Application of recombinant extracellular vesicle bioactive protein delivery system in anti-wrinkle and anti-aging

Abstract

The present disclosure provides the use of catalase or variants thereof or nucleic acid sequences or vectors or delivery systems in the preparation of pharmaceutical or cosmetic compositions for improving visible signs and / or biomarkers of skin photoaging.

Description

Applications of recombinant extracellular vesicle bioactive protein delivery systems in anti-wrinkle and anti-aging

[0001] This application claims priority to PCT application No. PCT / CN2025 / 081856, filed March 11, 2025. The contents of the earlier PCT application are considered part of this disclosure and are incorporated herein by reference in their entirety. Technical Field

[0002] This disclosure relates to the field of biomedicine, specifically to the use of recombinant extracellular vesicle bioactive protein delivery systems in anti-wrinkle and anti-aging. Background Technology

[0003] Skin aging and related cellular aging are caused by exogenous factors (such as ultraviolet radiation, smoking, and pollution) and endogenous factors (such as time, genetic factors, and hormones). Ultraviolet (UV) radiation is the strongest external driver of age-related changes in the skin (i.e., "photoaging"). Approximately 80% of facial aging can be attributed to photoaging. Clinically, photoaging of the skin mainly manifests as photoelastic tissue degeneration, wrinkles, and pigmentation.

[0004] Ultraviolet radiation triggers excessive production of reactive oxygen species (ROS) in the epidermis and dermis, with mitochondria being the primary site of ROS production in cells. ROS negatively impact skin cells, including keratinocytes and fibroblasts, by inducing oxidative damage and promoting inflammatory responses. These effects lead to decreased cell viability, accelerated collagen degradation, and upregulation of pro-inflammatory cytokines. Many features of skin aging are associated with inflammation. Elevated ROS levels are typically observed during the polarization of M0 macrophages into pro-inflammatory M1 macrophages. Excessive ROS further promotes M1 polarization. Cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), markers of M1 macrophages, are upregulated in photoaged skin.

[0005] Small molecules have limited therapeutic efficacy because they are ineffective at scavenging intracellular reactive oxygen species (ROS). Antioxidant enzymes, which react with these oxidants thousands to millions of times faster than small molecules, are the primary antioxidant defense mechanism. Catalase, the most active antioxidant enzyme in nature and the human body, plays a crucial role in mitigating oxidative stress by effectively scavenging ROS. Catalase's ability to break down hydrogen peroxide into water and oxygen significantly reduces oxidative damage caused by UV radiation. This enzymatic activity not only protects keratinocytes and fibroblasts from ROS-induced cellular damage but also helps maintain skin integrity and elasticity. Catalase can serve as an effective strategy for improving visible signs of photoaging, such as wrinkles and pigmentation.

[0006] Extracellular vesicles (EVs) are lipid bilayer nanoparticles released from all cell types into the extracellular space. EVs naturally transport essential cellular components (such as proteins or active enzymes, lipids, and nucleic acids like mRNA and microRNA) for intercellular communication. Therefore, EVs show great promise in delivering active ingredients, payloads, or therapeutic agents to target cells or intracellular spaces. As natural secretions of cells, EVs offer numerous advantages over synthetic drug delivery carriers (such as liposomes, lipid nanoparticles, and viral vectors) as drug delivery systems / carriers. EVs are naturally derived from cells, giving them inherent biocompatibility and making them less likely to trigger immune responses compared to synthetic carriers. This property is crucial for reducing potential side effects in therapeutic applications; EVs possess inherent tissue-targeting capabilities and the ability to cross biological barriers; EVs exhibit extremely high stability in biological fluids, which helps prolong their circulation time in the extracellular matrix and blood. EVs have been extensively validated in scientific research and clinical applications, demonstrating their effectiveness and safety in the field of medical aesthetics. Their potential therapeutic applications include anti-aging, anti-pigmentation, wound healing, and hair regeneration. However, the active ingredients in EVs derived from natural stem cells are very limited.

[0007] Catalase has a short half-life after entering the body due to rapid proteolytic degradation and immunogenicity. Therefore, encapsulating catalase in extracellular vesicles can enhance its stability, prolong its effective half-life, and allow it to better penetrate target tissues and cells, effectively scavenging reactive oxygen species. Currently, several methods have been developed for loading therapeutic agents into EVs, including sonication, electroporation, and passive incubation. However, these traditional methods involve multiple manufacturing steps, including catalase protein expression, EV preparation, and loading. They have several drawbacks, such as relatively low loading efficiency and high production costs.

[0008] Currently, there is a lack of low-cost, efficient catalase and its delivery system to improve photoaged skin conditions. Summary of the Invention

[0009] To address the aforementioned technical problems, this disclosure provides catalase and its delivery system using genetic engineering methods. By modifying production cells, co-expression of recombinant human catalase protein (rhCAT) and high-level recombinant human extracellular vesicles (rhEVs) is achieved, thereby simplifying the production and loading process into a one-step procedure. This not only improves loading efficiency but also significantly reduces overall production costs by streamlining the manufacturing workflow. Furthermore, the modified catalase can target mitochondria, making it more specific. The catalase and its delivery system disclosed herein offer a promising pathway for scavenging reactive oxygen species, alleviating oxidative stress, and improving visible signs and / or biomarkers of skin photoaging.

[0010] In a first aspect, this disclosure provides a catalase or a variant thereof that has at least one of the following biological activities: scavenging reactive oxygen species, alleviating oxidative stress, and / or improving visible signs and / or biomarkers of skin photoaging.

[0011] In some embodiments, the catalase or a variant thereof is a wild-type catalase or a modified catalase, wherein the modified catalase is selected from at least one group of: (i) a wild-type catalase having one or more amino acid mutations, insertions, deletions and / or additions, (ii) a wild-type catalase modified with a chemical group, or (iii) a fusion protein comprising (a) a wild-type catalase or a variant thereof, and (b) other functional proteins or peptides having a specific biological function; optionally, the functional protein or peptide has tissue-targeting activity; more preferably, it has mitochondrial-targeting activity; most preferably, the fusion protein is obtained by deleting the C-terminal amino acid residue “KANL” of the catalase and adding an N-terminal mitochondrial-targeting sequence.

[0012] In some embodiments, the catalase or a variant thereof is selected from at least one group of the following: (i) a catalase having the amino acid sequence shown in SEQ ID NO: 1, or a catalase having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 1; (ii) a mitochondrial-targeting catalase having the amino acid sequence shown in SEQ ID NO: 2, or a mitochondrial-targeting catalase having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 2.

[0013] Secondly, this disclosure provides a nucleic acid sequence encoding catalase or a variant thereof of the first aspect of this application.

[0014] Thirdly, this disclosure provides a vector containing the nucleic acid sequence of the second aspect of this application.

[0015] Fourthly, this disclosure provides a delivery system comprising: (i) catalase or a variant thereof of the first aspect of this application, and / or (ii) a nucleic acid sequence of the second aspect of this application or a vector of the third aspect of this application, and (iii) a delivery vector, wherein the delivery vector has the function of delivering catalase or a variant thereof to a specific tissue or cell.

[0016] In some embodiments, the delivery vector is selected from at least one of liposomes, extracellular vesicles (EVs), and virus-like particles (VLPs).

[0017] In some embodiments, the delivery vector is selected from at least one of general extracellular vesicles or modified extracellular vesicles; optionally, the extracellular vesicle is a general exosome or a modified exosome.

[0018] In some embodiments, extracellular vesicles are harvested from producing cells; optionally, the extracellular vesicles are exosomes, and the exosomes may be modified to enhance production, exposure duration, tissue-specific targeting, or endosome escape; more preferably, the exosomes comprise: (i) peptides and / or proteins containing GPI-anchored signal sequences, (ii) peptide / antibody fragment modifications, and / or (iii) other protein modifications.

[0019] In some embodiments, the production cells overexpress at least one protein or protein fragment selected from CD46, CD52, CD55, CD58, and CD59.

[0020] In some implementations, the production cells are non-human mammalian cell lines or human cell lines.

[0021] In some embodiments, the production cells are selected from the HEK 293F cell line, the HEK 293T cell line, stem cell lines, or any combination thereof.

[0022] Fifthly, this disclosure provides a pharmaceutical composition comprising: (i) catalase or a variant thereof of the first aspect of this application, or (ii) a nucleic acid sequence of the second aspect of this application or a vector of the third aspect of this application, or (iii) a delivery system of the fourth aspect of this application, and (iv) a pharmaceutically acceptable excipient.

[0023] In a sixth aspect, this disclosure provides a cosmetic composition comprising: (i) a catalase or a variant thereof of the first aspect of this application, or (ii) a nucleic acid sequence of the second aspect of this application or a carrier of the third aspect of this application, or (iii) a delivery system of the fourth aspect of this application, and (iv) an acceptable excipient.

[0024] In a seventh aspect, this disclosure provides a method for manufacturing a delivery system according to the fourth aspect of this disclosure, wherein the delivery system is formed by production cells and secreted / released into a culture medium, and then obtained by separation and purification, wherein the production cells are transfected with the vector of the third aspect of this application, and the delivery system is generated within the cells.

[0025] In some embodiments, the production cells are selected from the HEK 293F cell line, the HEK 293T cell line, stem cell lines, or any combination thereof.

[0026] Eighthly, this disclosure provides a method for manufacturing a pharmaceutical composition of the fifth aspect of this disclosure or a cosmetic composition of the sixth aspect of this disclosure, which uses catalase or a variant thereof of the first aspect of this application, or a nucleic acid sequence of the second aspect of this application, or a carrier of the third aspect of this application, or a delivery system of the fourth aspect of this application.

[0027] Ninthly, this disclosure provides a method of using a pharmaceutical composition of the fifth aspect of this disclosure or a cosmetic composition of the sixth aspect of this disclosure, the method comprising administering to a subject in need an effective amount of catalase or a variant thereof of the first aspect of this application, or a nucleic acid sequence of the second aspect of this application, or a carrier of the third aspect of this application, or a delivery system of the fourth aspect of this application.

[0028] Tenthly, this disclosure provides the use of catalase or a variant thereof of the first aspect of this application, or the nucleic acid sequence of the second aspect of this application, or the vector of the third aspect of this application, or the delivery system of the fourth aspect of this application, in the preparation of pharmaceutical or cosmetic compositions for scavenging reactive oxygen species, alleviating oxidative stress, and / or improving visible signs and / or biomarkers of skin photoaging.

[0029] In some embodiments, the pharmaceutical or cosmetic composition is used to improve photoaged skin conditions.

[0030] In some implementations, improving photoaged skin conditions includes: (i) promoting skin cell proliferation, optionally, the skin cells being human dermal fibroblasts and / or human keratinocytes; (ii) reducing ROS levels; (iii) enhancing collagen regeneration; (iv) reducing wrinkles; and / or (v) improving the inflammatory condition of photoaged skin.

[0031] In some embodiments, the pharmaceutical or cosmetic composition is used to treat photoaging, natural aging of the skin, melasma, pigmentation, and / or vitiligo. Attached Figure Description

[0032] The following is a brief description of the accompanying drawings, which are used only to illustrate the exemplary embodiments disclosed herein and are not intended to limit them.

[0033] Figure 1 shows a schematic diagram of the recombinant vector system. Figure 1A shows that the target gene is wild-type catalase. Figure 1B shows that the target gene is mitochondrial-targeted catalase.

[0034] Figure 2 shows the particle size distribution of extracellular vesicles.

[0035] Figure 3 shows a transmission electron microscope image of extracellular vesicles.

[0036] Figure 4 shows the Western blot analysis of positive and negative markers of purified extracellular vesicles.

[0037] Figure 5 shows the enzyme activities of recombinant human catalase (MCE), recombinant human catalase (Abcam), and 313EVCAT samples.

[0038] Figure 6 illustrates the protective effect of extracellular vesicles against protein degradation by catalase.

[0039] Figure 7 illustrates the cytotoxicity of blank extracellular vesicles, 313EVCAT, and mitoEVCAT.

[0040] Figure 8 shows the ability of 313EVCAT to remove hydrogen peroxide.

[0041] Figure 9 shows a comparison of the ability of 313EVCAT and extracellular vesicles derived from mesenchymal stem cells to scavenge hydrogen peroxide.

[0042] Figure 10 illustrates the ability of 313EVCAT to prevent UVB radiation damage.

[0043] Figure 11 shows the ability of 313EVCAT to scavenge extracellular hydrogen peroxide.

[0044] Figure 12 shows the relative average fluorescence intensity of UVB-irradiated cells pretreated with 313EVCAT, representing the intracellular hydrogen peroxide level.

[0045] Figure 13 shows the results of enzyme-linked immunosorbent assay (ELISA), demonstrating that 313EVCAT can enhance cell regeneration and promote collagen synthesis.

[0046] Figure 14 shows the results of real-time quantitative PCR analysis, demonstrating that 313EVCAT can enhance cell regeneration and promote collagen synthesis.

[0047] Figure 15 illustrates the ability of 313EVCAT to reduce the inflammatory cytokine IL-6.

[0048] Figure 16 illustrates the ability of 313EVCAT to reduce the inflammatory cytokine TNF-α.

[0049] Figure 17 shows the expression levels of SOD1, SOD2, and catalase in production cells and their secretory exosomes. Figure 17A shows the expression level of SOD1 in production cells and their exosomes. Figure 17B shows the expression level of SOD2 in production cells and their exosomes. Figure 17C shows the expression level of catalase in production cells and their exosomes.

[0050] Figure 18 shows a comparison of the average loading and enzyme activity of 313EVCAT with exogenously loaded catalase (electroporation and freeze-thaw cycling methods). Figure 18A shows the average catalase loading. Figure 18B shows the catalase activity.

[0051] Figure 19 shows the relative enzyme activities of mitoCAT protein loaded in mitoEVCAT and wtCAT protein loaded in 313EVCAT.

[0052] Figure 20 shows the ability of mitoEVCAT to remove H2O2.

[0053] Figure 21 illustrates the ability of mitoEVCAT to prevent UVB radiation damage.

[0054] Figure 22 shows the relative mean fluorescence intensity of UVB-irradiated cells pretreated with mitoEVCAT, representing the intracellular H2O2 level.

[0055] Figure 23 shows the red / green relative fluorescence ratio of UVB-irradiated cells pretreated with mitoEVCAT, reflecting the health status of mitochondria within the cells.

[0056] Figure 24 shows photographs of rats treated with UV irradiation using 313EVCAT, HA, or RA.

[0057] Figure 25 shows photographs of the anti-wrinkle effects of 313EVCAT, HA, or RA on UV-irradiated rats.

[0058] Figure 26 shows the wrinkle reduction rate of 313EVCAT, HA, or RA in UV-irradiated rats.

[0059] Figure 27 shows the skin thickness of rats treated with 313EVCAT, HA, or RA under UV irradiation.

[0060] Figure 28 shows H&E staining and Masson trichrome staining of UV-irradiated rat skin tissue treated with 313EVCAT, HA, or RA.

[0061] Figure 29 shows the collagen fiber area (%) of UV-irradiated rat skin tissue treated with 313EVCAT, HA, or RA.

[0062] Figure 30 shows the expression of type I collagen mRNA in UV-irradiated rats treated with 313EVCAT, HA, or RA.

[0063] Figure 31 shows the fluorescence results of dihydroethidine (DHE) staining in UV-irradiated rats treated with 313EVCAT, HA, or RA.

[0064] Figure 32 shows the average fluorescence intensity of dihydroethidium (DHE) staining in Figure 31.

[0065] Figure 33 shows the immunofluorescence results of CD86 and CD206 in UV-irradiated rats treated with 313EVCAT, HA, or RA.

[0066] Figure 34 shows the expression of IL-6 mRNA in UV-irradiated rats treated with 313EVCAT, HA, or RA.

[0067] Figure 35 shows the expression of TNF-α mRNA in UV-irradiated rats treated with 313EVCAT, HA, or RA.

[0068] Figure 36 shows the secretion of IL-1β in UV-irradiated rats treated with 313EVCAT, HA, or RA.

[0069] Figure 37 shows the secretion of IL-6 in UV-irradiated rats treated with 313EVCAT, HA, or RA.

[0070] Figure 38 shows the secretion of TNF-α in UV-irradiated rats treated with 313EVCAT, HA, or RA.

[0071] p<0.05, p<0.01, p<0.001, p<0.0001. Detailed Implementation

[0072] The present disclosure will be explained in more detail below. This description is not intended to exhaustively list all different ways in which the invention may be practiced or all features that may be added to the invention. For example, a feature described for one embodiment may be incorporated into other embodiments, while a feature described for a particular embodiment may be deleted from that embodiment. Furthermore, many variations and additions to the various embodiments described herein will be apparent to those skilled in the art based on the content of this disclosure without departing from the invention. Therefore, the following description is intended to illustrate some specific embodiments of the invention, rather than to exhaustively list all permutations, combinations, and variations thereof.

[0073] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains. These may vary for those skilled in the art as to the characteristics and effects desired by this disclosure, and each numerical parameter should be interpreted based on the number of significant figures and conventional rounding methods or as understood by one of skill in the art. Generally, the nomenclature used herein and the experimental procedures in organic chemistry, medicinal chemistry, and biology described herein are well-known and widely adopted in the art. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, preferred materials and methods are described herein. The following terms will be used in describing and claiming protection for this disclosure.

[0074] Unless the context clearly indicates otherwise, the terms “comprising,” “including,” or “containing” as used herein should be understood to imply inclusion of the stated elements, steps, or groups of elements, steps, but not to exclude any other elements, steps, or groups of elements, steps.

[0075] Unless expressly prohibited or otherwise stated in the context, the expression "A and / or B" includes three cases: (1) A, (2) B, and (3) A and B; the expression "A, B and / or C" includes seven cases: (1) A, (2) B, (3) C, (4) A and B, (5) A and C, (6) B and C, and (7) A, B and C. The meanings of similar expressions can be deduced accordingly.

[0076] As used in this article, the term "reactive oxygen species" (ROS) refers to peroxides, oxygen-containing free radicals, and substances that readily form free radicals in organisms and are involved in oxygen metabolism. It plays a crucial role in cell signaling and homeostasis. However, ROS levels can increase dramatically during environmental stress, such as ultraviolet radiation or heat exposure. This can cause severe damage to cell structure, a condition known as oxidative stress. One of the main sources of ROS in vivo is the substrate end of the mitochondrial inner membrane respiratory chain.

[0077] As used in this article, the term "wild type" refers to the most common phenotype observed in wild populations.

[0078] As used herein, the term "fusion protein" refers to a recombinant protein of two or more polypeptides. For example, fusion proteins can be produced by linking a nucleic acid sequence encoding one polypeptide with a nucleic acid sequence encoding another polypeptide or protein domain.

[0079] As used herein, the term "mitochondrial targeting" refers to a substance that can be specifically delivered to mitochondria. This substance is selected from at least one of small molecules, polymers, nucleic acids, peptides, proteins, and enzymes.

[0080] As used herein, the term "vector" is a nucleic acid molecule (preferably self-replicating) that transfers and / or replicates inserted nucleic acid molecules (such as transgenic or exogenous nucleic acids) into a host cell and / or replicates between host cells. It includes plasmids or viral chromosomes, recombinant DNA fragments inserted into their genomes, for the purpose of introducing transgenic recombinant DNA or polypeptides of the disclosed herein into host cells.

[0081] As used herein, the term “extracellular vesicle” (EV) should be understood in the sense known in the art as a vesicle containing a membrane-bound cytoplasmic portion that is released from the cell in the microenvironment, including exosomes, microvesicles, and apoptotic bodies.

[0082] As used in this article, the term "virus-like particle" (VLP) refers to a large particle assembled from one or more viral structural proteins, which does not contain viral nucleic acid, cannot replicate autonomously, and has an overall structure similar to that of a virus particle.

[0083] As used in this article, the term "exosome" refers to a small membrane vesicle (40-160 nm) containing complex RNA, DNA, lipids, metabolites and proteins.

[0084] As used herein, the term "GPI" refers to glycosylphosphatidylinositol, a GPI group added post-translationally to the C-terminus of a peptide. A GPI is a lipid moiety comprising a phosphoethanolamine linker, a glycan core, and a phospholipid tail. In some cases, the GPI group is covalently attached to a peptide as a post-translational modification marker to facilitate lipid raft partitioning, signal transduction, cell communication, or apical membrane targeting.

[0085] As used herein, “acceptable excipients” means compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues, organs, and / or bodily fluids of humans and animals, within the limits of reasonable medical judgment, without causing excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0086] As used in this article, "pharmaceuticalally acceptable excipients" refers to and includes all physiologically compatible solvents, dispersion media, coating materials, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents.

[0087] As used herein, the term “administration” refers to administering the composition to a subject or system to achieve delivery of catalase included in or as part of the composition.

[0088] As used herein, the term "effective dose" is defined as a dose sufficient to achieve or at least partially achieve the desired effect. The effective dose used for this purpose will depend on the severity of the disease being treated and the overall state of the patient's own immune system.

[0089] As used herein, the term "woodchuck hepatitisvirus posttranscriptional regulatory element" (WPRE) is a DNA sequence that, upon transcription, produces a tertiary structure that enhances gene expression. It is commonly used in molecular biology to increase the expression of genes delivered by viral vectors. A WPRE is a triple regulatory element comprising γ, α, and β components.

[0090] As used herein, the term "phenylmethylsulfonyl fluoride" (PMSF) refers to an irreversible, nonspecific inhibitor of serine proteases, papain, and acetylcholinesterase.

[0091] As used in this article, the term "peroxy orange-1" (PO-1) refers to an H2O2-specific fluorescent probe.

[0092] As used herein, the term “wtCAT” refers to wild-type catalase (NCBI accession number: NP_001743.1) having the amino acid sequence shown in SEQ ID NO.1 and the nucleic acid sequence shown in SEQ ID NO.5.

[0093] As used herein, the term “mitoCAT” refers to a modified catalase targeting mitochondria, having the amino acid sequence shown in SEQ ID NO.2 and the nucleic acid sequence shown in SEQ ID NO.6.

[0094] As used in this article, the term "313EVCAT" refers to wtCAT-rich EVs produced by HEK293 cells transfected with the wtCAT sequence, also known as evCAT.

[0095] As used herein, the term "rhCAT" (recombinant human catalase) refers to a commercially available recombinant catalase expressed by Escherichia coli, wherein rhCAT(Abcam) has the amino acid sequence shown in SEQ ID NO.3 and rhCAT(MCE) has the amino acid sequence shown in SEQ ID NO.4.

[0096] As used in this article, the term "mitoEVCAT" refers to mitoCAT-rich EVs produced by HEK293 cells transfected with the mitoCAT sequence.

[0097] As used in this article, the term “mesenchymal stem cell” (MSC) is a type of pluripotent stem cell that possesses all the common characteristics of stem cells, namely, the ability to self-renew and differentiate into multiple lineages.

[0098] As used in this article, the term "MSCEV" refers to an EV derived from MSC.

[0099] In a first aspect, this disclosure provides a catalase or a variant thereof that has at least one of the following biological activities: scavenging reactive oxygen species, alleviating oxidative stress, and / or improving visible signs and / or biomarkers of skin photoaging.

[0100] In some embodiments, the catalase or a variant thereof is a wild-type catalase or a modified catalase, wherein the modified catalase is a wild-type catalase having one or more amino acid mutations, insertions, deletions and / or additions.

[0101] In some embodiments, the catalase or a variant thereof is a wild-type catalase or a modified catalase, wherein the modified catalase is a wild-type catalase modified with chemical groups.

[0102] In some embodiments, the catalase or a variant thereof is a wild-type catalase or a modified catalase, wherein the modified catalase is a fusion protein comprising (a) a wild-type catalase or a variant thereof, and (b) other functional proteins or polypeptides having a specific biological function.

[0103] In some implementations, the functional protein or peptide has tissue-targeting activity.

[0104] In some implementations, the functional protein or peptide has mitochondrial-targeting activity.

[0105] In some implementations, the fusion protein is obtained by deleting the C-terminal amino acid residue "KANL" of catalase and adding an N-terminal mitochondrial targeting sequence.

[0106] In some embodiments, catalase or a variant thereof is a catalase having the amino acid sequence shown in SEQ ID NO: 1, or a catalase having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 1.

[0107] In some embodiments, the catalase or a variant thereof is a mitochondrial-targeted catalase having the amino acid sequence shown in SEQ ID NO: 2, or a mitochondrial-targeted catalase having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 2.

[0108] In a second aspect, this disclosure provides a nucleic acid sequence encoding a catalase or a variant thereof of the first aspect of this application.

[0109] Thirdly, this disclosure provides a vector containing the nucleic acid sequence of the second aspect of this application.

[0110] In a fourth aspect, this disclosure provides a delivery system comprising: (i) a catalase or a variant thereof of the first aspect of this application, and / or (ii) a nucleic acid sequence of the second aspect of this application or a vector of the third aspect of this application, and (iii) a delivery vector, wherein the delivery vector has the function of delivering the catalase or a variant thereof to a specific tissue or cell.

[0111] In some implementations, the delivery vehicle is an extracellular vesicle.

[0112] In some implementations, the delivery carrier is a liposome.

[0113] In some implementations, the delivery vector is a virus-like particle (VLP).

[0114] In some implementations, the delivery vector is a generic extracellular vesicle or a modified extracellular vesicle.

[0115] In some implementations, extracellular vesicles are generic exosomes or modified exosomes.

[0116] In some implementations, extracellular vesicles are harvested from producing cells.

[0117] In some implementations, exosomes are harvested from producing cells, and exosomes can be modified to enhance yield, exposure duration, tissue-specific targeting, or endosome escape.

[0118] In some embodiments, exosomes comprise: (i) peptides and / or proteins containing GPI-anchored signal sequences, (ii) peptide / antibody fragment modifications, and / or (iii) other protein modifications.

[0119] In some embodiments, the delivery system is separated from the conditioned medium by ultracentrifugation or tangential flow filtration (TFF).

[0120] In some embodiments, the particle size and distribution of the delivery system are analyzed by nanoflow cytometry (nFCM) or nanoparticle tracking analyzer (NTA).

[0121] In some embodiments, the producing cells overexpress at least one protein or protein fragment selected from CD46, CD52, CD55, CD58, and CD59. In some embodiments, the producing cells overexpress CD46. In some embodiments, the producing cells overexpress CD59. In some embodiments, the producing cells overexpress CD55.

[0122] In some embodiments, the production cells are non-human mammalian cell lines or human cell lines. In some embodiments, the production cells are HEK 293F cell lines. In some embodiments, the production cells are HEK 293T cell lines. In some embodiments, the production cells are stem cell lines. In some embodiments, the production cells are any combination of HEK 293F cell lines, HEK293T cell lines, and stem cell lines.

[0123] In some implementations, methods for transfecting production cells with expression vector systems include liposome transfection, viral infection, lentiviral transfection, calcium phosphate transfection, microwell transfection, and electroporation.

[0124] In a fifth aspect, this disclosure provides a pharmaceutical composition comprising: (i) a catalase or a variant thereof of the first aspect of this application, or (ii) a nucleic acid sequence of the second aspect of this application or a vector of the third aspect of this application, or (iii) a delivery system of the fourth aspect of this application, and (iv) a pharmaceutically acceptable excipient.

[0125] In some embodiments, the pharmaceutical composition comprises catalase or a variant thereof and a pharmaceutically acceptable excipient.

[0126] In some embodiments, the pharmaceutical composition comprises a nucleic acid sequence or carrier and a pharmaceutically acceptable excipient.

[0127] In some embodiments, the pharmaceutical composition comprises a delivery system and a pharmaceutically acceptable excipient.

[0128] In a sixth aspect, this disclosure provides a cosmetic composition comprising: (i) a catalase or a variant thereof of the first aspect of this application, or (ii) a nucleic acid sequence of the second aspect of this application or a carrier of the third aspect of this application, or (iii) a delivery system of the fourth aspect of this application, and (iv) an acceptable excipient.

[0129] In some embodiments, the cosmetic composition comprises catalase or a variant thereof and an acceptable excipient. In some embodiments, the cosmetic composition comprises a nucleic acid sequence or carrier and an acceptable excipient. In some embodiments, the cosmetic composition comprises a delivery system and an acceptable excipient.

[0130] In a seventh aspect, this disclosure provides a method for manufacturing a delivery system according to the fourth aspect of this disclosure, wherein the delivery system is formed and secreted / released into a culture medium by production cells, and then obtained through separation and purification. The production cells are transfected with the vector of the third aspect of this application, and the delivery system is generated within the cells. In some embodiments, the delivery system is obtained directly from the production cells.

[0131] The manufacturing method of the delivery system disclosed herein is referred to as "AesoBoost technology".

[0132] In some embodiments, the production cells are HEK 293T cells, HEK 293F cells, any HEK 293-derived cells, or any combination thereof. In some embodiments, the production cells are mammalian cells. In some embodiments, the production cells are genetically engineered stable cell lines. In some embodiments, the production cells are stable cell lines obtained through monoclonal selection.

[0133] Eighthly, this disclosure provides a method for manufacturing a pharmaceutical composition of the fifth aspect of this disclosure or a cosmetic composition of the sixth aspect of this disclosure, which uses catalase or a variant thereof of the first aspect of this application, or a nucleic acid sequence of the second aspect of this application, or a carrier of the third aspect of this application, or a delivery system of the fourth aspect of this application.

[0134] Ninthly, this disclosure provides a method of using a pharmaceutical composition of the fifth aspect of this disclosure or a cosmetic composition of the sixth aspect of this disclosure, the method comprising administering to a subject in need an effective amount of catalase or a variant thereof of the first aspect of this application, or a nucleic acid sequence of the second aspect of this application, or a carrier of the third aspect of this application, or a delivery system of the fourth aspect of this application.

[0135] Tenthly, this disclosure provides the use of catalase or a variant thereof of the first aspect of this application, or the nucleic acid sequence of the second aspect of this application, or the vector of the third aspect of this application, or the delivery system of the fourth aspect of this application, in the preparation of pharmaceutical or cosmetic compositions for scavenging reactive oxygen species, alleviating oxidative stress, and / or improving visible signs and / or biomarkers of skin photoaging.

[0136] In some implementations, pharmaceutical or cosmetic compositions are used to protect the structural stability and functionality of mitochondria.

[0137] In some embodiments, the pharmaceutical or cosmetic composition is used to improve photoaged skin conditions. In some embodiments, improving photoaged skin conditions includes promoting skin cell proliferation. In some embodiments, the skin cells are human dermal fibroblasts and / or human keratinocytes. In some embodiments, improving photoaged skin conditions includes reducing ROS levels. In some embodiments, improving photoaged skin conditions includes enhancing collagen regeneration. In some embodiments, improving photoaged skin conditions includes reducing wrinkles. In some embodiments, improving photoaged skin conditions includes improving the inflammatory condition of photoaged skin. In some embodiments, improving the inflammatory condition of photoaged skin includes reducing IL-6 and / or TNF-α.

[0138] In some embodiments, the pharmaceutical or cosmetic composition is used to treat photoaging, natural aging of the skin, melasma, pigmentation, and / or vitiligo.

[0139] The formation of melasma is related to a variety of factors, including genetic predisposition, ultraviolet radiation, endocrine changes, and the side effects of certain medications.

[0140] Skin pigmentation refers to the abnormal accumulation of melanin in the skin, forming dot-like or patchy lesions that differ from the normal skin color. Common causes include post-inflammatory hyperpigmentation, friction melanosis, and diseases such as cirrhosis.

[0141] Vitiligo is a disease caused by the destruction of melanocytes in the skin, leading to a lack of melanin and the formation of localized white patches. Oxidative stress is considered an important factor in the pathogenesis of vitiligo, potentially causing damage and dysfunction of melanocytes.

[0142] In some embodiments, for the treatment method, the delivery system, pharmaceutical composition, or cosmetic composition of this disclosure can be administered topically, subcutaneously, intravenously, intra-arterially, intranasally, or intraocularly.

[0143] In some implementations, for skin-related diseases or conditions, the administration method may be subcutaneous, intradermal, microneedling, or local treatment.

[0144] In some embodiments, the delivery systems, pharmaceutical compositions, or cosmetic compositions of this disclosure can be used alone or in combination with other clinically proven medical aesthetic treatments, including pharmaceuticals, medical devices, and active ingredients. Notable examples include dermal fillers composed of hyaluronic acid, collagen, botulinum toxin, biostimulants (such as poly-L-lactic acid and polycaprolactone), and technologies such as Thermage, Ultherapy, radiofrequency microneedling, and lasers, thereby producing synergistic effects.

[0145] In some embodiments, the delivery systems, pharmaceutical compositions, or cosmetic compositions of this disclosure may help alleviate certain treatment-induced inflammatory symptoms, such as redness and swelling, while enhancing wound healing throughout the treatment process.

[0146] The following examples, whether actual or contemplated, are provided in conjunction with the accompanying drawings to illustrate specific embodiments or features of the invention and are not intended to limit its scope.

[0147] Those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the spirit and scope of this disclosure. For the sake of brevity, descriptions of functions, structures, etc., well-known in the art are omitted in the following description.

[0148] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature or according to product specifications. Unless otherwise stated, the materials, reagents, etc., used in the following examples are commercially available.

[0149] Example 1: Genetically engineered production cells to overexpress catalase 1.1 Establishing a stable production cell line. The production cells were Expi293F cells transfected with a recombinant vector system. TM Cells (Thermo Fisher Scientific, A14527). A vector (Addgene plasmid #17448) was used to construct the recombinant vector system. The recombinant vector system contained a nucleic acid sequence encoding a target protein (PoI), more specifically, a wild-type catalase (wtCAT, Fig. 1A) having the amino acid sequence shown in SEQ ID NO. 1 and the nucleic acid sequence shown in SEQ ID NO. 5, or a mitochondrial-targeted catalase (mitoCAT, Fig. 1B) having the amino acid sequence shown in SEQ ID NO. 2 and the nucleic acid sequence shown in SEQ ID NO. 6. The recombinant vector system was configured from the 5'→3' end with the following: a constitutive cytomegalovirus promoter, the nucleic acid sequence of wtCAT or mitoCAT, and a post-transcriptional regulatory element (WPRE) of marmot hepatitis virus (WHP) for increasing the expression level of the target gene. The recombinant vector system also contained puromycin and penicillin resistance genes for selection purposes. The construction of the recombinant vector was performed by amplifying single fragments using polymerase chain reaction and then combining them seamlessly via enzyme ligation. Exogenous constructs can lead to overexpression of wtCAT or mitoCAT in producing cells and an increase in the concentration of wtCAT or mitoCAT.

[0150] The method of transfecting production cells with expression vector systems is lentiviral transfection via a three-plasmid lentiviral packaging system (Y. Mao, et. al., Lentiviral Vectors Mediate Long-Term and High Efficiency Transgene Expression in HEK 293T cells[J], International Journal of Medical Sciences, 2015, 12(5): 407-415. DOI:10.7150 / ijms.11270). Transfection may result in transient or stable transformation of production cells. Stable cell lines capable of stably expressing EV and wtCAT or mitoCAT were screened and selected from the transfected cell lines. Production cells encapsulated wtCAT or mitoCAT in recombinant EV. Monoclonal screening techniques were applied to stable cell lines to isolate monoclonal cell lines with enhanced stability and increased production of EV and wtCAT or mitoCAT.

[0151] 1.2 Production of extracellular vesicles loaded with wtCAT or mitoCAT 1.2.1 Cell culture Expi 293F TM Cells (Thermo Fisher Scientific, A14527) were cultured in DMEM medium containing 10% fetal bovine serum (Thermo Fisher Scientific) at 37°C under humidified conditions with 5% CO2. Cells were sporulated at 4 × 10⁶ cells / year. 5 Cells were seeded at 120 rpm / min on a shaker and cultured in serum-free medium containing 5% serum. Cells were incubated to achieve the maximum cell density with >85% cell viability. Cells were sequentially divided in the medium as the serum concentration decreased from 5% to 0% of the serum in the medium.

[0152] 1.2.2 Isolation and purification of extracellular vesicles: EVs were isolated from the conditioned medium by tangential flow filtration (TFF). For the TFF (Repligen, KMPi, and KrosFlo KR2i) method, cells containing EVs of this disclosure were filtered through a filter with a suitable mesh or pore size, and the filter molecular weight cutoff (MWCO) was 750 kDa or less / about 750 kDa.

[0153] The separated and concentrated EV solution is purified. Common purification methods for EV include size exclusion chromatography (SEC, Cytiva, AKTA Pure 25 M1).

[0154] Clean the hollow fiber filter (Repligen) with ultrapure water until the pH at the reflux end is neutral. Rinse the filter with PBS, then concentrate the EV solution by percolation, reducing the sample to approximately 1 / 100 to 1 / 25 of its original volume.

[0155] The concentrated solution was subjected to a second TFF to further concentrate it to approximately 1 / 20 to 1 / 5 for SEC purification.

[0156] The column (Cytiva, HiScale 16 / 40) was washed sequentially with water and PBS until conductivity and UV curves stabilized. Samples were loaded into loading cups, the run was started, and centrifuge tubes were placed in the collector to collect purified EVs. The purified samples were filtered, sterilized, and aliquoted. Samples were collected for exosome safety assessment (sterility, mycoplasma, and bacterial endotoxin detection) and characterization.

[0157] Example 2: Characterization and Validation of EVs Loaded with wtCAT or mitoCAT 2.1 Characterization of Extracellular Vesicles The particle size and distribution of EVs were analyzed by nanoflow cytometry (nFCM). The uniformity and size of EVs were measured by transmission electron microscopy (TEM).

[0158] The concentration of purified EV particles, measured by Apogee Micro-GxP nanoflow cytometry, was 7.95 × 10⁻⁶. 11 Particles / mL. After treatment with 0.1% Triton, the particle concentration was 8.63 × 10⁻⁶. 10 Analysis showed that the EV percentage in the sample was 89.15%. The particle size distribution is shown in Figure 2.

[0159] The purified EV sample was further diluted to 0.1 μg / μL. An equal volume of 4% paraformaldehyde was added to the sample, and the mixture was incubated for 2 hours. 3 μL of the mixture was added to a TEM grid, washed with 1× PBS, and fixed with 1% glutaraldehyde. After washing with PBS and ddH2O, the grid was stained on ice. The EVs were examined under a JEOL 1200EX transmission electron microscope at 100 kV. Figure 3 shows that the EV samples exhibited a typical cup-shaped appearance.

[0160] Figure 4 shows the results of Western blotting analysis of purified EV positive markers. ALIX, TSG101, and CD9 are positive markers that should be present in EVs, while CYC1 is a negative marker that should not be present in EVs.

[0161] Therefore, when considering the above results (nFCM, TEM, and WB), it can be seen that the particles in the sample meet the characteristics of extracellular vesicles and exosomes.

[0162] 2.2 Measurement of wtCAT or mitoCAT loading: ELISA and enzyme activity assays were used to assess whether wtCAT or mitoCAT was successfully loaded into EVs, the loading efficiency, and whether the loaded wtCAT or mitoCAT maintained its enzyme activity.

[0163] The particle concentration / mL and particle number / mL of EVs were detected using a human catalase ELISA kit (Abcam, ab277396) and a microplate reader (BioTek). The loading of wtCAT or mitoCAT was 210.7 copies / EV and 274.9 copies / EV, respectively (Table 1).

[0164] Table 1 Loading capacity of wtCAT or mitoCAT in EV

[0165] The enzyme activity of wtCAT encapsulated in EVs was measured using the Amplex Red Catalase Assay Kit (Thermo Fisher Scientific, A22180). 313EVCAT is an EV loaded with wtCAT, also known as evCAT. For a concentration of 1×10⁻⁶... 7 The 313EVCAT particles / mL had an enzyme activity exceeding 800,000 U / mg. The concentration of wtCAT could be calculated based on the average wtCAT loading per EV measured by ELISA. Recombinant catalase (rhCAT, Abcam, ab286037) (2 ng / mL) and recombinant catalase (rhCAT, MCE, HY-P7744) (2 ng / mL) were also measured using the Amplex Red Catalase Assay Kit (Thermo Fisher Scientific, A22180). Compared with recombinant catalase (rhCAT, Abcam, ab286037), the catalase loaded in 313EVCAT showed nearly 7.7 times higher enzyme activity (Figure 5). The enzyme activity of recombinant catalase (rhCAT, MCE, HY-P7744) was 683.6 mU / ng, the enzyme activity of recombinant catalase (rhCAT, Abcam, ab286037) was 106.3 mU / ng, and the enzyme activity of wtCAT loaded in 313EVCAT was 817.6 mU / ng.

[0166] Based on the loading amounts and enzyme activities of 313EVCAT and mitoEVCAT, the specific enzyme activity was calculated. As shown in Figure 19, the specific enzyme activity of mitoCAT protein loaded in mitoEVCAT is approximately twice that of wtCAT protein loaded in 313EVCAT. The modified mitoCAT protein exhibits a significantly stronger specific enzyme activity.

[0167] 2.3 EV Protection from Proteolytic Degradation Due to rapid proteolytic degradation in the extracellular space, catalase has a short half-life. EV can protect PoI from protease attack, enhance its stability, and prolong its effective half-life.

[0168] In this study, rhCAT (Abcam) and 313EVCAT were treated with 5 μL of 50 μg / mL proteinase K (Pro K, a serine protease, Thermo Fisher Scientific) at 50°C for 90 min. After cooling to 4°C, phenylmethanesulfonyl fluoride (PMSF) was added. The membrane containing the 313EVCAT solution was dissolved, centrifuged, and the supernatant was extracted. Enzyme activity was measured (Ample Red Hydrogen Peroxide Assay Kit, Thermo Fisher Scientific, A22180) to assess the ability of EV to protect catalase from protease degradation.

[0169] The results showed that the enzyme activity of rhCAT (Abcam) decreased significantly to 33.03% compared with the untreated control group (PBS). In contrast, 313EVCAT retained 83.05% of its activity, preserving most of the enzyme function (Figure 6).

[0170] Example 3: In vitro evaluation of photoaging and inflammation models 3.1 Cytotoxicity assay To assess the cytotoxicity of EVs, human skin fibroblast HFF-1 cells (ATCC, SCRC-1041) were used. TM ) using 1×10 9 Particle count / mL (measured by nFCM) of naked EVs without target protein, 1×10 9 and 1×10 10 Particle count / mL of purified 313EVCAT and mitoEVCAT were used for treatment, followed by cell viability assay.

[0171] HFF-1 cells were cultured at 37°C in DMEM containing 10% fetal bovine serum under humidified conditions balanced with 5% CO2. After 24 hours, the medium was removed, and the cells were treated with different concentrations of prepared EVs. Cell viability was then assessed after 24 and 48 hours of culture. Cell viability was measured using the CCK-8 reagent (Beyotime, C0039).

[0172] Cells in the control group were cultured in the same medium without EVs. It has been confirmed that the naked EVs, 313EVCAT, and mitoEVCAT of this disclosure did not exhibit cytotoxicity within the concentration range used in the experiments (Figure 7).

[0173] 3.2 Evaluation of the effects of 313EVCAT and mitoEVCAT on improving oxidative stress cell proliferation. Hydrogen peroxide and hydroxyl radicals are the main reactive oxygen species leading to intracellular and extracellular oxidative stress and damage. Therefore, the effects of 313EVCAT and mitoEVCAT on improving hydrogen peroxide-induced oxidative stress were evaluated.

[0174] HFF-1 cells were cultured at 37°C in DMEM containing 10% fetal bovine serum under humidified conditions balanced with 5% CO2. Cells were then cultured with 1×10⁻⁶ cells / mL. 10 Cells were incubated with purified 313EVCAT or mitoEVCAT for 24 hours. After incubation, the EVs were removed, and the cells were treated with 150 μM hydrogen peroxide for 6 hours. When H2O2 treatment was complete, the cells were replaced with EV-free medium and cultured for 24 and 48 hours. Cell viability was measured using the CCK-8 reagent (Beyotime, C0039). The results showed that 1 × 10⁻⁶ EVCAT was effective. 10 Pretreatment with 313EVCAT or mitoEVCAT at particle count / mL for 24 hours effectively protected HFF-1 cells from hydrogen peroxide-induced oxidative damage, highlighting the efficacy of 313EVCAT (Figure 8) or mitoEVCAT (Figure 20) in the oxidative stress model. “NC” refers to the negative control.

[0175] As shown in Figure 8, compared with NC, the cell viability after 24 hours of pretreatment with 313EVCAT was 0.9685, while that of the control group was 0.5678; the cell viability after 48 hours of pretreatment with 313EVCAT was 0.9725, while that of the control group was 0.6536.

[0176] As shown in Figure 20, compared with NC, the cell viability after 24 hours of pretreatment with mitoEVCAT was 0.3614, while that of the control group was 0.0350; the cell viability after 48 hours of pretreatment with mitoEVCAT was 0.4229, while that of the control group was 0.0279.

[0177] Currently, exosomes derived from mesenchymal stem cells (MSCs) have been widely used for research and commercial purposes. MSC-derived exosomes have antioxidant capabilities, including but not limited to superoxide dismutase (SOD) 1-3, glutathione peroxidase (GPX), and catalase. MSC-derived exosomes can alleviate cellular oxidative stress damage (Y. Yan et al., HucMSCexosome-derived GPX1 is required for the recovery of hepatic oxidant injury[J], Mol. Ther., 2017, 25: 465-479) and exert local anti-inflammatory effects (U. Pivorait et al., Exosomes from human dental pulp stem cells suppress carrageenan-induced acute inflammation in mice[J], Inflammation, 2015, 38(5): 1933-1941). MSC-derived exosomes stimulate the proliferation of H9C2 cardiomyocytes, inhibit H2O2-induced apoptosis, and suppress cardiac fibrosis and inflammation (L. Shao et al., MiRNA-sequence indicates that mesenchymal stem cells and exosomes have similar mechanism to enhance cardiac repair [J / OL], Biomed. Res. Int., 2017, 2017: 4150705).

[0178] Cells were treated with different concentrations of hydrogen peroxide for 6 hours. The results showed that 313EVCAT was superior to MSC-derived exosomes (MSCEVs) (Echo Biotech) in improving oxidative stress (Figure 9, Table 2).

[0179] Table 2 Cell viability after pretreatment with 313EVCAT or MSCEV

[0180] 3.3 Evaluation of the effects of UVB irradiation on cell proliferation, ROS clearance, mitochondrial structure maintenance, and collagen production: HFF-1 cells were cultured at 37°C in DMEM containing 10% fetal bovine serum under humidified conditions balanced with 5% CO2. Cells were then cultured with 1×10⁶ cells / mL of UVB irradiated with UVB. 10 The purified 313EVCAT or mitoEVCAT was incubated for 24 hours at a particle count / mL. Then the EVs were removed from the culture medium and treated with 150 mJ / cm² water.2 Cells were irradiated with UVB. Cells were replaced with EV-free medium and cultured for 24 and 48 hours. Cell viability was measured using the CCK-8 reagent (Beyotime, C0039).

[0181] The results showed that 313EVCAT (Figure 10) or mitoEVCAT (Figure 21) could improve cell survival and prevent UVB irradiation damage.

[0182] As shown in Figure 10, compared with NC, the cell viability after 24 hours of pretreatment with 313EVCAT was 0.6121, while that of the control group was 0.5106; the cell viability after 48 hours of pretreatment with 313EVCAT was 0.8938, while that of the control group was 0.6218.

[0183] As shown in Figure 21, compared with NC, the cell viability after 24 hours of pretreatment with mitoEVCAT was 0.6860, while that of the control group was 0.4718; the cell viability after 48 hours of pretreatment with mitoEVCAT was 0.5212, while that of the control group was 0.3359.

[0184] Ultraviolet radiation primarily causes cell damage by generating reactive oxygen species (ROS). Hydrogen peroxide can be transported intracellularly and extracellularly via diffusion. The efficacy of 313EVCAT in scavenging ROS both intracellularly and extracellularly was evaluated.

[0185] Cells with 1×10 10 The purified 313EVCAT particles were incubated for 24 hours. Then, the EVs were removed from the culture medium and treated with 300 mJ / cm² water. 2 Cells were irradiated with UVB. Extracellular hydrogen peroxide levels were measured using the Ample Red Hydrogen Peroxide Detection Kit (Thermo Fisher Scientific, A22188) (Figure 11). Nearly half of the H2O2 was eliminated compared to the control group. Cells were then subjected to 1×10⁶ UVB irradiation. 10 The purified 313EVCAT or mitoEVCAT was incubated for 24 hours at a particle count / mL. Then the EVs were removed from the culture medium and treated with 150 mJ / cm² water. 2 Cells were irradiated with UVB. Intracellular hydrogen peroxide levels were measured 4 h or 24 h post-irradiation using Peroxy Orange-1 (PO-1) reagent (Macklin Biochemical, P991796). The results showed that 313EVCAT (Figure 12) or mitoEVCAT (Figure 22) could consistently and stably reduce UVB-induced extracellular and intracellular excess hydrogen peroxide.

[0186] As shown in Figure 12, compared with NC, 4 hours after irradiation, the relative average fluorescence intensity of the pretreated group with 313EVCAT was 0.9985, while that of the control group was 1.2281; 24 hours after irradiation, the relative average fluorescence intensity of the pretreated group with 313EVTAT was 1.0121, while that of the control group was 1.2376, which means that the control group contained more hydrogen peroxide.

[0187] As shown in Figure 22, compared with NC, the relative average fluorescence intensity of the mitoEVCAT pretreated group was 1.0610 24 hours after irradiation, while that of the control group was 1.2357, which means that the control group contained more hydrogen peroxide.

[0188] Under ultraviolet radiation, the high-intensity energy often acts directly on mitochondria, damaging their structure and thus accelerating human aging. One hallmark of mitochondrial damage is the collapse of the mitochondrial membrane potential. Healthy mitochondria typically exhibit a high membrane potential, while damaged mitochondria typically show a low membrane potential. When detected using the JC-1 reagent, a high membrane potential usually appears as red fluorescence, while a low membrane potential appears as green fluorescence. The ratio of red to green fluorescence accurately reflects the health status of mitochondria within the cell.

[0189] Cells with 1×10 10 The purified mitoEVCAT was incubated for 24 hours at a particle count / mL. Then, the EVs were removed from the culture medium and treated with 300 mJ / cm² water. 2 Cells were irradiated with UVB. Mitochondrial membrane potential was measured using JC-1 reagent (Beyotime, C2003S). The results showed that mitoEVCAT (Figure 23) can effectively resist damage to mitochondria caused by high-intensity UVB, protecting mitochondrial structural stability and function.

[0190] As shown in Figure 23, compared with NC, the relative fluorescence (red / green ratio) of the pretreated group with mitoEVCAT was 5.5312, while that of the control group was 3.4748, which means that the mitochondrial structure of the pretreated group was more intact.

[0191] Cells with 1×10 10 The purified 313EVCAT particles were incubated for 24 hours. Then, the EVs were removed from the culture medium and treated with 300 mJ / cm² water. 2 Cells were irradiated with UVB. After culturing for 24 or 48 hours, the supernatant was collected for ELISA detection, and the cells were harvested for qPCR analysis. ELISA (MultiSciences, EK1C01) (Figure 13) and qPCR (Roche, LC480) (Figure 14) analyses showed that 313EVCAT can enhance cell regeneration capacity and regenerate collagen.

[0192] As shown in Figure 13, compared with NC, the concentration of pro-col 1a1 pretreated with 313EVCAT for 24 hours was 0.4129, while that in the control group was 0.2563; the concentration of pro-col 1a1 pretreated with 313EVCAT for 48 hours was 0.5099, while that in the control group was 0.2833.

[0193] As shown in Figure 14, compared with the control group, the relative expression of Col 1a1 mRNA was 0.7716 after 24 hours of pretreatment with 313EVCAT, while it was 0.5799 in the control group; after 48 hours of pretreatment with 313EVCAT, the relative expression of Col 1a1 mRNA was 0.6833, while it was 0.4673 in the control group.

[0194] 3.4 Evaluation of the effect of reducing inflammatory cytokines: Mouse macrophage Raw264.7 cells (Procell, CL-0190) were cultured in DMEM containing 10% fetal bovine serum. Cells were cultured at a rate of 6 × 10⁶ cells / year. 4 Cells were seeded into 12-well plates at a density of 1 × 10⁶ cells / well. 9 Particle count / mL (measured by nFCM) and 1×10 10 The purified 313EVCAT particles were incubated at 37°C under humidified conditions of 5% CO2 for 24 hours. EVs were then removed by washing with PBS.

[0195] Raw264.7 cells were treated with a combination of 100 ng / mL lipopolysaccharide (LPS) and 20 ng / mL interferon-γ (IFN-γ) for 24 hours. The culture medium was then collected, and the inflammatory cytokine IL-6 was measured using an ELISA kit (MultiSciences, EK206), and the inflammatory cytokine TNF-α was measured using an enzyme-linked immunosorbent assay kit (MultiSciences, EK282).

[0196] The results showed that the secretion of cytokines IL-6 (Figure 15) and TNF-α (Figure 16) was significantly increased in the control group, with IL-6 and TNF-α levels being 6.7-fold and 13.7-fold higher, respectively, than in the negative control group. However, incubation with 313EVCAT effectively reduced the secretion of inflammatory cytokines. Compared with the control group, the secretion of IL-6 and TNF-α in the M1 313EVCAT group was reduced by approximately 16% and 13%, respectively.

[0197] Comparative Example 1: EV Loading with SOD1 or SOD2 Besides catalase, other important enzymes in the reactive oxygen species scavenging pathway include SOD1 and SOD2. Recombinant vectors were constructed with SOD1 (amino acid sequence as shown in SEQ ID NO. 7, nucleic acid sequence as shown in SEQ ID NO. 9) or SOD2 (amino acid sequence as shown in SEQ ID NO. 8, nucleic acid sequence as shown in SEQ ID NO. 10) as the target proteins. Production cells containing the recombinant vectors were obtained using lentiviral transfection. Production cells were cultured, and then EVs were isolated and purified (see Example 1). The expression of SOD1 or SOD2 was tested, and the effect of loading SOD1 or SOD into EVs was also tested. The expression of catalase in Example 1 and the effect of loading catalase into EVs were also tested. Naked cells refer to cells without the recombinant vector (Expi293F). TM (Cells), naked EVs are exosomes secreted by naked cells.

[0198] Western blotting was used to detect target proteins in cells and EVs. After lysing cells and EV membranes using a Triton-100, total protein was collected and detected using the BCA method. Each sample was electrophoresed with the same protein content and detected with the antibody corresponding to the target protein; analysis was then performed using grayscale values.

[0199] The results, as shown in Table 3, Figures 17A and 17B, indicate that despite a significant increase in the expression levels of SOD1 or SOD2 in cells, these two enzymes were difficult to package into exosomes. In Figure 17C, the expression levels of catalase were significantly increased in both cells and exosomes. Therefore, this demonstrates that loading catalase using the delivery system of this disclosure is efficient and advantageous, and that loading different proteins, such as SOD1 and SOD2, using the same method may lead to significantly different results due to differences in intracellular expression levels, modifications, and localization.

[0200] Table 3. Expression of SOD1, SOD2 and CAT proteins

[0201] Comparative Example 2: Exogenous Loading of Catalase Exogenous loading typically requires the use of physical, chemical, or biological methods to incorporate the target protein into naked extracellular vesicles (naked EVs). In this application, two methods were used: electroporation and freeze-thaw cycling (see US20020409535A1) to test the effectiveness of exogenously incorporating catalases (rhCAT, MCE, HY-P7744) into naked EVs.

[0202] Referring to Example 2.2, the average catalase loading and catalase activity of 313EVCAT (generated by the delivery system of this disclosure) and exogenously incorporated catalase (electroporation and freeze-thaw cycles) were measured.

[0203] As shown in Figures 18A and 18B, the results indicate that the 313EVCAT produced by the delivery system of this disclosure is far superior to electroporation and freeze-thaw in terms of catalase loading capacity and enzyme activity.

[0204] The average catalase loading of 313EVCAT was 171.0723 / particle, 1.3788 / particle by electroporation, and 1.6048 / particle by freeze-thaw cycling. The enzyme activity of 313EVCAT was 430.7895 mU / 1E7 particles, 89.7872 mU / 1E7 particles by electroporation, and 121.3596 mU / 1E7 particles by freeze-thaw cycling.

[0205] This indicates that the endogenous incorporation of catalase has significant advantages, and the manufacturing method of the delivery system, named "AesoBoost technology" in this disclosure, is suitable for large-scale and efficient production of highly active extracellular vesicles loaded with catalase.

[0206] Example 4: In vivo evaluation of a photoaging model 4.1 Establishment of an animal model of photoaging Female SD rats (6 weeks old, average weight approximately 150 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. The rats were acclimatized for one week in the SPF-grade animal facility of the College of Life Sciences, Jilin University, with the ambient temperature controlled at 25°C and free access to food and water. The fur on the backs of the SD rats was removed. Except for the control group (n=4), the remaining rats were exposed to UV irradiation to simulate photoaged skin. The total dose of UVA and UVB was set at 14.07 J / cm². 2 and 11.66 J / cm 2 The UV lamp was positioned 30 cm away from the irradiated area. Rats were irradiated once daily for 7 consecutive weeks. EV treatment groups (313EVCAT and naked EV) and the hyaluronic acid (HA) treatment group received intradermal injections, while 0.5% retinoic acid (RA) was applied topically to the back skin (RA treatment group). The condition of the rats' back skin was photographed and recorded regularly throughout the experiment. At the end of the experiment, rats were euthanized by cervical dislocation; blood was collected via the retroorbital venous plexus, and back skin tissue was harvested for subsequent analysis.

[0207] 4.2 Evaluation of the effect of reducing UV-induced dermal wrinkles: Rat back skin photographs were imported into image analysis software (Image Pro Plus), and wrinkles were identified using the software. Statistical analysis was performed on the obtained wrinkle data to evaluate the construction of the photoaging model and the effect of different treatments on the number of wrinkles.

[0208] After successful model establishment, drug treatment was administered on days 1, 5, 8, 15, 22, and 28. Rats were photographed and weighed at each time point (Figure 24). At the end of the study, skin samples were collected from the back for histological analysis and skin replication. From day 22 onwards, the high-dose 313EVCAT group (1×10⁻⁶) received treatment. 10 The number of particles per mL (particles / mL) showed a more significant reduction in wrinkles compared to the naked EV group and the hyaluronic acid (HA) group, with further improvement by day 28. These results indicate that intradermal administration of 313EVCAT can effectively improve skin condition and reduce photoaging-induced wrinkles (Figure 26, Table 4). Skin replica results obtained on day 28 further confirmed that 313EVCAT had a superior anti-wrinkle effect compared to the UV control group, naked EV group, HA group, and retinoic acid (RA) control group (Figure 25). Notably, high-dose 313EVCAT (1×10⁻⁶) significantly reduced wrinkles compared to the naked EV group and the hyaluronic acid (HA) group. 10 Treatment with 313EVCATs for 28 consecutive days (particle count / mL) significantly reduced dermal thickening, and its therapeutic effect was superior to that of the RA group (Figure 27, Table 5), suggesting that 313EVCATs have a strong anti-photoaging effect.

[0209] Table 4. Wrinkle reduction rate (% on day 0)

[0210] Table 5 Skin thickness (mm)

[0211] 4.3 Evaluation of the Effects of 313EVCAT on Pathological Features and Collagen Content: To evaluate the histopathological effects of 313EVCAT treatment on photoaged skin, hematoxylin-eosin (H&E) staining was performed on dorsal skin samples from SD rats. After sacrificing the rats, dorsal skin tissue was harvested using surgical scissors, excess adipose tissue was removed, and the skin samples were fixed in centrifuge tubes containing 4% paraformaldehyde solution for 24 h. The fixed skin tissue was then embedded and paraffin sections were prepared. H&E staining was performed on the sections, and the structure of each skin layer, as well as the infiltration of collagen and inflammatory cells, were observed under an optical microscope. Paraffin sections were also prepared and Masson's trichrome staining was performed. The morphology and distribution of collagen fibers were observed under an optical microscope, and the collagen content was assessed. ImageJ software was used for quantitative analysis of collagen content in the tissues.

[0212] In the UV irradiation control group, obvious epidermal erosion, necrosis, and extensive inflammatory cell infiltration were observed, consistent with typical characteristics of photoaging and photodamage. In contrast, 313EVCAT (1×10⁻⁶) showed significantly better results. 10Treatment with 313EVCAT (particle count / mL) significantly alleviated the aforementioned pathological changes, reduced epidermal damage and inflammation, and restored dermal collagen fiber structure and density (Figure 28). To assess collagen deposition, skin samples collected on day 28 post-treatment were stained with Masson's trichrome and collagen content was quantitatively analyzed. Compared with HA and RA treatments, 313EVCAT administration significantly promoted collagen synthesis and remodeling, demonstrating stronger tissue repair and matrix regeneration capabilities (Figure 28). Immunohistochemical analysis was further used to assess collagen content in skin sections. Compared with the untreated group, the 313EVCAT-treated group showed significantly improved Col 1a1 regeneration, and its therapeutic effect on skin and collagen structure was superior to that of the HA and RA-treated groups (Figure 29, Table 6). In addition, RT-qPCR results showed that the Col 1a1 mRNA expression level in the high-dose 313EVCAT-treated group was 4.38 times higher than that in the control group, further confirming enhanced collagen synthesis (Figure 30, Table 6).

[0213] Table 6. Collagen fiber area (%) and expression of type I collagen mRNA

[0214] Tissue sections were immersed in dihydroethidium (DHE) working solution, ensuring the dye evenly covered the sections; then incubated in the dark for a certain period of time to allow DHE to enter the cells and be oxidized by ROS. After incubation, the sections were washed with PBS or other appropriate buffers to remove unbound DHE dye.

[0215] In ROS detection, appropriate excitation and emission wavelengths were set under a fluorescence microscope to detect the fluorescence signal generated by oxidized DHE. The intensity and distribution of intracellular fluorescence signals were observed and recorded, and the fluorescence intensity was proportional to the ROS level. ImageJ software was used to quantitatively analyze the fluorescence signals to assess the intracellular ROS level. The ROS level in skin tissue was detected by dihydroethidium (DHE) staining to evaluate the antioxidant properties of 313EVCAT (Figure 31). The results of fluorescence imaging and quantitative analysis showed that ROS accumulation was significantly reduced in the 313EVCAT treatment group, especially in the high-dose group (313EVCAT (1×10⁻⁶)). 10 Particle count / mL) Lower dose group (313EVCAT (1×10) 9 The particle count ( / mL) showed stronger ROS scavenging ability and exhibited a dose-dependent antioxidant effect (Figure 32, Table 7).

[0216] Table 7 Quantitative Analysis of Fluorescence Imaging

[0217] 4.4 Evaluation of the Anti-inflammatory Effect of 313EVCAT To investigate the anti-inflammatory effect of 313EVCAT, immunofluorescence was used to detect the expression levels of CD206 (Anti-CD206 purchased from PROTEINTECH, 18704-1-AP) and CD86 (Anti-CD86 purchased from Thermo Fisher Scientific, RAB02398) in skin sections. Compared with the untreated group, the green fluorescence signal of CD206 in the 313EVCAT-treated group was significantly enhanced, while the expression of CD86 was significantly reduced. Compared with other treatment groups, the green fluorescence of CD206 in the 313EVCAT-treated group was stronger, while that of CD86 showed the opposite trend, suggesting that 313EVCAT treatment can promote the transformation of pro-inflammatory M1 macrophages into anti-inflammatory M2 macrophages. The results indicate that low-dose 313EVCAT (1×10⁻⁶) is effective in promoting the transformation of pro-inflammatory M1 macrophages into anti-inflammatory M2 macrophages. 9 The treatment group (particle count / mL) also showed significant efficacy. Compared with commonly used drugs HA and RA, the 313EVCAT treatment group showed superior therapeutic effects in reversing macrophage phenotype (Figure 33).

[0218] Furthermore, to evaluate the therapeutic effect of 313EVCAT in vivo, its effect on anti-inflammatory response was systematically analyzed in a UV-induced photoaging rat model. RT-qPCR (Bio-Rad T100) results showed that, compared with the control group, 313EVCAT treatment significantly downregulated the mRNA expression of pro-inflammatory cytokines IL-6 (Figure 34) and TNF-α (Figure 35). Notably, low-dose 313EVCAT (1×10⁻⁶) significantly reduced the mRNA expression of these pro-inflammatory cytokines. 9 Treatment with (particle count / mL) also significantly reduced the expression of inflammation-related genes; in the high-dose group (1×10⁻⁶), the expression of inflammation-related genes was significantly reduced. 10 In terms of particle count / mL, the IL-6 mRNA level was similar to that in the NC group, suggesting effective inhibition of UV-induced inflammation. Consistent with the mRNA results, ELISA results showed that 313EVCAT treatment dose-dependently reduced the protein levels of IL-1β (Figure 36), IL-6 (Figure 37), and TNF-α (Figure 38) in serum. IL-1β was detected using ELISA kit (Elabscience, E-EL-R0012), IL-6 using ELISA kit (Elabsscience, E-EL-R0015), and TNF-α using ELISA kit (ElabsScience, E-EL-R2856).

[0219] Table 8 Evaluation of the anti-inflammatory effect of 313EVCAT

[0220] The results above and Table 8 indicate that 313EVCAT has a robust anti-inflammatory effect and can alleviate the increase of cytokines associated with UV-induced photoaging.

[0221] Those skilled in the art will readily recognize that other suitable modifications and alterations to the present disclosure described herein are obvious and can be made using suitable equivalents without departing from the scope of the disclosure or the embodiments disclosed herein.

[0222] According to this disclosure, all articles and methods disclosed and claimed herein can be prepared and performed without excessive experimentation, guided by the content of this disclosure. While the articles and methods of this disclosure have been described with reference to preferred embodiments, those skilled in the art will understand that variations can be made to the articles and methods without departing from the spirit and scope of this disclosure. All such variations and equivalents, whether present or developed hereafter, are considered to be within the spirit and scope of the disclosure as defined in the appended claims. All patents, patent applications, and publications mentioned in the specification demonstrate the level of skill of one ordinary person in the art to which this disclosure pertains. All patents, patent applications, and publications are incorporated herein by reference in their entirety as if each publication were expressly and individually indicated as being incorporated herein by reference in its entirety for any and all purposes. The exemplary descriptions herein may be appropriately implemented in the absence of any elements not specifically disclosed herein. Therefore, it should be understood that although this disclosure has been specifically disclosed through preferred embodiments and optional features, modifications and variations of the concepts disclosed herein can be made by those skilled in the art, and such modifications and variations are considered to be within the scope of this disclosure as defined in the appended claims.

Claims

1. Use of catalase or a variant thereof, or a nucleic acid sequence, or a vector or delivery system, in the preparation of a pharmaceutical composition or cosmetic composition for improving visible signs and / or biomarkers of photoaging of the skin.

2. The use according to claim 1, characterized in that, The catalase or its variants thereon have at least biological activity that improves visible signs and / or biomarkers of skin photoaging.

3. The use according to claim 1 or 2, characterized in that, The catalase or its variants are wild-type catalases or modified catalases, wherein the modified catalase is selected from at least one group of the following: (i) wild-type catalases having one or more amino acid mutations, insertions, deletions and / or additions; or (ii) wild-type catalases modified with chemical groups.

4. The use according to any one of claims 1-3, characterized in that, The catalase or a variant thereof is selected from at least one group of the following: (i) a catalase having the amino acid sequence shown in SEQ ID NO: 1, or a catalase having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity with SEQ ID NO:

1.

5. The use according to any one of claims 1-4, characterized in that, The nucleic acid sequence encodes catalase or a variant thereof as defined in any one of claims 2-4.

6. The use according to any one of claims 1-5, characterized in that, The vector contains the nucleic acid sequence as defined in claim 5.

7. The use according to any one of claims 1-6, characterized in that, The delivery system comprises: (i) a catalase or a variant thereof as defined in any one of claims 2-4; and / or, (ii) a nucleic acid sequence as defined in claim 5 or a vector as defined in claim 6; and (iii) a delivery vector, wherein the delivery vector has the function of delivering catalase or a variant thereof to a specific tissue or cell.

8. The use according to claim 7, characterized in that, The delivery vector is selected from at least one of liposomes, extracellular vesicles (EVs), and virus-like particles (VLPs).

9. The use according to claim 7 or 8, characterized in that, The delivery vector is selected from at least one of general extracellular vesicles or modified extracellular vesicles. Optionally, the extracellular vesicle is a general exosome or a modified exosome.

10. The use according to claim 9, characterized in that, The extracellular vesicles are harvested from producing cells; optionally, the extracellular vesicles are exosomes, and the exosomes may be modified to enhance production, exposure duration, tissue-specific targeting, or endosome escape; more preferably, the exosomes comprise: (i) peptides and / or proteins containing GPI-anchored signal sequences, (ii) peptide / antibody fragment modifications, and / or (iii) other protein modifications.

11. The use according to claim 10, characterized in that, The production cells thereon overexpress at least one protein or protein fragment selected from CD46, CD52, CD55, CD58 and CD59.

12. The use according to claim 10 or 11, characterized in that, The production cells mentioned therein are non-human mammalian cell lines or human cell lines.

13. The use according to any one of claims 10-12, characterized in that, The production cells are selected from HEK293F cell line, HEK 293T cell line, stem cell line or any combination thereof.

14. The use according to any one of claims 1-13, characterized in that, The delivery system is formed by production cells and secreted / released into a culture medium, and then separated and purified. The production cells are transfected with the vector as defined in claim 6, and the delivery system is generated within the cells.

15. The use according to claim 14, characterized in that, The production cells are selected from HEK 293F cell line, HEK 293T cell line, stem cell line or any combination thereof.

16. The use according to any one of claims 1-15, characterized in that, The pharmaceutical composition comprises: (i) a catalase or a variant thereof as defined in any one of claims 2-4; or, (ii) a nucleic acid sequence as defined in claim 5 or a vector as defined in claim 6; or, (iii) a delivery system as defined in any one of claims 7-15; and, (iv) a pharmaceutically acceptable excipient.

17. The use according to any one of claims 1-15, characterized in that, The cosmetic composition comprises: (i) a catalase or a variant thereof as defined in any one of claims 2-4; or, (ii) a nucleic acid sequence as defined in claim 5 or a vector as defined in claim 6; or, (iii) a delivery system as defined in any one of claims 7-15; and, (iv) an acceptable excipient.

18. The use according to any one of claims 1-17, characterized in that, The pharmaceutical or cosmetic composition is used to improve photo-aged skin conditions.

19. The use according to claim 18, characterized in that, The improvement of photoaged skin conditions mentioned herein includes: (i) promoting skin cell proliferation, optionally, the skin cells being human dermal fibroblasts and / or human keratinocytes; (ii) enhancing collagen regeneration; and (iii) reducing wrinkles.

20. The use according to any one of claims 1-19, characterized in that, The pharmaceutical or cosmetic composition described herein is used to improve and / or treat photoaging, natural aging of the skin, and / or melasma.

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