Pharmaceutical Research

, Volume 29, Issue 7, pp 1949–1959

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

  • Andrea Orthmann
  • Reiner Zeisig
  • Regine Süss
  • Dorothea Lorenz
  • Margit Lemm
  • Iduna Fichtner
Research Paper

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.

KEY WORDS

brain metastases LRP targeting transcytosis uptake 

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

Supplementary material

11095_2012_723_MOESM1_ESM.pdf (12 kb)
S1Vesicle stabilityover timeduring storagein 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)
11095_2012_723_MOESM2_ESM.pdf (5 kb)
S2LRP receptorexpression bycells 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)
11095_2012_723_MOESM3_ESM.pdf (19 kb)
S3MTO concentrationversustime inthe 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)
11095_2012_723_MOESM4_ESM.pdf (18 kb)
S4MTO concentrationversustime inthe kidney. For details see S3 (PDF 18 kb)
11095_2012_723_MOESM5_ESM.pdf (18 kb)
S5MTO concentrationversustime inthe liver. For details see S3 (PDF 18 kb)
11095_2012_723_MOESM6_ESM.pdf (21 kb)
S6MTO concentrationversustime inthe s.c. tumor. For details see S3 (PDF 21 kb)
11095_2012_723_MOESM7_ESM.pdf (20 kb)
S7MTO concentrationversustime inthe spleen. For details see S3 (PDF 19 kb)
11095_2012_723_MOESM8_ESM.pdf (20 kb)
S8MTO concentrationversustime inthe lung. For details see S3 (PDF 19 kb)
11095_2012_723_MOESM9_ESM.pdf (36 kb)
S9Concentration 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)
11095_2012_723_MOESM10_ESM.pdf (6 kb)
S10Tumor growthversustime ofs.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)
11095_2012_723_MOESM11_ESM.pdf (8 kb)
S11Body weightchange overtime. 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 Lfluid-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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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Andrea Orthmann
    • 1
  • Reiner Zeisig
    • 2
  • Regine Süss
    • 3
  • Dorothea Lorenz
    • 4
  • Margit Lemm
    • 1
  • Iduna Fichtner
    • 1
  1. 1.Experimental PharmacologyMax Delbrück Center for Molecular MedicineBerlin-BuchGermany
  2. 2.Experimental Pharmacology & Oncology Berlin-Buch GmbHBerlin-BuchGermany
  3. 3.Department of Pharmaceutical Technology and BiopharmacyAlbert-Ludwigs UniversityFreiburgGermany
  4. 4.Cellular Imaging Leibniz-Institut für Molekulare Pharmakologie (FMP)BerlinGermany

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