Frank Caruso is a Professor and Federation Fellow in the Department of Chemical and Biomolecular Engineering at the University of Melbourne. He previously led an international research group at the Max Planck Institute of Colloids and Interfaces in Germany. He has been awarded numerous prizes for his scientific achievements: Alexander von Humboldt Research Fellowship (1997); Max Planck Institute of Colloids and Interfaces award for research excellence (1998); German Federal Ministry of Education, Science, Research and Technology bioscience research award (1999); Royal Australian Chemical Institute Rennie Memorial Medal/Royal Society of Chemistry – Royal Australian Chemical Institute Exchange Medal (2000/2001); ARC Federation Fellowship (2001). He serves on the International Advisory Boards of the journals Advanced Functional Materials and Chemistry of Materials. His main interests are in combining recent new developments in bioscience with those in nanotechnology, in order to produce novel biofunctional materials for potential applications in medical diagnostics, pharmaceutics and catalysis.

Nanoengineered smart particles
by Professor Frank Caruso

I would like to take you through some of our work in the area of nanoengineering smart particles. The point has been driven home quite impressively already that advances in nanoscience and nanotechnology are basically linked with our ability to prepare nanomaterials with flexible and facile strategies. I am going to address both of those points.

The strategy that I am going to present to you is one that allows us to take particles and to modify their surfaces, as shown in Figure 1. This is a bare particle; this is a particle coated with very thin layers of polymer. Then we can go on and deposit nanoparticles onto that core system. I am going to focus on that strategy, and also specifically talk about particle science – colloid and interface science, as such.

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Colloids can be classified as particles in the size range of about one nanometre to a micron, or even more, in size, but the important thing is that they are dispersed in a different phase. Recent interest has been in going down in size of the particles, and the reason for that is that when you get to less than about 100 nanometres or, more particularly, less than about 10 nanometres, what you find is that the particles start to exhibit unique properties – magnetic, optical, catalytic and so on.

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Figure 2 shows 15-nanometre gold nanoparticles which are dispersed in water. They appear red to the eye. The dispersion is stable, but if you add a small amount of salt, or polymer – in this particular case, it is citrate stabilised gold nanoparticle dispersions – what you find is that you induce a colour change. This colour change is not due to change in the actual size of the individual entities or particles in solution, but the particles have clumped together. They have aggregated.

What we, in my group, do in modifying the surfaces of particles is to try and prevent such aggregation.

Colloids have been around for a very long time, and many of you will be familiar with the range of areas where they have been applied – for example as components in cosmetics, in paints, all the way through to agriculture and the mining industries. But really the underpinning theme and motivation of my group is that the demands placed on materials performance by nanotechnology and biotechnology require tailormade particles or materials. That is where we are focusing: to prepare materials that have significant functions and properties that we can introduce controllably.

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Figure 3 summarises my group's different areas of research.

The first illustration is of blood cells, biological cells, and an example of biosciences. I am going to be showing you how we can actually take biological specimens – in this case they are enzyme crystals – and encapsulate those, using self assembly.

Then I will move on and present to you a second example, of hollow materials. The second illustration is of capsular colloids or hollow spheres that have been dried onto a solid support. I will show you how these molecularly engineered polymer capsules can be utilised for drug delivery purposes.

We are also interested in the area of colloidal crystals, or artificial opals. These are basically close-packed spheres in a periodic arrangement, typically silica spheres or polystyrene beads, and when you shine light on these, you have wonderful properties that originate due to Bragg diffraction. The third illustration, which I have taken from Orlin Velev’s work, is that of silica spheres – in these cases they are ~270 nanometres in diameter and ~320 nanometres in diameter. What I would like to point out is that each sphere is composed of many smaller spheres. But they are assembled in a close-packed configuration, and you can see the wonderful colours that originate from those.

The interest in this area lies in the promise that if you can controllably pack these spheres, defect free, you should in principle be able to manipulate light. That is where a lot of the work in that area is heading.

If you look at nature, the classic example of diffraction of light is that of the butterfly. The wonderful colour originates from a periodic spacing of structures on the surface of the wings of the butterfly.

I am going to present to you the layer-by-layer processing technology that we use to modify particles, and apply that to micrometre-sized particles and then to nano-sized particles, and go through the examples of encapsulation and also drug delivery. If I have time, I will touch also on biological assays.

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How do we modify the surface of particles? Typically what we do is to take a dispersion with a concentration of less than about 10 weight per cent. The particles inherently have a charge, positive or negative. That is why they are in a dispersed state. All cases that I will be presenting are particles in water.

For example, if the charge on the particle is negative, we simply add a solution of oppositely charged polymer, of polycation. What you find is that one layer, and one layer only – a monomolecular layer – adsorbs onto the surface of the particle. Because we are working in excess conditions, we remove excess polymer, we have a surface charge reversal here, and then we can continue this process: second layer, multiple layers, on the particles.

What I would like to highlight is that the core can be anything from a gold nanoparticle through to a polymer latex bead, all the way up into the millimetre regime. It is a facile and flexible strategy, and so you can prepare composite materials, building blocks, which you can then go on and prepare advanced structures with.

In a subsequent step, what you can do is to remove this core material, if it is decomposable, simply by using acid if it is a weakly cross-linked polymer. Because these semipermeable shells here are indeed permeable, the components are actually removed from the interior and you end up with hollow polymer spheres.

What I should point out at this point is that the shells basically comprise alternating layers of polycation and polyanions. They are held together by multiple electrostatic interactions – self-assembled layers, and they are held together through electrostatics. They are very different from liposomes, in that they are stable over a wide pH range of about 1 to 12 when you are using strongly charged polymer systems. You can heat these up, you can expose them to a range of harsh chemicals, and they are stable. So they represent excellent containers that can be used for drug delivery and/or as confined environments for chemistry. And we can make them in the nano-size regime, below 100 nanometres, so you can do nanochemistry within them as well.

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Figure 5 shows examples of the polymers that we typically use: polyamines and polystyrene sulfonates. They are not restricted to these polymers. You can use biocompatible materials, you can use biodegradable materials. The only prerequisite is that you have a charge on each polymer. So it is a very facile and flexible strategy to that end.

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Figure 6 shows polymer latex beads which are about 650 nanometres in diameter. We deposited nine alternating polymer layers on the surface, and one of those polymers is fluorescently labelled. What you find under an electron microscope is that indeed you see a diameter increase, so it goes from about 650 to 680 nanometres. You cannot visualise the polymer on the surface because the electron contrast is similar. But if you look under a fluorescence microscope, you will see the fluorescence originating from the doped polymer in the FM images.

This fluorescence intensity in the Z direction is proportional to the number of layers that we deposit. That is proof in itself that we can deposit these layers.

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What has interested us in the last couple of years is: can we go down in size? I have given you the example of about half a micron, or 500 nanometres. Can we go down in size to 50 nanometres and 5 nanometres? As many of you may know, working at the micron scale is very different from working at the nanoscale. It took us a couple of years to learn how we could modify nanoparticles controllably, using the self assembly approach. We knew it was possible because if you look at nature, nature effectively wraps DNA around a 5-nanometre protein known as histone. So it is possible to wrap very small nanoparticles with DNA, which is a polyelectrolyte. For us the important point was that we controlled the solution processing conditions.

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If you have a polymer and it has a negatively charged backbone such as DNA, it is somewhat rigid. But if you add salt to that solution, you can screen the electrostatics and that polymer becomes more flexible, so you induce some coiling and much more flexibility, which allows it to wrap. In the result shown in Figure 8, these are 7-nanometre gold nanoparticles. You can’t see the polymer again, but we have confirmed it using other techniques. But what I would like to highlight here is that these nanoparticles are single nanoparticles, they are not aggregated, and they are indeed coated.

This is a significant result, because it tells us that we can virtually coat particles in the nanometre regime – whether it is a gold nanoparticle as in this example, a semiconductor nanocrystal or a variety of others – and once we have coated these, we know the functional groups on the surface on which we can then go on and couple proteins, for example. The reason why it is significant is that when you have nanoparticles, often it is very difficult to ascertain the functionality, or the functional groups, at the surface of those particles. Currently what we are doing is labelling these nanoparticles with proteins, more specifically Fab fragments of immunoglobulin, IgG.

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As a model example, just to show you that we can monitor the binding or attachment or coupling of proteins to nanoparticles, Figure 9 is an example of analytical ultracentrifugation. What is important here is that you see the difference, not in shape but in displacement of the curve to higher particle size, and this tell us that we can monitor very small differences in changes of the particle size using this technique of analytical ultracentrifugation. We are currently conducting more experiments in that area.

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The example I am going to give you in terms of a biomaterial is that of an enzyme. Figure 10 shows a catalase crystal, which is about 10 microns in diameter, and it has been dried on a solid support – this is an AFM (atomic force microscope) image. We have encapsulated this crystal, as a dispersion, with thin layers of polymers again in this alternating sequence and one of them has been fluorescently labelled. What you see under a fluorescence micrograph is that fluorescence originating from the surface of these particles.

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Figure 11 represents the highest loading of biomaterial in encapsulated form possible, because it is a crystal. So it and related systems could be attractive for delivery purposes, because you have a high payload factor. The coatings are very thin, about 20 nanometres, but look at the size: they are about 10 microns. You have these very thin polymer coats which encapsulate this enzyme.

Because it is only a crystal in a certain pH range, 4 to 6, when you change the pH – when you go below 4 or above 6 – you solubilise the enzyme. You dissolve it. If you dissolve it, you find that it is actually retained within these very thin polymer coats; however, you induce a morphology change in the polymer capsule itself. The reason for that is that when you dissolve it, there is an increase in osmotic pressure, and you get a change in the morphology of the polymer coatings. But, importantly, catalase – which is about 10 nanometres – does not permeate outside. It is kept within.

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We actually released the enzyme under certain pH conditions to measure its activity, and it was close to 100 per cent active, which tells us that the method that we have used to encapsulate the enzyme has not denatured it. More importantly, we can keep the catalase in but can we keep other material out.

The example shown in Figure 12 is that of a protease. We have taken this encapsulated enzyme system and we have added protease. Protease degrades the solubilised enzyme within two hours. Here we have activity as a function of time and you can see that the enzyme has lost its activity in a couple of hours. However, when we have assembled these very thin polymer coats, what we find is that the protease cannot actually get through to degrade the enzyme. So it is a powerful example of what we can do through self assembling polymer layers: we keep the enzyme in and we keep the protease out – wonderful for biological applications when you want to use protective coatings as such.

We can also tune the mesh size or the permeability of these layers, depending on the solution processing conditions and the chemistry concerning the functional groups of the polyelectrolytes.

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When you remove that enzyme, the polymer capsules or shells are left behind. They have been dried on a solid support and so you see folds and creases. Note the large size, about 20 microns, and the wall thickness again is about 20 nanometres. So it is a nanolayering strategy and each layer is about one to two nanometres thick.

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You can introduce some chemistry here. I am just illustrating this because, if you take polymers that you can cross-link, ultimately what you end up with is systems that have elasticity moduli greater than about 750 megapascals, so these are ultra-stable, ultra-tough systems that you can use for a whole variety of purposes, for chemistry and/or drug delivery.

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The example of drug delivery in Figure 15 concerns doxorubicin, an anti-cancer drug. We have taken polymer capsules, hollowed, prefabricated or pre-assembled them, and what you see is a cross-section of those. Figure 15 is a confocal microscopy image in which the shell has been labelled; that is why it shows up. I will show you how, in a two-step process, we can bring drugs into the interior of these.

The first step involves bringing a polymer into the core of the capsules. Because they are polymer layers which are held together through polycation and polyanion interactions, if you change the pH you can change the pore size, because the electrostatics change. In changing that pore size, the dextran sulfate, which is just a model polymer, actually diffuses in. And then, simply, we change the pH back so we lock it in, and we remove residual polymer from the surrounding solution. At that point we have got a polymer inside and we add the doxorubicin, which is a very small molecular weight material. That permeates through and binds, through an ion-exchange process, to the polymer which is within.

So we have a loading which is about 30 to 40 weight per cent – very simple, very straightforward. We can also prepare these systems from biodegradable and decomposable materials.

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If you look at the release under physiological conditions, what you find when we expose these systems to 154 millimols of sodium chloride is that indeed we can release the anti-cancer drug. The reason why it is released is that the salt displaces the doxorubicin through an ion-exchange process. We can control this release, depending on the thickness of these polymer layers and also depending on how we tailor the surfaces, whether we deposit lipid bilayers or nanoparticles. We are currently attaching proteins for targeting of these systems.

I have shown you how we can prepare a whole range of coated and encapsulated particles. A lot of the interest in my group is focusing on how we can use these as normal building blocks for the construction of advanced structures, simply because the core shell particles themselves have unique properties.

I showed you how we could use particles in encapsulation and also for sustained delivery. We expect these systems to impact a variety of areas.

I would like to thank the many people who have contributed to the work – members of the group, collaborators across the world, and the funding bodies.

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I will just leave you with a nano-biotechnology example of a 'Gold Nano-fish'. Each dot represents a gold nanoparticle, about 15 nanometres in diameter. It looks like a fish – there is an eye missing – but equally, you can rotate this 90° counterclockwise and it can be a nano-kangaroo.

Questions/discussion

Question: Could I start by asking about the example you gave us with the catalase crystal. Catalase is very well known, extremely tough, robust, tiny, two-dimensional crystals that electron microscopists have studied for a long time. So is that whole process that you described there likely to be limited in its applicability to other crystalline substrates?

FC: No, we don’t expect it to be limited to catalase alone. There are 100 or so protein crystals that one can try this on. The reason for that is that we can deposit these polymer layers out of varying conditions: with or without salt, in different pH conditions, in aqueous based systems, and so on. Because of that generality, we do expect it to be applicable to other systems as well. We find that it is simply adsorption onto the surface of materials, and so it doesn’t actually destroy the morphology of those.

Question: Is this a laboratory experiment that with considerable expense you can produce in the laboratory, or is this really commercially realisable drug delivery technology?

FC: I would agree to the latter point. We are using water-based chemistry here, or water-based science. It is cheap, it is versatile, it can be scaled up and indeed we have demonstrated that. We formed a start-up company, as such. The crucial point is more in the area of application. Once that is sorted out, then you can go in and, because it is a step-wise layering strategy, effectively like a Lego approach, you can introduce various functions. You can even tailor the surface in order to prevent fouling, for example, of biomaterials, and we have done some experiments there. But we have not done any in vivo experiments as such, although we are currently looking for interested collaborators to take on some of this work.

Question: What are the advantages of micro-encapsulation of your kind, versus macro-encapsulation of the traditional sort?

FC: Flexibility, versatility, stability; we can control the thickness of the layers, the composition, the structure, the permeability; it is water-based – control, control, control, very much so. We have done this for a whole range of different components, and it is the flexibility behind the approach which is its power.

Question: And is that important because of the ability to design-in a release feature, dependent on the physiological environments you are looking to do that in?

FC: Yes. For example, if you are looking at a certain property, such as if you want a thermo-responsive system, you would start with a polymer that displays some temperature sensitivity upon increasing or decreasing the temperature. If you want to design a system where you have a pH dependency, you simply take a polymer that has ionisable groups. Indeed, we have shown examples of both of these.

Question: Do these hollow sphere things that you are making have any use other than chemical release?

FC: Yes. One interest that we have is assembling them into layered structures. If you can assemble them into layered structures – for example, if you consider where you have layer 1 of a system that has component A on the inside, and then layer 2 where you have a system that has component B – you can make these into multilayered structures themselves and that is great for catalysis experiments. For example, you can assemble them onto a membrane and you can control the coating and the thickness, and then you can do sequential catalytic reactions through these systems.

The other thing that we have become interested in recently is using these as containers to perform PCR, polymerised chain reactions, so coupling this with molecular biology.