|“Lead Agency” N2-0027 (2015-2017)|
|Austrian partner:||Prof. Eva-Kathrin Sinner, Institute for Synthetic Bioarchitectures, University of Natural Resources and Life Sciences, Vienna, Austria|
|Slovenian partner:||Prof. Damijan Miklavcic, Faculty of Electrical Eng., University of Ljubljana|
|Funding:||Austrian Science Fund (FWF), Austria and Slovenian Research Agency (ARRS), Slovenia|
Research project co-funded by the Slovenian Research Agency.
- Member of University of Ljubljana
University of Ljubljana, Faculty of Electrical Engineering
- Electroporation as method for inserting functional membrane proteins in mammalian cells without the use of genetic manipulation
- 1.1.2015 – 31.12.2017
- Amount of financing
- 1,62 FTE
- Research activity
- 2.06.07 Engineering sciences and technologies / Systems and cybernetics / Biomedical technics
- Research Organisation
Gene transfection of a cell is a commonly employed tool of cell biology, which allows the cell to produce a foreign protein. The result is that the cell acquires a new function, as performed by the protein, and is described as a gain-of-function. If the genetic material integrates into the host genome, it becomes stably expressed, whereas on the opposite, the cell produces the protein only as long as the genetic material remains intact inside the cell. In either case, the foreign gene is typically controlled by a powerful promoter that ensures a high yield of protein production by the cell. This incurs a metabolic load on the cells since they will be driven to divert resources into production of the foreign protein. The result is poor health or even death of cells, if the metabolic load becomes overwhelming. Additional challenge in gene transfection is also its uncontrollability, which is also a known issue in therapeutic strategies based on gene delivery (gene therapies).
An attractive alternative to gene transfection is to introduce the desired protein, readymade, directly into the cells. This effectively circumvents the need for the cells to spend energy to produce target proteins and, moreover, to “understand” the coding genetic information correctly. Various physical methods have been developed to do this, including microinjection, osmotic shock, and trypsinization. However, this problem becomes complex when the protein in question is a membrane protein. Membrane protein functions tend to be highly delicate and finely tuned and their failure or absence is linked to a plethora of human disease, including diabetes, Alzheimer’s disease and Parkinson’s disease. Cancer cell proliferation and metastasis are also related to membrane proteins and those mechanisms that regulate their function. Not surprisingly, half of available pharmaceutical products target membrane proteins.
An important and interesting challenge, therefore, is how to introduce membrane proteins into the cell membrane without using gene transfection.
We propose to develop a method of delivering membrane proteins to the cell membrane using electroporation instead of genetic manipulation. This method will combine and exploit three powerful technologies currently used in the field of synthetic biology: (i) in vitro protein synthesis; (ii) the use of polymers in creating artificial membranes as scaffolds for protein folding and integration; (iii) the use of electroporation to fuse artificial membranes with cell membranes. Our aim is to tailor functional membrane proteins, immersed into membrane matrices, and deliver them by membrane fusion into living cells, acting as molecular, intracellular implants.
For synthesis of such “integrable” membrane protein implants, our partners from Vienna have developed a novel strategy, namely the cell-free synthesis of membrane proteins in membrane architectures; phospholipid vesicles as well as vesicles made from synthetic amphiphilic block-copolymers (polymersomes). The use of well-characterized polymers is preferred, since it also allows tuning of the polymersome physical and chemical properties over a wide range. In the presence of a cell-lysate with the necessary ribosomes/chaperones/building and regulating blocks, the respective DNA, coding for the membrane protein of interest, is spontaneously transcribed and finally translated into the membrane protein of interest – to be found in the resulting membrane architecture.
Membrane fusion by means of electroporation is also an established method, where short high-voltage electric pulses are used to transiently increase membrane permeability. Increased membrane permeability is furthermore correlated with membrane fusogenicity, which makes it possible to use electroporation for artificially inducing fusion between cell membranes, but also other artificial bilayered membranes. To achieve successful fusion while avoiding excessive membrane damage leading to cell death, the number, duration, and amplitude of electric pulses need to appropriately chosen. We recently employed nanosecond-duration electric pulses, which can only target the membrane areas relevant for fusion. This provides the possibility to efficiently fuse cells and vesicles with different size, which is an important advantage for our proposed method.
Development of our proposed method will involve establishing protocols for electroporation (electrofusion), then tracing and understanding the fate of membrane proteins delivered to cell membranes. Once the critical parameters have been identified and the method established, we will deliver the dopamine receptor D2L (DRD2L) to the membrane of P19 (embryonal mouse teratocarcinoma) cells. We will then ascertain by conventional cell biological parameters if differentiation of P19 toward a neurogenic lineage results from this process. In addition, the DRD2L will be tagged with the Vesicular Stomatitis Virus (VSV) glycoprotein. This will allow cells that have been modified successfully, to be purified using fluorescent antibody staining and fluorescence-activated cell sorting.
- The phases of the project and their realization
Task 1: Preparation of different polymersomes (PS) for fusion with cells
In the first task, we have set two research hypotheses: 1. We can prepare different polymersomes, vesicles from amphiphilic block copolymers, to detect electroporation and fusion, and 2. Polymerosomes (PS) can be porated with nanosecond electric pulses.
In our and partner laboratories we optimized the preparation of PS (large and giant vesicles) from basic polymer units of different lengths (diblock copolymers polybutadiene-polyethylene oxide), because the structure of the PS affects their stability and poration. Different methods of vesicle electroformation were used (with electrodes on the slides, on slides covered with a conductive substance ITO – indium-tin oxide). Polymersomes were labeled with different fluorescence markers for observation under a fluorescence microscope: NBD, lysamine rhodamin, DNS or Di-8-ANEPPS were incorporated into the vesicle membrane to determine cell fusion, and some PSs were filled with calcein to observe the shape and poration of vesicles exposed to electric field. By observing the release of the calcein from vesicles under the fluorescence microscope, it has been found that a PS can be porated with nanosecond electric pulses (nsEP), the advantage of which is to allow the fusion of cells and vesicles that vary considerably in size. The conclusion that PS can be porated with nsEP is the first step in achieving fusion of PS and cells, as two conditions must be met to fuse cells with PS: 1. cells and PS must be in contact, and 2. cells and PS must be porated at the point of contact. For this reason, we studied the PS poration for different pulse parameters (amplitude, duration and number of pulses), since in order to achieve the fusion of cells and PS, it is necessary to find a cross section of parameters where both cells and PS are porated and the cells must survive (the electroporation must be reversible). We have found that PS, probably due to their stability and rigidity, are porated at higher amplitudes/duration/number of pulses than cells.
We worked closely with the partner on the preparation protocols, and visits enabled us to present and analyze new findings, to constantly identify the essential problems that arise in research, and to find solutions to them by optimizing research methods and planning new experiments. We also met in the framework of the First World Congress on Electroporation in Portorož and the international workshop Electroporation-Based Techniques and Treatments in Ljubljana.
- Batista Napotnik, Tina, Rems, Lea, Bello, Gianluca, Sinner, Eva-Kathrin, Miklavčič, Damijan. Preparing polymersomes and cells for electrofusion. Programme and book of abstracts, 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies incorporating Bioelectrics. Portorož, Slovenia, 2015, 86, COBISS.SI-ID 11147604
- Bello, Gianluca, Batista Napotnik, Tina, Rems, Lea, Huber, Christoph, Küpcü, Seta, Angjeli, Belinda, Tan, Cherng-Wen Darren, Miklavčič, Damijan, Sinner, Eva-Kathrin. Electroporation as method for inserting functional membrane proteins in mammalian cells. Programme and book of abstracts, 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies incorporating Bioelectrics. Portorož, Slovenia, 2015, 63-64, COBISS.SI-ID 11150164
- Bello, Gianluca, Batista Napotnik, Tina, Rems, Lea, Miklavčič, Damijan, Sinner, Eva-Kathrin. Polymeric membranes in the form of giant lamellar vesicles as possible carrier of functional membrane proteins into viable cells via electrofusion. Proceedings of the Electroporation based technologies and treatments: international scientific workshop and postgraduate course. Ljubljana, Slovenia, 2015, 149, COBISS.SI-ID 11218260
Task 2: Fusion of polymerosomes (PS) with cells
In the second task, we have set up a research hypothesis: with nanosecond electric pulses we can achieve the fusion of polymersomes with CHO cells.
Using the Hoechst 33342 fluorescent dye, the effects of the electrical pulses of different parameters on the CHO fusion of the cells between each other were determined and found to be at lower pulses than the PS poration. Nevertheless, we determined the cross section of the pulses, which could lead to the poration of both cells and PS, thus allowing the fusion of cells and PS in close contact. However, we did not succeed.
For the fusion of cells and PS, a close contact between the cells and the PS must be first ensured. We did this with dielectrophoresis, but the cells migrated into in the chains first, and then the PS, so the chains were not mixed, and thus we did not establish enough contacts between the cells and the PS. This could be the reason why the fusion was not achieved. In studying the effects of the parameters of nanosecond electric pulses (nsEP), it was found that the repetition frequency of electric pulses in combination with solutions of different conductivities inside and outside the vesicles affect the shape of polymersomes, similar to that of the sinus electric field on liposomes. At high repetition frequency and more conductive medium inside the PS as compared to outside, we achieved an elongation of the PS in the direction of the electric field, which could further increase the contact of the cells and PS in the chains, thus providing better conditions for the fusion of cells with the PS. Despite the preparation of different PS in collaboration with a partner laboratory (size, length of the basic polymer units, different conductive solutions inside and outside the PS), the presence/absence of serum in the medium, the addition of polyethylene glycol to PS solution did not achieve the fusion of cells and PS. That is why we used another cell line, mouse melanoma cells B16F1, which in the past proved to be more fusogenic than CHO. In doing so, we first investigated if the PS were toxic for this line and found that they were not for the B16F1, nor for the CHO cells. However, also with this line we were unable to provide fusion with the PS.
- Batista Napotnik, Tina, Bello, Gianluca, Sinner, Eva-Kathrin, Miklavčič, Damijan. The effect of nanosecond, high-voltage electric pulses on the shape and permeability of polymersome GUVs. The Journal of Membrane Biology, 2017, 250, 5, 441-453, COBISS.SI-ID 11785300
Since we were unable to fuse cells with PS, we have been in constant contact with our partner research team and we have discussed about other ways of lowering the PS poration threshold and establishing contact between the cells, but we were not successful. This is probably due to the high stability and rigidity of the PS, since the elongated shape due to electric pulses with high pulse repetition frequency does not contribute to a higher rate of PS poration. Due to the use of different methods of detection of electroporation, we also wrote a review article on these methods, which will enable researchers in this field to facilitate the selection of an appropriate method for their specific experiments.
- Batista Napotnik, Tina, Miklavčič, Damijan. In vitro electroporation detection methods: an overview. Bioelectrochemistry, 2018, 120, 166-182, COBISS.SI-ID 11931476
Task 3: Delivery of the dopamine D2L receptor into cell membranes by the fusion of cells with proteopolymersomes
In the third assignment, the following hypothesis was made: by fusing cells with polymersomes with the incorporated protein, a transmembrane protein can be introduced into the cell membrane.
Task 2, to fuse cells with polymersomes (PS) without embedded protein, was not successful. Therefore, we wanted to test whether the cells can be fused with polymersomes that carry a transmembrane protein in their membranes (proteopolymersomes). Proteins in the membrane affect the local environment of the protein in the membrane, which could lead to easier poration and thereby successful fusion of proteopolymersomes with cells. In the partner laboratory, they are engaged in in vitro synthesis of proteins and the insertion of so-formed proteins into vesicles. However, when preparing PS with integrated D2L, they encountered problems with the incorporation of proteins into the PS, their orientation, and the purification of the thus prepared proteopolymersomes. These vesicles were thus not suitable for cell fusion tests.
- Citations for bibliographic records