Introduction
Nanoparticles including liposomes are increasingly used for delivery of drug molecules. During formulation of a liposome around a drug particle, a relatively large amount of free drug may remain unencapsulated and therefore not available for drug delivery via the liposomal carrier. It is important to quantify the amount of free vs encapsulated drug in a product in order to accurately know the delivered dose of drug to a patient [1]. In this study, Centrifugal Field-Flow Fractionation (CF3) was used to separate free drug from liposomes and drug-containing liposomes in order to quantify the free drug.
Principle of CF3
The CF3 fractionation channel is mounted on a centrifuge, which spins to generate a centrifugal field perpendicular to the channel flow. Less massive particles can diffuse against the centrifugal field and elute sooner than more massive particles, resulting in a mass separation.
Experimental Details and Results
Optical microscopy image of the liposome-drug product (Figure 2) shows a mix of three species: free drug (black specks), empty liposomes (bubble-like structures), and liposome-encapsulated drug. The goal of the study was to quantify the amount of free drug using CF3 coupled with diode array UV-Vis detection and verify the separation with optical microscopy.
The CF3-UV fractogram (Figure 3) shows clear separation of two populations in the sample. As CF3 separates by mass, the very light drug particles are likely to elute at the beginning of the separation, and the heavier liposome-encapsulated drugs will elute later. Fractions were collected every 15 seconds (0.25 minutes) and imaged using optical microscopy. Representative microscopy images of the fractions are shown in Figure 4 and Figure 5. Figure 4 shows a fraction collected in the intial peak from Figure 3, at 0.75 to 1 minutes, and only free drug particles are observed in this fraction. Figure 5 shows a fraction collected in the second peak from Figure 3, at 2.25 to 2.5 minutes, and both empty liposomes and liposome-encapsulated drug are observed in this fraction. Integration of the peak areas allowed calculation of the amount of free drug, at 29.0 ± 1.6 % of peak area.
Conclusion
Centrifugal Field-Flow Fractionation coupled with UV-Vis detection was able to separate and quantify the amount of free drug in a liposome-drug product. The separation was verified using optical microscopy. This is a promising application using FFF for separation and quantifi cation of drug particles, as the field of nanomedicine and liposomal drug delivery grows.
References
[1] Mayer L.D., St. -Onge G., Analytical Biochemistry, 1995, 232(2), 149-157.
