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Quantitative In Vitro Assessment of Liposome Stability and Drug Transfer Employing Asymmetrical Flow Field-Flow Fractionation (AF4)

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Abstract

Purpose

In the present study we introduce an efficient approach for a size-based separation of liposomes from plasma proteins employing AF4. We investigated vesicle stability and release behavior of the strongly lipophilic drug temoporfin from liposomes in human plasma for various incubation times at 37°C.

Methods

We used the radioactive tracer cholesteryl oleyl ether (COE) or dipalmitoyl-phosphocholine (DPPC) as lipid markers and 14C-labeled temoporfin. First, both lipid labels were examined for their suitability as liposome markers. Furthermore, the influence of plasma origin on liposome stability and drug transfer was investigated. The effect of membrane fluidity and PEGylation on vesicle stability and drug release characteristics was also analyzed.

Results

Surprisingly, we observed an enzymatic transfer of 3H-COE to lipoproteins due to the cholesterol ester transfer protein (CETP) in human plasma in dependence on membrane rigidity and were able to inhibit this transfer by plasma preincubation with the CETP inhibitor torcetrapib. This effect was not seen when liposomes were incubated in rat plasma. DPPC labels suffered from hydrolysis effects during preparation and/or storage. Fluid liposomes were less stable in human plasma than their PEGylated analogues or a rigid formulation. In contrast, the transfer of the incorporated drug to lipoproteins was higher for the rigid formulations.

Conclusions

The observed effects render COE-labels questionable for in vivo studies using CEPT-rich species. Here, choline labelled 14C-DPPC was found to be the most promising alternative. Bilayer composition has a high influence on stability and drug release of a liposomal formulation in human plasma.

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Abbreviations

AF4:

Asymmetrical flow field-flow fractionation

CETP:

Cholesteryl ester transfer protein

COE:

Cholesteryl oleyl ether

DLS:

Dynamic light scattering

DPPC:

1,2-Dipalmitoyl-sn-glycero-3-phosphocholine

DPPG:

1,2-Dipalmitoyl-sn-glycero-3-phosphoglycerol, sodium salt

EPC:

L-α-phosphatidylcholine (Egg Chicken)

EPG:

L-α-phosphatidylglycerol, sodium salt (Egg, Chicken)

EPR:

Enhanced permeation and retention

HDL:

High density lipoprotein

LDL:

Low density lipoprotein

MALLS:

Multi angle laser-light scattering

mTHPC:

Temoporfin

PCS:

Photon correlation spectroscopy

RES:

Reticuloendothelial system

VLDL:

Very low density lipoprotein

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ACKNOWLEDGMENTS AND DISCLOSURES

The authors thank biolitec biomedical technology GmbH (Jena, Germany) for kindly providing mTHPC and 14C-mTHPC, Lipoid GmbH (Ludwigshafen, Germany) for donation of EPC and EPG as well as Phospholipid Research Center (Heidelberg, Germany) for financial support.

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Authors and Affiliations

  1. Department of Pharmaceutical Technology, Friedrich Schiller University Jena, Lessingstraße 8, D-07743, Jena, Germany

    Stephan Holzschuh, Kathrin Kaeß, Alfred Fahr & Christiane Decker

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  1. Stephan Holzschuh
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  2. Kathrin Kaeß
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  3. Alfred Fahr
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  4. Christiane Decker
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Correspondence to Christiane Decker.

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Holzschuh, S., Kaeß, K., Fahr, A. et al. Quantitative In Vitro Assessment of Liposome Stability and Drug Transfer Employing Asymmetrical Flow Field-Flow Fractionation (AF4). Pharm Res 33, 842–855 (2016). https://doi.org/10.1007/s11095-015-1831-y

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  • DOI: https://doi.org/10.1007/s11095-015-1831-y

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