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Treatment of Experimental Brain Metastasis with MTO-Liposomes: Impact of Fluidity and LRP-Targeting on the Therapeutic Result

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ABSTRACT

Purpose

To test targeted liposomes in an effort to improve drug transport across cellular barriers into the brain.

Methods

Therefore we prepared Mitoxantrone (MTO) entrapping, rigid and fluid liposomes, equipped with a 19-mer angiopeptide as ligand for LDL lipoprotein receptor related protein (LRP) targeting.

Results

Fluid, ligand bearing liposomes showed in vitro the highest cellular uptake and transcytosis and were significantly better than the corresponding ligand-free liposomes and rigid, ligand-bearing vesicles. Treatment of mice, transplanted with human breast cancer cells subcutaneously and into the brain, with fluid membrane liposomes resulted in a significant reduction in the tumor volume by more than 80% and in a clear reduction in drug toxicity. The improvement was mainly depended on liposome fluidity while the targeting contributed only to a minor degree. Pharmacokinetic parameters were also improved for liposomal MTO formulations in comparison to the free drug. So the area under the curve was increased and t1/2 was extended for liposomes.

Conclusion

Our data show that it is possible to significantly improve the therapy of brain metastases if MTO-encapsulating, fluid membrane liposomes are used instead of free MTO. This effect could be further enhanced by fluid, ligand bearing liposomes.

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Abbreviations

BBB:

blood–brain barrier

DMEM:

Dulbecco’s Modified Eagle Medium

FCS:

foetal calf serum

LDL:

low-density lipoprotein

LRP:

LDL-lipoprotein receptor related protein

LUV:

large unilamellar vesicles

MDCK:

Madin-Darby canine kidney

MTO:

mitoxantrone

PIT:

post insertion technology

RTV:

relative tumor volume

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ACKNOWLEDGMENTS & DISCLOSURES

This project was financed in part by a grant of the Federal Ministry of Economics and Technology of Germany, PRO INNO II (KA0453401UL7). The authors gratefully acknowledge the excellent technical support of Annika Hoffmann (EPO GmbH, Berlin) and the valuable discussions about post insertion technology with Ronny Rüger (Friedrich-Schiller-Universität Jena). We also thank Lipoid GmbH Ludwigshafen for providing us with egg phosphatidylcholine.

Author information

Authors and Affiliations

  1. Experimental Pharmacology, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125, Berlin-Buch, Germany

    Andrea Orthmann, Margit Lemm & Iduna Fichtner

  2. Experimental Pharmacology & Oncology Berlin-Buch GmbH, Robert-Rössle-Str. 10, 13125, Berlin-Buch, Germany

    Reiner Zeisig

  3. Department of Pharmaceutical Technology and Biopharmacy, Albert-Ludwigs University, Stefan-Meier-Str. 19, 79104, Freiburg, Germany

    Regine Süss

  4. Cellular Imaging Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, 13125, Berlin, Germany

    Dorothea Lorenz

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  1. Andrea Orthmann
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Corresponding author

Correspondence to Reiner Zeisig.

Electronic supplementary material

Below is the link to the electronic supplementary material.

S1

Vesicle stability over time during storage in PBS. Changes in vesicle diameter were followed by dynamic light scattering (PCS) measurements. Stock solutions of liposomes were appropriately diluted and measured in triplicate at each predefined time point. Mean unimodal diameters ± S.D. are shown. (PDF 11 kb)

S2

LRP receptor expression by cells used. Cells were incubated with 50 μg/ml of the fluorescence labeled peptide 2 for indicated times at 37°C and 4°C. Two-color immunofluorescence cytometry was used to quantify the expression of LRP receptor on cellular surface using 10000 cells. Cell populations were gated and the percentage of cells positive for LRP receptor was calculated based on the mean fluorescence intensity (mFI) from histogram plot. (PDF 4 kb)

S3

MTO concentration versus time in the heart. NMRI:nu/nu mice were injected with 5 mg/kg MTO as solution or encapsulated in liposomes at t = 0 and mice were sacrificed at pre-defined time points. MTO concentration was determined as described in Material and Methods by HPLC. All data represent the mean ± S.D. for 3 samples, each determined in duplicate. (PDF 19 kb)

S4

MTO concentration versus time in the kidney. For details see S3 (PDF 18 kb)

S5

MTO concentration versus time in the liver. For details see S3 (PDF 18 kb)

S6

MTO concentration versus time in the s.c. tumor. For details see S3 (PDF 21 kb)

S7

MTO concentration versus time in the spleen. For details see S3 (PDF 19 kb)

S8

MTO concentration versus time in the lung. For details see S3 (PDF 19 kb)

S9

Concentration of MTO in different organs at t = 15 min. NMRI:nu/nu mice were injected with 5 mg/kg MTO as solution or encapsulated in liposomes at t = 0 and mice were sacrificed at pre-defined time points. MTO concentration was determined as described in Material and Methods by HPLC. All data represent the mean ± S.D. for 3 samples, each determined in duplicate. Insert: Concentration of MTO in the brain. (PDF 36 kb)

S10

Tumor growth versus time of s.c. tumor. MT-3 cells were transplanted s.c. into the left flank (5*106) and into the brain (5*103) of each nude mouse. Mice were treated i.v. with liposomes containing MTO or with free MTO, each in a dose of 4 mg/kg at day 3, 7 and 10. Control mice received saline solution. Diameter of subcutaneously growing tumor was measured twice weekly. Mice were sacrificed at day 22. Data were obtained from two independently performed experiments and are given as mean values +/− S.D (n: 5–16). (PDF 6 kb)

S11

Body weight change over time. Body weight of mice, which were treated as described in Fig. S9, was measured twice a week. Given is the relative change in body weight (in%) as compared to the start of the experiment. *: significantly different to saline treated group. **: significantly different to saline treated and to L fluid-LG treated group. #: three mice died after the third treatment because of drug related toxicity. (P < 0.05) (PDF 8 kb)

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Orthmann, A., Zeisig, R., Süss, R. et al. Treatment of Experimental Brain Metastasis with MTO-Liposomes: Impact of Fluidity and LRP-Targeting on the Therapeutic Result. Pharm Res 29, 1949–1959 (2012). https://doi.org/10.1007/s11095-012-0723-7

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  • DOI: https://doi.org/10.1007/s11095-012-0723-7

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