Internalisation of RGD-Engineered Extracellular Vesicles by Glioblastoma Cells
Glioblastoma multiforme (GBM) is the most aggressive CNS tumour with no efficient treatment, partly due to the retention of anticancer drugs by the blood–brain barrier (BBB) and their insufficient concentration in tumour cells. Extracellular vesicles (EVs) are attractive drug carriers because of the...
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| Format: | Article |
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MDPI AG
2022-10-01
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| Series: | Biology |
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| Online Access: | https://www.mdpi.com/2079-7737/11/10/1483 |
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| author | Dovydas Gečys Arūnas Kazlauskas Emilija Gečytė Neringa Paužienė Deimantė Kulakauskienė Indrė Lukminaitė Aistė Jekabsone |
| author_facet | Dovydas Gečys Arūnas Kazlauskas Emilija Gečytė Neringa Paužienė Deimantė Kulakauskienė Indrė Lukminaitė Aistė Jekabsone |
| author_sort | Dovydas Gečys |
| collection | DOAJ |
| description | Glioblastoma multiforme (GBM) is the most aggressive CNS tumour with no efficient treatment, partly due to the retention of anticancer drugs by the blood–brain barrier (BBB) and their insufficient concentration in tumour cells. Extracellular vesicles (EVs) are attractive drug carriers because of their biocompatibility and ability to cross the BBB. Additional efficiency can be achieved by adding GBM-cell-specific ligands. GBM cells overexpress integrins; thus, one of the most straightforward targeting strategies is to modify EVs with integrin-recognising molecules. This study investigated the therapeutic potential of genetically engineered EVs with elevated membrane levels of the integrin-binding peptide RGD (RGD-EVs) against GBM cells in vitro. For RGD-EV production, stable RGD-HEK 293FT cells were generated by using a pcDNA4/TO-Lamp2b-iRGD-HA expression vector and performing antibiotic-based selection. RGD-EVs were isolated from RGD-HEK 293FT-cell-conditioned medium and characterised by size (Zetasizer), specific markers (ELISA) and RGD expression (Western Blot). Internalisation by human GBM cells HROG36 and U87 MG and BJ-5ta human fibroblasts was assessed by fluorescent EV RNA labelling. The effect of doxorubicin-loaded RGD-EVs on GBM cells was evaluated by the metabolic PrestoBlue viability assay; functional GAPDH gene knockdown by RGD-EV-encapsulated siRNA was determined by RT-qPCR. RGD-EVs had 40% higher accumulation in GBM cells (but not in fibroblasts) and induced significantly stronger toxicity by loaded doxorubicin and GAPDH silencing by loaded siRNA compared to unmodified EVs. Thus, RGD modification substantially increases the specific delivery capacity of HEK 293FT-derived EVs to GBM cells. |
| first_indexed | 2024-03-09T20:40:20Z |
| format | Article |
| id | doaj.art-da17629574e34d2baf4761651c8e2ee3 |
| institution | Directory Open Access Journal |
| issn | 2079-7737 |
| language | English |
| last_indexed | 2024-03-09T20:40:20Z |
| publishDate | 2022-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biology |
| spelling | doaj.art-da17629574e34d2baf4761651c8e2ee32023-11-23T23:00:19ZengMDPI AGBiology2079-77372022-10-011110148310.3390/biology11101483Internalisation of RGD-Engineered Extracellular Vesicles by Glioblastoma CellsDovydas Gečys0Arūnas Kazlauskas1Emilija Gečytė2Neringa Paužienė3Deimantė Kulakauskienė4Indrė Lukminaitė5Aistė Jekabsone6Faculty of Pharmacy, Institute of Pharmaceutical Technologies, Lithuanian University of Health Sciences, LT-50162 Kaunas, LithuaniaLaboratory of Molecular Neurooncology, Neuroscience Institute, Lithuanian University of Health Sciences, LT-50161 Kaunas, LithuaniaLaboratory of Molecular Cardiology, Institute of Cardiology, Lithuanian University of Health Sciences, LT-50162 Kaunas, LithuaniaInstitute of Anatomy, Faculty of Medicine, Lithuanian University of Health Sciences, LT-44307 Kaunas, LithuaniaFaculty of Pharmacy, Institute of Pharmaceutical Technologies, Lithuanian University of Health Sciences, LT-50162 Kaunas, LithuaniaFaculty of Pharmacy, Institute of Pharmaceutical Technologies, Lithuanian University of Health Sciences, LT-50162 Kaunas, LithuaniaFaculty of Pharmacy, Institute of Pharmaceutical Technologies, Lithuanian University of Health Sciences, LT-50162 Kaunas, LithuaniaGlioblastoma multiforme (GBM) is the most aggressive CNS tumour with no efficient treatment, partly due to the retention of anticancer drugs by the blood–brain barrier (BBB) and their insufficient concentration in tumour cells. Extracellular vesicles (EVs) are attractive drug carriers because of their biocompatibility and ability to cross the BBB. Additional efficiency can be achieved by adding GBM-cell-specific ligands. GBM cells overexpress integrins; thus, one of the most straightforward targeting strategies is to modify EVs with integrin-recognising molecules. This study investigated the therapeutic potential of genetically engineered EVs with elevated membrane levels of the integrin-binding peptide RGD (RGD-EVs) against GBM cells in vitro. For RGD-EV production, stable RGD-HEK 293FT cells were generated by using a pcDNA4/TO-Lamp2b-iRGD-HA expression vector and performing antibiotic-based selection. RGD-EVs were isolated from RGD-HEK 293FT-cell-conditioned medium and characterised by size (Zetasizer), specific markers (ELISA) and RGD expression (Western Blot). Internalisation by human GBM cells HROG36 and U87 MG and BJ-5ta human fibroblasts was assessed by fluorescent EV RNA labelling. The effect of doxorubicin-loaded RGD-EVs on GBM cells was evaluated by the metabolic PrestoBlue viability assay; functional GAPDH gene knockdown by RGD-EV-encapsulated siRNA was determined by RT-qPCR. RGD-EVs had 40% higher accumulation in GBM cells (but not in fibroblasts) and induced significantly stronger toxicity by loaded doxorubicin and GAPDH silencing by loaded siRNA compared to unmodified EVs. Thus, RGD modification substantially increases the specific delivery capacity of HEK 293FT-derived EVs to GBM cells.https://www.mdpi.com/2079-7737/11/10/1483extracellular vesiclesEVsexosomesglioblastomatargeted delivery |
| spellingShingle | Dovydas Gečys Arūnas Kazlauskas Emilija Gečytė Neringa Paužienė Deimantė Kulakauskienė Indrė Lukminaitė Aistė Jekabsone Internalisation of RGD-Engineered Extracellular Vesicles by Glioblastoma Cells Biology extracellular vesicles EVs exosomes glioblastoma targeted delivery |
| title | Internalisation of RGD-Engineered Extracellular Vesicles by Glioblastoma Cells |
| title_full | Internalisation of RGD-Engineered Extracellular Vesicles by Glioblastoma Cells |
| title_fullStr | Internalisation of RGD-Engineered Extracellular Vesicles by Glioblastoma Cells |
| title_full_unstemmed | Internalisation of RGD-Engineered Extracellular Vesicles by Glioblastoma Cells |
| title_short | Internalisation of RGD-Engineered Extracellular Vesicles by Glioblastoma Cells |
| title_sort | internalisation of rgd engineered extracellular vesicles by glioblastoma cells |
| topic | extracellular vesicles EVs exosomes glioblastoma targeted delivery |
| url | https://www.mdpi.com/2079-7737/11/10/1483 |
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