Water-in-Oil 2-Hr Protocol

[OneScientista]

Build-A-Cell POPCCholesterol 11.pdf

[zjuradoq]

Is there a way to determine if the vesicles are the intended internal solution? Also if the vesicles are bi-layered or residues of oil.

Are you also proposing to change the composition to 1:1 and skip making lipid film, step 0, altogether?

[OneScientista]

Is there a way to determine if the vesicles are the intended internal solution?

• You could use a fluorescent label in the inner solution. Vesicles contain the inner solution that was used to make droplets in the suspension. Also, note in Build-A-Cell-POPC.pdf (below) the repeat of the microscopy of these vesicles. This was done about 24 hours after formation. This is another piece of evidence that these are vesicles. It is my understanding that water-in-oil droplets are not stable over long periods of time.

Also if the vesicles are bi-layered or residues of oil.

• Multilamellar vesicles are sometimes observed. They are typically not round, but appear as vesicle clusters/aggregates with noticeable irregularities. Electron microscopy could be used to confirm the thickness of the vesicle membrane.

Are you also proposing to change the composition to 1:1 and skip making lipid film, step 0, altogether?

• Yes! One of the considerations for this protocol was the time it takes to prepare the vesicles. Omitting formation of the lipid film greatly reduces the time requirement.

[OneScientista]

Build-A-Cell-POPC.pdf

[murrayrm]

@gregor0607 Can you remind us of the status of the "unit test" for testing whether or not we have functional extract inside of a vesicle?

@BuildACell/liposome-curators Note that there is a proposal to change the standard protocol based on experience in implementation. If the curators agree this is something that should be done, it should be discussed (either here or in a new issue) and decided upon.

[zjuradoq]

@OneScientista Thank you for sharing the florescence images. I can see what you mean but I also wonder if you have used the internal solution check of 2 uM HPTS, 250 uM HEPES. This way the vesicles will also be seen in the GFP channel. Also, was wondering if your outer solutions differ from the posted protocol?

I look forward to trying this method and will let you you know what I get.

[OneScientista]

@zjuradoq These are all good questions. Below are the composition and concentration of inner and outer solution I've used so far. I will amend my report to include this information. I may try 2 uM HPTS in the inner solution in the future. Did you use HPTS or calcein?

Inner solution:
200 mM sucrose
100 mM HEPES pH 8

Outer solution:
200 mM glucose
100 mM HEPES pH 8

[PaolaTorre]

@OneScientista
I doubt that bright field or DIC images could truly lead you to the conclusion that you have vesicle.
I think that you have very nice water in oil emulsion droplets, stabilized at the interface with lipid!
My comments are the following:

1. I think we included the presence of the 18:1 Liss PE to have a clear distinction among the kind of spherical objects you may have at the end of the protocol. If you exclude this lipid for your formulation, you are basically excluding your only way to understand if you have vesicle or not.
2. I tried in the past to evaporate the chloroform solely by heating at 80C deg. I don't think is a good way to really get rid of the chloroform . I mean it is if we think that the melting point is around 60C deg.
   But again you are adding too many variables to the protocol. Think about it, you add the lipid in chloroform to the oil, already after this step you may have formation of phases. Than you evaporate at 80C deg and you are melting the chloroform, who is going to tell you that really you eliminate all the residual?

[OneScientista]

@PaolaTorre Hi Paola, please take a look at Build-A-Cell-POPC.pdf (above). There are images of fluorescently-labeled vesicles. Comments?

[PaolaTorre]

@OneScientista
Errata Corrige, that it is -60C deg, but as you said that the T of boiling is 60Cdeg.
If you want to be sure that you really have vesicle, as we said, you shoul probably od some confocal microscopy.
My suggestions are the following:

1. Combine the two protocols.
   2)Try to be more patient and careful with the evaporation of the chloroform.This means don't aim to prepare an oil in 5 mL. My preference is to prepare many oils of 1 mL. I always prefer to evaporate the chloroform overnight.

[p-stano]

Hi, to reply the initial question:

Is there a way to determine if the vesicles are the intended internal solution? Also if the vesicles are bi-layered or residues of oil.

as mentioned the easiest way is to include a fluorescent marker in the I-solution.

Determining the vesicle lamellarity is not straightforward, and needs more work. See the following two articles:

• Akashi, K.; Miyata, H.; Itoh, H.; Kinosita, K. Preparation of Giant Liposomes in Physiological Conditions and Their Characterization under an Optical Microscope. Biophys. J. 1996, 71 (6), 3242–3250.
• Chiba, M.; Miyazaki, M.; Ishiwata, S. Quantitative Analysis of the Lamellarity of Giant Liposomes Prepared by the Inverted Emulsion Method. Biophys J 2014, 107 (2), 346–354.

Determining the residual oil (meaning: "traces") solubilized inside the lipid bilayer is also difficult. In one work the C. Sykes group has looked at the membrane mechanical properties, and compare them with those of "pure" membranes. But this cannot be done in a normal preparative lab.

If the presence of residual oil hamper the scope of the experiment, a suggestion, already present in the Weitz 2003 papers is to shift from "mineral oil" to squalene, which is almost insoluble in the lipid bilayer.

[OneScientista]

@murrayrm @zjuradoq @PaolaTorre @p-stano
Hello All. Based on the comments and communication with the members of the containers group, the main issue appears to be the result variability between users. I've heard a lot of comments to the effect that: " this didn't work in our hands". This needs to be addressed.

To address the issue of variability, my intern Theresa Chu and I undertook preparation of vesicles side by side from the same lipid/oil stock (made fresh most days) for three days. We both observed vesicles, but also observed differences. The only part of the protocol that was different was our handling of the emulsification process. This was not intended, just minor differences in how we each handle tubes, how we use the vortex, how long we keep them at room temperature, how much they warm up etc... The next three reports include both Theresa's and my results. Note that we only used POPC for this exercise. Since then, I've used different POPC:cholesterol combinations as well, more reports to come...

Here is what we learned:

Emulsification process is critical to variability in the number and size of droplets produced. Lipid composition, concentration, temperature, and type and intensity of shear forces make the most impact on the outcomes.

What is clear is that the process of emulsification needs to be standardized. We have purchased a vortex head adapter that, if successful, should remove the operator factor from the emulsification process.

It also appears that we will need slight variations to this protocol for different lipid compositions.

Looking forward to devising a robust vesicle-making protocol, and unleashing it onto the world in the near future!!!

Build-A-Cell_082218_Theresa started w Lipids.pdf

Build-A-Cell_08-23-18_POPC.pdf

Build-A-Cell_08-27-18_POPC.pdf

[OneScientista]

@zjuradoq Hi Zoila, were you able to try the short protocol?

[p-stano]

Hi Milena, the emulsification seems to be indeed a key moment in this preparation method. As you said it depends on chemical factors (type of oil, type and conc. of lipids, type of I-solution, and ratio between these 3 things) and mechanical ones.

Your experiments shows that between-operator reproducibility is not so good as expected, but at the same time you have GVs consistently, every time you want. I would take this part of the story as the positive one.

The differences between preparations are visible in the size distribution, and maybe the subtler lamellarity. Your idea about standardization of emulsification sounds good. But to extend this to everyone probably the same vortex machine should be used too.

One can be happy to have a reproducible, yet wide, size distribution.
But surely the dream is to have monodispersed w/o droplets (and thus monodispersed GVs). In my experience this never happens with POPC in manual emulsification.
But w/o droplets could be made by microfluidics for instance, and centrifuged manually later on. See here.

My comments to images and text in the pdf files:

• there is no size bar, but I have the impression that vesicles are smaller than what one would like to have. Hint to make larger GVs: reduce lipids and/or reduce shearing force.
• for homogeneity in size: if we think to reverse micelles (5-20 nm), they are rather homogeneous is size because they are at the thermodynamic equilibrium. All states are accessible and easily convert into each other. This does not happen so well in POPC w/o droplets, and not at all in POPC vesicles. Following this hypothesis, perhaps doping the lipid with a small amount of a non-lipid surfactant can be something to try. Which surfactant? Several studies on w/o emulsion have been done by using pure Tween/Span-stabilized droplets, after Tawfik and Griffiths. They are compatible with gene expression, see also here. Would a POPC/Tween/Span mix give a more homogeneous w/o droplet size distribution? I never tried - but I would be curious to know the result.