Chymotrypsin compound preparation injected under ultrasound and used for intervertebral disc degeneration and preparation method of chymotrypsin compound preparation

Abstract

The invention discloses a chymotrypsin compound preparation for intervertebral disc degeneration through ultrasonic injection and a preparation method, and relates to the technical field of chymotrypsin compound preparation preparation. Comprising freeze-dried recombinant chymotrypsin powder, biodegradable nanoparticles loaded with chymotrypsin and a matrix synthesis promoting factor, and a microenvironment response type hydrogel precursor solution, the freeze-dried recombinant chymotrypsin powder comprises chymotrypsin, trehalose, mannitol and a nonionic surfactant, wherein the mass ratio of the chymotrypsin to the trehalose to the mannitol to the nonionic surfactant is 1: 10: 5: 1; freeze-dried chymotrypsin powder, ultrasonic response type PLGA nano-microspheres loaded with chymotrypsin and matrix synthesis promoting factors and a microenvironment response hydrogel precursor solution containing a sound-sensitive cross-linking agent are organically combined, and removal-regeneration synergistic treatment is achieved after percutaneous injection is conducted to a degenerated intervertebral disc.

Description

Technical Field

[0001] This invention relates to the field of chymotrypsin complex preparation technology, and in particular to a chymotrypsin complex preparation for intervertebral disc degeneration administered via ultrasound injection and its preparation method. Background Technology

[0002] The preparation technology of rennet complex formulations generally refers to the formulation process of combining rennet with one or more excipients, carriers or active ingredients through physical mixing, encapsulation, cross-linking or freeze-drying to improve its stability, control its release behavior or expand its application functions. Common methods include loading the enzyme into liposomes, microspheres, nanoparticles or hydrogels, or co-freezing with protective agents such as sugars and polyols to improve storage stability. However, traditional technologies are mostly used in the fields of food, leather or laboratory modeling, and a controllable, synergistic and responsive complex formulation system for the treatment of intervertebral disc degeneration has not yet been formed.

[0003] In existing model studies, papain is only used to induce irreversible intervertebral disc damage by injecting high doses of non-selectively degrading proteoglycans. Its mechanism of action is destructive, uncontrollable, and without repair guidance, making it unsuitable for clinical treatment. Furthermore, the use of papain alone in existing technologies can cause excessive matrix degradation, accelerating intervertebral disc collapse. The use of growth factors such as TGF-β3 alone is difficult to effectively reach target cells due to the presence of dense cross-linked matrix and inflammatory barriers in the degenerated microenvironment, resulting in extremely low bioavailability. Summary of the Invention

[0004] In view of the aforementioned existing problems, the present invention is proposed.

[0005] Therefore, this invention provides a chymotrypsin complex preparation for intervertebral disc degeneration administered via ultrasound injection. This addresses the problems in existing model studies where chymotrypsin is only used to induce irreversible intervertebral disc damage through high-dose injection of non-selectively degraded proteoglycans. Its mechanism of action is destructive, uncontrollable, and lacks repair guidance, making it unsuitable for clinical treatment. Furthermore, existing technologies using chymotrypsin alone can cause excessive matrix degradation, accelerating disc collapse. Additionally, the use of growth factors such as TGF-β3 alone results in extremely low bioavailability due to the dense cross-linked matrix and inflammatory barrier in the degenerated microenvironment, hindering effective contact with target cells.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: In a first aspect, the present invention provides an ultrasound-injected chymotrypsin complex preparation for intervertebral disc degeneration, made of the following materials: Freeze-dried recombinant chymotrypsin powder, biodegradable nanospheres loaded with chymotrypsin and matrix synthesis promoters, and microenvironment-responsive hydrogel precursor solutions.

[0007] As a preferred embodiment of the ultrasound-guided injection of chymotrypsin complex for intervertebral disc degeneration according to the present invention, wherein: the freeze-dried recombinant chymotrypsin powder comprises chymotrypsin, trehalose, mannitol and nonionic surfactant, wherein the mass ratio of chymotrypsin, trehalose, mannitol and nonionic surfactant is 1:10:5:1.

[0008] As a preferred embodiment of the chymotrypsin complex preparation for intervertebral disc degeneration administered via ultrasound under the present invention, wherein: the matrix synthesis promoting factor is selected from one or more of TGF-β3, BMP-7, or SOX9 activating peptide; the nanospheres are composed of polylactic-co-glycolic acid copolymer (PLGA) with a particle size of 200–400 nm, and cavitation microbubble precursors or acoustic liposomes are incorporated into the PLGA matrix to enhance ultrasound responsiveness.

[0009] As a preferred embodiment of the ultrasound-injected chymotrypsin complex formulation for intervertebral disc degeneration described in this invention, the microenvironment and ultrasound dual-responsive hydrogel precursor solution comprises methacrylamide hyaluronic acid, thioketal diacrylate, acetal-modified gelatin, and an acoustically sensitive crosslinking agent that can be triggered by ultrasonic shear force or cavitation effect; the lyophilized recombinant chymotrypsin powder is reconstituted before use and dispersed together with the nanospheres in the hydrogel precursor solution to form an injectable composite precursor solution. This precursor solution can form a resident gel through in-situ crosslinking in the intervertebral disc, and under external low-intensity focused ultrasound (LIFU) irradiation, the initial burst release of chymotrypsin is accelerated, followed by the sustained release of matrix synthesis-promoting factors dependent on the microenvironment response.

[0010] Secondly, the present invention provides a method for preparing a chymotrypsin complex preparation for intervertebral disc degeneration via ultrasound injection, comprising: The chymotrypsin, trehalose, mannitol and nonionic surfactant were dissolved in sterile water at a mass ratio of 1:10:5:1 to form a homogeneous mixed solution. The resulting mixed solution was freeze-dried to obtain freeze-dried recombinant chymotrypsin powder; Encapsulating chymotrypsin and matrix synthesis promoters in polylactic acid-glycolic acid copolymer (PLGA) containing acoustic components, and preparing ultrasonically responsive biodegradable nanospheres with a particle size of 200–400 nm by a double emulsification-solvent evaporation method combined with ultrasound-assisted emulsification. Methacrylamide hyaluronic acid, thioketone diacrylate, acetal-modified gelatin, and a sound-sensitive crosslinking agent were dissolved in a phosphate buffer solution at pH 7.4 to prepare a microenvironment and ultrasound-responsive hydrogel precursor solution. The lyophilized recombinant chymotrypsin powder was reconstituted and mixed with the nanospheres, and then subjected to low-frequency ultrasound (20–50 kHz, power 0.5–2 W / cm²). 2 With the assistance of [unclear], it is uniformly dispersed in the hydrogel precursor solution to form an injectable composite precursor solution.

[0011] As a preferred embodiment of the ultrasound-guided injection of the chymotrypsin complex preparation for intervertebral disc degeneration according to the present invention, wherein the freeze-drying process includes: The mixed solution was kept at -40°C. Pre-freeze at °C for 2 hours, then freeze under vacuum of no more than 0.1 mbar at a temperature of -25 °C. Dry at °C for 24 hours, then at 25 °C. The lyophilized recombinant chymotrypsin powder with a loose and porous structure was obtained by secondary drying at °C and a vacuum degree not exceeding 0.05 mbar for 6 hours.

[0012] As a preferred embodiment of the chymotrypsin complex preparation for intervertebral disc degeneration administered via ultrasound under the present invention, wherein: the ultrasound-assisted emulsification step in the dual emulsification-solvent evaporation method includes: During the formation of W / O colostrum and W / O / W re-emulsion, pulsed ultrasound (frequency 20 kHz, power 100–300 W, pulse duration 5 s / interval 5 s) was applied to improve droplet uniformity and reduce particle size distribution. After the organic solvent evaporation stage, the precipitate was collected by centrifugation, washed, and freeze-dried to obtain ultrasonically responsive nanospheres with high encapsulation efficiency.

[0013] As a preferred embodiment of the chymotrypsin complex preparation for intervertebral disc degeneration administered via ultrasound under the present invention, wherein: the matrix synthesis-promoting factor is any one of TGF-β3, BMP-7, or SOX9 activating peptide; the encapsulation rate of the nanospheres for chymotrypsin is 65% to 75%, and the encapsulation rate for the matrix synthesis-promoting factor is 55% to 65%; and the nanospheres are at 1 MHz, 0.5–1.5 W / cm². 2 Under intense ultrasound irradiation, 30%–50% of chymotrypsin can be released within 5 minutes.

[0014] As a preferred embodiment of the chymotrypsin complex preparation for intervertebral disc degeneration administered via ultrasound under the present invention, wherein: in the microenvironment and ultrasound dual-responsive hydrogel precursor solution, the mass concentration of methacrylamide hyaluronic acid is 3%, thioketal diacrylate is 0.5%, acetal-modified gelatin is 2%, and the acoustic crosslinking agent is an acoustically cleavable crosslinking molecule containing peroxy bonds or ester bonds, with a concentration of 0.1%–0.3%; the pH value of the phosphate buffer is 7.4.

[0015] As a preferred embodiment of the ultrasound-guided injection of the chymotrypsin complex preparation for intervertebral disc degeneration described in this invention, wherein: after the injectable complex precursor fluid is percutaneously injected into the nucleus pulposus region of the intervertebral disc, in-situ cross-linking and drug release can be triggered by any of the following methods: Irradiate with 365 nm ultraviolet light for 5 seconds; It depends on the spontaneous cross-linking of local high reactive oxygen species and low pH microenvironment in intervertebral disc degeneration; By applying external low-intensity focused ultrasound, the simultaneous promotion of gel network reconstruction and rapid initial release of chymotrypsin through ultrasonic cavitation and thermal effects can achieve a spatiotemporally controllable therapeutic response.

[0016] The beneficial effects of this invention are as follows: By organically combining lyophilized chymotrypsin powder, ultrasound-responsive PLGA nanospheres loaded with chymotrypsin and matrix synthesis-promoting factors, and a microenvironment-responsive hydrogel precursor solution containing a sound-sensitive crosslinking agent, a synergistic treatment of clearance and regeneration is achieved: After percutaneous injection into the degenerated intervertebral disc, the rapid initial release of chymotrypsin can be precisely triggered by external low-intensity focused ultrasound (LIFU) to efficiently degrade the abnormal extracellular matrix. Subsequently, the matrix synthesis-promoting factors are released slowly in a local microenvironment of high reactive oxygen species / low pH to promote the regeneration of functional nucleus pulposus matrix. At the same time, the ultrasound-assisted preparation process effectively improves the uniformity and encapsulation rate of the nanospheres. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a flowchart of the preparation method of the chymotrypsin complex preparation for intervertebral disc degeneration by ultrasound injection in Example 1. Detailed Implementation

[0019] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0020] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0021] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0022] Example 1, referring to Figure 1 This is the first embodiment of the present invention, which provides an ultrasound-guided injection of a chymotrypsin complex preparation for intervertebral disc degeneration, comprising: Freeze-dried recombinant chymotrypsin powder, biodegradable nanospheres loaded with chymotrypsin and matrix synthesis promoters, and microenvironment-responsive hydrogel precursor solutions.

[0023] Furthermore, the freeze-dried recombinant chymotrypsin powder contains chymotrypsin, trehalose, mannitol, and a nonionic surfactant, wherein the mass ratio of chymotrypsin, trehalose, mannitol, and the nonionic surfactant is 1:10:5:1.

[0024] It should be noted that this specific ratio of lyophilization protection system can effectively maintain the spatial conformation and catalytic activity of chymotrypsin during freeze-drying and long-term storage, significantly inhibiting inactivation caused by interfacial adsorption, dehydration or oxidation, and ensuring that the enzyme activity retention rate after reconstitution is not less than 90%, thereby ensuring the accuracy of therapeutic dosage and the shelf stability of the formulation.

[0025] Furthermore, the matrix synthesis promoter is selected from one or more of TGF-β3, BMP-7, or SOX9 activating peptides, and the nanospheres are composed of polylactic acid-glycolic acid copolymer (PLGA) with a particle size of 200–400 nm.

[0026] It should be noted that co-loading matrix-promoting factors and chymotrypsin into PLGA nanospheres with a particle size range of 200–400 nm not only facilitates their uniform dispersion and stable embedding in the hydrogel network, but also utilizes the controllable degradation characteristics of PLGA materials to achieve delayed release of growth factors, avoiding their premature consumption during the clearance stage, thereby providing a continuous and effective biological signal for subsequent ECM synthesis in nucleus pulposus cells.

[0027] Furthermore, the lyophilized recombinant chymotrypsin powder is reconstituted before use and dispersed together with the nanospheres in the hydrogel precursor solution. After in-situ cross-linking, an injectable composite gel is formed, which can realize the on-demand release of chymotrypsin under the stimulation of the local microenvironment of intervertebral disc degeneration, and subsequently release matrix synthesis-promoting factors to synergistically achieve the controllable clearance and functional regeneration of abnormal extracellular matrix.

[0028] It should be noted that this composite gel structure integrates enzyme-controlled degradation, factor-induced synthesis, and intelligent response. In the low pH and high reactive oxygen microenvironment unique to degenerated intervertebral discs, it triggers the dynamic degradation of the hydrogel network, preferentially releasing chymotrypsin to selectively remove aged and cross-linked proteoglycans without damaging healthy collagen fibers. Subsequently, it releases factors such as TGF-β3 to activate endogenous repair pathways, achieving an orderly transformation from pathological matrix clearance to functional matrix reconstruction. This overcomes the technical bottlenecks of traditional single enzyme injection leading to irreversible damage or the failure of simple growth factor therapy due to microenvironment inhibition.

[0029] This embodiment also provides a method for preparing a chymotrypsin complex preparation for intervertebral disc degeneration via ultrasound injection, comprising: S1. Dissolve chymotrypsin, trehalose, mannitol and nonionic surfactant in sterile water at a mass ratio of 1:10:5:1 to form a homogeneous mixed solution.

[0030] Furthermore, the mass ratio of chymotrypsin, trehalose, mannitol, and nonionic surfactant is 1:10:5:1.

[0031] It should be noted that in this specific combination, trehalose, as a glass-forming agent, effectively encapsulates chymotrypsin molecules, inhibiting protein conformational unfolding during freezing and drying; mannitol provides a rigid framework to prevent freeze-drying collapse; and nonionic surfactants (such as Poloxamer188) shield the adsorption stress at the gas-liquid interface. The synergistic effect of the three significantly improves the structural integrity and catalytic activity retention of the enzyme during subsequent freeze-drying and reconstitution processes.

[0032] S2. The resulting mixed solution is freeze-dried to obtain freeze-dried recombinant chymotrypsin powder.

[0033] Furthermore, the mixed solution was kept at -40°C. Pre-freeze at °C for 2 hours, then freeze under vacuum of no more than 0.1 mbar at a temperature of -25 °C. Dry at °C for 24 hours, then at 25 °C. The lyophilized recombinant chymotrypsin powder with a loose and porous structure was obtained by secondary drying at °C and a vacuum degree not exceeding 0.05 mbar for 6 hours.

[0034] It should be noted that this programmed freeze-drying process, by precisely controlling the pre-freezing rate, the sublimation conditions of the first drying, and the desorption temperature and vacuum degree of the second drying, ensures that water is fully removed to prolong the stability of the formulation, while avoiding protein aggregation or inactivation caused by high temperature or excessively rapid dehydration. The result is a freeze-dried powder with high porosity, easy resolubility, and enzyme activity retention of ≥90%, which meets the clinical need for immediate preparation and use.

[0035] S3. Encapsulate chymotrypsin and matrix synthesis promoters in polylactic acid-glycolic acid copolymer (PLGA) containing acoustic components, and prepare ultrasonically responsive biodegradable nanospheres with a particle size of 200–400 nm by a double emulsification-solvent evaporation method combined with ultrasonic-assisted emulsification.

[0036] Furthermore, during the formation of W / O colostrum and W / O / W re-emulsion, pulsed ultrasound (frequency 20kHz, power 100–300 W, pulse duration 5 s / interval 5 s) was applied to improve droplet uniformity and reduce particle size distribution. After the organic solvent evaporation stage, the precipitate was collected by centrifugation, washed, and then freeze-dried to obtain ultrasonically responsive nanospheres with high encapsulation efficiency. The matrix synthesis-promoting factor is any one of TGF-β3, BMP-7, or SOX9 activating peptide; the nanospheres have an encapsulation efficiency of 65% to 75% for chymotrypsin and 55% to 65% for the matrix synthesis-promoting factor; and the nanospheres operate at 1 MHz and 0.5–1.5 W / cm². 2 Under intense ultrasound irradiation, 30%–50% of chymotrypsin can be released within 5 minutes.

[0037] It should be noted that by applying pulsed ultrasound (20 kHz, 100–300 W, 5 s / 5 s) during the formation of W / O colostrum and W / O / W re-emulsion, not only was the stability and homogeneity of the emulsion system significantly improved, effectively controlling the nanosphere particle size within the therapeutic window of 200–400 nm, but the encapsulation efficiency of chymotrypsin and matrix synthesis-promoting factors (such as TGF-β3, BMP-7, or SOX9 activating peptide) in the PLGA matrix was also enhanced, achieving encapsulation efficiencies of 65%–75% and 55%–65%, respectively. More importantly, the introduced acoustic component endowed the nanospheres with ultrasonic-responsive release capabilities at 1 MHz and 0.5–1.5 W / cm². 2 Ultrasonic irradiation can rapidly release 30%–50% of chymotrypsin within 5 minutes, thereby achieving precise and on-demand removal of abnormal matrix in degenerated intervertebral discs and avoiding the risks of systemic exposure and non-targeted degradation.

[0038] S4. Methacrylamide hyaluronic acid, thioketone diacrylate, acetal-modified gelatin, and acoustic crosslinking agent were dissolved in phosphate buffer solution at pH 7.4 to prepare a microenvironment and ultrasound-responsive hydrogel precursor solution.

[0039] Furthermore, in the microenvironment and ultrasound dual-response hydrogel precursor solution, the mass concentration of methacrylamide hyaluronic acid is 3%, thioketal diacrylate is 0.5%, acetal-modified gelatin is 2%, and the acoustic crosslinking agent is an acoustically cleavable crosslinking molecule containing peroxy bonds or ester bonds, with a concentration of 0.1%–0.3%; the pH value of the phosphate buffer is 7.4.

[0040] It should be noted that the preparation of a hydrogel precursor solution at pH 7.4 by co-concentrating methacrylated hyaluronic acid (3%), thioketal diacrylate (0.5%), acetal-modified gelatin (2%), and a sonically cleavable acoustic crosslinking agent (0.1%–0.3%) not only endows the system with intelligent responsiveness to the intervertebral disc degeneration microenvironment (such as low pH and high ROS), but also enables the system to undergo controllable cleavage under external ultrasound stimulation by introducing acoustically cleavable crosslinking structures containing peroxy bonds or ester bonds, thereby achieving dynamic reconstruction of the gel network. This synergistic effect of the dual response mechanism of microenvironment and ultrasound not only ensures the long-term residence of the preparation in the nucleus pulposus, but also provides multiple triggering pathways for the sequential release of subsequent drugs, significantly improving the precision and adaptability of treatment.

[0041] S5. Reconstitute the lyophilized recombinant chymotrypsin powder and mix it with the nanospheres, then sonicate at low frequency (20–50 kHz, power 0.5–2 W / cm²). 2 With the assistance of [unclear], it is uniformly dispersed in the hydrogel precursor solution to form an injectable composite precursor solution.

[0042] Furthermore, after the injectable composite precursor solution is percutaneously injected into the nucleus pulposus region of the intervertebral disc, in-situ cross-linking and drug release can be triggered through any of the following methods: Irradiate with 365 nm ultraviolet light for 5 seconds; It depends on the spontaneous cross-linking of local high reactive oxygen species and low pH microenvironment in intervertebral disc degeneration; By applying external low-intensity focused ultrasound, the simultaneous promotion of gel network reconstruction and rapid initial release of chymotrypsin through ultrasonic cavitation and thermal effects can achieve a spatiotemporally controllable therapeutic response.

[0043] It should be noted that low-frequency ultrasound (20–50 kHz, 0.5–2 W / cm²) was used during the preparation of the complex precursor solution. 2The auxiliary dispersion can effectively prevent the aggregation of nanospheres and reconstituted chymotrypsin in the high-viscosity hydrogel precursor, ensuring the uniformity and stability of the system and improving the smoothness of injection and the consistency of distribution. After the final injectable composite precursor fluid is injected into the intervertebral disc, in-situ cross-linking and drug release can be flexibly triggered by three methods: ultraviolet light, pathological microenvironment, or external low-intensity focused ultrasound. In particular, the ultrasound triggering mode can use its cavitation and mild thermal effects to simultaneously promote the rapid formation of gel and induce the explosive release of chymotrypsin, realizing "instant injection and control" spatiotemporal precision treatment, which greatly enhances the flexibility of clinical operation and the potential for individualized treatment.

[0044] In summary, this invention achieves a synergistic treatment of decomposition and regeneration by organically combining lyophilized chymotrypsin powder, ultrasound-responsive PLGA nanospheres loaded with chymotrypsin and matrix synthesis-promoting factors, and a microenvironment-responsive hydrogel precursor solution containing a sound-sensitive crosslinking agent. After percutaneous injection into the degenerated intervertebral disc, the rapid initial release of chymotrypsin can be precisely triggered by external low-intensity focused ultrasound (LIFU) to efficiently degrade the abnormal extracellular matrix. Subsequently, the matrix synthesis-promoting factors are released slowly in a local microenvironment of high reactive oxygen species / low pH to promote the regeneration of functional nucleus pulposus matrix. At the same time, the ultrasound-assisted preparation process effectively improves the uniformity and encapsulation efficiency of the nanospheres.

[0045] Example 2 is the second embodiment of the present invention. This embodiment provides the preparation and in vitro release performance verification of an ultrasound-responsive composite formulation using TGF-β3 as a matrix synthesis promoter, including: A 4 mg / mL mixed solution was prepared by dissolving chymotrypsin, trehalose, mannitol, and Poloxamer 188 in sterile water at a mass ratio of 1:10:5:1; and then subjected to -40°C sterilization. Pre-freeze at °C for 2 hours, -25°C °C / 0.08 mbar, one drying time 24 h, 25 The powder was dried for 6 hours at °C / 0.04 mbar to obtain a loose and porous freeze-dried powder.

[0046] Separately, rennet (2 mg) and TGF-β3 (100 ng) were dissolved in 1 mL of the inner aqueous phase. Dichloromethane (2 mL) containing PLGA (50:50, 15 kDa, 50 mg) and 1% cavitation microbubble precursor (such as perfluoropentane / phospholipid mixture) was added to form a W / O primary emulsion under pulsed ultrasound (20 kHz, 200 W, 5 s on / 5 s off, 30 s total). The emulsion was then added dropwise to an outer aqueous phase (10 mL) containing 1% PVA and ultrasound was applied again with the same parameters to form a W / O / W secondary emulsion. The solvent was stirred and evaporated for 4 h. The microspheres were collected by centrifugation, washed, and lyophilized to obtain ultrasound-responsive nanospheres with a particle size of 320 nm, a rennet encapsulation rate of 70%, and a TGF-β3 encapsulation rate of 62%.

[0047] Methacrylamide hyaluronic acid (30 mg), thioketal diacrylate (5 mg), acetal-modified gelatin (20 mg), and a sound-sensitive crosslinking agent (diacrylate containing peroxy bonds, 3 mg) were dissolved in 1 mL of pH 7.4 PBS to obtain a microenvironment and ultrasound-responsive hydrogel precursor solution.

[0048] After reconstitution of the freeze-dried powder, it was combined with nanospheres (1×10⁻⁶). 9 The mixture was blended with particles / mL and subjected to low-frequency sonication (30 kHz, 1.0 W / cm²). 2 With the assistance of 30 s, it is uniformly dispersed in the precursor fluid to form an injectable composite precursor fluid.

[0049] Three sets of conditions were set up for the in vitro release experiment (37) °C): (i) PBS + 1 mM H2O2, pH 6.2 (simulating the pathological microenvironment); (ii) Same as (i) and superimposed with 1 MHz, 1.0 W / cm 2 Ultrasonic irradiation for 5 minutes; (iii) Normal PBS, pH 7.4 (control).

[0050] (iv) The results showed that under (i) conditions, 41% of chymotrypsin was released cumulatively in 24 h, and TGF-β3 reached its peak on day 4 (38% daily release); while in (ii) ultrasound-triggered group, 45% of chymotrypsin was released within 5 min, accelerating the initial clearance phase, confirming the realization of ultrasound-enhanced temporal synergistic release.

[0051] Example 3, the third embodiment of the present invention, provides an ultrasound-responsive compound preparation of BMP-7 as a substitute for TGF-β3 and its nucleus pulposus cell activity evaluation, including: Except for replacing the matrix synthesis promoter with BMP-7 (100 ng), the other operations were the same as in Example 2, and an ultrasound-responsive compound formulation containing BMP-7 was prepared.

[0052] The composite precursor solution was injected into the upper chamber of the Transwell, and human degenerated nucleus pulposus cells were seeded in the lower chamber to simulate the intervertebral disc microenvironment (1% O2, pH 6.5).

[0053] Some experimental groups were treated with 1 MHz, 1.0 W / cm² after sample addition. 2 LIFU irradiation for 10 seconds was used to trigger gel cross-linking and drug release.

[0054] After 7 days of culture, the expression of type II collagen mRNA was upregulated by 3.2 times, the content of aggregated proteoglycans was increased by 2.8 times compared with the control group, and cell viability (CCK-8 assay) was increased by 45%. There was no significant difference between the ultrasound-triggered group and the non-triggered group, indicating that ultrasound did not produce cytotoxicity and that BMP-7 effectively activated the synthesis and metabolism of nucleus pulposus cells.

[0055] Example 4 is the fourth embodiment of the present invention. This embodiment provides a single microenvironmentally responsive composite formulation that does not contain acetal-modified gelatin but retains the acoustic crosslinking agent, comprising: Except that the hydrogel precursor solution contains only methacrylamide hyaluronic acid (3%), thioketone diacrylate (0.5%), and acoustic crosslinking agent (0.3%), and does not contain acetal-modified gelatin, the remaining steps are the same as in Example 2.

[0056] In vitro degradation experiments showed that under H2O2 (1 mM) conditions, the gel lost 65% of its weight after 7 days; however, in a low pH (6.2) ROS-free environment, the weight loss was only 28% after 7 days. Under 1 MHz ultrasound irradiation, even under neutral pH / ROS-free conditions, the gel swelling rate increased by 40% within 5 minutes, significantly accelerating drug release. These results indicate that although a pH-responsive unit is lacking, the acoustic crosslinking agent endows it with ultrasound-triggered capability, enabling controlled release through external intervention in non-ideal microenvironments. While the release precision is slightly lower than that of dual-response systems, it is still superior to traditional single-response systems.

[0057] Example 5, the fifth example of the present invention, provides an animal model validation of an ultrasound-responsive composite formulation using SOX9 activating peptide as a matrix synthesis promoter, including: A model of intervertebral disc degeneration was established by papain puncture of the caudal vertebrae of SD rats, and the rats were randomly divided into groups 2 weeks after the operation.

[0058] The experimental group received a percutaneous injection of the compound preparation of this invention (containing SOX9 activating peptide, 10 μg / disc), followed immediately by low-intensity focused ultrasound (LIFU, 1 MHz, 1.0 W / cm²). 2(15 s) to promote in situ cross-linking and initial release of chymotrypsin; the control group was injected with physiological saline.

[0059] Eight weeks later, T2-weighted MRI showed that the Pfirrmann score in the experimental group improved from grade IV to grade II–III, and the intervertebral disc height preservation rate was 82%. Histological staining (Safranin O / Fast Green) showed significant recovery of proteoglycans and a 3.5-fold increase in the area of ​​Col2a1 immunopositive, confirming that ultrasound-assisted delivery significantly enhanced the SOX9-activated peptide-driven nucleus pulposus phenotype maintenance and matrix regeneration effect.

[0060] Comparative Example 1: Conventional chymotrypsin solution (without complex structure) The chymotrypsin (4 mg / mL) was dissolved directly in physiological saline without lyophilization, encapsulation, or hydrogel composition, serving as Comparative Example 1.

[0061] In vitro stability test: 4 After storage at °C for 7 days, the enzyme activity retention rate was only 58%; 37 Completely inactivated after 24 hours at °C.

[0062] Following injection into the intervertebral discs of rats, MRI revealed irreversible collapse of the intervertebral space height, and histological examination showed extensive matrix dissolution and cell apoptosis, confirming that its destructive effects were uncontrollable and lacked therapeutic value. Even with the application of ultrasound, diffusion and release could not be controlled, resulting in poor efficacy and safety.

[0063] Comparative Example 2: Contains only TGF-β3 nanospheres + ordinary hydrogel (no chymotrypsin, no acoustically sensitive design) PLGA microspheres loaded only with TGF-β3 (without chymotrypsin or cavitation precursor) were prepared and dispersed in a non-responsive methacrylamide hyaluronic acid hydrogel (without thioketal, acetal, or acoustic crosslinking agent).

[0064] In vitro release showed sustained release of TGF-β3, but the efficacy was limited in the rat degeneration model: due to the failure to clear the aging cross-linked matrix, TGF-β3 could not effectively contact cell receptors, and Col2a1 expression was only increased by 1.3 times, which was significantly lower than that in Example 2 (3.2 times); even with the application of ultrasound, it was impossible to trigger the release of chymotrypsin or gel remodeling, proving that "barrier clearance" and "ultrasound response" are both indispensable.

[0065] Comparative Example 3: Freeze-dried powder + nanospheres but without microenvironment / Ultrasonic responsive hydrogel The lyophilized powder and nanospheres (containing cavitation precursors) obtained in Example 2 were directly suspended in physiological saline without adding any responsive hydrogel precursor solution.

[0066] Following percutaneous injection, the preparation rapidly diffused and leaked within the intervertebral disc, with 90% of its contents seeping into surrounding tissues within 24 hours. Although the nanospheres exhibited ultrasound responsiveness, the lack of a retention carrier prevented effective drug accumulation in the target area under ultrasound irradiation. MRI and histological examination revealed a weak therapeutic effect and mild inflammatory response in adjacent nerve roots, demonstrating that in situ retention is a prerequisite for precise ultrasound control.

[0067] Technical Effect Comparison Summary Table:

[0068] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A chymotrypsin complex preparation for intervertebral disc degeneration administered via ultrasound injection, characterized in that: Made from the following materials: Freeze-dried recombinant chymotrypsin powder, biodegradable nanospheres loaded with chymotrypsin and matrix synthesis promoters, and microenvironment-responsive hydrogel precursor solutions.

2. The chymotrypsin complex preparation for intervertebral disc degeneration administered via ultrasound injection as described in claim 1, characterized in that: The freeze-dried recombinant chymotrypsin powder contains chymotrypsin, trehalose, mannitol, and a nonionic surfactant, wherein the mass ratio of chymotrypsin, trehalose, mannitol, and the nonionic surfactant is 1:10:5:

1.

3. The chymotrypsin complex preparation for intervertebral disc degeneration administered via ultrasound injection as described in claim 2, characterized in that: The matrix synthesis promoting factor is selected from one or more of TGF-β3, BMP-7 or SOX9 activating peptide; the nanospheres are composed of polylactic acid-glycolic acid copolymer (PLGA) with a particle size of 200–400 nm, and cavitation microbubble precursors or acoustic liposomes are incorporated into the PLGA matrix to enhance ultrasonic responsiveness.

4. The chymotrypsin complex preparation for intervertebral disc degeneration administered via ultrasound under claim 3, characterized in that: The microenvironment and ultrasound dual-response hydrogel precursor solution contains methacrylamide hyaluronic acid, thioketone diacrylate, acetal-modified gelatin, and an acoustically sensitive crosslinking agent that can be triggered by ultrasonic shear force or cavitation effect. The lyophilized recombinant chymotrypsin powder is reconstituted before use and dispersed together with the nanospheres in the hydrogel precursor solution to form an injectable composite precursor solution. This precursor solution can form a resident gel in situ via crosslinking within the intervertebral disc and accelerate the initial burst release of chymotrypsin under external low-intensity focused ultrasound (LIFU) irradiation, followed by a microenvironment-dependent slow release of matrix synthesis-promoting factors.

5. A method for preparing a chymotrypsin complex preparation for intervertebral disc degeneration via ultrasound-guided injection, as described in any one of claims 1-4, characterized in that: include: The chymotrypsin, trehalose, mannitol and nonionic surfactant were dissolved in sterile water at a mass ratio of 1:10:5:1 to form a homogeneous mixed solution. The resulting mixed solution was freeze-dried to obtain freeze-dried recombinant chymotrypsin powder; Encapsulating chymotrypsin and matrix synthesis promoters in polylactic acid-glycolic acid copolymer (PLGA) containing acoustic components, and preparing ultrasonically responsive biodegradable nanospheres with a particle size of 200–400 nm by a double emulsification-solvent evaporation method combined with ultrasound-assisted emulsification. Methacrylamide hyaluronic acid, thioketone diacrylate, acetal-modified gelatin, and sound-sensitive crosslinking agent were dissolved in pH [a solution]. A microenvironment and ultrasound dual-response hydrogel precursor solution was prepared in phosphate buffer at 7.4°C. The lyophilized recombinant chymotrypsin powder was reconstituted and mixed with the nanospheres, and then subjected to low-frequency ultrasound (20–50 kHz, power 0.5–2 W / cm²). 2 With the assistance of [unclear], it is uniformly dispersed in the hydrogel precursor solution to form an injectable composite precursor solution.

6. The method for preparing the chymotrypsin complex preparation for intervertebral disc degeneration by ultrasound injection as described in claim 5, characterized in that: The freeze-drying process includes: The mixed solution was kept at -40°C. Pre-freeze at °C for 2 hours, then freeze under vacuum of no more than 0.1 mbar at a temperature of -25 °C. Dry at °C for 24 hours, then at 25 °C. The lyophilized recombinant chymotrypsin powder with a loose and porous structure was obtained by secondary drying at °C and a vacuum degree not exceeding 0.05 mbar for 6 hours.

7. The method for preparing the chymotrypsin complex preparation for intervertebral disc degeneration by ultrasound injection as described in claim 6, characterized in that: The ultrasonic-assisted emulsification step in the dual emulsification-solvent evaporation method includes: During the formation of W / O colostrum and W / O / W re-emulsion, pulsed ultrasound (frequency 20 kHz, power 100–300 W, pulse duration 5 s / interval 5 s) was applied to improve droplet uniformity and reduce particle size distribution. After the organic solvent evaporation stage, the precipitate was collected by centrifugation, washed, and freeze-dried to obtain ultrasonically responsive nanospheres with high encapsulation efficiency.

8. The method for preparing the chymotrypsin complex preparation for intervertebral disc degeneration by ultrasound injection as described in claim 7, characterized in that: The matrix synthesis-promoting factor is any one of TGF-β3, BMP-7, or SOX9 activating peptide; the nanospheres have an encapsulation efficiency of 65% to 75% for chymotrypsin and 55% to 65% for the matrix synthesis-promoting factor; and the nanospheres operate at 1 MHz and 0.5–1.5 W / cm². 2 Under intense ultrasound irradiation, 30%–50% of chymotrypsin can be released within 5 minutes.

9. The method for preparing the chymotrypsin complex preparation for intervertebral disc degeneration by ultrasound injection as described in claim 8, characterized in that: In the microenvironment and ultrasound dual-response hydrogel precursor solution, the mass concentration of methacrylamide hyaluronic acid is 3%, thioketone diacrylate is 0.5%, acetal-modified gelatin is 2%, and the acoustic crosslinking agent is an acoustically cleavable crosslinking molecule containing peroxy bonds or ester bonds, with a concentration of 0.1%–0.3%; the pH value of the phosphate buffer is 7.

4.

10. The method for preparing the chymotrypsin complex preparation for intervertebral disc degeneration by ultrasound injection as described in claim 9, characterized in that: After the injectable composite precursor fluid is percutaneously injected into the nucleus pulposus region of the intervertebral disc, in-situ cross-linking and drug release can be triggered through any of the following methods: Irradiate with 365 nm ultraviolet light for 5 seconds; It depends on the spontaneous cross-linking of local high reactive oxygen species and low pH microenvironment in intervertebral disc degeneration; By applying external low-intensity focused ultrasound, the simultaneous promotion of gel network reconstruction and rapid initial release of chymotrypsin through ultrasonic cavitation and thermal effects can achieve a spatiotemporally controllable therapeutic response.

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