Pharmaceutical Research

, Volume 30, Issue 7, pp 1883–1895 | Cite as

Improving Intracellular Doxorubicin Delivery Through Nanoliposomes Equipped with Selective Tumor Cell Membrane Permeabilizing Short-Chain Sphingolipids

  • Lília R. Cordeiro Pedrosa
  • Albert van Hell
  • Regine Süss
  • Wim J. van Blitterswijk
  • Ann L. B. Seynhaeve
  • Wiggert A. van Cappellen
  • Alexander M. M. Eggermont
  • Timo L. M. ten Hagen
  • Marcel Verheij
  • Gerben A. Koning
Research Paper

Abstract

Purpose

To improve nanoliposomal-doxorubicin (DoxNL) delivery in tumor cells using liposome membrane-incorporated short-chain sphingolipids (SCS) with selective membrane-permeabilizing properties. DoxNL bilayers contained synthetic short-chain derivatives of known membrane microdomain-forming sphingolipids; C8-glucosylceramide (C8-GluCer), C8-galactosylceramide (C8-GalCer) or C8-lactosylceramide (C8-LacCer).

Methods

DoxNL enriched with C8-GluCer or C8-GalCer were developed, optimized and characterized with regard to size, stability and drug retention. In vitro cytotoxic activity was studied in a panel of human tumor cell lines and normal cells. Intracellular Dox delivery was measured by flow cytometry and visualized by fluorescence microscopy. For a further understanding of the involved drug delivery mechanism confocal microscopy studies addressed the cellular fate of the nanoliposomes, the SCS and Dox in living cells.

Results

C8-LacCer-DoxNL aggregated upon Dox loading. In tumor cell lines SCS-DoxNL with C8-GluCer or C8-GalCer demonstrated strongly increased Dox delivery and cytotoxicity compared to standard DoxNL. Surprisingly, this effect was much less pronounced in normal cells. Nanoliposomes were not internalized, SCS however transfered from the nanoliposomal bilayer to the cell membrane and preceded cellular uptake and subsequent nuclear localization of Dox.

Conclusion

C8-GluCer or C8-GalCer incorporated in DoxNL selectively improved intracellular drug delivery upon transfer to tumor cell membranes by local enhancement of cell membrane permeability.

KEY WORDS

doxorubicin-nanoliposome short chain sphingolipid Tumor cell membrane permeabilization 

Abbreviations

C8-GluCer

C8-glucosylceramide

C8-GalCer

C8-galactosylceramide

C8-LacCer

C8-lactosylceramide

D:PL ratio

Drug to phospholipid initial mass ratio

Dox

Doxorubicin

FCS

Fetal calf serum

HSPC

Hydrogenated soy phosphatidylcholine

DSPE-PEG2000

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG2000

MIF

Mean of fluorescence intensity

NBD PE

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)

NL

Nanoliposomes

pdi

Polydispersity index

SCS

Short chain sphingolipids

SEM

Standard error of the mean

TCMM

Tumor cell membrane modulation

Supplementary material

11095_2013_1031_MOESM1_ESM.avi (44.1 mb)
Movie 1Localization of NBD-PE labeled C8-GalCer-DoxNL (green) and Dox (red) by confocal microscopy. BLM melanoma cell line was treated with 5 μM Dox formulated in NBD labelled- C8-GalCer-DoxNL and fluorescence of SCS and Dox was followed in time for a total of 40 min (AVI 45116 kb)
11095_2013_1031_Fig9_ESM.jpg (16 kb)
Supplemental Figure 1a

Stability at 37°C in the presence of 10%FCS of enriched C8-GluCer-DoxNL (■) was measured for 24 h (A) and enriched C8-GalCer-DoxNL (▲) for 15 h (B). Measurements were based on doxorubicin fluorescence following time and were performed continuously. Both enriched-DoxNL presented a better stability profile than non enriched-DoxNL (●) (JPEG 16 kb)

11095_2013_1031_Fig10_ESM.jpg (17 kb)
Supplemental Figure 1a

Stability at 37°C in the presence of 10%FCS of enriched C8-GluCer-DoxNL (■) was measured for 24 h (A) and enriched C8-GalCer-DoxNL (▲) for 15 h (B). Measurements were based on doxorubicin fluorescence following time and were performed continuously. Both enriched-DoxNL presented a better stability profile than non enriched-DoxNL (●) (JPEG 16 kb)

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High Resolution Image(TIFF 2211 kb)
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High Resolution Image(TIFF 2211 kb)
11095_2013_1031_Fig11_ESM.jpg (133 kb)
Supplemental Figure 2a

In vitro drug efficacy toward tumor cells (BLM, Mel57 melanoma, MCF7 and SKBR3 breast carcinoma, Panc1 and ASPC1 pancreatic carcinoma) and non-tumor cells (HUVEC and 3 T3). (A) Cells were treated with Doxil (●), non enriched-DoxNL (■), 10% C8-GluCer-DoxNL (▲) and 10% C8-GalCer-DoxNL (▼) for 24 h at 37°C. Cell survival was quantified by colorimetric SRB assay. (B) Cellular morphology was analyzed by microscopy. Values represent the mean ± SEM of at least 3 experiments (JPEG 132 kb)

11095_2013_1031_MOESM4_ESM.tif (138 kb)
High Resolution Image(TIFF 138 kb)
11095_2013_1031_MOESM5_ESM.doc (273 kb)
Supplemental Figure 2b(DOC 273 kb)
11095_2013_1031_MOESM6_ESM.ppt (1.3 mb)
Supplemental Figure 3Co-localization of NBD-PE labeled liposomes (green) and Dox (red) by Confocal Microscopy. After nucleus staining with Hoechst (blue), BLM melanoma cell line was treated with 10 μM Dox in form of C8-GluCer-DoxNL (A) and C8-GalCer-DoxNL (B). ASPC1 pancreatic carcinoma cells were equally treated with 10 μM Dox in form of C8-GluCer-DoxNL (C) and C8-GalCer-DoxNL (D). After treatment cells were incubated for 4 h at 37°C. Nuclear and cytoplasmatic drug uptake was analysed (PPT 1299 kb)
11095_2013_1031_Fig12_ESM.jpg (53 kb)
Supplemental Figure 4

Intracellular Dox uptake after treatment with DoxNL, 1 μM was quantified by flow cytometry in BLM melanoma, MCF7 breast carcinoma and ASPC1 pancreatic carcinoma (A). Doxorubicin was formulated in non enriched-DoxNL (open), 10% C8-GluCer-DoxNL (dark grey) and 10% C8-GalCer-DoxNL (black). Fluorescence of non treated cells was measured as a control (bright grey). At least three independent experiments were performed and values represent the mean ± SEM; *, P < 0.001; **, P < 0.01 and #, P < 0.05 versus non enriched-DoxNL at the same time point. (B) Overview of Dox uptake by 3 different tumor cell lines incubated with 1 μM C8-GluCer or C8-GalCer-DoxNL after 4 h of incubation. C8-GluCer seemed to increase Dox uptake in BLM (open) cells when compared to MCF7 (grey) and ASPC1 (black) cells. C8-GalCer-DoxNL gives similar results in all three cell lines. (JPEG 3.44 mb)

11095_2013_1031_MOESM7_ESM.tif (12.6 mb)
High Resolution Image(TIFF 12946 kb)
11095_2013_1031_MOESM8_ESM.doc (30 kb)
Supplemental Table 1In vitro IC50 values toward tumor cells BLM, melanoma and SKBR3, breast carcinoma, after treatment with C8-GluCer-DoxNL following different drug to phospholipid ratios: 0.25:1; 0.2:1; 0.15:1; 0.1:1 (w/w) (DOC 30.5 kb)
11095_2013_1031_MOESM9_ESM.doc (30 kb)
Supplemental Table 2Stability at 4°C after 6 months of different glycoceramide-enriched liposomes in a drug to phospholipid ratios of 0.1:1 (w/w). Size and pdi were analyzed. (DOC 30 kb)
11095_2013_1031_MOESM10_ESM.doc (34 kb)
Supplemental Table 3In vitro IC50 values of doxorubicin in different tumor cell lines, BLM, melanoma, MCF7 breast carcinoma and ASPC, pancreatic carcinoma, following different densities for C8-GalCer-DoxNL (DOC 34 kb)

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Lília R. Cordeiro Pedrosa
    • 1
  • Albert van Hell
    • 3
  • Regine Süss
    • 4
  • Wim J. van Blitterswijk
    • 5
  • Ann L. B. Seynhaeve
    • 1
  • Wiggert A. van Cappellen
    • 6
  • Alexander M. M. Eggermont
    • 1
    • 7
  • Timo L. M. ten Hagen
    • 1
  • Marcel Verheij
    • 3
    • 8
  • Gerben A. Koning
    • 2
  1. 1.Laboratory Experimental Surgical Oncology Section Surgical Oncology, Department of SurgeryErasmus Medical CenterRotterdamThe Netherlands
  2. 2.Laboratory Experimental Surgical Oncology Section Surgical Oncology, Department of SurgeryErasmus Medical CenterRotterdamThe Netherlands
  3. 3.Division of Biological Stress ResponseThe Netherlands Cancer Institute - Antoni van Leeuwenhoek HospitalAmsterdamThe Netherlands
  4. 4.Department of Pharmaceutical Technology and BiopharmacyAlbert-Ludwigs UniversityFreiburgGermany
  5. 5.Division of Cellular BiochemistryThe Netherlands Cancer Institute - Antoni van Leeuwenhoek HospitalAmsterdamThe Netherlands
  6. 6.Optical Imaging CentreErasmus Medical CenterRotterdamThe Netherlands
  7. 7.Institut de Cancerologie Gustave RoussyParisFrance
  8. 8.Department of RadiotherapyThe Netherlands Cancer Institute - Antoni van Leeuwenhoek HospitalAmsterdamThe Netherlands

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