Methods for making giant vesicles and their use
This application is directed to methods which allow for encapsulation of an array of biological materials under physiological conditions that are relevant given a biological context and the compositions made using those methods. These reconstituted biological materials encompass: a) purified proteins that can bind to the lipid membranes inside and outside of vesicles based on the electrostatic charge; b) purified cytosolic proteins that position themselves in the lumen of the vesicles, c) mammalian cell extracts with an array of cytosolic protein content, among other contents and d) small biological molecules such as DNA and RNA as well as fluorescent dyes/probes. These vesicles can be used to simulate cells in drug discovery methods as well as useful in administering drugs and other compositions to cells in vitro and in vivo.
1. A giant unilamellar vesicle (GUV) comprising a unilamellar lipid bilayer, wherein the unilamellar lipid bilayer includes the outer surface of the GUV and encloses the inner lumen of the GUV;
wherein the unilamellar lipid bilayer comprises at least a first lipid and second lipid selected from a phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol 4,5-biphosphate (PIP2), and DGS-NTA (Ni), and combinations thereof wherein the first and second lipid and combinations thereof may be the same or different;
and wherein the GUV comprises
a chemically inducible dimerization protein in the inner lumen and a binding partner protein on the outer surface; wherein the binding partner protein on the outer surface is fused with myristoylated alanine-rich protein kinase C substrate (MARCKS) or a C2 domain of lactadherin (lact-C2).
2. The GUV of claim 1 , wherein the inner lumen comprises a lipid selected from phosphatidylserine (PS), phosphatidylinositol 4,5-biphosphate (PIP2), and phosphatidylglycerol (PG): phosphatidylcholine (PC).
3. The GUV of claim 1 , wherein the outer surface comprises a lipid selected from phosphatidylcholine (PC), phosphatidylcholine (PC):phosphatidylserine (PS), and combinations thereof.
4. The GUV of claim 1 , wherein the diameter of the GUV is about 1 nm to about 1000 μm.
5. The GUV of claim 1 , wherein the first lipid and second lipid of the unilamellar lipid bilayer further comprises PEG2000.