ENCAPSULATED TARGET-MOLECULE AND THEIR USE

FIELD OF THE INVENTION

The present invention relates to a metal-organic framework with encapsulated target- molecule, a process for the preparation of a metal-organic framework with encapsulated target-molecule, as well as the metal-organic framework with encapsulated target-molecule obtained by the process, and the use of the metal-organic framework with encapsulated target-molecule.

TECHNICAL BACKGROUND

Metal-organic frameworks (MOFs) are coordination networks consisting of metal ions and organic linkers that are coordinated to the metal ions. MOFs have high porosity, large surface areas and tuneable functionality, and have great promise for applications in sorption and separation, catalysis, sensing and drug delivery. MOFs have been used as carriers of metal nanoparticles for catalysis (Jiang, et a I, J. Am. Chem. Soc. 131, 11302- 11303 (2009); and Dhakshinamoorthy, et al., Chem. Soc. Rev. 41, 5262-5284 (2012)) and of organic molecules such as drugs (US 2015/0150981; Horcajada, P., et al. Chem. Rev. 112, 1232-1268 (2011); and Vasconcelos et al., RSC Advances 2, 9437-9442 (2012)), proteins and DNA for medical applications (Lyu, et al., Nano Lett. 14, 5761-5765 (2014); and Liang et al., Nature Communications 6, Article number 7240, (2015)). WO 2012/020214 discloses a material possessing antimicrobial properties wherein both the framework itself as well as incorporated guest molecules are active against microbes. The potential applications of MOFs can be developed and extended by the introduction of hierarchical structures containing both micropores and mesopores. However, post- functionalisation strategies involve multiple steps, which are both costly and produce large amount of waste. Another drawback with current MOFs is the small pore window that limits their potential to encapsulate large molecules. In addition, compared with other inorganic carriers, for example mesoporous silica, the current loading capacity of large molecules in MOFs is rather low. One strategy to overcome these drawbacks is to combine MOF synthesis and molecule encapsulation into a one-pot process (Lyu, et al., Nano Lett. 14, 5761-5765 (2014); and Liang et al., Nature Communications 6, Article number 7240, (2015); Abstracts presented at International Conference on Nanospace Materials (Taipei, Taiwan; 23-25 June 2015) and Gordon Research Conference (Biddeford, ME, USA; June 28-July 3, 2015)).

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a metal-organic framework (MOF) comprising a core containing an encapsulated target-molecule, and a shell that is substantially free of target-molecules, and to provide a convenient process for the preparation of MOFs with tailored properties and improved loading capacity, also for large molecules. The present invention relates to a metal-organic framework with encapsulated target- molecule, characterized in that the MOF has a core-shell structure, wherein the core comprises the MOF and the encapsulated target-molecule, and the shell comprises the MOF and is substantially free of target-molecules. The present invention further relates to a process for the preparation of a metal-organic framework (MOF) with encapsulated target-molecule, comprising the steps of:

a) mixing a target-molecule with a metal salt in a suitable solvent and adjusting the pH of the solution to a value between 6 and 12;

b) adding a solution of organic linkers; and

c) collecting the MOF crystals formed.

The present invention also provides a metal-organic framework with encapsulated target-molecule obtained by the process according to the present invention. Further, the present invention provides use of a metal-organic framework with an encapsulated target-molecule obtained by the process according the present invention in separation, catalysis, for gas separation, removal of organic pollutants, bio-imaging, drug delivery, and sensing.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows pH-induced one-pot synthesis of MOFs with encapsulated target- molecules.

Figure 2 shows a SEM image of DOX@ZIF-8 with 20% DOX loadings (a), and powder X- ray diffraction pattern (PXRD, b), colour (c) and TEM images (d) of the DOX@ZIF-8 crystals intensifying with higher DOX loading (0, 4, 14 and 20% DOX loadings).

Figure 3 shows UV-Vis spectra of a DOX solution, the mixed solution of DOX molecule and zinc nitrate and solid-state UV-Vis spectra of DOX@ZIF-8.

Figure 4 shows the TEM image and the electron diffraction (ED) pattern (inset) of a single DOX@ZIF-8 crystal.

Figure 5 shows the distribution of mesopores in DOX@ZIF-8 crystals (with 20% DOX loading) illustrated by electron tomography, (a) TEM image of a DOX@ZIF-8 single crystal, (b) Cross-section of the electron tomogram with the mesopores marked by dark- grey areas, (c) 3D distribution of the mesopores in the DOX@ZIF-8 crystal. The DOX molecules are present in the mesopores. A mesopore-free shell is clearly seen, indicating that the shell is substantially free of the DOX molecules.

Figure 6 shows the particle size distribution of the DOX@ZIF-8 crystals with different DOX loadings, 0% (a), 4% (b), 14% (c), and 20% (d), obtained by dynamic light scattering (DLS).

Figure 7 shows the UV-Vis absorbance spectra of organic dye target molecular solution and the mixed solution of target-molecule and zinc nitrate.

Figure 8 shows PXRD patterns of hierarchical micro-/meso-structured ZIF-8 crystals in which different type of organic dyes have been encapsulated. Figure 9 shows PXRD pattern (a), SEM image (b) and TEM image (c) of ZIF-67 crystals (synthesized using Co as the metal instead of Zn in ZIF-8) in which DOX has been encapsulated.

Figure 10 shows a TEM image of (Pd&DOX)@ZIF-8.

Figure 11 shows in (a) the N ₂ adsorption/desorption isotherm, (b) pore size distribution of the DOX@ZIF-8 crystals after treatment.

Figure 12 shows the pH-responsive release of DOX from DOX@ZIF-8 crystals (a) and (b) and time dependent dissolving profiles of free DOX at different pH values (c) as determined by UV-Vis spectrophotometry. At high pH (6.5-7.4), there is virtually no release (< 1%). At low pH (5.0-6.0), > 95% of DOX is released during 7-9 days. There is an induction period of ca two days during which the DOX release was very low (< 2%), which shows that the shell is substantially free of DOX.

Figure 13 shows a comparison of mitochondrial functions of macrophages and breast cancer cells exposed to DOX@ZIF-8, a mixture of ZIF-8 and DOX (ZIF-8+DOX), pure ZIF-8 and free DOX.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a metal-organic framework (MOF) with an encapsulated target-molecule, characterized in that the MOF has a core-shell structure, wherein the core comprises the MOF and the encapsulated target-molecule, and the shell comprises the MOF and is substantially free of target-molecules.

Throughout the present description the term "shell" is used for the peripheral shell that surrounds the core in a core-shell structure. In the core-shell structure according to the present invention both the core and the shell contain the MOF. The core of the MOF further comprises one or more target molecules. The term "target-molecule", as used herein, denotes a molecule, material or a particle that is encapsulated in a MOF. The target-molecule for encapsulation in a MOF in the process according to the present invention may be a small organic molecule, such as: a drug, for example an anti-cancer drug; a dye, such as rhodamine B, methyl orange, methylene blue, or a mixture thereof; a vitamin; or a flavour. The target-molecule may also be a biomacromolecule, such as: a protein or DNA; an organometallic catalyst; a magnetic particle; an inorganic material, such as graphene or carbon nanotube; or a metal nanoparticle. In a specific embodiment, the target-molecule is an organic molecule, such as small organic molecule or a biomacromolecule.

MOFs can be porous and may contain intrinsic pores. The shell of the MOF according to the present invention is substantially free from pores equal to or larger than the encapsulated target-molecules, or pores larger than 1 nm. Preferably, the shell does not contain pores in the range 1-100 nm. More preferably, the shell does not contain pores in the range 1-50 nm, or 2-50 nm. The term "substantially free of target-molecules" intends to mean that <2 % of the total weight of the encapsulated target-molecules are encapsulated in the shell of the MOF according to the present invention. A MOF with a shell according to the present invention, enables the safe storage of the encapsulated target-molecule under physiological conditions and an induction release period in acidic media. The release of the encapsulated target-molecules is associated with the decomposition of the MOF at low pH, and especially by first decomposition of the shell. The shell preferably has a thickness that is at least twice the size of intrinsic pores of the MOF. Preferably, the shell has a thickness of at least 1 nm, at least 2 nm, at least 5 nm, at least 10 nm, or at least 15 nm. If the thickness is smaller than the intrinsic pores, the encapsulated molecules may diffuse out through the pores. The upper limit for the thickness of the shell depends on the size of the encapsulated target-molecule. The thickness of the shell may be up to 20% of the diameter of the MOF crystal. Crystals of MOF with encapsulated target-molecules according to the present invention may have a diameter in the range of 30-2000 nm, 30-500 nm, 50-500 nm, or 70-300 nm. The specific surface area (BET area) may be at least 500 m ² /g, or at least 1000 m ² /g-

The present invention also provides a process for the preparation of a metal-organic framework (MOF) with encapsulated target-molecule, comprising the steps of: a) mixing a target-molecule with a metal salt in a suitable solvent and adjusting the pH of the solution to a value between 6 and 12;

b) adding a solution of organic linkers; and

c) collecting the MOF crystals formed.

With the process according to the present invention it is possible to prepare a MOF comprising a core comprising the MOF and the encapsulated target-molecule and a shell comprising the MOF and that is substantially free of the target-molecules. The preparation is made as a one-pot process, which permits for less use of solvents and less amounts of waste.

The target molecule and metal salt are both soluble in the suitable solvent used for mixing in step (a). Suitable solvents may be water, methanol, dimethylformamide (DMF), ethyl acetate, diethyl ether, acetone acetonitrile, dimethyl sulfoxide (DMSO), and tetrahydrofuran (THF), or a mixture of one or more of these. The solvent is preferably selected from water and methanol, more preferably water. Performing step (a) at a pH of 6-12, or preferably a pH of 7-11, or a pH of 8-11, promotes the interaction between the target-molecules and the metal ions prior to the formation of the MOF crystals. The pH may be adjusted by a suitable base; such as NaOH, triethylamine (TEA), piperidine, ethanolamine, and sodium formate; preferably NaOH or triethylamine.

By mixing the target-molecule and the metal salt before the solution of organic linkers is added according to the present invention the loading of one or more types of target- molecules can be controlled by using the interactions between target-molecules and metal ions in the steps (a) to (b). Step (a) is preferably performed for at least 1 minute, at least 2, at least 5, or at least 10 minutes. Further, the time for step (a) is preferably not longer than 30 minutes, or 1 hour. The time allowed for the reaction of target- molecule, the metal salt and the organic linker in step (b) affects the thickness of the shell. The target-molecule, metal salt and organic linkers should be allowed to react for at least 1 minute, or at least 2, at least 5, at least 10, or at least 15 minutes, in step (b). The reaction time provides a possibility to adjust the thickness of the shell of the resulting MOF crystal. This enables control of the release of the encapsulated target- molecule. The time for step (b) is preferably not longer than 45 minutes, or 30 minutes. By using a very short reaction time it may be possible to prepare a MOF without a shell. The metal ions and organic linkers may be present in excess amount so that the crystallisation of the MOF can still occur even after the target-molecules have been depleted and a shell can continue to be formed. In a MOF according to the present invention, the encapsulated target-molecules are expected to be homogeneously distributed within the core of the MOF, while the shell is substantially free from target- molecules. An advantage with a shell that is substantially free from target-molecules is that the initial release of the target-molecules can be delayed, such that an induction period may be obtained even though the surrounding conditions are kept constant, for example without changing the pH. This is because the shell, which surrounds the core, should decompose before the encapsulated target-molecules may be released from the core.

The process according to the present invention provides for an easy preparation and enables easy control of the process as well as loading of target-molecules into the framework. The loading amount can be tuned by the concentration of the target- molecules in the solution used in the process. The loading of the target-molecule may be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10 %, or at least 15%, of the total weight of the metal-organic framework with the encapsulated target- molecule. The loading of a target-molecule is suitably not more than 50% of the total weight of the metal-organic framework with encapsulated target-molecule. The loading of an organic target-molecule is suitably not more than 35% of the total weight of the metal-organic framework with encapsulated target-molecule. High loadings, for example at least 5 wt%, or at least 10 wt%, of the target-molecule enables homogeneous distribution of the target-molecules within the core of the MOF. Another advantage with the method according to the present invention is that it facilitates encapsulation of large molecules in metal-organic frameworks. Further, the process according to the present invention also makes it possible to obtain multi-functionalized materials by encapsulation of two or more target-molecules in a one-pot process. If two or more different target-molecules are encapsulated, they may be chosen to have an activity that is either complementary to or is different from each other, which enables the production of a material with tailored properties. The target- molecules that may be encapsulated in a MOF according to the present invention may have different functionalities and may be selected from organic molecules, magnetic nanoparticles, quantum dots, metal oxides, biomolecules and enzymes. Thus, the process according to the present invention enables construction of multi-functional delivery systems for a wide range of applications by adding target-molecules with different functionalities in step (a). This opens new opportunities in developing multifunctional materials. The materials obtained by the process according to the present invention may be used for controlled and targeted release of drugs and other molecules; as heterogeneous catalysts; for gas separation, for example C0 ₂ separations; removal of organic pollutants; bio-imaging; sensing, such as in biosensors, etc. The synthesis is simple, green and scalable, and has strong potential for industrial applications.

The process according to the present invention also enables control of the mesoporosity of the final products. Mesopores typically have a diameter of from 2 to 50 nm. Removal of encapsulated target-molecules from a metal-organic framework prepared according to the present invention provides for the introduction of homogeneously distributed mesopores in the MOF. When encapsulated target-molecules that are homogeneously distributed in the core of a MOF are removed, the mesopores thus induced may be homogeneously distributed within said core. Removal of encapsulated target-molecules can be made after step (c) in the process according to the present invention, for example by dispersing MOF with encapsulated target-molecules in ethanol solution, and reflux for 0.5-2 h. Removal of the encapsulated target-molecules provides a MOF material with a hierarchical porosity, which can improve the diffusion and mass transfer properties. Thus, one embodiment of the present invention is a metal-organic framework with hierarchical porosity prepared by the process for preparing a MOF with an encapsulated molecule according to the present invention, and further comprising a step of removing the encapsulated target-molecules from the obtained MOF crystals. The term "hierarchical porosity", as used herein, denotes a porous system comprising i) pores that are intrinsic in a MOF, and ii) pores that are induced in the MOF by the target- molecule or by a subsequent removal of the target-molecule. The induced pores may occur only in the core of the MOF, while the shell is substantially free from induced pores. Intrinsic pores of MOFs are ordered and typically have a diameter in the range of 0.3 - 2.0 nm. The size of the induced pores that are introduced in the MOF can be tuned by the loading amount of encapsulated target-molecule. Removal of an encapsulated target-molecule in a process according to the present invention may induce pores with a diameter of 1-100 nm, 1-50 nm, 2-50 nm, or 10-20 nm. Thus, in a MOF having hierarchical porosity according to the present invention, the core may have pores induced by the target-molecules, which pores may have a diameter of 1-100 nm, 1-50 nm, 2-50 nm, or 10-20 nm while the shell is substantially free from such pores. Preferably, <1 % of the amount of the pores with a diameter of 1-100 nm, 1-50 nm, 2- 50 nm, or 10-20 nm occur in the shell. The induced pores may be interconnected. Further, the induced pores can be accessible for other molecules.

The collection of the MOF crystals in step (c) of the process according to the present invention may be performed by conventional methods, such as centrifugal separation, filtration and freeze-drying. The present invention further provides a metal-organic framework with encapsulated target-molecule, obtained by the process according to the present invention.

Suitable metal salts for use in the process according to the present invention comprises metal ions selected from the group consisting of lUPAC Groups 3 and 6-15, and combinations thereof. Preferably, the metal ions are selected from tetrahedrally- coordinated transition metal ions, such as iron, cobalt, copper and zinc, and their metal complexes; more preferably cobalt or zinc. Typical salt-forming anions are selected from the group consisting of acetate, sulphate, nitrate, phosphate, sulphide, halides, sulphite, carbonate and citrate. Specific examples of suitable metal salts are selected from ZnCI ₂ , Zn(CH ₃ COO) ₂ , ZnS0 ₄ , Zn(N0 ₃ ) ₂ , Zn ₃ (P0 ₄ ) ₂ or CoCI ₂ , Co(CH ₃ COO) ₂ , CoS0 ₄ , Co(N0 ₃ ) ₂ ; nitrate salts of cobalt and zinc are preferred.

The organic linker may be selected from a compound of formula (I), (II), (III), or any combination of the connection structure:

wherein

A ¹ , A ² , A ³ and A ⁴ are each independently selected from C, N, P and B, and

when A ¹ is N, P or B, then R ⁵ is absent, and

when A ⁴ is N, P or B, then R ⁸ is absent;

A ⁵ , A ⁶ and A ⁷ are each independently selected from C and N, and

when A ⁶ is N, then R ¹¹ is absent, and

when A ⁷ is N, then R ¹² is absent;

R ¹ , and R ⁴ and R ⁹ each individually comprise an electron withdrawing, or weakly electron donating, and non-sterically hindering group that does not interfere with transition metals that may become linked to any or both of the nitrogen atoms adjacent to the carbon atom in the ring to which R ¹ , and R ⁴ or R ⁹ , respectively, is attached;

R ² , R ³ , R ⁶ , and R ⁷ are each independently selected from electron withdrawing or weakly electron donating groups, such as hydrogen, Ci-6 alkyl, halo, cyano and nitro; R ¹⁰ is an electron withdrawing or a weakly electron donating group, such as a hydrogen, Ci-6 alkyl, halo, cyano or nitro;

when one or both of A ¹ and A ⁴ are C, then R ⁵ and R ⁸ are each independently selected from hydrogen, Ci-6 alkyl, halo, cyano and nitro;

when A ⁶ is C, then R ¹¹ is an electron withdrawing or a weakly electron donating group, such as a hydrogen, Ci-6 alkyl, halo, cyano or nitro;

when A ⁷ is C, then R ¹² is an electron withdrawing or a weakly electron donating group, such as a hydrogen, Ci-6 alkyl, halo, cyano or nitro. Preferably, A ¹ , A ² , A ³ - A ⁴ , A ⁵ , A ⁶ and A ⁷ are each independently selected from C and N, and

when A ¹ is N, then R ⁵ is absent,

when A ⁴ is N, then R ⁸ is absent,

when A ⁶ is N, then R ¹¹ is absent, and

when A ⁷ is N, then R ¹² is absent;

R ¹ , R ⁴ , and R ⁹ each individually are selected from hydrogen, Ci-6 alkyl, formyl, halo, cyano and nitro;

R ² , R ³ , R ⁶ , and R ⁷ are each independently selected from hydrogen, Ci-6 alkyl, halo, cyano and nitro;

R ⁵ and R ⁸ are each independently selected from hydrogen, Ci-6 alkyl, halo, cyano and nitro;

R ¹⁰ is hydrogen, Ci-6 alkyl, halo, cyano or nitro;

R ¹¹ is hydrogen, Ci-6 alkyl, halo, cyano or nitro; and

R ¹² is hydrogen, Ci-6 alkyl, halo, cyano or nitro.

More specifically, the organic linker may be selected from a compound of formula (I) or (II), wherein

A ¹ , A ² , A ³ and A ⁴ are C;

R ¹ is hydrogen, methyl or formyl;

R ² and R ³ are hydrogen; R ⁴ is hydrogen or amino; and

R ⁵ , R ⁶ , R ⁷ , and R ⁸ are hydrogen.

Preferably, the organic linker is selected from the group consisting of 2-methylimidazole, imidazole, 2-aminobenzimidazole, benzimidazole and carboxyaldehyde-2-imidazole.

The MOF prepared with the process according to the present invention may be a zeolitic imidazolate framework (ZIF), HKUST-1, MOF-5, MIL-53, MIL-101, UIO-66, NOTT-400, ZnBT or MOP-1. Preferably, the MOF is a ZIF. Zeolitic imidazolate frameworks (ZIFs) have the advantage that they possess high thermal and hydrothermal stabilities. Examples of zeolitic imidazolate frameworks are ZIF-1, ZIF-2, ZIF-3, ZIF-4, ZIF-5, ZIF-6, ZIF-7, ZIF-8, ZIF- 9, ZIF-10, ZIF-11, ZIF-12, ZIF-14, ZIF-20, ZIF-21, ZIF-22, ZIF-23, ZIF-60, ZIF-61, ZIF-62, ZIF- 64, ZIF-65, ZIF-67, ZIF-68. ZIF-69, ZIF-70, ZIF-71, ZIF-72, ZIF-73, ZIF-74, ZIF-75, ZIF-76 and ZIF-77. Preferably, the ZIFs are selected from ZIF-8, ZIF-67 and ZIF-70. ZIFs may be used in applications for gas separation, and as carriers for metal nanoparticles and drugs. Preferably, the metal-organic framework in the present invention is ZIF-8. Among the ZIFs, ZIF-8, built from zinc ion and 2-methylimidazolate, has the further advantage that it is non-toxic and biocompatible. ZIF-8 is also stable under physiological conditions (pH = 7.4) and decomposes under acidic conditions, which can be used to construct pH responsive drug delivery systems.

The present invention provides use of a metal-organic framework (MOF) with encapsulated target-molecule the materials according to the present invention, for controlled and targeted release of drugs and other molecules, as heterogeneous catalysts, for gas separation, removal of organic pollutants, bio imaging, or sensing. More specific, the present invention provides use of a metal-organic framework (MOF) with encapsulated target-molecule for drug delivery in cancer therapy via pH responsive release. The present invention thus provides for a method for drug delivery in cancer therapy by using a metal-organic framework (MOF) with encapsulated target-molecule. A pH-responsive drug delivery system enables a drug delivery system with no release of drugs under physiological condition in circulation. This is especially important for anticancer drugs, where no release is required during systemic circulation, but where controlled release of the drugs will be allowed after intracellular uptake of the systems in the cancer cells. Such a system would reduce systemic side effects during chemotherapy, as the active components are released only in the tumour region and not in general circulations.

A specific example of a target-molecule for encapsulation in a MOF according to the present invention is an anti-cancer drug, such as anti-cancer drugs selected from the group consisting of doxorubicin (DOX), daunorubicin, mitoxantrone, methotrexate, idarubicin, 5-fluorouracil, epirubicin, aclarubicin, pirarubicin, duazomycin, duazomycin, mitomycin, medorubicin, rodorubicin and bleomycin. Preferred anti-cancer drugs are selected from the group consisting of DOX, daunorubicin, mitoxantrone, methotrexate, and 5-fluorouracil. A more preferred anti-cancer drug for use in the present invention is DOX. DOX, daunorubicin, mitoxantrone, methotrexate, and 5-fluorouracil molecules have functional groups that form weak coordination bonds with Zn ²⁺ ions in aqueous medium. A metal-organic framework with encapsulated target-molecules according to the present invention is DOX@ZIF-8. DOX@ZIF-8 according to the present invention may have a DOX loading of at least 1 wt%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10 %, or at least 15 wt%, based on the total weight of the loaded MOF. Since ZIF-8 is non-toxic, biocompatible and stable under physiological conditions while it decomposes under acidic conditions, ZIF-8 with encapsulated doxorubicin ("DOX@ZIF- 8") can be utilised as an efficient drug delivery vehicle in cancer therapy via pH responsive release. DOX@ZIF-8 according to the present invention shows a higher efficacy on three different breast cancer cell lines compared to (non-encapsulated) doxorubicin.

Another important advantage with metal-organic frameworks with encapsulated target- molecules prepared according to the present invention is that there is virtually no release of the encapsulated target-molecules at high pH and a slower release of the target-molecules at low pH compared with other metal-organic frameworks with encapsulated target-molecules. As a specific example, the drug release for DOX@ZIF-8 is associated with the decomposition of ZIF-8 at low pH values. One important advantage is that DOX molecules are safely stored in DOX@ZIF-8 carriers prepared according to the present invention. After 15 days at a pH > 6.5, DOX@ZIF-8 prepared with the method according to the present invention only releases < 1% doxorubicin of the total amount of encapsulated doxorubicin. Decomposition of the drug-free shell of ZIF-8, which acts as a protective capsule for the drug, provides for an induction period during which the DOX release may be very low (< 1%). This unique release property of DOX@ZIF-8 may be used in a pH-responsive drug delivery system in cancer therapy. The DOX molecules may be retained in the ZIF-8 crystals during the circulation in blood stream (physiological conditions, pH ~ 7.4) and slowly released after its accumulation in tumour cells where the pH values are even lower at intracellular organelles (pH = 5-6). In general, organic dyes undergo photo-bleaching or fading due to the photochemical alteration of the dye molecules. This process leads to that the light emission capacity of the dye molecules decreases with time. An advantage with encapsulation of dyes in a MOF according to the present invention is that it increases the rigidity of the dye which significantly increases the lifetime of the dye molecules. The increase of the long lifetime by encapsulation of dye molecules in a MOF is important for tissue and biological applications involving fluorescence energy transfer, for example fluorescence lifetime imaging microscopy (FILM) and lifetime biosensors, where auto-fluorescence precludes the imaging. The dye molecules encapsulated in a MOF according to the present invention are protected by the MOF and may be insensitive to surrounding environment such as water vapor, oxygen, C0 ₂ , and others gases. It may also be less sensitive to the viscosity and polarity of solvents. Dye molecules encapsulated in a MOF according to the present invention may be used in solid state dyes (SSD), laser dyes, and as a probe for sensors. The synthesis used in the process according to the present invention is schematically shown in Figure 1. Metal ions and target-molecules (a) self-assemble into coordination polymers (b) in an aqueous solution with a pH value between 6 and 12. Organic linkers disassemble the metal ions from target-molecules and assemble the metal ions with the linkers into hierarchical MOFs (c). The resulting crystals consist of a core with isolated and homogeneously distributed target-molecules within a MOF, and a shell comprising MOF and that is substantially free of target-molecules. The loading amount, the thickness of the shell, and the hierarchical porosity of the crystals can be controlled by the concentration of the target-molecules in the solution.

EXAMPLES

Materials

The tested organic linker is 2-methylimidazole, and the metal ions are Zn ²⁺ and Co ²⁺ resulting in ZIF-8 and ZIF-67, respectively. Four different target-molecules are used: doxorubicin (DOX), rhodamine B (RhB), methyl orange (MO), and methylene blue (MB).

Chemicals and characterizations

The following reagents were purchased and used without purification: water, fetal bovine serum (FBS), phosphate- buffered saline (PBS), sodium dodecyl sulphate (SDS), polyvinylpyrrolidone (PVP), L-ascorbic acid, rhodamine B, methyl orange, methylene blue, 3-(4,5-dimethylthiazol-2-yl))-2,5-diphenyl tetrazolium bromide (MTT, Sigma- Aldrich), 4',6-diamidino-2-phenylindole (DAPI, Eugene, OR), DOX (Yinghuan Chempharm), Ζη(Νθ3)2·6Η2θ„ Co(N03)2-6H20, 2-methylimidazole (2-mim), and triethylamine (TEA), were purchased from Sigma Aldrich (Germany).

The PXRD patterns were collected at a speed of 3° (20)/min on a PANalytical X'Pert Pro diffracto meter equipped with a pixel detector using Cu Καΐ (λ = 1.5406 A) radiation. The morphology of DOX@ZIF-8 was observed by SEM (JEOL JSM-7401F) at an accelerating voltage of 2.0 kV. The HRTEM images were taken on a JEOL JEM-2100LaB6 microscope operating at 200 kV. The particle size distribution of DOX@ZIF-8 was determined by dynamic light scattering (DLS, Malvern Zetasizer Nano Series). The N2 adsorption- desorption isotherm was recorded at 77 K on a Micromeritics ASAP2020 analyzer. The MTT results, fluorescence intensity and protein quantification were obtained using a BioTek Synergy™ MX multi-mode micro plate reader operated by the Gen5™ software. The 570 nm wavelength was set in the UV/Vis absorbance model to determine the MTT reading, and 562 nm was set for the protein quantification. The fluorescence intensity was measured at excitation/emission wavelengths of 494/516 nm. The localizations of DOX and DOX@Z!F-8 were observed by confocal laser scanning microscopy (CLSM, Olympus FV1000) at an excitation/emission wavelengths of 405/461 nm for DAPI and 559/572 nm for DOX.

Example 1

Synthesis of DOX@ZIF-8. DOX@ZIF-8 crystals were synthesised in pure aqueous solutions. First 0.2 g of Zn(N03) ₂ -6H ₂ 0 (0.66 mmol) was dissolved in 0.8 g of deionized water, and the pH of the solution was adjusted to pH= 8 using a NaOH solution. Then 4 mL of DOX stock solution (0-10 mg/ml) were added and stirred for 1 min. Afterwards 10 g of a solution containing 2 g of 2-methylimidazole (2-mim, 24.36 mmol) as organic linker and 8 g of deionized water was added dropwise. The reaction mixture was stirred for 15 min at room temperature, and DOX@ZIF-8 crystals were collected by centrifugal separation (13000 rmp, 30 min) and washed at least three times with a mixture of ethanol and water. The powder product was dried at room temperature under vacuum. The loading amount of DOX was tuned by changing the concentration of the DOX stock solution. For comparison, a DOX-free ZIF-8 was synthesised in a similar way, using 4 ml of deionised H ₂ 0. See Table 1 for detailed descriptions. DOX@ZIF-8 crystals with 0, 4, 14 and 20 wt% loadings were achieved (Figure 2a-d), per weight of MOF with encapsulated target-molecule DOX.

DOX molecules were coordinated with Zn ²⁺ ions by examining the red shift of the UV-Vis spectrum, and comparing it with that of a free DOX solution, while the solid state UV- Vis spectrum of DOX@ZIF-8 showed that there was no coordination bond between DOX and Zn ²⁺ ions in DOX@ZIF-8 (Figure 3). Table 1 Synthesis conditions of ZIF-8 encapsulated with different target-molecules.

determined by UV-Vis spectroscopy from the concentration of target-molecules after the target- molecule@ZIF-8 materials have been completely dissolved in an acidic medium.

Characterization of DOX@ZIF-8 with SEM, TEM, PXRD, and dynamic light scattering.

The DOX@ZIF-8 materials prepared according Example 1 consisted of isolated crack-free crystals of diameter 70-300 nm as shown with scanning electron microscopy (SEM) (Figure 2(a)). Powder X-ray diffraction (PXRD) showed that the DOX@ZIF-8 nanocrystals were of high crystallinity with sharp diffraction peaks (Figure 2(b)). The DOX molecules were well dispersed, as there were no diffraction peaks from the free DOX molecules. Target-molecules were successfully loaded into the ZIF nanocrystals, as indicated by the colour of the DOX@ZIF-8 nanocrystals becoming more intense with higher DOX concentration (Figure 2(c)). Transmission electron microscopy (TEM) images were collected by tilting the crystal along two perpendicular axes in steps of 1° per step, covering angular range of 118° and 125°, respectively. The two series of TEM images obtained in this way were then used for tomographic reconstruction. TEM shows that the morphology of ZIF-8 nanocrystals did not change with increasing loading of DOX (from 0% to 20% loading) (Figure 2(d)) and that the presence of well-defined mesopores that were homogeneously distributed within the ZIF-8 nanocrystals when the loadings were high (≥ 14%). The size of the mesopores increased with the loading of DOX. Each ZIF-8 particle is a single crystal as confirmed by electron diffraction (Figure 4). Each ZIF-8 nanocrystal consisted of a core with isolated and homogeneously distributed mesopores and a mesopore-free shell as shown by electron tomography (Figure 5). For DOX@ZIF-8 with 20% loadings, the diameter of the mesopores, in which the DOX molecules were located, was 5-15 nm. The mesopore-free shell was approximately 20 nm thick, which was substantially free of DOX molecules. The particle size increases and the size distribution broadened (from 100 ± 30 nm to 230 ± 150 nm, respectively) with increased loading of DOX, as shown by dynamic light scattering (DLS, Figure 6, 0% DOX (a), 4% DOX (b), 14% DOX (c), and 20% DOX (d)). All these nanoparticles were highly dispersed and stable in water and PBS (pH 7.4), as a result of their high zeta-potentials (+30.5, +30.1, +30.7 and +31.1 mV for 0, 4, 14 and 20 wt% loadings, respectively). Example 2

Synthesis of dye@ZIF-8. The synthesis of ZIF-8 nanocrystals into which rhodamine B, methyl orange or methylene blue had been encapsulated was performed in a similar way as for Example 1, using 4 ml of stock solution of the respective dye. Stock solutions (10 mg/ml) of rhodamine B, methyl orange, and methylene blue, respectively, were prepared in deionized H ₂ 0. The same process was used as that for Example 1, with the 4 ml of stock solution of DOX replaced by 4 ml of stock solution of the respective dye. The amount of loading was determined by UV-Vis spectroscopy. The target- molecules@ZIF-8 nanocrystals were dissolved in a nitric acid solution, and the concentration of the target-molecules was determined by UV-Vis spectrophotometry (Perkin Elmer Lambda 19 UV-Vis-NIR spectrometer). The loading amount was determined from the UV-Vis absorbance at 479 nm for DOX, 554 nm for rhodamine B, 460 nm for methyl orange and 665 nm for methylene blue. The loadings of rhodamine B, methyl orange and methylene blue, were 15, 14 and 17%, respectively (Table 1, Figures 7 and 8). Example 3

Synthesis of DOX@ZIF-67. Hierarchical micro-/rneso-structured DOX@ZIF-67 material with 15% DOX loading was synthesised in aqueous solution (Figure 9). 1.77 g of cobalt nitrate hexahydrate (6 mmol) was dissolved in 30 ml of deionized water. 15 ml of stock solution (10 mg/ml DOX) was added into cobalt nitrate aqueous solution. The pH was adjusted to 8 using a NaOH solution. 1 g of 2-methylimidazole (12.18 mmol) was added to 30 ml of deionized water under stirring, during which 5.7 g of ammonium hydroxide (28-30% aqueous solution) was added. The cobalt nitrate aqueous solution was then added to the 2-methylimidazole solution, and the mixture immediately became a milky suspension. The precipitate was collected by centrifugation (13000 rmp, 30 min) and washed at least three times with a mixture of ethanol and H ₂ 0. The powder product was dried at room temperature under vacuum.

Example 4

Several components encapsulated in ZIF-8. Pd nanoparticles of diameter 10 nm stabilised with polyvinylpyrrolidone (PVP) were synthesised following a previously published method by Jin, et al., (2011). Briefly, 8.0 ml of aqueous solution containing 105 mg PVP (Mw=55,000), 60 mg of L-ascorbic acid and 300 mg of KBr in a 20-ml vial was heated for 10 min at 80 °C under magnetic stirring. Subsequently, 3.0 ml of an aqueous solution of Na ₂ PdCl4 (57 mg) was added with a pipette. The reaction was allowed to continue at 80 °C for 3 h. The product was collected by centrifugation, washed three times with water and dispersed in 10 ml of water. Typically, a solution of 0.2 g of (0.66 mmol) Ζη(Ν0 ₃ ) ₂ ·6Η ₂ 0, 4 ml of (10 mg/ml) DOX/rhodamine B solution and 1 ml stock solution of Pd nanoparticles was prepared. The pH was adjusted to 8 using a NaOH solution. After a short time of stirring (1 min), a 10 g solution containing 2 g (24.36 mmol) 2-mim and 8 g deionized H ₂ 0 was added dropwise. The reaction mixture was stirred for 15 minutes. The precipitate was collected by centrifugal separation and washed at least three times with a mixture of ethanol and H ₂ 0. The powder product was dried at room temperature under vacuum. The samples in which DOX and rhodamine B are the target-molecules are denoted as (Pd&DOX)@ZIF-8 and (Pd&rhodamine B)@ZIF- 8, respectively. Both mesopores and Pd nanoparticles were observed in the same ZIF nanoparticle (Figure 10).

Example 5

Synthesis of dye@ZIF-8 using triethylamine (TEA) as a base. 0.2 g of Ζη(Ν0 ₃ ) ₂ ·6Η ₂ 0 (1 mmol) was dissolved in 0.8 g of deionized water in a glass vial. A certain amount of TEA (0.003, 0.1, 1, 2 ml corresponding to 0.02, 0.7, 7, 14 mmol, respectively) was added to the Zn ²⁺ solution. The pH of the Zn ²⁺ solution was increased from ca 5.9-6.9 to 10.4 after the addition of TEA. A white precipitate of ZnO was observed. A stock solution (10 mg/ml) of rhodamine B was prepared in deionized H ₂ 0. A certain volume (1, 4, 5, 10, 15 ml) of the rhodamine B stock solution (corresponding to 2, 8, 10, 20 and 30 μιηοΙ rhodamine B, respectively) was added to the previous Zn ²⁺ solution. Then, a solution containing 2 g (24.69 mmol) of 2-mim and 8 g of deionized H ₂ 0 was added. The reaction vial was stirred for 30 min at room temperature. The particles were collected using centrifugation (13000 rmp, 30 min). The product was washed several times by water, and ethanol (2x40 ml). TEM images showed mesopores in RhB@ZIF-8. The fluorescence lifetime of the rhodamine B after encapsulation was measured using a Varian Cary Eclipse Fluorescence spectrophotometer at room temperature with an excitation wavelength of 500 nm and observing the fluorescence emission at 750, 620, 620, 600 nm. The lifetime (τ) of a fluorophore (e.g. dye molecules) represents an average value of time spent at the excited state. The lifetime of the Rhodamine B after encapsulation increased 2-27 folds compared to free Rhodamine B.

Example 6

Extraction of DOX from DOX@ZIF-8. 0.1 g of DOX@ZIF-8 was dispersed in 100 ml of ethanol solution (H ₂ 0:ethanol (v/v)=l:l), and the mixture was refluxed at 85 °C for 2h. The solid was recovered by centrifugation, washed with ethanol, and dried. The above extraction procedure was repeated four times. A hierarchical micro- and meso- porous ZIF-8 material was provided. The BET area is 1245 m ² /g (Figure 11). Example 7

Release of DOX from DOX@ZIF-8 nanocrystals at different pH. The potential of DOX@ZI F-8 as a drug delivery system for ca ncer therapy was studied. A typical release system was prepared by suspending 10 mg of DOX@ZI F-8 nanoparticles into 20.0 ml of pH 5.0 to 7.4 buffer solutions with 10% (v/v) fetal bovine serum (FBS). (pH 7.4, 6.5, 6.0, 5.0 and 4.0, respectively). Then the release system was kept at 37 °C under shaking (shaking frequency = 150 rpm). 1 ml of release medium was sampled at each time point and UV/Vis spectrophotometry was used to determine the percentage of DOX that had been release according to the formula,

Release percentage (%) = m _r /mi,

where m _r is the amount of released DOX while mi is the total amount of loaded DOX. After the measurement the sample was returned to the original release system. As shown by the pH-responsive cumulative release profiles of DOX@ZI F-8 with 20% DOX loading in Figure 12 (a), there is virtually no release of DOX (< 1%) from DOX@ZI F-8 even after 15 days at pH 6.5 in phosphate- buffered saline (PBS) with 10% (v/v) fetal bovine serum (FBS) at 37 °C. At low pH that there was an induction period of about 2 days, during which the DOX release was very low (< 2%).

The process of transfer from the circulation in the bloodstream to the endosome and lysosome compartments was mimicked, DOX@ZI F-8 nanoparticles were tested in a stepped release experiment. 10 mg of DOX@ZI F-8 was added into 20.0 ml of 7.4 buffer solutions with 10% (v/v) FBS and kept at 37 °C for 7 days. The pH of the solution was then adjusted to 6.5 with dilute HCI (0.6 M) and kept for another 7 days. The pH was again adjusted stepwise over 3 days to 6.0, 5.5 and 5.0 by adding dilute HCI (0.6 M). The adding time of acid is indicated by a rrows in Figure 12(b). The amount of loading was determined from the UV-Vis absorbance at 479 nm for DOX. From pH 7.4 to 6.5, the cumulated release of DOX remained very low (< 1%). When the pH value further decreased to 6.0, 5.5 and finally 5.0, a sharp increase of the released DOX was observed (Figure 12(b)). Results in Figure 12 (a) and (b) are presented as mean ± standard deviation (s.d.) (n=3). Free DOX completely dissolved in less than an hour at 37 °C in PBS at the pH range of 5.0-7.4 (Figure 12c).

Example 8

Cytotoxicity tests

The cytotoxicities of DOX@ZI F-8, free DOX, pure ZI F-8, and a mixture of free DOX and ZI F-8 were evaluated by determining the cellular viability using an MTT ((3-(4,5,- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, which measures the mitochondrial function of the cells. The comparison was made with the same concentrations of DOX and ZI F-8. For example 1 μg/ml DOX@ZI F-8 contains 0.8 μg of ZI F-8 and 0.2 μg of DOX, and is compared with 0.8 μg/ml ZI F-8, 0.2 μg/ml DOX, and a solution containing 0.8 μg/ml ZI F-8 and 0.2 μg/ml DOX. ZnO 100 μg/ml was used as a positive control for cell death in the primary macrophage experiments.

Human Cell lines. Human breast cancer cell lines, MCF-7, MDA-MB-231, and MDA-MB- 468 were purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U m l penicillin and 100 mg/ml streptomycin at 37 °C with 5% C0 ₂ in the atmosphere. The medium was changed twice a week. The cells were harvested with trypsin.

Apoptosis/Necrosis analysis. Apoptosis/necrosis of the cells was analysed by fluorescence activated cell sorting (FACS) with fluorescein isothiocyanate (FITC) Annexin V/Dead cell apoptosis kit staining. Briefly, the macrophages and breast cancer cells were seeded onto 96 well plates at a concentration of lxlO ⁶ cells/well a nd treated with PBS (control), 0.5 μg/ml DOX@ZIF-8, 0.4 μg/ml ZIF, 0.1 μg/ml DOX or a mixture of 0.4 μg/ml ZI F and 0.1 μg/ml DOX respectively for the desired time period (12 and 24 h for the macrophages, 24 and 48 h for the cell lines). Both the medium and the ha rvested cells were collected by centrifugation at 2000 rpm for 5 min and washed twice. The cells were suspended in a 100 μΙ lx annexin-binding buffer and stained with 5 μΙ of FITC-labeled Annexin V and 1 μΙ of 100 μg/ml propidium iodide (PI) at room temperature for 15 min. Then 400 μΙ of lx annexin-binding buffer was added, and the cells were analysed using a BD LSRFortessa flow cytometer with FITC and PETexas Red channel (detect PI). The data was analysed with the FCS Express 4 software.

Study of the uptake of ZIF-8 nanoparticles by TEM. The cells were fixed in 2.5% glutaraldehyde in a 0.1 M phosphate buffer (pH 7.4) at room temperature and transferred to an Eppendorf tube to be further fixed overnight in a refrigerator. After the fixation, the cells were rinsed with a 0.1 M phosphate buffer and centrifuged. The cell pellets were postfixed in 2% osmium tetroxide in a 0.1 M phosphate buffer (pH 7.4) at 4 °C for 2 h, dehydrated in ethanol followed by acetone and embedded in LX-112 (Ladd, Burlington, Vermont, USA). Ultrathin sections (of thickness 50-60 nm) were cut by a Leica Ultracut UCT (Leica, Wien, Austria). Sections were contrasted with uranyl acetate followed by lead citrate and examined in a Tecnai 12 Spirit Bio TWIN transmission electron microscope (FEI Company, Eindhoven, Netherlands) at 100 kV. Digital images were recorded by using a Veleta camera (Olympus Soft Imaging Solutions, GmbH Munster, Germany).

Measurement of cellular Zn ²⁺ level. Breast cancer cells were seeded onto 24 well plates at a density of lxlO ⁶ cells per well and pre-cultured for 24 h. The medium was removed and refreshed with new DMEM medium containing PBS (control), 0.5 μg/ml DOX@ZIF- 8, 0.4 ng/ml ZIF-8, 0.1 ^ιηΙ DOX or (0.4+0.1) §/ιτιΙ ZIF-8+DOX separately. Four parallel wells were set for each sample concentration. After a 2 h incubation, the medium was discarded and cells were washed with PBS twice, before being loaded with 2 μΜ fluorescent zinc indicator FlouZinc-3 (with 0.2% Pluronic F127) and kept in the dark for 30 min. The cells were then washed twice and lysed in 280 μΙ of cell lysis buffer for 30 min. Cell lysates from each well were transferred into 96 well black plates to measure the fluorescence intensity (100 μΙ of cell lysates) and quantify the protein concentration with a bicinchoninic acid (BCA) protein assay (25 μΙ cell lysates) respectively. The fluorescence intensity was determined at excitation/emission wavelengths of 494/516 nm, and the protein concentration was measured by the absorbance at 562 nm. The relative cellular Zn ²⁺ fluorescence intensity was calculated as:

Relative Zn ²⁺ level = Sample's (Fluorescence Intensity/ Protein Mass)] xlOO/ Control's (Fluorescence Intensit / Protein Mass) (1) Statistical analysis. The MTT assays, relative cellular Zn ²⁺ level and apoptosis/necrosis analysis are presented as mean values with standard deviations (s.d.). Statistical data analysis was performed by ANOVA, followed by a post hoc test (Tukey HSD, alpha 0.05) using KaleidaGraph v4.1. The significance level was set to be ***p<0.001.

The effect of ZI F-8 nanocrystals on mitochondrial function of breast cancer cell lines MCF-7, MDA-MB-231, and MDA-MB-468 was tested. The ZI F-8 nanocrystals had been degraded for 24 h in PBS at pH = 5 and 37 °C with the incubation time of 3 and 11 days, respectively.

Comparative toxicity studies of DOX@ZI F-8, a mixture of ZI F-8 and free DOX (denoted ZI F-8+DOX ), pure ZI F-8 and free DOX on primary macrophages and three breast cancer cell lines (MCF-7, MDA-MB-231 and MDA- MB-468) treated for 24 h (Figure 13) were performed. Figure 13(a) presents the mitochondrial functions in the macrophages for 12 and 24 h, respectively. Figure 13(b) shows the mitochondrial functions in the breast cancer cell lines MCF-7, MDA-MB-231 and MDA-M B-468 for 24 h. Results are presented as mean ± s.d. (n=6), *** p<0.001 (p is the probability), compared to the negative control in Figure 13(a) and compared to other treatments in Figure 13(b). Dose dependent toxic effects of DOX@ZI F-8 in the macrophages (Figure 13(a)) as well as in the three breast cancer cell lines (Figure 13(b)) were observed. After treatment with 0.5 μg/ml of DOX@ZI F-8 (equivalent to 0.1 μg/ml of DOX) for 24 h, the mitochondrial function fell significantly from 100% of the baseline level to 43% in MCF-7, to 16% in MDA-MB-231, and to 20% in MDA-MB-468 cells (Figure 13(b)). After treating the cells with 1 ug/ml of DOX@ZI F-8, the mitochondrial function fell to less than 10% of the baseline level (Figure 13(b)).