Protein carrier-based active ingredient transdermal delivery system, preparation method therefor, and use thereof

Abstract

A protein carrier-based active ingredient transdermal delivery system, which is a small molecule active ingredient-protein-polymer composite structure and comprises a small molecule active ingredient, a protein loaded with the small molecule active ingredient, and a polymer coating a surface of the protein. The protein is modified with a polymerizable double-bond compound. After the loading of the small molecule active ingredient, a polymerization reaction is initiated on the surface of the protein in situ, so that the surface of the protein is coated with a polymer layer. The polymer layer is biologically friendly, and the surface properties of the polymer layer can be regulated and controlled according to the transdermal depth requirement, thereby achieving the targeted transdermal delivery of proteins and active small molecules while breaking through the skin barrier, and significantly overcoming the defect of poor delivery efficiency of previous protein-based delivery systems. The transdermal delivery system has a simple preparation method with a high yield, and thus can be industrially produced on a large scale.

Description

A protein carrier-based active ingredient transdermal delivery system and its preparation method and application Technical Field

[0001] The present invention belongs to the field of drug delivery, and in particular relates to a protein carrier-based active ingredient transdermal delivery system and a preparation method and application thereof. Background Art

[0002] Compared to oral administration and intravenous injection, transdermal delivery offers advantages such as convenience and non-invasiveness, avoidance of the extreme pH environment of the gastrointestinal tract, and localized targeted delivery. It is particularly suitable for the treatment of skin diseases and the management of other skin problems. The skin is the largest organ in the human body, covering its entire surface and providing direct contact with the external environment and substances. The skin's powerful barrier function prevents external substances and environmental influences (including radiation damage and material intrusion) from damaging the body's stable physiological functions. However, this barrier function also severely hinders the penetration of active ingredients, resulting in extremely low bioavailability of transdermally delivered drugs. Furthermore, due to the skin's high barrier efficiency, simply increasing the concentration of small molecule drugs in transdermal formulations not only has little effect on the body but may also cause skin irritation. Transdermal delivery of active macromolecules is even more challenging. Due to their size, hydrophilicity, and other limitations, large molecule drugs not only have difficulty penetrating the skin structure but are also more likely to be recognized and directly eliminated by immune cells within the skin's immune barrier.

[0003] Currently reported or commercialized transdermal delivery strategies fall into two main categories: 1) using permeation enhancers to facilitate drug transdermal penetration; and 2) utilizing liposomes to load drugs for permeation. However, the use of permeation enhancers such as ethanol may affect the activity of some small molecules, and the addition of permeation enhancers increases the risk of skin irritation. Liposome-encapsulated drugs are typically 200-500 nm in size, making the stability of liposome emulsion formulations difficult to achieve for long-term use.

[0004] Human skin, with a thickness ranging from 90 to 120 μm, is a complex, multi-layered structure composed of different cell types, each with a specific function. For example, melanocytes produce pigment to protect against UV radiation, while fibroblasts synthesize the extracellular matrix to support the skin's structure. These various cells collectively form the skin's barrier function: the outermost layer of anucleated keratinocytes and the intercellular lipid environment form the first physical barrier, preventing water penetration and the intrusion of external substances such as pathogens. Because this first physical barrier shields the vast majority of foreign substances, existing transdermal delivery technologies primarily focus on breaching this barrier. Currently, the more established liposome delivery technology, due to its large particle size, only penetrates to a depth close to the initial barrier, preventing deeper delivery. However, beneath this first physical barrier, nucleated keratinocytes and tight junction proteins work together to regulate the intercellular spaces and further restrict the penetration of solute molecules by selecting for size and charge.

[0005] In view of the structure of the skin and the working principle of the skin barrier, the applicant uses protein as a template material, forms a polymer shell on the protein surface through in situ polymerization technology, and controls the shell properties (including size and surface charge, etc.) through copolymerization of multiple monomers, forming a series of protein nanogels with protein as the core and polymer as the shell, and the shell thickness and properties are controllable. Using the above-mentioned protein nanogels, the optimal size and material property combination for permeation through the skin barrier performance was studied and determined; further, the hydrophobic domain in the core protein was used as a loading site to adsorb and load small molecule hydrophobic drugs / active molecules to achieve transdermal delivery of small molecule hydrophobic drugs / active substances; further, using the universality of the in situ polymerization method, the protein was replaced with an enzyme with catalytic activity, achieving transdermal delivery of protein drugs (enzymes).

[0006] The inventor's previous research has developed an in situ polymerization technology for forming a polymer shell on the outer layer of the protein. The specific operations are summarized as 1) modifying reactive double bonds on the protein surface as polymerization sites; 2) adding acrylate / methyl acrylate monomers; 3) adding an initiator to form free radicals, which in turn trigger the polymerization of polymerizable monomers on the protein surface, and ultimately forming a polymer network to wrap the protein (referred to as protein nanogel). Based on the above research, a protein nanogel delivery system for gout, glioma and Parkinson's disease was successfully developed (article). The above studies all used intravenous injection as the mode of administration, and drug delivery was carried out through the blood. The delivery barrier faced by the design of delivery materials is the recognition, phagocytosis and clearance of foreign substances by immune cells in the blood and major organs. Therefore, it is necessary to obtain a protein nanogel (such as polypyrrolidone, polyphosphorylcholine, etc.) with electrical neutrality (Zeta potential as close to 0 as possible) and a thick hydration layer.

[0007] Transdermal delivery requires overcoming the skin barrier, which differs significantly from the systemic immune barrier, making traditional delivery systems unsuitable for transdermal drug delivery. A large number of delivery technologies suitable for intravenous injection have been developed in existing research and pharmaceutical applications, but the efficiency and effectiveness of transdermal drug delivery remain very low, and new, efficient transdermal delivery solutions are also relatively rare in basic research.

[0008] Summary of the Invention

[0009] In order to overcome the problem that the comprehensive performance of the transdermal delivery system for active pharmaceutical ingredients in the prior art cannot meet the needs, and cannot have both high efficiency in active ingredient delivery, biofriendliness, good stability, and predictable and controllable penetration depth to achieve targeted transdermal delivery. The present invention uses protein molecules as nano templates, compounds proteins with polymers, obtains nano-sized protein-polymer particles, and constructs a highly efficient and safe transdermal delivery system. It overcomes the problem that macromolecules including naked proteins, natural polymers (cyclodextrins), and synthetic polymers (PCL, PLGA) are difficult to penetrate the skin's efficient barrier due to their size and surface properties, resulting in lower utilization efficiency. In addition, during storage and administration, large fluctuations in temperature and pH environment will also cause damage to active substances (such as enzymes); during the penetration process, the fluctuation of pH in the skin layer with the penetration depth will also affect the physiological efficacy of the active substance. In the absence of protection, even if the active ingredient can penetrate the skin barrier, it cannot exert its physiological efficacy. In order to solve the above technical problems, the present invention provides the following technical solutions:

[0010] A protein carrier-based active ingredient transdermal delivery system is a small molecule active ingredient-protein-polymer composite structure, including a small molecule active ingredient, a protein loaded with the small molecule active ingredient, and a polymer coated on the surface of the protein.

[0011] In the present invention, after the protein is coated with a polymer, the thickness of the polymer shell and the properties of the polymer shell (surface active functional groups, surface potential, surface hydrophilicity and hydrophobicity) can be controlled by adjusting the polymerization time, monomer concentration and monomer ratio, thereby achieving control and regulation of the nanometer size and surface properties, and further achieving control of the penetration depth of the complex.

[0012] Furthermore, in the transdermal delivery system of the present invention, small molecule active ingredients can bind to protein molecules through adsorption, hydrophilic-hydrophobic interactions, electrostatic interactions, hydrogen bonding, and other methods. Furthermore, small molecules possess certain beneficial physiological activities. For example, resveratrol and curcumin are used to treat inflammation in the body; sirolimus, tacrolimus, and ruxolitinib are used to treat autoimmune diseases; fluconazole and itraconazole are used to treat fungal infections; and niacinamide, ascorbic acid, and α-arbutin are used to treat diseases caused by excessive pigmentation.

[0013] Furthermore, the protein is a protein with physiological activity and therapeutic efficacy, including delivery of etanercept for rheumatoid arthritis, plaque psoriasis, psoriatic arthritis, and ankylosing spondylitis; delivery of insulin glargine for type I and type II diabetes; delivery of pegfilgrastim for neutropenia; delivery of octreotide for gigantism, acromegaly, and thyrotropinoma; delivery of liraglutide for type II diabetes; delivery of desmopressin for nocturnal enuresis, transient diabetes insipidus, and von Willebrand disease; delivery of cyclosporine A for psoriasis; delivery of catalase for inflammatory diseases; and delivery of antigenic proteins and antigenic peptides for vaccination needs for disease prevention. In the present invention, the protein plays multiple roles. On the one hand, as a carrier of small molecule active ingredients, it can smoothly deliver them to the target location deep in the skin; on the other hand, the protein itself has physiological activity, such as therapeutic efficacy or the ability to play the catalytic role of protease.

[0014] Furthermore, the selection of polymers needs to take into account several aspects: first, it needs to be biofriendly and have little irritation to the skin; second, the polymer can have a certain affinity with the protein, including hydrogen bonds, electrostatic attraction, chemical bonds, etc.; third, the formation conditions of the polymer shell need to be mild to avoid drastic changes in temperature, pH and other conditions during the preparation process or the inactivation of active substances (including proteins and small molecules) caused by the introduction of too many organic solvents; fourth, the charge carried by the polymer as the coating layer should be controllable, by changing the ratio of different substances in the polymer and the thickness of the polymer shell, so that the surface potential of the protein nanogel changes within the range of -10mV to +10mV without affecting the stability of the system, and optimizing its penetration ability to break through the skin barrier. The above conditions can be met, and the polymer used in the present invention is selected from at least one of polyols (polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol condensation polymer), polyvinyl pyrrolidone, polyphosphorylcholine, polycarboxylic acid betaines, and polysulfonic acid betaines. The polymer is obtained by a polymerization method known in the art, such as polymerization of monomers containing carbon-carbon unsaturated double bonds under the action of an initiator, or condensation polymerization of substances having functional groups, or ring-opening polymerization.

[0015] Furthermore, a polymer shell is formed by polymerization reaction, and the optional monomers include at least one of vinylpyrrolidone, N-propylene succinimide, 2-methacryloyloxyethyl phosphorylcholine, polyethylene glycol methyl ether acrylate, and [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl) ammonium hydroxide; the cross-linking agent is selected from at least one of N,N'-methylenebisacrylamide, ethylene glycol diacrylate, polyethylene glycol diacrylate, and glycerol dimethacrylate.

[0016] In addition to initiating polymerization to form a polymer shell, at least one positively charged polyelectrolyte and one negatively charged polyelectrolyte can also be selected to wrap around the outside of the protein through electrostatic interactions to form a protein nanogel. Optional positively charged polyelectrolytes include: poly(propyleneamine hydrochloride), poly(L-lysine), polyethyleneimine, poly(L-histidine), poly(N,N-dimethylaminoethyl methacrylate), poly(methacrylamidopropyltrimethylammonium chloride), and natural or synthetic polysaccharides such as chitosan; optional negatively charged polyelectrolytes include: polyacrylic acid, polystyrene sulfonate, alginate, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dextran sulfate, polymethacrylic acid, oxidized cellulose, carboxymethyl cellulose, polyaspartic acid, and polyglutamic acid.

[0017] Furthermore, in the protein nanogel, the size of the protein is 2-15 nm, and the thickness of the polymer layer is 1 to 50 nm. The mass ratio of the small molecule active ingredient to the protein is 1:1-100, such as 1:2-10, and such as 1:2-5. The mass ratio of the small molecule active ingredient to the protein mainly depends on the amount of the small molecule active ingredient that the protein can load, and the ratio varies depending on different small molecule active ingredients and the type of protein carrier selected.

[0018] Furthermore, in the protein nanogel, the mass ratio of the small molecule active ingredient to the protein is 1:10-50, the size of the protein is 5-10 nm, and the thickness of the polymer layer is 2 to 30 nm. Preferably, when the active protein is an enzyme that needs to contact a substrate (such as tyrosinase), the thickness of the polymer layer is 7 to 22 nm.

[0019] When preparing protein nanogels using active proteins as templates, it is necessary to consider the effects of the presence and thickness of the polymer shell on the protein's activity and stability. A prerequisite for proteins / enzymes to exhibit activity is contact with substrate molecules, but the presence of a polymer shell hinders this contact. Furthermore, active proteins have complex structures and are easily inactivated. Polymer encapsulation can improve the protein's structural stability, thereby protecting its activity. Therefore, to achieve the objectives of the present invention, controlling the thickness of the polymer shell is crucial. On the one hand, it must protect the protein and promote penetration efficiency, but on the other hand, it must not shield the protein and the release and activity of the small molecule active ingredients loaded in the protein. By controlling polymerization conditions, the present invention prepared a series of protein nanogels with shell thicknesses ranging from 7 to 100 nm. It was found that polymer shells with shell thicknesses exceeding 50 nm would hinder the transport of substrate molecules, preventing the protein / or protein-loaded small molecules in the gel core from exerting their active effects, resulting in lower overall activity of the nanogel. Protein nanogels with shell thicknesses ranging from 7 to 22 nm can protect protein activity at pH values ​​between 4 and 8 and below 60°C.

[0020] The present invention optimizes the size and surface properties of protein nanogels and explores the relationship between the penetration depth of protein nanogels and their size and surface properties. Using the inert protein mouse serum albumin (MSA) as a template and acrylamide and aminopropylmethacrylamide hydrochloride as comonomers, a series of positively charged protein nanogels and negatively charged protein nanogels were synthesized, whose diameters were controllable between 20-200nm. The penetration depth of protein nanogels within 24 hours of transdermal administration was examined and it was found that when the gel size was the same, the penetration depth of protein nanogels with positive surface charge was deeper than that of negatively charged protein nanogels; when the surface potential was close, the penetration ability of protein nanogels with positive surface charge of different sizes was different. When the diameter of the protein nanogel was in the range of 20nm to 150nm, the relationship between the penetration depth D of the nanogel at 24 hours and its diameter d can be approximately fitted into the equation D=97.3+2.05d–0.0239d 2 +7.08E-5d 3 .

[0021] Based on this, the second object of the present invention is to provide a method for controlling the transdermal depth of an active ingredient transdermal delivery system based on a protein carrier for non-therapeutic purposes, which is based on the equation D = 97.3 + 2.05d - 0.0239d 2 +7.08×10 -5 d 3, by regulating the size d of the transdermal delivery system and then regulating the delivery depth D. Wherein D is the transdermal depth, unit μm; d is the size of the active ingredient transdermal delivery system of the present invention, unit nm. The scope of application of this equation is to predict the penetration depth of protein nanogels on skin tissue. Protein nanogels should have similar polymer shell physicochemical properties (such as the material's deformability, roughness, etc. should be similar to polyacrylamide, the overall shape of the nanogel should be close to spherical, the shell surface zeta potential should be +3mV to +7mV, such as about +4mV, about +5mV, about +6mV), and the nanogel size should be within the range of 20-150nm. This formula is universal for different protein cores. By controlling the protein size and the polymer shell thickness, in particular by controlling the polymerization reaction parameters such as polymerization time, monomer concentration and monomer ratio to control the polymer shell thickness, the purpose of controlling the delivery depth by regulating the size of the transdermal delivery system is achieved. For different diseases, the targets of transdermal drug delivery are different, resulting in different delivery depths required for transdermal delivery. For example, the delivery targets for fungal skin diseases are keratinocytes and keratinocytes in the epidermis, the delivery targets for vitiligo are melanocytes at the junction of the epidermis and dermis, and the delivery targets for transdermal vaccination are antigen-presenting cells in the epidermis and dermis. Therefore, it is of great significance to construct a transdermal delivery system with controllable transdermal depth. However, existing transdermal delivery technologies are not universally applicable to different proteins and small molecules, and there is little research on regulating transdermal depth. The penetration behavior of large and small molecules in different skin layers is still unclear, making it difficult to establish a formula to describe the efficiency of drug transdermal delivery.

[0022] If the deeper the transdermal penetration, the better, the size of the active ingredient transdermal delivery system of the present invention is 40-70 nm.

[0023] The third object of the present invention is to provide a method for preparing the above-mentioned active ingredient transdermal delivery system based on a protein carrier, comprising the following steps:

[0024] (1) adding a small molecule with a polymerizable double bond to a protein solution to obtain a surface-modified protein solution;

[0025] (2) uniformly mixing the small molecule active ingredient solution and the surface-modified protein solution to obtain a protein solution loaded with the small molecule active ingredient;

[0026] (3) The protein solution loaded with small molecule active ingredients, monomers, and cross-linking agents are mixed, diluted, and an initiator is added to initiate a polymerization reaction to obtain an active ingredient transdermal delivery system based on a protein carrier.

[0027] Furthermore, in step (1), the solvent of the protein solution is selected from a phosphate buffer solution or an organic solvent; further, the pH of the phosphate buffer solution is 5-8; the phosphate buffer solution is NaH2PO4-Na2HPO4; the organic solvent is selected from at least one of ethanol, methanol, acetone, and dimethyl sulfoxide; and the concentration of protein in the protein solution is 1-100 mg / L, preferably 1-10 mg / L.

[0028] Furthermore, in step (1), the small molecule with a polymerizable double bond is adsorbed on the surface of the protein molecule by electrostatic interaction or covalent crosslinking. Examples of the small molecule with a polymerizable double bond include, but are not limited to, at least one of N-propylene succinimide and maleimide. The mass ratio of the small molecule with a polymerizable double bond to the protein is 1:1-50, preferably 1:5-20.

[0029] Furthermore, in step (2), the small molecule active ingredient is attached to the protein through electrostatic interactions, hydrophilic-hydrophobic interactions, hydrogen bonds, cross-linking, etc. The concentration of the small molecule active ingredient solution is 1-100 mg / L, preferably 1-10 mg / L; the solvent of the small molecule active ingredient solution is selected from at least one of water, ethanol, propanol, ethylene glycol, and dimethyl sulfoxide.

[0030] Furthermore, in step (3), the monomer is selected from at least one of 2-methacryloyloxyethyl phosphorylcholine, vinylpyrrolidone, acrylamide, polyethylene glycol methyl ether acrylate, and N-(3-aminopropyl) methacrylamide hydrochloride, and the cross-linking agent is selected from at least one of N,N'-methylenebisacrylamide, ethylene glycol diacrylate, polyethylene glycol diacrylate, and glycerol dimethacrylate. Preferably, the monomer includes N-(3-aminopropyl) methacrylamide hydrochloride. When N-(3-aminopropyl) methacrylamide hydrochloride is used as a monomer, the surface of the resulting polymer carries a positive charge, which is conducive to interacting with the lipid components in the skin, weakening the barrier effect of the lipid bilayer on the protein nanogel, and promoting the intercellular penetration of the protein nanogel.

[0031] Furthermore, in step (3), the molar ratio of protein, monomer, and cross-linking agent is 1:100-100000:10-10000, preferably 1:2000-20000:200-2000.

[0032] Furthermore, in step (3), the initiator is selected from at least one of sodium persulfate, ammonium persulfate, and potassium persulfate, and the mass ratio of the initiator to the protein in step (1) is 1-3.8:1. Preferably, a co-initiator is also added, the co-initiator comprising at least one of sodium bisulfite and tetramethylethylenediamine, and the amount of the co-initiator is 1-3 times the mass of the initiator, such as 2 times.

[0033] Furthermore, in step (3), before polymerization, the protein concentration is diluted to 1-2 mg / mL using a buffer solution or an organic solvent.

[0034] Furthermore, the preparation method of the active ingredient transdermal delivery system based on a protein carrier further comprises a post-processing step (4): after the polymerization reaction is completed, the complex solution is subjected to ultrafiltration purification, and the purified mother liquor is refrigerated and stored.

[0035] The third object of the present invention is to provide the use of the above-mentioned protein carrier-based active ingredient transdermal delivery system in the preparation of medicines, cosmetics, and health products, which are absorbed and utilized by organisms in a transdermal form.

[0036] The organisms include humans and animals.

[0037] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0038] First, the present invention modifies proteins with polymerizable double-bond compounds, loads them with small-molecule active ingredients, and then initiates an in-situ polymerization reaction on the protein surface. This results in a biocompatible, non-irritating or minimally irritating polymer layer covering the protein surface. During transdermal delivery, the surface properties and thickness of the polymer layer are manipulated to effectively control the transdermal depth of the protein nanogel, allowing the nanogel to effectively penetrate the skin and reach the desired location for protein and small-molecule active ingredient release. This significantly improves the poor delivery efficiency of previous protein-based delivery systems.

[0039] 2. The preparation method of the transdermal delivery system of the present invention is simple, has a high yield, and can be industrialized and produced on a large scale.

[0040] 3. The raw materials used in the present invention are all bio-friendly and can be used as pharmaceutical reagents, and therefore can be used in transdermal delivery systems of drugs and other ingredients beneficial to the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG1 is a fitting curve of the penetration depth D and the particle size d of the protein nanogel in Example 1 at 24 hours;

[0042] FIG2 is a TEM image of the composite (PMPC-nEN@Cur) obtained in Example 2;

[0043] FIG3 is a confocal image of the permeation of polymer-protein complexes. DETAILED DESCRIPTION

[0044] The present invention will be further described below with reference to specific examples, but the present invention is not limited to the contents of the specification. All reagents used are commercially available reagents in the art.

[0045] Example 1 Preparation of polyacrylamide-mouse serum protein nanogel

[0046] (1) Dissolve 2 mg of mouse serum albumin in 2 mL of PBS buffer at pH 7.4 to obtain a 1 mg / mL MSA solution. Add a dimethyl sulfoxide solution containing 0.25 mg of N-propylene succinimide (mass concentration 1%) to the 2 mL 1 mg / mL mouse serum albumin (MSA) solution, mix thoroughly and react for 2 hours to obtain mouse serum albumin (aMSA) with a polymerizable double bond modified on its surface.

[0047] (2) aMSA, acrylamide (AAm), aminopropylmethacrylamide hydrochloride (APM), and N,N'-methylenebisacrylamide (BIS) were mixed at the specified molar ratio and diluted to 1 mg / mL with pH 7.4 phosphate buffer. Initiators were then added at a ratio of 3.8 mg of ammonium persulfate and 7.6 mg of tetramethylethylenediamine per mg of protein. In situ free radical polymerization was initiated at room temperature. After the reaction time was specified, polyacrylamide-mouse serum protein nanogels (nMSA) of varying sizes were obtained. The preparation conditions and the measured final sizes and surface zeta potentials are shown in Table 1.

[0048] Table 1 Preparation conditions, final sizes and surface potentials of protein nanogels of various sizes

[0049] In the table, N represents the particle size of MSA-NB, which ranges from 20 to 200 nm, and B represents the sign + or -, indicating the positive or negative surface zeta potential. It can be seen that when a certain proportion of aminopropylmethacrylamide hydrochloride (APM) monomer is added, the surface zeta potential of the resulting nanogel (nMSA) shifts from negative to positive.

[0050] Application Example 1: A series of protein nanogel transdermal experiments

[0051] 4 mL of 1 mg / mL of the various-sized protein nanogels nMSA from Example 1 was mixed with 1 mL of 1 mg / mL fluorescein isothiocyanate and reacted overnight. The mixture was dialyzed overnight in a 14,000 Da dialysis bag to remove unreacted small molecules, yielding fluorescently labeled nMSA. Fresh pigskin was washed with PBS and mounted, stratum corneum facing upward, in a transdermal diffusion apparatus and incubated at 37°C. 0.5 mL of a 1 mg / mL solution of the fluorescently labeled complex was dripped onto the pigskin tissue. After 24 hours, the tissue was cryosectioned and the nanocapsule penetration observed under a confocal microscope. The penetration depth is summarized in Table 2.

[0052] Table 2 Penetration depth of a series of protein nanogels through the skin for 24 hours

[0053] By comparing the penetration depths of protein nanogels of the same size but different surface potentials, it was found that the penetration depth of protein nanogels with positive zeta potential was deeper, indicating that the positive surface charge is conducive to the interaction between the nanogel and the skin to weaken the barrier. By comparing the penetration depths of protein nanogels of different sizes with positive surface potentials (about +5mV), it was found that when the particle size was between 20nm and 150nm, the protein nanogels were able to penetrate the epidermis and reach the vicinity of the dermis within 24 hours, and as the gel particle size increased, the penetration depth first increased and then decreased; while protein nanogels larger than 200nm mostly stayed in the stratum corneum above the epidermis. The penetration depths of nMSA-20+, nMSA-50+, nMSA-100+, and nMSA-150+ were fitted, and the fitting curve of the penetration depth D and particle size d of the protein nanogel with positive surface potential at 24 hours was shown in Figure 1. D=97.3+2.05d-0.0239d 2 +7.08E-5d 3 ,d∈[20,150], where the unit of D is μm and the unit of d is nm.

[0054] This expression can be used to preliminarily predict the permeability of protein nanogels with a particle size in the range of 20-150 nm and a positive surface potential, providing a reference for the design of systems with different delivery depth requirements in different application scenarios.

[0055] Example 2 Preparation of Polyphosphorylcholine-Etanercept Complex:

[0056] (1) Dissolve 2 mg of etanercept (EN) in 2 mL of PBS buffer (pH 7.4) to obtain a 1 mg / mL EN solution. Add a solution of 0.4 mg of N-propylene succinimide in dimethyl sulfoxide (mass concentration 1%) to the 2 mL of 1 mg / mL etanercept (EN) solution, mix thoroughly and react for 2 h to obtain etanercept (aEN) with a polymerizable double bond modified on its surface.

[0057] (2) To 2 mL of a 1 mg / mL surface double bond-modified protein solution, 0.2 mL of a 2 mg / mL ethanol solution of curcumin was added dropwise while stirring at 300 r / min. After thorough mixing, curcumin-loaded etanercept (aEN@Cur) was obtained.

[0058] (3) aEN@Cur, 2-methacryloyloxyethyl phosphorylcholine (MPC) and N,N'-methylenebisacrylamide (BIS) were mixed at a molar ratio of aEN@Cur:MPC:BIS = 1:20000:4000, and the protein concentration was diluted to 1 mg / mL with pH 7.4 phosphate buffer. Then, an initiator was added at a ratio of 3.8 mg of ammonium persulfate and 7.6 mg of tetramethylethylenediamine per mg of protein. In situ free radical polymerization was initiated at room temperature. After 4 h of reaction, a retinol-loaded polyphosphorylcholine-etanercept complex (PMPC-nEN@Cur) was obtained.

[0059] (4) Post-treatment: The obtained complex solution was ultrafiltered in an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa at 8000 rpm for 10 minutes to obtain a purified high-concentration mother solution with a concentration of 5.6 mg / mL, which was refrigerated for storage.

[0060] The stock solution of the complex (PMPC-nEN@Cur) obtained in Example 2 and etanercept (EN) were diluted to 1 mg / mL and their sizes were measured by dynamic light scattering (DLS). The particle size of the complex was measured at room temperature using a Malvern Nano Zs90 nanometer particle size analyzer, and the zeta potential was measured using a Malvern Nano Zs90 nanometer particle size analyzer by electrophoresis. The 1 mg / mL etanercept (EN) solution and the complex (PMPC-nEN@Cur) solution exhibited particle sizes of 6 nm and 53 nm, respectively, and zeta potentials of -3.5±1.7 mV and -2.4±0.8 mV, respectively. This indicates that a phosphorylcholine polymer shell approximately 23.5 nm thick was formed on the outer layer of etanercept (EN), resulting in the polyphosphorylcholine-etanercept complex (PMPC-nEN@Cur).

[0061] Figure 2 is a TEM photograph of the complex (PMPC-nEN@Cur) obtained in Example 2. The morphology and dispersion of the polyphosphorylcholine-etanercept complex (PMPC-nEN@Cur) were observed under a transmission electron microscope. It was found that its particle size was about 50 nm and the polymer shell thickness was about 22-24 nm, which was consistent with the DLS test results. The complex also had good dispersibility and did not show obvious aggregation.

[0062] Example 3 Preparation of Polyethylene Glycol-Cyclosporin A Complex:

[0063] (1) 2 mg of cyclosporin A (CsA) was dissolved in 2 mL of PBS buffer (pH 7.4) to obtain a 1 mg / mL CsA solution. 0.1 mg of N-acryl succinimide dissolved in dimethyl sulfoxide was added to the 2 mL 1 mg / mL cyclosporin A (CsA) solution, and the mixture was thoroughly mixed and reacted for 2 h to obtain cyclosporin A (aCsA) with a polymerizable double bond modified on its surface.

[0064] (2) To a 2 mL solution of 1 mg / mL cyclosporin with double bonds modified on the surface, 0.25 mL of a 0.2 mg / mL lycopene solution in acetone was added dropwise with stirring at 350 r / min. After thorough mixing, lycopene-loaded cyclosporin A (aCsA@Lyc) was obtained.

[0065] (3) aCsA@Lyc, polyethylene glycol methyl ether acrylate (PMEA), and N,N'-methylenebisacrylamide (BIS) were mixed at a molar ratio of aCsA@Lyc:PMEA:BIS = 1:3000:300. The protein concentration was diluted to 1 mg / mL with pH 7.4 phosphate buffer. Then, an initiator was added at a ratio of 3.8 mg of ammonium persulfate and 7.6 mg of tetramethylethylenediamine per mg of protein. In situ free radical polymerization was initiated at room temperature. After 4 h of reaction, lycopene-loaded polyethylene glycol-cyclosporin A complex (PEG-nCsA@Lyc) was obtained.

[0066] (4) Post-treatment: The obtained complex solution was ultrafiltered in an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa to obtain a purified high-concentration mother solution with a concentration of about 5 mg / mL, which was then refrigerated for storage.

[0067] The stock solution of the complex (PEG-nCsA@Lyc) obtained in Example 3 and cyclosporin A (CsA) were diluted to 1 mg / mL and their sizes were measured by dynamic light scattering (DLS). The particle size of the complex was measured at room temperature using a Malvern Nano Zs90 nanometer. The zeta potential was also measured by electrophoresis using a Malvern Nano Zs90 nanometer. The cyclosporin A (CsA) solution and the complex (PEG-nCsA@Lyc) solution, both at a concentration of 1 mg / mL, had particle sizes of 5 nm and 37 nm, respectively, and zeta potentials of -6.0±2.3 mV and -5.9±1.1 mV, respectively.

[0068] The particle size and Zeta potential results showed that a polyethylene glycol shell with a thickness of about 15 nm was formed on the outer layer of cyclosporine A (CsA), and a polyethylene glycol-cyclosporine A complex (PEG-nCsA@Lyc) was obtained.

[0069] Application Example 2

[0070] 2 mL of a 5.6 mg / mL polymer-protein complex was placed in a dialysis bag with a molecular weight cutoff of 3000. The bag was then immersed in phosphate buffer at pH 6, and the entire system was shaken at 37°C. The sustained release period was 48 hours. Samples were taken periodically over the 48 hours, and the concentration of the loaded small molecule drug was measured using a UV spectrophotometer. The sustained release of the small molecule drug from the complex system was calculated. The results are summarized in Table 3. The sustained release results show that the complex system exhibits a sustained release effect on the encapsulated functional small molecule within 24 hours, facilitating the sustained physiological effect of the small molecule in the delivery region over an extended period of time.

[0071] Table 3 Cumulative release rate

[0072] From the data in Table 3, it can be seen that the protein nanogel slowly releases the functional small molecules within 24 hours, which is beneficial for the functional small molecules to play a role after reaching the deep layer of the skin, thereby improving the utilization rate.

[0073] Application Example 3

[0074] Example 3 Determination of the antioxidant capacity of the prepared polyethylene glycol-cyclosporin A complex (PEG-nCsA@Lyc): The ROS level in human epidermal HaCaT cells was detected using a ROS kit. HaCaT cells (1*10 4 Cells were irradiated with a UVB irradiator for 12 hours to induce ROS production. Untreated cells were retained as negative controls. Following irradiation, the culture medium was removed from all wells and 100 μL of serum-free medium was added (serum was omitted to prevent serum from affecting cellular uptake of the complex during subsequent drug additions). The positive control group received 10 μL of PBS buffer, the low-concentration group received 10 μL of a 0.01 mg / mL complex solution, the medium-concentration group received 10 μL of a 0.02 mg / mL complex solution, and the high-concentration group received 10 μL of a 0.04 mg / mL complex solution. After 24 hours of incubation, the culture medium was removed from all wells and replaced with 100 μL of serum-free medium and 10 μL of DCFH-DA culture medium. The cells were incubated at 37°C in the dark for 20 minutes. The cells were then washed three times with PBS, and the fluorescence intensity of the DCF in the cells was detected using a microplate reader. The results are shown in Table 4.

[0075] Table 4 Antioxidant capacity test of polyethylene glycol-cyclosporin A complex

[0076] As can be seen from the data in Table 4, polyethylene glycol-cyclosporin A protein nanogel has good antioxidant capacity, and this capacity increases with increasing concentration.

[0077] Application Example 4

[0078] 4 mL of 1 mg / mL polymer-protein complex solution from Examples 1 and 2 was mixed with 1 mL of 1 mg / mL fluorescein isothiocyanate and reacted overnight. The mixture was dialyzed overnight in a 1000 Da dialysis bag to remove unreacted small molecules, yielding a fluorescently labeled complex. Fresh pigskin was washed with PBS and fixed with the stratum corneum facing upward in a transdermal diffusion apparatus, incubated at 37°C. 0.5 mL of 1 mg / mL fluorescently labeled complex solution was added dropwise to the pigskin tissue. Frozen sections were taken at 24, 4, and 1 hour, and the penetration of the nanocapsules was observed under a confocal microscope. The penetration depth is statistically reported in Table 5. Confocal images of the polymer-protein complex penetration observed at 24 hours are shown in Figure 3. It was found that the protein nanogels penetrated the skin barrier and reached the dermis at 24 hours.

[0079] Table 5 Transdermal performance of polymer-protein complexes

Claims

1. A protein carrier-based active ingredient transdermal delivery system, characterized in that: It is a small molecule active ingredient-protein-polymer composite structure, including a small molecule active ingredient, a protein loaded with the small molecule active ingredient, and a polymer coated on the surface of the protein.

2. [Corrected 07.06.2024 according to Rule 26] The active ingredient transdermal delivery system according to claim 1, characterized in that: The small molecule active ingredient can be combined with the protein molecule by at least one of adsorption, hydrophilic-hydrophobic interaction, electrostatic interaction and hydrogen bonding; and the small molecule has beneficial physiological activity.

3. The active ingredient transdermal delivery system according to claim 1, characterized in that: The small molecule active ingredient is selected from at least one of resveratrol, curcumin, sirolimus, tacrolimus, ruxolitinib, fluconazole, itraconazole, nicotinamide, ascorbic acid, and α-arbutin.

4. The active ingredient transdermal delivery system according to claim 1, characterized in that: The size of the protein is 3-20 nm and the isoelectric point is 3-9.

5. The active ingredient transdermal delivery system according to claim 1, characterized in that: The protein is selected from at least one of tyrosinase, etanercept, insulin glargine, pegfilgrastim, octreotide, liraglutide, desmopressin, cyclosporine A, catalase, antigenic protein, antigenic peptide, human serum albumin, glucose oxidase, lactate dehydrogenase, oxalate decarboxylase, urate oxidase, ethanol oxidase, and alcohol dehydrogenase.

6. The active ingredient transdermal delivery system according to claim 1, characterized in that: The polymer is selected from at least one of polyols (polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol condensation product), polyvinyl pyrrolidone, polyphosphorylcholine, polycarboxylic acid betaines, and polysulfonic acid betaines.

7. The active ingredient transdermal delivery system according to claim 1, characterized in that: The polymer is formed by a polymerization reaction of monomers to form a polymer shell on the surface of the protein, and the monomers are selected from at least one of vinyl pyrrolidone, N-acrylsuccinimide, 2-methacryloyloxyethyl phosphorylcholine, polyethylene glycol methyl ether acrylate, and [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl) ammonium hydroxide.

8. The active ingredient transdermal delivery system according to claim 1, characterized in that: The polymer is selected from polyelectrolytes, and the polyelectrolyte is a positively charged polyelectrolyte or a negatively charged polyelectrolyte.

9. The active ingredient transdermal delivery system according to claim 8, characterized in that: The positively charged polyelectrolyte is selected from at least one of poly(propyleneamine hydrochloride), poly(L-lysine), polyethyleneimine, poly(L-histidine), poly(N,N-dimethylaminoethyl methacrylate), poly(methacrylamide propyltrimethylammonium chloride), and natural or synthetic polysaccharides such as chitosan; the negatively charged polyelectrolyte is selected from at least one of polyacrylic acid, polystyrene sulfonate, alginate, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dextran sulfate, polymethacrylic acid, oxidized cellulose, carboxymethyl cellulose, polyaspartic acid and polyglutamic acid.

10. The active ingredient transdermal delivery system according to claim 1, characterized in that: The protein is selected from at least one of tyrosinase, etanercept, insulin glargine, pegfilgrastim, octreotide, liraglutide, desmopressin, cyclosporine A, catalase, antigenic protein, antigenic peptide, human serum albumin, glucose oxidase, lactate dehydrogenase, oxalate decarboxylase, urate oxidase, ethanol oxidase, and alcohol dehydrogenase; The small molecule active ingredient is at least one selected from resveratrol, curcumin, sirolimus, tacrolimus, ruxolitinib, fluconazole, itraconazole, nicotinamide, ascorbic acid, and α-arbutin; The polymer is selected from at least one of polyols (polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol condensation product), polyvinyl pyrrolidone, polyphosphorylcholine, polycarboxylic acid betaines, and polysulfonic acid betaines; or the polymer is selected from polyelectrolytes, and the polyelectrolyte is a positively charged polyelectrolyte or a negatively charged polyelectrolyte.

11. The active ingredient transdermal delivery system according to claim 1, characterized in that: The mass ratio of the small molecule active ingredient to the protein is 1:10-1000, the size of the protein is 2-15 nm, and the thickness of the polymer layer is 1 to 50 nm.

12. The active ingredient transdermal delivery system according to claim 11, characterized in that: The mass ratio of the small molecule active ingredient to the protein is 1:10-50, the size of the protein is 5-10 nm, and the thickness of the polymer layer is 7 to 22 nm.

13. A method for controlling the transdermal depth of an active ingredient transdermal delivery system based on a protein carrier for non-therapeutic purposes according to claim 1, characterized in that: It is based on the fitting equation D = 97.3 + 2.05d – 0.0239d 2 +7.08×10 -5 d 3 , by regulating the size d of the transdermal delivery system, the delivery depth D can be regulated; wherein D is the transdermal depth, in μm; d is the size of the transdermal delivery system for the active ingredient, in nm.

14. The method for controlling skin penetration depth according to claim 13, characterized in that: The protein is selected from at least one of tyrosinase, etanercept, insulin glargine, pegfilgrastim, octreotide, liraglutide, desmopressin, cyclosporine A, catalase, antigenic protein, antigenic peptide, human serum albumin, glucose oxidase, lactate dehydrogenase, oxalate decarboxylase, urate oxidase, ethanol oxidase, and alcohol dehydrogenase; The small molecule active ingredient is at least one selected from resveratrol, curcumin, sirolimus, tacrolimus, ruxolitinib, fluconazole, itraconazole, nicotinamide, ascorbic acid, and α-arbutin; The polymer is selected from at least one of polyols (polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol condensation product), polyvinyl pyrrolidone, polyphosphorylcholine, polycarboxylic acid betaines, and polysulfonic acid betaines; or the polymer is selected from polyelectrolytes, and the polyelectrolyte is a positively charged polyelectrolyte or a negatively charged polyelectrolyte.

15. The method for controlling skin penetration depth according to claim 14, characterized in that: The polymer is formed by a polymerization reaction of monomers to form a polymer shell on the surface of the protein, and the monomers are selected from at least one of vinyl pyrrolidone, N-acrylsuccinimide, 2-methacryloyloxyethyl phosphorylcholine, polyethylene glycol methyl ether acrylate, and [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl) ammonium hydroxide.

16. The method for preparing the active ingredient transdermal delivery system based on a protein carrier according to claim 1, characterized in that: The following steps are involved: (1) adding a small molecule substance with a polymerizable double bond to a protein solution to obtain a surface-modified protein solution; (2) mixing the small molecule active ingredient solution and the surface-modified protein solution uniformly to obtain a protein solution loaded with the small molecule active ingredient; (3) The protein solution loaded with small molecule active ingredients, monomers, and cross-linking agents are mixed, diluted, and an initiator is added to initiate a polymerization reaction to obtain an active ingredient transdermal delivery system based on a protein carrier.

17. The preparation method according to claim 16, characterized in that: In step (1), the solvent of the protein solution is selected from a phosphate buffer solution or an organic solvent; the pH of the phosphate buffer solution is 5-8; the solute of the phosphate buffer solution is NaH2PO4-Na2HPO4; the organic solvent is selected from at least one of ethanol, methanol, acetone, and dimethyl sulfoxide; the concentration of protein in the protein solution is 1-100 mg / L; The small molecule with polymerizable double bonds includes at least one of N-propylene succinimide and maleimide; the mass ratio of the small molecule with polymerizable double bonds to the protein is 1:1-50.

18. The preparation method according to claim 16, characterized in that: In step (3), the monomer is selected from at least one of 2-methacryloyloxyethyl phosphorylcholine, vinyl pyrrolidone, acrylamide, polyethylene glycol methyl ether acrylate, and N-(3-aminopropyl) methacrylamide hydrochloride, and the cross-linking agent is selected from at least one of N,N'-methylenebisacrylamide, ethylene glycol diacrylate, polyethylene glycol diacrylate, and glycerol dimethacrylate; The molar ratio of the protein, monomer and cross-linking agent is 1:100-100000:10-10000.

19. Use of the protein carrier-based active ingredient transdermal delivery system according to claim 1 in the preparation of medicines, cosmetics, and health products, wherein the medicines, cosmetics, and health products are absorbed and utilized by organisms in a transdermal form.

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