Measuring the surface charge of nanoparticles in biological fluids is critical for predicting their stability, cellular interactions, and biodistribution. The zeta potential, derived from electrophoretic mobility, serves as a key indicator of nanoparticle surface properties. However, obtaining accurate measurements in complex biological media such as serum, plasma, or cell culture media presents significant challenges due to the dynamic interplay of proteins, ions, and other biomolecules with nanoparticle surfaces.
Biological fluids contain high concentrations of proteins, electrolytes, and organic molecules that rapidly adsorb onto nanoparticle surfaces, forming a protein corona. This corona alters the effective surface charge, often masking the original zeta potential of the bare nanoparticle. For example, studies show that polystyrene nanoparticles initially possessing a negative zeta potential of -50 mV in water can shift to near-neutral values (-5 to +5 mV) after incubation in fetal bovine serum due to protein adsorption. The magnitude and direction of this shift depend on the nanoparticle's composition, size, and surface chemistry, as well as the specific proteins present in the biological fluid.
The ionic strength of biological media further complicates measurements by compressing the electrical double layer around nanoparticles. This compression reduces the measurable zeta potential magnitude, even if the intrinsic surface charge remains unchanged. Phosphate-buffered saline (PBS), commonly used as a diluent, can introduce artifacts due to its high ionic strength, leading to underestimation of zeta potential values. Alternative low-ionic-strength buffers, such as 1 mM NaCl or 5 mM HEPES, may provide more reliable measurements but risk altering the native state of the nanoparticle-protein complex.
Dynamic light scattering (DLS) instruments equipped with phase analysis light scattering (PALS) are typically employed for zeta potential measurements in biological media. However, the presence of proteins and other macromolecules increases the viscosity and refractive index of the medium, requiring careful calibration. The following table summarizes key adjustments needed for accurate measurements in biological fluids:
Parameter Adjustment Required
Medium Viscosity Use measured viscosity values instead of water
Refractive Index Input correct dispersant refractive index
Conductivity Account for high ionic strength effects
Temperature Maintain 37°C for physiological relevance
Protein adsorption is time-dependent, with rapid initial binding occurring within minutes and slower reorganization over hours. This temporal evolution means that zeta potential measurements must be performed at consistent time points post-incubation to enable meaningful comparisons. Immediate measurements after nanoparticle introduction to biological fluids may reflect only partial corona formation, while delayed measurements capture equilibrium states but may miss transient interactions relevant to in vivo behavior.
To mitigate these challenges, several methodological approaches have been developed. One strategy involves pre-coating nanoparticles with defined protein layers before measurement, creating a more reproducible system. For instance, pre-incubation with human serum albumin allows controlled study of how this dominant plasma protein affects zeta potential. Another approach uses density gradient centrifugation to isolate nanoparticle-protein complexes from unbound proteins prior to measurement, though this risks disturbing the corona structure.
The pH of biological fluids significantly influences zeta potential measurements. Blood plasma maintains a pH of 7.4, while endosomal compartments can drop to pH 5.0-6.0. Since protein adsorption and nanoparticle surface chemistry are pH-dependent, measurements should always report the pH conditions and, where possible, replicate physiological pH gradients encountered in vivo. Automated titration systems can map zeta potential versus pH profiles, revealing isoelectric points that predict nanoparticle aggregation tendencies.
Nanoparticle concentration in the measurement cell must be optimized to balance signal quality against artifactual interactions. Too high concentrations promote particle-particle interactions that skew results, while overly dilute samples yield poor signal-to-noise ratios. For gold nanoparticles in serum, concentrations between 10-50 μg/mL typically provide reliable measurements without interference effects.
Advanced techniques such as tunable resistive pulse sensing (TRPS) and microfluidic electrophoresis offer alternative approaches to conventional DLS-based zeta potential analysis in biological media. These methods can resolve subpopulations within heterogeneous nanoparticle samples and may better handle the high ionic strength of biological fluids. However, they require specialized instrumentation and validation against established techniques.
Control experiments are essential for interpreting zeta potential data in biological fluids. Measurements should include:
- Nanoparticles in buffer alone (no biological fluid)
- Biological fluid alone (no nanoparticles)
- Time-course measurements after mixing
- Multiple batches of biological fluid to account for donor variability
For cell culture media, the presence of phenol red indicator and varying nutrient compositions between formulations (DMEM vs. RPMI) can introduce additional variability. Serum-free media often yield more consistent results but lack physiologically relevant protein interactions.
Standardization remains a major hurdle in comparing zeta potential data across studies. Differences in instrument calibration, measurement parameters, and sample preparation protocols can lead to substantial variations in reported values. Recent efforts by organizations such as the International Organization for Standardization (ISO) aim to establish guidelines for nanoparticle characterization in biological matrices, including zeta potential measurement protocols.
The temperature at which measurements are conducted also affects results. While room temperature (25°C) measurements are common, physiological temperature (37°C) better reflects in vivo conditions but may require instrument modifications to maintain stable thermal control during analysis. Temperature fluctuations as small as 1°C can alter viscosity enough to impact calculated zeta potential values.
In conclusion, meaningful zeta potential characterization of nanoparticles in biological fluids requires careful consideration of medium composition, time-dependent protein interactions, measurement parameters, and appropriate controls. While no single protocol fits all applications, systematic approaches that account for these variables can yield data that better predicts nanoparticle behavior in biological systems. Future methodological developments should focus on real-time monitoring of zeta potential changes and improved standardization across research laboratories.