Limits...
Enhanced Specificity and Drug Delivery in Tumors by cRGD - Anchoring Thermosensitive Liposomes.

Dicheva BM, Ten Hagen TL, Seynhaeve AL, Amin M, Eggermont AM, Koning GA - Pharm. Res. (2015)

Bottom Line: Cytotoxic effect of TSL and RGD-TSL was studied on B16Bl6 melanoma, B16F10 melanoma and HUVEC.High resolution intravital microscopy demonstrated specific accumulation of RGD-TSL to the tumor vasculature.Moreover, application of hyperthermia resulted in massive drug release from RGD-TSL.

View Article: PubMed Central - PubMed

Affiliation: Laboratory Experimental Surgical Oncology, Section Surgical Oncology Department of Surgery, Erasmus MC Cancer Center, Rotterdam, The Netherlands. b.dicheva@erasmusmc.nl.

ABSTRACT

Purpose: To develop RGD-targeted thermosensitive liposomes with increased tumor retention, improving drug release efficiency upon mild hyperthermia (HT) in both tumor and angiogenic endothelial cells.

Methods: Standard termosensitive liposomes (TSL) and TSL containing a cyclic Arg-Gly-Asp (cRGD) pentapeptide with the sequence Arg-Cys-D-Phe-Asp-Gly (RGDf[N-Met]C) were synthetized, loaded with Dox and characterized. Temperature- and time-dependent drug release profiles were assessed by fluorometry. Intracellular Dox delivery was studied by flow cytometry and confocal microscopy. Cytotoxic effect of TSL and RGD-TSL was studied on B16Bl6 melanoma, B16F10 melanoma and HUVEC. Intravital microscopy was performed on B16Bl6 tumors implanted in dorsal-skin fold window-bearing mice. Pharmacokinetic and biodistribution of Dox-TSL and Dox-RGD-TSL were followed in B16Bl6 tumor bearing mice upon normothermia or initial hyperthermia conditions.

Results: DLS and cryo-TEM revealed particle homogeneity and size of around 85 nm. Doxorubicin loading efficiency was >95%as assessed by spectrofluorometry. Flow cytometry and confocal microscopy showed a specific uptake of RGD-TSL by melanoma and endothelial cells when compared to TSL and an increased doxorubicin delivery. High resolution intravital microscopy demonstrated specific accumulation of RGD-TSL to the tumor vasculature. Moreover, application of hyperthermia resulted in massive drug release from RGD-TSL. Biodistribution studies showed that initial hyperthermia increases Dox uptake in tumors from TSL and RGD-TSL.

Conclusion: RGD-TSL have potency to increase drug efficacy due to higher uptake by tumor and angiogenic endothelial cells in combination with heat-triggered drug release.

No MeSH data available.


Related in: MedlinePlus

(a). Confocal microscopy on melanoma B16Bl6 and B16F10 cells and HUVEC incubated with NBD-PE (green) labelled RGD-TSL for 3 h at 37°C and lysotracker (red). Unbound liposomes were removed by washing 3× with medium without FCS. After washing, B16Bl6 and B16F10 were immediately imaged at 37°C, whereas HUVEC were followed up to 7 h. Internalized liposomes in the cytosol can be observed in all the cell lines (white arrows). Non-internalized in the lysosomes liposomes were also visible (blue arrows). B16Bl6 and B16 internalized liposomes immediately in the lysosomes after the 3 h of incubation period (yellow colocalization of green liposomes and red lysotracker), whereas this process happened in HUVEC after 7 h. Images were taken by confocal microscope (40×, 2,5 μm pinhole, 2× zoom). Scale bar applies for all images, 20 μm. (b). Doxorubicin release (red) from DiD-labelled RGD-TSL (purple) in B16Bl6, B16F10 and HUVEC upon HT trigger. Cells were incubated with 50 μM Dox for 3 h at 37°C, after which cells were washed 3× with medium without FCS. Images were taken right after this incubation at 37°C. Then, HT at 42°C for 1 h was applied and images in the end of the HT treatment were recorded. Images were taken by confocal microscope. Scale bar applies for all images, 50 μm. (c). Colocalization of RGD-TSL (purple) with lysotracker (green) and Dox release (red) in the lysosomes. Scale bar applies for all images, 10 μm.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4628091&req=5

Fig4: (a). Confocal microscopy on melanoma B16Bl6 and B16F10 cells and HUVEC incubated with NBD-PE (green) labelled RGD-TSL for 3 h at 37°C and lysotracker (red). Unbound liposomes were removed by washing 3× with medium without FCS. After washing, B16Bl6 and B16F10 were immediately imaged at 37°C, whereas HUVEC were followed up to 7 h. Internalized liposomes in the cytosol can be observed in all the cell lines (white arrows). Non-internalized in the lysosomes liposomes were also visible (blue arrows). B16Bl6 and B16 internalized liposomes immediately in the lysosomes after the 3 h of incubation period (yellow colocalization of green liposomes and red lysotracker), whereas this process happened in HUVEC after 7 h. Images were taken by confocal microscope (40×, 2,5 μm pinhole, 2× zoom). Scale bar applies for all images, 20 μm. (b). Doxorubicin release (red) from DiD-labelled RGD-TSL (purple) in B16Bl6, B16F10 and HUVEC upon HT trigger. Cells were incubated with 50 μM Dox for 3 h at 37°C, after which cells were washed 3× with medium without FCS. Images were taken right after this incubation at 37°C. Then, HT at 42°C for 1 h was applied and images in the end of the HT treatment were recorded. Images were taken by confocal microscope. Scale bar applies for all images, 50 μm. (c). Colocalization of RGD-TSL (purple) with lysotracker (green) and Dox release (red) in the lysosomes. Scale bar applies for all images, 10 μm.

Mentions: The intracellular localization of liposomes was studied by live cell imaging in B16Bl6, B16F10 and HUVEC (Fig. 4a). Cells were incubated with NBD-PE labelled (green) RGD-TSL and lysotracker (red, to stain lysosomes) for 3 h at 37°C, after which the unbound liposomes were removed by washing. B16Bl6 and B16F10 were able to localize the RGD-TSL in the cytoplasm after 3 h of incubation and those liposomes were colocalized with the red lysotracker, seen as yellow fluorescence signal (white arrows). Still, there were some RGD-TSL (green fluorescence spots), which were not concentrated in the acidic compartments (blue arrows). By contrast, in HUVEC sequestering of RGD-TSL in the lysosomes occurred at a slower pace. After 3 h of incubation at 37°C, liposomes and lysotracker were observed as green and red separated fluorescence signals, showing no entrapment of the liposomes in the lysosomes. However, when cells were followed for a prolonged period, colocalization (in yellow) started to be visible. After 7 h of incubation, there was an abundant amount of RGD-TSL localized in the lysosomes but also some non-entrapped liposomes in lysosomes could be observed.Fig. 4


Enhanced Specificity and Drug Delivery in Tumors by cRGD - Anchoring Thermosensitive Liposomes.

Dicheva BM, Ten Hagen TL, Seynhaeve AL, Amin M, Eggermont AM, Koning GA - Pharm. Res. (2015)

(a). Confocal microscopy on melanoma B16Bl6 and B16F10 cells and HUVEC incubated with NBD-PE (green) labelled RGD-TSL for 3 h at 37°C and lysotracker (red). Unbound liposomes were removed by washing 3× with medium without FCS. After washing, B16Bl6 and B16F10 were immediately imaged at 37°C, whereas HUVEC were followed up to 7 h. Internalized liposomes in the cytosol can be observed in all the cell lines (white arrows). Non-internalized in the lysosomes liposomes were also visible (blue arrows). B16Bl6 and B16 internalized liposomes immediately in the lysosomes after the 3 h of incubation period (yellow colocalization of green liposomes and red lysotracker), whereas this process happened in HUVEC after 7 h. Images were taken by confocal microscope (40×, 2,5 μm pinhole, 2× zoom). Scale bar applies for all images, 20 μm. (b). Doxorubicin release (red) from DiD-labelled RGD-TSL (purple) in B16Bl6, B16F10 and HUVEC upon HT trigger. Cells were incubated with 50 μM Dox for 3 h at 37°C, after which cells were washed 3× with medium without FCS. Images were taken right after this incubation at 37°C. Then, HT at 42°C for 1 h was applied and images in the end of the HT treatment were recorded. Images were taken by confocal microscope. Scale bar applies for all images, 50 μm. (c). Colocalization of RGD-TSL (purple) with lysotracker (green) and Dox release (red) in the lysosomes. Scale bar applies for all images, 10 μm.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4628091&req=5

Fig4: (a). Confocal microscopy on melanoma B16Bl6 and B16F10 cells and HUVEC incubated with NBD-PE (green) labelled RGD-TSL for 3 h at 37°C and lysotracker (red). Unbound liposomes were removed by washing 3× with medium without FCS. After washing, B16Bl6 and B16F10 were immediately imaged at 37°C, whereas HUVEC were followed up to 7 h. Internalized liposomes in the cytosol can be observed in all the cell lines (white arrows). Non-internalized in the lysosomes liposomes were also visible (blue arrows). B16Bl6 and B16 internalized liposomes immediately in the lysosomes after the 3 h of incubation period (yellow colocalization of green liposomes and red lysotracker), whereas this process happened in HUVEC after 7 h. Images were taken by confocal microscope (40×, 2,5 μm pinhole, 2× zoom). Scale bar applies for all images, 20 μm. (b). Doxorubicin release (red) from DiD-labelled RGD-TSL (purple) in B16Bl6, B16F10 and HUVEC upon HT trigger. Cells were incubated with 50 μM Dox for 3 h at 37°C, after which cells were washed 3× with medium without FCS. Images were taken right after this incubation at 37°C. Then, HT at 42°C for 1 h was applied and images in the end of the HT treatment were recorded. Images were taken by confocal microscope. Scale bar applies for all images, 50 μm. (c). Colocalization of RGD-TSL (purple) with lysotracker (green) and Dox release (red) in the lysosomes. Scale bar applies for all images, 10 μm.
Mentions: The intracellular localization of liposomes was studied by live cell imaging in B16Bl6, B16F10 and HUVEC (Fig. 4a). Cells were incubated with NBD-PE labelled (green) RGD-TSL and lysotracker (red, to stain lysosomes) for 3 h at 37°C, after which the unbound liposomes were removed by washing. B16Bl6 and B16F10 were able to localize the RGD-TSL in the cytoplasm after 3 h of incubation and those liposomes were colocalized with the red lysotracker, seen as yellow fluorescence signal (white arrows). Still, there were some RGD-TSL (green fluorescence spots), which were not concentrated in the acidic compartments (blue arrows). By contrast, in HUVEC sequestering of RGD-TSL in the lysosomes occurred at a slower pace. After 3 h of incubation at 37°C, liposomes and lysotracker were observed as green and red separated fluorescence signals, showing no entrapment of the liposomes in the lysosomes. However, when cells were followed for a prolonged period, colocalization (in yellow) started to be visible. After 7 h of incubation, there was an abundant amount of RGD-TSL localized in the lysosomes but also some non-entrapped liposomes in lysosomes could be observed.Fig. 4

Bottom Line: Cytotoxic effect of TSL and RGD-TSL was studied on B16Bl6 melanoma, B16F10 melanoma and HUVEC.High resolution intravital microscopy demonstrated specific accumulation of RGD-TSL to the tumor vasculature.Moreover, application of hyperthermia resulted in massive drug release from RGD-TSL.

View Article: PubMed Central - PubMed

Affiliation: Laboratory Experimental Surgical Oncology, Section Surgical Oncology Department of Surgery, Erasmus MC Cancer Center, Rotterdam, The Netherlands. b.dicheva@erasmusmc.nl.

ABSTRACT

Purpose: To develop RGD-targeted thermosensitive liposomes with increased tumor retention, improving drug release efficiency upon mild hyperthermia (HT) in both tumor and angiogenic endothelial cells.

Methods: Standard termosensitive liposomes (TSL) and TSL containing a cyclic Arg-Gly-Asp (cRGD) pentapeptide with the sequence Arg-Cys-D-Phe-Asp-Gly (RGDf[N-Met]C) were synthetized, loaded with Dox and characterized. Temperature- and time-dependent drug release profiles were assessed by fluorometry. Intracellular Dox delivery was studied by flow cytometry and confocal microscopy. Cytotoxic effect of TSL and RGD-TSL was studied on B16Bl6 melanoma, B16F10 melanoma and HUVEC. Intravital microscopy was performed on B16Bl6 tumors implanted in dorsal-skin fold window-bearing mice. Pharmacokinetic and biodistribution of Dox-TSL and Dox-RGD-TSL were followed in B16Bl6 tumor bearing mice upon normothermia or initial hyperthermia conditions.

Results: DLS and cryo-TEM revealed particle homogeneity and size of around 85 nm. Doxorubicin loading efficiency was >95%as assessed by spectrofluorometry. Flow cytometry and confocal microscopy showed a specific uptake of RGD-TSL by melanoma and endothelial cells when compared to TSL and an increased doxorubicin delivery. High resolution intravital microscopy demonstrated specific accumulation of RGD-TSL to the tumor vasculature. Moreover, application of hyperthermia resulted in massive drug release from RGD-TSL. Biodistribution studies showed that initial hyperthermia increases Dox uptake in tumors from TSL and RGD-TSL.

Conclusion: RGD-TSL have potency to increase drug efficacy due to higher uptake by tumor and angiogenic endothelial cells in combination with heat-triggered drug release.

No MeSH data available.


Related in: MedlinePlus