Final Report Summary - DYNAMEM (Dynamic Combinatorial Chemistry at Lipid Membranes)
Great interest lies in the chemistry inside the lipidic cell membrane as membrane proteins, ion transporters and antibacterial drugs work inside and at the interface of the cell membrane. The goal of this project was to use dynamic combinatorial chemistry as a tool to study the dynamic behavior of molecules in a cell membrane mimic, namely vesicles.
To achieve this goal three ingredients were necessary:
1) A synthetic vesicle stable for the duration of the experiment.
2) Building blocks capable of interacting with the membrane and with functionality for achieving exchange reactions.
3) A driving force, i.e. osmotic pressure.
The first two points were achieved in the timeframe estimated in the proposal:
1) During the first part of the grant we tested the stability of different vesicle formulations. Most of the synthetic vesicles present in literature are only stable for few hours; timeframe that is not compatible with the dynamic exchange reaction we planned to use (the equilibration reaction of disulfides may require days). We discovered that a formulation with POPC (lipid) mixed with 5 mol% of cholesterol and 5 mol% of cetrimonium bromide was able to form vesicles that were stable for months. The cholesterol helped the packing of the lipids, making the membrane more compact, while the positively charged cetrimonium bromide provided a charge on the membrane surface making the vesicles repel one another preventing aggregation and precipitation. The stability of the vesicles was tested with DLS and cryo-TEM over time.
2) Two building blocks were synthesized, both composed of an aromatic dithiol moiety for achieving the disulfide exchange. The difference between the two molecules resides in the long tail attached for interacting with the membrane: in one case it was completely alkylic (hydrophobic) and in the other was mixed alkyl/ester (hydrophobic/hydrophilic). Both building blocks were fully characterized by standard methodology.
The third point of the plan was the most problematic, and the problems raised during the experiments have not been solved (yet).
3) Both building blocks were tested for reactivity inside the vesicle membranes. The building blocks were dissolved separately during the preparation of the lipid film in a concentration of 1 mol% (with respect to the lipids). Then vesicles were formed by standard procedures involving the hydration of the lipidic film and subsequent freeze-thaw cycles. Different salts (LiCl, NaCl, KCl, RbCl, CsCl) at different concentrations were added to the vesicle suspensions to create an osmotic pressure. All these experiments plus the one with no salt added were then analyzed by HPLC/UPLC.
Unfortunately, the synthetic building blocks and the lipids used have similar polarity and, although many different HPLC/ULPC columns/eluents were tested, no system was able to separate the two analytes. Along with this one should also consider that the amount of building block is only 1 mol% with respect to the lipids. If the building block form tetramers the concentration of the analyte drops to 0.25 mol% in respect to the lipids. Due to such situation, it was impossible to use the standard HPLC/UPLC analytical method for screening this particular dynamic combinatorial library.
This issue can be, in future, solved with the use of a SDS-PAGE for separating the analytes from the lipids as the electromobility of the two is different.
At the same time of the main project another one was started in parallel. Scope of this new project was to check if two dynamic combinatorial libraries are free to “communicate” through the exchange of only part of the dynamic library diffusing through the membrane. The experiment was set up such to have two different dynamic combinatorial libraries inside and outside the vesicle.
This first experiment was a success and the dynamic libraries were screened after the purification of the vesicles, making possible to check the “inside” and “outside” solution of the vesicles.
Using the HPLC-MS it was clear that the exchange between the two dynamic library was achieved even if only one of the component of the two libraries was free to pass through the membrane.
In a second experiment the two different libraries were enclosed into two families of vesicles, and these two families were separated by a dialysis membrane. In this way the two different families were not allowed to “touch” each other and the exchange of material was possible only by passing through the first family membrane and then diffusing into the second family membrane. The main concept is that a chemical produced inside one of the two families would trigger an effect inside the second family of vesicles.
However also in this case, the analytes were too low in concentration for achieving a good response from the HPLC-MS.
The optimization of this project is currently in the hands of other group members in the host laboratory.