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

Abstract

The present application relates to a kind of targeted supramolecular BAPs-PLGA nanoparticles and its preparation method and application.The nanoparticles are prepared by microfluidic technology and ultrasonic emulsification technology, using PLGA as oil phase, supramolecular BAPs as water phase to form primary emulsion, and then multiple emulsion is formed with water phase containing emulsifier and supramolecular RGD.The technology combines BAPs with low eutectic solvent (TG-DES) to form a stable supramolecular structure, significantly improving the solubility and stability of BAPs.Supramolecular RGD is attached to the surface of nanoparticles, giving it targeting ability, and can improve the cellular uptake capacity of BAPs through NRP-1 mediated transcytosis.The present application can improve the bioavailability of BAPs, and provides a new solution for drug delivery system and skin penetration enhancement.In addition, the nanoparticle preparation method of the present application is simple, the process is controllable, suitable for industrial production, and has wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine and cosmetics, and in particular to targeted supramolecular BAPs-PLGA nanoparticles and a preparation method and application thereof. Background Art

[0002] Bioactive peptides (BAPs), molecules with a wide range of bioactivities, have been widely used in clinical treatments and cosmetics. BAPs have demonstrated remarkable efficacy in promoting collagen synthesis, aiding wound healing, smoothing wrinkles, providing antioxidant, antibacterial, and whitening properties. However, BAPs also face numerous challenges, including: 1) physicochemical instability and susceptibility to oxidation and hydrolysis; 2) short half-lives and rapid clearance from the body; and 3) difficulty in penetrating the skin and cell membranes. Therefore, improving the stability of BAPs, extending their half-lives, and enhancing their permeability are key to addressing these challenges.

[0003] Betaine-glycerol deep eutectic solvent (TG-DES) is a deep eutectic solvent composed of betaine and glycerol. It exhibits excellent biocompatibility, a low melting point, and strong dissolving power. Existing technologies typically improve the stability of BAPs through chemical modification, metal ion complexation, liposome technology, and nanotechnology. However, these approaches have not effectively addressed the permeability issues of BAPs across biological barriers. Furthermore, the preparation process is complex, the cost is high, and industrialization is difficult.

[0004] Arginine-glycine-aspartic acid (Arg-Gly-Asp, RGD) is a tripeptide sequence widely used in targeted delivery systems. Its ability to specifically recognize and bind to integrin receptors on cell surfaces makes it crucial in drug delivery and tissue engineering. Currently, RGD is often chemically conjugated to the surface of nanocarriers to enhance targeting, but this approach suffers from low modification efficiency and poor RGD stability, and has not been fully effective in the delivery of BAPs.

[0005] To address the above problems, the present invention provides a targeted supramolecular BAPs-PLGA nanoparticle and a preparation method thereof, aiming to provide a more stable, more efficient, and easier to pass through the skin barrier BAPs carrier by combining supramolecular technology with poly(lactic acid-co-glycolic acid) (PLGA) nanoparticle technology, thereby improving the bioavailability and clinical application effect of BAPs. Summary of the Invention

[0006] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a targeted supramolecular BAPs-PLGA nanoparticle and a preparation method and application thereof, aiming to solve the application problems of poor skin permeability and stability of BAPs.

[0007] The technical solutions of the present invention are as follows:

[0008] In a first aspect of the present invention, a targeted supramolecular BAPs-PLGA nanoparticle is provided, wherein the nanoparticle is composed of supramolecular RGD, supramolecular BAPs and PLGA;

[0009] The supramolecular BAPs are prepared by mixing TG-DES and BAPs in a mass ratio of 1:(50-200), and stirring for 2-12 hours under a nitrogen pressure of 5-20 MPa;

[0010] The supramolecular RGD is prepared by mixing TG-DES and RGD in a mass ratio of 1:(50-200), and stirring for 2-12 hours under a nitrogen pressure of 5-20 MPa.

[0011] Furthermore, in the targeted supramolecular BAPs-PLGA nanoparticles, the BAPs are a composite peptide composed of acetyl hexapeptide-8, acetyl tetrapeptide-9 and hexapeptide-11;

[0012] The mass ratio of the acetyl hexapeptide-8, acetyl tetrapeptide-9 and hexapeptide-11 is (1-2):(1-2):(1-2).

[0013] Furthermore, the targeted supramolecular BAPs-PLGA nanoparticles have an average particle size of 100-200 nm and a polydispersity index of less than 10%.

[0014] The second aspect of the present invention provides a method for preparing targeted supramolecular BAPs-PLGA nanoparticles, comprising the following steps:

[0015] (1) Dissolve PLGA in dichloromethane to form a primary oil phase;

[0016] (2) using supramolecular BAPs as the primary water phase to perform primary emulsification with the primary oil phase in step (1) to form a W / O primary emulsion;

[0017] The mass ratio of the primary oil phase to the primary water phase is 1:(3-7);

[0018] (3) The primary emulsion obtained in step (2) and supramolecular RGD are used as a secondary aqueous phase for secondary emulsification to form a W / O / W multiple emulsion;

[0019] The mass ratio of the primary emulsion to the secondary aqueous phase is 1:(5-15);

[0020] (4) The multiple emulsion obtained in step (3) is stirred to evaporate the dichloromethane to obtain targeted supramolecular BAPs-PLGA nanoparticles.

[0021] Furthermore, the stirring temperature is 20-40° C., the stirring time is 4-24 h, and the stirring speed is 300-600 rpm.

[0022] Furthermore, the primary emulsification and the secondary emulsification are both performed using microfluidics technology and ultrasonic emulsification technology under the protection of inert gas.

[0023] Furthermore, the high-pressure homogenization pressure of the microfluidic technology is 2000-8000 bar, and the homogenization time is 20-60 minutes.

[0024] The power of the phacoemulsification technique is 100-600 W, the frequency is 10-60 kHz, the ultrasonic treatment is followed by a 2-second pause every 2 seconds, and the duration is 0.5-2 hours;

[0025] The inert gas is nitrogen, and the pressure is 5-20 MPa.

[0026] The third aspect of the present invention provides the use of the above-mentioned targeted supramolecular BAPs-PLGA nanoparticles in the preparation of pharmaceutical preparations and cosmetic products.

[0027] Beneficial Effects: The present invention combines BAPs with a deep eutectic solvent to form supramolecular BAPs through weak interactions such as hydrogen bonding, van der Waals interactions, and electrostatic interactions, thereby increasing the solubility and stability of BAPs. The BAPs are then encapsulated in PLGA nanoparticles, reducing the freedom of BAP molecular movement and the risk of enzymatic degradation, thereby improving their stability. Furthermore, the deep eutectic solvent forms a supramolecular structure with RGD, enhancing RGD stability. The RGD is attached to the PLGA nanoparticles through intermolecular interactions, providing targeting for the nanoparticles, targeting the cell membrane, and initiating NRP-1-mediated transcytosis, thereby resolving the problem of BAPs' difficulty in crossing the cell membrane and improving the bioavailability of BAPs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The method of the present invention and its beneficial effects are described in detail below with reference to the accompanying drawings and specific embodiments.

[0029] Figure 1 Schematic diagram of the preparation process of targeted supramolecular BAPs-PLGA nanoparticles.

[0030] Figure 2 Schematic diagram of molecular docking of BAPs components in the TG-DES system.

[0031] Figure 3 Schematic diagram of the weak interactions among the BAPs components in the TG-DES system.

[0032] Figure 4Schematic diagram of the electrostatic potential (ESP) of each BAPs component in the TG-DES system.

[0033] Figure 5 Ultraviolet absorption spectra of supramolecular BAPs, supramolecular RGD, TG-DES, BAPs and RGD.

[0034] Figure 6 Comparison of the hydrated particle size distribution of BAPs and supramolecular BAPs-PLGA nanoparticles.

[0035] Figure 7 Transmission electron microscopy observation images of nanoparticles (the left is the transmission electron microscopy observation image of PLGA nanoparticles; the right is the transmission electron microscopy observation image of BAPs-PLGA nanoparticles).

[0036] Figure 8 Transmission electron microscopy observation of BAPs.

[0037] Figure 9 Transmission electron microscopy observation of targeted supramolecular BAPs-PLGA nanoparticles.

[0038] Figure 10 Figure 3 is a graph showing the particle size changes of targeted supramolecular BAPs-PLGA nanoparticles within 30 days.

[0039] Figure 11 Schematic diagram of the relative survival rate of HaCat cells under the action of concentration gradient targeted supramolecular BAPs-PLGA nanoparticles and BAPs.

[0040] Figure 12 Schematic diagram of the relative survival rate of HFF-1 cells under the action of concentration gradient targeted supramolecular BAPs-PLGA nanoparticles and BAPs.

[0041] Figure 13 Schematic diagram of the uptake results of HaCat cells for targeted supramolecular BAPs-PLGA nanoparticles and the components in BAPs.

[0042] Figure 14 Schematic diagram of the fluorescence imaging results of each component in BAPs after 24 hours of transdermal penetration.

[0043] Figure 15 Schematic diagram of the fluorescence imaging results of each component in the targeted supramolecular BAPs-PLGA nanoparticles after 24 hours of transdermal penetration.

[0044] Figure 16 Statistical graph of the average fluorescence intensity of each component in BAPs and targeted supramolecular BAPs-PLGA nanoparticles after 24 hours of transdermal delivery.

[0045] Figure 17This is a comparison chart of the absorbance of BAPs and targeted supramolecular BAPs-PLGA nanoparticles after enzymatic hydrolysis. DETAILED DESCRIPTION

[0046] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the 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 in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within 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 those commonly understood by those skilled in the art in the technical field of the present invention. The terms used in the description of the present invention in this specification are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials in the following examples, unless otherwise specified, are purchased from conventional biochemical reagent stores.

[0047] The poly(lactic-co-glycolic acid) (PLGA) used in the present invention was purchased from Jinan Daigang Biotechnology Co., Ltd. with the trade name of ester-terminated poly(lactic-co-glycolic acid) PLGA75 / 25COOR, batch number 2024121709, and a molecular weight of 3.7-5.2 kd. Poly(vinyl alcohol) (PVA) was purchased from Guangzhou Weike Chemical Co., Ltd. with the batch number RA22080811 and a degree of hydrolysis of 88.2 mol%.

[0048] Embodiment 1:

[0049] This embodiment provides a method for preparing targeted supramolecular BAPs-PLGA nanoparticles, such as Figure 1 The detailed steps are as follows:

[0050] S1) Under nitrogen protection at 10 MPa pressure, at 37°C and with continuous stirring at 300 rpm, the composite peptide (acetyl hexapeptide-8, acetyl tetrapeptide-9, and hexapeptide-11 in a mass ratio of 1:1:1) and the deep eutectic solvent betaine-glycerol (TG-DES) in a mass ratio of 1:99 were uniformly mixed and stirred for 2 h to prepare supramolecular BAPs.

[0051] S2) Under nitrogen protection at 10 MPa pressure, RGD and TG-DES were uniformly mixed at a mass ratio of 1:99 at 37°C and continuously stirred at 300 rpm for 2 h to prepare supramolecular RGD.

[0052] S3) In a microfluidic operating system, at 37°C and a homogenizing 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 consisted of a 15% supramolecular BAPs aqueous solution with a mass ratio of 5:1. This cycle was repeated twice and emulsified under ultrasound at 300 W power and 20 kHz frequency. Ultrasonic treatment was repeated for 2 s followed by a 2 s pause, and alternating treatments were continued for 0.5 h to obtain a W / O primary emulsion.

[0053] S4) In a microfluidic system, at 37°C and a homogenizing pressure of 6000 bar, the primary emulsion (oil phase) was injected into a secondary aqueous phase (9:1 weight ratio) containing 0.2% PVA and 5% supramolecular RGD. This cycle was repeated twice, followed by ultrasonication at 300 W and 20 kHz, with a 2-s pause followed by a 2-s interval. This alternating treatment lasted for 0.5 h, resulting in a W / O / W multiple emulsion. Finally, the nanoparticles were stirred at room temperature for 8 h to evaporate the organic solvent, yielding targeted supramolecular BAPs-PLGA nanoparticles with supramolecular RGD attached to their surfaces and BAPs encapsulated within them.

[0054] For the supramolecular BAPs involved in step S1 of this embodiment, molecular docking was performed in the TG-DES system. The most stable conformation among the multiple conformations generated was selected as a reference, and only the most likely hydrogen bonds were displayed. The results are shown in FIG. Figure 2 As shown in the figure, based on the three-dimensional structure and computational simulation results, there are six hydrogen bonds between acetyl hexapeptide-8 and acetyl tetrapeptide-9, with hydrogen bond lengths of 2.9 Å, 2.9 Å, 3.0 Å, 3.0 Å, 3.2 Å, and 3.4 Å, respectively; there are two hydrogen bonds between acetyl hexapeptide-8 and hexapeptide-11, with hydrogen bond lengths of 2.9 Å and 3.0 Å, respectively; and there are two hydrogen bonds between hexapeptide-11 and acetyl tetrapeptide-9, with hydrogen bond lengths of 2.9 Å and 3.2 Å, respectively. The presence of hydrogen bonds enhances the stability of each component in the composite peptide and reduces the risk of enzymatic degradation of each component as a free peptide during transdermal delivery.

[0055] On this basis, weak interaction and electrostatic potential calculations were performed on the composite peptide in the TG-DES system, and the calculation results are as follows: Figure 3 、 Figure 4 As shown. Schematic diagram of weak interaction ( Figure 3 ) The color of the medium value surface (the pie-shaped structure in the figure) is obviously blue, indicating that there is a significant attraction, such as hydrogen bonds and halogen bonds of average strength; the isovalue surface is completely blue, indicating that there is a strong weak interaction here; the isovalue surface is green, indicating that there is a certain van der Waals force; the color of the isovalue surface is obviously red, indicating that there is a certain steric hindrance here, and if it is bright red, it means that the steric hindrance is very strong; Figure 3 As shown in 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 supramolecular structure.

[0056] The electrostatic potential of the complex peptide in the TG-DES system is as follows Figure 4 The electrostatic potential value of the blue area is negative, which means that this area is more likely to donate electrons, or is more nucleophilic than other areas (referring to the ability of a substance to donate electrons to other molecules or ions), laying a theoretical foundation for the co-assembly of the composite peptide and TG-DES into a supramolecular structure.

[0057] Example 2:

[0058] This embodiment provides a method for preparing targeted supramolecular BAPs-PLGA nanoparticles, and the detailed steps are as follows:

[0059] S1) Under nitrogen protection at 5 MPa pressure, at 5°C and 400 rpm, the composite peptide (acetyl hexapeptide-8, acetyl tetrapeptide-9, and hexapeptide-11 in a mass ratio of 2:1:1) and the deep eutectic solvent betaine-glycerol (TG-DES) in a mass ratio of 1:50 were uniformly mixed and stirred for 4 h to prepare supramolecular BAPs.

[0060] S2) Under nitrogen protection at 5 MPa pressure, RGD and TG-DES were uniformly mixed at a mass ratio of 1:50 at 5°C and 400 rpm with continuous stirring for 4 h to prepare supramolecular RGD.

[0061] S3) In a microfluidic operating system, at 5°C and a homogenizing pressure of 2000 bar, 1.5% PLGA in dichloromethane was injected as the primary oil phase into the primary aqueous phase. The primary aqueous phase consisted of a 15% supramolecular BAPs aqueous solution with a mass ratio of 3:1. This cycle was repeated twice and emulsified under ultrasound at 100 W power and 10 kHz frequency. Ultrasonic treatment was repeated for 2 s followed by a 2 s pause, and alternating treatments were continued for 1 h to obtain a W / O primary emulsion.

[0062] S4) In a microfluidic system, at 5°C and a homogenizing pressure of 2000 bar, the primary emulsion (oil phase) was injected into a secondary aqueous phase (an aqueous solution containing 0.2% PVA and 5% supramolecular RGD) at a mass ratio of 8:1. This cycle was repeated twice, followed by emulsification under ultrasound at 100 W power and 10 kHz frequency, with 2-s intervals followed by 2-s pauses, alternating for 1 hour. This resulted in a W / O / W multiple emulsion. Finally, the mixture was stirred at room temperature for 4 hours to evaporate the organic solvent, yielding targeted supramolecular BAPs-PLGA nanoparticles with supramolecular RGD attached to the surface and supramolecular BAPs encapsulated within.

[0063] Example 3:

[0064] This embodiment provides a method for preparing targeted supramolecular BAPs-PLGA nanoparticles, and the detailed steps are as follows:

[0065] S1) Under nitrogen protection at 15 MPa pressure, at 15°C and with continuous stirring at 500 rpm, the composite peptide (acetyl hexapeptide-8, acetyl tetrapeptide-9, and hexapeptide-11 in a mass ratio of 1:2:1) and the deep eutectic solvent betaine-glycerol (TG-DES) in a mass ratio of 1:150 were uniformly mixed and stirred for 8 h to prepare supramolecular BAPs.

[0066] S2) Under nitrogen protection at 15 MPa pressure, RGD and TG-DES were uniformly mixed at a mass ratio of 1:150 at 15°C and 500 rpm with continuous stirring for 8 h to prepare supramolecular RGD.

[0067] S3) In a microfluidic operating system, at 15°C and a homogenizing pressure of 4000 bar, 1.5% PLGA in dichloromethane was injected as the primary oil phase into the primary aqueous phase. The primary aqueous phase consisted of a 15% supramolecular BAPs aqueous solution with a mass ratio of 4:1. This cycle was repeated twice and emulsified under ultrasound at 200 W power and 20 kHz frequency. Ultrasonic treatment was repeated for 2 s followed by a 2 s pause, and alternating treatments were continued for 2 h to obtain a W / O primary emulsion.

[0068] S4) In a microfluidic system, at 15°C and a homogenizing pressure of 4000 bar, the primary emulsion (oil phase) was injected into a secondary aqueous phase (an aqueous solution containing 0.2% PVA and 5% supramolecular RGD) at a mass ratio of 12:1. This cycle was repeated twice, followed by emulsification under ultrasound at 200 W power and 20 kHz frequency, with alternating sonication cycles of 2 s followed by 2 s pauses, for 2 hours. This resulted in a W / O / W multiple emulsion. Finally, the mixture was stirred at room temperature for 12 hours to evaporate the organic solvent, yielding targeted supramolecular BAPs-PLGA nanoparticles with supramolecular RGD attached to the surface and supramolecular BAPs encapsulated within.

[0069] Example 4:

[0070] This embodiment provides a method for preparing targeted supramolecular BAPs-PLGA nanoparticles, and the detailed steps are as follows:

[0071] S1) Under nitrogen protection at 20 MPa pressure, at 40°C and 600 rpm, the composite peptide (acetyl hexapeptide-8, acetyl tetrapeptide-9, and hexapeptide-11 in a mass ratio of 1:1:2) and the deep eutectic solvent betaine-glycerol (TG-DES) in a mass ratio of 1:200 were uniformly mixed and stirred for 12 h to prepare supramolecular BAPs.

[0072] S2) Under nitrogen protection at 20 MPa pressure, 40°C, and continuous stirring at 600 rpm, RGD and TG-DES were uniformly mixed at a mass ratio of 1:200 and stirred continuously for 12 h to prepare supramolecular RGD.

[0073] S3) In a microfluidic operating system, at 40°C and a homogenizing pressure of 8000 bar, 1.5% PLGA in dichloromethane was injected as the primary oil phase into the primary aqueous phase. The primary aqueous phase consisted of an aqueous solution containing 15% supramolecular BAPs. The mass ratio of the primary aqueous phase to the primary oil phase was 7:1. This cycle was repeated twice and emulsified under ultrasound at a power of 500 W and a frequency of 50 kHz. Each ultrasonic treatment was followed by a 2-s pause, and alternating treatments were continued for 4 h to obtain a W / O primary emulsion.

[0074] S4) In a microfluidic system, at 40°C and a homogenizing pressure of 8000 bar, the primary emulsion (oil phase) was injected into a secondary aqueous phase (an aqueous solution containing 0.2% PVA and 5% supramolecular RGD) at a mass ratio of 15:1. This cycle was repeated twice, followed by emulsification under ultrasonication at 500 W and 50 kHz, with a 2-s pause followed by a 2-s interval. This alternating treatment lasted for 4 hours, resulting in a W / O / W multiple emulsion. Finally, the nanoparticles were stirred at room temperature for 24 hours to evaporate the organic solvent, yielding targeted supramolecular BAPs-PLGA nanoparticles with surface-attached supramolecular RGD and internally encapsulated supramolecular BAPs.

[0075] Example 5:

[0076] The preparation steps of PLGA nanoparticles without supramolecular BAPs encapsulated inside and without supramolecular RGD attached to the surface are as follows:

[0077] S1) In a microfluidic system, 1.5% PLGA in dichloromethane was injected as the primary oil phase into the primary water phase at a mass ratio of 5:1 at 37°C and a homogenizing pressure of 6000 bar. This cycle was repeated twice and emulsified under ultrasound at 300 W power and 20 kHz frequency. Ultrasonic treatments were repeated for 2 s followed by a 2 s pause, and alternating treatments were continued for 0.5 h to obtain a W / O primary emulsion.

[0078] In a microfluidic system (S2), at 37°C and 6000 bar homogenizing pressure, the primary emulsion (oil phase) was injected into a secondary aqueous phase (a 0.2% PVA-containing aqueous solution) at a mass ratio of 10:1. This cycle was repeated twice, followed by emulsification under ultrasound at 300 W power and 20 kHz frequency. Ultrasonic treatments were repeated for 0.5 h, followed by a 2-s pause between each 2-s treatment. This alternating treatment was repeated for 8 h at room temperature to evaporate the organic solvent, resulting in unloaded PLGA nanoparticles.

[0079] Example 6:

[0080] The preparation steps of BAPs-PLGA nanoparticles with supramolecular BAPs encapsulated inside and no supramolecular RGD attached to the surface are as follows:

[0081] S1) Under nitrogen protection at 10 MPa pressure, at 37°C and with continuous stirring at 300 rpm, the composite peptide (acetyl hexapeptide-8, acetyl tetrapeptide-9, and hexapeptide-11 in a mass ratio of 1:1:1) and the deep eutectic solvent betaine-glycerol (TG-DES) in a mass ratio of 1:99 were uniformly mixed and stirred for 2 h to prepare supramolecular BAPs.

[0082] S2) In a microfluidic operating system, at 37°C and a homogenizing 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 consisted of a 15% supramolecular BAPs aqueous solution with a mass ratio of 5:1. This cycle was repeated twice and emulsified under ultrasound at 300 W power and 20 kHz frequency. Ultrasonic treatment was repeated for 2 s followed by a 2 s pause, and alternating treatments were continued for 0.5 h to obtain a W / O primary emulsion.

[0083] S4) In a microfluidic system, at 37°C and a homogenizing pressure of 6000 bar, the primary emulsion (the oil phase) was injected into a secondary aqueous phase (a 9:1 mass ratio of secondary aqueous phase to primary emulsion) at 6000 bar. This cycle was repeated twice, followed by ultrasonication at 300 W and 20 kHz, with a 2-s pause followed by a 2-s interval. This alternating treatment lasted for 0.5 h, resulting in a W / O / W multiple emulsion. Finally, the mixture was stirred at room temperature for 8 h to evaporate the organic solvent, yielding BAPs-PLGA nanoparticles encapsulating supramolecular BAPs.

[0084] Comparative Example 1:

[0085] S1) Under nitrogen protection at 10 MPa pressure, at 37°C and with continuous stirring at 300 rpm, the composite peptide (acetyl hexapeptide-8, acetyl tetrapeptide-9, and hexapeptide-11 in a mass ratio of 1:1:1) and the deep eutectic solvent betaine-glycerol (TG-DES) in a mass ratio of 1:99 were uniformly mixed and stirred for 2 h to prepare supramolecular BAPs.

[0086] S2) Under nitrogen protection at 10 MPa pressure, RGD and TG-DES were uniformly mixed at a mass ratio of 1:99 at 37°C and continuously stirred at 300 rpm for 2 h to prepare supramolecular RGD.

[0087] S3) In a microfluidic operating system, at 37°C and a homogenizing 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 consisted of an aqueous solution containing 15% supramolecular BAPs. The mass ratio of the primary aqueous phase to the primary oil phase was 1:1. This cycle was repeated twice and emulsified under ultrasound at a power of 300 W and a frequency of 20 kHz. Each ultrasonic treatment was followed by a 2-s pause, and alternating treatments were continued for 0.5 h to obtain a W / O primary emulsion.

[0088] In a microfluidic control system (S4), at 37°C and a homogenizing pressure of 6000 bar, the primary emulsion (oil phase) was injected into a secondary aqueous phase containing 0.2% PVA and 5% supramolecular RGD in an aqueous solution with a mass ratio of 1:1. This cycle was repeated twice, followed by ultrasonication at 300 W and 20 kHz. Ultrasonication was repeated for 0.5 h, with a 2-s pause followed by a 2-s interval. This alternating treatment resulted in a W / O / W multiple emulsion. During stirring at room temperature, the organic solvent evaporated, and the excess PLGA in the oil phase precipitated, forming a white flocculent suspended in the nanosolution. After filtration to remove the precipitated PLGA, the nanosolution still exhibited large particle size and poor polydispersity, likely due to the increased viscosity of the system caused by the excessively high oil phase ratio, which was unfavorable for nanodispersion formation.

[0089] Comparative Example 2:

[0090] S1) Under nitrogen protection at 10 MPa pressure, 37°C, and continuous stirring at 300 rpm, the composite peptide (acetyl hexapeptide-8, acetyl tetrapeptide-9, and hexapeptide-11 in a mass ratio of 1:1:1) was uniformly mixed with a deep eutectic solvent betaine-glycerol (TG-DES) in a mass ratio of 1:10 and stirred continuously for 12 h. Some peptides remained undissolved and suspended in the system, exceeding the maximum amount of peptides that could be dissolved in the system, and no supramolecular system could be formed.

[0091] S2) Under nitrogen protection at 10 MPa pressure, RGD and TG-DES were uniformly mixed at a mass ratio of 1:10 at 37°C and continuously stirred at 300 rpm for 12 h. The results were consistent with those in S1, and no supramolecular system could be formed.

[0092] Comparative Example 3:

[0093] S1) Under nitrogen protection at 10 MPa pressure, at 37°C and with continuous stirring at 300 rpm, the composite peptide (acetyl hexapeptide-8, acetyl tetrapeptide-9, and hexapeptide-11 in a mass ratio of 1:1:1) and the deep eutectic solvent betaine-glycerol (TG-DES) in a mass ratio of 1:99 were uniformly mixed and stirred for 2 h to prepare supramolecular BAPs.

[0094] S2) Under nitrogen protection at 10 MPa pressure, RGD and TG-DES were uniformly mixed at a mass ratio of 1:99 at 37°C and continuously stirred at 300 rpm for 2 h to prepare supramolecular RGD.

[0095] S3) In a microfluidic operating system, at 37°C and a homogenizing pressure of 6000 bar, a 1.5% PLGA-containing dichloromethane solution was injected as the primary oil phase into the primary aqueous phase (15% supramolecular BAPs in aqueous solution). The mass ratio of the primary aqueous phase to the primary oil phase was 1:5. This cycle was repeated twice and emulsified under ultrasound at 300 W power and 20 kHz frequency. Each ultrasonic treatment was followed by a 2-s pause, and alternating treatments were continued for 0.5 h to obtain a W / O primary emulsion.

[0096] In a microfluidic control system (S4), at 37°C and a homogenizing pressure of 6000 bar, the primary emulsion was injected into a secondary aqueous phase consisting of a 5% supramolecular RGD aqueous solution. The mass ratio of the secondary aqueous phase to the primary emulsion was 1:10. This cycle was repeated twice, followed by ultrasonication at 300 W power and 20 kHz frequency. Ultrasonication was repeated for 0.5 h, with a 2-s pause followed by a 2-s interval. This alternating treatment resulted in a W / O / W multiple emulsion. During stirring at room temperature, as the organic solvent evaporated, a small amount of white flocculent material remained suspended in the nanosolution, similar to Comparative Example 1. After filtering to remove the precipitated PLGA, the particle size distribution of the nanosolution showed multiple peaks, indicating extremely poor polydispersity. With prolonged standing time, significant particle precipitation occurred. The primary function of an emulsifier is to reduce the surface tension at the oil-water interface and enhance the stability of the emulsion. The lack of an emulsifier can lead to emulsion instability, prone to delamination or rupture, thus impairing the formation and dispersibility of the nanoparticles and causing the aforementioned phenomenon.

[0097] Example 7:

[0098] This embodiment provides a method for preparing BAPs:

[0099] Under nitrogen protection at a pressure of 10 MPa, acetyl hexapeptide-8, acetyl tetrapeptide-9, and hexapeptide-11 were added to water at 37°C and 300 rpm with continuous stirring to achieve a concentration of 50 ppm for each peptide. The mixture was evenly mixed and stirred continuously for 12 h to prepare BAPs.

[0100] Test Example 1: The UV absorption spectra of supramolecular BAPs, supramolecular RGD, TG-DES, BAPs, and RGD were measured using an UV spectrophotometer (UV-1900i) to verify the formation of hydrogen bonds, thereby proving the formation of supramolecular structures.

[0101] The formation of hydrogen bonds affects the electronic structure of molecules, thereby changing their absorption spectra. In the UV spectrum, the π → π transition and n → π transition of solute molecules may be affected by the formation of hydrogen bonds. When the concentration is kept the same, the UV absorption spectra of supramolecular BAPs, supramolecular RGD and TG-DES, BAPs, RGD are as follows: Figure 5As shown in the figure, after TG-DES forms supramolecular BAPs and supramolecular RGD with BAPs and RGD, respectively, the UV absorption spectrum of TG-DES exhibits a red shift, meaning that the absorption peak of TG-DES moves to a lower wavenumber. This is likely because the formation of hydrogen bonds reduces the bond constant between hydrogen atoms and the atoms they are connected to, lowering the vibration frequency.

[0102] Test Example 2: The hydrated particle size distribution of the targeted supramolecular BAPs-PLGA nanoparticles prepared in Example 1, the PLGA nanoparticles prepared in Example 5, the BAPs-PLGA nanoparticles prepared in Example 6, and the BAPs prepared in Example 7 were tested.

[0103] The hydrated particle size, typically referring to the size of nanoparticles in aqueous solution, is measured using dynamic light scattering (DLS). This technique measures the Brownian motion of particles in a liquid, yielding the particle's hydrodynamic diameter, also known as the hydrated particle size. This diameter includes the nanoparticle core and the hydrated layer formed by water or other solvent molecules that may be adsorbed on its surface.

[0104] Experimental Method: After fully dispersing the sample to be tested and setting the solvent, temperature and other conditions, the particle size distribution was measured three times using a Malvern Panalytical's Mastersizer. The particle size distribution test results are shown in Tables 1-4:

[0105] Table 1. Particle size distribution of PLGA nanoparticles

[0106]

[0107] Table 2. Particle size distribution of BAPs-PLGA nanoparticles

[0108]

[0109] Table 3. Particle size distribution of targeted supramolecular BAPs-PLGA nanoparticles

[0110]

[0111] Table 4. Particle size distribution of BAPs

[0112]

[0113] BAPs is an aqueous solution of acetyl hexapeptide-8, acetyl tetrapeptide-9 and hexapeptide-11. The hydrated particle size test results are shown in Table 4. The average hydrated particle size in this system is 335.5 nm, and the polydispersity index is 36.5%. The results show that the particle size distribution uniformity of BAPs is poor and the polydispersity is poor.

[0114] Tables 1-3 show the hydrated particle size test results for PLGA nanoparticles, BAPs-PLGA nanoparticles, and targeted supramolecular BAPs-PLGA nanoparticles. PLGA nanoparticles had the smallest hydrated particle size, with an average value of 111.9 nm; BAPs-PLGA nanoparticles had the largest hydrated particle size, with an average value of 182.8 nm; and the average hydrated particle size of the targeted supramolecular BAPs-PLGA nanoparticles was 168.4 nm. The encapsulation of supramolecular BAPs resulted in a larger particle size than the PLGA nanoparticles. The supramolecular RGD adhered to the nanoparticle surface through intermolecular interactions, resulting in a more compact nanoparticle structure and a slightly smaller hydrated particle size than the BAPs-PLGA nanoparticles. Furthermore, the polydispersity index of the targeted supramolecular BAPs-PLGA nanoparticles was the smallest, at only 8.36%. This result indicates that the size distribution of the targeted supramolecular BAPs-PLGA nanoparticles is highly uniform and exhibits excellent polydispersity.

[0115] In summary, the targeted supramolecular BAPs-PLGA nanoparticles had the smallest particle size among the BAPs-containing groups, increasing their specific surface area, thereby improving their contact area with the skin and penetrability, achieving better transdermal efficacy. Furthermore, their polydispersity index was the lowest, indicating that the targeted supramolecular BAPs-PLGA nanoparticles possessed better dispersibility and higher particle size consistency.

[0116] After further analysis and processing of the data, the fitting results of the particle size distribution of the four groups of samples are as follows: Figure 6 As shown in the particle size distribution diagram, compared with BAPs-PLGA nanoparticles and BAPs, the high and narrow peak of the targeted supramolecular BAPs-PLGA nanoparticles can also explain their better dispersibility and particle size consistency.

[0117] Test Example 3:

[0118] The particle sizes of the targeted supramolecular BAPs-PLGA nanoparticles prepared in Example 1, the PLGA nanoparticles prepared in Example 5, the BAPs-PLGA nanoparticles prepared in Example 6, and the BAPs prepared in Example 7 were tested.

[0119] Particle size is a key factor influencing the performance of transdermal drug delivery systems. Smaller particles generally improve drug transdermal permeability because they more easily penetrate the tiny gaps in the skin's stratum corneum. Studies have shown that liposomes with a particle size less than 70 nm have superior transdermal performance, with deeper penetration and higher fluorescence retention within the skin. Therefore, particle size optimization is necessary to achieve optimal drug delivery.

[0120] Experimental method: Take 100 μL of the sample to be tested and drop it on the copper mesh support film. Let it stand and dry in a 50℃ oven for 2 hours. After it is completely dried, add another 100 μL of the sample and continue to stand and dry in a 50℃ oven for 2 hours to prepare the transmission electron microscopy observation sample.

[0121] PLGA nanoparticles ( Figure 7 Left), BAPs-PLGA nanoparticles ( Figure 7 right), BAPs ( Figure 8 ) and targeted supramolecular BAPs-PLGA nanoparticles ( Figure 9 ) showed that the PLGA nanoparticles had the smallest particle size, 20-30 nm; after internal encapsulation of BAPs, the particle size increased to approximately 50 nm; the targeted supramolecular BAPs-PLGA nanoparticles were evenly distributed in the solution, with no obvious agglomeration, and the single particle size was approximately 40 nm, showing excellent polydispersity. Moreover, after the supramolecular RGD was attached to the surface, the nanoparticle structure became more compact, manifested as a dark black color; the three polypeptide molecules in the BAPs system formed aggregates or clusters in the solution, with a particle size of approximately 200 nm. This aggregation may be caused by hydrophobic interactions, hydrogen bonds, or ionic interactions between the polypeptide molecules;

[0122] The particle size observed by transmission electron microscopy is significantly larger than the hydrated particle size, mainly because the former is obtained by directly observing the morphology and size of the particles through electron microscopy, while the hydrated particle size 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. In addition, the transmission electron microscopy observation results are consistent with the Malvern particle size analyzer test results, that is, the targeted supramolecular BAPs-PLGA nanoparticles show a smaller particle size and better polydispersity and particle size consistency compared to BAPs and BAPs-PLGA nanoparticles. Therefore, subsequent tests mainly use targeted supramolecular BAPs-PLGA nanoparticles as the research object.

[0123] Test Example 4:

[0124] The particle size stability of the targeted supramolecular BAPs-PLGA nanoparticles prepared in Example 1 was tested.

[0125] Particle size stability is crucial in the field of nanotechnology. It is not only related 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, and plays a significant role in improving drug bioavailability 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 impact. In terms of regulations and standards, particle size stability is also a key factor in nano-products complying with regulations and industry standards, and is crucial for market access and consumer trust. Therefore, the research and control of particle size stability is an important part of the development of nanotechnology that cannot be ignored.

[0126] The particle size changes of the 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 that of Test Example 2. The particle size measurement results of the targeted supramolecular BAPs-PLGA nanoparticles within 30 days are shown in Table 5. The particle size changes are shown in Table 5. Figure 10 shown.

[0127] Table 5. Particle size changes of targeted supramolecular BAPs-PLGA nanoparticles after 30 days

[0128]

[0129] As shown in Table 5, Figure 10 As shown in the data, the particle size of the targeted supramolecular BAPs-PLGA nanoparticles did not change much within 30 days, fluctuating around 169 nm. The polydispersity index tended to increase, but remained within 10%, demonstrating good polydispersity and excellent particle size stability, laying the foundation for the application of targeted supramolecular BAPs-PLGA nanoparticles.

[0130] Test Example 5:

[0131] The biosafety of the targeted supramolecular BAPs-PLGA nanoparticles prepared in Example 1 and the BAPs prepared in Example 7 were tested.

[0132] CCK-8, short for Cell Counting Kit-8, is a reagent that allows for simple and accurate cell proliferation and toxicity analysis. Its basic principle is that the reagent contains the water-soluble tetrazolium salt WST-8 [chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonate)-2H-tetrazolium monosodium salt]. This salt is reduced by cellular dehydrogenases to a highly water-soluble yellow formazan dye under the action of the electron carrier 1-methoxy-5-methylphenazinium methyl sulfate (1-Methoxy PMS). The amount of formazan generated is proportional to the number of viable cells. This property allows direct analysis of cell proliferation and toxicity. The specific experimental steps are as follows:

[0133] I (Cell Preparation): Resuscitate cells and subculture them to the logarithmic growth phase. On the day of the experiment, digest and count the cells and adjust the cell density to 1-5 x 10 ^4 pieces / mL.

[0134] II (Cell Seeding): Inoculate 100 μL of cell suspension into a 96-well plate, with 3-5 replicate wells per group. Incubate the cells in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere.

[0135] III (Drug Treatment): After 24 hours of cell culture, add the test substance at different concentrations and continue culturing for 24-72 hours. Add an equal volume of culture medium to the control group.

[0136] IV (CCK-8 Assay): Add 10 μL of CCK-8 solution to each well, mix gently to avoid bubbles, and incubate the cells in a 37°C, 5% CO2 incubator for 1-4 hours. After incubation, measure the absorbance (OD) of each well at 450 nm using a microplate reader and compare the results to those of the untreated control group.

[0137]

[0138] Where:

[0139] Relative survival rate - relative OD 450 nm , %;

[0140] Test OD 450 nm ——Average OD of the test substance 450 nm ;

[0141] Neg OD 450 nm ——Average OD of negative control 450 nm .

[0142] Human immortalized keratinocytes HaCaT were used as test cells to test the effects of targeted supramolecular BAPs-PLGA nanoparticles or BAPs on the survival rate of HaCaT cells at concentrations of 6.25%, 12%, 25%, 50% and 100%. Figure 11 As shown, under the action of different concentrations of targeted supramolecular BAPs-PLGA nanoparticles or BAPs, the survival rate of HaCaT cells was greater than 95%, and the targeted supramolecular BAPs-PLGA nanoparticles or BAPs did not cause obvious toxicity to HaCaT cells, that is, the targeted supramolecular BAPs-PLGA nanoparticles and BAPs had good biosafety.

[0143] Comparative test example 1:

[0144] The difference between comparative test example 1 and test example 5 is that the HaCaT cells in test example 5 are replaced with human foreskin fibroblasts HFF-1.

[0145] The effects of targeted supramolecular BAPs-PLGA nanoparticles or BAPs on the survival rate of HFF-1 cells at concentrations of 6.25%, 12%, 25%, 50% and 100% were tested. Figure 12 As shown, under the action of different concentrations of targeted supramolecular BAPs-PLGA nanoparticles or BAPs, the survival rate of HFF-1 cells was greater than 95%. Similar conclusions can be drawn as in Test Example 5, that targeted supramolecular BAPs-PLGA nanoparticles or BAPs did not cause significant toxicity to HFF-1 cells, indicating that targeted supramolecular BAPs-PLGA nanoparticles and BAPs have good biosafety.

[0146] Test Example 6:

[0147] The cell uptake ability of the targeted supramolecular BAPs-PLGA nanoparticles prepared in Example 1 and the BAPs prepared in Example 7 was tested.

[0148] A major goal of drug delivery systems is to effectively deliver therapeutic agents to specific cells or tissues. Cellular uptake is a critical step in this process, as drugs can only take effect within the cells once they are taken up by the target 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 drug uptake. The specific experimental steps are as follows:

[0149] I (Cell Culture): Resuscitate HaCaT cells, observe their cell morphology, and expand the culture to the desired number, with the cell density reaching 70% to 80% of the well area.

[0150] Ⅱ (Establishment of groups): Set up two groups for experiments: targeted supramolecular BAPs-PLGA nanoparticles and composite peptides.

[0151] III (slide): add Cells were plated.

[0152] IV (Co-incubation): Equal amounts of fluorescently labeled targeted supramolecular BAPs-PLGA nanoparticles and BAPs (AMC-labeled acetyl tetrapeptide-9, FIC-labeled hexapeptide-11, and RB-labeled acetyl hexapeptide-8) were added at pre-set concentrations and co-incubated for 48 hours. Finally, cells were carefully washed twice with PBS to terminate cellular uptake.

[0153] V: Stain the skeleton protein, use a water-soluble mounting medium for sealing, use a fluorescence microscope to take pictures, and choose four colors: red (skeleton protein), green (hexapeptide-11), orange (acetyl hexapeptide-8), and blue (acetyl tetrapeptide-9).

[0154] Tubulin is the basic protein unit that makes up microtubules. Microtubules are the primary components of the cytoskeleton and are present in almost all eukaryotic cells. In cellular uptake experiments, staining cytoskeletal proteins can help observe changes in the cytoskeleton during the uptake process, which is important for understanding how cells take in foreign substances through mechanisms such as endocytosis.

[0155] Cell uptake was observed under a fluorescence microscope. Figure 13 As shown, the backbone protein is red, hexapeptide-11 is green, acetyl hexapeptide-8 is orange, and acetyl tetrapeptide-9 is blue. The intensity of the green, orange, and blue fluorescence on the backbone protein reflects the cellular uptake capacity of hexapeptide-11, acetyl hexapeptide-8, and acetyl tetrapeptide-9. The three fluorescence intensities of the targeted supramolecular BAPs-PLGA nanoparticle group were significantly higher than those of BAPs, indicating that cells have a stronger ability to take up the bioactive peptides in the targeted supramolecular BAPs-PLGA nanoparticles than BAPs. This enhanced uptake capacity is attributed to the targeting effect of the supramolecular RGD on the nanoparticle surface.

[0156] Test Example 7:

[0157] In vitro transdermal effect of the BAPs prepared in Example 7

[0158] The skin is composed of the stratum corneum, epidermis, dermis, and subcutaneous tissue. After a drug is placed on the skin surface, it penetrates into the skin, passing through the epidermis to reach the dermis. Due to the 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, which meets the so-called "sink" condition, and the drug concentration is close to 0. In vitro transdermal diffusion experiments can predict the rate of drug transdermal absorption and study the effects of media, formulation composition, and transdermal absorption enhancers on drug transdermal rate. It is a prerequisite for ensuring the effectiveness and safety of transdermal drug preparations. The specific experimental procedures are as follows:

[0159] Ⅰ (Microscopic examination): Select undamaged pig skin under a dissecting microscope, cut 6 pieces of skin of equal size, wash once with physiological saline, and absorb the surface moisture with filter paper.

[0160] II (Fixed skin): Fix the skin on the Franz diffusion cell with the stratum corneum facing the dosing chamber and the dermis facing the receiving cell. Add 17 mL of normal saline to the receiving cell and remove bubbles to ensure that there are no bubbles between the dermis and the receiving solution.

[0161] III (Dosing): Open the instrument in advance and adjust the water bath temperature to 32 ± 1°C. Add 0.5 mL of fluorescent dye-labeled BAPs into the dosing chamber. Seal with parafilm and tin foil to prevent liquid evaporation. The effective penetration area is 0.36πcm 2 .

[0162] IV (Infiltration): Set the stirring speed to 300 rpm and keep it away from light.

[0163] V (Sampling): Incubate for 24 hours, then collect the skin at the 24th hour point. Then, perform tissue imaging and take photos to observe the penetration of the active ingredients in the sample into the skin.

[0164] VI (frozen section): The skin was fixed in 4% paraformaldehyde overnight, washed with PBS and quickly frozen in a -80°C freezer. The skin was then vertically embedded in OCT and full-thickness sectioned using a freezing microtome.

[0165] VII (Tissue Imaging Photography): The sliced ​​tissues were observed and photographed using a fluorescence inverted microscope at three excitation wavelengths. The transdermal effect of AMC-labeled acetyl tetrapeptide-9 was observed at the excitation wavelength of blue (377, 477); the transdermal effect of Fitc fluorescent labeled hexapeptide-11 was observed at the excitation wavelength of green (469, 525); and the transdermal effect of RB-labeled acetyl hexapeptide-8 was observed at the excitation wavelength of red (586, 647).

[0166] The imaging results after 24 hours of BAPs transdermal administration are as follows: Figure 14The fluorescence quantitative data are shown in Table 6:

[0167] Table 6. Fluorescence quantitative data statistics of BAPs after 24 hours of percutaneous penetration

[0168]

[0169] Combine Figure 14 As shown in Table 6, the average fluorescence intensities of acetyl tetrapeptide-9, hexapeptide-11, and acetyl hexapeptide-8 in the three parallel groups of BAPs were 1127±303.8 au, 2386±199.3 au, and 2269±259.1 au, respectively. All three had a small amount of penetration, but the fluorescence was basically concentrated in the stratum corneum. The subcutaneous fluorescence intensity was very low, and the transdermal effect was poor.

[0170] Comparative test example 2:

[0171] The difference between Comparative Test Example 2 and Test Example 5 is that the fluorescent dye-labeled BAPs in the administration step of Test Example 5 are replaced with a fluorescent dye-labeled targeted supramolecular BAPs-PLGA nanoparticle aqueous solution.

[0172] The imaging results of targeted supramolecular BAPs-PLGA nanoparticles after 24 hours of transdermal permeation are as follows: Figure 15 The fluorescence quantitative data are shown in Table 7:

[0173] Table 7. Fluorescence quantitative data statistics of targeted supramolecular BAPs-PLGA nanoparticles aqueous solution after 24 h of transdermal penetration

[0174]

[0175] According to Tables 6 and 7, the fluorescence quantitative graphs of BAPs and targeted supramolecular BAPs-PLGA nanoparticles after 24 h of transdermal penetration were drawn. The results are shown in Figure 6. Figure 16 As 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 were 4564±235.6 au, 10313±277.4 au and 12288±289.3 au, respectively, which were 4.05, 4.32 and 5.41 times the average fluorescence intensity of each BAPs component, respectively. Moreover, the fluorescence was not only concentrated in the stratum corneum, but obvious fluorescence could also be observed subcutaneously, showing an excellent transdermal effect.

[0176] 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, respectively. This indicates that the targeted supramolecular BAPs-PLGA nanoparticles have excellent penetration-enhancing ability.

[0177] Test Example 8:

[0178] Test the anti-enzymatic ability of BAPs prepared in Example 7

[0179] The primary site of action of pepsin is the peptide bond formed by the amino groups of aromatic amino acids (mainly tyrosine Tyr, phenylalanine Phe, and tryptophan Try) or acidic amino acids (mainly glutamic acid Glu and aspartic acid Asp). The amino acid sequences of hexapeptide-11, acetyl tetrapeptide-9, and acetyl hexapeptide-8 contain Phe, Asp, and Glu, respectively. Pepsin has a certain degree of enzymatic hydrolysis ability on all three peptides. Therefore, using pepsin as an example, the resistance of BAPs to enzymatic hydrolysis was observed. The specific experimental procedures are as follows:

[0180] I (enzymatic hydrolysis): 20 g of BAPs were placed in a constant temperature water bath. 300 mg of pepsin was added to initiate the hydrolysis reaction. The reaction was continued for 4 h. The reaction solution was removed and immediately poured into 20 g of strong alkaline solution (1 mol / L NaOH) to terminate the reaction.

[0181] II (Measure absorbance): Take 5 mL of reaction solution and add 1 mL of ninhydrin dropwise. Heat to develop color. To ensure the accuracy of the measurement, dilute the heated reaction gradient solution and measure its light absorbance at 570 nm. Ensure that the absorbance value is within the range of 0-1 to evaluate the free amino acid content, thereby reflecting the degree of enzymatic hydrolysis of the polypeptide.

[0182] Reaction Mechanism: A ninhydrin solution is heated with amino acids to generate ammonia. Ammonia reacts with ninhydrin and reduced ninhydrin to form a purple compound. The color of this compound is proportional to the amino acid content. The free amino acid content can be assessed by measuring absorbance at 570 nm. The absorbance values ​​after enzymatic hydrolysis of BAPs are shown in Table 8.

[0183] Table 8. Statistics of absorbance values ​​of BAPs and targeted supramolecular BAPs-PLGA nanoparticles after enzymatic hydrolysis

[0184]

[0185] Comparative test example 3:

[0186] Improving the in vivo stability of peptides primarily involves modifying their structure (forming cyclic peptides, using D-amino acids, or altering single or multiple amino acids), modifying their ends (hydrophobic and hydrophilic), and modifying them with biomacromolecules. These chemical modifications stabilize bioactive peptides, mitigating degradation and inactivation mediated by blood or tissue proteases, thereby enhancing the drug-like properties of peptide drug candidates. In addition to modifying the peptides themselves, polymers can be used to encapsulate them, forming a barrier between them and proteases and protecting them from protease cleavage. Targeted supramolecular BAPs-PLGA nanoparticles utilize microfluidics to encapsulate bioactive peptides in PLGA to enhance their resistance to enzymatic degradation.

[0187] The difference between Test Example 3 and Test Example 6 is that the BAPs in the enzymatic hydrolysis step in Test Example 6 are replaced with targeted supramolecular BAPs-PLGA nanoparticles, and the contents of acetyl tetrapeptide-9, hexapeptide-11, and acetyl hexapeptide-8 in the two remain consistent, and the absorbance values ​​after enzymatic hydrolysis are also shown in Table 8.

[0188] Comparison of absorbance of BAPs and targeted supramolecular BAPs-PLGA nanoparticles after the same enzymatic hydrolysis Figure 17 As shown, the UV absorbance of BAPs and targeted supramolecular BAPs-PLGA nanoparticles was 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 was higher in BAPs than in targeted supramolecular BAPs-PLGA nanoparticles. These results indicate that after enzymatic hydrolysis, the number of free amino acids in targeted supramolecular BAPs-PLGA nanoparticles decreased, demonstrating a certain degree of resistance to enzymatic hydrolysis.

[0189] Test Example 9:

[0190] To test the safety of formulated products containing targeted supramolecular BAPs-PLGA nanoparticles.

[0191] The targeted supramolecular BAPs-PLGA nanoparticles prepared in Example 1 and the BAPs prepared in Example 7 were respectively formulated into targeted supramolecular BAPs-PLGA nano essence emulsion and BAPs essence emulsion. The compositions thereof by mass percentage are shown in Table 9:

[0192] Table 9. Targeted supramolecular BAPs-PLGA nano-essence and BAPs essence

[0193]

[0194] Test Method: Using a qualified patch test device, perform a closed patch test. Place 0.020–0.025 g of the test substance in the device and apply it to the flexed forearm of the subject using hypoallergenic tape. After 24 hours, remove the test substance. Observe skin reactions 0.5, 24, and 48 hours after removal. Record the skin reaction grading criteria in the "Safety Technical Specifications for Cosmetics" (2015 edition).

[0195] Table 10. Summary of human patch test results

[0196]

[0197] Note: The skin reactions of 30 subjects at different observation times are shown in Table 12.

[0198] Table 11. Skin reaction grading standards for occlusive patch test

[0199]

[0200] Table 12. Skin reactions of subjects at different observation times

[0201]

[0202] The results of the human skin occlusive patch test are shown in Tables 10 and 12. No adverse reactions occurred on the skin of the 30 subjects in the targeted supramolecular BAPs-PLGA nano-essence emulsion group and the BAPs essence emulsion group, indicating good safety.

[0203] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still 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 RGD, supramolecular BAPs and PLGA; The supramolecular BAPs are prepared by mixing TG-DES and BAPs in a mass ratio of 1:(50-200), and stirring for 2-12 hours under a nitrogen pressure of 5-20 MPa; The supramolecular RGD is prepared by mixing TG-DES and RGD in a mass ratio of 1:(50-200), and stirring for 2-12 hours under a nitrogen pressure of 5-20 MPa; The BAPs are composite peptides 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); The nanoparticles have an average particle size of 100-200 nm and a polydispersity index of less than 10%; The nanoparticles are prepared by a W / O / W multiple emulsion method, that is, the oil phase of PLGA is emulsified with the aqueous phase containing supramolecular BAPs to form a W / O colostrum, which is then secondary emulsified with the aqueous phase containing an emulsifier and supramolecular RGD to form a W / O / W emulsion, and then the organic solvent is volatilized to solidify the nanoparticles.

2. The method for preparing targeted supramolecular BAPs-PLGA nanoparticles according to claim 1, characterized in that: The following steps are involved: (1) Dissolve PLGA in dichloromethane to form a primary oil phase; (2) using the supramolecular BAPs as a primary aqueous phase and performing a 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) The primary emulsion obtained in step (2) and the supramolecular RGD are used as a 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) The multiple emulsion obtained in step (3) is stirred to evaporate the dichloromethane to obtain targeted supramolecular BAPs-PLGA nanoparticles.

3. The method for preparing targeted supramolecular BAPs-PLGA nanoparticles according to claim 2, characterized in that: The stirring temperature is 20-40° C., the stirring time is 4-24 h, and the stirring speed is 300-600 rpm.

4. The method for preparing targeted supramolecular BAPs-PLGA nanoparticles according to claim 2, characterized in that: The primary emulsification and the secondary emulsification are both performed under the protection of inert gas using microfluidics technology and ultrasonic emulsification technology.

5. The method for preparing targeted supramolecular BAPs-PLGA nanoparticles according to claim 4, 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 phacoemulsification technique is 100-600 W, the frequency is 10-60 kHz, the ultrasonic treatment is followed by a 2-second pause every 2 seconds, and the duration is 0.5-2 hours; The inert gas is nitrogen, and the pressure is 5-20 MPa.

6. Use of the targeted supramolecular BAPs-PLGA nanoparticles according to claim 1 in the preparation of pharmaceutical preparations and cosmetic products.

Images