HK1230093A1 - In vivo and in vitro use of graphene - Google Patents

Description

In vivo and in vitro uses of graphene

Cross Reference to Related Applications

This application claims the benefit of U.S. provisional application 61/951,926 filed on 12/3/2014, which is incorporated herein by reference in its entirety.

Background

The present disclosure relates generally to the transport and delivery of substances in biological environments, and more particularly, to methods and devices for the transport and delivery of substances using carbon nanomaterials.

The delivery of drugs and cells in immunocompetent and immunocompromised organisms is a real current problem in medical research and practice today. The present study used a polymeric device and hydrogel as the delivery vehicle. Some examples include polytetrafluoroethylene with a non-woven polyester mesh backing, silicone, hydrogels, alginates, cellulose sulfate, collagen, gelatin, agarose, chitosan, and the like. Current delivery vehicles and devices are challenged by biofouling, biocompatibility issues, and delayed response. The thickness of prior art devices may limit efficacy because the limited diffusion of nutrients may kill the cells contained therein, or delay the bidirectional transport of the drug or molecule being sensed. Low permeability can also be problematic, at least in part due to thickness and mechanical stability in view of physical and osmotic stress.

In view of the above, improved techniques for transporting and delivering substances under various conditions, particularly in a biological environment, would be of considerable benefit in the art. The present disclosure satisfies this need and provides related advantages as well.

Summary of The Invention

The present disclosure describes an enclosure formed from perforated graphene or other perforated two-dimensional material. The enclosure may contain a plurality of substances therein that allow selected substances to move bi-directionally back and forth within the enclosure, retaining other selected substances therein, and preventing other selected substances from entering the enclosure. The encapsulates of the invention may be employed to release one or more selected substances into an environment external to the encapsulate, to allow one or more selected substances to enter the encapsulate from an environment external to the encapsulate, to inhibit and preferably prevent one or more selected substances from entering the encapsulate from an external environment, to retain (inhibit or preferably prevent withdrawal of) one or more selected substances within the encapsulate, or a combination of these applications. The size or range of sizes of the pores or openings is selected based on the particular application of the encapsulant. The term "encapsulate" refers to a space for containing one or more substances, which is formed at least in part from a porous two-dimensional material, such as a graphene-based material, wherein the one or more substances in the encapsulate can exit the encapsulate through channels of the porous two-dimensional material. Likewise, in certain embodiments, one or more substances from the external environment may enter the enclosure through the channels of the porous two-dimensional material. In particular embodiments, the external environment is a biological environment, which may be an in vivo biological environment or an in vitro biological environment.

In embodiments, the enclosure comprises one or more sub-compartments, each sub-compartment comprising an apertured two-dimensional material such that a wall or side forming at least a portion of the sub-compartment is an apertured two-dimensional material. Fluid communication is achieved by the selective penetration and/or exit of one or more substances into and/or out of the enclosure or sub-compartments thereof. The fluid may be a liquid or a gas, and includes fluids with entrained gas. The substance may be dissolved or suspended, or otherwise carried in the fluid. The fluid may be aqueous. The sub-compartments may be in direct fluid communication with adjacent sub-compartments or the external environment (where adjacent sub-compartments share at least one wall or side). In embodiments, one or more sub-compartments may be in direct fluid communication with an adjacent sub-compartment, but not with the external environment. At least one sub-compartment in the enclosure is in direct fluid communication with the external environment. The enclosure may have sub-compartments of different configurations. The sub-compartments may have any shape. The sub-compartments may for example be spherical, cylindrical or rectilinear. In embodiments, the sub-compartments may be nested. In embodiments, the enclosure may have a central sub-compartment that shares a wall or side with a plurality of surrounding sub-compartments. In embodiments, the sub-compartments may be arranged linearly within the enclosure. In embodiments, the encapsulate contains two sub-compartments. In embodiments, the encapsulate contains three, four, five or six sub-compartments. In an embodiment, the sub-compartments may be completely contained within another sub-compartment, wherein the inner sub-compartment is in direct fluid communication with the outer sub-compartment and the outer sub-compartment is in direct fluid communication with the external environment. In this embodiment, the internal sub-compartments are in indirect rather than direct fluid communication with the external environment. In embodiments where the enclosure comprises a plurality of sub-compartments, at least one sub-compartment is in direct fluid communication with the external environment and the remaining sub-compartments are in direct fluid communication with adjacent sub-compartments, but not all sub-compartments are in direct fluid communication with the external environment. In embodiments where the enclosure contains multiple sub-compartments, all of the sub-compartments are in direct fluid communication with the external environment.

The encapsulant encapsulates at least one substance. In embodiments, the encapsulate may contain more than one different substance. The different substances may be in the same or different sub-compartments. In embodiments, the different substances in the encapsulate are not all released to the environment outside the encapsulate. In embodiments, all of the different substances in the encapsulate are released to the external environment. In embodiments, the release rates of the different substances from the encapsulate into the external environment are the same. In embodiments, the rate of release of different substances from the encapsulate into the external environment is different. In embodiments, the relative amounts of the different substances released from the encapsulate are the same or different. The rate of release of the substance from the encapsulate can be controlled by the selected pore size, functionalization of the pores, or both.

Also described herein are methods for transporting and delivering a substance in a biological environment. In some embodiments, the method may comprise: the method includes introducing an encapsulate formed from graphene or other two-dimensional material into a biological environment, and releasing at least a portion of a substance in the encapsulate to the biological environment. In some or other embodiments, the method may include introducing an encapsulate formed from graphene into a biological environment, and migrating a substance from the biological environment into the encapsulate.

In an embodiment, the present invention provides a method comprising the steps of:

introducing an enclosure comprising a porous two-dimensional material to an environment, the enclosure containing at least one substance; and

releasing at least a portion of the at least one substance through the pores of the two-dimensional material to an environment external to the enclosure. Any of the encapsulates herein may be employed in the method.

In an embodiment, the present invention provides a method comprising the steps of:

introducing an enclosure comprising a porous two-dimensional material to an environment, the enclosure containing at least one first substance; and migrating the second substance from the environment into the enclosure. In embodiments, the first substance is a cell, the second substance is a nutrient, and the other second substance is oxygen.

The foregoing has outlined rather broadly the features of the present disclosure in order that the detailed description that follows may be better understood. Additional features and advantages of the disclosure will be described hereinafter. These and other advantages and features will become more apparent from the following description.

Brief Description of Drawings

For a more complete understanding of the present disclosure and the advantages thereof, reference is now made to the following descriptions taken in conjunction with the accompanying drawings which describe specific embodiments of the present disclosure, in which:

figure 1 shows a schematic diagram demonstrating the thickness of graphene-based materials compared to common drug delivery vehicles and devices. This figure also illustrates an embodiment of the present invention in contact with biological tissue in a biological environment, wherein an enclosure is provided with one or more support materials that are further external (re external) to the porous two-dimensional material and indicate possible capillary vascularization into such support materials.

Fig. 2A-D show schematic diagrams of different configurations of an encapsulant configuration made from useful two-dimensional materials according to various embodiments of the present disclosure.

Figures 3A and 3B are schematic illustrations of an encapsulate embodying the invention for immunoisolating living cells.

Fig. 4A-C illustrate an exemplary preparation of an encapsulate of the present invention.

Detailed Description

The present disclosure relates in part to methods of transporting and delivering substances in biological environments using graphene-based materials and other two-dimensional materials. The present disclosure also relates in part to encapsulates formed from graphene-based and other two-dimensional materials on or suspended from a suitable substrate or substrates, which may be porous or non-porous, that can serve as delivery vehicles in the environment external to the encapsulate, particularly the biological environment. The present disclosure also relates in part to encapsulates containing cells, drugs, and other drugs formed from graphene-based or other two-dimensional materials.

Graphene has gained widespread interest for use in many applications due to its good mechanical and electronic properties. Graphene represents an atomically thin layer of carbon in which carbon atoms are present as closely spaced atoms at regular lattice positions. The regular lattice sites may have a plurality of defects present therein, which may be naturally present or intentionally introduced to the basal plane of the graphene. Such defects are also referred to herein equivalently as "openings," perforations, "or" holes. The term "graphene with pores" is used herein to indicate that a graphene sheet has defects in its basal plane, regardless of whether the defects are naturally occurring or intentionally created. In addition to such openings, graphene and other two-dimensional materials may represent impermeable layers for many substances. Thus, when sized appropriately, an opening in an impermeable layer of such materials can be used to access an enclosure formed by the impermeable layer.

The present disclosure contemplates a variety of graphene-based encapsulates capable of delivering a target to an in vivo or in vitro location in an organism or similar biological environment while maintaining a barrier (e.g., an immunoisolation barrier). Encapsulation of molecules or cells that are transported bilaterally across a semi-permeable membrane (e.g., porous graphene or other two-dimensional material), while insulating cells in a biological environment (e.g., in an organism), etc., can enable the treatment to overcome graft rejection, repeated dosage requirements for drugs, and excessive surgical intervention. The above may be achieved by: techniques are provided that allow for xenogenic and allogeneic tissue transplants, long-term low dose therapeutic levels of drugs, and even sensory response paradigms to address the nutrients behind surgical intervention, thereby reducing complications from multiple surgical procedures at the same site. It should be recognized that the foregoing is merely representative of particular advantages of the present disclosure and should not be taken as limiting the scope of the embodiments described herein.

The present inventors have recognized that perforated graphene and other two-dimensional materials can readily facilitate the aforementioned performance while being superior to current delivery vehicles and devices, particularly immunoisolation devices. Graphene can accomplish this due to its unique thinness, strength, electrical conductivity (for potential electrical stimulation), and permeability in the form of perforations therein. The subsequent sieve-like transport properties, thin and transverse to the graphene membrane surface, can allow for a subversive time response compared to the long diffusion seen with thicker polymer membranes with comparable size properties.

Two-dimensional materials are most often atomically thin materials, ranging from a monolayer sub-nanometer thick to a few nanometers thick, and they typically have a high surface area. Two-Dimensional Materials include metal chalcogenides (e.g., transition metal dichalcogenides), transition metal oxides, hexagonal boron nitride, Graphene, silicone, and germane (see: Xu et al (2013) "Graphene-like Two-Dimensional Materials" Chemical Reviews 113: 3766-) -3798). Graphene represents a form of carbon in which carbon atoms are present in a single atomically thin layer or in several thin layers (e.g., about 20 layers or less) that form a fused six-membered ring that extends an sp 2-hybridized carbon planar lattice. Among its various forms, graphene has gained widespread interest for use in many applications, primarily due to its high electrical and thermal conductivity values, good in-plane mechanical strength, and unique advantageous combinations of optical and electrical properties. Other two-dimensional materials having a thickness of a few nanometers or less and an extended planar lattice have also gained interest for a variety of applications. In an embodiment, the two-dimensional material has a thickness of 0.3nm to 1.2 nm. In other embodiments, the two-dimensional material has a thickness of 0.3nm to 3 nm.

In various embodiments, the two-dimensional material comprises a thin layer of graphene-based material. In embodiments, the thin layer of graphene-based material is a thin layer of single-or multi-layer graphene or a thin layer comprising a plurality of interconnected single-or multi-layer graphene domains. In embodiments, the multi-layered graphene domains have 2 to 5 layers, or 2 to 10 layers. In an embodiment, the layer comprising the thin layer of graphene-based material further comprises a non-graphene carbon-based material located on a surface of the thin layer of graphene-based material. In embodiments, the amount of non-graphenic carbon-based material is less than the amount of graphene. In embodiments, the amount of graphene in the graphene-based material is from 60% to 95%, or from 75% to 100%.

In embodiments, the characteristic dimension of the perforations is 0.3nm to 10nm, 1nm to 10nm, 5nm to 20nm, 10nm to 50nm, 50nm to 100nm, 50nm to 150nm, 100nm to 200nm, or 100nm to 500 nm. In embodiments, the average pore size is within a specified range. In embodiments, 70% to 99%, 80% to 99%, 85% to 99%, or 90% to 99% of the perforations in a lamina or layer fall within the specified ranges, but other apertures fall outside the specified ranges.

The techniques used to form the graphene or graphene-based materials in the embodiments described herein are not considered to be particularly limited. For example, in some embodiments, CVD graphene or graphene-based materials may be used. In various embodiments, CVD graphene or graphene-based materials may be released from the substrate (e.g., Cu) on which they are grown and transferred to a polymer backing. Likewise, the techniques for introducing perforations into graphene or graphene-based materials should not be considered to be particularly limited, except that the technique is selected to produce perforations within a desired size range. Determining a suitable size of the perforations as described herein provides a desired selective permeability of a substance (atom, molecule, protein, virus, cell, etc.) for a given application. Selective permeability refers to the tendency of a porous material or a porous two-dimensional material to allow one or more substances to pass through (or be transported) more easily or more quickly than other substances. Selective permeability allows for the separation of substances that exhibit different passage or transport rates. In two-dimensional materials, the selective permeability is related to the size or dimension (e.g., diameter) of the opening and the relative effective size of the substance. The selective permeability of the perforations in two-dimensional materials, such as graphene-based materials, may also depend on the functionalization of the perforations (if any) and the specific species to be separated. The separation of two or more substances in a mixture includes the change in the ratio (weight ratio or molar ratio) of the two or more substances in the mixture after the mixture has passed through the porous two-dimensional material.

Graphene-based materials include, but are not limited to: single layer graphene, multilayer graphene, or interconnected single or multilayer graphene domains, and combinations thereof. In embodiments, the graphene-based material further includes a material formed by stacking single or multiple graphene thin layers. In embodiments, the multi-layer graphene includes 2 to 20 layers, 2 to 10 layers, or 2 to 5 layers. In embodiments, graphene is the predominant material in the graphene-based material. For example, the graphene-based material comprises at least 30% graphene, or at least 40% graphene, or at least 50% graphene, or at least 60% graphene, or at least 70% graphene, or at least 80% graphene, or at least 90% graphene, or at least 95% graphene. In embodiments, the graphene-based material comprises graphene selected from the following ranges: 30% to 95%, 40% to 80%, 50% to 70%, 60% to 95%, or 75% to 100%.

As used herein, "crystalline domain" refers to a region of a material in which atoms are uniformly ordered into a crystal lattice. The domains are uniform within their boundaries, but are different from adjacent regions. For example, ordered atoms of a single crystalline material have a single crystalline domain. In embodiments, at least some of the graphene domains are nanocrystals having a domain size of 1 to 100nm, or 10-100 nm. In embodiments, at least some of the graphene domains have a domain size of greater than 100nm to 1 μ ι η, or 200nm to 800nm, or 300nm to 500 nm. The "grain boundaries" formed by the crystalline defects at the edges of each domain distinguish adjacent crystal lattices. In some embodiments, the first lattice may be rotated relative to the second lattice by rotation about an axis perpendicular to the plane of the lamellae, such that the two lattices differ in "lattice orientation".

In embodiments, the thin layers of graphene-based material comprise single or multiple graphene thin layers, or a combination thereof. In embodiments, the thin layers of graphene-based material are single or multi-layer graphene thin layers, or a combination thereof. In another embodiment, the thin layer of graphene-based material is a thin layer comprising a plurality of interconnected single-or multi-layer graphene crystalline domains. In embodiments, interconnected domains are covalently bonded together to form a thin layer. Thin layers are polycrystalline when the domains in the thin layer differ in lattice orientation.

In embodiments, the thin layer thickness of the graphene-based material is 0.34 to 10nm, 0.34 to 5nm, or 0.34 to 3 nm. In embodiments, the thin layer of graphene-based material includes intrinsic defects. In contrast to the selective introduction of graphene-based material lamellae or perforations of graphene lamellae, inherent drawbacks result from the preparation of graphene-based materials. Such inherent deficiencies include, but are not limited to: lattice anomalies, pinholes, tears, cracks or wrinkles. Lattice anomalies may include, but are not limited to: carbocyclic rings having other than 6-membered rings (e.g., 5-, 7-, or 9-membered rings), vacancies, interstitial defects (which include the incorporation of non-carbon atoms in the crystal lattice), and grain boundaries.

In embodiments, the layer comprising the thin layer of graphene-based material further comprises a non-graphene carbon-based material located on a surface of the thin layer of graphene-based material. In embodiments, the non-graphene carbon-based material does not have long-range order and may be classified as amorphous. In embodiments, the non-graphene carbon-based material further comprises an element other than carbon and/or a hydrocarbon. Non-carbon elements that may be incorporated into the non-graphenic carbon include, but are not limited to: hydrogen, oxygen, silicon, copper and iron. In embodiments, the non-graphene carbon-based material comprises a hydrocarbon. In embodiments, the carbon is the predominant material in the carbon-based material that is not graphene. For example, the non-graphene carbon-based material comprises at least 30% carbon, or at least 40% carbon, or at least 50% carbon, or at least 60% carbon, or at least 70% carbon, or at least 80% carbon, or at least 90% carbon, or at least 95% carbon. In embodiments, the non-graphene carbon-based material comprises a range of carbons selected from: 30% to 95%, or 40% to 80%, or 50% to 70%.

Such nanomaterials, in which pores are intentionally created, are referred to herein as "porous graphene", "porous graphene-based materials" or "porous two-dimensional materials". The present disclosure also relates in part to perforated graphene, perforated graphene-based materials, and other perforated two-dimensional materials containing a plurality of perforations of a size (or size range) suitable for a given encapsulant application. The pore size distribution may be narrow, for example, limited to a size deviation of 1-10%, or a size deviation of 1-20%. In an embodiment, the reference size of the hole is selected for the application. For a circular hole, the reference dimension is the diameter of the hole. In embodiments related to non-circular apertures, the reference dimension may be considered as the maximum distance across the aperture, the minimum distance across the aperture, the average of the maximum and minimum distances across the aperture, or an equivalent diameter based on the in-plane area of the aperture. As used herein, a graphene-based material having pores includes a material in which non-carbon atoms are incorporated at the edges of the pores.

In various embodiments, the two-dimensional material comprises graphene, molybdenum sulfide, or boron nitride. In a more specific embodiment, the two-dimensional material may be graphene. The graphene according to embodiments of the present disclosure may include single-layer graphene, multi-layer graphene, or any combination thereof. Other nanomaterials with extended two-dimensional molecular structures may also constitute two-dimensional materials in various embodiments of the present disclosure. For example, molybdenum sulfide is a representative chalcogenide with a two-dimensional molecular structure, and other multiple chalcogenides may constitute two-dimensional materials in embodiments of the present disclosure. The selection of a suitable two-dimensional material for a particular application may be determined by a variety of factors, including the chemical or physical environment in which the graphene or other two-dimensional material is ultimately to be deployed. For use in the present invention, the materials employed in preparing the encapsulate are preferably biocompatible or can be made biocompatible.

The process of forming pores in graphene and other two-dimensional materials is referred to herein as "perforation", and such nanomaterials are referred to herein as "porous". In graphene thin layers, interstitial openings are formed by the structure of each six carbon atom ring in the thin layer and have a span of less than one nanometer. Specifically, the gap opening is considered to be about 0.3 nanometers across its longest dimension (the center-to-center distance between carbon atoms is about 0.28nm, and the opening is slightly smaller than this distance). Perforation of a sheet comprising a two-dimensional network generally means the formation of pores in the network that are larger than the interstitial openings.

Due to the atomic level thinness of graphene and other two-dimensional materials, it is possible to achieve high liquid flux rates during separation or filtration processes, even with pores in the 1-20nm range.

Chemical techniques can be used to create pores in graphene and other two-dimensional materials. Exposure of graphene or another two-dimensional material to ozone or atmospheric pressure plasma (e.g., oxygen/argon, or nitrogen/argon plasma) can affect perforation. Physical techniques such as ion bombardment can also be used to remove material from a planar structure of two-dimensional material in order to create pores. All such physical or chemical methods are applicable to the preparation of the porous two-dimensional materials used herein, depending on the pore size or range of pore sizes desired for a given application.

In various embodiments of the present disclosure, the size of the pores produced in graphene or other two-dimensional materials may range from about 0.3nm to about 50 nm. In a more specific embodiment, the pore size may range from 1nm to 50 nm. In a more specific embodiment, the pore size may range from 1nm to 10 nm. In a more specific embodiment, the pore size may range from 5nm to 10 nm. In a more specific embodiment, the pore size may range from 1nm to 5 nm. In a more specific embodiment, the pore size ranges from about 0.5nm to about 2.5 nm. In further embodiments, the pore size is from 0.3nm to 0.5 nm. In other embodiments, the pore size is 0.5nm to 10 nm. In a further embodiment, the size of the pores is from 5nm to 20 nm. In other embodiments, the pore size is 0.7nm to 1.2 nm. In further embodiments, the pore size is from 10nm to 50 nm. In embodiments where larger pore sizes are preferred, the pore size is from 50nm to 100nm, from 50nm to 150nm, or from 100nm to 200 nm.

The term "substance" is used generically herein to refer to atoms, molecules, viruses, cells, particles, and aggregates thereof. Substances of particular interest are molecules of different sizes, including biomolecules, such as proteins and nucleic acids. Substances may include drugs, pharmaceuticals, medicaments, and therapeutics, including biologies and small molecule drugs.

Figure 1 shows a schematic diagram demonstrating the thickness of graphene compared to common drug delivery vehicles and devices. The biocompatibility of graphene can also facilitate this application, in particular, by functionalizing graphene to be compatible with a particular biological environment (e.g., via available edge bonding, global surface functionalization, pi-bonding, etc.). Functionalization can provide membranes of increased complexity for the treatment of local and systemic diseases. FIG. 1 illustrates the walls of an enclosure formed with a porous two-dimensional material having a pore size in the range of 400-700nm, which will entrap viable cells. The external biological environment adjacent to the enclosure (complete enclosure not shown) is exemplified by an optional porous support structure (polymer or ceramic) adjacent to and external to the porous two-dimensional material and an optional textile support material external to the porous two-dimensional material. As shown, implantation of such an encapsulation allows for vascularization into any such external support material. In embodiments intended to provide immunoisolation, a smaller pore size is generally preferred to prevent the antibody from entering the encapsulate.

In various embodiments, the present disclosure describes sealed enclosures formed primarily from two-dimensional materials, such as graphene, that retain the ability to transport materials bi-directionally. In various embodiments, at least one section or face of the enclosure contains perforations of appropriate size in the two-dimensional material to allow the desired size of material to pass in and out, respectively, from the interior of the enclosure.

In some embodiments, a two-dimensional material such as graphene may be adhered to a suitable porous substrate. Suitable porous substrates may include, for example, thin film polymers and ceramics.

In embodiments, the enclosure may have a plurality of sub-compartments within the main enclosure, each sub-compartment comprising an apertured two-dimensional material to allow passage of one or more substances into or out of the sub-compartment. In such embodiments, the sub-compartments may have any useful shape or size. In specific embodiments, there are 2 or 3 sub-compartments. Several examples of encapsulant sub-compartments are illustrated in fig. 2A-2D. In fig. 2A, illustrating a nested configuration, the primary enclosure B contains entirely smaller enclosures a, such that the substance in the centermost enclosure a can enter the primary enclosure B and potentially react with or within the primary compartment during ingress and egress therefrom. In this embodiment, one or more of the substances in a may enter B, and one or more of the substances in a may be retained in a and not enter B. Two sub-compartments, in which one or more substances may pass directly between the sub-compartments, are in direct fluid communication. The passages between the sub-compartments and between the enclosure and the external environment are passages through pores of a perforated two-dimensional material. The barrier (membrane, i.e. perforated two-dimensional material) between compartments a and B may be permeable to all substances in a or selectively permeable to some substances in a. The barrier (membrane) between B and the external environment may be permeable to all substances in B or selectively permeable to some substances in B. In fig. 2A, sub-compartment a is in direct fluid communication with sub-compartment B, which in turn is in direct fluid communication with the external environment. Compartment a in this nested configuration can only be in indirect fluid communication with the external environment via an intermediate passage into sub-compartment B. The two-dimensional materials employed in the different sub-compartments of a given encapsulate may be the same or different materials, and the size of the perforations and apertures in the two-dimensional materials of the different sub-compartments may be the same or different, depending on the substance and application involved.

In fig. 2B, the enclosure is bisected by an impermeable wall (e.g., formed of a non-porous or impermeable sealant) to form sub-compartments a and B, with the two portions independently entering the exit location, but with no direct or indirect passage of the substance from a to B (however, it is understood that the substance exiting a or B may indirectly enter the other sub-compartment via the external environment).

In fig. 2C, the main enclosure is also divided into two sub-compartments a and B, but the barriers between the sub-compartments are formed by a perforated material. In an embodiment, the two sub-compartments not only enter the outlet position independently, but may also interact with each other, i.e. the sub-compartments are in direct fluid communication. In embodiments, the barrier (membrane) between compartments a and B is selectively permeable, e.g. it allows at least one substance in a to enter B, but does not allow substances originating in B to pass through to a.

Figure 2D illustrates an enclosure having three compartments. The enclosure is exemplified with sub-compartment a having an outlet into sub-compartment B, which in turn has an outlet to sub-compartment C, which in turn has an outlet into the outside environment. Compartments a and B do not have an outlet to the external environment, i.e. they are not in direct fluid communication with the external environment. Adjacent sub-compartments a and B and adjacent sub-compartments B and C are each separated by an apertured two-dimensional material and are thus in direct fluid communication with each other. The sub-compartment a can only be in indirect fluid communication with the compartment C and the external environment via sub-compartment B, or B and C, respectively. Various other combinations of semi-permeable barriers (membranes) or impermeable barriers may be employed to separate the compartments in the enclosures herein. Different perforation size limitations may vary depending on how the envelopes are ultimately shaped (e.g., whether one envelope is inside the other, relative to a side-by-side format). Regardless of the configuration chosen, the boundary of the encapsulate, or at least a portion thereof, may be constructed from a two-dimensional material in order to achieve its benefits, particularly with the thickness of the active membrane being less than the diameter of the target to be selectively passed through the membrane. In some embodiments, the pore size of the two-dimensional material may range in size from about 0.3nm to about 10 nm. Larger pore sizes are also possible.

It should also be noted that in some embodiments, the enclosure may be supported by one or more support structures. In embodiments, the support structure itself may have a porous structure, wherein the pores are larger than the pores of the two-dimensional material. In embodiments, the support structure is completely porous. In embodiments, the support structure is at least partially non-porous.

The various physical embodiments of the encapsulates and their uses described herein may allow for different levels of interaction and proportional complexity of the problem to be solved. For example, a single encapsulate may provide drug elution for a given period of time, or there may be perforations of multiple sizes to limit or allow movement of different targets, each of a particular size.

The increased complexity of the embodiments described herein having multiple sub-compartments may allow interactions between target compounds to catalyze or activate secondary responses (i.e., the "perceptual response" paradigm). For example, if there are two portions of the encapsulate that enter the outlet independently, exemplary compound a may undergo constant diffusion into the body, or after a period of time or only in the presence of a stimulus from the body. In such embodiments, exemplary compound a may activate exemplary compound B or inactivate its function and block exemplary compound B to prevent extravasation. The combination that produces the above effect may be reversible or irreversible. Furthermore, in other embodiments, exemplary compound a may interact with a chemical cascade produced outside the encapsulation, and the metabolite following the interaction may release exemplary compound B (by inactivating the functionalization). Other examples of producing effects in a similar manner, including the use of source cells (non-host, allogeneic) contained in an encapsulate within which secretions from the cells can produce a "sensory response" paradigm.

In other embodiments, growth factors may be loaded in the encapsulant to promote angiogenesis (see fig. 1). In the foregoing embodiments, cell survival may be far superior to the results of bidirectional transport of nutrients and waste products.

In other embodiments, the relative thinness of graphene may enable bidirectional transport across membrane envelopes in close proximity to blood vessels (particularly capillaries) and other target cells. The present embodiment utilizing a graphene-based encapsulant can provide a distinction from other solutions that achieve the same effect, as the graphene membrane does not significantly limit permeability. In contrast, diffusion of molecules through the medium or gap junctions can limit the movement of the target.

With respect to the foregoing, any "perceptual response" paradigm is made possible with graphene through excellent temporal response. The biocompatibility of graphene can also enhance this application, in the treatment of local and systemic diseases, to functionalized graphene membranes of increasing complexity, with expected lower levels of biofouling (due to functionalization or charging). Furthermore, the mechanical stability of graphene may make it suitable to withstand physical and osmotic stresses in vivo.

Figures 3A and 3B provide schematic illustrations of immunoisolation of the encapsulates of the invention. The enclosure is illustrated as having a single compartment. It is to be understood that the enclosure may have a plurality of sub-compartments, for example, two or three sub-compartments. The enclosure (30) of fig. 3A is shown in cross-section formed from an inner sheet or layer (31) comprising a porous two-dimensional material, such as a graphene-based material, and an outer sheet or layer (32) of a support material. The support material may be porous, selectively permeable, or non-porous and impermeable. However, at least a portion of the support material is porous or selectively permeable, suitable for the application of the encapsulate. The support sheet or layer may be, for example, a polymer or ceramic. The encapsulate contains selected living cells (33) for a given use. Fig. 3B provides an alternative cross-section of the enclosure of fig. 3A, showing a space or cavity formed between the first and second composite layers (32/31), wherein the sealant 34 is illustrated as sealing the edges of the composite layers. It will be appreciated that the seal at the edge of the composite layer may be formed using a physical method of clamping or crimping. The method and materials used to form the edge seal are not particularly limited, but a non-porous and impermeable seal or closure must be provided.

If cells are contained within the enclosure, at least a portion of the enclosure may be permeable to oxygen and nutrients sufficient for cell growth and maintenance, and may be permeable to waste products. The encapsulant is impermeable to cells, particularly immune cells. Cells from the external environment cannot enter the encapsulate and the cells in the encapsulate are retained. The encapsulant is impermeable to viruses or bacteria. The encapsulant is impermeable to the antibody. In contrast, depending on the application, the encapsulate may be permeable to the desired product, such as growth factors produced by the cell. The cells within the encapsulation are immunoisolated. In a specific embodiment, the pore size in the porous two-dimensional material for immunoisolation ranges from 1 to 10nm, more preferably from 3 to 10nm, and still more preferably from 3 to 5 nm.

Fig. 4A-4C illustrate exemplary methods for forming the encapsulates of the invention and introducing selected substances (e.g., cells) therein. The method is exemplified in connection with the use of a sealant to form the encapsulant. The exemplary encapsulate has no sub-compartments. An enclosure having sub-compartments (e.g., nested or adjacent sub-compartments) can be readily prepared using the exemplary methods. As illustrated in fig. 4A, the first composite layer or thin layer is formed by laying down a thin layer or layer of two-dimensional material, in particular a thin layer of graphene-based material or a thin layer (41) of graphene, in contact with a support layer (42). At least a portion of the support layer (42) of the first composite is porous or permeable. The pore size of the support layer is typically larger than the pores or openings in the two-dimensional material used and can be adjusted to the environment (e.g., a body cavity). A layer (44) of sealant (e.g., silicone) is applied over the thin layer or layers of perforated two-dimensional material outlining the compartments of the enclosure, wherein the sealant will form an impermeable seal around the perimeter of the enclosure. The formation of a single compartment is illustrated in fig. 4A-4C, however, it is understood that multiple independent compartments within an enclosure may be formed by similar methods. A thin layer or layer of apertured two-dimensional material is then contacted with the sealant to prepare a second composite layer in the same manner as the first composite layer and to position it (alternatively, the sealant may be applied to a portion of the composite layer and the layer may be folded up in contact with the sealant to form an enclosure). A seal is then formed between the two composite layers. Appropriate pressure can be applied to facilitate sealing without damaging the two-dimensional material or its supporting layer. It will be appreciated that an alternative enclosure may be formed by applying a thin layer or layer of non-porous and impermeable support material in contact with the sealant. In this case, only a portion of the enclosure is porous and permeable. The sealed composite layer is illustrated in fig. 4B, which shows that the sealing layer can be trimmed to size around the sealant to form an encapsulant. The resulting enclosure is shown with an outer porous support layer 42, a thin layer or layer of perforated two-dimensional material (41) placed as an inner layer, and a sealant 44 around the perimeter of the enclosure. As illustrated in fig. 4C, injection through the sealant layer after the encapsulant is formed may introduce cells or other substances into the encapsulant that are excluded by the channels of the perforated two-dimensional thin layer or layers. Any perforations formed by such injection may be sealed, if desired. It will be appreciated that the substance and cells may be introduced into the encapsulant prior to forming the seal. Those skilled in the art will appreciate that contemplated sterilization methods suitable for this application may be employed during or after preparation of the encapsulate.

In embodiments, the present invention provides an encapsulate of a porous two-dimensional material encapsulating a substance such that the substance is released to the environment outside the encapsulate through the passage of pores in the porous two-dimensional material. In embodiments, the encapsulant encapsulates more than one different substance. In embodiments, the different substances are not all released to the environment outside the enclosure. In embodiments, all of the different substances are released into the environment outside the enclosure. In embodiments, different substances are released into the environment outside the enclosure at different rates. In embodiments, different substances are released into the environment outside the enclosure at the same rate.

In an embodiment, the enclosure comprises two or more sub-compartments, wherein at least one sub-compartment is in direct fluid communication with the environment outside the enclosure through an aperture in the two-dimensional material of the sub-compartment. In an embodiment, each sub-compartment comprises an apertured two-dimensional material, and each sub-compartment is in direct fluid communication with the environment outside the enclosure through an aperture in the two-dimensional material of each sub-compartment.

In an embodiment, the enclosure is subdivided into two sub-compartments, which are separated from each other at least in part by a perforated two-dimensional material, such that the two sub-compartments are in direct fluid communication with each other through the perforations in the two-dimensional material. In an embodiment, the enclosure is subdivided into two sub-compartments each comprising a two-dimensional material, the sub-compartments being in direct fluid communication with each other through apertures in the two-dimensional material, and only one sub-compartment being in direct fluid communication with the environment outside the enclosure. In an embodiment, the enclosure is subdivided into two sub-compartments each comprising a two-dimensional material, the sub-compartments being in direct fluid communication with each other through pores in the two-dimensional material, and both sub-compartments also being in direct fluid communication with the environment outside the enclosure.

In an embodiment, the enclosure has an inner sub-compartment and an outer sub-compartment each comprising an apertured two-dimensional material, wherein the inner sub-compartment is completely enclosed within the outer sub-compartment, the inner and outer sub-compartments are in direct fluid communication with each other through the apertures in the two-dimensional material, and the inner sub-compartment is not in direct fluid communication with the environment outside the enclosure.

In embodiments wherein the enclosure has a plurality of sub-compartments each comprising a two-dimensional material, the sub-compartments are nested sub-compartments within one another, the sub-compartments each being in direct fluid communication with its adjacent sub-compartments through apertures in the two-dimensional material, the outermost sub-compartment being in direct fluid communication with the environment external to the enclosure, the remaining plurality of sub-compartments not being in direct fluid communication with the environment external to the enclosure.

In embodiments wherein the enclosure is subdivided into a plurality of sub-compartments each comprising two-dimensional material, each sub-compartment is in direct fluid communication with one or more adjacent sub-compartments, and only one sub-compartment is in direct fluid communication with the environment outside the enclosure.

In any of the encapsulate-configured embodiments herein, the at least one substance within the encapsulate that is released to the environment outside the encapsulate through the pores in the two-dimensional material is a drug, therapeutic agent, or medicament. In embodiments, wherein the substance released is a drug, therapeutic agent or drug, the two-dimensional material of the encapsulate for releasing the substance comprises pores ranging in size from 1-50 nm. In embodiments where the substance to be released is a drug, therapeutic agent or drug, the two-dimensional material of the encapsulate for releasing the substance comprises pores ranging in size from 1-10 nm.

In any of the encapsulate embodiments herein, the substance within the encapsulate is a cell, and the size of the pores in the two-dimensional material is selected to retain the cell within the encapsulate and to exclude immune cells and antibodies from the environment external to the encapsulate from entering the encapsulate. In particular embodiments, for use with a cell, the encapsulate is divided into a plurality of sub-compartments, and one or more of the sub-compartments contain the cell. The encapsulates may contain different cells within one sub-compartment or different cells within different sub-compartments of the same encapsulate. In a specific embodiment, for use of a cell, the encapsulate is a nested encapsulate in which the cell is within an internal sub-compartment.

In an embodiment, the enclosure has an inner sub-compartment and an outer sub-compartment each comprising an apertured two-dimensional material, wherein the inner sub-compartment is completely enclosed within the outer sub-compartment, the inner and outer sub-compartments are in direct fluid communication through the apertures in the two-dimensional material of the inner sub-compartment, the inner sub-compartment is not in direct fluid communication with the environment external to the enclosure, and the outer sub-compartment is in direct fluid communication with the environment external to the enclosure.

In embodiments, for use with a cell, the encapsulate has a plurality of sub-compartments each comprising a porous two-dimensional material and each in direct fluid communication with one or more adjacent sub-compartments, the cell being within one or more cell-containing sub-compartments each not in direct fluid communication with the environment external to the encapsulate.

In embodiments of the cell-containing encapsulate, the cell is a yeast cell or a bacterial cell. In embodiments of the encapsulate comprising a cell, the cell is a mammalian cell. In embodiments of the cell-containing encapsulate, the pores in the two-dimensional material of the encapsulate or sub-compartment range in size from 1-10nm, from 3-10nm, or from 3-5 nm.

In any of the encapsulate embodiments herein, the two-dimensional material in the encapsulate is supported on a porous substrate. In embodiments, the porous substrate may be a polymer or a ceramic.

In an embodiment of any of the encapsulates herein, the two-dimensional material is a graphene-based material. In an embodiment of any of the encapsulates herein, the two-dimensional material is graphene.

In an embodiment of any of the encapsulates herein, at least a portion of the pores in the two-dimensional material of the encapsulate are functionalized.

In embodiments of any of the encapsulates herein, at least a portion of the two-dimensional material is electrically conductive, and a voltage may be applied to at least a portion of the electrically conductive two-dimensional material. The voltage may be an AC or DC voltage. The voltage may be applied from a power source external to the enclosure. In embodiments, the enclosure device of the present invention further comprises a connector and directs the application of a voltage to the two-dimensional material from an external power source.

The present invention provides methods of using any of the encapsulates herein in a selected environment for delivery of one or more substances to the environment. In a particular embodiment, the environment is a biological environment. In embodiments, the encapsulate is implanted into a biological tissue. In embodiments, the encapsulate is employed for delivery of a drug, drug or therapeutic agent.

In embodiments, the present invention provides methods comprising: introducing an enclosure comprising a porous two-dimensional material to an environment, the enclosure containing at least one substance; and releasing at least a portion of the at least one substance to an environment external to the enclosure through the pores of the two-dimensional material. In embodiments, the encapsulate contains cells that are not released from the encapsulate, and a portion of the at least one substance released is a substance produced by the cells in the encapsulate.

In embodiments, the present invention provides methods comprising: introducing an enclosure comprising a porous two-dimensional material to an environment, the enclosure containing at least one first substance; and migrating a second substance from the environment into the enclosure. In embodiments, the first substance is a cell, the second substance is a nutrient, and the other second substance is oxygen.

In embodiments, the support layer may be a polymer or a ceramic material. Useful exemplary ceramics include nanoporous silica or SiN. Useful porous polymeric supports include track etched polymers, foamed polymers or non-woven polymers. The support material may be porous or permeable. A portion of the enclosure or sub-compartment, e.g., a wall, side, or portion thereof, may be a non-porous polymer or ceramic. Biocompatible polymers and ceramics are preferred. A portion of the enclosure may be formed from an encapsulant such as silicone, epoxy, polyurethane, or similar material. Biocompatible sealants are preferred.

In addition, the conductivity of graphene-based films, or films of other two-dimensional materials, may allow charging to occur from an external power source. In an exemplary embodiment, an AC or DC voltage may be applied to the conductive two-dimensional material of the encapsulant. The conductive properties of graphene may provide additional gating (gating) to charged molecules. The charging may occur permanently or only for a while to affect gating. The directed gating of charged molecules can be directed not only through the pores (or restricted through the pores), but also to the surface of the graphene to adsorb or bind and promote growth, promote the formation of protective layers, or provide a basis or mechanism for other biochemical effects on the body.

In such embodiments, both permanent and temporary bonding to graphene is possible. In addition to the foregoing advantages, the embodiments described herein may also have the advantage that they not only represent a subversive technology for prior art vehicles and other devices, but they also allow for the use of these vehicles and devices in new ways. For example, cell line development, therapeutic release agents, sensing paradigms (e.g., MRSw, NMR-based magnetic relaxation switching technology, see: Kohet al (2008) ang. chem. int' l Ed. Engl,47(22)4119-4121) can be used within the encapsulants described herein for reducing biofouling and oxidative damage (bioactivity), delivering superior permeability with less response delay, and providing mechanical stability. That is, the encapsulates described herein may allow existing technologies to be implemented in new ways that are not currently possible.

In addition to the in vivo and in vitro uses described above, the embodiments described herein may be utilized in other fields. The encapsulates described herein may also be used in non-therapeutic applications, for example, dosages of probiotics in dairy products (as opposed to currently used microencapsulation techniques that increase the viability of delivery to the gastrointestinal tract during processing). In this and other respects, it should be noted that the encapsulants and devices described herein formed thereby can span several orders of magnitude depending on manufacturing techniques and different target use requirements. However, it is believed that the envelope can be made small enough to circulate through the bloodstream. Alternatively, the encapsulation can be made large enough to implant (on the order of a few inches or more). These characteristics can result from the two-dimensional nature of graphene and its growth over a large surface area.

While the present disclosure has been described with reference to the disclosed embodiments, those skilled in the art will readily appreciate that these are only examples of the present disclosure. It will be understood that various modifications may be made without departing from the spirit of the disclosure. The disclosure can be modified to incorporate any number of variations, alterations, substitutions or equivalent arrangements not heretofore described, but which are commensurate with the spirit and scope of the disclosure. Additionally, while various embodiments of the disclosure have been described, it is to be understood that aspects of the disclosure may include only some of the described embodiments. Accordingly, the disclosure should not be construed as limited by the foregoing description.

Each concept or combination of components described or illustrated can be used to practice the invention unless otherwise specified. The specific names of the compounds are intended to be exemplary, as it is known that one skilled in the art may name the same compound differently. When a compound is described herein, for example, by formula or chemical name, without specifying a particular isomer or enantiomer of the compound, such description is intended to include each isomer and isomer or any combination of the compounds described individually. Those skilled in the art will appreciate that methods, apparatus elements, starting materials, and synthetic methods can be used in the practice of the invention without undue experimentation, except as specifically illustrated. All art-known functional equivalents of any such methods, apparatus elements, starting materials, and synthetic methods are intended to be included herein. Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the given range, are intended to be included in the disclosure. As used herein, a markush group or other grouping is intended to be individually included in the disclosure as well as all combinations and possible subcombinations of the group.

As used herein, "comprising" is synonymous with "including", "containing", or "characterized by" and is inclusive or open-ended, and does not exclude additional unrecited elements or method steps. As used herein, "consisting of …" excludes any element, step, or ingredient not specified in a claim element. As used herein, "consisting essentially of …" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claims. In particular in the description of the components of the compositions or in the description of the elements of the devices, any recitation herein of the term "comprising" is to be understood to encompass such compositions and methods as consist essentially of, and such compositions and methods as consist of, the recited components or elements. The invention illustratively described herein suitably may be practiced in the absence of any element, limitation or limitations which is not specifically disclosed herein.

The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Generally, the terms and phrases used herein have their art-recognized meanings and may be found by reference to standard texts, journal references, and backgrounds known to those skilled in the art. The foregoing definitions are provided to clarify their specific use in the context of the present invention.

All references, such as patent documents (including issued or granted patents or equivalents; patent application publications); and non-patent document documents or other starting materials are herein incorporated by reference in their entirety to the same extent as if each reference were individually incorporated by reference to the extent that each reference was at least partially inconsistent with the disclosure of this application (e.g., except for partially inconsistent portions of the references, such references are incorporated by reference as if each were partially inconsistent with the disclosure of this application).

All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. The references cited herein are hereby incorporated by reference in their entirety to indicate the state of the art, in some cases since the date of their application, and it is intended that this information be available herein (if necessary) to the exclusion of (e.g., disclaim) specific embodiments of the prior art. For example, when protecting a compound, it is to be understood that compounds known in the art, including certain compounds disclosed in the references disclosed herein (especially in the cited patent documents), are not intended to be included in the claims.

Claims (37)

1. An enclosure comprising a porous two-dimensional material encapsulating a substance such that the substance is released to an environment external to the enclosure through channels of pores in the porous two-dimensional material.

2. An enclosure according to claim 1, encapsulating more than one different substance, wherein the different substances are not all released to the environment outside the enclosure.

3. An encapsulate according to claim 2 wherein different substances are released into the environment external to the encapsulate at different rates and/or at different relative concentrations.

4. An enclosure according to claim 1, encapsulating more than one different substance, wherein all of the different substances are released into the environment outside the enclosure.

5. An encapsulate according to claim 4 wherein different substances are released at different rates and/or at different relative concentrations.

6. The enclosure of claim 1, wherein the enclosure comprises two or more sub-compartments, wherein at least one sub-compartment is in direct fluid communication with the environment external to the enclosure through an aperture in the two-dimensional material of the sub-compartment.

7. An enclosure according to claim 6, wherein each sub-compartment comprises an apertured two-dimensional material and each sub-compartment is in direct fluid communication with the environment external to the enclosure through an aperture in the two-dimensional material of each sub-compartment.

8. An enclosure according to claim 1, wherein the enclosure is subdivided into two sub-compartments which are at least partially separated from each other by an apertured two-dimensional material, such that the two sub-compartments are in direct fluid communication with each other through apertures in the two-dimensional material.

9. An enclosure according to claim 1, wherein the enclosure is subdivided into two sub-compartments each comprising a two-dimensional material, the sub-compartments being in direct fluid communication with each other through apertures in the two-dimensional material, and only one of the sub-compartments being in direct fluid communication with the environment external to the enclosure.

10. An enclosure according to claim 1, wherein the enclosure is subdivided into two sub-compartments each comprising a two-dimensional material, the sub-compartments being in direct fluid communication with each other through apertures in the two-dimensional material, and both sub-compartments also being in direct fluid communication with the environment external to the enclosure.

11. The enclosure of claim 1, having an inner sub-compartment and an outer sub-compartment each comprising an apertured two-dimensional material, wherein the inner sub-compartment is completely enclosed within the outer sub-compartment, the inner and outer compartments are in direct fluid communication with each other through apertures in the two-dimensional material, and the inner sub-compartment is not in direct fluid communication with an environment external to the enclosure.

12. The enclosure of claim 1, having a plurality of sub-compartments each comprising a two-dimensional material, the sub-compartments being nested one within the other, the sub-compartments each being in direct fluid communication with its adjacent sub-compartment through an aperture in the two-dimensional material, the outermost sub-compartment being in direct fluid communication with the environment outside the enclosure, the remaining plurality of sub-compartments not being in direct fluid communication with the environment outside the enclosure.

13. The enclosure of claim 1, subdivided into a plurality of sub-compartments each comprising two-dimensional material, wherein each sub-compartment is in direct fluid communication with one or more adjacent sub-compartments, but wherein only one sub-compartment is in direct fluid communication with the environment external to the enclosure.

14. The encapsulate of any one of claims 1-13, wherein at least one substance within the encapsulate that is released to the environment outside the encapsulate through pores in the two-dimensional material is a drug.

15. The encapsulate of any one of claims 1-13, wherein at least one substance within the encapsulate that is released to the environment outside the encapsulate through pores in the two-dimensional material is a drug, and wherein the pores in the two-dimensional material range in size from 1-50 nm.

16. The encapsulate of any one of claims 1-13, wherein at least one substance within the encapsulate that is released to the environment outside the encapsulate through pores in the two-dimensional material is a drug, and wherein the pores in the two-dimensional material range in size from 1-10 nm.

17. The encapsulate of claim 1, wherein the substance within the encapsulate is a cell, and the size of the pores in the two-dimensional material is selected to retain the cell within the encapsulate and exclude immune cells and antibodies from entering the encapsulate from an environment external to the encapsulate.

18. An encapsulate according to claim 17 wherein the encapsulate is divided into a plurality of sub-compartments and one or more sub-compartments contain cells.

19. The enclosure of claim 17 having an inner sub-compartment and an outer sub-compartment each comprising an apertured two-dimensional material, wherein the inner sub-compartment is completely enclosed within the outer sub-compartment, the inner and outer sub-compartments are in direct fluid communication through apertures in the two-dimensional material of the inner sub-compartment, the inner sub-compartment is not in direct fluid communication with an environment external to the enclosure, and the outer sub-compartment is in direct fluid communication with an environment external to the enclosure.

20. The enclosure of claim 17, having a plurality of sub-compartments each comprising an apertured two-dimensional material and each in direct fluid communication with one or more adjacent sub-compartments, the cells being within one or more cell-containing sub-compartments each not in direct fluid communication with an environment external to the enclosure.

21. The encapsulate of any one of claims 17-20, wherein the cell is a yeast or bacterial cell.

22. The encapsulate of any one of claims 17-20, wherein the cell is a mammalian cell.

23. The encapsulate of any one of claims 1-13 or 17-20, wherein the size of the pores in the two-dimensional material is in the range of 3-10 nm.

24. The encapsulate of any one of claims 1-13 or 17-20, wherein the size of the pores in the two-dimensional material is in the range of 3-5 nm.

25. The encapsulate of any one of claims 1-13 or 17-20, wherein the two-dimensional material is supported on a porous substrate.

26. The encapsulate of any one of claims 1-13 or 17-20, wherein the two-dimensional material is graphene.

27. The encapsulate of any one of claims 1-13 or 17-20, wherein the two-dimensional material is a graphene-based material.

28. The encapsulate of any one of claims 1-13 or 17-20, wherein at least a portion of the pores in the two-dimensional material are functionalized.

29. The enclosure of any one of claims 1-13 or 17-20, wherein at least a portion of the two-dimensional material is electrically conductive and a voltage is applied to at least a portion of the electrically conductive two-dimensional material.

30. A method, comprising: introducing an enclosure comprising a porous two-dimensional material to an environment, the enclosure containing at least one substance; and releasing at least a portion of at least one substance to an environment external to the enclosure through the pores of the two-dimensional material.

31. The method of claim 30, wherein the environment is a biological environment.

32. The method of claim 30 or 31, wherein the at least one substance a portion of which is released is a drug.

33. The method of claim 30 or 31, wherein the encapsulate contains cells that are not released from the encapsulate, and the at least one substance a portion of which is released is a substance produced by the cells in the encapsulate.

34. A method, comprising: introducing an encapsulate according to any one of claims 1 to 13 into an environment, the encapsulate containing at least one substance; and releasing at least a portion of at least one substance to an environment external to the enclosure through the pores in the two-dimensional material.

35. A method, comprising: introducing the encapsulate of any one of claims 17-20 into an environment; and releasing at least a portion of at least one substance to an environment external to the enclosure through the pores of the two-dimensional material, wherein the at least one substance is a substance produced by the cells within the enclosure.

36. A method, comprising: introducing an enclosure comprising a porous two-dimensional material to an environment, the enclosure containing at least one first substance; and migrating a second substance from the environment into the enclosure.

37. The method of claim 36, wherein the first substance is a cell, the second substance is a nutrient, and the other second substance is oxygen.