Single particle characterisation of lipidbased nanoparticles using automated Raman trapping analysis

Abstract

Liposomes are self-assembled lipid structures comprising a lipid membrane bilayer encapsulating an aqueous core. They have for a long time been acknowledged as appropriate delivery vectors for the delivery of both hydrophobic and hydrophilic cargo. However, they are not able to achieve active targeting of specific cells or tissues. Extracellular vesicles (EVs) are lipid membrane-bound vesicles ranging in diameter from 10s to 1000s of nm which are produced by almost all cells in the body. They have shown effective targeting ability and are innately biocompatible. However, they are heterogeneous and hard to purify in large enough amounts for use in drug delivery. It has been suggested that designing liposomes, which can mimic extracellular vesicles, might overcome these issues and allow better drug delivery vectors to be designed. In this thesis, a protocol to characterize liposomes on the single particle level was developed, using a single particle automated Raman trapping analysis (SPARTA) instrument which combines Raman spectrometry with optical trapping. As a model system, liposomes of varying lipids with varying degrees of cholesterol were studied, since high cholesterol content has often been reported for extracellular vesicles. This leads to the report having two goals, the first being to optimize and learn SPARTA in order to study particle on a single particle level, with a high throughput. The second goal was to characterize the efficiency of cholesterol encapsulation at high mol % and investigate how cholesterol affects the phase behavior of particles on the single particle level using SPARTA as the main method. Large unilamellar vesicles were formulated using three different phospholipids DPPC, POPC and DOPC. The difference between the three lipids are the amount of double bond unsaturations on the lipid tails; DPPC have zero, POPC have 1 and DOPC have 2. These three phospholipids were used to formulate vesicles with different amounts of cholesterol ranging from 0-70 mol % cholesterol for each lipid. The different compositions were then studied primarily using SPARTA with verification of lipid phase behavior using small and wide angle X-ray scattering (SAXS/WAXS) as a complimentary method. To experimentally determine maximum encapsulation of cholesterol into DPPC, POPC and DOPC liposomes, liposomes with compositions ranging from 40-70 mol % cholesterol were also analyzed using 1H − NMR. Initial experiments allowed the concentration range for single particle measurements to be determined. It was also shown that the particles studied in this thesis were homogeneous in composition, since only the signal intensity decreased upon increasing dilution. Next, it was shown that SPARTA can be used effectively to show cholesterol saturation which was confirmed by proton NMR, and double bond ratio and conformational order which were corroborated with SAXS/WAXS measurements. WAXS also showed formation of cholesterol crystals at higher cholesterol amount. SAXS/WAXS also allowed changes in D-spacing for specific lipid mixtures to be characterized with increased cholesterol. Together, these results showcase the possibles to characterise lipid vesicles on the single particle level using Raman spectroscopy, and specifically, how the effect of cholesterol on lipid phase behavior can be quantified on a single particle level. The findings here on these model systems allow us to better apply single particle automated Raman trapping analysis to study more complex, biological samples.