Container therapeutics at point of care

Abstract

Provided herein are modular enclosures and related methods for the manufacture of therapeutic delivery systems, including methods of preparing therapeutic delivery systems using any of the modular enclosures described herein. The modular enclosure contains various modules for plant growth, isolation of plant delivery components and transduction of plant delivery components with any of a wide range of biological materials depending on the end-application of interest. The self-contained nature of the module enclosures provides point-of-care capability with minimal to no human intervention needed to generate the therapeutic delivery system.

Description

Aty Ref. 340466: 63-24 WOCONTAINER THERAPEUTICS AT POINT OF CARECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority from United States Provisional Patent Application No. 63 / 715,325, filed Nov. 1, 2024, which is incorporated by reference herein to the extent not inconsistent herewith.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No. AI067773 awarded by National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] There is a need in the art for reliable, efficient and low-cost production of therapeutics, including a platform level of a carrier that can be tailored to any range of cargos and clinical applications. One challenge is in the obtaining of relevant carriers. The methods and systems provided herein address these needs by generation of a delivery component, including an extracellular vesicle, that can be used to deliver a therapeutic, wherein the delivery component is plant-based. The methods and enclosure modules described herein provide controlled environmental conditions for high production yield of a therapeutic product that is low cost, biocompatible and biodegradable, and whose production can be automated and self-sufficient. Use of plants and plant constituents as a starting material has numerous advantages over other synthetic methods, including viral or bacterial vehicles, animal based or chemically synthesized methodology.SUMMARY OF THE INVENTION

[0004] Provided herein are methods and systems suitable for integrating the workflow preparation of novel therapeutic delivery systems from raw materials collection, processing and production into a mobile infrastructure with controlled environmental conditions that can be mobile for operation at any point of care at the doorsteps of patients and heath care providers. A special embodiment comprises installing a shipping container like infrastructure that integrates the controlled automation of vertical plant farming, isolation of plant-based vesicles, purification, characterization, loading with a natural or synthetic agents but alsoAty Ref. 340466: 63-24 WO integration of a biological materials (e.g. gene, protein...), sterilization, packaging and delivery of a treatment or vehicle to a cellular therapy directly on site in a clinical setting (e.g. hospital parking lot) or any point of care close to patients and providers. The entire container space is designed to follow quality control and regulatory rules allowing the integrated processing of customized therapies that can satisfy FDA or other regulatory body for good manufacturing protocols (or GMP conditions). The container space has a green room that can be designed under BSL-1 safety for growing plants under controlled environmental agriculture in combination with a cleanroom processing room under ISO8 or even ISO5 standards in alignment with cGMP policies suitable to obtain a finished product readily available for on-site delivery in most clinical settings that may not have access to a dedicated environment for mRNA or protein-based treatment or gene or cellular therapies. This integrated capacity can also be applied to rural health, global health, or future space health with minimum modifications as recommended in some of the embodiments described in the present invention.

[0005] This infrastructure provides an in-situ resource utilization means or a self- sustainable resource for extending or managing resources for sustaining life conditions from nutrition, medication to energy production, including requiring minimal to no human intervention, except for the harvesting of an end product, including an end product relevant for therapeutic use.

[0006] Provided herein are modular enclosures for the preparation of therapeutic delivery systems and related methods of using, including to prepare a therapeutic delivery system, any of the modular enclosures described herein.

[0007] Generally, provided herein is a process spanning from plant growth, extracellular vesicle (Ev) extraction, then in parallel collection of cells from a patient, activating the cell cultures (hours), transduct the cells with the Evs (e.g., up to about 1 day), expand the cells (e.g. on the order of days, such as between 1 day and 10 days, including about 7 days), harvest the product and then generate final product, thereby making a therapeutic delivery system. As various protocols are tailored or optimized within the modular enclosure, including based on an application of interest, the turn-around time can be further minimized.

[0008] In an embodiment, provided is a modular enclosure for the manufacture of therapeutic delivery systems and, more generally, for plant components useful in a range ofAty Ref. 340466: 63-24 WO applications, including for batteries or for therapeutic delivery systems. The modular enclosure comprises a plant nursery container configured to generate a plant delivery component. The plant nursery container is any of a variety of growth platforms suitable for plant growth. It may be hydroponic media, artificial Light Emitting Diodes (LED) lighting across multiple intensities and wavelengths suitable for optimizing circadian rhythms, temperature and / or humidity sensors, automated delivery system of pre-formulated composition of nutrients or other climate means to control associated desirable plant traits (e.g. size, stress tolerance). It may comprise a vertical tower so as to maximize growth to floor footprint area. An isolator is operably connected to the plant nursery container, configured to receive and / or isolate the plant delivery components, including plant vesicles and / or plant cellulose scaffolds as building blocks for therapeutic carrier, for example. An analyzer is operably connected to the isolator, configured to analyze a plant processing parameter from the isolated plant delivery component. In this manner, the efficacy and properties of the plant delivery component can be assessed and characterized to confirm they are suitable for use in the application of interest. The analyzer may be configured or selected to analyze a plurality of plant processing parameters. A conditioner is operably connected to the analyzer, configured to collect a biological material, transduct the plant delivery component with the collected biological material, and extract a biological product from the transducted plant delivery component. For example, for a plant scaffold, the conditioner may provide to the plant scaffold a pharmaceutical drug for an incubation time to provide the transducted plant delivery component that comprises the plant scaffold with the pharmaceutical drug adsorbed thereto. For a PEV application, the biological material can be loaded into the PEV to provide a PEV loaded cargo ready for application as a therapeutic delivery system. Accordingly, the actual analyzer will depend on the application of interest.

[0009] The plant delivery component will depend on the end application of interest. Of particular interest herein are plant delivery components that comprise plant vesicles (PEVs) and / or a plant cellulose scaffolds. For leaf-based battery systems, the plant delivery component may correspond to PEVs that are high in metals facilitating their use for assembling an electrode device.

[0010] Any of the modular enclosures may further comprise a storage container to store the isolated PEVs and / or plant cellulose scaffolds. The isolated PEVs and / or plant cellulose scaffolds may be in a more unprocessed state (such as without any added biological material)Aty Ref. 340466: 63-24 WO or may be further downstream the processing, such as in a more ready-for-use format, including with a biological material added thereto.

[0011] Any of the modular enclosures may correspond to a shipping container. In other words, the modular enclosure may be contained within a shipping container, including a standard-sized shipping container that can be readily positioned on a tractor-trailer, railroad car, within a spacecraft, and the like. This provides a number of important benefits, including the ability to pre-position the modular enclosures and rapidly move them to areas of need, all with minimal to no human intervention. For example, the modular enclosure may be communicatively coupled and controlled to an external controller, including by a wired or wireless communication connection. . Energy power can be generated to the container by solar panels and supplies or reagents may be provided to the modular enclosure, including via access ports through the shipping container or there may be secured doors for ready access to a service worker to any points within the modular enclosure. The shipping container configuration ensures the modular enclosure is configured for transport without adversely impacting a plant vesicle and / or plant cellulose scaffold activity parameter.

[0012] Any of the modular enclosures may have the plant nursery container divided into a green room and a clean room. The green room may conform to BSL-1 safety guidelines and the clean room conform to at least ISO8 or at least ISO5 standards. Again, depending on the application of interest, the various sub-modules are selected to achieve certain sterility, safety standards, and the like.

[0013] The plant nursery container may comprise a vertical plant farming tower, wherein the vertical plant farming tower comprises at least an upper level and a lower level, each level being configured for the farming of plants. This is advantageous for utilizing full 3-D space in the nursery container sub-module of the modular enclosure.

[0014] Any of the modular enclosures may comprise a controller for automated control of each of the plant nursery containers, isolator and analyzer, including a controller operably connected to the vertical plant farming tower to at least partially automate or completely automate the vertical plant farming. This controller may be located on-site within the modular enclosure or may be at a physical distance from the modular disclosure, with communicative coupling between the controller and the modular enclosure. In this manner, the modular enclosure may be effectively monitored and controlled, as desired, by an individual that isAty Ref. 340466: 63-24 WO physically separated from the physical modular enclosure components. Controller is used broadly herein and can include computer systems that, via processer-implemented instructions, control various devices in the modular enclosure, including illumination, temperature, moisture, humidity, fertilization, pH and the like for plant growth and maintenance. Similarly, a controller is relevant for other sub-modules, such as the handling and processing of plant-based materials throughout the process, centrifugation, separation, filtration, sterilization, containment, introducing of reagents and biological material, incubation and the like.

[0015] The transducted plant delivery component, including PEVs and plant scaffolds, undergo an expansion reaction while in the conditioner, and wherein the biological product exhibits pharmaceutical properties suitable for just-in-time delivery of a therapeutic system specific to an individual condition at a site of care. For example, the modular enclosure may be positioned in a location where care is desired, thereby avoiding shipping of end-products from the modular enclosure to a remote location. This avoids the need to take care of potentially degradable end-products, such as degradation that may occur simply due to a shipment time without, for example, active cooling during shipment.

[0016] Any of the modular enclosures may further comprise: a loader comprising a natural or synthetic agent, wherein the loader is operably connected to the isolator, wherein the loader is configured to load the plant delivery component with the natural or synthetic agent. The agent may be a biologically-relevant material that affects a biological change to a patient exposed to the loaded plant delivery component, such as PEVs or plant scaffolds.

[0017] The natural or synthetic agent may comprise a nutrient, a medication, an antibody, a polynucleotide, a polypeptide, a protein, a carrier, or a combination thereof. The medication may comprise molecules such as cannabidiol, carboplatin, Withaferin A, Withaferin B, Withaferin C, Withaferin D, acetaminophen, ibuprofen, codeine, a volatile drug, a gas marble, a liquid marble (including a medical marble), an antibody, a nanobody, an aptamer, a nucleic acid, a flavonoid, or a combination thereof. The gas marble may comprise gaseous metabolic byproducts released from a mammalian body (e.g. oxygen, hydrogen, carbon monoxide, methane), or an element (e.g. noble gases such as Xe, Ar).

[0018] Any of the modular enclosures may further comprise an integrator comprising the biological product, wherein the integrator is operably connected to the loader, wherein theAty Ref. 340466: 63-24 WO integrator is configured to integrate the biological product into the loaded plant delivery component. A biological product may comprise a cell, a gene, a nucleic acid, a protein, fragments thereof, any antibody, or any combination thereof.

[0019] Any of the modular enclosures may further comprise: a sterilizer operably connected to the loader, wherein the sterilizer is configured to sterilize the integrated loaded plant delivery component. This can help ensure any end product that leaves the modular enclosure, including for therapy, is sterile. The sterilization may comprise steam, chemical (e.g. ethylene oxide, vaporized hydrogen peroxide) or radiation (e.g. ionizing irradiation, microwave energy) treatment. Furthermore, quality-control tests by the analyzer can confirm activity and efficacy.

[0020] Any of the modular enclosures may further comprise a packager operably connected to the sterilizer, wherein the packager is configured to package the sterilized plant delivery component, wherein the package is configured for delivery of the sterilized plant delivery component. For example, if the desired therapeutic is in pill form, the packager provides the end-product into pill form. Similarly, for IV infusion, as a cream or foam, injection, sublingual or nasal spraying, encapsulation and the like.

[0021] Provided herein are methods of making a therapeutic delivery system, including by using any of the modular enclosures provided herein. The method may comprise the steps of providing a source of plant delivery components to a plant nursery container; growing the source of plant delivery components in the plant nursery container for a grow time; obtaining an isolated plant delivery component with an isolator configured to isolate plant delivery components; and characterizing the isolated plant delivery components with an analyzer configured to characterize and / or preserve plant vesicles and / or plant cellulose scaffolds. Optionally, the analyzer may be connected to a storage container to store the isolated plant delivery components, including for reliable preservation and on-demand requirements.

[0022] The growing step may comprise growing a whole plant or culturing plant cells. The growing step may comprise vertical plant farming, including at least partially automated or completely automated vertical plant farming.

[0023] The method may further relate to: the obtaining of the plant delivery components occurs within a BSL-1 green room; the characterizing of the isolated plant delivery components occurs within the BSL-1 green room; and the method further comprise any oneAty Ref. 340466: 63-24 WO or more of: collecting a biological material from a plant source; transducting the isolated plant delivery components with the biological material within an ISO8 clean room to make transducted plant delivery components; expanding the transducted plant delivery components within the ISO8 clean room to make expanded plant delivery components; harvesting a biological product from the expanded plant delivery components within the ISO8 clean room; loading the isolated delivery vehicle with a natural or synthetic agent within the ISO8 clean room; integrating the biological product into the loaded plant delivery vehicle within the ISO8 clean room to make an integrated and loaded plant delivery vehicle; sterilizing the integrated and loaded plant delivery vehicle within the ISO8 clean room to make a sterilized plant delivery vehicle; and packaging the sterilized plant delivery vehicle within the ISO8 clean room for delivery of the sterilized plant delivery vehicle.

[0024] An advantage of the methods and modular enclosures provided herein, is that they may be fully automated, so that not active user-intervention is required in order to generate the therapeutic, and preferably at a location in proximity to a patient.BRIEF DESCRITPION OF THE DRAWINGS

[0025] FIG. 1 illustrates a process for generating a plant-based scaffold that provides high porosity (to support cellular growth) and various hydrophilicity properties, useful in a range of applications.

[0026] FIG. 2A illustrates an application of a plant scaffold (such as from FIG. 1) for drug delivery, FIGs 2B-2D illustrate various quantitative analysis of a plant scaffold used for drug delivery, including: drug adsorption onto plant scaffold (FIG. 2B); drug release from plant scaffold (FIG. 2C); and the biological efficacy of released drug as assessed by cell viability (FIG. 2D)

[0027] FIG. 3 summarizes the plant components and steps useful for making a leaf-based batter.

[0028] FIG. 4 illustrates various advantages of a leaf-based battery for energy production.

[0029] FIG. 5 illustrates a modular enclosure that is in the footprint of a shipping container (top left panel) and the various sub-modules illustrated in a cut-away view of the shipping container (top right panel). The bottom panel summarizes the various processes that can beAty Ref. 340466: 63-24 WO performed within the modular enclosure, from plant selection / growth to harvesting of a biotherapeutic for subsequent patient treatment.

[0030] FIG. 6 is a schematic illustration of the various processes that can be employed using plant starting materials with the modular enclosure.

[0031] FIG. 7 is a top cut-away view of an exemplified modular enclosure with the various modules contained therein to facilitate plant growth through to manufacture of an end product, such as a leaf battery or a therapeutic delivery system.

[0032] FIG. 8 is a schematic illustrate of the modular enclosure and related process of making a therapeutic delivery system or other plant-based system, such as a leaf battery.DETAILED DESCRIPTION OF THE INVENTION

[0033] Provided herein is a modular enclosure for the preparation of therapeutic delivery systems. The modular enclosure has a number of sub-modules (also referred herein as components) that provide different functionality so that there can be automated production of plant constituents used in the therapeutic delivery system. For example, a plant nursery container is an upstream component where a sufficient plant-based component is generated under controlled parameters (e.g., culture nutrient delivery assisted by artificial intelligence), such as plant vesicles and / or cellulose scaffold.

[0034] “Plant nursery container” is used broadly herein to refer to a device that can support and maintain plant growth, with examples including a vertical farming apparatus, hydroponic devices, a pot, a greenhouse and / or a planter. The plant nursery container may, more generally, be formed from a shipping container.

[0035] “Isolator” is used broadly herein to refer to components that are able to process from a plant source material a desired plant constituent and reflects that there are many different instruments capable of isolating plant vesicles and / or plant cellulose scaffolding. In some examples, an isolator can include any one or more of a centrifuge, filter, a gel separator, a separator funnel, a gradient, including a sucrose gradient, or an electromagnetic field condition for the active manipulation of said plant source material or components thereof, including use of a dielectrophoretic vesicle concentration trap.Aty Ref. 340466: 63-24 WO

[0036] “Analyzer” is used broadly herein to refer to components used to characterize the output from the isolator, as well as aspects that may be upstream, such as growing plant or plant culture, and reflects that there are many different instruments capable of analyzing plant vesicles and / or plant cellulose scaffolding. In some examples, an analyzer can include gas chromatography (for analyzing volatiles), liquid chromatography, mass spectrometry, FT-IR spectroscopy, Raman spectroscopy, a nanoparticle tracking analysis (NTA) or nanomaterials analyzer using dynamic light scattering (DLS), electrophoretic light scattering (ELS), or static light scattering (SLS), or optical detectors, including optical detectors comprising a photonic device, a near-field probe, or electron microscopy, including scanning, transmission, and cryogenic microscopy modalities, microscopes (including for fluorescence microscopy) or a combination thereof. The analyzer may include optical components, including optical dyes such as fluorescent molecules, and / or biological tags, such as antibodies, polynucleotide or protein probes, which in turn may have or be labelled with an optical tag.

[0037] “Integrator” is used broadly herein to refer to a device to integrate a biological material into the plant delivery component, reflecting that there are many different instruments capable of integrating biological materials into plant vesicles and / or plant cellulose scaffolding. In some examples, an integrator can include a microfluidic mixing chip, an incubation chamber, an electroporation system, a sonicator, an extruder, a freezethaw cycling device, a device for surfactant treatment, a transfection electroporation system, a biolistic system, or a dialysis system.

[0038] “ Sterilizer” is used broadly herein and reflects that there are many different instruments capable of sterilizing plant vesicles and / or plant cellulose scaffolding. In some examples, a sterilizer can include an optical source, such as for ultraviolet, gamma, electron beam, or X-ray radiation, including an ultraviolet, gamma, electron beam, or X-ray irradiator, an ozone generator, a supercritical carbon dioxide sterilizer, a chemical generator, an applicator, or a heater.

[0039] “Packager” is used broadly herein and reflects that there are many different instruments capable of packaging plant vesicles and / or plant cellulose scaffolding for delivery. In some examples, a packager can include a desiccator to provide the therapeutic into a solid that can be later dissolved in liquid, a dispenser and vial for dispensing liquid therapeutic into a vial for later use, a 3D-printing system for fuse deposition modeling, inkjet printing, selective laser sintering, semi-solid extrusion, direct powder extrusion,Aty Ref. 340466: 63-24 WO stereolithography, tablet manufacture components (compressors, granulators, mixing equipment, drying machinery, coating systems), such as via wet granulation, dry granulation, direct compression, IV bags for containing the therapeutic for later IV administration, attendant ingredients such as useful for formulating the therapeutic (binders, excipients, preservatives, etc.), a topical bandage-like patch device for transdermal or sub-cutaneous administration, an injector for intramuscular or intravenous injection, an inhaler, or an applicator for rectal, vaginal, or sub-lingual administration.

[0040] “ Conditioner” is used broadly herein and reflects that there are many different instruments capable of transducting loaded plant delivery components and expansion of the transducted component, including plant vesicles and / or plant cellulose scaffolds, with a biological material. In some examples, a conditioner can include an electroporation device, phage therapy device, an incubator, a syringe, a cell expansion and media exchange system, temperature regulators (e.g., heaters), fluidic controllers and associated fluid-containing reservoirs, and the like. The biological material is optionally from a subject. Alternatively, the biological material can be a manufactured material, such as a protein, polypeptide, nucleic acid sequence, gene, antibody, and the like. The common aspect around biological material is that there is an impact on a biological outcome from the therapeutic delivery system.

[0041] “Loader” is used broadly herein and reflects the portion of the process wherein additional ingredients are added to the isolated plant material relevant for the desired application in which the end therapeutic is intended to be used. There are many different instruments capable of loading plant vesicles and / or plant cellulose scaffolding with natural or synthetic agents, and can have some overlap with the conditioner, as both processes rely on instruments to combine a desired substance with the plant delivery component. Use of both conditioner and loader can reflect the addition of more than one substance to the plant delivery component. In some examples, a loader can include one or more of an incubator, electrochemical devices for introduction of materials via electrochemistry, a hydrogel, a conductive gel, an electrolyte, a battery, a dispenser, pumps, fluid conduits, a supercritical fluid system, a reactor, an ultrasonic homogenizer system, a random positioning machine, an electroporation system, and a spray coating system.

[0042] “Operably connected” refers to the configuration of two components that connect, either directly or indirectly, but in a manner that maintains operability and functionality of each component.Aty Ref. 340466: 63-24 WO

[0043] A “plant processing parameter” is used broadly herein to refer to any number of parameters that can be analyzed to determine the quality or quantity of plant vesicles and / or plant cellulose scaffolds. Examples include, but are not limited to, a measure of purity, a measure of size (e.g., average diameter), a measure of amount, a measure of concentration, a measure of activity, stability, omics analyses of composition (e.g., lipidomic characterization of the membrane composition, proteomic characterization of the membrane composition, metabol omics profiling of cargoes), electric potential measurement (e.g., zeta potential), encapsulation efficiency, loading efficiency, stability, shelf life, storage conditions, biodegradation, temperature resistance, sterility, or other quality attributes required for a specific targeted product and the like, depending on the application of interest. The desired plant processing parameter(s) to be measured informs analyzer selection (e.g., microscopes, imagers, electrical counters, assay, etc.).

[0044] A “plant vesicle and / or plant cellulose scaffold activity parameter” is used broadly herein to refer to any number of parameters that can be analyzed to determine the quality or quantity of plant vesicles and / or plant cellulose scaffolds. Examples include, but are not limited to, morphological homogeneity, mechanical properties (e.g., stiffness, roughness), electrostatic charge, pH stability, temperature stability, tolerance to freezing storage conditions (e.g., -200 °C to 60 °C), stability in biological fluids (e.g., blood, saliva), low toxicity in biological fluids, and the like, depending on the application of interest.

[0045] A “gas marble” or “liquid marble” is used broadly herein to refer to any number of spherical, quasi-spherical, or non-spherical objects containing an inner cargo or a gas (gas marble) or liquid (liquid marble) volume on the order of a few hundred mm3. A marble generally refers to a nanoparticle sized material (e.g., less than 1 mm in effective diameter) that envelops gases or liquids and that can withstand external forces and internal pressures, including greater than atmospheric pressure (e.g., up to 10 atmospheres), without a significant change in size or shape (e.g., expansion). Optionally, the inner volume of the marble may be less than 500 mm3, 200 mm3, 100 mm3, 50 mm3, 20 mm3, 10 mm3, or 1 mm3, including optionally, greater than 0.1 mm3, 0.5 mm3, 1 mm3, 5 mm3, 10 mm3, 50 mm3, 100 mm3, 200 mm3, 300 mm3, or any subranges thereof. In some examples, a marble comprises a biodegradable shell capable of storing the cargo for a time period and that can controllably degrade to facilitate release of the stored cargo. See, e.g., PCT / US24 / 27766 filed May 3, 2024 (Atty Ref. 339986: 6-23 WO), specifically incorporated by reference herein for gasAty Ref. 340466: 63-24 WO marble compositions and methods of making gas marbles. The gas marble may contain a noble gas or a mixture of noble gases.

[0046] A “medical marble” is used broadly herein to refer to any number of gas or liquid marbles wherein the inner volume of the marble comprises a gas or a liquid that can be used for a medical purpose. For example, the cargo within a medical gas or liquid marble may include a natural or synthetic agent, a radio-tracer, an antibiotic, a nutrient, a medication, an antibody, a polynucleotide, a polypeptide, a protein, a carrier, cannabidiol, carboplatin, Withaferin A, Withaferin B, Withaferin C, Withaferin D, acetaminophen, ibuprofen, codeine, a volatile drug, a nanobody, an aptamer, a flavonoid, or a combination thereof. Placement of these medical gas marbles into foam or gel materials can also be used for the preparation of pills or suppositories for oral, vaginal, topical, or rectal administration.

[0047] A “nanobody” is used broadly herein to refer to any number of particles classified as a single-domain antibody (sdAb). For example, a nanobody may comprise a single monomeric variable antibody domain. Optionally, a nanobody is able to bind selectively to a specific antigen. In some cases, a nanobody has a smaller mass than a typical antibody. For example, a nanobody may have a molecular weight of 12 kDa to 15 kDa.

[0048] The modular enclosures and related methods are useful in a range of applications. For example, in long duration missions where terrestrial earth is not readily accessible, including space or underwater travel (or even at remote sites or large number of different sites on Earth), the module enclosures provided herein can be a reliable and efficient platform for ensuring biotherapeutics are available. For example, to support human life in interplanetary habitats, the plant-based platforms provided herein is useful for radiation protection, as food, to provide energy (plant-based batteries), medical care, and for gas mixture production, including for air and atmosphere. Starting with plant seeds, hydroponic gardens can be used to grow the plants, and thereby the relevant delivery components, including vesicles and / or scaffolds. Other applications for the plant delivery components including radiation protection, neuroprotection and cognitive enhancers, gas therapies, tissue and cell regeneration, antibiotics, wound healing. Of course, the devices and methods provided herein are not limited to non-terrestrial earth missions. In fact, there can be valuable applications for these portable-type systems that may readily positioned anywhere on earth with efficient and easy start-up through to plant component harvesting and processing to generate a desired output; all with minimal human intervention.Aty Ref. 340466: 63-24 WO

[0049] Plant-based tissue engineering is relevant because the plant components can provide ideal scaffold properties.

[0050] Example 1: Plant based technology.

[0051] Key needs for human life interplanetary habitat includes: air and atmosphere (gas mixtures), water, food, radiation protection, gravity simulation, energy, psychological and social factors and emergency medical care. Plants can have a key role in addressing many of these needs (e.g., gas mixtures, food, radiation protection, energy and emergency medical care. Plant growth is compatible with “green” manufacturing and in-situ resource utilization (ISRU), and can occur from plant seeds via vertical farming and hydroponics. Plants can be utilized to grow and / or express various biotherapeutics, ranging from radiation protection and assessment, neuroprotection, cognitive enhancers, gas therapy, tissue and cell regeneration, antibiotics and wound healing. New access and engineering opportunities are provided by plants, including plants grown to hyperaccumulate relevant materials, such as chemicals that may be biologically relevant.

[0052] With respect to plant-based tissue engineering, plants can provide an ideal scaffold for biomaterials, as a plant scaffold can be biocompatible, non-toxic upon degradation, non- immunogenic, relevant mechanical properties, with 3D architecture, high porosity and physical / structural support to provide a template guide and direct cell behavior accommodation. FIG. 1 is an example of a process to provide a plant delivery component that is a plant scaffold template, by digestion of plant(s) to provide a cellulose-based scaffold. The scaffold has high porosity (middle panel) and different hydrophobicity properties depending on the plant type (bottom panel).

[0053] FIGs 2A-2D summarize the application of plant scaffolds for drug delivery, including for a representative pharmaceutical compound. The drug delivery may occur via a relatively simple incubation of a drug with a plant scaffold, as shown in FIG. 2A (including a scaffold that may be produced via the process summarized in FIG. 1). Drug adsorption onto the plant scaffold can be confirmed and assessed by liquid chromatography, including ultraperformance liquid chromatography. Efficacy of such incubation can be experimentally confirmed, including as illustrated by the experimental data of FIGs 2B-2D. Representative drugs include, but are not limited to, acetaminophen, (RS)-Ibuprofen and Codeine. ForAty Ref. 340466: 63-24 WO example, Codeine release were examined for various plant sources of the scaffold, including apple, celery, pepper and spinach leaves.

[0054] Another relevant plant delivery component are extracellular vesicles, including plant vesicles. Plant extracellular vesicles (PEVs) can have applications as therapeutic nanocarriers. PEVs can provide a drug delivery system with less toxicity to the patient, including less liver toxicity. PEVs may be in the form a lipid bilayer of between about 30 nm - 100 nm in diameter, with proteins, bioactive compounds, receptors and the like (generally hydrophobic materials) in or on the bilayer. Nucleic acids, proteins and bioactive compounds may also be positioned in the inner volume defined by the PEV lipid bilayer (generally hydrophilic materials). The PEVs may be used for drug loading, with production, purification, drug loading, bioengineering, and delivery to target (e.g., tumor) for therapy. PEVs are advantageous in that they can have a high production yield at low cost, while being biocompatible and biodegradable.

[0055] PEV preparation may begin with a plant library (e.g., ginger roots, olives, cloves, duckweeds, etc.) to help select a plant source having desired characteristics, followed by blending of the selected plant source and centrifugation to provide a raw pellet. The raw pellet may be subject to a gradient, including a sucrose gradient, with one exemplary spin being 50,000 to 150,000 g for 2 hours at 4°C. The desired fraction may be taken corresponding to the PEVs. In this manner, the obtained PEVs may be stained (e.g., olive EVs in cells), including with F-acting stain by Phalloidin 555, particle membranes by pKH67, etc. to confirm good PEV yield.

[0056] Another application for plant leaf is as a battery. For example, FIG. 3 summarizes the various steps, including a plant metal hyperaccumulator combined with PEVs, a plant cellulose scaffold, and hydrogel filling (e.g., polyacrylamide) + electrolyte (e.g., KOH / LiOH) or conductive gel (e.g., PEDOT). In this manner, a battery that is leaf-based (with the PEVs delivering the necessary plant metal portions of the battery). FIG. 4 summarizes the basic structure of a leaf-based battery, with a plant-based scaffold tending to have high porosity filled with a hydrogel / electrolyte solution (e.g., NaCl, KOH, ZnO). Such a battery advantageously is recyclable, low cost, has high volumetric capacity, rechargeable and can be part of a sustainable electronics platform. The scaffold properties can be tuned, to provide desired flexibility.Aty Ref. 340466: 63-24 WO

[0057] A fundamental unifying need for these applications is the ability to reliably generate the necessary plant components. Such a platform to facilitate desired generation is provided herein in the form of a “modular enclosure”, including as provided in the examples below.

[0058] Example 2: Making plant delivery compounds in a modular container platform.

[0059] Provided herein are modular enclosures configured to manufacture, for example, a therapeutic delivery system. FIG. 5 (top-right panel) and FIG. 8, for example, illustrates a modular enclosure 10 in that various sub-systems may be introduced / removed. This includes a plant nursery 20, an isolator 30, an analyzer 40, and a conditioner 50 (optionally comprising a loader 55), integrator 60, a sterilizer 70, a packager 80. These various sub-systems, together form the modular enclosure 10 illustrated in FIG. 5 (top-left panel) as a shipping container 11, with a cut-away view in the top-right panel. In this manner, the modular system may be readily scaled and transported in a reliable manner, even during plant growth and isolation of the plant delivery component. This is achieved by ensuring the shipping container is air-tight, with the different modules secured in place with appropriate shocks, dampers and the like. Within each of the modules, the various devices, starting materials, produced product, can likewise be secured.

[0060] The analyzer 40 may be used to assess a parameter, including a quality control parameter relevant for the plant delivery component. If there are any out of range results, the upstream growth 20 and / or isolation 30 may be adjusted. This provides a type of feedback loop, where the analyzer can help inform conditions in plant nursery container 20 so as to ensure appropriate plant delivery component from the isolator 30. As illustrated in FIG. 8, results from analyzer may also be used to inform isolator parameters to ensure good isolation of plant delivery component from whole plant starting material.

[0061] A storage container 61 may store the plant delivery component 41, including isolated PEVs and / or plant cellulose scaffolds.

[0062] If the results from analyzer suggest the plant delivery components are satisfactory, conditioner 50 and / or loader 55 may be used to provide a biological material and / or an agent material (synthetic or natural) to the plant delivery component. In this manner, the plant delivery component 41 is transducted to a transducted plant delivery component 42 having the biological material, and optionally another material, connected thereto. Optionally, the plant delivery component can be expanded. The conditioner optionally extracts a biologicalAty Ref. 340466: 63-24 WO product from the transducted plant delivery component or prepares the transducted plant delivery component for subsequent processing.

[0063] The integrator 60 takes the extracted biological product and integrates it into a loaded plant vesicle (PEV) and / or a plant cellulose scaffold, including as obtained from plant nursery container 20, to provide an integrated and loaded plant delivery component 43. The product may be sterilized via a sterilizer 70 to provide a sterilized integrated and loaded plant vesicles and / or plant cellulose scaffolds 44 and made ready for packaging via packager 80 o provide ready-to-use therapeutic delivery system 45 for on demand use. The on demand use, because of the modular enclosure platform, can be on-site, with minimal shipment as the modular enclosure can be readily positioned at the treatment location. As desired, the therapeutic delivery system made by the modular enclosure may also be stored in the modular enclosure for subsequent use.

[0064] The plant nursery 20 may be further divided into a green room 21 and a clean room 22, with different sterilization standards for each of those rooms. The plants may be grown in a vertical plant farming tower 23 having various levels, including an upper level 24 and a lower level 25 (FIG. 8). An output of the modular enclosure may be PEVs that have been suitably processed and modified. As illustrated in FIG. 6, the PEVs may be used for drug delivery or for a leaf battery (such as by the provision of hyperconcentrated metals). Controllers 26 may electronically provide automated control. The controller may comprise computer hardware and processors to implement a processing scheme, including for managing watering, fertilization, humidity, temperature and the like. Similarly, the processing scheme may be implemented for other sub-modules, such as by control of centrifuges sucrose gradients, fluidics, assays, incubation time and conditions, packaging and the like. A controller 26 may be onsite within the enclosure module, including separate controllers for separate instruments, or offsite, and in wireless and wired communication with controllable devices within the enclosure module, as reflected by dashed line 27 representing two-way communication between controller 26 and enclosure module 10, including to any controllable devices therein.

[0065] The engineering of the drug delivery may include the reliable release of drug via optical excitation, such as blue light illumination to break bonds and release a cargo protein. In this manner, the drug loading may comprise forming an optically-sensitive bond between the PEV and the drug cargo, including as mediated by CRY2-CIBN. The ability to isolateAty Ref. 340466: 63-24 WO compounds may be magnetically facilitated, including by a magnetically coupled bead. The delivery may utilize any of a variety of receptors on the PEV surface, to facilitate guided delivery of the PEV to a target.

[0066] Production may utilize a BSL-1 green clean room and ISO 8 delivery prep cleanroom. Relevant instruments include a plant grinder (e.g., Ball Mill), benchtop ultracentrifuge for density gradient purification, and liquid handler for automated gradient preparation. The analyzer for characterization may include an NTA nanomaterials analyzer, LC / MS-MS for lipidomics / cargo, and any of a variety of potency assays.

[0067] Example 3: PEVs (plant-derived extracellular vesicles).

[0068] As noted, PEVs have a number of advantages over animal vesicles. The PEVs have a lipid bilayer and are compatible with drug delivery via internally-positioned compounds surrounded by the lipid bilayer, or can be in or at the bilayer, including via surface modification. The PEVs are durable and stable, with good resistance to physical stress, and with applications ranging from therapeutic agents, cellular communication, and drug delivery systems (nanocarriers). The PEVs can be used with siRNA and RNA therapy. The PEVs can be used for gene therapy or cell therapy. Another configuration of the modular enclosure is provided in FIG. 7, with a cut-away top-view to illustrate the various sub-modules.STATEMENTS REGARDING INCORPORATION BY REFERENCE AND VARIATIONS

[0069] All references throughout this application, for example patent documents including issued or granted patents or equivalents; patent application publications; and nonpatent literature documents or other source material; are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference, to the extent each reference is at least partially not inconsistent with the disclosure in this application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference).

[0070] The terms and expressions which have been employed herein 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 theAty Ref. 340466: 63-24 WO invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, exemplary 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. The specific embodiments provided herein are examples of useful embodiments of the present invention and it will be apparent to one skilled in the art that the present invention may be carried out using a large number of variations of the devices, device components, methods, or steps set forth in the present description. As will be obvious to one of skill in the art, methods and devices useful for the present methods can include a large number of optional composition and processing elements and steps.

[0071] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art. As well, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably. The expression “of any of claims XX- YY” (wherein XX and YY refer to claim numbers) is intended to provide a multiple dependent claim in the alternative form, and in some embodiments is interchangeable with the expression “as in any one of claims XX- YY.”

[0072] When a group of substituents is disclosed herein, it is understood that all individual members of that group and all subgroups, are disclosed separately. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure. Specific names of compounds are intended to be exemplary, as it is known that one of ordinary skill in the art can name the same compounds differently.

[0073] Every monomer, polymer, system, structure, geometry, feature, combination thereof, or method described or exemplified herein can be used to practice the invention, unless otherwise stated.

[0074] Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition or concentration range, all intermediate ranges and subranges,Aty Ref. 340466: 63-24 WO as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the claims herein.

[0075] All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. References cited herein are incorporated by reference herein in their entirety to indicate the state of the art as of their publication or filing date and it is intended that this information can be employed herein, if needed, to exclude specific embodiments that are in the prior art. For example, when compositions of matter are claimed, it should be understood that compounds known and available in the art prior to Applicant's invention, including compounds for which an enabling disclosure is provided in the references cited herein, are not intended to be included in the composition of matter claims herein.

[0076] 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 the 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 claim. In each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

[0077] One of ordinary skill in the art will appreciate that starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents of any such materials and methods are intended to be included in this invention. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that 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 andAtty Ref. 340466: 63-24 WO 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.

[0078] Without wishing to be bound by any particular theory, there may be discussion herein of beliefs or understandings of underlying principles relating to the devices and methods disclosed herein. It is recognized that regardless of the ultimate correctness of any mechanistic explanation or hypothesis, an embodiment of the invention can nonetheless be operative and useful.

[0079] In general, the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references and contexts known to those skilled in the art.

Claims

Aty Ref. 340466: 63-24 WOCLAIMS1. A modular enclosure for the manufacture of therapeutic delivery systems comprising: a plant nursery container configured to generate a plant delivery component ; an isolator operably connected to the plant nursery container, configured to receive and / or isolate the plant delivery component; an analyzer operably connected to the isolator, configured to analyze a plant processing parameter from the plant delivery component; and a conditioner operably connected to the analyzer and / or isolator, configured to transduct the plant delivery component with a biological material, and extract a biological product from the transducted plant delivery component.

2. The modular enclosure of claim 1, wherein the plant delivery component comprises plant vesicles and / or a plant cellulose scaffolds.

3. The modular enclosure of claim 2, further comprising a storage container to store the isolated plant vesicles and / or plant cellulose scaffolds.

4. The modular enclosure of any one of claims 1-3, wherein the modular enclosure is formed from a shipping container configured for transport without adversely impacting a plant vesicle and / or plant cellulose scaffold activity parameter.

5. The modular enclosure of any one of claims 1-4, wherein: the plant nursery container is divided into a green room and a clean room; the green room conforms to BSL-1 safety guidelines; and the clean room conforms to at least ISO8 or at least ISO5 standards.

6. The modular enclosure of any one of claims 1-5, wherein the plant nursery container comprises a vertical plant farming tower, wherein the vertical plant farming tower comprises at least an upper level and a lower level, each level being configured for the farming of plants.

7. The modular enclosure of any one of claims 1-6, further comprising one or more controllers for automated control of each of the plant nursery container, isolator and analyzer, including a controller operably connected to the vertical plant farming tower to at least partially automate or completely automate the vertical plant farming.

8. The modular enclosure of any one of claims 1-7, wherein the transducted plant delivery component undergo an expansion reaction while in the conditioner, and wherein the biological product exhibits pharmaceutical properties suitable for just-in- time delivery of a therapeutic system specific to an individual condition at a site of care.Aty Ref. 340466: 63-24 WO9. The modular enclosure of any one of claims 1-8, further comprising: a loader comprising a natural or synthetic agent, wherein the loader is operably connected to the isolator, wherein the loader is configured to load the plant delivery component with the natural or synthetic agent.

10. The modular enclosure of claim 9, wherein the natural or synthetic agents comprises a nutrient, a medication, an antibody, a polynucleotide, a polypeptide, a protein, a carrier, or a combination thereof.

11. The modular enclosure of claim 10, wherein the medication comprises cannabidiol, carboplatin, Withaferin A, Withaferin B, Withaferin C, Withaferin D, acetaminophen, ibuprofen, codeine, a volatile drug, a gas marble, a liquid marble (including a medical marble), an antibody, a nanobody, an aptamer, a nucleic acid, a flavonoid, or a combination thereof.

12. The modular enclosure of claim 11, wherein the gas marble comprises a noble gas.

13. The modular enclosure of any one of claims 9-12, further comprising an integrator comprising the biological product, wherein the integrator is operably connected to the loader, wherein the integrator is configured to integrate the biological product into the loaded plant delivery component that is a loaded PEV and / or a plant cellulose scaffold.

14. The modular enclosure of claim 13, wherein the biological product comprises a cell, a gene, a nucleic acid, a protein, fragments thereof, any antibody, or any combination thereof.

15. The modular enclosure of any one of claims 1-14, further comprising: a sterilizer operably connected to the loader, wherein the sterilizer is configured to sterilize the integrated loaded plant delivery component.

16. The modular enclosure of claim 15, further comprising: a packager operably connected to the sterilizer, wherein the packager is configured to package the sterilized plant delivery component, wherein the package is configured for delivery of the sterilized plant delivery component.

17. A method of making a therapeutic delivery system, the method comprising the steps of: providing a source of plant delivery components to a plant nursery container; growing the source of plant delivery components in the plant nursery container for a grow time;Aty Ref. 340466: 63-24 WO obtaining an isolated plant delivery component with an isolator configured to isolate plant delivery components; and characterizing the isolated plant delivery components with an analyzer configured to characterize the plant delivery components; thereby making the therapeutic delivery system.

18. The method of claim 17, wherein the growing step comprises growing a whole plant or culturing plant cells.

19. The method of claim 18, wherein the growing step comprises vertical plant farming, including at least partially automated or completely automated vertical plant farming.

20. The method of any one of claims 17-19, wherein: the obtaining of the plant delivery components occurs within a BSL-1 green room; the characterizing of the isolated plant delivery components occurs within the BSL-1 green room; and the method further comprises: collecting a biological material from a plant source; transducting the isolated plant delivery components with the biological material within an ISO8 clean room to make transducted plant delivery components; expanding the transducted plant delivery components within the ISO8 clean room to make expanded plant delivery components; harvesting a biological product from the expanded plant delivery components within the ISO8 clean room; loading the isolated delivery vehicle with a natural or synthetic agent within the ISO8 clean room; integrating the biological product into the loaded plant delivery vehicle within the ISO8 clean room to make an integrated and loaded plant delivery vehicle; sterilizing the integrated and loaded plant delivery vehicle within the ISO8 clean room to make a sterilized plant delivery vehicle; and packaging the sterilized plant delivery vehicle within the ISO8 clean room for delivery of the sterilized plant delivery vehicle.

21. The method of any one of claims 17-20, that is automated and does not require any active user-intervention.

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