Dear David,

Without any specifics about your recipe and preparation I can only make some general remarks that may help.

Zeta potential is governed by ionic strength but mostly by pH. In order measure consistent zeta potentials you have to accurate measure pH and keep a fixed ionic strength. Practically I have seen that ionic strength has an optimum value to achieve the highest zeta potential, this is normally close to 1 mM for KCl. Lower ionic strength leads to an extended double layer and slipping plane thus lowering the zeta potential. Higher ionic strength depresses the double layer so that it may be closer to the particle surface so that zeta potential, measured at the slipping plane, does not express it correctly. If you want to retain high positive charge from amines it is needed to bring down the pH into the neutral or even acidic region. Please note that the proximity of the amine groups in your liposome may cause the pKa of the amine to be lowered by as much as two pH-points.

What may be the real problem here is that your drug is partly hydrophobic as well as negatively charged. It will therefore electrostatically interact with your liposome former (the stearyl amine) and also want to sit between the stearyl tails to establish hydrophobic interactions. Therefore your drug's position would be in the interface where it influences the zeta potential, charge and stability of the liposomes. There seems almost no way out of that with your current recipe. Therefore is it an option for you to use another liposome-former, preferably one that is mixed with a polycation, like chitosan or a Polylysine-derivative in case the positive charge is crucial for your application.

Anyway if acidic pH is possible this would be the first test to do as it is fast. Keep the ionic strength low to about 1-5 mM.

I hope this helps, kind regards, Leo

Hi,

To have stable liposomes, Zeta potential must be greater than +30mV, or less than -30mV. 

Also, utilization of cholesterol in optimum concentration, an antioxidant such as Vitamin E, in the structure of liposomes, will increase their stability.

Other parameters are ionic strength and pH of the medium (as our friend Leo said).

Have a look at this publication:

The Influence of Ionic Strength and Lipid Bilayer Charge on the Stability of Liposomes.

Article in: Journal of Colloid and Interface Science 164(1):78–87 · April 1994

DOI: 10.1006/jcis.1994.1145

Dear Leo.

First, our formula is DSPC:SA:Chol 7:3:2. So DSPC is the main lipid, SA is the cationic lipid with a pKa of 10 aprox so it's charged in neutral to acidic pH. And we add cholesterol to improve stability and rigidity. We are performed assays with different pH. 7,4 for saline; 6,8 for Tris and 5,8 for PBS. We want to use mild acidic conditions because of two reasons. First we use curcumin in another preparation and curcumin is degraded in neutral to basic conditions. Second, to charge stearylamine. We have been using 10mM of Tris-Cl in this case to achieve 50-75 mV. This is our measurement today. I told in the post that the liposomes in Tris looked better and effectively they have a +50mV zeta potential so I guess that's the different between Tris and NaCl 0,9%. 

Thanks a lot in your advice about Dexa which is, as you say, hydrophobic and negatively charged at the same time. We tried to encapsulate the pure hydrophobic Dexa but it was a failure since it is no that hydrophobic and our main lipid is very rigid (DSPC). 

How can I measure ionic strenght? I have different buffer with different pH and different concentrations:

• PBS 0,1X (15mM) pH 5,8
• Tris-Cl 10mM pH 6,8
• NaCl 0,9% pH 7,4

We tried with PBS and water but with 25mg/mL Dexa (failure, low zeta potential: +20mV). With NaCl 0,9% and 5mg/mL Dexa zeta potential was +35mV and with Tris (5mg/mL Dexa), zeta potential was +50mV. So that's the effect of Tris versus NaCl but I am not sure if it's because of the nature of Tris or because its pH or ionic strenght. 

Thank a lot Leo.

________

Dear Mozafari, we indeed use cholesterol and we want a zeta potential above +30mV for stability and biological purposes. I'm gonna look at that paper. Thanks a lot.

Dear David,

Ionic strength cannot be measured but is calculated. It is the addition of all ionic concentrations multiplied with their valencies. The most simple explanation how to calculate it, with an example, is: https://en.wikipedia.org/wiki/Ionic_strength.

From your data the TRIS and PBS, are pH-buffers and the NaCl is not. Since the trend seen in zeta potential versus pH is not linear (otherwise PBS at pH= 5.8 should be better), I suspect it is not pH but the type of ion present. TRIS will form a cation at pH= 6.8, whereas phosphate will be an anion. It is these small ions that not just help buffer, they may adhere to your liposomes and cause a positive increase of your zeta potential.

Perhaps adding another primary amine with a bit longer alkyl-chain will improve zeta potential even more as these substances generally are forced to the interface.

Hoping this helps !! Kind regards, Leo

Thanks a lot.  I will let you know what's working better. For now it's Tris 10mM pH 6,8 but we are going to change pH to examine if the zeta potential is better. We have to use a buffer that is compatible with celullar assays.