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Molecular Imprinting mediated regulation of cell behavior and related biomedical applications

Periodic Reporting for period 1 - MIP4CELL (Molecular Imprinting mediated regulation of cell behavior and related biomedical applications)

Reporting period: 2016-03-01 to 2018-02-28

The applications of the research carried out in this project are found in medicine – in tissue engingeering, reproductive medicine and stem cell therapies. This refers to replacement of cells or tissue e.g. in cancer treatment due to loss of healthy cells hit by cytostatic agents or in brain or spinal cord injury. To produce the tissue or cells requires a medium mimicking the native environment around the cell and this is what this project aims to do. Cellular processes are hence crucially dependent on dynamic receptor-ligand interactions occurring at the interface between the cell membrane and the extracellular matrix (ECM) (J. Robertus, W. R. Browne, B. L. Feringa, Chem. Soc. Rev. 2010, 39, 354 – 378.) Changes in these interactions as a consequence of ECM remodeling, give rise to specific cell signaling and intracellular cascades. These processes are central in the physiology and pathological processes like tissue self-repair and tumorigenesis. As mimics of such dynamic interactions, artificial matrices with reversible display of bioactive ligands have attracted much attention. Surfaces capable of modulating cell-biomaterial interactions are commonly exploited for in-situ cell biology experimentation and in tissue engineering. Current methods to control reversible ligand presentation on biomaterial interfaces mainly rely on surface functionalization with reversible linkers (e.g. noncovalent or reversible covalent interactions) to which the bioactive ligand is tethered (Fig.1). For example, by means of host-guest chemistry, reversible covalent chemistry, molecular assembly or other multiple non-covalent interactions, the integrin-targeted cell adhesive peptide RGD (Arg-Gly-Asp) could be dynamically and reversibly immobilized on the biointerfaces to regulate cell adhesion behavior. These approaches towards simulating the reversible ligand presentation in a biological system have greatly promoted the development of dynamic biointerfaces and a new generation of artificial ECM materials. However, to date, only a few reversible linkage chemistries have been exploited and even fewer that can be geometrically adressed.
The overall objective of this project was to develop a new dynamic and geometrically addressable methodology for biofunctionalization and apply it to reversible cell adhesion and tissue engineering.
Topic 1. An epitope imprinted surface for switchable and addressable cell adhesion

We have employed a combined epitope and surface imprinting strategy for introducing the cell adhesive peptide RGD in a fully exposed form. The principle is based on the use of a short peptide (the epitope) as template during the imprinting process, and subsequently to use this peptide sequence as an RGD-tag for anchoring the RGD sequence to the substrate surface (Fig. 2). In this design, the epitope peptide could act as a reversible anchor of the bioactive RGD peptide leaving the latter exposed for interacting with the cell surface integrin receptors. Moreover, addition of an appropriate amount of the epitope peptide to the system would induce a gradual release of the bound RGD-based peptides through competitive molecular exchange i.e. the bioactivity on the material interface can be dynamically as well as spatially controlled.
This strategy was successfully realized. Careful optimization resulted in hydrated polymer films featuring a very high but switchable affinity for the epitope peptide. This switchable property was used in cell adhesive tests on mouse 3T3 fibroblasts. This showed that cell adhesion only occurred on the epitope-RGD modified imprinted surface but that the cells could subsequently be released by addition of the epitope peptide as competitive displacer.

The results of this work has been published in the high impact journal Angewandte Chemie:
Pan, G. et al. An Epitope-Imprinted Biointerface with Dynamic Bioactivity for Modulating Cell–Biomaterial Interactions. Angewandte Chemie International Edition, n/a-n/a, doi:10.1002/anie.201710972.
It was rated as a Very Important Paper (VIP) and highlighted by an inside back cover

Topic 2. Switchable cell adhesion using reversible self assembled monolayers (rSAMs)

As a second approach to introduce dynamic bioactivity we have used reversible self-assembled monolayers (rSAMs). This new surface modification approach utilizes noncovalent amidinium- carboxylate ion pairs for building up stable two-dimensional assemblies, akin to lipid bilayers but with a simple preparation process, enhanced rinsing stability and fast on/off rates (Yeung, S. Y. et al. Reversible Self-Assembled Monolayers (rSAMs): Adaptable Surfaces for Enhanced Multivalent Interactions and Ultrasensitive Virus Detection. ACS Central Science, doi:10.1021/acscentsci.7b00412 (2017)). We have demonstrated the use of mixed rSAMs as a tunable platform for reversible cell adhesion using an RGD peptide. A series of mixed rSAMs was prepared containing varying mole fractions of RGD and evaluated the surfaces for their ability to modulate cell adhesive behaviour. With the incorporation of RGD in the layers, the number of adhered cells increased. Reversibility was demonstrated by competitive displacement with soluble benzamidine amphiphile or RGD. The method is unique with respect to its dynamic character i.e. the rSAM layers display lateral diffusion coefficients in the order of lipid bilayer membranes.
This work is summarized in a manuscript and a patent application:
Yeung et al. Reversible Self Assembled Monolayer as a fluidic platform for stimulated cell adhesion. Manuscript in preparation.
Sellergren, B.; Yeung, S. Y.; Pan, G. Lipid bilayer membrane mimics. Swedish patent application 1830098-8; 2018-03-24
In summary, we introduced two promising approaches to reversible presentation of bioactive ligands and a dynamic control of cell-material interactions. The project has advanced the state of the art in the following ways:

1) By combining epitope and surface imprinting we have shown a unique approach to reversibly introduce bioactive ligands at a synthetic biointerface, displaying high accessibility and enhanced cell adhesion.

2) We have introduced the first addressable cell adhesive surfaces where different cells can be cultured and released on demand in spatially addressable locations by choosing different epitope anchors for RGD or other bioactive ligands.

3) Using a robust lipid-bilayer membrane mimic we have demonstrated the first lipid bilayer like dynamic platform for tunable and reversible cell adhesion.

The development of tools for regenerative medicine and cancer treatment are of fundamental importance to the health care industry. After decades of research, many regenerative medicine technologies are in development and approaching commercialization stage. The huge market, with an estimated US$ 7.2 Billion market size in 2010, will progress at a rapid pace in the coming future and witness the advent of new generation of treatments across the globe. The results of MIP4CELL may impact biomedicine and tissue engineering in a major way. Our approach to dynamic biofunctionalization can change the way cells are grown, offer a new level of control of cell fate, and provide a novel technique for capture and release of cells in a controlled manner. These features are highly desired in the field of regenerative medicine and cancer management.
Fig. 1
Fig. 2