NZ616780B2 - A method for the treatment of a solid tumour - Google Patents

Abstract

Disclosed herein is the use of two separate formulations of a particulate material, and a cellular toxin, effective to penetrate a solid tumour and induce cytotoxicity, in the manufacture of a medicament for the treatment of a solid tumour, wherein said particulate material is in the form of a dispersion in a liquid carrier, the particulate material being maintained in the dispersed state by a steric stabiliser; wherein said steric stabiliser comprises an anchoring portion and a steric stabilising polymeric segment, wherein the anchoring portion is different from the steric stabilising polymeric segment; and wherein the anchoring portion has an affinity towards the surface of the particulate material and secures the stabiliser to the particulate material. rsion in a liquid carrier, the particulate material being maintained in the dispersed state by a steric stabiliser; wherein said steric stabiliser comprises an anchoring portion and a steric stabilising polymeric segment, wherein the anchoring portion is different from the steric stabilising polymeric segment; and wherein the anchoring portion has an affinity towards the surface of the particulate material and secures the stabiliser to the particulate material.

Description

A METHOD FOR THE TREATMENT OF A SOLID TUMOUR . 1 - A METHOD OF TREATMENT AND AGENTS USEFUL FOR SAME FIELD OF THE ION The present invention relates generally to a method of treating a neoplastic condition and to agents useful for same. More ularly, the present invention is directed to a method of facilitating the treatment of a solid tumour in a sed manner via. the co-administration of particulate material and a cellular toxin. The method of the present invention is useful in a of primary and metastatic s. range of therapeutic treatments including the treatment l0 BACKGROUND OF THE INVENTION Bibliographic details of the ations referred to by the author in this specification are ted alphabetically at the end of the description.

The reference in'this specification to any prior publication (or ation derived from it), or to any matter which is known, is not, and should not be taken as an ‘ ledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Malignant tumours, or cancers, grow in an uncontrolled manner, invade normal tissues, and often metastasize and grow at sites distant from the tissue of origin. In general, cancers are derived from one or only a few normal cells that have undergone a poorly understood process called malignant transformation. Cancers can arise from almost any tissue in the body.

Those derived from epithelial cells, called carcinomas, are the most common kinds of cancers.

Sarcomas are malignant tumours of mesenchymal tissues, arising from cells such as fibroblasts, muscle cells, and fat cells. Solid malignant tumours of lymphoid tissues are called lymphomas, and marrow and blood-borne malignant tumours of lymphocytes and other hematopoietic cells are called leukaemias.

Cancer is one of the three leading causes of death in industrialised nations. As treatments for infectious es and the prevention of cardiovascular disease ues to improve, and the average life expectancy ses, .cancer is likely to become the most common fatal disease in these countries. ore. successfully treating cancer requires that cells be removed or destroyed without killing the An ideal way to . all the malignant patient. achieve this would be to induce an immune response against the tumour that would W0 2012/142669 discriminate between the cells of the tumour and their normal cellular counterparts. r, immunological approaches to the treatment of cancer have been attempted for over a century with unsustainable results.

Accordingly, current methods of treating cancer continue to follow the long used protocol of surgical excision (if possible) followed by radiotherapy and/or chemotherapy, if ary. The success rate of this rather crude form of treatment is extremely variable but generally decreases significantly as the tumour becomes more advanced and metastasises.

Further, these treatments are associated with severe side effects ing disfigurement and scarring from y (cg. mastectomy or limb amputation), severe nausea and vomiting from chemotherapy, and most significantly, the damage to normal tissues such as the hairfollicles, gut and bone marrow which is induced result of the relatively nonospecific targeting ‘ as a mechanism of the toxic drugs which form part of most cancer treatments and is a major limiting factor for dosage Still further, common chemotherapy drugs do not significantly penetrate into tissue further than about 70 microns from the blood supply (Primeau er 0]. Clin. Cane. Res. 2005, llz8782-8788; nton et al. Nat. Rev. Cancer‘ 2006, 6:583-592). The rapid growth and poor vascular development of most solid tumours puts many tumour cells well beyond the capacity of the drugs to penetrate the tissue. Critically, many cells experience sub-lethal doses. allowing them to survive and to develop drug resistance.

Solid tumours cause the greatest number of deaths from cancer and mainly comprise tumours of the linings of the bronchial tree and the alimentary tract that are knOWn as carcinomas. in the year 2000 in Australia. cancer ted for 30% of male deaths and 25% of female deaths r in Australia 2000, 2003) and it accounted for 24% of male and 22% of female deaths in the US in year 2001 (Arias et a1. 2003,.Naliona! Vital Statistics Reports 52:|1 l-llS). Solid tumours are not usually curable once they have spread or ‘metastasised’ hout the body. The prognosis of atic solid tumours has improved only marginally in the last 50 years. The best chance for the cure of a solid tumour remains in the use of local treatments such as surgery and/or radiotherapy when the solid tumour is sed to its originating lining ~and has not spread either to the lymph nodes that drain the tumour or elsewhere. Nonetheless, even at this early stage, and particularly if the tumour has spread to the draining lymph nodes, microscopic deposits of cancer known as etastases may have y spread throughout the body and will subsequently lead to the death of the patient. In WO 42669 this sense. cancer is a systemic disease that requires systemically stered treatments. Of the patients who receive surgery and/or herapy as definitive local treatment for their primary tumour and who have micrometastases, a minor proportion may be cured or at least achieve a durable remission from cancer by the addition of adjuvant systemic ents such I ' as cytotoxic chemotherapy er hormones.

Conventionally, solid cancer has been treated locally with surgery and/or radiotherapy, and during its metastatic stage with systemically administered cytotoxic drugs, which often interfere with the cell cycle of both normal and malignant cells. The relative selectivity of this approach for the treatment of malignant tissues is based to some extent on the more rapid of recovery of normal tissues from xic drug damage. More recently, the targeted therapy cancer has aimed to improve the therapeutic ratio of cancer treatment by enhancing its specificity and/or precision of delivery to malignant tissues while minimising adverse consequences to normal non-malignant tissues. Two of the major classes of targeted therapy are (i) the small molecule tors such as the tyrosine kinase inhibitors imatinib mesylate (Glivece’), ib a®) and erlotinib (Tarceva®), and (ii) the monoclonal antibodies (mAb) such as rituximab (Mabthera®) and trastuzumab (Herceptin®). in parallel to the development of targeted therapies, combining at least two conventional anti-cancer treatments such as chemotherapy and radiotherapy in novel ways has been another approach to the development of cancer eutics. By ting synergistic interactions between the different modalities of treatment, ed modality ent seeks to improvo treatment efficacy so that the therapeutic. ratio for the combined treatment is superior to that for each of the individual treatments.

Combined» modality treatment using external beam radiation and radiosensitising chemotherapeutic drugs such as 5-fluorouracil and cisplatin (chemoradiotherapy) has improved survival in a number of solid tumours such as those of head and neck, lung, oesophagus, stomach, pancreas and rectum because of both ed local tumour control and reduced rates of distant failure (TS ce. Oncology (Humington) 17:23—28. 2003).

Although radiosensitising drugs increase tumour response, they also increase toxicity to adjacent normal tissues, which is ally true of the potent new generation radiosensitisers, gemcitabine and docetaxel. However, decreasing the ion volume allows cytotoxic doses of gemcitabine to be better tolerated clinically (Lawrence 2003, supra). adiotherapy may overcome mutually reinforcing resistance mechanisms, which may only manifest in vivo.

PCT/AU2012l000414 Radioimmunotherapy (RlT) is a systemic treatment that takes age of the specificity and avidity of the antigen-antibody interaction to deliver lethal doses of radiation to cells that bear the target antigen. Radio-isotopes that emit B-particles (e.g. 13‘Iodine, ”Yttrium. '88%enium, and 67Copper) are usually used to label monoclonal antibodies (mAb) for eutic applications. The energy from B-radiation is released at relatively low intensity over distances measured in millimeters (Waldmann, Science 252:1657-l662, l991; Bender er al., Cancer Research 52:121-126, 1992; O'Donoghue el al. Journal of Nuclear Medicine 36:]902-1909, I995; hs et al. International Journal of Cancer 81:985—992, 1999). Thus, nergy B-emitters such as ium are useful for the treatment of larger and heterogeneous solid tumours (Liu er al. Bioconjugaie try 12:7—34. 200i). Research interest‘in radioimmunotherapy has been reawakened because in spite of the low radiation doses delivered, significant and cted ical effects of Ri'f upon surrounding host cells have been observed (Xue er al. Proceedings of the National Academy of es of the United States of America 9921376543770, 2002). Furthermore, the lower but biologically effective dose of radiation delivered by RIT had greater cytocidal effects than a larger dose of radiation conveyed as external beam radiotherapy hova et al., PNAS l0] :l4865-l4870, 2004). Nonetheless, the efficiency of RlT as a treatment for solid tumours may be hampered by the low penetration of antibody through the tissue barriers that surround the target antigen in the tumour, which will uently extend circulatory half life of the antibody (Britz- Cunningham er al. Journal of Nuclear Medicine 44:]945-1961, 2003). Furthermore, RIT is often impeded by the heterogeneity of the target antigen’s expression within the tumour. Thus, gh RlT affords lar ing of tumour cells. the major limitation of RlT remains the toxicity that may result from large .doses of radiation that are delivered systemically in order to achieve sufficient targeting —Cunningham et al. 2003. supra; Christiansen et al.

' Molecular Cancer Therapy 3:]493-1501, 2004). Altogether, a useful therapeutic index using Rl'l‘ has proven difficult to achieve clinically (Sellers 2! al. Journal of Clinical investigation 104:1655-1661, 1999).

Tumour associated antigens, which would allow differential targeting of tumours. while sparing normal cells, have also been the focus of cancer research. gh abundant ubiquitous antigens may provide a more concentrated and accessible target for RlT, studies adopting this have been extremely limited.

The development of nanoparticle technology was also hailed as an exciting new W0 20 669 frontier in terms of the development of new and effective cancer treatments. However, although previous attempts at using particulate material, such as nanoparticles, to target s for either diagnostic or therapeutic purposes have been extensive. in the context of therapeutics there has. disappointingly, been minimal success. With stics. relatively shallow ation of the particles into the tumour has been sufficient to achieve the objective of visualising the tumour. However, in terms of the delivery ofa therapeutic agent, such shallow penetration has not been sufficient to ively deliver the agent throughout the tumour, in particular to the interior of the tumour, as is required if tumour destruction is to be achieved. in relation to therapeutics, specifically, conjugation of particles to a wide variety of different materials has so far failed to live up to the promise of achieving effective tumour penetration, this being an essential prerequisite for a therapeutic to have any chance of iveness.

Significant effort has also been made to take advantage of the enhanced permeability and retention (EPR) effect of tumours as a means to develop an ive therapeutic. t limiting the present invention to any one theOry or mode of action, this is a well described phenomenon based on the notion that certain sizes of molecules, typically liposomes or macromolecular drugs, tend to preferentially accumulate in tumour tissue. The general explanation for this phenomenon is that. in order fdr tumour cells to grow quickly, they must stimulate the production of blood vessels. VEGF and other growth factors are involved in cancer angiogenesis. Tumour cell aggregates of sizes as small as l50-200um become dependent on blood supply carried by neovasculature for their nutritional and oxygen supply.

These newly formed tumour vessels are usually al in form and ecture. They comprise -aligned defective endothelial cells with wide fenestrations, lacking a smooth muscle layer, or innervation with a wider lumen, and impaired functional receptors for angiotensin 11. Furthermore, tumour tissues usually lack effective lymphatic drainage. All ' these factors will lead to abnormal molecular and fluid transport cs, especially for macromolecular drugs. Accordingly, it has been thought that one way to achieve ive drug targeting to solid tumours is to exploit these abnormalities of tumour vasculature in terms of active and selective delivery of anticancer drugs to tumour tissues, notably defining the EPR effect of macromolecular drugs in solid tumours. Due to their large lar size, nanosized macromolecular anticancer drugs administered intravenously escape renal clearance. Often they cannot penetrate the tight endotheiial junctions of normal blood vessels, but they can WO 42669 extravasate in tumour vasculature and become trapped in the tumour vicinity. Nevertheless, the EPR effect has not been efficiently or successfully harnessed.

Various rticles have been designed which are directed to ing efficient ar endocytosis. However, even if this is achievable, the issue of tissue penetration is still The general notion of the a separate one which, to date, has not been successfully overcome. use of a nanoparticle as a vector for delivery of a drug is widely sed in the literature but. in the absence of achieving deep tumour penetration, is of limited value.

Even where effective tumour distribution of a drug is achieved (by whatever means) a further problem has been the fact that neoplastic cells within solid tumours can exhibit a slowed metabolism. This means that even if a cytotoxic drug ates to these cells. if it is not effectively metabolised'it will have a limited impact on the viability of the tumOur.

Accordingly, there is an urgent and ongoing need to develop ed systemic therapies for solid cancers, in particular metastatic cancers.

In work leading up to the present invention it has been determined that particulate material which is ined in a sed state by a stabiliser is able to achieve deeper penetration into solid tumour models than has previously been achievable using nanoparticle technology. This has enabled the development of an effective means for treating solid tumours, both primary and metastatic, based on the co-administration of a cellular toxin with the particulate material. By either sequentially or simultaneously delivering this toxin, deeper ation and therefore more extensiVe cellular exposure to the toxin is achieved. By virtue of the less effective reticuloendothelial clearance which is associated with s, a form of targeted treatment is effectively achieved. Still r, it has been observed that the toxin uptake by tumours penetrated by the particles of the present invention is effective, suggesting upregulation of tumour cell metabolism. Accordingly, the method of the present invention provides a means for achieving a more effective localised delivery and uptake of a- cellular~ toxin to a tumour and its metastases in a manner which is characterised by significantly ed outcomes and/or d side effects ve to those which would'normally be expected in the context of conventional treatment of an equivalent type of tumour. This is an extremely significant development since current protocols directed to treating metastatic disease are based on the non-targeted systemic delivery of chemotherapeutic agents.

W0 2012/142669 SUMMARY OF THE INVENTION Throughout this specification and the claims which follow. unless the t es ise, the word “comprise”, and variations such as “comprises" and “comprising", will be understood to imply the inclusion of a stated r or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

As used herein, the term “derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified. but has not necessarily been obtained directly from the Specified source. r. as used herein the singular forms of “a”, “and" and “the” include plural referents unless the context clearly dictates otherwise.

' Unless ise d, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

One aspect of the present invention is directed to a method of treating a solid tumour in a subject, said method comprising co-administering to said subject an effective amount of particulate material and a\celiular toxin for a time and under conditions sufficient to facilitate distribution of said particulate material and toxin to said tumour wherein: (i) said particulate material is administered in the form ofa dispersion in a liquid carrier, the particulate material being maintained in the dispersed state by a stabiliser; and (ii) said stabiliser comprising an anchoring portion that (a) anchors the iser to the' particulate material, and (b) is different from the remainder of the iser; and wherein said particulate materiai and toxin penetrate said solid tumour.

For ience, said particulate material that is maintained in the dispersed state by a stabiliser may herein be referred to as lised particulate material".

In one embodiment, the stabiliser is a steric stabiliser, said steric stabiliser comprising a steric ising polymeric segment and an anchoring portion, wherein the steric stabilising polymeric segment is different from the anchoring portion. and wherein the anchoring portion anchors the stabiliser to the particulate material The method may ore comprise co-administering to said subject an effective amount of particulate al and a cellular toxin for a time and under conditions sufficient to facilitate distribution of said particuiate material and toxin to said tumour wherein: (i) said particulate al is administered in the form of a dispersion in a liquid carrier, the particulate material being maintained in the dispersed state by a steric stabiliser; 2012/000414 (ii) said steric stabiliser Comprising a steric stabilising polymeric segment and an anchoring portion, wherein the steric stabilising polymeric segment is different from the anchoring portion, and wherein the anchoring portion s the stabiliser to the particulate material; and wherein said particulate al and toxin penetrate said solid tumour.

In r embodiment, said solid tumour is benign.

In a further embodiment said tumour is malignant. in yet r embodiment, said anchoring portion is an anchoring ric segment.

In that case, said stabiliser comprises an anchoring polymeric segment, or said steric stabiliser comprises a steric stabilising polymeric segment and an anchoring polymeric segment; in a further embodiment, said stabiliser comprises an anchoring portion, one or both of the stabiliser or anchoring portion being derived from one or more ethylenically unsaturated monomers that have been polymeriscd by a living polymerisation que, wherein the anchoring portion is different from the remainder of the stabiliser, and wherein the anchoring portion anchors the iser to the particulate material. According to this embodiment, the anchoring portion may be referred to as an anchoring polymeric segment.

In another ment, said steric iser comprises a steric ising polymeric segment and an anchoring polymeric segment, one or both of which are derived from one or more ethylenically unsaturated monomers that have been polymerised by a living polymerisation technique, wherein the steric stabilising polymeric segment is different from the anchoring polymeric segment, and wherein the anchoring polymeric segment anchors the stabiliser to the particulate al.

In another aspect the present invention provides a method of treating a malignant solid tumour in a subject, said method comprising co-administering to said t an effective amount of particulate material and a ar toxin for a time and under conditions sufiicienl to facilitate distribution of said particulate al and toxin to said tumour wherein: (i) said particulate material is administered in the form of a dispersion in a liquid carrier, the ulate al being maintained in the dispersed state by a iser; and (ii) said stabiliser comprising an anchoring portion that (a) anchors the stabiliser to the particulate material. and (b) is different from the remainder of the stabiliser; and wherein said particulate material and toxin penetrate said solid tumour.

Where the stabiliser is a steric stabiliser comprising a steric stabilising polymeric segment and an anchoring portion, wherein the steric stabilising polymeric segment is different W0 2012/142669 from the ing portion, the method of treating a malignant solid tumour in a subject comprises co-administering to said subject an effective amount of particulate material and a cellular toxin for a time and under conditions sufficient to facilitate distribution of said particulate material and toxin to said tumour wherein: (i) said partiCulate material is administered in the form of a dispersion in a liquid carrier, the particulate material being maintained in the dispersed state by a steric stabiliser; (ii) said steric stabiliser comprising a steric stabilising polymeric segment and an anchoring portion, n the steric stabilising polymeric segment is different from the anchoring portion, and wherein the anchoring portion anchors the stabiliser to the particulate material; and wherein said particulate material and toxin ate said solid tumour.

In one ment, said malignant solid tumour is a metastatic malignant solid tumour. Reference to “metastatic” should be understood as a reference to a tumour which either has one metastatisation or may have one metastatisation.

In another embodiment, said malignant solid tumour is a central s system tumour. retinoblastoma, neuroblastoma, paediatric tumour, head and neck cancer such as squamous cell cancer, breast and prostate cancer, lung , kidney cancers, such as renal cell adenocarcinoma, oesophagogastric , hepatocellular oma, pancreaticobiliary neoplasia, such as adenocarcinomas and islet cell tumours. colorectal cancer, cervical cancer. anal cancer, uterine or other reproductive tract , urinary tract cancer, such as of the ureter or bladder. germ cell tumour such as a testicular germ cell tumour or ovarian germ cell tumour, ovarian cancer, such as an ovarian epithelial cancer, oma of unknOWn primary, hilman immunodeficiency associated malignancy, such as Kaposi's sarcoma, lymphoma. leukemia, malignant ma, sarcoma, endocrine , such as of the thyroid gland. mesothelioma or other pleural or peritoneal tumour, neuroendocrine tumour or carcinoid By “co-administration” is meant that the stabilised particulate material and the cellular toxin are administered as separate entities in their own right. In other words, at the time of administration the stabilised particulate material and the cellular toxin are not covalently or chemically coupled to each other.

Co-administration of the stabilised particulate al and the cellular toxin in the context of the present invention includes both simultaneous and sequential administratiOn.

Simultaneous administration includes where the ised ulate material and the eeiiular toxin are present in the same formulation or in two different formulations,_but each are nevertheless administered at substantially the same time. In the case of sequential administrationpa multi-step procedure is used where the stabilised particulate material is administered in one step and the cellular toxin is administered at a different time in a separate step. The cellular toxin may be administered prior to administration of the stabilised particulate material. The time difference between administration of the stabilised ulate al and the ar toxin in sequential administration can vary. but will generally range from about 1 minute to about 4 days, for example from about l minute to about 2 hours. or item about 1 minute to about 24 hours, or from about 1 minute to about l2 hours, or from about 1 minute to about 6 hours, or from about I minute to about 3 hours, or from about 1 minute to about i hour.

In a sequential administration, the stabilised particulate material will generally be stered prior to the cellular toxin.

The particulate material and the cellular toxin may be administered by the same or different routes.

Without ng the present invention to any one theory or mode of action, once the particulate material has penetrated the tumour, effective ation of the administered celluiar toxin is aiso. achieved. it will be appreciated that it is well within the skills of the person in the art, and in light of the teaching provided herein, to select and design an administration protocol for the elements herein described.

In a further aspect there is provided a method of treating a solid tumour in a subject, said method comprising: (a) administering to said subject an effective amount of particulate. material and for a time and under conditions sufficient to facilitate distribution of said particulate material to said tumour wherein: (i) said particulate material is administered in the form of a dispersion in a liquid carrier, the particulate material being maintained in the dispersed state by a stabiliser; and (ii) said stabiliser comprising an ing portion that (a) anchors the stabiliser to the particulate material, and (b) is different from the remainder of the stabiliser; W0 42669 (b) administering to to said subject an effective amount of a ar toxin subsequently administration of said particulate material; and wherein said particulate material and toxin penetrate said solid tumour.

Where the stabiliser is a steric iser comprising a steric stabilising polymeric is ent segment and an anchoring n. wherein the steric stabilising polymeric segment from the anchoring portion, the method of treating a solid tumour in a subject comprises: (a) stering to said subject an effective amount of particulate material and for a time and under conditions sufficient to facilitate distribution of said particulate material to said tumour wherein: (i) said particulate material is administered in the form of a sion in a liquid carrier, the particulate al being maintained in the dispersed state by a steric iser; and (ii) said steric stabiliser comprising a steric stabilising polymeric segment and an anchoring portion, wherein the steric stabilising polymeric segment is different [5 from the anchoring portion, and wherein the anchoring portion anchors the stabiliser to the particulate material; and (b) administering to said subject an effective amount ofa cellular toxin subsequently to administration of said particulate al; and wherein said particulate material and toxin ate said solid tumour.

In yet another aspect there is provided a method of treating a solid tumour in a subject, said method comprising co-administering to said subject an ive amount of particulate material and a cytostatic or cytocidal agent for~a time and under conditions sufficient to facilitate distribution of said particulate material and toxin to said tumour, wherein: (i) said particulate material is administered in the form of a dispersion'in a liquid carrier, the particulate al being maintained in the sed state by a stabiliser; and (ii) said stabiliser comprising an anchoring portion that (a) anchors the stabiliser to the particulate material, and (b) is different from the remainder of the stabiliser; and wherein said particulate material and said cytostatic or cytocidal agent penetrate said solid tumour.

Where the stabiliser is a steric stabiliser comprising a steric stabilising polymeric segment and an anchoring n, wherein the steric stabilising polymeric segment is different from the anchoring portion, the method of treating a solid tumour in a subject ses co— administering to said subject an effective amount of particulate material and a cytostatic or 2012/000414 cytocidal agent for a time and under conditions sufficient to tate bution of said particulate material and toxin to said tumour, n: (i) said particulatemateriai is administered in the form of a dispersion in a liquid carrier, the particulate material being maintained in'the dispersed state by a steric stabiliser; (ii) said steric stabiliser comprising a sterie stabilising polymeric segment and an anchoring portion, wherein the steric stabilising polymeric segment is different from the ing portion, and wherein the anchoring portion anchors the stabiliser to the particulate material; and wherein said particulate al and said cytostatic or cytocidai agent penetrate said solid tumour.

Examples of cytotoxic agents include, but are not iimited to, Actinomycin D, Adriamycin, Arsenic Trioxide, Asparaginase, Bleomycin, Busulfan. sar, latinum Carmustine, Chiorambucil, Cisplatin, Corticosteroids, Colieheamicin, Cyclophosphamide, Daunorubicin, Docetaxel. Doxombicin, Epirubicin‘ Etoposide.

Fludarabine, Fluorouracii, Gemcitabina, Gemcitabine, Gemzar, Hydroxyurea, ldarubicin, lfosfamide. lrinotecan, Lomustinei lan, Mercaptomurine, Methotrexate. Mitomycin, Mitoxantrone, Oxaliplatin, Paciitaxei, Platinol, Platinex, Proearbizines Raltitrexeel. Rixin, Steroids, Streptozocin, Taxol, re, Thioguanine. Thiotepa, Tomudex, Topotecan, ‘ Treosulfan, Trihydrate, Vinblastine, Vincristine, Vindesine, Vinorelbina, Vinorelbine, duanomycin, dactinomysin, esorubisin, mafosfamide, ne arabinoside, bis« chloroethylnitrosurea, Mitomycin C, mithramycin, prednisomz, hydroxyprogesterone, testosterone. fen, dacarbazine, hexamethylmeiamine, pentamethylmelamine, amsacrine, chlorambudil, methylcyclohcxylnitrosurea, nitrogen mustards, Cyclophosphamide, 6- mercaptopurine, 6-thioguanine, cytarabine. S-azacytidine. deoxyco-i‘ormycin‘ 4- ,hydroxyperoxycyclophosphoramide, S-fluorouracil , S-fluorodeoxyuridine (S-FUdR), colchicine, trimetrexate, oside, diethylstilbestrol.

Reference to “cellular toxin" should also be understood to extend to any other molecule which is perhaps not traditionally regarded as a cytotoxic agent but heless falls 3O within the scope of the present definition on the basis that it induces cellular damage, for example DNA damage, such as nucleophosmin or agents which induce cellular damage as part of a synergistic process with another agent. Examples include tic dies, prodrugs, CHK [/2 inhibitor (such as CBP-SOI or AZD7762), histone deacetyiase inhibitor (such as W0 2012/142669 -[3_ mimetic (such as vorinostat), tumour necrosis factor d apoptosis inducing ligand or BH3 ABT737), small molecule inhibitors such as the tyrosine kinase inhibitors imatinib mesylate c‘D), gefitinib (lressaQ) and erlotinib (Tarceva®), and the monoclonal antibodies (mAb) such as mab (Mabthera®) and trastuzumab (Herceptin®). in yet another embodiment, combination treatments may include, for example. gemcitabine together with a CHK1/2 inhibitor or irinotecam together with a CHK l/2 inhibitor.

The particulate al and/or the stabiliser may be d to a ligand to effectfmore specific targeting to a . This will not necessarily be applicable in every situation but, to the extent that an appropriate target molecule exists for a given tumour, this may provide additional useful Specificity.

According to such an embodiment, there is provided a method of treating a solid tumour in a subject, said method comprising co-administcring to said subject an effective amount of particulate material and a ar toxin for a time and under conditions ent to facilitate distribution of said particulate material and toxin to said tumour, wherein: (i) said particulate material is administered in the form of a dispersion in a liquid carrier, the particulate material being maintained in the diSpersed state by a stabiliser; and (ii) said iser comprising an anchoring portion that (a) anchors the stabiliser to the particulate material, and (b) is different from the remainder of the stabiliser; wherein the particulate material and/or the stabiliser is linked, bound or otherwise associated with a ligand directed to a tumour le and wherein said' particulate material and toxin penetrate said solid tumour.

Where the stabiliser is a steric stabiliser comprising a steric stabilising polymeric segment and an anchoring portion, wherein the steric stabilising polymeric segment is different from the anchoring portion, there is also provided a method of treating a solid tumour in a t, said method comprising co-administering to said t an effective amount of particulate material and a cellular toxin for a time and under conditions sufficient to facilitate distribution of said particulate material and toxin to said tumour, wherein: (i) said particulate al is administered in the form of a dispersion in a liquid carrier, the particulate material being maintained in the dispersed state by a steric stabiliser; and (ii) said steric stabiliser comprising a steric ising ric segment and an anchoring portion, wherein the steric stabilising polymeric segment is different from W0 2012/142669 the stabiliser to the the anchoring portion, and wherein the anchoring portion anchors particulate material; wherein the particulate material and/or the steric stabiliser is linked, bound or otherwise material associated with a ligand directed to a tumour molecule and wherein said particulate and toxin penetrate said solid tumour. in yet another aspect, there isprovided the use of particulate material and a cellular wherein: toxin in the manufacture of a medicament for the treatment of a solid tumour (i) said particulate material is in the form of a dispersionin a liquid carrier, the particulate al being maintained in the dispersed state by a stabiliser; and (ii) said stabiliser comprising an anchoring portion that (a) anchors the stabiliser to the particulate al, and (b) is different from the remainder of the stabiliser; and wherein said particulate al and toxin penetrate said solid tumour.

Where the stabiliser is a steric stabiliser comprising a steric stabilising polymeric t and an anchoring portion, wherein the steric stabilising polymeric segment is ' from the anchoring n, there is provided the use of particulate material and a ar toxin in the manufacture of a medicament for the ent of a solid tumour wherein: (i) said particulate material is in the form of a dispersion in a liquid Icarrier, the particulate material being maintained in the dispersed state by a steric iser; and (ii) said steric stabiliscr comprising a steric ising polymeric segment and an anchoring portion, wherein the steric stabilising polymeric segment is different from the ing portion, and wherein the anchoring portion anchors the stabiliser to the particulate material; and wherein said particulate material and toxin penetrate said solid tumour.

Further s and/or embodiments of the invention are discussed in more detail below. 2012/000414 BRIEF DESCRIPTION OF THE DRAWINGS Figure l: Sterically stabilised nanoparticles are able to penetrate into spheroids.

TEM images of the accumulation of NPZ particles in Spheroids. Arrows indicate areas of tticle accumulation. Boxed regiort is enlarged and shown in the image on right. Scale bars as indicated.

Figure 2: Nanoparticles can influence the diffusion of fluorescent active nds.

Co-administration of the fluorescent active compounds a) doxorubicin and b) mitoxantrone with nanoparticles from examples 2, 3, and 5. Single confocal images of fluorescent drug diffusion into DLD-l spheroids. Scale bar 200 pm.

ED Figure 3: The majority of rticles tested did not affect cellular outgrowth from spheroids. Plot of normalised cellular outgrowth as described in example 29 of the nanoparticles listed in examples 1, 2. 4, 8. 9, 12. l3. l5, l6, and IS. Error bars represent rd error.

Figure 4: Composition of the ,nan0particle core does not influence nanoparticle '15 iveness, Plot of normalised cellular outgrowth as described in example 29 of the nanoparticles from examples 2, 4, 6. 7, 9, 10, ll. [2, l3, I4. 16, 17, and 18. co-administered with doxorubicin. The untreated control Spheroids had a normalised outgrowth value of 331% +/- 23. Error bars represent standard error.

Figure 5: Nanoparticle size does not correlate with nanoparticle effectiveness. Plot of - normalised cellular outgrowth as described in e 29'of the nanoparticles listed in examples 1,2, 4, 7, 9, 10, 11, 12, 13, i4, 16, 17, and 18 co-administered with doxorubicin. The untreated control spheroids had a ised outgrowth value of 331% +/- 23. Error bars represent standard error.

Figure 6: Nanopartieles stabilised with 5-10% amine onalised polymer increase the effectiveness of ‘doxorubicin. Plot of normalised cellular wth as described in example 29 of the nanoparticles listed in examples 2, 3. 4. 5, 20. 21, 22, and 24. co-administered with doxorubicin. The untreated control spheroids had a ised outgrowth value of 33l% +/- 23. Error bars represent standard error.

Figure 7: Effectiveness of co—administration of NPs with 5% amine functionalised stabiliser end group coatings with different cores and doxorubicin compared to doxoru bicin alone. Plot of normalised cellular outgrowth as describedm example 29 of the nanoparticles listed in examples 2, 8, 9, and 12 co-administered with doxorubicin. The W0 2012/142669 ~16- bars untreated control ids had +/- 23. Error a ised outgrowth value of 331% represent standard error.

Figure 8: The efl'ect of'the active compounds when co-administered with nanoparticles on the viability of spheroids made from two different cancer cell lines. Plot of normalised cellular outgrowth as described in example 29 of the nanoparticles listed in examples 2, 3, 4, and 5 co-administered with active compounds (Table 2). The untreated DLD-l control ids had a normalised outgrowth value of 331% +/- 23. The untreated PA-l control spheroids had a normalised outgrowth value of 294% +/- 2i. Error bars represent standard error.

Figure 9: Effect of delayed administration of active compound compared to co- administration of active compound and nanoparticlcs. Plot of normalised cellular outgrowth as described in example 29 of the nanoparticles listed in examples 2, 3, 4, and 5 DLD—l spheroids were either co-administered nanoparticles and active nd (light grey bars) or administered nanoparticles, then 24 hours later treated with active compound (dark grey bars). The untreated DLD-l l spheroids had a normalised outgrowth value of 331% +/- 23. Error bars represent standard error.

Figure 10: Effect of delayed administration of active cOmpound compared to co- administration of active compound and nanOparticles. Plot of normalised cellular outgrowth as described in example 29 of the rticles listed in examples 2, 3, 4, and 5.

PA-l spheroids were either co-administered nanoparticles and active compound (light grey bars) or administered nanoparticles, then 24 hours later treated with active compound (dark grey bars).The untreated PA-l control spheroids had a ised outgrowth value of 294% +/- 21. Error bars represent standard error.

Figure 11: The most effective inistered nanoparticle and active combinations for DLD-l and PA-l cells. Plot of ised cellular outgrowth as described in example 29 of the nanoparticles listed in es 2. 5. I4, 20, 2]. and 22, co-administered with active compounds in DLD-l ids (A) and PA-l Spheroids (B). Error bars represent standard error.

Figure 12: The covadministration of NPZ but not NP19 or NPZS with doxorubicin 3‘0 promotes doxorubicin ion througimut the spheroid. Cenfocal images of doxorubicin diffusion in spheroids treated with 1 pM Doxorubicin and the nanoparticlesas indicated. Scale bar 200 pm.

Figure 13 shows a schematic illustration of stabilised ulate material that may be used in -17. accordance with the present invention.

Figure 14 shows a schematic illustration of stabilised ulate material that may be used in accordance with the present invention.

Figure 15 shows a schematic illustration showing the hydrodynamic volume of a stabilised particulate material.

DETAILED PTION OF THE INVENTION The present invention is predicated, in part, on the determination that particulate material which is ined in a sed state by a certain type of stabiliscr can achieve a deeper and more effective penetration into a solid tumour than has been previously achievable using particle technology. The nature of the penetration effected by these particulate materials has achieved both a cantly wider cellular distribution, within the tumour, of the tbxin co- administered with the particulate material and, further, more effective ion of cellular toxicity: Since reticuloendothelial clearance from the sites of tumours is significantly less effective than in normal tissue. the method of the invention enables not only more effective tumour ation but, further, the delivery of lower concentrations of cellulai toxins which are enabled to localise, and thereby concentrate. at tumour sites. This reduces the side effects which would be apparent in the context of conventidnal systemic chemotherapy where such treatment would be delivered at the highest dose which can be tolerated by the patient and, further, often in the context of multiple repeated rounds over a period of months. This development now provides a realistic means of moving away from the treatment of primary tumours and metastatic disease via the rgeted, systemic delivery of chemotherapy.

Reference to a “solid tumour” herein should be understood as a reference to an encapsulated or unencapsulated mass or other form of growth or cellular aggregate which comprises neoplastic cells. nce to a “neoplastic cell" should be understood as a reference to a cell exhibiting abnormal growth. The term “growth" should be understood in its st sense and includes nce to proliferation. The phrase “abnormal growth” in this context is intended as a reference to cell growth which, relative to normal cell growth. exhibits- one or more of an se in the rate of cell divisiori, an increase in the number of cell ons, a decrease in the length of the period of cell division, an increase in the frequency of periods of cell on or uncontrolled proliferation and evasion of apoptosis. Without limiting the present ion in any way, the common medical meaning of the term “ne0piasia” refers to new cell growth that results as a loss‘of responsiveness to normal growth -18. controls, e.g. to neoplastic cell growth. Neoplasias include “tumours" which may be either benign, pro-malignant or malignant. The term “neoplasm” should be understood as a reference form of growth or to a lesion, tumour or other encapsulated or psulated mass or other cellular aggregate which comprises neoplastic cells.

The term “neoplasm”, in the t of the present invention should be understood to include reference to all types of cancerous growths or oncogenic processes, metastatic tissues or state or malignantly transformed cells, tissues or organs irrespective of histOpathologic type of invasiveness.

The term “carcinoma” is recognised by those skilled in the an and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, urinary system omas, testicular carcinomas, breast carcinomas, prostate carcinomas, endocrine system carcinomas and melanomas.

Exemplary carcinomas include those g from tissue of the breast. The term also es carcinosarcomas, e.g. which include malignant tumours composed of carcinomatous and sarcomatous s. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumour cells form recognisable lar structures.

The neoplastic cells comprising the neoplasm may be any cell type, derived from any tissue, such as an epithelial‘or non-epithelial cell. Examples of neoplasms and stic Cells encompassed by the t invention include, but are not limited to central nervous system tumours, retinoblastoma, neuroblastoma and other paediatric tumours, head and neck cancers (e.g. squamous cell cancers), breast and prostate cancers, lung cancer (both small and non- small cell lung ), kidney cancers (cg. renal cell adenocarcinoma), oesophagogastric cancers, hepatocellular carcinoma, pancreaticobiliary neoplasias (e.g. adenocarcinomas and islet cell tumours). ctal cancer, cervical and anal cancers, uterine and other reproductive tract cancers, urinary tract cancers (e.g. of ureter and bladder), germ cell tumours (e.g. testicular germ cell tumours or ovarian germ cell tumours), ovarian cancer (e.g. n epithelial cancers), carcinomas of n primary, human immunodeficiency associated malignancies (cg. 's sarcoma), mas, malignant melanomas, sarcomas, endocrine tumours (e.g. of thyroid gland), mesothelioma and other pleural or peritoneal tumours, neuroendocrine s and carcinoid tumours.

Preferably, the present invention is directed to the treatment of a malignant neoplastic condition and even more preferably a metastatic neoplastic condition. it would‘be appreciated that although the method of the invention can be applied to the treatment of any neoplasm, it is particularly useful in terms of the treatment of metastasised neoplasms. Without limiting the present invention to any one theory or mode of action, non-metastasised primary tumours are treatable either by the method of the invention or by conventional treatment regimes such as surgical on of the tumour or radiotherapy. r, tumours which have metastasised are not curable by either of these conventional treatment regimes due to the often extensive spread and growth of metastatic nodules. Accordingly, such conditions are currently only treatable by the administration of systemic chemotherapy, this treatment regime often g severe side effects for limited curative potential. Still further, even in the context of primary tumours which appear not to have asised, chemotherapy is still often recommended following surgery and radiation in case metastatictsmead has occurred but is not yet detectable.

This is a particularly common practice in the context of cancers which are traditionally regarded as aggressive, such as breast and colon s. ‘The method of sent invention now provides an alternative to the application of aggressive systemic chemotherapy treatment regimes. Since the systemic administration of the cytotoxic agent of the present invention is able to be delivered in a more localised fashion to tumours and is more effectively metabolised by the neoplastic cells, the occurrence of side effects can be minimised via the administration of lower doses of the cellular toxin. in one embodiment, said solid tumour is benign. in r embodiment, said solid tumour is ant.

Preferably, said malignant solid tumour is a metastatic malignant soiid tumour.

Reference to “metastatic” should be understood as a nce to a tumour which either has undergone metastatisation or may have undergone metastatisation. in one embodiment, said malignant solid tumour is a central s system tumour, biastoma, neurobiastoma, paediatric tumour. head and neck cancer such as squamous cell cancer, breast and prostate , lung cancer, kidney cancers, such as renal cell areinoma, oesophagogastric , hepatocellular oma, pancreaticobiliary neOplasia, such as adenocarcinomas and islet cell tumours, colorectal cancer, cervical cancer, anal cancer, uterine or other reproductive tract cancer, urinary tract cancer, such as of the ureter or bladder, germ cell tumour such as a testicuiar germ cell tumour or ovarian germ cell , ovarian cancer, such as an ovarian epithelial cancer, carcinoma of unknown primary human immunodeficiency associated malignancy, such as Kaposi's sarcoma, lymphoma. leukemia, malignant melanoma, sarcoma, endocrine , such as of the thyroid gland, mesothelioma or other pleural or peritoneal tumour, neuroendocrine tumour or carcinoid -20.. tumour.

As detailed hereinbefore, the method of the present invention is based on the co— administration of a cellular toxin with stabilised particulate material. Previous attempts at using particulate material, such as nanoparticles. to target tumours for either diagnostic or therapeutic purposes have been extensive but, in the context of therapeutics, of minimal success} With diagnostics, relatively shallow penetration of the particles into the tumour has been sufficient to achieve the objective of visualisin'g the tumour. However, in terms of the delivery of a therapeutic agent, such shallow penetration has not been sufficient to effectively deliver the agent hout the tumour, in particular to the or of the tumour. In on to therapeutics, specifically, conjugation of particles to a wide variety of different materials has so far failed to live up to the promise of achieving effective tumour penetration, this being an essential prerequisite for a eutic to have any chance of effectiveness. icant effort has also been made to take advantage of the enhanced permeability and retention (EPR) effect of tumours as a means to develop an effective therapeutic. Without limiting the present invention to any one theory or mode of action, this is a well described phenomenon based on the notion that certain sizes of molecules, typically liposomes or macromolecular drugs, tend to preferentially accumulate in tumour . The general explanation for this phenomenon is that, in order for tumour cells to grow y, they must stimulate the production of blood vessels. VEGF and other growth factors are involved in cancer angiogenesis. Tumour cell aggregates of sizes as small as I50~200um become dependent on blood supply carried by culature for their nutritional and oxygen supply.

These newly formed tumour vessels are usually abnormal in form and architecture. They comprise poorly-aligned defective endothelial cells with wide fenestrations. lacking a smooth muscle layer, or innervation with a wider lumen, and impaired functional receptors for angiotcnsin Il. rmore,tumour tissues y'lack effective tic ge. All these .factors will lead to abnormal molecular and fluid transport dynamics, especially for macromolecular drugs. Accordingly, it has been t that one way to achieve selective drug targeting to solid tumours is to exploit these abnormalities of tumour vasculature in terms of active and selective ry of anticancer drugs to tumour tissues, notably defihing the EPR effect of macromolecular drugs in solid tumours. Due to their large molecular size, nanosized macromolecular anticancer drugs administered intravenously escape renal clearance. Often they cannot penetrate the tight endothelial junctions of normal blood vessels. but they can W0 2012/142669 2012/000414 extravasate in tumour vasculature and become trapped in the tumour vicinity. Nevertheless. the EPR effect has not been efficiently or successfully harnessed.

Various nanOparticles have been designed which are directed to achieving efficient cellular endocytosis. However, even if this is able, the issue of tissue penetration is still a separate one which, to date, has not been successfully overcome. The general notion of the in the literature but, use of a nanOparticle as a vector for delivery of a drug is widely sed in the e of achieving deep tumour penetration, is of limited value.

Even where effective tumour distribution of a drug is achieved (by whatever means) a further problem has been the fact that neoplastic cells within solid tumours can exhibit a slowed metabolism. This means that even if a cytotoxic drug penetrates to these cells, if it is not effectively metaboliscd it will have a limited impact on the viability of the . t limiting the present invention to any one theory or mode of action. the method of the present invention is thought to e its therapeutic es by both deep penetration of the tumour by the particulate material, which y enables simultaneous or sequential IS penetration by a cellular toxin, and enabling ive metabolism of the toxin so as to achieve cell death. Still without limiting the present ion in any way, it is thought that this may be due to the particulate material defined herein, by virtue of their design, acting to upregulate cellular metabolism which has become slowed ordonnant.

The cellular toxin of the present invention should be understood as any proteinaceous or non—proteinaceous molecule or group of molecules which will either retard cell growth or induce cell death, for example either by directly killing the cell or else delivering a signal which induces apoptosis. That is, the agent may be either cytostatic or cytocidal, it would be appreciated by the persowof skill in the an that the method of the present invention can be designed to deliver one cellular toxin or multiple cellular toxins (Le. a “cocktail” of drugs).

The decision in relation to how best to proceed can be made by the person of skill in the art as a matter cf routine procedure. For example, depending on the tumour type, certain'specil‘tc drugs or combinations of drugs are regarded as particularly desirable to use. it would be appreciated that the body of knowledge in relation to the teristics and use of cytotoxic agents is ive and the person of skill in the art could design an administration protocol to meet the parameters of the t invention as a matter of routine ure.

Reference to “cellular toxin” herein should therefore be understood as a reference to any agent which acts to damage or destroy cells. Without limiting the present invention to any one theory or mode of action, many such agents function via the induction of apoptotic W0 2012/142669 is not the only mechanism by which such agents function and‘it is processes. r, this vable that the subject damage or cell death may be d by some other mechanism.

Examples of cytotoxic agents include, but are not limited to, Actinomycin D. Adriamycin, Arsenic Trioxide, Asparaginase, Bleomycin, Busulfan, Camptosar, Carboplatinum.

Carmustine, Chlorambucil. Cisplatin, Corticosteroids, eamicin, Cyclophosphamide, Daunorubicin, Docetaxcl, Doxorubicin, Epirubicin, Etoposide, Fludarabine, Fluorouracil.

Gemcitabina, Gemcitabine, Gemzar, Hydroxyurea. ldarubicin, lfosfamide, lrinotecan, Lomustine, Melphalan, Mercaptomurine, Methotrexate, cin, Mitoxantrone.

Oxaliplatin, Paclitaxel, ol, Platinex, Procarbizine. Raltitrexeel. Rixin. Steroids, Streptozocin, Taxol, Taxotere, Thioguanine, Thiotepa, x, Topotecan, Treosulfan, Trihydrate, stine, Vincristine, Vindesine, Vinorelbina, lbine, ycin, dactinomysin, esorubisin, mafosfamide, cytosine arabinoside, bis~chloroethyInitrosurea, Mitomycin C, mithramycin, prednisone. hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, hexamethylmelamine, pentamethylmelaminc, amsacrine, chlorambudil, methylcyclohexylnitrosurea, nitrogen ds, Cyclophosphamide, 6-mercaptopurine. 6- thioguanine, bine, S—azacytidine, deoxyco-formycin, 4~ hydroxyperoxycyclophosphoramide, S-fluorouracil (S-FU), S-fluorodcoxyuridine (S-FUdR), colchicine, trimetrexate, teni-poside, diethylstilbestrol.

However, reference to “cellular toxin” should also be understood to extend to any other 2O molecule which is perhaps not traditionally regarded as a cytotoxic agent but nevertheless falls within the SCOpe of the present definition on the basis that it induces cellular damage, for example DNA damage, such as nucleophosmin or agents which induce cellular damage as part of a synergistic process with another agent. Examples include catalytic antibodies, gs.

CHKl/Z inhibitor (such as CBP-SOI or AZD7762). e deacetylase inhibitor (such as vorinostat), tumour necrosis factor related apoptosis inducing ligand or BH3 mimetic (such as ABT737), small molecule inhibitors such as the tyrosine kinase inhibitors-imatinib mesylate (GlivecQ), gefitinib (lressa‘m) and erlotinib va®), and the monoclonal antibodies (mAb) such as rituximab era®) and trastuzumab (Herceptinw). in yet another embodiment, combination treatments may include, for example, gemcitabine together with a CHKI/Z inhibitor or irinotecam er with a CHKI/2 inhibitor. in a still further embodiment, the cellular toxin may be a molecule which functions as an RNA interference mechanism. Without limiting the present invention to any One theory or ~23- mode of action “RNA interference" broadly describes a methanism of gene ing which based on degrading or otherwise ting the translation of mRNA in a sequence specific manner. in terms of the application of this technology to selectively knocking down gene expression, exogenous double stranded RNA (dsRNA) specific to a gene sought to be d down can be introduced into the ellular nment. Once the dsRNA enters the cell, it is cleaved by an RNaselllelike enzyme‘ Dicer, into double stranded small interfering RNAs (siRNAs) 2i-23 nucleotides in length that contain 2 nucleotide overhangs on the 3' ends. in an ATP dependent step. the siRNAs become integrated into a'multi-subunit protein complex known as the RNAi induced silencing complex (RISC), which guides the siRNAs to the target RNA sequence. The siRNA unwinds and the antisense strand s bound to RISC and directs degradation of the complementary target mRNA ce by a combination of endo- and exonucleases. However, whereas the RNAi mechanism was ally identified in the context of its role as a microbial defence mechanism in higher eukaryotes, it is also known that RNAi based gene expression knockdown can also function as a mechanism to regulate endogenous gene sion. Specifically, microRNA (miRNA) is a form of endogenous single-stranded RNA which is typically 20-25 nucleotides and is nously transcribed from DNA, but not translated into protein. The DNA sequence that codes for an miRNA gene generally es the miRNA sequence and an approximate e cempiement. When this DNA sequence is transcribed into a single—stranded RNA molecule. the miRNA sequence and its reverse-complement base pair to form a double stranded RNA hairpin l00p, this forming the primary miRNA ure (pri-miRNA). A nuclear enzyme cleaves the base of the hairpin to form RNA. The pre-miRNA molecule‘is then actively transported out ”of the nucleus into the cytoplasm where the Dicer enzyme cuts20-25 nucleotides from the base of the hairpin to release the mature miRNA.

Although both of the RNA interference mechanisms detailed above effectively achieve the same outcome, being selective gene expression knockdowm RNAi based on the use of exogenously administered dsRNA generally results in mRNA degradation while RNAi based on the s of miRNAs generally s in translational repression by a mechanism which does not involve mRNA degradation. The RNA interference which is contemplated in the context of the present invention should be understood to encompass reference to both of these RNAi gene knockdowri mechanisms. The induction of this miRNA based knockdown mechanism could be achieved by administering. in accordance with the method of the invention, exogenous RNA oligonucleotides of the same sequence as an miRNA. pre-miRNA should understood that these exogenOus RNA or RNA molecules. r, it be oligonucleotides may lead to either mRNA degradation (analogous to that observed with the introduction of an exogenous siRNA pepulation) or mRNA translateral repression, this being akin to the mechanism by which the endogenous miRNA molecules function. in terms of the objective of the. present invention, the occurrence of either gene knockdown mechanism is able.

The RNA interference mechanism herein discussed is effected via the use of an RNA oligonucleotide which can induce an RNA interference mechanism. Reference to an “RNA oligonucleotide" should thereforebe understood as a reference to an RNA nucleic acid l0 molecule which is double stranded or single stranded and is capable of either effecting the induction of an RNA interference mechanism directed to knocking down the expression of a in this regard, gene targeted or downregulating or preventing the onset of such a mechanism. the t oligonucleotide may be capable of directly modulating an RNA interference mechanism or it may require further sing, such as is teristic of hairpin double stranded RNA which requires on of the hairpin region, longer double stranded RNA les which require cleavage by dicer or sor molecules such as RNA which similarly require cleavage: The subject oligonucleotide may be double stranded (as is typical in the context of effecting RNA interference) or single stranded (as may be the case if one is seeking only to produce a RNA oligouucleotide suitable for binding to an endogenously sed gene). Examples of RNA oligonucieotides suitable for. use in the context of the present invention include, but are not limited to: _ (i) long double stranded RNA (dsRNA) — these are generally produced as a result of the hybridisation of a sense RNA strand and an antisense RNA strand which are each separately transcribed by their own vector. Such double stranded molecules are not characterised by a hairpin loop. These molecules are required to be cleaved by an enzyme such as Dicer in order to generate short interfering RNA (siRNA) duplexes.

This cleavage event preferably occurs in the cell in which the dsRNA is transcribed. (ii) hairpin double stranded RNA (hairpin dsRNA) — these molecules exhibit a 00p configuration and are generally the result of the transcription of a construct with inverted repeat sequences which are separated by a nucleotide spacer region, such as an intron. These molecules are generally of longer RNA molecules which require both 'the hairpin loop to be cleaved off and the resultant linear double stranded molecules to be cleaved by Dicer in order to te siRNA. This type of molecule has the -25.. advantage of being expressible by a single . (iii) short interfering RNA (siRNA) — these can be synthetically generated or, recombinantly expressed by the promoter based expression of a vector comprising tandem sense and antisense strands each terised by its .own promoter and a 4-5 thymidine transcription termination site. This enables the generation of 2 te transcripts which subsequently anneal. These transcripts are generally of the order of 20-25 nucleotides in length. Accordingly, these molecules require no further ge to enable their functionality in the RNAi pathway. (iv) short hairpin RNA (shRNA) - these les are also known as “small hairpin RNA” and are similar in length to the siRNA molecules but with the exception that they are expressed from a vector comprising inverted repeat sequences of the 20-25 nucleotide RNA molecule, the inverted repeats being separated by a nucleotide spacer.

Subsequently to cleavage of the hairpin (loop) region. there is generated a functional siRNA molecule.

(V) micro RNA/small temporal RNA (miRNA/srRNA) —‘miRNA and stRNA are generally understood to ent naturally ing endogenously expressed molecules.

' Accordingly, although the design and administration of a molecule intended to mimic the activity of a miRNA will take the form of a synthetically generated or rccombinantly expressed siRNA' molecule, the method of the t invention nevertheless extends to the design and expression of oligonucleotides intended to mimic miRNA, pri-miRNA or pre-miRNA molecules by virtue of exhibiting essentially identical RNA sequences and overall structure. Such recombinantly generated les may be referred to as either miRNAs or siRNAs. (vi) miRNAs which mediate l development (stNAs). the stress se (srRNAs) or cell cycle (ccRNAs). (vii)? RNA oligonucleotides designed to ise and prevent the functioning of endogenously sed miRNA or stRNA or exogenously introduced siRNA. It would be appreciated that these molecules are not designed to invoke the RNA interference mechanism but, rather, prevent the upregulation of this pathway by the miRNA and/or siRNA molecules which are present in the intracellular environment, In terms oftheir effect on the miRNA to which they hybridise, this is reflective of more classical antisense inhibition. it .will be appreciated that the person of skill in the art can determine the most suitable W0 2012/142669 —26— RNA oligonucleotide for use in any given situation. For example, although it is preferable that the subject oligonucleotide exhibits l00% complementarity to its target nucleic acid molecule. nevertheless exhibit some degree of mismatch to the extent that the oiigonucleotide may hybridisation sufficient to induce an RNA interference response in a ce specific manner is enabled. Accordingly, it is red that the oligonucleotide of the present invention ses least 70% complementarity, more preferably at least 90% at sequence complementarity and even more preferably, 95%, 96%, 97%, 98% 99% or 100% sequence complementarity.

In another example pertaining to the design of oligonucleotides suitable for use in the to determine the present invention, it is within the skill of the person of skill in the art particular structure and length of the subject oligonucleotide, for example r it takes the form of dsRNA, hairpin dsRNA, siRNA, shRNA, miRNA, pre-miRNA. pri-miRNA etc. For example, it is generally tood that oop RNA structures, such as hairpin dsRNA and shRNA, are more efficient in terms of achieving gene knockdown than, for e, double stranded DNA which is generated utilising two constructs separately coding the sense and antisense RNA strands. Still further, the nature and length of the intervening spacer region can impact on the functionality of a given stem-loop RNA molecule. In yet still another example, the choice of long dsRNA, which requires cleavage by an enzyme such as Dicer. or short dsRNA (such as siRNA or shRNA) can be relevant ifthere is a risk that in the context ofthe particular cellular environment an interferon response could be generated. this being a more icant risk where long dsRNA is used than where short dsRNA molecules are utilised. in still yet another example, r a single stranded or double stranded nucleic acid molecule is required to be used will also depend on the functional outcome which is sought. For example. to the extent that one is targeting an endogenously expressed miRNA with an antisense molecule. it would generally be appropriate to design a single ed RNA oligonucleotide suitable for specifically hybridising to the t miRNA. However, to the extent that it is sought to induce RNA interference, a double stranded siRNA molecule is required. This may be designed as a long dsRNA molecule which undergoes further cleavage or an siRNA. Still further, the final present invention. is preferably ed to result in the generation of a effector RNA oligonucleotide (i.e. a siRNA or miRNA le) which is preferably leSS than nucleotides in length, more ably l5~25 nucleotides in length and most preferably l9, ,21, 22 or 23 nucleotides in length.

Stabilised particulate material in accordance with the invention can advantageously be 2012/000414 maintained in a diSpersed state at low concentrations. The ability for the particulate material to ‘in a dispersed state in a diverse array of liquid carriers (including body fluids) at relatively low tration, coupled with the ability to tailor the design of the stabiliser on a molecular level (e.g. its composition and molecular weight), may, without wishing to be limited by theory; play a role in enabling the particulate material to achieve deep penetration of solid tumours.

As used herein, the expression “particulate material" is intended to embrace al - that is capable of being dispersed throughout the liquid carrier and that presents a surface to which the stabiliser maybe associated.

The particulate material will generally be of a size that is less than about 500 nm. less than about 350 nm, less than about 250 nm. less than about l00 nm, less than about 50 nm, less than about 25 nm or less than about 15 nm.

In one embodiment, said particulate material is about: 2, 4, 6, 8, IO, 20, 30, 40, 50, 60. 70, 80, 90, too, 110, no, I30, 140, ISO, 160, I70. 180, l90, 200. 2l0. 220, 230. 240, 250. 260, 270, 280, 290, or 300 nm.

By having an ability to be dispersed throughout the liquid carrier, it will be appreciated that the particulate material will be sufficiently insoluble in the liquid carrier so as to enable the dispersion to have effective application.

The particulate material may be in the form of primary particles, or in the form or an aggregation of primary particles.

For avoidance of any doubt, reference herein to the “size” of the ulate material is intended to denote an average size (at least about 50 number %) of the particles based on the largest dimension of ‘a given particle. The size of the particulate material per se is ined herein by Transmission Electron Microscopy (TEM).

For avoidance of any doubt. when the particulate material is. in the form of an aggregation of primary particles, reference to the size of such material is intended to be a reference to the largest ion of the aggregate not the primary particles that form the aggregate.

‘ Apart from having medicinal utility in the context of the present application, there is no ular limitation on composition of the ulate al. The particulate material may have an organic composition or an inorganic composition or a combination thereof. The ulate material may be inorganic, organic or a combination thereof.

Examples of ulate material include one or more of a metal, a metal alloy, a metal salt, oxide, an inorganic oxide, a radioactive isotope, a polymer a metal complex, a metal particle, and/or combinations thereof. and salts, More specific examples of particulate materials include gold, silver, boron, chromium oxide, complexes or oxides thereof, calcium carbonate, barium sulphate, iron oxide, cobalt oxide, manganese oxide, n oxide, iron oxyhydroxide, chromium oxyhydroxide, cobalt oxyhydroxidc, ese oxyhydroxide, chromium dioxide, other transition metal oxides, polymers such as polystyrene, p0|y(methyl methacrylate) and poly(butadiene).

In some embodiments of the invention, it is red that the particulate material is magnetic. ic particulate al that may be used in accordance with the invention will generally be of a size of less than about 350nm. Those skilled in the art will appreciate that the composition and/or size of the particles can influence their magnetic properties. The magnetic particulate al will generally t ferromagnetic, ferrimagnetic or superparamagnetic preperties.

The specific size of the magnetic particulate material used will generally be dictated by the intended application of the compositions. For some applications, it may be desirable for the magnetic particulate material to be of a size of less than about 300 nm, for example less than about 100 nm, or less than about 50 nm.

There is no particular tion on the type of magnetic particulate material that may be used in ance with the invention. Examples of suitable magnetic materials include, but are not limited to, iron. nickel, chromium, cobalt, oxides thereof or mixtures of any of these. Preferred iron oxide magnetic particulate materials include Hon oxide (i.e. y-FezOg, also known as maghemite) and magnetite (Fe304). in that is some applications, it may be desirable to use magnetic material superparamagnetic (i.e. uperparamagnetic panicles). As used herein, the term "superparamagnetic” is intended to mean magnetic particles that do not have the following properties; (i) coercivity, (ii) remanence, or (iii) a hysteresis loop when the rate of change of an applied magnetic field is quasi static.

The ic material is preferably selected from ferrites of general a 03 where M is a bivalent metal such as Fe, Co, Ni, Mn, Be, Mg, Ca, Ba, Sr, Cu, Zn, Pt or mixtures thereof, or oplumbite type oxides of the general formula MO.6Fe203 where M is a large bivalent ion, ic iron, cobalt or nickel. Additionally, they could be particles of pure Fe, Ni, Cr or C0 or oxides of these. ‘Altematively they could be mixtures of any of these.

I’CT/AU2012/000414 In one embodiment, the magnetic particulate material is or comprises iron oxide such less than 50 nm, as magnetite (F6304) or maghemite (y-Fe203) with a particle size preferably for example between 2 and 40 nm.

Particulate material used in accordance with the invention may conveniently be prepared using ques knowrt in the art.

In accordance with the invention, the particulate al is maintained in the dispersed state by a stabiliser. By being “maintained" in this context is meant that in the absence of the stabiliser the particulate material would otherwise late or settle out from the liquid_ carrier as sediment. in other words, the stabiliser functions to retain the particulate material in the dispersed state.

The alate al is in the form of a dispersion within a liquid carrier, the particulate material being maintained in the dispersed state by a stabiliser. By “stabiliser" is meant an agent that associates with the particulate material and assists with preventing it from flocculating or otherwise becoming non-dispersed within the liquid carrier.

The stabiliser used in accordance with the inventiOn comprises an anchoring portion that (a) anchors the stabiliser to the particulate material, and (b) is different from the remainder of the iser.

By an “anchoring portiOn" is meant a moiety such as an atom or group of covalently d atoms that functions to anchor the stabiliser to the particulate material.

By an anchoring portion that “anchors” the stabiliser to the particulate material is meant it is the anchoring portion per se that directly tethers or binds the stabiliser to the particulate material.

The ing portion therefore binds the stabiliser to the ulate material.

There is no particular limitation on the way in which the stabiliser is anchored to the particulate material. For example, it may be covalently coupled to the particulate material. and/or secured to the ulate material through electrostatic forces, hydrogen bonding, ionic charge, van der Waals forces, or any combination thereof.

The stabiliser functions to prevent the particulate material from flocculating or otherwise becoming nonvdispersed (i.e. aggregated) within the liquid carrier h known mechaniSms such as steric repulsion, osteric repulsion and/or electrostatic repulsion.

Without wishing to be limited by theory, use of a stabiliser in accordance with the invention is believed to facilitate (i) transport of the ulate al in vivo to the site of the solid tumour, and/or (ii) penetration the particulate material throughout the solid tumour, W0 2012/142669 and/or (iii) uptake by subpopulations of cells within the tumour that would not otherwise accumulate effective doses of the cellular toxin.

One or more stabiliser can be uSed in accordance with the present invention.

By the anchoring portion being “different” to the remainder of the stabiliser is meant that the anchoring portion has a different ure or molecular composition to the rest of stabiliser. In other Words, the stabiliser will have a ising portion (i.e. the portion that functions as a stabilising moiety) and an anchoring portion (i.e. the portion that functions to secure or bind the stabiliser to the particulate material). The stabilising portion and the anchoring portion are ent.

By providing the stabiliser with different ural features that give rise to the stabilising and anchoring functions, it has been found that practical effect of both functions can be enhanced. Without wishing to be limited by theory, a strong'association between the ulate al and‘the stabiliser (provided by the anchoring portion). in ation with dedicated ising moiety is believed to enable the particulate material to be maintained in a dispersed state throughout a diverse array of liquid carriers at very low trations. Such properties make the particulate material well suited to being maintained in a dispersed statc post stration within body fluids.

Those skilled in the art will appreciate that stabilisers with a unique anchoring portion can function differently to stabilisers without such a unique anchoring portion, For example, a stabiliser such as polyethylene glycol (PEG) can adsorb to the surface of a particulate material and on as a stabiliser. In that case, any part(s) of the PEG chain, which does not have an anchoring portion that is different from the remainder of the stabiliser, will adsorb in a random fashion giving rise to a non-uniform surface stabilising layer.

In contrast, by ing a stabiliser with a stabilising portion and different anchoring n arrangement a more controlled .and uniform surface stabilising layer can advantageously be formed. For e the presence of the unique anchoring portion can promote on the surface of the particulate material a brush stabilising layer. where the anchoring n is d to the surface of the particulate material and the remainder ofthe stabiliser (he. the stabilising. portion) s out from the surface of the particulate material into the liquid carrier akin to the bristles extending from the surface of a brush (hence the name "brush" stabilising layer). As ahcase in point, the PEG stabiliser mentioned above might be functionalised with one or more carboxylic acid groups at the end of the PEG chain to provide for an anchoring portion. The acid functionalised anchoring portion, which is different from W0 2012/142669 the remainder of the stabiliser, can then Secure or bind the PEG chain to the particulate material and allow it to extend freely into the carrier liquid.

Suitable stabilisers may be nonionic, anionic, cationic, or zwitterionic. in one embodiment, the particulate material is maintained in the dispersed state by a steric stabiliser, wherein the steric stabiliser comprises a steric ising polymeric segment and an anchoring portion, wherein the steric stabilising polymeric segment is different from the anchoring portion, and wherein the anchoring n binds the stabiliser to the ulate material.

In a similar manner to that outlined above, steric stabilising ric segment functions to stabilise the particulate material within the liquid carrier, and the anchoring portion functions to secure the stabiliser to the particulate material. By providing the stabiliser with different Structural features that give rise to the steric stabilising and anchoring functions. it has been found that practical effect of both functions can be enhanced.

Examples of le stabilisers include, but are not limited to, those having a polymeric stabilising segment.

The stabilising segment will be soluble in the liquid carrier. in one embodiment, the polymeric ising segment cemprises polymer selected from polyacrylamide, polyethylene oxide, polyhydroxyethylacrylate, poly N: isopropylacrylamide, polydirnethylaminoethyImethacrylate, polyvinyl p‘yrrolidOne and copolymers thereof.

In another embodiment, the anchoring portion comprises one or more ylic acid groups, One or more phosphate , one or more phosphonate groups. one or more phosphinate groups, one or more thiol groups. one or more thiocarbonylthio groups. one or more sulfonic acid groups, one or more ethoxysilyl , and combinations f. ln one embodiment, the anchoring portion is an anchoring polymeric segment and at least one of the steric stabilising and anchoring polymeric ts comprise polymerised residue of one or more ethylenically unsaturated monomers. Employing at least one such ric t is believed to enhance the stabilising pr0perties of the steric stabiliser. in one embodiment. the anchoring portion is an anchoring polymeric t and at least one of the steric stabilising and anchoring polymeric segments'is derived from one or more ethylenicaily unsaturated monomers that have been “polymerised by a living polymerisation technique. Employing at least one such polymeric'segment is ed to e the stabilising properties of the steric stabiliser.

By being a “steric” 'stabiliser is meant that stabilisation of the particulate material throughout the liquid carrier occurs as a result of steric repulsion forces. Having said this, the steric stabiliser may present electrostatic repulsion forces that also assist with stabilisation the particulate material. The steric stabilising function of the iser'used in aCcordancc with the ion therefore plays an important role in enabling the particulate material to be maintained in a dispersed state hout a diverse array of liquid carriers, including body fluids.

In one embodiment, the stabiliser used in accordance with the invention comprises an ionisabie functional group that does not form part of the anchoring portion and presents within the carrier liquid a cationic or c charge. Stabiliser comprising such ble functional group(s) (e.g. amine or carboxylic acid) may be present in an amount ranging from about 2 wt% to about 50 wt%, or about 5 wt % to about 40 wt%, relative to the total wt % of stabiliser used. The use of this type of stabiliser provides for electrosteric isation. The ce of such stabiliser has surprisingly been found to e penetration of the particulate material. in one embodiment, the stabiliser comprises a steric ising polymeric segment having a terminal (i.e. at the end of the r segment or chain) functional group. The onal group may be an ionisable functional group, such as one that can provide for a cation (e.g. amine) or an anion (carboxylic acid). In one embodiment the ionisable functional group provides for a cation.

In a further embodiment, the stabiliser comprises a steric stabilising polymeric segment having a terminal functional group selected from an amine a carboxylic acid and an alcohol The amount of stabiliser used relative to the particulate material will vary depending on the nature of the particulate material, particularly its size. For example, lg of Snm particulate material will require more stabiliser than i g of 1 micron particulate material due to its increased surface area. Those skilled in the art will be able to determine the ed amount of stabiliser for a given. particulate material.

For avoidance of any doubt, reference herein to specific features of the “stabiliscr” is intended to embrace all forms of stabilisers contemplated for use in ance with the invention (i.e. where the stabiliser comprises an anchoring portion that is different form the remainder of the stabiliser, or where the stabiliser is a steric stabiliser sing a steric stabilising polymeric segment and an anchoring portion. Wherein the steric stabilising polymeric segment is different from the anchoring portion). in one embodiment, the stabiliser used in ance with the invention comprises a polymeric structure. There is no particular limitation on the molecular weight of the stabiliser, and this feature of the stabiliser may be dictated in part on the mode by which the sion to be administered to a subject. The stabiliser may. for example. have a number average molecular weight of up to about 160,000, or up to about 150,000, or up to about 100,000, or up to about 50,000. in one embodiment. the isers used in ance with the present invention will haVe a relatively low number average molecular weight compared with Stabilisers conventionally used to stabilise particulate material. in some embodiments of the invention, it may be preferable that the number average molecular weight of the stabiliser is less than about 30,000, or less than about 20,000, or less than abOut , or even less than about 5,000. The number average molecular weight of‘ the stabiliser may also range from about 1,000 to about 3,000.

Stabilisers used in accordance with the invention having. a quite low number average IS molecular weight (e.g. less than about 5,000, preferably in the range of from about 1,000 to about 3000) have been found to be particularly effective at stabilising particulate al in viva. lar weight values referred to herein are number average molecular weight values (Mn). if appropriate, the molecular weight is to be determined using gel permeation chromatography (GPC). GPC can be performed using polystyrene standards for hydrophobic polymers and polyethylene oxide standards for hilic polymers.

Those skilled in the art will appreciate that determination of the lar weight for a block copolymer may e additional procedures. For example, it may useful to determine the molecular weight of the first block before the second block is added. If a block is less than about 3000 molecular weight this can be determined by ospray mass spectroscopy (EMS). For higher molecular weigh , GPC can be employed using polystyrene standards for hydrophobic blocks and polyethylene oxide standards for hydrophilic blocks.

Determining the molecular weight of an overall block copolymer will typically depend on the length of the two blocks and their solubility characteristics. The molecular weight of low molecular weight block copolymers can be determined by EMS as mentioned above. For higher molecular weight block copolymers for which suitable solvents and standards can be found, GPC may be used. For example, if both blocks are hydrophobic and soluble in, for example, tetrahydrofuran (THF), GPC can be carried out against polystyrene standards; if both WO 42669 blocks are hydrophilic and soluble in, for example, THF, it may be useful to use hylene oxide standards, rather than polystyrene standards. However, it may be that the blocks of a block copolymer are too dissimilar to allow for a common dissolving solvent; poly acrylamide- b-polystyrene is an example of such a block copoiymer. in that case, it will generally be necessary to first block and then grow the second block and prepare and characterise the calculate the molecular weight of the second block on the basis of the degree of conversion monomer to polymer.

Stabilisers used in accordance with the invention can advantageously exhibit highly efficient stabilising properties in abilisation ofthe particulate material can be achieved at both low and high concentrations of the particulate material within a liquid carrier. The stabilisers can also provide for stable dispersions of the particulate material throughout a diverse array of liquid carriers, such as those having a high ionic strength (e.g. 0.!5 M NaCl solution, and even as high as in a saturated NaCl solution at room ature), and also over a wide pH range. Such properties make the dispersions particularly suitable for in vivo IS applications.

Without wishing to be d by theory, the highly efficient stabilising properties that can be provided by the stabilisers are believed to stem at least in part from isers comprising an ing portion that is separate to and different from the stabilising portion and securely s the stabiliser to the particulate material.

By nce the stabiliser being “anchored" to the particulate material, or wherein the anchoring portion “anchors" the stabiliser to the particulate material, is meant that the stabiliser is securely attached to the paniculate material within a liquid carrier and‘can remain so attached in the absence of free stabiliser in the liquid carrier, where the liquid r has a high ionic strength (cg. saturated aqueous sodium chloride solution), and/or where the liquid carrier has a low ionic strength (cg. pure water).

Anchoring of the stabiliser to the particulate material may be achieved as a result of the anchoring portion being (I) covalently coupled to the particulate al, and/or (2) secured to the particulate material through electrostatic forces, hydrogen bonding, ionic charge, Van 'der Waals forces, or any combination thereof.

As a result of the isers being ed to the particular material, the ulate material can be maintained in a diSpersed state within the liquid carrier despite it being present at low or high concentration and/or the liquid carrier having low or high ionic strength.

Accordingly, a dispersion of the particulate material in a liquid carrier in accordance -35. under conditions that with the ion can be advantageously stable (Le. does not flocculate) conventionallystabilised particulate material would be le (i.e. would flocculate).

Those skilled in the art will appreciate that stabilisers that are not covalently bound to particulate al generally stabilise and in particulate material in a dispersed state by ng in from a state of equilibrium of being adsorbed to and ed the particulate material. Accordingly, where a stabiiiser is present at a relatively low concentration in a given liquid carrier. the equilibrium is generally shifted in favour of the stabiliser'being desorbed from the ulate material, which in turnresults in flocculation of the particulate material.

Where anchoring occurs by the stabiliser being covalently coupled to the particulate material, there can of course be no desorption of the stabiliser. Where anchoring occurs by other means, the stabilisers used in accordance with the present invention are nevertheless securely attached to the particulate material and therefore undergo iittle if no desorption from the particulate material even when present at low concentration within the liquid carrier. In other words, when present at low concentration» within the liquid carrier the equilibrium of adsorbed stabilisers used in accordance with the invention is strongly in favour of the stabiliser being adsorbed to the particulate material, which in turn facilitates the particulate material being maintained in a diSpersed state.

A convenient test to confirm the anchoring characteristic of 'stabilisers used in ance with the invention, which in turn may also reflect their ability to maintain the particulate material in the required dispersed state, can be performed by diluting the steric stabilised particulate material to l% solids using a suitable liquid carrier (typically water), centrifuging this solution so that the solids form a plug, and then ng the supernatant liquid to isolate the solid plug. The solid plug is then combined with a suitable liquid carrier (typically water) without adding more iser so as to again form l% solids. Sodium de is then added to the resulting solution to yield 10% by‘weight sodium chloride. lithe particulate material can be redispersed in this final solution and remains diSpersed for at least 1 hour, the steric stabilisers are regarded as being ed to the particulate material.

By “steric stabilising polymeric segment" is meant a segment or region of the steric stabiliser that is polymeric (Le. formed by the polymerisation of at least one type of monomer) and that provides for the steric stabilising function of the steric stabiliser. For convenience, the steric stabilising polymeric segment may hereinafter be referred to as polymeric segment A‘ As mentioned. the steric stabilising polymeric segment ons to stabilise the particulate al throughout the liquid r by providing, steric repulsion forces.

By being polymeric, it will be appreciated that the steric stabilising segment ses polymerised monomer residues. Thus, the segment will comprise polymerised monomer residues that give rise to the required steric stabilising properties. The polymerised menomer residues that make up the steric ising polymeric t may be the same or different.

The steric stabilising polymeric segment may be substituted with a moiety (eg. an optional substituent as herein defined), or contain a polymerised monomer residue, that gives rise to electrostatic ising properties.

To provide the desired stabilising effect, the stabilising portion will be soluble in at least the liquid carrier. The solubility ofa given stabilising portion in a given liquid carrier can readily be determined by simply preparing the stabilising portion in isolation and conducting a suitable solubility test in the chosen liquid carrier.

Similarly. to provide the d steric stabilising effect, the steric stabilising polymeric segment will be soluble in at least the liquid carrier. The solubility ofa given steric stabilising polymeric segment in a given liquid carrier can readily be determined by simply preparing the polymeric segment in isolation and conducting a le solubility test in the chosen liquid carrier.

The stabiliser as a whole; may or may not be soluble in the given carrier liquid. but will nonethelesspresent a stabilising n that is soluble.

Those skilled in the art will have an understanding of polymeric materials that may be ed as the steric ising polymeric segment, as to the monomers that may be polymerised to form such polymers. For example, suitable polymeric als include, but are not limited to, polyacrylamide, polyethylene oxide, polyhydroxyethylacrylate. poly N- 'isopropylacrylamide, polydimethylaminoethylmethacrylate, polyvinyl pyrrolid0nc and copolymers thereof. Thus, suitable monomers that may be used to form the stabilising polymeric segment' include, but are not limited to, acrylamide, ethylene oxide, hydroxyethylacrylater ropylacryiamide, dimethylaminoethylmethacrylate, vinyl idone and combinations f.

The particular steric stabilising polymeric segment used as part of the steric stabiliser will of course depend upon the nature of the liquid carrier. For example. if an aquedus liquid carrier is used, the steric stabilising polymeric segment should be e in the aqueous media. Those skilled in the art will be able to select an appropriate steric stabilising ric segment for the chosen liquid carrier.

By being able to select a specific steric stabilising polymeric segment independent of W0 42669 portion, steric stabilisers used in accordance with the ion the anchoring can advantageously be tailor designed to suit a particular liquid carrier and thereby maximise the stabilising properties of the steric stabiliser.

There is no particular limitation concerning the polymerisation that technique may be used to prepare the steric stabilising polymeric t. Living polymerisation techniques have been found particularly useful in that . Those skilled in the art will appreciate that ~ “living polymerisation" is a form of radical addition polymerisation whereby chain growth that give rise to pr0pagates with essentially no chain transfer and essentially no termination dead polymer . By a “dead r chain” is meant one that can not undergo further addition of monomers. in a living polymerisation, typically all polymer chains are initiated at the start of the polymerisation with minimal new chains being initiated in latter stages of the polymerisation.

After this initiation process, all the polymer chains in effect grow at the same rate.

Characteristics and properties of a living polymerisation generally include (i) the molecular weight of the polymer increases with conversion, (ii) there is a narrow distribution of polymer chain lengths (i.e. they are of similar molecular weight), and (iii) additional monomers can be added to the polymer chain to create block co~polymer structures. Thus living polymerisation enables excellent control over molecular weight, polymer chain architecture and polydispersity of the resulting polymer that can not be achieved with non-living polymerisation s.

Suitable living polymerisation techniques may be selected from ionic polymerisation and controlled radical polymerisation (CRP). Examples of CR? include, but are not limited to, iniferter polymerisation, stable free radical mediated polymerisation (SFRP). atom transfer radical polymerisation (ATRP), and reversible on fragmentation chain transfer (RAFT) polymerisation.

The steric stabilising polymeric t may be formed by the polymerisation of one type of monomer or a combination of two or more ent monomers. ingly. the steric stabilising polymeric segment may be a homopolymeric segment or a copolymeric segment.

Given that the stabilising polymeric segment forms only part of the steric iser, rather than defining the steric stabilising polymeric t in terms of its number average lar weight, it can instead be useful to make reference to the number of polymerised monomeric units that collectively form the segment. Thus, although there is no particular tion on the number of such units that collectively form the steric stabilising polymeric segment. in some embodiments of the invention it may be desirable that the steric stabiliser has in that case, it is preferable that the steric a relatively low number average molecular weight. stabilising polymeric t has less than about 100, more preferably less than about 50. most preferably from about 10 to about 30 polymerised monomer e units that make up the overall segment.

The steric stabilisers used in ance with the ion also comprise an anchoring portion. The function of the anchoring portion has been mentioned. Provided that the stabiliser can be suitably anchored to the particulate material and is different to the steric stabiliser, there is no particular limitation concerning the form of the anchoring portion.

The anchoring portion will be covalently coupled to the steric stabilising segment. For convenience. the anchoring portion may be represented as “B“. The steric stabilising polymeric segment and the anchoring portion may be covalently coupled by any le means. For example. the steric stabiliser may be described as or comprising the structure A-C‘- B, where A represents the steric stabilising polymeric segment, B represents the anchoring portion and C represents a coupling moiety. Alternatively, the steric ising polymeric t and the anchoring portion may be directly covalently coupled and therefore the ‘stabiliser can be simplistically described as or comprising the structure AB. in that case, A ents the steric ising polymeric segment and 8 represents the anchoring portion.

The Specific anchoring portion used will generally be dictated by the nature of the particulate material to which it is to be anchored. Those skilled in the art will be able to select an appropriate ing portion to bind with the surface of a given particulate material.

When selecting the steric stabilising segment and anchoring portion. it may be desirable to consider the properties of these respective ents in the context of the intended application of the sion. For example, one or both of the steric stabilising segment and anchoring portion may be selected such that they are biodegradable and/or biocompatible, The anchoring n may be present as one or more moieties that form a 00valent bond with the particulate material so as to covalently couple the stabiliser to the particulate material. For example, the anchoring portion (in an anchored state). may be derived from a thin! moiety (-SH) that ntly couples the stabiliser to the particulate material via a -S- linkage. in other words, the stabiliser used comprises a thiol , but it will be ntly coupled to the particle via a -S— linkage. Accordingly, reference to a stabiliser “used“ in accordance with the invention is ed to be a reference to the form of the stabiliser prior to W0 2012/142669 PCT/AU2012l0004l4 it being anchored to the particulate material.

The anchoring portion may be a polymeric t, or in other words an anchoring will ric segment. in this form, anchoring of the stabiliser to the particulate material generally of covalent coupling rather by way of electrostatic not be by way but forces, hydrogen bonding, ionic charge, Van der Waals forces, or any combination thereof.

By an “anchoring polymeric segment" is meant a segment or region of the steric stabiliser that is polymeric and that has an affinity toward the e of the particulate material and functions to anchor Or bind the steric stabiliser to the particulate material. For convenience, the anchoring polymeric t may also be represented as “B”. .10 By being polymeric, it will be iated that the anchoring segment comprises polymerised monomer residues. The segment will se polymerised monomer residues that give rise to the ed anchoring to the particulate material. The polymerised monomer residues that make up the anchoring polymeric segment may be the same or ent.

The anchoring polymeric segment can present multiple sites for binding interactions with the particulate material and it is believed that this property enables the stabiliser to be‘ anchored ly to the ulate material despite not being covalently coupled thereto.

Generally, the anchoring polymeric segment will have at least two polymerised r residues that each provides a site for binding with the particulate material, preferably at least three, more preferably at least five, still more preferably at least seven, most preferably at least ten of such polymerised monomer residues. Not all of the polymerised monomer residues that make up the anchoring ric segment are necessarily required to give rise to a binding interaction with the particulate material, but it is generally preferred that the majority if not all of the polymerised monomer residues that make up the anchoring polymeric segment do give rise to a binding interaction with the ulate material.

The anchoring ric t may therefore be described as having le sites that collectively anchor the stabiliser to the particulate material. Even where a given binding site only provides a relatively weak interaction with the particulate material, the presence of les of such sites within the segment enables it as a whole to bind securely with the particulate material.

The anchoring polymeric segment can also be substituted with a moiety (cg. an optional substituent as herein defined) that may or may not give rise to a binding interaction with the particulate material.

The Specific anchoring polymeric segment used will generally be dictated by the nature of the particulate material to which it is to bind. _ When bing the interaction of the anchoring polymeric segment with the particulate material, it can be convenient to refer to the hilic and hydrOphobic character of the segment and the particuiate material. Thus, in l. suitable g interactions will or hydrophobic occur when the segment and the ulate material have similar hilic character. For example, where the particulate material has a relatively hydrophilic surface (e.g. its surface can be wetted with water), then good binding should be attained using an anchoring ric segment that has hydrophilic character (e.g. in its ed form the segment would be soluble in an aqueous medium).

Such an example might be realised where the particulate material is of a type that can form a charge on its surface. in that case, it may be ble for the segment to se polymerised residues of monomers that can also form a charge (e.g. residues of an ionisable monomer) so as to promote ionic binding between the segment and the particulate al.

Promoting the formation of such charged species might be facilitated by adjusting the pH of the liquid carrier in which the stabiliser and particulate material reside.

By the term “ionisable monomer” is meant that the monomer comprises a functional group which can be ionised in solution to form .a cationic or anionic group.

Such functional loss groups will generally be capable of being ionised under acidic or basic conditions through or acceptance of a proton. Generally, the ional groups are acid groups or basic groups (Le. groups that can donate or accept a H atom, respectively). For example, a carbox‘ylic acid onal group may form a carboxylate anion under basic conditions, and an amine functional group may form a quaternary ammonium cation under acidic conditions. The functional groups may also be capable of being ionised through an ion exchange process.

Examples of suitable ionisable monomers having acid groups include, but are not limited to, methacrylic acid, acrylic acid, itaconic acid, ene carboxylic acids, p-styrene suifonic acids, vinyl sulfonic acid, vinyl phosPhonic acid, ryloxyethyl ate, 2- (methacryloyloxy) ethyl phOSphate, ethacrylic acid, alpha-chloroacrylic acid, crotonic acid, fumaric acid, citraconic acid, mesaconic acid, and maleic acid. Examples of suitable ionisable monomers which have basic groups include, but are not limited to, 2-(dimethyl amino) ethyl and propyl acryiates and methacrylates, and the corresponding 3u(diethylamino) ethyl and propyl acrylates and methacryiates.

Those skilled in the art will be able to select an appropriate anchoring polymeric segment to bind with the surface of a given particulate material. -4].

By being able to select a specific anchoring polymeric segment independent of the steric stabilising polymeric segment, the steric stabilisers used in accordance with the invention can advantageously be tailor designed to suit a particular particulate material and thereby maximise the anchoring properties of the steric stabiliser. For example, it may be desirable that the anchoring polymeric t comprise carboxylic acid, phosphinale, phosphonate and/or phosphate functional groups. Where the particulate material to which anchoring segment binds ses iron (e.g. magnetic iron oxide particulate material), it may be desirable for the segment to comprise phosphinate, onate, and/or phosphate functional groups. Such segments will lly be formed using monomers that comprise the phosphorous onal groups.

Those skilled in the art will appreciate the variety of polymeric materials that may be employed as the anchoring polymeric segment, as to the rs that may be polymerised to form such polymers. For e, suitable polymeric materials include, but are not d to, polyacrylic acid, thacrylic acid, polystyrene, polyitaconic acid, poly-p-styrene carboxylic acids, poiy-p—styrene suifonic acids,~polyvinyl sulfonic acid, polyvinyl phosphonic acid, poly monoacryloxyethyl phosphate. poly-Z-(methylacryloyloxy) ethyl phosphate, polyethacryiic acid, poly-alpha-chloroacrylic acid, poiycrotonic acid,—~poiyfumaric acid, polycitraconic acid, polymesaconic acid, poiymaleic' acid, poly-Z-(dimethyi amino) ethyl and propyl acrylates and methacrylatcs, the corresponding poly(diethylamino) ethyl and propyi tes and methacrylates, hydrophobic acryiate and methacrylate polymers, methylaminoethyimethacrylate, and copolymers thereof. Thus, suitable monomers that may be used to form the anchoring polymeric segment include. but are not limited to, acrylic acid, methacrylic acid, itaconic acid, p-styrene ylic acids, ene sulfonic acids, vinyl sulfonic acid, Ivinyl phosphonic acid, monoacryioxyethyl phosphate, 2-(methylacryloyloxy) ethyl phOSphate, ethacrylic acid, alpha-chloroacrylic acid, crotonic acid, fumaric acid, onic acid, mesaconic acid, maleic acid, 2-(dimethyl amino) ethyl and propyl tes and methacrylates, the corresponding 3-(diethylamino) ethyl and prOpyl acrylates and methacrylates, styrene, hydr0phobic acrylate and methacrylate monomers, dimethylaminoethylmethacrylate, and combinations thereof. _ Living polymerisation techniques such as those herein described have been found particularly useful in preparing the anchoring polymeric segment. v.Where the anchoring portion is an anchoring polymeric segmentpat least one of the steric stabilising and anchoring polymeric segments may be derived from one or more ethylenically unsaturated monomers that have been polymerised by a living polymerisation technique. Where only one of the segments is d in this manner, it will ably be the anchoring polymeric t.

The anchoring polymeric segment may be formed by the polymerisation of one type of monomer or a combination of two or more different monomers. Accordingly, the ing polymeric segment may be a homopolymeric t or a copolymeric segment.

Given that the anchoring polymeric segment may form only part of the steric stabiliser, rather than defining the anchoring polymeric segment in terms of its number average molecular weight, it can instead be useful to make reference to the number of polymerised monomeric units that collectively form the segment. Thus, although there is no particular limitation on the number of such units that collectively form the anchoring polymeric segment. in some embodiments of the invention it may be desirable that the steric stabiliser has a relatively low number average lar weight. In that cases it is rable'that the anchoring polymeric segment has-less than about IOO. more ably less than about 40, still more preferably less than about 30, even more preferably from about 5 to about 25. most preferably from about 5 to about 15 polymerised monomer residue units that make up the overall segment.

When selecting the steric stabilising and anchoring ric segment, or the: monomers that may be used to prepare them, it may be desirable to consider the properties of the» respective polymeric segments in the context of the intended application of the dispersion.

For example, oneor both polymeric segment may be selected such that they are biodegradable and/or patible.

Provided that the stabiliser ons as herein described there is no particular limitation on how the ising polymeric segment and the anchoring polymeric segment are to be spatially arranged.

I'I‘he steric stabilising polymeric segment and the anchoring polymeric segment may be coupled to each other by any suitable means to form the steric stabiliser used in accordance with invention. For example. the steric stabiliser may be described as or sing the structure A-C-B, where A represents the steric stabilising polymeric segment, B represents the anchoring polymeric segment and C represents a coupling moiety. Alternatively. the steric stabilising polymeric segment and the anchoring polymeric t may be directly coupled to each other via a covalent bond and therefore the stabiliser can be simplistically described as or comprising an A-B block copolymer. In that case, A represents the steric stabilising 2012/000414 polymeric t and B ents the anchoring polymeric segment. lt wiil be appreciated from the description above that each of A and B can independently be (e.g. random, block, tapered, etc.). The a homdpolymcr or a copolymer stabiliser may comprise more than one'steric stabilising polymeric segment (A) and more than one anchoring polymeric segment (B). For example, the stabiliser may be described as or comprising an A-B-A block copolymer. In that case, each A represents the steric ising polymeric segment, which may be the same or different, and B represents the anchoring polymeric segment. The stabiliser might aiso be described as or comprising a B-A-B biock copolymer, where each B represents the anchoring polymeric segment. which may be the same or different. and A represents the steric stabilising polymeric segment that is of sufficient chain length such that it forms a “loop" that extends into the liquid carrier and performs its ising role.

The stabiliser may also have more complex structures such as star and comb polymer structures. in that case, the anchoring polymeric Segment B might represent the main polymer backbone of such structures, with multiple .steric stabilising ric segments A being attached thereto.

The interaction of a steric stabiliser used in accordance with the invention (in the form of an A-B block copoiymer structure) with particulate material in the liquid carrier might be illustrated in the not to scale simplified schematic shown in Figure l3. ; with reference to Figure l3, the c stabiliser represented by an A-B block copoiymer exhibits an affinity toward the surface of the particulate al (P) through the anchoring polymeric segment (B). The anchoring polymeric segment (B) therefore secures the steric stabiliser‘to the particulate material. The anchoring polymeric segment (B) provides multiple sites for binding interactions between the segment and the particuiate material. The steric stabilising polymeric segment (A), which is ent to t (B). is soluble in the liquid carrier and functions to maintain the ulate material diSpersed hout the liquid carrier. it will be appreciated that in practice the e of the particulate material will have illustration in many steric isers secured thereto, and that these haye been omitted from the Figure l3 for clarity.

A similar illustration to that in Figure 13 is shown in Figure l4 where the steric stabiliser used in accordance with the invention is in the form of an A-B-A block copoiymer.

At least one of the steric ising and anchoring polymeric segments may be derived from one or more ethylenically unsaturated rs that have been p‘olymerised by a living YCT/AU2012/000414 polymerisation technique such as ionic polymerisation, iniferter polymerisation, SFRP, ATRP, and RAFT risation. Of these living polymerisation techniques. RAFT risation is preferred.

The stabiliser used ing to the invention may be prepared and then used to stabilise the particulate material. atively, a moiety may be anchored to the particulate material and that moiety used to tate polymerisation of monomer so as to grow the stabiliser out from the particulate al.

Those skilled in the art will appreciate that the stabiliser selected for use in accordance with the invention may depend on the nature of the particulate material being stabilised and the way in which it is to be administered to a subject. For example, if the particulate material is to be administered intravenously and is required to remain in circulation for some time, the stabiliser may need to be a steric stabiliser as herein described.

If the particulate material is to be administered orally and needs to remain stable in the high acid conditions of the stomach, the stabiliser may also need to be a steric stabiliser as herein described. for example a steric iser comprising poly acrylamlde.

By the particulate material being “dispersed throughout” a liquid carrier is meant that the particulate material presents as a dESpersed phase throughout the liquid r which itself, relative to the particulate material, presents as a continuous liquid medium or phase. ln other words. the composition might be described as comprising a suspension or diSpersion of the particulate material throughout the liquid r.

As used herein. the term “liquid” in the t of the liquid carrier is intended to mean a vehicle in which the particulate material is dispersed throughout and which is in a liquid state at least at the temperature of intended use in the methods of the invention. Typically, a liquid carrier will be considered to be in a “liquid” state if, in the absence of astabiliser, particulate material dispersed throughout the carrier can ate or settle out from the carrier to form sediment. In other words, if the particulate material can move relatively freely in the e, then it is considered “liquid”.

The liquid carrier may be made up of one or more different liquids. Suitable pharmacologically acceptable liquid carriers are described in Martin, Remington's Pharmaceutical Sciences, 18‘h Ed, Mack Publishing Co., Easton, PA, (1990). Generally, the liquid carrier will be an s liquid carrier. Water or soluble saline solutions and aqueous dextrose and glycerol solutions are preferably ed as liquid carriers. ularly for injectable solutions.

W0 2012/142669 pharmacologically acceptable ves The dispersion may comprise one or more known to those in the art. For example. the liquid carrier may comprise one or more ves and preservatives. such as wetting agents, devfoaming agents, surfactants, buffers, electrolytes, liquid carrier and any additive therein (if present) will in The particular nature of the Those d in the art will be part depend upon the intended application of the composition. able to select a le liquid carrier and additive (if present) for the intended application of the dispersion.

It should also be understood that the particulate material and/or (steric) stabiliser may also be coupled to a ligand to effect more specific targeting, to a tumour. This will not necessarily be applicable in every situation but, to the extent that an appropriate target molecule exists for a given tumour, this may provide additional useful specificity.

Although the general notion of targeted therapy is not new, to date the s of targeted therapy has been limited by virtue of meeting the criteria which have been required of a potential target le, these being: (i) cell surface location (ii) high cell surface molecule density (iii) lack of internalisation of the molecule; and (iv) lack of appreciable antigen shedding from the cell surface.

Limitations do exist in terms of the fication of such molecules, in particular antigens which are also ideally tumour~specific. However, to the extent that such s are known for a given situation, they may be usefully exploited.

To this end, reference to a “ligand" should be tood as a reference to any molecule having specificity (not necessarily exclusive specificity, although this is preferable) 'and binding affinity for a tumour molecule. Examples of ligands include immunointeractive 2-5 molecules, peptidomimetic agents, lanthamide metals (which interact with RNA species), enzymatic substrates (which interact with cell death-related enzymes) and putrescine (which interacts with tissue lutaminase). In one embodiment, the ligand is an immunointeractive molecule. Although a preferred immunointeractive molecule is an immunoglobulin molecule, the present invention extends to other immunointeractive molecules such as antibody nts, single chain antibodies. nized antibodies including humanized antibodies and T-cell ated antigen-binding molecules (TABMS).

Most ably, the immunointeractive molecule is an antibody such as a polyCIOnal or monoclonal antibody. It should be understood that the t ligand may be linked, bound or cell. otherwise associated to any other proteinaceous or non-proteinaceous molecule or The ligand is “directed to" the tumour molecule. it should be understood that the ligand may not necessarily exhibit complete ivity, although this is preferable. For example, antibodies are known to sometimes crossreact with other antigens.

An nic determinant or epitope includes that part of the molecule to which an immune response can directed. The antigenic determinant or epitope may be a B-cell epitope or where riate a T~cell receptor binding molecule.

The t invention may also be designed such that a ligand is dir’ected to one or more tumour molecules. Accordingly. the present invention may be designed to administer two or more ligands directed to different targets, for example as a means of increasing the time of cellular toxin which is delivered to a population of neoplastic cells. It also provides a convenient means of simultaneously delivering two different toxins.

Where used, the ligand may be ly bound to the particulate material or indirectly bound to the particulate material by fonning part of the steric iser. For example. the ligand may be bound to the steric stabiliser.

Those skilled in the art will appreciate that the dispersed ulate material used in accordance with the invention will present a hydrodynamic diameter within the liquid carrier.

The hydrodynamic diameter is the distance or size that is d from the ulate material per se and the steric stabilisers associated with the particulate material. This can be more clearly explained with reference to Figure 15 where the particulate material per se (IO) is dispersed within a carrier liquid (not shown) by (steric) stabilisers (20). The hydrodynamic diameter (30) of the dispersed particulate material can therefore be seen to represent the diameter afforded by a combination of the particulate material and the (steric) stabilisers.

Where the sed particulate al does not have a symmetrical shape, the hydrodynamic er will be considered to be that of the largest hydrodynamic diameter presented by the dispersed particulate material.

Without wishing to be limited by theory, it is believed that the hydrodynamic diameter of the dispersed particulate material may also play a role in facilitating deep penetration ofthe particulate material within s.

In one embodiment, the hydrodynamic diameter of the dispersed particulate al is less than about 500 nm. is less than about 350 nm, less than about 250 nm, less than about 100 nm, less than about 50 nm, less than about 25 nm or less than about IS nm.

In a further embodiment, the hydrodynamic diameter of the dispersed particulate W0 42669 material is about: 10, 20, 30, 40, 50, 60, 70, 80,90, IOO, 110, 120, 130, I40, 150,- 160, I70, 180, 190, 200, 210, 220, 230. 240, 2,50, 260, 270, 280, 290, or 300 nm.

For avoidance of any doubt, reference herein to the “the hydrodynamic diameter“ of the dispersed particulate material is intended to denote an average diameter (at least about 50 number °/o) of the dispersed particulate material. The ynamic diameter of dispersed particulate material is determined herein by Hydrodynamic Chromatography (HDC, PL-PSDA (Polymer Laboratories».

Reference herein to a “subject” should be understood to encompass , primates, livestock animals (e.g. sheep, pigs, cattle, horses, donkeys). laboratory test animals (eg. mice, rabbits, rats. guinea pigs), companion animals (e.g. dogs, cats) and captive wild animais (cg. foxes, kangaroos, deer). Preferably, the mammal is a human. it should be tood that the term “treatment” does not necessarily imply that a subject is treated until total recovery. Accordingly, treatment includes reducing the severity of an existing condition, amelioration of the symptoms of a particular condition or ting or ' otherwise reducing the risk of developing a particular condition.

An “effective amount” means an amount necessary at least partly to attain the desired response, or to delay the onset or t progression or halt altogether, the onset or progression of a particular condition being treated. The amount varies depending upon the health and physical'COndition of the individual to be treated, the taxonomic group of individual to be d, the degree of tion desired. the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

In a related aSpect of the present invention, the subject oing treatment may be any human or animal in need of therapeutic treatment. in this regard, reference herein to “treatment" is to be considered in its broadest context. The term “treatment" does not necessarily imply that a mammal is treated until total recovery. ingly, treatment includes amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. "Treatment" may also reduce the severity of an ng condition.

Administration of the ulate material and cellular toxin, in the form of pharmaceutical compositions, may be performed by any convenient means. The pharmaceutical c0mposition is plated to exhibit therapeutic ty when administered in an amount which depends on the particular case. The variation depends, for example. on the -43. for use. A human or animal and the particular agent, particulate material and texin ed applicable. Dosage regimes may beadjusted to provide the broad range of doses may be optimum therapeutic response.

Routes of administratiOn include, but are not limited to, respiratorally, racheally, nasopharyngeally, intravenously, intraperitoneally, subcutaneously, intracranially, intradermally, intramuscularly, intraoccularly, intrathecally, intracereberally, intranasally, infusion, orally, rectally, via lV drip patch and implant. The particle may also be administered directly to the tumour.

The compositions in accordance with the invention comprise pharmacologically acceptable particulate material dispersed throughout a pharmacologically'acceptable liquid carrier. By “pharmacologically acceptable” is meant that the particulate material, liquid r, or other constituent of‘the compositiOn (cg. the steric iser) is suitable for stration to a subject in their own right. ln other words, administration of the particulate material, liquid carrier or other tuent of the composition to a subject will not result in unacceptable toxicity, including allergenic ses and disease states.

As a guide only, a person skilled in the art may consider “pharmacologically acceptable" as an entity approved by a regulatory agency of a federal or state government or listed in the US Pharmacopeia or other generally recognised pharmacopeia for use in animals, and more particularly humans.

Having said this, those skilled in the art will appreciate that the suitability of a ition for administration to a subject and whether or not a given particulate material or liquid carrier Would be considered pharmacologically acceptable, will to some extent depend upon the mode of administration selected. Thus. the mode of administration may need to be considered when evaluating whether a given composition-is suitable for administration to a subject or pharmacologically acceptable.

The ceutical forms are preferably suitable for injectable use and include sterile sterile aqueous solutions (where water soluble) or dispersions and powders for the stable oraneous preparation of sterile injectable solutions or sion. It must be under the conditions of manufacture and storage and must be preserved against the 3O contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium ning, for example, water, ethanol, polyol (for example, glycerol, ene glycol and liquid polyethylene glycol, and the like), suitable es thereof, and vegetable oils: The proper fluidity can be ined, for example, by the use of a W0 2012/142669 in case of coating such as lecithin. by the maintenance of the ed particle size the dispersion and by the use of actants. The preventions of the action of micrOOrganisms for example, parabens, can be brought about by variOus antibacterial and ngal agents, chlorobutanol, , sorbic acid, thimerosal and the like. In many cascs, it will be preferable to include isotonic agents, for example. sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for e, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ients enumerated above, as required. followed by filtered sterilisation. Generally. dispersions are prepared by incorporating the various ised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. in the case of sterile powders for the preparation of sterile able solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a l5 powder of the active ingredient plus any additional desired ingredient from previously sterile- filtered solution thereof. ln one embodiment, the stabilised particulate material and cellular toxin may be formulated in a single formulation. In an alternative embodiment. said stabilised ulate material and cellular toxin are formulated in two separate ations.

The present invention is further described by reference to the following non-limiting examples.

EXAMPLE 1 Steric stabilization of iron oxide rticles in aqueous dispersion using poly(monoacryloxyethyl phOSphate)w-block—poly(acrylamide)zu macro raft agent.

Part (a): Preparation ofdiluted aqueousferrofluid stable in acidic medium.

Magnetite nanopanicles were produced following the method of Massar’t ration of aqueous magnetic liquids in alkaline and acidic media. [BEE Transactions on Magnetics, l98i. MAG-”(2): p. 1247—1248). In a typicalreaction, 80 ml of 1M FeCl3.6H20 in 2M HCl and 40 ml of 1M FeClel-le in 2M HCI were mixed in a 2 Litre beaker and the mixture d to 1.2 Litre with MQ-water. 250 ml H (28% (w/w)) was then quickly added to the beaker and the mixture vigorously stirred for 30 minutes. Upon adding NH4OH, the colour . of the mixture immediately turned from orange to black suggesting the formation of magnetite. 2012/000414 .50- Magnetite was then oxidized in acidic medium to maghemite by heating at 90°C with iron reddish brown. e for about an hour. The colour of the suspension changed from black to then decanted, washed acetone and Maghemite particles were magnetically with finally about] .5 peptized in water yielding a stable dispersion (5 wt %). The pH of the sion was — 2.

Part (b): Preparation of a potflmonoacryloxyethy! ate)[0-b!ack~p0/y(acrylamide)20 macro-RAFT agent using: 2-{I(dodecylsulfanyl)carbonorhioyllsulfanyl} succinic acid.

A solution of odecylsulfanyl)carbonothioyl]sulfanyl} succinic acid (0.8l g, 2.0 mmol), 4,4’-azobis(4~cyanovaleric acid) (0.09 g, 0.3 mmol), acrylamide (2.87 g, 40.3 mmol) in dioxane (15 g) and water (i S g) was prepared in a 100 'mL round bottom flask, This was stirred magnetically and sparged with nitrogen for 15 minutes. The flask was then heated at 70°C for 4 hrs. At the end of this period, monoacryloxyethyl phosphate (3.98 g. 20 mmol) and 4.4’~ azobis(4-cyanovaleric acid) , 0.3 mmol) were added to the flask. The mixture was deoxygenated and heating was. continued at 80°C for a further 12 hours. The copolymer solution had 23.6% solids. The copolymer solution was then diluted with MQ water to 0.7 wt% and the pH ed to 5 using 0.IM NaOH.‘ .

Part (a): Preparation of sterically stabilized iron oxide nanoparticles from the aqueous ferroflm‘d ple 1, part (a) and the macro~RAFT agent ofexample 1. part (b).

Nanoparticle dispersion prepared in example 1 part (a) (27 g) was d with MQ water (200g) to yield a 2 wt% dispersion of the nanoparticles. The pH of this nanoparticle sion was then raised to 5 using 0.1 M sodium hydroxide. The 2 wt% dispersion of the iron oxide dispersion was then added to the macro—RAFT copolymer solutiOn from example l, part (b) (100 g). The mixture was stirred vigorously with an overhead stirrer for 45minutes before the pH was adjusted to pH 7 using sodium hydroxide solution. The mixture was then left to stir vigorously for r 2 hours at room temperature. The nanoparticle dispersion was then dialysed to remove salts, residual solvents, unwanted low molecular weight reaction side, products and unbound polymer. Bigger particles in the dispersion were removed by ultracentrifugation. The purified nanoparticlc sion was then distilled to increase the solids loading in the aqueous ferrofluid dispersion to about 70 wt%. The resulting aqueous ferrofluid was found to be stable in 60% ammonium nitrate solution.

EXAMPLE 2 Steric stabilization of iron oxide nanoparticles in aqueous dispersion using 95% poly(monoacryloxyethyl ate)lO—block-poly(ethylene oxide)” macro raft agent and W0 2012/142669 macro raft agent % onoacryloxyethyl phosphate)I0-block-poly(acrylamide)25 monometftyl with 2- Part ,(a): Estenfication of poly(ethy1ene glycol) ether {[(butylsulfany!)carbonothioyllsuyanylfpropanoic acid it. and MethoxyPEG (Mn ~798) was warmed and stirred to liquefy and homogenize bottom flask, and then 19.95 g (25.0 mmol) was then weighed into a 250 mL 3-necked round d to solidify. utylsulfanyl)carbonothioyl]sulfanyl}propanoic acid (6.96 g, 29.3 mmol) and 4-dimethylaminopyridine (360 mg, 2.9 mmol) were added to the flask, a magnetic stirbar was introduced, and the flask was purged with nitrogen. Dry dichloromethane (75 mL) all dissolved. The flask was then was added and the mixture was d until the solids had ' cooled in an ice bath and a Solution of N,N'-dicyclohexylcarbodiimide (6.03 g, 29.3 mmol) dry dichloromethane (25 mL) was then added dropwise over 1 h. The reaction was d in the-ice-bath for a further 10 min, then at room ature for 24 h. The resulting yellow slurry was diluted with 1:1. hexane-ether (I00 mL) and filtered through a sintered glass funnel.

The filter residue was washed with further small portions of H -ether until it was i5 white, and the combined filtrates were evaporated to give a cloudy and gritty dull orange oil.

The crude product was dissolve in dichloromethane (75 mL) and stirred with solid oxalic acid (4 g) for l h. then diluted with hexane (70 mL) and allowed to settle, producing a flocculent white precipitate. The mixture was filtered and evaporated. and the crude oil was dissolved in 2:1 hexane-dichloromethane (150 mL) and passed through a plug of alumina (40 g). Elution with further 2:] hexane~dichloromethanc was continued until the eluate was colourless. The combined eluates were dried with sodium te, filtered, and evaporated to give a clear pale orange oil, 24.69 g, 97%.

Part (b): Preparation of a poly(ethylene oxide)! 7-block-poiy(monoacryloxyethy! phosphate)10 macro-Rafi agent based on (he macro—RA FT ple 2 part (a).

A on of RAFT-PEG from example 2 part (a) (3.60 g, 3.5 mmol), 4,4’-azobis(4- cyanovaleric acid) (0.20 g, 0.7 mmol), monoacryloxyethyl ate (689 g, 35 mmol) in dioxane (45 g) and water (22.5 g) was prepared in a 250 mL round bottom flask. This was stirred magnetically and sparged with nitrogen for IS minutes and the reaction was carried out at 70°C for 12 hours. The copolymer solution had l5.l% solids. The copolymer solution was then diluted with MQ water to 0.7 wt% and the pH adjusted to 5 using 0.1M NaOH.

Part (C): Preparation of a po(y(manoacryloxyethyl phasphatefl0-block-poly(acrylamide)20 ‘ macro-RAFT agent using 2-{[(butylsulfanyI)carbonor}:ioyl/suVanyipr-opanoic acid. acid (3.2 g, 13.6 A solution of 2-{[(butylsulfanyl)carbonothioyl]sulfanyl}propanoic mmol), zobis(4—cyanovaleric acid) (0.19 g. 0.7 mmol), acrylamide (19.27 g, 271.1 round bottom flask. This mmol) in dioxane (45 g) and water (22.5 g) was prepared in a 250 mL for 15 minutes. The flask was then placed was d magnetically and d with nitrogen in’ a 70°C for 4 hrs. The homopolymer solution had 32.0% solids. 15g of the obtained lymer solution, monoacryloxyethyl phosphate (4.50 g, 22.9 mmol) and 4,4’-azobis(4- cyanovaleric acid) (0.043, 0.2 mmol) were added to a lOOmL round bottom flask. The mixture flask stirred in a 70°C oil bath was deoxygenated by en sparging for 15 minute and the for 12 hours. The copolymer solution, which contained 40.4% , was then diluted with MQ water to 1.2 wt%. The pH of the diluted copolymer solution was adjusted to S with 0.1M NaOH.

Parr (d): Preparation of ally stabilized iron oxide nanopartr‘cles from the aqueous ferrofluld of example 1 part (a) and a 95:5 blend of the macro-RAFT agent of example 2 part (b)aml lhe macro-RAFT agent ofexample 2 part (c).

A nanopartlcle dispersion prepared ing to example 1, part (a) was d with MQ water to yield a 2 Wt% dispersion of the nanoparticles. The pH of this prepared nanoparticle dispersion was then raised to S. A blend of macro-RAFT which consist of 50 g of‘ a 0.7 wt% solution of example 2 part (b) and 50 g of 1.2 wt% solution of example 2 part (c) were mixed together and the pH adjusted to 5 using 0.1M NaOH. The 2 wt % dispersion iron oxide maintained at the same pH was then added to the macro-RAFT blend. The mixture was vigorously stirred for 2 hours at room temperature before the pH was adjusted to 7.0. mixture was then left stirring for another 3 hours. At this pH the 00polymer remained partially neutralized while the nanoparticles were sufficiently above their point of zero charge to also be stable. The dispersion was then dialysed to remove salts, residual solvents. unwanted low molecular weight reactiOn side products and unbound polymer. Bigger particles in the dispersion Were removed by ultracentrifugation. The d nanoparticle dispersion was then distilled to increase the solids loading in the aqueous ferrofluid dispersion to about 70 wt%.

The resulting aqueous ferrofluid was found to be stable in phosphate buffered saline solution.

Part (e): Modification ofstabllisersfor iron oxide particles ofexample 2 par! (d) Into coated nanoparticles prepared from example 2 part (d) (7.8 g), N- hydroxysuccinimide (NHS, 14.4mg) and then 1-Ethyl(3-Dimethylamino- propyl)carbodiimide (EDAC, 20 mg) were added, mixed by shaking and allowed to react for 2 hours at room temperature. A solution of e (90 mg of 2,2’-(Ethylenedioxy)bis- -53.. (ethylamine) in 1 ml of water) was then added to the reaction mixture and allowed to react for dialysed water with numerous a further 3.5 hours. The on was then against excess changes, to remove free EDAC and the reaction lay—products.

EXAMPLE 3 Steric stabilization of iron oxide nanoparticles in aqueous dispersion using the uid of example 1 part (a) and the poly(monoacryloxyethyl phosphate)w~block-poly(ethylene oxide)" macro raft agent of e 2 part (b) Nanopanicle dispersion (8.0 g) prepared according to example i part (a) was diluted with 50g of MQ water to yield a 0.5 wt% sion of the nanoparticles. The pH of this prepared nanoparticle dispersion was then raised to S. The 0.5 wt °/o dispersion of iron oxide maintained at the same pH was then added to the 50 g of macro~RAFT agent from example 2 the pH was part (b). The mixture was vigorously stirred for 2 hours at room temperature before adjusted to 7.0. The mixture was then left ng for another 3 hours. At this pH the mer remained partially neutralized while the nanopartieies were sufficiently above their point of zero charge to also be stable. The sion was then ed to remove salts. residual solvents. unwanted low molecular weight reaction side products and unbound polymer. The final solids of the sion was 0.74%.

EXAMPLE 4 Steric stabilization of iron oxide nanoparticles of the aqueous ferrofluid of example 1 part (3) using the poly(monoacryloxyethyl phosphate ),o.block-poly(acrylamideizo macro- ‘raft agent of example 2 part (c) Nanoparticle dispersion prepared in example I part (a) (6.!9 g)‘ was diluted with MQ water (100 g) to yield a l wt% dispersion of the nanoparticles, The pH of this nanoparticle sion was then raised to 5 using 0.l M sodium hydroxide. The 2 WI% dispersion of the iron oxide dispersion was then added to the macro-RAFT copolymer soluti0n from e 2 before the part (c) (50 g). The mixture was stirred vigorously with an overhead stirrer 2 hours pH was adjusted to pH 7 using sodium hydroxide solution. The mixture was then left to stir vigorously for another '12 hours at room temperature. The nanoparticle dispersion was then dialysed to remove salts, residual solvents, unwanted low molecular weight reaction side 3O products and unbound polymer. The solid content of the final dispersion is 0.71%.

EXAMPLE 5 the aqueous Steric stabilization oi'iron Oxide nanoparticles in aqueous dispersion from ferrofluid of example 1 part (a) using 100% amine modified poly(monoacryloxyethyl phosphate)w-block-poly(acrylamide)zo Part (a): Preparation of amine modified pab:(mon0acryloxyethyl ate)!0-block- . poly(acrylamide)20 macro-RAFT agentfrom the macro-RAFT agent ofexample 2 part (c) N-hydroxy succinimide 98% (0.64 g), 2. 2’«(Ethylenedioxy)bis—(ethylamine), _98% (0.54 g) was added to 30.0 g of poly(monoacryloxyethyl phosphateho-blockpoly (acrylamide)zo block copolymer of example 2 part (c) at pH 6.25 in a 100 mL glass .

The bottle was sealed with parafilm and placed on a roller for mixing for 2 hours. After 2 hours, the mixture had a pH of 8.l2 to yield the Nl-lS-activated carboxyl groups which are reactive towards primary amine. l.26 g of N-(3~Dimethylaminopropyl)-N'-ethy|carbodiimide hydrochloride was then added to the mixture, which was left on the roiler for r 12 hours.

The excess N-(3~Dimethylaminopropyl)-N'-ethyIcarbodiimi'de hydrochloride was removed by dialysis.

Part (b): Preparation .of sterically stabilized iron oxide nanoparticles from the aqueous ferrofluid ofexample 1, part (a) and the macro—RAFT agent ofexample 5, part (a) NanOparticle dispersion prepared in example 1 part (a) (8.38 g) was diluted with MQ water (50 g) to yield a 0.5 wt% dispersion of the nanoparticles. The pH of this nanOparticle diSpérsion was then raised to 5 using 0.1 M sodium ide. The 0.5 wt% dispersion of the iron oxide nanoparticles was then added to the macro-RAFT copolymer solution, from example 5 part (a) (22.6 g) and 50 g of MQ'water. The mixture was stirred vigorously with an overhead stirrer for 2 hours before the pH was ed to pH 7 using sodium hydroxide on The mixture was then left to stir vigorously for r 10 hours at room temperature.

The nanoparticle dispersion was then dialysed to remove salts, residual solvents, ed low molecular weight on side products and unbound polymer. The solids content of the dialysed aqueous ferrofluid sion was 0.36%.

EXAMPLE 6 Steric stabilization of iron oxide nanoparticles in aqueOUS dispersion using 95% poly(monoacryl0xyethyi phosphate)w-block-poly(acrylamidem macro-raft agent of example 2 part (c) and 5% amine modified poly(monoacryloiryethyl phosphate)m-block- poiy(acrylamide)m of example 5 part (a) Nanoparticle dispersion ed in example l part (a) (8.09 g) was d with MQ water (50 g) to yield a 0.5 wt% dispersion of the nanoparticles. The pH of this nanoparticle 0.5 wt% dispersion of the dispersion was then raised to 5 using 0.1 M sodium hydroxide. The solution which was pH iron oxide dispersion was then added to the RAFT copolymer .0 from example 5 part (a) (1.7 g), example 2 part (c) (1.0 g) and 50 g of MQ water. The the pH was adjusted mixture was stirred vigorously with an overhead stirrer for 2 hours before solution. The mixture was then left to stir vigorously for to pH 7 using sodium hydroxide then dialysed to remove r 3 hours at room ature. The rticle diSpersion was and unbound salts, residual solvents, unwanted low molecular weight reaction side products polymer. The solids content of the ed aqueous ferrofluid dispersion is 0.53%. ‘ EXAMPLE 7 Steric stabilization of iron oxide nanoparticles in aqueous di5persion using poly(monoacryloxyethyl phosphate)w-block-polflacrylamidem macro raft agent Part (a): Preparation ofdiluted aqueous/errofluid stable in acidic medium Magnetite nanoparticles were produced following the method of Massart (Preparation of aqueous magnetic liquids in alkaline and acidic media. lEEE Transactions on ics, 1981. MAG-l7(2): p. 1247-1248). An s e of ferric and ferrous chlorides was added to ammonia solution. The resulting precipitate was isolated by centrifugation then oxidised to maghemite by mixing with iron e solution and heating. The precipitate was then washed in 2 molar nitric acid then finally peptised by water to form a dilute aqueous ferrofluid of approximately 5 wt% solids.

Part (1)): Preparation of a poly(monoacryloayetltyl phosphate)10~block-poly(acrylamide)60 RAFT agent using 2-{[(butylsuyanyvcarbonothtoyllsulfanylf propanoic acid.

A solution of 2-{[(butylsulfanyl)carbonothioyI]sulfanyl} propanoic acid (0.26 g, Ll mmol), 4,4‘-azobis(4-cyanovaleric acid) (0.06 g. 0.2 mmol). acrylamide (4.73 g, 66 mmol) in dioxane (10 g) and water (10 g) was prepared in a 100 mL round bottom flask. This was stirred magnetically and sparged with nitrogen for IS minutes. The flask was then heated at 70°C for 4 hrs. At the end of this period, monoacryloxyethyl phosphate (2.17 g, 11.! mmol) and 4.4’- azobis(4-cyanovaleric acid) (0.06 g, 0.2 mmol) were added to the flask. The mixture was deoxygenated and heating was continued at 80°C tor a further 12 hours. The copolymer solution had 24% solids. The mer solution was then d with MQ water to 0.7 wt% 3O and the'pH adjusted to 5 using 0.1M NaOH.

- Part (c): Preparation of sterically stabilized iron oxide nanoparticles from the aqueous ferrofluid ofpart (a) and the macro-RAFT agent ofexample 7part (b).

Nanoparticle dispersion prepared in example 7 pan (a) (40 g) was diluted with MQ water (200g) to yield 'a l wt% dispersion of the nanoparticles. The pH of this nanoparticic dispersion was then raised to 5 using 0.l M sodium hydroxide. The lwt°/o sion of the iron oxide dispersion was then added to the macro—RAFT mer solution from part (b) (200 g). The mixture was stirred vigorously with an overhead stirrer for 45minutes before pH was ed to pH 7 using sodium hydroxide solution. The mixture was then left to stirr vigorously for another 2 hours at room temperature. The nanoparticle sion was then dialysed to remove salts, residual solvents, unwanted low molecular weight reaction side products and unbound polymer. Bigger particles in the dispersion were removed by ultiacentrifugation. The purified nanoparticie dispersion was then distilled to increase the solids loading in the aqueous luid dispersion to about 70 wt%.

Steric stabilization of sigma ludox as 30 silica particles using 95% poly/[2- (dimethylamino)ethyl methacrylatelio-bloek-polflethylene oxide)" macro raft agent and 5% poly(2-(dimethylamino)ethyl rylate)w~block-poly(acrylamidem macro raft agent Part (0): Preparation ofa poly/2-(dimetlxylamino)ethyl methacrylate]!0-block-pobr(erhylene ! 7 macro-RA‘FT agent based on 2-{/butylsulfany!)carbonothioyll-suq'anylfpropanoic acid.

A solution of 80 from example 2 part (a) (l .38 g, 1.4 mmol), 4.4‘-azobis(4- cyanovaleric acid) (0.08 g, 0.3 mmol), ethylamino)ethyl methacrylate (2.l3 g, 13.6 mmol) in dioxane (10 g) and water (5 g) was prepared in a 100 mL round bottom flask. This was stirred magnetically and sparged with nitrogen for 15 minutes and the reaction was carried out at 70°C for a 12 hours. The copolymer solution had 18.4% .

Part (b): Selective quaternization of a poly[2-(dimerhylaminokrhyl methacrylate/ID—block- poly(ethylene oxide)! 7 macro-RAFT agent based on 2-{{butylsulfanyl)carbonolhioyll— sulfanyl}propan0ic acid.

Example 8 part (a), l6.5 g was diluted with MQ water (17 g) and methyl iodide (0.7 g) added. The mixture was stirred at room temperature for 1 hour before being partially dried 3O using a rotary evaporator. The dried samples were then placed in the vacuum oven to dry the macro raft agent which yielded l00% solids.

Part (0): Preparation of a poly(2-(Dimerhylamino)ethyl methacrylare) 10-bIock- crylamide)25 macro-RAFT agent based on tylsulfanyl)carbonothioyl]; sulfanylfpropanoic acid. .

A on of 2-{[(butylsulfanyl)carbonothioyllsulfanylipropanoic acid (0.6 g. 2.6 mmol), zobis(4—cyanovaleric acid) (0.11 g, 0.4 mmol), acrylamide (4.45 g, 62.7 mmol) in dioxane (18.8 g) and water (10.5 g) was prepared in a 100 mL round bottom flask. This was stirred magnetically and sparged with nitrogen for IS minutes. The flask was then placed in a 709C oil bath for 4 hrs. The homopolymer solution had 32.7% solids. All of the hom0polymer solution obtained, 2-(Dimethylamino)ethyl methacrylate (3.94 g, 25.1 mmol) and 4.4‘- (4-cyanovaleric acid) (0.088 g, 0.32 mmol) were added to a mom. round bottom flask.

The mixture was deoxygenated for £5 minute and placed in a 70°C oil bath for 12 hours. The final solids ofcopolymer solution was 26.9%.

Part (d): Selective quaternization of a poly(2~(dimethylamino)ethyl methacrylateNU-ltlock- pobr(acrylamide)25 RAFT agent based on 2-{/butylsulfanyl)carbonothioyll- sulfanyl}propanoic acid. e 8 part (c) (I93 g) was diluted with MQ water (20 g) and methyl iodide (0.78 g) was added. The mixture was stirred at room temperature for l hour be partially dried using a rotary evaporator.. The partially dried samples was then placed in the vacuum oven to dry the macro rah agent which yield l00% solids.

Part (e): Preparation ofsterically stabilized Sigma Ludox A530 silica particles using a 95:5 blend ofthe macro-RAFT agents ofexample 8 part (b) and example 8 part (d) Ludox A830 from Sigma Aldrich (2.5 g) was diluted with MQ water (100 g) to yield a 2 wt% dispersion of the nanOparticles and the pH is 9.62. A mixture of example 8 part (b) (0.96 g) and of example 8 part (d) (0.0653 g) was dissolved in MQ water (50 g) and the pH was 7.59. The 2 wt% dispersion was then poured into the e of macro-Raft agents. The mixture was vigorously stirred for 5 hours at room temperature. The dispersion was then ed to remove salts, residual solvents. unwanted low molecular weight reaction side products and unbound polymer. The solid content of the dialysed silica sol sion was 0.69%. The pH ofthe sample was adjusted to 6.76 with sodium hydroxide solution.

Part 0): Modification ofstabilisersfor silica particles ofexample 8 part (e) [EP341070A] Sterically stabilised silica sol particles prepared from example 8 part (e) (60g). N- hydroxysuccinimide (NI-ls, 39.4mg) and then l-Ethyl(3-Dimethylamino- pr0pyl)carbodiimide (EDAC, 56.2mg) were added, mixed by shaking and allowed to react for 2 hours at room temperature. 37mg of2,2’-(Ethylenedioxy)bis-(ethylaminc) was then added to W0 2012/142669 the on mixture and allowed to react for a further 12 hours. The solution was then dialysed against excess water with numerous changes, to remove free EDAC and the reaction by- products.

EXAMPLE 9 Steric stabilization of sigma ludox as40 silica sol using 95% polyl2—(dimethylamino)ethyi methacryiate]io—block—polflethylene oxide)" macro raft agent and 5% poly(2- hylamino)ethyl methacrylate).o-block-poly(acrylamide)2s macro raft agent Part (0): Preparation of sterically stabilized Sigma Ludox A530 Silica particles and a 95:5 blend oftlre RAFT agents ofexample «Spar! (b) and e 8 part (d) Ludox A840 from Sigma Aldrich (5.0g) was diluted with M0 water lOOg to yield a 2 wt% sion of the nanoparticles and the pH is 9.97. A mixture of macro-RAFT agents, which consisted of example 8 pan (1)) (1.72 g) and example 8 part (d) (0.13 g) was dissolved in 100 g of M0 water and the pH was 7.95. The 2 wt% dispersion was then poured into the mixture of macro-RAFT agents. The mixture was vigorously stirred for 5 hours at room temperature. The dispersion was then dialysed to remove salts, residual solvents, unwanted low lar weight reaction side products and unbound polymer. The solid content of the dialysed silica sol dispersion is 1.45 %. The pH of sample was 7.65.

Parr (b): Modification ofstabilisers ofsilica particles ofexample 9 part (a) To sterically stabilised silica sol particles prepared in example .9 part (a) (30 g), N- hydroxysuccinimidc (NHS, 11.6 mg) and i—Ethyl(3-Dimethylamino-propyl)carbodiimide ‘(EDAC. 16.6 mg) were added, mixed by shaking and allowed to react for 2 hours at room temperature. 2,2'-(Ethylenedioxy)bis-(ethyIamine) (45.1 mg) was then added to the reaction mixture and d to react for a further 12 hours. The solution was then dialysed against excess water with numerous changes. to remove free EDAC and the reaction by-products.

EXAMPLE 10 Steric stabilization of 130 nm silica particles using poly(2-(dimethylamino)ethyl methacrylate)m-block-poly(acrylamide)25 macro raft agent Part (a): Silica les were prepared using the s of Costa et at.

(Carlos A. R. Costa, Carlos A. P. Leite. and do Galembeck J. Phys. Chem. B. 2003, 107 (20), 4747-4755.) to obtain l30 nm diameter silica particles at 0.18 % solids in wate i'. part Part (b): Steric stabilization of the 130 nm diameter silica particles of example (it) using the RA FT agent ofexample 8 part (d) Silica particle sion of example 10 part (a) (l l .IS g) was diluted with MQ water (20 g) Macro-RAFT to yield a 0.! wt% dispersion of the nanopanicles with a pH of 9.26. in 25g of M0. water (25 g) to yield a agent of example 8 part (d) (0.023 g) was ved solution of pH 5.80. The silica sion and the macro—RAFT solution were then blended and vigorously stirred for 5 hours at room temperature. The dispersion was then centrifuged to remove salts, residual solvents, unwanted low molecular weight reaction side products and unbound polymer. The solid content of the sterically stabilised silica dispersion is 0.8] %.

EXAMPLE 11 Steric ization of the 130 nm diameter silica particles of example 10 part (3) using the “grow from” approach.

The silica particles of example 10 part (a) were RAFT functionalised using 6- (Triethoxysilyl)hexyl 2-(((Methylthio)carbonothioyi)—2-phenylacetate and r chains comprising poly methoxy—PEG acrylate (Aldrich .454 g/ mol) were grown from the surface of the particles according to the methods of Ohno et al. (Kohji Ohno. Ying Ma, Yun Huang, Chizuru Mori, Yoshikazu Yahata, Yoshinobu Tsujii, Thomas eyer, John Moraes. and Sébastien Perrier oleCules, 20] l, 44 (22), pp 894443953.) The molecular weight obtained for each ed chain was approximately 56.000 g/mol. The final particles were obtained in water at a solids content of to mg/mL and the particle size was 258 nm. as measured by DLS.

EXAMPLE 12 Steric stabilization of 10—15 nm gold nanoparticles in aqueous diSpersion using 95% thylene oxide)" macro raft agent and 5% poly(acrylamide)m macro raft agent Part (a): Synthesis of 10—15 am citrate stabilized gold nanopartr‘cles stable in aqueous medium Citrate-stabilized gold nanoparticles (IO-l5 nm) were prepared using Frens' method (Frens, G. Nat. Phys. Sci. 1973, 24!, 20-2.) Briefly, all glassware was first washed with an aqua regia solution (25 vol % trated nitric acid and 75 vol % concentrated hydrochloric acid), then rinsed with Milli-Q water several times, and dried. 100 ml of an aqueous solution containing tertrachloroaureic(lll) acid rate (0.0!g, 0.025mmol) was refluxed in a 500 mL 3-necked round bottom flask. 2 ml solution of trisodium citrate dihydrate (0.02 g. 0.068 mmol) was added to it. The solution was heated to boiling point vigorous stirring. Boiling and 2012/000414 vigOrous stirring was maintained for 30 min. A progressive change of colour from yellow to wine red was observed. The solution was cooled down. dialysed to get rid of excess sodium citrate and stored in at 5°C. The rticie concentration in the dispersion was 50 ppm.

Part‘ (b): Preparation of a crylamide)20 macro—RA FT agent using: 2- {llbutylsulfanybcarbonothioyl/salfanyl}propanoic acid.

A solution of 2-{[(butyisulfanyl)carbonothioyl]sulfanyl} propanoic acid (0.7l g, 3.0 mmol), zobis(4-cyanovaleric acid) (0.04 g, 0.15 mmol), acrylamide (4.28 g, 60.2 mmol) in dioxane (7.5 g) and water (7.5 g) was prepared in a 100 mL round bottom flask. This was stirred magnetically and sparged with nitrogen for 15 minutes. The flask was then placed in a 70°C oil bath with continued stirring for 4 hrs. The polymer solution had 25. l7% .

Part (c): Preparation of sterically stabilized 10-15 am gold nanoparti'cles from the citrate stabilised gold nanoparticles of example 12 part (a) and a 95:5 blend of the macro-RAFT . agent of example 2 part (a) and the macro-RAFT agent ofexample 12 part (b). lOO ml gold nanoparticle dispersion (50 ppm) of e 12 part (a) was transferred to a 250 ml round bottom flask. A 10 ml solution ning 0.0l2 g of the RAFT agent of example 2 part (a) and 0.l5 g the macro-RAFT agent of example 12 part (b) was then added.

The mixture was stirred vigorously with a magnetic stirrer bar for 2 hours at room temperature and then ed to remove salts, residual solvents, unwanted low molecular weight reaction side products and unbound polymer. The purified nanoparticle dispersion was at a concentration of 50 ppm and was stored in the fridge at 5°C. The resulting aqueous nanoparticle dispersion was found to be stable in phosphate buffer saline solution, Part (d): Modification ofstabilisersfor gold nanoparticles ofexample 12 part (c) Into coated nanoparticles prepared from example 12 part (c) (IOO ml), N- hydroxysuccinimide (NHS, 4mg) and then l~Ethyl(3-Dimethylamino-propyl)carbodiimide (EDAC. 4.l mg) were added, mixed by shaking and allowed to react for 2 hours at room temperature. A solution of diamine (21mg of2,2’-(Ethylenedioxy)bis-(ethylamine) in 2 ml of water) was then added to the reaction mixture and allowed to react for a further 3.5 hours. The solution was then dialysed against excess water with numerous changes, to remove free EDAC and the reaction by-products.

EXAMPLE 13 Steric stabilization of 3-8 nm gold nanoparticles dispersed in aqueous medium using thiol modified poly(acrylamide)20 Part (a): Thiol modification ofpoly(acrylamide)20 macro-RAFT agent ofpart example 12 part (b) using isopropyl amine.

A solution of the poly(acrylamide)2o macro-RAFT agent of example l2 part (b) (lg, 0.6 mmol), isopropyl a'm'me (L77g, 30 mmol) in dioxane (7.5 g) and water (7.5 g) was prepared in a 100 mL round bottom flask. This was stirred ically and sparged with en for l5 minutes, then d to react for a 24 hours at 25°C. At the end ofthis period, the polymer was precipitated in diethyl ether (50 ml). The precipitates were separated from the on e by filtration and dried under yacuum using a rotary evaporator. The dried thioi terminated poly(acrylamide)zo was d with nitrogen for IS minutes and stored in an airtight container at 20°C.

Parr (b): Preparation ofsterically stabilized 3-8 nm gold nanoparticles in aqueous dt'Spersr'on using thiol ed poly(acntlamide)20 ofexample 13 part (a).

Milli-Q water (250 mL) was refluxed in a 500 mL ed round bottom flask. 25 mL of a aqueOUS solution containing tertrachloroaureic(lll) acid trihydrate (0.057! g, 0.1444 mmol) was then added and the solution heated to boiling. Then, a solution in water (25 mL) of tris‘odium citrate dihydrate (0.5 g, 1.7 mmol) and thiol modified poly(acrylamide)zo (O.l2 g, 0.0779 mmol) of example .13 part (a) was added and the reaction carried out for 2 hours at °C. By the end of this period, the colour of the on had turned from yellow to wine red.

The molar ratio of steric stabilizer to the tettrachloroaureicflli) acid trihydrate in this case is 0.5. The gold nanoparticles were ted from the dispersion by centrifugation at 52,000 g for 30 min. The rticles were redispersed in MillioQ water at a concentration of I90 ppm.

The size of gold nanoparticles obtained from TEM was 3-8 nm.

EXAMPLE l4 Steric stabilizatiOn of 8-10 nm gold nanoparticles dispersed in aqueous medium using thiol modified poly(acrylamide)zo of example 13 part (a) Milli-Q water (250 mL) was refluxed in a 500 mL 3-necked round bottom flask. 25 mL of an aqueous solution containing tertrachloroaureic(lll) acid trihydrate (0.0652 g, 0.16 mmol) was added and the solutiOn was heated to boiling. Then, a solution in water (25 mL) of trisodium citrate dihydrate (0.5 g, l.7 mmol) and thiol modified poly(acrylamide)zo (0.022 g, 0.0l42 mmol) of example 13, part (a) was added and allowed to react for 2 hours at 25°C. By the end ‘30 of this period, the colour of the solution had turned from yellow to wine red. The molar ratio of steric stabilizer to the tertrachloroaureic(lll) acid trihydrate in this case was 0.09. The gold nanoparticles were separated from the dispersion by centrifugation at 52.000 g for 30 min. The W0 2012/142669 2012/000414 -52. nanoparticles were redispersed in Milli-Q water at a concentration of 390ppm. The size of gold nanoparticles obtained from TEM was 8-10 nm.

EXAMPLE )5 Steric ization of 30—40 nm gold nanoparticles dispersed in aqueous medium using thiol modified poly(acrylamide)2o of example l3 part (a) Part (a): Synthesis of 30—40 am citrate stabilized gold nanoparticles stable in aqueous medium.

Citrate-stabilized gold nanoparticles (30-40 nm) were prepared using Frens method (Frans. G. Nat. Phys. Sci. 1973, 241, 20—2.) Briefly, all glassware was first washed with an aqua regia solution (25 vol % concentrated nitric acid and 75 vol % concentrated hydrochloric acid), then rinsed with Milli-Q water several times. and dried. 100 ml of an aqueous solution containing tertrachloroaureic(lll) acid trihydrate (0.0lg, mol) was refluxed in a 500 mL 3-necked round bottom flask. 1 ml solution of ium citrate dihydrate (0.0] g, 0.034 mmol) was then added. The solution was heated to boiling with vigorous ng. Boiling and vigorous stirring was maintained for 30 min. A progressive change of colour from yellow to winered was observed. The solution was cooled to ambient, ed to get rid of excess sodium citrate and stored at 5°C. The nanoparticle concentration in the dispersion was 50 ppm.

Part ([2): Preparation ically stabilized 30-40 am gold nanoparticles from the aqueous gold nanoparticle dispersion ofexample {5 part (a) and thiol ed poly(acrylamide)20 of example 13 part (a). l00 ml gold nanOparticle dispersion (50 ppm) of example 15 part (a) was taken in a 250 ml round bottom flask. 10 ml solution of aqueous solution of example l3 part (a) containing thiol modified poly(acrylamide}zo 8 g, 0.0044 mmol) was then added. The mixture was stirred vigorously with a magnetic stirrer bar for 2 hours at room ature and then dialysed to remove salts, residual solvents, unwanted low molecular weight reaction side products and unbound polymer. The purified rticle dispersion was then distilled to increase the solids loading in the aqueous nanoparticle dispersion to l92ppm. The resulting aqueous nanoparticle dispersion was found to be stable in phosphate buffer saline solution, EXAMPLE 16 3O Synthesis of polystyrene nanOparticles in s dispersion using poly(styrene)9-b- poly(acrylamide).5 macro raft agent Part (a): Preparation of self assembled poly(styrene)9-b-poly(acrylamide)15 macro-RAFT agent using: 2—{[(butylsalfanyl)carbonothioyllsulfanylfpropanoic acid.

WO 42669 . 3.36 A solution of 2—{[(buty1sulfanyl)carbonothioy1]suli‘anyl)propanoic acid (0.80 g, mmol). 4.4’-azobis(4-cyanovaleric acid) (0.10 g, 0.36 mmol). acrylamide (3.71 g, 52.06 mmol) in dioxane (6.61 g) and water (4.41 g) was prepared in a 50 mL round bottom flask.

This was stirred magnetically and sparged with nitrogen for 10 s. The flask was then heated at 70°C for 5 hrs to produce the clear homopolymer solution. At the end of this , styrene (3.16 g, 30.3 mmol), '4,4’-azobis(4-cyanovaleric acid) (0.19 g, 0.69 mmol), dioxane (21.15 g) and water (614 g) were added to the flask. The mixture was Stirred, dcoxygenated with nitrogen for 10 minutes. The flask was then immersed back in an oil bath at 70°C for overnight with constant stirring.

Part (6): Synthesis styrene uanopartt‘cles using the selfassembled macro-RAFT agent prepared in example 16 part (a) _ To a clear dispersion of macro-RAFT agent from example 16 part (a) (1.00 g) in a 50 mL round bottom flask on a magnetic stirrer, sodium hydroxide solution (1.94 g of 0.3% solution, 0.15 mmol with water (22.1 g)) was added drop wise. To this mixture styrene (1.109 g, 10.5 mmoi) Was added and stirred ght. 4,4'-azobis(4-cyanovaleric acid) (15.5 mg, 0.055 mmol) and sodium hydroxide solution (1.04 gof 3% soiution, 0.78 mmol) were added.

The flask was stirred for 2 hours, then sealed and subsequently deoxygenated with nitrogen sparging for 10 minutes. The whole flask was immersed in an oil bath with a temperature setting of 80°C and maintained at that temperature for 5 hours under constant magnetic stirring.

The latex contained particles with average diameter of 15 nm by zer light scattering. The latex was ed against Q water to remove impurities.

EXAMPLE 17 Synthesis of polystyrene nanoparticles in aqueous dispersion using self assembled ' poly(styrene)9-b-poly(acrylamideho macro raft agent of example 16 part (a) Part (0): Further growth of the selfassembled macro-RAFT agent ofexample I6 part (a) to form poly(styrene)52~b-poly(acrylamide)20 macro-RAFT. , To a clear dispersion of RAFT agent from example 16 part (a) (1.02 g) in a 25 mL round bottom flask on a magnetic'stirrer. sodium hydroxide solution (0.44 g of 3% on, 0.33 mmol), zobis(4-cyanovaieric acid) (14.1 mg, 0.05 mmol) and water (14.0 '30 g) were added and stirred to dissolved. To this mixture styrene (0.61 g, 5.85 mmol) was added and stirred overnight. The flask was then sealed and subsequently deoxygenated with nitrogen sparging‘ for 10 minutes. The whole flask was immersed in an oil bath with a temperature ~64~ setting of 70°C and maintained at that temperature for 6 hours under constant magnetic stirring.

A clear diSpersion was obtained Part (b): Synthesis of polystyrene nanOpam‘cIes using the‘macro-RAFT agent dispersion prepared in example 17 part (a) To a clear solution of macro-RAFT agent from e l7 part (a) (6.09 g) in a 50 mL round bottorn flask on a magnetic stirrer, sodium hydroxide on (0.36 g of 3% solution, 0.27 mmol), 4,4’-azobis(4-cyanovaleric acid) (26.6 mg, 0.095 mmol), styrene (0.45 g, 4.37 mmol) and water (8.31 g) were added. The flask was stirred for 5 hours, then sealed and subsequently deoxygenated with nitrogen ng for 10 minutes. The whole flask was immersed in an oil bath with a temperature setting of 70°C and maintained at that temperature for overnight under constant magnetic stirring. The latex contained particles with mean diameter of 47 nm by Zetasizer light scattering. The latex was ed against miIIi-Q water to remove impurities.

EXAMPLE 18 Synthesis of polystyrene nanoparticles in aqueous sion using po|y(acrylamide)zo macro raft agent Part (a): Preparation of poly(acrylamide)20 RAFT agent using: 2- {[(butylsulfanylkarbonothioyljsuyanylfpropanoic acid.

A solution of 2~{[(butylsulfanyl)carbonothioyI]sulfanyl}propanoic acid (0.73 g, 3.08 mmol), 4,4’-azobis(4-cyanovaleric acid) (0.07 g, 0.3 mmol), acrylamide (4.30 g, 60.5 mmol) in dioxane (15 g) and water (7.5 g) was prepared in a 100 mL round bottom flask. This was stirred magnetically and sparged with nitrogen for 15 minutes. The flask was then heated at 70°C for 4 hrs to produce the clear homopolyrner soiution.

Parr(b): Synthesis ofpolystyrene nanopam'cles using the maéro-RAFT agent prepared in example 18 part (a) A clear solution of macro-RAFT agent from example 18 part (a) (1.05 g), sodium hydroxide (2.07 g of 3% solution, 1.55 mmol) and water (12.16 g) was prepared in a 25 mL . round bottom flask, stirring on a magnetic stirrer. To this solution 4,4’-azobis(4-cyanova|eric acid) (13.6 mg, 0.049 mmol), dioxane (1.1 g) and styrene (1.125 g, 10.8 mmol) were added.

The mixture was stirred for 2 hours to obtain an emulsion like mixture. The flask was sealed and subsequently enated with nitrogen sparging for 10 s. The whole flask Was immersed in an oil bath with a temperature setting of 70°C and ined at that temperature for overnight under constant magnetic stirring. The latex contained les with average :65. diameter of 200nm by Zetasizer light scattering. The latex was dialysed against milli-Q water to remove impurities.

EXAMPLE l9 Stabilisation of iron oxide nanoparticles with dextran from leuconostoc mesenteroides (average molecular weight of 9000-1 1,000, sigma aldrich) coated particles. (example 19 is a comparative example) ml of 0.5 M FeCl2/4H20 and 25ml of IM FCC13/6H20 were mixed and magnetically stirred in a 500 ml 3 neck round bottom flask. The resulting solution was diluted by adding 100 ml of MQ water and placed in an oil bath at 70°C. Dextran solution (50 ml of IO lS% solids in water) was added and the solution maintained in the oil bath for 10 minutes.

Ammonia solution (30 ml, 28%) was then added and the mixture kept at 70°C for a further 45 minutes. The reaction product was cooled to room temperature and dialysed t MQ water to remove excess ammonia. The water was changed at least three times. Larger aggregates were removed by magnetic ntation. Volume was reduced to about 100 ml by removing water on rotary evaporator. The final dispersion was sonicated at 70% AMP using an ultrasonicator for 10 minutes and at also at 30% AMP for 30 minute.

EXAMPLE 20 Steric stabilization of iron oxide nanoparticles of example 1 part (a) using 50% poly(monoacryioxyethyl phosphate)io~block—poly(ethylene oxideln macro raft agent of example 2 part (b) and 50% amine d poly(monoacryloxyethyl phosphate).o-block- poly(acrylamid8)zo macro raft agent Part (a): ation of stert'cally stabilized iron oxide nanopartt'cles from the aqueous ferroflm'd of example 1 part (a) and a 50:50 blend of the macro-RAFT agent of example 2 part (b) and the macro-RAFT agent ple 2 part (c).

Aqueous ferrofluid prepared according to example 1, part (a) (8.!0 g) was diluted with MQ water (50 g) to yield a 0.5 wt% diSpersion of the nanoparticles. The pH of'this prepared rticle dispersion was then raised to 5. A blend of macro-RAFT which consist of 50 g of at 5-.l wt% , 3.3 wt% of which was the macro-RAFT agent of example 2 part (b) and i8 wt% of which was the macro-RAFT agent of example 2 part (c) were mixed together and the pH adjusted to 5 using 0.iM NaOH. The dimension of iron oxide. maintained at the same pH was then added to the RAFT blend. The mixture was usly stirred for 2 hours at r00m ature before the pH was adjusted to 7.0. The mixture was then left stirring for another 12 hours. At this pH the copolymer remained partially neutralized while the 2012/000414 nanoparticles were sufficiently above their point ’of zero charge to also be . The diSpersion was then dialysed to remove salts, residual solvents, unwanted low molecular weight on side products and unbound polymer. The solid content of the dialysed aqueous uid dispersion is 0.6%.

Part (f): Modification ofstabilisersfor iron oxide particles ofexample 20 part (a) Into coated nanoparticles prepared from example 20 part (a) (703), N- hydroxysuccinimide (NHS, 89.3mg) and then l-Ethyl-3—(3-Dimethylamino- propyl)carbodiimide (EDAC, 127mg) were added, mixed by g and allowed to react for 2 hours at room temperature. 29lmg of 2,2’-(Bthylenedioxy)bis-(ethylamine) was then added to. the reaction mixture and allowed to react for a further 12 hours. The solution was then dialysed t excess water with numerous changes, to remove free EDAC and the reaction byproducts.

EXAMPLE 2! Steric stabilization of iron oxide nanoparticles of example 1 part (a) using 80% poly(monoacryloxyethyl phosphate)w-block—poly(ethylene oxide)” macro raft agent of example 2 part (b) and 20% amine modified poly(monoacryloxyethyl phosphatem'block- . poly(acrylamide)2o macro raft agent Part (11): Preparation of stert‘cally ized iron oxide nanopam’ctes from the aqueous ferrofluid ofexample 1 part (a) and a 80:20 blend of the RAFT agent of example 2 part (b) and the macro-RAFT agent ofexample 2 part (c).

Aqueous ferrofluid prepared'according to example 1, part (a) (8. l0 g) was diluted with MQ water (50 g) to yield a 0.5 wt% dispersion of the nanoparticles. The pH of this prepared rticle dispersion was then raised to 5. A blend of macro-RAFT which consist of 50 g of at 6.0 wt% solids, 5.28 wt% of which was the macro-RAFT agent of example 2 part (b) and 0.72 wt% of which was the macro-RAFT agent of example 2 part (c) were mixed together and the pH adjusted to 5 using 0.lM NaOH. The sion of iron oxide, maintained at the same pH was then added to the macro-RAFT blend. The e was vigorously stirred for 2 hours at room temperature before the pH was ed to 7.0. The mixture was then left Stirring for another 12 hours. ,At this pH the cepolymer remained partially neutralized while the 3c nanoparticles were sufficiently above their point of zero charge to also be stable. The dispersion was then dialysed to 'remove salts, residual solvents, unwanted low molecular weight reaction side products and unbound polymer. The solid content of the dialysed aqueous ferrofluid dispersion is 0.7%.

WO 42669 Part (b): cation ofstabilisersfor iron oxide particles of example 21 part (a) late coated nanoparticles prepared from example 2, Part (C) (60g), N- hydroxysuccinimide (NHS, 39.4mg) and then l(3 -Dimethylamino~ pr0pyl)carbodiimide (EDAC, 56.2mg) were added, mixed by shaking and allowed to react for 2 hours at room temperature. 130mg of 2,2‘-(Ethylenedioxy)bis-(ethylamine) was then added to the reaction mixture and allowed to react for a further l2 hours. The solution was then dialysed against excess water with numerous changes, to remove free EDAC and the reaction by-products.

E 22 Steric stabilization of iron oxide nanoparticles of example 1 part (a) using 90% poly(monoacryloxyethyl phosphate).o-block~poly(ethylene oxidchv macro raft agent of example 2» part (b) and 10% amine modified poly(monoacryloxyethyl ate)w-block- cry_lamide)m macro raft agent Part (a): Preparation of stericaliy stabilized iron oxide nanoparticies from the aqueous ferrofluid of example ‘1 part (a) and a 90:10 blend of the macro-RAFT agent of example 2 part (b) and the macro-RAFT agent ofexample 2 part (c).

Aqueous ferrofluid prepared according to example I, part (a) (8.10 g) was d with MQ water (50 g) to yield a 0.5 wt% dispersion of the nanoparticles. The pH of this prepared nanoparticle dispersion was then raised to S. A blend of RAFT which consist of 50 g of at 6.3 wt% solids, 6.4 wt% of which was the macro-RAFT agent of e 2 part (b) and 05.9 wt% of which was the macro-RAFT agent of example 2 part (c) were mixed together and the pH adjusted to 5 using 0.1M NaOH. The dESpersion of iron oxide. ined at the same pH was then added to' the macro-RAFT blend. The mixture was vigorously stirred for 2 hours at room temperature before the pH was ed to 7.0. The mixture was then left stirring for another l2 hours. At this pH the copolymer remained partially neutralized while the .nanoparticles were sufficiently above their point of zero charge to also be stable. The dispersion was then dialysed to remove salts, residual solvents, unwanted low molecular weight reaction side products and unbound polymer. The solid content of the dialysed aqueous ferrofluid dispersion is 0.87%.

Part (b): Amine modification ofstabilisersfor iron oxide particles ofexample 22 part (a into coated nanoparticles prepared from example 2 part (a) (60g). N- hydroxysuccinimide (NHS, 24.7mg) and then l-Ethyl(3-Dimethylamino- prepyl)carbodiimide (EDAC, 34mg) were added, mixed by shaking and allowed to react for 2 .68.. hours at room temperature. l8.2mg of 2,2‘-(Ethylenedioxy)br‘s—(ethylaminc) was then added to the reaction mixture and allowed to react for a further i2 hours. The solution was then dialysed against excess water with numerous changes, to remove free EDAC and the reaction by- products.

EXAMPLE 23 Stabilisation of iron oxide nanoparticles with leuconostoc mesenteroides dextran (average molecular weight 35,000-45,000 from sigma aldrich) (example 23 is a comparative . example) . ml of 0.5 M FeCl2/4H20 in solutions and 25ml of 1M FeCl3/6H20 in solution was l0 magnetically stirred in a 500 ml 3 neck round bottom flask. The solution mixture was d by adding 100 ml of Mili-Q water and the resulting on placed in an oil bath at 70°C.

After 10 minutes dextran solution (15%. 50 ml) was then added followed by ammonia solution (28% 30 ml). The e was kept at 70°C for a further 45 minutes. The reaction mixture was cooled to room ature and dialysed against MQ water to remove excess l5 ammonia. The water was changed at least three times. Larger aggregates were d by magnetic sedimentation. The volume of the dispersion was reduced to about lOO ml by using a rotary evaporator. The final dispersion was sonicated at 70% AMFusing an ultrasoriicator for l0 s followed by sonication at 30% AMP for 30 minute.

EXAMPLE 24 Steric stabilization of iron oxide rticles of example 1 part (3) using 98% poly(monoacryloxyethyl phosphateho-block-polflethylene oxide)” macro raft agent and 2% amine modified poly(monoacryloxyethyl phosphate)m~hlock—poly(acrylamideho macro raft agent Part (a): Preparation of slerically stabilized iron oxide nanoparticles from the aqueous ferrofluid of example 1 part (a) and a 98:2 blend of (he macro-RAFT agent of example 2 part (b) and the macro-RAFT agent ofexample 2 part (c). [EP341063] Aqueous ‘luid prepared according to example 1, part (a) (8. 10 g) was diluted with MQ} water (50 g) to yield a 0.5 wt% diSpersion of the nanoparticles. The pH of this ed nanoparticle dispersion was then raised to 5. A blend of macro-RAFT which consist of 50 g of at 6.48 wt% solids, 6.4 wt% of which was the macro-RAFT agent of example 2 part (b) and 0.08 wt% of which was the macro~RAFT agent of example 2 part (c) were mixed together and the pH adjusted to 5 using 0.1M NaOH. The dispersion of iron oxide. maintained at the same pH was then added to the macro-RAFT blend. The e was vigorously stirred for 2 hours .69- left stirring for at room temperature before the pH was adjusted to 7.0. The mixture was then another 12 hours: At this pH the copolymer remained partially neutralized while the nanoPanicles were Sufficiently above their point of zero charge to also be stable. The sion was then ed to remove salts. residual ts. ed low molecular weight reaction side products and unbOUnd polymer. The solid content of the dialysed aqueous uid dispersion is 0.8%.

Part (b): Amine modification ofstabilisers ofiron oxide particles of example 24 part (0) Into coated nanoparticles prepared from example 24. Part (a) (55g), N- ysuccinimide (NHS. 5.1 'mg) and then I -Ethyl(3—Dimethylamino- propyl)carbodiimide (EDAC, 6.8 mg) were added, mixed by shakinglandallowed to react for 2 hours at room temperature. 2,2’-(Ethylenedioxy)bis-(ethylamine) (18.2 mg) was then added to the reaction mixture, which was allowed to react for a further 12 hours. The solution was then dialysed against excess water with numerous changes, to remove free EDAC and the reaction by-products.

EXAMPLE 25 General method for preparation. of spheroids Human DLD-l colon cancer cells and human PA-i ovarian cancer cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were ined in complete media (Advanced DMEM (lnvitrogen) and supplemented with 2% foetal bovine Serum (Sigma) and 2mM axTM (lnvitrogen)) at 37°C in a humidified, 5% C02 atmosphere. Spheroids were formed by g lthlOs cells/ml onto agarose coated 96 well imaging plates (BD Bioscicnces) and the cells allowed to aggregate for 72 hrs at 37°C in a humidified, 5% C02 atmosphere resulting in the formation of single id per well.

EXAMPLE 26 Assessment of cytotoxicity of active compounds and nanoparticles Active compounds and/or nanoparticles were diluted as required in cell media immediately prior to the assay. Cytotoxicity was determined using the MTT assay. as follows. i x 105 cells were seeded onto each well of flat bottomed 96-well plates and allowed to attach overnight. Solutions of compounds +/- nanOparticles were added to triplicate wells at concentrations spanning a 4—log range and incubated for 72 hrs. MTT (3»(4. 5- dimethylthiazolyl~2)-2, S»diphenyltetrazoiium e) (1.0 mM) was added to each well and were incubated for a further 4 hrs. The culture medium was removed from each well, DMSO (150 uL) was added, the plate shaken for 5 seconds and the absorbance measured immediately "at 600 nm in a Victor’V micrOplate reader (Perkin Elmer). {C50 values were determined as the drug concentration that reduced the absorbance to 50% of that in untreated control wells. At ,least three independent experiments were performed for each compound with triplicate readings in each experiment. Cytotoxicity values for all active compounds used are listed in Table 2.

EXAMPLE 27 l method for treating the cancer spheroids of e 5 with (a) na'noparticles alone, (b) inistration of nanopa rticles and active compound or (c) time course ent of nanoparticle first then active compound All nanoparticles were sterilised either by ion through a 0.22 pm filter or by autoclaving at l20°C, 2 KPa for 20 min in a Tomy high pressure steam sterilizer ES-3l5 before use in cellular assays. (a) To the suspension of the 3 day old spheroids from e 25, 100 pl of a solution containing nanoparticles incomplete media was added to each spheroid, to yield a final concentration of particles of lOppm- in 200 pl total volume. he spheroids were replaced in an incubator at 37°C. 5% 002. After 24 hours incubation, the nanoparticles in the media were removed by washing with excess ate buffered saline prior to further experimentation. (b) To the suspension of the 3 day old spheroids from example 25, 100 pl of a solution containing active compound and nanoparticles lete media was added to each spheroid, to yield a ‘final concentration of particles of lOppm. The tration of the active compounds used is defined in Table 3. The spheroids were replaced in an incubator 37°C. 5% C02 atmosphere. After 24 hours incubation, the free active compound and nanoparticles in the media were removed by washing with excess ate buffered saline. (0) To the su5pension of the 3 day old spheroids from example 25, l00 pl of a solution containing nanoparticles in complete media was added to each spheroid, to yield a final concentration of particles of l0ppm. The spheroids were replaced in an incubator at 37°C, 5% C02. After 24 hours incubation, the spheroids were closed with an active compound at the concentration listed in Table 2 and incubated for a further 24 hours at 37°C, 5% C02. The free 3O active nd and nanOparticles in the media were removed by washing with excess phosphate buffer saline prior to further mentation.

EXAMPLE 28 W0 2012/142669 .71.

General method for imaging spheroids treated with nanoparticles and a fluorescent active compound by confocal microscopy.

Spheroids from example 25 were d as per example 27b and 27c then transferred to a glass bottomed 35 mm dish (Mattek) and imaged on an Olympus FVIOOO confocal micrOSCOpe using an Olympus UPLAPO [Ox/0.40 air objective lens. Single confocal images h the central region of the Spheroid were taken. Excitation and on settings were fluorophore dependent: Doxorubicin ex.:559nm em:575-675; ntrone ex:405nm, em2575- EXAMPLE 29 General method for ing effectiveness of active compound +l- nanoparticles in spheroids (outgrowth assay) Spheroids from example 25 were treated as per example 27b and 27c. The spheroids were then transferred to a 24 well plate using a wide bore transfer pipette and the medium replaced with } mL of fresh media in each well. The spheroids were then incubated for 48 hours at 37°C in a 5% C02 humidified environment, allowing the spheroid to attach to the plate and the cells to grow out from the spheroid onto the surface of the plate. Hoechst 33342 was then added to the wells and incubated for 30 minutes at 37°C in a 5% C02 humidified environment. Widefield fluorescence images of the brightfield and Hoechst 33342 stained nuclei were taken ofthe cells that had grown out from the spheroid (Olympus CellR)‘ To quantitate the outgrowth, the number of nuclei within a 60° angle from the edge of the id was counted. These values were then plotted in a graph ised to ids treated with active compound alone or untreated control spheroids for comparison.

EXAMPLE 30 Sterically stabilised nanoparticles are able to penetrate into spheroids ids from example 25 were treated asper e 27a with les from example 2 and washed with phosphate buffered saline, followed by primary fixation with 2.5% glutaraldehyde solutionand secondary fixation with 1% osmium tetroxide, The spheroids were washed then dehydrated in a gradient of ethanol and infiltrated with Spurr’s Resin. Ultra-thin sections with a nominal thickness of 95 nm were cut. placed on mesh grids and post stained with uranyl acetate and lead e. TEM images of the spheroid sections were obtained using a JEOL I400 TEM at 120 kV.

The images in Figure l were taken from the central regiOn ofthe id and show an acoumulation of nanoparticles (darker stained areas as indicated with arrows) within the cytoplasm of the cells. The enlarged region indicated by the box shows the well dispersed individual'nanoparticles.

EXAMPLE 31 The effect of nanoparticles on drug diffusion DLD-l spheroids prepared as were dosed the protocol in per example 25 as per example 27a with nanoparticles from es 2, 3, and _S) and imaged under ions described in example 28. Dox0rubicin alone diffused approximately 70 um into the spheroid.

Co-administration of doxorubicin and NP3 or 5 enhanced the id penetration of doxorubicin to approximately IOO pm. in contrast; co-administration ofNP2 and doxorubiein resulted in doxorubiein diffusion throughout the entire spheroid (Figure 2A). Mitoxantrone aloue also diffused approximately 70 um into the spheroid. Cooadministration of NP3 and mitoxantrone had little effect on mitoxantrone diffusion, whereas co-administration of mitoxantrone and NP2 or NPS significantly enhanced the diffusion of mitoxantrone into’ the spheroid (Figure 23).

EXAMPLE 32 The effect of nanoparticles on spheroid viability DLD—l spheroids ed as per example 25 were dosed as per the protocol in e 27a with nanopaniclesfrom examples 1, 2, 4, 8, 9, l2, l3. 15, l6, and l8. The effectiveness of nanoparticles alone in spheroids was assessed as per example 29. it was found that the majority of nanoparticles tested had little cytotoxic effect as shown in Figure 3.

EXAMPLE 33 The effect of nanoparticies with different core types on the viability of spheroids when co-administered with doxorubiein.

DLD-l spheroids prepared as per e 25 were dosed as per the protocol in example 27b with nanoparticles from examples 2, 4, 6, 7, 9, 10, i1, 12, l3, I4, 16, 17, and 18 and doxorubicin. Effectiveness was determined as per e 29. Figure 4 shows that inistration of nanoparticles‘NP2 (iron core), NPl 1(silica core), NPIZ (gold core), and NP18 (polystyrene core) with doxombicin was more effective than doxorubicin treatment alone as shown by the decreased cellular wth from the ids. The composition of the nanopatticle core does not ate with effectiveness, EXAMPLE 34 W0 2012/142669 s73. when The effect of nanoparticles with ent core sizes on the viability of spheroids ninistered with doxorubicin.

DLD-l spheroids prepared as per example 25 were dosed as per the protocol in example 27b with nanoparticles from examples I, 2, 4, 7f, 9, 10, ll, 12, l3, l4, l6, l7, and 18 and doxorubicin. Effectiveness was determined as per example 29. Several different nanoparticles with a range of core sizes from 10 rim to 200nm when inistered with doxorubicin were shown to be more effective than doxorubicin alone (Figure 5).It was shown that co-administration of les NP]. NP2, NPIZ and NP! 8 co~administered with doxorubicin was approximately 50% more effective than doxorubicin treatment alone.

‘ EXAMPLE 35 The effect of the functionalised stabiliser end group on spheroid viability when co- administered with doxorubicin.

DLD-I ‘spheroids prepared as per e 25 were dosed as per the protocol in example 27b with nanopanicles listed in examples 2, 3, 4, 5, 20, 21, 22, and 24 and doxorubicin. Effectiveness was determined as per e 29. It was found thatthe amine functionalised end group effected spheroid viability when co-administered with doxorubicin. By varying the tage of amine functionalised groups on the surface of the nanoparticles, we found that particles containing between 5-20% amine functionalised end groups were the most effective when co—administered with doxorubicin. Doxorubicin ' was the most ive when co-administered with nanopanicles containing stabilisers with % amine onalised end groups.

EXAMPLE 36 Nanoparticles of different cores stabilised with 5% amine functionalised end groups co-administered to spheroids with doxorubicin.

DLD~1 spheroids prepared as per example 25 were dosed as per the protocol in example 271) with nanoparticles listed in examples 2, 8, 9 and 12 and doxorubicin.

Effectiveness was determined as per example 29. Nanoparticles stabilised with 5% amine functionalised end groups were made with different cores and it was shown that all were more effective than doxorubicin alone and had a similar'level of iveness when co- 3O administered with doxorubicin (Figure 7).

EXAMPLE 37 2012/000414 The effect of the active compounds when co-administered with nanOparticles on the viability of spheroids made from two different cancer cell lines.

DLD~l and PA-l spheroids were prepared as per example 25 and dosed as per the _ protocol in example 271) with nanoparticles listed in examples 2. 3, 4, and 5 and active cornpounds. Effectiveness was determined as per example 29. The majority of particles and active compounds had similar effectiveness between the two cell lines, with the exception of mitOXantrone. inistration of mitoxantrone and nanoparticles was significantly more effective in the PA~l cell ovarian cancer cell line compared to the 01D- 1 colorectal cancer line.

EXAMPLE 38 Comparative example between co-administration of rticles and active nds, and administration of rticles with delayed administration of. active compounds in two different cell lines.

DLD-l and PA-l spheroids were prepared as per example 25 and closed as per the protocol in example 27b and 270 with the rticles listed in examples 2. 3, 4, and 5 and active compounds. Effectiveness was determined as per example 29. Figures 9 and 10 show that for some le and active combinations e.g. 5FU+NP3 there is no difference in effectiveness in either cell line for either mode of treatment. In general however, there is little correlation n ent schedule and effectiveness n the two cell lines tested. It will be important to determine which nanoparticle/active combination is most effective for each cancer type. It should be noted that mitoxantrone requires the co- administration of nanoparticles in PA-l cells for greatest effectiveness.

EXAMPLE 39 Examples of the most effective co-administered combination of rticlcs and ‘ active compound for each active compound tested.

DLD-l and PA-l spheroids were prepared as per example 25 and dosed as per the protocol in example 27b with the nanoparticles listed in examples 2, 5, M, 20, 2}, and 22 and active compounds. Effectiveness was determined as per example 29. The results presented are for the most effective nanoparticle(s) co-administered with each active compound in both DLD-l spheroids (Figure HA) and PA-l spheroids (Figure 118).

EXAMPLE 40 Treating the cancer spheroids of example 25 with the iron oxide rticles of es 1 and 2 to enable spheroid penetration by cisplatin.

DLD-l spheroids prepared as per example 25 were dosed as with 100 pl of solution of complete media containing cisplatin and iron oxide nanoparticles from examples 1 and 2 to yield a final tration of both tin and iron oxide of 6ppm, The spheroids with iron oxide particles and cisplatin were replaced in the tor and maintained at 37°C in a 5% C02 atmosphere. After 48 hours incubation, the free cisplatin and nanoparticles in the media were washed with excess phosphate buffered saline. Analysis by atomic absorption spectroscopy showed that after 48 hours incubation the concentration of ciSplatin in ”the spheroids with NP] nanoparticles, NPZ nanoparticles and without iron oxide particles was 0.60, 0.63 and 020 ppb, respectively, a 3-fold increase in cisplatin accumulation when nanoparticles were present.

E 4! Comparative example: Doxorubicin penetration into spheroids when co-ad ministered with anchored sterically'stabilised particles compared to co-administration with unanchored sterically stabilised particles.

DLD-l spheroids from example 25 were either dosed with NP2 le 2), which are particles coated with a stabiliser containing a ate anchoring group or NPl9 or NP23 les l9 and 23), which are particles coated with a stabiliser that has no anchoring portion as per example 27b. The spheroid was then imaged by confocal micr05copy (as per example 28) to visualise doxorubicin fluorescence. Spheroids treated with doxorubicin and NH had significantly more doxorubicin fluorescence in the centre of the spheroid compared to the spheroids treated with doxorubicin alone and to spheroids treated with doxorubicin co—administered with the unanchored sterically-stabilised particles NPl9 and NP23 (Figure :2).

EXAMPLE 42 Potential testing regime to determine the most effective nanoparticle and active compound for patient tumours.

To identify which ) of nanoparticles and which ) of active drug and an optimum combination of nanoparticles and drug were the most ive for an individual patient, tumour biopsies would initially be tested. Several core tumour biopsies would be ~76 - taken from a patient, dissected into smaller s (approx lmml) and closed with selected nanopanicle/drug combinations. Each dosed sample would be flanked by an untreated sample and a drug only to control for intra-tumour variability. After 24hrs= the sample would be ted to an outgrowth assay to measure the efficacy of the tumour treatments with nanoparticles/drug to determine the most ive compOSition and administration of nanoparticles and drug: Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described, it is to ‘ be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, es, compositions and compounds referred to or indicated in this cation, individually or collectively, and any and all combinations of any two or more of said steps or features. -77.

Table 1. LIST OF NANOPARTICLES USED TO EXEMPLIFY THIS PATENT NP core, diameter Fe203, IO-ISQm F6203, 10-15nm I Fc203,10-15nm NP4 F6203, 10-1Snm F6203, 10-15nm 100% NH; 2"Uas F6203, 10»15nm m Fezol, 30.4mm i"Uas 3:02, msnm —-ssoz, m “m 8102, I30nm 100% C00 u- 8102, 130nm 100% PEGAcr late I—I N Zw I- N Goid, 10—15nm 95%PEG 5%NH2 Gold, 3»8nm ’ 100% C00 NPI4 Gold, 10~15nm . 100% C00 NPIS Gold, m E vs: ,~nsnm Pst ,~4onm PSI , ~200nm F6203, 1045“!“ mo}, no-lsnm F6203» 1045"?" F8203, 10-15nm 90% PEG 10% NH: Fe203, IO-ISnm 40K Dextran Fe203, 10-15nm 98% PEG 2% NH; WO 42669 .73-~ Table 2: 72hr ICso values for the active compounds used in this study.

*Paclitaxcl was cytotoxic at concentrations as low as 0.] nM in this assay.

Table 3: Concentrations of active compounds used for dosin‘g in each cell line.

W0 2012/142669 BIBLIOGRAPHY Arias et al. 2003. National Vital Statistics Reports 5231 I M I 5 Bender et al., Cancer Research 52:121-126, I992 Bria-Cunningham ei al, Journal ofNuclear Medicine 44: I 945- I 96l , 2003 Christiansen et al. Molecular Cancer Therapy 3: I493- I 501, 2004 Dadachova ei al. PNAS [01:I4865-i4870, 2004 Griffiths el al. International Journal of Cancer 8 I 92. 1999 Lawrence TS. Oncology (Huntington) I7:23-28. 2003 Liu et al. Bioconjugaie Chemistry 12:7-34, 2001 Massarl, Preparation ofaqueous ic liquids in alkaline and acidic . IEEE Transactions on Magnetics, I981. MAG—17(2): p. 1247-1248 Minchinton. A. l. and Tannock, I. F., Nat. Rev. Cancer 2006, 6:583-592 O’Donoghue et al. Journal ofNuclear Medicine 36: I 902-] 909, I995 Primeau el al. Clin. Canc. Res. 2005, 11:8782-8788 Sellers et al. Journal ofClinical Investigation I0421655-166I, I999 Waldmann. Science 252: I 657-1662, I991 Xue et al. PNAS 99: 13765-1 3770, 2002 - 30 _

Claims (23)

CLAIMS 1.: cellular toxin effective

1 . Use oftwo separate formulations of a particulate material and a in the manufacture ofa medicament for the to penetrate a solid tumour and induce xicity is in the form of a dispersion in a treatment ofa solid tumour n said particulate material the diSpersed state by a steric liquid carrier, the particulate material being maintained in and a steric stabilising stabiliser; wherein said steric stabiliser comprises an anchoring portion polymeric segment, wherein the anchoring portion is different from the steric ising towards the e of polymeric segment; and wherein the anchoring portion has an affinity material. the ulate material and secures the stabiliser to the particulate

2. The use according to claim 1, wherein the steric stabiliser has an average molecular weight of from about 1,000 to about 60,000 average molecular weight (Mn).

3. The use ing to claim 2, wherein the steric stabiliser has an average molecular weight ranging from about 1,000 to about 30,000 Mn.

4. The use according to claim 3, wherein the steric stabiliser has an average lar weight ranging from about 1,000 to about 5,000 Mn.

5. The use according to any one ofclaims 1 to 4, n the steric stabilising polymeric segment comprises a terminal ionic functional group.

6. The use according to claim 5, wherein the ionic onal group is a cation.

7. The use according to any one ofclaims 1 to 6, wherein the steric stabilising polymeric segment of the stabiliser comprises polymer selected from poly(acrylamide), thylene oxide), poly(hydroxyethylacrylate), poly(N—isopropylacry1a1nide), poly(dimethy1arninoethyl methacrylate), polyvinyl pyrrolidone), and copolymers thereof.

8. The use according to any one ms 1 to 7, wherein the steric stabilising polymeric segment has no more than about 50 polymerised monomer units that collectively form the H.\l'ml\[mcnruvcxuNRPanthCOFMNZbZ78L! docs~l7l09f2015 .8], segment.

9. The use according to any one of claims 1 to 8, wherein the anchoring portion one or more comprising one or more carboxylic acid groups, one or more phosphate groups, one or phosphinate groups, one or more thiol groups, one or more thiocarbonylthio , or combinations thereof. more sulfonic acid groups, ethoxysilyl ,

10. The use according to any one of claims 1 to 9, wherein one or both of the steric e of one or stabilising and ing polymeric ts comprise the polymerised more ethylenically unsaturated monomers.

11. The use ing to any one of claims 1 to 10, wherein the particulate material is selected from a metal, a metal alloy, a metal salt, a metal complex, a metal oxide, an inorganic oxide, a radioactive isotope, a polymer particle, and combinations thereof.

12. The use according to any one ofclaims 1 to 1 1, wherein the particulate material ranges in size from about IOnm to about 350nm.

13. The use according any one ofclaims 1 to 12, wherein said particulate material is iron.

14. The use according any one ofclaims 1 to 12, wherein said particulate material is gold.

15. The use according any one of claims 1 to 12, wherein said particulate al is siliCOn oxide.

16. The use according any one of claims 1 to 12, wherein said particulate material is yrene ranging in size from about lOnm to about 15nm.

17. The use according to any one of Claims 13 to 16, wherein said particulate material ranges in size from about 10 nm to about 15 nm.

18. The use according to Claim 15, wherein said particulate material ranges in size from H:\fnn\lnlcmot'anRPoflleCOFMT‘RZGZ73L] docs-I‘lltlv/mls , 32 _ about 30 nm to about 40 nm. material is co-

19. The use ing to any one ofClaims 1 to 18, wherein said particulate administered with from the one or more ar toxins ed group comprising: and/0r RNA interference herapy, targeted therapy, immunotherapy, radiotherapy therapy.

20. The use according to claim 19, wherein said chemotherapy is selected from actinomycin D, adriamycin, arsenic de, asparaginase, bleomycin, busulfan, camptosar, carboplatinum, carmustine, chlorambucil, cisplatin, corticosteroids, colicheamicin, cyclophosphamide, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabina, gemcitabine, gemzar, hydroxyurea, idarubicin, ifosfamide, ecan, lomustine, lan, mercaptomurine, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, platinol, platinex, procarbizine, raltitrexeel, rixin, steroids, streptozocin, taxol, taxotere, thioguanine, thiotepa, tomudex, topotecan, treosulfan, trihydrate, vinblastine, vincristine, vindesine, vinorelbina, vinorelbine, duanomycin, dactinomysin, esorubisin, mafosfamide, cytosine arabinoside, bis~chlor0ethylnitrosurea, mitomycin C, mitln'amycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, hexamethylmelamine, pentamethylmelamine, amsacrine, chlorambudil,, methylcyclohexylnitrosurea, nitrogen mustards, cyclophosphamide, aptopurine, 6~thioguanine, cytarabine, S—azacytidine, deoxyco-formycin, 4-hydroxyperoxycyclophosphoramide, S-fiuorouracil (S-FU), S-fluorodeoxyuridine (S-FUdR), cine, trimetrexate, teni-poside, and lstilbestrol.

21. The use according to claim 20, wherein said chemotherapy is selected from the group comprising doxorubicin, mitoxantrone, cisplatin, paclitaxel and S-FU.

22. The use according to claim 19, wherein said RNA erence therapy is an RNA oligonucleotide.

23. The use according to any one of claims 1 to 22, wherein said particulate material or steric stabiliser r comprises a ligand directed to said solid tumour. Ht\fuu\lmumor:u\NRPunbl\DCC\FMm2627XZ_ | .docx-