Summary
Liposomes are a common system used to encapsulate and deliver bioactive cargo molecules. In contrast to simple emulsions (oil-in-water or water-in-oil), these lipid vesicles are composed of well-defined bilayer structures and have aqueous interiors. These bilayers encircle the molecule of interest, often proteins or oligonucleotides such as DNA or RNA. These vesicle structures are inherently biocompatible and can be brought into the interior of living cells via endocytosis, where gradients in pH or ionic strength result in the targeted release of a molecule with biological activity. Since both the self-assembly and release mechanisms rely strongly on electrostatics, zeta potential and hydrodynamic size can play a role in characterizing these structures.
Introduction
Liposomes are a commonly used tool for encapsulating biological materials. These self-assembled structures are composed of lipid bilayers that encircle a given cargo molecule, frequently charged biomolecules such as oligonucleotides. These structures can serve as mimics of cell membranes, exosomes, and other biological vesicle structures and, as such, provide a high level of biocompatibility and can serve as a targeted mechanism to deliver a biologically relevant cargo material to a cell.
Like many self-assembled structures, these liposomes, or lipid vesicles, are often electrostatically stabilized, and when prepared from crude lipid stock can be heterogeneous in size and structure.
Self-assembled structures may be complex and can depend on preparation, purity of starting materials, and sample history. In the absence of mechanical homogenization, lipid vesicles and lipid nanoparticles can form higher order structures; for instance, ‘vesicle-in-vesicle’ structures such as multilamellar vesicles, often containing variable numbers of layers. A commercially available liposome composed of a known mixture of lipids was used to simplify this process and produce a more uniform starting material.
Sample Preparation
The starting material was a lyophilized powder containing a known ratio of L-α-Phosphatidylcholine (PC) and stearylamine (SA), the latter having a cationic headgroup, and the former having a zwitterionic head and branched tail. These freeze-dried lipid vesicles were then resuspended in 10 mL of triple filter deionized water, yielding a stock concentration of 6.3 mM phosphatidylcholine and 1.8 mM stearylamine. This liposome stock was then diluted 20:1 in phosphate buffered saline (PBS) to prepare a working solution. A 153 μM (10mg/mL) stock solution of serum albumin (BSA) was also prepared.
Results
Five different mole fractions of this ternary mixture were examined in order to determine the effect of protein-loading on premixed liposomes. The mole fractions for the ternary mixture are defined as follows:
X BSA = moles BSA / ( moles BSA + moles PC + moles SA )
X PC = moles PC / ( moles BSA + moles PC + moles SA )
X SA= moles SA / ( moles BSA + moles PC + moles SA ) For simplicity we can choose to reduce this to a binary mixture, since the ratio of phosphatidylcholine to stearylamine is kept constant. Thus, the two relevant mole fractions become X BSA and X Lipids.
Liposomes assembled from mixtures of phosphatidylcholine and stearylamine are expected to be cationic, so it follows that when there is an excess of liposomes relative to our cargo molecule, in this case serum albumin (BSA), the zeta potential is positive (ca. +20 mV). At physiological pH, BSA is weakly negatively charged, consistent with the roughly -15 mV zeta potential measured in the absence of any lipids.
Similarly, the average size of vesicles can be observed to decrease in the presence of excess cargo which may reflect a change in the structure of the liposomes, although the potentially enormous heterogeneity of these lamellar structures makes it difficult to draw such conclusions from scattering alone.
Recap
This is a useful starting point which suggests the need for complimentary techniques such as confocal fluorescence microscopy, small angle X-ray scattering, or assays that can be used to confirm colocalization of cargo molecules, to characterize higher order vesicle structure, or to confirm the successful protection and delivery of cargo molecules.
- The NanoBrook can be used to measure size and zeta potential of self-assembled systems including liposomes as well as other vesicle structures. These measurements can be made at physiological pH and ionic strength.
- Effective diameter and zeta potential can be measured rapidly, allowing for quick screening of a wide range of different encapsulation conditions.
- Liposomes and other lipid-based nanomaterials are essential for delivery of bioactive materials to drug targets.
Instruments: