Green Synthesis of Zinc Oxide Nanoparticles: Preparation, Characterization, and Biomedical Applications - A Review

Mohamed T El-Saadony; Guihong Fang; Si Yan; Samar Sami Alkafaas; Mahmoud A El Nasharty; Sohila A Khedr; Aya Misbah Hussien; Soumya Ghosh; Mthokozisi Dladla; Sara Samy Elkafas; Essam H Ibrahim; Heba Mohammed Salem; Walid FA Mosa; Ahmed Ezzat Ahmed; Dina Mostafa Mohammed; Sameh A Korma; Marawan K El-Tarabily; Ahmed M Saad; Khaled A El-Tarabily; Synan F AbuQamar

doi:10.2147/IJN.S487188

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Review

Green Synthesis of Zinc Oxide Nanoparticles: Preparation, Characterization, and Biomedical Applications - A Review

Authors El-Saadony MT , Fang G, Yan S, Alkafaas SS , El Nasharty MA , Khedr SA, Hussien AM, Ghosh S, Dladla M , Elkafas SS, Ibrahim EH, Salem HM , Mosa WF, Ahmed AE, Mohammed DM, Korma SA, El-Tarabily MK, Saad AM, El-Tarabily KA , AbuQamar SF

Received 16 July 2024

Accepted for publication 17 October 2024

Published 3 December 2024 Volume 2024:19 Pages 12889—12937

DOI https://doi.org/10.2147/IJN.S487188

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 3

Editor who approved publication: Prof. Dr. RDK Misra

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Mohamed T El-Saadony,^1,^* Guihong Fang,^2,^3,^* Si Yan,³ Samar Sami Alkafaas,⁴ Mahmoud A El Nasharty,⁵ Sohila A Khedr,⁶ Aya Misbah Hussien,⁷ Soumya Ghosh,⁸ Mthokozisi Dladla,⁹ Sara Samy Elkafas,^10,¹¹ Essam H Ibrahim,^12,¹³ Heba Mohammed Salem,¹⁴ Walid FA Mosa,¹⁵ Ahmed Ezzat Ahmed,¹² Dina Mostafa Mohammed,¹⁶ Sameh A Korma,¹⁷ Marawan K El-Tarabily,¹⁸ Ahmed M Saad,¹⁹ Khaled A El-Tarabily,²⁰ Synan F AbuQamar²⁰

¹Department of Agricultural Microbiology, Faculty of Agriculture, Zagazig University, Zagazig, 44511, Egypt; ²School of Public Health, Heinz Mehlhorn Academician Workstation, Hainan Medical University, Haikou, Hainan, 571199, People’s Republic of China; ³Qionghai People’s Hospital, Qionghai, Hainan, 571400, People’s Republic of China; ⁴Molecular Cell Biology Unit, Division of Biochemistry, Department of Chemistry, Faculty of Science, Tanta University, Tanta, 31527, Egypt; ⁵Department of Chemistry, Faculty of Science, Ain Shams University, Cairo, 11566, Egypt; ⁶Industrial Biotechnology Department, Faculty of Science, Tanta University, Tanta, 31733, Egypt; ⁷Biotechnology Department at Institute of Graduate Studies and Research, Alexandria University, Alexandria, 21531, Egypt; ⁸Natural and Medical Sciences Research Center, University of Nizwa, Nizwa, 616, Oman; ⁹Human Molecular Biology Unit (School of Biomedical Sciences), Faculty of Health Sciences, University of the Free State, Bloemfontein, 9300, South Africa; ¹⁰Production Engineering and Mechanical Design Department, Faculty of Engineering, Menofia University, Shebin El Kom, Menofia, 32511, Egypt; ¹¹Faculty of Control System and Robotics, ITMO University, Saint-Petersburg, 197101, Russia; ¹²Biology Department, Faculty of Science, King Khalid University, Abha, 61413, Saudi Arabia; ¹³Blood Products Quality Control and Research Department, National Organization for Research and Control of Biologicals, Cairo, 12611, Egypt; ¹⁴Department of Poultry Diseases, Faculty of Veterinary Medicine, Cairo University, Giza, 12211, Egypt; ¹⁵Plant Production Department (Horticulture-Pomology), Faculty of Agriculture, Saba Basha, Alexandria University, Alexandria, 21531, Egypt; ¹⁶Nutrition and Food Sciences Department, National Research Centre, Dokki, Giza, 12622, Egypt; ¹⁷Department of Food Science, Faculty of Agriculture, Zagazig University, Zagazig, 44511, Egypt; ¹⁸Faculty of Medicine, University of Debrecen, Debrecen, 4032, Hungary; ¹⁹Department of Biochemistry, Faculty of Agriculture, Zagazig University, Zagazig, 44511, Egypt; ²⁰Department of Biology, College of Science, United Arab Emirates University, Al Ain, 15551, United Arab Emirates

*These authors contributed equally to this work

Correspondence: Si Yan, Qionghai People’s Hospital, Qionghai, Hainan, 571400, People’s Republic of China, Email [email protected] Synan F AbuQamar, Department of Biology, College of Science, United Arab Emirates University, Al Ain, 15551, United Arab Emirates, Tel +971 3 713 6733, Fax +971 3 713 4927, Email [email protected]

Abstract: Over the last decade, biomedical nanomaterials have garnered significant attention due to their remarkable biological properties and diverse applications in biomedicine. Metal oxide nanoparticles (NPs) are particularly notable for their wide range of medicinal uses, including antibacterial, anticancer, biosensing, cell imaging, and drug/gene delivery. Among these, zinc oxide (ZnO) NPs stand out for their versatility and effectiveness. Recently, ZnO NPs have become a primary material in various sectors, such as pharmaceutical, cosmetic, antimicrobials, construction, textile, and automotive industries. ZnO NPs can generate reactive oxygen species and induce cellular apoptosis, thus underpinning their potent anticancer and antibacterial properties. To meet the growing demand, numerous synthetic approaches have been developed to produce ZnO NPs. However, traditional manufacturing processes often involve significant economic and environmental costs, prompting a search for more sustainable alternatives. Intriguingly, biological synthesis methods utilizing plants, plant extracts, or microorganisms have emerged as ideal for producing ZnO NPs. These green production techniques offer numerous medicinal, economic, environmental, and health benefits. This review highlights the latest advancements in the green synthesis of ZnO NPs and their biomedical applications, showcasing their potential to revolutionize the field with eco-friendly and cost-effective solutions.

Keywords: antimicrobial, antioxidant, green synthesis, nanomedicine, nanoparticle, zinc oxide

Introduction

Zinc (Zn) is essential to human, animal, and plant metabolism, playing a key role in muscles, bones, skin, and brain function.¹ As an essential trace mineral, zinc oxide (ZnO) is commonly used in dietary supplements, cosmetics, and medical products. While most commercially available ZnO is synthetically produced, it also naturally occurs in the earth’s crust as the mineral zincite. ZnO is known for being safe and gentle on human skin, making it ideal for various topical applications.¹ ZnO nanoparticles (NPs) have diverse applications across multiple industries, including use as photocatalysts,² ethanol gas sensors,^1,3 ultraviolet (UV) light-emitting devices,^4,5 and in the pharmaceutical,⁶ and cosmetics sectors.⁷ Metal and metal oxide NPs, including ZnO NPs are integral to advancements in textiles, medicine, catalysis, pharmaceuticals, agriculture, heavy industrial consumer products, and antimicrobial testing.^8–11 This versatility highlights the significant role of ZnO and its nanoparticulate forms in driving innovation and improving products that touch our daily lives.^9–11

ZnO NPs offer a unique blend of safety, affordability, and versatility, making them a standout among metal oxide NPs.¹² Their strong UV absorption and transparency to visible light make them highly effective as sunblock agents.¹³ In addition, their ability to generate reactive oxygen species (ROS) has spurred research into their antibacterial and anticancer properties.¹⁴ ZnO NPs are increasingly recognized for their biomedical applications, particularly in drug delivery systems and bioimaging.¹⁵ They can target and release drugs at specific sites, enhancing therapeutic outcomes, and their bioimaging capabilities improve the visualization of biological processes and disease states.¹³ The United States Food and Drug Administration (FDA) has approved ZnO in bulk as a generally recognized safe substance, and ZnO NPs larger than 100 nm are deemed to be compatible with biological systems,¹⁵ highlighting their potential in pharmaceutical delivery applications. Moreover, ZnO NPs are being explored for wound healing due to their antimicrobial properties and ability to promote tissue regeneration. This multifunctionality positions ZnO NPs as a promising tool in various medical and pharmaceutical applications, driving innovation and improving patient care.¹⁵

Recent research highlights the advantages of using biological sources as reducing agents in the synthesis of ZnO NPs.¹⁶ This eco-friendly approach is favored for its non-hazardous simplicity, low energy consumption, and cost-effectiveness. Plant-derived compounds, such as terpenoids, alkaloids, polyphenols, saponins, flavanones, and tannins, have been shown to effectively reduce Zn precursors. In addition, plant extracts have demonstrated superior efficacy against bacterial and fungal infections.¹⁷ This review provides a comprehensive overview of the latest advancements in the green synthesis of ZnO NPs and explores their promising medical applications.

Transition from Conventional to Green Synthesis of ZnO NPs

ZnO NPs offer a balanced combination of low toxicity and high biodegradability compared to other nanomaterials, such as gold nanoparticles (AuNPs), which are non-biodegradable and can accumulate in tissues.^18,19 While liposomes also possess non-toxic and biodegradable characteristics, ZnO NPs provide additional therapeutic benefits due to their antibacterial and anti-inflammatory properties.²⁰ Moreover, ZnO NPs are multifunctional and can selectively generate ROS in cancer cells, a significant advantage in targeted cancer therapy.²¹ Their potential applications in imaging and diagnostics further enhance their appeal.²¹

NPs can be generated through biological, chemical, and physical methods.²² However, physical methods are not ideal for large-scale production due to low yield, high energy requirements, and increased input costs.^23,24 While chemical methods have become the preferred choice for NP synthesis,^22,23,25 they are time-consuming, labor-intensive, and pose hazardous effects on humans and the environment.^22,23,25 In addition, chemo-synthesized NPs often exhibit instability and toxicity, limiting their biomedical applications.^26,27 Therefore, developing efficient, reliable, safe, and environmentally friendly techniques for NP synthesis is crucial.²⁷

Recently, green biological-mediated approaches have gained attention.^28,29 Plants and microorganisms can generate NPs that are safe, environmentally sustainable, and cost-effective.^30,31 Biological agents, such as algae, bacteria, fungi, and plants, can serve as solvents and stabilizers, creating a green synthetic pathway for NPs and reducing the toxicity of the end product.³² This ability not only diminishes environmental pollution but also transforms heavy metals (HMs) from industrial wastes into safer compounds. The distinctive biochemical mechanisms of these agents offer novel and untapped avenues for converting inorganic metallic ions into metal NPs.^30,33,34 Plants contain diverse metabolites and biomolecules, including proteins, vitamins, coenzyme-based intermediates, phenols, flavonoids, and carbohydrates, which can interact with metal ions and reduce their sizes to the nano range.³⁵ For example, flavonoids, characterized by their polyphenolic structures and multiple hydroxyl (-OH) groups attached to aromatic rings, can donate electrons to reduce metal ions such as Zn²⁺ and Ag⁺ to their corresponding metallic NPs. Similarly, phenolic compounds, with their hydroxyl groups, act as electron donors, facilitating the reduction of metal ions.^17,36

Once the metal ions are reduced, flavonoids and phenolic compounds further contribute to the stabilization and capping of the NPs.³⁷ The hydroxyl and other functional groups in these compounds form strong interactions, such as hydrogen bonding and van der Waals forces, with the metal surface, preventing NP aggregation and enhancing their stability in suspension.^37,38 This capping process not only stabilizes NPs but also improves their biocompatibility, making them suitable for various biomedical applications.³⁸

For instance, the flavonoid quercetin has been shown to effectively synthesize silver nanoparticles (AgNPs).³⁹ Quercetin, with its multiple hydroxyl groups, reduces Ag⁺ to Ag and subsequently caps the AgNPs, enhancing their stability. Another example involves the use of Aloe vera extract in the green synthesis of ZnO NPs.⁴⁰ A. vera extract, rich in polyphenols, vitamins, enzymes, and amino acids, acts as both a reducing and capping agent. The polyphenolic compounds, such as flavonoids and tannins, reduce Zn²⁺ ions from zinc nitrate to ZnO NPs.^40,41 Simultaneously, the organic molecules, including polysaccharides and proteins in the extract, cap the NPs, preventing aggregation and ensuring stability.⁴⁰ This dual action of reduction and capping by A. vera extract not only prevents ZnO NPs from clumping together but also enhances their biocompatibility and dispersibility in aqueous solutions, making this method environmentally friendly and suitable for various applications, including antibacterial and anticancer therapies.⁴¹

Similar to plants, green algae (Chlorophyceae), blue-green algae (Cyanophyceae), brown algae (Phaeophyceae), and red algae (Rhodophyceae) have secondary metabolites and demonstrate remarkable efficiency in producing NPs of metals and metal oxides.^42–44 Algae contain cytotoxic substances, such as laminarians, terpenoids, and fucoidans, which can combat cancer, inhibit proliferation, and suppress tumors.^45–48 Due to their lack of external reducing or capping agents, high energy efficiency, affordability, safety, and simplicity, algae are highly recommended for green NP synthesis in the pharmaceutical and biomedical sectors.^30,48

The biological synthesis of NPs using microorganisms has gained interest as a sustainable approach for NP production. Bacteria, which can be easily cultured, generate bioactive molecules in liquid form that convert metal ions into metallic NPs.^28,49,50 The cellular mass of bacteria can serve as miniature factories for producing metal oxide NPs.^50–52 These “nano-factories” hold great potential in modern nanotechnology, facilitating the production of various types of NPs. Typically, green NPs are derived from live cells, bioactive molecules isolated from biological systems, or cell-free supernatants.⁵³ Although the precise production process is not yet fully understood, essential enzymes have been found to contain amino and carboxylic groups that bind to metal ions and subsequently reduce them into metal NPs.^54,55 Biological green approaches use plants, plant extracts, or microorganisms to generate NPs as environmentally friendly alternatives to chemical and physical processes. Utilizing microorganisms in NPs production requires complex procedures for maintaining cell cultures and several purification steps.⁵⁶

Besides being expensive and time-consuming, common methods like chemical precipitation generate dangerous chemical species that stick to surfaces and harm healthcare applications.^56,57 Solvent-based techniques, such as solvothermal,⁵⁸ hydrothermal,^59,60 sol-gel,^61,62 chemical precipitation,⁶³ are among the most frequently employed strategies for producing ZnO NPs. Some reactions require air and heat to initiate, while others need a nonreactive environment and employ lethal compounds like hydrogen sulfide (H₂S) and toxic stabilizers. These compounds used to stabilize NPs are poisonous and have harmful consequences.⁶⁴ Hazardous substances generated through chemical processes are surface-absorbable and negatively affect medical applications.⁶⁴

To address these issues, eco-friendly NP production technology has been developed. Green NPs utilize harmless, non-hazardous, and eco-friendly compounds.⁵⁶ Gelatin is used as a stabilizing agent when the sol-gel method is modified for producing ZnO NPs.⁶⁵ These ZnO NPs display a hexagonal (Wurtzite) morphology and range from 30–60 nm in diameter. Studies in the impact of varying oxidation temperatures on the structure of ZnO NPs suggest that gelatin holds significant potential as a stabilizing agent in the sol-gel method for generating ZnO particles at the nanoscale.^63,65 Due to the high costs and requirements associated with chemical solvents as reducing agents in conventional methods,⁶⁶ green methods have been used to synthesize ZnO NPs in a flower-shaped form that has garnered increased attention. ZnO NPs have been exploited in various industrial sectors, including pharmaceuticals, cosmetics, antibacterial products, textiles, and automotive industries.^56,67 Recent studies indicate that ZnO NPs have superior antibacterial capabilities compared to microparticles.⁶⁸

Eco-friendly materials like algae, bacteria, fungi, plant leaf extract, natural materials, and biopolymers, offer several advantages in the production of ZnO NPs by leveraging the inherent qualities of microorganisms and plant, especially for pharmaceutical and medical applications.^56,67 ZnO NPs serve as fillers in medicinal products, cosmetics, and medication carriers.⁶⁹ Compared to AgNPs, ZnO NPs are cost-effective and have a desirable white appearance.⁷⁰ Plant extract are widely used methods to manufacture ZnO NPs. Aloe barbadensis leaf extract was used for fabricating ZnO NPs, yielding spherical and highly stable particles with sizes ranging from 25–40 nm. Adjusting the quantity of leaf broth solution can modulate particle size.⁷¹ Algal-based synthesis is another method, where biological catalysts offer greater specificity and control, allowing customization of NP properties.^72,73 Overall, green synthesis techniques for producing ZnO NPs offer sustainable methods utilizing enzymatic, plant, microbial, and algae systems, highlighting their potential for eco-friendly nanomaterials and encouraging further research for diverse applications in biomedical sciences, sensor technology, catalysis, and other fields.⁷³

Biosynthesis of ZnO NPs Using Bacteria

Biological methods offer a promising alternative for synthesizing NPs, presenting clear benefits over other approaches due to their safety, simplicity, non-toxic nature, eco-friendliness, biocompatibility, and cost-effectiveness.⁷⁴ This process utilizes biologically active materials from microorganisms or plant extracts to create ZnO NPs. These materials serve dual roles in NP synthesis, acting as both reducing and capping agents. Typically, the biosynthesis of ZnO NP synthesis involves a metal precursor, such as soluble salts, into prepared biological extracts.⁷⁴ The reaction leads to a color change, resulting in ZnO NPs powder.⁷⁴

Microbial fabrication of ZnO NPs can be classified into intracellular and extracellular synthesis. In intracellular synthesis, Zn precursor molecules are taken up by microbial cells, reduced and then extruded as ZnO NPs.⁷² For instance, Lactobacillus paracasei from dairy products can produce spherical ZnO NPs intracellularly, while Bacillus licheniformis and Bacillus subtilis can synthesize them extracellularly.^72–79

The process begins with bacterial isolation and purification on nutrient agar plates using the serial dilution method, followed by incubation at 35–37°C for 24 hours.⁷³ Pure colonies are then cultured for ZnO NPs production. Each bacterial strain is in nutrient broth (at 35–37°C for 24 hours) under shaking conditions. The supernatant is then used for ZnO NP synthesis, where zinc sulfate (ZnSO₄) and sodium hydroxide are mixed with the culture filtrate, followed by heating, microwaving, and cooling to facilitate NP formation.⁷³ The resulting white deposition is then washed, centrifuged, and oven-dried at 40°C for 8 hours to obtain powdered ZnO NPs. The NPs are stored for further study, and the most potent bacterial strain is identified.^72,75 Table 1 illustrates the various bacterial strains suitable for environmentally friendly ZnO NPs synthesis.

Table 1 Green Synthesis of ZnO NPs Using Bacteria

Biosynthesis of ZnO NPs Using Fungi

Fungal biomass serves as a readily available and renewable resource for producing nanostructured ZnO using an eco-friendly synthesis method.⁷⁶ This fungal synthesis technique is similar to microbiological procedures, including the isolation and culture of fungi in typical fungal extract preparations.⁷⁶ During cultivation, the fungal strain is maintained in a sterile environment at a controlled temperature. After a set period, the liquid medium, free of cells but rich in fungal byproducts, is separated by filtration and centrifugation. This solution is then used to produce ZnO NPs.⁷⁷

Among various fungi, Aspergillus niger is frequently used as the eco-friendly synthesis of ZnO NPs. Successful syntheses have been achieved using A. niger and Aspergillus terreus, which act as biological reducing and capping agents.⁷⁵ Biomass from fungal strains such as Fusarium oxysporum, Penicillium citrinum, Aspergillus fumigatus, Aspergillus tubulin, and A. niger, can be cultivated in malt glucose yeast peptone (MGYP) broth. These strains are isolated from soil with high HM contamination, unlike standard strains of A. tubulin, A. fumigatus, F. oxysporum, and P. citrinum. The fungi are grown in 250 mL Erlenmeyer flasks containing 50 mL liquid medium with glucose (15.0 g), yeast extract (1.0 g), (NH₄)₂SO₄ (1.0 g), MgSO₄7H₂O (0.1 g), K₂HPO₄ (2.0 g), and KH₂PO₄ (7.0 g) per liter.⁷⁸ Incubation occurred at 28 ± 2°C and 180 rpm for 5 days. The fungal biomass is then washed and transferred to deionized water for further incubation. After this period, the biomass is filtered, and the cell-free filtrate is used for ZnO NP biosynthesis.⁷⁸ Each treatment involves mixing 10 mL of 3.0 mM ZnSO₄ solution with 10 mL of fungal filtrate, adjusting the pH to 6.5, and incubating in an orbital shaker for 72 h in the dark.⁷⁸ The formation of NPs is indicated by a whitish precipitate at the flask’s base, which is separated by centrifugation at 10,000 rpm for 10 min. Positive and negative controls are maintained by incubating fungal mycelium with deionized water and ZnSO₄ solution, respectively.⁷⁸

This promising, eco-friendly, and cost-effective approach in nanotechnology faces several limitations and challenges. One significant issue is the extended incubation times required for fungal cultures, which generally grow more slowly than bacterial cultures.⁷⁹ This necessitates longer periods for NP synthesis and involves extensive trial and error to optimize conditions for maximum yield and desired NP properties.⁷⁹ In addition, fungal synthesis requires specific environmental conditions, including precise temperature and pH ranges, as well as a balanced supply of nutrients. Deviations from these conditions can lead to suboptimal synthesis or even complete failure of the process.⁷⁹

Yield variations also pose a challenge, as different fungal strains exhibit varying capabilities for synthesizing ZnO NPs.^100,101 These results leads to inconsistencies in yield and NP properties. Moreover, factors such as medium composition, aeration, and light exposure significantly affect the yield and quality of ZnO NPs, making reproducibility difficult.¹⁰² These biological variabilities complicate the scaling up of the process for industrial applications.^100,102

Post-synthesis, the purification and processing of NPs to remove biological contaminants is often complex and involves multiple steps. Controlling the size and shape of ZnO NPs is crucial for their applications; However, achieving uniformity remains challenging with biological synthesis methods.¹⁰²

Environmental and health concerns must also be addressed, necessitating strict biosafety measures to prevent contamination and mitigate potential health risks associated with handling live fungal cultures.¹⁰¹ Although the method is environmentally friendly, the disposal of fungal biomass and by-products must be managed carefully to avoid environmental contamination.^101,102 Addressing these limitations requires ongoing research and development to optimize conditions, improve yields, and ensure the consistency and scalability of fungal synthesis of ZnO NPs.¹⁰² Table 2 summarizes studies on the green production of ZnO NPs utilizing fungi.

Table 2 Green Synthesis of ZnO NPs Using Fungi

Biosynthesis of ZnO NPs Using Plants

The biological synthesis of NPs offers a viable alternative to conventional chemical or physical fabrication techniques.^24,56,129 Most studies focus on eco-friendly methods for producing metal and oxide NPs, highlighting the efficiency and safety of plant-based synthesis.¹³⁰ Factors like pH levels and annealing temperature significantly influence the size and morphology of the ZnO NPs. Similarly, gelatin was used as a stabilizing agent in the sol-gel method to produce hexagonal ZnO NPs, which are calcined at various temperatures.¹³¹ Jiménez-Rosado et al¹³² have produced ZnO NPs from pepper extracts high in polyphenols, finding that green synthesis may yield pure smaller NPs than chemical methods.¹³²

Further studies demonstrated the synthesis of ZnO using plant extracts like Solanum rantonnetii and thyme, with varying calcination temperatures affecting the NPs’ characteristics.¹³³ For instance, thyme-synthesized ZnO NPs may exhibit optimal quality of 450°C.¹³⁴ Leaf extracts of Turkish pine (Pinus Brutia) were also used, showing that the pH levels can significantly alter the morphology and size of ZnO NPs.¹³⁵ MuthuKathija et al¹³⁶ have utilized Pisonia alba leaf extract to produce ZnO NPs with notable ultraviolet–visible (UV-Vis) spectroscopy at 375 nm.

Additional research explored various plant extracts for ZnO NP synthesis, including Vitex negundo, Trifolium pratense, Lagenaria siceraria, and green tea leaves, each demonstrating specific applications from antibacterial properties to supercapacitor potential.¹³⁷ Other studies highlight the effectiveness of ZnO NPs in treating urinary tract infections and other medical conditions.¹³⁸ Table 3 depicts plant species suited for ecologically friendly ZnO NPs production.

Table 3 Green Synthesis of ZnO NPs Using Plants

Biosynthesis of ZnO NPs Using Algae

The eco-friendly production of ZnO NPs through algae represents a sustainable method with potential applications across diverse sectors.^157,158 Algae, abundant and cost-effective biological resources, present a distinct advantage for synthesizing ZnO NPs due to their inherent capacity to generate bioactive substances.¹⁵⁸ This procedure utilizes the bioactive elements in algae, such as pigments and proteins, to reduce Zn precursors and stabilize the resulting NPs. This green method eliminates the need for hazardous chemicals and energy-intensive processes, thus reducing environmental impact.¹⁵⁸

In addition, the ZnO NPs produced demonstrate improved compatibility with biological systems, showing promise for various applications in medical settings, agriculture, and environmental remediation.¹⁵⁹ The creation of ZnO NPs through green synthesis using algae showcases the seamless fusion of biotechnology and nanotechnology, presenting an innovative and environmentally friendly approach to develop novel nanomaterials.^158,159 Table 4 illustrates the types of algae that can be utilized in the successful green synthesis of ZnO NPs.

Table 4 Green Synthesis of ZnO NPs Using Algae

Traditional versus Green Methods in Producing ZnO NPs

Both traditional and green methods have their advantages and challenges in the production of ZnO NPs. Traditional methods offer high yield, consistency, and scalability, but they come with a higher environmental cost.¹⁷² In contrast, green methods, while environmentally friendly and sustainable, face challenges related to scalability and consistency. Addressing these challenges through standardization, optimization, and technological integration can enhance the industrial applicability of green methods, making them a viable alternative to traditional approaches.¹⁷²

Traditional methods, such as chemical precipitation, the sol-gel method, and hydrothermal synthesis, are well-established and widely used in industry.^173,174 These methods are highly effective, providing controlled size distribution and high purity of ZnO NPs. For instance, chemical precipitation involves reacting zinc salts with alkaline agents to precipitate ZnO NPs, resulting in fast reaction rates and high output.¹⁷⁵ Similarly, the sol-gel method produces uniform and pure ZnO NPs through hydrolysis and condensation of Zn precursors.¹⁷³ Hydrothermal synthesis, which crystallizes ZnO NPs under high pressure and temperature, allows for excellent control over particle size and morphology.¹⁷⁶ However, these traditional methods often require significant energy consumption to maintain precise temperature and pH conditions, contributing to a substantial carbon footprint.^172,175

The environmental impact of traditional methods is further compounded by the production of hazardous chemical by-products, necessitating careful waste management.¹⁷² Despite these drawbacks, traditional methods are highly scalable, with established protocols and infrastructure that ensure consistent and reproducible properties of ZnO NPs across batches.¹⁷⁷ However, the costs associated with energy consumption and waste management remain high.

On the other hand, green methods for producing ZnO NPs emphasize sustainability and environmental friendliness. Prominent green methods include microbial synthesis and the use of waste materials.¹⁷⁷ Microbial synthesis employs bacteria, fungi, or algae, harnessing their metabolic activity to biosynthesize ZnO NPs with specific shapes and sizes.^177,178 In addition, utilizing waste materials such as agricultural or industrial by-products can yield functional ZnO NPs, promoting sustainable resource use. These green methods generally require lower energy consumption, resulting in a reduced carbon footprint.¹⁷⁸ They also minimize hazardous chemical waste by utilizing renewable and biodegradable materials, enhancing their sustainability.¹⁷⁸

Despite their environmental benefits, green methods face significant challenges in industrial applicability.¹⁷⁹ Maintaining consistency and reproducibility at larger scales is difficult due to the inherent variability in biological materials and processes. To overcome this, standardizing biological materials and developing robust protocols are essential.¹⁷⁹ Furthermore, green methods often yield lower outputs compared to traditional methods.¹⁸⁰ Optimizing biological conditions and scaling up cultivation processes can help address this issue. Process control in biological systems is another challenge, which can be mitigated by integrating advanced monitoring and control systems.¹⁷⁹ Although the initial costs for research and development of green methods can be high, investing in pilot projects and forming public-private partnerships can share the costs and risks, making green methods economically viable.^179,180

Applications of ZnO NPs in Biomedicine

ZnO NPs, a newer of cost-effective and less hazardous nanomaterial have garnered significant interest in various biomedical fields, including anticancer, antioxidant, anti-inflammatory, antibacterial, and anti-diabetes applications, as well as bioimaging and drug delivery.¹⁸¹ This section highlights current developments in the biological uses of ZnO NPs. Nanotechnology in medical research has facilitated a deeper understanding of molecular biology, potentially enabling the design of novel therapies for diseases that were previously difficult to target due to size limitations.¹⁸² The creation of bio-functional NPs is crucial for biomedical purposes, attracting significant interest from numerous research teams in recent years.¹⁸³

Various materials and chemical manufacturing techniques for biomedical applications of ZnO NPs are currently under investigation.^182,183 Zn, a natural element found in all living organisms, is essential for the metabolic processes of humans, animals, and plants.² All living organisms require exposure to the biosphere’s normal background levels of Zn. ZnO is extensively used in the medical, pharmaceutical, and cosmetic sectors and is recognized for its beneficial use as a dietary supplement.¹⁸³ Although inhaling ZnO dust and fumes is generally considered harmless, precautions must be taken to avoid it. Consequently, regulations have been established to limit potential exposure.¹⁸⁴ The biological activities of various green ZnO nanoparticles are outlined in Table 5.

Table 5 Biological Activities of Different Green ZnO NPs

Anticancer Activity of ZnO NPs

Cancer treatment has traditionally involved surgery, radiation, and chemotherapy.^221–223 However, these treatments often come with severe side effects.²²⁴ ZnO NPs have shown promise due to their selective cytotoxicity towards malignant cells in vitro. Their surfaces can be modified to enhance this selective cytotoxicity, leading to the elimination of cancerous cells without harming healthy cells.²²⁵ Siddiqi et al²²⁶ have reported that ZnO NPs are toxic to both Gram-positive and Gram-negative bacteria and can affect primary human T-cells. ZnO NPs also considered safe to living organisms due to the essential nature of Zn(II) ions for adults. These advantages have spurred research into ZnO NPs for cancer treatment, highlighting their potential as biodegradable and biocompatible nanoplatforms.²²⁴

ZnO NPs combat tumors by increasing ROS generation and promoting apoptosis.²²⁷ Their electrostatic properties are also beneficial for anticancer effects. Neutral hydroxyl groups attached to ZnO NPs change their surface charge behavior. In a high-pH solution, protons move away from the particle surface, giving the surface oxygen atoms a negative charge. At lower pH values, positively charged zinc hydroxide (ZnOH₂⁺) forms on the particle surface. ZnO NPs have a positive surface charge and an isoelectric point of 6.4–6.75 in healthy conditions.²²⁸ Cancer cell membranes, however, have a markedly negative potential and contain many anionic phospholipids, such as phosphatidylserine.²²⁹

The positive charge ZnO NPs enhances their interactions with cancer cells, increasing cellular absorption, cytotoxicity, and phagocytosis.²³⁰ Studies have shown that NPs are harmless to mature human dermal fibroblasts and arterial endothelial cells but are damaging to metastatic tumor cells, and they increase apoptosis in neural stem cells.²³¹ Nanomedicine based on ZnO NPs offers high biocompatibility, cancer targeting ability, ease of surface functionalization, and drug delivery capability, addressing many of the drawbacks of traditional treatments.²²⁵ However, challenges remain, such as the need for biocompatible dispersion techniques and a better understanding of the specific cytotoxic mechanisms.²²⁵

Autophagy, a process of cellular self-consumption, can be triggered by stimuli like ROS, dysfunctional organelles, protein aggregation, and certain anticancer drugs. This process can lead to apoptosis in cancer cells by promoting self-degradation.²³² Therefore, autophagy plays a crucial role in NP-induced cytotoxicity by enhancing cancer cell viability and activating death pathways.²³²

Hussein and Mohammed¹⁵¹ have synthesized ZnO NPs using grape (Vitis vinifera) extract and demonstrated their significant inhibitory effect against the bacteria Staphylococcus aureus and Klebsiella pneumoniae. These ZnO NPs have also shown notable cytotoxic effects against MCF-7 and AMGM5 human cancer cell lines,¹⁵¹ highlighting their potential in treating both bacterial infections and cancer. Chandrasekaran et al²³³ have synthesized ZnO NPs chemically and using plant leaves, and evaluated their anticancer, antidiabetic, and antibacterial properties. The plant-derived green-synthesized ZnO NPs exhibit the highest α-amylase inhibition efficiency and significant cytotoxicity against the MCF7 cell line.²³³ In terms of antibacterial activity, green-synthesized ZnO NPs showed stronger effects against Salmonella typhi and B. subtilis than to chemically synthesized.²³³ This underscores the enhanced antimicrobial efficacy of green-synthesized ZnO NPs, making them promising for further applications in cancer treatment and beyond.²³³ The anticancer mechanisms of ZnO NPs are illustrated in Figure 1.

Figure 1 The anticancer mechanisms of ZnO NPs.

Abbreviations: ZnO NPs, zinc oxide nanoparticles; EPR, endoplasmic reticulum.

Delivery of Cancer Drugs Using ZnO NPs

Incorporating ZnO NPs into therapeutic formulations significantly enhances the potential for safer and more efficient cancer therapies. By utilizing NP-based drug delivery to target specific sections of cancer cells, it is feasible to reduce the total quantity of drugs used and minimize unwanted side effects.²³⁴ ZnO NPs are preferable to other nanomaterials because they are less toxic and more biodegradable. There is considerable interest in using ZnO NPs for cancer treatments.¹⁸¹ Loading drugs such as doxorubicin (DOX), paclitaxel, curcumin, and baicalin onto ZnO NPs can improve their solubility, toxicity, and distribution within cancer cells.²³⁵ Previous studies have suggested that both ROS and autophagy influence ZnO NPs’ cytotoxicity, although the specific mechanisms regulating ROS and autophagy remain unidentified.²³⁶

Using ZnO NPs, researchers have explored the mechanisms governing autophagy and the correlation between ROS and autophagy in lung epithelial cells.²³⁶ Batool and colleagues have synthesized ZnO NPs using A. barbadensis leaf extract for stabilization and capping purposes.²³⁷ They determine the drug loading capacity (LC) and loading efficiency (LE) of un-stabilized and polyethylene glycol (PEG)-ZnO NPs with DOX and gemcitabine (GEM). DOX exhibits better LE at 65% (650 mg/g) and LC 32% (320 mg/g) on ZnO NPs than GEM, which showed LE 30.5% (30 mg/g) and LC 16.25% (162 mg/g).²³⁷ Similar findings have been observed for PEG-ZnO NPs, with DOX showing 68 and 35% increases in LE (680 mg/g) and LC (350 mg/g), respectively, compared to GEM, whose LE and LC values increased by 35 (350 mg/g) and 19% (190 mg/g), respectively. Using the 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay, DOX was chosen to encapsulate NPs to assess their in vitro antiproliferative capability against the triple-negative breast cancer (TNBC) cell line (MDA-MB-231).²³⁷

Gomathi et al²³⁸ have infused DOX into ZnO NPs using the sol-gel process. Various pH conditions (3.0, 6.0, 8.2, and 10.0) have been used to load the drug with a pH of 6.0 determined to be optimal based on UV and SEM analyses. The in vitro cytotoxicity of DOX, ZnO, and ZnO-DOX against HeLa cells is evaluated using the MTT colorimetric cell viability test. ZnO-DOX cells are suppressed by 99.4% at a concentration of 100 g/mL. These findings provide compelling evidence that green biosynthesized ZnO may function well as a potential nano-drug carrier for the targeted drug delivery system.²³⁸ Figure 2 depicts anticancer drug delivery using ZnO NPs in cancer treatment.

Figure 2 Anticancer drug delivery of ZnO NPs in cancer treatment.

Abbreviation: ZnO NPs, zinc oxide nanoparticles.

ZnO NPs Cytotoxic Effect on Cancer Cells

ZnO NPs induce the death of cancer cells without harming healthy cells.²³⁹ However, before ZnO NPs can be used in medicine, several challenges must be addressed, such as developing biocompatible dispersion methods and understanding the mechanisms behind their selective cytotoxicity.¹⁸¹ Research on the cytotoxic effects of ZnO NPs on mammalian cells is limited, and experts do not agree on the significance of existing findings.¹⁸¹

Studies have shown that ZnO NPs reduce the viability of primary human T cells at concentrations lethal to both Gram-negative and Gram-positive bacteria.²⁴⁰ Despite numerous reports indicating that these NPs do not harm cultured human dermal fibroblasts, they are toxic to cancerous and vascular endothelial cells and induce apoptosis in brain stem cells.¹³⁷ The size of NPs also influences their impacts on cell survival. Van Giau et al¹³⁸ found that ZnO NPs measuring 8 nm were more toxic to S. aureus than between 50–70 nm. Youssef et al²³² observed an inverse relationship between NP size and toxicity in certain cells, specifically noting that smaller NPs generate more ROS.²³² Sudhakaran et al²⁴¹ demonstrated that ZnO NPs are toxic to neural stem cells regardless of particle size, with toxicity varying based on dosage.²⁴¹

Achieving precise targeting of ZnO NPs to cancer cells without affecting normal cells remains a significant challenge. It is essential to enhance targeting mechanisms to increase specificity and reduce off-target effects.²⁴² Furthermore, large-scale synthesis of ZnO NPs with consistent quality and reproducibility is crucial for clinical applications, as variability in NP size, shape, and surface properties can impact their therapeutic efficacy and safety. The regulatory pathway for the approval of nanomedicine, including ZnO NPs, is complex and requires comprehensive evaluation of their safety, efficacy, and quality.²⁴²

Future research directions should prioritize conducting in-depth toxicological studies to understand the long-term effects of ZnO NPs in vivo, focusing on toxicity mechanisms, biodistribution, and clearance from the body.²⁴³ It is also important to explore advanced surface modification techniques that enhance the stability, targeting specificity, and therapeutic efficacy of ZnO NPs. Functionalizing the surface with targeting ligands, polymers, and other biomolecules can significantly improve their performance.²⁴³

In addition, investigating the use of ZnO NPs in combination with other treatment modalities, such as immunotherapy, can enhance anticancer efficacy; thus, the synergistic effects with existing treatments should be thoroughly evaluated.²⁴³ Bridging the gap between laboratory research and clinical application through well-designed pre-clinical and clinical studies is critical. These studies should aim to establish safety, optimal dosing, and therapeutic protocols for ZnO NP-based treatments.²⁴³

Developing personalized nanomedicine strategies that utilize ZnO NPs tailored to individual patient profiles could maximize therapeutic efficacy while minimizing adverse effects.²⁴³ Finally, research into novel synthesis methods to produce ZnO NPs with controlled size, shape, and surface properties should prioritize green synthesis approaches that utilize eco-friendly materials and processes, thereby enhancing biocompatibility and reducing environmental impact.²⁴³

Targeted NPs offer several therapeutic benefits, such as multidrug conjugation, high payload capacity, adjustable discharge kinetics, selective localization, and the ability to evade multidrug resistance mechanisms.²⁴⁴ Various NP functionalization approaches have been developed to enhance the selectivity and specificity of anti-cancer activity. For instance, modifying the surface of ZnO NPs has significantly improved their ability to target specific cancer cells and increased their resilience over time. Research has focused on altering of ZnO NP surfaces using various biological elements, including proteins, hyaluronan, nucleic acids, peptides, and folic acid.²³² This biocompatible coating did not affect the anticancer effectiveness of ZnO NPs but also increased their selective targeting of cancer cells while sparing normal cells.²³²

Anti-Diabetic Activity of ZnO NPs

Research has explored the anti-diabetic effects of ZnO NPs due to their role in facilitating insulin storage, production, and release, given the essential nature of Zn in these processes.²⁴⁵ Studies have shown that ZnO NPs can significantly increase insulin levels, enhance glucose elimination, and improve Zn status, exhibiting higher anti-diabetic activity than ZnSO₄.²⁴⁵ For instance, Gadoa et al²⁴⁶ demonstrated that ZnO NPs could restore the cellular structure, improve structural integrity, and normalize biochemical markers such as serum insulin and blood glucose levels, thus proving effective in managing diabetes-induced pancreatic disorders.²⁴⁶ Further trials combined ZnO NPs with diabetic medications like red sandalwood and vildagliptin to enhance efficacy.²⁴⁷ These medications inhibit pancreatic amylase and colonic-glucosidase, enzymes that break down carbohydrates into glucose.²⁴⁸

Diabetes results from a metabolic imbalance due to inadequate insulin production or effectiveness, leading to inefficient carbohydrate processing and persistently high blood sugar levels.²⁴⁹ Inhibiting enzymes like glucosidase and amylase can help regulate blood glucose levels. Current drugs can inhibit these enzymes but often have drawbacks.²⁵⁰ However, Ci-ZnO NPs have demonstrated promising results in suppressing the activities of amylase and glucosidase by interacting with their active and allosteric sites.²⁵⁰ This interaction leads to competitive and non-competitive inhibition, with inhibition percentages ranging between 20–74% for amylase and 36–82% for glucosidase, the latter having a higher inhibition rate.²⁵⁰

For glucosidase, ZnO NPs can bind directly to the active site, preventing substrate access, or to allosteric sites, causing conformational changes that reduce enzyme activity.²⁵¹ Similarly, for amylase, ZnO NPs can compete with the substrate for the active site or bind allosterically, altering the enzyme’s shape and functionality.²⁵¹

These interactions result in structural changes, including alterations in the secondary and tertiary structures of the enzymes, which affect their stability and flexibility, ultimately reducing their catalytic efficiency.²⁵² Experimental evidence, including spectroscopic and molecular docking studies, supports these mechanisms, demonstrating that ZnO NPs can induce significant conformational alterations and modify the stability of enzyme molecules.^252,253 According to Nazarizadeh and Asri-Rezaie²⁵⁴, over 400 million people worldwide had diabetes in 2014, making it a significant public health concern. Diabetes mellitus arises from metabolic dysfunction where the body either fails to produce enough insulin or utilizes it ineffectively.²⁵⁵ Zn plays a crucial role in insulin storage, synthesis, and secretion and is essential for maintaining insulin’s structural integrity.²⁵⁶ ZnO NPs have been developed as a novel method for Zn administration, and their anti-diabetic benefits have been investigated. For example, red sandalwood extract with ZnO NPs were used as an anti-diabetic medication, finding that the combination was more effective in inhibiting pancreatic glucosidase and amylase than the components alone.²³³

In another study, Nazarizadeh and Asri-Rezaie²⁵⁴ examined ZnSO₄ and ZnO NPs’ antioxidant activity in diabetic rats, finding that small ZnO NPs had a significantly larger antidiabetic impact at higher doses, demonstrated by decreased blood sugar levels, increased insulin levels, and enhanced serum Zn status. Higher doses also aggravated oxidative stress, indicated by increased malondialdehyde (MDA) production and decreased total antioxidant capacity.²³³

ZnO NPs enhance insulin signaling pathways and intracellular glucose transport primarily by mimicking insulin action and stimulating pancreatic β-cells.²⁵⁷ They activate the insulin receptor, initiating a signaling cascade that includes the phosphorylation of insulin receptor substrates (IRS), activation of phosphatidylinositol-3-kinase (PI3K), and protein kinase B (Akt).²⁵⁸ Akt activation promotes the translocation of glucose transporter 4 (GLUT4) to the cell membrane, facilitating increased glucose uptake.²⁵⁸ In addition, ZnO NPs stimulate β-cells to secrete more insulin and reduce oxidative stress, thereby improving β-cell function and overall glucose homeostasis under diabetic conditions.²⁵⁹

Elevated blood sugar levels can enhance inflammation by modulating the production of C-reactive protein (CRP) and interleukins, which are associated with cardiovascular conditions.²⁴⁹ Rehana et al²⁴⁹ synthesized ZnO NPs using hydroxyethyl cellulose as a stabilizer to alleviate diabetic complications. These NPs reduced levels of asymmetric dimethylarginine (ADMA), fasting blood sugar, MDA, and inflammatory markers like interleukin-1 (IL-1) and CRP in diabetic rats while increasing nitric oxide (NO) levels and antioxidant enzyme PON-1.²⁴⁹

In addition, Eswari et al²⁶⁰ used leaf extract of teak (Tectona grandis) and Indian abutilon (Abutilon indicum) to produce ZnO NPs. The X-ray diffraction (XRD) analysis confirmed the effective creation of wurtzite ZnO NPs with average crystalline sizes of 17 nm for teak and 22 nm for Indian abutilon. Optical assessments revealed absorption bands around the 350 nm UV range, indicating band gap values of 3.0 eV and 3.1 eV.²⁶⁰ The anti-diabetic and anti-inflammatory properties of these ZnO NPs were examined using bovine albumin serum (BSA) denaturation and amylase inhibition techniques, achieving inhibition percentages of 95.42 and 94.82%, respectively.²⁶⁰ Furthermore, MTT tests indicated a reduction in viability among MCF-7 breast cancer cell lines.²⁶⁰ In summary, studies suggest that ZnO NPs hold significant potential for treating diabetes and its complications, due to their ability to improve insulin activity, regulate blood glucose levels, and reduce inflammation.

However, several regulatory challenges must be addressed before ZnO NPs can be integrated into clinical practice. Safety concerns, such as potential cytotoxicity and genotoxicity, require thorough assessment, and long-term exposure studies are necessary to ensure safety.²⁶¹ Establishing safe and effective dosage ranges is another critical regulatory hurdle.²⁶² In addition, ensuring consistent quality and reproducibility in the production of ZnO NPs is essential for regulatory approval; thus, detailed characterization including size, shape, surface charge, and coating is necessary for regulatory compliance. Ethical considerations, such as informed consent and long-term monitoring of patients, must also be addressed.²⁶²

Integrating ZnO NPs into existing diabetes treatment protocols could involve using them as an adjunct therapy alongside current antidiabetic medications, potentially enhancing their effects and reducing required dosages.^263,264 Personalized approaches that consider individual patient responses to ZnO NP therapy could be developed for more effective diabetes management. Furthermore, combining ZnO NPs with other therapeutic agents, such as antioxidants, anti-inflammatory drugs, or insulin sensitizers, may provide synergistic benefits.²⁶⁵ While ZnO NPs hold significant promise for diabetes treatment, comprehensive research and rigorous regulatory scrutiny are essential to ensure their safe and effective integration into clinical practice.²⁶⁵

Antimicrobial Activity of ZnO NPs

ZnO NPs exhibit promising antibacterial properties due to their substantial surface area and ability to combat various diseases. Recent studies have highlighted their antimicrobial properties, making them a reliable therapeutic option in medical technology at both micro and nanoscale levels.^266–268 Despite their advantages over microparticles, the exact mechanisms underlying their efficacy remain unclear. Notably, ZnO NPs are effective against both Gram-negative and Gram-positive bacteria, as well as spores resistant to high pressure and temperature.²²⁶

The concentration and size of ZnO NPs significantly impact their pharmacological behavior. The effectiveness of these NPs correlates with the dimensions and quantity, though the precise therapeutic mechanisms are still not fully understood.²⁶⁹ Some hypotheses suggest that the particle adhesion to microbial surfaces due to constant pressure plays a role, while others propose that the hydrogen peroxide (H₂O₂) production is a key factor. Increased dosages of ZnO NPs enhance their potency, treatment time, and efficacy,²⁶⁹ partly due to variations in particle size and surface area-to-volume ratio.²⁶⁹

Escherichia coli, Vibrio cholerae, and other Gram-negative bacteria are common models for studying ZnO NPs’ antibacterial effectiveness,²⁷⁰ along with Gram-positive bacteria such as S. aureus.²⁷⁰ Research has also included Proteus vulgaris, Pseudomonas aeruginosa, B. subtilis, and Enterococcus faecalis.⁷⁰ Studies have shown ZnO NPs exhibit significant antibacterial action against these bacteria, often linked to the production of ROS, which disrupt cell membranes and compromise their integrity.^201,271

In specific studies, ZnO with an average size of around 13 nm disrupted bacterial cell membranes through direct contact, while other findings indicated that Zn(II) ions from ZnO NP suspensions did not exhibit antibacterial effects.²⁷² For instance, ZnO NPs inhibited E. coli at approximately 3.4 mM concentrations and S. aureus at concentrations below 1 mM.²⁷² ZnO NPs have also been studied for their potential in treating cholera, a severe intestinal illness caused by V. cholerae.²⁷³ Research by Sarwar et al²⁷⁴ revealed that ZnO NPs effectively inhibited the growth of the El Tor (N16961) variant of V. cholerae by inducing ROS overproduction, leading to bacterial membrane damage and increased permeability. In mouse models, ZnO NPs demonstrated the ability to impede cholera toxin’s attachment to the GM1 gangliosides receptor, causing the toxin’s structural collapse.²⁷⁵

The antibacterial effects of ZnO NPs are believed to stem from their ability to generate oxidative stress, disrupt cell membranes, and impede respiratory enzymes through interaction with Zn(I) ions. This leads to ROS and free radical production, causing irreversible damage to bacterial mitochondria, DNA, and membranes.²²⁶ NPs can penetrate cell membranes more easily than larger particles, allowing direct interaction with intracellular components, which increases ROS production and subsequent cellular damage.²⁷⁶ The size of NPs also affects their uptake by cells through endocytosis, with smaller ZnO NPs being more readily absorbed and distributed throughout the cytoplasm and organelles, including mitochondria.²⁷⁷ Once inside, these NPs can cause mitochondrial dysfunction, leading to further ROS production and triggering cell death pathways such as apoptosis or necrosis.^277,278 The primary mechanism of ZnO NPs toxicity is oxidative stress induced by ROS, with smaller NPs generating higher levels of ROS, overwhelming the cell’s antioxidant defenses.²⁷⁷ This imbalance causes oxidative damage to lipids, proteins, and DNA.²⁷⁷

Elevated ROS levels can also activate inflammatory pathways. Due to their enhanced ROS production, smaller ZnO NPs provoke stronger inflammatory responses, leading to the release of pro-inflammatory cytokines.²⁷⁹ Chronic inflammation can result in tissue damage and contribute to diseases, such as cancer. Furthermore, the ROS generated by smaller ZnO NPs can induce DNA damage, leading to mutations and chromosomal aberrations.²⁸⁰ This genotoxicity may result in cell cycle arrest, apoptosis, or uncontrolled cell proliferation, all of which contribute to carcinogenesis.²⁸⁰

In summary, the toxicity of ZnO NPs is strongly influenced by their size. Smaller NPs, with their higher surface area-to-volume ratio, produce more ROS, leading to increased oxidative stress, cellular damage, and inflammation.⁶⁴ Understanding these size-dependent effects is crucial for evaluating the safety of ZnO NPs in biomedical and industrial applications. Strategies to mitigate their toxicity may include controlling particle size, surface modifications, or using antioxidants to neutralize ROS.

Studies by Ghasemi and Jalal²⁸¹ indicated that ZnO NPs enhance the effectiveness of antibiotics like ceftazidime and ciprofloxacin against Acinetobacter baumannii, a pathogen responsible for infections such as pneumonia and meningitis.²⁸¹ When combined with antibiotics, improved antibiotic absorption and altered bacterial shape, demonstrating the potential for combined treatments.²⁸² ZnO NPs also enhance the antibacterial efficacy of the photosensitizer crystal violet.^99,283 Research by Chen et al²⁸⁴ using surface enhanced Raman spectroscopy (SERS) showed that ZnO NPs antibacterial activity varies with dosage and duration, with smaller doses over extended periods mimicking the effects of higher doses.²⁸⁴ The effectiveness of ZnO NPs as antibacterial agents is illustrated in Figure 3.

Figure 3 The antibacterial activity of ZnO NPs.

Abbreviations: ZnO NPs, zinc oxide nanoparticles; ROS, reactive oxygen species.

In summary, ZnO NPs possess significant antibacterial properties, and hold promises for various medical and industrial applications. Their dual functionality, including the ability to induce oxidative stress and disrupt cell membranes, makes them potent antibacterial agents suitable for future research and development in combating bacterial infections.

Impact on NPs Modifications on Cellular Uptake and Targeted Interactions with Cancerous Vs Normal Cells

Modifications to NPs significantly influence their cellular uptake and interactions with both cancerous and normal cells.²⁷⁶ These modifications can be optimized to improve targeting, minimize side effects, and enhance therapeutic efficacy.²⁸⁵ Surface modifications play a crucial role in this process. Surface modifications are key, especially when functionalization with targeting ligands such as antibodies, peptides like arginine-glycine-aspartic acid (RGD), aptamers, or small molecules like folic acid. These enable precise targeting of specific antigens overexpressed on cancer cells.²⁸⁵ For instance, HER2 in breast cancer can be targeted with antibodies, while integrins on tumor vasculature can be targeted with RGD peptides.²⁸⁶ Aptamers, which are single-stranded DNA or RNA molecules, bind to specific proteins on cancer cells, and folic acid-conjugated NPs can target folate receptors commonly overexpressed in cancer cells.²⁸⁷

The surface charge of NPs also influences their interaction with cells. Cationic NPs, which are positively charged, interact more readily with the negatively charged cell membranes, enhancing uptake, but may also increase toxicity.²⁸⁸ In contrast, anionic and neutral NPs tend to be less toxic and can be modified with stealth properties, such as PEGylation (attachment of PEG) to evade the immune system and increase circulation time.^288,289 The hydrophilicity or hydrophobicity of NP coatings is also crucial; hydrophilic coatings like PEG reduce opsonization and immune recognition²⁹⁰, while hydrophobic coatings enhance interaction with cell membranes but may result in rapid clearance from the bloodstream.²⁹¹

NPs interaction with cellular receptors is another critical factor. Cancer cells often overexpress receptors, such as transferrin or folate receptors, which can be targeted by corresponding ligands on NPs.²⁹² Mutated receptors in cancer cells present novel targets that normal cells do not have, enabling selective targeting. Normal cells, with tightly regulated receptor expression, provide an opportunity to design NPs that minimize off-target effects by focusing on receptors overexpressed in cancer cells.²⁹²

NPs uptake occurs through various cellular pathways. Endocytosis, including clathrin-mediated (CME), caveolae-mediated (CavME), and macropinocytosis, is a primary mechanism for internalization.²⁹³ In CME, NPs bind to receptors that cluster into clathrin-coated pits, leading to internalization. CavME involves internalization through caveolin-enriched plasma membrane invaginations, while macropinocytosis allows larger particles or aggregates to be taken up by membrane ruffling and engulfment.²⁹⁴ Small and lipophilic NPs can also passively diffuse through the cell membrane without receptor-mediation.²⁹⁴

Cancerous and normal cells differ significantly in how they interact with and uptake NPs. The enhanced permeability and retention (EPR) effect, characterized by leaky vasculature and poor lymphatic drainage in tumors, allows for passive NP targeting, a feature typically absent in normal tissues.^295,296 Tumors also have an acidic microenvironment and higher redox potential compared to normal tissues, which supports the use of pH-sensitive and redox-sensitive NPs for targeted release within the tumor.²⁹⁷ Moreover, hypoxic conditions and the metabolic reprogramming (Warburg effect) in tumors provide further strategies for targeting hypoxia-sensitive and metabolically tuned NPs.²⁹⁸

The Multifaceted Antimicrobial Mechanisms of ZnO NPs

Next-generation nano-antibiotics using ZnO NPs have been developed to combat drug resistance in various treatments.²⁹⁹ These NPs are distinct in their size, crystalline structure, porosity, shape, and content,²³⁹ which confer broad antibacterial action against a range of pathogens, including P. aeruginosa, S. aureus, and E. coli.¹³⁷

In both clinical and non-clinical settings, ZnO NPs can be combined with antibiotics and anti-inflammatory drugs to enhance their effectiveness against harmful microorganisms, while reducing the risk of antibiotic resistance.^137,138 Despite the unclear specific mechanisms of their medicinal activity, ZnO is being explored as a pharmacological agent at micro- and nanoscale levels. It is suggested that cell swelling occurs primarily due to ROS generation on particle surfaces, Zn ion release, membrane dysfunction, and NP uptake.³⁰⁰ High temperature processing of ZnO NPs significantly affects their therapeutic efficacy, whereas lower temperature processing has a lesser effect.³⁰⁰ ZnO NPs are also being studied in conjunction with medical ablation techniques and their potential in anti-cancer treatments when exposed to heat.³⁰¹

ZnO materials exhibit antimicrobial effects through several mechanisms, including the release of Zn(II) ions, adsorption abilities, ROS generation, reactions within microorganisms, induction of lipid peroxidation, interference with DNA replication, and DNA fragmentation,³⁰² Zn(II) ions produced by ZnO NPs/microparticles (MPs) affect metabolic processes and enzyme systems in microorganisms, inducing antibacterial responses.¹⁵⁹ Under UV and visible light, ZnO NPs/MPs act as photocatalysts, generating ROS and attracting particles to the bio-membrane through charge interactions.^303,304

Furthermore, ZnO NPs can release Zn(II) ions, which interact with the bacterial cell membrane and intracellular components.^305,306 These ions bind to negatively charged membrane sites, disrupting membrane potential and inhibiting vital functions like nutrient transport and energy production.³⁰⁵ In addition, ZnO NPs and Zn(II) ions interfere with cellular respiration by inhibiting respiratory enzymes essential for the electron transport chain, leading to reduced ATP production and energy depletion.^307,308 Furthermore, ROS generated by ZnO NPs oxidize proteins, disrupting metabolic enzymes and structural proteins crucial for cell integrity.²⁸³ ROS can also induce DNA strand breaks, resulting in mutations and impaired replication and transcription.¹²⁹

ZnO NPs/MPs interact with microorganisms by adsorbing onto their positively charged surfaces and negatively charged cell walls or bio-membranes.³⁰⁹ Their penetration into the membrane causes physical damage, creating pits and perforations that compromise its integrity, leading to leakage and cell death.³¹⁰ They also mediate lipid peroxidation-induced oxidative stress, which damages DNA. The effectiveness of ZnO NPs/MPs against pathogenic microorganisms varies depending on their porosity, particle size, and shape.^309,310

ZnO NPs/MPs exhibit enhanced antibacterial action even against deadly pathogens and show broad antibacterial properties when combined with other antibiotics. This adaptability makes them promising platforms for commercial and clinical applications. Other biomaterials, metal doping, and metal oxide NPs/MPs are also being explored for similar purposes.³⁰²

ZnO NPs are not only effective against bacteria but also against fungi, as shown in Figure 4. Abd-Elmaqsoud et al³¹¹ found that ZnO NPs derived from Moringa oleifera were detrimental to the plant pathogens, Alternaria saloni and Sclerrotium rolfii. Similarly, Alhazmi and Sharaf³¹² explored the effectiveness of ZnO NPs against various fungal strains, noting significant impact on both plant and food pathogens, particularly Aspergillus nidulans, Trichoderma harzianum, Rhizopus stoloniferous, and Aspergillus flavus. Consequently, the researchers suggested that ZnO NPs could be utilized in the food and agriculture industries.³¹² In addition, ZnO NPs exhibit a concentration-dependent effect on the survival of Candida albicans. At a concentration of 0.1 mg/mL, ZnO NPs significantly reduced C. albicans viability. When combined with visible light, the frequency of yeast cell death increased.³¹²

Figure 4 The antifungal activity of ZnO NPs.

Abbreviation: ZnO NPs, zinc oxide nanoparticles.

Perveen et al³¹³ produced ZnO NPs using vegetable seed extracts from Brassicaceae plants, including sarson, turnip, white radish, red radish, and cauliflower. These ZnO NPs demonstrated antibacterial activity, with inhibition zones ranging between 10–20 mm, compared to 25 mm for streptomycin.³¹³ This indicates that red radish, white radish, turnip, sarson, and cauliflower can be sources of ZnO NPs with active antibacterial properties.³¹³

The response of bacterial and fungal cell membranes to ZnO NPs differ due to their distinct membrane compositions. In bacterial cells, ZnO NPs primarily disrupt the membrane and increase permeability through electrostatic interactions between the negatively charged membrane components and the positively charged ZnO NPs.^314,315 This is further intensified by the generation of ROS, which exacerbates membrane damage, leading to cell lysis and death. In contrast, fungal membranes, rich in sterols like ergosterol, respond to ZnO NPs by increasing membrane permeability and causing cellular contents leakage.³¹⁶ Although ROS generation also contributes to membrane damage in fungi, the effect is less pronounced due to their stronger antioxidative defenses compared to bacteria.³¹⁶

Anti-Inflammatory Activity of ZnO NPs

Inflammation is a complex response of body tissues to various potential threats, including irritants, cellular damage, or pathogens.³¹⁷ The biological functions of Zn ions and the creation of NPs have highlighted the anti-inflammatory capabilities of ZnO NPs. Atopic dermatitis (AD), a chronic inflammatory skin disorder, is characterized by a decreased skin barrier function and a complex interaction between genetic and environmental factors.³¹⁷

Textiles, which are in close contact with the skin, have been explored for their potential therapeutic effects. Kahru and Mortimer³¹⁸ investigated the impact of ZnO-enhanced textile fibers on oxidative stress in individuals with AD. Their results indicated that wearing ZnO-induced fabrics overnight for three consecutive days significantly improved pruritus and sleep quality, likely due to the antibacterial and antioxidant properties of ZnO fabrics.³¹⁸

Youssef et al²³² studied the effect of ZnO NPs of varying sizes on allergic skin in a mouse model of AD. They found that the bulk-sized ZnO (bZnO) remained on the skin’s surface layers, while nanoscale ZnO (nZnO) could penetrate the deeper layers of sensitive skin prone to allergic reactions..²³² In an animal model of Alzheimer’s disease, nZnO significantly decreased pro-inflammatory cytokines and exerted greater anti-inflammatory effects than bZnO, as evidenced by reductions in Th2 cytokines, IL-13, IFN-, and IL-10.²³² Such findings demonstrate that ZnO NPs significantly reduce skin inflammation in AD models.

These interactions primarily occur through the inhibition of key inflammatory signaling pathways. ZnO NPs are taken up by immune cells through endocytosis, where they release Zn ions into the cellular environment.³¹⁹ These ions disrupt signaling cascades that lead to cytokine production, specifically by reducing the activation of nuclear factor-kappa B (NF-kB), a critical transcription factor involved in pro-inflammatory cytokine expression.²¹³

The NF-κB signaling pathway plays a central role in regulating inflammation and immune responses.³²⁰ Normally, NF-κB is kept inactive in the cytoplasm by inhibitor proteins (IκBs).³²⁰ Upon activation by pathogens or inflammatory signals, IκB are phosphorylated and degraded, allowing NF-κB to move into the nucleus and trigger the transcription of target genes, including IL-6 and TNF-α.^320,321 ZnO NPs inhibit this process by two main mechanisms: First, Zn ions prevent IκB phosphorylation and degradation, thereby blocking NF-κB activation.²¹³ Second, ZnO NPs scavenge ROS, which are involved in NF-κB activation.³²² By reducing ROS levels and oxidative stress, ZnO NPs further inhibit NF-κB activation, leading to lower transcription of pro-inflammatory cytokines and reduced inflammation. This modulation of the NF-κB pathway by ZnO NPs highlights their potential as therapeutic agents in controlling inflammation and related diseases.³²²

The anti-inflammatory capabilities of ZnO NPs extend beyond AD, proving effective for various inflammatory diseases. Imraish et al³²³ assessed the anti-inflammatory potential of ZnO NPs on RAW 264.7 macrophages stimulated by LPS, noting their superior ability to decrease NO production and suppress the expression of associated proteins such as inducible NO synthase (iNOS), COX-2, IL-1, IL-6, and TNF in a dose-dependent manner.³²³ This study highlights the broad spectrum of anti-inflammatory effects of ZnO NPs, emphasizing their potential therapeutic applications in various inflammatory conditions.³²³ Further supporting the anti-inflammatory role of ZnO NPs, Abdelbaky and colleagues³²⁴ synthesized ZnO NPs using a solution of Pelargonium odoratissimum leaf extract as a reducing agent.³²⁴ In vitro models using the human red blood cells (HRBC) membrane stabilization method (MSM), such as hypotonicity-induced hemolysis, were employed to assess the anti-inflammatory properties of both the ZnO NPs and the water-based extract from P. odoratissimum leaves.³²⁴ Compared to conventional indomethacin at a dose of 1000 µg/mL, a maximum membrane stabilization of 95.6% was observed.³²⁴

The effectiveness of bulk and nano-ZnO was examined, revealing that only nano-sized ZnO particles can infiltrate the innermost layers of allergic skin, reducing localized skin irritation while promoting the synthesis of IgE antibodies. Ilves et al¹⁵⁷ hypothesized that this is due to non-specific processes caused by liberated Zn (II) that limit B-cell IgE synthesis. These findings suggest that ZnO NPs could potentially be used to treat inflammation. The anti-inflammatory impact of ZnO NPs is illustrated in Figure 5.

Figure 5 Anti-inflammatory effect of ZnO NPs.

Abbreviations: ZnO NPs, zinc oxide nanoparticles; ROS, reactive oxygen species.

Treatment of Skin Disorders Using ZnO NPs

ZnO is commonly found in medications such as diaper rash powders, barrier creams, antimicrobial ointments, hemimorphite cream, and antidandruff shampoos. Athletes also use ZnO tape to protect soft tissue during exercise.²³² Incorporating ZnO NPs into creams, ointments, and lotions provides protection against sunburn and other UV-related skin issues. Due to its excellent UV-A (320–400 nm) and UV-B (280–320 nm) reflectance and perfect photostability, the Bureau has officially approved ZnO for use in sunscreen.²³²

Studies have shown that ZnO NPs can downregulate type I collagen in skin tissue. Results indicate that ZnO NPs creams at low (1%) and high (6%) concentrations offer preventive benefits against oxidative damage and allergic dermatitis induced by lead oxide, likely due to the anti-inflammatory and antioxidant properties of ZnO NPs.³²⁵ At lower concentrations, such as 1%, ZnO NPs can effectively reduce inflammation and oxidative stress, promoting skin health and providing a barrier against environmental pollutants.³²⁶ The antioxidant properties neutralize free radicals, reducing oxidative damage to the skin. In addition, their anti-inflammatory effects can mitigate allergic responses and dermatitis by decreasing the production of pro-inflammatory cytokines like IL-6 and TNF-α.³²⁷

Conversely, higher concentrations of ZnO NPs, such as 6%, while still offering protection against oxidative stress and inflammation, may pose risks with frequent and prolonged exposure.³²⁶ Research shows that in cases of epidermal barrier dysfunction, such as eczema or psoriasis, high concentrations of ZnO NPs increase the risk of adverse effects, including melanoma.³²² In mouse models with epidermal barrier failure, topical application of ZnO NPs led to penetration into deeper skin layers, reaching the stratum basale, causing skin lesions resembling melanoma.³²² This suggests that high doses of ZnO NPs can disrupt normal cellular functions when the skin barrier is compromised.

Furthermore, both in vitro and in vivo studies have shown that ZnO NPs can exert anti-apoptotic effects on melanocytes by activating the NF-κB pathway through oxidative stress, leading to increased cell survival and proliferation.³²² In compromised skin, this could contribute to malignancies like melanoma.³²²

The distinction between the effects of different ZnO NP concentrations highlights the importance of dosage. While low concentrations primarily offer protective and benefits by reducing oxidative damage and inflammation, high concentrations can pose significant risks, especially in compromised skin conditions.³²⁶ The ability of ZnO NPs to penetrate deeper skin layers and activate pathways that prevent apoptosis and promote cell proliferation highlights the need for careful consideration of concentration and exposure duration. Balancing the benefits and risks of ZnO NPs is crucial, particularly in therapeutic and cosmetic applications where long-term skin health is a priority.^322,326 The skin-protecting impacts of ZnO NPs are displayed in Figure 6.

Figure 6 Skin protective effect of ZnO NPs.

Abbreviations: ZnO NPs, zinc oxide nanoparticles; ROS, reactive oxygen species; UV, ultraviolet.

ZnO NPs and Drug Delivery

Nanotechnology has numerous applications, one of the most important being drug delivery, which has proven effective in treating various diseases, including cancer.^328–330 NPs are crucial in medication transportation.²²¹ Many researchers have utilized ZnO NPs for drug delivery across different diseases.¹⁸¹ For instance, Badıllı et al³³¹ employed ZnO quantum dots (QDs) to deliver DOX precisely to HeLa cells. To enhance the stability of these nanomaterials, chitosan was applied to ZnO NPs.¹⁸¹ Their research demonstrated that this drug delivery technology could successfully transport DOX to cancer cells.¹⁸¹ In addition, NPs are significant in gene delivery methods for various cells, particularly malignant cells.¹⁸¹ This gene transfer method offers several advantages, including the effective and secure delivery plasmid-containing genes to target tissues.¹⁸¹

The differences between drug and gene delivery using NPs are significant in terms of mechanisms, requirements, and necessary modifications to ensure efficacy and stability within the body.³³² In drug delivery, NPs act as carriers for therapeutic agents like DOX, enabling targeted and controlled release.³³³ The primary goal is to enhance the drug’s solubility, stability, and bioavailability while minimizing side effects.^332,333 For instance, ZnO NPs used for DOX delivery can be modified with chitosan, which not only improves stability but also enhances cellular uptake and controlled release at the target site, ensuring efficient delivery to cancer cells.³³³

In contrast, gene delivery involves transporting genetic material, such as DNA or RNA, into cells to modify gene expression and treat diseases at the genetic level.³³⁴ NPs in gene delivery must protect the genetic material from degradation by nucleases and facilitate its entry into target cells.^334,335 Common modifications include adding cationic polymers or lipids to form complexes with negatively charged nucleic acids, protecting them from enzymatic degradation and promoting cellular uptake.^336,337 For example, coating NPs with chitosan enhances their ability to bind with DNA or RNA and facilitates endocytosis by target cells.³³⁸

Stability within the body is crucial for both drug and gene delivery systems.³³⁹ For drug delivery, NPs are often modified with biocompatible and biodegradable materials to prevent premature degradation and ensure sustained release of the therapeutic agent.^339–341 In gene delivery, stability is enhanced through protective coatings and stabilizing agents that shield the genetic material from the biological environment.³⁴² In addition, targeting ligands can be attached to the surface of NPs to direct them to specific cell types, improving the efficiency of gene transfer and reducing off-target effects.³⁴²

Bio-Imaging and ZnO NPs

This method of gene dispersion has several advantages. Genes generated on the surface of NPs encoded by plasmids can be safely and efficiently transferred to target tissues.^343,344 Recent research has explored ZnO nanostructures such as, nanorings, nanorods, nanotubes, and NPs, particularly for their potential in biological imaging.³⁴⁴ This characteristic has several biological and therapeutic uses and applies to varied degrees.³⁴⁴ Luminescent ZnO NPs, also known as ZnO QDs, possess advantageous photophysical properties and are widely recognized for their safety.³⁴⁴ ZnO is used as a food preservative and a component in sunscreen products, making its luminescent properties applicable in various natural and medicinal contexts.³⁴⁴ ZnO is used as fluorescence imaging often used in preclinical research due to its cost-effectiveness and practicability, benefits from these properties.²⁴⁹

Several studies have highlighted the importance of ZnO NPs in cellular imaging. These NPs can emit green fluorescence due to oxygen vacancies and other processes, facilitating the visualization of cancer cells via minimally invasive methods.³⁴⁴ Green, fluorescent ZnO NPs conjugated with transferrin have been used for this purpose. Additionally, the optical properties of ZnO nanomaterials can be modified by incorporating cations like Ni, Cu, or Co, stabilized in aqueous colloidal solutions for various imaging purposes.³⁴⁴ These small ZnO NPs can infiltrate the cell nucleus, and hetero-structural ZnO/Au nanocomposites have been developed and studied for their optical properties and biocompatibility.³⁴⁴

ZnO nanorods can form Au nanoclusters on their tips and surfaces. ZnO nanorods coated with antibodies against the epidermal growth factor receptor have been used to scan cancer cells in vitro.³¹⁸ QDs are favored for optical imaging due to their attractive optical characteristics.³⁴⁵ ZnO QDs used in in vitro cell imaging showed stable luminescence without cytotoxicity under UV illumination. These QDs have also been evaluated in mice through intradermal and intravenous injections.²⁰⁹

Each imaging method has its own advantages and disadvantages.³⁴⁶ Functionalizing nanomaterials to be detectable by multiple imaging modalities offers synergistic benefits.³⁴⁶ Nanomaterials are more suitable for multimodal imaging than small molecules due to their larger surface areas, providing more sites for functionalization and allowing customization for multimodal detection.³⁴⁷ For instance, sub-6 nm-diameter Gd-doped ZnO QDs have been generated for optical and magnetic resonance imaging (MRI). Fe₃O₄-ZnO core-shell magnetic QDs have also been investigated for potential cancer imaging and therapy.³⁴⁸

In clinical settings, radionuclide-based imaging techniques like PET and single-photon emission computed tomography (SPECT) are more frequently than optical imaging due to their unlimited tissue penetration, high sensitivity, and quantitative capabilities.^346,348 Recent studies have shown that ZnO NPs available in various morphologies, can be used as bioimaging materials.³⁴⁹ Hyperbranched polymers have been employed to produce amphibious ZnO QDs with blue fluorescence, demonstrating their bioimaging applicability.³⁴⁹ ZnO NP surfaces are easily modifiable and durable in aqueous solutions, with water-soluble ZnO enhanced by hyperbranched polyethylenimine compounds performing well in bioimaging.³⁴⁹

Masar et al³⁵⁰ investigated pure n-type ZnO NPs for bioimaging using standard fluorescence microscopy methods. Typically, NPs require UV excitation sources to emit light. However, this study shows that by reducing the energy gap, a 405 nm laser may sufficiently excite NPs for emissions observable during confocal microscope live-cell imaging.³⁵⁰ This research lays the foundation for using these NPs in various bioimaging applications, allowing to study interactions between pristine n-type ZnO NPs and human cells using fluorescence-driven imaging techniques.³⁵¹ Their developing production process also controls specific defects in pure n-type ZnO NPs for bioimaging.³⁵¹

ZnO NPs Based Biosensors

Biosensors have broad applications in various fields, including the food industry, healthcare, environmental monitoring, and biological or chemical assessment.³⁵² They are classified based on their detection principles into electrochemical, photometric, piezoelectric, and calorimetric devices.³⁵² Nanomaterials are gaining significant attention due to their unique features, whether used alone or in conjunction with biologically active substances.³⁵³ These properties make them a robust foundation for designing high-performance biosensors. The expanded surface area of nanomaterials facilitates the attachment of a wide range of biomolecules, such as antibodies, enzymes, and other proteins.³⁵³ Additionally, they enable direct electron transfer from the electrodes to the active regions of the biomolecules.³⁵³

ZnO nanomaterials, in particular, have several desirable properties, including high isoelectric point (IEP; 9.5), strong adsorption capacity, excellent biosensing, and high catalytic efficiency.¹⁵⁶ These properties make ZnO suitable for electrostatic adsorption of specific proteins like enzymes and antibodies, which have lower IEPs.¹⁵⁶ Nanomaterials with higher electron transfer capacities, larger surface areas, and improved biocompatibility or stability are especially beneficial for use in biosensors.³⁵⁴ ZnO-based biosensors are commonly used to detect various small-molecule analytes, such as cholesterol, glucose, H₂O₂, phenol, and urea. There are numerous biosensors available for detecting specific chemical and physical properties, like pH.³⁵⁵

Dönmez³⁵⁶ created an amperometric glucose biosensor employing ZnO NPs and the root of Zingiber officinale. Glucose oxidase (GOx) was anchored onto a carbon paste electrode (CPE) modified with ZnO through glutaraldehyde cross-linking.²⁹⁸ The resulting biosensor (GOx-ZnO/CPE) demonstrated excellent electrocatalytic glucose measurement capabilities. It featured a low detection limit (14.7 uM), rapid response time (less than 1 second), high sensitivity (15.98 A/mM.cm²), and high biological affinity (Michaelis-Menten constant of 0.99 mM). Additionally, the biosensor showed excellent resistance to interference from uric and ascorbic acids.²⁹⁸

The large surface area and excellent electronic transport properties of ZnO NPs significantly enhance sensor sensitivity by providing more active sites for biomolecule attachment and facilitating efficient electron transfer.^348,357 The increased surface area allows for greater adsorption of biomolecules such as enzymes, antibodies, and proteins, improving the chances of interactions with target analytes.³⁵⁸ These interactions are essential for detection, as they enhance the binding affinity and specificity of the sensor.³⁵⁸ In addition, ZnO NPs enable direct electron transfer between the biomolecules and the electrode, leading to faster and more accurate responses.^357,358 Together, these combined properties make ZnO NPs highly effective in developing sensitive and reliable biosensors.³⁵⁸

Toxicological Effects of ZnO NPs

The release of Zn-ions from ZnO NPs highlights their potentially hazardous nature, despite being typically considered insoluble in water.³⁵⁹ The pathways through which NPs are taken up by cells influence their surface, shape, size, and properties.³⁵⁹ Enhanced understanding of NP toxicity in both environmental and biological contexts has prompted nano-toxicologists to call for deeper insights into the atomic interactions between NPs and organic structures.³⁵⁹

Aravantinou et al³⁴⁵ investigated the enduring harmful effects of ZnO NPs on microalgae within a reconstructed traditional water treatment setup, incorporating a continuous provision of NPs. Like other metal oxide NPs, ZnO NPs are known for generating ROS and cause apoptosis, in addition to their extraordinary healing capabilities. Due to their properties, ZnO NPs serve as antimicrobial, antibacterial, and anticancer agents.³⁴⁵ They have been shown to produce synergistic benefits when used with various therapy regimens. ZnO NPs are employed for clinical diagnostics and targeted medicine administration, having several applications in the medical field and being environmentally safe.³⁴⁵ Additionally, they are economically viable as they are inexpensive to produce.³⁴⁵

Furthermore, ZnO NPs exhibit differential effects on cancerous versus healthy cells, which is crucial for their safe therapeutic use. In cancerous cells, ZnO NPs induce oxidative stress and apoptosis more effectively due to increased ROS production and an altered redox state.^19,360 This targeted effect enhances their potential as anticancer agents. In contrast, healthy cells are generally more resilient to oxidative stress and less susceptible to ZnO NP-induced toxicity.³⁶¹ However, high concentrations or prolonged exposure can still harm healthy cells, potentially causing inflammation or cellular damage.³⁶² Therefore, precise dosage and targeted delivery are essential to optimize therapeutic benefits while minimizing toxicity risks to healthy tissues.³⁶²

Challenges and Future Perspectives

The green synthesis of ZnO NPs faces several challenges that must be overcome for effective biomedical application. A key issue is the variability in biological sources.³⁴² The biochemical compositions of plants, microorganisms, and algae varies significantly, which affects the size, shape, and functionality of the resulting NPs.³⁴² This inconsistency complicates standardization, which is essential for ensuring consistent quality in biomedical applications.³⁴² In addition, scaling up green synthesis from laboratory settings to industrial production remains a significant obstacle.³⁶³

Another challenge is the limited understanding of the biochemical mechanisms involved in reducing and stabilizing metal ions through biological agents.³⁵⁹ This knowledge gap makes it difficult to control and optimize the synthesis process, resulting in issues with reproducibility and NP uniformity.³⁵⁹

Regulatory and safety concerns further complicate the application of green synthesis in biomedicine. Although eco-friendly, these methods still require extensive testing for toxicity, biocompatibility, and long-term safety to gain regulatory approval.¹⁷² The regulatory landscape for nanomaterials is continually evolving with stringent safety standards necessary for clinical and commercial use.¹⁷² Additionally, the cost-effectiveness of green synthesis relies on the availability and accessibility of biological sources.³⁶⁴ Seasonal variations and geographical limitations can impact the supply of raw materials, affecting production and cost.³⁶⁴

Despite these challenges, the future of green synthesis of ZnO NPs is promising. Advancements in characterization techniques, such as high-resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FTIR) are offering deeper insights into the mechanisms of synthesis.³⁶⁵ These tools enable better control over NP production by revealing the interactions between metal ions and biological molecules. Furthermore, integrating green synthesis methods with other eco-friendly technologies like renewable energy sources and biodegradable materials can enhance the sustainability and environmental benefits of ZnO NP production, contributing to a more sustainable nanotechnology industry.³⁴²

Functionalizing ZnO NPs with specific biomolecules, ligands, or polymers could enhance targeted delivery in biomedical applications, leading to improved drug delivery systems, better imaging techniques, and more effective therapies.³⁶⁶ Exploring a wider variety of biological sources, such as rare plants, extremophiles, and marine organisms, could also open new avenues for green synthesis of NPs with unique and desirable properties.³⁶⁷

Interdisciplinary collaboration among material scientists, biologists, chemists, and engineers will be critical in overcoming these challenges. Such partnerships can drive innovation and optimize green synthesis methods. Finally, developing comprehensive regulatory frameworks specific to nanomaterials can ensure the safe and sustainable production of ZnO NPs, with clear guidelines to safeguard both environmental and human health.

Conclusion

The green synthesis of ZnO NPs represents a major advancement in biomedical nanotechnology. Over the past decade, their unique properties have been widely explored, particularly in antibacterial treatments, drug and gene delivery, anticancer therapies, cell imaging, and biosensing. While traditional synthetic methods are effective, they pose significant economic and environmental challenges. In contrast, green synthesis method, using plants, plant extracts, and microorganisms, offers a sustainable and eco-friendly alternative that reduces environmental impact, lowers production costs, and minimizes health risks, making it suitable for large-scale production.

Green synthesis harnesses the natural biochemical processes of biological agents, such as plant-derived compounds, to reduce metal ions and stabilize NPs. This method not only adheres to green chemistry principles but also enhances the biocompatibility and therapeutic potential of ZnO NPs. Recent studies emphasize the superior antibacterial and anticancer properties of ZnO NPs produced via green synthesis, highlighting their potential in medical and pharmaceutical applications.

However, several challenges remain before the full benefits of green synthesis can be realized. These include variability in biological sources, difficulties in scaling up production, limited understanding of the underlying biochemical mechanisms, stability concerns, and regulatory barriers. Overcoming these hurdles will require advancements in characterization techniques, integration of green synthesis with other sustainable technologies, and interdisciplinary research efforts. Establishing comprehensive regulatory frameworks is essential for ensuring the safe and effective use of ZnO NPs in biomedical applications.

Despite these obstacles, the future of green synthesis for ZnO NPs is promising. Improved characterization methods, such as HRTEM and FTIR, can provide deeper insights into synthesis mechanisms and better control over production. Functionalizing ZnO NPs with specific biomolecules or polymers can enhance their targeted delivery and therapeutic effectiveness. In addition, exploring a broader range of biological sources may reveal unique properties for NP synthesis. This review highlights the potential of green synthesis to revolutionize nanotechnology, providing eco-friendly, cost-effective solutions that align with global sustainability goals.

Acknowledgments

The authors extend their appreciation to the Deanship of Research and Graduate Studies at King Khalid University for funding this work through Large Research Project under grant number (R.G.P. 2/491/44).

Funding

This work was financially supported by the Finance Science and Technology Project of Hainan Province (ZDYF2019031); UAEU program of Advanced Research (Grant number: 21S169) and Khalifa Center for Biotechnology and Genetic Engineering-UAEU (Grant #: 31R286).

Disclosure

The authors report no conflict of interest in this work.

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