Protein carrier-based active ingredient transdermal delivery system as well as preparation method and application thereof

Abstract

The invention provides an active component transdermal delivery system based on a protein carrier, which is of a small molecule active component-protein-polymer composite structure and comprises a small molecule active component, a protein loaded with the small molecule active component and a polymer coated on the surface of the protein. After the protein is modified with a polymerizable double-bond compound and loaded with small-molecule active components, the polymerization reaction is initiated in situ on the surface of the protein through the polymerization reaction, the surface of the protein is coated with a polymer protection layer, the polymer protection layer is biologically friendly, and the surface property of the polymer layer can be regulated and controlled according to the transdermal depth requirement; the targeted transdermal delivery of protein and active small molecules is realized while the skin barrier is broken through, and the defect of poor delivery efficiency based on a protein delivery system in the past is remarkably improved. The transdermal delivery system is simple in preparation method and high in yield, and industrial large-scale production can be achieved.

Description

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 with oral administration and intravenous injection, transdermal delivery has the advantages of convenience and non-invasiveness, avoiding the extreme pH environment of the gastrointestinal tract, and local 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 of the human body and covers the surface of the human body. It is also the organ of direct contact between the human body and the external environment and substances. The strong barrier function of the skin can prevent the damage of external substances and environmental influences (including radiation damage and material invasion, etc.) to the stable physiological functions of the organism. However, the existence of this barrier function also seriously hinders the penetration of active ingredients, making the bioavailability of transdermally delivered drugs extremely low. Moreover, due to the high barrier efficiency of the skin, simply increasing the concentration of small molecule drugs in transdermal preparations is not only of little help to the body, but also may cause irritation to the skin; and for active macromolecules, transdermal delivery is more difficult. Macromolecule drugs are limited by factors such as their size. It is not only difficult to penetrate the skin structure, but also more likely to be recognized by immune cells in the skin immune barrier and directly cleared.

[0003] The transdermal delivery strategies that have been reported or commercialized are mainly divided into two ideas: 1) using penetration enhancers to help drugs penetrate the skin; 2) using liposomes to load drugs for penetration. However, the use of penetration enhancers such as ethanol may affect the activity of some small molecule ingredients, and the addition of penetration enhancers increases the risk of skin irritation. The size of liposome-encapsulated drugs is generally 200-500nm, and the stability of liposome emulsion preparations is difficult to meet the requirements for long-term use.

[0004] The thickness of human skin ranges from 90 to 120 μm. It is a multi-layered complex structure composed of different types of cells. Each type of cell has a specific function, such as melanocytes producing pigments to resist ultraviolet radiation, and fibroblasts synthesizing extracellular matrix to support the skin structure. There are many types of cells that together form the barrier function of the skin: the outermost layer of anucleated keratinocytes and the lipid environment between cells constitute the first physical barrier to prevent water penetration and invasion of external substances such as pathogens. Since the first layer of physical barrier shields most foreign substances, the existing transdermal delivery technology mainly focuses on breaking through the first layer of barrier. The current more mature liposome delivery technology has a large particle size, and its penetration depth is basically close to the first barrier, and it cannot be delivered at a deeper level. However, under the first physical barrier, there are also nuclear keratinocytes and tight junction proteins that work together to regulate the gap between cells, and will further limit the penetration of solute molecules by screening size and charge properties.

[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 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 protein nanogels, the optimal size and material property combination for permeating 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, thereby initiating the polymerization of polymerizable monomers on the protein surface, and finally 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 has been successfully developed (article). The above studies all use intravenous injection as the mode of administration, and drug delivery is carried out through the blood. The delivery barrier faced by the design of delivery materials is the recognition, phagocytosis and removal 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 needs to overcome the skin barrier, which is very different from the systemic immune barrier. In existing research and drug applications, a large number of delivery technologies that can be used for intravenous injection have been developed, but the efficiency and effectiveness of transdermal drug delivery are still very low, and there are few new and efficient transdermal delivery solutions in basic research. Summary of the invention

[0008] In order to overcome the fact that the comprehensive performance of the transdermal delivery system of active pharmaceutical ingredients in the prior art cannot meet the needs, and cannot have both high efficiency of active ingredient delivery, bio-friendliness, 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 an efficient and safe transdermal delivery system. It overcomes the high-efficiency barrier effect of macromolecules including naked proteins, natural polymers (cyclodextrins), and synthetic polymers (PCL, PLGA) due to their size and surface properties, which is difficult to penetrate the skin, resulting in lower utilization efficiency. In addition, during the administration and penetration process, large fluctuations in temperature and pH environment will also cause the destruction of active substances (such as enzymes). In the absence of protection, even if the active ingredients can penetrate the skin barrier, they cannot exert their physiological effects. In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] 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.

[0010] In the present invention, after the protein is coated with the 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 nanosize and surface properties.

[0011] Furthermore, in the transdermal delivery system of the present invention, the small molecule active ingredients can be combined with protein molecules by adsorption, hydrophilic-hydrophobic interaction, electrostatic interaction, hydrogen bonding, etc.; at the same time, the small molecules have certain beneficial physiological activities. For example, resveratrol, curcumin, etc. are selected for inflammation in the body, sirolimus, tacrolimus, ruxolitinib, etc. are selected for autoimmune diseases, fluconazole, itraconazole, etc. are selected for fungal infections, and nicotinamide, ascorbic acid, α-arbutin, etc. are selected for hyperpigmentation diseases.

[0012] Further, the protein is a protein with physiological activity and therapeutic efficacy, including delivery of etanercept for rheumatoid arthritis, plaque psoriasis, psoriatic arthritis, 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; delivery of antigenic proteins and antigenic peptides for vaccination needs for disease prevention. In the present invention, the protein plays a variety of 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 it has therapeutic efficacy, or can play the catalytic role of protease.

[0013] Further, the selection of polymers needs to take into account several aspects: first, it needs to be bio-friendly and have little irritation to the skin; secondly, the polymer can have a certain affinity with the protein, including hydrogen bonds, electrostatic attraction, chemical bonds, etc.; finally, the charge carried by the polymer as the coating layer should be controllable, and by changing the ratio of different substances in the polymer and the thickness of the polymer shell, 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 product), polyvinyl pyrrolidone, polyphosphorylcholine, polycarboxylic acid betaine, and polysulfonic acid betaine. The polymer is obtained by a polymerization method known in the art, such as polymerization of a monomer containing a carbon-carbon unsaturated double bond under the action of an initiator, or polycondensation of a substance with a functional group, or ring-opening polymerization.

[0014] Furthermore, a polymer shell is formed by polymerization reaction, and the optional monomers include 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; the cross-linking agent is selected from at least one of N,N'-methylenebisacrylamide, ethylene glycol diacrylate, polyethylene glycol diacrylate, and glycerol dimethacrylate.

[0015] 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 interaction to form a protein nanogel. Optional positively charged polyelectrolytes include: poly (propyleneamine hydrochloride), poly (L-lysine), polyethylene imine, poly (L-histidine), poly methacrylate N, N-dimethylaminoethyl ester, polymethacrylamide propyl trimethyl ammonium chloride, and natural or synthetic polysaccharides such as chitosan, etc.; 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, etc.

[0016] Further, 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, 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 according to different small molecule active ingredients.

[0017] 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, the thickness of the polymer layer is 7 to 22 nm.

[0018] When preparing protein nanogels with active proteins as templates, it is necessary to consider the effects of the presence and thickness of the polymer shell on the activity and stability of the protein. The premise for the protein / enzyme to show activity is contact with the substrate molecules, but the presence of the polymer shell will hinder the contact between the two; at the same time, the structure of the active protein is relatively complex and easy to inactivate. The polymer wrapping can improve the structural stability of the protein, thereby protecting its activity. Therefore, to achieve the purpose of the present invention, the control of the thickness of the polymer shell is crucial. On the one hand, it is necessary to achieve the protective effect on the protein, but it is not so far as to shield the protein and the release and action of the small molecule active ingredients loaded in the protein. The present invention prepares a series of protein nanogels with a shell thickness of 7-100nm by controlling the polymerization conditions. It is found that the polymer shell with a shell thickness of more than 50nm will hinder the transmission of substrate molecules, so that the protein / or protein-loaded small molecules at the core of the gel cannot play an active role, and show a lower overall activity of the nanogel. The protein nanogel with a shell thickness of 7-22nm can protect the activity of the protein in the pH range of 4 to 8 and below 60°C.

[0019] 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 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, and their diameters were controllable between 20-200nm. The penetration depth of protein nanogels within 24 hours of transdermal penetration was investigated and it was found that when the gel size was the same, the penetration depth of protein nanogels with positive charges on the surface was deeper than that of negatively charged protein nanogels; when the surface potential was close, the penetration abilities of protein nanogels with positive charges on the surface of different sizes were 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 .

[0020] 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 fitted 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 is regulated. Wherein D is the transdermal depth, in μm; d is the size of the active ingredient transdermal delivery system of the present invention, in nm. The scope of application of this equation is to predict the penetration depth of protein nanogel on skin tissue. The protein nanogel should have similar polymer shell physicochemical properties (such as the deformation ability and roughness of the material should be similar to polyacrylamide, the overall shape of the nanogel should be close to a spherical shape, and the shell surface zeta potential should be +3mV to +7mV, such as about +4mV, about +5mV, about +6mV), and the size of the nanogel 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 the 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 of fungal skin diseases are keratinocytes and keratinocytes in the epidermis, the delivery targets of vitiligo are melanocytes at the junction of the epidermis and dermis, and the delivery targets of 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, the existing transdermal delivery technology is not universal for different proteins and small molecules, and there are few studies on regulating transdermal depth; the penetration behavior of large molecules and small molecules in different skin layers is still unclear, and it is difficult to establish a formula to describe the efficiency of drug transdermal delivery.

[0021] If the larger the penetration depth, the better, the size of the active ingredient transdermal delivery system of the present invention is 40-70 nm.

[0022] 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:

[0023] (1) adding a small molecule with a polymerizable double bond to a protein solution to obtain a surface-modified protein solution;

[0024] (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;

[0025] (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.

[0026] 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.

[0027] Furthermore, in step (1), the small molecule with a polymerizable double bond is adsorbed on the surface of the protein molecule by electrostatic interaction, covalent crosslinking, etc. 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.

[0028] Furthermore, in step (2), the small molecule active ingredient is attached to the protein by electrostatic interaction, hydrophilic-hydrophobic interaction, hydrogen bonding, 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.

[0029] Furthermore, 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. Preferably, the monomer includes N-(3-aminopropyl) methacrylamide hydrochloride. With N-(3-aminopropyl) methacrylamide hydrochloride as a monomer, the surface of the obtained 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.

[0030] 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.

[0031] 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, and the co-initiator includes 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.

[0032] Furthermore, in step (3), before polymerization, the protein concentration is diluted to 1-2 mg / mL using a buffer solution or an organic solvent.

[0033] Furthermore, the method for preparing the active ingredient transdermal delivery system based on a protein carrier further comprises a post-treatment step (4): after the polymerization reaction is completed, the complex solution is purified by ultrafiltration, and the purified mother solution is refrigerated and stored.

[0034] 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 drugs, cosmetics, and health products, wherein the drugs, cosmetics, and health products are absorbed and utilized by organisms in a transdermal form.

[0035] The organisms include humans and animals.

[0036] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0037] 1. The present invention modifies polymerizable double bond compounds on proteins, loads small molecule active ingredients, and then initiates polymerization reaction in situ on the protein surface through polymerization reaction, and coats the protein surface with a polymer protective layer, which is bio-friendly and has no or little irritation to the skin. During the transdermal delivery process, the surface properties and thickness of the polymer protective layer can be regulated to effectively regulate the transdermal depth of the protein nanogel, so that the nanogel as a whole can effectively penetrate the skin to reach the position where the protein and small molecule active ingredients need to be released. This significantly improves the defect of poor delivery efficiency of previous protein delivery systems.

[0038] 2. The preparation method of the transdermal delivery system of the present invention is simple, has a high yield, and can be industrialized and mass-produced.

[0039] 3. The raw materials used in the present invention are all bio-friendly and can be used as drug 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

[0040] Figure 1 It is the fitting curve of the penetration depth D and the particle size d of the protein nanogel in Example 1 at 24 hours.

[0041] Figure 2 This is a TEM image of the composite (PMPC-nEN@Cur) obtained in Example 2.

[0042] Figure 3 is a confocal image of the permeation of polymer-protein complexes. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with specific examples, but is not limited to the contents of the specification. All reagents used are commercially available reagents in the art.

[0044] Example 1 Preparation of polyacrylamide-mouse serum protein nanogel

[0045] (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-acrylsuccinimide (mass concentration 1%) to 2 mL of the 1 mg / mL mouse serum albumin (MSA) solution, mix thoroughly and react for 2 hours to obtain a mouse serum albumin (aMSA) with a polymerizable double bond modified on the surface.

[0046] (2) aMSA, acrylamide (AAm), aminopropyl methacrylamide hydrochloride (APM) and N,N'-methylenebisacrylamide (BIS) were mixed according to the specified molar ratio, and the protein concentration was diluted to 1 mg / mL with pH 7.4 phosphate buffer. Then, 3.8 mg of ammonium persulfate and 7.6 mg of tetramethylethylenediamine were added to the initiator according to each 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 different sizes were obtained. The preparation conditions and the measured final size and surface zeta potential are shown in Table 1.

[0047] Table 1 Preparation conditions and final sizes and surface potentials of protein nanogels of a series of sizes

[0048]

[0049] In the table, MSA-NB, N is the particle size, which is 20-200nm, and B is the symbol + or -, indicating the positive or negative of its surface Zeta potential. When a certain proportion of aminopropyl methacrylamide hydrochloride (APM) monomer is added, the surface Zeta potential of the obtained nanogel (nMSA) changes from negative potential to positive potential.

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

[0051] 4 mL of the protein nanogel nMSA of different sizes in Example 1 with a concentration of 1 mg / mL was mixed with 1 mL of fluorescein isothiocyanate with a concentration of 1 mg / mL and reacted overnight, and dialyzed overnight in a dialysis bag with a molecular weight cutoff of 14000 Da to remove unreacted small molecules to obtain fluorescently labeled nMSA. Fresh pigskin was washed with PBS, and the stratum corneum was fixed upward in a transdermal diffusion instrument and kept warm at 37°C. 0.5 mL of a fluorescently labeled complex solution with a concentration of 1 mg / mL was dripped onto the pigskin tissue, and frozen sections were made after 24 hours. The penetration of the nanocapsules was observed under a confocal microscope, and the results of the statistical penetration depth are shown in Table 2.

[0052] Table 2 The penetration depth of a series of protein nanogels in 24 hours

[0053] Sample name nMSA-20- nMSA-50- nMSA-100- nMSA-150- nMSA-200- Penetration depth (μm) 85.0±15.7 89.5±8.1 80.6±14.0 71.3±11.9 60.4±5.2 Sample name nMSA-20+ nMSA-50+ nMSA-100+ nMSA-150+ nMSA-200+ Penetration depth (μm) 120.7±8.3 166±15.9 111.5±6.7 104.9±6.8 67.5±2.8

[0054] By comparing the penetration depths of protein nanogels of the same size but different surface potentials, it was found that the protein nanogels with positive zeta potential had a deeper penetration depth, 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 could 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 most of the protein nanogels larger than 200nm 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 curves of the penetration depth D and particle size d of the protein nanogels with positive surface potential at 24 hours were obtained as shown in the figure. Figure 1 shown.

[0055] 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.

[0056] This expression can be used to preliminarily predict the permeability of protein nanogels with a particle size in the range of 20-150nm and a positive surface potential, providing a reference for the design of systems with different delivery depth requirements in different application scenarios.

[0057] Example 2 Preparation method of polyphosphorylcholine-etanercept complex:

[0058] (1) Dissolve 2 mg of etanercept (EN) in 2 mL of PBS buffer at pH 7.4 to obtain a 1 mg / mL EN solution. Add a dimethyl sulfoxide solution containing 0.4 mg of N-propylene succinimide (mass concentration 1%) to 2 mL of the 1 mg / mL etanercept (EN) solution, mix thoroughly and react for 2 hours to obtain etanercept (aEN) with a polymerizable double bond modified on the surface;

[0059] (2) To 2 mL of a 1 mg / mL surface double bond-modified protein solution, 0.2 mL of a 2 mg / mL curcumin ethanol solution was added dropwise while stirring at 300 r / min, and the mixture was thoroughly mixed to obtain curcumin-loaded etanercept (aEN@Cur).

[0060] (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, and in situ free radical polymerization was initiated at room temperature. After 4 h of reaction, a polyphosphorylcholine-etanercept complex loaded with retinol (PMPC-nEN@Cur) was obtained.

[0061] (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 stored in a refrigerator.

[0062] The mother solution of the complex (PMPC-nEN@Cur) obtained in Example 1 and etanercept (EN) were diluted to 1 mg / mL, and their sizes were tested by dynamic light scattering (DLS). The particle size of the complex in the system was tested using a Malvern Nano Zs90 nanoparticle size potentiometer at room temperature; the Zeta potential was tested using a Malvern Nano Zs90 nanoparticle size potentiometer by electrophoresis. The particle sizes of the etanercept (EN) solution and the complex (PMPC-nEN@Cur) solution, both at a concentration of 1 mg / mL, were 6 nm and 53 nm, respectively, and the Zeta potentials were -3.5±1.7 mV and -2.4±0.8 mV, respectively.

[0063] Figure 2This 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, and it was found that its particle size was about 50 nm and the thickness of the polymer shell was about 22-24 nm, which was consistent with the DLS test results; and the complex had good dispersibility and no obvious aggregation occurred.

[0064] Example 3 Preparation method of polyethylene glycol-cyclosporine A complex:

[0065] (1) 2 mg of cyclosporine A (CsA) was dissolved in 2 mL of PBS buffer at pH 7.4 to obtain a 1 mg / mL CsA solution. 0.1 mg of N-acryl succinimide was added to 2 mL of the 1 mg / mL cyclosporine A (CsA) solution, wherein N-acryl succinimide was dissolved in dimethyl sulfoxide, and the mixture was mixed thoroughly and reacted for 2 hours to obtain cyclosporine A (aCsA) with a polymerizable double bond modified on the surface.

[0066] (2) To 2 mL of 1 mg / mL surface double-bond modified glargine insulin solution, 0.25 mL of 0.2 mg / mL lycopene in acetone solution was added dropwise with stirring at 350 r / min, and the mixture was thoroughly mixed to obtain lycopene-loaded cyclosporin A (aCsA@Lyc).

[0067] (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, 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, and 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.

[0068] (4) Post-treatment: The obtained complex solution was subjected to ultrafiltration treatment 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 stored in a refrigerator.

[0069] The mother solution of the complex (PEG-nCsA@Lyc) obtained in Example 3 and cyclosporine A (CsA) were diluted to 1 mg / mL, and their sizes were tested by dynamic light scattering (DLS). The particle size of the complex in the system was tested using a Malvern Nano Zs90 nanoparticle size potentiometer at room temperature; the Zeta potential was tested using a Malvern Nano Zs90 nanoparticle size potentiometer by electrophoresis. The cyclosporine A (CsA) solution and the complex (PEG-nCsA@Lyc) solution, both with 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.

[0070] The results of particle size and Zeta potential showed that a phosphorylcholine polymer shell with a thickness of about 25 nm was formed on the outer layer of etanercept (EN), and a polyphosphorylcholine-etanercept complex (PMPC-nEN@Cur) was obtained; 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.

[0071] Application Example 2

[0072] Take 2mL of polymer-protein complex with a concentration of 5.6mg / mL and put it into a dialysis bag with a molecular weight cutoff of 3000, immerse the dialysis bag in a phosphate buffer with a pH of 6, and oscillate the entire system at 37°C. Sustained release time 48h. Within 48h, samples were taken regularly to measure the concentration of loaded small molecule drugs under an ultraviolet spectrophotometer, and the sustained release of small molecule drugs by the composite system was calculated. The results are statistically shown in Table 3. The sustained release results show that the composite system has a sustained release effect on the encapsulated functional small molecules within 24 hours, which is conducive to the continued physiological effect of small molecules on the delivery part for a longer period of time.

[0073] Table 3 Cumulative release rate

[0074]

[0075] It can be seen from the data in Table 3 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 layers of the skin, thereby improving the utilization rate.

[0076] Application Example 3

[0077] Example 3 Determination of the antioxidant capacity of the prepared polyethylene glycol-cyclosporin A complex (PEG-nCsA@Lyc): The ROS level in human epidermal cells HaCaT cells was detected using a ROS kit. HaCaT cells (1*10 4 / well), after 12h of complete cell attachment, the cells were irradiated with UVB ultraviolet irradiator to induce internal ROS production, and cells not treated with ultraviolet radiation were left as negative controls. After irradiation, the original culture medium was removed from all wells and 100μL serum-free culture medium was added (the culture medium was not added with serum to avoid the effect of serum on the cell uptake of the complex during the subsequent drug addition process), 10μL PBS buffer was added to the positive control group, 10μL 0.01mg / mL complex solution was added to the low concentration experimental group, 10μL 0.02mg / mL complex solution was added to the medium concentration experimental group, and 10μL 0.04mg / mL complex solution was added to the high concentration experimental group. After incubation for 24h, the original culture medium was removed from all cells and 100μL serum-free culture medium and 10μL DCFH-DA culture solution were added, and incubated at 37°C in the dark for 20min. The cells were then washed three times with PBS, and the fluorescence intensity of the cell DCF was detected using an enzyme marker. The results are statistically shown in Table 4.

[0078] Table 4 Antioxidant capacity test of polyethylene glycol-cyclosporin A complex

[0079]

[0080] It can be seen from the data in Table 4 that the polyethylene glycol-cyclosporin A protein nanogel has good antioxidant capacity, and this capacity increases with increasing concentration.

[0081] Application Example 4

[0082] 4 mL of 1 mg / mL polymer-protein complex solution of Example 1 and Example 2 was mixed with 1 mL of 1 mg / mL fluorescein isothiocyanate and reacted overnight. The mixture was dialyzed overnight in a dialysis bag with a molecular weight cutoff of 14,000 Da to remove unreacted small molecules to obtain a fluorescently labeled complex. Fresh pig skin was washed with PBS, fixed in a transdermal diffusion instrument with the stratum corneum facing upward, and kept warm at 37°C. 0.5 mL of 1 mg / mL fluorescently labeled complex solution was dripped onto the pig skin tissue, and frozen sections were taken at 24 h, 4 h, and 1 h. The penetration of the nanocapsules was observed under a confocal microscope, and the results of the statistical penetration depth are shown in Table 5. The confocal image of the penetration of the polymer-protein complex observed at 24 hours is shown in Table 5. Figure 3 In the study, it was found that the protein nanogel penetrated the skin barrier and reached the dermis within 24 hours.

[0083] Table 5 Transdermal penetration of polymer-protein complexes

[0084]

[0085]

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. The active ingredient transdermal delivery system according to claim 1, characterized in that: The small molecule active ingredient is capable of combining 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; further, 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; The common properties of the protein are: size 3-20 nm, isoelectric point 3-9; preferably water-soluble protein; preferably, the protein is selected from at least one of tyrosinase, etanercept, insulin glargine, pegfilgrastim, octreotide, liraglutide, desmopressin, cyclosporine A, catalase, antigen protein, antigen peptide, human serum albumin, glucose oxidase, lactate dehydrogenase, oxalate decarboxylase, urate oxidase, ethanol oxidase, and alcohol dehydrogenase.

3. 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 betaine, and polysulfonic acid betaine; further, the polymer shell is formed by polymerization reaction, and the optional monomers include at least one of vinyl pyrrolidone, 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.

4. The active ingredient transdermal delivery system according to claim 1, characterized in that: The polymer is selected from polyelectrolytes; preferably, 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.

5. The active ingredient transdermal delivery system according to claim 1, characterized in that: The mass ratio of small molecule active ingredient to protein is 1:10-1000, the size of protein is 2-15 nm, and the thickness of polymer layer is 1 to 50 nm; Furthermore, 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; further, the thickness of the polymer layer is 7 to 22 nm.

6. 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 any one of claims 1 to 5, 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 is regulated; wherein D is the transdermal depth, in μm; d is the size of the transdermal delivery system of the active ingredient of the present invention, in nm.

7. The method for preparing the active ingredient transdermal delivery system based on a protein carrier according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) adding a small molecule 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.

8. The preparation method according to claim 7, characterized in that: 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; the concentration of protein in the protein solution is 1-100 mg / L, preferably 1-10 mg / L; Furthermore, 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, preferably 1:5-20. Furthermore, in step (2), 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.

9. The preparation method according to claim 7, 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; preferably, the monomer includes N-(3-aminopropyl) methacrylamide hydrochloride; Further, the molar ratio of protein, monomer and cross-linking agent is 1:100-100000:10-10000, preferably 1:2000-20000:200-2000; Furthermore, 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 includes at least one of sodium bisulfite and tetramethylethylenediamine, and the amount of the co-initiator is 1-3 times the mass of the initiator; Furthermore, the method further comprises a post-treatment step (4): after the polymerization reaction is completed, the complex solution is purified by ultrafiltration, and the purified mother liquor is refrigerated and stored.

10. Use of the protein carrier-based active ingredient transdermal delivery system according to any one of claims 1 to 5 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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