Lifespan extension and anti-aging activation delivery system using three-dimensional bioprinting technology

Abstract

The present invention relates to a drug delivery system using three-dimensional bioprinting technology, and more specifically, to a sustained-release delivery system capable of time-release sleep-inducing components, sleep-maintaining components, anti-aging active components, and lifespan-extending active components synchronized with human biological rhythms through a core-shell-multilayer type delivery system having a multi-compartment structure, and a method for manufacturing the same. The present invention also relates to a delivery system that provides a lifespan-extending effect at the cellular level through telomere protection, inhibition of cellular aging, improvement of mitochondrial function, and maintenance of protein homeostasis.

Description

Technology Field

[0001] The present invention relates to a drug delivery system using three-dimensional bioprinting technology, and more specifically, to a sustained-release delivery system capable of time-release sleep-inducing components, sleep-maintaining components, anti-aging active components, and lifespan-extending active components synchronized with human biological rhythms through a core-shell-multilayer type delivery system having a multi-compartment structure, and a method for manufacturing the same. The present invention also relates to a delivery system that provides a lifespan-extending effect at the cellular level through telomere protection, inhibition of cellular aging, improvement of mitochondrial function, and maintenance of protein homeostasis. Background Technology

[0002] Aging is a complex biological process in which biological functions gradually decline over time. Characteristics of aging include genomic instability, telomere shortening, epigenetic changes, loss of protein homeostasis, dysregulation of trophic sensing, mitochondrial dysfunction, cellular senescence, stem cell depletion, and changes in intercellular communication. These characteristics of aging are interconnected, and intervention regarding one characteristic can affect others.

[0003] Telomeres are repetitive DNA sequences located at the ends of chromosomes that maintain chromosomal stability and prevent the loss of genetic information during DNA replication. Telomeres gradually shorten with cell division, and reaching a critical length induces cellular senescence or apoptosis. Telomerase is a reverse transcriptase that extends telomeres and exhibits activity in stem cells and germ cells. Cycloastrazenol, astragaloside IV, and TA-65 are known telomerase activators.

[0004] Cellular senescence is a state in which cells irreversibly cease the cell cycle and exhibit a characteristic phenotype. Senescent cells display an senescence-related secretory phenotype and secrete inflammatory cytokines, chemokines, growth factors, and matrix metalloproteinases. The accumulation of senescent cells is associated with tissue decline and aging-related diseases. Senolics are drugs that selectively eliminate senescent cells, including fisetin, quercetin, dasatinib, and nabitoclax.

[0005] 3D bioprinting is a technology that fabricates tissue structures by stacking biocompatible materials and cells in three dimensions. Bioprinting methods include extrusion-based, inkjet, and laser-assisted methods, with extrusion-based methods being the most widely used. Extrusion using a coaxial nozzle enables the simultaneous formation of core-shell structures.

[0006] Sleep is an essential process for maintaining homeostasis and physiological recovery in the human body. During sleep, the secretion of growth hormone, enhancement of immune function, memory consolidation, and cell regeneration proceed actively. Human sleep has a structure in which non-REM and REM sleep repeat in cycles of approximately 90 to 120 minutes, and 4 to 6 sleep cycles occur during the night. Slow-wave sleep is dominant in the early stages of sleep, while the proportion of REM sleep increases in the later stages.

[0007] There is a close link between sleep and lifespan extension. During sleep, the glymphatic system is activated to remove metabolic waste and amyloid beta from the brain. Sleep deprivation accelerates telomere shortening, increases oxidative stress, and promotes the secretion of inflammatory cytokines. Quality sleep is essential for cell repair and DNA repair, and sleep disorders have been reported to be associated with accelerated aging and shortened lifespan.

[0008] 3D bioprinting is a technology that fabricates tissue structures by stacking biocompatible materials and cells in three dimensions. Bioprinting methods include extrusion-based, inkjet, and laser-assisted methods, with extrusion-based methods being the most widely used. Extrusion using a coaxial nozzle enables the simultaneous formation of core-shell structures.

[0009] Methacrylated gelatin is a photocrosslinkable hydrogel precursor in which methacrylic groups are introduced by reacting methacrylic anhydride with the amine groups of gelatin. Methacrylated gelatin undergoes radical polymerization upon irradiation with ultraviolet or visible light to form a crosslinked network. The crosslinking density and mechanical properties can be controlled according to the degree of methacrylation.

[0010] Alginates are anionic polysaccharides extracted from brown algae. Alginates have a block copolymer structure of mannuronic acid and gluturonic acid and rapidly gel through ionic crosslinking with divalent cations. Alginate hydrogels exhibit excellent biocompatibility and are suitable for drug encapsulation and controlled release.

[0011] Nanocellulose is a nanomaterial obtained by breaking down cellulose to a nanoscale. Cellulose nanofibrils are fibrous materials with a diameter of 5 nm to 50 nm and a length of several micrometers. Through TEMPO oxidation, carboxyl groups are introduced to the surface of cellulose, improving water dispersibility. Nanocellulose can be utilized as a barrier layer that limits molecular diffusion by forming a dense network.

[0012] Poly-N-isopropylacrylamide is a representative temperature-sensitive polymer. Poly-N-isopropylacrylamide has a lower critical dissolution temperature of approximately 32°C, maintains a hydrophilic swelling state below the lower critical dissolution temperature, and transitions to a hydrophobic shrinkage state above the lower critical dissolution temperature. By copolymerizing with acrylic acid, the lower critical dissolution temperature can be controlled to a physiological temperature range.

[0013] Polydopamine is a melanin-like polymer formed by the oxidative self-polymerization of dopamine under alkaline conditions. Inspired by the adhesive proteins of shellfish, polydopamine exhibits strong adhesion to various substrate surfaces. The catechol / quinone redox cycle of polydopamine provides reactive oxygen species scavenging capabilities.

[0014] Cholesteryl esters are ester complexes of cholesterol and fatty acids. Cholesteryl esters exhibit thermotropic liquid crystal properties and undergo phase transitions between smectic, cholesteric, and isotropic phases depending on temperature. The phase transition temperature is controlled by the chain length and degree of unsaturation of the fatty acids.

[0015] Metal-phenolic networks are supramolecular coating layers formed by coordination bonds between polyvalent metal ions and polyphenols. Tannic acid is a natural polyphenol with 25 galloyl groups that forms coordination bonds with metal ions such as Fe, Ti, and Al. Metal-phenolic networks are formed by self-assembly via sequential immersion and possess a uniform thickness at the nanometer level.

[0016] Cyclodextrin is a cyclic oligosaccharide in which 6 to 8 glucopyranose units are linked by α-1,4-glycosidic bonds. Beta-cyclodextrin consists of 7 glucopyranose units and has hydrophobic cavities with a diameter of approximately 6.0 Å to 6.5 Å. Adamantane is a guest molecule with high binding affinity to the cavities of beta-cyclodextrin. The host-guest bond between beta-cyclodextrin and adamantane is utilized in the formation of supramolecular cross-linked structures.

[0017] Zeolitic imidazolate frameworks are a type of metal-organic framework composed of metal ions and imidazolate ligands. Zeolitic imidazolate framework-8 is formed from zinc ions and 2-methylimidazole and has a topology similar to the sodalite structure of zeolites. Zeolitic imidazolate framework-8 exhibits pH reactivity, being stable in acidic environments and decomposing in neutral to weakly acidic environments.

[0018] Ferrocene is an organometallic compound in which an iron ion is sandwiched between two cyclopentadienyl rings. Ferrocene exhibits reversible redox reactions, and electron transfer between Fe₂ and Fe₃ is easy. Polymers containing ferrocene can exhibit structural changes in response to the in vivo redox environment.

[0019] Freeze-drying is a drying method that removes moisture from a frozen sample by sublimation. Freeze-drying provides a dried form that allows for long-term storage while maintaining the stability of heat-sensitive biologically active substances. Freeze-dried hydrogels maintain a porous sponge structure and can be restored to their original form upon hydration.

[0020] Enteric coatings are coatings designed to provide resistance to gastric acid and dissolve in the intestines. Copolymers of methacrylic acid and methyl methacrylate have the characteristic of ionizing and dissolving at a pH of 5.5 or higher. Enteric coatings prevent drug degradation by gastric acid and enable intestinal-specific drug delivery.

[0021] Microneedles are micro-needle structures that penetrate the stratum corneum of the skin to deliver drugs to the dermis. The length of microneedles is generally 100 to 1,000 meters, and painless administration is possible because they do not reach the depth where pain receptors are present. Dissolvable microneedles release encapsulated drugs as they dissolve in moisture within the skin. Prior art literature

[65535] U.S. Patent Application Publication US2021 / 0093687 (April 1, 2021) U.S. Patent Publication US7914826 (March 29, 2011) J Polym Sci. Vol.60, pp.1700-1709 (2021.)Materials. Vol.13, No.5270 (2020.)Polymers. Vol.13, No.4367 (2021.) The problem to be solved

[0022] The problem that the present invention aims to solve is to provide a delivery vehicle capable of synchronizing with human biological rhythms and releasing sleep-inducing components, sleep-maintaining components, anti-aging active components, and life-extending active components at different times by forming a multi-compartment structure using 3D bioprinting technology.

[0023] Another problem that the present invention aims to solve is to provide a carrier in which a shell portion, an intermediate layer portion, a diffusion barrier layer, and a core portion form physically separated independent compartments, and the timing of component release is controlled by the material and structural characteristics of each compartment.

[0024] Another problem that the present invention aims to solve is to provide a delivery vehicle capable of simultaneously achieving improved sleep quality and extended lifespan by delivering telomere-protecting components, cell aging-inhibiting components, sirtuin-activating components, mitochondrial function-improving components, and autophagy-inducing components in synchronization with the cell repair and regeneration processes activated during sleep.

[0025] Another problem that the present invention aims to solve is to provide a carrier capable of releasing components in response to changes in the in vivo environment by including various stimulus-responsive components such as a temperature-sensitive polymer, a cholesteryl ester liquid crystal layer, a metal-phenolic network coating layer, a supramolecular cross-linked structure, a metal-organic framework nanoparticle, and a redox-responsive cross-linked structure.

[0026] Another problem that the present invention aims to solve is to provide a method for reproducibly manufacturing the multi-compartment structured carrier through a manufacturing method including coaxial nozzle printing, multi-stage crosslinking, and freeze-drying. means of solving the problem

[0027] According to one aspect of the present invention, a carrier having a multi-compartment structure formed by three-dimensional bioprinting may be provided, comprising: a shell portion having micropores formed on its surface and containing a sleep-inducing component and a hydrogel; an intermediate layer portion disposed inside the shell portion and containing a sleep-maintaining component and a biodegradable polymer; a diffusion barrier layer disposed inside the intermediate layer portion; and a core portion disposed inside the diffusion barrier layer and containing an anti-aging active component and a sustained-release matrix.

[0028] In one embodiment, the hydrogel of the shell portion may include one or more of alginate and hyaluronic acid, the biodegradable polymer of the intermediate layer may include one or more of polylactic-co-glycolic acid and cross-linked gelatin, the diffusion barrier layer may include nanocellulose, and the sustained-release matrix of the core portion may include one or more of methacrylated gelatin and polycaprolactone.

[0029] In one embodiment, the sleep-inducing component may include one or more of melatonin, GABA, and L-theanine, the sleep-maintaining component may include one or more of a magnesium compound, a herbal extract, and sustained-release melatonin, and the anti-aging active component may include one or more of nicotinamide mononucleotide, exosomes, resveratrol, and antioxidant peptides.

[0030] In one embodiment, the shell portion may have a fractal structure or a gyroid structure, the core portion may have a spherical shape, and the carrier may include a three-dimensional lattice structure or a microchannel inside.

[0031] In one embodiment, one or more of the shell portion, the intermediate layer portion, and the core portion may comprise a temperature-sensitive polymer, and the temperature-sensitive polymer may comprise poly-N-isopropylacrylamide or a copolymer thereof. The temperature-sensitive polymer may be a copolymer of N-isopropylacrylamide and acrylic acid and may have a lower critical melting temperature of 32 to 34°C.

[0032] In one embodiment, the micropores of the shell portion may have a size of 50 to 200 Ìm, and the porosity of the shell portion may be 40 to 70%. The thickness of the shell portion may be 200 to 1,000 Ìm, the thickness of the intermediate layer portion may be 500 to 2,000 Ìm, the thickness of the diffusion barrier layer may be 10 to 100 Ìm, and the diameter of the core portion may be 1,000 to 5,000 Ìm.

[0033] In one embodiment, the alginate may have a concentration of 1 to 4 weight% and may be sodium alginate having a mannuronic acid / gluuronic acid ratio of 1.0 to 1.5. The methacrylated gelatin may have a degree of methacrylation of 40 to 80% and may be included in the core portion at a concentration of 5 to 15 weight%. The nanocellulose may be TEMPO oxidized cellulose nanofibrils and may have a diameter of 5 to 20 nm.

[0034] In one embodiment, based on the total weight of the carrier, the content of melatonin may be 0.1 to 5.0 weight%, the content of GABA may be 1.0 to 10.0 weight%, and the content of nicotinamide mononucleotide may be 0.5 to 10.0 weight%.

[0035] In one embodiment, the polydopamine coating layer formed on the outer surface of the shell portion may be further included. The polydopamine coating layer may have a thickness of 10 to 50 nm. The polydopamine coating layer may include a copolymer of dopamine and 5,6-dihydroxyindole-2-carboxylic acid. The polydopamine coating layer may provide improved mucosal adhesion and an active oxygen species scavenging effect.

[0036] In one embodiment, a cholesteryl ester liquid crystal layer disposed between the shell portion and the intermediate layer portion may be further included. The cholesteryl ester liquid crystal layer may include a mixture of cholesteryl oleate and cholesteryl nonanoate, and the mixture may have a phase transition temperature of 35 to 37°C. The cholesteryl oleate and the cholesteryl nonanoate may be mixed in a weight ratio of 7:3 to 9:1, and the thickness of the cholesteryl ester liquid crystal layer may be 20 to 100°C. The liquid crystal layer may provide an emission gating effect due to a change in viscosity around the phase transition temperature.

[0037] In one embodiment, the diffusion barrier layer may include a metal-phenolic network coating layer formed by the coordination bonding of metal ions and tannic acid. The metal ions are Ti 4+ , Fe 3+ and Al 3+ It may include one or more of the above, and the thickness of the metal-phenolic network coating layer may be 10 to 30 nm.

[0038] In one embodiment, the core portion may comprise methacrylated gelatin conjugated with beta-cyclodextrin and multi-arm polyethylene glycol having adamantan ends, and the methacrylated gelatin conjugated with beta-cyclodextrin and the multi-arm polyethylene glycol-adamantan may form a supramolecular cross-linked structure by host-guest bonding. The beta-cyclodextrin conjugation rate of the methacrylated gelatin conjugated with beta-cyclodextrin may be 5 to 15 mol%, and the multi-arm polyethylene glycol-adamantan may be 4-arm polyethylene glycol-adamantan carboxylic acid and may have a molecular weight of 8,000 to 12,000 Da.

[0039] In one embodiment, the intermediate layer may comprise 1-aminoadamantan or a pharmaceutically acceptable salt thereof. The content of the 1-aminoadamantan or its salt may be 0.1 to 1.0 weight% based on the total weight of the intermediate layer. The 1-aminoadamantan may trigger the disintegration of the supramolecular cross-linked structure as a competitive guest molecule.

[0040] In one embodiment, the metal-organic framework nanoparticles dispersed within the core portion may be further included, and the metal-organic framework nanoparticles may be zeolic imidazolate framework-8 formed from zinc ions and 2-methylimidazole, and some or all of the anti-aging active ingredients may be supported on the zeolic imidazolate framework-8 nanoparticles. The zeolic imidazolate framework-8 nanoparticles may have a particle size of 50 to 200 nm and a polydispersity index of 0.3 or less, and the drug loading rate of the zeolic imidazolate framework-8 nanoparticles may be 10 to 30 weight%.

[0041] In one embodiment, the core portion may include a redox-reactive cross-linked structure formed by Schiff base bonding of ferrocene carboxyaldehyde and branched polyethyleneimine. The branched polyethyleneimine may have a molecular weight of 20,000 to 30,000 Da, and the conjugation rate of the ferrocene carboxyaldehyde may be 3 to 10 mol%. The redox-reactive cross-linked structure may react with glutathione in vivo to provide cross-link disintegration and component release.

[0042] In one embodiment, the core portion may further include a lifespan-extending active ingredient, and the lifespan-extending active ingredient may include one or more of a telomere-protecting ingredient, a senolic ingredient, an autophagy-inducing ingredient, an AMPK-activating ingredient, and a mitochondrial-targeted antioxidant ingredient.

[0043] In one embodiment, the telomere protective component may include one or more of cycloastragenol, astragaloside IV, and astragalus extract. The content of cycloastragenol may be 0.01 to 1.0 weight% based on the total weight of the carrier, and the content of astragaloside IV may be 0.1 to 5.0 weight% based on the total weight of the carrier.

[0044] In one embodiment, the senolitic component may include one or more of fisetin, quercetin, and combinations thereof. The content of fisetin may be 0.5 to 10.0 weight% based on the total weight of the carrier, and the content of quercetin may be 0.5 to 10.0 weight% based on the total weight of the carrier.

[0045] In one embodiment, the autophagy-inducing component may include one or more of spermidine, trehalose, and urolitin A. The content of spermidine may be 0.1 to 5.0 weight% based on the total weight of the carrier, and the content of trehalose may be 1.0 to 15.0 weight% based on the total weight of the carrier.

[0046] In one embodiment, the AMPK activating component may include one or more of berberine and ginsenoside Rg3. The content of the berberine may be 0.5 to 5.0 weight% based on the total weight of the carrier.

[0047] In one embodiment, the mitochondrial-targeted antioxidant component may include one or more of coenzyme Q10, pyrroloquinoline quinone, and alpha-lipoic acid. The content of coenzyme Q10 may be 0.5 to 5.0 weight% based on the total weight of the carrier, and the content of pyrroloquinoline quinone may be 0.01 to 0.5 weight% based on the total weight of the carrier.

[0048] In one embodiment, the lifespan-extending active ingredient may further include one or more of glycine and taurine. The content of glycine may be 1.0 to 10.0 weight% based on the total weight of the carrier, and the content of taurine may be 1.0 to 10.0 weight% based on the total weight of the carrier.

[0049] In one embodiment, the lifespan-extending active ingredient may further include alpha-ketoglutarate or a salt thereof. The content of the alpha-ketoglutarate or a salt thereof may be 0.5 to 5.0 weight% based on the total weight of the carrier.

[0050] In one embodiment, the core portion may include nicotinamide mononucleotide and resveratrol as the anti-aging active ingredients, and spermidine and fisetin as the lifespan-extending active ingredients. The combination may provide a lifespan-extending effect through multiple pathways of NAD+ supplementation, sirtuin activation, autophagy induction, and senescent cell removal. The total content of the anti-aging active ingredients and the lifespan-extending active ingredients in the core portion may be 5.0 to 25.0 weight% based on the total weight of the delivery vehicle.

[0051] In one embodiment, the intermediate layer may further include a NAD+ precursor auxiliary component, and the NAD+ precursor auxiliary component may include one or more of nicotinamide riboside and tryptophan. The NAD+ precursor auxiliary component may be released prior to the release of nicotinamide mononucleotide from the core portion to prepare a NAD+ synthesis pathway.

[0052] In one embodiment, the delivery body may have the form of an oral multi-pellet, a skin-attachable microneedle patch, and an in-body insertable scaffold.

[0053] According to another aspect of the present invention, a method for manufacturing a carrier having a multi-compartment structure based on three-dimensional bioprinting may be provided, comprising: a bioink preparation step of preparing a first bioink comprising an anti-aging active ingredient and a polymer for forming a sustained-release matrix, and a second bioink comprising a sleep-inducing ingredient and a polymer for forming a hydrogel; a coaxial nozzle printing step of forming a core-shell structure by simultaneously extruding the first bioink to the inside and the second bioink to the outside using a coaxial nozzle; a multi-stage crosslinking step of sequentially performing ionic crosslinking and photocrosslinking on the core-shell structure; and a freeze-drying step of freeze-drying the crosslinked structure.

[0054] In one embodiment, the coaxial nozzle printing step may include a step of adjusting the ratio of the inner diameter to the outer diameter of the coaxial nozzle so that the thickness ratio of the core portion and the shell portion of the core-shell structure is in the range of 1:2 to 1:5.

[0055] In one embodiment, the first bio-ink may have a viscosity of 10,000 to 50,000 cP at 25°C, and the second bio-ink may have a viscosity of 1,000 to 5,000 cP at 25°C.

[0056] In one embodiment, the coaxial nozzle printing step may include the step of using a coaxial nozzle comprising an inner nozzle having an inner diameter of 200 to 500 Ìm and an outer nozzle having an inner diameter of 500 to 1,000 Ìm. The coaxial nozzle printing step may be performed at a temperature of 20 to 37°C at a nozzle movement speed of 5 to 30 mm / s.

[0057] In one embodiment, the multi-stage crosslinking step comprises: an ionic crosslinking step of immersing the core-shell structure in an aqueous calcium chloride solution of 0.5 to 2.0 weight% for 1 to 10 minutes; and 5 to 20 mW / cm² in the presence of a photoinitiator. 2 The method may sequentially include a photocrosslinking step of irradiating ultraviolet rays with an intensity of 30 seconds to 5 minutes. The photoinitiator may be 2-hydroxy-4'-hydroxyethoxy-2-methylpropiophenone or lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and the concentration of the photoinitiator may be 0.05 to 0.5 weight%.

[0058] In one embodiment, the freeze-drying step may include a pre-freezing step of pre-freezing the cross-linked structure at -70 to -80°C; and a sublimation drying step of drying for 24 to 48 hours at a pressure of 0.1 mbar or less and a temperature of -40 to -50°C.

[0059] In one embodiment, after the freeze-drying step, an enteric coating step of forming an enteric coating layer on the surface of the carrier may be further included.

[0060] In one embodiment, after the coaxial nozzle printing step, a metal-phenolic network coating step may be further included, wherein a metal ion solution and a tannic acid solution are sequentially immersed on the surface of the core portion of the core-shell structure to form a metal-phenolic network coating layer. The metal-phenolic network coating step may include: a step of immersing the surface of the core portion in a metal ion solution containing titanium butoxide; a step of immersing it in an aqueous tannic acid solution after withdrawing it from the metal ion solution; and a step of repeating the immersion in the metal ion solution and the immersion in the aqueous tannic acid solution 3 to 5 times. The metal ion solution may be a solution in which 0.05 to 0.2 mg / mL of titanium butoxide is dissolved in a 1:1 volume ratio mixed solvent of ethanol and water, and the aqueous tannic acid solution may contain 0.2 to 0.6 mg / mL of tannic acid.

[0061] In one embodiment, after the freeze-drying step, the method may further include a polydopamine coating step of forming a polydopamine coating layer by immersing the carrier in a coating solution comprising dopamine hydrochloride and a buffer. The polydopamine coating step may include the step of immersing the carrier in a Tris-hydrochloride buffer containing 1.5 to 2.5 mg / mL of dopamine hydrochloride and adjusted to a pH of 8.0 to 9.0; and the step of stirring in an open container for 2 to 6 hours.

[0062] In one embodiment, the coaxial nozzle printing step may include the step of forming a liquid crystal layer by applying a cholesteryl ester mixture to the inner surface of the shell portion after forming the shell portion. The cholesteryl ester mixture may be applied in a molten state at 50 to 70°C, and after application, slowly cooled to 20 to 30°C to form a smectic liquid crystal phase.

[0063] In one embodiment, the bio-ink preparation step may include a drug loading step of loading the anti-aging active ingredient onto zeolic imidazolate framework-8 nanoparticles; and a nanoparticle dispersion step of dispersing the zeolic imidazolate framework-8 nanoparticles loaded with the drug into the first bio-ink. The drug loading step may include a step of dissolving Zn(NO3)2O6H2O, 2-methylimidazole, and the anti-aging active ingredient in methanol; a step of letting the solution stand at room temperature for 20 to 28 hours; and a step of centrifuging to obtain zeolic imidazolate framework-8 nanoparticles loaded with the drug.

[0064] In one embodiment, the bio-ink preparation step may further include the step of adding a lifespan-extending active ingredient to the first bio-ink. The lifespan-extending active ingredient may include one or more of a telomere-protecting ingredient, a senolic ingredient, an autophagy-inducing ingredient, an AMPK-activating ingredient, and a mitochondrial-targeted antioxidant ingredient. Effects of the invention

[0065] The delivery vehicle according to the present invention has a multi-compartment structure formed by three-dimensional bioprinting, thereby enabling the release of sleep-inducing components, sleep-maintaining components, anti-aging active components, and life-extending active components at different times. Due to the time-lag release characteristics, it is possible to deliver components synchronized with the human sleep cycle and circadian rhythm.

[0066] The carrier according to the present invention can prevent interaction between components enclosed in each compartment by the physical separation of the shell portion, the intermediate layer portion, the diffusion barrier layer, and the core portion, and can improve the stability of each component.

[0067] The delivery vehicle according to the present invention can simultaneously achieve improved sleep quality and anti-aging effects by delivering life-extending active ingredients in synchronization with the glymphatic system, growth hormone secretion, DNA repair, and cell regeneration processes that are activated during sleep.

[0068] The carrier according to the present invention can contribute to delaying telomere shortening due to cell division and extending the replication lifespan of cells by including a telomere-protecting component.

[0069] The carrier according to the present invention can contribute to the selective removal of accumulated senescent cells by including a senolic component and can contribute to alleviating chronic inflammation caused by aging-related secretory phenotypes.

[0070] The delivery vehicle according to the present invention can increase intracellular nicotinamide adenine dinucleotide levels by including nicotinamide mononucleotide or nicotinamide riboside and contribute to metabolic improvement through sirtuin activation.

[0071] The delivery vehicle according to the present invention can contribute to the removal of damaged proteins and mitochondria and the maintenance of protein homeostasis by including an autophagy-inducing component.

[0072] The carrier according to the present invention comprises a temperature-sensitive polymer, thereby allowing the release rate of components to be controlled in response to changes in body temperature. Due to the temperature responsiveness, release control synchronized with minute changes in body temperature during sleep may be possible.

[0073] The carrier according to the present invention may have improved mucosal adhesion by including a polydopamine coating layer and may provide an active oxygen species scavenging effect.

[0074] The carrier according to the present invention includes a cholesteryl ester liquid crystal layer, thereby enabling emission gating by a change in viscosity around the phase transition temperature. Due to the gating effect, emission automatically synchronized with the biological rhythm without external stimulation may be possible.

[0075] The carrier according to the present invention may form a uniform diffusion barrier at the nano level by including a metal-phenolic network coating layer, and may provide an additional antioxidant effect by tannic acid.

[0076] The carrier according to the present invention comprises a beta-cyclodextrin-adamantan supramolecular crosslinking structure, thereby enabling programmed crosslinking de-crosslinking and component release by competitive guest molecules.

[0077] The delivery vehicle according to the present invention may have an increased drug loading capacity by including zeolic imidazolate framework-8 nanoparticles, and may enable intestinal-specific release due to pH responsiveness.

[0078] The carrier according to the present invention may enable the release of a component in response to an in vivo redox environment by including a ferrocene-polyethyleneimine redox crosslinking structure.

[0079] The manufacturing method according to the present invention can simultaneously form a core-shell structure by coaxial nozzle printing and can independently control the decomposition rate of each section by multi-stage crosslinking. Brief explanation of the drawing

[0080] FIG. 1 is a flowchart illustrating a method for manufacturing a carrier according to one embodiment of the present invention. Specific details for implementing the invention

[0081] In one aspect of the present invention, a carrier according to the present invention may have a multi-compartment structure formed by three-dimensional bioprinting. The multi-compartment structure may include a shell portion, an intermediate layer portion, a diffusion barrier layer, and a core portion, and each compartment may be physically separated to form an independent space. The multi-compartment structure may have a core-shell-multilayer form, and each compartment may be arranged concentrically.

[0082] In one embodiment, the shell portion may be located in the outermost region of the carrier. The shell portion may include a sleep-inducing component and a hydrogel. The sleep-inducing component may exist in a dispersed or dissolved state within the hydrogel. Micropores may be formed on the surface of the shell portion, and the micropores may provide a pathway for body fluids to penetrate into the shell portion. The hydrogel may have a low cross-linking density and may hydrate, swell, or dissolve in an in vivo environment. Upon administration into the body, the shell portion may rapidly release the sleep-inducing component, thereby rapidly increasing the concentration of the sleep-inducing component in the blood to induce sleep.

[0083] In one embodiment, the intermediate layer may be disposed on the inner side of the shell portion. The intermediate layer may include a sleep-maintaining component and a biodegradable polymer. The biodegradable polymer may have a higher crosslinking density than the hydrogel of the shell portion and may have hydrophobic properties. The intermediate layer may undergo degradation after the shell portion has dissolved, and the sleep-maintaining component may be released at a time after the sleep-inducing component of the shell portion has been released. The intermediate layer may contribute to preventing night awakenings during the mid-sleep phase and extending the depth of sleep.

[0084] In one embodiment, the diffusion barrier layer may be disposed on the inner side of the intermediate layer. The diffusion barrier layer may have a thin film form surrounding the core portion. The diffusion barrier layer can control the diffusion of a component contained in the core portion toward the intermediate layer. The diffusion barrier layer can protect the core portion for a certain period of time even after the shell portion and the intermediate layer portion have decomposed, and can delay the timing of component release from the core portion.

[0085] In one embodiment, the core portion may be disposed inside the diffusion barrier layer to form a central region of the carrier. The core portion may include an anti-aging active ingredient and a sustained-release matrix. The anti-aging active ingredient may exist in a dispersed or encapsulated state within the sustained-release matrix. The sustained-release matrix may have a dense polymer network structure and may be composed of a material with the slowest biodegradation rate. The release of the anti-aging active ingredient from the core portion may proceed intensively during the period of active cell regeneration in the latter part of sleep.

[0086] For example, the sleep-inducing component from the shell portion may be released at least 80% within 30 minutes after administration, the sleep-maintaining component from the middle layer may be released 2 to 4 hours after administration, and the anti-aging active component from the core portion may be released 4 to 8 hours after administration. The time-lag release characteristics may be synchronized with the human sleep cycle and hormone secretion time.

[0087] In one aspect of the present invention, the shell portion, the intermediate layer portion, the diffusion barrier layer, and the core portion may each be composed of different materials. The materials of each section may differ in their decomposition rate, crosslinking density, and hydrophilic / hydrophobic characteristics, and accordingly, the release timing of the components contained in each section may be controlled.

[0088] In one embodiment, the hydrogel of the shell portion may comprise one or more of alginate and hyaluronic acid. The alginate may be a natural polysaccharide derived from seaweed and may gradually disintegrate by reacting with ions in the body. The hyaluronic acid may be a glycosaminoglycan with excellent biocompatibility and may enable rapid hydration and dissolution due to its high water retention capacity.

[0089] In one embodiment, the biodegradable polymer of the intermediate layer may comprise one or more of polylactic-co-glycolic acid and cross-linked gelatin. The polylactic-co-glycolic acid may be a hydrophobic polymer and may degrade by hydrolysis. The cross-linked gelatin may have a high cross-linking density and may have a slower degradation rate than the hydrogel of the shell.

[0090] In one embodiment, the diffusion barrier layer may comprise nanocellulose. The nanocellulose may be cellulose fibers having a diameter on a nanometer scale and may form a dense network structure to restrict the diffusion of molecules.

[0091] In one embodiment, the sustained-release matrix of the core portion may comprise one or more of methacrylated gelatin and polycaprolactone. The methacrylated gelatin may be photocrosslinked by irradiation with ultraviolet or visible light, and the crosslinking density may be controlled according to the irradiation intensity. The polycaprolactone may be a biodegradable polyester and may degrade slowly over several months. The sustained-release matrix may form a rigid lattice structure.

[0092] For example, the shell portion may be composed of a mixed hydrogel of alginate and hyaluronic acid, the intermediate layer may be composed of a mixture of polylactic-co-glycolic acid and cross-linked gelatin, and the core portion may be composed of a composite matrix of methacrylated gelatin and polycaprolactone.

[0093] In one aspect of the present invention, the active ingredients included in the carrier may be classified into a sleep-inducing ingredient, a sleep-maintaining ingredient, and an anti-aging active ingredient. Each of the active ingredients may be placed in different compartments of the carrier and, accordingly, released at different times.

[0094] In one embodiment, the sleep-inducing component may include one or more of melatonin, GABA, and L-theanine. The melatonin may be a hormone secreted by the pineal gland and may be involved in regulating circadian rhythms. The GABA may be an inhibitory neurotransmitter and may induce a state of relaxation by inhibiting the excitation of the nervous system. The L-theanine may be an amino acid derived from tea leaves and may provide a relaxation effect through an increase in alpha waves.

[0095] In one embodiment, the sleep maintenance component may include one or more of a magnesium compound, a herbal extract, and sustained-release melatonin. The magnesium compound may be magnesium oxide, magnesium citrate, or magnesium glycinate, and may contribute to muscle relaxation and nerve stabilization. The herbal extract may be valerian extract, chamomile extract, or lavender extract. The sustained-release melatonin may be formulated to be released more slowly than the melatonin in the shell portion.

[0096] In one embodiment, the anti-aging active ingredient may include one or more of nicotinamide mononucleotide, exosomes, resveratrol, and antioxidant peptides. The nicotinamide mononucleotide may be a NAD+ precursor and may be involved in cellular energy metabolism and DNA repair. The exosomes may be stem cell-derived extracellular vesicles and may contribute to intercellular signaling and tissue regeneration. The resveratrol may be a polyphenol compound and may delay cellular aging through sirtuin activation. The antioxidant peptide may be glutathione or carnosine.

[0097] For example, the shell portion may contain a combination of melatonin, GABA, and L-theanine, the middle layer may contain a combination of a magnesium compound and sustained-release melatonin, and the core portion may contain a combination of nicotinamide mononucleotide, exosomes, and antioxidant peptides.

[0098] In one aspect of the present invention, the carrier may have a structural form with a surface area and volume ratio controlled. The structural form may affect the component release rate from each compartment.

[0099] In one embodiment, the shell portion may have a fractal structure or a gyroid structure. The fractal structure may include a repeating pattern having self-similarity and may increase the surface area. The gyroid structure may be a three-dimensional periodic structure having a minimum surface area and may provide a complex porous structure that can only be realized by 3D printing. Maximizing the surface area of ​​the shell portion may contribute to the rapid dissolution and release of the water-inducing component.

[0100] In one embodiment, the core portion may have a spherical shape. The spherical shape may provide the minimum surface area relative to the same volume. Minimizing the surface area of ​​the core portion may reduce the erosion and release rate of the anti-aging active ingredient and enable delayed release until the latter part of the water surface.

[0101] In one embodiment, the carrier may include a three-dimensional lattice structure or microchannels inside. The three-dimensional lattice structure may form regular empty spaces inside the carrier. The microchannels may be printed in a shape similar to capillaries and may provide a pathway for the efficient delivery of active ingredients into the bloodstream. The size and spacing of the microchannels may be adjusted to control the penetration rate of body fluids.

[0102] For example, the shell portion may have a gyroid structure to maximize the surface area to volume ratio, the core portion may have a shape close to a sphere to minimize the surface area to volume ratio, and microchannels with a diameter of 100 to 500 Ìm may be formed in a network form inside the carrier.

[0103] In one aspect of the present invention, the carrier may include a temperature-sensitive polymer. The physical properties of the temperature-sensitive polymer may change in response to changes in body temperature.

[0104] In one embodiment, one or more of the shell portion, the intermediate layer portion, and the core portion may comprise a temperature-sensitive polymer. The temperature-sensitive polymer may comprise poly-N-isopropylacrylamide or a copolymer thereof. The poly-N-isopropylacrylamide may have a lower critical dissolution temperature, exhibit hydrophilicity below the lower critical dissolution temperature, and exhibit hydrophobicity above the lower critical dissolution temperature.

[0105] For example, during sleep, peripheral body temperature may decrease during the early stages of sleep and may increase during the early morning hours. The temperature-sensitive polymer may contract or expand in response to minute changes in body temperature, and accordingly, the release rate of the active ingredient may be controlled.

[0106] FIG. 1 is a flowchart illustrating a method for manufacturing a carrier according to the present invention. Referring to FIG. 1, the manufacturing method may include a bio-ink preparation step (S10), a coaxial nozzle printing step (S20), a multi-stage crosslinking step (S30), and a freeze-drying step (S40). The manufacturing method may optionally further include an enteric coating step (S50). Each of the steps may be performed sequentially.

[0107] In one aspect of the present invention, the present invention may relate to a method for manufacturing a carrier having a multi-compartment structure based on three-dimensional bioprinting. As illustrated in FIG. 1, the manufacturing method may include a bio-ink preparation step (S10), a coaxial nozzle printing step (S20), a multi-stage crosslinking step (S30), and a freeze-drying step (S40).

[0108] In one embodiment, the bio-ink preparation step (S10) may include preparing a first bio-ink and a second bio-ink. The first bio-ink may include an anti-aging active ingredient and a polymer for forming a sustained-release matrix. The second bio-ink may include a sleep-inducing ingredient and a polymer for forming a hydrogel. The first bio-ink may have a higher viscosity than the second bio-ink, thereby ensuring structural stability during printing.

[0109] In one embodiment, the coaxial nozzle printing step (S20) may include forming a core-shell structure by simultaneously discharging the first bio-ink to the inside and the second bio-ink to the outside using a coaxial nozzle. The coaxial nozzle may operate in a coaxial extrusion manner that enables a dual discharging process. The ink for the core may be discharged from the center of the coaxial nozzle, and the ink for the shell may be discharged from the outer part of the coaxial nozzle.

[0110] In one embodiment, the multi-stage crosslinking step (S30) may include sequentially performing ionic crosslinking and photocrosslinking on the core-shell structure. The ionic crosslinking can fix the shape of the shell portion. The photocrosslinking can densify the polymer network inside the core portion to impart sustained-release properties.

[0111] In one embodiment, the freeze-drying step (S40) may include freeze-drying the cross-linked structure. The freeze-drying can remove moisture while preserving the activity of the active ingredient. After freeze-drying, the carrier can maintain a porous framework and can be stored for a long period.

[0112] For example, in the bio-ink preparation step (S10), the first bio-ink may be prepared by mixing nicotinamide mononucleotide and exosomes with methacrylated gelatin, and the second bio-ink may be prepared by mixing melatonin, GABA and L-theanine with alginate.

[0113] In one aspect of the present invention, the coaxial nozzle printing step (S20) may include adjusting the thickness ratio of the core portion and the shell portion of the core-shell structure. The thickness ratio may be controlled by adjusting the ratio of the inner diameter to the outer diameter of the coaxial nozzle.

[0114] In one embodiment, the thickness ratio of the core portion and the shell portion may be in the range of 1:2 to 1:5. As the thickness ratio is adjusted within the above range, the delay time until the component is released from the core portion after the shell portion is dissolved can be adjusted.

[0115] For example, when the thickness ratio is 1:2, the release delay time from the core portion may be relatively short, and when the thickness ratio is 1:5, the release delay time may be relatively long.

[0116] In one aspect of the present invention, the manufacturing method may further include an enteric coating step (S50) after the freeze-drying step (S40). The enteric coating step (S50) may include forming an enteric coating layer on the surface of the carrier.

[0117] In one embodiment, the enteric coating layer may have resistance to gastric acid and may be configured to dissolve in the intestine. The enteric coating layer may protect the shell portion while the delivery vehicle passes through the stomach upon oral administration, and may dissolve after reaching the intestine to initiate the dissolution of the shell portion.

[0118] For example, the enteric coating layer may include a copolymer of methacrylic acid and methyl methacrylate and may have the characteristic of being soluble at pH 5.5 or higher.

[0119] In one aspect of the present invention, the delivery vehicle may have various end forms. The end form may be selected depending on the administration route and the site of application.

[0120] In one embodiment, the delivery body may have the form of an oral multi-pellet. The oral multi-pellet may be in the form of a plurality of small capsules produced by 3D printing and may be taken orally.

[0121] In one embodiment, the delivery body may have the form of a skin-attachable microneedle patch. The microneedle patch may include a plurality of microneedles, and layers may be separated according to the length of the microneedles. The microneedle patch can deliver ingredients to the dermis layer of the skin in stages.

[0122] In one embodiment, the delivery body may have the form of an implantable scaffold. The scaffold may be implanted into the body during plastic surgery or treatment and may function as a support for managing sleep cycles and aging over the long term.

[0123] For example, the oral multi-pellet may be spherical or elliptical with a diameter of 3 to 15 mm, the length of the microneedle may be 100 to 1,000 m, and the scaffold may be printed in a shape suitable for the application site.

[0124] In one aspect of the present invention, the micropores formed on the surface of the shell portion may have a specific size and porosity. The size and porosity of the micropores may affect the penetration rate of body fluids and the release rate of the sleep-inducing component.

[0125] In one embodiment, the micropores may have a size of 50 to 200 Ìm. If the pore size is less than 50 Ìm, the penetration of body fluid may be delayed, and if the pore size exceeds 200 Ìm, the structural stability of the shell portion may be reduced.

[0126] In one embodiment, the porosity of the shell portion may be 40 to 70%. If the porosity is less than 40%, the rapid release of the sleep-inducing component may be limited, and if the porosity exceeds 70%, the mechanical strength of the shell portion may be insufficient.

[0127] For example, the micropores may have a size of 80 to 150 Ìm, and the porosity of the shell portion may be 50 to 60%.

[0128] In one aspect of the present invention, the shell portion, the intermediate layer portion, the diffusion barrier layer, and the core portion may each have a specific thickness range. The thickness of each section may affect the component release timing and release duration.

[0129] In one embodiment, the thickness of the shell portion may be 200 to 1,000 Ìm. The thickness of the intermediate layer portion may be 500 to 2,000 Ìm. The thickness of the diffusion barrier layer may be 10 to 100 Ìm. The diameter of the core portion may be 1,000 to 5,000 Ìm.

[0130] For example, in the case of a carrier for 8 hours of sleep, the thickness of the shell portion may be 300 to 500 Ìm, the thickness of the intermediate layer portion may be 800 to 1,200 Ìm, the thickness of the diffusion barrier layer may be 30 to 50 Ìm, and the diameter of the core portion may be 2,000 to 3,000 Ìm.

[0131] In one aspect of the present invention, the alginate included in the shell portion may have a specific concentration and composition ratio. The characteristics of the alginate may affect the viscosity, gelation characteristics, and decomposition rate of the shell portion.

[0132] In one embodiment, the alginate may have a concentration of 1 to 4 weight percent. If the concentration is less than 1 weight percent, it may be difficult to maintain the shape during printing, and if the concentration exceeds 4 weight percent, the viscosity may be excessively high, making it difficult to extrude.

[0133] In one embodiment, the alginate may be sodium alginate having a mannuronic acid / gluuronic acid ratio of 1.0 to 1.5. The mannuronic acid / gluuronic acid ratio may affect the flexibility and gel strength of the alginate.

[0134] For example, the alginate may have a concentration of 2 to 3 weight%, and the mannuronic acid / gluuronic acid ratio may be 1.2 to 1.4.

[0135] In one aspect of the present invention, the methacrylated gelatin included in the core portion may have a specific degree of methacrylation and concentration. The degree of methacrylation may affect the crosslinking density of the network formed after photocrosslinking.

[0136] In one embodiment, the methacrylated gelatin may have a degree of methacrylation of 40 to 80%. If the degree of methacrylation is less than 40%, the crosslinking density is low and sustained-release properties may be insufficient, and if the degree of methacrylation exceeds 80%, brittleness may increase due to excessive crosslinking.

[0137] In one embodiment, the methacrylated gelatin may be included in the core portion at a concentration of 5 to 15 weight percent. Within the concentration range, the core portion may have appropriate mechanical strength and sustained-release properties.

[0138] For example, the methacrylated gelatin may have a degree of methacrylation of 50 to 70% and may be included at a concentration of 8 to 12 weight%.

[0139] In one aspect of the present invention, the nanocellulose included in the diffusion barrier layer may have a specific type and specification. The characteristics of the nanocellulose may affect the density and diffusion control ability of the diffusion barrier layer.

[0140] In one embodiment, the nanocellulose may be a TEMPO-oxidized cellulose nanofibrill. The TEMPO oxidation can facilitate nanofibrillation by introducing carboxyl groups to the surface of the cellulose.

[0141] In one embodiment, the cellulose nanofibrils may have a diameter of 5 to 20 nm. Nanofibrils within this diameter range can form a dense network to effectively limit molecular diffusion.

[0142] For example, the cellulose nanofibrils may have a diameter of 8 to 15 nm and may be included in the diffusion barrier layer at a concentration of 0.5 to 2 weight%.

[0143] In one aspect of the present invention, the temperature-sensitive polymer may have a specific composition and a lower critical melting temperature. The lower critical melting temperature may determine responsiveness to changes in body temperature.

[0144] In one embodiment, the temperature-sensitive polymer may be a copolymer of N-isopropylacrylamide and acrylic acid. The lower critical dissolution temperature of the copolymer may be controlled according to the content of acrylic acid.

[0145] In one embodiment, the copolymer may have a lower critical dissolution temperature of 32 to 34°C. The lower critical dissolution temperature may correspond to the range of changes in peripheral body temperature during sleep, and accordingly, the release of components in response to changes in body temperature may be controlled.

[0146] For example, the copolymer may be copolymerized with N-isopropylacrylamide and acrylic acid in a molar ratio of 90:10 to 85:15 and may have a lower critical dissolution temperature around 33°C.

[0147] In one aspect of the present invention, the active ingredient included in the carrier may have a specific content range. The content may be determined based on the total weight of the carrier.

[0148] In one embodiment, the content of melatonin may be 0.1 to 5.0 weight%. The content of GABA may be 1.0 to 10.0 weight%. The content of nicotinamide mononucleotide may be 0.5 to 10.0 weight%.

[0149] For example, the content of the melatonin may be 0.5 to 2.0 weight%, the content of the GABA may be 3.0 to 7.0 weight%, and the content of the nicotinamide mononucleotide may be 2.0 to 5.0 weight%.

[0150] In one aspect of the present invention, the bio-ink prepared in the bio-ink preparation step (S10) may have a specific viscosity range. The viscosity may affect the ejection characteristics and shape retention ability during printing.

[0151] In one embodiment, the first bio-ink may have a viscosity of 10,000 to 50,000 cP at 25°C. The high viscosity can ensure the structural stability of the core portion.

[0152] In one embodiment, the second bio-ink may have a viscosity of 1,000 to 5,000 cP at 25°C. The relatively low viscosity may contribute to rapid diffusion and release characteristics.

[0153] For example, the first bio-ink may have a viscosity of 20,000 to 35,000 cP at 25°C, and the second bio-ink may have a viscosity of 2,000 to 3,500 cP at 25°C.

[0154] In one aspect of the present invention, the coaxial nozzle used in the coaxial nozzle printing step (S20) may have a specific specification. The specification of the coaxial nozzle may affect the dimensions of the core-shell structure.

[0155] In one embodiment, the coaxial nozzle may include an inner nozzle and an outer nozzle. The inner nozzle may have an inner diameter of 200 to 500 Ìm. The outer nozzle may have an inner diameter of 500 to 1,000 Ìm.

[0156] For example, the inner nozzle may have an inner diameter of 300 to 400 Ìm, and the outer nozzle may have an inner diameter of 600 to 800 Ìm. The inner nozzle and the outer nozzle may be made of stainless steel or polyetheretherketone.

[0157] In one aspect of the present invention, the coaxial nozzle printing step (S20) may be performed under specific temperature and speed conditions. These conditions may affect the ejection characteristics of the bio-ink and the stability of the active ingredient.

[0158] In one embodiment, the coaxial nozzle printing step (S20) may be performed at a temperature of 20 to 37°C. The temperature range can prevent thermal decomposition of the active ingredient while maintaining the fluidity of the bio-ink.

[0159] In one embodiment, the coaxial nozzle printing step (S20) may be performed at a nozzle movement speed of 5 to 30 mm / s. Within the speed range, uniform layer thickness and precise shape control may be possible.

[0160] For example, the coaxial nozzle printing step (S20) can be performed at a temperature of 25 to 30°C and a nozzle movement speed of 10 to 20 mm / s.

[0161] In one aspect of the present invention, the multi-stage crosslinking step (S30) may sequentially include an ionic crosslinking step and a photocrosslinking step. The conditions of each crosslinking step may affect the crosslinking density and sustained-release characteristics.

[0162] In one embodiment, the ion crosslinking step may include immersing the core-shell structure in an aqueous calcium chloride solution of 0.5 to 2.0 weight% for 1 to 10 minutes. Calcium ions in the aqueous calcium chloride solution may form ionic bonds with the carboxyl groups of the alginate to induce gelation.

[0163] In one embodiment, the photocrosslinking step is 5 to 20 mW / cm² in the presence of a photoinitiator. 2 It may include irradiating with ultraviolet light at an intensity of 30 seconds to 5 minutes. Radical polymerization between methacrylate groups of the methacrylated gelatin may be initiated by the ultraviolet irradiation, thereby forming a covalent bond network.

[0164] For example, the ion crosslinking step may include immersion in a 1.0 wt% aqueous calcium chloride solution for 3 to 5 minutes, and the photocrosslinking step may be 10 mW / cm² 2 It may include irradiating ultraviolet light with a wavelength of 365 nm at an intensity for 2 to 3 minutes.

[0165] In one aspect of the present invention, the photoinitiator used in the photocrosslinking step may have a specific type and concentration. The type of photoinitiator may affect photocrosslinking efficiency and cell compatibility.

[0166] In one embodiment, the photoinitiator may be 2-hydroxy-4'-hydroxyethoxy-2-methylpropiophenone or lithium phenyl-2,4,6-trimethylbenzoylphosphinate. The photoinitiators may be water-soluble and biocompatible.

[0167] In one embodiment, the concentration of the photoinitiator may be 0.05 to 0.5 weight%. If the concentration is less than 0.05 weight%, the photocrosslinking efficiency may be insufficient, and if the concentration exceeds 0.5 weight%, cytotoxicity may increase.

[0168] For example, the photoinitiator may be lithium phenyl-2,4,6-trimethylbenzoylphosphinate and may be included at a concentration of 0.1 to 0.3 weight%.

[0169] In one aspect of the present invention, the freeze-drying step (S40) may include a preliminary freezing step and a sublimation drying step. The freeze-drying conditions may affect the preservation of the activity of the active ingredient and the maintenance of the porous structure.

[0170] In one embodiment, the pre-freezing step may include freezing the cross-linked structure at -70 to -80°C. Rapid freezing at the low temperature can prevent damage to the active ingredient by minimizing the size of the ice crystals.

[0171] In one embodiment, the sublimation drying step may be performed for 24 to 48 hours at a pressure of 0.1 mbar or less and a temperature of -40 to -50°C. Under these conditions, ice may be sublimated to remove moisture, and the porous framework may be maintained.

[0172] For example, the above preliminary freezing step can be performed at -80°C for 12 hours, and the above sublimation drying step can be performed at a pressure of 0.05 mbar and a temperature of -50°C for 36 hours.

[0173] In one aspect of the present invention, the carrier may further include a polydopamine coating layer formed on the outer surface of the shell portion. The polydopamine coating layer may improve the surface characteristics of the carrier.

[0174] In one embodiment, the polydopamine may be formed by the self-polymerization of dopamine under alkaline conditions. The dopamine comprises catechol groups and amine groups, and may form a melanin-like structure through the oxidation and polymerization of the catechol groups. The polydopamine coating layer may have adhesive properties mimicking clam adhesive proteins and may prevent detachment when applied to a mucosal-adherent delivery system.

[0175] In one embodiment, the catechol / quinone redox cycle of the polydopamine can scavenge reactive oxygen species. The reactive oxygen species scavenging action can contribute to promoting sleep onset by alleviating oxidative stress during the early stages of sleep.

[0176] For example, the polydopamine coating layer may include an indole framework having structural similarity to melatonin contained in the shell portion, and may create an affinity environment for melatonin receptors.

[0177] In one aspect of the present invention, the polydopamine coating layer may have a specific thickness range. The thickness may be controlled by the coating time and the dopamine concentration.

[0178] In one embodiment, the polydopamine coating layer may have a thickness of 10 to 50 nm. If the thickness is less than 10 nm, the continuity of the coating may be insufficient, and if the thickness exceeds 50 nm, it may affect the dissolution rate of the shell portion.

[0179] For example, the polydopamine coating layer may have a thickness of 20 to 35 nm.

[0180] In one aspect of the present invention, the polydopamine coating layer may comprise a copolymer of dopamine and 5,6-dihydroxyindole-2-carboxylic acid. The 5,6-dihydroxyindole-2-carboxylic acid may be a eumelanin precursor.

[0181] In one embodiment, the copolymer may be a copolymer of the dopamine and the 5,6-dihydroxyindole-2-carboxylic acid. The copolymer may have a structure more similar to melanin than the polydopamine, and may have increased structural similarity to melatonin.

[0182] For example, the molar ratio of the dopamine and the 5,6-dihydroxyindole-2-carboxylic acid may be 8:2 to 5:5.

[0183] In one aspect of the present invention, the carrier may further include a cholesteryl ester liquid crystal layer disposed between the shell portion and the intermediate layer portion. The cholesteryl ester liquid crystal layer may have thermotropic liquid crystal characteristics.

[0184] In one embodiment, the cholesteryl ester may be an ester conjugate of cholesterol and a fatty acid. The cholesteryl ester may exhibit a phase transition depending on the temperature, maintaining a smectic high-viscosity phase below the phase transition temperature and transitioning to a cholesteric low-viscosity phase above the phase transition temperature. The change in viscosity due to the phase transition may affect the component diffusion rate from the intermediate layer.

[0185] In one embodiment, the cholesteryl ester may have structural similarity to ceramide in the stratum corneum of the skin. This structural similarity may promote penetration into the stratum corneum when applied via transdermal delivery.

[0186] For example, during sleep, the peripheral body temperature may drop to 34 to 35°C, and the cholesteryl ester liquid crystal layer may maintain a high viscosity phase to suppress the release of components from the intermediate layer. During the latter part of sleep or just before waking up, the body temperature may rise to 36.5 to 37°C, and the cholesteryl ester liquid crystal layer may transition to a low viscosity phase, thereby initiating the release of the anti-aging active component.

[0187] In one aspect of the present invention, the cholesteryl ester liquid crystal layer may have a specific composition and a phase transition temperature. The composition may be set to control the phase transition temperature.

[0188] In one embodiment, the cholesteryl ester liquid crystal layer may comprise a mixture of cholesteryl oleate and cholesteryl nonanoate. The cholesteryl oleate may be an ester of oleic acid and cholesterol, and the cholesteryl nonanoate may be an ester of nonanoic acid and cholesterol.

[0189] In one embodiment, the mixture may have a phase transition temperature of 35 to 37°C. The phase transition temperature may correspond to the range of changes in body temperature during sleep, and may enable the release of components automatically synchronized with the biological rhythm without external stimulation.

[0190] For example, the above mixture may have a phase transition temperature of around 36°C, and the phase transition may proceed in response to a minute change of 0.5 to 1°C in body temperature.

[0191] In one aspect of the present invention, the cholesteryl ester liquid crystal layer may have a specific mixing ratio and thickness. The mixing ratio may be set to finely control the phase transition temperature.

[0192] In one embodiment, the cholesteryl oleate and the cholesteryl nonanoate may be mixed in a weight ratio of 7:3 to 9:1. Depending on the mixing ratio, the phase transition temperature may be controlled within the range of 35 to 37°C.

[0193] In one embodiment, the thickness of the cholesteryl ester liquid crystal layer may be 20 to 100 Ìm. If the thickness is less than 20 Ìm, the phase transition effect may be insufficient, and if the thickness exceeds 100 Ìm, the overall size of the carrier may increase excessively.

[0194] For example, the cholesteryl oleate and the cholesteryl nonanoate may be mixed in a weight ratio of 8:2, and the thickness of the cholesteryl ester liquid crystal layer may be 40 to 60 Ìm.

[0195] In one aspect of the present invention, the diffusion barrier layer may comprise a metal-phenolic network coating layer. The metal-phenolic network coating layer may be formed by the coordination bonding of metal ions and tannic acid.

[0196] In one embodiment, the tannic acid may be a polyphenol having 25 galloyl groups. The catechol structure of the galloyl group may form a coordination bond with a metal ion and may form a network structure by bonding with a number of metal ions.

[0197] In one embodiment, the metal-phenolic network can be formed by self-assembly. The self-assembly can be carried out by sequentially immersing in a metal ion solution and a tannic acid solution, and can form a uniform coating layer at the nano level.

[0198] In one embodiment, the tannic acid may have antioxidant activity due to the catechol structure of the galloyl group. The antioxidant activity may provide an additional antioxidant effect independently of the anti-aging active ingredient included in the core portion. In addition, the protein binding ability of the tannic acid may stabilize the surface of the exosome.

[0199] For example, the metal-phenolic network can be maintained stably under physiological conditions of pH 7.4 and can decompose under acidic conditions of pH 6.5 or lower. The metal-phenolic network exposed after the shell part is dissolved can protect the core part at physiological pH.

[0200] In one aspect of the present invention, the metal-phenolic network coating layer may have specific metal ions and a thickness. The type of metal ions may affect the stability and decomposition characteristics of the coating layer.

[0201] In one embodiment, the metal ion is Ti 4+ , Fe 3+ and Al 3+ It may include one or more of the above Ti. 4+ The ion can have a high binding affinity with the above tannic acid and can form a stable coating layer.

[0202] In one embodiment, the thickness of the metal-phenolic network coating layer may be 10 to 30 nm. The thickness may be thinner and more uniform compared to a nanocellulose barrier, and precise diffusion control may be possible.

[0203] For example, the above metal ion is Ti 4+ It may be, and the thickness of the metal-phenolic network coating layer may be 15 to 25 nm.

[0204] In one aspect of the present invention, the core portion may include a supramolecular cross-linked structure formed by a host-guest bond. The supramolecular cross-linked structure may be formed by methacrylated gelatin conjugated with beta-cyclodextrin and a multi-arm polyethylene glycol having adamantan ends.

[0205] In one embodiment, the beta-cyclodextrin may be a cyclic oligosaccharide composed of seven glucopyranose units. The beta-cyclodextrin may have a hydrophobic cavity, and the cavity may accommodate a hydrophobic guest molecule of a suitable size.

[0206] In one embodiment, the adamantane may be a cage-structured hydrocarbon composed of three fused cyclohexane rings. The adamantane may have a high binding affinity to the cavity of the beta-cyclodextrin, and about 10 5 M -1 It can represent the coupling constant of.

[0207] In one embodiment, the host-guest bond between the methacrylated gelatin conjugated with the beta-cyclodextrin and the multi-arm polyethylene glycol having the adamantane terminus may be reversible. Due to the reversible bond, the crosslink may be dismantled in the presence of a competitive guest molecule, thereby initiating the decomposition of the core portion and the release of components.

[0208] For example, unlike covalent crosslinking, the supramolecular crosslinking structure described above may enable programmed crosslinking based on molecular recognition and can contribute to precise control of the timing of component release.

[0209] In one aspect of the present invention, the methacrylated gelatin conjugated with the beta-cyclodextrin and the multi-arm polyethylene glycol-adamantane may have specific specifications.

[0210] In one embodiment, the beta-cyclodextrin conjugation rate of the methacrylated gelatin conjugated with the beta-cyclodextrin may be 5 to 15 mol%. If the conjugation rate is less than 5 mol%, the supramolecular crosslinking density may be insufficient, and if the conjugation rate exceeds 15 mol%, the mechanical properties of the methacrylated gelatin may be degraded.

[0211] In one embodiment, the multi-arm polyethylene glycol-adamantan may be a 4-arm polyethylene glycol-adamantan carboxylic acid. The 4-arm structure may have adamantan groups at four ends and may form multipoint crosslinks. The multi-arm polyethylene glycol-adamantan may have a molecular weight of 8,000 to 12,000 Da.

[0212] For example, the beta-cyclodextrin conjugation rate may be 8 to 12 mol%, and the molecular weight of the multi-arm polyethylene glycol-adamantan may be 10,000 Da.

[0213] In one aspect of the present invention, the intermediate layer may comprise 1-aminoadamantan or a pharmaceutically acceptable salt thereof. The 1-aminoadamantan may function as a competitive guest molecule for dismantling the supramolecular cross-linked structure of the core.

[0214] In one embodiment, the 1-aminoadamantan may also be referred to as amantadine. The 1-aminoadamantan may have a cage structure similar to that of the adamantan and may have binding affinity for the cavity of the beta-cyclodextrin.

[0215] In one embodiment, when the 1-aminoadamantan is released from the intermediate layer, the 1-aminoadamantan can competitively bind to the beta-cyclodextrin cavity of the core. Through the competitive binding, the multi-arm polyethylene glycol-adamantan can be dissociated from the beta-cyclodextrin, and the supramolecular cross-linked structure can be sequentially dismantled.

[0216] In one embodiment, the 1-aminoadamantan may have NMDA receptor antagonistic action and dopamine release-promoting action. The above action may contribute to preparation for the transition to wakefulness in the latter part of sleep and to the enhancement of freshness upon waking.

[0217] For example, as the intermediate layer decomposes, the 1-aminoadamantan may be gradually released, thereby triggering the disintegration of the crosslinking of the core. The process can implement a programmed release mechanism based on molecular recognition rather than simple diffusion control.

[0218] In one aspect of the present invention, the 1-aminoadamantan or its salt may be included in a specific content range. The content may affect the efficiency of the supramolecular crosslinking dismantling.

[0219] In one embodiment, the content of 1-aminoadamantan or its salt may be 0.1 to 1.0 weight% based on the total weight of the intermediate layer. If the content is less than 0.1 weight%, the competitive binding effect may be insufficient, and if the content exceeds 1.0 weight%, an excessive pharmacological effect may occur.

[0220] For example, the content of the 1-aminoadamantan may be 0.3 to 0.7 weight%.

[0221] In one aspect of the present invention, the carrier may further comprise metal-organic framework nanoparticles dispersed within the core portion. The metal-organic framework nanoparticles may function as a carrier for the anti-aging active ingredient.

[0222] In one embodiment, the metal-organic framework nanoparticle may be a zeolitic imidazolate framework-8 formed from zinc ions and 2-methylimidazole. The zeolitic imidazolate framework-8 may have a porous crystal structure formed by zinc ions forming coordination bonds with 2-methylimidazole.

[0223] In one embodiment, the zeolic imidazolate framework-8 is 1,000 to 3,000 m 2 It can have an ultra-high specific surface area of ​​1 / g. The above ultra-high specific surface area can increase the drug loading capacity compared to a conventional polymer matrix.

[0224] In one embodiment, the zeolic imidazolate skeleton-8 may have pH reactivity that is stable under gastric acid conditions and decomposes in the intestinal environment. The zinc-imidazole coordination bond of the zeolic imidazolate skeleton-8 may be maintained under gastric acid conditions of pH 1 to 2, but may dissociate in the intestinal environment of pH 6.5 to 7.4. Due to the pH reactivity, intestinal-specific release upon oral administration may be achieved.

[0225] In one embodiment, some or all of the anti-aging active ingredients may be supported on the zeolic imidazolate framework-8 nanoparticles. The nicotinamide mononucleotide or the exosome may be adsorbed or encapsulated in the pores or on the surface of the nanoparticles.

[0226] For example, the zeolic imidazolate framework-8 nanoparticles can be dispersed within the sustained-release matrix of the core portion, and can improve the stability and loading amount of the anti-aging active ingredient.

[0227] In one aspect of the present invention, the zeolic imidazolate framework-8 nanoparticles may have a specific particle size and drug loading rate.

[0228] In one embodiment, the zeolic imidazolate framework-8 nanoparticles may have a particle size of 50 to 200 nm. If the particle size is less than 50 nm, the drug loading amount may be limited, and if the particle size exceeds 200 nm, uniform dispersion within the core may be difficult.

[0229] In one embodiment, the zeolic imidazolate framework-8 nanoparticles may have a polydispersity index of 0.3 or less. The polydispersity index may indicate the size uniformity of the nanoparticles.

[0230] In one embodiment, the drug loading rate of the zeolic imidazolate framework-8 nanoparticles may be 10 to 30 weight%. The drug loading rate may be achieved by the ultra-high specific surface area.

[0231] For example, the zeolic imidazolate framework-8 nanoparticles may have a particle size of 80 to 150 nm and a drug loading rate of 15 to 25 weight%.

[0232] In one aspect of the present invention, the core portion may include a redox-reactive crosslinking structure. The redox-reactive crosslinking structure may be formed by Schiff base bonding between ferrocene carboxyaldehyde and branched polyethyleneimine.

[0233] In one embodiment, the ferrocene may be an organometallic compound in which an iron ion is sandwiched between two cyclopentadienyl rings. The ferrocene is Fe 2+ / Fe 3+ It can exhibit a reversible oxidation-reduction reaction. When the ferrocene is oxidized and converted to ferrocenium, hydrophilicity may increase due to cationization, and it may escape from the hydrophobic matrix of the core part, causing structural relaxation.

[0234] In one embodiment, the ferrocene carboxyaldehyde may be a compound in which an aldehyde group is substituted on ferrocene. The aldehyde group may form a Schiff base bond with the amine group of the branched polyethyleneimine, and a cross-linked network may be formed by said bond.

[0235] In one embodiment, the redox-reactive cross-linked structure may respond to an increase in intracellular glutathione concentration during sleep. The glutathione may act as a reducing agent and affect the redox-reduction state of the ferrocene. During sleep, a reducing environment may be created within the cell, and the redox-reactive cross-linked structure may undergo disintegration in response to the change in the environment.

[0236] For example, the above redox-reactive cross-linked structure can synchronize drug release with the in vivo redox state, and may enable the release of components tailored to the metabolic state of the cell.

[0237] In one aspect of the present invention, the component forming the redox-reactive crosslinked structure may have specific specifications.

[0238] In one embodiment, the branched polyethyleneimine may have a molecular weight of 20,000 to 30,000 Da. The branched structure may include a plurality of primary, secondary, and tertiary amine groups, and the amine groups may provide binding sites with the ferrocene carboxyaldehyde.

[0239] In one embodiment, the conjugation rate of the ferrocene carboxyaldehyde may be 3 to 10 mol%. If the conjugation rate is less than 3 mol%, the redox reactivity may be insufficient, and if the conjugation rate exceeds 10 mol%, the water solubility of the polyethyleneimine may be reduced.

[0240] For example, the molecular weight of the branched polyethyleneimine may be 25,000 Da, and the conjugation rate of the ferrocene carboxyaldehyde may be 5 to 8 mol%.

[0241] In one aspect of the present invention, the manufacturing method may further include a metal-phenolic network coating step after the coaxial nozzle printing step (S20). The metal-phenolic network coating step may include forming a metal-phenolic network coating layer on the surface of the core portion of the core-shell structure.

[0242] In one embodiment, the metal-phenolic network coating step may include sequentially immersing the surface of the core portion with a metal ion solution and a tannic acid solution. By the sequential immersion, a coordination bond may be formed between the metal ion and the tannic acid, and the coating layer may self-assemble through the bond.

[0243] For example, the metal-phenolic network coating step may be performed after printing the core portion and before printing the intermediate layer portion, and may form the diffusion barrier layer between the core portion and the intermediate layer portion.

[0244] In one aspect of the present invention, the metal-phenolic network coating step may include a plurality of detailed steps.

[0245] In one embodiment, the metal-phenolic network coating step may include the step of immersing the surface of the core portion in a metal ion solution containing titanium butoxide. The titanium butoxide is Ti 4+ It can be a precursor of ions.

[0246] In one embodiment, the metal-phenolic network coating step may include a step of immersing in an aqueous tannic acid solution after withdrawal from the metal ion solution. During the immersion, the Ti 4+ A coordination bond can be formed between the ion and the tannic acid.

[0247] In one embodiment, the metal-phenolic network coating step may include the step of repeating the immersion of the metal ion solution and the immersion of the tannic acid aqueous solution 3 to 5 times. By repeating, the thickness of the coating layer may be increased, and a thickness of 10 to 30 nm may be achieved.

[0248] For example, each of the above immersions may be performed for 1 minute, and between the immersions, a step of washing with deionized water may be performed.

[0249] In one aspect of the present invention, the solution used in the metal-phenolic network coating step may have a specific composition.

[0250] In one embodiment, the metal ion solution may be a solution in which 0.05 to 0.2 mg / mL of titanium butoxide is dissolved in a mixed solvent of ethanol and water in a 1:1 volume ratio. The mixed solvent may enable contact with the surface of the core while controlling the hydrolysis of the titanium butoxide.

[0251] In one embodiment, the aqueous tannic acid solution may contain 0.2 to 0.6 mg / mL of tannic acid. A uniform coating layer may be formed within the concentration range.

[0252] For example, the metal ion solution may contain 0.1 mg / mL of titanium butoxide, and the tannic acid aqueous solution may contain 0.4 mg / mL of tannic acid.

[0253] In one aspect of the present invention, the manufacturing method may further include a polydopamine coating step after the freeze-drying step (S40). The polydopamine coating step may include immersing the carrier in a coating solution comprising dopamine hydrochloride and a buffer solution to form a polydopamine coating layer.

[0254] In one embodiment, the polydopamine coating layer may be formed by self-polymerization of the dopamine under alkaline conditions. Under the alkaline conditions, the catechol group of the dopamine may be oxidized and converted into a quinone, and a melanin-like polymer may be formed by a subsequent polymerization reaction.

[0255] For example, the polydopamine coating step can be performed before the enteric coating step (S50).

[0256] In one aspect of the present invention, the polydopamine coating step may include a plurality of detailed steps.

[0257] In one embodiment, the polydopamine coating step may include the step of immersing the carrier in a Tris-hydrochloride buffer containing 1.5 to 2.5 mg / mL of dopamine hydrochloride and adjusted to a pH of 8.0 to 9.0. The concentration of the Tris-hydrochloride buffer may be 10 mM.

[0258] In one embodiment, the polydopamine coating step may include a step of stirring in an open container for 2 to 6 hours. The open container may enable oxygen supply to promote the oxidation and polymerization of the dopamine.

[0259] For example, the polydopamine coating step can be performed at 25°C for 4 hours in a Tris-hydrochloride buffer with a pH of 8.5 containing 2 mg / mL of dopamine hydrochloride.

[0260] In one aspect of the present invention, the coaxial nozzle printing step (S20) may include the step of forming a liquid crystal layer by applying a cholesteryl ester mixture.

[0261] In one embodiment, the liquid crystal layer forming step may include applying the cholesteryl ester mixture to the inner surface of the shell portion after the shell portion is formed. The application may be performed by dip coating or spray coating.

[0262] For example, the liquid crystal layer formation step may be performed after printing the intermediate layer and before printing the shell, and the cholesteryl ester liquid crystal layer may be formed between the intermediate layer and the shell.

[0263] In one aspect of the present invention, the cholesteryl ester mixture may be applied in a molten state, and a liquid crystal phase may be formed by slow cooling.

[0264] In one embodiment, the cholesteryl ester mixture may be applied in a molten state at 50 to 70°C. At the above temperature, the cholesteryl ester mixture may be in an isotropic liquid phase.

[0265] In one embodiment, a smectic liquid crystal phase can be formed by slowly cooling to 20 to 30°C after coating. The slow cooling rate may affect the orientation and uniformity of the liquid crystal phase.

[0266] For example, the above cholesteryl ester mixture can be applied in a molten state at 60°C and can be slowly cooled to 25°C at a rate of 1 to 2°C / min.

[0267] In one aspect of the present invention, the bio-ink preparation step (S10) may include a drug loading step of loading the anti-aging active ingredient onto zeolic imidazolate framework-8 nanoparticles and a nanoparticle dispersion step of dispersing the nanoparticles loaded with the drug into the first bio-ink.

[0268] In one embodiment, the drug loading step may be performed using a one-pot synthesis method in which the drug is encapsulated simultaneously with the synthesis of the zeolic imidazolate framework-8 nanoparticles. In the one-pot synthesis method, the anti-aging active ingredient may be encapsulated inside the nanoparticles during the nanoparticle formation process.

[0269] In one embodiment, the nanoparticle dispersion step may include dispersing the drug-loaded nanoparticles in a polymer solution of the first bio-ink. The dispersion may be performed by ultrasonic treatment or mechanical stirring.

[0270] For example, the zeolic imidazolate framework-8 nanoparticles loaded with the above drug may be dispersed in an amount of 1 to 10 weight percent of the weight of the first bio-ink.

[0271] In one aspect of the present invention, the drug loading step may be performed under specific synthesis conditions.

[0272] In one embodiment, the drug loading step may include the step of dissolving Zn(NO3)2O6H2O, 2-methylimidazole, and the anti-aging active ingredient in methanol. The molar ratio of the zinc ion to the 2-methylimidazole may be 1:4 to 1:10.

[0273] In one embodiment, the drug loading step may include the step of leaving the solution at room temperature for 20 to 28 hours. During the leaving period, a coordination bond may be formed between the zinc ion and the 2-methylimidazole so that the zeolic imidazolate framework-8 crystal may grow, and the anti-aging active ingredient may be encapsulated inside the crystal.

[0274] In one embodiment, the drug loading step may include the step of obtaining drug-loaded zeolic imidazolate framework-8 nanoparticles by centrifugation. The centrifugation may be performed at 8,000 to 12,000 × g for 10 to 20 minutes.

[0275] For example, the above drug loading step may include dissolving 0.3 g of Zn(NO3)2o6H2O, 0.66 g of 2-methylimidazole, and 50 mg of nicotinamide mononucleotide in 20 mL of methanol, letting it stand at room temperature for 24 hours, and centrifuging at 10,000 × g for 15 minutes.

[0276] In one aspect of the present invention, the anti-aging active ingredient or lifespan-extending active ingredient may include one or more of a telomere-protecting ingredient, a cell aging-inhibiting ingredient, a sirtuin-activating ingredient, a NAD+ precursor, an autophagy-inducing ingredient, an mTOR-inhibiting ingredient, an AMPK-activating ingredient, a mitochondrial-targeted antioxidant ingredient, and a senolic ingredient.

[0277] In one aspect of the present invention, based on the total weight of the carrier, the content of spermidine may be 0.1 to 5.0 weight%, the content of fisetin may be 0.5 to 10.0 weight%, the content of quercetin may be 0.5 to 10.0 weight%, and the content of cycloastragenol may be 0.01 to 1.0 weight%.

[0278] In one aspect of the present invention, the delivery body may be configured to simultaneously achieve improvement in sleep quality and extension of lifespan. A sleep-inducing component released from the shell portion may promote the onset of sleep to induce high-quality sleep, and a lifespan-extending active component may be released from the core portion in synchronization with the growth hormone secretion, glymphatic system, and DNA repair processes activated during sleep.

[0279] In one embodiment, growth hormone secretion may increase during a sleep state induced by the release of the sleep-inducing component, and the growth hormone may promote cell regeneration and tissue repair. At the above time, nicotinamide mononucleotide released from the core may increase intracellular NAD+ levels, thereby enhancing the cell regeneration effect caused by the growth hormone.

[0280] In one embodiment, the glymphatic system may be activated during the latter part of sleep to remove metabolic waste from the brain, and the antioxidant component and autophagy-inducing component released from the core at that time may contribute to maintaining homeostasis of brain cells.

[0281] In one embodiment, the telomere protective component may include one or more of cycloastrazenol, astragaloside IV, and TA-65.

[0282] In one embodiment, the senolitic component may include one or more of fisetin, quercetin, and combinations thereof.

[0283] In one embodiment, the autophagy-inducing component may include one or more of spermidine and trehalose.

[0284] In one embodiment, the AMPK activating component may include berberine.

[0285] In one embodiment, the mitochondrial-targeted antioxidant component may include one or more of coenzyme Q10, pyrroloquinoline quinone, and alpha-lipoic acid.

[0286] For example, the core portion may include a combination of nicotinamide mononucleotide, resveratrol, spermidine, and fisetin, and this combination may provide a lifespan extension effect through multiple pathways of NAD+ supplementation, sirtuin activation, autophagy induction, and senescent cell removal.

[0287] In one aspect of the present invention, the core portion may further include a lifespan-extending active ingredient in addition to the anti-aging active ingredient. The lifespan-extending active ingredient may include a component that contributes to delaying aging and extending lifespan at the cellular level. The lifespan-extending active ingredient may include one or more of a telomere-protecting component, a senolic component, an autophagy-inducing component, an AMPK-activating component, and a mitochondrial-targeted antioxidant component.

[0288] In one embodiment, the telomere protective component may be a component that delays telomere shortening through telomerase activation. The telomere is a repetitive DNA sequence located at the end of a chromosome, which gradually shortens with cell division, and when it reaches a critical length, cell aging or apoptosis may be induced. The telomere protective component may contribute to maintaining or extending telomere length by increasing telomerase activity.

[0289] In one embodiment, the senolitic component may be a component that selectively removes senescent cells. The senescent cells are cells whose cell cycle has been irreversibly arrested, exhibiting an aging-related secretory phenotype and capable of secreting inflammatory cytokines, chemokines, and growth factors. The accumulation of the senescent cells may be associated with tissue deterioration and chronic inflammation, and the senolitic component may contribute to the recovery of tissue function through the selective removal of the senescent cells.

[0290] In one embodiment, the autophagy-inducing component may be a component that promotes the degradation of damaged proteins and organelles within the cell. The autophagy may be an essential process for maintaining protein homeostasis, removing damaged mitochondria, and maintaining intracellular homeostasis. Autophagy function may decline with aging, and the autophagy-inducing component may contribute to maintaining cell health by restoring autophagy activity.

[0291] In one embodiment, the AMPK activating component may be a component that activates AMPK, an intracellular energy-sensing kinase. The AMPK activation can promote catabolism and inhibit anabolism, and induce mitochondrial biosynthesis and autophagy. The AMPK activation can provide metabolic effects similar to calorie restriction.

[0292] In one embodiment, the mitochondrial-targeted antioxidant component may be a component that selectively accumulates in mitochondria and scavenges reactive oxygen species within the mitochondria. As aging progresses, mitochondrial function may decline and the production of reactive oxygen species may increase, and the mitochondrial-targeted antioxidant component may contribute to protecting mitochondrial function.

[0293] For example, the core portion may include a combination of nicotinamide mononucleotide, resveratrol, spermidine, and fisetin, and the combination may provide a lifespan extension effect through multiple pathways of NAD+ supplementation, sirtuin activation, autophagy induction, and senescent cell removal.

[0294] In one aspect of the present invention, the telomere protective component may comprise one or more of cycloastrazenol, astragaloside IV, and astragalus extract.

[0295] In one embodiment, the cycloastragenol may be a triterpenoid sapogenin derived from Astragalus membranaceus. The cycloastragenol may contribute to maintaining telomere length through telomerase activation. The cycloastragenol is known as the main active ingredient of TA-65.

[0296] In one embodiment, the astragaloside IV may be a saponin glycoside derived from Astragalus membranaceus. The astragaloside IV may exhibit telomerase activation, antioxidant, anti-inflammatory, and immunomodulatory effects.

[0297] In one embodiment, the astragalus extract may be extracted from the root of Astragalus membranaceus and may be a complex extract comprising the cycloastrazenol and the astragaloside IV.

[0298] For example, the telomere protective component may contain cycloastrazenol and astragaloside IV in a weight ratio of 1:10 to 1:50.

[0299] In one aspect of the present invention, the telomere protective component may be included in a specific content range. The content may be set considering the telomerase activation effect and safety.

[0300] In one embodiment, the content of the cycloastrazenol may be 0.01 to 1.0 weight% based on the total weight of the carrier. If the content is less than 0.01 weight%, the telomerase activation effect may be insufficient, and if the content exceeds 1.0 weight%, excessive telomerase activation may be a concern.

[0301] In one embodiment, the content of astragaloside IV may be 0.1 to 5.0 weight% based on the total weight of the carrier.

[0302] For example, the content of the cycloastrazenol may be 0.05 to 0.5 weight%, and the content of the astragaloside IV may be 0.5 to 3.0 weight%.

[0303] In one aspect of the present invention, the senolitic component may include one or more of fisetin, quercetin, and combinations thereof.

[0304] In one embodiment, the fisetin may be a flavonoid contained in strawberries, apples, and onions. The fisetin may exhibit sirtuin-activating, antioxidant, anti-inflammatory, and senolytic effects. The fisetin has been shown to improve health lifespan in aging mice.

[0305] In one embodiment, the quercetin may be a flavonoid contained in onions, apples, and tea. The quercetin may exhibit antioxidant, anti-inflammatory, and senolytic effects. The quercetin may be used to remove senescent cells, either alone or in combination with other senolytic components.

[0306] In one embodiment, the combination of fisetin and quercetin may exhibit a synergistic senolytic effect. The combination may contribute to the removal of various types of senescent cells by targeting different senescent cell survival pathways.

[0307] For example, the fisetin and the quercetin can be combined in a weight ratio of 1:1 to 1:3.

[0308] In one aspect of the present invention, the senolitic component may be included in a specific content range.

[0309] In one embodiment, the content of fisetin may be 0.5 to 10.0 weight% based on the total weight of the carrier. If the content is less than 0.5 weight%, the senolytic effect may be insufficient, and if the content exceeds 10.0 weight%, toxicity to normal cells may be a concern.

[0310] In one embodiment, the content of the quercetin may be 0.5 to 10.0 weight% based on the total weight of the carrier.

[0311] For example, the content of the fisetin may be 2.0 to 5.0 weight%, and the content of the quercetin may be 2.0 to 5.0 weight%.

[0312] In one aspect of the present invention, the autophagy-inducing component may include one or more of spermidine, trehalose, and urolitin A.

[0313] In one embodiment, the spermidine may be a natural polyamine and may be contained in wheat germ, soybeans, and aged cheese. The spermidine may exhibit autophagy induction, anti-inflammatory, and cardiovascular protective effects. The spermidine may promote the expression of autophagy-related genes through the inhibition of histone acetyltransferase.

[0314] In one embodiment, the trehalose may be a disaccharide formed by combining two molecules of glucose. The trehalose can induce autophagy through an mTOR-independent pathway and may contribute to the removal of protein aggregates.

[0315] In one embodiment, the uroritin A may be an intestinal metabolite of ellagic acid. The uroritin A can selectively remove damaged mitochondria by inducing mitophagy.

[0316] For example, the spermidine and the trehalose can be combined in a weight ratio of 1:5 to 1:15.

[0317] In one aspect of the present invention, the autophagy-inducing component may be included in a specific content range.

[0318] In one embodiment, the content of spermidine may be 0.1 to 5.0 weight% based on the total weight of the carrier. If the content is less than 0.1 weight%, the autophagy induction effect may be insufficient, and if the content exceeds 5.0 weight%, excessive autophagy may be a concern.

[0319] In one embodiment, the content of the trehalose may be 1.0 to 15.0 weight% based on the total weight of the carrier.

[0320] For example, the content of the spermidine may be 0.5 to 2.0 weight%, and the content of the trehalose may be 3.0 to 8.0 weight%.

[0321] In one aspect of the present invention, the AMPK activating component may include one or more of berberine and ginsenoside Rg3.

[0322] In one embodiment, the berberine may be an isoquinoline alkaloid contained in Coptis japonica, Phellodendron amurense, and Barberry. The berberine may exhibit AMPK activation, hypoglycemic, anti-inflammatory, and antibacterial effects. The berberine may activate AMPK by inhibiting mitochondrial respiratory chain complex I and increasing the AMP / ATP ratio.

[0323] In one embodiment, the ginsenoside Rg3 may be a saponin derived from ginseng. The ginsenoside Rg3 may exhibit AMPK activation, anticancer, anti-inflammatory, and immunomodulatory effects.

[0324] For example, the berberine and the ginsenoside Rg3 can be combined in a weight ratio of 1:0.5 to 1:2.

[0325] In one aspect of the present invention, the AMPK activating component may be included in a specific content range.

[0326] In one embodiment, the content of the berberine may be 0.5 to 5.0 weight% based on the total weight of the carrier. If the content is less than 0.5 weight%, the AMPK activation effect may be insufficient, and if the content exceeds 5.0 weight%, gastrointestinal side effects may be a concern.

[0327] For example, the content of the berberine may be 1.0 to 3.0 weight%.

[0328] In one aspect of the present invention, the mitochondrial-targeted antioxidant component may include one or more of coenzyme Q10, pyrroloquinoline quinone, and alpha-lipoic acid.

[0329] In one embodiment, the coenzyme Q10 may be in the form of ubiquinone or ubiquinol. The coenzyme Q10 is a component of the mitochondrial electron transport chain, is involved in ATP production, and can exhibit antioxidant activity. As aging progresses, the level of coenzyme Q10 in the body may decrease, and mitochondrial function may be improved through supplementation.

[0330] In one embodiment, the pyrroloquinoline quinone may be a redox cofactor and may exhibit mitochondrial biosynthesis promoting and neuroprotective effects. The pyrroloquinoline quinone may promote the generation of new mitochondria by increasing PGC-1á expression.

[0331] In one embodiment, the alpha-lipoic acid may be a sulfur-containing antioxidant and may function as a cofactor for pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase within mitochondria. The alpha-lipoic acid may exhibit both water-soluble and fat-soluble antioxidant activity.

[0332] For example, the above coenzyme Q10, the above pyrroloquinoline quinone, and the above alpha-lipoic acid may be combined in a weight ratio of 10:1:5.

[0333] In one aspect of the present invention, the mitochondrial-targeted antioxidant component may be included in a specific content range.

[0334] In one embodiment, the content of the coenzyme Q10 may be 0.5 to 5.0 weight% based on the total weight of the carrier.

[0335] In one embodiment, the content of the pyrroloquinoline quinone may be 0.01 to 0.5 weight% based on the total weight of the carrier. The pyrroloquinoline quinone can be effective even at low doses.

[0336] For example, the content of the above coenzyme Q10 may be 1.0 to 3.0 weight%, and the content of the above pyrroloquinoline quinone may be 0.05 to 0.2 weight%.

[0337] In one aspect of the present invention, the life-extending active ingredient may further include one or more of glycine and taurine.

[0338] In one embodiment, the glycine is the simplest amino acid and may be a major component of collagen. The glycine may contribute to the regulation of methionine metabolism, glutathione synthesis, and anti-inflammatory effects. The glycine may also exhibit an effect of improving sleep quality, thereby linking sleep with life extension.

[0339] In one embodiment, the taurine is a sulfur-containing amino acid and may be present in high concentrations in the heart, brain, and muscles. The levels of the taurine in the body may decrease with aging, and health lifespan may be improved through supplementation. The taurine may contribute to antioxidant, anti-inflammatory, improvement of mitochondrial function, and regulation of osmotic pressure.

[0340] For example, the glycine and taurine can be combined in a weight ratio of 1:1 to 2:1.

[0341] In one aspect of the present invention, the glycine and the taurine may be included in a specific content range.

[0342] In one embodiment, the content of the glycine may be 1.0 to 10.0 weight% based on the total weight of the carrier.

[0343] In one embodiment, the content of the taurine may be 1.0 to 10.0 weight% based on the total weight of the carrier.

[0344] For example, the content of the glycine may be 3.0 to 7.0 weight%, and the content of the taurine may be 3.0 to 7.0 weight%.

[0345] In one aspect of the present invention, the lifespan-extending active ingredient may further comprise alpha-ketoglutarate or a salt thereof.

[0346] In one embodiment, the alpha-ketoglutarate may be an intermediate metabolite of the Krebs cycle and may play a key role in energy metabolism. The alpha-ketoglutarate has been confirmed to have a lifespan-extending effect in nematodes and mice. The alpha-ketoglutarate may also be involved in promoting collagen synthesis and regulating immune function.

[0347] In one embodiment, the salt of the alpha-ketoglutarate may be calcium alpha-ketoglutarate or sodium alpha-ketoglutarate. The calcium alpha-ketoglutarate may have excellent stability and high bioavailability.

[0348] For example, the above-mentioned calcium alpha-ketoglutarate may be included in the core portion and released during the latter part of sleep, and may contribute to the improvement of cellular energy metabolism.

[0349] In one aspect of the present invention, the alpha-ketoglutarate or its salt may be included in a specific content range.

[0350] In one embodiment, the content of the alpha-ketoglutarate or its salt may be 0.5 to 5.0 weight% based on the total weight of the carrier.

[0351] For example, the content of the calcium alpha-ketoglutarate may be 1.0 to 3.0 weight%.

[0352] In one aspect of the present invention, the core portion may include an integrated combination of the anti-aging active ingredient and the lifespan-extending active ingredient. The integrated combination may provide a synergistic effect through multiple pathways.

[0353] In one embodiment, the core portion may include nicotinamide mononucleotide and resveratrol as the anti-aging active ingredients. The nicotinamide mononucleotide can increase intracellular NAD+ levels, and the resveratrol can activate sirtuins. The combination of the two ingredients can provide an anti-aging effect through the NAD+ / sirtuin pathway.

[0354] In one embodiment, the core portion may include spermidine and fisetin as the lifespan-extending active ingredients. The spermidine can induce autophagy, and the fisetin can remove senescent cells. The combination of the two ingredients can provide a lifespan-extending effect through a cell cleansing pathway.

[0355] For example, the nicotinamide mononucleotide, the resveratrol, the spermidine, and the fisetin may be combined in a weight ratio of 5:1:1:2.

[0356] In one aspect of the present invention, the total content of the anti-aging active ingredient and the lifespan-extending active ingredient may be set to a specific range.

[0357] In one embodiment, the core portion may have a total content of the anti-aging active ingredient and the lifespan-extending active ingredient of 5.0 to 25.0 weight% based on the total weight of the carrier. If the content is less than 5.0 weight%, an effective dose may not be reached, and if the content exceeds 25.0 weight%, the structural stability of the carrier may be reduced.

[0358] For example, the total content of the anti-aging active ingredient and the lifespan-extending active ingredient may be 10.0 to 18.0 weight%.

[0359] In one aspect of the present invention, the intermediate layer may further include a NAD+ precursor auxiliary component. The NAD+ precursor auxiliary component may be released prior to the release of nicotinamide mononucleotide from the core to prepare a NAD+ synthesis pathway.

[0360] In one embodiment, the NAD+ precursor auxiliary component may include one or more of nicotinamide riboside and tryptophan. The nicotinamide riboside may be an alternative precursor of nicotinamide mononucleotide. The tryptophan may be a precursor of the de novo NAD+ synthesis pathway.

[0361] For example, the nicotinamide riboside may be released from the intermediate layer during the middle part of the sleep, and the nicotinamide mononucleotide may be released from the core part during the latter part of the sleep, thereby enabling a continuous supply of NAD+.

[0362] In one aspect of the present invention, the lifespan-extending active ingredient may mean an ingredient that contributes to delaying aging at the cellular level and extending healthy lifespan. The lifespan-extending active ingredient may target one or more of telomere shortening, cellular aging, loss of protein homeostasis, mitochondrial dysfunction, and trophic sensing dysregulation, which are known characteristics of aging.

[0363] In one embodiment, the telomere protective component may be a component that delays telomere shortening through telomerase activation. The telomere is a repetitive DNA sequence at the end of a chromosome and may have a structure in which the TTAGGG sequence is repeated thousands of times. The telomere may be shortened by 50 to 200 base pairs due to the limitations of the DNA replication mechanism during cell division, and cell aging or apoptosis may be induced when a critical length is reached. The telomerase is a ribonucleoprotein complex having reverse transcriptase activity and can maintain telomere length by synthesizing the TTAGGG repetitive sequence at the end of the telomere.

[0364] In one embodiment, the senolitic component may be a component that selectively removes senescent cells. The senescent cells are cells whose cell cycle has been irreversibly arrested and may be characterized by increased expression of p16INK4a and p21CIP1, increased senescence-related beta-galactosidase activity, and a senescence-related secretory phenotype. The senescence-related secretory phenotype may include the secretion of interleukin-6, interleukin-8, monocyte chemotactic protein-1, and matrix metalloproteinases, and may contribute to chronic inflammation and functional decline of surrounding tissues.

[0365] In one embodiment, the autophagy-inducing component may be a component that promotes the degradation of damaged proteins and organelles within the cell. The autophagy may be a process in which cytoplasmic substances are isolated into autophagosomes, which are double-membrane vesicles, and then fused with lysosomes to degrade them. The autophagy may be classified into macroautophagy, microautophagy, and chaperone-mediated autophagy, and macroautophagy may be the most studied form. The autophagy may be induced by mTOR inhibition or AMPK activation, and the ATG protein family may mediate the formation of autophagosomes.

[0366] For example, the lifespan-extending active ingredient may include a combination of ingredients targeting multiple pathways, and the combination may provide a synergistic lifespan-extending effect by simultaneously regulating two or more of the NAD+ / sirtuin pathway, mTOR / autophagy pathway, telomerase activation pathway, and senolitic pathway.

[0367] In one aspect of the present invention, the delivery vehicle may be configured to simultaneously achieve improvement in sleep quality and extension of lifespan. There may be a biological correlation between sleep and lifespan extension.

[0368] In one embodiment, the glymphatic system may be activated during sleep. The glymphatic system may be a system in which cerebrospinal fluid flows into the brain parenchyma through the perivascular space to remove metabolic waste. The glymphatic system may be activated as the extracellular space expands due to the contraction of glial cells during sleep, and may contribute to the removal of amyloid beta and tau proteins.

[0369] In one embodiment, sleep deprivation can accelerate telomere shortening. When sleep duration is short or sleep quality is poor, oxidative stress increases and the secretion of inflammatory cytokines may be promoted, which can accelerate telomere shortening. Quality sleep can support antioxidant defense and DNA repair processes essential for telomere maintenance.

[0370] In one embodiment, growth hormone secretion may increase during sleep. The growth hormone may be secreted intensively during slow-wave sleep and may contribute to cell regeneration, tissue repair, and protein synthesis. The growth hormone may promote the production of IGF-1, and the IGF-1 may regulate cell growth and survival.

[0371] For example, the sleep-inducing component released from the shell can promote sleep onset and induce high-quality sleep, and the lifespan-extending active component can be released from the core in synchronization with the glymphatic system, growth hormone secretion, and DNA repair processes activated during sleep. Through this synchronized release, the sleep recovery function can be maximized and the lifespan-extending effect can be enhanced.

[0372] In one aspect of the present invention, the lifespan-extending active ingredient may be included in the core portion and released during the latter part of sleep. The release timing may coincide with a period of active cell regeneration.

[0373] In one embodiment, the nicotinamide mononucleotide may be released at a time when 4 to 8 hours have elapsed after administration. This time may correspond to a period when slow-wave sleep decreases and REM sleep increases, and may be a period when cellular energy metabolism is active. The release of the nicotinamide mononucleotide may increase intracellular NAD+ levels and contribute to sirtuin activation and improvement of mitochondrial function.

[0374] In one embodiment, the spermidine may be released at a time when 5 to 8 hours have elapsed after administration. By inducing autophagy at this time, the removal of damaged proteins and organelles accumulated during sleep may be promoted.

[0375] In one embodiment, the fisetin may be released at a time when 6 to 8 hours have elapsed since administration. At this time, the senolytic effect may be exerted, thereby contributing to the removal of senescent cells.

[0376] For example, the lifespan-extending active ingredients included in the core portion may be released sequentially in the order of nicotinamide mononucleotide, spermidine, and fisetin, and through this sequential release, a continuous process of NAD+ supplementation -> autophagy induction -> senescent cell removal may proceed.

[0377] In one aspect of the present invention, a carrier produced by the manufacturing method illustrated in FIG. 1 may include a lifespan-extending active ingredient. In the bio-ink preparation step (S10), the lifespan-extending active ingredient may be added to the first bio-ink. The lifespan-extending active ingredient may be mixed with the anti-aging active ingredient in the first bio-ink and may be encapsulated within the core portion through the coaxial nozzle printing step (S20), the multi-stage crosslinking step (S30), and the freeze-drying step (S40).

[0378] The present invention will be explained in more detail below using examples. However, the following examples are intended only to explain the structure and effects of the present invention and are not intended to limit the scope of the invention.

[0379] Examples 1-1 to 1-5 and Comparative Examples 1-1 to 1-2

[0380] A second bio-ink was prepared by dissolving 2.5 wt% sodium alginate, 0.5 wt% hyaluronic acid, 1.0 wt% melatonin, 5.0 wt% GABA, and 3.0 wt% L-theanine in phosphate-buffered saline. A first bio-ink was prepared by dissolving 10 wt% methacrylated gelatin, 5 wt% polycaprolactone, 3.0 wt% nicotinamide mononucleotide, 1.0 wt% resveratrol, and 0.2 wt% lithium phenyl-2,4,6-trimethylbenzoylphosphinate as a photoinitiator in phosphate-buffered saline. The viscosity of the first bio-ink was 25,000 cP at 25°C, and the viscosity of the second bio-ink was 2,500 cP at 25°C.

[0381] An ink for a diffusion barrier layer was prepared by dispersing 1.0 wt% of TEMPO oxidized cellulose nanofibrils in deionized water. An ink for an intermediate layer was prepared by dissolving 15 wt% of polylactic-co-glycolic acid, 5.0 wt% of magnesium glycinate, and 0.5 wt% of sustained-release melatonin in a mixed solvent of dichloromethane and dimethyl sulfoxide.

[0382] Using a coaxial nozzle bioprinter, the first bio-ink was simultaneously ejected through an inner nozzle with an inner diameter of 300 Ìm and the second bio-ink through an outer nozzle with an inner diameter of 700 Ìm. The printing temperature was set to 28℃ and the nozzle travel speed to 15 mm / s. The thickness ratio of the core part to the shell part was controlled to 1:3. After printing the core part, the ink for the diffusion barrier layer was applied to a thickness of 50 Ìm, and then the ink for the intermediate layer was printed to a thickness of 1,000 Ìm.

[0383] To control the porosity of the shell portion, the spacing of the printing path was changed. Example 1-1 was printed to have a porosity of 40%, Example 1-2 to have a porosity of 50%, Example 1-3 to have a porosity of 55%, Example 1-4 to have a porosity of 65%, and Example 1-5 to have a porosity of 70%. Comparative Example 1-1 was printed to have a porosity of 30%, and Comparative Example 1-2 to have a porosity of 80%. The pore size was all set to the same value of 100 µm.

[0384] After printing is complete, ionic crosslinking is performed by immersion in a 1.0 wt% aqueous calcium chloride solution for 5 minutes, followed by irradiation with 365 nm wavelength ultraviolet light at 10 mW / cm² 2 Photocrosslinking was performed by irradiating with an intensity of 3 minutes. The crosslinked structure was pre-frozen at -80°C for 12 hours, and then freeze-dried for 36 hours at a pressure of 0.05 mbar and a temperature of -50°C to obtain a carrier. The diameter of the obtained carrier was approximately 8 mm.

[0385] Examples 2-1 to 2-3 and Comparative Examples 2-1 to 2-2

[0386] The same bio-ink composition and manufacturing method as in Examples 1-3 were used, but the shell thickness was changed by adjusting the external nozzle discharge amount of the coaxial nozzle. Example 2-1 was manufactured to have a shell thickness of 200 Ìm, Example 2-2 to have a shell thickness of 600 Ìm, and Example 2-3 to have a shell thickness of 1,000 Ìm. Comparative Example 2-1 was manufactured to have a shell thickness of 100 Ìm, and Comparative Example 2-2 to have a shell thickness of 1,200 Ìm. Other process conditions were applied identically to those in Examples 1-3.

[0387] Examples 3-1 to 3-4

[0388] The same manufacturing method as in Examples 1-3 was used, but the content of the active ingredients was changed. Example 3-1 used 0.1 wt% melatonin, 1.0 wt% GABA, and 0.5 wt% nicotinamide mononucleotide. Example 3-2 used 2.5 wt% melatonin, 5.0 wt% GABA, and 5.0 wt% nicotinamide mononucleotide. Example 3-3 used 5.0 wt% melatonin, 10.0 wt% GABA, and 10.0 wt% nicotinamide mononucleotide. Example 3-4 added 0.05 wt% stem cell-derived exosomes and 2.0 wt% glutathione to the composition of Example 3-2. Other process conditions were applied identically to those in Examples 1-3.

[0389] Examples 4-1 to 4-3 and Comparative Examples 4-1 to 4-2

[0390] The same bio-ink composition and printing conditions as in Examples 1-3 were used, but the concentration of the calcium chloride aqueous solution was changed during ionic crosslinking. Example 4-1 was immersed in a 0.5 wt% calcium chloride aqueous solution for 5 minutes, Example 4-2 in a 1.0 wt% calcium chloride aqueous solution for 5 minutes, and Example 4-3 in a 2.0 wt% calcium chloride aqueous solution for 5 minutes. Comparative Example 4-1 was immersed in a 0.3 wt% calcium chloride aqueous solution for 5 minutes, and Comparative Example 4-2 in a 2.5 wt% calcium chloride aqueous solution for 5 minutes. The photocrosslinking and freeze-drying conditions were applied in the same way as in Examples 1-3.

[0391] Examples 5-1 to 5-3

[0392] N-isopropylacrylamide and acrylic acid were mixed in a molar ratio of 90:10, and 1.0 mol% of ammonium persulfate and 1.0 mol% of tetramethylethylenediamine were added to perform radical polymerization at 25°C for 8 hours. After the reaction was complete, the mixture was dialyzed for 48 hours using a dialysis membrane with a molecular weight cutoff of 12,000 Da and freeze-dried to obtain a poly-N-isopropylacrylamide-co-acrylic acid copolymer. The lower critical melting temperature of the copolymer was 33°C.

[0393] In Example 5-1, the copolymer was added to the second bio-ink at 5 wt% and applied to the shell portion. In Example 5-2, the copolymer was added to the ink for the intermediate layer at 5 wt% and applied to the intermediate layer portion. In Example 5-3, the copolymer was added to the first bio-ink at 3 wt% and applied to the core portion. Other manufacturing conditions were applied in the same manner as in Example 1-3.

[0394] Examples 6-1 to 6-3

[0395] The carriers prepared according to Examples 1-3 were immersed in a coating solution containing 2 mg / mL of dopamine hydrochloride and 10 mM Tris-hydrochloride buffer. The pH of the coating solution was adjusted to 8.5. Polydopamine coating was performed in an open container at 25°C under gentle stirring conditions. Coating was performed for 2 hours for Example 6-1, 4 hours for Example 6-2, and 6 hours for Example 6-3. After coating was completed, the carriers were washed three times with deionized water and dried at room temperature.

[0396] Examples 7-1 to 7-3 and Comparative Examples 7-1 to 7-2

[0397] Cholesteryl oleate and cholesteryl nonanoate were melt-mixed at 60°C. In Example 7-1, the two components were mixed in a weight ratio of 7:3, in Example 7-2 in a weight ratio of 8:2, and in Example 7-3 in a weight ratio of 9:1. In Comparative Example 7-1, a weight ratio of 5:5 was used, and in Comparative Example 7-2, cholesteryl oleate was used alone.

[0398] After completing printing up to the intermediate layer according to Examples 1-3, the molten cholesteryl ester mixture was applied by a dip coating method while maintaining the temperature at 37°C. The application amount was 1.0 mg / cm² relative to the surface area of ​​the carrier. 2 After coating, the smectic liquid crystal phase was formed by slow cooling to 25°C at a rate of 1°C / min. Subsequently, shell printing, subsequent crosslinking, and freeze-drying processes were performed in the same manner as in Examples 1-3.

[0399] Examples 8-1 to 8-3 and Comparative Examples 8-1 to 8-2

[0400] A metal ion solution was prepared by dissolving 0.1 mg / mL of titanium butoxide in a 1:1 volume ratio mixed solvent of ethanol and water. An aqueous tannic acid solution was prepared by dissolving 0.4 mg / mL of tannic acid in deionized water.

[0401] After completing the core printing according to Examples 1-3, one cycle was defined as immersing the surface of the core in the metal ion solution for 1 minute, washing with deionized water, immersing it in the aqueous tannic acid solution for 1 minute, and washing with deionized water. Example 8-1 was performed for 3 cycles, Example 8-2 for 4 cycles, and Example 8-3 for 5 cycles. Comparative Example 8-1 was performed for 2 cycles, and Comparative Example 8-2 for 7 cycles. After the metal-phenolic network coating was completed, it was dried by nitrogen blowing, and then the printing of the intermediate layer and shell, and subsequent processes, were performed in the same manner as in Examples 1-3.

[0402] Examples 9-1 to 9-3 and Comparative Example 9-1

[0403] 100 mg of mono-6-deoxy-6-amino-beta-cyclodextrin was dissolved in 10 mL of 2-morpholinoethanesulfonic acid buffer with 60 mg of 1-ethyl-3-dimethylaminopropylcarbodiimide and 36 mg of N-hydroxysuccinimide and activated at room temperature for 30 minutes. 1 g of methacrylated gelatin was dissolved in 50 mL of phosphate-buffered saline at 40°C, after which the activated beta-cyclodextrin solution was added and reacted at 40°C for 12 hours. After the reaction was complete, the gelatin was dialyzed at 40°C for 3 days using a dialysis membrane with a molecular weight cutoff of 12,000 Da and freeze-dried to obtain methacrylated gelatin conjugated with beta-cyclodextrin.

[0404] 1 g of 4-arm polyethylene glycol-amine was dissolved in dichloromethane, and 40 mg of 1-adamantan carboxylic acid was activated with 45 mg of dicyclohexylcarbodiimide and 25 mg of N-hydroxysuccinimide and added, and the mixture was reacted at room temperature for 24 hours. 4-arm polyethylene glycol-adamantan was obtained by filtration and ether precipitation.

[0405] Example 9-1 used beta-cyclodextrin-methacrylated gelatin with a beta-cyclodextrin conjugation rate of 5 mol%. Example 9-2 used a conjugation rate of 10 mol%, and Example 9-3 used a conjugation rate of 15 mol%. Comparative Example 9-1 used a conjugation rate of 3 mol%. The above beta-cyclodextrin-methacrylated gelatin and the above 4-arm polyethylene glycol-adamantan were mixed in a 1:1 molar ratio to prepare a bio-ink for the core, and a carrier was prepared in the same manner as in Example 1-3.

[0406] Examples 10-1 to 10-3

[0407] When preparing a carrier containing a core portion of a supramolecular cross-linked structure prepared according to Example 9-2, 1-aminoadamantan hydrochloride was added to the ink for the intermediate layer. 0.1 wt% of 1-aminoadamantan hydrochloride was added in Example 10-1, 0.5 wt% in Example 10-2, and 1.0 wt% in Example 10-3. Other manufacturing conditions were applied in the same manner as in Example 9-2.

[0408] Examples 11-1 to 11-3 and Comparative Examples 11-1 to 11-2

[0409] Solution A was prepared by dissolving 0.3 g of Zn(NO3)2O6H2O and 50 mg of nicotinamide mononucleotide in 10 mL of methanol. Solution B was prepared by dissolving 0.66 g of 2-methylimidazole in 10 mL of methanol. Solution B was added dropwise to Solution A while stirring, and then left to stand at room temperature for 24 hours. The mixture was centrifuged at 10,000 × g for 15 minutes, washed three times with methanol, and vacuum dried at 60°C to obtain ZIF-8 nanoparticles loaded with nicotinamide mononucleotide.

[0410] Nanoparticles of different particle sizes were prepared by changing the synthesis conditions. Nanoparticles with a particle size of 80 nm were obtained in Example 11-1, 120 nm in Example 11-2, and 180 nm in Example 11-3. Nanoparticles with a particle size of 40 nm were obtained in Comparative Example 11-1 and 250 nm in Comparative Example 11-2. The above nanoparticles were dispersed in 5 wt% of the first bio-ink, and a carrier was prepared in the same manner as in Example 1-3.

[0411] Examples 12-1 to 12-3 and Comparative Example 12-1

[0412] 500 mg of branched polyethyleneimine was dissolved in 20 mL of anhydrous ethanol, ferrocene carboxyaldehyde was added, and the mixture was reacted at 50°C for 6 hours to form Schiff base bonds. 30 mg of sodium cyanoborohydride was added, and the mixture was reduced and stabilized at room temperature for 2 hours. The mixture was dialyzed for 24 hours using a dialysis membrane with a molecular weight cutoff of 3,500 Da and freeze-dried to obtain a ferrocene-polyethyleneimine crosslinking agent.

[0413] Example 12-1 used a crosslinking agent with a ferrocene carboxyaldehyde bonding rate of 3 mol%. Example 12-2 used a bonding rate of 6 mol%, and Example 12-3 used a bonding rate of 10 mol%. Comparative Example 12-1 used a bonding rate of 2 mol%. 2 wt% of the above crosslinking agent was added to the first bio-ink, and a carrier was prepared in the same manner as in Example 1-3.

[0414] Example 13

[0415] A first bio-ink was prepared using nicotinamide mononucleotide-supported ZIF-8 nanoparticles prepared according to Example 11-2, beta-cyclodextrin-methacrylated gelatin and 4-arm polyethylene glycol-adamantan synthesized according to Example 9-2, and a ferrocene-polyethyleneimine crosslinking agent synthesized according to Example 12-2. The first bio-ink contained 10 wt% beta-cyclodextrin-methacrylated gelatin, 2 wt% 4-arm polyethylene glycol-adamantan, 5 wt% ZIF-8 nanoparticles, 2 wt% ferrocene-polyethyleneimine crosslinking agent, 1.0 wt% resveratrol, and 0.2 wt% photoinitiator.

[0416] A second bio-ink was prepared containing 5 wt% of poly-N-isopropylacrylamide-co-acrylic acid copolymer synthesized according to Example 5-1. An ink for an intermediate layer was prepared containing 0.5 wt% of 1-aminoadamantan hydrochloride according to Example 10-2.

[0417] After forming the core portion by coaxial nozzle printing, a metal-phenolic network coating was performed for 4 cycles according to Example 8-2. Subsequently, an intermediate layer was printed, and a cholesteryl ester liquid crystal layer was formed according to Example 7-2, after which the shell portion was printed. Ionic crosslinking, photocrosslinking, and freeze-drying were performed in the same manner as in Example 1-3. Finally, a polydopamine coating was performed for 4 hours according to Example 6-2 to obtain a composite carrier.

[0418] Examples 14-1 to 14-3

[0419] Various final forms of carriers were prepared by using the same bio-ink composition as in Examples 1-3, but changing the printing shape and post-treatment.

[0420] In Example 14-1, spherical oral multi-pellets with a diameter of 5 mm were prepared. After freeze-drying, they were immersed in Eudragit L100-55 coating solution to form an enteric coating layer with a thickness of 80 Ìm.

[0421] In Example 14-2, a skin-attachable microneedle patch was manufactured. A conical microneedle array with a length of 600 Ìm and a base diameter of 300 Ìm was formed using a silicone mold. The shell composition was sequentially filled in the tip of the microneedle, the middle layer composition in the middle, and the core composition in the base.

[0422] In Example 14-3, an implantable scaffold was manufactured. It was printed in the shape of a rectangular prism measuring 20 mm × 10 mm × 5 mm, and a microchannel with a diameter of 300 Ìm was formed in a grid shape inside.

[0423] Examples 15-1 to 15-3 and Comparative Example 15-1

[0424] The same manufacturing method as in Examples 1-3 was used, but a telomere protective component was added to the first bio-ink. A telomere protective component composition was prepared by mixing cycloastragenol and astragaloside IV in a weight ratio of 1:20.

[0425] In Example 15-1, the telomere protective component composition was added to the first bio-ink at 0.01 wt% based on cycloastragenol and 0.2 wt% based on astragalloside IV. In Example 15-2, it was added at 0.1 wt% based on cycloastragenol and 2.0 wt% based on astragalloside IV. In Example 15-3, it was added at 0.5 wt% based on cycloastragenol and 5.0 wt% based on astragalloside IV. In Comparative Example 15-1, it was added at 0.005 wt% based on cycloastragenol and 0.05 wt% based on astragalloside IV. Other process conditions were applied in the same manner as in Example 1-3.

[0426] Examples 16-1 to 16-4 and Comparative Examples 16-1 to 16-2

[0427] The same manufacturing method as in Examples 1-3 was used, but a senolic component was added to the first bio-ink.

[0428] In Example 16-1, 2.0 wt% of fisetin was added to the first bio-ink. In Example 16-2, 5.0 wt% of fisetin was added. In Example 16-3, 3.0 wt% of quercetin was added. In Example 16-4, 3.0 wt% of fisetin and 3.0 wt% of quercetin were added in combination at a weight ratio of 1:1. In Comparative Example 16-1, 0.3 wt% of fisetin was added. In Comparative Example 16-2, 12.0 wt% of fisetin was added. The fisetin and quercetin were pre-dissolved in dimethyl sulfoxide and then dispersed in the first bio-ink. Other process conditions were applied in the same manner as in Example 1-3.

[0429] Examples 17-1 to 17-4 and Comparative Example 17-1

[0430] The same manufacturing method as in Examples 1-3 was used, but an autophagy-inducing component was added to the first bio-ink.

[0431] Example 17-1 added 0.5 wt% of spermidine trihydrochloride to the first bio-ink. Example 17-2 added 2.0 wt% of spermidine trihydrochloride. Example 17-3 added 5.0 wt% of trehalose. Example 17-4 added 1.0 wt% of spermidine trihydrochloride and 8.0 wt% of trehalose in combination. Comparative Example 17-1 added 0.05 wt% of spermidine trihydrochloride. Other process conditions were applied in the same manner as in Example 1-3.

[0432] Examples 18-1 to 18-3 and Comparative Example 18-1

[0433] The same manufacturing method as in Examples 1-3 was used, but an AMPK activating component was added to the first bio-ink.

[0434] A 10 wt% solution was prepared by pre-dissolving berberine hydrochloride in deionized water. In Example 18-1, 0.5 wt% of berberine hydrochloride was added to the first bio-ink. In Example 18-2, 2.0 wt% of berberine hydrochloride was added. In Example 18-3, 3.0 wt% of berberine hydrochloride and 1.0 wt% of ginsenoside Rg3 were added in combination. In Comparative Example 18-1, 0.2 wt% of berberine hydrochloride was added. Other process conditions were applied in the same manner as in Example 1-3.

[0435] Examples 19-1 to 19-4 and Comparative Example 19-1

[0436] The same manufacturing method as in Examples 1-3 was used, but a mitochondrial-targeted antioxidant component was added to the first bio-ink.

[0437] A 20 wt% suspension was prepared by pre-dispersing Coenzyme Q10 (ubiquinol form) in medium-chain triglycerides. In Example 19-1, 1.0 wt% of Coenzyme Q10 was added to the first bio-ink. In Example 19-2, 3.0 wt% of Coenzyme Q10 was added. In Example 19-3, 0.1 wt% of pyrroloquinoline quinone disodium salt was added. In Example 19-4, 2.0 wt% of Coenzyme Q10, 0.1 wt% of pyrroloquinoline quinone disodium salt, and 1.0 wt% of alpha-lipoic acid were added in combination. In Comparative Example 19-1, 0.2 wt% of Coenzyme Q10 was added. Other process conditions were applied in the same manner as in Example 1-3.

[0438] Examples 20-1 to 20-3

[0439] The same manufacturing method as in Examples 1-3 was used, but an amino acid-based lifespan-extending component was added to the first bio-ink.

[0440] In Example 20-1, 5.0 wt% of glycine was added to the first bio-ink. In Example 20-2, 5.0 wt% of taurine was added. In Example 20-3, 3.0 wt% of glycine and 3.0 wt% of taurine were added in combination. The glycine and taurine were pre-dissolved in phosphate-buffered saline and then mixed into the first bio-ink. Other process conditions were applied in the same manner as in Examples 1-3.

[0441] Examples 21-1 to 21-3 and Comparative Example 21-1

[0442] The same manufacturing method as in Examples 1-3 was used, but alpha-ketoglutarate or a salt thereof was added to the first bio-ink.

[0443] In Example 21-1, 1.0 wt% of calcium alpha-ketoglutarate was added to the first bio-ink. In Example 21-2, 3.0 wt% of calcium alpha-ketoglutarate was added. In Example 21-3, 2.0 wt% of sodium alpha-ketoglutarate was added. In Comparative Example 21-1, 0.2 wt% of calcium alpha-ketoglutarate was added. Other process conditions were applied in the same manner as in Example 1-3.

[0444] Example 22

[0445] Based on the method for manufacturing an integrated composite delivery system prepared according to Example 13, a lifespan-extending active ingredient was added to the first bio-ink. The first bio-ink contained 10 wt% beta-cyclodextrin-methacrylated gelatin, 2 wt% 4-arm polyethylene glycol-adamantan, 5 wt% nicotinamide mononucleotide-supported ZIF-8 nanoparticles, 2 wt% ferrocene-polyethyleneimine crosslinking agent, 1.0 wt% resveratrol, and 0.2 wt% photoinitiator.

[0446] 1.0 wt% spermidine trihydrochloride, 3.0 wt% fisetin, 0.1 wt% cycloastragenol, 2.0 wt% coenzyme Q10, and 3.0 wt% glycine were additionally added to the first bio-ink. 0.5 wt% 1-aminoadamantan hydrochloride and 1.0 wt% nicotinamide riboside were added to the ink for the intermediate layer.

[0447] After forming a core portion by coaxial nozzle printing, a metal-phenolic network coating was performed for 4 cycles, and an intermediate layer was printed. After forming a cholesteryl ester liquid crystal layer, a shell portion was printed, followed by ion crosslinking, photocrosslinking, and freeze-drying. Finally, a polydopamine coating was performed for 4 hours to obtain a composite carrier incorporating a life-extending active ingredient.

[0448] Examples 23-1 to 23-2

[0449] The same manufacturing method as in Examples 1-3 was used, but a NAD+ precursor auxiliary component was added to the ink for the intermediate layer.

[0450] In Example 23-1, 1.0 wt% of nicotinamide riboside chloride was added to the ink for the intermediate layer. In Example 23-2, 0.5 wt% of nicotinamide riboside chloride and 2.0 wt% of L-tryptophan were added in combination. The first bio-ink contained 3.0 wt% of nicotinamide mononucleotide, configured so that nicotinamide riboside in the intermediate layer is released first, followed by nicotinamide mononucleotide in the core. Other process conditions were applied in the same manner as in Example 1-3.

[0451] Experimental Example 1

[0452] The differential release characteristics of the carriers prepared in Examples 1-1 to 1-5 and Comparative Examples 1-1 to 1-2 were evaluated. The carriers were immersed in an artificial serous solution at 37°C and stirred at 50 rpm using the paddle method. Test samples were collected at 0.5, 1, 2, 4, 6, and 8 hours, and the concentrations of melatonin and nicotinamide mononucleotides were quantified using high-performance liquid chromatography. Each test was repeated three times.

[0453] In Example 1-1, the melatonin release rate was 72.3 ± 3.1% at 30 minutes, and the nicotinamide mononucleotide release rate was 78.5 ± 4.2% at 8 hours. In Example 1-2, the melatonin release rate was 78.6 ± 2.8% at 30 minutes, and the nicotinamide mononucleotide release rate was 82.1 ± 3.5% at 8 hours. In Example 1-3, the melatonin release rate was 83.2 ± 2.5% at 30 minutes, and the nicotinamide mononucleotide release rate was 85.7 ± 3.1% at 8 hours. In Example 1-4, the melatonin release rate was 86.8 ± 2.2% at 30 minutes, and the nicotinamide mononucleotide release rate was 88.3 ± 2.8% at 8 hours. In the case of Examples 1-5, the melatonin release rate was 89.1 ± 2.0% at 30 minutes, and the nicotinamide mononucleotide release rate was 87.2 ± 3.3% at 8 hours.

[0454] In the case of Comparative Example 1-1, the melatonin release rate at 30 minutes was 58.4 ± 4.5%, which was lower than that of the above examples, and this was observed to be due to delayed penetration of body fluids caused by the low porosity. In the case of Comparative Example 1-2, the melatonin release rate at 30 minutes was high at 95.2 ± 1.8%, but due to the structural collapse of the shell part, the core part component was released early, and the nicotinamide mononucleotide release rate reached 95.8 ± 2.1% at 8 hours, and 82.3 ± 3.5% was already released at 4 hours, so it was observed that the differential release characteristics were degraded.

[0455] Experimental Example 2

[0456] The structure of the carriers prepared in Examples 2-1 to 2-3 and Comparative Examples 2-1 to 2-2 was observed using a scanning electron microscope, and the shell dissolution time was measured.

[0457] The shell thickness of Example 2-1 was measured as 198 ± 12 Ìm, and the time for complete dissolution of the shell was 18 ± 2 minutes. The shell thickness of Example 2-2 was measured as 605 ± 25 Ìm, and the time for complete dissolution of the shell was 32 ± 3 minutes. The shell thickness of Example 2-3 was measured as 1,015 ± 38 Ìm, and the time for complete dissolution of the shell was 48 ± 4 minutes.

[0458] The shell thickness of Comparative Example 2-1 was measured to be 95 ± 8 Ìm, and the time for complete dissolution of the shell was short at 8 ± 1 min. Under the above conditions, it was predicted that the increase in blood concentration of the sleep-inducing component would be excessively rapid. The shell thickness of Comparative Example 2-2 was measured to be 1,210 ± 45 Ìm, and the time for complete dissolution of the shell was long at 65 ± 5 min, so a delay in the sleep-inducing effect was predicted.

[0459] Experimental Example 3

[0460] The compressive strength and decomposition time of the carriers prepared in Examples 4-1 to 4-3 and Comparative Examples 4-1 to 4-2 were measured.

[0461] The compressive strength of Example 4-1 was 15.3 ± 1.2 kPa, and the complete decomposition time was 7.2 ± 0.5 hours. The compressive strength of Example 4-2 was 28.5 ± 2.1 kPa, and the complete decomposition time was 8.5 ± 0.4 hours. The compressive strength of Example 4-3 was 42.1 ± 3.5 kPa, and the complete decomposition time was 10.2 ± 0.6 hours.

[0462] The compressive strength of Comparative Example 4-1 was low at 8.2 ± 0.8 kPa, making it difficult to maintain its shape during handling, and the complete decomposition time was short at 5.8 ± 0.4 hours. The compressive strength of Comparative Example 4-2 was high at 55.3 ± 4.2 kPa, but due to excessive crosslinking, the complete decomposition time was extended to 14.5 ± 0.8 hours, making it difficult to synchronize with an 8-hour sleep cycle.

[0463] Experimental Example 4

[0464] The lower critical melting temperature of the poly-N-isopropylacrylamide-co-acrylic acid copolymer used in Examples 5-1 to 5-3 was measured using a UV-visible spectrophotometer. The temperature at which the transmittance at a wavelength of 500 nm becomes 50% was defined as the lower critical melting temperature. As a result of the measurement, the lower critical melting temperature of the copolymer was 33.2 ± 0.3℃.

[0465] Release tests were performed on the carrier prepared in Example 5-1 in artificial serous fluid at 32°C and 37°C, respectively. The melatonin release rate at 30 minutes at 32°C was 68.5 ± 3.2%, and at 37°C it was 88.3 ± 2.5%. An increase in release rate with increasing temperature was observed, which was determined to be due to changes in the pore structure caused by the phase transition of the copolymer.

[0466] Experimental Example 5

[0467] The thickness of the polydopamine coating layer of the carriers prepared in Examples 6-1 to 6-3 was measured by ellipsometry, and the contact angle and 2,2-diphenyl-1-picrylhydrazyl radical scavenging ability were evaluated.

[0468] The thickness of the polydopamine coating layer in Example 6-1 was 15 ± 2 nm, the contact angle was 52 ± 3°, and the 2,2-diphenyl-1-picrylhydrazil radical scavenging activity was 28.5 ± 2.1%. The thickness of the polydopamine coating layer in Example 6-2 was 32 ± 3 nm, the contact angle was 45 ± 2°, and the 2,2-diphenyl-1-picrylhydrazil radical scavenging activity was 45.2 ± 3.5%. The thickness of the polydopamine coating layer in Example 6-3 was 48 ± 4 nm, the contact angle was 38 ± 2°, and the 2,2-diphenyl-1-picrylhydrazil radical scavenging activity was 58.7 ± 4.2%.

[0469] The contact angle of the uncoated carrier was 78 ± 4°, and it was observed that the surface hydrophilicity increased due to the polydopamine coating.

[0470] Experimental Example 6

[0471] The phase transition temperature of the cholesteryl ester mixture used in Examples 7-1 to 7-3 and Comparative Examples 7-1 to 7-2 was measured using a differential scanning calorimeter. The heating rate was set to 2°C / min.

[0472] The phase transition temperature of Example 7-1 was 35.2 ± 0.2℃. The phase transition temperature of Example 7-2 was 36.1 ± 0.2℃. The phase transition temperature of Example 7-3 was 37.3 ± 0.3℃.

[0473] The phase transition temperature of Comparative Example 7-1 was 32.5 ± 0.3℃, which is lower than body temperature; therefore, it was predicted that when administered into the body, it would already transition to a low-viscosity phase at room temperature, and thus no temperature gating effect would occur. The phase transition temperature of Comparative Example 7-2 was 41.2 ± 0.4℃, which is higher than body temperature; therefore, it was predicted that the high-viscosity phase would be maintained within the normal body temperature range, and thus release would be excessively delayed.

[0474] Release tests were performed on the carrier prepared in Example 7-2 in artificial serous fluids at 34°C and 37°C, respectively. The release rate of nicotinamide mononucleotide at 4 hours at 34°C was 12.3 ± 1.5%, and at 37°C it was 45.8 ± 3.2%. A difference in release rate was observed around the phase transition temperature.

[0475] Experimental Example 7

[0476] The thickness of the metal-phenolic network coating layer formed in Examples 8-1 to 8-3 and Comparative Examples 8-1 to 8-2 was measured by ellipsometry, and the stability according to pH was evaluated.

[0477] The coating layer thickness of Example 8-1 was 12 ± 1 nm. The coating layer thickness of Example 8-2 was 18 ± 2 nm. The coating layer thickness of Example 8-3 was 25 ± 2 nm. The coating layer thickness of Comparative Example 8-1 was 7 ± 1 nm. The coating layer thickness of Comparative Example 8-2 was 38 ± 3 nm.

[0478] The retention rate of the coating layer was measured after immersion in phosphate-buffered saline at pH 7.4 for 24 hours. Example 8-1 showed a retention rate of 85.2 ± 3.5%, Example 8-2 showed 92.1 ± 2.8%, and Example 8-3 showed 95.3 ± 2.1%. Comparative Example 8-1 showed a low retention rate of 68.5 ± 4.2%, indicating that the diffusion barrier function was insufficient. Comparative Example 8-2 showed a retention rate of 97.8 ± 1.5%, but due to excessive thickness, the release of the core component was delayed, and the nicotinamide mononucleotide release rate was only 62.3 ± 4.5% after 8 hours.

[0479] Experimental Example 8

[0480] The supramolecular crosslinking characteristics of the carriers prepared in Examples 9-1 to 9-3 and Comparative Example 9-1 were evaluated. The carriers were immersed in a 0.5 mM solution of 1-aminoadamantan, and the degree of swelling of the core portion over time was measured.

[0481] In the case of Example 9-1, the swelling degree of the core part was 125 ± 5% after 4 hours. In the case of Example 9-2, the swelling degree of the core part was 145 ± 6% after 4 hours. In the case of Example 9-3, the swelling degree of the core part was 168 ± 8% after 4 hours. In the case of Comparative Example 9-1, the swelling degree of the core part was low at 108 ± 4% after 4 hours, indicating that the supramolecular crosslinking density was insufficient.

[0482] In phosphate-buffered saline without 1-aminoadamantan, the swelling degree of the core portion at 4 hours of Example 9-2 was 112 ± 4%, which was observed to be different from when a competitive guest was present.

[0483] Experimental Example 9

[0484] The particle size, polydispersity index, and drug loading rate of ZIF-8 nanoparticles prepared in Examples 11-1 to 11-3 and Comparative Examples 11-1 to 11-2 were measured.

[0485] The particle size of Example 11-1 was 82 ± 8 nm, the polydispersity index was 0.18, and the drug loading rate was 18.5 ± 1.2 wt%. The particle size of Example 11-2 was 118 ± 12 nm, the polydispersity index was 0.15, and the drug loading rate was 22.3 ± 1.5 wt%. The particle size of Example 11-3 was 175 ± 18 nm, the polydispersity index was 0.21, and the drug loading rate was 25.8 ± 1.8 wt%.

[0486] The particle size of Comparative Example 11-1 was 42 ± 5 nm, and the drug loading rate was low at 8.5 ± 0.8 wt%. The particle size of Comparative Example 11-2 was 255 ± 25 nm, and the polydispersity index was high at 0.35, indicating reduced uniformity.

[0487] To evaluate pH responsiveness, the nanoparticles of Example 11-2 were immersed in artificial gastric fluid at pH 1.2 and artificial intestinal fluid at pH 6.8, and the drug release rate was measured. The release rate at 2 hours at pH 1.2 was 8.2 ± 1.1%, and the release rate at 2 hours at pH 6.8 was 65.3 ± 4.2%. Selective release in the intestinal environment was observed.

[0488] Experimental Example 10

[0489] The glutathione reactivity of the carriers prepared in Examples 12-1 to 12-3 and Comparative Example 12-1 was evaluated. The carriers were immersed in a 10 mM glutathione solution and a phosphate-buffered saline solution without glutathione, and the nicotinamide mononucleotide release rate was measured.

[0490] Under the glutathione 10 mM condition, the release rate at 6 hours of Example 12-1 was 52.3 ± 3.5%, Example 12-2 was 68.5 ± 4.2%, and Example 12-3 was 78.2 ± 4.8%. The release rate at 6 hours of Comparative Example 12-1 was low at 35.2 ± 2.8%.

[0491] The release rate at 6 hours in Example 12-2 in phosphate-buffered saline without glutathione was 42.1 ± 3.2%, which was different from the case with glutathione. This was determined to be due to redox-reactive crosslinking by glutathione.

[0492] Experimental Example 11

[0493] The cytotoxicity and antioxidant activity of the components released from the carriers prepared in Examples 3-4 and Example 13 were evaluated. Cell viability was measured using the MTT assay with human skin fibroblasts, and intracellular NAD+ levels and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical scavenging activity were measured.

[0494] The viability of cells treated with the release solution of Examples 3-4 was 95.2 ± 3.5%, the intracellular NAD+ level increased by 1.35 ± 0.12 times compared to the untreated control group, and the 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical scavenging activity was 42.5 ± 3.8%.

[0495] The viability of cells treated with the release solution of Example 13 was 93.8 ± 4.1%, the intracellular NAD+ level increased by 1.52 ± 0.15 times compared to the untreated control group, and the 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical scavenging activity was 58.3 ± 4.5%.

[0496] The viability of cells treated with the release solution of an empty carrier that does not contain active ingredients was 98.5 ± 2.1%, and no cytotoxicity of the carrier material itself was observed.

[0497] Experimental Example 12

[0498] The 8-hour time-lag release profile of the integrated composite delivery system prepared in Example 13 was evaluated. The release rates of melatonin, magnesium, and nicotinamide mononucleotides over time were measured in an artificial serous fluid at 37°C.

[0499] For melatonin, the release rate was 82.5 ± 2.8% at 30 minutes and reached 92.1 ± 2.1% at 1 hour. For magnesium, the release rate was 35.2 ± 2.5% at 2 hours, 72.8 ± 3.2% at 4 hours, and reached 88.5 ± 2.8% at 6 hours. For nicotinamide mononucleotide, the release rate was 8.5 ± 1.2% at 2 hours, 28.3 ± 2.5% at 4 hours, 58.2 ± 3.5% at 6 hours, and reached 85.3 ± 3.1% at 8 hours.

[0500] The above release profile exhibited a three-stage time-lag release pattern of rapid release of the sleep-inducing component, mid-sleep release of the sleep-maintaining component, and delayed release of the anti-aging active component in the latter part of sleep. This pattern was observed to be temporally consistent with the sleep latency, deep sleep, and growth hormone secretion phases of the human sleep cycle.

[0501] Experimental Example 13

[0502] The telomerase activating effect of the components released from the carriers prepared in Examples 15-1 to 15-3 and Comparative Example 15-1 was evaluated. Human skin fibroblasts (HDFs) were induced to age by 10 passages and then treated with the released solution of the carriers for 72 hours. Telomerase activity was measured using the TRAP assay, and relative telomere length (T / S ratio) was measured using quantitative real-time polymerase chain reaction. Each experiment was repeated three times.

[0503] The telomerase activity of cells treated with the release solution of Example 15-1 increased by 1.25 ± 0.08 times compared to the untreated control group, and the relative telomere length increased by 1.12 ± 0.05 times. The telomerase activity of cells treated with the release solution of Example 15-2 increased by 1.58 ± 0.12 times compared to the control group, and the relative telomere length increased by 1.28 ± 0.08 times. The telomerase activity of cells treated with the release solution of Example 15-3 increased by 1.82 ± 0.15 times compared to the control group, and the relative telomere length increased by 1.35 ± 0.10 times.

[0504] The telomerase activity of cells treated with the release solution of Comparative Example 15-1 was 1.08 ± 0.05 times that of the control group, with no significant increase observed, and the relative telomere length was 1.03 ± 0.04 times that of the control group, which was similar to the control group. From the above results, it was observed that a telomerase activating effect occurred at a content of 0.01 wt% or more of cycloastrazenol and 0.2 wt% or more of astragaloside IV.

[0505] Experimental Example 14

[0506] The senolytic effects of the carriers prepared in Examples 16-1 to 16-4 and Comparative Examples 16-1 to 16-2 were evaluated. Human skin fibroblasts were irradiated with 10 Gy to induce aging and cultured for 7 days. After aging induction, the cells were treated with the release solution of the carriers for 48 hours, and aging-related beta-galactosidase staining, measurement of p16INK4a expression levels, and evaluation of cell viability were performed.

[0507] The proportion of beta-galactosidase-positive cells in cells treated with the release solution of Example 16-1 decreased to 68.5 ± 4.2% compared to untreated senescent cells, and p16INK4a expression decreased to 0.72 ± 0.08 times. The proportion of beta-galactosidase-positive cells in cells treated with the release solution of Example 16-2 decreased to 52.3 ± 3.8%, and p16INK4a expression decreased to 0.55 ± 0.06 times. The proportion of beta-galactosidase-positive cells in cells treated with the release solution of Example 16-3 decreased to 58.2 ± 4.5%, and p16INK4a expression decreased to 0.62 ± 0.07 times. The proportion of beta-galactosidase-positive cells in cells treated with the release solution of Example 16-4 decreased to 45.8 ± 3.2%, and p16INK4a expression decreased to 0.48 ± 0.05 times, which suggested a synergistic effect from the combined use of fisetin and quercetin.

[0508] The proportion of beta-galactosidase-positive cells in cells treated with the release solution of Comparative Example 16-1 was 92.5 ± 3.5%, indicating an insufficient effect in removing senescent cells. In the case of cells treated with the release solution of Comparative Example 16-2, cytotoxicity to normal cells was observed, and the overall cell viability decreased to 65.2 ± 5.8%, which was determined to be due to the excessive fisetin content.

[0509] Experimental Example 15

[0510] The autophagy-inducing effect of the carriers prepared in Examples 17-1 to 17-4 and Comparative Example 17-1 was evaluated. Human skin fibroblasts were treated with the release solution of the carriers for 24 hours, after which the LC3-II / LC3-I ratio and p62 protein levels were measured using Western blot. An increase in the LC3-II / LC3-I ratio and a decrease in p62 were used as indicators of autophagy activation.

[0511] The LC3-II / LC3-I ratio of cells treated with the release solution of Example 17-1 increased by 1.45 ± 0.12 times compared to the untreated control group, and the p62 level decreased by 0.78 ± 0.06 times. The LC3-II / LC3-I ratio of cells treated with the release solution of Example 17-2 increased by 2.15 ± 0.18 times compared to the control group, and the p62 level decreased by 0.58 ± 0.05 times. The LC3-II / LC3-I ratio of cells treated with the release solution of Example 17-3 increased by 1.85 ± 0.15 times compared to the control group, and the p62 level decreased by 0.65 ± 0.06 times. The LC3-II / LC3-I ratio of cells treated with the release solution of Example 17-4 increased by 2.52 ± 0.22 times compared to the control group, and the p62 level decreased by 0.45 ± 0.04 times, suggesting a synergistic effect from the combined use of spermidine and trehalose.

[0512] The LC3-II / LC3-I ratio of cells treated with the release solution of Comparative Example 17-1 was 1.12 ± 0.08 times that of the control group, and no significant autophagy induction effect was observed. From the above results, it was observed that an autophagy induction effect occurs at a spermidine content of 0.1 wt% or more.

[0513] Experimental Example 16

[0514] The AMPK activating effect of the delivery agents prepared in Examples 18-1 to 18-3 and Comparative Example 18-1 was evaluated. Human liver cancer cell lines (HepG2) were treated with the release solution of the delivery agents for 6 hours, and then the phosphorylated AMPK (Thr172) / total AMPK ratio and the phosphorylated ACC (Ser79) / total ACC ratio were measured using Western blot. ACC (acetyl-CoA carboxylase) is a downstream target of AMPK and is phosphorylated and inactivated by AMPK.

[0515] The p-AMPK / AMPK ratio of cells treated with the release solution of Example 18-1 increased by 1.52 ± 0.10 times compared to the untreated control group, and the p-ACC / ACC ratio increased by 1.45 ± 0.12 times. The p-AMPK / AMPK ratio of cells treated with the release solution of Example 18-2 increased by 2.25 ± 0.18 times compared to the control group, and the p-ACC / ACC ratio increased by 2.08 ± 0.15 times. The p-AMPK / AMPK ratio of cells treated with the release solution of Example 18-3 increased by 2.65 ± 0.22 times compared to the control group, and the p-ACC / ACC ratio increased by 2.42 ± 0.18 times, suggesting a combined effect of berberine and ginsenoside Rg3.

[0516] The p-AMPK / AMPK ratio of cells treated with the release solution of Comparative Example 18-1 was 1.18 ± 0.08 times that of the control group, indicating an insufficient AMPK activation effect. From the above results, it was observed that an AMPK activation effect occurred at a berberine content of 0.5 wt% or more.

[0517] Experimental Example 17

[0518] The mitochondrial function-improving effect of the carriers prepared in Examples 19-1 to 19-4 and Comparative Example 19-1 was evaluated. Human skin fibroblasts, in which oxidative stress was induced by treatment with 200 ÌM hydrogen peroxide for 4 hours, were treated with the release solution of the carriers for 24 hours. Basal oxygen consumption rate, maximum oxygen consumption rate, and reserve respiratory capacity were measured using a Seahorse XF analyzer, and mitochondrial membrane potential (Km) was measured using JC-1 staining.

[0519] The basal oxygen consumption rate of cells treated with the release solution of Example 19-1 recovered to 1.28 ± 0.08 times compared to the untreated control group after inducing oxidative stress, and the mitochondrial membrane potential recovered to 1.32 ± 0.10 times. The basal oxygen consumption rate of cells treated with the release solution of Example 19-2 recovered to 1.52 ± 0.12 times compared to the control group, and the mitochondrial membrane potential recovered to 1.55 ± 0.12 times. The basal oxygen consumption rate of cells treated with the release solution of Example 19-3 recovered to 1.35 ± 0.10 times compared to the control group, and the mitochondrial membrane potential recovered to 1.38 ± 0.11 times. The basal oxygen consumption rate of cells treated with the release solution of Example 19-4 recovered by 1.78 ± 0.15 times compared to the control group, the mitochondrial membrane potential recovered by 1.82 ± 0.14 times, and the respiratory reserve capacity increased by 1.65 ± 0.12 times. The above results suggested a synergistic effect from the combined use of coenzyme Q10, pyrroloquinoline quinone, and alpha-lipoic acid.

[0520] The basal oxygen consumption rate of cells treated with the release solution of Comparative Example 19-1 was 1.08 ± 0.05 times that of the control group, indicating that the effect of improving mitochondrial function was insufficient.

[0521] Experimental Example 18

[0522] The effect of the delivery system prepared in Examples 21-1 to 21-3 and Comparative Example 21-1 on improving cellular energy metabolism was evaluated. Human skin fibroblasts were treated with the release solution of the delivery system for 24 hours, and then intracellular ATP levels, NAD+ / NADH ratio, and alpha-ketoglutarate levels were measured.

[0523] The ATP levels of cells treated with the release solution of Example 21-1 increased by 1.18 ± 0.06 times compared to the untreated control group, and the NAD+ / NADH ratio increased by 1.15 ± 0.08 times. The ATP levels of cells treated with the release solution of Example 21-2 increased by 1.35 ± 0.10 times compared to the control group, and the NAD+ / NADH ratio increased by 1.28 ± 0.10 times. The ATP levels of cells treated with the release solution of Example 21-3 increased by 1.28 ± 0.08 times compared to the control group, and the NAD+ / NADH ratio increased by 1.22 ± 0.09 times.

[0524] The ATP levels of cells treated with the release solution of Comparative Example 21-1 were 1.05 ± 0.04 times compared to the control group, and no significant improvement in energy metabolism was observed. From the above results, it was observed that an improvement in energy metabolism occurs at a content of 0.5 wt% or more of calcium alpha-ketoglutarate.

[0525] Experimental Example 19

[0526] The glutathione synthesis-promoting and antioxidant effects of the carriers prepared in Examples 20-1 to 20-3 were evaluated. Human skin fibroblasts were treated with the release solution of the carriers for 48 hours, after which intracellular reduced glutathione (GSH) levels, oxidized glutathione (GSSG) levels, the GSH / GSSG ratio, and intracellular reactive oxygen species levels were measured. Reactive oxygen species were measured using DCFDA fluorescence staining.

[0527] The GSH levels of cells treated with the release solution of Example 20-1 increased by 1.42 ± 0.10 times compared to the untreated control group, the GSH / GSSG ratio increased by 1.35 ± 0.12 times, and the reactive oxygen species levels decreased by 0.75 ± 0.06 times. The GSH levels of cells treated with the release solution of Example 20-2 increased by 1.28 ± 0.08 times compared to the control group, the GSH / GSSG ratio increased by 1.22 ± 0.10 times, and the reactive oxygen species levels decreased by 0.82 ± 0.05 times. The GSH levels of cells treated with the release solution of Example 20-3 increased by 1.55 ± 0.12 times compared to the control group, the GSH / GSSG ratio increased by 1.48 ± 0.14 times, and the reactive oxygen species levels decreased by 0.68 ± 0.05 times. The above results suggested that glycine acts as a direct precursor of glutathione, taurine provides an antioxidant effect, and the combination of the two components exhibits a synergistic effect.

[0528] Experimental Example 20

[0529] The comprehensive anti-aging effect of a composite delivery system incorporating the lifespan-extending active ingredient prepared in Example 22 was evaluated. Human skin fibroblasts were induced to replicate aging by 10 passages and then treated with the release solution of the delivery system for 7 days. Telomerase activity, the ratio of beta-galactosidase-positive cells, the LC3-II / LC3-I ratio, intracellular NAD+ levels, mitochondrial membrane potential, and cell proliferation rate were comprehensively measured.

[0530] Telomerase activity in cells treated with the release solution of Example 22 increased by 1.65 ± 0.12 times compared to the untreated senescent cell control group. The proportion of beta-galactosidase-positive cells decreased to 48.5 ± 3.5% compared to the senescent cell control group. The LC3-II / LC3-I ratio increased by 2.35 ± 0.18 times compared to the control group. Intracellular NAD+ levels increased by 1.72 ± 0.14 times compared to the control group. Mitochondrial membrane potential recovered by 1.68 ± 0.12 times compared to the control group. Cell proliferation rate increased by 1.45 ± 0.10 times compared to the control group.

[0531] From the above results, it was confirmed that the combination of nicotinamide mononucleotide, resveratrol, spermidine, fisetin, cycloastrazenol, coenzyme Q10, and glycine exhibits a synergistic anti-aging effect by simultaneously regulating the NAD+ / sirtuin pathway, autophagy pathway, senolic pathway, telomerase activation pathway, and mitochondrial function improvement pathway.

[0532] Experimental Example 21

[0533] The differential release characteristics of the lifetime-extending active ingredients of the carriers prepared in Examples 22 and 23-1 were evaluated. The release rates of spermidine, fisetin, nicotinamide mononucleotide, and nicotinamide riboside over time in an artificial serous solution at 37°C were measured by high-performance liquid chromatography.

[0534] In Example 22, for spermidine, the release rate was 25.3 ± 2.2% at 4 hours, 52.8 ± 3.5% at 6 hours, and reached 78.5 ± 4.2% at 8 hours. For fisetin, the release rate was 18.5 ± 1.8% at 4 hours, 45.2 ± 3.2% at 6 hours, and reached 72.3 ± 3.8% at 8 hours. For nicotinamide mononucleotide, the release rate was 22.5 ± 2.0% at 4 hours, 55.3 ± 3.5% at 6 hours, and reached 82.8 ± 4.0% at 8 hours.

[0535] In Example 23-1, the release rate of nicotinamide riboside in the middle layer was 35.2 ± 2.5% after 2 hours and reached 68.5 ± 3.8% after 4 hours. The release rate of nicotinamide mononucleotide in the core layer was 15.8 ± 1.5% after 4 hours and reached 80.2 ± 4.0% after 8 hours. From the above results, it was confirmed that nicotinamide riboside is released first during the middle of sleep to prepare the NAD+ synthesis pathway, and nicotinamide mononucleotide is released during the latter part of sleep to enable a continuous supply of NAD+.

[0536] The above release profile exhibited a four-stage time-lag release pattern of sleep-inducing component (80% release within 30 minutes) -> sleep-maintaining component (2–4 hours) -> NAD+ precursor co-component (2–4 hours) -> life-extending active component (4–8 hours), which was observed to be suitable for providing a life-extending effect synchronized with the sleep cycle.

[0537] Although embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and it will be obvious to those skilled in the art that various modifications and variations are possible within the scope of the technical concept of the present invention as described in the claims.

Claims

Claim 1 A carrier having a multi-compartment structure formed by 3D bioprinting, comprising: a shell portion having micropores formed on its surface and containing a sleep-inducing component and a hydrogel; an intermediate layer portion disposed inside the shell portion and containing a sleep-maintaining component and a biodegradable polymer, comprising 1-aminoadamantan; a diffusion barrier layer disposed inside the intermediate layer portion; and a core portion disposed inside the diffusion barrier layer and containing an anti-aging active component and a sustained-release matrix. A carrier having a multi-compartment structure formed by 3D bioprinting, comprising a polydopamine coating layer formed on the outer surface of the shell portion, wherein the core portion comprises methacrylated gelatin conjugated with beta-cyclodextrin and multi-arm polyethylene glycol having adamantan terminals, wherein the methacrylated gelatin conjugated with beta-cyclodextrin and the multi-arm polyethylene glycol-adamantan form a supramolecular cross-linked structure through host-guest bonding, and wherein the polydopamine coating layer comprises a copolymer of dopamine and 5,6-dihydroxyindole-2-carboxylic acid and has a thickness of 10 to 50 nm. Claim 2 A carrier having a multi-compartment structure formed by three-dimensional bioprinting, wherein the hydrogel of the shell portion comprises one or more of alginate and hyaluronic acid, the biodegradable polymer of the intermediate layer comprises one or more of polylactic-co-glycolic acid (PLGA) and cross-linked gelatin, the diffusion barrier layer comprises nanocellulose, and the sustained-release matrix of the core portion comprises one or more of methacrylated gelatin (GelMA) and polycaprolactone (PCL). Claim 3 A carrier having a multi-compartment structure formed by three-dimensional bioprinting, wherein the sleep-inducing component comprises one or more of melatonin, GABA, and L-theanine, the sleep-maintaining component comprises one or more of a magnesium compound, a herbal extract, and sustained-release melatonin, and the anti-aging active component comprises one or more of nicotinamide mononucleotide (NMN), exosomes, resveratrol, and antioxidant peptides. Claim 4 A carrier having a multi-compartment structure formed by 3D bioprinting, wherein the shell portion has a fractal structure or a gyroid structure, the core portion has a spherical shape, and the carrier includes a 3D lattice structure or microchannels inside. Claim 5 A carrier having a multi-compartment structure formed by three-dimensional bioprinting, wherein at least one of the shell portion, the intermediate layer portion, and the core portion comprises a temperature-sensitive polymer, and the temperature-sensitive polymer comprises poly(N-isopropylacrylamide) (PNIPAM) or a copolymer thereof. Claim 6 In claim 1, the carrier has a multi-compartment structure formed by three-dimensional bioprinting, having one of the forms of an oral multi-pellet, a skin-attachable microneedle patch, and an in vivo-implantable scaffold. Claim 7 delete Claim 8 delete Claim 9 delete

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