The more we know about pathophysiological processes in the human body, the more points of attack we can identify in the fight against diseases. Having discovered the vesicle-based intercellular communication system, we now have new options for the targeted transport of molecular information and drugs over great distances in the human body.

Key biological concepts

Vesicles are a transport system that has been developed in the course of evolution and can be found in all forms of life, cells and tissues. We can find them within cells, in the cytoplasm and in the extracellular space. Depending on their specific type and function, vesicles have a diameter ranging from less than 50 up to 1,000 nanometres. They can thus only be seen under high-resolution microscopes (see figures). Their name “nano”vesicles is also derived from their size.

The formation of vesicular structures creates separate compartments within a cell, so that vesicles can shield their content from the surrounding environment. They are covered by a double lipid layer similar to other membrane structures like the external cell membrane or numerous intracellular organelles. The vesicles’ coat protects the molecular cargo inside them during transport. At the same time, other membrane components which are an integral part of the coat are exposed on the surface and allow for interactions with the surrounding environment, for example, with receptors.

Under certain conditions, vesicles can fuse with other membrane structures. This is important for intracellular transport, for example when membrane proteins are transported from the place where they are produced inside a cell to the external cell membrane, or vice versa.

The exchange of information at the synapses of neurons is a well-known example of the role of vesicles in intercellular communication. Vesicles loaded with neurotransmitters are stored at the synapses. As soon as an electrical signal is received there, these vesicles fuse with the cell membrane in the blink of an eye, secreting the neurotransmitters in this process and transmitting the signal to the next cell. The release of hormones is another example of the secretory function of vesicles.

The diversity of vesicles

In 1967, extracellular vesicles were described for the first time. Back then, it was discovered that blood platelets (thrombocytes) secrete minute particles which were initially termed “platelet dust” or “cellular dust” and considered not to have any function. It took decades until the effect of these vesicles on the immune system was discovered and they were identified as facilitators of the intercellular exchange of information, making it clear that they played a significant role within the human organism and held high potential for biomedical applications.

Extracellular vesicles that are actively and continuously secreted can be specifically loaded with different macromolecular cargos, including ribonucleic acids (RNAs), proteins, lipids and other bioactive molecules, and transport them. As soon as extracellular vesicles reach their target cell, they are absorbed by this cell and release their cargo inside it. Depending on the type of message, they may initiate reactions in the receiving cell, affect its function or control its development.

Cells can also use the compartments isolated by vesicles, so that cellular processes can take place separately within them. There are special vesicles, the so-called lysosomes, in which cell components that are no longer needed are digested or lysed in an acid milieu with a pH of 4.5 to 5. Peroxisomes digest lipids and metabolise alcohol. In the process of programmed cell death (apoptosis), vesicles with particularly large diameters of up to 500 nanometres pinch off from the cells. These apoptotic bodies fragment the cell and have surface markers which signal to the immune system’s phagocytes that they have to remove them.

Biomedical potential

Today, vesicles are studied in connection with various applications. First of all, liposomes, which are very simple vesicles, are used as models for drug envelopes which have already been approved for years and are used in anaesthetics and to treat infections and different types of cancer such as leukaemia or breast cancer.

Another interesting discovery is the role that vesicles play in the extracellular space, where they serve as communication hubs and can, for example, secrete cytokines, i.e., signal proteins. Another line of research addresses the use of natural extracellular vesicles as transport systems and ways of leveraging their inherent effect on the immune system for therapeutic purposes. Generally speaking, vesicles perform a previously unforeseen range of mediating functions within the human organism. We have only just started to understand them, but research promises to open up great new potential for precision medicine.

What is the role of precision in medicine and what are the approaches of precision research?

Precision means that we can tailor drugs to individual needs, which allows us to provide the right drug in the right mode of administration and application. The most pressing issue in the process of drug development is that many promising substances eventually fail, because they are not well absorbed or are spread too widely in the body. They are not transported to the desired location in sufficient amounts.

That’s why, on the one hand, we try to improve the targeting of existing drugs. On the other hand, precision medicine is about enabling the use of new classes of drugs, such as RNA-based therapeutics. This includes vaccines based on messenger-RNA (mRNA), for example, like the COVID-19 vaccine. These drugs are not well absorbed in the human body, or they are rapidly degraded. In cases like this, nanovesicles allow for efficient packaging of the drugs.

Can you briefly take us through the evolution of nanovesicles as a research field?

In the beginning, vesicles were regarded as the cellular waste disposal service. Their active role as part of the immune system was only recognised in the 1980s. Since then, interest in this field of research has steadily increased. The actual breakthrough was then achieved between 2007 and 2009, when two important flagship research papers showed that vesicles are absorbed by cells and can release their cargo within target cells. This made it very clear that vesicles are part of a fundamentally important communication system.

What does the future hold for nanovesicle research?

My vision is to develop a comprehensive tool box which enables us to package novel drugs in vesicles, and to find the right vesicles for the right applications. We are particularly interested in vesicles from well-tolerated and readily available sources. For example, we’re working on obtaining vesicles from natural foods or by-products, such as whey. Together with one of our partners from the biotech industry, we cooperate with dairy factories producing Grana Padano and large quantities of whey that we can use to obtain vesicles.

How do you then get the active agents into the isolated vesicles?

This is one of the greatest challenges. We are testing different approaches. One option is to chemically modify drugs so that they can pass through the membrane into a vesicle. Producing hybrid vesicles is another promising option. In this case, the drug is packed into a synthetic vesicle which is then fused with a natural vesicle.

How does the LBI for Nanovesicular Precision Medicine contribute to this field of research?

Many questions in the field of nanovesicles still remain unanswered, in particular when it comes to translating the results of basic research into practical applications. At the Institute, we can bring together academic research and certain aspects of industrial development.

On the one hand, we have the necessary time horizon for planning; on the other hand, the entire research group can work on finding answers to one and the same question, which is often impossible in an academic setting. Our interdisciplinary team includes experts from various fields such as physics, chemistry, molecular biology and pharmacology. We also work together with clinicians, tech companies and psychologists, who carry out structured surveys for us. This kind of cooperation involving many different experts enables us to solve questions which research is currently still failing to answer.

Where do you see your field of research in ten years’ time?

In just ten years’ time, we are going to have the first clinical data collected by the Research Program Line on “Native Nanovesicular Therapeutics” and concrete clinical candidates identified by the Research Program Line on “Drug Shuttles”. We will then be able to focus on commercial development; our work will certainly not be finished in ten years.

What do nanovesicles look like and what makes them special?

Nanovesicles are not empty vessels but contain bioactive molecules. They can be used for communication and help cells to recognise the tissue surrounding them. We study vesicles with a diameter of approx. 100 nanometres. They are quite hard, like squash balls, and covered by a double membrane which comprises different surface markers and receptors and protects the soluble cargo inside the vesicles.

Why are nanovesicles so promising for therapeutic applications?

For a long time, we placed our hopes in stem cell therapy to enable us to restore damaged or destroyed tissue structures. We found that vesicles secreted by stem cells also have positive regenerative effects. So we are now studying the characteristics of vesicles from cells of the umbilical cord. Mothers giving birth at University Hospital Salzburg have donated umbilical cord tissue, which can then be used to produce vesicular therapeutics. We use the vesicles isolated from umbilical cord tissue without any chemical or genetic modification. A different line of research studies nanovesicles loaded with specific drugs.

How do nanovesicles facilitate the regeneration of tissues?

The greatest problem is that scar tissue is formed faster than injured tissue is repaired or regenerated. Take injuries of the spinal cord, for example. If we do not intervene within 72 hours, a scar will have formed, which will then interfere with the healing process. We also see this in connection with implants such as hip joints, cochlear implants or pacemakers.

Our tests have shown that vesicles from stem cells from umbilical cord blood reduce the formation of scars. In tests with sheep, we were able to show that a single injection of vesicles can almost completely prevent scar formation after a traumatic tendon injury by modulating individual inflammatory factors in a targeted way instead of suppressing all of them. We have not yet understood the exact mechanism, but we think that adenosine, a signal molecule that activates inflammatory cells, may play an essential role.

Are there any clinical success stories you can share with us?

We have successfully cooperated with Prof. Matthias Krause and his team from the department of paediatric neurosurgery at the Landeskliniken Salzburg to treat two children with spina bifida, which is a “split spine”. This deformity is normally treated by surgery to push the spinal cord back into the body. However, scar formation interferes with the children’s further development. The two children have both made good progress after the treatment. Nevertheless, these are just individual cases of expanded access. The next step would be to run clinical trials. We can only follow up on the results if they can be reproduced in 100 per cent of cases.

Why is it important to involve many different fields right from the beginning?

My task is to build bridges. I want to communicate the benefits of the biomedical products we study to authorities, medical doctors and patients, for example. At the same time, we have to comply with the legal provisions to be able to successfully develop new products.

It is important to involve experts from various fields because safety plays such an important role in clinical research. Pharmaceutical products have to be produced according to the strict rules of Good Manufacturing Practice (GMP). Basic research often fails to take account of these rules from the outset, even though they are crucial for the practical implementation of the results. Our first product is currently going through the clinical trial approval process. This is a really important milestone!

What about the general public?

We endeavour to involve those affected right from the outset. One patient, for example, a healthcare professional herself who had participated in an expanded access programme, expressed an interest in getting involved at the Institute. That’s a perfect example of “Open Innovation in Science”. We involve people from many different fields in our research planning. This helps us to improve our research work and achieve our goals faster.

How long will it take until your research results can be applied in practice?

We have made good progress so far. Our greatest asset with nanovesicles is that we already have a product that we can use. Using them as “drug shuttles” is still challenging, though. There are still many fundamental questions to be answered concerning their absorption and distribution in the human body, as well as safety aspects such as toxicity and mother-to-child transmission.

Where do you see the greatest challenges?

Acceptance in society is definitely a huge challenge. The COVID-19 crisis has fuelled growing scepticism towards science, in spite of, or perhaps even because of, the increased communication on the topic of “clinical research”. Our goal is to build trust in science and research, especially among the affected patients. I think that a German documentary for kids, “Die Sendung mit der Maus” (The Show with the Mouse), which conveys information to children in a simple and straightforward way, could be a model for explaining complex and scientific topics in a way that is easy to understand and builds trust.