Measuring the zeta potential of liposomal nanoparticles presents unique considerations due to their phospholipid bilayer structure, dynamic surface properties, and sensitivity to measurement conditions. Unlike rigid inorganic nanoparticles, liposomes exhibit complex interfacial behavior influenced by composition, environmental factors, and measurement techniques. The following discussion focuses on critical aspects of zeta potential analysis specific to liposomal systems.

The phospholipid bilayer introduces several measurement complexities. Liposomes possess a soft, fluid surface where the shear plane—the boundary used to calculate zeta potential—is less defined than in solid nanoparticles. The mobility of phosphate head groups and acyl chains affects the positioning of this plane, leading to potential variability in measurements. Phosphatidylcholine-based liposomes typically exhibit negative zeta potentials in physiological pH ranges due to the ionization of phosphate groups, with values often falling between -10 mV to -30 mV. However, this varies significantly with lipid composition. Incorporating cationic lipids like DOTAP can shift potentials to +30 mV to +60 mV, while anionic lipids such as phosphatidylserine yield more negative values (-40 mV to -60 mV).

Cholesterol incorporation alters zeta potential through multiple mechanisms. At concentrations of 30-50 mol%, cholesterol condenses the bilayer, reducing phospholipid head group mobility and increasing surface charge density. This typically enhances the magnitude of zeta potential by 5-15% compared to cholesterol-free liposomes at the same lipid composition. Cholesterol also decreases membrane permeability to ions, which can affect the stability of the electrical double layer during measurement. However, cholesterol does not contribute direct charge effects since it lacks ionizable groups.

Maintaining vesicle integrity during measurement requires careful optimization of several parameters. The electric field strength in electrophoretic light scattering must balance between sufficient particle mobility and avoidance of liposome deformation. Field strengths above 10 V/cm can induce bilayer stretching or rupture, particularly for large unilamellar vesicles (LUVs) below 200 nm diameter. Temperature control is equally critical, as lipid phase transitions dramatically affect zeta potential. For example, DPPC liposomes show a 20-30% change in zeta potential magnitude when measured above versus below their phase transition temperature (41°C).

Sample preparation introduces additional challenges. Buffer ionic strength must be optimized—typically between 1-10 mM for liposomes—to prevent both double layer compression (at high ionic strength) and excessive particle aggregation (at low ionic strength). Common buffers like PBS are problematic due to their high conductivity; lower-conductivity alternatives such as HEPES or Tris-HCl are preferred. The dilution process itself can alter liposome properties; excessive dilution may destabilize vesicles, while insufficient dilution leads to multiple scattering effects. A dilution factor of 1:100 to 1:1000 in matching buffer is generally effective.

Measurement interpretation requires formulation-specific considerations. For PEGylated liposomes, the zeta potential reflects the hydrodynamic slip plane within the polymer brush layer rather than the actual lipid surface. This results in lower apparent zeta potentials compared to non-PEGylated equivalents, with reductions of 5-15 mV depending on PEG chain length and density. Multilamellar vesicles (MLVs) present another complication, as their zeta potential represents an average of all lamellae interfaces rather than just the outermost surface.

The pH dependence of liposomal zeta potential follows different patterns than solid nanoparticles. Phospholipids exhibit broad titration curves rather than sharp inflection points due to distributed charge across the bilayer. For example, the zeta potential of POPC liposomes changes gradually from +5 mV at pH 3 to -25 mV at pH 9, without distinct isoelectric points. This behavior contrasts with metal oxide nanoparticles that show abrupt potential reversals at specific pH values.

Several artifacts can occur during liposomal zeta potential measurement. Vesicle deformation in the electric field may cause anomalous mobility values, detectable through abnormal electrophoretic mobility distributions. Sample turbidity affects measurement accuracy; liposome concentrations should maintain count rates between 50-500 kcps in typical instruments. Air bubbles introduced during sample loading can adhere to liposomes, drastically altering their apparent mobility. Centrifugation or filtration pre-treatments may be necessary for extruded liposome preparations to remove dust or lipid aggregates.

The relationship between zeta potential and liposome stability follows different thresholds than other nanoparticles. While colloidal theory suggests |30 mV| indicates stability, liposomes often remain stable at |15-20 mV| due to steric stabilization from protruding lipid headgroups. However, this depends on storage conditions—lyophilized liposomes require higher initial zeta potentials to maintain stability upon reconstitution than aqueous suspensions.

Advanced measurement techniques can provide additional insights. Phase analysis light scattering (PALS) improves accuracy for low-mobility liposomes compared to fast field reversal methods. Laser Doppler electrophoresis with multi-angle detection helps compensate for the size-dependent mobility variations inherent in polydisperse liposome samples. For temperature-sensitive formulations, simultaneous dynamic light scattering and zeta potential measurements track size and potential changes during thermal cycling.

Data interpretation should account for time-dependent effects. Liposome zeta potentials often drift during measurement as ions redistribute across the bilayer. Recording initial values within the first 30 seconds of measurement provides the most representative data for freshly prepared samples. Long-duration measurements may show potential shifts of 2-5 mV/min due to ion adsorption or pH changes from electrode reactions.

The following table summarizes key measurement parameters for different liposome types:

Liposome Type | Optimal Field Strength | Buffer Ionic Strength | Temperature Range | Expected Potential Range
Conventional | 5-8 V/cm | 1-5 mM | 20-25°C | -20 to -40 mV
Cationic | 4-6 V/cm | 1-3 mM | 20-25°C | +30 to +60 mV
PEGylated | 6-10 V/cm | 5-10 mM | 20-25°C | -5 to -20 mV
Thermosensitive | 5-7 V/cm | 1-5 mM | 4-50°C | -15 to -30 mV

Understanding these formulation-specific behaviors allows proper design of zeta potential measurement protocols for liposomal nanoparticles. The data obtained must be interpreted within the context of the liposome's composition, preparation method, and intended application environment to yield meaningful characterization results.