A protein carrier-based active ingredient transdermal delivery system, and a preparation method and application thereof

Abstract

The application provides 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 coated on the surface of the protein. The application modifies a polymerizable double bond compound on the protein, loads the small-molecule active ingredient, and then initiates a polymerization reaction in situ on the surface of the protein after loading the small-molecule active ingredient, so as to coat a polymer protective layer on the surface of the protein. The polymer protective layer is biocompatible, and the surface properties of the polymer layer can be regulated according to the transdermal depth requirement. The protein and the active small molecule are targeted to be delivered transdermally while breaking through the skin barrier, and the defects of poor delivery efficiency of the previous protein delivery system are significantly improved. The preparation method of the transdermal delivery system is simple, the yield is high, and the system can be produced on a large scale.

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 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] Based on the structure of skin and the mechanism of skin barrier, the applicant uses protein as template material, forms polymer shell layer on the surface of protein by in-situ polymerization technology, and controls the shell layer properties by copolymerization of various monomers, to form a series of protein nanogels with protein as core and polymer as shell layer, and the thickness and properties of the shell layer are controllable. Using the above protein nanogel, the optimal size and material property combination of the skin barrier permeation performance are studied and determined; further, using the hydrophobic domain in the core protein as a loading site, adsorbing and loading small molecule hydrophobic drugs / active molecules, realizing the transdermal delivery of small molecule hydrophobic drugs / active substances; further, using the universality of in-situ polymerization method, replacing protein with catalytically active enzyme, realizing the transdermal delivery of protein drugs (enzyme).

[0006] The inventor's previous research developed an in-situ polymerization technology to form a polymer shell layer on the outside of the protein. The specific operation is summarized as follows: 1) modifying the protein surface with reactive double bonds as polymerization sites; 2) adding acrylate / methyl acrylate monomers; 3) adding an initiator to form free radicals, which then initiate the polymerization of polymerizable monomers on the surface of the protein, and finally form a polymer network to encapsulate the protein (referred to as protein nanogel). Based on the above research, protein nanogel delivery systems for gout, brain glioma and Parkinson's disease have been successfully developed (articles). The above research all use intravenous injection as the administration method, and the drug delivery is carried out through the blood. The delivery barrier faced by the design of the delivery material is the recognition, phagocytosis and elimination of foreign substances by immune cells in the blood and various organs. Therefore, protein nanogels with neutral electric charge (zeta potential close to 0 as much as possible) and thick hydration layer (such as polypyrrolidone, polyphosphocholine, etc.) are needed.

[0007] Transdermal delivery needs to overcome the skin barrier, and the difference between the skin barrier and the systemic immune barrier is great. In existing research and drug applications, a large number of delivery technologies capable of intravenous injection have been developed, but the efficiency and effectiveness of transdermal administration are still very low, and new and efficient transdermal delivery schemes are also less in basic research. SUMMARY

[0008] In order to overcome the fact 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 of 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 nanosized protein-polymer particles, and constructs a highly 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 are 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 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 nanosize and surface properties.

[0011] 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; 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 hyperpigmentation.

[0012] 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.

[0013] 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; 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. By changing the ratio of different substances in the polymer and the thickness of the polymer shell, the surface potential of the protein nanogel is changed in the range of -10mV to +10mV without affecting the stability of the system, thereby optimizing its ability to penetrate the skin barrier. To meet the above conditions, 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 betaines, and polysulfonic acid betaines. The polymer is obtained by a polymerization method well known in the art, such as by polymerization of a monomer containing a carbon-carbon unsaturated double bond under the action of an initiator, or by polycondensation of a substance with a functional group, or by ring-opening polymerization.

[0014] 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.

[0015] In addition to initiating polymerization to form a polymer shell, at least one positively charged polyelectrolyte and one negatively charged polyelectrolyte can be selected to form a protein nanogel by electrostatic interaction on the outside of the protein. The optional positively charged polyelectrolyte includes: poly(acrylamide hydrochloride), poly(L-lysine), polyethyleneimine, poly(L-histidine), poly(N,N-dimethylaminoethyl methacrylate), poly(methacrylamide propyl trimethyl ammonium chloride), and natural or synthetic polysaccharides such as chitosan and the like; the optional negatively charged polyelectrolyte includes: poly(acrylic acid), polystyrene sulfonate, alginate, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dextran sulfate, poly(methacrylic acid), oxidized cellulose, carboxymethyl cellulose, polyaspartic acid, and polyglutamic acid, and the like.

[0016] Further, in the protein nanogel, the size of the protein is 2-15 nm, and the thickness of the polymer layer is 1-50 nm. The mass ratio of the small molecule active ingredient to the protein is 1:1-100, such as 1:2-10, and at 1:2-5. The mass ratio of the small molecule active ingredient to the protein mainly depends on the amount of small molecule active ingredient that the protein can load, and the ratio is different according to different small molecule active ingredients.

[0017] Further, 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 2-30 nm. Preferably, the thickness of the polymer layer is 7-22 nm.

[0018] When preparing a protein nanogel using an active protein as a template, the influence of the presence and thickness of the polymer shell on the activity and stability of the protein needs to be considered. The prerequisite for the activity of a protein / enzyme is to contact with a substrate molecule, but the presence of the polymer shell hinders the contact between the two. At the same time, the structure of the active protein is relatively complex and is easy to be inactivated, and the polymer wrapping can improve the structural stability of the protein, thereby protecting its activity. Therefore, to achieve the purpose of the present application, the control of the thickness of the polymer shell is crucial. On the one hand, the protective effect on the protein needs to be achieved, but on the other hand, the release and action of the protein and the small molecule active ingredient loaded in the protein should not be shielded. The present application has prepared a series of protein nanogels with a shell thickness of 7-100 nm by controlling the polymerization conditions, and it is found that the polymer shell with a shell thickness of more than 50 nm hinders the transmission of the substrate molecule, so that the protein or the small molecule loaded in the protein at the core of the gel cannot exert active effect, and the overall activity of the nanogel is low. The protein nanogel with a shell thickness of 7-22 nm can protect the activity of the protein under the condition of pH 4-8 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 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 .

[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 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.

[0021] 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.

[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) 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;

[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] Further, 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, dimethyl sulfoxide; the concentration of the protein in the protein solution is 1-100 mg / L, preferably 1-10 mg / L.

[0027] Further, in step (1), the small molecule with polymerizable double bonds is adsorbed on the surface of the protein molecule by electrostatic interaction, covalent crosslinking, etc. Examples of the small molecule with polymerizable double bonds include but are not limited to at least one of N-acrylsuccinimide, maleimide. The mass ratio of the small molecule with polymerizable double bonds to the protein is 1:1-50, preferably 1:5-20.

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

[0029] Further, in step (3), the monomer is selected from at least one of 2-methacryloyloxyethylphosphocholine, vinylpyrrolidone, acrylamide, polyethylene glycol methyl ether acrylate, N-(3-aminopropyl) methacrylamide hydrochloride, and the crosslinking agent is selected from at least one of N,N'-methylene bisacrylamide, ethylene glycol diacrylate, polyethylene glycol diacrylate, glycerol dimethyl acrylate. Preferably, the monomer includes N-(3-aminopropyl) methacrylamide hydrochloride. With N-(3-aminopropyl) methacrylamide hydrochloride as the monomer, the obtained polymer has a positive charge on the surface, which is conducive to interaction with the lipid components in the skin, weakens the hindering effect of the lipid bilayer on the protein nanogel, and promotes the penetration of the protein nanogel through the intercellular pathway.

[0030] Further, in step (3), the mass ratio of the protein, the monomer, and the crosslinking agent is 1:100-100000:10-10000, preferably 1:2000-20000:200-2000.

[0031] Further, in step (3), the initiator is selected from at least one of sodium persulfate, ammonium persulfate, 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 tetramethyl ethylenediamine, and the amount of the co-initiator is 1-3 times, such as 2 times, the amount of the initiator.

[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 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.

[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 medicines, cosmetics, and health products, which 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] 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 polymer protective layer that is biofriendly and non-irritating or minimally irritating to the skin. During transdermal delivery, the surface properties and thickness of the polymer protective layer can be 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.

[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 produced on a large scale.

[0039] 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

[0040] Figure 1 d is the fitting curve of the penetration depth D and 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 Confocal images of the permeation of polymer-protein complexes. DETAILED DESCRIPTION

[0043] 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.

[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-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.

[0046] (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.

[0047] Table 1 Preparation conditions, final sizes and surface potentials of protein nanogels of various sizes

[0048]

[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. The addition of a certain proportion of aminopropylmethacrylamide hydrochloride (APM) monomer can shift the surface zeta potential of the resulting nanogel (nMSA) 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] 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 penetration depth of protein nanogels with positive zeta potential was deeper, indicating that the positive surface charge was 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 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 nanogel with positive surface potential at 24 hours were obtained as shown below. 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 20-150 nm 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 (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.

[0059] (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.

[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. In situ free radical polymerization was initiated at room temperature. After 4 h of reaction, a curcumin-loaded polyphosphorylcholine-etanercept complex (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 refrigerated for storage.

[0062] The stock solution of the complex (PMPC-nEN@Cur) obtained in Example 1 and etanercept (EN) were diluted to 1 mg / mL and their sizes were measured using dynamic light scattering (DLS). The particle size of the complex was measured using a Malvern Nano Zs90 nanometer at room temperature. The zeta potential was also measured using a Malvern Nano Zs90 nanometer using 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, with zeta potentials of -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. It was found that its particle size was approximately 50 nm and the polymer shell thickness was approximately 22-24 nm, which was consistent with the DLS test results. The complex also had good dispersion and showed no obvious aggregation.

[0064] Example 3 Preparation of Polyethylene Glycol-Cyclosporin A Complex:

[0065] (1) 2 mg of cyclosporine 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 cyclosporine A (CsA) solution, and the mixture was thoroughly mixed and reacted for 2 h to obtain cyclosporine A (aCsA) with a polymerizable double bond modified on its surface.

[0066] (2) To 2 mL of a 1 mg / mL solution of surface double-bond-modified insulin glargine, 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.

[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. 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.

[0068] (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.

[0069] The stock 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 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 cyclosporine A (CsA) solution and the complex (PEG-nCsA@Lyc) solution, both at a concentration of 1 mg / mL, exhibited particle sizes of 5 nm and 37 nm, respectively, with zeta potentials of -6.0±2.3 mV and -5.9±1.1 mV, respectively.

[0070] The particle size and zeta potential results 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; and 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] 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.

[0073] Table 3 Cumulative release rate

[0074]

[0075] 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.

[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 HaCaT cells was detected using a ROS kit. HaCaT cells (1*10 4Cells 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.

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

[0079]

[0080] 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.

[0081] Application Example 4

[0082] 4 mL of the polymer-protein complex solution of Example 1 and Example 2 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. The unreacted small molecules were removed by dialyzing overnight in a dialysis bag with a molecular weight cutoff of 14,000 Da to obtain a fluorescently labeled complex. Fresh pig skin 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 the fluorescently labeled complex solution with a concentration of 1 mg / mL was added dropwise on the pig skin tissue, and frozen sections were taken at 24h, 4h and 1h. The penetration of the nanocapsules was observed under a confocal microscope, and the results of the penetration depth were statistically 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 protein nanogels penetrated the skin barrier and reached the dermis within 24 hours.

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

[0084]

[0085]

Claims

1. A transdermal delivery system for active ingredients based on a protein carrier, characterized in that: It is a small molecule active ingredient-protein-polymer composite structure, comprising a small molecule active ingredient, a protein loaded with the small molecule active ingredient, and a polymer coated on the surface of the protein; the small molecule active ingredient-protein-polymer composite structure is a polyphosphocholine-etanercept complex loaded with curcumin or a polyethylene glycol-cyclosporin A complex loaded with lycopene; The preparation method of the active ingredient transdermal delivery system based on a protein carrier comprises the following steps: (1) adding a small molecule with a polymerizable double bond to a protein solution to obtain a surface-modified protein solution; the small molecule with a polymerizable double bond includes N-propylene succinimide; (2) uniformly mixing a small molecule active ingredient solution and a surface-modified protein solution to obtain a protein solution loaded with a small molecule active ingredient; (3) mixing the protein solution loaded with a small molecule active ingredient, a monomer, and a cross-linking agent, diluting the mixture, adding an initiator, and initiating a polymerization reaction to obtain an active ingredient transdermal delivery system based on a protein carrier; the monomer is selected from 2-methacryloyloxyethyl phosphorylcholine or polyethylene glycol methyl ether acrylate, and the cross-linking agent is N,N'-methylenebisacrylamide; the molar ratio of protein, monomer, and cross-linking agent is 1:100-100000:10-10000.

2. 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.

3. The active ingredient transdermal delivery system according to claim 2, 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 2 to 30 nm.

4. The active ingredient transdermal delivery system according to claim 3, characterized in that The polymer layer thickness ranged from 7 to 22 nm.

5. The active ingredient transdermal delivery system according to claim 1, characterized in that In step (1), the solvent of the protein solution is selected from a phosphate buffer solution or an organic solvent; and the concentration of the protein in the protein solution is 1-100 mg / L.

6. The active ingredient transdermal delivery system according to claim 5, characterized in that 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 1-10 mg / L.

7. The active ingredient transdermal delivery system according to claim 5, characterized in that The mass ratio of the small molecule with polymerizable double bonds to the protein is 1:1-50.

8. The active ingredient transdermal delivery system according to claim 7, characterized in that The mass ratio of the small molecule with polymerizable double bonds to the protein is 1:5-20.

9. The active ingredient transdermal delivery system according to claim 1, characterized in that In step (2), the concentration of the small molecule active ingredient solution is 1-100 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.

10. The active ingredient transdermal delivery system according to claim 9, characterized in that In step (2), the concentration of the small molecule active ingredient solution is 1-10 mg / L.

11. The active ingredient transdermal delivery system according to claim 1, characterized in that In step (3), the molar ratio of protein, monomer and cross-linking agent is 1:100-100000:10-100001:2000-20000:200-2000.

12. The active ingredient transdermal delivery system according to claim 1, characterized in that 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.

13. The active ingredient transdermal delivery system according to claim 12, characterized in that A co-initiator is also added, which 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.

14. The active ingredient transdermal delivery system according to claim 12, characterized in that The method for preparing 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.

15. Use of the active ingredient transdermal delivery system based on a protein carrier according to any one of claims 1 to 14 in the preparation of a drug that is bioabsorbed and utilized in a transdermal form.

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