Nanoengineering of the Next Generation of Biological Surfaces

Nanoscience and nanotechnology involve the study, imaging, measuring, modelling, or manipulation of matter at the molecular and nanometre scale. The application of nanotechnology and nanoscience to biological systems - known as bionanotechnology¹ - holds the promise of solving many of today's challenges in healthcare. Dramatic breakthroughs are expected in life sciences research that could contribute to sensitive and early detection of a number of human diseases, such as cancer, Alzheimer's disease, multiple sclerosis or rheumatoid arthritis.

Therapeutic fields such as drug delivery, tissue engineering, and drug discovery, will also benefit greatly from advances in bionanotechnology. Although often considered¹ one of the key technologies of the 21st century, bionanotechnology is still in a fairly embryonic state and much of its potential is yet to be realised. Surface bionanotechnology - the science and technology of understanding and precisely controlling and manipulating surface biological properties at the molecular and nanometre scale - is one of most exciting and potentially important areas of bionanotechnology.

The objective of Dr. Mendes' research at the University of Birmingham is to further develop the interdisciplinary surface bionanotechnology field both on a fundamental level and towards biological and medical applications. Dr. Mendes' research group aims to generate surface materials with biological properties that are precisely controlled and manipulated at the molecular and nanometre length scales by interfacing nanotechnology with biological systems.

This field of research is one of most exciting and potentially important areas of bionanotechnology, but so far limited progress has been made partly due to the complexity involved in the design and fabrication processes. In order to tackle this ambitious challenge, we have been developing breakthrough methodologies for engineering patterned² and stimuli-responsive³ surfaces. Within the field of patterned biological surfaces, Dr. Mendes' research group in collaboration with Machesky group (The Beatson Institute for Cancer Research, UK) has developed⁴ a methodology to control the spatial immobilisation of cell-adhesive (fibronectin) and non-cell-adhesive (bovine serum albumin - BSA) proteins in specific micro-areas of a glass surface in order to gain valuable insights into how cells explore their environment and form new adhesion contacts for motility and spreading.

Dr. Mendes used micro-contact printing (µCP) and mouse embryonic fibroblast (MEF) cells for the study of both filopodia and filopodial/lamellipodial transitions. µCP was employed to create fibronectin linear patterns with widths varying from 10 µm to 2.5 µm and spacings between 10 µm and 0.5 µm that were backfilled with BSA. Fibronectin regions provided an activating signal and an adhesive surface, while denaturated BSA regions enabled us to examine how cells interact with non-adhesive areas. MEF cells were able to spread on these patterned surfaces, and for wider fibronectin widths and BSA gaps (5 µm x 5 µm and 10 µm x 10 µm patterned surfaces) the orientation of spreading was always in the direction of the striped pattern (Figure 1).

Figure 1: Images are single frames from a timelapse of a MEF spreading on 10 µm fibronectin stripes.

By using these wider linear patterned surfaces, it was also possible to observe that the size of lamellipodia was not dependant on the width of the stripe and that lamellipodia, but not filopodia, require adhesion for persistent protrusion. These studies also allowed us to learn more about the involvement of Arp2/3 complex in cell spreading, and in particular, that the localisation of Arp2/3 complex to filopodia is independent of adhesion. More recently, Dr. Mendes has developed⁵ a protocol to create robust and high resolution patterns of bacteria onto material surfaces, allowing arrays of viable cells to be developed with controlled spatial structure for a variety of experimental procedures including cell-to-cell communication studies.

Within the field of stimuli-responsive surfaces, Dr. Mendes' research group has developed⁶ electro-active surfaces that have been successfully employed to switch on functionalities in situ, offering an unprecedented ability to manipulate the interactions of proteins with surfaces. A new class of switchable biological surfaces - surface-confined electro-switchable peptides - have been also generated⁷ that have the capacity to regulate biomolecular interactions in response to an applied electrical potential.

This system is based upon the conformational switching of positively charged oligolysine peptides that are tethered to a gold surface, such that bioactive molecular moieties - biotin - incorporated on the end of the oligolysines can be exposed (bio-active state) or concealed (bio-inactive state) on demand (Figure 2), as a function of surface potential. The oligolysine peptides exhibit protonated amino side chains at pH =7, providing the basis for the switching "On" and "Off" of the biological activity on the surface.

For instance, upon application of a negative potential, the positively charged molecular system experiences an electrostatic attraction to the surface, leading to a mechanical molecular motion that shields the bioactive moiety (Figure 2). In order to test the viability of the proposed switching mechanism we selected an end functionalised biotinylated peptide as the biorecognition motif, four lysine residues as the switching unit and a terminal cysteine for self-assembled monolayer (SAM) formation on gold surfaces - biotin-Lys-Lys-Lys-Lys-Cys (Biotin-KKKKC).

Figure 2. Schematic representation of the switching of mixed TEGT-biotinylated peptide SAMs between a bio-active and bio-inactive state. Depending on the electrical potential applied, the peptide can expose or conceal the biotin site and regulate its binding to NeutrAvidin.

In order to provide sufficient spatial freedom for each biotinylated peptide on the surface, such that it can undergo conformational changes upon switching without steric hindrance from neighbour molecules, gold surfaces were functionalised with a two-component, mixed SAM of the biotin-KKKKC peptide and tri(ethylene glycol)-terminated thiol (TEGT) (Figure 2).

Apart from ensuring sufficient spatial freedom for synergistic molecular reorientation of the surface-bound biotinylated peptide, the short oligo(ethylene glycol) groups prevent nonspecific interaction from the proteins. As a control, a two-component, mixed SAM was also prepared using TEGT and a peptide without the biotin moiety - KKKKC. The dynamics of switching the biological properties was studied by observing the binding events between biotin and fluorescently labeled NeutrAvidin.

Fluorescence microscope images and SPR spectral data clearly revealed binding at + 0.3 V, reduced binding at - 0.4 V, and intermediate binding in the at open circuit (OC) conditions (no applied potential). Depending on the electrical potential applied to the mixed SAMs, bioactive molecules incorporated onto the SAM can be fully exposed for binding (+ 0.3 V, bio-active state) or concealed (- 0.4 V, bio-inactive state) to the extent that binding affinity can be reduced to over 90% of its bio-active state (Figure 3). Furthermore, reversibility studies by SPR also demonstrated that the developed switchable surface allows reversible control of biomolecular interactions.

Figure 3. SPR sensorgram traces showing the binding of NeutrAvidin to the Biotin-KKKKC:TEGT mixed SAMs and KKKKC:TEGT mixed SAMs under OC conditions and an applied positive (+ 0.3 V) and negative (- 0.4 V) potential.

This surface technology takes advantage of the unique dynamic properties of surface-confined charged peptide linkers at the nanometer scale to induce On-Off switching of specific biomolecular interactions, setting the stage for advances in biological research, medicine, biotechnology, and bioengineering.

References

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3. P. M. Mendes, Chemical Society Reviews 2008, 37, 2512.
4. S. A. Johnston, J. P. Bramble, C. L. Yeung, P. M. Mendes, L. M. Machesky, BMC Cell Biology 2008, 9, 65.
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7. C. L. Yeung, P. Iqbal, M. Allan, M. Lashkor, J. A. Preece, P. M. Mendes, Advanced Functional Materials, 2010, 20, 2657.