WO2010139942A2

WO2010139942A2 - Nanoparticle for biomolecule delivery

Info

Publication number: WO2010139942A2
Application number: PCT/GB2010/001070
Authority: WO
Inventors: Duncan Graham; Nial Joseph Wheate; Sarah Brown; Gemma Craig
Original assignee: University Of Strathclyde
Priority date: 2009-06-02
Filing date: 2010-06-01
Publication date: 2010-12-09
Also published as: GB0909435D0; WO2010139942A3

Abstract

A nanoparticle comprises a metal nanoparticle, e.g. gold, functionalised with at least one linker, the at least one linker comprising at least one first moiety capable of interacting with the metal nanoparticle, and at least one second moiety capable of interacting with at least one platinum group metal-based biologically active compound, e.g. a drug, wherein the molar ratio or capacity of platinum group metal-based biologically active compounds per nanoparticle is greater than (150). The present invention also relates to a method of preparing a nanoparticle according to the present invention. The present invention also relates to a biologically active nanoparticle comprising a nanoparticle according to the present invention, and at least one platinum group metal-based biologically active compound. The biologically active nanoparticle is more efficiently targeted to specific parts of the body, e.g. cancer cells, while reducing the side effects of the biomolecules being delivered.

Description

NANOPARTICLE FOR BIOMOLECULE DELIVERY

FIELD OF INVENTION

The present invention relates to a nanoparticle comprising a metal nanoparticle, e.g. gold, functionalised with one or more linkers comprising moieties capable of interacting with the metal nanoparticle and moieties capable of interacting with a biologically active substance, e.g. a drug, and in particular a platinum group metal-based anti-cancer drug. The present intention also relates to a method of preparing such a metal nanoparticle comprising reacting a metal nanoparticle with a linker.

The present intention also relates to a biologically active nanoparticle comprising the functionalised nanoparticle, and a biologically active compound.

The present intention also relates to a method of preparing such a biologically active nanoparticle comprising reacting the functionalised nanoparticle with a biologically active compound.

BACKGROUND TO INVENTION

Cancer is believed to account for more than 12% of all deaths worldwide.

With the ageing populations of many Western countries its incidence is increasing. Accordingly, there is an international need to develop more effective drug treatments. Running parallel to drug discovery is considerable interest in the development of drug delivery vehicles for drugs that are approved or currently under clinical evaluation. Cisplatin, or cis-diamminedichloroplatinum(II) is an isomeric, square planar, neutral complex which is used to treat a variety of human cancers, including ovarian, testicular, lung, bladder, colorectal, head and neck cancer. Unfortunately, cisplatin has several major drawbacks. Many human cancer cell lines have intrinsic resistance to the drug, and in others where cisplatin does have some initial activity, many rapidly acquire a resistance. Acquired resistance is thought to arise through four possible mechanisms, including: reduced cellular uptake; enhanced DNA adduct identification and repair; increased DNA adduct tolerance; and elevated glutathione levels leading to increased deactivation before cisplatin reaches its cellular target. Recently, elevated intracellular chloride concentrations have been shown as another possible mechanism of resistance. Cisplatin is also limited because of its severe dose-limiting side-effects which include nephrotoxicity, ototoxicity and neurotoxicity. Many of the side-effects of cisplatin are due to its non-specific attack of all rapidly dividing cells and therefore, platinum-based chemotherapy can be greatly improved through enhanced drug delivery.

Carboplatin, or diammine[l,2-cyclobutanedicarboxylato]platinum(II), is one drug that was designed specifically to reduce the side-effects of platinum treatment. Carboplatin was given FDA approval in March 1989 on the basis of its different toxicity profile to cisplatin. Patients experience little, to no, nephrotoxicity, therefore removing the need for hydration, and much reduced oto-, neuro- and gastrointestinal toxicity. At doses of around 900 mg/m² in human patients, bone marrow toxicity (including leukopenia, neutropenia and thrombocytopenia) is the DLT. Carboplatin is currently sold by Bristol Myers-Squibb under the brand name Paraplatin™, and under the generic name of Carboplatin by at least 5 other companies. The drug is used in the treatment of advanced ovarian carcinoma, including ovarian carcinomas which have recurred after previous treatment with cisplatin. Carboplatin is used either as a single agent or in combination therapy with other oncology drugs. Newer drugs combining decreased side-effects and activity in cisplatin resistant cells are now being developed. One such drug, recently approved for use by the FDA, is oxaliplatin, or trans-L-diaminocyclohexaneoxalatoplatinum(II). While the major adducts formed with DNA by cisplatin and oxaliplatin are very similar, it is thought the latter drug is able to overcome cisplatin resistance because the bulky and hydrophobic DACH ligand assists in inhibiting DNA transcription. Oxaliplatin is currently sold in the USA under the brand name Eloxatin™, by Sanofi-Aventis, for the treatment of advanced carcinomas of the rectum or colon.

Cisplatin, carboplatin, and oxaliplatin are defined respectively by the following formulae:

cisplatin carboplatin oxaliplatin Platinum drugs can be passively targeted to solid tumours through the enhanced permeability and retention effect. Alternatively, platinum drugs can be actively targeted to both solid tumours and leukaemias through the use of cancer related substrates (like folate), aptamers, peptides and antibodies. Over the last decade gold nanoparticles have been developed that demonstrate a wide variety of applications, including catalysis, improving computer memory and in bioanalysis and imaging. Gold is also known to be largely non-toxic and immunogenic. Gold particles can be readily functionalised with multiple targeting molecules and have already shown potential for the delivery of other non-platinum based drugs.

It is believed attachment of platinum drugs to gold nanoparticles may have two benefits over existing drugs. Firstly, they can be better targeted to cancer cells through passive (enhanced permeability and retention effect) or through active targeting (further attachment of substrates, aptamers, peptides or antibodies to the gold nanoparticles as well). Better targeting of cancer cells will reduce the type and severity of the side-effects associated with chemotherapy. Secondly, better targeting will increase the dose delivered to cells, thereby preventing the development of resistance (through better kill rates). In cells that have already developed resistance (through reduced uptake), better targeting may overcome drug resistance. In addition, the existing drug delivery systems capable of delivering biomolecules, e.g. drugs, to specific parts of the human body, show limited capacity in terms of the number of drug molecules the system is capable of delivering. For example, the capacity of carbon nanotubes is typically 82 drug molecules per NT, that of hyper-branched polymers is typically 3-6 drug molecules per polymer, and that of dendrimers is typically 30-104 per dendrimer depending on dendrimer generation/size.

US Patent Application Publication No. US 2006/0099146 discloses a Near Infrared Sensitive (NIR-sensitive) nanoparticle complex comprising a NIR-sensitive nanoparticle and surfactant(s) adsorbed on the nanoparticle, wherein the surfactant comprises one or more thiol groups and one or more carboxy, amine, and/or hydroxy groups Further, it is provided a NIR-sensitive nanoparticle complex(es) having biomolecule(s), for example drug(s), loaded on the surfactant(s).

US Patent Application Publication No. US 2006/222595 discloses materials and methods relating to nanoparticles, for example, nanoparticle compositions, methods for making nanoparticle compositions, and methods for using nanopaiticle compositions. In some cases, the nanoparticles are gold (e.g., colloidal gold) nanoparticles. A nanoparticle can include one or more agents linked to its surface, such as therapeutic and/or diagnostic agents, and can be from about 1 nm to about 10 nm in size.

International Patent Application Publication No. WO 2009/062138 (Virginia Tech Intellectual Properties, Inc.) discloses thiolated taxane derivatives for reaction with gold nanoparticles for use as antitumor agents. In WO 2009/062138 the preparation of metallic nanoparticle-paclitaxel derivative complexes exclusively involves the use of paclitaxel derivatives, and the binding of the nanoparticle to the drug is dictated and optimised to suit this particular type of drug.

Therefore, there is a need in the prior art to develop new substances and/or methods for delivering platinum group metal-based biologically active molecules such that these are more efficiently targeted to specific parts of the body, e.g. cancer cells, while reducing the side effects of the biomolecules being delivered.

SUMMARY OF INVENTION

According to a first aspect of the present invention there is provided a nanoparticle comprising a metal nanoparticle functionalised with at least one linker, the at least one linker comprising at least one first moiety capable of interacting with the metal nanoparticle, and at least one second moiety capable of interacting with at least one platinum group metal-based biologically active compound, wherein the molar ratio or capacity of platinum group metal-based biologically active compounds per nanoparticle is greater than 150, and wherein the linker has the following formula:

(1) R₁-Y-R₂ wherein: Y is a linking moiety;

Ri is a first moiety capable of interacting with a metal nanoparticle; and

R₂ is a second moiety capable of interacting with at least one platinum group metal-based biologically active compound. A platinum group metal is understood in the art as referring to any one of six particular Group VIII transition metals, namely ruthenium, rhodium, palladium, osmium, iridium and platinum.

Typically, the platinum group metal-based biologically active compound is a platinum-based biologically active compound.

Typically, Ri may comprise a hydrophobic moiety. By such provision the stability of the functionalised nanoparticle may be improved or optimised in an aqueous medium, e.g. an aqueous dispersion.

Advantageously, the first moiety Ri may interact with the metal nanoparticle through co-ordination bonding.

Alternatively, the first moiety Rj may interact with the metal nanoparticle through shelling in an organic or inorganic polymer, or through hydrophobic interactions.

Typically, the first moiety Ri may comprise an optionally substituted 5 or 6- membered sulphur-containing ring structure.

Preferably, the first moiety Rj may comprise an optionally substituted 5 or 6- membered disulfϊde-containing heterocycle.

Typically, the second moiety R₂ may comprise a hydrophilic moiety. Typically also, the second moiety R₂ may comprise a reactive moiety comprising, e.g. a Lewis base such as a weak acid group.

Preferably, the second moiety R₂ may comprise a carboxylic acid group. Alternatively, the second moiety R₂ may comprise other reactive groups such as a dicarboxylic acid group or a thiol group.

Y may be an organic linking moiety, or an inorganic linking moiety comprising e.g. silane.

Typically, Y may be a hydrophilic linking moiety. By such provision solvation or dispersibility of the functionalised nanoparticle may be improved or optimised in an aqueous medium.

Typically, Y may be a linking moiety having the following formula: (2) X₂-L₁-X, wherein: Lj is an optionally substituted linking structure;

> Xi, X₂ are modifying linking groups. Preferably, Li may comprise an optionally substituted polymeric structure. A polymer is herein defined as a compound comprising a minimum of two repeating structural units.

Typically, L] may comprise a linear polymeric chain, e.g. polyethylene glycol (PEG).

Alternatively, Li may comprise a non-linear polymeric structure, e.g. a dendritic structure.

Alternatively, Li may comprise a Ci₀-C₂O₀, e.g. a C_2O-Ci_2O, optionally substituted alkyl or hereroalkyl chain. It is believed that the relatively long linking moiety provided in the linker as disclosed above may enhance stability of the functionalised nanoparticle, in particular against in vivo degradation.

Typically, Xi and X₂ may independently comprise, e.g., an amide, ester, ether, sulphate, sulfonate, imide, urethane and/or urea group. Conveniently, the metal nanoparticle may be surface-functionalised with the at least one linker.

Conveniently, the ratio of reactive moieties capable of interacting with the at least one platinum group metal-based biologically active compound per metal nanoparticle may be in the range of 150 - 30,000, preferably approximately 200 - 30,000.

Typically, when the linker comprises a linear polymeric chain, the ratio of reactive moieties capable of interacting with the at least one platinum group metal- based biologically active compound per metal nanoparticle may be approximately 200 - 1000, preferably approximately 250 - 300. Beneficially, the at least one platinum group metal-based biologically active compound may be a cell-deliverable compound.

Typically, the at least one platinum group metal-based biologically active compound may be a DN A-interactive component, and/or a pharmaceutical compound, e.g. a drug. Preferably, the drug may be an anti-cancer drug.

Typically, the drug may be a mononuclear platinum-based anti-cancer drug, e.g. cisplatin, picoplatin, satraplatin or preferably oxaliplatin, or a multinuclear platinum-based anti-cancer drug, e.g. BBR3464 or CT-3610. The at least one platinum group metal-based biologically active compound may further comprise at least one compound, e.g. at least one compound for actively targeting cancer cells which may be e.g. a vitamin such as folate, a hormone such as estrogen, a prostate or leukaemia-targeting aptamer, a lung cancer-targeting peptide, and/or a B-cell lymphoma-targeting antibody.

Typically, the metal nanoparticle may be noble metal nanoparticle, e.g. a gold nanoparticle. It is understood that the term nanoparticle covers all 'nano' modalities where one dimension is sub 100 nm, e.g. nanoshells, hollow nanoparticles, nanoprisms, nanocubes, nanorods, etc. Advantageously, the metal nanoparticle may comprise at least one first portion having magnetic properties. By such provision the movement of the nanoparticle, e.g. inside a patient's body, may be directed by use of a controllable magnetic field. This may eliminate the side-effects of the platinum group metal- based biologically active compound intended to be delivered by the nanoparticle. Typically, the at least one first portion may comprise iron.

The at least one first portion may comprise an inner or core portion of the nanoparticle.

Conveniently, the at least one first portion may comprise an iron core.

The nanoparticle may further comprise at least one second portion provided on at least part of an outer portion of the nanoparticle.

The at least one second portion may be devoid of magnetic properties.

Typically, the at least one second portion may comprise a noble metal, e.g. gold.

Conveniently, the at least one second portion may be coated onto at least part of the at least one first portion and may preferably cover substantially the whole surface of the at least one first portion. By such provision the exposed surface of the nanoparticle may comprise an outer surface of the at least one second portion which may comprise a biologically suitable material, e.g. a noble metal such as gold, while ensuring the nanoparticle exhibits advantageous magnetic properties. Preferably, the linker may be a compound of formula (3): (3)

According to a second aspect of the present invention there is provided a method of preparing a nanoparticle according to the first aspect of the invention comprising reacting a metal nanoparticle with a linker comprising at least one first moiety capable of interacting with the metal nanoparticle, and at least one second moiety capable of interacting with at least one platinum group metal-based biologically active compound.

Typically, the metal nanoparticle may be a noble metal nanoparticle, e.g. a gold nanoparticle.

The method may further comprise preparing the metal nanoparticle, e.g. gold nanoparticle, by dissolving a metal salt in water and adding a reducing agent so as to form colloidal nanoparticles, e.g. colloidal gold nanoparticles.

The method of preparing the metal nanoparticle may comprise dissolving sodium tetrachloroaurate (III) in water, heating, stirring, and adding a reducing agent, e.g. tribasic sodium citrate, so as to form colloidal gold nanoparticles.

Preferably, the colloidal gold nanoparticles may have a substantially uniform size distribution.

The linker may be a linker as defined in the first aspect of the invention. The method may further comprise preparing the linker by reacting a first compound of formula (4), with a second compound of formula (5), then with a third compound of formula (6). (4) A₂-R₂-B₂ wherein A₂ is a protective group or end-capping group, e.g. Wang resin, which may temporarily protect second reactive moiety R₂;

R₂ is a second moiety capable of interacting with a platinum group metal-based biologically active compound; and B₂ is a reactive moiety, e.g. a carboxylic acid-containing moiety, capable of reacting with a group Z₂ of the second compound of formula (5). (5) Z₁-L₁-Z₂ wherein Zi is a reactive moiety, e.g. an amine-containing moiety, capable of reacting with a group Wi of the third compound of formula (6);

Li is an optionally substituted linking structure, e.g. a linear polymeric chain such as polyethylene glycol (PEG), a non-linear polymeric structure such as a dendritic structure, or a Ci₀-C₂₀₀, e.g. a C₂₀-Ci₂₀, optionally substituted alkyl or hereroalkyl chain; and

Z₂ is a reactive moiety, e.g. an amine-containing moiety, capable of reacting with a group B₂ of the first compound of formula (4). (6) W,-R, wherein W] is a reactive moiety capable of reacting with a group Zj of the second compound of formula (5); and

Ri is a first moiety capable of interacting with a metal nanoparticle. Typically, Ri may comprise a hydrophobic moiety. By such provision the stability of the functionalised nanoparticle may be improved or optimised in an aqueous medium, e.g. an aqueous dispersion.

Advantageously, the first moiety Ri may interact with the metal nanoparticle through co-ordination bonding. Alternatively, the first moiety Ri may interact with the metal nanoparticle through shelling in an organic or inorganic polymer, or through hydrophobic interactions.

Typically, the first moiety Ri may comprise an optionally substituted 5 or 6- membered sulphur-containing ring structure. Preferably, the first moiety Ri may comprise an optionally substituted 5 or 6- membered disulfide-containing heterocycle.

Typically, the second moiety R₂ may comprise a hydrophilic moiety. Typically also, the second moiety R₂ may comprise a reactive moiety comprising, e.g. a Lewis base such as a weak acid group. Preferably, the second moiety R₂ may comprise a carboxylic acid group.

Alternatively, the second moiety R₂ may comprise other reactive groups such as a dicarboxylic acid group or a thiol group. Typically, the method of preparing a linker may comprises reacting a first compound of formula (4a) with a second compound of formula (5a), then with a third compound of formula (6a)

wherein A is a protective or end-capping group, e.g. Wang resin. Typically, the method may comprise reacting a gold nanoparticle with the linker of formula (3). (3)

According to a third aspect of the invention there is provided a dispersion comprising a nanoparticle according to the first aspect of the invention, and a dispersing medium.

Typically, the dispersing medium is water.

Preferably, the dispersion is a stable aqueous dispersion, i.e. an aqueous dispersion exhibiting long storage stability.

According to a fourth aspect of the present invention there is provided a biologically active nanoparticle comprising a nanoparticle according to the first aspect of the invention, and at least one platinum group metal-based biologically active compound. Beneficially, the at least one platinum group metal-based biologically active compound may be a cell-deliverable compound.

Typically, the at least one platinum group metal-based biologically active compound may be a DNA-interactive component, and/or a pharmaceutical compound, e.g. a drug.

Preferably, the drug may be an anti-cancer drug.

Typically, the drug may be a mononuclear platinum-based anti-cancer drug, e.g. cisplatin, picoplatin, satraplatin or preferably oxaliplatin, or a multinuclear platinum-based anti-cancer drug, e.g. BBR3464 or CT-3610. The at least one platinum group metal-based biologically active compound may further comprise at least one compound, e.g. at least one compound for actively targeting cancer cells which may be e.g. a vitamin such as folate, a hormone such as estrogen, a prostate or leukaemia-targeting aptamer, a lung cancer-targeting peptide, and/or a B-cell lymphoma-targeting antibody. Typically, the biologically active nanoparticle may be approximately 10 - 300 nm in diameter, preferably approximately 25 - 200 nm in diameter.

Typically, the biologically active nanoparticle may be an oxaliplatin-tethered gold nanoparticle comprising a gold nanoparticle functionalised with at least one linker of formula (3), and oxaliplatin. (3)

According to a fifth aspect of the present invention there is provided a method of preparing the biologically active nanoparticle according to the fourth aspect of the invention comprising reacting a nanoparticle according to the first aspect of the invention with at least one platinum group metal-based biologically active compound. The method of preparing the biologically active nanoparticle may further comprise preparing a nanoparticle according to the first aspect of the invention by the method according to the second aspect of the invention. Typically, the method comprises reacting a nanoparticle comprising a gold nanoparticle surfaced-modified by the linker of formula (3), with oxaliplatin. (3)

According to a sixth aspect of the present invention, there is provided a pharmaceutical composition comprising a compound according to the fourth aspect of the invention, together with a pharmaceutically acceptable excipient therefor.

According to a seventh aspect of the present invention, there is provided the use of a compound according to the fourth aspect of the invention for the preparation of a medicament for therapeutic application in the treatment of a disease, e.g. cance *"r^•.

According to an eighth aspect of the present invention, there is provided the use of a compound according to the fourth aspect of the invention for the treatment of a disease, e.g. cancer.

According to a ninth aspect of the present invention there is provided a method of treating a disease, e.g. cancer, the method comprising delivering an effective amount of a compound according to the fourth aspect of the invention, or of a pharmaceutical composition according to the sixth aspect of the invention, to a subject in need thereof.

The method may further comprise exposing a treated region to radiation, e.g. near Infra-Red (NIR). NIR radiation, in particular, may activate, complement or assist the compound according to the fourth or fifth aspect of the invention. It has been suggested that when NIR is applied, the drug may be released from the nanoparticles to destroy cancerous cells. Such irradiation may further improve efficiency of the biologically active nanoparticle, reduce its toxicity and improve patient compliance and convenience compared to conventional cancer treatments.

Subjects may be a mammal, preferably a human. The subject may be a non- human primate or non-primate such as used in animal model testing. While it is particularly contemplated that the method is suitable for use in medical treatment of humans, it is also applicable to veterinary treatment, including treatment of companion animals such as dogs, cats, rabbits and the like, and domestic animals such as horses, ponies, donkeys, mules, llama, alpaca, pigs, cattle and sheep, or zoo animals such as primates, felids, canids, bovids, and ungulates.

Suitable mammals include members of the Orders Primates, Rodentia, Lagomorpha, Cetacea, Carnivora, PerissodacLyla and ArtiodacLyla.

Pharmaceutical formulations include those suitable for oral, topical (including dermal, buccal and sublingual), rectal or parenteral (including subcutaneous, intradermal, intramuscular and intravenous), nasal and pulmonary administration e.g., by inhalation. The formulation may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association an active compound with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.

Pharmaceutical formulations suitable for oral administration wherein the carrier is a solid are most preferably presented as unit dose formulations such as boluses, capsules or tablets each containing a predetermined amount of active compound. A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine an active compound in a free-flowing form such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, lubricating agent, surface-active agent or dispersing agent. Moulded tablets may be made by moulding an active compound with an inert liquid diluent. Tablets may be optionally coated and, if uncoated, may optionally be scored. Capsules may be prepared by filling an active compound, either alone or in admixture with one or more accessory ingredients, into the capsule shells and then sealing them in the usual manner. Cachets are analogous to capsules wherein an active compound together with any accessory ingredient(s) is sealed in a rice paper envelope. An active compound may also be formulated as dispersible granules, which may for example be suspended in water before administration, or sprinkled on food. The granules may be packaged, e.g., in a sachet. Formulations suitable for oral administration wherein the carrier is a liquid may be presented as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water liquid emulsion. Formulations for oral administration include controlled release dosage forms, e.g., tablets wherein an active compound is formulated in an appropriate release - controlling matrix, or is coated with a suitable release - controlling film. Such formulations may be particularly convenient for prophylactic use. Pharmaceutical formulations suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories may be conveniently formed by admixture of an active compound with the softened or melted carrier(s) followed by chilling and shaping in moulds. Pharmaceutical formulations suitable for parenteral administration include sterile solutions or suspensions of an active compound in aqueous or oleaginous vehicles.

Injectible preparations may be adapted for bolus injection or continuous infusion. Such preparations are conveniently presented in unit dose or multi-dose containers which are sealed after introduction of the formulation until required for use. Alternatively, an active compound may be in powder form which is constituted with a suitable vehicle, such as sterile, pyrogen-free water, before use.

An active compound may also be formulated as long-acting depot preparations, which may be administered by intramuscular injection or by implantation, e.g. subcutaneously or intramuscularly. Depot preparations may include, for example, suitable polymeric or hydrophobic materials, or ion-exchange resins. Such long-acting formulations are particularly convenient for prophylactic use.

Formulations suitable for pulmonary administration via the buccal cavity are presented such that particles containing an active compound and desirably having a diameter in the range of 0.5 to 7 microns are delivered in the bronchial tree of the recipient.

As one possibility such formulations are in the form of finely comminuted powders which may conveniently be presented either in a pierceable capsule, suitably of, for example, gelatin, for use in an inhalation device, or alternatively as a self- propelling formulation comprising an active compound, a suitable liquid or gaseous propellant and optionally other ingredients such as a surfactant and/or a solid diluent. Suitable liquid propellants include propane and the chlorofluorocarbons, and suitable gaseous propellants include carbon dioxide. Self-propelling formulations may also be employed wherein an active compound is dispensed in the form of droplets of solution or suspension.

Such self-propelling formulations are analogous to those known in the art and may be prepared by established procedures. Suitably they are presented in a container provided with either a manually-operable or automatically functioning valve having the desired spray characteristics; advantageously the valve is of a metered type delivering a fixed volume, for example, 25 to 100 microlitres, upon each operation thereof.

As a further possibility an active compound may be in the form of a solution or suspension for use in an atomizer or nebuliser whereby an accelerated airstream or ultrasonic agitation is employed to produce a fine droplet mist for inhalation.

Formulations suitable for nasal administration include preparations generally similar to those described above for pulmonary administration. When dispensed such formulations should desirably have a particle diameter in the range 10 to 200 microns to enable retention in the nasal cavity; this may be achieved by, as appropriate, use of a powder of a suitable particle size or choice of an appropriate valve. Other suitable formulations include coarse powders having a particle diameter in the range 20 to 500 microns, for administration by rapid inhalation through the nasal passage from a container held close up to the nose, and nasal drops comprising 0.2 to 5% w/v of an active compound in aqueous or oily solution or suspension.

It should be understood that in addition to the aforementioned carrier ingredients the pharmaceutical formulations described above may include, an appropriate one or more additional carrier ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.

Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.

Therapeutic formulations for veterinary use may conveniently be in either powder or liquid concentrate form. In accordance with standard veterinary formulation practice, conventional water soluble excipients, such as lactose or sucrose, may be incorporated in the powders to improve their physical properties. Thus particularly suitable powders of this invention comprise 50 to 100% w/w and preferably 60 to 80% w/w of the active ingredient(s) and 0 to 50% w/w and preferably 20 to 40% w/w of conventional veterinary excipients. These powders may either be added to animal feedstuffs, for example by way of an intermediate premix, or diluted in animal drinking water.

Liquid concentrates of this invention suitably contain the compound or a derivative or salt thereof and may optionally include a veterinarily acceptable water- miscible solvent, for example polyethylene glycol, propylene glycol, glycerol, glycerol formal or such a solvent mixed with up to 30% v/v of ethanol. The liquid concentrates may be administered to the drinking water of animals.

BRIEF DESCRIPTION OF DRAWINGS

Embodiments of the invention will now be given by way of example only, and with reference to the accompanying drawings, which are:

Figure 1 A schematic representation of the chemical synthesis of a modified PEG-functionalised gold nanoparticle according to a first embodiment of the first aspect of the present invention, and of an oxaliplatin-tethered gold nanoparticle according to a first embodiment of the third aspect of the present invention;

Figure 2 A modified PEG-functionalised cisplatin-tethered gold-coated iron nanoparticle according to a second embodiment of the first aspect of the present invention;

Figure 3 A graph showing the size distribution of a colloidal gold nanoparticle prepared by a method according to a first embodiment of the second aspect of the present invention; Figure 4 A graph showing the size distribution of a colloidal gold nanoparticle prepared by a method according to a second embodiment of the second aspect of the present invention;

Figure 5 Scanning electron microscope (SEM) images showing (a) the gold nanoparticles of Figure 3, and (b) the gold nanoparticles of

Figure 4;

Figure 6 Scanning electron microscope (SEM) images representing (a) naked gold nanoparticles, (b) gold nanoparticles functionalised with modified PEG and (c) oxaliplatin-tethered gold nanoparticles;

Figure 7 An electron probe microanalysis spectrum of oxaliplatin-tethered gold nanoparticles; Figure 8 A table showing the zeta potential and particle size of naked gold nanoparticles, gold nanoparticles functionalised with a modified

PEG linker, gold nanoparticles and linker with added diisopropylamine base to deprotonate the carboxylic acid groups, and gold-oxaliplatin nanoparticles, as determined by dynamic light scattering;

Figure 9 A transmission Electron Microscope (TEM) image showing both the intracellular and intranuclear uptake of the gold-oxaliplatin nanoparticles into A549 lung cancer cells; and

Figure 10 A graph displaying the relationship between the concentration of cisplatin added to a tethered pegylated gold nanoparticle and the zeta potential measured of the final product.

EXAMPLES

Materials

Potassium tetrachloroplatinate (II), (\R, 2i?)-(-)-l,2-diaminocyclohexane, silver nitrate, sodium tetrachloroaurate (III), sodium citrate, sodium cyanide, Wang resin, succinic anhydride, Jeffamine® ED-2003, dimethylaminopyridine, N, N- diisopropylcarbodiimide, N,N-diisopropylethylamine, 1 ,3-dimethyl-3,4,5,6- tetrahydro-2(lH)-pyrimidone, dichloromethane and methanol were purchased from Sigma Aldrich.

Thioctic acid was purchased from Acros Organics.

Analytical techniques

UV- Visible Spectrometry

U V- Visible data was acquired using a Varian Cary 300 Bio Spectrometer. Concentrations of samples were determined using the molar extinction coefficient of 2.7 x 10⁸ at 520 nm.

Mass spectrometry

Mass spectrometry was carried out as a service by Swansea EPSRC mass spectrometry centre.

Inductively coupled plasma - mass spectrometry (ICP-MS) experiments were carried out on a Thermo Electron Corp. X-Series II quadrupole ICP-MS. The instrument used a concentric nebuliser with a Peltier cooled conical single-pass spray chamber with impact bead and had an integral peristaltic pump for sample uptake from a Cetac ASX-520 autosampler. A hexapole for CCT ED (Collision Cell

Technology with Energy Discrimination) mode was used to remove polyatomic interferences. Instrumental operating conditions used were 1400W RF forward power, 13 L/min plasma flow, 1.0 L/min nebulizer flow and 0.8 L/min auxiliary flow. A sample flush time of 60 s, a wash time of 60 s and a peak jump mode was used with a dwell time per isotope of 10 ms. Pt was determined using the ¹⁹⁴Pt and ¹⁹⁵Pt isotopes. Calibration solutions were prepared from a Spex "CertiPrep" certified standard diluted as required with 2% Fisher "Primar Plus" nitric acid. Gold nanoparticles and gold nanoparticles modified with oxaliplatin (900 μL, 26.7 nM based on extinction coefficient of gold) were digested by addition of sodium cyanide solution ( 100 μL, 0.067M) for 24 hours. Scanning electron microscopy

1 μL of sample was dried onto a silicon substrate and placed under vacuum. SEM images were collected using a FEI Sirion 200 ultra high resolution Schottky field emission scanning electron microscope running FEI software. Accelerating voltages of 5 or 9.55 kV were applied to each sample and a spot size of 3 used.

Electron Probe Microanalyser (EPMA)

1 μL of sample was dried onto a silicon substrate and placed under vacuum. The wavelength-dispersive X-ray (WDX) spectrum acquired in a Cameca SXlOO electron probe microanalyser (EPMA) using a pentaerythritol (PET) crystal with a lattice spacing of 2d=8.75 Angstroms. A 20 keV, 40 nA electron beam was used.

Dynamic light scattering

Dynamic light scattering and zeta potential experiments were conducted on a Malvern Zetasizer Nano ZS. Nanoparticles, nanoparticles with linker, nanoparticles with linker and DIPEA and oxaliplatin- or cisplatin-modified nanoparticle samples were prepared at 5 nM. The machine was calibrated using a 60 nm polystyrene standard. 1 mL of sample was loaded into a cell and particle size and zeta potential was measured simultaneously. Samples of each kind were prepared in triplicate and data for each sample acquired three times.

In vitro growth inhibition assays

A549 lung epithelial cancer cells were grown in Dulbecco's Modified Eagles medium (DMEM) containing 10% foetal calf serum at 37 ⁰C and in a 5% CO₂ atmosphere. A549 cells were trypsinised, counted and adjusted to 500,000 cells/mL and 90 μL aliquots added to a 96 well plate. From stock solutions 10 μL naked nanoparticle, nanoparticle with polyether linker, oxaliplatin tethered gold nanoparticle and cisplatin (as a control comparison) were added, in triplicate, to each well. The plate was then cultured for 24h, the cells were fixed with 4% parafomaldehyde and crystal violet was used to stain the viable cells. Absorbance of the crystal violet stain was measured using a plate reader at 540 nm. IC5₀ values (μM) were then determining by plotting cell viability, as a percentage compared to untreated cells, as a function of drug concentration. TEM

A549 lung epithelial cancer cells were grown in Dulbecco's Modified Eagles medium (DMEM) containing 10% foetal calf serum at 37 ⁰C and in a 5% CO₂ atmosphere. A549 cells were trypsinised, counted and adjusted to 100,000 cells/mL and 2 mL added per plate. Stock solutions of oxaliplatin modified nanoparticles were diluted using DMEM and added to the plate in 800 μL quantities. Cells were cultured for 24 hours and fixed.

Preparation

Synthesis of naked gold nanoparticles

Referring to Figure 1 , the synthesis of oxaliplatin-tethered gold nanoparticles began with the synthesis of naked gold nanoparticles from sodium tetrachloroaurate(III) . According to a first synthesis method, sodium tetrachloroaurate(III) hydrate

(50 mg, 0.14 mmol ) was dissolved in distilled water (500 mL) and then heated to 100 ⁰C with continuous stirring. Upon boiling, tribasic sodium citrate solution (1 % m/v, 7.5 mL) was added and the solution was maintained at boiling for 15 mins under continuous stirring before allowing to cool to room temperature. UV -Visible spectra were collected of the nanoparticles to ascertain their quality. A λ max of 519 nm -

523 nm indicated successful formation of the colloid. Dynamic Light Scattering (DLS) was used to determine particle size, as illustrated in Figure 3.

According to a second synthesis method, sodium tetrachloroaurate(III) hydrate (50 mg, 0.14 mmol ) was dissolved in distilled water (500 mL) and then heated to 100 ⁰C with continuous stirring. Upon boiling, tribasic sodium citrate solution (1 % m/v,

20 mL) was added and the solution was maintained at boiling for 15 mins under continuous stirring before allowing to cool to room temperature. UV-Visible spectra were collected of the nanoparticles to ascertain their quality. A λ max of 519 nm - 523 nm indicated successful formation of the colloid. Dynamic Light Scattering (DLS) was used to determine particle size, as illustrated in Figure 4.

Synthesis of the polyethyleneglycol linker

The polyethylene glycol (PEG) linker was made using solid phase synthesis. Wang resin (0.5 g, 1 mmol/ g loading) was suspended in DCM (50 mL). Succinic anhydride (0.25 g, 5 eq.) and dimethylaminopyridine (0.3 g, 5 eq.) were added. This was then refluxed at 60 ⁰C for 6 hours. The resin was then separated from the mixture by filtration and washed several times with copious DCM and MeOH. The acid functionalisation of the resin was tested with Malachite Green. The resin was then resuspended in DCM (50 mL). Jeffamine® ED-2003 (4.25 g, 5 eq.) and N, N-

Diisopropylcarbodiimide (387 μL, 5 eq.) were added and the mixture agitated overnight. The resin was then separated from the mixture as before, using filtration and washed several times with copious DCM and MeOH. The resin was once again suspended in DCM (50 mL) and to this N, N-Diisopropylcarbodiimide (387 μL, 5 eq.) and thioctic acid (0.516 g, 5 eq.) was added. The mixture was agitated overnight in dark conditions and then washed as before with DCM and MeOH. The resin was suspended in 10 % trifluoroacetic acid in DCM at room temperature for 3 hours and agitated to cleave the desired compound. The resin was removed by filtration and filtrate concentrated under reduced pressure. The remaining liquid was then washed with ether which was subsequently decanted off and the product was left to dry under high vacuum overnight. Yield: 386 mg, 17.8 %.

Assembly of oxaliplatin-modified nanoparticles

The active component oxaliplatin was synthesised by published methods (Wheate, N.J. et al., Novel platinum(II)-based anticancer complexes and molecular hosts as their drug delivery vehicles, Dalton Trans., 5055-5064 (2007)).

To an eppendorf containing gold nanoparticles (1 mL, 17 nM), the linker was added (100 μL, 1 mM) and was allowed to react with nanoparticles for 4 hours. To wash away unbound linker, the nanoparticles were centrifuged at 7000 rpm for 20 mins, the supernatant removed and the remaining pellet dissolved in MiIIiQ water (1 mL). This was repeated once further. ΛζTV-Diisopropylethylamine (100 μL, 0.1 mM) was added to the nanoparticles.

The active component of oxaliplatin, dichloro-li?,27?- diamonocyclohexaneplatinum(II) was activated using AgNO_3. Oxaliplatin (10 mg) was dissolved in l,3-Dimethyl-3,4,5,6-tetrahydro-2(lH)-pyrimidone (1 mL), added to the nanoparticles in 100 μL aliquots and left overnight. These were then centrifuged as before, at 7000 rpm for 20 mins. The supernatant containing unbound oxaliplatin was removed and the remaining pellet redissolved in MiIIiQ water (1 rnL). This wash was repeated once further.

Assembly of cisplatin-modified nanoparticles Cisplatin-modified nanoparticles were prepared according to a method as described above, but using cisplatin instead of oxaliplatin.

Assembly of gold-coated iron cisplatin-modified nanoparticles

Cisplatin-modified nanoparticles as shown in Figure 2 were prepared according to a method as described above, but using a gold-coated iron nanoparticle instead of a gold nanoparticle.

Characterisation

Oxaliplatin-tethered gold nanoparticles were characterised by scanning electron microscope (SEM), inductively coupled plasma mass spectrometry (ICP-

MS), dynamic light scattering, and X-ray fluorescence.

Cytotoxicity, cellular uptake and localisation in A549 epithelial lung cancer cells was examined using in vitro growth inhibition assays and transmission electro microscopy (TEM). Cisplatin-tethered gold nanoparticles were characterised by dynamic light scattering.

Results

As shown in Figure 3, the size distribution of naked gold nanoparticles when using 7.5 mL 1% w/v solution Of Na₃Ct produced a polydisperse colloid with two distinct peaks. In one example, 77.7 % of the colloid was 43.13 nm in size and the average particle size was 16.12 nm.

As shown in Figure 4, the size distribution of naked gold nanoparticles when using 20 mL 1% w/v solution Of Na₃Ct produced a monodisperse colloid having an average particles size of 22.44 nm. The preparation of a monodisperse nanoparticle size distribution is particularly advantageous because of the desired application in the field of biodiagnostics.

Figure 5 shows scanning electron microscope (SEM) images depicting (a) the gold nanoparticles prepared according to the first synthesis method described above (using 7.5 mL 1% w/v solution of Na₃Ct), and (b) the gold nanoparticles gold nanoparticles prepared according to the second synthesis method described above (using 20 mL 1% w/v solution of Na₃Ct). For both syntheses, spherical particles were observed. The oxaliplatin-tethered gold nanoparticles (made using gold nanoparticles prepared according to the first synthesis method described above) were first characterised using an Electron Probe Microanalyser (EPMA). The nanoparticles were confirmed as containing both gold and platinum through the observance of type peaks for these elements at 2.12 and 2.05 eV, respectively, as shown in Figure 7. Whilst the amount of gold and platinum in the sample could not be determined quantitatively, due to scattering effects from the rough surface of the sample, the spectra clearly indicated that each nanoparticle contained a much larger percentage of gold compared to platinum.

The number of oxaliplatin molecules per gold nanoparticle, after digestion with sodium cyanide, was determined by ICP-MS against a standard curve. The results indicate around 260-300 drug molecules per nanoparticle, which significantly higher than the number of drugs molecules able to be delivered by other drug delivery systems like carbon nanotubes (82 drug molecules per NT), hyper-branched polymers (3-6 drug molecules), or dendrimers (30-104 depending on the dendrimer generation/size).

Figure 8 shows the size and charge of the gold nanoparticles (made using gold nanoparticles prepared according to the first synthesis method described above), the nanoparticles with the linker only and the gold-oxaliplatin nanoparticles. It can be seen from Figure 8 that, upon addition of the PEG linker, the nanoparticles increase in size slightly but maintain their slightly negative charge, which is consistent with the deprotonation of the terminal carboxylic acid groups. Upon addition of oxaliplatin the nanoparticles increase significantly in size (4.5-fold) and change from a net negative to a net positive charge. The increase in size upon addition of oxaliplatin was unexpected. It is believed to occur through two possible mechanisms, as depicted in Figure 1. The first mechanism is thought to represent aggregation through hydrophobic interactions between the cyclohexane components of the drugs (structure IA). The second mechanism is thought to represent crosslinking of multiple nanoparticles using oxaliplatin as a bridging ligand (structure 1 B). To examine if aggregation was the reason for the increase in particle size the nanoparticles were examined using scanning electron microscopy (SEM), as shown in Figure 6. The naked nanoparticles showed nanoparticles approximately 21-29 nm in diameter and the nanoparticles with linker only were roughly 30-40 nm and with some evidence of aggregation. The gold-oxaliplatin nanoparticles appear to be roughly the same size by now highly aggregated microparticles up to 1.6 microns in length. Figure 6 clearly shows the increase in size of the nanoparticles with functionalisation and the high propensity of the oxaliplatin-tethered nanoparticles to aggregate in both the solution and solid state. The ability of the oxaliplatin-tethered gold nanoparticles to induce cellular apoptosis was determined using growth inhibition assays with the A549 lung epithelial cancer cell line. Both the naked nanoparticle and the nanoparticles with the PEG linker demonstrated no cytotoxicity at concentrations up to 170 nM. In contrast, the oxaliplatin-tethered gold nanoparticles had an IC₅₀ of 20 nM (based on nanoparticle concentration), which is significantly more active than cisplatin (IC₅₀ =

15.7 μM). Even when the cytotoxicity was calculated based on the concentration of oxaliplatin on the nanoparticles (IC₅₀ = 2.3 μM), the drug was still 3-fold more active than cisplatin. It was thought that the benefit of the oxaliplatin-tethered gold nanoparticles would come from their enhanced plasma retention and circular times and better uptake into solid tumours, in vivo, compared with cisplatin. Therefore, the enhanced in vitro cytotoxicity was unexpected and suggests that gold nanoparticle- based delivery of platinum drugs may significantly improve their potency.

Given the enhanced cytotoxicity, the uptake and localisation of the oxaliplatin- tethered nanoparticles was then examined using transmission electron microscopy (TEM). The A549 lung cells were incubated with 225 nM oxaliplatin-tethered nanoparticle for 24 h then analysed. Referring to Figure 9, the following features can be identified: (1) extracellular nanoparticles; (2) intracellular nanoparticle; (3) intranuclear nanoparticle; (4) contracted cell wall;(5) double envelope nuclear membrane;(6) endosome; and (7) lysosome. At the nanoparticle concentration tested, cell growth was very significantly hindered, as demonstrated by a large contraction of the cell wall. Gold nanoparticles were observed both inside and outside the cell, but more importantly also within the cell nucleus. Gold nanoparticles are not known to be able to penetrate the cell nucleus and neither the naked nanoparticles nor the nanoparticles tested under the same conditions as the oxaliplatin-tethered nanoparticles were observed in the nucleus. This result implies some sort of oxaliplatin-mediated uptake mechanism into the nucleus, where the drug is then released to bind with DNA. The uptake of gold nanoparticles into mammalian cells and macrophages is usually via endocytosis. Whilst both endosomes and lysosomes were observed in the TEM images, no nanoparticles were located within them. Some cationic nanoparticles have previously been reported to enter cells by passing through the cell membrane by generating transient holes. It is believed that the oxaliplatin- tethered nanoparticles of the present invention may be taken up by the cancer cells in a similar manner. Figure 10 displays the relationship between the concentration of cisplatin in a tethered pegylated gold nanoparticle and the zeta potential measured of the final product. This experiment aimed at determining the optimum cisplatin concentration in the system. As shown in Figure 10, at higher concentrations, as the zeta potential approached and then surpassed zero, the system was unstable and the nanoparticles aggregated, crashed out and adhered to the inside walls of the eppendorf sample tube.

UV-Visible spectroscopy was used to confirm that cisplatin had been tethered to the nanoparticle. This was evidenced by a characteristic significant increase in the absorbance in the ultra-violet region, at 230-240 nm. This distinct peak was present for both 25 μL and 50 μL. The cisplatin-modified gold-coated iron nanoparticles as shown in Figure 2 were characterised using an Electron Probe Microanalyser (EPMA). The nanoparticles were confirmed as containing iron, gold and platinum through the observance of type peaks for these elements at 6.40, 2.12 and 2.05 eV, respectively.

Claims

1. A nanoparticle comprising a metal nanoparticle functionalised with at least one linker, the at least one linker comprising at least one first moiety capable of interacting with the metal nanoparticle, and at least one second moiety capable of interacting with at least one platinum group metal-based biologically active compound, wherein the molar ratio or capacity of platinum group metal-based biologically active compounds per nanoparticle is greater than 150, and wherein the linker has the following formula:

(1) R₁-Y-R₂ wherein: Y is a linking moiety;

Ri is a first moiety capable of interacting with a metal nanoparticle; and R₂ is a second moiety capable of interacting with at least one platinum group metal-based biologically active compound.

2. The nanoparticle of claim 1, wherein Ri comprises a hydrophobic moiety.

3. The nanoparticle of claim 1 or claim 2, wherein Ri comprises an optionally substituted 5 or 6-membered disulfide-containing heterocycle.

4. The nanoparticle according to any of the preceding claims, wherein the second moiety R₂ comprises a hydrophilic moiety.

5. The nanoparticle according to any of the preceding claims, wherein the second moiety R₂ comprises a reactive moiety comprising, e.g. a Lewis base such as a weak acid group.

6. The nanoparticle according to any of the preceding claims, wherein Y is a hydrophilic linking moiety.

7. The nanoparticle according to any of the preceding claims, wherein Y is a linking moiety having the following formula: (2) X₂-L₁-X₁ wherein: L₁ is an optionally substituted linking structure; and

X₁, X₂ are modifying linking groups.

8. The nanoparticle of claim 7, wherein L) comprises an optionally substituted polymeric structure.

9. The nanoparticle of claim 8, wherein Li comprises a linear polymeric chain, e.g. polyethylene glycol (PEG).

10. The nanoparticle of claim 8, wherein L₁ comprises a non-linear polymeric structure, e.g. a dendritic structure.

1 1. The nanoparticle according to any of the preceding claims, wherein the ratio of reactive moieties capable of interacting with the at least one platinum group metal-based biologically active compound per metal nanoparticle is in the range of 150 - 30,000.

12. The nanoparticle of claim 1 1, wherein the ratio of reactive moieties capable of interacting with the at least one platinum group metal-based biologically active compound per metal nanoparticle is in the range of 200 - 30,000.

13. The nanoparticle according to claim 9, wherein the ratio of reactive moieties capable of interacting with at least one platinum group metal-based biologically active compound per metal nanoparticle is in the range of 200 - 1000.

14. The nanoparticle of claim 13, wherein the ratio of reactive moieties capable of interacting with at least one platinum group metal-based biologically active compound per metal nanoparticle is in the range of 250 - 300.

15. The nanoparticle of claim 1, wherein the linker is a compound of formula (3): (3)

16. The nanoparticle according to any of the preceding claims, wherein the at least one platinum group metal-based biologically active compound comprises an anti-cancer drug, e.g. a mononuclear platinum-based anti-cancer drug, e.g. cisplatin, picoplatin, satraplatin or preferably oxaliplatin, or a multinuclear platinum-based anti-cancer drug, e.g. BBR3464 or CT- 3610.

17. The nanoparticle according to any of the preceding claims, wherein the at least one platinum group metal-based biologically active compound further comprises at least one compound for actively targeting cancer cells, e.g. a vitamin such as folate, a hormone such as estrogen, a prostate or leukaemia-targeting aptamer, a lung cancer-targeting peptide, and/or a B- cell lymphoma-targeting antibody.

18. The nanoparticle according to any of the preceding claims, wherein the metal nanoparticle is a noble metal nanoparticle, e.g. a gold nanoparticle.

19. The nanoparticle according to any of the preceding claims, wherein the metal nanoparticle comprises at least one inner portion having magnetic properties.

20. A method of preparing a nanoparticle according any of claims 1 to 19, comprising reacting a metal nanoparticle with a linker comprising at least one first moiety capable of interacting with the metal nanoparticle, and at least one second moiety capable of interacting with a platinum group metal-based biologically active compound.

r>^ /nnoocΛi7r\

21. The method of claim 20, further comprising preparing the metal nanoparticle by dissolving a metal salt in water and adding a reducing agent so as to form colloidal nanoparticles have a substantially uniform size distribution.

22. The method of claim 20 or claim 21 , further comprising preparing a linker by reacting a first compound of formula A₂-R₂-B_2. wherein A₂ is a protective group or end-capping group, e.g. Wang resin, which may temporarily protect second reactive moiety R₂;

R₂ is a second moiety capable of interacting with a platinum group metal- based biologically active compound; and

B₂ is a reactive moiety, e.g. a carboxylic acid-containing moiety, capable of reacting with a group Z₂ of a second compound of formula Zi-L]-Z₂, with a second compound of formula Zi-L]-Z_2> wherein Zi is a reactive moiety, e.g. an amine-containing moiety, capable of reacting with a group Wi of a third compound of formula Wi-Ri;

L] is an optionally substituted linking structure; and

Z₂ is a reactive moiety, e.g. an amine-containing moiety, capable of reacting with a group B₂ of the first compound of formula A₂-R₂-B₂, followed by a third compound of formula W]-R] wherein W, is a reactive moiety capable of reacting with a group Zi of the second compound of formula Z]-Li-Z₂; and

Ri is a first moiety capable of interacting with a metal nanoparticle.

23. The method of claim 22, wherein Ri, R₂ and L are defined according to any of claims 2 to 5 and/or 7 to 10.

24. The method of claim 22, wherein the method of preparing a linker comprises reacting a first compound of formula (4a) with a second compound of formula (5a), then with a third compound of formula (6a).

wherein A is a protective or end-capping group, e.g. Wang resin.

25. The method according to any of claims 20 to 24, comprising reacting a gold nanoparticle with the linker of formula (3). (3)

26. A dispersion comprising a nanoparticle according to any of claims 1 to 19, and a dispersing medium.

27. The dispersion of claim 26, wherein the dispersing medium is water.

28. A biologically active nanoparticle comprising a nanoparticle according to any of claims 1 to 19, and at least one platinum group metal-based biologically active compound.

29. The biologically active nanoparticle of claim 28, wherein the biologically active nanoparticle is approximately 10 - 300 nm in diameter.

30. The biologically active nanoparticle of claim 29, wherein the biologically active nanoparticle is approximately 25 - 200 nm in diameter.

31. A method of preparing the biologically active nanoparticle according to any of claims 28 to 30, comprising reacting a nanoparticle according to any of claim 1 to 19 with at least one platinum group metal-based biologically active compound.

32. A pharmaceutical composition comprising a biologically active nanoparticle according to any of claim 28 to 30, together with a pharmaceutically acceptable excipient therefor.

33. Use of a biologically active nanoparticle according to any of claim 28 to 30 for the preparation of a medicament for therapeutic application in the treatment of a disease, e.g. cancer.

34. A biologically active nanoparticle according to any of claim 28 to 30 for the treatment of a disease, e.g. cancer.

35. The use of claim 33 or the biologically active nanoparticle of claim 34, wherein the at least one platinum group metal-based biologically active compound comprises an anticancer drug, e.g. a mononuclear platinum-based anti-cancer drug, e.g. cisplatin, picoplatin, satraplatin or preferably oxaliplatin, or a multinuclear platinum-based anti-cancer drug, e.g. BBR3464 or CT-3610.

36. A method of treating a disease, e.g. cancer, the method comprising delivering an effective amount of a biologically active nanoparticle according to any of claim 28 to 30, or of a pharmaceutical composition according to claim 32, to a subject in need thereof.

37. The method of claim 36, further comprising exposing a treated region to radiation, e.g. near Infra-Red (NIR).