Engineered exosomes, compositions, nucleic acid constructs and applications for skin rejuvenation

Abstract

This invention discloses an engineered exosome comprising (a) a KGF peptide fused to a first anchoring peptide, (b) an EGF peptide fused to a second anchoring peptide, and (c) an FGF-2 peptide fused to a third anchoring peptide, wherein (a), (b), and (c) are anchored to the membrane of the exosome via the first, second, and third anchoring peptides, respectively, and wherein the KGF peptide, the EGF peptide, and the FGF-2 peptide are exposed on the outer surface of the exosome membrane. The composition, nucleic acid construct, and use of the exosome for skin rejuvenation are also disclosed.

Description

Engineered exosomes for skin rejuvenation The present disclosure relates to engineered exosomes, and more particularly to exosomes carrying different fusion proteins anchored to the exterior of the exosome membrane. The present disclosure also relates to compositions comprising the engineered exosomes, nucleic acid constructs comprising polynucleotides encoding the fusion proteins, and uses of the engineered exosomes for skin rejuvenation. Sun exposure, skin conditions, aging, and even genetics can cause abnormalities in the skin on your face and other parts of your body. These abnormalities include texture changes like wrinkles and acne scars, pigmentation changes like freckles and sun spots, or visible blood vessels. Additionally, your skin may lose its radiance, feel less firm, and lose the healthy glow of youthful skin. Skin rejuvenation refers to any procedure designed to improve the appearance of the skin. Different treatment modalities can be used to treat different aspects of skin damage. Laser resurfacing, mechanical peels, chemical peels, and injectable products can improve the appearance of fine lines and wrinkles across the entire face or in specific areas of the face, such as the upper lip and around the eyes. These treatments can also be used to address pigmentation disorders, such as sun spots and age spots, and they can be used to improve the appearance of acne scars or other skin conditions. Products based on regenerative medicine can be divided into the following two categories: cell-based therapies and cell-free therapies. These two types can be autologous or allogeneic. The most common products based on regenerative medicine include cell-based methods using autologous adult stem cells (such as mesenchymal stem cells (MSC), fibroblasts), emerging blood-derived cell therapies (such as platelet-rich plasma (PRP) products), and the clinical application of induced pluripotent stem cells (iPSC) is constantly developing, among which cell therapies for a variety of diseases are in the clinical research stage, especially those cell therapies that meet the lower risk of use of iPSC-derived products (such as iPSC-derived MSC (iPSC-MSC)). Cell-free products are mainly based on secretory components of MSC, such as MSC-derived exosomes (MSC-exo), MSC-derived conditioned medium (MSC-CM) and MSC-derived extracellular vesicles (MSC-EV). Autologous transplantation is performed by isolating and culturing MSCs from the patient's own tissue sources, such as adipose-derived MSCs (AD-MSCs), bone marrow-derived MSCs (BM-MSCs), umbilical cord-derived MSCs (UC-MSCs), fetal dermal MSCs (FD-MSCs), endometrium-derived MSCs, and menstrual blood (eMSCs / MenSCs). Autologous PRP is obtained by collecting and centrifuging the patient's own blood. The molecular mechanisms behind the effects of regenerative medicine-based therapies are still under investigation. However, the unique properties of MSCs, such as self-renewal, multidirectional differentiation, inflammation regulation, immunomodulation, angiogenesis, and the hematopoietic capacity of PRP, manifested by the release of cytokines and various growth factors, are believed to play an important role in skin regeneration. Therefore, there is a need for additional and improved methods and compositions for skin rejuvenation. In one aspect of the present invention, an engineered exosome is provided, comprising (a) a KGF polypeptide fused to a first anchor polypeptide, (b) an EGF polypeptide fused to a second anchor polypeptide, and (c) an FGF-2 polypeptide fused to a third anchor polypeptide, wherein (a), (b) and (c) are anchored to the membrane of the exosome through the first, second and third anchor polypeptides, respectively, and wherein the KGF polypeptide, the EGF polypeptide and the FGF-2 polypeptide are exposed on the outer surface of the membrane of the exosome. In some embodiments, the engineered exosome further comprises (d) a PDGF-BB polypeptide, the PDGF-BB polypeptide is fused to a fourth anchor polypeptide, and the PDGF-BB polypeptide is exposed on the outer surface of the membrane of the exosome. In some embodiments, the KGF polypeptide is a KGF-1 polypeptide. Preferably, the KGF-1 polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the EGF polypeptide is a human EGF polypeptide or an ortholog or paralog thereof. Preferably, the EGF polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the FGF-2 polypeptide is human FGF-2 or an ortholog or paralog thereof; more preferably, the FGF-2 polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:3. In some embodiments, the PDGF-BB polypeptide is a human PDGF-BB dimer; more preferably, the PDGF-BB dimer comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:4. In some embodiments, these anchoring polypeptides are exosomal membrane proteins, membrane targeting sequences, or anchoring functional fragments thereof. Exemplary exosomal membrane proteins include, but are not limited to, lamp2b, tetraspanins such as CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactadherin (C1C2 domain), vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative regulator (PTGFRN), and any combination thereof. Exemplary membrane targeting sequences include, but are not limited to, glycosylphosphatidylinositol (GPI) anchors and lipid-anchored proteins. In preferred embodiments, the anchor polypeptides comprise full-length CD63 or truncated CD63 that retains the TM3 domain. In preferred embodiments, each of the first, second, third, and fourth anchor polypeptides (when present) comprises the TM3 domain of CD63. In preferred embodiments, each of the first, second, third, and fourth anchor polypeptides (when present) is the TM3 domain of CD63. In some embodiments, each of the anchor polypeptides is the TM3 domain of CD63; preferably, the TM3 domain of CD63 comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the KGF polypeptide, the EGF polypeptide, the FGF-2 polypeptide, and the PDGF-BB polypeptide (when present) are optionally fused to the N-terminus or C-terminus of the corresponding anchor polypeptide via a peptide linker. Preferably, the peptide linker consists of glycine and serine, such as (G4S)n, where n is an integer from 1 to 3. In some embodiments, the exosomes are not derived from mesenchymal stem cells; preferably, the exosomes are not derived from stem cells. In some embodiments, the exosomes (i) enhance fibroblast proliferation, (ii) upregulate COL2A1, COL3A1, fibronectin, TIMP1, and / or COL1A1B genes, (iii) downregulate MMP7, MMP9, and / or MMP16B genes, and / or (iv) downregulate melanin synthesis. Another aspect of the present disclosure relates to a composition comprising the exosomes as described herein, and a carrier; preferably, the composition is a liquid preparation; more preferably, the composition is formulated for topical or subcutaneous administration. In some embodiments, the composition does not contain KGF polypeptide (e.g., KGF-1 polypeptide), EGF polypeptide, FGF-2 polypeptide (e.g., human FGF-2 polypeptide), or PDGF-BB polypeptide (e.g., human PDGF-BB dimer polypeptide) that is not attached to the membrane of the exosome. Another aspect of the present disclosure relates to a nucleic acid construct comprising a polynucleotide encoding: (a) a KGF polypeptide fused to a first anchor polypeptide, (b) an EGF polypeptide fused to a second anchor polypeptide, and (c) an FGF-2 polypeptide fused to a third anchor polypeptide. In some embodiments, the nucleic acid construct further comprises a polynucleotide encoding (d) a PDGF-BB polypeptide fused to a fourth anchor polypeptide. In some embodiments, the KGF polypeptide is a KGF-1 polypeptide. Preferably, the KGF-1 polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the EGF polypeptide is a human EGF polypeptide or an ortholog or paralog thereof. Preferably, the EGF polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the FGF-2 polypeptide is human FGF-2 or an ortholog or paralog thereof; more preferably, the FGF-2 polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:3. In some embodiments, the PDGF-BB polypeptide is a human PDGF-BB dimer; more preferably, the PDGF-BB dimer comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:4. In some embodiments, the polynucleotide encoding the KGF polypeptide has the nucleotide sequence shown in SEQ ID NO: 5 or a degenerate sequence thereof. In some embodiments, the polynucleotide encoding the EGF polypeptide has the nucleotide sequence shown in SEQ ID NO: 6 or a degenerate sequence thereof. In some embodiments, the polynucleotide encoding the FGF-2 polypeptide has the nucleotide sequence shown in SEQ ID NO: 7 or a degenerate sequence thereof. In some embodiments, the polynucleotide encoding the PDGF-BB polypeptide has the nucleotide sequence shown in SEQ ID NO: 8 or a degenerate sequence thereof. In some embodiments, these anchoring polypeptides are exosomal membrane proteins, membrane targeting sequences, or anchoring functional fragments thereof. Exemplary exosomal membrane proteins include, but are not limited to, lamp2b, tetraspanins such as CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactadherin (C1C2 domain), vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative regulator (PTGFRN), and any combination thereof. Exemplary membrane targeting sequences include, but are not limited to, glycosylphosphatidylinositol (GPI) anchors and lipid-anchored proteins. In preferred embodiments, the anchor polypeptides comprise full-length CD63 or truncated CD63 that retains the TM3 domain. In preferred embodiments, each of the first, second, third, and fourth anchor polypeptides (when present) comprises the TM3 domain of CD63. In preferred embodiments, each of the first, second, third, and fourth anchor polypeptides (when present) is the TM3 domain of CD63. In some embodiments, the polynucleotide encoding the TM3 domain of CD63 has a nucleotide sequence as set forth in SEQ ID NO: 10 or a degenerate sequence thereof. In some embodiments, the polynucleotide is a single polynucleotide comprising nucleotide segments encoding polypeptides (a), (b), (c), and optionally (d), wherein each of polypeptides (a), (b), (c), and optionally (d) is linked to an anchor polypeptide; preferably, polypeptides (a), (b), (c), and optionally (d) are separated by self-cleaving peptides; optionally, these self-cleaving peptides are 2A peptides, such as T2A, E2A, P2A, or any combination thereof. Another aspect of the present disclosure relates to a vector comprising a nucleic acid construct as described in any one of the nucleic acid constructs described herein. Another aspect of the present disclosure relates to a cell transduced with a vector described herein, wherein the polynucleotide is integrated into the genome of the cell. In some embodiments, the cell is not a mesenchymal stem cell; preferably, the cell is not a stem cell; preferably, the cell is a mammalian cell; or more preferably, the cell is a HEK293 or CHO cell. Another aspect of the present disclosure relates to a method of producing engineered exosomes as disclosed herein, comprising (a) transducing a cell as described above with a vector as described above; (b) culturing the cell under conditions that allow exosomes to be secreted from the cell; and (c) collecting and purifying the exosomes. In some embodiments, the method further comprises adapting the cells to serum-free conditions during step (b). Yet another aspect of the present disclosure relates to the use of the engineered exosomes or compositions as disclosed herein in the manufacture of a medicament for skin rejuvenation; preferably, the skin rejuvenation comprises treating any one of the following: sun damage; sensitive skin; scars such as acne or stretch marks such as stretch marks or puberty marks, chicken pox or wounds; wrinkles such as static or dynamic wrinkles; dark circles; hyperpigmentation such as skin darkening or freckles, skin sagging, uneven or dull skin tone, or any combination thereof. Yet another aspect of the present disclosure relates to a method for skin rejuvenation; preferably, the skin rejuvenation comprises treating any of the following: sun damage; sensitive skin; scars such as acne or stretch marks such as stretch marks or puberty marks, chicken pox or wounds; wrinkles such as static or dynamic wrinkles; dark circles; hyperpigmentation such as skin darkening or freckles, skin sagging, uneven or dull skin tone, or any combination thereof. Yet another aspect of the present disclosure relates to the engineered exosomes or compositions as disclosed herein for use in skin rejuvenation; preferably, the skin rejuvenation comprises treating any of the following: sun damage; sensitive skin; scars such as acne or stretch marks such as stretch marks or puberty marks, chicken pox or wounds; wrinkles such as static or dynamic wrinkles; dark circles; hyperpigmentation such as skin darkening or freckles, skin sagging, uneven or dull skin tone, or any combination thereof. These and other aspects and advantages of the present disclosure will be apparent from the detailed description provided below. Figure 1 shows the construction of a stable cell line that can secrete engineered exosomes loaded with functional proteins. Mammalian cells (e.g., HEK293 cells) were cultured and infected with a lentivirus that packaged the functional genes EGF, FGF-2, KGF-1, and PDGF-BB. The stable cell line was then selected using antibiotics. After three passages, expression of the functional gene was found in both the cell pellet and the exosomes. After confirming expression, the stable cell line was adapted to culture under serum-free conditions. Finally, the culture supernatant of the serum-free stable cell line was collected, and the exosomes purified by ultracentrifugation were identified. Figure 2 shows the characterization of engineered exosomes derived from stable cell lines. Figure 2A shows the particle size and concentration of exosomes derived from stable cell lines (NO: 6 and NO: 8) analyzed by NanoFCM. Figure 2B shows further examination of exosomes from stable cell lines (NO: 6 and NO: 8) by transmission electron microscopy (TEM). Figure 2C shows immunoblotting analysis of exosomes using an antibody against CD63, a scaffold protein fused to exosomes. Figure 3 shows that engineered exosomes exhibit enhanced cell proliferation activity in fibroblasts. Two fibroblast cell lines, HSF (human skin fibroblasts) (Figure 3A) and HFF (human foreskin fibroblasts) (Figure 3B), were cultured and incubated with exosomes (Nos. 6 and 8) at varying concentrations (0.5 μg, 1.0 μg, and 2.0 μg). The functional protein KGF (KGF-1) was used as a positive control. After 48 hours of incubation, cell viability was assessed using a CCK8 assay. Figure 4 shows that engineered exosomes regulate skin regeneration-related genes in dermal fibroblasts. HSFs were cultured and incubated with exosomes (No. 6 or No. 8) for 48 hours. RNA was then extracted from the cell pellets, and RT-qPCR was used to detect the expression of skin regeneration-related genes, including COL2A1 (Figure 4A), COL3A1 (Figure 4B), fibronectin (Figure 4C), TIMP1 (Figure 4D), and MMP7 (Figure 4E). Figure 5 shows that engineered exosomes regulate genes related to skin regeneration in a zebrafish model. Zebrafish embryos (4 dpf, dpf refers to days post fertilization) were prepared with yolk sacs. Exosomes (No. 6 or No. 8) were injected. Twenty-four hours after injection, gene expression analysis was performed using RT-qPCR to detect genes related to skin regeneration, including COL1A1B (Figure 5A), MMP9 (Figure 5B), and MMP16B (Figure 5C). Figure 6 shows that engineered exosomes help downregulate melanin synthesis. Figure 6A shows that human melanocytes were cultured and incubated with exosomes (NO: 6 or NO: 8) for 48 hours. The cell pellet was then used for melanin extraction, and the concentration of melanin was measured using a melanin assay kit (Fluorometric). Figure 6B shows that in a zebrafish model, zebrafish embryos (4 hpf, hpf refers to hours after fertilization) were cultured in exosomes (NO: 8) at 28°C for 45 hours in the dark. Images were then captured under a dissecting microscope and analyzed to evaluate melanin. Whitening efficacy (%) = (Mock group (Mock) - NO: 8) / Mock * 100%. definition When used with the word "comprising" in this specification, including the claims, the words "a," "an," and "an" mean "one or more." As used herein, the terms "or" and "and / or" are used to describe combinations of multiple components or mutually exclusive combinations. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." Specifically, x, y, or z can be explicitly excluded from an embodiment. In this application, the term "about" is used according to its ordinary and customary meaning in the fields of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences varies depending on the number of matching or homologous positions the sequences have. An "unrelated" or "non-homologous" sequence has less than 40% identity, but preferably less than 25% identity, to one of the sequences of the present disclosure. When a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a particular percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of "sequence identity" to another sequence, it means that when aligned, the percentage of bases (or amino acids) that are the same when the two sequences are compared. Such alignments and homology or sequence identity percentages can be determined using software programs known in the art. As used herein, the term "linker" refers to a short stretch of amino acids (AA) or a nucleotide sequence containing two or more identical or different amino acids or nucleotides. As used herein, "cell line" refers to a cell population formed through one or more subcultures of a primary cell culture. Each round of subculture is called a passage. After cells have been subcultured, they are referred to as "passaged" cells. A particular cell population or cell line is sometimes referred to or characterized by its number of passages. For example, a cultured cell population that has been passaged ten times may be referred to as a P10 culture. The primary culture, i.e., the first culture after the cells are isolated from the tissue, is designated P0. After the first subculture, the cells are described as a second-generation culture (P1 or passage 1). After the second subculture, the cells become a third-generation culture (P2 or passage 2), and so on. Those skilled in the art will understand that there may be many population doublings during the passage period; therefore, the number of population doublings of a culture is greater than the number of passages. The expansion of cells during passage (e.g., the number of population doublings) depends on many factors, including but not limited to the inoculation density, substrate, culture medium, growth conditions, and the time between passages. The terms "reduce," "inhibit," "alleviate," "reduce," "decrease," "prevent," and their grammatical equivalents (including "lower," "lesser," etc.), when referring to any symptom in an untreated subject compared to a treated subject, mean that the amount and / or extent of the symptom in the treated subject is less than the amount and / or extent of the symptom in the untreated subject, and that this difference is clinically relevant in the eyes of any medical professional. In one embodiment, the amount and / or extent of the symptom in the treated subject is at least 10% less, at least 25% less, at least 50% less, at least 75% less, and / or at least 90% less than the amount and / or extent of the symptom in the untreated subject. As used herein, the term "therapeutically effective amount" is synonymous with "effective amount," "therapeutically effective dose," and / or "effective dose," and refers to the amount of a compound that elicits the biological, cosmetic, or clinical response sought by the practitioner in an individual in need thereof. As an example, an effective amount is an amount sufficient to reduce hair loss. For a particular application of the disclosed methods, the appropriate effective amount to administer can be determined by one skilled in the art using the guidance provided herein. For example, the effective amount can be inferred from in vitro and in vivo assays as described herein. One skilled in the art will recognize that the individual's condition can be monitored throughout the course of treatment, and the effective amount of the exosomes or compositions disclosed herein administered can be adjusted accordingly. As used herein, the term "treatment" refers to intervention intended to alter the natural course of the individual or cell being treated and can be performed either prophylactically or during the pathological progression of a disease or condition. Treatment can achieve one or more of a variety of desired outcomes, including, for example, preventing the onset or recurrence of the disease, alleviating symptoms, diminishing any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, ameliorating or palliating the disease state, or providing remission or improved prognosis. As used herein, the term "subject" is used interchangeably with the term "individual" or "patient" and generally refers to an individual in need of treatment. The subject can be a mammal, such as a human, dog, cat, horse, pig, or rodent. As used herein, "carrier" refers to a diluent, adjuvant, excipient, or vehicle administered with a therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as saline solutions and oils, including oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the composition is administered intravenously, saline solutions are preferred carriers. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, and the like. If desired, the composition may also contain a small amount of a wetting agent or emulsifier or a pH buffer. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, and a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. The term "N-terminal amino acid residue" or "N-terminus" refers to the first amino acid residue (amino acid 1) of a polypeptide or peptide. The term "C-terminal amino acid residue" or "C-terminus" refers to the last amino acid residue (amino acid n, where n = the total number of residues in the peptide or polypeptide) of a polypeptide or peptide. The term "KGF" refers to keratinocyte growth factor, a member of the epithelial mitogen and fibroblast growth factor (FGF) family. In vertebrates, the FGF family comprises 22 members that are essential for regulating many developmental processes. KGF includes two functionally similar variants, KGF-1 (also known as FGF-7) and KGF-2 (also known as FGF-10). Characterization of recombinant human fibroblast growth factor FGF-10 has revealed functional similarities to keratinocyte growth factor (FGF-7). Both growth factors interact with the same high-affinity receptor (KGFR, an isoform of FGFR2) that differs from FGFR2 in the latter half of the third immunoglobulin loop and is encoded by an alternative exon. The ability of KGF-1 and KGF-2 to bind KGFR with high affinity distinguishes them from other members of the FGF-10 family. KGF-2 is highly related to KGF-1, binding to the same receptor as KGF-1 and sharing 57% sequence homology. Human KGF-2 is 96% identical to rat KGF-2 and specifically stimulates the growth of normal human epidermal keratinocytes. The amino acid sequence of human KGF-1 can be obtained from UniProtKB / Swiss-Prot: P21781.1, and the mature chain of human KGF-1 is shown in SEQ ID NO: 1 of the present disclosure. The amino acid sequence of human KGF-2 can be obtained from UniProtKB / Swiss-Prot: O15520.1, with the mature chain located between amino acids 38 and 208. The term "FGF-2" is also referred to as basic FGF ("bFGF"). It is known that both FGF1 and FGF2 are released in large quantities by damaged endothelial cells and macrophages at the wound site, and if FGF2 activity is blocked, wound angiogenesis is almost completely impaired. FGF2 is also known to induce scarless healing. However, due to the short half-life of free FGF, the use of delivery systems has been proposed. Among FGFs, research on their application in wound healing and skin regeneration has primarily focused on FGF2. Because acidic gelatin is highly negatively charged, it is able to retain FGF2 well by forming an ionic complex with gelatin. The amino acid sequence of human KGF-2 can be obtained from UniProtKB / Swiss-Prot: P09038.3, and its mature chain is located between amino acids 143 and 288, which is shown in this disclosure as SEQ ID NO: 3. The term "EGF" refers to epidermal growth factor, a growth factor that stimulates cell growth, proliferation, and differentiation by binding to its receptor, EGFR. It has been shown to act as a potent mitogen by stimulating mRNA, DNA, and protein synthesis in epithelial cells. It is a single-chain polypeptide composed of 53 amino acids, which are derived from cleavage of a larger precursor, the proprotein EGF. EGF is now known to be the prototypical member of the group I EGF family, which also includes transforming growth factor-α (TGF-α), heparin-binding EGF (HB-EGF), amphiregulin, betacellulin, epiregulin, and epidermal growth factor. Structurally, they all contain one or more EGF repeats (EGF motifs) in their extracellular domains. These repeats are sequences of 35 to 40 amino acids separated by six conserved cysteines in the following pattern: CX7CX3-5CX10-12CXCX5GXRC (C, cysteine; G, glycine; R, arginine; X, other amino acids). One glycine and one arginine in this sequence are also conserved in all EGF-related growth factors, but are not conserved in proteins containing the EGF motif but without growth factor activity. These six cysteines pair and form three intramolecular disulfide bonds with the following interactions: C1-C3, C2-C4, and C5-C6 (numbered according to their order in the sequence), which is crucial for maintaining their biological activity. Functionally, these growth factors have the ability to bind to the same receptor, the EGF receptor (EGFR, ErbB1), activate its intrinsic tyrosine kinase activity, and couple the receptor to downstream signaling pathways that control cell proliferation, differentiation, survival, or motility. The amino acid sequence of human EGF can be obtained from GenBank: AAS83395.1, which is also shown in this disclosure as SEQ ID NO: 2. The term "PDGF" refers to platelet-derived growth factor, which is a potent mitogen and chemoattractant for many cells of mesenchymal origin, such as fibroblasts, activated macrophages, and smooth muscle cells. PDGF plays a key role in angiogenesis, embryonic development, inflammation, and cell differentiation; therefore, it can benefit wound healing. The PDGF family of growth factors consists of four different polypeptide chains encoded by four different genes: the classical PDGF-A and PDGF-B chains, and the more recently discovered PDGF-C and PDGF-D. These four PDGF chains assemble into disulfide-bonded dimers by homodimerization or heterodimerization, and five different dimer isoforms have been described to date: PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD. Two different PDGF receptors ( α and β) mediate the effects of PDGF on target cells. PDGF-A and PDGF-C chains selectively bind α receptors, while PDGF-D preferentially binds to β receptors, and PDGF-B exhibits similar affinity for both receptors. Receptor activation requires PDGF-induced receptor dimerization, which leads to tyrosine transphosphorylation. PDGF AA only induces α / α receptor dimer, PDGF AB induced α / α dimer and α / β dimer, and PDGF BB induced all 3 combinations. Although PDGF-C only α receptor binding, but it can produce through the trans-activation of β receptors α-β heterodimer. A potential application of PDGF-BB protein is to induce wound healing in diabetic patients. Furthermore, compositions containing PDGF can promote the healing of damaged or failing bones by promoting the growth of connective tissue, bone growth, and by stimulating collagen synthesis in damaged bone areas. The amino acid sequence of human PDGF-B can be obtained from GenBank: CAA02294.1, where amino acids 82 to 190 represent the mature peptide, which is also shown in SEQ ID NO: 4 of the present disclosure. COL2A1 (collagen type II The α1 gene provides instructions for making a component of type II collagen called the procollagen α1(II) chain. Type II collagen adds structure and strength to the connective tissue that supports the body's muscles, joints, organs, and skin. Type II collagen is found primarily in cartilage, a solid, flexible tissue that makes up the majority of the skeleton during early development. Most cartilage later turns into bone, with the exception of cartilage that continues to cover and protect the ends of bones and is found in the nose and outer ear. Type II collagen is also part of the clear gel that fills the eyeball (vitreous humor), the inner ear, and the central part of the discs between the vertebrae (nucleus pulposus). To build type II collagen, three precursors are needed. The α1(II) chains entwine to form three-stranded, rope-like procollagen molecules. These procollagen molecules are then processed by enzymes within the cell. After processing, these molecules leave the cell and arrange themselves into long, thin fibrils that connect (crosslink) to each other in the spaces surrounding the cell. Crosslinking results in the formation of very strong, mature type II collagen fibers. COL3A1 (collagen type III The α1 chain) gene provides instructions for making type III collagen. Collagen is a family of proteins that strengthen and support many tissues in the body. Type III collagen is found in the skin, lungs, intestinal lining, and blood vessel walls. The α1(III) chain is composed of Produced by the COL3A1 gene. Each molecule of type III procollagen consists of three copies of this chain. These three-chain, rope-like procollagen molecules are enzymatically processed outside the cell to produce mature type III collagen. The collagen molecules then arrange themselves into long, thin fibrils, which form stable interactions (crosslinks) with each other and with other types of collagen in the spaces between cells. Crosslinking results in the formation of very strong collagen fibers. The term "fibronectin" refers to the fibronectin 1 gene or its product. This gene encodes fibronectin, a glycoprotein that exists as a soluble dimer in plasma and as dimers or multimers on the cell surface and in the extracellular matrix. The encoded preproprotein undergoes proteolytic processing to produce the mature protein. Fibronectin is involved in cell adhesion and migration processes, including embryonic development, wound healing, coagulation, host defense, and metastasis. This gene has three regions that undergo alternative splicing, potentially generating 20 different transcript variants, at least one of which encodes an isoform that undergoes proteolytic processing. TIMP1, or TIMP metallopeptidase inhibitor 1, belongs to the TIMP gene family. The proteins encoded by this gene family are natural inhibitors of matrix metalloproteinases (MMPs), a group of peptidases involved in extracellular matrix degradation. In addition to its inhibitory effects on most known MMPs, the encoded protein promotes cell proliferation in a variety of cell types and also exhibits anti-apoptotic functions. Transcription of this gene is highly inducible in response to numerous cytokines and hormones. Furthermore, expression on a partially, but not fully, inactivated X chromosome suggests that gene inactivation is polymorphic in human females. The COL1A1B gene is specific to zebrafish. In tetrapods, collagen type I is composed mainly of two α1 chain and one The trimer composed of α2 chains α1 chain and The α2 chain is encoded by the COL1A1 and COL1A2 genes. α1 (I), α3(I) and The three collagen type I genes of the α2(I) chain, col1a1a, col1a1b, and col1a2, exhibit similar spatial-temporal expression patterns during embryonic and larval development, suggesting that they are co-regulated and interdependent during these stages. The presence of α(I) chains, although in embryos α1(I) exists in two different glycosylation states, indicating a developmentally specific collagen composition. Although present in equal amounts in adult bone, skin, and scales, α1(I), α3(I) and α2(I) chain, but the data presented suggest tissue-specific stoichiometry and / or post-translational modification states of collagen type I. Matrix metalloproteinases (MMPs) belong to a family of zinc-dependent extracellular matrix (ECM) remodeling endopeptidases capable of degrading virtually every component of the ECM. ECM degradation is crucial because it is involved in embryonic development and angiogenesis. It is also involved in cell repair and tissue remodeling. Altered MMP expression leads to abnormal ECM degradation, a primary cause of the development of chronic degenerative diseases and vascular complications caused by diabetes. Furthermore, this process has been implicated in neurodegeneration and cancer progression. Within the ECM, tissue inhibitors of MMPs (TIMPs) inhibit the proteolytic activity of MMPs. TIMPs are important regulators of ECM turnover, tissue remodeling, and cell behavior. Consequently, TIMPs (like MMPs) regulate angiogenesis, cell proliferation, and apoptosis. Disruptions in the balance between MMPs and TIMPs are implicated in the pathophysiology and progression of several diseases. MMPs belong to a family of endopeptidases comprising 23 members. They contain zinc, are calcium-dependent, and are capable of degrading and remodeling proteins that form the ECM. They are also involved in diverse biological and physiological processes regulated by hormones, growth factors, and cytokines. Based on their subcellular distribution and specificity for ECM components, MMPs are classified as membrane-type matrix metalloproteinases (MT-MMPs), collagenases, gelatinases, stromelysins, and matrilysins. Collagenases (MMP-1, MMP-8, MMP-13, and MMP-18) degrade triple-helical fibrillar collagen, a major component of bones and ligaments. Gelatinases (MMP-2 and MMP-9) are involved in various cellular processes, including angiogenesis and neurogenesis; these proteases alter the molecules of the basement membrane, subsequently leading to cell death. Matrixlysins (MMP-3, MMP-10, and MMP-11) are small proteases that degrade ECM fragments. Matrixlysins (MMP-7 and MMP-26) process cell surface molecules and digest ECM components. Members of the tetraspanin family (such as CD63, CD81, and CD9) are ubiquitously expressed on exosomes and are widely used as exosome biomarkers, participating in physiological processes such as cell adhesion, cell motility, and signal transduction. CD63 (the first characteristic is a tetraspanin) has two extracellular loops of varying sizes and two short cytoplasmic domains, which are involved in the signal transduction process of various types of immune cells. Continuous domain deletion has shown that transmembrane helix 3 (TM3) is necessary and sufficient for membrane anchoring and exosome targeting. The amino acid sequence of human CD63 can be obtained from, for example, GenBank: AHI51903.1, and the amino acid sequence of its TM3 corresponds to amino acids 70 to 133, which is also shown in the present disclosure as SEQ ID NO: 9. The term "anchor polypeptide" refers to a polypeptide that is anchored to the exosome membrane when the exosome is produced by the cell. Transmembrane proteins are typical anchor polypeptides in this disclosure. "Anchored" or its grammatical variations refers to at least one fragment of a polypeptide being embedded in the exosome membrane. The anchor polypeptide may be fully or partially embedded in the exosome membrane. In this disclosure, the anchor polypeptide is fused to a polypeptide heterologous to the exosome naturally produced by the same cell, such as the KGF-1 polypeptide. Exemplary anchor polypeptides are exosome membrane proteins, membrane targeting sequences, or anchoring functional fragments thereof. Exemplary exosome membrane proteins include, but are not limited to, LAMP2b, tetraspanins such as CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactadherin (C1C2 domain), vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative regulator (PTGFRN), and any combination thereof. Exemplary membrane targeting sequences include, but are not limited to, glycosylphosphatidylinositol (GPI) anchors and lipid-anchored proteins. A detailed review of GPI anchors in exosomes can be found, for example, in Michel Vidal, “Exosomes and GPI-anchored proteins: Judicious pairs for investigating biomarkers from body fluids”. Advanced Drug Delivery Reviews, Vol. 161-162, 2020 (incorporated herein by reference in its entirety). In a preferred embodiment of the present disclosure, the anchoring polypeptide comprises or consists of transmembrane helix 3 (TM3) of the CD63 protein. Engineered exosomes One aspect of the present disclosure relates to an engineered exosome comprising (a) a KGF polypeptide fused to a first anchor polypeptide, (b) an EGF polypeptide fused to a second anchor polypeptide, and (c) an FGF-2 polypeptide fused to a third anchor polypeptide, wherein (a), (b) and (c) are anchored to the membrane of the exosome through the first, second and third anchor polypeptides, respectively, and wherein the KGF polypeptide, the EGF polypeptide and the FGF-2 polypeptide are exposed on the outer surface of the membrane of the exosome. In a preferred embodiment, the engineered exosomes provided by the present disclosure comprise (a) a KGF polypeptide fused to a first anchor polypeptide, (b) an EGF polypeptide fused to a second anchor polypeptide, (c) an FGF-2 polypeptide fused to a third anchor polypeptide, and (d) a PDGF-BB polypeptide fused to a fourth anchor polypeptide, wherein (a), (b), (c) and (d) are anchored to the membrane of the exosome through the first, second, third and fourth anchor polypeptides, respectively, and wherein the KGF polypeptide, the EGF polypeptide, the FGF-2 polypeptide and the PDGF-BB polypeptide are exposed on the outer surface of the membrane of the exosome. In the present disclosure, the KGF polypeptide can be a KGF-1 polypeptide or a KGF-2 polypeptide. In preferred embodiments, the KGF polypeptide is a KGF-1 polypeptide. In some embodiments, the KGF-1 polypeptide is a human EGF polypeptide or an ortholog or paralog thereof. In some embodiments, the KGF-1 polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the KGF-1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the KGF-1 polypeptide consists essentially of the amino acid sequence set forth in SEQ ID NO:1. In preferred embodiments, the EGF polypeptide is a human EGF polypeptide or an ortholog or paralog thereof. In some embodiments, the EGF polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the EGF polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the EGF polypeptide consists essentially of the amino acid sequence set forth in SEQ ID NO:2. In preferred embodiments, the FGF-2 polypeptide is a human FGF-2 polypeptide or an ortholog or paralog thereof. In some embodiments, the FGF polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the FGF-2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the FGF-2 polypeptide consists essentially of the amino acid sequence set forth in SEQ ID NO:3. In preferred embodiments, the PDGF-BB polypeptide is a human PDGF-BB dimer or an ortholog or paralog thereof. In some embodiments, the PDGF-BB polypeptide is a human PDGF-BB dimer. In some embodiments, the PDGF-BB polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the PDGF-BB polypeptide comprises the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the PDGF-BB polypeptide consists essentially of the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, these anchoring polypeptides are exosomal membrane proteins, membrane targeting sequences, or anchoring functional fragments thereof. Exemplary exosomal membrane proteins include, but are not limited to, lamp2b, tetraspanins such as CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactadherin (C1C2 domain), vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative regulator (PTGFRN), and any combination thereof. Exemplary membrane targeting sequences include, but are not limited to, glycosylphosphatidylinositol (GPI) anchors and lipid-anchored proteins. In preferred embodiments of the present disclosure, the first, second, third, and fourth anchoring polypeptides comprise the TM3 domain of CD63. In preferred embodiments, each of the first, second, third, and fourth anchoring polypeptides comprises the TM3 domain of CD63. In some embodiments, the anchor polypeptides are full-length CD63 proteins, such as full-length human CD63 (see, e.g., UniProtKB / Swiss-Prot: F8VZE2, P08962, Q5TZP3, Q8N6Z9, or Q9UCG6). In some embodiments, the anchor polypeptides are truncated CD63 proteins comprising a TM3 domain and at least one of a TM1, TM2, and TM4 domain. For example, the anchor polypeptides can consist of TM2 and TM3 of CD63; TM3 and TM4 of CD63; or TM1, TM2, and TM3 of CD63. In some embodiments, the anchor polypeptides consist of the TM3 domain of CD63. In the present disclosure, the first, second, third, and fourth anchor polypeptides can be different or identical. In preferred embodiments, the first, second, third, and fourth anchor polypeptides are identical and consist of the TM3 domain of CD63. In preferred embodiments, the TM3 domain of CD63 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9. In preferred embodiments, the TM3 domain of CD63 comprises the amino acid sequence of SEQ ID NO: 9. In preferred embodiments, the TM3 domain of CD63 consists essentially of the amino acid sequence of SEQ ID NO: 9. Therefore, in a preferred embodiment, an engineered exosome is provided, comprising (a) a KGF-1 polypeptide fused to a first anchor polypeptide, (b) an EGF polypeptide fused to a second anchor polypeptide, and (c) an FGF-2 polypeptide fused to a third anchor polypeptide, wherein each of the first, second, and third anchor polypeptides comprises the TM3 domain of CD63, wherein (a), (b), and (c) are anchored to the membrane of the exosome through the first, second, and third anchor polypeptides, respectively, and wherein the KGF-1 polypeptide, the EGF polypeptide, and the FGF-2 polypeptide are exposed on the outer surface of the membrane of the exosome. In a preferred embodiment, an engineered exosome is provided, comprising (a) a human KGF-1 polypeptide fused to a first anchor polypeptide, (b) a human EGF polypeptide fused to a second anchor polypeptide, and (c) a human FGF-2 polypeptide fused to a third anchor polypeptide, wherein each of the first, second, and third anchor polypeptides comprises the TM3 domain of CD63, wherein (a), (b), and (c) are anchored to the membrane of the exosome through the first, second, and third anchor polypeptides, respectively, and wherein the human KGF-1 polypeptide, the human EGF polypeptide, and the human FGF-2 polypeptide are exposed on the outer surface of the membrane of the exosome. In a preferred embodiment, an engineered exosome is provided, comprising (a) a KGF-1 polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 1, wherein the KGF-1 polypeptide is fused to a first anchor polypeptide, (b) an EGF polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 2, wherein the EGF polypeptide is fused to a second anchor polypeptide, and (c) an FGF-2 polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 3, wherein the EGF polypeptide is fused to a second anchor polypeptide. NO: 3 has an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, the FGF-2 polypeptide is fused to a third anchor polypeptide, wherein each of the first, second and third anchor polypeptides comprises the TM3 domain of CD63, wherein (a), (b) and (c) are anchored to the exosome membrane through the first, second and third anchor polypeptides, respectively, and wherein the KGF-1 polypeptide, the EGF polypeptide and the FGF-2 polypeptide are exposed on the outer surface of the exosome membrane. In a preferred embodiment, an engineered exosome is provided, comprising (a) a KGF-1 polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 1, wherein the KGF-1 polypeptide is fused to a first anchor polypeptide, (b) an EGF polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 2, wherein the EGF polypeptide is fused to a second anchor polypeptide, and (c) an FGF-2 polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 3, the FGF-2 polypeptide is fused to a third anchor polypeptide, wherein each of the first, second, and third anchor polypeptides comprises a TM3 domain of CD63 comprising an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9, wherein (a), (b), and (c) are anchored to the exosome membrane through the first, second, and third anchor polypeptides, respectively, and wherein the KGF-1 polypeptide, the EGF polypeptide, and the FGF-2 polypeptide are exposed on the outer surface of the exosome membrane. In a preferred embodiment, an engineered exosome is provided, comprising: (a) a KGF-1 polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, fused to a first anchor polypeptide; (b) an EGF polypeptide having an amino acid sequence as shown in SEQ ID NO: 2, fused to a second anchor polypeptide; and (c) an FGF-2 polypeptide having an amino acid sequence as shown in SEQ ID NO: 3, fused to a third anchor polypeptide, wherein each of the first, second, and third anchor polypeptides comprises the TM3 domain of CD63 having an amino acid sequence as shown in SEQ ID NO: 9, wherein (a), (b), and (c) are anchored to the membrane of the exosome through the first, second, and third anchor polypeptides, respectively, and wherein the KGF-1 polypeptide, the EGF polypeptide, and the FGF-2 polypeptide are exposed on the outer surface of the membrane of the exosome. In a preferred embodiment, an engineered exosome is provided, comprising (a) a KGF-1 polypeptide fused to a first anchor polypeptide, (b) an EGF polypeptide fused to a second anchor polypeptide, (c) an FGF-2 polypeptide fused to a third anchor polypeptide, and (d) a PDGF-BB polypeptide fused to a fourth anchor polypeptide, wherein each of the first, second, third, and fourth anchor polypeptides comprises the TM3 domain of CD63, wherein (a), (b), (c), and (d) are anchored to the membrane of the exosome through the first, second, third, and fourth anchor polypeptides, respectively, and wherein the KGF-1 polypeptide, the EGF polypeptide, the FGF-2 polypeptide, and the PDGF-BB polypeptide are exposed on the outer surface of the membrane of the exosome. In a preferred embodiment, an engineered exosome is provided, comprising (a) a human KGF-1 polypeptide fused to a first anchor polypeptide, (b) a human EGF polypeptide fused to a second anchor polypeptide, (c) a human FGF-2 polypeptide fused to a third anchor polypeptide, and (d) a human PDGF-BB polypeptide fused to a fourth anchor polypeptide, wherein each of the first, second, third, and fourth anchor polypeptides comprises the TM3 domain of CD63, wherein (a), (b), (c), and (d) are anchored to the membrane of the exosome through the first, second, third, and fourth anchor polypeptides, respectively, and wherein the human KGF-1 polypeptide, the human EGF polypeptide, the FGF-2 polypeptide, and the PDGF-BB polypeptide are exposed on the outer surface of the membrane of the exosome. In a preferred embodiment, an engineered exosome is provided, comprising (a) a KGF-1 polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 1, wherein the KGF-1 polypeptide is fused to a first anchor polypeptide, (b) an EGF polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 2, wherein the EGF polypeptide is fused to a second anchor polypeptide, and (c) an FGF-2 polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 3. (a) (b) (c) (d) an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the first, second, third and fourth anchor polypeptides, the FGF-2 polypeptide being fused to a third anchor polypeptide, and (d) a PDGF-BB polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 4, the PDGF-BB polypeptide being fused to a fourth anchor polypeptide, wherein each of the first, second, third and fourth anchor polypeptides comprises the TM3 domain of CD63, wherein (a), (b), (c) and (d) are anchored to the exosome membrane through the first, second, third and fourth anchor polypeptides, respectively, and wherein the KGF-1 polypeptide, the EGF polypeptide, the FGF-2 polypeptide and the PDGF-BB polypeptide are exposed on the outer surface of the exosome membrane. In a preferred embodiment, an engineered exosome is provided, comprising (a) a KGF-1 polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 1, wherein the KGF-1 polypeptide is fused to a first anchor polypeptide, (b) an EGF polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 2, wherein the EGF polypeptide is fused to a second anchor polypeptide, and (c) an FGF-2 polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 3, wherein the EGF polypeptide is fused to a second anchor polypeptide, NO:3, the FGF-2 polypeptide being fused to a third anchor polypeptide, and (d) a PDGF-BB polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:4, the PDGF-BB polypeptide being fused to a fourth anchor polypeptide, wherein each of the first, second, third and fourth anchor polypeptides comprises a TM3 domain of CD63 comprising a TM3 domain identical to the amino acid sequence of SEQ ID NO:5. NO: 9 has an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, wherein (a), (b), (c) and (d) are anchored to the exosome membrane through the first, second, third and fourth anchor polypeptides, respectively, and wherein the KGF-1 polypeptide, the EGF polypeptide, the FGF-2 polypeptide and the PDGF-BB polypeptide are exposed on the outer surface of the exosome membrane. In a preferred embodiment, an engineered exosome is provided, comprising (a) a KGF-1 polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, wherein the KGF-1 polypeptide is fused to a first anchor polypeptide, (b) an EGF polypeptide having an amino acid sequence as shown in SEQ ID NO: 2, wherein the EGF polypeptide is fused to a second anchor polypeptide, (c) an FGF-2 polypeptide having an amino acid sequence as shown in SEQ ID NO: 3, wherein the FGF-2 polypeptide is fused to a third anchor polypeptide, and (d) a PDGF-BB polypeptide having an amino acid sequence as shown in SEQ ID NO: 4, wherein the PDGF-BB polypeptide is fused to a fourth anchor polypeptide, wherein each of the first, second, third, and fourth anchor polypeptides consists of the TM3 domain of CD63 having an amino acid sequence as shown in SEQ ID NO: NO: The amino acid sequence shown in 9, wherein (a), (b), (c) and (d) are anchored to the exosome membrane through the first, second, third and fourth anchor polypeptides, respectively, and wherein the KGF-1 polypeptide, the EGF polypeptide, the FGF-2 polypeptide and the PDGF-BB polypeptide are exposed on the outer surface of the exosome membrane. In any of the above embodiments, the KGF polypeptide, the EGF polypeptide, the FGF-2 polypeptide, and the PDGF-BB polypeptide (when present) are fused directly or via a peptide linker to the C-terminus of the first, second, third, and fourth anchor polypeptides, respectively. The peptide linker can be any peptide linker available in the art for linking different domains or functional regions in a fusion protein. In a preferred embodiment, the peptide linker is composed of glycine and serine, such as (G4S)n, where n is an integer from 1 to 3. In the present disclosure, preferably, the exosomes are not derived from mesenchymal stem cells. More preferably, the exosomes are not derived from stem cells. In preferred embodiments, the exosomes provided by the present invention are derived from non-stem cells, such as CHO or HEK293 cells. In the present disclosure, preferably, the exosomes are purified and / or isolated from the cells from which they are derived. In some embodiments, the exosomes provided herein enhance fibroblast proliferation. In some embodiments, the exosomes provided herein upregulate COL2A1, COL3A1, fibronectin, TIMP1, and / or COL1A1B genes. In some embodiments, the exosomes provided herein downregulate MMP7, MMP9, and / or MMP16B genes. In some embodiments, the exosomes provided herein downregulate melanin synthesis. In some embodiments, the exosomes provided herein enhance fibroblast proliferation; upregulate COL2A1, COL3A1, fibronectin, TIMP1, and / or COL1A1B genes; downregulate MMP7, MMP9, and / or MMP16B genes; and downregulate melanin synthesis. Methods and compositions for skin rejuvenation Various aspects of the present disclosure relate to methods and compositions for skin rejuvenation. In preferred embodiments, the skin rejuvenation comprises treating any of the following: sun damage; sensitive skin; scars such as acne or stretch marks such as stretch marks or puberty marks, chicken pox, or wounds; wrinkles such as static or dynamic wrinkles; dark circles; hyperpigmentation such as skin darkening or freckles, skin laxity, uneven or dull skin tone, or any combination thereof; the method comprising administering to a subject in need thereof a pharmaceutically or cosmetically effective amount of an engineered exosome or composition disclosed herein. In a preferred embodiment, a method for combating wrinkles, including static and dynamic wrinkles, such as on the neck and / or face, is provided, comprising administering to a subject in need thereof a pharmaceutically or cosmetically effective amount of an engineered exosome or composition disclosed herein. In a preferred embodiment, a method for treating hyperpigmentation, such as on the neck and / or face, is provided. In a preferred embodiment, a method of treating scars, including scars from acne or stretch marks such as stretch marks or puberty marks, for example, on the abdomen and / or legs, is provided, comprising administering to a subject in need thereof a pharmaceutically or cosmetically effective amount of an engineered exosome or composition disclosed herein. In the present disclosure, provided compositions comprise the engineered exosomes and a carrier. In preferred embodiments, the compositions are liquid formulations. In preferred embodiments, the compositions are formulated for topical or subcutaneous administration. Compositions comprising the engineered exosomes are contemplated for use, including, for example, soaps, shampoos, ointments, and other such formulations. In preferred embodiments, the composition does not contain KGF polypeptides (e.g., KGF-1 polypeptides), EGF polypeptides, FGF-2 polypeptides, or PDGF polypeptides (e.g., PDGF-BB polypeptides) that are not attached to the membrane of the exosomes (i.e., free polypeptides). For example, other than the polypeptides anchored to the membrane of the engineered exosomes, no additional KGF polypeptides (e.g., KGF-1 polypeptides), EGF polypeptides, FGF-2 polypeptides, or PDGF polypeptides (e.g., PDGF-BB polypeptides) are added or supplemented to the composition. In some embodiments, the composition is a cosmetic composition. In some embodiments, the composition is a non-cosmetic composition. In some embodiments, the composition is a pharmaceutical composition. Nucleic acid constructs, vectors, cells and production methods Various aspects of the present disclosure also relate to nucleic acid constructs encoding polypeptides anchored to exosomes. In some embodiments, a nucleic acid construct is provided, comprising a polynucleotide encoding: (a) a KGF polypeptide fused to a first anchor polypeptide, (b) an EGF polypeptide fused to a second anchor polypeptide, and (c) an FGF-2 polypeptide fused to a third anchor polypeptide. In some embodiments, a nucleic acid construct is provided, comprising a polynucleotide encoding: (a) a KGF polypeptide fused to a first anchor polypeptide, (b) an EGF polypeptide fused to a second anchor polypeptide, (c) an FGF-2 polypeptide fused to a third anchor polypeptide, and (d) a PDGF-BB polypeptide fused to a fourth anchor polypeptide. In some embodiments, a set of three nucleic acid constructs is provided, wherein the first nucleic acid construct comprises a polynucleotide encoding (a), the second nucleic acid construct comprises a polynucleotide encoding (b), and the third nucleic acid construct comprises a polynucleotide encoding (c), wherein (a), (b) and (c) are as defined above. In some embodiments, a set of four nucleic acid constructs is provided, wherein the first nucleic acid construct comprises a polynucleotide encoding (a), the second nucleic acid construct comprises a polynucleotide encoding (b), the third nucleic acid construct comprises a polynucleotide encoding (c), and the fourth nucleic acid construct comprises a polynucleotide encoding (d), wherein (a), (b), (c) and (d) are as defined above. In some embodiments, a set of two nucleic acid constructs is provided, wherein one nucleic acid construct comprises a polynucleotide encoding two of (a), (b), and (c), and the other nucleic acid construct comprises a polynucleotide encoding the remaining polypeptide, wherein (a), (b), and (c) are as defined above. In some embodiments, a set of two nucleic acid constructs is provided, wherein one nucleic acid construct comprises a polynucleotide encoding two of (a), (b), (c), and (d), and the other nucleic acid construct comprises a polynucleotide encoding the other two, wherein (a), (b), (c), and (d) are as defined above. In some embodiments, a set of three nucleic acid constructs is provided, wherein the first nucleic acid construct comprises a polynucleotide encoding two of (a), (b), (c), and (d), the second nucleic acid construct comprises a polynucleotide encoding one of the remaining polypeptides, and the third nucleic acid construct comprises a polynucleotide encoding the other of the remaining polypeptides, wherein (a), (b), (c), and (d) are as defined above. In a preferred embodiment, a single nucleic acid construct is provided comprising a polynucleotide encoding (a), (b) and (c), wherein (a), (b) and (c) are as defined above. In a preferred embodiment, a single nucleic acid construct is provided comprising a polynucleotide encoding (a), (b), (c) and (d), wherein (a), (b), (c) and (d) are as defined above. In some embodiments, these anchoring polypeptides are exosomal membrane proteins, membrane targeting sequences, or anchoring functional fragments thereof. Exemplary exosomal membrane proteins include, but are not limited to, lamp2b, tetraspanins such as CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactadherin (C1C2 domain), vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative regulator (PTGFRN), and any combination thereof. Exemplary membrane targeting sequences include, but are not limited to, glycosylphosphatidylinositol (GPI) anchors and lipid-anchored proteins. In some embodiments, each of these anchor polypeptides may be located at the N-terminus of the polypeptide to be presented on the surface of the exosome (e.g., EGF, KGF, FGF-2, and PDGF-BB) to ensure that the polypeptide to be presented is exposed on the surface of the exosome. In some embodiments, each of these anchor polypeptides may be located at the C-terminus of the polypeptide to be presented on the surface of the exosome to ensure that the polypeptide to be presented is exposed on the surface of the exosome. For example, when lamp2b is used as one of these anchor polypeptides, the polypeptide to be presented on the surface of the exosome (e.g., EGF, KGF, FGF-2, or PDGF-BB) may be located at the N-terminus of lamp2b. For example, when the TM3 domain of CD63 is used as one of these anchor polypeptides, the polypeptide to be presented on the surface of the exosome (e.g., EGF, KGF, FGF-2, or PDGF-BB) may be located at the C-terminus of the TM3 domain of CD63. In some embodiments, when different anchor polypeptides are used, the polypeptide to be presented on the surface of the exosomes can be located at the N-terminus or C-terminus of the anchor polypeptides, depending on the type of anchor polypeptide used. In preferred embodiments, the anchor polypeptides comprise full-length CD63 or a truncated CD63 that retains the TM3 domain. In preferred embodiments, each of the anchor polypeptides comprises the TM3 domain of CD63. In preferred embodiments, each of the first, second, third, and fourth anchor polypeptides is the TM3 domain of CD63. In a preferred embodiment, the polynucleotide may comprise a nucleotide fragment encoding the following substances from 5' to 3': (i) [first anchor polypeptide]-[KGF]-[self-cleaving peptide]-[second anchor polypeptide]-[EGF]-[self-cleaving peptide]-[third anchor polypeptide]-[FGF-2], (ii) [first anchor polypeptide]-[KGF]-[self-cleaving peptide]-[second anchor polypeptide]-[FGF-2]-[self-cleaving peptide]-[third anchor polypeptide]-[EGF], (iii) [first anchor polypeptide]-[EGF]-[self-cleaving peptide]-[second anchor polypeptide]-[KGF]-[self-cleaving peptide]-[third anchor polypeptide]-[FGF-2], (iv) [first anchor polypeptide]-[EGF]-[self-cleaving peptide]-[second anchor polypeptide]-[FGF-2]-[self-cleaving peptide]-[third anchor polypeptide]-[KGF], (v) [first anchor polypeptide]-[FGF-2]-[self-cleaving peptide]-[second anchor polypeptide]-[KGF]-[self-cleaving peptide]-[third anchor polypeptide]-[EGF], or (vi) [first anchor polypeptide]-[FGF-2]-[self-cleaving peptide]-[second anchor polypeptide]-[EGF]-[self-cleaving peptide]-[third anchor polypeptide]-[KGF], wherein [ ] represents a single polypeptide, and ]-[ represents a linker or bond. In preferred embodiments, the self-cleaving peptide is a 2A peptide, such as T2A, E2A, P2A, or any combination thereof. For example, the self-cleaving peptide is a T2A peptide. After translation of the polynucleotide, the self-cleaving peptide is cleaved to produce three or four separate fusion proteins, each comprising a single polypeptide to be presented on the surface of exosomes and a single anchor polypeptide. In a preferred embodiment, a single nucleic acid construct is provided comprising a polynucleotide encoding (a), (b) and (c), wherein (a), (b) and (c) are as defined above, and wherein each of the first, second and third anchor polypeptides comprises the TM3 domain of CD63, and the polynucleotide may comprise, from 5' to 3', a nucleotide fragment encoding one of: (i)[TM3]-[KGF]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[FGF-2], (ii)[TM3]-[KGF]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[EGF], (iii)[TM3]-[EGF]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[FGF-2], (iv)[TM3]-[EGF]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[KGF], (v)[TM3]-[FGF-2]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[EGF], and (vi)[TM3]-[FGF-2]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[KGF], wherein [] represents a single polypeptide, and ]-[ represents a linker or bond; and wherein TM3 represents the TM3 domain of CD63; and T2A represents the self-cleaving peptide T2A. In a preferred embodiment, the polynucleotide may comprise a nucleotide fragment encoding the following substances from 5' to 3': (a) [first anchor polypeptide]-[KGF]-[self-cleaving peptide]-[second anchor polypeptide]-[EGF]-[self-cleaving peptide]-[third anchor polypeptide]-[FGF-2]-[self-cleaving peptide]-[fourth anchor polypeptide]-[PDGF-BB], (b) [first anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[second anchor polypeptide]-[KGF]-[self-cleaving peptide]-[third anchor polypeptide]-[EGF]-[self-cleaving peptide]-[fourth anchor polypeptide]-[FGF-2], (c) [first anchor polypeptide]-[KGF]-[self-cleaving peptide]-[second anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[third anchor polypeptide]-[EGF]-[self-cleaving peptide]-[fourth anchor polypeptide]-[FGF-2], (d) [first anchor polypeptide]-[KGF]-[self-cleaving peptide]-[second anchor polypeptide]-[EGF]-[self-cleaving peptide]-[third anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[fourth anchor polypeptide]-[FGF-2], (e) [first anchor polypeptide]-[KGF]-[self-cleaving peptide]-[second anchor polypeptide]-[FGF-2]-[self-cleaving peptide]-[third anchor polypeptide]-[EGF]-[self-cleaving peptide]-[fourth anchor polypeptide]-[PDGF-BB], (f) [first anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[second anchor polypeptide]-[KGF]-[self-cleaving peptide]-[third anchor polypeptide]-[FGF-2]-[self-cleaving peptide]-[fourth anchor polypeptide]-[EGF], (g) [first anchor polypeptide]-[KGF]-[self-cleaving peptide]-[second anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[third anchor polypeptide]-[FGF-2]-[self-cleaving peptide]-[fourth anchor polypeptide]-[EGF], (h) [first anchor polypeptide]-[KGF]-[self-cleaving peptide]-[second anchor polypeptide]-[FGF2]-[self-cleaving peptide]-[third anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[fourth anchor polypeptide]-[EGF], (i) [first anchor polypeptide]-[EGF]-[self-cleaving peptide]-[second anchor polypeptide]-[KGF]-[self-cleaving peptide]-[third anchor polypeptide]-[FGF-2]-[self-cleaving peptide]-[fourth anchor polypeptide]-[PDGF-BB], (j) [first anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[second anchor polypeptide]-[EGF]-[self-cleaving peptide]-[third anchor polypeptide]-[KGF]-[self-cleaving peptide]-[fourth anchor polypeptide]-[FGF-2], (k) [first anchor polypeptide]-[EGF]-[self-cleaving peptide]-[second anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[third anchor polypeptide]-[KGF]-[self-cleaving peptide]-[fourth anchor polypeptide]-[FGF-2], (1) [first anchor polypeptide]-[EGF]-[self-cleaving peptide]-[second anchor polypeptide]-[KGF]-[self-cleaving peptide]-[third anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[fourth anchor polypeptide]-[FGF-2], (m) [first anchor polypeptide]-[EGF]-[self-cleaving peptide]-[second anchor polypeptide]-[FGF-2]-[self-cleaving peptide]-[third anchor polypeptide]-[KGF]-[self-cleaving peptide]-[fourth anchor polypeptide]-[PDGF-BB], (n) [first anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[second anchor polypeptide]-[EGF]-[self-cleaving peptide]-[third anchor polypeptide]-[FGF-2]-[self-cleaving peptide]-[fourth anchor polypeptide]-[KGF], (o) [first anchor polypeptide]-[EGF]-[self-cleaving peptide]-[second anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[third anchor polypeptide]-[FGF-2]-[self-cleaving peptide]-[fourth anchor polypeptide]-[KGF], (p) [first anchor polypeptide]-[EGF]-[self-cleaving peptide]-[second anchor polypeptide]-[FGF-2]-[self-cleaving peptide]-[third anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[fourth anchor polypeptide]-[KGF], (q) [first anchor polypeptide]-[FGF-2]-[self-cleaving peptide]-[second anchor polypeptide]-[KGF]-[self-cleaving peptide]-[third anchor polypeptide]-[EGF]-[self-cleaving peptide]-[fourth anchor polypeptide]-[PDGF-BB], (r) [first anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[second anchor polypeptide]-[FGF-2]-[self-cleaving peptide]-[third anchor polypeptide]-[KGF]-[self-cleaving peptide]-[fourth anchor polypeptide]-[EGF], (s) [first anchor polypeptide]-[FGF-2]-[self-cleaving peptide]-[second anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[third anchor polypeptide]-[KGF]-[self-cleaving peptide]-[fourth anchor polypeptide]-[EGF], (t) [first anchor polypeptide]-[FGF-2]-[self-cleaving peptide]-[second anchor polypeptide]-[KGF]-[self-cleaving peptide]-[third anchor polypeptide]-[PDGF-BB] [self-cleaving peptide]-[fourth anchor polypeptide]-[EGF], (u) [first anchor polypeptide]-[FGF-2]-[self-cleaving peptide]-[second anchor polypeptide]-[EGF]-[self-cleaving peptide]-[third anchor polypeptide]-[KGF]-[self-cleaving peptide]-[fourth anchor polypeptide]-[PDGF-BB], (v) [first anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[second anchor polypeptide]-[FGF-2]-[self-cleaving peptide]-[third anchor polypeptide]-[EGF]-[self-cleaving peptide]-[fourth anchor polypeptide]-[KGF], (w) [first anchor polypeptide]-[FGF-2]-[self-cleaving peptide]-[second anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[third anchor polypeptide]-[EGF]-[self-cleaving peptide]-[fourth anchor polypeptide]-[KGF], or (x) [first anchor polypeptide]-[FGF-2]-[self-cleaving peptide]-[second anchor polypeptide]-[EGF]-[self-cleaving peptide]-[third anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[fourth anchor polypeptide]-[KGF], wherein [ ] represents a single polypeptide, and ]-[ represents a linker or bond. In a preferred embodiment, the self-cleaving peptide is a 2A peptide, such as T2A, E2A, P2A or any combination thereof. For example, the self-cleaving peptide is a T2A peptide. In other embodiments, a single polynucleotide encodes at least one anchor polypeptide located at the C-terminus of the polypeptide to be presented on the surface of the exosomes. In some embodiments, each of the first, second, third, and fourth anchor polypeptides are different, and thus these anchor polypeptides can be located at the N-terminus or C-terminus of the polypeptides KGF, EGF, FGF-2, and optionally PDGF-BB. In a preferred embodiment, a single nucleic acid construct is provided comprising a polynucleotide encoding (a), (b), (c) and (d), wherein (a), (b), (c) and (d) are as defined above, and wherein each of the first, second, third and fourth anchor polypeptides comprises the TM3 domain of CD63, and the polynucleotide may comprise, from 5' to 3', a nucleotide fragment encoding one of the following: [TM3]-[KGF]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[FGF-2]-[T 2A]-[TM3]-[PDGF-BB], [TM3]-[PDGF-BB]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[FGF-2], [TM3]-[KGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[FGF-2], [TM3]-[KGF]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[FGF-2], [TM3]-[KGF]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[PDGF-BB], [TM3]-[PDGF-BB]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[EGF], [TM3]-[KGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[EGF], [TM3]-[KGF]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[EGF], [TM3]-[EGF]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[PDGF-BB], [TM3]-[PDGF-BB]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[FGF-2], [TM3]-[EGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[FGF-2], [TM3]-[EGF]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[PDGF-BB] -[T2A]-[TM3]-[FGF-2], [TM3]-[EGF]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[PDGF-BB], [TM3]-[PDGF-BB]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[KGF], [TM3]-[EGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[KGF], [TM3]-[EGF]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[KGF], [TM3]-[FGF-2]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[PDGF-BB], [TM3]-[PDGF-BB]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[EGF], [TM3]-[FGF-2]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[EGF], [TM3]-[FGF-2]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[EGF], [TM3]-[FGF-2]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[PDGF-BB], [TM3]-[PDGF-BB]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[KGF], [TM3]-[FGF-2]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[EG F]-[T2A]-[TM3]-[KGF], or [TM3]-[FGF-2]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[KGF] wherein [] represents a single polypeptide, and ]-[ represents a linker or bond; and wherein TM3 represents the TM3 domain of CD63; and T2A represents the self-cleaving peptide T2A. In this section, KGF, EGF, FGF-2 and PDGF-BB polypeptides have the meanings and preferred embodiments given above in connection with the section entitled Engineered Exosomes. In some embodiments, the polynucleotide encoding the KGF polypeptide has the nucleotide sequence shown in SEQ ID NO: 5 or a degenerate sequence thereof. In some embodiments, the polynucleotide encoding the EGF polypeptide has the nucleotide sequence shown in SEQ ID NO: 6 or a degenerate sequence thereof. In some embodiments, the polynucleotide encoding the FGF-2 polypeptide has the nucleotide sequence shown in SEQ ID NO: 7 or a degenerate sequence thereof. In some embodiments, the polynucleotide encoding the PDGF-BB polypeptide has the nucleotide sequence shown in SEQ ID NO: 8 or a degenerate sequence thereof. In some embodiments, the polynucleotide encoding the TM3 domain of CD63 has the nucleotide sequence shown in SEQ ID NO: 10 or a degenerate sequence thereof. Also provided is a vector comprising the nucleic acid construct. In some embodiments, the vector is a viral vector. In preferred embodiments, the vector is a lentiviral vector or an adeno-associated viral vector. The vectors provided herein facilitate integration of a polynucleotide encoding a polypeptide anchored to the membrane of the engineered exosome into the genome of the cell producing the exosome. Also provided are cells transduced with the vector. In preferred embodiments, the cells are not mesenchymal stem cells. In preferred embodiments, the cells are not stem cells. In preferred embodiments, the cells are non-stem cells, such as HEK293 or CHO cells. The present disclosure also provides a method for producing the engineered exosomes provided herein, comprising transducing the above-mentioned cells, such as HEK293 cells, with the above-mentioned vector; culturing the cells under conditions that allow the engineered exosomes to be secreted from the cells; and collecting and purifying the engineered exosomes. In some embodiments, the method comprises adapting the cells from serum-containing conditions to serum-free conditions during the culture period. The adaptation may comprise stepwise adaptation, i.e., gradually reducing the proportion of complete medium while increasing the proportion of serum-free medium. Sequence Listing AA : Amino acid sequence Example Example 1. Construction of engineered exosomes derived from stable cell lines. Materials and methods Materials: HEK293 cell line (human embryonic kidney 293 cells, CRL-1573 TM ) were purchased from ATCC and maintained in DMEM (high glucose) containing 10% (vol / vol) FBS, supplemented with 100 U / mL penicillin and 100 μg / mL streptomycin. CHO-K1 cell line (Chinese hamster ovary cell) was purchased from BeNa Culture Collection (Beijing, China). CHO-K1 cells were maintained in F-12K (31765035, Thermo Fisher Scientific, USA) containing 10% (vol / vol) FBS, supplemented with 100 U / mL penicillin and 100 μg / mL streptomycin. HSF (human skin fibroblasts) and HFF (human foreskin fibroblasts) were purchased from Mingzhou Biotechnology Co., Ltd. (Ningbo, China) and cultured in DMEM (high glucose) containing 10% FBS. The cells were incubated at 37°C with 5% CO The antibody used in this study was anti-CD63 antibody (Cat. No. MA5-32085, Invitrogen). pGOI Plasmid Construction: The amino acid sequences of all target genes (including KGF-1, EGF, FGF-2, and PDGF-BB) were derived from Uniprot, and the corresponding DNA sequences (see SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively) were synthesized by General Biotechnology (Chuzhou, China) using the plasmid pCDH-CMV-MCS-EF1a-GFP+BSD (System Biosciences). A T2A peptide (see SEQ ID NO: 13 and SEQ ID NO: 14) was used to dissociate all target proteins into individual proteins after translation. Four plasmids from the third-generation system (i.e., pGOI, pGag / pol, pRev, and pVSV-G) were used to generate lentivirus, with pCDH-CMV-MCS-EF1a-GFP+BSD (blasticidin resistance) serving as the lentiviral packaging plasmid. Lentiviruses were packaged by WZ Biotechnology (Jinan, China). Payload genes constructed in two pGOI plasmids: NO: 6: CD63-TM3-linker-KGF-1-T2A-CD63-TM3-linker-EGF-T2A-CD63-TM3-linker-FGF-2, wherein each CD63-TM3 is the TM3 domain of CD63, encoded by the DNA sequence shown in SEQ ID NO: 10, each linker is encoded by the DNA sequence shown in SEQ ID NO: 12, and each T2A is encoded by the DNA sequence shown in SEQ ID NO: 14. NO: 8: CD63-TM3-linker-KGF-1-T2A-CD63-TM3-linker-EGF-T2A-CD63-TM3-linker-FGF-2-T2A-CD63-TM3-linker-PDGF-BB, wherein each CD63-TM3 is the TM3 domain of CD63, encoded by the DNA sequence shown in SEQ ID NO: 10, each linker is encoded by the DNA sequence shown in SEQ ID NO: 12, and each T2A is encoded by the DNA sequence shown in SEQ ID NO: 14. Stable cell line generation: Stable HEK293 cell lines expressing target proteins (including KGF-1, EGF, FGF-2, and PDGF-BB) were generated by plasmid transfection or infection with the corresponding lentivirus. Forty-eight hours after transfection or infection, cells were selected by adding antibiotics, such as blasticidin (Solarbio Life Sciences), to a final concentration of 6 μg / ml. Single-cell colonies expressing green fluorescent protein (GFP) were selected and cultured in complete medium containing 6 μg / ml blasticidin. Stable cell lines were monitored for GFP and corresponding target protein expression. Adaptation of cell culture to SFM (serum-free medium): After three initial passages in FM (complete medium), HEK293 stable cell lines were adapted to serum-free culture starting from the 4th passage. Cells were subcultured using the medium composition in Table 1. To establish a fully serum-free culture, cells should be cultured in SFM (HyClone TM The cells were subcultured for at least three times in the presence of adenovirus (Cytiva Life Sciences, SH31193.02, peak expression). Table 1. Adaptation culture medium FM: complete medium; SFM: serum-free medium Exosome isolation: Stable cell lines were seeded in T150 flasks, cultured for 24 hours, rinsed extensively with PBS, and incubated in SFM for an additional 48 hours. Cells were removed by centrifugation at 300 × g for 10 minutes, and the cell-free extracellular medium containing exosomes was harvested. Dead cells and cellular debris were then removed by centrifugation at 10,000 × g for 30 minutes. Finally, the clarified supernatant was centrifuged twice at 100,000 × g for 70 minutes to pellet the exosomes. The exosome pellet was then resuspended. All centrifugation steps were performed at 4°C. FIG1 schematically shows a flow chart of a process for producing exosomes derived from HEK293 cells according to an exemplary embodiment of the present disclosure. Example 2. Characterization of exosomes Analysis of exosome particle concentration and size distribution Exosomes derived from stable cell lines NO:6 or NO:8 (transduced with pGOI NO:6 and NO:8, respectively) were analyzed for particle concentration and size distribution using NanoFCM (NanoFCM, Xiamen, China). NanoFCM analysis uses two single-photon counting avalanche photodiodes (APDs) to simultaneously detect side scatter (SSC) and fluorescence of individual particles. First, an exosome pellet was prepared for analysis. Then, 200 nm polystyrene beads conjugated with PE and AF488 fluorophores were used for particle concentration, and a silica nanosphere mixture (NanoFCM, Xiamen, China) was used for fineness distribution. The detector recorded the number of particles transmitted during a 1-minute interval during each test. Each sample was diluted to achieve an optimal particle count range of 3,000 to 9,000 particles / minute. Flow rate and side scatter intensity were converted to vesicle concentration and size using NanoFCM software (NanoFCM Professional V2.0). Figure 2A shows the particle size and particle concentration of exosomes derived from stable cell lines NO: 6 and 8 analyzed by NanoFCM. The exosomes of NO: 6 had an average particle size of 71.3 nm, and the exosomes of NO: 8 had an average particle size of 72.9 nm. Identify target proteins on exosomes by immunoblotting (WB) or ELISA To identify target protein expression on exosomes, purified exosomes were lysed with RIPA lysis buffer (Beyotime) supplemented with 1 mM protease inhibitor phenylmethylsulfonyl fluoride (PMSF; Beyotime) and phosphatase inhibitors (Beyotime), followed by heat denaturation, separation by SDS-PAGE, and transfer to a PVDF membrane (Millipore, MA, USA). Proteins were detected by incubation with a primary antibody against CD63 (an exosomal scaffold protein) followed by incubation with an HRP-conjugated secondary antibody (Invitrogen). The membrane was then visualized using an enhanced chemiluminescence reagent (Millipore, MA, USA). To measure target protein concentrations, purified exosomes were prepared and analyzed for target protein expression on exosomes using appropriate ELISA kits (Solarbio, SEKH-0051; SEKH-0220; SEKH-0052; SEKH-0290) according to the manufacturer's instructions. Transmission electron microscopy (TEM) analysis of exosomes TEM was used to confirm the presence of exosomes. Approximately 20 μL of exosomes were individually added to a copper grid. Any excess liquid was removed using filter paper, and the sample was negatively stained with 2% uranyl acetate for 30 seconds. The grid was rinsed with deionized water and allowed to dry overnight. The sample was then air-dried using an incandescent lamp and observed using an electron microscope (Hitachi, S-3000N). Figure 2B shows transmission electron microscopy (TEM) images of exosomes produced by stable cell lines NO:6 and 8. Figure 2C shows immunoblot analysis of exosomes from cell line NO:8 using an antibody against CD63-TM3 (a scaffold protein fused to the exosomes). As expected, the engineered exosome-associated target proteins KGF (KGF-1), FGF-2, PDGF-BB, and EGF were present in purified exosomes from stable cell line NO:8. The concentration of functional proteins in exosomes was measured using an ELISA kit. Table 2 shows the results of different protein concentrations measured by ELISA. Table 2. Concentration of effective factors determined by ELISA Example 3. Engineered exosomes exhibit enhanced cell proliferation activity on fibroblasts. Exosomes incubated with fibroblasts HSF or HFF HSF or HFF cells were incubated at a density of 4000 cells / well in a 96-well plate with various concentrations (0.5 μg, 1.0 μg, and 2.0 μg) of exosomes derived from NO:6 or NO:8 for 48 hours. KGF was used as a positive control. After 48 hours, cell viability was assessed using a cell counting kit (CCK8) assay (MCE, HY-K0301). Figure 3 shows that the engineered exosomes exhibited enhanced cell proliferation activity on both HSF and HFF fibroblasts. Moreover, the cell proliferation activity was enhanced in a dose-dependent manner, especially when HSF cells were treated with exosome NO:8. Example 4. Quantification of skin regeneration-related genes in fibroblasts after treatment with exosomes by RT-qPCR Total RNA was isolated from fibroblasts treated as described above using TRIzol LS reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. ® The tested RNA was reverse transcribed using the III One Step RT-qPCR SYBR Green Kit (Yeasen, HB220624) and measured by qPCR. RNA copy number was normalized to the cellular 18S rRNA copy number. Specific primers for genes involved in skin regeneration are shown in Table 3. Table 3. RT-qPCR primers in fibroblasts Figures 4A to 4E show the fold changes in gene copy number for the genes tested. Both NO: 6 exosome treatment and NO: 8 exosome treatment significantly increased the expression of proteins involved in extracellular matrix maintenance (such as collagen (COL1A2, COL3A1), fibronectin, and TIMP1) and inhibited the expression of MMP7. Example 5. Evaluation of anti-wrinkle efficacy in a zebrafish model Zebrafish embryos (4 dpf, dpf refers to days post fertilization) were prepared with yolk sacs. Exosomes (No. 6 or No. 8) were injected. Twenty-four hours after injection, gene expression analysis was performed using RT-qPCR to detect genes associated with skin regeneration (Hunter Biotech). Specific primers are shown in Table 4 below. Table 4. RT-qPCR primers in zebrafish Figure 5 demonstrates that engineered exosomes regulate genes involved in skin regeneration in a zebrafish model. Treatment with exosomes containing exosomes NO:8 significantly increased collagen (COL1A1B) synthesis in vivo and inhibited the expression of MMP9 and MMP16B, which degrade the extracellular matrix. Exosomes containing exosomes NO:6 significantly increased collagen (COL1A1B) synthesis in vivo and inhibited the expression of MMP9. Example 6. Engineered exosomes help downregulate melanin synthesis. In vitro melanin synthesis analysis Human melanocytes were cultured and incubated with exosomes from the NO:6 or NO:8 stable cell lines for 48 hours. The cell pellets were then used for melanin extraction, and melanin concentration was measured using a Fluorometric Melanin Assay Kit (ABN-KA6030, Abnova). Evaluating hyperpigmentation inhibitory efficacy in zebrafish models Zebrafish embryos (4 hpf, hpf refers to hours post fertilization) were cultured in 6-well plates with 3 ml of exosomes (No. 8) at 28°C for 45 hours. All wells (15 zebrafish embryos per well) were incubated in the dark. Images were then captured and analyzed under a dissecting microscope to assess the inhibitory efficacy against hyperpigmentation. The calculation was as follows: Whitening efficacy (%) = (Mock - No. 8) / Mock * 100%. Figures 6A and 6B show that engineered exosomal NO:8 significantly downregulated melanin synthesis in both melanocyte and zebrafish models. In the zebrafish model, when treated with NO:8, the whitening efficacy reached 48% (P<0.001). [Sequence Listing] See electronic copy of the sequence listing. TW202528538A_114100614_SEQ.xml

Claims

1. An engineered exosome comprising (a) a human KGF-1 peptide fused to a first anchoring peptide, (b) a human EGF peptide fused to a second anchoring peptide, and (c) a human FGF-2 peptide fused to a third anchoring peptide, wherein (a), (b), and (c) are anchored to the membrane of the exosome via the first, second, and third anchoring peptides, respectively, wherein the human KGF-1 peptide, the human EGF peptide, and the human FGF-2 peptide are exposed on the outer surface of the membrane of the exosome, and the anchoring peptide is selected from lamp2b, CD63, CD9, CD81, platelet-derived growth factor receptor (PDGFR), lactin C1C2 domain, vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative regulator (PTGFRN), glycosylphosphatidylinositol (GPI) anchors, lipid anchoring proteins, and any combination thereof.

2. The engineered exosome according to claim 1, wherein the engineered exosome further comprises (d) a human PDGF-BB peptide fused to a fourth anchoring peptide, and wherein the human PDGF-BB peptide is exposed on the outer surface of the membrane of the exosome, wherein the fourth anchoring peptide is selected from lamp2b, CD63, CD9, CD81, platelet-derived growth factor receptor (PDGFR), lactobacin C1C2 domain, vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative regulator (PTGFRN), glycosylphosphatidylinositol (GPI) anchors and lipid anchoring proteins, and any combination thereof.

3. The engineered exosome according to claim 1, wherein the human KGF-1 polypeptide comprises an amino acid sequence as shown in SEQ ID NO:

1.

4. The engineered exosome according to claim 1, wherein the human EGF polypeptide comprises an amino acid sequence as shown in SEQ ID NO:

2.

5. The engineered exosome according to claim 1, wherein the human FGF-2 polypeptide comprises an amino acid sequence as shown in SEQ ID NO:

3.

6. The engineered exosome according to claim 2, wherein the human PDGF-BB polypeptide is a human PDGF-BB dimer, and wherein the human PDGF-BB dimer comprises an amino acid sequence as shown in SEQ ID NO:

4.

7. The engineered exosome according to claim 1 or 2, wherein the anchoring polypeptide comprises full-length CD63 or truncated CD63 retaining the TM3 domain.

8. The engineered exosomes according to claim 2, wherein each of the anchored peptides is a TM3 domain of CD63.

9. The engineered exosome according to claim 8, wherein the TM3 domain of said CD63 comprises an amino acid sequence as shown in SEQ ID NO:

9.

10. The engineered exosome according to claim 2, wherein the human KGF-1 peptide, the human EGF peptide, the human FGF-2 peptide, and the human PDGF-BB peptide are fused to the C-terminus of the first, second, third, and fourth anchoring peptides, respectively, through peptide linkers.

11. The engineered exosome according to claim 10, wherein the peptide linker is (G4S)n, where n is an integer from 1 to 3.

12. The engineered exosomes according to claim 1, wherein the exosomes are not derived from stem cells.

13. The engineered exosomes according to claim 1, wherein the engineered exosomes: (i) enhance fibroblast proliferation, (ii) upregulate COL2A1, COL3A1, fibronectin, TIMP1 and / or COL1A1B genes, (iii) downregulate MMP7, MMP9 and / or MMP16B genes, and / or (iv) downregulate melanin synthesis.

14. A composition comprising an exosome according to any one of claims 1 to 13, and a carrier.

15. The composition according to claim 14, wherein the composition is formulated for topical or subcutaneous application.

16. The composition according to claim 14, wherein the composition is a cosmetic composition or a non-cosmetic composition.

17. The composition according to claim 14, wherein the composition is a pharmaceutical composition.

18. The composition according to claim 14, wherein the composition does not contain human KGF-1 peptide, human EGF peptide, human FGF-2 peptide or human PDGF-BB dimer that is not attached to the membrane of the exosome.

19. A nucleic acid construct comprising a polynucleotide encoding: (a) a human KGF-1 polypeptide fused to a first anchoring polypeptide, (b) a human EGF polypeptide fused to a second anchoring polypeptide, and (c) a human FGF-2 polypeptide fused to a third anchoring polypeptide; wherein any of the anchoring polypeptides is selected from lamp2b, CD63, CD9, CD81, platelet-derived growth factor receptor (PDGFR), lactobacin C1C2 domain, vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative regulator (PTGFRN), glycosylphosphatidylinositol (GPI) anchors and lipid anchoring proteins, and any combination thereof.

20. The nucleic acid construct according to claim 19, wherein the polynucleotide further encodes (d) a human PDGF-BB polypeptide, the human PDGF-BB polypeptide being fused with a fourth anchoring polypeptide, wherein the fourth anchoring polypeptide is selected from lamp2b, CD63, CD9, CD81, platelet-derived growth factor receptor (PDGFR), lactobacin C1C2 domain, vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative regulator (PTGFRN), glycosylphosphatidylinositol (GPI) anchors and lipid anchoring proteins, and any combination thereof.

21. The nucleic acid construct according to claim 20, wherein (i) the human KGF-1 polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 1; (ii) the human EGF polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 2; (iii) the human FGF-2 polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 3; (iv) the human PDGF-BB polypeptide is a human PDGF-BB dimer; the human PGDF-BB dimer comprises an amino acid sequence as shown in SEQ ID NO: 4; and (v) any of the anchoring polypeptides comprises a full-length CD63 or a truncated CD63 retaining the TM3 domain.

22. The nucleic acid construct according to claim 21, wherein (i) the polynucleotide encoding the human KGF-1 polypeptide has a nucleotide sequence as shown in SEQ ID NO: 5 or a degenerate sequence thereof; (ii) the polynucleotide encoding the human EGF polypeptide has a nucleotide sequence as shown in SEQ ID NO: 6 or a degenerate sequence thereof; (iii) the polynucleotide encoding the human FGF-2 polypeptide has a nucleotide sequence as shown in SEQ ID NO: 7 or a degenerate sequence thereof; (iv) the polynucleotide encoding the human PDGF-BB polypeptide has a nucleotide sequence as shown in SEQ ID NO: 8 or a degenerate sequence thereof; and / or (v) each of the first, second, third and fourth anchoring polypeptides is a TM3 domain of CD63.

23. The nucleic acid construct according to claim 22, wherein the polynucleotide encoding the TM3 domain of said CD63 has a nucleotide sequence as shown in SEQ ID NO: 10 or a degenerate sequence thereof.

24. The nucleic acid construct according to claim 20, wherein the polynucleotide is a single polynucleotide comprising a nucleotide fragment encoding the polypeptides (a), (b), (c), and (d), wherein each of the polypeptides (a), (b), (c), and (d) is linked to the anchoring polypeptide.

25. The nucleic acid construct according to claim 24, wherein the polypeptides (a), (b), (c) and (d) are separated by self-cleaving peptides.

26. The nucleic acid construct according to claim 25, wherein the self-cleaving peptide is T2A, E2A, P2A or any combination thereof.

27. A vector comprising a nucleic acid construct according to any one of claims 19 to 26.

28. A cell transduced using the vector according to claim 27, wherein the polynucleotide is integrated into the genome of the cell.

29. The cell according to claim 28, wherein the cell is not a stem cell.

30. The cell according to claim 28, wherein the cell is a mammalian cell.

31. Use of an engineered exosome according to any one of claims 1 to 13 in the manufacture of a medicament for skin rejuvenation.

32. The use as described in claim 31, wherein the skin rejuvenation includes treating any of the following: sun damage, sensitive skin, scars, wrinkles, dark circles, or hyperpigmentation.

33. The use as described in claim 32, wherein: (i) the scar is acne or stretch marks; (ii) the wrinkle is static or dynamic; and / or (iii) the hyperpigmentation is darkening of the skin or freckles, loose skin, uneven skin tone or dullness, or any combination thereof.

34. The use as described in claim 32, wherein the scar is a stretch mark, puberty mark, chickenpox mark, or wound.

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