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

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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.

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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

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Acknowledgments and Disclosures

This work was financed by the Dutch Cancer Society.

Author information

Authors and Affiliations

  1. Laboratory Experimental Surgical Oncology Section Surgical Oncology, Department of Surgery, Erasmus Medical Center, PO Box 2040, Rotterdam, 3000 CA, The Netherlands

    Lília R. Cordeiro Pedrosa, Ann L. B. Seynhaeve, Alexander M. M. Eggermont & Timo L. M. ten Hagen

  2. Laboratory Experimental Surgical Oncology Section Surgical Oncology, Department of Surgery, Erasmus Medical Center, Room Ee151b, PO Box 2040, Rotterdam, 3000 CA, The Netherlands

    Gerben A. Koning

  3. Division of Biological Stress Response, The Netherlands Cancer Institute - Antoni van Leeuwenhoek Hospital, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands

    Albert van Hell & Marcel Verheij

  4. Department of Pharmaceutical Technology and Biopharmacy, Albert-Ludwigs University, Freiburg, 79104, Germany

    Regine Süss

  5. Division of Cellular Biochemistry, The Netherlands Cancer Institute - Antoni van Leeuwenhoek Hospital, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands

    Wim J. van Blitterswijk

  6. Optical Imaging Centre, Erasmus Medical Center, PO Box 2040, Rotterdam, 3000 CA, The Netherlands

    Wiggert A. van Cappellen

  7. Institut de Cancerologie Gustave Roussy, Villejuif, Paris, 94800, France

    Alexander M. M. Eggermont

  8. Department of Radiotherapy, The Netherlands Cancer Institute - Antoni van Leeuwenhoek Hospital, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands

    Marcel Verheij

Authors
  1. Lília R. Cordeiro Pedrosa
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Corresponding author

Correspondence to Gerben A. Koning.

Electronic supplementary material

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Movie 1

Localization 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)

Supplemental Figure 1aSupplemental Figure 1b

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)

(JPEG 16 kb)

High Resolution Image

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High Resolution Image

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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)

High Resolution Image

(TIFF 138 kb)

Supplemental Figure 2b

(DOC 273 kb)

Supplemental Figure 3

Co-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)

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)

High Resolution Image

(TIFF 12946 kb)

Supplemental Table 1

In 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)

Supplemental Table 2

Stability 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)

Supplemental Table 3

In 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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Pedrosa, L.R.C., van Hell, A., Süss, R. et al. Improving Intracellular Doxorubicin Delivery Through Nanoliposomes Equipped with Selective Tumor Cell Membrane Permeabilizing Short-Chain Sphingolipids. Pharm Res 30, 1883–1895 (2013). https://doi.org/10.1007/s11095-013-1031-6

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