The zeta potential or viscosity/conc of your suspension can affect the agglomeration. You could add a 1:10 molar ratio of PS to your main phospholipid to your formulation. PS will be incorporated mainly in the outer monolayer and is more negatively charged so would increase stability in that respect by minimising fusion of other vesicles.

In terms of the drug "settling" this sounds like the drug could be precipitating which if it is then isn't incorporated into Liposomes and crystallising in your suspension and thus not actually soluble.

This is just speculation though, hopefully someone more experience can chime in, though what methods did you use and what is your formulation?

As mentioned earlier, the composition of lipid-based formulations depend upon the lipid composition and surfactant employed. The ratio of lipids, addiition of PEG or other surfactant and optimal concentration of lipids may solve the problem.

For details, you might refer our publications which are related to phospholipid-shelled nanobubbles and doxorubicin delivery, but they might help you in understanding the issues.

Article Surface Composition and Preparation Method for Oxygen Nanobu...

Article Anti-Tumor Drug-Loaded Oxygen Nanobubbles for the Degradatio...

Hi Mehrnoush Mehrarya

Check the following points:

1. Cholesterol amount in your liposomes;

2. Their zeta potential;

3. Phase transition temperature;

4. pH of the suspension medium;

Some lines relevant to "aggregation" issue from our book:

" ... We have found that both anionic (with dicetylphosphate, DCP, as negatively charged ingredient) and neutral (without DCP) liposomes containing PC can interact with model membrane systems (in the form of fusion/aggregation) when containing a 10% molar ratio of cholesterol (Mozafari and Hasirci 1998).

Anionic vesicles containing 10% cholesterol were also able to incorporate DNA

molecules in the presence of Ca2+. Increasing the cholesterol content of

these vesicles from 10% to 40%, however, rendered them not only unable

to undergo fusion/aggregation with model membranes but also unable to

incorporate the DNA molecules. ..."

" ... This is due to the fact that the phase behaviour of a liposomal membrane determines such properties as permeability, fusion, aggregation and protein binding, all of which can markedly affect the stability of liposomes and their behaviour in biological systems (New 1990).

".... Application of similarly charged ingredients in liposome structure hence reduces aggregation and fusion of the vesicles and results in increased stability of the liposomal suspension. This is because liposomes are thermodynamically unstable. The lipid vesicles will aggregate, fuse, flocculate and precipitate during storage. Increasing interparticle repulsion, either electrostatic or steric, can

enhance their stability. The former has been achieved by increasing the

liposome surface zeta potential, while for the latter, bulky nonionic groups

such as polyethylene glycol are included into the lipid bilayer (Keller

2001). ..."

Mozafari, M. R., & Mortazavi, S. M. (2005). Nanoliposomes: from fundamentals to recent developments. Trafford Pub. Ltd. Oxford, UK.

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Also, you may find this publication relevant to your research:

Mourtas, S., Lazar, A. N., Markoutsa, E., Duyckaerts, C., & Antimisiaris, S. G. (2014). Multifunctional nanoliposomes with curcumin–lipid derivative and brain targeting functionality with potential applications for Alzheimer disease. European journal of medicinal chemistry, 80, 175-183.

Tony Tiller thanks a lot. it was useful.

Muhammad Saad Khan thanks a lot. it was useful.

professor M. R. Mozafari thank you for taking the trouble to help me. it was very useful.