Targeted supramolecular BAPs-PLGA nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to a targeted supramolecular BAPs-PLGA nanoparticle as well as a preparation method and an application of the targeted supramolecular BAPs-PLGA nanoparticle. The nanoparticle is prepared through a microfluidic technology and an ultrasonic emulsification technology, PLGA is used as an oil phase, supramolecular BAPs is used as a water phase to form a primary emulsion, and then the primary emulsion and a water phase containing an emulsifier and supramolecular RGD form a multiple emulsion. According to the technology, the BAPs and the eutectic solvent (TG-DES) are combined to form a stable supramolecular structure, so that the solubility and the stability of the BAPs are remarkably improved. The supramolecular RGD is attached to the surface of the nanoparticle and endows the nanoparticle with targeting property, and the cellular uptake ability of the BAPs can be improved through NRP-1 mediated transendocytosis. According to the invention, the bioavailability of the BAPs can be improved, and a new solution is provided for a drug delivery system and skin penetration enhancement. In addition, the preparation method of the nanoparticles is simple, controllable in process, suitable for industrial production and wide in application prospect.

Description

Technical Field

[0001] The present invention relates to the technical fields of biomedicine and cosmetics, and particularly to a targeted supramolecular BAPs-PLGA nanoparticle and its preparation method and application. Background Art

[0002] Bioactive peptides (BAPs), as a kind of molecules with extensive biological activities, have been widely used in clinical treatment and cosmetics. BAPs have remarkable effects in promoting collagen synthesis, assisting wound healing, smoothing wrinkles, antioxidation, antibacterial, whitening, etc. However, BAPs also face many challenges, including: 1) unstable physicochemical properties, being prone to oxidation and hydrolysis; 2) short half-life and fast in vivo clearance rate; 3) being difficult to penetrate through the skin or cell membrane. Therefore, enhancing the stability of BAPs, prolonging their half-life, and improving their permeability are the keys to solving such problems.

[0003] Betaine-glycerol deep eutectic solvent (TG-DES) is a deep eutectic solvent composed of betaine and glycerol, having good biocompatibility, a relatively low melting point, and strong dissolving ability. Existing technologies usually improve the stability of BAPs by means of chemical modification, metal ion complexation, liposome technology, and nanotechnology, etc. However, these methods still fail to effectively solve the permeability problem of BAPs when passing through biological barriers, and the preparation process is complex, the preparation cost is high, and industrialization is difficult.

[0004] Arginine-glycine-aspartic acid (Arg-Gly-Asp, abbreviated as RGD) is a tripeptide sequence widely used in targeted delivery systems. Because it can specifically recognize and bind to integrin receptors on the cell surface, it plays an important role in the fields of drug delivery and tissue engineering. In the existing technologies, RGD is often chemically conjugated to the surface of nanocarriers to enhance the targeting property, but this method has problems such as low modification efficiency and poor RGD stability, and fails to fully play a role in the delivery of BAPs.

[0005] In view of the above deficiencies of the existing technologies, the purpose of the present invention is to provide a targeted supramolecular BAPs-PLGA nanoparticle and its preparation method and application, aiming to solve the application problems of poor skin permeability and poor stability of BAPs. Summary of the Invention

[0006] In view of the deficiencies of the above-mentioned existing technologies, the object of the present invention is to provide a targeted supramolecular BAPs-PLGA nanoparticle and its preparation method and application, aiming to solve the application problems of poor skin permeability and poor stability of BAPs.

[0007] The technical solution of the present invention is as follows: In the first aspect of the present invention, a targeted supramolecular BAPs-PLGA nanoparticle is provided, and the nanoparticle is composed of supramolecular RGD, supramolecular BAPs, and PLGA; The supramolecular BAPs are prepared by mixing TG-DES and BAPs at a mass ratio of 1:(50-200) and stirring for 2-12 hours under the protection of a nitrogen pressure of 5-20 MPa; The supramolecular RGD is prepared by mixing TG-DES and RGD at a mass ratio of 1:(50-200) and stirring for 2-12 hours under the protection of a nitrogen pressure of 5-20 MPa.

[0008] Further, for the targeted supramolecular BAPs-PLGA nanoparticle, the BAPs is a composite peptide composed of acetyl hexapeptide-8, acetyl tetrapeptide-9, and hexapeptide-11; The mass ratio of acetyl hexapeptide-8, acetyl tetrapeptide-9, and hexapeptide-11 is (1-2):(1-2):(1-2).

[0009] Further, for the targeted supramolecular BAPs-PLGA nanoparticle, the average particle size of the nanoparticle is 100-200 nm, and the polydispersity index is less than 10%.

[0010] In the second aspect of the present invention, a preparation method of a targeted supramolecular BAPs-PLGA nanoparticle is provided, including the following steps: (1) Dissolve PLGA in dichloromethane to form a primary oil phase; (2) Use supramolecular BAPs as the primary aqueous phase and perform primary emulsification with the primary oil phase in step (1) to form a W / O primary emulsion; The mass ratio of the primary oil phase to the primary aqueous phase is 1:(3-7); (3) Use the primary emulsion obtained in step (2) and supramolecular RGD as the secondary aqueous phase for secondary emulsification to form a W / O / W multiple emulsion; The mass ratio of the primary emulsion to the secondary aqueous phase is 1:(5-15); (4) Stir and volatilize dichloromethane from the multiple emulsion obtained in step (3) to obtain the targeted supramolecular BAPs-PLGA nanoparticle.

[0011] Further, the temperature of the stirring is 20-40°C, the time of the stirring is 4-24 h, and the rotation speed of the stirring is 300-600 rpm.

[0012] Further, both the primary emulsification and the secondary emulsification adopt microfluidic technology and ultrasonic emulsification technology and are carried out under the protection of an inert gas.

[0013] Further, the high-pressure homogenization pressure of the microfluidic technology is 2000 - 8000 bar, and the homogenization time is 20 - 60 min.

[0014] The power of the ultrasonic emulsification technology is 100 - 600 W, the frequency is 10 - 60 kHz, and it pauses for 2 seconds after every 2 seconds of ultrasonic treatment, with a duration of 0.5 - 2 h; The inert gas is nitrogen, and the pressure is 5 - 20 MPa.

[0015] In the third aspect of the present invention, there is provided an application of the above-mentioned targeted supramolecular BAPs-PLGA nanoparticles in the preparation of pharmaceutical preparations and products in the field of cosmetics.

[0016] Beneficial effects: In the present invention, BAPs are combined with a deep eutectic solvent to form supramolecular BAPs through weak interactions such as hydrogen bonds, van der Waals interactions, and electrostatic interactions, increasing the solubility and stability of BAPs and encapsulating them in PLGA nanoparticles, reducing the degree of freedom of molecular motion of BAPs and the risk of enzymatic hydrolysis, thereby improving their stability. On the other hand, the deep eutectic solvent forms a supramolecular structure with RGD, enhancing the stability of RGD and attaching it to the PLGA nanoparticles through intermolecular forces, providing targeting for the nanoparticles, targeting the cell membrane, and initiating NRP-1-mediated transcytosis to solve the problem that BAPs are not easily able to cross the cell membrane, thereby improving the bioavailability of BAPs. Brief Description of the Drawings

[0017] The methods and their beneficial effects of the present invention will be described in detail below in conjunction with the drawings and specific embodiments.

[0018] Figure 1 It is a schematic diagram of the preparation process of the targeted supramolecular BAPs-PLGA nanoparticles.

[0019] Figure 2 It is a schematic diagram of molecular docking of each component of BAPs in the TG-DES system.

[0020] Figure 3 It is a schematic diagram of the weak interactions of each component of BAPs in the TG-DES system.

[0021] Figure 4 It is a schematic diagram of the electrostatic potential (ESP) of each component of BAPs in the TG-DES system.

[0022] Figure 5 It is an ultraviolet absorption spectrum diagram of supramolecular BAPs, supramolecular RGD, and TG-DES, BAPs, and RGD.

[0023] Figure 6It is a comparison chart of the hydrodynamic particle size distributions of BAPs and supramolecular BAPs-PLGA nanoparticles.

[0024] Figure 7 It is a transmission electron microscope observation image of the nanoparticles (the left is the transmission electron microscope observation image of PLGA nanoparticles; the right is the transmission electron microscope observation image of BAPs-PLGA nanoparticles).

[0025] Figure 8 It is a transmission electron microscope observation image of BAPs.

[0026] Figure 9 It is a transmission electron microscope observation image of targeted supramolecular BAPs-PLGA nanoparticles.

[0027] Figure 10 It is a graph of the particle size change of targeted supramolecular BAPs-PLGA nanoparticles within 30 d.

[0028] Figure 11 It is a schematic diagram of the relative survival rates of HaCat cells under the action of targeted supramolecular BAPs-PLGA nanoparticles and BAPs with concentration gradients.

[0029] Figure 12 It is a schematic diagram of the relative survival rates of HFF-1 cells under the action of targeted supramolecular BAPs-PLGA nanoparticles and BAPs with concentration gradients.

[0030] Figure 13 It is a schematic diagram of the uptake results of each component in targeted supramolecular BAPs-PLGA nanoparticles and BAPs by HaCat cells.

[0031] Figure 14 It is a schematic diagram of the fluorescence imaging results after 24 h of transdermal penetration of each component in BAPs.

[0032] Figure 15 It is a schematic diagram of the fluorescence imaging results after 24 h of transdermal penetration of each component in targeted supramolecular BAPs-PLGA nanoparticles.

[0033] Figure 16 It is a statistical chart of the average fluorescence intensity after 24 h of transdermal penetration of each component in BAPs and targeted supramolecular BAPs-PLGA nanoparticles.

[0034] Figure 17 It is a comparison chart of the absorbance of BAPs and targeted supramolecular BAPs-PLGA nanoparticles after enzymatic hydrolysis. Specific implementation manners

[0035] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in this specification in the description of the present invention are only for the purpose of describing specific embodiments and are not used to limit the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The experimental materials in the following embodiments are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0036] : The poly(lactic-co-glycolic acid) (PLGA) used in the present invention was purchased from Jinan Daigang Biotechnology Co., Ltd., with the product name of ester-terminated poly(lactic-co-glycolic acid) PLGA75 / 25COOR, batch number 2024121709, molecular weight of 3.7 - 5.2 kD. Polyvinyl alcohol (PVA) was purchased from Guangzhou Weike Chemical Co., Ltd., batch number RA22080811, and the degree of hydrolysis was 88.2 mol%.

[0037] Example 1: This example provides a method for preparing targeted supramolecular BAPs-PLGA nanoparticles, as Figure 1 shown. The detailed operation steps are as follows: S1) Under the protection of nitrogen at a pressure of 10 MPa, at 37 °C, with continuous stirring at 300 rpm, the composite peptide (acetyl hexapeptide-8, acetyl tetrapeptide-9, hexapeptide-11 in a mass ratio of 1:1:1) and the eutectic solvent betaine-glycerol (TG-DES) were uniformly mixed at a mass ratio of 1:99 and continuously stirred for 2 h to prepare supramolecular BAPs; S2) Under the protection of nitrogen at a pressure of 10 MPa, at 37 °C, with continuous stirring at 300 rpm, RGD and TG-DES were uniformly mixed at a mass ratio of 1:99 and continuously stirred for 2 h to prepare supramolecular RGD; S3) In a microfluidic operating system, at 37 °C and a homogenization pressure of 6000 bar, 1.5% PLGA in dichloromethane was injected as the primary oil phase into the primary aqueous phase. The primary aqueous phase was a 15% aqueous solution of supramolecular BAPs. The mass ratio of the primary aqueous phase to the primary oil phase was 5:1. It was circulated 2 times and emulsified under ultrasound at a power of 300 W and a frequency of 20 kHz. After each 2 s of ultrasound treatment, it was paused for 2 s, and the alternating treatment continued for 0.5 h to obtain a W / O primary emulsion.

[0038] S4) In the microfluidic operating system, at a homogenization pressure of 6000 bar and 37 °C, the primary emulsion was injected into the secondary aqueous phase as the oil phase. The secondary aqueous phase was an aqueous solution containing 0.2% PVA and 5% supramolecular RGD. The mass ratio of the secondary aqueous phase to the primary emulsion was 9:1. It was circulated 2 times and emulsified under ultrasound at a power of 300 W and a frequency of 20 kHz. After each 2 s of ultrasonic treatment, it was paused for 2 s, and the alternating treatment continued for 0.5 h to obtain a W / O / W multiple emulsion. Finally, it was stirred at room temperature for 8 h to volatilize the organic solvent, obtaining targeted supramolecular BAPs-PLGA nanoparticles with supramolecular RGD attached to the surface and supramolecular BAPs encapsulated inside.

[0039] For the supramolecular BAPs involved in step S1 in this example, molecular docking was performed in the TG-DES system. The most stable conformation was selected as the reference from the multiple generated conformations, and only the most likely hydrogen bonds were shown. The results are as Figure 2 shown. According to the three-dimensional structure and computational simulation results, there are 6 hydrogen bonds between acetyl hexapeptide-8 and acetyl tetrapeptide-9, and the hydrogen bond lengths are 2.9 Å, 2.9 Å, 3.0 Å, 3.0 Å, 3.2 Å, and 3.4 Å respectively; there are 2 hydrogen bonds between acetyl hexapeptide-8 and hexapeptide-11, and the hydrogen bond lengths are 2.9 Å and 3.0 Å respectively; there are 2 hydrogen bonds between hexapeptide-11 and acetyl tetrapeptide-9, and the hydrogen bond lengths are 2.9 Å and 3.2 Å respectively. The existence of hydrogen bonds enhances the stability of each component in the composite peptide and reduces the risk of each component being enzymatically hydrolyzed in the form of free peptides during the transdermal process.

[0040] On this basis, calculations of weak interactions and electrostatic potential were performed on the composite peptide under the TG-DES system, and the calculation results are respectively as Figure 3 、 Figure 4 shown. In the schematic diagram of weak interactions ( Figure 3 ), the color of the isosurface (the disc-shaped structure in the figure) is significantly blue, indicating the existence of significant attractive interactions, such as hydrogen bonds and halogen bonds of general strength; the isosurface is completely blue, indicating a very strong weak interaction here; the isosurface is green, indicating the existence of certain van der Waals forces; the color of the isosurface is significantly red, indicating the existence of certain steric hindrance effects, and if it is bright red, it indicates strong steric hindrance; as Figure 3 shown, under the TG-DES system, hydrogen bonds and van der Waals forces exist between the components of the composite peptide as the main non-covalent interactions, promoting the formation of the supramolecular structure.

[0041] The electrostatic potential of the composite peptide under the TG-DES system is as Figure 4As shown, it is divided into two regions, blue and red. The electrostatic potential value of the blue region is negative, indicating that this region is more likely to donate electrons, or in other words, it is more nucleophilic (referring to the ability of a substance to contribute electrons to other molecules or ions) than other regions, laying a theoretical foundation for the co-assembly of the composite peptide and TG-DES into a supramolecular structure.

[0042] Example 2: This example provides a method for preparing targeted supramolecular BAPs-PLGA nanoparticles. The detailed operation steps are as follows: S1) Under nitrogen protection at a pressure of 5 MPa, at 5 °C, with continuous stirring at 400 rpm, the composite peptide (acetyl hexapeptide-8, acetyl tetrapeptide-9, and hexapeptide-11 with a mass ratio of 2:1:1) and the deep eutectic solvent betaine-glycerol (TG-DES) are uniformly mixed at a mass ratio of 1:50 and continuously stirred for 4 h to prepare supramolecular BAPs. S2) Under nitrogen protection at a pressure of 5 MPa, at 5 °C, with continuous stirring at 400 rpm, RGD and TG-DES are uniformly mixed at a mass ratio of 1:50 and continuously stirred for 4 h to prepare supramolecular RGD. S3) In a microfluidic operating system, at 5 °C and a homogenization pressure of 2000 bar, 1.5% PLGA in dichloromethane is injected as the primary oil phase into the primary aqueous phase. The primary aqueous phase is a 15% aqueous solution of supramolecular BAPs. The mass ratio of the primary aqueous phase to the primary oil phase is 3:1. It is circulated 2 times and emulsified under ultrasound at a power of 100 W and a frequency of 10 kHz. After each 2 s of ultrasound treatment, it is paused for 2 s, and the alternating treatment lasts for 1 h to obtain a W / O primary emulsion.

[0043] S4) In a microfluidic operating system, at 5 °C and a homogenization pressure of 2000 bar, the primary emulsion is injected as the oil phase into the secondary aqueous phase. The secondary aqueous phase is an aqueous solution containing 0.2% PVA and 5% supramolecular RGD. The mass ratio of the secondary aqueous phase to the primary emulsion is 8:1. It is circulated 2 times and emulsified under ultrasound at a power of 100 W and a frequency of 10 kHz. After each 2 s of ultrasound treatment, it is paused for 2 s, and the alternating treatment lasts for 1 h to obtain a W / O / W multiple emulsion. Finally, it is stirred at room temperature for 4 h to volatilize the organic solvent, obtaining targeted supramolecular BAPs-PLGA nanoparticles with supramolecular RGD attached to the surface and supramolecular BAPs encapsulated inside.

[0044] Example 3: This example provides a method for preparing targeted supramolecular BAPs-PLGA nanoparticles. The detailed operation steps are as follows: S1) Under the protection of nitrogen gas at a pressure of 15 MPa, at 15 °C, with continuous stirring at 500 rpm, the composite peptide (acetyl hexapeptide-8, acetyl tetrapeptide-9, hexapeptide-11 with a mass ratio of 1:2:1) and the eutectic solvent betaine-glycerol (TG-DES) were uniformly mixed at a mass ratio of 1:150 and continuously stirred for 8 h to prepare supramolecular BAPs; S2) Under the protection of nitrogen gas at a pressure of 15 MPa, at 15 °C, with continuous stirring at 500 rpm, RGD and TG-DES were uniformly mixed at a mass ratio of 1:150 and continuously stirred for 8 h to prepare supramolecular RGD; S3) In a microfluidic operating system, at 15 °C and a homogenization pressure of 4000 bar, 1.5% PLGA in dichloromethane was injected into the primary aqueous phase as the primary oil phase. The primary aqueous phase was a 15% aqueous solution of supramolecular BAPs. The mass ratio of the primary aqueous phase to the primary oil phase was 4:1. It was circulated 2 times and emulsified under ultrasound at a power of 200 W and a frequency of 20 kHz. After each 2 s of ultrasound treatment, it was paused for 2 s, and the alternating treatment continued for 2 h to obtain a W / O primary emulsion.

[0045] S4) In a microfluidic operating system, at 15 °C and a homogenization pressure of 4000 bar, the primary emulsion was injected into the secondary aqueous phase as the oil phase. The secondary aqueous phase was an aqueous solution containing 0.2% PVA and 5% supramolecular RGD. The mass ratio of the secondary aqueous phase to the primary emulsion was 12:1. It was circulated 2 times and emulsified under ultrasound at a power of 200 W and a frequency of 20 kHz. After each 2 s of ultrasound treatment, it was paused for 2 s, and the alternating treatment continued for 2 h to obtain a W / O / W multiple emulsion. Finally, it was stirred at room temperature for 12 h to volatilize the organic solvent, and targeted supramolecular BAPs-PLGA nanoparticles with supramolecular RGD attached to the surface and supramolecular BAPs encapsulated inside were obtained.

[0046] Example 4: This example provides a method for preparing targeted supramolecular BAPs-PLGA nanoparticles. The detailed operation steps are as follows: S1) Under the protection of nitrogen gas at a pressure of 20 MPa, at 40 °C, with continuous stirring at 600 rpm, the composite peptide (acetyl hexapeptide-8, acetyl tetrapeptide-9, hexapeptide-11 with a mass ratio of 1:1:2) and the eutectic solvent betaine-glycerol (TG-DES) were uniformly mixed at a mass ratio of 1:200 and continuously stirred for 12 h to prepare supramolecular BAPs; S2) Under the protection of nitrogen gas at a pressure of 20 MPa, at 40 °C, with continuous stirring at 600 rpm, RGD and TG-DES were uniformly mixed at a mass ratio of 1:200 and continuously stirred for 12 h to prepare supramolecular RGD; S3) In the microfluidic operating system, at a homogenization pressure of 8000 bar and 40 °C, 1.5% PLGA in dichloromethane was injected as the primary oil phase into the primary aqueous phase. The primary aqueous phase was an aqueous solution containing 15% supramolecular BAPs. The mass ratio of the primary aqueous phase to the primary oil phase was 7:1. This was cycled 2 times and emulsified under ultrasound at a power of 500 W and a frequency of 50 kHz. After each 2 s of ultrasonic treatment, it was paused for 2 s, and the alternating treatment continued for 4 h to obtain a W / O primary emulsion.

[0047] S4) In the microfluidic operating system, at a homogenization pressure of 8000 bar and 40 °C, the primary emulsion was injected as the oil phase into the secondary aqueous phase. The secondary aqueous phase was an aqueous solution containing 0.2% PVA and 5% supramolecular RGD. The mass ratio of the secondary aqueous phase to the primary emulsion was 15:1. This was cycled 2 times and emulsified under ultrasound at a power of 500 W and a frequency of 50 kHz. After each 2 s of ultrasonic treatment, it was paused for 2 s, and the alternating treatment continued for 4 h to obtain a W / O / W multiple emulsion. Finally, it was stirred at room temperature for 24 h to allow the organic solvent to volatilize, obtaining targeted supramolecular BAPs-PLGA nanoparticles with supramolecular RGD attached to the surface and supramolecular BAPs encapsulated inside.

[0048] Example 5: The preparation steps of PLGA nanoparticles without supramolecular BAPs encapsulated inside and without supramolecular RGD attached to the surface are as follows: S1) In the microfluidic operating system, at a homogenization pressure of 6000 bar and 37 °C, 1.5% PLGA in dichloromethane was injected as the primary oil phase into the primary aqueous phase. The mass ratio of the primary aqueous phase to the primary oil phase was 5:1. This was cycled 2 times and emulsified under ultrasound at a power of 300 W and a frequency of 20 kHz. After each 2 s of ultrasonic treatment, it was paused for 2 s, and the alternating treatment continued for 0.5 h to obtain a W / O primary emulsion.

[0049] S2) In the microfluidic operating system, at a homogenization pressure of 6000 bar and 37 °C, the primary emulsion was injected as the oil phase into the secondary aqueous phase. The secondary aqueous phase was an aqueous solution containing 0.2% PVA. The mass ratio of the secondary aqueous phase to the primary emulsion was 10:1. This was cycled 2 times and emulsified under ultrasound at a power of 300 W and a frequency of 20 kHz. After each 2 s of ultrasonic treatment, it was paused for 2 s, and the alternating treatment continued for 0.5 h to obtain a W / O / W multiple emulsion. Finally, it was stirred at room temperature for 8 h to allow the organic solvent to volatilize, obtaining empty PLGA nanoparticles.

[0050] Example 6: The preparation steps of BAPs-PLGA nanoparticles with supramolecular BAPs encapsulated inside and without supramolecular RGD attached to the surface are as follows: S1) Under the protection of nitrogen gas at 10 MPa, at 37 °C, with continuous stirring at 300 rpm, the composite peptide (acetyl hexapeptide-8, acetyl tetrapeptide-9, hexapeptide-11 with a mass ratio of 1:1:1) and the eutectic solvent betaine-glycerol (TG-DES) were uniformly mixed at a mass ratio of 1:99 and continuously stirred for 2 h to prepare supramolecular BAPs; S2) In a microfluidic operating system, at 37 °C and a homogenization pressure of 6000 bar, 1.5% PLGA in dichloromethane was injected as the primary oil phase into the primary aqueous phase. The primary aqueous phase was a 15% aqueous solution of supramolecular BAPs. The mass ratio of the primary aqueous phase to the primary oil phase was 5:1. It was circulated 2 times and emulsified under ultrasound at a power of 300 W and a frequency of 20 kHz. After each 2 s of ultrasound treatment, it was paused for 2 s, and the alternating treatment continued for 0.5 h to obtain a W / O primary emulsion.

[0051] S4) In a microfluidic operating system, at 37 °C and a homogenization pressure of 6000 bar, the primary emulsion was injected as the oil phase into the secondary aqueous phase. The secondary aqueous phase was an aqueous solution containing 0.2% PVA. The mass ratio of the secondary aqueous phase to the primary emulsion was 9:1. It was circulated 2 times and emulsified under ultrasound at a power of 300 W and a frequency of 20 kHz. After each 2 s of ultrasound treatment, it was paused for 2 s, and the alternating treatment continued for 0.5 h to obtain a W / O / W multiple emulsion. Finally, it was stirred at room temperature for 8 h to volatilize the organic solvent, and BAPs-PLGA nanoparticles with supramolecular BAPs encapsulated inside were obtained.

[0052] Comparative Example 1: S1) Under the protection of nitrogen gas at 10 MPa, at 37 °C, with continuous stirring at 300 rpm, the composite peptide (acetyl hexapeptide-8, acetyl tetrapeptide-9, hexapeptide-11 with a mass ratio of 1:1:1) and the eutectic solvent betaine-glycerol (TG-DES) were uniformly mixed at a mass ratio of 1:99 and continuously stirred for 2 h to prepare supramolecular BAPs; S2) Under the protection of nitrogen gas at 10 MPa, at 37 °C, with continuous stirring at 300 rpm, RGD and TG-DES were uniformly mixed at a mass ratio of 1:99 and continuously stirred for 2 h to prepare supramolecular RGD; S3) In a microfluidic operating system, at 37 °C and a homogenization pressure of 6000 bar, 1.5% PLGA in dichloromethane was injected as the primary oil phase into the primary aqueous phase. The primary aqueous phase was an aqueous solution containing 15% of supramolecular BAPs. The mass ratio of the primary aqueous phase to the primary oil phase was 1:1. It was circulated 2 times and emulsified under ultrasound at a power of 300 W and a frequency of 20 kHz. After each 2 s of ultrasound treatment, it was paused for 2 s, and the alternating treatment continued for 0.5 h to obtain a W / O primary emulsion.

[0053] S4) In the microfluidic operating system, at a homogenization pressure of 6000 bar and 37 °C, the primary emulsion was injected into the secondary aqueous phase as the oil phase. The secondary aqueous phase was an aqueous solution containing 0.2% PVA and 5% supramolecular RGD. The mass ratio of the secondary aqueous phase to the primary emulsion was 1:1. It was circulated 2 times and emulsified under ultrasound at a power of 300 W and a frequency of 20 kHz. After every 2 s of ultrasonic treatment, it was paused for 2 s, and the alternating treatment continued for 0.5 h to obtain a W / O / W multiple emulsion. During the stirring process at room temperature, as the organic solvent volatilized, the excessive PLGA in the oil phase precipitated out and formed white flocculates suspended in the nano-solution. After filtering off the precipitated PLGA, the nano-solution still exhibited a relatively large particle size and poor polydispersity, probably because the too-high oil-phase ratio increased the viscosity of the system and was not conducive to the formation of nano-dispersion.

[0054] Comparative Example 2: S1) Under the protection of nitrogen gas at a pressure of 10 MPa, at 37 °C, and under continuous stirring at 300 rpm, the composite peptide (acetyl hexapeptide-8, acetyl tetrapeptide-9, and hexapeptide-11 with a mass ratio of 1:1:1) and the deep eutectic solvent betaine-glycerol (TG-DES) were uniformly mixed at a mass ratio of 1:10 and continuously stirred for 12 h. There was still some polypeptide that was not completely dissolved and was suspended in the system, exceeding the maximum amount of polypeptide that could be dissolved in this system, and a supramolecular system could not be formed. S2) Under the protection of nitrogen gas at a pressure of 10 MPa, at 37 °C, and under continuous stirring at 300 rpm, RGD and TG-DES were uniformly mixed at a mass ratio of 1:10 and continuously stirred for 12 h. The result was the same as that of S1, and a supramolecular system could not be formed. Comparative Example 3: S1) Under the protection of nitrogen gas at a pressure of 10 MPa, at 37 °C, and under continuous stirring at 300 rpm, the composite peptide (acetyl hexapeptide-8, acetyl tetrapeptide-9, and hexapeptide-11 with a mass ratio of 1:1:1) and the deep eutectic solvent betaine-glycerol (TG-DES) were uniformly mixed at a mass ratio of 1:99 and continuously stirred for 2 h to prepare supramolecular BAPs. S2) Under the protection of nitrogen gas at a pressure of 10 MPa, at 37 °C, and under continuous stirring at 300 rpm, RGD and TG-DES were uniformly mixed at a mass ratio of 1:99 and continuously stirred for 2 h to prepare supramolecular RGD. S3) In the microfluidic operating system, at a homogenization pressure of 6000 bar and 37 °C, 1.5% PLGA in dichloromethane was injected as the primary oil phase into the primary aqueous phase, which was an aqueous solution of 15% supramolecular BAPs. The mass ratio of the primary aqueous phase to the primary oil phase was 1:5. This was cycled 2 times and emulsified under ultrasound at a power of 300 W and a frequency of 20 kHz. After every 2 s of ultrasonic treatment, it was paused for 2 s, and the alternating treatment continued for 0.5 h to obtain a primary W / O emulsion.

[0055] S4) In the microfluidic operating system, at a homogenization pressure of 6000 bar and 37 °C, the primary emulsion was injected as the oil phase into the secondary aqueous phase, which was an aqueous solution of 5% supramolecular RGD. The mass ratio of the secondary aqueous phase to the primary emulsion was 1:10. This was cycled 2 times and emulsified under ultrasound at a power of 300 W and a frequency of 20 kHz. After every 2 s of ultrasonic treatment, it was paused for 2 s, and the alternating treatment continued for 0.5 h to obtain a W / O / W multiple emulsion. During the stirring process at room temperature, as the organic solvent volatilized, similar to Comparative Example 1, a small amount of white flocculants were suspended in the nano-solution. After filtering out the precipitated PLGA, there were multiple peaks in the particle size distribution diagram of the nano-solution, showing extremely poor polydispersity. With the extension of the standing time, obvious particles precipitated. The main role of the emulsifier is to reduce the surface tension at the oil-water interface and increase the stability of the emulsion. The lack of an emulsifier will cause the emulsion to be unstable, prone to stratification or rupture, thus affecting the formation and dispersibility of the nanoparticles, resulting in the above phenomena.

[0056] Example 7: This example provides a method for preparing BAPs: Under the protection of nitrogen gas at a pressure of 10 MPa, at 37 °C, with continuous stirring at 300 rpm, acetyl hexapeptide-8, acetyl tetrapeptide-9, and hexapeptide-11 were added to water to make the concentration of each peptide reach 50 ppm, and they were uniformly mixed and continuously stirred for 12 h to prepare BAPs.

[0057] Test Example 1: By using a UV spectrophotometer (UV-1900i) to test the UV absorption spectra of supramolecular BAPs, supramolecular RGD, and TG-DES, BAPs, and RGD, the formation of hydrogen bonds was verified, thereby corroborating the formation of the supramolecular structure.

[0058] The formation of hydrogen bonds will affect the electronic structure of molecules, thereby changing their absorption spectra. In the UV spectrum, due to the formation of hydrogen bonds, the π → π transition and n → π transition of solute molecules may be affected. Under the condition of keeping the concentration the same, the UV absorption spectra of supramolecular BAPs, supramolecular RGD, and TG-DES, BAPs, and RGD are as Figure 5As shown, after TG-DES forms supramolecular BAPs and supramolecular RGD with BAPs and RGD respectively, a red shift phenomenon occurs in the ultraviolet absorption spectrum of TG-DES, that is, the absorption peak of TG-DES moves towards the lower wavenumber direction. This may be because the formation of hydrogen bonds reduces the bond force constant between hydrogen atoms and the atoms they are connected to, resulting in a decrease in the vibration frequency.

[0059] Test Example 2: Test the hydrodynamic diameter distribution of the targeted supramolecular BAPs-PLGA nanoparticles prepared in Test Example 1, the PLGA nanoparticles prepared in Example 5, the BAPs-PLGA nanoparticles prepared in Example 6, and the BAPs prepared in Example 7.

[0060] Hydrodynamic diameter generally refers to the particle size of nanoparticles in an aqueous solution, which is measured by dynamic light scattering (DLS) technology. This technology measures the Brownian motion of particles in a liquid to obtain the hydrodynamic diameter of the particles, that is, the hydrodynamic diameter. This diameter includes the core of the nanoparticle and the hydration layer formed by water molecules or other solvent molecules that may be adsorbed on its surface.

[0061] Experimental method: After fully dispersing the sample to be tested, set the conditions such as solvent and temperature, and then use a Malvern particle size analyzer (Malvern Panalytical's Mastersizer) to measure it three times repeatedly. The test results of its particle size distribution are shown in Tables 1-4 respectively: Table 1. Particle size distribution of PLGA nanoparticles

[0062] Table 2. Particle size distribution of BAPs-PLGA nanoparticles

[0063] Table 3. Particle size distribution of targeted supramolecular BAPs-PLGA nanoparticles

[0064] Table 4. Particle size distribution of BAPs

[0065] BAPs is an aqueous solution of acetyl hexapeptide-8, acetyl tetrapeptide-9, and hexapeptide-11. The test results of its hydrodynamic diameter are shown in Table 4. The average value of the hydrodynamic diameter in this system is 335.5 nm, and the polydispersity index is 36.5%. This result indicates that the particle size distribution uniformity of BAPs is poor and the polydispersity is poor.

[0066] Table 1-3 shows the test results of the hydrated particle sizes of PLGA nanoparticles, BAPs-PLGA nanoparticles, and targeted supramolecular BAPs-PLGA nanoparticles, respectively. Among them, PLGA nanoparticles have the smallest hydrated particle size, with an average value of 111.9 nm; BAPs-PLGA nanoparticles have the largest hydrated particle size, with an average value of 182.8 nm; while the average hydrated particle size of targeted supramolecular BAPs-PLGA nanoparticles is 168.4 nm. The internal encapsulation of supramolecular BAPs makes their particle size larger than that of PLGA nanoparticles. The supramolecular RGD attaches to the surface of the nanoparticles through intermolecular interactions, making the nanoparticle structure more compact, thus making its hydrated particle size slightly smaller than that of BAPs-PLGA nanoparticles. Moreover, the polydispersity index of targeted supramolecular BAPs-PLGA nanoparticles is only 8.36% at the minimum. This result indicates that the size distribution of targeted supramolecular BAPs-PLGA nanoparticles has good uniformity and excellent polydispersity.

[0067] In summary, targeted supramolecular BAPs-PLGA nanoparticles have the smallest particle size in the group containing BAPs, increasing the specific surface area, thereby improving their contact area and penetrability with the skin and achieving a better transdermal effect. Moreover, its polydispersity index is the smallest, indicating that targeted supramolecular BAPs-PLGA nanoparticles have better dispersibility and higher particle size consistency.

[0068] Further analysis and processing of the data showed that the fitting results of the particle size distributions of the four groups of samples are as Figure 6 shown. In the particle size distribution diagram, compared with BAPs-PLGA nanoparticles and BAPs, the higher and narrower peak shape of targeted supramolecular BAPs-PLGA nanoparticles can also illustrate its better dispersibility and particle size consistency.

[0069] Test Example 3: The particle sizes of the targeted supramolecular BAPs-PLGA nanoparticles prepared in Test Example 1, the PLGA nanoparticles prepared in Example 5, the BAPs-PLGA nanoparticles prepared in Example 6, and the BAPs prepared in Example 7.

[0070] Particle size is one of the key factors affecting the performance of transdermal drug delivery systems. Smaller particle sizes can usually improve the transdermal permeability of drugs because they are more likely to pass through the tiny gaps in the skin stratum corneum. Research has shown that liposomes with a particle size smaller than 70 nm have more advantages in transdermal performance, with a deeper transdermal depth and higher fluorescence retention in the skin. Therefore, it is necessary to optimize the particle size to achieve the best drug delivery effect.

[0071] Experimental method: 100 μL of the sample to be tested was respectively dropped onto the copper mesh-supported film, and left to dry statically in an oven at 50 °C for 2 h. After thorough drying, an additional 100 μL of the sample was added and left to dry statically in the oven at 50 °C for another 2 h to prepare the sample for transmission electron microscopy observation.

[0072] PLGA nanoparticles ( Figure 7 left), BAPs-PLGA nanoparticles ( Figure 7 right), BAPs ( Figure 8 ), and targeted supramolecular BAPs-PLGA nanoparticles ( Figure 9 ) showed in the transmission electron microscopy observation that the PLGA nanoparticles had the smallest particle size, 20 - 30 nm; after encapsulating BAPs internally, the particle size increased to about 50 nm; the targeted supramolecular BAPs-PLGA nanoparticles were uniformly distributed in the solution, no obvious aggregation was found, and the single particle size was about 40 nm, showing excellent polydispersity. And after attaching the supramolecular RGD on the surface, the nanoparticle structure was more compact, manifested as the nanoparticles showing a deep black color; the three polypeptide molecules in the BAPs system formed aggregates or clusters in the solution, with a particle size of about 200 nm. This aggregation might be caused by hydrophobic interactions, hydrogen bonds or ionic interactions between the polypeptide molecules; The particle size observed by transmission electron microscopy was significantly larger than the hydrodynamic diameter, mainly because the former was obtained by directly observing the morphology and size of the particles through an electron microscope, while the hydrodynamic diameter included the core of the nanoparticles and the hydration layer formed by water molecules or other solvent molecules that might be adsorbed on their surface. In addition, the transmission electron microscopy observation results were consistent with the test results of the Malvern particle size analyzer, that is, compared with BAPs and BAPs-PLGA nanoparticles, the targeted supramolecular BAPs-PLGA nanoparticles showed a smaller particle size, better polydispersity and particle size consistency. Therefore, the subsequent tests mainly used the targeted supramolecular BAPs-PLGA nanoparticles as the research object.

[0073] Test Example 4: Particle size stability of the targeted supramolecular BAPs-PLGA nanoparticles prepared in Test Example 1.

[0074] Particle size stability is crucial in the field of nanotechnology. It not only relates to the efficiency and safety of drug delivery systems but also directly affects the performance of materials science and industrial applications. Particle size stability ensures the effective release and absorption of drugs in the body, playing a significant role in improving the bioavailability of drugs and reducing side effects. In industrial applications, particle size stability is of great significance for ensuring product quality and consistency, improving production efficiency, and reducing environmental impacts. In terms of regulations and standards, particle size stability is also a key factor for nano-products to comply with regulations and industry standards, which is crucial for market access and consumer trust. Therefore, the research and control of particle size stability are an indispensable part of the development of nanotechnology.

[0075] The particle size changes of targeted supramolecular BAPs-PLGA nanoparticles were continuously measured by dynamic light scattering (DLS) technology within one month to evaluate their stability. The specific test method was consistent with Test Example 2. The particle size measurement results of targeted supramolecular BAPs-PLGA nanoparticles within 30 d are shown in Table 5, and the particle size changes are as Figure 10 shown.

[0076] Table 5. Particle size changes of targeted supramolecular BAPs-PLGA nanoparticles within 30 d

[0077] As shown in Table 5, Figure 10 the particle size changes of targeted supramolecular BAPs-PLGA nanoparticles within 30 d were not significant, fluctuating around 169 nm. The polydispersity index showed an increasing trend but still remained within 10%, demonstrating good polydispersity and excellent particle size stability, laying a foundation for the application of targeted supramolecular BAPs-PLGA nanoparticles.

[0078] Test Example 5: The biosafety of the targeted supramolecular BAPs-PLGA nanoparticles prepared in Test Example 1 and BAPs prepared in Example 7.

[0079] CCK-8, full name Cell Counting Kit-8 reagent, can be used for simple and accurate cell proliferation and toxicity analysis. Its basic principle is as follows: This reagent contains a water-soluble tetrazolium salt WST-8 [chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt], which is reduced by dehydrogenases in cells to a highly water-soluble yellow formazan product (Formazan dye) under the action of the electron carrier 1-methoxy-5-methylphenazinium sulfate dimethyl ester (1-Methoxy PMS). The amount of formazan produced is proportional to the number of live cells. Therefore, this property can be used to directly perform cell proliferation and toxicity analysis. The specific experimental steps are as follows: Ⅰ (Cell preparation): Resuscitate the cells and subculture them to the logarithmic growth phase. On the day of the experiment, digest the cells and count them, and adjust the cell density to 1-5 x 10 ^4 cells / mL.

[0080] Ⅱ (Cell seeding): Seed the cell suspension into a 96-well plate at a volume of 100 μL / well, and set 3-5 parallel wells for each group. Place the cells in an incubator at 37°C and 5% CO2 for 24 hours to allow the cells to adhere to the plate.

[0081] Ⅲ (Drug treatment): According to the experimental design, after culturing the cells for 24 hours, add test substances at different concentrations and continue culturing for 24-72 hours. Add an equal volume of culture medium to the control group.

[0082] Ⅳ (CCK-8 detection): Add 10 μL of CCK-8 solution to each well, gently mix it to avoid generating bubbles, and place the cells in an incubator at 37°C and 5% CO2 for 1-4 hours. After the incubation, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value (OD value) of each well at a wavelength of 450 nm, compare it with the control group not treated with the test substance, and calculate the results.

[0083]

[0084] Where: Relative survival rate - relative OD 450 nm , %; Test OD 450 nm - The average OD of the test substance 450 nm ; Neg OD 450 nm - The average OD of the negative control 450 nm .

[0085] Using the human immortalized keratinocyte HaCaT as the test cells, the effects of targeted supramolecular BAPs-PLGA nanoparticles or BAPs on the viability of HaCaT cells were tested at concentrations of 6.25%, 12%, 25%, 50% and 100%. The results are as Figure 11 shown. Under the action of targeted supramolecular BAPs-PLGA nanoparticles or BAPs at different concentrations, the viability of HaCaT cells was greater than 95%, and targeted supramolecular BAPs-PLGA nanoparticles or BAPs did not cause obvious toxicity to HaCaT cells, that is, targeted supramolecular BAPs-PLGA nanoparticles and BAPs have good biosafety.

[0086] Comparative test example 1: The difference between comparative test example 1 and test example 5 is that the HaCaT cells in test example 5 were replaced with human foreskin fibroblasts HFF-1.

[0087] The effects of targeted supramolecular BAPs-PLGA nanoparticles or BAPs on the viability of HFF-1 cells were tested at concentrations of 6.25%, 12%, 25%, 50% and 100%. The results are as Figure 12 shown. Under the action of targeted supramolecular BAPs-PLGA nanoparticles or BAPs at different concentrations, the viability of HFF-1 cells was greater than 95%. A conclusion similar to that of test example 5 can be obtained, that is, targeted supramolecular BAPs-PLGA nanoparticles or BAPs did not cause obvious toxicity to HFF-1 cells, that is, targeted supramolecular BAPs-PLGA nanoparticles and BAPs have good biosafety.

[0088] Test example 6: Test the uptake ability of the test cells for the targeted supramolecular BAPs-PLGA nanoparticles prepared in Example 1 and the BAPs prepared in Example 7.

[0089] A major goal of drug delivery systems is to effectively deliver therapeutic agents to specific cells or tissues. Cellular uptake is a key step in this process because only when the drug is taken up by the target cells can it exert its effect inside the cells. By designing drug delivery systems that can specifically target certain cell types, drug side effects can be reduced and therapeutic efficacy can be improved. By modifying the surface of PLGA nanoparticles with supramolecular RGD, active targeting of specific cell types is achieved, thereby promoting cellular uptake of the drug. The specific experimental steps are as follows: Ⅰ (Cell culture): Resuscitate HaCaT cells, observe their cell morphology and expand the culture to the required number, and the cell density reaches 70% - 80% of the well area.

[0090] Ⅱ (Grouping): Two groups, namely targeted supramolecular BAPs-PLGA nanoparticles and composite peptides, were set up for the experiment.

[0091] Ⅲ (Cover slips): Add cells to each well for cover slips.

[0092] Ⅳ (Co-incubation): Add equal amounts of fluorescently labeled targeted supramolecular BAPs-PLGA nanoparticles and BAPs (AMC-labeled acetyl tetrapeptide-9, Fitc-labeled hexapeptide-11, and RB-labeled acetyl hexapeptide-8) according to the preset concentrations for co-incubation for 48 h. Finally, carefully wash the cells twice with PBS to terminate cell uptake.

[0093] Ⅴ: Stain the cytoskeletal proteins, perform cover slip mounting with a water-soluble mounting medium, take pictures using a fluorescence microscope, and select four colors: red (cytoskeletal proteins), green (hexapeptide-11), orange (acetyl hexapeptide-8), and blue (acetyl tetrapeptide-9).

[0094] Tubulin is the basic protein unit that constitutes microtubules. Microtubules are the main components of the cytoskeleton and exist in almost all eukaryotic cells. In the cell uptake experiment, staining the cytoskeletal proteins can help observe the changes in the cytoskeleton during the uptake process, which is important for understanding how cells take up external substances through mechanisms such as endocytosis.

[0095] Observation of cell uptake under a fluorescence microscope showed that Figure 13 as shown, the cytoskeletal proteins were red, hexapeptide-11 was green, acetyl hexapeptide-8 was orange, and acetyl tetrapeptide-9 was blue. The intensities of the green, orange, and blue fluorescence on the cytoskeletal proteins reflected the cell's uptake ability of hexapeptide-11, acetyl hexapeptide-8, and acetyl tetrapeptide-9. The fluorescence intensities of the three in the targeted supramolecular BAPs-PLGA nanoparticle group were significantly higher than those of BAPs, that is, compared with BAPs, the cell's uptake ability of the bioactive peptides in the targeted supramolecular BAPs-PLGA nanoparticles was stronger. This improvement in ability benefited from the targeting effect of the supramolecular RGD on the nanoparticle surface.

[0096] Test Example 7: In vitro transdermal effect of the BAPs prepared in Test Example 7 The skin is composed of the stratum corneum, epidermis, dermis, subcutaneous tissue, etc. After the drug is placed on the skin surface, it penetrates into the skin, reaches the dermis through the epidermis. Since there are abundant capillaries in the dermis, the drug can be quickly absorbed and then enter the systemic circulation. Therefore, the concentration of the drug on the skin surface is very low, that is, it meets the so-called "sink" condition, and the drug concentration is close to 0. In vitro transdermal diffusion experiments can predict the transdermal absorption rate of drugs, study the effects of media, formulation components, transdermal absorption promoters, etc. on the transdermal rate of drugs, and are the prerequisite guarantees for the effectiveness and safety of transdermal drug preparations. The specific experimental operations are as follows: Ⅰ (Microscopic examination): Select intact porcine skin under a dissecting microscope, cut 6 pieces of skin of the same size, wash it once with normal saline, and blot the surface moisture with filter paper.

[0097] Ⅱ (Fixing the skin): Fix the skin on the Franz diffusion cell, with the stratum corneum facing the dosing chamber and the dermis facing the receiving chamber. Add 17 mL of normal saline to the receiving chamber and remove the air bubbles to ensure that there are no air bubbles between the dermis of the skin and the receiving liquid.

[0098] Ⅲ (Dosing): Turn on the instrument in advance, adjust the water bath temperature to 32 ± 1 °C, add 0.5 mL of BAPs labeled with fluorescent dye to the dosing chamber, seal it with a sealing film, and cover it with tin foil to prevent liquid evaporation. The effective permeation area is 0.36π cm 2 .

[0099] Ⅳ (Permeation): Set the stirring speed to 300 rpm and keep it in the dark.

[0100] Ⅴ (Sampling): Incubate for 24 h, collect the skin at the 24 h time point. Then perform tissue imaging and photography to observe the permeation of the active ingredient of the sample in the skin.

[0101] Ⅵ (Freezing section): Put the skin into 4% paraformaldehyde for fixation overnight, wash it clean with PBS, then quickly freeze it in a -80 °C refrigerator, and then perform OCT vertical embedding and use a cryostat for full-thickness sectioning.

[0102] Ⅶ (Tissue imaging and photography): Use a fluorescence inverted microscope to observe and photograph the sectioned tissue at three excitation wavelengths. Observe the transdermal effect of acetyl tetrapeptide-9 labeled with AMC under the excitation wavelength of Blue (377,477); observe the transdermal effect of hexapeptide-11 labeled with Fitc fluorescence under the excitation wavelength of Green (469,525); observe the transdermal effect of acetyl hexapeptide-8 labeled with RB under the excitation wavelength of Red (586,647); The imaging results after 24 h of BAPs transdermal penetration are as Figure 14 shown, and the fluorescence quantitative data are shown in Table 6: Table 6. Statistical Table of Fluorescence Quantitative Data after 24-hour Percutaneous Penetration of BAPs

[0103] Combined Figure 14 With Table 6, it can be seen that the average fluorescence intensities of acetyl tetrapeptide-9, hexapeptide-11, and acetyl hexapeptide-8 in the three parallel groups of BAPs are 1127±303.8 a.u., 2386±199.3 a.u., and 2269±259.1 a.u., respectively. A small amount of all three penetrated, but the fluorescence was basically concentrated in the stratum corneum, and the subcutaneous fluorescence intensity was very low, with poor transdermal effect.

[0104] Comparative Test Example 2: The difference between Comparative Test Example 2 and Test Example 5 is that the BAPs labeled with fluorescent dye in the drug administration step of Test Example 5 were replaced with an aqueous solution of targeted supramolecular BAPs-PLGA nanoparticles labeled with fluorescent dye.

[0105] The imaging results after 24-hour transdermal penetration of targeted supramolecular BAPs-PLGA nanoparticles are as Figure 15 shown, and the fluorescence quantitative data are shown in Table 7: Table 7. Statistical Table of Fluorescence Quantitative Data after 24-hour Percutaneous Penetration of Aqueous Solution of Targeted Supramolecular BAPs-PLGA Nanoparticles

[0106] According to Table 6 and Table 7, the fluorescence quantitative graphs after 24-hour percutaneous penetration of BAPs and targeted supramolecular BAPs-PLGA nanoparticles were drawn, and the results are as Figure 16 shown. The average fluorescence intensities of acetyl tetrapeptide-9, hexapeptide-11, and acetyl hexapeptide-8 in the three parallel groups of targeted supramolecular BAPs-PLGA nanoparticles are 4564±235.6 a.u., 10313±277.4 a.u., and 12288±289.3 a.u., respectively, which are 4.05, 4.32, and 5.41 times the average fluorescence intensities of each component of BAPs. Moreover, the fluorescence is not only concentrated in the stratum corneum, but obvious fluorescence can also be observed subcutaneously, showing excellent transdermal effect.

[0107] In summary, the fluorescence intensity directly reflects the penetration ability of the polypeptide. The fluorescence intensities of acetyl tetrapeptide-9, hexapeptide-11, and acetyl hexapeptide-8 in the targeted supramolecular BAPs-PLGA nanoparticles are significantly stronger than those of BAPs. That is, the penetration abilities of acetyl tetrapeptide-9, hexapeptide-11, and acetyl hexapeptide-8 in the targeted supramolecular BAPs-PLGA nanoparticles are stronger than those of BAPs, which are 4.05, 4.32, and 5.41 times that of BAPs, indicating that the targeted supramolecular BAPs-PLGA nanoparticles have excellent penetration-enhancing ability.

[0108] Test Example 8: The anti-proteolytic ability of BAPs prepared in Test Example 7 The main sites of pepsin action are the peptide bonds formed by the amino groups of aromatic amino acids (mainly including tyrosine Tyr, phenylalanine Phe, and tryptophan Try) or acidic amino acids (mainly glutamic acid Glu and aspartic acid Asp). The amino acid sequences of hexa-peptide-11, acetyl tetra-peptide-9, and acetyl hexa-peptide-8 contain Phe, Asp, and Glu respectively, and pepsin has a certain proteolytic ability for these three peptides. Therefore, taking pepsin as an example, the anti-proteolytic ability of BAPs was observed. The specific experimental operations are as follows: Ⅰ (Enzymatic hydrolysis): Take 20 g of BAPs and place them in a constant temperature water bath. After adding 300 mg of pepsin, start the hydrolysis reaction and continue the reaction for 4 h. Take out the reaction solution and immediately pour it into 20 g of strong alkali solution (1 mol / L NaOH) to terminate the reaction.

[0109] Ⅱ (Measuring absorbance): Take 5 mL of the reaction solution and add 1 mL of ninhydrin dropwise, heat for color development. To ensure the accuracy of the measurement, dilute the heated reaction gradient solution and measure its light absorption at 570 nm to ensure that the absorption value is within the range of 0-1, so as to evaluate the free amino acid content and thus reflect the degree of polypeptide enzymatic hydrolysis.

[0110] Reaction mechanism: Ninhydrin solution reacts with amino acids under heating to generate ammonia. Ammonia reacts with ninhydrin and reduced ninhydrin to form a purple compound. The intensity of the color of this compound is proportional to the content of amino acids. The content of free amino acids can be evaluated by measuring the light absorption at 570 nm. The absorbance values after the enzymatic hydrolysis of BAPs are shown in Table 8.

[0111] Table 8. Statistical table of absorbance values after enzymatic hydrolysis of BAPs and targeted supramolecular BAPs-PLGA nanoparticles

[0112] Comparative Test Example 3: To improve the in vivo stability of polypeptides, there are mainly modifying the polypeptide structure (forming cyclic peptides, using D-amino acids, changing single or multiple amino acids), modifying both ends of the polypeptide (hydrophobic modification and hydrophilic modification), and modifying the polypeptide with biomacromolecules. By using these chemical modifications to stabilize bioactive peptides, it can alleviate the degradation and inactivation mediated by blood or tissue proteases, thereby enhancing the drug-likeness of polypeptide candidate drugs. In addition to modifying the polypeptide itself, high molecular substances can also be used to encapsulate the polypeptide to form a barrier between the polypeptide and proteases, which can also protect it from protease cleavage. The targeted supramolecular BAPs-PLGA nanoparticles are prepared by microfluidic technology to encapsulate bioactive peptides in PLGA to enhance their anti-proteolytic ability.

[0113] The difference between Comparative Test Example 3 and Test Example 6 is that the BAPs in the enzymatic hydrolysis step of Test Example 6 were replaced with targeted supramolecular BAPs-PLGA nanoparticles. The contents of acetyl tetrapeptide-9, hexapeptide-11, and acetyl hexapeptide-8 in both were kept consistent, and the absorbance values after enzymatic hydrolysis are shown in Table 8.

[0114] The comparison of the absorbance of BAPs and targeted supramolecular BAPs-PLGA nanoparticles after the same enzymatic action is as Figure 17 shown. The ultraviolet light absorptions of BAPs and targeted supramolecular BAPs-PLGA nanoparticles were 0.252 ± 0.0025 Abs and 0.182 ± 0.0021 Abs respectively, BAPs > targeted supramolecular BAPs-PLGA nanoparticles, that is, after pepsin hydrolysis, the number of free amino acids in each group, BAPs > targeted supramolecular BAPs-PLGA nanoparticles. The results showed that after enzymatic action, the number of free amino acids in the targeted supramolecular BAPs-PLGA nanoparticles decreased, demonstrating a certain anti-enzymatic ability.

[0115] Test Example 9: Test the safety of the formulated product containing targeted supramolecular BAPs-PLGA nanoparticles.

[0116] Using the targeted supramolecular BAPs-PLGA nanoparticles prepared in Example 1 and the BAPs prepared in Example 7, they were respectively formulated into targeted supramolecular BAPs-PLGA nano essence milk and BAPs essence milk. By mass percentage, their formula compositions are shown in Table 9: Table 9. Targeted supramolecular BAPs-PLGA nano essence milk and BAPs essence milk

[0117] Test method: Select qualified patch test equipment. Using the closed patch test method, place 0.020 - 0.025 g of the test substance in the patch test equipment and apply it to the flexor side of the forearm of the subject with a low-sensitization tape. After 24 h, remove the test substance, and observe the skin reaction at 0.5, 24, and 48 h after removal respectively. Record the results according to the skin reaction grading standard in the "Cosmetics Safety and Technology Specifications" (2015 edition).

[0118] Table 10. Summary of the results of human patch tests

[0119] Note: The skin reaction conditions of 30 subjects at different observation times are shown in Table 12.

[0120] Table 11. Skin reaction grading standard for skin closed patch test

[0121] Table 12 Skin reaction conditions of subjects at different observation times

[0122] The results of the human skin occlusive patch test are shown in Table 10 and Table 12. No adverse reactions occurred in the skin of 30 subjects in the targeted supramolecular BAPs-PLGA nano essence milk group and the BAPs essence milk group, showing good safety.

[0123] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments by using the disclosed technical content within the scope of the technical solution of the present invention. However, as long as it does not depart from the content of the technical solution of the present invention, any modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A targeted supramolecular BAPs-PLGA nanoparticle, characterized in that, The nanoparticles are composed of supramolecular arginine-glycine-aspartic acid (RGD), supramolecular bioactive peptides (BAPs), and poly(lactic-co-glycolic acid) (PLGA); The supramolecular BAPs are prepared by mixing betaine-glycerol deep eutectic solvent (TG-DES) and BAPs at a mass ratio of 1:(50 - 200) and stirring for 2 - 12 hours under the protection of a nitrogen pressure of 5 - 20 MPa; The supramolecular RGD is prepared by mixing TG-DES and RGD at a mass ratio of 1:(50 - 200) and stirring for 2 - 12 hours under the protection of a nitrogen pressure of 5 - 20 MPa.

2. The targeted supramolecular BAPs-PLGA nanoparticles according to claim 1, characterized in that, The BAPs are a composite peptide composed of acetyl hexapeptide-8, acetyl tetrapeptide-9, and hexapeptide-11; The mass ratio of acetyl hexapeptide-8, acetyl tetrapeptide-9, and hexapeptide-11 is (1 - 2):(1 - 2):(1 - 2).

3. The targeted supramolecular BAPs-PLGA nanoparticles according to claim 1, wherein The average particle size of the nanoparticles is 100 - 200 nm, and the polydispersity index is less than 10%.

4. A method for preparing a targeted supramolecular BAPs-PLGA nanoparticle according to any one of claims 1-3, characterized in that, It includes the following steps: (1) Dissolve PLGA in dichloromethane to form a primary oil phase; (2) Use the supramolecular BAPs as the primary aqueous phase and conduct primary emulsification with the primary oil phase in step (1) to form a W / O primary emulsion; The mass ratio of the primary oil phase to the primary aqueous phase is 1:(3 - 7); (3) Conduct secondary emulsification with the primary emulsion obtained in step (2) and the supramolecular RGD as the secondary aqueous phase to form a W / O / W multiple emulsion; The mass ratio of the primary emulsion to the secondary aqueous phase is 1:(5 - 15); (4) Evaporate dichloromethane from the multiple emulsion obtained in step (3) by stirring to obtain the targeted supramolecular BAPs-PLGA nanoparticles.

5. The preparation method of a targeted supramolecular BAPs-PLGA nanoparticle according to claim 4, characterized in that, The temperature of the stirring is 20 - 40 °C, the time of the stirring is 4 - 24 h, and the rotation speed of the stirring is 300 - 600 rpm.

6. The preparation method of a targeted supramolecular BAPs-PLGA nanoparticle according to claim 4, characterized in that, Both the primary emulsification and the secondary emulsification are carried out under the protection of an inert gas using microfluidic technology and ultrasonic emulsification technology.

7. The preparation method of a targeted supramolecular BAPs-PLGA nanoparticle according to claim 6, characterized in that, The high-pressure homogenization pressure of the microfluidic technology is 2000 - 8000 bar, and the homogenization time is 20 - 60 min; The power of the ultrasonic emulsification technology is 100 - 600 W, the frequency is 10 - 60 kHz, pause for 2 seconds after every 2 seconds of ultrasonic treatment, and the duration is 0.5 - 2 h; The inert gas is nitrogen, and the pressure is 5 - 20 MPa.

8. Use of a targeted supramolecular BAPs-PLGA nanoparticle according to any one of claims 1 - 3 in the preparation of pharmaceutical preparations and products in the field of cosmetics.

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