Hello there,
I am trying to coat plastic beads (spherotech beads) with cationic lipid bilayers.
There are several protocols for neutral and negatively-charged bilayer formation on polystyrene beads, normally using divalent cations (ie. calcium), but I am not aware of bilayer formation when positively charged membranes are used.
More specifically, I want to form supported lipid bilayers on polystyrene beads using as much as 50mol% DOTAP lipids.
If I repeat the protocols use for neutral/negative membranes, bilayers are not formed.
I would be really grateful if you could help me with some hints, protocols or papers in that direction =)
Rafa
The protocol for anionic lipids will fail for cationic lipids. You will need to find an anion that can perform the same role as the Ca+2 ions.
What if you go to a sulfonated PS in the anionic form? You can get beads of these that are used as ion exchange resins. If you treat it with base, the sulfonate anions will pair with the cation head groups of the lipids to initiate double layer formation.
Dear Rafael Lira ,
The behaviour of cationic lipids depend on numerous factors. See for a nice overview:
https://www.researchgate.net/profile/Henrique_Faneca/publication/7471375_Cationic_liposomes_for_gene_delivery/links/549560650cf29b944823add3/Cationic-liposomes-for-gene-delivery.pdf?origin=publication_detail
You mentioned 50% DOTAP, often DOPE is used as colipid. For DOPC an excellent paper with a lot of details and good references is:
Jurkiewicz, P., Olżyńska, A., Langner, M., & Hof, M. (2006). Headgroup hydration and mobility of DOTAP/DOPC bilayers: a fluorescence solvent relaxation study. Langmuir, 22(21), 8741-8749.
(see Google Scholar for a pdf file through academia.edu)
Basically the message is: At low DOTAP concentrations it is thought that the electrostatic repulsion between cationic TAP groups and the positive charged group of the choline moiety causes reorientation of the PC headgroups to a more vertical position. While cationic TAP attracts the negatively charged group of the choline moiety. Both result in membrane compression. At larger DOTAP concentrations, the bilayer expand due to the cationic charges repulsion . It has been shown that when the extent of such an expansion is large enough, a transition to the interdigitated phase is possible:
Ryhänen, S. J., Alakoskela, J. M. I., & Kinnunen, P. K. (2005). Increasing surface charge density induces interdigitation in vesicles of cationic amphiphile and phosphatidylcholine. Langmuir, 21(13), 5707-5715.
(see Google Scholar for a pdf file through academia.edu)
Interdigitation is rather complex but the following paper gives some good ideas:
Kranenburg, M., Vlaar, M., & Smit, B. (2004). Simulating induced interdigitation in membranes. Biophysical journal, 87(3), 1596-1605.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1304565/pdf/1596.pdf
Hopefully one of the above mentioned references might help you to get an idea how to resolve your problem.
Best regards.
Added:
Possibly the following reference (with mentioned references herein) helps you when it comes to supported bilayers.
Weingart, J., Vabbilisetty, P., & Sun, X. L. (2013). Membrane mimetic surface functionalization of nanoparticles: Methods and applications. Advances in colloid and interface science, 197, 68-84.
https://europepmc.org/articles/pmc3729609
on my side I know that the presence of clusters of positively charged amino acid residues on the protein surface allowing one protein molecule to interact simultaneously with several anionic lipids.
Dear all,
thank you for all the input and literature.
Dear James E Hanson , I thought something in that direction... because the promoter of bilayer formation with neutral and negative lipids is a divalent cation, I thought about using a divalent anion. This is something I'll try in the following days. But by looking at literature, I'm surprised there are not many papers studying this.
Also, when you say "sulfonated PS", by PS you mean polystyrene right (I'm not familiar with the terms yet)?
I'm sure I can find them, but do you know any paper where they use a similar protocol? Else, do you know any provider?
Thank you very much for the comments and I'm looking forward to hear from you.
Dear Rob Keller , unfortunately I wasn't able to open some of the links you sent.
I would be really glad if you check them again.
Also from the publications, I think all the studies are performed with free-standing bilayers? Perhaps you came across a study where they also used supported bilayers?
Once again thank you for the time you took.
Yes - PS is polystyrene.
One brand is amberlyst (from dow)
basically they make beads by polymerizing styrene sulfonic acid with divinylbenzene to crosslink.
Anything that is a strong acid ion exchange resin should work.
To my understanding the reason people use cations to spread negatively charged lipids is the negative surface charge of most substrates. So positively charged vesicles should spread more easily, which is also observed for example in this article:
Article Supported lipid membranes
So the idea to use negatively charged beads sounds very promising, but the problem might also be something completely different. Do you get nice bilayers with neutral lipids?
One unrelated idea: Sometimes microbeads a stabilized by detergent, that might mess with the lipids.
Dear Hannes Witt ,
thanks for the input.
So far I have only used published protocols that were devised for neutral or negative membranes. I do get nice coating, which I assume by using fluorescently labelled lipids and the membranes are pretty homogeneous. I know people also usually do tests to check for membrane fluidity (using FRAP for example) which I haven't yet.
Because this protocol didn't work for my positive membranes, i will now try a few things here and there, including the suggestions from the answers above.
Perhaps they will even be formed with no salt, as you pointed out that the support surface is negative.
I will also be careful with possible detergent contamination in case I ever used beads that came in contact with detergents.
Thanks =)
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