An ultrasonic response type curcumin / perfluoropentane co-loaded liposome nanoparticle, a preparation method and application thereof

Abstract

The application discloses an ultrasonic response type curcumin / perfluoropentane co-loaded liposome nanoparticle and a preparation method and application thereof. The application constructs a liposome nanoparticle system loaded with curcumin and a PFP phase change response mechanism, and realizes controllable release under the action of LIFU. The platform combines a natural active small molecule with a physical response mechanism, and provides a new strategy for improving the local utilization efficiency of curcumin. The ultrasonic response type curcumin / perfluoropentane co-loaded liposome nanoparticle is combined with low-intensity ultrasound, a 'drug delivery + physical assistance' combined intervention mode is proposed, the synergistic effect is verified in in-vivo and in-vitro models, it is proved that on the basis of traditional single drug intervention, the physical regulation dimension of the liposome nanoparticle system is increased, the treatment effect is remarkable, and a new idea is provided for the local treatment strategy of osteoarthritis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an ultrasound-responsive curcumin / perfluoropentane co-loaded liposome nanoparticle, its preparation method, and its application. Background Technology

[0002] Osteoarthritis (OA) is a chronic degenerative joint disease characterized by degeneration of articular cartilage, subchondral bone remodeling, and synovial inflammation. Its onset is closely related to age; as the body ages, the metabolic function of chondrocytes declines and tissue repair capacity weakens, making the elderly the primary patient group for OA. In recent years, with the deepening of population aging, the prevalence of OA has shown a continuous upward trend, becoming one of the important diseases affecting the mobility and quality of life of middle-aged and elderly people. Joint pain, stiffness, and limited function not only reduce the independence of daily life in the elderly but also increase the medical burden and social care pressure.

[0003] The occurrence and development of osteoarthritis (OA) is highly complex, involving metabolism, nutrition, and inflammation. Currently, clinical treatment for OA primarily focuses on symptom relief, commonly employing methods such as nonsteroidal anti-inflammatory drugs (NSAIDs), intra-articular injections, and physical therapy. However, while NSAIDs mainly alleviate pain by inhibiting inflammatory mediators, they cannot halt the degenerative process of cartilage, and long-term use in the elderly may increase the risk of gastrointestinal and cardiovascular adverse reactions. For advanced-stage patients, joint replacement surgery is the main way to restore joint function, but its significant trauma and high risk of postoperative complications limit its widespread clinical application. Overall, there is still a lack of effective interventions to slow disease progression; therefore, exploring safe treatment strategies with disease-modifying effects remains of great practical significance.

[0004] In recent years, with the deepening research into the pathogenesis of osteoarthritis (OA), oxidative stress has been identified as one of the important pathological factors driving disease progression. Against the backdrop of aging, the level of reactive oxygen species (ROS) in chondrocytes gradually increases, while their antioxidant defense capacity decreases, leading to an imbalance in intracellular redox homeostasis. Excessive ROS can damage cell membrane lipids, proteins, and DNA structure, further inducing chondrocyte apoptosis or dysfunction, and promoting the expression of matrix-degrading enzymes, thereby accelerating the cartilage degeneration process. Mitochondria, as the core organ for cellular energy metabolism and ROS generation, directly affect the survival and metabolism of chondrocytes. When mitochondrial function is impaired, ATP production decreases, membrane potential declines, and ROS accumulates excessively, creating a vicious cycle that exacerbates cell damage. Multiple studies have shown significant mitochondrial structural and functional abnormalities in OA cartilage tissue, suggesting that mitochondrial dysfunction plays a crucial role in the development and progression of the disease. Therefore, intervention studies focusing on mitochondrial homeostasis and oxidative stress regulation may provide new insights for delaying OA progression.

[0005] Curcumin, a natural polyphenolic compound derived from the rhizome of turmeric, possesses broad-spectrum anti-inflammatory, antioxidant, and immunomodulatory activities. In recent years, with the deepening of research into natural drugs, curcumin has gradually gained attention in the field of osteoarthritis. In vitro and animal experiments have shown that curcumin can inhibit the expression of inflammatory factors and matrix-degrading enzymes, reduce chondrocyte damage, and improve joint structural degeneration to some extent. However, curcumin still faces significant limitations in practical applications. On the one hand, its poor water solubility, low bioavailability, and rapid in vivo metabolism affect the maintenance of effective drug concentrations; on the other hand, current research on the mechanism of action of curcumin mainly focuses on single inflammatory or antioxidant indicators, lacking a systematic mechanistic analysis, and exhibiting a certain degree of "multi-target superposition." Because the pathway of action is not yet fully elucidated, there are significant differences in efficacy between different studies, which also limits its further translational application.

[0006] Low-intensity focused ultrasound (LIFU), as a non-invasive physical stimulation method, has been used to promote tissue repair and improve drug delivery. Ultrasound can enhance cell membrane permeability through mechanical vibration and cavitation effects, and to some extent promote the local release and tissue penetration of nanoparticles.

[0007] Currently, there are few reports on the use of liposome-encapsulated curcumin combined with low-intensity ultrasound for the treatment of osteoarthritis. Summary of the Invention

[0008] The purpose of this invention is to address the above-mentioned problems by providing an ultrasound-responsive curcumin / perfluoropentane co-loaded liposome nanoparticle, its preparation method, and its application.

[0009] To achieve its objective, the present invention employs the following technical solution:

[0010] The first aspect of this invention provides a method for preparing ultrasound-responsive curcumin / perfluoropentane co-supported liposome nanoparticles, comprising the following steps:

[0011] S1. Preparation of curcumin liposome membranes: Curcumin liposome membranes were prepared by rotary evaporation-membrane hydration method;

[0012] S2. Hydration and PFP emulsification: PBS buffer was added to the curcumin liposome film prepared in step S1, and hydration was carried out at 35-40°C with stirring. The resulting lipid suspension was transferred to a centrifuge tube, and perfluoropentane PFP was added dropwise under ice bath conditions. Subsequently, ultrasonic emulsification was performed, followed by low-temperature high-speed centrifugation. The supernatant was discarded, and the suspension was resuspended in PBS and washed to obtain ultrasonically responsive curcumin / perfluoropentane co-loaded liposome nanoparticles Cur-NPs.

[0013] Preferably, S1, preparation of curcumin liposome film: prepared by rotary evaporation-film hydration method, DPPC, DSPE, cholesterol and curcumin are weighed and added to a rotary evaporation flask, anhydrous chloroform is added, and ultrasonically dissolved under light-protected conditions until completely clear, and rotary evaporation is performed to form a uniform liposome film. Rotary evaporation conditions: 35~37 ℃, 120~150 rpm, -0.07~-0.09 MPa; then vacuum drying is continued to remove residual solvent; wherein, the ratio of DPPC, DSPE, cholesterol, curcumin and anhydrous chloroform is: 5~7 mg : 1.5~2.5 mg : 1.5~2.5 mg : 1 mg : 4~6 mL.

[0014] Preferably, S2, hydration and PFP emulsification: PBS buffer is added to the curcumin liposome film prepared in step S1, and hydration is carried out for 25-35 min under stirring conditions of 35-40 °C and 120-150 rpm. The resulting lipid suspension is transferred to a centrifuge tube, and perfluoropentane PFP is added dropwise under ice bath conditions. Then, emulsification is carried out using an ultrasonic disruptor. The ultrasonic conditions are 140-160W, 35-45% power, 4-6 s working / 4-6 s intermittent, for a total of 5-7 min. After emulsification, the mixture is centrifuged at 1-4 °C and 8000-12000 rpm for 8-12 min. The supernatant is discarded, and the mixture is resuspended in PBS and washed to obtain ultrasound-responsive curcumin / perfluoropentane co-loaded liposome nanoparticles Cur-NPs.

[0015] More preferably, in step S2, perfluoropentane PFP is added dropwise to the lipid suspension at a volume ratio of 8~12:1, wherein the lipid suspension is prepared by adding 3~5 mL of PBS buffer to the curcumin liposome membrane prepared in step S1 using 1 mg of curcumin.

[0016] A second aspect of the present invention provides an ultrasound-responsive curcumin / perfluoropentane co-supported liposome nanoparticle, which is prepared by the method described in any of the above-mentioned methods.

[0017] A third aspect of the present invention provides a hydrogel containing co-loaded curcumin / perfluoropentane lipid nanoparticles, which is obtained by mixing the above-mentioned ultrasound-responsive curcumin / perfluoropentane co-loaded liposome nanoparticles (Cur-NPs) with sodium alginate (ALG) solution under low-speed stirring in an ice bath to form a composite system.

[0018] The fourth aspect of the present invention comprises the following steps in the preparation method of the above-mentioned co-loaded curcumin / perfluoropentane lipid nanoparticle hydrogel: the above-mentioned ultrasound-responsive curcumin / perfluoropentane co-loaded liposome nanoparticles Cur-NPs are mixed with sodium alginate ALG solution under low-speed stirring in an ice bath to form a composite system, thereby obtaining the co-loaded curcumin / perfluoropentane lipid nanoparticle hydrogel Cur-ALG; the concentration of Cur-NPs in the composite system is 0.5~2 mg / mL, and the concentration of ALG is 5~20 mg / mL.

[0019] Preferably, in the above preparation method, the low-speed stirring is 100~300 rpm, the stirring time is 3~5 min, the concentration of Cur-NPs in the composite system is 1~2 mg / mL, and the concentration of ALG is 10~20 mg / mL.

[0020] The fifth aspect of the present invention relates to the use of the above-described ultrasound-responsive curcumin / perfluoropentane co-loaded liposome nanoparticles Cur-NPs or co-loaded curcumin / perfluoropentane lipid nanoparticle hydrogels Cur-ALG in the preparation of a medicament for treating osteoarthritis.

[0021] Preferably, the application involves combining Cur-NPs or Cur-ALG with LIFU, with the subject receiving local LIFU intervention immediately after each intra-articular injection of Cur-NPs or Cur-ALG.

[0022] Based on the high prevalence of osteoarthritis (OA) in the elderly and the limitations of current treatments, and considering the important roles of oxidative stress and mitochondrial dysfunction in disease progression, this invention constructs a liposome-encapsulated curcumin nanodelivery system and combines it with LIFU technology to explore its application effects and potential mechanisms in OA intervention. Furthermore, its effects on inflammatory responses, oxidative stress levels, and joint structural degeneration are observed in in vitro chondrocyte models and in vivo DMM mouse models. Experiments demonstrate that Cur-NPs can significantly improve IL-1β-induced cell viability decline, reduce ROS levels, restore mitochondrial membrane potential, and partially reverse the abnormal expression of degeneration-related proteins such as MMP-13 and PTGS2; the protective effect is further enhanced after combined LIFU intervention. In vivo experiments show that Cur-NPs combined with LIFU can significantly reduce osteophyte formation, lower OARSI scores, and reduce synovitis scores in DMM mice. Curcumin nanoparticles combined with low-intensity focused ultrasound can improve osteoarthritis-related pathological changes in in vitro and in vivo models, and its effect may be related to the regulation of mitochondrial function and oxidative stress.

[0023] The beneficial effects of this invention are:

[0024] (1) Construction of a curcumin nanodelivery platform with acoustic response characteristics: In this study, a liposome nanosystem loaded with curcumin and PFP phase transition response mechanism was constructed, and controllable release was achieved under the action of LIFU. This platform combines natural active small molecules with physical response mechanisms, providing a new strategy for improving the local utilization efficiency of curcumin.

[0025] (2) The ultrasound-responsive curcumin / perfluoropentane co-loaded liposome nanoparticles of the present invention are combined with low-intensity ultrasound to propose a "drug delivery + physical assistance" joint intervention mode. The synergistic effect is verified in in vivo and in vitro models, proving that the liposome nanosystem of the present invention has a significant therapeutic effect when the physical regulation dimension is added on the basis of traditional single drug intervention, providing a new idea for local treatment strategy of osteoarthritis. Attached Figure Description

[0026] Figure 1 The preparation and physicochemical characterization of Cur-NPs are as follows: (A) Transmission electron microscopy (TEM) images of Lip NPs; (B) TEM images of Cur-NPs; (C) Comparison of average particle size between Lip NPs and Cur-NPs; (D) Zeta potential measurements of Lip NPs and Cur-NPs; (E) Particle size changes of Lip NPs stored at 4°C for 7 days; (F) Particle size changes of Cur-NPs stored at 4°C for 7 days. Data are expressed as mean ± SD, and ns indicates no statistically significant difference.

[0027] Figure 2The drug loading performance and UV absorption characteristics of Cur-NPs are shown: (A) Encapsulation efficiency of Cur-NPs under different curcumin dosages; (B) Drug loading rate of Cur-NPs under different curcumin dosages; (C) UV absorption spectra of Cur-NPs after rupture at different concentrations; (D) Curcumin standard curve and linear regression analysis results.

[0028] Figure 3 The formulation screening and macroscopic morphology of the Cur-ALG hydrogel system are shown: (A) The effects of different Cur-NPs concentrations, ALG concentrations and stirring conditions on the formation of the Cur-ALG system; (B) Comparison of the macroscopic appearance of Cur-NPs solution, ALG solution and Cur-ALG mixture system.

[0029] Figure 4 Cryo-SEM images of Cur-ALG hydrogels are shown: (AB) Microstructure of ALG hydrogels at different magnifications; (CD) Microstructure of Cur-ALG hydrogels at different magnifications.

[0030] Figure 5 The in vitro stability of Cur-ALG hydrogel in different solutions was observed.

[0031] Figure 6 The effects of Cur-NPs and LIFU on chondrocyte viability were as follows: The changes in cell viability in each group after 24 h of IL-1β (10 ng / mL) stimulation were detected by the CCK-8 assay. Data are expressed as mean ± SD (n = 3); ****P < 0.0001, ***P < 0.001, *P < 0.05.

[0032] Figure 7 The results showed that Cur-NPs combined with LIFU reduced IL-1β-induced ROS levels in chondrocytes: (A) DCFH-DA fluorescence staining was used to detect intracellular ROS levels, with green fluorescence indicating ROS signal; (B) Quantitative analysis of ROS fluorescence intensity in each group; data are expressed as mean ± SD (n = 3); ***P < 0.001; **P < 0.01, *P < 0.05.

[0033] Figure 8 The results showed that Cur-NPs combined with LIFU improved mitochondrial membrane potential in chondrocytes: (A) TMRE fluorescence staining to detect mitochondrial membrane potential (red), Hoechst staining of nuclei (blue); (B) Quantitative analysis of TMRE fluorescence intensity in each group; data are expressed as mean ± SD (n = 3); ***P < 0.001; **P < 0.01, *P < 0.05.

[0034] Figure 9 This study demonstrated that Cur-NPs combined with low-intensity ultrasound improved the expression of proteins related to chondrocyte degeneration phenotype: (A) Western blot analysis of NOS2, PTGS2, MMP-13, ACAN, and COL2A1 protein expression levels in each group, with GAPDH as an internal control; (B) Quantitative analysis of protein grayscale values; data are expressed as mean ± SD (n = 3); ***P < 0.001; ****P < 0.0001, ***P < 0.001, **P < 0.01; ns indicates no statistically significant difference.

[0035] Figure 10 The results show that Cur-ALG combined with low-intensity ultrasound improves osteophyte formation and joint degeneration in DMM mice: (A) Micro-CT three-dimensional reconstruction images of the knee joints of mice in each group and three-dimensional extraction images of osteophytes; the red area represents osteophytes; (B) Quantitative analysis results of osteophyte volume; data are expressed as mean ± SD (n = 6); ****P < 0.0001; *P < 0.05; ns indicates no statistical significance.

[0036] Figure 11 The results show that Cur-ALG combined with low-intensity ultrasound improved the histological damage of joints in DMM mice: (A) Representative images of Safranin O-Fast Green staining and H&E staining of the knee joints of mice in each group; (B) Quantitative analysis results of OARSI score and synovial inflammation score; Data are expressed as mean ± SD (n = 6); ****P < 0.0001, **P < 0.01, *P < 0.05; ns indicates no statistical significance. Detailed Implementation

[0037] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0038] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0039] Example 1

[0040] 1. Materials and Methods

[0041] 1.1 Main Reagents

[0042] Table 1 Main Reagents

[0043]

[0044] 1.2 Preparation and Characterization of Curcumin Phase Change Liposome Nanoparticles (Cur-NPs)

[0045] This study constructed an ultrasound-responsive curcumin / perfluoropentane co-loaded liposome nanoparticle (Cur-NPs) and further compounded it with sodium alginate (ALG) to form an in-situ gelling hydrogel nanocomposite system for the treatment of osteoarthritis (OA).

[0046] 1.2.1 Preparation of Cur-NPs

[0047] (1) Preparation of lipid films

[0048] Liposomes were prepared using a rotary evaporation-thin-film hydration method. DPPC (dispalmitoylphosphatidylcholine) (6 mg), DSPE (1,2-distearate-3-phosphatidylethanolamine) (2 mg), cholesterol (2 mg), and curcumin (1 mg) were weighed and added to a 50 mL round-bottom rotary evaporator flask. 5 mL of anhydrous chloroform was added, and the mixture was sonicated until completely clear under light-protected conditions. The rotary evaporation conditions were set to 37 °C, 150 rpm, and −0.08 MPa to form a uniform lipid film. The film was then vacuum dried for 1 h to remove residual solvent.

[0049] (2) Hydration and PFP emulsification

[0050] Add 4 mL of PBS to the lipid membrane and hydrate for 30 min at 40 °C and 150 rpm. Transfer the resulting lipid suspension to a centrifuge tube and add PFP (lipid:PFP = 10:1 by volume) dropwise under ice bath conditions. Then emulsify using an ultrasonic homogenizer (150 W, 40% power, 5 s on / 5 s off, 6 min total). Centrifuge at 10,000 rpm for 10 min at 4 °C, discard the supernatant, resuspend in PBS, and repeat the washing process three times to obtain Cur-NPs. The preparation method for Lip NPs is the same, except that the curcumin addition step is omitted. For fluorescently labeled nanoparticles (DiI / DiR), add 10 μL of dye during the lipid dissolution stage, and the remaining steps are the same.

[0051] 1.2.2 Characterization of the physicochemical properties of nanoparticles

[0052] 1.2.2.1 Morphological observation (TEM)

[0053] After dilution, Cur-NPs were dropped onto a copper mesh and allowed to dry naturally before their microstructure was observed using a transmission electron microscope.

[0054] 1.2.2.2 Particle size and zeta potential

[0055] The particle size and surface potential of Lip NPs and Cur-NPs were determined using a dynamic light scattering instrument.

[0056] 1.2.2.3 Drug loading and encapsulation efficiency

[0057] Cur-NPs were prepared by adding different amounts of curcumin (0.25–2 mg). After centrifugation, DMF was added to break the emulsion, and the curcumin concentration was determined by ultraviolet spectrophotometry.

[0058] Calculation formula:

[0059] Encapsulation efficiency (EE%) = (Mass of curcumin in nanoparticles / Total dosage) × 100%

[0060] Drug loading (DL%) = (Mass of curcumin in nanoparticles / Total mass of nanoparticles) × 100%

[0061] 1.2.2.4 Ultraviolet Absorption Spectrum

[0062] Different concentrations of Cur-NPs were demulsified with DMF and their ultraviolet absorption spectra were measured to confirm the characteristic peak of curcumin.

[0063] 1.2.2.5 Stability Testing

[0064] Particle size changes were measured at 0, 1, 3, and 7 days to evaluate storage stability.

[0065] 1.3 Preparation and Characterization of Cur-ALG Hydrogel Nanocomposites

[0066] 1.3.1 Preparation of Cur-ALG

[0067] Cur-NPs were mixed with ALG solution (final concentration 20 mg / mL) under low-speed stirring in an ice bath to form a composite system, namely co-loaded Cur / PFP lipid nanoparticles alginate (Cur-ALG).

[0068] 1.3.2 Optimization of gelation conditions

[0069] (1) Optimize Cur-NPs concentration (0–10 mg / mL)

[0070] (2) Optimize ALG concentration (5–20 mg / mL)

[0071] (3) Optimize the stirring speed

[0072] By injecting the mixture into a Ca-containing 2+ / Mg 2+ Observe the gelation process in (1.8 mM / 1.5 mM) solutions to screen for the optimal ratio.

[0073] 1.3.3 Characterization of Cur-ALG

[0074] 1.3.3.1 Macroscopic morphology

[0075] The appearance of Cur-NPs, ALG, and the complex was photographed.

[0076] 1.3.3.2 Microstructure

[0077] The hydrogel structure was observed using cryo-scanning electron microscopy; the porous structure was observed using SEM after lyophilization.

[0078] 1.3.3.3 Fluorescence Distribution

[0079] Confocal observation was used to observe the dispersion of DiI-labeled Cur-NPs in ALG.

[0080] 1.3.3.4 LIFU Response Phase Transition

[0081] Low-intensity focused ultrasound (4 W / cm) was used 2 Stimulation was performed at 50% duty cycle for 3 minutes, and the liquid-gas phase transition phenomenon was observed under a microscope.

[0082] 1.3.3.5 In vitro stability

[0083] Cur-ALG was immersed in DMEM, PBS and physiological saline, and structural changes were observed at different time points.

[0084] 1.3.3.6 In-situ gelation properties

[0085] Injected into Ca 2+ / Mg 2+ Observe the gel-forming ability in solution.

[0086] 1.3.3.7 Rheological Testing

[0087] The storage modulus (G') and loss modulus (G'') are measured to evaluate the gelation performance.

[0088] 1.4 In vitro drug release experiment

[0089] 1.4.1 Routine Release Experiment

[0090] The in vitro release behavior of Cur-NPs was evaluated using the dialysis bag method. A certain volume of Cur-NPs suspension was placed in a dialysis bag with a molecular weight cutoff of 8-14 kDa, and then placed in a release system containing 50 mL of PBS buffer (pH 7.4). The release experiment was conducted under isothermal shaking at 37 °C. 1 mL samples were taken at preset time points (0, 2, 4, 8, 12, 24, 48, and 72 h), and an equal volume of fresh PBS was added to maintain a constant system volume. The concentration of curcumin in the release solution was determined by ultraviolet spectrophotometry, and the cumulative release rate was calculated based on a standard curve. All experiments were repeated three times.

[0091] 1.4.2 LIFU-triggered release experiment

[0092] To evaluate the effect of LIFU on drug release behavior, an ultrasound intervention group was set up in addition to the conventional release system. The ultrasound intervention was performed according to the set parameters (frequency 1 MHz, sound intensity 0.5 W / cm²). 2 The release system was subjected to intermittent sonication (duty cycle 20%, treatment time 5 min). The release solution was collected at the same time points, and the curcumin concentration was measured. The cumulative release curves of the sonication group and the conventional release group were compared to evaluate the promoting effect of ultrasound on drug release.

[0093] 1.5 Cell Culture and In Vitro Experiment Design

[0094] 1.5.1 Chondrocyte Culture

[0095] The chondrocytes used in this study were provided by the Institute of Ultrasound Imaging, Chongqing Medical University. The cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and incubated at 37°C with 5% CO2. Cells were passaged when they reached 80%–90% confluence. Cells from passages 3–5 were used for subsequent experiments in this study.

[0096] 1.5.2 IL-1β-induced in vitro model of osteoarthritis

[0097] To establish an in vitro model of inflammatory osteoarthritis, chondrocytes were stimulated with recombinant human IL-1β (10 ng / mL) for 24 h to simulate the inflammatory microenvironment. Preliminary experiments verified that this concentration and treatment time could significantly induce an inflammatory response and increase oxidative stress levels.

[0098] 1.5.3 Group Design

[0099] The in vitro experiments were divided into the following four groups: control group, IL-1β group (10 ng / mL), IL-1β + Cur-NPs group, and IL-1β + Cur-NPs + LIFU group. After 24 h of treatment according to the experimental protocol, each group was subjected to subsequent detection, including cell viability, ROS level, mitochondrial membrane potential and protein expression analysis.

[0100] 1.5.4 Cell viability assay (CCK-8)

[0101] Conventional method.

[0102] 1.5.5 ROS Level Detection

[0103] Chondrocytes were 1×10 6 Cells were seeded per well in 6-well plates and cultured overnight at 37°C and 5% CO2. The culture medium was changed to contain the corresponding treatment factor according to the experimental group, and incubation continued for 24 h. After treatment, intracellular ROS levels were detected using the DCFH-DA probe. DCFH-DA was diluted to a working concentration of 10 μM, the culture medium was discarded, and 1.5 mL of probe working solution was added to each well. The cells were incubated at 37°C in the dark for 30 min. After incubation, the cells were washed three times with PBS to remove any probes that had not entered the cells. Images were then observed and captured under an inverted fluorescence microscope, and ROS fluorescence signals were detected using the FITC channel. Fluorescence intensity was semi-quantitatively analyzed using ImageJ software.

[0104] 1.5.6 Detection of mitochondrial membrane potential (ΔΨm)

[0105] The mitochondrial membrane potential (ΔΨm) of chondrocytes was detected using a tetramethylrhodamine ethyl ester (TMRE) fluorescent probe. Chondrocytes were cultured at a concentration of 1 × 10⁻⁶ cells / year. 6 100 cells / well were seeded in 6-well plates and cultured overnight. Membrane potential was measured 24 h after each treatment. After discarding the culture medium, pre-prepared TMRE working solution (final concentration 100 nM) was added, and the plates were incubated at 37°C in the dark for 20-30 min. After incubation, the plates were gently washed 2-3 times with PBS to remove unbound dye. Fluorescence images were then observed and captured under an inverted fluorescence microscope, and the TMRE fluorescence signal was detected using the TRITC channel. Fluorescence intensity was semi-quantitatively analyzed using ImageJ software to reflect changes in mitochondrial membrane potential.

[0106] 1.5.7 Western blot analysis

[0107] Conventional method.

[0108] 1.6 Animal Experiments

[0109] The experimental animals used in this study were 8-week-old female C57BL / 6 mice, purchased from Guangdong Yaokang Biotechnology Co., Ltd. All animal husbandry, modeling, and material collection procedures were conducted at the Experimental Animal Center of Sun Yat-sen University.

[0110] 1.6.1 Establishment of DMM mouse model of osteoarthritis

[0111] A mouse osteoarthritis (OA) model was established using the medial meniscus destabilization (DMM) method. All animal experiments were conducted in an animal testing center in accordance with relevant animal ethics guidelines. Mice were anesthetized via intraperitoneal injection (sodium pentobarbital, 0.6 mL / 10 g). After adequate anesthesia, the mice were fixed on the operating table, and both knee joints were routinely disinfected. A longitudinal skin incision of approximately 0.5–1 cm was made along the medial side of the knee joint, and the subcutaneous tissue and joint capsule were bluntly dissected to expose the knee joint structures. Under microscopic conditions, the medial meniscus and tibial ligament were severed to disrupt medial meniscus stability, thereby inducing joint degeneration. After hemostasis, the joint capsule and skin were sutured layer by layer. Postoperatively, the mice were placed in a warm environment for recovery and returned to their cages for free movement after regaining consciousness. Postoperative wound healing and activity levels were routinely observed. This study used bilateral knee joint modeling.

[0112] 1.6.2 Grouping and Dosing

[0113] After DMM modeling was completed, the mice were randomly divided into four groups: Sham group (only the joint was exposed, without cutting the medial meniscus and tibial ligament), DMM group (model control group), DMM + Cur-ALG group, and DMM + Cur-ALG + LIFU group, with 8 mice in each group.

[0114] Intervention began in the first week post-surgery. The Cur-ALG group and the combined treatment group received intra-articular injections of 10 μL Cur-ALG (with final concentrations of Cur-NPs and ALG in the combined system of 2 mg / mL and 20 mg / mL, respectively) per knee, once weekly for 6 weeks. The DMM group received an equal volume of PBS as a control. Both knees underwent injection. The combined treatment group received local LIFU intervention immediately after each intra-articular injection. Ultrasound parameters were set as follows: frequency 1 MHz, sound intensity 0.5 W / cm². 2The duty cycle was 20%, with each irradiation lasting 5 minutes, once a week. Coupling agent was applied to the knee joint surface before irradiation. Mice were sacrificed 8–10 weeks post-surgery, and knee joint specimens were collected for Micro-CT scanning, histological staining, and subsequent analysis.

[0115] 1.6.3 Micro-CT Scanning and 3D Reconstruction Analysis

[0116] High-resolution scanning of mouse knee joint specimens was performed using a small animal Micro-CT system (Inveon, Siemens). After collection, knee joint specimens were fixed in 4% PFA and brought to room temperature before scanning. Scanning parameters were set as follows: tube voltage 80 kV, tube current 500 μA, and resolution 10–20 μm. The region of interest (ROI) was the articular region of the distal femur and proximal tibia, including the medial and lateral subchondral bone and osteophyte formation areas. Three-dimensional reconstruction of the images was performed using Inveon's built-in Multimodal 3D Visualization software, and quantitative analysis was conducted on each group of samples. Measurement parameters included osteophyte volume (mm²). 3 The data included the joint space width (mm). All analyses were performed under uniform threshold conditions to ensure data consistency.

[0117] 1.6.4 Histological staining and scoring

[0118] 1.6.4.1 Tissue decalcification and paraffin embedding

[0119] Knee joint specimens were fixed in 4% PFA for 48 h, then placed in 0.5 M EDTA decalcification solution at 4°C for decalcification. The decalcification solution was changed every 2–3 days for 4–8 weeks until complete decalcification. After decalcification, the specimens were dehydrated with graded ethanol, cleared, and embedded in paraffin to prepare 4–5 μm serial sections for later use.

[0120] 1.6.4.2 Safranin O–Fast Green Staining

[0121] Paraffin sections were dewaxed, rehydrated with graded ethanol, and stained using the Safranin O-Fast Green kit. The sections were then stained with Fast Green and Safranin sequentially, dehydrated with graded ethanol, cleared with xylene, and mounted. The staining of cartilage matrix proteoglycans was observed under a microscope, and images were acquired for analysis.

[0122] 1.6.4.3 H&E staining

[0123] Paraffin sections were dewaxed, rehydrated, and then stained with hematoxylin and eosin (H&E). After staining, they underwent gradient dehydration, clearing, and mounting. The articular cartilage structure and synovial inflammation were observed under a microscope and photographed for documentation.

[0124] 1.6.4.4 OARSI Score

[0125] The degree of cartilage degeneration was assessed using the OARSI-recommended scoring criteria on Safranin O–Fast Green stained sections. The scoring criteria included cartilage surface integrity, the degree of matrix staining loss, and changes in cartilage thickness, with a score range of 0-6. Three to five consecutive sections from each sample were evaluated, and the average score was used as the final score. All scores were completed by two independent observers under blinded conditions, meaning the raters interpreted the scores without knowing the grouping information. If there were significant discrepancies in the scores, a consensus was reached through discussion.

[0126] 1.6.4.5 Synovial Inflammation Score

[0127] H&E stained sections were used to assess the degree of synovial inflammation. A semi-quantitative scoring system was established based on synovial cell layer thickening, the degree of inflammatory cell infiltration, and changes in synovial tissue structure. The scoring criteria referenced previous literature, classifying synovial inflammation into a score of 0–3 (0 for no significant inflammation, 3 for significant inflammatory cell infiltration and synovial hyperplasia). Representative fields of view were selected for scoring from each sample, and a blinded assessment method was used. The average value was then used for statistical analysis.

[0128] 1.7 Statistical Analysis

[0129] All experimental data are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was used for comparisons between groups. If the differences were statistically significant, Tukey's method was used for subsequent multiple comparisons. Independent samples t-tests were used for comparisons between two groups. Non-parametric tests were used for non-normally distributed data. Statistical analysis was performed using SPSS software (27.0.1) and R software. A p-value < 0.05 was considered statistically significant.

[0130] 2. Results

[0131] 2.1 Construction and Physicochemical Characterization of Cur-NPs

[0132] To construct curcumin liposome nanoparticles (Cur-NPs) with ultrasonic response potential, this study prepared a phase change liposome system using a rotary evaporation-thin film hydration combined with ultrasonic emulsification method, and systematically characterized its morphology, particle size, potential and stability.

[0133] Transmission electron microscopy results showed that the unloaded curcumin liposome nanoparticles (Lip NPs) exhibited a regular spherical structure with clear boundaries and good dispersibility. Figure 1 A). After being loaded with curcumin, Cur-NPs also maintained a relatively uniform spherical morphology ( ). Figure 1 (B) No obvious aggregation or structural collapse was observed overall, suggesting that the drug loading did not disrupt the basic structure of the liposomes.

[0134] Dynamic light scattering results showed that the average particle size of Lip NPs was 102.1 ± 0.3 nm; after loading curcumin, the particle size of Cur-NPs increased to 163.7 ± 11.9 nm. Figure 1 The increased particle size suggests that curcumin has successfully intercalated into the lipid bilayer or internal structure. Zeta potential analysis showed that the Lip NPs potential was -1.11 ± 0.39 mV, while the Cur-NPs potential was -4.97 ± 0.62 mV. Figure 1 The presence of a .D indicates that the surface charge of the nanoparticles changed somewhat after drug loading, but the overall structure remained negatively charged and dispersed. To assess the storage stability of the system, this study further examined the particle size changes of the two types of nanoparticles stored at 4°C for 7 days. The results showed that neither Lip NPs nor Cur-NPs exhibited significant changes in particle size over the 0–7 days. Figure 1 (E–F, P>0.05), indicating that the nanosystem has good particle size stability under short-term storage conditions.

[0135] In summary, this study successfully constructed Cur-NPs with complete structure, moderate particle size, and good stability, providing a reliable material basis for subsequent in vitro and in vivo experiments.

[0136] To further evaluate the drug loading capacity of Cur-NPs, this study prepared nanoparticles under different curcumin dosages (0.1–1.0 mg) and measured their encapsulation efficiency and drug loading rate. The results showed that within the dosage range of 0.1–1.0 mg, the encapsulation efficiency of Cur-NPs remained at a high level (approximately 85%–90%). Figure 2 The liposome system (.A) indicates that it has a good loading capacity for curcumin. The loading rate gradually increases with increasing feed amount. Figure 2 The value of .B) reaches its maximum at a feed amount of 1.0 mg, indicating that the system has room for improvement in load capacity.

[0137] UV-Vis spectroscopy analysis showed that the Cur-NPs solution after DMF sphere breaking treatment exhibited a significant absorption peak at approximately 430 nm. Figure 2The absorbance wavelength (.C) is consistent with the characteristic absorption wavelength of curcumin, further confirming the successful loading of curcumin into liposomes. Standard curve results show that within the detected concentration range, absorbance exhibits a good linear relationship with concentration (R² = 0.9994). Figure 2 The results (.D) provide a reliable basis for subsequent drug loading calculations. In summary, the Cur-NPs constructed in this study have high encapsulation efficiency and stable drug loading capacity, and the UV spectral characteristics and standard curve verification support their successful drug loading.

[0138] 2.2 Formulation optimization and characterization of Cur-ALG hydrogel composite system

[0139] 2.2.1 Cur-ALG Formulation Optimization

[0140] To construct a Cur-ALG hydrogel nanocomposite system with in-situ gelation capability, this study optimized and screened the Cur-NPs / ALG concentration and stirring conditions.

[0141] First, under the condition of a fixed final ALG concentration of 20 mg / mL, the dispersion of Cur-NPs at different concentrations (0, 0.5, 1, 1.5, and 2 mg / mL) in the system was investigated. The results showed that Cur-NPs could be uniformly dispersed in the ALG solution within all concentration ranges. Figure 3 No obvious precipitation or stratification was observed in the initial sample (.A), indicating that the system has good nanoparticle loading capacity. Subsequently, under the condition of a fixed final concentration of Cur-NPs of 2 mg / mL, the effects of different ALG concentrations (5, 10, 20 mg / mL) on gelation properties were compared. The results showed that with the increase of ALG concentration, the formed hydrogel structure became more stable and the toughness was enhanced. Considering both gelation effect and operability, 20 mg / mL ALG was finally selected as the optimal formulation concentration. In addition, different stirring speeds also had a certain impact on the stability of the system. Higher stirring speeds may induce local phase transitions and form bubbles. Therefore, low-speed stirring (about 100 rpm) under ice bath conditions was used for 3-5 minutes until completely mixed to prepare the Cur-ALG system.

[0142] In summary, the optimized formulation for Cur-ALG was determined to be: Cur-NPs 2 mg / mL, ALG 20 mg / mL (final concentration), prepared by low-speed stirring in an ice bath. Macroscopic observation showed that the Cur-NPs solution was a uniform yellow liquid.

[0143] ALG solution is a transparent, viscous liquid. When the two are mixed, they form a pale yellow, homogeneous, viscous system. Figure 3 (B) No obvious stratification or precipitation was observed in the system, suggesting that Cur-NPs can be stably dispersed in the ALG matrix.

[0144] 2.2.2 Microstructure characterization of Cur-ALG hydrogel

[0145] To further observe the microstructure of Cur-ALG hydrogels after gelation, this study used cryo-scanning electron microscopy (Cryo-SEM) to analyze the internal morphology of the hydrogels. The ALG hydrogels without curcumin loading exhibited a regular porous three-dimensional network structure. Figure 4 (A–B) The pore size distribution is relatively uniform, and the pore wall structure is continuous and intact, exhibiting typical characteristics of cross-linked polysaccharide gels. The Cur-ALG hydrogel formed after loading Cur-NPs also maintains a porous network structure. Figure 4 The overall pore morphology (C–D) showed no significant collapse or disruption, indicating that the introduction of nanoparticles did not significantly affect the basic framework structure of the hydrogel. Meanwhile, local structural changes were observed on the pore walls and inside the pores, suggesting that the nanoparticles were successfully embedded in the hydrogel network. These results demonstrate that Cur-NPs can be uniformly distributed within the hydrogel matrix without disrupting the three-dimensional structure of the ALG, providing a structural basis for subsequent drug release and ultrasonic response functions.

[0146] 2.2.3 In vitro stability of Cur-ALG under different physiological environments

[0147] To evaluate the stability of Cur-ALG hydrogels in simulated physiological environments, this study used Cur-ALG hydrogels after gelation.

[0148] ALG was immersed in DMEM, PBS, and physiological saline, and macroscopic morphological changes were observed over 7 days. The results showed that Cur-ALG hydrogels maintained their basic morphological structure stability under all three solution conditions. Figure 5 During the observation period, the hydrogel did not exhibit significant swelling, collapse, or structural disintegration, and its overall outline remained intact. Different solution environments did not significantly affect the appearance of the hydrogel, suggesting that the Cur-ALG system possesses good in vitro stability. These results demonstrate that the Cur-ALG hydrogel can maintain good structural integrity under common culture environments and simulated physiological conditions, providing a material stability basis for subsequent in vivo applications.

[0149] 2.2.4 Rheological Characterization

[0150] To further evaluate the gelation behavior and mechanical properties of the Cur-ALG system, we performed time-scan rheological tests on ALG solution, Cur-ALG solution, ALG gel, and Cur-ALG gel. The results showed that in the solution state (ALG solution and Cur-ALG solution), the loss modulus (G″) was higher than the storage modulus (G′), indicating that the system exhibits predominantly viscous behavior and fluid-like mechanical characteristics. However, with the addition of Ca... 2+ In the subsequently formed ALG gel and Cur-ALG gel, the storage modulus (G′) is significantly higher than the loss modulus (G″), indicating that the system exhibits predominantly elastic behavior and typical gel-like mechanical characteristics. Furthermore, the G′ values ​​of Cur-ALG gel and ALG gel are on the same order of magnitude, demonstrating that the introduction of Cur-NPs did not disrupt the gel-forming ability of the ALG network structure. These results prove that the Cur-ALG system can successfully form a stable three-dimensional cross-linked network structure under ionic cross-linking conditions, possessing excellent gel mechanical support capabilities, providing a materials science basis for subsequent in-situ injection and controlled-release applications.

[0151] 2.3 Effects of Cur-NPs on chondrocyte viability

[0152] CCK-8 assay results showed that, compared with the control group, chondrocyte viability was significantly decreased after 24 h of IL-1β stimulation (P < 0.0001), indicating that the in vitro injury model was successfully established by inflammatory stimulation. Compared with the IL-1β group, Cur-NPs treatment significantly improved cell survival (P < 0.001), indicating that it has a certain protective effect against inflammatory damage. Furthermore, the combination of LIFU further increased cell viability (P < 0.05), suggesting that LIFU can enhance the protective effect of CurNPs. Figure 6 This result indicates that Cur-NPs have a protective effect on chondrocytes under inflammatory conditions, and LIFU can further enhance their biological effects.

[0153] 2.4 Cur-NPs combined with LIFU reduce oxidative stress levels

[0154] DCFH-DA fluorescence staining results showed that, compared with the control group, the green fluorescence intensity in chondrocytes was significantly enhanced after IL-1β stimulation, indicating a significant increase in ROS levels. Compared with the IL-1β group, the ROS fluorescence intensity was significantly reduced after Cur-NPs treatment. Combined LIFU treatment further decreased ROS levels. Quantitative fluorescence intensity analysis results were consistent with the microscopic images; the ROS level in the IL-1β group was significantly higher than that in the control group (P < 0.001), the Cur-NPs group significantly reduced ROS levels (P < 0.01), and the combined LIFU treatment group further reduced ROS levels (P < 0.05). Figure 7 These results suggest that Cur-NPs can alleviate inflammatory-induced oxidative stress damage, and that LIFU-assisted antioxidant activity can enhance their antioxidant effects.

[0155] 2.5 Cur-NPs combined with LIFU ameliorate the IL-1β-induced decrease in mitochondrial membrane potential

[0156] To assess the effects of Cur-NPs and LIFU on mitochondrial function, this study used TMRE fluorescent probes to detect mitochondrial membrane potential (ΔΨm) in chondrocytes. Fluorescence microscopy revealed that, compared to the control group, IL-1β stimulation significantly reduced intracellular TMRE red fluorescence, indicating a significant decrease in mitochondrial membrane potential. Cur-NPs treatment significantly enhanced the red fluorescence signal, indicating partial recovery of mitochondrial membrane potential. Combined LIFU treatment further enhanced TMRE fluorescence, bringing it close to the control group level. Figure 8 As a positive control, the FCCP-treated group almost completely lost TMRE fluorescence signal, verifying the effectiveness of the experimental system. Quantitative fluorescence intensity analysis results were consistent with image observation. The membrane potential level in the IL-1β group was significantly lower than that in the control group (P < 0.001); Cur-NPs treatment significantly improved the decrease in membrane potential (P < 0.01); and the recovery effect was further enhanced after combined LIFU treatment (P < 0.05). These results indicate that Cur-NPs can alleviate mitochondrial functional damage caused by inflammatory stimulation, and LIFU can enhance its protective effect.

[0157] 2.6 Cur-NPs combined with LIFU regulate the expression of inflammation and matrix degradation-related proteins

[0158] To further evaluate the effects of Cur-NPs and LIFU on cartilage degeneration phenotypes, this study used Western blot to detect the expression levels of matrix synthesis-related proteins (ACAN, COL2A1) and inflammation and matrix degradation-related proteins (NOS, PTGS2, MMP-13). Figure 9The results showed that, compared with the control group, IL-1β stimulation significantly upregulated the expression of NOS2, PTGS2, and MMP-13 proteins, while significantly downregulating the expression of ACAN and COL2A1 (P < 0.001), indicating that inflammatory stimulation successfully induced the cartilage degeneration phenotype. Compared with the IL-1β group, Cur-NPs treatment significantly reduced the expression of NOS2, PTGS2, and MMP-13, while partially restoring the expression levels of ACAN and COL2A1 (P < 0.05). Combined LIFU treatment further enhanced these effects, with a further decrease in the expression of inflammation-related proteins and a further upregulation of matrix synthesis proteins.

[0159] The above results indicate that Cur-NPs can effectively alleviate the metabolic imbalance of chondrocyte matrix caused by inflammatory stimulation, and LIFU can enhance its protective effect.

[0160] 2.7 Cur-ALG combined with LIFU improves joint structural degeneration in DMM mice

[0161] To evaluate the therapeutic effect of Cur-ALG combined with low-intensity ultrasound on an in vivo model of osteoarthritis, this study established a mouse OA model using DMM surgery, and performed Micro-CT scanning and three-dimensional reconstruction analysis after the intervention. Figure 10 Micro-CT three-dimensional reconstruction results showed that, compared with the Sham group, the DMM group mice exhibited significant osteophyte formation and irregular changes in the joint margin structure. Cur-ALG intervention significantly reduced the number and volume of osteophytes; combined with low-intensity ultrasound treatment, osteophyte formation was further alleviated, and the overall joint structure remained relatively intact. Quantitative analysis of osteophyte volume showed that the osteophyte volume in the DMM group was significantly higher than that in the Sham group (P < 0.001); Cur-ALG intervention significantly reduced osteophyte volume (P < 0.05); and combined with low-intensity ultrasound treatment, the osteophyte volume showed a further decreasing trend. These results indicate that Cur-ALG combined with low-intensity ultrasound can effectively alleviate DMM-induced joint structural degeneration in mice.

[0162] 2.8 Histological staining and scoring results

[0163] To further evaluate the effects of Cur-ALG combined with low-intensity ultrasound on joint tissue pathological changes, this study performed Safranin O-Fast Green staining and H&E staining on the knee joints of mice in each group, and analyzed OARSI scores and synovial inflammation scores. Figure 11 ).

[0164] Histological results showed that the Sham group had intact cartilage structure, smooth cartilage surface, uniform safranin staining, and clear joint space. The DMM group showed significant cartilage surface damage, weakened matrix staining, and thinning of the cartilage layer, with osteophyte formation and synovial hyperplasia at the joint margins. After Cur-ALG intervention, the cartilage surface structure was relatively intact, and matrix staining was partially restored; combined with low-intensity ultrasound treatment, cartilage matrix staining was further improved, and the cartilage layer structure became more continuous. OARSI scores showed that the DMM group score was significantly higher than the Sham group (P < 0.001); Cur-ALG treatment significantly reduced the OARSI score (P < 0.01); and the score further decreased after combined low-intensity ultrasound (P < 0.05). Synovial inflammation score analysis showed that the DMM group had significant synovial hyperplasia and inflammatory cell infiltration; the degree of inflammation decreased after Cur-ALG intervention; the combined low-intensity ultrasound group showed further improvement compared to the DMM group, but there was no statistically significant difference compared to the Cur-ALG alone group. The above results indicate that Cur-ALG combined with low-intensity ultrasound can effectively reduce DMM-induced cartilage tissue degeneration in mice and alleviate synovial inflammation to some extent.

[0165] 3. Analysis and Summary

[0166] This study systematically investigated the interventional effects and potential mechanisms of curcumin nanoparticles combined with LIFU on osteoarthritis (OA). Results showed that Cur-ALG combined with LIFU could ameliorate IL-1β-induced chondrocyte inflammatory damage in vitro, manifested as increased cell viability, decreased ROS levels, and restored mitochondrial membrane potential, while also regulating the expression of matrix metabolism-related proteins. In a DMM mouse model, the combined intervention significantly reduced osteophyte formation and cartilage structure destruction, suggesting a similar protective effect in vivo. Further SMR analysis revealed a potential association between multiple curcumin-related targets and OA risk at the genetic level, some of which are closely related to mitochondrial homeostasis regulation. Based on these clues, this study conducted preliminary validation at the protein level, providing support for the hypothesis that "mitochondrial function participates in the material intervention process."

[0167] Overall, this study has formed a relatively complete chain of evidence, from material construction and in vitro and in vivo functional verification to genetically assisted screening, providing experimental evidence for the application of curcumin nanodelivery systems in OA and its potential mechanism of action.

[0168] 3.1 The rationale and potential advantages of the combined strategy of curcumin nanodelivery and low-intensity focused ultrasound

[0169] The results of this study show that Cur-ALG combined with LIFU exhibits a superior improvement trend compared to single interventions in both in vitro and in vivo models. This suggests that drug intervention alone may be insufficient to achieve ideal results in the complex joint microenvironment, while a combined physical and drug strategy may be more helpful in enhancing the treatment response.

[0170] Previous studies have shown that curcumin possesses anti-inflammatory and antioxidant effects, but its in vivo application is limited by factors such as poor solubility, low stability, and limited tissue penetration, especially in the context of intra-articular local administration, where maintaining a sustained effective concentration is difficult. Therefore, nanodelivery systems have been widely used to improve the bioavailability and release characteristics of curcumin. However, some studies have also indicated that while nanocarriers can improve drug stability, their distribution and release in complex tissue environments may still be limited by diffusion barriers and cellular uptake efficiency.

[0171] Low-intensity focused ultrasound (HIFU), as a non-invasive physical stimulation method, has been reported to enhance cell membrane permeability, promote drug release, and improve the local microcirculation environment. In joint disease research, ultrasound-assisted drug delivery has gained increasing attention, but its specific mechanisms of action and synergistic effects with specific drugs remain unclear. Some studies suggest that ultrasound may enhance drug entry efficiency into cells through mechanical vibration or cavitation effects, but other literature indicates that inappropriate ultrasound parameter selection may have stress effects on cells. Therefore, the effectiveness of combined strategies depends on reasonable dosage and parameter control. Based on preliminary experiments optimizing ultrasound parameters, this study observed a more significant improvement trend in ROS levels, mitochondrial membrane potential, and histological scores in the combined intervention group, consistent with the expectation of "physical enhancement of drug effects." This suggests that under appropriate conditions, HIFU may amplify the anti-inflammatory and antioxidant effects of curcumin by promoting the release of nanocarriers or enhancing cellular uptake of curcumin. Of course, this study has not directly verified the specific physical mechanism of ultrasound enhancement, therefore, other possible biological regulatory effects cannot be ruled out. Future research could further combine real-time release detection or cellular uptake experiments to more precisely elucidate the synergistic mechanism between the two.

[0172] 3.2 Significance of Curcumin's Anti-inflammatory Effects in Osteoarthritis

[0173] In an in vitro model, this study observed that IL-1β stimulation significantly upregulated the expression of NOS2, PTGS2, and MMP-13, while inhibiting the expression of ACAN and COL2A1. These trends were significantly improved after Cur-ALG combined with low-intensity ultrasound intervention. These results suggest that curcumin-based materials can regulate the matrix metabolism of chondrocytes under inflammatory conditions.

[0174] The anti-inflammatory effects of curcumin have been reported in various disease models, exerting a protective effect by inhibiting the expression of inflammatory factors and the activity of related signaling pathways. Consistent with previous studies, this study also observed that the curcumin nanodelivery system inhibited inflammation-related proteins. However, explaining its protective effect solely by "anti-inflammatory" may be insufficient. On the one hand, inflammation regulation has received considerable attention in research; on the other hand, simply inhibiting inflammation cannot completely block the OA process, suggesting the existence of deeper cellular function regulatory mechanisms.

[0175] Notably, in this study, the improvement in inflammatory markers was accompanied by a decrease in ROS levels and a recovery in mitochondrial membrane potential. This suggests that the effects of material intervention may not be limited to the level of inflammatory signaling, but may involve cellular energy metabolism and mitochondrial homeostasis regulation.

[0176] 3.3 The effects of curcumin on osteoarthritis may go beyond simple anti-inflammatory mechanisms.

[0177] In recent years, osteoarthritis has gradually shifted from a simple "inflammatory degeneration" model to a "metabolic-inflammatory coupling imbalance" model. Chondrocytes, as cells with low turnover rates, are highly dependent on mitochondrial homeostasis. Multiple studies have indicated the widespread accumulation of ROS, decreased mitochondrial membrane potential, and suppressed mitochondrial biogenesis in OA cartilage, suggesting that energy metabolism disorders may precede obvious structural damage. Therefore, understanding OA progression at the mitochondrial level may be more explanatory than simply inhibiting inflammation. The antioxidant effect of curcumin has long been attributed to its free radical scavenging ability, but increasing evidence suggests that its regulation may involve mitochondrial functional remodeling, rather than just passive antioxidant activity. This study observed changes in molecules related to mitochondrial biogenesis and antioxidant defense in an inflammatory model, suggesting that material intervention may touch upon the mitochondrial regulatory axis. This level of action differs from the traditional approach of simple anti-inflammatory treatment and is closer to the reconstruction of cellular metabolic homeostasis.

[0178] Notably, genetic analysis also suggests a link between some mitochondrial-related genes and OA risk. While genetic associations do not directly equate to drug targets, they indicate the potential role of mitochondrial homeostasis in disease susceptibility. This evidence from different levels corroborates each other to some extent, making "mitochondrial regulation" a direction worthy of further investigation.

[0179] From a treatment strategy perspective, if inflammation is a superficial phenomenon, while mitochondrial dysfunction represents an earlier or more fundamental change, then interventions targeting mitochondrial homeostasis may have longer-term significance in delaying disease progression. This may provide new theoretical support for the combined application of material delivery systems and physical stimulation.

Claims

1. A method for preparing ultrasound-responsive curcumin / perfluoropentane co-supported liposome nanoparticles, characterized in that, The steps include the following: S1. Preparation of curcumin liposome membranes: Curcumin liposome membranes were prepared by rotary evaporation-membrane hydration method; S2. Hydration and PFP emulsification: PBS buffer was added to the curcumin liposome film prepared in step S1, and hydration was carried out at 35-40°C with stirring. The resulting lipid suspension was transferred to a centrifuge tube, and perfluoropentane PFP was added dropwise under ice bath conditions. Subsequently, ultrasonic emulsification was performed, followed by low-temperature high-speed centrifugation. The supernatant was discarded, and the suspension was resuspended in PBS and washed to obtain ultrasonically responsive curcumin / perfluoropentane co-loaded liposome nanoparticles Cur-NPs.

2. The preparation method according to claim 1, characterized in that: S1. Preparation of curcumin liposome membrane: A rotary evaporation-membrane hydration method was used. DPPC, DSPE, cholesterol, and curcumin were weighed and added to a rotary evaporation flask. Anhydrous chloroform was added, and the mixture was sonicated until completely clear under light-protected conditions. A uniform liposome membrane was formed by rotary evaporation under the following conditions: 35~37 ℃, 120~150 rpm, -0.07~-0.09 MPa. Subsequent vacuum drying was performed to remove residual solvent. The ratio of DPPC, DSPE, cholesterol, curcumin, and anhydrous chloroform was 5~7 mg : 1.5~2.5 mg : 1.5~2.5 mg : 1 mg : 4~6 mL.

3. The preparation method according to claim 2, characterized in that: S2. Hydration and PFP Emulsification: PBS buffer was added to the curcumin liposome membrane prepared in step S1, and hydration was carried out at 35-40 °C and 120-150 rpm for 25-35 min. The resulting lipid suspension was transferred to a centrifuge tube, and perfluoropentane PFP was added dropwise under ice bath conditions. Emulsification was then carried out using an ultrasonic disruptor at 140-160 W, 35-45% power, 4-6 s working time / 4-6 s interval, for a total of 5-7 min. After emulsification, the mixture was centrifuged at 1-4 °C and 8000-12000 rpm for 8-12 min. The supernatant was discarded, and the mixture was resuspended in PBS and washed to obtain ultrasound-responsive curcumin / perfluoropentane co-loaded liposome nanoparticles Cur-NPs.

4. The preparation method according to claim 3, characterized in that: In step S2, perfluoropentane PFP is added dropwise to the lipid suspension at a volume ratio of 8~12:

1. The lipid suspension is prepared by adding 3~5 mL of PBS buffer to the curcumin liposome membrane prepared in step S1 using 1 mg of curcumin.

5. An ultrasound-responsive curcumin / perfluoropentane co-supported liposome nanoparticle, characterized in that: It is prepared by the method described in any one of claims 1 to 4.

6. A hydrogel co-loaded with curcumin / perfluoropentane lipid nanoparticles, characterized in that: The composite system is obtained by mixing the ultrasound-responsive curcumin / perfluoropentane co-supported liposome nanoparticles (Cur-NPs) as described in claim 5 with sodium alginate (ALG) solution under low-speed stirring in an ice bath.

7. The method for preparing the co-loaded curcumin / perfluoropentane lipid nanoparticle hydrogel according to claim 6, characterized in that, The method includes the following steps: mixing the ultrasound-responsive curcumin / perfluoropentane co-loaded liposome nanoparticles (Cur-NPs) as described in claim 5 with sodium alginate (ALG) solution under low-speed stirring in an ice bath to form a composite system, thereby obtaining the co-loaded curcumin / perfluoropentane lipid nanoparticle hydrogel Cur-ALG; the concentration of Cur-NPs in the composite system is 0.5~2 mg / mL, and the concentration of ALG is 5~20 mg / mL.

8. The preparation method according to claim 7, characterized in that: The low-speed stirring is 100~300 rpm, the stirring time is 3~5 min, the concentration of Cur-NPs in the composite system is 1~2 mg / mL, and the concentration of ALG is 10~20 mg / mL.

9. The use of the ultrasound-responsive curcumin / perfluoropentane co-loaded liposome nanoparticles Cur-NPs as described in claim 5 or the co-loaded curcumin / perfluoropentane lipid nanoparticle hydrogel Cur-ALG as described in claim 6 in the preparation of a medicament for treating osteoarthritis.

10. The application according to claim 9, characterized in that: The application involves combining Cur-NPs or Cur-ALG with LIFU, with the subject receiving local LIFU intervention immediately after each intra-articular injection of Cur-NPs or Cur-ALG.

Images