Active Tumor-Targeting Nano-formulations Containing Simvastatin and Doxorubicin Inhibit Melanoma Growth and Angiogenesis

Primary melanoma aggressiveness is determined by rapid selection and growth of cellular clones resistant to conventional treatments, resulting in metastasis and recurrence. In addition, a reprogrammed tumor-immune microenvironment supports melanoma progression and response to therapy. There is an ur...

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Main Authors: Giorgiana Negrea, Valentin-Florian Rauca, Marta Szilvia Meszaros, Laura Patras, Lavinia Luput, Emilia Licarete, Vlad-Alexandru Toma, Alina Porfire, Dana Muntean, Alina Sesarman, Manuela Banciu
Format: Article
Language:English
Published: Frontiers Media S.A. 2022-04-01
Series:Frontiers in Pharmacology
Subjects:
Online Access:https://www.frontiersin.org/articles/10.3389/fphar.2022.870347/full
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author Giorgiana Negrea
Valentin-Florian Rauca
Valentin-Florian Rauca
Marta Szilvia Meszaros
Laura Patras
Lavinia Luput
Emilia Licarete
Emilia Licarete
Vlad-Alexandru Toma
Vlad-Alexandru Toma
Alina Porfire
Dana Muntean
Alina Sesarman
Manuela Banciu
author_facet Giorgiana Negrea
Valentin-Florian Rauca
Valentin-Florian Rauca
Marta Szilvia Meszaros
Laura Patras
Lavinia Luput
Emilia Licarete
Emilia Licarete
Vlad-Alexandru Toma
Vlad-Alexandru Toma
Alina Porfire
Dana Muntean
Alina Sesarman
Manuela Banciu
author_sort Giorgiana Negrea
collection DOAJ
description Primary melanoma aggressiveness is determined by rapid selection and growth of cellular clones resistant to conventional treatments, resulting in metastasis and recurrence. In addition, a reprogrammed tumor-immune microenvironment supports melanoma progression and response to therapy. There is an urgent need to develop selective and specific drug delivery strategies for modulating the interaction between cancer cells and immune cells within the tumor microenvironment. This study proposes a novel combination therapy consisting of sequential administration of simvastatin incorporated in IL-13-functionalized long-circulating liposomes (IL-13-LCL-SIM) and doxorubicin encapsulated into PEG-coated extracellular vesicles (PEG-EV-DOX) to selectively target both tumor-associated macrophages and melanoma cells. To this end, IL-13 was conjugated to LCL-SIM which was obtained via the lipid film hydration method. EVs enriched from melanoma cells were passively loaded with doxorubicin. The cellular uptake of rhodamine-tagged nano-particles and the antiproliferative potential of the treatments by using the ELISA BrdU-colorimetric immunoassay were investigated in vitro. Subsequently, the therapeutic agents were administered i.v in B16.F10 melanoma-bearing mice, and tumor size was monitored during treatment. The molecular mechanisms of antitumor activity were investigated using angiogenic and inflammatory protein arrays and western blot analysis of invasion (HIF-1) and apoptosis markers (Bcl-xL and Bax). Quantification of oxidative stress marker malondialdehyde (MDA) was determined by HPLC. Immunohistochemical staining of angiogenic markers CD31 and VEGF and of pan-macrophage marker F4/80 was performed to validate our findings. The in vitro data showed that IL-13-functionalized LCL were preferentially taken up by tumor-associated macrophages and indicated that sequential administration of IL-13-LCL-SIM and PEG-EV-DOX had the strongest antiproliferative effect on tumor cells co-cultured with tumor-associated macrophages (TAMs). Accordingly, strong inhibition of tumor growth in the group treated with the sequential combination therapy was reported in vivo. Our data suggested that the antitumor action of the combined treatment was exerted through strong inhibition of several pro-angiogenic factors (VEGF, bFGF, and CD31) and oxidative stress-induced upregulation of pro-apoptotic protein Bax. This novel drug delivery strategy based on combined active targeting of both cancer cells and immune cells was able to induce a potent antitumor effect by disruption of the reciprocal interactions between TAMs and melanoma cells.
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spelling doaj.art-8b0acd811112429abc5b0a7d281a4fb32022-12-22T02:50:33ZengFrontiers Media S.A.Frontiers in Pharmacology1663-98122022-04-011310.3389/fphar.2022.870347870347Active Tumor-Targeting Nano-formulations Containing Simvastatin and Doxorubicin Inhibit Melanoma Growth and AngiogenesisGiorgiana Negrea0Valentin-Florian Rauca1Valentin-Florian Rauca2Marta Szilvia Meszaros3Laura Patras4Lavinia Luput5Emilia Licarete6Emilia Licarete7Vlad-Alexandru Toma8Vlad-Alexandru Toma9Alina Porfire10Dana Muntean11Alina Sesarman12Manuela Banciu13Doctoral School in Integrative Biology, Faculty of Biology and Geology, “Babes-Bolyai” University, Cluj-Napoca, RomaniaDepartment of Molecular Biology and Biotechnology, Center of Systems Biology, Biodiversity and Bioresources, Faculty of Biology and Geology, “Babes-Bolyai” University, Cluj-Napoca, RomaniaDepartment of Dermatology and Allergology, School of Medicine, Technical University of Munich, Munich, GermanyDepartment of Molecular Biology and Biotechnology, Center of Systems Biology, Biodiversity and Bioresources, Faculty of Biology and Geology, “Babes-Bolyai” University, Cluj-Napoca, RomaniaDepartment of Molecular Biology and Biotechnology, Center of Systems Biology, Biodiversity and Bioresources, Faculty of Biology and Geology, “Babes-Bolyai” University, Cluj-Napoca, RomaniaDepartment of Molecular Biology and Biotechnology, Center of Systems Biology, Biodiversity and Bioresources, Faculty of Biology and Geology, “Babes-Bolyai” University, Cluj-Napoca, RomaniaDepartment of Molecular Biology and Biotechnology, Center of Systems Biology, Biodiversity and Bioresources, Faculty of Biology and Geology, “Babes-Bolyai” University, Cluj-Napoca, RomaniaMolecular Biology Centre, Institute for Interdisciplinary Research in Bio-Nano-Sciences, Babes-Bolyai University, Cluj-Napoca, RomaniaDepartment of Molecular Biology and Biotechnology, Center of Systems Biology, Biodiversity and Bioresources, Faculty of Biology and Geology, “Babes-Bolyai” University, Cluj-Napoca, RomaniaDepartment of Experimental Biology and Biochemistry, Institute of Biological Research, Branch of NIRDBS Bucharest, Cluj-Napoca, RomaniaDepartment of Pharmaceutical Technology and Biopharmaceutics, Faculty of Pharmacy, University of Medicine and Pharmacy “Iuliu Hatieganu”, Cluj-Napoca, RomaniaDepartment of Pharmaceutical Technology and Biopharmaceutics, Faculty of Pharmacy, University of Medicine and Pharmacy “Iuliu Hatieganu”, Cluj-Napoca, RomaniaDepartment of Molecular Biology and Biotechnology, Center of Systems Biology, Biodiversity and Bioresources, Faculty of Biology and Geology, “Babes-Bolyai” University, Cluj-Napoca, RomaniaDepartment of Molecular Biology and Biotechnology, Center of Systems Biology, Biodiversity and Bioresources, Faculty of Biology and Geology, “Babes-Bolyai” University, Cluj-Napoca, RomaniaPrimary melanoma aggressiveness is determined by rapid selection and growth of cellular clones resistant to conventional treatments, resulting in metastasis and recurrence. In addition, a reprogrammed tumor-immune microenvironment supports melanoma progression and response to therapy. There is an urgent need to develop selective and specific drug delivery strategies for modulating the interaction between cancer cells and immune cells within the tumor microenvironment. This study proposes a novel combination therapy consisting of sequential administration of simvastatin incorporated in IL-13-functionalized long-circulating liposomes (IL-13-LCL-SIM) and doxorubicin encapsulated into PEG-coated extracellular vesicles (PEG-EV-DOX) to selectively target both tumor-associated macrophages and melanoma cells. To this end, IL-13 was conjugated to LCL-SIM which was obtained via the lipid film hydration method. EVs enriched from melanoma cells were passively loaded with doxorubicin. The cellular uptake of rhodamine-tagged nano-particles and the antiproliferative potential of the treatments by using the ELISA BrdU-colorimetric immunoassay were investigated in vitro. Subsequently, the therapeutic agents were administered i.v in B16.F10 melanoma-bearing mice, and tumor size was monitored during treatment. The molecular mechanisms of antitumor activity were investigated using angiogenic and inflammatory protein arrays and western blot analysis of invasion (HIF-1) and apoptosis markers (Bcl-xL and Bax). Quantification of oxidative stress marker malondialdehyde (MDA) was determined by HPLC. Immunohistochemical staining of angiogenic markers CD31 and VEGF and of pan-macrophage marker F4/80 was performed to validate our findings. The in vitro data showed that IL-13-functionalized LCL were preferentially taken up by tumor-associated macrophages and indicated that sequential administration of IL-13-LCL-SIM and PEG-EV-DOX had the strongest antiproliferative effect on tumor cells co-cultured with tumor-associated macrophages (TAMs). Accordingly, strong inhibition of tumor growth in the group treated with the sequential combination therapy was reported in vivo. Our data suggested that the antitumor action of the combined treatment was exerted through strong inhibition of several pro-angiogenic factors (VEGF, bFGF, and CD31) and oxidative stress-induced upregulation of pro-apoptotic protein Bax. This novel drug delivery strategy based on combined active targeting of both cancer cells and immune cells was able to induce a potent antitumor effect by disruption of the reciprocal interactions between TAMs and melanoma cells.https://www.frontiersin.org/articles/10.3389/fphar.2022.870347/fullmelanomadrug delivery systemsliposomesextracellular vesiclessimvastatindoxorubicin
spellingShingle Giorgiana Negrea
Valentin-Florian Rauca
Valentin-Florian Rauca
Marta Szilvia Meszaros
Laura Patras
Lavinia Luput
Emilia Licarete
Emilia Licarete
Vlad-Alexandru Toma
Vlad-Alexandru Toma
Alina Porfire
Dana Muntean
Alina Sesarman
Manuela Banciu
Active Tumor-Targeting Nano-formulations Containing Simvastatin and Doxorubicin Inhibit Melanoma Growth and Angiogenesis
Frontiers in Pharmacology
melanoma
drug delivery systems
liposomes
extracellular vesicles
simvastatin
doxorubicin
title Active Tumor-Targeting Nano-formulations Containing Simvastatin and Doxorubicin Inhibit Melanoma Growth and Angiogenesis
title_full Active Tumor-Targeting Nano-formulations Containing Simvastatin and Doxorubicin Inhibit Melanoma Growth and Angiogenesis
title_fullStr Active Tumor-Targeting Nano-formulations Containing Simvastatin and Doxorubicin Inhibit Melanoma Growth and Angiogenesis
title_full_unstemmed Active Tumor-Targeting Nano-formulations Containing Simvastatin and Doxorubicin Inhibit Melanoma Growth and Angiogenesis
title_short Active Tumor-Targeting Nano-formulations Containing Simvastatin and Doxorubicin Inhibit Melanoma Growth and Angiogenesis
title_sort active tumor targeting nano formulations containing simvastatin and doxorubicin inhibit melanoma growth and angiogenesis
topic melanoma
drug delivery systems
liposomes
extracellular vesicles
simvastatin
doxorubicin
url https://www.frontiersin.org/articles/10.3389/fphar.2022.870347/full
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