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Hyaluronan-rich matrices crosslinked with collagen-like peptides for 3D culture of ovarian cancer cells

Periodic Reporting for period 1 - MHAtriCell (Hyaluronan-rich matrices crosslinked with collagen-like peptides for 3D culture of ovarian cancer cells)

Reporting period: 2015-06-15 to 2017-06-14

What is the problem/issue being addressed

We have created synthetic biomimetic soft but robust biomaterials without involving complex chemical crosslinking procedures. These biomaterials can be used for a variety of potential applications such as creating 3D in vitro models mimicking the complex microenvironment that persists in vivo and as artificial constructs in tissue engineering. Recent literature reviews are reporting a dramatic decrease in success rate of therapeutics progressing to the clinic due to lack of efficacy in phase 3 trials. Since the efficacy of these drugs are usually tested in preclinical models before progressing toward phase 3 trials, therefore we can reason that data obtained at preclinical stages are not reliable and a wealth of these information are generated using 2D cell culture models. It is well known that cells in 3D display very different morphology and gene expression patterns. These complex model systems are not routinely incorporated in drug discovery regime. Moreover, published reports portraying detailed comparison between 2D and 3D cultures are very few. Another important drawback is the use of cell culture scaffold materials, which are commonly derived from animal proteins such as collagen type I. These proteins often vary in composition that can limit the reproducibility of experimental results. Additionally, the mechanical properties and gelation kinetics of commonly used cell culture support materials are difficult to control. Hence, synthetic peptide or polymeric hydrogels have now paved the way toward fabricating matrices that can provide support to the cells as well as promote specific cell-matrix cross-talk. However, stand-alone peptide hydrogels even though mimic the nanofibrous morphology of cell extracellular matrix (ECM), are less stable in buffers and have poor mechanical properties. Polymeric gels on the other hand, lack the nanoarchitecture of the ECM. We have addressed this important issue by developing a hybrid system of peptide-polymer hydrogels that can provide the nanofibrous architecture with superior mechanical strength.

Why is it important for society

The fundamental beneficial impact that our project can bring is the reduction of overall animal use. Since 3D models can more realistically mimic the 3D in vivo tissue environment, therefore we can eliminate the need of elaborate studies in mouse, rats or rabbits and thus reduce the number of animals to be sacrificed. This in turn can reduce the time and cost of animal studies and thus can be overall beneficial to the pharmaceutical industry. Moreover, careful selection of biological epitopes can also provide a scope for customisation of our formulations for studying specific interactions between cells and the ECM. This will help in understanding the underlying mechanisms of certain deadly diseases such as cancer. Our data indicate that our formulations can be customized to mimic ECM of different tissues in the body.

What are the overall objectives

The overall objectives of the project achieved

• To design synthesize and characterise different classes of peptides
• To study their non-covalent self-assembly behaviour with HA
• Characterise the formulations eg 3D hydrogel, membranes, sacs etc obtained from self-assembly of HA and peptides. Characterisations would include the microstructure of the gels/membranes, rheological properties indicating mechanical stiffness, viscoelastic behaviour and the stability of the formulations
• Contribute toward a suitable application for the developed formulations

During the course of our project, we have fully achieved the first three objectives. We have identified a suitable area for applying our formulations and we are working toward drafting a proposal based on this objective. We will also seek collaboration for completing this part of the objective.
We have designed, synthesized and characterised peptides with novel sequences that can be classified into seven different classes depending on their amino acid composition. We evaluated the secondary structure that the synthesised peptides tend to adopt in aqueous solution or in PBS buffer. Then, we used these peptides to study their self-assembly behaviour when mixed with HA solution. We have used different molecular weight HA, varying temperature, concentration and pH to optimise the best conditions of self-assembly. We have been successfully in obtaining 3D hydrogels and sac-like membranes using our peptide class II. We have characterised the gels and other structures with scanning electron microscope (SEM), stress strain rheological measurements and stability studies in buffer and in presence of an enzyme hyaluronidase, which is known to degrade HA in vivo.
In conclusion, we have identified with de-novo sequences that can form 3D hydrogels by noncovalent supramolecular self-assembly with HA. These polysaccharide-peptide hybrid gels display the nanoarchitecture of tissues, are stable in aqueous buffer, enzymatically degradable and possesses superior mechanical strength. We are in the process of writing a manuscript to disseminate the results achieved.
Although there are many reports of peptide gels in literature, usually they are considerably weak and not robust. We have developed strong robust gels using a peptide-polymer hybrid system. Moreover, our building blocks are carefully chosen. The peptide sequences are very short, easy to synthesize and scale-up. Our system incorporates a natural polymer (HA) as the backbone of the supramolecular gel for increasing mechanical strength. In addition, HA is the natural component of ECM surrounding most tissues and it can be easily obtained through bacterial fermentation. Till today, no literature reports have reported formation of robust and stable hydrogels with ECM-like fibrous architecture from very short peptides via non-covalent interaction with high molecular weight HA.
Depending on how intelligently we apply our formulations, in future it can open up new opportunities of collaboration with industries and a possible patent filing.
Research concept developed by the MHAtriCell project