SELF ASSEMBLED CARBON BASED STRUCTURES AND RELATED METHODS

FIELD OF THE INVENTION The present invention relates generally to novel layered graphene and related structures and materials; methods for producing, through self-assembly, novel layered graphene and related structures or materials of controlled pore size.

BACKGROUND TO THE INVENTION

Graphene and related materials such as, for example, graphene oxide (GO), reduced graphene oxide (rGO) and chemically-doped graphene have attracted considerable attention due to their exceptional inherent properties that result from, amongst other things, their two dimensional character. ¹ For example, the exceptional electrical properties of graphene ¹ has made it a target of interest in the development of various electrical components such as batteries and sensors. As one of the strongest materials known, considerable research has also been directed towards use of graphene in composite materials. ² In addition, graphene and related materials have been a target of interest in the biomedical field with suggestions that they may prove useful in the field of drug delivery and biosensors.

Due to the hydrophobic character of graphene and some related materials such as highly reduced GO sheets, they quickly 're-stack' (i.e. aggregate) when placed in aqueous solutions to form layered, near-graphitic structures with sub-nanometre inter- sheet spacing. Whilst such restacking may often be desirable for applications, the loss of the space between the sheets is a major barrier for many potential applications. Many potential applications of graphene and related materials require or would benefit from the individual sheets of graphene or related materials being assembled into a layered structure with inter-sheet spacing (i.e. pores) that is greater than that found in the simple aggregated form.

A number of alternative approaches to production of layered graphene and graphene- related materials with pores between the layers have been researched. The first main approach for producing such materials is through insertion of inorganic spacing elements such as carbon black, ³ randomly ^4"10 and vertically aligned ^{11 ,12} carbon nanotubes, mesoporous carbon nanoparticles, ¹⁴ iron oxide nanorods, ¹⁵ ruthenium dioxide nanoparticles, ¹⁶ gold nanoparticles ^17"21 or tin oxide nanoparticles ²² or nanorods, ²³ either in the solution or vapour phase. A second approach is through insertion of polymers between the sheets, including those that react with the graphene-based material such as polyaniline, ²⁴ polyallylamine, ²⁵ and regenerated cellulose ²⁶ and those that are polymerized between the sheets such as polypyrrole. ²⁷ None of these approaches provide a high degree of control over the inter-layer spacing or its uniformity due to the challenges in precisely controlling the size of the spacing units.

A third approach is use of metal ions that not only push apart the individual graphene or graphene-related sheets, but also cross-link them laterally. ²⁸ Whilst use of ions gives tighter control over the pore size, the range of pore sizes is very limited indeed and still of the order of 1 nm. The intercalated ions are also easily removed depending on the solution conditions.

A fourth approach is the insertion of molecular linkers between sheets of graphene- related materials via chemical reaction between the linker molecules and some of the oxygen and other functionalities on the graphene-related sheets. This has been realized in a number of ways including p-phenylenediamine (PPD), ²⁹ Ni(ll) aza-macrocyclic complexes, ³⁰ benzenediboronic acid, ³¹ and 3,3'-diaminobenzidine and related compounds. ³² The pore sizes accessible here are small, however, due to the constraint on the size of the molecules involved. Where pores sizes much larger than the molecule are claimed, as in the case of PPD, ²⁹ this reflects an unpredictable process over which one would anticipate major issues with control of pore size.

A fifth approach is through use of a monolayer of aryl azobenzene linkers with one end of the linker chemical bound to a graphene sheet and the other physically adsorbed to the adjacent graphene sheet. ³³ Although this approach gives finer control over the distance between two successive graphene sheets in this example, the size range is severely limited by the lack of rigidity in the molecules beyond the short scale. The above review demonstrates that there are two major issues with the state of the art to date in the field. The first is the inability to control to a high degree the spacing between the individual layers of graphene or graphene-related material (i.e. pore size) for anything other than the smallest spacing where use of metal ions facilitate finer control, albeit only under certain controlled conditions. The degree of control over the pore size and its dispersion is poor for anything beyond 1 nm and well into the mesopore size range as defined by lUPAC. This lack of consistency and ability to vary the inter-sheet distance in a systematic way presents as an impediment to the use of graphene and graphene-based materials in a number of fields where it is important to control or dictate in a consistent and controlled way this inter-sheet distance (i.e. pore size).

Due to the use of designed peptides in the invention presented here, it is worthy of mention that some have also used biomolecules to form constructs composed of graphene and graphene-related materials. Xu et a/. ³⁴ used DNA to yield a three dimensional GO-based material. There is, however, no claim that the DNA act to separate the GO layers, and this is unlikely to be the case given the claimed material formation mechanism. Others ³⁵ have used amino acids to achieve some form of spacing, but this was felt to be via electrostatic repulsion/attraction between the charged amino acids akin to the mechanism associated with metal ions. Finally, Wu et a/. ³⁶ have proposed the use of a peptide to self-assemble a GO hydrogel for drug delivery applications. Once again, however, there is no claim that the peptides create pores of a given size by sitting between the individual GO layers, with the peptide design being such as to induce the random assembly of the GO.

Many of the approaches mentioned above involve layer-by-layer manufacturing where the macroscopic material is built up by putting down on a substrate successive layers of graphene or graphene-related material and spacer in an alternating fashion. This process leads to only a small fraction of the graphene or graphene-related material being separated by the spacers, with the remainder being essentially multi-layer graphene or graphene-related material or even more disordered than this. It also has the disadvantage that it is a cyclic process requiring significantly more than one cycle to be undertaken to achieve the final product. Only a small fraction of the prior art involves a self-assembly process in the manufacture and, even then, they do not lead to structures akin to those proposed here in any way, nor provide the degree of control over the pore size gained here.

It is an object of the present invention to provide a layered graphene or graphene-related material structure in which the space between the layers of graphene or graphene- related materials can be controlled over a wide range of pore sizes.

It is an object of the present invention to overcome, or at least substantially ameliorate, the disadvantages and shortcomings of the prior art. STATEMENTS OF THE INVENTION

According to an aspect of the invention there is provided a spacer molecule comprising: at least first and second binding parts adapted to non-covalently bond at least a first graphene sheet to a second graphene sheet, said spacer comprising at least 9 amino acid residues and wherein when in a solution comprising two or more graphene sheets said spacer molecule contacts first and second graphene sheets to provide a layered structure and a predetermined space between at least the first graphene sheet and the at least second graphene sheet to provide a porous layered graphene structure.

Reference to "graphene" includes graphene oxide, reduced graphene, doped graphene and related carbon based materials.

In a preferred embodiment of the invention said spacer molecule comprises:

i) a first end group adapted to non-covalently bond a first graphene sheet; ii) a second end group adapted to non-covalently bond a second graphene sheet;

iii) a spacer middle portion separating first and second end groups comprising at least 9 amino acid residues, wherein when in a solution comprising two or more graphene sheets said spacer molecule contacts first and second graphene sheets to provide a layered structure and a predetermined space between at least the first graphene sheet and the at least second graphene sheet to provide a porous layered graphene structure.

In a preferred embodiment of the invention said first end group comprises one or more amino acids or modified amino acids.

In a preferred embodiment of the invention said second end group comprises one or more amino acids or modified amino acids.

In a preferred embodiment of the invention said first and/or said second end group comprises one or more aromatic amino acids or modified amino acids. In a preferred embodiment of the invention said aromatic amino acid is selected from the group consisting of: histidine, phenylalanine, tryptophan, or tyrosine. In a preferred embodiment of the invention said spacer middle portion comprises 9 to at least 50 amino acids or modified amino acids.

The spacer molecule can include one or more natural or modified amino acids. Modified amino acids can be naturally or non-naturally occurring. The spacer molecule can be polar or non-polar and can include polar or non-polar amino acids or modified amino acids to alter the hydrophobicity or lipophilicity of the porous layered graphene structure. Examples of naturally occurring polar amino acids include glutamine, asparagine, histidine, serine, threonine, tyrosine, cysteine, methionine and tryptophan and modified amino acid variants thereof. Examples of non-polar, or hydrophobic amino acids, include alanine, isoleucine, leucine, phenylalanine, valine, proline and glycine and including modified amino acid variants thereof. The combinations of polar and non-polar amino acids can be used to alter the hydrophobicity or lipophilicity of the porous layered graphene structure.

In a preferred embodiment of the invention said spacer middle portion comprises one or more polar amino acids.

In an alternative preferred embodiment of the invention said spacer middle portion comprises one or more non-polar amino acids.

In a preferred embodiment of the invention said spacer middle portion comprises at least 4, 8, 12, 16, 20, 24, 28, 32, 36, 40 or at least 44 amino acids. In a preferred embodiment of the invention said spacer middle portion comprises over at least part of its length an alpha helix.

In an alternative preferred embodiment of the invention said spacer middle portion comprises over at least part of its length a beta sheet.

In a further embodiment of the invention said alpha helix comprises the same amino acid residues, for example amino acids selected from the group consisting of: methionine, alanine, leucine, glutamine, lysine or arginine. It will be apparent this embodiment comprises an alpha helix comprising essentially of, for example poly-arginine or poly-lysine. In an alternative preferred embodiment of the invention said alpha helix comprises alternating, different amino acids selected from the group consisting of: methionine, alanine, leucine, glutamine, lysine or arginine. It will be apparent this embodiment comprises an alpha helix with, for example, alternating arginine and lysine amino acids.

In a preferred embodiment of the invention said spacer molecule comprises amino acids selected from the amino acid sequence:

Xaai Xaa ₂ Xaa ₃ Xaa ₄ Xaas Xaa6 Xaa ₇ Xaas Xaag Xaa-io Xaa-n Xaa-i ₂ Xaai ₃ Xaa-i ₄ [SEQ ID NO: 1 ] wherein

Xaa-ι and/or Xaa ₂ is an aromatic amino acid

Xaa ₃ and/or Xaa ₄ is glycine;

Xaa ₅ to Xaa ₁₀ comprise at least 5 amino acids selected from the group: methionine, alanine, leucine, glutamine, lysine or arginine;

Xaan and/or Xaa ₁₂ is glycine; and

Xaa ₁₃ and/or Xaa ₁₄ is an aromatic amino acid.

In an alternative preferred embodiment of the invention said spacer molecule comprises amino acids selected from the amino acid sequence: Xaai Xaa ₂ Xaa ₃ Xaa ₄ Xaa ₅ Xaa ₆ Xaa ₇ Xaa ₈ Xaa ₉ Xaa ₁₀ Xaan Xaa ₁₂ Xaa ₁₃ Xaa ₁₄ [SEQ ID NO: 1 ]

wherein

Xaa _! and/or Xaa ₂ is an aromatic amino acid

Xaa ₃ and/or Xaa ₄ is glycine;

Xaa ₅ to Xaa ₁₀ comprise at least 5 amino acids selected from the group: tyrosine, phenylalanine, tryptophan, threonine, valine or isoleucine, optionally Xaa ₅ to Xaa ₁₀ can include one or more aromatic amino acid residue

Xaan and/or Xaa-| ₂ is glycine; and

Xaa ₁₃ and/or Xaa ₁₄ is an aromatic amino acid. In an alternative embodiment of the invention of the invention said spacer comprises the amino acid sequence:

Xaai Xaa2 Xaa3 Xaa ₄ Xaas Xaag Xaa ₇ Xaa8 Xaag Xaa-io Xaa-n Xaa-i2 Xaa-i3 Xaa-i ₄ [SEQ ID NO: 1 ]

wherein Xaa ₅ to Xaa ₁₀ comprises the same amino acid residues wherein said amino acid residues are selected from the group consisting of: methionine, alanine, leucine, glutamine, lysine or arginine. In an alternative preferred embodiment of the invention said spacer comprises the amino acid sequence:

Xaa-| Xaa2 Xaas Xaa ₄ Xaas Xaag Xaa ₇ Xaa8 Xaag Xaa-io Xaan Xaa-12 Xaa-i3 Xaa-| ₄ wherein Xaa ₅ to Xaa ₁₀ comprises alternating amino acid residues wherein said amino acid residues are selected from the group consisting of: methionine, alanine, leucine, glutamine, lysine or arginine.

In a preferred embodiment of the invention said spacer comprises the amino acid sequence: FFGGEEEEEEGGFF [SEQ ID NO: 3], or a modified amino acid sequence wherein said modified amino acid sequence is at least 75% identical to the amino acid sequence FFGGEEEEEEGGFF [SEQ ID NO: 3] and that retains or has enhanced graphene binding. In addition, the invention features peptide sequences having at least 80% identity with FFGGEEEEEEGGFF [SEQ ID NO: 3], or fragments and functionally equivalent peptides thereof. In one embodiment, the peptides have at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, still more preferably at least 97% or 98% identity, and most preferably at least 99% identity with the amino acid sequence set for as FFGGEEEEEEGGFF [SEQ ID NO: 3]. A spacer comprising FFGGEEEEEEGGFF [SEQ ID NO: 3] can be modified by one or more substitutions, additions, deletions, truncations which may be present in any combination. Among preferred modifications are those that vary by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid by another amino acid of like characters. Most highly preferred are modified peptides which retain the same function and activity as the reference peptide from which it varies. In a preferred embodiment of the invention said peptide comprising or consisting of an amino acid sequence selected from the group consisting of:

i) FFGGEEEEGGFF [SEQ ID NO: 2];

ii) FFGGEEEEEEGGFF [SEQ ID NO: 3];

iii) FFGGEEEEEEEEEEEEGGFF [SEQ ID NO: 4];

iv) FFGGQQQQQQGGFF [SEQ ID NO: 5];

v) FFGGKKKKKKGGFF [SEQ ID NO: 6];

vi) WWGGEEEEEEGGWW [SEQ ID NO: 7];

vii) FFGGEKEKEKGGFF [SEQ ID NO: 8];

viii) FFGGEQKEQKGGFF [SEQ ID NO: 9];

ix) FFGGEMEMEMGGFF [SEQ ID NO: 10]; and

x) FFGGQMQMQMGGFF [SEQ ID NO: 1 1].

According to an aspect of the invention there is provided a layered graphene structure comprising one or more spacers according to the invention.

In a preferred embodiment of the invention said layered graphene structure, includes: at least a first graphene layer and at least a second graphene layer, at least one spacer layer, wherein said at least one spacer layer includes at least one spacer unit, the spacer unit including a middle portion and an upper end and a lower end, so as to provide a desired predetermined space between the at least first graphene layer and the at least second graphene layer.

In preference, the layered graphene structure is a hydrogel. In preference, the spacer is a peptide. In preference, the middle portion is hydrophilic.

In preference, the middle portion is a substantially rigid structure. In preference, the middle portion has an alpha-helix structure.

In preference, the middle portion has a beta-sheet structure. In preference, the upper end and lower end are connected to the middle portion flexible connecting units.

In preference, the flexible connecting units are glycine.

In preference, the upper end and lower end are amino acids.

In preference, the upper end and lower end are selected from the group of amino acids consisting of histidine, tyrosine, phenylalanine and tryptophan.

In preference, the upper end and lower end are the same.

In an alternative preference, the upper end and lower end are the not the same. In a preferred embodiment of the invention said structure is porous.

In a preferred embodiment of the invention said structure is mesoporous, for example the pores are at least 2nm in diameter and less than 50nm in diameter. In alternative preferred embodiment of the invention said structure is macroporous, for example the pores are greater than 50nm in diameter.

In a preferred embodiment of the invention said layered structure is at least 10nm thick. Preferably said layered structure is between 10-100nm thick.

In an alternative embodiment of the invention said layered structure is greater than 100nm thick. In a further alternative embodiment of the invention said layered structure is between 100 to 1000nm thick.

In an embodiment of the invention said layered structure is greater than l OOOnm thick. In a further preferred embodiment of the invention said layered structure comprises at least 2 graphene layers. In a preferred embodiment of the invention said layered structure comprises at least 2 to 100 graphene layers.

In an alternative embodiment of the invention said layered structure comprises greater than 100 graphene layers.

According to a further aspect of the invention there is provided a device comprising a graphene structure according to the invention. In a preferred embodiment of the invention said device is a drug delivery device wherein said device is further modified to include at least one biologically active agent.

A "drug delivery device" is a generic term to include structures that facilitate the controlled release of a therapeutic agent. Typically, drug delivery devices are adapted to deliver a drug of a particular dosage via a particular route of administration, for example intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, transdermal or trans-epithelial. Drug delivery devices typically are engineered to release the associated therapeutic agent in a controlled release, for example immediate delayed or sustained release or combinations thereof. A drug delivery device as a gel can be applied directly to, for example, a tumour or other diseased tissue to allow direct release of the drug at or on the diseased tissue. A drug delivery device could also be a bandage or hydrogel adapted to contact a wound to administer a wound healing agent and/or an anti-microbial agent. In an alternative preferred embodiment of the invention said device is a medical device.

Medical devices are implanted into patients to treat a variety of diseases and conditions. Medical devices include catheters, stents [ureteral or prostatic stents], cannulas, prosthesis and implants, gels. The surfaces of medical devices can be adapted to release agents, for example therapeutic agents, that treat disease or reduce the likelihood of infection. The implantation of a medical device necessarily requires the exposure of the patient to both immune rejection of the implanted device and also an increased probability of an adventitious infection by a microbial pathogen. The invention therefore anticipates a device according to the invention that includes an agent to prevent immune rejection and/or an antimicrobial agent such as an antibiotic or heavy metal such as silver, gold or copper. In an alternative preferred embodiment of the invention said therapeutic agent is an anticancer agent.

In a preferred embodiment of the invention said drug delivery device comprises a gel.

According to the present invention, although this should not be seen as limiting the invention in any way, there is provided a method of producing a layered graphene structure the method including the steps of: mixing graphene and a solution of spacer units having a first end group and a second end group in a solution; non-covalently bonding a first end group of the spacer unit to a first side of at least a first graphene sheet; non-covalently bonding a second end group of the spacer unit to a first side of at least a second graphene sheet; wherein the spacer units form a spacer layer, so as to provide a desired predetermined space between the at least first graphene sheet and the at least second graphene sheet.

In preference, the layered graphene structure is self-assembled. In preference, the spacer units facilitates the self-assembly and stabilisation of the layered graphene structure.

In preference, the spacer unit is at least one amino acid. In preference, the spacer unit is at least one peptide.

In preference, the spacer unit is at least one polypeptide.

In preference, the first end group and the second end group of the spacer units are selected from the group of functional groups including aromatic and non-aromatic functional groups. According to a further aspect of the invention there is provided a process for the purification of one or more molecules comprising the steps:

i) providing a mixture comprising a molecule to be purified;

ii) contacting said mixture with a layered graphene structure according to the invention and provide conditions to allow association of said one or more molecules with said layered graphene structure to provide a graphene structure comprising said molecule[s];

iii) contacting the layered graphene comprising molecule[s] with a wash buffer to remove non-specifically associated molecules; optionally iv) repeating step ii) and/or iii);

v) eluting associated molecule[s] from said layered graphene to provide a purified solution of said molecule[s]; and optionally

vi) repeating step v). In a preferred method of the invention said layered graphene is part of a column and said molecules are contacted with said layered graphene.

In an alternative preferred method of the invention said layered graphene is contacted with a solution comprising a mixture of said molecules.

"Molecules" refer to organic or inorganic compounds and encompasses proteins [e.g. enzymes, hormones, antibodies or antibody fragments, antigenic proteins and polypeptides found in vaccines, peptides such as peptide hormones, nucleic acids, [e.g. DNA fragments, cDNA, mRNA, plasmid and vector nucleic acid]. Also included are small organic molecules such as chemotherapeutic agents, antibiotics, anti-inflammatories.

The properties of the compositions and layered materials described herein may be tuned or customised based on the substitution or variation of the spacer units. Those skilled in the art would recognize and understand that variations to the spacer units could be readily synthesised using conventional approaches to provide a particular property, for example the ability of the layered structure to act as a filter or detector of an analyte.

In some embodiments, by varying the distance between the sheets of graphene, the resultant material may be used to trap or sequester a desired target molecule within a solution or mixture. In addition, the spacer unit may be further functionalised to interact with the desired target molecule. In further embodiments, the compositions and layered materials of the present invention may be useful as electron transport materials in photovoltaic devices, such as solar cells. They may be combined with other materials such as electron conducting materials. Alternatively, compositions and layered materials of the present invention may be useful in applications such as conductive coatings, energy storage materials such as secondary batteries and supercapacitors, artificial photosynthetic devices for producing hydrogen from water and other nano-reactor applications.

In yet further embodiments of the present invention, the spacer units may be designed to respond or react to external stimuli so that the spacing between the graphene sheets are affected, for example, by changes in pH to the liquid in which the present invention is immersed. Changes in pH may change the length of the spacer units resulting in the space between the graphene sheets either increasing or decreasing. The spacer units may also be designed to be able to reversibly or irreversibly disengage with the adjacent graphene sheets after formation of the composition of the present invention. In this way it may be possible to predetermine a point at which any components held within the present invention are released into its immediate environment or alternatively define a point at which the functioning of the present invention may be halted or adjusted.

The present invention provides a way in which the spacing between the graphene sheets can be controlled by way of modification of the size, shape and composition of the peptide.

Some Specific Embodiments of the Invention

Therapeutic Agents

Small Organic Molecules A general definition of "chemotherapeutic agent" is an agent that typically is a small chemical compound that preferably kills cells in particular diseased cells or tissue or is at least cytostatic. Agents can be divided with respect to their structure or mode of action. For example, chemotherapeutic agents include alkylating agents, anti-metabolites, anthracyclines, alkaloids, plant terpenoids and toposisomerase inhibitors. Chemotherapeutic agents typically produce their effects on cell division or DNA synthesis. Examples of alkylating agents are cisplatin, carboplatin or oxaliplatin. Examples of anti-metabolites include purine or pyrimidine analogues. Purine analogues are known in the art. For example thioguanine is used to treat acute leukaemia. Fludarabine inhibits the function of DNA polymerases, DNA primases and DNA ligases and is specific for cell-cycle S-phase. Pentostatin and cladribine are adenosine analogues and are effective against hairy cell leukaemias. A further example is mercaptopurine which is an adenine analogue. Pyrimidine analogues are similarly known in the art. For example, 5-fluorouracil (5-FU), floxuridine and cytosine arabinoside. 5-FU has been used for many years in the treatment of breast, colorectal cancer, pancreatic and other cancers. 5-FU can also been formed from the pro-drug capecitabine which is converted to 5-FU in the tumour. Leucovorin, also known as folic acid, is administered as an adjuvant in cancer chemotherapy and which enhances the inhibitory effects of 5-FU on thymidylate synthase. Alkylating agents are also known in the art and include vinca alkaloids, for example vincristine or vinblastine. Terpenoids have been used for many years and include the taxanes, for example, palitaxel. In a preferred embodiment said agent is doxorubicin. Prodrugs are also within the scope of the invention. A prodrug is a substance that is converted from an inactive or partially active agent by chemical conversion, for example enzymatic conversion, to an active or more active drug.

The spacer according to the invention can be designed to accept small organic molecules as herein disclosed.

Moreover, antibiotics and antiviral agents are effective in the treatment of microbial, for example bacterial and parasitic pathogens and pathogenic viruses. Examples of classes of antibiotics effective in the control of bacterial pathogens include, by example only, penicillins, cephalosporins, rifamycins, sulphonamides, macrolides and tetracyclines. Also included within the scope of the invention are antibacterial peptides such as dermicidins, cecropins and defensins. Antiviral agents include anti-retroviral drugs such as zidovudine, lamivudine, efavirenz and abacavir; and anti-viral drugs such as ganciclovir, aciclovir and oseltamivir. Anti-protozoan agents include lumefantrine, mefloquine, amodiaquine, sulfadoxine, chloroquine used in the treatment of malaria and also combination therapies that use these agents in combination with artemisinin. These are additional non-limiting examples of agents that can be used with the device according to the invention.

Antibodies

Antibodies include polyclonal and monoclonal antibodies, prepared according to conventional methodology. Typically antibodies are directed to cell surface proteins, for example receptors. However, intracellular delivery of antibodies and antibody fragments is known, for example see WO2007/064727; WO2004/030610; WO03/095641 ; WO02/07671 ; WO01/43778; WO96/40248; and WO94/01 131 each of which is incorporated by reference in their entirety.

Chimeric antibodies are recombinant antibodies in which all of the V-regions of a mouse or rat antibody are combined with human antibody C-regions. Humanised antibodies are recombinant hybrid antibodies which fuse the complementarity determining regions from a rodent antibody V-region with the framework regions from the human antibody V- regions. The C-regions from the human antibody are also used. The complementarity determining regions (CDRs) are the regions within the N-terminal domain of both the heavy and light chain of the antibody to where the majority of the variation of the V- region is restricted. These regions form loops at the surface of the antibody molecule. These loops provide the binding surface between the antibody and antigen.

Antibodies from non-human animals provoke an immune response to the foreign antibody and its removal from the circulation. Both chimeric and humanised antibodies have reduced antigenicity when injected to a human subject because there is a reduced amount of rodent (i.e. foreign) antibody within the recombinant hybrid antibody, while the human antibody regions do not ellicit an immune response. This results in a weaker immune response and a decrease in the clearance of the antibody. This is clearly desirable when using therapeutic antibodies in the treatment of human diseases. Humanised antibodies are designed to have less "foreign" antibody regions and are therefore thought to be less immunogenic than chimeric antibodies.

Various fragments of antibodies are known in the art. A Fab fragment is a multimeric protein consisting of the immunologically active portions of an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, covalently coupled together and capable of specifically binding to an antigen. Fab fragments are generated via proteolytic cleavage (with, for example, papain) of an intact immunoglobulin molecule. A Fab ₂ fragment comprises two joined Fab fragments. When these two fragments are joined by the immunoglobulin hinge region, a F(ab') ₂ fragment results. An Fv fragment is multimeric protein consisting of the immunologically active portions of an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region covalently coupled together and capable of specifically binding to an antigen. A fragment could also be a single chain polypeptide containing only one light chain variable region, or a fragment thereof that contains the three CDRs of the light chain variable region, without an associated heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multi specific antibodies formed from antibody fragments, this has for example been described in US patent No 6,248,516. Fv fragments or single region (domain) fragments are typically generated by expression in host cell lines of the relevant identified regions. These and other immunoglobulin or antibody fragments are within the scope of the invention and are described in standard immunology textbooks such as Paul, Fundamental Immunology or Janeway et al. Immunobiology. Molecular biology now allows direct synthesis (via expression in cells or chemically) of these fragments, as well as synthesis of combinations thereof. A fragment of an antibody or immunoglobulin can also have bispecific function as described above. The device according to the invention can be adapted by manipulation of pore size to carry antibodies and antibody fragments as herein disclosed. In general, doses of antibodies (or fragments thereof) of between 10 and 500 μι |/ιτιΐ generally will be formulated and administered according to standard procedures. Exemplary doses can range from 10 to 250 μ9/ΓηΙ, 30 μ9/ιηΙ to 250 g/ml, 50 ng/ml to 250 ng/ml, 30 such as 10 μg/ml, 20

40 μg/ml, 50 g/ml, 60 μg/ml, 70 μg/ml, 80

μg/ml or 500 Other protocols for the administration of compositions will be known to one of ordinary skill in the art, in which the dose amount, schedule of injections, sites of injections, mode of administration and the like vary from the foregoing. The administration of compositions to mammals other than humans, (e.g. for testing purposes or veterinary therapeutic purposes), is carried out under substantially the same conditions as described above. A subject, as used herein, is a mammal, preferably a human, and including a non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent.

Pharmaceutical Proteins & Peptides

Similarly, so called protein or peptide biologies can be associated with the device according to the invention. This includes pharmaceutically active proteins.

Examples of pharmaceutical proteins include "cytokines". Cytokines are involved in a number of diverse cellular functions. These include modulation of the immune system, regulation of energy metabolism and control of growth and development. Cytokines mediate their effects via receptors expressed at the cell surface on target cells. Examples of cytokines include the interleukins such as: IL1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32 and 33. Other examples include growth hormone, leptin, erythropoietin, prolactin, tumour necrosis factor [TNF] granulocyte colony stimulating factor (GCSF), granulocyte macrophage colony stimulating factor (GMCSF), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1 ), leukemia inhibitory factor (LIF) and oncostatin M (OSM), interferon a, interferon β, interferon ε, interferon κ and ω interferon. Examples of pharmaceutically active peptides include GLP-1 , anti-diuretic hormone; oxytocin; gonadotropin releasing hormone, corticotrophin releasing hormone; calcitonin, glucagon, amylin, A-type natriuretic hormone, B-type natriuretic hormone, ghrelin, neuropeptide Y, neuropeptide YY ₃ _ _36, growth hormone releasing hormone, somatostatin; or homologues or analogues thereof.

The term "chemokine" refers to a group of structurally related low-molecular weight factors secreted by cells having mitogenic, chemotactic or inflammatory activities. They are primarily cationic proteins of 70 to 100 amino acid residues that share four conserved cysteine residues. These proteins can be sorted into two groups based on the spacing of the two amino-terminal cysteines. In the first group, the two cysteines are separated by a single residue (C-x-C), while in the second group they are adjacent (C-C). Examples of member of the 'C-x-C chemokines include but are not limited to platelet factor 4 (PF4), platelet basic protein (PBP), interleukin-8 (IL-8), melanoma growth stimulatory activity protein (MGSA), macrophage inflammatory protein 2 (MIP-2), mouse Mig (m1 19), chicken 9E3 (or pCEF-4), pig alveolar macrophage chemotactic factors I and II (AMCF-I and -II), pre-B cell growth stimulating factor (PBSF).and IP10. Examples of members of the 'C-C group include but are not limited to monocyte chemotactic protein 1 (MCP-1 ), monocyte chemotactic protein 2 (MCP-2), monocyte chemotactic protein 3 (MCP-3), monocyte chemotactic protein 4 (MCP-4), macrophage inflammatory protein 1 a (MIP-1- a), macrophage inflammatory protein 1 β (MIP-1 -β), macrophage inflammatory protein 1-γ (MIP-1 -γ), macrophage inflammatory protein 3 a (MIP-3-a, macrophage inflammatory protein 3 β (MIP-3-β), chemokine (ELC), macrophage inflammatory protein-4 (MIP-4), macrophage inflammatory protein 5 (MIP-5), LD78 β, RANTES, SIS-epsilon (p500), thymus and activation-regulated chemokine (TARC), eotaxin, I-309, human protein HCC- 1/NCC-2, human protein HCC-3.

A number of growth factors have been identified which promote/activate endothelial cells to undergo angiogenesis. These include vascular endothelial growth factor (VEGF A), VEGF B, VEGF C, and VEGF D, transforming growth factor (TGFb), acidic and basic fibroblast growth factor (aFGF and bFGF), and platelet derived growth factor (PDGF).

VEGF is an endothelial cell-specific growth factor which has a very specific site of action, namely the promotion of endothelial cell proliferation, migration and differentiation. VEGF is a complex comprising two identical 23 kD polypeptides. VEGF can exist as four distinct polypeptides of different molecular weight, each being derived from an alternatively spliced mRNA. bFGF is a growth factor that functions to stimulate the proliferation of fibroblasts and endothelial cells. bFGF is a single polypeptide chain with a molecular weight of 16.5Kd. Several molecular forms of bFGF have been discovered which differ in the length at their amino terminal region. However the biological function of the various molecular forms appears to be the same.

Pro-drug activating polypeptides are also within the scope of the invention. The term pro- drug activating genes refers to nucleotide sequences, the expression of which, results in the production of proteins capable of converting a non-therapeutic compound into a therapeutic compound, which renders the cell susceptible to killing by external factors or causes a toxic condition in the cell. An example of a prodrug activating gene is the cytosine deaminase gene. Cytosine deaminase converts 5-fluorocytosine to 5 fluorouracil, a potent anti-tumour agent. The lysis of the tumour cell provides a localized burst of cytosine deaminase capable of converting 5FC to 5FU at the localized point of the tumour resulting in the killing of many surrounding tumour cells. Additionally, the thymidine kinase (TK) gene (see US5,631 ,236 and US5,601 ,818) in which the cells expressing the TK gene product become susceptible to selective killing by the administration of ganciclovir may be employed. Other examples of pro-drug activating enzymes are nitroreductase and cytochrome p450's (e.g. CYP1A2, CYP2E1 or CYP3A4).

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps. "Consisting essentially" means having the essential integers but including integers which do not materially affect the function of the essential integers.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

An embodiment of the invention will now be described by example only and with reference to the following figures:

Figure 1 summarizes the principles of the invention (the solution molecules and ions required to maintain an appropriate solution condition (e.g. neutral) have been omitted for clarity);

Figure 2 shows a view of an example of the present invention, a reduced graphene oxide (rGO) layered hydrogel construct of approximately 5 μΐη thickness obtained via peptide- directed self-assembly using the peptide FFGGEEEEEEGGFF [SEQ ID NO: 3] and the method described herein (see Methods for details);

Figure 3 shows a view of another example of the present invention, a reduced graphene oxide layered hydrogel construct of approximately 70 nm thickness obtained via peptide- directed self-assembly using the peptide FFGGEEEEEEGGFF [SEQ ID NO: 3] and supported on a glass slide (see Methods for details) that is sat on top of some written text on a piece of paper;

Figure 4 shows an SEM image of a freeze dried sample of the hydrogel shown in Figure 2 edge on (see Methods for details); Figure 5 (top) shows on an AFM image two independent paths A-B and C-D taken by the AFM probe for a sample of the material shown in Figure 2 (see Methods for details), and the AFM probe height along the two paths (middle and bottom) showing the distance between successive rGO sheets separated by the peptide FFGGEEEEEEGGFF [SEQ ID NO: 3] (i.e. the pore size) that has been designed to yield a pore size of 2.6 nm;

Figure 6 shows the variation with time of doxorubicin (DOX) anti-cancer drug uptake (see Methods for details) for different rGO hydrogels formed using the peptide FFGGEEEEEEGGFF [SEQ ID NO: 3] where the degree of reduction of the rGO is different (the take-up for the rGO hydrogel formed in the absence of the peptide is also shown for reference); Figure 7 shows the DOX release profile at different pH values (see Methods for details) for the optimal rGO hydrogel formed using the peptide FFGGEEEEEEGGFF [SEQ ID NO: 3];

Figure 8 shows the results of an MTT assay based analysis (see Methods for details) to assess the toxicity of different concentrations of the rGO hydrogel formed using the peptide FFGGEEEEEEGGFF [SEQ ID NO: 3];

Figure 9 shows the results of an MTT assay based analysis (see Methods for details) to assess the toxicity of DOX alone and DOX released from the hydrogel formed using the peptide FFGGEEEEEEGGFF [SEQ ID NO: 3]; and

Figure 10 shows the variation with time of uptake of three different sized dextran molecules (see Methods for details) for an rGO hydrogel formed using the peptide FFGGEEEEEEGGFF [SEQ ID NO: 3].

DETAILED DESCRIPTION OF THE INVENTION Methods Preparation of graphene oxide

Graphene oxide (GO) was prepared from natural or synthetic graphite according to the improved Hummer's method. ³⁷ Briefly, a 9:1 mixture of concentrated sulphuric acid and phosphoric acid (120:13 mL) was cooled overnight to 4 °C. The already cooled acid mixture was slowly added to the graphite powder (1 g) and potassium permanganate (6 g) under stirring at room temperature. Then the mixture was heated to 50 °C for 12 h to form a thick paste. The paste was then cooled to room temperature and then poured onto ice cubes (150 mL of Milli-Q water) with 30% hydrogen peroxide (1 mL). The mixture was then washed and filtered with distilled water and hydrochloric acid (32 %) followed by repeated washing with ethanol and eventually with Milli-Q water. For each successive wash the obtained brown dispersion was centrifuged at 4400 rpm for 2 h to remove residual salts and any un-exfoliated graphite oxide, which is usually present in a very small amount. The obtained GO was vacuum dried overnight at room temperature.

Preparation of reduced graphene oxide

One instance of reduced GO (rGO) was prepared from 25 imL of a homogeneous dispersion of GO (0.5 mg/ml). After adding to this solution in a volumetric flask 25 μΙ of hydrazine solution (35 wt% in water) and 75.0 μΙ of ammonia solution (28 wt% in water), the dispersion was vigorously stirred for a few minutes before being placed in a silicon oil bath (~95 °C) for 1 hr. Other instances of rGO with different levels of GO reduction from ~20 wt% oxygen down to ~8% wt% oxygen were obtained by varying the hydrazine volume between 25 and 75 μΙ and the reaction temperature between 85 and 95 °C.

Preparation of layered GO and rGO hydrogel

One instance of the layered rGO hydrogel material with a dominant inter-sheet spacing (i.e. pore size) of 2.6 nm was produced by adding 25 mL of rGO dispersion (0.5 mg/mL) to 25 mL of a solution of the peptide FFGGEEEEEEGGFF [SEQ ID NO: 3] (0.10 μΜ) and then stirring for 30 min before filtering it through a mixed cellulose ester filter membrane (47mm in diameter, 0.05 μιη pore size, Millipore) by vacuum filtration. The resultant hydrogel film was then washed three times and immediately transferred to a Petri dish and immersed in Milli-Q water overnight to remove the remaining unbound peptide.

Other instances of the rGO hydrogel material with a dominant pore size of 2.6 nm were obtained by using rGO dispersions with concentrations varying from 0.0025 mg/mL upwards in the aforementioned process. The thickness of the resultant hydrogel films varied from 10s of nm, which meant they needed to be supported; an as-received glass microscope slide was used for this purpose, but it is anticipated that other materials could also be used. The hydrogel films were transferred from the filter membrane to the glass slide support by tightly clamping the filter membrane supported hydrogel film between two glass slides for 12 h at room temperature before dissolving away the filter membrane with acetone.

GO-based variants of the hydrogel material were also similarly made, although the drainage time was greater for the same applied vacuum pressure due to the greater hydrophilicity of the GO. Characterization of the hydrogel material mesoscale morphology and pore size

The morphologies of the graphene hydrogel films prepared as described above were investigated using a scanning electron microscope (SEM; QUANTA 450). The hydrogels were cut using a razor blade then dried in a freeze dryer (188K at 10 ² Pa for 24hrs),and then mounted in a cross-sectional sample holder before being imaged in the SEM.

The spacing between the individual rGO or GO sheets (i.e. the pore size) was assessed by atomic force microscopy (AFM; NT-MDT Ntegra Solaris) in tapping mode. The AFM samples were prepared by drop-casting a diluted suspension of crushed hydrogel film onto a cleaned mica substrate. They were imaged immediately after preparation.

Characterization of molecular separation capability of hydrogel material

To demonstrate the capacity of the hydrogel films to separate molecules based on their size, it was exposed to solutions of FITC-labelled dextrans of three different diameters (they are generally claimed to be spherical in nature): FITC-dextran-4k (FD4, Sigma- Aldrich; mol. wt. 4000, Stokes radius -1.4 nm); FITC-dextran-10k (FD10, Sigma- Aldrich; mol. wt. 10,000, Stokes radius -2.3 nm); and FITC-dextran-20k (FD20, Sigma-Aldrich; mol. wt. 20,000, Stokes radius -3.3 nm). The experiments involved placing 20 mg of the hydrogel into three different 3 mL cuvettes containing 20 μιη solutions of FD4, FD10 and FD20 respectively and then monitoring in real time the concentration of the molecule in the solution using a UV-vis spectrometer (CHEMUSB4, Ocean Optics,) operating at 490 nm.

Characterization of drug loading into and release from the graphene hydrogels

The loading of doxorubicin (DOX) anti-cancer drug into the rGO hydrogels was assessed using a technique similar to that used to assess the filtration capability of the hydrogel. After adding 15 mg of the hydrogel to 3 mL of the DOX solution (50 g/mL) in a cuvette, the concentration of the latter was monitored in real time for 24 h by a UV-VIS spectrometer (USB4000-UV-VIS, Ocean Optics) operating at 490 nm. The variation of the drug loading in the hydrogel with time was estimated from this.

To characterise the release of DOX from a hydrogel, the DOX-loaded film was first removed from the drug loading cuvette and rinsed several times with deionized water to remove unbound drug and drug attached to the outer surface of the hydrogels. The film was then divided into three roughly equal parts before then being immersed in three separate cuvettes containing a 3 ml. aqueous PBS solution at 37 °C and pH 5.4, 7.4 and 9.4 respectively to mimic the release profile in physiological acidic, neutral and basic environments. The solutions in the cuvettes were constantly stirred whilst being maintained at 37 °C. At predetermined time intervals, 1 ml. of the solution from the cuvettes was withdrawn (with 1 mL of fresh PBS solution replacing it) to determine the DOX release using UV-Vis spectroscopy. Cell viability assessments

The toxicities of the graphene hydrogel, DOX and DOX-loaded hydrogel were assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium assay in a microplate reader. The step-by-step experimental details for each assessment are:

Cell culturing: Mesenchymal stem cells (MSC) were first seeded in 96-well microplate at a density of 5.0x 10 ⁴ cells/mL in 200 μΙ_ DMEM supplemented with 10% FBS, 100 U mL ¹ of penicillin, 100 mg mL ¹ of streptomycin and 2 mM L L glutamine for 24 hours and incubated at 37 °C in a 5% C0 ₂ humidified incubator.

Cell treatment with target to be assessed: Cells were then cultured in medium with the target to be assessed (hydrogel, DOX, DOX-loaded hydrogel) for 24 hours. A control containing only the cells and no hydrogel was also included. Three replicate wells were used per sample, including the control.

MTT assay: 10 μΙ_ of MTT (5 mg/ml in PBS) were added to each well, including both samples and controls, and then incubated for 4 h at 37 °C. All the liquid was then removed from wells, transferred into new microplate and 150 μΙ_ dimethyl sulfoxide (DMSO) was added to each well to ensure complete solubilization of formazan crystals.

Cell viability measurement: After 1 h further incubation, the absorbance was measured at 595 nm using a microplate reader (BioTek, USA). Cell viability was expressed as a percentage of the control cell culture value. Example 1

As illustrated in Figure 1 , the invention here centres on a peptide that is designed to self-assemble graphene sheets with a specific distance between the sheets, h, and which is composed of the following:

the end groups (at least two of) that non-covalently bind to the graphene;

the middle part (at least one of) that prefers to sit in the solution phase between the two graphene sheets (e.g. it is overall hydrophilic when the solution is aqueous); and

the flexible connection between the end groups and the middle (at least two of) that provide the right balance of flexibility to ensure the middle prefers to stay in the space between the layers and the end groups attached to the graphene

Example 2

Figure 2 shows an example of an instance of an rGO hydrogel dominated by pores of 2.6 nm width produced using 25 mL of a 0.5 mg/mL rGO dispersion and 25 ml. of a 0.10 μΜ solution of the peptide FFGGEEEEEEGGFF [SEQ ID NO: 3], which was designed to yield a pore size of 2.6 nm. The hydrogel film shown here is an approximately 5 μιη thick paper-like material that is flexible and non-brittle.

Example 3

Figure 3 shows an example of an instance of an rGO hydrogel dominated by pores of 2.6 nm width produced using 100 mL of 0.0025 mg/mL rGO dispersion and 100 mL of a 0.0005 μΜ solution of the peptide FFGGEEEEEEGGFF [SEQ ID NO: 3], which was designed to yield a pore size of 2.6 nm. The hydrogel film is approximately 70 nm thick and, thus, requires support on a surface (a microscope glass slide in this example). Example 4

The lamellar nature of the hydrogel material at the mesoscale is illustrated by the SEM image in Figure 4. Example 5

The pore size in the hydrogel made using the peptide FFGGEEEEEEGGFF [SEQ ID NO: 3] (i.e. that shown in Figures 2 and 3) is revealed in Figure 5, which shows that the AFM probe experiences vertical displacements of around 2.6 nm as it crosses the edges of the rGO sheets revealed when the hydrogel is fractured.

Example 6 Figure 6 shows that the amount of doxorubicin (DOX) anti-cancer drug that can be loaded into a hydrogel made using the peptide FFGGEEEEEEGGFF [SEQ ID NO: 3] is a strong function of the level of reduction of the rGO: whilst the highly reduced rGO-based hydrogel is marginally better than the mildly-reduced rGO, significantly higher loadings can be achieved by identifying the optimal degree of reduction. This figure also shows that the pores created by use of the peptide increases substantially the DOX capacity of the hydrogel.

Example 7 Figure 7 shows that after an initial low level burst effect (less than 10%), the DOX only continues to be released to any significant extent under acidic conditions akin to those typical of tumour sites. The release rate is sustained at an essentially constant value for around 3.5 days before release halts. Release beyond this point could possibly be enhanced by further optimisation of the hydrogel.

Example 8

The hydrogel by itself appears to be non-toxic as shown in Figure 8, which shows that cell viability over a 24 hour period was statistically invariant from the control (no hydrogel) for the case where 5, 10, 15, 20 and 25 mg of hydrogel were present.

Example 9

Figure 9 compares the cell toxicity of 5 μg of DOX in 24 hours provided via 100 μΙ_ of a 50 μg/mL DOX solution (DOX-only) and 15 mg of a hydrogel loaded with approximately 150 g of DOX that releases 5 μg in the 24 hours assessed (DOX-loaded hydrogel). This shows that the hydrogel-loaded DOX possesses a toxicity that is similar to that of DOX- only. As the hydrogel contains around 30 times this dose with approximately 60% of that being released based on the current realisation (see Figure 7), the hydrogel can deliver the required does in a sustained way for around 3.5 days without intervention. Longer periods could be achieved by increasing the mass of hydrogel or improving its formulation to ensure more than 60% of the DOX were released from the sample.

Example 10

The ability of the hydrogel to separate bio- and other larger molecules by size is illustrated in Figure 10, which shows the variation through time of the bulk phase concentrations of dextran molecules of three different sizes in solution with the hydrogel shown in Figure 2. As expected, the rate of uptake of FD4, which is around half the size of the pores in the hydrogel (1.4 nm vs. 2.6 nm), is taken up more quickly than that of FD10, which is only slightly smaller than the pore size (2.3 nm vs. 2.6 nm) - this demonstrates kinetics-based separation of bio- and other large molecules. The 3.3 nm size of the FD20 means it cannot enter the 2.6 nm pores that dominate the hydrogel's porosity - this demonstrates separation based on size exclusion.

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