A short peptide dimer and its nanocomplex

Abstract

The present application belongs to the field of polypeptide drug preparation, and particularly relates to a kind of short peptide dimer and its nanocomposite.The tripeptide (Ser-Cys-Arg) is assembled to form short peptide dimer by hydrogen bond in aqueous solution, which first discloses that short peptide forms cyclic dimer structure through intermolecular hydrogen bond, without using chemical crosslinking agent, oxidizing agent or protection / deprotection step, and the process is more simple and mild, and more suitable for synergistic assembly with drug molecules to form nanocomposite.The short peptide dimer provided by the present application can efficiently encapsulate model drugs to form "peptide-drug" nanocomposite, which can diffuse in gel matrix to epidermis, form multiple non-covalent interactions with skin lipids and keratin, significantly improve the transdermal permeability of drugs through dual pathways of cell penetration and intercellular gap transport, and reach the action site to realize efficient targeted drug release.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide drug preparation, and specifically relates to a short peptide dimer and its nanocomplex. Background Technology

[0002] As a non-invasive drug delivery method, transdermal drug delivery systems have gradually become an important research direction in the field of drug delivery due to their advantages such as non-invasiveness, ease of use, ability to bypass the first-pass effect of the liver, and ability to maintain stable blood drug concentrations. This method of drug delivery is particularly suitable for patients with chronic diseases who need long-term medication, thereby significantly improving patient adherence and medication safety. However, the stratum corneum of the skin has a very dense structure, forming a natural physiological barrier. This barrier severely limits the transdermal penetration efficiency of most drug molecules (especially hydrophilic drugs and macromolecular drugs), thus posing a significant challenge to transdermal drug delivery technology.

[0003] To overcome this obstacle, various techniques to enhance penetration have been proposed and applied. These techniques, including physical, chemical, and biological strategies, collectively constitute the modern transdermal drug delivery system. Among physical methods, iontophoresis uses microcurrents to drive charged drugs into the skin; electroporation uses high-voltage pulses to create transient hydrophilic channels in the stratum corneum; and microneedle arrays physically penetrate the stratum corneum through micron-sized needles to establish microchannels, opening feasible pathways for the delivery of large molecule drugs. In addition, techniques such as ultrasound delivery (acoustic effect) and thermal ablation temporarily alter the properties of the skin barrier through physical means. Chemical methods mainly rely on penetration enhancers. Traditional chemical enhancers (such as nitroglycerin, menthol, and dimethyl sulfoxide) reversibly disrupt the ordered arrangement of the lipid bilayer of the stratum corneum, increasing its fluidity and thus promoting drug diffusion. However, these small-molecule penetration enhancers often cause skin irritation, and their mechanisms of action are not specific. Biological strategies represent a new generation of penetration-enhancing technologies, among which flexible liposomes with hyperdeformability and skin-penetrating peptides (SPPs) are the most promising. Flexible liposomes utilize their hyperdeformability to squeeze through the narrow lipid channels of the stratum corneum, thereby improving penetration and delivery efficiency; while SPPs have attracted much attention due to their excellent biocompatibility and unique synergistic delivery mechanism. SPPs mainly interact with skin lipids or keratin through mechanisms such as electrostatic attraction, hydrogen bonding, and regulation of lipid fluidity. This interaction temporarily and reversibly disrupts the stratum corneum structure, thereby promoting efficient, controlled, and non-invasive drug penetration through the skin barrier. They show particularly promising applications in the transdermal delivery of macromolecular drugs and localized precise targeting.

[0004] Chinese patent CN119504927A discloses a transdermal tripeptide and its application, designing and synthesizing a novel transdermal tripeptide, Ser-Cys-Arg. This transdermal tripeptide exhibits significant transdermal activity. However, as a transdermal permeability enhancer, the function of S3 peptide (Ser-Cys-Arg) is mainly limited to passive diffusion of drugs through physical mixing. Although it has a permeation-enhancing effect, it has significant disadvantages compared to the cyclic dimer it self-assembles into: a single S3 peptide lacks a nanostructure for actively encapsulating drugs and cannot form a stable "peptide-drug" complex; its mechanism of action is relatively traditional, making it difficult to effectively solubilize poorly soluble drugs; at the same time, this linear structure does not possess reversible assembly characteristics and cannot respond to the lesion microenvironment to achieve intelligent and controllable drug release.

[0005] To address the aforementioned problems, existing research has elucidated the construction of dimers; however, the synthesis of dimers presents significant difficulties. Current techniques for preparing dimers include the following: The first type of method is the covalent cyclization strategy, which introduces stable covalent links between peptide molecules through chemical or enzymatic methods, thereby obtaining cyclic dimers with well-defined structures and rigid conformations. Typical methods include: 1. Head-tail cyclization between terminal amino and carboxyl groups; 2. Oxidative coupling of disulfide bonds between cysteine ​​residues; 3. Intermolecular crosslinking using click chemistry (such as azido-alkyne cycloaddition), amide condensation, or thioether bond formation; 4. Site-directed cyclization reactions catalyzed by enzymes such as transglutaminase and sortase. Cyclic dimers obtained by this type of method have stable structures and good reproducibility, but usually require protection / deprotection steps, specific reaction conditions, or catalytic systems, making the process relatively complex and costly.

[0006] The second type of method is a template- or scaffold-assisted cyclization strategy. This involves introducing metal ions, small organic molecules, polymers, or protein templates to guide the spatial orientation of short peptides and form cyclic dimer structures. For example, using Zn²⁺... + Cu² + The coordination of metal ions with histidine and cysteine ​​side chains, or the use of scaffolds such as surfactants, nanoparticles, and cyclodextrins to achieve conformational constraint, can reduce the reaction energy barrier to some extent. However, the complex system composition and the potential for template residue or removal issues may affect its biological applications. Summary of the Invention

[0007] To overcome the aforementioned problems, this invention provides a short peptide dimer and its nanocomposite. By mixing the tripeptide (S3) with water, it assembles into a cyclic dimer structure via hydrogen bonding. This is further combined with a model drug to form a "peptide-drug" nanocomposite, achieving a triple mechanism of drug encapsulation, enhanced permeation, and controlled release. The S3 dimer provided by this invention self-assembles into a highly ordered structure under mild conditions, actively encapsulating and solubilizing drugs, synergistically performing the triple functions of encapsulation, permeation enhancement, and controlled release. This represents a technological leap from simple permeation enhancers to intelligent delivery systems.

[0008] A short peptide dimer, wherein the short peptide dimer is formed by the assembly of a tripeptide (S3) via hydrogen bonds; the amino acid sequence of the tripeptide is Ser-Cys-Arg; The short peptide dimer is formed by the reaction and assembly of 1-10 wt% S3 in an aqueous solution.

[0009] In aqueous systems, S3 can self-assemble through intermolecular hydrogen bonding, hydrophobic interactions, and synergistic effects between polar groups to form a stable cyclic dimer structure. This cyclic dimer is a non-covalent self-assembled structure, and its formation does not depend on chemical cross-linking or covalent bonding. It is characterized by mild formation conditions, strong reversibility, and good biocompatibility.

[0010] Specifically, the reaction assembly temperature is 20–30℃ and the pH is 6.0–7.5, which allows the guanidino group of arginine to be fully protonated and become positively charged, while the thiol group of cysteine ​​is partially deprotonated, thereby effectively promoting the formation of hydrogen bond network.

[0011] Preferably, the reaction assembly temperature is 25°C.

[0012] The reaction assembly time shall not be less than 24 hours.

[0013] The reaction assembly is carried out by stirring at a speed of 50–300 rpm. The stirring speed should not be too high to prevent the weak hydrogen bond interactions from being destroyed by shear force.

[0014] To avoid charge shielding effects interfering with intermolecular interactions, the ionic strength of the aqueous solution is 0.001–0.010 mol·L⁻¹. -1 .

[0015] The present invention also provides a nanocomposite comprising the above-mentioned short peptide dimer and a drug.

[0016] The drugs include diclofenac sodium and baicalin.

[0017] The mass ratio of S3 to the drug is 1:5-20.

[0018] The nanocomposite is prepared by adding S3 and the drug to an ionic strength of 0.001–0.010 mol·L⁻¹. -1 Nanocomposites can be obtained by stirring in an aqueous NaCl solution at a pH of 6.0-7.5 and a temperature of 20-30℃ for at least 24 hours at 50-300 rpm. The obtained nanocomposites are then freeze-dried to obtain a stable solid powder.

[0019] Beneficial effects: 1. This invention is the first to reveal that short peptides form cyclic dimer structures through intermolecular hydrogen bonds. The short peptide dimer structure provided by this invention mainly relies on the self-assembly of intermolecular hydrogen bonds and hydrophobic interactions. The dynamic reversibility of this interaction allows the dimer to efficiently encapsulate drug molecules in a more flexible manner, forming stable nanocomposites. In addition, this dynamic structure can disassemble in vivo in response to stimulation by the lesion microenvironment (such as elevated reactive oxygen species levels), thereby achieving intelligent and controllable drug release at the target site, improving therapeutic efficacy and reducing systemic side effects.

[0020] 2. The short peptide dimers provided by this invention can be formed by molecular self-assembly under aqueous conditions without the use of chemical cross-linking agents, oxidants, or protection / deprotection steps. The process is simpler and milder, and more suitable for co-assembly with drug molecules to form nanocomposites.

[0021] 3. The short peptide dimer structure provided by the present invention has hydrophilic / hydrophobic domains, which can induce drug molecules to attach to the aggregate and promote drug solubilization. Attached Figure Description

[0022] Figure 1 The short peptide structure of S3 in Example 1 and its FTIR and mass spectrometry characterization are shown below. Figure 2 The short peptide structure of S2 in Comparative Example 1 and its FTIR and mass spectrometry characterization are shown below. Figure 3 The short peptide structure of S4 in Comparative Example 2 and its FTIR and mass spectrometry characterization are shown below. Figure 4 The short peptide structure of S7 in Comparative Example 3 and its FTIR and mass spectrometry characterization are shown. Figure 5 This is a schematic diagram of the nanocomposite formed by S3 and sodium dichlorophenate in Example 2; Figure 6 The conformation of S3 in the aqueous system in Example 1 is a cyclic dimer structure; Figure 7 The radial distribution function between S3 molecules in Example 1; Figure 8 The radial distribution function between S3 molecules and water molecules in Example 1; Figure 9 This is a transient distribution diagram of S3 molecules and diclofenac sodium molecules in Example 2; Figure 10 The radial distribution function between S3 molecules and diclofenac sodium molecules in Example 2; Figure 11 This is a schematic diagram of the simulated docking of S3 in Example 2; Figure 12 Transmission electron microscopy (TEM) images of S3, DS, and S3-DS nanocomposites in Example 2; Figure 13 The mass spectra of S3, DS, and S3-DS nanocomposites in Example 2 are shown. Figure 14 The infrared spectrum of the S3 and S3-DS nanocomposite in Example 2; Figure 15 The ¹H NMR spectra of S3, DS, and S3-DS nanocomposites in Example 2 are shown below. Figure 16 AFM image of the S3-DS nanocomposite in Example 2; Figure 17 This is a mass spectrometry image of the skin on the back of the mouse in Experiment Example 1; Figure 18 The inhibitory rates of different gel patches on xylene-induced ear swelling in mice; Figure 19 The analgesic rate of different gel patches on sodium acetate-induced writhing response in mice; Figure 20 Changes in swelling rate of sodium urate-induced paw edema in mice using different gel patches; Figure 21 To investigate the effects of different gel patches on the urate-induced paw inflammatory index in mice; Figure 22 Histopathological sections of mouse paw skin treated with different gel patches; Figure 23 The results are from a skin irritation test of the gel patch. Detailed Implementation

[0023] Example 1: Preparation of short peptide dimers At an ionic strength of 0.005 mol·L -1 1 wt% S3 (SCR) was added to the NaCl aqueous solution and stirred at 100 rpm for 24 h at 25 °C, while the pH was maintained at 7.0 using a 10 mM HEPES buffer system.

[0024] The preparation method of S3 is described in Example 1 of CN119504927A.

[0025] Example 2: Preparation of nanocomposites using diclofenac sodium as a drug Add S3 to the aqueous solution at a mass ratio of 1:8 to diclofenac sodium, bringing the volume to 0.005 mol·L⁻¹. -1 The nanocomposite S3-DS was obtained by stirring in an aqueous NaCl solution at 25°C and 100 rpm for 24 h, while maintaining the pH at 7.0 using a 10 mM HEPES buffer system. The obtained nanocomposite was further freeze-dried to obtain a stable solid powder.

[0026] Example 3: Preparation of nanocomposites using baicalin as a drug Add S3 to the aqueous solution at a mass ratio of 1:8 to baicalin, bringing the volume to 0.005 mol·L⁻¹. -1 The nanocomposite S3-BG was obtained by stirring in an aqueous NaCl solution at 25°C and 100 rpm for 24 h, while maintaining the pH at 7.0 using a 10 mM HEPES buffer system. The obtained nanocomposite was further freeze-dried to obtain a stable solid powder.

[0027] Example 4 Based on the mass of the paste mixture, the composition is as follows: sodium polyacrylate 7%, carbomer 980 0.45%, sodium carboxymethyl cellulose 3%, gelatin 2%, the nanocomposite S3-DS prepared in Example 2 1.08%, glycerol 30.0%, PEG600 0.8%, bentonite 1.2%, EDTA 0.1%, ethanol 0.5%, and the remainder is water.

[0028] Sodium polyacrylate, carbomer 980, sodium carboxymethyl cellulose, and gelatin were added to a solution of water to form a gel-like substance, which swelled overnight. Sodium diclofenac sodium was added to solution A with ethanol. Then, glycerol, PEG600, and bentonite were added sequentially to form a suspension, solution B. EDTA was added to solution B with water. S3 was added to solution B with water. The pH was adjusted to 6.5, and the mixture was stirred until a paste-like consistency was achieved. This paste was then coated onto a non-woven fabric and cured at 40°C in a drying oven for 20 minutes. After cooling to room temperature, the mixture was covered with a polyethylene film and left to stand for 48 hours to obtain a 1% sodium diclofenac sodium patch.

[0029] Based on the mass of the paste mixture, the first addition of water was 30.0%; the second addition was 5.87%; and the third addition was 18.0%.

[0030] Example 5 Based on the mass of the paste mixture, the following components were present: sodium polyacrylate 7.0%, carbomer 980 0.45%, sodium carboxymethyl cellulose 3%, gelatin 2%, the nanocomposite S3-BG prepared in Example 3 1.08%, glycerol 30.0%, PEG600 0.8%, bentonite 1.2%, EDTA 0.1%, ethanol 0.5%, and the remainder was water.

[0031] Sodium polyacrylate, carbomer 980, sodium carboxymethyl cellulose, and gelatin were added to a solution of water to form a gel-like substance, which swelled overnight. Baicalin was added to solution A with an appropriate amount of ethanol. Then, glycerol, PEG 600, and bentonite were added sequentially to form a suspension, solution B. EDTA was added to solution B with water. S3 was added to solution B with water. The pH was adjusted to 6.5, and the mixture was stirred until a paste-like consistency was achieved. This paste was then coated onto a non-woven fabric and cured at 40°C in a drying oven for 20 minutes. After cooling to room temperature, the mixture was covered with a polyethylene film and left to stand for 48 hours to obtain a 1% baicalin patch.

[0032] Based on the mass of the paste mixture, the first addition of water was 30.0%; the second addition was 5.87%; and the third addition was 18.0%.

[0033] Comparative Example 1 In this comparative example, S3 in Example 1 was replaced with S2 (RP), and all other conditions were the same as in Example 1. Short peptide dimers could not be prepared.

[0034] The preparation method of S2 refers to Example 1 of patent CN119504925A.

[0035] The reason is speculated to be that S2 (RP) consists only of Arg and Pro, lacking key residues such as cysteine ​​and serine that can provide a stable hydrogen bond network and conformational matching, and also lacking the sequence basis for the "multi-point cooperative non-covalent assembly" required by this invention. In particular, the cyclic side chain of Pro significantly restricts the conformational freedom of the main chain, weakens the establishment of a regular hydrogen bond network, and is not conducive to the formation of a stable, closed cyclic dimer structure. Therefore, theoretically, S2 is unlikely to self-assemble into a stable short peptide dimer under the conditions of Example 1.

[0036] Comparative Example 2 In this comparative example, S3 in Example 1 was replaced with S4 (ARCG, SEQ ID NO.1), and all other conditions were the same as in Example 1. Short peptide dimers could not be prepared.

[0037] The preparation method of S4 refers to Example 1 of patent CN119504928A.

[0038] The reason is speculated to be that although S4 (ARCG) contains Cys and Arg and has a certain basis for electrostatic and hydrogen bonding interactions, its sequence length, amino acid arrangement, and the introduction of Gly significantly change the local conformational flexibility and intermolecular matching mode. Compared with S3 (Ser-Cys-Arg) in Example 1, S4 is more likely to form disordered aggregates or weakly interacting clusters, rather than forming stable cyclic dimers with specific geometric constraints.

[0039] Comparative Example 3 In this comparative example, S3 in Example 1 was replaced with S7 (RRFHLLP, SEQ ID NO.2), and all other conditions were the same as in Example 1. Short peptide dimers could not be prepared.

[0040] The preparation method of S7 refers to Example 1 of patent CN119504931A.

[0041] The reason is speculated to be that S7 (RRFHLLP) is a relatively long sequence, rich in hydrophobic residues (Phe, His, Leu, Leu) and lacking cysteine, thus lacking the key non-covalent interaction combination required for the formation of the dimer of this invention. This sequence is more likely to produce hydrophobically driven non-specific aggregation rather than forming regular, reversible cyclic dimers with specific topologies. Furthermore, the terminal Pro residue also affects the conformational continuity of the main chain, further weakening regular assembly. Therefore, theoretically, S7 is not suitable as a building block for short peptide dimers.

[0042] Comparative Example 4 In this comparative example, the S3-DS nanocomposite in Example 4 was replaced with diclofenac sodium, and all other conditions were the same as in Example 4, to prepare the DS patch.

[0043] Without the addition of short peptide dimers, diclofenac sodium exists only in a free state or a simple physical mixture, and cannot be encapsulated, confined, or stabilized by the dimer nanostructure, thus making it difficult to form nanocomposites. The resulting product is merely a conventional DS patch, lacking the drug molecular-level encapsulation, solubilization, sustained release, and enhanced transdermal delivery mechanisms provided by short peptide dimers. Therefore, short peptide dimers are essential structural units for achieving nano-scale formation, controlled release, and enhanced transdermal delivery.

[0044] like Figure 1-4 The figures shown are the structural formulas of the short peptides prepared in Example 1 and Comparative Examples 1-3, and their FTIR and mass spectrometry characterizations. Figure 5-10 The simulation results for Examples 1 and 2 are shown.

[0045] in, Figure 5This is a schematic diagram of the nanocomposite formed by S3 and the drug molecule diclofenac sodium in Example 2, illustrating the assembly process of S3 and diclofenac sodium (DS). There are intermolecular hydrogen bonds between the C-terminus and N-terminus of the S3 molecule, which facilitates intermolecular assembly. The DS molecule contains active groups such as -Cl, -NH, benzene ring and -COONa, which can form hydrogen bonds, electrostatic and hydrophobic interactions with the hydrophilic / hydrophobic groups on the S3 molecule.

[0046] To study the interaction between S3 and water molecules in solution, the conformation of S3 was analyzed, such as... Figure 6 The figure shows the conformation of S3 in an aqueous system – a cyclic dimer structure. Hydrogen bonds are represented by black dashed lines. The figure depicts the water distribution around the dimer structure. Hydrogen bonds stabilize the dimer structure, and hydrogen bonds are also formed between the cyclic dimers. S3 interacts with water molecules through hydrogen bonds, further stabilizing the dimer structure. In this system, the intermolecular self-assembly of S3 leads to the formation of a cyclic dimer structure.

[0047] To analyze the intermolecular interactions of S3, several radial distribution functions (RDFs) were calculated. These functions reflect the local structural characteristics of the material, reveal atomic distribution patterns, and help determine the effective intermolecular potential energy and probability distribution between interacting substances. Figure 7 The radial distribution function (RDF) between S3 molecules illustrates the RDF distribution between nitrogen (N) and oxygen (O) atoms in the two tripeptide molecules. Figure 7 The significant peak observed at approximately 2.5–4 Å indicates a significant intermolecular interaction between the two S3 molecules, suggesting the formation of a stable dimer structure within the system. Figure 8 The radial distribution function (RDF) between S3 and water molecules is shown, revealing the RDF distribution of nitrogen and oxygen atoms between them, clearly demonstrating the interaction between S3 and water molecules. Figure 9 (Instantaneous distribution diagram of S3 molecules and diclofenac sodium molecules) and Figure 10 The results of the radial distribution function (between S3 molecules and diclofenac sodium molecules) show that S3 molecules self-assemble into a stable cyclic dimer structure in the system, while water molecules further stabilize the conformation of the nanocomposite through solvation. In addition to water molecules, DS was also observed to be distributed near the S3 dimer. Figure 9 This illustrates the spatial distribution of DS around the cyclic dimer structure (surrounding water molecules are hidden in the figure for easier observation), as shown below. Figure 9 As shown, the drug molecules are mainly enriched in the region near the sulfur (S) atom of cysteine ​​in S3.

[0048] To further elucidate the interaction mechanism between DS and the cyclic dimer structure, the radial distribution function (RDF) between the dichloroaniline (DCA) center of DS and the sulfur atom in S3, as well as the radial distribution function between the carboxylate (-COO-) group of DS and the sulfur atom, were calculated. The results are as follows: Figure 10 As shown, a distinct peak is observed within a range of approximately 3-5 Å from the sulfur atom, indicating that the aromatic hydrophobic groups and charged groups of DS are preferentially distributed in the cysteine-adjacent region within the cyclic dimer. This spatial distribution characteristic suggests that the stable localization of DS in the cyclic dimer is primarily driven by hydrophobic interactions and van der Waals interactions. Simultaneously, electrostatic interactions and hydrogen bonding between the carboxylate group and adjacent positively charged residues further enhance its binding stability, thereby synergistically promoting the formation of stable peptide-drug non-covalent nanocomposites. These interactions are crucial.

[0049] like Figure 11 AutoDock simulations of intermolecular docking showed that hydrogen bonds constitute the main interaction between S3 and DS. Theoretical simulations revealed a significant hydrogen bond interaction between S3 and DS, promoting the formation of the S3-DS nanocomposite. To confirm the existence of this nanocomposite structure, the S3-DS nanocomposite prepared in Example 2 was analyzed for verification, and the results are as follows: Figures 12-16 As shown.

[0050] First, a transmission electron microscope (TEM) was used for observation. Figure 12 The image shows transmission electron microscopy (TEM) images of S3, DS, and S3-DS (scale bar 100 nm). The image reveals that S3 exhibits numerous spherical aggregates with a particle size of approximately 2 nm, while DS appears as oily droplets. Upon recombination of S3 and DS, spherical composites with sizes ranging from 40 to 200 nm are observed.

[0051] Subsequently, mass spectrometry was used to study the aggregation behavior of S3 and DS in aqueous solution. Figure 13 Mass spectrometry results for S3, DS, and S3-DS are presented. Both DS and S3 carry a single charge, while the S3-DS complex exhibits a double charge peak with a mass-to-charge ratio of 341, indicating a significant interaction between the two components, resulting in the formation of a dimer.

[0052] FT-IR and ¹H NMR also provide strong evidence for the formation of the S3-DS complex. Figure 14 The image shows the FT-IR of the S3-DS nanocomposite. The characteristic absorption peaks of the S3-DS nanocomposite show significant changes compared to the free S3 peptide. Firstly, at 3351 cm⁻¹... -1 and 3032cm -1The absorption peaks at 1541 cm⁻¹ (corresponding to the stretching vibrations of the NH bond in -CO=NH⁻, NH₂, and the combined -NH⁻ bond, respectively) exhibit significant broadening and redshift. This phenomenon indicates that the formation of the S3-DS nanocomposite promotes the expansion and strengthening of the intermolecular hydrogen bond network, thereby leading to a decrease in the force constant of the NH bond. Secondly, at 1541 cm⁻¹... -1 and close to 1670cm -1 The significant redshift of the amide II peak observed at 1670 cm⁻¹ (originating from NH bending and CN stretching vibrations) directly reveals the involvement of amide bonds in the interaction between the peptide backbone and the drug, possibly accompanied by local adjustments to the peptide secondary structure. Meanwhile, at 1670 cm⁻¹... -1 The characteristic absorption peaks appearing nearby show significant broadening. This strongly confirms the presence of -COO in DS. - Strong ionic interactions and hydrogen bonds are formed between the group and the guanidino cation of the arginine side chain.

[0053] like Figure 15 The ¹H NMR spectra of S3, DS, and S3-DS nanocomposites are shown. The β-, δ-, and γ-CH2 signals of the arginine residue side chains in S3 all exhibit significant low-field shifts. This phenomenon ultimately demonstrates a close-range, high-intensity ionic and hydrogen bond interaction between the positively charged guanidino group and the negatively charged carboxylic acid group. This leads to a decrease in the electron density of adjacent protons and enhances the shielding effect. Furthermore, the chemical shift of the Hα protons along the peptide backbone is also extensively affected, with some signal peaks changing shape and overlapping. These results indicate that DS binding not only occurs at specific sites but also induces conformational rearrangement and adaptive adjustments throughout the peptide backbone, resulting in a more stable composite structure.

[0054] like Figure 16 This is an AFM image of the S3-DS nanocomposite, showing the spherical complex formed between the two components, with sizes ranging from 40 to 200 nanometers. These results indicate that the S3 dimer carrier can enter the skin via both intercellular gap channels and transcellular channels.

[0055] Experimental Example 1 In this experiment, six hairless mice were divided into three groups: the first group served as a blank control group; the second group received only the DS patch prepared in Example 4 as a control group; and the third group received the 1% diclofenac sodium patch prepared in Example 4 as an experimental group. After 8 hours of transdermal penetration, the mice were euthanized, and the patches were excised from a specific skin area (i.e., the skin area under the patch) for mass spectrometry imaging analysis. MALDI-MSI was used to visualize and study the transdermal penetration and metabolism of S3-DS in mouse skin.

[0056] The blank control group refers to a hydrogel containing no drugs.

[0057] like Figure 17 The image shows a mass spectrometry image of mouse dorsal skin. The results indicate that MALDI-MSI technology successfully detected the specific spatial distribution of two endogenous lipid molecules in blank skin tissue (blank control group). Cholesterol sulfate mainly accumulated in the stratum corneum region, a distribution pattern highly consistent with its physiological functions in maintaining skin barrier integrity, regulating keratinocyte differentiation, and retaining moisture. In contrast, phosphatidylglycerol was widely distributed throughout the entire skin structural layer, reflecting its ubiquitous role as a fundamental phospholipid component in cell membrane metabolism. These findings confirm the reliability and applicability of the established MALDI-MSI method for locating endogenous lipid molecules in the skin.

[0058] In the control and experimental groups treated with diclofenac sodium, the spatial distribution patterns of cholesterol sulfate and phosphatidylglycerol were basically consistent with those in the blank group, indicating that the drug intervention did not significantly change the spatial conformation of the main skin lipid structure.

[0059] Regarding the target drug, diclofenac sodium, it was detected in the treated skin tissue in both groups. However, because its concentration was controlled within a safe range, its signal intensity was significantly lower than that of the two endogenous lipids. In the control group, where the skin tissue was relatively thin, the drug entered and distributed primarily via passive diffusion, with significant accumulation in the outermost layer of the skin, indicating a tendency for retention in this area. In the experimental group, despite the thicker skin tissue, the drug carrier, aided by the S3 peptide, enhanced penetration, resulting in deeper distribution. This led to a more uniform distribution of diclofenac sodium in the skin and reduced accumulation in the outermost layer. These results suggest that S3 can alter transdermal penetration behavior, thereby enhancing drug delivery and distribution to deeper skin tissues.

[0060] Experiment Example 2 The experimental cases were the normal control group, the model group, the 1% baicalin patch group (S3-BG-GP), the 1% diclofenac sodium patch group (S3-DS-GP), and the Sakigawa gout patch group (SGP). The normal control group refers to the normal mouse group; The model group refers to the mouse group after successful modeling without any drug treatment; The 1% baicalin patch group refers to the 1% baicalin patch prepared using Example 5; Sakikawa Gout Patches are gout-type acupressure stimulation patches purchased from Sakakawa Hisa Pharmaceutical Co., Ltd. in Japan. The 1% diclofenac sodium patch set refers to the 1% diclofenac sodium patch prepared using Example 4.

[0061] like Figure 18 As shown, the inhibitory effects of the Sakikawa gout patch group, the 1% baicalin patch group, and the 1% diclofenac sodium patch group on xylene-induced ear swelling in mice were evaluated: compared with the Sakikawa gout patch group (inhibition rate of 19%), the inhibition rates of the 1% baicalin patch group and the 1% diclofenac sodium patch group were increased by 15% and 10%, respectively. This indicates that the 1% baicalin patch and the 1% diclofenac sodium patch showed highly significant (P<0.001) and significantly (P<0.05) anti-inflammatory effects compared with the Sakikawa gout patch group.

[0062] Figure 19 This study demonstrated the analgesic effects of the Sakigawa gout patch group, the 1% baicalin patch group, and the 1% diclofenac sodium patch group on acetic acid-induced writhing responses in experimental mice. The analgesic efficacy was repeatedly verified in three independent experiments, recording the number of writhing responses in mice within 30 minutes after injection of 0.7% acetic acid solution (0.1 mL / 10 g). The analgesic rates of the Sakigawa gout patch group, the 1% baicalin patch group, and the 1% diclofenac sodium patch group reached 47.1%, 72.3%, and 77.4%, respectively. A highly significant difference was found between the 1% baicalin patch group and the 1% diclofenac sodium patch group compared to the Sakigawa gout patch group (P < 0.01). However, no significant difference was found between the 1% baicalin patch group and the 1% diclofenac sodium patch group. The results indicate that both the 1% baicalin patch and the 1% diclofenac sodium patch exhibit significant analgesic effects.

[0063] In the experiment evaluating the effects of 1% baicalin gel patches and 1% diclofenac sodium patches on natriuretic acid-induced acute gouty arthritis in mice, the daily relative body weight changes of the mice are shown in Table 1. There were no significant differences in body weight changes between the model groups and the normal control group. The 1% baicalin patch group (S3-BG-GP), the Sakigawa gout patch group (SGP), and the 1% diclofenac sodium patch group (S3-DS-GP) showed significant or highly significant differences on days 2, 3, 4, 5, 6, and 7. The effects of 1% diclofenac sodium patches on paw swelling rate and paw inflammation index in mice with acute gouty arthritis are shown in Table 1. Figure 20 , 21 As shown in the figure, the 1% diclofenac sodium patch group showed significant differences compared to the model group.

[0064] Table 1. Daily relative body weight changes in mice (x±s, n=10)

[0065] like Figure 22The image shows the effects of each group on the histopathological changes of the skin tissue of the mouse paws induced by sodium urate. From left to right, the groups are: normal control group, model group, Sakikawa gout patch group, 1% diclofenac sodium patch group, and 1% baicalin patch group. The image shows that the subcutaneous tissue thickness of the normal group mice was normal, with no neutrophil infiltration or proliferation. The model group mice showed significant subcutaneous tissue proliferation and a large number of neutrophil infiltrations. Compared with the model group, the 1% baicalin patch group significantly reduced the inflammatory response, with a small number of scattered inflammatory cells. The 1% diclofenac sodium patch group and the Sakikawa gout patch group also significantly reduced the inflammatory response, but the effect was lower than that of the 1% baicalin patch group.

[0066] like Figure 23 The results of the skin irritation test for each group are shown below. From top to bottom, they are the normal group, the Sakikawa gout patch group, the 1% diclofenac sodium patch group, and the 1% baicalin patch group. As can be seen from the figure, the normal group, the Sakikawa gout patch group, the 1% baicalin patch group, and the 1% diclofenac sodium patch group did not irritate the mouse skin. The data in Table 2 also verify this conclusion.

[0067] Table 2. Mouse skin irritation test

[0068] Comparative Example 5 In this comparative example, the aqueous solution in Example 1 was replaced with a 45 wt% aqueous ethanol solution, while all other conditions remained the same as in Example 1. Short peptide dimers could not be formed.

[0069] The reason is speculated to be that the introduction of ethanol significantly changed the solvation environment of the system, reduced the hydrogen bonding and hydrophobic synergistic driving force in the aqueous phase which are conducive to non-covalent assembly, and weakened the directional interaction between S3 molecules. Therefore, S3 is unlikely to maintain the regular conformation and intermolecular coordination mode in Example 1, and it is expected that it will not be able to form a stable short peptide dimer structure.

[0070] Comparative Example 6 In this comparative example, the aqueous solution in Example 1 was replaced with PBS buffer, and all other conditions were the same as in Example 1. Short peptide dimers could not be formed.

[0071] The preparation method of PBS buffer is as follows: Weigh 8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4 and dissolve them in 800mL distilled water. Adjust the solution to 7.4 with HCl, and finally add distilled water to make up to 1L to obtain 0.01M PBS buffer.

[0072] The presumed reason is that S3 cannot form a cyclic dimer in the PBS buffer system, due to the high concentration of inorganic salt ions (such as Na+) in PBS. + Cl- It can have a significant shielding effect on the electrostatic interactions and hydrogen bond networks between peptide molecules, thereby weakening the effective recognition and self-assembly driving force between molecules; in addition, the complex ionic environment of the solution in the PBS system may change the hydration state and conformational distribution of short peptide molecules, making the non-covalent cyclic dimer structure that can be formed in the low ionic strength aqueous phase tend to dissociate or be difficult to maintain stably.

[0073] Comparative Example 7 In this comparative example, the pH in Example 1 was adjusted to 8, while all other conditions remained the same as in Example 1. Short peptide dimers could not be formed.

[0074] The reason is speculated to be that cysteine ​​thiol groups are more prone to deprotonation under alkaline conditions, which weakens the intermolecular hydrogen bond network and directional non-covalent interactions in the system. At the same time, the tendency of thiol oxidation and side reactions increases, making it difficult for S3 to maintain the stable conformation and co-assembly mode formed in Example 1. Therefore, it is expected that it will be difficult to form a regular and stable short peptide cyclic dimer structure.

[0075] Comparative Example 8 In this comparative example, the mass fraction of S3 in Example 1 was adjusted to 12 wt%, while all other conditions remained the same as in Example 1. Short peptide dimers could not be formed.

[0076] The reason is speculated to be that: as the concentration of the system increases, non-specific collisions between molecules will be significantly enhanced, and excessive aggregation and local phase separation may be triggered. Under these conditions, S3 is expected to be difficult to form a stable and uniform cyclic dimer structure.

Claims

1. A nanocomposite, characterized in that, Includes short peptide dimers and drugs; The drug in question is diclofenac sodium; The short peptide dimer is formed by the assembly of tripeptides through hydrogen bonds; the amino acid sequence of the tripeptide is Ser-Cys-Arg. The nanocomposite is prepared by adding the tripeptide and drug to an ionic strength of 0.001–0.010 mol·L⁻¹. -1 The nanocomposite was obtained by stirring at 50-300 rpm for no less than 24 h in an aqueous NaCl solution at a pH of 6.0-7.5 and a temperature of 20-30℃.

2. The nanocomposite according to claim 1, characterized in that, The mass ratio of the tripeptide to the drug is 1:5-20.

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