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WO2017010945A1 - Micro-encapsulation de composés à l'intérieur de spores naturelles et de graines de pollen - Google Patents

Micro-encapsulation de composés à l'intérieur de spores naturelles et de graines de pollen Download PDF

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Publication number
WO2017010945A1
WO2017010945A1 PCT/SG2016/050333 SG2016050333W WO2017010945A1 WO 2017010945 A1 WO2017010945 A1 WO 2017010945A1 SG 2016050333 W SG2016050333 W SG 2016050333W WO 2017010945 A1 WO2017010945 A1 WO 2017010945A1
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WIPO (PCT)
Prior art keywords
spore
whole
compound
substance
spores
Prior art date
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Ceased
Application number
PCT/SG2016/050333
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English (en)
Inventor
Nam-Joon Cho
Michael Graeme POTROZ
Raghavendra Chaluvayya MUNDARGI
Jae Hyeon Park
Joshua Alexander JACKMAN
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Nanyang Technological University
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Nanyang Technological University
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Publication of WO2017010945A1 publication Critical patent/WO2017010945A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
    • A23P10/00—Shaping or working of foodstuffs characterised by the products
    • A23P10/30—Encapsulation of particles, e.g. foodstuff additives
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/08—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing solids as carriers or diluents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/0241—Containing particulates characterized by their shape and/or structure
    • A61K8/0279—Porous; Hollow
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/11—Encapsulated compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/92—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof
    • A61K8/922—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof of vegetable origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9706—Algae
    • A61K8/9722—Chlorophycota or Chlorophyta [green algae], e.g. Chlorella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9728—Fungi, e.g. yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9755—Gymnosperms [Coniferophyta]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9794—Liliopsida [monocotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/10—Washing or bathing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/10—General cosmetic use
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/20—Chemical, physico-chemical or functional or structural properties of the composition as a whole
    • A61K2800/28—Rubbing or scrubbing compositions; Peeling or abrasive compositions; Containing exfoliants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/41—Particular ingredients further characterized by their size
    • A61K2800/412—Microsized, i.e. having sizes between 0.1 and 100 microns
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/56—Compounds, absorbed onto or entrapped into a solid carrier, e.g. encapsulated perfumes, inclusion compounds, sustained release forms

Definitions

  • the compound or substance may be, e.g., a therapeutic agent, herb, nutraceutical, food substance, food supplement, herbicide, pesticide, cosmetic (e.g., a fragrance), disinfectant, cleaning agent, diagnostic agent, ink, antimicrobial substance, fuel.
  • the whole spore encapsulating the compound(s) or substance(s) is coated with or co-encapsulated with a hydrogel or other agent(s) to control the rate of release of the compound(s) or substance(s) from the spore.
  • provided herein are methods of producing whole spores encapsulating a compound(s) or substance(s).
  • the method further comprises coating the whole spore, or encapsulating the compound(s) or substance(s) with a hydrogel or other agent(s) to control the rate of release of the compound(s) or substance(s) from the spore.
  • formulations comprising either a whole spore, or a whole spore encapsulating a compound(s) or substance(s), and uses of those formulations.
  • Plant based spores, algae, and pollen grains represent a form of natural
  • Such natural packaging means are effective in protecting sensitive biological materials from environmental extremes in the form of prolonged desiccation, UV exposure, and predatory organisms.
  • a range of plants produce spores as a form of seed, which contains all the genetic material necessary to produce a new plant.
  • Such spores provide a ready-made capsule scaffold with high structural uniformity and a large internal cavity which may be used to encapsulate a wide range of materials. Human consumption of natural spores and pollen grains as biosupplements, homoeopathy medicine pave a way to explore these materials for encapsulation applications specific to therapeutic loading and release.
  • lycopodium clavatum is one species of the genus Lycopodium which produces spores and which has been identified to contain a range of promising phytochemicals for therapeutic applications ranging from stomach ailments to Alzheimer's disease.
  • Lycopodium spores provide a robust capsule structure and are commercially available in large quantities across globe and these spores are often used in traditional herbal medicine with a wide range of therapeutic benefits including improved osteogenesis, cognitive function, treatment of gastrointestinal disorders,
  • a major challenge in producing microencapsulated products is ensuring size monodispersity, which can have a large effect on drug release characteristics with respect to an intended target organ.
  • size monodispersity In addition to size monodispersity, having well-defined microstructures plays an important role in exploring widespread applications.
  • Most conventional materials processing techniques used for encapsulation such as emulsion solvent evaporation, spray drying, and chemical conjugation fail to reliably provide either size monodispersity or well- defined microstructures.
  • prior arts reported the use of processed empty exine microcapsules of spores and pollen for the encapsulation of drugs, vaccines, and MRI contrast agents. Producing these empty capsules is very tedious involving harsh chemical processing for prolonged days highly affecting the industrial costing inclusive of manpower, process and time duration. Thus, there is a need for new methods for microencapsulating various compounds and substances.
  • Camellia oil also known as tea seed oil is the actual green tea oil. Tea seed oil is a potentially healthy in more ways than one. It is great for cooking, and from nutritional point of view. Tea seed oil is used in a number of beauty products. This oil has been used as a cooking for centuries in Southern China and they make many more uses with it. The oil helps to prevent and smooth wrinkles and stretch marks. It is also used to strengthen and promote healthy growth of fingernails by massaging the oil into the nail. This product is also suitable for the formulation of cosmetic products designed to condition hair, and to treat and prevent hair damages. [0006] Camellia oil is extracted from the seeds of the tea plant. That makes it the real tea oil. Tea tree oil on the other hand does not come from the tea plant. It comes from the tree called Melaleuca alternifolia, which is native to Australia. There are some varieties of Camellia oil.
  • Camellia japonica oil also known as Japanese tea oil. However, this plant does not produce tea leaves. It is a flowering plant with red blooms. Its oil is known as tsubaki oil and it is used heavily in cosmetic applications.
  • Camellia sinensis oil This is the tea seed oil.
  • Camellia oleifera oil This is known as tea oil or Camellia oil.
  • the oil is extracted using solvent extraction or cold processing.
  • solvent extraction or cold processing One might hear about cold filtered oil, but that does not mean cold pressed oil. If the contents used to make oil are heated prior to oil extraction, it may change the chemical composition and properties of nutrients in that oil, which is often not natural.
  • tea tree oil the increasingly popular remedy for everything from spots to insect bites and vapour rubs, is under threat of being banned by the European Union. The EU has said that even small amounts of the undiluted oil could be unsafe and unstable after clinical trials found users risked rashes and allergies.
  • microbeads Today, a significant number of personal care products such as scrubs and toothpastes are known to contain thousands of minuscule balls of plastic called microplastics, or more specifically, microbeads. Over the years, microbeads have replaced traditional,
  • microbeads used in personal care products are mainly made of polyethylene (PE), but can be also be made of polypropylene (PP), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA) and nylon. Where products are washed down the drain after use, microbeads flow through sewer systems around the world before making their way into rivers and canals and ultimately, straight into the seas and oceans, where they contribute to the plastic soup.
  • microplastics are defined as: plastic pieces or fibres measuring less than 5 mm. The microbeads found in personal care products are almost always smaller than 1 mm
  • Plastic is produced in large quantities each year primarily because its applications in the modern world are infinite. According to the New York Times, approximately 300 million tons of plastic are produced globally each year. Due to its variety of uses and relatively low cost, plastic production is going to continue to increase for the future. It has reached the point where plastic pollution is accounting for an estimated $13 billion dollars of damage. In addition, micro-plastic beads have become a significant issue in the world's marine environments.
  • microplastics are a large industry that often goes unnoticed. They are used in many everyday items such as switches, sensors, and in lighting. Additionally, they are used as exfoliants in everyday cosmetic products such as face wash, moisturizer, lipstick, and toothpaste.
  • the term "microplastics” specifically refers to small pieces of plastic material that are found in the marine environment. In general, microplastics range in size from a few ⁇ to 500 ⁇ (or 0.50 mm), which is almost microscopic. Microplastics can originate from a variety of sources, including the production of plastic microbeads often found in cosmetics.
  • Cosmetic products containing microbeads are popular all around the world because consumers enjoy the clean feeling that they provide.
  • microbeads act as good exfoliants because they can be shaped into small spheres which are effective at removing excess oil and dirt on the surface of skin without being harsh or stripping the skin of its essential oils. It is important to note that a typical facial scrub contains approximately 350,000 microbeads. However, these microbeads, made of synthetic polymers like polyethylene and/or polypropylene plastic, are having an adverse effect on the environment.
  • plastic microbeads which are composed of organic polymers of polyethylene and/or polypropylene compounds.
  • plastics are non-degradable, which contributes to the problem of pollution in aquatic systems.
  • one solution is to incorporate the use of biodegradable plastics in the production of microbeads.
  • the process to make these plastics into spherically shaped microbeads is very similar to that of synthetic microbeads.
  • microbeads varying in composition, size, and shape, they also differ in hardness. The hardness depends on the particular application, however they should be sufficiently hard so that they cleanse the skin as desired.
  • traditional polypropylene and polyethylene microbeads are no longer an appropriate option for cosmetic products.
  • a whole spore described herein is engineered to encapsulate a compound or substance of interest and coated with an agent to facilitate controlled release of the compound or substance of interest from the whole spore.
  • an whole spore described herein is engineered to encapsulate a compound or substance of interest and an agent to facilitate controlled release of the compound or substance of interest from the whole spore
  • an whole spore is a Abies spore, a Agrocybe spore, a
  • an whole spore has a size in the range of 0.5 ⁇ to 300 ⁇ . In another embodiment, an whole spore has a size in the range of 40 ⁇ to 100 ⁇ . In another embodiment, an whole spore has a size in the range of 1 ⁇ to 40 ⁇ . In another embodiment, an whole spore has a size in the range of 1 ⁇ to 80 ⁇ .
  • the compound or substance of interest is a therapeutic agent.
  • the therapeutic agent is a small organic molecule, a peptide, a nucleic acid, a protein, a polymer, a biologies, a medicinal preparation of proteins, a herbal medicine, an inorganic compound, an organometallic compound, lithium, a platinum-based agent, or gallium.
  • the compound or substance of interest is an oil.
  • the compound or substance of interest is a fragrance.
  • the compound or substance of interest is a cleaning agent.
  • the compound or substance of interest is a disinfectant agent.
  • the compound or substance of interest is a pesticide.
  • the compound or substance of interest is a herb. In another embodiment, the compound or substance of interest is a food ingredient. In one embodiment, the food ingredient is a caffeine. In another embodiment, the compound or substance of interest is a herbicide. In another embodiment, the compound or substance of interest is a fuel.
  • a formulation comprising the whole spore and a diluent or carrier.
  • a formulation comprising the whole spore and a diluents or pharmaceutically acceptable carrier.
  • the formulation is for topical administration.
  • the formulation is for parenteral administration.
  • a method for of treating a disease or condition in a subject comprising the formulation, wherein the therapeutic agent encapsulated in the whole spore is beneficial for treating the disease or condition.
  • provided herein is a cosmetic product or personal care product comprising the whole spore.
  • a food or drink product comprising the whole spore.
  • a herbal product comprising the whole spore.
  • a pesticide In another aspect, provided herein is a herbicide.
  • a method for masking the taste of a compound or substance comprising encapsulating the compound or substance in a whole spore and formulating that in a drink or food product.
  • the encapsulation comprises contacting the compound or substance with the whole spore.
  • the encapsulation comprises contacting the compound or substance with the whole spore under vacuum pressure.
  • the method further comprises coating the whole spore with agent for controlling the release of the compound or substance from the spore.
  • a method of improving the stability of a compound or substance comprising encapsulating the compound or substance in a naturally occurring whole spore.
  • the encapsulation comprises contacting the compound or substance with the whole spore.
  • the encapsulation comprises contacting the compound or substance with the whole spore under vacuum pressure.
  • the method further comprises coating the whole spore with an agent for controlling the release of the compound or substance from the whole spore.
  • a method of exfoliating skin comprising contacting the skin of a subject with a formulation comprising a whole spore.
  • a method of exfoliating skin comprising contacting the skin of a subject with a formulation comprising a whole spore engineered to encapsulate a compound or substance that is beneficial or useful in a cosmetic or personal care product.
  • a method of reducing the toxicity of a compound or substance comprising encapsulating the compound or substance in a naturally occurring whole spore.
  • the encapsulation comprises contacting the compound or substance with the whole spore.
  • the encapsulation comprises contacting the compound or substance with the whole spore.
  • the method further comprises coating the whole spore with an agent for controlling the release of the compound or substance from the whole spore.
  • a method of preparing a formulation comprising a compound or substance of interest and the whole spore comprising encapsulating the compound or substance of interest in the whole spore.
  • FIGS. 1A-1F Schematic of natural lycopodium spores and processing techniques to encapsulate biomacromolecules.
  • FIG. 1A Spore microstructure depicting uniform ridges distributed on the surface with natural sporoplasm constituents contained inside, these spores originate from a vascular plant with spirally arranged leaves.
  • FIG. IB Natural spores suspended in a biomacromolecule solution for the uptake of macromolecules, the enlarged insert depicts macromolecule entry via nanochannels located within the lycopodium microstructure.
  • FIG. 1C Spores encapsulating biomacromolecules are indicated along with the natural sporoplasm constituents. The three different microencapsulation techniques used are represented.
  • FIG. 1A Spore microstructure depicting uniform ridges distributed on the surface with natural sporoplasm constituents contained inside, these spores originate from a vascular plant with spirally arranged leaves.
  • FIG. IB Natural spores suspended in a biomacromolecule solution
  • FIG IE Passive macromolecule loading technique involving the incubation of natural spores in the aqueous macromolecule solution at 4°C under stirring at 500 rpm.
  • FIG IE Compression technique involving the compression of a dry spore powder and incubating the resulting spore tablet in the macromolecule solution for the uptake of macromolecules by the spores.
  • FIG IF Vacuum loading technique involving the application of a vacuum to a suspension containing natural spores and macromolecules, whereby the biomacromolecules enter the spores through the nanochannels located within the surface microstructures of natural spores.
  • FIG. 2A-2D FlowCam measurements: Polymer microspheres standard (50 ⁇ 1 ⁇ ) (Thermoscientific, USA).
  • FIG. 2A Representative histogram of equivalent spherical diameter vs. frequency using 1000 highly focused image analysis after measurement with particle count of 5000 with ESD of 49.65 ⁇ 0.91 ⁇ .
  • FIG. 2B Representative graph from histogram of circularity vs. frequency indicating microspheres very near to ideal circle value (1).
  • FIG. 2C Histogram of edge gradient vs. frequency indicating highly focused microspheres.
  • FIG. 2D Representative image of microspheres at 20 X magnification with FC200 flow cell at flow rate of 0.1 ml/min.
  • FIGS. 3A-3H Characterization of natural lycopodium spores before and after biomacromolecule loading by FlowCam ® : Size and circularity by dynamic imaging particle analysis (DIPA, FlowCam ® ) with a particle count of 10,000 spores before and after BSA- loading. Representative graphs from curve fitting to histograms for equivalent spherical diameter, circularity and edge gradient for spores before and after encapsulation of
  • FIGS. 3E, 3F, 3G, and 3H represent natural spores before loading, as well as, passive, compression, and vacuum loading techniques captured by FlowCam ® at 20x magnification, respectively.
  • FIG. 4 Characterization of natural lycopodium spores before and after
  • FIG. 4 A, FIG. 4 B, FIG. 4C, and FIG. 4D represent natural spores before loading, as well as, passive, compression, and vacuum loading techniques captured by FESEM (JEOL, Japan).
  • FIGS. 5A-5D Confocal microscopy analysis of natural lycopodium spores before and after biomacromolecule loading:
  • CLSM images in the row of FIG. 5A are natural lycopodium spores before BSA-loading. These natural spores exhibit autofluorescence due to the presence of terpenoid, phenolic, and carotenoid molecules.
  • the spore's natural sporoplasm constituents are observed as microglobules inside the spore in both the blue and red channel along with the overlaid image of the natural spore without biomacromolecule loading, and there is also a clear absence of any green fluorescence.
  • FIG. 5B depicts BSA-loaded spores using the passive loading technique.
  • the row of FIG. 5C depicts BSA-loaded spores using the compression loading technique.
  • the row of FIG. 5D depicts BSA-loaded spores using the vacuum loading technique. All of these spore microparticles exhibit a green colour due to the presence of FITC-BSA in the green channel, and the overlaid images indicate the presence of spore constituents along with encapsulated biomacromolecules. (Scale bars are 10 ⁇ ).
  • FIGS. 6A-6B Z-stack images from confocal laser scanning microscopy (CLSM) showing 35 optical sections of an L. clavatum spore after FITC-BSA loading (FIG. 6A) and before FITC-BSA loading (FIG. 6B).
  • CLSM confocal laser scanning microscopy
  • FIGS. 8A-8C CLSM images after FITC-BSA release from natural spores prepared by different techniques in pH 7.4 media: the row of FIG. 8A: Passive loading technique; the row of FIG. 8B: Compression loading technique; and the row of FIG. 8C: Vacuum loading technique, (scale bars are 10 ⁇ ).
  • FIGS. 9A-9C Scanning electron microscopic images of L. clavatum spores after coating. Images represent 0.5% alginate coated spores (FIG. 9A), 1% alginate coated spores (FIG. 9B), and 2% alginate coated spores (FIG. 9C), respectively.
  • FIGS. 10A-10D Schematic of natural sunflower pollen grains processing to encapsulate macromolecules by different techniques.
  • FIG. 10A Dried natural pollen grains exhibiting a characteristic oval shape with uniform spikes on the surface.
  • FIG. 10B Pollen grains suspended in an aqueous solution of macromolecules for encapsulation by passive, compression and vacuum techniques.
  • FIG. IOC A fully hydrated natural pollen grain loaded with macromolecules are indicated along with the original pollen contents.
  • FIG. 10D A fully hydrated natural pollen grain after the release of macromolecules from the natural pores within the pollen grain walls.
  • FIGS. 1 lA-11C Characterization of natural sunflower pollen before and after BSA-loading by FlowCam : Size and circularity by dynamic imaging particle analysis (DIPA, FlowCam ® ) with a particle count of 10,000 pollen grains before and after BSA-loading.
  • DIPA dynamic imaging particle analysis
  • FIG. 11 A Equivalent spherical diameter vs. Frequency
  • FIG. 11B Circularity vs. Frequency
  • FIG. 11C Edge gradient vs. Frequency.
  • FIGS. 12A-12D Characterization of natural sunflower pollen before and after BSA-loading by SEM: Images in FIGS. 12A, 12B, 12C, and 12D represent natural pollen grains before loading as well as, after passive, compression, and vacuum loading techniques captured in FlowCam ® at 20x magnification, respectively.
  • FIGS. 13A-13D Characterization of natural sunflower pollen before and after BSA-loading by SEM: FIG. 13 A represents natural pollen grains before loading and the images in FIGS. 13B, 13C, and 13D, respectively, indicate macromolecule loaded pollen grains by passive, compression, and vacuum loading techniques captured by FESEM (JEOL, Japan).
  • FIGS. 14A-14D Confocal microscopy analysis of natural sunflower pollen grains before and after macromolecule loading: CLSM images in the row of FIG. 14A are natural sunflower pollen grains before BSA-loading. In the row of FIG. 14B, BSA-loaded pollen grains using the passive loading technique. In the row of FIG. 14C, BSA-loaded pollen grains using the compression loading technique. In the row of FIG. 14D, BSA-loaded pollen grains using the vacuum loading technique. (Scale bars are 10 ⁇ ).
  • FIGS. 15A-15B Z-stack images from confocal laser scanning microscopy showing 50 optical sections of a pollen grain after FITC-BSA loading (FIG. 15A), and before FITC-BSA loading (FIG. 15B).
  • FIGS. 16A-16C In-vitro release profiles: Simulated intestinal fluid, pH 7.4 media (FIG. 16A), Simulated gastric fluid, pH 1.2 media (FIG. 16B), Release profile of BSA-loaded pollen grains coated with alginate (FIG. 16 C).
  • FIGS. 17A-17D Scanning electron microscope images of pollen grains after alginate coating. Images in the rows of FIGS. 17A and 17B represent 0.1 % alginate coated pollen grains after 1 min and 10 min incubation times and images in the rows of FIG. 17C and FIG. 17D represent 0.5 % alginate coated pollen grains after 1 min and 10 min incubation times.
  • FIGS. 18A-18D Scanning electron microscope images of pollen grains before and after 2 % alginate coating.
  • FIGS. 18A and 18B Natural pollen grain before coating process.
  • FIG. 18C Intact pollen grain after alginate coating.
  • FIG. 18D Represents pollen surface covered with alginate.
  • FIGS. 19A-19C CLSM images after FITC-BSA release from pollen grains prepared by different techniques in pH 7.4 media: Passive technique (row in FIG. 19A),
  • FIGS. 20A-20C Characterization of 5-FU loaded L. clavatum spore formulations. Diameter, circularity, aspect ratio and edge gradient were analyzed by dynamic imaging particle analysis (DIPA) with a 1000 particle count. Representative graphs with standard deviation from three measurements and curve fitting to histograms are presented as diameter vs. frequency (FIG. 20A), circularity vs. frequency (FIG. 20B), aspect ratio v. frequency (FIG. 20C), and edge gradient vs. frequency (FIG. 20D).
  • DIPA dynamic imaging particle analysis
  • FIGS. 21A-21D Dynamic imaging particle analysis images of 5-FU loaded L. clavatum spores. Images in FIGS. 21A, 21B, 21C, and 21D represent L. clavatum spores before and after 5-FU loading by passive, compressive, and vacuum loading techniques, respectively.
  • FIGS. 22A-22D Characterization of 5-FU loaded L. clavatum spores by SEM.
  • SEM images in FIGS. 22A, 22B, 22C and 22D represent L. clavatum spores before loading (FIG. 22A) and after loading by passive (FIG. 22B), compression (FIG. 22C), and vacuum (FIG. 22D) loading techniques.
  • FIGS. 23A-23B Characterization of Eudragit RS 100-coated L. clavatum spores by SEM.
  • FIG. 23A 5-FU loaded spores after coating with 2.5% Eudragit RS 100.
  • FIG. 23B 5-FU loaded spores after coating with 10% Eudragit RS 100.
  • FIGS. 24A-24C In vitro release profiles of 5-FU loaded L. clavatum spores.
  • FIG. 24 A Cumulative release profiles of 5-FU loaded spores by passive, compression, and vacuum loading (FIG 24 A) in simulated gastric fluid (SGF pH 1.2) and (FIG. 24B) simulated intestinal fluid (SIF), pH 7.4 phosphate buffered saline.
  • FIGS. 25A-25C Characterization of BSA loading in natural pine pollen based on conventional vacuum loading protocols.
  • FIG. 25A SEM images depicting surface cleanliness of BSA loaded pine pollen in relation to washing.
  • FIG. 25B Encapsulation efficiency and loading efficiency in relation to washing.
  • FIG. 25C CLSM images depicting localization of FITC-BSA.
  • FIG. 26 CLSM images depicting short-term passive loading trends for FITC-BSA in natural pine pollen.
  • FIG. 27 Long-term passive loading trends for BSA / FITC-BSA in natural pine pollen. CLSM images depicting uptake and localization of FITC-BSA during passive loading.
  • FIG. 28 Confocal laser scanning microscopy (CLSM) depicting short-term passive loading trends for FITC-BSA in natural camellia pollen.
  • FIGS. 29 A- 29B Confocal laser scanning microscopy (CLSM) analysis of L. clavatum spores before and after calcein loading.
  • FIG. 29A CLSM images in the first row indicate spores with sporoplasm.
  • FIG. 29B The CLSM images in the second row indicate calcein loading into spores. Scale bars are 10 ⁇ .
  • FIG 30 Confocal laser scanning microscopy (CLSM) images of Camellia seed oil and nile read dye blend in Camellia pollen based formulation.
  • FIGS. 31A-31B Size and morphological characterization of Camellia pollen grains and sporopollenin exine capsules (SECs).
  • FIG. 31 A FlowCam analysis of Camellia pollen and SECs.
  • FIG. 3 IB FlowCam analysis of Camellia pollen (left) and SEC (right).
  • FIGS. 32A-32B Characterization of caffeine (CF)-loaded L. clavatum spores by SEM.
  • FIG. 32A Spores before CF-loading.
  • FIG. 32B Spores after CF-loading with coencapsulant ERS. Scale bars are 10 ⁇ .
  • FIGS. 33A-33B Confocal laser imaging microscopy (CLSM) analysis of L.
  • FIG. 33A depict spores with sporoplasm.
  • FIG. 33B depicts CF-Calcein loading into spores with coencapsulant ERS.
  • FIG. 34 In vitro release profiles of caffeine (CF) from L. clavatum spore formulations. Spores-CF physical mixture and CF-loaded with co-encapsulant ERS in simulated salvia fluid (SSF).
  • FIGS 35A-35B Taste masking evaluation of caffeine formulations.
  • FIG. 35A Bitterness score from human volunteers during bitterness threshold test.
  • FIG. 35B Human volunteer score with CF formulated with a physical mixture L. clavatum spores and ERS.
  • FIG. 36 Contact angle data for UV-Ozone treated camellia pollen showing a decrease in contact angle with increasing UV-Ozone treatment duration.
  • FIG. 37 Scanning electron microscopy images of Camellia pollen grains treated or untreated with UV/Ozone. Treated means that the pollen has been defatted and treated with UV- Ozone exposure.
  • FIG. 38 Aqueous suspensions of untreated and of UV-Ozone treated Camellia pollen.
  • FIG. 39 Scanning electron microscopy images of untreated and UV-Ozone treated Camellia SECs. Treated means that the pollen has been defatted and treated with UV-Ozone exposure.
  • FIG. 40 Aqueous suspensions of untreated and of UV-Ozone treated Camellia SECs.
  • FIG. 41A-41C Aqueous suspensions.
  • FIG. 41A Camellia seed oil and water.
  • FIG. 41B Camellia SECs oil loaded, ethanol washed and UV-Ozone treated.
  • FIG. 41C Aqueous suspensions.
  • FIG. 41A Camellia seed oil and water.
  • FIG. 41B Camellia SECs oil loaded, ethanol washed and UV-Ozone treated.
  • FIG. 41C Aqueous suspensions.
  • FIG. 41A Camellia seed oil and water.
  • FIG. 41B Camellia SECs oil loaded, ethanol washed and UV-Ozone treated.
  • FIG. 41C Aqueous suspensions.
  • FIG. 41A Camellia seed oil and water.
  • FIG. 41B Camellia SECs oil loaded, ethanol washed and UV-Ozone treated.
  • FIG. 41C Aqueous suspensions.
  • FIG. 41A Camellia seed oil and water.
  • FIG. 41B Camellia SECs oil loaded,
  • FIG. 42 Macromolecular encapsulation in Camellia pollen grains and SECs.
  • FIG. 43 Process schematic to obtain defatted natural pollen grains.
  • FIG. 44 SEM images of whole spore microbead examples.
  • whole spores engineered to encapsulate a compound or substance of interest In another aspect, provided herein are whole spores engineered to encapsulate a compound or substance of interest and coated with an agent to facilitate controlled release of the compound or substance from the whole spores. In another aspect, provided herein are whole spores engineered to encapsulate a compound or substance of interest and an agent with the compound or substance to facilitate controlled release of the compound or substance from the whole spores.
  • a whole spore engineered to encapsulate a compound or substance of interest has no significant amount of the compound or substance adhered to the surface of the whole spore. "No significant amount” in this context means that the exterior of the whole spore maintains its natural architectural features and surface appearance on a microscale (e.g., surface roughness) as assessed with a degree of high confidence using standard measuring techniques known in the art, for example, scanning electron microscopy.
  • a whole spore engineered to encapsulate a compound or substance of interest with a percentage of the compound or substance adhered to the surface of the whole spore.
  • the whole spore engineered to encapsulate a compound or substance of interest has more than 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the compound or substance adhered to the surface of the whole spore.
  • a whole spore engineered to encapsulate a compound or substance of interest maintains the general size, shape and morphology of the whole spore without encapsulation of the compound or substance.
  • a whole spore engineered to encapsulate a compound or substance of interest maintains the general morphology of the whole spore without encapsulation of the compound or substance.
  • a whole spore engineered to encapsulate a compound or substance of interest maintains the general size of the whole spore without encapsulation of the compound or substance.
  • a whole spore engineered to encapsulate a compound or substance of interest maintains the general shape of the whole spore without encapsulation of the compound or substance.
  • a whole spore engineered to encapsulate a compound or substance of interest swells in size relative to the whole spore without
  • the whole spore engineered to encapsulate a compound or substance of interest swells to more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 125%, 150%, 175% or 200% the size of the whole spore without encapsulation of the compound or substance.
  • a whole spore is engineered to encapsulate a compound or substance of interest to localize the effect of the compound or substance to the site where the whole spore is applied.
  • a whole spore is used to encapsulate a compound or substance of interest to protect the compound or substance from harsh conditions
  • a whole spore is used to encapsulate a compound or substance of interest in order to stabilize the compound or substance.
  • a whole spore is used to encapsulate a compound or substance of interest to reduce the toxicity of the compound or substance in a subject.
  • a whole spore is used to encapsulate a compound or substance of interest to mask the taste of the compound or substance.
  • a whole spore is used to encapsulate a compound or substance of interest in order to control the release of the compound or substance.
  • a whole spore is used to co-encapsulate a compound or substance of interest and an agent that allows for a modified release rate of the compound or substance.
  • a whole spore is used to encapsulate a compound or substance of interest and is coated with an agent that allows for a modified release rate of the compound or substance.
  • biologically active substances such as potent pesticides, herbicides, and fertilizers that are used in agriculture, require methods that allow for their stability and release rates to be modified to minimize their environmental impact.
  • whole spores derived from naturally occurring sources are readily available, at low cost, and in large quantities. While whole spores derived from naturally occurring sources typically sell for about $20 to $30 per kg, extracting exine capsules raises the production costs to about $3500 to $35,000 per kg. Commercially available extracted exine capsules sell typically for about $200,000 per kg. [0084] Other advantages and improvements of whole spores over existing methods, devices or materials, for example, include:
  • Camellia oil can be encapsulated in aqueous formulations.
  • Plant-based spores and pollen grains from different types of specific plant species are microscale particles that are naturally produced, abundant in renewable supply, highly monodisperse per species, mechanically strong, chemically resilient, biodegradable,
  • the plant-based spores and pollen grains are biodegradable and permit organic recycling.
  • ⁇ Hydrophilic/hydrophobic properties of the surface coating can be controlled in order to support water filtration and prevent clogging.
  • ⁇ The surface roughness of spores and grains from different species is variable and can be used in different cosmetic applications.
  • Personal care products such as lip balm, deodorant, eyeliner, lipstick, lotion, mouthwash, shampoo, conditioner, make up, shaving cream, toothpaste, and numerous others are all are used on the human body for personal hygiene and/or beatification.
  • Many of these products contain synthetic plastics, which have already been proven to negatively affect the environment. Incorporating biodegradable and/or natural microbeads into personal care products will reduce aquatic pollution while serving the same purpose as synthetic plastics.
  • Plant-based spores and pollen grains have very similar properties to plastic microbeads, which give them potential as a natural microbead in cosmetics. Indeed, microbeads must be spherically shaped in order to properly exfoliate and cleanse the skin of the consumer.
  • non-biodegradable plastics that are currently being used in personal care products range in size anywhere from 10 ⁇ to 100 ⁇ (0.01 mm-0.10 mm). All of these features lend plant-based spores and pollen grains great potential as substitutes for the aforementioned commercial applications.
  • the term "whole” in the context of a spore means a spore that comprises an exine shell, an intine layer and cytoplasmic organelles therein.
  • the term "whole” in the context of a spore excludes any spore consisting of or consisting essentially of only an exine shell or a fragment thereof. In other words, a whole spore contains additional components that are missing from an exine shell alone.
  • a whole spore retains more than 50% (preferably, more than 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) of the main components of a spore, e.g., extine shell, intine layer, and cytoplasmic organelles.
  • the whole spore comprises an exine shell, an intine layer, cytoplasmic organelles and other components found in nature associated with a spore (e.g., endexine, nexine, proteins, lipids, nucleic acids, etc.).
  • a whole spore has a size in the range of 0.5 ⁇ to 300 ⁇ . In some embodiments, a whole spore has a size in the range of 1 ⁇ to 100 ⁇ . In certain embodiments, a whole spore has a size in the range of 10 ⁇ to 100 ⁇ . In some embodiments, a whole spore has a size in the range of 3 ⁇ to 80 ⁇ . In certain embodiments, a whole spore has a size in the range of 0.5 ⁇ to 40 ⁇ . In some embodiments, a whole spore has a size in the range of 40 ⁇ to 300 ⁇ .
  • a whole spore has a size in the range of 0.5 ⁇ to 300 ⁇ . In some embodiments, a whole spore has a size in the range of 40 ⁇ to 100 ⁇ . In certain embodiments, a whole spore has a size in the range of 1 ⁇ to 40 ⁇ .
  • a whole spore comprises a component(s) that is beneficial to a subject.
  • a whole spore comprises a component(s) that is of therapeutic value.
  • a whole spore has detoxification properties.
  • exine shell means the acetolysis-resistant biopolymeric (e.g., sporopollenin) outer coating of a spore or pollen grain.
  • the exine shell of a spore can be isolated by techniques known in the art, including, e.g., successive treatments with organic solvents, alkali, acid and/or enzymes so as to remove the other components of the spore, such as the cellulosic initine layer and lipid, protein and nucleic acid components that may be attached to or contained within the exine shell.
  • the exine shell takes the form of an essentially hollow capsule, which typically comprises sporopollenin.
  • spore refers not only to true spores, commonly defined as a unit of asexual reproduction, including endospores, and as such as are produced by nonflowering plants, bacteria, fungi, algae, ferns, and mosses, but also pollen grains, commonly defined as a unit of sexual reproduction, and as such are produced by seed-bearing plants (spermatophytes).
  • pollen grains commonly defined as a unit of sexual reproduction, and as such are produced by seed-bearing plants (spermatophytes).
  • the spore is pollen.
  • the spore is bee pollen, tree pollen, flower pollen, pine pollen or grass pollen.
  • the spore is a plant spore.
  • the spore is a fungal spore.
  • the spore is a bacterial spore.
  • the spore is an Abies spore, Agrocybe spore, Aspergillus niger spore, Bacillus subtilis spore, Cantharellus minor spore, Epicoccum spore, Cuburbita spore, Cucurbitapapo spore, Ganomerma spore, Lycopodium clavatum spore, Myosotis spore, Penicillium spore, Periconia spore, ryegrass spore, Timothy grass spore, maize spore, hemp spore, rape hemp spore, wheat spore, Urtica dioica spore, sunflower spore ⁇ e.g., Helianthus annuus spore), pine spore ⁇ e.g., Pinus taeda spore), corn spore ⁇ e.g., Zea mays spore), cattail spore ⁇ e.g., Typha angusti
  • a whole spore used in accordance with the disclosure herein is a naturally occurring spore.
  • naturally occurring in the context of a spore means that the spore is produced by a living organism found in nature.
  • a whole spore used in accordance with the disclosure herein is derived from a naturally occurring source(s).
  • naturally occurring source(s) is a living organism found in nature.
  • the naturally occurring source(s) is a plant(s), bacteria, fungi, algae, fern(s), moss(es) or other spore-producing organism(s), whether prokaryotic or eukaryotic.
  • the naturally occurring organism is a plant(s), fern(s) or moss(es). In another embodiment, the naturally occurring organism is a bacteria. In another embodiment, the naturally occurring organism is algae. In another embodiment, the naturally occurring organism is fungi. In some embodiments, the naturally occurring organism is Abies, Agrocybe, Aspergillus niger, Bacillus subtilis, Cantharellus minor, Epicoccum,
  • Cuburbita Cucurbitapapo , Ganomerma, Lycopodium clavatum, Myosotis, Penicillium,
  • Baccharis
  • the naturally occurring organism is a species described in the Example Section, infra. In one specific embodiment, the naturally occurring organism is Lycopodium clavatum or another species from the same family. In another specific embodiment, the naturally occurring organism is camellia. In another specific embodiment, the naturally occurring organism is pine.
  • a whole spore used in accordance with the disclosure herein is derived from a genetically engineered living organism that produces spores.
  • the genetically engineered living organism is a plant(s), bacteria, fungi, algae, fern(s), moss(es) or other spore-producing organism(s), whether prokaryotic or eukaryotic.
  • the genetically engineered living organism is a plant(s), fern(s) or moss(es).
  • the genetically engineered living organism is a bacteria.
  • the genetically engineered living organism is algae.
  • the genetically engineered living organism is fungi.
  • the genetically engineered living organism is a genetically engineered version of Abies, Agrocybe, Aspergillus niger, Bacillus subtilis, Cantharellus minor, Epicoccum, Cuburbita, Cucurbitapapo ,
  • Ganomerma Lycopodium clavatum, Myosotis, Penicillium, Periconia, ryegrass, Timothy grass, maize, hemp, rape hemp, wheat, Urtica dioica, sunflower (e.g., Helianthus annuus), pine (e.g., Pinus taeda), corn (e.g., Zea mays), cattail (e.g., Typha angustifolia), rape (e.g., Brassica napus), dandelion (e.g., Taraxacum offinale), rye (e.g., Secale cereale), Eastern Baccharis (e.g.,
  • the genetically engineered living organism is a genetically engineered version of a species described in the Example Section, infra.
  • the genetically engineered living organism is a genetically engineered version of Lycopodium clavatum or another species from the same family.
  • the genetically engineered living organism is a genetically engineered version of camellia.
  • the genetically engineered living organism is a genetically engineered version of pine pollen.
  • the genetically engineered organism may be a naturally occurring organism that has been genetically engineered to have a beneficial property.
  • the genetically engineered organism is a naturally occurring organism that has been genetically engineered to reduce production one or more allergens (e.g. , allergenic proteins).
  • the genetically engineered organism is a naturally occurring organism that has been genetically engineered to have an altered form of a protein known to be an allergen.
  • the genetically engineered organism is a naturally occurring organism that has been genetically engineered to produce a higher than normal amount of spores.
  • a whole spore used in accordance with the disclosure herein is isolated from a naturally occurring source or a genetically engineered organism that produces spores.
  • the whole spore is isolated from a biological matrix containing non-spore contaminants as well.
  • non-spore contaminants include, but are not limited to plant-based or natural debris such as fragments of soil, stone, branches, leaves, flower petals, waxes, resins, nectar, and the like.
  • Techniques for isolating of a whole spore are known to those of skill in the art and include, e.g. , sieving the matrix to isolate the spores and remove the non-spore contaminants.
  • the isolation of a whole spore includes cleaning the spore of contaminants and cleaning any surface-adhered compounds of the spore.
  • the isolated whole spore is 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% free of non-spore contaminants.
  • a whole spore used in accordance with the disclosure herein has been modified structurally.
  • the whole spore may be modified after isolation from a naturally occurring source or a genetically engineered organism that produces spores.
  • one, two, three or all of the following structural features of a whole spore may be modified: (i) the surface of the spore may modified (e.g., the surface roughness may be altered), (ii) the size of the spore may be modified, (iv) the shape of the spore may be modified and/or (v) the spore's structural robustness may be modified (e.g., the spore's resistance to mechanical pressure has been strengthened or weakened).
  • Structural features of the spore may be modified using any technique known in the art so long as the components of the whole spore remain.
  • structural features of a spore may be modified by exposure to a buffer, a certain pH or pH range, or a certain temperature or temperature range. See, e.g. , Section 5.4 and the Example Section, infra, for methods for modifying the structural features of a whole spore as well as whole spores that have undergone such modifications.
  • a whole spore used in accordance with the disclosure herein has been subjected to processing either prior to, during or post-isolation from a naturally occurring source or a genetically engineered organism that produces spores.
  • the spore may be processed in any way so long as the components of a whole spore remain.
  • a whole spore used in accordance with the disclosure herein has been subjected to exposure to a solvent.
  • the solvent is an organic or inorganic solvent.
  • the organic solvent is methyl ether, ethyl ether, diethyl ether, acetone, ethanol, methanol, N-methyl pyrrolidone, dimethyl formamide, dichlorome thane, ethylene glycol dimethyl ether, dimethylformamide, methyl sulfoxide, ethyl acetate, trifluoroacetic acid, tetrahydrofuran, any likewise organic solvent, or combinations thereof.
  • the solvent is water, and the processing of the spore includes washing the spore after the whole spore has been isolated.
  • a whole spore used in accordance with the disclosure herein has been subjected to a washing step.
  • the washing step may occur prior to or after the spore has been subjected to a treatment, such as a chemical treatment (e.g., a solvent).
  • the washing step may occur after isolation of the whole spore from a naturally occurring source or a genetically engineered organism that produces spores.
  • the washing includes removing surface adhered contaminants and/or naturally occurring surface adhered lipid-like compounds, typically referred to as pollenkitt, from the spore.
  • a whole spore used in accordance with the disclosure is defattened to minimize the spore's allergenicity.
  • a "defattened" spore(s) refers to a spore(s) that has its surface proteins or other surface adhered contaminants removed.
  • defattened spores are obtained by washing the spores in organic solvent, for example, ethyl ether.
  • camellia oil and camellia pollen grains or derivatives thereof are dissolved in water or other aqueous suspension at an oikpollen mass ratio of 10: 1 or lower.
  • the oil and dry pollen grains can be mixed until a homogenous slurry is formed.
  • the oil is encapsulated inside the pollen grains which can be achieved by freeze-drying of the sample or other loading method known in the art.
  • the pollen grains can also be treated with ultraviolet (UV) light and ozone in order to render them hydrophilic and therefore soluble in aqueous suspensions.
  • UV ultraviolet
  • the preferred UV light and ozone treatment occurs at atmospheric pressure with UV light generated at both 185 nm and 254 nm wavelengths.
  • the oil encapsulated inside the pollen grains can be mixed with water in order to permit aqueous suspensions of Camellia green tea oil or other oil of choice.
  • the pollen grains are first treated with UV-ozone before loading occurs in order to optimize loading.
  • a whole spore used in accordance with the disclosure herein has not been subjected to processing either prior to, during or post-isolation from a naturally occurring source or a genetically engineered organism that produces spores.
  • a whole spore used in accordance with the disclosure herein has not been subjected to exposure to a solvent, such as an organic solvent (e.g., methyl ether, ethyl ether, diethyl ether, acetone, ethanol, methanol, N-methyl pyrrolidone, dimethyl formamide, dichloromethane, ethylene glycol dimethyl ether, dimethylformamide, methyl sulfoxide, ethyl acetate, trifluoroacetic acid, tetrahydrofuran, any likewise organic solvent, or combinations thereof) or inorganic solvent.
  • an organic solvent e.g., methyl ether, ethyl ether, diethyl ether, acetone, ethanol, methanol, N-methyl pyrrolidone, dimethyl formamide, dichloromethane, ethylene glycol dimethyl ether, dimethylformamide, methyl sulfoxide, ethyl acetate, trifluoroace
  • a whole spore used in accordance with the disclosure herein is not considered allergic.
  • pollen allergies are due to a high level of airborne exposure to pollen combined with a genetic tendency of the allergic subject.
  • Pollen grains used in food products typically result in minimal cases of allergic response, since they are ingested orally, rather than through the respiratory system.
  • pollens are generally recognized as safe (GRAS) by the U.S. Food and Drug Administration.
  • a whole spore selected for encapsulation depends upon, inter alia, the compound or substance to be encapsulated in the whole spore, the formulation comprising the whole spore, and the intended use of the formulation.
  • a whole spore larger than 40 ⁇ is used to encapsulate a food product or component thereof, or an herbal medicine.
  • pine pollen, corn pollen, or rye pollen is used as a whole spore to encapsulate a food product or component thereof, or an herbal medicine.
  • a whole spore selected for encapsulation depends upon, inter alia, the compound or substance to encapsulated in the whole spore, the formulation comprising the whole spore, and the intended use of the formulation.
  • a whole spore smaller than 40 ⁇ (but larger than zero) is used to encapsulate a therapeutic.
  • a Lycopodium clavatum spore, sunflower pollen, or Camellia pollen is used as the whole spore to encapsulate a therapeutic.
  • sunflower pollen is used as the whole spore to encapsulate a compound or substance of interest ⁇ e.g., a therapeutic) for targeted intestinal delivery.
  • a compound or substance of interest e.g., a therapeutic
  • any compound or substance of interest may be encapsulated in whole spores.
  • the term "encapsulate” and cognates thereof in the context of the whole spore means to take up a compound or substance by sorption, adhesion or bond, whether or not chemical or physical in nature, within the inner core of the whole spore.
  • the term “encapsulate” is used interchangeably used with the terms “load” or “take up” and cognates thereof.
  • the term “sorption” and cognates thereof refer to absorption and adsorption.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is a therapeutic(s), a cosmetic product(s) or a component thereof, a personal care product(s) or a component thereof, a processed food(s) or a component thereof, a processed drink(s) or a component thereof, an agricultural product(s) or a component thereof, a household product(s) or a component thereof, toiletry product(s) or a component thereof, or a probe(s).
  • the compound or substance of interest is a therapeutic.
  • therapeutics include, but are not limited to small organic molecules, biologies, medicinal preparation of proteins, herbal medicines, inorganic and organometallic compounds (such as, lithium, platinum-based agents, gallium, and heavy metals), wound or burn healing agents, antiinflammatory agents, anti-irritants, antimicrobial agents (which can include antifungal and antibacterial agents), vitamins, vasodilators, topically effective antibiotics and antiseptics, or any other medicine.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is hormone, antibody, cytokine, chemotherapeutic agent, or other agent useful or beneficial for treating a disease.
  • the therapeutic is 5 -fluorouracil.
  • the therapeutic is not 5-fluorouracil.
  • a compound(s) and/or substance(s) for encapsulation in the whole spores is a therapeutic other than 5- fluorouracil.
  • a compound(s) and/or substance(s) for encapsulation in the whole spores is a therapeutic agent other than a chemotherapeutic agent.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is an ink.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is diagnostic agent.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is an antimicrobial substance. In some embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is a small molecule. In certain embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is biomolecule. In certain embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is a macromolecule.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is a cosmetic product or a component thereof.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is a personal care product or a component thereof.
  • Different spores can be selected for use in cosmetic and person care products, as e.g. , microbeeads, depending, inter alia, on the type of product, because different whole spores have different surface roughness.
  • cosmetic and personal care products include, but are not limited to, makeup products (for example, foundations, powders, blushers, eye shadows, eye and lip liners, lipsticks, other skin colourings and skin paints), skin care products (for example, cleansers, moisturisers, emollients, skin tonics and fresheners, exfoliating agents and rough skin removers), fragrances, perfume products, essential oils, sunscreens, UV protective agents other than sunscreens, self tanning agents, after-sun agents, anti-ageing agents, anti- wrinkle agents, skin lightening agents, topical insect repellants, hair removing agents, hair restoring agents, or nail care products (such as nail polishes or polish removers).
  • a perfume product may comprise more than one fragrance.
  • a cosmetic and personal care substance includes a high quality bioactive ingredients or compounds having cosmetic and personal care properties.
  • a cosmetic and personal care substance encapsulated in a whole spore is a compound that protects a subject from oxidation or UV light.
  • a cosmetic and personal care substance encapsulated in a whole spore is flavor, aroma, nutrient, fragrance, phytochemical, or therapeutic.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is a processed food or a component thereof, or a processed drink or a component thereof.
  • Processed foods or drinks include, for example, food additives, health food and supplements, flavours, aromas, nutrients, bioactives, or phytochemicals.
  • health food and supplements include nutrients or dietary supplements (such as vitamins, minerals, folic acid, omega-3 oils, fish oils, fibres, and so-called "probiotics" or "prebiotics”).
  • one, two or more of the following compounds or substances are encapsulated in a whole spore: a food additive(s), a health food, a food
  • a compound(s) and/or substance(s) for encapsulation in whole spores is a food additive.
  • food additives include acids, acidity regulators, anticaking agents, antifoaming agents, antioxidants, bulking agents, food coloring, color retention agents, emulsifiers, flavors, flavor enhancers, flour treatment agents, glazing agents, humectants, tracer gas, preservatives, stabilizers, sweeteners, thickeners, or thickening agents. Accordingly, in certain embodiments, one, two or more of these compounds or substances are encapsulated in a whole spore.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is a flavour.
  • flavours include natural flavoring substances, nature-identical flavoring substances, or artificial flavoring substances.
  • flavours are selected from a group of flavorings consisting of diacetyl, acetylpropionyl, acetoin, isoamyl acetate, benzaldehyde, cinnamaldehyde, ethyl propionate, methyl anthranilate, limonene, ethyl decadienoate, allyl hexanoate, ethyl maltol, ethylvanillin, and methyl salicylate.
  • flavours include salts, sugars or artificial sweeteners.
  • flavours include savory flavorants, for example, amino acids and nucleotides, in the form of sodium or calcium salts.
  • flavours are sour additives, such as organic and inorganic acids.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is caffeine. In other embodiments, a compound(s) and/or substance(s) for encapsulation in whole spores is not caffeine.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is an aroma(s).
  • aromas include esters, linear-terpenes, cyclic-terpenes, aromatic, amines, alcohols, aldehydes, ketones, lactones, or thiols.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is a nutrient.
  • nutrients include carbohydrates, proteins, fats, dietary minerals, vitamins, or dietary fiber. Accordingly, in certain embodiments, one, two or more of these compounds or substances are encapsulated in a whole spore
  • a compound(s) and/or substance(s) for encapsulation in whole spores is a bioactive compound or substance.
  • bioactives include fatty acids, flavonoids, caffeine, carotenoids, carnitine, choline, coenzyme Q, creatine, dithiolthiones, phytosterols, polysaccharides, phytoestrogens, glucosinolates, polyphenols, lipids, anthocyanins, prebiotics, or taurine. Accordingly, in certain embodiments, one, two or more of these compounds or substances are encapsulated in a whole spore
  • a compound(s) and/or substance(s) for encapsulation in whole spores is a phytochemical.
  • phytochemicals include, but are not limited to, terpenoids (for example carotenoids, triterpenoid, monoterpenes, steroids), phenolic compounds (for example natural monophenols, polyphenols, aromatic acids, phenylethanoids),
  • glucosinolates for example, isothiocyanate precursors, aglycone derivatives, organosulfides, organosulfur compounds, and indoles
  • betalains for example betacyanins, betaxanthins
  • chlorophylls organic acids, amines, carbohydrates (for example, monosaccharides, and polysaccharides) and protease inhibitors. Accordingly, in certain embodiments, one, two or more of these compounds or substances are encapsulated in a whole spore
  • a compound(s) and/or substance(s) for encapsulation in whole spores is a fuel. In some embodiments, a compound(s) and/or substance(s) for
  • encapsulation in whole spores is a disinfectant or cleaning agent.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is a lipid, proteinaceous agent (e.g., protein, polypeptide or peptide), fatty acid or carbohydrate.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is an agricultural product or a component thereof.
  • the agricultural product is a pesticide or a fertilizer.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is a pesticide.
  • pesticides include chemically-related pesticides or pest specific formulations. Chemically-related pesticides include, for example, organophosphate pesticides, carbamate pesticides, organochlorine insecticides, pyrethroid pesticides, or sulfonylurea herbicides.
  • Pest specific formulations include, for example, algicides, antifouling agents, antimicrobials, attractants, biopesticides, biocides, disinfectants, sanitizers, fungicides, fumigants, herbicides, insecticides, miticides, microbial pesticides, molluscicides, nematicides, ovicides, pheromones, repellents, or rodenticides. Accordingly, in certain embodiments, one, two or more of these pesticides are encapsulated in a whole spore.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is a fertilizer.
  • fertilizers include nitrogen fertilizers, phosphate fertilizers, potassium fertilizers, compound fertilizers, organic fertilizers or elemental compounds, for example, calcium, magnesium, and sulfur.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is a compound and/or substance found in a household product.
  • household products include surface cleaners, disinfectants and other antimicrobial agents, fragrances, perfume products, air fresheners, insect and other pest repellants, laundry products (e.g. , washing and conditioning agents), fabric treatment agents (including dyes), cleaning agents, UV protective agents, dishwashing products, paints, varnishes, inks, dyes and other colouring products, and adhesive products.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is a compound and/or substance found in a toiletry product.
  • toiletry products include soaps; detergents and other surfactants; deodorants and antiperspirants;
  • a compound(s) and/or substance(s) for encapsulation in whole spores is a probe.
  • probes include fluorescence-tagged molecules.
  • probes include bovine serum albumin (BSA), calcein, or fluorescein isothiocyanate (FITC)-conjugated.
  • BSA bovine serum albumin
  • FITC fluorescein isothiocyanate
  • a compound(s) and/or substance(s) for encapsulation in whole spores is a plant extract.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is a lipophilic compound(s) (e.g. , an oil(s)).
  • a compound(s) and/or substance(s) for encapsulation in whole spores is a traditional herbal medicine.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is a modern pharmaceutical(s).
  • a compound(s) and/or substance(s) for encapsulation in whole spores is an oil.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is an oil.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is an oil.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is an oil.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is an oil.
  • Camellia japonica oil Japanese tea oil
  • Camellia sinensis oil tea seed oil
  • Camellia oleifera oil tea oil
  • a compound(s) and/or substance(s) for encapsulation in whole spores is a compound or substance disclosed in the Example Section, infra.
  • a compound(s) and/or substance(s) for encapsulation in whole spores is not found in nature to be associated with the whole spore.
  • a whole spore is loaded with any suitable amount of the compound or substance of interest.
  • the suitable amount of a compound or substance will depend on, inter alia, the intended use of a formulation comprising the whole spore and the compound or substance encapsulated in the whole spore.
  • the formulation includes the whole spore and the compound or substance at a weight ratio of from 100: 1 to 1: 1.
  • a larger whole spore may be needed to encapsulate a larger amount of the compound or substance.
  • only one compound or substance of interest is encapsulated in the whole spore. In other embodiments, two or more compounds or substances of interest are encapsulated in the whole spore.
  • the encapsulated compound or substance is retained within cavities of the whole spore. In some embodiments, the encapsulated compound or substance is preferably retained within a central cavity of the whole spore. In some embodiments, a percentage of the encapsulated compound or substance is attached to a surface of the whole spore. In some embodiments, the percentage of encapsulated compound or substance attached to the surface of the whole spore is less than 5% by weight of the entire encapsulated amount of the compound or substance.
  • agents for controlling the rate of release of a substance or compound of interest encapsulated in a whole spore include coating agents or co-loading agents.
  • coating agents include waxes, butters, starches, rosins, resins, hydrogels, alginate and polysaccharides.
  • agents include hydroxypropyl methyl cellulose, methyl cellulose, sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate, polyethylene oxide, xanthan gum, Eudragit, Carbomers, oils and waxes and methacrylate copolymers.
  • the coating may be a natural coating, such as starches, waxes, resins, rosins, etc.
  • a synthetic polymer coating is used for controlled release and improved product stability.
  • co-loading agents include glycerol, hydrogels, glucose and oils.
  • the co-loading agent is a viscous loading solution having a viscosity that is greater than the viscosity of water.
  • hydrogels for controlling the rate of release of a substance or compound of interest include water- swelling polymers.
  • ionic hydrogel polymers as well as non-ionic hydrogel polymers (e.g., non-ionic hydrophilic hydrogel polymers) can be used.
  • a pharmaceutical-suitable homo-polymer hydrogel such as a polymer polymerized from the same type of monomers without cross-linking to two or more different kinds of monomers, a polymer with the same kind of side chains, a non-copolymer
  • a formulation includes the whole spore, a substance or compound of interest, and about 4% to 80% by weight of the non-cross-linked, water-swelling homo-polymer.
  • non-cross-linked, water-swelling homo-polymer examples include, but are not limited to, hydroxypropyl methylcellulose (HPMC, e.g., METHOCELTM, etc.), alginate, sodium alginate, cellulose hydrogel, polyvinylpyrrolidone, hydroxypropyl cellulose (HPC; e.g., KLUCELTM, etc.), nitrocellulose, hydroxypropyl ethylcellulose, hydroxypropyl butylcellulose, hydroxypropyl pentylcellulose, methyl cellulose, hydroxyethyl cellulose, alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose acetate, carboxymethyl cellulose, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, poly-hydroxyalkyl methacrylate, polymethacrylic acid, polymethylmethacrylate, poly vinyl alcohol, sodium polyacrylic acid, calcium polyacrylic acid, polyacrylic acid, acidic acidic
  • the agent for controlling the release rate of a compound or substance of interest from a whole spore is an agent described in the Example Section, infra.
  • the agent for controlling the release rate of a compound from a whole spore is alignate.
  • the agent for controlling the release rate of a compound or substance of interest from a whole spore decreases the rate of release of the compound or substance from the spore.
  • the presence of the agent reduces the rate of release of the compound by 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 percent relative to the rate of release of the compound or substance from a whole spore not encapsulated or coated with the agent under identical conditions.
  • the presence of the agent results in a release rate within 1 hour that is reduced by 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 percent relative to the rate of release of the compound or substance within 1 hour from a whole spore not encapsulated or coated with the agent under identical conditions.
  • the presence of the agent reduces the cumulative release of the compound by 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 percent relative to the cumulative release of the compound or substance from a whole spore not encapsulated or coated with the agent under identical conditions.
  • the presence of the agent results in cumulative release within 1 hour that is reduced by 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 percent relative to the cumulative release of the compound or substance within 1 hour from a whole spore not encapsulated or coated with the agent under identical conditions.
  • the agent for controlling the release rate of a compound or substance of interest from a whole spore prolongs the release time of the compound or substance from the spore.
  • the presence of the agent prolongs the release time of the compound by 20 min, 30 min, 40 min, 50 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 15 h, 20 h , 25 h, or 30 h relative to the release time of the compound or substance from a whole spore not encapsulated or coated with the agent under identical conditions.
  • the total release time of the compound or substance from the spore encapsulated or coated with the agent is 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 15 h, 20 h , 25 h, or 30 h.
  • Total release time in this context refers to the time period of continuous release until at least 95% of the encapsulated compound or substance are released from the spore.
  • the total release time of the compound or substance from the spore encapsulated or coated with the agent is 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 15 h, 20 h, 25 h, or 30 h without a burst effect.
  • Burst effect in this context refers to a release of at least 90% of the encapsulated compound or substance within the first 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 min from the onset of the release.
  • the coating agent, co-loading agent, hydrogel or other agent for controlling the release rate of a compound or substance of interest is engineered to co- encapsulate with the substance or compound in the whole spore.
  • a formulation includes a whole spore, an compound or substance of interest encapsulated within the whole spore, and a co-encapsulated agent for controlling the rate of release of the substance or compound from the whole spore.
  • the formulation includes one or more co-encapsulated release controlling agents.
  • the co-encapsulated agent is retained within cavities of the whole spore. In some embodiments, the co-encapsulated agent is preferably retained within a central cavity of the whole spore. In some embodiments, a percentage of the encapsulated agent is attached to a surface of the whole spore. In some embodiments, the percentage of co- encapsulated agent attached to the surface of the whole spore is less than 5% by weight of the entire encapsulated amount of the compound or substance.
  • the step of co-encapsulating the agent is performed as a concurrent or separate step to encapsulating the compound or substance in the whole spore.
  • co-encapsulating includes one or more distinct processing steps.
  • the agent for controlling the rate of release of a compound or substance of interest from a whole spore coated on the whole spore includes the whole spore, a compound or substance of interest encapsulated in the whole spore, and an agent for controlling the rate of release of the substance or compound from the whole spore, wherein the whole spore is coated with the agent.
  • the coat of a whole spore includes a microbead coat.
  • the microbead coat includes alginate microbeads.
  • modifying the properties of a whole spore includes modifying the structural features of the spore.
  • Structural features of the spore include, for example, the size, shape or composition of the spore.
  • modifying structural features of the spore includes modifying the surface of spore, for example, the surface roughness, altering the size or shape of the spore, or modifying the spore's structural robustness, for example by strengthening or weakening the spore's resistance to mechanical pressure.
  • modifying the mechanical robustness of the spore comprises using chemical processing.
  • chemical processes that structurally modify spores includes controlled application of acids, alkalis, oxidative processes, and solvents.
  • chemical processing that exposes the spore to acid or alkali compounds that alters the exine polymer structure, causing the exine shell to fracture more easily may be used. If the mechanical robustness of the spores is decreased it may allow for more rapid spore breakdown, and more rapid release of the loaded compounds.
  • the chemical processing alters outer exine shell polymer structure of the spore, while maintaining the structural integrity of the spore. For example, oxidative processes degrade the exine polymer and cause the exine to fracture more easily.
  • Other processing examples are treatments with fused potassium hydroxide, and in oxidizing mixtures such as hypochlorite/hydrochloric acid, potassium dichromate/sulphuric acid, hydrogen peroxide/sulphuric acid, and ozone.
  • Other examples includes solvents (e.g. , 2-aminoethanol, 3-animopropanol, 2,2'2"-nitriltriethanol, and 4-methylmorpholine-N-oxide) that soften and eventually dissolve the exine polymer shell of the spores.
  • modifying the properties of a whole spore includes exposing the whole spore to UV light to increase hydrophilicity of the whole spores.
  • exposure to UV light may alter the spore's hydrophilicity by changing its surface chemistry by converting hydrophobic surface proteins into their hydrophilic counterparts. See, e.g., the Example Section, infra, regarding UV light exposure.
  • hydrophilic and/or hydrophobic properties of a whole spore are controlled and modified by coatings in order to support water filtration and prevent clogging of the spore.
  • encapsulating a compound and/or substance of interest in a whole spore as well as co-encapsulating a compound and/or substance of interest and an agent that controls the release rate of the compound and/or substance from the whole spore.
  • Any technique known to one of skill in the art may be used to encapsulate a compound and/or substance of interest in whole spore or co-encapsulate a compound and/or substance of interest and an agent that controls the release rate of the compound and/or substance from the whole spore.
  • a technique described in the Example Section, infra is used to encapsulate a compound and/or substance of interest in a whole spore, or co- encapsulate a compound and/or substance of interest and an agent that controls the release of the compound and/or substance from the whole spore.
  • a technique described in the Example Section, infra for a method for coating a whole spore with an agent that controls the release rate of an encapsulated compound and/or substance from the whole spore.
  • a method of encapsulating a compound or substance of interest in a whole spore comprises contacting the compound or substance with the whole spore.
  • the step of contacting the compound or substance with the whole spore comprises dissolving the compound or substance in a solvent, suspending the whole spore in the solution, and allowing the whole spore to encapsulate the compound or substance for a specific duration.
  • the method further comprises upon encapsulating of the compound or substance in the whole spore, removing the whole spore from the solution.
  • the method further comprises upon removing the whole spore from the solution, freezing and freeze-drying the whole spore.
  • the step of allowing the whole spore to encapsulate the compound or substance comprises mixing the solution and cooling the solution below room temperature.
  • the cooling temperature is about 4° Celsius.
  • the specific duration for allowing the whole spore to encapsulate the compound or substance is 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 2 hours, 3 hours, 4 hours, 5 hours or more, or 1 to 2 hours, 1 to 5 hours, 2 to 3 hours or 2 to 4 hours.
  • the removing the whole spore from the solution comprises centrifuging the solution. In some embodiments, centrifuging the solution is performed at 12000 rpm for a duration of 4 minutes.
  • a method for encapsulating a compound or substance of interest in a whole spore comprises compressing the whole spore into a tablet and contacting the tablet with the compound or substance.
  • the step of contacting the tablet with the compound or substance comprises dissolving the compound or substance in a solvent, soaking the tablet of the whole spore in the solution, and allowing the whole spore to encapsulate the compound or substance for a specific duration.
  • the method further comprises upon encapsulating of the compound or substance in the whole spore, removing the whole spore from the solution.
  • the method further comprises upon removing the whole spore from the solution, freezing and freeze-drying the whole spore.
  • the step of allowing the whole spore to encapsulate the compound or substance comprises mixing the solution and cooling the solution below room temperature. In some embodiments, the cooling temperature is about 4° Celsius. In some embodiments, the specific duration for allowing the whole spore to encapsulate the compound or substance is 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 2 hours, 3 hours, 4 hours, 5 hours or more, or 1 to 2 hours, 1 to 5 hours, 2 to 3 hours or 2 to 4 hours. In some embodiments, the step of removing the whole spore from the solution comprises centrifuging the solution. In some embodiments, the step of centrifuging the solution comprises the solution being centrifuged at 12000 rpm for a duration of 4 min.
  • the step of compressing the whole spore into a table comprises applying a compression pressure of 5 ton or at least 1 ton for a duration of at least 10 sec or 20 sec. In some embodiments, the step of compressing the whole spore into a table further comprises filling the whole spore into die and applying the compression pressure to the die.
  • a method for encapsulating a compound or substance of interest in a whole spore comprises contacting the compound or substance with the whole spore under vacuum pressure.
  • the step of contacting of the compound or substance with the whole spore under vacuum pressure comprises dissolving the compound or substance in a solvent, suspending the whole spore in the solution, applying a vacuum to the suspension, and allowing the whole spore to encapsulate the compound or substance for a specific duration.
  • the method further comprises upon encapsulating of the compound or substance in the whole spore, removing the whole spore from the solution.
  • the method further comprises upon removing the whole spore from the solution, freezing and freeze-drying the whole spore.
  • the step of allowing the whole spore to encapsulate the compound or substance comprises mixing the solution and cooling the solution below room temperature. In some embodiments, the cooling temperature is about 4° Celsius. In some embodiments, the specific duration for allowing the whole spore to encapsulate the compound or substance is 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 2 hours, 3 hours, 4 hours, 5 hours or more, or 1 to 2 hours, 1 to 5 hours, 2 to 3 hours or 2 to 4 hours. In some embodiments, the step of removing the whole spore from the solution comprises centrifuging the solution. In some embodiments, centrifuging the solution is performed at 12000 rpm for a duration of 4 min.
  • the step of applying a vacuum to the suspension comprises using a freeze-drier.
  • the vacuum includes a pressure of 2 mbar or at least less than 5 mbar.
  • a coating is generally used in the context of applying an agent to the whole spore's surface, while co-encapsulation includes filling at least some of spore's cavities with the agent. Any technique known to one of skill in the art can be used to coat a whole spore with an agent that controls the release rate of a compound or substance of interest from the whole spore.
  • a method for coating a whole spore with an agent that controls release of a compound or substance of interest from the whole spore comprises using individual particle coating or agglomerate particle coating to coat the agent on the whole spore.
  • individual particle coating includes spray coating, sputtering, or applying vapor deposition.
  • agglomerate particle coating includes pressing spore pellets and dip coating, spray coating, sputtering, or applying vapor deposition.
  • agglomerate particle coating comprises mixing the whole spores with a co- encapsulating compound or substance and solidifying the mixture, by various techniques, to form agglomerates that contain the whole spores and the compound or substance.
  • a method of assessing the encapsulation of a compound or substance in a whole spore comprises using a dynamic image particle analyzer to assess structural characteristics of the whole spore.
  • the assessed structural characteristics include uniformity, size, shape and micromeritic properties of the whole cell.
  • a dynamic image particle analyzer uses a high-resolution digital camera and objective lens to capture images of the particles, i.e. , the whole spore encapsulated with the compound or substance, flowing through a thin transparent flow cell. Particle size uniformity data is then generated based on digital signal processing of the images. Besides size determination, the digital particle images allows obtaining additional information including edge gradient, circularity, and the shape of whole spores.
  • size, edge gradient and circularity analysis by the DIPA is performed with an initial particle count of 10,000 whole spores for all the batch formulations and images are processed using software to obtain 1000 well focused whole spores.
  • representative data is plotted as a histogram and fitted with a Gaussian curve and values are reported with standard deviations.
  • DIPA is used as described in the Example Section, infra.
  • a method of assessing the encapsulation of a compound or substance in a whole spore comprises using a confocal laser scanning microscope to visualize the whole spore.
  • the method comprises mounting whole spores encapsulating the substance or compound on a sticky slide.
  • the method comprises measuring the fluorescence from the compound or substance encapsulated in the whole spore.
  • the compound or substance is a fluorescence probe or a fluorescence probe- tagged molecule.
  • the compound or substance is a fluorescently labeled version of a compound described in Section 5.2, supra.
  • the compound or substance is FITC-conjugated BSA, fluorescein, 5 -fluorouracil, or calcein.
  • the compound or substance is not is FITC-conjugated BSA, fluorescein, 5- fluorouracil, or calcein.
  • confocal laser scanning microscopy is used as described in the Example Section, infra.
  • a method for determining the amount of compound or substance encapsulated in a whole spore comprises: (1) rupturing the compound or substance loaded whole spores; (2) incubating the ruptured whole spores in a solution to allow for maximum compound release into the solution; (3) separating the mass of the whole spore from the solution containing the compound by filtration; (4) using spectrographic analysis of the solution containing the compound (for example, UV spectroscopy) to determine light absorption properties of the solution containing the compound; and (5) comparing the determined light absorption properties against a standard absorption curve to determine the amount of compound or substance, wherein standard absorption curve is obtained from light absorption data collected from a series of solutions with a known amount of the compound.
  • the method for determining the amount of compound or substance encapsulated in a whole spore further comprises repeating steps (l)-(5) using a placebo and subtracting the determined light absorption properties of the placebo from the determined light absorption properties of the whole spore prior to comparing the determined light absorption properties against a standard absorption curve to determine the amount of compound or substance.
  • the additional step ensures an increased accuracy in determining the amount of compound or substance.
  • the amount of compound or substance in the whole spore, the percentage of compound or substance loading, and the percentage of encapsulation efficiency are determined by:
  • a method for determining a weight ratio of a whole spore to a compound or substance encapsulated in the whole spore comprises using the method for determining the amount of compound or substance encapsulated in the whole spore to determine an amount of compound or substance and an amount of the whole spore, wherein the whole spore amount is measured from the separated mass of the whole spore in the above step (3).
  • the ratio is given by the amount of compound or substance : the amount of the whole spore. For example, 4 mg of compound and 6 mg of whole spore gives a ratio of 1 : 1.5.
  • a method for assessing the controlled release rate of a compound or substance encapsulated in a whole spore comprises incubating a formulation of the whole spore in a solution, allowing release of the encapsulated compound or substance into the solution, and determining the amount of released compound using standard analytical chemistry techniques.
  • standard analytical chemistry techniques include, for example, UV spectrometry.
  • a method for assessing the controlled release rate of a compound or substance encapsulated in a whole spore comprises performing steps (2)-(5) of the method for determining the amount of compound or substance encapsulated in a whole spore, wherein the incubation of the whole spores is stopped at a fixed time point.
  • a method for assessing allergies in subjects comprises performing allergy blood testing or skin prick testing.
  • the method further comprises exposing the subject to a whole spore or a compound or substance-encapsulated whole spore. Exposure, for example, includes skin contact, inhalation or ingestion. Most pollen allergies are typically related to inhalation exposure.
  • the method comprises determining the response of a subject's skin upon contacting the compound or substance with the skin.
  • formulations comprising whole spores, whole spores encapsulating a compound or substance of interest, or whole spores co-capsulating a compound or substance of interest and an agent that facilitates controlled release of the compound or substance, and uses thereof.
  • a formulation comprises whole spores encapsulating a compound or substance of interest.
  • a formulation comprises whole spores co-encapsulating a compound or substance and an agent to facilitate controlled release of the compound or substance from the whole spore.
  • a formulation comprises whole spores encapsulating a compound or substance and coated with an agent to facilitate controlled release of the compound or substance from the whole spore.
  • a formulation described herein further comprises one or more additional agents, such as a fluid vehicle(s), an excipient(s), a diluents(s), a carrier(s), a stabilizer(s), a surfactant(s), a penetration enhancer(s) or other agents for targeting delivery of the whole spore and/or the compound or substance of interest to the intended site of
  • additional agents such as a fluid vehicle(s), an excipient(s), a diluents(s), a carrier(s), a stabilizer(s), a surfactant(s), a penetration enhancer(s) or other agents for targeting delivery of the whole spore and/or the compound or substance of interest to the intended site of
  • a formulation comprises a whole spore(s) and a diluent or carrier. In another embodiment, a formulation comprises a whole spore(s) and a diluents(s) or pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to non-toxic carrier.
  • a formulation comprises whole spores encapsulating a compound or substance of interest and a diluents or pharmaceutically acceptable carrier.
  • a formulation comprises whole spores co-encapsulating a compound or substance and an agent to facilitate controlled release of the compound or substance from the whole spore and a diluents(s) or pharmaceutically acceptable carrier.
  • a formulation comprises whole spores encapsulating a compound or substance and coated with an agent to facilitate controlled release of the compound or substance from the whole spore and a diluents(s) or pharmaceutically acceptable carrier.
  • a formulation described herein may comprise a weight ratio of compound or substance of interest to whole spore of from 0.0001 : 1 to 50: 1, such as from 0.001: 1 to 5: 1, 0.01: 1 to 5: 1, 0.1: 1 to 5: 1, or 0.5: 1 to 50: 1.
  • suitable formulations are prepared by methods commonly employed using conventional, organic or inorganic additives or carriers, such as an excipient (e.g., sucrose, glucose, lactose, cellulose, sorbitol, talc, mannitol, calcium phosphate, starch, or calcium carbonate), a binder (e.g., cellulose, hydroxymethylcellulose, methylcellulose, polyvinylpyrrolidone, polypropylpyrrolidone, gum arabic, gelatin, polyethyleneglycol, starch, or sucrose), a disintegrator (e.g., starch, hydroxypropylstarch, carboxymethylcellulose, low substituted hydroxypropylcellulose, calcium phosphate, sodium bicarbonate, or calcium citrate), a lubricant (e.g., magnesium stearate, talc, light anhydrous silicic acid, or sodium lauryl sulfate), a flavoring agent (e.g., citric acid
  • an excipient e
  • a whole spore, a whole spore encapsulating a compound or substance, a whole spore co-encapsulating a compound or substance and coated with an agent to facilitate controlled release of the compound or substance from the whole spore, or a whole spore encapsulating a compound or substance and coated with an agent to facilitate controlled release of the compound or substance from the whole spore will vary depending on the intended use.
  • a formulation for administration to a subject may vary depending upon the route of administration to a subject.
  • the formulations described herein can be administered by any route known to one of skill in the art.
  • a formulation described herein can be orally, parenterally, intradermally, intramuscularly, intraperitoneally, percutaneously, intravenously, subcutaneously, intranasally, epidurally, sublingually, intracerebrally, intravaginally,
  • a formulation described herein is administered to a subject orally.
  • a formulation described herein is administered to a subject parenterally (e.g., subcutaneously, intramuscularly or intravenously).
  • administration is left to the discretion of the health-care practitioner, and can depend in-part upon the site of the medical condition or the type of whole spore or the compound or substance.
  • a formulation may for example take the form of a lotion, cream, ointment, paste, gel, foam, a hydrogel lotion, a skin patch or any other physical form known for topical administration, including for instance a formulation which is, or may be, applied to a carrier such as a sponge, swab, brush, tissue, skin patch, dressing or dental fibre or tape to facilitate its topical administration. It may take the form of a viscous or semi-viscous fluid, or of a less viscous fluid such as might be used in sprays (for example nasal sprays), drops (e.g. eye or ear drops), aerosols or mouthwashes.
  • a topical formulation is a cosmetic or therapeutic lotion.
  • the whole spore described herein may be formulated as a composite powder-like material. This composite powder-like material is incorporated into a wide range of foods or drinks, processed foods, food supplements, etc.
  • provided herein are products comprising a formulation described herein. In yet another aspect, provided herein are products comprising the whole spore.
  • formulations for certain types of products such as, e.g. , pharmaceutical products; herbal or nutraceutical products; personal healthcare products; cosmetics and personal care products (e.g. bath products, soaps, hair care products; nail care products, and dental products such as toothpastes, dentifrices, mouthwashes and dental flosses); food and drink products (including food and beverage additives and ingredients); and pesticides, herbicides and fertilizers; household products (whether for internal or external use, including surface cleaners, disinfectants and other antimicrobial agents, fragrances, perfume products, air fresheners, insect and other pest repellants, pesticides, laundry products (e.g. , washing and conditioning agents), fabric treatment agents (including dyes), cleaning agents, UV protective agents, dishwashing products, paints, varnishes, inks, dyes and other colouring products, and adhesive products); agricultural and horticultural products
  • toiletry products including soaps; detergents and other surfactants; deodorants and antiperspirants; lubricants; fragrances; perfume products; dusting powders and talcum powders; hair care products such as shampoos, conditioners and hair dyes; and oral and dental care products such as toothpastes, mouth washes and breath
  • a whole spore, a whole spore encapsulating a compound or substance, a whole spore co-encapsulating a compound or substance and coated with an agent to facilitate controlled release of the compound or substance from the whole spore, or a whole spore encapsulating a compound or substance and coated with an agent to facilitate controlled release of the compound or substance from the whole spore may be added or included in any formulation known to one of skill in the art, including those described herein.
  • formulations of a whole spore encapsulating a compound or substance of interest are used in shower gels, toothpastes, mouthwash and face cleansers, to cure skin complaints, aging and stretch marks, to treat cuts and burns, and/or as an insect and lice repellent.
  • a formulation of whole spores encapsulating a compound or substance of interest comprises a plurality of Camellia pollen and oils for cosmetic and dermatology applications.
  • the formulation comprises an effective quantity of Camellia japonica pollen grains or equivalent with one or more types of Camellia oil in order to reduce skin irritation and optimize therapeutic properties.
  • the natural microsphere carrier for the pollen carries the added benefit of providing a therapeutic effect due to the components found in its natural composition
  • formulations for personal care products as described herein comprise a whole spore or a whole spore encapsulating a compound or substance of interest.
  • a method of treating a disease or condition in a subject comprising administering to the subject a formulation comprising a whole spore encapsulating a compound or substance beneficial to treating the disease or condition (e.g., a therapeutic, herbal medicine or nutraceutical).
  • a formulation comprising a whole spore encapsulating a compound or substance beneficial to treating the disease or condition (e.g., a therapeutic, herbal medicine or nutraceutical).
  • encapsulated in the whole spore is beneficial for treating the disease or condition.
  • the formulations described herein are for treating treating skin or skin structure conditions (for example, acne, psoriasis or eczema), wound or burn healing, treating anti-inflammatory diseases or conditions, and/or use as anti-irritants or antimicrobial agents (including antifungal and antibacterial agents).
  • subject refers to a patient, such as an animal, a mammal or a human, who has been the object of treatment, observation or experiment and is at risk of (or susceptible to) developing a disease or condition.
  • a method for protecting a compound or substance of interest from heat, light (including UV light), water, oxygen, oxidizing agents or conditions, and other environmental hazards examples include: (1) protection from atmospheric effects, in particular from light and/or oxygen, and therefore from premature degradation; (2) physical protection to help reduce loss of the compound or substance by for instance evaporation, diffusion or leaching; (3) good uniformity in size, shape and surface properties, unlike typical synthetic encapsulating entities; (4) significant variation in spore size and shape between different species, allowing a formulation to be tailored dependent on the nature and desired concentration of the compound or substance, the site and manner of its intended application, the desired release rate, the likely storage conditions prior to use; (5) granularity providing an exfoliating effect; (6) protection against toxic or adverse effect of compound or substance by physically shielding the compound or substance from contact until release commences; (7) antioxidant for encapsulated compound or substance; and (8) tastelessness allowing taste masking of the compound or
  • the use of a whole spore encapsulating a compound or substance of interest in a formulation modifies the hydrophobicity, nitrogen/oxgen plasma, etc. of the compound or substance. In some embodiments, the use of a whole spore encapsulating a compound or substance of interest in a formulation improves the dispersion characteristics of the compound or substance.
  • kits for improving the stability of a compound or substance of interest comprising encapsulating the compound or substance in a whole spore. Stability of a compound or substance can be assessed by technology known in the art.
  • oxidation includes aerial oxidation.
  • oxidative stability may be measured by measuring the rate of change in a parameter such as peroxide value. Additionally or alternatively, oxidative stability may be measured by measuring the rate of change of redox potential, thiobarbituric acid value, iodine value, anisidine value, TOTOX value (defined as two times the peroxide value added to the anisidine value) and/or free fatty acid content, and/or by the RANCIMAT, active oxygen or Schaal oven test methods, or by any other suitable test method.
  • Other methods for determining oxidative stability includes using an oxidative stability instrument (OSI) or an oxidograph, which are automated versions of the more complicated AOM (active oxygen method).
  • OSI oxidative stability instrument
  • AOM active oxygen method
  • RANCIMAT method has become the most established and accepted into a number of national and international standards.
  • kits for reducing the toxicity of a compound or substance comprising encapsulating the compound or substance in a naturally occurring whole spore.
  • the method allows for targeting a location of a subject's body for release of the compound or substance.
  • the methods allows for lowering the required amount of compound or substance to be administered to a subject.
  • the whole spore is co-encapsulated with an agent that controls the rate of release of the compound or substance spore.
  • the whole spore is coated with an agent that controls the rate of release of the compound or substance from the spore. Any technique known to one of skill in the art can be used to assess the ability of the compound or substance-encapsulated whole spore to reduce the toxicity of the compound or substance.
  • allergies to a formulation for administration is tested before use.
  • a compound or substance of interest e.g., a nutrient, phytochemical or bioactive molecule
  • methods for masking the taste of a compound or substance of interest comprising encapsulating the compound or substance in a naturally occurring whole spore and formulating the whole spore in a drink or food.
  • the whole spore is co- encapsulated with an agent that controls the rate of release of the compound or substance spore.
  • the whole spore is coated with an agent that controls the rate of release of the compound or substance from the spore.
  • Any technique known to one of skill in the art e.g., surveys
  • described herein see, e.g., the Example Section, infra
  • the method of encapsulating hydrophobic materials comprises:
  • provided herein are methods for exfoliating skin comprising contacting the skin of a subject with a formulation comprising a whole spore.
  • methods for exfoliating skin comprising contacting the skin of a subject with a formulation comprising a whole spore engineered to encapsulate a compound or substance that is beneficial or useful in a cosmetic or personal care product. Any technique known to one of skill in the art can be used to assess the ability of a whole spore or a compound or substance- encapsulated whole spore to exfoliate skin.
  • a whole spore is used as a microbead.
  • a microbead may be used for any application in which plastic microbeads are used, e.g., cosmetics, toothpastes, hair products, etc.
  • the whole spore used as a microbead is engineered to encapsulate a compound or substance of interest (e.g., a compound or substance that is beneficial or useful in a cosmetic or personal care product). SEM images of examples of whole spore microbeads are illustrated in FIG. 44.
  • biomacromolecules in addition, tunable release has been achieved by various alginate coatings of spores to achieve several release profiles.
  • This study provides a unique approach to utilize natural spores with unique materials properties, such as size uniformity and well-defined microstructures, as an advanced material for biomacromolecules encapsulation for controlled and targeted release applications.
  • Plant based spores represent one form of natural encapsulation, and a wide range of specific plant species which produce spores are commonly found in the natural world. ⁇ 1 ' 21 Such natural packaging means are effective in protecting sensitive biological materials from environmental extremes in the form of prolonged desiccation, UV exposure, and predatory organisms. [3] A range of plants produce spores as a form of seed, which contains all the genetic material necessary to produce a new plant. [4 ' 5] Such spores provide a ready-made capsule scaffold with high structural uniformity and a large internal cavity which may be used to encapsulate a wide range of materials.
  • Lycopodium clavatum is one species of the genus Lycopodium which produces spores and which has been identified to contain a range of promising phytochemicals for therapeutic applications ranging from stomach ailments to Alzheimer's disease.
  • Lycopodium spores provide a robust capsule structure and are commercially available in large quantities across globe.
  • Lycopodium spores are often used in traditional herbal medicine with a wide range of therapeutic benefits including improved osteogenesis, improved cognitive function, [12] treatment of gastrointestinal disorders, [8] hepatoprotective activity, [13] and antioxidative properties.
  • a major challenge in producing microencapsulated products is ensuring size monodispersity, [26 ' 27] which can have a large effect on drug release characteristics with respect to
  • biomacromolecule-loaded spores using three different microencapsulation techniques.
  • the techniques we have developed to utilize natural spores are simple, cost effective, and versatile and can be applied to the development of several encapsulation products to overcome limitations of current encapsulated products while providing well-defined micromeritic properties.
  • the specific scientific rationalities of the present work are i). Encapsulation of macromolecules into natural spores as biomaterials and the retention of natural spores constituents, ii).
  • this study demonstrates the use of natural spores as a novel encapsulating material and this research provides a new dimension in the use of spores, which strongly supported by the use of lycopodium spores as plant-based medicine [8 10] for various ailments due to the intrinsic therapeutic benefits of spore constituents.
  • our studies demonstrate that these medicinal spores can be encapsulated with molecules of interest for tailored applications.
  • Encapsulation of macromolecules into natural lycopodium spores Dissolve 75 mg BSA into 0.6 mL purified water in a 1.5 mL polypropylene tube and suspend 150 mg of spores in the BSA solution. Mix the suspension by vortexing (VWR, Singapore) for 5 min and transfer the tube to a thermoshaker (Hangzhou Allsheng Inst. Singapore) at 4°C and 500 rpm for passive loading. In the case of compression loading, prepare a compressed tablet by using a hydraulic press at 5 ton pressure for 20 sec, soak the spore tablet in a BSA solution and allow for BSA uptake by the spore particles (Dimensions of spore tablets are provided in supporting
  • Passive loading technique Dissolve 75 mg BSA in to 0.6 mL purified water in a 1.5 mL polypropylene tube and suspend 150 mg natural spores into BSA solution. Mix the suspension by using vortex mixer (VWR, Singapore) for 5 min and transfer the tube to thermoshaker (Hangzhou Allsheng Inst. Singapore) set at 4°C, 500 rpm for 2 h incubation. Stop the process and collect the BSA-loaded spores by centrifugation at 12000 rpm for 4 min. Wash the spores quickly using 0.5 ml water and centrifuge to remove surface adhered BSA. Freeze the spores in freezer at -70°C for 30 min and freeze dry for 24 h. The resultant macromolecule loaded spores are stored in -20°C until further in-vitro characterizations. Prepare the placebo spores using similar procedure without BSA and preserve in -20°C.
  • Compression loading technique Fill 150 mg natural spores into 12 mm die and compressed to form a tablet of around 10 - 12 mm dia. under hydraulic press to provide 5 ton load for 20 sec. using FTIR pellet maker. The dimensions of the spores tablet are mentioned in Table 2 and these tablets are soaked in 0.6 mL of 75 mg BSA containing aqueous solution in a 20 mL flat glass bottle for 2 h at 4°C to allow uptake of BSA molecules. Stop the process and collect BSA-loaded spores by centrifugation at 12000 rpm for 4 min. Wash quickly using 0.5 ml water and centrifuge to remove surface bound BSA. Freeze the spores in freezer at -70°C for 30 min and freeze dry for 24 h. The resultant spores are stored in -20°C until further
  • Vacuum loading technique Dissolve 75 mg BSA in to 0.6 mL purified water in a
  • Natural lycopodium spores and macromolecule-loaded spores (2 mg/ml) with a pre-run volume of 0.5 mL were primed manually into the flow cell and were analyzed with a flow rate of 0.1 ml/min, camera rate of 10 frames/s leading to a sampling efficiency of about 9 %.
  • a minimum of 10,000 particles were fixed as count for each measurement and three separate measurements were performed and data analysis was carried out using highly focused 1000 spores segregated by edge gradient. Instrument was calibrated using polystyrene microspheres (50 ⁇ 1 ⁇ ,
  • SEM imaging was performed using a FESEM 7600F (JEOL, Japan). Samples were coated with platinum at a thickness of 10 nm by using a JFC-1600 (JEOL, Japan) (20mA, 60 sec) and images were recorded by employing FESEM with an acceleration voltage of 5.00 kV at different magnifications to provide morphological information/to predict morphological observations.
  • micrographic analysis was performed using a Carl Zeiss LSM700 (Germany) confocal microscope.
  • Laser excitation lines 405 nm (6.5 %), 488 nm (6 %) and 633 nm (6 %) with DIC in an EC Plan-Neofluar lOOx 1.3 oil objective M27 lens were used. Fluorescence from natural and macromolecule loaded lycopodium spores were collected in photomultiplier tubes equipped with the following emission filters; 416-477, 498-550, 572-620.
  • the laser scan speed was set at 67 sec per each phase (1024x1024:84.94 ⁇ sizes) and plane mode scanning with a 3.15 pixel dwell was used, and at least three images were captured for each sample and all images were processed and converted under the same conditions using software ZESS 2008 (ZEISS, Germany). See the next paragraph below for more details regarding the confocal laser scanning microscopy analysis.
  • Confocal laser scanning microscopy analysis of natural and macromolecule loaded natural spores Confocal laser scanning micrographic analysis were done using a Carl Zeiss LSM700 (Germany) confocal microscopy equipped with three spectral reflected/fluorescence detection channels, six laser lines (405/458/488/514/543/633 nm) and connected to Zl inverted microscope (Carl Zeiss, Germany). Natural and macromolecule-loaded spores were mounted on sticky slides (Ibidi, Germany), a drop of mounting medium (Vectashield®) was added and spore particles were covered with another sticky slide.
  • the iris was set as optimal for the sample conditions and all images were captured at mid of the particle (optical section) and other settings fixed same for all samples and at least three images were captured for each different sample and all images were processed at same conditions using ZESS 2008 software (ZEISS, Germany).
  • Encapsulation efficiency Suspend 5 mg BSA-loaded lycopodium spores in 1.4 mL PBS, vortex for 5 min and probe sonicate for 10 sec (3 cycles, 40 % amplitude). Filter the solution to collect extracted BSA using 0.45 ⁇ PES syringe filters (Agilent, USA). Measure the absorbance at 280 nm (Boeco-S220, Germany) using a placebo extract as a blank to compute the amount of BSA in the spore particles. In particular, measure the absorbance at 280 nm using placebo extract as blank to compute amount of BSA in the natural spores as below:
  • Amount of BSA (mg) Absorbance x dilution factor
  • In-vitro drug release evaluation in simulated gastric fluid (0.1 M HCl, pH 1.2): Suspend 5 mg BSA-loaded spores and placebo in 1.4 ml media and incubate at 37°C, 50 rpm. Collect 1 ml release samples at specified time intervals by centrifugation at 14000 rpm 30 sec and replenish with fresh 1 ml release media. Filter the release sample using PES membrane filters (Agilent, USA) and measure absorbance at 280 nm using placebo as blank. Compute amount of BSA released using BSA standard curve.
  • In-vitro drug release evaluation in simulated intestinal fluid PBS pH 7.4: Suspend 5 mg BSA-loaded spores and placebo in 1.4 ml media and incubate at 37°C, 50 rpm. Collect 1 ml release samples at specified time intervals by centrifugation at 14000 rpm 30 sec and replenish with fresh 1 ml release media. Filter the release sample using PES membrane filters (Agilent, USA) and measure absorbance at 280 nm using placebo as blank. Compute amount of BSA released using BSA standard curve.
  • FIGS. 1A-1E shows a schematic of the different encapsulation techniques developed to utilize natural spores as advanced encapsulating materials. Attractive features of our techniques include both versatility and simplicity with the potential to allow for application to a variety of small or large biomolecules under ambient processing conditions.
  • FIG. 1A shows the origin of natural spores from the vascular plant lycopodium, these spores exhibit both well-defined size and microstructures. When these spores are suspended in a biomacromolecule solution (FIG. IB), the biomacromolecules enter the internal spore cavities through natural nanochannels in the spore wall of approximately 40 nm size.
  • FIG. IB biomacromolecule solution
  • FIGS. ID, IE, and IF represent the three different microencapsulation techniques passive, compression, and vacuum loading, respectively.
  • the spores are incubated in a biomacromolecule solution, with additional external forces being applied in the compression and vacuum processes for the encapsulation of biomacromolecules.
  • FIGS. 3A-3E show representative histogram data with Gausian curve fitting of equivalent spherical diameter (ESD) vs. Frequency, with an average ESD of 30.31 ⁇ 1.87 ⁇ for (FIG. 3A) natural spores and an ESD of 30.63 ⁇ 1.92 ⁇ , 30.61 ⁇ 1.92 ⁇ , and 30.56 ⁇ 1.88 ⁇ respectively for (FIG. 3B) passive, (FIG. 3C) compression, and (FIG. 3D) vacuum loaded spores.
  • ESD equivalent spherical diameter
  • FIGS. 3A-3E The size uniformity and circularity of natural spores was supported by ESD data before and after biomacromolecule loading.
  • the data is represented by curve fitting to histograms of circularity vs. frequency as shown in FIGS. 3A-3E.
  • the quality of the images used for data analysis is evident from the edge gradient vs. frequency data which indicates that well focused spores formulations were used during FlowCam analysis.
  • FIGS. 3E, 3F, 3G, and 3H suggest the structural similarity of spores before loading, as well as after passive, compression, and vacuum loading techniques, respectively.
  • FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D are displayed as FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D, respectively, for spores before loading, as well as after passive, compression, and vacuum loading techniques.
  • Structural and morphological observations show that natural spores formulations have maintained their structural integrity without any denaturation, and exhibit size uniformity after biomacromolecule encapsulation using the three different microencapsulation techniques.
  • an encapsulation material it is of the utmost importance to retain structural integrity after material processing at ambient temperatures.
  • Theoretical loading is based on 50% weight of natural lycopodium spores: ⁇ c) BSA encapsulation efficiency is determined using 5 mg of BS A-loaded natural spore particles.
  • Lycopodium is stated to have therapeutic effects on biliary stones and liver failure, and due to its diverse importance, application of these spores is emerging as a potential new treatment modality in health care.
  • These proven therapeutic benefits have led to the commercialization of lycopodium based oral herbal formulations for the treatment of diverse health conditions such as anxiety, albuminuria, constipation, dysentery, gallstones, heartburn, hemorrhoids, impotence, indigestion, irritability, prostatitis, renal colic, and rheumatism.
  • FIG. 7A indicates 90% biomacromolecules release in the first 5 minutes and complete release was observed in 30 to 60 minutes. There was no significant difference among the release from BSA-loaded spores prepared using different techniques (p > 0.05). In case of intestinal conditions (FIG. 7B), a similar burst release was observed with spore formulations prepared by three different loading techniques suggesting no significant release differences in simulated gastric and intestinal conditions. The observed release trend indicates fast release in both simulated conditions and is evident due to the high aqueous solubility of BSA resulting in rapid release from nanodomains of natural spores.
  • biomacromolecules into natural spores by three different microencapsulation techniques viz., passive, compression and vacuum loading.
  • DIPA dynamic image particle analysis
  • bovine serum albumin (BSA) was loaded into natural Helianthus annuus (sunflower) pollen employing the same three encapsulation techniques (passive, compression, and vacuum loading) .
  • the genetic material is stored within the cytoplasmic core of the pollen grain and surrounded by a double layer shell consisting of an in tine and an exine layer.
  • the outermost exine layer contains the sporopoUenin biopolymer, which is considered to be one of nature's most resilient materials.
  • sporopoUenin biopolymer which is considered to be one of nature's most resilient materials.
  • both the sporopoUenin exine and cellulosic intine layers are permeable and undergo dehydration and hydration which facilitates materials loading as the surrounding fluid is drawn into the internal pollen cavity.
  • the potential advantages of natural sunflower pollen grains as a drug delivery vehicle are enormous; (1) Proven track record as safe for human oral consumption due to use as a biosupplement and in herbal medicine. (2) Common constituent of 'bee pollen' for human consumption for nutritional and therapeutic benefits.
  • biomacromolecules Through the comparison of three different encapsulation strategies (passive hydration, hydraulic compression, and vacuum-assisted), we demonstrate multiple routes to achieve high-efficiency protein loading with bovine serum albumin (BSA) as a model biomacromolecule. Importantly, the methods used are environmentally friendly and preserve the complex architecture of natural pollen grains, including size, uniformity, and surface features. Furthermore, we demonstrate that a controlled release profile is achievable by encapsulating pollen grains inside alginate hydrogel beads. Taken together, our findings offer compelling evidence that natural pollen grains are excellent drug delivery vehicles.
  • BSA bovine serum albumin
  • bovine serum albumin BSA
  • FITC-conjugated BSA FITC-conjugated BSA
  • H-1000 Vectashield (H-1000) medium was procured from Vector labs (CA,
  • Encapsulation of macromolecules into natural pollen grains Dissolve 75 mg BSA into 0.5 mL purified water in a 1.5 mL polypropylene tube and suspend 150 mg of natural pollen grains in the BSA solution. Mix the suspension by vortexing (VWR, Singapore) for 5 min and transfer the tube to a thermoshaker (Hangzhou Allsheng Inst. Singapore) at 4°C and 500 rpm for passive loading.
  • VWR Vortexing
  • thermoshaker Hangzhou Allsheng Inst. Singapore
  • passive loading prepare compressed tablet by using a hydraulic press at 5 ton pressure for 20 sec, soak the tablet in BSA solution and allow for BSA uptake by the pollen grains (Dimensions of compressed tablets are provided in supporting information).
  • the 5 ton compression pressure employed is able to retain intact sunflower pollen structure with some portion of pollen cytoplasmic constituents, as indicated by red and blue channel CLSM autofluorescence.
  • For the vacuum loading technique use a BSA and pollen grains suspension, and slowly apply a 2 mbar vacuum in a freeze dryer (Labconco, MO, USA). Maintain the quantity of BSA, pollen grains and incubation time (2 hour) constant for all the batches, and after incubation collect the BSA-loaded pollen grains by centrifugation at 12000 rpm for 4 min and wash using 0.5 ml water, then centrifuge to remove surface adhered BSA.
  • Compression filling Fill 150 mg natural sunflower pollen grains into 12 mm die and compress to form a tablet of around 10 - 12 mm dia. under hydraulic press to provide 5 ton load for 20 sec. using FTIR pellet maker. The pellet formed in this method is soaked in 0.5 mL of 75 mg BSA containing aqueous solution in 20 mL flat glass bottle for 2 h at 4°C to allow swelling of pollen grains thereby BSA is entrapped in the pollen grains. Stop the process and collect the BSA-loaded particles by centrifugation at 12000 rpm for 4 min. Wash using 0.5 ml water and centrifuge to remove surface bound BSA.
  • Vacuum filling Dissolve 75 mg BSA into 0.5 mL purified water in a 1.5 mL centrifuge tube and suspend 150 mg pollen grains and vortex for 5 min to homogenize. Apply a vacuum at 2 mbar for 2 h using a freeze-drier. Stop the process and collect BSA-loaded pollen grains by centrifugation at 12000 rpm for 4 min. Wash using 0.5 ml water and centrifuge to remove surface bound BSA, then freeze the pollen grains in a freezer at -70°C for 30 min and freeze dry for 24 h. The resultant particles are stored in -20°C for further characterization. Prepare the placebo pollen grains with the same procedure without BSA and preserve in -20°C.
  • FITC-conjugated BSA was encapsulated by three different techniques as mentioned in section 1.1. with a batch size of 22.5 mg containing 7.5 mg FITC-BSA per batch of natural pollen grains. 6.2.2.3 Characterization of natural and macromolecule-loaded natural pollen grains
  • FlowCam® FlowCam VS benchtop system (FlowCam® VS, Fluid Imaging Technologies, Maine, USA) was equipped with a 200 ⁇ flow cell (FC-200), a 20X magnification lens (Olympus®, Japan) and controlled by the visual spreadsheet software version 3.4.11. The system was flushed with 1 mL deionized water (Millipore, Singapore) at a flow rate of 0.5 ml/min and flow cell cleanliness was monitored visually before each sample run.
  • FC-200 200 ⁇ flow cell
  • 20X magnification lens Olympus®, Japan
  • Natural sunflower pollen grains and macromolecule-loaded grains of 0.5 mL (2 mg/ml) with a pre -run volume of 0.5 mL (primed manually into the flow cell) were analyzed with a flow rate of 0.1 ml/min and a camera rate of 10 frames/s leading to a sampling efficiency of about 9 %.
  • a minimum of 10,000 particles were fixed as the count for each measurement and three separate measurements were performed and data analysis was carried out using 1000 well focused pollen grains segregated by edge gradient.
  • the instrument was calibrated using polystyrene microspheres (50 ⁇ 1 ⁇ , Thermoscientific, USA)
  • SEM imaging was performed using a FESEM 7600F (JEOL, Japan). Samples were coated with platinum at a thickness of 10 nm by using a JFC-1600 (JEOL, Japan) (20mA, 60 sec) and images were recorded by employing FESEM with an acceleration voltage of 5.00 kV at different magnifications to provide morphological information/ observe morphological characteristics.
  • micrographic analysis was performed using a Carl Zeiss LSM700 (Germany) confocal microscope. Laser excitation lines 405 nm (6.5 %), 488 nm (6 %) and 633 nm (6 %) with DIC in an EC Plan-NeofluarlOOxl.3 oil objective M27 lens were used. Fluorescence from natural and macromolecule loaded pollen grains were collected in photomultiplier tubes equipped with the following emission filters; 416-477, 498-550, 572-620.
  • the laser scan speed was set at 67 sec per each phase (1024x1024:84.94 ⁇ sizes) and plane mode scanning with a 3.15 pixel dwell was used and at least three images were captured for each sample and all images were processed and converted under the same conditions using software ZESS 2008 (ZEISS, Germany). See the next paragraph for additional information regarding the confocal laser scanning microscopy analysis.
  • Confocal laser scanning microscopy analysis of natural and macromolecule loaded natural pollen grains Confocal laser scanning micrographic analysis was performed using a Carl Zeiss LSM700 (Germany) confocal microscope equipped with three spectral
  • photomultiplier tubes equipped with the following emission filters; 416-477, 498-550, 572-620.
  • the laser scan speed was set at 67 sec per each phase (1024x1024: 84.94 ⁇ 2 sizes) and plane mode scanning with 3.15 of pixel dwell.
  • the iris was set as optimal for the sample conditions and all images were captured at the mid region of the particle (optical sections) and other settings were fixed the same for all samples and at least three images were captured for each different sample and all images were processed and converted under the same conditions using software ZESS 2008 (ZEISS, Germany).
  • Amount of BSA (mg) Absorbance x dilution factor
  • centnfugation at 14000 rpm for 30 s, and replenish with fresh release media Filter the release sample using PES filters and measure absorbance at 280 nm using a placebo release sample as a blank. Compute the amount of BSA released using a BSA standard curve.
  • In-vitro drug release evaluation in simulated gastric fluid 0.1 M HCl, pH 1.2
  • In- vitro drug release evaluation in simulated intestinal fluid pH 1.2
  • Suspend 5 mg BSA-loaded pollen grains and placebo in pH 1.2 media Incubate at 37°C at 50 rpm and collect the release samples at 5 min, 15 min and 30 min by centrifugation at 14000 rpm for 30 sec, replenish with fresh release media and continue the release study. Filter the release sample using PES membrane filters and measure absorbance at 280 nm using a placebo release sample as a blank. Compute the amount of BSA released using a BSA standard curve.
  • FIGS. 10A-10D outline the basic hydration and encapsulation process, then subsequent release. Hydration begins with taking dried sunflower pollen grains with the cytoplasmic material intact (FIG. 10A) and combining with a BSA solution (FIG. 10B). The BSA solution is absorbed into the pollen grain as the pollen grain swells and the BSA solution fills the additional volume created by the swelling process (FIG. IOC). This process may be natural, as in the passive loading method, or assisted, as in the compression or vacuum loading methods. After the BSA is released in the simulated intestinal or gastric fluid, the pollen grains remain swollen although all BSA is released (FIG. 10D).
  • FIG. 11 A shows representative data by curve fitting histograms of equivalent spherical diameter (ESD) vs. frequency with average an ESD of 37.93 ⁇ 1.41 ⁇ for natural pollen grains and an ESD of 36.54 ⁇ 1.45 ⁇ , 36.95 ⁇ 1.35 ⁇ , and 36.17 ⁇ 1.36 ⁇ respectively for passive, compression, and vacuum loaded pollen grains.
  • ESD equivalent spherical diameter
  • the pollen circularity was measured before and after BSA loading and the data is represented by curve fitting to histograms of circularity vs. frequency as shown in FIG. 1 IB.
  • the quality of the image focus of the images used for data analysis is evident from FIG. 11C, and the edge gradient vs. frequency data which is represented indicates that highly focused pollen grains were used during DIPA analysis.
  • FIGS. 12A, 12B, 12C, and 12D indicate the structural similarity of pollen before loading as well as passive, compression, and vacuum loading techniques, respectively.
  • DIPA scanning electron microscopy
  • FIGS. 12A, 12B, 12C, and 12D indicate the structural similarity of pollen before loading as well as passive, compression, and vacuum loading techniques, respectively.
  • SEM scanning electron microscopy
  • Compression loading 50 18.8 ⁇ 1.5 37.8 ⁇ 3.2
  • BSA encapsulation efficiency is determined using 5 mg BSA-loaded natural pollen grains.
  • FIGS. 19A, 19B, and 19C clearly indicate the release of FITC-BSA from pollen grains prepared using three different techniques and the pollen structure was found to remain intact. It is also evident from the CLSM images that a low amount of BSA binding to the exine has occurred and is clearly visible by the resulting 'green ring'.
  • This example demonstrates a cost-effective, simple approach to produce oral- controlled release formulations of 5-fluorouracil (5-FU) based on natural L. clavatum spores.
  • the data provided in this example demonstrates that the vacuum loading technique provides the highest encapsulation efficiency of 49% compared to the passive and compression loading techniques.
  • Micrometric properties of the 5-FU loaded spores confirmed a uniform size distribution, and surface characterization of 5-FU spores verified no evidence of residual 5-FU, indicating encapsulation of 5-FU inside spores.
  • Uniform Eudragit RS100 coatings (ERS) on 5- FU loaded spores provide a controlled release of 5-FU for up to 30 hours.
  • the demonstrated features of 5-FU loaded spores indicate a potential oral drug delivery system for gastrointestinal cancer treatment and other maladies.
  • 5-Fluorouracil solution was prepared by dissolving 75 mg of drug in a 1.8 mL mixture of ethanol and 1 N ammonium hydroxide (1: 1) solution.
  • Whole L. clavatum spores (150 mg) were suspended in the prepared solution. The suspension was vortexed for 5 min and the tube was transferred to a thermoshaker (Hangzhou Allsheng Inst. Singapore) set at 500 rpm for 2 hours incubation at room temperature.
  • the 5-FU loaded spores were collected by centrifugation at 4500 rpm for 3 min. The spores were washed using 4 mL deionized water and centrifuged to remove surface adhered 5-FU.
  • the 5-FU loaded spores were placed in a freezer at -70° Celsius for 30 min and freeze-dried for 24 hours.
  • the resulting 5-FU loaded spores were stored in a dry cabinet at room temperature until further characterization.
  • the placebo passive-loaded spores without 5-FU were prepared by using the same procedure as described above.
  • the spores were placed in a freezer at -70° Celsius for 30 min and freeze-dried for 24 hours. The resulting spores were stored in a dry cabinet until further characterization.
  • the placebo compression-loaded spores without 5-FU were prepared by using the same procedure as described above.
  • Vacuum-assisted 5-FU loading was performed by suspending 150 mg of L.
  • clavatum spores in 1.8 mL of 5-FU solution. The suspension was vortexed for 5 min. The sample was placed in a freeze-drier (Lanconco, USA) and a 1 mbar vacuum was applied for 2 hours. The process was stopped and the 5-FU loaded L. clavatum spores were washed using 4 mL water and centrifuged to remove surface bound drug. The spores were placed in a freezer at -70° Celsius for 30 min and freeze-dried for 24 hours. The resulting spore particles were stored in a dry cabinet until further characterization. The placebo vacuum-loaded spores without 5-FU were prepared by using the same procedure without 5-FU as described above.
  • the benchtop system (FlowCamVS, Fluid Imaging Technologies, Maine, USA) was installed with a visual spreadsheet software version 3.4.11., 200 ⁇ flow cell (FC-200) and a 20x magnification lens (Olympus , Japan).
  • the flow cell was cleaned by flushing the system with 1 mL of deionized water at a flow rate of 0.5 mL/min.
  • the instrument was calibrated using polystyrene microspheres (50 ⁇ 1 ⁇ ) and a pre -run volume of 0.5 mL of whole L. clavatum spores and 5-FU loaded spores were primed and transferred into the flow cell.
  • 5-FU loaded L. clavatum spores were coated using Eudragit RS100 (ERS) at two different ERS concentrations (2.50% w/v and 10.0% w/v).
  • the coating solutions were prepared by slowly dissolving Eudragit RS100 in acetone.
  • 150 mg of 5-FU loaded (vacuum method) spores were added to 1.2 mL of Eudragit RS100 solution in a PFA round bottom flask and the solvent was evaporated in a vacuum desiccator for 1 hour. Further, spores were dried in vacuum oven (Memmert GmbH, Germany) at 1 mbar for 1 hour. The dried spore formulation was then gently powdered using an agate pestle and mortar and stored in a dry cabinet until further characterization.
  • clavatum spores were suspended in 10 mL of release media and incubated at 37° Celsius while stirring at 50 rpm in an orbital shaker incubator (LM-450D, Yihder, Taiwan). At predetermined time points, 1 mL of release media was collected and replenished with fresh release media. The absorbance in the release sample was measured using a UV spectrometer (Boeco-S220,
  • 5-FU encapsulation efficiency is determined using 10 mg of 5-FU loaded whole L. clavatum spore.
  • FIGS. 20A-20D illustrate the results from the DIP A. It is evident from diameter measurements (FIG. 20A) that the whole spores with a native diameter of 30 ⁇ 0.45 mm remain unchanged after 5-FU encapsulation by all three encapsulation techniques. The diameter of spores before and after 5-FU loading is listed in Table 10 . The 5-FU loaded spores retained the intact microstructure with uniform size distribution. In order to investigate the uniform shape of 5-FU loaded spores, the circularity and aspect ratio were measured and the data are illustrated in FIGS.
  • FIGS. 21A-21D The images captured during DIPA are presented in FIGS. 21A-21D for spores before 5-FU loading, as well as after loading by passive, compression and vacuum loading techniques, respectively.
  • the DIPA images indicate that all spores after 5-FU loading retained well-defined microstructures supporting the DIPA data for the uniform size distributions.
  • clavatum spores before and after 5-FU loading were analyzed by SEM as described in Section 6.3.1.5.
  • the SEM images after 5-FU loading by passive, compression and vacuum are presented in FIGS. 22A-22D, respectively.
  • the structural and morphological data for spores before 5-FU loading showed characteristic well-defined ornamentation with reticulate structure and uniform size distribution.
  • the spore's native microstructure and ornamentation were retained.
  • the 5-FU encapsulated spores showed no detrimental effect to the spore microstructure by drug loading even after the use of external factors such as compression at 5 ton and with 1 mbar vacuum.
  • the surface of the 5-FU loaded spore was clean without any evidence of residual drug aggregation suggesting the encapsulated drug was principally inside the spore's internal cavity.
  • the data for 5-FU loaded spores supports that the disclosed methods to encapsulate 5-FU in whole L. clavatum spores offers excellent potential as a multiparticulate oral delivery system with uniform size distribution and well-defined surface morphology.
  • FIGS. 23A and 23B illustrated the SEM images of ERS-coated spores using 2.5% and 10% ERS concentrations, respectively.
  • the surface morphology of whole spores after coating indicates that spores were coated with ERS, and that the ERS coating was higher in the case of 10% ERS-coated spores.
  • the muri located on the spores was filled with the coating material which acts as a barrier for 5-FU release.
  • FIGS. 24A and 24B illustrate the 5-FU release profiles in SGF (pH 1.2) and SIF (pH 7.4), respectively. High release rates of up to 90% were observed in the initial 10 min and complete 5-FU release was observed within 60 min due to exit via the nanochannels in the exine wall (Diego-Taboada et al., Pharmaceutics 6 (2014) 80-96). Similarly higher 5-FU release in SGF for stomach-targeted release was reported by Bhardwaj et al.
  • Eudragit RS 100 is a copolymer of ethyl acrylate, methyl methacrylate and is widely used as a coating material to develop controlled release formulations (Alai et al. , J. Microencapsul. 30 (2013) 519-529; Piao et al, AAPS PharmSciTech. 11 (2010) 630-636).
  • the initial coating and in-vitro release studies in simulated gastrointestinal conditions using different concentrations of Eudragit RS 100 indicates that coatings with 2.5% w/v and 10% w/v ERS provided a suitable coating on whole L. clavatum spores.
  • FIG. 24C illustrates the data for in-vitro release profiles using ERS coated spores, indicating that the ERS coating significantly (p ⁇ 0.05) retarded 5-FU release under simulated gastrointestinal conditions.
  • the inset (FIG. 24C) indicates around 70% of 5-FU was released in the initial 2 hours and by increasing the ERS concentration to 10% the 5-FU release was reduced to 50%. Further, in-vitro 5-FU release was extended up to 30 hours and a significant (p ⁇ 0.05) difference in 5-FU release was observed with 10% ERS- coated spores in comparison to 2.5% coating, suggesting that 10% ERS coating is beneficial to achieve controlled 5-FU release from whole spores.
  • the in-vitro release data indicates that 5-FU release from the ERS coated spores was a result of polymer erosion from the surface of spores, as the enteric coating was higher the 5-FU release was lowered during 30 hours.
  • the possible mechanism of 5- FU release from enteric coated spores was a combination of dissolution, diffusion erosion and is consistent with previous finding (Piao et al., AAPS PharmSciTech. 11 (2010) 630-636).
  • in-vitro release of 5-FU from L. clavatum spores can be controlled in gastrointestinal conditions by ERS coating.
  • Similar 5-FU release profiles from modified sodium alginate microspheres were reported by Sanli et al. (Sanli et al., Drug Deliv. 21 (2014) 213-220) with controlled release up to 12 hours under simulated gastrointestinal conditions.
  • the controlled gastrointestinal release of 5-FU is highly beneficial in the treatment of breast, stomach and colon cancer, possibly avoiding furthermore repeated doses.
  • the disclosed results for 5-FU loaded L. clavatum whole spores indicate that whole spores could encapsulate and control the release of 5-FU under gastrointestinal conditions.
  • bovine serum albumin (BSA) was loaded into whole pine pollen grains employing three different encapsulation techniques (passive, compression, and vacuum loading).
  • BSA was loaded into whole unprocessed pine pollen grains by utilizing the vacuum loading technique as described above. After encapsulation of BSA, surface cleanliness was observed in relation to the number of washings. One water wash was determined to be adequate to remove residual surface adhered BSA (see FIG. 25 A, which shows the surface cleanliness of BSA-loaded pine pollen grains after zero, one, two or three washing steps).
  • Loading efficiency (LE) and encapsulation efficiency (EE) data was also measured for the BSA- loaded pine pollen formulations after each washing step. As shown in FIG. 25B, at zero washings, immediately after encapsulation, approximately 80% of the BSA was still present in the formulation, and that approximately 27 wt.% of the formulation comprised BSA, resulting in a loading ratio of about 1:3 (BSA:pollen grain). After one wash step approximately 40% of the BSA remained in the formulation, and approximately 13 wt.% of the formulation comprised BSA, resulting in a loading ratio of about 1 :7 (FIG. 28B). Subsequent washing resulted in further reductions in both LE and EE (FIG. 28B).
  • FITC-BSA FITC-conjugated BSA
  • CLSM confocal laser scanning microscopy
  • bovine serum albumin (BSA) was load into whole camellia pollen grains employing a passive loading technique.
  • Calcein (pharma grade), whole L. clavatum spores, and other solvents were purchased from Sigma-Aldrich (Singapore). Polystyrene microspheres (50 ⁇ 1 ⁇ ) were purchased from Thermoscientific (CA, USA).
  • Calcein-loaded formulations were transferred to a 250 ml beaker washed using 40 ml hot water (45° Celsius), and collected. The collected formulations were frozen at -20° Celsius for 1 hour, and freeze-dried (Labconco, MO, USA) for 24 hours. The spore formulations were collected, weighed and stored in a dry cabinet until further characterizations.
  • Confocal laser scanning micro graphic ( CLSM) analysis Confocal laser scanning micrographic (CLSM) analysis was performed using a Carl Zeiss LSM700 (Germany) confocal microscope. Laser excitation lines at 405 nm (6.5 %), 488 nm (6 %), and 633 nm (6 %) with differential inference contrast (DIC) in an EC Plan-NeofluarlOOxl.3 oil objective M27 lens were used. Fluorescence from calcein-loaded spores were collected in photomultiplier tubes equipped with the following emission filters: 416-477 nm, 498-550 nm, and 572-620 nm.
  • the laser scan speed was set at 67 sec per each phase (1024x1024:84.94 ⁇ sizes) and a plane mode scanning with a 3.15 pixel dwell was used. At least three images were captured for each sample and all images were processed and converted under the same conditions using software ZESS 2008 (ZEISS, Germany).
  • camellia oil was loaded into whole camellia pollen grains employing the vacuum loading technique.
  • FIGS. 31A and 3 IB illustrate that the size and morphology of whole Camellia pollen grains significantly differ from the size and morphology of Camellia pollen grain's isolated sporopollenin exine capsules (SEC) as measured by DIPA.
  • SEC isolated sporopollenin exine capsules
  • caffeine was load into whole L. clavatum spores employing a modified passive loading technique.
  • Caffeine (pharma grade), L. clavatum spores and other solvents were purchased from Sigma- Aldrich (Singapore). Polystyrene microspheres (50 ⁇ 1 ⁇ ) were purchased from Thermoscientific (CA, USA).
  • Encapsulation of caffeine ( CF) into L. clavatum spores Caffeine loading into spores was performed using a modified passive loading technique. CF equivalent to 50 % theoretical loading was dissolved in 11 mL dichloromethane with or without co-encapsulant (1.8 % w/v, Eudragit RS 100). Spores (1 g) were suspended in CF solution in 50 mL polypropylene tubes. The suspension was mixed for 10 min using a vortex mixer (IKA, Staufen, Germany) to form a homogeneous suspension. The CF suspension was incubated at room temperature overnight with intermittent stirring at 200 rpm for 5 hours. The suspension was filtered by using vacuum filtration.
  • CF-loaded formulations were then transferred to a 250 ml beaker and washed using 40 ml hot water (45° Celsius). After collecting, formulations were frozen at -20° Celsius for 1 hour and freeze-dried (Labconco, MO, USA) for 24 hours. The spore formulations were collected, weighed and stored in a dry cabinet until further characterizations. Placebo spores were prepared with the same procedure, except CF, and also stored in the dry cabinet at room temperature. [00322] To investigate CF encapsulation by CLSM, a fluorescent calcein-CF mixture was loaded according to the same procedure by dissolving 22 mg calcein in 2.2 mL DMSO and uniformly mixing with above CF solution.
  • DIP A Dynamic image particle analysis
  • magnification lens Olympus ® , Japan.
  • the system was flushed with 1 mL deionized water (Millipore, Singapore) at a flow rate of 0.5 ml/rnin and flow cell cleanliness was visually inspected before each sample run. Spores before and after CF loading with a concentration of 2 mg/ml were primed manually into the flow cell (a pre-run volume of 0.5 mL) and were analyzed with a flow rate of 0.1 ml/min and a camera rate of 14 frames/s leading to a sampling efficiency of approximately 12.2 %. A minimum of 10,000 spores were fixed as the particle count for each measurement and three independent measurements were performed.
  • the laser scan speed was set at 67 sec per each phase (1024x1024:84.94 ⁇ sizes) and plane mode scanning with a 3.15 pixel dwell was used and at least three images were captured for each sample and all images were processed and converted under the same conditions using software ZESS 2008 (ZEISS,
  • In-vitro release studies of CF-loaded spores In order to predict in-vitro release profiles of CF-loaded spores formulations, the release studies were performed in simulated saliva fluid pH 6.8 (SSF) for up to 5 min. 10 mg CF-loaded spores were suspended in 20 mL SSF and incubated at 37° Celsius, 50 rpm in a orbital shaker incubator LM-450D (Yihder, Taiwan). At predetermined time points 1 ml of release sample was collected and replenished with fresh release fluids. The absorbance of release sample was measured using UV spectrometer (Boeco- S220, Germany) at 275 nm with placebo as blank. [00328] Statistical Analysis: Statistical analysis was performed using two-tailed t-tests and p ⁇ 0.05 was considered as statistically significant. Encapsulation efficiency and in vitro release data are reported as mean values ⁇ standard deviation of three independent experiments.
  • FIGS. 32A-32B shows the L. clavatum spores before CF loading (FIG. 32A) and after CF-loading with co-encapsulant Eudragit RS 100 (ERS) by SEM. Micromeritic properties of CF-loaded spores confirmed a uniform size distribution, indicating monodisperse
  • FIGS. 33A-33B show CLSM images of spores with sporoplasm before CF- Calcein loading (FIG. 33 A) and after CF-loading into spores with coencapsulant ERS.
  • the CF encapsulated with Eudragit RS 100 (ERS) as coencapsulant provide highest encapsulation efficiency, 12 %.
  • In-vitro release profiles in simulated saliva fluid confirmed lower release profiles compared to physical mixture of CF with spores.
  • the controlled release of CF from CF-loaded spores with ERS as coencapsulant confirmed extended CF release for up to 24 hours indicating
  • Table 11 provides the percentages of caffeine loading into L. clavatum spores using the modified passive loading technique described above.
  • CF encapsulation efficiency is determined using 10 mg of CF loaded L. clavatum spores.
  • the human volunteers were administered orally 2 mL of pure caffeine solution starting with water (blank) and different CF dose (0.5, 1, 5, 10 mg).
  • the volunteers were requested to score the bitterness on a scale of 0 to 5 for each solution, where 0 indicates none and 5 indicates strong bitterness.
  • the bitterness recognition threshold for all the human volunteers was assessed.
  • the volunteers were requested to place test products (physical mixture of CF with spores and CF loaded spores) on their tongue for duration of 30 sec. Both the products were
  • Evaluation score is based on scale 0 to 5: 0 is no bitterness and 5 is highest bitterness.
  • Taste threshold of the human volunteers reached with 1 mg CF.
  • Table 12 lists the individual bitterness evaluation scores of all human volunteers for formulations including: water, CF at 0.5 mg, 1 mg, 5mg, and 10 mg in 2 ml water, a physical mixture of CF and L. clavatum spores (negative control), and CF-loaded/ERS-coencapsulated L. clavatum spores (lead formulation).
  • FIGS. 35A and 35B show the corresponding bitterness score histograms for these formulations.
  • the bitter taste of caffeine can be effectively masked by encapsulating CF into L. clavatum spores by the modified passive loading technique or by coencapsulating with ERS.
  • the results from the human trials confirmed taste masking of CF from CF encapsulated spores formulations, making them suitable for masking the bitter taste of commercial food supplements and pharmaceuticals.
  • UV Ozone Cleaner Surface Modification Using Ultraviolet (UV) Ozone Cleaner.
  • the surface of the pollen grains were modified by exposure to UV-Ozone cleaner.
  • a thin layer of whole Camellia pollen grains (approximately 50 mg) was spread evenly on a 90 x 15 mm petri dish and UV- Ozone treated using a benchtop PSD Series UV-Ozone cleaner (Novascan, United States).
  • the UV-Ozone treatment of the pollen grains ranged from 30 sec to 120 min.
  • Contact Angle Measurements A thin layer of the UV-Ozone treated pollen was spread out on self-adhesive carbon tape on a glass slide. A 2 ⁇ L ⁇ bead of water was slowly lowered onto the pollen layer. The contact angle was measured using Attension Theta Optical Tensiometer (Biolin Scientific Holding AB, Sweden) with OneAttension 1.0 software.
  • FIG. 36 illustrates the contact angle data for UV-Ozone treated Camellia pollen grains showing a decrease in contact angle with increasing UV-Ozone treatment duration.
  • Treatment of Camellia pollen with UV-Ozone produced a decrease in contact angle with increasing UV-Ozone treatment duration.
  • the observed decrease in contact angle indicates a decrease in hydrophilicity of the pollen grains upon modifying the pollen surface through exposure to UV-Ozone.
  • UV-Ozone treatment improved the hydrophilicity of pollen grains and sporopollenin exine capsules (SECs) in order to aid aqueous dispersion of the pollen grains and encapsulated materials therein.
  • FIG. 37 shows SEM images of the UV-Ozone treated and untreated pollen grains, indicating improved surface roughness in the case of the UV-Ozone treated pollen grains.
  • FIG. 39 shows SEM images of the UV-Ozone treated and untreated Camellia SECs.
  • FIG. 38 shows aqueous solutions of the UV-Ozone treated and untreated Camellia pollen grains.
  • FIG. 40 shows aqueous solutions of the UV-Ozone treated and untreated Camellia SECs.
  • FIG. 41 A shows an aqueous solution of Camellia seed oil.
  • FIG. 41B shows an aqueous solution of unloaded and untreated Camellia SECs.
  • FIG. 41C shows an aqueous solution of Camellia SECs that were oil loaded, ethanol washed and UV-Ozone treated.
  • FIG. 42 shows CLSM images illustrating macromolecular encapsulation in Camellia pollen grains and SECs.

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Abstract

Selon un aspect, l'invention concerne des spores entières modifiées pour encapsuler un ou plusieurs composés ou une ou plusieurs substances. Dans certains modes de réalisation, la spore entière encapsulant le ou les composés, ou la ou les substances, est revêtue ou co-encapsulée avec un hydrogel, ou un ou plusieurs autres agents, pour réguler la vitesse de libération du ou des composés ou de la ou des substances de la spore. Selon un autre aspect, l'invention concerne des procédés de production des spores entières encapsulant un ou plusieurs composés ou une ou plusieurs substances. Selon un autre aspect, l'invention concerne des formulations comprenant soit une spore entière, soit une spore entière encapsulant un ou plusieurs composés ou une ou plusieurs substances, et des utilisations de ces formulations.
PCT/SG2016/050333 2015-07-16 2016-07-15 Micro-encapsulation de composés à l'intérieur de spores naturelles et de graines de pollen Ceased WO2017010945A1 (fr)

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WO2018146365A1 (fr) * 2017-02-09 2018-08-16 Universidade De Santiago De Compostela Particules purifiées de pollen et leurs utilisation pour administrer des nanosystèmes
WO2018089924A3 (fr) * 2016-11-11 2019-06-13 The Trustees Of Columbia University In The City Of New York Systèmes et procédés de fabrication d'actionneurs sensibles à l'eau
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WO2022180407A1 (fr) * 2021-02-26 2022-09-01 Botanical Solutions Limited Constructions d'exine
CN116019846A (zh) * 2023-02-14 2023-04-28 北京安吉贝玛健康科技有限公司 一种用于治疗心脑血管病的配方及其制备方法
EP4114479A4 (fr) * 2020-03-02 2024-04-03 Sporogenics Pte. Ltd. Composition de barrière anti-adhérence, ses procédés de fabrication et son utilisation
WO2025012506A1 (fr) * 2023-07-11 2025-01-16 Universidade De Santiago De Compostela Particules de pollen utilisées en tant que véhicules pour l'administration pulmonaire

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