Wow, that’s a long story! After getting a PhD in physics, I decided that the problems outside physics looked a lot more interesting than problems in physics. So, I switched to biochemistry as a postdoc in Oxford, knowing nothing about lipids or membranes, but a bit about nuclear magnetic resonance (NMR). There I met Ben de Kruijff, a postdoc from Utrecht, and we used 31P NMR to study the structural properties of lipids found in biological membranes. I became fascinated by lipid polymorphism, which involves the preference of some membrane lipids for non-bilayer structures, such as the hexagonal HII phase. Our work led to the conclusion that such lipids play essential roles in membrane fusion processes that are basic to life, such as those that occur during fertilization and trafficking of intracellular vesicles.
Credit: Blake Camden
After establishing my laboratory at the University of British Columbia (UBC) in the 1980s, I also became interested in the functional consequences of lipid asymmetry; the fact that the lipid composition on one side of a biological membrane is different to the lipid composition on the other side. I wondered whether we could generate lipid asymmetry in response to trans-bilayer ion gradients, such as pH gradients. We developed ways to make bilayer vesicles with a pH gradient and then generated lipid asymmetry by including an ‘ionizable cationic lipid’ that can exist in a neutral form or a positively charged (protonated) form, depending on the pH. In vesicles exhibiting a pH gradient, this lipid preferentially distributes to the lipid monolayer experiencing the lower pH, thus producing lipid asymmetry.
We got a bit distracted at this point, as we found out that we could also load anticancer drugs into bilayer vesicles exhibiting a pH gradient, leading me and four senior postdocs to co-found Inex Pharmaceuticals to develop liposomal anti-cancer drugs. However, in the mid-1990s, the CEO complained to me that he couldn’t raise money putting old drugs into liposomes. We needed to be doing gene therapy! The only way we could efficiently encapsulate highly negatively charged nucleic acid-based drugs was to use cationic lipids. The cationic lipids then available were permanently positively charged and were highly toxic in vivo, so we had to find an alternative. Luckily, we tried the ionizable cationic lipid used for our lipid asymmetry studies and found that if we loaded the nucleic acid polymer at low pH, where the ionizable lipid was positively charged, the nucleic acid payload was retained when the pH was raised to physiological pH values. And the systems were much less toxic.
