Throughout our project, we had meetings with numerous experts of different scientific fields in order to receive advice on multiple ideas and questions regarding our project. In the finding phase of our project, we consulted several scientists from a variety of research topics, in order to gain many different perspectives on our initial ideas. Dr. Katharina Achazi, Prof. Dr. Gert Fricker (both drug delivery), Prof. Dr. Friedrich Frischknecht (parasitism), Dr. Sadaf Pashapour (microfluidics), Prof. Dr. Dirk Grimm (virology) and Prof. Dr. Stephan Wölfl (molecular cell biology) were the ones who supported us most with valuable feedback regarding our first project idea.
The first version of our project idea did not focus on the herpes simplex virus as our target. Instead, we wanted to produce siRNA against the Tick borne encephalitis virus (TBEV), since Heidelberg is within an area with the highest TBEV rates in Germany. Dr. Achazi pointed out that the TBEV virus can enter the body at any part, usually from a tick bite on the extremities, and highlighted that by the time the virus shows first effects in the brain area, the viral load is already so high that it is very difficult to transport sufficient amounts of siRNA through the nose-to-brain route. Prof. Dr. Grimm suggested considering the SARS-CoV-2 virus as a target since it attacks in the nose and in the lung respiratory system. Those proposals brought us to the idea of targeting the herpes simplex virus, due to its properties of also attacking the mouth and nose area, as well as the fact that no vaccines or other satisfying treatment options currently exist.
Another question was which kind of drug delivery to opt for. While we first thought of an adeno-associated virus transporting our siRNA, encouraged by Prof. Dr. Grimm, we soon realised that the realisation of this method would be very difficult and take a lot of time. But then, Prof. Dr. Fricker recommended using solid lipid nanoparticles or cationic polymers as a delivery method instead, as they are easier to produce and are very flexible in terms of their lipid composition and possible surface modifications. In a later interview with Dr. Ulrich Massing, we realised that in contrast to lipid nanoparticles, liposomes are easier to produce and also more suitable for upscaling, resulting in our final choice of using liposomes as our drug delivery method. Our project presentation and enthusiasm even convinced Prof. Dr. Gert Fricker to support our work by letting us work in his lab and develop the optimal encapsulation for our siRNAs, and he became our second principal investigator of the 2022 iGEM Team Heidelberg besides Prof. Dr. Wölfl.
Since we first considered using a nasal spray to guarantee an easy application, Prof. Dr. Fricker suggested using computational models to aid and guide the development of this device. He for instance proposed to computationally simulate and evaluate different parameters of the sprayed droplets such as their size, liposome concentration and pressure applied in the application device, to create droplets with the highest penetration efficiency of the olfactory epithelium cells. Our drylab team was inspired by this idea and, although we decided to change the way of application during our project, also designed a Molecular Dynamics simulation to model the formation process of liposomes depending on their individual lipid composition, thereby enabling new insights for the liposome wetlab team into their experimental work.
Furthermore, we were interested in going even further and creating lipid nanoparticles (LNPs) using microfluidic chips. Dr. Pashapour kindly gave us a lot of insight into the prerequisites for producing such devices. We discussed how stencils for these chips are created, how PDMS is applied and activated, as well as what kind of equipment we would need to efficiently produce LNPs. Additionally, we received recommendations concerning which chemicals and vendors work best for our project. She even went as far as offering to produce the stencil for our first designs, as we did not have the possibilities to do that on our own.
We were very lucky in being able to get in touch with Prof. Dr. Ulrich Massing, the inventor of the dual centrifuge with which the Fricker Lab produces its liposomes, because we wanted to learn more about this new invention and in which ways it is best applied. We had two online meetings with him, and in both sessions, we were able to gain many new insights into how lipid nanoparticles and liposomes can be produced with this machine, as well as a first feedback on how the development from our lab experiments to a final drug would look like, and how we should adjust our liposome production method accordingly.
In our first meeting, we presented both iGEM itself as well as our project idea. The first thing that Prof. Massing pointed out was the fact that we have to be careful of choosing the N to P ratio high enough (e.g. more positive than negative charges in total) , so that no “zebra pattern” occurs, where a negative RNA-layer is surrounded by a positive lipid layer which is again surrounded by a negative RNA-layer and so on, creating giant lipid nanoparticles with a very low transfection efficiency. We also came to the conclusion that, although both options are possible with the DC, liposomes (fluid-filled) are significantly easier to produce than lipid nanoparticles (core made out of lipid) and still very effective when penetrating into a cell. Another important point he made was the emphasis on clinical applicability, i.e. whether the current laboratory experiments are designed in such a way that the next steps of drug development in the clinical direction can be undertaken or not. Considering this aspect, Prof. Massing also praised our idea of a microfluidics device, as this creates liposomes by injecting lipids dissolved in ethanol into an aqueous solution with the siRNA, which means that no toxic organic solvents are involved, making this method a lot safer compared to any production technique involving a lipid film (such as our current protocol). Furthermore, he stated it fulfils the three most important aspects of medical devices: It is quick, easy to handle, and safe, because it is an entirely sterile system; making the microfluidics device a good candidate for further development.
The second meeting started off with an update of our project, of our recent results and a few questions regarding these. One big question which we were still asking ourselves was the reason behind the negative surface potential of our liposomes, which in theory should be positively charged because the cationic lipids in the liposomal membrane should shield the negative charge of the nucleic acids. There, Prof. Massing suggested that the oligonucleotides may get caught on the surface between the PEG-tails, leading to a net negative surface charge; which would need further research to be confirmed. He also explained to us that we shouldn’t try to make the liposomes more positive through the addition of magnesium ions, as these do not shield the charge of the nucleic acids, but rather connect liposomes to each other, eventually leading to them fusioning and thereby many big liposomes with a low cell penetrating efficiency.
Finally, we also discussed what next steps would need to be taken for a final approved drug based on the molecules that the liposomes contain. Prof. Massing first pointed out that a cytotoxicity assay would definitely be advisable, because cationic lipids always have a possibility of being somewhat cytotoxic, and he also suggested to test the mucosal toxicity, because the nose as the area of application can be very sensitive, especially near the central nervous system. Lastly, we were thinking about how to get the unencapsulated siRNA and maybe even the siRNA stuck to the surface of the liposomes out of the solution without losing too many liposomes. When e.g. passing the solution through a filter, lots of liposomes get permanently stuck and clog the filter after a while. But Prof. Massing stated that as long as no different clinical active profile is observed between the filtered and the unfiltered versions (which is very likely due to unencapsulated siRNA being most likely degraded in the mucus very quickly), this cleanup step is not necessary, meaning that for the final drug, more liposomes can be produced per batch.
To conclude, the different expert interviews gave us many excellent new ideas and valuable information, thereby helping us greatly in finalising our project as well as planning for possible next steps beyond.
To find out about the market viability and the requirements of future users for our microfluidics device,
we conducted an interview with Dr. Sebastian Rupiani, who works as a Research Technology Specialist at Merck KGaA.
Through this interview, we gained valuable information about what future customers will pay attention to and how we
can successfully compete with other suppliers on the market. For instance, Dr. Rupiani sees the greatest strength of
our device in its ease of use, as it does not require trained personnel to use: "Microfluidics is a popular method with
many applications. Few companies have brought solutions for nanoparticle manufacturing to market yet, that's why the need
for a method with such increased usability is currently high." At the same time, he said, few companies have yet developed
and brought to market a device that can reproducibly produce nanoparticles as drug carriers. When asked what later
customers will primarily look for in our device, Dr. Rupiani mentioned to us, on the one hand, fast and reliable
production to avoid supply bottlenecks. In this context, the speed of replacement deliveries could already decide whether
our product can prevail over the competitors.
Concerning the needs of the end user, it was also made clear to us during the interview that the speed and efficiency
of a process alone are not enough for the end user to decide in favor of our device ultimately: "It's not just about
efficiency, but also about other factors such as price, availability, and reproducibility of the results. In any case,
a much more efficient process will obviously be interesting, especially if that significantly reduces the number of
users or person-hours." When asked about large-scale industrial use, Dr. Rupiani advised us that the output volume of
our device is too small for industrial use and that we should therefore focus on research use.
At the end of the interview, we asked for an evaluation of the technical planning of our device. He pointed out that in microfluidic technology, the channels of the device must always remain free so that no precipitation can occur. This requires a suitable cleaning procedure, which must be described in the instructions for use. We were also advised not to make our system too complicated technically, as this is always a classic source of errors ("less is more").
Through the interview with Dr. Sebastiano Rupiani, we received the feedback that our microfluidics device has the potential to prevail on the market against other products and suppliers. He has already told us some of the requirements that future end users will place particular emphasis on and that we will, therefore, definitely take into account in the future. At the same time, the interview also gave us suggestions on how we can further develop our device on a technical level to ensure that it functions smoothly. After the interview, the section on proper cleaning of the device was expanded in the preliminary instructions for use of our microfluidics device with information on suitable solvent, flow rate and duration of the cleaning process.
During our project, we discovered a newly published paper in March 2022 which described a similar approach to ours to treat herpes: “Liposomal siRNA Formulations for the Treatment of herpes simplex virus-1: In Vitro Characterization of Physicochemical Properties and Activity, and In Vivo Biodistribution and Toxicity Studies”. They had also used siRNA encapsulated in liposomes to cancel the gene expression of an HSV protein. However, they used IP and IV injections on mice which led to accumulations of the liposomes in the visceral organs, instead of using a nose spray as we had planned. We immediately scheduled a meeting with the PIs of the involved groups, Prof. Golomb and Prof. Rupp, to receive their perspective and new insights into our project.
Until our discussion with Golomb and Rupp, we had planned on targeting the olfactory nerves in the nose with a nasal spray. The professors advised us against using a nose spray, as the dosage is difficult to control, resulting in most of the solution ending up in the lungs and in the stomach instead of the brain. After further consideration and an interview with the doctor Prof. Haefeli, we adapted the application to an aerosol applicator introduced as two sondes into each nostril. This approach facilitates dosage and droplet size control to optimize the uptake of the drug into the nerve cells. (Read more in Interview with Haefeli & Proposed Implementation) Furthermore, they drew our attention to the trigeminal ganglia as another target path, as these are the hiding places of latent Herpes simplex viruses (HSV) after an infection. Thus, we expanded our target to both olfactory neurons and trigeminal ganglia.