WO2018000043A1 - Coating material for cells - Google Patents

Abstract

The present invention relates to a cell coated with a layer of crystalline Metal Organic Framework (MOF).

Description

COATING MATERIAL FOR CELLS

FIELD OF THE INVENTION The invention relates in general to coating materials for cells. In particular, the invention relates to cells coated with non-biological material and to a method for preparing the same.

BACKGROUND OF THE INVENTION The handling and preservation of cells, for example single cells, without adversely affecting their viability is essential for the study of their complex biological functions, and for the development of cell-based clinical and industrial applications. Notions derived from the study of isolated cells are critical for the development of cell -based therapy of diseases, cell-based diagnostics, drug screening, and large scale development of products in the food and beverage industry.

However, the lipid bi-layer membrane encasing a single cell offers limited, if any, protection from environmental stresses. As a result, strategies for providing artificial long- term protection and preservation of the cells are often employed.

Prolonged cell viability and protection from environmental stresses may be afforded by the deposition of durable, synthetic coatings on a cell. A number of protective coating materials for cells have been proposed. For example, microencapsulation of whole population of cells in polymer membranes has been studied since the 1930s, and remains an important technological development in the field of tissue engineering and regenerative medicine.

On the other hand, encapsulation of single cells has shown promise in other cell-based therapies that require long term preservation and use on demand, although these extra requirements present even more challenges to ensure the coating is robust, stable but degradable when required. To that end a number of protective coating materials have been proposed, including silica, silica-titania, graphene, and polydopamine.

However, despite providing a certain degree of protection conventional cell coatings preclude the possibility to recover the cell in a viable state after it is coated, for example by removing the coating. Removal of conventional cell coatings typically requires noxious chemicals and harsh conditions, inevitably leading to cell death. In the cases of tissue engineering and regenerative medicine removal of the coating from the population of cells is not even necessary contemplated. Also, conventional cell coating materials are often not ideal in that they provide for an indiscriminate barrier against diffusion of cytotoxic molecules and cell nutrients equally. This in turn limits long-term preservation of cell viability.

Accordingly, there remains the opportunity to develop techniques for coating cells that provide for cell protection against environmental stresses, long-term preservation of cell viability, and/or post-coating recovery of the cell in a viable state for further use.

SUMMARY OF THE INVENTION The present invention provides a cell coated with a layer of crystalline Metal Organic Framework (MOF).

It has surprisingly now been found that coating a cell with a layer of crystalline MOF provides the cell not only with enhanced resistance to a variety of physicochemical environmental stresses, such as enzymatic attacks, chemical attacks, irradiation and/or heat, but that coating can advantageously be removed on-demand without adversely affecting viability of the cell. Furthermore, the unique porosity of crystalline MOF provides for a coating layer that preserves cell viability by acting as a size-selective permeable physical barrier. Specifically, the layer of crystalline MOF allows diffusion of nutrients into the cytoplasm while protecting the cell from the attack of cytotoxic agents. The present invention further provides a method of coating a cell with a layer of crystalline Metal Organic Framework (MOF), the method comprising combining in a solution the cell and MOF precursor compounds, wherein the cell promotes formation of the layer of crystalline MOF.

It has also surprisingly now been found that when in a solution with MOF precursor compounds, cells can promote formation of MOF in crystalline form. By being crystalline and forming around the cell, the MOF provides for a coating that is selectively permeable to nutrients, thus supporting cell viability while protecting the cell from cytotoxic agents.

MOFs are hybrid coordination structures formed by metal clusters comprising metal ions, e.g. metal ions or metal oxides, coordinated by multi-functional organic ligands. This results in the formation of one-, two- or three-dimensional structures that can be highly porous. The crystalline nature of a MOF arises from a regular and spatially ordered distribution of intrinsic cavities within the framework. The size of the intrinsic cavities is characteristic of each specific crystalline MOF and may range from 5 to 500 angstroms (A). The size distribution of the intrinsic cavities is extremely narrow, lending such materials to applications that require, for example, precise size selectivity of filtered or absorbed matter.

Advantageously, the layer of crystalline MOF is selectively permeable. Specifically, since the size distribution of a crystalline MOF's intrinsic cavities is narrow, the layer of crystalline MOF allows diffusion of cell nutrients from the external environment into the cell cytoplasm while protecting the cell from multiple external cytotoxic aggressors. This can significantly improve cell viability and reduce malnutrition of the coated cells.

The present invention can advantageously make use of a variety of different MOFs and a diverse range of cells. For example, the crystalline MOF may be a meso- or micro- MOF, and the cell may be a eukaryotic or a prokaryotic cell. As a result, a large number of cell/MOF combinations are available to address application-specific requirements. In some embodiments, the cell is one that has been isolated from a living organism (e.g. an animal, including a human, or plant). In other embodiments, the cell is isolated from a line of cells grown in an in vitro culture. In further embodiments, the cell is an artificial cell. Advantageously, the layer of crystalline MOF around the cell can be removed on-demand under physiochemical conditions that do not adversely affect cell viability. It will be appreciated that the programmed breaking-up of the cytoprotective coating is pivotal for the practical use of cells in sensors, drug delivery systems, cell therapy, or regenerative medicine.

The invention further provides for use of Metal Organic Framework (MOF) precursor compounds in coating a cell, wherein the MOF precursor compounds form a layer of crystalline MOF around the cell. 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 a schematic cross-section of a cell coated with a layer of crystalline ZIF-8;

Figure 2 shows a schematic illustration of a cyclic procedure involving the coating of a cell with a layer of MOF and cell separation from the crystalline MOF;

Figure 3 shows synchrotron small-angle X-ray scattering (SAXS) diffraction patterns of standard ZIF-8 crystals (black) and Saccharomyces cerevisiae yeast cells coated with ZIF- 8 (grey). The inset in Figure 3 shows the 2D SAXS pattern recorded for the cells coated with ZIF-8 (measured using a 0.5 mm capillary as a sample holder); Figure 4 shows SEM images of Saccharomyces cerevisiae yeast cells after incubation in solution with ZIF-8 precursor compounds for (a) 1 minutes, (b) 5 minutes, (c) 10 minutes, and (d) 20 minutes; Figure 4(e) shows an SEM image of a fragment of a layer of ZIF-8; Figure 5 shows (a) differential interference contrast (DIC) and (b) fluorescent microscopy images of Saccharomyces cerevisiae yeast cells coated with a layer of ZIF-8 using FDA as a fluorescent and viability indicator, and (c) FDA and (d) resazurin cell viability assay on the non-coated cells and cells coated with a layer of ZIF-8; Figure 6 shows (a) data from flow cytometry tests performed measuring the ATP metabolic activity of (b) non-coated Saccharomyces cerevisiae yeast cells and (c) Saccharomyces cerevisiae yeast cells coated with a layer of ZIF-8 over time, using resazurin as a fluorescent indicator; Figure 7 shows cell viability (%) of (1) non-coated Saccharomyces cerevisiae yeast cells (dark grey), and (2) non-coated Saccharomyces cerevisiae yeast cells in the presence of dispersed (i.e. non-coating) ZIF-8 (patterned dark grey) when exposed to cell lysis enzyme lyticase for 3 h, and yeast cells coated with a layer of ZIF-8 in the presence lyticase for 3 h (light grey) and 24 h (patterned light grey);

Figure 8 shows cell viability data relative to non-coated Saccharomyces cerevisiae yeast cells and Saccharomyces cerevisiae yeast cells coated with a layer of ZIF-8 after 24 h exposure to filipin, an anti-fungal drug; Figure 9 shows data relative to Saccharomyces cerevisiae yeast cells growth measurement (OD₆oo) for non-coated Saccharomyces cerevisiae yeast cells (black circles) and Saccharomyces cerevisiae yeast cells coated with a layer of ZIF-8 (grey circles) before and after dissolution of the layer of MOF by EDTA; Figure 10 shows SEM images of a cracked ZIF-8 layer from coated yeast cells obtained after (a) 1, (b) 2, (c) 3 and (d) 4 coating cycles, and (e) thickness values of the corresponding coating layer plotted against the cycle number; and

Figure 11 shows (a) an SEM image of Micrococcus Luteus coated with a layer of ZIF-8, (b) SAXS diffraction patterns of standard ZIF-8 crystals (bottom line, black), native Micrococcus Luteus (middle line, light grey), and Micrococcus Luteus coated with ZIF-8 (top line, dark grey), and in the inset of (b) a 2D representation of SAXS patterns generated by the ZIF-8 coated bacteria, (c) a fluorescent microscopy image of Micrococcus Luteus coated with a layer of ZIF-8, obtained using FDA as a fluorescent cell viability indicator, and (d) FDA cell viability assay on uncoated Micrococcus Luteus (naked) and Micrococcus Luteus coated with a layer of ZIF-8.

Some Figures contain colour representations or entities. Coloured versions of the Figures are available upon request. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a cell coated with a layer of crystalline Metal Organic Framework (MOF). By the cell being 'coated' with a layer of crystalline MOF is meant that the layer of MOF is provided around the entire cell such that, as a result of the cell being coated, a continuous layer (i.e. a continuous coating) of MOF exists around the cell. Accordingly, for an external molecule to enter into the cytoplasm of the coated cell it would have to first diffuse through the intrinsic cavities of crystalline MOF forming the coating layer. In other words, the invention provides a cell encapsulated (e.g. encased, or enclosed) within a layer of crystalline MOF.

A schematic cross-section representation of a cell coated with a layer of MOF is shown in Figure 1. Provided the crystalline MOF is formed as a coating layer around the cell, there is no particular restriction on the composition of a MOF useful for the invention. Accordingly, the present invention is applicable to a variety of different MOFs. As a skilled person would know, MOFs are porous materials. The porosity of a MOF can be visualised as a spatial arrangement of cavities in the form of cages connected by channels. MOFs according to the present invention include those having at least two metal clusters coordinated by at least one organic ligand. Depending on the particular choice of metal ions and organic ligands, MOFs having cavities in the form of open micro- and mesopores are available.

As used herein, the expression 'metal cluster' is intended to mean a chemical moiety that contains at least one atom or ion of at least one metal or metalloid. This definition embraces single atoms or ions and groups of atoms or ions that optionally include organic ligands or covalently bonded groups. Accordingly, the expression 'metal ion' includes, for example, metal ions, metalloid ions and metal oxides.

Suitable metal ions that form part of a MOF structure can be selected from Group 1 through 16 metals of the IUPAC Periodic Table of the Elements including actinides, and lanthanides, and combinations thereof. The metal ion may be selected from Li⁺, Na⁺, K⁺ Rb⁺, Be²⁺, Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Sc³⁺, Y³⁺, Ti⁴⁺, Zr⁴⁺, Hf⁴*, V⁵⁺, V⁴⁺, V³⁺, V²⁺, Nb³⁺ Nb⁵⁺, Ta⁵⁺, Cr⁶⁺, Cr³⁺, Mo⁶⁺, Mo³⁺, W⁶⁺, W³⁺, Mn⁴⁺, Mn³⁺, Mn²⁺, Re⁷⁺, Re²⁺, Fe³⁺, Fe²⁺ Ru⁴⁺, Ru³⁺, Ru²⁺, Os³⁺, Os²⁺, Co³⁺, Co²⁺, Rh³⁺, Rh²⁺, Rh⁺, Ir⁴⁺, Ir²⁺, Ir⁺, Ni²⁺, Pd⁴⁺, Pd²⁺ Pt⁴⁺, Pt²⁺, Cu²⁺, Cu⁺, Ag⁺, Au⁺, Zn²⁺, Cd²⁺, Hg²⁺, B³⁺, Al³⁺, Ga³⁺, In³⁺, TI³⁺, Si⁴⁺, Si²⁺ Ge⁴⁺, Ge²⁺, Sn⁴⁺, Sn²⁺, Pb⁴⁺, Pb²⁺, As⁵⁺, As³⁺, Sb⁵⁺, Sb³⁺, Bi⁵⁺, Bi³⁺, La³⁺, Ce³⁺, Ce⁴⁺, Pr³⁺ Pr⁴⁺, Nd³⁺, Sm³⁺, Sm²⁺, Eu³⁺, Eu²⁺, Gd³⁺, Tb³⁺, Tb⁴⁺, Dy³⁺, Ho³⁺, Er³⁺, Tm³⁺, Tm²⁺, Yb³⁺ Yb²⁺, Lu³⁺, Th⁴⁺, U⁶⁺, U⁵⁺, U⁴⁺, U³⁺, and a combination thereof.

Suitable metal ion coordinating organic ligands can be derived from oxalic acid, malonic acid, succinic acid, glutaric acid, phtalic acid, isophtalic acid, terephthalic acid, citric acid, trimesic acid, 1,2,3-triazole, pyrrodiazole, or squaric acid. Organic ligands suitable for the purpose of the invention comprise organic ligands listed in WO 2010/075610 and Filipe A. Almeida Paz, Jacek Klinowski, Sergio M. F. Vilela, Joao P. C. Tome, Jose A. S. Cavaleiro, Joao Rocha, 'Ligand design for functional metal-organic frameworks', Chemical Society Reviews, 2012, Volume 41, pages 1088-1110, the contents of which are included herein in their entirety.

In some embodiments, the MOF is a lanthanide (Ln) MOF, for example Er(bdc), Dy(bdc), Tb(bpdc), mixed Gd(bpdc) and Tb(bpydc), Tb(bdc), Eu(bdc), Gd(bdc) or Ln(bpedc), in which bdc = 1,4-benzenedicarboxylate, bpdc = 4,4'-biphenyldicarboxylate and bpydc = 2,2'-bipyridine-5,5'-dicarboxylate, and bpedc = biphenylethene-4,4'-dicarboxylate.

The organic ligand and the source of metal ions make up the MOF precursor compounds. In some embodiments, the MOF is selected from mixed component MOFs, known as MC- MOFs. MC-MOFs have a structure that is characterised by more than one kind of organic ligand and/or metal. MC-MOFs can be obtained by using different organic ligands and/or metals directly in the solution into which MOF precursor compounds and the cell are combined, or by post-synthesis substitution of organic ligands and/or metals species of formed MOFs. Specific examples of MC-MOFs can be found in A.D. Burrows, CrystEngComm 2011, Volume 13, pages 3623-3642, which content is included herein in its entirety.

In some embodiments, the MOF is a zinc imidazolate framework (ZIF). ZIFs are a sub- class of MOFs that are particularly suited to biologic applications because of (i) their prolonged stability in physiological conditions, (ii) the pH responsive nature of their metal- organic ligand bonds, which can be used as a trigger for pH-induced drug delivery applications, and (iii) negligible cytotoxicity. In addition, ZIFs can be synthesized in water and are chemically stable in water even at high temperatures (e.g. at boiling point) for prolonged periods of time (e.g. several weeks). The stability of ZIFs in water makes them preferred matrices for providing a layer for cells. ZIFs feature tetrahedrally-coordinated transition metal ions (e.g. Fe, Co, Cu, or Zn) connected by organic imidazolate organic ligands, resulting in three-dimensional porous solids. Similarly to zeolites, ZIFs have great thermal and chemical stability. Depending on the choice of ligand and metal ions, provided by the precursor compounds, many ZIF topologies can be synthesized.

Accordingly, a MOF that may be made in accordance with the invention may be a carboxylate -based MOF, a heterocyclic azolate-based MOF, or a metal-cyanide MOF. Specific examples of MOFs that may be made according to the present invention include those commonly known in the art as CD-MOF-1, CD-MOF-2, CD-MOF-3, CPM-13, FJI- 1, FMOF-1, HKUST-1, IRMOF-1, IRMOF-2, IRMOF-3, IRMOF-6, IRMOF-8, IRMOF-9, IRMOF-13, IRMOF-20, JUC-48, JUC-62, MIL-101, MIL-100, MIL-125, MIL-53, MIL-88 (including MIL-88A, MIL-88B, MIL-88C, MIL-88D series), MOF-5, MOF-74, MOF-177, MOF-210, MOF-200, MOF-205, MOF-505, MOROF-2, MOROF-1, NOTT-100, NOTT- 101, NOTT-102, NOTT-103, NOTT-105, NOTT-106, NOTT-107, NOTT-109, NOTT- 110, NOTT-111, NOTT-112, NOTT-113, NOTT-114, NOTT-140, NU-100, rho-ZMOF, PCN-6, PCN-6', PCN9, PCN10, PCN12, PCN12', PCN14, PCN16, PCN-17, PCN-21, PCN46, PCN66, PCN68, PMOF-2(Cu), PMOF-3, SNU-5, SNU-15', SNU-21S, SNU-21H, SNU-50, SNU-77H, UiO-66, UiO-67, soc-MOF, sod-ZMOF, TUDMOF-1, UMCM-2, UMCM-150, UTSA-20, ZIF-2, ZIF-3, ZIF-4, ZIF-8, ZIF-9, ZIF- 10, ZIF-11, ZIF-12, ZIF- 14, ZIF-20, ZIF-21, ZIF-23, ZIF-60, ZIF-61, ZIF-62, ZIF-64, ZIF-65, ZIF-67, ZIF-68, ZIF-69, ZIF-70, ZIF-71, ZIF-72, ZIF-73, ZIF-74, ZIF-75, ZIF-76, ZIF-77, and ZIF-90. In certain preferred embodiments, the MOF is selected from ZIF-8, HKUST-1, IRMOF-1, MIL-53, MIL-88,MIL-88A, MIL-88B, MIL-88C, MIL-88D, MOF-5, MOF-74, NOTT- 100, Ln-bdc, ZIF-67, ZIF-90, ZIF-67, and a combination thereof.

The MOF according to the invention is crystalline. For avoidance of doubt, any reference made herein to a 'MOF' is therefore to be intended as reference to a 'crystalline MOF' . In a crystalline MOF the metal clusters are coordinated by the organic ligands to form a geometrically regular network made of repeating units of cluster/organic ligand arrangements. The crystalline nature of a MOF arises from regular and spatially ordered distribution of intrinsic cavities forming the MOF framework.

As used herein the expression 'intrinsic cavities' is intended to mean the ordered network of interconnected voids that is specific to a crystalline MOF by the very nature of the MOF. As it is known in the art, the intrinsic cavity network of a MOF results from the specific spatial arrangement of the MOF's metal clusters and organic ligands and is unique to any pristine crystalline MOF.

The intrinsic cavities of a crystalline MOF can be visualised as being formed by regularly distributed cages interconnected by windows or channels. The specific shape of cages and window/channels in a crystalline MOF is determined by the spatial arrangement of the chemical species forming the MOF framework. Accordingly, the expression 'intrinsic cavities' specifically identifies the overall ordered network of cages and window/channels of the native MOF framework.

A crystalline MOF generates diffraction patterns when characterized by commonly known crystallographic characterization techniques. These include, for example, X-ray powder diffraction (XPD), 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 regular network of intrinsic cavities provides a crystalline MOF with unique porosity characteristics. That is, a crystalline MOF is inherently porous. By being inherently porous, a crystalline MOF is inherently permeable. In other words, the present invention provides a cell coated with a permeable layer of crystalline Metal Organic Framework (MOF). Accordingly, the present invention also provides a method of coating a cell with a permeable layer of crystalline Metal Organic Framework (MOF), the method comprising combining in a solution the cell and MOF precursor compounds, wherein the cell promotes formation of the permeable layer of crystalline MOF. Similarly, the present invention provides use of Metal Organic Framework (MOF) precursor compounds in coating a cell, wherein the MOF precursor compounds form a permeable layer of crystalline MOF around the cell.

In the present invention a layer of crystalline MOF coats a cell.

As used herein, the term 'cell' means a biological unit comprising a membrane-bound cytoplasm.

That is, a cell suitable for use in the invention is a cell provided with all structural features of a living cell, i.e. a whole cell. In other words, the present invention provides a whole cell coated with a layer of crystalline Metal Organic Framework (MOF).

The present invention may therefore be described as providing a method of coating a whole cell with a layer of crystalline Metal Organic Framework (MOF), the method comprising combining in a solution the cell and MOF precursor compounds, wherein the cell promotes formation of the layer of crystalline MOF.

Accordingly, the invention further provides use of Metal Organic Framework (MOF) precursor compounds in coating a whole cell, wherein the MOF precursor compounds form a layer of crystalline MOF around the cell. In the context of the invention the cell will be understood as being provided in a viable state. By the cell being 'viable' is meant that the cell is a living cell as opposed to a dead cell. In other words, the present invention provides a viable cell coated with a layer of crystalline Metal Organic Framework (MOF). The present invention may therefore be described as providing a method of coating a viable cell with a layer of crystalline Metal Organic Framework (MOF), the method comprising combining in a solution the cell and MOF precursor compounds, wherein the cell promotes formation of the layer of crystalline MOF.

Accordingly, the invention further provides use of Metal Organic Framework (MOF) precursor compounds in coating a viable cell, wherein the MOF precursor compounds form a layer of crystalline MOF around the cell.

Cell viability can be determined by any means known to persons skilled in the art. For the purpose of this invention reference is made to the standard viability assay based on resazurin reduction, as described in Riss T.L., Moravec R.A., Niles A.L., Benink H.A., Worzella T.J., Minor L., et al. (2004). 'Cell Viability Assays', in Assay Guidance Manual, Eds Sittampalam G.S., Coussens N.P., Nelson H., Arkin M., Auld D., Austin C, et al., editors. (Bethesda, MD: Eli Lilly & Company). Provided the cell promotes formation of a layer of crystalline MOF (discussed in more detail below), a cell suitable for the purposes of the present invention may be any type of cell, whether modified or not, and derived from any source. In that context, such a cell may be, for example, a eukaryotic cell or a prokaryotic cell, including a genetically-modified cell, a cell containing the same genetic material as a naturally-occurring cell, a cell from a line of cells, or one isolated from an organism.

In some embodiments, the cell is a eukaryotic cell. A eukaryotic cell comprises genetic material that is enclosed within a nuclear envelope (also known as nuclear membrane, nucleolemma or karyotheca). Multiple eukaryotic cells can organise into complex structures and are the characteristic cells of animals (including humans), plants, fungi, and Protista.

Accordingly, in some embodiments the cell is a eukaryotic cell belonging to kingdom Animalia, for example an animal cell or a human cell. Such a cell has characteristically no cell wall or chloroplasts. Examples of eukaryotic cells belonging to kingdom Animalia include exocrine secretory epithelial cells, hormone-secreting cells, keratinizing epithelial cells, wet stratified barrier epithelial cells, neurons (e.g. sensory transducer cells, autonomic neuron cells, sense organ and peripheral neuron supporting cells, central nervous system neurons, glial cells, or lens cells), metabolism and storage cells, barrier function cells, extracellular matrix cells, contractile cells (e.g. skeletal muscle cells, red skeletal muscle cells, white skeletal muscle cells, intermediate skeletal muscle cells, nuclear bag cells of muscle spindle, nuclear chain cells of muscle spindle, satellite cells such as stem cells, heart muscle cells, ordinary heart muscle cells, nodal heart muscle cells, purkinje fiber cells, smooth muscle cells, myoepithelial cells of iris, myoepithelial cell of exocrine glands), blood and immune system cells (e.g. erythrocytes, leukocytes, platelets, megakaryocytes, monocytes, connective tissue macrophage cells, epidermal Langerhans cells, osteoclast cells, dendritic cells of lymphoid tissues, microglial cells of central nervous system, neutrophil granulocytes, eosinophil granulocytes, basophil granulocytes, hybridoma cells, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural Killer T cells, B cells, natural killer cells, reticulocytes), stem cells (e.g. totipotent stem cells, pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent cells and including embryonic stem cells, fetal stem cells, adult stem cells, and amniotic stem cells), germ cells (e.g. oogonium/oocytes, spermatids, spermatocytes, spermatogonium cells, spermatozoons), nurse cells, interstitial cells, and bone marrow cells. In some embodiments, the cell is not derived from a human embryo. In these instances, the present invention therefore provides a cell coated with a layer of crystalline Metal Organic Framework (MOF), the cell being one that is not derived from a human embryo. In line with these embodiments the invention also provides a method of coating a cell with a layer of crystalline Metal Organic Framework (MOF), the cell being one that is not derived from a human embryo, and the method comprising combining in a solution the cell and MOF precursor compounds, wherein the cell promotes formation of the layer of crystalline MOF. Accordingly, the invention also provides for use of Metal Organic Framework (MOF) precursor compounds in coating a cell, wherein the cell is not derived from a human embryo, and the MOF precursor compounds form a layer of crystalline MOF around the cell. In line with these embodiments the invention therefore provides a whole cell coated with a layer of crystalline Metal Organic Framework (MOF), the cell being one that is not derived from a human embryo. Also in accordance with these embodiments the invention provides a method of coating a whole cell with a layer of crystalline Metal Organic Framework (MOF), the cell being one that is not derived from a human embryo, and the method comprising combining in a solution the cell and MOF precursor compounds, wherein the cell promotes formation of the layer of crystalline MOF. Accordingly, in line with these embodiments the invention also provides for use of Metal Organic Framework (MOF) precursor compounds in coating a whole cell, wherein the cell is not derived from a human embryo, and the MOF precursor compounds form a layer of crystalline MOF around the cell. In other words, according to these embodiments the present invention provides a viable cell coated with a layer of crystalline Metal Organic Framework (MOF), the cell being one that is not derived from a human embryo. In line with these embodiments the invention also provides a method of coating a viable cell with a layer of crystalline Metal Organic Framework (MOF), the cell being one that is not derived from a human embryo, and the method comprising combining in a solution the cell and MOF precursor compounds, wherein the cell promotes formation of the layer of crystalline MOF. Accordingly, in line with these embodiments the invention also provides for use of Metal Organic Framework (MOF) precursor compounds in coating a viable cell, wherein the cell is not derived from a human embryo, and the MOF precursor compounds form a layer of crystalline MOF around the cell.

In some embodiments the cell is an anchorage-independent cell such as a hepatocyte. As a skilled person would know, a hepatocyte is particularly environmentally sensitive.

In some embodiments, the cell is a hormone producing cell. Examples of a hormone inducing cell include a cell of the anterior pituitary gland, which can be harnessed to provide artificial organs.

In some embodiments, the cell is an insulin-producing cell. Examples of an insulin- producing cell include a mammalian pancreatic alpha cell, beta cell and intact islet. Those cells may be used to provide artificial pancreas. In some embodiments, the cell is a tumor cell.

A tumor cell for use in accordance to the invention may be a cell associated with any type of cancer. For example, a tumor cell suitable for use in the invention may be a modified cytokine secreting cell, such as a myoblast or a xenogenic cell. Examples of a tumor cell suitable for use in the invention also include a cell associated with a cancer such as bladder cancer, bone cancer, bowel cancer, brain cancer, breast cancer, kidney cancer, leukaemia, liver cancer, lung cancer, lymphoma, mesothelioma, myeloma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, or uterine cancer.

In some embodiments, the cell is a eukaryotic cell belonging to kingdom Plantae. Such cells have chloroplasts, cellulose cell walls, and large central vacuoles. Examples of eukaryotic cells belonging to kingdom Plantae include plant cells such as parenchyma cells, collenchyma cells, sclerenchyma cells, meristematic cells, xylem cells, plant epidermal cells, and plant stem cells (e.g. cambium cells and callus cells).

In some embodiments, the cell is a eukaryotic cell belonging to kingdom Fungi (i.e. eukaryotic Fungal hypha cells). Such cells are typically tubular, multinucleated, and have a chitinous cell wall. Examples of eukaryotic cells belonging to kingdom Fungi include yeast cells (e.g. yeast cells of the species Saccharomyces cerevisiae, Cryptococcus, and Candida).

In some embodiments, the cell is a eukaryotic cell belonging to kingdom Protista. Such cells include all eukaryotic cells that do not belong to kingdom Animalia, Plantae or Fungi. In particular, kingdom Protista includes all organisms which are unicellular or unicellular-colonial and which form no tissues.

In some embodiments, the cell is a prokaryotic cell. Unlike eukaryotic cells, a prokaryotic cell lacks a nuclear envelope separating genetic material from the cytoplasm. Examples of prokaryotic cells include bacterial cells (i.e. unicellular microorganisms belonging to the Domain Bacteria), and Archea cells (i.e. unicellular microorganisms belonging to the Domain Archea).

In some embodiments, the cell is an artificial cell. By the expression 'artificial cell' is meant an engineered particle that mimics one or many functions of a naturally-occurring eukaryotic or prokaryotic cell.

In other embodiments, the cell is a genetically-modified cell. By the expression 'genetically-modified cell' is meant a cell containing genetic material that has been altered in a way that does not occur naturally. Methods for genetically-modifying a cell would be known to the skilled person, and include methods based on the alteration of the genetic material of the cell by removing heritable material or by introducing exogenous DNA.

In some embodiments, the cell is one that has been isolated from a living organism, such as a human, an animal or a plant. In some instances, subsequent to isolation the cell is subjected to ex vivo manipulation (e.g. activation, genetic modification, culture, expansion etc.) before being coated with a layer of crystalline MOF. A skilled person would be aware of available techniques and procedures to isolate a living cell from a living organism and optionally to manipulate the cell.

In further embodiments, the cell is isolated from a line of cells grown in an in vitro culture. A skilled person would be aware of available techniques and procedures to isolate a cell from an in vitro line of cells. The invention also provides a method of coating a cell with a layer of crystalline Metal Organic Framework (MOF). Specifically, the method of the invention comprises combining in a solution the cell and MOF precursor compounds.

MOF precursor compounds include those compounds known in the art that provide the metal ions listed herein in the solution within a suitable solvent. Those compounds may be salts of the relevant metal ions, including metal-chlorides, -nitrates, -acetates -sulphates, - hydrogen sulphates, -bromides, -carbonates, -phosphates, and derivatives thereof, including mono- and poly- hydrate derivatives.

Examples of suitable metal salt precursor compounds include, but are not limited to, cobalt nitrate (Co(N03)2 xH20), zinc nitrate (Ζη(Νθ3)2· χΗ2θ), iron(III) nitrate (Fe(N03)3 xH20), aluminium nitrate (Α1(Νθ3)3· χΗ2θ), magnesium nitrate (Mg(N03)2-xH20), calcium nitrate (Ca(N03)2-xH20), beryllium nitrate (Be(N03)2-xH20), europium nitrate (Eu(N03)3-xH20), terbium nitrate (Tb(N03)3-xH20), ytterbium nitrate (Yb(N03)3-xH20), dysprosium nitrate (Dy(N03)3-xH20), erbium nitrate (Er(N03)3 xH20), gallium nitrate (Ga(N03)3 xH20), gadolinium nitrate (Gd(N03)3-xH20), nickel nitrate (Νί(Νθ3)2· χΗ2θ), lead nitrate (Pb(N03)2-xH20), cadmium nitrate (Cd(N03)2-xH20), manganese(II) nitrate (Μη(Νθ3)2·χΗ2θ), cobalt chloride (CoCb x fcO), zinc chloride (ZnCb x fcO), iron(III) chloride (FeCb xfbO), iron(II) chloride (FeCb xfbO), aluminium chloride (AlCb-xfbO), magnesium chloride (MgCb xfbO), calcium chloride (CaCb xfbO), beryllium chloride (BeCb xfbO), europium chloride (EuCb xfkO), terbium chloride (TbCb xfbO), ytterbium chloride (YbCb xfbO), dysprosium chloride (DyCb xfbO), erbium chloride (ErCb xfkO), gallium chloride (GaCb xfbO), gadolinium chloride (GdCb xfbO), nickel chloride (NiCb-xH20), lead(II) chloride (PbCb-xH20), cadmium chloride (CdCb-xH20) ), manganese(II) chloride (MnCb-xH20), cobalt acetate (Co(CH₃COO)2-xH20), zinc acetate (Zn(CH₃COO)2-xH20), iron(III) acetate (Fe(CH₃COO)3-xH20), iron(II) acetate (Fe(CH₃COO)2-xH20), aluminium acetate (Al(CH₃COO)3-xH20), magnesium acetate (Mg(CH₃COO)2-xH20), calcium acetate (Ca(CH₃COO)2-xH20), beryllium acetate (Be(CH₃COO)2-xH20), europium acetate (Eu(CH₃COO)3-xH20), terbium acetate (Tb(CH₃COO)3-xH20), ytterbium acetate (Yb(CH₃COO)3-xH20), dysprosium acetate (Dy(CH₃COO)3-xH20), erbium acetate (Er(CH₃COO)3-xH20), gallium acetate (Ga(CH₃COO)3-xH20), gadolinium acetate (Gd(CH₃COO)3-xH20), nickel acetate (Ni(CH₃COO)2-xH20), lead(II) acetate (Pb(CH₃COO)2-xH20), cadmium acetate (Cd(CH₃COO)2-xH20) ), manganese(II) acetate (Mn(CH₃COO)2-xH20), cobalt sulphate (CoS04-xH20), zinc sulphate (ZnS04-xH20), iron(III) sulphate (Fe2(S04)3-xH20), iron(II) sulphate (FeS04-xH20), aluminium sulphate (A12(S04)3-xH20), magnesium sulphate (MgS04-xH20), calcium sulphate (CaS04-xH20), beryllium sulphate (BeS04-xH20), europium sulphate (Eu2(S04)3-xH20), terbium sulphate (Tb2(S04)3-xH20), ytterbium sulphate (Yb2(S04)3 xH20), dysprosium sulphate (Dy2(S04)3-xH20), erbium sulphate (Er2(S04)3-xH20), gallium sulphate (Ga2(S04)3-xH20), gadolinium sulphate (Gd2(S04)3 xH20), nickel sulphate (NiS04-xH20), lead sulphate (PbSCW xFhO), cadmium sulphate (CdSCW xFhO), manganese(II) sulphate (MnS0₄ xH20), cobalt hydroxide (Co(OH)2 xH20), zinc hydroxide (Ζη(ΟΗ)2·χΗ2θ), iron(III) hydroxide (Fe(OH)3 xH20), iron(III) oxide:hydroxide (FeO(OH) xH20), Iron(II) hydroxide (Fe(OH)2 xH20), aluminium hydroxide (Α1(ΟΗ)3·χΗ2θ), magnesium hydroxide (Mg(OH)2 xH20), calcium hydroxide (Ca(OH)2-xH20), beryllium hydroxide (Be(OH)2 xH20), europium hydroxide (Eu(OH)3-xH20), terbium hydroxide (Tb(OH)3 xH20), ytterbium hydroxide (Yb(OH)3-xH20), dysprosium hydroxide (Dy(OH)3 xH20), erbium hydroxide (Er(OH)3-xH20), gallium hydroxide (Ga(OH)3 xH20), gadolinium hydroxide (Gd(OH)3-xH20), nickel hydroxide (Ni(OH)2-xH20), lead hydroxide (Pb(OH)2-xH20), cadmium hydroxide (Cd(OH)2-xH20), manganese(II) hydroxide (Μη(ΟΗ)2·χΗ2θ), cobalt bromide (CoBr2-xH20), zinc bromide (ZnBr2-xH20), iron(III) bromide (FeBr3-xH20), iron(II) bromide (FeBr2-xH20), aluminium bromide (AlBr3-xH20), magnesium bromide (MgBr2-xH20), calcium bromide (CaBr2-xH20), beryllium bromide (BeBr2-xH20), europium bromide (EuBr3-xH20), terbium bromide (TbBr3-xH20), ytterbium bromide (YbBr3-xH20), dysprosium bromide (DyBr3-xH20), erbium bromide (ErBr3-xH20), gallium bromide (GaBr3-xH20), gadolinium bromide (GdBr3-xH20), nickel bromide (NiBr2-xH20), lead bromide (PbBr2-xH20), cadmium bromide (CdBr2-xH20), manganese(II) bromide (MnBr2-xH20), cobalt carbonate (CoC0₃-xH20), zinc carbonate (ZnC0₃-xH20), iron(III) carbonate (Fe₂(C03)₃-xH20), aluminium carbonate (Ai₂(C0₃)₃-xH20), magnesium carbonate (MgC0₃-xH20), calcium carbonate (CaC03-xH20), beryllium carbonate (BeC0₃-xH20), europium carbonate (Eu₂(C0₃)₃-xH20), terbium carbonate (Tb₂(C0₃)₃-xH20), ytterbium carbonate (Yb₂(C0₃)₃-xH20), dysprosium carbonate (Dy₂(C0₃)₃-xH20), erbium carbonate (Er₂(C0₃)₃-xH20), gallium carbonate (Ga₂(C0₃)₃-xH20), gadolinium carbonate (Gd₂(C0₃)₃-xH20), nickel carbonate (NiCOyxFhO), lead carbonate (PbCOyxFhO), cadmium carbonate (CdC0₃ xH20), manganese(II) carbonate (MnC0₃ xH20), and mixtures thereof, where x ranges range from 0 to 12.

MOF precursor compounds also include organic ligands of the kind described herein that coordinate the metal ion clusters in the MOF framework. The organic ligands include molecules that have at least two chemical moieties capable of coordinating a metal ion. In some embodiments, these groups comprise carboxylates, phosphonates, sulphonates, N- heterocyclic groups, and combinations thereof. Suitable organic ligands include those ligands listed in WO 2010/075610 and Filipe A. Almeida Paz, Jacek Klinowski, Sergio M. F. Vilela, Joao P. C. Tome, Jose A. S. Cavaleiro, Joao Rocha, Ligand design for functional metal-organic frameworks, Chemical Society Reviews, 2012, Volume 41, pages 1088-1110, the contents of which are included herein in their entirety.

Examples of organic ligand precursor compounds include, but are not limited to, 4,4',4"- [benzene-l,3,5-triyl-tris(ethyne-2,l-diyl)]tribenzoate, biphenyl-4,4'-dicarboxylate, 4,4',4"- [benzene-l,3,5-triyl-tris(benzene-4,l-diyl)]tribenzoate, 1,3,5-benzenetribenzoate, 1,4- benzenedicarboxylate, benzene-l,3,5-tris(lH-tetrazole), 1,3,5-benzenetricarboxylic acid, terephthalic acid, imidazole, benzimidazole, 2-nitroimidazole, 2-methylimidazole (Hmlm), 2-ethylimidazole, 5-chloro benzimidazole, purine, fumaric acid, a-cyclodextrin, β- cyclodextrin, γ-cyclodextrin l,4-Bis(l-imidazolyl)benzene), 4,4'-Bispyridyl, 1,4- Diazabicyclo[2.2.2]octane, 2-amino-l,4-benzenedicarboxylate, 2-amino-l,4- benzenedicarboxylic acid, 4,4'-Azobenzenedicarboxylate, 4,4'-Azobenzenedicarboxylic acid, Aniline-2,4,6-tribenzoate, Aniline-2,4,6-tribenzic acid, Biphenyl-4,4'-dicarboxylic acid, l, -Biphenyl-2,2',6,6'-tetracarboxylate, l, -Biphenyl-2,2',6,6'-tetracarboxylic acid, 2,2'-Bipyridyl-5,5'-dicarboxylate, 2,2'-Bipyridyl-5,5'-dicarboxylic acid, 1,3,5-Tris(4- carboxyphenyl)benzene, l,3,5-Tris(4-carboxylatephenyl)benzene, 1,3,5-

Benzenetricarboxylate, 2,5-Dihydroxy- 1 ,4-benzenedicarboxylate, 2,5-Dihydroxy- 1 ,4- benzenedicarboxylic acid, 2,5-Dimethoxy-l,4-benzenedicarboxylate, 2,5-Dimethoxy-l,4- benzenedicarboxylic acid, 1,4-Naphthalenedicarboxylate, 1,4-Naphthalenedicarboxylic acid, 1,3-Naphthalenedicarboxylate, 1,3-Naphthalenedicarboxylic acid, 1,7- Naphthalenedicarboxylate, 1,7-Naphthalenedicarboxylic acid, 2,6-

Naphthalenedicarboxylate, 2,6-Naphthalenedicarboxylic acid, 1,5-

Naphthalenedicarboxylate, 1,5-Naphthalenedicarboxylic acid, 2,7- Naphthalenedicarboxylate, 2,7-Naphthalenedicarboxylic acid, 4,4',4"-Nitrilotrisbenzoate, 4,4',4"-Nitrilotrisbenzoic acid, 2,4,6-Tris(2,5-dicarboxylphenylamino)-l,3,5-triazine, 2,4,6-Tris(2,5-dicarboxylatephenylamino)-l,3,5-triazine, 1,3,6,8-Tetrakis(4- carboxyphenyl)pyrene, l,3,6,8-Tetrakis(4-carboxylatephenyl)pyrene, 1,2,4,5-Tetrakis(4- carboxyphenyl)benzene, l,2,4,5-Tetrakis(4-carboxylatephenyl)benzene, 5,10,15,20- Tetrakis(4-carboxyphenyl)porphyrin, 5,10,15,20-Tetrakis(4-carboxylatephenyl)porphyrin, adenine, adeninate, fumarate, 1,2,4,5-benzenetetracarboxylate, 1,2,4,5- benzenetetracarboxylic acid, 1,3,5-benzenetribenzoic acid, 3-amino-l,5- benzenedicarboxylic acid, 3-amino-l,5-benzenedicarboxylate, 1,3-benzenedicarboxylic acid, 1,3-benzenedicarboxylate, 4,4',4"-[benzene-l,3,5-triyl-tris(ethyne-2,l- diyl)]tribenzoic acid, 4,4',4"-[benzene-l,3,5-triyl-tris(benzene-4,l-diyl)]tribenzoic acid, oxalic acid, oxalate, fumaric acid, fumarate, maleic acid, maleate, trans, trans -mucomc acid, trans, trans -mucon&tc, cis,trans-mucomc acid, cis, trans -mucon&tc, cis, czs-muconic acid, cis,cis-mnconate, pyrazole, 2,5-dimethylpyrazole, 1,2,4-triazole, 3,5-dimethyl-l,2,4- triazole, pyrazine, 2,5-dimethylpyrazine, hexamethylentetraamine, nicotinic acid, nicotinate, isonicotinic acid, isonicotinate, 4-(3,5-dimethyl-lH-pyrazole)-benzoic acid, 2,5- furandicarboxylic acid, 2,5-furandicarboxylate, 3,5-dimethyl-4-carboxypyrazole, 3,5- dimethyl-4-carboxylatepyrazole, 4-(3,5-dimethyl-lH-pyrazol-4-yl)-benzoic acid, 4-(3,5- dimethyl-lH-pyrazol-4-yl)-benzoate, and mixtures thereof. It will be understood that the organic ligands can also be functionalised organic ligands. For example, any one of the organic ligands listed herein may be additionally functionalised by amino-, such as 2-aminoterephthalic acid, urethane-, acetamide-, or amide-. The organic ligand can be functionalised before being used as precursor for MOF formation, or alternatively the assembled MOF itself can be chemically treated to functionalise its bridging organic ligands. A skilled person will be aware of suitable chemical protocols that allow functionalizing a MOF with functional groups, either by pre-functionalizing organic ligands used to synthesize the MOF or by post-functionalizing a pre-formed MOF. Suitable functional groups that may be provided on the MOF include -NHR, -N(R)₂, -NH₂, -N0₂, -NH(aryl), halides, aryl, aralkyl, alkenyl, alkynyl, pyridyl, bipyridyl, terpyridyl, anilino, -O(alkyl), cycloalkyl, cycloalkenyl, cycloalkynyl, sulfonamido, hydroxyl, cyano, - (CO)R, -(S0₂)R, -(C0₂)R, -SH, -S(alkyl), -S0₃H, -S0^3"M⁺, -COOH, COO^"M⁺, -P0₃H₂, - P0₃H^"M⁺, -P03^2"M²⁺, -C0₂H, silyl derivatives, borane derivatives, ferrocenes and other metallocenes, where M is a metal atom, and R is Ci_io alkyl.

There is no particular restriction on the nature of the solvent that can be used to prepare the solution in which MOF precursor compounds and a cell are combined, provided that (i) the MOF precursor compounds are soluble in the solvent, and (ii) the cell is compatible with the solvent. That is, the solvent will typically be one that does not adversely affect the cell viability.

Examples of solvent that may be used include dimethylformamide, tetrahydrofuran, methanol, ethanol, dimethyl sulfoxide (DMSO), acetone, water and mixtures thereof.

In some embodiments, the solution into which the cell and MOF precursor compounds are combined is an aqueous solution, for example a deionised water solution, or a physiological buffered solution (i.e. water comprising one or more salts such as KH₂P0₄, NaH₂P0₄, K₂HP0₄, Na₂HP0₄, Na₃P0₄, K₃P0₄, NaCl, KC1, MgCl₂, CaCl₂, etc.).

Provided the layer of MOF forms, there is no particular limitation regarding the concentration of MOF precursor compounds present in the solution.

Concentrations of MOF precursor compounds in the solution can include a range between about 0.001 M and 10 M, between about 0.01 M and 5 M, between about 0.01 M and 5 M, between about 0.02 M and 1 M, between about 0.02 M and 0.5 M, between about 0.05 M and 0.25 M, or between about 0.08 M and 0.16 M. The values refer to concentration of organic ligand as well as concentration of metal salt, relative to the total volume of the solution containing the MOF precursor compounds and the cell. The ratio between the concentration of organic ligands and the concentration of metal salts is not limited, provided the ratio is adequate for the formation of a layer of MOF promoted by the combination with the cell in accordance to the invention. In some embodiments, the organic ligand to metal salt ratio may range from about 1000: 1 to about 1:1000 (mohmol), from about 500: 1 to about 1:500, from about 100:1 to about 1: 100, from about 70: 1 to about 1:70, from about 30: 1 to about 1:30, from about 10: 1 to about 1: 10, from about 5: 1 to about 1:5, from about 2.5: 1 to about 1:2.5, from about 2: 1 to about 1:2, or from about 1.5: 1 to about 1: 1.5.

According to the method of the invention, the cell promotes formation of a layer of crystalline MOF.

By the cell 'promotes' formation of the layer of crystalline MOF is meant the cell per se causes, induces or triggers formation of the crystalline MOF upon combination with the MOF precursor compounds in a solution. As a result of the cell promoting formation of the layer of MOF, the MOF grows around the cell to eventually coat it entirely.

Without being limited by theory, it is believed the ionic species-rich environment of a cell membrane acts to locally concentrate MOF precursor compounds thanks to the chelating ability of membrane species. It is believed that formation of a crystalline MOF is facilitated by membrane species affinity towards MOF precursor compounds arising, for example, from intermolecular hydrogen bonding and hydrophobic interactions. This in turn favours localised formation of the MOF leading to the growth of a porous exoskeleton encasing the cell. The resulting increase in the local concentration (i.e. in the immediate surroundings of the cell) of both metal cations (deriving from the dissolution of the metal salt precursor) and organic ligands would facilitate pre-nucleation clusters of the MOF framework. It has been found that hydrophilic molecules and molecules having negatively charged domains or moieties (e.g. carboxyl groups, hydroxyl groups, amino groups etc.) show improved ability to nucleate a MOF over molecules with more hydrophobic character and positively charged moieties. It may therefore be postulated that negatively charged domains in the cell membrane attract the positive metal ions provided by the MOF metal precursor in solution and contribute to stabilize the metal-organic ligand clusters at the early stages of MOF formation. Provided crystalline MOF forms, there is no particular limitation regarding the amount of cells present in the solution with the MOF precursor compounds.

In some embodiments, the solution contains a number of cells between about 1 and about lOxlO ¹⁰ per ml of solution, between about 1 and about 10x10 ^s per ml of solution, between about 1 and about lOxlO ⁶ per ml of solution, between about 1 and about lOxlO ⁴ per ml of solution, between about 1 and about 10x10 per ml of solution, between about 1 and about 100 per ml of solution, between about 1 and about 50 per ml of solution, between about 1 and about 25 per ml of solution, or between about 1 and about 10 per ml of solution.

Combining the MOF precursor compounds in solution with the cell is surprisingly sufficient to cause formation of the MOF framework. There is no need to apply other factors or reagents to trigger formation of the MOF. For example, it is not necessary to apply heat to the solution as conventionally done in traditional solvothermal MOF synthesis methods (which typically require use of a heat source such as an oven, for example a microwave oven, a hot plate, or a heating mantel).

Accordingly, in some embodiments formation of the layer of crystalline MOF is effected 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 about -50°C and about 75°C, between about -50°C and about 50°C, or between about -50°C and about 30°C. In some embodiments, formation of the layer of crystalline MOF is effected at a solution temperature between about 0°C and about 10°C, between about 0°C and about 8°C, or between about 2°C and about 6°C. In a preferred embodiment, formation of the layer of crystalline MOF is effected at a solution temperature of about 4°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 cells.

There is no particular limitation on the order in which the MOF precursor compounds and the cell may be combined into the solution. For example, a solution containing a metal precursor may be first mixed with a solution containing an organic ligand, and a separate solution containing a cell is subsequently introduced into the solution containing the metal salt and the organic ligand.

Alternatively, a solution containing a cell and an organic ligand may be first prepared, and subsequently introduced into a separate solution containing a metal precursor.

Also, a solution containing a cell and a metal precursor may be first prepared, and subsequently introduced into a separate solution containing an organic ligand. Still further, separate solutions each individually containing a metal precursor, an organic ligand and a cell, respectively, may be mixed together at the same time.

Formation of the MOF according to the method of the invention is advantageously fast. Depending on the type of cell used and the type of MOF precursor compounds used, it has been found that upon bringing the cell and the MOF precursor compounds together in a solution the layer of crystalline MOF may form within about 1 second, 10 seconds, 1 minute, 10 minutes, 30 minutes, 60 minutes or 2 hours. In some embodiments, the cell promotes formation of the layer of crystalline MOF in less than about 1 minute, less than about 5 minutes, less than about 10 minutes, less than about 15 minutes, less than about 30 minutes, less than about 60 minutes, or less than about 120 minutes. Under the same conditions of time, temperature and concentration of MOF precursor compounds, it was found in a solution containing only MOF precursor compounds (i.e. with no cell) the MOF would not form. In other words, the cell per se has been found to promote formation of the MOF. Coating a cell with a layer of crystalline MOF advantageously hinders cell proliferation, yet maintaining cell viability. In other words, the coating layer of crystalline MOF prevents cell division by inducing an artificial hibernation state of the cell, while at the same time allowing transport of nutrients or chemical stimulants necessary for cell viability. Without being restricted by theory, it is believed that the layer of MOF acts as a physical restriction suppressing cells from budding. Accordingly, a cell coated with a layer of crystalline MOF does not self-reproduce, and can be advantageously stored in a coated form over days, weeks, months or years without adversely affecting its viability.

Provided storage does not adversely affect cell viability, there is no limit as to the temperature at which a coated cell can be stored. In some embodiments, the coated cell is stored at a temperature of between about 0°C and about 10°C, between about 0°C and about 8°C, or between about 2°C and about 6°C. The coated cell may be stored at storage temperature for a period of time of at least a week, at least a month or at least a year, for example a week, a month or a year. In an embodiment, the coated cell is stored for about 2 months at about 4°C.

Additionally, the layer of MOF can advantageously act as a selectively permeable physical barrier that allows diffusion of nutrients and substrates into the cytoplasm, that is, one can control the microenvironment for optimal cellular function. Simultaneously, the layer can also protect the cell from the attack of cytotoxic agents, macrophages or immunoglobulins. Since the layer of MOF can impede diffusion of molecules that are larger than the MOF intrinsic cavities, large cytotoxic bio-molecules (e.g. cytotoxic enzymes) cannot diffuse through the layer to attack the cell. This is schematically represented in Figure 2, showing a cell being coated with a layer of MOF that allows diffusion of nutrients while acting as physical barrier for large molecules.

Further advantageously, cell self-reproduction (i.e. cell proliferation) can be restored when the cell separates from the crystalline MOF. For example, the cell in a viable state separates from the crystalline MOF as a resolute of the MOF dissolving, degrading, disintegrating, rupturing, or deteriorating. As a result of the cell separating in a viable state from the crystalline MOF, all biological functions of the cell prior to being coated are reinstated. For example, upon separation from the crystalline MOF the cell recuperates its ability to self-replicate.

In some embodiments, the cell in a viable state separates from the MOF as a result of the layer of MOF dissolving. This may be achieved, for example, by inducing a variation of the pH of the solvent or by adding into the dispersion medium a compound that dissolves the layer of MOF.

Examples of MOFs that may be used in applications based on pH-triggered cell release include MOFs that are stable at certain pH values, but dissolve at certain other pH values. For example, the MOF may be stable above a threshold pH value. In that case there is no detectable release of the cell into the dispersion medium. However, the MOF may dissolve when the pH drops below the threshold, resulting in the release of the cell into the dispersion medium.

For example, certain MOFs can be stable at pH higher than physiological pH (about 7.4), but dissolve when the pH drops to physiological pH. This can result in the release of the cell into the dispersion medium. In some embodiments, the cell separates from the crystalline MOF as a result of the layer of MOF being dissolved by a compound that is added to the dispersion medium. Examples of such compounds include ethylenediamine tetra-acetic acid (EDTA), Diethylenetriaminepentaacetic acid (DTPA), N,N-bis(carboxymethyl)glycine; Triglycollamic acid (NT A), and phosphate buffer. Provided the addition of the compound into the dispersion medium results in the dissolution of the layer of crystalline MOF, there is no limitation as to the amount of compound that can be added. For example, the compound may be added in an amount sufficient to bring its concentration in the dispersion medium to between about 0.001 mol/L and about 10 mol/L, between about 0.01 mol/L and about 10 mol/L, between about 0.1 mol/L and about 10 mol/L, between about 0.5 mol/L and about 10 mol/L, between about 1 mol/L and about 10 mol/L, between about 1 mol/L and about 8 mol/L, between about 1 mol/L and about 5 mol/L, or between about 1 mol/L and about 2 mol/L.

Provided the layer of crystalline MOF coats the cell and that the cell can subsequently separate in a viable state from the crystalline MOF, there is no limitation as to the largest thickness of the layer of crystalline MOF. By the 'largest thickness' of the layer of crystalline MOF is meant the maximum thickness of the layer measured by SEM along a radial direction perpendicular to the cell external membrane. In some embodiments, the largest thickness of the layer of crystalline MOF ranges from about 10 nm to about 500 μιη, from about 25 nm to about 250 μιη, from about 50 nm to about 200 μιη, from about 50 nm to about 100 μιη, from about 100 nm to about 250 nm from about 50 nm to about 25 μιη, from about 50 nm to about 10 μιη, from about 50 nm to about 5 μιη, from about 50 nm to about 2.5 μιη, from about 50 nm to about 1 μιη, or from about 50 nm to about 0.5 μιη. Specific embodiments of the invention will now be described with reference to the following non-limiting examples. EXAMPLES Materials Saccharomyces cerevisiae (Baker's yeast), Micrococcus Luteus, yeast extract, zinc acetate dihydrate, 2-methylimidazole (Hmlm), D-(+)-glucose, lyticase from Bacillus subtilis (>500 U mL^"1), resazurin sodium salt, filipin, and methylene blue were purchased from Sigma Aldrich. Fluorescein diacetate (FDA) was purchased from Life Technologies (Australia). All the other reagents were purchased from Sigma Aldrich (Australia) and used without further modification.

Instrumentation

Optical micrographs (DIC and fluorescence) were obtained using an Olympus BX60M microscope. Scanning electron microscope (SEM) images of samples were taken on a Zeiss MERLIN SEM at an accelerating voltage of 5.0 kV. Confocal microscopy images were acquired via a Nikon AIR confocal laser scanning microscope. Synchrotron SAXS data were collected at the SAXS/WAXS beamline at the Australian Synchrotron.⁵ Diffraction patterns were collected using a Pilatus 1M detector.

EXAMPLE 1

Encapsulation of yeast cells and bacteria cells with a layer of ZIF-8 2 mg dry yeast cells were cultured in the yeast culture media containing Saccharomyces cerevisiae yeast cells extract (10 mg mL^"1) and glucose (20 mg mL^"1) with continuous shaking at 30 °C for 18 h. The yeast cells (were washed with deionized (DI) water three times and finally suspended in 5 mL aqueous solution of Hmlm (160 mM). 5 mL aqueous solution of zinc acetate dihydrate (40 mM) was then added into the Hmlm solution containing the yeast cells. The mixture was placed on a shaking stage (300 rpm) for 10 min for the formation of the layer of ZIF-8. The coated cells were washed with DI water three times to remove the excess ZIF-8 precursor compounds, and finally suspended in DI water.

The formation of a layer of ZIF-8 encapsulating Micrococcus Luteus was obtained according to an analogous procedure, except that cells of Micrococcus Luteus (a bacterium) were used instead of yeast cells.

Characterisation of the layer of MOF

The micro structure of the layer of MOF was analysed by synchrotron small-angle X-ray scattering (SAXS). The resulting scattering pattern was comprised of peaks that were analogous in position and relative intensity to pure ZIF-8 (Figure 3), thus confirming the nature, structure and crystallinity of the layer.

The morphology and elemental distribution of the ZIF-8 layer was also assessed by scanning electron microscopy (SEM) and Energy-dispersive X-ray spectroscopy (EDS), respectively. SEM imaging of c.a. 500 yeast cells coated within a layer of ZIF-8 revealed that each individual cells was coated with a homogeneous layer of ZIF-8. That is, SEM images showed that each discrete cell was individually and entirely coated with a layer of ZIF-8. The analysis did not reveal, for example, aggregates of cells coated within the same layer of ZIF-8, or partially non-coated cells.

Elemental analysis performed using high-magnification SEM was consistent with a homogeneous distribution of Zn, O and C (the main components of ZIF-8) on the cell surface. This strongly supports the formation of a continuous layer of ZIF-8 on individual yeast cells.

SEM images of Saccharomyces cerevisiae yeast cells after incubation in solution with ZIF- 8 precursor compounds for (a) 1 minutes, (b) 5 minutes, (c) 10 minutes, and (d) 20 minutes are shown in Figure 4. Analysis of SEM images taken on fragments of a crushed layer of ZIF-8 revealed an average layer thickness of approximately 100 nm (Figure 4(e)). It was also found that further exposure to ZIF-8 precursor compounds would result in further growth of the layer of MOF up to 260 nm depending on the exposure length.

The thickness of the ZIF-8 coatings could be tuned in the 100-250 nm range by carrying out sequential ZIF-8 coating steps (Figure 10). Figure 10 shows SEM images of cracked ZIF-8 coating layer which were used to measure the thickness of ZIF-8 coatings on yeast cells after 1 to 4 coating cycles. In particular, (a) relates to 1 coating cycle, resulting in a coating layer thickness of 104nm, (b) relates to 2 coating cycles, resulting in a thickness of the coating layer of 148nm, (c) relates to 3 coating cycles, resulting in a thickness of the coating layer of 210nm, and (d) relates to 4 coating cycles, resulting in a thickness of the coating layer of 257nm. Figure 10 (e) shows a plot of the ZIF-8 coating thickness against the number of subsequent coating cycles.

Confocal scanning laser microscopy (CLSM) was also employed to assess the homogeneity of the layer of ZIF-8. For this experiment the yeast cells and ZIF-8 layer were labelled with FDA (green) and Alexa Fluor 647 (red), respectively. The high- resolution optical cross-section is consistent with the SEM/EDS data and provides further evidence of a continuous coating of ZIF-8 over the entire cell.

In order to assess the generality of this approach we examined the formation of ZIF-8 coatings on the bacterium Micrococcus Luteus that possess a peptidoglycans-based outer membrane. Micrococcus Luteus can survive in oligotrophic (nutrient deficient) environments and is of interest for biotechnological applications (e.g. terpenes biosynthesis). Based on our investigation a ZIF-8 coating was successfully formed on these gram-positive cocci without affecting cell viability, as shown by the data shown in Figure 11. In particular, Figure 11 shows (a) an SEM image of Micrococcus Luteus coated with a layer of ZIF-8, (b) SAXS diffraction patterns of standard ZIF-8 crystals (bottom line, black), native Micrococcus Luteus (middle line, dark grey), and Micrococcus Luteus coated with ZIF-8 (top line, grey), and in the inset of (b) a 2D representation of SAXS patterns generated by the ZIF-8 coated bacteria, (c) a fluorescent microscopy image of Micrococcus Luteus coated with a layer of ZIF-8, obtained using FDA as a fluorescent cell viability indicator, and (d) FDA cell viability assay on uncoated Micrococcus Luteus (naked) and Micrococcus Luteus coated with a layer of ZIF-8.

The data highlights the potential of employing MOFs such as ZIF-8 as layer for a variety of basic functional biological units.

EXAMPLE 2

Viability test

Cell viability was studied by the FDA and resazurin assay independently. 1—3 Metabolically active cells can hydrolyze FDA into fluorescently bright fluorescein by esterases, while resazurin measures the mitochondrial activity within the cells. FDA stock solution was prepared by dissolving 5 mg of FDA in 1 mL acetone. To each 0.2 mL of the yeast suspension, 2 μΐ. of the FDA stock solution was added and incubated at 30 °C for 20 min.

The cells were then washed three times with DI water to remove free dyes in the solution.

For resazurin assay, 20 μΐ. resazurin solution (0.15 mg mL^"1 in DPBS) was added into each 0.2 mL yeast suspension and incubated at 30 °C for 2 h. The cells were then washed three times with DI water to remove free dyes in the solution.

For both the FDA (Figures 5(a), (c)) and resazurin assays (Figures 5(b), (d)) the fluorescent intensity remained unchanged before and after formation of the MOF coating, indicating that the ZIF-8 coating is essentially non-toxic to yeast cells. To confirm that ZIF-8 is non-toxic to yeast cells we performed a control test, where free ZIF-8 particles (average diameter c.a. 500 nm) and yeast cells were combined in solution. After a 24 hr incubation period the viability of the ZIF-8 treated yeast cells was found to be essentially identical to that of untreated cells. This data confirms that ZIF-8 particles do not adversely affect yeast cells. Data collected from Flow cytometry tests performed measuring the ATP metabolic activity of non-coated Saccharomyces cerevisiae yeast cells and (c) Saccharomyces cerevisiae yeast cells coated with a layer of ZIF-8 over time using resazurin as a fluorescent indicator are shown in Figures 6(a) -(c).

EXAMPLE 3

Lyticase assay Lyticase (MW = 54.6 kDa, about 5.3 nm maximum dimension) is widely used as cell lytic enzymes by enzymatic digestion of cell wall. 2000 U lyticase was added into each 1 mL of DPBS solution containing non-coated or ZIF-8 coated yeast cells. The mixture solution was placed onto a shaking stage (300 rpm) at 30 °C. The cell viability was monitored for 24 h using a resazurin assay.

Over the course of the experiment the ZIF-8 coated yeast did not give rise to a significant change in the fluorescent emission (5.3% and 19% loss in 3 and 24 hours, respectively, as shown in the right plots of Figure 7), while for the unprotected, non-coated, yeast a 95% reduction in fluorescence was detected within 3 h (Figure 7, left plots).

This result suggests that lyticase catalyzes cell lysis of the non-coated yeast cells and demonstrates that the ZIF-8 coating functions as a protective layer against this cytotoxic enzymatic agent. Filipin anti-fungal assay

Anti-fungal drug filipin (MW = 655 Da, 1x1.3x1.9 nm) was dissolved in DMSO and added to each wells containing the yeast cells in the yeast growth media in a 96-well plate to a final concentration of 200 μΜ filipin. This antifungal drug was selected as the molecule is slightly bigger (3- to 5-fold) than the ZIF-8 interconnecting cavities. The plate was incubated at 30 °C for 24 h under constant, soft shaking. After 24 h incubation, FDA was used as an indicator for viable yeast cells. The ZIF-8 coated yeast cells were firstly treated with EDTA (100 niM, 10 μί) to dissolve the ZIF-8 coatings before the addition of FDA. To each well containing 200 μL· yeast solution, FDA solution (2 μί, 5 mg niL^"1) was then added and incubated for 20 min. The cells were then washed three times with DI water to remove free dyes in the solution and finally observed under a fluorescent microscope.

After 24 h incubation in the presence of filipin the control sample (non-coated yeast) showed almost 100% mortality, while ZIF-8 coated yeast cells showed a minimal reduction in cell viability with less than 10% cells killed by the anti-fungal drug (Figure 8). These results indicate that the ZIF-8 coating is homogenous and can protect cells from both relatively small molecules, such as anti-fungal chemicals and large cytotoxic proteins.

EXAMPLE 4 Cell self-reproduction

Both ZIF-8 coated and non-coated yeast cells were suspended and diluted in the yeast culture media containing yeast extract (10 mg mL^-1) with or without glucose (20 mg mL^-1) at 30°C, with appropriate yeast concentration to yield OD₆oo (optical density at 600 nm) value of 0.2 - 0.3. OD₆oo experiments quantitatively measure cell proliferation by determining turbidity increases after division and is widely used to study the stage of cultured cells, i.e. whether they are in a lag phase, growth phase, stationary phase, or death phase. A standard procedure for OD₆oo determination is described in A. L. Koch, Biochim. Biophys. Acta 1961, 51, 429, the entire content of which is herein incorporated in its entirety. 200 μΐ_^ of the yeast suspension was transferred into each well of a 96-well microplate. The microplate was inserted into a microplate reader with constant shaking at 250 rpm, and the OD₆oo value was recorded continuously at 30 min interval.

For native yeast, the OD₆oo remained stable for 6 h before exponential growth was observed (Figure 9). This data indicates that the yeast cells were initially in a dormant/hibernation state (lag phase for 6 h) before entering a rapid cell proliferation (budding) state. In contrast, the OD₆oo measurements of ZIF-8 coated yeast showed no obvious growth even after 6 hours. Accordingly, it can be concluded that the ZIF-8 layer maintained the yeast in an extended lag phase. We postulate the ZIF-8 layer acts as a physical restriction suppressing the yeast cells from budding.

Since ethylenediamine tetra-acetic acid (EDTA) can dissolve ZIF-8, its potential to 'switch-off the artificially induced cell dormancy state was investigated. Figure 9 shows that subsequent to the addition of EDTA the yeast cells quickly moved to growth and germination states. We note that when compared to the incubation period of the non-coated cells (c.a. 6 h), ZIF-8 protected yeast is essentially instantaneous (c.a. a few minutes). This suggests that glucose, an essential nutrient, was able to diffuse through the ZIF-8 coating and maintain cell viability.

Furthermore, the OD₆oo data also showed that the growth rate and final cell number of the yeast, after the removal of the ZIF-8 layer, reached a similar level to non-coated yeast. Thus it can be concluded that the ZIF-8 coatings have no measurably adverse impact on the yeast cells. In summary, our results show that a ZIF-8 layer can extend the cell's lifetime, by artificially supressing cell division, without significantly affecting the activity of cells in the growth state.

While the invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. Every formulation or combination of components described or exemplified can be used to practice the invention, unless otherwise stated. Specific names of compounds are intended to be exemplary, as it is known that one of ordinary skill in the art can name the same compounds differently.

Throughout this specification and the claims which 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.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS

1. A cell coated with a layer of crystalline Metal Organic Framework (MOF).

2. The cell according to claim 1, which is a eukaryotic cell selected from an animal cell, a plant cell, a fungi cell, and a Protista cell.

3. The cell according to claim 1, which is a prokaryotic cell selected from a bacterial cell, and an Archea cell.

4. The cell according to any one of claims 1 to 3, wherein the crystalline MOF is selected from ZIF-8, HKUST-1, IRMOF-1, MIL-53, MIL- 88, MIL- 88 A, MIL-88B, MIL- 88C, MIL-88D, MOF-5, MOF-74, NOTT-100, Ln-bdc, ZIF-67, ZIF-90, ZIF-67, and a combination thereof.

5. The cell according to any one of claims 1 to 4, wherein the layer of crystalline MOF has a largest thickness of from about 10 nm to about 500 μιη.

6. A method of coating a cell with a layer of crystalline Metal Organic Framework (MOF), the method comprising combining in a solution the cell and MOF precursor compounds, wherein the cell promotes formation of the layer of crystalline MOF.

7. The method according to claim 6, wherein the cell is a eukaryotic cell selected from an animal cell, a plant cell, a fungi cell, and a Protista cell.

8. The method according to claim 6, wherein the cell is a prokaryotic cell selected from a bacterial cell, and an Archea cell.

9. The method according to any one of claims 6 to 8, wherein the solution contains a number of cells between about 1 and about lOxlO ¹⁰ per ml of solution.

10. The method according to any one of claims 6 to 9, wherein the MOF precursor compounds have a concentration in the solution of between about 0.001 M and 10 M.

11. The method according to any one of claim 6 to 10, wherein formation of the encapsulating framework is effected at a solution temperature that is lower than 75°C.

12. The method according to any one of claims 6 to 11, wherein the cell promotes formation of the layer of crystalline MOF in less than 15 minutes.

13. The method according to any one of claims 6 to 12, wherein the layer of crystalline MOF has a largest thickness of from about 10 nm to about 500 μιη.

14. The method according to any one of claims 6 to 13, wherein the crystalline MOF is selected from ZIF-8, HKUST-1, IRMOF-1, MIL-53, MIL- 88, MIL- 88 A, MIL-88B, MIL- 88C, MIL-88D, MOF-5, MOF-74, NOTT-100, Ln-bdc, ZIF-67, ZIF-90, ZIF-67, and a combination thereof.

15. Use of Metal Organic Framework (MOF) precursor compounds in coating a cell, wherein the MOF precursor compounds form a layer of crystalline MOF around the cell.