Technical Field of the Invention

The present invention relates to a plasmonic nanoparticle assembly.

In particular, the present invention relates to a plasmonic nanoparticle assembly for use in inter alia biomedical applications, e.g. biomedical theranostics; energy applications, e.g. as near-infrared and/or visible absorbers and solar cells; and metamaterial applications.

Background to the Invention

In recent years, there has been a growing interest in potential uses of metal nanoparticles in biomedical applications, e.g. targeted drug delivery, photoablation therapeutics, cancer therapeutics, biosensors, bioimaging and bio-diagnostics; energy applications; and metamaterial applications.

Metal nanoparticles can exhibit an optical phenomenon known as localised surface plasmon resonance (LSPR). This phenomenon occurs when light interacts with metal nanoparticles that are smaller than the incident wavelength of light.

LSPR enhances the electromagnetic field around metal nanoparticles, which can be utilised to enhance spectroscopic signals such as during surface-enhanced Raman scattering (SERS). As a result of this enhancement in combination with suitable detection techniques, metal nanoparticles can work effectively as theranostic agents.

The optical properties of metal nanoparticles can be tailored towards a particular application, e.g. by varying the morphology of nanoparticles and interaction between nanoparticles to achieve a specific LSPR wavelength of maximum absorption (kmax).

For example, near-infrared (NIR) absorbance is important for in vivo biomedical diagnostics and therapeutics, as NIR light photons have high penetration into biological tissue and low absorbance by biological tissue.

Various nanoparticle assemblies have been developed. For example, a prior art plasmonic nanoparticle assembly comprises a core metal nanoparticle and a halo of outer metal nanoparticles. Another prior art plasmonic nanoparticle assembly comprises a core metal nanoparticle and outer metal nanoparticles, each outer nanoparticle attached to the core metal nanoparticle by a single linker molecule.

However, there are problems with many nanoparticle assemblies of the prior art. For example, many prior art assemblies require elaborate synthesis, purification and characterisation. In addition, many prior art assemblies are synthesised using processes which are not environmentally friendly. These problems mean that many prior art assemblies are not suitable for scale-up and therefore are not attractive for large-scale field applications.

It is an object of the present invention to provide a plasmonic nanoparticle assembly that overcomes or ameliorates one or more of the above-mentioned or other problems.

Summary of the Invention

According to a first aspect of the present invention there is provided a plasmonic nanoparticle assembly comprising: a core metal nanoparticle having a first median average particle size; and at least two arms, each arm comprising: at least one linking molecule and at least two outer metal nanoparticles, the or each linking molecule in each arm directly or indirectly connecting the outer metal nanoparticles to the core metal nanoparticle, wherein the metal of the core metal nanoparticle and the metal of the outer metal nanoparticles are independently selected from a noble metal and copper, the outer metal nanoparticles have a second median average particle size which is smaller than the first median average particle size, and the plasmonic nanoparticle assembly has a total mean average particle size of less than 150 nm.

In some embodiments the nanoparticle assembly has a total mean average particle size of less than 125 nm, less than 110 nm or no more than 100 nm. The total mean average particle size is suitably determined using dynamic light scattering (DLS), or transmission electron microscopy (TEM).

In embodiments, the plasmonic nanoparticle assembly exhibits broadband near- infrared (NIR) absorbance, e.g. across the ‘optical window’ of 650 nm to 1100 nm; and colloidal stability, rendering it particularly suitable for use in in vivo diagnostic applications and tracking applications. The assembly may be biocompatible.

The assembly may be operable to be resonant with, or absorb, electromagnetic radiation across the near-infrared (NIR) region of the electromagnetic spectrum. In this way, the assembly may be detectable across the near-infrared (NIR) region of the electromagnetic spectrum using spectroscopic techniques.

The assembly may be operable to be resonant with and/or absorb electromagnetic radiation across the 650 nm to 1100 nm region of the electromagnetic spectrum. In this way, the assembly may be detectable across the 650 nm to 1100 nm region of the electromagnetic spectrum using spectroscopic techniques.

The assembly may be operable to be resonant with and/or absorb electromagnetic radiation across the visible region of the electromagnetic spectrum. In this way, the assembly may be detectable across the visible (VIS) region of the electromagnetic spectrum using spectroscopic techniques.

The core nanoparticle may be made of metal selected from any noble metals and copper, as desired or required for a given application.

The core nanoparticle may comprise, substantially comprise, consist essentially of or consist of one or more metals selected from the group of ruthenium, rhodium, palladium, silver, osmium, iridium, platinum, gold and copper. The core nanoparticle may comprise at least 90 wt.%, suitable at least 95 wt.%, suitably at least 99 wt.%, suitably at least 99.5 wt.% of one or more metals selected from the group of ruthenium, rhodium, palladium, silver, osmium, iridium, platinum, gold and copper, based on the total weight of the core nanoparticle.

In some embodiments the core nanoparticle comprises, substantially comprises, consists essentially of or consists of gold, silver or copper. The core nanoparticle may comprise at least 90 wt.%, suitable at least 95 wt.%, suitably at least 99 wt.%, suitably at least 99.5 wt.% of one or more of gold, silver and copper, based on the total weight of the core nanoparticle.

The first median average particle size may be up to about 100 nm, suitably up to about 80 nm, suitably up to about 60 nm, suitably up to about 50 nm, suitably up to about 40 nm, suitably up to about 30 nm, suitably up to about 20 nm. The first median average particle size may be about 10 nm to about 90 nm, suitably about 8 nm to about 50 nm, suitably about 12 nm to about 40 nm, suitably about 15 nm, such as in the range 10 nm to 25 nm or 10 nm to 20 nm. The first median average particle size is suitably determined using DLS or TEM.

Each outer nanoparticle may be made of any metal selected from any noble metals and copper, as desired or required for a given application.

Each outer nanoparticle may comprise, substantially comprise, consist essentially of or consist of one or more metal selected from the group of ruthenium, rhodium, palladium, silver, osmium, iridium, platinum, gold and copper. The outer nanoparticles may comprise at least 90 wt.%, suitable at least 95 wt.%, suitably at least 99 wt.%, suitably at least 99.5 wt.% of one or more metal selected from the group of ruthenium, rhodium, palladium, silver, osmium, iridium, platinum, gold and copper, based on the total weight of the outer nanoparticles.

In some embodiments the core nanoparticle comprises, substantially comprises, consists essentially of or consists of gold, silver or copper. The outer nanoparticle may comprise at least 90 wt.%, suitable at least 95 wt.%, suitably at least 99 wt.%, suitably at least 99.5 wt.% of one or more of gold, silver and copper, based on the total weight of the outer nanoparticles.

In some embodiments, both the core nanoparticle and outer nanoparticles independently comprise, substantially comprise, consist essentially of or consist of gold or silver, preferably gold when used for biomedical applications. Both the core nanoparticle and the outer nanoparticles may independently comprise at least 90 wt.%, suitable at least 95 wt.%, suitably at least 99 wt.%, suitably at least 99.5 wt.% of one or more of gold, silver and copper, preferably gold when used for biomedical applications, based on the total weight of the core nanoparticle and outer nanoparticles.

The second median average particle size may be up to about 30 nm, suitably up to about 20 nm, suitably up to about 16 nm, suitably up to about 12 nm, suitably up to about 8 nm, suitably up to about 6 nm. The second median average particle size may be from about 1 nm to about 10 nm, suitably about 2 nm to about 8 nm, suitably about 4 nm to about 6 nm, suitable about 5 nm. The second median average particle size may suitably be determined using Dynamic Light Scattering (DLS) or Transmission Electron Microscopy (TEM).

The second median average particle size may be up to about 70%, suitably up to about 60%, suitably up to about 50%, suitably up to about 40%, suitably up to about 30%, suitably up to about 20%, of the first median average particle size. The second median average particle size may be from about 5% to about 70%, suitably from about 10% to about 60%, suitably from about 15% to about 50%, suitably from about 20% to about 40%, suitably from about 25% to about 35%, of the first median average particle size.

The arms may have a median average length of from about 5 nm to about 50 nm, suitably about 6 nm to about 40 nm, suitably about 8 nm to about 30 nm, suitably about 10 nm to about 25 nm. The mean/median average length of the arms is suitably determined using DLS or TEM.

In some embodiments the, more or each arm independently has a median length and/or median width of at least 5 nm, at least 6 nm, at least 7 nm, at least 8 nm, at least 9 nm or at least 10 nm. In some embodiments, each arm independently has a median length and median width of at least 7 nm or at least 8 nm. The median average length and/or median average width of the, more or each arm is suitably determined using DLS or TEM.

Each linking molecule may comprise three, four or more nanoparticle connection groups. Each nanoparticle connection group of each linking molecule may be connected to a core nanoparticle or an outer nanoparticle.

One or more of the nanoparticle connection groups may be independently provided towards or at a terminal region of each linking molecule.

One, more or each arm may comprise two or more linking molecules. In some embodiments each arm comprises a first linking molecule connected to a core nanoparticle and one or more outer nanoparticles, and a second linking molecule being connected to the same outer nanoparticle as the first linking molecule and at least one further outer nanoparticle. One, more or each arm may comprise a series or chain of linking molecules, each connected via a shared outer nanoparticle. Each linking molecule in the series or chain may be connected to a further outer nanoparticle not connected to any other linking molecule, and/or connected to a branch linking molecule.

Alternatively, or additionally, one more or each arm may comprise a two- dimensional or three-dimensional network of linking molecules and outer nanoparticles; such as at least one linking molecule connected to a core nanoparticle and a first outer nanoparticle, the first outer nanoparticle being connected to a two- dimensional or three-dimensional network of further linking molecules connected to nodes comprising an outer nanoparticle.

Examples of suitable nanoparticle connection groups include one or more of amine (-NEE) groups, nitro (-NO ₂ ) groups, alkyne groups, thiol (-SH) groups (e.g. dithio groups and trithio groups), azide groups and carboxyl (-COOH) groups. The SH group can be advantageous for connection to gold nanoparticles. The -COOH group can be advantageous for connection to silver nanoparticles.

Each linking molecule may be flexible.

Each linking molecule may be a polymer. Any suitable polymer may be used, as are known to those skilled in the art. The polymer may be a linear polymer. Alternatively, the polymer may be a branched polymer.

Where each linking molecule is a polymer, one or more of the nanoparticle connection groups may be independently provided towards or at a terminal region of the polymer.

Where each linking molecule is a branched polymer, one or more of the nanoparticle connection groups may be independently provided towards or at a terminal region of a branch of the branched polymer.

Each linking molecule may be independently selected from the group comprising a polyester, a polyether, a polyurethane and a vinyl polymer. Examples of suitable polyesters include polylactides, polyacrylates (e.g. polymethyl methacrylate, methyl acrylate and ethyl acrylate) and any combination thereof.

Examples of suitable polyethers include polyalkylene glycols (e.g. polyethylene glycol, polyethylene glycol methacrylate).

Suitably, the polyether is 4arm-PEG5K-SH of the general formula:

Examples of suitable vinyl polymers include polyacrylonitriles and polyalkylenes (e.g. polypropylene and polystyrene) and any combination thereof. Each linking molecule may have a median average particle size of from 0.2 nm to about 10 nm, suitably about 0.5 nm to about 7 nm, suitably about 0.8 nm to about 4 nm, suitably about 1 nm to about 3 nm, suitably about 2 nm. The mean average particle size of the linking molecule may be determined using DLS or TEM.

Each arm may comprise three, four, five, six, seven, eight, nine, ten or more outer noble metal and/or copper nanoparticles.

The plasmonic nanoparticle assembly may comprise one or more functional agents connected to the assembly.

Non-limiting examples of functional agents include pharmaceuticals, therapeutic or therapy-related molecules, radioactive agents, diagnostic labels, targeting ligands (e.g. antibodies, aptamers, deoxyribonucleic acid (DNA) and micro- ribonucleic acid (micro-RNA), biocompatible polymers (e.g. polymers for enhancing biocompatibility), biomarkers and any combination thereof.

Examples of suitable diagnostic labels include surface enhanced Raman spectroscopy (SERS) labels e.g. 2-quinolinethiol (QTH) and biphenylthiol (BPT) and 4-MBA. The skilled person will appreciate that many other functional agents can be used, as desired or required for a given application.

According to a second aspect of the present invention, there is provided a composition comprising the plasmonic nanoparticle assembly of the first aspect of the present invention.

The composition may be an aqueous composition. Alternatively, the composition may be an organic-carrier based composition (i.e. a composition having an organic solvent).

The composition may be one or more of a nanoplasmonic composition, a biomarker composition, a pharmaceutical composition, a radio-pharmaceutical composition and a biological theranostic composition.

The composition may comprise the plasmonic nanoparticle assembly at a concentration of about 100 wt.%, suitably from about 50 wt.% to about 90 wt.%, suitably from about 60 wt.% to about 80 wt.%, based on the total weight of the composition.

The composition may further comprise one or more stabilising agents, biocompatibility agents, targeting agents, labelling agents, functional agent conjugating molecules.

According to a third aspect of the present invention, there is provided the plasmonic nanoparticle assembly according to the first aspect of the present invention, or a composition according to the second aspect of the present invention, for use as a medicament.

The plasmonic nanoparticle assembly according to the first aspect of the present invention, or the composition according to the second aspect of the present invention, may be for use as a biological agent, diagnostic agent, therapeutic agent, theranostic agent and/or biosensor (e.g. optical biosensor).

According to a fourth aspect of the present invention, there is provided a method of preparing a plasmonic nanoparticle assembly of the first aspect of the present invention, the method comprising the steps of: (i) providing core metal nanoparticles, outer metal nanoparticles and linking molecules; and

(ii) mixing the core metal nanoparticles, the outer metal nanoparticles and the linking molecules; or

(iii) (a) mixing the core metal nanoparticles or the outer metal nanoparticles, and the linking molecules to form functionalised metal nanoparticles, and (b) mixing the functionalised metal nanoparticles and the other of the core metal nanoparticles and the outer metal nanoparticles.

In step (i), the core metal nanoparticles may be provided in a composition. The composition may be an aqueous composition. Alternatively, the composition may be an organic carrier-based composition (i.e. a composition wherein the solvent is an organic solvent).

In step (i), the outer metal nanoparticles may be provided in a composition. The composition may be an aqueous composition. Alternatively, the composition may be an organic carrier-based composition.

In step (i), the linking molecules may be provided in a composition. The composition may be an aqueous composition. Alternatively, the composition may be an organic carrier-based composition.

In step (ii) or (iii)(a), the linking molecules may be in excess relative to the metal nanoparticles (outer metal nanoparticles and core metal nanoparticles). In step (ii) or (iii)(a), the ratio of the number of the linking molecules to the total number of metal nanoparticles may be from 2: 1 to 100000: 1, suitably from 50: 1 to 1000: 1, suitably from 300:1 to 600:1.

In embodiments, step (iii)(a) may comprise mixing the core metal nanoparticles and the linking molecules to form functionalised core metal nanoparticles. In such embodiments, step (iii)(b) may comprise mixing the functionalised core metal nanoparticles and the outer metal nanoparticles. In such embodiments, in step (iii)(b), the outer metal nanoparticles may be in excess relative to the core metal nanoparticles. In such embodiments, in step (iii)(b), the ratio of the number of outer metal nanoparticles to the number of core metal nanoparticles may be at least about 8:1, suitably at least about 20:1, suitably at least about 25:1, suitably at least about 30:1, suitably at least about 35:1, suitably at least about 40:1. In such embodiments, in step (iii)(b), the ratio of the number of outer metal nanoparticles to the number of core metal nanoparticles may be from about 10:1 to about 60:1, suitably from about 30:1 to about 50:1, suitably from about 36: 1 to about 44: 1, suitably from about 38:1 to about 42: 1.

In other embodiments, step (iii)(a) may comprise mixing the outer metal nanoparticles and the linking molecules to form functionalised outer metal nanoparticles. In such embodiments, step (iii)(b) may comprise mixing the functionalised outer metal nanoparticles and the core metal nanoparticles. In such embodiments, in step (iii)(b), the outer metal nanoparticles may be in excess relative to the core metal nanoparticles. In such embodiments, in step (iii)(b), the ratio of the number of outer metal nanoparticles to the number of core metal nanoparticles may be at least about 8:1, suitably at least about 20:1, suitably at least about 25:1, suitably at least about 30:1, suitably at least about 35:1, suitably at least about 40:1. In such embodiments, in step (iii)(b), the ratio of the number of outer metal nanoparticles to the number of core metal nanoparticles may be from about 10:1 to about 60:1, suitably from about 30:1 to about 50:1, suitably from about 36:1 to about 44:1, suitably from about 38 : 1 to about 42 : 1.

The method may comprise providing one or more functional agents.

In embodiments, the method may comprise, during step (ii), during step (iii)(a), between step (iii)(a) and step (iii)(b) or during step (iii)(b), mixing in the or each functional agent.

In embodiments, the method may comprise, after step (ii) or after step (iii(b)), mixing in the or each functional agent.

Mixing in the or each functional agent during step (ii), during step (iii)(a), between step (iii)(a) and step (iii)(b) or during step (iii)(b), can be described as ‘pre- labelling’. Advantageously, pre-labelling can increase the likelihood of the functional agent reaching Raman hotspots on the plasmonic nanoparticle assembly. In such embodiments, step (iii)(b) may comprise mixing the functionalised metal nanoparticles labelled with the or each functional agent and the other of the core metal nanoparticles and the outer metal nanoparticles.

Mixing in the or each functional agent after step (ii) or after step (iii(b)), can be described as ‘post-labelling’. Post-labelling can be useful in applications wherein a low amount of functional agent is required.

The or each functional agent may be provided at any suitable concentration as desired or required for a given application.

The or each functional agent may be present in an amount of up to 0.0001 wt.%, suitably up to 0.001 wt.%, suitably up to 0.01 wt.%, suitably up to 0.1 wt.%, suitably up to 1 wt.%, suitably up to 5 wt.%, suitably up to 10 wt.%, suitably up to 20 wt.%, suitably up to 40 wt.%, suitably up to 60 wt.%, suitably up to 80 wt.%, based on the total weight of the plasmonic nanoparticle assembly.

The ratio of the number of the or each functional agent to the number of the core metal nanoparticles may be from 1 : 1 to 100000: 1, suitably from 10: 1 to 5000: 1, suitably from 20:1 to 2000:1.

The ratio of the number of the or each functional agent to the number of the plasmonic nanoparticle assemblies may be from 1:1 to 100000:1, suitably from 10:1 to 5000:1, suitably from 20:1 to 2000:1.

The skilled person will appreciate that any feature or features of any aspect of the present of the invention may be combined with any other feature or features of any other aspect of the present invention.

Detailed Description of the Invention

In order that the invention may be more clearly understood one or more embodiments thereof will now be described, by way of example only, with reference to the accompanying drawings, of which:

Figure 1A shows a UV-Visible-NIR spectrum of (i) 15 nm Au NPs, (ii) 5 nm Au NPs and (iii) multi-tentacle (multi-arm) nanoparticle assemblies;

Figure IB shows three vials, one containing an aqueous suspension of the 5 nm Au NPs, the second containing an aqueous suspension of the 15 nm Au NPs and the third containing an aqueous suspension of the multi-tentacle (multi-arm) nanoparticle assemblies;

Figure 1C shows a dynamic light scattering (DLS) size distribution of the multi arm nanoparticle assembly; Figure ID shows transmission electron micrographs (TEMs) of the multi-arm nanoparticle assembly;

Figure IE shows tilted TEMs of the multi-arm nanoparticle assembly, at tilt angles of -30°, 0° and +30°;

Figure IF shows statistical analysis of the number of arms per NP core of the multi arm nanoparticle assemblies, obtained using ImageJ on conventional TEM micrographs;

Figure 1G shows statistical analysis of the arm length of the multi-arm nanoparticle assemblies, obtained using ImageJ on conventional TEM micrographs;

Figure 1H shows TEM-energy dispersive x-ray spectroscopy (EDS) mapping of sulfur present in the linking polymers in the multi-arm nanoparticle assemblies;

Figure II shows TEM-selected area electron diffraction (SAED) of the multi-arm nanoparticle assemblies;

Figure 2 shows a schematic representation of formation of the multi-arm nanoparticle assemblies;

Figure 3 A shows a schematic representation of a gold NP core functionalised with linking polymers;

Figure 3B shows a graphical representation of the change in morphology of the nanoparticle assemblies as a function of the ratio of the number of reactant core NPs (X) and reactant outer NPs (Y);

Figure 3C shows a UV-Visible spectrum of a (i) control, and (ii-iv) nanoparticle assemblies formed at different ratios of the number of reactant core NPs (X) and reactant outer NPs (Y); Figure 4A shows a surface enhanced Raman spectroscopy (SERS) spectrum of multi-arm nanoparticle assemblies measured with a 830 nm laser line: (i) multi-arm nanoparticle assemblies with no label (= 0 M); (ii) linking polymer- functionalised core Au NP labelled with 2-quinolinethiol (QTH) ([QTH]=4.76xlO ^_8 M); and (iii) multi-arm nanoparticle assemblies pre-labelled with QTH ([QTH]=1.7xlO ^-7 M); intensity scale bar = 100 counts, accumulation = 60 s; and a graphical representation of (i), (ii) and (iii);

Figure 4B shows a SERS spectrum of multi-arm nanoparticle assemblies post- labelled with QTH ([QTH]=1.7xlO ^-7 M); intensity scale bar = 1000 counts, accumulation = 50 s; and a graphical representation of the multi arm nanoparticle assemblies;

Figure 4C shows a SERS spectrum of multi-arm nanoparticle assemblies post- labelled with biphenylthiol (BPT): (i) [BPT]=1.4xlO ^-5 M, (ii) [BPT]=1.25xlO^ M and (iii) [BPT]=2.22xlO- ⁴ M; intensity scale bar =

5000 counts, accumulation = 10 s; and a graphical representation of the multi-arm nanoparticle assemblies;

Figure 5 shows a TEM micrograph of a linking polymer- functionalised 15 nm Au NP positioned in a hole of a holey carbon coated copper grid; Figure 6 shows a UV-Visible-NIR spectrum of: (i) linking polymer- functionalised 15 nm Au NPs, (ii) after overnight incubation of 15 nm Au NPs and 5 nm Au NPs in volume ratios of 6 (15 Au NPs 6:1 5 nm Au NPs), and (iii) multi-arm nanoparticle assemblies at the same volume ratio; Figure 7 shows linking polymer-induced aggregation behaviour of 5 nm NPs (A) UV-Vis spectrum of (i) citrate-stabilised 5 nm Au NP exhibiting LSPR = 510 nm, and (ii) linking polymer mediated random aggregation of 5 nm NPs exhibiting LSPR = 525 nm negligible NIR absorbance and (B) TEM micrographs of linking-molecule mediated random aggregation of 5 nm NPs in low and high magnification; and Figure 8 shows a DLS plot of 4-arm PEG thiol linking polymer 1 mM in milli-Q water wherein the solution was filtered via a syringe filter of 0.2 pm before analysis.

Instruments

UV Vis Spectroscopy. Ultraviolet-visible spectra were acquired using an Evolution Array UV-Visible spectrophotometer in the range of 400-1100 nm with a 1 cm path length cell with baseline correction.

Dynamic Light Scattering (DLS). DLS measurements were performed using a Malvern Zetasizer Ultra running DTS software and a 4 mW He-Ne laser at 633 nm. Analysis was performed at an angle of 90° and a constant temperature of 25 °C. Dilute particle concentrations were used to ensure that multiple scattering and particle-particle interactions could be considered to be negligible. Disposable cuvettes of 1 cm pathlength was employed for size measurements, whereas, surface charge zeta potential measurements were performed in a capillary folded disposable cuvette. Three repeats of each were collected and an average and median value obtained from them.

Transmission Electron Microscopy. A JEOL 2100 transmission electron microscope (TEM) was used to study the NP morphologies on 200 mesh holey C-coated copper grids at 100 kV. As-prepared hybrid samples were diluted 100-fold and deposited on TEM grids so as to minimize the drying artifacts. The sizes were determined using ImageJ software by measuring ~50-70 individual assemblies per sample. Additionally, energy-dispersive X-ray spectrometer (EDS) and selected-area electron diffraction (SAED) was measured on the TEM grid with the same sample on which TEM was performed. EDS was performed with an Oxford Instrument X-MAXN EDS detector, it allows chemical analysis of features as small as a few nanometers. The SAED measurements was performed at 200kV.

Raman Spectroscopy. SERS spectra were recorded with in the spectral range of 200-2000 cm ^-1 with a Renishaw model In Via micro Raman spectrometer equipped with 830 nm excitation from a diode laser, a single diffraction grating, and an electrically cooled CCD detector. The laser power was 30 mW at the sample. Accumulations from 10-60 s were used for various measurements. Low-volume quartz cuvettes were used as sample holders.

Synthesis of Gold Nanoparticles

Two batches of citrate-stabilised gold nanoparticles (Au NPs) were synthesised using traditional methods of reducing chloroauric acid by sodium citrate and sodium borohydride. The first batch comprised citrate-stabilised Au NPs having a median average particle size of 15 nm, synthesised via reducing gold salt with sodium citrate at boil. The second batch comprised citrate-stabilised Au NPs having a median average particle size of 5 nm. The 5 nm Au NPs included of a range of sizes, from about 2 nm to 6 nm. Purification or segregation to obtain narrower particle size distributions was avoided to maintain scalability. Commercially available 15 nm and 5 nm Au NPs have similar particle size distributions and hence could be used as “off-the-shelf’ reactant. Although the nanoparticles herein were synthesized in a laboratory, they could alternatively be purchased with similar specifications.

Using UV-Visible spectroscopy, it was determined that the 15 nm citrate-stabilised Au NPs exhibited LSPR _ma\ at 515 nm, and the 5 nm citrate-stabilised Au NPs exhibited LSPR _max at 510 nm, as shown in Fig. 1A.

Using transmission electron microscopy (TEM), it was determined that the 15 nm citrate-stabilised AuNPs had a TEM size of 16 ± 1 nm, and the 5 nm citrate-stabilised Au NPs had a TEM size of 4 ± 2 nm (averaged from 90-100 population using ImageJ).

Dynamic light scattering (DLS) indicated median average particle sizes of 15 nm for the 15 nm citrate-stabilised AuNPs and 5 nm for the 5 nm citrate-stabilised AuNPs,

Functionalisation of 15 nm Gold Nanoparticles with Linking Polymers

The 15 nm Au NPs (which served as core NPs) were functionalised with a linking polymer, i.e. a four-arm PEG5k-SH polymer (purchased from Sigma- Aldrich and used as received) having a number average molecular weight (M„) of 5000 Da and the below general chemical structure. An aliquot of 150 pL from a 10 mM aqueous solution of the linking polymer was added to 3 mL of 15 nm Au NPs (containing approximately 8.3 x 10 ¹¹ NPs/mL; calculated from absorbance at 450 nm in accordance with ‘ Haiss , W.; Nguyen I K., I; Jenny, A.; David G., F. Determination of Size and Concentration of Gold Nanoparticles from Extinction Spectra. Anal. Chem. 2008, 80 (17), 6620 6625 as would be known by a person skilled in the art) suggesting a theoretical availability of 3.6 x 10 ⁵ polymer macromolecules/NP (« 509 polymer molecules/nm ² ).

This ensured a full coverage of linking polymer onto the core 15 nm Au NPs. Equilibrium was reached by standing the solution to rest overnight, then excess linking polymer was removed by centrifugation, then the sample was re-suspended in milli-Q water.

The linking polymer- functionalised 15 nm Au NPs showed an LSPR redshift of 2 nm relative to the citrate-stabilised 15 nm Au NPs. Moreover, the linking polymer- functionalised 15 nm Au NPs exhibited a zeta potential of -12 mV whereas the citrate- stabilised 15 nm Au NPs exhibited a zeta potential of -48 mV. Further increase in linking polymer concentration did not affect the surface charge of the sample, indicating that full coverage was reached.

TEM micrographs of the linking polymer- functionalised 15 nm Au NPs presented a polymer “halo” around each NP as observed on the holey carbon-coated TEM grid (see Fig. 5).

The TEM micrographs, along with the LSPR shift and zeta potential change, confirmed the ligand exchange and linking polymer functionalisation.

Formation of the Multi- Arm NP Assemblies

An excess of citrate-capped 5 nm Au NPs (denoted by Y) was mixed with the prepared linking polymer- functionalised 15 nm core Au NPs (denoted by X), corresponding to a volume ratio of Y/X of 6. This resulted in a ratio of the number of 15 nm core Au NPs to 5 nm outer Au NPs of approximately 31 (i.e. net NPs of 5 nm ÷ net NPs of 15 nm).

The resultant colloid sample was left overnight and characterized by UV-Vis spectroscopy. Although the absorbance profile of the product showed negligible change in the primary LSPR peak, an increase in the NIR absorbance of approximately 33% of that of its LSPR (Xmax) was observed in the region of 650-1100 nm (see Fig. 6 (ii)). The colloid sample was then centrifuged to separate the unanchored 5 nm Au NPs, the supernatant was removed and the sample was resuspended in milli-Q water. The colloid sample suspension was purple in colour.

The absorbance profile of the resultant nanoparticle assembly is shown in Fig 6 (iii), as well as Fig. lA(iii). The UV-Vis-NIR spectrum of the fully formed gold nanoparticle assembly featured an LSPR primary peak at approximately 540 nm with a significant increase in the broadband NIR absorbance of ca. 83% of that at its l,,, _ac in the entire optical window of 650-1100 nm.

Further analysis of the physical properties of the multi-arm nanoparticle assembly revealed the characteristic morphological features thereof which are shown in Figs. 1C- G. In particular, the gold nanoparticle assembly was characterised by a DLS median average particle size of 69 nm (see Fig. 1C) and a TEM median average particle size of 62±4 nm measured from 87 individual gold nanoparticle assembly TEM images using ImageJ (a number of which TEM images are shown in Fig. ID). The multi-arm nanoparticle assembly with a sub- 100 nm total mean average particle size is beneficial for in vivo diagnostics applications, as larger nanostructures would not preferentially be taken up by tumor cells by virtue of the enhanced penetration and retention (EPR) effect. As the process is entirely water-based, the process is environment-friendly and inherently less toxic to living cells.

Morphology of the Multi-Arm Nanoparticle Assembly

To obtain a better understanding of the morphology of the multi-arm nanoparticle assembly, tilted TEM was performed thereon, spanning a total angle of 60° with measurements at +30°, 0° and -30° tilts. The tilted TEM images are shown in Fig. IE (row-wise, different gold nanoparticle assemblies) at the three tilt angles (column- wise). This confirms that the multi-arm nanoparticle assemblies are unique 3-D nanostructures each with multiple ‘tentacles’ (or arms) anchored tightly onto the core NP and projecting in different directions.

Further analysis of the TEM micrographs, employing ImageJ and measuring approximately 50-70 individual multi-arm nanoparticle assemblies, revealed that the multi-arm nanoparticle assemblies each had an average of 4-5 arms per core (see. Fig. IF) with a median average arm length of approximately 14-20 nm (see Fig. 1G), suggesting that most arms are longer in length than width. It was observed that all arms were >8 nm in both the median average length and median average width, which suggested that neither the length nor the width was composed of a single 5 nm NP (corresponding to actual size of 4 ± 2, i.e. from 2 nm to 6 nm. Additionally, the arms could not have been composed of 15 nm NPs as that would require the length and width both to be above 15 nm, which was not the case. This was surprising as the freely available linker end groups of the linking polymers would typically attach to one or two 5 nm Au NPs providing a morphology structurally similar to core-satellites.

TEM-energy dispersive x-ray spectroscopy (EDS) was used to map the sulfur of the thiol end group of linking polymer on the nanoparticle assembly and confirmed that the arms were formed of 5 nm NPs inter-linked with the linking polymer.

The multi-arm nanoparticle assemblies were further analysed using selected area electron diffraction (SAED)-TEM (see Fig. II). This showed ring patterns consistent with poly crystalline spherical Au NPs corresponding to diffraction rings of 111, 200, 220 and 311 planes. The most prominent ring of the low energy uniform facet of 111 confirmed that the multi-arm nanoparticle assemblies were composed of the octahedral facet of single nanoparticles. The other 200, 220 and 311 reflections also correlate to facets of trapezohedron-type structures present in smaller fractions in pseudo-spherical nanoparticles. The absence of 100 and 110 facet confirmed the absence of any rod-like or cube-like structures. The above observations thus ruled out the likelihood of any crystal overgrowth in the multi-arm nanoparticle assembly morphology.

The experimental data suggests formation of core multi-arm nanoparticle assemblies, comprising the 15 nm Au NP as the core and multiple 5 nm Au NPs with the linking polymers to form each arm. The flexible ester bonds of the polyethylene glycol branches of the linking polymer makes it feasible for one linking polymer to anchor one to three 5 nm Au NPs.

The linking polymer featuring a DLS median average size of 2.5 ± 1.5 (i.e., 1-4 nm) (see Fig. 8) creates a scenario of size overlap with some population of the 5 nm NP (i.e., 2-6 nm). The average linking polymer footprint is approximately 5 nm ² (2.5 nm diameter) with a range of 0.8-12 nm ² (1-4 nm diameter) projecting onto a 5 nm NP with average surface area of 80 nm ² within the range of 12-113 nm ² (2-6 nm diameter). The steric hindrance of close packing of thiol end groups onto the high curvature of 5 nm NP, in addition to the linking polymer-NP size and footprint overlap, might hinder anchoring of multiple end groups onto one NP. In contrast, there is an increased probability for the linking polymer to bind to two or more 5 nm NPs, preferably of the smaller dimensions. These therefore allow for multiple anchoring aiding the formation of multi-tentacles.

In a separate experiment, 5 nm Au NPs were reacted with the linking polymer. The 5 nm NPs assembled together forming tentacular structures without being able to observe a clear periphery of the individual 5 nm NPs comprising the tentacular nano-structure (see Fig. 7). This confirms that the tentacles or arms are inherently composed of multiple 5 nm NPs. Such tentacle nanostructures presented a single peak LSPR at 525 nm, devoid of any NIR absorbance. This indicates superiority of the developed core multi-arm nanoparticle assembly structure.

Fig. 2 illustrates the formation of the multi-arm nanoparticle assembly. The notable 3- dimensional morphologies are near-isotropic due to the random arrangement of arms on the core. This would thus favor polarization-independent absorbance and SERS behavior similar to nano-branched assemblies, as opposed to polarization dependency of anisotropic gold nanorods and linear nanochain assemblies. This behavior is especially important for in vivo applications where the orientation of the nanostructures in the media cannot be controlled.

The Effect of Varying the Ratio of 15 nm Au NPs to 5 nm Au NPs

Two further ratios of the number (ratio of net number of NPs) of linking polymer functionalised 15 nm Au NPs (denoted by X) and citrate stabilised 5 nm Au NPs (denoted by Y) were studied, corresponding to Y/X =1.3 and Y/X=5 (in addition to Y/X=31, as above). Further nanoparticle assemblies were prepared using those ratios and employing an analogous method to that described hereinabove.

The three different ratios yielded categorically different nano-assembly morphologies with distinctly different absorbance profiles, as shown in Figs 3 A-C. As a control (see Fig. 3C (i)), citrate stabilized 15 and 5 nm Au NPs were mixed in absence of the linking polymer.

The control presented a LSPR peak at 514 nm, as shown in Fig. 3C (i), which indicates that no assembly is formed in absence of the linking polymer.

The Y/X=l .3 ratio resulted in a nanoparticle assembly with few 5 nm Au NPs to anchor onto the core 15 nm Au NP, and a core-satellite gold nanoparticle assembly morphology, with 5 nm Au NP satellites sparsely positioned onto the core (see Fig. 3B, top row). The UV-Vis spectrum of the core-satellite morphology (Fig. 3C, (ii)) exhibited an LSPR peak at 534 nm with negligible NIR absorbance. This is considered to be due to the poor plasmon coupling between X-Y and low Y density, along with the wider nano-gap due to the polymer size separating them.

The Y/X=5 ratio resulted in a nanoparticle assembly with a 15 nm Au NP core densely covered with 5 nm Au NP satellites, visually imitating a core-shell type morphology (see Fig. 3B, middle row). The UV-Vis spectrum of the core-shell type morphology (Fig. 3C, (iii)) exhibited an LSPR red-shift to 557 nm, which suggests improved plasmon coupling due to the presence of higher numbers of 5 nm Au NPs.

On moving from the gold nanoparticle assembly morphology from core-satellite to core-shell and core multi-arm (i.e. with the increase in Y:X) there is an increased hot spot density and maximized plasmon coupling. The core multi-tentacle gold nanoparticle assembly benefits from the hot-spots of both X-Y and dramatically increased Y-Y, whereas Y-Y nano-junctions are rare in the other morphologies. Furthermore, although the linking polymer is a relatively large macromolecule (>1 nm), the flexibility of the polymer chains allows for much closer packing of Y-Y, which in turn aids in reducing the gap of X-Y.

Surface Enhanced Raman Scattering The surface enhanced resonant Raman scattering (SERS) performance was studied for the multi-arm nanoparticle assemblies (Y/X=31), which absorbed significantly in the broad NIR “optical window” region, a useful laser excitation range for in vivo diagnostics.

2-quinolinethiol (QTH) was employed as a SERS label and functionalised via its thiol end group onto the already formed multi-arm nanoparticle assemblies. The prepared colloid samples were filled in quartz cuvettes and analysed with a Renishaw In Via Raman spectrometer at 830 nm.

Although ideally a high concentration of label would provide better signal and hence be efficient as a tracking agent for diagnostics, the leaching out of the label to the surrounding cellular matrix causing toxicity is a noted concern hindering its real-life applications. Thus, we explored the potential of the multi-arm nanoparticle assemblies in providing detectable SERS signals {i.e., signal to noise ratio SNR>2) in a wide range of concentrations. With significantly lower label concentration of the order of nM, it becomes important to position them at hot-spots. This was attempted by employing a pre-labelling methodology where the label molecule was incorporated during the nano assembly formation. This was achieved by incorporating QTH after the 15 nm core NP was functionalised with the linking polymer and before the addition of the 5 nm NPs, thereby guiding most of the QTH molecules to position themselves at the core surface and around the core-tentacle juncture.

Fig. 4A shows a comparison of the multi-arm nanoparticle assembly (no label, graphical representation (i)), a linking polymer- functionalised core Au NP labelled with QTH (no 5 nm NPs) (graphical representation (ii)) and the multi-arm assembly formed from the labelled core, i.e. a pre-labelled multi-arm nanoparticle assembly (graphical representation (iii)).

Both DLS data (median size=68 nm) and UV-Vis plots confirmed that the pre-labelled assemblies were similar to the unlabelled assemblies, hence making comparisons meaningful. The core multi-arm nanoparticle assemblies (no label) did not exhibit any prominent Raman peaks of the polyethylene glycol polymer (see spectrum (i) in Fig. 4A) due to its low Raman scattering cross-section, which also suggested that upon tagging them with SERS labels there would not be any Raman signal interference from the linking polymer.

The polymer functionalised core with QTH (see spectrum (ii) in Fig. 4A) was compared to nano-assemblies pre-labelled with QTH (see spectrum (iii) in Fig. 4A) both having a low concentration of QTH of 4.76 x 10 ⁸ M, i.e. 47 nM. Spectrum (iii) in Fig. 4A shows peaks of QTH at 634, 667, 1083, 1115, 1363, 1425, 1542 cm ¹ . The signature peak of QTH at 1363 cm ¹ assigned to the aromatic C-C stretching, v(CC), is prominent.

Upon comparison of spectra (ii) and (iii), it is evident that hot-spots created due to assembly formation makes the nM label concentration readily detectable in nano assemblies although it remains undetectable for the core-labelled sample. Therefore, the multi-arm nanoparticle assemblies are intensely SERS-active.

A post-labelling methodology was also studied wherein QTH was loaded onto formed multi-arm nanoparticle assemblies (i.e. post labelling). As higher concentrations of QTH could not be used for pre-labelled strategies due to destabilization of the core Au NP, higher QTH concentrations for post-labelled multi-arm nanoparticle assemblies were studied (i.e. QTH added after nano-assembly formation). A QTH concentration of 1.7 x KG ⁷ M (0.17 mM) was easily detectable with a high SNR (signal to noise ratio) as shown in Fig. 4B. The spectrum presented peaks 375, 598, 772, 877, 1085, 1315, 1363, 1441 cm ¹ . The signature peaks at 1363 and 772 cm ^-1 assigned to the aromatic v(CC) and ring breathing mode are prominent.

As shown in the graphical representations of pre- and post-labelled nanoparticles (see Figs. 4A-C), QTH molecules with higher concentration and added at a later stage (post label) would be distributed mostly on the outer surface as compared to the pre-labelled nanoparticles.

Typically, higher SERS label concentrations are applied for medical tracking purposes as a maximised signal is essential for detection from within deep tissues. Another SERS label biphenylthiol (BPT) with similar structure to that of QTH was also studied at different label concentrations with post-labelling methodology, corresponding to (i) [BPT]=1.4xlO ^-5 M, (ii) [BPT]=1.25xl(T ⁴ M and (iii) [BPT]=2.22xlO ⁴ M. Fig. 4C shows the SERS spectra of BPT post-labelled nano-assemblies with signature peaks at 1580, 1278, 1074, 877, 473 and 287 cm ^-1 . An increase in SERS signal is observed from spectra (i) to (iii) due to the increase in BPT concentration. Overall, we can conclude that core multi-arm nanoparticle assembly related hot-spots are important to amplify SERS signals, allowing detection of multiple SERS labels in the concentration range of nM to mM.

Summary

We have demonstrated a non-complex methodology for preparing multi-arm nanoparticle assemblies with a distinct 3D morphology of multiple arms anchored onto a core NP. Advantageously, off-the-shelf reactants such as Au NPs (15 nm and 5 nm) and commercially available linking molecules can be used, without involving elaborate linking molecule or nanoparticle synthesis. Importantly, the multi-arm nanoparticle assemblies exhibited significant broadband NIR absorbance in addition to visible absorbance, making them highly suitable for biomedical diagnostic and tracking applications in the optical window of 650-1100 nm. The total mean average size of the multi-arm nanoparticle assemblies of approximately 70 nm comprised of biocompatible gold nanoparticles and polyethylene glycol linking polymers in aqueous media, with good colloidal stability. We investigated the 3D nano-morphology and deduced that on average 4-5 arms, each arm formed of multiple 5 nm NPs, were anchored to the 15 nm NP core. The flexible linking polymer (DLS median average size of 2.5 nm) served as the sole linker between 15 nm and 5 nm NPs, as well as between several 5 nm NPs. This indicated that linkers need not be sub-1 nm for maximizing plasmon coupling and boosting NIR absorbance. Finally, the morphology of the nanoparticle assemblies was highly dependent on the ratios between reactant NPs, and by altering the ratios, distinctly different morphologies such as core-satellite, core-shell and core multi-arm were observed. Among all such morphologies, only the core multi-arm nanoparticle assemblies provided the much desired broadband NIR absorbance. The NIR-SERS behavior of these systems labelled with multiple SERS labels was studied using 830 nm laser excitation, which provided detectable signals in the wide dynamic range of nM-mM. The developed multi-arm nanoparticle assemblies thus demonstrate potential as novel medical theranostics agents. The one or more embodiments are described above by way of example only. Many variations are possible without departing from the scope of protection afforded by the appended claims.