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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

Pharmacokinetics (a and b) and biodistribution (c and d) of Dox-TSL and Dox-RGD-TSL in B16BL6 tumor bearing mice upon NT or initial HT conditions. A,C. At NT condition, mice were injected with 3 mg/kg Dox and blood sampling was performed at the indicated time points and organs collected 24 h after liposomes injection. At HT condition (b and d), tumors in mice were preheated for 1 h at 41°C and cooled down for 15 min, in order to allow for liposome extravasation. Then, liposomes were injected at 3 mg/kg Dox and blood samples were collected up to 24 h, after which the organs were removed. The Dox concentration in the blood and organs was analyzed by HPLC.
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Fig8: Pharmacokinetics (a and b) and biodistribution (c and d) of Dox-TSL and Dox-RGD-TSL in B16BL6 tumor bearing mice upon NT or initial HT conditions. A,C. At NT condition, mice were injected with 3 mg/kg Dox and blood sampling was performed at the indicated time points and organs collected 24 h after liposomes injection. At HT condition (b and d), tumors in mice were preheated for 1 h at 41°C and cooled down for 15 min, in order to allow for liposome extravasation. Then, liposomes were injected at 3 mg/kg Dox and blood samples were collected up to 24 h, after which the organs were removed. The Dox concentration in the blood and organs was analyzed by HPLC.

Mentions: To follow up Dox clearance from circulation, its distribution in healthy organs and tumors and to be able to quantify Dox concentrations in tumors and organs, the pharmacokinetic and biodistribution profiles of Dox in TSL or RGD-TSL were studied (Fig. 8) under NT or initail HT conditions. At NT condition (Fig. 8a and c), Dox from both formulations seemed to clear from circulation quite fast in the first 1 h, as it was faster for RGD-TSL than for TSL (27% v/s 52% remaining Dox respectively). After 2 h of liposome circulation, the trend was the same showing lower remaining Dox from RGD-TSL than from TSL (10% v/s 20% respectively). At later time points (4,6,24 h) there was barely any Dox present in circulation from TSL and RGD-TSL. The application of initial HT (Fig. 8b) seemed to increase the presence of Dox from RGD-TSL in circulation at 1 h time point, after which its clearance was the same as at NT conditions. Besides, upon initial HT conditions, clearance of Dox from TSL and RGD-TSL was similar. Considering the biodistribution of Dox (Fig. 8c and d), at both NT and HT conditions, there was a significant uptake of Dox from both formulations in the spleen as it was higher for Dox from RGD-TSL than TSL (11.3 respectively v/s 8.3% ID/g at NT; and 11 v/s 6% ID/g at HT). Similar high Dox accumulation in the kidney was observed from both formulations. Dox accumulated in the liver was similar for RGD-TSL and TSL under NT (3.9%ID/g respectively v/s 2.7%ID/g). The higher Dox uptake in spleen and liver from RGD-TSL is due to most probably opsonization of RGD-TSL by proteins in these organs. There was a minimal uptake of Dox from TSL and RGD-TSL in the heart, lungs and muscle upon NT and HT. No Dox was detected in the brain from neither of the formulations. At NT, the tumor uptake of Dox was similar for both formulations. Application of initial HT for 1 h at 41°C was able to cause ~ 3.7 fold increase of Dox delivery to the tumor from RGD-TSL (1.6 v/s 6% ID/g) and ~ 2.3 fold increased Dox amount to the tumor from TSL (1.7 v/s 4% ID/g). The amount of Dox delivered to the tumor upon initial HT conditions from RGD-TSL was not significantly different from Dox delivered from TSL (p-value 0.1).Fig. 8


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)

Pharmacokinetics (a and b) and biodistribution (c and d) of Dox-TSL and Dox-RGD-TSL in B16BL6 tumor bearing mice upon NT or initial HT conditions. A,C. At NT condition, mice were injected with 3 mg/kg Dox and blood sampling was performed at the indicated time points and organs collected 24 h after liposomes injection. At HT condition (b and d), tumors in mice were preheated for 1 h at 41°C and cooled down for 15 min, in order to allow for liposome extravasation. Then, liposomes were injected at 3 mg/kg Dox and blood samples were collected up to 24 h, after which the organs were removed. The Dox concentration in the blood and organs was analyzed by HPLC.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4628091&req=5

Fig8: Pharmacokinetics (a and b) and biodistribution (c and d) of Dox-TSL and Dox-RGD-TSL in B16BL6 tumor bearing mice upon NT or initial HT conditions. A,C. At NT condition, mice were injected with 3 mg/kg Dox and blood sampling was performed at the indicated time points and organs collected 24 h after liposomes injection. At HT condition (b and d), tumors in mice were preheated for 1 h at 41°C and cooled down for 15 min, in order to allow for liposome extravasation. Then, liposomes were injected at 3 mg/kg Dox and blood samples were collected up to 24 h, after which the organs were removed. The Dox concentration in the blood and organs was analyzed by HPLC.
Mentions: To follow up Dox clearance from circulation, its distribution in healthy organs and tumors and to be able to quantify Dox concentrations in tumors and organs, the pharmacokinetic and biodistribution profiles of Dox in TSL or RGD-TSL were studied (Fig. 8) under NT or initail HT conditions. At NT condition (Fig. 8a and c), Dox from both formulations seemed to clear from circulation quite fast in the first 1 h, as it was faster for RGD-TSL than for TSL (27% v/s 52% remaining Dox respectively). After 2 h of liposome circulation, the trend was the same showing lower remaining Dox from RGD-TSL than from TSL (10% v/s 20% respectively). At later time points (4,6,24 h) there was barely any Dox present in circulation from TSL and RGD-TSL. The application of initial HT (Fig. 8b) seemed to increase the presence of Dox from RGD-TSL in circulation at 1 h time point, after which its clearance was the same as at NT conditions. Besides, upon initial HT conditions, clearance of Dox from TSL and RGD-TSL was similar. Considering the biodistribution of Dox (Fig. 8c and d), at both NT and HT conditions, there was a significant uptake of Dox from both formulations in the spleen as it was higher for Dox from RGD-TSL than TSL (11.3 respectively v/s 8.3% ID/g at NT; and 11 v/s 6% ID/g at HT). Similar high Dox accumulation in the kidney was observed from both formulations. Dox accumulated in the liver was similar for RGD-TSL and TSL under NT (3.9%ID/g respectively v/s 2.7%ID/g). The higher Dox uptake in spleen and liver from RGD-TSL is due to most probably opsonization of RGD-TSL by proteins in these organs. There was a minimal uptake of Dox from TSL and RGD-TSL in the heart, lungs and muscle upon NT and HT. No Dox was detected in the brain from neither of the formulations. At NT, the tumor uptake of Dox was similar for both formulations. Application of initial HT for 1 h at 41°C was able to cause ~ 3.7 fold increase of Dox delivery to the tumor from RGD-TSL (1.6 v/s 6% ID/g) and ~ 2.3 fold increased Dox amount to the tumor from TSL (1.7 v/s 4% ID/g). The amount of Dox delivered to the tumor upon initial HT conditions from RGD-TSL was not significantly different from Dox delivered from TSL (p-value 0.1).Fig. 8

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