Carthamidin-mediated silver nanoparticles synthesis, characterization and anticancer activity-Invitro Approach

doi:10.21203/rs.3.rs-4356996/v1

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Carthamidin-mediated silver nanoparticles synthesis, characterization and anticancer activity-Invitro Approach

https://doi.org/10.21203/rs.3.rs-4356996/v1

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The synthesis of silver nanoparticles from biological sources is been fascinated by the research fraternities owing to the distinctive feature of non-toxicity and benign synthesis methodologies. In this pipeline, we have synthesized silver nanoparticles (AgNPs) from flavonoid Carthamidin (CT) pigment. The yellow colored water soluble pigment reduces the silver nitrate into silver ions in a simple one pot method. The CTAgNPs were exclusively characterized by UV-Vis spectroscopy, X-ray Diffraction (XRD), Transmision Electron Microscopy (TEM), Scanning Electron Microscopy (SEM) and Energy Dispersive Analysis Spectroscopy (EDAX). The characterization techniques infer that CTAgNPs are spherical in shape with an average size of 59nm and face centered cubic with crystalline lattice. The CTAgNPs evaluated as a cytotoxic agent in the MCF 7 cells and molecularly annotated the CTAgNPs apoptosis induction by AO/EB staining, Reactive Oxygen Species (ROS) assay, quantification of lactate Dehydrogenase (LDH) and arresting of cell cycle by flow cytometry. Finally, the CTAgNPs possess a remarkable cytotoxicity in the MCF 7 cells and induce the apoptosis mechanism. Henceforth, CTAgNPs can be promulgated as a nanodrug for the treatment of breast cancer therapy.

Breast cancer

Chemotherapy

Betanin

Apoptosis

flow cytometry

Emerging as a groundbreaking field, nanotechnology has the potential to revolutionize several industries, including materials research, electronics, healthcare, and energy [1]. Nanotechnology is the study of controlling and manipulating matter at the nanoscale, which is typically defined as 1 to 100 nm. Nano-biotechnology, or the combination of nanotechnology and biotechnology, is a rapidly emerging field with great potential in environmental research, agriculture, medicine, and diagnostics, among other fields [2]. By utilizing the unique properties of nanomaterials with biotechnology tools, nano-biotechnology opens up new opportunities for disease diagnostics, customized therapy, biosensors, bio-imaging, and other applications [3]. Nanomaterials, such as nanoparticles, nanotubes, and nanocomposites, are widely used in biotechnology because of their small size, high surface area to volume ratio, and unique physicochemical properties [4]. These properties make it possible to precisely interact with biological molecules, cells, and tissues, creating new opportunities in tissue engineering, drug delivery, and diagnostics [5].

Nanomaterials were created by chemical-based reduction, vapor condensation, evaporated form, laser ablation, ball milling, and the sol-gel technique [6]. There are numerous drawbacks to the traditional approaches. The green synthesis process uses stabilizing and reducing agents such as enzymes, plant extracts, and algae to create nanoparticles with specific composition and surface properties [7]. The subject of organic synthesis for nanoparticles has attracted a lot of attention lately because of its affordability, versatility, and eco-friendliness. As reduction and stabilization agents, hazardous chemicals and solvents are used in the physical and chemical processes of conventional Ag NP production, which poses a health risk to humans and the environment [8]. Conventional methods provide lower nanomaterial yields, making them unsuitable for large-scale manufacture. To adjust the chemical, biological, and physical characteristics of silver nanoparticles, various synthesis methods are used. Different compounds of flowers, plants, seeds, algae, microorganisms, etc. can be used in the process of organic synthesis of nanomaterials as capping, stabilization, and reducing agents [9]. Many biogenic chemical compounds are excellent reducing and capping agents for silver nanoparticle formation. In addition, compared to both plants and microorganisms, chemical compounds are fairly simple for the synthesis of NPs [10].

The search for new natural therapeutics has focused more attention on a bioactive compound called carthamidin, which is present within Carthamus tinctorius L. Scientific research has focused on carthamidin, a flavonoid molecule found in large quantities in safflower petals, because of its possible health-promoting qualities. Research indicates that carthamidin possesses noteworthy antioxidant properties, potentially accounting for its capacity to alleviate inflammation and oxidative stress inside the organism [11]. This substance has the potential for several health benefits, including cardiovascular protection as well as anti-cancer activities; nevertheless, more research is required to completely understand its mechanisms of action and medicinal potential [12]. As more information about carthamidin's bioactivity is gathered, it looks like a strong contender for investigations into the drug's potential for use in treating or preventing human health issues. Carthamidin has been used for hundreds of years in traditional medicine and is a viable choice for many pharmacological uses.Variety of literature reports suggested the use of natural phytocompound Quercetin, Chrysin, Curcumin, Gallic acid as a reducing agent for the synthesis of metallic nanoparticles [13–16]. The current study aimed to synthesize CTAgNPs with enhanced anticancer activity by using the bioactive flavanoid compound carthamidin. The effective utilization of carthamidinas a bio-resource for synthesizing silver nanoparticles is what makes this study innovative.

Reagents and Chemicals

Carthamidin was friendly gifted from a Natural Chemicals and Dyeing Industry, Kerala India and 99.8% pure silver nitrate (AgNO₃) were purchased from Sigma-Aldrich Chemicals. MTT, LDH, and acridine orange/ethidium bromide stain were purchased from Hi-Media. For this current research, all further chemicals were of analytical grade.

Synthesis of AgNPs

The Carthamidin is a yellow colored soluble pigment dissolved in water (Fig. 1). AgNO₃ was used as a precursor in the synthesis of AgNPs, and a 0.1-molar silver nitrate solution was prepared using 100 milliliters of de-ionized water. Ag NPs were created by mixing the carthamidin mixture and the solution for the precursor in a 1:1 ratio. The downpour turned out to be brown. Next, it was centrifuged at 10,000 rpm for 10 minutes. The pellet has been retrieved and again cleaned with double-distilled water to get rid of impurities. After that, it was dried at 80°C in a hot-air oven. The dried powder was stored in a container that was sterilized so that further characterization and investigation could be carried out.

Characterization

Using a UV–visible spectrophotometer, the bio-reduction of metallic ions in solution was determined. X-ray diffraction (XRD) investigation was performed to ascertain the size and crystal structure of Ag NPs. The gathered data were assessed using ORIGIN software (version 9.0). The morphology and elemental content of the Ag NPs were investigated and analyzed using SEM and TEM with energy-dispersive X-ray spectroscopy, SEM, TEM, EDAX) [17].AgNP formation was aided by the reducing and stabilizing effects of the biochemicals in the carthamidin aqueous solution. Carthamidin's aqueous solution's biological ingredients serve as a biological reductant, reducing silver ions to their zerovalent state.

Invitro Anticancer Activities of AgNPs

Cell Culture

MCF-7. The human breast cancer cell line MCF-7 was maintained in DMEM using L-glutamine and 1000 mg/L glucose (DMEM), supplemented with 10% FBS, streptomycin sulfate (0.1 mg/mL), and penicillin G (100 units/mL), in a humidified environment with 5% CO₂ at 37°C. The cell line has receptors for estrogen, progesterone, and glucocorticoids [18].

MTT assay

For assessing MTT, a colorimetric assay, AgNPs were added to 96-well plates, and the yellow compound tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Hi-Media) was incubated immediately. The plate had been incubated for 24 and 48 hours at 37°C in a humidified environment with 5% CO₂. Following a PBS wash, 100 µL of MTT (5 mg/mL) reagent was added to the cells, and they were incubated for four hours at 37°C. After removing the MTT dye, 100 µL of DMSO was applied to the culture. At 576 nm, the optical density of the sample was then measured. The impact of AgNPs on MCF-7 cells and their viability percentage was ascertained by contrasting the outcomes with the control group.

AO/EB staining

The use of the AO/EB double labeling approach allowed researchers to investigate how AgNPs induced apoptosis in MCF-7 cells. The MCF-7 cells that were cultivated were propagated (1 × 105 cells/well) into a 12-well chamber plate and kept at 37°C for 12 hours in an atmosphere that was humidified with 5% CO₂. Following treatment with the appropriate AgNPs IC50 values, the seeding cells were cultured for 24 hours at 37°C. Ultimately, 50 µL of the dye combination (AO-EB) was given to the cells, and any apoptotic cell death was detected using a fluorescent microscope [19].

ROS assay

In the cytotoxic mechanism of metallic nanoparticles, the formation of ROS is recognized as a crucial death process. Cell cycle arrest, apoptosis/necrosis, DNA damage, and suppression of cell proliferation are all brought on by this oxidative cell damage. Following cell internalization, cellular esterases convert the non-fluorogenic dye 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) to its derivatives, 2′,7′-dichlorodihydrofluorescein (H₂DCF). ROS and other intracellular oxidants convert H₂DCF to 2′,7′-dichlorofluorescein (DCF). When DCF is excited at 490 nm, it increases in luminescence at 530 nm, indicating that it has accumulated in the cell. In 6-well plates, MCF-7 cells were planted at a density of 3.0 × 105 cells/well in growth media. Following a 24-hour incubation period, cells were subjected to a 50 µM dose of each AgNP sample. Insteadof an AgNP sample, deionized water was used as the control. The cells were then cultivated in an environment with 5% CO₂ at 37°C. Following a 24-hour incubation period, trypsinization was used to gather each cell. Three rounds of washings in Dulbecco's saline solution with phosphate buffer (pH 7.4) were performed on the cell pellet. Following the addition of the H₂DCFDA reagent (2µM, 1 mL) into every cell pellet, a further hour of dark, room-temperature incubation was carried out. We used Dulbecco's saline solution with phosphate buffered (pH 7.4) to wash the cell pellet. The fluorescence level (FL1-H) of each cell pellet was then measured using a flow cytometer for analysis. Ten thousand gate interactions were measured for every analysis. CellQuest software was used to examine the results [20].

LDH Assay

Using the previously described LDH leakage test, the cytotoxicity of AgNPs was investigated. In 96-well plates, MCF 7 cells were planted at a density of 1 × 10⁴ cells/well and allowed to develop for a full day. AgNPs were added to cells by replacing culture media with 200 L of dilute mineral oil that had been mixed with AgNPs and then incubated for a full day. To remove AgNPs and cell debris for the LDH leakage assay, 1.5 mL microtubes containing the cell culture medium were centrifuged at 16,000 g for 20 minutes. In the wells of 96-well plates, a 200-L aliquot of working reagent containing 2.5 mM sodium pyruvate and 0.2 mM NADH was combined with 20 microliters of supernatant. Using a SpectraMax M3 microplate reader, the absorbance at 340 nm was measured for three minutes. The slope of decreasing absorbance was used to compute the relative LDH activity. The cells treated with 1% Triton X-100 and the cells treated with the untreated medium were considered to have 100% and 0% viability, respectively, based on LDH activity measurements. Every sample had two assays performed on it. AgNP-treated cells' viability was reported as a percentage of untreated cells' viability. Bovine serum albumin (0.01%) was added to the reaction mixture in recombinant LDH tests to enhance LDH stability and disperse AgNPs [21].

Flow Cytometry analysis

The FACS analysis was performed to ascertain the DNA content in the CTAgNPs treated cells. The methodology was performed according to our protocol [22]

Statistical Analysis

The tables and figures display the mean values as well as the standard deviations (±) of each assay, which were analyzed for a minimum of three replicates. A percentage of viability was obtained using the MTT assay, with concentrations expressed with the control viability. The Student's t-test was utilized for statistical analysis of the obtained results, and a p-value of less than 0.05 was deemed significant.

UV–visible spectrophotometer

After diluting the carthamidin solution with silver nitrate, the mixture was allowed to react for 30 minutes at room temperature for the production of CTAgNPs (Fig. 2). Eventually, the combination above witnessed a color change from yellow to a darker brown, signifying the production of CTAgNPs (Fig. 3a). CTAgNPs surface plasmon resonance (SPR) is demonstrated by the UV–Vis absorption peak that occurs at 431 nm (Fig. 3b). To verify the production of CTAgNPs, we compared the UV results with the silver nanoparticles synthesized from Ulva rigida [23]. Similarly, a single peak appears in the UV–Vis spectra of biogenic AgNPs mediated by chitosan between 400 and 480 nm [24]. In previous investigations, the synthesis of silver nanoparticles was confirmed by reporting UV–Vis absorption peaks at 400–450 nm, which were synthesized using palm date fruit extract [25].

XRD analysis

XRD analysis was used to investigate the crystalline structure of cantharidin-mediated Ag NPs, as shown in Fig. 4. The planes (122), (200), (220), and (311) facets of the silver crystal are responsible for the hard peaks at 2 theta = 37.9°, 45.0°, 64.2°, and 78°, as well. (JCPDS Card File No. 003–0921). At a 2θ value of 37.9°, the peak was reached, which matched the structure of the lattice plane at (122). The gained XRD spectrum validates the crystalline nature of the developed Ag NPs. Using Scherrer's equation; the average size of the CTAgNPs formed by carthamidin extracts was ascertained. It was discovered that the produced nanoparticles had average diameters of 52 nm. CTAgNPs were identified as FCC in XRD spectra by the presence of planes of (111), (200), (220), and (311) [26]. In addition to this, a few other distinct peaks were observed; these could be the result of the organic chemicals present in the carthamidin extract.

TEM, SEM WITH EDX

EM and TEM pictures of CTAgNPs made using carthamidin are displayed in the Fig. 5a-e. CTAgNPs with an average size of 59nm were spherical and stabilized by carthamidin. TEM analysis was used to observe the size and form of the produced CTAgNPs. Transmission electron microscopy research revealed that CTAgNPs were extremely crystalline, aggregated, and spherically formed, in line with earlier literature investigations. The size of the CTAgNPs was determined to be 52.26 nm by using a transmission electron microscope micrograph [27].In the earlier study, Ag NPs were synthesized and their elemental and structural compositions were determined using EDX analysis. Moreover, EDX analysis was used to determine the elemental composition; the spectra demonstrate the presence of Ag as a metal constituent element peak. The presence of silver in EDX spectra has been confirmed [28]. The presence of organic molecules formed from carthamidin was confirmed by the observation of additional carbon peaks.

MTT assay

The MTT test has been used to evaluate the cytotoxic properties of the carthamidin on MCF 7 cells at various doses (20–100 µg/mL). The cells treated with CTAgNPs were exclusively observed in the microscopy for the morphological changes. In the Fig. 6-a –f it is noted that CTAgNPs cause membrane damage, the collapse of membrane structure integrity, internal organelle damage and leakage. These indications of structural abnormalities in the MCF 7 cells treated by CTAgNPs might occur due to the phenomenon of apoptosis as reported in various literature [29–33]. Therefore, further molecular studies are extended to annotate the apoptosis induction by CTAgNPs in MCF 7 cells.

The vitality of the cells was assessed after a 24-hour exposure and contrasted with control (Fig. 7). As the concentration of the CTAgNPs increased, the results showed a dosage-dependent decrease in cell viability. Particularly, cell viability held steady at the lowest tested concentration (20 µg/mL), with an average viability of almost 80%. The carthamidin concentration was shown to cause a progressive decrease in cell viability. Cell viability was significantly reduced at the maximum concentration examined (100 µg/mL), suggesting a notable cytotoxic effect. Moreover, at the 24-hour time point, the half-maximal inhibitory concentration (IC50) was determined to be 112 µg/mL. The Carthamidin-mediated silver nanoparticles exhibited stronger anticancer activity than bare Carthamidin. In our previous study, Carthamidin performed superior anticancer activity (results under communication). According to these results; the Carthamidin-mediated CTAgNPs have enhanced anticancer activity due to the nano-sized and coating of Carthamidin silver core.

AO/EB staining

The changes in morphology in MCF-7 cells were seen using the AO/EB fluorescent microscopic staining test. The AO/EB staining method distinguishes between normal and apoptotic cells. After a day, the Fig. 8a, b displays the cells that were left untreated, exposed to CTAgNPs at doses of 112 µg/mL for CTAgNP. The microscopic image demonstrates how the treated cells have changed color (to orange), signifying apoptotic cells, while the control cells remained green following staining. In addition, there is nuclear breakdown shrinkage and blebbing of the membranes in the treated cells [33].With the microscopic images, concluding that the the carthamidin-mediated AgNPs induce apoptosis.

ROS staining

By using ROS staining, the measurement of intracellular reactive oxygen species (ROS) is qualitatively assessed in the treated cells. Notably, the concentration of the CTAgNPs under examination and ROS production were found to have a dose-dependent connection. When compared to both lower and higher concentrations, there was a notable increase in ROS levels at the IC50 value of 112 µg/mL (Fig. 8c -e). Interestingly, our control group showed very little fluorescence intensity, suggesting that under normal physiological environments, very little ROS is produced. This highlights the compound's selectivity in inducing ROS and confirms its function as a strong regulator of oxidative stress pathways. The lack of fluorescent in the control cells emphasizes the validity of our findings and confirms the dependability of our experimental setup. The concentration below which a 50% reduction of cell viability happens is represented by the determined IC₅₀ value of 112 µg/mL, which is closely correlated with the peak ROS generation. The build-up of ROS in a dose-dependent manner is consistent with other research that suggests oxidative stress is a major mechanism behind the cytotoxic effects of several pharmaceutical drugs [34].

Moreover, further research is necessary to clarify the particular molecular pathways by which the chemical induces ROS. Comprehending the subsequent signaling pathways triggered by reactive oxygen species (ROS) production may offer significant perspectives on its possible therapeutic applications and expedite the creation of focused therapies for illnesses associated with oxidative stress [356]. In summary, our ROS staining research reveals that ROS generation is elicited at an IC₅₀ concentration of 112 µg/mL. The compound's ability to specifically induce ROS is demonstrated by the lack of fluorescence in control cells, and the cantharidin-mediated AgNPs treated cells emit green fluorescence indicating the generation of ROS which promises as a therapeutic drug that targets oxidative stress pathways is indicated by the observed dose-response relationship. To understand the molecular mechanisms behind ROS-mediated cytotoxicity and to take advantage of the therapeutic potential of ROS regulation, more investigation is necessary.

LDH assay

The LDH assay aimed to measure the release of lactate dehydrogenase (LDH) from injured cells to assess the effect of the test chemical on cellular membrane integrity [36]. Various concentrations of the CTAgNPs (20–100 µg/mL) were applied to the cells, and after the predetermined incubation time, LDH activity was measured. At every tested concentration, a rise in LDH activity was seen that was concentration-dependent. A marginal increase in LDH activity was observed at the test compound's lowest dose (20 µg/mL) as compared with the control group, suggesting small cellular membrane injury (Fig. 8f). An increasing rise in LDH activity was noted with increasing CTAgNPs concentration, indicating greater cellular membrane permeability and cell injury. Interestingly, a notable increase in LDH activity was seen at the highest quantity tested (100 µg/mL), indicating severe breakdown of the cellular membrane and widespread cell injury. This result supports the MTT assay's dose-dependent cytotoxic effects, suggesting a relationship between the CTAgNPs concentration and its effects on membrane integrity and cellular viability. These findings highlight the test compound's possible cytotoxicity, as demonstrated by the concentration-dependent rise in LDH activity, a sign of damage to cellular membranes [37]. To assess the therapeutic relevance of these findings and to clarify the underlying mechanisms leading to this observed effect, more research is necessary.

Flow cytometry analysis

Metallic nanoparticles regulate the cell cycle checkpoints and inhibit the cycle at various phases. Our recent studies of copper oxide and silver nanoparticles from Beta vulgaris and Polianthes tuberosa proved that nanoparticles arrest the cell cycle due to the induction of apoptosis. [38–39]. In the study, we annotate the response of CTAgNPs in MCF 7 cells by flow cytometry analysis. The method depends on the staining of DNA by propidium iodide to determine the percentage of cells that undergo apoptosis at a concentration of 112 µg/mL of CTAgNPs in 24 h. After the incubation period, the CTAgNPs treated MCF 7 cells were analysed by flow cytometry. As a result, the CTAgNPs treated cells showed later apoptotic cells than control cells. The cell cycle analysis chromatogram is displayed in Fig. 9a-f. As it represented the CTAgNPs treated MCF 7 cells showed an increased accumulation of DNA in GO/G1 phase by 91%; S-6%, G2/M 2.4%. In control cells the DNA content noted was GO/G1 phase by 78%; S-5%, G2/M 15%. This remarkably indicates that treated cells indicate that there is an increase in cell population at GO/G1 phase whereas there is a decrease in the cell population in other phases. Therefore the CTAgNPs arrest the cell cycle at the GO/G1 phase and it’s a process of apoptosis.

The preliminary results of the CTAgNPs cytotoxicity in MCF 7 cells decipher that CTAgNPs cause dose dependent toxicity in the MCF 7 cells. The CTAgNPs induce apoptosis process which is evidently observed by the AO/EB staining method and ROS generation. The generated ROS subsequently cause the mitochondrial membrane damage and release of LDH. In addition the CTAgNPs arrest the cell cycle at GO/G1 phase and induce the apoptosis process of cell death. Overall, it is notably observed that CTAgNPs induce apoptosis mediated cell death in the MCF 7 cells. Thus conclusively it can be further extend into intrinsic and extrinsic studies for developing CTAgNPs as a nano based drug in cancer chemotherapy.

Acknowledgements

The authors thank the Department of Science and Technology for providing FIST facilities (SR/FST/LS-I/2018/187 dt 20.12.2018) in the Department of Biotechnology, KAHE.

Data Availability

Data will be made available on request.

Funding

This project was financially supported by the University Seed Money Grant (KAHE/R-Acad/A1/Seed Money/002), Karpagam Academy of Higher Education, Coimbatore.

Author Contribution

John Joseph– Methodology Selva Kumar TWriting, Muthukumar Krishnan – methodology, Data Curation, Investigation, Rajkuberan Chandrasekaran- Writing – review & editing, Writing – original draft, Visualization, Supervision, Project administration, Methodology, Funding acquisition, Formal analysis, Conceptualization.

Competing Interests

The authors declare no competing interests.

Ethical Approval

Not applicable.

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No competing interests reported.

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Carthamidin-mediated silver nanoparticles synthesis, characterization and anticancer activity-Invitro Approach

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Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Reagents and Chemicals

Synthesis of AgNPs

Characterization

Invitro Anticancer Activities of AgNPs

Cell Culture

MTT assay

AO/EB staining

ROS assay

LDH Assay

Flow Cytometry analysis

Statistical Analysis

RESULTS AND DISCUSSION

UV–visible spectrophotometer

XRD analysis

TEM, SEM WITH EDX

MTT assay

AO/EB staining

ROS staining

LDH assay

Flow cytometry analysis

CONCLUSION

Declarations

References

Additional Declarations

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