WO2020252536A1 - Systèmes de réseaux organiques à liaisons hydrogène - Google Patents

Systèmes de réseaux organiques à liaisons hydrogène Download PDF

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WO2020252536A1
WO2020252536A1 PCT/AU2020/050624 AU2020050624W WO2020252536A1 WO 2020252536 A1 WO2020252536 A1 WO 2020252536A1 AU 2020050624 W AU2020050624 W AU 2020050624W WO 2020252536 A1 WO2020252536 A1 WO 2020252536A1
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hof
bio
molecule
solution
biohof
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Nicholas White
Christian DOONAN
Christopher SUMBY
Weibin LIANG
Paolo Falcaro
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The University Of Adelaide
Graz University Of Technology
The Australian National University
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Application filed by The University Of Adelaide, Graz University Of Technology, The Australian National University filed Critical The University Of Adelaide
Publication of WO2020252536A1 publication Critical patent/WO2020252536A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
    • B01J31/02—Catalysts comprising hydrides, coordination complexes or organic compounds containing organic compounds or metal hydrides
    • B01J31/04—Catalysts comprising hydrides, coordination complexes or organic compounds containing organic compounds or metal hydrides containing carboxylic acids or their salts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C257/00—Compounds containing carboxyl groups, the doubly-bound oxygen atom of a carboxyl group being replaced by a doubly-bound nitrogen atom, this nitrogen atom not being further bound to an oxygen atom, e.g. imino-ethers, amidines
    • C07C257/10—Compounds containing carboxyl groups, the doubly-bound oxygen atom of a carboxyl group being replaced by a doubly-bound nitrogen atom, this nitrogen atom not being further bound to an oxygen atom, e.g. imino-ethers, amidines with replacement of the other oxygen atom of the carboxyl group by nitrogen atoms, e.g. amidines
    • C07C257/18—Compounds containing carboxyl groups, the doubly-bound oxygen atom of a carboxyl group being replaced by a doubly-bound nitrogen atom, this nitrogen atom not being further bound to an oxygen atom, e.g. imino-ethers, amidines with replacement of the other oxygen atom of the carboxyl group by nitrogen atoms, e.g. amidines having carbon atoms of amidino groups bound to carbon atoms of six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
    • C07C51/43—Separation; Purification; Stabilisation; Use of additives by change of the physical state, e.g. crystallisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/04—Peptides being immobilised on, or in, an organic carrier entrapped within the carrier, e.g. gel, hollow fibre
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
    • B01J31/003—Catalysts comprising hydrides, coordination complexes or organic compounds containing enzymes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03013—Alcohol oxidase (1.1.3.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/01—Peroxidases (1.11.1)
    • C12Y111/01006—Catalase (1.11.1.6)

Definitions

  • the invention relates to hydrogen-bonded organic framework (HOF) and methods for producing the same.
  • Hydrogen-bonded organic frameworks 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.
  • 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.
  • HEF crystalline hydrogen-bonded organic framework
  • 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.
  • the bio-molecule is selected from a polyamino acid, a penicillin, a peptide, a protein, a nucleic acid, a nucleocapsid, and a combination thereof.
  • bioactivity of bio-molecules is strongly related to their spatial conformation.
  • encapsulation of a bio-molecule according to the invention can retain the native conformation of the bio-molecule.
  • the encapsulated bio-molecule can maintain its native bioactivity.
  • 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.
  • 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.
  • 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.
  • Metal species e.g. metal ions
  • the present invention also provides crystalline HOF having a framework that defines intrinsic cavities and encapsulates a bio-molecule.
  • the bio-molecule has a smallest dimension that is larger than the size of any intrinsic cavity of the framework.
  • 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.
  • 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.
  • 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.
  • FIG. 5 shows PXRD patterns for CC44 and CS144 HOFs, and corresponding HOFs encapsulating the enzyme catalase (CAT), i.e. CAT@CC44, and CAT@CSi44,
  • CAT enzyme catalase
  • 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,
  • CAT catalase
  • 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 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.
  • the present invention provides a method for producing a hydrogen-bonded organic framework (HOF).
  • HEF hydrogen-bonded organic framework
  • Hydrogen-bonded organic frameworks are frameworks of organic linkers interconnected through hydrogen-bonding interactions to form an open cage/scaffold-like structure that defines spatially ordered cavities.
  • 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.
  • HOFs 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.
  • hydrophilicity 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.
  • 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.
  • 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.
  • DAT 2,4-diaminotriazine
  • amide amino, cyano, benzimidazolone
  • imide imidazole
  • amidinium boronic acid
  • resorcinol pyridine
  • guanidinium sulfonate
  • 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.
  • the organic core is an aromatic organic core.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the HOF of the invention is crystalline.
  • 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.
  • HOFs arises from regular and spatially ordered distribution of intrinsic cavities forming the framework.
  • 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.
  • 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.
  • 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.
  • 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.
  • the average dimension of the intrinsic cavities of a crystalline HOF are quantified by gas absorption measurements.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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-diaminostria
  • 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)
  • the HOF precursor is made of identical organic linkers.
  • the resulting HOF is a“single component” HOF.
  • the HOF precursor comprises a combination of at least two types of organic linkers.
  • the resulting HOF is a“mixed-component” HOF.
  • the HOF precursor is used in solution with a deprotonating agent.
  • 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 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.
  • suitable solvents for use in the invention include methanol, ethanol, dimethyl sulfoxide (DMSO), acetone, water, and mixtures thereof.
  • the solution into which the bio-molecule and HOF precursor are combined is an aqueous solution.
  • 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)).
  • 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-(
  • the HOF precursor may be present in the solution in any amount that ensures formation of HOF.
  • 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).
  • 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.
  • the weight ratio between the different types of organic linkers may be any weight ratio that is adequate for the formation of HOF.
  • the HOF precursor is made of two types of organic linkers.
  • the weight ratio between the two types of organic linkers may be any weight ratio that is adequate for the formation of HOF.
  • 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.
  • 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.
  • the HOF encapsulates the bio-molecule.
  • the HOF encapsulating" the bio-molecule, the molecular structure of the HOF surrounds the entire bio-molecule.
  • 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.
  • 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.
  • 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.
  • the HOF may form first, and "encapsulation" of the (small) bio molecule results from the bio-molecule spontaneously infiltrating the formed HOF.
  • 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.
  • 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.
  • the solution temperature may be between -50°C and 75°C, between -50°C and 50°C, or between -50°C and 30°C.
  • the method is performed at room temperature.
  • 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.
  • 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.
  • the bio-molecule may be added directly to a solution of the HOF precursor.
  • the HOF precursor may be added to a solution of the bio-molecule.
  • the bio-molecule may be introduced at any stage of the procedure.
  • 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.
  • the HOF precursor 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.
  • 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.
  • 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.
  • CLSM confocal scanning laser microscope
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • organic solvents e.g. urea
  • 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.
  • 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.
  • HEF crystalline hydrogen- bonded organic framework
  • the present invention also provides crystalline HOF having a framework that defines intrinsic cavities and encapsulates a bio-molecule.
  • 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.
  • 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.
  • PDB Protein Data Bank
  • 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.
  • 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.
  • the "smallest dimension" of the protein or nucleic acid is the smallest of a, b and c.
  • 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.
  • 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.
  • the smallest dimension of the bio-molecule can advantageously be any degree larger than the size of any intrinsic cavity of the HOF.
  • 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.
  • 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.
  • the bio-molecule is an amino acid.
  • amino acid refers to an organic acid containing both a basic amino group (NH2) and an acidic carboxyl group (COOH).
  • NH2 basic amino group
  • COOH acidic carboxyl group
  • 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.
  • 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.
  • 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.
  • the bio-molecule is a peptide.
  • 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.
  • the peptide is a modified peptide.
  • 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.
  • 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.
  • the bio-molecule is a protein.
  • 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.
  • 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.
  • plasma proteins include, but are not limited to Albumin (HSA), haemoglobin, thrombin, fibronectin, fibrinogen, immunoglobulins, coagulation factors (FX, FVIII, FIX)).
  • extracellular proteins include, but are not limited to collagen, elastin, keratin, actin, tubulin, myosin, kinesin and dynein.
  • hormones and growth factors include, but are not limited to insulin, EGF, VEGF, FGF, insulin like growth factor, androgens, estrogens.
  • antigen proteins include, but are not limited to ovalbumin (OVA), keyhole limpet hemocyanin and bovine serum albumin (BSA) and immunoglobulins.
  • OVA ovalbumin
  • BSA bovine serum albumin
  • Proteins that can be used in the invention include enzymes.
  • 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.
  • enzymes used in the textile industry include, but are not limited to endoglucases, oxidases, amylases, proteases cellulases and xylanases.
  • 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.
  • HRP horse radish peroxidase
  • urease urease
  • RNA or DNA enzymes such as ribonuclease.
  • 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.
  • the bio-molecule is a nucleic acid.
  • nucleic acid 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), micro RNAs (miRNAs), micro RNAs (miRNAs), micro
  • nucleic acid includes non-naturally occurring modified forms, as well as naturally occurring forms.
  • the nucleic acid molecule comprises from about 8 to about 80 nucleobases ⁇ i.e. from about 8 to about 80 consecutively linked nucleic acids).
  • 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,
  • 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.
  • the bio-molecule is a composite bio-molecule.
  • the bio-molecule is a bioactive entity resulting from the combination of two or more types of bio-molecules described herein.
  • the bio-molecule may be the combination of a protein and a nucleic acid in the form of a nucleocapsid.
  • 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.
  • 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.
  • Fluorescein isothiocyanate 0.5 mg
  • CAT catalase
  • bovine liver 2000-5000 units mg 1 protein, 40 mg
  • carbonate -bicarbonate aqueous buffer solution 0.1 M, pH 9.2, 4 mF
  • the FITC-tagged CAT 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.
  • 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. 3 ⁇ 42 (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.
  • 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.
  • PXRD powder X-Ray Diffraction
  • Fluorescein-tagged CAT was used in the synthesis of FCAT@BioHOF-l to study the spatial localization of the enzyme within the HOF structure.
  • FCAT- on-BioHOF-1 sample was also synthesized via surface adsorption of the FCAT on as- synthesized FCAT @ BioHOF-1.
  • CLSM technique Olempus 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.
  • 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.
  • HNO3/HCI 0.25 mL of 70% HNO3 (Ajax) and 0.25 mL of 37% HC1 (Chem- supply)
  • 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 3 ⁇ 40 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.
  • 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.
  • 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.
  • 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).
  • reaction rate ( obs , mM s 1 ) is defined as the initial H2O2 decomposition velocity of the enzymatic assay.
  • H2O2 standard solution 50 pL
  • 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.
  • V7 / « The H2O2 decomposition rate
  • neat BioHOF-1 crystals do not catalyze the decomposition of H2O2 .
  • the supernatant of the FCAT@BioHOF-l composite showed negligible enzymatic acitivty which suggests that enzyme leaching is minimal.
  • 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.
  • 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 is an iron-heme enzyme that catalyses the decomposition of hydrogen peroxide to water and oxygen.
  • CAT Catalase
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • BioHOF-1 coating was encapsulating alcohol oxidase (AOx, from Pichia pastoris), which catalyzes the oxidation of short aliphatic alcohols to formaldehyde with concomitant production of H2O2.
  • AOx alcohol oxidase
  • 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 3 ⁇ 40 (1 mL).
  • HRP horseradish
  • Milli-Q 3 ⁇ 40 1.8 mL
  • solution B (0.01 mL)
  • 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.
  • 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).
  • 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).
  • 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.
  • SAXS Synchrotron Small Angle X-ray Scattering
  • 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.
  • pure HOF obey only to a Power law, which is related to the aggregation of the large HOF particles.
  • 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 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.
  • FCAT-on-BioHOF-1 pre-formed HOFs
  • 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.
  • Fresh comparative samples of FCAT-on-BioHOF-1 were also exposed to a proteolytic agent (trypsin) and the biological activity of the samples monitored.
  • trypsin proteolytic agent
  • 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.
  • 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.
  • 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.
  • 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)
  • 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 devistern of the resulting samples are shown on Figure 24(a).
  • 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)
  • 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 devistern of the resulting samples are shown on Figure 25(b).

Abstract

L'invention concerne un procédé de production d'un réseau organique à liaisons hydrogène (HOF) cristallin qui encapsule une bio-molécule, ce procédé comprenant l'association de cette bio-molécule et d'un précurseur de HOF dans une solution, le précurseur de HOF s'auto-assemblant pour former un HOF qui encapsule la bio-molécule.
PCT/AU2020/050624 2019-06-19 2020-06-19 Systèmes de réseaux organiques à liaisons hydrogène WO2020252536A1 (fr)

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