Characterizing the size and concentration of liposomes using NanoSight Pro

Introduction

Liposomes are spherical-shaped vesicles composed of one or more phospholipid bilayers. They are often used as drug delivery vehicles for the administration of pharmaceutical drugs and nutrients; as such, it is essential that their size distribution is well characterized for manufacturing and batch release. Nanoparticle Tracking Analysis (NTA)  is a powerful technique for the high-resolution analysis of particle size distributions and concentration. This application note will demonstrate how the new Malvern Panalytical NanoSight Pro can measure the particle concentration as well as the particle size of liposome samples over a range of dilutions. The samples were also measured on a Zetasizer Ultra Red to compare their working concentration ranges. 

Experimental

The liposomes used were Formumax HSPC/CHOL liposomes (Cat. No.: F0102) and were diluted using Gibco PBS at pH 7.2 (Cat No.: 20012019) using a gravimetric dilution protocol. Nominal dilutions from 100x to 5,000,000x were measured. 

A NanoSight Pro,  equipped with a 488nm laser module, and a Zetasizer Ultra Red were used to measure the liposomes at 25°C. On the NanoSight Pro, six dilutions from x10,000 to x5,000,000 were each measured in triplicate, with each measurement performed under flow at 1.5 µL/min made of 5 videos of 750 frames each. Using the Zetasizer, six dilutions from x100 to x500,000 were each measured in triplicate using a particle concentration MADLS measurement, using an RI of 1.46 with an absorption of 0.001. 

Results

The size results across the dilution range, measured on both the NanoSight Pro and the Zetasizer using MADLS, show a consistent mode size of 82-93nm with no significant change across the whole range, as can be seen in Figure 1. 

Figure 2, below, shows the size distributions for the 50,000x dilution and we can see how closely aligned they are - although the high resolution of the NanoSight Pro provides a narrower distribution.

The average concentrations from each instrument are shown below in Figure 3, demonstrating strong concordance between the two techniques for the 50,000x and 100,000x dilutions. This agreement indicates that these dilution levels fall within a suitable concentration range for both techniques. At lower dilution factors, the sample concentration becomes too high for NTA to resolve all particle tracks. Conversely, when using the Zetasizer Ultra at higher dilution factors, the sample's concentration becomes too low, resulting in insufficient scattering signals to accurately determine the concentration. While each technique excels within its specific optimum concentration measurement range, applying both techniques expands the scope of linearity studies. When used together, both methods collectively span the entire linearity range. 

Conclusion

In this study, we have demonstrated that the NanoSight Pro can accurately measure the size and concentration of liposomes across a variety of dilutions, showing a strong correlation with the Zetasizer within their overlapping ranges. Although the operational ranges of the instruments differ, both can be measured across a broad spectrum of magnitudes. Notably, the NanoSight Pro excels in detecting particles at lower concentrations and offers higher resolution, enabling it to assess the size distribution of samples with enhanced precision. When used in tandem, data from both NanoSight Pro and Zetasizer Ultra provide a comprehensive view of liposome concentration linearity, coupled with precise size measurements to confirm sample stability.