Pharmaceutical composition for drug delivery

Abstract

The present invention relates to a pharmaceutical composition comprising nanoparticles of a cyanine dye and a therapeutic agent. The present invention also relates to the use of said pharmaceutical composition in therapy.

Description

PHARMACEUTICAL COMPOSITION FOR DRUG DELIVERY

[0001] The present invention relates to a pharmaceutical composition comprising nanoparticles of a cyanine dye and a therapeutic agent. The present invention also relates to the use of said pharmaceutical composition in therapy.BACKGROUND

[0002] Senescence is a cellular damage and stress response that triggers a halt in division and the secretion of proinflammatory cytokines and chemokines to aid in the repair of tissue. However, in instances where senescent cells are not cleared by the immune system or upon persistent cellular damage, organ and organismal dysfunction can occur. Senescent cells have been associated with the ageing process as well as the progression of pathological manifestations of multiple diseases such as fibrotic disorders (e.g., pulmonary and kidney fibrosis), cardiovascular diseases (e.g., atherosclerosis), neurological disorders (Alzheimer's and Parkinson disease), skeletomuscular disorders (osteoarthritis and sarcopenia), diabetes 1 and 2, and many different cancer types. The presence of senescent cells has been demonstrated in all stages of cancer development, including initiation, although these cells can commonly play different roles, depending on the context. For example, in pre-cancerous lesions such as prostatic intraepithelial neoplasia (PIN) or lung adenomas, they are believed to halt the progression into full blown cancer. However, these cells can also create pro-tumourigenic environments and can contribute to relapse I formation of metastatic cancer cells in tumours by modulating the microenvironment. Such roles, commonly related to the persistence of uncleared senescent cells, have also been demonstrated in cancer tissue after chemotherapy treatment where the accumulation of senescent cells leads to development of a pro-tumourigenic environment, often leading to the cancer relapse after treatment. Furthermore, the unspecific nature of current chemotherapeutic strategies means that senescent cells can also be formed outside the initial tumor site, which might lead to undesired side effects and secondary cancer formation, even later in life. Thus, senescent cells are becoming an important target not only for treatment of age-related diseases but also to reduce harmful side effects of chemotherapies and prevent the formation of secondary cancers post treatment, and even as a cancer preventative strategy in groups at high cancer risk.

[0003] Reports have shown that in some types of cancers, Idiopathic pulmonary fibrosis and Alzheimer’s disease, the elimination of senescent cells can lead to more desirable treatment outcomes and health spans. For example, senescent cells targeted by a combination ofDasatinib and quercetin led to an improvement in physical function of patients as well as a reduction in senescent markers. Moreover, it has been shown in mouse models of aging that targeting and eliminating senescent cells improved lifespan by 30% while at the same time reducing the mortality hazard by 65%. In spite of this, there remains a number of challenges hindering therapeutic interventions at the senescent cell level, such as the accuracy of senescent cell targeting strategies as well as therapeutic efficacy once at the target site.

[0004] There is therefore a need to improve senescent cell treatment, in particular the ability for a therapeutic agent to selectively target senescent cells. The present invention was devised with the foregoing in mind.BRIEF SUMMARY OF THE DISCLOSURE

[0005] According to a first aspect, the present invention provides a pharmaceutical composition comprising: nanoparticles of a cyanine dye; and a therapeutic agent, wherein the therapeutic agent is associated to the nanoparticles.

[0006] According to a further aspect, the present invention provides a pharmaceutical composition according to the first aspect for use in therapy.

[0007] According to a further aspect, the present invention provides a pharmaceutical composition according to the first aspect for use as a medicament.

[0008] According to a further aspect, the present invention provides a pharmaceutical composition according to the first aspect for use as a senolytic.

[0009] According to a further aspect, the present invention provides a pharmaceutical composition according to the first aspect for use in the treatment of a disease or disorder in which the presence of senescent cells is implicated.

[0010] According to a further aspect, the present invention provides a pharmaceutical composition according to the first aspect for use in the treatment of a proliferative disorder.

[0011] According to a further aspect, the present invention provides a pharmaceutical composition according to the first aspect for use in the treatment of cancer.

[0012] According to a further aspect, the present invention provides a pharmaceutical composition according to the first aspect for use in the treatment of a pre-malignant lesion, a tumour (e.g., a pre-malignant tumour, a malignant tumour or a tumour following treatment-induced (e.g., chemotherapy) cellular senescence), an age-related disorder (e.g., chronic disorders such as cancer, cardiovascular diseases (e.g., atherosclerosis), fibrosis (e.g., pulmonary fibrosis, idiopathic pulmonary fibrosis and kidney fibrosis), neurological disorders (e.g., Alzheimer’s and Parkinson’s disease), Type 1 & 2 diabetes, skeletomuscular disorders (e.g., sarcopenia, osteoarthritis and osteoporosis), inflammatory diseases, chronic obstructive pulmonary disease (COPD), infarction, aneurysm, cataracts, post-infarction tissues, frailty, chronic kidney disease, diabetic macular degeneration etc.) or a wound.

[0013] According to a further aspect, the present invention provides a method of treating a disease or disorder in which the presence of senescent cells is implicated in a patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition according to the first aspect.

[0014] According to a further aspect, the present invention provides a method of treating a proliferative disorder in a patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition according to the first aspect.

[0015] According to a further aspect, the present invention provides a method of treating cancer in a patient in need of such treatment, said method comprising administering to said patient in need of such treatment a therapeutically effective amount of a pharmaceutical composition according to the first aspect.

[0016] According to a further aspect, the present invention provides a method of treating a pre-malignant lesion, a tumour (e.g., a pre-malignant tumour, a malignant tumour or a tumour following treatment-induced (e.g., chemotherapy) cellular senescence), an age- related disorder (e.g., chronic disorders such as cancer, cardiovascular diseases (e.g., atherosclerosis), fibrosis (e.g., pulmonary fibrosis, idiopathic pulmonary fibrosis and kidney fibrosis), neurological disorders (e.g., Alzheimer’s and Parkinson’s disease), Type 1 & 2 diabetes, skeletomuscular disorders (e.g., sarcopenia, osteoarthritis and osteoporosis), inflammatory diseases, chronic obstructive pulmonary disease (COPD), infarction, aneurysm, cataracts, post-infarction tissues, frailty, chronic kidney disease, diabetic macular degeneration etc.) or a wound in a patient in need of such treatment, said method comprising administering to said patient in need of such treatment a therapeutically effective amount of a pharmaceutical composition according to the first aspect.

[0017] According to a further aspect, the present invention provides the use of a pharmaceutical composition according to the first aspect in the manufacture of a medicament for the treatment of a disease or disorder in which the presence of senescent cells is implicated.

[0018] According to a further aspect, the present invention provides the use of a pharmaceutical composition according to the first aspect in the manufacture of a medicament for the treatment of a proliferative disorder.

[0019] According to a further aspect, the present invention provides the use of a pharmaceutical composition according to the first aspect in the manufacture of a medicament for the treatment of cancer.

[0020] According to a further aspect, the present invention provides the use of a pharmaceutical composition according to the first aspect in the manufacture of a medicament for the treatment of a pre-malignant lesion, a tumour (e.g., a pre-malignant tumour, a malignant tumour or a tumour following treatment- induced (e.g., chemotherapy) cellular senescence), an age-related disorder (e.g., chronic disorders such as cancer, cardiovascular diseases (e.g., atherosclerosis), fibrosis (e.g., pulmonary fibrosis, idiopathic pulmonary fibrosis and kidney fibrosis), neurological disorders (e.g., Alzheimer’s and Parkinson’s disease), Type 1 & 2 diabetes, skeletomuscular disorders (e.g., sarcopenia, osteoarthritis and osteoporosis), inflammatory diseases, chronic obstructive pulmonary disease (COPD), infarction, aneurysm, cataracts, post-infarction tissues, frailty, chronic kidney disease, diabetic macular degeneration etc.) or a wound.

[0021] According to a further aspect, the present invention provides a process for preparing a pharmaceutical composition comprising nanoparticles of a cyanine dye and a therapeutic agent, the process comprising the steps of:(i) providing an aqueous solution of the cyanine dye;(ii) maintaining the aqueous solution of the cyanine dye at a temperature of between 15 °C to 85 °C, preferably with agitation, to allow the cyanine dye molecules to aggregate and form nanoparticles;(iii) collecting and purifying the resultant nanoparticles present in the solution; and(iv) mixing the purified nanoparticles with a therapeutic agent, wherein step (iii) comprises at least one separation step wherein the nanoparticles formed in step (ii) are separated from the aqueous solution and re-suspended or dispersed in a different aqueous medium. Suitably, step (iii) comprises at least one centrifugation step wherein the solution is centrifuged to form a pellet of the nanoparticles, removing the supernatant, optionally washing the pellet, and re-suspending the pellet in a different aqueous medium.

[0022] In the above-outlined aspects of the invention, the proliferative disorder is suitably cancer and the cancer is suitably a human cancer. Most suitably, the cancer includes those in which cellular senescence has developed (e.g., following treatment, such as chemotherapy, of a tumour). Any suitable cancer may be targeted (e.g., adenoid cystic carcinoma, adrenal gland tumour, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia-telangiectasia, Beckwith-Wiedemann Syndrome, bile duct cancer (cholangiocarcinoma), Birt-Hogg-Dube Syndrome, bladder cancer, bone cancer, brain stem glioma, brain tumour, breast cancer, Carney Complex, central nervous system tumours, cervical cancer, colorectal cancer, Cowden Syndrome, craniopharyngioma, desmoplastic infantile ganglioglioma, ependymoma, oesophageal cancer, Ewing sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, familial GIST, familial malignant melanoma, familial non-VHL clear cell renal cell carcinoma, familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumour - GIST, germ cell tumour, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary diffuse gastric cancer, hereditary leiomyomatosis and renal cell cancer, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papillary renal carcinoma, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumour, laryngeal and hypopharyngeal cancer, leukaemia (acute lymphoblastic leukaemia (ALL), acute myeloid leukaemia (AML), B-cell prolymphocytic leukaemia, hairy cell leukaemia, chronic lymphocytic leukaemia (CLL), chronic myeloid leukaemia (CML), chronic T-cell lymphocytic leukaemia, eosinophilic leukaemia), Li-Fraumeni Syndrome, liver cancer, lung cancer (non-small cell lung cancer, small cell lung cancer), Lymphoma (Hodgkin, non-Hodgkin), Lynch Syndrome, mastocytosis, medulloblastoma, melanoma, meningioma, mesothelioma, multiple endocrine neoplasia Type 1 & 2, multiple myeloma, MUTYH (or MYH)-associated polyposis, myelodysplastic syndromes (MDS), nasal cavity and paranasal sinus Cancer, nasopharyngeal Cancer, neuroblastoma, neuroendocrine tumours (e.g., of the gastrointestinal tract, lung or pancreas), neurofibromatosis Type 1 & 2, nevoid basal cell carcinoma syndrome, oral and oropharyngeal cancer, osteosarcoma, ovarian / fallopian tube I peritoneal cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers Syndrome, pheochromocytoma, paraganglioma, pituitary gland tumour, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., Kaposi or soft tissue), skin cancer, small bowel cancer, stomach cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis complex, uterine cancer, vaginal cancer, Von Hippel-Lindau syndrome, vulvar cancer, Waldenstrom’s macroglobulinemia, Werner syndrome, Wilms Tumour and xeroderma pigmentosum). Particular cancers of interest include haematological cancers such as lymphomas (including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL),Burkitt lymphoma (BL) and angioimmunoblastic T-cell lymphoma (AITL)), leukaemia’s (including acute lymphoblastic leukaemia (ALL) and chronic myeloid leukaemia (CML)), multiple myeloma, breast cancer, non-small cell lung cancer (NSCLC), colorectal cancer, endometrial cancer, gastro-oesophageal cancer, neuroendocrine cancers, osteosarcomas, prostate cancer, pancreatic cancer, small intestine cancer, bladder cancer, rectal cancer, cholangiocarcinoma, CNS cancer, thyroid cancer, head and neck cancer, oesophageal cancer, and ovarian cancer.DETAILED DESCRIPTIONDefinitions

[0023] Unless otherwise stated, the following terms used in the specification and claims have the following meanings set out below.

[0024] It will be understood that the terms “pharmaceutical composition” and “composition” can be used interchangeably, with both referring to the pharmaceutical composition of the present invention.

[0025] The term "aryl" or “aromatic” as used herein means an aromatic ring system comprising 6, 7, 8, 9 or 10 ring carbon atoms. Aryl is often phenyl but may be a polycyclic ring system, having two or more rings, at least one of which is aromatic. This term includes reference to groups such as phenyl, naphthyl and the like.

[0026] The term “heteroaryl” or “heteroaromatic” means an aromatic mono-, bi-, or polycyclic ring incorporating one or more (for example 1-4, particularly 1 , 2 or 3) heteroatoms selected from nitrogen, oxygen or sulfur. Examples of heteroaryl groups are monocyclic and bicyclic groups containing from five to twelve ring members, and more usually from five to ten ring members. The heteroaryl group can be, for example, a 5- or 6-membered monocyclic ring or a 9- or 10- membered bicyclic ring, for example a bicyclic structure formed from fused five and six membered rings or two fused six membered rings. Each ring may contain up to about four heteroatoms typically selected from nitrogen, sulfur and oxygen. Typically, the heteroaryl ring will contain up to 3 heteroatoms, more usually up to 2, for example a single heteroatom.

[0027] The term “carbocyclyl”, “carbocyclic” or “carbocycle” means a non-aromatic saturated or partially saturated monocyclic, or a fused, bridged, or spiro bicyclic carbocyclic ring system(s). Monocyclic carbocyclic rings contain from about 3 to 12 (suitably from 3 to 7) ring atoms. Bicyclic carbocycles contain from 7 to 17 carbon atoms in the rings, suitably 7 to 12 carbon atoms, in the rings. Bicyclic carbocyclic rings may be fused, spiro, or bridged ring systems.

[0028] The term “heterocyclyl”, “heterocyclic” or “heterocycle” means a non-aromatic saturated or partially saturated monocyclic, fused, bridged, or spiro bicyclic heterocyclic ring system(s). Monocyclic heterocyclic rings contain from about 3 to 12 (suitably from 3 to 7) ring atoms, with from 1 to 5 (suitably 1 , 2 or 3) heteroatoms selected from nitrogen, oxygen or sulfur in the ring. Bicyclic heterocycles contain from 7 to 17 member atoms, suitably 7 to 12 member atoms, in the ring. Bicyclic heterocyclic(s) rings may be fused, spiro, or bridged ring systems.

[0029] The term “substituted” as used herein in reference to a moiety means that one, two, three, four or more positions on the moiety are substituted. Preferably, “substituted” as used herein in reference to a moiety means that 1 , 2 or 3, of the hydrogen atoms in said moiety are replaced independently of each other by the corresponding number of substituents. Even more preferred, “substituted” as used herein in reference to a moiety means that 1 or 2, of the hydrogen atoms in said moiety are replaced independently of each other by the corresponding number of substituents.

[0030] It will, of course, be understood that substituents are only at positions where they are chemically possible, the person skilled in the art being able to decide (either experimentally or theoretically) without inappropriate effort whether a particular substitution is possible.

[0031] Throughout the entirety of the description and claims of this specification, where subject matter is described herein using the term “comprise” (or “comprises” or “comprising”), the same subject matter instead described using the term “consist of” (or “consists of” or “consisting of’) or “consist essentially of” (or “consists essentially of’ or “consisting essentially of’) is also contemplated.

[0032] Furthermore, throughout the entirety of the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0033] When a component is described as “consisting essentially of’ a subsequently recited material, it is to be understood that the component concerned may contain 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, of the subsequently recited material.

[0034] Features described in conjunction with a particular aspect, embodiment or example of the present invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless clearly incompatible therewith. All of thefeatures disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The present invention is not restricted to the details of any of the specific embodiments recited herein. The present invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.Pharmaceutical compositions

[0035] In accordance with a first aspect, the present invention provides a pharmaceutical composition comprising: nanoparticles of a cyanine dye; and a therapeutic agent, wherein the therapeutic agent is associated to the nanoparticles.

[0036] Through rigorous investigations, the inventors have found that pharmaceutical compositions of the present invention are particularly well suited for targeting senescent cells and treating diseases I disorders associated with senescent cells. In particular, it has been found that nanoparticles of a cyanine dye can be used as efficient drug-delivery systems due to their high affinity for senescent cells. Association of a therapeutic agent to the nanoparticles allows for the therapeutic agent to be efficiently transported to senescent cells. Once at the desired location, the therapeutic agent dissociates from the nanoparticles of the cyanine dye, thereby releasing the therapeutic agent.

[0037] Cyanine dyes are known in the art and will be understood to relate to compounds which typically comprise a polymethine chain attached to at least one organic moiety such as a heterocycle, heteroaryl, aryl and / or a carbocycle. It will, of course, be understood that these are merely exemplary organic moieties, and the invention is not limited in this regard. Rather, the term cyanine dye will be understood to encompass compounds which typically comprise a polymethine chain (of any length) attached to at least one organic moiety. For example, it may be that the polymethine chain is attached to at least one organic moiety such as pyrrole, imidazole, thiazole, pyridine, quinoline, indole and / or benzothiazole, any of which may be optionally substituted. It may also be that the polymethine chain is attached to at least one organic moiety such as an aryl, a heteroaryl, a carbocycle or a heterocycle, any of which may be optionally substituted. The at least one organic moiety may be attached to the polymethine chain in any manner and on any atom of the polymethine chain. Typically,the at least one organic moiety is a terminal group on the polymethine chain. When the at least one organic moiety is a terminal group on the polymethine chain, the cyanine dyes can be classified as closed chain cyanines (two ring organic moieties (e.g., heterocycles) are present at the terminal ends of the polymethine chain), hemicyanines (one ring organic moiety and one non-ring organic moiety are present at the terminal ends of the polymethine chain), streptocyanines (two non-ring organic moieties are present at the terminal ends of the polymethine chain) and merocyanines (one amino group and one carbonyl group are present at the terminal ends of the polymethine chain). Suitably, the cyanine dye is a closed chain cyanine, a hemicyanine, a streptocyanine or a merocyanine. Most suitably, the cyanine dye is a closed chain cyanine.

[0038] The cyanine dye may comprise a polymethine chain of any length. Suitably, the cyanine dye comprises a polymethine chain of 1-20 chain carbon atoms. Chain carbon atoms refer to the minimum number of carbon atoms in the chain directly linking the organic moieties. More suitably, the cyanine dye comprises a polymethine chain of 1-10 chain carbon atoms. Yet more suitably, the cyanine dye comprises a polymethine chain of 1-8 chain carbon atoms. Yet even more suitably, the cyanine dye comprises a polymethine chain of 3-8 chain carbon atoms. Most suitably, the cyanine dye comprises a polymethine chain of 7 chain carbon atoms.

[0039] In an embodiment, the cyanine dye is a closed chain cyanine, a hemicyanine, a streptocyanine or a merocyanine, and the cyanine dye comprises a polymethine chain of 1- 10 chain carbon atoms. In an embodiment, the cyanine dye is a closed chain cyanine, and the cyanine dye comprises a polymethine chain of 3-8 chain carbon atoms.

[0040] Alternatively, the cyanine dye does not comprise a polymethine chain and the organic moieties are directly linked. Such a cyanine dye is classified as an apocyanine. Suitably, the cyanine dye is an apocyanine.

[0041] The cyanine dye may be selected from the group consisting of indocyanine green (ICG), IR-140, IR-820, IR-806, IR-783, IR-780, Cy3, Cy3 NHS, Cy3 carboxylic acid, Cy5, Cy5 NHS, Cy5 carboxylic acid, Cy7, Cy7 NHS, Cy7 carboxylic acid, Cy7.5, Cy7.5 NHS, Cy7.5 carboxylic acid, DiOC6, JC-1 and cypate. Suitably, the cyanine dye is ICG or cypate. Most suitably, the cyanine dye is ICG.

[0042] The cyanine dye may be modified by any suitable means and all modified cyanine dyes are encompassed within the scope of the present invention. For example, it may be that any of the cyanine dyes discussed herein may be modified by any suitable process with any suitable moiety (i.e., polyamine modification). Suitably, the cyanine dye is a modified cyanine dye. More suitably, the cyanine dye is modified ICG, modified IR-140, modified IR- 820, modified IR-806, modified IR-783, modified IR-780, modified Cy3, modified Cy3 NHS,modified Cy3 carboxylic acid, modified Cy5, modified Cy5 NHS, modified Cy5 carboxylic acid, modified Cy7, modified Cy7 NHS, modified Cy7 carboxylic acid, modified Cy7.5, modified Cy7.5 NHS, modified Cy7.5 carboxylic acid, modified DiOC6, modified JC-1 or modified cypate. More suitably, the cyanine dye is modified cypate or modified ICG. Most suitably, the cyanine dye is modified ICG.

[0043] Any of the cyanine dyes discussed herein, including modified cyanine dyes, are suitable for nanoparticle formation (i.e., the formation of nanoparticles of a cyanine dye). Furthermore, any of the cyanine dyes discussed herein can aggregate to form J-aggregates (i.e., the nanoparticles of a cyanine dye aggregate to form J-aggregate nanoparticles).

[0044] In embodiments where the cyanine dye is ICG (or modified ICG), the nanoparticles may be further characterised by a characteristic J-absorption band at 895nm.

[0045] The nanoparticles may have a particle size (i.e., particle diameter) of less than 200 nm. Suitably, the nanoparticles have a particle size of less than 180 nm. More suitably, the nanoparticles have a particle size of less than 160 nm. Even more suitably, the nanoparticles have a particle size of less than 140 nm. Still even more suitably, the nanoparticles have a particle size of less than 120 nm. Yet still even more suitably, the nanoparticles have a particle size of less than 100 nm. Most suitably, the nanoparticles have a particle size of 80- 100 nm.

[0046] In an embodiment, the cyanine dye is selected from the group consisting of ICG, IR- 140, IR-820, IR-806, IR-783, IR-780, Cy3, Cy3 NHS, Cy3 carboxylic acid, Cy5, Cy5 NHS, Cy5 carboxylic acid, Cy7, Cy7 NHS, Cy7 carboxylic acid, Cy7.5, Cy7.5 NHS, Cy7.5 carboxylic acid, DiOC6, JC-1 and cypate, and the nanoparticles have a particle size of less than 200 nm. In an embodiment, the cyanine dye is ICG or cypate, and the nanoparticles have a particle size of less than 140 nm. In an embodiment, the cyanine dye is ICG, and the nanoparticles have a particle size of less than 100 nm.

[0047] In the pharmaceutical compositions, the nanoparticles may be dispersed in an aqueous medium. In such embodiments, the pharmaceutical composition may be substantially free of nanoparticles in a non-aggregated form. The pharmaceutical composition may therefore comprise 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0% of nanoparticles in a non-aggregated form.

[0048] As previously mentioned, the nanoparticles of a cyanine dye have a high affinity for senescent cells. Suitably, the nanoparticles selectively target I are capable of selectively targeting senescent cells. Accordingly, in the pharmaceutical compositions, the therapeutic agent is efficiently transported to senescent cells by their association with the nanoparticlesof a cyanine dye. Suitably, the nanoparticles transport I are capable of transporting the therapeutic agent associated therewith to senescent cells. Once at the senescent cells, the therapeutic agent can be released by dissociating from the nanoparticles, thereby exerting their therapeutic activity (e.g., on the senescent cells in cell lysis). The pharmaceutical composition is therefore suitable for treating diseases I disorders associated with senescent cells and senescent cell build-up. Suitably the therapeutic agent is cytotoxic to senescent cells. More suitably, the therapeutic agent is a senolytic and / or a chemotherapeutic agent. Most suitably, the therapeutic agent is a senolytic.

[0049] The therapeutic agent may target specific sites in senescent cells, such as proteins and / or DNA in senescent cells. For example, it may be that the therapeutic agent targets BCL proteins (e.g., BCL2, BCL3, BCL5, BCL6, BCL7A, BCL9 or BCL10) in senescent cells. Suitably, the therapeutic agent targets BCL2, BCLXL or BCLw proteins in senescent cells.

[0050] Typically, the pharmaceutical composition comprises one therapeutic agent. However, it may be that the pharmaceutical composition comprises more than one (e.g., two, three, four, five, six...) therapeutic agents. Suitably, the pharmaceutical composition comprises one or two therapeutic agents. In embodiments where the pharmaceutical composition comprises two or more therapeutic agents, each therapeutic agent will be different from one another. Most suitably, the pharmaceutical composition comprises one therapeutic agent.

[0051] The therapeutic agent may be substantially water soluble (e.g., doxorubicin and cisplatin) or substantially water insoluble (e.g., navitoclax and paclitaxel). Suitably, the therapeutic agent is selected from the group consisting of navitoclax, venetoclax, dasatinib, quercetin, doxorubicin, cisplatin, paclitaxel, fisetin, chlorambucil, cyclophosphamide, carboplatin, obatolax, vincristine, topotecan, bleomycin, mitoxantrone and docetaxel. More suitably, the therapeutic agent is selected from the group consisting of navitoclax, venetoclax, dasatinib, quercetin, doxorubicin, cisplatin and paclitaxel. Most suitably, the therapeutic agent is navitoclax or doxorubicin.

[0052] In the pharmaceutical composition, the therapeutic agent is associated to the nanoparticles of a cyanine dye. The term “associated” may be understood to mean that the therapeutic agent is encapsulated within the nanoparticles and / or bound to surface of the nanoparticles such that it can be transported to senescent cells and subsequently released (i.e. , the therapeutic agent is loaded into I onto the nanoparticles). The term “association” encompasses any means by which the therapeutic agent may be associated to the nanoparticles, including, for example, one or more of the following: hydrogen bonding, dipole-dipole forces, ion-dipole forces, Van der Waals forces, London dispersion forces and / or non-covalent interactions, such as TT-TT stacking.

[0053] Suitably, the therapeutic agent is encapsulated (i.e., enclosed) within the nanoparticles. Most suitably, the therapeutic agent is encapsulated within the nanoparticles by non-covalent interactions, such as TT-TT stacking.

[0054] In the pharmaceutical composition, the therapeutic agent may be present at a loading of about 1 weight % to about 40 weight % relative to the total weight of the composition. Suitably, the therapeutic agent is present at a loading of about 3 weight % to about 35 weight % relative to the total weight of the composition. More suitably, the therapeutic agent is present at a loading of about 5 weight % to about 30 weight % relative to the total weight of the composition. Even more suitably, the therapeutic agent is present at a loading of about 5 weight % to about 10 weight % relative to the total weight of the composition. Alternatively, the therapeutic agent is present at a loading of about 20 weight % to about 30 weight % relative to total the weight of the composition.

[0055] In an embodiment, the pharmaceutical composition comprises one therapeutic agent, and the therapeutic agent is selected from the group consisting of navitoclax, venetoclax, dasatinib, quercetin, doxorubicin, cisplatin, paclitaxel, fisetin, chlorambucil, cyclophosphamide, carboplatin, obatolax, vincristine, topotecan, bleomycin, mitoxantrone and docetaxel. In an embodiment, the pharmaceutical composition comprises one therapeutic agent, wherein the therapeutic agent is navitoclax or doxorubicin.

[0056] In an embodiment, in the pharmaceutical composition, the therapeutic agent is present at a loading of about 1 weight % to about 40 weight % relative to the total weight of the composition, and the therapeutic agent is selected from the group consisting of navitoclax, venetoclax, dasatinib, quercetin, doxorubicin, cisplatin, paclitaxel, fisetin, chlorambucil, cyclophosphamide, carboplatin, obatolax, vincristine, topotecan, bleomycin, mitoxantrone and docetaxel. In an embodiment, in the pharmaceutical composition, the therapeutic agent is present at a loading of about 5 weight % to about 30 weight % relative to the total weight of the composition, and the therapeutic agent is selected from the group consisting of navitoclax, venetoclax, dasatinib, quercetin, doxorubicin, cisplatin and paclitaxel. In an embodiment, in the pharmaceutical composition, the therapeutic agent is present at a loading of about 5 weight % to about 10 weight % relative to the total weight of the composition, and the therapeutic agent is navitoclax or doxorubicin.

[0057] In an embodiment, the cyanine dye is selected from the group consisting of ICG, IR- 140, IR-820, IR-806, IR-783, IR-780, Cy3, Cy3 NHS, Cy3 carboxylic acid, Cy5, Cy5 NHS, Cy5 carboxylic acid, Cy7, Cy7 NHS, Cy7 carboxylic acid, Cy7.5, Cy7.5 NHS, Cy7.5 carboxylic acid, DiOC6, JC-1 and cypate, and the therapeutic agent is selected from the group consisting of navitoclax, venetoclax, dasatinib, quercetin, doxorubicin, cisplatin, paclitaxel, fisetin, chlorambucil, cyclophosphamide, carboplatin, obatolax, vincristine,topotecan, bleomycin, mitoxantrone and docetaxel. In an embodiment, the cyanine dye is ICG or cypate, and the therapeutic agent is selected from the group consisting of navitoclax, venetoclax, dasatinib, quercetin, doxorubicin, cisplatin and paclitaxel. In an embodiment, the cyanine dye is ICG, and the therapeutic agent is navitoclax or doxorubicin. In these embodiments, the therapeutic agent is optionally present at a loading of about 1 weight % to about 40 weight %, of about 3 weight % to about 35 weight %, of about 5 weight % to about 30 weight %, of about 5 weight % to about 10 weight %, or of about 20 weight % to about 30 weight % relative to total the weight of the composition. In these embodiments, it may optionally be (inclusive with aforementioned loading or in isolation) that the nanoparticles have a particle size of less than 200 nm, less than 180 nm, less than 160 nm, less than 140 nm, less than 120 nm, less than 100 nm or 80-100 nm.

[0058] As set out herein, the therapeutic agent is transported to senescent cells by association to nanoparticles of a cyanine dye. Once at senescent cells, the therapeutic agent dissociates (i.e., is released) from the nanoparticles of the cyanine dye. Suitably, the therapeutic agent dissociates from the nanoparticles following administration.

[0059] Suitably, the pharmaceutical composition releases in vivo. i) more than 5% of the therapeutic agent after 12 hours; ii) more than 10% of the therapeutic agent after 24 hours; iii) more than 15% of the therapeutic agent after 36 hours; and / or iv) more than 20% of the therapeutic agent after 48 hours; relative to the total amount of therapeutic agent.

[0060] More suitably, the pharmaceutical composition releases in vivo. i) more than 10% of the therapeutic agent after 12 hours; ii) more than 20% of the therapeutic agent after 24 hours; iii) more than 30% of the therapeutic agent after 36 hours; and / or iv) more than 40% of the therapeutic agent after 48 hours; relative to the total amount of therapeutic agent.

[0061] Even more suitably, the pharmaceutical composition releases in vivo. i) more than 20% of the therapeutic agent after 12 hours; ii) more than 40% of the therapeutic agent after 24 hours; iii) more than 60% of the therapeutic agent after 36 hours; and / oriv) more than 80% of the therapeutic agent after 48 hours; relative to the total amount of therapeutic agent.

[0062] The pharmaceutical compositions may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular, intraperitoneal or intramuscular dosing or as a suppository for rectal dosing).

[0063] Suitably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0064] The pharmaceutical compositions may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art. Thus, compositions intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and / or preservative agents.Therapeutic uses and applications

[0065] The present invention provides a pharmaceutical composition as defined herein for use in therapy.

[0066] The present invention provides a pharmaceutical composition as defined herein for use as a medicament.

[0067] The present invention provides a pharmaceutical composition as defined herein for use as a senolytic.

[0068] The present invention provides a pharmaceutical composition as defined herein for use in the treatment of a disease or disorder in which the presence of senescent cells is implicated.

[0069] The present invention provides a pharmaceutical composition as defined herein for use in the treatment of a proliferative disorder.

[0070] The present invention provides a pharmaceutical composition as defined herein for use in the treatment of cancer.

[0071] The present invention provides a pharmaceutical composition as defined herein for use in the treatment of a pre-malignant lesion, a tumour (e.g., a pre-malignant tumour, amalignant tumour or a tumour following treatment-induced (e.g., chemotherapy) cellular senescence), an age-related disorder (e.g., chronic disorders such as cancer, cardiovascular diseases (e.g., atherosclerosis), fibrosis (e.g., pulmonary fibrosis, idiopathic pulmonary fibrosis and kidney fibrosis), neurological disorders (e.g., Alzheimer’s and Parkinson’s disease), Type 1 & 2 diabetes, skeletomuscular disorders (e.g., sarcopenia, osteoarthritis and osteoporosis), inflammatory diseases, chronic obstructive pulmonary disease (COPD), infarction, aneurysm, cataracts, post-infarction tissues, frailty, chronic kidney disease, diabetic macular degeneration etc.) or a wound.

[0072] The present invention provides a method of treating a disease or disorder in which the presence of senescent cells is implicated in a patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition as defined herein.

[0073] The present invention provides a method of treating a proliferative disorder in a patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition as defined herein.

[0074] The present invention provides a method of treating cancer in a patient in need of such treatment, said method comprising administering to said patient in need of such treatment a therapeutically effective amount of a pharmaceutical composition as defined herein.

[0075] The present invention provides a method of treating a pre-malignant lesion, a tumour (e.g., a pre-malignant tumour, a malignant tumour or a tumour following treatment-induced (e.g., chemotherapy) cellular senescence), an age-related disorder (e.g., chronic disorders such as cancer, cardiovascular diseases (e.g., atherosclerosis), fibrosis (e.g., pulmonary fibrosis, idiopathic pulmonary fibrosis and kidney fibrosis), neurological disorders (e.g., Alzheimer’s and Parkinson’s disease), Type 1 & 2 diabetes, skeletomuscular disorders (e.g., sarcopenia, osteoarthritis and osteoporosis), inflammatory diseases, chronic obstructive pulmonary disease (COPD), infarction, aneurysm, cataracts, post-infarction tissues, frailty, chronic kidney disease, diabetic macular degeneration etc.) or a wound in a patient in need of such treatment, said method comprising administering to said patient in need of such treatment a therapeutically effective amount of a pharmaceutical composition as defined herein.

[0076] The pharmaceutical compositions may be administered to a subject by any convenient route of administration, whether systemically, peripherally or topically (i.e., at the site of desired action). Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray);ocular (e.g., by eye drops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including intratumoral, subcutaneous, intradermal, intramuscular, intravenous, intra-arterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot or reservoir, for example, subcutaneously or intramuscularly.

[0077] An effective amount of the therapeutic agent for use in therapy is an amount sufficient to treat or prevent a disease I disorder referred to herein, slow its progression and / or reduce the symptoms associated with the condition.

[0078] The amount of therapeutic agent that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the individual treated and the particular route of administration. For example, a formulation intended for oral administration to humans will generally contain, for example, from 0.5 mg to 0.5 g of the therapeutic agent (more suitably from 0.5 mg to 100 mg, for example from 1 mg to 30 mg) compounded with an appropriate and convenient amount of excipients.

[0079] The size of the dose for therapeutic or prophylactic purposes of the therapeutic agent will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well-known principles of medicine.

[0080] In using the therapeutic agent for therapeutic or prophylactic purposes it will generally be administered so that a daily dose in the range, for example, 0.1 mg / kg to 75 mg / kg body weight is received, given if required in divided doses. In general, lower doses will be administered when a parenteral route is employed. Thus, for example, for intravenous or intraperitoneal administration, a dose in the range, for example, 0.1 mg / kg to 30 mg / kg body weight will generally be used. Similarly, for administration by inhalation, a dose in the range, for example, 0.05 mg / kg to 25 mg / kg body weight will be used. Oral administration may also be suitable, particularly in tablet form. Typically, unit dosage forms will contain about 0.5 mg to 0.5 g of the therapeutic agent.

[0081] The present invention also provides the use of a pharmaceutical composition as defined herein in the manufacture of a medicament for the treatment of a disease or disorder in which the presence of senescent cells is implicated.

[0082] The present invention provides the use of a pharmaceutical composition as defined herein in the manufacture of a medicament for the treatment of a proliferative disorder.

[0083] The present invention provides the use of a pharmaceutical composition as defined herein in the manufacture of a medicament for the treatment of cancer.

[0084] The present invention provides the use of a pharmaceutical composition as defined herein in the manufacture of a medicament for the treatment of a pre-malignant lesion, a tumour (e.g., a pre-malignant tumour, a malignant tumour or a tumour following treatment- induced (e.g., chemotherapy) cellular senescence), an age-related disorder (e.g., chronic disorders such as cancer, cardiovascular diseases (e.g., atherosclerosis), fibrosis (e.g., pulmonary fibrosis, idiopathic pulmonary fibrosis and kidney fibrosis), neurological disorders (e.g., Alzheimer’s and Parkinson’s disease), Type 1 & 2 diabetes, skeletomuscular disorders (e.g., sarcopenia, osteoarthritis and osteoporosis), inflammatory diseases, chronic obstructive pulmonary disease (COPD), infarction, aneurysm, cataracts, post-infarction tissues, frailty, chronic kidney disease, diabetic macular degeneration etc.) or a wound.

[0085] The term "proliferative disorder", “proliferative condition” and “proliferative disease” are used interchangeably herein and pertain to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo.

[0086] The proliferative disorder is suitably cancer (e.g., human cancer). Most suitably, the cancer includes those in which cellular senescence has developed (e.g., following treatment, such as chemotherapy, of a tumour). Any suitable cancer may be targeted (e.g., adenoid cystic carcinoma, adrenal gland tumour, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia-telangiectasia, Beckwith-Wiedemann Syndrome, bile duct cancer (cholangiocarcinoma), Birt-Hogg-Dube Syndrome, bladder cancer, bone cancer, brain stem glioma, brain tumour, breast cancer, Carney Complex, central nervous system tumours, cervical cancer, colorectal cancer, Cowden Syndrome, craniopharyngioma, desmoplastic infantile ganglioglioma, ependymoma, oesophageal cancer, Ewing sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, familial GIST, familial malignant melanoma, familial non-VHL clear cell renal cell carcinoma, familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumour - GIST, germ cell tumour, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary diffuse gastric cancer, hereditary leiomyomatosis and renal cell cancer, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papillary renal carcinoma, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumour, laryngeal and hypopharyngeal cancer, leukaemia (acute lymphoblastic leukaemia (ALL), acute myeloid leukaemia (AML), B-cell prolymphocytic leukaemia, hairy cell leukaemia, chronic lymphocytic leukaemia (CLL), chronic myeloid leukaemia (CML), chronic T-cell lymphocytic leukaemia, eosinophilic leukaemia), Li-Fraumeni Syndrome, liver cancer, lung cancer (non-small cell lung cancer,small cell lung cancer), Lymphoma (Hodgkin, non-Hodgkin), Lynch Syndrome, mastocytosis, medulloblastoma, melanoma, meningioma, mesothelioma, multiple endocrine neoplasia Type 1 & 2, multiple myeloma, MUTYH (or MYH)-associated polyposis, myelodysplastic syndromes (MDS), nasal cavity and paranasal sinus Cancer, nasopharyngeal Cancer, neuroblastoma, neuroendocrine tumours (e.g., of the gastrointestinal tract, lung or pancreas), neurofibromatosis Type 1 & 2, nevoid basal cell carcinoma syndrome, oral and oropharyngeal cancer, osteosarcoma, ovarian / fallopian tube I peritoneal cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers Syndrome, pheochromocytoma, paraganglioma, pituitary gland tumour, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., Kaposi or soft tissue), skin cancer, small bowel cancer, stomach cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis complex, uterine cancer, vaginal cancer, Von Hippel-Lindau syndrome, vulvar cancer, Waldenstrom’s macroglobulinemia, Werner syndrome, Wilms Tumour and xeroderma pigmentosum). Particular cancers of interest include haematological cancers such as lymphomas (including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Burkitt lymphoma (BL) and angioimmunoblastic T-cell lymphoma (AITL)), leukaemia’s (including acute lymphoblastic leukaemia (ALL) and chronic myeloid leukaemia (CML)), multiple myeloma, breast cancer, non-small cell lung cancer (NSCLC), colorectal cancer, endometrial cancer, gastro-oesophageal cancer, neuroendocrine cancers, osteosarcomas, prostate cancer, pancreatic cancer, small intestine cancer, bladder cancer, rectal cancer, cholangiocarcinoma, CNS cancer, thyroid cancer, head and neck cancer, oesophageal cancer, and ovarian cancer.

[0087] The therapeutic agent may also be used in combination with one or more additional treatments. For example, the therapeutic agent may be used in combination with one or more additional therapeutic agents (as defined herein) and / or therapies (i.e., radiotherapy). Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment. Such combination products employ the therapeutic agent within the dosage range defined herein. The term “combination” will be understood to refer to simultaneous, separate or sequential administration. Where the administration is sequential or separate, the delay in administering the second component should not be such as to lose the beneficial effect of the combination. Combination treatment involves the combined treatment in a treatment regimen or course of treatment and would not cover the situation where a patient has been treated with one agent in the past and then is treated with the second agent at a much later time (and in a different treatment regimen) when the effects of the first agent have passed.Process for preparing a pharmaceutical composition

[0088] The present invention also provides a process for preparing a pharmaceutical composition comprising nanoparticles of a cyanine dye and a therapeutic agent, the process comprising the steps of:(i) providing an aqueous solution of the cyanine dye;(ii) maintaining the aqueous solution of the cyanine dye at a temperature of between 15 °C to 85 °C, preferably with agitation, to allow the cyanine dye molecules to aggregate and form nanoparticles;(iii) collecting and purifying the resultant nanoparticles present in the solution; and(iv) mixing the purified nanoparticles with a therapeutic agent, wherein step (iii) comprises at least one separation step wherein the nanoparticles formed in step (ii) are separated from the aqueous solution and re-suspended or dispersed in a different aqueous medium.

[0089] In step (i) and / or step (ii), the pH of the aqueous solution may be maintained within an appropriate range. Suitably the pH of the aqueous solution in step (i) and / or step (ii) is within the range of 2 to 9. More suitably, the pH of the aqueous solution in step (i) and / or step (ii) is within the range of 2 to 8. Even more suitably, the pH of the aqueous solution in step (i) and / or step (ii) is within the range of 2 to 7. Yet even more suitably, the pH of the aqueous solution in step (i) and / or step (ii) is within the range of 2 to 6.

[0090] The concentration of cyanine dye in the aqueous solution in step (i) may be within the range of 0.01 mM to 10 mM. More suitably, the concentration of cyanine dye in the aqueous solution in step (i) is within the range of 0.1 mM to 7.5 mM. Even more suitably, the concentration of cyanine dye in the aqueous solution in step (i) is within the range of 0.25 mM to 5 mM. Yet even more suitably, the concentration of cyanine dye in the aqueous solution in step (i) is within the range of 0.5 mM to 2 mM. Yet still even more suitably, the concentration of cyanine dye in the aqueous solution in step (i) is within the range of 0.6 mM to 0.9 mM. Yet still even more suitably, the concentration of cyanine dye in the aqueous solution in step (i) is within the range of 0.7 mM to 0.8 mM. Most suitably, the concentration of cyanine dye in the aqueous solution in step (i) is 0.75 mM.

[0091] In step (i), the cyanine dye is provided in an aqueous solution. The aqueous solution of the cyanine dye may comprise water. Suitably, the aqueous solution of the cyanine dye is a solution of the cyanine dye in water.

[0092] In step (ii) the aqueous solution of the cyanine dye is maintained at a temperature of between 15 °C to 85 °C. It will be appreciated that the cyanine dye may be maintained at any temperature within this range for any appropriate period of time. Suitably, in step (ii), the solution is heated to a temperature within the range of 40 °C to 85 °C, optionally for 0.5 to 48 hours. More suitably, in step (ii), the solution is heated to a temperature within the range of 45 °C to 85 °C, optionally for 0.5 to 48 hours. Yet more suitably, in step (ii), the solution is heated to a temperature within the range of 55 °C to 75 °C, optionally for 0.5 to 48 hours. Yet still even more suitably, in step (ii), the solution is heated to a temperature range of 60 °C to 70 °C, optionally for 0.5 to 48 hours. Most suitably, in step (ii), the solution is heated to a temperature of 65 °C, optionally for 0.5 to 48 hours. In any of the aforementioned embodiments, in step (ii), the solution may be maintained within the stated temperature range defined herein for 0.5 to 36 hours, and optionally for 0.5 to 24 hours.

[0093] In step (ii), the process may further comprise monitoring the formation of nanoparticles, optionally by monitoring the depletion of the non-aggregated cyanine dye from the aqueous solution and / or the formation of the nanoparticles. Typically, the formation of nanoparticles is monitored by a UV-Vis spectrophotometer. In embodiments where the cyanine dye is ICG, the formation of nanoparticles is monitored by the UV-Vis transition from 780 nm to 895 nm.

[0094] In step (iii), the at least one separation step separates the nanoparticles formed in step (ii) from the aqueous solution to remove any non-aggregated cyanine dye from the aqueous medium. The separation step may be carried out multiple times to ensure the nanoparticles are substantially free from any non-aggregated cyanine dye.

[0095] In step (iii), any suitable separation technique that isolates the nanoparticles formed in step (ii) from the remainder of the aqueous solution (and any non-aggregated cyanine dye) may be used. Suitable techniques include centrifugation, filtration and / or dialysis. It will be appreciated that any suitable method of dialysing the solution of nanoparticles may be used. It will also be appreciated that any suitable method of filtering the solution of nanoparticles may also be used.

[0096] In step (iii), the collection and purification of the nanoparticles may comprise two or more, or three or more, independent separation (e.g., centrifugation, filtration and / or dialysis) steps.

[0097] In some embodiments, in step (iii), the collection and purification of the nanoparticles comprises one, two or three centrifugation steps. In some embodiments, the collection and purification of the nanoparticles comprises one centrifugation step.

[0098] Suitably, step (iii) comprises at least one centrifugation step wherein the solution is centrifuged to form a pellet of the nanoparticles, removing the supernatant, optionally washing the pellet, and re-suspending the pellet in a different aqueous medium.

[0099] In embodiments where the separation technique in step (iii) includes at least one centrifugation step, it will be appreciated that any suitable centrifugal force may be used. Suitably, the centrifugal force is sufficient enough to form a pellet of the nanoparticles. Suitably, the centrifugal force is 20,000 x g (RCF) to 50,000 x g (RCF). More suitably, the centrifugal force is 25,000 x g (RCF) to 45,000 x g (RCF). Yet more suitably, the centrifugal force is 27,500 x g (RCF) to 42,500 x g (RCF). In an embodiment, the centrifugal force is 30,000 x g (RCF) to 40,000 x g (RCF). Suitably, the centrifugation step is performed at 10,000 rpm to 25,000 rpm. More suitably, the centrifugation step is performed at 12,500 rpm to 22,500 rpm. Even more suitably, the centrifugation step is performed at 15,000 rpm to 20,000 rpm. In an embodiment, the centrifugation step is performed at 17,000 rpm to 18,000 rpm. In such embodiments, the collection and purification of the nanoparticles may further comprise additional steps of dialysing the solution of nanoparticles and / or filtering the solution of nanoparticles. The centrifugation step may be performed at a temperature of 0 °C to 10 °C, optionally for 30 seconds to 1 hour. Suitably, the centrifugation step is performed at a temperature of 1 °C to 7 °C, optionally for 1 minute to 1 hour. More suitably, the centrifugation step is performed at a temperature of 3 °C to 5 °C, optionally for 20 minutes to 40 minutes.

[0100] In step (iv), the purified nanoparticles are mixed with a therapeutic agent. The molar ratio of therapeutic agent to purified nanoparticles in step (iv) may be 20:1 to 1 :20. Suitably, the molar ratio of therapeutic agent to purified nanoparticles in step (iv) is 15:1 to 1 :5. More suitably, the molar ratio of therapeutic agent to purified nanoparticles in step (iv) is 12:1 to 1 :2. Even more suitably, the molar ratio of therapeutic agent to purified nanoparticles in step (iv) is 10: 1 to 1 : 1. Most suitably, the molar ratio of therapeutic agent to purified nanoparticles in step (iv) is 10:1 , 5:1 or 1 :1.

[0101] Suitably, step (iv) is performed at a temperature of 10 °C to 40 °C. More suitably, step (iv) is performed at a temperature of 15 °C to 35 °C. Even more suitably, step (iv) is performed at a temperature of 20 °C to 30 °C. Most suitably, step (iv) is performed at a temperature of 25 °C.

[0102] The therapeutic agent in step (iv) may optionally be contacted with (i.e., dissolved in) a solvent before mixing with the purified nanoparticles. The solvent may be an organic solvent. The organic solvent may comprise any one or more of the following: acetic acid, acetone, acetonitrile, methanol, ethanol, propanol, butanol, chloroform, 1 ,2- dichloroethane, ethylene glycol, diethylene glycol, dimethyl formamide, dimethylsulfoxide,1 ,4-dioxane, ethyl acetate, hexane, heptane, benzene, toluene, naphthalene, xylene and / or tetrahydrofuran. Suitably, the organic solvent comprises methanol, acetone and / or tetrahydrofuran.

[0103] In embodiments wherein the therapeutic agent in step (iv) is contacted with a solvent before mixing with the purified nanoparticles. The therapeutic agent may be dissolved in the solvent (e.g. ethanol or DMSO) prior to mixing with the aqueous nanoparticles. Alternatively, the solvent comprising the therapeutic agent may be evaporated (e.g., by rotary evaporation) to obtain a film of the therapeutic agent before mixing with the purified nanoparticles.

[0104] Suitably, step (iv) comprises at least one separation step wherein the pharmaceutical composition formed by mixing the purified nanoparticles with a therapeutic agent is separated (i.e., isolated) from the aqueous medium of step (iii). Optionally, the isolated pharmaceutical composition is re-suspended or dispersed in a different aqueous medium.

[0105] The at least one separation step may be carried out multiple times. Any suitable separation technique that isolates the pharmaceutical composition formed in step (iv) may be used. Suitable techniques include centrifugation, filtration and / or dialysis. Suitably, step (iv) comprises at least one separation step, wherein the at least one separation step is centrifugation. The at least one separation step may comprise two or more, or three or more, independent separation (e.g., centrifugation, filtration and / or dialysis) steps.

[0106] In embodiments where step (iv) comprises at least one separation step and the separation technique is centrifugation, it will be appreciated that any suitable centrifugal force may be used. Suitably, the centrifugal force is sufficient enough to form a pellet of the pharmaceutical composition. Suitably, the centrifugal force is 20,000 x g (RCF) to 50,000 x g (RCF). More suitably, the centrifugal force is 25,000 x g (RCF) to 45,000 x g (RCF). Yet more suitably, the centrifugal force is 27,500 x g (RCF) to 42,500 x g (RCF). In an embodiment, the centrifugal force is 30,000 x g (RCF) to 40,000 x g (RCF). Suitably, the centrifugation step is performed at 10,000 rpm to 25,000 rpm. More suitably, the centrifugation step is performed at 12,500 rpm to 22,500 rpm. Even more suitably, the centrifugation step is performed at 15,000 rpm to 20,000 rpm. In an embodiment, the centrifugation step is performed at 17,000 rpm to 18,000 rpm. The centrifugation step may be performed at a temperature of 0 °C to 10 °C, optionally for 30 seconds to 1 hour. Suitably, the centrifugation step is performed at a temperature of 1 °C to 7 °C, optionally for 1 minute to 1 hour. More suitably, the centrifugation step is performed at a temperature of 3 °C to 5 °C, optionally for 20 minutes to 40 minutes.

[0107] Suitably, step (iv) comprises at least one centrifugation step to form a pellet of the pharmaceutical composition, removing the supernatant, optionally washing the pellet, and re-suspending the pellet in a different aqueous medium.

[0108] The present invention also provides a process for preparing a pharmaceutical composition as defined herein comprising nanoparticles of a cyanine dye and a therapeutic agent, the process comprising the steps of:(i) providing an aqueous solution of the cyanine dye;(ii) maintaining the aqueous solution of the cyanine dye at a temperature of between 15 °C to 85 °C, preferably with agitation, to allow the cyanine dye molecules to aggregate and form nanoparticles;(iii) collecting the resultant nanoparticles present in the solution;(iv) dissolving the resultant nanoparticle to form dimer molecules of the cyanine dye;(v) mixing the dimer molecules of the cyanine dye with a therapeutic agent;(vi) forming nanoparticles of the dimer molecules cyanine dye that comprise the therapeutic agent; and(vii) collecting the nanoparticles formed in step (vi).

[0109] Suitably, step (iii) comprises at least one separation step wherein the nanoparticles formed in step (ii) are separated from the aqueous solution and resuspended or dispersed in a different aqueous medium.

[0110] Suitably, steps (i) to (iii) are as defined in any one of statements 70 to 105 set out below.

[0111] Suitably, the cyanine dye is ICG and dimers of ICG are formed in step (iv) of the process.

[0112] Further features of this process are defined in statements 148 to 156 below.

[0113] In an embodiment, the pharmaceutical composition of the process of the invention is as defined anywhere herein.

[0114] In an embodiment, the nanoparticles of a cyanine dye of the process of the invention are as defined herein.

[0115] In an embodiment, the cyanine dye of the process of the invention are as defined anywhere herein.

[0116] In an embodiment, the therapeutic agent of the process of the invention is as defined herein.

[0117] In an embodiment, the pharmaceutical composition, the nanoparticles of a cyanine dye and the therapeutic agent of the process of the invention are as defined herein.

[0118] In an embodiment, the pharmaceutical composition, the nanoparticles of a cyanine dye, the cyanine dye itself and the therapeutic agent of the process of the invention are as defined herein.

[0119] The following numbered statements 1 to 143 are not claims, but instead define particular aspects and embodiments of the claimed invention:1. A pharmaceutical composition comprising: nanoparticles of a cyanine dye; and a therapeutic agent, wherein the therapeutic agent is associated to the nanoparticles.2. The pharmaceutical composition of statement 1, wherein the cyanine dye is a closed chain cyanine, a hemicyanine, a streptocyanine or a merocyanine.3. The pharmaceutical composition of statement 1 or 2, wherein the cyanine dye is a closed chain cyanine.4. The pharmaceutical composition of any one of the preceding statements, wherein the cyanine dye comprises a polymethine chain of 1-20 chain carbon atoms.5. The pharmaceutical composition of any one of the preceding statements, wherein the cyanine dye comprises a polymethine chain of 1-10 chain carbon atoms.6. The pharmaceutical composition of any one of the preceding statements, wherein the cyanine dye comprises a polymethine chain of 1-8 chain carbon atoms.7. The pharmaceutical composition of any one of the preceding statements, wherein the cyanine dye comprises a polymethine chain of 3-8 chain carbon atoms.8. The pharmaceutical composition of any one of the preceding statements, wherein the cyanine dye comprises a polymethine chain of 7 chain carbon atoms.9. The pharmaceutical composition of statement 1, wherein the cyanine dye is an apocyanine.10. The pharmaceutical composition of any one of the preceding statements, wherein the cyanine dye is selected from the group consisting of ICG, IR-140, IR-820, IR-806, IR-783, IR-780, Cy3, Cy3 NHS, Cy3 carboxylic acid, Cy5, Cy5 NHS, Cy5 carboxylic acid, Cy7, Cy7 NHS, Cy7 carboxylic acid, Cy7.5, Cy7.5 NHS, Cy7.5 carboxylic acid, DiOC6, JC-1 and cypate.11. The pharmaceutical composition of any one of the preceding statements, wherein the cyanine dye is ICG or cypate.12. The pharmaceutical composition of any one of the preceding statements, wherein the cyanine dye is ICG.13. The pharmaceutical composition of any one of the preceding statements, wherein the cyanine dye is a modified cyanine dye.14. The pharmaceutical composition of any one of the preceding statements, wherein the cyanine dye is modified ICG, modified IR-140, modified IR-820, modified IR-806, modified IR-783, modified IR-780, modified Cy3, modified Cy3 NHS, modified Cy3 carboxylic acid, modified Cy5, modified Cy5 NHS, modified Cy5 carboxylic acid, modified Cy7, modified Cy7 NHS, modified Cy7 carboxylic acid, modified Cy7.5, modified Cy7.5 NHS, modified Cy7.5 carboxylic acid, modified DiOC6, modified JC-1 and modified cypate.15. The pharmaceutical composition of any one of the preceding statements, wherein the cyanine dye is modified ICG or modified cypate.16. The pharmaceutical composition of any one of the preceding statements, wherein the cyanine dye is modified ICG.17. The pharmaceutical composition of any one of the preceding statements, wherein the cyanine dye is suitable for nanoparticle formation.18. The pharmaceutical composition of any one of the preceding statements, wherein the cyanine dye can aggregate to form J-aggregates.19. The pharmaceutical composition of any one of the preceding statements, wherein the cyanine dye is ICG (or modified ICG), and the nanoparticles are further characterised by a characteristic J-absorption band at 895nm.20. The pharmaceutical composition of any one of the preceding statements, wherein the nanoparticles have a particle size of less than 200 nm.21. The pharmaceutical composition of any one of the preceding statements, wherein the nanoparticles have a particle size of less than 180 nm.22. The pharmaceutical composition of any one of the preceding statements, wherein the nanoparticles have a particle size of less than 160 nm.23. The pharmaceutical composition of any one of the preceding statements, wherein the nanoparticles have a particle size of less than 140 nm.24. The pharmaceutical composition of any one of the preceding statements, wherein the nanoparticles have a particle size of less than 120 nm.25. The pharmaceutical composition of any one of the preceding statements, wherein the nanoparticles have a particle size of less than 100 nm.26. The pharmaceutical composition of any one of the preceding statements, wherein the nanoparticles have a particle size of 80-100 nm.27. The pharmaceutical composition of any one of the preceding statements, wherein the nanoparticles are dispersed in an aqueous medium.28. The pharmaceutical composition of statement 27, wherein the pharmaceutical composition is substantially free of nanoparticles in a non-aggregated form.29. The pharmaceutical composition of statement 27 or 28, wherein the pharmaceutical composition comprises 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0% of nanoparticles in a nonaggregated form.30. The pharmaceutical composition of any one of the preceding statements, wherein the nanoparticles selectively target I are capable of selectively targeting senescent cells.31. The pharmaceutical composition of any one of the preceding statements, wherein the nanoparticles transport I are capable of transporting the therapeutic agent associated therewith to senescent cells.32. The pharmaceutical composition of any one of the preceding statements, wherein the therapeutic agent is cytotoxic to senescent cells.33. The pharmaceutical composition of any one of the preceding statements, wherein the therapeutic agent is a senolytic and / or a chemotherapeutic agent.34. The pharmaceutical composition of any one of the preceding statements, wherein the therapeutic agent is a senolytic.35. The pharmaceutical composition of any one of the preceding statements, wherein the therapeutic agent targets I is capable of targeting proteins and / or DNA in senescent cells.36. The pharmaceutical composition of any one of the preceding statements, wherein the therapeutic agent targets / is capable of targeting BCL proteins (e.g., BCL2, BCL3, BCL5, BCL6, BCL7A, BCL9 or BCL10) in senescent cells.37. The pharmaceutical composition of any one of the preceding statements, wherein the therapeutic agent targets I is capable of targeting BCL2, BCLXL or BCLw proteins in senescent cells.38. The pharmaceutical composition of any one of the preceding statements, wherein the composition comprises more than one (e.g., two, three, four, five, six...) therapeutic agents.39. The pharmaceutical composition of any one of the preceding statements, wherein the composition comprises one or two therapeutic agents.40. The pharmaceutical composition of any one of the preceding statements, wherein the composition comprises one therapeutic agent.41. The pharmaceutical composition of any one of the preceding statements, wherein the therapeutic agent is selected from the group consisting of navitoclax, venetoclax, dasatinib, quercetin, doxorubicin, cisplatin, paclitaxel, fisetin, chlorambucil, cyclophosphamide, carboplatin, obatolax, vincristine, topotecan, bleomycin, mitoxantrone and docetaxel.42. The pharmaceutical composition of any one of the preceding statements, wherein the therapeutic agent is selected from the group consisting of navitoclax, venetoclax, dasatinib, quercetin, doxorubicin, cisplatin and paclitaxel.43. The pharmaceutical composition of any one of the preceding statements, wherein the therapeutic agent is navitoclax or doxorubicin.44. The pharmaceutical composition of any one of the preceding statements, wherein the therapeutic agent is encapsulated (i.e. , enclosed) within the nanoparticles.45. The pharmaceutical composition of any one of the preceding statements, wherein the therapeutic agent is encapsulated within the nanoparticles by non-covalent interactions, such as TT-TT stacking.46. The pharmaceutical composition of any one of the preceding statements, wherein the therapeutic agent is present at a loading of about 1 weight % to about 40 weight % relative to the total weight of the composition.47. The pharmaceutical composition of any one of the preceding statements, wherein the therapeutic agent is present at a loading of about 3 weight % to about 35 weight % relative to the total weight of the composition.48. The pharmaceutical composition of any one of the preceding statements, wherein the therapeutic agent is present at a loading of about 5 weight % to about 30 weight % relative to the total weight of the composition.49. The pharmaceutical composition of any one of the preceding statements, wherein the therapeutic agent is present at a loading of about 5 weight % to about 10 weight % relative to the total weight of the composition.50. The pharmaceutical composition of any one of statements 1-48, wherein the therapeutic agent is present at a loading of about 20 weight % to about 30 weight % relative to total the weight of the composition.51. The pharmaceutical composition of any one of the preceding statements, wherein the pharmaceutical composition releases in vivo. i) more than 5% of the therapeutic agent after 12 hours; ii) more than 10% of the therapeutic agent after 24 hours; iii) more than 15% of the therapeutic agent after 36 hours; and / or iv) more than 20% of the therapeutic agent after 48 hours; relative to the total amount of therapeutic agent.52. The pharmaceutical composition of any one of the preceding statements, wherein the pharmaceutical composition releases in vivo. i) more than 10% of the therapeutic agent after 12 hours; ii) more than 20% of the therapeutic agent after 24 hours; iii) more than 30% of the therapeutic agent after 36 hours; and / or iv) more than 40% of the therapeutic agent after 48 hours; relative to the total amount of therapeutic agent.53. The pharmaceutical composition of any one of the preceding statements, wherein the pharmaceutical composition releases in vivo. i) more than 20% of the therapeutic agent after 12 hours; ii) more than 40% of the therapeutic agent after 24 hours; iii) more than 60% of the therapeutic agent after 36 hours; and / or iv) more than 80% of the therapeutic agent after 48 hours; relative to the total amount of therapeutic agent.54. The pharmaceutical composition of any one of the preceding statements, wherein the composition further comprises one or more pharmaceutically acceptable excipients.55. The pharmaceutical composition of any one of the preceding statements for use in therapy.56. The pharmaceutical composition of any one of statements 1-54 for use as a medicament.57. The pharmaceutical composition of any one of statements 1-54 for use as a senolytic.58. The pharmaceutical composition of any one of statements 1-54 for use in the treatment of a disease or disorder in which the presence of senescent cells is implicated.59. The pharmaceutical composition of any one of statements 1-54 for use in the treatment of a proliferative disorder.60. The pharmaceutical composition of any one of statements 1-54 for use in the treatment of cancer.61. The pharmaceutical composition of any one of statements 1-54 for use in the treatment of a pre-malignant lesion, a tumour (e.g., a pre-malignant tumour, a malignant tumour or a tumour following treatment- induced (e.g., chemotherapy) cellular senescence), an age-related disorder (e.g., chronic disorders such as cancer, cardiovascular diseases (e.g., atherosclerosis), fibrosis (e.g., pulmonary fibrosis, idiopathic pulmonary fibrosis and kidney fibrosis), neurological disorders (e.g., Alzheimer’s and Parkinson’s disease), Type 1 & 2 diabetes, skeletomuscular disorders (e.g., sarcopenia, osteoarthritis and osteoporosis), inflammatory diseases, chronic obstructive pulmonary disease (COPD), infarction, aneurysm, cataracts, post-infarction tissues, frailty, chronic kidney disease, diabetic macular degeneration etc.) or a wound.62. A method of treating a disease or disorder in which the presence of senescent cells is implicated in a patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition as defined in any one of statements 1-54.63. A method of treating a proliferative disorder in a patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition as defined in any one of statements 1-54.64. A method of treating cancer in a patient in need of such treatment, said method comprising administering to said patient in need of such treatment a therapeutically effective amount of a pharmaceutical composition as defined in any one of statements 1-54.65. A method of treating a pre-malignant lesion, a tumour (e.g., a pre-malignant tumour, a malignant tumour or a tumour following treatment-induced (e.g., chemotherapy) cellularsenescence), an age-related disorder (e.g., chronic disorders such as cancer, cardiovascular diseases (e.g., atherosclerosis), fibrosis (e.g., pulmonary fibrosis, idiopathic pulmonary fibrosis and kidney fibrosis), neurological disorders (e.g., Alzheimer’s and Parkinson’s disease), Type 1 & 2 diabetes, skeletomuscular disorders (e.g., sarcopenia, osteoarthritis and osteoporosis), inflammatory diseases, chronic obstructive pulmonary disease (COPD), infarction, aneurysm, cataracts, post-infarction tissues, frailty, chronic kidney disease, diabetic macular degeneration etc.) or a wound in a patient in need of such treatment, said method comprising administering to said patient in need of such treatment a therapeutically effective amount of a pharmaceutical composition as defined in any one of statements 1-54.66. Use of a pharmaceutical composition as defined in any one of statements 1-54 in the manufacture of a medicament for the treatment of a disease or disorder in which the presence of senescent cells is implicated.67. Use of a pharmaceutical composition as defined in any one of statements 1-54 in the manufacture of a medicament for the treatment of a proliferative disorder.68. Use of a pharmaceutical composition as defined in any one of statements 1-54 in the manufacture of a medicament for the treatment of cancer.69. Use of a pharmaceutical composition as defined in any one of statements 1-54 in the manufacture of a medicament for the treatment of a pre-malignant lesion, a tumour (e.g., a pre-malignant tumour, a malignant tumour or a tumour following treatment- induced (e.g., chemotherapy) cellular senescence), an age-related disorder (e.g., chronic disorders such as cancer, cardiovascular diseases (e.g., atherosclerosis), fibrosis (e.g., pulmonary fibrosis, idiopathic pulmonary fibrosis and kidney fibrosis), neurological disorders (e.g., Alzheimer’s and Parkinson’s disease), Type 1 & 2 diabetes, skeletomuscular disorders (e.g., sarcopenia, osteoarthritis and osteoporosis), inflammatory diseases, chronic obstructive pulmonary disease (COPD), infarction, aneurysm, cataracts, post-infarction tissues, frailty, chronic kidney disease, diabetic macular degeneration etc.) or a wound.70. A process for preparing a pharmaceutical composition comprising nanoparticles of a cyanine dye and a therapeutic agent, the process comprising the steps of:(i) providing an aqueous solution of the cyanine dye;(ii) maintaining the aqueous solution of the cyanine dye at a temperature of between 15 °C to 85 °C, preferably with agitation, to allow the cyanine dye molecules to aggregate and form nanoparticles;(iii) collecting and purifying the resultant nanoparticles present in the solution; and(iv) mixing the purified nanoparticles with a therapeutic agent, wherein step (iii) comprises at least one separation step wherein the nanoparticles formed in step (ii) are separated from the aqueous solution and re-suspended or dispersed in a different aqueous medium.71. The process of statement 70, wherein the pH of the aqueous solution in step (i) and / or step (ii) is within the range of 2 to 9.72. The process of statement 70 or 71, wherein the pH of the aqueous solution in step (i) and / or step (ii) is within the range of 2 to 8.73. The process of statement 70, 71 or 72, wherein the pH of the aqueous solution in step (i) and / or step (ii) is within the range of 2 to 7.74. The process of any one of statements 70-73, wherein the pH of the aqueous solution in step (i) and / or step (ii) is within the range of 2 to 6.75. The process of any one of statements 70-74, wherein the concentration of cyanine dye in the aqueous solution in step (i) is within the range of 0.01 mM to 10 mM.76. The process of any one of statements 70-75, wherein the concentration of cyanine dye in the aqueous solution in step (i) is within the range of 0.1 mM to 7.5 mM.77. The process of any one of statements 70-76, wherein the concentration of cyanine dye in the aqueous solution in step (i) is within the range of 0.25 mM to 5 mM.78. The process of any one of statements 70-77, wherein the concentration of cyanine dye in the aqueous solution in step (i) is within the range of 0.5 mM to 2 mM.79. The process of any one of statements 70-78, wherein the concentration of cyanine dye in the aqueous solution in step (i) is within the range of 0.6 mM to 0.9 mM.80. The process of any one of statements 70-79, wherein the concentration of cyanine dye in the aqueous solution in step (i) is within the range of 0.7 mM to 0.8 mM.81. The process of any one of statements 70-80, wherein the concentration of cyanine dye in the aqueous solution in step (i) is 0.75 mM.82. The process of any one of statements 70-81, wherein the aqueous solution of the cyanine dye comprises water.83. The process of any one of statements 70-82, wherein the aqueous solution of the cyanine dye is a solution of the cyanine dye in water.84. The process of any one of statements 70-83, wherein in step (ii), the solution is heated to a temperature within the range of 40 °C to 85 °C, optionally for 0.5 to 48 hours.85. The process of any one of statements 70-84, wherein in step (ii), the solution is heated to a temperature within the range of 45 °C to 85 °C, optionally for 0.5 to 48 hours.86. The process of any one of statements 70-85, wherein in step (ii), the solution is heated to a temperature within the range of 55 °C to 75 °C, optionally for 0.5 to 48 hours.87. The process of any one of statements 70-86, wherein in step (ii), the solution is heated to a temperature range of 60 °C to 70 °C, optionally for 0.5 to 48 hours.88. The process of any one of statements 70-87, wherein in step (ii), the solution is heated to a temperature of 65 °C, optionally for 0.5 to 48 hours.89. The process of any one of statements 70-88, wherein in step (ii), the process further comprises monitoring the formation of nanoparticles, optionally by monitoring the depletion of the non-aggregated cyanine dye from the aqueous solution and / or the formation of the nanoparticles.90. The process of any one of statements 70-89, wherein in step (iii), the at least one separation step separates the nanoparticles formed in step (ii) from the aqueous solution to remove any non-aggregated cyanine dye from the aqueous medium.91. The process of any one of statements 70-90, wherein in step (iii), the at least one separation step is centrifugation, filtration and / or dialysis.92. The process of any one of statements 70-91, wherein in step (iii), the collection and purification of the nanoparticles comprises two or more, or three or more, independent separation (e.g., centrifugation, filtration and / or dialysis) steps.93. The process of any one of statements 70-92, wherein step (iii) comprises at least one centrifugation step wherein the solution is centrifuged to form a pellet of the nanoparticles, removing the supernatant, optionally washing the pellet, and re-suspending the pellet in a different aqueous medium.94. The process of any one of statements 91-93, wherein the centrifugal force is 20,000 x g (RCF) to 50,000 x g (RCF).95. The process of any one of statements 91-94, wherein the centrifugal force is 25,000 x g (RCF) to 45,000 x g (RCF).96. The process of any one of statements 91-95, wherein the centrifugal force is 27,500 x g (RCF) to 42,500 x g (RCF).97. The process of any one of statements 91-96, wherein the centrifugal force is 30,000 x g (RCF) to 40,000 x g (RCF).98. The process of any one of statements 91-97, wherein the centrifugation step is performed at 10,000 rpm to 25,000 rpm.99. The process of any one of statements 91-98, wherein the centrifugation step is performed at 12,500 rpm to 22,500 rpm.100. The process of any one of statements 91-99, wherein the centrifugation step is performed at 15,000 rpm to 20,000 rpm.101. The process of any one of statements 91-100, wherein the centrifugation step is performed at 17,000 rpm to 18,000 rpm.102. The process of any one of statements 70-101, wherein the collection and purification of the nanoparticles further comprises additional steps of dialysing the solution of nanoparticles and / or filtering the solution of nanoparticles.103. The process of any one of statements 91-102, wherein the centrifugation step may be performed at a temperature of 0 °C to 10 °C, optionally for 30 seconds to 1 hour.104. The process of any one of statements 91-103, wherein the centrifugation step is performed at a temperature of 1 °C to 7 °C, optionally for 1 minute to 1 hour.105. The process of any one of statements 91-104, wherein the centrifugation step is performed at a temperature of 3 °C to 5 °C, optionally for 20 minutes to 40 minutes.106. The process of any one of statements 70-105, wherein the molar ratio of therapeutic agent to purified nanoparticles in step (iv) is 20:1 to 1 :20.107. The process of any one of statements 70-106, wherein the molar ratio of therapeutic agent to purified nanoparticles in step (iv) is 15:1 to 1 :5.108. The process of any one of statements 70-107, wherein the molar ratio of therapeutic agent to purified nanoparticles in step (iv) is 12:1 to 1 :2.109. The process of any one of statements 70-108, wherein the molar ratio of therapeutic agent to purified nanoparticles in step (iv) is 10:1 to 1 :1.110. The process of any one of statements 70-109, wherein the molar ratio of therapeutic agent to purified nanoparticles in step (iv) is 10:1, 5:1 or 1:1.111. The process of any one of statements 70-110, wherein step (iv) is performed at a temperature of 10 °C to 40 °C.112. The process of any one of statements 70-111, wherein step (iv) is performed at a temperature of 15 °C to 35 °C.113. The process of any one of statements 70-112, wherein step (iv) is performed at a temperature of 20 °C to 30 °C.114. The process of any one of statements 70-113, wherein step (iv) is performed at a temperature of 25 °C.115. The process of any one of statements 70-114, wherein the therapeutic agent in step (iv) is contacted with (i.e., dissolved in) a solvent before mixing with the purified nanoparticles.116. The process of statement 115, wherein the solvent is an organic solvent.117. The process of statement 116, wherein the organic solvent comprises any one or more of the following: acetic acid, acetone, acetonitrile, methanol, ethanol, propanol, butanol, chloroform, 1 ,2-dichloroethane, ethylene glycol, diethylene glycol, dimethyl formamide, dimethylsulfoxide, 1 ,4-dioxane, ethyl acetate, hexane, heptane, benzene, toluene, naphthalene, xylene and / or tetrahydrofuran.118. The process of statement 116 or 117, wherein the organic solvent comprises methanol, acetone and / or tetrahydrofuran.119. The process of any one of statements 115-118, wherein the organic solvent is evaporated (e.g., by rotary evaporation) to obtain a film of the therapeutic agent before mixing with the purified nanoparticles, or the therapeutic agent is dissolved in the organic solvent (alone) prior to mixing with the aqueous nanoparticles.120. The process of any one of statements 70-119, wherein step (iv) comprises at least one separation step wherein the pharmaceutical composition formed by mixing the purified nanoparticles with a therapeutic agent is separated (i.e., isolated) from the aqueous medium of step (iii).121. The process of statement 120, wherein the isolated pharmaceutical composition is re-suspended or dispersed in a different aqueous medium.122. The process of statement 120 or 121 , wherein the at least one separation step is carried out multiple times.123. The process of statement 120, 121 or 122, wherein the at least one separation step is centrifugation, filtration and / or dialysis.124. The process of statement 123, wherein the at least one separation step is centrifugation.125. The process of any one of statements 120-124, wherein the at least one separation step comprises two or more, or three or more, independent separation (e.g., centrifugation, filtration and / or dialysis) steps.126. The process of any one of statements 123, 124 or 125, wherein the centrifugal force is 20,000 x g (RCF) to 50,000 x g (RCF).127. The process of any one of statements 123-126, wherein the centrifugal force is25,000 x g (RCF) to 45,000 x g (RCF).128. The process of any one of statements 123-127, wherein the centrifugal force is27,500 x g (RCF) to 42,500 x g (RCF).129. The process of any one of statements 123-128, wherein the centrifugal force is30,000 x g (RCF) to 40,000 x g (RCF).130. The process of any one of statements 123-129, wherein the centrifugation step is performed at 10,000 rpm to 25,000 rpm.131. The process of any one of statements 123-130, wherein the centrifugation step is performed at 12,500 rpm to 22,500 rpm.132. The process of any one of statements 123-131, wherein the centrifugation step is performed at 15,000 rpm to 20,000 rpm.133. The process of any one of statements 123-132, wherein the centrifugation step is performed at 17,000 rpm to 18,000 rpm.134. The process of any one of statements 123-133, wherein the centrifugation step is performed at a temperature of 0 °C to 10 °C, optionally for 30 seconds to 1 hour.135. The process of any one of statements 123-134, wherein the centrifugation step is performed at a temperature of 1 °C to 7 °C, optionally for 1 minute to 1 hour.136. The process of any one of statements 123-135, wherein the centrifugation step is performed at a temperature of 3 °C to 5 °C, optionally for 20 minutes to 40 minutes.137. The process of any one of statements 70-136, wherein step (iv) comprises at least one centrifugation step to form a pellet of the pharmaceutical composition, removing the supernatant, optionally washing the pellet, and re-suspending the pellet in a different aqueous medium.138. The process of any one of statements 70-137, wherein the pharmaceutical composition is as defined in any one of statements 1-54.139. The process of any one of statements 70-138, wherein the nanoparticles of a cyanine dye are as defined in any one of statements 1-54.140. The process of any one of statements 70-139, wherein the cyanine dye is as defined in any one of statements 1-54.141 . The process of any one of statements 70-140, wherein the therapeutic agent is as defined in any one of statements 1-54.142. The process of any one of statements 70-141 , wherein the pharmaceutical composition, the nanoparticles of a cyanine dye and the therapeutic agent are independently as defined in any one of statements 1-54.143. The process of any one of statements 70-142, wherein the pharmaceutical composition, the nanoparticles of a cyanine dye, the cyanine dye itself and the therapeutic agent are independently as defined in any one of statements 1-54.144. A process for preparing a pharmaceutical composition comprising nanoparticles of a cyanine dye and a therapeutic agent, the process comprising the steps of:(i) providing an aqueous solution of the cyanine dye;(ii) maintaining the aqueous solution of the cyanine dye at a temperature of between 15 °C to 85 °C, preferably with agitation, to allow the cyanine dye molecules to aggregate and form nanoparticles;(iii) collecting the resultant nanoparticles present in the solution;(iv) dissolving the resultant nanoparticle to form dimer molecules of the cyanine dye;(v) mixing the dimer molecules of the cyanine dye with a therapeutic agent;(vi) forming nanoparticles of the dimer molecules cyanine dye that comprise the therapeutic agent; and(vii) collecting the nanoparticles formed in step (vi).145. A process of statement 144, wherein step (iii) comprises at least one separation step wherein the nanoparticles formed in step (ii) are separated from the aqueous solution and re-suspended or dispersed in a different aqueous medium.146. A process of statement 144 or 145, wherein steps (i) to (iii) are as defined in any one of statements 70 to 105 above.147. A process of any one of statements 144 to 146, wherein the cyanine dye is ICG and dimers of ICG are formed in step (iv) of the process.148. A process of any one of statements 144 to 147, wherein in step (iv) the nanoparticles are dissolved in a suitable alcohol, e.g. ethanol.149. A process of any one of statements 144 to 148, wherein in step (v) the dimers of the cyanine dye are mixed with the therapeutic agent with a molar ratio of therapeutic agent to cyanine dye dimer of 20:1 to 1 :20, 15:1 to 1 :15, 12:1 to 1 :12, 10:1 to 1 :10, 10:1 , 5:1 , 1 :1 , 1 :5 or 1 :10.150. A process of any one of statements 144 to 149, wherein in step (vi) the nanoparticles are formed by maintaining an aqueous solution of the cyanine dye dimer and therapeutic agent at a temperature of between 15 °C to 85 °C, preferably with agitation, to allow the cyanine dye dimer molecules to aggregate and form nanoparticles comprising the therapeutic agent.151. A process of any one of statements 144 to 150, wherein in step (vi) the pH of the aqueous solution is within the range of 2 to 9, 2 to 8, 2 to 7, or 2 to 6.152. A process of any one of statements 144 to 151 , wherein in step (vi) the concentration of cyanine dye dimer in the aqueous solution in step (i) is within the range of 0.01 mM to 10 mM. 0.1 mM to 7.5 mM, 0.25 mM to 5 mM, 0.5 mM to 2 mM, 0.6 mM to 0.9 mM, or 0.7 mM to 0.8 mM.153. A process of any one of statements 144 to 152, wherein in step (vi) the solution is heated to a temperature within the range of:(i) 40 °C to 85 °C, optionally for 0.5 to 48 hours;(ii) 45 °C to 85 °C, optionally for 0.5 to 48 hours;(iii) 55 °C to 75 °C, optionally for 0.5 to 48 hours;(iv) 60 °C to 70 °C, optionally for 0.5 to 48 hours; or(v) 65 °C, optionally for 0.5 to 48 hours.154. A process of any one of statements 144 to 153, wherein in step (vi) the therapeutic agent in step (iv) is contacted with (i.e., dissolved in) a solvent before mixing with the purified nanoparticles.155. The process of statement 154, wherein the solvent is an organic solvent, optionally selected from any one of the following: acetic acid, acetone, acetonitrile, methanol, ethanol, propanol, butanol, chloroform, 1 ,2-dichloroethane, ethylene glycol, diethylene glycol, dimethyl formamide, dimethylsulfoxide, 1 ,4-dioxane, ethyl acetate, hexane, heptane, benzene, toluene, naphthalene, xylene and / or tetrahydrofuran.156. A process of any one of statements 144 to 155, wherein in step (vii) the nanoparticles are collected by separating the nanoparticles from the aqueous medium in the manner defined in statements 123 to 137.EXAMPLESFiguresFigure 1 A shows the chemical structure of rhodamine, which was used as a proof of concept in the pharmaceutical compositions of the present invention. Rhodamine was associated to J-aggregate nanoparticles (in replacement of the therapeutic agent), to see whether it could be associated and subsequently dissociated.Figure 1 B shows the colour changes of the rhodamine-loaded J-aggregate nanoparticles in water (before disruption) and ethanol (after disruption). The colour change is indicative of rhodamine dissociating from the J-aggregate nanoparticles.Figure 1C shows the absorbance spectra of the rhodamine-loaded J-aggregate nanoparticles in water and in ethanol. The restoration of the rhodamine absorbance indicates its release from the J-aggregate supramolecular structure.Figure 1 D shows a solution of rhodamine loaded J-aggregate nanoparticles being added to ethanol. The composition, when rhodamine is incorporated into the structure of the J- aggregate nanoparticles, is green (left). When the composition contacts ethanol (middle) the structure is disrupted and the rhodamine dissociates, resulting in a purple solution (right).Figure 2A shows the chemical structure of doxorubicin.Figure 2B (top) shows absorbance spectra for doxorubicin-loaded J-aggregate nanoparticles in water and ethanol. Figure 2B also shows (bottom) fluorescence spectra for doxorubicin-loaded J-aggregate nanoparticles in water and ethanol. The fluorescence of doxorubicin was quenched when incorporated into the structure of the J-aggregate nanoparticles (bottom left). When the structure of the J-aggregate nanoparticles is disrupted with ethanol, doxorubicin is released and its fluorescence restored (bottom right).Figure 3 shows the characterisation of cellular senescence in A549 and SK-MEL-103 cells. Figures 3A and 3B show western blots of cellular senescence markers, demonstrating a reduction in pRB, and increased p21 levels in senescent cells compared to controls. Figures 3C and 3D show senescence-associated p-Galactosidase staining using a commercial p- Galactosidase staining kit.Figure 4 shows the cell viability of J-aggregate nanoparticles without a therapeutic agent in control and senescent A549 and SK-MEL-103 cells. This demonstrates that the J-aggregate nanoparticles show no toxicity on their own.Figure 5A shows the cell viability of navitoclax without J-aggregate nanoparticles in control and senescent cells for the SK-MEL-103 cell line.Figure 5B shows the cell viability of navitoclax-loaded J-aggregate nanoparticles in control and senescent cells for the SK-MEL-103 cell line.Figure 6A shows the cell viability of navitoclax without J-aggregate nanoparticles in control and senescent cells for the A549 cell line.Figure 6B shows the cell viability of navitoclax-loaded J-aggregate nanoparticles in control and senescent cells for the A549 cell line.Figure 7 shows the effect of navitoclax-loaded J-aggregate nanoparticles and navitoclax without J-aggregate nanoparticles on control cells at 0 days (Figures 7A and 7B). Also shown is the effect of navitoclax-loaded J-aggregate nanoparticles and navitoclax without J- aggregate nanoparticles on senescent SK-MEL-103 cells at 0 days (Figures 7C and 7D).Figure 8 shows the effect of navitoclax-loaded J-aggregate nanoparticles and navitoclax without J-aggregate nanoparticles on control cells at 12 hours (Figures 8A and 8B). Also shown is the effect of navitoclax-loaded J-aggregate nanoparticles and navitoclax without J- aggregate nanoparticles on senescent SK-MEL-103 cells at 12 hours (Figures 8C and 8D).Figure 9 shows the effect of navitoclax-loaded J-aggregate nanoparticles and navitoclax without J-aggregate nanoparticles on control cells at 1 day (24 hours) (Figures 9A and 9B). Also shown is the effect of navitoclax-loaded J-aggregate nanoparticles and navitoclax without J-aggregate nanoparticles on senescent SK-MEL-103 cells at 1 day (24 hours) (Figures 9C and 9D).Figure 10 shows the effect of navitoclax-loaded J-aggregate nanoparticles and navitoclax without J-aggregate nanoparticles on control cells at 1 day and 12 hours (36 hours) (Figures 10A and 10B). Also shown is the effect of navitoclax-loaded J-aggregate nanoparticles and navitoclax without J-aggregate nanoparticles on senescent SK-MEL-103 cells at 1 day and 12 hours (36 hours) (Figures 10C and 10D).Figure 11 shows images of melanoma SK-Mel-103 control (non-senescent) cells incubated with doxorubicin (Dox) alone and doxorubicin-loaded J-aggregate nanoparticles after 16 hours of incubation. The cells were imaged at different wavelengths to show the distribution of Hoerscht stain, doxorubicin (Dox) and the J-aggregate nanoparticles (jagg) and the images entitled “merge” show the amalgamation of the separate Hoerscht stain, doxorubicin (Dox) and the J-aggregate nanoparticles (jagg) images.Figure 12 shows images of melanoma SK-MEL-103 senescent cells (induced with 5 .M of Palbociclib) incubated with doxorubicin (Dox) alone and doxorubicin-loaded J-aggregate nanoparticles after 16 hours of incubation. The cells were imaged at different wavelengths to show the distribution of Hoerscht stain, doxorubicin (Dox) and the J-aggregatenanoparticles (jagg) and the images entitled “merge” show the amalgamation of the separate Hoerscht stain, doxorubicin (Dox) and the J-aggregate nanoparticles (jagg) images..Figure 13 shows a schematic representation of the formation of J-aggregate nanoparticles (labelled “NanoJagg”) from ICG and the subsequent treatment of these nanoparticles with ethanol / drying to form ICG dimers. The ICG dimers can be mixed with doxorubicin or other therapeutics and used to form drug-loaded J-aggregate nanoparticles (labelled “Loaded NanoJagg”), which may then be used for therapeutic applications.Figure 14 shows a plot of absorbance versus wavelength (nm) to illustrate the formation of J-aggregate nanoparticles from ICG dimers at different time points (0, 0.5, 2, 4 and 16 hours).Figure 15A shows a plot of absorbance versus wavelength (nm) of the NanoJagg (J- aggregate Nanoparticle) formed from Doxorubicin and ICG dimers (1 :5 ratio).Figure 15B shows the fluorescence interpolated concentration of doxorubicin in NanoJagg (J-aggregate nanoparticle) in water where its nanostructure is intact versus methanol, where the J-aggregate nanoparticle structure is dissembled thus releasing doxorubicin. The fluorescence signal was used to calculate percent loading of 7.19%.Figure 16 shows images of non-senescent MDA-MB-231 cells incubated with doxorubicin doxorubicin-loaded J-aggregate nanoparticles after 48 hours of incubation. The cells were imaged at different wavelengths to show the distribution of the nuclei, lysosomes, the J- aggregate nanoparticles (Nanojagg) and doxorubicin. The images entitled “merge” show the amalgamation of all of the aforementioned images, demonstrating enhanced lysosome localization in NanoJagg encapulsulated doxorubicin.Figure 17 shows that effect of doxorubicin-loaded J-aggregate nanoparticles (NanoDox) and a doxorubicin alone on MDA-MB-231 control cells (A) and senescent cells (B).Materials and methodsJ-aggregate nanoparticle synthesis

[0120] An aqueous ICG (Acros Organics, 10321541) solution (0.75 mM, 10 mL) was sonicated for 10 minutes and then heated to 65 °C under stirring (500 rpm). The reaction was monitored by UV-Vis spectrophotometer to assess J-aggregate nanoparticle formation (indicated by a transition of 780nm to 895nm). Upon reaction completion (~24 h) the mixture was centrifuged and washed three times at 17000 rpm (31 ,000 x g) at 4 °C for 30 minutes in a Sorvall LYNX 4000 high speed centrifuge. The obtained pellet of this reaction wasredispersed in deionised water and filtered through a 0.2 pm filter and lyophilized to obtain dark green nanoparticles (5.4 mg, 54% yield).

[0121] Lyophilization was carried out using a Telstar LyoQuest benchtop freeze dryer (0.008 mBar, -70 °C). The hydrodynamic size and zeta potential of the nanoparticles were measured using a Zetasizer Nano Range instrument (Malvern Panalytical). To determine the chemical composition LC-MS and1H NMR were conducted on the obtained nanoparticles and the supernatant obtained during the centrifugationPharmaceutical composition synthesisW / ater soluble therapeutic agents

[0122] J-aggregate nanoparticles of ICG 1 mL (2 mg / mL), as prepared above, were suspended in deionised water and mixed with various concentrations of water soluble therapeutic agents. In this example, doxorubicin was the chosen therapeutic agent and different molar ratios of doxorubicin : nanoparticles were investigated (10:1 , 5:1 and 1 :1). Doxorubicin was added to the nanoparticles and the mixture was left to stir at 400 rpm overnight at 25 °C. Nanoparticles of the pharmaceutical composition were obtained by centrifugation and washed three times at 17000 rpm (31 ,000 x g) at 4 °C for 30 minutes in a Sorvall LYNX 4000 high speed centrifuge. The obtained pellet of this reaction was redispersed in deionised water and filtered through a 0.2 pm filter for cell culture work.Water insoluble therapeutic agents

[0123] In this example, water insoluble therapeutic agents were used, specifically navitoclax and paclitaxel. The water insoluble therapeutic agent was first dissolved in an appropriate solvent (methanol, acetone, orTHF) at molar ratios of (10:1 , 5:1 , and 1 :1 relative to the nanoparticles). These solutions were then evaporated by rotary evaporation to obtain a thin film. To this film, 1 mL of 2 mg / mL ICG J-aggregate nanoparticle solution, as prepared above, was added. This mixture was left to stir at 400 rpm overnight at 25 °C. In some cases these solutions of therapeutic agents in solvents (DMSO or methanol), were added directly to a solution 1 mL of 2 mg / mL ICG J-aggregate nanoparticle solution, as prepared above. This mixture was left to stir at 400 rpm overnight at 25 °C. In both cases, nanoparticles of the pharmaceutical composition were obtained by centrifugation and washed three times at 17000 rpm (31000 x g) at 4 °C for 30 minutes in a Sorvall LYNX 4000 high speed centrifuge. The obtained pellet of this reaction was redispersed in deionised water and filtered through a 0.2 pm filter for cell culture work.Encapsulation of Drugs in J-aggregate nanoparticles of ICG dimer

[0124] Using the above method we create NanoJagg J-aggregates of ICG dimer nanoparticles. After synthesis and purification, we dissolve the nanoparticles in ethanol triggering their de-aggregation into the dimers of Indocyanine green. The ethanol is then removed by roto-evaporation. The created dimers are then mixed with Doxorubicin (ApexBio, A3966) in molar equivalents ranging from 20:1 to 1 :1 (ICG:Doxorubicin). This solution is then heated at 65°C to reform the NanoJagg J-aggregate ICG dimer nanoparticles and encapsulate the drug in 2-4 hours. The resulting Nanoparticles are washed three times in deionized water.

[0125] In the case of Navitoclax (Stratech, HY-10087 and ApexBio, A3007) a 1- 10% DMSO solution is used to enable the drug to be soluble. In molar equivalents ranging from 20:1 to 1 :1 (ICG:Navitoclax). This solution is then heated at 65°C to reform the NanoJagg J-aggregate ICG dimer nanoparticles and encapsulate the drug in 2-4 hours. The is washed three times in the same solution, then twice with deionized water. In both cases the J-aggregate absorbance peak at 895 nm is monitored to ensure synthesis is completed.Dissociation of the therapeutic agent

[0126] J-aggregate nanoparticles are a supramolecular structure which can be dissembled to its component parts. To demonstrate this concept, the J-aggregate nanoparticles were tested in the presence of organic solvents (e.g., acetone, acetonitrile, dimethyl formamide, dimethylsulfoxide, methanol, ethanol, isopropanol, tetrahydrofuran etc.). When the solvents are added to the nanoparticles, the supramolecular structure falls apart, and only the monomeric unit remains, previously determined as a dimer of ICG. This process also allows for the abrupt release of any cargo.Determination of therapeutic agent loading concentrationDoxorubicin

[0127] Due to doxorubicin’s fluorescent properties, a standard curve can be created plotting concentration of therapeutic agent vs. its fluorescent emission. The slope of this curve can be used to determine the concentration of doxorubicin released from the washed and pelleted J-aggregate nanoparticles after its disassembly in the solvents.Navitoclax and pacliatxel

[0128] The loading concentration of these non-fluorescent therapeutic agents was determined by High-performance liquid chromatography (HPLC) using an Agilent 1260 Infinity Quaternary LC equipped using an Agilent Zorbax SB-C18. The mobile phase consisted of H2O : acetonitrile (50 / 50) and the flow rate was set at 1.0 mL / min. The retention time of navitoclax was found to be 5 minutes and the total run time was 15 minutes.Cell lines

[0129] The A549 (human lung adenocarcinoma) cell line was obtained from the European Collection of Authenticated Cell Cultures (ECACC). The SK-MEL-103 (human melanoma) cancer cell line was acquired from the American Type Culture Collection (ATCC). MIA PaCA-2 and PANC-1 were purchased from American Type Culture Collection (ATCC). MDA-MB-231 cells were purchased from American Type Culture Collection (ATCC).

[0130] These cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, 11965092) and supplemented with 10% Fetal Bovine Serum (FBS, Gibco, 26140087). All cell lines were incubated in 20% O2 and 5% CO2 at 37 °C. Cells were routinely tested for mycoplasma using the universal Mycoplasma Detection Kit (ATCC) or by RNA- capture ELISA.Senescence induction

[0131] For senescence induction, SK-MEL-103 cells were supplemented with the same media containing Palbociclib (PD0332991 , MCE) at 5 pM for 7 days. A549 cells were supplemented with the same media containing 15 pM Cisplatin (Stratech) for 10 days or 10 pM Palbociclib Cisplatin (Stratech) was reconstituted in sterile PBS and Palbociclib in dimethyl sulfoxide.Senescence characterisation

[0132] Cell lysis was performed using RIPA buffer (Sigma) supplemented with phosphatase inhibitors (PhosSTOPTM EASYpak Phosphatase Inhibitors Cocktail, Roche) and protease inhibitors (cOmpleteTM Protease Inhibitor Cocktail, Roche). Proteins were quantified using bicinchoninic acid (BCA) assay and separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore) according to standardprotocols. Membranes were immunoblotted with antibodies against p21 (556430) from BD Pharmingen, phospho-Rb (pRBS807 / 822) from Cell Signaling and normalised to GAPDH (ab9485) from Abeam. After incubation with the primary antibody overnight at 4 °C, membranes were washed and incubated with secondary HRP-conjugated AffiniPure antibodies (Jackson ImmunoResearch) for 1 hour at room temperature and subsequently incubated with Enhanced Chemiluminescence Detection solution (Amersham). Skim milk was used as the blocking solution, and media for all antibodies.

[0133] SA-p-Gal staining was performed using the Senescence p-Galactosidase Staining kit (Cell Signaling), following the manufacturer instructions. Briefly, cells were fixed at room temperature for 15 minutes with 2% formaldehyde, washed with PBS and incubated overnight at 37 °C with the staining solution containing X-gal in N,N-dimethylformamide (pH 6.0). The next day cells were washed three times with PBS for 2 minutes, and finally PBS was added to the cells for imaging. Pictures were taken using a Wide Field Zeiss Axio Observer 7. For tissue cryosections this method was repeated, however the tissue was incubated with X-gal for 4-6 hours.Cell viability assays

[0134] Cell viability was determined using CellTiter-Blue assay (Promega). CellTiter Blue uses the reduction of resazurin to resorufin to measure cell viability. Cells were seeded in 96-well plates (Eppendorf), 3,500 control cells / well and 5,000 senescent cells / well. After 24 hours, J-aggregate nanoparticles loaded with therapeutic agents were added to the cells for 72 hours at a range of 1 pg / mL to 100 pg / mL. After 72 hours, 4 pL CellTiter-Blue reagent was added to each well. After incubation for 2 hours the absorbance and fluorescence were recorded for each well on the Infinite 200 Pro (Tecan) using 560 / 590 excitation emission. The viability studies were conducted in 4 technical replicates and 3 biological replicates. The viability was calculated according to the following equation: 100 x ((Sample - Negative Control) / (Untreated Cells - Negative Control)). This was shown as percent viability.Incucyte

[0135] Cells were seeded in 96-well plates (Eppendorf), 3,500 control cells / well and 5,000 senescent cells / well. After 24 hours, J-aggregate nanoparticles loaded with therapeutic agents were added to the cells for 72 hours at a range of 1 pg / mL to 100 pg / mL. Images were taken every 2 hours, at both Orange (ext:536-568 / ems:576-639) and near infrared (NIR) (ext:648-674 / ems:685-771). Images (Figures 7-10) were acquired on an Incucyte Sx5(Essen Bioscience). A total of 4 pictures per well were analysed using the IncuCyte ZOOM™ software analyzer and used to create images and videos.Confocal microscopy

[0136] Cells were seeded in 96-well plates (Eppendorf), 3,500 control cells / well and 5,000 senescent cells / well in 96-well p-clear plates (Greiner Bio-One). After 24 hours cells were incubated with J-aggregate nanoparticles at concentrations of 10-100 pg / mL. Confocal images were acquired on a Leica SP5 confocal microscope using a 20X HCX PL APO 0.5 NA dry objective or a 40X HCX PL APO 1.3 NA oil immersion objective. Hoechst (ThermoFischer) was used to specifically dye the nucleus at 5 pg / mL. Lysotracker were detected by using excitation wavelength of 488 nm (Argon laser) and with a detection window between 510 and 530 nm. The organelle specific dyes were used according to their manuals. Doxorubicin was detected by using excitation wavelength of 488 nm (Argon laser) and with a detection window between 580 and 600 nm. J-aggregate nanoparticles were detected by using excitation wavelength of 633 nm (argon laser) and with a detection window between 680-720 nm. Cells without dyes and J-aggregate nanoparticles were used in parallel as autofluorescence controls using the corresponding excitation and detection wavelengths.

[0137] Images (Figures 11 and 12) were obtained on a Leica SP5 confocal microscope analyzed with LAS AF Lite (Leica). For Figure 16, images were captured on Zeiss Axio Observer Z1 , and images were analyzed with Zeiss Zen Lite.Organelle colocalization analysis by confocal microscopy

[0138] Cells were trypsinized and seeded in a flat-bottom p-clear 96-well plates (Greiner Bio-One, #655087) at a density of 3,500-5,000 control and 4,000-6,000 senescent cells / well. Once the cells were attached, culture medium was changed to DM EM supplemented with 0.2% FBS and incubated with cyanine dye nanoparticles (100 pg / mL - 10 pg / mL) for 12-16 h. Afterward, cells were washed 3xwith PBS for 5 min. Specific organelle stains LysoTracker Green DND-26 (Cell Signaling Technology, FM8783).Immunoblotting

[0139] Cell lysis was performed using RIPA buffer (Sigma) supplemented with phosphatase inhibitors (PhosSTOP™ EASYpak Phosphatase Inhibitors Cocktail, Roche) and protease inhibitors (complete™ Protease Inhibitor Cocktail, Roche). Proteins werequantified and separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore) according to standard protocols.

[0140] Membranes were immunoblotted with antibodies against p21 (556430) from BD Pharmingen, phospho-Rb (pRBS807 / 822) from Cell Signalling and normalized to GAPDH (ab9485) from Abeam. After incubation with the primary antibody overnight, membranes were washed and incubated with secondary HRP-conjugated AffiniPure antibodies (Jackson ImmunoResearch) for 1 h at room temperature and subsequently incubated with Enhanced Chemiluminescence Detection solution (Amersham).MaterialsDoxorubicin (APExBIO, A3966)Navitoclax (Stratech, HY- 10087)Paclitaxel (TCI, P1632).Results and discussion

[0141] Rhodamine B (Figure 1A) was used as a model drug to demonstrate loading of a compound into the J-aggregate nanoparticles. Visibly when rhodamine-loaded J-aggregates are added to ethanol, the rhodamine is released and the solution appears pink (Figure 1 B). Additionally, UV-Vis analysis demonstrates this phenomenon (Figure 1C). In water the rhodamine-loaded J-aggregate nanoparticles demonstrate the characteristic J-band at 895nm. Upon ethanol addition the J-aggregate supramolecular structure is disrupted, and both the rhodamine (560 nm) and dimeric-ICG (780 nm) become present. This process is shown pictorially (Figure 1 D). The disappearance of the rhodamine B max absorbance (560nm) when the J-aggregate nanoparticles are formed indicates that rhodamine B has associated with the nanoparticle structure.

[0142] The same J-aggregate nanoparticles were then loaded with doxorubicin (Figure 2A). These were evaluated with UV-Vis absorbance (Figure 2B - top) and fluorescence emission (Figure 2B - bottom). Upon disruption of the J-aggregate nanoparticles structure in ethanol, the characteristic dimer of ICG absorbance (780nm), and a slight absorbance increase at doxorubicin’s absorbance maximum (490 nm) is seen. The fluorescence emission spectrum is more obvious, where when loaded into the structure of the J-aggregate nanoparticles, doxorubicin’s fluorescence is quenched (Figure 2B - bottom left). Upon dissociation of the nanoparticles supramolecular structure, doxorubicin’s fluorescence is restored emission max (590 nm) (Figure 2B - bottom right). These results indicate thatdoxorubicin is incorporated into the structure of the J-aggregate nanoparticle, and, once released, regains its fluorescent properties.

[0143] Characterisation of cellular senescence in A549 and SK-Mel-103 cell lines. A549 cells were induced to become senescent with 15 pM of cisplatin for 10 days, and SK-MEL- 103 cells were incubated with 5 pM of Palbociclib for 7 days. After which the expression of phospho-RB a marker of cell cycle progression, p21 a marker of the senescent program, and Senescence p-Galactosidase activity was evaluated. In both cases the treatment led to a decreased expression of phospho-RB indicating a halt in the cell cycle (Figures 3A and 3B). Just as well p21 is upregulated in both cases indicating an initiation of the senescence program (Figures 3A and 3B). Senescence p-Galactosidase activity was found to be increased following both treatments (Figures 3C and 3D). Taken together this indicates that cells used in this study are bona fide senescent cells.

[0144] Cell viability was assessed using the CellTiterBlue assay where senescent and non-senescent human lung adenocarcinoma (A549) and human melanoma (SK-MEL-103) cell cultures were supplemented with a wide range of J-aggregate nanoparticle concentrations (0.09 - 100 pg / mL) (Figure 4). These results indicate that the J-aggregate nanoparticles on their own, do not reduce cell viability up to 100 pg / mL.

[0145] When loaded with navitoclax, J-aggregate nanoparticles display similar effectiveness to free navitoclax in SK-MEL-103 cells (Figures 5A and 5B). However, they slightly reduce toxicity to the proliferating control cells.

[0146] When loaded with navitoclax, J-aggregate nanoparticles display similar effectiveness to free navitoclax in A549 cells (Figures 6A and 6B). However, they slightly reduce toxicity to the proliferating control cells.

[0147] Figures 7-10 demonstrate that navitoclax-loaded J-aggregate nanoparticles have an improved senescent cell selectivity to navitoclax alone. Additionally, uptake can be tracked by increase in NIR fluorescence (blue), which indicates J-aggregate nanoparticle uptake and subsequent apoptosis. These results show that navitoclax can be delivered to senescent cells, and is still in its active state, and when encapsulated it reduces the toxicity to control cells.

[0148] Figure 11 demonstrates different colocalization patterns of doxorubicin and encapsulated doxorubicin in control non-senescent cells. When doxorubicin is encapsulated in the j-aggregate nanoparticle there is less localized with the nucleus. This could explain the reduced toxicity observed for these cells when doxorubicin is encapsulated. When doxorubicin isn’t encapsulated more localization with the nucleus is observed.

[0149] Figure 12 demonstrates localization patterns of doxorubicin and J-aggregate nanoparticle encapsulated doxorubicin in senescent cells. In both cases much of the signal is located away from the nucleus.

[0150] Figure 13 is a schematic of the ICG dimer loaded method. Firstly, indocyanine green (ICG) is heated at 65 °C to form the J-aggregate nanoparticles (NanoJagg). These nanoparticles are then dissolved into their component parts (ICG dimers) using ethanol or another solvent. The dimers are then mixed with doxorubicin or the therapeutic of choice, at certain ratios, and again heated to reform the NanoJaggs loaded with doxorubicin or the therapeutic of choice. These can then be applied to senescent cells, and the diseases that they cause.

[0151] Figure 14 demonstrates J-aggregate nanoparticle (NanoJaggs) formation over time from dimer. Already in 2 hours we find the J-aggregate nanoparticle (NanoJaggs) are reformed.

[0152] Figure 15A demonstrates the disassembly process of the doxorubicin loaded J- aggregate nanoparticles (NanoJaggs). When a solvent in this case methanol is added the supramolecular structure disassembles and the dimers are reformed. Triggering the release of doxorubicin. Figure 15B demonstration of doxorubicin fluorescence (excitation 495nm, emission 590 nm) when J-aggregate nanoparticle is formed (in water) compared to when it is disassembled (in methanol). Monitored doxorubicin fluorescence signal increases when nanoparticle structure is dissembled and its released.

[0153] Figure 16 demonstrates lysosomal colocalization of doxorubicin loaded J- aggregate nanoparticles (NanoJaggs) when compared to free doxorubicin which colocalizes in the nucleus. This could explain the reduced toxicity seen in Figure 17.

[0154] Figure 17A are IC50 curves of MDA-MB-231 control (non-senescent) cells. Here loaded doxorubicin J-aggregate nanoparticles (NanoJaggs) (IC50 of 2.08 .M). This was 17 times less toxic than was free doxorubicin (IC50 of 0.12 .M). Likely due to the enhance lysosome accumulation as noted in Figure 16. Figure 17B are IC50 curves of MDA-MB-231 senescent cells. Here loaded doxorubicin J-aggregate nanoparticles (NanoJaggs) (IC50 of 11.6 jiM). was almost the as free doxorubicin (IC50 of 9.16 .M). Where no reduction of toxicity was seen.

[0155] While specific embodiments of the invention have been described herein for the purpose of reference and illustration, various modifications will be apparent to a person skilled in the art without departing from the scope of the invention as defined by the appended claims.

Claims

CLAIMS1. A pharmaceutical composition comprising: nanoparticles of a cyanine dye; and a therapeutic agent, wherein the therapeutic agent is associated to the nanoparticles.

2. The pharmaceutical composition of claim 1 , wherein the cyanine dye comprises a polymethine chain of 1-20 chain carbon atoms.

3. The pharmaceutical composition of claim 1 or 2, wherein the cyanine dye is selected from the group consisting of ICG, IR-140, IR-820, IR-806, IR-783, IR-780, Cy3, Cy3 NHS, Cy3 carboxylic acid, Cy5, Cy5 NHS, Cy5 carboxylic acid, Cy7, Cy7 NHS, Cy7 carboxylic acid, Cy7.5, Cy7.5 NHS, Cy7.5 carboxylic acid, DiOC6, JC-1 and cypate.

4. The pharmaceutical composition of any one of the preceding claims, wherein the cyanine dye can aggregate to form J-aggregates.

5. The pharmaceutical composition of any one of the preceding claims, wherein the nanoparticles have a particle size of less than 200 nm.

6. The pharmaceutical composition of any one of the preceding claims, wherein the nanoparticles have a particle size of 80-100 nm.

7. The pharmaceutical composition of any one of the preceding claims, wherein the therapeutic agent is cytotoxic to senescent cells.

8. The pharmaceutical composition of any one of the preceding claims, wherein the therapeutic agent is a senolytic and / or a chemotherapeutic agent.

9. The pharmaceutical composition of any one of the preceding claims, wherein the composition comprises one therapeutic agent.

10. The pharmaceutical composition of any one of the preceding claims, wherein the therapeutic agent is selected from the group consisting of navitoclax, venetoclax, dasatinib, quercetin, doxorubicin, cisplatin, paclitaxel, fisetin, chlorambucil, cyclophosphamide, carboplatin, obatolax, vincristine, topotecan, bleomycin, mitoxantrone and docetaxel.

11. The pharmaceutical composition of any one of the preceding claims, wherein the therapeutic agent is encapsulated (i.e. , enclosed) within the nanoparticles.

12. The pharmaceutical composition of any one of the preceding claims, wherein the therapeutic agent is present at a loading of about 1 weight % to about 40 weight % relative to the total weight of the composition.

13. The pharmaceutical composition of any one of the preceding claims, wherein the therapeutic agent is present at a loading of about 5 weight % to about 10 weight % relative to the total weight of the composition.

14. The pharmaceutical composition of any one of claims 1-12, wherein the therapeutic agent is present at a loading of about 20 weight % to about 30 weight % relative to total the weight of the composition.

15. The pharmaceutical composition of any one of the preceding claims, wherein the pharmaceutical composition releases in vivo. i) more than 5% of the therapeutic agent after 12 hours; ii) more than 10% of the therapeutic agent after 24 hours; iii) more than 15% of the therapeutic agent after 36 hours; and / or iv) more than 20% of the therapeutic agent after 48 hours; relative to the total amount of therapeutic agent.

16. The pharmaceutical composition of any one of the preceding claims, wherein the pharmaceutical composition releases in viva. i) more than 20% of the therapeutic agent after 12 hours; ii) more than 40% of the therapeutic agent after 24 hours; iii) more than 60% of the therapeutic agent after 36 hours; and / or iv) more than 80% of the therapeutic agent after 48 hours; relative to the total amount of therapeutic agent.

17. The pharmaceutical composition of any one of the preceding claims for use in therapy.

18. The pharmaceutical composition of any one of claims 1-16 for use in the treatment of a disease or disorder in which the presence of senescent cells is implicated.

19. The pharmaceutical composition of any one of claims 1-16 for use in the treatment of a pre-malignant lesion, a tumour (e.g., a pre-malignant tumour, a malignant tumour or a tumour following treatment-induced (e.g., chemotherapy) cellular senescence), an age- related disorder (e.g., chronic disorders such as cancer, cardiovascular diseases (e.g., atherosclerosis), fibrosis (e.g., pulmonary fibrosis, idiopathic pulmonary fibrosis and kidney fibrosis), neurological disorders (e.g., Alzheimer’s and Parkinson’s disease), Type 1 & 2 diabetes, skeletomuscular disorders (e.g., sarcopenia, osteoarthritis and osteoporosis), inflammatory diseases, chronic obstructive pulmonary disease (COPD), infarction, aneurysm, cataracts, post-infarction tissues, frailty, chronic kidney disease, diabetic macular degeneration etc.) or a wound.

20. A process for preparing a pharmaceutical composition comprising nanoparticles of a cyanine dye and a therapeutic agent, the process comprising the steps of:(i) providing an aqueous solution of the cyanine dye;(ii) maintaining the aqueous solution of the cyanine dye at a temperature of between 15 °C to 85 °C, preferably with agitation, to allow the cyanine dye molecules to aggregate and form nanoparticles;(iii) collecting and purifying the resultant nanoparticles present in the solution; and(iv) mixing the purified nanoparticles with a therapeutic agent, wherein step (iii) comprises at least one separation step wherein the nanoparticles formed in step (ii) are separated from the aqueous solution and re-suspended or dispersed in a different aqueous medium.

21. The process of claim 20, wherein the pH of the aqueous solution in step (i) and / or step (ii) is within the range of 2 to 9.

22. The process of any one of claim 20 or 21 , wherein the concentration of cyanine dye in the aqueous solution in step (i) is within the range of 0.01 mM to 10 mM.

23. The process of any one of claims 20-22, wherein in step (iii), the at least one separation step is centrifugation, filtration and / or dialysis.

24. The process of any one of claims 20-23, wherein the molar ratio of therapeutic agent to purified nanoparticles in step (iv) is 20:1 to 1 :20.

25. The process of any one of claims 20-24, wherein the molar ratio of therapeutic agent to purified nanoparticles in step (iv) is 10:1 to 1 :1.

26. A process for preparing a pharmaceutical composition comprising nanoparticles of a cyanine dye and a therapeutic agent, the process comprising the steps of:(i) providing an aqueous solution of the cyanine dye;(ii) maintaining the aqueous solution of the cyanine dye at a temperature of between 15 °C to 85 °C, preferably with agitation, to allow the cyanine dye molecules to aggregate and form nanoparticles;(iii) collecting the resultant nanoparticles present in the solution;(iv) dissolving the resultant nanoparticle to form dimer molecules of the cyanine dye;(v) mixing the dimer molecules of the cyanine dye with a therapeutic agent;(vi) forming nanoparticles of the dimer molecules cyanine dye that comprise the therapeutic agent; and(vii) collecting the nanoparticles formed in step (vi).

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