To detect the entrapment of the Doxorubicine in liposomal formulation first you have to separate entrapped drug using Sephadex column and then you have to use some disrupting agents like protamine, triton- X or ethanol that can disrupts the lipid bi-layer and drug can come out, then centrifuge for 2000 rpm for 20 min. and Analyze the superntent using UV or HPLC.

Release study of doxorubicin from liposomes can be easily analysed by Dialysis method.

Different liposome sizes take different time and speed to separate accurately by centrifugation.

Try different speeds and time and did a stewart assay just to make sure that you have accurate separation.

Otherwise try 60,000 rpm for 30 min at 4 °C (Ultracentrifuge) and 14,000 × g for 30 min at 4 °C controlled temperature centrifuge (Mikro 220R centrifuge (Hettich centrifuges, Germany)

Hi Dr Haniyeh Abdollahzadeh "Separation Technology" is a very complicated area of science. Although there are lots of mathematical equations and physics around it, however, particularly in the area of drug delivery it is mainly based on trial and error.

With respect of liposomal doxorubicin studies, similar to your project, you may find these publications useful:

Gabizon, A., Shmeeda, H., & Barenholz, Y. (2003). Pharmacokinetics of pegylated liposomal doxorubicin. Clinical pharmacokinetics, 42(5), 419-436.

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Abraham, S. A., Waterhouse, D. N., Mayer, L. D., Cullis, P. R., Madden, T. D., & Bally, M. B. (2005). The liposomal formulation of doxorubicin. In Methods in enzymology (Vol. 391, pp. 71-97). Academic Press.

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Gabizon, A., Catane, R., Uziely, B., Kaufman, B., Safra, T., Cohen, R., ... & Barenholz, Y. (1994). Prolonged circulation time and enhanced accumulation in malignant exudates of doxorubicin encapsulated in polyethylene-glycol coated liposomes. Cancer research, 54(4), 987-992.

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Harris, L., Batist, G., Belt, R., Rovira, D., Navari, R., Azarnia, N., ... & TLC D‐99 Study Group. (2002). Liposome‐encapsulated doxorubicin compared with conventional doxorubicin in a randomized multicenter trial as first‐line therapy of metastatic breast carcinoma. Cancer, 94(1), 25-36.

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Muggia, F. M., Hainsworth, J. D., Jeffers, S., Miller, P., Groshen, S., Tan, M., ... & Burnett, A. (1997). Phase II study of liposomal doxorubicin in refractory ovarian cancer: antitumor activity and toxicity modification by liposomal encapsulation.Journal of Clinical Oncology, 15(3), 987-993.

Dear Han Abd,

To separate liposomes from released Dox, you may centrifuge the sample in Eppendorf tube at 13,000 X g for 20 min at 20 °C using a refrigerated centrifuge (NF 800R, NUVE, Turkey).

For the purification of liposomes, a centrifuged Giant Vesicle suspension containing supernatant is filtered through a nucleopore polycarbonate membrane with 10 μm diameter pores (Nomura Micro Science, Atsugi, Japan) using a homemade apparatus. Please see this for more details. Best.. Article A membrane filtering method for the purification of giant un...