Dynamic Light Scattering (DLS) Service

Case Integration of Standardized Dispersion Protocols and Dynamic Light Scattering for Improved Nanomaterial Hazard Assessment in Cell Media

Background

The assessment of nanomaterial (NM) hazards lacks consistency across laboratories, necessitating the development of standardized protocols. Realistic conditions for NM exposure, influenced by diverse physicochemical properties, present challenges. Dispersion protocols, crucial for toxicological tests, vary based on NM characteristics, and understanding their impact on stability and cellular responses is vital.

Samples

The study focuses on diverse NMs, including nanometric silica (NM-101, NM-200, NM-203), titanium dioxide (NM-100, NM-101), and cerium dioxide (NM-212). These NMs, with varying primary particle sizes and compositions, represent materials commonly encountered in industrial and environmental settings.

Technical Methods

Nanomaterial Selection:

• Diverse nanomaterials (NMs) were chosen, including nanometric silica (NM-101, NM-200, NM-203), titanium dioxide (NM-100, NM-101), and cerium dioxide (NM-212).
• NMs represented a range of primary particle sizes and compositions relevant to industrial and environmental exposure scenarios.

Dispersion Protocols:

Standardized Protocol:

• Utilized albumin as a dispersant.
• Employed a standardized sonication procedure.
• Based on protocols developed within EU NANOGENOTOX and NANoREG.

Traditional Protocol:

• Involved simple dispersion in water.
• Utilized brief sonication.

Dynamic Light Scattering (DLS) Measurements:

• DLS employed to monitor size distribution of NM dispersions.
• Consideration of limitations in DLS measurements, including artifacts and interference from proteins, particularly in dilute samples.
• Evaluation of factors such as count rate, repeatability, and overlapping of distribution curves to assess suspension stability.

Characterization of Stock Suspensions:

• Thermal Gravimetric Analysis (TGA) coupled with Fourier Transform Infrared (FTIR) detector conducted under nitrogen atmosphere to confirm stability and purity of NM batches.
• Analysis of weight loss and derivative curves to ascertain any variation in surface chemistry due to storage or manipulation.

Characterization of Stock Suspensions (continued):

• Differentiation between NM-200 and NM-203 based on synthetic methods, impacting surface chemistry and hydrophilicity.
• Characterization of titanium dioxide (NM-100, NM-101) and cerium dioxide (NM-212) NMs with focus on particle sizes and specific surface area.

Preparation for Toxicological Tests:

• Two protocols employed for NM preparation, differing in the type of solution and sonication procedure.
• Traditional protocol: NMs suspended in ultrapure water, de-agglomerated by vortexing and probe sonication.
• Standardized protocol: Small amount of ethanol introduced, powder suspended in water with 0.05% w/v albumin, followed by sonication.

Characterization of Stock Suspensions by DLS:

• Immediate DLS measurements post-sonication to assess hydrodynamic size distributions, average zeta size, and polydispersity index (PDI).
• Evaluation of dispersion patterns and stability in both media (RPMI and DMEM).

Characterization of NMs in Cell Media:

• DLS analysis of NMs in media supplemented with fetal bovine serum (FBS) at low (1 µg/mL) and high (100 µg/mL) concentrations.
• Examination of hydrodynamic size distributions, Zeta potential, and PDI in RPMI and DMEM.

Stability During Incubation:

• Evaluation of NM behavior in cell media (RPMI and DMEM) through DLS analysis post-dilution and after 48 hours of incubation at the highest concentration (100 µg/mL).
• Comparison of DLS patterns between traditional and standardized protocols.

Cytotoxicity Assessment:

• Measurement of lactate dehydrogenase (LDH) leakage as an indicator of cellular membrane damage.
• Assessment of cytotoxic effects on THP-1 (RPMI media) and RAW 264.7 (DMEM media) cell lines for silica NMs (NM-200, NM-203).

Activation of Cells:

• Evaluation of pro-inflammatory activation in RAW 264.7 cells through the release of nitric oxide (NO).
• Measurement of release of cytokine IL-1β in THP-1 cells as an indication of pro-inflammatory response.

Data Analysis and Correlation:

• Correlation of DLS data with cellular responses to understand the impact of dispersion protocols on NM behavior in cell media.
• Consideration of the role of protein corona formation in modifying cellular responses to NMs.

Results

Stability and Size Distribution:

• Standardized protocols enhance stability and reduce hydrodynamic diameters for nanometric primary particle NMs (NM-101, NM-200, NM-203).
• Negligible effects observed for sub-micrometric particle samples.

Correlation with Cellular Response:

• Complex correlation between DLS data and cellular responses.
• Standardized protocols mitigate cytotoxic effects in some cases but increase cellular activation in others.
• Protein corona formation influences material-dependent responses.

Material-Specific Impacts:

• Different materials exhibit varied responses to dispersion protocols.
• Silica NMs (NM-200, NM-203) show distinct behaviors, with the standardized protocol mitigating cytotoxicity and stabilizing dispersions.