With their high surface area and enhanced physicochemical properties, nanomaterials play a critical role in drug delivery, consumer products, and environmental technologies. However, their nanoscale dimensions enable interactions with cellular components in complex and sometimes unexpected ways, potentially inducing oxidative stress, inflammation, or bioaccumulation. As their use expands, understanding these risks through nanotoxicity testing becomes essential.1
Why Assess Nanotoxicity?
Assessing nanotoxicity helps ensure the safe use of nanomaterials while protecting human health and the environment. Nanomaterials can enter the body through inhalation, ingestion, or injection. Once inside, they may accumulate in organs and disrupt cellular functions. Their presence in cosmetics, pharmaceuticals, and household goods also raises concerns about environmental exposure. Reliable assessment methods help identify potential hazards before widespread use.2
Methods of Nanotoxicity Assessment
In Vitro Methods
In vitro methods are widely used to assess nanotoxicity through controlled experiments on cell cultures. Cytotoxicity assays such as MTT (tetrazolium-based assays) and LDH (lactate dehydrogenase) release assays evaluate cell viability and membrane integrity.3
Genotoxicity tests, including comet and micronucleus assays, examine DNA damage and chromosomal alterations caused by nanoparticle exposure. By exposing specific cell lines, such as epithelial cells that model the skin, lungs, or gastrointestinal tract, these methods provide critical insights into how nanomaterials interact with different biological barriers.3
For instance, Collins et al. provide key recommendations for conducting in vitro comet assays with mammalian cell cultures. They suggest using non-cytotoxic concentrations, defined as less than 20 % cell viability loss, and recommend concentrations below 100–150 μg/mL for non-cytotoxic nanomaterials.
The selection of cell lines should align with the target organ and exposure route, ensuring relevant biological insights. To capture the full spectrum of nanoparticle interactions, both short-term (2–3 hours) and long-term (24-hour) exposure studies are advised.
Additionally, distinguishing between direct DNA interactions and oxidative stress-induced genotoxicity remains a crucial consideration.4
In Vivo Methods
In vivo studies assess how nanomaterials behave in living organisms. Rodent models help researchers track bioaccumulation and long-term effects on organs like the liver, kidneys, and brain.3 These tests use exposure routes that mimic real-world scenarios, such as inhalation, ingestion, and injection.
While in vivo testing provides valuable data, ethical concerns and species differences highlight the need for alternatives. Regulatory efforts increasingly focus on reducing animal testing by improving in vitro and computational models.3, 5
Computational Methods
Computational toxicology applies in silico models to predict nanotoxicity by analyzing the physicochemical properties of nanoparticles. Techniques such as Quantitative Nanostructure-Toxicity Relationship (QNTR) and Quantitative Structure-Activity Relationship (QSAR) modeling rely on descriptors like particle size, surface charge, aggregation state, and solubility to estimate biological interactions and toxic potential.6
These models offer an efficient alternative to traditional toxicity assessments by reducing dependence on animal studies, minimizing costs, and enabling high-throughput screening. By incorporating data from in vitro experiments, bioinformatics, and machine learning algorithms, computational approaches refine toxicity predictions and enhance our understanding of nanoparticle behavior within biological systems.5
Surface Characterization Techniques
The size, shape, and surface chemistry of nanoparticles influence their interactions with biological systems. Several techniques help researchers analyze these properties:
Scanning Electron Microscopy (SEM): SEM provides high-resolution images of nanoparticles, allowing detailed analysis of their size, shape, and surface morphology. By scanning a focused electron beam across the sample surface, SEM generates images based on the interaction of electrons with the sample. This technique is especially useful for identifying surface features, defects, and coatings.7
Atomic Force Microscopy (AFM): AFM provides three-dimensional imaging and precise measurements of surface properties such as roughness, stiffness, and adhesion strength. Unlike SEM, AFM does not require extensive sample preparation and can operate under ambient or liquid conditions, preserving the native state of nanoparticles. This makes it particularly valuable for studying nanoparticle interactions with biological membranes and their penetration into cells. AFM also quantifies forces between nanoparticles and biological systems, providing insights into their physical interactions and toxicity mechanisms.7
X-Ray Photoelectron Spectroscopy (XPS):XPS is used to analyze the surface chemistry of nanoparticles, including their elemental composition, oxidation states, and surface coatings. This technique is highly sensitive to the outermost layers of nanoparticles, making it ideal for studying functional groups and ligands that influence toxicity.7
Torelli et al. developed an XPS data correction method for non-planar surfaces, improving accuracy when analyzing nanoparticles as small as 20 nm. Such refinements help predict how surface modifications affect biological interactions.8
Protocols for Nanotoxicity Testing
Standardized Guidelines
The OECD Sponsorship Programme has assessed various nanomaterials to refine test methodologies, while European initiatives like NANOHARMONY and Gov4Nano focus on standardizing protocols across different regulatory frameworks. These efforts aim to improve test reproducibility and promote global data acceptance under the Mutual Acceptance of Data (MAD) principle.9
Testing Procedures
Nanotoxicity assessments combine in vitro, in vivo, and computational approaches. Testing procedures vary based on exposure routes (oral, dermal, or inhalation) and duration (acute vs. chronic).10
Advanced in vitro assays measure cytotoxicity, oxidative stress, and DNA damage, while in vivo studies track bioaccumulation and organ-specific effects. Newer methods like microfluidic systems and co-culture models enhance test accuracy by mimicking real physiological conditions.10
What Does the Future of Nanotoxicity Testing Look Like?
Despite progress, testing nanotoxicity remains complex. Nanomaterials vary in size, shape, and surface chemistry, making it hard to develop universal protocols. A lack of standardization also leads to inconsistencies across studies.11
Future efforts will focus on integrating advanced technologies. Predictive in silico models and high-throughput in vitro systems will likely play a bigger role in screening nanomaterials. Organ-on-a-chip models could further improve accuracy by replicating human tissue environments.11
Reference and Further Readings
1. Savage, DT.; Hilt, JZ.; Dziubla, TD. (2019). In Vitro Methods for Assessing Nanoparticle Toxicity. Nanotoxicity: Methods and protocols. https://link.springer.com/protocol/10.1007/978-1-4939-8916-4_1
2. Huang, H.-J.; Lee, Y.-H.; Hsu, Y.-H.; Liao, C.-T.; Lin, Y.-F.; Chiu, H.-W. (2021). Current Strategies in Assessment of Nanotoxicity: Alternatives to in Vivo Animal Testing. International journal of molecular sciences. https://www.mdpi.com/1422-0067/22/8/4216
3. Roberto, MM.; Christofoletti, CA. (2019). How to Assess Nanomaterial Toxicity? An Environmental and Human Health Approach. [Online] IntechOpen. https://www.intechopen.com/chapters/68905
4. Collins, A.; El Yamani, N.; Dusinska, M. (2017). Sensitive Detection of DNA Oxidation Damage Induced by Nanomaterials. Free Radical Biology and Medicine. https://www.sciencedirect.com/science/article/pii/S089158491730062X
5. Budama-Kilinc, Y.; Cakir-Koc, R.; Zorlu, T.; Ozdemir, B.; Karavelioglu, Z.; Egil, AC., Kecel-Gunduz, S. (2018). Assessment of Nano-Toxicity and Safety Profiles of Silver Nanoparticles. [Online] IntechOpen. https://www.intechopen.com/chapters/60486
6. Fourches, D.; Pu, D.; Tassa, C.; Weissleder, R.; Shaw, SY.; Mumper, RJ. Tropsha, A. (2010). Quantitative Nanostructure− Activity Relationship Modeling. ACS nano. https://pubmed.ncbi.nlm.nih.gov/20857979/
7. Gunsolus, IL.; Haynes, CL. (2016). Analytical Aspects of Nanotoxicology. Analytical chemistry. https://pubs.acs.org/doi/full/10.1021/acs.analchem.5b04221
8. Torelli, MD.; Putans, RA.; Tan, Y.; Lohse, SE.; Murphy, CJ.; Hamers, RJ. (2015). Quantitative Determination of Ligand Densities on Nanomaterials by X-Ray Photoelectron Spectroscopy. ACS applied materials & interfaces. https://pubs.acs.org/doi/full/10.1021/am507300x
9. Krug, HF.; Nau, K. (2022). Methods and Protocols in Nanotoxicology. Frontiers Media. https://www.frontiersin.org/journals/toxicology/articles/10.3389/ftox.2022.1093765/full
10. Handy, RD.; van den Brink, N.; Chappell, M.; Mühling, M.; Behra, R.; Dušinská, M.; Simpson, P.; Ahtiainen, J.; Jha, A. N.; Seiter, J. (2012). Practical Considerations for Conducting Ecotoxicity Test Methods with Manufactured Nanomaterials: What Have We Learnt So Far? Ecotoxicology. https://link.springer.com/article/10.1007/s10646-012-0862-y
11. Patel, RJ.; Alexander, A.; Puri, A.; Chatterjee, B. (2021). Current Challenges and Future Needs for Nanotoxicity and Nanosafety Assessment. Nanotechnology in Medicine: Toxicity and Safety. https://onlinelibrary.wiley.com/doi/abs/10.1002/9781119769897.ch14

News
AI Predicts Sudden Cardiac Arrest Days Before It Strikes
AI can now predict deadly heart arrhythmias up to two weeks in advance, potentially transforming cardiac care. Artificial intelligence could play a key role in preventing many cases of sudden cardiac death, according to [...]
NanoApps Medical is a Top 20 Feedspot Nanotech Blog
There is an ocean of Nanotechnology news published every day. Feedspot saves us a lot of time and we recommend it. We have been using it since 2018. Feedspot is a freemium online RSS [...]
This Startup Says It Can Clean Your Blood of Microplastics
This is a non-exhaustive list of places microplastics have been found: Mount Everest, the Mariana Trench, Antarctic snow, clouds, plankton, turtles, whales, cattle, birds, tap water, beer, salt, human placentas, semen, breast milk, feces, testicles, [...]
New Blood Test Detects Alzheimer’s and Tracks Its Progression With 92% Accuracy
The new test could help identify which patients are most likely to benefit from new Alzheimer’s drugs. A newly developed blood test for Alzheimer’s disease not only helps confirm the presence of the condition but also [...]
The CDC buried a measles forecast that stressed the need for vaccinations
This story was originally published on ProPublica, a nonprofit newsroom that investigates abuses of power. Sign up to receive our biggest stories as soon as they’re published. ProPublica — Leaders at the Centers for Disease Control and Prevention [...]
Light-Driven Plasmonic Microrobots for Nanoparticle Manipulation
A recent study published in Nature Communications presents a new microrobotic platform designed to improve the precision and versatility of nanoparticle manipulation using light. Led by Jin Qin and colleagues, the research addresses limitations in traditional [...]
Cancer’s “Master Switch” Blocked for Good in Landmark Study
Researchers discovered peptides that permanently block a key cancer protein once thought untreatable, using a new screening method to test their effectiveness inside cells. For the first time, scientists have identified promising drug candidates [...]
AI self-cloning claims: A new frontier or a looming threat?
Chinese scientists claim that some AI models can replicate themselves and protect against shutdown. Has artificial intelligence crossed the so-called red line? Chinese researchers have published two reports on arXiv claiming that some artificial [...]
New Drug Turns Human Blood Into Mosquito-Killing Weapon
Nitisinone, a drug for rare diseases, kills mosquitoes when present in human blood and may become a new tool to fight malaria, offering longer-lasting, environmentally safer effects than ivermectin. Controlling mosquito populations is a [...]
DNA Microscopy Creates 3D Maps of Life From the Inside Out
What if you could take a picture of every gene inside a living organism—not with light, but with DNA itself? Scientists at the University of Chicago have pioneered a revolutionary imaging technique called volumetric DNA microscopy. It builds [...]
Scientists Just Captured the Stunning Process That Shapes Chromosomes
Scientists at EMBL have captured how human chromosomes fold into their signature rod shape during cell division, using a groundbreaking method called LoopTrace. By observing overlapping DNA loops forming in high resolution, they revealed that large [...]
Bird Flu Virus Is Mutating Fast – Scientists Say Our Vaccines May Not Be Enough
H5N1 influenza is evolving rapidly, weakening the effectiveness of existing antibodies and increasing its potential threat to humans. Scientists at UNC Charlotte and MIT used high-performance computational modeling to analyze thousands of viral protein-antibody interactions, revealing [...]
Revolutionary Cancer Vaccine Targets All Solid Tumors
The method triggers immune responses that inhibit melanoma, triple-negative breast cancer, lung carcinoma, and ovarian cancer. Cancer treatment vaccines have been in development since 2010, when the first was approved for prostate cancer, followed [...]
Scientists Uncover Hidden Protein Driving Autoimmune Attacks
Scientists have uncovered a critical piece of the puzzle in autoimmune diseases: a protein that helps release immune response molecules. By studying an ultra-rare condition, researchers identified ArfGAP2 as a key player in immune [...]
Mediterranean neutrino observatory sets new limits on quantum gravity
Quantum gravity is the missing link between general relativity and quantum mechanics, the yet-to-be-discovered key to a unified theory capable of explaining both the infinitely large and the infinitely small. The solution to this [...]
Challenging Previous Beliefs: Japanese Scientists Discover Hidden Protector of Heart
A Japanese research team found that the oxidized form of glutathione (GSSG) may protect heart tissue by modifying a key protein, potentially offering a novel therapeutic approach for ischemic heart failure. A new study [...]