Cascade response type nanoparticles for adenomyosis and preparation method thereof

Abstract

The invention relates to the technical field of biological medicine, and provides cascade response type nanoparticles for adenomyosis and a preparation method thereof. The core of the system is nanoparticles constructed based on a carrier PLGA-(S-S-PPS) 2, and the disulfide bond of the carrier can be specifically broken and disintegrated in high-concentration H2O2 of a focus. The nanoparticles are loaded with a pH responsive fluorescent-ketal-berberine prodrug, ketal bonds are hydrolyzed in an acidic microenvironment, and berberine is released to realize fluorescence visualization. Hyaluronidase is encapsulated in the nanoparticles and is used for degrading a hyaluronic acid matrix. The surface modified RGD peptide can be specifically combined with integrin alpha v beta 3 to realize active targeting. The synergistic action mechanism of the system is as follows: RGD-mediated targeted accumulation, the carrier disintegrates and releases the content under H2O2, hyaluronidase degrades the matrix to enhance permeation, the prodrug releases berberine in an acid environment, and cell proliferation is synergistically inhibited and angiogenesis is reduced. Experiments prove that the hydrogel has good ROS / pH / enzyme responsiveness, fluorescence visualization, targeted accumulation, high drug loading rate (18.7%) and biological safety.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a cascade-responsive nanoparticle for adenomyosis and its preparation method. Background Technology

[0002] Adenomyosis is a common gynecological disease, its pathological essence lying in the abnormal invasion of endometrial glands and stroma into the myometrium, forming diffuse lesions. This pathological process not only leads to the destruction and hypertrophy of the myometrium but also triggers a series of severe and progressively worsening clinical symptoms, including severe progressive dysmenorrhea, chronic pelvic pain, and significant impairment of fertility, greatly affecting patients' quality of life and fertility needs. In recent years, in-depth research has revealed a unique and active pathological microenvironment within adenomyosis lesions, providing a key breakthrough for a deeper understanding of the disease mechanism and the development of novel therapies. The core characteristics of this microenvironment are: abnormally elevated levels of reactive oxygen species (ROS), a significantly lower acidic microenvironment than the physiological state, and pathological angiogenesis driven by abnormally high expression of integrin αvβ3. The synergistic effect of this high oxidative stress state, acidic conditions, and angiogenesis network not only promotes lesion survival, persistent inflammatory response, and local infiltration but also constitutes a significant marker distinguishing it from normal myometrium. In addition, the natural compound berberine has shown therapeutic potential due to its multiple mechanisms that are highly compatible with the pathological microenvironment of adenomyosis, such as clearing highly reactive oxygen species, inhibiting integrin αvβ3-mediated angiogenesis, regulating inflammation and apoptosis, and utilizing the acidic environment of the lesion to achieve targeted enrichment.

[0003] Current clinical treatment of adenomyosis still faces significant challenges. Drug therapy (such as nonsteroidal anti-inflammatory drugs, hormonal contraceptives, and gonadotropin-releasing hormone agonists) primarily relieves symptoms temporarily by suppressing ovarian function or reducing inflammation, but it suffers from high relapse rates after discontinuation, side effects such as decreased bone density or menopausal symptoms with long-term use, and cannot cure the lesions. Surgical treatment (such as lesion resection or total hysterectomy) can effectively eliminate lesions, but the former is prone to recurrence due to unclear lesion boundaries, while the latter completely leads to permanent loss of fertility, neither of which can meet the fertility needs of patients of childbearing age. Conservative interventional treatments (such as uterine artery embolization and high-intensity focused ultrasound) can shrink lesions, but the efficacy varies greatly from person to person, and there are risks of ovarian function damage and pregnancy complications. Therefore, there is an urgent need for a precise targeted therapy strategy, developing an intelligent delivery system that can specifically release natural drugs in response to the lesion microenvironment to achieve effective inhibition and elimination of lesions while minimizing damage to normal tissues. Summary of the Invention

[0004] The purpose of this invention is to address the above-mentioned technical problems by providing a cascade-response nanoparticle for adenomyosis.

[0005] The core of the nanoparticles in this invention is composed of a ROS-responsive PLGA-(SS-PPS)2 carrier. The PLGA backbone provides biodegradability, while the internal disulfide bonds endow it with the ability to specifically break down and disintegrate in the high-concentration H2O2 microenvironment of adenomyosis lesions. The system is loaded with a pH-responsive fluorescent-ketal-berberine prodrug. In the acidic microenvironment of the lesion, the ketal bonds of this prodrug can be hydrolyzed, thereby precisely releasing the therapeutically active berberine active ingredient, and the drug release pathway can be visualized fluorescently. Hyaluronidase is also encapsulated within the particles to degrade the hyaluronic acid matrix at the lesion site, reducing the tissue barrier and significantly enhancing drug penetration. The nanoparticle surface is modified with RGD peptides, which specifically recognize integrin αvβ3 overexpressed in the vascular endothelium of the lesion, enabling active targeting and efficient accumulation of the nanoparticles at the lesion site. Furthermore, soybean lecithin and PVA serve as stabilizing components, jointly ensuring the integrity of the nanoparticle structure.

[0006] Currently, there are no research reports on technologies for highly effective targeted therapy of adenomyosis based on cascade-responsive nanoparticles integrating a four-fold synergistic mechanism of "targeted accumulation - carrier-responsive disintegration - enzymatic penetration - precise prodrug release" or the application of berberine in the treatment of adenomyosis. This nanoparticle drug delivery system achieves precise drug accumulation, deep penetration, and in-situ release at the lesion site through RGD peptide-mediated active lesion targeting, H2O2-responsive carrier disintegration, hyaluronic acid-mediated enzymatic penetration, and pH-responsive prodrug activation. Furthermore, this invention provides an innovative solution to overcome the challenges of insufficient active targeting efficiency and accumulation concentration, poor tissue penetration, and weak microenvironment responsiveness in adenomyosis drug delivery. Compared to traditional systemic drug delivery or passive targeting systems, this system significantly improves drug concentration and therapeutic specificity within the lesion, demonstrating outstanding therapeutic efficiency and promising clinical translation prospects.

[0007] This invention also provides a cascade-responsive nanoparticle for adenomyosis and a method for preparing the same, specifically including the following steps:

[0008] (1) Preparation of ROS-responsive carrier: In an anhydrous and oxygen-free environment, HO-PLGA-COOH (Mn=10 kDa) was first dissolved in anhydrous DMSO and added to the EDC / NHS activation system. After the carboxyl activation was completed, cystamine was added and reacted at 4 °C for 2 h to form PLGA-cystamine intermediate. After dialysis purification, HS-PPS-COOH (Mn=10 kDa) DMSO solution was added dropwise to the system and the temperature was raised to 25 °C for 36 h. A two-arm disulfide bond bridged structure (PLGA-(SS-PPS)2) was constructed by secondary amidation. After dialysis purification, the reaction solution was freeze-dried to obtain the ROS-responsive carrier PLGA-(SS-PPS)2.

[0009] (2) Preparation of fluorescent-ketal-berberine prodrug: Berberine (BBR) was dissolved in 15 mL of anhydrous acetone, and 2,2-dimethoxypropane and 10 mg of p-toluenesulfonic acid were added. The mixture was refluxed at 60 °C for 8 h under nitrogen protection to generate the ketalized intermediate KET-BBR. After the reaction system was cooled to room temperature, Cy5-NHS ester was added, and the pH was adjusted to 8.5 with 0.1 M NaOH. The mixture was stirred at room temperature for 12 h under light-protected conditions to connect the Cy5 fluorescent group through ester bonds, forming the target product Cy5-KET-BBR. The crude product was purified by silica gel column chromatography. The elution fraction was monitored under a 365 nm UV lamp. The dual-color fluorescent fraction was enriched based on the dual fluorescence signals of yellow autofluorescence of BBR and red fluorescence of Cy5. The solvent was removed by rotary evaporation under reduced pressure and then dried under vacuum.

[0010] (3) Assembly of nanoparticles: Thiolized chitosan and hyaluronidase (HAase) were mixed in a pH 5.0 acetate buffer, and 0.5 mM H2O2 was added to initiate disulfide crosslinking to form 200 nm HAase-chitosan submicron. Subsequently, 40 mg of PLGA-(SS-PPS)2, 15 mg of Cy5-KET-BBR, 8 mg of soybean lecithin and HAase-chitosan submicron were dissolved in 2 mL of dichloromethane to form an oil phase, which was injected into an ice bath aqueous phase containing 5% PVA. After ultrasonic emulsification, the solvent was evaporated under reduced pressure. Finally, RGD modification was performed. The nanoparticles were coupled with 1.5 mg of c(RGDfK) peptide in pH 7.4 PBS for 12 h by EDC / NHS. After purification by ultracentrifugation, the nanoparticles were freeze-dried to obtain RGD-modified nanoparticles.

[0011] Furthermore, the molar ratio of HO-PLGA-COOH to cystamine in step (1) is 1:1.2.

[0012] Furthermore, in step (1), the molar ratio of EDC·HCl to NHS in the EDC / NHS activation system is 1:0.8.

[0013] Furthermore, the molar ratio of PLGA to PPS in step (1) is 1:2.

[0014] Furthermore, the molecular weight cutoff of the dialysis bag used in step (1) is 3.5 kDa.

[0015] Furthermore, the molar ratio of BBR to 2,2-dimethoxypropane in step (2) is 1:5.

[0016] Furthermore, the molar ratio of KET-BBR to Cy5-NHS ester in step (2) is 1:1.2.

[0017] Furthermore, the λ of the dual-color fluorescent component in step (2) ex / em=650 / 670 nm.

[0018] Furthermore, the degree of thiol substitution of the thiolated chitosan in step (3) is 2.3 mmol / g.

[0019] Furthermore, the HAase-chitosan submicroparticles in step (3) contain 500 U of HAase.

[0020] Furthermore, the parameters for ultrasonic emulsification in step (3) are 200W, 70% amplitude, and 4 min.

[0021] Furthermore, the parameters for ultracentrifugation purification in step (3) are 25,000 rpm and 30 min.

[0022] The advantages of this invention are:

[0023] This invention is the first to assemble ROS-responsive PLGA-SS-PPS carrier, pH-responsive ketal-berberine prodrug, soybean lecithin and hyaluronidase into nanoparticles using a one-step ultrasonic emulsification method, and then modify the surface with RGD peptides to construct a cascade-responsive nanoparticle with a four-fold synergistic mechanism of "targeted accumulation - carrier-responsive disintegration - enzymatic penetration - precise prodrug release".

[0024] This nanoparticle enables cascade-responsive drug release. First, it accumulates at the lesion site via RGD peptide targeting; then, the carrier disintegrates under high concentrations of H2O2, releasing the prodrug and hyaluronidase; hyaluronidase degrades the matrix, enhancing permeability; finally, the prodrug hydrolyzes in an acidic microenvironment, precisely releasing berberine, thereby synergistically inhibiting lesion cell proliferation, reducing angiogenesis, and relieving pain. Experiments validated its excellent ROS / pH dual-responsiveness, fluorescent visualization of the drug release process, lesion-targeted accumulation, high drug loading rate (18.7%), and biocompatibility. Attached Figure Description

[0025] Figure 1 XPS is the carrier of the nanoparticles of this invention.

[0026] Figure 2 This is the prodrug characterization of the nanoparticles of this invention. Figure 2 (a) is a comparison of ultraviolet absorbance; Figure 2 (b) is a comparison of fluorescence absorbance.

[0027] Figure 3 This is a transmission electron microscope image of the nanoparticles of this invention.

[0028] Figure 4 This refers to the Zeta potential of the nanoparticles of this invention.

[0029] Figure 5 This refers to the ROS responsiveness of the nanoparticles of this invention. Figure 5 (a) shows the particle size variation under different conditions; Figure 5 (b) is a comparison of drug loading rates; Figure 5 (c) refers to drug release behavior under different conditions.

[0030] Figure 6 This refers to the enzyme responsiveness of the nanoparticles of this invention.

[0031] Figure 7 This refers to the pH responsiveness of the nanoparticles of this invention.

[0032] Figure 8 This is a diagram illustrating the in vivo targeted accumulation effect of the nanoparticles of this invention.

[0033] Figure 9 This relates to the cell proliferation properties of the nanoparticles of this invention.

[0034] Figure 10 This invention relates to the angiogenesis-inhibiting properties of the nanoparticles.

[0035] Figure 11 This relates to the in vivo biocompatibility of the nanoparticles of this invention. Detailed Implementation

[0036] The technical solutions described in this invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of this invention. Obviously, the embodiments described in this specification are only some feasible technical solutions of this invention. Other implementation methods obtained by those skilled in the art based on the embodiments of this invention without any creative effort should be considered to fall within the scope of protection of this invention.

[0037] Example 1: Preparation of Cascade-Responsive Nanoparticles

[0038] (1) Preparation of ROS-responsive carrier: In an anhydrous and oxygen-free environment, HO-PLGA-COOH (Mn=10 kDa) was first dissolved in anhydrous DMSO and added to an EDC / NHS activation system (the molar ratio of EDC·HCl to NHS was 1:0.8). After the carboxyl activation was completed, cystamine was added and reacted at 4 °C for 2 h to form PLGA-cystamine intermediate (the molar ratio of HO-PLGA-COOH to cystamine was 1:1.2). Then, HS-PPS-COOH (Mn=10 kDa, the molar ratio of PLGA to PPS was 1:2) in DMSO solution was added dropwise to the system and the temperature was raised to 25 °C for 36 h. A two-arm disulfide bond bridged structure (PLGA-(SS-PPS)2) was constructed by secondary amidation. The reaction solution was purified by dialysis (the molecular weight cutoff of the dialysis bag was 3.5 kDa) and then freeze-dried to obtain the ROS-responsive carrier PLGA-(SS-PPS)2.

[0039] (2) Preparation of fluorescent-ketal-berberine prodrug: Berberine was dissolved in 15 mL of anhydrous acetone, and 2,2-dimethoxypropane (molar ratio of BBR to 2,2-dimethoxypropane was 1:5) and 10 mg of p-toluenesulfonic acid were added. The mixture was refluxed at 60 °C for 8 h under nitrogen protection to generate the ketalized intermediate KET-BBR. After the reaction system was cooled to room temperature, Cy5-NHS ester (molar ratio of KET-BBR to Cy5-NHS ester was 1:1.2) was added, and the pH was adjusted to 8.5 with 0.1 M NaOH. The mixture was stirred at room temperature for 12 h under light-protected conditions to connect the indole-dicarboxycyanine (Cy5) fluorescent group through ester bonds to form the target product Cy5-KET-BBR. The crude product was purified by silica gel column chromatography. ex / em The dual-color fluorescent component, which simultaneously displays yellow (BBR fluorescence) and red (Cy5 fluorescence), was collected at 650 / 670 nm, and the solvent was removed by rotary evaporation under reduced pressure and then dried under vacuum.

[0040] (3) Assembly of nanoparticles: Thiolized chitosan (thiol substitution degree 2.3 mmol / g) and hyaluronidase (HAase) were mixed in a pH 5.0 acetate buffer, and 0.5 mM H2O2 was added to initiate disulfide crosslinking to form 200 nm HAase-chitosan submicron; then 40 mg of PLGA-(SS-PPS)2, 15 mg of Cy5-KET-BBR, 8 mg of soybean lecithin and HAase-chitosan submicron (containing 500 U HAase) were dissolved in 2 mL of dichloromethane to form an oil phase, which was injected into an ice bath aqueous phase containing 5% PVA, and ultrasonically emulsified (200 W, 70% amplitude, 4 min), and the solvent was evaporated under reduced pressure; finally, RGD modification was performed, and the nanoparticles were coupled with 1.5 mg of c(RGDfK) peptide in pH 7.4 PBS EDC / NHS for 12 h, and purified by ultracentrifugation (25000 rpm, 30 min). After freeze-drying, RGD-modified nanoparticles were obtained.

[0041] Comparative Example 1: Preparation of cascade-responsive nanoparticles based on PLGA-SS-PPS support

[0042] The difference between this comparative example and Example 1 is that the carrier for the nanoparticles is a single-arm PLGA-SS-PPS.

[0043] Preparation of ROS-responsive carrier: Under anhydrous and oxygen-free conditions, HO-PLGA-COOH (Mn=10 kDa) was first dissolved in anhydrous DMSO and added to an EDC / NHS activation system (EDC·HCl to NHS molar ratio of 1:0.8). After carboxyl activation, cystamine was added and the reaction was carried out at 4 °C for 1 h. Subsequently, a DMSO solution of HS-PPS-COOH (Mn=10 kDa, PLGA to PPS molar ratio of 1:1) was added dropwise to the system, and the temperature was raised to 25 °C for 36 h. A single-arm PLGA-SS-PPS structure was formed through an amidation reaction. The reaction solution was purified by dialysis (dialysis bag molecular weight cutoff of 3.5 kDa) and then freeze-dried to obtain the ROS-responsive carrier PLGA-SS-PPS. Comparative Example 2: Preparation of cascade-responsive nanoparticles based on free HAase

[0044] The difference between this comparative example and Example 1 is that the HAase was not coated, and the HAase-chitosan submicroparticles were replaced with free HAase.

[0045] Comparative Example 3: Preparation of cascade-responsive nanoparticles based on ketal-berberine

[0046] The difference between this comparative example and Example 1 is that the prodrug ketal-berberine was not fluorescently treated, and Cy5-KET-BBR was replaced with KET-BBR.

[0047] Comparative Example 4: Preparation of cascade-responsive nanoparticles without RGD modification

[0048] The difference between this comparative example and Example 1 is that the nanoparticles were not modified with RGD peptides.

[0049] Comparative Example 5: Preparation of HAase-free cascade-responsive nanoparticles

[0050] The difference between this comparative example and Example 1 is that the nanoparticles were not loaded with HAase.

[0051] Comparative Example 6: Preparation of RGD-PLGA-BBR Nanoparticles

[0052] Under ice bath conditions, 40 mg of PLGA-COOH (15 kDa), 15 mg of BBR technical grade, and 8 mg of soybean lecithin were dissolved in 2 mL of dichloromethane to form an organic phase, which was then injected into a 5% PVA aqueous solution. The mixture was ultrasonically emulsified (200 W, 70% amplitude, 4 min) to form a primary emulsion. Subsequently, the organic solvent was removed by rotary evaporation under reduced pressure at 40 °C for 30 min, and the mixture was filtered through a 0.45 μm filter to obtain drug-loaded nanoparticles. The nanoparticles were coupled with 1.5 mg of c(RGDfK) peptide in EDC / NHS in pH 7.4 PBS for 12 h, purified by ultracentrifugation (25000 rpm, 30 min), and freeze-dried to obtain RGD-modified nanoparticles.

[0053] Experimental Example 1: XPS characterization of the carrier for the nanoparticles prepared in Example 1

[0054] To verify the disulfide bonds in the nanoparticle support prepared in Example 1, the chemical state distribution of sulfur was quantitatively analyzed by XPS. The results are as follows: Figure 1 As shown, disulfide bonds (-SS-) account for 19.3% of the total sulfur in PLGA-(SS-PPS)2, while no disulfide bonds were detected in the control pure PPS and cystamine. Therefore, the carrier of the nanoparticles prepared in Example 1 has disulfide bond characteristics.

[0055] Experimental Example 2: Characterization of the prodrug of the nanoparticles prepared in Example 1

[0056] To understand the structure of the fluorescent-ketal-berberine prodrug, nanoparticles were dispersed in PBS solution, and their characteristic peaks were analyzed using a UV spectrophotometer and a fluorescence spectrophotometer. The results are as follows: Figure 2 As shown in (a), the nanoparticles exhibit characteristic UV peaks at 345 and 650 nm, with absorbances of 1.7 and 0.5 au, respectively. Figure 2 As shown in (b), fluorescence characteristic peaks appear at 540 and 670 nm, with absorbances of 18.4 and 5.3 au, respectively. Therefore, it is shown that the fluorescent-ketal-berberine prodrug in the nanoparticles of Example 1 was successfully prepared.

[0057] Experimental Example 3: Physical characterization of the nanoparticles prepared in Example 1

[0058] Microstructure: The nanoparticles were diluted and dropped onto a copper grid and allowed to air dry. The microstructure of the microspheres was characterized using transmission scanning electron microscopy (TEM). The results are as follows: Figure 3 As shown, the nanoparticles prepared in Example 1 have a uniform spherical structure of about 100 nm.

[0059] Zata potential: To characterize whether the RGD peptide modification of the nanoparticles was successful, the surface charge of the particles was characterized. The microspheres were ultrasonically dispersed in deionized water, and the zeta potential of the microspheres was evaluated using a zeta potential meter. Results are as follows: Figure 4 As shown, the Zeta potentials before and after modification are 20.7 and 3.4 mV, respectively. This is because the bare nanoparticles contain carboxyl groups (-COOH) and have a negative surface charge, while the RGD peptide (c(RGDfK)) contains lysine (-NH3) residues. + After modification, the nanoparticles tend to have a near-neutral charge. Therefore, the nanoparticles prepared in Example 1 were successfully modified with RGD peptides.

[0060] Experimental Example 4: Characterization of the ROS responsiveness of the nanoparticles prepared in Example 1

[0061] To verify the ROS responsiveness of the nanoparticles, the nanoparticles from Example 1 were co-incubated under different conditions (pH 7.4 PBS and 10 mM H₂O₂). DLS was used to detect particle size changes at different time points (0, 1, 4, 8, and 24 h). The results are as follows: Figure 5 As shown in (a), the particle size change of the nanoparticles was only 27% under PBS conditions, while it reached 130% under 10 mM H2O2. This is attributed to the cleavage of the ROS-responsive carrier of the nanoparticles under 10 mM H2O2. Therefore, the nanoparticles of Example 1 have good ROS responsiveness.

[0062] To verify the advantages of the PLGA-(SS-PPS)2 two-arm structure, the drug loading rate of the nanoparticles was characterized. After constructing a standard curve of concentration gradient, the nanoparticles obtained in Example 1 and Comparative Example 1 were dissolved in DMSO, and their drug loading rates were calculated using a UV spectrophotometer. The results are as follows: Figure 5 As shown in (b), the drug loading rate of the nanoparticles prepared in Example 1 reached 18.4%, while the drug loading rate of Comparative Example 1 was only 4.7%. This is because the carrier with a double-arm structure is more likely to encapsulate the drug, thus increasing its drug loading capacity. Therefore, the nanoparticles prepared in Example 1 have a very high drug loading rate.

[0063] To understand the effects of different drug release conditions and carrier structures on the drug release behavior of nanoparticles, nanoparticles were co-incubated under different conditions (pH 7.4 PBS and 10 mM H2O2), and the absorbance at 340 nm was measured at different time points (0, 1, 4, 8, and 24 h) using a UV spectrophotometer. Figure 5As shown in (c), the cumulative drug release rates of the nanoparticles in Example 1 and Comparative Example 1 were only 7% and 19.3% under PBS conditions, respectively, while reaching 90.2% and 42.9% under 10 mM H2O2. This difference between the two conditions is likely because the disulfide bonds in the nanoparticles can only break under ROS response. Furthermore, compared to Example 1, the comparative example showed a higher cumulative release rate under PBS and a lower cumulative release rate under 10 mM H2O2, likely due to the lower drug encapsulation capacity and weaker encapsulation ability of the single-arm carrier. Therefore, the nanoparticles prepared in Example 1 exhibit good ROS responsiveness.

[0064] In summary, the nanoparticles prepared in Example 1 exhibit excellent ROS responsiveness, and the double-arm structure of the carrier significantly improves the drug loading rate and enhances the ROS responsiveness.

[0065] Experimental Example 5: Characterization of the enzyme responsiveness of the nanoparticles prepared in Example 1

[0066] To evaluate the enzymatic responsiveness of the nanoparticles, the nanoparticles prepared in Examples 1, 2, and 5 were dispersed in a pH 5.0 acetate buffer (simulating the acidic environment of lysosomes) to prepare a 1 mg / mL solution. The absorbance of the solution was dynamically monitored at 600 nm wavelength under constant temperature of 25 °C using a microplate reader. Samples were taken every 2 h for 24 h, and the enzyme-triggered disintegration effect was evaluated by the turbidity change rate. Results are as follows: Figure 6 As shown, the absorbance at 0 h was 0.35 au, and after 24 h, the absorbances of Example 1, Comparative Example 2, and Comparative Example 5 were 0.05, 0.25, and 0.33 au, respectively, with turbidity reduction rates of 86%, 29%, and 6%, respectively. This is because the nanoparticles of Example 1 have a protective shell to prevent HAase from inactivation and efficiently degrade PPS, while the HAase in the nanoparticles of Comparative Example 2 lacks a protective shell, and its peptide bonds are hydrolyzed by serum proteases, failing to effectively trigger nanoparticle degradation. Comparative Example 5 lacks HAase, and therefore no degradation occurs. Thus, the nanoparticles prepared in Example 1 exhibit good enzyme responsiveness.

[0067] Experimental Example 6: Characterization of pH responsiveness and fluorescence visualization of the nanoparticles prepared in Example 1

[0068] To evaluate the pH responsiveness of the nanoparticles, the nanoparticles prepared in Example 1, Comparative Example 3, and Comparative Example 6 were dispersed in 10 mL of pH 5.0 acetate buffer (simulating the acidic environment of lysosomes) and pH 7.4 PBS (simulating the physiological environment), respectively, serving as the experimental and control groups. The BBR release channel was monitored using a fluorescence spectrophotometer under constant temperature and light-protected conditions at 25 °C: excitation was performed at 345 nm, the emission spectrum was scanned from 400 to 600 nm, and the fluorescence intensity at 450 nm was recorded at different times. The fluorescence intensity at 0 h was 150 au, and the fluorescence intensity at 24 h was as follows: Figure 7 As shown, under pH 5.0 conditions, the fluorescence intensity of Examples 1, 3, and 6 increased from 152 au to 463, 451, and 180 au, respectively, within 24 h, while under pH 7.4 conditions, the fluorescence intensity increased to 157, 155, and 154 au, respectively. This is because the fluorescent prodrug Cy5-KET-BBR of Examples 1 and the ketal prodrug KET-BBR of Comparative Example 1 both have ketal bonds and disintegrate under acidic conditions, releasing the original drug BBR, which exhibits fluorescence properties; while the drug in Comparative Example 6 is the original drug BBR, which has no pH-responsive properties. Therefore, the nanoparticles prepared in Example 1 have good pH-responsive properties.

[0069] To evaluate the fluorescence visualization properties of the nanoparticles, the nanoparticles prepared in Example 1, Comparative Example 3, and Comparative Example 6 were dispersed in 10 mL of pH 5.0 acetate buffer (simulating the acidic environment of lysosomes). After 24 h, 20 μL of the dispersion was dropped onto a glass slide. The excitation wavelength was 640 nm, and the emission wavelength was 670 nm. The red fluorescence was observed using a laser confocal microscope in the dark. The results are shown in Table 1. The sample of Example 1 showed obvious fluorescence, while Comparative Example 3 and Comparative Example 6 showed no fluorescence. This is because the prodrug in Example 1 was labeled with Cy5 fluorescence, thus the drug released by the nanoparticles prepared in the examples had a fluorescence visualization effect.

[0070] Table 1. Fluorescence phenomena of nanoparticles prepared in Example 1, Comparative Example 3, and Comparative Example 6

[0071] Group Fluorescence phenomenon Example 1 Red fluorescence Comparative Example 3 No red fluorescence Comparative Example 6 No red fluorescence

[0072] Experimental Example 7: In vivo targeted characterization of nanoparticles prepared in Example 1

[0073] Ten-week-old female adenomyosis model mice were selected, and lesions were established through surgical transplantation: unilateral uterine horn was removed, and tissue fragments were obtained using a 2 mm biopsy needle after longitudinal dissection. Three tissue fragments were sutured to both sides of the peritoneal wall (6 fragments / mouse). Postoperatively, estradiol (E2, 0.1 μg / mouse / day, for 3 consecutive days) was administered to synchronize estrous cycles and promote lesion growth. Mice were divided into an experimental group (tail vein injection of 50 μL of nanoparticle PBS solution prepared in Example 1, concentration 10 mg / L) and a control group (tail vein injection of an equal volume of nanoparticle PBS solution prepared with contrast agent 4). Twenty-four hours after injection, lesion tissue was harvested, embedded in OCT, and prepared into 10 μm thick frozen sections. After DAPI staining, the distribution of nanoparticles was observed using a fluorescence microscope (Cy5 channel: excitation / emission wavelength = 648 / 668 nm). The results are as follows: Figure 8 As shown, the accumulation of nanoparticles in the lesions of the experimental group was higher than that of the control group, indicating that RGD modification achieved active enrichment by targeting lesion angiogenesis (high expression of αvβ3 integrin). Therefore, the nanoparticles prepared in Example 1 have the potential to deliver drugs for targeted treatment of adenomyosis.

[0074] Experimental Example 8: Cell proliferation characterization of nanoparticles prepared in Example 1

[0075] The effect of the nanoparticles prepared in Example 1 on the proliferation of ectopic endometrial stromal cells (EESCs) was evaluated using the thymidine nucleoside analogue (BrdU) method. EESCs were exposed to a control group (pH 5.0 acetate buffer containing 5 mM H2O2) and an experimental group (the nanoparticles from Example 1 dissolved in the control group solution) for 72 hours. The experimental group was treated with different concentrations of nanoparticle solution (50, 200, and 400 μg / mL). Figure 9 As shown, the experimental group inhibited the proliferation of EESCs compared to the control group. Furthermore, the results showed that the proportion of BrdU-positive cells in EESCs was lower in the 200 and 400 μg / mL nanoparticle treatments than in the control group. This is attributed to the berberine prodrug released by the nanoparticles inhibiting cell proliferation. These results indicate that the nanoparticles prepared in Example 1 have an inhibitory effect on the proliferation of EESCs derived from adenomyosis.

[0076] Experimental Example 9: Characterization of the angiogenesis inhibition of the nanoparticles prepared in Example 1

[0077] To evaluate the ability of the nanoparticles prepared in Example 1 to inhibit angiogenesis, the effect of the nanoparticles on the secretion of the key pro-angiogenic factor VEGF by adenomyosis-related cells was determined using an enzyme-linked immunosorbent assay (ELISA). Primary endometrial stromal cells isolated from ectopic lesions of adenomyosis patients were used as a model. Control group cells were placed in PBS buffer at pH 7.4, while experimental group cells were treated with 200 μg / mL of the nanoparticles from Example 1 (using the same solvent as the control group) for 24 hours. After treatment, the cell culture supernatant was collected, and the VEGF protein concentration in the supernatant was quantitatively detected using a specific VEGF ELISA kit. Results are as follows: Figure 10 As shown, compared with the control group, the nanoparticle treatment in Example 1 reduced the VEGF protein level in the supernatant of primary endometrial stromal cells. The VEGF concentration in the experimental group was 25.3 pg / mL, which was 52% lower than that in the control group. These results indicate that the nanoparticles of Example 1 can inhibit the production of the pro-angiogenic factor VEGF, suggesting that they have the potential to inhibit angiogenesis.

[0078] Experimental Example 10: In vivo biosafety characterization of the nanoparticles prepared in Example 1

[0079] To assess the biosafety of the microspheres prepared in Example 1, we euthanized mice on the last day of the experiment and harvested vital organs (spleen, liver, heart, kidney, and lung). After H&E staining, the vital organs were fixed, embedded, and subjected to histological examination. In any treatment group, such as Figure 11 As shown, we did not detect necrosis or significant tissue damage (including apoptosis, nuclear cleavage, pyknosis, or inflammatory cell infiltration). This indicates that the dosage used was safe and there was no apparent toxicity.

[0080] In summary, the cascade-responsive nanoparticles for adenomyosis of the present invention possess a synergistic delivery capability of targeted accumulation at lesions, carrier-responsive disintegration, enzymatic penetration, and precise prodrug release. Experimental results show that the system achieves a drug loading rate of 18.7% and successfully achieves: good ROS-responsive carrier lysis, pH-responsive prodrug release, fluorescence visualization of the drug release process, targeted accumulation at lesion sites, inhibition of abnormal proliferation of myometrial lesion cells, reduction of pathological angiogenesis, and good biocompatibility. This system holds promise as a novel and efficient drug delivery strategy for patients with adenomyosis.

[0081] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A cascade-responsive nanoparticle for adenomyosis, characterized in that, The nanoparticles contain a PLGA-(SS-PPS)2 carrier, a fluorescent ketal-berberine prodrug, and hyaluronidase, and the surface of the nanoparticles is modified with RGD peptide.

2. The cascade-responsive nanoparticle for adenomyosis according to claim 1, characterized in that: The nanoparticles have a spherical structure and a size of 100 nm.

3. The cascade-responsive nanoparticle for adenomyosis according to claim 1, characterized in that: The PLGA-(SS-PPS)2 support was pyrolyzed under 10 mM H2O2.

4. The cascade-responsive nanoparticle for adenomyosis according to claim 1, characterized in that: The fluorescent-ketal-berberine prodrug releases at pH 5.

0.

5. A method for preparing cascade-responsive nanoparticles for adenomyosis as described in any one of claims 1-4, characterized in that, The preparation method includes the following steps: (1) Preparation of ROS-responsive carrier: In an anhydrous and oxygen-free environment, HO-PLGA-COOH with Mn=10 kDa was first dissolved in anhydrous DMSO and added to the EDC / NHS activation system. After the carboxyl activation was completed, cystamine was added and reacted at 4 °C for 2 h to form PLGA-cystamine intermediate. Then, DMSO solution of HS-PPS-COOH with Mn=10 kDa was added dropwise to the system and the temperature was raised to 25 °C for 36 h. A two-arm disulfide bond bridging structure was constructed by secondary amidation. The reaction solution was purified by dialysis using a dialysis bag with a molecular weight cutoff of 3.5 kDa and then freeze-dried to obtain ROS-responsive carrier PLGA-(SS-PPS)2. (2) Preparation of fluorescent-ketal-berberine prodrug: BBR was dissolved in 15 mL of anhydrous acetone, and 2,2-dimethoxypropane and 10 mg of p-toluenesulfonic acid were added. The molar ratio of BBR to 2,2-dimethoxypropane was 1:

5. The reaction was carried out under nitrogen protection at 60 °C for 8 h to generate the ketalized intermediate KET-BBR. After the reaction system was cooled to room temperature, Cy5-NHS ester was added. The molar ratio of KET-BBR to Cy5-NHS ester was 1:1.

2. The pH was adjusted to 8.5 with 0.1 M NaOH. The mixture was stirred at room temperature for 12 h under light-protected conditions to connect the Cy5 fluorescent group through ester bonds, forming the target product Cy5-KET-BBR. The crude product was purified by silica gel column chromatography. The elution fraction was monitored under a 365 nm UV lamp. The dual-color fluorescent fraction was enriched based on the dual fluorescence signals of yellow autofluorescence of BBR and red fluorescence of Cy5. The solvent was removed by rotary evaporation under reduced pressure and then dried under vacuum. (3) Assembly of nanoparticles: Thiolized chitosan and HAase were mixed in a pH 5.0 acetate buffer, and 0.5 mM H2O2 was added to initiate disulfide crosslinking to form 200 nm HAase-chitosan submicron. Subsequently, 40 mg of PLGA-(SS-PPS)2, 15 mg of Cy5-KET-BBR, 8 mg of soybean lecithin and HAase-chitosan submicron containing 500 U HAase were dissolved in 2 mL of dichloromethane to form an oil phase, which was injected into an ice bath aqueous phase containing 5% PVA. After ultrasonic emulsification, the solvent was evaporated under reduced pressure. The ultrasonic emulsification parameters were 200 W, 70% amplitude, and 4 min. Finally, RGD modification was performed. The nanoparticles were coupled with 1.5 mg of c(RGDfK) peptide in pH 7.4 PBS by EDC / NHS for 12 h, purified by ultracentrifugation at 25000 rpm for 30 min, and then freeze-dried to obtain RGD-modified nanoparticles.

6. The method for preparing cascade-responsive nanoparticles for adenomyosis according to claim 5, characterized in that, The molar ratio of HO-PLGA-COOH to cystamine in step (1) is 1:1.

2.

7. The method for preparing cascade-responsive nanoparticles for adenomyosis according to claim 5, characterized in that, In step (1), the molar ratio of EDC·HCl to NHS in the EDC / NHS activation system is 1:0.

8.

8. The method for preparing cascade-responsive nanoparticles for adenomyosis according to claim 5, characterized in that, The molar ratio of PLGA to PPS in step (1) is 1:

2.

9. The method for preparing cascade-responsive nanoparticles for adenomyosis according to claim 5, characterized in that, The λex / em of the dual-color fluorescent component in step (2) is 650 / 670 nm.

10. The method for preparing cascade-responsive nanoparticles for adenomyosis according to claim 5, characterized in that, The degree of thiol substitution of the thiolated chitosan in step (2) is 2.3 mmol / g.

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