NANOPARTICLES AND THEIR USE IN CANCER THERAPY

Abstract

The present invention provides a nanoparticle comprising a core comprising a metal; and a corona comprising a plurality of ligands covalently linked to the core, the plurality of ligands including at least a first species of ligand comprising an ethylene glycol portion and an amine group and at least a second species of ligand comprising a carbohydrate group, for use in a method of treating a cancer, particularly skin cancer, in a mammalian subject. Also disclosed are methods of treatment by administering the nanoparticles alone or in combination with radiotherapy.

Claims

1.-22. (canceled)

23. A method of treatment of a cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a nanoparticle comprising a core comprising a metal; and a corona comprising a plurality of ligands covalently linked to the core, the plurality of ligands including at least a first species of ligand comprising an ethylene glycol portion and an amine group and at least a second species of ligand comprising a carbohydrate group.

24. The method according to claim 23, wherein said cancer is selected from the group consisting of: squamous cell carcinoma (SCC), cervical cancer, skin cancer, oral cancer, glioma, lung cancer, bladder cancer, ocular cancer, stomach cancer, and esophageal cancer.

25. The method according to claim 24, wherein said cancer is oral or skin SCC.

26. The method according to claim 23, wherein said method further comprises administering radiotherapy to said subject.

27. (canceled)

28. The method according to claim 23, wherein said method of treating said cancer comprises administering said nanoparticle and subsequently or concurrently administering said radiotherapy to the subject.

29. The method according to claim 28, wherein said radiotherapy comprises x-ray radiation.

30. The method according to claim 23, wherein said first species of ligand comprises an amine-functionalised poly(ethylene glycol) or amine-functionalised oligo(ethylene glycol).

31. The method according to claim 30, wherein said first species of ligand comprises an amine-functionalised hexaethylene glycol.

32. The method according to claim 31, wherein said first species of ligand comprises a ligand according to formula (I): ##STR00005##

33. The method according to claim 23, wherein said second species of ligand comprises a monosaccharide.

34. The method according to claim 33, wherein said second species of ligand comprises galactose, glucose or N-acetylglucosamine.

35. The method according to claim 34, wherein said second species of ligand comprises alpha-galactose covalently linked to said core via a thioethyl group.

36. The method according to claim 23, wherein said first and second species of ligands are present on the nanoparticle in a molar ratio in the range 95:5 to 5:95, or 80:20 to 20:80, or 60:40 to 40:60.

37. The method according to claim 36, wherein said first and second species of ligands are present at a ratio in the range 45:55 to 55:45.

38. The method according to claim 23, wherein the only ligands covalently linked to the core are said first species of ligand and said second species of ligand.

39. A pharmaceutical composition for use in a method as defined in claim 23, the composition comprising one or more nanoparticles comprising a core comprising a metal; and a corona comprising a plurality of ligands covalently linked to the core, the plurality of ligands including at least a first species of ligand comprising an ethylene glycol portion and an amine group and at least a second species of ligand comprising a carbohydrate group.

40. A pharmaceutical composition according to claim 39, wherein the pharmaceutical composition is formulated for topical administration.

41. An article of manufacture comprising: a nanoparticle comprising a core comprising a metal; and a corona comprising a plurality of ligands covalently linked to the core, the plurality of ligands including at least a first species of ligand comprising an ethylene glycol portion and an amine group and at least a second species of ligand comprising a carbohydrate group; a container for housing the nanoparticle; and an insert or label with administration and/or dosage instructions for the treatment of a cancer, optionally wherein the cancer is selected from the group consisting of: squamous cell carcinoma (SCC), cervical cancer, skin cancer, oral cancer, glioma, lung cancer, bladder cancer, ocular cancer, stomach cancer, and esophageal cancer.

42. An article of manufacture comprising: a pharmaceutical preparation as defined in claim 39; a container for housing the pharmaceutical composition; and an insert or label with administration and/or dosage instructions for the treatment of a cancer, optionally wherein the cancer is selected from the group consisting of: squamous cell carcinoma (SCC), cervical cancer, skin cancer, oral cancer, glioma, lung cancer, bladder cancer, ocular cancer, stomach cancer, and esophageal cancer.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0043] FIG. 1 shows a schematic representation of a nanoparticle having a plurality of ligands in the ratio of 1:1 of alpha-galactose:PEGamine and a gold core.

[0044] FIG. 2 shows clonogenic assay triplicate results in the form of a photograph of two six-well plates, in which: the upper row of the upper plate has three wells with HSC-3 cells+NP37 nanoparticles (50:50 alpha-galactose-C2:PEGamine-GNP); the lower row of the upper plate has HaCaT cells+NP37 nanoparticles; the upper row of the lower plate has HSC3 cells without nanoparticles (control); and the lower row of the lower plate has HaCaT cells without nanoparticles (control). The plates were seeded at 1000 cells/well for 24 hours, then exposed to NP37 nanoparticles (15 μg/ml) or no nanoparticles for 3 hours, washed out, then cultured in normal medium for 7 days. The results show that the oral SCC tumour cell line, HSC3, when treated with NP37 nanoparticles exhibited near total cell death in comparison to untreated HSC3 cells and in comparison to keratinocyte, HaCaT, cells treated with NP37 nanoparticles or untreated.

[0045] FIG. 3 shows clonogenic assay duplicate results in the form of a photograph of two 24-well plates, in which: HSC-3 (upper plate) and HaCaT (lower plate) cells were treated with 3 (row 1), 10 (row 2), 15 (row 3) and 30 (row 4) μg/ml of MP253 (60:40 alpha-galactose-C2:PEGamine-GNP; columns 1 and 2), NP37 (50:50 alpha-galactose-C2:PEGamine-GNP; columns 3 and 4), and MP254 (40:60 alpha-galactose-C2:PEGamine-GNP; columns 5 and 6) nanoparticles. Plates were seeded with 500 cells/well and nanoparticles were applied for 3 hours, then washed out and replaced with fresh medium, then left for 7 days. The results show cell killing for HSC-3 cells by all three nanoparticle types in comparison to HaCaT cells. The order of potency (highest to lowest) was MP253>MP254>NP37.

[0046] FIG. 4 shows clonogenic assay results of various nanoparticles to HSC-3 cells (columns 1, 2 and 3) and HaCaT cells (columns 4, 5 and 6). All nanoparticle treatments were at 15 μg/ml for 3 hours. The top row treatments were (left to right): MP184 (50:50 alpha-galactose-C2:PEGamine-GNP), MP185 (50:50 beta-glucose-C2:PEGamine-GNP) and MP186 (50:50 N-acetyl-glucosamine-C2:PEGamine-GNP). The lower row treatments were (left to right): MP187 (100% alpha-galactose-C2-GNP), MP188 (100% PEGamine-GNP) and untreated control “zero”. The results show clear cell killing of HSC-3 cells in comparison to HaCaT cells by MP184, MP185, MP186 and MP188 at the concentration tested.

[0047] FIG. 5 shows a dose-response curve (colonies % control vs. concentration of NP37 nanoparticles μg/ml) derived from clonogenic assay results with three cell lines: HaCaT (upper line; red); HSC-3 (middle line, crossing to lower line above 10 μg/ml; green); and HeLa (lower line, crossing to middle line above 10 μg/ml; blue). The cells were incubated for 6 hours with NP37 nanoparticles at 1, 3, 10, 30 and 100 μg/ml. The data points at 15 μg/ml NP37 nanoparticles were from a separate experiment which used a 3-hour incubation time with NP37 nanoparticles.

[0048] FIG. 6 shows micrographs with silver enhancement of cellular uptake of NP37 nanoparticles. The black dots show gold nanoparticles; the bright green is a nuclear stain. Results are shown for HSC-3 cells and HaCaT cells at time zero (0 hours), 2 hours and 4 hours after treatment with NP37 nanoparticles. The results show accumulation of NP37 nanoparticles into cells, adjacent to the cell nucleus, which may represent accumulation within the Golgi.

[0049] FIG. 7 shows silver enhancement images for acute (3-hour) uptake of nanoparticles by HSC-3 (left) and HaCaT cells (right). The paired images show phase (left) and bright field (right). The nanoparticles treatments (all 15 μg/ml) were (top to bottom): MP184 (50:50 alpha-galactose-C2:PEGamine-GNP), MP185 (50:50 beta-glucose-C2:PEGamine-GNP), MP186 (50:50 N-acetyl-glucosamine-C2:PEGamine-GNP), MP187 (100% alpha-galactose-C2-GNP), MP188 (100% PEGamine-GNP), and zero (untreated control). The results indicate greater cell uptake of MP184-186, with lower uptake of MP187 and MP188. The location of the majority of the staining was found to be juxtanuclear.

[0050] FIG. 8 shows micrograph images of HSC-3 cells treated with NP37 nanoparticles (30 μg/ml for 3 hours) in the presence or absence of either sodium pyruvate (Pyr) of ascorbic acid (AA) as antioxidants. The Pyr concentrations tested were (left to right): zero, 0.1 mM, 1 mM, 2 mM and 5 mM. The AA concentrations tested were (left to right): zero, 0.05 mM, 0.1 mM, 0.5 mM and 1 mM. The results indicate that both antioxidant treatments were able to prevent NP37-mediated cell death of HSC-3 cells.

[0051] FIG. 9 shows NP37 nanoparticle murine skin penetration following 200 μg/ml topical application of nanoparticles for 4 hours. The skin section was fixed and stained with silver enhancer. The images show untreated control (left) and NP37 nanoparticles (right). On the left-hand image the black regions within the skin are melanin. On the right-hand image, the silver staining reveals the gold nanoparticles as an even grey/green stain, which is distinct from melanin. The results suggest penetration of murine skin by NP37 nanoparticles.

[0052] FIG. 10 shows NP37 nanoparticle skin penetration following 200 μg/ml topical application of nanoparticles for 4 hours. The skin samples were fixed, gelatine embedded, vibratome sectioned, then silver stained. The silver reaction product is brown. The upper row shows untreated control. The middle row shows skin treated with 200 μg/ml NP37+penetration enhancer (60 mg N-lauroylsarcosine and 40 mg sorbitan monolaurate (Span® 20) per 10 ml of phosphate buffer:ethanol (1:1)) for 4 hours. The bottom row shows 200 μg/ml NP37 for 4 hours. The results indicate that NP37 nanoparticles are rapidly absorbed into murine skin.

[0053] FIG. 11 shows dose-dependent toxicity (% control surviving) of various ratios of sugar:PEGamine nanoparticles against HSC-3 Oral SCC cells (left-hand panel) and HaCaT control keratinocytes. The key to the right of the right-hand panel shows (from top to bottom): alpha-galactose:PEGamine 100:0 (orange circles); 80:20 (red squares); 60:40 (green triangles); 50:50 (blue inverted triangles); 40:60 (blue diamonds); 20:80 (pink circles); 0:100 (black squares); xGal (grey triangles); and amino linker (AL) (grey circles). The IC.sub.50 of the 50:50 alpha-galactose:PEGamine nanoparticles was found to be 0.96 μg/ml (approximately 50 nM), which is much lower than the IC.sub.50 measured for the same nanoparticles against the HaCaT cells>10000 μg/ml, indicating selective anti-cancer toxicity. The 50:50 ratio alpha-galactose:PEGamine AuNPs were found to exhibit the greatest anti-cancer toxicity, followed by the 60:40 ratio alpha-galactose:PEGamine AuNPs as the next most potent.

[0054] FIG. 12 shows toxicity (surviving fraction; y-axis) plotted against radiation dose (Gray; x-axis) of HSC-3 cells (left-hand panel) and HaCaT cells (right-hand panel) for control (red line; no drug), 0.3 μg/ml gold nanoparticles with a corona of 50:50 alpha-galactose:PEGamine (blue), and 0.6 μg/ml gold nanoparticles with a corona of 50:50 alpha-galactose:PEGamine (green). The nanoparticle-treated HSC-3 cells exhibited enhancement of radiation-induced cell killing (approximately 1.4× for 0.3 μg/ml AuNPs; 2.8× for 0.6 μg/ml AuNPs). A much lower level of radiation potentiation was seen in the non-cancer HaCaT cells (approximately 1.0× for 0.3 μg/ml AuNPs; 1.1× for 0.6 μg/ml AuNPs).

DETAILED DESCRIPTION OF THE INVENTION

[0055] In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

[0056] Nanoparticles

[0057] As used herein, “nanoparticle” refers to a particle having a nanomeric scale, and is not intended to convey any specific shape limitation. In particular, “nanoparticle” encompasses nanospheres, nanotubes, nanoboxes, nanoclusters, nanorods and the like. In certain embodiments the nanoparticles and/or nanoparticle cores contemplated herein have a generally polyhedral or spherical geometry.

[0058] Nanoparticles comprising a plurality of carbohydrate-containing ligands have been described in, for example, WO 2002/032404, WO 2004/108165, WO 2005/116226, WO 2006/037979, WO 2007/015105, WO 2007/122388, WO 2005/091704 (the entire contents of each of which is expressly incorporated herein by reference) and such nanoparticles may find use in accordance with the present invention.

[0059] As used herein, “corona” refers to a layer or coating, which may partially or completely cover the exposed surface of the nanoparticle core. The corona includes a plurality of ligands which generally include at least one carbohydrate moiety, one surfactant moiety and/or one glutathione moiety. Thus, the corona may be considered to be an organic layer that surrounds or partially surrounds the metallic core. In certain embodiments the corona provides and/or participates in passivating the core of the nanoparticle. Thus, in certain cases the corona may include a sufficiently complete coating layer substantially to stabilise the semiconductor or metal-containing core. However, it is specifically contemplated herein that certain nanoparticles having cores, e.g., that include a metal oxide-containing inner core coated with a noble metal may include a corona that only partially coats the core surface. In certain cases the corona facilitates solubility, such as water solubility, of the nanoparticles of the present invention.

[0060] Nanoparticles are small particles, e.g. clusters of metal or semiconductor atoms, that can be used as a substrate for immobilising ligands.

[0061] Preferably, the nanoparticles have cores having mean diameters between 0.5 and 50 nm, more preferably between 0.5 and 10 nm, more preferably between 0.5 and 5 nm, more preferably between 0.5 and 3 nm and still more preferably between 0.5 and 2.5 nm. When the ligands are considered in addition to the cores, preferably the overall mean diameter of the particles is between 2.0 and 20 nm, more preferably between 3 and 10 nm and most preferably between 4 and 5 nm. The mean diameter can be measured using techniques well known in the art such as transmission electron microscopy.

[0062] The core material can be a metal or semiconductor (said semiconductor optionally comprising metal atoms or being an organic semiconductor) and may be formed of more than one type of atom. Preferably, the core material is a metal selected from Au, Fe or Cu. Nanoparticle cores may also be formed from alloys including Au/Fe, Au/Cu, Au/Gd, Au/Fe/Cu, Au/Fe/Gd and Au/Fe/Cu/Gd, and may be used in the present invention. Preferred core materials are Au and Fe, with the most preferred material being Au. The cores of the nanoparticles preferably comprise between about 100 and 500 atoms (e.g. gold atoms) to provide core diameters in the nanometre range. Other particularly useful core materials are doped with one or more atoms that are NMR active, allowing the nanoparticles to be detected using NMR, both in vitro and in vivo. Examples of NMR active atoms include Mn.sup.+2, Gd.sup.+3, Eu.sup.+2, Cu.sup.+2, V.sup.+2, Co.sup.+2, Ni.sup.+2, Fe.sup.+3 and lanthanides.sup.+3, or the quantum dots described elsewhere in this application.

[0063] Nanoparticle cores comprising semiconductor compounds can be detected as nanometre scale semiconductor crystals are capable of acting as quantum dots, that is they can absorb light thereby exciting electrons in the materials to higher energy levels, subsequently releasing photons of light at frequencies characteristic of the material. An example of a semiconductor core material is cadmium selenide, cadmium sulphide, cadmium tellurium. Also included are the zinc compounds such as zinc sulphide.

[0064] In some embodiments, the nanoparticle or its ligand comprises a detectable label. The label may be an element of the core of the nanoparticle or the ligand. The label may be detectable because of an intrinsic property of that element of the nanoparticle or by being linked, conjugated or associated with a further moiety that is detectable. Preferred examples of labels include a label which is a fluorescent group, a radionuclide, a magnetic label or a dye. Fluorescent groups include fluorescein, rhodamine or tetramethyl rhodamine, Texas-Red, Cy3, Cy5, etc., and may be detected by excitation of the fluorescent label and detection of the emitted light using Raman scattering spectroscopy (Y. C. Cao, R. Jin, C. A. Mirkin, Science 2002, 297: 1536-1539).

[0065] In some embodiments, the nanoparticles may comprise a radionuclide for use in detecting the nanoparticle using the radioactivity emitted by the radionuclide, e.g. by using PET, SPECT, or for therapy, i.e. for killing target cells. Examples of radionuclides commonly used in the art that could be readily adapted for use in the present invention include .sup.99mTc, which exists in a variety of oxidation states although the most stable is TcO.sup.4−; .sup.32P or .sup.33P; .sup.57Co; .sup.59Fe; .sup.67Cu which is often used as Cu.sup.2+ salts; .sup.67Ga which is commonly used a Ga.sup.3+ salt, e.g. gallium citrate; .sup.68Ge; .sup.82Sr; .sup.99Mo; .sup.103Pd; .sup.111In which is generally used as In.sup.3+ salts; .sup.125I or .sup.131I which is generally used as sodium iodide; .sup.137Cs; .sup.153Gd; .sup.153Sm; .sup.158Au; .sup.186Re; .sup.201Tl generally used as a Tl.sup.+ salt such as thallium chloride; .sup.39Y.sup.3+; .sup.71Lu.sup.3+; and .sup.24Cr.sup.2+. The general use of radionuclides as labels and tracers is well known in the art and could readily be adapted by the skilled person for use in the aspects of the present invention. The radionuclides may be employed most easily by doping the cores of the nanoparticles or including them as labels present as part of ligands immobilised on the nanoparticles.

[0066] Administration and Treatment

[0067] The nanoparticles and compositions of the invention may be administered to patients by any number of different routes, including enteral or parenteral routes. Parenteral administration includes administration by the following routes: intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraocular, transepithelial, intraperitoneal and topical (including dermal, ocular, rectal, nasal, inhalation and aerosol), and rectal systemic routes.

[0068] The nanoparticles of the invention may be formulated as pharmaceutical compositions that may be in the forms of solid or liquid compositions. Such compositions will generally comprise a carrier of some sort, for example a solid carrier or a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. Such compositions and preparations generally contain at least 0.1 wt % of the compound.

[0069] For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution or liquid which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, solutions of the compounds or a derivative thereof, e.g. in physiological saline, a dispersion prepared with glycerol, liquid polyethylene glycol or oils.

[0070] In addition to one or more of the compounds, optionally in combination with other active ingredient, the compositions can comprise one or more of a pharmaceutically acceptable excipient, carrier, buffer, stabiliser, isotonicising agent, preservative or anti-oxidant or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g., intramuscular injection.

[0071] Preferably, the pharmaceutically compositions are given to an individual in a prophylactically effective amount or a therapeutically effective amount (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. Typically, this will be to cause a therapeutically useful activity providing benefit to the individual. The actual amount of the compounds administered, and rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, N.Y., USA); Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994. By way of example, and the compositions are preferably administered to patients in dosages of between about 0.01 and 100 mg of active compound per kg of body weight, and more preferably between about 0.5 and 10 mg/kg of body weight.

[0072] The following is presented by way of example and is not to be construed as a limitation to the scope of the claims.

EXAMPLES

Example 1—Synthesis of Nanoparticles

[0073] Gold nanoparticles having a corona of carbohydrate ligands or glutathione ligands were synthesised essentially as described previously (WO 2011/154711; and Lund et al., 2011, Biomaterials Vol. 32 pp. 9776-9784, the entire contents of which are expressly incorporated herein by reference).

[0074] AL/α-Gal NPs

Preparation of 2-thio-ethyl-α-D-galactoside (α-galactose C2SH)

[0075] ##STR00002##

[0076] To a suspension of galactose (3 g, 16.65 mmol) in 2-bromoethanol (30 ml), acid resin Amberlite 120-H is added to reach pH 2. The reaction is stirred for 16 hours at 50-60° C. The reaction mixture is filtered and washed with MeOH. Triethylamine is added to reach pH 8. The crude of the reaction is concentrated and co evaporated 3 times with toluene. The reaction mixture is dissolved pyridine (75 mL) and Ac2O (35 mL) and a catalytic amount of DMAP are added at 0° C. and stirred for 3 h at rt. The mixture is diluted with AcOEt and washed with 1.H.sub.2O; 2.HCl (10%) 3. NaHCO.sub.3 dis 4.H.sub.2O. The organic layer is collected and dried over anhydrous Na.sub.2SO.sub.4. TLC (Hexane: AcOEt 3:1, 2 elutions) shows a major product (desired) and a lower Rf minority. The product is purified by flash chromatography using the mixture hexane: ethyl acetate 6:1 as eluent and the 2-bromoethyl-alpha-galactoside (2) is obtained.

[0077] The product of the previous reaction, 2 is dissolved in 27 ml of 2-butanone. To this solution, a catalytic amount of tetrabutylammonium iodide and 4 equivalents of potassium thioacetate are added. The resulting suspension is stirred for 2 hours at room temperature. Throughout this period the reaction is tested by TLC (hexane-AcOEt 2:1, 2 elutions) for the disappearance of the starting material. The mixture is diluted with 20 ml of AcOEt and washed with a saturated NaCl solution. The organic phase is dried, filtered and evaporated under vacuum. The product is purified in hexane/AcOEt 2:1.fwdarw.1:1 to obtain the acetylthio-alpha-galactoside 3.

[0078] The new product of the reaction, 3 is dissolved in a mixture dichloromethane-methanol 2:1. To this mixture a solution of 1N sodium methoxide (1 equivalent) is added and stirred for 1 hour at room temperature. Amberlite IR-120H resin is added to achieve pH 5-6. The resulting mixture is then filtered and concentrated to dryness to obtain the final product (α-galactose C2SH).

[0079] Preparation of Amino-Thiol Linker.

##STR00003##

[0080] To a solution of PPh.sub.3 (3 g, 11.4 mmol) in 20 ml dry THF, DIAC (2.3 g, 11.4 mmol) is added. The mixture is allowed to stir at 0° C. 15 min until the appearance of a white product. To this mixture a solution of hexaethyleneglycol (1.45 mL, 5.7 mmol) and HSAc (610 μl, 8.55 mmol) in dry THF (20 mL) is added dropwise (addition funnel). After 15 min the products begin to appear on TLC at Rf 0.2. The solution is concentrated in an evaporator. The crude of the reaction is dissolved in 50 ml of dichloromethane and washed with a solution of K.sub.2CO.sub.3 10%. The organic phase is dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. Flash chromatography of the crude using AcOEt: Hexane 1:1, AcOEt and finally DCM:MeOH 4:1 as eluent gave the acetyl-thio-hexaethyleneglycol derivative.

[0081] The reaction product is dissolved in 5 ml of DMF and PPh.sub.3 (2.25 g, 8.55 mmol), NaN.sub.3 (0.741 g, 11.4 mmol) and BrCl.sub.3C (0,845 ml, 8.55 mmol) are added and the solution subsequently stirred for 40 min at room temperature. The resulting product has a higher Rf than the starting product when performing TLC (DCM:MeOH 25:1). The reaction mixture is diluted with 100 ml of diethylether and washed three times with H.sub.2O. The organic phase is dried over anhydrous Na.sub.2SO.sub.4, filtered and evaporated under vacuum. The product is purified by flash chromatography using the mixture of eluents DMC/MeOH 200:1 and DCM/MeOH 40:1 to obtain the azido-acetylthio-hexaethyleneglycol derivative.

[0082] To remove the triphenyl phosphine oxide, the reaction product is dissolved in 10 ml of THF and 0.5 g of MgCl.sub.2 is added to this solution. The reaction is stirred for 2 h at 80° C. until a white precipitate appears and then is filtered through celite.

[0083] The product is dissolved in a mixture of ethanol:H.sub.2O 3:1 and added Zn dust (0.45 g, 6.84 mmol) and NH.sub.4Cl (0.6 g, 11.4 mmol). The reaction was stirred at reflux for 1 h until the presence of starting material is no longer detectable by TLC (DCM/MeOH 25:1). The reaction is filtered through celite and the solvent is evaporated. The crude de reaction is diluted with AcOEt and extract with 5 ml H.sub.2O. The aqueous phase is evaporated to dryness to obtain the amino-thiol-hexaethylenglycol product.

[0084] Alpha-galactose C2 derivative 3 and hexaethyleneglycol amine linker 6 were taken from Midatech Biogune stock. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC.HCl), HAuCl.sub.4, NaBH.sub.4 were purchased from Sigma-Aldrich Chemical Company. Imidazole-4-acetic acid monohydrochloride was purchased from Alfa Aesar. Company High quality MeOH and Nanopure water (18.1 mΩ) were used for all experiments and solutions.

##STR00004##

[0085] α-GalC2 (Alpha) [0086] 2′-thioethyl-α-D-galactopyranoside (alpha)

[0087] EG6NH2 [0088] 1-amino-17-mercapto-3,6,9,12,15,-pentaoxa-heptadecanol or [0089] 1-amino-6-mercapto-hexaethylenglycol (vulgar name)

[0090] Preparation of AL/α-Gal NPs: To a mix of amine-mercapto hexaethylenglycol linker 6 and alpha-galactose ligand 3 in a ratio 1:1 (0.58 mmol, 3 eq.) in MeOH (49 mL) was added an aqueous solution of gold salt (7.86 mL, 0.19 mmol, 0.025M). The reaction was stirred for 30 seconds and then, an aqueous solution of NaBH.sub.4 (1N) was added in several portions (4.32 mL, 4.32 mmol). The reaction was shaken for 100 minutes at 900 rpm. After this time, the suspension was centrifuged 1 minute at 14000 rpm. The supernatant is removed and the precipitated was dissolved in 2 mL of water. Then, 2 mL of the suspension were introduced in two filters (AMICON, 10 KDa, 4 mL) and were centrifuged 5 minutes at 4500 g. The residue in the filter was washed twice more with water. The final residue was dissolved in 80 mL of water.

[0091] For the preparation of gold NPs manufacture was under laminar flow cabinet. All glass and plastic material (such as eppendorfs, vials and bottles) and solvent (water, HAc) were first sterilized in an autoclave. All other disposables (such as tips and filters) came pre-sterilized.

Example 2—Bioactivity of Nanoparticles

[0092] Cell Killing

[0093] HSC-3, HaCaT, and HeLa cell lines are maintained in low glucose DMEM with 10% foetal calf serum.

[0094] For clonogenic assay, cells are seeded at a density of either 500 cells/well in 24-well plastic culture plates or at 1000 cells per well in 6-well culture plates. After allowing 24 hr for cells to attach, nanoparticles are added at the required concentration for 3 hr, then washed off and replaced with fresh medium. Cells are then maintained for 7 days to grow as individual colonies. Colonies are then stained with 0.5% w/v methylene blue in 50% ethanol. The number of colonies (>50 cells) are counted per condition and photographed under identical lighting.

[0095] For radiotherapy, the protocol is as above using 24-well plates. Cells are loaded for 3 hr with nanoparticles and then irradiated with 4Gy of 150 keV x-rays. Approximately 1 hour later the cells are washed and replaced with fresh medium and maintained for 7 days.

[0096] As shown in FIG. 2, clonogenic assay results show that the oral SCC tumour cell line, HSC-3, when treated with NP37 nanoparticles (50:50 alpha-galactose-C2:PEGamine-GNP) exhibited near total cell death in comparison to untreated HSC-3 cells and in comparison to keratinocyte, HaCaT, cells treated with NP37 nanoparticles or untreated.

[0097] The results shown in FIG. 3 indicate cell killing for HSC-3 cells by all three nanoparticle types, MP253 (60:40 alpha-galactose-C2:PEGamine-GNP), NP37 (50:50 alpha-galactose-C2:PEGamine-GNP), and MP254 (40:60 alpha-galactose-C2:PEGamine-GNP) in comparison to HaCaT cells. The order of potency (highest to lowest) was MP253>MP254>NP37.

[0098] A range of nanoparticle corona types were investigated for cancer cell killing activity. FIG. 4 shows clonogenic assay results of various nanoparticles to HSC-3 cells and HaCaT cells. The nanoparticle treatments were: MP184 (50:50 alpha-galactose-C2:PEGamine-GNP), MP185 (50:50 beta-glucose-C2:PEGamine-GNP), MP186 (50:50 N-acetyl-glucosamine-C2:PEGamine-GNP), MP187 (100% alpha-galactose-C2-GNP), MP188 (100% PEGamine-GNP) and untreated control “zero”. The results show clear cell killing of HSC-3 cells in comparison to HaCaT cells by MP184, MP185, MP186 and MP188 at the concentration tested (15 μg/ml for 3 hours).

[0099] As shown in FIG. 5, NP37 nanoparticles killed HSC-3, HeLa and (at higher concentrations) HaCaT cells in a dose-dependent manner. The LD.sub.50 values for NP37 nanoparticles against the three cell types are shown in Table 1. The sensitivity of the tumour cell lines HeLa (cervical) and HSC-3 (oral SCC) relative to HaCaT (keratinocytes) is indicative of therapeutic benefit in the treatment of cancer.

TABLE-US-00001 TABLE 1 LD.sub.50 for NP37 in 3 cell types Cell type LD 50 μg/ml HeLa 2 HSC 11 HaCaT 38

[0100] Potentiation of nanoparticle-induced HSC-3 cell killing was observed following irradiation with 4Gy 150 keV x-rays.

[0101] Mechanism of Cell Killing

[0102] The potential role for reactive oxygen species (ROS) in the nanoparticle-mediated cell death was investigated. HSC-3 and HaCaT cells were treated with 15 μg/ml NP37 nanoparticles for 3.5 hours. As shown in Table 2, the ROS-sensitive probe, DCFHDA, shows selectively higher fluorescence signal in the HSC-3 cells when NP37 nanoparticles are present. Results also indicate that nanoparticles that exhibit cell toxicity to cancer cells also cause a rise in intracellular calcium within seconds of addition. Without wishing to be bound by any particular theory, the inventors believe the calcium signalling may play a role in the mechanism of cell killing.

TABLE-US-00002 TABLE 2 NP37 increases reactive oxygen species (ROS) in cells HSC HaCaT HSC % max HaCaT % max DCFHDA only 31.1 6 67.7 61.8 NP37 + DCFHDA 42 7 91.5 72.1 DCF-ox (max) 45.9 9.7 100 100

[0103] As shown in FIG. 8, the antioxidants sodium pyruvate and ascorbic acid are able to prevent NP37-mediated HSC-3 cell death at concentrations in the range tested. HSC-3 cells were treated with NP37 nanoparticles (30 μg/ml for 3 hours) in the presence or absence of either sodium pyruvate (Pyr) of ascorbic acid (AA) as antioxidants. The Pyr concentrations tested were: zero, 0.1 mM, 1 mM, 2 mM and 5 mM. The AA concentrations tested were: zero, 0.05 mM, 0.1 mM, 0.5 mM and 1 mM.

[0104] Cell Uptake of Nanoparticles

[0105] As shown in FIG. 6, NP37 nanoparticles exhibit rapid uptake into HSC-3 cells and HaCaT cells, with accumulation of the nanoparticles adjacent to the cell nucleus, possibly within the Golgi.

[0106] The results shown in FIG. 7 indicate greater cell uptake by HSC-3 and HaCaT cells of the nanoparticles MP184-186, with lower uptake of MP187 and MP188. The location of the majority of the staining was again found to be juxtanuclear. The nanoparticles treatments (all 15 μg/ml) were: MP184 (50:50 alpha-galactose-C2:PEGamine-GNP), MP185 (50:50 beta-glucose-C2:PEGamine-GNP), MP186 (50:50 N-acetyl-glucosamine-C2:PEGamine-GNP), MP187 (100% alpha-galactose-C2-GNP), MP188 (100% PEGamine-GNP), and zero (untreated control).

[0107] Nanoparticle penetration of skin samples was also investigated. FIG. 9 shows NP37 nanoparticle murine skin penetration following 200 μg/ml topical application of nanoparticles for 4 hours. FIG. 10 shows NP37 nanoparticle skin penetration following 200 μg/ml topical application of nanoparticles for 4 hours. Shown are: untreated control, skin treated with 200 μg/ml NP37+penetration enhancer (60 mg N-lauroylsarcosine and 40 mg sorbitan monolaurate (Span® 20) per 10 ml of phosphate buffer:ethanol (1:1)) for 4 hours, and skin treated with 200 μg/ml NP37 for 4 hours. The results indicate that NP37 nanoparticles are rapidly absorbed into murine skin.

Example 3—Further Bioactivity Experiments

[0108] Further toxicity studies were performed using the same methodology as described above in Example 2. The results are shown in FIGS. 11 and 12.

[0109] FIG. 11 shows dose-dependent toxicity (% control surviving) of various ratios of sugar:PEGamine nanoparticles against HSC-3 Oral SCC cells (left-hand panel) and HaCaT control keratinocytes. The IC.sub.50 of the 50:50 alpha-galactose:PEGamine nanoparticles was found to be 0.96 μg/ml (approximately 50 nM), which is much lower than the IC.sub.50 measured for the same nanoparticles against the HaCaT cells>10000 μg/ml, indicating selective anti-cancer toxicity. The 50:50 ratio alpha-galactose:PEGamine AuNPs were found to exhibit the greatest anti-cancer toxicity, followed by the 60:40 ratio alpha-galactose:PEGamine AuNPs as the next most potent.

[0110] FIG. 12 shows the results of studies investigating the ability of the nanoparticles to potentiate radiation-induced cell killing, i.e. a chemoradiotherapeutic effect. The FIG. 12 graphs show toxicity (surviving fraction; y-axis) plotted against radiation dose (Gray; x-axis) of HSC-3 cells (left-hand panel) and HaCaT cells (right-hand panel). The nanoparticle-treated HSC-3 cells exhibited enhancement of radiation-induced cell killing (approximately 1.4× for 0.3 μg/ml AuNPs; 2.8× for 0.6 μg/ml AuNPs). A much lower level of radiation potentiation was seen in the non-cancer HaCaT cells (approximately 1.0× for 0.3 μg/ml AuNPs; 1.1× for 0.6 μg/ml AuNPs).

[0111] Electron microscopy studies (TEM; not shown) demonstrate that AuNPs selectively accumulate in skin cancer cells (i.e. higher uptake by

[0112] HSC cells compared with HaCaT cells). Without wishing to be bound by any particular theory, the present inventors believe that the selective toxicity of the AuNPs to the skin cancer cells may be attributable to the selective accumulation in the skin cancer cells.

[0113] These results demonstrate that the nanoparticles of the present invention are selectively toxic for cancer cells and selectively enhance radiotherapy.

[0114] All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0115] The specific embodiments described herein are offered by way of example, not by way of limitation. Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.