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). Binding of DiD-labeled RGD-TSL (purple) to tumor vasculature (green) of B16Bl6 window chamber bearing mice. Binding of liposomes to tumor endothelial cells started 20 min after injection and was followed in time up to 24 h. Representative images from intravital microscopy were selected. Scale bar applies to all images, 50 μm. (b). RGD-TSL and TSL appearance (DiD, in purple) in tumor vasculature (green) during 5 h at NT in B16Bl6 window bearing mice (Fig B left panels) and upon subsequent HT at 42°C for 1 h (Fig. 5bright panels). DiD-labelled RGD-TSL or TSL were injected i.v, after which they were allowed to circulate in blood stream at NT for 5 h in order to allow binding of RGD-TSL to angiogenic endothelial cells. Thereafter, HT at 42°C for 1 h was applied to promote extravasation of RGD-TSL and TSL. Scale bar applies to all images 50 μm. (c). In vivo quantification of DiD liposomal fluorescence before and during 1 h of HT, presented as integrated density (IntDen) in time, see Materials and methods for details.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4628091&req=5

Fig6: (a). Binding of DiD-labeled RGD-TSL (purple) to tumor vasculature (green) of B16Bl6 window chamber bearing mice. Binding of liposomes to tumor endothelial cells started 20 min after injection and was followed in time up to 24 h. Representative images from intravital microscopy were selected. Scale bar applies to all images, 50 μm. (b). RGD-TSL and TSL appearance (DiD, in purple) in tumor vasculature (green) during 5 h at NT in B16Bl6 window bearing mice (Fig B left panels) and upon subsequent HT at 42°C for 1 h (Fig. 5bright panels). DiD-labelled RGD-TSL or TSL were injected i.v, after which they were allowed to circulate in blood stream at NT for 5 h in order to allow binding of RGD-TSL to angiogenic endothelial cells. Thereafter, HT at 42°C for 1 h was applied to promote extravasation of RGD-TSL and TSL. Scale bar applies to all images 50 μm. (c). In vivo quantification of DiD liposomal fluorescence before and during 1 h of HT, presented as integrated density (IntDen) in time, see Materials and methods for details.

Mentions: In order to proof that RGD-TSL target angiogenic endothelial cells in vivo, intravital microscopy in B16Bl6 window chamber bearing mice was performed. To visualize circulating liposomes in the blood stream, liposomes were labelled with DiD (purple). In these mice tumor vasculature is visualized by the constitutive expression of a GFP-eNOS-tag fusion protein in endothelial cells (Fig. 6). Twenty minutes after injection of RGD-TSL, next to circulating liposomes in the lumen of the blood vessels, bound RGD-TSL could be observed (Fig. 6a yellow arrows). These liposomes can be visualized as patchy fluorescent spots on the vessel walls. Besides bound liposomes to the angiogenic endothelial cells, extravasated liposomes from tumor vasculature are visible already 20 min after injection (white arrows). They are visible as diffuse purple fluorescence outside of the green blood vessels. Binding continued in time and was pronounced 24 h after injection. In contrast, using DiD-labeled TSL, no binding to tumor vasculature was detected after 5 h of circulation. In the last time point (24 h), only extravasated TSL were visible (Fig. 6a, right panel).Fig. 6


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). Binding of DiD-labeled RGD-TSL (purple) to tumor vasculature (green) of B16Bl6 window chamber bearing mice. Binding of liposomes to tumor endothelial cells started 20 min after injection and was followed in time up to 24 h. Representative images from intravital microscopy were selected. Scale bar applies to all images, 50 μm. (b). RGD-TSL and TSL appearance (DiD, in purple) in tumor vasculature (green) during 5 h at NT in B16Bl6 window bearing mice (Fig B left panels) and upon subsequent HT at 42°C for 1 h (Fig. 5bright panels). DiD-labelled RGD-TSL or TSL were injected i.v, after which they were allowed to circulate in blood stream at NT for 5 h in order to allow binding of RGD-TSL to angiogenic endothelial cells. Thereafter, HT at 42°C for 1 h was applied to promote extravasation of RGD-TSL and TSL. Scale bar applies to all images 50 μm. (c). In vivo quantification of DiD liposomal fluorescence before and during 1 h of HT, presented as integrated density (IntDen) in time, see Materials and methods for details.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: (a). Binding of DiD-labeled RGD-TSL (purple) to tumor vasculature (green) of B16Bl6 window chamber bearing mice. Binding of liposomes to tumor endothelial cells started 20 min after injection and was followed in time up to 24 h. Representative images from intravital microscopy were selected. Scale bar applies to all images, 50 μm. (b). RGD-TSL and TSL appearance (DiD, in purple) in tumor vasculature (green) during 5 h at NT in B16Bl6 window bearing mice (Fig B left panels) and upon subsequent HT at 42°C for 1 h (Fig. 5bright panels). DiD-labelled RGD-TSL or TSL were injected i.v, after which they were allowed to circulate in blood stream at NT for 5 h in order to allow binding of RGD-TSL to angiogenic endothelial cells. Thereafter, HT at 42°C for 1 h was applied to promote extravasation of RGD-TSL and TSL. Scale bar applies to all images 50 μm. (c). In vivo quantification of DiD liposomal fluorescence before and during 1 h of HT, presented as integrated density (IntDen) in time, see Materials and methods for details.
Mentions: In order to proof that RGD-TSL target angiogenic endothelial cells in vivo, intravital microscopy in B16Bl6 window chamber bearing mice was performed. To visualize circulating liposomes in the blood stream, liposomes were labelled with DiD (purple). In these mice tumor vasculature is visualized by the constitutive expression of a GFP-eNOS-tag fusion protein in endothelial cells (Fig. 6). Twenty minutes after injection of RGD-TSL, next to circulating liposomes in the lumen of the blood vessels, bound RGD-TSL could be observed (Fig. 6a yellow arrows). These liposomes can be visualized as patchy fluorescent spots on the vessel walls. Besides bound liposomes to the angiogenic endothelial cells, extravasated liposomes from tumor vasculature are visible already 20 min after injection (white arrows). They are visible as diffuse purple fluorescence outside of the green blood vessels. Binding continued in time and was pronounced 24 h after injection. In contrast, using DiD-labeled TSL, no binding to tumor vasculature was detected after 5 h of circulation. In the last time point (24 h), only extravasated TSL were visible (Fig. 6a, right panel).Fig. 6

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