WO2010111741A1

WO2010111741A1 - Nanomaterials and methods of preparation therefor

Info

Publication number: WO2010111741A1
Application number: PCT/AU2010/000370
Authority: WO
Inventors: Liu Ruili; Qin Li; Dongqing Wu
Original assignee: Curtin University Of Technology
Priority date: 2009-03-31
Filing date: 2010-03-31
Publication date: 2010-10-07

Abstract

A method of preparing a nanomaterial comprising a plurality of discrete nanoparticles of a substance is provided. The method comprises the steps of: providing a functionalised carrier; associating the functionalised carrier with a precursor of said substance to produce a first composite material; converting the bound precursor to said substance in a manner to produce a second composite material comprising a plurality of nanoparticles of the substance supported on the carrier; and removing the carrier from the second composite material. The methods of the present invention may be employed to prepare nanoparticles of a substance comprising one or more metals, alloys, inorganic substances such as inorganic compounds, organic substances such as organic compounds, carbon-based materials or doped carbon-based materials, and composite materials thereof. Said composite materials comprise a plurality of discrete nanoparticles of said substance supported on a carrier. Carbon-based nanomaterials (and doped carbon-based materials) prepared from the present invention may be further passivated to prepare luminescent carbon-based nanomaterials.

Description

NANOMATERIALS AND METHODS OF PREPARATION THEREFOR

Field

The present invention relates to nanomaterials, precursor materials therefor, composite materials containing said nanomaterials and methods of preparing nanomaterials and composite materials containing said nanomaterials. The present invention also relates to use -of such nanomaterials as bio- imaging agents.

Background

There is considerable interest in the application of nanoparticles in electronic or optical materials, catalysis, as well as use as therapeutic and bio- imaging agents.

Synthetic strategies which facilitate preparation of nanoparticles with a controlled size and morphology are particularly attractive and much attention has focused on various methods for preparing metallic nanoparticles.

Schrinner et al recently reported in Science, Vol. 323, pp 617-620, a method for the preparation of single nanocrystals of platinum prepared by partial dissolution of Au-Pt nanoalloys. The metal nanoparticles produced by this method are firmly embedded in a network of polyelectrolyte chains grafted onto the surface of a solid polystyrene core. The embedded metal nanoparticles exhibit high catalytic activity. However, the method fails to provide a route by which the metal nanoparticles may be released from the polyelectrolyte chains as discrete metal nanoparticles, and so the difference in catalytic activity between the embedded particles and the discrete metal nanoparticles has been unable to be tested.

There has also been considerable interest directed to preparation of carbon-based nanomaterials (CNMs) comprising a plurality of discrete carbon-based nanoparticles (also referred to as 'carbon dots'), particularly photoluminescent CNMs. They are highly desirable in bioimaging applications, disease detection and drug delivery applications. Compared to fluorescent semiconductor nanocrystals (quantum dots, QDs) , photoluminescent CNMs are regarded as superior in chemical inertness, biocompatibility and potentially low- toxicity .

Nanodiamonds, synthesized by highly energetic processes, such as detonation, shock wave and high-energy electron beam, have demonstrated stable photoluminescence . CNMs have also been prepared by laser ablation of graphite followed by surface- passivation, and are capable of excitation by a broad range of wavelengths and emit bright photoluminescence. This method produces CNMs having a particle size > 5 nm in a broad particle size distribution and quantum yields of about 4-10%. Photoluminescent CNMs also extend to carbon nanoparticles separated from candle soot, albeit with a very low quantum yield of ~ 1%.

Accordingly, there is a need to develop further novel photoluminescent carbon-based nanomaterials with good biocompatibility and high photoluminescence efficiencies in a wide range of pH values .

In particular there is a need to develop a method of preparation of nanomaterials comprising a plurality of discrete nanoparticles, in particular photoluminescent CNMs, which does not require elaborate equipment and may be readily applied on an industrial scale.

Summary

According to a first aspect there is provided a method of preparing a nanomaterial comprising a plurality of discrete nanoparticles of a substance, said method comprising the steps of: a) providing a functionalised carrier; b) associating the functionalised carrier with a precursor of said substance to produce a first composite material; c) converting the bound precursor to said substance in a manner to produce a second composite material comprising a plurality of nanoparticles of the substance supported on the carrier; and d) removing the carrier from the second composite material.

In one embodiment of the invention the step of providing a functionalised carrier comprises modifying a surface of a carrier with one or more functionalisation agents.

The carrier may be a material capable of being removed from the second composite material under conditions which do not substantially physically and/or chemically degrade the nanoparticles supported thereon.

The methods of the present invention may be employed to prepare nanoparticles of a substance comprising one or more metals, alloys, inorganic substances such as inorganic compounds, organic substances such as organic compounds, or carbon-based materials, and composite materials thereof. Said composite materials comprise a plurality of discrete nanoparticles of said substance supported on a functionalised carrier. Generally, the discrete nanoparticles may be regularly distributed over the surface of the carrier.

In one form, the method of the present invention may be employed to prepare a photoluminescent carbon-based nanomaterial .

Accordingly, in a second aspect of the present invention there is provided a method of preparing a photoluminescent carbon- based nanomaterial comprising a plurality of photoluminescent carbon-based nanoparticles, the method comprising the steps of: a) providing a functionalized carrier; b) associating the functionalised carrier with a carbon- containing precursor to produce a first composite material ; c) converting the bound carbon-containing precursor to a carbon-based substance in a manner to produce a second composite material comprising a plurality of carbon- based nanoparticles supported on the carrier; d) removing the carrier from the second composite material; and e) passivating the carbon-based nanoparticles separated from the second composite material in step d) to produce the photoluminescent carbon-based material .

The method as defined above provides a photoluminescent carbon-based nanomaterial having a particle size distribution within a narrow size distribution range.

In a further aspect of the present invention there is provided a photoluminescent carbon-based nanomaterial comprising a plurality of surface-passivated carbon particles having a particle size distribution in a range of about 1 nm to about 10 nm.

In one embodiment the photoluminescent carbon-based nanomaterial comprises a plurality of surface-passivated amorphous carbon particles having a particle size distribution in a range of about 1 nm to about 10 nm.

In another embodiment, the photoluminescent carbon-based nanomaterial emits luminescence in a wavelength range from about 420 nm to about 650 nm under excitation in a wavelength range from about 300 nm to about 600 nm. The photoluminescent carbon-based nanomaterial may also emit luminescence in the wavelength range of 420 nm to 600 nm when excited by a near infrared pulse laser having a wavelength in a range of about 700 nm to about 1000 nm. In a further embodiment, the photoluminescent carbon-based nanomaterial exhibits a photoluminescent quantum yield of greater than 10%, in particular greater than 14%.

In a still further embodiment, the photoluminescent carbon- based nanomaterial is water-soluble.

The photoluminescent carbon-based nanomaterial of the present demonstrates good biocompatibility and offers single molecule resolution owing to the small particle size of about 1 nm to about 10 nm .

In another aspect of the present invention there is provided a bioimaging agent comprising a surface-passivated carbon particle having a particle size distribution in a range of about 1 to about 10 nm.

The method of the present invention may also be employed to prepare a doped carbon-based nanomaterial .

Accordingly, in a further aspect of the present invention there is provided a method for preparing a doped carbon-based nanomaterial comprising a plurality of doped carbon-based nanoparticles, the method comprising the steps of: a) providing a functionalized carrier; b) associating the functionalised carrier with a carbon- containing precursor containing a dopant to produce a first composite material; c) converting the bound carbon-containing precursor containing the dopant to a doped carbon-based substance in a manner to produce a second composite material comprising a plurality of discrete doped carbon-based nanoparticles supported on the carrier; and d) removing the carrier from the second composite material.

It will be appreciated that the doped carbon-based material of the present invention may undergo a passivating step to render the doped carbon-based material photoluminescent .

The invention also provides a method of preparing novel composite materials comprising a plurality of discrete nanoparticles of a substance supported on a carrier.

In a still further aspect of the present invention there is provided a method of preparing a composite material comprising a plurality of discrete nanoparticles of a substance supported on a carrier, the method comprising the steps of : a) providing a functionalized carrier; b) associating the functionalised carrier with a precursor of the substance to produce a first composite material; c) converting the bound precursor to the substance in a manner to produce a second composite material comprising a plurality of discrete nanoparticles of the substance supported on the carrier.

The composite materials may subsequently be used to prepare the nanomaterials of the present invention. According to another aspect of the invention there is provided a method of preparing a nanomaterial of a substance comprising a plurality of discrete nanoparticles of the substance, the method comprising the steps of: a) providing a first composite material comprising a precursor of said substance bound to a carrier; b) converting the bound precursor to the substance in a manner to produce a second composite material comprising a plurality of discrete nanoparticles of the substance supported on the carrier; and c) removing the carrier from the second composite material .

In an alternative form, the method of preparing a nanomaterial of a substance comprising a plurality of discrete nanoparticles of the substance comprises the steps of: providing a composite material comprising a plurality of discrete nanoparticles of the substance supported on the carrier; and removing the carrier from the composite material.

Brief Description of the Figures

Figure 1 is a schematic representation of a photoluminescent carbon-based nanomaterial in accordance with the present invention;

Figure 2 is a series of confocal microscopy images of E.coli ATCC 25922 cell labelled with the photoluminescent carbon-based nanomaterial of the present invention (A)_EX= 458 nm detected with 475 nm long pass filter; (B)_EX=488 nm detected with 505 nm long pass filter; (0)^=514 nm detected with 530 nm long pass filter;

Figure 3 are scanning electron microscopy (SEM) images (A and B) and a transmission electron microscopy (TEM) image (C) of a composite material comprising as-made polymer/F127/silica composites for use in preparing one embodiment of the photoluminescent carbon-based nanomaterial in accordance with the present invention, and a TEM image (D) of the resulting carbon dots derived from carbon/silica comosites calcined at 900⁰C in Ar and etched with NaOH (2M) at 40⁰C for 48 h but with some residue of silica spheros in accordance with one embodiment of the photoluminescent carbon-based nanomaterial of the present invention; Figure 4 is a series of Fourier transform infrared spectra of (a) carbon dots (CDs) derived from etching off the parent C/SiO₂ composite, (b) oxidized CDs after being oxidized with 3M HNO₃ acqueous solution, (c) passivation agent PEG_I50ON alone, and (d) photoluminecent CDs after being passivated with PEG_I50ON comprising carbon-based nanomaterial and photoluminescent carbon-based nanomaterial prepared in accordance with the present invention;

Figure 5 is a Raman spectrum for an amorphous carbon- based nanomaterial of the present invention comprising crude carbon dots derived from carbon/silica composites. It displays two broad peaks at around 1330 and 1600 cm^"1, attributed to the D-band (sp³) , respectively. The coexistence of both bands suggests that the crude carbon dots are amorphous ; - 9a-

Figure 6 is a series of UV-visible absorption spectra (A and C) and photoluminescence emission spectra (B and D) which were recorded for progressively longer excitation wavelengths from 320 to 500 nm in 20 nm increment of oxidized CDs (A and B) and 0.1 mg/ml PEG_I50ON (C and D) . Excitation scattering is abbreviated as ES and photoluminescence is abbreviated as PL. The black lines in Figure 6B and D are for eye guide: left peaks are due to the excitation scattering while right peaks come from photoluminescence. As shown by spectra B and D, no distinct photon emission can be observed for oxidized CDs or

Figure 7 is a series of UV-visible absorption spectra (A) and photoluminescence emission spectra (B) excited at 360nm of PEG_I50ON -passivated carbon dots in Milli-Q water at pH=7 (•) , pH=5 (HAc-NaAc 0.1 M) (A), pH=9 (NaHCO_3-NaC₂O₃, 0.1 M) (T) and quinine sulphate (■) in 0.1 M H₂SO₄ solution as the standard. The asymmetry of all CDs emission spectra may be caused by the re-absorption in the blue part of the spectra; Figure 8 is a series of confocal microscopy images of

Murine P19 progenitor cells labelled with the photoluminescent carbon-based nanomaterial of the present invention after 24 hours incubation time. The grey channel shows the transmission images, while the intensity coded (green) channel shows the fluorescence;

Figure 9 is a series of UV-visible absorption spectra and photoluminescence emission spectra of several photoluminescent carbon-based nanomaterials CD2 , CD3 , CD4 (a) and PL emission spectra of CD2 (b) , CD3 (c) , and CD4 (d) . Inserted photos were taken when the suspensions were excited - 9b-

with an UV lamp (365 nm) ;

Figure 10 is a series of photoluminescent excitation spectra of several photoluminescent carbon-based nanomaterials CD2 (a) , CD3 (b) , and CD4 (c) of the present invention;

Figure 11 is a series of confocal microscopy images of HeLa cells labelled with several photoluminescent carbon- based nanomaterials CD2 , CD3 , CD4 for 24h in accordance with the present invention. The results are the mean ± SD of 3 separate experiments.

Figure 12 is a series of confocal microscopy- transmission and photoluminescent images of HeLa cells which have internalised after 2h incubation several photoluminescent carbon-based nanomaterials (a) CD2 ; (b) CD3 ; (c) CD4; (d) Tf-CD2; (e) Tf-CD3; (f) Tf-CD4.

Photoluminescence (shown as green) and transmission images are merged;

Figure 13 is a further series of confocal microscopy transmission and photoluminescent images of HeLa cells which have internalised several transferrin conjugated photoluminescent carbon-based nanomaterials in accordance with the present invention. Comparison of cells treated with: (a) CD3 (left transmission and PL images) versus CD3-Tf (right transmission and PL images); (b) CD3-Tf incubated with cells pre-treated with free Tf for 1 hr (transmission and PL images) and cells non-pretreated with free Tf (right transmission and PL images) ; (c) CD4-Tf incubated with cells pre-treated with free Tf for 1 hr (left transmission and PL images) and cells non-pretreated with free Tf (right transmission and PL images) . - 10 -

Detailed Description

In one aspect, the present application relates to a method of preparing a nanomaterial comprising a plurality of discrete nanoparticles of a substance.

The term "substance" is used broadly to refer to any chemical or material. Examples of "substances" may include one or more metals, alloys, inorganic substances such as inorganic compounds, organic substances such as organic compounds, or carbon-based materials, including dopant-containing carbon- based materials. Illustrative examples of carbon-based materials include, but are not limited to, amorphous carbon, semi-crystalline carbon, crystalline carbon, graphitic carbon, and graphene-like carbon.

The nanoparticles prepared in accordance with the methods of the present invention may have a particle size in a range of about 1 nm to about 10 nm. Generally, said nanoparticles

- 11 -

have a substantially uniform particle size with a particle size distribution range of about 1 nm to 2 nm.

The method for preparing a nanomaterial comprising a plurality of discrete nanoparticles of a substance comprises the steps of: a) providing a functionalised carrier,- b) associating the functionalised carrier with a precursor of said substance to produce a first composite material; c) converting the precursor of the first composite material to said substance in a manner to produce a second composite material comprising a plurality of discrete nanoparticles of the substance supported on the carrier; and d) removing the carrier from the second composite material.

Functionalised carrier

The term "functionalised carrier" refers to a carrier whose surface has been modified with one or more functionalisation agents. In one embodiment of the invention the step of providing a functionalised carrier comprises modifying a surface of a carrier with one or more functionalisation agents .

Although the carrier may take any suitable size or form, for ease of handling it is preferred that the carrier is a regularly shaped particle, in particular a spherical particle, having a particle size of about 50 nm to several microns. In this way, the carrier provides a convenient - 12 -

substrate on which a plurality of discrete nanoparticles may^¬ be formed, so circumventing the tendency of nanoparticles to aggregate together.

Alternatively, the carrier may take the form of a planar substrate on which the plurality of nanoparticles may be discretely formed.

While not wishing to be bound by theory, the inventors opine that the size of the carrier and the relative degree of curvature influences the particle size of the nanoparticles which form on the surface of the carrier. The spacing between the functionalisation agents conjugated to the surface of the carrier and its radial dispersion thereon, in particular, are thought to influence the nanoparticle size.

The carrier may be a porous material. In particular, the carrier may be a microporous material or a mesoporous material .

The carrier may also be a material that is physically and/or chemically stable under the conditions used to convert the precursor which is bound to the modified surface of the carrier to discrete nanoparticles of said substance. - 13 -

The carrier may be formed from an inorganic material or an organic material, in particular a polymeric material.

Examples of suitable inorganic materials include, but are not limited to, silica, alumino-silicates, in particular zeolites, metal oxides such as aluminium oxide, iron oxide, zinc oxide or zirconium oxide, carbon, and calcium sulfate.

Silica colloid spheres, porous silica materials, and zeolites are particularly preferred as the carrier because they are thermally stable at temperatures greater than 600 ⁰C, capable of dissolution in basic or acidic solution, and relatively inexpensive. Further, terminal hydroxyl groups on the surface of such silica materials are capable of forming secondary bonding interactions, such as hydrogen bonds and/or electrostatic attractive forces with the functionalisation agents .

Examples of suitable organic materials include, but are not limited to polymeric materials, such as polystyrene or polymethymethacrylate (PMMA), and biodegradable materials, such as polysaccharides. The term "biodegradable" means that a material can be degraded, either enzymatically or hydrolytically, (including under physiological conditions) to smaller molecules. Polymeric materials such as polystyrene or PMMA can be provided which have negatively charged terminal groups, such as carboxyl , sulphate or sulphonate functional groups, on the surface of the carrier.

Alternatively, polymeric materials such as polystyrene or PMMA can be provided which have positively charged terminal - 14 -

groups, such as ammonium functional groups, on the surface of the carrier.

The surface of the carrier is modified with one or more functionalisation agents. Generally, an exterior surface of the carrier may be modified. Alternatively, in embodiments wherein the carrier is porous, in particular wherein the carrier is microporous or mesoporous, it will be appreciated that the surface of the micropores or mesopores, as well as the exterior surface of the carrier, may be modified. In some embodiments it is advantageous to use a mesoporous carrier as the pores of the carrier assist with localization of the precursor within a confined space of the pores.

The term "modified with one or more functionalisation agents" refers to terminal groups on the surface of the carrier that are bonded with the functionalisation agents via primary or secondary bonding interactions.

In one embodiment of the invention, the step of modifying the surface of the carrier with one or more functionalisation agents comprises forming primary or secondary bonding interactions between the surface of the carrier and the functionalisation agents. In one form this may involve conjugating terminal functional groups on the surface of the carrier with the functionalisation agents. In this way, the functionalisation agents becomes "anchored" or bound to the surface of the carrier. - 15 -

The functionalisation agents used to modify the surface of the carrier may be a long chain organic compound having functional groups and/or moieties capable of forming primary bonding and/or secondary bonding interactions with terminal groups on the surface of the carrier. Preferred types of secondary bonding interactions include hydrogen bonding, van der Waals interactions, dipole-dipole interactions, and electrostatic attraction, hydrophobic interactions. In general, such functional groups and/or moieties are located at or proximal to a terminal end of the long chain organic compound .

Additionally, the functionalisation agents may have further functional groups and/or moieties capable of forming secondary bonding interactions with the precursor. The further functional groups and/or moieties which are capable of forming secondary bonding interactions with the precursor may be the same or different from the functional groups and/or moieties which are capable of forming primary bonding and/or secondary bonding interactions with terminal groups on the surface of the carrier. In general, the further functional groups and/or moieties are located at one or more sites along the length of the long chain organic compound.

In one embodiment of the invention the functionalisation agents comprises an amphiphilic long chain organic compound having a plurality of functional groups and/or moieties capable of forming secondary bonding interactions with the surface of the carrier and with the precursor. - 16 -

Suitable examples of such amphiphilic long chain organic compounds include, but are not limited to, surfactants, in particular ionic or non- ionic surfactants, or surfactants containing a hydrophilic block, such as polyethyleneoxide (PEO) , polyacrylic acid (PAA) , polyethylenimine (PEI) , or polymethylmethacrylate (PMMA), polyelectrolytes, and amphiphilic linear or branched chain polymers, in particular homopolymers , diblock copolymers, triblock copolymers, and more particularly those amphiphilic linear or branched chain polymers containing at least one hydrophilic block.

In a preferred form of the invention, the functionalisation agent is amphiphilic triblock copolymer. Suitable examples of such amphiphilic triblock copolymers include, but are not limited to, F127 (EO_1O6PO_7OEO₁₀₆, M_W = 12600), P123 (EO₂₀PO₇₀EO₂₀) , P103 (EO₁₇PO₅₆EO₁₇) , L121 (EO₅PO₇₀EO₅) , P85 (EO₂₆PO₃₉EO₂₆) , P65 (EO₂₀PO₃₀EO₂₀), F88 (EO₁₀₀PO₃₉EO₁₀₀) , F98 (EO₁₂₃PO₄₇EO₁₂₃) , F108 (EO₁₃₂PO₅₀EO₁₃₂) , B50-6600 (EO₃₉BO₄₇EO₃₉) , B70-4600 (EO₁₅BO₄₅EO₁₅) , B40- 1900 (EO₁₃BO₁₁EO₁₃ ) , and B20-3800 (EO₃₄BO₁₁EO₃₄) .

The functionalisation agent may alternatively comprise a natural or a synthetic polyelectrolyte . Suitable examples of polyelectrolytes include, but are not limited to, poly(acrylic acid), poly(styrene sulfonate), poly (diallyldimethyl ammonium chloride), poly (allylamine hydrochloride) (PAH), or others. Preferred cationic groups is the amino group and preferred anionic groups are carboxylic acid, sulfonic acid phosphates and the like. - 17 -

Suitable examples of cationic polyelectrolytes include, but are not limited to, 2-aminoethylmethacrylate, poly (allylamine) , poly (ethyleneimine) , poly (diallyldimethylammonium chloride), poly (arginine) , chitosan, cationic collapsible proteins, cationic peptides, poly (methylacrylamideo propyl trimethyl ammonium chloride) and poly (lysine) .

Suitable examples of anionic polyelectrolytes include, but are not limited to, poly(acrylic acid), poly(styrene sulfonic acid), poly (glutamic acid), poly (methacryclic acide) , poly (aspartic acid), nucleic acid, nucleotides, anionic collapsible proteins, anionic peptides, poly (anetholesulfonic acid), cellulose, poly(maleic acid), poly (vinylphosphoric acide), and PEG.

It will be appreciated that modifying the surface of a carrier with one or more functionalisation agents may also encompass the in situ generation of one or more functionalisation agents bound to the surface of the carrier by reacting short chain oligomeric compounds with the carrier. For example, the surface of a polystyrene carrier may be modified with a polyelectrolyte by reacting the polystyrene carrier with a solution of short chain polyethylenimide oligomers, whereby electrostatic charge interactions generate positively charged polyelectrolytes bound to the surface of the carrier. - 18 -

Generally, the functionalisation agent is bound uniformly on the surface of the carrier. The long chains of the bound functionalisation agent extend outwardly from the surface of the carrier. The further functional groups and/or moieties of the functionalisation agent are selected to facilitate formation of primary or secondary bonding interactions with the precursor, depending on the chemical nature of the precursor. For example hydrophilic blocks or hydrophobic blocks within an amphiphillic co-block polymer may facilitate formation of secondary bonding interactions with a polymer precursor, depending on the chemical nature of the polymer precursor. The bonded precursor is immobilized by, or localized in the long chains of, the functionalisation agent, thereby increasing the loading of the precursor proximal to the functionalized surface of the carrier.

Precursor of the substance

The "precursor" of the substance is any chemical or material which can be converted by one or more chemical reactions to the substance. Examples of such chemical reactions include, but are not limited to, polymerisation, oxidation, reduction, thermal degradation by oxidative or reductive means, thermal degradation induced by microwave radiation, acid-base reactions, precipitation, metal-ligand complexation reactions, host-guest interactions, self-assembly reactions, supramolecular assembly, and so forth.

Suitable examples of precursors for metals or metal alloys - 19 -

include, but are not limited to, one or more metal ions, cationic complex ions of metals, anionic complex ions of metals, metal complexes, metal compounds, organometallic compounds including metallocenes, and so forth.

Suitable examples of precursors for inorganic compounds or organic compounds comprise solutions of said inorganic compounds or organic compounds, or one or more suitable reactants for preparation of said inorganic compounds or organic compounds. The reactants may be capable of associating with the functionalisation agent via primary or secondary bonding interactions prior to undergoing one or more chemical reactions to convert the reactants to the desired inorganic or organic compound.

Suitable examples of precursors for carbon-based materials, including dopant-containing carbon-based materials include, but are not limited to, a polymer precursor or any organic compound or material which is capable of binding to the functionalisation agent and converting to a carbon-based material, in particular by thermal degradation. Illustrative examples such organic compounds include, but are not limited to, surfactants such as glucose.

The polymer precursor may be any suitable polymer oligomer (M_w < 500) including N-, S-, B-, F-, Si-containing polymer oligomers (M_w < 500) . Illustrative examples of the polymer precursor include, but are not limited to, resol (phenol/formaldehyde resins, M_w < 500), pyrrole, polymeracryonitrile (PAN) , aniline, acetonitrille, - 20 -

thiphenemethanol , P- fluorphenol- formaldehyde, boronic ester, boron nitride and boron nitrogen polymers, polycarbosilane (PCS) .

Advantageously, when using a polymer precursor containing atoms in addition to C, H and 0, the method of the present invention provides a convenient route by which atoms of other elements (i.e. dopants) may be introduced into the carbon- based nanomaterials formed by the methods of the present invention. In other words, the polymer precursor with atoms in addition to C, H and 0 can be considered as a dopant- containing polymer precursor and it facilitates introduction of a dopant into the carbon-based nanoparticle . Thus, the method of the present invention provides a convenient route by which doped carbon-based nanoparticles may be prepared. The introduction of lattice defects associated with the presence of dopants in the lattice of doped carbon-based nanomaterials, enables variation in their optical properties such as the excitation and emission wavelengths and their quantum yield efficiencies. Doped carbon-based nanomaterials may also be employed as catalysts.

Associating the functionalised carrier with a precursor of said substance to produce a first composite material

The step of associating the functionalized carrier with the precursor to produce a first composite material comprises forming primary or secondary bonding interactions between the further functional groups and/or moieties of the functionalisation agents and the precursor. - 21 -

In one preferred form of the invention said step may comprise conjugating amphiphilic blocks of the functionalisation agent with the polymer precursor by means of secondary bonding interactions, in particular hydrogen bonding interactions or van der Waals interactions.

In another form of the invention said step may comprise forming electrostatic charge interactions between charged functional groups and/or moieties on the functionalisation agent and the precursor, in particular a charged precursor.

In a further form of the invention said step may comprise adsorption of the precursor onto the functionalized carrier.

The first composite material provided by the present invention comprises a precursor of said substance bound to the functionalized carrier. In some embodiments of the invention the first composite material may comprise nanoparticles of the precursor supported on the functionalized carrier.

Converting the bound precursor to said substance in a manner to produce a second composite material comprising a plurality of nanoparticles of the substance supported on the carrier;

The step of converting the bound precursor to said substance may comprise subjecting the precursor to one or more chemical reactions. Examples of such chemical reactions include, but are not limited to, polymerisation, oxidation, reduction, - 22 -

thermal degradation by oxidative or reductive means, acid-base reactions, precipitation, metal-ligand complexation reactions, host-guest interactions, self-assembly reactions, supramolecular assembly, and so forth.

In one particular embodiment of the invention, the step of converting the polymer precursor to carbon-based material in a manner to produce a second composite material comprising a plurality of carbon-based nanoparticles supported on the carrier comprises: polymerising the polymer precursor on the functionalized carrier, thereby producing a composite material comprising a plurality of discrete polymer nanoparticles supported on the carrier; and converting the polymer nanoparticles of the composite material to carbon-based nanoparticles.

As the polymer precursor is bound to the functionalisation agent, polymerization of the bound polymer precursor occurs on the functionalized carrier, rather than in solution, thereby producing the composite material in which the polymer particles are substantially evenly spaced over the surface of the carrier. In one embodiment of the invention, the polymer particles so formed have a particle size in a range of about 2 nm to about 100 nm.

It will be appreciated by a person skilled in the art that the step of polymerizing the polymer precursor is performed under conditions suitable to effect polymerization of the polymer precursor. - 23 -

In one embodiment of the invention, the step of converting the carbon-based precursor, in particular the polymer nanoparticles , of the, composite material to carbon-based nanoparticles comprises carbonizing the carbon-based precursor or polymer nanoparticles to carbon-based nanoparticles .

In one embodiment, carbonizing the carbon-based precursor or polymer nanoparticles comprises heating the composite material to a temperature at which the carbon-based precursor or polymer nanoparticles thermally decompose to carbon-based nanoparticles. It will be appreciated that during thermal decomposition, other chemical processes, such as dehydrogenation and further polymerization of the polymer nanoparticles, may concurrently take place.

In another embodiment, carbonizing the carbon-based precursor or the polymer nanoparticles comprises treating the carbon- based precursor or the polymer nanoparticles with microwave energy to induce thermal decomposition.

In still another embodiment, carbonizing the carbon-based precursor or the polymer nanoparticles comprises treating the carbon-based precursor or the polymer nanoparticles with concentrated sulfuric acid (H₂SO₄) to prepare carbon-based nanoparticles .

In one form of the invention the carbon-based precursor or polymer nanoparticles thermally decompose to amorphous, - 24 -

semicrystalline, or crystalline carbon nanoparticles . In another form, the particle size of the carbon-based nanoparticles is in a range of about 1 nm to about 10 nm.

Interestingly, although converting the polymer nanoparticles to carbon-based nanoparticles is accompanied by volume shrinkage, in other words, the carbon-based nanoparticles have a smaller particle size than the polymer nanoparticles, aggregation of the carbon-based nanoparticles into a larger aggregate particle during thermal decomposition is circumvented by employing the carrier as an "anchor" for the carbon-based nanoparticles. Generally, the particle size of the carbon-based nanoparticles may be in a range of about 1 nm to about 10 nm and with a narrow particle size distribution of about 1 nm to 2 nm. For example, carbon- based nanoparticles of the present invention have been prepared in a range of about 1.5 nm to about 2.5 nm.

The step of converting dopant-containing carbon-based precursors or dopant-containing polymer nanoparticles thus facilitates the inclusion of dopants in the lattice of the carbon-based nanoparticles of the present invention. Doped carbon nanoparticles prepared in accordance with the present invention, in particular N-doped carbon nanoparticles, may be employed as catalysts.

The present invention thus also provides a composite material comprising a plurality of nanoparticles of a substance supported on a carrier. - 25 -

Rewoving the carrier from the second composite material

The carrier may be removed from the second composite material by any means that will selectively degrade the carrier without substantially chemically or physically degrading the nanoparticles supported on the surface of the carrier. Removing the carrier from the second composite material thus comprises subjecting the second composite material to a selective degradation process.

Examples of such means of selectively degrading the carrier include, but are not limited to, selective dissolution of the carrier with a solvent in which the carrier is soluble, thermal degradation, microwave-induced degradation, plasma- induced degradation, chemical degradation by acid-base reactions or electrochemical reactions.

In this way, a nanomaterial comprising a plurality of discrete nanoparticles of a substance with a particle size of about 1 nm to about 10 nm may be prepared.

Passivating carbon-based nanoparticles

The carbon-based nanoparticles prepared in accordance with the present invention do not display photoluminescent properties until a surface of the carbon-based nanoparticles is passivated. Accordingly, the method of the present invention may further comprise passivating the carbon-based nanoparticles separated from the second composite material to produce a photoluminescent carbon-based material. - 26 -

Prior to passivating the carbon-based nanoparticles, the method further comprises functionalizing the carbon-based nanoparticles with terminal functional groups on a surface of the carbon-based nanoparticles. The terminal functional groups are selected to be capable of forming primary and secondary bonding interactions, such as for example, through amidation, hydrogen bonding, or electrostatic attraction, with the passivating agent. Suitable examples of such terminal functional groups include, but are not limited to, carbonyl groups, hydroxyl groups, amine groups, and so forth.

In one form of the invention, the step of functionalizing the carbon-based nanoparticles comprises reacting the carbon- based nanoparticles with a reagent capable of introducing terminal functional groups on the surface of the carbon-based nanoparticles. Suitable examples of such reagents include, but are not limited to, oxidants such as concentrated nitric acid, sulfuric acid, hydrogen peroxide, potassium permanganate, O₂ plasma, and a mixture of sulfuric acid and a nitrate salt .

The step of passivating the carbon nanoparticles comprises reacting the carbon nanoparticles with a passivating agent. The passivating agent may be any suitable long chain organic compound capable of binding to a surface of the carbon nanoparticles by primary or secondary bonding interactions, such as for example, through amidation, hydrogen bonding, or electrostatic attraction forces. Suitable examples of passivating agents include, but are not limited to, - 27 -

amphiphilic oligomeric polymers, long-chained surfactants, nucleotides, peptides, and so forth. In one preferred embodiment, the passivating agent comprises a diamine- terminated oligomeric polymeric poly (ethylene glycol), such as for example H₂NCH₂(CH₂CH₂O)_nCH₂CH₂CH₂NH₂ (average n ~ 35, PEG_I5OON) and PEI-PEG-PEI (polyehtylenimine-polyethylene glycol-polyethylenimine, 5k-5k-5k) triblock copolymer.

Photoluminescent carbon-based nanomaterial

The present invention thus provides a photoluminescent carbon-based nanomaterial comprising a surface-passivated carbon particle having a particle size in a range of about 1 to about 10 nm.

The carbon-based nanomaterial may comprise substantially amorphous carbon, semi-crystalline carbon, or crystalline carbon, depending on the polymer precursor. Also, depending on the polymer precursor used in its preparation it is possible to introduce atoms other than C, H, 0 into the lattice structure to vary the optical properties of the nanomaterial as described in the foregoing description.

Typically, the photoluminescent carbon-based nanomaterial emits luminescence in a wavelength range from about 420 nm to about 650 nm under excitation in a wavelength range from about 300 nm to about 600 nm. The photoluminescent carbon- based nanomaterial may also emit luminescence in the wavelength range of 420 nm to 600 nm when excited by a near - 28 -

infrared pulse laser having a wavelength in a range of about 700 nm to about 1000 nm.

The photoluminescent carbon-based nanomaterial may be two photon excitable.

Generally, the photoluminescent quantum yield of the photoluminescent carbon-based nanomaterial is over 10%, in particular over 14%.

The photoluminescent carbon-based nanomaterial may have one or more excitation centres and/or one or ore emission centres .

The photoluminescent properties of the passivated carbon- based nanomaterial may be dependent on the nature of the passivating agent employed to passivate the carbon nanoparticles .

As a result of the versatility in passivating agents that may be used to passivate the carbon-based nanomaterial of the present invention, these materials may be prepared to be water-soluble and to demonstrate good biocompatibility and low cytotoxicity. The small particle size of about 1.5 nm to about 3.5 nm affords single molecule resolution and thus they may be readily used as a bioimaging agent.

In particular, the passivated carbon-based nanomaterial may be further conjugated by primary and/or secondary bonding interactions, in particular by covalent bonding, with biochemical substances such as peptides, proteins, nucleic - 29 -

acids, nucleotides, and so forth. In this way, the biocompatibility of the photoluminescent carbon-based nanomaterials of the present invention may be further improved .

Laser scanning confocal microscopy studies have shown that such surface-passivated carbon nanoparticles of the present invention have very good biocompatibility. Figure 2 shows confocal microscopy images of E.coli cells labeled with the material of the present invention after a co-incubation of 24 h, where the E.coli cells appear to be completely covered by the surface-passivated carbon nanoparticles of the present invention.

The invention will be illustrated in greater detail with reference to the following examples.

Example 1

The photoluminescent carbon-based nanomaterial of the present invention comprises surface-passivated carbon nanoparticles having a particle size distribution in a range of about 1 to about 10 nm, as schematically shown in Figure 1.

Broadly, such photoluminescent carbon-based nanomaterials may be readily prepared in accordance with Scheme 1 below in which a composite material comprising a plurality of discrete polymer nanoparticles supported on a carrier is first prepared. The polymer nanoparticles supported on the carrier are then converted to carbon nanoparticles, after which time the carrier is removed leaving a monodisperse suspension of - 30 -

carbon nanoparticles . The carbon nanoparticles are then passivated to render them photoluminescent .

Reso!/F127/Siθ2 C/SiO. Photoluminescent ' composites composites carbon dots

Scheme 1

A specific example of how the invention may be employed is as follows.

A colloidal suspension of 10 wt% SiO₂ (5 g, ~ 120 nm) was added to an aqueous solution of F127 (EOio6P0₇₀EOio6/ M_w = 12600) (1.0 g in 10 mL) . The mixture was stirred overnight at room temperature. After the removal of excessive surfactant by centrifugation (7,000 rpm, 10 min) the surfactant-modified silica spheres were redispersed into 100 mL NaOH aqueous solution (pH 9) . Meanwhile, 6 mL of freshly prepared resol precursors in basic condition was added. The mixture was stirred at 66 ⁰C for 48 h to allow for the polymerization of the resol particles on the surfactant- modified silica spheres. After centrifugation (7,000 rpm, 10 min) and drying at room temperature, khaki-coloured as-made polymer/F127/silica composites were obtained. The satellite- like morphology of the as-made precursor composites are shown in the SEM and TEM images of Figure 3, confirming that resols were polymerized on the surface of the silica spheres. From - 31 -

the SEM and TEM images the diameter of the polymer particles was estimated to be in the range of 10-40 nm.

The composite material was calcined at 900 ⁰C in Ar for 2 h resulting in formatting of corresponding carbon nanoparticle/silica composites.

The carbon nanoparticles were released from the silica carrier by etching with 2M NaOH solution at 40 °C for 48 h. The excessive NaOH was first neutralized by nitric acid and then removed by dialyzing the supernatant against Milli-Q water through dialysis membrane (MWCO 1000) . Carbon nanoparticles were separated from any remaining carbon nanoparticle/silica composites by high speed centrifugation (10,000 rpm, 30 minutes) to provide a light yellow suspension containing crude carbon nanoparticles, as shown in Figure 3D. Fourier transform infrared (FTIR) spectroscopy (Figure 4) show the absence of characteristic peaks of silica, suggesting that silica was completely removed. Further Raman spectroscopy (Figure 5) reveals that the resultant crude carbon nanoparticles possess both sp² and sp³ bonds, indicating an amorphous nature. Elemental analysis shows that the crude carbon nanoparticles are composed of C 90.32 wt%, H 1.26 wt%, and 0 (calculated) 8.34 wt%.

The light yellow suspension containing crude carbon nanoparticles was added with a quarter volume of concentrated nitric acid (end concentration 3M) and the mixture was refluxed for 24 h. The excess acid was first neutralized by sodium carbonate, and then removed by dialyzing against

Milli-Q water. The suspension appeared as a homogeneous light-yellow solution, and FTIR spectroscopy (Figure 4) of the oxidized carbon nanoparticles shows a sharp peak at 1737 cm^"1 assigned to the stretching vibrations of C=O, indicating - 32 -

the presence of carbonyl groups. This is consistent with the increase of oxygen content as observed by the elemental analysis: C 78.17 wt%, H 5.08 wt% and 0 (calculated) 16.75 wt%.

A surface passivation process was carried out as follows: 50 mg diamine-terminated oligomeric polymeric poly (ethylene glycol), H₂NCH₂(CH₂CH₂O)_nCH₂CH₂CH₂NH₂ (average n ~ 35, PEG_1500N) was added into 20 mL oxidized carbon nanoparticle solution and then subjected to ultrasonocation for 10 min to form a homogenous suspension. The mixture was subsequently heated at 120 ⁰C for 72 h for surface passivation. The optically transparent and photoluminescent carbon nanoparticle suspension was purified by dialyzing against Milli-Q water with a cellulose ester membrane bag (Mw = 3500) , during which excess PEG_1500N was removed. After filtering through 0.2 μm Teflon, a clear light yellow aqueous suspension containing PEG_1500N passivated carbon nanoparticles was finally obtained.

As shown by the FTIR spectra (Figure 4), the broadened peak at 1737 cm indicates the co-existence of

suggesting a fraction of the carbonyl groups have been converted to amide groups during the passivation process. Elemental analysis reveals that the composition of carbon nanoparticles passivated with PEG_1500N is: C 64.65 wt%, H 7.67 wt%, N 1.13 wt%, and 0 (calculated) 26.55 wt% .

Strong photoluminescence was observed when this suspension was excited by a 365 nm UV lamp. As shown in Figure 6, the emission spectra of these carbon nanoparticles are broad, ranging from 430 nm (violet) to 580 nm (yellow) , with a dependence on the excitation wavelengths. By selecting quinine sulfate as the standard and 360 nm as the excitation wavelength, the photoluminescent quantum yield of the PEG_I50ON - 33 -

passivated carbon nanoparticles was measured and calculated to be 14.7%. Additionally, the carbon nanoparticles are stable in a wide pH range from 5 to 9 with only slight decrease in the photoluminescent quantum yields (11.0% and 12.1%, respectively) and no shift of the emission peak when excited at 360 nm (Figure 7) .

Example 2

Photoluminescent carbon-based nanomaterials of the present invention ("carbon dots") can be readily internalized in Murine P19 progenitor cells as described below and shown in Figure 8.

Murine P19 cells (ATCC/LGC Promochemc, Wesel, Germany) were cultured in 5 mL medium (MEM Alpha Modification with L- Glutamine and nucleosides, PAA Laboratories GmbH, Austria) in culture flasks (one for control, one for internalization analysis) at 37 ⁰C with 5% CO₂. A 1.1 μM carbon dot suspension (200 μL) was mixed with 1 mL medium. The carbon dots mixture was added into the test cell culture. After an incubation of 14 h, compared with the control culture, the cells did not show noticeable abnormality. These cells were washed with PB and detached by Trypsin at 37 ⁰C for 5 min. Medium (2mL) was then added at room temperature. The cells were harvested by centrifugation (800 rpm, 5 min) and re- suspended in 5 mL PBS solution. The PBS-cell suspension (1 mL) was added into a Willco-dish™ (Willco Wells BV, the Netherlands) for confocal microscopy analysis. Due to the high sensitivity of P19 cells to the solid surface, they did not favour the glass bottom of the Willco-dish. Therefore, - 34 -

these cells do not exhibit their usual fibroblastic morphology. Nevertheless, the image in Figure 8 clearly demonstrates the internalization of carbon-dots.

Example 3

Preparation of metallic Au nanocrystals

A carrier comprising a microsphere of a polymeric material such as polystyrene, PMMA, can be readily tuned to either become negatively charged (with carboxyl , or sulphate or sulphonate functional groups) or positively charged (with amine groups on surface) . In this example, negatively charged (with carboxyl groups) polystyrene submicron particles are mixed with polyethyleneimide short chain solution, whereupon the electrostatic charge interactions generate carrier whose surface is functionalized with polyelectrolytes . A solution of the precursor of the metal, for example, HAuCl₄, is added to a suspension of the functionalized carrier and the positive charge of the polyelectrolyte immobilises and confines the anionic complex ions of metals, e.g. the

[AuCl₄]^". Subsequent reduction with NaBH₄ under mild conditions results in the formation of gold nanoparticles on the carrier particle surface. Following treatment with acetone or tetrahydrofuran to dissolve the carrier the bound metallic Au nanoparticles are liberated as discrete metallic Au nanoparticles.

Example 4

Preparation of palladium nanoparticles - 35 -

An inorganic carrier comprising carbon was functionalized with hydrophilie-hydrophobic copolymers, such as polystyrene -co-PMMA, in a toluene suspension. A palladium precursor comprising palladium hexafluroacetylacetonate Pd(F₆AcAc)₂ is added in the suspension. The fluorinated organometallic, Pd(F₆AcAc)₂ preferentially adsorbs on the polystyrene segment of the functionalized carrier. After drying, the mixture was annealed at 200⁰C in vacuum for 24 hours. During the annealing, the Pd nanoparticles will be formed exclusively on the polystyrene domains on the carbon particle. The carbon carrier and copolymer matrix can then be removed by thermal oxidation.

Example 5

Preparation of graphitic nanoparticles

Silica submicron carrier particles are first treated with organo-silane for surface modification, e.g. N- trimethoxylsilylpropyl-N,N,N - trimethylammonium chloride. Subsequently, oppositely charged Fe₃O₄ or nickel nanoparticles are adsorbed on the silica carriers. Carbon precursors such as resols, polystyrene, or glucose are subsequently introduced in a relatively high concentration. After mixing well, either the suspension of the mixture or the dried mixture can be microwaved for a few seconds (Is - 100s) . Because the Fe₃O₄ or Ni nanoparticles act as the absorption centres of the microwave, the carbon precursors around the nanoparticles are carbonized into a graphitic structure. After collecting the solid particles and removing - 36 -

the carrier particle as well as the Fe₃O₄ or nickel nanoparticles by acids or bases, nano-graphitic materials (multilayered or single-layered) will be formed.

Example 6

Preparation of nanoparticles of pharmaceutical compounds

Crystal polymorphism and nanosized crystal for high drug potency are both very important areas for pharmaceutical industry. The described system can also be used for forming nanocrystals for some high value drugs which are administrated in solid form. Here we use paracetamol as a simple example to illustrate the application.

Biodegradable polymeric spheres can be used as the carrier particles. Polyelectrolytes such as short chains of polyethyleneimide can be added to modify the surface and form the polyelectrolyte functionalisation agents. Afterwards, a paracetamol solution is mixed with the functionalized carrier suspension. Due to the electrostatic interactions, the polyelectrolytes will attract and confine the paracetamol monomers, create a localised higher concentration on the functionalized carrier. When the paracetamol concentration in the suspension is further increased or the temperature is lowered to induce the supersaturation, precipitation first occurs at the carrier surface owing to the localised higher concentration of the paracetamol monomers, in other words the localised crystalisation occurs at the carrier surface. The carrier particles with fine crystals embedded in the polymer layer can then be easily separated from the bulk suspension. - 37 -

In the following step, the carrier particles may be removed by undergoing enzymatic digestion, or thermal degradation or plasma removal to release discrete nanoparticles of the organic compound.

Example 7

Preparation of different types of photoluminescent carbon- based materials (carbon dots, CDs) . CDs were prepared in accordance with the methods of the present invention. After being treated with concentrated nitric acid to introduce carboxyl groups, the CDs were mixed with three types of polymers, respectively: Polymer 1: poly (ethylene glycol), H₂NCH₂(CH₂CH₂O)_nCH₂CH₂CH₂NH₂ (average n ~ 35, PEG1500_N) ; Polymer 2: a triblock copolymer consisting of poly (ethylenimide) -jb-poly (ethyleneglycol) -b- poly (ethylenimide) (PEI-PEG-PEI, 5 k -5 k - 5k) ; and Polymer 3: a 4-arm, amine terminated PEG ( 4-arτn PEG, MW: 10 k) . Each mixture suspension of bare CDs and excessive polymer was heated at 120⁰C under reflux for 72 hours for surface passivation. Subsequently, the optically transparent and photoluminescent CD suspensions were purified by dialyzing against Milli-Q water with a cellulose ester membrane bag. The final three nanoformulations with the three different polymer coatings are referred to herein as CD2 (CD- PEG1500_N) , CD3 (CD- PEI-PEG-PEI) , and CD4 (CD- 4 arm PEG) .

Optical Characterizations. Optical absorption and PL spectroscopy were used to examine the spectral properties of the CDs. UV-Vis absorption spectra were recorded with a -38-

Shimadzu 3600 UV-VIS-NIR spectrophotometer, using a quartz cuvette with a 1 cm path length. PL excitation and emission spectra are acquired on a Fluorolog-3 spectrofluorimeter

(Jobin Yvon, Longjumeau, France) . The concentration and hydrodynamic diameter of the photoluminescent particles in the suspensions were characterized by fluorescence correlation spectroscopy (FCS) . FCS was performed with a commercial FCS setup (Carl Zeiss, Germany), consisting of the module ConfoCor 2 and an inverted microscope model Axiovert 200. Dynamic light scattering (Brookhaven 90PLUS with

ZetaPALS option) was used to determine the zeta potential of the 3 nanoformulations of carbon dots.

Conjugation of CDs with Transferrin. For targeted in vitro imaging studies, the NH₂ terminated CDs were conjugated with transferrin using EDC chemistry. In a typical protocol, 0.5 mg of transferrin was added to 0.5 ml of ultrapure water followed by addition of 25 μl of 0.1 M EDC solution and gently stirred for 30 min. Next, 1 mL of the nanoparticles was added and the mixture was incubated at room temperature for 2 h to allow the protein to covalently bond to the NH₂ groups of the CDs .

Studies of Cellular Uptake and Targeting. For in vitro imaging, HeLa cell line was cultured in Dulbecco minimum essential media (DMEM) with 10% fetal bovine serum (FBS) , 1% penicillin, and 1% amphotericin B. The day before treatment with the passivated CDs, cells were seeded in 35 mm culture dishes. On the treatment day, the cells, at a confluency of 50-60%, in serum-supplemented medium were treated with the - 39 -

passivated CD nanoparticles at a specific concentration (100 μL per 1 mL of medium) for two hours at 37 ⁰C.

Cellular Imaging. Confocal microscopy images were obtained using a Leica TCS SP2 AOBS spectral confocal microscope

(Leica Microsystems Semiconductor GmbH, Wetzler, Germany) with laser excitation at 442 nm. All confocal images, which were compared, were obtained at the same parameters of laser power, confocal pinhole, gain, offset, and scanning speed.

Cell Viability Assay. The cellular cytotoxicity of CD2 , CD3 , CD4 was tested on HeIa cells. The cell viability assay was performed using a CellTiter 96AQueous Non-Radioactive Cell Proliferation Assay (Pomega GPR G5421) based on reduction of a tetrazolium component [3- (4 , 5-dimethylthiazol- 2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H- tetrazolium, inner salt; MTS] into an insoluble formazan product by the mitochondria of viable cells. HeLa cells were seeded in a 96 well plate (-10,000 cells/ml/well) and maintained in culture medium for 24h at 37°C. Then lOμl of CD2/CD3/CD4 was added to the every well containing lOOμl of medium and cells were incubated for another 24h. After this, the cells were carefully rinsed, followed by treatment with MTS reagent for 2 hours. The produced formazan was quantified by measurement of its absorbance of at 490nm, using an Elisa plate reader.

The concentrations and the average hydrodynamic diameters of the as-prepared passivated CDs were obtained by FCS as: 1.1 μM, 2.6 nm for CD2 ; 1.4 μM, 1.84 nm for CD3 ; and 250 nM, 3.6 - 40 -

nm for CD4. Apparently, the mixture of Polymer 3 and nascent CDs yielded a 5 fold lower amidation efficiency, possibly due to steric hindrance caused by the large size of Polymer 3, the 4 -armed 1OkD PEG. The particle morphology was observed by TEM and AFM (see Supporting Information) , showing particle size in the range of 1.5 - 3 nm.

Optical Properties. All the three nanoformulations of CDs showed broad absorption in the UV-VIS range. As seen in Figure 9 (a) , CD3 solution absorbs more strongly than CD2 and CD4. There is also a shoulder on the CD3 absorption curve at about 370nm, which is different from the monotonically falling curves for CD2 and CD4. The difference in absorbance is much higher than would be expected from the difference in the CD concentration which leads us to assume that the passivation of the CDs surface with PEI-PEG-PEI results in the formation of a higher amount of the absorbing centers. Under excitation at 365 nm, both CD2 and CD3 suspensions exhibited blue luminescence, while emission from CD4 appears yellowish (see photos in Figure 9) . The PL spectra of CD2 and CD3 are shown in Figure 9 (b) and 9 (c) respectively. They both are multicolored where the emission maxima shifted bathochromically as the excitation wavelength increased from 320 nm to 600 nm step-wisely. There are also noticeable differences between the PL of CD2 and CD3. Whereas CD3 seems to emit more intensively than CD2 , one should note that the absorbance of CD3 is much higher. Furthermore, the most intensive PL from CD3 appears under 440 nm excitation and has a maximum at ~510 nm, while CD2 photoluminescence is the most intensive under excitation at 360 nm, with the peak - 41 -

positioned at 460 nm (Figure 9) . It is worth noting that the PL emission spectra of CD4 (Figure 9 (d) ) appear very- different from those of CD2 and CD3. The lowest light absorption and PL intensities are partly due to the lower concentration of CD4. However, when CD4 was excited at 320 nm, the PL spectrum was extremely broad, peaking at around 550nm, with the highest intensity among the series PL spectra. Secondly, when increasing the excitation wavelength, both the width and the intensity of the PL emission spectra decreased, and the maxima position is even blue- shifted (up to -510 nm) when the excitation wavelength is increased from 320 nm to 400 nm. When the excitation is further tuned towards longer wavelengths (>400 nm) , the PL emission band starts to move bathochromically up to -650 nm. This overall complex behavior of the PL emission spectra is apparently associated with a variety of emitting centers present in the CD suspensions. In support of this, the decays of PL emission are non-monoexponential for all the three types of CD suspensions, with the average lifetime in the range of a few nanoseconds (see Supporting Information) . An inspection of the PL excitation spectra (Figure 10) confirms the existence of multiple types of emitting centers; however, they can be assigned to a few distinct types. The PL excitation band of CD3 appears as continuously shifting but can be also presented as a combination of two PL excitation bands with maxima at - 410 and -460 nm. The PL excitation spectra of CD2 and CD4 are different from those of CD3 , but similar to each other. In particular, one can distinguish the PL excitation bands which are common to both CD2 and CD4 , with maxima at ~370nm, two other peaks at -420-430 nm and -500 nm. - 42 -

The differences in the optical properties of CD2 , CD3 and CD4 are merely caused by the difference in polymer chains bound to the nanoparticle surface, despite that all the three types of polymers were coupled on the nascent CDs by an identical amidation procedure. The PL excitation and emission spectra of the CD suspensions are determined by the type of polymer conjugated to CDs. Thus, a presence of PEI leads to the difference between the PL excitation and emission spectra of CD3 and the other two types of CDs, which do not contain PEI. Similarly, a structural difference between PEG and 4 -arm PEG in CD2 and CD4, correspondingly, determines the formation of the differing nanoparticle-polymer conjugation sites, which are specific to the type of polymer and responsible for optical absorption and photoluminescence . The hydrodynamic diameters characterized by FCS, that is 2.6 nm for CD2 , 1.84 nm for CD3 , and 2.9 nm for CD4 , also provide a clue to the polymer arrangement on the CD surface . When CD2 and CD4 are compared, their hydrodynamic sizes are in proportion to the molecular weight of the passivating polymers, i.e. Mw 1.5 k PEGl50O_N versus Mw 10 k 4-arm PEG. However, surprisingly, in spite of the covalently-bound Mw 15 k PEI-PEG-PEI polymers on the surface, CD3 appears even smaller than CD2. The reduced size of CD3 may be due to the multiple bonding points between the excessive amount of amine groups in the PEI segments and the abundant carboxyl groups on the nascent CDs. Therefore, PEI-PEG-PEI may wrap around the nascent CD surface through multiple amide bonds, deviating from the likely stretching- out morphology in the case of the diamine PEG chains . - 43 -

The FCS measurement also reveals the single nanoparticles brightness expressed in photon counts per particle for a given excitation laser intensity. CD3 has shown 11 kHz/particle compared to 4.4 of CD2. The much increased brightness of CD3 may be explained by the surface-wrapping of the triblock copolymer through the abundant amide coupling on the CD surface, which leads to better passivation. However, it is interesting to note that according to the FCS measurement, CD4 has a brightness of 20 kHz/particle, even higher than CD3. The causes of CD4 ' s highest single particle photon count as well as having a largest Stokes shift when excited by UV light remain elusive and are currently under further investigation.

Cytotoxicity. The CDs were introduced into the HeLa cell cultures to evaluate their potential as bioimaging agents. The cytotoxicity of the CDs was also evaluated. As can be seen in Figure 11, CD2 , CD3 and CD4 demonstrate low cytotoxicity. It is worthwhile to point out that the concentration used in this cytotoxicity experiment is twice as high than the CD concentration used for cell imaging.

Targeting Cancer Cells. For both the applications of disease detection and drug delivery, it is desirable that the photoluminescent nanoparticles can be tailored to actively target a specific type of cells or a particular intracellular compartment as rapid passive cellular uptake of these nanoparticles may not necessarily be advantageous in many biomedical applications. To test the usability of these CDs nanoformulation as targeting photoluminescent nanoprobes, all - 44 -

the three CDs were coupled with human transferrin (Tf) through carbodiimide chemistry following known techniques. Tf, a serum glycoprotein (80 kDa) , has been known for its ability in targeting cancer cells due to over-expression of Tf receptors on cancer cell membranes. The PL emission spectra of the Tf conjugated CDs remain the same as before conjugation (data not shown). 200 μL of CD2 , CD3 , CD4 , Tf- CD2 , Tf-CD3 and Tf-CD4 (Tf conjugated CDs are of equivalent concentrations to their corresponding parent suspensions) were added to dishes with HeLa cells, containing 2mL of the medium. After 2 hours of incubation, HeLa cells are shown to internalize the protein conjugated CD2 and CD4 much more efficiently than non-Tf-conjugated CD2 and CD4 (Figures 12a, 12e and 12b, 12f ) . CD3 demonstrated higher passive cellular uptake in comparison with the other two types of non-Tf- conjugated CDs by the HeLa cells (Figure 12c) . This outcome is consistent with the nanoparticles physical and chemical properties. Due to the excessive amine groups on the CD3 surface carried by the PEI-PEG-PEI polymer chains, CD3 is positively charged (zeta potential +3.35 mV) . Therefore, CD3 is more capable of binding with the cell membrane through electrostatic interactions. On the other hand, although CD2 and CD4 also have amine groups on the surface due to the amine terminated PEG chains, their overall surface charges are negative; the values of zeta potential are -14.51 mV for CD2 and -2.98 mV for CD4. The negative charge may be attributed to the density of polymer coverage on the surface as well as the charge negativity of the PEG segment, a widely accepted anti-biofouling polymer. It is also noted that CD3 is the smallest species (1.8 nm diameter) among the 3 types - 45 -

of CDs, which may also play a role in its more efficient cellular uptake. These cellular uptake experiments have shown that the design of surface functional groups of all three types of CDs permit efficient conjugation ability with biomolecules through carbodiimine chemistry. A selection of functional polymer groups for conjugation to the nanoparticle surface to a great extent determines the efficiency of the cellular uptake of the nanoparticles .

It is worth noting that the difference in the PL signal between the cells treated with Tf-conjugated and non-Tf- conjugated CDs looks more pronounced for CD2 and CD4 , than in the case of CD3. However, one can still discriminate the difference between non-Tf-conjugated and Tf-conjugated CD3 , if imaging conditions (e.g. gain of the confocal PL detector) are chosen in such a way that no PL signal is visible for passive cellular uptake (non-Tf-conjugated CD3 ) . Consequently, the preferential PL signal from the cells targeted with Tf-conjugated CD3 (active cellular uptake) would be easily seen (Figure 12b) .

To check whether the increased internalization of the Tf- conjugated CDs was indeed mediated by the Tf receptors which were over-expressed on cancer cell membranes, the cells were pre-treated with free Tf before treating them with Tf- conjugated CDs. As seen in Figure 13, pre-treatment of cells with free Tf causes a sharp decrease in the targeting efficiency for Tf-conjugated CDs, suggesting saturation of the Tf receptors with free Tf . These data strongly support a Tf-receptor-mediated cellular uptake of the Tf-conjugated -46-

CDs, confirming the cell targeting capability of the bioconjugated carbon dots.

In the description of the invention, except where the context requires otherwise due to express language or necessary- implication, the words "comprise" or variations such as "comprises" or "comprising" are used in an inclusive sense, i.e. to specify the presence of the stated features, but not to preclude the presence or addition of further features in various embodiments of the invention.

It is to be understood that, although prior art use and publications may be referred to herein, such reference does not constitute an admission that any of these form a part of the common general knowledge in the art, in Australia or any other country.

Numerous variations and modifications will suggest themselves to persons skilled in the relevant art, in addition to those already described, without departing from the basic inventive concepts. All such variations and modifications are to be considered within the scope of the present invention, the nature of which is to be determined from the foregoing description .

Claims

- 47 -CLAIMS :

1. A method of preparing a nanomaterial comprising a plurality of discrete nanoparticles of a substance, said method comprising the steps of : a) providing a functionalised carrier; b) associating the functionalised carrier with a precursor of said substance to produce a first composite material; c) converting the bound precursor to said substance in a manner to produce a second composite material comprising a plurality of nanoparticles of the substance supported on the carrier; and d) removing the carrier from the second composite material .

2. The method according to claim 1, wherein the carrier comprises a material capable of being removed from the second composite material under conditions which do not substantially physically and/or chemically degrade the nanoparticles supported thereon.

3. The method according to claim 1 or claim 2, wherein removing the carrier from the second composite material comprises subjecting the second composite material to a process that selectively degrades the carrier.

4. The method according to 3, wherein the process to selectively degrade the carrier comprises dissolving the carrier with a solvent in which the carrier is soluble, thermal degradation, microwave- induced degradation, plasma-induced degradation, or chemical degradation. - 48 -

5. The method according to any one of claims 1 to 4, wherein the carrier is a material that is physically and/or chemically stable under the conditions used to convert the precursor which is bound to the modified surface of the carrier to discrete nanoparticles of said substance.

6. The method according to any one of claims 1 to 5, wherein the carrier is an inorganic material.

7. The method according to claim 6, wherein the carrier comprises an inorganic material selected from a group comprising silica, alumino-silicates, in particular zeolites, metal oxides comprising aluminium oxide, iron oxide, zinc oxide or zirconium oxide, carbon, and calcium sulfate.

8. The method according to any one of claims 1 to 5, wherein the carrier is an organic material .

9. The method according to claim 8, wherein the organic material is a polymeric material.

10. The method according to any one of claims 1 to 9 , wherein providing a functionalised carrier comprises modifying a surface of a carrier with one or more functionalisation agents .

11. The method according to claim 10, wherein the step of modifying the surface of the carrier with one or more functionalisation agents comprises forming primary or - 49 -

secondary bonding interactions between a surface of the carrier and the functionalisation agents.

12. The method according to claim 10 or claim 11, wherein the functionalisation agents comprises a long chain organic compound having functional groups and/or moieties capable of forming primary bonding and/or secondary bonding interactions with terminal groups on the surface of the carrier.

13. The method according to claim 12, wherein the functionalisation agent is provided with further functional groups and/or moieties capable of forming secondary bonding interactions with the precursor.

14. The method according to claim 13, wherein the functionalisation agent comprises an amphiphilic long chain organic compound having functional groups and/or moieties capable of forming secondary bonding interactions with the surface of the carrier and with the precursor.

15. The method according to claim 14, wherein the functionalisation agent comprises an amphiphilic long chain organic compound selected from a group comprising surfactants, in particular ionic or non-ionic surfactants, or surfactants containing a hydrophilic block, such as polyethyleneoxide (PEO) , polyacrylic acid (PAA) , polyethylenimine (PEI) , or polymethylmethacrylate (PMMA) , polyelectrolytes, and amphiphilic linear or branched chain polymers, in particular homopolymers, diblock copolymers, - 50 -

triblock copolymers, and more particularly those amphiphilic linear or branched chain polymers containing at least one hydrophilic block.

16. The method according to claim 15, wherein the functionalisation agent is an amphiphilic triblock copolymer.

17. The method according to claim 13, wherein the functionalisation agent may comprises a cationic polyelectrolyte or an anionic polyelectrolyte .

18. The method according to any one of claims 1 to 17, wherein the precursor of the substance comprises a chemical or material which is capable of being converted to the substance by one or more chemical reactions.

19. The method according to claim 18, wherein the one or more chemical reactions are selected from a group comprising polymerisation, oxidation, reduction, thermal degradation by oxidative or reductive means, acid-base reactions, precipitation, metal-ligand complexation reactions, host- guest interactions, self-assembly reactions, supramolecular assembly, and so forth.

20. The method according to claim 18 or claim 19, wherein the precursor for metals or metal alloys comprises one or more metal ions, cationic complex ions of metals, anionic complex ions of metals, metal complexes, metal compounds, organometallic compounds including metallocenes, and so - 51 -

forth .

21. The method according to claim 18 or claim 19, wherein the precursor for inorganic compounds or organic compounds comprises solutions of said inorganic compounds or organic compounds, or one or more suitable reactants for preparation of said inorganic compounds or organic compounds .

22. The method according to claim 18 or claim 19, wherein the precursor for carbon-based materials, including dopant- containing carbon-based materials comprises a polymer precursor or any organic compound or material which is capable of binding to the functionalisation agent and converting to a carbon-based material, in particular by thermal degradation.

23. The method according to claim 22, wherein the polymer precursor comprises a polymer oligomer (M_w < 500) including N-, S-, B-, F-, Si-containing polymer oligomers (M_w < 500).

24. The method according to claim 23, wherein the polymer oligomer is selected from a group comprising resol

(phenol/formaldehyde resins, M_w < 500) , pyrrole, polymeracryonitrile (PAN) , aniline, acetonitrille, thiphenemethanol, P-fluorphenol- formaldehyde, boronic ester, boron nitride and boron nitrogen polymers, polycarbosilane (PCS) .

25. The method according to any one of claims 22 to 24, - 52 -

wherein the polymer precursor is a dopant-containing polymer precursor.

26. The method according to any one of claims 10 to 25, wherein the step of associating the functionalized carrier with the precursor to produce a first composite material comprises forming primary or secondary bonding interactions between the functional groups and/or moieties of the functionalisation agent and the precursor.

27. The method according to claim 26, wherein said step comprises conjugating amphiphilic blocks of the functionalisation agent with the polymer precursor.

28. The method according to claim 26, wherein said step comprises forming electrostatic charge interactions between charged functional groups and/or moieties on the functionalisation agent and a charged precursor.

29. The method according to claim 27, wherein said step comprises adsorption of the precursor onto the functionalized carrier.

30. The method according to any one of claims 1 to 29, wherein the step of converting the bound precursor to said substance comprises subjecting the bound precursor to one or more chemical reactions.

31. The method according to claim 30, wherein the one or more chemical reactions is selected from a group comprising - 53 -

polymerisation, oxidation, reduction, thermal degradation by oxidative or reductive means, acid-base reactions, precipitation, metal-ligand complexation reactions, host- guest interactions, self-assembly reactions, supramolecular assembly.

32. The method according to claim 30, wherein the step of converting the polymer precursor to carbon-based material in a manner to produce a second composite material comprising a plurality of carbon-based nanoparticles supported on the carrier comprises: polymerising the polymer precursor on the functionalized carrier, thereby producing a composite material comprising a plurality of discrete polymer nanoparticles supported on the carrier; and converting the polymer nanoparticles of the composite material to carbon-based nanoparticles .

33. The method according to claim 32, wherein the step of converting the polymer nanoparticles of the composite material to carbon-based nanoparticles comprises heating the composite material to a temperature at which the polymer nanoparticles thermally decompose to carbon-based nanoparticles .

34. The method according to claim 33, wherein the polymer nanoparticles thermally decompose to amorphous, semicrystalline, or crystalline carbon nanoparticles.

35. A composite material comprising a precursor of a substance - 54 -

bound to a functionalised carrier.

36. The composite material according to claim 35, wherein the composite material comprises nanoparticles of a precursor of the substance supported on the functionalised carrier.

37. The composite material according to claim 35, wherein the nanoparticles of the precursor of the substance have a particles size in a range of about 2 nm to about lOOnm.

38. A composite material comprising a plurality of discrete nanoparticles of a substance supported on a functionalised carrier.

39. A composite material according to any one of claims 35 to 38 when used to prepare a nanomaterial comprising a plurality of discrete nanoparticles of the substance.

40. A method of preparing a nanomaterial of a substance comprising a plurality of discrete nanoparticles of the substance, the method comprising the steps of: a) providing a first composite material comprising a precursor of said substance bound to a carrier; b) converting the bound precursor to the substance in a manner to produce a second composite material comprising a plurality of discrete nanoparticles of the substance supported on the carrier; and c) removing the carrier from the second composite material . - 55 -

41. A method of preparing a nanomaterial of a substance comprising a plurality of discrete nanoparticles of the substance comprises the steps of: providing a composite material comprising a plurality of discrete nanoparticles of the substance supported on the carrier; and removing the carrier from the composite material.

42. A method for preparing a doped carbon-based nanomaterial comprising a plurality of doped carbon-based nanoparticles, the method comprising the steps of: a) providing a functionalized carrier; b) associating the functionalised carrier with a carbon- containing precursor containing a dopant to produce a first composite material; c) converting the bound carbon-containing precursor containing the dopant to a doped carbon-based material in a manner to produce a second composite material comprising a plurality of discrete doped carbon-based nanoparticles supported on the carrier,- and d) removing the carrier from the second composite material.

43. A method of preparing a composite material comprising a plurality of discrete nanoparticles of a substance supported on a carrier, the method comprising the steps of: a) providing a functionalized carrier; b) associating the functionalised carrier with a precursor of the substance to produce a first composite material; and - 56 -

c) converting the bound precursor to the substance in a manner to produce a second composite material comprising a plurality of discrete nanoparticles of the substance supported on the carrier.

44. A method of preparing a photoluminescent carbon-based nanomaterial comprising a plurality of photoluminescent carbon-based nanoparticles, the method comprising the steps of : a) providing a functionalized carrier; b) associating the functionalised carrier with a carbon- containing precursor to produce a first composite material; c) converting the bound carbon-containing precursor to a carbon-based substance in a manner to produce a second composite material comprising a plurality of carbon-based nanoparticles supported on the carrier; d) removing the carrier from the second composite material; and e) passivating the carbon-based nanoparticles separated from the second composite material in step d) to produce the photoluminescent carbon-based material .

45. The method according to claim 44, wherein prior to passivating the carbon-based nanoparticles, the method further comprises functionalizing the carbon-based nanoparticles with terminal functional groups on a surface of the carbon-based nanoparticles. - 57 -

46. The method according to claim 45, wherein the step of functionalizing the carbon-based nanoparticles comprises reacting the carbon-based nanoparticles with a reagent capable of introducing terminal functional groups on the surface of the carbon-based nanoparticles.

47. The method according to any one of claims 44 to 46, wherein the step of passivating the carbon nanoparticles comprises reacting the carbon nanoparticles with a passivating agent.

48. The method according to claim 47, wherein the passivating agent comprises a long chain organic compound capable of binding to a surface of the carbon nanoparticles by primary or secondary bonding interactions.

49. The method according to claim 48, wherein the passivating agent is selected from a group of compounds comprising amphiphilic oligomeric polymers, long-chained surfactants, nucleotides, peptides.

50. The method according to claim 49, wherein the passivating agent comprises a diamine-terminated oligomeric polymeric poly (ethylene glycol), H₂NCH₂(CH₂CH₂O)_nCH₂CH₂CH₂NH₂ (average n ~ 35, PEG_ISOON) and PEI-PEG-PEI (polyehtylenimine- polyethylene glycol-polyethylenimine, 5k-5k-5k) triblock copolymer. - 58 -

51. A photoluminescent carbon-based nanomaterial comprising a surface-passivated carbon particle having a particle size distribution in a range of about 1 to about 10 nm.

52. The photoluminescent carbon-based nanomaterial according to claim 51, wherein the photoluminescent carbon-based nanomaterial emits luminescence in a wavelength range from about 420 nm to about 600 nm under excitation in a wavelength range from about 300 nm to about 540 nm.

53. The photoluminescent carbon-based nanomaterial according to claim 51 or claim 52, wherein the photoluminescent quantum yield of the photoluminescent carbon-based nanomaterial is greater than 10%.

54. The photoluminescent carbon-based nanomaterial according to any one of claims 51 to 53, wherein said nanomaterial has a particle size of about 1.5 nm to 2.5 nm.

55. A bioimaging agent comprising a surface-passivated carbon particle having a particle size distribution in a range of about 1 to about 10 nm.