WO2020180311A1 - Compositions d'exosomes à base de plantes et leur utilisation pour rajeunir la peau - Google Patents

Compositions d'exosomes à base de plantes et leur utilisation pour rajeunir la peau Download PDF

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Publication number
WO2020180311A1
WO2020180311A1 PCT/US2019/020920 US2019020920W WO2020180311A1 WO 2020180311 A1 WO2020180311 A1 WO 2020180311A1 US 2019020920 W US2019020920 W US 2019020920W WO 2020180311 A1 WO2020180311 A1 WO 2020180311A1
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WIPO (PCT)
Prior art keywords
skin
exosomes
plant
heat shock
response
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PCT/US2019/020920
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English (en)
Inventor
Benjamin M. Buehrer
John W. Ludlow
Peter E. PIERACCINI
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Zen-Bio, Inc.
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Priority to PCT/US2019/020920 priority Critical patent/WO2020180311A1/fr
Publication of WO2020180311A1 publication Critical patent/WO2020180311A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/886—Aloeaceae (Aloe family), e.g. aloe vera
    • 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
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/645—Proteins of vegetable origin; Derivatives or degradation products thereof
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine

Definitions

  • the present disclosure relates to plant exosome compositions, and preparation thereof, for uses including rejuvenating, regulating and conditioning skin.
  • Naturally derived botanical components including biologically active molecules such as polyphenols, polysaccharides, glycoconjugates and glycoproteins are isolated from plants and incorporated into skincare products to improve skin tone and quality; however, single components are largely ineffective by themselves. This result necessitates choosing several individual components to combine for efficacy.
  • Mammalian cells are known to secrete extracellular vesicles known as microvesicles or exosomes.
  • Exosomes were once thought of as contaminating debris in cell culture, however, it has become recognized that the secreted microvesicles are packed with protein and RNA cargos and play a role in cellular signaling and communication.
  • Exosomes contain functional mRNA, miRNA, DNA, and protein molecules that can be taken up by target cells and affect target cell biology.
  • Proteomic and genomic analysis of exosome cargo has revealed a broad range of signaling factors that are both cell type-specific as well as differentially regulated based on the secreting cells’ environment [2].
  • HSP70 has been previously shown to be a cargo constituent of exosomes [3, 4, 5].
  • the genetic information contained in exosomes may influence or even direct the fate of the target cell, for example by triggering target cell activation, migration, growth, differentiation or de-differentiation, or by promoting apoptosis or necrosis.
  • exosomes may provide additional cell factors for assistance in wound healing and epithelial remodeling.
  • exosomes have been identified from other organisms, such as certain bacteria and Drosophila, suggesting that they are an evolutionarily conserved method of secretion and intercellular communication [6, 7]. This has led to questioning whether plants also secrete exosome or exosome-like vesicles (ELVs) [8].
  • ELVs exosome-like vesicles
  • the recent identification of apoplastic vesicles and ELVs from fruit juices strongly supports the presence of exosome secretion in plants [9-12].
  • plant-derived exosome and ELV cargo may be regulated by the plant’ s environment.
  • Plants respond to environmental stressors in a similar manner as mammalian cells as exemplified by exposure to heat stress increasing heat shock protein gene expression (HSP70 and HSP100) in Aloe barbadensis Miller (Aloe vera) [13]. Herein, we provide evidence that these changes are translated into exosome or ELV cargo content.
  • a topical composition for regulating skin condition includes an effective amount of isolated exosomes having increased levels of heat shock stress-response molecules and a carrier.
  • the isolated exosomes are isolated from a plant’ s leaf flesh conditioned by growing the plant under conditions that include a heat shock of the plant at a temperature of from about 33°C to about 45°C for about 1 hour to about 3 hours.
  • the level of heat shock stress-response molecule in the heat-shocked plant exosome is from about 10 times to about 20 times higher than the level of heat shock stress-response molecule in the exosomes from non-heat shocked plants.
  • the heat shock stress-response molecule comprises HSP70.
  • the composition further includes from about 0.1 to about 20% of a moisturizing agent.
  • the moisturizing agent comprises one or more of panthenol, pantothenic acid derivatives, glycerin, glycerol, dimethicone, petrolatum, hyaluronic acid, or ceramides, and mixtures thereof.
  • the composition may also include one or more of a vitamin B3 compound, tocopherol or esters of tocopherol, and/or an anti-oxidant.
  • the composition may comprise tocopherol nicotinate.
  • the plant is from the Asphodelaceae family.
  • the plant is of the Aloe genus, including Aloe vera, Aloe barbadensis Miller, Aloe aborenscens or Aloe vera L.
  • the composition is in the form of a liquid, lotion, cream, gel, foam, mousse, spray, paste, powder, or solid.
  • a method for regulating skin condition comprises applying to human skin at least once a day over at least seven days a topical composition according to the first aspect, wherein regulating skin condition includes one or more of inducing increased skin integrity by cell renewal, enhancing water content or moisture of skin, reducing trans epidermal water loss, skin flaking, and scaling, improving skin thickness, enhancing skin tensile properties, reducing the appearance of dermal fine lines and wrinkles, improving skin texture, reducing skin pores size, enhancing skin smoothness, improving skin age spots, improving skin tone, or improving the appearance of scars and skin abrasions.
  • a glove for conditioning the skin has a coating composition on the inside thereof, wherein the coating composition includes isolated plant-derived exosomes having increased levels of heat shock stress-response molecules; and a powder carrier.
  • the plant-derived exosomes are isolated from a plant’s leaf flesh
  • a method of stimulating a skin rejuvenating response in human primary fibroblasts includes treating human primary fibroblasts with exosomes having increased levels of heat shock stress-response molecules.
  • the exosomes are obtained from a plant’ s leaf flesh conditioned by growing the plant under conditions that include a heat shock of the plant at a temperature of from about 33°C to about 45°C for about 1 hour to about 3 hours.
  • the heat shock temperature is from about
  • the heat shock temperature is from about 40°C to about 45 °C.
  • the skin rejuvenating response includes increasing production of collagen I in human primary fibroblasts by about 10% to about 80% relative to control.
  • the skin rejuvenating response may include increasing production of collagen I in human primary fibroblasts by at least 20% relative to control, by at least 40% relative to control and/or by at least 60% relative to control.
  • collagen I production in human primary fibroblasts may be increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, and/or 80% relative to control.
  • the skin rejuvenating response may comprise increasing production of hyaluronic acid in human primary fibroblasts by about 10% to about 50% relative to control.
  • the skin rejuvenating response may include increasing production of hyaluronic acid in human primary fibroblasts by at least 20%, by at least 30%, and/or by at least 40% relative to control.
  • hyaluronic acid production may increase by about 10%, 15%, 20%, 25%, 30%, 35%, and/or 40%, relative to control.
  • the skin rejuvenating response may comprise increasing production of elastin protein in human primary fibroblasts by about 100% to about 400% relative to control.
  • the skin rejuvenating response may include increasing production of elastin protein in human primary fibroblasts by at least 120%, by at least 150%, by at least 200%, and/or by at least 300% relative to control.
  • a method of regulating an inflammatory response in mammalian cells includes treating human primary fibroblasts with exosomes having increased levels of heat shock stress-response molecules.
  • the exosomes are obtained from a plant’ s leaf flesh conditioned by growing the plant under conditions that include a heat shock of the plant at a temperature of from about 33°C to about 45 °C for about 1 hour to about 3 hours.
  • the heat shock temperature is from about 33°C to about 37 °C. In another feature of the fifth aspect, the heat shock temperature is from about 40 °C to about 45 °C.
  • regulating the inflammatory response comprises reducing the amount of inflammatory response molecules in mammalian cells upon exposure of the cells to immune response inducing bacteria.
  • the amount of inflammatory response molecules is reduced by about 20% to about 50%.
  • the amount of inflammatory response molecules may be reduced by at least 30% or by at least 40%.
  • the inflammatory response molecule may comprise IL6 or TNFa.
  • FIGS. 1A and IB are graphs showing the size distribution of a representative sample of A) non-heat shocked (mean 156nm, mode 115nm) and B) 45°C for 3 hours heat-shocked (mean 156nm, mode 117nm) aloe vera-derived isolated exosomes according to one or more embodiments of the present disclosure.
  • FIG. 2 is a graph showing the relative changes in HSP70 mRNA transcript levels in exosomes isolated from aloe vera according to one or more embodiments of the present disclosure before and after exposure to heat shock. Values shown are Relative Quantification values determined by the AACt method relative to the non-heat shocked exosome values.
  • FIG. 3 is a bar graph showing the amount of cell proliferation in human primary fibroblasts after 3 day incubation with serum free medium, medium with 10% fetal bovine serum, or varying concentrations of aloe vera-derived exosomes isolated according to one or more embodiments of the present disclosure. Values shown are percent increase in proliferation relative to serum free medium.
  • FIG. 4 is a graph showing the amount of collagen I production in human primary fibroblasts after a 48 hour incubation with medium control, transforming growth factor b (TGF beta), or exosomes isolated from aloe vera according to one or more embodiments of the present disclosure. Values shown are ng/ml of collagen.
  • TGF beta transforming growth factor b
  • FIG. 5 is a graph showing the amount of hyaluronic acid produced by human primary fibroblasts after a 48 hour incubation with medium control, interleukin 1 (IL-1), or exosomes isolated from aloe vera according to one or more embodiments of the present disclosure. Values shown are ng/ml of hyaluronic acid.
  • IL-1 interleukin 1
  • FIG. 6 is a graph showing the amount of elastin protein produced by human primary fibroblasts after a 48 hour incubation with medium control, interleukin 1 (IL-1), or exosomes isolated from aloe vera according to one or more embodiments of the present disclosure. Values shown are ng/ml of elastin.
  • IL-1 interleukin 1
  • FIG. 7 is a graph showing quantified interleukin 6 (IL-6) secreted protein levels from human peripheral blood mononuclear cells after being incubated overnight with the following treatments: without LPS or exosomes (Media Only), with lpg/ml LPS and without exosomes (lpg/ml LPS), with lpg/ml LPS and lOOnM dexamethasone and without exosomes (Dex 100nM+LPS), with lpg/ml LPS in combination with exemplary aloe vera-derived exosomes (lpg/ml LPS + Aloe Exosomes). Values shown are pg/ml of IL-6.
  • FIG. 8 is a graph showing quantified tumor necrosis factor alpha (TNFa) secreted protein levels from human peripheral blood mononuclear cells after being incubated overnight with the following treatments: without LPS or exosomes (Media Only), with lpg/ml LPS and without exosomes (lpg/ml LPS), with lpg/ml LPS and lOOnM
  • TNFa tumor necrosis factor alpha
  • dexamethasone and without exosomes (Dex 100nM+LPS), with lpg/ml LPS in combination with exemplary aloe vera-derived exosomes (lpg/ml LPS + Aloe Exosomes). Values shown are pg/ml of TNFa.
  • Exosomes represent a compelling therapeutic for a range of indications, especially those requiring delivery to tissues with reduced vasculature or prominent necrosis.
  • Exosomes unlike stem cells, do not require an oxygenated blood supply to exert their impact. And, because exosomes fuse with cell membranes directly, there is no requirement for receptor mediated uptake of their pro-healing cargos. Accordingly, the isolated plant-derived exosomes produced according to the methods provided herein can have advantages over existing systemic pharmaceuticals or direct application of plant extracts for regulating skin conditions.
  • exosome loading can be engineered to result in exosomes having enhanced skin conditioning activities, such as and including, increased proliferative and anti-inflammatory activities.
  • isolated exosomes can be prepared from aloe vera leaf flesh in a controlled environment, wherein the plant is exposed to various stimuli to manipulate the exosomal cargo.
  • aloe vera leaves can be subjected to relatively high temperature (otherwise known as“heat shock”) to produce exosomes having increased levels of heat shock stress-response molecules, including stress-response proteins in the HSP70 protein family.
  • HSP70 proteins are a family of proteins expressed in response to heat stress or heat shock. HSP70 proteins have three major functional domains: N-terminal ATPase domain, substrate binding domain, and C-terminal domain.
  • the term“increased levels” of heat shock stress-response molecules means that the amount of stress-response molecules present in exosomes of a plant that has been subjected to a relatively high temperature (or heat shock) is higher than the amount of stress-response molecules present in exosomes of a plant subjected to conventional plant exposure temperatures (for example, room temperature, which is generally around 25 °C).
  • increased levels may include increases of 5% to 200% relative to plants having no heat shock treatment.
  • the level of heat shock stress-response molecules in exosomes of a heat shocked plant may be 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175% or 200% greater than the level of heat shock stress-response molecules in exosomes of non-heat shocked plants.
  • the level of heat shock stress-response molecules in exosomes of a heat shocked plant may be 2, 3, 4, 5, 10, 15, 20, 25, or 30 times greater than the level of heat shock stress-response molecules in exosomes of non-heat shocked plants.
  • stress-response molecules and“heat shock stress-response molecules” are used interchangeably herein for the purposes of the specification and claims. These terms are meant to include molecules present in exosomes that are secreted by plant cells subjected to high temperature (otherwise known as“heat shock”). Often, heat shock stress-response molecules are proteins. Similarly, the terms“exosomes” and“heat shock exosomes” and“heat shocked exosomes” are used interchangeably herein for the purposes of the specification and claims to represent exosomes that are secreted by plant cells subjected to high temperature (otherwise known as“heat shock”).
  • the term“about” when used in connection with one or more numbers or numerical ranges should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth.
  • the recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.
  • a plant-derived, exosome-containing composition comprising isolated plant-based exosomes containing heat shock stress-response molecules and a carrier.
  • the heat shock stress-response molecules can be any molecules present in the plant exosomes that are secreted by plant cells in response to being subjected to a growing temperature that is relatively higher than the growing temperature to which the plant was exposed previously.
  • Heat shock stress-response molecules are typically proteins produced by cells in response to exposure to stressful conditions, such as heat shock. Heat-shock proteins are named according to their molecular weight. For example, Hsp60, Hsp70 and Hsp90 refer to families of heat shock proteins on the order of 60, 70, and 90 kilodaltons in size, respectively.
  • a plant may be accustomed to being grown at a temperature of about room temperature, which is about 25°C. Thus, any growing temperature higher than 25°C could be a relatively higher temperature. A plant may also be accustomed to a growing temperature that is higher or lower than room temperature.
  • a relatively higher growing temperature may include a temperature at least
  • a relatively higher growing temperature may include 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50°C.
  • Increasing the growing temperature of the plant may include increasing the temperature to a range of about 30°C to about 45 °C, about 30°C to about 40°C, about 32°C to about 38°C, about 33°C to about 37°C, and/or about
  • a plant can be subjected to a relatively higher growing temperature for various periods of time.
  • a plant can be subjected to a relatively higher temperature for a period of time of about 30 minutes to about 4 hours, including, for example, 30 minutes, 60 minutes, 90 minutes, 2 hours, 2.5 hours, 3 hours, 3.5 hours, or 4 hours.
  • the plant may then be exposed to a relatively lower growing temperature for a period of time.
  • the plant may be exposed to a temperature of about 25 °C to about 27 °C for about 24 hours to about 72 hours subsequent to heat shocking.
  • the carrier may be an acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more exosomes to a subject.
  • Typical pharmaceutically acceptable carriers include, but are not limited to, binding agents (e.g. pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc); fillers (e.g. lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc); lubricants (e.g.
  • An exemplary embodiment of a plant-derived exosome-containing composition includes a composition comprising: i) isolated aloe vera plant exosomes having increased levels of heat shock stress-response molecules; and ii) a carrier.
  • the aloe vera exosomes can be produced by a process comprising: (a) growing the plant, wherein the growing includes a step of heat shocking the plant by increasing the growth temperature for a set period of time; and (b) isolating the exosomes having increased levels of heat shock stress-response molecules from the leaf flesh.
  • composition can be in any number of physical forms, including, but not limited to a liquid, lotion, cream, gel, foam, mousse, spray, paste, powder, or solid.
  • Isolation of the exosomes can be performed using various processes and/or combination of processes.
  • isolating the exosomes can be carried out by centrifugation, including one or more centrifugation steps.
  • the one or more centrifugation steps can include centrifugation at relatively lower speeds of 400 to 500 x g, at relatively moderate speeds of 10,000 to 20,000 x g, and relatively higher speeds of 80,000 to 100,000 x g or greater.
  • isolating the exosomes can further include freeze drying isolated exosomes. Freeze dried exosomes can be stored at room temperature.
  • the plant may be any plant believed to secrete heat shock response molecules in response to a heat shock process such as that described above.
  • the plant may be of the Asphodelaceae family.
  • the plant can further be of the genus Aloe, including, but not limited to, Aloe vera and Aloe barbadensis.
  • the heat shock stress-response molecules can include heat shock proteins, such as proteins from the following protein families: HSP60, HSP70, and HSP90.
  • a method for making plant-derived exosomes having increased levels of heat shock stress-response molecules including:
  • the growing condition includes a step of heat shocking the plant by increasing the growth temperature from a range of about 20°C to about 30°C, to about 33°C to about 37°C for about 1 hour to about 3 hours, and/or to about 40°C to about 45°C for about 1 hour to about 3 hours, and wherein the plant’s leaf flesh contains the exosomes having the increased levels of heat shock stress-response molecules.
  • heat shock stress does not affect the size distribution of plant-derived exosomes.
  • the results of the testing described in Example 2 indicate that exosomes produced after heat shock treatment are within the size range typically used for most applications.
  • FIG. 1A shows the size distribution of a representative sample of isolated exosomes from non-heat shocked aloe vera with mean of 156 nm and a mode of 115 nm.
  • FIG. IB shows the size distribution of a representative sample of isolated exosomes from the leaf flesh of an aloe vera plant 6 hours after a 3 hour 45 °C heat shock with a mean of 156nm and a mode of 116nm. As shown, there is no significant effect on the size distribution of exosomes following a heat shock stress.
  • heat shock increases the production of heat shock stress-response molecules in plant cells, thereby resulting in an increased amount of stress-response molecules in exosomes derived from the heat-shocked plant cells.
  • exposure to heat shock for 2 to 3 hours significantly upregulated the transcript levels of an exemplary heat shock stress-related molecule, HSP70, in the contents of the exosomes, and the upregulation persisted for 24 hours after removal of the heat shock stress.
  • HSP70 heat shock stress-related molecule
  • FIG. 3 shows that treatment with isolated exosomes from the plant leaf flesh of heat shocked plants significantly increased proliferation of human primary fibroblasts.
  • the level of proliferation of the human primary fibroblasts induced by the isolated exosomes approached that induced by fetal bovine serum.
  • Example 4 Experimental results shown in Example 4 demonstrate that isolated exosomes prepared from aloe vera leaf flesh of heat shocked plants can induce collagen I, hyaluronic acid and elastin synthesis in human primary fibroblasts.
  • FIG. 4 shows that treatment with isolated exosomes from heat shocked plants increased collagen I production of human primary fibroblasts. Specifically, exposure of the plant to heat shock increased the beneficial effects of the isolated exosomes on human primary fibroblast collagen I production by about 40% to about 80% and this exosome activity persisted up to 24 hours after heat shock exposure. In addition, the increase in collagen I production of the human primary fibroblasts induced by the isolated exosomes approached that of the individual growth factor, TGF-b.
  • FIG. 5 shows that treatment with the isolated exosomes from heat shocked plants increased hyaluronic acid production of human primary fibroblasts. Specifically, exposure of the plant to heat shock increased the effects of the isolated exosomes on human primary fibroblast hyaluronic acid production by about 31% to about 45% and this exosome activity persisted up to 24 hours after heat shock exposure. In addition, the increase in hyaluronic acid production of the human primary fibroblasts induced by the isolated exosomes approached that of the individual growth factor, IL-1.
  • FIG. 6 shows that treatment with the isolated exosomes from heat shocked plants increased elastin protein production of human primary fibroblasts by 140 - 375%.
  • the increase in elastin protein production of the human primary fibroblasts induced by the isolated exosomes surpassed that of the individual growth factor, IL-1 (35%).
  • Lypopolysaccharide is a major component of the outer membrane of
  • isolated exosomes from plants exposed to heat shock The elevation is reduced by 25-50% by isolated exosomes from plants exposed to heat shock.
  • a method for treating a skin condition including one or more of putting on, embedding into, or filling an area on the skin of a living body a composition of the present disclosure including isolated plant-derived exosomes having increased levels of heat shock stress-response molecules, wherein the condition of the skin is treated.
  • the skin condition can include, for example, one or more of a wound, a bum, a bum resulting from radiation treatment, a discoloration, a scar, and a keloid.
  • a topical composition for regulating skin condition, the composition comprising an effective amount of isolated exosomes having increased levels of heat shock stress-response molecules and a carrier.
  • a topical composition for regulating skin condition, the composition including: i) an effective amount of isolated exosomes having increased levels of heat shock stress-response molecules; and ii) a carrier, wherein the isolated exosomes are isolated from a plant’s leaf flesh conditioned by growing the plant under conditions that include a heat shock of the plant at a temperature of about 33°C to about 37°C for about 1 hour to about 3 hours, and/or about 40°C to about 45°C for about 1 hour to about 3 hours.
  • a topical composition for regulating skin condition comprising: i) an effective amount of isolated exosomes having increased levels of heat shock stress-response molecules; and ii) a carrier, wherein the isolated exosomes are produced by a process comprising: (a) growing a plant, wherein the growth condition includes a step of heat shocking plant by increasing the temperature from a range of about 20°C to about 30°C, to about 33 °C to about 37°C for about 1 hour to about 3 hours, and/or to about 40°C to about 45°C for about 1 hour to about 3 hours, and wherein the plant’ s leaf flesh contains the exosomes having the increased levels of heat shock
  • a method for making a topical composition for regulating skin condition including: combining isolated exosomes having increased levels of heat shock stress-response molecules with a carrier, wherein the exosomes are isolated from the flesh of a plant’ s leaf conditioned by growing the plant under conditions including a heat shock of the plant at a temperature of about 33°C to about 37°C for about 1 hour to about 3 hours, and/or of about 40°C to about 45°C for about 1 hour to about 3 hours.
  • compositions provided for regulating skin conditions can include one or more of inducing increased skin integrity by cell renewal; enhancing water content or moisture of skin; reducing trans epidermal water loss, skin flaking, and scaling; improving skin thickness; enhancing skin tensile properties; reducing the appearance of dermal fine lines and wrinkles; improving skin texture; reducing skin pores size; enhancing skin smoothness; improving skin age spots; improving skin tone; or improving the appearance of scars and skin abrasions.
  • the composition can further include from about 0.1 to about 20% of a moisturizing agent.
  • the moisturizing agent can include one or more of panthenol, pantothenic acid derivatives, glycerin, glycerol, dimethicone, petrolatum, hyaluronic acid, or ceramides, and mixtures thereof.
  • the composition can further include a vitamin B3 compound.
  • vitamin B3 compounds can include tocopherol nicotinate, niacinamide, and inositol hexanicotinate
  • the composition can further include an anti-oxidant.
  • the anti-oxidant can include one or a combination of tocopherol or esters of tocopherol.
  • the isolated exosomes can be freeze dried.
  • a method for regulating a human skin condition which includes applying to human skin at least once a day over at least seven days a topical composition as described herein comprising isolated exosomes having increased levels of heat shock stress-response molecules.
  • the method can further include applying the topical to human skin at least twice a day over at least fourteen days.
  • a coating composition for conditioning skin or hair, the coating composition including: i) isolated plant-derived exosomes having increased levels of heat shock stress-response molecules; and ii) a carrier, wherein the plant-derived exosomes are produced by a process including: (a) growing the plant, wherein the growth condition includes a step of heat shocking the plant by increasing the temperature from a range of about 20°C to about 30°C, to about 33 °C to about 37°C for about 1 hour to about 3 hours, and/or to about 40°C to about 45°C for about 1 hour to about 3 hours, and wherein the plant’ s leaf flesh contains the exosomes having the increased levels of heat shock
  • the process for producing the isolated plant-derived exosomes can further include freeze drying the isolated exosomes.
  • the process for producing the isolated plant-derived exosomes can further include freeze drying the isolated exosomes and the carrier can be a dry powder.
  • the coating compositions for conditioning skin or hair of the present disclosure can be a dry powder coating composition applied to the inside of a glove.
  • the coating compositions for conditioning skin or hair of the present disclosure can be in the form of a liquid, lotion, cream, gel, foam, mousse, spray, paste, powder, or solid.
  • a glove for conditioning the skin, the glove having a coating composition on the inside thereof, the coating composition including: i) isolated plant-derived exosomes having increased levels of heat shock stress-response molecules; and ii) a powder carrier, wherein the isolated plant-derived exosomes are produced by a process including: (a) growing the plant, wherein the growth condition includes a step of heat shocking the plant by increasing the temperature from a range of about 20°C to about 30°C, to about 33°C to about 37°C for about 1 hour to about 3 hours, and/or to about 40°C to about 45 °C for about 1 hour to about 3 hours, and wherein the plant’s leaf flesh contains the exosomes having the increased levels of heat shock stress-response molecules; (b) isolating the exosomes having increased levels of heat shock stress-response molecules from the plant’s leaf flesh; and (c) freeze drying the isolated exosomes.
  • a glove for conditioning the skin, the glove having a coating composition on the inside thereof, the coating composition comprising: i) isolated plant-derived exosomes having increased levels of heat shock stress-response molecules; and ii) a powder carrier, wherein the plant-derived exosomes are isolated from the plant’ s leaf flesh conditioned by growing the plant under conditions including a heat shock of the plant at a temperature of about 33°C to about 37 °C for about 1 hour to about 3 hours, and/or of about 40°C to about 45 °C for about 1 hour to about 3 hours.
  • a glove for conditioning the skin, the glove having a coating composition on the inside thereof, the coating composition comprising isolated plant-derived exosomes having increased levels of heat shock stress-response molecules and a powder carrier.
  • a method for making a glove for conditioning the skin comprising applying to the inside of the glove, a coating composition according to the present disclosure comprising isolated exosomes having increased levels of heat shock stress-response molecules and a powder carrier.
  • Aloe vera plants were grown and sampled under the following conditions.
  • the plants were maintained at room temperature, approximately 25 ° C with ample water and light.
  • Leaf samples from plants grown in the room temperature conditions were taken by removing a 3 inch leaf section for further preparation.
  • Leaf section preparation is described below.
  • the plant was then heat shocked by warming it to 35 ° C in an incubator for 2 hours. After the 2 hour incubation, 3 inch leaf sections from the heated plant were dissected for further preparation.
  • the inner leaf pulp containing parenchymal mesophyll cells was carefully removed to avoid collecting any outer leaf skin material and then homogenized. Homogenization was performed using a Precellys tissue homogenizer, ceramic beads and sterile PBS to disrupt the plant pulp tissue. The homogenate was transferred to a larger tube and the beads were washed 4 times with sterile PBS to collect all of the homogenate, which was also transferred to the larger tube for storage at -80 ° C until used for exosome isolation.
  • the exosomes produced by the plants were isolated from the inner leaf pulp homogenate using the following procedure.
  • the homogenate was diluted in phosphate-buffered-saline (PBS) at a ratio of about 16g of homogenate per 50 mL PBS.
  • PBS phosphate-buffered-saline
  • the diluted homogenate mixture was put through a freeze thaw cycle by freezing at -80 ° C and then allowing the mixture to thaw at room temperature, whereupon the volume was increased to 300 ml. by adding PBS.
  • the diluted homogenate mixture was then fractionated by differential centrifugation to isolate the exosomes produced by the plants.
  • the diluted homogenate mixture was subjected to centrifugation at 4,000 x g for 20 min at room temperature to pellet cell debris (in 50, 250, or 500 mL screw cap vessels).
  • the clarified supernatant was collected and subjected to centrifugation at 12,000 x g (Avg. RCF) for 30 minutes at room temperature to remove any intact chloroplasts.
  • the subsequent clarified supernatant was brought up to 360 mL with PBS and subjected to centrifugation at 100,000 x g (Avg. RCF) for 2 hrs at 4 ° C to pellet exosomes.
  • the supernatant was aspirated and the pellet(s) suspended in minimum volume of DPBS (300-1000 pL).
  • FIGs. 1A and IB show the resulting size distribution of representative samples from non-heat shocked and heat shocked aloe vera plants.
  • FIG. 1A shows the particle size distribution of exosomes derived from non-heat shocked aloe vera plants. As can be seen, the exosomes had a mean diameter of 156 nm and a mode diameter of 115 nm.
  • FIG. IB shows the particle size distribution of exosomes derived from an aloe vera plant 6 hours after it was heat shocked at 45 ° C for 3 hours. The heat shocked exosomes had a mean diameter of 156 nm and a mode diameter of 117 nm. As shown, exposure to heat shock stress did not significantly change the mean particle size of the isolated exosomes. Thus, heat shock treatment does not detrimentally alter or impact the applications in which heat shocked exosomes can be used.
  • RNA was isolated from exosomes derived from the leaf pulp of aloe vera plants exposed to no heat shock, 35 ° C heat shock for 2 hours, 45 ° C heat shock for 3 hours, and 24 hour room temperature recovery after 45 ° C heat shock for 3 hours.
  • 2 x 10 L 10 exosomes from each treatment were used to isolate total RNA using a micro RNeasy kit from Qiagen and the manufacturer’s protocol.
  • Total RNA was quantified spectrophometrically using manufacturer’s instructions and a NANODROP (THERMO FISHER, Corp) spectrophotometer.
  • cDNA was prepared using 300ng total RNA per reaction in 20 microliter final volume and qPCR performed using lOng of cDNA per triplicate, primers for HSP70 and 18s genes, and SYBR Green master mix in a final volume of 15 microliters. Relative gene expression was determined by the AACt method using 18s gene expression as the internal housekeeping gene and comparisons made to the no heat shock control exosomes.
  • HSP70 is a heat stress response molecule. In this testing, HSP70 was used as a marker to show that heat stress responses to the plant were transferred to the plant exosomes. One of skill in the art will understand that other stress response molecules can be transferred to the plant exosomes.
  • FIG. 2 shows the relative changes in HSP70 mRNA transcript levels in exosomes isolated from aloe vera plants for each of the treatments set forth above. Values shown are Relative Quantification values determined by the AACt method (Fold Change) relative to the non-heat shocked exosome values. Statistical significance compared to the non-heat shocked sample was determined using student’s t-test and a p-value ⁇ 0.05, significance is denoted by a As can be seen in FIG. 2, induction of a heat shock stress at both 35 ° C and 45 ° C increased the transcript level of the stress response molecule, HSP70, relative to that for non-heat treated plants.
  • the transcript level at a heat shock temperature of 35 °C was less than the transcript level at a heat shock temperature of 45 °C.
  • the data shows a 10X increase in transcript level of the stress response molecule HSP70 after a heat shock of 35 °C in comparison to the same for non-heat shocked exosomes.
  • Transcript level of HSP70 after a heat shock of 45 °C was about 17X that for non-heat shocked exosomes
  • transcript level of HSP70 after heat shock of 45 °C and 24 hour waiting period was about 22X that for non-heat shocked exosomes.
  • the increase in stress response molecules was maintained for at least 24 hours after removing the heat shock stress.
  • FIG. 3 graphically shows the results of the cell proliferation in the human primary fibroblasts. The values shown are percent increase in proliferation compared to media only treatment.
  • the data in FIG. 3 shows that treatment with the heat shocked isolated exosomes increased proliferation of human primary fibroblasts. For example, heat shocked exosomes produced up to 25% more fibroblasts than a media only control. While aloe vera-derived exosomes with no heat shock demonstrated a dose dependent induction of proliferation, many of the heat-shocked exosomes provided a greater increase in proliferation than the non-heat shocked exosomes. Moreover, the data shows that different concentrations of isolated exosomes have differing effects on the proliferation of human primary fibroblasts.
  • FIG. 4 is a graph showing the amount of collagen I production (in ng/mL) in human primary fibroblasts after a 48 hour incubation with medium control, transforming growth factor b (TGF beta), or exosomes isolated from aloe vera plants. Values shown are ng/ml of collagen.
  • TGF beta transforming growth factor b
  • treatment with the isolated exosomes only from heat shocked aloe vera plants increased collagen I production of human fibroblasts.
  • Exosome samples from heat-shocked plants were obtained at the following times: 1) after heat shock of 35°C for 2 hours, 2) after 3 hours of 45 °C heat shock, and 3) after 24 hours post 45°C heat shock.
  • Collagen I production was increased by 42 - 78% for all three heat-shocked aloe vera plant exosome types relative to media alone. Exosomes from aloe vera plants not receiving a heat shock stress did not increase collagen I production. The highest collagen I production was measured for exosomes obtained immediately after heat shock of 35°C for 2 hours. TGF -l is a known activator for collagen I synthesis;
  • TGF -l was included in the testing for comparison with exosomes from heat shocked aloe vera plants.
  • FIG. 6 is a graph showing the amount of elastin protein (in ng/mL) produced by human primary fibroblasts after a 48 hour incubation with medium control, interleukin 1 (IL-1), or exosomes isolated from aloe vera plants.
  • IL-1 interleukin 1
  • exosomes were isolated from heat-shocked plants at varying times: 1) immediately after heat shock of 35°C for 2 hours, 2) 6 hours post 45°C heat shock, and 3) 24 hours post 45 °C heat shock. Values shown are ng/ml of elastin.
  • FIG. 6 shows that isolated exosomes from heat shocked aloe vera plants greatly increased elastin synthesis, by 139 - 377% relative to media only samples (control), including above that induced by IL-1 (36%), and that this activity was maintained out to 24 hour following heat shock.
  • Exosomes from non-heat shocked aloe vera plants increased elastin protein production as well. The limit for clinically relevant elastin induction is currently unknown. Thus, it is beneficial to understand the effect on elastin production after treatment with exosomes from heat-shocked as well as non-heat shocked plants.
  • Example 5 it is beneficial to understand the effect on elastin production after treatment with exosomes from heat-shocke
  • PBMCs peripheral blood mononuclear cells
  • Lypopolysaccharide is a major component of the outer membrane of Gram-negative bacteria and is known to induce a strong immune response in mammalian cells promoting the secretion of inflammatory cytokines.
  • Previous reports indicate the induction of inflammatory cascades in PBMCs in response to LPS, including the
  • inflammatory molecules IL6 and TNFa [14-16].
  • human PBMCs were concomitantly exposed to LPS (lpg/ml) and exemplary isolated exosomes from heat shocked aloe vera plants.
  • Dexamethasone a synthetic glucocorticoid known to reduce inflammation in response to LPS, was included as a control comparator.
  • Cells were treated as follows: 1) without LPS and without exosomes (Media Only), 2) with lpg/ml LPS and without exosomes (lpg/ml LPS), 3) with lpg/ml LPS and lOOnM dexamethasone (Dex lOOnM + LPS), and 4) with lpg/ml LPS in combination with heat-shocked aloe vera-derived isolated exosomes (lpg/ml LPS + Aloe Exosomes).
  • FIG. 7 is a graph showing quantified interleukin 6 (IL-6) secreted protein levels from human peripheral blood mononuclear cells after being incubated overnight with the following treatments: without LPS or exosomes (Media Only), with lpg/ml LPS and lOOnM dexamethasone (Dex IOOhM+LPS) only, with lpg/ml LPS (lpg/ml LPS) only, and a series of exemplary samples with lpg/ml LPS in combination with aloe vera-derived exosomes (lpg/ml LPS + Aloe Exosomes).
  • IL-6 interleukin 6
  • the exemplary samples of aloe vera derived exosomes all used a concentration of 1 x 10 L 8 particles/mL of exosomes isolated from plants at varying times: 1) no heat shock, 2) immediately after heat shock of 35 °C for 2 hours, 3) immediately after heat shock of 45°C for 3 hours, 4) 6 hours post 45°C heat shock, and 5) 24 hours post 45 °C heat shock were used.
  • Y axis values shown are pg/ml of IL-6.
  • Treatment with aloe exosomes isolated immediately after heat shock of 45 °C for 3 hours and 6 hours post 45 °C heat shock provided decreased production of IL-6.
  • Aloe exosomes isolated 6 hours post 45°C heat shock provided the biggest decrease in production of IL-6, which most closely approached IL-6 production decrease achieved by treatment with lOOnM dexamethasone.
  • FIG. 8 is a graph showing quantified tumor necrosis factor alpha (TNFa) secreted protein levels from human peripheral blood mononuclear cells after being incubated overnight with the following treatments: without LPS or exosomes (Media Only), with lpg/ml LPS and without exosomes (lpg/ml LPS), with lpg/ml LPS and lOOnM
  • TNFa tumor necrosis factor alpha
  • dexamethasone and without exosomes (Dex 100nM+LPS), and a series of exemplary samples with lpg/ml LPS in combination with aloe vera-derived exosomes (lpg/ml LPS + Aloe Exosomes).
  • the exemplary samples of aloe vera derived exosomes all used a concentration of 1 x 10 L 8 particles/mL of exosomes isolated from plants at varying times: 1) no heat shock, 2) immediately after heat sho
  • ck of 35°C for 2 hours, 3) immediately after heat shock of 45°C for 3 hours, 4) 6 hours post 45°C heat shock, and 5) 24 hours post 45°C heat shock were used.
  • Y-axis values shown are pg/ml of TNFa.
  • Treatment with aloe exosomes isolated immediately after heat shock of 45 °C for 3 hours and 6 hours post 45 °C heat shock provided decreased production of TNFa relative to no treatment. The biggest decrease in production of TNFa was provided by treatment with exosomes isolated 6 hours post 45 °C heat shock.
  • the results indicate a significant elevation in IL-6 (87-fold) and TNFa (62-fold) protein secretion in PBMCs induced by LPS at lpg/ml.
  • the elevation is reduced by the isolated aloe vera exosomes from heat-shocked plants by about 40% for TNFa and about 50% for IL-6.
  • These data indicate that the isolated exosomes described herein can inhibit the production of inflammatory cytokines including IL6 and TNFa that act locally to recruit monocytes to the site of inflammation ⁇

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Abstract

L'invention concerne une composition topique pour la régulation de l'état de la peau. La composition comprend une quantité efficace d'exosomes isolés ayant des niveaux accrus de molécules de réponse au stress de choc thermique et un support. Les exosomes isolés sont obtenus à partir de la chair de feuille d'une plante conditionnée en faisant pousser la plante dans des conditions qui comprennent un choc thermique de la plante à une température allant d'environ 33 °C à environ 45 °C pendant environ 1 heure à environ 3 heures. Les utilisations des compositions contenant des exosomes pour réguler la peau humaine consistent à induire une intégrité de la peau accrue par le renouvellement cellulaire, à améliorer la teneur en eau ou l'humidité de la peau, à réduire la perte d'eau transépidermique, l'exfoliation de la peau et la desquamation, à améliorer l'épaisseur de la peau, à améliorer les propriétés d'élasticité de la peau, à réduire l'apparition de ridules et de rides du derme, à améliorer la texture de la peau, à réduire la taille des pores de la peau, à améliorer la douceur de la peau, à améliorer les taches de vieillesse de la peau, à améliorer le teint de la peau, et à améliorer l'aspect des cicatrices et des abrasions de la peau.
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