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Article

Biosynthesis of Zinc Oxide Nanoparticles Using Seaweed: Exploring Their Therapeutic Potentials

by
Sohaila I. Abotaleb
1,
Saly F. Gheda
2,
Nanis G. Allam
2,
Einas H. El-Shatoury
1,
João Cotas
3,
Leonel Pereira
3,* and
Ali M. Saeed
1
1
Microbiology Department, Faculty of Science, Ain Shams University, Cairo 11566, Egypt
2
Botany Department, Faculty of Science, Tanta University, Tanta 31527, Egypt
3
CFE—Centre for Functional Ecology, Science for People & Planet Marine Resources, Conservation and Technology—Marine Algae Lab, Department of Life Sciences, University of Coimbra, Calçada Martim de Freitas, 3000-456 Coimbra, Portugal
*
Author to whom correspondence should be addressed.
Appl. Sci. 2024, 14(16), 7069; https://doi.org/10.3390/app14167069
Submission received: 14 June 2024 / Revised: 20 July 2024 / Accepted: 25 July 2024 / Published: 12 August 2024
(This article belongs to the Section Applied Biosciences and Bioengineering)
Browse Figures
Figure 1
<p>Used seaweeds: <span class="html-italic">Ulva lactuca</span> (<b>A</b>), <span class="html-italic">Ulva intestinalis</span> (<b>B</b>) (Chlorophyta), and <span class="html-italic">Sargassum muticum</span> (<b>C</b>) (Phaeophyceae).</p> "> Figure 2
<p>UV spectra of ZnO nanoparticles synthesized using different seaweeds: <span class="html-italic">Ulva lactuca</span> (<b>A</b>), <span class="html-italic">Ulva intestinalis</span> (<b>B</b>), and <span class="html-italic">Sargassum muticum</span> (<b>C</b>).</p> "> Figure 3
<p>FTIR analysis of ZnO nanoparticles of the different seaweeds: <span class="html-italic">Ulva lactuca</span> (<b>A</b>), <span class="html-italic">Ulva intestinalis</span> (<b>B</b>), and <span class="html-italic">Sargassum muticum</span> (<b>C</b>).</p> "> Figure 4
<p>XRD analysis of ZnO nanoparticles prepared from tested seaweeds: <span class="html-italic">Ulva lactuca</span> (<b>A</b>), <span class="html-italic">Ulva intestinalis</span> (<b>B</b>), and <span class="html-italic">Sargassum muticum</span> (<b>C</b>).</p> "> Figure 5
<p>Transmission electron micrographs of ZnO-NPs biosynthesized using different seaweeds: <span class="html-italic">Ulva lactuca</span> (<b>A</b>), <span class="html-italic">Ulva intestinalis</span> (<b>B</b>), and <span class="html-italic">Sargassum muticum</span> (<b>C</b>).</p> "> Figure 6
<p>Zeta potential analysis of ZnO-NPs biosynthesized using different seaweeds: <span class="html-italic">Ulva lactuca</span> (<b>A</b>), <span class="html-italic">Ulva intestinalis</span> (<b>B</b>), and <span class="html-italic">Sargassum muticum</span> (<b>C</b>).</p> "> Figure 7
<p>Effects of <span class="html-italic">Ulva lactuca</span> ZnO-NP treatment on tumor volume.</p> "> Figure 8
<p>Effects of <span class="html-italic">Ulva lactuca</span> ZnO-NP treatment on tumor weight.</p> "> Figure 9
<p>Effects of <span class="html-italic">Ulva lactuca</span> ZnO-NP treatment on oxidative stress.</p> "> Figure 10
<p>Microscopic pictures of H&amp;E-stained, untreated Ehrlich ascites tumors (EAC) and the treated groups. (<b>A</b>) Ehrlich acetic carcinoma, (<b>B</b>) doxorubicin, (<b>C</b>) ZnO-NPs biosynthesized using <span class="html-italic">Ulva lactuca</span> extract. Low magnification 100×: bar 100; higher magnification 400×: bar 50.</p> ">
Versions Notes

Abstract

:
This study aimed to biosynthesize zinc oxide nanoparticles (ZnO-NPs) using extracts from various seaweeds, including Ulva lactuca, Ulva intestinalis (Chlorophyta), and Sargassum muticum (Phaeophyceae). The biosynthesized ZnO-NPs were characterized using UV spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, transmission electron microscopy, and zeta potential analysis. Their antimicrobial activity was assessed using the disk diffusion method, revealing significant efficacy against two bacterial species (Klebsiella pneumoniae and Escherichia coli) and two fungal species (Candida albicans and Aspergillus niger). Additionally, the antioxidant potential of the ZnO-NPs was evaluated based on the total antioxidant capacity, ferric reducing antioxidant power, and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assays. The antioxidant activity of the ZnO-NPs was confirmed using the three antioxidant assays. The ZnO-NPs of U. lactuca recorded the highest antioxidant activity. The cytotoxicity of the ZnO-NPs was tested on different cell lines using the MTT assay. The ZnO-NPs of U. lactuca showed very weak cytotoxic effects on WI 38 (84.98 ± 4.6 µg/mL) and 23, and this result confirmed its safety on normal cells. The ZnO-NPs of U. lactuca showed moderate cytotoxic effects on the HepG-2 (46.66 ± 2.8 µg/mL) and MCF-7 (30.60 ± 2.1 µg/mL) cell lines. In an in vivo study, the ZnO-NPs of U. lactuca showed a decrease in tumor volume, weight, and serum malondialdehyde in experimental mice, while the total antioxidant capacity of the serum was increased. Histopathological changes in ZnO-NPs indicated a reduction in tumor size, a lower number of mitosis divisions, and a rise in apoptosis correlated with the ZnO-NPs of the U. lactuca-treated groups. In conclusion, biosynthesized ZnO-NPs from seaweed showed potent antimicrobial, antioxidant, and antitumor activities, which can be used in the pharmaceutical industry.

1. Introduction

The creation of multifunctional nanomaterials is now a widespread trend due to their distinctive features, which may be reached by reducing particle size and increasing surface area [1]. ZnO nanoparticle applications (ZnO-NPs) are greatly expanded in this new technological era, including antimicrobial, anticancer, and antioxidant uses, among others [2]. As an n-type semiconductor, ZnO has a variety of appealing qualities, including being non-toxic, having a large band gap, giving strong chemical stability at an ambient temperature and having a high binding energy.
ZnO can be manufactured in the nanometer range using traditional methods on a large scale via a variety of chemical and mechanical procedures [3]. Several limits are apparent when using these traditional methodologies, notwithstanding their applicability in the business sector. For instance, the chemical synthesis of ZnO nanoparticles involves the use of poisonous and dangerous compounds as reductants and stabilizers, whilst other processes may demand high temperatures, which consume significant energy [4]. The public becomes more aware of environmental pollution as a result, and interest in the one-pot green synthesis method increases. The preference for synthesizing ZnO nanoparticles has emerged as a result of the benefits of green approaches, such as their simplicity and inexpensive operating costs.
Many naturally occurring, renewable resources, including yeast, bacteria, seaweed, and plant extract, are used for the environmentally friendly manufacture of ZnO nanoparticles in place of hazardous chemicals. These natural materials, known as “bioresources”, are exceptionally rich in bioactive components such as amino acids, carbohydrates, and polyphenolic compounds, which can act as reducing agents and prevent the creation of ZnO nanoparticles [5]. Compared to hazardous chemicals, biological substances are less toxic because they are natural and biodegradable [6]. Consequently, this green synthesis method will be considered a viable substitute for the commercial and industrial manufacture of ZnO nanoparticles in the future.
With regard to their high content of biomolecules such as sugars and proteins, seaweed (marine macroalgae) is an excellent choice for use as a bioresource in the effective green production of ZnO nanoparticles [7]. Seaweed nanoparticles are relatively stable in solutions, harmless to the environment, and largely safe in a variety of fields. By virtue of the presence of lipids, proteins, and polysaccharides with different functional groups on the surfaces of their cell walls, seaweed has a substantial metal-binding capacity [8].
According to published research by ref. [9], green synthesis utilizing alga extract as a bio-reductant successfully generated ZnO nanoparticles with rod-like shapes. The research found that interaction between the zinc precursor and the possibly bioactive compounds found in the green seaweed compounds, including amine, carbonyl, and nitro groups, led to the formation of ZnO nanoparticles. It was unexpected that the seaweed-mediated ZnO nanoparticles outperformed the chemically generated ZnO nanoparticles in terms of antibacterial activity.
Furthermore, ref. [10] used ZnO nanoparticles generated from Spirulina sp. (Cyanobacteria) on an ultrafiltration membrane to remove drugs. The results showed that green-produced ZnO nanoparticles improved membrane performance by increasing hydrophilicity and antifouling features. According to numerous research studies [11,12,13], green seaweed-derived ZnO-NPs can be utilized to inhibit the growth of bacteria and fungi that are resistant to antibiotics. According to [14], Ulva lactuca (Chlorophyta) extract was used to biosynthesize zinc oxide nanoparticles. As materials for antioxidant defense against free radicals generated during metabolism, ZnO-NPs are also essential, according to ref. [15].
The current study aimed to investigate green biosynthesis of ZnO-NPs from three seaweed species, as well as to explore their activity as antibacterial, antifungal, antioxidant, and anticancer agents.

2. Materials and Methods

2.1. Seaweed Sample Collection

Three seaweed species were gathered in summer 2020. Two seaweed species (Ulva lactuca and Ulva intestinalis—Chlorophyta) were harvested from the Rocky Bay of Abu Qir, Alexandria, Mediterranean Sea, Egypt, while the seaweed Sargassum muticum (Phaeophyceae) was collected from Hurghada in the Red Sea. The collected seaweed samples were immediately brought to the laboratory and cleaned of epiphytes and rocks using tap water. For taxonomical identification, some of the obtained samples were kept in 5% formalin. The other fraction of the seaweed was air-dried in the shade at 30 ± 2 °C. For subsequent research, the dried seaweed samples were crushed into a fine powder and stored in tightly covered containers according to refs [16,17,18,19].

2.2. Seaweed Extracts Preparation

Using a magnetic stirrer, 10 g of seaweed powder and 100 mL of distilled water were combined in a flask, heated to 60 °C for 30 min in a water bath, and shaken overnight using a shaker. After a thorough filtering through filter paper, the extract was chilled and kept for the biosynthesis procedure [20] The filtrate was utilized as a bio-reducing agent for the preparation of nanoparticles with zinc acetate dihydrate.

2.3. Biosynthesis of Zinc Oxide Nanoparticles (ZnO-NPs)

A 50 mL flask was filled with 5 mL of algae extract. Next, 15 mL of zinc precursor (Zn (CH3CO2)2, 0.02 M) was added. The reaction mixture’s pH was then brought to 12 by adding 2 M NaOH dropwise while stirring continuously for two hours at 60 °C. The cloudy white color of the reaction mixture indicated the production of ZnO-NPs. To remove any unreacted solutes and phytochemicals, the reaction product was centrifuged for 15 min at 3000 rpm. The white precipitate that was left behind was then washed five times using deionized water. Ultimately, the white precipitate underwent a 48 h oven-drying process at 60 °C [21].

2.4. Characterization of Biosynthesized Zinc Oxide Nanoparticles

The physicochemical characteristics of the ZnO-NPs produced from the seaweed samples were examined using a variety of characterization techniques. These techniques include ultraviolet (UV) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, zeta potential, X-ray diffraction (XRD), and transmission electron microscopy (TEM).

2.4.1. Ultraviolet Vis Spectrometry

Aliquots (0.2 mL) of the colloidal suspension were taken and diluted in 2 mL of deionized water. The UV-Vis spectra of the resultant diluents were then measured using a double-beam PC scanning spectrophotometer, (LAMBDA 365 UVVis Spectrophotometer, 190–1100 Nm with wavelength range 190–1100 nm, PerkiElmer, Mumbai, Maharashtra, India) carried out at the Scientific Research Center and Measurement-Tanta University. It was possible to identify the bio-reduction of the pure zinc ions in the aqueous solution. The absorption maxima were searched between 200 and 800 nm in wavelength. The absorbance peak for the ZnO nanoparticles was reported to occur at wavelengths of between 310 and 360 nm.

2.4.2. Fourier Transformer Infrared Analysis

The functional groups in the seaweed crude extracts that were responsible for the reduction of the zinc ions were discovered using a Fourier transformer infrared (FTIR) spectrophotometer (tensor 27 Bruker, Germany) carried out at the Scientific Research Center and Measurement-Tanta University.

2.4.3. X-ray Diffraction

An X-ray diffraction analysis of powder was performed, the spectra were recorded, and the region of 500 to 4000 cm−1 was included. The size and crystalline shape of the ZnO-NPs were determined based on the X-ray diffraction (XRD) dimensions (Philips-X’Pert MPD X-ray diffractometer, Naperville, IL, USA). A glass substrate was coated with the dried NP powder while being scanned in the vicinity of 2 θ, from 4 to 90° at 0.5°/min, and with a time constant of 2 s. Based on the XRD pattern and the line width of the maximum intensity reflection peak, Scherrer’s equation was used to determine the mean particle diameter of ZnO-NPs as follows: The values “°2” denotes the Bragg’s angle, is the corrected full width at half maximum (FWHM) in radians, “sample” and “ref.” are the FWHM of the sample and reference peaks, respectively, “D” represents the average crystal size, “K” is the Scherrer coefficient (0.89), and is the X-ray wavelength (1.5406).

2.4.4. Transmission Electron Microscopy

For examination using a transmission electron microscope (TEM), the nanoparticles were suspended in a solvent and processed using the coated gride technique. The coated gride was examined using a JEM-1400 plus (JEoL, Akishima, Tokyo, Japan) transmission electron microscope unit at the Faculty of Science, Alex, andria University, Egypt.

2.4.5. Zeta Potential Analysis

By putting 2 mL of the samples in a four-sided clear plastic cuvette and using a Malvern zeta sizer instrument (Malvern instruments Co., Malvern, UK) at 25 °C, the zeta potential of the biosynthesized ZnO-NP aqueous solutions was assessed. The transparent disposable zeta cell was used for the instant test.

2.5. Applications of Biosynthesized Zinc Oxide Nanoparticles

2.5.1. Antimicrobial Activity of Biosynthesized Zinc Oxide Nanoparticles

Two Gram-negative bacterial pathogenic strains (Escherichia coli and Klebsiella pneumoniae) were obtained from the Bacteriology Laboratory, Faculty of Science, Tanta University. Microbial suspensions were prepared in a sterile saline solution (0.9% NaCl) at a turbidity of 106 to 108 CFU/mL. The bacterial strains were cultivated in MacConkey solid medium. In addition, two fungal strain, Candida albicans and Aspergillus niger, were cultivated on Sabouraud Dextrose agar medium. The antimicrobial activities of the biosynthesized ZnO-NPs were evaluated using the disk diffusion method [22]. After being placed in Petri dishes that had already been pre-inoculated with 100 mL of each microbe suspension, sterilized paper disks loaded with 15, 30, and 60 g/disk of ZnO-NPs were incubated at 37 °C. Standard antibiotics were used as a positive control, and seaweed extracts and zinc acetate solution were used as negative controls. The inhibition zone widths were measured after the incubation period. The positive antibacterial controls were ciprofloxacin (CIP), amoxicillin (Ax), and ampicillin–sulbactam (SAM). The positive antifungal controls were fluconazole (FLU), itraconazole (ITC), and metronidazole (MT) [23].

2.5.2. Antioxidant Activity of Zinc Oxide Nanoparticles

DPPH Radical Scavenging Activity

The capacity of the ZnO-NPs to reduce the stable 2,2-diphenyl-picrylhydrazyl (DPPH) free radical was assessed as described in ref. [24]. Three hundred microliters of ZnO-NPs at various concentrations (3.37, 6.75, 12.5, 25, and 50) mg/mL were mixed with 2.7 mL of DPPH reagent solution and allowed to stand for 30 min in the dark at room temperature. At 517 nm, the samples’ absorbance was measured, and as a negative control, the absorbance of DPPH alone was also measured. Using ascorbic acid as a reference, the antioxidant capabilities of the samples were examined. Additionally, the ascorbic acid and DPPH IC50 values were computed. The following formula was used to determine the percentage of DPPH scavenging activity:
Reduction in DPPH activity (%)
= (Ac − As)/(Ac) × 100     
where Ac is the absorbance of the control and As is the absorbance of the sample.

Total Antioxidant Capacity Assay

The total antioxidant activity of the ZnO-NPs was evaluated using the phosphor-molybdenum method [25]. A known weight of ZnO-NPs was dissolved in 10 mL of 1 mM dimethyl sulfoxide to create various quantities of ZnO-NPs. A total of 300 microliters of each concentration was added to 3 mL of the phosphomolybdate reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). Following the reaction’s light shielding, the vials were incubated in a water bath at 95 °C for 90 min. The absorbance was measured at 695 nm in relation to a blank after the mixture was cooled to room temperature. An ascorbic acid reference was used. The findings were expressed as mg of ascorbic acid (mg AAE/g DW) per gram of the dry weight of the NPs.

Ferric Reducing Antioxidant Power Assay

The chelation ferric reducing antioxidant power assay of ferrous ions by ZnNPs was estimated as described in ref. [26]. To begin, 2.5 mL of sodium phosphate buffer (1%) was mixed with 1 mL of each of the three distinct ZnO-NP concentrations (12.5, 25 and 50 mg/mL), and the mixture was then incubated for 20 min at 50 °C in a water bath. After that, 2.5 mL of 10% trichloroacetic acid was added, and the mixed solution was then centrifuged at 500× g for 10 min. The supernatant was then combined with 1 mL of ferric chloride solution (0.1%) and 5 mL of distilled H2O. At 700 nm, the mixture’s absorbance was determined. An increased reducing power is shown by higher absorbance readings. The results were expressed as ascorbic acid equivalents (AAE) per gram of dried sample (mg AAE/g DW).

2.5.3. Cytotoxicity Assay of Biosynthesized Zinc Oxide Nanoparticles

The cell lines were received from ATCC via the Holding Company for Biological Products and Vaccines (VACSERA), Cairo, Egypt, for hepatocellular carcinoma (HEPG-2), mammary gland breast cancer (MCF-7), colorectal carcinoma colon cancer (HCT-116), and human lung fibroblast (WI-38) normal cell lines [27]. For comparison, doxorubicin, a typical anticancer medication, was used. Using the MTT test, these cell lines were employed to ascertain the inhibitory effects of zinc oxide nanoparticles on cell growth. This colorimetric assay relies on living cells’ mitochondrial succinate dehydrogenase to change the yellow tetrazolium bromide (MTT) into a purple formazan derivative. The cell lines were grown in RPMI-1640 media with 10% fetal bovine serum. At 37 °C in an incubator with 5% CO2, 100 units/mL of penicillin and 100 g/mL of streptomycin were introduced. At a density of 1.0 × 104 cells/well, the cell lines were planted in a 96-well plate for 48 h at 37 °C with 5% CO2. After 24 h of incubation, the cells were exposed to various concentrations of zinc oxide nanoparticles. Following a 24 h drug treatment period, 20 mL of a 5 mg/mL MTT solution was applied and incubated for 4 h. Each well received 100 mL of dimethyl sulfoxide (DMSO) to dissolve the produced purple formazan. An ELIZA Exlservice Holdings, Inc company (EXL 800), Jersey City, NJ, USA plate reader was used to measure and record the results of the colorimetric assay at an absorbance of 570 nm. By multiplying the A570 of the treated samples and the A570 of the untreated samples by 100, the relative cell viability was estimated as a percentage [28,29].

2.5.4. Antitumor Activity of Zinc Oxide Nanoparticles (In Vivo Study)

Experimental Animals

Swiss female albino adult mice weighing an average of 25 to 30 g were purchased from Theodore Bilharz Institute in Giza, Egypt, and housed at the Faculty of Science animal facility at Ain Shams University. The animals were kept in typical laboratory settings (26 ± 1 °C), 12 h light/12 h dark cycle in standard-sized polycarbonate cages with unlimited access to food and water. Prior to EAT induction, the animals were housed for a total of 10 days in these regular settings. The Research Ethics Committee of the Faculty of Science at Ain Shams University in Cairo, Egypt, approved of all the animal experiments conducted in this study, in accordance with the “Principles of Laboratory Animal Care” (ASU-SCI/MICR/2023/5/4).

Ehrlich Ascites Carcinoma Cells

The National Cancer Institute at Cairo University, Cairo, Egypt, provided the mouse Ehrlich ascites carcinoma (EAC) cells. Both in vivo and in vitro tests were performed using the EAC cells. They were kept alive by passing 1 × 106 cells intraperitoneally on a weekly basis in adult female Swiss albino mice [30]. The ascitic fluid was extracted from the peritoneal cavity of tumor-bearing mice seven days after inoculation using a needle under aseptic conditions. Using a Neubauer hemocytometer and the trypan blue dye (0.4%) exclusion method, ascitic tumor cell counts were carried out. The vitality of the cells was consistently determined to be about 95%.

Ehrlich Solid Tumor Model

All the mice (54) received a subcutaneous injection of 5 × 105 cells per 0.1 mL into their right hind limb (thigh) [31]. The solid tumor’s volume was measured using a digital vernier caliper and then calculated using the following formula: A × B 2 × 0.5, where A is the tumor’s greatest diameter and B is its narrowest perpendicular diameter. Five days following tumor injection (day 0), when the solid tumor became palpable (50–100 mm3), the animals were randomly assigned to five groups: the EAT control group (n = 13), which received no treatment throughout the experiment; the EAT/ZnO-NPs control group (n = 11), which received 0.1 mL of ZnO-NPs per day per mouse orally for 21 days starting on day 1; the PEITC-treated group (n = 11), which received ZnO-NPs at a dose of 60 mg kg−1 day−1 orally 20 for 21 days starting on day 1 for a total volume of 0.1 mL of ZnO-NPs per mouse; the doxorubicin-treated group (n = 11), which received dox at dose a of 2 mg kg−1 day−1 i.p.21 for 21 days starting on day 1 for a total volume of 0.1 mL per mouse; and the PEITC/dox combination-treated group (n = 11), which received ZnO-NPs (60 mg kg−1 per 0.1 mL ZnO-NPs per day, orally) 2 h prior to receiving dox (2 mg kg−1 per 0.1 mL per day, i.p.) for 21 days starting on day 1.
In addition, the normal control group (n = 8) received 0.1 mL of oil per day per mouse orally for 21 days starting on day 1. From the fifth day of tumor induction, the tumor volume was measured every fifth day for a total of 21 days. On day 22, the experiment’s final day, the mice were killed, and the tumors were removed, weighed, and kept in buffered formalin so they could be examined histopathologically in more detail. Three mice from each group were watched to see how long they lived. By allowing the animals to die naturally, the following formulas: MST = ∑Msurvival time (days) of each mouse in a group]/(total number of mice) and %ILS = [(MST of treated group)/(MST of control group)] × 100 were used to calculate the mean survival time (MST) and percentage increase in life span (%ILS):

Oxidative Stress Assay

Tests for the total antioxidant capacity and lipid peroxide were conducted. Spectrophotometric measurements were made of the serum malondialdehyde (MDA) levels and serum total antioxidant capacity (TAC) using commercially available MDA and TAC colorimetric test kits from Biodiagnostic Company (Giza, Egypt) in accordance with the protocols outlined in ref. [32].

Histopathological Analysis

The tumors were removed on day 22 and preserved in 10% neutral buffered formalin solution before being embedded in paraffin. Hematoxylin and eosin (H&E) staining was performed on five-micrometer slices. A pathologist who was blind to the treatment groups inspected the slides for histopathological alterations using light microscopy.

2.6. Statistical Analysis

The current study’s statistical presentation and analyses were carried out utilizing the mean and standard deviation of three replicates. The ANOVA test was performed using the computer application SPSS V.16 for Windows. SPSS was utilized to compare the different times within the same group of quantitative data [33].

3. Results

3.1. Identification of Seaweed Samples

The seaweed samples used in this study were identified as follows (Figure 1):
  • Ulva lactuca:
    • Kingdom: plantae
    • Phylum: Chlorophyta
    • Class: Ulvophyceae
    • Order Ulvales
    • Family: Ulvaceae
    • Genus: Ulva
    • Species: lactuca
  • Ulvaintestinalis (Chlorophyta):
    • Kingdom: plantae
    • Phylum: Chlorophyta
    • Class: Ulvophyceae
    • Order Ulvales
    • Family: Ulvaceae
    • Genus: Ulva
    • Species: intestinalis
  • Sargassum muticum (Phaeophyceae):
    • Kingdom: Chromista
    • Phylum: Heterokontophyta
    • Class: Phaeophyceae
    • Order: Fucales
    • Family: Sargassaceae
    • Genus: Sargassum
    • Species: muticum

3.2. Characterization of Biosynthesized Zinc Oxide Nanoparticles

3.2.1. UV–Vis Spectroscopic Analysis

Visual inspection was the first method used for the characterization of ZnO-NP synthesis, followed by UV measurements of this change at various wavelengths (200 to 800 nm) to identify the surface plasmon resonance (SPR). Figure 2 shows that the UV–visible spectra of the ZnO-NPs biosynthesized using the seaweed extracts (U. lactuca, U. intestinalis, and S. muticum) demonstrated significant peaks at 374, 375, and 376 nm, respectively, which were mostly distinguished by ZnO-NPs.

3.2.2. FTIR Analysis of Zinc Oxide Nanoparticles

FTIR measurements were performed to look into the possible biomolecules that could cap and stabilize the metal nanoparticles that the seaweeds produced. Additionally, it attests to the employed seaweeds’ ability to reduce metal ions. Figure 3 shows that ZnO-NPs were produced by U. lactuca at peaks of 410, 480, and 615 cm−1 and by U. intestinalis at approximately 412, 475, and 841 cm−1. On the other hand, the stretch for the ZnO-NPs synthesized by S. muticum was discovered to be approximately 425, 530, and 613 cm−1. The peak around 1320–1000 cm−1 were due to C-O stretching alcohols, carboxylic acids, esters, ethers. The peaks at 1500–1400 cm−1 were due to C-C stretch (in-ring) aromatics. The peak at around 3450 cm−1 indicates the O-H group. With the attachment of the various functional groups, proteins and metabolites such as terpenoids surrounded the produced ZnO nanoparticles. The results of the FTIR study show that the reduction, capping, and stability of the biosynthesized ZnO-NPs were all influenced by the organic compounds in the seaweed extract.

3.2.3. XRD Analysis of Zinc Oxide Nanoparticles

Figure 4 illustrates different intense peaks of Bragg reflections of ZnO-NPs formed using the seaweeds with Brags angles corresponding to zinc 2θ = 102. The XRD analysis verifies the wurtzite structure of the ZnO nanoparticles, and the narrow peak generation with a Bragg’s angle of 2θ = 102° indicates that the ZnO nanoparticles are crystalline. The ZnO nanoparticles are suggested to be face-centered cubic at a Bragg’s angle of 2θ = 100°. The prominent peaks show that the nanoparticles are stabilized by some capping agents. The strong Bragg’s angle reflection suggests that the capping agents caused strong X-ray scattering centers in the crystalline phase.

3.2.4. Transmission Electron Microscopy (TEM) of the Biosynthesized Zinc Oxide Nanoparticles

TEM is a powerful method that was used to determine the shape and size of the nanoparticles and their distribution in the colloidal solution. The particle size, as well as the distribution of the ZnO-NPs, were determined based on the TEM images (Figure 5). It is clear that the frequency size ranged from approximately 4.13 to 8.56 nm, with a mean particle size estimated to be 5.6 nm for the ZnO-NPs formed using the U. lactuca extract. Also, the ZnO-NPs formed using the S. muticum extract ranged from 15,66 to 27.15 nm, with a mean particle size of 20.39 nm, while the ZnO-NPs formed by the E. intestinalis extract ranged from 32.16 to 48.25 nm, with a mean particle size of 42.27 nm. Also, based on the TEM images, the ZnO-NPs formed using U. lactuca, U. intestinalis, and S. muticum were widely distributed.

3.2.5. Zeta Potential Analysis

The zeta potential is a physical feature displayed by any particle in a suspension that predicts the particles’ long-term stability, with the pH of the ZnO solution being 12. For U. lactuca ZnO-NPs, the zeta potential analysis revealed a net charge of −42.1 mV, a conductivity of 2.06 mS/cm, and a standard deviation of 4.10 mV. Additionally, the analysis demonstrated a conductivity of 1.92 mS/cm, a net charge of −25 mV, and a standard deviation of 4.10 mV for the U. intestinalis ZnO-NPs, as well as a conductivity of 4.10 mS/cm, a net charge of −37 mV, and a standard deviation of 4.84 mV for the S. muticum ZnO-NPs. These values can be characterized as high-quality potentials with high levels of particle stability because they fall within the known range of −30 mV to +30 mV (Figure 6).

3.3. Antimicrobial Activity of the Biosynthesized ZnO-NPs

The ZnO-NPs biosynthesized using crude extracts of U. lactuca, U. intestinalis, and S. muticum were evaluated against two pathogenic bacterial species, Escherichia coli and klebsiella pneumonia, and two fungal species, Aspergillus niger and Candida ablicans, using the disk diffusion method (Table 1). The diameter of the inhibitory zones ranged from (11 ± 0.21 mm to 21.3 ± 1.02 mm) for different ZnO-NP concentrations and other treatments. The highest inhibition zone of the Ulva lactuca ZnO-NPs was observed for the 60 mg/mL treatment against A. niger (21.3 ± 1.02 mm), followed by E. coli (20.7 ± 0.42 mm), C. ablicans (19.3 ± 0.36 mm), and then K. pneumonia (17.8 ± 0.02 mm). These results were followed by the treatment using 30 mg/mL, and finally, for the 15 mg/mL treatment as shown in Table 1.
The highest inhibition zone of the S. muticum ZnO-NPs was observed for the 60 mg/mL treatment against E. coli (19.1 ± 0.39 mm), followed by C. ablicans (18.6 ± 0.36 mm), then A. niger (17.7 ± 1.05 mm), and finally K. pneumonia (15.1 ± 0.02 mm). These results were followed by the treatment using 30 mg/mL, and finally, with 15 mg/mL treatment.
On the other hand, the highest inhibition zone of the U. intestinalis ZnO-NPs was observed in the 60 mg/mL treatment against K. pneumonia (17.6 ± 0.23 mm), followed by E. coli (16.9 ± 0.75 mm), then A. niger (16.2 ± 0.24 mm), and finally C. ablicans (15.8 ± 0.00 mm). These results were followed by the treatment using 30 mg/mL, and finally, with the 15 mg/mL treatment. Thus, by increasing the concentration of ZnO-NPs, the diameter of the inhibition zone also increased. The inhibition values of the standard antibiotics fluctuated depending on the bacterial and fungal strains.

3.3.1. Antioxidant Activity of the Biosynthesized ZnO-NPs

DPPH Radical Scavenging Activity

The IC50 value of the DPPH scavenging activity was considerably lower (12.6 μg/mL) for the U. lactuca ZnO-NPs than that of the ZnO-NPs synthesized using S. muticum and U. intestinalis extracts, which was 13.8 μg/mL and 15.2 μg/mL, respectively as shown in Table 2. Increasing concentrations of ZnO-NPs showed comparatively higher DPPH antioxidant activities (Table 2) compared to ascorbic acid (AA) as a standard antioxidant. The IC50 value of ascorbic acid was 2.4 ± 0.20 μg/mL.

Total Antioxidant Capacity (TAC)

The total antioxidant capacity increased as the concentration of ZnO-NPs increased. The biosynthesized ZnO-NPs exhibited substantial phosphor-molybdenum reduction activity, with 38 ± 0.01, 35 ± 0.06, and 29 ± 0.15 mg AAE/g DW for U. lactuca, S. muticum, and U. intestinalis ZnO-NPs crude extracts as shown in Table 3, respectively, at 50 μg/mL.

Ferric Reducing Antioxidant Power (FRAP)

The FRAP assay showed comparable results to the TAC assay, where the antioxidant activity for the U. lactuca, S. muticum, and U. Intestinalis ZnO-NPs exhibited significant difference values of 49.57 ± 0.98, 35.23 ± 0.27, and 26.4 ± 1.00 mg AAE/g DW, respectively, at a concentration of 50 μg/mL (Table 4).
As the seaweed U. lactuca exhibited the best antioxidant activity among the three tested seaweeds, the following experiments were performed to study its effects as a cytotoxic and anti-tumor agent.

3.3.2. Cytotoxicity Assay of Biosynthesized ZnO-NPs

The cytotoxic effects of the biosynthesized ZnO-NPs of U. lactuca at concentrations ranging from 1.56–100 μg/mL were detected using the WI38, HepG2, HCT116, and MCF7 cell lines based on the results of the MTT assay. The obtained results showed very weak effects of the biosynthesized ZnO-NPs on WI38 normal cells (84.98 ± 4.6). This result confirmed its safety and non-toxicity. The recorded IC50 value of the ZnO-NPs of U. lactuca showed moderate cytotoxic effects on HepG-2 cell line (46.66 ± 2.8 µg/mL) and MCF-7 cell line (30.60 ± 2.1 µg/mL), whereas the ZnO-NPs recorded weak cytotoxic effects on the HCT-116 cell line (58.69 ± 3.3 µg/mL) compared to the doxorubicin (a chemotherapeutic drug) (Table 5).

3.3.3. Antitumor Effects of ZnO-NPs

Effects of U. lactuca ZnO-NPs on Solid Tumor Growth Inhibition

The average tumor volume in the control mice increased from 78.62 mm3 on day 0 to 1597.16 mm3 after 21 days of treatment. The average tumor volume in the mice treated with the U. lactuca ZnO-NPs was 243.4 mm3 after 21 days of treatment, which was significantly different from the result of the Dox treatment, which was 203.12 mm3 after 21 days of treatment (Figure 7).
Quantitatively, the weight average of the tumors in the control mice was 4.33 gm. The average weight of the tumor in the mice treated with ZnO-NPs of U. lactuca was 1.9 gm after 21 days of treatment, which was significantly different compared to the result of the Dox treatment, which was 1.16 gm after 21 days of treatment (Figure 8).

Effects of Ulva Lactuca ZnO-NP Treatment on Oxidative Stress

The serum TAC and MDA levels were different in the control mice: 1.03 and 19.68, respectively. The serum TAC was increased in the Dox group and the mice treated with the U. lactuca ZnO-NPs: 1.91 and 1.63, respectively. On the other hand, the serum MDA was decreased in the Dox and ZnO-NPs of the U. lactuca ZnO-NP-treated mice: 7.81 and 9.95, respectively (Figure 9).

3.4. Effects of ZnO-NPs on Histopathological Changes

Tumor cross-sectional pictures were closely observed for large tumor cells, and a significant number of cells undergoing mitosis were visible in the control group. After receiving Dox and the ZnO-NP treatments, the tumor size and mitotic numbers were both decreased. Additionally, all the groups treated with the ZnO-NPs showed increased apoptosis when compared to the control group (Figure 10).
Figure 10 shows many unusual mitotic figures (black arrowheads), pleomorphic forms, hyperchromatic nuclei, and solid masses of large, round, polygonal, highly pigmented tumor cells. Minimal necrotic zones (N) with pyknosis, karyorrhexis (black arrows), and karyolysis, which manifested particularly in the center portions of the tumors, are interspersed between wide viable areas (V). In the middle of the viable patches, several newly developed blood capillaries are observed (red arrows). The growth inhibitory effects of the drugs are assessed based on their ability to increase areas of necrosis, decrease the density of newly formed blood capillaries, decrease the numbers and sizes of viable areas, and increase the numbers of ghost cells (black arrows) inside viable areas. Consequently, these effects were markedly induced in Dox group, slightly induced in the white treated group, and moderately induced in the brown treated group.

4. Discussion

Due to its advantages over conventional chemical synthesis processes regarding expenses and effects on the environment, green nanomaterials have attracted significant attention. One of the well-known metal oxide nanoparticles with considerable applicability in numerous sectors is zinc oxide nanoparticles (ZnO-NPs) [34].
The visual change as a white precipitate was initially used to pinpoint the biosynthesis of ZnO-NPs. The findings demonstrated that zinc acetate (Zn (CH3CO2))2 and seaweed crude extract from U. lactuca, S. muticum, and U. intestinalis effectively reacted to create ZnO-NPs via a reduction process within 2 h using a magnetic stirrer [35].
It was found that the biosynthesized ZnO-NPs’ surface plasmon band has a wavelength of between 310 and 360 nm. In the present study, the formation of ZnO-NPs synthesized using U. lactuca, S. muticum, and U. intestinalis was confirmed based on the UV–Vis spectrum, which showed emission peaks at 374, 375, and 376 nm, respectively. According to ref. [36], because the ZnO nanoparticles are situated significantly below the band gap wavelength of 358 nm, the produced ZnO nanopowder’s absorption spectra exhibited a strong absorption band at around 355 nm and an excitonic absorption peak at about 258 nm. Additionally, ref. [37] observed that ZnO-NPs displayed high UV absorption bands at 265 and 390 nm, indicating the synthesis of ZnO-NPs. According to various recent studies, the existence of bands between 230 and 390 nm is a sign of the development of ZnO-NPs [38].
The functional group that is in charge of reduction and serves as a capping agent in biosynthetic nanoparticles can be identified and characterized using an important technique known as FTIR [39]. In this study, the FTIR analysis confirmed the reduction of metal ions and determined the potential biomolecules in charge of the effective stability and capping of the metal nanoparticles formed by the seaweeds extracts. Similar studies have demonstrated that metal oxides exhibit absorption bands originating from interatomic vibrations in the fingerprint regions below 1000 cm−1 [40]. In order to reduce, cap, and stabilize biosynthesized ZnO-NPs, ref. [41] conducted an FTIR investigation using Ericaria crinita (formerly Cystoseira crinita) (Phaeophyceae) extract. They found that phenolic chemicals and proteins play a role in this process.
By passing X-rays through a pattern that produces nanoparticles, the XRD technique determines the size and shape of materials [38]. In the present study, ZnO-NPs were formed by seaweeds with Brags angles corresponding to zinc 2θ = 102. The XRD research indicated that the ZnO nanoparticles had a wurtzite structure, and the emergence of a narrow peak with a Bragg’s angle of 2 = 102 suggests that the ZnO nanoparticles were crystalline. The ZnO-NPs produced using E. crinita were subjected to XRD analysis, which revealed distinctive peaks at values of 31.78, 34.42, 36.26, 47.6, 56.6, and 62. Ref. [41] attributed these values to the (100), (002), (101), (102), (110), (103), and (112) planes, respectively. The current findings were consistent with many studies that reported XRD patterns of phyco-synthesized ZnO-NPs that were similar to the present results [11,42].
A transmission electron microscopy (TEM) analysis was used to determine the size and morphological characteristics of the generated ZnO-NPs. In this study, ZnO-NPs were determined based on the TEM image within a size range of approximately 4.13 to 8.56 nm, with a mean particle size estimated to be 5.6 nm for the ZnO-NPs formed using the U. lactuca extract. The size of the ZnO-NPs formed using the S. muticum extract ranged from 15.66 to 27.15 nm, with a mean particle size of 20.39 nm. The ZnO-NPs formed using the U. intestinalis extract ranged from 32.16 to 48.25 nm, with a mean particle size of 42.27 nm [43]. S. muticum aqueous extract is engaged in the synthesis of hexagonal ZnO-NPs with sizes ranging from 3 nm to 57 nm and an average size of 42 nm. In the study by ref. [41], a hexagonal wurtzite structure with an average size of 42.6 nm was visible in the TEM images of the biosynthesized ZnO-NPs. The morphology was composed of stacked rectangular particles without aggregation, which may be connected to the biochemical characteristics of Ericaria crinita (Phaeophyceae).
Any particle in suspension will display a zeta potential: a physical characteristic that indicates a particle’s long-term stability. The zeta potential, which measures a particle’s surface potential, is affected by changes at the particle’s interface with the dispersing medium. The adsorption of ionic species from the aqueous dispersion medium or the dissociation of functional groups on the particle’s surface can both cause these modifications [44]. Good physical and chemical stability of the colloidal solution depends on a relatively high value of the zeta potential, because large repulsive forces tend to prevent aggregation produced by inadvertent collisions of nearby nanoparticles. The zeta potential signal of highly cationic and highly anionic nanoparticles is characterized as being larger than +30 mV or less than 30 mV, respectively [45]. Based on this connection, the ZnO-NPs biosynthesized using U. lactuca, S. muticum, and U. intestinalis were thought to be substantially anionic, which is crucial for biological applications. Refs [46,47] reported that their zeta potential analysis verified the stability of nanoparticles that were biosynthesized using seaweed.
Numerous researchers have reported on the antibacterial properties of biologically produced ZnO-NPs [48]. The ZnO-NPs produced in the current study using different seaweeds demonstrated potent antifungal action against two fungal strains, Aspergillus niger and Candida albicans, as well as two multidrug-resistant bacterial strains, K. pneumonia and E. coli. This suggests that the results are noteworthy. This is important, since many bacterial and fungal strains have developed multidrug resistance as a result of the extensive usage of numerous synthetic antibacterial drugs. Generally, the ZnO-NP concentrations increased in tandem with the inhibitory zone’s diameter [10].
ZnO-NPs produced using seaweed have a large surface area-to-volume ratio, which may have an inhibitory effect. Alternatively, the positive charge of the ZnO-NPs may interact with the negatively charged microbial membrane, affecting intracellular protein leakage and cell membrane permeability, both of which inhibit growth [7]. Moreover, NPs inhibit cell proteins, stopping the growth of bacteria by combining with thiol (-SH) groups. Furthermore, this inhibition is most likely due to the interactions of the ZnO-NPs with the phosphorous group in DNA, which stops protein synthesis and DNA replication. In general, nanoparticles (NPs) can impact biological reactions by generating reactive oxygen species (ROS), which can damage cellular constituents such as proteins, DNA, and lipids, ultimately impeding growth [41].
Antioxidants diminish various diseases, including infections, ischemia, and infections caused by diabetes, cancer, and cardiovascular disease by converting reactive oxygen species (ROS) into non-toxic compounds, inhibiting their negative effects [49]. The DPPH scavenging method used in this work proved that ZnO-NPs have antioxidant activities because they have functional groups on their surface. The higher the sample’s DPPH radical scavenging activity, which is a stable radical with a nitrogen core, the greater the sample’s antioxidant activity [50]. The biosynthesized ZnO-NPs’ quantitative total antioxidant capacity (TAC) was ascertained using a phosphor-molybdenum assay. The amount of TAC measured was impacted by the molybdenum reduction [51]. The ability of ZnO-NPs to change from ferric (III) to ferrous (II) in a redox-linked colorimetric process involving a single electron transfer has been used to measure the antioxidant activity of these particles using FRAP [52]. In order to suppress reactive species, antioxidants biologically transfer an electron or hydrogen (H) atom to them [53].
Thus, the antioxidant potential can be evaluated using hydrogen atom transfer (HAT) and single-electron transfer-dependent assays (SET). SET assays are used to measure an antioxidant’s capacity to lessen an oxidant, which undergoes a color change when reduced. SET assays such as total antioxidant capacity (TAC) and ferric reducing antioxidant power (FRAP) tests could be used to quantify the reduced capacity. Nevertheless, the phosphor-molybdenum and FRAP experiments showed that the NPs had strong, considerable antioxidant potential. It should be emphasized that in the phosphor-molybdenum and ferrozine experiments, electron transfer occurs at different redox potentials, and in both cases, the structure of the antioxidant (ZnO-NPs) being studied controls the reducing activity.
To figure out whether the studied nanoparticles could be used safely for treatment, cytotoxicity studies of the ZnO-NPs were performed. Based on the decreased viability of the investigated cell lines following incubation with the nanoparticles at increasing concentrations, the cytotoxicity of the ZnO-NPs was calculated. First, the cytotoxicity was assayed using the MTT test. This investigation showed that the vitality of the treated cell lines was dose-dependent; it reduced with increasing NP concentrations. This result was consistent with those of earlier investigations [54]. The outcomes showed that at larger concentrations of the nanoparticles, i.e., 25, 50, and 100 g/mL, the viability of the cells considerably decreased. These outcomes were comparable to those obtained in ref. [55]. The biosynthesized ZnO-NPs of U. lactuca were more effective on the MCF-7 cell line than on the HePG-2 and HCT-116 cell lines, recording a cell viability percentage of 24.6%. Higher ZnO-NP concentrations had a stronger inhibitory impact, almost completely killing the cells. This showed that the release of zinc ions (Zn+) from the particle surface and their subsequent adsorption to the surfaces of the cells as a source of toxic Zn+ ions, or the formation of toxic Zn+ ions through oxidative dissolution in the presence of oxygen, ligands, or organisms, is the main mechanism of ZnO-NP cytotoxicity on MCF-7 [47]. The new biomaterial is safe to use as an alternative material for a new anti-tumor and anti-microbial medicine, according to ZnO-NPs’ cytotoxic impact [56].
A multi-step process, carcinogenesis involves both mutation and enhanced cell proliferation [57]. ROS overproduction compromises antioxidant defenses, and defective DNA repair processes can result in oxidative damage to cellular macromolecules [57]. ROS easily oxidize cellular fatty acids to create lipid peroxyl radicals and lipid hydroperoxides, which grow into MDA. Their suggested antioxidant activity is one of the potential methods through which ZnO-NPs exert their anti-tumor actions. This is supported by the results of the current study, which revealed that after the administration of ZnO-NPs, TAC greatly increased and MDA dramatically decreased compared to the control group [58]. Although the Dox treatment efficiently reduced tumor burden, it also resulted in much higher TAC and lower MDA levels when compared to the normal control group. This was consistent with the research in ref. [59], which showed that Dox generated oxidative stress throughout the body.
Apoptosis induction in various tumors is another potential method through which ZnO-NPs exercise their anti-tumor effects [60]. Apoptosis is a method through which cells die in a controlled manner. A characteristic of cancer is apoptosis resistance.
According to this study, ZnO-NPs have anti-proliferative and anti-tumor effects on many cancer types. According to ref. [61], ZnO-NPs were able to treat bladder cancer patients with Dox chemoresistance. However, in this investigation, we evaluated PEITC’s ability to effectively trigger apoptosis. Additionally, we examined for the first time how ZnO-NPs and Dox together could decrease tumor growth, both in vivo and in vitro. The EAT model was employed to accomplish this. When compared to the healthy control group, our findings showed that oral treatment of ZnO-NPs alone considerably reduced the burden of solid tumors without modifying the degree of oxidative stress, as seen by a non-significant rise in MDA and a non-significant decline in TAC. The anti-tumor impacts of the ZnO-NP therapy were comparable to those due to the Dox therapy. Interestingly, we discovered that when both treatments were used together, the tumor burden was dramatically reduced compared to when each treatment was used alone. These results were further supported by PEITC’s capacity to considerably reduce ILS and MST when used alone or in conjunction with Dox. Additionally, the tumor tissues examined based on their histopathology revealed that all the groups that received PEITC, either alone or in conjunction with other treatments, saw a drop in the number of mitosis divisions and an increase in the number of apoptotic bodies [62,63]. Remarkably, the maximum dose of ZnO-NPs and Dox showed a significant reduction in cellular viability, which was higher than the effects of both drugs alone. Overall, our results showed that the combination therapy was significantly more effective than ZnO-NPs or Dox alone at inhibiting cellular survival, both in vivo and in vitro. A multi-step process, carcinogenesis involves both mutation and enhanced cell proliferation [57]. Overproduction of reactive oxygen species (ROS), weak antioxidant defenses, and/or defective DNA repair processes can all lead to oxidative damage to cellular macromolecules [31]. ROS easily oxidize cellular fatty acids to create lipid peroxyl radicals and lipid hydroperoxides, which grow into MDA [64]. ZnO-NPs’ purported antioxidant action is one of the putative methods through which it exerts its anti-tumor effects. Ref. [65] showed that Dox produced oxidative stress in every part of the body. The ZnO-NP/Dox combination treatment significantly reduced the elevated MDA level caused by Dox treatment alone and greatly enhanced the lowered TAC. Consequently, the combination treatment provided the best tumor growth inhibition with the least amount of oxidative stress. This is an amazing finding. This can be explained by the fact that catalase, glutathione peroxidase, and superoxide dismutase are just a few of the many antioxidant enzymes that are thought to be strongly induced by ZnO-NPs [66]. Therefore, these enzymes may eliminate the ROS that causes oxidative damage, which preserves lipid molecules and lowers the production of MDA [67].

5. Conclusions

The present study confirmed the biosynthesis of zinc nanoparticles using extracts of three different seaweed species (Ulva lactuca, Ulva intestinalis, and Sargassum muticum). FTIR, XRD, TEM, and zeta potential studies were used to describe the ZnO-NPs synthesized using seaweed and validated the synthesis of zinc nanoparticles. The Ulva lactuca ZnO-NPs demonstrated the greatest antimicrobial effects among the three tested seaweeds against two bacterial and two fungal species. The antioxidant potentiality of the Ulva lactuca ZnO-NPs was confirmed by the three antioxidant assays. Moreover, the Ulva lactuca ZnO-NPs were found to have very weak cytotoxic effects on WI 38, indicating its safety for use on normal cells. The Ulva lactuca ZnO-NPs had moderate cytotoxic effects on the HepG2 and MCF7 cell lines.
Based on the results of this study, we recommend using seaweed-derived ZnO-NPs for pharmacological and biological purposes. Future research is still required to identify the mechanism(s) through which ZnO-NPs exercise their biological antimicrobial and anticancer potentiality as well as to determine their therapeutic safety limitations, particularly when used near people.

Author Contributions

Conceptualization, S.F.G., A.M.S., E.H.E.-S. and S.I.A.; methodology, S.I.A., S.F.G., N.G.A., E.H.E.-S. and A.M.S.; software, S.F.G.; validation, S.F.G.; formal analysis, S.I.A., S.F.G. and A.M.S.; N.G.A., E.H.E.-S. and L.P.; investigation, S.F.G., S.I.A. and S.F.G.; resources, S.F.G.; writing—original draft preparation, S.F.G., S.I.A., N.G.A., A.M.S. and E.H.E.-S.; writing—review and editing, S.F.G., S.I.A., J.C. and L.P.; visualization, L.P.; supervision, S.F.G. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by FCT-Fundação para a Ciência e Tecnologia, I.P., in the framework of the Project UIDB/04004/2020 and DOI identifier 10.54499/UIDB/04004/2020 (https://doi.org/10.54499/UIDB/04004/2020 accessed on 14 March 2024).

Institutional Review Board Statement

Swiss female albino adult mice, weighing 25 to 30 g on average, were purchased from Theodore Bilharz Institute in Giza, Egypt, and housed at the Faculty of Science animal facility at Ain Shams University. The research ethics committee of Faculty of Science at Ain Shams University in Cairo, Egypt approved all animal experiments conducted for this study in accordance with the principles of laboratory animal care. Code: ASU-SCI/MICR/2023/5/4.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data supporting the findings of this article are available within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Sun, C.; Hu, K.; Mu, D.; Wang, Z.; Yu, X. The Widespread Use of Nanomaterials: The Effects on the Function and Diversity of Environmental Microbial Communities. Microorganisms 2022, 10, 2080. [Google Scholar] [CrossRef] [PubMed]
  2. Hashem, A.H.; Rizk, S.H.; Abdel-Maksoud, M.A.; Al-Qahtani, W.H.; AbdElgawad, H.; El-Sayyad, G.S. Unveiling anticancer, antimicrobial, and antioxidant activities of novel synthesized bimetallic boron oxide–zinc oxide nanoparticles. RSC Adv. 2023, 13, 20856–20867. [Google Scholar] [CrossRef] [PubMed]
  3. Narjis, A.; El Aakib, H.; Boukendil, M.; El Hasnaoui, M.; Nkhaili, L.; Aberkouks, A.; Outzourhit, A. Controlling the structural properties of pure and aluminum doped zinc oxide nanoparticles by annealing. J. King Saud. Univ.-Sci. 2020, 32, 1074–1080. [Google Scholar] [CrossRef]
  4. Nagar, A.; Kumar, A.; Parveen, S.; Kumar, A.; Dhasmana, H.; Husain, S.; Verma, A.; Jain, V.K. Zinc oxide nanoflowers synthesized by sol-gel technique for field emission displays (FEDs). Mater. Today Proc. 2020, 32, 402–406. [Google Scholar] [CrossRef]
  5. Jayabalan, J.; Mani, G.; Krishnan, N.; Pernabas, J.; Devadoss, J.M.; Jang, H.T. Green biogenic synthesis of zinc oxide nanoparticles using Pseudomonas putida culture and its In vitro antibacterial and anti-biofilm activity. Biocatal. Agric. Biotechnol. 2019, 21, 101327. [Google Scholar] [CrossRef]
  6. Mohd Yusof, H.; Mohamad, R.; Zaidan, U.H.; Rahman, N.A.A. Sustainable microbial cell nanofactory for zinc oxide nanoparticles production by zinc-tolerant probiotic Lactobacillus plantarum strain TA4. Microb. Cell Factories 2020, 19, 10. [Google Scholar] [CrossRef] [PubMed]
  7. Mansour, A.T.; Alprol, A.E.; Khedawy, M.; Abualnaja, K.M.; Shalaby, T.A.; Rayan, G.; Ramadan, K.M.A.; Ashour, M. Green Synthesis of Zinc Oxide Nanoparticles Using Red Seaweed for the Elimination of Organic Toxic Dye from an Aqueous Solution. Materials 2022, 15, 5169. [Google Scholar] [CrossRef] [PubMed]
  8. Sharawy, Z.Z.; Ashour, M.; Labena, A.; Alsaqufi, A.S.; Mansour, A.T.; Abbas, E.M. Effects of dietary Arthrospira platensis nanoparticles on growth performance, feed utilization, and growth-related gene expression of Pacific white shrimp, Litopenaeus vannamei. Aquaculture 2022, 551, 737905. [Google Scholar] [CrossRef]
  9. Alprol, A.E.; Mansour, A.T.; El-Beltagi, H.S.; Ashour, M. Algal Extracts for Green Synthesis of Zinc Oxide Nanoparticles: Promising Approach for Algae Bioremediation. Materials 2023, 16, 2819. [Google Scholar] [CrossRef]
  10. Bhattacharya, P.; Mukherjee, D.; Deb, N.; Swarnakar, S.; Banerjee, S. Application of green synthesized ZnO nanoparticle coated ceramic ultrafiltration membrane for remediation of pharmaceutical components from synthetic water: Reusability assay of treated water on seed germination. J. Environ. Chem. Eng. 2020, 8, 103803. [Google Scholar] [CrossRef]
  11. Chandra, H.; Patel, D.; Kumari, P.; Jangwan, J.S.; Yadav, S. Phyto-mediated synthesis of zinc oxide nanoparticles of Berberis aristata: Characterization, antioxidant activity and antibacterial activity with special reference to urinary tract pathogens. Mater. Sci. Eng. C 2019, 102, 212–220. [Google Scholar] [CrossRef]
  12. Miri, A.; Mahdinejad, N.; Ebrahimy, O.; Khatami, M.; Sarani, M. Zinc oxide nanoparticles: Biosynthesis, characterization, antifungal and cytotoxic activity. Mater. Sci. Eng. C 2019, 104, 109981. [Google Scholar] [CrossRef]
  13. Rad, S.S.; Sani, A.M.; Mohseni, S. Biosynthesis, characterization and antimicrobial activities of zinc oxide nanoparticles from leaf extract of Mentha pulegium (L.). Microb. Pathog. 2019, 131, 239–245. [Google Scholar] [CrossRef]
  14. Ishwarya, R.; Vaseeharan, B.; Kalyani, S.; Banumathi, B.; Govindarajan, M.; Alharbi, N.S.; Kadaikunnan, S.; Al-anbr, M.N.; Khaled, J.M.; Benelli, G. Facile green synthesis of zinc oxide nanoparticles using Ulva lactuca seaweed extract and evaluation of their photocatalytic, antibiofilm and insecticidal activity. J. Photochem. Photobiol. B Biol. 2018, 178, 249–258. [Google Scholar] [CrossRef]
  15. Rudayni, H.A.; Shemy, M.H.; Aladwani, M.; Alneghery, L.M.; Abu-Taweel, G.M.; Allam, A.A.; Abukhadra, M.R.; Bellucci, S. Synthesis and Biological Activity Evaluations of Green ZnO-Decorated Acid-Activated Bentonite-Mediated Curcumin Extract (ZnO@CU/BE) as Antioxidant and Antidiabetic Agents. J. Funct. Biomater. 2023, 14, 198. [Google Scholar] [CrossRef] [PubMed]
  16. Aleem, A.A. The Marine Algae of Alexandria, Egypt; University of Alexandria: Alexandria, Egypt, 1993; ISBN 9789770052815/9770052817. [Google Scholar]
  17. Jha, B.; Reddy, C.; Thakur, M.; Rao, M. Seaweeds of India: The Diversity and Distribution of Seaweeds of Gujarat Coast; Springer Science & Business Media: New York, NY, USA, 2009; Volume 3. [Google Scholar] [CrossRef]
  18. Kanaan, H.; Belous, O. Marine Algae of the Lebanese Coast; Nova Science Publishers: New York, NY, USA, 2016; pp. 1–126. ISBN 978-1-53610-211-6. [Google Scholar]
  19. Guiry, M.; Guiry, G. Algaebase. World-Wide Electronic Publication, National University of Ireland. 2024. Available online: http://www.algaebase.org (accessed on 22 February 2024).
  20. Osman, M.; Abushady, A.; Elshobary, M. In vitro screening of antimicrobial activity of extracts of some macroalgae collected from Abu-Qir bay Alexandria, Egypt. Afr. J. Biotechnol. 2010, 9, 7203–7208. [Google Scholar]
  21. Akpomie, K.G.; Ghosh, S.; Gryzenhout, M.; Conradie, J. One-pot synthesis of zinc oxide nanoparticles via chemical precipitation for bromophenol blue adsorption and the antifungal activity against filamentous fungi. Sci. Rep. 2021, 11, 8305. [Google Scholar] [CrossRef] [PubMed]
  22. Vijaya, P.P. In vitro biocompatibility and antimicrobial activities of zinc oxide nanoparticles (ZnO NPs) prepared by chemical and green synthetic route—A comparative study. BioNanoScience 2020, 10, 112–121. [Google Scholar]
  23. Hassan, S.E.L.D.; Salem, S.S.; Fouda, A.; Awad, M.A.; El-Gamal, M.S.; Abdo, A.M. New approach for antimicrobial activity and bio-control of various pathogens by biosynthesized copper nanoparticles using endophytic actinomycetes. J. Radiat. Res. Appl. Sci. 2018, 11, 262–270. [Google Scholar] [CrossRef]
  24. Brand-Williams, W.; Cuvelier, M.E.; Berset, C. Use of a free radical method to evaluate antioxidant activity. LWT-Food Sci. Technol. 1995, 28, 25–30. [Google Scholar] [CrossRef]
  25. Ahmed, D.; Baig, H.; Zara, S. Seasonal variation of phenolics, flavonoids, antioxidant and lipid peroxidation inhibitory activity of methanolic extract of Melilotus indicus and its sub-fractions in different solvents. Int. J. Phytomedicine 2013, 4, 326–332. [Google Scholar]
  26. Do, Q.D.; Angkawijaya, A.E.; Tran-Nguyen, P.L.; Huynh, L.H.; Soetaredjo, F.E.; Ismadji, S.; Ju, Y.-H. Effect of extraction solvent on total phenol content, total flavonoid content, and antioxidant activity of Limnophila aromatica. J. Food Drug Anal. 2014, 22, 296–302. [Google Scholar] [CrossRef] [PubMed]
  27. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63. [Google Scholar] [CrossRef]
  28. Denizot, F.; Lang, R. Rapid colorimetric assay for cell growth and survival: Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods 1986, 89, 271–277. [Google Scholar] [CrossRef] [PubMed]
  29. Gheda, S.F.; Abo-Shady, A.M.; Abdel-Karim, O.H.; Ismail, G.A. Antioxidant and Antihyperglycemic Activity of Arthrospira platensis (Spirulina platensis) Methanolic Extract: In vitro and in vivo Study. Egypt. J. Bot. 2021, 61, 71–93. [Google Scholar] [CrossRef]
  30. Sathisha, M.P.; Revankar, V.K.; Pai, K.S.R. Synthesis, Structure, Electrochemistry, and Spectral Characterization of Bis-Isatin Thiocarbohydrazone Metal Complexes and Their Antitumor Activity Against Ehrlich Ascites Carcinoma in Swiss Albino Mice. Met.-Based Drugs 2008, 2008, 362105. [Google Scholar] [CrossRef]
  31. Suddek, G.M. Sunitinib improves chemotherapeutic efficacy and ameliorates cisplatin-induced nephrotoxicity in experimental animals. Cancer Chemother. Pharmacol. 2011, 67, 1035–1044. [Google Scholar] [CrossRef] [PubMed]
  32. Koracevic, D.; Koracevic, G.; Djordjevic, V.; Andrejevic, S.; Cosic, V. Method for the measurement of antioxidant activity in human fluids. J. Clin. Pathol. 2001, 54, 356–361. [Google Scholar] [CrossRef]
  33. Faisal, S.; Jan, H.; Shah, S.A.; Shah, S.; Khan, A.; Akbar, M.T.; Rizwan, M.; Jan, F.; Wajidullah; Akhtar, N.; et al. Green Synthesis of Zinc Oxide (ZnO) Nanoparticles Using Aqueous Fruit Extracts of Myristica fragrans: Their Characterizations and Biological and Environmental Applications. ACS Omega 2021, 6, 9709–9722. [Google Scholar] [CrossRef]
  34. Mishra, D.; Chitara, M.K.; Negi, S.; Pal singh, J.; Kumar, R.; Chaturvedi, P. Biosynthesis of Zinc Oxide Nanoparticles via Leaf Extracts of Catharanthus roseus (L.) G. Don and Their Application in Improving Seed Germination Potential and Seedling Vigor of Eleusine coracana (L.) Gaertn. Adv. Agric. 2023, 2023, 7412714. [Google Scholar] [CrossRef]
  35. Matur, M.; Madhyastha, H.; Shruthi, T.S.; Madhyastha, R.; Srinivas, S.P.; Navya, P.N.; Daima, H.K. Engineering bioactive surfaces on nanoparticles and their biological interactions. Sci. Rep. 2020, 10, 19713. [Google Scholar] [CrossRef] [PubMed]
  36. Mekky, A.; Farrag, A.; Hmed, A.; Sofy, A. Preparation of Zinc Oxide Nanoparticles using Aspergillus niger as Antimicrobial and Anticancer Agents. J. Pure Appl. Microbiol. 2021, 15, 1547–1566. [Google Scholar] [CrossRef]
  37. Jayappa, M.D.; Ramaiah, C.K.; Kumar, M.A.P.; Suresh, D.; Prabhu, A.; Devasya, R.P.; Sheikh, S. Green synthesis of zinc oxide nanoparticles from the leaf, stem and in vitro grown callus of Mussaenda frondosa L.: Characterization and their applications. Appl. Nanosci. 2020, 10, 3057–3074. [Google Scholar] [CrossRef] [PubMed]
  38. Sharma, A.; Sharma, S.; Sharma, K.; Chetri, S.P.K.; Vashishtha, A.; Singh, P.; Kumar, R.; Rathi, B.; Agrawal, V. Algae as crucial organisms in advancing nanotechnology: A systematic review. J. Appl. Phycol. 2016, 28, 1759–1774. [Google Scholar] [CrossRef]
  39. Naseer, M.; Aslam, U.; Khalid, B.; Chen, B. Green route to synthesize Zinc Oxide Nanoparticles using leaf extracts of Cassia fistula and Melia azadarach and their antibacterial potential. Sci. Rep. 2020, 10, 9055. [Google Scholar] [CrossRef] [PubMed]
  40. Elrefaey, A.A.K.; El-Gamal, A.D.; Hamed, S.M.; El-belely, E.F. Algae-mediated biosynthesis of zinc oxide nanoparticles from Cystoseira crinita (Fucales; Sargassaceae) and it’s antimicrobial and antioxidant activities. Egypt. J. Chem. 2022, 65, 231–240. [Google Scholar] [CrossRef]
  41. Mohamed, A.A.; Abu-Elghait, M.; Ahmed, N.E.; Salem, S.S. Eco-friendly Mycogenic Synthesis of ZnO and CuO Nano-particles for In Vitro Antibacterial, Antibiofilm, and Antifungal Applications. Biol. Trace Elem. Res. 2021, 199, 2788–2799. [Google Scholar] [CrossRef] [PubMed]
  42. Azizi, S.; Ahmad, M.B.; Namvar, F.; Mohamad, R. Green biosynthesis and characterization of zinc oxide nanoparticles using brown marine macroalga Sargassum muticum aqueous extract. Mater. Lett. 2014, 116, 275–277. [Google Scholar] [CrossRef]
  43. El-Naggar, N.E.-A.; Hussein, M.H.; El-Sawah, A.A. Phycobiliprotein-mediated synthesis of biogenic silver nanoparticles, characterization, in vitro and in vivo assessment of anticancer activities. Sci. Rep. 2018, 8, 8925. [Google Scholar] [CrossRef]
  44. Aiad, M.A.; Moursi, E.A.; El-Dissoky, R.A.; Amer, M.M. Response of Maize Crop to Irrigation Under Different Rates and Doses of Nitrogen Fertilization in the North Nile Delta Region. J. Soil Sci. Agric. Eng. 2014, 5, 77–92. [Google Scholar] [CrossRef]
  45. Ismail, G.A.; El-Sheekh, M.M.; Samy, R.M.; Gheda, S.F. Antimicrobial, Antioxidant, and Antiviral Activities of Biosynthesized Silver Nanoparticles by Phycobiliprotein Crude Extract of the Cyanobacteria Spirulina platensis and Nostoc linckia. BioNanoScience 2021, 11, 355–370. [Google Scholar] [CrossRef]
  46. Agarwal, H.; Menon, S.; Shanmugam, V.K. Functionalization of zinc oxide nanoparticles using Mucuna pruriens and its antibacterial activity. Surf. Interfaces 2020, 19, 100521. [Google Scholar] [CrossRef]
  47. Al-Dabbas, M.M. Antioxidant activity of different extracts from the aerial part of Moringa peregrina (Forssk.) Fiori, from Jordan. Pak. J. Pharm. Sci. 2017, 30, 2151–2157. [Google Scholar]
  48. Nagajyothi, P.C.; Cha, S.J.; Yang, I.J.; Sreekanth, T.V.M.; Kim, K.J.; Shin, H.M. Antioxidant and anti-inflammatory activities of zinc oxide nanoparticles synthesized using Polygala tenuifolia root extract. J. Photochem. Photobiol. B Biol. 2015, 146, 10–17. [Google Scholar] [CrossRef]
  49. Abifarin, T.O.; Afolayan, A.J.; Otunola, G.A. Phytochemical and Antioxidant Activities of Cucumis africanus L.f.: A Wild Vegetable of South Africa. J. Evid.-Based Integr. Med. 2019, 24, 2515690X19836391. [Google Scholar] [CrossRef]
  50. Sumayya, S.S.; Murugan, K. Antioxidant potentialities of marine red Seaweeds Gracillaria dura: A search. Pharma Innov. J. 2019, 8, 1157–1161. [Google Scholar]
  51. Apak, R.; Özyürek, M.; Güçlü, K.; Çapanoğlu, E. Antioxidant Activity/Capacity Measurement. 2. Hydrogen Atom Transfer (HAT)-Based, Mixed-Mode (Electron Transfer (ET)/HAT), and Lipid Peroxidation Assays. J. Agric. Food Chem. 2016, 64, 1028–1045. [Google Scholar] [CrossRef]
  52. Rajakumar, G.; Thiruvengadam, M.; Mydhili, G.; Gomathi, T.; Chung, I.-M. Green approach for synthesis of zinc oxide nanoparticles from Andrographis paniculata leaf extract and evaluation of their antioxidant, anti-diabetic, and anti-inflammatory activities. Bioprocess. Biosyst. Eng. 2018, 41, 21–30. [Google Scholar] [CrossRef]
  53. Paszek, E.; Czyz, J.; Woźnicka, O.; Jakubiak, D.; Wojnarowicz, J.; Łojkowski, W.; Stepień, E. Zinc oxide nanoparticles impair the integrity of human umbilical vein endothelial cell monolayer in vitro. J. Biomed. Nanotechnol. 2012, 8, 957–967. [Google Scholar] [CrossRef]
  54. Cierech, M.; Wojnarowicz, J.; Kolenda, A.; Krawczyk-Balska, A.; Prochwicz, E.; Woźniak, B.; Łojkowski, W.; Mierzwińska-Nastalska, E. Zinc Oxide Nanoparticles Cytotoxicity and Release from Newly Formed PMMA–ZnO Nano-composites Designed for Denture Bases. Nanomaterials 2019, 9, 1318. [Google Scholar] [CrossRef]
  55. Klaunig, J.E.; Kamendulis, L.M.; Hocevar, B.A. Oxidative Stress and Oxidative Damage in Carcinogenesis. Toxicol. Pathol. 2010, 38, 96–109. [Google Scholar] [CrossRef]
  56. Sanad, M.F.; Nabih, S.; Ma, G. A Lot of Promise for ZnO-5FU Nanoparticles Cytotoxicity against Breast Cancer Cell Lines. J. Nanomed. Nanotechnol. 2018, 9, 1–8. [Google Scholar] [CrossRef]
  57. Qin, X.-J.; He, W.; Hai, C.-X.; Liang, X.; Liu, R. Protection of multiple antioxidants Chinese herbal medicine on the oxidative stress induced by adriamycin chemotherapy. J. Appl. Toxicol. 2008, 28, 282. [Google Scholar] [CrossRef] [PubMed]
  58. Anjum, S.; Hashim, M.; Malik, S.A.; Khan, M.; Lorenzo, J.M.; Abbasi, B.H.; Hano, C. Recent Advances in Zinc Oxide Nanoparticles (ZnO NPs) for Cancer Diagnosis, Target Drug Delivery, and Treatment. Cancers 2021, 13, 4570. [Google Scholar] [CrossRef] [PubMed]
  59. Tang, K.; Lin, Y.; Li, L.-M. The Role of Phenethyl Isothiocyanate on Bladder Cancer ADM Resistance Reversal and Its Molecular Mechanism. Anat. Rec. 2013, 296, 899–906. [Google Scholar] [CrossRef] [PubMed]
  60. Owusu-Apenten, R.; Lam, Y. Enzyme Induction and Cytotoxicity of Phenethyl Isothiocyanate and its Glutathione Conjugate towards Breast Cancer Cells. Pure Appl. Chem. Sci. 2013, 1, 63–73. [Google Scholar] [CrossRef]
  61. Bansal, P.; Medhe, S.; Ganesh, N.; Srivastava, M.M. Isothiocyanates: Naturally occurring HepG2 human liver carcinoma inhibitor. Inter. J. Bioassays 2013, 2, 325–328. [Google Scholar]
  62. Rice-Evans, C.; Burdon, R. Free radical-lipid interactions and their pathological consequences. Prog. Lipid Res. 1993, 32, 71–110. [Google Scholar] [CrossRef]
  63. Okulicz, M.; Bialik, I.; Chichłowska, J. The time-dependent effect of gluconasturtiin and phenethyl isothiocyanate on metabolic and antioxidative parameters in rats. J. Anim. Physiol. Anim. Nutr. 2005, 89, 367–372. [Google Scholar] [CrossRef]
  64. Gharpure, S.; Yadwade, R.; Ankamwar, B. Non-antimicrobial and Non-anticancer Properties of ZnO Nanoparticles Biosynthesized Using Different Plant Parts of Bixa orellana. ACS Omega 2022, 7, 1914–1933. [Google Scholar] [CrossRef]
  65. Agrawal, S.S.; Saraswati, S.; Mathur, R.; Pandey, M. Antitumor properties of Boswellic acid against Ehrlich ascites cells bearing mouse. Food Chem. Toxicol. 2011, 49, 1924–1934. [Google Scholar] [CrossRef]
  66. Azarin, K.; Usatov, A.; Minkina, T.; Plotnikov, A.; Kasyanova, A.; Fedorenko, A.; Duplii, N.; Vechkanov, E.; Rajput, V.D.; Mandzhieva, S.; et al. Effects of ZnO nanoparticles and its bulk form on growth, antioxidant defense system and expression of oxidative stress related genes in Hordeum vulgare L. Chemosphere 2022, 287, 132167. [Google Scholar] [CrossRef] [PubMed]
  67. Bratovcic, A. Antioxidant enzymes and their role in preventing cell damage. Acta Sci. Nutr. Health 2020, 4, 1–7. [Google Scholar] [CrossRef]
Applsci 14 07069 g001
Figure 1. Used seaweeds: Ulva lactuca (A), Ulva intestinalis (B) (Chlorophyta), and Sargassum muticum (C) (Phaeophyceae).
Figure 1. Used seaweeds: Ulva lactuca (A), Ulva intestinalis (B) (Chlorophyta), and Sargassum muticum (C) (Phaeophyceae).
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Figure 2. UV spectra of ZnO nanoparticles synthesized using different seaweeds: Ulva lactuca (A), Ulva intestinalis (B), and Sargassum muticum (C).
Figure 2. UV spectra of ZnO nanoparticles synthesized using different seaweeds: Ulva lactuca (A), Ulva intestinalis (B), and Sargassum muticum (C).
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Figure 3. FTIR analysis of ZnO nanoparticles of the different seaweeds: Ulva lactuca (A), Ulva intestinalis (B), and Sargassum muticum (C).
Figure 3. FTIR analysis of ZnO nanoparticles of the different seaweeds: Ulva lactuca (A), Ulva intestinalis (B), and Sargassum muticum (C).
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Figure 4. XRD analysis of ZnO nanoparticles prepared from tested seaweeds: Ulva lactuca (A), Ulva intestinalis (B), and Sargassum muticum (C).
Figure 4. XRD analysis of ZnO nanoparticles prepared from tested seaweeds: Ulva lactuca (A), Ulva intestinalis (B), and Sargassum muticum (C).
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Figure 5. Transmission electron micrographs of ZnO-NPs biosynthesized using different seaweeds: Ulva lactuca (A), Ulva intestinalis (B), and Sargassum muticum (C).
Figure 5. Transmission electron micrographs of ZnO-NPs biosynthesized using different seaweeds: Ulva lactuca (A), Ulva intestinalis (B), and Sargassum muticum (C).
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Figure 6. Zeta potential analysis of ZnO-NPs biosynthesized using different seaweeds: Ulva lactuca (A), Ulva intestinalis (B), and Sargassum muticum (C).
Figure 6. Zeta potential analysis of ZnO-NPs biosynthesized using different seaweeds: Ulva lactuca (A), Ulva intestinalis (B), and Sargassum muticum (C).
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Figure 7. Effects of Ulva lactuca ZnO-NP treatment on tumor volume.
Figure 7. Effects of Ulva lactuca ZnO-NP treatment on tumor volume.
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Figure 8. Effects of Ulva lactuca ZnO-NP treatment on tumor weight.
Figure 8. Effects of Ulva lactuca ZnO-NP treatment on tumor weight.
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Figure 9. Effects of Ulva lactuca ZnO-NP treatment on oxidative stress.
Figure 9. Effects of Ulva lactuca ZnO-NP treatment on oxidative stress.
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Figure 10. Microscopic pictures of H&E-stained, untreated Ehrlich ascites tumors (EAC) and the treated groups. (A) Ehrlich acetic carcinoma, (B) doxorubicin, (C) ZnO-NPs biosynthesized using Ulva lactuca extract. Low magnification 100×: bar 100; higher magnification 400×: bar 50.
Figure 10. Microscopic pictures of H&E-stained, untreated Ehrlich ascites tumors (EAC) and the treated groups. (A) Ehrlich acetic carcinoma, (B) doxorubicin, (C) ZnO-NPs biosynthesized using Ulva lactuca extract. Low magnification 100×: bar 100; higher magnification 400×: bar 50.
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Table 1. Inhibition zone diameters (mm) of ZnO-NPs biosynthesized using different seaweeds.
Table 1. Inhibition zone diameters (mm) of ZnO-NPs biosynthesized using different seaweeds.
ZnO-NP TreatmentPotency
(mg/mL)		Zones of Inhibition (mm)f Value
Klebsiella pueumoniaeE. coliCandida albicansAspergillus niger
Ulva lactuca
ZnO-NPs		6017.8 ± 1.5420.7 ± 2.0619.3 ± 1.4221.3 ± 2.192.861 **
3014.2 ± 0.9713.3 ± 0.8315.2 ± 1.0513.4 ± 1.123.848 *
1511.7 ± 0.6812.6 ± 0.7611.4 ± 0.4111.2 ± 0.35.904 *
Sargassum muticum
ZnO-NPs		6015.1 ± 0.9519.1 ± 1.2318.6 ± 1.6317.7 ± 1.42.955 **
3013.3 ± 1.1112.5 ± 0.4215.7 ± 1.714.3 ± 0.883.871 *
1512.8 ± 0.8411.3 ± 0.312.2 ± 0.4911 ± 0.215.874 *
Ulva intestinalis
ZnO-NPs		6017.6 ± 1.3316.9 ± 1.6115.8 ± 0.5316.2 ± 1.093.018 **
3013.1 ± 0.7415.2 ± 1.2813.9 ± 0.9415.1 ± 0.743.83 *
1512.8 ± 0.9512.4 ± 0.4911.1 ± 0.2212.1 ± 0.515.812 *
AntibioticsCiprofloxacin13.4 ± 0.8712.8 ± 0.27
Amoxicillinax 25R20.2 ± 2.65
S-Adenosylmethionin R
Synthetic antifungalFluconazole R12.3 ± 0.56
Isothiocyanafcs R11.7 ± 0.63
Metronidazole R14.2 ± 1.12
Negative controlsZN Acetate13.6 ± 1.0211.1 ± 1.0313.4 ± 0.00
Ulva lactuca13.2 ± 0.8311.6 ± 0.5412.5 ± 0.65
Sargassum muticum13.1 ± 0.8NA11.9 ± 0.36
Ulva
intestinalis		12.6 ± 0.2411.7 ± 0.32NA
Each value is the mean of three replicates ± standard deviation. p > 0.05, nonsignificant; p < 0.05, * significant; p < 0.001, ** highly significant. R = Resistance, NA = no activity.
Table 2. DPPH radical scavenging activity (%) of the biosynthesized ZnO-NPs of different seaweeds.
Table 2. DPPH radical scavenging activity (%) of the biosynthesized ZnO-NPs of different seaweeds.
ZnO-NPs
Concentration (μg/mL)		Seaweedsf Value
Ulva lactucaSargassum
muticum		Ulva intestinalis
Radical scavenging %3.3721.23 ± 2.4520.15 ± 2.8419.61 ± 3.70.368 *
6.7537.1 ± 4.6634.2 ± 4.7831.09 ± 5.121.915 **
12.549.3 ± 5.4248.5 ± 5.9845.2 ± 6.120.691 *
2564.2 ± 6.3762.1 ± 6.5959.8 ± 8.470.466 *
5086.47 ± 7.5684.3 ± 8.4578.1 ± 6.981.596 *
IC50 (µg/mL)12.6 ± 1.4613.8 ± 1.4515.2 ± 2.562.355 **
Each value is the mean of three replicates ± standard deviation. p > 0.05, nonsignificant; p < 0.05, * significant; p < 0.001, ** highly significant.
Table 3. Total antioxidant capacity (TAC) of the ZnO-NPs biosynthesized using different seaweeds.
Table 3. Total antioxidant capacity (TAC) of the ZnO-NPs biosynthesized using different seaweeds.
ZnO-NP Concentration (mg/mL)Seaweedf Value
Ulva lactucaSargassum muticumUlva intestinalis
5038 ± 5.1235 ± 4.0829 ± 3.785.512 *
2526 ± 3.7825.3 ± 2.9821 ± 2.453.769 *
12.514 ± 2.812.9 ± 1.659.9 ± 1.155.684 *
Each value is the mean of three replicates ± standard deviation. p > 0.05, nonsignificant; p < 0.05, * significant.
Table 4. Ferric reducing antioxidant power (FRAP) of ZnO-NPs biosynthesized using different seaweeds.
Table 4. Ferric reducing antioxidant power (FRAP) of ZnO-NPs biosynthesized using different seaweeds.
ZnO-NP Concentration (mg/mL)Seaweedf Value
Ulva lactucaSargassum
muticum		Ulva intestinalis
5049.57 ± 6.1235.23 ± 5.1226.4 ± 2.8428.593 **
2534.52 ± 4.6323 ± 2.8421.3 ± 3.1419.712 **
12.518.2 ± 2.4515 ± 2.377.9 ± 2.1525.665 **
Each value is the mean of three replicates ± standard deviation. p > 0.05, nonsignificant; p < 0.001, ** highly significant.
Table 5. Cytotoxicity assay of biosynthesized ZnO-NPs against different cell lines.
Table 5. Cytotoxicity assay of biosynthesized ZnO-NPs against different cell lines.
No.TreatmentIn Vitro Cytotoxicity IC50 (µg/mL)f Value
HePG2HCT-116MCF-7WI-38
DOX4.50 ± 0.25.23 ± 0.34.17 ± 0.26.72 ± 0.530.932 **
1ZnO-NPs of Ulva lactuca46.66 ± 2.858.69 ± 3.330.60 ± 2.184.98 ± 4.6181.938 **
Each value is the mean of three replicates ± the standard deviation. p > 0.05, nonsignificant; p < 0.001, ** highly significant. IC50 (µg/mL): 1–10 (very strong), 11–20 (strong), 21–50 (moderate), 51–100 (weak), and above 100 (non-cytotoxic). DOX: doxorubicin.
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Abotaleb, S.I.; Gheda, S.F.; Allam, N.G.; El-Shatoury, E.H.; Cotas, J.; Pereira, L.; Saeed, A.M. Biosynthesis of Zinc Oxide Nanoparticles Using Seaweed: Exploring Their Therapeutic Potentials. Appl. Sci. 2024, 14, 7069. https://doi.org/10.3390/app14167069

AMA Style

Abotaleb SI, Gheda SF, Allam NG, El-Shatoury EH, Cotas J, Pereira L, Saeed AM. Biosynthesis of Zinc Oxide Nanoparticles Using Seaweed: Exploring Their Therapeutic Potentials. Applied Sciences. 2024; 14(16):7069. https://doi.org/10.3390/app14167069

Chicago/Turabian Style

Abotaleb, Sohaila I., Saly F. Gheda, Nanis G. Allam, Einas H. El-Shatoury, João Cotas, Leonel Pereira, and Ali M. Saeed. 2024. "Biosynthesis of Zinc Oxide Nanoparticles Using Seaweed: Exploring Their Therapeutic Potentials" Applied Sciences 14, no. 16: 7069. https://doi.org/10.3390/app14167069

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