To determine the content of EVs and their possible biological impact on different cells can be challenging. Interestingly, we have found some EVs can induce differentiation on certain specific cell lines but we wonder if the reduction of such EVs membrane fluidity can be performed by a standarized protocol (adding low unstaturated fatty acids, or decreasing temperature) and if so, if this would reduce their capacity for cell-membrane penetration?
Well, certainly you can decrease lipid membrane fluidity by decreasing the temperature, and this decrease in temperature of course will slow down any reaction that has an activation energy barrier to cross. So in essence you would not know why the reaction (of cell membrane penetration) decreased.
There certainly is no standardized protocol to decrease fluidity of biological membranes by changing the composition. if you can load high enough mole fraction of something like distearoylglycerophoshocholine or distearoylglycerophosphoethanolamine with gammacyclodextrin or lipid transfer proteins, or even cholesterol with betacyclodextrin, you will affect the fluidity. There are a few caveats, that can all be summarized that you will also also change other physical properties at the same time. For example you might induce lateral phase separation and lateral segregation of different types of EV membrane proteins. You will also change the curvature stresses and propensity to fusion though it may not be too important in your system if your EVs are internalized through pinocytosis. For small EVs bending modulus changes may not be important since they will be too stiff to bend significantly anyway, but under some conditions they me play a role. So, essentially you should think what are all the properties that you might change by loading the vesicles with one type of lipid, and then try to think what different lipid you could add that would also decrease fluidity and that have different effects on these other properties, and then also have a negative control with similar thickness but little effect on fluidity.
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