Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
  • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
  • A61K49/1887—Agglomerates, clusters, i.e. more than one (super)(para)magnetic microparticle or nanoparticle are aggregated or entrapped in the same maxtrix
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

• the present invention relates to clusters of nanoparticles, hereinafter referred to as nanoparticle clusters and particularly to methods useful in the formation of clusters of a predetermined size.
• the invention provides for magnetic nanoparticle clusters.
• the invention also provides for the use of such nanoparticle clusters in medical applications such as drug release, as mediators for hyperthermia and as contrast agents for magnetic resonance imaging.
• nanoparticle clusters for the production of contrast agents by simultaneous in situ growth and stabilisation of the nanoparticles in the presence of polymers (usually dextran) or polyelectrolytes.
• polymers usually dextran
• polyelectrolytes Such techniques are described, for example, in WO2005076938 which provides a description of multi-polymer- coated magnetic nanoparticle clusters, aqueous magnetic fluids comprising same, and methods of their use in separation procedures.
• the multi-polymer- coated magnetic nanoparticle clusters comprise a super paramagnetic core, with a first polymer attached thereto, which does not render the first polymer- super paramagnetic particle complex colloidally stable, and a second polymer attached thereto, which stabilizes the complex.
• Methods of separation including separation of expressed protein from cells and viruses expressing the same are all described. Although the described method has the ability to provide a stable cluster, the size control of the formed clusters is determined by the surface chemistry of the stabilising polymer used and the larger clusters are essentially polymers with low nanoparticle content. It is therefore difficult to control the size distribution of the formed clusters.
• the size of the cluster finally obtained is process controlled as opposed to user controlled. It can therefore be difficult to generate samples of reproducible size. Additionally, a broad range of cluster sizes is obtained
• a methodology is provided that enables a growth in suspension of clusters of nanoparticles.
• the size of the final clusters chosen may be selected with some precision. Similarly, control over size distribution may also be exerted.
• Monitoring techniques may be employed to monitor the growth of the clusters in suspension to determine when the required size has been achieved. Using this technique it is also possible to control the polydispersity of the suspension.
• Figure 1 is a pictorial representation of the steps involved in the preparation of nanoparticle clusters in accordance with an exemplary method according to the teaching of the invention.
• Figure 1a is a pictorial representation showing an alternative mechanism for provision of nanoparticle clusters in accordance with the teaching of the invention.
• Figure 2 is a schematic showing such steps associated with the representation illustrated in Figure 1.
• Figure 3 shows data from two typical PCS experiments with different ratios of nanoparticles to silica surface, the results demonstrating control over the rate of growth
• Figure 4 shows data recorded for a different series of preparations, providing further confirmation that the clusters grow by the addition of nanoparticles from the suspension.
• Figure 5 shows PCS data for ME addition of magnetite suspension H5a over silica-C18 showing the increase in Z-average of suspension with time in run 1.
• PCS diameter in the Y-axis is converted to the volume and shown as the inset.
• Figure 6 shows PCS data for ME addition of magnetite suspension H5a over silica-C18.
• Figure 7 shows the change of polydispersity index and the mean count rates for
• FIG. 9 shows Z-average size of H5b of slightly different size (11.8nm) for ME addition experiment during 4 consecutive runs.
• Figure 10 shows the change in mean count rate with time for 1 monolayer addition of magnetite suspension (H5b) over silica-C18.
• Figure 11 shows the change in PDI with time for ME addition of magnetite suspension (H5b) over silica-C18.
• Figure 12 shows PCS correlation data fits and the intensity size distribution for sample H5b in an ME experiment over silica-C18 after 5 hours(— ), 13 hours (— ) and at the end of the experiment 48 hours ( ⁇ —).
• the intensity distribution for the 48 hours (— ) data is zero in the range shown; larger particles only present.
• Figure 13 shows PCS data for suspension H5a for BE addition over silica-C18 showing the increase in Z-average of suspension with time for run 1 ( ⁇ »-»), and for run 2 (TM»). The parameters for the linear fits to the short (s) and long (I) time data are included for each run.
• Figure 14 shows polydispersity index (PDI) data and mean count rate in a BE experiment with sample H5a.
• PDI polydispersity index
• Figure 15 shows PCS data fit and intensity size distribution for suspension H5a over porous silica-C18 substrate from the first run of the BE experiment, after 14 hours (— ), 44 hours (— ) and 120 hours ( ⁇ —).
• Figure 16 shows Z-average data with time for three runs for an BE experiment( ⁇ ) H5a run 1 , (o) H5a run 2 and (V) H5b run 3.
• Figure 17 shows UV absorbance at 475 nm over time for a BE experiment with
• Figure 18 shows PCS data for ME adsorption of magnetite suspension onto silica-CN substrate.
• the Z-average size is indicated with a (D), the PDI with (®).
• Figure 19 is a scanning electron microscope image of fresh silica-C18 surface.
• Figure 20 is an electron micrograph of coated silica-C18 particles from a ME experiment.
• Figure 21 is a magnified image of the region of Figure 20 that was indicated by an arrow.
• Figure 22 is an electron micrograph of coated silica-C18 particles from a BE experiment.
• Figure 23 shows PCS data fit and polydispersity index for a suspension of oleate coated nanoparticles prepared by a different synthetic route and placed over an alkyl grafted -C18 substrate from a different source to that of the results provided in Figure 3.
• Figure 24 shows a mechanism whereby citrate coated gold nanoparticles are stripped of their citrate capping agent through interaction with NH 2 -modified silica particles to effect generation of gold nanoparticle clusters.
• Figure 25 shows examples of TEM images showing the growth of gold nanoparticle clusters.
• the present invention will now be described with reference to exemplary embodiments which are provided to assist in an understanding of the teaching of the invention.
• a methodology is provided that enables a growth in suspension of clusters of nanoparticles.
• the clusters are formed of a plurality of individual nanoparticles.
• the size of the final clusters chosen may be selected with some precision. Similarly, control over size distribution may also be exerted.
• Monitoring techniques may be employed to monitor the growth of the clusters in suspension to determine when the required size has been achieved.
• nanoparticles in suspension are exposed to an activation source which serves to stimulate the combination of two or more individual nanoparticles to create a nanoparticle cluster.
• the source of activation could be an activation substrate or indeed other activated particles or already formed clusters within the suspension. It does normally require for the activation source to be a material which provides an activation surface.
• the growth can be monitored and once clusters of a required size are obtained, then the further growth of the clusters can be restricted by removing the suspension from the contact with the activation source. In this way it is possible for the user to select the final dimensions of the clusters provided. In effect, the control of the dimension of the growth of the nanoparticle clusters is maintained through interaction between the nanoparticles in the suspension and the source of their activation.
• the source of activation is a substrate such that the interaction is between the nanoparticles in the first phase (the liquid phase) and the activating substrate in the second phase (the solid phase).
• a substrate that may be utilised is a silica substrate.
• silica is intended to include both pure silica (Si ⁇ 2) and other naturally occurring forms of silica which, as will be understood by the person skilled in the art, includes impurities in the form of one or more trace elements.
• One particularly useful form of silica is alkyl chain grafted silica, another is cyanated silica.
• the source of activation is another activated particle or cluster within the suspension.
• Such an arrangement is particularly useful in the context of seeding a suspension with already activated particles or clusters to enable continued growth of clusters within that suspension or indeed where a continuous cycle of formation of clusters is desired, and the number of activatable particles in the suspension has been diminished through the formation of clusters.
• the nanoparticles are stabilised through provision of a plurality of capping agents on the nanoparticle. It will be appreciated that the choice of capping agent selected will have to be made based on the capacity of the capping agent to bind onto the surface of the nanoparticle so as to stabilise the nanoparticle.
• the capping agent is subsequently at least partially stripped from the nanoparticle.
• the stripping of the capping agent changes the stability of the nanoparticle such that it is now reactive.
• the stripping may be effected through provision of an activating material such as a substrate that exhibits an affinity for the capping agent. Interaction between a capped nanoparticle and the activating agent initiates the stripping. Subsequent to their formation, reactive nanoparticles combine with one another to effect generation of nanoparticle clusters- i.e. two or more nanoparticles which are chemically of physically linked to one another.
• a methodology in accordance with the teaching of the invention is based on an understanding that bringing a suspension of dispersed nanoparticles into contact with an activating source such as a surface may lead to the growth of clusters of the nanoparticles.
• the contact with the surface leads to a gradual aggregation of individual particles into clusters.
• a user can terminate the interaction between the two once clusters of a desired size are predominant.
• a portion of the suspension could be removed, allowing the remainder to continue the process of nanoparticle cluster growth.
• a suspension of nanoparticles are brought into contact with a surface (Step 200).
• individual particles of the plurality of available particles in the suspension become activated through a loss of surfactant molecules on desorption from the surface (Step 205).
• Two or more of these activated particles combine, leading to the growth of clusters (Step 210).
• a repeat of this interaction between activated particles results in larger clusters which may precipitate from the suspension (Step 215).
• Both the primary particles in the suspension and/or the formed clusters have an affinity for the surface and in this exemplary embodiment where the substrate is provided below the suspension, precipitation from suspension may be on-going throughout the process. In this way, there is ongoing loss of material from the suspension. Once the clusters of useful dimensions are found then it is possible to remove the suspension, with those clusters, and then retrieve or stabilize the clusters.
• a contact between an individual nanoparticle which is in suspension with the surface of the substrate activates the previously stabilized nanoparticle which may then combine with a similarly activated particle.
• activated particles can activate unactivated or stabilized particles. It is thought however that a dominant source of activation, where there is an activating substrate, is the activating substrate.
• the process may be controlled. This control may be effected by either monitoring the process through a sampling method and terminating the process when the sampled distribution size meets predetermined requirements or, assuming that after a predetermined time that a predetermined size distribution has been achieved and using time as the terminating factor.
• these coated nanoparticles form the primary particles within the process and have a core with a coating or capping agent provided on the surface thereof, the coating being activatable, which will result in an activated nanoparticle.
• the nanoparticles are provided in a stabilized form, the stabilization being provided by the capping agent on the surface of the individual nanoparticles.
• the suspensions were placed over a C18-grafted silica substrate in a standard cuvette.
• the size of the suspended particles were then monitored by photon correlation spectroscopy, PCS.
• the particles were observed to gradually increase in size, due to aggregation, or clustering, of the primary particles.
• Figures 1 a illustrates an alternative mechanism through which interaction between the nanoparticles, the suspension and the substrate may result in the gradual aggregation of individual nanoparticles to form nanoparticle clusters.
• the substrate is again formed from silica and the nanoparticles are provided in a suspension of oleic acid which assists in maintaining the nanoparticles in a stable form.
• the suspension is brought into contact with the silica substrate.
• the silica interacted directly with the nanoparticles whereas in this indirect mechanism the silica slowly strips the suspending medium of oleic acid which is the surfactant on the primary nanoparticles.
• This surfactant which has provided the stability previously evident in the primary nanoparticles effects the generation of reactive nanoparticles within the suspension (Step 205) and results in cluster growth through interaction of two or more reactive nanoparticles (Step 210).
• This cluster growth further shifts the equilibrium between free surfactant in the suspending medium and nanoparticle-adsorbed surfactant, so that some surfactant desorbs creating active nanoparticles with incomplete surfactant layers, resulting in cluster growth.
• Once the clusters have reached a predetermined size it is possible to terminate the growth of clusters by removing the suspension from contact with the activating substrate (Step 215).
• FIG. 3 shows data from two typical PCS experiments with different ratios of nanoparticles to silica surface. The results demonstrate control over the rate of growth and that the cluster size may be selected in a range from 10 to 400nm, by simply removing the suspension from the silica substrate at the appropriate time. Experiments have also been performed that demonstrate that it is possible to grow the clusters off-line and to monitor their growth by taking samples intermittently for PCS.
• the stabilizing surfactant for example oleate
• volume of a sample for reaction is not limited to the volume of a cuvette (c. 4 ml).
• a technique in accordance with the teaching of the invention enables a continuous monitoring of the process in order to allow the user to select an appropriate time to terminate the interaction between the particles in suspension and the substrate.
• Figure 4 recorded for a different series of preparations, provides further confirmation that the clusters grow by the addition of nanoparticles from the suspension. Initially as larger clusters scatter light more strongly, the intensity of scattered light increases. However as the process continues, eventually there are fewer clusters present and the scattering intensity decreases.
• the suspensions may be particularly useful as contrast agents for magnetic resonance imaging, and as mediators in hyperthermia. These applications are particularly suitable because clusters can accumulate in specific sites due to their physical size. Once in situ, they may be detected (as in the case of MRI) or if the formed nanoparticle clusters have magnetic properties may be stimulated to produce heat (for hyperthermia applications).
• the actual dimensions of the clusters are important for effectiveness. Using clusters formed in accordance with the teaching of the invention therefore may be considered as extremely effective in that their size and distribution of size is controllable.
• the provision of particles of a controlled size should improve the physical targeting of the particles, which as mentioned above may allow a targeting of specific structures for MRI, e.g. the leaky vasculature of tumours.
• the same methodology can be used to produce different sized nanoparticle clusters, it may be possible to develop agents that target more than one disease. For instance, it is known that leaky vessels are also associated with autoimmune diseases, including arthritis.
• drug delivery or drug targeting within the context of the present invention is based on an understanding that particles in the body are rapidly recognised and attacked by the macrophages (white blood cells) and processed by the reticuloendothelial system.
• the blood circulation time can be increased by using smaller particles ( ⁇ 50nm is good) and/or generating what is termed stealth particles by for example grafting masking molecules such as polyethylene- glycol onto them.
• These particles will circulate many times and accumulate anywhere that they have a specific chemical interaction (e.g. if they are coated with an antigen and the antibody is present), or a physical preference.
• sub-1 OOnm particles eventually accumulate in the leaky vasculature of tumours (if present).
• This physical accumulation is essentially a passive process and while the word targeted suggests a more active direction of the specific drug to an area of interest it is sometimes used to describe this passive process.
• thermo-sensitive liposomes having nanoparticle clusters provided therein or polymeric coated nanoparticle clusters may be useful in drug sequestering and controlled release of the drug to a target site within the body.
• the liposome or polymer may be used to provide a matrix within which a particular drug or pharmaceutical composition is loaded. By encapsulating nanoparticle clusters within such a matrix and then introducing that combination into the body it is possible to deliver the pharmaceutical composition to the desired target site.
• the encapsulation combination is disrupted and the composition released to the surrounding tissue.
• a fluid formed from such a drug delivery unit has components of a controlled dimension and as such the ultimate destination of the components of that fluid within the body is determinable in a fashion that heretofore was not as known.
• the heating of the nanoparticle clusters may be effected in a number of suitable manners such as application of a magnetic or RF field, or indeed the targeting of the site where the drug delivery agent is located with a laser.
• nanoparticle clusters formed in accordance with the teaching of the invention includes the formation of catalytic surfaces.
• Nanoparticle clusters of for example metal oxide materials prepared by this process and deposited on a solid substrate would have high surface area to volume ratios and so may be usable catalysts (e.g. in catalytic converters).
• Most applications would require removal of the capping agents (fatty acids), but this could be achieved by heating them as part of an annealing step which would also serve the purpose of fixing the nanoparticle clusters to the substrate.
• Clusters formed using the techniques of the invention would be formed of very monodisperse primary particles and could therefore be considered as providing excellent catalytic properties.
• Such complex structures is predicated in having formed the initial nanoparticle clusters.
• the methodology of the present invention creates the clusters within a suspension. Once the desired size of nanoparticle clusters is obtained, stabilisation of the clusters may be effected. It will be understood that a number of different mechanisms for stabilisation of nanoparticle clusters may be utilised, and it is not intended that the present invention be limited to anyone specific mechanism. Nevertheless examples of possible approaches will now be described which are based on a cross-linking and phase transfer of the nanoparticle clusters from suspension in an organic solvent into a stable aqueous suspension.
• An alternative method could involve reacting the pre-formed clusters with aqueous polyelectrolytes, including polystyrene-sulphonate or one of the many poly(alkyl-cyanoacrylate)s. This approach, it will be appreciated, may result in stabilisation of the clusters and rendering them water soluble in one step.
• adsorption which involves separation of an atom, molecule, or particle from one phase accompanied by its accumulation or concentration on the surface of a second phase, in this example a silica substrate.
• Physical adsorption is caused mainly by van der Waals and electrostatic forces between adsorbate molecules and the atoms/molecules on the adsorbent surface.
• adsorbents are characterised by surface properties such as surface area and polarity. A large specific surface area is preferable for providing adsorption capacity.
• Porous carbon-grafted silica comes under the category of non polar adsorbents.
• adsorbents have more affinity with oil or hydrocarbons than water.
• chemical adsorption involves the formation of chemical bonds between atoms on the adsorbed molecule and atoms on the surface of the second phase. It will be appreciated that within the context of the present invention that any type of adsorption that enables an activation of the suspended particles can be considered useful and it is not intended to limit the invention to any one example or constituent of any one example of a suitable adsorbent, indeed distinctly different types of adsorption are clearly present in the examples presented.
• the experimental data is based on an investigation into the interaction of a nonaqueous magnetite suspension with macroporous silica.
• silica-C18 an octadecyl carbon loaded material
• silica-CN a negatively charged cyanated silica
• untreated silica and an APS modified silica an untreated silica and an APS modified silica.
• silica-C18 The substrate used in most of the experiments, silica-C18, was silica with 60A pores, 50 ⁇ m average particle size, and endcapping coated with C18 (Octadecyl, 6% carbon load) from Alltech Associates, Deerfield, IL. Silica-CN a negatively charged cyanated silica, and an untreated silica of the same dimensions from the same source were also used.
• the magnetite nanoparticles were synthesised by co-precipitation of mixed Fe 3 VFe 2+ salts with ammonium hydroxide. A monolayer of surfactant coating was applied by adding excess oleic acid during the iron oxide precipitation. The magnetite dispersion was precipitated with acetone and methanol and washed five times with acetone and lastly with ethanol. The precipitate was then phase transferred into heptane. The heptane suspension was centhfuged at 13000 rpm ( ⁇ 16000g rcf) for 40 min to remove any aggregated particles. Suspension H5a had a Z-average of 12.0 nm with a low PDI (0.09) and H5b had a Z- average size 11.8 nm Z-average and was also monodisperse PDI (0.07).
• the cuvette was placed in a PCS (Photon Correleation Spectroscopy) spectrometer with suitable care to avoid any agitation.
• the temperature of the cuvettes was maintained at 25°C.
• the quantity of silica was restricted so that the incident laser light was not occluded. In effect this limited the height of the silica to ⁇ 1 mm, which is equivalent to 50 mg of silica.
• the silica-C18 particles are spherical with diameter of 50 ⁇ m.
• the adsorbed nanoparticles form a sheet of hexagonal closed packed spheres on the silica-C18 surface to form a uniform monolayer, and A bilayer is composed of two identical monolayers.
• the onset of growth/aggregation of magnetite nanoparticles in suspension was monitored by PCS spectroscopy at an interval of 30 min for 1 -7 days (as long as there were particles in suspension).
• the standard operating procedure (SOP) used for the PCS measurements was for 200 measurements with a suitable delay such that successive measurements are separated by 30 min. Each measurement takes about 5 min which, consists of 20 runs of 10 sec each. For most experiments the cuvette position and the attenuation index remain constant throughout the experiment as all the data collected was with one SOP (standard operating procedure). Therefore the backscattered light intensity is representative of the volume weighted number of scatterers.
• the mean count rates also increased drastically to 4000 kcps in just 7 hours in case of the first run.
• the two runs were performed under identical conditions, as far as possible, using the same magnetite suspension, but 19 days apart.
• Such information would indicate that certain implementations of the methodology of the teaching of the invention may require a monitoring of the size distribution as an indicator as to when is a suitable time to terminate the interaction between the suspended nanoparticles and their source of activation whereas other implementations may obviate the need for such monitoring by making assumptions that after a prescribed time period that a certain size distribution will have been realised.
• the correlation functions for three selected times are shown in Figure 12.
• the quality of data fit is poorer for the later measurements.
• the PDI is high at ⁇ 12 hours, but there is still a good fit to the data, hence the Z-average may be considered a realistic average of the experimental distribution.
• the counts are highest in this region. But after about 24 hours the counts decrease to a small number.
• the fit in blue is only partly through the data points after 48 hours, hence the polydispersity index is 1.00, and the Z-average is a less reliable measure.
• the PCS software for all the ME experiments crashed, due to low scattering intensity, after 25-35 hours.
• ME is the speed of growth (but the dispersity is poor), at least over a standing suspension. Furthermore, one could utilise the differences in growth rates to use an initial ME approach to generate some clusters rapidly which are then fed through to another volume where they are used to seed particles which are being activated using a BE approach.
• Samples for electron microscopy were prepared by spreading a drop of liquid on an SEM stub and allowing it to dry. The liquid was taken from the bottom of a cuvette, using a glass pipette, once the PCS measurement had ended.
• An 'uncoated' silica-C18 sample was prepared by splashing a pinch of powder on the tip of a spatula onto an aluminium SEM stub fixed with conducting carbon tape.
• SEM images of the silica-C18 particles under low magnification indicated that the particles in the range of 25-50 ⁇ m; with a few larger particles of up to 100 ⁇ m.
• the SEM image in Figure 19 gives an insight of the fine morphology of the crystalline silica-C18 surface with very fine tiny white amorphous inclusions. Higher resolution images show the inclusions are in the size range from 50-200 nm.
• the micrographs of most of the regions of monolayer treated magnetite suspension indicated no coating of nanoparticles on the silica-C18 surface
• Magnetite nanoparticles were found to be deposited as large aggregates along edges, particularly in the vicinity of neighbouring silica particles.
• the arrow in Figure 20 indicates one of such few regions of nanoparticle deposits, which was selected for the image shown in Figure 21.
• the magnetic particle clusters are as big as 100-200 nm.
• the Z-average cluster size increased slowly in a linear fashion for the first few hours. This was followed by a transitional phase of faster growth, up to around 12 hours. This phase was associated with an increase in the backscattered light intensity and an increase in the PDI to around 0.2. At this time the clusters are in the region of 100nm diameter. There then followed a phase of very rapid linear growth (of the order of 100 nm/hour) that resulted in clusters in the ⁇ m range within a few hours. This phase was associated with a decrease in scattered light and an increase in the PDI up to the maximum value of 1.0. The data acquisition invariably stopped, due to low backscattered light intensity, within 24 hours.
• the cluster growth was generally slower, and was usually observed to occur in three stages. It started with a brief phase of slow linear growth, similar to the ME experiments, this was followed by another phase of linear growth at a faster rate (c. 0.5nm/hour) and ended with a phase of slower linear growth (c. 0.25nm/hour) in most cases. There were some experiments where growth continued more rapidly and in an anomalous fashion. As in the ME experiment the backscattered intensity increased, until the transition to the slower growth phase at about 40-60 hours, after which it began to decrease again. There are some common features to the ME and BE experiments, there is an induction phase during which there is little change in the particle size, after which the cluster growth accelerates significantly.
• the backscattered light intensity grows to a maximum in both cases before decreasing as the iron concentration decreases.
• the growth is slower and the clusters that remain in suspension do not grow to be as large.
• the growing clusters are also far more monodisperse for the BE experiment.
• the BE experiments if there is an induction phase, it is much shorter than for the ME experiments.
• silica may be responsible for the surface activity.
• impurities such as aluminium oxide
• clusters are observed to grow over C18-grafted silica of higher quality (lower impurity content), while no cluster growth is observed for untreated silica, but very rapid adsorption is observed for silica-CN. It should also be pointed out that while 11 nm magnetite particles will diffuse easily through the gaps between the silica particles, it is unlikely that the larger clusters will be able to do so.
• the population of surface activated nanoparticles in suspension exceeds a critical value.
• the duration of the induction phase varies somewhat, which may be related to the low probability of establishing nanoparticle deposits on the silica surface.
• the magnetite content is relatively low, there remains silica surface capacity for generating more surface activated nanoparticles.
• Figure 9 which probably arises due to the aggregation of clusters.
• the growing clusters precipitate, as is shown in Figure 1.
• Cluster growth is slower and more controlled.
• the magnetite content is relatively high, so there is less available silica surface capacity for generating surface activated nanoparticles, which therefore constitute a smaller fraction of the total suspended matter throughout.
• Cluster growth is due to the addition of individual nanoparticles to the growing clusters. There is a strong tendency for all the clusters in the population to grow together, which suggests that the clusters at the upper end of the distribution are somewhat less reactive.
• Figure 13 There is eventually a transition to a phase of slower cluster growth, Figure 13, which may be due to the complete depletion of the original 11 nm particles from the suspension.
• the curve at 44 hours still contains some nanoparticles, this corresponds to the time at which the initial growth begins to slow.
• the invention has been described with reference to a nanoparticles generated using a technique advocated by Sun et al.
• the Sun approach produces coated nanoparticles.
• the invention is not to be construed as being limited to the formation of the initial stabilized nanoparticles using the Sun technique.
• an experiment was performed where a sample of oleic acid coated nanoparticles, which were prepared by a different synthetic route, in heptane was placed over an alkyl grafted -C18 substrate obtained from a different source to that used in the previously described experiments.
• the nanoparticle preparation involved formation of a micro-emulsion and was based on a published procedure [Lee, H. S., Lee, W. C, Furubayashi, T.
• citrate coated gold particles are provided in a water solution (2400).
• the coated and stabilized nanoparticles are provided with a size distribution of about 16-17nm.
• the suspension is brought into contact with a (3-aminopropyl) triethoxysilane-modified (APS- modified) silica substrate or particles of such a silica having dimensions of about 300nm (2405).
• APS-modified silica results in formation of a layer of nanoparticles on the suface of the substrate and the generation of reactive gold nanopartiles in the suspension (2410).
• Two or more of these reactive nanoparticles combine with one another to effect generation of gold nanoparticle clusters in a water suspension(2415). It will be appreciated that this reinforces the general application of the teaching of the invention as to how controlled growth of nanoparticle clusters can be effed through provision of capped nanoparticles and a suitable activating agent that exhibits an affinity for the capping agent.
• Figure 25 shows examples of TEM images of the gold nanoparticle nanocluster growth. It is evident that after a period of 1 hour that the size distribution of the clusters has grown to about 300nm from less than the initial 100nm after 1 minute exposure of the capped gold nanoparticles to the activating agent- in this case (3-aminopropyl) triethoxysilane (APS)-modified silica.
• the activating agent in this case (3-aminopropyl) triethoxysilane (APS)-modified silica.
• This stripping is effected by providing an activating agent that exhibits an affinity for the capping agent and the stripping could be effected through either a direct, an indirect or a combination of the two mechanisms.
• the results or features described with reference to one figure or example could be interchanged with those of another figure or example without departing from the spirit or scope of the invention as it will be understood that an inter-changeability of integers or components may be possible and should be considered as falling within the teaching of the invention.
• results presented in the experimental data are exemplary of the types of results that may be obtained using the teaching of the invention. Where they are explained with reference to certain underlying beliefs or understandings it is not intended that the invention be limited to such beliefs or understandings.
• the activation of a nanoparticle in suspension using an activation agent and the subsequent growth of clusters as is provided by the teaching of present invention should not be limited in any fashion except as may be deemed necessary in the light of the appended claims.

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