WO2014197940A1

WO2014197940A1 - One step assembly of metal-polyphenol complexes for versatile film and particle engineering

Info

Publication number: WO2014197940A1
Application number: PCT/AU2014/000616
Authority: WO
Inventors: Frank Caruso; Hirotaka EJIMA; Joseph Jacob RICHARDSON; Martin Peter VAN KOEVERDEN
Original assignee: The University Of Melbourne
Priority date: 2013-06-12
Filing date: 2014-06-12
Publication date: 2014-12-18

Abstract

Engineered articles have a wide variety of applications due to the ability to vary their properties by variations in the functionality of the layers in the article walls and in the material (if any) contained in the core of the article. In particular, articles of this type such as capsules are of significant interest for a number of applications especially in the medical field where they have found to have uses as vehicles for drug and vaccine delivery. In addition the capsules have potential applications as diagnostics and in bio sensing research. The present invention relates to an improved method of forming a film or network on a substrate from contacting said substrate with a polyphenol compound and a metallic cation or a mixture of metallic cations for less than 30 minutes, which method can be utilised for the rapid synthesis of articles of this type.

Description

One Step Assembly of Metal-Polyphenol Complexes for Versatile Film and

Particle Engineering

Technical Field

[1] Advances in materials design and application are highly dependent on the development of versatile thin film and particle engineering strategies that can be used to fabricate articles such as particles in a rapid and reproducible manner. For example, supramolecular metal-organic thin films have attracted widespread interest due to their diverse properties, which include: (i) stimuli-responsiveness imparted by the dynamic nature of supramolecular coordination bonds; (ii) hybrid physicochemical properties of both metals and organic materials; and (iii) controlled structure and functionality achieved by variation of the molecular building blocks. Although metal-organic thin films show potential for sensing, separation processes and catalysis such films are fabricated with multiple, time-consuming steps. Moreover, biomedical applications of these films have thus far been limited because they are typically unstable in water or are toxic.

[2] In addition articles that can be fabricated using film forming technology may have a number of other potential uses depending on the nature of the article including the materials in the coating of the article and any materials that may be contained in the core of the article. An example of a well-known article is a capsule. Articles of this type have an almost universal number of applications due to the ability to vary the properties of the article by variations in the functionality of the materials in the article walls and in the material (if any) contained in the core of the article. In particular, articles of this type are of significant interest for a number of applications especially in the medical field where they have found to have uses as vehicles for drug and vaccine delivery. In addition articles of this type have potential applications as diagnostics and in bio sensing research. The present invention relates to a method of formation of a film or network on a substrate that can be extended to a method of making an article of the type previously referred to.

Background of Invention

[3] There are an almost limitless number of applications of articles such as capsules due to the ability to tailor an article for any desired end use application. It is possible, for example, to modify the contents of the core of an article to modify the properties of the final article thus produced. For example in imaging applications a detectable core may be utilised to allow imaging or detection of the article in location. In addition the properties of the layer(s) that make up the walls of the article allow the properties of the article to be modified. Thus, for example the article walls may be modified to break down under certain conditions of temperature, pH or the like. Alternatively the article walls may be modified to target certain biological systems in a drug delivery sense. In other embodiments articles of an appropriate type may be used as a catalyst in chemical reactions where the walls of the article are designed to degrade (and hence release the catalyst in the core) under certain predetermined conditions. As can be seen, therefore, articles of this type have a significant number of potentially useful applications.

[4] In addition articles in the nanometre to micrometre size range are an important subset of articles due to their inherent ability to be used in biological systems. They can be used for encapsulating and thereafter releasing various substances (e.g., drugs, cosmetics, dyes, pesticides, and inks), or in other diverse applications such as catalysis or sensing. A variety of chemical and physicochemical procedures have been employed to prepare inorganic, metallic, polymeric and composite articles including capsules, including spray pyrolysis, nozzle reactor processes, self-assembly of molecules (e.g., vesicles, dendrimers, and block copolymers), and sacrificial core substrate-assisted methods, such as Layer-by- Layer and surface precipitation.

[5] Notwithstanding the variety of possible production techniques the production of articles (especially nano and micro- articles and capsules) is typically accomplished by the template mediated assembly of robust polymer films. In this technique films are engineered around templates by polymerizing the film layers onto templates or by depositing multiple polymer layers onto templates using Layer-by- Layer assembly (LbL). There are a number of advantages in using LbL for article preparation as it is sufficiently flexible such that various templates and template sizes can be layered on, and different polymers with tuneable functionality, responsiveness and degradability can be used in various combinations in order to control the physical, chemical and biological properties of the final article. This versatility of chemical and physical properties has made multilayer articles such as LbL capsules and colloids particularly relevant in drug delivery and bio-sensing research and has thus made them very attractive targets for research and study.

[6] Layer-by- Layer (LbL) assembly in tandem with colloidal templating involves the alternate deposition of polymers on a colloidal surface driven by electrostatics, hydrogen bonding, covalent bonding and/or complementary base pairing, typically followed by subsequent removal of the core by chemical or thermal means. Although the chemical and physical properties of LbL capsules can be precisely tuned, colloidal LbL, the current polymer assembly technique is highly labour intensive and time consuming and thus is not commercially attractive. LbL is commonly achieved by dispersing substrates in polymer solution, spinning the coated substrates down and washing sufficiently to remove excess polymer from the solution whereby the next polymer of interest is added and the procedure is repeated. Accordingly, the film deposition process requires numerous centrifugation and wash steps, and is generally limited to templates either dense enough or large enough for centrifugal sedimentation. Even when small templates can be centrifuged the potential for aggregation is increased through longer and faster spinning.

[7] Accordingly the multiple centrifugation and wash steps required between layer depositions in traditional LbL techniques are not only time consuming, but have prevented colloidal LbL from being truly automated due to the inevitable variability of pellet formation associated with centrifugation. As such the technique has not been able to effectively scaled up to allow production in industrial quantities. The failure to provide a process that can be carried out on a commercial scale has effectively meant that articles such as multilayer capsules produced by LbL techniques has remained a laboratory curiosity rather than achieving widespread industrial application.

[8] Some systems have been developed for streamlining LbL by utilizing filters or micro- fluidic devices. However, these systems remain limited in certain aspects inherent to traditional LbL, such as the choice of template used or the exploitation of different polymer interactions. Furthermore, these systems can suffer from engineering issues such as the caking of membranes, clogging of channels or low-throughput of materials.

[9] As a result of these limitations there has been limited availability of articles such as capsules, especially nano- and microcapsules due to the difficulty in manufacturing them in significant amounts. These limitations have greatly hindered the application of well-defined template derived articles such as capsules in biological applications, particularly in delivery, where small sized capsules are preferred. If an alternative approach to the formation of film layers on templates or substrates could be developed there is the possibility that it could provide a rapid technique for the preparation of articles of this type.

[10] Accordingly it would be desirable to develop a method of production of a film or network on the surface of a template or a substrate that could be used for the rapid synthesis of articles of this type.

[1 1] The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application. mary of Invention

[12] As a result of research into improving the efficiency of film or network forming techniques, especially film or network forming techniques around substrates the present applicants have identified a rapid method for the formation of a film or network of controllable thickness utilising one step assembly of a coordination complex on the surface of the substrate. This technique allows for rapid film/network formation along with control over a number of properties of the film/network such as its breakdown and the like.

[13] Accordingly in one embodiment the present invention provides a method of forming a film or network on the surface of a substrate of less than 1 cm the method comprising the steps of (i) forming a solution containing the substrate and (ii) contacting the solution containing the substrate with a solution of a metallic cation or a mixture of metallic cations and a solution of a polyphenolic compound to form a film or network on the surface of the substrate, wherein the contacting occurs for less than 30 minutes.

[14] The method of the present invention is typically very rapid and therefore allows the rapid formation of a film or network on the surface of a substrate. As a result of the rapid nature of the film/network forming step the technique may be used to form articles that hitherto would have required a laborious synthetic technique to manufacture. This technique therefore has the potential to not only provide a rapid method for the formation of a film or network on a surface of a substrate but, in addition, allows for a rapid synthesis of articles as the substrate can act as a template for template mediated assembly using the film or network forming technique. Accordingly the film/network forming techniques of the present invention have allowed the rapid synthesis of a number of articles of significant biological interest.

[15] Accordingly in yet a further embodiment the present invention provides a method of manufacturing an article the method comprising the steps of (i) forming a solution containing a substrate of less than I cm (ii) contacting the solution containing the substrate with a solution of a metallic cation or a mixture of metallic cations and a solution of a polyphenolic compound to form a film or network on the surface of the substrate, wherein the contacting occurs for less than 30 minutes.

[16] As stated previously there are a number of applications of articles of this type including their use in delivery of active agents. In one aspect the present invention provides a method of delivering an active agent to a part of the body of a mammal, the method comprising encapsulating the active agent in an article formed from an interconnected network of a polyphenolic compound and a metal cation or mixture of metal cations, and administering the article containing the active agent to the mammal. [17] In addition in certain circumstances the articles may be used in imaging. This will occur in circumstances where either the article contains an imaging agent in its core or where the article itself contains a metallic cation that can be imaged. In yet an even further aspect the invention provides a method of diagnostic imaging of a part of the body of a mammal, the method comprising administering an article to the mammal, the article being formed from an interconnected network of a polyphenolic compound and a metal cation or mixture of metal cations and containing a detectable moiety, and detecting the presence of the detectable moiety in the mammal.

[18] The present applicants have also found that the metal contained in the film or network of the articles of the present invention are available for certain chemical reactions such as catalysis. Accordingly in yet an even further aspect the present invention provides a method of catalysis of a chemical reaction, the method comprising contacting the reaction mixture with an article formed from an interconnected network of a polyphenolic compound and a metal cation or mixture of metal cations.

Brief Description of Drawings

[19] Figure 1 shows Fe'"/TA films prepared on PS substrates with various shapes (planar, spherical and ellipsoidal) and sizes (D = 120 nm - 10 μηι). (A) Photograph of PS slides before (top) and after (bottom) Fe'"/TA coating. (B-K) Microscopy images of Fe'"/TA capsules. DIC (B,I,J), AFM (C), TEM (E,F,G,K), SEM (D), and fluorescent microscopy (H) images.

[20] Figure 2 shows the general structure of tannic acid and a postulated schematic of how a metallic cation such as iron and tannic acid can react to form a film on the substrate.

[21] Figure 3 shows (A) CLSM images of Fe'"/TA capsules incubated with FITC-dextran (4, 250, 2,000 kDa, from left to right). Scale bars are 10 μιη. (B) The percentage of permeable capsules plotted against different molecular weights of FITC-dextran.

[22] Figure 4 shows XPS spectra. XPS spectra of Fe'"/TA capsules. Capsules were prepared from the following Fe'" and TA concentrations. [FeCI₃-6H₂0] = (A) 0.06 mg ml_^"1, (B) 0.12 mg mL^"1, (C) 0.2 mg mL^"1. [TA] = 0.4 mg mL^"1 for (A-C).

[23] Figure 5 shows AFM force analysis of Fe'"/TA capsules. (A) Representative force- deformation curves for the small deformation regime of Fe'"/TA capsules. (B) Force- deformation approach curves for large deformations of the first, third and tenth force cycle on the same individual Fe'"/TA capsule, demonstrating reversible shell deformation/reformation. [24] Figure 6 shows Fe'"/TA film growth with sequential deposition cycles on template particles. (A) Film thickness of Fe'"/TA capsules prepared from 3.6 μιτι-diameter PS templates measured by AFM (mean ± S.D., N = 20). (B) Representative AFM images and corresponding height profiles of the Fe'"/TA capsules with the indicated number of deposition cycles.

[25] Figure 7 shows Fe'"/TA film growth with sequential deposition cycles on planar substrates. (A) UV-Vis absorption spectra for Fe'"/TA films (up to five deposition cycles) deposited on quartz. (B) AFM height image of a scratched zone of an Fe'"/TA film (five deposition cycles) on a quartz substrate showing bare substrate (left) and a 20 nm-thick Fe'"/TA film (right). (C) Ellipsometric thickness of Fe'"/TA films on Au substrates (mean ± S.D., N = 5).

[26] Figure 8 shows the effect of iron(lll) chloride hexahydrate concentration ([FeCI₃-6H₂0]) on film thickness. (A) Representative AFM height images (5 ^χ 5 μηι) of capsules prepared at different [FeCI₃-6H₂0]. (B) Film thickness of the capsules prepared at the indicated [FeCI₃-6H₂0]. Film thicknesses of 20 capsules were measured. The results are average values with standard deviations (mean ± S.D., N = 20).

[27] Figure 9 shows the film thicknesses of the capsules prepared at the indicated TA concentrations. The results are average values with standard deviations (mean ± S.D., N = 20).

[28] Figure 10 shows the Adsorption isotherm of TA. (A) A 3D structure of TA. The structure was modeled using Chem3D software (CambridgeSoft Corporation), and subjected to energy minimization by the molecular mechanics method (MM2). Red: oxygen; gray: carbon; white: hydrogen. (B) Adsorbed amount of TA onto PS particles (D = 836 nm) plotted against the initial concentration of TA. Approximately 10 s was allowed for TA adsorption. The plot was fitted to the following Langmuir equation; y = y_max ^χ ax I (1 + ax), where y is the adsorbed amount of TA, y_max is the maximum y, x is the concentration of TA, a is a constant. By assuming that a TA molecule is monodisperse (Mw = 1701.2 g mol^"1) and occupies 4 nm², the adsorption amount of 100% coverage was calculated to be 2.42 mg/g of PS. This value is similar to the y_max value, suggesting that TA molecules can form a densely packed layer on the template surface, even without Fe'".

[29] Figure 11 shows a Schematic illustration of the Fe'"/TA film formation processes at different Fe'" concentrations. Excess Fe'" results in the small aggregates in bulk solution. These small aggregates attach to the surface, leading to the increase in roughness of the capsules. [30] Figure 12 shows Fe'"/TA coating on various substrates. (A-C) Photograph of planar substrates before (upper) and after (lower) Fe'"/TA coating. Glass (A), Au (B), PDMS (C). (D) Zeta potential values of particulate substrates in water before and after the Fe'"/TA coating. The results are average values with standard deviations (mean ± S.D.). (E) A confocal laser scanning microscopy image of protein-loaded CaC0₃ particles (red) coated with Fe'"/TA films (green) (F,G) TEM images of Au NPs non-coated (F) and coated (G) with Fe'"/TA films. (H,l) Photographs of Fe'"/TA capsules loaded with Fe₃0₄ nanoparticles dispersed in water before (H) and after (I) applying a magnet.

[31] Figure 13 shows Fe'"/TA coating on various particles. (A) A 3D CLSM image of Rhodamine B-loaded CaC0₃ particles (red) coated with Fe'"/TA films (green). (B) A SEM image of mesoporous CaC0₃ templates. (C-E) DIC, SEM, and TEM images of Fe'"/TA replica particles. (F) A TEM image of Au NPs coated with Fe'"/TA films. (G) TEM images of Fe'"/TA capsules loaded with Fe₃0₄ nanoparticles.

[32] Figure 14 shows pH-responsive disassembly of Fe'"/TA capsules. (A) UV-Vis absorption spectra of spherical Fe'"/TA capsule dispersions (D = 3.6 μηι, 4.0 ^χ 10⁷ capsules ml_^"1) at various pH. Inset is a photograph of the capsule dispersions at the indicated pH. (B) pH-dependent transition of dominant Fe'"/TA complexation state. R represents the remainder of the TA molecule. (C) Plot of remaining capsule population (%) against time. The results are average values with standard deviations from three independent measurements (mean ± S.D.).

[33] Figure 15 shows A DIC image of Fe'"/TA capsules prepared from 3.6 μηΊ-diameter PS templates, showing that they shrink at pH 2.

[34] Figure 16 shows a representative AFM image (left) and height profile (right) of a Fe'"/TA capsule after incubation at pH 5.0 for two days.

[35] Figure 17 shows MTT assay of Fe'"/TA capsules on HeLa cells. Cells were incubated at the indicated capsule ratio for 72 h. The results are average values with standard deviations (mean ± S.D., N = 4).

[36] Figure 18 shows Capsules prepared from various metals. (A) AFM image of V'"/TA capsules. (B) EDS analysis of V'"/TA capsules. (C) DIC image of Gd^l"/Fe'"/TA hybrid capsules. (D) EDS analysis of Gd"^l/Fe"^l/TA hybrid capsules. (E) DIC image of C^/Fe^TA hybrid capsules. (F) EDS analysis of Cr^l"/Fe'"/TA hybrid capsules. The Cu signals in the EDS spectra are from the copper grids used for TEM. [37] Figure 19 shows TA capsules in solution produced with a variety of metals. The inset shows a photo of the respective TA capsule dispersions.

[38] Figure 20 shows film deposition of another polyphenol compound, (-)- Epigallocatechin gallate (EGCG), with Fe'". (A) A suspension of 3.6 μιη-diameter PS particles uncoated (left) and Fe'"/EGCG-coated (right). The zeta-potential shifted from -27.4 ± 2.8 to -37.1 ± 6.2 mV after Fe'"/EGCG coating, indicating successful film deposition. (B) Pellets of uncoated (left) and Fe'"/EGCG coated PS particles (right). (C) UV-Vis spectra monitoring of sequential Fe'"/EGCG coatings (seven coatings) on a quartz slide. (D) A photograph of an uncoated (left) and Fe'"/EGCG coated (right) quartz slide.

[39] Figure 21 Assembly of capsules from various metals, a, Schematic illustration of the assembly of TA and metal ions to form a MPN film on a particulate template, followed by the subsequent formation of a MPN capsule, b, Periodic table: metals highlighted in dark highlight are used to form MPN capsules in this study. Metals highlighted in light highlight show other metals that may be used for MPN capsule preparation based on previous studies on metal-coordination with phenolics in bulk solution, c, DIC images of MPN capsules prepared from different metals. Scale bars are 5 μηι. Inset images are photographs of MPN capsule suspensions.

[40] Figure 22 shows Film thickness and corresponding AFM images of different MPN capsules prepared from different metal concentrations. Data are means ± SD (N = 10).

[41] Figure 23 shows The dependence of fluorescence intensity on the feed concentration of Eu'" for Eu'"-TTA-TA capsules. The inset shows an image of the Eu'"-TTA-TA capsule dispersions.

[42] Figure 24 shows UV-Vis spectra of MPN capsule suspensions prepared from various metals.

[43] Figure 25 shows Fourier transformed infrared (FT-IR) spectra of (a) TA and (b) Fe'"- TA capsules.

[44] Figure 26 shows Survey scan spectra of X-ray photoelectron spectra (XPS) of MPN capsules coordinated with different metals.

[45] Figure 27 shows X-ray diffraction pattern of Cu"-TA capsules. [46] Figure 28 shows structural characterization of MPN capsules, a-c, SEM (a), TEM (b) and AFM (c) images of Cu"-TA capsules, d, EDX elemental mapping of Cu"-TA, Al'"-TA and Zr^lv-TA capsules. Scale bars are 2 μιη in (a)-(c), 200 nm in the inset of (b) and 1 μιη in (d).

[47] Figure 29 shows Morphology of Al'"-TA capsules obtained from D = 3.6 μιη templates, as characterized by SEM (a), TEM (b) and AFM (c). Scale bars are 2 μηι in (a) and (b), and 5 μηι in (c).

[48] Figure 30 shows the morphology of Zr^lv-TA capsules obtained from D = 3.6 μιη templates was characterized using SEM (a), TEM (b) and AFM (c). Scale bars are 2 μηι in (a) and (b), 5 μηι in (c).

[49] Figure 31 shows controlled disassembly of MPN capsules, a, pH-dependent disassembly kinetics of Cu"-TA, Al'"-TA and Zr^lv-TA capsules at different pH, as assessed by flow cytometry. Data are means ± SD (N = 3). b, Degradation of FITC-dextran loaded Al'"-TA capsules in JAWS II cells at different times, as imaged by deconvolution fluorescence microscopy. Green corresponds to FITC-dextran, red to the cell membrane staining and blue to nuclear staining. Scale bars are 10 μιη.

[50] Figure 32 shows MTT assay of Al'"-TA capsules using JAWS II cells. Cells were incubated with different numbers of capsules for 48 h. The results are average values with standard deviations (means ± SD, N = 4).

[51] Figure 33 shows engineering multifunctional MPN capsules for imaging, a, Normalized fluorescence spectra of capsule suspensions of Eu'"-TTA-TA and Tb'"-AA-TA excited at 360 nm. Insets are photographs of the corresponding capsule suspensions (top) and of PDMS coated with Eu'"-TTA-TA (bottom) excited at 365 nm. Scale bars are 1 cm. b, Pseudo-colour PET phantom of suspensions of ⁶ Cu"-TA capsules at different dilution ratios (stock suspension concentration is 3.2 ^χ 10⁸ capsules per ml with an activity of 1 MBq). c, Small-animal PET/CT image of an in vivo model 30 min after injection of 1 MBq ⁶ Cu"-TA capsules (maximum intensity projection (right), sagittal view (left)), d, MRI phantom images of capsule suspensions immobilized in agarose. Shown are T_x -weighted inversion recovery images (with inversion time Τ_γ = 1 ,375 ms) and T₂ -weighted spin echo images (with echo time T_E = 50 ms) of samples with three different metal concentrations: 0.02 mM, 0.1 mM and

0.3 mM (0.15 mM for Gd'"-TA). e, Table of MRI relaxivities for Fe'"-TA, Mn"-TA and Gd'"-TA capsule suspensions measured with a 9.4 T animal MRI system.

[52] Figure 34 shows Post-mortem biodistribution of ⁶ Cu"-TA capsules. Data are given as a % of injected dose per body weight (g) as measured 30 min after a lateral tail-vein injection. [53] Figure 35 shows Results of the MR relaxivity experiments with Gd'^-TA , Mn"-TA and Fe^Hi-TA capsules. The relaxation rates were measured on a 9.4 T Ri system. The fitted relaxivities are labelled in each panel The dashed lines show the linear fits and the dotted lines show the fit uncertainties.

[54] Figure 36 shows MPN capsules for catalysis, a, Schematic illustration of the hydrogenation of quinoline. b, Catalytic activities of Rhl!l-TA capsules and RhCI(CO)(TPPTS)2 for the hydrogenation of quinoline at different reaction temperatures.

[55] Figure 37 shows the Structural characterization of DOX-Alill-TA capsules, a) DIG, b) DM, c) TEM, and d) SEM images of DOX-AUIi-TA capsules, e) EDX elemental mapping of DOX-Allil-TA capsules. Scale bars represent 2 pm in a)-d), 0.2 pm in the inset of c) and d), and 1 pm in e).

[56] Figure 38 shows a) Degradation kinetics of AI^IM-TA capsules at pH 5.0, 6.0, and 7.4, as assessed by ftow cytometry, b) Time-dependent release of DOX from DOX-Ai^m-TA capsules at pH 5.0, 6.0, and 7.4. All data are means ± SD (n = 3).

[57] Figure 39 shows images of intracellular delivery of DOX-Al^m~TA capsules in a) MB231 cells and b) HeLa cells acquired by deconvolution microscope with a standard F!TC/TRITC/DAPI filter set. The blue nucleus stained with Hoechst 33342 was visualized with the DAPt filter, and green cell membrane stained with wheat germ agglutinin Alexa Fluor^® 488 was visualized with the FITC filter. The inherent red fluorescence of DOX were observed with the TRITC filter. All scale bars represent 10 pm. c) Cytotoxicity of DOX-Ai^m-TA capsules and free DOX as a function of DOX concentration evaluated by MTT assay. The cell viability of untreated cells was normalized as 100%. Data represent mean ± SD (n = 4). d) Percentage of apoptotic cell HeLa and M8231 cells after treatment with DOX-AI⁸ⁱ-TA or DOX at different dose for 48 h. All data represent mean ± SD (n = 3, Student's t-test, ^*P < 0.05, ^**P < 0.01. *^**P < 0.005 ).

[58] Figure 40 shows fluorescence spectra of Α -ΤΑ capsules loaded with different amount of DOX.

Detailed Description

[59] In this specification a number of terms are used which are well known to a skilled addressee. Nevertheless for the purposes of clarity a number of terms will be defined in order to inform the reader. [60] As used herein the term film refers to a thin coating or layer. A film may be a continuous film or discontinuous film in the sense that there may be gaps in the film as a result of the materials from which it is made. A film may be porous.

[61] As used herein the term network refers to an interconnected group of objects that form a structure significantly more extensive in size than the individual components from which it is made. In relation to the present invention a network refers to the structure formed by the complex between the polyphenolic compound and the metallic cation or mixture of metallic cations. If a network is sufficiently extensive it may be classified as a film.

[62] The applicants have found that the methods of the present invention are amenable to the rapid formation of a wide variety of metal containing films or networks on a wide variety of substrates which in itself means that the articles thus formed can be used in a number of applications. As previously stated the method can also be used in the production of a wide range of articles as in principle the process can be applied to a wide range of substrates of varying shapes, sizes and functionalities. The method of the invention may be used in circumstances where there is the intention that the substrate is intended to form part of the final article or in circumstances where it is the intention that the substrate does not form part of the final article.

[63] Specifically as the process for producing articles of the present invention allows for either the retention or removal of the substrate following completion of the process there is the ability to produce articles containing a solid core (by retention of the substrate with the film or network on the surface) or articles having a hollow or substantially hollow core (by removal of the substrate). In one embodiment the article is a particle. In one embodiment the article is a capsule.

[64] The first step in the processes of the present invention involves forming a solution containing the substrate as the process of film/network formation of the present invention is carried out in solution. In circumstances where only a portion of the substrate is to be covered with the film/network then all that is required is that the substrate be placed into a solution. In circumstances where the entire surface of the substrate is to be coated it is typical that the step of forming a solution containing the substrate comprises suspending the substrate in solution.

[65] The substrate may be of any suitable substrate material. In theory the substrate could be a solid, a liquid or a gas depending upon the exact nature of the substrate that is desired to be coated by the process of the invention. For example a liquid substrate (typically called an emulsion substrate) could be an oil particle (oil in water) or a silicon emulsion. In relation to gaseous substrates a suitable example would be an air bubble immobilised in a permeable matrix. In certain embodiments the substrate is a solid substrate. In certain embodiments the substrate a liquid substrate.

[66] The process of the present invention is found to be applicable to substrates made from a wide variety of materials. The nature of the substrate will depend on the identity of the substrate desired to be coated (in the film or network forming process of the invention) or the whether there is an intention to remove or retain the substrate (in the method of manufacturing an article process of the invention). In certain embodiments the substrate is selected from the group consisting of an organic particle, an inorganic particle, a biological particle and combinations thereof.

[67] The identity of the substrate may also vary depending upon the intended end use of the article of the invention. For example if the intended end use of the article is to deliver an agent to a part of the body then in some embodiments of the invention it is desirable to incorporate the agent to be delivered on or into the substrate prior to the formation of the film or network on the surface of the substrate. In these embodiments the substrate chosen is typically a porous substrate and the agent to be delivered is adsorbed/absorbed on or into the porous substrate. A skilled worker in the field would readily be able to determine a suitable porous substrate based on the desired agent to be incorporated in or onto the substrate.

[68] Examples of suitable materials include inorganic oxides such as ceria, zirconia, titania and silica, metals such as gold, and organics such as certain polymeric materials like polystyrene, melamine formaldehyde or biological materials like alginate. Emulsion substrates could also be used such as air bubbles, oil droplets or silicon based silane emulsions. Whilst the substrate may be made of any suitable material as discussed above it is commonly silica based due to the relative ease of access of materials of this type and their relatively low cost.

[69] In one embodiment the substrate is a solid substrate. The substrate may be a solid non porous substrate or a porous substrate. In one embodiment the substrate is a liquid substrate. In one embodiment the substrate is a porous substrate.

[70] In certain embodiments the substrate is selected from the group consisting of polystyrene, glass, gold (Au), polydimethylsiloxane (PDMS), poly(lactic-co-glycolic acid) (PLGA), melamine-formaldehyde resin (MF), low-molecular-weight PDMS emulsion, silica (Si0₂), aminated Si0₂, cetyltrimethylammonium bromide-capped Au nanoparticles (Au NPs), calcium carbonate (CaC0₃), Escherichia coli (E. coli), and Staphylococcus epidermidis (S. epidermidis). In one embodiment the substrate is a metallic particle. In one embodiment the substrate is a silica particle. [71] In one embodiment the substrate is made of a suitable material which allows for its subsequent removal during a substrate removal step. A skilled addressee in the art will readily understand the types of materials that can be used to form substrates of this type depending upon the layers of material to be coated on the substrate. As will be readily appreciated if an optional substrate removal step is contemplated then the metallic cation and the polyphenolic compound used to form the metal complex that forms the film layer(s) on the substrate must be compatible with (i.e. not degrade) under the conditions required for substrate removal.

[72] The substrates used may take any suitable shape and may be for example in the shape of, spheres, cubes, prisms, fibres, rods, tetrahedrons or irregular particles. Accordingly, the shape of each substrate is independently selected from the group consisting of a sphere, a cube, a prism, a fibre, a rod, a tetrahedron and an irregular shape. As will be appreciated by a skilled worker in the art the shape of the substrate will typically determine the shape of the article ultimately produced. It is typical, however, that the substrate is spherical or substantially spherical.

[73] It will be convenient to describe the invention in terms of a spherical substrate, but it shall be kept in mind that any article produced by the process of the invention may be of any shape depending on the shape of the substrate. Thus in general the final shape of the articles produced by the process of the invention will take the general shape of the substrate used in their production. Thus for example if the substrate is spherical then the final product will typically be spherical. If the substrate is a fibre then once again the final product will typically be a fibre. There may be some fluctuation in the size of the article compared to the size of the substrate, due to shrinkage and/or swelling of the article depending on the specific production conditions. A skilled worker in the field will typically be able to readily choose a suitable substrate shape for their desired end use application.

[74] The substrate may be of any suitable size with the size being determined, in part by the desired size of the final article to be produced and by the availability of the desired substrate. In particular the methods of the present invention are found to be particularly applicable to substrates of less than 1 cm. In one embodiment the substrate has a particle size of less than 1 mm. In one embodiment the substrate has a particle size of less than 500 μηι. In one embodiment the substrate has a particle size of less than 100 μηι. In one embodiment the substrate has a particle size of less than 1000 nm. In one embodiment the substrate has a particle size of less than 500 nm. In one embodiment the substrate has a particle size of less than 200 nm. In one embodiment the substrate has a particle size of less than 100 nm. In one embodiment the substrate has a particle size of less than 10 nm. In one embodiment the substrate has a particle size of less than 1 nm. In one embodiment the substrate has a particle size from 1 nm to 1 cm. In one embodiment the substrate has a particle size from 1 nm to 1 cm. In one embodiment the substrate has a particle size from 1 nm to 5 mm. In one embodiment the substrate has a particle size of from 1 nm to 1 mm. In one embodiment the substrate has a particle size of from 1 nm to 500 μηι. In one embodiment the substrate has a particle size of from 1 nm to 400 μηι. In one embodiment the substrate has a particle size of from 1 nm to 300 μηι. In one embodiment the substrate has a particle size of from 1 nm to 200 μηι. In one embodiment the substrate has a particle size of from 1 nm to 100 μηι. In one embodiment the substrate has a particle size of from 1 nm to 50 μηι. In one embodiment the substrate has a particle size of from 1 nm to 1000 nm. In one embodiment the substrate has a particle size of from 1 nm to 500 nm. In one embodiment the substrate has a particle size of from 1 nm to 400 nm. In one embodiment the substrate has a particle size of from 1 nm to 300 nm. In one embodiment the substrate has a particle size of from 1 nm to 200 nm. In one embodiment the substrate has a particle size of from 1 nm to 100 nm. In one embodiment the substrate has a particle size of from 1 nm to 50 nm. In one embodiment the substrate has a particle size of from 20 nm to 500 nm. In one embodiment the substrate has a particle size of from 20 nm to 400 nm. In one embodiment the substrate has a particle size of from 20 nm to 300 nm. In one embodiment the substrate has a particle size of from 20 nm to 200 nm. In one embodiment the substrate has a particle size of from 20 nm to 100 nm. In one embodiment the substrate has a particle size of from 50 nm to 400 nm. In one embodiment the substrate has a particle size of from 50 nm to 300 nm. In one embodiment the substrate has a particle size of from 50 nm to 200 nm. In one embodiment the substrate has a particle size of from 50 nm to 100 nm. In one embodiment the substrate has a particle size of from 30 nm to 30 μηι. In another embodiment the substrate has a particle size of from 200 nm to 6 μηι. In another embodiment the substrate has a particle size of from 400 nm to 5 μηι. In another embodiment the substrate has a particle size of from 500 nm to 4 μηι. Once again a skilled worker will readily be able to choose a suitable sized substrate based on the desired size of the final particle. In general one of the advantages of the method of the present invention in comparison to methods that do not use a substrate is that it provides for fine control over the final article geometry which allows for the controlled formation of articles of the desired size and shape with a high degree of certainty.

[75] The surface of the substrate may be modified by addition of functional moieties to enhance the binding of a metal complex to the surface of the substrate. Any of a number of functional moieties can be added onto the surface of the substrate with the choice of functional moiety being chosen to complement the metal complex being bound to the surface. As used herein the term complement is intended to mean that two materials "complement" each other if they have a binding affinity for each other.

[76] A skilled worker in the area will generally have little difficulty in choosing a functional moiety to introduce onto the surface of the substrate to complement the chosen metal complex. By way of example, one method of modifying the surface of a silica substrate is to graft a moiety such as 3-aminopropyltriethoxysilane (APTS) onto the surface of the silica particle. This introduces an amine surface functionality that can interact with any phenolic groups on a polyphenolic compound to host the polyphenolic compound on the surface. If it was desired to host a polyphenolic compound on the surface that contains amino moieties this could similarly be carried out by attaching phenolic moieties to the exposed surface of the substrate.

[77] In addition to the discussion of possible substrates above it will be appreciated that in the process of making an article the substrate may be either a functional substrate or a sacrificial substrate. In one embodiment the substrate is a functional substrate. In another embodiment the substrate is a sacrificial substrate.

[78] A functional substrate is a substrate that is intended to remain in the article after the article is produced to impart some functional property on the article. An example of such a functional substrate would be a metallic substrate that is used in imaging applications. Another example of a functional substrate may be a radioactive metal substrate used in targeted chemotherapy applications. A skilled worker in the art can readily identify suitable materials for use as functional substrates as the choice of functional substrate will typically be determined by the function required.

[79] A sacrificial substrate is a substrate that is used during the production of the article but which is designed to be removed after article formation to form a hollow article. The choice of a sacrificial substrate is general relatively straightforward as the main consideration is the ability to remove the substrate without causing any damage to the metal complex used in the film used in formation of the article.

[80] As discussed above once the substrate is chosen the first step in the processes of the present invention involves forming a solution containing the substrate as the process of film/network formation of the present invention is carried out in solution. In circumstances where only a portion of the substrate is to be covered with the film or network then all that is required is that the substrate be placed into a solution. In circumstances where the entire surface of the substrate is to be coated it is typical that the step of forming a solution containing the substrate comprises suspending the substrate in solution. In the processes of the invention the concentration of the substrate in the solution can vary very widely as all that is required is that the substrate be in solution to facilitate addition of the further solutions of the metallic cation and the polyphenolic compound. Nevertheless the solution concentration is typically relatively low being between 1 and 500 mg/mL. In some embodiments the solution is from 1 to 300 mg/mL. In some embodiments the solution is from 1 to 100 mg/mL. In some embodiments the solution is about 10mg/mL. A number of different solvents may be used to make up the solution containing the substrate but it is typical that the solution is an aqueous solution.

[81] Without wishing to be bound by theory it is believed that the films or networks formed by the process of the present invention are formed by a coordination complex between the metallic cation or mixture of metallic cations and the polyphenolic compound. This explains the rapid formation of the film or network on the surface of the substrate and also ultimately provides for rapid dis-assembly of the film or network if required as a coordination complex of this type can generally be rapidly broken down based on the pH of the solution it is exposed to or other factors.

[82] Once the solution containing the substrate has been prepared as discussed above the methods of the present invention involve contacting the solution containing the substrate with a solution of a metallic cation or a mixture of metallic cations and a solution of a polyphenolic compound. The contacting may involve simultaneous addition of both solutions to the solution containing the substrate or it may involve sequential addition of the two solutions.

[83] In certain embodiments the contacting comprises addition of a solution of a metallic cation or a mixture of metallic cations to the solution containing the substrate followed by addition of a solution of the polyphenolic compound. In certain embodiments the contacting comprises addition of a solution of the polyphenolic compound to the solution containing the substrate followed by addition of a solution of a metallic cation or a mixture of metallic cations.

[84] In the methods of the present invention any suitable metallic cation or a mixture of metallic cations may be used that is capable of forming a coordination complex with a polyphenolic compound. Examples of suitable metallic cations include cations from an alkali earth metal, a transition metal or a lanthanide metal.

[85] In certain embodiments the metallic cation or a mixture of metallic cations is selected from the group consisting of a magnesium cation, a calcium cation, a cadmium cation, a strontium cation, a barium cation, an iron cation, an aluminium cation, a ruthenium cation, a rhodium cation, a terbium cation, a vanadium cation, a chromium cation, a manganese cation, a zinc cation, a copper cation, a cobalt cation, a nickel cation, a molybdenum cation, a titanium cation, a zirconium cation, a cerium cation, a europium cation, a gadolinium cation and a mixture of two or more thereof.

[86] In general these metals can be used in a variety of oxidation states although it is preferred that the metallic cation is selected from the group consisting of Ba²⁺, Sr²*, Mg²⁺, Ca²⁺, Cd²⁺, Mn²⁺, Cu²⁺ Zn²⁺, Co²⁺, Ni²⁺, Mo²⁺, Fe³⁺, Al³⁺, Ru³⁺, Rh³⁺, Tb³⁺, V³⁺, Cr³⁺, Eu³⁺, Gd³⁺, Zr ⁺, Ti ⁺ and Ce ⁺.

[87] The metallic cation or mixture of metallic cations in solution may be used with any suitable counter anion that does not interfere with complex formation. In addition there is the requirement that the metallic species be water soluble and so the anion is chosen to create a water soluble species. In general a halogen counter anion is suitable with the chloride anion being particularly suitable. Examples of suitable metal species used in creating the solution of the metallic cation or mixture of metallic cations include BaCI₂, SrCI₂, MgCI₂, CaCI₂, MnS0₄, CuCI₂, ZnS0₄, Co(N0₃)₂, FeCI₃, AICI₃, VCI₃, CrCI₃, EuCI₃, GdCI₃, ZrCI₄, TiCI₄, and (NH₄)₂Ce(No₃)₆.

[88] In certain embodiments the metallic cation is an iron cation. In certain embodiments the metallic cation is iron (III).

[89] The solution of the metallic cation or mixture of metallic cations may be at any suitable concentration. Indeed the concentration of the solution of the metallic cation or mixture of metallic cations will be determined based on the nature of the substrate to be coated (and its concentration) as well as the concentration of the polyphenolic compound to be used. Typically however, the solution of the metallic cation or mixture of metallic cations is at a concentration of less than 1 mM. In certain embodiments the concentration of the solution of the metallic cation or mixture of metallic cations is from 0.1 to 0.9 mM. In certain embodiments the concentration of the solution of the metallic cation or mixture of metallic cations is from 0.2 to 0.8 mM. In certain embodiments the concentration of the solution of the metallic cation or mixture of metallic cations is from 0.22 to 0.74 mM.

[90] The selection of the cation (or mixture of cations) will depend upon a number of factors such as the desired metal (or metals) to be incorporated into the final article. Thus for example in certain applications such as catalysis as discussed below there is a requirement that a certain metal (the one that acts as the catalyst) be incorporated into the article so that it can act as a catalyst. In certain other applications, such as diagnostic imaging, the metal is a detectable moiety such as a radioactive metal that can be used in the imaging application. Once again a strength of the process of the present invention is the flexibility the process provide for the rapid synthesis of a wide variety of articles containing different metals with a diverse range of functionality. In general a skilled worker in the art can readily choose the desired metal (or metals) to achieve the desired properties in the finished article.

[91] A large number of different polyphenolic compounds may be used in the methods of the invention with the only real requirement being that the polyphenolic compound is capable of forming a coordination complex with a metallic cation.

[92] An example of a subset of polyphenolic compounds that are found to be particularly suitable are those that contain catechol groups, gallol groups or a mixture thereof. Catechol groups (ortho phenols) form strong complexes with metallic cations and any polyphenolic compound containing these groups will be applicable for use in the present invention. In a similar vein gallol groups (1 ,2,3 tri hydroxyl phenols) also form very strong coordination complexes and so any polyphenolic compound containing these groups will also be particularly suitable.

[93] An example of a particularly suitable set of compounds are tannins. Tannins are polyphenolic compounds that are extracted from various plants and trees and which are generally classified according to their chemical structures as being (a) hydrolysable tannins; (b) condensed tannins and (c) mixed tannins containing both hydrolysable and condensed tannins. Any of a wide range of tannins may be used although it is typically preferred that the naturally occurring plant tannins are used. Vegetable tannins of this type include tannins derived from Quebracho, mimosa, mangrove, spruce, hemlock, gabien, wattles, catechu, uranday, tea, larch, myrobalan, chestnut, wood, divi-divi, valonia, sumac, chinchona, oak and the like. These plant tannins are typically useful as the polyphenolic compounds used in the present invention as whilst they are not pure compounds with known structures they contain numerous components including phenolic moieties such as catechol and pyrogallol and the like condensed into a complicated structure. As such they make particularly suitable polyphenolic compounds.

[94] In certain embodiments the polyphenolic compound is selected from the group consisting of tannic acid and epigallocatechin. In one embodiment the polyphenolic compound is tannic acid. In one embodiment the polyphenolic compound is epigallocatechin.

[95] Although the chemical structure of tannic acid is usually given as a decagalloyl glucose (C76H52O46) it is actually a mixture of polygalloyi glucose molecules with different numbers of esterified gallic acid moieties. Tannic acid is particularly suitable as it rapidly forms coordination complexes with metallic cations. For example three galloyl groups from tannic acid can react with a metallic cation such as Fe'" ion to form a stable octahedral complex thus allowing each tannic acid molecule to react with several Fe'" centers to form a cross-linked film.

[96] The solution of the polyphenolic compound may be at any suitable concentration. Indeed the concentration of the solution of the polyphenolic compound will be determined based on the nature of the substrate to be coated (and its concentration) as well as the concentration of the metallic cation or mixture of metallic cations to be used. Typically however, the solution of the polyphenolic compound is at a concentration of less than 1 mM. In certain embodiments the concentration of the solution of the polyphenolic compound is from 0.1 to 0.9 mM. In certain embodiments the concentration of the solution of the polyphenolic compound is from 0.2 to 0.8 mM. In certain embodiments the concentration of the solution of the polyphenolic compound is from 0.22 to 0.74 mM.

[97] Indeed while the discussion above had focussed on the individual concentrations of the metallic cation and the polyphenolic compound a skilled addressee will readily appreciate that as the final film or network that is formed is a coordination complex then the important variable is the ratio of these two concentrations to each other. The ideal ratio of concentrations will be determined by the individual metallic cation or mixture of metallic cations and polyphenolic compound used in each instance as this will affect the reaction stoichiometry of the final complex formed. Whilst the reaction will proceed under a wide variety of conditions there is no doubt that for any given combination of metallic cation or mixture of metallic cations and polyphenolic compound there is an optimal ratio that can be determined by a skilled worker in the art

[98] Nevertheless the present applicants have found that a suitable ratio of metallic cation or mixture of metallic cations to polyphenolic compound is in the ratio of 0.5: 1 to 5: 1. In some embodiments the molar ratio of metallic cation or mixture of metallic cations to polyphenolic compound is in the ratio of 1 : 1 to 3: 1. In some embodiments the molar ratio of metallic cation or mixture of metallic cations to polyphenolic compound is in the ratio of about 1 : 1.

[99] The contacting may occur at any suitable temperature. In certain embodiments the contacting may occur at a temperature in the range of 10°C to 40°C. As previously stated the process is very rapid with film or network formation being completed very soon after addition of the second solution. As such in some embodiments the contacting occurs for less than 30 minutes. In some embodiments the contacting occurs for less than 25 minutes. In some embodiments the contacting occurs for less than 20 minutes. In some embodiments the contacting occurs for less than 15 minutes. In some embodiments the contacting occurs for less than 10 minutes. In some embodiments the contacting occurs for less than 5 minutes. In some embodiments the contacting occurs for less than 2 minutes. In some embodiments the contacting occurs for less than 1 minute. In some embodiments the contacting occurs for less than 50 seconds. In some embodiments the contacting occurs for less than 40 seconds. In some embodiments the contacting occurs for less than 30 seconds. In some embodiments the contacting occurs for less than 20 seconds. In some embodiments the contacting occurs for less than 10 seconds. In some embodiments the contacting occurs for less than 5 seconds. In some embodiments the contacting occurs for about 2 seconds.

[100] After the two solutions have been added to the solution containing the substrate the applicants have found that film or network formation occurs rapidly to form the film or network on the substrate. Where the method aims to produce an article it is typical that the substrate is suspended in solution and the solution now contains suspended substrate particles with a film or network on the surface. These can be analysed in solution or can be recovered from solution for further elaboration.

[101] As will be appreciated by a skilled addressee the present invention is sufficiently flexible that it allows for the formation of a film or network of a wide variety of thicknesses in a single step. This is very advantageous as it means that the process can be very rapid in the formation of films or networks on a substrate. Nevertheless the current process also contemplates situations where it is desirable to add multiple layers to the substrate. This might be, for example, where there is a desire to incorporate a different metal into each layer in order to provide a different functional effect. As will be readily appreciated this is easily managed by repeating the steps of adding a solution of a metallic cation and a solution of a polyphenolic compound to form a further layer. This step can be repeated any number of times until the desired number of layers have been applied.

[102] In certain embodiments the process of the invention for making articles may involve the step of removal of the substrate to produce an article having a hollow core. If desired the substrate may be removed by exposure to a suitable agent that is capable of degrading the substrate. In general the agent will be chosen such that it is able to degrade the substrate but such that it will not damage the film on the surface of the substrate. An example of a suitable agent is hydrofluoric acid or sodium hydroxide. It has been found that the silicone dioxide core of a silica substrate is readily degraded in hydrofluoric acid, and is converted to [SiF6]2- ion thereafter leaving the macromolecular structure.

[103] Typically the mixture containing the substrate is shaken when the substrate is exposed to the hydrofluoric acid. If hydrofluoric acid is used it is found that the silica can be dissolved using a wide range of concentrations of acid. The acid may be of any strength although it is convenient to use an acid strength of from 1 to 10 M, more preferably about 2 M. In some cases the hydrofluoric acid is applied as a buffered solution with ammonium fluoride. Whereas hydrofluoric acid is preferred as a solvent, other suitable solvents would be well appreciated by the skilled practitioner.

[104] In one embodiment removal of the substrate comprises dissolving the substrate. The substrate may be dissolved in a number of ways and the exact method chosen will depend on the nature of the substrate to be dissolved and the nature of the macromolecular layers. In one embodiment the substrate is dissolved by contacting the substrate with a solution of ammonium fluoride in HF.

[105] In principle any substance that can dissolve or degrade the substrate may be used in the step of removing the substrate and a skilled addressee would readily understand the required agent based on the substrate used at first instance.

[106] Following optional removal of the substrate the resultant article is then typically washed to remove any residual agent used in the removal of the substrate as well as any unbound materials. The articles may then be isolated by filtration and/or centrifugation.

[107] The articles that can be produced by the process of the present invention have a wide variety of potential applications depending upon the exact manner in which they have been formulated. For example they may be used in catalysis, imaging or in the delivery of active agents.

[108] The articles produced by the process of the present invention are formed from an interconnected network of a polyphenolic compound and a metal cation or mixture of metal cations. The interconnected network may be substantial enough to be classified as a film. The size of the substrate is typically determined by the size of the substrate used in the formation of the article. Nevertheless the article is typically less than 1 cm in size.

[109] As stated above in one embodiment the present invention therefore provides a method of delivering an agent active agent to a part of the body of a mammal, the method comprising encapsulating the active agent in an article formed from an interconnected network of a polyphenolic compound and a metal cation or mixture of metal cations, and administering the article containing the active agent to the mammal.

[1 10] The active agent may be any active agent that has a desired biological activity. The active agent may be a pharmaceutically active agent or a veterinary active agent. In principle any active agent that can be used can be delivered by the articles of the present invention. Potential active agents may include proteins or protein crystals, peptides, DNA, polymer-drug conjugates, hydrophobic drugs, nanoparticles e.g. magnetite, and quantum dots.

[1 11] The first step in the process involves encapsulation of the active agent in the article produced by the method of the present invention. Whilst this may be achieved in a number of ways it is typical that the encapsulation process takes advantage of the substrate used in the processes of the present invention. Accordingly in one embodiment the step of encapsulation of the active agent in the article of the present invention involves synthesis of the article around the agent. Accordingly in certain embodiments a porous substrate is chosen and the active agent in solution is bought into contact with the porous substrate such that a certain amount of the active agent in solution is adsorbed or absorbed into the pores of the porous substrate. Once the active agent is absorbed into the porous template the process of article formation as discussed previously is carried out and following removal of the template the final product is an active agent encapsulated in an article formed from an interconnected network of a polyphenolic compound and a metal cation or mixture of metal cations. Once encapsulated in this way the active agent is then ready to be delivered.

[1 12] The administration of the article containing the active agent to the mammal may be carried out in any way known in the art. Indeed given that the materials are generally quite small in size a suitable means of administration is by injection.

[1 13] Pharmaceutical compositions containing an active agent encapsulated as discussed above for parenteral injection typically comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[1 14] These compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminium monostearate and gelatin. [1 15] Once administered to the body it is desirable that the active agent be able to be controllably released at the site of interest. One advantage of the articles of the present invention is that they allow for release in this way as the complex between the metal and the polyphenolic compound is pH dependent. As such when the complex degrades it release the active agent at the site of interest.

[1 16] The articles of the present invention may also be used in diagnostic imaging. Accordingly in some embodiments the present invention also provides a method of diagnostic imaging of a part of the body of a mammal, the method comprising administering an article to the mammal, the article being formed from an interconnected network of a polyphenolic compound and a metal cation or mixture of metal cations and containing a detectable moiety, and detecting the presence of the detectable moiety in the mammal. The articles of the present invention can be used in a number of diagnostic imaging techniques including Positron emission topography (PET), magnetic resonance imaging (MRI) and fluorescence imaging. In one embodiment the imaging technique is PET imaging. In one embodiment the imaging technique is MRI imaging.

[1 17] As stated above in order to be used for diagnostic imaging it is necessary that the article formed from an interconnected network of a polyphenolic compound and a metal cation or mixture of metal cations contains a detectable moiety.

[1 18] In certain embodiments depending upon the choice of the metal used to form the interconnected network, the metal is a detectable moiety in its own right and can be used as the detectable moiety for the purposes of diagnostic imaging. For example, any radioisotope of a metal that may be imaged may be incorporated into the articles of the present invention for the purposes of radio-imaging. Examples of metals that may be incorporated into the articles of the invention for use in radioimaging, for example, include Bismuth 213, Cobalt 57, Cobalt 60, Holmium 166, Lutetium 177, Rhenium 186, Technetium 99, Coper 64, Gallium 67, indium 1 11 , and Thallium 201 is a detectable moiety for PET Imaging. In addition certain metals such as Fe'", Mn" and Gd" can be used as MRI contrast agents merely by way of example.

[1 19] Alternatively a substrate may be used (and retained) such that the encapsulated substrate acts as the detectable moiety. A skilled addressee would readily be able to identify suitable substrates that have this type of activity so that the final article also has the ability. In other embodiments it is necessary to modify the article produced by the process of the present invention so that the article contains a detectable moiety. There are a number of ways that the articles produced by the process of the invention may be modified to achieve this result. One way is to take advantage of excess binding spots on the metal to attach a further ligand to the metals that are detectable in an imaging technique.

[120] Nevertheless, utilising the articles of the invention in imaging typically relies on at least one of the metals present in the article being able to be detected by an imaging technique such as fluorescence or radioimaging or by incorporation of a ligand on the metal that is fluorescent. A skilled worker in the art will readily appreciate the properties of various metals in order to enable them to be used in imaging applications of these types.

[121] For example when the desired method of imaging is fluorescent imaging this is typically carried out by addition of a ligand to the surface of the article in order to enhance the flourescent properties of the article. Alternatively the fluorescence of the articles may be inherent to the metallic cations incorporated such Europium and Terbium. In some embodiments in order to achieve adequate fluorescence from the metal there is a requirement for the addition of a sensitising co-ligand which complexes with the metallic cation (in addition to the polyphenolic ligand) and enhances the energy absorbance and fluorescence of the metallic cation. In many instance the ligand is not fluorescent itself. If a sensitising co-ligand is used it is typically incorporated into the articles during film assembly (in the case of Europium, i.e. it is added to the coating solution), or a complex between the sensitising ligand and the metallic cation (in the case of Terbium) is pre-formed by mixing in solution; this solution is then added to a solution of polyphenolic compound and the substrates. As the fluorescence of the articles is achieved during the coating process; a separate coating step is not required.

[122] There are a wide variety of metal ligands that could be used to enhance flouresence of this type. Examples of ligands that can be incorporated to increase fluorescent imaging include the following

Ms

[123] !n addition to the ligands described you could also use co-ligands of the type given be!ow (pyridine dicarboxyiic acid and derivative esters and amides, and 2- hydroxyisophthalic acid and derivative esters and amides and pyridine derivatives such as 2,2'-bipyridine (bipy) and substituted derivatives, 2,2';6'.2"-terpyridine (terpy) and substituted derivatives and 1 ,10-phenanthroline and substituted derivatives (phen)) as these have been widely disclosed in the literature for the formation of fluorescent metal complexes. Examples of these include:

Pyridine dicarboxylic acid or a derivative

2-hydroxy isophthallic acid or a derivative X=OH, OR (R is alkyl, NH₂, NHR (Ris alkyl)

X=OH, OR (R is alkyl, NH₂, NHR (Ris alkyl)

[124] As would be appreciated by a skilled addressee any form of imaging is typically only useful if the imaging provides information to the person carrying out the analysis. As such in order to obtain the maximum amount of information using the articles of the present invention it is preferred that they are attached to a biological entity prior to use. As will be appreciated in these cases diagnostic imaging will rely on the binding to the biological entity being involved in facilitating the localisation of the article containing the metal in the desired tissues or organs of the subject being treated/imaged.

[125] Thus for example in relation to the use of the articles of the invention in imaging it is anticipated that these may be used by first binding them to a biological entity of interest followed by administration of an effective amount of the article to a subject followed by monitoring of the subject after a suitable time period to determine if the article has localised at a particular location in the body or whether the article is broadly speaking evenly distributed through the body. As a general rule where the article is localised in tissue or an organ of the body this is indicative of the presence in that tissue or organ of something that is recognised by the particular molecular recognition moiety used. [126] Accordingly judicious selection of a biological entity to connect the article is important in determining the efficacy of any of the articles of the invention in diagnostic imaging applications. In this regard a wide range of biological entities that can act as molecular recognition moieties are known in the art which are well characterised and which are known to selectively target certain receptors in the body. In particular a number of biological entities that can act as molecular recognition moieties or molecular recognition portions are known that target tissue or organs when the patient is suffering from certain medical conditions. Examples of biological entities that can act as molecular recognition moieties or molecular recognition portions that are known and may be used in this invention include Octreotate, octreotide, [Tyr³]-octreotate, [Tyr¹]-octreotate, bombesin, bombesin(7-14), gastrin releasing peptide, single amino acids, penetratin, annexin V, TAT, cyclic RGD, glucose, glucosamine (and extended carbohydrates), folic acid, neurotensin, neuropeptide Y, cholecystokinin (CCK) analogues, vasoactive intestinal peptide (VIP), substance P, alpha- melanocyte-stimulating hormone (MSH). For example, certain cancers are known to over express somatostatin receptors and so the molecular recognition moiety may be one which targets these receptors. An example of a molecular recognition moieties or molecular recognition portions of this type is [Tyr³]-octreotate. Another example of a molecular recognition moieties or molecular recognition portions is cyclic RGD which is an integrin targeting cyclic peptide. In other examples a suitable molecular recognition moieties or molecular recognition portions is bombesin which is known to target breast and pancreatic cancers.

[127] The monitoring of the subject for the location of the article will typically provide the analyst with information regarding the location of the detectable moiety and hence the location of any material that is targeted by the molecular recognition moiety (such as cancerous tissue). An effective amount of the article of the invention will depend upon a number of factors and will of necessity involve a balance between the amount of detectable moiety required to achieve the desired imaging effect and the general interest in not exposing the subject (or their tissues or organs) to any unnecessary levels of radiation which may be harmful.

[128] The articles may also be used in the catalysis of chemical reactions. In essence any chemical reaction that can be catalysed by a metal can be catalysed by the articles of the present invention. Examples of metal catalysed reactions include small molecule synthesis reactions (such as the formation of ammonia), hydrogenation of alkenes, alkynes, aromatics or hetroaromatics; hydroformylation of alkenes and alkynes; ring opening polymerization of lactones to produce polyesters and oxidation of various substrates merely by way of example. [129] In using the articles of the present invention in catalysis either the metal cation (or one of the metal cations in case more than one is used) is chosen based on its ability to catalyse the desired chemical reaction. Once the metal has been identified it is incorporated into the article of the invention in order to produce an article that is suitable for use as a catalyst. Examples of metals that have been used in hydrogenation, for example, include, platinum, palladium, rhodium, ruthenium and iridium.

[130] Examples of metals that have also been shown to have catalytic activity include Vanadium, Cobalt, Nickel, Zirconium, Molybdenum, Ruthenium and Rhodium.

[131] Once the article has been produced containing the desired metal for the catalytic reaction the desired amount of article (typically in solution) is added to the reaction mixture to be catalysed. The articles are used in much the same way that any normal catalytic material is used. A skilled worker in the art will readily be able to determine the desired metal to be incorporated into the article of the invention in order to catalyse the desired reaction.

[132] Examples of materials and methods for use with the process of the present invention will now be provided. In providing these examples, it is to be understood that the specific nature of the following description is not to limit the generality of the above description.

EXAMPLES

[133] The present invention will now be described with reference to the following examples. Materials

[134] Tannic acid (TA, ACS reagent), iron(lll) chloride hexahydrate (FeCI₃-6H₂0), vanadium(lll) chloride (VCI₃), gadolinium(lll) chloride hexahydrate (GdCI₃-6H₂0), chromium(lll) chloride hexahydrate (CrCI₃-6H₂0), 3-(N-morpholino)propanesulfonic acid (MOPS), poly(sodium 4-styrenesulfonate) (PSS, M_w -70,000), polyethyleneimine (PEI, M_w -10,000), sodium phosphate buffer saline (PBS), sodium acetate, glycine, dimethyldiethoxysilane (DMDES), iron oxide nanoparticles (Fe₃0₄) (D = 5 nm), ammonium hydroxide, fluorescein isothiocyanate (FITC), FITC-dextran with various average molecular weights (4, 10, 70, 250, 500, 2,000 kDa), bovine serum albumin (BSA), lysozyme, rhodamine B, (-)-Epigallocatechin gallate (EGCG), and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma-Aldrich. Tetrahydrofuran (THF) and ethanol were purchased from Chem Supply. Polystyrene (PS) particles (D = 0.124 ± 0.005, 0.836 ± 0.024, 3.55 ± 0.07, 10.02 ± 0.08 μιτι), silica particles (D = 3.08 ± 0.13 μηη), aminated silica particles (D = 3.07 ± 0.12 μιη), and melamine formaldehyde (MF) particles (D = 2.98 ± 0.06 μιτι) were purchased from Microparticles GmbH. Escherichia coli (ATCC number 14948) and Staphylococcus epidermidis (ATCC number 14990) were obtained from American Type Culture Collection (ATCC). Alexa Fluor 633 carboxylic acid succinimidyl ester, Dulbecco's modified eagle medium (DMEM), and GlutaMAX were obtained from Life Technologies. All these materials were used as received. Ellipsoidal PS particles were prepared according to the reported stretching method from commercially obtained spherical PS particles (D = 3.6 μιη). Fe₃0 - loaded and non-loaded low-molecular-weight polydimethylsiloxane (PDMS) emulsions were prepared by the base-catalyzed hydrolysis and polymerization of DIVIDES. The emulsion templates were dialyzed against water before use (pH 6.5). Rhodamine B or protein (Alexa 633-labeled lysozyme) loaded CaC0₃ particles were synthesized via a precipitation reaction in the presence of PSS. PSS was removed by thermal annealing (550°C, 6 h) for mesopore formation. High-purity water with a resistivity of 18.2 ΜΩ.αη was obtained from an inline Millipore RiOs/Origin water purification system.

[135] All other materials not expressly mentioned were readily available from commercial suppliers.

Characterization

[136] Differential interference contrast (DIC) and fluorescence microscopy images were taken with an inverted Olympus 1X71 microscope. Confocal laser scanning microscopy (CLSM) images were acquired with a Leica TCS SP2 laser scanning microscope. Atomic force microscopy (AFM) experiments were carried out with a JPK NanoWizard II BioAFM. Typical scans were conducted in intermittent contact mode with MikroMasch silicon cantilevers (NSC/CSC). The film thickness and roughness of the Fe'"/TA capsules (spherical, D = 3.6 μιη) were analyzed using JPK SPM image processing software (version V.3.3.32). Transmission electron microscopy (TEM) images and energy dispersive X-ray spectroscopy (EDS) profiles were acquired using a FEI Tecnai TF20 instrument with an operation voltage of 200 kV. Scanning electron microscopy (SEM) images were obtained on a FEI Quanta 200 field emission scanning electron microscope operated at an accelerating voltage of 10 kV. In AFM, TEM/EDS and SEM experiments, the capsule suspensions (2 μΙ_) were allowed to air- dry on PEI-coated glass slides, formvar-carbon coated copper grids and Piranha cleaned silicon wafers, respectively. The SEM samples were then sputter coated with Au. Zeta- potential measurements were carried out in water by using a Zetasizer Nano ZS (Malvern). UV-Vis absorption measurements were carried out on a Nano drop ND-1000 UV-Vis spectrophotometer (Thermo Scientific) or a Varian Cary 4000 UV-Vis spectrophotometer. Flow cytometry assays were performed on a Cyflow Space (Partec GmbH) flow cytometer. [137] X-ray photoelectron spectroscopy (XPS) was performed on a VG ESCALAB220i-XL spectrometer equipped with a hemispherical analyzer. The incident radiation was monochromatic Al Ka X-rays (1486.6 eV) at 220 W (22 mA and 10 kV). Survey (wide) and high resolution (narrow) scans were taken at analyzer pass energies of 100 eV and 50 eV, respectively. Survey scans were carried out with a 1.0 eV step size and a 100 ms dwell time. Narrow high resolution scans were run over a 20 eV binding energy range with a 0.05 eV step size and a 250 ms dwell time. Base pressure in the analysis chamber was below 8.0 ^χ 10^"9 mbar. A low energy flood gun was used to compensate the surface charging effect. All data were processed using CasaXPS software and the energy calibration was referenced to the C 1s peak at 285.0 eV. For the sample preparation, concentrated capsule suspensions were cast onto Piranha cleaned silicon wafers and allowed to air dry.

[138] Spectroscopic ellipsometry of air-dried films was performed using an Auto SE spectroscopic ellipsometer (Horiba Jovin Yvon). Spectroscopic data were acquired between 400 and 800 nm with a 2 nm increment over at least five spots on the wafer. Ellipsometric thicknesses were modeled using the integrated software (DeltaPsi 2) using a two layer optical model consisting of Fe'"/TA on Au. Optical parameters for Au, as provided by Horiba from published reference data, were used to model the substrate. The overlying Fe'"/TA film was modeled using a two oscillator Tauc Lorentz dispersion model.

Example 1. General procedure for the formation of a metal complex coating on a Planar Substrate

[139] PS, glass, Au, PDMS or quartz substrates were soaked in water in a 50 ml_ tube. Solutions of the suitable metal cation (in this case FeCI₃-6H₂0) and a suitable polyphenolic compound (in this case Tannic acid) were added to this aqueous solution to yield the following final concentrations (FeCI₃-6H₂0: 0.1 mg ml_^"1, Tannic acid: 0.4 mg ml_^"1 in 20 ml_ of water). The solution was vigorously mixed by a vortex mixer for 10 s immediately after the individual additions of FeCI₃-6H₂0 and Tannic acid. The pH of this solution was subsequently raised by adding 1 N NaOH solution to ca. pH 8. Then, the substrates were rinsed with water.

[140] For preparation of samples for ellipsometric analysis, sequential build up of Fe'"/Tannic acid layers was performed on Au-coated silicon wafers prepared immediately prior to use. Silicon wafers (<100> orientation, n-type, MMRC Pty. Ltd.) were cut to approximately 1.5 ^χ 3 cm slides and soaked for 20 min in Piranha solution (98% H₂SO₄:30% H₂0₂ (7:3)). The slides were then sonicated in isopropanol:water (1 : 1) solution for 20 min and finally heated to 60°C for 20 min in RCA SC-1 solution (H₂O:30% NH₄OH:30% H₂0₂ (5:1 : 1)). The slides were washed thoroughly with water after each step. The cleaned wafers were dried under a nitrogen stream then sputter-coated (Emitech K575X) with a ca. 20 nm Cr adhesion layer (50 mA sputter current, 2 min) followed by ca. 50 nm Au (50 mA sputter current, 3 min). The experiment was conducted using varying sizes of support to demonstrate the generality of the method.

Example 2. General procedure for the formation of a metal complex film on a particulate substrate

[141] The standard condition is described as follows. Aliquots (5 μΙ_) of FeCI₃-6H₂0 (10 mg ml_^"1) and then TA (40 mg ml_^"1) solutions were added to the aqueous 3.6 μιτι-diameter PS template suspension (490 μΙ_) to yield the following final concentrations (PS: 10 mg mL^"1, FeCI₃-6H₂0: 0.1 mg mL^"1, TA: 0.4 mg mL^"1 in 0.5 mL of water). The suspension was vigorously mixed by a vortex mixer for 10 s immediately after the individual additions of FeCI₃-6H₂0 and TA. The pH of this suspension was subsequently raised by adding 0.5 mL of MOPS buffer (20 mM, pH 7.4). The particles were washed with water three times to remove excess TA and FeCI₃. In the washing step, the particles were spun down by centrifugation (2,000 g, 30 s) and the supernatant was removed. The remaining pellet was redispersed in the desired solvents.

[142] The result of the experiments carried out in examples 1 and 2 is shown in Fig. 1. The figure illustrates the deposition of Fe'"/TA films on planar (Fig. 1A) and particulate (Fig. 1 , B to K) polystyrene (PS) templates. In carrying out these experiments the color of the template suspension immediately turned blue upon addition of Fe'" and TA solutions. Stirring times (20 sec and 1 h) had no effect on the color or on the resulting film thickness under standard conditions, implying that the film formation process was completed instantaneously. Formation of the Fe'"/TA films on the PS particles shifted the surface zeta-potential from -27.4 ± 2.8 to -64.0 ± 6.9 mV due to the acidic nature of the galloyl groups in TA. After removing the PS template, we obtained highly uniform microcapsules with a zeta-potential of -64.6 ± 7.3 mV, which was approximately the same value as before template removal.

[143] Differential interference contrast (DIC) microscopy, atomic force microscopy (AFM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) images of the Fe'"/TA capsules are shown in Fig. 1 , B to E. Monodisperse, spherical capsules were readily observed under DIC (Fig. 1 B). The permeability of these capsules is molecular weight dependent, and they are essentially impermeable to 2,000 kDa dextran (Fig. 3). The presence of Fe in the films was confirmed by energy dispersive X-ray spectroscopy (EDS) and X-ray photoelectron spectroscopy (XPS) (Fig. 4). The capsules observed by AFM, SEM and TEM (Fig. 1 , C to E), had folds and creases because these measurements were performed on dried samples. Different-sized templates (D = 120 nm, 840 nm, 3.6 μηι, and 10 μιτι) can be exploited for capsule preparation (Fig. 1 , F to I). Ellipsoidal PS templates, prepared by stretching spherical PS particles above their glass transition temperature, were also used to obtain ellipsoidal capsules (Fig. 1 , J and K).

[144] From AFM height analysis, the single-wall thickness of the capsules was determined to be half of the minimum height of the collapsed flat regions (10.4 ± 0.6 nm). The Young's modulus (Ey) of the D = 3.6 μιη capsules was estimated to be 1 .0 ± 0.2 GPa by AFM force measurements (Fig. 5). This E_Y is at the high end of the range observed for layer-by-layer (LbL) polyelectrolyte capsules (10 - 1000 MPa). Several groups have reported LbL capsules fabricated from TA and other polymers. For example, LbL capsules of TA/poly(/V- vinylpyrrolidone) (PVPON) exhibit a bilayer thickness of 1.0 - 2.2 nm depending on the molecular weight of the PVPON. The Fe'"/TA film, obtained through the one-step assembly, is thicker than four bilayers of TA/PVPON obtained through multi-step LbL assembly, demonstrating the efficiency of the one-step process.

Example 3. Sequential assembly of film layers

[145] In order to show that the technique could be repeated to from multilayer films if required a number of sequential assembly trials were conducted. For sequential assembly experiments the standard conditions described in examples 1 and 2 were repeated, although the order of reagent addition was reversed (i.e., TA and then Fe'"). We adsorbed TA and then Fe'" to avoid rapid depletion of free Fe'" from solution into the preformed film, as the Fe'" concentration is below that required for saturation of the film binding sites. This is confirmed by XPS data which show that the film stoichiometry is dependent on the [Fe^m] used. Analogous to LbL assembly, the thickness of Fe'"/TA films can be further increased by simply repeating the rapid coating procedure (Figs. 6 and 7).

Example 4. Varying concentration of metal cation/and/or polyphenolic compound

[146] The concentration of FeCI₃-6H₂0 (0.06 - 0.20 mg mL^"1) and TA (0.10 - 1 .80 mg mL^"1) were altered from the standard conditions described above in examples 1 and 2 while the other variables were kept constant. The starting amounts of the different templates were calculated so that their surface areas were constant. To obtain capsules, the PS, low- molecular-weight PDMS emulsion and CaC0₃ templates were removed by washing with THF, ethanol or 0.1 M EDTA (pH 7.4) five times, respectively. For fluorescent labeling of the Fe'"/TA film, the suspension was incubated with FITC-BSA (1 mg mL^"1) for 15 min. The effect of TA and FeCI₃-6H₂0 concentrations (hereafter denoted as [TA] and [FeCI₃-6H₂0], respectively) on the resulting film thickness and morphology were investigated by AFM using D = 3.6 μηι PS templated capsules.

[147] When [TA] was kept constant at 0.40 mg ml_^"1 (0.24 mM), capsules were obtained over a [FeCI₃-6H₂0] range of 0.06 to 0.20 mg ml_^"1 (0.22 to 0.74 mM), approximately corresponding to molar ratios of 1 : 1 to 3: 1 between Fe'" and TA. The resulting stoichiometries in the capsule walls were determined by XPS (Fig. 4) to be Fe'":TA = ca. 1 :4, 1 :3, and 1 :2 for feed [FeCI₃-6H₂0] of 0.06, 0.12, and 0.20 mg ml_^"1, respectively. This demonstrates that the feed concentrations influence the capsule stoichiometries. Above and below [FeCI₃-6H₂0] = 0.06 - 0.20 mg ml_^"1, aggregated capsules and few capsules were formed, respectively. In the absence of Fe'", no capsules were formed. As [FeCI₃-6H₂0] was increased in this concentration range, the Fe'"/TA film became thicker, from 7.7 ± 0.4 to 1 1.9 ± 1.2 nm, and exhibited increased roughness (Fig. 8). Increasing the amount of Fe'" to three molar equivalents of TA resulted in capsules that had a grainy surface because of the excess Fe'" (Fig. 8A). Values of RMS roughness (300 nm by 300 nm, fold-free flat region) were 1.3 ± 0.1 , 1.6 ± 0.1 and 7.7 ± 0.4 nm for [FeCI₃-6H₂0] of 0.06, 0.10 and 0.20 mg ml_^"1, respectively. In contrast to these observations based on Fe'", [TA] had minimal impact upon film assembly. Capsules with constant film thickness and roughness were obtained throughout a concentration range of [TA] = 0.10 to 1.80 mg ml_^"1 (0.06 to 1.06 mM), when [FeCI₃-6H₂0] was fixed at 0.10 mg ml_^"1 (0.37 mM) (Fig. 9). These results suggest that TA was relatively in higher excess than Fe'" under these conditions, and that Fe'", and not TA, influenced the film thickness.

Example 5. Variation of Surface of template

[148] To further investigate the mechanism of the Fe'"/TA film formation, polyethyleneimine (PEI)-coated PS templates were used for capsule preparation. The PEI coating changed the zeta-potential of the templates from negative (-27.4 ± 2.8 mV) to positive (36.9 ± 5.6 mV). Capsules were still formed with these positively charged templates, indicating that the surface charge is not an important factor for film deposition. We also determined the zeta-potential after incubating the bare PS particles with either TA or FeCI₃. The adsorption of TA reduced the zeta-potential value to -39.4 ± 3.6 mV, while the value was slightly changed after incubation with FeCI₃ (-33.1 ± 4.7 mV). Furthermore, rapid surface-adherence and formation of a TA layer on the template particle surface were confirmed by adsorption experiments (Fig. 10). Catechol-functionalized molecules and their derivatives have a high affinity for a wide variety of substrates with different surface charges. Thus, it is most likely that the free TA or small Fe'"/TA complexes initially adsorb onto the template surface and are subsequently cross-linked by further Fe'" complexation. The increase in the cross-linker concentration causes the attraction of more TA to the initially formed films, making them thicker. Film growth is completed when free Fe¹" in the bulk solution is consumed. Excess Fe¹" induces aggregation of Fe'"/TA complexes in the bulk solution. These small aggregates subsequently bind to the surface, leading to an increased roughness of the capsule films (Fig. 11). Unlike thin film formation using dopamine self-polymerization, this study relies on the complexation of TA with Fe'" through coordination bonds, which allows the films to form rapidly and disassemble in response to pH.

Example 6. Variation of template materials

[149] To show the versatility of this method, various planar and particulate substrates including glass, gold (Au), polydimethylsiloxane (PDMS), poly(lactic-co-glycolic acid) (PLGA), melamine-formaldehyde resin (MF), low-molecular-weight PDMS emulsion, silica (Si0₂), aminated Si0₂, cetyltrimethylammonium bromide-capped Au nanoparticles (Au NPs), calcium carbonate (CaC0₃), Escherichia coli (E. coli), and Staphylococcus epidermidis (S. epidermidis) with various surface properties (anionic, neutral and cationic) were coated with the Fe'"/TA films using the procedure described in example 1 or 2 and the results shown in Fig. 12. The color and zeta-potential values (Fig. 12, A to D) changed after the coating in all cases, demonstrating that Fe'"/TA films can be formed on a wide variety of substrates. Fig.1 2E and Fig. 13 show protein and rhodamine B loaded CaC0₃ coated with Fe'"/TA films, respectively. The replica particles were obtained by filling mesoporous CaC0₃ particles with Fe'"/TA complexes and dissolving the CaC0₃ cores (Fig. 13, B to E). After the coating, a Fe'"/TA shell layer was visible around the Au NP core (Fig. 12, F and G, Fig. 13F). Magnetic Fe₃0₄ nanoparticles were encapsulated by coating low-molecular-weight PDMS emulsion templates loaded with Fe₃0₄ nanoparticles (Fig. 13). Subsequent removal of the emulsion by ethanol produced magnetically active Fe'"/TA capsules (Fig. 12, H and I).

Example 7. Disassembly Experiments

[150] The 2 ml_ capsule suspensions prepared in example 2 (prepared from 3.6 μηι- diameter PS templates, 1.0 * 10⁷ capsules ml_^"1) in 20 mM MOPS (pH 7.4), 0.1 M EDTA (pH 7.4), 50 mM sodium acetate (pH 4.0, pH 5.0) or 50 mM Gly-HCI (pH 3.0) were incubated in a thermostated shaker bath at 37°C for the desired time. The suspensions were diluted with water and subjected to flow cytometry assays to count the number of capsules.

[151] The results show that the coordination between Fe'" and TA is pH-dependent, and the obtained capsules exhibit pH-dependent disassembly. The color of the capsule suspension was different at varying pH (Fig. 14A). The suspension was colorless at pH < 2, blue at 3 < pH < 6, and red at pH > 7. This color change is consistent with observations for analogous Fe'"/catechol complexes, which can be attributed to the transition between the mono-, bis- and tris-complex states (Fig. 14B). At pH 2.0, the Fe'"/TA capsules shrunk immediately (Fig.

15) and were disassembled afterward. At low pH, most of the hydroxyl groups are protonated, which leads to rapid de-cross-linking and disassembly of the films. Even above pH 3.0, Fe'"/TA capsules disassembled. Fig. 14C shows the disassembly kinetics of the Fe'"/TA capsules. At pH 3.0, all of the capsules had disassembled in 4 h, while at pH 4.0, 6 days incubation was required to disassemble the majority of the capsules. In contrast, ca. 70% and 90% of the capsules still remained intact after 10 days incubation at pH 5.0 and pH 7.4, respectively. The stability constants of Fe'"/TA are 1.5 ^χ 10⁵, 3.4 ^χ 10⁹, 2.8 ^χ 10¹⁷ at pH 2, 5, 8, respectively. Additionally, we carried out AFM experiments after incubation at pH 5.0 (Fig.

16) . The films became thinner (6.0 ± 1.4 nm) and rougher, confirming the gradual disassembly of the Fe'"/TA films. Ethylenediaminetetraacetic acid (EDTA) accelerated the disassembly because of its strong affinity for Fe'" (Fig. 14C).

Example 8. Permeability Tests

[152] The dispersion of Fe'"/TA capsules (prepared from PS templates, D = 3.6 μηι, 4.0 ^χ 10⁷ capsules mL^"1) prepared as in example 2 was mixed with an equal volume of FITC- dextran solution (1 mg mL^"1). CLSM images of the capsules were taken within 10 min after mixing and the capsules with dark interiors were considered to be impermeable, whereas capsules with interiors of similar fluorescent intensity as the outer environment were considered to be permeable. 100 capsules were examined. The results are shown in Fig. 3. The permeability of these capsules is molecular weight dependent, and they are essentially impermeable to 2,000 kDa dextran.

Example 9. Mechanical Testing of capsules

[153] AFM force spectroscopy measurements were performed in water with a JPK NanoWizard II BioAFM using colloidal probe modified cantilever tips (spring constant 36.4 mN m^"1, probe diameter 32.4 μιη). Glass slides and cantilevers were cleaned with 30 vol% isopropanol, water, and plasma treatment to remove any contaminant material. PEI was adsorbed to cleaned glass substrates prior to measurement for electrostatic immobilization of the capsules. For fabrication of the modified cantilevers, tipless cantilevers (MikroMasch) were first calibrated on a cleaned glass substrate to determine the inverse optical lever sensitivity (InvOLS), and then the spring constant was determined using a modified thermal noise method. A spherical glass bead (Polysciences) was attached to the calibrated cantilever using an epoxy resin (Selleys Araldite Super Strength, Selleys) via careful micromanipulation using the AFM and associated optics, and allowed to dry overnight. To obtain force-distance curves on Fe'"/TA capsules prepared from 3.6

PS templates, the probe was optically positioned above individual capsules, and an approach- retract cycle initiated with a constant piezo velocity of 750 nm s^"1. The temperature was regulated to 19 - 21 °C during force measurements, and monitored using a JPK BioCell. Collected force spectra were analyzed using JPK data processing software. A baseline was first subtracted from the non-contact z-range of the force-displacement data, a probe/surface contact point assigned, and the effect of cantilever bending subtracted to result in force- deformation (F-S) data. The E_Y of the spherical capsule could be estimated using the Reissner model for thin-walled spherical shells. A wall thickness (h) of 10.4 nm, a Poisson ratio (v) of 0.5, and an effective radius (R_eff) of 1 .60 μιη were used. Using the Reissner equation:

the Ey was estimated to be 1 .0 ± 0.2 GPa. Only deformation data over the capsule shell thickness was used (Fig. 5A). This result was also confirmed with measurements using a stiffer cantilever (spring constant 72.6 mN m^"1 , probe diameter 31 .9 μηι), and at least 10 single capsules were analyzed.

Example 10. Determination of Tannic acid Adsorption Isotherms

[154] Five mg of PS particles (D = 0.836 μηι) and TA (0.0005, 0.005, 0.01 , 0.02, 0.04, 0.05, 0.075, 0.125, 0.175, 0.2 mg) were mixed in 0.5 ml_ of water. After 10 s of vortex mixing to adsorb TA, the particle suspensions were centrifuged (10,000 g, 5 min). 50 μΙ_ of supernatant was transferred to new tubes. The absorbance at 275 nm of these solutions (2 μΙ_) was measured with a Nano drop ND-1000 UV-Vis spectrophotometer. Adsorbed amounts of TA (mg/g of PS) at each concentration were calculated from a separately constructed calibration curve.

Example 11. Cell Viability Assays

[155] In order to determine the toxicity of the particles early passage HeLa cells were seeded in a 96- well plate at a density of 1 ^χ 10⁴ cells well^"1 and cultured in DM EM supplemented with 10% FBS and 2 mM GlutaMAX at 37°C in 5% C0₂. The cells were exposed to varying amounts of Fe'"/TA capsules in a total volume of 200 μΙ_ for 72 h. After aspirating the supernatant of each well and washing three times with PBS, 180 μΙ_ of the medium and 20 μΙ_ of MTT solution (5 mg mL^"1 in PBS) were added to the wells. Plates were further incubated at 37°C in 5% C0₂ for 3 h. After the addition of 150 μΙ_ of solubilization mixture (0.04 N HCI in isopropanol), the absorbance at 560 nm (blue formazan) was measured with a plate reader (Multiskan Ascent, Thermo Scientific). The absorbance of control wells without MTT was subtracted. All experiments were performed in quadruplicate and the relative cell viability was normalized relative to the untreated control cells. The cytotoxicity of Fe'"/TA capsules was shown to be negligible (Fig. 17). Coupled with their pH- sensitive disassembly profile, Fe'"/TA capsules have potential biomedical application, because of the varying pH in different parts of the body, e.g. blood (pH 7.4), stomach (pH 1.0 - 3.0), duodenum (pH 4.8 - 8.2), etc.

Example 12. Procedure for making films using other metals

[156] Aliquots of TA and then metal salt solutions were added to the aqueous 3.6 μηι- diameter PS template suspension to yield the following final concentrations (PS: 10 mg mL^"1, TA: 0.4 mg mL^"1, metal to TA molar ratio: 1 : 1 , in 0.5 mL of water). The suspension was vigorously mixed by a vortex mixer for 10 s immediately after the individual additions of metals and TA. The pH of this suspension was subsequently raised by adding 0.1 mL of MOPS buffer (100 mM, pH 8.0). The particles were washed with water three times to remove excess TA and metals. In the washing step, the particles were spun down by centrifugation (2,000 g, 30 s) and the supernatant was removed. The remaining pellet was redispersed in the desired solvents. The results are shown in Fig. 18 and 19.

Example 13. Formation of film using (-)-Epigallocatechin gallate as the polyphenolic compound

[157] Fe'"/EGCG coatings were deposited in a slightly modified way as described above, with TA being replaced by EGCG. Aliquots of FeCI₃-6H₂0 (5 μΙ_, 10 mg mL^"1) and EGCG (20 μί, 10 mg mL^"1) solutions were added to the aqueous 3.6 μιτι-diameter PS template suspension (475 μί) to yield the following final concentrations (PS: 10 mg mL^"1, FeCI₃-6H₂0: 0.1 mg mL^"1, EGCG: 0.4 mg mL^"1 in 0.5 mL of water). The pH of this suspension was subsequently raised by adding 0.5 mL of MOPS buffer (20 mM, pH 7.4). The coated particles were washed three times with water and subjected to zeta-potential measurements. For planar substrates (quartz slide), solutions of FeCI₃-6H₂0 and EGCG were added to the aqueous solution containing the substrate to yield the following final concentrations (FeCI₃-6H₂0: 0.1 mg mL^"1, EGCG: 0.4 mg mL^"1 in 20 mL of water). The solution was vigorously mixed by a vortex mixer for 10 s immediately after the individual additions of FeCI₃-6H₂0 and EGCG. The pH of this solution was subsequently raised by adding 1 N NaOH solution to ca. pH 8. The substrates were then rinsed with water and dried under a nitrogen stream. The results are shown in Fig. 20. Example 14. Preparation of Monometallic MPN Capsules by Coating Particulate Substrates

[158] The standard preparation process is described as follows. Particulate templates (PS templates, D = 3.6 μηι) are first immersed in water (490 μΙ). After sequential addition of 5 μΙ of TA solution (24 mM) and metal precursor solutions (24 mM of MnS0₄, ZnS0 , CuCI₂, Co(N0₃)₂, NiCI₂, CdCI₂, Mo₂(OCOCH₃)₄, AICI₃, VCI₃, FeCI₃, CrCI₃, RhCI₃, RuCI₃, ZrCI₄, CeCI₃, EuCI₃, GdCI₃ or TbCI₃ solutions) was added and the dispersion was vortexed, the pH of this suspension was subsequently raised by adding 500 μΙ MOPS buffer (100 mM, pH 8.0). The particles were washed with water three times to remove excess TA and metal ions. In the washing step, the particles were spun down by centrifugation (3,000 g, 30 s) and the supernatant was removed and the pellets were vortexed for 10 s. To obtain capsules, the PS or CaC0₃ templates were removed by washing with THF four times. In each THF washing step, the pellets were first vortexed for 10 s and then 700μΙ of THF was added to the pellet and the pellet was resuspended through gentle pipetting (at least 20 times). This suspension was then kept on the rotator for 20 min. Finally the particles were centrifuged (3,000g, 30 s), the supernatant was removed, and the process was repeated 4 times. After the final THF washing step, 200μΙ of high purity water was used to resuspend the pellet through gentle pipetting. Samples for characterisation by SEM, TEM and AFM, removal of the PS templates was achieved by immersion in THF overnight with constant agitation (in roatator) during the third washing step followed with the fourth THF washing step the next day. Finally, the remaining pellet was washed by water again three times and redispersed in the desired solvent. The concentrations of metal solutions were altered (in the range of 0.06 to 0.72 mM [Cu", Af and Zr^lv] for Figure 22 and 0.24 to 1.5 mM [Eu^m] for Figure 23) from the standard conditions described above while the other variables were kept constant.

[159] The coordination between TA and metals was confirmed by UV-Vis absorption spectrophotometry, Fourier transform infrared spectrophotometry (FT-I R) and X-ray photoelectron spectroscopy (XPS). UV-Vis spectra of MPN capsule suspensions show new absorbance peaks after metal chelation, which suggests coordination between TA and the respective metals (Fig. 24). FT-I R spectra of the MPN capsules (air-dried) also indicate that the phenolic groups coordinated with metal ions, as evidenced by the reduced intensity of the HO-C stretching peak when compared with non-coordinated TA (Fig. 25). Furthermore, the XPS survey scan spectra confirmed the presence of each metal in the individual capsule shells (Fig. 26). TA showed a major peak at 533.5 eV and a relatively small peak at 529.4 eV, which can be ascribed to the C=0 and HO-C groups, respectively³⁵. After chelation with metal ions, the peak of the HO-C group in the Oi_s spectra shifted from 529.4 eV to a higher binding energy of 531.8 eV (Fig. 26), which suggests electron transfer from TA to the metals³⁶. The MPN films are amorphous materials, as confirmed by X-ray diffraction data (Fig. 27).

[160] The morphology of the capsules was characterized using differential interference contrast (DIC) microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM) (Fig. 28, Fig. 29 and 30). All MPN capsules were monodisperse and spherical, as observed by DIC microscopy and freeze-dried SEM (Fig. 28a, inset). SEM, TEM and AFM showed collapsed capsules, with folds and creases similar to air-dried polymeric hollow capsules. No excess coordination cluster aggregates were observed on the surface of the MPN capsules in TEM (Fig. 28b) or AFM (Fig. 28c). Energy dispersive X-ray (EDX) mapping analysis of MPN capsules demonstrated that the metal distribution patterns matched well with high-angle annular dark-field images (HAADF) and the distribution patterns of C and O maps (Fig. 28d). This confirms that the metals were associated with TA and well distributed in the MPN films. AFM was then used to probe the material differences between MPN capsules prepared from metals of different oxidation numbers: Cu"-TA, Al'"-TA and Zr^lv-TA (Fig. 22). The thickness and stability of the MPN films could be tuned by the choice of metal and the metal feed concentration (hereafter denoted [M]). Notably, comparison of film thicknesses among these three MPN capsules showed that Zr^lv-TA capsules were the thickest, and Cu"-TA capsules the thinnest. The film thickness of Cu"-TA capsules increased from 9.0 ± 0.8 nm to 10.7 ± 1.5 nm when the feed [Cu^M] changed from 0.06 (Cu":TA=3: 1) to 0.72 mM (Cu":TA=1 :3). Similarly, the minimum thickness of Zr^lv-TA capsules was 11.9 ± 1.2 nm (Zr^lv:TA=3: 1), and raising [Zr^lv] increased the film thickness to 15.4 ± 2.2 nm (Zr^lv:TA=1 :3). The film thicknesses of Al'"-TA films exhibited a similar trend to Cu"-TA and Zr^lv-TA. We note that below a [M] of 0.06 mM, capsules for all three metals were difficult to form, while above 0.72 mM the capsules aggregated.

Example 15. Preparation of Monometallic MPN Films on PDMS (polydimethylsiloxane) Substrates

[161] PDMS substrates were soaked in water in a 50 ml tube. Solutions of TA, metal precursors (EuCI₃ solutions) and 2-thenoyltrifuoroacetone (TTA solution) were added to achieve the final concentrations of 0.24 mM for TA, Eu and TTA. The solutions were vigorously mixed by a vortex mixer for 5 min after the individual additions. The pH of this solution was subsequently raised by adding 1 N NaOH solution to ca. pH 8.0. Finally, the substrates were rinsed with water. This coating process was repeated ten times to enhance the fluorescence.

Example 16. Preparation of Bimetallic MPN Capsules by Coating Particulate

Substrates

[162] For the preparation of (⁶ Cu"/Eu'"-TTA)-TA capsules, after the addition of TA solution 5 _μΙ (24 mM), 5 μΙ of Cu(C0₂CH₃)₂ (24 mM), 5 MBq ⁶ Cu, 30 μΙ of EuCI₃ (24 mM), and 60 μΙ of TTA (24 mM) were added to the solution sequentially. The suspension was immediately mixed by a vortex mixer for 10 s. The pH of this suspension was subsequently raised by adding 100 μΙ MOPS buffer (100 mM, pH 8.0). The particles were washed first with 500 μΙ of water three times to remove the excess TA and ⁶ Cu", and afterwards three times with 500 μΙ THF to remove the PS cores. In the washing step, the particles were spun down by centrifugation (3,000 g, 30 s) and the supernatant was removed. The radioactivity of the suspension was counted using a gamma counter (Wizard single-detector gamma-counter, Perkin Elmer).

[163] Before the preparation of (Eu^l"-TTA/Tb'"-AA)-TA capsules, the preparation of the capsules the Tb'"-AA complex was synthesized in a 1.5 ml_ Eppendorf tube. 50 μΙ_ of Tb"'CI₃ (240 mM, ethanol) was added first, then 100 μΙ_ of AA (240 mM, ethanol) was added, and finally 30 μΙ_ of 1 M NaOH was added. The mixture was vortexed immediately and heated at 45 °C under sonication for at least 2 h until it became transparent for maximum fluorescence intensity. In this experiment, PS particles were added to 330 μΙ_ of water instead of 440 μΙ_. The addition order was 5 μΙ_ of TA solution (24 mM), 15 μΙ_ of Eu'"CI₃ (24 mM), 30 μΙ_ of TTA (24 mM, ethanol), and 60 μΙ_ of Tb'"-AA complex (ethanol). The particles were only washed once with 500 μΙ_ water to remove the excess TA and metals. The supernatant was removed after this washing step and the pellet was vortexed, and 300 μΙ_ of THF was added to the pellet to remove the core and suspend the capsules for imaging. Note that the fluorescence reduced over time after the addition of THF. Finally, the concentrations of Eu'" and Tb'" were altered to tune the fluorescence of the capsules. Example 17. pH-Degradation Experiments

[164] Capsule disassembly experiments were carried out in PBS buffer solutions at different pH values (7.4, 6.0 and 5.0). The capsule/PBS solutions were constantly incubated on a thermostated shaker (Eppendorf Thermomixer Comfort) at 37°C at 800 rpm. At different time points, 10 μΙ of the capsule dispersion (~10⁶ capsules) was collected and diluted with 990 ml of deionized water for flow cytometric analysis. Samples were measured on a Partec CyFlow Space (Partec GmbH, Germany) flow cytometer to count the number of capsules. Data were analysed to give the mean ± SD from three independent measurements.

[165] The pH-disassembly kinetics of the MPN capsules prepared from each of the three model metals (Cu", Al'", Zr^lv) were examined. (A moderate [M] of 0.24 mM (M:TA=1 : 1) was used to prepare these capsules.) The stability of the MPN capsules decreased as the pH decreased from 7.4 to 5.0 (Fig. 31 a). The stability of the capsules is directly related to film thickness. At pH 5.0, over 25% of the relatively thin Cu"-TA capsules (9.7 ± 1.0 nm) disassembled within 1 h, whereas it took roughly 6 h for 25% of the Al'"-TA capsules (10.8 ± 1.3 nm) to disassemble, and even after 168 h, less than 25% of the relatively thick Zr^lv-TA capsules (14.2 ± 1.5 nm) had disassembled. The intermediate disassembly kinetics for the Al'"-TA capsules correspond to a desirable profile for drug delivery, as the capsules are relatively stable at the pH of the bloodstream (7.4) and gradually disassemble at lower pH values that correspond to those in endosomal and lysosomal compartments (5.0-6.0). .

Example 18. Capsule Cytotoxicity Assay

[166] HeLa cells were used to evaluate the cytotoxicity of Al'"-TA capsules. The cells were cultured in Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 units ml^"1 penicillin and 100 μg ml^"1 streptomycin at 37°C in an incubator with 5% C0₂ atmosphere and 95% relative humidity. When the cell confluency reached -70%, 100 μΙ of cells were seeded into 96-well plates (Costar 3596, Corning, USA) at a density of 1 ^χ 10⁴ cells ml^"1. After 24 h, culture media were replaced with fresh media containing a different number of capsules. After another 24 h of incubation, 10 μΙ of 5 mg ml^"1 filtered MTT stock solution was added to each well, and unreacted dye was removed by aspiration after 4 h. The formazan crystals were dissolved in 100 μΙ well^"1 DMSO and measured on an Infinite M200 microplate reader (Tecan, Switzerland) at a wavelength of 570 nm. The cell viability was expressed as a relative percentage of the untreated cells.

[167] The cytotoxicity of the Al'"-TA capsules was determined to be negligible (Fig. 32), hence the drug delivery properties of the Al'"-TA capsules were investigated with model cargo (dextran-fluorescein isothiocyanate, FITC-dextran). These capsules were incubated with JAWS II cells for different times. The cellular membranes were stained with AlexaFluor (AF) 594-wheat germ agglutinin, and internalization of these capsules was verified by deconvolution fluorescence microscopy (Fig. 31 b). At the time interval of 16 h, most internalized Al'"-TA capsules were intact, retaining their original spherical shape (see yellow arrows in Fig. 31b). With increasing incubation time (to 24 h), a larger number of deformed and disassembled Al'"-TA capsules were observed (see white arrows in Fig. 31 b). The intracellular disassembly of Al'"-TA capsules provides the potential for tailored drug release, which is of importance for advanced drug delivery⁴⁰.

Example 19. Intracellular Drug Release Experiments

[168] JAWS II cells were seeded at 3 ^χ 10⁴ cells well^"1 into 8-well Lab-Tek I chambered coverglass slides (Thermo Fisher Scientific, Rochester) and allowed to grow for 24 h. Afterwards, cells were incubated with capsules (at capsule-to-cell ratio of 100: 1) for 16 h and 24 h followed by washing with PBS three times. Cells were first fixed with 4% paraformaldehyde for 15 min at 37°C and washed with PBS twice. Afterwards, the cells were stained with wheat germ agglutinin (5 μg ml^"1) at room temperature for 15 min, followed by Hoechst 33342 (2 μg ml^"1) at room temperature for 20 min.

Example 20. Fluorescence Experiments

[169] Particulate templates (PS, D = 3.6 μηι) were firstly immersed in water (485 μΙ). For the preparation of Eulll-TTA-TA capsules, 5 μΙ of TA solution (24 mM), EuCI3 solution (24 mM) and TTA solution (24 mM) were added to the template suspension. The pH of this suspension was subsequently raised by adding 100 μΙ MOPS buffer (100 mM, pH 8.0). For the preparation of Tblll-AA-TA capsules, 5 μΙ of TA (24 mM) then 60 μΙ Tb'"-AA solution (preparation described in Example 16, [184]) were added to the template suspension. The pH of this suspension was subsequently raised by adding 100 μΙ MOPS buffer (100 mM, pH 8.0). The particles were washed with water three times to remove excess TA and metal ions. In the washing step, the particles were spun down by centrifugation (3,000 g, 30 s) and the supernatant was removed. To obtain capsules, the PS templates were removed by washing with THF five times. Finally, the remaining pellet was washed by water again three times and redispersed in the desired solvents. The feed concentration of Eulll was altered (in the range of 0.24 to 1.5 mM) from the conditions described above while the other variables were kept constant. For the fluorescence spectral measurements, 120 μΙ of the obtained suspension was added to the micro quartz cells (1-mm path width) equipped with thermostatic cell holders and the measurements were performed at 20°C. [170] Accordingly to impart imaging properties to the MPN capsules, we employed 2- thenoyltrifluoroacetone (TTA) and acetylacetone (AA) as coligands to enhance the fluorescence intensities of Eu'"-TA and Tb'"-TA capsules, respectively, and to demonstrate that guest functional ligands can be easily incorporated into MPN films. Fig. 33a shows the fluorescence spectra of these capsules. The red fluorescence observed in the Eu'"-TTA-TA capsules is mainly attributed to the ⁵D₀ → ⁷F₂ transition around 613 nm⁴¹ and the green fluorescence of the Tb'"-AA-TA capsules is ascribed to the ⁵D₄→ ⁷F₅ transition around 545 nm⁴². The fluorescence intensity of the Eu'"-TTA-TA capsules is dependent on the [Eu^IM], with an increase in fluorescence intensity dependent on the [Eu^m] (Fig. 23). The insets of Fig.33a show photographs of the Tb'"-AA-TA and Eu'"-TTA-TA capsule suspensions (upper) and polydimethylsiloxane (PDMS) coated with an Eu'"-TTA-TA film excited at 365 nm (lower).

Example 21. Positron Emission Tomography (PET) of Phantom

[171] The 64Cull-TA capsules were placed on the mouse bed and a 10 min PET scan using NanoPET/CT In Vivo Preclinical Imager (Bioscan, Washington DC, USA) with a PET acquisition time 10 min and coincidence relation, 1-3 was performed. Image reconstruction was performed with the following parameters: OSEM with SSRB 2D LOR, energy window, 400-600 keV; filter Ramlak cut off 1 , number of iteration/subsets, 8/6. In order to add anatomical information to the molecular PET, computer tomography (CT) scans were acquired directly after the PET scan. For the scans, an X-ray voltage of 45 kVp, an exposure time of 900 ms and a pitch of 0.5 were used. A total projection of 240 projects over 360° of rotation was acquired. Projection data were rebinned by 1 :4 and reconstructed, using a RamLak filter, into a matrix having an isotropic voxel size of 96 μηι. Image files of PET and CT scan were fused and analysed using the analysis software InVivoScope version 2.00. The radioactivity concentrations in the PET images of the phantom were recalculated and the results are expressed as Bq ml-1.

Example 22. PET/CT Image and Post-Mortem Biodistribution

[172] Animals were anesthetized with ketamine (50 mg kg-1 ; Parnell Laboratories, New South Wales, Australia) and xylazine (10 mg kg-1 ; Troy Laboratories, New South Wales, Australia) and placed on a 37°C heater mat to prevent hypothermia. The animals were injected with 64Cull-TA capsules, corresponding to an activity of 1 MBq, via a lateral tail vein and a 30 min PET scan using NanoPET/CT In Vivo Preclinical Imager (Bioscan, Washington DC, USA) with a PET acquisition time 30 min and coincidence relation, 1-3 was performed. Image reconstruction was performed with the following parameters: OSEM with SSRB 2D LOR, energy window, 400-600 keV; filter Ramlak cut off 1 , number of iteration/subsets, 8/6. During the imaging, the animals were placed in a Minerve imaging chamber and anesthetized with a mixture of 2.5% Isoflurane in oxygen (1 I min-1). Anaesthesia was monitored by measuring respiratory frequency and body temperature was kept at 37°C with a heating pad underneath the animal. In order to add anatomical information to the molecular PET, computer tomography (CT) scans were acquired directly after the PET scan. For the scans, an X-ray voltage of 45 kVp, an exposure time of 900 ms and a pitch of 0.5 were used. A total projection of 240 projects over 360° of rotation was acquired. Projection data were rebinned by 1 :4 and reconstructed using a RamLak filter, into a matrix having an isotropic voxel size of 96 μηι. Image files of PET and CT scan were fused and analysed using the analysis software InVivoScope version 2.00. After the CT scan the animals were perfused with PBS, the organs were removed and measured with an aliquot of injected solution as standard in the gamma counter (Perkin Elmer) using an energy window between 450 and 650 keV. Results are expressed as % injected dose per gram of tissue (% ID g-1).

Example 23. Magnetic Resonance Imaging (MRI) Experiments

[173] For the MRI relaxivity experiments, 200 μΙ with different concentrations of one compound (either Fe'"-TA, Mn"-TA or Gd'"-TA) were mixed with 500 μΙ of a 2% agarose gel, resulting in a dilution series with different metal concentrations ranging from 0.02 mM to 0.3 mM for each compound. The corresponding relaxation times T_x , T₂ and T^* were measured with an inversion recovery sequence with different inversion times Τ_τ , a spin echo and a gradient echo sequence with varying echo times T_E , respectively. All MRI experiments were performed on a 9.4 T high field animal MRI system (Bruker Biospin 94/20, Bruker Biospin, Ettlingen, Germany). The subsequent data evaluation was performed using the software MATLAB. The measured MR signal S(T_E) from the spin echo sequence was fitted using the formula:

S(T_E) = S₀ e

(1 ) where S₀ and T₂ are free fit parameters with S₀ being the maximum signal amplitude (for a theoretical echo time Γ_Ε = 0 ) and T₂ being the transverse relaxation time. The same formula but with T^* instead of T₂ was used for the gradient echo sequence. The measured MR signal S(7J) from the inversion recovery sequence was fitted using the formula:

(2) where S₀ , q and T_x are free fit parameters, with S₀ being the maximum signal amplitude (for a theoretical inversion time T_l→∞) and T_x being the longitudinal relaxation time. For a perfect inversion pulse the factor q is well defined by q = 2. However, for non-perfect inversion pulses the parameter q has smaller values ( q < 2 ) and should therefore be a free fit parameter. All relaxation times 7 (either T_x , T₂ or T^*) were determined for each metal concentration c of a given compound and the corresponding relaxation rates i¾ = 1/7^ were calculated and fitted according to:

(3) ^ = ^o + ^ ^c where R_i0 and r_{ are free fit parameters, with R_i0 being the relaxation rate of the agarose gel without contrast agent (thus for c = 0 ) and r_{ (either r_x or r₂ or r^*) being the desired relaxivity of the compound.

[174] Accordingly we extended the investigation of MPN capsules for biomedical imaging by incorporating metals useful for positron emission tomography (PET) and magnetic resonance imaging (MRI). Radioactive ⁶ Cu"-TA capsules were prepared by adding 5 MBq of ⁶ Cu during MPN film assembly. Fig. 33b shows the corresponding PET phantom image, which suggests that the ⁶ Cu"-TA capsules are efficient PET-active vehicles, and useful for tracking the biodistribution of both loaded drugs and the carrier itself. Therefore, ⁶ Cu"-TA capsules were injected into healthy BALB/c mice. PET/Computed Tomography (CT) scans were acquired and the biodistribution of the capsules was evaluated after 30 min. The PET/CT images showed significant uptake of the capsules in the liver, spleen and kidney (Fig. 33c), which was further confirmed by the post-mortem biodistribution data (see Supplementary Fig. 34). This gamma-counter data set revealed general tissue distribution and demonstrated that the ⁶ Cu"-TA capsules mainly accumulated in the liver and spleen, as a consequence of the reticuloendothelial system that processes microparticles¹⁹. It is plausible that the biodistribution of ⁶ Cu"-TA capsules can be tailored by controlling capsule properties (e. g., size, shape and surface chemistry) given the versatility of MPN materials.²⁹ We then explored the possibility of generating MRI contrast agents by the coordination of Fe'", Mn" and Gd'" into MPN capsules. We performed MR relaxometry experiments with these capsules and determined their relaxivities on a 9.4 T animal MRI system (Fig. 33d-e and Fig. 35). Among all three samples the Mn"-TA capsules displayed the highest relaxivity r₂ , which is of the order of 60 s^"1 mM^"1 and generally sufficient for in vivo use⁴³.

Example 24 Amphiphilic Catalytic Experiments [175] 5 ml of the of Rhlll-TA capsule dispersion (the concentration of Rhlll was determined to be 0.2 mM by ICP-AES) and 0.5 mmol of quinoline was added to a steel reactor (Parr reactor, Series 4590). Before the reaction, the hydrophilic Rhlll-TA capsules reside in the aqueous phase and cannot react with the hydrophobic quinoline. In order to progress the reaction, the aqueous and organic phases were mixed together by vigorous stirring, which allows the quinoline to react with the Rhlll-TA capsules, where the active atomic H dissociates from quinoline over the Rhlll-TA capsules, resulting in the hydrogenation of quinoline. Catalytic hydrogenation was conducted under a constant H2 pressure of 2.0 MPa and the temperature was incrementally changed from 30 to 70°C. When the reaction was completed, the organic phase was collected by decantation and analysed by GC. Analyses of the hydrogenated quinoline (HQ) and residual substrate (Q) were conducted by calculating the area of each signal peak in GC. The turnover frequency (TOF) of the catalyst in the hydrogenation experiments was calculated using the following equation2:

TOFimol -mo -h-^ *^Qi→

' Rh^m (mol)x t(h)

[176] This example demonstrated the catalytic function of Rh'"-TA capsules for the hydrogenation of quinoline versus a commercial catalyst, RhCI(CO)(TPPTS)₂ (TPPTS = triphenylphosphine-trisulfonated). As shown in Fig.36, the initial hydrogenation activities (turnover of frequency, TOF) of both Rh'"-TA capsules and RhCI(CO)(TPPTS)₂ are quite low when the reaction temperature is below 50°C. However, a dramatic increase in hydrogenation activity can be observed in Rh'"-TA capsules when the reaction temperature changes from 50°C to 70°C; 16 to 103 mol mol^"1 h^"1, respectively. The catalytic activity of Rh'"-TA capsules was significantly greater than RhCI(CO)(TPPTS)₂ between 50°C and 70°C, where the maximum initial TOF of RhCI(CO)(TPPTS)₂ was only 44 mol mol^"1 h^"1, which is 43% of the TOF value for the Rh'"-TA capsules.

Example 25. Loading Doxorubicin with MPN Capsules

[177] PSS-doped CaC0₃ was first prepared according to the method below: 2.4 mL of sodium carbonate (1 M) and 200 mL of PSS (1 mg/mL) was mixed in a 200 mL of beaker, in which 4.8 mL of CaCI₂ solution (1 M) was instantly added under vigorous stirring. After 30 s, the resulted CaC0₃ precipitate was washed for three times to remove any unreacted substance and was redissolved in 24 mL of Dl water. The porous, spherical CaC0₃ particles with the average diameter of 2.5 μιη were obtained. Afterwards, 0.1 mL of doxorubicin (DOX) hydrochloride solution (10 mg/mL) was used to mix with 0.5 mL of PSS-doped CaC0₃ template for 30 min, following by extensive wash to remove excessive DOX. The coating of metal-polyphenol complexes was conducted by sequential addition of 2 μΙ of TA solution (2.4 mM) and 2 μΙ_ of metal precursor solutions (2.4 mM of AICI₃, MnS0₄ or GdCI₃ solutions) in the presence of 400 μΙ_ of DOX-deposited CaC0₃ solution (1.0 mg/mL). The solution mixture was vigorously vortexed for 10 s and was added with 100 μΙ_ of MOPS buffer (pH 8.0, 100 mM). The solution mixture was washed three times and the CaC0₃ template was removed by tris- acetate buffer (2 M, pH 8.0). The resulted DOX-MPN capsules were washed by water three times and redispersed in Dl water. PSS-doped CaC0₃ without preloading DOX was used as the template for the blank MPN capsule preparation.

Example 26. DOX-MPN loading capacity measurement

[178] 10 uL of DOX-Al'"-TA capsules were first degraded by incubating with equal volume of 0.2 M HCI for 30 minutes, and the resulted solution was subsequently diluted with 90 μΙ_ of PBS buffer (pH 7.4). The fluorescence intensity was measured on an Infinite M200 microplate reader (Tecan, Switzerland) at an excitation wavelength of 480 and emission wavelength of 570 nm. The encapsulated DOX concentration was calculated with a standard curve calibrated with DOX samples of known concentration according to the previous reports.'²¹ Meanwhile, 10 uL of DOX-Al'"-TA capsules were measured on a Partec CyFlow Space (Partec GmbH, Germany) flow cytometer to count the number of capsules. The loading capacity was expressed as expressed as pigogram of DOX per capsule.

Example 27. Capsule Degradation and In Vitro DOX Release

[179] Degradation study of blank MPN capsule (without loading DOX) was carried out in PBS buffer solutions at different pH values (7.4, 6.0 and 5.0). The capsule/PBS solutions were constantly incubated on a thermostated shaker (Eppendorf Thermomixer Comfort) at 37 °C at 800 rpm. At different time points, 10 μΙ_ of the capsule dispersion (~10⁶ capsules) was collected and diluted with 990 ml of deionized water for flow cytometric analysis. Samples were measured on a Partec CyFlow Space (Partec GmbH, Germany) flow cytometer to count the number of capsules. Data were analysed to give the mean ± SD from three independent measurements. The in vitro release of DOX from Al'"-TA capsules was studied as follow: DOX-Al'"-TA capsules were resuspended in 400 μΙ_ of PBS buffer solutions of different pH (7.4, 6.0, 5.0) and were shaked at 37 °C at 800 rpm. At predefined time points, the capsules in the buffered solutions were centrifuged and the 350 μΙ_ of the supernatant was carefully collected for DOX fluorescence quantification. The fluorescence intensity of 100 μΙ_ of supernatant was measured on an Infinite M200 microplate reader (Tecan, Switzerland) at an excitation wavelength of 480 and emission wavelength of 570 nm. The DOX concentration in the supernatant was calculated based on the standard curve calibrated with DOX solutions of known concentrations.

Example 28. Capsule Permeability Evaluation

[180] The evaluation of capsule permeability was conducted according to the method below: the dispersion of AI(I II)-MPN capsules (~ 2.0 ^χ 10⁷ capsules/mL) was mixed with an equal volume of FITC-dextran solution (5 mg/ml_). Deconvolution microscope images of the capsules were taken within 10 min after incubation of capsules with FITC-dextran solutions for 15 min in dark. Capsules were randomly divided into 3 groups, and each group contains 50 to 70 capsules. Capsules with interiors of similar fluorescent intensity as the outer environment were considered to be permeable, whereas dark interiors were considered to be impermeable.

Example 29. Intracelluar DOX Delivery

[181 ] For DOX intracellular release and cell uptake visualization, HeLa and MB231 cells were seeded at 2 ^χ 10⁴ cells per well into 8-well Lab-Tek chambered coverglass slides (Thermo Fisher Scientific, Rochester) and allowed to grow for 24 h. Afterwards, cells were incubated with capsules (at capsule-to-cell ratio of 20: 1) for 24 h followed by washing with PBS three times. Cells were first fixed with 4% paraformaldehyde for 15 min at 37 °C, washed with PBS twice and stained with wheat germ agglutinin (0.25 ig/mL) at room temperature for 10 min, followed by staining with Hoechst 33342 (2 ig/mL) at room temperature for 15 min.

Example 30. Effectiveness of DOX-loaded MPN Capsules in Killing Cancer Cell Lines

[182] HeLa (human cervical carcinoma cell line) and MDA-MB-231 (Human breast carcinoma cell line) cells were used to evaluate the cytotoxicity of DOX-Al'"-TA capsules by MTT assay. When the cell confluency reached -70%, 100 μΙ_ of cells were seeded into 96- well plates (Costar 3596, Corning, USA) at a density of 1 ^χ 10⁴ cells/mL. After 24 h, culture media were replaced with fresh media containing a different number of capsules. After another 48 h of incubation, 10 μί of 5 mg/mL filtered MTT stock solution was added to each well, and unreacted dye was removed by aspiration after 4 h. The formazan crystals were dissolved in 100 μΙ_ per well DMSO and were measured on an Infinite M200 microplate reader (Tecan, Switzerland) at a wavelength of 570 nm. The cell viability was expressed as a relative percentage of the untreated cells. Example 31. Apoptosis Assay

[183] Apoptosis of HeLa and MB231 cells induced by DOX-Al^li!-TA capsules was evaluated through Annexin V/propidium iodide (Pi) double staining assay, in brief, cells were seeded in 6-weli plate at the cell density of 3 * 10⁵ per well. After incubation of DOX or DOX- Al^m-TA at an equivalent DOX concentration of 0.5 and 2.5 pg/mL for 48 h, the cells were gentiy washed with cold PBS three times and were harvested by trypsinization. The cells were then stained with Alexa Fluor® 488 annexin V and Pi for flow cytometry analysis by using Alexa Fluor® 488 annexin V/Dead Ceil Apoptosis Kit (Life technologies, Australia) according to the manufacturer's instruction. The apoptottc ceils were then analyzed by using A50 Apogee flow cytometer (Apogee Flow System, UK) to measure the fluorescence emission at 530 nm and 575 nm using 488 nm excitation.

Example 32. Statistical Analysis

[184] All the results are expressed as mean ± standard deviation. Student's t-test was used to assess statistical significance of difference between group means.

[185] Finally, it will be appreciated that various modifications and variations of the methods and compositions of the invention described herein will be apparent to those skiiled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are apparent to those skilled in the art are intended to be within the scope of the present invention.

Claims

1. A method of forming a film or network on the surface of a solid substrate of less than 1 cm in size the method comprising the steps of (i) forming a solution containing the substrate (ii) contacting the solution containing the substrate with a solution of a metallic cation or a mixture of metallic cations and a solution of a polyphenolic compound to form a film or network on the surface of the substrate, wherein the contacting occurs for less than 30 minutes.

2. A method according to claim 1 wherein the step of forming a solution containing the substrate comprises suspending the substrate in solution.

3. A method according to claim 1 or 2 wherein the film or network is formed by a coordination complex between the metallic cation and the polyphenolic compound.

4. A method according to any one of claims 1 to 3 wherein the metallic cation is a cation from an alkali earth metal, a transition metal or a lanthanide metal.

5. A method according to any one of claims 1 to 4 wherein the metallic cation is selected from the group consisting of a magnesium cation, a calcium cation, a cadmium cation, a strontium cation, a barium cation, an iron cation, an aluminium cation, a ruthenium cation, a rhodium cation, a terbium cation, a vanadium cation, a chromium cation, a manganese cation, a zinc cation, a copper cation, a cobalt cation, a nickel cation, a molybdenum cation, a titanium cation, a zirconium cation, a cerium cation, a europium cation , a gadolinium cation and a mixture of two or more thereof.

6. A method according to any one of claims 1 to 5 wherein the metal is iron.

7. A method according to any one of claims 1 to 6 wherein the metallic cation is iron (III).

8. A method according to any one of claims 1 to 7 wherein the polyphenolic compound contains catechol groups, gallol groups or a mixture thereof.

9. A method according to any one of claims 1 to 8 wherein the polyphenolic compound is selected from the group consisting of tannic acid and epigallocatechin.

10. A method according to any one of claims 1 to 9 wherein the polyphenolic compound is tannic acid.

1 1. A method according to any one of claims 1 to 10 wherein the contacting comprises addition of a solution of a metallic cation to the solution containing the substrate followed by addition of a solution of the polyphenolic compound.

12. A method according to any one of claims 1 to 10 wherein the contacting comprises addition of a solution of the polyphenolic compound to the suspension of the substrate in solution to the solution containing the substrate followed by addition of a solution of a metallic cation.

13. A method according to any one of claims 1 to 12 wherein the molar ratio of metallic compound to polyphenolic compound is in the ratio of 0.5: 1 to 5: 1.

14. A method according to any one of claims 1 to 13 wherein the molar ratio of metallic compound to polyphenolic compound is in the ratio of 1 :1 to 3: 1.

15. A method according to any one of claims 1 to 14 wherein the contacting occurs at a temperature in the range of 10°C to 40°C.

18. A method according to any one of claims 1 to 17 wherein the substrate is selected from the group consisting of an organic particle, an inorganic particle, a biological particle and combinations thereof.

19. A method according to any one of claims 1 to 18 wherein the substrate is selected from the group consisting of polystyrene, glass, gold (Au), polydimethylsiloxane (PDMS), poly(lactic-co-glycolic acid) (PLGA), melamine-formaldehyde resin (MF), low-molecular- weight PDMS emulsion, silica (Si0₂), aminated Si0₂, cetyltrimethylammonium bromide- capped Au nanoparticles (Au NPs), calcium carbonate (CaC0₃), Escherichia coli (E. coli), and Staphylococcus epidermidis (S. epidermidis).

20. A method according to any one of claims 1 to 19 wherein the substrate is a metallic particle.

21. A method according to any one of claims 1 to 19 wherein the substrate is a silica particle.

22. A method according to any one of claims 1 to 21 wherein the substrate is porous or non-porous.

23. A method according to any one of claims 1 to 22 wherein the shape of the substrate is selected from the group consisting of a sphere, a cube, a prism, a fibre, a rod, a tetrahedron and an irregular shape.

25. A method of manufacturing an article the method comprising the steps of (i) forming a solution containing a solid substrate of less than 1 cm in size (ii) contacting the solution containing the substrate with a solution of a metallic cation or a mixture of metallic cations and a solution of a polyphenolic compound to form a film or network on the surface of the substrate, wherein the contacting occurs for less than 30 minutes.

26. A method according to claim 25 wherein the step of forming a solution containing the substrate comprises suspending the substrate in solution.

27. A method according to claim 25 or 26 wherein the film or network is formed by a coordination complex between the metallic cation and the polyphenolic compound.

28. A method according to any one of claim 25 to 27 wherein the metallic cation is a cation from an alkali earth metal, a transition metal or a lanthanide metal.

29. A method according to any one of claims 25 to 28 wherein the metallic cation is selected from the group consisting of a magnesium cation, a calcium cation, a cadmium cation, a strontium cation, a barium cation, an iron cation, an aluminium cation, a ruthenium cation, a rhodium cation, a terbium cation, a vanadium cation, a chromium cation, a manganese cation, a zinc cation, a copper cation, a cobalt cation, a nickel cation, a molybdenum cation, a titanium cation, a zirconium cation, a cerium cation, a europium cation, a gadolinium cation and a mixture of two or more thereof. .

30. A method according to any one of claims 25 to 29 wherein the metal is iron.

31. A method according to any one of claims 25 to 30 wherein the metallic cation is iron (III).

32. A method according to any one of claims 25 to 31 wherein the polyphenolic compound contains catechol groups, gallol groups or a mixture thereof.

33. A method according to any one of claims 25 to 32 wherein the polyphenolic compound is selected from the group consisting of tannic acid and epigallocatechin

34. A method according to any one of claims 25 to 33 wherein the polyphenolic compound is tannic acid.

35. A method according to any one of claims 25 to 34 wherein the contacting comprises addition of a solution of a metallic cation to the suspension of the substrate in solution followed by addition of a solution of the polyphenolic compound.

36. A method according to any one of claims 25 to 35 wherein the contacting comprises addition of a solution of the polyphenolic compound to the suspension of the substrate in solution followed by addition of a solution of a metallic cation.

37. A method according to any one of claims 25 to 36 wherein the molar ratio of metallic compound to polyphenolic compound is in the ratio of 0.5: 1 to 5: 1.

38. A method according to any one of claims 25 to 37 wherein the molar ratio of metallic compound to polyphenolic compound is in the ratio of 1 :1 to 3: 1.

39. A method according to any one of claims 25 to 38 wherein the contacting occurs at a temperature in the range of 10°C to 40°C.

42. A method according to any one of claims 25 to 41 wherein the substrate is selected from the group consisting of an organic particle, an inorganic particle, a biological particle and combinations thereof.

43. A method according to any one of claims 25 to 42 wherein the substrate is selected from the group consisting of glass, gold (Au), polydimethylsiloxane (PDMS), poly(lactic-co- glycolic acid) (PLGA), melamine-formaldehyde resin (MF), low-molecular-weight PDMS emulsion, silica (Si0₂), aminated Si0₂, cetyltrimethylammonium bromide-capped Au nanoparticles (Au NPs), calcium carbonate (CaC0₃), Escherichia coli (E. coli), and Staphylococcus epidermidis (S. epidermidis).

44. A method according to any one of claims 25 to 42 wherein the substrate is a metallic particle.

45. A method according to any one of claims 25 to 42 wherein the substrate is a silica particle.

46. A method according to any one of claims 25 to 42 wherein the substrate is porous or non-porous.

47. A method according to any one of claims 25 to 46 wherein the shape of the substrate is selected from the group consisting of a sphere, a cube, a prism, a fibre, a rod, a tetrahedron and an irregular shape.

49. A method according to any one of claim 25 to 48 wherein the method further comprises the step of removing the substrate to produce an article having a hollow core.

50. A method according to claim 49 wherein removing the substrate comprises dissolving the substrate.

51. A method according to claim 49 or 50 wherein the substrate is removed by subjecting the article to a solution to dissolve the substrate.

52. A method according to claim 51 wherein the solution is a solution of ammonium fluoride in HF.

53. A method according to any one of claims 25 to 52 wherein the article is a particle.

54. A method according to any one of claims 25 to 53 wherein the article is a capsule.

55. A method of delivering an active agent to a part of the body of a mammal, the method comprising encapsulating the active agent in an article formed from an interconnected network of a polyphenolic compound and a metal cation or mixture of metal cations, administering the article containing the active agent to the mammal.

56. A method according to claim 55 wherein the metallic cation is a cation from an alkali earth metal, a transition metal or a lanthanide metal.

57 A method according to claim 55 or 56 wherein the metallic cation is selected from the group consisting of a magnesium cation, a calcium cation, a cadmium cation, a strontium cation, a barium cation, an iron cation, an aluminium cation, a ruthenium cation, a rhodium cation, a terbium cation, a vanadium cation, a chromium cation, a manganese cation, a zinc cation, a copper cation, a cobalt cation, a nickel cation, a molybdenum cation, a titanium cation, a zirconium cation, a cerium cation, a europium cation , a gadolinium cation and a mixture of two or more thereof.

58. A method according to any one of claims 55 to 57 wherein the metal is iron.

59. A method according to claim 58 wherein the metallic cation is iron (III).

60. A method according to any one of claims 55 to 59 wherein the polyphenolic compound contains catechol groups, gallol groups or a mixture thereof.

61. A method according to any one of claims 55 to 60 wherein the polyphenolic compound is selected from the group consisting of tannic acid and epigallocatechin

62. A method according to any one of claims 55 to 61 wherein the polyphenolic compound is tannic acid.

63. A method of diagnostic imaging of a part of the body of a mammal, the method comprising administering an article to the mammal, the article being formed from an interconnected network of a polyphenolic compound and a metal cation or mixture of metal cations and containing a detectable moiety, and detecting the presence of the detectable moiety in the mammal.

64. A method according to claim 63 wherein the metallic cation is a cation from an alkali earth metal, a transition metal or a lanthanide metal.

65 A method according to claim 63 or 64 wherein the metallic cation is selected from the group consisting of a magnesium cation, a calcium cation, a cadmium cation, a strontium cation, a barium cation, an iron cation, an aluminium cation, a ruthenium cation, a rhodium cation, a terbium cation, a vanadium cation, a chromium cation, a manganese cation, a zinc cation, a copper cation, a cobalt cation, a nickel cation, a molybdenum cation, a titanium cation, a zirconium cation, a cerium cation, a europium cation , a gadolinium cation and a mixture of two or more thereof.

66. A method according to any one of claims 63 to 65 wherein the metal is Europium or Terbium

67. A method according to any one of claims 63 to 66 wherein the polyphenolic compound contains catechol groups, gallol groups or a mixture thereof.

68. A method according to any one of claims 63 to 67 wherein the polyphenolic compound is selected from the group consisting of tannic acid and epigallocatechin

69 A method according to any one of claims 63 to 68 wherein the polyphenolic compound is tannic acid.

70. A method according to claim 63 wherein the imaging is carried out by magnetic resonance imaging (MRI), Positron emission topography (PET) or fluorescence imaging.

71. A method of catalysis of a chemical reaction, the method comprising contacting the reaction mixture with an article formed from an interconnected network of a polyphenolic compound and a metal cation or mixture of metal cations.

72. A method according to claim 71 wherein the reaction is an oxidation or a reduction reaction.

73. A method according to claim 71 or 72 wherein the reaction is a hydrogenation reaction.

74. A method according to claim 71 wherein the metallic cation is a cation from an alkali earth metal, a transition metal or a lanthanide metal.

75 A method according to claim 74 wherein the metallic cation is selected from the group consisting of a magnesium cation, a calcium cation, a cadmium cation, a strontium cation, a barium cation, an iron cation, an aluminium cation, a ruthenium cation, a rhodium cation, a terbium cation, a vanadium cation, a chromium cation, a manganese cation, a zinc cation, a copper cation, a cobalt cation, a nickel cation, a molybdenum cation, a titanium cation, a zirconium cation, a cerium cation, a europium cation , a gadolinium cation and a mixture of two or more thereof.

76. A method according to claim 71 wherein the metal is selected from the group consisting of platinum, palladium, rhodium, ruthenium, iridium, vanadium, cobalt, nickel, zirconium, and molybdenum.

77. A method according to any one of claims 71 to 76 wherein the polyphenolic compound contains catechol groups, gallol groups or a mixture thereof.

78. A method according to any one of claims 71 to 77 wherein the polyphenolic compound is selected from the group consisting of tannic acid and epigallocatechin

79 A method according to any one of claims 71 to 78 wherein the polyphenolic compound is tannic acid.