GB2532449A - Use of Nanomaterials in treating cancer - Google Patents

Abstract

A nanomaterial for use in the treatment, prophylaxis, or prevention of cancer by inhibiting the proliferation of cancer stem cells, where the nanomaterial is functionalised by at least one functional group comprising a heteroatom selected from O, S, and N. Preferably the functional group is selected from one or more of: carbonyl, carboxyl, expoxide, hydroxyl, amino, amido, imino, oximo, ether, ester, guanidine, and hydroxylamine. Preferably the nanomaterial is a carbon nanomaterial, especially one based on mono-layer graphene, few-layer graphene, nano-graphite, single-wall or multi-wall carbon nanotubes, fullerenes, carbon nano-horns, carbon nano-fibres, or amorphous or partially amorphised nano-carbons or mixtures thereof Preferably, the nanomaterial is graphene oxide. The graphene oxide may be produced by a modified Hummers method. Preferably the use of the nanomaterial is in combination with another form of cancer treatment selected from one or more of: administration of one or more chemotherapeutic agents, radiotherapy, or surgery.

Description

Use of Nanomaterials in Treating Cancer [0001] This invention relates to methods of treating cancer, preventing cancer and preventing recurrence of cancer. The methods involve the use of nanomaterials, e.g. graphene oxide, to inhibit cancer stem cell proliferation and to promote cancer stem cell differentiation.

BACKGROUND

[0002] Cancer stem cells (CSCs), or tumor-initiating cells (TICs), are thought to be resistant to most conventional anti-cancer therapies, and have been implicated in cancer treatment failure, tumor recurrence and distant metastasis. Thus, residual treatment-resistant cancer stem cells are believed to be responsible for poor clinical outcome in most cancer types. Since CSCs are relatively rare and elusive, very little is known about them, especially regarding their physiology and metabolic phenotype.

[0003] Consistent with the idea that CSCs are resistant to cellular stress, they are able to undergo anchorage-independent growth in low-attachment plates, allowing the formation of 3D spheroids with the properties of cancer stem cells and/or progenitor cells. Under these suspension conditions, most epithelial cancer cells undergo a specialized form of cell death/apoptosis, termed anoikis. Therefore, the preparation of 3D spheroid cultures provides a functional assay to enrich for a population of cells with an epithelial stem cell-like phenotype. In this regard, the behavior of 3D spheroids (also known as mammosphere cultures) prepared from primary breast cancer cells or breast cancer epithelial cell lines are the most well-characterized.

[0004] If a means could be found for the effective targeting of CSCs it would offer the possibility of improved outcomes in cancer treatment. It would find use in the treatment of cancer, in cancer prevention, in preventing the recurrence of cancer and in preventing the metastatic spread of cancer.

[0005] The uses of graphene and other nanomaterials such as nanotubes and their functionalised derivatives in medicine have been investigated. Nanomaterials have been shown to selectively gain access to cancer cells.

[0006] Graphene oxide and other water soluble graphene derivatives have been used to deliver anticancer drug molecules and/or genes to tumours. Often the graphene oxide is stabilised in solution by the addition of functionalities such as polyethylene glycol (PEG) and polyethylenimine (PEI). Illustrative examples include: Zhang et al, Small, 2011, 7, 460-464; Bao et al, Small, 2011, 7, 1569-1578.

[0007] PEGylated graphene oxides have also been used to target tumours in photothermal therapy (Yang et al, Nano Lett., 2010, 10, 3318-3323; Robinson et al, J Am. Chem. Soc., 2011, 133, 6825-6831).

[0008] Graphene oxide and bacterially reduced graphene oxide have been implicated as being cytotoxic in their own right to cancer cells (Gurunathan et al, International Journal of Nanomedicine, 2013, 8, 1015-1027; Chen et al, Adv. Healthcare Mater., 2014, 3, 14861495). PEGylated graphene oxide nanosheets have been shown to inhibit breast cancer cell metastasis. The mechanism of this inhibition was the down regulation of the expression of genes involved in energy metabolism (Zhou et al., Biomaterials, 2014, 35, 9833-9843).

[0009] It is an aim of certain embodiments of this invention to provide materials for use in inhibiting CSC proliferation and, in particular, for selectively promoting CSC differentiation.

[0010] It is an aim of certain embodiments of this invention to provide materials for use in treating cancer.

[0011] It is an aim of certain embodiments of this invention to provide materials for use in preventing cancer.

[0012] It is an aim of certain embodiments of this invention to provide materials for use in preventing the recurrence of cancer.

[0013] It is an aim of certain embodiments of this invention to provide materials for use in preventing the metastatic spread of cancer.

[0014] Another aim of certain embodiments of this invention is to provide materials that can be used to treat a variety of different cancer types.

[0015] It is an aim of certain embodiments of this invention to provide materials that can be effective in treating cancer cells with mutations, e.g. those causing drug resistance.

[0016] A further aim of certain embodiments of the invention is to provide a treatment which offers reduced secondary side effects. Thus, it is also an aim that bulk cancer cells and normal tissue cells should not be affected by the treatment.

[0017] Certain embodiments of the invention achieve one or more of the above aims. BRIEF SUMMARY OF THE DISCLOSURE [0001] In a first aspect of the invention there is provided a nanomaterial for use in the treatment, prophylaxis or prevention of cancer.

[0002] Preferably, the nanomaterial comprises at least one functional group that comprises a heteroatom selected from 0, S and N. [0003] The functionalised nanomaterial is active by inhibiting the proliferation of cancer stem cells (CSCs), and in particular by promoting the differentiation of CSCs.

[0004] The efficacy of the materials of the present invention in treating cancer is due to the novel approach for the treatment. Selective targeting of the differentiation and proliferation of stem cells enables an effective treatment to be deployed. The treatment may arise by inhibiting the proliferation (e.g. the anchorage independent growth) of cancer stem cells (CSCs).

[0005] As described below, a carbon-based functionalised nanomaterial such as graphene oxide is preferably used in the treatment of cancers. Thus, in this case, the activity of the carbon nanomaterial arises because it is able to inhibit the proliferation (e.g. the anchorage independent growth) of CSCs in the treatment of cancer.

[0006] The present invention adopts a treatment of cancer which is a mutation independent approach. This phenotype approach targets the physical behaviour of the cells and as a result is applicable to a range of different cancer types. The material of the invention does not target a specific mutation. One benefit of the invention is that it precludes the problem of drug resistance which, otherwise, arises due to mutation of the target cancer cells.

[0007] The compounds of the invention may be for use in inhibiting cancer stem cell production, for use in inhibiting stem cell renewal and/or for use in promoting and/or modulating stem cell differentiation.

[0008] The use may be in the treatment, prophylaxis or prevention of a metastatic cancer. The use may be in preventing cancer. The use may be in preventing or reducing the likelihood of the recurrence of cancer.

[0009] The use may be in combination with another form of cancer treatment. This other form of cancer treatment may be administration of one or more chemotherapeutic agents, radiotherapy or surgery.

[0010] One potentially very beneficial application of invention would be to administer the nanomaterial as a lavage in surgery. Thus, the use may be as a lavage applied, after surgical removal of a tumour, to the site formerly occupied by the tumour. This will help reduce the likely incidence of regrowth of the tumour or of secondary tumours spreading to other sites.

[0011] In a second aspect of the invention is provided a method of inhibiting the proliferation, production or renewal of CSCs; the method comprising: contacting the CSCs or a region where it is possible that CSCs may be present with a nanomaterial [0012] The invention also provides a method of promoting and/or modulating cancer stem cell differentiation, the method comprising: contacting the CSCs or a region where it is possible that CSCs may be present with a nanomaterial.

[0013] Preferably, the nanomaterial comprises at least one functional group that comprises a heteroatom selected from 0, S and N. [0014] In a third aspect of the invention is provided a method of treating or preventing cancer the method comprising: administering to a subject in need thereof a therapeutically effective amount of a nanomaterial.

[0015] Preferably, the nanomaterial comprises at least one functional group that comprises a heteroatom selected from 0, S and N. [0016] The cancer may be a metastatic cancer. The method may be a method of preventing cancer. The method may be a method of preventing or reducing the likelihood of the recurrence of cancer.

[0017] The method may be a method of inhibiting the anchorage independent growth of CSCs. The inventors have surprisingly found that nanomaterials such as those described selectively inhibit the growth of cancer stem cells without inhibiting the growth of other fibroblasts. The nanomaterials also do not directly damage cancer tumour cells.

[0018] The nanomaterial may comprise a plurality of functional groups, each independently comprising a heteroatom selected from 0 and N. Exemplary functional groups include: carboxyl, carbonyl, epoxide, hydroxyl, amino, amido, imino, oximo, ether, ester, guanidine, hydroxylamine. The nanomaterial may comprise a plurality of functional groups selected from amine, carboxyl, epoxy, hydroxyl and carbonyl. The nanomaterial may therefore comprises a plurality of functional groups selected from: carboxyl, epoxy, hydroxyl and carbonyl. The nanomaterial may comprise a plurality of functional groups that comprise an oxygen atom. The nanomaterial may comprise a plurality of the same functional group, e.g. a plurality of carboxyl groups. More preferably, the plurality of functional groups comprises two or more different groups, e.g. the nanomaterial may comprise a plurality of carboxylic acid groups and a plurality of amine groups or the nanomaterial may comprise a plurality of carboxylic acid group, a plurality of epoxide groups and a plurality of carbonyl groups. It may be that the plurality of functional groups render the nanomaterial water soluble. The functional groups may be connected to the carbon nanomaterial either directly or indirectly through covalent or non-covalent means. Preferably however they are covalently attached to the carbon nanomaterial. Preferably they are directly attached to the carbon nanomaterial.

[0019] Preferably, the nanomaterial is a carbon nanomaterial. The carbon nanomaterial may be based on a scaffold selected from: mono layer graphene, few-layer graphene, nano-graphite, single-wall and multi-wall carbon nanotubes, fullerenes, carbon nano-horns, carbon nano-fibers, amorphous and partially amorphised nano-carbons and mixtures thereof.

[0020] Preferably, the carbon nanomaterial is a functionalised graphene. Thus, it may be graphene which is substituted with a plurality of functional groups, each independently comprising a heteroatom selected from 0 and N. Thus the nanomaterial may be graphene which is substituted by a plurality of functional groups selected from amine, carboxyl, epoxy, hydroxyl and carbonyl. Thus, it may be graphene which is substituted with a plurality of functional groups, each independently comprising an oxygen atom. Thus the nanomaterial may be graphene which is substituted by a plurality of functional groups selected from amine, carboxyl, epoxy, hydroxyl and carbonyl. The graphene may be substituted with a plurality of the same functional group, e.g. a plurality of carboxyl groups.

The graphene may be substituted with a plurality of two or more different functional groups, e.g. the nanomaterial may comprise a plurality of carboxylic acid groups and a plurality of amine groups or the nanomaterial may comprise a plurality of carboxylic acid group, a plurality of epoxide groups and a plurality of carbonyl groups.

[0021] The functionalised graphene may be monolayer functionalised graphene or few layer functionalised graphene or a mixture thereof [0022] The functionalised graphene may be a graphene oxide. Graphene oxide has the benefit of being non-toxic. We have found that it is selective against the growth of stem cells which give rise to a number of different cancers. The functionalised graphene may be a reduced graphene oxide. The process of reducing graphene oxide does not typically remove all of the oxygen functional groups. The oxygen content of both graphene oxide and reduced graphene oxide can be varied depending on the method used to make it.

[0023] The functionalised graphene may have an average oxygen:carbon weight ratio in the range of from 0.01:1 to 1:2. For example the range may be from 0.2:1 to 1:1.25 (this range is typical for graphene oxide). The range may be from 0.75:1 to 1.25:1 (this range is typical for graphene oxide formed by oxidising graphite in such a way that the oxidising agent can intercalate between the graphite layers). The range may be from 0.2:1 to 0.5:1 (this range is typical for graphene oxide formed by oxidising graphite in such a way that the oxidising agent cannot intercalate between the graphite layers).

[0024] The individual graphite oxide flakes typically contain predominantly carboxylic groups functional groups at the edges and epoxide, hydroxide and ketone functional groups in the central regions, on both faces of the graphene sheet. Where graphene oxide has been formed by oxidising graphite in such a way that the oxidising agent cannot intercalate between the graphite layers, the functionalisation will predominantly be around the edges of the flakes. Where graphene oxide has been formed by oxidising graphite in such a way that the oxidising agent can intercalate between the graphite layers, there will be significant amounts of functionalisation in the central regions of the flakes.

[0025] The functionalised graphene may have an average oxygen:carbon weight ratio in the range of from 0.1:1 to 0.25:1 (this range is typical for reduced graphene oxide).

[0026] Functionalised hexagonal boron nitride (hBN) is another 2-D material that could find application in the treatment of cancer in accordance with the present invention.

Hexagonal boron nitride is similar in structure to graphene.

[0027] The nanomaterial may be modified to improve its aqueous stability, e.g. to reduce or prevent aggregation and/or precipitation. Thus, the nanomaterial may be modified with water soluble polymer substituents. Examples of such polymers include polyethylene glycol, polyethylenimine and hyaluronic acid. The water soluble polymer substituents may be covalently bonded to the nanomaterial. Illustrative covalent linker groups include: ester linkers (e.g. formed from carboxylic acid groups on the nanomaterial), imine (e.g. formed from amine or carbonyl groups on the nanomaterial), amide groups (e.g. formed from amine or carboxylic acid groups on the nanomaterial) or ether linkages (e.g. formed from hydroxyl or epoxy groups on the nanomaterial). In an embodiment, the nanomaterial is not modified with water soluble polymer substituents.

[0028] The cancer may be selected from: sarcoma, carcinoma, blastoma, lymphoma and leukemia, basal cell carcinoma, medulloblastoma, rhabdomyosarcoma, chondrosarcoma, melanoma, small-cell lung cancer, non-small-cell lung cancer, B-cell lymphoma, multiple myeloma, brain cancer, esophagus cancer, breast cancer, ovarian cancer, stomach cancer, colorectal cancer, liver cancer, kidney cancer, head and neck cancer, mesothelioma, soft tissue sarcomas, bone sarcomas, testicular cancer, prostate cancer, pancreatic cancer, bone cancer, bone metastasis, acute leukemia, chronic leukemia, glioma, bladder cancer, endocrine system cancer, parathyroid gland cancer, thyroid gland cancer, cervical cancer, endometrium cancer, ovarian cancer, skin cancer, renal cell carcinoma, pituitary adenoma, spinal axis tumours, uterine cancer, gastric cancer and biliary tract cancer..

[0029] The nanomaterials may be formulated in a pharmaceutically acceptable organic solvent, e.g. DMSO, propylene glycol, NMP.

[0030] The nanomaterials may be formulated in a mixture of a pharmaceutically acceptable organic solvent (e.g. DMSO, propylene glycol, NMP) and a sterile aqueous medium.

[0031] Preferably, the nanomaterials are formulated in a sterile aqueous medium.

Exemplary, aqueous media would be one or more of: deionised water, saline (e.g. 0.9% w/v NaCI in water), phosphate buffered saline, glucose solutions (e.g. 5% w/v dextrose in water), glucose saline mixed solutions (e.g. 5% w/v dextrose and 0.9% w/v NaCI in water or 5% w/v dextrose and 0.45% w/v NaCI in water). Such aqueous sterile solutions can be used for intravenous (parenteral) administration or as a lavage for use in combination with a surgical method of cancer treatment.

[0032] Preferably, the nanomaterials are formulated as a solution in the aqueous medium. However, it is also within the scope of this invention that they are formulated as a suspension in the aqueous medium or that they are only partially dissolved in the aqueous medium, forming a mixture that is simultaneously a solution and a suspension of the nanomaterial.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which: Figure 1 shows atomic force microscopy (AFM) images showing the size distribution of flakes in the tested GO dispersions (a) big (5-20pm), (b) medium (1-3pm) and (c) small (0.1-1 pm).

Figure 2 shows the UV-Vis absorption spectra of the different flake-sizes in the tested GO dispersions, used to determine their concentration.

Figure 3 shows representative Raman spectra of the GO flakes in the tested dispersions.

Figure 4 shows that GO selectively targets cancer stem cells (CSCs) in breast cancer cells. Upper panels show inhibition data for large and small GO flakes of the anchorage-independent proliferation of MCF7 CSCs, as evidenced by inhibition of mammosphere formation. Lower panels show cell viability of the total MCF7 cell population when treated with GO (large and small flakes). An * indicates p < 0.05 (Student's t-test).

Figure 5 shows inhibition data for large GO flakes against the anchorage-independent proliferation of SKOV3 ovarian cancer cells (A), U87 glioblastoma cells (B), PC3 prostate cancer cells (C), A549 lung cancer cells (D) and pancreatic cancer cells (E). An * indicates p < 0.05 (Student's t-test).

Figure 6 shows representative images of cancer stem cells which have (right hand panels) and which have not (left hand panels) been exposed to GO dispersion (large flakes; 25 pg/mL). CSCs shown are MCF7 breast cancer cells, SKOV3 ovarian cancer cells, PC3 prostate cancer cells, U87 glioblastoma cells, A549 lung cancer cells and MIA-PaCa-2 pancreatic cancer cells.

Figure 7 shows cell viability of the total population of cancer cells as assessed using an SRB assay when treated with GO dispersion (large flakes): SKOV3 ovarian cancer cells (A), U87 glioblastoma cells (B), PC3 prostate cancer cells (C), A540 lung cancer cells (D) as well as MIA-PaCa-2 pancreatic cancer cells.

Figure 8 shows the cell viability of normal fibroblasts (hTERT-BJ1 fibroblasts) when treated with GO dispersion as assessed using an SRB assay.

Figure 9 shows the effect of GO on the differentiation of CSCs. An * indicates p < 0.05 (Student's t-test).

DETAILED DESCRIPTION

[0034] Cancer stem cells (CSCs) may also be referred to as tumorigenic stem cells or stem cell like tumorigenic cells.

[0035] Graphene consists of a planar lattice of sp2 carbon atoms, with each carbon atom bonded to three neighbouring carbons and forming a corner of three separate tessellated hexagons. A single electron (formally from a carbon p-orbital) from each carbon atom form a delocalised "sea" of electrons. Monolayer graphene consists of a single isolated layer and is a single atom thick. The term graphene is also applied to few layer graphene materials which have properties which are closer to monolayer graphene than they are to graphite. Few layer graphene is from 2 to 10 layers thick. Few layer graphene is known as graphene because its properties tend to be closer to that of monolayer graphene than to graphite. The term few layer' as used herein refers to a functionalised graphene material which is from 2 to 10 graphene layers think.

[0036] When graphene is functionalised, covalent bonds are typically formed between carbon atoms and the new functionality. The lattice becomes locally non-planar (i.e non-planar in those regions in which the functionalisation is present). The process of functionalisation can often break carbon-carbon bonds in the graphene lattice and/or can lead to the formation of tetrahedrally orientated carbon atoms in that lattice. For example, graphene oxide is considered to comprise epoxide, hydroxyl, carbonyl and carboxylic acid groups. The formation of both carbonyl and carboxylic acid groups comprise the cleaving of carbon-carbon bonds. Depending on the method used to make graphene oxide, graphene oxide can still comprise regions in which the sp2 carbon lattice is undisturbed and regions which are highly oxygenated. The functionalisation may be on the basal plane of the graphene sheet, the edge of the graphene sheet or both.

[0037] Thus functionalised monolayer graphene is a single layer of interlinked carbon atoms with substituent functional groups. Few layer functionalised graphene comprises particles with from 2 to 10 such layers.

[0038] The graphene oxide for use in this application can be made by any means known in the art. In one illustrative method, graphite oxide can be prepared from graphite flakes (e.g. natural graphite flakes) by treating them with potassium permanganate and sodium nitrate in concentrated sulphuric acid. This method is called Hummers method. Another method is the Brodie method, which involves adding potassium chlorate (KCIO3) to a slurry of graphite in fuming nitric acid.

[0039] Individual graphene oxide sheets can then be exfoliated by dissolving graphite oxide in water or other polar solvents with the help of ultrasound, and bulk residues can then be removed by centrifugation and optionally a dialysis step to remove additional salts.

[0040] A higher oxygen content can be achieved by subjecting the exfoliated graphene oxide sheets to a further oxidation step, e.g using more potassium permanganate and sodium nitrate in concentrated sulphuric acid.

[0041] Another method of obtaining higher oxygen content is to oxidise worm-like graphite rather. Worm-like graphite is graphite that has been treated with concentrated sulphuric acid and hydrogen peroxide at 1000C to convert graphite into an expanded "worm-like" graphite. When this expanded graphite undergoes an oxidation reaction it exhibits a higher increase the oxidation rate and efficiency (due to a higher surface area available in expanded graphite as compared to pristine graphite) and the resultant graphene oxide contains more oxygen containing functional groups than graphene oxide prepared from natural graphite.

[0042] Reduced graphene oxide is made by reducing graphene oxide. The method of reduction will influence both the oxygen content of the resultant reduced graphene oxide and the amount of defects in the graphene lattice. Graphene oxide can be reduced by heating to produce thermally reduced graphene oxide and it can be reduced by bacterial action to produce bacterially reduced graphene oxide. More preferably it can be reduced by subjecting it to a chemical reducing agent. Illustrative examples include: hydrazine, HI, HBr, ascorbic acid, NaBH4, LiAIH4 etc. [0043] For a review of the methodologies available for both producing and reducing graphene oxide see Dreyer et al. The chemistry of graphene oxide, Chem. Soc. Rev., 2010, 39, 228-240. The methodology is disclosed in that reference are applicable to the present invention.

[0044] The nanomaterial may be used a sole therapy or as a combination therapy with an additional active agent. Thus the method of treatment may involve administration of the nanomaterial on its own or in combination with an additional active agent. Optionally, the additional active agent may be an anti-tumour agent selected from the list below.

[0045] Such chemotherapy may include one or more of the following specific anti-cancer agents listed below or anti-cancer agents from one or more of the categories of listed below:- (i) antiproliferative/antineoplastic drugs and combinations thereof, such as alkylating agents (for example cis-platin, oxaliplatin, carboplatin, cyclophosphamide, nitrogen mustard, bendamustin, melphalan, chlorambucil, busulphan,capecitabine temozolamide, ifosamide, mitobronitol, carboquone, thiotepa, ranimustine, nimustine, AMD-473, altretamine, AP-5280, apaziquone, brostallicin, carmustine, estramustine, Is fotemustine, gulfosfamide, KW-2170, mafosfamide, mitolactol, etaplatin, lobaplatin, nedaplatin, strrplatin and nitrosoureas); antimetabolites (for example gemcitabine and antifolates such as fluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed, methotrexate, pemetrexed, cytosine arabinoside, 6-mercaptopurine riboside, leucovarin, UFT, doxifluridine, carmoflur, cytarabine, enocitabine S-1, 5-azacitidine, cepecitabine, clofarabine, decitabine, eflornithine, ethynlcytidine, TS-1, nelarabine, nolatrexed, ocosfate, pelitrexol, triapine, trimetrexate, vidarabine, and hydroxyurea); antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin, mithramycin, aclarubicin, actinomycin D, amrubicin, annamycin, elsamitrucin, galarubicin, nemorubicin, neocarzinostatin, peplomycin, piarubicin, rebeccamycin, stimalamer, streptozocin, valrubicin and zinostatin); antimitotic agents (for example vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine and taxoids like taxol, docetaxol (Taxotere), and paclitaxel and polokinase inhibitors); proteasome inhibitors, for example carfilzomib and bortezomib; interferon therapy; and topoisomerase inhibitors (for example epipodophyllotoxins like etoposide and teniposide, aclarubicin, amonafide, belotecan, 10-hydroxycamptothecin, 9-aminocamptothecin, diflomotecan, edotecarin, exatecan, gimatecan, lurtotecan, pirarubicin, pixantrone, rubitecan, sobuzoxane, SN-38, tafluposide, amsacrine, topotecan, mitoxantrone and camptothecin) and adjuvants used in combination with these therapies, for example folinic acid; (ii) cytostatic agents such as antioestrogens (for example tamoxifen, fulvestrant, toremifene, raloxifene, droloxifene, lasofoxifeneand iodoxyfene), antiandrogens (for example bicalutamide, mifepristone, flutamide, nilutamide, casodex and cyproterone acetate), LHRH antagonists or LHRH agonists (for example goserelin, leuprorelin and buserelin), progestogens (for example megestrol acetate), aromatase inhibitors (for example as anastrozole, letrozole, vorazole and exemestane) and inhibitors of 5 a-reductase such as finasteride; (iii) anti-invasion agents, for example dasatinib and bosutinib (SKI-606), and metalloproteinase inhibitors, inhibitors of urokinase plasminogen activator receptor function or antibodies to Heparanase; (iv) inhibitors of growth factor function: for example such inhibitors include growth factor antibodies and growth factor receptor antibodies, for example the anti-erbB2 antibody trastuzumab [Herceptin'"'], the anti-EGFR antibody panitumumab, the anti-erbB1 antibody cetuximab, tyrosine kinase inhibitors, for example inhibitors of the epidermal growth factor family (for example EGFR family tyrosine kinase inhibitors such as gefitinib, erlotinib and 6-acrylamido-N-(3-chloro-4-fluorophenyI)-7-(3-morpholinopropoxy) -quinazolin-4-amine (CI 1033), erbB2 tyrosine kinase inhibitors such as lapatinib); ErbB2 inhibitors (for example GW-28297, Herceptin, 2C4, pertuzumab, TAK-165, GW-572016, AR-209, and 2B-1); inhibitors of the hepatocyte growth factor family; inhibitors of the insulin growth factor family; modulators of protein regulators of cell apoptosis (for example Bcl-2 inhibitors); inhibitors of the platelet-derived growth factor family such as imatinib and/or nilotinib (AMN107); inhibitors of serine/threonine kinases (for example Ras/Raf signalling inhibitors such as farnesyl transferase inhibitors, for example sorafenib, tipifarnib and lonafarnib), inhibitors of cell signalling through MEK and/or AKT kinases, c-kit inhibitors, abl kinase inhibitors, PI3 kinase inhibitors, Plt3 kinase inhibitors, CSF-1R kinase inhibitors, IGF receptor, kinase inhibitors; aurora kinase inhibitors and cyclin dependent kinase inhibitors such as CDK2 and/or CDK4 inhibitors; (v) antiangiogenic agents such as those which inhibit the effects of vascular endothelial growth factor, [for example the anti-vascular endothelial cell growth factor antibody bevacizumab (AvastinTM); COXII inhibitors (for example Arcoxia (etoricoxib), Bextra (valdecoxib), Celebrex (celecoxib), Paracoxib Vioxx (rofecoxib)); MMP inhibitors (for example MMP-2 inhibitors, MMP-9 inhibitors, AG-3340, RO 32-3555, and RS 13-0830); thalidomide; lenalidomide; and for example, a VEGF receptor (for example SU-11248, SU-5416, SU-6668, and angiozyme) tyrosine kinase inhibitor (such as vandetanib, vatalanib, sunitinib, axitinib and pazopanib); acitretin; fenretinide; zoledronic acid; angiostatin; aplidine; cilengtide; A-4; endostatin; halofuginome; rebimastat; removab; revlimid; squalamine; ukrain; and vitaxincombretastatin; (vi) gene therapy approaches, including for example approaches to replace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2; (vii) immunotherapy approaches, including for example antibody therapy such as alemtuzumab, rituximab, ibritumomab tiuxetan (Zevalin®) and ofatumumab; interferons such as interferon a; interleukins such as IL-2 (aldesleukin); interleukin inhibitors for example I RAK4 inhibitors; cancer vaccines including prophylactic and treatment vaccines such as HPV vaccines, for example Gardasil, Cervarix, Oncophage and Sipuleucel-T (Provenge); interferons, such as interferon alpha, interferon alpha-2a, interferon alpha-2b, interferon beta, interferon gamma-la, and interferon gamma-n; PF3512676; Filgrastim (Neupogen); lentinan; sizofilan; TheraCys; ubenimex; WF-10; BAM-002; dacarbazine; daclizumab; denileukin; gemtuzumab; ozogamicin; imiquimod; lenograstim; melanoma vaccine (Corixa); molgramostim; OncoVAX-CL; sargramostim; tasonermin; tecleukin; thymalasin; tositumomab; Virulizin; Z-100; epratuzumab; mitumomab; oregovomab; pemtumomab; and toll-like receptor modulators for example TLR-7 or TLR-9 agonists; and (viii) cytotoxic agents for example fludaribine (fludara), cladribine, pentostatin (NipentTM), edotecarin, SU-11248, paclitaxel, Erbitux, and irinotecan; (ix) steroids such as corticosteroids, including glucocorticoids and mineralocorticoids, for example aclometasone, aclometasone dipropionate, aldosterone, amcinonide, beclomethasone, beclomethasone dipropionate, betamethasone, betamethasone dipropionate, betamethasone sodium phosphate, betamethasone valerate, budesonide, clobetasone, clobetasone butyrate, clobetasol propionate, cloprednol, cortisone, cortisone acetate, cortivazol, deoxycortone, desonide, desoximetasone, dexamethasone, dexamethasone sodium phosphate, dexamethasone isonicotinate, difluorocortolone, fluclorolone, flumethasone, flunisolide, fluocinolone, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluorocortisone, fluorocortolone, fluocortolone caproate, fluocortolone pivalate, fluorometholone, fluprednidene, fluprednidene acetate, flurandrenolone, fluticasone, fluticasone propionate, halcinonide, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone aceponate, hydrocortisone buteprate, hydrocortisone valerate, icomethasone, icomethasone enbutate, meprednisone, methylprednisolone, mometasone paramethasone, mometasone furoate monohydrate, prednicarbate, prednisolone, prednisone, tixocortol, tixocortol pivalate, triamcinolone, triamcinolone acetonide, triamcinolone alcohol and their respective pharmaceutically acceptable derivatives. A combination of steroids may be used, for example a combination of two or more steroids mentioned in this paragraph; (x) targeted therapies, for example PI3Kd inhibitors, for example idelalisib and perifosine; (xi) and additional active agents such as estramustine phosphate, fludarabine phosphate, farnesyl transferase inhibitors, PDGFr, streptozocin, strontium-89, suramin, hormonal therapies (for example Lupron, doxercalciferol, fadrozole, formestane and trelstar), supportive care products (for example, Filgrastim (Neupogen), ondansetron (Zofran), Fragmin, Procrit, Aloxi and Emend), biological response modifiers (e.g. Krestin, lentinan, sizofiran, picibanil and ubenimex), alitretinoin, ampligen, atrasenten, bexarotene, bosentan, calcitriol, exisulind, fotemustine, ibandronic acid, miltefosine, 1-asparaginase, procarbazine, dacarbazine, hydroxycarbamide, pegaspargase, tazarotne, TLK-286, Velcade, Tarceva, tretinoin.

[0046] Alternatively or additionally, the compound of the invention may be administered in combination with at least one additional agent having activity other than anticancer, e.g. having an activity selected from anti-inflammatory, anti-emetic, antibiotic, antiviral, and anaesthetic.

[0047] The combination therapies defined above may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment.

Such combination products may employ the nanomaterials within a therapeutically effective dosage range and the other pharmaceutically-active agent within its approved dosage range.

[0048] Where the nanomaterials are administered with at least one other active agent, e.g. anticancer agent, that agent or at least one of those agents may be associated with the nanomaterial. Thus, at least one other active agent may be bonded to the nanomaterial by covalent bonds or it may be held in association with the nano-material by Van der Waals, hydrogen bonding and/or ri--rr interactions. Thus, the nanomaterials may act both as an inhibitor of CSC growth and as a means for delivery of another active agent.

[0049] The nanomaterials may be used in combination with conventional surgery. A particular application of invention would be to administer the nanomaterial as a lavage during or after surgery. Thus, the use may be as a lavage applied, during and/or after surgical removal of a tumour, to the site of the tumour and/or formerly occupied by the tumour.

[0050] The nanomaterials may be used in combination with chemotherapy, radiotherapy, phototherapy, photothermal therapy, hormone therapy, gene therapy, and any other conventional means of treating cancer.

[0051] The use may be in combination with another form of cancer treatment. This other form of cancer treatment may be administration of one or more chemotherapeutic agents, radiotherapy or surgery. A particular application of invention would be to administer the nanomaterial as a lavage in surgery. Thus, the use may be as a lavage applied, after surgical removal of a tumour, to the site formerly occupied by the tumour.

[0052] The nanomaterial may be in the form of a salt. For a review on suitable salts, see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002). Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 1 5-naphthalenedisulfonate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate, stearate, succinate, tartrate, tosylate, trifluoroacetate salts and mixtures thereof. The nanomaterials may comprise amine functionalities. Where this is the case, the amines may be in the form of the free base or they may be in the form of acid addition salts. Where the nanomaterials comprise amine functionalities, the nanomaterials will typically comprise a plurality of amine functionalities. Thus, it may be that all of the amine functionalities are in the form of a free base or all of the amine functionalities are in the form of acid addition salts. Typically, however, a portion of the amine functionalities will be in the form of a free base and a portion of the amine functionalities will be in the form of acid addition salts.

[0053] Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts and mixtures thereof. For a review on suitable salts, see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002). The nanomaterials may comprise carboxylic acid functionalities. Where this is the case, the carboxylic acids may be in the form of the free acid or they may be in the form of base addition salts. Where the nanomaterials comprise carboxylic acid functionalities, the nanomaterials will typically comprise a plurality of carboxylic acid functionalities. Thus, it may be that all of the amine functionalities are in the form of a free acid or all of the amine functionalities are in the form of base addition salts. Typically, however, a portion of the carboxylic acid functionalities will be in the form of a free acid and a portion of the carboxylic acid functionalities will be in the form of base addition salts.

[0054] The dosage of nanomaterial administered will, of course, vary with the compound employed, the mode of administration, the treatment desired and the disorder indicated.

For example, if the nanomaterial is administered orally, then the daily dosage of the compound of the invention may be in the range from 0.01 micrograms per kilogram body weight (fag/kg) to 100 milligrams per kilogram body weight (mg/kg).

[0055] The nanomaterial may be used on its own but will generally be administered in the form of a pharmaceutical composition in which the nanomaterial is in association with a pharmaceutically acceptable adjuvant, diluent or carrier. Conventional procedures for the selection and preparation of suitable pharmaceutical formulations are described in, for example, "Pharmaceuticals -The Science of Dosage Form Designs", M. E. Aulton, Churchill Livingstone, 1988.

[0056] Depending on the mode of administration of the nanomaterials, the pharmaceutical composition which is used to administer the nanomaterials will preferably comprise from 0.05 to 99 %w (per cent by weight) nanomaterials, more preferably from 0.05 to 80 %w nanomaterials, still more preferably from 0.10 to 70 %w nanomaterials, and even more preferably from 0.10 to 50 %w nanomaterials, all percentages by weight being based on total composition.

[0057] The pharmaceutical compositions may be administered topically (e.g. to the skin) in the form, e.g., of creams, gels, lotions, solutions, suspensions, or systemically, e.g. by oral administration in the form of tablets, capsules, syrups, powders or granules; or by parenteral administration in the form of a sterile solution, suspension or emulsion for injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion); by rectal administration in the form of suppositories; or by inhalation in the form of an aerosol.

[0058] For oral administration the nanomaterials may be admixed with an adjuvant or a carrier, for example, lactose, saccharose, sorbitol, mannitol; a starch, for example, potato starch, corn starch or amylopectin; a cellulose derivative; a binder, for example, gelatine or polyvinylpyrrolidone; and/or a lubricant, for example, magnesium stearate, calcium stearate, polyethylene glycol, a wax, paraffin, and the like, and then compressed into tablets. If coated tablets are required, the cores, prepared as described above, may be coated with a concentrated sugar solution which may contain, for example, gum arabic, gelatine, talcum and titanium dioxide. Alternatively, the tablet may be coated with a suitable polymer dissolved in a readily volatile organic solvent.

[0059] For the preparation of soft gelatine capsules, the nanomaterials may be admixed with, for example, a vegetable oil or polyethylene glycol. Also liquid or semisolid formulations of the nanomaterials may be filled into hard gelatine capsules. Liquid preparations for oral application may be in the form of syrups or suspensions, for example, solutions containing the compound of the invention, the balance being sugar and a mixture of ethanol, water, glycerol and propylene glycol. Optionally such liquid preparations may contain colouring agents, flavouring agents, sweetening agents (such as saccharine), preservative agents and/or carboxymethylcellulose as a thickening agent or other excipients known to those skilled in art.

[0060] Preferably, however, as mentioned elsewhere in this specification, the nanomaterials are formulated for intravenous (parenteral) administration, e.g. as a sterile aqueous or oily solution.

[0061] The size of the dose for therapeutic purposes of compounds of the invention will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well known principles of 10 medicine.

[0062] Dosage levels, dose frequency, and treatment durations of compounds of the invention are expected to differ depending on the formulation and clinical indication, age, and co-morbid medical conditions of the patient. The standard duration of treatment with compounds of the invention is expected to vary between one and seven days for most clinical indications. It may be necessary to extend the duration of treatment beyond seven days in instances of recurrent infections or infections associated with tissues or implanted materials to which there is poor blood supply including bones/joints, respiratory tract, endocardium, and dental tissues.

[0063] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of them mean "including but not limited to", and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0064] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments.

The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0065] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

EXAMPLES

Example 1 -Preparation and characterisation of araphene oxide suspensions [0066] Graphene oxide was prepared by using the Hummers method with modifications. The advantage of using the modified Hummers method is the safety of the reaction and the effectiveness of the oxidation, exfoliation and purification of the reaction products. Specifically, 22.5 g KM nat were slowly added to the mixture of natural flakes graphite (5g), KNO3 (4.5 g) and concentrated H2SO4 (169 mL) at -0° C while maintaining permanent stirring. The mixture was then kept at room temperature for 7 days. During this step the KNO3 along with H2SO4 and KM nO4 promote the oxidation of graphite through the specific Mn-based oxidizing agent species. The oxidizing species attack the graphite by intercalation, oxidation and expansion of the layers. With the extent of oxidation, the mixture became thick and dark brown. The dark brown mixture is then slowly diluted with 5 wt% H2SO4 (550 mL). After 3 hours, H202 (15g, 30 vol%) is gradually added leading to a considerable effervescence. The light yellow resulting suspension is stirred for another 2 hours and further diluted with a mixture of 3 wt% H2SO4 -0.5 wt % H202 (500 mL). During the above step, the residual permanganate and manganese dioxide is reduced to colourless manganese sulphate. The reaction by-products are then removed by washing the mixture with a mixture of 3 wt% H2SO4 and 0.5 wt% H202 and then deionised water followed by centrifugation. After washing and centrifugation the supernatant was discarded and the brown viscous fraction redispersed by vigorous shaking. The final remaining mixture was brown-orange, with a gel-like consistency and free of any visible particles. The resulted gel was partially exfoliated graphite oxide. The individual graphite oxide flakes contain carboxylic groups mainly at the edges, and epoxides, hydroxides and ketones groups mainly on the both planar sides. The C to 0 ratio varies from batch to batch but is typically slightly lower or slightly higher than 1.

[0067] A certain amount of partially exfoliated graphite gel of was diluted with deionised water and sonicated in an Elmasonic P7OH sonicator at 50% sonication power for 30 min to complete the exfoliation process of graphite oxide into graphene oxide monolayers.

[0068] The graphene oxide flakes of different sizes were separated by centrifuging graphene oxide suspension at various rpm and collecting different phases of the suspension to provide three different suspensions, each containing different size flakes: (a) big (5-20pm), (b) medium (1-3pm) and (c) small (0.1-1 pm). The GO flake sizes in each dispersions were estimated by spin-casting the graphene dispersion on a Si/SiO2 substrate to yield a sub-monolayer film, followed by atomic force microscopy imaging. The flake size distribution was estimated from visual inspection of 10 AFM images per each sample of 20pm scan. Representative AFM images are shown in Figure 1B. The AFM characterization of graphene oxide flakes was performed on a Bruker Dimension FastScan AFM system by using taping mode. The substrates were prepared by spin-casting the suspension on a Si/SiO2 substrate to yield monolayer film, followed by AFM imaging.

[0069] The concentration of the GO dispersions was determined by employing UV-Vis spectroscopy. The Figure 2 shows the UV-Vis absorption spectra of the different flake-sizes GO dispersions. All spectra show characteristic absorption of GO, with a peak at -230nm and a shoulder at -300nm assigned to the Tr-us transitions of C=C and n-n* of C=0 bonds respectively. The spectra were measured by diluting the initial GO dispersions with deionised (DI) water in the ratio DI:GO of 5:0.1, 15:0.1 and 5:0.5 for big, medium and small flakes dispersions, respectively. The dispersions were diluted to give an absorption intensity lower than 1. The concentrations of the dispersions were then determined from the absorption intensity at 230nm by using the Beer-Lambert law: A=c/c, where A is the absorption value, c is the mass extinction coefficient and / is the path length (0.01m). The E value for GO dispersed in water was estimated to be 4185 mL.mg-l.m-1 and was determined from the calibration curve of a GO suspension with a known concentration (data not shown here). The concentrations of the initial GO suspensions were estimated to be 0.94 mg/mL, 5.51 mg/mL and 0.26 mg/mL for the big, medium and small flakes, respectively. The UV-Vis spectra were recorded in 10 mm path length quartz cells using a PerkinElmer Lambda -1050 UV-Vis-NIR spectrometer.

[0070] The GO dispersions were characterised by Raman spectroscopy using a 633nm excitation wavelength and a laser spot-size of 700nm. Representative spectra are provided in Figure 3. Raman spectral maps were obtained on GO flakes coated on Si/SiO2 substrates, as for AFM. Raman spectra of GO is characterised by 3 peaks, the G peak and 2D peak being representative of the pristine graphene structure and the D peak arising from the defects induced in the graphene structure by oxidation. All three GO dispersions yield identical Raman spectra indicating similar extents of oxidation. The Raman spectra were taken with a Renishaw inVia confocal Raman microscope, equipped with Leica microscope and CCD detector.

Example 2 -Activity of graphene oxide against CSCs [0071] A single cell suspension was prepared using enzymatic (lx Trypsin-EDTA, Sigma Aldrich, #T3924), and manual disaggregation (25 gauge needle) to create a single cell suspension. Cells were plated at a density of 500 cells/cm2 in mammosphere medium (DMEM-F12/B27/20ng/m1EGF/PenStrep) in non-adherent conditions, in culture dishes coated with (2-hydroxyethylmethacrylate) (poly-HEMA [Sigma]). Cells were grown for 5 days and maintained in a humidified incubator at 37°C at an atmospheric pressure in 5% (v/v) carbon dioxide/air. After 5 days for culture, spheres >50mm were counted using an eye piece graticule, and the percentage of cells plated which formed spheres was calculated and is referred to as percentage mammosphere formation, and was normalized to one (1 = 100 %MSF).

[0072] Figure 4 (upper panels) shows that GO (both large and small flakes) inhibits the anchorage-independent proliferation of MCF7 CSCs, as evidenced by inhibition of mammosphere formation. Figure 5 shows that GO (large flakes) inhibits the anchorage-independent proliferation of SKOV3 ovarian cancer cells (A), U87 glioblastoma cells (B), PC3 prostate cancer cells (C), A549 lung cancer cells (D), as well as pancreatic cancer cells (E), in a concentration-independent manner. Figure 6 shows representative images of the spheroid assays, the numerical data for which is provided in Figure 5.

[0073] These results indicate that GO inhibits sphere formations of a range of cancer types. This suggests that the GO targets the physical behaviour of the cells rather than any specific receptor and as such could offer a mutation independent approach of treating cancer which might be expected to be effective against a range of different cancer types.

[0074] Example 3 -Activity of graphene oxide against cancer cell populations and normal fibroblasts [0075] A Sulforhodamine B (SRB) Assay system measures total biomass by staining cellular proteins with Sulforhodamine B. Cells were seeded in 96-well plates and incubated in a 5% CO2 atmosphere at 37°C. After cells were attached to the bottom of the plates, they were treated with graphene oxide at the indicated concentrations. After 3-5 days of treatment, media was removed, and cells were rinsed with PBS and fixed in 10% tricloroacetic acid for 1 hour at 4°C. Then, cells were washed carefully and all PBS was removed to allow cells to dry. SRB was then added to the cells for 15 minutes at room temperature. Cells were washed with 1% acetic acid and the incorporated dye was solubilized from the cells with 10 mM Tris Base solution at pH 8.8. The absorbance was spectrophotometrically measured at 540 nm in a plate reader. Background measurements were subtracted from all values.

[0076] This methodology was used to test the effect of GO on the cell viability of a range of cancer cells and of hTERT-BJ1 fibroblasts. All the cells were purchased from ATCC.

[0077] Figure 4 (lower panels) shows that GO (large and small flakes) does not affect cell viability of the total MCF7 cell population. Likewise, Figure 7 shows that GO does not affect cell viability of the total population of SKOV3 ovarian cancer cells (A), U87 glioblastoma cells (B), PC3 prostate cancer cells (C), A540 lung cancer cells (D) and MIA-PaCa-2 pancreatic cancer cells. Cell viability was assessed using an SRB assay.

[0078] Figure 8 shows that GO does not affect the cell viability of hTERT-BJ1 fibroblasts (again, this was assessed using an SRB assay). hTERT-BJ1 fibroblasts can be considered to be representative of the cell population of normal fibroblasts.

[0079] These results indicate that graphene oxide's activity against CSCs is unusually selective.

Example 4 -The effect of GO on the differentiation of CSCs.

[0080] MCF7 cells were treated as monolayer cultures with small or large GO (50pg/m1) for 48 hours or left untreated (vehicle alone control). Then, cells were trypsinised and plated on low-attachment plates for 10 hours to induce anoikis and enrich for cancer stem cells. Single cells were then analysed by FACS to quantitate the CD44(+)CD24-/low population, which represents the cancer stem cells. The results are shown in Figure 9.

[0081] Figure 9A. -Note that, as expected, the CD44(+)CD24-/low population (corresponding to the population of CSCs) is greatly enriched after 10 hours in low-attachment conditions (vehicle alone control).

[0082] Figure 9B. -Interestingly, GO does not reduce the total number of anoikisresistant cells (data not shown), but rather induces the expression of CD24, thereby significantly reducing the CD44(+)CD24-/low population.

[0083] Results: Consistent with decreased stemness, MCF7 CSCs show a shift towards differentiation upon GO treatment and re-express CD24, which is low/negative in bona-fide breast CSCs [0084] Thus, GO appears to inhibit mammosphere formation (i.e. stem cell proliferation) by promoting stem cell differentiation.