Hydrogel compositions

Abstract

The present invention relates to compositions comprising metal-organic framework (MOF) nanoparticle or nanoparticles, methods of preparing said compositions, methods of treatment using said compositions, and uses of said compositions.

Description

Hydrogel CompositionsField of the Invention

[0001] The present invention relates to compositions comprising metal-organic framework (MOF) nanoparticle or nanoparticles, methods of preparing said compositions, methods of treatment using said compositions, and uses of said compositions.Background of the Invention

[0002] Photodynamic therapy (PDT) has emerged as an important tool for cancer treatment due to its non-invasive nature, high selectivity for the target tissue, and minimal side effects as compared to conventional medical treatments, such as chemotherapy and radiotherapy.

[0003] PDT typically involves the administering of a photosensitizer (PS) directly into a tumour, followed by its direct or indirect activation by electromagnetic (EM) radiation to generate highly cytotoxic reactive oxygen species (ROS), particularly singlet oxygen (1O2), within the tumour. This then triggers cell apoptosis and necrosis within the tumour. The selectivity of PDT treatments can be enhanced by the ability to simultaneously restrict the location of both the PS and the EM radiation exposure within the tumour region.

[0004] Hence, with relative safety, PDT can be used as an adjuvant therapy in combination with other treatments in recurrent tumours. The use of PDT for treating cancers in the head and neck is particularly advantageous as the high localisation of PDT causes less damage to surrounding areas and hence reduces aesthetic and functional impairments.

[0005] Most PDT processes heavily depend on an adequate oxygen supply within the target cell for the generation of ROS. Due to the rapid cell proliferation typical of tumour cells and the distance of such cells from functional blood vessels, the microenvironment within a tumour cell is typically highly hypoxic. This can then limit the therapeutic efficacy of PDTs, including those based on MOF nanoparticles, that depend on the in-situ production of ROS.

[0006] Different strategies have been developed to mitigate this issue and improve the PDT effect, for example, by increasing the intracellular O2concentration, including hyperbaric oxygen inhalation, reoxygenation using oxygen carriers, or in situ O2generation. One known approach to alleviate hypoxia is the use of MOFs with inherent catalytic sites or nanoparticle@MOF composites to catalyse the intracellular transformation of H202to O2, such that O2can further be reacted to produce ROS. Another approach targets the presence ofROS scavengers, such as glutathione (GSH), within a cell so as to maximise the efficacy of the limited ROS that is produced. MOFs bearing high valence metal ions, such as Cu (II), Mn (III) and Mn (IV) have been reported to eliminate intracellular GSH.

[0007] Despite significant progress achieved with such approaches, the relatively low intracellular H2O2levels and high expression of GSH within cells can affect the cells reoxygenation and ROS generation capability. PDTs using MOF nanoparticle compositions have had minimal success to date. Therefore, novel strategies to improve the efficiency and efficacy of PDT treatments for treating cancer are still required.

[0008] The present invention addresses these and other problems associated with known PDT therapeutics.Summary of the Invention

[0009] Accordingly, in a first aspect, the present invention provides a composition comprising a metal-organic framework (MOF) nanoparticle or nanoparticles and a hydrogel. Each nanoparticle comprises a MOF comprising a plurality of metal ions (inorganic nodes) and a plurality of organic ligands. Each MOF nanoparticle comprises a photosensitiser and one or more moieties capable of associating to oxygen.

[0010] The one or more moieties capable of associating to oxygen may each have a molecular weight of about 1000 g / mol or less. Preferably, the one or more moieties capable of associating to oxygen each have a molecular weight of from about 200 g / mol to about 1000 g / mol, or about 300 g / mol to about 900 g / mol, or about 400 g / mol to about 800 g / mol. Most preferably, the one or more moieties capable of associating to oxygen each have a molecular weight of about 819 g / mol.

[0011] Preferably, each of the one or more moieties capable of associating to oxygen comprises a perfluorocarbon moiety. Preferably, the one or more moieties capable of associating to oxygen are one or more perfluorocarbon moieties. More preferably, each of the one or more moieties capable of associating to oxygen is a perfluorocarbon moiety. Perfluorocarbon moieties having a carbon backbone chain of about 6 to about 16 carbon atoms are preferred. Preferably, the perfluorocarbon moiety is a perfluorooctyl group.

[0012] Each of the one or more moieties capable of associating to oxygen may be a 1 ,3-diene. In some embodiments, each of the one or more moieties capable of associating to oxygen isone or more aromatic moieties selected from the group consisting of: anthracene or a derivative thereof, naphthalene or a derivative thereof, and 2-pyridone or a derivative thereof; preferably anthracene or a derivative thereof. In certain embodiments, each of the one or more moieties capable of associating to oxygen is a moiety selected from anthracene, naphthalene and 2-pyridone; preferably anthracene.

[0013] Preferably, the MOF nanoparticles have an enhanced oxygen carrying capacity of greater than about 5 mg / mL as measured according to the Oxygen Carrying Capacity Test Method defined herein. Preferably, the MOF nanoparticles have an enhanced oxygen carrying capacity of greater than about 6 mg / mL, greater than about 8 mg / mL, or greater than about 10 mg / mL as measured according to the Oxygen Carrying Capacity Test Method defined herein.

[0014] Accordingly, in a related aspect, the present invention provides a composition comprising a metal-organic framework (MOF) nanoparticle or nanoparticles and a hydrogel wherein each MOF nanoparticle comprises: a MOF comprising a plurality of metal ions and a plurality of organic ligands; a photosensitiser, and one or more perfluorocarbon moieties.

[0015] Perfluorocarbon moieties having a carbon backbone chain of about 6 to about 16 carbon atoms are preferred. Preferably, the perfluorocarbon moiety is a perfluorooctyl group.

[0016] Preferably, the MOF nanoparticles have an enhanced oxygen carrying capacity of greater than about 5 mg / mL as measured according to the Oxygen Carrying Capacity Test Method defined herein. Preferably, the MOF nanoparticles have an enhanced oxygen carrying capacity of greater than about 6 mg / mL, greater than about 8 mg / mL, or greater than about 10 mg / mL as measured according to the Oxygen Carrying Capacity Test Method defined herein.

[0017] Accordingly, in a further related aspect, the present invention provides a composition comprising a metal-organic framework (MOF) nanoparticle or nanoparticles and a hydrogel. Each MOF nanoparticle comprises: a MOF comprising a plurality of metal ions and a plurality of organic ligands; and a photosensitiser; wherein the MOF nanoparticles have an enhanced oxygen carrying capacity of greater than about 5 mg / mL measured according to the Oxygen Carrying Capacity Test Method defined herein.

[0018] Preferably, the MOF nanoparticles have an enhanced oxygen carrying capacity of greater than about 6 mg / mL, greater than about 8 mg / mL, or greater than about 10 mg / mL as measured according to the Oxygen Carrying Capacity Test Method defined herein.

[0019] Preferably, each MOF nanoparticle comprises one or more perfluorocarbon moieties. Perfluorocarbon moieties having a carbon backbone chain of about 6 to about 16 carbon atoms are preferred. Preferably, the perfluorocarbon moiety is a perfluorooctyl group.

[0020] Accordingly, in another related aspect, the present invention provides a composition comprising a metal-organic framework (MOF) nanoparticle or nanoparticles and a hydrogel. Each MOF nanoparticle comprises: a MOF comprising a plurality of metal ions and a plurality of organic ligands; a photosensitiser; and a 1 ,3-diene. In some embodiments, the 1 ,3-diene is one or more aromatic moieties selected from the group consisting of: anthracene or a derivative thereof, naphthalene or a derivative thereof, and 2-pyridone or a derivative thereof; preferably anthracene or a derivative thereof. In certain embodiments, the MOF nanoparticles comprise a moiety selected from anthracene, naphthalene and 2-pyridone; preferably anthracene. Moieties such as anthracene, naphthalene and 2-pyridone can react with oxygen in the presence of a photosensitiser and EM radiation to form endoperoxides (i.e. these moieties are “moieties capable of associating to oxygen”). These endoperoxides can then release singlet oxygen when exposed to thermal energy, such as body temperature (37 °C), when injected into a tumour.

[0021] Preferably, the MOF nanoparticles have an enhanced oxygen-carrying capacity of greater than about 5 mg / mL as measured according to the Oxygen Carrying Capacity Test Method defined herein. Preferably, the MOF nanoparticles have an enhanced oxygen-carrying capacity of greater than about 6 mg / mL, greater than about 8 mg / mL, or greater than about 10 mg / mL as measured according to the Oxygen Carrying Capacity Test Method defined herein.

[0022] The present invention provides hydrogel compositions comprising PS-containing MOF nanoparticles that may display an enhanced oxygen-carrying capability. The MOF nanoparticles may comprise a high oxygen affinity moiety, such as a perfluoro moiety. Any inclusion of a high oxygen affinity moiety may be controlled to provide improved oxygencarrying capability.

[0023] PDT therapies that depend on the generation of ROS require an oxygen supply within the target cell. The microenvironment within a tumour cell is typically highly hypoxic, limiting the therapeutic efficacy of PDTs that depend on the in situ production of ROS. The present invention addresses this problem by providing a MOF suitable for PDT therapy that may deliver oxygen to the tumour cell, including deeper more inaccessible tumours. The MOF mayprovide a source of oxygen for ROS generation by the photosensitiser, preferably even within the hypoxic microenvironment of a tumour cell.

[0024] The present invention provides improved MOF-based PDT treatments by the use of MOF nanoparticles (comprising suitable PS and metal cations) hydrogel compositions having the ability to carry oxygen to within a tumour cell. The use of the inventive MOF nanoparticles hydrogel compositions enables the increased generation of ROS even within the hypoxic microenvironment of a tumour cell. The localisation of the PDT MOF nanoparticles within a tumour may be enhanced by dispersing the MOF nanoparticles within compositions, such as hydrogels, to facilitate localisation of the MOF composition close to the point of injection. The hydrogel may provide both spatial control of the MOF nanoparticles and a mechanism for controlled release of a drug.

[0025] Furthermore, the use of a modified MOF nanoparticle to deliver the PS to within the tumour cells has many advantages. The size of the nanoparticle can be tuned so as to cross a target cell membrane. The nanoparticle can be coated or treated to improve its biocompatibility. Many nanoparticles comprising PS tend to aggregate in aqueous suspension due to the hydrophobicity of the PS. The incorporation of the PS in a suitably modified MOF nanoparticle can be used to address the tendency of the nanoparticles to aggregate.

[0026] The following embodiments relate to each of the foregoing aspects.

[0027] Preferably, the plurality of organic ligands are selected from 2',3”-dimethyl- [1 ,1’:4',1 ”:4",1 ”’-quaterphenyl]-4,4”’-dicarboxylic acid (Li), tetrakis (4- carboxyphenyl)porphyrin (TCPP), 1 ,4-benzenedicarboxylic acid (BDC), 1 ,3,5- benzenetricarboxylic acid (H3BTC), 1 ,3, 5, 8- (p-benzoate)pyrene linkers (H4TBAPy), 4’,4”’,4””’,4”””’-(ethene-1 ,1 ,2,2- tetrayl)tetrakis(([1 ,1'-biphenyl]-3-carboxylic acid)) (H4ETTC), a ligand of Formula I, a ligand of Formula II, a ligand of Formula III, a ligand of Formula IV, a ligand of Formula V, a ligand of Formula VI, and chlorin-based ligands.

[0028] Preferably, the plurality of metal ions are metal ions having an atomic weight of greater than 40 g / mol. Preferably, the plurality of metal ions may (as a plurality) consist essentially of (that is to say substantially all of the metal ions within the MOF nanoparticle are the same), or consist of, ions of a metal selected from the group consisting of: zirconium, iron, thorium, hafnium, gadolinium, neodymium, ytterbium and erbium; more preferably the plurality of metal ions may consist essentially of ions, or consist of ions, of a metal selected from the group consisting of: zirconium, hafnium and ruthenium; most preferably the plurality of metal ionsmay consist essentially of ions, or consist of ions, of a metal selected from the group consisting of zirconium and hafnium. .In some embodiments, the plurality of metal ions consists essentially of zirconium; and the organic ligand is the photosensitiser l_2or the photosensitiser l_3, preferably wherein the organic is the photosensitiser l_2In some such embodiments, the MOF nanoparticle further comprises a phosphate-terminated mPEG according to Structure A (below), wherein m is from about 40 to about 250.

[0029] The use of high atomic number metal cations in the MOF nanoparticles means that high energy, penetrating EM radiation can be used, if required, for less accessible, deeper tumours to activate a suitable PS by a two-step process. The metal cation can be excited / activated by high energy EM radiation (e.g. Gamma rays or X-rays) so as to emit photoelectrons. The photoelectrons then excite / activate the PS to generate ROS, such as singlet oxygen. This allows treatment of tumours further away from the skin or accessible surfaces. Typically, the metals in the MOF have an atomic number greater than 40. Such metals are referred to as “high-Z metals”. Typically, the metal species are biocompatible. For example, the skilled person will appreciate that a metal species such as Cadmium may be toxic and undesirable. The chemistries of some metals, such as rare earth metals (e.g. yttrium), are so different from those involved in any biological processes that the metal is essentially biologically inert. Such metals are typically also suitable for the present invention due to their high ability to absorb high-energy EM radiation. High Z metals such as zirconium, hafnium and thorium are preferred, in particular for the treatment of deep tumours (e.g. those that cannot be treated with visible light) with Gamma rays or X-rays. The MOF nanoparticles of the invention are also suitable for shallower tumours, such as skin cancer, by use of varying frequencies of EM radiation.

[0030] EM radiation includes energy which is part of the electromagnetic spectrum, such as radio waves, microwaves, infrared, (visible) light, ultraviolet, X-rays, and gamma rays. The frequency of radio waves is between 3 Hz and 1 GHz. The frequency of microwaves is between 1 GHz and 300 GHz. The frequency of infrared is between 300 GHz and 400 THz. The frequency of visible light is between 400 THz and 790 THz. The frequency of ultraviolet is between 800 THz and 30000 THz. The frequency of X-rays is between 30000 THz and 30000000 THz. The frequency of gamma rays is above 30000000 THz.

[0031] The photosensitiser may be selected from the group consisting of: porphyrins, chlorins, bacteriochlorins, phthalocyanines, boron-dipyrromethene (BODIPY) derivatives, disalycilidene-1 ,2-cyclohexylidenediamine derivatives, gemcitabine, or combinations thereof, preferably wherein the photosensitiser is a BODIPY derivative. In some embodiments, thephotosensitiser is disclosed in U.S. Pat. 10,206,871 , which is incorporated herein by reference in its entirety.

[0032] The photosensitiser may also be selected from the group consisting of: 5, 15-di(p- benzoato)porphyrin (DBP) and derivatives thereof; 5,15-di(p-benzoato)chlorin (DBC) or derivatives thereof; 5, 15-di(p-benzoato)bacteriochlorin (DBBC) and derivatives thereof; 5, 10, 15, 20-tetra(p-benzoato)porphyrin and derivatives thereof; 5, 10, 15, 20-tetra(p- pyridyl)porphyrin, phthalocyanine-octacarboxylic acid and derivatives thereof and di(5'- benzoatosalycylidene)-1 ,2-cyclohexylidenediamine and derivatives thereof, and combinations thereof. The term “derivative” includes metal complexes of a species.

[0033] Preferably, the photosensitiser is selected from the group consisting of: BODIPY- based ligands of Formula I, Aza-BODIPY-based ligands of Formula II, porphyrin-based ligands of Formula III, bacteriochlorin-based ligands of Formula IV, phthalocyanine-based ligands of Formula V, M(bpy)s ligands of Formula VI, and combinations thereof. Photosensitisers of Formula I and Formula VI are particularly preferred.Iormu a

[0034] Preferably, when the photosensitiser is one of Formula I to Formula VI, each of the plurality of organic ligands is photosensitiser.

[0035] Preferably, the photosensitiser is BODIPY-based ligand or a derivative thereof.Preferably, the photosensitiser is selected from the group consisting of: l_2and l_3

[0036] The photosensitiser may be attached to or otherwise associated with each MOF nanoparticle by any appropriate means. The skilled person would be aware of attachment / association modalities. Preferably, in each MOF nanoparticle (a) at least one of the plurality of organic ligands comprises photosensitiser; (b) each of the plurality of organic ligands comprises or consists of photosensitiser; (c) the photosensitiser is attached to one or more metal ions of the nanoparticle, optionally by phosphate-metal coordination; or (d) the photosensitiser is non-covalently attached to the MOF nanoparticle. Most preferably, (a) at least one of the plurality of organic ligands comprises photosensitiser; or (b) each of the plurality of organic ligands comprises photosensitiser. Appropriate ligands include those selected from the group consisting of: porphyrins, chlorins, bacteriochlorins, large-ring TT- conjugation systems, phthalocyanines, boron-dipyrromethene (BODIPY) derivatives or a disalycilidene- 1 ,2-cyclohexylidenediamine derivative.

[0037] In some embodiments, when the photosensitiser is selected from the group consisting of l_2 and l_3, either (a) at least one of the plurality of organic ligands comprises photosensitiser; or (b) each of the plurality of organic ligands comprises photosensitiser. Preferably, at least one of or each of the plurality organic ligands comprises photosensitiser l_2. Preferably, at least one of or each of the plurality organic ligands is photosensitiser l_2.

[0038] Preferably, each MOF nanoparticle comprises one or more hydrophilic polymers.

[0039] The moiety capable of associating to oxygen or the PFC may exhibit poor aqueous solubility, for example, PFCs may be formed as emulsions. In addition, photosensitisers can display hydrophobic properties resulting in aggregation in aqueous solution. Thus, there may be a need for moieties within the MOF nanoparticle which improve aqueous solubility, and / or biocompatibility and / or reduce aggregation due to excessive hydrophobicity. The presence of a hydrophilic polymer in MOF nanoparticles comprising perfluorocarbon moieties can improve the aqueous solubility of MOF nanoparticles that may otherwise display poor aqueous solubility due to the presence of perfluorocarbon moieties. Thus, hydrophilic polymer and moiety capable of associating to oxygen or PFC moiety within the MOF can simultaneously provide oxygen-carrying capability, biocompatibility and reduce excessive hydrophobicity.

[0040] Preferably, the hydrophilic polymer is selected from the group consisting of: polyethylene glycol (PEG) or a derivative thereof, dioleoylphosphatidylcholine (DOPC) or a derivative thereof, 1 ,2-Dioleoyl-3-trimethylammonium propane (DOTAP) or a derivative thereof, polyvinyl alcohol (P A) or a derivative thereof, poly (lactic-co-glycolic acid) (PLGA) or a derivative thereof, heparin or a derivative thereof, chitosan or a derivative thereof, pluronic F-127, and phospholipids. More preferably, the hydrophilic polymer comprises polyethylene glycol or a derivative thereof. In some embodiments, the hydrophilic polymer is linear methoxy polyethylene glycol (mPEG).

[0041] Preferably, the polyethylene glycol or derivative thereof comprises from about 40 to about 250 repeating units. PEGs display good hydrophilicity and biocompatibility. Typically, the PEG may have a molecular weight of from about 2000 g / mol to about 10000 g / mol (PEG2000 to PEG 10000), preferably from about 3000 g / mol to about 9000 g / mol (PEG3000 to PEG9000), from about 4000 g / mol to about 8000 g / mol (PEG4000 to PEG8000), from about 5000 g / mol to about 7000 g / mol (PEG5000 to PEG7000), from about 5000 g / mol to about 6000 g / mol (PEG5000 to PEG6000). Preferably, the PEG has a molecular weight of about 5000 g / mol (PEG5000).

[0042] The size of the hydrophilic polymer may affect the biocompatibility and stability of the MOF nanoparticle.

[0043] In embodiments, when the hydrophilic polymer comprises polyethylene glycol or a derivative thereof, preferably the polyethylene glycol is linear polyethylene glycol or a derivative thereof. Preferably the polyethylene glycol is predominantly linear polyethylene glycol or a derivative thereof, more preferably the polymer may consist essentially of, or consists of, linear polyethylene glycol or a derivative thereof. Preferably, the hydrophilic polymer is not or is free from branched polyethylene glycol or branched derivatives thereof.Preferably, the polymer comprises, or consists essentially of, a linear alkoxy polyethylene glycol (PEG), wherein the alkoxy group is preferably a Ci to C5alkoxy, more preferably a Ci to C3alkoxy. Even more preferably, the hydrophilic polymer comprises, or consists essentially of, a phosphate-terminated linear methoxy polyethylene glycol (mPEG).

[0044] The one or more moieties capable of associating to oxygen or the one or more perfluorocarbon moieties or the one or more aromatic moieties may be attached to or otherwise associated with the MOF nanoparticle by any appropriate means. The skilled person would be aware of attachment / association modalities. Preferably, (a) at least one of the plurality of organic ligands comprises at least one of the one or more moieties capable of associating to oxygen or at least one of the one or more perfluorocarbon moieties or at least one of the aromatic moieties; or (b) each of the plurality of organic ligands comprises the one or more moieties capable of associating to oxygen or the one or more perfluorocarbon moieties; or (c) the one or more moieties capable of associating to oxygen or the one or more perfluorocarbon moieties or the one or more aromatic moieties are non-covalently attached to the MOF nanoparticle; or (d) the one or more moieties capable of associating to oxygen or the one or more perfluorocarbon moieties or the one or more aromatic moieties are attached to the one or more hydrophilic polymers; or (e) the one or more moieties capable of associating to oxygen or the one or more perfluorocarbon moieties or the one or more aromatic moieties is attached to one or more metal ions of the nanoparticle, optionally by phosphate-metal coordination.

[0045] Preferably, when the one or more moieties capable of associating to oxygen or the one or more perfluorocarbon moieties or the one or more aromatic moieties are attached to the one or more hydrophilic polymers, they form one or more polymers and each of the one or more polymers are attached to one or more metal ions of the nanoparticle. Preferably, the one or more polymers comprise a phosphate group and the one or more polymers are attached to one or more metal ions of the nanoparticle by phosphate-metal coordination.

[0046] Typically, the mPEG may have a molecular weight of from about 2000 g / mol to about 10000 g / mol (mPEG2000 to mPEG 10000), preferably from about 3000 g / mol to about 9000 g / mol (mPEG3000 to mPEG9000), from about 4000 g / mol to about 8000 g / mol (mPEG4000 to mPEG8000), from about 5000 g / mol to about 7000 g / mol (mPEG5000 to mPEG7000), from about 5000 g / mol to about 6000 g / mol (mPEG5000 to mPEG6000). Preferably, the mPEG has a molecular weight of about 5000 g / mol (mPEG5000).

[0047] Highly fluorinated compounds such as perfluorocarbons (PFCs) show high affinity for oxygen molecules and have exhibited promise as oxygen carriers. Thus, the Inventors have found that addition of a PFC group to the MOF increases the oxygen-carrying capability of the nanoparticle. As described herein, PFCs may have poor aqueous solubility and so hydrophilic polymers may be incorporated in the MOF to improve aqueous solubility.

[0048] In embodiments, the polymer is a phosphate-terminated mPEG according to Structure AStructure A wherein n is from about 40 to about 250.

[0049] F-PEG is provided as an illustration of a preferred embodiment. F-PEG allows for straightforward attachment of both the moiety capable of associating to oxygen or PFC moiety and hydrophilic polymer to the metal ions of the MOF nanoparticle by phosphate coordination chemistry. The phosphate-metal coordination bond is robust in the presence of phosphate ions, such as those in phosphate buffered saline (PBS) or in vivo.

[0050] Other PFC / PEG phosphate molecules are suitable for use in the present invention. Indeed, it is well within the skilled person’s abilities to prepare alternative PFC / PEG phosphates that display the desired oxygen carrying capacity and hydrophilicity.

[0051] In a preferred embodiment, the invention provides a composition comprising a polymer-coated metal-organic framework (MOF) nanoparticle or nanoparticles and a hydrogel. Each nanoparticle comprising a MOF comprising a plurality of metal ions and a plurality of organic ligands; wherein each MOF nanoparticle comprises photosensitiser; wherein the polymer coating comprises a hydrophilic polymer and a moiety capable of associating to oxygen; and wherein the polymer coating is attached to one or more metal ions of each nanoparticle. The embodiments described herein in relation to all aspects of the invention apply mutatis mutandis to this preferred embodiment.

[0052] In a further preferred embodiment, the invention provides a composition comprising a polymer-coated metal-organic framework (MOF) nanoparticle or nanoparticles and a hydrogel. Each nanoparticle comprising a MOF comprising a plurality of metal ions and a plurality of organic ligands; wherein each MOF nanoparticle comprises photosensitiser; wherein the polymer coating comprises a hydrophilic polymer and a perfluorocarbon moiety; and wherein the polymer coating is attached to one or more metal ions of each nanoparticle. The embodiments described herein in relation to all aspects of the invention apply mutatis mutandis to this preferred embodiment.

[0053] The compositions according to the foregoing aspects of the invention are particularly advantageous as they may facilitate increased oxygen carrying within or on the MOF nanoparticles and consequently an improved ability to carry oxygen into tumours during PDT, thus providing an improved cancer treatment. The use of a hydrogel increases the localisation of the MOF nanoparticles when injected into a tumour. The advantages of the compositions according to the first aspect of the invention are demonstrated in the Examples and Figures described herein.

[0054] Increasing the oxygen-carrying capacity of the MOF nanoparticles of the present invention has been observed to increase performance even in the hypoxic environment of a cancerous cell (tumour cell). Interestingly, such nanoparticles also exhibit generally higher performance than equivalents that do not contain oxygen carrying moieties, even when they have not been pre-oxygenated. This is believed to be due to the concentration of the low levels of oxygen available within the cell around the moiety capable of associating to oxygen or the PFC moiety and hence the proximity to the PS.

[0055] Preferably, the composition may comprise between about 0.001 wt% and about 10 wt% of the composition MOF nanoparticle by weight, or between about 0.001 wt% and about 1 wt% of the composition MOF nanoparticle by weight, or between about 0.001 wt% and about 0.1 wt% of the composition MOF nanoparticle by weight.

[0056] Preferably the composition is a pharmaceutical composition. Preferably, the composition may comprise one or more pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients may be selected from the group consisting of: solvents, co-solvents, buffers, stabilisers, antioxidants, preservatives, chelating agents, emulsifiers, flavourings, lubricants, suspending agents, tonicity adjusting agents, surfactants, solubilisers, suspending aids, dispersion agents, humectants, thickeners, colouring agent, wetting agent, anti-foaming agent, viscosity modifier, sweeteners and combinations thereof.

[0057] The composition may be substantially saturated with oxygen or the composition may be saturated with oxygen.

[0058] The hydrogel may be a biocompatible hydrogel. For example, the hydrogel may be selected from the group consisting of: collagen, polyvinyl alcohol, oxidised polysaccharides, cellulose derivatives, such as hydroxyethylcellulose, and biocompatible organic polymers and copolymers. Preferably, the biocompatible hydrogel is selected from the group consisting of alginate, RGD(arginine-glycine-aspartate)-modified alginate, amylase, amylpectin, cellularose, chitosan, collagen, dextran, fibrin, gelatin, glycogen, heparin, hyaluronic acid, poly(acrylamide), poly(P-aminoester), poly(caprolactone), matrigel, multi-arm polyethylene glycol, poly-hydroxyethyl acrylate, poly(hydroxyethyl methacrylate), poly(N- isopropylacrylamide), poly(glycolic acid), poly(lactic acid), poly(lactic acid-glycolic acid), oligo(poly(ethylene glycol)fumarate), poly(vinyl alcohol), poly(vinyl acid), and combinations thereof.

[0059] The hydrogel may be shear thinning, that is the non-Newtonian behaviour of fluids whose viscosity decreases under shear strain. The use of shear thinning hydrogels is with low shear viscosity can help reduce the re-aggregation of nanoparticles in the composition following their dispersion, they may also aid in injection of the composition.

[0060] At very low shear rates, such as in vivo, the higher kinematic viscosity of the composition may help prevent unwanted movement of the nanoparticles away from the treatment area. It may also prevent aggregation of the nanoparticles in the composition prior to use. A lower viscosity, during injection when the composition is subjected to higher shear, may help ease of injection and reduce discomfort. The most viscous medical compositions routinely used in medical treatments are typically based on oils such as sesame oil and its derivatives and typically have a viscosity of between 30 and 50 Pa.s at temperatures around 37.5 °C. Water has a kinematic viscosity of ~ 1 Pa.s.

[0061] Preferably, the hydrogel has a viscosity of less than about 15.0 Pascal seconds (Pa.s), when measured at 25 s-1and 20 °C according to the Viscosity Test Method defined herein. Preferably, the hydrogel has a viscosity of less than about 12 Pa.s, less than about 10 Pa.s, less than about 8 Pa.s. Preferably, the hydrogel has a viscosity of about 2 Pa.s or greater, or about 3 Pa.s or greater, or about 4 Pa.s or greater.

[0062] Embodiments related to each of the first and related aspects of the invention apply mutatis mutandis to the second aspect of the invention.

[0063] In a second aspect, the present invention provides the composition as defined in the first or related aspect of the invention for use as a medicament.In particular, the invention provides a composition for use as a medicament, the composition comprising a metal-organic framework (MOF) nanoparticle or nanoparticles, wherein the or each nanoparticle comprises a MOF comprising a plurality of metal ions (inorganic nodes) and a plurality of organic ligands, wherein the or each MOF nanoparticle comprises photosensitiser, and wherein the or each MOF nanoparticle is configured to deliver oxygen to the photosensitiser for use therewith. The or each MOF nanoparticle may comprise one or more moieties capable of associating with oxygen as described in the first aspect or related aspect. Preferably, the or each MOF nanoparticle comprises a perfluorocarbon moiety.Additionally or alternatively, the or each MOF nanoparticle may comprise moieties selected from the group consisting of anthracene, naphthalene and 2-pyridone.

[0064] In a third aspect, the present invention provides the composition as defined in the first or related aspects for use in the treatment of cancer by photodynamic therapy.In particular, the invention provides a composition for use in the treatment of cancer by photodynamic therapy, the composition comprising a metal-organic framework (MOF) nanoparticle or nanoparticles, wherein the or each nanoparticle comprises a MOF comprising a plurality of metal ions (inorganic nodes) and a plurality of organic ligands, wherein the or each MOF nanoparticle comprises photosensitiser, and wherein the or each MOF nanoparticle is configured to deliver oxygen to the photosensitiser for use therewith. The or each MOF nanoparticle may comprise one or more moieties capable of associating with oxygen as described in the first aspect or related aspect. Preferably, the or each MOF nanoparticle comprises a perfluorocarbon moiety.Additionally or alternatively, the or each MOF nanoparticle may comprise moieties selected from the group consisting of anthracene, naphthalene and 2-pyridone.

[0065] The cancer is preferably a solid cancer or tumour. The cancer may be selected from the group consisting of: breast cancer, prostate cancer, lung cancer, brain tumours (including glioblastoma), bladder cancer, cervical cancer, head and neck cancers, Hodgkin's lymphoma,Non-Hodgkin's Lymphoma, melanoma, pancreatic cancer, rectal cancer, skin cancer (including Basal Cell Carcinoma and Squamous Cell Carcinoma) esophageal cancer, thyroid cancer, testicular cancer, mesothelioma, ovarian cancer, kidney cancer, liver cancer, gastric (stomach) cancer, bone cancer (including osteosarcoma and Ewing sarcoma), soft tissue sarcomas, endometrial (uterine) cancer, spinal tumour, nasopharyngeal cancer, salivary gland cancer, childhood cancers (certain types such as some leukaemias, neuroblastoma, Wilms tumour), colon cancer. The cancer is preferably a solid cancer or tumour.

[0066] As described herein, the photodynamic therapy is carried out using EM radiation. For example, EM radiation selected from the group consisting of radio waves, microwaves, infrared, (visible) light, ultraviolet, X-rays, and gamma rays. Gamma and X-rays may be preferred for treating deep tumours (e.g. where visible light cannot be used).

[0067] The second and third aspects of the invention are particularly advantageous as they provide MOF nanoparticles having improved oxygen carrying capacity for use in treatment. In particular, the treatment of cancer by photodynamic therapy. Such treatments may reduce the need for invasive therapies, such as surgery. The advantages of the second and third aspects of the invention are demonstrated in the Examples and Figures described herein.

[0068] In a fourth aspect, the invention provides a method of preparing a composition as defined the first or related aspects of the invention. The method comprising the steps of: (a) mixing a plurality of metal ions, a plurality of organic ligands, photosensitiser and one or more moieties capable of associating to oxygen in a solvent to form a suspension of one or more MOF nanoparticles, (b) optionally additionally mixing one or more hydrophilic polymers in step a., (c) optionally drying the one or more MOF nanoparticles by lyophilisation, (d) dispersing the one or more MOF nanoparticles in a hydrogel to provide the composition, and (e) optionally mixing one or more pharmaceutically acceptable excipients into the composition. Preferably, the one or more moieties capable of associating to oxygen are perfluorocarbon moieties. In some embodiments, the plurality of organic ligands is the photosensitiser, wherein the photosensitiser is of Formula I, Formula II, Formula III, Formula IV, Formula V or Formula VI.

[0069] In a fifth aspect, the invention provides a method of preparing a composition as defined in the first or related aspects of the invention. The method comprising the steps of: (a) mixing a plurality of metal ions and a plurality of organic ligands in a solvent to form a suspension of one or more initial MOF nanoparticles, (b) encapsulating photosensitiser within and / or locating photosensitiser on the one or more initial MOF nanoparticles to provide a suspension of one or more photosensitiser-containing MOF nanoparticles, (c) subsequently mixing thesuspension of one or more photosensitiser-containing MOF nanoparticles with the polymer coating and allowing the polymer coating to attach to one or more metal ions of the one or more photosensitiser-containing MOF nanoparticles to provide a suspension of one or more polymer-coated MOF nanoparticles, (d) optionally drying the one or more polymer-coated MOF nanoparticles by lyophilisation, (e) dispersing the one or more polymer-coated MOF nanoparticles in a hydrogel to provide the composition, and (f) optionally mixing one or more pharmaceutically acceptable excipients into the composition. The polymer is as defined in relation to the first and related aspects of the invention.

[0070] Preferably, step (c) is carried out in water at about 10 °C to about 30 °C, or about 15 °C to about 25 °C, or about 20 °C. Typically, step (c) lasts less than about 24 hours, preferably less than about 18 hours, preferably from about 1 hour to about 24 hours, preferably from about 8 hours to about 20 hours, preferably from about 10 hours to about 16 hours.

[0071] Typically, the coordination step (c) is performed for a period of time (the coating time) sufficient for the metal sites on the external surface of the MOF nanoparticles to be substantially saturated.

[0072] Preferably, the polymer coating makes up less than about 40 % by weight of the polymer-coated nanoparticle MOF, preferably less than about 30 % by weight, more preferably from about 10 % by weight to about 40 % by weight, preferably from about 20 % by weight to about 35 % by weight of the polymer-coated nanoparticle amorphous MOF.

[0073] In the methods of the fourth and fifth aspects, step a. may be carried out at a temperature of from about 80 °C to about 140 °C, or from about 100 °C to about 120 °C. Preferably, step a. is carried out at a temperature of about 120 °C. Preferably, step a. is carried out over about 12 h to about 48 h, or about 20 h to about 40 h.

[0074] The methods of the fourth and fifth aspects of the invention are particularly advantageous because the methods produce stable MOF nanoparticles having improved oxygen affinity. The advantages of the methods according to the seventh and eighth aspects of the invention are demonstrated in the Examples and Figures described herein.

[0075] In a sixth aspect, the present invention provides a method of preparing one or more MOF nanoparticles as defined in the first or related aspects of the invention, the method comprising the steps of: (a) mixing a plurality of metal ions, a plurality of organic ligands and one or more moieties capable of associating to oxygen in a solvent to form a suspension of one or more initial MOF nanoparticles, (b) mixing the suspension of one or more initial MOFnanoparticles with photosensitiser to replace one or more of the plurality of organic ligands with photosensitiser by post-synthetic ligand exchange to form the one or more MOF nanoparticles, (c) optionally additionally mixing one or more hydrophilic polymers in step a., (d) optionally drying the one or more MOF nanoparticles by lyophilisation, (e) dispersing the one or more MOF nanoparticles in a hydrogel to provide the composition, and (f) optionally mixing one or more pharmaceutically acceptable excipients into the composition. . Preferably, the one or more moieties capable of associating to oxygen are perfluorocarbon moieties.

[0076] Step (a) may be carried out at a temperature of from about 80 °C to about 140 °C, or from about 100 °C to about 120 °C. Preferably, step (a) is carried out at a temperature of about 120 °C. Preferably, step (a) is carried out over about 12 h to about 48 h, or about 20 h to about 40 h. Preferably, step (b) is carried out at a temperature of from about 50 °C to about 120 °C, or from about 60 °C to about 100 °C. Preferably, step a. is carried out at a temperature of about 70 °C. Preferably, step (b) is carried out over about 2 days to about 8 days, or about 4 days to about 6 days. Preferably, step (b) is carried out over about 4 days.

[0077] The methods of the fourth to sixth aspects may be used to prepare one or more MOF nanoparticles of the first or related aspects of the invention. For example, the one or more moieties capable of associating to oxygen in the methods of the fourth to sixth aspects of the invention may be replaced with perfluorocarbon moieties or aromatic moieties.

[0078] The Inventors have found that BODIPY derivatives usually have a high tendency to form aggregates in aqueous solutions, creating low intersystem crossing efficiency that is detrimental in PDT. Attempts to form MOF nanoparticles, especially Zr-containing MOF nanoparticles, using BODIPY derivatives as ligands by direct solvothermal methods were unsuccessful, likely due to the limited stability of the BODIPY core in the long-term presence of the strong acids and high temperature typically required for MOF nanoparticle synthesis.

[0079] Thus, the use of BODIPY-derivatives as ligands for MOF nanoparticles has been limited. Therefore, there is a need for improved methods for the preparation of such MOF nanoparticles as part of a preferred aspect of the present invention.

[0080] The inventors have discovered that it is possible to introduce a BODIPY-derivative ligand into a MOF nanoparticle having a ligand of identical length by a post-synthetic exchange strategy. A preferred MOF is derived from Zr UiO-69 (referred to herein as 69-Me2) wherein the ligand Li (shown below and Figure 10a) of 69-Me2has been replaced by the BODIPY derivative l_2by a post-synthesis ligand exchange. The l_2BODIPY derivative ligand has a verysimilar length to Li and identical bidentate functionality. In a similar manner the BODIPY derivative l_3can also be used for post-synthesis ligand exchange of Li in 69-Me2to form 69- l_3. In some embodiments, in step b. the organic ligand Li is replaced with photosensitiser l_2or L3.

[0081] Other suitable MOF nanoparticles which have ligands of identical sizes to BODIPY derivatives and which could be modified to have BODIPY functionality include PCN-222. Furthermore, the method of the ninth aspect can be used to substitute any ligands having similar size to a desired photosensitiser.

[0082] Thus, the method of the sixth aspect of the invention is particularly advantageous as the method provides an alternative way of introducing ligands that are otherwise difficult to introduce into MOF nanoparticles. The advantages of the method according to the sixth aspect of the invention are demonstrated in the Examples and Figures described herein.

[0083] Preferably, the methods of the fourth, fifth and sixth aspects of the invention are carried out in a medium selected from the group consisting of water, dimethylformamide (DMF), saline, dimethylsulfoxide (DMSO) or acetic acid. Preferably, the methods of the seventh, eighth and ninth aspects of the invention are carried out in an aqueous medium.

[0084] In a seventh aspect, the invention provides a composition manufactured according to the method as defined in fourth, fifth or sixth aspects of the invention.

[0085] In an eighth aspect, the invention provides a method of treating cancer by photodynamic therapy, the method may comprise administering to a patient the composition as defined in the first or related aspects of the invention.In particular, the invention provides a method of treating cancer by photodynamic therapy comprising administering to a patient a composition comprising metal-organic framework (MOF) nanoparticle or nanoparticles, wherein each nanoparticle comprises a MOF comprising a plurality of metal ions (inorganic nodes) and a plurality of organic ligands, wherein each MOF nanoparticle comprises photosensitiser, and wherein the or each MOF nanoparticle is configured to deliver oxygen to the photosensitiser for use therewith. Each MOF nanoparticle may comprise one or more moieties capable of associating with oxygen as described in the first aspect or related aspect. Preferably, each MOF nanoparticle comprises a perfluorocarbon moiety.Additionally or alternatively, each MOF nanoparticle may comprise moieties selected from the group consisting of anthracene, naphthalene and 2-pyridone.

[0086] In a ninth aspect, the invention provides the use of the composition as defined in the first or related aspects of the invention for the manufacture of a medicament for the treatment of cancer by photodynamic therapy.

[0087] For the avoidance of doubt, embodiments related to each aspect of the invention apply mutatis mutandis to the other aspects of the invention. Further aspects and embodiments of the present invention will be evident from the discussion herein.Brief Description of the DrawingsFigure 1 Illustration of a possible disordered orientation of L2.Figure 21H NMR spectrum of F-PEG.Figure 313C NMR spectrum of F-PEG.Figure 419F NMR spectrum of F-PEG.Figure 531P NMR spectrum of F-PEG.Figure 6 GPC trace of F-PEGFigure 7 a. The structures of tetrahedral cavities, and b, two-fold interpenetrated frameworks of 69-Me2, 69-1.2, and 69-L3. Hydrogen atoms are omitted for clarity.Figure 8 Optical microscope image of Me2(left) and 69-L2(right) crystalsFigure 91H NMR spectrum of the digested SC 69-L2.Figure 10 a, Illustration of the synthesis through PSE. b, Octahedral cavity, c, Tetrahedral cavity, d, Their packing mode in the non-interpenetrated feu network, e, Structure of the twofold interpenetrated network in which the parent and second interpenetrating networks are depicted as large sphere and small sphere, respectively. Hydrogen atoms, guest solvents, and disorder are omitted for clarity.Figure 11 a, Representation of the PSE and PEGylation. b, Simulated and experimental PXRD patterns, c and d, Experimental N2isotherm at 77K, inset, N2isotherm at 77 K plotted with a logarithmic x-axis to highlight the differences in the low-pressure region, e, PSD obtained with the NLDFT method, f, Intensity-average diameter, and g, Zeta potential of aqueous suspensions of 69-Me2, 69-L2, 69-L2@P, and 69-L2@F (n = 3). h, HAADF-STEM image and, i, EDS mapping of 69-L2. Scale bar 50 nm. j, TEM image of 69-L2@F. Scale bar: 500 nm. k, UV-vis (solid line) and fluorescence (dashed line, Aexc = 395 nm) spectra of the aqueous solutions of 69-L2, 69-L2@P, and 69-L2@F. I, time-dependent changes of dissolved oxygen concentrations in 69-L2@F or 69-L2@P at the same 69-L2concentrations of 2 mg mL-1. m, the DPBF degradation rate curves with 69-L2@F and 69-L2@P in MeOH.Figure 12 Synthesis of L3Figure 131H NMR spectrum of S6Figure 141H NMR spectrum of S7Figure 1513C NMR spectrum of S7Figure 161H NMR spectrum of l_3Figure 1713C NMR spectrum of l_3Figure 18 The lengths of Li, l_2, and l_3Figure 19 SEM images and particle size analysis of a. 69-Me2, b. 69-L2, c. 69-L2@P and d. 69-l_2@F. Scale bar 1 pmFigure 20 TEM (left) and HAADF-STEM (right) images of a. 69-Me2and b. 69-L2Figure 21 TGA profiles of a. 69-L2and 69-L2@P, b. 69-L2and 69-L2@PFigure 22 Simulated and experimental PXRD patternsFigure 23 FT-IR spectra of PEG in 69-L2@P and 69-L2@FFigure 24 TEM (left) and HAADF-STEM (right) images of a. 69-L2@P and b. 69-L2@F.Figure 25 Long-term dispersity of 69-L2and 69-L2@F in water or PBS (pH = 7.4).Figure 26 TEM images of a. 69-L2and b. 69-L2@F in water after 3 weeks. Scale bar: 100 nmFigure 27 Time-dependent TEM bare 69-L2and 69-L2@F in PBS (pH =7.4) monitored by TEM. Scale bar: 100 nm.Figure 28 Normalized absorbance (solid line) and emission spectra (dashed line, Aex = 395 nm) of 69-L2in different solvents with varying polaritiesFigure 29 The detection of ROS generation using DPBF as1O2indicator in water, a, 69-L2@F. b, 69-L2@P. C, DPBF.Figure 30 Cytotoxicity of 69-L2, 69-L2@P and 69-L2@F under dark conditions. MDA-MB-231 cells' viability was measured by MTS assay after 72 h incubation.Figure 31 a, CLSM images of MDA-MB-231 cells after incubating with 69-L2, 69-L2@P, and 69-L2@F for 24 h at a concentration of 100 pg / mL; scale bar, 20 pm. MDA-MB-231 cells' viability was measured using the methyl thiazolyl tetrazolium (MTT) assay, b, cells were incubated under normoxic conditions, and c, under the hypoxic condition with 1 % O2after LED light irradiation. Concentrations are based on bare 69-L2. Data are presented as mean ± SD (n=3; **** p<0.001 , *** p<0.001 , ** p<0.01 , * p<0.05, two-way ANOVA followed by a Sidak's test for multiple comparisons).Figure 32 3D z-stack CLSM of a. 69-L2, b, 69-L2@P and c, 69-L2@F. Scale bar: 10 pmFigure 33 TEM images of the stained MDA-MB-231 cells after incubation with BODIPY-MOF samples, a, Untreated, b, 69-L2@P. c, 69-L2@F. Arrows denote MOF nanoparticles.Figure 342D CLSM observation of ROS generation of MDA-MB-231 cells incubated with 69- L2, 69-L2@P, and 69-L2@F (25 pg / mL and 100 pg / mL based on the concentration of 69-L2) under dark or LED light irradiation in normoxic conditions. When exposed to ROS, the nonfluorescent DCFH-DA could be efficiently converted into fluorescent DCF. Scale bar: 10 pmFigure 35 ROS generation of 69-L2@P and 69-L2@F. MDA-MB-231 cells were treated with 69-L2@P or 69-L2@F for 24 h at a concentration of 25 pg / mL based on 69-L2, followed by green LED light irradiation. ROS generation was measured by DCF fluorescence intensityusing flow cytometry, a, Normalized DCF fluorescent intensity (n=5; ** p<0.01 , * p<0.05, two- way ANOVA followed by a Sidak's test for multiple comparisons), b, Overlapped histograms of DCF intensity for normoxic and hypoxic cells, c, Flow cytometry analysis of cells with MOF intensity vs DCF intensity.Figure 36 Overlapped histograms of DCF intensity for normoxic and hypoxic MDA-MB-231 cells incubated with 69-L2@P and 69-L2@F with light irradiationFigure 37 Relative cell death of MDA-MB-231 cells after incubation with the control, 69-L2, 69-L2@P or 69-L2@F a, without, and b, with LED light irradiationFigure 38 Live cell images of PDT treated MDA-MB-231 cells by IncuCyte before and after LED light treatment.Figure 39 a, Schematic showing the preparation of 69-L2@F-Gel and their photodynamic therapeutic mechanism, b, In vivo experimental set-up. c, Tumour size (n=5) following treatment (statistical analysis was performed with multiple t-tests, **P<0.01). d, Fluorescence signal of 69-L2@F after intratumoral injections, e, Kaplan-Meier survival curve after treatment (statistical analysis was performed with Log-rank (Mantel-Cox) test, ****p<0.0001). Survival cutoff criteria included tumour ulceration or compassionate euthanasia, when the aggregate tumour burden >50% difference between treated groups and controls, or if the tumour impeded eating, urination, defecation or ambulation.Figure 40 SEM imaging of only hydrogel (upper image) and 69-L2@F-Gel (lower image).Scale bar: 100 pm (inset: 1 pm)Figure 41 Energy-dispersive X-ray spectroscopy (EDS) mapping of 69-L2@F-GelFigure 42 Body weight monitoring. Body weight is depicted as the mean of each treatment group. No decrease or changes in body weight were found for all treatment mice groups (n= 5)Figure 43 a, Live imaging of athymic Balb / C female nude mice with triple-negative breast tumor xenograft implanted with hydrogel or 69-L2@F-Gel with or without green LED irradiation (n=5 per group). Ex vivo images of breast tumours and whole organs (T, tumour; Lv, liver; K, kidneys; S, spleen; H, heart; L, lung) are also presented, b, Luminescence signal of luciferase-expressing MDA-MB-231 cells in breast tumours and whole organs at Day 7 post in situ hydrogel injection, c, Fluorescence signal of 69-L2@F in breast tumours and whole organs at Day 7 post in situ hydrogel injection, d, Haematoxylin and eosin (H&E) stains of tumours from treated groups with hydrogel or 69-L2@F-Gel with or without green LED irradiation. Tumour front, bracket; Adipocytes, arrow; Necrotic tissue, asterisk. Scalebar: 200 pm. e, Immunohistochemistry staining of KI-67 (proliferation marker) of tumours from treated groups with hydrogel or 69-L2@F-Gel after green LED irradiation. Scale bar: 50 pm. f, Percentage of inflammatory foci / tumour in the four groups tested (statistical analysis was performed with one-way ANOVA test, ***P<0.001). g, Percentage of necrosis / tumour in the four groups tested (statistical analysis was performed with one-way ANOVA test, *P<0.05).Figure 44 Haematoxylin and eosin (H&E) stains of tumours from treated groups with hydrogel or 69-L2@F-Gel with or without green LED irradiation. Tumour front (Bracket), Adipocytes (Arrow), Necrotic tissue (Asterisk). Scale bar: 200 pmDetailed Description of the Invention

[0088] References herein to a singular of a noun encompass the plural of the noun, and vice- versa, unless the context implies otherwise.

[0089] Throughout this specification the word ‘comprise’, or variations such as ‘comprises’ or ‘comprising’, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. The term ‘comprising’ includes within its ambit the term ‘consisting’ or ‘consisting essentially of’.

[0090] The term ‘consisting’ or variants thereof is to be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, and the exclusion of any other element, integer or step or group of elements, integers or steps.

[0091] The term ’consisting essentially of’ or variants thereof is to be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, and that further components may be present, but only those not materially affecting the essential characteristics of the embodiment of the invention.

[0092] The term ‘about’ herein, when qualifying a number or value, is used to refer to values that lie within ± 5% of the value specified. For the avoidance of doubt, where a number or value is specified herein in the absence of the term ‘about’, the number or value should be understood according to standard numeric rounding conventions according to the number of decimal places. For example, a whole number, such as 6, is understood to encompass values > 5.5 and < 6.5. Likewise, a number specified to one decimal place, such as 5.3, is understood to encompass values > 5.25 and < 5.35.

[0093] Where a range of values is provided, the range includes the end point values. For example, the range 20 to 28, or from 20 to 28, would include the values 20, 21 , 22, 23, 24, 25, 26, 27 and 28; the range 5 to 15, or from 5 to 15, would include the values 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 and 15.

[0094] The present invention relates to metal-organic framework (MOF) nanoparticles, methods of manufacturing said MOF nanoparticles, and uses of said MOF nanoparticles.

[0095] Metal-organic frameworks (MOFs) are crystalline materials consisting of coordination bonds between metal ions, e.g. metal cations, and multidentate organic ligands. The MOF structure is characterised by an open framework that may be porous.

[0096] MOF nanoparticles comprise metal-organic frameworks and may display 3D architectures. MOF nanoparticles disclosed herein may be generally rod-shaped, spherical, or cuboid. Polymer-coated MOF nanoparticles of the present invention may each comprise a MOF comprising a plurality of metal ions (inorganic nodes) and a plurality of organic ligands, wherein each MOF nanoparticle comprises photosensitiser and one or more moieties capable of associating to oxygen, preferably one or more perfluorocarbon moieties.

[0097] Preferred parent MOFs for the MOF nanoparticles according to the present invention may be UiO-69 (referred to as 69-Me2 herein), UiO-66, UiO-68, or Mx-M(bipy)s (where bipy is 2,2'-bipyridine, Mxcan be zirconium, iron, thorium, and hafnium , ruthenium, gadolinium, neodymium, ytterbium and erbium, and M can be ruthenium (Ru), iridium (Ir), and osmium (Os)). The structure of M(bipy)s is shown below:M(bipyh

[0098] . For example, 69-Me2where the ligand is replaced with l_2is denoted as 69-L2. Nomenclature for MOF nanoparticles having polymer attached is MOF@polymer. For example, 69-L2wherein the mPEG polymer is attached via phosphate coordination is denotedas 69-l_2@P. 69-1.2 wherein the mPEG polymer and perfluorooctyl group are attached via phosphate coordination is denoted as 69-L2@F.

[0099] “Hydrophilic polymer” refers to a polymer having a strong affinity for water. For example, hydrophilic polymers may interact with water via hydrogen bonding.

[0100] “Perfluorocarbon” or “perfluorocarbon moiety” or “PFC” (used interchangeably herein) refers to chemical groups composed of only carbon and fluorine atoms. All available bonding sites of the carbon atoms are occupied by fluorine atoms, one bonding site is available to associate to the or each MOF. Examples of perfluorocarbon groups suitable for use in the present invention include those having from six to 16 carbon atoms, preferably perfluorohexyl, perfluorooctyl, or perfluorononyl groups.

[0101] “Moiety” or “moieties” refers to a distinct part, structural feature, or functional group within a larger molecule, i.e. a part of a larger molecule that has its own characteristic properties. For example, a perfluorocarbon moiety refers to the part of the MOF nanoparticle composed of only carbon and fluorine atoms, such as a perfluorooctyl group.

[0102] “Photosensitiser” refers to a molecule that can be raised to an excited electronic state, either by direct excitation by the absorption of EM radiation or indirect excitation by activation of a neighbouring entity such as a metal cation absorbing EM radiation and emitting photoelectrons. When in the excited electronic state, the photosensitiser can react with molecular oxygen to form ROS, such as singlet oxygen. For example, in its excited state, a photosensitiser can undergo intersystem crossing and transfer energy to oxygen to produce ROS, such as singlet oxygen, that can absorb light and transfer its energy to other molecules.

[0103] “Hypoxia” or “hypoxic” refers to a condition in which tissue is deprived of an adequate supply of oxygen. The oxygen supply to tumour cells can be insufficient due to the rapid growth of the tumour cells without the equivalent rapid growth of blood vessels to supply oxygen to all parts of the tumour.

[0104] “Normoxia” or “normoxic” refers to cells having a normal level of oxygen availability. The concentration of oxygen within each cell is within the expected physiological range.

[0105] “Cancer” refers to a group of diseases characterised by uncontrolled growth and spread of abnormal cells.

[0106] Photodynamic therapy” refers to a therapy involving the use of a photosensitiser and EM radiation to generate singlet oxygen from molecular oxygen to promote cell death. Photodynamic therapy typically involves the administering of a photosensitizer directly into a tumour, followed by its direct or indirect activation by electromagnetic (EM) radiation to generate highly cytotoxic reactive oxygen species, particularly singlet oxygen, within the tumour. This then triggers cell apoptosis and necrosis within the tumour.

[0107] “Hydrogel” refers to gels comprising water within a cross-linked polymer network. The levels of water are typically between about 70 wt% and about 99 wt% based on the weight of the hydrogel. A number of different chemistries can be used to form hydrogels. The skilled person is well aware of these and the appropriate hydrogel suitable for an application.

[0108] The term “hydrogel” includes macroscopic hydrogels, microgels and nanogels. A microgel comprises fragments of hydrogel having dimensions of the order of microns. A nanogel comprises fragments of hydrogel having nanometre-scale dimensions. The mesh size of the hydrogel needs to be large enough size to physically entrap the MOF nanoparticles. The term “hydrogel” also includes mixes and blends which can undergo a sol-gel transition inside the human body after injection to form the hydrogel in situ.

[0109] The particle size of the one or more MOF nanoparticles may be from about 25 nm to about 500 nm. Preferably the particle size of the one or more MOF nanoparticles is from about 25 nm to about 300 nm, or from about 50 nm to about 200 nm, or from about 100 nm to about 250 nm, or from about 100 nm to about 200 nm.

[0110] The particle size of the one or MOF nanoparticles may be measured according to the dynamic light scattering (DLS) method disclosed herein. The particle size refers to the hydrodynamic diameter, which can be defined as the radius of a hypothetical sphere that diffuses at the same rate as the particle under investigation.

[0111] Typically, a MOF nanoparticle contains only a single species of the metal ion. Accordingly, the plurality of metal ions (inorganic nodes) may consist essentially of (or consist of) ions of a metal selected from the group consisting of zirconium, iron, thorium, hafnium, ruthenium, gadolinium, neodymium, ytterbium and erbium. The plurality of metal ions (inorganic nodes) may consist essentially of (or consist of) an ion of a metal selected from the group consisting of zirconium, hafnium and ruthenium. Preferably, the plurality of metal ions consists essentially of (or consists of) an ion of a metal selected from the group consisting of zirconium and hafnium. Typically, the metal ion selected will be biocompatible / pharmaceutically acceptable.

[0112] In embodiments, the metal ions are capable of absorbing x-rays. Such metal ions comprise hafnium (Hf), lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Gd), cadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), barium (Ba), tantalum (Ta), tungsten (W,) rhenium (Re), iridium (Ir), lead (Pb), and bismuth (Bi). The plurality of metal ions may consist essentially of (or consist of) ions of a metal selected from the group consisting of: hafnium (Hf), lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Gd), cadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), barium (Ba), tantalum (Ta), tungsten (W,) rhenium (Re), iridium (Ir), lead (Pb), and bismuth (Bi).

[0113] The organic ligands in the MOFs according to the present invention may be multidentate. A multidentate organic ligand is an organic ligand capable of donating two or more pairs of electrons in a complexation reaction to form two or more coordinate bonds. The organic ligands may comprise two or more oxygen and / or nitrogen atoms suitable for donating a pair of electrons to form the two or more coordinate bonds. Preferably, the oxygen atoms may be present as carboxylate or nitro groups. Preferably, the nitrogen atoms may be present as amine groups. Typically, the plurality of organic ligands may be an aromatic carboxylate, an aromatic amine and / or an aromatic nitro. Preferably, the plurality of organic ligands may be selected from the group consisting of: Li, tetrakis (4-carboxyphenyl)porphyrin (TCPP), 1 ,4- benzenedicarboxylic acid (BDC), 1 ,3,5-benzenetricarboxylic acid (H3BTC), 1 ,3, 5, 8- (p- benzoate) pyrene linkers (H4TBAPy), 4’,4”’,4””’,4”””’-(ethene-1 ,1 ,2,2- tetrayl)tetrakis(([1 , 1 '- biphenyl]-3-carboxylic acid)) (H4ETTC), M(bipy)3-based ligands (where bipy is 2,2'-bipyridine, and M can be ruthenium (Ru), iridium (Ir), and osmium (Os)) (photosensitiser of Formula VI), photosensitiser of Formula I, photosensitiser of Formula II, photosensitiser of Formula III, photosensitiser of Formula IV, photosensitiser of Formula V and chlorin-based ligands. Typically, the organic ligand selected will be biocompatible / pharmaceutically acceptable.

[0114] Optionally, the photosensitiser or moiety capable of associating to oxygen or perfluorocarbon moiety may be attached to the plurality of organic ligands. Optionally, the photosensitiser, as defined herein, may be one or more of the plurality of organic ligands in the MOF nanoparticle.

[0115] Moieties such as anthracene, naphthalene and 2-pyridone can react with oxygen in the presence of a photosensitiser and EM radiation to form endoperoxides. Theseendoperoxides can then release singlet oxygen when exposed to thermal energy, such as body temperature (37 °C), when injected into a tumour. The endoperoxide may be formed prior to injection into the tumour when the or each MOF nanoparticle is exposed to air and, therefore, oxygen. Additionally, or alternatively, the endoperoxide may be formed in situ in the tumour and may be formed repeatedly while a source of oxygen is available. For example, the MOF nanoparticle may be injected into a tumour comprising an endoperoxide and, when singlet oxygen is released at 37 °C, the MOF nanoparticle can again be exposed to EM radiation to regenerate the endoperoxide for release of singlet oxygen.

[0116] In embodiments, the electromagnetic radiation is X-ray radiation. MOFs and methods using X-ray radiation, which can be combined with the moiety capable of associating to oxygen disclosed herein, are described in US 10,206,871 , which is incorporated by reference in its entirety. In such embodiments, the patient can be irradiated with X-rays in any suitable manner and / or using any suitable equipment, such as that currently being used for delivering X-rays in a medical or veterinary setting. In some embodiments, the X-ray source and / or output can be refined to enhance disease treatment. For instance, the X-rays can be generated using a peak voltage, current and / or, optionally, a filter chosen to minimize DNA damage in the patient due to X-ray irradiation and maximize X-ray absorption by the scintillator.

[0117] In some embodiments, the irradiating can comprise generating X-rays using a tungsten or another metal target, Cobalt-60 sources (cobalt unit), linear accelerators (linacs), lr-192 sources, and Cesium-137 sources. In some embodiments, the irradiating comprises passing the X-rays (e.g., the X-rays generated using a tungsten target) through a filter prior to irradiation the patient. In some embodiments, the filter can comprise an element with an atomic number of at least 20. In some embodiments, the filter comprises copper (Cu). In some embodiments, the filter can have a thickness that is less than about 5 millimeters (mm). In some embodiments, the filter can have a thickness of less than about 4 mm (e.g., less than about 3 mm, less than out 1 mm, less than about 0.5 mm, less than about 0.4 mm, less than about 0.3 mm, less than about 0.2 mm, or less than about 0.1 mm).

[0118] The X-rays can be generated using a peak voltage, current and / or, optionally, a filter chosen to minimize DNA damage in the patient due to X-ray irradiation and maximize X-ray absorption by the scintillator. In some embodiments, the X-rays are generated using a peak voltage that is less than about 230 kVp. In some embodiments, the peak voltage is less than about 225 kVp, less than about 200 kVp, less than about 180 kVp, less than about 160 kVp, less than about 140 kVp, less than about 120 kVp, less than about 100 kVp, or less than about80 kVp. In some embodiments, the X-rays are generated using a peak voltage that is about 120 kVp.

[0119] Any suitable scintillator can be used. In some embodiments, the scintillator comprises a lanthanide (i.e., La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, or Lu). The scintillator can be, for example, a lanthanide nanoparticle (e.g., co-administered with the MOF nanoparticles disclosed herein comprising the photosensitizer moiety capable of associating to oxygen). For example, the lanthanide nanoparticles can be trapped in cavities or pores within the MOF comprising the photosensitizer. In some embodiments, the lanthanide is the metal of the SBUs of the MOF comprising the photosensitizer. In some embodiments, the lanthanide nanoparticle can be a lanthanide core-shell nanoparticle, further optionally wherein the shell of the lanthanide core-shell nanoparticle comprises a lanthanide chalcogenide. In some embodiments, the scintillator comprises a lanthanide aluminum garnet or a lanthanide fluoride.

[0120] Other suitable scintillators include, but are not limited to, carbon dots; core-shell nanoparticles wherein the shell comprises zinc sulfide and the core comprises a transition metal or lanthanide metal; and / or nanoparticles comprising gold, platinum, or iridium.

[0121] Lyophilisation (also known as freeze drying or cryodesiccation) is a drying process carried out at low temperature. Lyophilisation generally involves reducing temperature and pressure to below the substance's triple point and removing the frozen solvent (e.g. water ice) by sublimation. For aqueous compositions, such as those disclosed herein, lyophilisation may be carried out at temperatures of from about -50 °C to -80 °C, preferably about -70 °C, and pressures of from about 1000 Pa (0.01 bar) to about 100 Pa (0.001 bar), preferably about 800 Pa (0.008 bar).

[0122] The composition may comprise dissolved oxygen. The composition may comprise dissolved oxygen levels greater than those found at atmospheric temperature and pressure. The dissolved oxygen level may be from about 12 mg / L to about 25 mg / L, or from about 12 mg / L to about 25 mg / L for the fluorinated and non- fluorinated MOFs. Dissolved oxygen levels may be measured using Hanna Instruments HI-2004-02 Edge® Dissolved Oxygen Meter. Compositions comprising PFC-containing MOF nanoparticles displayed a slower decrease in dissolved oxygen concentrations relative to compositions comprising non-PFC-containing MOF nanoparticles.

[0123] Methods of administration of a pharmaceutical composition according to the present invention include oral, parenteral (for example, intravenous, subcutaneous, intramuscular or intraarticular, intratumoral), cutaneous (for example, topical) or inhalation administration. Intratumoral administration is particularly preferred. The pharmaceutical composition may be a liquid composition.

[0124] The MOF nanoparticles and compositions of the invention may be used in conjunction with other cancer therapies. For example, the MOF nanoparticles and compositions may be used in conjunction with chemotherapy or radiotherapy. For example, gemcitabine may be used as both photosensitiser and chemotherapy.

[0125] The MOF nanoparticles of the invention may comprise a pharmaceutically active ingredient located on and / or within the MOF nanoparticles. For example, the pharmaceutically active ingredient may be encapsulated within the MOF nanoparticle. The pharmaceutically active ingredient may be a chemotherapeutic agent.Examples

[0126] The invention will now be demonstrated by reference to the following non-limiting examples.

[0127] Unless otherwise mentioned, room temperature and pressure are 20 °C (293.15 K, 68 °F) and 1 atm (14.696 psi, 101.325 kPa), respectively.Experimental MethodsPowder X-ray diffraction (PXRD)

[0128] PXRD data were collected on a Bruker D8 DAVINCI diffractometer at 298 K (24.85 °C) using Cu Ka radiation. The calculated PXRD patterns were produced using the Mercury program and single crystal reflection data.Thermogravimetric analysis (TGA)

[0129] TGA measurements were carried out using a TA Instruments Discovery SDT650. Measurements were collected from room temperature to 800 °C with a heating rate of 5 °C / min under nitrogen.Fourier-transform infrared spectroscopy (FT-IR)

[0130] FT-IR was carried out using a Bruker Tensor 27 FTIR with the attenuated total reflectance (ATR) method.Gas uptake

[0131] N2adsorption isotherm measurements were performed on a Micromeritics 3-Flex analyzer at 77 K (-196.15 °C). Around 80 mg samples were used for each measurement, Prior to the measurement, all the samples were degassed under vacuum at 50 °C for 24 hours using the internal turbopump. Gas uptakes were performed on the air-dried samples. The BET areas were estimated by applying the Brunauer-Emmett-Teller (BET) equation.Dynamic light scattering (DLS) and zeta potential

[0132] Measurements were recorded in an aqueous solution with the sample concentration of around 0.2 mg / mL by a Zetasizer Nano ZS, (Malvern Instrument Ltd., U.K.) equipped with a He-Ne laser operating at 633 nm at 25 °C. Zeta potential was measured in a folded capillary Zeta cell DTS1070. The Smoluchowski equation was used to calculate the zeta potential. Measurements were performed three times with over 10 subruns for each sample. Error bars represent the standard deviation of three measurements.Inductively coupled plasma-optical emission spectroscopy (ICP-OES)

[0133] ICP-OES was performed using a Perkin Elemer ICP-OES Optima 2100DV. Samples were dispersed in 2 mL of nitric acid and 6 mL of hydrochloric acid (CAUTION!) and left to stand at room temperature in the fume cupboard for at least 2 h until all reactions have ceased. After that, samples were heated at 120 °C for 10 h to digest the sample completely. After this time, the clear solution was obtained, which was then diluted 50 times with Millipore water and analyzed for P and Zr content by comparing it to the standard P and Zr solutions. 3 repeats were performed for each sample.UV-Vis spectroscopy

[0134] UV-vis and fluorescence spectra were recorded using a Tecan Spark® Multimode Microplate Reader.Liquid nuclear magnetic resonance spectroscopy (NMR)

[0135] Liquid NMR was carried out using a Bruker 400 MHz Avance III HD Smart Probe Spectrometer.Scanning electron microscopy (SEM)

[0136] The samples for SEM tests were coated with Pt or Au for 40 seconds and imaged using a FEI Nova Nano SEM 450.Transmission electron microscopy (TEM)

[0137] The samples were prepared by dispersing the samples in ethanol using ultrasonication. After that, a small number of suspensions were drop-casted on a copper grid with a carbon support film. A Thermo Scientific (FEI) Talos F200X G2 TEM operating at 200 kV was utilized for TEM, HAADF-STEM and EDS analysis. TEM images were acquired using a Ceta, 4k x 4k CMOS camera. EDS data was collected in STEM mode using the HAADF detector with EDS spectra and maps collected using the Super-X EDS detector system, which consists of 4 windowless silicon drift detectors.Transmission electron microscopy (TEM) of the stained cells

[0138] MDA-MB-231 cells were fixed in 2% glutaraldehyde / 2% formaldehyde in 0.05 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCI2 at 4 °C for at least 4 h . Samples were washed 3x in 0.05 M sodium cacodylate buffer pH 7.4 and osmicated for 2h at room temperature with 1% osmiumtetroxide / 1 .5% potassium ferricyanide in 0.05 M sodium cacodylate buffer pH 7.4. Samples were washed 3x in deionized water and dehydrated in a series of ethanol solutions (50% / 70% / 95% / 100% / 100% dry ethanol), 2x in each, followed 2x with acetonitrile. The samples were resin embedded in Quetol resin, with fresh resin each day for 5 days. The resin mix was: 12 g Quetol 651 , 15.7 g nonenyl succinic anhydride (NSA), 5.7 g methyl nadic anhydride (MNA) and 0.5 g benzyldimethylamine (BDMA) (all from TAAB). The resin was cured in an embedding oven at 60 °C for 2 days. Thin sections (~ 70 nm) were cut using an ultramicrotome (Leica Ultracut E) and placed on bare 300 mesh copper TEM grids. Samples were post-stained with 2% uranyl acetate / 50% methanol for 3 min, followed by Reynold's lead citrate for 6 min. Samples were imaged in a Tecnai G20 TEM (FEI / Thermo Fisher Scientific) run at 200 keV using a 20 urn objective aperture to improve contrast. Images were acquired using an ORCA HR high-resolution CCD camera (Advanced Microscopy Techniques Corp, Danvers USA).Single crystal X-ray diffraction

[0139] Single crystal X-ray diffraction was performed on the PROXIMA 2A micro-focused beamline in SOLEIL synchrotron (A = 0.729319 A) using an EIGER X9M 2D hybrid photon counting detector. Data were collected at 100 K. Data integration and reduction were undertaken with Xia21. No corrections for solvent scattering have been made. The structure suffers from a significant amount of disorder, some of which is included in the presented model and some which could not be modelled with standard tools. The conformation for the L2 modelled in the structure represents the majority contributions, as evidenced by the acceptable R1 value. It is possible that, beyond the currently modelled orientations of L2, it also exists in reverse orientation, slightly offset from the modelled position. An illustration of this possibility is given in Figure 1. Due to the pseudo-symmetry of the core of L2, it is not possible to distinguish this from the modelled orientation at the resolution obtainable at the beamline PROXIMA 2A (approximately 0.7 A). In addition, it is possible to identify two weak residual electron density peaks at (0.353, 0.652, 0.152) and (0.318, 0.625, 0.125), which may represent the two fluorine atoms in this reverse orientation. When only the reverse orientation is modelled, the R1 and wR2 values are higher than the presented model. This kind of disorder cannot be modelled at the same time as the two existing orientations using the standard SHELX tools. This explains the combination of poor refinement statistics and thermal ellipsoids with very good integration statistics, a stable and chemically sensible model and an absence of confidently identifiable electron density peaks for the other orientations or ligands. The structure was solved using the direct method and refined by full-matrix least-squares on F2 by the SHELXTL-2014 software package. The disorder was modelled using standard crystallographic methods, including constraints, restraints and rigid bodies where necessary. All carbon-bound hydrogen atoms were added in idealized positions and refined using a riding model. DFIX, DELU, SIMU, SADI and SIMU restraints were used to obtain reasonable parameters. H-atoms were refined isotropically, while the other atoms were refined anisotropically. All the phenyl rings are constrained to the ideal six-membered ring. Crystal data and details of the data collection are given in Table 1 , while the selected bond distances and angles are presented in Tables 2-3.Table 1. Crystal data and structure refinement for 69-L2 and 69-L3Table 2 Selected bond lengths [A] and angles [°] for 69-L2Table 3 Selected bond lengths [A] and angles [°] for 69-L3Size exclusion chromatography with multi-angle light scattering (SEC-MLAS)

[0140] SEC-MLAS was performed on a Shimadzu HPLC system consisting of an LC-20AD Pump, SIL-20A autosampler, CTO-20A column oven and CBM-20A control unit. The column set was made up of 1 x PSS SUPREMA analytical 100 A (8 x 300 mm) and 2 x PSS SUPREMA analytical 3000A (8 x 300 mm). Dual detection was achieved via a Wyatt DAWN HELEOS-II multiangle light scattering (MALS) detector (laser at A = 658 nm), and Wyatt Optilab rEX differential refractive index (DRI) detector with a 658 nm light source. MilliQ Water containing 0.1 mol / L sodium nitrate and 0.01 mol / L sodium azide was used as the eluent at a flow rate of 1.0 mL / min. The column temperature and the detector temperature were kept at 30 °C. All data analysis was performed using Wyatt Astra 6.1.1 software. A literature value for the dn / dc of polyethylene glycol) in water (0.134 mL / g)2 was used to determine the molecular weight of all samples.Cell culture

[0141] MDA-MB-231 cells were chosen as our in vitro and in vivo cellular system to evaluate the PDT efficacy of 69-L2@P and 69-L2@F against breast cancer cells. MDA-MB-231 cells were maintained at 37°C, and 5% CO2in high-rich glucose (4500 mg / L) DMEM (Gibco Dulbecco's Modified Eagle Medium, Gibco® 41965039) supplemented with 10% (v / v) fetal bovine serum (Sigma-Aldrich F9665), 100 units / mL penicillin, and 100 pg / mL streptomycin (Life Technologies 15140122). The supplemented medium is then named the 'complete' medium. PBS (Sigma D8537) and trypsin-EDTA (Life Technologies 25300054) were used to maintain the cell line. For hypoxic cultures, cells were split and incubated under normal conditions (with approximately 18.6% O2concentration) for at least 8 h to allow cells to adhereto the bottom of the plates before they were put in a designated hypoxic incubator with 1 % O2 concentration.MTS cytotoxicity assay

[0142] The concentration (based on 69-L2)-dependent viability of 69-1.2, 69-l_2@P and 69- L2@F on MDA-MB-231 cell line were evaluated using MTS assay (Promega, USA). Cells were seeded into 96-well plates at a density of 10 000 cells / well in 100 pL of complete growth medium and incubated at 37 °C, 5% CO2for 24 h. Subsequently, 69-L2, 69-L2@P and 69- L2@F were dispersed and diluted in complete medium in a range of concentrations accordingly, of which 100 pL were added to each well and incubated for 72 h at 37 °C, 5% CO2in the dark. At the end of the incubation period for 72 h, cells were washed once with PBS followed by 2 h incubation of diluted MTS solution that was prepared according to manufacturer protocol. The absorbance of each well was measured at 490 nm using a Spark plate reader (TECAN, CH). Control measurements included negative control of cells with DMEM, cells with DMEM containing 1% of water, and cell-free cultured media (blank). All experiments were conducted in biological triplicates.MTT cytotoxicity assay

[0143] The concentration-dependent viability of 69-L2@P and 69-L2@F was investigated using the Thiazolyl Blue Tetrazolium Bromide (Sigma, M2128-5G, dissolved as 40 mg / mL stock in DMSO). Briefly, MDA-MB-231 cells were seeded on a 96-well plate at a density of 5000 cells / well for approximately 24 h. 69-l_2@P and 69-l_2@F were dispersed in a complete medium, and a range of concentrations was prepared accordingly, of which 100 pL were added to each well and incubated at either normoxic or hypoxic condition for 24 h. Then, plates were treated with LED light irradiation (525 nm high-power LED, 3.1 W, SOLIS-525C, ThorLabs) for 10 min. Following a further incubation of 48 h, the treatment solutions were removed, and cells were washed once with PBS. 2 mg / mL of final MTT working solution was added to each well, and the contents were incubated for 4 h at 37°C / 5% CO2. After the incubation, the supernatant was removed, and 150 pL of DMSO was added to dissolve the formazan crystals. The resulting solution was then transferred to a clean 96-well plate, and the absorbance was recorded by UV-Vis SPECTROstar Nano at 570 nm. All experimental results were independently carried out three times with five technical repeats in each experiment.Live cytotoxicity assay

[0144] The time-dependent cytotoxicity of 69-L2, 69-L2@P, and 69-L2@F (100 pg / mL based on 69-L2) in MDA-MB-231 cell line was evaluated using the lncuCyte®S3 Live Cell Analysis System (Sartorius). Cells were seeded into two 96-well plates at a density of 10000 cells / well in 100 pL of complete growth medium and incubated at 37 °C, 5% CO2for 24 h. Subsequently, different concentrations of 69-L2, 69-L2@P, and 69-L2@F were diluted in a complete medium containing 250 nM of Incucyte® Cytotox Dye and incubated at 37 °C, 5% CO2. After 24h, one plate was treated with LED light irradiation using Lumidox® Gen II 96- Well LED Arrays at 527 nm wavelength for 10 minutes. Both light and non-light-treated plates were imaged every 3 h for 7 days under cell culture conditions with 10* objective using the brightfield and the red channels. Mean cell confluence was calculated using the images taken from 3 random fields of view per well using the IncuCyte S3 v2022A software. All Incucyte experiments were performed in triplicate. Relative confluence values were obtained by dividing the Cytotox red signal by the total confluence and normalizing each value to the time zero value in each sample.Flow cytometry

[0145] MDA-MB-231 cells were seeded on to 6-well plate the day before they were treated with 69-L2@P and 69-L2@F at a concentration of 25 pg / mL (based on 69-L2) to each well for 24 h. Then, 10 pM of DCF-DA were added to each well and incubated for 2 h before their LED light (3.1 W, SOLIS-525C, ThorLabs) irradiation for 10 min. Following their light irradiation, cells were washed with PBS and detached from the plate using trypsin for 3 min at 37°C / 5% CO2. The cell pellets were then collected and washed with PBS twice by centrifugation and fixed on ice with 4% paraformaldehyde (PFA) for immediate acquisition on the same day. Unstained and single stain compensation tubes, with and without MOFs treatment, were also prepared for spectral overlap compensation. The samples are then acquired using BD LSRFortessa which is equipped with three lasers (405, 488 and 642 nm) in standard configuration and standard filter sets. FlowJo v10.8.1 was used for analysis.Confocal microscopy

[0146] MDA-MB-231 Cells were seeded into an 8-well Nunc™ Lab-Tek™ II Chamber Slide™ System Measurements at a concentration of 100 000 cells / mL and incubated at 37 °C overnight. Subsequently, the cells were treated with water (control) or 69-L2, 69-L2@P and 69- L2@F at a concentration of 100 pg / mL based on 69-L2for 24 h. For the internalization experiment, cells were then washed twice with 1xPBS and stained with CellMask™ Red(Thermo Fischer) Plasma membrane stain and Hoechst 33342 (Thermo Fisher) according to the manufacturer's instructions. For the ROS generation confocal experiment, cells were washed with 1xPBS and incubated with DCF for 2 h. Subsequently, one plate was left in dark conditions for the duration of the experiment, while another plate was treated with LED light irradiation for 10 minutes. Cells were washed three times with 1xPBS, left in growth media, and imaged using a confocal microscope (Axio Observer Z1 LSM 800, Zeiss). The 405 nm, 488 nm, 561 nm, and 640 nm lasers were used to excite MOFs and stains. Images were collected using an oil immersion 63X / 1.4NA lens. Zen software (Zeiss) was used for acquisition and image processing.Oxygen Carrying Capacity Test Method

[0147] The oxygen-carrying capacity of MOF nanoparticles was measured as follows.

[0148] Ultra-pure water, Milli-Q water (18.2 MO. cm resistivity at 25 °C), was saturated with oxygen at 20 °C by extended bubbling of pure oxygen through the water until the oxygen content as measured by a dissolved oxygen probe shows no further change. Suitable equipment includes the Hanna Instruments HI-2004-02 Edge® Dissolved Oxygen Meter.

[0149] The MOF nanoparticles being tested were then dispersed into a volume of water saturated with oxygen to form a MOF nanoparticle suspension having a solids concentration of 0.2 wt%. Water volumes of 200 mL are preferred, but other volumes are suitable. Oxygen was bubbled through the suspension to keep the oxygen level saturated.

[0150] The suspension was then exposed to the atmosphere and the oxygen content of the MOF nanoparticle suspension monitored over time at 20 °C with agitation at 500 rpm. Free oxygen will diffuse out from the suspension to the surrounding atmosphere. The oxygencarrying capacity of the MOF nanoparticles is the difference in dissolved oxygen level after 30 minutes at 20 °C between the test MOF nanoparticle suspension and a sample of pure water saturated with oxygen and prepared and tested in an identical manner.

[0151] The more oxygen is associated with the nanoparticles, the less oxygen will be lost to the surrounding atmosphere and the higher the measurement of dissolved oxygen in the suspension.Materials and Synthesis

[0152] All reagents unless otherwise stated were obtained from commercial sources and were used without further purification. 2',3”-dimethyl-[1 ,1 ’:4',1 ”:4",1 ”’-quaterphenyl]-4,4”’- dicarboxylic acid (Li) was synthesised according to the procedure reported in Lippke et al., Inorg. Chem. 2017, 56, 748-761. S1 was synthesised according to the procedure reported in Nepomnyashchii et al. J. Am. Chem. Soc. 2011 , 133, 8633-8645 Milli-Q water (18.2 MQ.cm resistivity at 25 °C) was used throughout the experiment. Dialysis tubing (Molecular Weight Cut-off; MWCO 3,500 and 12,000 - 14,000 Daltons) was obtained from Medicell Membranes Ltd. 525 nm high-power LED (3.1 W, SOLIS-525C, ThorLabs) was used for light irradiation. The concentrations of MOF samples used in the in vitro and in vivo studies were calculated based on the amount of bare 69-L2.Synthesis of mPEG5K-phosphate (P-PEG)

[0153] To a flame-dried two-neck flask, phosphorus oxychloride (POCh) (100 mmol) in 10 mL of dry dichloromethane (DCM) was added, followed by the addition of dry triethylamine (300 mmol) at 0 °C. After that, polyethylene glycol) methyl ether (Mn = 5000, 30 mmol) was added to the mixture in 100 mL of dry DCM over 30 min. The resulting solution was stirred at room temperature for 10 h, then 40 mL of water was added, the mixture was reacted for an additional 3 h. DCM was removed under a vacuum, and the raw product was purified by dialysis (MWCO 3,500) followed by removing the residual water with lyophilization to give mPEG-POs as a white solid (127.5 g, 25.5 mmol, 85%).1H NMR (D2O, 400 MHz), 5: 3.63 (s, 448H, - CH2OCH2-), 3.31 (s, 3H, -OCH3)31P NMR 5: (D2O, 162 MHz) 5: 0.22.Synthesis of Zr6cluster

[0154] The Zr6cluster was synthesized according to the reported procedure (Noh et al., Chem. Mater. 2018, 30 (7), 2193-2197). 15 ml of a 80 % solution of Zr(OBu)4 (5 mmol) in n- Butanol was added into a solution of 100 g (818.8 mmol) benzoic acid in 300 ml of n-propanol, resulting in a clear mixture solution. The mixture is further refluxed for overnight and white solid precipitation appears. After filtration, 10.2 g (yield: 63 %) solid was separated, washed by anhydrous n-propanol five times and dried under vacuum.Synthesis of L2Synthesis of S2

[0155] To a flame-dried flask was added with S1 (1.05 g, 4 mmol), NIS (1.8 g, 8 mmol), and anhydrous DCM (120 mL). The mixture was stirred at room temperature for 24 h. Afterward, the mixture was concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel (DCM / cyclohexane, 1 :1 v / v) to give compound S2 as a red solid (1.75 g, 85%).1H NMR (400 MHz, CDCI3): 52.62 (s, 3H), 2.60 (s, 6H), 2.46 (s, 6H).Synthesis of S4

[0156] A 100 mL flame-dried round-bottom flask was charged with S2 (1.29 g, 2.5 mmol), S3 (1.94 g, 8.75 mmol, 3.5 equiv), K2CO3(2.07 g, 15 mmol, 6 equiv) and Pd(PPh3)4(289 mg, 0.25 mmol), followed by addition of the degassed dioxane (30 mL) and H2O (6 mL). The resulting suspension was heated at 90 °C for 24 h. After cooling to room temperature, water was added. The resulting mixture was then extracted with DCM 3 times and washed with water. The combined organic extracts were dried over Na2SO4and then concentrated under reduced pressure. The crude solid was purified by column chromatography on silica gel (EtOAc / cyclohexane, 1 :10, v / v) to afford S4 as a red solid (936 mg, 61%).1H NMR (400 MHz, CDCI3): 5 8.07 (d, J = 8.3 Hz, 4H), 7.29 (d, J = 8.4 Hz, 4H), 2.73 (s, 3H), 2.49 (s, 6H), 2.35 (s, 6H), 1.62 (s, 18H). 13C NMR (101 MHz, CDCI3): 5 165.56, 152.36, 142.27, 138.15, 137.30, 132.87, 132.40, 130.79, 130.22, 129.52, 81.12, 28.23, 17.35, 15.52, 13.28. HRMS-ESI (m / z): Calculated for S4: C36H4I BN2O4F2: 614.3127, Found: 614.3148.Synthesis of L2

[0157] To a solution of S4 (660 mg, 1.07 mmol) in dry CH2CI2(35 mL) was added trifluoroacetic acid (15 mL) at 0 °C. The reaction mixture was allowed to stand at 0 °C for 5 h, and then stirred at room temperature overnight. Afterward, the solvent was removed withflowing nitrogen. The resulting residue was then washed with Et2O (30 mL) two times, affording L2as a red solid (483 mg, 90 %).1H NMR (400 MHz, DMSO-d6): 6 8.04 (d, J = 8.3 Hz, 4H), 7.45 (d, J = 8.3 Hz, 4H), 2.79 (s, 3H), 2.43 (s, 6H), 2.39 (s, 6H). 13C NMR (126 MHz, DMSO- d6): 0 167.55, 151.68, 144.48, 138.36, 138.04, 132.40, 132.23, 130.82, 129.93, 129.90, 17.68, 15.62, 13.57. HRMS-ESI (m / z): Calculated for [L2+ H]+, C28H26BF2N2O4: 503.1954, Found: 503.1954.Synthesis of L3Synthesis of S6

[0158] To a flame-dried flask was added with S5 (648 mg, 2 mmol), NIS (900 mg, 4 mmol), and anhydrous DCM (80 mL). The mixture was stirred at room temperature for 24 h. Afterward, the mixture was concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel (DCM / cyclohexane, 1 :2 v / v) to give compound S6 as a red solid (933 mg, 81%).1H NMR (700 MHz, CDCI3): 5 7.56 - 7.53 (m, 3H), 7.28 - 7.26 (m, 2H), 2.67 (s, 6H), 1.41 (s, 6H).Synthesis of S7

[0159] A 150 mL flame-dried round-bottom flask was charged with S6 (1.29 g, 4.3 mmol), S3 (5.3 g, 4 equiv), K2CO3(3.6 g, 6 equiv) and Pd(PPh3)4(497 mg, 0.43 mmol), followed by addition of the degassed dioxane (60 mL) and H2O (12 mL). The resulting suspension was heated at 90 °C for 36 h. After cooling to room temperature, water was added. The resulting mixture was then extracted with DCM two times and washed with water. The combined organic extracts were dried over Na2SO4and then concentrated under reduced pressure. The crude solid was purified by column chromatography on silica gel (EtOAc / DCM / cyclohexane, 1 :1 :30, v / v / v) to afford S7 as a red solid (1.42 g, 49%).1H NMR (700 MHz, CDCI3): 5 8.04 (d, J = 8.0Hz, 4H), 7.52 (dd, J = 13.1 , 7.1 Hz, 3H), 7.39 - 7.37 (m, 2H), 7.25 (d, J = 8.0 Hz, 4H), 2.57 (s, 6H), 1.63 (s, 18H), 1.34 (s, 6H). 13C NMR (176 MHz, CDCI3): 0 165.56, 154.21 , 142.59, 139.47, 138.04, 135.13, 133.00, 131.46, 130.72, 130.23, 129.99, 129.45, 129.36, 129.23, 127.92, 81.10, 28.21 , 13.40, 12.74. HRMS-ESI (m / z): Calculated for S7: C4I H43BF2N2O4: 676.3284, Found: 676.3273.Synthesis of l_3

[0160] To a solution of S7 (846 mg, 1.5 mmol) in dry CH2CI2(50 mL) was added trifluoroacetic acid (20 mL) at 0 °C. The reaction mixture was allowed to stand at 0 °C for 5 h, and then stirred at room temperature overnight. Afterward, the solvent was removed with flowing nitrogen. The resulting residue was then washed with Et2O (50 mL) two times, affording L3as a red solid (719 mg, 85 %).1H NMR (500 MHz, DMF-d7): 13.33 (s, 2H), 8.11 (d, J = 8.0 Hz, 4H), 7.66 (dd, J = 14.7, 7.1 Hz, 3H), 7.62 - 7.58 (m, 2H), 7.48 (d, J = 8.0 Hz, 4H), 2.59 (s, 6H), 1 .42 (s, 6H).13C NMR (126 MHz, DMF-d7) 6 167.41 , 154.18, 143.60, 139.74, 138.02, 134.98, 132.92, 131.50, 130.53, 130.11 , 129.82, 129.81 , 129.76, 128.44, 13.19, 12.64. HRMS-ESI (m / z): Calculated for [L3+ H]+, C33H28BF2N2O4: 565.2110, Found: 565.2082.Synthesis of F-PEG

[0161] The synthesis of F-PEG was adapted from a reported method with slight modification (Keiper et al., J. Am. Chem. Soc. 2002, 124, 1834-5). To a flame-dried two-neck flask, phosphorus oxychloride (1 .6 mL, 17.2 mmol) in 40 mL of dry DCM was added, and the mixture was cooled to 0 °C. Afterward,1H,1H,2H,2H-perfluoro-1-decanol (7.89 g, 17.0 mmol) and dry triethylamine (51.6 mmol) in 20 mL DCM were slowly added via syringe, leading to the formation of white precipitate. The mixture was then stirred at 0 °C for 1.5 h. After that, polyethylene glycol) methyl ether (Mn = 5000, 17 mmol) in dry DCM (20 mL) was added. The resulting solution was allowed to warm to room temperature and stirred under N2overnight. The white precipitate was filtered and washed with dry DCM (50 mL). Water (20 mL) was added to the filtrate and then was stirred at 40 °C overnight. Afterward, DCM was removed using flowing N2, and purified by dialysis (MWCO: 1 ,000), followed by removing the residualwater with lyophilization. The crude solid was purified by column chromatography on silica gel (DCM / MeOH, 10:1 , v / v) to afford F-PEG as a white solid (10 mmol, 59%).1H NMR (400 MHz, CDCh): 5 4.20 (q, J = 7.0 Hz, 2H), 3.55 (s, 471 H), 3.28 (s, 3H), 2.45 (ddd, J = 18.4, 12.2, 6.5 Hz, 2H).13C NMR (CDCh, 101 MHz): 5 70.54.31P NMR (CDCh, 162 MHz): 5 -4.04.19F NMR (CDCh, 376 MHz): 5 -85.53, -118.33, -126.58, -127.47, -128.37, -130.86. NMR spectra and size exclusion chromatography with multi-angle light scattering (SEC-MALS) confirm the formation and purity of F-PEG (Figures 2 to 6).Synthesis of BO DIPY-based MOFsSynthesis of UiO-69 (69-Me2) single crystal (SC 69-Me2)

[0162] 69-Me2was synthesized following a previously reported procedure (Lippke et al., Inorg. Chem. 2017, 56, 748-761). To a 20 mL vial was added with Li (36 mg), ZrOCI28H2O (28 mg), acetic acid (0.982 mL) and DMF (5 mL). The resulting mixture was sonicated for 10 min and then heated at 120 °C for 48 h. After cooling down to room temperature, colourless crystals of 69-Me2were harvested by filtration.

[0163] 69-Me2is a two-fold interpenetrated feu network with identical ligand-metal node connectivity to that of the UiO-66 to UiO-68 series MOFs (Figure 7).Synthesis of 69-Me2nanoparticle (69-Me2)

[0164] To a 20 mL vial was added with Li (18 mg), Zr6O8cluster (30 mg), acetic acid (150 pL) and DMF (8 mL). The resulting mixture was sonicated for 10 min and then heated at 120 °C for 12 h. After cooling to room temperature, the obtained sample was collected by high-speed centrifugation (15,000 rpm, 35 min), followed by washing with hot DMF 3 times and exchanging with ethanol 3 times. The final product was dried or re-dispersed in ethanol for further use.Synthesis of 69-L2and 69-Ls

[0165] 69-L2and 69-L3were synthesised by post-synthetic ligand exchange. Briefly, crystals or powders of 69-Me2(30 mg) were incubated in the DMF solution (4 mL) of L2or L3(100 mg) at 70°C for 4 days. SC 69-Me2was used to prepare SC 69-L2or SC 69-L3. 69-Me2nanoparticle was used to prepare 69-L2nanoparticle or 69-L3nanoparticle. The supernatant was exchanged with the fresh solution of L2or L3(100 mg) in DMF (4 mL) every 12 h. 69-L2and 69-L3in red colour was harvested by centrifugation (Figure 8). The colour change fromcolourless 69-Me2 (denoted as SC 69-Me2) to red after incubation in a DMF solution of l_2 at 70 °C (Figure 8), indicates the successful incorporation of L2.

[0166] An1H NMR study on a digested sample shows that l_2entirely replaced Li in 69-Me2after 4 days (Figure 9). This new material is denoted as 69-1.2 and its crystals as SC 69-1.2. Most importantly, this transformation process takes place in a SC-SC manner, where the single-crystal X-ray diffraction (SCXRD) analysis clearly shows the successful replacement of Li by L2. Identical to the parent 69-Me2, SCXRD reveals that 69-L2, formulated as [Zr6O4(OH)4(L2)6], is also a two-fold interpenetrated network, and crystallises in the cubic space group Fd-3m with a = 38.395 A. Each hexanuclear [Zr6(p3-O)4(p3-OH)4] cluster is connected to twelve bidentate carboxylate groups from twelve independent L2, where the neighbouring [Zr6(p3-O)4(p3-OH)4] clusters are linked by one l_2ligand, thereby forming an independent 3D framework in feu topology with two types of cavities (Figure 10d): 128 octahedral (2.26 nm, pale yellow, Figure 10b) and tetrahedral (1.31 nm, cyan, Figure 10c).

[0167] Notably, each l_2is disordered on the crystallographic symmetry axis. Two such identical networks are staggered with respect to one another, affording the final 69-1.2, in which the [Zr6(p3-O)4(p3-OH)4] clusters and three l_2ligands within the same trigonal face of the second network partially interpenetrate the tetrahedral and octahedral cavities of the first network, respectively (Figure 10e); 69-L2possesses compromised cavities with diameters of 1.31 nm (Figure 7a). Despite the presence of two-fold interpenetration, PLATON calculation (http: / / www.platonsoft.nl / platon / ) suggests a 54.5% void volume available for guest inclusion in 69-L2. Figures 11 b-11e show the powder X-ray diffraction (PXRD) patterns, 77 K N2 isotherms, and DFT pore size distribution (PSD) of SC 69-Me2and SC 69-L2.

[0168] Further to the utilisation of the meso-methyl BODIPY-based ligand L2, this SC-SC transformation methodology was successfully extended to incorporate the comparably bulky meso-phenyl BODIPY-based ligand L3, obtaining the single crystal, termed 69-L3(Figure 12, Figures 7, 13 to 17, 18 and Tables 1 and 3). To the best of the Inventors’ knowledge, this represents the first example of Zr-based MOFs constructed exclusively from BODIPY-derived ligands through a SC-SC process, since most of the reported BODIPY-containing Zr-based MOFs have been prepared via post-synthetic modification.

[0169] PXRD shows good agreement within 69-L2, 69-Me2, their single crystal analogues, and the simulated pattern, indicating the phase purity (Figure 11 b). Figures 11 c-11 d show typical Type I isotherms of microporous materials, with a sharp uptake at low relative pressure (P / Po< 0.01) for SC 69-L2and the related nanoparticles, 69-L2, consistent with their parent 69-Me2material. It was noted that N2 uptake at P / Po = 0.8 decreases from 428 to 393 cm3g-1(SC 69- Me2to SC 69-L2) and from 407 to 327 m3g-1(69-Me2to 69-L2nanoparticles) for micro-and nanosized MOFs in each case after the PSE process with Brunauer-Emmett-Teller (BET) areas - calculated using BETSI54 as described in Osterrieth, J. W. M. et al., Advanced Materials, 2022, 34, 2201502 - decreasing from 1639 to 1522 m2g-1and from 1388 to 1197 m2g-1, respectively. The lower N2uptakes and BET areas are attributed to the slightly increased volume and molecular weight of l_2ligands (Figure 18), which could be further confirmed by the decrease in PSD from 1.38 to 1.20 nm (Figure 11 e). Additionally, dynamic light scattering (DLS) of an aqueous suspension of 69-L2indicates z-average size and zeta potential of 171 ± 5 nm and -26 ± 5 mV, respectively, identical to the parent 69-Me2(Figures 11f and 11g).

[0170] It is also noted that the particle size obtained through DLS is 2~3 times larger than the transmission electron microscopy (TEM) and the scan electron microscopy (SEM) sizes, due to the presence of aggregates (Figures 11 h, 19 and 20). Specifically, TEM shows that 69-L2nanoparticles exist in the form of aggregation consisting of a few nanoparticles, and each particle exhibits distorted octahedral morphology with a diameter of around 80 nm (Figure 11 h). Moreover, high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) imaging reveals the existence of highly oriented channels with a lattice spacing of 1.2 nm (Figure 20b), which is slightly smaller than 1.3 nm of the parent 69-Me2(Figure 20a), consistent with the single-crystal structure and the decreased PSD obtained by 77 K N2isotherm (Figures 7a, 10e, 11e). Further energy-dispersive X-ray spectroscopy (EDS) mapping analysis confirms the uniformly distributed B and F elements within the 69-L2nanoparticles (Figure 11 i).Synthesis of 69-L2@P

[0171] A PEGylation strategy was pursued to relieve the hypoxia issue of solid tumours in future in vivo studies, for translational feasibility, and to improve the colloidal stability of the MOF system. The introduction of a phosphate-functionalized methoxy polyethylene glycol (P- PEG) onto Zr-based MOFs can simultaneously enhance their colloidal and chemical stability (WO 2022 / 122983 incorporated by reference herein in its entirety). P-PEG was synthesised as described herein and 69-L2@P was prepared as a control using P-PEG.

[0172] P-PEG solution (15 mL, 25 mg / mL in H2O) was added into the aqueous suspension of 69-L2(5 mL, 10 mg / mL). After stirring at room temperature overnight, the reaction mixture was centrifuged (25 min, 16,000 rpm) to remove the unreacted P-PEG, and washed with freshwater three times. The final product, denoted as 69-l_2@P, was kept in water. The amount of P-PEG (wt %) = mass of encapsulated P-PEG / 69-L2@P * 100. The amount of P-PEG in 69- l_2@P is 21.7 wt%, determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES) through measuring the ratio of P to Zr (Table 4), and confirmed using thermogravimetric analysis (TGA) (Figure 21).Table 4 ICP-OES analysis for 69-L2@P and 69-L2@FSample P Zr PEG loading(ppm) (ppm) (wt%)69-l_2@P batch 1 0.037549096 3.833998622 19.469-l_2@P batch 2 0.029658185 2.563730218 22.169-l_2@P batch 3 0.025422343 2.011467253 23.769-l_2@F batch 1 0.021017144 2.594717573 16.969-l_2@F batch 2 0.019827533 1.95927668 20.269-l_2@F batch 3 0.02381091 3.101977208 16.1PEG loading = Mass of PEG loaded / (Mass of MOF + Mass of PEG loaded)

[0173] The crystallinity of 69-L2is preserved after performing the PEGylation using P-PEG, with a new peak centred at 29 = 19° (Figures 11 b and 22). In addition, 77 K N2isotherms show that 69-l_2@P adsorbs 24 cm3g-1N2at P / Po= 0.8, with BET area decreasing from 1197 to 38 m2g-1, suggesting the blockage of nitrogen at 77 K towards the internal porosity of 69-L2. Fourier-transform infrared (FT-IR) spectra indicate the existence of PEG within 69-L2@P (Figure 23). TEM images show the well-preserved morphology after PEGylation with P-PEG, consisting of a few aggregated nanoparticles with diameters of 170 nm, consistent with the parent 69-L2(Figure 11j and 24). Moreover, HAADF-STEM imaging demonstrates the preservation of highly ordered mesopores after PEGylation (Figure 24). The z-average size of 69-l_2@P is 188 ± 3 nm, obtained by DLS analysis (Figure 11f). Furthermore, 69-L2@P displays a more negative zeta potential of -41 ± 1 mV compared to the parent 69-L2(Figure ng).Synthesis of 69-L2@F

[0174] Given the high oxygen affinity of fluorinated polymers, it was envisioned that integrating fluorinated polymer into P-PEG would combine the merits of fluorinated polymers and P-PEG. To validate this hypothesis, a perfluorooctyl segment was introduced into the P-PEG, yielding a double-tailed phosphate known as F-PEG.

[0175] PEGylation of 69-L2was then performed using F-PEG under the protocol reported in (WO 2022 / 122983 incorporated by reference herein in its entirety) and described herein.

[0176] F-PEG was synthesised as described herein. F-PEG solution (15 ml_, 25 mg / mL in H2O) was added into the aqueous suspension of 69-1.2 (5 ml_, 10 mg / mL). After stirring at room temperature overnight, the reaction mixture was centrifuged (25 min, 16,000 rpm) to remove the unreacted F-PEG, and washed with fresh water three times. The final product, denoted as 69-L2@F, was kept in water. The amount of F-PEG (wt %) = mass of encapsulated F-PEG / 69- L2@F x 100. The amount of F-PEG in and 69-L2@F is 17.7 wt%, determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES) through measuring the ratio of P to Zr (Table 4), and confirmed using thermogravimetric analysis (TGA) (Figure 21).

[0177] The crystallinity of 69-L2is preserved after performing the PEGylation using F-PEG, with a new peak centred at 29 = 23° (Figures 1 1 b and 22). In addition, 77 K N2 isotherms show that 69-L2@F adsorbs 37 cm3g-1N2 at P / Po = 0.8, respectively, with BET area decreasing from 1 197 to 104 m2g-1, suggesting the blockage of nitrogen at 77 K towards the internal porosity of 69-L2. Fourier-transform infrared (FT-IR) spectra indicate the existence of PEG within 69-L2@F (Figure 23). TEM images show the well-preserved morphology after PEGylation with F-PEG, consisting of a few aggregated nanoparticles with diameters of 170 nm, consistent with the parent 69-L2(Figures 1 1j and 24). Moreover, HAADF-STEM imaging demonstrates the preservation of highly ordered mesopores after PEGylation (Figure 24). The z-average sizes of 69-L2@F are 171 ± 1 nm, obtained by DLS analysis (Figure 1 1f). Furthermore, 69-L2@F displays more negative zeta potential of -45 ± 1 mV compared to the parent 69-L2(Figure 1 1 g).

[0178] Following the surface modification of 69-L2with F-PEG, its impact on the colloidal stability of 69-L2was assessed. Briefly, bare 69-L2and 69-L2@F were treated in water and PBS (pH = 7.4), then hydrodynamic size changes were monitored using DLS. As shown in Figure 25, 69-L2@F remained stable for up to 8 days, with negligible changes in hydrodynamic size. Although the size of 69-L2@F began to increase after 8 days, they still outperformed the untreated 69-L2, which experienced a rapid increase in size to approximately 400 nm within just 1 day. When treated in PBS (pH = 7.4), 69-L2@F maintained its hydrodynamic sizes for up to 30 h, after which it underwent rapid aggregation. In contrast, the bare 69-L2aggregated dramatically under identical conditions. Morphological changes for MOF particles were examined using TEM. Both bare 69-L2 and 69-L2@F retained their morphologies when suspended in water for up to 3 weeks (Figure 26). However, when exposed to PBS (pH = 7.4), the bare 69-L2decomposed rapidly after 4 h and degraded completely at 16 h, while the morphologies of 69-L2@F remained intact for up to 48 h before particles began to degrade oraggregate (Figure 27). These findings highlight the pronounced improvement in both colloidal stability and chemical stability conferred by F-PEG.The digestion of 69-L2samples

[0179] To a 6 mL vial, the activated 69-1.2 (10 mg) was stirred with 10X PBS (4 mL) at 50 °C for 7 days. Afterward, the solvent was removed under the vacuum. The residue was then incubated with 5M NF F (5 mL) at 50 °C for another 3 days. DMSO-de (0.7 mL) was added after removing the water, and the mixture was sonicated until fully dissolved, which was used for 1 H NMR measurement.Evaluation of the Oxygen Loading Capability of 69-L2@P and 69-L2@F

[0180] Milli-Q water was firstly bubbled with the flowing O2to saturate the content of O2. 69- L2@P and 69-L2@F were then diluted with the above Milli-Q water to 220 mL with a concentration of 2 mg / mL in terms of 69-L2. It was assumed that 69-L2@F, 69-L2@P, and water had the same initial amount of O2, which decreases over time due to the diffusion of dissolved O2into the air. The capped suspensions were then stabilized for 1 min. Afterward, the diluted suspensions were uncapped and stirred mildly, where the O2concentration was monitored using a portable dissolved oxygen meter (Hanna Instruments HI-2004-02 Edge® Dissolved Oxygen Meter) for 30 min. The values were recorded automatically every 30 s.Evaluation of ROS Generation Capability of 69-L2@P and 69-L2@F

[0181] The time-dependent ROS generation capabilities of 69-L2@P, 69-L2@F, and the control group were determined by a chemical acceptor, 1 ,3-diphenylisobenzofuran (DPBF, 97%, Acros Organics), under the LED light treatment (525 nm high-power LED, 3.1 W, SOLIS- 5250, ThorLabs). The solvents were bubbled with flowing oxygen for 30 min before sample preparation. Briefly, DPBF (50 pM) was irradiated with stirring at 525 nm in the presence of the sample (20 pM based on 69-L2) in MeOH (20 mL) at room temperature with the reaction vial open to the air. Aliquots of the reaction (100 pL) were taken every 30 s and measured by UV-Vis spectroscopy. The decrease of the Amax of DBPF (410 nm) was then evaluated and plotted according to A / A0 vs. time.

[0182] As shown in Figure 28, solvents have negligible influence on the position of the absorbance spectra of 69-L2, which possess identical absorbance bands in DMF, EtOH, and H2O, with two typical peaks centred at 530 and 380 nm, originating from the TT-TT* transitionsand the charge-transfer transitions of the BODIPY ligands. In comparison, solvents appear to have a minor impact on the emission spectra, where the related emission maximum exhibits slight shifts depending on the solvent used (Figure 28). In addition, 69-L2, 69-L2@P, and 69- L2@F have nearly identical absorbance and fluorescence spectra (Figure 11k), suggesting the negligible effect of the PEGylation process on emission.

[0183] Considering the excellent oxygen affinity of the perfluorooctyl segment, the oxygencarrying ability of 69-L2@F in water was then evaluated, using water and 69-L2@P as controls. The changes in dissolved oxygen concentration were monitored using a dissolved oxygen meter. Figure 111 shows the time-resolved dissolved O2concentration as a function of the increased time. 69-L2@F suspension exhibited a noticeably higher O2concentration compared to those of 69-L2@P and pure water, where the presence of PEG component has a negligible effect on O2solubilisation. More specifically, the initial dissolved O2concentration of around 25 mg / mL decreases, after 30 minutes, to 21.46, 12.36, and 11.31 mg / mL for 69- L2@F, 69-L2@P, and 69-L2, respectively (Figure 111). The dissolved oxygen content of water was 9.61 mg / mL. Thus, the oxygen carrying capacities of 69-L2@F, 69-L2@P, and 69-L2, were 11.85 mg / mL, 2.75 mg / mL and 1.7 mg / mL, respectively. These results demonstrate the excellent oxygen-carrying capability of 69-L2@F and MOFs containing perfluorocarbon moieties, which can potentially enhance the therapeutic effect of PDT.

[0184] The ROS generation ability of 69-L2@F and 69-L2@P in air-saturated MeOH was assessed using a commercial trapping agent, 1 ,3-diphenylisobenzofuran (DPBF). Figure 29 shows the degradation rate curves of DPBF, a common ROS quencher, when exposed to 69- L2@F and 69-L2@P in MeOH under continuous LED light irradiation (525 nm, high-power LED, 3.1 W, SOLIS-525C, 257 ThorLabs), whereas 69-L2@F shows enhanced ROS generation capability with a 80% decrease in the relative absorbance at 410 nm after 120s (Figure 11 m). For comparison, 69-L2@F takes 270 s to achieve an 80% decrease, whereas the control group exhibits a negligible decline of DPBF absorbance under the same irradiation treatment (Figure 29c). Overall, these results demonstrate that 69-L2@F could act as an efficient PS for PDT applications while simultaneously holding the capability of carrying O2.Preparation of 69-L2@F-Hydrogel (69-l_2@F-Gel)

[0185] Hydroxyethylcellulose (Natrosol 250 from HX Pharma) was mixed with ultrapure water to give a 1.25 wt% solution and sterilised under UV light for 15 minutes. Then 1 mL of a suspension of 69-L2@F having a concentration of 5 mg / mL was added to the hydrogel with intense mixing to give a composition comprising MOF nanoparticles and hydrogel with a 69- L2@F concentration of 2.5 mg / mL (or ~ 0.0025 wt %).In Vitro Study

[0186] Cytotoxicity of 69-L2, 69-L2@P and 69-L2@F was assessed under dark conditions. MDA-MB-231 cells viability was measured by MTS cytotoxicity assay after 72 h incubation. Cell culture of MDA-MB-231 cells and MTS cytotoxicity assay methods are described herein.

[0187] Breast cancer is one of the most commonly diagnosed cancers worldwide. Triplenegative breast cancer, a subset of breast cancer, is considered the most aggressive and invasive. Here, the PDT effect of BODIPY-based MOFs was investigated in the triple-negative breast cancer cells MDA-MB-231. First, the biocompatibility and intracellular uptake of 69-L2, 69-L2@P, and 69-L2@F was examined. As shown in Figure 30, 69-L2@P and 69-L2@F exhibit negligible cytotoxicity after incubation for 24 h in dark conditions, even at concentrations of 500 pg / mL. In comparison, bare 69-L2shows a reduction of over 30% in viability under the same concentration of 500 pg / mL. Altogether, this is consistent with the knowledge that the presence of PEG around the nanosized MOFs could significantly improve their biocompatibility due to the limited aggregation of the nanoparticles.

[0188] To validate the cellular uptake of the BODIPY-based MOFs, confocal laser scanning microscopy (CLSM) imaging was performed. Figure 31a shows increased fluorescence signals of the BODIPY core in MDA-MB-231 cells after incubation cells with 69-L2, 69-L2@P or 69-L2@F for 24 h; Figure 32 show the 3D z-stack CLSM, where 69-L2, 69-L2@P, and 69- L2@F are internalized and accumulated inside the cells. Figure 33 shows the TEM imaging of the heavy-metal-stained MDA-MB-231 cells where the presence of the cell membrane and the nucleus was observed, as well as the internalized 69-L2@P and 69-L2@F nanoparticles. Taken together, these results suggest excellent biocompatibility and successful internalization of 69-L2@P and 69-L2@F required for PDT.

[0189] Next, the PDT efficacy of 69-L2@F under both normoxia and hypoxia conditions in vitro was evaluated, using 69-L2@P as a control. Figure 31 b shows that, upon light irradiation, MDA-MB-231 cells incubated with 69-L2@P and 69-L2@F exhibit more than 40% reduction in viability at MOF concentrations as low as 10 pg / mL under normoxic conditions. When increasing the concentration to 25-100 pg / mL, 69-L2@F shows a significant reduction in cell viability compared to 69-L2@P, suggesting its superior in vitro PDT efficacy. The difference in cell viability could be attributed to the O2enrichment of fluorous tags around 69-L2, which would attract the O2within the media, thus improving the ROS generation and PDT effect. Unlike in normoxia, 69-L2@P and 69-L2@F show a similar trend in viability but to a lesserextent at low concentrations under hypoxic conditions - with 69-l_2@F having a consistently higher efficacy than 69-L2@P. Cell viabilities under hypoxic conditions are slightly higher than the same concentrations under normoxia (Figure 31c). This finding is attributed to the insufficient O2supply in hypoxia for ROS generation. Notably, at concentrations over 25 pg / mL, 69-L2@F shows higher toxicity against MDA-MB-231 cells in hypoxia than those treated with 69-L2@P. Again, the excellent oxygen-carrying capability provided by perfluorooctyl-containing 69-L2@F could lead to higher concentrations of oxygen and hence higher PDT efficacy against MDA-MB-231 cells in vitro when irradiated by LED light, favouring PDT treatment with deep-seated tumours.

[0190] To assess the intracellular ROS generation capability of 69-L2@P and 69-L2@F, 2', 7’- dichlorodihydrofluorescein diacetate (DCF-DA) was selected as a probe to detect intracellular ROS levels. Briefly, MDA-MB-231 cells were incubated with 25 and 100 pg / mL of either 69- L2@P or 69-L2@F for 24 h before being irradiated with LED light for 10 min. Figure 34 shows how all the MDA-MB-231 cells treated with 69-L2, 69-L2@P, or 69-L2@F at 25 and 100 pg / mL concentrations exhibit dim-green fluorescence from DCF before light irradiation. After 10 minutes of LED light treatment, the intracellular fluorescence becomes bright-green, demonstrating successful generation of ROS. Figure 35a shows the intracellular ROS level when treated with 69-L2@P and 69-L2@F, quantified by the fluorescence intensity of DCF; ROS levels are 3.5- and 4-fold higher than the control group in normoxia. This further suggests that the photocytotoxicity observed in normoxia was due to the significant increase in intracellular ROS generated by the BODYIPY-core (Figure 35b), leading to enhanced oxidative toxicity.

[0191] Figure 35a shows the normalized DCF fluorescent intensity of approximately 175 and 225 for 69-L2@P and 69-L2@F in the case of hypoxic cells. A ca. 1.8- and 2.3-fold increase for 69-L2@P and 69-L2@F compared to the control was observed, but to a lesser extent when compared to normoxia. Again, it is noted that 69-L2@F generated more intracellular ROS than 69-L2@P under both normoxia and hypoxia conditions (Figures 35a, 35b, and 36). Indeed, the difference in ROS generation between 69-L2@F and 69-L2@P under hypoxia is approximately 12% higher than normoxia, possibly due to a slight increase in O2concentration caused by the oxygen-carrying 69-L2@F at the cellular level. Figure 35c shows the DCF intensity as a function of BODIPY-MOFs intensity quantified by flow cytometry; 69-L2@P and 69-L2@F had similar cellular uptakes under both normoxia and hypoxia compared to the related controls. In addition, IncuCyte was used to perform real-time monitoring of the live MDA-MB-231 cells, which were treated with 69-L2, 69-L2@P or 69-L2@F (see Supporting Information for full details). As shown in Figures 37 and 38, the untreated cells proliferated throughout theobservation period, regardless of light irradiation. Similar trends were also found in the treated cells without light irradiation (Figures 37 and 38). In contrast, MDA-MB-231 cells exhibited significant changes in morphology and movement after light treatment (Figure 38). All these solidly showcase the promising potential of 69-L2@F for PDT against hypoxic tumours in real setups.In Vivo StudyIn vivo tumour growth and PDT Method

[0192] Athymic Balb / C female nude mice (5 weeks old, average weight = 20 g) were purchased from Charles River Laboratories (France) and acclimated for 1 week before the experiments, in aseptic conditions with water and standard pellet food provided ad libitum and with light / dark cycles of 12 h.

[0193] For breast tumour induction, luciferase-expressing MDA-MB-231 cells (5x106 / mouse) were injected subcutaneously on the right flank. The tumour volumes were measured every other day with a calliper and calculated by: tumour volume (mm3) = width. (length2) / 2. When they reached approximately 30 mm3, mice were distributed in four groups (5 mice / group): 1) Hydrogel, 2) 69-L2@F-Hydrogel (69-L2@F-Gel), 3) Hydrogel + LED, 4) 69-L2@F-Hydrogel (69-L2@F-Gel) + LED.

[0194] After being anesthetized with isoflurane (IsoFlo 100%p / p), 50 pL per mouse of the hydrogel only or the 69-L2@F-hydrogel were injected intratumorally (1.25% w / v HEC hydrogel or 2.5 mg / mL 69-L2in 1.25% w / v HEC hydrogel, respectively, in groups 1) and 3) or groups 2) and 4). ). Mice from groups 3) and 4) were irradiated with a green LED (525 nm high-power LED, 3.1 W, SOLIS-525C, ThorLabs) for 10 minutes. After receiving the treatment, fluorescence imaging was performed on each group using the Newton FT500 imaging system (Vilber) after excitation at 540 nm.

[0195] Following the fluorescence imaging, mice were injected subcutaneously with D- Luciferin (ABP Biosciences, 15 mg / mL in DPBS, 150 pL / mouse). After 10 minutes, the same system was used for bioluminescence imaging. The LED irradiation procedure was repeated on days 2 and 4 post intratumoral injection of the hydrogel or 69-L2@F-Gel, and the fluorescence and bioluminescence imaging procedures were repeated on days 2, 4 and 7. At the endpoint (day 7), mice were euthanized by cervical dislocation.

[0196] The tumours and the organs (liver, kidneys, spleen, heart, and lungs) were collected and imaged for fluorescence and bioluminescence and then fixed in 10% formalin for further histology studies. Histological sections of the tumours were stained with haematoxylin and eosin. The quantification was done with the Visiopharm Integrator System software (VIS; Visiopharm A / S, Hoersholm, Denmark) and a NanoZoomer-SQ Digital slide scanner (Hamamatsu Photonics). For the quantitative measurements, on each tumour, a systematic uniform random sampling (meander sampling) was carried out for each slide. Step-lengths of 1134.70 pm were used in both x-and y-directions, enabling the acquisition of 80% of the total area using an objective of 10x. The meander sampling generated an average of 50 fields for each tumour, which were overlapped using a test system. A total of 25 grid points were regularly arranged, covering 41201.51 pm2 per point (area per point; a / p). The sectional area of the tumour and of the necrosis were estimated by an unbiased, stereological technique based on point-counting (Howard and Reed, 2004), in which the total number of grid points in a section hitting the structures of interest (p structure) was calculated: Sectional area per structure = (p structure) * (a / p) * 2.

[0197] For immunohistochemical analysis, the tumours were stained with the antibody anti- Ki67. A representative picture at 40x per section was taken and the immunoratio was calculated using the immunoratio plugin on FIJI. Areas of necrosis were avoided. All the animal experiments were approved by the Ethical Committee and the Animal Welfare and Ethics Body of the Nova Medical School (21_01_ORBEA.1) and followed the Animal Research guidelines of Nova Medical School and of the Directorate-General for Food and Veterinary Medicine.In vivo tumour growth and PDT Results

[0198] The inventive hydrogel scaffold allowed the delivery of a higher effective local dose of the MOF. This not only enhances the MOF’s therapeutic stability but also minimises sideeffects and reduces clearance of the therapeutic agent by the body’s metabolic and excretory systems. Consequently, the treatment remains at the tumour site for a longer duration, improving its therapeutic effect.

[0199] The systemic administration of the MOFs disclosed herein may lead to their nonspecific accumulation in various organs, resulting in unwanted off-target effects. More importantly, their intrinsic photodynamic capacity, crucial to their therapeutic function, might be at risk of compromise if there is widespread distribution of the MOFs in the body. With this in mind, 69-L2@F was loaded in a hydrogel as a localized delivery depot, denoted as 69-I_2@F-Gel, and mice received a single intratumoral injection of the treatment or control (Figure 39a).

[0200] SEM imaging shows the well-maintained morphology after forming the 69-L2@F-Gel composite (Figure 40); energy-dispersive X-ray spectroscopy (EDS) mapping confirms the homogenous distribution of 69-L2@F within the hydrogel (Figure 41). Following intratumoral injection of 69-L2@F-Gel, mice were exposed to a green LED (69-L2@F-Gel + LED). Figure 39b depicts the therapeutic scheme, with the green LED exposure repeated on days 2 and 4 following intratumoral injection Using a live imaging system, tumour progression inhibition by luciferase expression was investigated.

[0201] Body weight remained unaltered during the experiment (Figure 42), suggesting the biocompatibility of the treatment without associated toxicity or side effects. Figure 39c shows the evolution of tumour size following the treatment; a tumour region of interest (ROI) of 1.5 x 108 and 2.9 x 108 were obtained for the 69-L2@F-Gel + LED and the 69-L2@F-Gel treatments, respectively. The 69-L2@F-Gel + LED treatment significantly reduced the tumour size at day 4 (n = 5, P = 0.002) compared to the control hydrogel + LED, with more than 80% (n = 5, P = 0.001) reduction at day 7 after intratumoral injection. The tumour regression is consistent with the hypoxia, in vitro results (Figure 31c), where MDA-MB-231 cell viability significantly decreased at day 3 of treatment with 69-L2@F + LED. In the in vivo studies, 69-L2@F fluorescently was tracked fluorescently, showing that 69-L2@F remained at the tumour site for 7 days after its injection (Figure 39d).

[0202] Conde et al. (Nat. Mater. 2016, 15, 1128-1138, Nat. Mater. 2016, 15, 353-363, Proc. Natl. Acad. Sci. U.S.A. 2015, 112, E1278-E1287 and ACS Cent. Sci. 2021 , 7, 868-881) have shown that hydrogels are able to sense and differentially react with the disease microenvironment, potentiating targeted drug release and uptake in certain disease settings. These hydrogels also prove to be incredibly efficient for tumour size reduction (~80-85%) 14 days post-gel implantation. Taking one step forward, the cellulose-based hydrogels developed in this study were injected inside the tumour tissue, leading to the in situ activation of 69-L2@F with LED light, allowing the production of ROS from the inside to the outside of the tumour microenvironment.

[0203] The 69-L2@F-Gel + LED treatment inhibited tumour growth significantly more than the control groups, resulting in a more potent effect in terms of speed, reducing more than 80% of the tumour in only 7 days. Remarkably, tumour regression occurs after a single injection with no adverse effects in mice. Moreover, the inhibition of tumour growth significantly increasedmice survival (P < 0.0001) up to day 11 in the 69-l_2@F-Gel + LED group (Figure 39e), when compared to the other groups, which survived up to day 7, reinforcing the effectiveness of the treatment.

[0204] Live imaging of the treated mice supports the inhibition of tumour growth by demonstrating clear tumour regression (Figures 43a and 43b) from day 0 to day 7 following in situ treatment injection and LED illumination. This information also demonstrates the presence of 69-L2@F at the tumour site throughout the experiment (Figures 43a and 43c). In contrast, no fluorescence signal was found in any of the major organs, meaning that the hydrogel enabled the exclusive accumulation of 69-L2@F in the tumour tissue, as demonstrated by the ex vivo images of the organs (Figure 43a).

[0205] Moreover, breast tumours treated either with the hydrogel (with or without LED irradiation) or with 69-L2@F-Gel without LED irradiation present adipocytes localized at the invasive tumour front (Figures 43d and 44). Cancer cells often invade the adipose tissue and induce adipocytes to release free fatty acids, which are absorbed by cancer cells and used to produce ATP, thus facilitating tumour growth. Note that, at the invasive front, the size and number of adipocytes (arrows in Figures 43d and 44) are reduced with 69-L2@F-Gel + LED. It is also important to highlight that the close localization between adipocytes and invasive cancer cells (adipocytes in the vicinity of cancer cells) displays profound phenotypic and functional alterations. The role of adipocytes relies on their support and promotion of tumour growth. Furthermore, when compared to mice treated with 69-L2@F-Gel + LED irradiation, histological images of these solid tumours consistently showed an increase in the invasive front (brackets in Figures 43d and 44) of the tumour with a higher accumulation of active and dividing cells. There are also multifocal to coalescing areas of necrosis (asterisks in Figure 43d and 44) in the tumours treated with 69-L2@F-Gel with LED irradiation, corroborating the role of the LED-activated 69-L2@F in promoting a PDT that employs exogenously produced ROS to kill breast cancer cells by light activation.

[0206] Furthermore, tumours from the 69-L2@F-Gel + LED-treated mice show much less actively proliferating cells when compared to the Hydrogel + LED group (Figure 43e). This is observed as a decrease in the brown colour, which identifies the presence of the Ki-67 protein, a marker for cellular proliferation and ribosomal RNA transcription. Likewise, the percentage of inflammatory foci / tumour in the 69-L2@F-Gel + LED group is highly significantly increased (P < 0.001) when compared to the controls (Figure 43f). This is consistent with ROS production in the treated group, as these molecules can trigger cell death-associated pathways throughnecrosis, as we can observe by the significant (P < 0.05) increase in % necrosis when compared to the controls (Figure 43g).

[0207] Every document cited herein, including any cross-referenced or related patent or application, is hereby incorporated by reference in its entirety unless expressly excluded or otherwise limited.

[0208] It will be appreciated that various modifications may be made to the embodiments shown without departing from the spirit and scope of the invention as defined by the accompanying claims.

[0209] The project leading to this application has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 726380).The invention is further described with reference to the following numbered embodiments:1. A composition comprising a metal-organic framework (MOF) nanoparticle or nanoparticles and a hydrogel; wherein the or each nanoparticle comprises a MOF comprising a plurality of metal ions and a plurality of organic ligands; wherein the or each MOF nanoparticle comprises photosensitiser and one or more perfluorocarbon moieties.2. The composition according to embodiment 1 , wherein the or each MOF nanoparticle has an enhanced oxygen carrying capacity of greater than 5 mg / mL measured according to the Oxygen Carrying Capacity Test Method defined herein.3. A composition comprising a metal-organic framework (MOF) nanoparticle or nanoparticles and a hydrogel; wherein the or each nanoparticle comprises a MOF comprising a plurality of metal ions and a plurality of organic ligands; wherein the or each MOF nanoparticle comprises photosensitiser; wherein the or each MOF nanoparticle has an enhanced oxygen carrying capacity of greater than about 5 mg / mL measured according to the Oxygen Carrying Capacity Test Method defined herein.4. The composition according to embodiment 3, wherein the or each MOF nanoparticle comprises one or more perfluorocarbon moieties.5. The composition according to any one of embodiments 1 to 4:(i) wherein the hydrogel has a viscosity of less than about 15.0 Pascal seconds (Pa.s), when measured at 25 s-1and 20 °C according to the Viscosity Test Method defined herein; and / or(ii) wherein the hydrogel is a biocompatible hydrogel, preferably the hydrogel is selected from the group consisting of: collagen, polyvinyl alcohol, polysaccharides, cellulose derivatives and biocompatible organic polymers and copolymers, preferably wherein the biocompatible hydrogel is selected from the group consisting of alginate, RGD-modified alginate, amylase, amylpectin, cellularose, chitosan, collagen, dextran, fibrin, gelatin, glycogen, heparin, hyaluronic acid, poly(acrylamide), poly(P-aminoester), poly(caprolactone), matrigel, multi-arm polyethylene glycol, poly-hydroxyethyl acrylate, poly(hydroxyethyl methacrylate), poly(N-isopropylacrylamide), poly(glycolic acid), poly(lactic acid), poly(lactic acid-glycolic acid), oligo(poly(ethylene glycol)fumarate), poly(vinyl alcohol), poly(vinyl acid), and combinations thereof; and / or(iii) comprising one or more pharmaceutically acceptable excipients selected from the group consisting of: solvents, co-solvents, buffers, stabilisers, antioxidants, preservatives, chelating agents, emulsifiers, flavourings, lubricants, suspending agents, tonicity adjusting agents, surfactants, solubilisers, suspending aids, dispersion agents, humectants, thickeners, colouring agent, wetting agent, anti-foaming agent, viscosity modifier, sweeteners and combinations thereof; and / or(iv) wherein the composition comprises between about 0.001 wt% and about 0.1 wt% of the composition MOF nanoparticle by weight.6. The composition according to any one of embodiments 1 to 5, wherein each MOF nanoparticle comprises one or more hydrophilic polymers, preferably wherein the hydrophilic polymer is selected from the group consisting of: polyethylene glycol (PEG) or a derivative thereof, polyvinyl alcohol (P A) or a derivative thereof, poly (lactic-co-glycolic acid) (PLGA) or a derivative thereof, Heparin or a derivative thereof, Chitosan or a derivative thereof, pluronic F-127, and phospholipids, dioleoylphosphatidylcholine (DOPC) or a derivative thereof, and 1 ,2-Dioleoyl-3-trimethylammonium propane (DOTAP) or a derivative thereof, more preferably wherein the hydrophilic polymer is linear methoxy polyethylene glycol (mPEG)7. The composition according to any one of embodiments 1 to 6, whereina. at least one of the plurality of organic ligands comprises at least one of the one or more perfluorocarbon moieties ; or b. each of the plurality of organic ligands comprises the one or more perfluorocarbon moieties ; or c. the one or more perfluorocarbon moieties are non-covalently attached to the or each MOF nanoparticle; or d. the one or more perfluorocarbon moieties are attached to the one or more hydrophilic polymers to form one or more polymers.8. The composition according to embodiment 7, wherein each of the one or more polymers are attached to one or more metal ions of each nanoparticle, preferably wherein the one or more polymers comprises a phosphate group and wherein the one or more polymers are attached to one or more metal ions of each nanoparticle by phosphate-metal coordination, more preferably wherein the polymer is a phosphate-terminated mPEG according to Structure AStructure A wherein n is from about 40 to about 250.9. The composition according to any one of embodiments 1 to 8, wherein a. at least one of the plurality of organic ligands comprises photosensitiser; b. each of the plurality of organic ligands comprises photosensitiser; or c. the photosensitiser is non-covalently attached to the or each MOF nanoparticle.10. The composition according to any one of embodiments 1 to 9, wherein the photosensitiser is selected from the group consisting of: chlorin-based ligands, BODIPY- based ligands of Formula I, Aza-BODIPY-based ligands of Formula II, porphyrin-based ligands of Formula III, bacteriochlorin-based ligands of Formula IV, phthalocyanine-based ligands of Formula V, M(bpy)3ligands of Formula VI, and combinations thereofFormula VI, preferably wherein the photosensitiser is a BODIPY-based ligand, more preferably wherein the photosensitiser is selected from the group consisting of: l_2and l_311. The composition according to any one or embodiments 1 to 10, wherein the plurality of metal ions are metal ions having an atomic weight of greater than 40, preferably wherein the plurality of metal ions consist essentially of a metal ion selected from the group consisting of: zirconium, iron, thorium, hafnium , ruthenium, gadolinium, neodymium, ytterbium and erbium.12. The composition according to any one of embodiments 1 to 11 for use as a medicament.13. The composition according to any one of embodiments 1 to 11 for use in the treatment of cancer by photodynamic therapy, preferably wherein cancer is selected from the group consisting of: breast cancer, prostate cancer, lung cancer, brain tumours (including glioblastoma), bladder cancer, cervical cancer, head and neck cancers, Hodgkin's lymphoma, Non-Hodgkin's Lymphoma, melanoma, pancreatic cancer, rectal cancer, skin cancer (including Basal Cell Carcinoma and Squamous Cell Carcinoma) esophageal cancer, thyroid cancer, testicular cancer, , mesothelioma, ovarian cancer, kidney cancer, liver cancer, gastric (stomach) cancer, bone cancer (including osteosarcoma and Ewing sarcoma), soft tissue sarcomas, endometrial (uterine) cancer, spinal tumour, nasopharyngeal cancer, salivary gland cancer, childhood cancers (such as leukaemia, neuroblastoma, Wilms tumour), colon cancer.14. A method of manufacturing the composition as defined in embodiments 1 to 11 , the method comprising the steps of: a. mixing a plurality of metal ions, a plurality of organic ligands, photosensitiser and one or more perfluorocarbon moieties in a solvent to form a suspension of one or more MOF nanoparticles, b. optionally additionally mixing one or more hydrophilic polymers in step a., c. optionally drying the one or more MOF nanoparticles by lyophilisation, d. dispersing the one or more metal-organic framework (MOF) nanoparticles in a hydrogel to provide the composition, e. optionally mixing one or more pharmaceutically acceptable excipients into the composition.15. A method of manufacturing the composition as defined in any one of embodiments 1 to 11 , the method comprising the steps of: a. mixing a plurality of metal ions, a plurality of organic ligands and one or more perfluorocarbon moieties in a solvent to form a suspension of one or more initial MOF nanoparticles, b. mixing the suspension of one or more initial MOF nanoparticles with photosensitiser to replace one or more of the plurality of organic ligands with photosensitiser by post-synthetic ligand exchange to form the one or more MOF nanoparticles, c. optionally additionally mixing one or more hydrophilic polymers in step a., d. optionally drying the one or more MOF nanoparticles by lyophilisation,e. dispersing the one or more metal-organic framework (MOF) nanoparticles in a hydrogel to provide the composition, f. optionally mixing one or more pharmaceutically acceptable excipients into the composition.

Claims

Claims1. A composition comprising a metal-organic framework (MOF) nanoparticle or nanoparticles and a hydrogel; wherein the or each nanoparticle comprises a MOF comprising a plurality of metal ions and a plurality of organic ligands; wherein the or each MOF nanoparticle comprises a photosensitiser and one or more moieties capable of associating to oxygen.

2. The composition according to claim 1, wherein the one or more moieties capable of associating to oxygen are one or more perfluorocarbon moieties.

3. The composition according to claim 2 wherein one or more perfluorocarbon moieties have a carbon backbone of about 6 to 16 carbon atoms.

4. The composition according to claim 3 wherein the perfluorocarbon moiety is a perfluorooctyl group.

5. The composition according to claim 1 wherein the one or more moieties capable of associating to oxygen is a 1,3-diene.

6. The composition according to claim 5, wherein the one or more moieties capable of associating to oxygen are one or more aromatic moieties selected from the group consisting of anthracene or a derivative thereof, naphthalene or a derivative thereof, and 2-pyridone or a derivative thereof.

7. The composition according to claim 6, wherein the one or more moieties capable of associating to oxygen are selected from anthracene, naphthalene and 2-pyridone.

8. The composition according to claim 7, wherein the one or more moieties capable of associating to oxygen are anthracene or a derivative thereof.

9. The composition according to claim 7 or 8, wherein each of the one or more moieties capable of associating to oxygen are anthracene.

10. The composition according to any one of claims 1-9, wherein each MOF nanoparticle has an enhanced oxygen carrying capacity of greater than about 5mg / mL measured according to the Oxygen Carrying Capacity Test Method defined herein.

11. The composition according to any one of claims 1-10, wherein a. at least one of the plurality of organic ligands comprises at least one of the one or more moieties capable of associating to oxygen; or b. each of the plurality of organic ligands comprises the one or more moieties capable of associating to oxygen; or c. the one or more moieties capable of associating to oxygen are non-covalently attached to each MOF nanoparticle.

12. The composition according to any one of claims 1 to 11:(i) wherein the hydrogel has a viscosity of less than about 15.0 Pascal seconds (Pa.s), when measured at 25 s1and 20 °C according to the Viscosity Test Method defined herein; and / or(ii) wherein the hydrogel is a biocompatible hydrogel, preferably the hydrogel is selected from the group consisting of: collagen, polyvinyl alcohol, polysaccharides, cellulose derivatives and biocompatible organic polymers and copolymers, preferably wherein the biocompatible hydrogel is selected from the group consisting of alginate, RGD- modified alginate, amylase, amylpectin, cellularose, chitosan, collagen, dextran, fibrin, gelatin, glycogen, heparin, hyaluronic acid, poly(acrylamide), poly( - aminoester), poly(caprolactone), matrigel, multi-arm polyethylene glycol, polyhydroxyethyl acrylate, poly(hydroxyethyl methacrylate), poly(N- isopropylacrylamide), poly(glycolic acid), poly(lactic acid), poly(lactic acid-glycolic acid), oligo(poly(ethylene glycol)fumarate), poly(vinyl alcohol), poly(vinyl acid), and combinations thereof; and / or(ill) comprising one or more pharmaceutically acceptable excipients selected from the group consisting of: solvents, co-solvents, buffers, stabilisers, antioxidants, preservatives, chelating agents, emulsifiers, flavourings, lubricants, suspending agents, tonicity adjusting agents, surfactants, solubilisers, suspending aids, dispersion agents, humectants, thickeners, colouring agent, wetting agent, anti-foaming agent, viscosity modifier, sweeteners and combinations thereof; and / or(iv) wherein the composition comprises between 0.001 wt% and 0.1 wt% of the composition MOF nanoparticle by weight.

13. The composition according to any one of claims 1 to 12, wherein each MOF nanoparticle comprises one or more hydrophilic polymers.

14. The composition according to claim 13, wherein the hydrophilic polymer is selected from the group consisting of: polyethylene glycol (PEG) or a derivative thereof, polyvinyl alcohol (PVA) or a derivative thereof, poly (lactic-co-glycolic acid) (PLGA) or a derivative thereof, Heparin or a derivative thereof, Chitosan or a derivative thereof, pluronic F-127, and phospholipids, dioleoylphosphatidylcholine (DOPC) or a derivative thereof, and l,2-Dioleoyl-3- trimethylammonium propane (DOTAP) or a derivative thereof.

15. The compositions according to claim 14, wherein the hydrophilic polymer is linear methoxy polyethylene glycol (mPEG)16. The composition according to any one of claims 13-15, wherein the one or more moieties capable of associating to oxygen are attached to the one or more hydrophilic polymers to form one or more polymers.

17. The composition according to claim 16, wherein each of the one or more polymers are attached to one or more metal ions of each MOF nanoparticle.

18. The composition according to claim 17, wherein the one or more polymers comprises a phosphate group and wherein the one or more polymers are attached to the one or more metal ions of each MOF nanoparticle by phosphate-metal coordination.

19. The composition according to claim 18, wherein the polymer is a phosphate-terminated mPEG according to Structure AStructure A wherein n is from about 40 to about 250.

20. The composition according to any one of claims 13 to 19, wherein each MOF nanoparticle is coated with a polymer coating comprising the hydrophilic polymer and a moiety capable of associating to oxygen; and wherein the polymer coating is attached to one or more metal ions of each MOF nanoparticle.

21. The composition according to claim 20, wherein the polymer coating comprises the hydrophilic polymer and a perfluorocarbon moiety.

22. The composition according to any one of claims 1 to 20, wherein the photosensitiser is selected from the group consisting of porphyrins, chlorins, bacteriochlorins, phthalocyanines, boron-dipyrromethene (BODIPY) derivatives, disalycilidene-l,2-cyclohexylidenediamine derivatives, gemcitabine, and combinations thereof.

23. The composition according to any one of claims 1 to 22, wherein a. at least one of the plurality of organic ligands comprises photosensitiser or b. each of the plurality of organic ligands comprises photosensitiser; or c. the photosensitiser is non-covalently attached to the or each MOF nanoparticle.

24. The composition according to any one of claims 1 to 23, wherein the photosensitiser is selected from the group consisting of: chlorin-based ligands, BODIPY-based ligands of FormulaI, Aza-BODIPY-based ligands of Formula II, porphyrin-based ligands of Formula III, bacteriochlorin-based ligands of Formula IV, phthalocyanine-based ligands of Formula V,M(bpy)3ligands of Formula VI, and combinations thereofFormu a VI25. The composition according to claim 24, wherein the photosensitiser is a BODIPY-based ligand.

26. The composition according to claim 25, wherein the photosensitiser is selected from the group consisting of: L2and L327. The composition according to any one of claims 24-26, wherein when the photosensitiser is of Formula I, Formula II, Formula III, Formula IV, Formula V or Formula VI, each of the plurality of organic ligands is photosensitiser.

28. The composition according to any one of claims 1-27, wherein the plurality of organic ligands are selected from 2',3"-dimethyl-[l,l':4',l":4",l"'-quaterphenyl]-4,4"'-dicarboxylic acid (Li), tetrakis (4-carboxyphenyl)porphyrin (TCPP), 1,4-benzenedicarboxylic acid (BDC), 1,3,5- benzenetricarboxylic acid (H3BTC), 1,3, 5, 8- (p-benzoate)pyrene linkers (H4TBAPy), 4',4"',4""',4"""'-(ethene-l,l,2,2- tetrayl)tetrakis(( [l,l’-biphenyl]-3-ca rboxylic acid)) (H4ETTC), a ligand of Formula I, a ligand of Formula II, a ligand of Formula III, a ligand of Formula IV, a ligand of Formula V, a ligand of Formula VI, and chlorin-based ligands.

29. The composition according to any one or claims 1 to 28, wherein the plurality of metal ions are metal ions having an atomic weight of greater than 40, preferably wherein the plurality of metal ions consist essentially of a metal ion selected from the group consisting of: zirconium, iron, thorium, hafnium , ruthenium, gadolinium, neodymium, ytterbium and erbium.

30. The composition according to claim 28, wherein the plurality of metal ions consists essentially of zirconium; and the organic ligand is the photosensitiser L2or the photosensitiser L3.

31. The composition according to claim 29, wherein the organic ligand is the photosensitiser L2.

32. The composition according to claim 30, further comprising a phosphate-terminated mPEG according to Structure AStructure A wherein n is from about 40 to about 25033. The composition according to any one of claims 1 to 32 for use as a medicament.

34. The composition according to any one of claims 1 to 32 for use in the treatment of cancer by photodynamic therapy,35. The composition according to claim 34, wherein cancer is selected from the group consisting of: breast cancer, prostate cancer, lung cancer, brain tumours (including glioblastoma), bladder cancer, cervical cancer, head and neck cancers, Hodgkin's lymphoma, Non-Hodgkin's Lymphoma, melanoma, pancreatic cancer, rectal cancer, skin cancer (including Basal Cell Carcinoma and Squamous Cell Carcinoma) esophageal cancer, thyroid cancer, testicular cancer, , mesothelioma, ovarian cancer, kidney cancer, liver cancer, gastric (stomach) cancer, bone cancer (including osteosarcoma and Ewing sarcoma), soft tissue sarcomas, endometrial (uterine) cancer, spinal tumour, nasopharyngeal cancer, salivary gland cancer, childhood cancers (such as leukaemia, neuroblastoma, Wilms tumour), and colon cancer.

36. A method of manufacturing the composition as defined in any one of claims 12 to 32, the method comprising the steps of: a. mixing a plurality of metal ions, a plurality of organic ligands, photosensitiser and one or more moieties capable of associating to oxygen in a solvent to form a suspension of one or more MOF nanoparticles, b. optionally additionally mixing one or more hydrophilic polymers in step a., c. optionally drying the one or more MOF nanoparticles by lyophilisation,d. dispersing the one or more metal-organic framework (MOF) nanoparticles in a hydrogel to provide the composition, e. optionally mixing one or more pharmaceutically acceptable excipients into the composition.

37. The method of claim 36, wherein the one or more moieties capable of associating to oxygen are one or more perfluorocarbon moieties.

38. The method of claim 36 or claim 37, wherein the plurality of organic ligands is the photosensitiser, and wherein the photosensitiser is of Formula I, Formula II, Formula III, Formula IV, Formula V or Formula VIFormu a VI39. A method of manufacturing the composition as defined in any one of claims 1 to 32, the method comprising the steps of: a. mixing a plurality of metal ions, a plurality of organic ligands and one or more moieties capable of associating to oxygen in a solvent to form a suspension of one or more initial MOF nanoparticles, b. mixing the suspension of one or more initial MOF nanoparticles with photosensitiser to replace one or more of the plurality of organic ligands with photosensitiser by postsynthetic ligand exchange to form the one or more MOF nanoparticles, c. optionally additionally mixing one or more hydrophilic polymers in step a., d. optionally drying the one or more MOF nanoparticles by lyophilisation, e. dispersing the one or more metal-organic framework (MOF) nanoparticles in a hydrogel to provide the composition,f. optionally mixing one or more pharmaceutically acceptable excipients into the composition.

40. The method of claim 39, wherein the one or more moieties capable of associating to oxygen are one or more perfluorocarbon moieties.

41. The method of claim 39 or claim 40, wherein in step b. the organic ligand 2',3"-dimethyl- [l,l':4',l":4",l"'-quaterphenyl]-4,4"'-dicarboxylic acid (Li) is replaced with photosensitiser L2or L3:

42. A method of preparing a composition as defined in any one of claims 16-32, the method comprising the steps of: a. mixing a plurality of metal ions and a plurality of organic ligands in a solvent to form a suspension of one or more initial MOF nanoparticles, b. encapsulating photosensitiser within and / or locating photosensitiser on the one or more initial MOF nanoparticles to provide a suspension of one or more photosensitiser-containing MOF nanoparticles, c. subsequently mixing the suspension of one or more photosensitisercontaining MOF nanoparticles with the polymer coating and allowing the polymer coating to attach to one or more metal ions of the one or more photosensitisercontaining MOF nanoparticles to provide a suspension of one or more polymer-coated MOF nanoparticles,d. optionally drying the one or more polymer-coated MOF nanoparticles by lyophilisation; e. dispersing the one or more polymer-coated MOF nanoparticles in a hydrogel to provide the composition; and f optionally mixing one or more pharmaceutically acceptable excipients into the composition.

Images