Nantotechniques and approaches in biotechnology

TRENDS in Biotechnology Vol.19 No.3 March 2001

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Opinion

The nanoworld has been discovered in the past decadeor so and this discovery has depended to a large extenton the invention of the atomic force microscope and ofa variety of methods of fabricating nanostructures.

The nanoworld – natural ‘nanoness’

The Princess in the fairy story was so sensitive thatshe could feel a pea through many layers of sheet;the scale relationships are about 400 to 1. Cells seemto be even more sensitive to their environmentbecause they can react to objects as small as 5 nm,which are some 1000–5000 times smaller thanthemselves. Of course cells have to get closer to theobject and many adhesions of cells are made overdistances of 3–15 nm from the plasmalemma lipidbarrier to the surrounding objects. In vivo there isconsiderable detail in the surrounding environmentof the cell, for instance the 66 nm banding oncollagen fibres amongst which many cells live.

When cells are taken out of the body to becultured, or when prosthetic devices, such as bonepins, are implanted, the cells might encountera very unfamiliar nanoworld in which nanodetail ischaotic or random or even a world that offers the cellsopposite cues to those that they usually receive. Forexample, the average plastic culture dish has ridges~10 nm high on the culture surface (Fig. 1). Thepolished metal surface of the metal ‘ball’ on a ball andsocket hip joint has a scratched surface with groovesand ridges ~20–50 nm deep. Indeed, making a truelyflat surface is difficult but some crystal cleavagesurfaces, such as silicon and mica, can be flat overlarge areas to a few nanometres and happily, ifsurprisingly, glass coverslips are relatively flat, butpolishing or similar processes do not yield good quality surfaces. The surface chemistry, as well as thetopography, might also be chaotic on the molecular

scale. For these reasons alone we need to know moreabout the reactions of cells to the nanoworld and howto control them.

Small is odd – an unexpected world

The macroscopic world can be scaled down throughorders of magnitude to the microscopic scale with littleor no change in expected properties. Knowing themechanical properties of a 1 mm cube of glass allows theprediction of the mechanical behaviour of a large sheetof plate glass or even of a glass mountain (if there wereone). This is not the case as you enter the nanoworld;the gate to which is ~100–300 nm in dimension.Interfacial forces begin to become of great importance,quantization effects emerge and everything has to berethought. Indeed, our experiences in this world havemade us realize that our conventional knowledge,derived appreciably from colloid chemistry, is notadequate for understanding nanostructures. Theseexperiences suggest that nanostructures will provide avery effective experimental tool for studying thephysical chemistry of complex systems such as colloids.Gleiche et al.1 and Fradin et al.2 have producedexamples of different types of interface effects on thenanoscale. Interestingly, several attempts have beenmade to define the dimensional limits of objects thatshould interest the student of the nanoworld andseveral have opted for the 100 nm limit, but perhaps itshould be larger at the 300 nm gateway.

Do cells react?

Despite the obvious opportunities for cells to havean interesting nanometric environment in which tolive, do they respond to such features? The evidenceis becoming increasingly clear that they do for manyvarieties of animal cell but the evidence for bacteriais less clear. The answer to this question could notbe sought until reliable ways of reproduciblyproducing relatively large areas of nanopatternedsurfaces were discovered. By the late 1980s, it wasalready known that many vertebrate cell typesresponded to micrometric topography but effects forsome cell types were larger for deep structures inthe 5–10 µm depth ranges than they were forshallower structures. There was therefore an

Nantotechniques and

approaches in

biotechnology

Adam Curtis and Chris Wilkinson

Nanotechnology has enabled the development of an amazing variety of

methods for fabricating nanotopography and nanopatterned chemistry in

recent years. Some of these techniques are directed towards producing single

component particles, as well as multi-component assembly or self-assembly.

Other methods are aimed at nanofeaturing and patterning surfaces that have a

specific chemistry or topography. This article concentrates mainly on surface-

directed nanobiotechnologies because they are nearer to commercial

realisation, such as use in tissue engineering, control of biofouling and cell

culture, than those directed at producing nanoparticles.

Adam Curtis*

Chris Wilkinson

Centre for CellEngineering, University ofGlasgow, Glasgow, UK G12 8QQ.*e-mail: [email protected]

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Fig. 1. The surface of a polystyrene tissue culture dish viewed withthe aid of a Nanoscope IIIa atomic force microscope used in thecontact mode. Dark areas are depressed, white areas raised. Verticalscale range (black to white) 30 nm.


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expectation that the effects might disappear atapproximately the 1 µm level.Clark3 had an inherent scepticism about this limitand using holographic photolithography produced asurface with grooves 130 nm wide and 9 µm deep.Epithelial cells cultured on this surface respondedby alignment of isolated cells and of the cytoskeletonto the grooves even when grown in sheets ofadherent cells, although the cell outlines were notaligned. This aroused interest in the possibility thatnanofeatures might affect cells but at that datenanofabrication was poorly developed and so

progress was slow. However, Wojciak-Stothard4

found a simple way to produce features ofnanometric depth by reducing the dry etch time.This method produced grooves that weremicrometrically wide, millimetrically long andnanometrically deep. Cells of fibroblastic,endothelial, epithelial and macrophage typesresponded to these grooves by increased adhesionand orientation. At that date the shallowest grooveswere 44 nm deep. Later, Rajnicek5 used some ofthese structures and nerve cells responded to 5 nm steps. These cell types reacted to thenanometric structures by changes in cell adhesion,alignment and orientation of the cells, cytoskeletalorientation and changes in cell activation3,4,6–9.

As a result of collaborations between cellbiologists and electronic engineers, we have begun toproduce nanofeatured surfaces in silica. Regulararrays, such as that shown in Fig. 2, reduced theadhesion of several cell types even though the flatunfeatured surface outside the ‘etched’ area allowedgood adhesion of the same cells (Fig. 3). To replicatethese structures cheaply and quickly, a reversedmaster in silica was produced so that casts,embossing or injection moulded polymers would havethe desired structure. Thus, if large areas of nano-pitsare needed the master is made with nanopillars orprojections. Interstage secondary masters can beproduced using a variety of methods so that the finalproduct is identical to the original master. Thetechniques used are identical to those involved inthe production of long runs of a CD and the accuracyof replication can lie at the 2 nm level. However,these are not the only ways of making masters butare the preferred ways of copying a structure.

The interesting feature of the silica dots is that thiswas probably the first structure for biological use thatwas made on the nanoscale in x, y and z dimensions.Previous structures had usually been on thenanoscale in only one or two of the three dimensions.

Alternative methods of nanofabrication

The main current methods of nanofabrication are listedin Table 1 but it is important to note that the pace ofdevelopment is so fast that new methods are emergingon a monthly basis. Colloidal resists are colloidalmaterials, such as gold metal sols or other very smallparticulate materials, which if spread on a surface willform a random or semi-random distribution of particlesthat can then be used as an etch resist and laterremoved (or left if desired). The pattern of the particlesis transferred into the substratum by the etch.

Microcontact printing10,11 is so simple in concept andso familiar to children that most of us are amazed thatit works so well. A photolitho-graphically patternedstamp, usually made from polydimethylsiloxane, isfabricated with the desired pattern etched to a veryshallow depth in the surface. This stamp is then loadedfrom a protein, polysaccharide or other large molecule-bearing surface carrying these molecules in a weakly or

Opinion

Fig. 2. Scanning electronmicrograph of silicapillars. Fabricated in fusedquartz using electron-beam lithography. Centreto centre spacing is50 nm. (Courtesy ofB. Casey, GlasgowUniversity, UK.)

Fig. 3. Low adhesion on a nanopatterned surface. Low-power light micrograph of rat tendon cells(dark stained), grown on a polycaprolactone surface cast from solvent solution of a 300 nm centre tocentre silica structure. The clear area free from cells is the surface structured with nanopits, shown indetail in Fig. 6. The culture period was 21 days. No cells are found on the nanostructured area. Thescale-bar represents 10 microns. (Courtesy of J. Gallagher, Glasgow University, UK.)



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unattached form. If hydrophilic proteins or peptides areto be printed then the surface of the stamp needs to bemade hydrophilic, for example by a very short-termetch with oxygen plasma. The stamp is then broughtinto contact with the surface that will carry the print.That surface already bears a cross-linking reagent,such as glutaraldehyde, attached, for example, toaminopropyl triethoxy silane. The stamp is left incontact for about an hour and then removed. Theprotein is then found to be transferred to the surface,for example an appropriate polymer bearing theaminopropyl triethoxy silane (Figs 4 and 5).

Monolayer assembly of thiol compounds on a goldsubstratum12 provides an interesting researchprocess but is unlikely to be of commercial potentialbecause it does not allow transfer to an embossing,casting or moulding system.

Recently, a range of new technologies hasappeared. These include ‘rapid prototyping’13,self-assembling proceses14 and developments ofdiaelectrophoretic patterning15.

Characterizing the surfaces

Unfortunately, the majority of methods forcharacterizing surfaces are ones that average overfairly large sampling areas. However, scanning electronmicroscopy, atomic force microscopy (AFM) and otherscanning probe microscopies give good images ofsurface topography at high resolution (Fig. 6) and thelatter two microscopies can work in many differentmodes, such as force modes that allow force patternor surface mechanical properties to be measured.AFM has the potential for direct chemical mapping ofthe surface but this has not yet been taken very far.

Chemistry or topography?

One area of controversy is whether the cells react tolocal patterns of chemical difference or to features

such as topography. Obviously, microcontactprinting is intended to produce patterns ofchemistry and the self-assembling systems shoulddo the same. But the direct writing of a pattern intoan isotropic substratum, such as fused quartz(silica), should in theory produce a surface that lackspatterned chemical features and the replication ofsuch a surface by embossing into polymer shouldhave a similar result. It is possible that slightdefects or anisotropies in the etch process or

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TRENDS in Biotechnology

(a)

(g)

(h)

(e)(d)

(f)

(b)

(c)

Fig. 4. Microcontact printing. The method of printing strips of protein.(a) A thick (>5 µm) photolithographic resist is illuminated through amask with the desired design. (b) The illuminated areas are specificallydissolved. (c) A polysiloxane polymer (Sylgard 187 Dow Corning) iscast onto the resist and cured at 90°C. (d) The surface to be patterned iscleaned and prepared (i.e. glass with 3-aminopropyl, 3-aminoethyltriethoxysilane). (e) The sylgard stamp (made with or without abacking) is removed and cleaned. (f) The surface is chemicallyactivated by using a crosslinking agent (i.e. glutaraldehyde). (g) Thestamp is inked with a solution of the desired peptide or after thoroughcleaning, the stamp can be reused several times. (Courtesy ofDr M. Riehle, Glasgow University, UK.)

Fig. 5. Phase contrast view of mouse endothelial cells grown for16 hours on a glass coverslip, microcontact printed with alternatestripes of albumin (no adhesion) and beta-thymosin sulfoxide. The cellsadhere to the beta-thymosin sulfoxide. The cells line up 50 µm apart,corresponding to the width of the albumin stripes.

Table 1. Current methods of nanofabrication

Type Materials Resolution

Electron beam lithographya Silica, silicon x, y and z to 10 nmSilicon nitrideSilicon carbide

Colloidal resistsa On any of above x, y and z to 5 nm

Self-organizing (assembling) Polymer demixing In 10 nm rangessystems Self-assembling particles and

monolayersOther self-assembling systems

Microcontact printing Any fairly large molecule x and y to 200 nmz to one monolayer

Embossing, casting or Most polymers Down to 21 nminjection moulding

From master made by Some metalsabove methods

Particle synthesis Many materials Upwards from (e.g. superparamagnetic beads) 3 nm diameter

a Followed by dry or wet etch.



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embossing process produce small very localizedchemical differences, but this is at present a slightly sterile argument because we do not yetpossess the chemical means to make surfaceanalyses with such high resolution.

One argument favouring the existence oftopographic reactions of cells runs as follows. Cellsreact in similar ways to the same topography onsurfaces that are chemically very different. Forexample, silica dots (such as those in Fig. 2) havemuch the same effect on cell adhesion as the samesurface made in polycaprolactone or polyurethanes.It can of course be argued that the cells are reactingto a layer of protein adsorbed onto the surface andthat this accounts for the similarities, but this leavesthe question of why the topography should producedifferences in adsorption.

Britland et al.16 competed topography againstchemistry to see which produces the greatest alignmentof neurites. The extending neurites of nerve cells wereoffered a choice of aligning to chemically printed stripsof the protein laminin or to grooves crossing the lamininstrips at 90 degrees. The shallowest grooves lost out tothe chemical tracks in this competition but when thegrooves were deeper than 500 nm topographic effectsoverwhelmed the chemical ones.

Chemical tracks appear to give cell alignmentcues but the premises of the argument can bereversed and the statement made that tracks ofchemicals do have a finite thickness and the cellsmight be reacting to the topography of the track. It isnoteworthy that cells aligning on tracks are oftenaligned to the edge of the track.

What do cells react to?

If the cells are reacting to physical forces in andinduced by the substratum rather than to specificchemical bonding, then a large range of possibilitiesshould be considered (Box 1).

Products and possible products

At the moment this area is one of promise andperception and not of complete achievement.Starting by considering effects of nanotopography,any system where you wish to have very lowadhesion or very high adhesion, especially in anembossable non-degradable or bio-degradablepolymer, is potentially useful in a wide variety ofbiotechnological devices or biomedical uses. Thesystems depending on surface topography or surfaceimmobilization of chemicals are likely to berelatively durable because the features are stronglyattached to the surface and relatively cheap toproduce because printing or embossing processescan be used. There will probably be two types ofproducts: (1) those whose primary purpose is toprovide high or low adhesion, for example in devicesto prevent tissue adhesion and (2) those productswhose primary purpose is different but where low(or high) adhesion is required to improveperformance of the device, for instance to preventfouling of biosensors.

However, many other types of nanodevice forbiomedical and biological use can and have beenenvisaged. Adhesion might be important in thesedevices but nano-detection of single or very smallnumbers of molecules, nanometric movement andcharge flow devices will also be important. Box 2reviews the products that are being realized or aregenerally accepted as goals that should be achieved.

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Nanoscope Contact AFMScan size 5.000 µMSetpoint 0 VScan rate 1.001 HzNumber of samples 256

Fig. 6. The figure showsatomic force microscopicviews of nanostructures.(a) Polycaprolactone pits(as shown in low power inFig. 3) after 21 daysculture. (Nanoscope IIIacontact mode imagecourtesy of J. Gallagher.)(b) Microcontact printedalbumin strip viewedusing AFM. Note the largedifference betweenhorizontal and verticalmagnifications wheninterpreting this image.The ‘globular’ structuresare appreciably largerthan single albuminmolecules.

• Imbalanced or asymmetric distribution ofinterfacial forces across the nanostructure sothat concentrations of attractive or repulsiveforces occur at particular places, for instanceelectrostatic forces of repulsion might beconcentrated at sharp projections.

• Strains within the cells induced by theirattempt to conform to the non-planar surface.

• Stress relief in the substratum surface itselfinduced partly by random thermal events andpartly by the cells themselves. This is likely toexert mechanical forces on cells.

• Patterns of surface chemistry possiblydisplaying binding sites for specific molecules.

Box 1.What features of nanopattern do cells react to?

Conclusion

Two years ago, a review of a meeting on‘nanotechnology in biotechnology’17 described several speakers likening nanobiological devices to Rube Goldberg’s (devices) as ‘inelegantbut ingeniously baroque’. I find this a curiousstatement. Cells live in a nano- or micro-featuredenvironment. Natural selection would act to remove redundant reactions and redundancy is thespirit of the baroque. Devices we make are likely to operate correctly only if they interact withcells in much the same general way as theenvironment does. So the route into this world ought to be one in which we systematically examine the reactions of cells to simple well-defined structures.

Moreover, this area of scientific endeavour is atruly interdisciplinary one and any effectiveprogress, even on a single project, needscontributions from physicists, engineers, material chemists, biophysicists, cell and molecular biologists, as well as end users such asorthopaedic surgeons.



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References

1 Gleiche, M. et al. (2000) Nanoscopic channellattices with controlled anisotropic wetting.Nature 403, 173–175

2 Fradin, C. et al. (2000) Reduction in the surfaceenergy of liquid interfaces at short length scales.Nature 403, 871–874

3 Clark, P. et al. (1991) Cell guidance by ultrafinetopography in vitro. J. Cell Sci. 99, 73–77.

4 Wojciak-Stothard, B. et al. (1996) Guidance andactivation of murine macrophages by nanometric scale topography. Exp. Cell Res. 223,426–435.

5 Rajnicek, A.M. and McCaig, C.D. (1997) Contact guidance of CNS neurites on groovedquartz : influence of groove dimensions,neuronal age and cell type. J. Cell Sci.110, 2915–2924

6 Curtis, A. and Wilkinson, C. (1997) Reactions ofCells to Nanotopography. In Cellular &

Molecular Biology Letters: Biophysics ofMembrane Transport: XIII School ProceedingsPt. 1 Membrane Transport, (K.K.J. Kuczera,B. Rozycka-Roszak, S. Przestalski, J. Szopa,A. Kozubek, A.F. Sikorski eds) Ladekzdroj,Poland, pp. 9–18

7 Curtis, A. and Wilkinson, C. (1999) Reactions ofCells to Nanotopography (Lackie J.M., et al. eds)Portland Press, London, UK

8 Wilkinson, C.D.W. and Curtis, A.S.G. (1996)Nanofabrication and its applications inmedicine and biology. Dev. in Nanotechnology3, 19–31

9 Wilkinson, C.D.W. et al. (1998) Nanofabricationin cellular engineering J.Vac. Sci. Technol. B.16, 3132–3136

10 Jackman, R.J. et al. (1995) Fabrication ofsubmicrometer features on curved substrates by microcontact printing. Science269, 664–666

11 Brittain, S. et al. (1998) Soft lithography andmicrofabrication. Physics World 11, 31–36

12 Patel, N. et al. (1998) Spatially controlled cellengineering on biodegradable polymer surface.FASEB J. 12, 1447–1454

13 Fan,H. et al. (2000) Rapid prototyping of patterned functional nanostructures.Nature 405, 56–60

14 Kazmaier, P. and Chopra, N. (2000) Bridgingsize scales with self-assembling supramolecularmaterials.MRS Bulletin 25, 30–35

15 Schaffer, E. et al. (2000) Electrically inducedstructure formation and pattern transfer. Nature 403, 874–877

16 Britland, S. et al. (1996) Morphogenetic guidancecues can interact synergistically andhierarchically in steering nerve cell growth.Experimental Biology Online EBO 1:2.

17 Lee, S.C. (1998) Biotechnology fornanotechnology. Trends Biotechnol. 16, 230–240

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Examples of devices for controlling cells where

nanofeatures form part of the device

• Cell traps (prisons and death cells)• Cell orienters and movement stimulators• Collagen aligners• Biosensors (not yet made at the nanoscale)• Nanoanalysers (e.g. for DNA and specific proteins).

Examples of complete devices

• Nanosamplers (e.g. of cell surface; to be fullyrealized)

• Nanoimplanters (e.g. of artificial surfaces, DNA,drugs)

• Magnetic nanoparticles for drug delivery, celldestruction

• Cell moving and assembling devices• Low adhesion/high adhesion surfaces for

biomedical uses (e.g. stents)• Nanomachines

Box 2. Biotechnological uses of nanotopography:

main areas

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Nantotechniques and approaches in biotechnology

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