The ‘Regenerative Medicine I’ research team at the Reutlingen-based NMI Natural and Medical Sciences Institute at the University of Tübingen (NMI Reutlingen) develops technologies that enable the clinical application of siRNA for the regeneration of tissue. By interfering with complementary mRNA, siRNA, i.e. small, interfering RNA molecules, can prevent the process of translation. While the genetic information stored in the DNA is still transcribed into mRNA, it is eventually blocked and destroyed, with the result that the message is lost and the cellular protein synthesis machinery is no longer able to produce a protein. Researchers at the NMI Reutlingen are aiming to use siRNA for turning off proteins that prevent the application of neuroregenerative therapies as well as preventing fibrotic connective tissue alterations from occurring at sites where implants have been inserted. 

Injured peripheral nerves can – at least in principle – grow again. The ‘only’ thing they require to do this is the release of the growth brake that the body forms during early human development in the form of inhibitory proteins. These inhibitory proteins stop nerves from growing any more than they need to. The use of siRNA to counteract the inhibition of cells at the site of injury would create an important prerequisite for the medically controlled regeneration of nerves. However, one thing still needs to be taken care of: the severed nerves must grow in a specific direction and towards a specific target. Only then are they able to establish the lost connection. Researchers at the NMI Reutlingen are testing nerve guidance conduits and tubular implants for the potential to guide growing nerves to their particular targets. Their work within a large-scale EU project gives the NMI researchers and their partners the perfect opportunity to be able to couple siRNA nanoparticles to implants, thereby providing the implants with the means to release the growth brake.

Dr. Hanna Hartmann, who works in an NMI team led by Prof. Dr. Burkhard Schlosshauer and coordinates the NMI researchers’ work, explains the concept: “We are specifically focused on the protein RhoA, which inhibits the growth of axons. We take several commercially available siRNAs that are complementary to the mRNA of the protein and use cell cultures to identify the sequences that lead to a reduction in the amount of RhoA.” Two years into the three-year project, the researchers have identified sequences that lead to the outgrowth of neurites. They will now package the siRNA into nanoparticles in a way that enables the delayed release of the former. The nanoparticles must not release their cargo too soon as the neurons first need to grow towards the siRNA, which is quite a long process. The EU project involves several partners: the coupling of siRNA to nanoparticles is done in cooperation with Danish nanoparticle experts and French pharmacologists; suitable packaging polymers are developed by the Baden-Württemberg institute ITV Denkendorf; and Swedish neurosurgeons provide the know-how required for future clinical application.

The partners work hand in hand: the French partners encapsulated the siRNA into nanoparticles and they were sent to the NMI where the function of the particles is currently being tested in cell cultures. Following the satisfactory conclusion of these experiments, the nanoparticles will be sent to Sweden and tested in animals. “We are developing specific assays for this project that not only provide us with information on whether the cells survive, but also on functional effects,” said Hartmann. These functional effects depend on how well the RNA is able to enter cells. Key in this process is the electrical charge of the nanoparticles, which are between 150 and 200 nanometres in size. The electrical charge can be controlled by the ratio between the ‘packaging material’ (in this case chitosan) and siRNA. Chitosan is produced from chitin and has a positive charge. The siRNA is negatively charged, which is why it is normally unable to penetrate hydrophobic barriers (e.g. cell membranes).

The properties of chitosan can be altered by chemical modification. The NMI has set up an in-house tandem project with the goal of using chemically modified chitosan to optimise the nanoparticles used. “In cooperation with the institute’s biomaterials specialists led by Dr. Simona Margutti, we alter the degree of chitosan acetylation, characterise the charge of the particles and test whether this has an effect on transfection,” said Hartmann explaining that specific connective tissue cells (i.e. fibroblasts) are used for these tests. This is why the team of researchers can use the results from this project directly in another project dealing with the prevention of fibroses.

Fibrosis is the formation of excess connective tissue in a reparative or reactive process (e.g. a reaction to implants). This process can lead to the undesired encapsulation of implants, especially subcutaneous ones. Fibrotic tissue can also disturb the function of drug-releasing and sensory implants. “We have plans to use siRNA in order to reduce the synthesis of collagen in the fibroblasts,” said Hartmann. Fibroses may of course also develop with nerve guidance conduits, which could be prevented from using nanoparticles coupled with siRNA that impairs collagen production. Another option is to couple two types of nanoparticles to the implant – one type that impairs growth-inhibiting processes and another that prevents fibroses from occurring. Hartmann finds both options rather interesting. Future research will reveal the potential advantages of combining siRNA with nanoparticles.

A general problem is related to how the nanoparticles can be securely attached to the implant. The NMI researchers and their partners had an idea that was both simple and smart: they added anti-freezing sugar in order to lyophilise the nanoparticles in the presence of the implant material at -80°C. Vacuum freeze-drying improves the adhesion of the particles to the implant material. The method has already been successfully tested with nerve guidance conduits developed in cooperation with the ITV Denkendorf. The tests will now be expanded to other materials in the hope that the researchers will be able to develop a suitable tool for many implant purposes. The NMI hopes to be able to do this in cooperation with industry partners. “We would like to establish contact with further implant manufacturers and specifically elucidate the reaction to foreign bodies,” said Hartmann.

Further information:
NMI Natural and Medical Sciences Institute at the University of Tübingen
Dr. Hanna Hartmann
Markwiesenstraße 55

72770 Reutlingen
Tel.: +49 (0)7121/ 51 530 - 872
E-mail: hanna.hartmann(at)nmi.de