Researchers examine the application and usefulness of nanoparticle tracking analysis and light scattering techniques in the characterization of liposomes used as drug delivery vehicles.


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Figure 1: NTA measurement set up. Images: Malvern Instruments

When considering potential drug delivery vehicles, liposomes are an important option and have already been approved for use with a number of therapeutic formulations. Liposomes are comprised of phospholipids and may be single- or multi-layered, can be produced in different sizes and have a hydrophilic interior and hydrophobic shell. They are biodegradable and essentially non-toxic and, importantly, are capable of encapsulating both hydrophilic and hydrophobic materials. In addition, the surface of the liposome can be modified in order to enable targeting of drugs at specific biological sites, promote longevity of the liposome in vivo or engineer a diagnostic tool.

As with all such developments, it’s essential to ensure that the physical properties of the liposomes are suitable for their application. How will they behave once they are in the body, and are they stable enough to ensure the drug is delivered to the required site? Are the particles sized correctly for clinical applications, or will they be lost from the bloodstream?

Knowing the size, concentration and zeta potential of a liposome preparation can help predict its fate in vivo, while the association of charged liposomes with oppositely charged molecules can be monitored by measuring the zeta potential of the resulting complex. These factors can have a marked effect on the efficiency of drug delivery, and their analysis can assist formulators when considering a specific liposome as a suitable vehicle. Reliable analytical systems that provide robust data make an important contribution to the formulation process. Nanoparticle tracking analysis and dynamic light scattering techniques are two approaches that provide essential and complementary information.

Figure 2: Particles are seen as light points moving under Brownian motion. Nanoparticle tracking analysis
Nanoparticle tracking analysis (NTA) uses laser light scattering to examine nanoparticles in solution (Figure 1). It enables the visualization of individual particles and tracking of their Brownian motion so that size distributions, based on individual particles, are built up in a matter of seconds.

Light scattered by the particles in solution is captured using a scientific digital camera and the motion of each particle is tracked from frame to frame by the instrument software (Figure 2).

This rate of particle movement is related to a sphere equivalent hydrodynamic radius as calculated through the Stokes-Einstein equation (Figure 3). The technique calculates particle size on a particle-by-particle basis and, because video clips form the basis of the analysis, it’s possible to accurately characterize real-time events.

Figure 3: The Stokes-Einstein equation.Since NTA technology allows the visualization of nanoparticles simultaneously but separately, it’s possible to obtain additional information. One such possibility is measurement of the relative light scattering intensity of a particle. The resulting data can be plotted against the independently obtained measurement of particle size, allowing particles of different refractive index (RI) or material composition to be resolved in even greater detail. This unique ability potentially allows the user to probe whether nanoscale drug delivery structures such as liposomes vary in their contents: Empty liposomes may have a lower RI (light scattering power) than those loaded with a higher RI material. Such differences could allow their discrimination even if they were of very similar size.

This single particle detection system also allows the measurement of particle concentration.

Particle size and zeta potential
While the sites of action of liposome-cell interactions are largely determined by size, knowing the zeta potential of a liposome preparation can help predict the fate of the liposomes in vivo. The zeta potential of a particle is the overall charge that the particle acquires in a particular medium. In gene therapy for instance, zeta potential measurements can be used to optimize the ratio for particular liposomes with various DNA plasmids in order to minimize aggregation (Figure 4).

The well-established technique of dynamic light scattering (DLS) is widely used in the characterization of liposomes and, since zeta potential is also such a vitally important parameter, analytical systems, such as the Zetasizer Nano from Malvern Instruments, that offer the measurement of both, are finding widespread application. While DLS is used to measure particle size, the technique of laser doppler microelectrophoresis is used for zeta potential measurement.

Figure 4: Cationic liposomes (positively charged) are complexed with DNA (plasmids).Once again, light scattering resulting from the Brownian motion of the particles is at the heart of the DLS technique. DLS measures the time-dependent fluctuations in the intensity of the scattered light and determines the diffusion coefficients of the particles. Again using the Stokes-Einstein equation, this information is converted into a particle size distribution.

Using laser doppler microelectrophoresis to measure zeta potential, an electric field is applied to a solution of molecules or a dispersion of particles, which then move with a velocity related to their zeta potential. Measurement of this velocity enables the calculation of electrophoretic mobility and from this the zeta potential and zeta potential distribution.

In conclusion
The physical characterization of liposomes is of great importance in understanding their suitability for a range of applications with rapid, repeatable characterization, an important consideration as part of the development and quality control processes. The techniques described above provide complementary information about the size, concentration and zeta potential of liposome preparations.