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Small, round, versatile: cyclic dipeptides in hydrogels

Researchers from the University of Tübingen have discovered a new type of hydrogelators: cyclic peptides consisting of amino acid building blocks that are also found in the human body. Preliminary work has shown that these hydrogels are excellently suited as carriers for medical substances. In addition, mechanically damaged hydrogels have excellent self-healing capacity and are therefore suitable for injection-based drug delivery.

The consistency of the gels depends on the blend of substances. The one shown here is stronger than jelly bears. © Nachtsheim, University of Tübingen

Prof. Dr. Boris J. Nachtsheim originally planned to start a research project on abiogenesis. He wanted to study the chemical evolution of amino acids and small peptides into complex organic systems. “Some of the steps involved in the transformation of amino acids and peptides into organic systems are known from primordial soup experiments. It has also been demonstrated that certain chemicals react to form di- and tripeptides. However, this knowledge alone does not produce functional units. We therefore wanted to join amino acids into higher order structures with some function,” says Nachtsheim. What happened next is a prime example of how university research can unexpectedly lead to new application perspectives. It is also an argument in favour of basic research. 

The initial experiments were relatively simple: Nachtsheim and his team succeeded in producing cyclic dipeptides from the amino acid phenylalanine in combination with other proteinogenic amino acids. They heated an aqueous suspension with dipeptides to a temperature of 50 to 90 degrees until all solids completely dissolved. The peptide-containing solution was subsequently cooled, resulting in a more or less solid gel-like substance. “The cyclic dipeptides self-assemble into higher order structures, which form hydrogels through cross-linking. We had found the smallest peptide units that have ever been described to form higher order structures in aqueous environments,” says Nachtsheim. In chemical nomenclature, cyclic dipeptides are diketopiperazines, DKPs, as Nachtsheim calls them. The researchers then decided to take a closer look at the DKPs that had come to the fore during abiogenesis research. The project was funded by the Baden-Württemberg government as part of a junior professor programme. “We had not done any preliminary investigations and DFG funding would have been difficult to obtain. So we applied for Baden-Württemberg Stiftung funding and were successful,” says Nachtsheim.

In the construction kit: a new class of hydrogelators

The gel’s microstructure can be clearly discerned under the microscope. Left: dipeptide gel consisting of the amino acids phenylalanine and histidine. Right: gel consisting of phenylalanine and serine. © Nachtsheim, University of Tübingen

The research has been funded since 2011 with 100,000 euros over three years, providing Nachtsheim with funds for a doctoral student. The researchers found that their innovative hydrogels were very stable, more stable, in fact, than hydrogels that consisted of more complex molecules. In addition, the researchers were able to produce gels of different strengths by using different DKP blends and concentrations. Nachtsheim compares this with a water colour box. Different shades (gels) and intensities (gel firmness) can be obtained by mixing two colours (amino acids) with water in a specific ratio. The material properties can be fine-tuned by changing the solution’s pH and the type of buffer used. “Acetate or phosphate buffers are used for this purpose because buffer systems such as these are also found in the human body. However, we still need to closely examine the materials’ technical potential,” says Nachtsheim. 

Only proteinogenic amino acids are used – phenylalanine is either combined with serine, cysteine, glutamate, histidine or lysine – which ensures that the resulting hydrogels are biocompatible and suitable for use in the human body. The consistency of the gel determines which medical agents can be incorporated. The drugs are added to the DKP mixture at room temperature. Provided that the drug tolerates heating, the procedure leads to a drug-containing hydrogel that transports the drug to a specific site in the body.

Carrier material for medical agents

Following mechanical destruction (using an oscillating stamp; thixotropy), the hydrogels regenerate within minutes or hours. The regeneration time depends on the gels’ composition. © Nachtsheim, University of Tübingen
Mixing ratio, amino acid concentration and buffers have an impact on the time frame during which the drug is released from the hydrogel. “With an anionic gel, negatively charged protein drugs are released relatively quickly because of the repulsive interactions; a cationic gel releases the drug more slowly,” says Nachtsheim. The researchers were able to show that the anionic properties of the gel depended on the pH and the blend of DKPs used. For example, glutamate has a negative charge in a neutral solution due to its carboxylic acid side chain; the basic amino acids lysine and histidine are cationic in neutral solution, i.e. have a positive charge. The pH also determines where the drug is released, at least when the drug contained in the hydrogel is administered orally. “The hydrogel can also be used as a material for delayed drug release. We are able to control the composition of the gel so that it does not dissolve in the acidic environment of the stomach, but travels further into the intestines where basic conditions prevail,” explains Nachtsheim.

Hydrogels totally regenerate after mechanical destruction

The hydrogels have one more property that makes them highly interesting for pharmaceutical companies as it is a feature that offers completely new drug delivery options: the gels have the capacity to rebuild the cross-links and form functional hydrogels again. Hydrogels that are mechanically destroyed, for example with the oscillating stamp that is used in thixotropy* experiments, tend to form new cross-links within minutes or hours. Their regenerative capacity depends on which DKP blend is selected. “This is exactly what we need in order to be able to inject a gel. The process of injection inevitably leads to the destruction of the gel,” says Nachtsheim.

The best thing about the method is its simplicity. The production of drug-containing hydrogels is based on heating and cooling the desired mixture. It all works without the need for complicated synthesis steps and modifications. It is also possible to attach drugs chemically to DKPs. However, the potential benefits are not yet known and need to be investigated. One idea is to colonise the hydrogels with living cells. Nachtsheim explains: “DKPs are very small and connected to one another by hydrogen bonds. This makes the system relatively flexible. The matrix is elastic and adapts to the growing cells. Traditional biocompatible gels consist of much larger molecules and polymers, which makes the system a lot more rigid.” 

The researchers will now have to investigate how the hydrogels react to living cells and vice versa. In order to do this, Nachtsheim is looking for cooperation partners with biomedical or cell biological know-how. “We can measure what the gels are able to do in terms of the material used. However, we will need to work with biochemists and medical specialists in order to develop the hydrogels into commodities suitable for medical application,” concludes Nachtsheim. 

*Thixotropy refers to the property of materials to change their degree of viscosity. Materials that are viscous under static conditions become less viscous over time when exposed to mechanical impact. They return to their original state when no longer agitated or otherwise stressed. A materials' thixotropic properties can be measured with an oscillating stamp that agitates the material under investigation.

Further information:

University of Tübingen
Institute of Organic Chemistry
Prof. Dr. Boris J. Nachtsheim
Auf der Morgenstelle 18
72076 Tübingen
Tel.: +49 (0)7071 29-72035
E-mail: boris.nachtsheim@uni-tuebingen.de

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/small-round-versatile-cyclic-dipeptides-in-hydrogels