Final Report Summary - SMART (Shedding microvesicles as drug loaded Trojan horses (SMART) – an exploration of unique drug carriers using a versatile enzyme nano assay)
The development of efficient drug carriers is a major challenge in the field of drug delivery. Most existing delivery systems do not deliver their cargo efficiently or are rapidly inactivated by the immune system, leading to unwanted side effects or reduced efficacy. One example of where such selective drug delivery is very important is cancer therapy. The drugs that are used to treat this life-threatening disease are highly effective but they also have severe side-effects (e.g. nausea or hair loss) which are debilitating for the patients and they can interfere with the treatment. In order to bring those drugs where they should be, they should be packed into a kind of envelope that navigates the medicine to its desired target. In this project, so-called extracellular vesicles (EVs) were used as such novel envelopes to deliver drugs in a highly controlled and specific manner. These EVs are very tiny (about 10,000 times smaller than a pinhead) and they are produced by many types of cells. In nature, cells use those EVs to transfer information from one cell to another, i.e. EVs have the characteristic to go to one specific cell (e.g. cancer cell) but not to any other (healthy) cells, which makes them ideal delivery candidates. The main goal of this project was to harness this body's own intercellular “shuttle service” to create new carrier systems that are physiologically compatible and show high stability in biological liquids. In SMART, there has been significant and excellent progress towards achieving the objectives of the project and the research in the Stevens lab has significantly advanced the state-of-the-art of the fields of extracellular vesicles and therapeutic delivery. The significant results obtained during this Marie Sklodowska-Curie fellowship are the following key novelties:
1. Detailed physicochemical and biochemical characterisation of EVs from various cell sources
2. Introduction of novel loading methods to encapsulate drugs of different hydrophobicities into EVs
3. Detailed analysis of the uptake of porphyrin loaded EVs using high-content imaging using in vitro cell models
4. Improved cellular delivery of small molecule drugs (photoactivatable porphyrins) compared to existing carrier systems, such as liposomes
A detailed progress towards the objectives for each task can be described as follows:
1. In this project, different types of mammalian cells were grown in culture and the EVs produced by these cells were isolated. The isolation was undertaken by differential centrifugation, a well- established standard technique. EVs were characterised physico-chemically for size, shape, morphology, and long term stability. Such analysis helped to determine differences between vesicles from different cells which will be highly beneficial for developing them as drug carriers. The identification of a characteristic “fingerprint” of the different cell-derived vesicles will add significant knowledge to the research community in this relatively new field and will open potential new applications. The floating (buoyant) density of EVs was determined after sucrose gradient centrifugation, a well-established method. A biochemical analysis of EVs’ surface markers was conducted using flow cytometry after labelling of EVs with fluorochrome-labelled antibodies. Buoyant density and surface markers are an important measure to distinguish EV populations and provide important knowledge for their future development as clever drug system.
2. The next challenge was to incorporate a drug into the inside of these EV without destroying them.
Various methods have been assessed during drug loading of EVs: passive incubation of drug was reported in the literature and was found to work well for encapsulation of drugs into EVs, in particular for hydrophobic compounds. We have tested different additional methods to cross that barrier and two of them have proven to be very efficient. The first one was using an electric pulse (e.g. a brief electric shock) to open up the wall/shell of EVs for a very short period so that drugs could enter. The other technique was employing a surfactant to solubilise the EVs’ membranes just enough so that the drug could enter but not so much that the whole EV was destroyed. Both methods (electric pulse and surfactant) were very efficient at loading the drug into the EVs.
3. and 4. The drug that was used in these experiments had a special property: when inside the cell and light is shone on the cell, then the drug can kill the cell. In order to analyse whether the EVs are a suitable envelope and whether they can bring that drug inside cells, cancer cells were safely grown in a small plate and the drug loaded EVs or free (i.e. non-encapsulated) drug were added to the plate. We applied high-content imaging to assess the global EV uptake in a large number of cells. Moreover, this imaging technique could be performed in real time and using live cells. When the cells were irradiated with light, we noticed that only those cells that had been previously incubated with the drug-containing EVs, but not those with free drug, died. Microscopy enabled us to look at these cells, and we saw that the EVs were highly efficient to bring their cargo (i.e. the drug) into the cells.
Indeed they were much more efficient than other artificial carriers such as liposomes that we used for comparison.
This work has already cumulated in a manuscript by G. Fuhrmann, A. Serio, M. Mazo, R. Nair, M.M. Stevens “Active loading into extracellular vesicles significantly improves the cellular uptake and photodynamic effect of porphyrins” Journal of Controlled Release. 2015. 205: 35-44. The Journal of Controlled Release is one of the most important journals in pharmaceutics, biomaterials, and drug delivery. Our manuscript showcases the first comprehensive assessment of various loading techniques to incorporate model drugs of different hydrophobicity into EVs. Active loading methods substantially augmented the drug encapsulation into EVs and significantly improved cellular uptake and therapeutic efficiency compared to free or liposomal drugs. We also published two other articles based on this work (G. Fuhrmann, I.K. Herrmann, M.M. Stevens “Cell-derived vesicles for drug therapy and diagnostics: opportunities and challenges” Nano Today. 2015. 10: 397-409; L. Santos, G. Fuhrmann, M. Juenet, N. Amdursky, C. Horejs, P. Campagnolo, M.M. Stevens “Extracellular stiffness modulates the expression of functional proteins and growth factors in endothelial cells” Advanced Healthcare Materials. 2015. 4: 2056-2063.) Our results are the first step in developing a smart system that brings the drugs where they are most needed: the diseased part of the body. The use of EVs is a promising approach for the future that may prove fruitful for the healthcare system, which will be gauged by conducting intensive research within this area. The development of novel, selective and biocompatible delivery systems will hugely strengthen the biomedical sector in Europe; its potential translation will impact on cost and outcome in healthcare services and will thus be a strong benefit for the society in general. This Marie Sklodowska-Curie Fellowship was extremely successful, leading to our better understanding of extracellular vesicle-based drug delivery systems and resulted in 3 high quality manuscripts and a further 3 manuscripts in preparation.
There is no issue on management or project planning; experimental procedures and training activities are planned at least 1-2 months in advance. There is no change on legal status. Planned milestones and deliverables of the project are not affected; in fact, improvements to the concept of the project are implemented. Project website is currently under construction. There is no gender or ethical issues. There is no subcontracting. Regarding management costs, as previously described in part 1, there is no deviation between actual and planned researcher-months.
1. Detailed physicochemical and biochemical characterisation of EVs from various cell sources
2. Introduction of novel loading methods to encapsulate drugs of different hydrophobicities into EVs
3. Detailed analysis of the uptake of porphyrin loaded EVs using high-content imaging using in vitro cell models
4. Improved cellular delivery of small molecule drugs (photoactivatable porphyrins) compared to existing carrier systems, such as liposomes
A detailed progress towards the objectives for each task can be described as follows:
1. In this project, different types of mammalian cells were grown in culture and the EVs produced by these cells were isolated. The isolation was undertaken by differential centrifugation, a well- established standard technique. EVs were characterised physico-chemically for size, shape, morphology, and long term stability. Such analysis helped to determine differences between vesicles from different cells which will be highly beneficial for developing them as drug carriers. The identification of a characteristic “fingerprint” of the different cell-derived vesicles will add significant knowledge to the research community in this relatively new field and will open potential new applications. The floating (buoyant) density of EVs was determined after sucrose gradient centrifugation, a well-established method. A biochemical analysis of EVs’ surface markers was conducted using flow cytometry after labelling of EVs with fluorochrome-labelled antibodies. Buoyant density and surface markers are an important measure to distinguish EV populations and provide important knowledge for their future development as clever drug system.
2. The next challenge was to incorporate a drug into the inside of these EV without destroying them.
Various methods have been assessed during drug loading of EVs: passive incubation of drug was reported in the literature and was found to work well for encapsulation of drugs into EVs, in particular for hydrophobic compounds. We have tested different additional methods to cross that barrier and two of them have proven to be very efficient. The first one was using an electric pulse (e.g. a brief electric shock) to open up the wall/shell of EVs for a very short period so that drugs could enter. The other technique was employing a surfactant to solubilise the EVs’ membranes just enough so that the drug could enter but not so much that the whole EV was destroyed. Both methods (electric pulse and surfactant) were very efficient at loading the drug into the EVs.
3. and 4. The drug that was used in these experiments had a special property: when inside the cell and light is shone on the cell, then the drug can kill the cell. In order to analyse whether the EVs are a suitable envelope and whether they can bring that drug inside cells, cancer cells were safely grown in a small plate and the drug loaded EVs or free (i.e. non-encapsulated) drug were added to the plate. We applied high-content imaging to assess the global EV uptake in a large number of cells. Moreover, this imaging technique could be performed in real time and using live cells. When the cells were irradiated with light, we noticed that only those cells that had been previously incubated with the drug-containing EVs, but not those with free drug, died. Microscopy enabled us to look at these cells, and we saw that the EVs were highly efficient to bring their cargo (i.e. the drug) into the cells.
Indeed they were much more efficient than other artificial carriers such as liposomes that we used for comparison.
This work has already cumulated in a manuscript by G. Fuhrmann, A. Serio, M. Mazo, R. Nair, M.M. Stevens “Active loading into extracellular vesicles significantly improves the cellular uptake and photodynamic effect of porphyrins” Journal of Controlled Release. 2015. 205: 35-44. The Journal of Controlled Release is one of the most important journals in pharmaceutics, biomaterials, and drug delivery. Our manuscript showcases the first comprehensive assessment of various loading techniques to incorporate model drugs of different hydrophobicity into EVs. Active loading methods substantially augmented the drug encapsulation into EVs and significantly improved cellular uptake and therapeutic efficiency compared to free or liposomal drugs. We also published two other articles based on this work (G. Fuhrmann, I.K. Herrmann, M.M. Stevens “Cell-derived vesicles for drug therapy and diagnostics: opportunities and challenges” Nano Today. 2015. 10: 397-409; L. Santos, G. Fuhrmann, M. Juenet, N. Amdursky, C. Horejs, P. Campagnolo, M.M. Stevens “Extracellular stiffness modulates the expression of functional proteins and growth factors in endothelial cells” Advanced Healthcare Materials. 2015. 4: 2056-2063.) Our results are the first step in developing a smart system that brings the drugs where they are most needed: the diseased part of the body. The use of EVs is a promising approach for the future that may prove fruitful for the healthcare system, which will be gauged by conducting intensive research within this area. The development of novel, selective and biocompatible delivery systems will hugely strengthen the biomedical sector in Europe; its potential translation will impact on cost and outcome in healthcare services and will thus be a strong benefit for the society in general. This Marie Sklodowska-Curie Fellowship was extremely successful, leading to our better understanding of extracellular vesicle-based drug delivery systems and resulted in 3 high quality manuscripts and a further 3 manuscripts in preparation.
There is no issue on management or project planning; experimental procedures and training activities are planned at least 1-2 months in advance. There is no change on legal status. Planned milestones and deliverables of the project are not affected; in fact, improvements to the concept of the project are implemented. Project website is currently under construction. There is no gender or ethical issues. There is no subcontracting. Regarding management costs, as previously described in part 1, there is no deviation between actual and planned researcher-months.