Active oxygen degradable polydiacetylene prodrug as well as preparation and application thereof

Abstract

The invention relates to an active oxygen degradable polydiacetylene prodrug as well as preparation and application thereof, and belongs to the technical field of medicines. By utilizing the unique property that a main chain of a polydiacetylene polymer is completely broken into carboxyl-containing small molecules when the active oxygen level is increased, a carboxyl-containing drug is used as a raw material to construct a polydiacetylene prodrug, so that long-acting on-demand drug release of active oxygen response is realized. According to the prodrug, active oxygen is utilized to trigger a conjugated main chain to break so as to release an active drug, and meanwhile, a biocompatible small molecule by-product is generated. According to the polydiacetylene polymer side chain covalent connection medicine carrying system, the phenomenon of burst release of the medicine is effectively avoided, continuous administration as long as several months is achieved, and the polydiacetylene polymer side chain covalent connection medicine carrying system has high focus site selectivity and extremely low systemic toxicity. The active oxygen responsive degradation process and drug release cooperate to play a therapeutic role, and a negative feedback regulation drug release loop is formed, so that excessive administration and whole body exposure are avoided, on-demand remodeling of a microenvironment is realized, and the therapeutic effect is improved.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and more specifically, relates to a reactive oxygen species degradable polydiacetylene prodrug, its preparation and application. Background Technology

[0002] Osteoarthritis (OA), a chronic disease, is characterized by its long course and recurrent flare-ups, often requiring lifelong management and imposing a significant clinical burden. Patients frequently experience persistent joint pain, swelling, and decreased mobility, severely impacting their quality of life. Although existing medications are available, traditional therapies with short half-lives typically require frequent dosing and are accompanied by systemic side effects, posing challenges to long-term treatment. For such chronic diseases, an ideal long-acting drug delivery system should maintain effective drug concentrations at the lesion site, reduce dosing frequency, and improve patient compliance. However, the pathogenesis of OA is complex, exhibiting significant heterogeneity among individuals and at different stages of the disease. This necessitates treatment strategies that not only possess sustained-release capabilities but also achieve on-demand dosing—intelligently responding to dynamic fluctuations in the local inflammatory microenvironment to regulate drug release, thereby effectively controlling symptoms while minimizing systemic exposure and toxicity risks.

[0003] To address these challenges, extensive research has focused on developing drug delivery materials capable of achieving long-acting and controlled release. Microenvironment-responsive delivery systems designed to address pathological signals such as altered pH levels at inflammatory sites, elevated reactive oxygen species (ROS), or enzyme overexpression have significantly improved drug selectivity and reduced systemic toxicity. However, achieving precise controlled release over weeks or even months remains a major obstacle. Traditional carriers relying on physical encapsulation typically exhibit release modes primarily based on passive diffusion or bulk material degradation. These mechanisms are inherently difficult to control with high precision—especially under the dynamic and heterogeneous conditions of a chronic inflammatory microenvironment, leading to unpredictable release behavior and reduced long-term treatment consistency.

[0004] To overcome the limitations of physical encapsulation, covalently coupling drugs to responsive chemical structures within a carrier matrix has become an effective strategy for achieving controlled and selective release. By utilizing stimulus-responsive chemical bonds (such as reactive oxygen species-sensitive bonds or acid-labile bonds) that specifically break in the pathological microenvironment, this method can effectively prevent burst release and improve the precision of targeted drug release. However, despite its advantages in improving selectivity and reducing systemic toxicity, this strategy faces new challenges: drug release efficiency often depends on the rate and completeness of bond breaking under physiological conditions, a process that exhibits individual variability and disease stage heterogeneity. An ideal treatment strategy for chronic inflammation requires not only responsive release but also negative feedback regulation capabilities—that is, drug release itself synchronously regulates the microenvironment, attenuates inflammatory signals, and thus delays subsequent release, forming a self-regulating on-demand treatment cycle. Furthermore, the fate of chemically modified carrier molecules in vivo after drug release remains a concern, including their biocompatibility, degradation pathways, and potential long-term cumulative toxicity. Therefore, there is an urgent need to develop drug carriers that combine high efficiency, covalent responsiveness, complete degradability, and long-term self-regulating drug release capabilities, so as to achieve precise and long-acting drug delivery while ensuring safety and reducing metabolic burden. Summary of the Invention

[0005] This invention provides a reactive oxygen species (ROS)-degradable poly(diacetylene) prodrug, its preparation, and its application. Utilizing the unique property of poly(diacetylene) polymers where the main chain completely breaks down into carboxyl-containing small molecules when ROS levels rise, it achieves highly efficient ROS-triggered release of the active parent drug, while simultaneously generating biocompatible, non-toxic small molecule byproducts. This poly(diacetylene) polymer side chain covalently links to the drug-loaded system, effectively avoiding drug burst release and enabling continuous drug delivery for several months, with high site selectivity and extremely low systemic toxicity. Its release process is directly coupled with local ROS levels, forming an endogenous negative feedback regulatory loop: ROS triggers drug release and carrier degradation; the released drug and degradation products synergistically exert the original drug's pharmacological and antioxidant effects; thereby reducing local ROS levels and slowing subsequent release, achieving on-demand remodeling of the microenvironment and improving therapeutic efficacy. This solves the technical problems of drug burst release or the inability to achieve long-term self-regulated on-demand drug release in existing technologies, and is completely degradable with good biocompatibility.

[0006] According to a first aspect of the present invention, a polydiacetylene prodrug is provided, the polydiacetylene prodrug having the structural formula shown in Formula I: ; Where R represents the portion of the drug containing a carboxyl group that is linked to the carboxyl group.

[0007] Preferably, the carboxyl-containing drug is an arylpropionic acid nonsteroidal anti-inflammatory drug.

[0008] Preferably, the aryl propionic acid nonsteroidal anti-inflammatory drug is naproxen, ketoprofen, flurbiprofen, or ibuprofen.

[0009] According to another aspect of the present invention, a method for synthesizing the polydiacetylene prodrug is provided, comprising the following steps: a) Add the drug containing a carboxyl group and the amide reaction catalyst to an organic solvent to activate the carboxyl group; then add propargylamine dropwise to induce an amide reaction and obtain a terminal alkyne intermediate; b. Add the terminal alkyne intermediate obtained in step a and the terminal alkyne oxidative coupling reaction catalyst to an organic solvent, continuously introduce oxygen, and undergo an alkyne oxidative coupling reaction to obtain a diyne monomer. c. Method 1: The diacetylene monomer obtained in step b is induced to undergo topological polymerization by heating or ultraviolet light to obtain a polydiacetylene prodrug; Alternatively, in method two: the diacetylene monomer and amphiphilic surfactant obtained in step b are added to an organic solvent and ground until the solvent evaporates to form a homogeneous paste. Then water is added and ground to disperse the mixture to obtain an aqueous dispersion of the diacetylene monomer. Then, the diacetylene monomer is induced to undergo topological polymerization by ultraviolet light to obtain an aqueous dispersion of the polyacetylene prodrug.

[0010] Preferably, the carboxyl-containing drug is an arylpropionic acid nonsteroidal anti-inflammatory drug.

[0011] Preferably, the aryl propionic acid nonsteroidal anti-inflammatory drug is naproxen, ketoprofen, flurbiprofen, or ibuprofen.

[0012] Preferably, in step a, the catalyst for the amide reaction is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole; In step b, the catalyst for the terminal alkyne oxidative coupling reaction is cuprous chloride and N,N,N',N'-tetramethylethylenediamine.

[0013] Preferably, in step c, before heating or ultraviolet irradiation, a step of static crystallization is included to allow the diyne monomers to arrange in an orderly manner. In step c, the heating temperature is 50-200℃, and the wavelength of the ultraviolet light irradiation is 200-400 nm.

[0014] According to another aspect of the present invention, the application of the aforementioned polydiacetylene prodrug in the preparation of long-acting controlled-release drugs for chronic diseases is provided.

[0015] Preferably, the chronic disease is osteoarthritis, rheumatoid arthritis, immune system disease, or chronic pain.

[0016] In summary, compared with the prior art, the above-described technical solutions conceived by this invention mainly possess the following technical advantages: (1) This invention achieves ultra-long-acting, on-demand precise drug delivery. The prodrug system covalently links drug molecules to the polydiacetylene backbone, effectively avoiding the initial burst release problem caused by traditional physical encapsulation. Its unique reactive oxygen species-responsive degradation mechanism directly links the drug release process to the inflammation level at the lesion site, realizing microenvironment-responsive intelligent on-demand drug delivery. A single dose can maintain efficacy for several months in animal models.

[0017] (2) The prodrug of this invention utilizes reactive oxygen species to trigger the breakage of the conjugated main chain to release the active drug, while simultaneously generating biocompatible small molecule byproducts. This poly(diacetylene) polymer side chain covalently linked drug delivery system effectively avoids drug burst release, enabling continuous drug administration for several months, and exhibits high lesion site selectivity and extremely low systemic toxicity. Its reactive oxygen species-responsive degradation process synergistically exerts a therapeutic effect with drug release, forming a negative feedback regulation drug release loop, thereby avoiding over-administration and systemic exposure, achieving on-demand remodeling of the microenvironment and improving therapeutic efficacy.

[0018] (3) This invention achieves synergistic therapy of drug release and microenvironment regulation. The prodrug system not only serves as a drug reservoir but is also a therapeutic component itself. Simultaneously with reactive oxygen species triggering drug release, the polydiacetylene backbone undergoes complete degradation. This process simultaneously removes excess reactive oxygen species from the microenvironment, exerting an antioxidant therapeutic effect. Drug release and reactive oxygen species scavenging work synergistically to jointly reshape the microenvironment.

[0019] (4) The polydiacetylene prodrug of the present invention achieves a self-regulating negative feedback treatment cycle. The locally elevated reactive oxygen species trigger drug release and carrier degradation. The released drug and its degradation products work together to exert pharmacological and antioxidant effects, thereby reducing the local reactive oxygen species level. The reduction in the reactive oxygen species level, in turn, slows down the subsequent drug release rate. This self-regulating characteristic greatly improves the safety and long-term controllability of the treatment.

[0020] (5) The polydiacetylene prodrug of this invention combines high therapeutic efficacy with high safety. This is due to the stability of the polydiacetylene backbone under physiological conditions and its complete degradation only under pathologically high levels of reactive oxygen species. The prodrug exhibits high selectivity at its target site, minimizing off-target effects and systemic exposure. Its final degradation products are biocompatible small molecules, avoiding the long-term cumulative toxicity risks associated with non-degradable carrier materials, thus providing crucial safety assurance for clinical translation.

[0021] (6) This invention provides a scalable modular drug delivery platform technology with a universal design strategy. The controlled polymerization of polydiacetylene and the ROS-responsive degradation backbone can serve as a universal platform, covalently linking different therapeutic drug molecules, providing a novel approach and solution for developing new long-acting formulations for various chronic diseases requiring long-term on-demand treatment. Attached Figure Description

[0022] Figure 1 Synthesis and characterization of naproxen prodrug PDANPX. a) Chemical reaction equation for the synthesis of PDANPX from naproxen. b) Mass spectrum of ANPX. c) Mass spectrum of PDANPX. d) Fourier transform infrared spectra of NPX, ANPX, and PDANPX. e) Side view of the crystal structure of PDANPX determined by microcrystalline electron diffraction. f) Top view of the crystal structure of PDANPX. Color scheme: red spheres represent oxygen atoms; blue spheres represent nitrogen atoms; gray spheres represent carbon atoms; hydrogen atoms are omitted. g) Chemical structural formula of PDANPX. h) Fluorescence emission spectrum of PDANPX (excitation wavelength = 488 nm).

[0023] Figure 2 ¹H NMR spectra of ANPX and DANPX. a) ¹H NMR characterization of ANPX. ¹H NMR (400 MHz, CDCl₃) δ 7.84 – 7.05 (m, 6H), 5.58 (s, 1H), 4.09 – 3.94 (m, 2H), 3.94 – 3.90 (m, 3H), 3.80 – 3.59 (m, 1H), 2.14 (t, J = 2.5 Hz, 1H), 1.59 (t, J = 7.5 Hz, 3H).b) ¹H NMR characterization of DANPX. ¹H NMR (400 MHz, DMSO) d6) δ 8.48 (s, 2H), 8.00 – 7.00 (m, 12H), 4.05 – 3.89 (m, 4H), 3.86 (s, 6H), 3.77 – 3.65 (m, 2H), 1.60 – 1.15(m, 6H). Figure 3 NPX, ANPX, and DANPX 13 C NMR characterization. The selected area highlights the characteristic changes in the C≡C signal.

[0024] Figure 4Characterization of ANPX, DANPX, and PDANPX. a) Transmission electron microscopy (TEM) image of DANPX. b) TEM image of PDANPX. c) Powder X-ray diffraction (XRD) patterns of DANPX and PDANPX. d) Raman spectra of DANPX and PDANPX. e) Topological polymerization of PDANPX under UV irradiation, monitored by UV-Vis spectroscopy. (Inset: Change in absorbance of PDANPX at 532 nm with UV irradiation time. f) Changes in hydrodynamic diameter and count rate of DANPX and PDANPX dispersions before and after acetone treatment (n = 3). The image shows the Tyndall effect of the PDANPX dispersion after monomer dissolution. g) Hydrodynamic diameter of PDANPX dispersion. h) Zeta potential of PDANPX dispersion. i) Changes in the hydrodynamic diameter of PDANPX over 30 days, monitored by dynamic light scattering. Data are expressed as mean ± standard deviation.

[0025] Figure 5 In vitro ROS-responsive degradation and ROS scavenging of PDANPX. a) Raman spectral monitoring of the degradation process of PDANPX under HClO treatment (Raman intensity at 0.4 M + 4,6-diynediic acid at 2264 cm⁻¹ over 7 days). - ¹ The C≡C peak at this location was normalized as an internal reference. b) The percentage change in relative intensity of the C=C and C≡C Raman peaks of PDANPX during 7 days of HClO treatment. c) Monitoring the degradation of PDANPX-15 under HClO treatment for 7 days using UV-Vis spectroscopy (inset: absorbance at 532 nm as a function of time). d) The concentration-dependent HClO scavenging capacity of PDANPX quantified by the TMB method (n = 3). e) Scanning electron microscopy images of PDANPX. f) Scanning electron microscopy images of the degradation products. g) Changes in hydrodynamic diameter and zeta potential of PDANPX before and after degradation (n = 5). h) Single-photon dual-channel imaging analysis of the relative intensity of the C=C and C≡C Raman peaks before and after degradation (left). Figure 1 Optical microscope image; left Figure 2 Enlarged view of the area within the red box; Left Figure 3 : Relative intensity of C=C Raman peak; Left Figure 4 (Relative intensity of C≡C Raman peak). Data are expressed as mean ± standard deviation.

[0026] Figure 6. In vitro long-acting on-demand sustained-release of PDANPX as the active drug naproxen. a) Schematic diagram of the ROS-triggered degradation mechanism of PDANPX. b) Liquid chromatography of PDANPX degradation products. The red arrow indicates the elution peak of naproxen in the degradation products, with a retention time of 14.81 min. c) Liquid chromatography showing the elution curve of the degradation product with the same mass-to-charge ratio as naproxen. d) Mass spectrum of the degradation product with the same mass-to-charge ratio as naproxen. e) Elution curve of the degradation product with the same mass-to-charge ratio as the amide-containing intermediate. f) Mass spectrum of the degradation product with the same mass-to-charge ratio as the amide-containing intermediate. g) Elution curve of the degradation product with the same mass-to-charge ratio as glycine. h) Mass spectrum of the degradation product with the same mass-to-charge ratio as glycine. i) NPX, DANPX, PDANPX, and PDANPX degradation products in naproxen IC50. 50 Relative COX-2 enzyme activity at concentration (10 μM). j) Cumulative percentage of naproxen released from PDANPX over 31 days of ROS and protease treatment. Data are expressed as mean ± standard deviation.

[0027] Figure 7 . PDANPX endocytosis and cell safety. a) Representative confocal microscopy images of PDANPX endocytosis in RAW 264.7 cells. b) Representative confocal microscopy images of PDANPX endocytosis in primary rat chondrocytes. c) Survival rate of RAW 264.7 cells after co-culturing with different concentrations of PDANPX for 24 hours. d) Survival rate of primary chondrocytes after co-culturing with different concentrations of PDANPX for 24 hours. Data are expressed as mean ± standard deviation.

[0028] Figure 8. In vitro synergistic antioxidant and anti-inflammatory effects of PDANPX. a) Schematic diagram of the regulatory effects of different treatments on oxidative stress levels and polarization direction in RAW 264.7 cells. b) Changes in intracellular H2O2 concentration in RAW 264.7 cells after 24 hours of PDANPX incubation (n=3). c) Relative mRNA expression levels of iNOS and Arg-1 in RAW 264.7 cells after 24 hours of PDANPX incubation (n=3). d) Relative mRNA expression levels of IL-1β and TGF-β in RAW 264.7 cells after 24 hours of incubation. e) IL-1β secretion in RAW 264.7 cell supernatant after 48 hours of incubation. f) TGF-β secretion in RAW 264.7 cell supernatant after 48 hours of incubation. g) Representative results of Western blots on the expression levels of CD86, CD206, and CD11B proteins in RAW 264.7 cells after 48 hours of PDANPX incubation. h) Quantitative analysis of protein expression in Figure g (protein levels were standardized using β-actin as an internal reference) (n=3). Data are expressed as mean ± standard deviation, and statistical analysis was performed using one-way ANOVA combined with Tukey's post-hoc test.

[0029] Figure 9 Direct and indirect chondrogenic effects of PDANPX. a) Relative mRNA expression levels of iNOS, COX-2, and IL-6 in primary chondrocytes after 24 hours of PDANPX incubation (n=3). b) Representative results of Western blots on iNOS and COX-2 protein expression levels in primary chondrocytes. c) Quantitative analysis of protein expression in Figure b (protein levels normalized with GAPDH as internal control) (n=3). d) Schematic diagram of the experimental design for indirect co-culture of primary chondrocytes with RAW 264.7 cell supernatant. e) Relative mRNA expression levels of iNOS, COX-2, and IL-6 in primary chondrocytes after 24 hours of incubation with different RAW 264.7 cell supernatants (n=3). f) Representative results of Western blots on iNOS and COX-2 protein expression levels in primary chondrocytes after 24 hours of incubation with different RAW 264.7 cell supernatants. g) Quantitative analysis of protein expression in Figure f (protein levels standardized using GAPDH as an internal reference) (n=3). Data are expressed as mean ± standard deviation, and statistical analysis was performed using one-way ANOVA combined with Tukey's post-hoc test.

[0030] Figure 10Long-acting analgesic effect of PDANPX in a rat model of osteoarthritis. a) Experimental procedure for rat osteoarthritis research. b) Mechanical pain threshold measured by electronic Von Frey after treatment (n=3). c) Representative force-time curves for each experimental group. d) Comparison of mechanical pain thresholds between the ipsilateral and contralateral paws at the experimental endpoint (n=3). e) Changes in stride length assessed by a computerized gait analysis system. f) Changes in swing time assessed by a computerized gait analysis system. g) Changes in footprint area assessed by a computerized gait analysis system. Data are expressed as mean ± standard deviation and statistical analysis was performed using one-way ANOVA combined with Tukey's post-hoc test.

[0031] Figure 11 . Joint-protective effect and persistent intra-articular retention of PDANPX in a rat model of osteoarthritis. a) Micro-CT images of the knee joint in each group. First row: 3D reconstruction of the knee joint; second row: wear pattern of the medial tibial plateau (enlarged area in red box shows articular surface damage); third row: representative two-dimensional coronal section. Micro-CT quantitative analysis of subchondral bone parameters of the medial tibial cartilage: b) bone volume fraction (BV / TV), c) bone surface area to volume ratio (BS / BV), d) trabecular thickness (Tb.Th), e) trabecular pattern factor (Tb.Pf) (n=5). f) Raman spectral analysis of articular cartilage surface in the PDANPX-treated group. Panels from left to right: Left 1 is bright-field image of cartilage surface (red PDANPX residue is visible in the treated group); Left 2 is Raman intensity map of C=C stretching vibration; Left 3 is Raman intensity map of C≡C stretching vibration; Left 4 is the average Raman spectrum of the selected area. Data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA combined with Tukey post-hoc test.

[0032] Figure 12 PDANPX provides long-term relief of cartilage degeneration and synovitis in rats with osteoarthritis. a) Representative coronal section of the knee joint stained with H&E (top image) and magnified view of the selected area (bottom image), showing inflammatory infiltration in the synovial tissue. b) Synovitis score based on H&E stained sections (n=5). c) Coronal section stained with SO / FG (top image) and magnified view of the selected area (bottom image), showing cartilage degeneration. d) OARSI score of articular cartilage degeneration based on SO / FG staining (n=5). e) Quantitative analysis of articular cartilage thickness (n=5). f) CD86 in synovial tissue. + With CD206 + Double immunofluorescence staining of macrophages. g) Synovial CD86 calculated based on immunofluorescence staining. + / CD206 +Cell ratio (n=3). h) Immunohistochemical staining of the pro-inflammatory cytokine IL-6 in the knee joint. i) Quantitative analysis of IL-6 expression in the knee joint based on immunohistochemical staining (n=3). Data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA and Tukey's post-hoc test.

[0033] Figure 13 Joint-protective effect and sustained intra-articular retention of PDANPX in a rabbit osteoarthritis model. a) Experimental procedure for rabbit osteoarthritis research. b) Long-term intra-articular retention of PDANPX (marked in red). c) Degeneration of medial tibial plateau cartilage. Top image: 3D reconstructed image from mini-CT. Bottom image: Macroscopic view of the joint surface. Red box indicates magnified area of ​​cartilage wear. d) Mini-CT images of knee joints in each group. First row: 3D reconstructed image of knee joint; Second row: Representative 2D coronal section; Third row: Local magnified view (red arrow: osteophyte formation). Quantitative mini-CT analysis of subchondral bone parameters of the medial tibial plateau: e) Bone volume fraction (BV / TV); f) Bone specific surface area (BS / BV); g) Trabecular bone thickness (Tb.Th); h) Trabecular bone pattern factor (Tb.Pf) (n=3). Data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA and Tukey post-hoc test.

[0034] Figure 14 PDANPX provides long-term relief of cartilage degeneration in rabbit osteoarthritis. a) Representative H&E and SO / FG stained sections of femoral articular cartilage. b) Representative H&E and SO / FG stained sections of tibial plateau articular cartilage. c) OARSI score of femoral cartilage degeneration (n=3). d) OARSI score of tibial plateau cartilage degeneration (n=3). e) Elevation of joint skin temperature. Data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA and post-hoc Tukey's test.

[0035] Figure 15MRI evaluation of PDANPX's long-term effect in alleviating osteoarthritis in rabbits. a) MRI evaluation: First row: T2-weighted image showing joint effusion (high signal indicated by red arrow). Second row: Fat-suppressed T2-weighted imaging (red arrow: cartilage thinning / defect). Third row: Color-coded cartilage thickness map; the selected area (white arrow) with blue / black tones indicates cartilage thinning / defect. b) Quantitative analysis of the change in representative rabbit joint effusion volume over time as assessed by T2-weighted MRI. c) Evaluation of typical trends in ipsilateral articular cartilage thickness from lateral to medial in each group using multi-slice MRI analysis. d) Quantitative analysis of cartilage thickness based on MRI (n=3). Data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA and post-hoc Tukey's test.

[0036] Figure 16 PDANPX provides long-term relief of synovitis in rabbit osteoarthritis. a) Representative H&E-stained sections of synovial tissue. The figure below shows a magnified view of the selected area, with red arrows indicating inflammatory cell infiltration within the synovium. b) Synovitis scores based on H&E staining (n=3). Data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA and post-hoc Tukey's test.

[0037] Figure 17 Biosafety assessment of PDANPX in rats.

[0038] Figure 18 Biocompatibility assessment of PDANPX in rabbits. a) White blood cell count. b) Neutrophil percentage. c) Red blood cell count. d) Hemoglobin concentration. e) Platelet count. f) Alanine aminotransferase level. g) Aspartate aminotransferase level. h) Blood urea nitrogen concentration. i) Creatinine level. j) Representative sections of major organs stained with H&E.

[0039] Figure 19 Characterization of AKP, DAKP, and PDAKP. a) Chemical equation for the synthesis of DAKP from ketoprofen. b) Mass spectra of AKP and c) DAKP. d) Fourier transform infrared spectra of KP, AKP, and DAKP. e) Chemical structure of PDAKP. f) Raman spectra of DAKP and PDAKP.

[0040] Figure 20 1H NMR spectra of AKP and DAKP. a) AKP 1 ¹H NMR characterization. ¹H NMR (400 MHz, CDCl₃) δ 7.60 (dddd, J= 18.7, 15.4, 9.8, 7.4 Hz, 9H), 5.71 (s, 1H), 4.00 (qdd, J = 17.6,5.3, 2.6 Hz, 2H), 3.64 (q, J = 7.1 Hz, 1H), 2.18 (t, J = 2.5 Hz, 1H), 1.55 (d, J =7.2 Hz, 3H). b) DAKP's 1 ¹H NMR characterization. ¹H NMR (600 MHz, CDCl₃) δ 7.83 – 7.39 (m, ¹⁸H), 6.02 (d, δ ) J = 4.2 Hz, 2H), 4.08 – 3.92 (m, 4H), 3.67 – 3.61 (m, 2H), 1.52(d, J = 7.1 Hz, 6H). Figure 21 KP, AKP, and DAKP 13 C NMR characterization. The selected area highlights the characteristic changes in the C≡C signal.

[0041] Figure 22 Characterization of AFP, DAFP, and PDAFP. a) Chemical equation for the synthesis of DAFP from flurbiprofen. b) Mass spectra of AFP and c) DAFP. d) Fourier transform infrared spectra of FP, AFP, and DAFP. e) Chemical structure of PDAFP. f) Raman spectra of DAFP and PDAFP.

[0042] Figure 23 1H NMR spectra of AFP and DAFP. a) AFP 1 ¹H NMR characterization. ¹H NMR (400 MHz, CDCl₃) δ 7.58 – 7.33 (m, 6H), 7.18 – 7.08 (m, 2H), 5.70 (s, 1H), 4.04 (qdd, J = 17.6,5.3, 2.5 Hz, 2H), 3.60 (q, J = 7.1 Hz, 1H), 2.21 (t, J = 2.5 Hz, 1H), 1.56 (d, J =7.2 Hz, 3H).b) DAFP of 1 ¹H NMR characterization. ¹H NMR (400 MHz, DMSO) d6) δ 8.55 (t, J = 5.1 Hz,2H), 7.57 – 7.33 (m, 12H), 7.25 – 7.15 (m, 4H), 4.05 – 3.90 (m, 4H), 3.67 (q, J = 7.0 Hz, 2H), 1.35 (d, J = 7.0 Hz, 6H). Figure 24 FP, AFP, and DAFP 13 C NMR characterization. The selected area highlights the characteristic changes in the C≡C signal.

[0043] Figure 25 This is a schematic diagram illustrating the mechanism of action of the polydiacetylene prodrug of the present invention. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0045] This invention discloses a method for synthesizing a polydiacetylene prodrug, the reaction formula of which is as follows:

[0046]

[0047]

[0048] The synthesis method includes the following steps: a. The drug R-COOH containing a carboxyl group as shown in Formula 1 and the amide reaction catalyst are added to an organic solvent. Preferably, the amide reaction catalyst is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBT). Preferably, the organic solvent is dichloromethane. The mixture is stirred in an ice-water bath for 30 min to activate the carboxyl group. Then, propargylamine as shown in Formula 2 is slowly added dropwise, and the mixture is stirred at room temperature. An amide reaction occurs under the action of the catalyst to obtain the terminal alkyne intermediate shown in Formula 3. b. Add the terminal alkyne intermediate shown in Formula 3 and the terminal alkyne oxidative coupling reaction catalyst to an organic solvent. Preferably, the terminal alkyne oxidative coupling reaction catalyst is cuprous chloride (CuCl) and N,N,N',N'-tetramethylethylenediamine (TMEDA). Preferably, the organic solvent is acetone. Oxygen is continuously introduced and the mixture is stirred at room temperature. Under the action of the catalyst, an alkyne oxidative coupling reaction occurs to obtain the diyne monomer shown in Formula 4. c. Method 1: The diacetylene monomer shown in Formula 4 is subjected to topological polymerization by heating or ultraviolet irradiation, preferably irradiated with 365 nm ultraviolet light or heated to 120 °C, to obtain the polydiacetylene prodrug with the structure shown in Formula I; preferably, the wavelength range of ultraviolet irradiation is 200-400 nm and the heating temperature is 50-200 °C, more preferably, the wavelength range of ultraviolet irradiation is 365 nm and the heating temperature is 120 °C; Method 2: Add the diacetylene monomer shown in Formula 4 and the amphiphilic surfactant to an organic solvent. Preferably, the amphiphilic surfactant is soybean lecithin or DSPE-PEG. Preferably, the organic solvent is chloroform or acetone. Grind until the solvent evaporates to form a homogeneous paste. Then add water and grind to disperse, to obtain an aqueous dispersion of the diacetylene monomer. Then, induce the diacetylene monomer to undergo topological polymerization by ultraviolet light to obtain an aqueous dispersion of the polyacetylene prodrug with the structure shown in Formula I.

[0049] The following are specific examples.

[0050] Example 1: Synthesis and Characterization of Naproxen Polydiacetylene Prodrug Based on the aforementioned synthetic route, this invention uses naproxen (chemical structural formula as shown in Formula II) as a representative model drug for the synthesis of a polydiacetylene prodrug. The chemical equation for the synthetic route is as follows: Figure 1 First, naproxen (NPX) reacts with propargylamine via an amide reaction catalyst, preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBT), to generate ANPX. Subsequently, ANPX undergoes an oxidative coupling reaction catalyzed by cuprous chloride (CuCl) and N,N,N',N'-tetramethylethylenediamine (TMEDA) in an oxygen atmosphere to yield the diacetylene monomer DANPX of the naproxen prodrug.

[0051]

[0052] II By nuclear magnetic resonance hydrogen spectrum ( Figure 2 a is the proton spectrum of ANPX, b is the proton spectrum of DANPX, and carbon spectrum ( Figure 3 ) and mass spectrometry ( Figure 1(b is the mass spectrum of ANPX, c is the mass spectrum of DANPX) The synthesized ANPX and DANPX were structurally characterized to verify the correctness of the synthesized product structures. Fourier transform infrared spectra of the products ANPX and DANPX ( Figure 1 (d) shows characteristic absorption peaks for alkynyl and amide groups.

[0053] The crystal structure of monomer DANPX was determined by microcrystalline electron diffraction. Figure 1 'e' is a side view. Figure 1 (f is a top view). The structure clearly shows that the π-π stacking between the naphthalene rings arranges the amide groups at a fixed spacing to form a hydrogen-bonded framework. Cell parameters (monoclinic P21): a = 4.841 Å, b = 10.321 Å, c = 26.902 Å, α = 90°, β = 93.82°, γ = 90°, V = 1341.3 ų, Z = 2. This ordered arrangement orients the diyne groups at a repeating spacing of 4.84 Å, which closely matches the ideal spacing of 4.9 Å for topological polymerization, thus providing favorable structural geometry for the reaction. The predicted hydrogen bonds between the amide groups are confirmed by the OH distance of 1.82 Å and the NH…O bond angle of 175.35°.

[0054] DANPX exhibits a strong naphthalene ring stacking effect, enabling it to undergo efficient polymerization under heating, ultraviolet light irradiation, or crystallization conditions to yield naproxen polydiacetylene prodrug (PDANPX), with the chemical structural formula shown below. Figure 1 The resulting polymer PDANPX has a typical polydiacetylene C=C and C≡C alternating conjugated main chain structure, with naproxen groups as side chains, thus forming a highly simplified prodrug with extremely high drug content.

[0055] Given the poor water solubility of polydiacetylene, this invention employs a dispersion polymerization strategy to prepare a PDA prodrug suspension with uniform size. DANPX and soybean lecithin were mixed and ground at a 1:1 mass ratio and dispersed in water, followed by topological polymerization induced by 365 nm ultraviolet light.

[0056] Under 488 nm laser excitation, PDANPX exhibited strong green autofluorescence ( Figure 1 h). Transmission electron microscopy images show DANPX ( Figure 4 a) and PDANPX ( Figure 4 (b) Both form short, rod-shaped particles with regular shapes. The powder X-ray diffraction (XRD) patterns of DANPX and PDANPX almost overlap, exhibiting sharp Bragg diffraction peaks without significant broadening, indicating that they have high crystallinity. Figure 4c). Raman spectroscopy of PDANPX further confirmed the ordered polymerization of the monomers, characterized by the appearance of the C=C characteristic peak and the shift of the C≡C peak position. Figure 4 The polymerization process was monitored using UV-Vis spectroscopy, and the gradual increase in characteristic absorption at 532 nm confirmed the continuous formation of the conjugated poly(diacetylene) backbone. Figure 4 Solubility tests in acetone, a good solvent for monomers, showed that unpolymerized DANPX dissolved completely, while polymerized PDANPX exhibited a red colloidal dispersion and a significant Tyndall effect, confirming the formation of an acetone-insoluble conjugated polydiacetylene backbone. Figure 4 Dynamic light scattering analysis showed that PDANPX formed a homogeneous dispersion system with a hydrodynamic average diameter of approximately 562 nm. Figure 4 The zeta potential is approximately -20 mV (g). Figure 4 These fluid dynamic parameters can be maintained stably for at least 30 days. Figure 4 (i), exhibiting excellent dispersion stability.

[0057] Example 2: In vitro ROS-responsive degradation and ROS scavenging of naproxen polydiacetylene prodrug Under acidic conditions (pH 4.0), by administering 50 μg mL daily for 7 consecutive days... - The ROS-responsive degradation behavior of PDANPX was evaluated by adding 0.2% HClO to a PDANPX prodrug suspension¹. The degradation of PDANPX was tracked using Raman spectroscopy. Raman testing was performed using a 532 nm laser (1.5 mW power, 0.1 s exposure time, 10 acquisitions in total), with the C≡C peak of 0.4 M decacarbon-4,6-diynediic acid used as an internal standard for spectral normalization. During degradation, the relative intensity of the PDANPX Raman spectrum gradually decreased (…). Figure 5 (a), corresponding to the characteristic Raman peak intensity of C=C and C≡C bonds gradually decreases ( Figure 5 (b). The absorbance at 532 nm was tracked using UV-Vis spectroscopy, and the absorbance decreased synchronously with increasing degradation time. Figure 5 (c). These phenomena confirm that the poly(diacetylene) conjugated backbone underwent ROS-triggered degradation.

[0058] The scavenging ability of PDANPX against ROS was evaluated using the 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric method. The results showed that PDANPX could efficiently scavenge ROS in a concentration-dependent manner. Figure 5 (d), further supporting its inherent antioxidant capacity.

[0059] Scanning electron microscopy further revealed that PDANPX degrades from a uniform short rod-shaped structure during the degradation process. Figure 5 e) is transformed into nanoparticles with heterogeneous morphology ( Figure 5 f), whose hydrodynamic diameter decreases from 562 nm to 150 nm, and the zeta potential changes from -20 mV to -4 mV ( Figure 5 g). Single-photon two-channel Raman imaging shows strong C=C and C≡C characteristic peaks in intact PDANPX, while these signals almost completely disappear after degradation despite the presence of residual microparticles ( Figure 5 h).

[0060] The above results indicate that PDANPX can undergo ROS-triggered complete backbone cleavage, gradually degrade and has the effect of scavenging ROS.

[0061] Example 3 Naphroxen polydiacetylene prodrug for long-term in vitro on-demand sustained release of the original drug naproxen The polydiacetylene prodrug PDANPX can undergo stepwise cleavage of the conjugated backbone in response to high levels of reactive oxygen species. This ROS-triggered degradation process produces intermediates containing amide bonds that are susceptible to protease cleavage. Under the action of proteases, the intermediates are further degraded, releasing the original drug naproxen (NPX) and the biocompatible by-product glycine. The degradation mechanism is as Figure 6 a.

[0062] The degradation products under the combined treatment of ROS and protease were analyzed by liquid chromatography-mass spectrometry, and multiple chromatographic peaks were detected ( Figure 6 b). Among them, a peak signal consistent with the mass-to-charge ratio of naproxen was identified, and the retention time of the elution peak was 14.81 min ( Figure 6 c), and the mass-to-charge ratio was consistent with the theoretical molecular weight ( Figure 6 d), confirming the release of the original drug naproxen in PDANPX; an elution peak of the intermediate containing an amide bond was detected, and the retention time was as Figure 6 e, and the mass-to-charge ratio was consistent with the theoretical molecular weight ( Figure 6 f); an elution peak of the small molecule by-product glycine was detected, and the retention time was as Figure 6 g, and the mass-to-charge ratio was consistent with the theoretical molecular weight ( Figure 6 h), further providing evidence for the stepwise degradation mechanism.

[0063] Under the condition that the equivalent molar concentration of naproxen is 10 μM, neither the monomer DANPX containing the naproxen structural unit nor the polydiacetylene prodrug PDANPX showed inhibitory activity against COX-2 enzyme. In contrast, the degradation products of PDANPX successfully inhibited 69.4% of COX-2 activity, strongly proving that the degradation of PDANPX can generate pharmacologically active naproxen ( Figure 6 i).

[0064] The cumulative release of naproxen from PDANPX over 31 days was quantitatively analyzed by high-performance liquid chromatography (HPLC). PDANPX remained stable in the presence of proteases, indicating that protease-mediated hydrolysis alone could not trigger drug release. Conversely, the combined action of ROS and proteases enabled sustained release of naproxen, with approximately 21.7% of the total drug released over 31 days. Figure 6 (j). This long-term and conditionally dependent release characteristic confirms its potential for self-regulating drug release in response to inflammation levels. The structural basis of this behavior lies in the stacking of naproxen substituents on the side chains of the poly(diacetylene) conjugated backbone, resulting in a significant steric hindrance effect that prevents the protease from directly contacting the amide bond of naproxen. Therefore, in a normal physiological microenvironment with low ROS concentrations, non-specific degradation of PDANPX is effectively avoided.

[0065] In summary, PDANPX enables ultra-long-acting, reactive oxygen species-triggered, and protease-assisted naproxen release while simultaneously scavenging reactive oxygen species. This process forms a negative feedback loop, slowing down subsequent drug release and ultimately achieving on-demand dosing synchronized with inflammation. Therefore, this mechanism provides a theoretical basis for potential synergistic antioxidant and anti-inflammatory therapies, while establishing a self-regulating therapeutic cycle adapted to inflammatory activity.

[0066] Example 4: Cellular uptake and cellular biosafety of naproxen polydiacetylene prodrug Cellular uptake of PDANPX was investigated in both macrophages and primary chondrocytes. Confocal imaging of DiD-labeled lecithin confirmed that red-fluorescent lecithin was uniformly coated on the surface of green-fluorescent PDANPX particles, indicating a stable nucleoshell structure. RAW 264.7 cells and rat primary chondrocytes were co-incubated with DiD-labeled PDANPX (10 μg mL⁻¹) for 24 hours. After washing with PBS to remove free particles, cells were fixed with 4% paraformaldehyde and stained with DAPI. Cell localization was observed using confocal microscopy at excitation wavelengths of 488 nm (PDANPX autofluorescence), 640 nm (DiD), and 360 nm (DAPI). In macrophages (… Figure 7 a) and chondrocytes ( Figure 7 Highly efficient cellular uptake of PDANPX was observed in both of the results (b).

[0067] The cellular biocompatibility of PDANPX was assessed using the MTT assay. Cells were treated with different concentrations of PDANPX for 24 hours, and then incubated at 37°C with 0.5 mg / mL... -¹ MTT co-incubated for 4 hours. The resulting formazan crystals were dissolved in DMSO, and the absorbance was measured at 570 nm. The results showed that, over a wide concentration range, PDANPX was effective against macrophages (…). Figure 7 c) and chondrocytes ( Figure 7 (d) None of them showed significant cytotoxicity.

[0068] Example 5: Antioxidant and anti-inflammatory effects of naproxen polydiacetylene prodrug in macrophages To further elucidate its biological functions at the cellular level, this invention investigated the antioxidant and anti-inflammatory activities of PDANPX in macrophages, as illustrated in the experimental diagram below. Figure 8 a. It is known that oxidative stress caused by the imbalance between reactive oxygen species (ROS) production and the antioxidant defense system plays a central role in the pathogenesis of osteoarthritis. Excessive ROS disrupts redox signaling, damages biomolecules such as proteins, lipids, and DNA, and accelerates cartilage degradation, thereby exacerbating joint inflammation. To evaluate the antioxidant capacity of PDANPX, this invention established an LPS-stimulated RAW 264.7 macrophage model to simulate an inflammatory oxidative environment. PDANPX treatment significantly reduced intracellular H2O2 levels by 80.6%, almost restoring them to baseline, while free naproxen only achieved a 52.8% reduction (…). Figure 8 (b). These results indicate that PDANPX can effectively alleviate oxidative stress in inflammatory macrophages.

[0069] Synovial macrophages are a key driver of osteoarthritis pathology; polarization of the pro-inflammatory M1 phenotype promotes synovitis, cartilage destruction, and pain. Conversely, M2 macrophages secrete anti-inflammatory and regenerative cytokines such as TGF-β, promoting tissue repair. This invention evaluated the ability of PDANPX to regulate macrophage phenotype. After 24 hours of PDANPX treatment, the mRNA expression level of the M2 marker Arg1 was significantly upregulated by 3.48 times compared to the LPS group. Figure 8 (c). Simultaneously, PDANPX significantly inhibited the expression of the pro-inflammatory cytokine IL-1β at the transcriptional level and promoted the expression of the anti-inflammatory cytokine TGF-β. Figure 8 (d). Extending the treatment time to 48 hours significantly downregulated IL-1β secretion. Figure 8 (e), and increased TGF-β secretion by 1.49 times compared to the LPS group ( Figure 8 This enhances its anti-inflammatory and cartilage repair effects. Furthermore, the M2 surface marker CD206 was increased to 2.58 times that of the LPS group (f), thus strengthening its anti-inflammatory and cartilage repair effects. Figure 8The g and h values ​​confirmed the sustained enhancement of M2 polarization. These results indicate that PDANPX exerts a synergistic anti-inflammatory effect by simultaneously scavenging reactive oxygen species and releasing naproxen, reprogramming macrophages from a pro-inflammatory M1 phenotype to a reparative M2 phenotype.

[0070] Example 6: Direct protective effect of naproxen polydiacetylene prodrug in primary chondrocytes Given the central role of chondrocytes in the pathology and pain mechanisms of osteoarthritis, this invention further explores the direct protective effect of PDANPX on primary chondrocytes. IL-1β is one of the most critical cytokines in the OA process, driving chondrocyte catabolism and inflammation while inhibiting anabolistic repair. Furthermore, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are key mediators of OA-related pain and inflammation. After IL-1β stimulation of primary chondrocytes, PDANPX treatment significantly inhibited the expression of iNOS, COX-2, and IL-6 at both the mRNA and protein levels. At 24 hours, naproxen and PDANPX produced comparable transcriptional repression effects. Figure 9 (a); However, at 48 hours, PDANPX showed a stronger inhibitory effect—reducing iNOS and COX-2 protein expression by 52.1% and 45.4%, respectively, while naproxen only inhibited them by 8.57% and 13.2% (a). Figure 9 (b, c). These results indicate that PDANPX provides more durable and significant anti-inflammatory protection to chondrocytes compared to free naproxen.

[0071] Example 7: Naproxen polydiacetylene prodrug exerts indirect chondrogenic protective effects through intercellular communication between macrophages and chondrocytes. Macrophages are a major source of pro-inflammatory cytokines in osteoarthritis joints. To investigate transcellular communication between macrophages and chondrocytes, this study established an indirect co-culture system. A schematic diagram of the experimental procedure is shown below. Figure 9 The conditioned medium for M1 macrophages induced by lipopolysaccharide significantly increased the expression levels of inducible nitric oxide synthase, cyclooxygenase-2, and interleukin-6 in chondrocytes, confirming that M1 macrophages have a strong pro-inflammatory paracrine effect. Figure 9 (e). In contrast, conditioned medium for macrophages treated with PDANPX significantly inhibited the expression of these inflammatory markers. Compared with the lipopolysaccharide group, the mRNA levels of iNOS, COX-2, and IL-6 in co-cultured chondrocytes decreased by 86.9%, 67.2%, and 57.7%, respectively, and the inhibitory effect was significantly better than that of the naproxen group (e). Figure 9 (e). Meanwhile, PDANPX-treated macrophage conditioned medium reduced the expression levels of iNOS and COX2 proteins in chondrocytes by 48.1% and 67.5%, respectively. Figure 9 f, g). These results indicate that PDANPX not only exhibits direct antioxidant and anti-inflammatory effects on chondrocytes but also indirectly exerts chondroprotective functions by reprogramming the polarization state of macrophages and altering the cytokine profile in the joint microenvironment.

[0072] In summary, PDANPX achieves multi-level anti-inflammatory effects through a synergistic mechanism. In addition to scavenging reactive oxygen species and controlling the release of naproxen, PDANPX can reprogram macrophages into the M2 phenotype, reshape the inflammatory factor landscape, and inhibit the direct and paracrine activation of chondrocyte inflammation and pain-related signaling pathways. This multi-level synergistic effect highlights the important potential of PDANPX as a treatment strategy for osteoarthritis.

[0073] Example 8: Long-term alleviation of osteoarthritis in rats by naproxen polydiacetylene prodrug To evaluate the in vivo therapeutic effect and long-term drug retention characteristics of PDANPX, the present invention established a chronic osteoarthritis model in 6-week-old male Sprague-Dawley rats by transection of the anterior cruciate ligament. One week after surgery, the animals were randomly grouped and received single intra-articular injections of 1% hyaluronic acid, free naproxen, or PDANPX, respectively. During the 8-week observation period, the analgesic effect, structural changes, and histological outcomes were systematically evaluated. The schematic diagram of the experimental procedure is as shown in Figure 10 a.

[0074] Behavioral evaluations showed that PDANPX continuously exhibited analgesic effects throughout the study period. Although naproxen has inherent anti-inflammatory and analgesic effects, its short in vivo half-life limits the sustained efficacy. During the eight-week study, the mechanical pain threshold in the OA+NPX group decreased to a level comparable to that of the OA+HA group within one week, and both groups were significantly lower than the healthy control group and the PDANPX group ( Figure 10 b). Although the OA+HA group and the OA+NPX group showed a shortened paw withdrawal latency indicating hyperalgesia, the PDANPX group maintained a normal force-time response ( Figure 10 c). At week 8, the pain thresholds of the affected and contralateral limbs in the PDANPX group were comparable, while significant inter-limb differences were observed in the OA+HA group and the OA+NPX group ( Figure 10 d). Gait analysis also indicated that PDANPX could significantly increase the stride length of rats ( Figure 10 e), reduce the swing time of the affected limb ( Figure 10 f), and increase the contact area of the affected paw with the ground ( Figure 10 g), reflecting reduced joint pain and enhanced motor function.

[0075] Microcomputed tomography further confirmed the arthroprotective effect of PDANPX. In a rat model of osteoarthritis, the surfaces of the medial femoral condyle and tibial plateau became rough and irregular, accompanied by osteophyte formation and cartilage erosion. PDANPX treatment significantly alleviated these structural lesions, restoring joint morphology to near-normal levels. Figure 11 (a). To assess subchondral bone changes in osteoarthritis, we performed a quantitative analysis of bone parameters in the medial subchondral region 22 of the tibia. Osteoarthritis induces a significant decrease in bone volume fraction (BV / TV) (a). Figure 11 (b) while increasing the bone surface area to volume ratio (BS / BV) Figure 11 c), indicating bone loss and increased surface complexity. This is accompanied by a decrease in trabecular thickness (Tb. Th). Figure 11 d) and trabecular pattern factor (Tb.Pf) Figure 11 An increase in (e) indicates an osteoporotic transformation of bone trabeculae from plate-like to rod-like structures. A single intra-articular injection of PDANPX can significantly delay the progression of osteoarthritis and restore the above parameters to near-normal levels. Figure 11 (be).

[0076] To investigate intra-articular retention, Raman spectroscopy imaging analysis was performed on knee joint sections 8 weeks after injection. Significant characteristic Raman peaks corresponding to C=C and C≡C bonds were still clearly detected in the PDANPX-treated joint. Figure 11 The f) indicates that it has a persistent local retention in the synovial cavity.

[0077] Histological analysis further confirmed the therapeutic efficacy of PDANPX. Hematoxylin and eosin (H&E) staining showed significant cartilage degeneration, synovial hyperplasia, and inflammatory cell infiltration in both the OA+HA and OA+NPX groups. In contrast, the OA+PDANPX group showed only mild inflammatory infiltration, and the joint structure remained intact. Figure 12 (a). Quantitative scoring showed that synovitis symptoms in rats treated with PDANPX were significantly reduced ( Figure 12 (b). Safranin O / Fixed Green staining showed that PDANPX significantly improved cartilage thinning and fibrosis, with a smaller area of ​​fibrosis repair. Figure 12 The result (c) indicates reduced cartilage wear and enhanced repair capacity. Correspondingly, the OARSI score confirms a significant improvement in cartilage integrity. Figure 12 (d), and the PDANPX treatment group maintained a thicker articular cartilage layer ( Figure 12 (e).

[0078] Immunofluorescence and immunohistochemical analysis provided an in-depth understanding of the anti-inflammatory mechanism of PDANPX. Dual staining with CD86 and CD206 revealed significantly enhanced polarization of M2 macrophages in the synovial tissue. Figure 12 f, g). Meanwhile, immunohistochemical staining results showed that PDANPX could effectively inhibit the expression of inflammatory cytokine IL-6 in synovial tissues ( Figure 12 h, i).

[0079] In summary, in the rat osteoarthritis model, single intra-articular injection of PDANPX could achieve persistent analgesia and joint structure protection. Its long-term therapeutic effect was due to the stable retention of the preparation in the joint cavity, continuous drug release, and active regulation of the local immune microenvironment.

[0080] Example 9 Ultra-long-acting alleviation of rabbit osteoarthritis by naproxen polydiacetylene prodrug To further evaluate the sustained-release behavior and long-term therapeutic effect of PDANPX, the present invention established an osteoarthritis model induced by transection of the anterior cruciate ligament in rabbits. Compared with rodents, rabbits have higher similarities with humans in joint anatomical structure, biomechanical properties, and synovial fluid metabolism, so they are particularly suitable for evaluating the effects of drugs on the progression of osteoarthritis and the retention effect in the joint. Their larger joint size is more conducive to longitudinally tracking dynamic pathological changes such as joint effusion and soft tissue lesions through magnetic resonance imaging. After single intra-articular injection of PDANPX, the experimental rabbits were monitored for four months, and their retention in the joint and the inhibitory effect on the progression of osteoarthritis were comprehensively evaluated through CT and MR imaging, histology, and hematology analysis. The schematic diagram of the experimental procedure is as Figure 13 a.

[0081] During the four-month treatment monitoring period, macroscopic anatomical examination showed that red PDANPX continuously remained in the joint cavity, confirming the long-term retention and stability of the preparation, as well as the formation of a persistent intra-articular drug depot ( Figure 13 b).

[0082] micro-CT results showed that typical osteoarthritic changes occurred in both the OA+HA group and the OA+NPX group, including irregular surfaces of the medial femoral condyle and tibial plateau, accompanied by obvious osteophyte formation at the joint edge, and cartilage erosion with loss of normal luster visible on the medial tibial plateau. In contrast, the joints in the PDANPX treatment group maintained a smooth and intact cartilage surface and a nearly normal joint morphology ( Figure 13 c). Three-dimensional reconstruction and two-dimensional coronal sections further confirmed these findings, showing that the joint edges in the PDANPX group were significantly smoother and osteophyte formation was reduced ( Figure 13 d). Quantitative analysis of bone microstructure parameters showed that single intra-articular injection of PDANPX could significantly delay the progression of osteoarthritis, and multiple measurement parameters, such as bone volume fraction (BV / TV) ( Figure 13 e), bone surface area to volume ratio (BS / BV) ( Figure 13f), trabecular bone thickness (Tb. Th) ( Figure 13 g) and trabecular pattern factor (Tb. Pf) Figure 13 The h values ​​were all close to normal levels.

[0083] Histological analysis supports these imaging findings: femur in the PDANPX treatment group ( Figure 14 a) and tibial plateau cartilage ( Figure 14 (b) remained morphologically intact with less fibrosis, and its OARSI score was significantly lower than that of the control group ( Figure 14 c represents the OARSI score of the femur. Figure 14 d represents the OARSI score of the tibia. These results indicate that PDANPX can effectively protect subchondral bone structure through its long-acting controlled release and local therapeutic effects. Furthermore, no temperature difference was observed between the treated limb and the contralateral healthy joint. Figure 14 The result (e) reflects stable local inflammation control and sustained treatment efficacy.

[0084] MRI evaluation further clarified the therapeutic effect of PDANPX on joint inflammation and cartilage pathology. T2-weighted MRI showed non-cystic, poorly defined high-signal areas in the bone marrow region after osteoarthritis induction, consistent with bone marrow edema, and correlated with reduced cartilage volume in the medial tibial plateau and condyle. Compared with the OA+HA group and the OA+NPX group, PDANPX treatment significantly reduced high signal intensity and intra-articular effusion, indicating that this agent can alleviate inflammatory exudation and cartilage damage. Figure 15 (a, b). Fat-suppressed T2-weighted sequences showed that the cartilage layer in the OA+HA and OA+NPX groups was thinned and discontinuous, while the PDANPX group maintained a continuous and uniform cartilage surface without visible defects, and its average cartilage thickness was comparable to that of the healthy contralateral limb. Figure 15 (c, d). These findings demonstrate that the reactive oxygen species-responsive controlled-release properties of PDANPX can effectively reduce inflammatory exudation and cartilage degeneration during long-term treatment. H&E staining of synovial tissue showed that inflammatory cell infiltration and synovial hyperplasia were significantly reduced in the PDANPX group (c, d). Figure 16 (a) Quantitative scores showed that the symptoms of synovitis in rabbits treated with PDANPX were significantly reduced ( Figure 16 (b) indicates that it has sustained anti-inflammatory activity during the 4-month treatment period.

[0085] In summary, these results indicate that in a large animal rabbit model of osteoarthritis, a single administration of PDANPX can provide sustained, inflammatory-responsive drug release and reactive oxygen species scavenging activity, thereby jointly inhibiting joint effusion, protecting cartilage integrity, and maintaining therapeutic effects for several months.

[0086] Example 10 Biosafety assessment of naproxen polydiacetylene prodrug In rat experiments, pathological examination of vital organ tissues such as the heart, liver, spleen, lungs, and kidneys of rats two months after drug administration, using HE staining, revealed no signs of tissue damage or inflammation. Figure 17 This confirms the excellent biocompatibility of PDANPX.

[0087] In rabbit experiments, blood biochemical analysis of complete blood count and biochemical parameters after 4 months of drug administration showed that all parameters remained within the normal range after long-term treatment. Figure 18 AE is a blood routine indicator. Figure 18 Fi is a blood biochemical marker). HE staining results showed that histological examination of the rabbit's major organs revealed no pathological changes. Figure 18 (j), confirming that PDANPX maintains excellent biocompatibility even when retained in the joint cavity for extended periods.

[0088] Example 11 Synthesis and Characterization of Ketoprofen Polydiacetylene Prodrug To verify the generality of the aforementioned synthetic route, ketoprofen (chemical structural formula as shown in Formula III) was used as another representative model drug for the synthesis of a polydiacetylene prodrug. The chemical equation for the synthetic route is as follows: Figure 19 First, ketoprofen (KP) reacts with propargylamine via an amide reaction catalyst, preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBT), to generate AKP. Subsequently, AKP undergoes an oxidative coupling reaction catalyzed by cuprous chloride (CuCl) and N,N,N',N'-tetramethylethylenediamine (TMEDA) in an oxygen atmosphere to yield the diacetylene monomer DAKP, a prodrug of ketoprofen.

[0089]

[0090] III By nuclear magnetic resonance hydrogen spectrum ( Figure 20 a is the 1H NMR spectrum of AKP, b is the 1H NMR spectrum of DAKP, and carbon NMR spectrum ( Figure 21 ) and mass spectrometry ( Figure 19 (b is the mass spectrum of AKP, c is the mass spectrum of DAKP) The synthesized AKP and DAKP were structurally characterized to verify the correctness of the synthesized product structures. Fourier transform infrared spectra of the products AKP and DAKP (…). Figure 19 (d) shows characteristic absorption peaks for alkynyl and amide groups.

[0091] DAKP can polymerize under heating, ultraviolet light, or crystallization conditions to yield ketoprofen polydiacetylene prodrug (PDAKP), with the chemical structural formula shown below. Figure 19The resulting polymer, PDAKP, possesses a typical polydiacetylene alternating C=C and C≡C conjugated main chain structure, with ketoprofen groups as side chains, thus forming a highly simplified prodrug with extremely high drug content. Raman spectroscopy of PDAKP further verified the ordered polymerization of monomer DAKP, characterized by the appearance of a C=C characteristic peak and the shift of the C≡C peak position. Figure 19 f).

[0092] Example 12 Synthesis and Characterization of Flurbiprofen Polyacetylene Prodrug To verify the generality of the aforementioned synthetic route, flurbiprofen (chemical structural formula as shown in Formula IV) was used as another representative model drug for the synthesis of a polydiacetylene prodrug. The chemical equation for the synthetic route is as follows: Figure 22 First, flurbiprofen (FP) reacts with propargylamine via an amide reaction catalyst, preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBT), to generate AFP. Subsequently, AFP undergoes an oxidative coupling reaction catalyzed by cuprous chloride (CuCl) and N,N,N',N'-tetramethylethylenediamine (TMEDA) under an oxygen atmosphere to yield the diacetylene monomer DAFP of the flurbiprofen prodrug.

[0093]

[0094] IV By nuclear magnetic resonance hydrogen spectrum ( Figure 23 a is the proton spectrum of AFP, b is the proton spectrum of DAFP, and carbon spectrum ( Figure 24 ) and mass spectrometry ( Figure 22 (b is the mass spectrum of AFP, c is the mass spectrum of DAFP) The synthesized AFP and DAFP were structurally characterized to verify the correctness of the synthesized product structures. Fourier transform infrared spectra of products AFP and DAFP ( Figure 22 (d) shows characteristic absorption peaks for alkynyl and amide groups.

[0095] DAFP can polymerize under heating, ultraviolet light, or crystallization conditions to obtain flurbiprofen poly(diacetylene) prodrug (PDAFP), with the chemical structure shown below. Figure 22 The resulting polymer PDFP possesses a typical polydiacetylene alternating C=C and C≡C conjugated main chain structure, with flurbiprofen groups as side chains, thus forming a highly simplified prodrug with extremely high drug content. Raman spectroscopy of PDAFP further verified the ordered polymerization of monomer DAFP, characterized by the appearance of a C=C characteristic peak and the shift of the C≡C peak position. Figure 22 f).

[0096] A schematic diagram illustrating the mechanism of action of the polydiacetylene prodrug of this invention is shown below. Figure 25 As shown, by Figure 25It is evident that at sites of inflammation with elevated reactive oxygen species (ROS) levels, the polydiacetylene backbone undergoes oxidative cleavage to release the active drug while simultaneously scavenging ROS. This process forms a negative feedback loop: drug release and antioxidant activity reduce ROS levels, thereby slowing down subsequent release. The remodeled oxidative microenvironment promotes reparative M2 macrophage polarization and TGF-β secretion, supporting tissue repair.

[0097] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A polydiacetylene prodrug, characterized in that, The structural formula of the polydiacetylene prodrug is shown in Formula I: ； Formula I Where R represents the portion of the drug containing a carboxyl group that is linked to the carboxyl group.

2. The polydiacetylene prodrug as described in claim 1, characterized in that, The drug containing a carboxyl group is an arylpropionic acid nonsteroidal anti-inflammatory drug.

3. The polydiacetylene prodrug as described in claim 1, characterized in that, The aryl propionic acid nonsteroidal anti-inflammatory drugs are naproxen, ketoprofen, flurbiprofen, or ibuprofen.

4. The method for synthesizing the polydiacetylene prodrug according to any one of claims 1-3, characterized in that, Includes the following steps: a) Add the drug containing a carboxyl group and the amide reaction catalyst to an organic solvent to activate the carboxyl group; then add propargylamine dropwise to induce an amide reaction and obtain a terminal alkyne intermediate; b. Add the terminal alkyne intermediate obtained in step a and the terminal alkyne oxidative coupling reaction catalyst to an organic solvent, continuously introduce oxygen, and undergo an alkyne oxidative coupling reaction to obtain a diyne monomer. c. Method 1: The diacetylene monomer obtained in step b is induced to undergo topological polymerization by heating or ultraviolet light to obtain a polydiacetylene prodrug; Alternatively, in method two: the diacetylene monomer and amphiphilic surfactant obtained in step b are added to an organic solvent and ground until the solvent evaporates to form a homogeneous paste. Then water is added and ground to disperse the mixture to obtain an aqueous dispersion of the diacetylene monomer. Then, the diacetylene monomer is induced to undergo topological polymerization by ultraviolet light to obtain an aqueous dispersion of the polyacetylene prodrug.

5. The method for synthesizing the polydiacetylene prodrug as described in claim 4, characterized in that, The drug containing a carboxyl group is an arylpropionic acid nonsteroidal anti-inflammatory drug.

6. The method for synthesizing the polydiacetylene prodrug as described in claim 5, characterized in that, The aryl propionic acid nonsteroidal anti-inflammatory drugs are naproxen, ketoprofen, flurbiprofen, or ibuprofen.

7. The method for synthesizing the polydiacetylene prodrug as described in claim 4, characterized in that, In step a, the catalyst for the amide reaction is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole; In step b, the catalyst for the terminal alkyne oxidative coupling reaction is cuprous chloride and N,N,N',N'-tetramethylethylenediamine.

8. The method for synthesizing the polydiacetylene prodrug as described in claim 4, characterized in that, In step c, method one, before heating or ultraviolet irradiation, a step of static crystallization is also included to allow the diyne monomers to arrange in an orderly manner; In step c, the heating temperature is 50-200℃, and the wavelength of the ultraviolet light irradiation is 200-400 nm.

9. The application of the polydiacetylene prodrug as described in any one of claims 1-3 in the preparation of long-acting controlled-release drugs for chronic diseases.

10. The application as described in claim 9, characterized in that, The chronic diseases mentioned are osteoarthritis, rheumatoid arthritis, immune system disorders, or chronic pain.

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