FIELD OF THE INVENTION

The invention relates to hydrogen-bonded organic framework (HOF) and methods for producing the same.

BACKGROUND OF THE INVENTION

Hydrogen-bonded organic frameworks (HOFs) represent a new and emerging class of porous material that comprise frameworks of organic linkers interconnected through hydrogen-bonding interactions to form an open cage/scaffold-like structure that defines spatially ordered cavities. The unique size characteristics and spatial distribution of the cavities provide HOFs with a surface area in the order of thousands of square meters per gram.

HOFs, their properties and applications thereof remain largely unexplored. An opportunity therefore exists to research those materials with a mind to developing new products, methods and/or applications.

SUMMARY OF THE INVENTION

The present invention provides a method for producing a crystalline hydrogen-bonded organic framework (HOF) that encapsulates a bio-molecule, the method comprising combining in a solution the bio-molecule and a HOF precursor, wherein the HOF precursor self-assembles to form HOF that encapsulates the bio-molecule.

The invention stems from a surprising observation that HOFs can form in the presence of a bio-molecule resulting in the encapsulation of the bio-molecule within the HOF. Without wishing to be limited by theory, it is believed that hydrogen bonds forming between the constituting organic linkers of the HOF precursor can also form between the organic linkers and certain chemical sites of the bio-molecule.

The present invention can advantageously provide for a uniform distribution of bio molecules within the HOF framework, which in turn can enable the so formed HOFs to contain a large amount of encapsulated bio-molecules per unit mass or unit volume.

A diverse range of bio-molecules can be used according to the invention. In some embodiments, the bio-molecule is selected from a polyamino acid, a penicillin, a peptide, a protein, a nucleic acid, a nucleocapsid, and a combination thereof.

Those skilled in the art will appreciate that the bioactivity of bio-molecules such as proteins and nucleic acids is strongly related to their spatial conformation. Advantageously, encapsulation of a bio-molecule according to the invention can retain the native conformation of the bio-molecule. As a result, the encapsulated bio-molecule can maintain its native bioactivity. That is, the encapsulating framework advantageously provides a protective support for the bio-molecules. The protective capability of the framework is believed to derive from charge-based interactions between the framework and the guest bio molecule, resulting in significant enhancement of the bio-molecule stability.

The present invention also advantageously allows for encapsulation within a crystalline HOF of a bio-molecule irrespective of the relative dimension between intrinsic cavities of the HOF and the bio-molecule. Accordingly, in some embodiments the bio-molecule has a smallest dimension that is larger than the size of any intrinsic cavity of the HOF. For example, the method of the invention allows encapsulating within a crystalline HOF a bio molecule such as a peptide, a protein, a nucleic acid, or a nucleocapsid that is considerably larger than any intrinsic cavity of the framework. This approach can provide for unique HOFs to be used, the likes of which are precluded by post-synthesis infiltration methods.

The present invention therefore also provides a method of producing crystalline HOF having a framework that (i) defines intrinsic cavities, and (ii) encapsulates a bio-molecule, said method comprising combining in a solution the bio-molecule and a HOF precursor, wherein the bio-molecule has a smallest dimension that is larger than the size of any intrinsic cavity of the framework, and the HOF precursor self-assembles to form HOF that encapsulates the bio-molecule.

In some embodiments, the HOF precursor is a non-metallic precursor. That is, the HOF precursor does not contain any metal element in its chemical structure. Metal species (e.g. metal ions) may be potentially toxic in biologic environments. As such, the possibility to obtain a metal-free porous host guest system furthers opportunities for biotechnology applications. These may include for example the transport, storage and in vitro delivery of protein-based therapeutics, and obviating the cold chain in the transport and storage of vaccines for delivery to remote or underdeveloped locations.

The present invention also provides crystalline HOF having a framework that defines intrinsic cavities and encapsulates a bio-molecule.

In some embodiments, the bio-molecule has a smallest dimension that is larger than the size of any intrinsic cavity of the framework.

Accordingly, the invention also provides a crystalline HOF having a framework that (i) defines intrinsic cavities and (ii) encapsulates bio-molecule, wherein the bio-molecule has a smallest dimension that is larger than the size of any intrinsic cavity of the framework.

HOF formed according to the invention can be visualised as being a unique bio-composite in which the HOF forms a continuous host matrix phase within which guest bio-molecules are uniformly dispersed. As the biochemical characteristics of the encapsulated bio-molecule can advantageously be preserved, the HOF/bio-molecule systems of the invention can advantageously possess high bioactivity, exceptional protective abilities and trigger-release properties, and offer potential applicability in industrial-scale enzymatic catalysis, enzyme industrial remediation, drug-delivery systems, high sensitivity bioassays and biosensors. The HOF/bio-molecule systems of the invention may also find application in the medical field and research in general.

Further aspects and/or embodiments of the invention are discussed in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will be now described with reference to the following non limiting drawings, in which:

Figure 1 shows examples of moieties of the HOF precursor that form inter-molecular hydrogen bonds essential to the structural integrity of the HOF,

Figure 2 shows the geometry of typical hydrogen-bonding units assembled from organic linkers through multiple intermolecular hydrogen-bonds, serving as the building blocks for HOF construction,

Figure 3 shows an example HOF structure obtained using hexakis-[4-(2,4-diamino-l,3,5- triazin-6-yl) phenyl] benzene as HOF precursor,

Figure 4 shows the chemical structure of (a) tetrakis(4-carboxyphenyl)methane and (b) tetrakis (4-[aminomethaniminium]phenyl) methane and tetrakis (4-[aminomethaniminium] phenyl) silane HOF precursors used in the Examples to form C44 and CS144 HOFs.

Figure 5 shows PXRD patterns for CC44 and CS144 HOFs, and corresponding HOFs encapsulating the enzyme catalase (CAT), i.e. CAT@CC44, and CAT@CSi44,

Figure 6 shows confocal laser scanning micrographs showing the fluorescence, bright field, and overlay images of fluorescein tagged catalase (FCAT) encapsulated by CC44 HOF (FCAT@CC44), compared to samples obtained by adsorbing FCAT on the external surface of CC44 HOF (FCAT-on-CC44), Figure 7 shows the biological activity of (a) free FCAT and (b) FCAT@BioHOF-l in different pH conditions, Figure 8 shows the catalytic activity of catalase (CAT) before and after (a) exposure to DMF for 15 minutes, and (b) heat treatment at 70°C for 5 minutes,

Figure 9 shows the catalytic activity of catalase (CAT) encapsulated within CC44 HOF (CAT@CC44) before and after (a) exposure to DMF for 15 minutes, and (b) heat treatment at 70 °C for 5 minutes,

Figure 10 shows the enzymatic activity for free FCAT and FCAT@BioHOF-l treated at 60°C for 30 minutes, Figure 11 shows the relative activity (%) of free FCAT (bottom data) and FCAT@BioHOF- 1 (top data) as a function of incubation time at 60 °C,

Figure 12 shows the biological activity of (a) free FCAT and (b) FCAT@BioHOF-l after proteolytic agent treatment,

Figure 13 shows the relative activity (%) of free FCAT (bottom data) and FCAT@BioHOF- 1 (top data) as a function of incubation time in Trypsin in phosphate buffer,

Figure 14 shows the biological activity of (a) free FCAT and (b) FCAT@BioHOF-l after treatment with an unfolding agent in phosphate buffer,

Figure 15 shows simulated and experimental powder X-ray diffraction patterns of the as- synthesized FCAT@BioHOF-l and composites after testing their stability, Figure 16 shows the catalytic activity of (a) fluorescein-tagged alcohol oxidase (FAOx) and (b) FAOx @BioHOF-l after thermal treatment (60, 70, and 80 °C for 10 min) in water, Figure 17 shows the catalytic activity of (a) FAOx and (b) FAOx@BioHOF-l before and after proteolytic agent treatment in phosphate buffer, Figure 18 shows simulated and experimental PXRD patterns of the as-synthesized FAOx@BioHOF-l and composites after thermal, proteolytic agent treatment in phosphate buffer, or unfolding agent treatment in phosphate buffer,

Figure 19 shows the integrated intensity of the (100) diffraction peak of BioHOF-1 at 6.1 nm ¹ (I (q= 6.1 nm ¹ )) and Invariant calculated from Time-Resolved SAXS for the synthesis of BioHOF-1 and bovine serum albumin (BSA)@ BioHOF-1,

Figure 20 shows the biological activity of FCAT-on-BioHOF-1 before and after unfolding agent treatment in phosphate buffer,

Figure 21 shows the catalytic activity of FAOx-on-BioHOF-1 before (top data) and after (bottom data) unfolding agent treatment in phosphate buffer,

Figure 22 shows the catalytic activity of FAOx-on-BioHOF-1 before and after proteolytic agent treatment (trypsin (2 mg mL ¹ ) in phosphate buffer,

Figure 23 shows the catalytic activity of (a) catalase (CAT) and (b) CAT encapsulated within CC44 (CAT@CC44) before and after trypsin treatment, Figure 24 shows XRD patterns of (a) FITC-BSA@(4+2)HOF and (b) yeast@(4+2) HOF, and

Figure 25 shows XRD patterns of (a) yeast@(4+4) HOF and (b) insulin@(4+2) HOF. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for producing a hydrogen-bonded organic framework (HOF).

Hydrogen-bonded organic frameworks (HOFs) are frameworks of organic linkers interconnected through hydrogen-bonding interactions to form an open cage/scaffold-like structure that defines spatially ordered cavities. As used herein, the expression“organic linker” means a chemical compound having a chemical structure comprising carbon atoms and at least two moieties capable of promoting hydrogen bonds with at least one other organic linker. The organic linker may comprise hetero atoms, for example, oxygen, nitrogen, silicon etc.

To realize permanent porosity in HOFs, stable and robust open frameworks can be constructed by judicious selection of the organic linkers, which act as rigid molecular building blocks hydrogen-bonded with strong hydrogen-bonding interactions. Framework stability might be further enhanced through framework interpenetration and other types of weak intermolecular interactions such as p···p interactions. Owing to the reversible and flexible nature of hydrogen-bonding connections, HOFs show high crystallinity, solution processability, easy healing and purification.

By“hydrogen bond” is meant herein an attractive interaction between (i) a hydrogen atom from a molecule and/or a molecular fragment X-H in which X is more electronegative than H and (ii) an atom and/or moiety of at least one other organic linker. Typically, moieties capable of promoting a hydrogen bond between organic linkers forming the HOFs of the invention include high electronegative elements such as oxygen, nitrogen, fluorine, and a combination thereof. Suitable examples of the chemical structure of such moieties are shown in Figure 1.

The moieties capable of promoting a hydrogen bond may be any moiety known to the skilled person to promote formation of hydrogen bonds with corresponding moieties of at least another organic linker. Examples of such suitable moieties include carboxylic acid, pyrazole, 2,4-diaminotriazine (DAT), amide, amino, cyano, benzimidazolone, imide, imidazole, amidinium, boronic acid, resorcinol, pyridine, guanidinium, sulfonate and 2,6- diaminopurine. The chemical structure of at least some of those moieties is shown in Figure 1.

The organic core of the organic linkers forming the HOFs of the invention may be any carbon-based structure capable of having attached thereto at least two moieties capable of promoting a hydrogen bond. For example, the organic core is an aromatic organic core. In those instances, the organic core may comprises one or more Ce aromatic ring(s) having at least two moieties capable of promoting a hydrogen bond attached thereto. Presence of at least one Ce aromatic ring ensures that the at least two moieties attached thereto promote hydrogen bonds along specific spatial directions. A skilled person would be able to devise specific spatial arrangements of the organic core based on one or more Ce aromatic ring(s), such that the attached moieties can promote formation of at least two hydrogen bonds along specific directions. For example, the organic core includes Ce aromatic ring structures having at least two moieties capable of promoting hydrogen bonds along a linear direction, trigonal planar directions, square planar directions, tetrahedral directions, trigonal pyramidal directions, trigonal bi-pyramidal directions, and/or octahedral directions.

Suitable examples of organic linkers making the HOFs of the invention include compounds having at least two moieties capable of promoting a hydrogen bond, each of which comprises (i) hydrogen and (ii) oxygen and/or nitrogen. The HOFs of the present invention will comprise a suitable number of organic linkers required to form the framework. Such linker will typically comprise nitrogen and/or oxygen moieties as donors or acceptors for highly directional hydrogen-bonding.

For example, HOFs according to the present invention include those having at least two or at least three organic linkers interconnected at their extremities through hydrogen-bonding interactions. In some embodiments, the HOF of the invention is made from organic linkers having an equal number of hydrogen-bonding donors and acceptors. Those organic linkers are particularly suitable for generation of stable HOFs because the hydrogen-bonding donors/acceptors can distinctly form certain inherent hydrogen-bonding units, which can be dimers, trimers, and even chain structures, as exemplified in Figure 2. The Figure shows the geometry of typical hydrogen-bonding units assembled from organic linkers through multiple intermolecular hydrogen-bonds, serving as the building blocks for HOF construction.

The HOF may be a single component HOF, or a mixed-component HOF. While a“single component” HOF is formed by identical organic linkers linked together through hydrogen bonds, a“mixed-component” HOF is formed by a combination of at least two types of organic linkers linked together through hydrogen bonds.

In some embodiments, the HOF is selected from a DAT derivative HOF, a carboxylic acid HOF, an azole derivative HOF, and amide derivative HOF, a pyridine derivative HOF, a benzoin derivative HOF, a Pyridine-carboxylic derivative HOF, and an ammonium sulfonate derivative HOF.

Specific examples of suitable HOFs that may be made according to the present invention include those commonly known in the art as HOF-la (C ₃₇ H ₃₂ N ₂₀ ), HOF-2a (C ₃₆ H ₃₄ N ₂₀ O ₂ ), HOF-3a (C33H27N15), HOF-4a (C ₆ IH ₄₈ N2O), HOF-5a (C38H32N20), HOF-6a (C56H42N24), HOF-7a (C ₅₆ H ₄₂ N ₂₄ 0Zn), HOF-9a (CseBd^OZn), HOF- 10a (C ₅₆ H ₄₂ N ₂₄ 0Zn), IISERP- HOF1 (C ₂₁ H ₁₅ NO ₆ ), HOF-11, Tcpb/H ₃ BTB (C ₂₇ HI ₈ 0 ₆ ), HOF-BTB, HOF-TCBP (C4 _O H ₂₆ 0 ₈ ), PFC- I/H ₄ TB APy (C ₄₄ H ₂₆ 0 ₈ ), CoTCPp (C ₇₂ H ₄₄ N ₄ O ₁₂ C0), TCF-1 (C ₂₉ H ₂₀ O ₈ ), Tp-1 (C60H36O12), T12-1 (C66H36O12), T18-1 (C72H36O12), Ex-1 (C _9O H ₄₈ OI ₂ ), TpMe-1 (C72H60O12), TpF-1 (C60H24F12O12), CPHAT-la (C72H44N4O12), CBPHAT-la (C72H44N4O12), PETHOF-1 (C ₆₂ H ₈ 0i2), PETHOF-2 (C ₆₂ H ₈ 0i2), Trispyrazole 1 (C33H12F12N6), Trispyrazole 16 (C30H9F12N9), Tristetrazole 23 (C27H6F12N12), Trispyrazole 25 (C51H12F24N6), Benzotrisimidazole (C12H3F9N6), FDM-15 (C ₂₃ HI ₄ N ₆ ), TTBI

(C23H14N6O3), T2-b (C23H14N6O3), T2-g (C23H14N6O3), T2-d (C23H14N6O3), NPSU-3 (C46H60N6O5), HOF-8 (C24H18N6O3), SOF-la (C60H36N10), Cyclotetrabenzoin (C32H24O8), CB[6] (C36H36N24O12L2H2O, TTP (C18H12N3O6P3), CC3 (C72H84N12), CC44, CS144, and TBC[4]DHQ (C38H44O6).

The HOF of the invention is crystalline. In a crystalline HOF the organic linkers form a geometrically regular three-dimensional network. A crystalline HOF generates diffraction patterns when characterized by commonly known crystallographic characterization techniques. These include, for example, powder X-ray diffraction (PXRD), grazing incidence X-ray diffraction, small angle X-ray scattering (SAXS), single crystal X-Ray diffraction, electron diffraction, neutron diffraction and other techniques that would be known to the skilled person in the field of crystallography of materials.

The crystalline nature of HOFs arises from regular and spatially ordered distribution of intrinsic cavities forming the framework. As used herein the expression "intrinsic cavities" is intended to mean the ordered network of interconnected voids that is specific to a crystalline HOF by the very nature of the HOF. As it is known in the art, the intrinsic cavity network of a HOF results from the specific spatial arrangement of the HOF's organic linkers and is unique to any pristine crystalline HOF.

The intrinsic cavities of crystalline HOF can have any shape obtainable by the ordered spatial arrangements of the organic linkers. For example, the intrinsic cavities of crystalline HOF may be in the form of uniaxial cylindrical channels, which are spatially arranged to form an ordered geometrical pattern. As a further example, the intrinsic cavities of crystalline HOF may be the form of regularly distributed cages interconnected by windows or channels. The specific shape of cages and window/channels in crystalline HOFs is determined by the spatial arrangement of the chemical species forming the HOF framework. Accordingly, the expression "intrinsic cavities" specifically identifies the overall ordered network of cages and window/channels of the native HOF framework.

According to the present invention, the average dimension of the intrinsic cavities of a crystalline HOF are quantified by gas absorption measurements. Specifically, reference herein to the dimension (or size) of the intrinsic cavity of a HOF will be understood to be the size value determined by X-ray crystallographic techniques.

The intrinsic cavities of the HOF may have any average size compatible with a stable HOF structure obtainable using organic linkers of the kind described herein. For example, the intrinsic cavities of the HOF may have an average size of from about 5 A to about 500 A, from about 5 A to about 100 A, from about 5 A to about 50 A, from about 5 A to about 40 A, from about 5 A to about 30 A, from about 5 A to about 20 A, from about 5 A to about 15 A, from about 5 A to about 12 A, from about 5 A to about 10 A, from about 5 A to about 9 A, from about 5 A to about 8 A, from about 5 A to about 7 A, or from about 5 A to about 6 A.

There is no particular limitation on the size of HOF, provided it encapsulates the bio molecule. In some embodiments, HOF is provided in the form of particles which largest dimension ranges from about 0.1 pm to about 5 mm, from about 1 pm to about 5 mm, from about 10 pm to about 5 mm, from about 50 pm to about 5 mm, from about 100 pm to about 5 mm, from about 250 pm to about 5 mm, or from about 500 pm to about 5 mm.

The HOF of the invention encapsulates a bio-molecule.

As used herein, the term "bio-molecule" and its variants comprise any bio-active compound isolated from a living organism, as well as synthetic or recombinant analogs or mimics, derivatives, mutants or variants and/or bioactive fragments of the same. As used herein, the term "bioactive" and its variants such as "bioactivity" used in reference to a bio-molecule refer to any in vivo or in vitro activity that is characteristic of the bio -molecule itself, including the interaction of the bio -molecule with one or more targets.

For example, bioactivity can include the selective binding of an antibody to an antigen, the enzymatic activity of an enzyme, and the like. Such activity can also include, without limitation, binding, fusion, bond formation, association, approach, catalysis or chemical reaction, optionally with another bio-molecule or with a target molecule. Examples of suitable bio-molecules for use in the invention include a protein, a peptide, a nucleic acid, a nucleotide, or an amino acid.

The method of the invention comprises combining in a solution the bio-molecule and a HOF precursor.

Suitable HOF precursor for use in the method of the invention include any one of the organic linkers described herein, a salt thereof, and a combination thereof. In some embodiments, the HOF precursor is selected from one or more of hexakis[4-(2,4-diamino-l,3,5-triazin-6- yl)phenyl]benzene, 3 ,3 ',5,5 '-tetrakis-(4-carboxyphenyl)- 1 , 1 '-biphenyl (H ₄ TCBP), triptycene trisbenzimidazolone (TTBI), 9,10-bis(4-((3,5-dicyano-2,6- dipyridyl)dihydropyridyl)phenyl)-anthracene, l,3,5-tri(4-carboxyphenyl)benzene (TCPB), 4,4',4'',4"'-tetra(4,6-diaminostriazin-2-yl)tetraphenylmetha ne, Ni,N ₃ ,N ₅ -tri(pyridin-4- yl)benzene- 1,3, 5-tricarboxamide (TPBTC), 4', 4", 4"'-tetra(2, 4-diamino- 1,3, 5-triazin-6-yl)- tetraphenylethene, 1, 3, 5-tris(2, 4-diamino- 1, 3, 5-triazin-6-yl)-2, 4, 6-trimethyl-benzene

(TDTTB), tetrakis(4'-(2,4-diamino-l,3,5-triazin-6-yl)-[l,l '-biphenyl] -4-yl)methane, tris(4- carboxyphenyl)amine (TCPA), 5,10,15,20-tetrakis(4-(2,4diaminotriazinyl)phenyl)- porphyrin (H2TDPP), 4,4'-biphenyldisulfonic acid, 1,5-napthalenedisulfonic acid, naphthalene- 1, 4:5, 8-bis(dicarboximide) (NDI), 2,5,8, 1 l-tetrahexylperylene-3,4:9,10- bis(dicarboximide) (PDI), tetraphenylethylene-DAT (TPE-DAT), and 5,10,15,20-tetra(4-(4- acetateethyl)phenoxy)phenylporphyrin.

Design principles, precursors, and synthesis procedures of HOFs may be found, for example, in Jie Luo et al.,“Hydrogen-bonded organic frameworks: design, structures and potential applications”, CrystEngComm, 2018, 20, 5884; Rui-Biao Lin et al.,“Multifunctional porous hydrogen-bonded organic framework materials”, Chem. Soc. Rev., 2019, 48, 1362; Dong- Dong Zhou et al.,“High- symmetry hydrogen-bonded organic frameworks: air separation and crystal-to-crystal structural transformation”, Chem. Commun., 2016, 52, 4991-4994; and Yi-Fei Han et al.,“Porous Hydrogen-Bonded Organic Frameworks”, Molecules, 2017, 22, 266, the content of all of which is incorporated herein in its entirety. An example HOF obtained using hexakis[4-(2,4-diamino-l,3,5-triazin-6-yl)phenyl]benzene as HOF precursor is shown in Figure 3.

In some embodiments, the HOF precursor is made of identical organic linkers. When identical organic linkers are used as the HOF precursor, the resulting HOF is a“single component” HOF.

In alternative embodiments, the HOF precursor comprises a combination of at least two types of organic linkers. When at least two types of organic linkers are used as the HOF precursor, the resulting HOF is a“mixed-component” HOF.

In some embodiments, the HOF precursor is used in solution with a deprotonating agent. The use of a deprotonating agent facilitates formation of intermolecular hydrogen bonds between molecules of the HOF precursor. The use of a deprotonating agent is particularly useful, for example, when an organic linker used as HOF precursor has a weak acidic activity (such as in the case of carboxylic -based organic linkers). A skilled person would be aware of procedures to provide a HOF precursor in deprotonated form. For example, the HOF precursor may be provided in solution with a deprotonating compound, such as ammonia, Na2C03, NaHC03, K2CO3, NH4OH, or a combination thereof. The deprotonating agent may be used in any amount that would be effective to deprotonate the HOF precursor. In some embodiments, the deprotonating agent is used in an amount sufficient to bring the solution of HOF precursor and bio-molecule to a pH of between about 7 and about 9.

The bio-molecule and the HOF precursor may be combined in a solution using any solvent that (i) solubilise the HOF precursor, and (ii) is compatible with the bio-molecule. That is, the solvent will typically be one that does not adversely affect the bioactivity of the bio molecule. Examples of suitable solvents for use in the invention include methanol, ethanol, dimethyl sulfoxide (DMSO), acetone, water, and mixtures thereof.

In some embodiments, the solution into which the bio-molecule and HOF precursor are combined is an aqueous solution. For example, the method of the invention may comprise combining in a solution the bio-molecule and a HOF precursor using a solvent selected from water (e.g. deionised water), and a physiological buffered solution (e.g. water comprising one or more salts such as KH2PO4, NaH2P04, K2HPO4, Na2HP04, Na3P04, K3PO4, NaCl, KC1, MgCh, CaCF, 3-(N-morpholino)propanesulfonic acid (MOPS), tris(hydroxymethyl)aminomethane (TRIS), (4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid) (HEPES), and 2-(N-morpholino)ethanesulfonic acid (MES)).

The HOF precursor may be present in the solution in any amount that ensures formation of HOF. For example, suitable amounts of HOF precursor in the solution can include a range from about 0.1 mg to about 50 mg, from about 0.1 mg to about 25 mg, from about 0.1 mg to about 10 mg, from about 1 mg to about 10 mg, or from about 2.5 mg to about 5 mg, relative to 1 ml of solvent (e.g. water). The values refer to the total amount of HOF precursor, either when made by a single type of organic linker or by a mixture of two or more types of organic linkers, relative to the total volume of solvent used to form the solution containing the HOF precursor and the bio-molecule.

When the HOF precursor is made of a mixture of two or more types of organic linkers, the weight ratio between the different types of organic linkers may be any weight ratio that is adequate for the formation of HOF. In some embodiments, the HOF precursor is made of two types of organic linkers. In those instances, the weight ratio between the two types of organic linkers may be any weight ratio that is adequate for the formation of HOF. For example, the two organic linkers may be used in a 1: 1, 1.25: 1, 1.5: 1, 2: 1, or 5: 1 weight ratio.

The HOF precursor self-assembles to form HOF that encapsulates the bio-molecule. By the HOF precursor "self-assembling" to form HOF is meant that organic linkers making the precursor autonomously (i.e. spontaneously) bond to one another by inter-molecular hydrogen bonds without external assistance, resulting in formation of a discrete non-random HOF aggregate structure. Such bonding occurs through random movement of the linkers in the solution due only to the inherent chemical nature and structure of the linkers in the HOF precursor. By the method of the invention, the HOF encapsulates the bio-molecule. By the HOF "encapsulating" the bio-molecule, the molecular structure of the HOF surrounds the entire bio-molecule. Without wanting to be bound by theory, the mechanism by which the HOF encapsulates the bio-molecule may depend on the size of the bio-molecule relative to the size of the HOF cavities. For example, the HOF may "encapsulate" the bio-molecule by forming around the bio-molecule. This mechanism may be postulated irrespective of whether the bio-molecule is larger or smaller than the HOF cavities. In those instances, it is believed the organic linkers also promote hydrogen bonds with the bio-molecule. In that context, formation of encapsulating HOF may be facilitated by the bio -molecule's affinity towards the HOF precursor arising, for example, from intermolecular hydrogen bonding and hydrophobic interactions. However, for bio-molecule that are smaller than the HOF cavities it is not excluded that the HOF may form first, and "encapsulation" of the (small) bio molecule results from the bio-molecule spontaneously infiltrating the formed HOF.

Surprisingly, irrespective of the mechanism by which the HOF encapsulates the bio molecule, the nature of the bio-molecule, and the size of the bio-molecule, presence of the bio-molecule in the solution of HOF precursor does not disrupt the formation of the encapsulating HOF. That is, HOF that forms in the presence of the bio-molecule can surprisingly present the same structure and micro structure characteristics of the same HOF forming absent the bio-molecule.

Combining the HOF precursor in solution with the bio-molecule is sufficient to ensure that HOF forms around the bio-molecule to encapsulate it. There is no need to apply other external factors to trigger formation of the encapsulating HOF. For example, it is not necessary to apply heat to the solution. Accordingly, in some embodiments formation of the encapsulating framework is performed at a solution temperature that is lower than 100°C, 90°C, 75°C, 50°C, or 35°C. Thus, the solution temperature may be between -50°C and 75°C, between -50°C and 50°C, or between -50°C and 30°C.

In some embodiments, the method is performed at room temperature. As used herein, the expression "room temperature" will be understood as encompassing a range of temperatures between about 20°C and 25°C, with an average of about 23°C. Performing the method at these lower temperatures is advantageous for heat sensitive proteins such as antibodies, fibronectin glycoproteins, proteolytic enzymes and collagens.

There is no particular limitation on the order in which the HOF precursor and bio -molecule may be combined into the solution.

For example, a solution containing a HOF precursor may be first made, and a separate solution containing a bio-molecule is subsequently introduced into the solution containing the HOF precursor.

Alternatively, the bio-molecule may be added directly to a solution of the HOF precursor. Conversely, the HOF precursor may be added to a solution of the bio-molecule.

When the HOF precursor is made by at least two types of organic linkers, individual solutions of each organic linker may be first made and the solutions combined to form a solution of the HOF precursor. In that context, the bio-molecule may be introduced at any stage of the procedure. For example, the bio-molecule may be introduced either directly or in solution form into one (or more) of the initial solutions of the organic linkers, or to the solution of the HOF precursor.

For example, when the HOF precursor is made of two types of organic linkers, two solutions each containing one type of organic linker may be first made, and the solutions combined to form a solution of the HOF precursor. In that context, the bio-molecule may be introduced either directly or in solution form to either (or both) of the two initial solutions of the organic linkers, or to the solution of the HOF precursor.

Formation of HOF according to the method of the invention is advantageously fast. Depending on the type of bio-molecule used and the type of HOF precursor used, it has been found that upon bringing the bio-molecule and the HOF precursor together in a solution HOF may form within about 30 seconds, 1 minute, 10 minutes, 30 minutes, 60 minutes, 2 hours, or 4 hours.

Bio-molecules encapsulated within the HOF may be advantageously uniformly distributed throughout the entire volume of the HOF. The distribution profile of bio-molecules within the framework can be determined by confocal laser scanning microscopy emission measurements. The distribution of bio-molecules will be considered "uniform" throughout the volume of the framework if the intensity of the emission signal recorded using a confocal scanning laser microscope (CLSM) scanning across any plane of a HOF having encapsulated bio-molecules labelled with a fluorescent dye does not vary more than 10% when measured at the optimum emission wavelength of the dye, when scanning at the optimum excitation wavelength of the dye using 0.1 micrometre linear increments.

In addition, uniform distribution of bio-molecules encapsulated within the HOF obtained by the method of the invention may inherently provide for a large amount of bio-molecules encapsulated within the HOF framework per unit volume. For example, the method of the invention may provide HOFs encapsulating from about 1% wt to about 32% wt bio molecule, from about 5% wt to about 30% wt bio-molecule, or from about 10% wt to 20% wt bio-molecule, expressed as the ratio between the amount (in milligram) of encapsulated protein and the weight (in milligram) of the resulting HOF. Typically, the amount of encapsulated protein is derived from the UV-Vis spectroscopy absorbance measurements of proteins in solution, performed on samples of liquid solution before and after encapsulation. Other available procedures include fluorescence and inductively coupled plasma mass spectrometry (ICP-MS).

Advantageously, the method of the invention allows for HOFs having a framework that encapsulates a bio-molecule in its native conformation. The expression "native conformation" is used herein to indicate the three-dimensional conformation that gives rise to a bio-molecule's bioactivity. For example, the native conformation of a bio-molecule such as a peptide, protein or a nucleic acid results from the spontaneous or assisted folding of the polypeptide or the polynucleotide to assume the lowest enthalpy molecular conformation. Such conformation results from the specific chemical characteristics and sequence of the amino acids and the nucleotides that form the polypeptide and the polynucleotide, respectively.

By encapsulating a bio-molecule in its native conformation, the HOF can advantageously preserve the bio-activity of the bio-molecule. This means that either (i) the encapsulated bio molecule shows bio-activity characteristics identical to those of the free bio-molecule, or (ii) the encapsulated bio-molecule shows masked bio-activity because it is physically isolated from the external environment. In (ii), however, the bio-activity of the bio-molecule can be advantageously harnessed upon dissolution/destmction of the framework.

The bio-molecule encapsulated within the framework may be released into a solvent by dissolving the HOF suspended within the solvent, for example by inducing a variation of the pH of the solvent. According to this approach, the HOFs may be good candidates for pH- induced targeted release of the encapsulated bio-molecule, useful for example in drug delivery applications into living organisms. Alternatively, the application of light can trigger a conformational change of the ligand-metal stereochemistry which may thus result in a change in the intrinsic cavity size and so release the bio-molecular cargo. Examples of HOFs that may be used in applications based on pH-triggered release of a bio-molecule include HOFs that are stable at certain pH values, but dissolve at certain other pH values. For example, the HOF may be stable above a threshold pH value. In that case there is no detectable release of the bio-molecule into the solution within which the HOF is suspended. However, the HOF may dissolve when the pH drops below the threshold, resulting in the release of the bio-molecule into the solution.

The HOF framework can advantageously protect the encapsulated bio-molecule from environmental conditions that would otherwise destroy the bio-molecule in its free form, i.e. not encapsulated within the HOF. That is, the encapsulating framework improves the stability of the bio-molecule in a diversity of environmental conditions. For example, it was found that encapsulated bio-molecules preserve their bio-activity even after the HOF is exposed to temperatures up to 90°C, for example from about 60°C to about 80°C, for periods of time up to and exceeding 1 hour, for example about 30 minutes. Further, encapsulated bio-molecules preserve their bio-activity even after the HOF is exposed to organic solvents (e.g. urea) that would otherwise decompose the bio-molecule. Similarly, encapsulated bio molecules preserve their bio-activity even after the HOF is exposed to proteolytic agents that would normally lead to decomposition of the bio-molecule structure and loss of bio-activity.

The invention also provides a HOF encapsulating a bio-molecule as obtained by the method disclosed herein.

The present invention also advantageously allows for encapsulation within a crystalline HOF of a bio-molecule irrespective of the relative dimension between the intrinsic cavities and the bio-molecule. For example, the method of the invention allows encapsulating within a crystalline HOF a bio-molecule that is considerably larger than the intrinsic cavities of the framework. This approach can provide for unique HOFs, the likes of which are precluded by post-synthesis infiltration methods.

The present invention therefore also provides a method of producing crystalline hydrogen- bonded organic framework (HOF) that defines intrinsic cavities and encapsulates a bio molecule, said method comprising combining in a solution a HOF precursor and a bio molecule, wherein the bio-molecule has a smallest dimension that is larger than the size of any intrinsic cavity of the HOF, and the HOF precursor self-assembles to form HOF that encapsulates the bio-molecule.

The present invention also provides crystalline HOF having a framework that defines intrinsic cavities and encapsulates a bio-molecule.

In some embodiments, the bio-molecule has a smallest dimension that is larger than the size of any intrinsic cavity of the HOF. The smallest dimension of a bio-molecule can be determined by using techniques well known to those skilled in the art. Where the bio- molecule is a protein or a nucleic acid that can be crystallised for the purpose of XRD characterisation, the expression "smallest dimension" means the smallest value of any dimension (as opposed to molecular weight) of the protein or nucleic acid that is obtained from the corresponding Protein Data Bank (PDB) file of the protein or nucleic acid.

As it is known in the art, a PDB file of a protein or nucleic acid encodes the spatial distribution of each atom forming the protein or nucleic acid as determined by XRD and NMR characterisations performed on the protein or the nucleic acid in their crystallised form. Crystallisation of a protein or a nucleic acid is achieved according to procedures that would be known to a skilled person.

As it is known in the art, PDB files can be read by 3D editing software to obtain a 3D visualisation of the resulting protein or nucleic acid structure. The 3D visualisation software allows for accurate determination of the geometric size of the modelled protein or nucleic acid by way of a string of 3 lengths values in a "a x b x c" format. Thus, in this context the "smallest dimension" of the protein or nucleic acid is the smallest of a, b and c.

In the case of a bio-molecule such as a protein or nucleic acid that cannot be crystallised for the purpose of XRD and NMR characterisation, the expression "smallest dimension" refers to the Stokes radius of the protein or nucleic acid determined according to the procedure described in detail in Harold P. Erickson, "Size and Shape of Protein Molecules at the Nanometer Level Determined by Sedimentation, Gel Filtration, and Electron Microscopy", Biological Procedures Online, Volume 11, Number 1.

In those embodiments where the bio-molecule has a smallest dimension that is larger than the size of any intrinsic cavity of the HOF, the bio-molecule may be of any size provided the smallest dimension of the bio-molecule is larger than the size of any intrinsic cavity of the HOF. For example, the smallest dimension of the bio-molecule can advantageously be any degree larger than the size of any intrinsic cavity of the HOF. In some embodiments, the smallest dimension of the bio-molecule may be at least 1.5, 2, 5, 10, 25, 50, 75, 100, 250, 500, 750, or 1000 times larger than the size of any intrinsic cavity of the HOF. The bio-molecule may be relatively tightly encapsulated within the HOF framework such that, for example, relative movement between the bio-molecule and the encapsulating framework is impeded. In those instances, the bio-molecule is believed to sit within the HOF framework as a heterogeneous and discontinuous guest phase within a self-defined cavity. That would be the case, for example, of a bio-molecule having a smallest dimension that is larger than the size of any intrinsic cavity of the HOF.

In one embodiment, the bio-molecule is an amino acid.

As used herein, the expression "amino acid" refers to an organic acid containing both a basic amino group (NH2) and an acidic carboxyl group (COOH). The expression is used in its broadest sense and may refer to an amino acid in its many different chemical forms including a single administration amino acid, its physiologically active salts or esters, its combinations with its various salts, its tautomeric, polymeric and/or isomeric forms, its analog forms, its derivative forms, and/or its decarboxylation products.

Examples of amino acids useful in the invention comprise, by way of non-limiting example, Agmatine, Beta Alanine, Arginine, Asparagine, Aspartic Acid, Cysteine, Glutamine, Glutamic Acid, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, PhenylBeta Alanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, and Valine.

Provided the HOF forms, there is no particular limitation regarding the concentration of amino acids present in the solution with the HOF precursor.

Suitable concentrations of amino acids in the solution can include a range of between about 0.1 and 100 mg/mL, between about 0.1 and 75 mg/mL, between about 0.1 and 50 mg/mL, between about 0.1 and 25 mg/mL, between about 0.2 and 25 mg/mL, between about 0.25 and 25 mg/mL, between about 0.25 and 20 mg/mL, between about 0.25 and 15 mg/mL, between about 0.25 and 10 mg/mL, and between about 0.025 and 1.5 mg/mL. In one embodiment, the bio-molecule is a peptide.

As used herein, and as it would be known to a skilled person, the term "peptide" identifies a sequence of amino acids made up of a single chain of amino acids joined by peptide bonds. The amino acids may be any amino acid described herein.

The peptide may be any peptide that can be encapsulated within the HOF. In some embodiments, the peptide is a modified peptide. By the expression "modified peptide" is meant a peptide having incorporated non-natural amino acids, including synthetic non-native amino acids, substituted amino acids, or one or more L- and D-amino acids. In some embodiments, the bio-molecule is a peptide hormone, such as insulin.

Suitable concentrations of peptides in the solution can include a range of between about 0.1 and 100 mg/mL, between about 0.1 and 75 mg/mL, between about 0.1 and 50 mg/mL, between about 0.1 and 25 mg/mL, between about 0.2 and 25 mg/mL, between about 0.25 and 25 mg/mL, between about 0.25 and 20 mg/mL, between about 0.25 and 15 mg/mL, between about 0.25 and 10 mg/mL, and between about 0.025 and 1.5 mg/mL.

In one embodiment, the bio-molecule is a protein.

As used herein, the term "protein" refers to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers. As used herein, the term "protein" also embraces an enzyme.

A protein commonly folds into a unique 3-dimensional structure. A protein may assume many 3 -dimensional shapes. The overall shape of a single protein molecule is identified as "tertiary structure". The basic bio-activity function of a protein is determined / controlled by its tertiary structure. In accordance with the invention the protein may be selected from therapeutic or prophylactic proteins. These may include plasma proteins, hormones and growth factors, extracellular proteins, and protein antigens for vaccines. They may also be selected from structurally useful proteins for use in cosmetics and foods.

Examples of plasma proteins include, but are not limited to Albumin (HSA), haemoglobin, thrombin, fibronectin, fibrinogen, immunoglobulins, coagulation factors (FX, FVIII, FIX)). Examples of extracellular proteins (and in some case these are also described as structural proteins) include, but are not limited to collagen, elastin, keratin, actin, tubulin, myosin, kinesin and dynein.

Examples of hormones and growth factors include, but are not limited to insulin, EGF, VEGF, FGF, insulin like growth factor, androgens, estrogens.

Examples of antigen proteins include, but are not limited to ovalbumin (OVA), keyhole limpet hemocyanin and bovine serum albumin (BSA) and immunoglobulins.

Proteins that can be used in the invention include enzymes. As used herein, the term "enzyme" refers to a protein originating from a living cell or artificially synthesised that is capable of producing chemical changes in an organic substance by catalytic action.

Enzymes are industrially useful in many areas such as food, textiles, animal feed, personal care and detergents, bioremediation and catalysis. In these application areas, conservation of conformation and activity, bioavailability and release profile and the adoption of an encapsulation carrier all play some role in their industrial utility. Enzymes are also useful in biomedical devices and sensors, owing to their high selectivity.

Enzymes useful in the invention thus can be categorised according to their end use application. Examples of enzymes used in the food industry include, but are not limited to pectinases, renin, lignin- modifying enzymes, papain, lipases, amylases, pepsin and trypsin.

Examples of enzymes used in the textile industry include, but are not limited to endoglucases, oxidases, amylases, proteases cellulases and xylanases.

Examples of enzymes used in the biomedical/sensor industry include, but are not limited to dehydrogenases, lipases, horse radish peroxidase (HRP), urease and RNA or DNA enzymes such as ribonuclease.

Provided the HOF forms from the precusors, there is no particular limitation regarding the concentration of proteins present in the solution with the HOF precursor.

Suitable concentrations of protein in the solution can include a range of between about 0.1 and 20 mg/mL, between about 0.15 and 10 mg/mL between about 0.15 and 7.5 mg/mL, between about 0.2 and 5 mg/mL, between about 0.25 and 5 mg/mL, between about 0.03 and 5 mg/mL, between about 0.025 and 2.5 mg/mL, between about 0.025 and 2 mg/mL, between about 0.025 and 1.5 mg/mL, or between about 0.025 and 1.25 mg/mL.

In one embodiment, the bio-molecule is a nucleic acid.

As used herein, the expression "nucleic acid", synonym of the term "polynucleotide", refers to polymeric macromolecules, or large biological molecules, essential for all known forms of life which may include, but are not limited to, DNA (cDNA, cpDNA, gDNA, msDNA, mtDNA), oligonucleotides (double or single stranded), RNA (sense RNAs, antisense RNAs, mRNAs (pre-mRN A/hnRN A) , tRNAs, rRNAs, tmRNA, piRNA, aRNA, RNAi, Y RNA, gRNA, shRNA, stRNA, ta-siRNA, SgRNA, Sutherland RNA, small interfering RNAs (siRNAs), double-stranded RNAs (dsRNA), short hairpin RNAs (shRNAs), piwi-interacting RNAs (PiRNA), micro RNAs (miRNAs), small nucleolar RNAs (SnoRNAs), small nuclear (SnRNAs) ribozymes, aptamers, DNAzymes, ribonuclease-type complexes and other such molecules as herein described. For the avoidance of doubt, the expression "nucleic acid" includes non-naturally occurring modified forms, as well as naturally occurring forms. In some embodiments, the nucleic acid molecule comprises from about 8 to about 80 nucleobases {i.e. from about 8 to about 80 consecutively linked nucleic acids). One of ordinary skill in the art will appreciate that the present invention embodies nucleic acid molecules of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,

29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,

77, 78, 79, or 80 nucleobases in length.

Provided the HOF forms, there is no particular limitation regarding the concentration of nucleic acid present in the solution with the HOF precursor.

Suitable concentration of nucleic acids in the solution include a range of between about 0.001 to 100 mM, between about 2 to 50 pM, between about 2 to 10 pM, between about 3 to 5 pM, between about 3.45 to 5 pM, or between about 3.45 to 4 pM relative to the total volume of solution containing the HOF precursor and the nucleic acids.

In some embodiments, the bio-molecule is a composite bio-molecule. By being“composite”, the bio-molecule is a bioactive entity resulting from the combination of two or more types of bio-molecules described herein. For example, the bio-molecule may be the combination of a protein and a nucleic acid in the form of a nucleocapsid. As used herein, and as it would be known to a skilled person, a "nucleocapsid" is a constitutional unit of a virus, i.e. a composite bio-molecule resulting from the combination of (i) a nucleic acid genome and (ii) a protein capsid that covers the genome. For instance, the bio-molecule may be a virus, or the nucleocapsid component of a vaccine. Specific embodiments of the invention will now be described with reference to the following non-limiting examples.

EXAMPLES

All chemicals and solvents used in the Examples described below were purchased from commercial sources and used as received without further purification.

EXAMPLE 1

Preparation of fluorescein-tagged enzymes

Fluorescein isothiocyanate (FITC, 0.5 mg) and catalase (CAT; Sigma- Aldrich, catalase from bovine liver, 2000-5000 units mg ¹ protein, 40 mg) were dissolved in carbonate -bicarbonate aqueous buffer solution (0.1 M, pH 9.2, 4 mF) and left for two hours in darkness at room temperature under gentle stirring. The FITC-tagged CAT (FCAT) was recovered by passing the reaction mixture through an Illustra NAP-25 column (GE Healthcare Fife Sciences, NSW, Australia).

The crude FCAT solution was concentrated through a 10 K membrane by centrifugation at 4°C (4,000 rpm for 20 min), followed by solvent-exchange with ultrapure water. The concentration-solvent-exchange process was repeated two times to ensure the buffer salts were completely removed from the solution. Thereafter, the concentrated FCAT aqueous solution was passed through an NAP-25 column again to ensure the completely removal of unreacted FITC. The obtained FCAT solution was stored in darkness at 4°C.

A similar method was used to prepare fluorescein-tagged alcohol oxidase (FAOx, Sigma- Aldrich, alcohol oxidase solution from Pichia pastoris, buffered aqueous solution, 10-40 units/mg protein). Synthesis method for Hydrogen-bonded Organic Frameworks (HOFs)

The chemical structure of HOF precursors LI, L2, and L3 used in this Example are shown in Figure 4. In a typical synthesis, / e l ra /;/.s(4-amidini u m p h c n y 1 ) m c t h a n c (l-CL, 4 mg) was dissolved in ¾0 (1 mL) to form solution A. 7h/ra :/.s(4-carboxylphcnyl) methane (H42, 3 mg) was dispersed in Milli-Q H2O (950 pL), followed by the addition of aqueous ammonium hydroxide solution (1% v/v, 50 pL) to deprotonate 2 (solution B). Thereafter, solution B was added to solution A under stirring conditions at room temperature. Precipitates were formed immediately upon mixing. The reaction mixture was left to gently stir in the dark for 1 h. The BioHOF-1 material was then recovered by centrifugation, and then washed, dispersed, and centrifuged three times each in Milli-Q H2O to remove any unreacted precursors.

Synthesis of FC AT @ BioHOF-1 biocomposite

The typical synthesis procedure for the FCAT@ BioHOF-1 biocomposite was carried out as follows: l-CL (4 mg) was dissolved in H2O (0.5 mL) to form solution A. An aqueous solution of FCAT (1 mg, 0.5 mL of 2 mg mL ¹ stock solution) was added to solution A and stirred at room temperature for 10 min to form solution B. ¾2 (3 mg) was dissolved in H ₂ q/NH ₄ qH(1%) (19: 1)(1 mL) to form solution C. Solution C was then added dropwise to solution B under stirring. The mixture was then left to gently stir for another 1 h to ensure the completion of the synthesis. Thereafter, the FCAT@BioHOF-l composite was collected by centrifugation and then washed, dispersed, and centrifuged three times each in Milli-Q H2O to remove any unreacted precursors and loosely adsorbed FCAT.

Synthesis of the FAOx@ BioHOF-1 biocomposite l-CL (4 mg) was dissolved in H2O (0.75 mL) to form Solution A. An aqueous solution of FAOx (0.5 mg, 0.25 mL of 2 mg mL ¹ stock solution) was added to solution A and stirred at room temperature for 10 min to form solution B. ¾2 (3 mg) was dissolved in H ₂ q/NH ₄ qH(1%) (19: 1)(1 mL) to form solution C. Solution C (20 pL) was then added to solution B under gentle stirring every 20 s. The mixture was gently stirred for an additional 1 h to ensure the completion of the synthesis. Thereafter, the FAOx@BioHOF-l composite was collected by centrifugation and then washed, dispersed, and centrifuged three times each in Milli-Q FhO to remove any unreacted precursors and loosely adsorbed FAOx.

EXAMPLE 2

Characterisation of the FCAT@BioHOF-l and FAOx@BioHOF-l

The as-synthesized samples were examined by powder X-Ray Diffraction (PXRD), which confirmed that the samples shared the same crystalline topology, as shown in the diffraction patterns of Figure 5. PXRD patterns were obtained using a D4 ENDEAVOR X-ray Diffractometer from Bruker. A Co anode was used to produce K _a radiation (l = 1.78897 A). Flat plate diffraction data was collected over the range 2Q = 5-40°. The PXRD data were modified by PowDLL Converter (version 2.68.0.0) and expressed as the copper-source irradiated patterns (l = 1.54056 A). Inspection of the PXRD showed peaks characteristic of BioHOF-1 at 20 values of 8.6 and 17.4°, indicating that the HOF forms in the presence of the enzyme.

Fluorescein-tagged CAT (FCAT) was used in the synthesis of FCAT@BioHOF-l to study the spatial localization of the enzyme within the HOF structure. For comparison, an FCAT- on-BioHOF-1 sample was also synthesized via surface adsorption of the FCAT on as- synthesized FCAT @ BioHOF-1. The presence and spatial location of the fluorophore-tagged biomolecules in (or on) the HOF composites was determined using CLSM technique (Olympus FV3000 Confocal Laser Scanning Microscope, OLYMPUS). The fluorescein- tagged biomolecules were excited at 488 nm and the fluorescence signal was collected in a window from 495 to 545 nm. As shown in Figure 6 (top row), the FCAT was homogeneously distributed throughout the FCAT @ BioHOF-1 crystal. In contrast, for the FCAT-on- BioHOF-1, the FCAT was just located on the HOF surface (Figure 6, bottom row). These results confirmed the successful encapsulation of FCAT within the HOF, which we attributed to the hydrogen bonding interaction between the HOF ligands and the enzyme surface. Scanning electron microscopy (SEM) images were collected using a Philips XL30 Field Emission Scanning Electron Microscope (FESEM). Prior to analysis, the samples were dispersed in ethanol by sonication, drop-cast on an aluminum SEM stage (12 mm), and sputter-coated with platinum to form a thin film (5 nm).

The loading of FCAT in the FCAT@BioHOF-l biocomposite was determined to be 6.0 ± 0.5 wt% by Inductively Coupled Plasma Mass Spectrometer (ICP-MS). ICP-MS was performed on an Agilent 8900x QQQ-ICP-MS. The free enzyme or enzyme @BioHOF-l composites (approximately 1 mg) were dispersed in a solution of HNO3/HCI (0.25 mL of 70% HNO3 (Ajax) and 0.25 mL of 37% HC1 (Chem- supply)) and stored in Eppendorf tubes at room temperature overnight. The mixture was then centrifuged to remove any particulates in the supernatant. Thereafter, the clear supernatant (0.4 mL) was diluted to a final volume of 5 mL with Milli-Q H2O for ICP-MS analysis. The amount of enzyme within the sample was calculated according to a standard calibration curve for sulfur (prepared from sulfur standard solution from Sigma-Aldrich, Sulfur Standard for ICP (TraceCERT®, 1000 mg/L S in H2O)). FAOx@BioHOF-l (FAOx = fluorescein tagged AOx) composites had an enzyme loading of 2.9 ± 0.6 wt%. Time-resolved SAXS data was collected on the SAXS beamline at the ELETTRA synchrotron light source. Operation occurred at a photon energy of 8 keV covering the range of momentum transfer, q = 4p sin(0)/k, between 0.1 and 7.7 nm ¹ . The kinetics of the MOF nucleation and growth was monitored using a commercial stopped flow apparatus (Bio- Logic, Grenoble, France) especially designed for synchrotron radiation SAXS investigations. Two independently controlled syringes were filled with the carboxylate ligand solution and the amidinium ligand (or amidinium ligand + BSA) solution, respectively. Two step-motors controlled the volumes that were mixed and injected into a quartz capillary (1 mm) placed in the X-Ray beam (the mixing/injection process lasts a few ms). The volume ratio between the two solutions and their concentration was set to maintain the conditions used for the syntheses in batch. A total volume of 900 pL was injected for each experiment. The start of the mixing sequence was triggered from the X-ray data- acquisition system. Images were taken with a time resolution of 100 ms (detector: Pilatus3 1M, Dectris Ltd, Baden, Switzerland; sample to detector distance: 1260 mm, as determined with a silver behenate calibration sample). All experiments were performed at room temperature. The resulting two-dimensional images were radially integrated to obtain a ID pattern of normalized intensity versus scattering vector q. The background was collected using Milli-Q ¾0 and subtracted as background from the normalized data.

Solid-state UV-visible spectra were measured for alcohol oxidase (AOx), AOx@BioHOF- 1, and BioHOF-1. AOx exhibits a pair of peaks at 376 and 466 nm representing the FAD and FADFb groups. In the case of AOx@BioHOF-l composites, the 466 nm peak does not shift dramatically, representing the unchanged binding environment around FADFb during the synthesis of the composites. The incorporation of FCAT into the composite is supported by solid-state UV-visible spectroscopy data that show the Soret absorption band at 407 nm (p-p*), due to the iron-heme cofactor in CAT. Furthermore, the congruence of the Soret absorption peak in free FCAT and FCAT@BioHOF-l is indicative that the encapsulation process did lead to significant structural changes at the enzyme active site.

EXAMPLE 3

Catalytic performance of FCAT and the FCAT@BioHOF-l composite

The Ferrous Oxidation in Xylenol orange (FOX) assay was applied to quantify the concentration of produced H2O2. FCAT or the FCAT @ BioHOF-1 composite was added into phosphate buffer (100 mM, pH 8, 0.2 mL). Thereafter, H2O2 stock solution (1 mM in H2O, 0.15 mL) was added. The volume of the reaction mixture was adjusted to 1 mL by H2O. The catalyst dosage (based on FCAT) in the enzymatic reactions were 2.6 and 2.9 pg for FCAT and FCAT @ BioHOF-1, respectively (determined by ICP-MS). At different time intervals, aliquots of the mixtures (50 pL) were sampled and mixed with FOX reagent (950 pL) in an Eppendorf tube and then incubated for at least 30 min at room temperature. After incubation, the samples were centrifuged. The UV-visible absorbances at 560 nm for the supernatant were recorded to calculate the H2O2 concentration. The reaction rate ( _obs , mM s ¹ ) is defined as the initial H2O2 decomposition velocity of the enzymatic assay.

For calibration, H2O2 standard solution (50 pL) at various concentrations were mixed with of FOX reagent (composed of 250 pM ammonium ferrous sulfate, 100 pM xylenol orange, 100 mM sorbitol in 25 mM FhSO ₄ )(950 pL) and incubated for 30 min at room temperature before reading absorbance at 585 nm. The concentration of H2O2 was calibrated using an extinction coefficient of 39.4 M ¹ cm ¹ at 240 nm.

The H2O2 decomposition rate ( V7 _/« ) was quantified via the FOX assay which measures the consumption of H2O2. As a control experiment we confirmed that neat BioHOF-1 crystals do not catalyze the decomposition of H2O2 _. Conversely, when exposed to H2O2, at room temperature and pH 8 FCAT@BioHOF-l retained activity ( V,,bs = 0.27 pM s ¹ pg _FCAT ¹ ). Also the supernatant of the FCAT@BioHOF-l composite showed negligible enzymatic acitivty which suggests that enzyme leaching is minimal.

A significant motivation for the encapsulation of enzymes within porous frameworks is to protect the biomacromolecule from conditions that would typically lead to a partial or complete loss of its native activity. Thus, we compared the rate of H2O2 decomposition catalyzed by free FCAT and FCAT@BioHOF-l in a variety of conditions, as described in the following Examples.

EXAMPLE 4

Protective capacity afforded to FCAT by the encapsulating HOF pH

The biological activity of free FCAT and FCAT @ BioHOF-1 in different pH conditions was monitored. We assessed the capacity of the HOF coating to protect CAT over the pH range 5-10 as it is known that the optimal activity for free CAT is at pH of 7-8. The enzymatic assays were performed in citrate buffer (pH 5, 0.1 M), phosphate buffer (pH 6-8, 0.1 M), or glycine-HCl buffer (pH 9-10, 0.1 M) with H2O2 (0.15 ruM). The total volume of the reaction mixture was fixed to 1 ruL. The values of reaction rate (V ₀ bs) is summarized in the inset of Figures 7(a) and 7(b), and outlined in Table 1. Numbers in brackets indicate the standard deviation of measurements. The error bars indicate the standard deviation of two independent measurements. The FCAT dosage in the assay for FCAT and FCAT@BioHOF-l were 2.6 and 2.9 pg (based on FCAT, determined by ICP-MS), respectively. These crystalline materials are stable in water over a wide pH range (5 to 10). Table 1 - Reaction rates from Figures 7(a) and 7(b)

Figures 7(a) and 7(b) show the optimal activity for the biocomposite is significantly broadened, indeed, >90% of the maximum activity is retained for FCAT@BioHOF-l operating over the pH range 5-10. This represents a notable advantage for HOF biocomposites as access to mildly acidic pH conditions is not readily achievable for ZIF- based biocomposites, which are unstable in acidic conditions.

EXAMPLE 5

Protective capacity afforded to FCAT by the encapsulating HOF temperature and solvent

To demonstrate the catalytic activity and protecting effects of the catalase @ HOF (CAT @ HOF) biocomposites, we measured hydrogen peroxide (H2O2) decomposition (the enzymatic reaction of catalase) for the samples using the Ferrous Oxidation in Xylenol orange (FOX) assay. The FOX assay is used to measure the residue H2O2 in solution that is based on the oxidation of ferrous (Fe ²⁺ ) to ferric (Fe ³⁺ ) ions by H2O2 with the subsequent binding of the Fe ³⁺ ion to the ferric- sensitive dye xylenol orange, yielding an orange to purple complex (colour dependent on the amount of H2O2 present), which is measured at 560 nm.

Catalase (CAT) is an iron-heme enzyme that catalyses the decomposition of hydrogen peroxide to water and oxygen. Thus in the assay, if the materials are catalytically active, we would expected the H2O2 in solution would be consumed by the enzyme and no colour change for the FOX reagent (the assay solution would stay yellow and no peak would be seen at 585 nm in the UV-vis analysis). The assay in this study was performed with an initial H2O2 concentration of 0.2 mM. The catalyst concentration for the same sample under different treatments was kept roughly the same. Further work is on-going to determine the enzyme loading in different samples using ICP-MS.

As shown in Figures 8 and 9, the free catalase (CAT) loses much of its activity after DMF treatment for 15 min (Figure 8(a)), whilst the catalytic activity of the CAT@HOF biocomposites is almost unaffected (Figure 9(a)). Similarly, the HOF shell provides a certain amount of protection for the encapsulated CAT for heat treatment at 70 °C for 5 minutes (Figure 9(b)) whereas free CAT is completely inactivated by the same heat treatment at 70 °C for 5 minutes (Figure 8(b)).

For comparison, the concentration of the CAT for all the tests was kept the same. The assay giving the data shown in Figure 8 was performed with initial H2O2 concentration of 0.20 mM. After 5 min of reaction with H2O2, 50 pL of reaction solution was added to 950 pL of FOX solution and left at room temperature for 30 min before the UV-vis analysis.

Also, for comparison the concentration of the CAT@CC44 for all the experiments was kept the same. The assay giving the data shown in Figure 9 was performed with initial H2O2 concentration of 0.20 mM. After 5 min of reaction with H2O2, 50 pL of reaction solution was added to 950 pL of FOX solution and left at room temperature for 30 min before the UV-vis analysis.

EXAMPLE 6

Protective capacity afforded to FCAT by the encapsulating HOF temperature

Enzymes are also known to be sensitive to elevated temperatures. Thus, we assessed the enzymatic activity for free FCAT and FCAT@BioHOF-l treated at 60 °C over a 30 min period. The enzymatic assays were performed in phosphate buffer (pH 8, 0.1 M) with H2O2 (0.15 mM). The total volume of the reaction mixture was fixed to 1 mL. V ₀ bs is summarized in the inset of Figures 10(a), 10(b), and 11, and in Table 2. Numbers in brackets indicate the standard deviation of measurements. The error bars indicate the standard deviation of two independent measurements. The FCAT dosage in the assay for FCAT and FCAT@BioHOF- 1 were 2.6 and 2.9 pg (based on FCAT, determined by ICP-MS), respectively.

Table 2 - Reaction rates from Figures 10(a) and 10(b)

The respective activites are shown in Figures 10 and 11 as a percentage of the activity of a control experiment carried out at room temperature (RT, 25 + 1 °C). Free FCAT rapidly lost activity from 1.23 mM s ¹ pgFCAT ¹ at RT to 0.02 pM s ¹ pgFCAT ¹ after exposure to 60 °C temperatures for 30 min (S13, and S14). However, the BioHOF-1 coating significantly enhanced the thermostability of the encapsulated enzyme maintaining 79% of its initial activity after 30 min at 60 °C EXAMPLE 7

Protective capacity afforded to FCAT by the encapsulating HOF proteolytic agent We also checked the protection effect of the CAT/HOF composite over proteolytic agent (trypsin). Trypsin is a serine protease, which hydrolyses proteins and makes them lose their biological function. As shown in Figure 23(a), CAT loses part of its enzymatic activity after exposure to 2 mg mL ¹ trypsin for 6 h, which is indicated by the slower H2O2 decomposition rate shown in Figure 23(a).

In contrast, the enzymatic function of the CAT@CC44 system remains unchanged upon exposure to the proteolytic reagent trypsin, as shown in Figure 23(b). This protective effect is attributed to the small pore aperture size of the HOF shell which would size-exclude the trypsin and avoid its contact with the encapsulated CAT.

For comparison, the concentration of the CAT for all the tests was the same. The assay providing the data shown in Figure 23 was performed with initial H2O2 concentration of 0.20 mM. After 5 min of reaction with H2O2, 50 pL of reaction solution was added to 950 pL of FOX solution and left at room temperature for 30 min before the UV-vis analysis.

EXAMPLE 8

Protective capacity afforded to FCAT by the encapsulating HOF proteolytic agent Biological activity of (a) free FCAT and (b) FCAT@BioHOF-l after proteolytic agent treatment (2 mg mL ¹ trypsin in 0.1 M pH 8 phosphate buffer for 10-120 min at room temperature). The enzymatic assays were performed in phosphate buffer (pH 8, 0.1 M) with H2O2 (0.15 mM). The total volume of the reaction mixture was fixed to 1 mL. V ₀ bs is summarized in the inset of Figures 12(a) and 12(b), and in Table 3. Numbers in brackets indicate the standard deviation of measurements. The error bars indicate the standard deviation of two independent measurements. The FCAT dosage in the assay for FCAT and FCAT@BioHOF-l were 2.6 and 2.9 mg (based on FCAT, determined by ICP-MS), respectively.

Table 3 - Reaction rates from Figures 12(a) and 12(b)

FCAT@BioHOF-l retains 78% of its original activity while free FCAT is completely deactivated (Figures 12 and 13). This result can be explained as the ca. 6.4 A pore diameters of BioHOF-1 would restrict access of trypsin (from Porcine pancreas , 23.8 kDa, approximated as an ellipsoid with dimensions of 4.8 x 3.7 x 3.2 nm) to the embedded enzyme. It is noteworthy that after exposure to trypsin the initial activity of FCAT @ BioHOF-1 decreases then remains constant after 30 min, a possible explanation being that the initial loss of activity is due to the decomposition of surface bound enzyme. Figure 13 shows the relative activity (%) of free FCAT and FCAT @ BioHOF-1 as a function of incubation time in Trypsin (2 mg mL ¹ ) in phosphate buffer (pH 8, 0.1 M). In the enzymatic tests, of H2O2 (0.15 mM) was used as a substrate.

EXAMPLE 9

Protective capacity afforded to FCAT by the encapsulating HOF unfolding agent

Biological activity of (a) free FCAT and (b) FCAT@BioHOF-l after treatment with an unfolding agent (urea (6 M) in phosphate buffer (pH 8, 0.1 M) for 30 min). The enzymatic assays were performed in phosphate buffer (pH 8, 0.1 M) with H2O2 (0.15 mM). The total volume of the reaction mixture was fixed to 1 mL. V,,bs is summarized in the inset of Figures 14(a) and 14(b) and in Table 4. Numbers in brackets indicate the standard deviation of measurements. The error bars indicate the standard deviation of two independent measurements. The FCAT dosage in the assay for FCAT and FCAT@BioHOF-l were 2.6 and 2.9 pg (based on FCAT, determined by ICP-MS), respectively. After urea (6 M) treatment for 30 min, FCAT and FCAT@BioHOF-l retained 6.08 ± 0.18 and 74.37 ± 2.17% of their original activity, respectively.

Table 4 - Reaction rates from Figures 14(a) and 14(b)

EXAMPLE 10

Figure 15 shows simulated and experimental powder X-ray diffraction patterns of the as- synthesized FCAT @ Bio FIOF-1 and composites after testing their stability as described in Examples 4-9. The PXRD patterns of FCAT@BioHOF-l composites after exposure to elevated temperature, a pH range of 5-10 and trypsin each possess the characteristic HOF peaks at 20 values of 8.6 and 17.4 ⁰ confirming retention of crystallinity (Figure 15).

EXAMPLE 11

Catalytic performance ofFAOx and FAOx@ BioHOF-1 composite, and protective capacity afforded to FAOx by the encapsulating HOF - temperature

We further explored the protective capacity of the BioHOF-1 coating by encapsulating alcohol oxidase (AOx, from Pichia pastoris), which catalyzes the oxidation of short aliphatic alcohols to formaldehyde with concomitant production of H2O2. Given that the FAOx @ BioHOF-1 was enzymatically active, we examined the HOF’s protective capacity when composites were exposed to elevated temperature, trypsin and urea. Notably, the BioHOF-1 coating offered significant protection to the embedded enzyme compared to free FAOx.

The enzymatic activity of FAOx and the FAOx@BioHOF-l composite were measured according to a modified protocol from Sigma- Aldrich. One tablet of 2,2'-azino-/?;.s-(3- ethylbenzothiazoline-6-sulfonic acid) (ABTS, Sigma-Aldrich, 10 mg substrate per tablet) was dissolved in potassium phosphate buffer (pH 7.5, 0.1 M, 10 mL) to form solution A. Oxygen gas was bubbled through solution A for ~5 minutes before use. Solution B (-250 units mL ¹ of peroxidase solution) was prepared by dissolving Peroxidase from horseradish (HRP, Sigma-Aldrich, 148 units mg ¹ , 1.7 mg) in Milli-Q ¾0 (1 mL). In a typical assay test, solution A (1 mL), Milli-Q ¾0 (1.8 mL), solution B (0.01 mL), and aqueous methanol solution (methanol (0.01 mL) in Milli-Q H2O (1 mL), 0.1 mL) were mixed in a cuvette (light path = 1 cm) under magnetic stirring. Thereafter, free FAOx or FAOx@BioHOF-l stock solution (0.1 mL) was introduced and the reaction mixture was monitored continuously at 405 nm under stirring conditions. The reaction rate was determined using the maximum linear rate of the increase of A405 _nm .

In the total reaction mixture (3.01 mL), the final chemical concentrations are: potassium phosphate (33 mM), 0.66 mM ABTS (0.66 mM), aqueous methanol (0.033% (v/v)), and HRP (2.5 units). The generation of H2O2 in the system was calculated according to the formation of oxidized ABTS in the system (ABTS, extinction coefficient at 405 nm = 36.8 mM ¹ cm ¹ ).

Methanol

H ₂ 0 ₂ + ABTS 2H ₂ 0 + ABTS (oxidized)

Figure 16 shows the activity of the FAOx@BioHOF-l which showed that the composite retained 58% of the activity of free FAOx (3.6 ± 0.2 and 2.1 ± 0.2 mM min ¹ pg _FAOx ¹ for free FAOx and FAOx@BioHOF-l, respectively) (Figures 16(a) and 16(b), and summarized in Table 5). Table 5 - Reaction rates from Figures 14(a) and 14(b)

Catalytic activity of FAOx (a) and FAOx@BioHOF-l(b) after thermal treatment (60, 70, and 80°C for 10 min) in water. After thermal treatment, the solution of FAOx or FAOx@BioHOF-l was cooled down on ice before the measurement of their activity. The error bars indicate the standard deviation of two independent measurements. The dosage of FAOx and FAOx@BioHOF-l in the assay test is 9.4 and 11.2 pg (based on the amount of FAOx, calculated from ICP-MS), respectively. A noteworthy result of this current work is that, in our hands, alcohol oxidase retains enzymatic activity within the HOFs; however, the same enzyme shows no activity when adsorbed on, or encapsulated in ZIF materials. In summary, the composite maintained 85% of its original activity after heating at 60 °C (compared to 20% for the free enzyme).

EXAMPLE 12

Protective capacity afforded to FAOx by the encapsulating HOF - proteolysis

The catalytic activity of (a) FAOx and (b) FAOx@BioHOF-lbefore and after proteolytic agent treatment (trypsin (2 mg mL ¹ ) in phosphate buffer (pH 8, 0.1 M) for 2 h) is shown in Figures 17(a) and 17(b). The error bars indicate the standard deviation of two independent measurements. The dosage of FAOx and FAOx@BioHOF-l in the assay test is 9.4 and 11.2 pg (based on the amount of FAOx, calculated by ICP-MS), respectively. The values of V _obs for FAOx and FAOx@BioHOF-l after proteolysis were 0.5 ± 0.1 and 1.5 ± 0.1 pM min ¹ pgFAOx ¹ , respectively. Figure 17. In summary, the composite maintained 70% of its original activity after exposure to trypsin (compared to 14% for the free enzyme).

EXAMPLE 13

Protective capacity afforded to FAOx by the encapsulating HOF unfolding agent

Catalytic activity of (a) FAOx and (b) FAOx @BioHOF-lbef ore (black) and after (red) unfolding agent treatment (urea (6 M) in phosphate buffer (pH 8, 0.1 M) for 30 min). The error bars indicate the standard deviation of two independent measurements. The dosage of FAOx and FAOx@BioHOF-l in the assay test is 9.45 and 11.2 pg (based on the amount of FAOx, calculated by ICP-MS), respectively. The values of V _0bs for FAOx and FAOx@BioHOF-l after treatment were 0.54 ± 0.07 and 1.52 ± 0.09 mM min ¹ pg _FAOx ¹ , respectively. In summary, the composite maintained about 71% of its original activity after exposure to urea (compared to 15% for the free enzyme).

EXAMPLE 14

Figure 18 shows simulated and experimental PXRD patterns of the as-synthesized FAOx@BioHOF-l and composites after thermal (60, 70, or 80 °C for 10 min), proteolytic agent treatment (Trypsin (2 mg mL ¹ ) in phosphate buffer(pH 8, 0.1 M) or unfolding agent treatment (urea (6 M) in phosphate buffer (pH 8, 0.1 M)), as described in Examples 11-13.

EXAMPLE 15

Synchrotron X-ray diffraction formation kinetics of HOF biocomposites

Time-resolved SAXS experiments were performed using the stop-flow setup available at the SAXS beamline. The kinetics of the nucleation, growth and crystallization of the HOF particles were monitored in solution (time resolution 100 ms). In this study, BSA was used as a model biomolecule. The growth of bioHOF-1 particles was studied in presence and absence of the protein to investigate the role of BSA in their nucleation and growth.

The time evolution the integrated intensity of the (100) diffraction peak at 6.1 nm ¹ (I (q=6.1 nm ¹ )) and Invariant (Q) for both the bare BioHOF-1 and for the BSA@BioHOF-l composite material, with Q in the integral scattering intensity of an ensemble of particles and is calculated in the q range from 0.1 nm ¹ to 0.7 nm ¹ . As evidenced in Figure 19, the presence of BSA does not significantly influence the trend of Q and I (q=6.1 nm ¹ ).

In the presence or absence of BSA, the nuclei appear to have already formed within the first 100 ms. Then, Q slightly increases during the first 10 s of the reaction suggesting a predominant process related to the reorganization of already formed particles. ⁷ After 10 seconds, Q started to decrease and plateau after 100 s. This trend could be related the amorphous particles formed within the first 10 s crystallizing in the remaining 100 s. This hypothesis is supported by the analysis of the evolution of the intensity of the (110) diffraction peak of the BioHOF-1 at 6.1 nm ¹ . In fact, within the first 50 s of measurements, no diffraction peaks were observed. Conversely, after 50 s, it was possible to clearly observe the (110) diffraction peak of the BioHOF-1 and to monitor the increasing of its integrated area over time. In both cases, the integrated area of the (110) peak reached its maximum after around 200 seconds.

We monitored the kinetics of the nucleation, growth and crystallization of HOF particles (time resolution 100 ms) in the presence and absence of bovine serum albumin (BSA). Our data show that nuclei formed within the first 100 ms both in the presence and absence of BSA. Subsequently, in both cases, the nuclei form amorphous particles which crystallize within 100 s. This interpretation is supported by monitoring the intensity of the (110) diffraction peak of BioHOF-1 at 6.1 nm ¹ . After 50 s of mixing, it was possible to clearly observe the (110) diffraction peak of the BioHOF-1 which increased in intensity until it reached a maximum after ca. 200 s. These experiments suggest that on time scales > 100 ms, HOF growth is not influenced by the presence of BSA and that encapsulation occurs via particle aggregation. The kinetic of formation of BioHOF-1 crystals was further investigated via optical microscopy. The solutions of the two ligands were mixed in a vial and then 5 pL of the mixture was immediately drop-cast on a microscope slide. Several images of the drop-casted mixture were collected at different growth times using a 20x objective (not shown). It is possible to identify the presence of a large number of small particles after 30 s. Micrometries BioHOF-1 crystals with needle-like morphology could be observed after 60 s. The number of these needle-like crystals rapidly increased in the first 120 s of the synthesis. Afterwards, the number of visible crystals slowly grew until the apparent completion of the synthesis after 300 seconds. These images further support the conclusion made from the analysis of the time-resolved SAXS data. With the employed set-up and in the investigated timeframe, the presence of BSA did not play a significant role in the kinetic of the particle growth. The mechanism of formation of BSA@BioHOF-l composite material could be defined as an encapsulation process.

EXAMPLE 16

Synchrotron Small Angle X-ray Scattering (SAXS) porosity characterisation To further investigate the hierarchical pore structure of the BSA@BioHOF-l biocomposite, the SAXS patterns were analyzed after 15 min of growth. The fitted the SAXS patterns of both the BioHOF-1 and BSA@BioHOF-l were fitted with a hierarchical model of large structures represented by the HOF crystals with non-correlated pores replicated by the BSA into the HOF crystals. Considering the large size of the HOF particles, the BSA@BioHOF- 1 data fit showed the presence of two distinctive components: 1) a Power Law component related to the large particles (>300 nm) and 2) a Guinier knee that is related to the presence of mesopores. This Guinier knee has been modeled by non-interacting spherical pores following a Schulz distribution for their number size distribution. The Guinier Radius has been calculated from the mean size of the volume size distribution. The calculated values for the radius of gyration (R _g ) of the mesopores is 4.9 ± 0.5 nm. The size of these mesopores is sufficient to accommodate isolated BSA molecules (R _g = 3.02 nm ).

Conversely, pure HOF obey only to a Power law, which is related to the aggregation of the large HOF particles. Thus, mesopores are absent in pure HOF. This hierarchical pore structure is compatible with the one reported models of porous biocomposites encapsulating BSA. Further, the sample possess pore dimensions (limiting pore diameter ca. 6.4 A) that significantly exceed those found ZIF-based materials of sodalite topology (3.4 A).

COMPARATIVE EXAMPLE 1

For comparison with the HOF biocomposites obtained with the procedures described in the previous Examples, comparative samples were prepared by adsorbing FCAT onto the external surface of pre-formed HOFs (herein "FCAT-on-BioHOF-1"). The comparative samples were then exposed to an unfolding agent (urea) and the biological activity of the samples monitored.

Biological activity of FCAT-on-BioHOF-1 before and after unfolding agent treatment (urea (6 M) in phosphate buffer (pH 8, 0.1 M) for 30 min). The enzymatic assays were performed in phosphate buffer (pH 8, 0.1 M) with H2O2 (0.15 mM). The total volume of the reaction mixture was fixed to 1 mL. V,,bs is summarized in the inset of Figure 20 and in Table 6. Numbers in brackets indicate the standard deviation of measurements. The error bars indicate the standard deviation of two independent measurements. The FCAT dosage in the assay for FCAT-on-BioHOF-1 was 5 pg (calculated by Bradford assay). After urea (6 M) treatment for 30 min, FCAT-on-BioHOF-1 retained 28.3 ± 3.3% of its original activity. As expected, the activity of the surface bound FCAT is significantly reduced after treatment with urea. Table 6 - Reaction rates from Figure 20

FAOx-on-BioHOF-1 was synthesized by mixing FAOx (0.2 mL of 2 mg mL ¹ FAOx stock solution) with the as-synthesized BioHOF-1 (5 mg) in FhO at room temperature for 1 h. Thereafter, the solid was recovered by centrifugation and washed with FhO (3 times) to remove the excess FAOx. All supernatants were collected. The concentration of FAOx in the supernatant was determined by the Bradford assay. The amount of FAOx adsorbed on FAOx-on-BioHOF-1 was calculated by the difference between the FAOx used in the synthesis and that in the collected supernatant.

Figure 21 shows the catalytic activity of FAOx-on-BioHOF-1 before (black) and after (red) unfolding agent treatment (urea (6 M) in phosphate buffer (pH 8, 0.1 M) for 30 min). The error bars indicate the standard deviation of two independent measurements. The dosage of FAOx-on-BioHOF-1 in the assay test is 10.5 pg (based on the amount of FAOx, calculated by Bradford assay). The values of Vobs for FAOx-on-BioHOF-1 before and after treatment were calculated to be 1.53 ± 0.07 and 0.26 ± 0.02 mM min ¹ pg _FAOx ¹ , respectively. After trypsin treatment, only 17.08 ± 1.33% of the original activity was retained for FAOx-on- BioHOF-1 sample.

COMPARATIVE EXAMPLE 2

Fresh comparative samples of FCAT-on-BioHOF-1 were also exposed to a proteolytic agent (trypsin) and the biological activity of the samples monitored.

Biological activity of FCAT-on-BioHOF-1 before and after proteolytic agent treatment (trypsin (2 mg mL ¹ ) in phosphate buffer (pH 8, 0.1 M) for 30 min). The enzymatic assays were performed in phosphate buffer (pH 8, 0.1 M) with H2O2 (0.15 mM). The total volume of the reaction mixture was fixed to 1 mL. The FCAT dosage in the assay for FCAT-on- BioHOF-1 was 5 mg (calculated by Bradford assay). After trypsin treatment, 24.8 ± 1.6% of its original activity was retained for FCAT-on-BioHOF-1 sample. As expected, the activity of the surface bound FCAT is significantly reduced after treatment with tripsin.

Figure 22 shows the catalytic activity of FAOx-on-BioHOF-1 before and after proteolytic agent treatment (trypsin (2 mg mL ¹ ) in phosphate buffer (pH 8, 0.1 M) for 2 h). The error bars indicate the standard deviation of two independent measurements. The dosage of FAOx-on-BioHOF-1 in the assay test is 10.5 pg (based on the amount of FAOx, calculated by Bradford assay). The values of V _obs for FAOx-on-BioHOF-1 before and after proteolysis were calculated to be 1.53 ± 0.07 and 0.11 ± 0.0 pM min ¹ pg _FAOx ¹ , respectively. After trypsin treatment, only 7.11 ± 0.42% of the original activity was retained for FAOx-on- BioHOF-1 sample.

EXAMPLE 17

Synthesis of rigid (4+4) HOF systems

Tetraamidinium(4 mg) was dissolved in H2O (2: 1) (1.00 mL) to form Solution A. The dicarboxylic acid (3 mg) was dissolved in H ₂ q/NH ₄ qH(1%) (38: 18:2)( 1 mL) to form solution B. Solution B (50 pL)was added to stirring solution A over 20 s intervals. The mixture was then left stirring after addition for 1 h to ensure the completion of the synthesis.

Synthesis of rigid (4+2) HOF systems

Tetraamidinium (4 mg) was dissolved in Acetone/H ₂ 0 (2: 1) (1.00 mL) to form Solution A. The dicarboxylic acid (3 mg) was dissolved in Acetone/H ₂ 0/NH ₄ 0H(l%) (38: 18:2)(1 mL) to form solution B. Solution B (50 pL) was added to stirring solution A over 20 s intervals. The mixture was then left stirring after addition for 1 h to ensure the completion of the synthesis Synthesis of flexible (4+2) HOF systems

Tetraamidinium (4 mg) was dissolved in Acctonc/FLO (2:1)(1.00 mL) to form Solution A. The dicarboxylic acid (4-12 eq.) was dissolved in Acetone/H ₂ 0/NH ₄ 0H(28%) (40: 19: 1)(1 mL) to form solution B. Solution B (50 pL) was added to stirring solution A over 20 s intervals. The mixture was then left stirring after addition for 1 h to ensure the completion of the synthesis.

Synthesis of Protein@(4+2)HOF

Tetraamidinium (4 mg) was dissolved in Acctonc/FLO (2:1) (0.75 mL) to form Solution A. BSA enzyme (0.5 mg, 0.25 mL of 2 mg/mL stock solution in Acctonc/FLO (2:1)) was added into solution A and stirred for around 10 min at room temperature to form solution B. Tetracarboxylate(3 mg) was dissolved in Acetone/H ₂ 0/NH ₄ 0H(l%) (40:18:2)(1 mL) to form solution C. Solution C (50 pL) was then added to a stirring solution B over 20 s intervals. The mixture was then left stirring after addition for 1 h to ensure the completion of the synthesis. The XRD poattern of the resulting samples are shown on Figure 24(a).

Synthesis of Yeast@(4+2)HOF

Cultured yeast (2 mg) was added to biphenyldicarboxylate(3 mg) that was dissolved in Acetone/H ₂ 0/NH ₄ 0H(l%) (40:18:2)(1 mL) to form solution A. Tetraamidinium (4 mg) was dissolved in Acetone/FLO (2:1) (1 mL) to form Solution B. The two solutions were combined and stirred for 10 minutes to ensure the coating of yeast cells. The XRD poattern of the resulting samples are shown on Figure 24(b).

Synthesis of Yeast@ (4+4 )HOF

Cultured yeast (2 mg) was added to biphenyldicarboxylate(3 mg) that was dissolved in H ₂ q/NH ₄ qH(1%) (40: 18:2)(1 mL) to form solution A. Tetraamidinium(4 mg) was dissolved in FLO (1 mL) to form Solution B. The two solutions were combined and stirred for 10 minutes to ensure the coating of yeast cells. The XRD poattem of the resulting samples are shown on Figure 25(a).

Synthesis of Insulin@(4+2)HOF

Tetraamidinium(4 mg) was dissolved in Acetone/FbO (2:1) (0.75 mL) to form Solution A. The enzyme (0.5 mg, 0.25 mL of 2 mg/mL stock solution in Acctonc/FLO (2:1)) was added into solution A and stirred for around 10 min at room temperature to form solution B. Tetracarboxylate(3 mg) was dissolved in Acetone/H ₂ 0/NH ₄ 0H(l%) (40: 18:2)(1 mL) to form solution C. Solution C (50 pL)was then added to a stirring solution B over 20 s intervals. The mixture was then left stirring after addition for 1 h to ensure the completion of the synthesis. The XRD poattern of the resulting samples are shown on Figure 25(b).

Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

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