A pentapeptide-based supramolecular assembly particle and its use as a drug delivery carrier

Abstract

The application discloses a kind of pentapeptide-based supramolecular assembly particles and its application as drug delivery carrier, belong to the field of biological assembly material.The TGCP of the present application, TGCP / nHA are prepared from TP5, HA and GA into carrier particles with drug loading performance and potential anti-inflammatory performance, it has spontaneous fluorescence performance, and can exist stably in neutral and alkaline environment, can be decomposed in acidic environment, can be loaded with small molecules with different solubility, and has certain programmed response performance at physiological and pathological pH.The present application is found through in-vitro experiment exploration that the carrier particles have good biocompatibility, and for in-vitro induced inflammatory cell experiment, it is found that TGCP and TGCP / nHA both have certain anti-inflammatory activity, can inhibit the expression of cell inflammatory factor IL-6, and play the role of inhibiting inflammation.

Description

Technical Field

[0001] This invention belongs to the field of bioassembly materials, specifically relating to a pentapeptide-based supramolecular assembly particle and its application as a drug delivery carrier. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Inflammation is the body's defensive response to stimuli and a common clinicopathological process. If the immune response is uncontrolled, it can lead to autoimmune or autoinflammatory diseases. In recent years, novel functional biomaterials with the potential to reduce inflammation levels have been rapidly developed. However, developing biocompatible drug delivery vectors with anti-inflammatory functions, controllable structure, stimulus responsiveness, and specific targeting remains challenging.

[0004] Dynamic covalent chemistry is a powerful technique that can be combined with non-covalent interactions to guide molecular assembly and create supramolecular systems for self-healing materials, biomolecular modification, and drug delivery, exhibiting reversibility under specific stimuli. Among these, the Schiff base reaction, involving the formation of imine bonds (-C=N-), has attracted increasing attention due to its mild reaction conditions, rapid reaction rate, and the autofluorescence and pH-responsive properties of the assembled systems.

[0005] Glycosaminoglycan-based assemblies have shown great potential in promoting wound healing due to their moisturizing and anti-inflammatory effects. Glycosaminoglycans with carboxyl and hydroxyl groups can form non-covalent interactions with other functional supramolecular assemblies. Studies have shown that self-assembled peptide-based materials can be used as drug delivery carriers with high targeting and delivery efficiency. Short peptides possess strong assembly capabilities and programmability. Over the past few decades, various short peptides have been synthesized and utilized to construct supramolecular assemblies with different morphologies and specific biological functions. However, there are currently no reports on the preparation of biocompatible drug delivery carriers with anti-inflammatory functions, controllable structures, stimulus responsiveness, and specific targeting using short peptides combined with Schiff base supramolecular assembly technology. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a pentapeptide-based supramolecular assembled particle and its application as a drug delivery carrier. The pentapeptide-based supramolecular assembled particle provided by the present invention has anti-inflammatory activity, good biocompatibility, and specific drug loading capacity for both water-soluble and lipid-soluble drugs. Furthermore, drugs with different solubilities are released at different rates under the same conditions.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides a pentapeptide-based supramolecular assembled particle comprising an immunologically active short biopeptide and a crosslinking agent.

[0009] The short biopeptide and cross-linking agent undergo a Schiff base reaction in the reaction system and obtain pentapeptide-based supramolecular assembled particles through supramolecular self-assembly.

[0010] In some embodiments of the present invention, the short biopeptide includes thymopentin (TP5), and the crosslinking agent is a small molecule aldehyde.

[0011] In some embodiments of the present invention, the reaction system is water or water containing an organic solvent.

[0012] In some embodiments of the present invention, the pentapeptide-based supramolecular assembled particles are spherical.

[0013] In some embodiments of the present invention, the pentapeptide-based supramolecular assembled particles further include glycosaminoglycan compounds.

[0014] Preferably, the short biopeptide is thymopentin, the glycosaminoglycan compound is hyaluronic acid (HA), and the crosslinking agent is glutaraldehyde (GA); the molar ratio of thymopentin to hyaluronic acid is 1:0 to 1:8, and the molar ratio of thymopentin to glutaraldehyde is 1:1 to 1:3.

[0015] A second aspect of the present invention provides a method for preparing the pentapeptide-based supramolecular assembled particles, comprising the following steps:

[0016] A cross-linking agent solution was added to a mixed solution containing short biopeptides and glycosaminoglycans, and the mixture was allowed to stand to obtain pentapeptide-based supramolecular assembled particles.

[0017] The short biopeptide is thymopentin, the glycosaminoglycan compound is hyaluronic acid, and the crosslinking agent is glutaraldehyde; the molar ratio of thymopentin to hyaluronic acid is 1:0 to 1:8, and the molar ratio of thymopentin to glutaraldehyde is 1:1 to 1:3.

[0018] In some embodiments of the present invention, the molar ratio of thymopentin to hyaluronic acid is preferably 1:1, 1:4 or 1:8.

[0019] In some embodiments of the present invention, the molar ratio of thymopentin to glutaraldehyde is preferably 1:2.

[0020] In some embodiments of the present invention, the concentration of thymopentin in the mixed solution is 1 mg / mL to 4 mg / mL, preferably 2 mg / mL;

[0021] The concentration of the glutaraldehyde solution is 8%-50%, and the mass percentage is preferably 25%.

[0022] In some embodiments of the present invention, the molecular weight of the hyaluronic acid is 40-1000 kDa, preferably 780 kDa.

[0023] The pentapeptide-based supramolecular assembled particles provided by this invention possess biocompatibility, autofluorescence properties, and pH responsiveness, and can be used as drug delivery carriers. Therefore, a third aspect of this invention provides an application of the aforementioned pentapeptide-based supramolecular assembled particles as a delivery carrier.

[0024] In some embodiments of the present invention, the application as a delivery carrier includes, but is not limited to, any of the following:

[0025] (1) As a drug delivery carrier;

[0026] (2) As a tool for studying the delivery performance of guest molecules; the guest molecules include drugs or model drugs; wherein the model drugs are drugs used to achieve disease modeling effects;

[0027] (3) As a means of exploring the controlled release performance of drugs and a programmed drug release model;

[0028] (4) As a delivery carrier for anti-inflammatory drugs.

[0029] In a fourth aspect, the present invention provides a pharmaceutical formulation with inherent fluorescent properties, wherein the pharmaceutical formulation is provided by encapsulating the drug to be delivered using the aforementioned pentapeptide supramolecular assembled particles.

[0030] The drugs include both hydrophobic and hydrophilic drugs.

[0031] In some embodiments of the present invention, the method for preparing the pharmaceutical preparation includes the following steps:

[0032] The drug to be delivered is added to a mixed solution containing thymopentin and hyaluronic acid, followed by the addition of glutaraldehyde solution. The mixture is then allowed to stand to obtain the drug formulation.

[0033] A fifth aspect of the present invention provides the use of the aforementioned pentapeptide supramolecular assembled particles in the preparation of anti-inflammatory drugs.

[0034] In some embodiments of the present invention, the anti-inflammatory drug has the effect of reducing the concentration of inflammatory factors; preferably, the inflammatory factor is IL-6.

[0035] The beneficial effects of this invention are as follows:

[0036] This invention discloses a biocompatible and autofluorescent thymopentin-based carrier particle (TGCP) and a hyaluronic acid composite carrier particle (TGCP / HA). The carrier particles are sustained-release carriers containing the immunomodulatory short peptide thymopentin (TP5), the glycosaminoglycan compound hyaluronic acid (HA) with moisturizing, lubricating, and potential anti-inflammatory properties, and glutaraldehyde (GA), a commonly used cross-linking agent for compound cross-linking. This invention prepares TP5, HA, and GA into carrier particles TGCP and TGCP / nHA with drug-carrying and potential anti-inflammatory properties. These particles exhibit autofluorescence and are stable in neutral and alkaline environments, but decompose in acidic environments. They can also encapsulate small molecules with different solubility properties and exhibit programmed response performance under physiological and pathological pH conditions. In vitro experiments have shown that these carrier particles have good biocompatibility. Furthermore, in in vitro inflammatory cell induction experiments, both TGCP and TGCP / nHA demonstrate anti-inflammatory activity, inhibiting the expression of the inflammatory cytokine IL-6 and thus exerting an anti-inflammatory effect.

[0037] Compared to the complex preparation process and stringent storage and usage conditions of previous drug delivery carriers, the pentapeptide-based supramolecular assembled particles provided by this invention have a simple preparation process, relatively simple structural components, good biocompatibility, and are easy to store. Attached Figure Description

[0038] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0039] Figure 1 The properties of TGCP prepared in Example 1 of this invention are characterized; wherein, A is a scanning electron microscope (SEM) image of TGCP carrier particles dispersed in an aqueous system; B is a SEM image of TGCP particles after ultrasonication; C is a histogram and fitting curve of TGCP particle size distribution plotted based on statistical data measured by SEM; D is the particle size distribution of TGCP measured by DLS.

[0040] Figure 2 The images show SEM images of the TGCP / nHA carrier particles prepared in Example 1 of this invention; wherein, A is the SEM image of TGCP / 1HA prepared when the molar ratio of TP5 to HA is 1:1; B is the SEM image of TGCP / 4HA prepared when the molar ratio of TP5 to HA is 1:4; and C is the SEM image of TGCP / 8HA prepared when the molar ratio of TP5 to HA is 1:8.

[0041] Figure 3The images shown are fluorescence spectra and laser confocal imaging images of the carrier particles prepared in Examples 1 and 2 of this invention. A represents the fluorescence spectrum of TGCP carrier particles dispersed in water, and images acquired in the blue, green, and red channels at a fixed excitation wavelength; B represents the fluorescence spectrum of TGCP / 1HA carrier particles dispersed in water, and images acquired in the blue, green, and red channels at a fixed excitation wavelength; C represents the fluorescence spectrum of TGCP / 4HA carrier particles dispersed in water, and images acquired in the blue, green, and red channels at a fixed excitation wavelength; D represents the fluorescence spectrum of TGCP / 8HA carrier particles dispersed in water, and images acquired in the blue, green, and red channels at a fixed excitation wavelength. The scale line in the laser confocal imaging image is 25 μm.

[0042] Figure 4 The stability of TGCP prepared in Example 1 of this invention under different pH conditions is shown below; where A is the SEM image of TGCP in solutions with pH values ​​of 2.48, 3.76, 5.15, 7.44, 9.81 and 11.70 respectively; B is the SEM and TEM images of TGCP incubated in PBS buffer solution with pH value of 5.0 for 1, 5 and 10 days respectively; C is the SEM and TEM image of TGCP incubated in PBS buffer solution with pH value of 7.4 for 1, 5 and 10 days respectively.

[0043] Figure 5 The images show the SEM images and drug release curves of TGCP loaded with small molecule drugs prepared in Example 1 of this invention; wherein, A is the SEM image of TGCP@AHI after loading small molecule AHI; B is the SEM image of TGCP@PCA after loading small molecule PCA; C is the drug release curve of TGCP@AHI; and D is the drug release curve of TGCP@PCA.

[0044] Figure 6To evaluate the biocompatibility of the pentapeptide-based carrier particles prepared in Examples 1 and 2 of this invention, the in vitro cell viability (n=3) of NIH-3T3 and RAW264.7 cells was determined using the CCK-8 assay under different culture conditions; where A represents the viability of NIH-3T3 cells with different concentrations of TGCP; B represents the viability of NIH-3T3 cells with different concentrations of TGCP / 4HA; C represents the viability of NIH-3T3 cells with different concentrations of TGCP / 8HA; and D represents the viability of NIH-3T3 cells with different concentrations of TGCP / 8HA. Viability of TGCP@PCA against RAW264.7 cells; E represents the viability of different concentrations of TGCP / 4HA@PCA against RAW264.7 cells; F represents the viability of different concentrations of TGCP / 8HA@PCA against RAW264.7 cells; G represents the viability of different concentrations of TGCP@AHI against RAW264.7 cells; H represents the viability of different concentrations of TGCP / 4HA@AHI against RAW264.7 cells; I represents the viability of different concentrations of TGCP / 8HA@AHI against RAW264.7 cells.

[0045] Figure 7 The concentration of IL-6 (n=3) after culturing RAW264.7 cells with the TGCP particles prepared in Example 1 of the present invention for 24 hours;

[0046] Figure 8 The graphs show the anti-inflammatory performance evaluation of the pentapeptide-based carriers prepared in Examples 1 and 2 of this invention; where A represents the IL-6 concentration (n=3) of RAW264.7 cells after 24 hours of culture with TGCP@AHI; B represents the IL-6 concentration (n=3) of RAW264.7 cells after 24 hours of culture with TGCP / 4HA@AHI; C represents the IL-6 concentration (n=3) of RAW264.7 cells after 24 hours of culture with TGCP / 8HA@AHI; D represents the IL-6 concentration (n=3) of RAW264.7 cells after 24 hours of culture with TGCP@PCA; E represents the IL-6 concentration (n=3) of RAW264.7 cells after 24 hours of culture with TGCP / 4HA@PCA; and F represents the IL-6 concentration (n=3) of RAW264.7 cells after 24 hours of culture with TGCP / 8HA@PCA. Detailed Implementation

[0047] This invention provides a pentapeptide-based supramolecular assembled particle and its application as a drug delivery carrier. Compared with the complex preparation process and stringent storage and usage conditions of previous drug delivery carriers, the pentapeptide-based supramolecular assembled particle of this invention has a simple preparation process, relatively simple structural components, and good biocompatibility.

[0048] A first typical embodiment of the present invention provides a pentapeptide-based supramolecular assembled particle, which includes an immunologically active short biopeptide and a cross-linking agent;

[0049] The short biopeptide and cross-linking agent undergo a Schiff base reaction in the reaction system and obtain pentapeptide-based supramolecular assembled particles through supramolecular self-assembly.

[0050] In some embodiments of this implementation, the short biopeptide includes thymopentin (TP5), and the cross-linking agent is a small molecule aldehyde. TP5 (Arg-Lys-Asp-Val-Tyr, RKDVY) is a polypeptide hormone secreted by the thymus gland, composed of five amino acids, and is an immunomodulatory pentapeptide with good biocompatibility and biodegradability.

[0051] In some embodiments of this implementation, the reaction system is water or water containing an organic solvent.

[0052] The organic solvents include, but are not limited to, methanol, ethanol, or dimethyl sulfoxide. The addition of the organic solvents can regulate the particle assembly process.

[0053] The reaction system is water, preferably ultrapure water.

[0054] In some embodiments of this implementation, the pentapeptide-based supramolecular assembly particles are spherical.

[0055] In some embodiments of this implementation, the pentapeptide-based supramolecular assembled particles further include glycosaminoglycan compounds.

[0056] The bio-short peptide is thymopentin, the glycosaminoglycan compound is hyaluronic acid (HA), and the cross-linking agent is glutaraldehyde (GA); the molar ratio of thymopentin to hyaluronic acid is 1:0 to 1:8, and the molar ratio of thymopentin to glutaraldehyde is 1:1 to 1:3.

[0057] This invention provides two types of pentapeptide-based supramolecular assembled particles. One type is a pentapeptide-based supramolecular assembled particle (TGCP) obtained by Schiff base reaction and supramolecular self-assembly of the immunologically active short peptide TP5 and a common chemical cross-linking agent in a reaction system. The other type is a pentapeptide-based supramolecular assembled particle (TGCP / HA) obtained by Schiff base reaction and supramolecular self-assembly of the immunologically active short peptide TP5, hyaluronic acid (HA), a glycosaminoglycan compound with moisturizing, lubricating, and potential anti-inflammatory properties, and a common chemical cross-linking agent in a reaction system. Both types of pentapeptide-based supramolecular assembled particles are sustained-release drug delivery carrier particles.

[0058] This invention prepares the above three substances into drug-loading and potential anti-inflammatory carrier particles TGCP and TGCP / HA. These particles exhibit autofluorescence, are stable in neutral and alkaline environments but decompose in acidic environments, and can encapsulate small molecules with varying solubility. They also demonstrate programmed response under physiological and pathological pH conditions. In vitro experiments revealed good biocompatibility of these carrier particles. Furthermore, in in vitro inflammatory cell induction experiments, both TGCP and TGCP / HA exhibit anti-inflammatory activity, inhibiting the expression of the inflammatory cytokine IL-6 and thus exerting an anti-inflammatory effect.

[0059] A second typical embodiment of the present invention provides a method for preparing the pentapeptide-based supramolecular assembled particles, comprising the following steps:

[0060] A cross-linking agent solution was added to a mixed solution containing short biopeptides and glycosaminoglycans, and the mixture was allowed to stand to obtain pentapeptide-based supramolecular assembled particles.

[0061] The short biopeptide is thymopentin, the glycosaminoglycan compound is hyaluronic acid, and the crosslinking agent is glutaraldehyde; the molar ratio of thymopentin to hyaluronic acid is 1:0 to 1:8, and the molar ratio of thymopentin to glutaraldehyde is 1:1 to 1:3.

[0062] This invention provides a method for preparing the aforementioned pentapeptide-based supramolecular assembled particles. By adding glycosaminoglycan compounds such as hyaluronic acid (HA) to adjust the size and dispersibility of TGCP, TGCP / HA exhibits excellent injectability and synergistic anti-inflammatory activity.

[0063] In some embodiments of this implementation, the molar ratio of thymopentin to hyaluronic acid is preferably 1:1, 1:4, or 1:8.

[0064] In some embodiments of this implementation, the molar ratio of thymopentin to glutaraldehyde is preferably 1:2.

[0065] In some embodiments of this implementation, the concentration of thymopentin in the mixed solution is 1 mg / mL-4 mg / mL, preferably 2 mg / mL;

[0066] The concentration of the glutaraldehyde solution is 8%-50%, and the mass percentage is preferably 25%.

[0067] In some embodiments of this implementation, the molecular weight of the hyaluronic acid is 40-1000 kDa, preferably 780 kDa.

[0068] In some embodiments of this implementation, the reaction environment is static, including static in a light-proof environment, constant temperature preparation at a fixed speed in a shaker, and magnetic stirring preparation, with constant temperature preparation at a fixed speed in a shaker being preferred.

[0069] In some embodiments of this implementation, for the preparation of TGCP, a certain proportion of GA aqueous solution is added to TP5 aqueous solution, mixed evenly, and allowed to stand to obtain a turbid suspension with yellow precipitate. The precipitate is washed by centrifugation with ultrapure water and stored in Milli-Q water at room temperature to obtain TGCP.

[0070] In some embodiments of this implementation, for the preparation of TGCP / HA, TP5 and HA are dissolved in a certain molar ratio, and then a certain proportion of GA aqueous solution is added. After mixing evenly and standing at room temperature in the dark, a turbid suspension colloid is obtained with a yellow precipitate (a yellow precipitate appears when the molar ratio of HA to TP5 is small). The suspension colloid is dialyzed at room temperature in a dialysis membrane with a fixed molecular weight cutoff, and then freeze-dried to obtain TGCP / HA.

[0071] The fixed molecular weight cutoff of the dialysis membrane is 100-7000 Da, preferably 3500 Da.

[0072] The pentapeptide-based supramolecular assembled particles provided by this invention possess biocompatibility, autofluorescence properties, and pH responsiveness, and can be used as drug delivery carriers. Therefore, a third typical embodiment of this invention provides an application of the aforementioned pentapeptide-based supramolecular assembled particles as a delivery carrier.

[0073] In some embodiments of this implementation, the application as a delivery carrier includes, but is not limited to, any of the following:

[0074] (1) As a drug delivery carrier;

[0075] (2) As a tool for studying the delivery performance of guest molecules; the guest molecules include drugs or model drugs; wherein the model drugs are drugs used to achieve disease modeling effects;

[0076] (3) As a means of exploring the controlled release performance of drugs and a programmed drug release model;

[0077] (4) As a delivery carrier for anti-inflammatory drugs.

[0078] A fourth typical embodiment of the present invention provides a pharmaceutical formulation with inherent fluorescent properties, wherein the pharmaceutical formulation is used to encapsulate the drug to be delivered using the aforementioned pentapeptide supramolecular assembled particles.

[0079] The drugs include both hydrophobic and hydrophilic drugs.

[0080] In some embodiments of this implementation, the method for preparing the pharmaceutical preparation includes the following steps:

[0081] The drug to be delivered is added to a mixed solution containing thymopentin and hyaluronic acid, followed by the addition of glutaraldehyde solution. The mixture is then allowed to stand to obtain the drug formulation.

[0082] In a fifth typical embodiment of the present invention, the application of the pentapeptide-based supramolecular assembled particles in the preparation of anti-inflammatory drugs is provided.

[0083] In some embodiments of this implementation, the anti-inflammatory drug has the effect of reducing the concentration of inflammatory factors; preferably, the inflammatory factor is IL-6.

[0084] This invention also provides a method for loading model dyes and small drug molecules onto pentapeptide supramolecular assembled particles. A variety of hydrophilic and hydrophobic model dye molecules are selected, and the loading performance of TGCP or TGCP / HA is explored by co-precipitation method.

[0085] The loading method includes the following steps: preparing an aqueous solution of TP5 and a model dye or model drug small molecule, or preparing an aqueous solution of TP5, HA and a model dye or model drug small molecule; adding GA solution, allowing it to stand, and forming a colored precipitate. The molecular weight of the dye or drug loaded with TGCP or TGCP / HA is determined using UV-Vis absorption spectroscopy, high-performance liquid chromatography, or other methods, combined with a standard curve.

[0086] The model dye can be cationic, anionic, water-soluble, or water-insoluble, and is preferably a small molecule water-insoluble dye.

[0087] The model drug molecules can be water-soluble or lipid-soluble, preferably lipid-soluble.

[0088] The method for measuring the molecular weight of the dye or the molecular weight of the drug can be ultraviolet-visible absorption spectroscopy, fluorescence spectroscopy, high performance liquid chromatography, etc., preferably ultraviolet-visible absorption spectroscopy and high performance liquid chromatography.

[0089] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0090] Unless otherwise specified, the reagents used in the following examples are all commercially available products that can be purchased.

[0091] Example 1 Preparation of TGCP

[0092] For TGCP: Dissolve 1 mg TP5 in 500 μL H2O. Add a certain proportion of 25 wt% GA aqueous solution to the TP5 solution, with TP5 / GA molar ratios of 1:1, 1:2, and 1:3. Vortex the mixture for 15 seconds and let it stand at room temperature in the dark for one day. A turbid suspension with a yellow precipitate appears at the bottom of the centrifuge tube. Centrifuge the precipitate and wash it three times with ultrapure water, then place it in Milli-Q pure water at room temperature to obtain the TGCP carrier particles.

[0093] When the TP5 / GA molar ratio is 1:2, the resulting TGCP carrier particles are characterized by scanning electron microscopy, such as... Figure 1 As shown in Figure A, the precipitate consists of uniform spherical particles with a smooth and flat surface. After ultrasonic treatment, it was observed that the interior is porous and the edges are relatively loose. Figure 1 B). This indicates that TGCPs can provide a considerable amount of space for the loading of guest molecules. Statistical analysis shows that the average diameter of TGCPs is approximately 979.9 nm ( Figure 1 C). The size measured by DLS is approximately 1046 nm. Figure 1 D), In addition, the zeta potential of the TGCP carrier particles was measured to be -35.35±2.92mV.

[0094] Example 2 Preparation of TGCP / HA

[0095] For TGCP / nHA: Dissolve 1 mg TP5 in 500 μL H2O, then add HA powder. Determine the mass of HA based on the molar ratio of TP5 to HA repeating units (1:0.1 to 1:8). Add a 25 wt% GA aqueous solution with a TP5 to GA molar ratio of 1:2, and vortex for 60 seconds. After standing at room temperature in the dark for 1 day, a yellow precipitate will form when the HA content is low; when the HA content is high, the solution viscosity will be high, resulting in a yellow suspension. To remove residual GA, the sample was dialyzed at room temperature using a dialysis bag with a molecular weight cutoff of 3500 Da for 3 days, with the water changed every 6 hours. The purified sample was freeze-dried to obtain the solid product TGCP / nHA, which was then stored at -80°C until further use.

[0096] Among them, TGCP / nHA with TP5 / HA molar ratios of 1:1, 1:4, and 1:8 were characterized by scanning electron microscopy, such as... Figure 2 As shown, uniform spherical particles were also obtained. Figure 2It can be seen that as the HA content increases, the particle size of the TGCP / nHA carrier particles gradually increases. When the TP5 / HA molar ratio is 1:1, the particle size of the obtained TGCP / 1HA carrier particles is around 284 nm; when the TP5 / HA molar ratio is 1:4, the particle size of the obtained TGCP / 4HA carrier particles is around 374 nm; and when the TP5 / HA molar ratio is 1:8, the particle size of the obtained TGCP / 8HA carrier particles is around 445 nm. Therefore, the particle size of the pentapeptide supramolecular assembly particles can be adjusted by controlling the amount of HA.

[0097] Note: To distinguish TGCP / HA carrier particles obtained with different TP5 / HA molar ratios, the carrier particles with a TP5 / HA molar ratio of 1:1 are named TGCP / 1HA, the carrier particles with a TP5 / HA molar ratio of 1:4 are named TGCP / 4HA, and the carrier particles with a TP5 / HA molar ratio of 1:8 are named TGCP / 8HA.

[0098] The following tests were conducted using the TGCP carrier particles obtained in Example 1 with a TP5 / GA molar ratio of 1:2 and the TGCP / 1HA, TGCP / 4HA, and TGCP / 8HA prepared in Example 2.

[0099] Example 3 Autofluorescence performance testing of TGCP and TGCP / HA

[0100] The carrier particles TGCP and TGCP / nHA in this embodiment have certain autofluorescence properties. The fluorescence spectra and CLSM images of the samples were obtained by testing with a fluorescence spectrophotometer and a confocal laser scanning microscope (CLSM), respectively.

[0101] The autofluorescence properties of TGCP and TGCP / HA were studied using fluorescence spectroscopy and CLSM. The detection results are as follows: Figure 3 As shown. For TGCP ( Figure 3 A) Five broad emission peaks were observed at wavelengths of 448, 469, 520, 607, and 658 nm, indicating that the particles exhibit multi-wavelength fluorescence emission in blue, green, and red. This phenomenon is due to the n-π* transition of the Schiff base bond, indicating that the support particles possess autofluorescence functionality. Multi-wavelength fluorescence emission was also observed after non-covalent binding with HA, but the emission wavelengths were slightly different. Figure 3 B. Figure 3 C Figure 3 D). Autofluorescence can be further confirmed in CLSM images. When the excitation wavelength is 405 nm, the multi-wavelength autofluorescence characteristics of TGCP and TGCP / HA in the blue (430-480 nm), green (500-570 nm), and red (590-690 nm) bands are as follows: Figure 3As shown, the addition of HA did not affect the autofluorescence properties of TGCP. Furthermore, the particle size decreased after adding HA, consistent with the results of the scanning electron microscopy images. Compared to carrier particles requiring fluorescent labeling, this invention avoids interference from external chemical reagents, making the carrier particles of this invention more suitable as potential fluorescent carriers.

[0102] Example 4 Acid-base responsiveness of TGCP

[0103] This embodiment takes into account that the pH value of the inflamed site is usually lower than that of normal tissue, so constructing a pH-responsive carrier will be more attractive.

[0104] First, 100 μL of a 2 mg / mL TGCP suspension was added to aqueous solutions with different pH values ​​(0.48, 2.48, 3.76, 5.15, 7.44, 9.81, 11.70, and 13.24, adjusted by 0.1 M NaOH and HCl) and incubated for at least 15 days. Alternatively, 100 μL of the 2 mg / mL TGCP suspension was placed in buffer solutions with a pH of 5.0 or 7.4 and incubated for at least 10 days. The samples were characterized at fixed time points.

[0105] The prepared TGCP was added to acid and alkaline solutions of different pH values ​​prepared with HCl and NaOH and mixed thoroughly. The morphological changes of TGCP with varying pH values ​​were observed using scanning electron microscopy. Figure 4 A) When the pH of the system reached 2.48 and 3.76, the structure of TGCP was significantly disrupted, forming incomplete and broken spherical structures. At pH 5.15, partial structural damage and particle adhesion were observed. When the pH increased from 7.44 to 11.70, no significant structural changes were observed. This indicates that the TGCP prepared in this invention is more stable in neutral and alkaline environments, while it easily decomposes in acidic systems, exhibiting a certain degree of acid responsiveness.

[0106] Example 5 Stability of TGCP in buffer solutions

[0107] To further investigate the stability of TGCP in neutral and acidic environments, this example uses SEM and TEM to characterize the microstructural changes of TGCP dispersed in PBS buffer solutions at different pH values ​​after 1, 5, and 10 days of incubation.

[0108] After dispersing TGCP in a PBS buffer solution with a pH of 5.0 ( Figure 4(B) Some particles are destroyed within a day, leading to adhesion and accumulation. Simultaneously, smooth, perfectly spherical TGCPs can be observed in the TEM field of view. After five days of cultivation, almost all TGCPs have formed an aggregated state, making it difficult to distinguish individual particles. The external structure of the particles observed in the corresponding TEM field appears to be damaged, and the outermost layer is loosened. By the tenth day of cultivation, the shape of the TGCPs becomes increasingly difficult to discern; most TGCPs are amorphous stacks or fragments composed of assembly units. This characteristic of decomposition in an acidic environment indicates that the TGCP carrier particles prepared in this invention can be used as a drug container, capable of releasing loaded drug molecules under pathological pH conditions.

[0109] No significant changes were observed in the morphology of TGCP after one day of incubation in neutral PBS (pH 7.4) buffer solution. Figure 4 C). TEM images also showed a smooth, intact spherical morphology. Over time, slight dissociation occurred on the particle surface, and the stacking of TGCPs on the surface became loose. After 10 days of incubation in neutral PBS buffer, the structure gradually loosened due to the insufficiently tight stacking of individual TGCP units. The results indicate that a small amount of H... + This can trigger the structural destruction of TGCP. Therefore, the prepared carrier has the potential to release drugs sustainably under physiological pH conditions.

[0110] Example 6 Encapsulation properties of TGCP for small molecule dyes

[0111] Loading of model dye molecules: Thioflavin T (ThT), Rhodamine B (RhB), Toluidine Blue (TB), and Nile Red (NR) were selected as hydrophilic and hydrophobic dye molecule models, and the loading performance of TGCP was investigated by co-precipitation method. 500 μL aliquots containing 1 mg TP5 were prepared. Alcoholic solutions of NR (0.5 mg / mL, 60 μL), RhB (0.5 mg / mL, 60 μL), TB (0.85 mg / mL, 50 μL), or ThT (5 mg / mL, 50 μL) were added to the TP5 solution, respectively. Then, 25 wt% GA solution was added to each mixture, and the mixture was vortexed for 30 seconds. After standing for 24 hours, the supernatant was centrifuged at 8000 rpm for 30 minutes to separate the precipitate and dissolve it in DMSO (1 mL). The amounts of guest molecules ThT, TB, NR and RhB encapsulated in TGCP were determined using UV-Vis absorption spectroscopy (Agilent Cary 100) combined with the calibration absorption curve.

[0112] The formulas for calculating drug loading (DL) and encapsulation efficiency (EE) are as follows:

[0113] DL% = (W tm -Wum / W tt )×100%

[0114] EE% = (W tm -W um / W tm )×100%

[0115] Among them, W tm It is the total weight of the guest molecules; W um It is the weight of the unbound guest molecules; W tt It is the total weight of the bound guest molecule and TGCP. W um The value is obtained by measuring the supernatant.

[0116] Encapsulating small molecules into carrier particles reflects the material's properties and expands its potential applications. This embodiment utilizes small molecule dyes including the amphoteric cationic dye RhB, the water-soluble cationic dyes ThT and TB, and the water-insoluble dye NR. Under the same conditions, the encapsulation efficiency (EE) and loading capacity (LC) of TGCP for RhB, ThT, TB, and NR were evaluated using standard curves. The EEs for RhB, ThT, TB, and NR were 92.3%, 81.6%, 71.2%, and 99.6%, respectively. The LCs for RhB, ThT, TB, and NR were 5.27%, 4.08%, 3.44%, and 6.16%, respectively.

[0117] In addition, hydrophobic small drug molecules (curcumin and nifedipine) were encapsulated using TGCP. A 500 μL aliquot containing 1 mg TP5 was prepared. An alcoholic solution of curcumin (1 mg / mL, 50 μL) or an alcoholic solution of nifedipine (4 mg / mL, 150 μL) was added to the TP5 solution, and then 25 wt% GA solution was added to each mixture and vortexed for 30 seconds. After standing for 24 h, the supernatant was centrifuged at 8000 rpm for 30 min. The precipitate was separated and dissolved in DMSO (1 mL). The amount of hydrophobic small drug molecules (curcumin and nifedipine) encapsulated in the TGCP was determined using UV-Vis absorption spectroscopy (Agilent Cary 100) combined with a calibrated absorption curve.

[0118] TGCP showed promising results for encapsulating hydrophobic small drug molecules (curcumin and nifedipine), with EE values ​​of 90.8% and 89.0%, respectively. The LC values ​​for curcumin and nifedipine were 5.75% and 4.45%, respectively. This demonstrates that TGCP can effectively encapsulate both hydrophilic and hydrophobic guest molecules.

[0119] Example 7 Loading of model drug molecules with TGCP

[0120] Traditional Chinese herbal medicines contain bioactive components that have significant effects in reducing inflammation and promoting cartilage regeneration. Protocatechuic acid (PCA) and icariin (AHI), as two hydrophilic and hydrophobic model drugs, have been shown to have significant anti-inflammatory activity. This example prepared TGCP@drugs encapsulating these two drugs. First, 1 mg of TP5 was accurately weighed and dissolved in 500 μL of H2O. Then, 5 μL of AHI dimethyl sulfoxide solution (300 μM) or 50 μL of PCA aqueous solution (3 mM) was added. After mixing for 60 seconds, 25 wt% GA aqueous solution was added at a TP5 / GA molar ratio of 1:2. The mixture was vortexed for 15 seconds and incubated at room temperature in the dark for one day. A turbid suspension and a pale yellow precipitate appeared at the bottom of the centrifuge tube. The samples were TGCP@AHI or TGCP@PCA, respectively.

[0121] To prepare TGCP / nHA@AHI and TGCP / nHA@PCA (n = 4 or 8), TP5 and HA powders were dissolved in equal volumes of H2O at molar ratios of 1:4 and 1:8, respectively. 5 μL of AHI alcohol solution (300 μM) or 50 μL of PCA aqueous solution (3 mM) was added and mixed for 120 seconds. A 25 wt% GA aqueous solution was added at a TP5 / GA molar ratio of 1:2. After standing at room temperature in the dark for 1 day, a turbid suspension was obtained. The sample was freeze-dried to obtain a solid product, which was stored at -80°C until further use.

[0122] To determine drug concentration and packaging details, high-performance liquid chromatography (HPLC, Agilent 1220 Infinity II) was used to analyze loading efficiency. HPLC was performed using a ZORBAX SB-C18 column (4.6 × 250 mm) at a flow rate of 0.5 mL / min. -1 For AHI, phase A was a 0.1% aqueous phosphoric acid solution, and phase B was methanol. The gradient of phase B was 80%–100%, and elution was performed for 20 min. The eluent was monitored at a wavelength of 270 nm using a UV detector. For PCA, the mobile phase consisted of solvent A being a 0.1% aqueous phosphoric acid solution and solvent B being methanol. The gradient of phase B was 10%–50%, and elution was performed for 30 min. The elution process was monitored at a wavelength of 260 nm using a UV detector.

[0123] The TGCP prepared in this invention has a certain carrying capacity for both hydrophilic and hydrophobic drug molecules. Figure 5 A, Figure 5B represents SEM images of TGCP@AHI and TGCP@PCA, respectively. The encapsulation amount and efficiency of PCA and AHI were determined using HPLC. The encapsulation efficiency (EE) and LC of PCA were 39.7% and 9.2%, respectively. The EE and LC of AHI were 61.9% and 3.4%, respectively. TGCP showed a higher encapsulation efficiency for the water-insoluble small molecule AHI than for the water-soluble PCA.

[0124] Example 8 Drug release of TGCP under different pH conditions

[0125] The pH-responsive release of TGCP (i.e., TGCP@AHI and TGCP@PCA) coated with drug molecules (PCA, AHI) prepared in Example 7 was investigated in PBS buffer solutions with pH values ​​of 7.4 and 5.0. After centrifugation of the prepared samples (8000 rpm / min, 15 min), drug molecules not coated with TGCP remained in the supernatant. 0.5 mL of the supernatant was taken for HPLC detection (using the same HPLC detection method as in Example 7), and then an equal volume of fresh PBS buffer solution was added. Drug molecules were continuously released for 30 days in a constant temperature shaker at 37.0 °C and 100 rpm. The release of drug molecules was quantitatively analyzed by high performance liquid chromatography according to the standard curve, using the same HPLC detection method as in Example 7.

[0126] Compared to neutral conditions, the release rate of lipid-soluble AHI was significantly higher in acidic environments, reaching release equilibrium in PBS at pH 5.0 in approximately 11 days. Figure 5 C). However, under physiological pH conditions, the release continued for approximately 22 days. Hydrophilic small molecule PCA, on the other hand, showed a faster release rate in both acidic and neutral environments. Figure 5 (D) The slow release of hydrophobic drugs from TGCPs may be due to the presence of hydrophobic spaces within the particles. These drugs are adsorbed both inside and on the outer surface of the particles. Therefore, the release of the drug is slow after the carrier particles loosen. In contrast, TGCPs have a weaker loading capacity for hydrophilic drugs than hydrophobic drugs, and thus their release rate is relatively faster.

[0127] Example 9 In vitro biocompatibility of TGCP

[0128] In vitro cytotoxicity assays are commonly used to assess the biocompatibility of biological materials because they are short-cycle, highly sensitive, and allow for large-scale sample screening. Mouse embryonic fibroblasts (NIH-3T3) and mouse mononuclear macrophages (RAW264.7) were cultured in complete medium and passaged every 1.5 days at a fixed passage ratio. NIH-3T3 and RAW264.7 cells were seeded in 96-well plates. Compounds AHI, PCA, TGCP, TGCP / 4HA, TGCP / 8HA, TGCP@PCA, TGCP / 4HA@PCA, TGCP / 8HA@PCA, TGCP@AHI, TGCP / 4HA@AHI, and TGCP / 8HA@AHI prepared in Example 7 were diluted to appropriate concentration gradients with medium. A fixed amount of drug-containing medium was added to each well. Cells were incubated at 37.0°C for 24 and 48 hours. After incubation, CCK-8 solution was added, and the 96-well plates were incubated in a cell culture incubator in the dark for a certain period to allow for color development. The absorbance values ​​at a fixed wavelength were read using a microplate reader, and the data were processed using GraphPad Pism software. Each experiment was repeated at least three times.

[0129] The results of the in vitro cytotoxicity assay for TGCP are as follows: Figure 6 As shown in A, even at a co-culture concentration of approximately 5 mg·mL⁻¹ -1 No significant cytotoxicity was observed under these conditions. These results indicate that TGCP has high biocompatibility and is suitable for biomedical applications. Under the same experimental conditions, the effects of TGCP / 4HA and TGCP / 8HA on NIH-3T3 cell viability were investigated at concentrations up to 5 mg / mL. -1 No significant cytotoxicity was observed at that time. Figure 6 (BC). The results showed that the prepared particles had good biocompatibility with normal cells.

[0130] To explore the anti-inflammatory effects of drug-loaded particles, RAW264.7 cells were used in this experiment. To prevent the survival rate of drug-loaded particles on RAW264.7 macrophages from affecting the anti-inflammatory results, this example tested the cell viability of the drug-loaded particles after encapsulation. Figure 6 Taking D as an example, a blank control group and a free drug control group were set up. The concentration of PCA was 0.3 mM, and the concentration of AHI was 3 μM. After culturing cells in a medium containing a certain amount of TGCP@PCA drug-loaded particles for 24 and 48 hours, cell viability was assessed using the CCK-8 assay. At a concentration of 500 μg / mL... -1At that time, the survival rate of RAW264.7 cells exceeded 90%, indicating no significant cytotoxicity. Under the same experimental conditions, TGCP@PCA, TGCP / 4HA@PCA, TGCP / 8HA@PCA, TGCP@AHI, TGCP / 4HA@AHI, and TGCP / 8HA@AHI were also tested. Figure 6 D、 Figure 6 E, Figure 6 F, Figure 6 G, Figure 6 H, Figure 6 As shown in Figure I, the prepared particles did not exhibit significant toxicity to RAW264.7 cells. This indicates that the particles have good biocompatibility and confirms that the drug-loaded particles do not affect cell viability.

[0131] Example 10 In vitro anti-inflammatory activity of TGCP

[0132] The in vitro anti-inflammatory activity of TGCP was tested using an enzyme-linked immunosorbent assay (ELISA). RAW264.7 cells were seeded in 24-well plates and cultured overnight to allow cell adhesion. Cells were then treated with lipopolysaccharide (LPS) to activate macrophage polarization. After incubation for a period of time, DMEM medium containing different formulations was added according to the grouping criteria (untreated control group, LPS-induced positive control group, AHI, TGCP / nHA@AHI, TGCP / nHA@AHI). To measure the extracellular levels of pro-inflammatory factors, the supernatant was collected, and IL-6 levels were measured using an appropriate ELISA kit according to the reagent company's protocol. Normal macrophages and untreated LPS-activated macrophages served as the NT group and control group, respectively.

[0133] The experiment used LPS-induced macrophages to detect the levels of inflammatory factors in the cell supernatant. The NT group consisted of macrophages not induced by LPS, while the control group consisted of macrophages induced by LPS (5 μg / mL). -1 The positive control group was TGCP@AHI(l) and TGCP@AHI(h) after LPS induction. -1 ) and high concentration (250 μg·mL) -1 The treatment group. TGCP alone showed some anti-inflammatory activity at high concentrations. Figure 7 This may be related to the properties of TP5. Since TP5 was originally used as a short, immunologically active peptide for the treatment of immunodeficiency, its half-life was prolonged after being converted into TGCP in this embodiment. This may result in the release of a certain amount of TP5 during administration as the TGCP degrades, exhibiting a specific anti-inflammatory effect.

[0134] Experimental results showed that even at low concentrations, TGCP@AHI was significantly different from the control group. Figure 8 A). At high concentrations (250 μg·mL⁻¹) -1 The differences were even more pronounced under these conditions. This indicates that TGCP@AHI can inhibit the production of inflammatory factors or reduce their concentrations. PCA, which also has anti-inflammatory potential, yielded similar results. Figure 8 (D) However, at the same concentration, TGCP@AHI was less effective than TGCP@PCA in inhibiting the inflammatory factor IL-6. This may be because free PCA is hydrophilic, which promotes the release of the TGCP carrier, thus releasing more drug within 24 hours. Furthermore, after loading the drug into TGCP / 4HA and TGCP / 8HA, inflammatory factors in the cell supernatant were measured. The results still showed some anti-inflammatory effects. The anti-inflammatory effect was enhanced at higher concentrations of TGCP / 4HA@drug. The results for TGCP / 8HA@drug also showed a specific concentration dependence. These results indicate that TGCP, TGCP / 4HA, and TGCP / 8HA can maintain their original inhibitory effect on IL-6 after encapsulating small drug molecules. As a supplement, they can reduce the concentration of inflammatory factors and are potential sustained-release anti-inflammatory carriers for the treatment of inflammation-related diseases.

[0135] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A pentapeptide-based supramolecular assembled particle, characterized in that, It includes short bioactive peptides and cross-linking agents; The bio-short peptide and cross-linking agent undergo a Schiff base reaction in the reaction system and obtain pentapeptide-based supramolecular assembled particles through supramolecular self-assembly. The short biopeptide is thymopentin, and the cross-linking agent is glutaraldehyde; The reaction system is water or water containing an organic solvent; The pentapeptide-based supramolecular assembled particles are spherical; The pentapeptide-based supramolecular assembly particles also include hyaluronic acid, with a molar ratio of thymopentin to hyaluronic acid of 1:0 to 1:8 and a molar ratio of thymopentin to glutaraldehyde of 1:1 to 1:

3.

2. A method for preparing the pentapeptide-based supramolecular assembled particles according to claim 1, characterized in that, Includes the following steps: Glutaraldehyde solution was added to a mixed solution containing thymopentin and hyaluronic acid, and the mixture was allowed to stand to obtain pentapeptide-based supramolecular assembled particles. The molar ratio of thymopentin to hyaluronic acid is 1:0 to 1:8, and the molar ratio of thymopentin to glutaraldehyde is 1:1 to 1:

3.

3. The method for preparing pentapeptide-based supramolecular assembled particles as described in claim 2, characterized in that, The concentration of thymopentin in the mixed solution is 1 mg / mL to 4 mg / mL; The concentration of the glutaraldehyde solution is 8%-50% by mass.

4. The method for preparing pentapeptide-based supramolecular assembled particles as described in claim 2, characterized in that, The molecular weight of the hyaluronic acid is 40-1000 kDa.

5. The use of the pentapeptide-based supramolecular assembly particles of claim 1 in the preparation of a delivery carrier.

6. The application as described in claim 5, characterized in that, The application includes any of the following: (1) Preparation of drug delivery carriers; (2) Prepare a tool for studying the delivery performance of guest molecules; the guest molecules include drugs or model drugs; wherein the model drug is a drug used to achieve the effect of disease modeling; (3) To study the controlled release performance of drugs and to develop a programmed drug release model; (4) Preparation of anti-inflammatory drug delivery carriers.

7. A pharmaceutical formulation with inherent fluorescent properties, characterized in that, The pharmaceutical formulation is encapsulated with the pentapeptide-based supramolecular assembled particles as described in claim 1; The drugs include both hydrophobic and hydrophilic drugs; The preparation method of the pharmaceutical formulation includes the following steps: The drug to be delivered is added to a mixed solution containing thymopentin and hyaluronic acid, followed by the addition of glutaraldehyde solution. The mixture is then allowed to stand to obtain the drug formulation.

8. The use of the pentapeptide-based supramolecular assembled particles of claim 1 in the preparation of anti-inflammatory drugs; The anti-inflammatory drug has the effect of reducing IL-6 concentration.

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