Exosomic aloe-emodin hydrogel carrying palmatine hydrochloride based on deconstruction and reconstruction of ancient prescription and preparation method and application thereof

Abstract

The application discloses a kind of based on ancient prescription deconstruction and reconstruction of carrying salt hydrochloride palmitine-Haizhou drynaria exosome rheum acid hydrogel and its preparation method and application, belong to biological medicine and nanometer preparation technical field.The preparation method of the hydrogel includes the following steps: after mixing Haizhou drynaria exosome with salt hydrochloride palmitine, drug loading is carried out using ultrasonic method to obtain EVs-Pal suspension, rheum acid is added to NaHCO3 buffer solution of pH 8-9 to prepare precursor solution, precursor solution and EVs-Pal suspension are fully mixed after natural cooling.The application has the advantages that: it solves the technical problems of poor targeting of existing osteoarthritis treatment drugs, single treatment level, and short in vivo retention time of osteoarthritis joint cavity drug administration, repeated puncture clinical pain points, and also solves the problems of traditional Chinese medicine compound dosage form, such as extensive, mechanism is fuzzy, cannot accurately control immune metabolic disorder.

Description

Technical Field

[0001] This invention relates to a hydrogel, its preparation method, and its application. Specifically, it relates to a hydrogel loaded with palmatine hydrochloride-Drynaria fortunei exosomes and rhein based on the deconstruction and reconstruction of an ancient formula, its preparation method, and its application in the immune metabolic remodeling of osteoarthritis. This invention belongs to the field of biomedicine and nanoparticle technology. Background Technology

[0002] Osteoarthritis (OA) is a common chronic degenerative joint disease and a core contributing factor to limb disability in middle-aged and elderly individuals. It is characterized by its protracted and difficult-to-treat nature, high disability rate, and severe impact on patients' quality of life, while also increasing the social burden on healthcare. The pathological mechanism of osteoarthritis is complex, not simply a matter of mechanical wear and tear on the joints. Instead, it is characterized by progressive degradation of articular cartilage, chronic and persistent inflammation of the synovium, abnormal subchondral bone remodeling, and abnormal osteophyte formation, accompanied by an immune-metabolic disorder involving multiple cells, factors, and signaling pathways. Modern research has confirmed that in the lesion microenvironment of osteoarthritis (OA), chondrocytes and synovial cells can secrete excessive amounts of reactive oxygen species (ROS) and pro-inflammatory factors such as IL-1β, TNF-α, and IL-6, which continuously induce chondrocyte apoptosis, inhibit chondrocyte proliferation, and accelerate the degradation of extracellular matrix such as collagen and proteoglycans. At the same time, it can mediate the abnormal polarization of joint macrophages towards the pro-inflammatory M1 phenotype, further amplifying the local inflammatory cascade reaction and forming a vicious pathological cycle of "oxidative stress damage - inflammation aggravation - cartilage degeneration", ultimately leading to joint structural destruction and loss of function.

[0003] Currently, the mainstream clinical treatments for osteoarthritis mainly include oral nonsteroidal anti-inflammatory drugs (NSAIDs), intra-articular injections of corticosteroids, and sodium hyaluronate. These methods can only provide short-term symptomatic intervention and cannot fundamentally reverse cartilage degeneration and microenvironmental disorders. Furthermore, they have significant technical limitations and clinical drawbacks: First, systemic oral administration has significant toxic side effects. Long-term use of NSAIDs can easily induce gastrointestinal ulcers, cardiovascular damage, and metabolic damage to liver and kidney function, resulting in poor drug safety. Second, intra-articular administration has a very short retention time. The joint cavity has rapid synovial fluid exchange and lymphatic drainage mechanisms, and free small-molecule drugs are rapidly metabolized and eliminated after injection, with a half-life of only 1-2 hours in the body. This makes it difficult to maintain an effective therapeutic concentration, requiring repeated punctures for administration, significantly increasing the risk of joint infection and patient suffering. Third, existing treatments have single targets and limited efficacy. Most drugs only have single anti-inflammatory or analgesic effects and lack the comprehensive intervention capabilities to simultaneously clear oxidative stress, regulate joint immune metabolic homeostasis, and repair degenerated cartilage. Therefore, they cannot block the vicious cycle of OA and are difficult to cure or reverse the disease progression.

[0004] Rhein is the core active monomer of rhubarb, a traditional Chinese medicine. It possesses multiple pharmacological activities, including anti-inflammatory, antioxidant, chondrocyte protection, and inhibition of degenerative damage, making it an excellent active ingredient for treating bone pain and joint swelling. However, rhein's strong hydrophobicity and extremely poor water solubility result in low bioavailability, poor water dispersibility, and insufficient formulation stability, making it impossible to directly prepare injectable formulations. This significantly limits its precise intra-articular local administration and long-term clinical application.

[0005] Drynaria fortunei is a classic Chinese medicine used to tonify the kidneys, strengthen bones, heal injuries, and relieve pain. It is traditionally used to treat conditions such as weakness of tendons and bones, traumatic injuries, and bone degeneration. Existing research often uses Drynaria fortunei from southern China, as described in the pharmacopoeia. This invention, however, selects Drynaria fortunei from the coastal area of ​​Kunyu Mountain in Yantai, a species known for its superior active ingredients and biological functions due to the unique stress environment of the coastal region. The derived plant exosomes possess low immunogenicity, excellent biocompatibility, natural nanoparticle size characteristics, and tissue penetration ability. They can also regulate osteogenic differentiation, inhibit abnormal osteoclast activation, and protect cartilage metabolism. These natural biological nanocarriers combine delivery and therapeutic activity, effectively compensating for the poor biocompatibility, lack of repair activity, and susceptibility to immune rejection inherent in synthetic nanocarriers.

[0006] Palmatine hydrochloride (Pal) is the core active alkaloid of Coptis chinensis, possessing significant broad-spectrum anti-inflammatory effects, scavenging oxygen free radicals, and inhibiting the release of inflammatory factors, enabling precise targeted intervention of joint inflammatory responses. However, palmatine hydrochloride suffers from rapid in vivo metabolism, poor stability of the free drug, and low accumulation in the joint cavity, making it difficult to achieve long-term continuous treatment when administered alone.

[0007] The traditional Qing Dynasty formula, Da Huo Luo Dan, is a renowned prescription for treating bone injuries and arthralgia. Its core formulation theory is "tonifying bones and strengthening the foundation, clearing heat and removing dampness, and removing blood stasis and unblocking the meridians." However, this ancient formula suffers from problems such as outdated dosage form, complex ingredients, poor targeting, and unclear mechanism of action, making it unsuitable for modern precision and minimally invasive treatment needs. Addressing the technical bottlenecks of existing osteoarthritis treatments, including insufficient long-term efficacy, single target, irreversible degeneration, and poor carrier safety, this study aims to develop a composite nanodelivery system based on the innovative deconstruction and reconstruction of the ancient formula. This system combines long-term sustained release, immune metabolic microenvironment remodeling, and simultaneous anti-inflammatory, antioxidant, and cartilage repair effects, possessing significant clinical value and industrial implications for achieving precise, safe, long-lasting, and minimally invasive treatment of osteoarthritis. Summary of the Invention

[0008] This invention addresses the shortcomings of existing osteoarthritis treatments, such as rapid drug clearance in vivo, a single mechanism of action, limited anti-inflammatory effects without reversing cartilage degeneration, poor biocompatibility and lack of repair activity of artificial nanocarriers, and the fixed dosage forms, unclear effective substances, and lack of precise regulatory mechanisms of traditional Chinese medicine compound prescriptions. Based on the core compatibility theory of Da Huo Luo Dan, a famous prescription for treating arthritis from the Qing Dynasty, this invention deconstructs and modernizes the traditional ancient prescription, providing a ternary integrated long-acting sustained-release hydrogel composite nanodelivery system that can be formed in situ in the joint cavity. This aims to overcome the technical bottlenecks of the extensive application of traditional compound prescriptions, outdated dosage forms, and broad mechanisms of action.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing a hydrogel loaded with palmatine hydrochloride-Drynaria fortunei exosomes and rhein based on ancient formula deconstruction and reconstruction includes the following steps: (1) Extract exosomes of Drynaria fortunei from fresh rhizomes of Drynaria fortunei, then mix the exosomes of Drynaria fortunei with palmatine hydrochloride, load the drug by sonication, incubate in a water bath at 37°C for 1 h, then centrifuge to collect the precipitate to obtain exosomes EVs-Pal loaded with palmatine hydrochloride, resuspend EVs-Pal in PBS to prepare an EVs-Pal suspension with a concentration of 0.2 mg / mL; (2) Add rhein to NaHCO3 buffer solution with pH 8-9 to prepare a precursor solution with rhein content of 4 mg / mL, then sonicate to completely dissolve the rhein, and then let it cool naturally. (3) When the in vivo solution is naturally cooled to 37°C, the above-mentioned EVs-Pal suspension with a concentration of 0.2 mg / mL is quickly and gently added. After thorough mixing, the solution is allowed to cool naturally to form a hydrogel Rh Gel@EVs-Pal loaded with barmatine hydrochloride-Drynaria fortunei exosome rhein.

[0010] Preferably, in step (1), the method for extracting *Drynaria fortunei* exosomes from the rhizome of fresh *Drynaria fortunei* is as follows: crush the rhizome of fresh *Drynaria fortunei*, soak it in pure water for 2 hours, then centrifuge at low speed to remove large particles of impurities, then filter it with a 0.22 μm filter membrane to remove small particles of impurities, and finally centrifuge at high speed to retain the precipitate to obtain *Drynaria fortunei* exosomes.

[0011] Preferably, in step (1), the exocrine secretions of Drynaria fortunei are suspended in PBS to prepare an RD-EVs suspension with a concentration of 0.2 mg / mL, and the palmatine hydrochloride is dissolved in methanol to prepare a palmatine hydrochloride solution with a concentration of 10 μg / mL. The RD-EVs suspension and the palmatine hydrochloride solution are added at a mass ratio of 1:1.

[0012] Preferably, in step (1), the method of drug loading by ultrasound is as follows: using an ultrasound instrument equipped with a 0.25mm probe, setting the amplitude to 30%, performing ultrasound processing according to a pulse mode of 3s ultrasound and 5s interval, and immediately placing the sample in an ice bath to cool for 2 minutes after each 6 pulse cycles, the ultrasound-cooling step is repeated for a total of 6 rounds.

[0013] Preferably, in step (3), the total protein concentration of *Drynaria fortunei* exosomes in the final system is controlled to reach a preset therapeutic dose of 100 μg / mL.

[0014] A hydrogel containing berberine hydrochloride-Drynaria fortunei exosomes and rhein based on ancient formula deconstruction and reconstruction was prepared using the aforementioned method.

[0015] The aforementioned application of hydrogel containing palmatine hydrochloride-Drynaria fortunei exosomes and rhein based on ancient formula deconstruction and reconstruction in the preparation of drugs for treating osteoarthritis.

[0016] The advantages of this invention are: (1) This invention solves the technical problems of poor targeting and single treatment level of existing osteoarthritis treatment drugs. This invention breaks through the inherent defects of traditional inert drug carriers that only have the function of carrying and have no pharmacological activity. Based on the core compatibility theory of the ancient formula of Da Huo Luo Dan, a three-element active synergistic delivery system is constructed. Among them, rhein has the effects of clearing heat and detoxifying, inhibiting abnormal synovial proliferation, and downregulating the expression of inflammatory factors. Palmatine hydrochloride has broad-spectrum anti-inflammatory, oxygen free radical scavenging, analgesic and anti-inflammatory activities. Haizhou bone drynaria exosomes have both natural targeted delivery characteristics and biological activities that promote cartilage repair and regulate immune metabolism. After encapsulating palmatine hydrochloride-loaded *Drynaria fortunei* exosomes within a rhein-based self-assembled hydrogel and injecting them into the joint cavity, the outer rhein hydrogel rapidly acts on the surface of the synovial membrane, quickly suppressing the acute inflammatory response. Subsequently, the sustained-release *Drynaria fortunei* exosomes deeply penetrate the cartilage lesion area, precisely releasing palmatine hydrochloride to achieve deep and potent anti-inflammatory and analgesic effects. Simultaneously, relying on the exosomes' own bioactivity, they continuously repair degenerated cartilage and correct joint immune metabolic disorders. These three components form a step-by-step closed-loop treatment system of "surface anti-inflammatory and damage control - deep and precise anti-inflammatory - long-term repair and remodeling," achieving multi-dimensional synergistic effects and obtaining unexpected technical results that are significantly superior to the 1+1+1>3 of single-component superposition.

[0017] (2) This invention addresses the clinical pain points of short retention time and repeated punctures required for intra-articular drug administration in osteoarthritis. Existing small-molecule anti-inflammatory and antioxidant drugs are easily metabolized and cleared rapidly in the joint cavity through synovial fluid exchange and lymphatic drainage, resulting in short half-lives and difficulty in maintaining stable and effective therapeutic concentrations. The hydrogel containing palmatine hydrochloride-Drynaria fortunei exosomes and rhein constructed in this invention can be stably formed in situ in the acidic pathological microenvironment of osteoarthritis. Relying on the dense network characteristics of the supramolecular self-assembly structure, it achieves graded, controllable, and long-lasting sustained release of drugs. A single intra-articular injection can achieve continuous drug release for up to 7 days, greatly prolonging the retention time of active components in the joint lesion, effectively avoiding the risk of joint infection and patient treatment pain caused by frequent puncture administration, and significantly improving the long-term efficacy and compliance of minimally invasive treatment for osteoarthritis.

[0018] (3) This invention solves the technical defects of traditional artificial nanocarriers, such as poor biocompatibility, potential immune risks, and lack of repair activity. Currently, artificially synthesized nanocarriers generally suffer from limited biocompatibility, are prone to inducing mild local immune rejection, and can only carry drugs but cannot participate in tissue repair. This invention innovatively selects exosomes derived from the coastal plant Drynaria fortunei as a natural biological carrier. Compared with the conventional Drynaria fortunei from the Southern Pharmacopoeia, the coastal stress environment gives it superior antioxidant, immunomodulatory, and cartilage-protective activities. It also has the advantages of low immunogenicity, high biocompatibility, and degradability without residue. It can stably load small molecule drugs, improve drug stability, and achieve targeted enrichment of lesions. It can also actively participate in the regulation of the joint microenvironment and the repair of cartilage damage, realizing the dual function of "carrier carrying + active treatment". This completely overcomes the defects of traditional inert carriers, such as lack of pharmacological activity and insufficient biological safety.

[0019] (4) This invention solves the problems of crude dosage forms, unclear mechanisms, and inability to precisely regulate immune and metabolic disorders in traditional Chinese medicine compound prescriptions. Traditional ancient prescriptions for treating arthralgia, such as Da Huo Luo Dan, are mostly oral crude preparations, which have problems such as complicated ingredients, unclear effective substances, weak targeting, large systemic side effects, and inability to precisely reverse cartilage degeneration. This invention deconstructs and modernizes the core drug group of classic ancient prescriptions, removes redundant and ineffective components, and precisely separates four functional units: anti-inflammatory, antioxidant, repair-promoting, and long-acting sustained-release. These are assembled into a standardized composite nano-delivery system, which can precisely target and regulate multiple pathological mechanisms such as oxidative stress damage, inflammatory cascade reaction, macrophage polarization imbalance, and cartilage matrix metabolic disorders in osteoarthritis. It systematically reshapes the immune and metabolic homeostasis of joints, breaking through the dual limitations of traditional Chinese medicine compound prescriptions that "treat the symptoms but not the root cause" and modern Western medicine that "intervene at a single target". This invention provides a brand-new technical solution for the modern and precise transformation of ancient prescriptions and the root cause treatment of osteoarthritis. Attached Figure Description

[0020] Figure 1 These are TEM observation results of RD-EVs; Figure 2This is a graph showing the DLS measurement results of RD-EVs; Figure 3 This is a TEM observation image of EVs-Pal; Figure 4 This is a graph showing the DLS measurement results of EVs-Pal; Figure 5 This is a graph showing the in vitro release rates of EVs-Pal in PBS buffer at different pH values; Figure 6 This is a comparison chart of the gelation behavior of rhein precursor solutions of different concentrations; Figure 7 The graph shows the zeta potential detection results of rhein precursor solutions of different concentrations after gelation. Figure 8 This is a graph showing the results of AM / PI double staining; Figure 9 These are the results of a hemolysis experiment. The top row shows photos before the addition of red blood cells, and the bottom row shows photos after incubation and centrifugation. Figure 10 This is a graph showing the results of the hemolysis rate calculation; Figure 11 This is a graph showing the fluorescence detection results; Figure 12 This is a Western Blot result image, where A is the control group, B is the IL-1β induced group, C is the Rh Gel group, D is the Pal gel group, E is the RD-EVs gel group, and F is the Rh Gel@EVs-Pal group. Figure 13 This is an image showing the results of in vivo imaging observation; Figure 14 This is a Micro-CT scan result image; Figure 15 This is a graph showing the results of bone mineral density (BMD) testing of the knee joint. *** indicates p<0.001. Figure 16 This is a graph showing the results of the knee joint bone volume fraction (BV / TV) test. *** indicates p<0.001. Figure 17 This is a graph showing the results of the knee joint trabecular bone count (Tb.N), where ** indicates p<0.01; Figure 18 This is a graph showing the results of the trabecular separation (Tb.Sp) test of the knee joint. *** indicates p<0.001. Figure 19 This is a graph showing the results of trabecular bone thickness (Tb.Th) measurement in the knee joint. *** indicates p<0.001. Figure 20 This is a diagram showing the H&E staining results of the heart, liver, spleen, lungs, and kidneys. Detailed Implementation

[0021] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0022] I. Extraction of exosomes from *Drynaria fortunei* (RD-EVs) In response to the specific nature of the complex pathological microenvironment in osteoarthritis, this invention selects *Drynaria fortunei*, a unique coastal herb from Kunyu Mountain in Yantai, Shandong, as a substitute for the conventional *Drynaria fortunei* found in the Southern Pharmacopoeia, and extracts highly active plant exosomes to construct a natural bio-nanocarrier system.

[0023] Weigh 500g of fresh rhizomes of Drynaria fortunei, crush them, soak them in purified water for 2 hours, then centrifuge at low speed (300×g, 10min) to remove large particles of impurities, then filter them with a 0.22μm filter membrane to remove small particles of impurities, and finally centrifuge at high speed (110000×g, 70min) to retain the precipitate, and obtain Drynaria fortunei exosomes (RD-Evs).

[0024] Unlike all existing technologies related to Drynaria fortunei that use Southern Chinese Pharmacopoeia fructus, this invention exclusively selects Drynaria fortunei from Haizhou, Kunyu Mountain, Yantai. The coastal stress environment gives its exosomes stronger antioxidant and immunomodulatory activities, which are suitable for the core of Da Huo Luo Dan (a traditional Chinese medicine formula) which clears damp heat, removes blood stasis, and strengthens tendons and bones. The carrier also has drug therapeutic functions, which solves the defects of poor biocompatibility and lack of repair activity of artificial carriers.

[0025] RD-EVs were resuspended in PBS, and the suspension was dropped onto a copper grid for transmission electron microscopy (TEM). Morphological observation was performed after negative staining. Results showed that RD-EVs exhibited a typical cup-shaped vesicle structure with intact structure. Figure 1 ).

[0026] RD-EVs were resuspended in PBS, and the suspension was injected into the sample cell of a dynamic light scattering (DLS) instrument. After adjusting the instrument parameters, measurement data were collected. The results showed that the particle size distribution of RD-EVs was 143.7 ± 10 nm. Figure 2 ).

[0027] II. Preparation of exosomes loaded with palmatine hydrochloride (EVs-Pal) First, we investigated the effect of drug loading treatment methods on drug loading efficiency (encapsulation rate and drug loading amount).

[0028] RD-EVs were resuspended in PBS to prepare an RD-EVs suspension with a concentration of 0.2 mg / mL. Paltine hydrochloride was dissolved in methanol to prepare a Pal solution with a concentration of 10 μg / mL. The RD-EVs suspension and Pal solution were added at a mass ratio of 1:1 to obtain a mixture. Subsequently, drug loading was carried out using three strategies: co-incubation, sonication, and repeated freeze-thaw cycles. Specifically: Co-incubation method: The mixture is placed in a 25°C constant temperature water bath shaker and incubated at 150 rpm for 10 min. The concentration gradient difference is used to promote the passive diffusion of the drug palmatine hydrochloride into the exosomes of Drynaria fortunei.

[0029] Ultrasonic method: The mixture was placed in an ultrasonic instrument equipped with a 0.25mm probe, and the amplitude was set to 30%. Ultrasonic treatment was performed using a pulse pattern of 3 seconds of ultrasound followed by a 5-second interval. To prevent the heat generated by ultrasound from damaging the exosome activity, the sample was immediately placed in an ice bath for 2 minutes after every 6 pulse cycles. The ultrasound-cooling step was repeated for a total of 6 cycles. After treatment, the mixture was incubated in a 37°C water bath for 1 hour (to promote the repair and closure of the exosome membrane structure).

[0030] Repeated freeze-thaw method: The mixture was frozen in a -80℃ freezer for 1 hour, then thawed at room temperature for 10 minutes. The freeze-thaw process was repeated 3 times. After treatment, the mixture was incubated in a 37℃ water bath for 1 hour (to promote the repair and closure of exosome membrane structures).

[0031] After drug loading treatment, each group was ultracentrifuged (100,000 × g, 70 min) to remove free drug, and the precipitate was collected to obtain exosomes loaded with palmatine hydrochloride, denoted as EVs-Pal. The encapsulation efficiency and drug loading of each group were determined.

[0032] The encapsulation efficiency and drug loading results for each group are shown in Table 1.

[0033] Table 1. Results of Encapsulation Efficiency and Drug Loading under Different Drug Loading Methods

[0034] The results showed that the ultrasonic method had the best drug loading effect.

[0035] Considering both drug loading efficiency and ease of operation, ultrasonic loading was chosen as the drug loading process for EVs-Pal.

[0036] Next, we will investigate the effect of the ratio of RD-EVs to Pal on drug delivery efficiency.

[0037] RD-EVs suspension (concentration of 0.2 mg / mL) and Pal solution (concentration of 10 μg / mL) were added at mass ratios of 1:1, 1:2, and 2:1, respectively. Drug loading was carried out using the aforementioned ultrasonic method. After drug loading, the unencapsulated free drug was removed by ultracentrifugation (100,000 × g, 70 min). The precipitate was collected, and the encapsulation efficiency and drug loading of each group were determined.

[0038] The encapsulation efficiency and drug loading results for each group are shown in Table 2.

[0039] Table 2. Results of Encapsulation Efficiency and Drug Loading under Different Feed Ratios

[0040] The results showed that the drug loading effect was optimal when the RD-EVs suspension (concentration of 0.2 mg / mL) and Pal solution (concentration of 10 μg / mL) were added at a mass ratio of 1:1; neither too high a ratio (2:1) nor too low a ratio (1:2) could achieve both encapsulation efficiency and drug loading.

[0041] Considering both drug utilization and carrier loading capacity, the RD-EVs suspension (concentration of 0.2 mg / mL) and Pal solution (concentration of 10 μg / mL) were added at a mass ratio of 1:1 during the subsequent preparation of EVs-Pal.

[0042] EVs-Pal were prepared by ultrasonication with an appropriate amount of Pal solution (concentration of 10 μg / mL) and RD-EVs suspension (concentration of 0.2 mg / mL) at a mass ratio of 1:1. The resulting suspension was then dropped onto a copper grid of a transmission electron microscope (TEM) and negatively stained for morphological observation. The results showed that EVs-Pal exhibited a typical and complete cup-shaped vesicle structure. Figure 3 This confirms that the drug loading did not disrupt the integrity of the exosomes.

[0043] An appropriate amount of Pal solution (concentration 10 μg / mL) and RD-EVs suspension (concentration 0.2 mg / mL) were added at a mass ratio of 1:1 and ultrasonically prepared to form EVs-Pal. The resulting suspension was injected into the sample cell of a dynamic light scattering (DLS) instrument. After adjusting the instrument parameters, measurement data were collected. The results showed that the particle size distribution of EVs-Pal was 151.8 ± 10 nm. Figure 4 The average particle size is slightly larger than that of RD-EVs.

[0044] Finally, the in vitro drug release behavior of EVs-Pal was examined.

[0045] The EVs-Pal prepared by feeding Pal solution (concentration of 10 μg / mL) and RD-EVs suspension (concentration of 0.2 mg / mL) at a mass ratio of 1:1 was placed in PBS buffer at pH 7.4 (simulating physiological environment) and pH 5.5 (simulating pathological microenvironment) to investigate the in vitro drug release behavior of EVs-Pal.

[0046] The results showed that EVs-Pal drug release was significantly time-dependent; a burst release effect was observed in the initial stage, with approximately 35% of palmatine hydrochloride released within 2 hours, a phenomenon attributed to the dissociation of drug adsorbed on the vesicle surface; subsequently, the release rate leveled off, entering a sustained-release phase, with a cumulative release rate of approximately 50% at 6 hours and over 60% at 24 hours, indicating that EVs-Pal possesses good drug delivery potential. Figure 5 ).

[0047] III. Investigating the optimal gelling concentration for rhein self-assembled hydrogels (Rh Gel) Rhein self-assembled hydrogels (RhGel) were prepared by utilizing the self-assembly property of rhein under weakly alkaline conditions. Specifically: Rhein was added to a NaHCO3 buffer solution with a pH of 8-9 (pH 8.5 in this specific embodiment) to prepare precursor solutions with rhein concentrations of 2 mg / mL, 4 mg / mL and 8 mg / mL, respectively. The rhein was completely dissolved by ultrasonic heating and then allowed to cool naturally to allow the rhein to self-assemble into a hydrogel with a three-dimensional network structure.

[0048] The gelation of precursor solutions with different rhein contents is shown in the figure. Figure 6 .Depend on Figure 6 It can be seen that 4 mg / mL is the optimal concentration for the formation of stable RhGel.

[0049] The zeta potential detection results of precursor solutions with different rhein contents after gelation are shown in the figure. Figure 7 .Depend on Figure 7 It can be seen that the zeta potential of stable Rh Gel (rhein content 4 mg / mL) is -22.1 mV.

[0050] IV. Constructing Rh Gel, Pal gel, RD-EVs gel, and Rh Gel@EVs-Pal Rhein hydrogels (Rh Gel), pal gels (Rh Gel), exosomes of *Drynaria fortunei* loaded with palmatine hydrochloride, RD-EVs gel (Rh Gel@EVs-Pal) (Rh Gel@EVs-Pal) (Rh Gel@EVs-Pal) were prepared using supramolecular self-assembly technology with rhein. The specific preparation methods are as follows: Rhein was added to a NaHCO3 buffer solution at pH 8.5 to prepare a precursor solution with a rhein concentration of 4 mg / mL. The solution was then sonicated to completely dissolve the rhein and allowed to cool naturally to form a rhein hydrogel, denoted as Rh Gel (as a control).

[0051] Rhein was added to a NaHCO3 buffer solution at pH 8.5 to prepare a precursor solution with a rhein concentration of 4 mg / mL. The solution was sonicated to completely dissolve the rhein and then allowed to cool naturally. When the precursor solution cooled to 37°C, a 10 μg / mL Pal solution (palmatine hydrochloride dissolved in methanol) was slowly added dropwise to the precursor solution at a volume ratio of 1:19. The two solutions were then gently mixed by pipetting and allowed to cool naturally to form a rhein hydrogel loaded with palmatine hydrochloride, denoted as Pal gel (as a control).

[0052] Rhein was added to a NaHCO3 buffer solution at pH 8.5 to prepare a precursor solution with a rhein concentration of 4 mg / mL. The solution was sonicated to completely dissolve the rhein and then allowed to cool naturally. When the precursor solution cooled to 37°C, a 0.2 mg / mL suspension of RD-EVs (RD-EVs resuspended in PBS) was quickly and gently added to control the total protein concentration of RD-EVs in the final system to reach the preset therapeutic dose of 100 μg / mL. The two solutions were then gently mixed by pipetting up and down, and allowed to cool naturally to form a rhein hydrogel loaded with bone fragmentation exosomes, denoted as RD-EVs gel (as a control).

[0053] Rhein was added to a NaHCO3 buffer solution at pH 8.5 to prepare a precursor solution with a rhein concentration of 4 mg / mL. The solution was sonicated to completely dissolve the rhein and then allowed to cool naturally. When the precursor solution cooled to 37°C, an EVs-Pal suspension (resuspended in PBS at a concentration of 0.2 mg / mL) obtained by sonication of Pal solution (concentration 10 μg / mL) and RD-EVs suspension (concentration 0.2 mg / mL) at a mass ratio of 1:1 was quickly and gently added. The total protein concentration of RD-EVs in the final system was controlled to reach the preset therapeutic dose of 100 μg / mL. The two were gently mixed by pipetting up and down, and then allowed to cool naturally to form a rhein hydrogel loaded with palmatine hydrochloride-Drynaria fortunei exosomes, denoted as Rh Gel@EVs-Pal.

[0054] V. Cell compatibility and blood compatibility of Rh Gel@EVs-Pal 1. Cell compatibility To verify the cell compatibility of Rh Gel@EVs-Pal, the viability of SW1353 cells was assessed using a Calcein-AM / PI double staining kit. Specifically: SW1353 cells were loaded at 5 × 10 4Cells were seeded at a density of 100 cells / well in 24-well plates and cultured for 4 hours. The medium was then replaced with extracts of different hydrogels (Rh Gel, Pal Gel, RD-EVs gel, or Rh Gel@EVs-Pal) and incubated for another 24 hours. Subsequently, cells were washed with PBS to remove serum interference and incubated with a Calcein-AM / PI mixed staining solution in the dark. Cell status was observed using a fluorescence microscope; live cells showed green fluorescence (Calcein), and dead cells showed red fluorescence (PI). The effect of each hydrogel on cell viability was evaluated by comparing the distribution of live and dead cells in each group.

[0055] The AM / PI (calcein / propidium iodide) double staining results for each group are shown in the figure. Figure 8 The results showed that most cells in each group exhibited green fluorescence (living cells), while very few exhibited red fluorescence (dead cells).

[0056] This indicates that Rh Gel@EVs-Pal has good biocompatibility within the dosing concentration range (100 μg / mL).

[0057] 2. Blood compatibility analysis Hemolysis experiments were conducted by co-incubating Rh Gel@EVs-Pal with red blood cells to evaluate the blood compatibility of Rh Gel@EVs-Pal.

[0058] Fresh blood was collected from the posterior venous plexus of rats, placed in an anticoagulant tube, centrifuged (1000 rpm, 10 min), and repeatedly washed with physiological saline. The blood was then resuspended in physiological saline to prepare a 2 wt% red blood cell suspension.

[0059] In the experimental group, red blood cell suspension was mixed with Rh Gel hydrogel extract, EVs-Pal hydrogel extract, or Rh Gel@EVs-Pal hydrogel extract (the preparation method of hydrogel extract is as follows: 1 g of Rh Gel, EVs-Pal, or Rh Gel@EVs-Pal is mixed with 10 mL of sterile physiological saline, and the mixture is shaken and extracted at 37°C for 24 h. The supernatant is collected and filtered through a 0.22 μm microporous membrane to obtain the corresponding hydrogel extract) at a volume ratio of 1:1 and incubated at 37°C for 4 h.

[0060] In the positive control group, red blood cell suspension was mixed with deionized water at a volume ratio of 1:1 and incubated at 37°C for 4 hours.

[0061] In the negative control group, red blood cell suspension was mixed with physiological saline at a volume ratio of 1:1 and incubated at 37°C for 4 hours.

[0062] After incubation, the material was centrifuged (1000 rpm, 10 min) and the supernatant was collected. The absorbance (OD) of the hemoglobin release was measured at a wavelength of 540 nm to assess the risk of hemolysis.

[0063] The hemolysis rate is calculated using the following formula: Hemolysis rate (%) = (OD) 实验组 -OD 阴性对照组 ) / (OD 阳性对照组 -OD 阴性对照组 ) × 100%.

[0064] The results of the hemolysis test for each group are shown below. Figure 9 The hemolysis rate calculation results are shown in [link to calculation]. Figure 10 .

[0065] The results showed that the positive control group experienced a severe hemolytic reaction with a hemolysis rate greater than 95%; the supernatant of the Rh Gel@EVs-Pal group was clear and transparent with no obvious hemoglobin release. The quantitative measurement of the hemolysis rate of Rh Gel@EVs-Pal was about 2%, which is within the non-hemolytic range. This indicates that the Rh Gel@EVs-Pal prepared by this invention does not damage the red blood cell membrane, has good blood compatibility, and is suitable for local intra-articular injection.

[0066] VI. In vitro antioxidant activity, cartilage synthesis-promoting ability, and cartilage degradation-inhibiting ability of Rh Gel@EVs-Pal 1. Antioxidant activity The DCFH-DA fluorescent probe was used to assess the level of oxidative stress in RAW264.7 cells. Specifically: RAW264.7 cells were fed at a concentration of 1×10⁻⁶. 5 Seeds were inoculated at a density of [number] cells / dish in 35mm glass-bottom confocal culture dishes and incubated overnight. The experiment was divided into three groups: control group, model group, and hydrogel treatment group. Among them: Control group: No treatment was given; Model group: 1 μg / mL lipopolysaccharide (LPS) was added to the culture medium for stimulation to construct an in vitro inflammation and oxidative stress model; Hydrogel treatment group: 1 μg / mL lipopolysaccharide (LPS) was added to the culture medium for stimulation, and different hydrogel extracts (Rh Gel, Pal Gel, RD-EVs gel or Rh Gel@EVs-Pal) were added for co-incubation.

[0067] After 12 hours of culture, the supernatant was discarded and the cells were washed with PBS. Serum-free medium containing 5 μM DCFH-DA fluorescent probe was added, and the cells were incubated at 37°C in the dark for 30 minutes. During this period, the DCFH-DA fluorescent probe entered the cells, was hydrolyzed by esterases to non-fluorescent DCFH, and then oxidized by intracellular ROS to DCF with high fluorescence intensity. After staining, the cells were washed three times with sterile PBS to remove any residual extracellular probe. The cells were then immediately observed under a laser confocal scanning microscope, and images were acquired to analyze the intracellular ROS fluorescence intensity.

[0068] The fluorescence intensity detection results for each group are shown below. Figure 11 .Depend on Figure 11 It can be seen that the green fluorescence intensity of the LPS-induced group was significantly enhanced, while the green fluorescence intensity of the Rh Gel@EVs-Pal group was significantly reduced, indicating that Rh Gel@EVs-Pal can effectively remove excess reactive oxygen species in cells.

[0069] 2. Promotes cartilage synthesis and inhibits cartilage degradation. The experiment was divided into three groups: control group, IL-1β induction group, and hydrogel treatment group. The treatment was performed at a rate of 1×10⁻⁶. 6 SW1353 cells were seeded into six-well plates at a density of cells / well. After the cells reached a suitable degree of confluence: (1) The control group was not treated; (2) The IL-1β-induced group was stimulated with IL-1β; (3) The hydrogel-treated group was stimulated with the same amount of IL-1β as the IL-1β-induced group, and different hydrogel (Rh Gel, Pal Gel, RD-EVs gel or Rh Gel@EVs-Pal) extracts were added.

[0070] After 24 hours of treatment, cells were washed with pre-cooled PBS, and total cell protein was extracted using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. The supernatant was collected by low-temperature high-speed centrifugation, and the protein concentration was determined using a BCA protein assay kit. 5× loading buffer was added and the mixture was denatured in a 100°C metal bath for 6 minutes.

[0071] Equal amounts of protein samples (30 μg) were separated by SDS-PAGE gel electrophoresis (150 V, 60 min). After electrophoresis, the proteins were transferred to a PVDF membrane using a wet transfer method under ice-water bath conditions (400 mA, 30 min). After transfer, the membrane was blocked with 5% skim milk at room temperature for 4 h to block non-specific binding. Subsequently, the membrane was incubated overnight at 4 °C with diluted NF-κB pathway-related primary antibody and internal control antibody. The next day, the membrane was washed three times with TBST (10 min each time), and incubated with HRP-labeled secondary antibody of the corresponding species at room temperature for 1.5 h. After washing again, ECL chemiluminescence detection reagent was added, and protein band signals were acquired using a gel imaging system. Finally, the gray values ​​of each protein band were analyzed using ImageJ software, and the relative expression levels of the target proteins were calculated using the internal control protein as a standard. The relative expression levels of COL-II, SOX9, MMP13, and Adamts5 were specifically detected and normalized using the internal control protein.

[0072] The Western blot results for each group are shown below. Figure 12 See Tables 3 and 4.

[0073] Table 3. Calculation results of protein expression levels in each group

[0074] Table 4. Calculation results of relative protein expression levels in each group

[0075] The results showed that Rh Gel@EVs-Pal treatment significantly upregulated the expression levels of SOX9, a key transcription factor in cartilage formation, and type II collagen (COL-II), an extracellular matrix component, while significantly downregulating the expression of matrix metalloproteinase MMP13 and agglutinase Adamts5.

[0076] It is evident that Rh Gel@EVs-Pal can effectively inhibit cartilage degradation and promote matrix synthesis by clearing intracellular ROS, upregulating the expression of SOX9 and COL-II, and downregulating the expression of MMP13 and Adamts5.

[0077] VII. Efficacy of Rh Gel@EVs-Pal in the treatment of osteoarthritis in vivo 1. Establish a model A rat model of knee arthritis was obtained by intra-articular injection of sodium iodoacetate (MIA) into the knee joint.

[0078] 2. In vivo imaging Free DIR group: After modeling, free DIR dye was injected into the knee joint cavity of the rat knee arthritis model.

[0079] Rh Gel@EVs-Pal group: After modeling, Rh Gel@EVs-Pal loaded with DIR dye was injected into the knee joint cavity of the rat knee arthritis model.

[0080] The retention time of Rh Gel@Evs-Pal in the knee joint cavity of rats was observed using a small animal in vivo imaging system. The results are shown below. Figure 13 .Depend on Figure 13 It can be seen that the fluorescence intensity of the free DIR group decayed rapidly within a short period of time (3 days); while the fluorescence signal of the Rh Gel@Evs-Pal group remained in the rat knee joint cavity for a significantly longer time, and a clear fluorescence signal could still be observed until the 7th day.

[0081] 3. Micro-CT Image Analysis The experiment was divided into three groups: control group, model group, and hydrogel treatment group.

[0082] Control group rats received no treatment. Other groups underwent MIA modeling. Treatment began on day 3 after MIA modeling. The modeling group received weekly intra-articular injections of 100 μL of physiological saline as a negative control, while the hydrogel treatment groups received weekly intra-articular injections of 100 μL of the corresponding drug-loaded hydrogel. These treatments were administered weekly for 8 weeks. At the end of the treatment, the heart, liver, spleen, lung, and kidney were harvested (for subsequent H&E staining). The right knee joint tissue was completely dissected, trimmed, and fixed in 4% paraformaldehyde solution (for subsequent Micro-CT scanning and histopathological examination).

[0083] The knee joints of rats in each group were scanned using Micro-CT. 2D and 3D scan results are shown below. Figure 14 .Depend on Figure 14 It was found that the model group exhibited severe osteoarthritis (OA) characteristics, with rough joint surfaces and extensive exophytic osteophyte formation. Coronal imaging showed severe erosion and thinning of the subchondral bone plate, and a sparse and disordered internal trabecular network, confirming the bone destruction caused by OA. Treatment with different drug-loaded hydrogels alleviated these pathological changes to varying degrees. The joint surface smoothness of rats in the hydrogel-treated groups was significantly improved, and osteophyte formation was significantly reduced. In particular, the Rh Gel@Evs-Pal group showed relatively intact subchondral bone plate structure and a more compact and orderly arrangement of trabecular bone, indicating that this treatment strategy can effectively inhibit bone destruction and abnormal remodeling during the OA process.

[0084] The microstructural parameters of the knee joint bones of rats in each group were detected using Avatar analysis software. The results are shown in Tables 5 to 9. Figures 15 to 19 .

[0085] Table 5. Results of bone mineral density (BMD) of the knee joint in rats of each group.

[0086] Table 6. Results of knee joint bone volume fraction (BV / TV) detection in rats of each group

[0087] Table 7. Results of trabecular bone number (Tb.N) in the knee joint of rats in each group.

[0088] Table 8. Results of trabecular separation (Tb.Sp) of the knee joint in rats of each group.

[0089] Table 9. Results of trabecular bone thickness (Tb.Th) in the knee joint of rats in each group.

[0090] Depend on Figures 15 to 19 It can be seen that compared with the model group, the bone microstructure parameters of the rat knee joint in the Rh Gel@Evs-Pal group were significantly improved, specifically: bone volume fraction (BV / TV), number of trabeculae (Tb.N), trabecular thickness (Tb.Th) and bone mineral density (BMD) were significantly increased; and trabecular separation (Tb.Sp) was significantly decreased.

[0091] 4. Biocompatibility analysis The H&E staining results of the heart, liver, spleen, lungs, and kidneys of rats in each group are shown in the figure. Figure 20 The results showed that the structures of all organs were clear and there was no obvious pathological damage.

[0092] This confirms that Rh Gel@EVs-Pal has good biocompatibility within the dosing concentration range (100 μg / mL).

[0093] In summary, the core formulation system of this invention is strictly traced back to the renowned Qing Dynasty formula for treating bone injuries and arthralgia, Da Huo Luo Dan (from *Lantai Guifan*). It uses the core formulation concept of this formula for treating chronic degenerative bone arthralgia as the sole theoretical support of the ancient formula. Through precise functional deconstruction and modern reconstruction of the ancient formula, it overcomes the technical problems of outdated traditional Chinese medicine compound dosage forms, unclear material basis, and broad target of action. This achieves a high-quality transformation of traditional Chinese medicine experience in treating arthralgia into modern precision nano-preparations. The specific ancient formula basis is as follows: (1) Tracing the origins and core compatibility of the ancient formula of Da Huo Luo Dan Da Huo Luo Dan is a classic formula for treating arthralgia recorded by Xu Dachun, a physician of the Qing Dynasty. It is a representative formula for treating knee pain, bone and joint swelling and pain, and chronic degeneration of muscles and bones in ancient times. It is specifically designed for the core complex pathogenesis of bone arthralgia in middle-aged and elderly people, which is rooted in liver and kidney deficiency, and manifested by damp-heat stagnation and meridian obstruction. This is highly consistent with the pathological characteristics of modern knee osteoarthritis, which is "kidney deficiency and degeneration + chronic low-grade inflammation + oxidative metabolic disorder". The original formula contains more than fifty herbs, but its core effective herb group for treating bone arthralgia is clearly fixed as follows: Drynaria fortunei, Coptis chinensis, and Rheum palmatum. Among them, Drynaria fortunei tonifies the kidney and strengthens bones, heals injuries and consolidates the foundation, and is specifically used to treat degeneration of muscles and bones; Coptis chinensis clears away latent damp-heat and blood stasis in the meridians and relieves joint swelling and pain; Rheum palmatum cools the blood and removes blood stasis, dredges meridian stagnation and clears metabolic accumulation. The three herbs work together with cold and hot properties, tonify and clear, and treat both the root cause and the symptoms, forming the core functional unit of the ancient formula for treating chronic bone arthralgia with mixed deficiency and excess.

[0094] (2) Inherent defects of traditional ancient prescriptions Traditional Da Huo Luo Dan is an oral pill, which has significant technical problems: systemic administration has extremely poor targeting, the drug is difficult to accumulate in the joint lesion site, the local effective concentration is low and the onset of action is slow; the formula has a complex composition, the effective substances are unclear, and the mechanism of action is broad and vague, making it impossible to achieve precise treatment; long-term oral administration can easily cause metabolic burden on the liver and kidneys, making it difficult to meet the modern osteoarthritis treatment needs of long-term, minimally invasive and safe treatment. The traditional dosage form and application mode seriously limit its precise clinical application.

[0095] (3) The innovative logic of this invention is uniquely deconstructed and reconstructed. This invention abandons the redundant medicinal ingredients and traditional crude oral dosage form of Da Huo Luo Dan, and for the first time, it functionally deconstructs, purifies, and reconstructs the system of its core three-herb combination for treating arthralgia: the traditional efficacy of the ancient formula of Drynaria fortunei (Gu Sui Bu) in "tonifying the kidney and promoting bone regeneration, strengthening the foundation and repairing the body" is deconstructed into a Drynaria fortunei exosome targeting carrier + cartilage repair functional unit; the efficacy of Coptis chinensis (Huang Lian) in "clearing heat and drying dampness, detoxifying and anti-inflammatory" is deconstructed into a palmatine immune metabolism anti-inflammatory unit; and the efficacy of Rheum palmatum (Da Huang) in "removing blood stasis, anti-oxidation, and promoting joint mobility" is deconstructed into a rhein supramolecular self-assembled long-acting sustained-release matrix unit. Through the integrated assembly of the three functional units, a novel composite nanodelivery system is constructed, which solves the technical pain points of traditional ancient formulas, such as weak targeting, short in vivo retention time, broad mechanism of action, and inability to reverse cartilage degeneration. This achieves a modern, precise, and long-lasting innovative upgrade of the traditional ancient formula compatibility theory.

[0096] Furthermore, different from the traditional single nano-drug loading and passive slow-release repair system, the present invention is based on the core compatibility of Da Huo Luo Dan, and originally constructs an integrated composite nano-delivery system of "Rhizoma Drynariae from Haizhou exosomes - palmatine - rhein hydrogel", which is not a simple physical mixture. Relying on the natural targeted loading characteristic of Haizhou Rhizoma Drynariae exosomes, the specific anti-inflammatory advantage of palmatine and the supramolecular in-situ gel-forming long-acting slow-release characteristic of rhein, an integrated technical system of targeted drug loading, in-situ gel formation, long-acting local retention, and precise microenvironment regulation is formed, which effectively solves the clinical technical pain points of rapid drug metabolism and clearance in the joint cavity and the need for repeated puncture administration.

[0097] In addition, the present invention strictly inherits the core pathogenesis of the ancient prescription for treating arthralgia of Da Huo Luo Dan, which is "tonifying bone and consolidating the root, clearing heat and eliminating dampness, removing blood stasis and dredging collaterals", and breaks the limited treatment ideas of existing researches that are single anti-inflammatory, simple anti-oxidation or single bone repair promotion. Relying on the multi-target synergistic feature of the ternary integrated nano-delivery system, it systematically regulates the joint immune metabolic microenvironment at multiple levels: clearing excessive oxidative stress, blocking the inflammatory cascade amplification effect, reshaping the immune polarization balance of macrophages, inhibiting chondrocyte degeneration and apoptosis, and correcting the metabolic disorder of cartilage matrix, thus forming a full-chain treatment mechanism of "stopping loss of oxidative damage - regulating immune inflammation - repairing degenerated tissue". Multi-omics and in vivo and in vitro experiments have verified that the overall therapeutic effect of the combined use of the three components is significantly better than the individual action and simple superposition of each single component, producing an unexpected synergistic effect.

[0098] It should be noted that the above embodiments are only examples for clearly illustrating the present invention, and are not intended to limit the implementation manners of the present invention. For those of ordinary skill in the art, on the basis of the above description, other variations or modifications of different forms can be made. It is impossible to exhaust all the embodiments here. All obvious variations or modifications derived from the technical solution of the present invention still fall within the protection scope of the present invention.

Claims

1. A method for preparing a hydrogel loaded with palmatine hydrochloride-Drynaria fortunei exosomes and rhein based on ancient formula deconstruction and reconstruction, characterized in that, Includes the following steps: (1) Extract exosomes of Drynaria fortunei from fresh rhizomes of Drynaria fortunei, then mix the exosomes of Drynaria fortunei with palmatine hydrochloride, load the drug by sonication, incubate in a water bath at 37°C for 1 h, then centrifuge to collect the precipitate to obtain exosomes EVs-Pal loaded with palmatine hydrochloride, resuspend EVs-Pal in PBS to prepare an EVs-Pal suspension with a concentration of 0.2 mg / mL; (2) Add rhein to NaHCO3 buffer solution with pH 8-9 to prepare a precursor solution with rhein content of 4 mg / mL, then sonicate to completely dissolve the rhein, and then let it cool naturally. (3) When the in vivo solution is naturally cooled to 37°C, the above-mentioned EVs-Pal suspension with a concentration of 0.2 mg / mL is quickly and gently added. After thorough mixing, the solution is allowed to cool naturally to form a hydrogel RhGel@EVs-Pal loaded with barmatine hydrochloride-Drynaria fortunei exosome rhein.

2. The preparation method according to claim 1, characterized in that, In step (1), the method for extracting *Drynaria fortunei* exosomes from the fresh rhizome of *Drynaria fortunei* is as follows: The rhizomes of fresh Drynaria fortunei were crushed, soaked in purified water for 2 hours, then centrifuged at low speed to remove large particles of impurities. After that, they were filtered through a 0.22μm filter membrane to remove small particles of impurities. Finally, they were centrifuged at high speed to retain the precipitate and obtain Drynaria fortunei exosomes.

3. The preparation method according to claim 1, characterized in that, In step (1), the exocrine secretions of Drynaria fortunei were suspended in PBS to prepare a suspension of RD-EVs with a concentration of 0.2 mg / mL. Palmatine hydrochloride was dissolved in methanol to prepare a solution of palmatine hydrochloride with a concentration of 10 μg / mL. The suspension of RD-EVs and the solution of palmatine hydrochloride were added at a mass ratio of 1:

1.

4. The preparation method according to claim 1, characterized in that, In step (1), the method of drug loading using ultrasound is as follows: Using an ultrasonic instrument equipped with a 0.25mm probe, the amplitude was set to 30%, and ultrasonic treatment was performed according to a pulse pattern of 3 seconds of ultrasound followed by 5 seconds of intermittent pulse. After each 6 pulse cycles, the sample was immediately placed in an ice bath to cool for 2 minutes. The ultrasound-cooling step was repeated for a total of 6 cycles.

5. The preparation method according to claim 1, characterized in that, In step (3), the total protein concentration of *Drynaria fortunei* exosomes in the final system is controlled to reach the preset therapeutic dose of 100 μg / mL.

6. A hydrogel loaded with palmatine hydrochloride-Drynaria fortunei exosomes and rhein based on ancient formula deconstruction and reconstruction, characterized in that, It is prepared by the method described in any one of claims 1 to 5.

7. The application of the hydrogel containing berberine hydrochloride-Drynaria fortunei exosomes and rhein based on ancient formula deconstruction and reconstruction as described in claim 6 in the preparation of a drug for treating osteoarthritis.

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