inhibition gradually diminished. At 1nM, no notable inhibition was observed as shown in Table.3.The data exhibited consistent reliability, with low standard deviation values across concentrations. These findings underscore the compound's potential as a cytotoxic agent against SKOV-3 cells, emphasizing the importance of dosage for therapeutic efficacy.
Table.2.Cytotoxicity of Mn/ZnO-NCs against BXPC-3 cells and OVCAR-3 cells
Concentration
|
BXPC-3 Cells
% inhibition
|
Std
|
OVCAR-3 Cells
% inhibition
|
Std
|
3000nM
|
92.2
|
0.2
|
91.5
|
0.4
|
1000nM
|
81.2
|
1.4
|
84.0
|
0.0
|
300nM
|
63.7
|
1.9
|
66.2
|
1.6
|
100nM
|
28.9
|
5.2
|
36.4
|
0.3
|
30nM
|
15.7
|
3.6
|
17.2
|
2.6
|
10nM
|
4.5
|
3.1
|
17.7
|
1.4
|
3nM
|
6.6
|
6.7
|
15.5
|
8.8
|
1nM
|
1.0
|
1.2
|
0.0
|
0.0
|
Table.3.Cytotoxicity of Mn/ZnO-NCs against MDA-MB231cells and SKOV-3 Cells
Concentration
|
MDA-MB231 Cells
% inhibition
|
Std
|
SKOV-3 Cells
% inhibition
|
Std
|
3000nM
|
92.2
|
0.0
|
94.3
|
0.3
|
1000nM
|
88.5
|
0.6
|
93.7
|
1.0
|
300nM
|
73.0
|
1.8
|
84.1
|
0.1
|
100nM
|
45.7
|
3.0
|
60.3
|
1.1
|
30nM
|
24.2
|
9.0
|
28.5
|
1.6
|
10nM
|
22.3
|
3.5
|
13.7
|
1.8
|
3nM
|
18.4
|
1.7
|
6.8
|
1.9
|
1nM
|
0.0
|
0.0
|
0.0
|
0.0
|
The study showed that the synthesised NPs significant antitumor activity against all the cell lines studied. The concentration of compound where percent inhibition is equal to 50 is called the IC50 value. The NPs showed IC50 values of 195.5nM against BXPC3 cells, IC50 values of 178.10 nM against OVCAR3 cell lines, IC50 values of 128.1nM against MDA-MB231 cells, IC50 values of 65.08 nM against SKOV3 cells. The synthesised NPs showed better inhibition of SKOV3 cell lines in comparison to other cell lines studied.