WO2018178213A1

WO2018178213A1 - A method for the surface-modification of metal nanoparticles and uses thereof

Info

Publication number: WO2018178213A1
Application number: PCT/EP2018/058031
Authority: WO
Inventors: Leona MCGIRR; Steven Bell; Christopher Scott
Original assignee: The Queen's University Of Belfast
Priority date: 2017-03-28
Filing date: 2018-03-28
Publication date: 2018-10-04
Also published as: GB201704952D0

Abstract

A method for the surface-modification of metal nanoparticles, which functionalised metal nanoparticles can be encapsulated in a biocompatible polymer, and to which a binding agent can be conjugated is disclosed. The disclosure further relates to metal nanoparticles produced by said methods and uses thereof. Thus, the present disclosure provides a method for modifying the surface of metal nanoparticles, said method comprising: contacting the surface of the metal nanoparticles with a linking agent to produce functionalised metal nanoparticles, wherein the linking agent is X-Y-Z, in which X is a sulphur containing group, Y is a bond or a linking group, and Z is a carboxy or amino group, and wherein the surface of the metal nanoparticles is contacted with ≤ 8.197 x 10-21 moles, or ≤ 1000-fold excess, of linking agent per square nanometer (nm2) of the metal nanoparticle surface; and contacting the functionalised metal nanoparticles with a hydrophobic group to produce surface-modified metal nanoparticles.

Description

A method for the surface-modification of metal nanoparticles and uses thereof Technical Field

This invention relates to a method for the surface-modification of metal nanoparticles, which functionalised metal nanoparticles can be encapsulated in a biocompatible polymer, and to which a binding agent can be conjugated. The invention further relates to metal nanoparticles produced by said methods and uses thereof.

Background Art

Metal nanoparticles, and in particular gold nanoparticles (Au NPs), have a combination of physical, chemical, optical and electronic properties which distinguishes them from other biomedical nanotechnologies and therefore provide a highly multifunctional platform for cancer therapeutics and cancer cell imaging.

Au NPs can be functionalized with antibodies and biomolecules allowing for selective targeting and reduction in adverse effects caused to healthy cells. Thus, Au NPs have been widely used in anti- cancer drug delivery and have been shown to deliver a higher concentration of nanoparticles and therapeutic drugs compared to un-modified gold nanoparticles.

Au NPs have been widely used as contrast agents for cancer imaging in surface enhanced Raman spectroscopy (SERS), optical coherence tomography (OCT), biosensing and photoacoustic imaging. Furthermore, Au NPs have been of great interest for photothermal therapy due to their strong and tuneable linear absorption in the near-infrared (NIR) region (tissue window). This allows for deep penetration in living tissues. Direct absorption of 800 nm light will cause the aggregated Au NPs to increase in temperature and therefore these nanoparticles can be used to give a photothermal effect in tissues. However, the physiochemical properties of these Au NPs make them difficult to control in vivo, with poor bioavailability, pharmacokinetic profiles and toxicity issues. To address this issue, the Au NPs can be coated in a biocompatible layer forming a hybrid nanoparticle.

Hybrid nanoparticles which combine multiple functions in a single nanosystem have been extensively researched. By combining two materials, it is possible to overcome the shortcomings of one material and give additional functionality to the original nanoparticle. Polymers are widely used to improve biocompatibility and reduce cytotoxicity of Au NPs. Furthermore, the polymer provides specific bonds that allow the surface to be functionalized with antibodies, for selective targeting. The combination of gold and polymer in one nanoparticle gives the possibility of creating a superior hybrid nanoparticle with both biocompatibility and useful optical properties, making them very promising platforms for the treatment of cancer. Poly(lactic-co-glycolic acid) (PLGA) is one of the most successfully used biodegradable polymers because its hydrolysis leads to metabolite monomers, lactic acid and glycolic acid. PLGA is approved by the US Food and Drug Administration (FDA) and European Medicine Agency (EM) in various drug delivery systems in humans. However, PLGA nanoparticles are hydrophobic and can be eliminated from the blood stream by the reticulo-endothehtlial system (RES). Advantageously, the nanoparticles can be coated in molecules which increase the hydrophilicity of the surface. The most common moiety used for surface modification is the hydrophilic and non-ionic polymer;

polyethylene glycol (PEG). PLGA-PEG nanoparticles have increased hydrophilicity and have shown an increase in blood circulation half-life by several orders of magnitude compared to PLGA nanoparticles. These nanoparticles can passively accumulate in the tumour bed due to the enhanced permeation and retention (EPR) effect. However, the EPR effect can have a relatively poor specificity of 20-30% in delivery to cancer cell when compared to normal healthy cells.

Selectivity of the PLGA-PEG nanoparticles can be improved with antibody conjugation. For this to occur, the nanoparticle surface may have free carboxyl groups present and hence PLGA-PEG- COOH is advantageously used. Antibodies can bind to receptors over-expressed on cancer cells. Several over-expressed receptors found on cancer cells including the transferrin receptor, the folate receptor, glycoproteins, the epidermal growth factor receptor (EGFR) and integrins have been extensively researched. EGFR has proven to be one of the most promising receptors for targeting several types of cancers, as it is over-expressed in a variety of human tumours including head and neck, breast, lung, colorectal, prostate, kidney and bladder cancer. Nanoparticles with EGFR- specific antibodies attached to their surface have shown increased specify and internalization.

Here, we describe a method for the synthesis of surface-modified of metal nanoparticles, which metal nanoparticles can be encapsulated in a biocompatible polymer, and to which a binding agent can be conjugated.

Summary of the Invention

Accordingly, in one aspect, the invention provides a method for modifying the surface of metal nanoparticles, said method comprising:

contacting the surface of the metal nanoparticles with a linking agent to produce functionalised metal nanoparticles, wherein the linking agent is X-Y-Z, in which X is a sulphur containing group, Y is a bond or a linking group, and Z is a carboxy or amino group, and wherein the surface of the metal nanoparticles is contacted with < 8.197 x 10^~21 moles of linking agent per square nanometer (nm²) of the metal nanoparticle surface; and

contacting the functionalised metal nanoparticles with a hydrophobic group to produce surface-modified metal nanoparticles.

Optionally, the metal nanoparticles are selected from gold, copper, platinum, iron, zinc, titanium, cadmium, selenium, tellurium, and silver nanoparticles. Optionally, the metal nanoparticles are selected from alloys of gold, copper, platinum, iron, zinc, titanium, cadmium, selenium, tellurium, and silver. Optionally, the metal nanoparticles comprise or consist of gold, copper, platinum, iron, zinc, titanium, cadmium, selenium, tellurium, or silver, or alloys thereof, as well as metal oxides thereof, such as, monoxides, dioxides, and trioxides. Optionally, the metal nanoparticles are colloidal metal nanoparticles. Optionally, prior to

functionalization with the linking group, the metal nanoparticles are suspended in a suitable buffer. Optionally, prior to functionalization, the metal nanoparticles are suspended in a sodium citrate buffer. Optionally, prior to functionalization, the metal nanoparticles are reduced and stored in a sodium citrate buffer. Optionally, each metal nanoparticle has a shape selected from a sphere, rod, a polygonal rod, rectangular block, cube, tetrapod, and pyramid. Optionally, each metal nanoparticle is in the shape of a sphere or a rod. Optionally, each metal nanoparticle is in the shape of a nanostar, nanourchin, nanocube, nanocage, nanoshell, or hollow gold nanosphere.

Optionally, the metal nanoparticles have a diameter of about 10-200 nm, optionally about 20-200 nm, optionally about 30-200 nm, optionally about 40-200 nm, further optionally about 50-200 nm.

Optionally, the metal nanoparticles have a diameter of about 30-80 nm, optionally about 40-60 nm, further optionally about 50 nm. Alternatively, the metal nanoparticles can be described as having a diameter of > 10 nm, optionally > 20 nm, optionally > 30 nm, optionally > 40 nm, optionally > 45 nm, further optionally > 50 nm, and said metal nanoparticles can, optionally, have a diameter of < 200 nm, optionally < 200 nm, optionally < 190 nm, optionally < 180 nm, optionally < 170 nm, optionally < 160 nm, optionally < 150 nm, optionally < 140 nm, optionally < 130 nm, optionally < 120 nm, optionally < 110 nm, optionally < 100 nm, optionally < 90 nm, optionally < 80 nm, optionally < 70 nm, optionally < 60 nm, further optionally < 55 nm. Without wishing to be bound by theory, it is understood that small (<10 nm) metal nanoparticles, such as small Au NPs which may be synthesised directly in toluene using the so-called Brust method, have optical properties such that they do not absorb at the required wavelength (ca. 800 nm) which is set by the "tissue window", i.e. wavelengths suitable to penetrating body tissue. Larger particles, such as particles greater than 200 nm in diameter, have reduced cellular uptake and are therefore unsuitable for therapy and imaging applications.

Optionally, sulphur-containing group X is selected from a thiol, sulphide, disulphide, thiocyanate, thione, thial, thioester group. Optionally, sulphur-containing group X is selected from a thiol, and a disulphide group. Optionally, the linking group Y is a chemical bond, such as a covalent bond, optionally a covalent bond between two carbon atoms. Optionally, the linking group Y is a single, double or triple bond, such as a single, double or triple bond between two carbon atoms.

Alternatively, the linking group Y is a substituted or unsubstituted aliphatic, cycloaliphatic, aryl or heteroaryl group. Optionally, Y is a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heteroaryl group. In other words, Y is, optionally, a substituted or unsubstituted group selected from an alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heteroaryl group. Optionally, Y is a substituted or unsubstituted alkyl, aryl or heteroaryl group. In other words, Y is, optionally, a substituted or unsubstituted group selected from an alkyl, aryl or heteroaryl group.

Optionally, Y is a substituted or unsubstituted group selected from an aryl or heteroaryl group. Optionally, Y is a substituted or unsubstituted aryl group.

Optionally, Y is a substituted or unsubstituted aryl group selected from phenyl, 1-naphthyl, 2- naphthyl, 1-anthracyl and 2-anthracyl. Optionally, Y is substituted or unsubstituted phenyl.

Optionally, Y is phenyl optionally substituted with R⁺, -OR⁺, -N(R⁺)₂, -C(0)R⁺, -C0₂R⁺, -N0₂, or- S0₂R⁺; wherein R⁺ is hydrogen, or an optionally substituted aliphatic group, such as an optionally substituted C1-C6 aliphatic group.

Optionally, Y is phenyl or phenyl substituted with N0₂.

Optionally, the linking agent is selected from mercaptoacetic acid, optionally dilute mercaptoacetic acid; 4-mercaptobenzoic acid; 5,5'-dithiobis-(2-nitrobenzoic acid); co-substituted alkyl thiols of the formula SH(CH₂)_nCOOH, such as mercaptopropionic acid, mercaptobutanoic acid, and

mercaptopentanoic acid; substituted forms of co-substituted aryl thiols of the formula SH-Y-COOH where Y is an aryl or substituted aryl group, such as 2-mercaptopropionic acid, 3-mercaptopropionic acid, 3-mercaptobutanoic acid, and 4-mercaptopropionic acid. Optionally, the hydrophobic group is a substituted or unsubstituted aliphatic, cycloaliphatic, aryl or heteroaryl group. Optionally, the hydrophobic group is a phenyl group or dicyclohexyl group.

Optionally, the hydrophobic group is a substituted or unsubstituted alkane, aryl or heteroaryl group. Optionally, the surface of the metal nanoparticles is contacted with the linking agent for sufficient time to allow a layer of linking agent to form on the surface of the metal nanoparticles, thus producing functionalised metal nanoparticles. Optionally, the surface of the metal nanoparticles is contacted with the linking agent for sufficient time to allow a layer, optionally a monolayer, of linking agent to form on the surface of the metal nanoparticles, without large amounts of particle aggregation, thus producing functionalised metal nanoparticles. Optionally, the surface of the metal nanoparticles is contacted with the linking agent for at least 30 seconds, optionally 30 seconds to 3 hours, optionally 30 seconds to 1 hour, optionally 30 seconds to 30 minutes, further optionally about 30 minutes.

By "functional ised metal nanoparticles" it is meant that the metal nanoparticles comprise on their surface at least one linking group comprising a free carboxy group which may be contacted with at least one hydrophobic group comprising an amino group or, alternatively, the metal nanoparticles comprise on their surface at least one linking group comprising a free amino group which may be contacted with at least one hydrophobic group comprising a carboxy group. By "surface-modified metal nanoparticles", it is meant that the functionalised metal nanoparticles as described herein comprise at least one hydrophobic group on their surface, which hydrophobic group allows the metal nanoparticle to be dissolved in organic solvent.

The surface of the metal nanoparticles is contacted with < 8.197 x 10^~21 moles of linking agent per square nanometer (nm²) of the metal nanoparticle surface. In other words, the surface of the metal nanoparticles is contacted with < 1000-fold excess of the linking agent. Optionally, the surface of the metal nanoparticles is contacted with < 8.197 x 10^~22 moles (equivalent to < 100-fold excess), optionally < 7.377 x 10^~22 moles (equivalent to < 90-fold excess), optionally < 6.558 x 10^~22 moles (equivalent to < 80-fold excess), optionally < 5.738 x 10^~22 moles (equivalent to < 70-fold excess), optionally < 4.918 x 10^~22 moles (equivalent to < 60-fold excess), optionally < 4.099 x 10^~22 moles (equivalent to < 50-fold excess), optionally < 3.279 x 10^~22 moles (equivalent to < 40-fold excess), optionally < 2.459 x 10^~22 moles (equivalent to < 30-fold excess), optionally < 1 .639 x 10^~22 moles (equivalent to < 20-fold excess), optionally < 8.197 x 10^~23 moles (equivalent to < 10-fold excess), optionally < 4.099 x 10^~23 moles (equivalent to < 5-fold excess), optionally < 8.197 x 10^~24 moles (equivalent to < 0-fold excess), optionally < 8.197 x 10^~25 moles (equivalent to < 0.1 -fold excess), optionally < 8.197 x 10^~26 moles (equivalent to < 0.01-fold excess), of linking agent per square nanometer (nm²) of the metal nanoparticle surface.

Optionally, the surface of the metal nanoparticles is contacted with between about 8.197 x 10^~21 moles to about 8.197 x 10^~26 moles of the linking agent, optionally between about 8.197 x 10^~22 moles to about 8.197 x 10^~26 moles of the linking agent, optionally between about 8.197 x 10^~23 moles to about 8.197 x 10^~26 moles of the linking agent, optionally between about 8.197 x 10^~23 moles to about 8.197 x 10^~26 moles of the linking agent, optionally between about 8.197 x 10^~24 moles to about 8.197 x 10^~26 moles of the linking agent, optionally between about 8.197 x 10^"24 moles to about 8.197 x 10^"25 moles of the linking agent, further optionally about 8.197 x 10^"24 moles, per square nanometer (nm²) of the metal nanoparticle surface.

Optionally, the surface of the metal nanoparticles is contacted with < 100-fold excess, optionally < 90-fold excess, optionally < 80-fold excess, optionally < 70-fold excess, optionally < 60-fold excess, optionally < 50-fold excess, optionally < 40-fold excess, optionally < 30-fold excess, optionally < 20- fold excess, optionally < 10-fold excess, optionally < 5-fold excess, optionally < zero fold (0-fold) excess, optionally about a zero fold (0-fold) excess, optionally < 0.1 fold excess, further optionally < 0.01 fold excess of the linking agent. It will be understood that fold excess refers to the excess amount of linking agent required to form a monolayer of linking agent molecules on the surface of a metal nanoparticle. For example, 100-fold excess corresponds to amount of linking agent which is 100 times than the amount required to form a monolayer of linking agent molecules on the surface of a metal nanoparticle.

Optionally, the surface of the metal nanoparticles is contacted with between 100- to 0.01-fold excess, optionally between 10- to 0.01 fold excess of the linking agent, optionally between a 10- to 0.1-fold excess of the linking agent, further optionally between zero- to 0.1 -fold excess of the linking agent.

Optionally, unbound linking agent is removed before adding the hydrophobic groups to the functionalised metal nanoparticles. Optionally, the unbound linking agent is removed by centrifugation at > 3000 g, optionally > 4000 g, optionally > 5000 g, further optionally > 5500 g, and discarding the supernatant comprising the unbound linking agent. Optionally, the unbound linking agent is removed by centrifugation at about 4,000 g and discarding the supernatant comprising the unbound linking agent. Optionally, said centrifugation comprises a centrifugation cycle of > 10 minutes, optionally > 15 minutes, further optionally about 15 minutes. Optionally, said centrifugation comprises 1 , 2, 3 or more centrifugation cycles. Optionally, the centrifugation is carried out as a single centrifugation cycle at about 4,000 g for about 15 minutes.

Optionally, the hydrophobic group is added to the surface of the functionalised metal nanoparticles to produce surface-modified metal nanoparticles. Optionally, the hydrophobic group is added to the surface of the functionalised metal nanoparticles by mixing the functionalised metal nanoparticles in a phase transfer solution comprising an amine, optionally wherein the amine is dicyclohexylamine. Optionally, the phase transfer solution further comprises a carbodiimide, optionally wherein the carbodiimide is dicyclohexylcarbodiimide. Optionally, the phase transfer solution further comprises a drying reagent, optionally wherein the drying reagent is sodium sulphate.

Optionally, the surface-modified metal nanoparticles are isolated from the phase transfer solution. Optionally, the surface-modified metal nanoparticles are isolated from the phase transfer solution via a Schlenk line.

It will be understood that the above-described method for modifying the surface of metal nanoparticles is carried out in two steps of contacting the surface of the metal nanoparticles with a linking agent to produce functionalised metal nanoparticles, and contacting the functionalised metal nanoparticles with a hydrophobic group to produce surface-modified metal nanoparticles. However, it is possible to synthesise functionalised thiols which already carry a hydrophobic group and that may be reacted directly with the metal nanoparticles thus producing surface-modified metal nanoparticles in a single step.

Optionally, prior to modifying the surface of the metal nanoparticles, the metal nanoparticles are contained in an aqueous solution. Optionally, after modifying the surface of metal nanoparticles according to the methods described herein, the surface-modified metal nanoparticles are transferred to a non-aqueous solution, optionally an organic solvent, further optionally dimethyl sulfoxide (DMSO). In a further aspect, the invention provides a method for encapsulating a metal nanoparticle in a biocompatible polymer, said method comprising:

(i) contacting a surface-modified metal nanoparticle, produced according to the method described herein for modifying the surface of metal nanoparticles, with a biocompatible polymer; and

(ii) contacting the product of step (i) with an emulsifier to produce an encapsulated metal nanoparticle.

Optionally, the biocompatible polymer is contained in a non-aqueous solution, optionally an organic solvent, further optionally DMSO. Optionally, the surface-modified metal nanoparticles are contained in a non-aqueous solution, optionally an organic solvent, further optionally DMSO. Optionally, the organic solvent in which the biocompatible polymer is contained is the same type of solvent as the organic solvent in which the surface-modified metal nanoparticles are contained. In other words, the biocompatible polymer and the surface-modified metal nanoparticles are contained in the same type of solvent which has the advantage that, when the surface-modified metal nanoparticles are contacted with the biocompatible polymer, there is no requirement to change the organic solvent in which one or other of the surface-modified metal nanoparticles and the biocompatible polymer are contained such that they can be mixed and subsequently contacted with the emulsifier.

Optionally, the biocompatible polymer is selected from poly(lactic-co-glycolic acid) (PLGA), poly(lactide) (PLA), poly(glycolide) (PGA), poly(butyl cyanoacrylate) (PBCA), and N-(2- hydroxypropyl)methacrylamide (HPMA) copolymers. Optionally, the biocompatible polymer is PLGA. PLGA is synthesized by means of ring-opening co-polymerization of two different monomers, the cyclic dimers (1 ,4-dioxane-2,5-diones) of glycolic acid and lactic acid. Polymers can be synthesized as either random or block copolymers thereby imparting additional polymer properties. Optionally, the PLGA comprises hydrophilic group, optionally wherein the hydrophilic group is polyethylene glycol (PEG). Optionally, the PLGA form is PLGA-PEG-COOH.

Optionally, step (ii) comprises stirring the product of step (i) and the emulsifier. Optionally, said stirring is on ice. That is, the product of step (i) and the emulsifier are stirred while maintaining the mixture on ice at a temperature of from about 4 to 21 C. Optionally, said stirring further comprises agitation. Suitable means of agitation are known in the art. Optionally, said agitation is sonication. Without wishing to be bound by theory, it is understood that during agitation such as sonication, the temperature of the sample can increase and therefore the sample is kept on ice during the agitation to prevent this heating.

Optionally, step (ii) comprises contacting the product of step (i) with the emulsifier and an immiscible solvent to produce an encapsulated metal nanoparticle. Optionally, the immiscible solvent is water. Optionally, or alternatively, the emulsifier is polyvinyl alcohol.

Optionally, the method further comprises (iii) removing the organic solvent. Optionally, the organic solvent is suitable to dissolve the biocompatible polymer. Suitable solvents for dissolving biocompatible polymers are known in the art. Optionally, the organic solvent comprises, or consists of, dichloromethane or acetone. Optionally, the organic solvent is

dichloromethane or acetone. In a further aspect, the invention provides a method for conjugating a binding agent to an encapsulated metal nanoparticle, said method comprising:

conjugating the binding agent via a linker to an encapsulated metal nanoparticle produced according to the method described herein for encapsulating a metal nanoparticle in a biocompatible polymer.

Optionally, the binding agent is selected from a binding agent selected from a drug, a protein, a carbohydrate, a nucleotide sequence and a combination thereof. Optionally, the binding agent is a protein. Optionally, the binding agent is an antibody. Optionally, the binding agent is an antibody or an antibody fragment selected from a monoclonal antibody, a polyclonal antibody, a single-chain antibody (scFv), a recombinant heavy-chain-only antibody (VHH), an Fv, a Fab, a Fab', and a F(ab')2.

Optionally, the linker is selected from a carbodiimide linker such as a carbodiimide-amine

(EDC/NHS) linker, an aldehyde linker, a maleimide linker, and a linker comprising a reactive moiety for use in click-like chemistry.

Optionally, the linker is coupled to the encapsulated metal nanoparticle by contacting the

encapsulated metal nanoparticle with the linker. Optionally, the linker is a carbodiimide linker and the linker is coupled to the encapsulated metal nanoparticle by contacting the encapsulated metal nanoparticle with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS).

In a further aspect, the invention provides a surface-modified metal nanoparticle, wherein said metal nanoparticle is produced according to the method described herein for modifying the surface of metal nanoparticles. In a further aspect, the invention provides an encapsulated metal nanoparticle, wherein said encapsulated metal nanoparticle is produced according to the method described herein for an encapsulating metal nanoparticle in a biocompatible polymer. In a further aspect, the invention provides an encapsulated metal nanoparticle conjugated to a binding agent, wherein the binding agent-encapsulated metal nanoparticle is produced according to the method described herein for conjugating a binding agent to an encapsulated metal nanoparticle.

In a further aspect, the invention provides a binding agent-encapsulated metal nanoparticle, produced according to the method described herein for conjugating a binding agent to an encapsulated metal nanoparticle, for use in therapy. Optionally, the binding agent-encapsulated metal nanoparticle is for use in photothermal therapy.

Optionally, the binding agent-encapsulated metal nanoparticle, produced according to the method described herein for conjugating a binding agent to an encapsulated metal nanoparticle, is for use in imaging. Optionally, the binding agent-encapsulated metal nanoparticle is for use in therapeutic imaging. Optionally, the binding agent-encapsulated metal nanoparticle is for use in imaging, optionally therapeutic imaging, applications such as surface enhanced Raman spectroscopy (SERS), optical coherence tomography (OCT), biosensing and photoacoustic imaging. Optionally, binding agent-encapsulated metal nanoparticles may be used in a method of imaging target cells or a target tissue in a sample or in a human or animal body, the method comprising: contacting the target cells or the target tissue with binding agent-encapsulated metal nanoparticles that bind to or are phagocytosed by the target cells or the target tissue, irradiating the sample, and imaging the cells or tissue by scatter imaging. Optionally, the target cells or target tissue are cells or tissue in, or taken from, a human or animal, such as cells or tissue selected from cancerous, non-cancerous, epithelial, hematopoietic, stem, spleen, kidney, pancreas, prostate, liver, neuron, breast, glial, muscle, sperm, heart, lung, ocular, brain, bone marrow, foetal, blood, leukocyte, lymphocyte cells or tissue, or a combination thereof. Optionally, the irradiation is an x-ray beam and the scatter imaging is X-ray scatter imaging.

Optionally, the binding agent-encapsulated metal nanoparticle produced according to the method described herein for conjugating a binding agent to an encapsulated metal nanoparticle, is for use in the treatment of cancer. Optionally, the treatment of cancer comprises photothermal therapy. Optionally, the binding agent-encapsulated metal nanoparticle is for use in causing cell death.

Optionally, the binding agent-encapsulated metal nanoparticle is for use in causing apoptotic or necrotic cell death, or variations of apoptotic/necrotic cell death such as necroptotic cell death.

Optionally, the binding agent-encapsulated metal nanoparticle is for use in a method of treating cancer in a subject, the method comprising contacting the cancer with the binding agent- encapsulated metal nanoparticle, irradiating the binding agent-encapsulated metal nanoparticle to produce a photothermal effect, which photothermal effect causes cell death in the cancer.

Optionally, said contacting the cancer with the binding agent-encapsulated metal nanoparticle comprises administering the binding agent-encapsulated metal nanoparticle to the subject, optionally by administering the binding agent-encapsulated metal nanoparticle to the subject via local, intratumoral, intraarterial, intravenous, intrathecal, intracavitary subcutaneous, intramuscular injection, or oral delivery. Optionally, the irradiation is with a laser, optionally a laser emitting light with a wavelength of about 400-1 100 nm, optionally about 750-850 nm, optionally about 775-800 nm, further optionally about 785 nm. Optionally, the subject is a human or animal body. Optionally, the cancer comprises a pre-cancer or cancer such as melanoma, leukemia, ovarian cancer, colon cancer, prostate cancer, lung cancer, liver cancer, pancreatic cancer, bladder cancer, breast cancer, gastric cancer, colon cancer, head and neck cancer, esophagus cancer, synovium cancer, brain cancer, or bronchus cancer, in particular, chronic myelogenous leukemia (CML) or chronic lymphocytic leukemia (CLL). By "about", as used herein, it is meant that the recited value may be precisely the recited value, optionally ± 5% of the recited value, optionally ± 10% of the recited value, further optionally ± 20% of the recited value.

Brief Description of the Drawings

The embodiments of the present invention will now be described, by way of example, with reference to the accompanying drawings, in which:

Figure 1 is a schematic representation of an embodiment of the present invention. Gold

nanoparticles (Au NPs) are encapsulated in PLGA-PEG-COOH with an antibody conjugated to the surface, incubated with A549 lung cancer cells and irradiated with a 785nm laser which results in increased cell death to the A549 lung cancer cells.

Figure 2 is a schematic representation of surface modification of gold nanoparticles, encapsulation of gold nanoparticles via oil in water emulsion and antibody conjugation on the gold/PLGA-PEG-COOH nanoparticle surface.

Figure 3 depicts an a) environmental SEM image, b) UV-Vis spectra, c) Zetasizer, d) brightfield STEM image, e) AFM and f) Poisson distribution of the gold nanoparticles encapsulated in PLGA.

Figure 4 depicts environmental SEM showing white dots in a light grey surrounding, which are indicative of the clusters of hybrid polymer/gold nanoparticles (AuP NPs) inside a cell. The STEM image b) is the same as image a) but the AuP NPs are black inside a cell which is grey; c) a bar graph of cell viability for A549 cells with increasing concentrations of AuP NPs, analysed by MTT assay after 24hr incubation, n = 5 SD; and d) a standard curve for the % of antibody present plotted against absorbance. The % of antibody present is calculated from the amount of antibody added to the nanoparticle sample i.e. 100% is the total amount of antibody added to the nanoparticles.

Figure 5 depicts a) the bottom-up laser system with the laser beam pathway indicated by the red arrows, b) a photograph of the 785 nm laser passing through a rectangular slit and into the bottom of a well in a 96-well plate, containing a circular piece of white paper. A second well without the laser beam is also shown and c) a representative image of what was observed with a fluorescent microscope after photothermal irradiation of the A549 cells containing the T-NPs and stained with propidium iodide.

Figure 6 depicts statistical analysis in the form of a) one-way ANOVA of EGFR-positive cells (A549), b) t-test of EGFR-positive cells (A549), c) t-test of EGFR-negative cells (A2780) and d) flow cytometry results of cell death induced in EGFR-positive cells (A549) when treated with T-NPs and photothermal irradiation. The figure demonstrates that a large increase in cell death of the A549 cells is observed when the T-NPs are conjugated with Cetuximab compared to non-modified T-NPs. An increase in cell death is also observed with the EGFR-positive cells (A549) over the EGFR- negative cells (A2780) when given the same treatment of T-NPs and photothermal irradiation.

Furthermore, this increase in cell death to the EGFR-positive cells is mostly necrotic cell death. Cells were incubated for 30 mins with 50 μΙ of 0.125 mg/ml NP formulations, cells were washed twice and then irradiated with a 785 nm laser for 360 sec. Cell death was evaluated using propidium iodide staining (n=6).

Figure 7 depicts UV Vis absorption spectra of gold nanoparticles modified with neat MAA in water after 4 days and non-modified gold nanoparticles in water. In the spectra of Au colloid with neat MAA, the second peak at ca. 758 nm is characteristic of aggregated particles, which are

electronically coupled because they are very close in proximity to each other.

Figure 8 depicts UV/Vis absorption spectra of gold nanoparticles modified with neat MAA in water after 0 mins, 30 mins and 60 mins. It is demonstrated that even 30 minutes after thiol addition, the reaction and its associated aggregation were almost complete and there was marginal reduction in the extent of aggregation.

Figure 9 depicts the UV/Vis spectra of a) gold nanoparticles in water, b) gold nanoparticles with 20 μΙ of 10^~2 M MAA added in water, and c) gold nanoparticles with 20 μΙ of neat MAA added in water, and confirms that there was less aggregation in the gold colloid with dilute MAA, compared to the gold colloid with neat MAA, as the peak at 758 nm was significantly smaller.

Figure 10 depicts UV/Vis spectra of Au NPs treated with neat MAA. At 0 mins, there is a peak present at 530nm, which is indicative of individual 50nm spherical gold nanoparticles. There is also a peak at 650nm, which suggests the presence of two 50nm spherical gold nanoparticles attached to each other (dinners). As the time increases to 30 mins, up to 96 hours, the relative absorbance of the gold nanoparticles is about 0.2, indicating that the gold nanoparticles have precipitated.

Figure 1 1 depicts UV Vis spectra of Au NPs treated with MAA at 10^"1 M. At 0 mins, 30 mins, 1 hour and 2 hours, two peaks are present at 530nm and 650nm. The relative absorbance of both peaks decrease as the time increases. The spectra at 24 hours shows a large reduction in the relative absorbance of the peaks at 530nm and 630nm indicating the precipitation of the gold nanoparticles. In the 96 hour spectra, the relative absorbance is 0.2, thereby indicating that the gold nanoparticles have completely precipitated.

Figure 12 depicts UV Vis spectra of Au NPs treated with MAA 10^~2 M. The spectra are similar to the spectra of Au NPs treated with MAA 10^~ M. However, at 96 hours, the peaks at 530nm and 650nm can still be observed. Therefore, the gold nanoparticles treated with 10^~2 M MAA do not fully precipitate at 96 hours.

Figure 13 depicts SEM image of gold nanoparticles and PLGA nanoparticles, as indicated by the arrows. The SEM image shows the gold nanoparticles are completely separate to the PLGA nanoparticles. The encapsulation of the gold nanoparticles relies mostly on the emulsion step of the gold nanoparticles with the PLGA, where the gold nanoparticles in DMSO / DCM were mixed with PLGA in DCM. The presence of some DMSO in the solution may have caused the gold nanoparticles to stay at the interphase of the two solutions and therefore not mix together.

Figure 14 depicts the zeta potential of the hybrid gold/PLGA-PEG-COOH nanoparticles, which was found to be -26 mV due to the negative carboxyl groups on the nanoparticle's surface. This indicated that the hybrid nanoparticles should have a good stability.

Figure 15 depicts the characteristic 4-mercaptobenzoic acid (MBA) peaks at 1589 and 1085 cm^"1 present in MBA-modified Au NPs spectra and the MBA-modified Au NPs encapsulated in PLGA spectra.

Figure 16 depicts the characteristic 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) peak at 1347 cm^"1 present in the DTNB-modified Au NPs spectra and DTNB-modified Au NPs encapsulated in PLGA.

Figure 17 depicts a Au NP having a linking group comprising a carboxy group may be contacted with a hydrophobic group comprising an amino group, and a Au NP having a linking group comprising an amino group may be contacted with a hydrophobic group comprising a carboxy group, to produce surface-modified gold nanoparticles.

Detailed Description MATERIALS & METHODS Materials

Gold (in) chloride trihydrate (> 99.9%), trisodium citrate, mercaptoacetic acid, dimethyl sulfoxide, dicyclohexylamine, anhydrous sodium sulphate and dichloromethane were purchased from Sigma- Aldrich. PLGA-PEG-COOH was purchased from PolySciTech. 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysulfosuccinimide and propidium iodide 1.Omg/mL solution in water were purchased from Thermo Fischer Scientific. The A549 lung cancer cells were kindly provided by Dr. Daniel Longley (Queen's University Belfast) and cultured in DMEM medium supplemented with 10% FBS (Foetal Bovine Serum) and 10% Penicillin-Streptomycin.

Gold nanoparticle synthesis

The citrate reduced Au nanoparticles were prepared by using a variation of Frens citrate reduction (Frens, G., "Particle Size and Stability in Metal Colloids". Colloid. Polym. Sci. 1972, 250, 736-741 ). A solution of HAuCI₄ (50ml of 0.1 % w/v in DDI water) was brought to boil and a solution of trisodium citrate (5.6ml of 1 % w/v) was rapidly added to the boiling solution; after 1 minute the solution was left to cool at room temperature. The colloid was further diluted in trisodium citrate (10^"4M; typically 15 mL colloid with 15mL citrate and 1 mL water) and then centrifuged for 3hrs at 3500 rpm (2054 g). The supernatant was removed and replaced with fresh trisodium citrate (10^"4M) solution. This formed hydrophilic gold nanoparticles.

Surface Modification of gold nanoparticles The citrate reduced gold nanoparticle surface was modified with a covalent amide coupling reaction to enable phase transfer of the hydrophilic gold nanoparticles into dichloromethane. This was performed by a variation of a previously reported by McMahon et al. ("Phase transfer of large gold nanoparticles to organic solvents with increased stability". Langmuir 2007, 23 (3), 1414-1418). Mercaptoacetic acid (MAA) was added to citrate reduced gold nanoparticles at room temperature and stirred to enable a monolayer of mercaptoacetic acid to form on the surface of the gold nanoparticles. 0.02 ml of neat MAA was added to 10ml of gold colloid, 0.02 ml of 10 M MAA was added to 10ml of gold colloid, or 0.02 ml of 10^"2 M MAA was added to 10ml of gold colloid.

Solutions were then centrifuged for 15 minutes at between 1000 g to 5500 g only. Centrifuged solutions were concentrated by a factor of 15 which left 1 ml remaining. A fresh phase transfer solution comprising 30 mM dicyclohexylcarbodiimide (DCC) (0.06 g in DMSO) and 30 mM of dicylohexylamine (DCHA) (0.06 ml in 10 ml of DMSO) was made. 10 ml of this phase transfer solution was added to 0.5 g of sodium sulphate and after a few minutes, the solution was decanted from the sodium sulphate. The DMSO was removed with a Schlenk line and resuspended in DCM. Alternatively, the gold nanoparticles may be surface modified as described above with using, in place of MAA, 4-mercaptobenzoic acid (MBA) or 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) or other suitable modifier having a sulphur containing group and an amine or carboxylic acid group (Figure 17). Figures 15 and 16 depict the results of successful surface-modification of gold nanoparticles using MBA and DTNB, respectively. These surface-modified gold nanoparticles can then be contacted with a suitable hydrophobic group containing a carboxy group (Figure 17).

Encapsulation The modified gold nanoparticles were encapsulated in PLGA-PEG-COOH with an oil-in-water emulsion technique. 5 mg of PLGA-PEG-COOH was dissolved in 1 ml dichloromethane (DCM). 1.5 ml of modified gold nanoparticles in DCM was then added. This was then introduced to 10 ml 2.5% PVA (MW 13,000-23,000, 87-89% hydrolysed) in MES buffer (50Mm, pH 5.0) whilst stirring on ice. This was sonicated for 60 pulses and left stirring in a fume hood to allow DCM to evaporate. After approximately 2 hours the solvent had evaporated and the solution was centrifuged for 15 minutes, 4 °C at 20850 g. The supernatant was removed, 1 ml of water was added, allowed to rehydrate and was then resuspended using a sonic probe, as shown in Figure 3.

Antibody conjugation

Cetuximab was attached to the PEG-PLGA-COOH nanoparticles with the use of carbodiimide chemistry, as shown in Figure 4. The hybrid nanoparticles were centrifuged at 14,000 rpm (20850 g) for 15 minutes and the supernatant was replaced with 2-(N-morpholino)ethanesulfonic acid, also known as MES buffer. This process was repeated three times. The hybrid nanoparticles were then passed through a 0.2μιη sterile filter and diluted with MES buffer to a 1 mg/ml concentration. A 19 mg/ml solution of 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) in MES buffer was prepared and passed through a 0.2μιη sterile filter. An 82 mg/ml of N-hydroxysuccinimide (NHS) in MES buffer was also prepared and filtered. 5μΙ of EDC followed by 5μΙ of NHS were added to the 1 mg/ml solution of hybrid nanoparticles. This was left to stir for 20 minutes. Afterwards, the nanoparticles were centrifuged 14,000 rpm (20850 g) for 15 minutes and resuspended in MES buffer. 10μΙ of Cetuximab (5mg/ml) was added to the hybrid nanoparticles and left to stir for 1 hr. This was then centrifuged for 15 minutes at 14,000 rpm (20850 g) and resuspended in phosphate buffered saline solution (PBS). Characterisation of T-NPs

The zeta potential and size measurements recorded in this work have been measured by dynamic light scattering and laser Doppler electrophoresis, using a Malvern Zetasizer Nano ZS system equipped with a 633 nm laser. All atomic force microscope results recorded in this work were carried out on an easy Scan 2 AFM with Nanosurf software. SEM/STEM images were obtained using a Quanta FEI 250 equipped with a field emission gun (FEG). To calculate the Poisson distribution, 100 PLGA nanoparticles were imaged with the STEM and the number of particles they each contained was recorded. The distribution was then compared to a series of Poisson distributions created in Microsoft Excel with different average particle contents.

Toxicity of T-NPs to A549 cells

The toxicity of the T-NPs within the cells was examined with an MTT assay. 1 x 10⁴ A549 cells were seeded in a 96-well plate and then incubated for 24 hours. After this time, a uniform layer of cells had adhered to the bottom of the wells. The T-NPs were filtered with a 0.45 μιτι sterile filter to ensure no bacteria were present before they were introduced into the cells. 50 μΙ of 0.13, 0.14, 0.17, 0.20, 0.25 and 0.33 mg/ml of T-NPs were added to the A549 cells and the cells were then incubated for 24 hours. The supernatant was removed and 100 μΙ of MTT added to each well. The enzymes reduced the MTT to purple formazon crystals that were dissolved in DMSO. This was then read using a plate reader at 570 nm and the results were collected.

Cellular uptake

1 x 10⁴ A549 cells were seeded into a 96-well plate and incubated for 24hrs. 50 μΙ of the T-NPs were then added and incubated for 24hrs. The cells were detached from the bottom of the well with 100 μΙ of trypsin and added on top of a copper TEM grid. The cells on the copper TEM grid were imaged with STEM.

Photothermal irradiation setup

A bottom-up laser system was built which used a 60 mW 785nm laser. In this system, the laser beam is reflected at a right angle with a prism, which then goes through a safety shutter. The beam is then reflected at a right angle by another prism, which brings the beam through an iris and access hole to the bottom of the well plate, as shown in Figure 5a. The laser power was controlled using a rotatable polarizer and initial experiments were set to provide irradiance of 1W/cm². As the laser beam passes through the bottom of the 96-well plate, it is likely to scatter as it hits the plastic. To image how much scattering occurs, a small circular piece of white paper was cut to fit the bottom of a well in the 96-well plate. The 96-well plate was placed on top of the bottom-up laser system and a camera was attached facing down on the well. The camera was necessary as it would be too bright to view with the naked eye and could cause permanent eye damage. The laser was turned on and a photo was taken, as shown in Figure 5b. The photograph shows the whole well illuminated with light and not just the small rectangular area where the laser passes through. This would suggest that a large amount of scattering occurs as the beam enters into the well. Therefore, the whole well and all the cells within the well are affected by the laser.

Photothermal irradiation The A549 cells were prepared by seeding 14 x 10⁴ cells into a 96-well plate and were left to adhere to the surface for 24 hours. The medium was then replaced with 100 μΙ of serum-free DMEM medium and left to 'starve' for 3 hours. The antibody conjugated T-NP solution was diluted to 0.125 mg/ml with PBS. These solutions were then added to the cells. The negative control was prepared by adding PBS and the positive control was heated to 70^°C for 30 minutes. The medium was replaced with DMEM containing no phenol red, the cells were then irradiated for 360 seconds with a 32mW 785nm laser, through a 7 x1 mm slit. The cells were then incubated for 1 hour at 37 °C with a C0₂ concentration of 5%. The cells were stained with propidium iodide (PI) and imaged with a Nikon fluorescence microscope with a 35mm Nikon SLR camera attached to the front port of the instrument. The camera was set to maximum sensitivity and Nikon ACT-1 software was used. A 10x objective was used to image the cells. The cells that were stained red, and therefore dead, were counted and recorded. A representative sample of what was observed with the fluorescence microscope is shown in Figure 5c.

Flow cytometry

The A549 cells were prepared, as described above for the photothermal irradiation. The cells were stained with Annexin V/PI and added to the flow cytometer. BD FACE Diva software was used and the gates were set according to the compensation.

RESULTS AND DISCUSSION

Metal nanoparticles, in particular gold nanoparticles, are known to have a combination of physical, chemical optical and electronic properties which provide a highly multifunctional platform for cancer therapeutics and cancer cell imaging. In particular, gold rods and spherical gold nanoparticles have been widely used in photothermal therapies as both nanoparticle shapes can be manipulated so that their surface Plasmon corresponds to the wavelength of commercially used in photothermal therapies such as 785 nm.

Gold nanorods display two Plasmon bands that are tuneable, depending on the dimensions of the nanorod. One Plasmon band corresponds to oscillations along the length of the gold nanorods (longitudinal Plasmon band) and the other along the width of the gold nanorods (transverse Plasmon band). These Plasmon bands can lie between 500 and 1600 nm, their positions vary with the aspect ratio so they can be adjusted to make the Plasmon shift towards the near-IR region where tissue absorption is low.

Aggregation of gold nanoparticles with sodium chloride causes interparticle surface Plasmon coupling resulting in a significant colour change from red to purple due to a shift in the wavelength of the Plasmon from 520 nm to 730 nm. Aggregation of the spherical gold nanoparticles enables them to also be used as targets as in photothermal therapy as it shifts the wavelength of the Plasmon to the near-IR region (i.e. "tissue window"). This allows for deep penetration in living tissues. Direct absorption of 800 nm light will cause the aggregated spherical gold nanoparticles to increase in temperature and, therefore, these spherical gold nanoparticles can be used to provide a

photothermal effect in tissues. In the present study, spherical gold nanoparticles were used as photothermal targets due to ease of preparation over then gold nanorods.

50 nm aqueous gold nanoparticles were synthesised using the Frens method. Since the encapsulation process used required that the gold nanoparticles were dispersed in organic solvents, it was necessary to phase-transfer them from the aqueous into the organic phase. Phase-transfer of gold nanoparticles greater than 20 nm in diameter has proven to be problematic, as these particles are electrostatically stabilized in aqueous, but not organic, solvents. In recent years, there has been significant research in the phase-transfer of large nanoparticles but few have been successful in transferring large gold nanoparticles, from aqueous to organic solvent without aggregation. In this research, we describe a method to minimise nanoparticle aggregation and successfully phase-transfer 50 nm gold nanoparticles from the aqueous into the organic phase. At the start of this work, McMahon et al.'s literature procedure was followed ("Phase transfer of large gold nanoparticles to organic solvents with increased stability". Langmuir 2007, 23 (3), 1414-1418). However, the first step of adding neat mercaptoacetic acid (MAA) to the gold colloid and leaving it to stir for 4 days produced highly aggregated gold nanoparticles (see Figure 7). It was found that even 30 minutes after the thiol addition, the reaction and its associated aggregation were almost complete and there was a marginal reduction in the extent of aggregation (see Figure 8). The relative size of the surface area of the gold nanoparticles and the amount of MAA added were calculated, as indicated below.

Actual amount of gold:

Atomic mass of gold 197

x Mass of hydrated gold chloride = x 0.05

Molecular mass of hydrated gold chloride ^{y 5} 394

= 0.025g

Volume of 60 nm gold nanoparticle sphere:

Mass of 1 particle:

Volume x density of gold = (1.1x10^s) x (19.30xl0^"21 ) = 2.12 x l0^"15 g

Number of particles in solution: 1.18 10¹³

Number of particles in 10ml used: = 2.12xl0¹² inlOml

^ 5.56

Surface area of gold nanoparticle:

4m-² = 4 π30² = l.lOxlO⁴ nm²

Total surface area of gold nanoparticles in 10ml:

Surface area x total number of nanoparticles = (l.lOxlO⁴ )x(2.12 x 10¹²)

= 2.33 x lO¹⁶ nm²

Number of mercaptoacetic acid molecules required for monolayer on gold nanoparticle surface:

Surface area of gold nanoparticles 2.33x10¹⁶

= 1.15x10

Surface area of mercaptoacetic acid 0.202

Moles required for monolayer:

Number of particles 1.15xl0¹⁷

= 1.91xl0^-7 moles

Avogadro's constant 6.02xl0²³

98% MAA used in the McMahon et al. literature = 98g/100ml , density = 1.32 g/ml

1000x1.32

Moles in 1000ml= = 14.33 moles

92.12

Moles of MAA used = volume x concentration

= (2xl0^"5) x 14.33 = 2.88 x10^"4 moles

It was calculated that the McMahon et al. procedure using neat MAA used approximately 1508 times more MAA than was required to cover the surface area of the gold nanoparticles with a single monolayer. Therefore, the neat MAA was diluted to 10^~2 M, which dilution was found to significantly reduce the aggregation of the nanoparticles (see Figure 9). It will further be understood that, if a linking agent other than MAA is to be used, e.g. a linking agent with a smaller/larger surface area, everything in the calculation would remain the same except the surface area of the linking agent, which would be known or readily determined by the person skilled in the art. Therefore, this calculation can be used as a template for different linking agents and has successfully been used by the present inventors with respect to other linking agents including 4-Mercaptobenzoic acid and 5,5- dithio-bis-(2-nitrobenzoic acid) to determine the appropriate excess of linking agent (and avoid unwanted aggregation).

To further elucidate the effect of neat MAA on nanoparticles aggregation, gold particles were treated with neat, 10^~1 M and 10^~2 M MAA and monitored over a 96 hour period. As demonstrated from the calculations above, the number of moles of neat MAA added to the gold nanoparticles was 2.88 x 10 ⁴ moles (i.e. 1508-fold excess). The number of moles of 10^"1 M MAA and 10^"2 M MAA which was added to the same quantity (surface area) of gold nanoparticles was 2 x 10^~6 moles (10-fold excess) and 2 x 10^"7 moles (zero excess), respectively. For nanoparticles treated with neat MAA, UV Vis spectral analysis shows a peak present at 530nm, which is indicative of individual 50nm spherical gold nanoparticles (Figure 10). There is also a peak at 650nm, which suggests the presence of two 50nm spherical gold nanoparticles attached to each other (dimers). As the time increases to 30 mins, and up to 96 hours, the relative absorbance of the gold nanoparticles is about 0.2, indicating that the gold nanoparticles have precipitated. For nanoparticles treated with MAA at 10^~1 M, two peaks are present, at 530nm and 650nm, up to 2 hours. The relative absorbance of both peaks decrease as the time increases. The spectra at 24 hours shows a large reduction in the relative absorbance of the peaks at 530nm and 630nm indicating the precipitation of the gold nanoparticles. In the 96 hour spectra, the relative absorbance is 0.2, thereby indicating that the gold nanoparticles have completely precipitated. For nanoparticles treated with MAA at 10^~2 M, the spectra are similar to the spectra of Au NPs treated with MAA 10^" M. However, at 96 hours, the peaks at 530nm and 650nm can still be observed. Therefore, the gold nanoparticles treated with 10^~2 M MAA do not fully precipitate at 96 hours. These data confirm that by diluting MAA to, for example, 10^~1 M, 10^~2 M, or even less, reduces or avoids the aggregation of gold nanoparticles.

In the next step, the modified gold nanoparticles were centrifuged three times and resuspended in deionised water. Due to the presence of thiol on the surface of the nanoparticle, the gold

nanoparticles stuck to the side of the glass centrifuge tubes and did not fully resuspend into deionised water after each wash. As a result, the nanoparticle solution went from a dark pink/purple to light pink/purple colour after centrifugation. To help the resuspension of the nanoparticles, the centrifuge tubes were placed in a sonication bath for 5 minutes but this was not fully successful. Since thiols have a lower affinity to plastic than glass, the centrifuge tubes were changed from Nalgene plastic. This significantly reduced the proportion of gold nanoparticles lost during centrifugation. Centrifuge rotation speed and number of centrifugation cycles were also investigated. It was found that reducing the centrifuge rotation speed to about 4000 g, and the centrifugation cycles to one, improved the recovery of resuspendable nanoparticles was achieved - see Table 1.

Table 1

No. of cycles g-force Resuspendable Colourless supernatant

3 5500 yes yes

2 5500 yes yes

¹ 5500 yes yes

¹ 4000 yes yes

¹ 3000 no yes

¹ 2000 no no

¹ 1500 no no

¹ 1000 no no The final step in this method was to transfer the gold nanoparticles in DMSO to DCM. The McMahon et al. method diluted the gold nanoparticles in DMSO with DCM but this formed a low concentration of nanoparticles in DCM and it was later found that the residual DMSO caused problems with the encapsulation process (see Figure 13). As a result, the DMSO was removed on a Schlenk line and the gold nanoparticles were re-suspended in DCM. This combination of parameters resulted in the high concentration of gold nanoparticles maintained throughout the surface modification process. Pre-aggregation was monitored with UV Vis absorption at each step of the surface modification process, as shown in Figure 3b.

The modified gold nanoparticles in DCM were then encapsulated in PLGA-PEG-COOH with a solvent evaporation method. This formed gold nanoparticles entrapped in PLGA-PEG-COOH. The schematic representing the surface modification and encapsulation is shown in Figure 2. Characterisation

T-NPs were characterised with scanning electron microscopy (SEM), scanning transmission electron microscopy (STEM), atomic force microscopy (AFM) and dynamic light scattering (DLS) methods to determine their size, surface charge and number of encapsulated metal NPs, as shown in Figure 3. The T-NPs had a zeta potential of -26m V (see Figure 14) and, therefore, had good stability and low cytotoxicity. The SEM, STEM, AFM and DLS results all gave an approximate hybrid nanoparticle size of 150 nm, as shown in Figure 3a, c, d and e. The SEM image in Figure 3a reveals the cluster of 50 nm Au nanoparticles surrounded by the organic PLGA-PEG-COOH corona. The coating of PLGA-PEG-COOH polymer maintains the biocompatibility of the nanoparticle and allows for subsequent surface modification. The overall size of the hybrid nanoparticles is dependent on how many gold nanoparticles are encapsulated. The AFM image in Figure 3e provides an average size distribution of 200 nm but does not suggest how many gold nanoparticles are encapsulated within the polymer. Therefore, the number of individual gold nanoparticles encapsulated within the polymer, and the frequency of occurrence, was investigated.

As a result of the preparation method, where a small number of gold nanoparticles are distributed within solvent droplets in the emulsion formed by the sonication process, it would be expected that the same number of gold nanoparticles would not be encapsulated within each sphere. This would be true even if every droplet were exactly the same volume. In fact, the number of nanoparticles trapped within each droplet should follow a Poisson distribution. Approximately 100 PLGA-PEG- COOH nanoparticles were imaged with the STEM and the number of particles they contained was recorded, as shown in Figure 3d. The distribution was then compared to a series of Poisson distributions created in Microsoft Excel with different average particle contents. The best fit with the experimental data was with the function corresponding to 2.6 particles per droplet, as shown in Figure 3f. The reasonable fit between the experimental data and the Poisson distribution suggests that the assumptions in the model are reasonable and in particular, that the emulsion droplet volumes were reasonably similar, since variation in that volume would severely distort the distribution. Similarly, the fit also shows that pre-aggregation of the Au nanoparticles was also negligible since that too would distort the distribution. It is useful to note that although each hybrid nanoparticle contained an average of 2.6 Au particles, the most likely situation was for the hybrid nanoparticles to contain two gold nanoparticles but examples with one and three particles were also common.

Cellular uptake and cytotoxicity Cellular uptake of the T-NPs was confirmed with SEM and STEM, as depicted in Figure 4a and b. In Figure 4, the SEM image (a) shows a large grey object with white dots, which is indicative of a large cell containing many clusters of T-NPs. This is also confirmed in the STEM image (b) where the large grey cell contains many black dots. The diameter of the A549 cells typically range from 10-20 μιη; however, in these images, the cell's diameter is only 6 μιτι, suggesting that cell shrinkage may have occurred when the cells were removed from the medium and added on to the grid. The black dots that are indicative of the T-NPs were measured and it was calculated that approximately 100 Au NPs in total were present within the cell.

The cytoxicity of the T-NPs to the A549 cells was measured with an MTT assay. Figure 4c shows that the variation of cell viability between T-NP concentrations in the range of 0.1 mg/ml to 0.333 mg/ml was relatively small. A concentration of 0.2 mg/ml was optimum since this was the best compromise between using the highest possible particle concentration and cell viability.

Cetuximab antibodies were then conjugated onto the T-NP surface to increase selectivity and enhance receptor-mediated uptake T-NPs to the cancer cells. A bicinchoninic acid (BCA) assay was performed to quantify how much antibody was present on the T-NPs. It was found that 15 μg of cetuximab was attached per 1 mg of polymer, as shown in Figure 4d.

Photothermal irradiation results

The combined treatment of T-NPs and photothermal irradiation caused an increase in cell death to the A549 cells, as shown in Figure 6 a and b. When the T-NPs were functionalised with the Cetuximab antibodies (Cetux T-NPs), incubated with the A549 cells and irradiated with the laser, the 'Cetux T-NPs laser' had 7 times more cell death present than the non-modified 'T-NPs laser', as shown in Figure 6a.

When the EGFR positive A549 lung cancer cells were compared to the EGFR negative A2780 ovarian cancer cells, it was expected the Cetux T-NPs would give a negative result with the photothermal therapy. This was confirmed, as shown in Figure 6c. It was found that more cell death for EGFR positive cells (A549) compared to EGFR negative cells (A2780) when treated with Cetux T-NPs and irradiated with a laser. This result implies that the combined treatment of T-NPs and photothermal irradiation works; moreover, when the T-NPs are targeted with Cetuximab, the treatment is further enhanced.

The photothermal therapy results showed an increase in cell death to the A549 cancer cells.

However, it was not understood what type of cell death the photothermal therapy caused; necrosis or apoptosis. Flow cytometry analysis of the cell death caused by the photothermal and T-NP treatment was carried out, the results of which are shown in Figure 6d. It was found that 'No NPs no laser' had the largest number of viable cells present (76%) and 'Cetux T-NPs laser' had the smallest number of viable cells present (52%), indicating that the combined treatment of nanoparticles and laser irradiation caused a reduction in viable cells. 'Cetux T-NPs laser' had the largest necrotic cell death present of 20% and 'No NPs laser' has the least amount of necrotic cell death present of 2%. This confirms that the combined treated of Cetux T-NPs and laser irradiation causes a large increase in necrotic cell death (18%). Overall, the cells containing 'Cetux T-NPs laser' had the smallest amount of viable cells present (52%) and the largest necrotic cell death (18%). This confirms that when Cetux T-NPs are added to the cells and irradiated with the laser, a photothermal effect causes an increase in necrotic cell death and a decrease in viable cells present.

Conclusion In this research, the effect of T-NPs and photothermal therapy on A2780 breast cancer cells lines was investigated. MTT assays demonstrated the low toxicity of the T-NPs to the A549 cells. A combination of scanning and transmission electron microscopy (STEM) confirmed cellular uptake of the T-NPs into the A549 cells. Photothermal therapy has proven to be an effective method for rapidly inducing cancer cell death. A 785 nm laser with a surface power density of 1 W/cm² has been shown to be sufficient to causing increase cell death to the A549 with T-NPs present. When an antibody was conjugated to the surface of the T-NPs, the speed of cellular uptake increased. An increase in cell death after photothermal therapy was also observed when the T-NPs had an antibody attached. Flow cytometry suggested that the combined treatment of T-NPs and photothermal irradiation caused increased necrotic cell death.

To conclude, this research has brought together several different methods to test the T- NPs biocompatibility, cellular uptake and optical properties for photothermal therapy. It has successfully provided a method to increase in cancer cell death to A549 lung cancer cells. The invention is not limited to the embodiments described herein but can be amended or modified without departing from the scope of the present invention.

Claims

1. A method for modifying the surface of metal nanoparticles, said method comprising:

contacting the surface of the metal nanoparticles with a linking agent to produce

functionalised metal nanoparticles, wherein the linking agent is X-Y-Z, wherein X is a sulphur containing group, Y is a bond or a linking group, and Z is a carboxy or amino group, and wherein the surface of the metal nanoparticles is contacted with < 8.197 x 10^~21 moles of linking agent per square nanometer (nm²) of the metal nanoparticle surface; and

2. A method for modifying the surface of metal nanoparticles, said method comprising:

functionalised metal nanoparticles, wherein the linking agent is X-Y-Z, wherein X is a sulphur containing group, Y is a bond or a linking group, and Z is a carboxy or amino group, and wherein the surface of the metal nanoparticles is contacted with < 1000-fold excess of the linking agent per square nanometer (nm²) of the metal nanoparticle surface; and

3. The method of Claim 1 or 2, wherein the metal nanoparticles are selected from gold, copper, platinum, iron, zinc, titanium, cadmium, selenium, tellurium, and silver nanoparticles and

nanoparticles comprising alloys of gold, copper, platinum, iron, zinc, titanium, cadmium, selenium, tellurium, and silver.

4. The method of any one of Claims 1 to 3, wherein the metal nanoparticles have a diameter of about 10-200 nm, optionally about 20-100 nm, optionally about 30-70 nm, optionally about 40-60 nm, further optionally about 50 nm.

5. The method of any one of Claims 1 to 3, wherein the metal nanoparticles have a diameter of > 40 nm, optionally > 45 nm, further optionally > 50 nm.

6. The method of any one of the preceding claims, wherein the linking group Y is a substituted or substituted aliphatic, cycloaliphatic, aryl or heteroaryl group.

7. The method of Claim 6, wherein Y is a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heteroaryl group.

8. The method of Claim 6, wherein Y is a substituted or unsubstituted alkyl, aryl or heteroaryl group.

9. The method of any one of the preceding claims, wherein the linking agent is selected from mercaptoacetic acid, optionally dilute mercaptoacetic acid; 4-mercaptobenzoic acid, 5,5'-dithiobis-(2- nitrobenzoic acid); co-substituted alkyl thiols of the formula SH(CH₂)_nCOOH, such as

mercaptopropionic acid, mercaptobutanoic acid, mercaptopentanoic acid; substituted forms of co- substituted aryl thiols of the formula SH-Y-COOH where Y is an aryl or substituted aryl group, such as 2-mercaptopropionic acid, 3-mercaptopropionic acid, 3-mercaptobutanoic acid, 4- mercaptopropionic acid.

10. The method of any one of the preceding claims, wherein the hydrophobic group is a substituted or unsubstituted aliphatic, cycloaliphatic, aryl or heteroaryl group, optionally wherein the hydrophobic group is a phenyl group or dicyclohexyl group.

1 1. The method of Claim 10, wherein the hydrophobic group is a substituted or unsubstituted aryl or heteroaryl group.

12. The method of any one of the preceding claims, wherein the surface of the metal nanoparticles is contacted with the linking agent for at least 30 seconds, optionally 30 seconds to 3 hours, optionally 30 seconds to 1 hour, optionally 30 seconds to 30 minutes, further optionally about 30 minutes.

13. The method of any one of the preceding claims, wherein the surface of the metal nanoparticles is contacted with < 8.197 x 10^~22 moles, optionally < 8.197 x 10^~23 moles, further optionally < 8.197 x 10^~24 moles of linking agent per square nanometer (nm²) of the metal nanoparticle surface.

14. The method of any one of Claims 1-12, wherein the surface of the metal nanoparticles is contacted with between about 8.197 x 10^~21 moles to about 8.197 x 10^~26 moles of the linking agent, optionally between about 8.197 x 10^~22 moles to about 8.197 x 10^~26 moles of the linking agent, optionally between about 8.197 x 10^~23 moles to about 8.197 x 10^~26 moles of the linking agent, optionally between a about 8.197 x 10^~23 moles to about 8.197 x 10^~26 moles of the linking agent, optionally between about 8.197 x 10^~24 moles to about 8.197 x 10^~26 moles of the linking agent, further optionally about 8.197 x 10^"24 moles, per square nanometer (nm²) of the metal nanoparticle surface.

15. The method of any one of the preceding claims, wherein unbound linking agent is removed before adding the hydrophobic groups to the functionalised metal nanoparticles.

16. The method of Claim 15, wherein the unbound linking agent is removed by centrifugation at > 3000 g, optionally > 4000 g, further optionally about 4,000 g, for > 10 minutes, optionally > 15 minutes, further optionally about 15 minutes.

17. The method of Claim 16, wherein the centrifugation is carried out as a single centrifugation step at about 4,000 g for about 15 minutes.

18. The method of any one of the preceding claims, wherein the hydrophobic group is added to the surface of the functionalised metal nanoparticles by incubating the functionalised metal nanoparticles in a phase transfer solution comprising dicyclohexylamine.

19. The method of Claim 18, wherein said phase transfer solution further comprises

dicyclohexylcarbodiimide.

20. The method of Claim 18 or 19, wherein said phase transfer solution further comprises a drying reagent, optionally wherein the drying reagent is sodium sulphate.

21. The method of any one of Claims 17-20, wherein the surface-modified metal nanoparticles are isolated from the phase transfer solution, optionally wherein said nanoparticles are isolated from the phase transfer solution via a Schlenk line.

22. The method of any preceding claim, wherein modifying the surface of metal nanoparticles takes place in an aqueous solution.

23. The method of any preceding claim, wherein the surface-modified metal nanoparticles are transferred to a non-aqueous solution, optionally wherein the non-aqueous solution is an organic solvent, further optionally dimethyl sulfoxide (DMSO).

24. A method for encapsulating a metal nanoparticle in a biocompatible polymer, said method comprising:

(i) contacting the surface-modified metal nanoparticles, produced according to the method of any one of the Claims 1 to 23, with a biocompatible polymer; and

25. The method of Claim 24, wherein the surface-modified metal nanoparticles are provided in a non-aqueous solution, optionally wherein the non-aqueous solution is an organic solvent, further optionally DMSO.

26. The method of Claim 24 or 25, wherein the biocompatible polymer is provided in a non-aqueous solution, optionally wherein the non-aqueous solution is an organic solvent, further optionally DMSO.

27. The method of Claim 26, wherein the non-aqueous solution, optionally organic solvent, in which the biocompatible polymer is contained is the same type of solvent as the non-aqueous solution, optionally organic solvent, in which the surface-modified metal nanoparticles are contained.

28. The method of any of Claims 24 to 27, wherein the biocompatible polymer is selected from poly(lactic-co-glycolic acid) (PLGA), poly(lactide) (PLA), poly(glycolide) (PGA), poly(butyl cyanoacrylate) (PBCA), and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, optionally wherein the PLGA is PLGA-PEG-COOH.

29. The method of any one of Claims 24 to 28, wherein step (ii) comprises stirring the product of step (i) and an emulsifier, optionally wherein said stirring is on ice, further optionally wherein said stirring further comprises agitation, optionally herein said agitation is sonication.

30. The method of any one of Claims 24 to 29, wherein step (ii) further comprises contacting the product of step (i) with an immiscible solvent and the emulsifier, and/or wherein the emulsifier is polyvinyl alcohol.

31. The method of any one of Claims 24 to 30, further comprising:

(iii) removing the organic solvent.

32. The method of any one of Claims 24 to 31 , wherein the organic solvent is suitable to dissolve the biocompatible polymer, optionally wherein the organic solvent comprises

dichloromethane or acetone.

33. A method of conjugating a binding agent to an encapsulated metal nanoparticle, said method comprising:

conjugating the binding agent via a linker to an encapsulated metal nanoparticle produced according to the method of any one of Claims 24-32.

34. The method of Claim 33, wherein the binding agent is a protein, optionally an antibody.

35. The method of Claim 33 or 34, wherein the linker is selected from a carbodiimide linker, an aldehyde linker, a maleimide linker and a linker comprising a reactive moiety for use in click-like chemistry.

36. The method of Claim 35, wherein the linker is a carbodiimide linker and wherein the linker is coupled to the encapsulated metal nanoparticle by contacting the encapsulated metal nanoparticle with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

37. A metal nanoparticle, wherein said metal nanoparticle is produced according to the method of any one of Claims 1-23.

38. An encapsulated metal nanoparticle, wherein said encapsulated metal nanoparticle is produced according to the method of any one of Claims 24-32.

39. An encapsulated metal nanoparticle conjugated to a binding agent, wherein the binding agent-encapsulated metal nanoparticle is produced according to the method of any one of Claims 33-36.

40. An encapsulated metal nanoparticle and binding agent according to Claim 39, wherein said binding agent-encapsulated metal nanoparticle is for use in therapy, optionally photothermal therapy.

41. An encapsulated metal nanoparticle and binding agent according to Claim 39, wherein said binding agent-encapsulated metal nanoparticle is for use in imaging, optionally therapeutic imaging.

42. An encapsulated metal nanoparticle and binding agent according to Claim 39, wherein said binding agent-encapsulated metal nanoparticle is for use in the treatment of cancer.

43. The encapsulated metal nanoparticle and binding agent for use according to Claim 42, wherein said treatment comprises photothermal therapy.