cytotoxic effects of zinc nanoparticles synthesized with the hydroalcoholic extract of lavender plant (Lavandula Stoechas) on human breast cancer (MCF7) and human colon cancer (HT29) cell lines

doi:10.21203/rs.3.rs-4811373/v1

Download PDF

Research Article

cytotoxic effects of zinc nanoparticles synthesized with the hydroalcoholic extract of lavender plant (Lavandula Stoechas) on human breast cancer (MCF7) and human colon cancer (HT29) cell lines

https://doi.org/10.21203/rs.3.rs-4811373/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Introduction

Green synthesis is the production of metal nanoparticles (MNPs) with biological agents, including plant extracts, without special physical and chemical solutions and conditions. Due to their unique properties, MNPs are used in various sciences and industries. The study aimed to investigate the cytotoxic effects of zinc nanoparticles (ZnNPs) synthesized by the hydroalcoholic extract of lavender plant against human breast cancer cell lines (MCF7) and human colon cancer cell lines (HT29) with the approach of identifying the ability of these NPs to produce anticancer drugs.

Method and Materials:

ZnNPs are synthesized using the hydroalcoholic extract of the lavender plant. Evaluate and characterize nanoparticles (NPs) by Tyndall effect, UV-Vis, DLS, FT-IR, Zeta-P, Raman spectroscopy, SEM, AFM, and XRD methods. The cytotoxic effects of produced NPs against cancer cell lines MCF7 and HT29 and the healthy cells MCF10a and HGF were measured using the MTT method.

Results:

According to the findings of DLS analysis, the average size of ZnNPs is 40 and 50, respectively, at a concentration of 3 mM. The cytotoxicity of nanoparticles on MCF7 and HT29 cell lines produces the highest effect after 48 hours.

Conclusion:

The lavender plant is a suitable option for the green synthesis of ZnNPs and can be used as a stable source for production MNPs. Also, the synthesized ZnNPs showed cytotoxic effects against the examined cancer cells, while they did not cause toxicity to healthy human cells.

lavender

hydroalcoholic extract

zinc nanoparticles

green synthesis

Nanotechnology examines the properties of materials and compounds in 1 to 100 nanometers. Materials in nano size have many applications in various industries, including medicine [1]. In the past years, synthetic compounds synthesized by physical methods have been the primary approach in industries. Still, due to identifying their toxic effects on human health, the approach to using these artifacts for compounds synthesized by natural methods has been changed [2]. Green Synthesis refers to producing NPs using plants or their metabolites, ultimately reducing the toxicity of produced NPs [2].

Plants are the largest and most widely used source for the green synthesis of NPs due to their diverse and wide range of phytochemicals that act as reducing and stabilizing agents for forming NPs. Various phytochemicals found in plants, such as organic acids, amines, amides, aldehydes, ketones, and flavonoids, are responsible for the reduction of metal ions and the formation of NPs, and various proteins found in metabolites and plant extracts are responsible for the stability of synthesized NPs [3]. Biosynthesis using different species leads to the production of durable NPs with safe performance. In past research, various plant species and microorganisms have succeeded in producing NPs with various abilities [4, 5].

The structure of NPs synthesized by the green method includes shape, size, and morphology depending on the type of metabolism used. Factors such as chemical interactions, pH, and temperature effects impact the final result. According to the type of organism used, NPs are formed intracellularly or extracellularly [6–8].

The green synthesis of NPs resulting from using plant or microbial extracts has various advantages, including compatibility with the environment, simplicity, and low cost of the process. In contrast, the chemical and physical methods of NP synthesis have complex, expensive processes that lead to the production of waste becomes dangerous [9]. Green Synthesis using plant extracts due to the use of secondary metabolites as reducing and masking agents ultimately leads to the formation and stabilization of NPs, while green synthesis using microbial extracts requires aseptic cultivation and production; the commercialization of these NPs is expensive, and the green synthesis of NPs with algae is also not economical due to its time-consuming nature [9]. Different parts of plants, including stems, leaves, roots, flowers, seeds, and metabolites, make NPs [10–12]. One of the advantages of green synthesis of NPs by plants is less energy consumption, no need for high temperature and pressure, production of fewer by-products, low final cost, and the possibility of large-scale production. Synthesized green NPs are not limited in clinical applications due to their high stability and low toxicity compared to non-biological chemical NPs [13].

Lavender plants are one of the flowering plants in the mint family (Lamiaceae). It is a sturdy and full-stemmed plant whose height reaches 80–120 cm. The leaves are small, oval-triangular, with deep comb divisions. This plant has compounds such as diterpenes, cyclic alcohols, flavonoids, organic acids, methylcarnosic acid, and saponins. Among other known important chemical compounds of lavender are cineol, linalool, volatile oils, tannin, essential oil, linalyl acetate, camphor, and geraniol. One of the most important members of the lavender plant family is the genus Lavandula, which has more than 35 species worldwide, spread from Key Verde and the Canary Islands to the Mediterranean region, India, and Southwest Asia. Lavandula stoechas essential oil and extract have many biological activities and medicinal properties, including antispasmodic, sedative, anti-inflammatory, antioxidant, antimicrobial, antifungal, insecticidal, and larvicidal activities [14].

Metal zinc is an essential trace element found in muscle, bone, skin, and dental tissues, as well as in the hard part. It plays a vital role in numerous metabolic processes, from protein synthesis to immunity building to gene expression. As a structural and regulatory component of more than 300 enzymes in the body and more than 2000 transcription factors, zinc has antioxidant properties that, in addition to physiological functions, can help reduce the damage caused by ultraviolet rays and reduce the risk of cancer. Zinc Oxide Nanoparticles (ZnONPs) are white, odorless powders with a molecular weight of 81.38 g/mol. The US Food and Drug Administration (FDA) considers it a generally recognized safe substance (GRAS) [15].

This research aims to investigate the ability of the lavender plant to synthesize ZnNPs, evaluate the structure of the synthesized NPs, and investigate their cytotoxic effects on human breast cancer (MCF7) and human colon cancer (HT29) cell lines with the approach of identifying the ability of these NPs to produce drugs. It has been anticancer.

The plant used

The lavender plant was collected from the pastures and agricultural lands around the city of Qom (Iran) and registered as a reference sample after identification in the herbarium section of the Faculty of Pharmacy of Shahid Beheshti University (Tehran, Iran) with the specific numbers of lavender (SBMU-8303). Also, the name of the identified species of lavender (Lavandula Stoechas) was determined; it is necessary to mention that Prof Kamali Nejad registered the above strains.

Cell lines

Cell lines, human breast cancer cells (MCF7), human colon cancer cells (HT29), normal human gingival fibroblasts (HGF), and healthy breast cells (MCF10a) were obtained from the National Center for Genetic and Biological Resources of Iran (IBRC) has been prepared and used.

Hydroalcoholic extract

Two hundred grams of dried and ground lavender leaves were poured into a sterile Erlenmeyer flask containing one liter of solvent (distilled water-ethanol) with a ratio of 50/50 and heated for ten days at 25⁰C in the dark [16]. Then, the extract was passed through a filter paper, and the resulting solution was placed in an oven at 45⁰C to evaporate the solvent.

Preparation of salt storage solution

Different concentrations of zinc salt were required, and for their preparation, a concentrated solution with a specific purity percentage and standard of one molar was used. To prepare 50 mL, zinc sulfate and zinc nitrate solutions with one molar concentration of 12.05, 12.45, 14.35, 14.85 grams of ZnSO₄.4H₂O and Zn(No₃)2.6H₂O salt powder (Merck, Germany), was mixed with deionized water to a volume of 50 ml. The prepared solutions were filtered with a 0.22-micron filter and stored at 4⁰C.

Synthesis of NPs

Metal powder was prepared in 20 mL of extracts with different concentrations, including (1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 mM) at 25⁰C, pH = 10, and was mixed at 120 rpm for 24 hours.

Isolation and purification of NPs

Separation of NPs was done with an ultracentrifuge device for 10 minutes at 4⁰C and 11000 rpm. To wash the surface of deposited NPs, after dispersing them in deionized distilled water, they were centrifuged under the previous conditions. In total, this process is repeated three times to separate the ZnNPs and thoroughly wash the surfaces of the NPs.

Characterization of NPs

Various tests were used to identify and confirm the synthesized NPs. The Tyndall effect test, which is known to verify the formation of colloidal particles, was used. Ultraviolet-visible spectroscopy (UV-Vis) was used to investigate the synthesis spectrum of NPs. For this analysis, 0.01 grams of NPs were dissolved in ethanol and dispersed well under ultrasonic waves for 30 minutes. Then, their absorption spectrum was measured by the spectrometer of an Optizon 3230 UV device at a wavelength of 200 to 800 nm.

Dynamic Light Diffraction (DLS)

0.001 g of dried powder of NPs was mixed in 5 ml of deionized water for 30 minutes under ultrasonic waves. Then, using a DLS device (Cordouan3 Vasco), the size of homogenized NPs and their size distribution pattern (PDI) were determined. Also, the dispersion index of NPs was determined. DLS test was performed in Shahid Beheshti Faculty of Pharmacy (Tehran, Iran) with a Zeta sizer device (Malvern, England).

Fourier Transform Infrared Spectroscopy (FT-IR)

The dried powder of NPs was used in combination with potassium bromide (KBr) in tablet form. Then, the metal mold carrying the pill inside the Nicolet Avatar spectrometer, USA. It was placed in the FTIR laboratory belonging to Diptronic Laboratory (Tehran, Iran), and the spectrum of the samples was measured in the range of 400–4000 cm^− 1 with a resolution of 4 cm^− 1.

Zeta Potential Analysis (Zeta-P)

The amount of 0.001 g of dried powder of NPs was suspended in 5 ml of deionized water for 30 minutes under ultrasonic waves, and the zeta potential for NPs was determined at room temperature and pH = 7.4 by the device available in the central laboratory zeta compact (CAD, France) Shahid Beheshti Faculty of Pharmacy (Tehran, Iran) was measured.

Raman Spectroscopy

To confirm the synthesis of NPs, Raman spectroscopy was used, and Raman spectra were recorded with Almega Dispersive spectrometer manufactured by USA-Thermo Nicolet company with a 532 nm output laser beam and 100 mW power. This analysis was done in the Dipetronic Laboratory (Tehran, Iran).

Scanning Electron Microscope (SEM)

The morphology of NPs was investigated by SEM electron microscope. To prepare the samples for photographing with an electron microscope, the samples are placed inside the Sonicator device (30 minutes), and then a drop of the colloid of each of the ZnNPs is placed on the copper grids covered with carbon. After drying them at ambient temperature to create an electric current, the surface of the sample is covered with an ultra-thin layer of gold and palladium alloy. After preparing the desired sample, a 15 kW SEM electron microscope belonging to DiPetronic Laboratory (Tehran, Iran) was used to take pictures.

Atomic Force Microscope (AFM)

To determine the morphology of ZnNPs, photographs were taken with an AFM microscope. To prepare the samples, after placing each sample in the sonicator (30 minutes), a drop of colloid of ZnNPs was placed on the silicon nitride tip, and it was placed near the ambient temperature until drying. Photographs were taken in the field using an AFM electron microscope belonging to DiPtronic Laboratory (Tehran, Iran) with non-contact mode.

Energy Dispersive Spectroscopy (XRD)

To determine the elemental composition of the produced colloidal sample, a scanning electron microscope coupled with a MIRA 3 coupled electron microscope belonging to DiPtronic Laboratory (Tehran, Iran) was used. The sample preparation method is the same as the SEM sample.

Cell Culture

Human colon cancer (HT 29) and breast cancer (MCF7) cell lines, healthy gum fibroblast (HGF) cell lines, and healthy breast cells (MCF10a) were used in this study. These cells were cultured respectively in Dulbecco's Modified Eagle's Medium DMEM - Ham's F12 and DMEM high glucose containing 10% fetal bovine serum, penicillin (100 units/ml) and streptomycin (100 mg/ml). Then, they were placed in an incubator containing 5% CO₂ and 95% humidity at 37°C. Flasks were examined daily by an inverted microscope in terms of cell growth and density, morphology, and contamination control. The complete culture medium containing 10% FBS was changed at the required time.

MTT assay

Investigation of the cytotoxicity effect of ZnNPs (A2) and its control (C2) by colorimetric method, using the MTT staining 3-[4,5-dimethylthiazol-2-yl]− 2,5-diphenyl tetrazolium bromide) water-soluble salt.

This method is based on the activity of succinate dehydrogenase enzyme in the mitochondria of living cells and purple insoluble formazan crystals. This compound is soluble in DMSO. Since dead cells cannot convert MTT into formazan, the level of formazan created is proportional to the number of living cells. To experiment, HT 29, MCF7, MCF10a, and HGF cells were planted in several 5000 cells in each well of a 96-well plate. Forty-eight hours after seeding the cells, the old culture medium was removed from the wells, and the cells were treated with different concentrations (50, 100, 150, 250, and 300 µg/mL) of NPs and chemotherapy drug cisplatin (1.25, 5 2, 5, 10, 20, 40 µg/mL) were treated. Four wells were considered for Hertimar, and four wells were assigned as controls for the whole culture medium in each experiment. The plates were incubated in an incubator containing 5% CO2, 95% humidity, and 37°C for 24, 48 and 72 hours. After the incubation time, the supernatant of each well was carefully removed and replaced with a complete culture medium containing MTT solution and placed in the incubator for 4 hours; then, the supernatant of each well was drained and 200 microliters of DMSO solution was replaced with the previous solution to dissolve the formazan crystals. After 15 minutes, the optical density was read using an ELISA device (USA, Bio Tek, Synergy/HTX) at a wavelength of 570 nm. The amount of absorption is directly proportional to the number of living cells. The results were calculated in terms of the percentage of live cells compared to the control from the following equation:

100 x (average absorption of control cells/average absorption of treated cells) = percentage of living cells

Statistical Analysis

Data analysis was done by Excel and GraphPad Prism 8.2.1 software. The drug concentration required to inhibit cell growth by 50% (IC50) was calculated and plotted by PRISM software and by non-linear regression analysis using dose-response curves.

To determine the best time and conditions for the synthesis of ZnNPs, after confirming the initial results related to the color change and the Tyndall effect, as well as the DLS test, several analytical tests were performed, and the data obtained related to the synthesis of ZnNPs by the hydroalcoholic extract of the lavender plant. It shows that the best concentration of synthesized NPs was determined to be 3 mM.

Amount of plant extract and the production of ZnNPs

To achieve optimal NPs in terms of diameter and particle size distribution, the optimal synthesis time for producing ZnNPs is determined. Determining this time is related to the amount of proteins produced and phenolic compounds in the plant and will play an influential role in the production of NPs.

The color change due to the Tyndall effect indicates the formation of colloidal particles and the synthesis of ZnNPs (Figure 1).

UV-Vis

UV-Vis spectrometer images of ZnNPs synthesized by the lavender plant, the obtained absorption curve shows the synthesis of ZnNPs.

The synthesis of ZnNPs is carried out in the UV-Vis spectroscopic spectrum at a wavelength of 300 to 400 nm, and the obtained curve shows the formation of NPs in the above range; it can be stated that ZnNPs (Figure 2) have been synthesized.

Evaluation of the production of ZnNPs

DLS

The hydrodynamic diameter size of synthesized NPs at pH=10 and with a final concentration of 3 mM of zinc salt in lavender plant extract was recorded as 50 nm (Figure 3). Also, the drawn peak shape shows the uniform dispersion of produced NPs. In addition, the dispersion index of NPs produced by plant extract was calculated as 0.308, and their Zeta-P was calculated as 1.72 mV.

FT-IR

Based on the analysis of the results obtained from the infrared Fourier transform spectroscopy, the binding of different functional groups to the surface of NPs was proved (Figure 4). Although this type of spectrometry cannot provide a clear and precise structure of the conjugated compounds of ZnNPs, it can reveal the kinds of functional groups present in the sample.

Image analysis shows that Peaks 3424.92 and 2921.38 are respectively associated with O–H bonds and alcohol group, peaks 1580.19 are associated with N–H bonds or C=O of the amino group, peak 1407.73 are associated with C–H bonds and alkane group, and finally, peak 1026.63 are associated with C–N bonds. Like the rest of the peaks, the amino group indicates the presence of intermediary groups in the NPs composition. The presence of alcohol, amine, and alkane groups in the analysis confirmed the synthesis of NPs; also, the above links are used as practical tools in the synthesis of NPs to improve the stability, active compounds, and physicochemical properties of NPs and the existence of these groups. The test results show the quality and stability of the synthesized NPs.

Zeta potential (Zeta-P)

Zeta potential analysis is used to check the stability of particles (Figure 5). In zeta potential analysis, particles with zeta potential more positive than +30 mV or negative than -30 mV are usually considered stable. However, if the particle density is greater than the dispersant, even though they are dispersed, they will eventually settle and form a closed bed (i.e., a hard cake).

The data obtained from examining the zeta potential of ZnNPs synthesis showed that this particle has relative stability with a zeta potential of -27 mV.

Investigating the synthesis of ZnNPs using Raman spectroscopy

Raman spectroscopy determines the material's structure by using light radiation on the material, which is one of the most critical applications of this measurement to check the size of particles and the crystallization of NPs. The Raman spectrum describes matter using light scattering. The Raman spectrum of the ZnNPs (Figure 6) shows the size of the formed NPs, and in Table 1, the specifications of the ZnNPs, including the wavelength and size of the synthesized NPs, are given in the range of nanometers. According to the obtained graph of the Raman spectrum of ZnNPs synthesis, it is impossible to say in what range the largest amount of NPs is formed, but it can be said that the particles are formed in the range of 3 to 25 nm.

Table 1. Interpretation of the Raman graph of the synthesized ZnNPs

NPs size	wavelength
3.90	2113.7961
8.72	2559.0518
7.61	2413.1362
18.30	2600.7688
23.69	2774.0364
20.58	2678.0928

SEM and AFM

The SEM images of the produced ZnNPs show that the synthesis NPs were spherical, homogeneous, and of the same size (Figure 7). Also, particles can accumulate in some areas; however, the particles in the accumulated areas are separate, and the border between them is apparent. The presence of these boundaries in the range of particle accumulation indicates the favorable stability of NPs synthesized with masking agents. In addition, the AFM images also confirmed the SEM image and reported the size of the produced NPs below 100 nm (Figure 8).

In Figure 7, the formed NPs can be seen in different sizes between 10 and 50 nm, which proves that ZnNPs were synthesized by the lavender plant in the nanometer range.

AFM images related to the synthesis of ZnNPs (Figures 8-10) show the formation of NPs in the nanometer range in the lavender plant.

Based on the obtained results, it can be seen that the size of NPs calculated in SEM photography and dynamic light diffraction analysis (DLS) were not equal. The particles in the solution have a surface charge; in any case, they cover the surrounding particles. In addition, ions with the opposite charge can be seen on the surface of the particles. Therefore, the particle has two layers: one on the surface and the other around it. These outer and inner layers move along with the particle when the particle moves in the solution. Therefore, a hypothetical distance between the particle and the fluid medium can be imagined. This distance is called hydrodynamic distance, and it is known as zeta potential. Therefore, according to the zeta potential of the produced ZnNPs, it can be concluded that the electrostatic agent alone could not ensure the stability of the NPs. However, apart from repulsive and attractive forces, other effects are also involved in the stability of NPs. In this regard, we can mention the effect of steric hindrance 1 as one of the most important factors affecting the stability of NPs. This agent is formed by the absorption of polymer materials on the surface of NPs. The mentioned agent formed on the particle acts like a resistant barrier and prevents the particles from coming together and causing coagulation. Unlike electrostatic stability, which is seen in the range of zeta potential greater than +30 and less than -30, there is not much repulsive force in this case. When molecules come into contact, the particles are exposed to the attraction force. This is even though the electrostatic repulsion force typically repels them from each other.

Therefore, according to the observations, a combined state resulting from electrostatic repulsion and steric hindrance has been created in the produced ZnNPs. Hence, the difference in the average size of the particles reported in the SEM images compared to the zeta size of each plant extract was related to the difference caused by the hydrodynamic diameter of the NPs. The actual diameter of the NPs has been displayed in the presence of the dynamic diameter. Based on this figure, electron imaging has calculated the actual diameter of NPs, and dynamic light diffraction analysis has reported the diameter of NPs along with the bio-stabilizing factors attached to NPs.

XRD

The spectrum of energy distribution of synthesized ZnNPs is also shown in SEM and AFM images (Figures 7-10). Also, this figure shows the peak related to the Zinc element.

ZnNPs, due to their surface plasmon resonance properties, have a distinct optical absorption peak in the range of 2.2 keV, and the formation of a peak in this range indicates the formation of NPs. The peaks formed in Figure 11 indicate the formation of ZnNPs.

Cytotoxicity of ZnNPs

The cytotoxic effect of NPs (A2) on the proliferation of HT 29, MCF7, MCF10a, and HGF cells was evaluated using the MTT method. These cells were treated with six different concentrations of NPs (50, 100, 150, 250, and 300 µg/ml) for 24, 48 and 72 hours.

Considering that in 24 hours, the drugs did not have enough time to affect the cells, and in 72 hours, due to the consumption of the culture medium by the cells, the obtained data could not be relied upon, here Only the results of the test are reported within 48 hours.

As shown in Figure 12, A2 inhibits the growth of different cells in a concentration-dependent and time-dependent manner. By examining the results of the MTT test, it was observed that with the increase in the concentration of NPs and with the rise in the treatment time, the survival percentage of cancer cells has decreased significantly compared to the control. IC₅₀ values calculated from dose-response curves in different cell lines are shown in Figures 12-15.

In Table 2, the IC₅₀ value in the examined cell lines during the use of A2, C2, and Cisplatin after 48 hours is given in order.

Table 2. IC₅₀ value in the examined cell lines during the use of A2, C2, and Cisplatin after 48 hours is given in order

IC 50 (µg/ml)	Cell line
140.2	A2	HGF
155.2	C2
76.38	Cisplatin
65.42	A2	HT29
94.68	C2
155.2	Cisplatin
54.65	A2	MCF 7
168.9	C2
65.19	Cisplatin
100.1	A2	MCF 10a
132.8	C2
55.48	Cisplatin

By examining the results of the MTT test, it was observed that the effect of A2 NPs on normal cells is much lower than on cancer cells. A2 NPs had significant cytotoxic effects in most tested concentrations in human breast cancer (MCF7) and human colon cancer (HT29) cells and inhibited the growth of cancer cells. Normal human gingival fibroblasts (HGF) and healthy breast cells (MCF10a) showed more resistance to NPs than human breast and colon cancer cells. As a result, the above NPs can find many biomedical applications with therapeutic importance to deal with breast and colon cancer.

In this research, the proof of the ability to produce ZnNPs by the hydroalcoholic extract of the lavender medicinal plant was investigated to find a new way to improve the characteristics of the synthesized NPs in terms of particle size and distribution.

In the first step, after the preparation of hydroalcoholic extract and synthesis of NPs, the effect of time on the amount and size of NPs production was evaluated, and it was shown that the synthesis of NPs depends on time and other conditions. In other words, this is related to the higher amount of secreted proteins and phenolic compounds in the obtained extract, and based on macroscopic observations, out of the five time periods, the final period considered for the synthesis of ZnNPs is more favorable. Is. As a result, based on the macroscopic and microscopic results, at the end of the synthesis process and with equal volume ratios of the extract to the metal salt solution, they could produce ZnNPs. Based on the results, the pH value influences the size and distribution of the particles. Also, increasing or decreasing the concentration of zinc metal salts has changed the size of NPs. These observations are related to the ratio of metal salt concentration to regenerating and stabilizing proteins in the supernatant. In addition, the plant extract showed the highest color intensity at pH = 10. In other words, it can be said that the mentioned pH provides more reducing agents to metal ions to produce more ZnNPs. Also, the performance of the functional groups in proteins has been improved at the mentioned pH for the stability of NPs.

Examining the results of this research shows that the average diameter of ZnNPs produced in lavender extract at a temperature of 25ºC, a pH = 10, a final concentration of 3 mM, and a speed of 120 rpm was equal to 27.62 nm. The zeta potential of NPs has been reported as -27 mV.

In this study, the formation of NPs was measured using the Tyndall effect, UV-Vis, DLS, FTIR, Zeta-P, Raman spectroscopy, SEM, AFM, and XRD methods, and all the mentioned measurements confirmed the synthesis of ZnNPs. they did

Also, this research showed that the proteins released by the lavender plant extract could regenerate zinc ions by the extracellular method. In addition, considering the lack of reports that the lavender plant is pathogenic, this plant can be regarded as a suitable option for entering the industry to produce MNPs.

In this research, the cytotoxic effect of ZnNPs on the proliferation of human breast cancer cells (MCF7), human colon cancer cells (HT29), normal human gingival fibroblasts (HGF), and healthy breast cells (MCF10a) was evaluated using the MTT method. Took These cells were treated with six different concentrations of NPs (50, 100, 150, 250, and 300 µg/ml) for 24, 48 and 72 hours. The data of the investigation of the cytotoxic effect of the present study show that the synthesized ZnNPs had significant cytotoxic effects in most of the tested concentrations in human breast cancer cells (MCF7) and human colon cancer cells (HT29) and inhibited the growth of cancer cells. As a result, the above NPs can find many biomedical applications with therapeutic importance to deal with breast and colon cancer.

In 2019, Fakhari et al. conducted a study to produce ZnONPs by green synthesis method using the aqueous extract of the plant (Laurus nobilis L) and with two salts (zinc acetate and zinc nitrate) as precursors. Synthesized NPs were characterized by UV-Vis, FT-IR, XRD, EDX, and SEM analyses, and the results showed a hexagonal wurtzite structure; SEM analyses showed a spherical shape with a medium size (21.49 and 25.26 nm) for the synthesized NPs. This is demonstrated by zinc acetate and zinc nitrate, respectively. EDX analysis confirmed the high purity of the synthesized NPs [17]. Patrón-Romero et al. 2020 produced ZnONPs using green synthesis, and after characterizing the NPs with different analyses, they investigated the cytotoxic effects of NPs on two cell lines, which did not have toxic effects [18]. Salim et al. 2020 synthesized ZnONPs nanoparticles using Deverra tortuosa extract. The anticancer activity of the synthesized NPs in vitro against two cancer cell lines (human colon adenocarcinoma "Caco-2" and human lung adenocarcinoma "A549") was compared with their activity on human lung fibroblast cell line (WI38) using MTT method was investigated and the synthesized NPs showed significant selective cytotoxicity against the two investigated cancer cell lines [19]. Umamaheswari et al. 2021 Investigating the ability of green synthesis of ZnONPs with the extract of Raphanus sativus var. Longipinnatus did. The results showed that the synthesized ZnONPs have a cytotoxic effect on the lung cancer cell line (A549) [20].

In past research, ZnNPs were synthesized using the green method and characterized by various spectroscopic techniques. Then, the properties created by the synthesized NPs, such as the different properties and cytotoxic effects, were measured. In the current research, all the above steps have been carried out, and the NPs produced by the green synthesis method, in addition to having all the characteristics of NPs, have strong cytotoxic effects.

According to the findings obtained in the current research from the biosynthesis of ZnNPs by the lavender plant, it can be concluded that the green synthesis created from the obtained data can create cytotoxic effects against the examined cancer cell lines. Which can be used in treating diseases or in combination with new drugs by continuing to do more research in the future.

Ethical Approval

Human and animal samples were not used in this research.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Funding

This work was funded by a grant (grant no: IR.MUQ.REC.1401.200) from the Vice for Research and Technology of Qom University of Medical Sciences, Qom, Iran.

Author Contribution

Aida Mehdipour, Project administration, Funding acquisitionMahdieh Ameri Shah Reza, Data curation, Formal Analysis, Conceptualization, Investigation, Project administration, Resources, Software, Supervision, Visualization, Writing – original draft, Writing – review & editingMohammad Hossein Jafari Baravati, Investigation, MethodologyAlireza Rasouli, Writing – original draft, Writing – review & editingGholam Ali Jafari, MethodologyFatemeh Heidari, Methodology

Acknowledgement

The authors would like to thank all staff in the Labs where the investigations were done. Indeed, the authors’ special gratitude goes to the laboratory staff at the Department of Biotechnology and Medicinal Chemistry especially all staff in the Fermentation Lab at Shahid Beheshti University of Medical Sciences, and Cellular and Molecular Research Center, and the Vice for Research and Technology of Qom University of Medical Sciences, Qom, Iran, for their technical assistance and support during this study. The author would like to acknowledge Prof Kamali Nejad and Mrs. Leili Astaraki in the School of Pharmacy, Shahid Beheshti for their helpful suggestions, and guidance.

Data Availability

All data are given in the article.

Rafique, M., et al., A review on green synthesis of silver nanoparticles and their applications. Artificial cells, nanomedicine, and biotechnology, 2017. 45(7): p. 1272-1291.
Gour, A. and N.K. Jain, Advances in green synthesis of nanoparticles. Artificial cells, nanomedicine, and biotechnology, 2019. 47(1): p. 844-851.
Kavitha, A., et al., A mini review on plant-mediated zinc oxide nanoparticles and their antibacterial potency. Biocatalysis and Agricultural Biotechnology, 2023. 48: p. 102654.
Alsharif, S.M., et al., Multifunctional properties of spherical silver nanoparticles fabricated by different microbial taxa. Heliyon, 2020. 6(5).
Salem, S.S. and A. Fouda, Green synthesis of metallic nanoparticles and their prospective biotechnological applications: an overview. Biological trace element research, 2021. 199(1): p. 344-370.
Saied, E., et al., The catalytic activity of biosynthesized magnesium oxide nanoparticles (MgO-NPs) for inhibiting the growth of pathogenic microbes, tanning effluent treatment, and chromium ion removal. Catalysts, 2021. 11(7): p. 821.
Salem, S.S., Baker’s yeast-mediated silver nanoparticles: Characterisation and antimicrobial biogenic tool for suppressing pathogenic microbes. BioNanoScience, 2022. 12(4): p. 1220-1229.
Suba, S., et al., Microbial mediated synthesis of ZnO nanoparticles derived from Lactobacillus spp: Characterizations, antimicrobial and biocompatibility efficiencies. Sensors International, 2021. 2: p. 100104.
Javed, R., et al., Role of capping agents in the application of nanoparticles in biomedicine and environmental remediation: recent trends and future prospects. Journal of Nanobiotechnology, 2020. 18: p. 1-15.
Abdelghany, T.M., et al., Phytofabrication of zinc oxide nanoparticles with advanced characterization and its antioxidant, anticancer, and antimicrobial activity against pathogenic microorganisms. Biomass Conversion and Biorefinery, 2023. 13(1): p. 417-430.
Al-Zahrani, F.A., et al., Green synthesis and antibacterial activity of Ag/Fe2O3 nanocomposite using Buddleja lindleyana extract. Bioengineering, 2022. 9(9): p. 452.
Aref, M.S. and S.S. Salem, Bio-callus synthesis of silver nanoparticles, characterization, and antibacterial activities via Cinnamomum camphora callus culture. Biocatalysis and Agricultural Biotechnology, 2020. 27: p. 101689.
Vijayaraghavan, K. and T. Ashokkumar, Plant-mediated biosynthesis of metallic nanoparticles: A review of literature, factors affecting synthesis, characterization techniques and applications. Journal of environmental chemical engineering, 2017. 5(5): p. 4866-4883.
Ez zoubi, Y., D. Bousta, and A. Farah, A Phytopharmacological review of a Mediterranean plant: Lavandula stoechas L. Clinical Phytoscience, 2020. 6: p. 1-9.
Yagoub, A.E.A., et al., Antimicrobial properties of zinc oxide nanoparticles synthesized from lavandula pubescens shoot methanol extract. Applied Sciences, 2022. 12(22): p. 11613.
Rashan, L.J., et al., Characterization of the anticancer properties of monoglycosidic cardenolides isolated from Nerium oleander and Streptocaulon tomentosum. Journal of ethnopharmacology, 2011. 134(3): p. 781-788.
Fakhari, S., M. Jamzad, and H. Kabiri Fard, Green synthesis of zinc oxide nanoparticles: a comparison. Green chemistry letters and reviews, 2019. 12(1): p. 19-24.
Patrón-Romero, L., et al., Synthesis, characterization and cytotoxicity of zinc oxide nanoparticles by green synthesis method. Journal of Drug Delivery Science and Technology, 2020. 60: p. 101925.
Selim, Y.A., et al., Green synthesis of zinc oxide nanoparticles using aqueous extract of Deverra tortuosa and their cytotoxic activities. Scientific reports, 2020. 10(1): p. 3445.
Umamaheswari, A., et al., Green synthesis of zinc oxide nanoparticles using leaf extracts of Raphanus sativus var. Longipinnatus and evaluation of their anticancer property in A549 cell lines. Biotechnology Reports, 2021. 29: p. e00595.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

cytotoxic effects of zinc nanoparticles synthesized with the hydroalcoholic extract of lavender plant (Lavandula Stoechas) on human breast cancer (MCF7) and human colon cancer (HT29) cell lines

Status:

Version 1

Abstract

Introduction

Method and Materials:

Results:

Conclusion:

Figures

Introduction

Method and Materials

The plant used

Cell lines

Hydroalcoholic extract

Preparation of salt storage solution

Synthesis of NPs

Isolation and purification of NPs

Characterization of NPs

Dynamic Light Diffraction (DLS)

Fourier Transform Infrared Spectroscopy (FT-IR)

Zeta Potential Analysis (Zeta-P)

Raman Spectroscopy

Scanning Electron Microscope (SEM)

Atomic Force Microscope (AFM)

Energy Dispersive Spectroscopy (XRD)

Cell Culture

MTT assay

Statistical Analysis

Results

Discussion

Conclusion

Declarations

Ethical Approval

Funding

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Status:

Version 1