Periodic Reporting for period 1 - RNAhello (Exosome inspired RNA communication between HiErarchicaL poLymer prOtocells)
Berichtszeitraum: 2021-06-01 bis 2023-05-31
The project sought to mimic biological communication and information transfer in artificial multicellular systems. Development of artificial cell systems has the potential to increase our understanding of cellular processes and biological functions, produce therapeutic technologies for advancement of global healthcare, and give rise to further understanding into how life evolved on earth.
Firstly, the effect of coacervate artificial cell constituents on enzymatic activity was investigated. This project has resulted in a manuscript which is ready to be submitted for publication. Secondly, in order to achieve RNA communication between artificial cells and living cells, a UV responsive polymersome system was developed as a shuttling vehicle. siRNA transfer from coacervates to HELA cells was successfully observed, and further experiments are investigating gene knockdown in living cells, based on this exosome inspired system. The development of protocell based communication with exosome-inspired RNA containing polymersome transfer, is a significant advancement in the artificial cell and systems chemistry field. This second project has also resulted in a manuscript which is almost ready to be submitted for publication.
The project has concluded that RNA transfer between artificial cells and living cells is possible when encapsulating the RNA in UV responsive polymersomes. The results pave the way for artificial cells to be used as therapeutic devices in the future, due to protection of typically unstable RNA molecules inside polymersomes.
Firstly, the effect of coacervate artificial cell constituents on enzymatic activity was investigated. This project has resulted in a manuscript which is ready to be submitted for publication. Secondly, in order to achieve RNA communication between artificial cells and living cells, a UV responsive polymersome system was developed as a shuttling vehicle. siRNA transfer from coacervates to HELA cells was successfully observed, and further experiments are investigating gene knockdown in living cells, based on this exosome inspired system. The development of protocell based communication with exosome-inspired RNA containing polymersome transfer, is a significant advancement in the artificial cell and systems chemistry field. This second project has also resulted in a manuscript which is almost ready to be submitted for publication.
The project has concluded that RNA transfer between artificial cells and living cells is possible when encapsulating the RNA in UV responsive polymersomes. The results pave the way for artificial cells to be used as therapeutic devices in the future, due to protection of typically unstable RNA molecules inside polymersomes.
Part 1: Encapsulation and responsive release of polymersomes from protocells
Components of the previously established protocell system were successfully synthesized, including modified amyloses and membrane forming terpolymer. The amphiphilic polymers for forming the polymersomes were synthesized following a ring opening polymerization of caprolactone and trimethylene carbonate monomers. To increase nucleic acid encapsulation, one solution is the incorporation of imidazole containing groups in the hydrophobic block. To achieve this an activated ester monomer was synthesized, para-pentafluorophenol-trimethylene carbonate (TMC(OPhF5)), which was then polymerized in the same conditions as previously, to give PEG-p(CL-TMC(OPhF5)). The activated ester was successfully modified with imidazolepropylamine. At a 50:50 ratio of PEG-P(CL-TMCimid) and NBenz-PEG-P(CL-TMCimid) in the polymersome formulation, stable polymersomes formed and the surface charge of the polymersomes increased from from negative to positive after UV irradiation. Encapsulation studies with cy5-siRNA in polymersomes showed between 15-20% encapsulation efficiency. Polymersomes were incorporated into the established coacervate system, and were released from coacervates after UV irradiation as followed by confocal microscopy. The supernatant from the release experiment was also analysed by DLS, which also indicated release of nanoparticles by an increasing count rate over time.
Exploitation and dissemination: This work will be published in a journal article, and was presented at CHAINS chemistry conference 2022 and PTN Dutch Polymer Days 2023. A review article was published as part of this work in Accounts of Materials Research: ‘Complex Coacervate Materials as Artificial Cells’ 10.1021/accountsmr.2c00239.
Part 2: exosome-like RNA transfer between protocells and living cells
Initial studies incubating coacervates containing polymersomes with HeLa cells have been performed . Cells were incubated in microscopy slides and coacervates added. The coacervates and cells were irradiated with UV light for 10 min, and then imaged. An increase in intracellular fluorescent signal was seen with the UV responsive polymersomes compared to the non-responsive polymersomes. These results indicate that the UV responsive polymersomes are able to be uptaken into living cells after UV charge switching is triggered. Further studies are being carried out to determine if the RNA remains functional, by repeating the experiments with eGFP-siRNA. These results will be a first example of RNA signalling between artificial cells and living cells. The research article resulting from these studies will be of high impact.
Exploitation and dissemination: This work will be published in a journal article, and was presented at Advanced Materials and Nanotechnology (Rotorua, NZ) and CHAINS chemistry conference 2023.
Components of the previously established protocell system were successfully synthesized, including modified amyloses and membrane forming terpolymer. The amphiphilic polymers for forming the polymersomes were synthesized following a ring opening polymerization of caprolactone and trimethylene carbonate monomers. To increase nucleic acid encapsulation, one solution is the incorporation of imidazole containing groups in the hydrophobic block. To achieve this an activated ester monomer was synthesized, para-pentafluorophenol-trimethylene carbonate (TMC(OPhF5)), which was then polymerized in the same conditions as previously, to give PEG-p(CL-TMC(OPhF5)). The activated ester was successfully modified with imidazolepropylamine. At a 50:50 ratio of PEG-P(CL-TMCimid) and NBenz-PEG-P(CL-TMCimid) in the polymersome formulation, stable polymersomes formed and the surface charge of the polymersomes increased from from negative to positive after UV irradiation. Encapsulation studies with cy5-siRNA in polymersomes showed between 15-20% encapsulation efficiency. Polymersomes were incorporated into the established coacervate system, and were released from coacervates after UV irradiation as followed by confocal microscopy. The supernatant from the release experiment was also analysed by DLS, which also indicated release of nanoparticles by an increasing count rate over time.
Exploitation and dissemination: This work will be published in a journal article, and was presented at CHAINS chemistry conference 2022 and PTN Dutch Polymer Days 2023. A review article was published as part of this work in Accounts of Materials Research: ‘Complex Coacervate Materials as Artificial Cells’ 10.1021/accountsmr.2c00239.
Part 2: exosome-like RNA transfer between protocells and living cells
Initial studies incubating coacervates containing polymersomes with HeLa cells have been performed . Cells were incubated in microscopy slides and coacervates added. The coacervates and cells were irradiated with UV light for 10 min, and then imaged. An increase in intracellular fluorescent signal was seen with the UV responsive polymersomes compared to the non-responsive polymersomes. These results indicate that the UV responsive polymersomes are able to be uptaken into living cells after UV charge switching is triggered. Further studies are being carried out to determine if the RNA remains functional, by repeating the experiments with eGFP-siRNA. These results will be a first example of RNA signalling between artificial cells and living cells. The research article resulting from these studies will be of high impact.
Exploitation and dissemination: This work will be published in a journal article, and was presented at Advanced Materials and Nanotechnology (Rotorua, NZ) and CHAINS chemistry conference 2023.
It is anticipated that the project will generate interest and stimulate further research in artificial-cell systems chemistry. In the long term, the project could lead to insights and possible medical technology for gene therapies, and therefore has the potential to have an impact on patients and their quality of life. Thanks to this fellowship I will publish a number of articles in high impact journals, and expand the fields knowledge on how polymersome compartments can be employed in artificial cell research as shuttling vehicles for biomolecular cargo. The project has already lead to the initiation of further research ideas in our department, and as the outcomes of the project become available as open access publications, will inspire further research throughout Europe. In addition the high school educational dissemination project from this MSCA fellowship is available on a dedicated website, and will be published in an education research journal soon.