Your description of the experiments and difficulties (you had) is too limited. In order to analyse your preparation procedure an accurate description of the entire procedure is needed.

I'l try to add as an attachment a paper in which liposome preparation has been described in detail. You have only to omit the clodronate suspension

3.1 Preparation of clodronate liposomes and control liposomes

1. Add 4.30 ml phosphatidylcholine solution to 4.00 ml cholesterol solution in a 0.5 liter round bottom flask (see Note 9,10).

2. Remove the ethanol by low vacuum (58 mbar) rotary (150 rpm) evaporation at 40 ºC. At the end a thin phospholipid film will form against the inside of the flask. Remove the condensed ethanol by aerating the flask three times.

3. Disperse the phospholipid film in 20 ml clodronate solution (for clodronate liposomes) or 20 ml PBS (for empty liposomes) by gentle rotation (max.100 rpm) at room temperature. Development of foam should be avoided by reducing the speed of rotation.

4. Keep the milky white suspension at room temperature for about 2 hr (see Note 12).

5. Shake the solution gently (development of foam should be avoided). Put the suspension in a 50 ml plastic tube and sonicate in a water bath (55 kHz) for 3 minutes.

6. Keep the suspension at room temperature for 2 hr (or over night at 4°C). In order to limit the maximum diameter of the liposomes for intravenous injection, the suspension can be filtered using sterile membrane filters with 3.0 μm pores (Millipore).

7. Before using the clodronate liposomes:

(a) Remove the non-encapsulated clodronate by centrifuging the liposomes at 24,000 x g and 10°C for 60 min.. The clodronate liposomes will form a white band at the top of the suspension, whereas the suspension itself will be nearly clear.

(b) Carefully remove the clodronate solution under the white band of liposomes with a 10 ml pipet (about 1 % will be encapsulated). Resuspend the liposomes in approximately 45 ml PBS (see Note 7, 13).

8. Wash the liposomes 4-5 times using centrifugation at 24,000 x g and 10°C for 25 min. Remove each time the upper solution and resuspend (see Note 7) the pellet in approximately 45 ml PBS.

9. Resuspend (see Note 7) the final liposome pellet in PBS and adjust to a final volume of 20.0 ml. The suspension should be shaken (gently) before administration to animals or before dispensing, in order to achieve a homogeneous distribution of the liposomes in suspension.

Thank you so much for your help. we wanted to study the uptake assay and we simply used phosphotidylcholine and we re-suspended in tris buffer. We solved the problem as my colleague seemed to prepare very concentrated solution in chloroform at the beginning made the layer too thick to re-suspend.

After chloroform evaporation, the lipid film could be dettached from the walls of round bottom flask by means of some glass beads (with diameters of a few millimeters) during the hydration step. Thereafter, the glass beads must be removed and the milky suspension will be subjected to mechanical stirring at high turation.

Thank you so much

Dera Muthanna,

I use Rotavapor ( Rotavapor® R-215 | buchi.com ) in order to emilinate all the solvent from the lipidic solution, and after I introduce water and glass beads.  Glass beads lead you to remove the lipid film and the water to hydrate it to form  multilamellar liposomes.

Goog Luck,