Analysis of Liposomes and other Drug Delivery Systems by NTA
A wide variety of nanostructures can be utilised as drug delivery vehicles and can be designed and constructed to target specific receptors, to exhibit increased payload and circulatory lifetime by operating in ‘stealth-mode’, reduce side-effects, improve uptake and efficacy, etc.
Liposomes are one such type of structure that have been the subject of significant R&D for many years. The use and potential of liposomes (Figure 1) as drug deliverers continues to grow in importance. The reasons are clear:
- Drugs delivered via liposomes may be protected from the actions of metabolizing enzymes
- Lipophillic drugs may be made soluble
- Drugs can be targeted to specific areas by attaching ligands to the liposome
- Liposomes are readily absorbed by cells
- The rate of drug release may be controlled by the selection of liposome
- Using liposomes as a drug deliverer allows potentially lower doses of drug to be used, reducing toxicity and side-effects. Furthermore, it is possible that gene therapy drugs may be delivered by liposomes.
The size of the liposomes is increasingly being recognised as an important factor in treatment efficacy. The size of the liposome used in drug delivery may affect its circulation and residence time in the blood, the efficacy of the targeting, the rate of cell absorption (or endocytosis) and, ultimately, the successful release of its payload. Such size considerations are also hugely important to nanoscale polymer-encapsulated drug delivery systems. Accurate measurement of the particles being administered is therefore imperative.
Figure 1: Typical Liposome Structure.
Sizing Liposomes by NanoSight NTA
NanoSight instruments accurately and rapidly size liposomes in water requiring only small volumes and very little sample preparation. The system enables individual liposomes in suspension to be visualised and their Brownian motion tracked – enabling particle size distributions, based on individual particles, to be built up in a matter of seconds.
Figure 2: Typical image of liposomes provided by a NanoSight instrument.
Whilst the NanoSight viewing unit provides a unique view of the nanoparticles (Figure 2), the Nanoparticle Tracking and Analysis software suite (NTA 2.0) is used to accurately size the liposomes (Figure 3).
Figure 3: NTA Results page from bimodal nanoparticle sample.
Simultaneous Multi-paramete Analysis of Nanoparticles in Real-time
Nanoparticle Refractive Index
Uniquely, and because NanoSight’s technology allows nanoparticles to visualised simultaneously but separately, it is possible to obtain more information about any given nanoparticle than merely determining its size through Brownian motion analysis.
The NanoSight system provides the unique ability to for provide a true number-based size distribution of the sample along with a range of statistical measurements.
It is also possible to measure the relative light scattering intensity of a particle and plot this data against the independently obtained measurement of its size. This allows particles of different refractive index or material composition to be resolved in even greater detail than is afforded by NTA’s already exceptional size resolving capabilities.
The following example shows a mixture of 100nm polymeric nanoparticle of relatively low refractive index (and therefore scattering power) can be easily resolved from a mixture of 30nm and 60nm metallic nanoparticles which can be seen to be smaller.
Figure 4: Results from NTA showing 3 peaks; 30nm Gold, 60nm Gold and 100nm Polystyrene
Note that the 60nm peak is more highly scattering. The vertical axis on these plots is of particle concentration allowing numbers frequency distributions to be obtained directly from the sample.
This unique ability potentially allows the user to probe whether nanoscale drug delivery structures such as liposomes vary in their contents, i.e. empty liposomes may have a lower Ri (light scattering power) than those loaded with a higher Ri material. This would allow them to be discriminated even though they may be of very similar size.
By using different illumination laser wavelengths (405nm and 532nm blue and green lasers) instead of the standard 635nm red laser, it is possible to excite suitable fluorophores attached to, or contained within, liposomes and similar drug delivery vehicles.
In the following example, micro-and nanovesicles have been labelled with suitable fluorophores through the use of surface-specific antibodies, thus allowing a particular sub-population of labelled nanovesicle particles to be discriminated from unlabelled.
The similarly sized peaks shown below indicate that nearly all the vesicles seen under light scattering mode (green line) have been labelled with fluorophores (red line, as visualised under a suitable excitation wavelength).
Figure 6: Results from NTA in light-scattering mode (green line) and fluorescent mode (red line).
For further information, contact NanoSight or your local distributor, listed at www.nanosight.com