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CN-122005769-A - Chymosin compound preparation for intervertebral disc degeneration by ultrasonic injection and preparation method thereof

CN122005769ACN 122005769 ACN122005769 ACN 122005769ACN-122005769-A

Abstract

The invention discloses a chymosin composite preparation for disc degeneration and a preparation method thereof, and relates to the technical field of chymosin composite preparation, comprising freeze-dried recombinant chymosin powder, biodegradable nano-microspheres loaded with chymosin and matrix-promoting synthetic factors and a microenvironment responsive hydrogel precursor solution, wherein the freeze-dried recombinant chymosin powder comprises chymosin, trehalose, mannitol and a nonionic surfactant, the mass ratio of chymosin to trehalose to mannitol to the nonionic surfactant is 1:10:5:1, and the removal-regeneration cooperative treatment is realized by organically combining the freeze-dried chymosin powder, the ultrasonic responsive PLGA nano-microspheres loaded with chymosin and matrix-promoting synthetic factors and the microenvironment responsive hydrogel precursor solution containing a sound-sensitive crosslinking agent after the freeze-dried recombinant chymosin powder is percutaneously injected into an degenerated disc.

Inventors

  • DU YUXUAN
  • ZHAN JIAWEN
  • SI JIANGTAO
  • YANG HONGYU
  • LU JIAJIN
  • LIU LI
  • ZHAO KE

Assignees

  • 中国中医科学院望京医院(中国中医科学院骨伤科研究所)

Dates

Publication Date
20260512
Application Date
20260323

Claims (10)

  1. 1. A chymosin composite preparation for disc degeneration is injected under ultrasound, which is characterized by being prepared from the following materials: Freeze-dried recombinant chymosin powder, biodegradable nano-microspheres loaded with chymosin and matrix-promoting synthetic factors, and microenvironment responsive hydrogel precursor solution.
  2. 2. The ultrasonic injection chymosin complex preparation for disc degeneration according to claim 1, wherein the freeze-dried recombinant chymosin powder comprises chymosin, trehalose, mannitol and a nonionic surfactant, wherein the mass ratio of chymosin, trehalose, mannitol and nonionic surfactant is 1:10:5:1.
  3. 3. The method for preparing chymosin complex for disc degeneration by ultrasonic injection according to claim 2, wherein the matrix-promoting synthesis factor is selected from one or more of TGF-beta 3, BMP-7 or SOX9 activation peptide, the nanoparticle is composed of polylactic acid-glycolic acid copolymer (PLGA) with particle size of 200-400 nm, and cavitation microbubble precursor or sonoliposome is incorporated into PLGA matrix to enhance ultrasonic responsiveness.
  4. 4. The method of claim 3, wherein the microenvironment and ultrasound dual-response type chymosin precursor solution comprises methacrylic acid acylated hyaluronic acid, thioketal diacrylate, acetal modified gelatin, and an acoustically sensitive cross-linking agent capable of being triggered to break by ultrasonic shear force or cavitation effect, wherein the freeze-dried recombinant chymosin powder is jointly dispersed in the hydrogel precursor solution with the nano-microspheres after reconstitution before use to form an injectable composite precursor solution, wherein the precursor solution can be subjected to in-situ cross-linking to form a resident gel in the intervertebral disc, and the initial burst release of chymosin is accelerated under the irradiation of external low-intensity focused ultrasound (LIFU), and the matrix-promoting factor is slowly released depending on the microenvironment response.
  5. 5. A method of preparing a chymosin complex formulation for disc degeneration by ultrasonic injection, the chymosin complex formulation for disc degeneration according to any one of claims 1-4, characterized in that: comprising the following steps: chymosin, trehalose, mannitol and a nonionic surfactant are dissolved in sterile water according to the mass ratio of 1:10:5:1 to form a uniform mixed solution; Freeze-drying the obtained mixed solution to obtain freeze-dried recombinant chymosin powder; The chymosin and matrix-promoting synthetic factors are jointly contained in polylactic acid-glycolic acid copolymer (PLGA) containing sound-sensitive components, and ultrasonic response type biodegradable nano-microspheres with the particle size of 200-400 nm are prepared through a double-emulsion-solvent volatilization method and ultrasonic auxiliary emulsification; Dissolving methacryloyl hyaluronic acid, thioketal diacrylate, acetal modified gelatin and a sound-sensitive cross-linking agent in phosphate buffer solution with pH of 7.4 to prepare micro-environment and ultrasonic dual-response hydrogel precursor solution; and (3) re-dissolving the freeze-dried recombinant chymosin powder, mixing with the nano-microspheres, and uniformly dispersing in the hydrogel precursor solution under the assistance of low-frequency ultrasound (20-50 kHz and 0.5-2W/cm 2 ) to form the injectable composite precursor solution.
  6. 6. The method for preparing a chymosin complex formulation for disc degeneration by ultrasonic injection according to claim 5, wherein the freeze-drying process comprises: mixing the solution at a temperature of-40 Pre-freezing at-25deg.C for 2 hr, and vacuum maintaining at-25 at a vacuum level of not more than 0.1mbar Drying at C for 24 hr at 25 deg.C And (3) performing secondary drying for 6 hours under the condition that the degree of vacuum is not more than 0.05mbar at the C and the degree of vacuum to obtain freeze-dried recombinant chymosin powder with loose porous structure.
  7. 7. The method for preparing a chymosin complex formulation for disc degeneration by ultrasonic injection according to claim 6, wherein the step of ultrasonic assisted emulsification in the double emulsification-solvent evaporation method comprises: In the process of forming the W/O colostrum and the W/O/W compound emulsion, pulse ultrasonic (frequency 20 kHz, power 100-300W, pulse time 5 s/interval 5 s) is respectively applied to improve the uniformity of emulsion drops and reduce the particle size distribution; After the volatilizing stage of the organic solvent is finished, centrifugally collecting the precipitate, washing and freeze-drying to obtain the ultrasonic response type nanometer microsphere with high encapsulation efficiency.
  8. 8. The method for preparing a chymosin complex preparation for disc degeneration by ultrasonic injection according to claim 7, wherein the matrix-promoting synthesis factor is any one of TGF-beta 3, BMP-7 or SOX9 activation peptide, the encapsulation rate of the nano-microsphere to chymosin is 65% to 75%, the encapsulation rate of the matrix-promoting synthesis factor is 55% to 65%, and 30% -50% of chymosin can be released within 5 minutes under the ultrasonic irradiation of intensities of 1 MHz, 0.5-1.5W/cm 2 .
  9. 9. The method for preparing a chymosin composite preparation for disc degeneration by ultrasonic injection according to claim 8, wherein the mass concentration of the methacryloyl hyaluronic acid in the micro-environment and ultrasonic double-response hydrogel precursor solution is 3%, the thioketal diacrylate is 0.5%, the acetal modified gelatin is 2%, the acoustic-sensitive crosslinking agent is an acoustic-cleavable crosslinking molecule containing a peroxy bond or an ester bond, the concentration is 0.1% -0.3%, and the pH value of the phosphate buffer solution is 7.4.
  10. 10. The method for preparing chymosin complex formulation for disc degeneration by ultrasonic injection according to claim 9, wherein the injectable complex precursor solution is triggered in situ crosslinking and drug release after percutaneous puncture injection into nucleus pulposus region of an intervertebral disc by any one of the following means: 365 Irradiating the ultraviolet light for 5 seconds; self-crosslinking of local high active oxygen and low pH microenvironment depending on intervertebral disc degeneration; External low-intensity focused ultrasound is applied, and ultrasonic cavitation and thermal effect are utilized to synchronously promote gel network reconstruction and rapid initial release of chymosin, so that time-space controllable therapeutic response is realized.

Description

Chymosin compound preparation for intervertebral disc degeneration by ultrasonic injection and preparation method thereof Technical Field The invention relates to the technical field of chymosin composite preparation, in particular to chymosin composite preparation for intervertebral disc degeneration by ultrasonic injection and a preparation method thereof. Background The chymosin composite preparation technology generally refers to a preparation technology that chymosin is combined with one or more auxiliary materials, carriers or active ingredients through physical mixing, embedding, crosslinking or freeze-drying and other means to improve the stability, control the release behavior or expand the application function of chymosin, and common methods comprise loading enzymes into liposomes, microspheres, nanoparticles or hydrogels or co-freeze-drying with protective agents such as saccharides, polyalcohols and the like to improve the storage stability, but the traditional technology is mainly used in the fields of food, leather or laboratory modeling, and no controllable, synergistic and responsive composite preparation system for intervertebral disc degeneration treatment has been formed yet. In the prior model research, chymosin (papain) is only used for non-selectively degrading proteoglycan through high-dose injection to induce irreversible intervertebral disc injury, the action mechanism is destructive, uncontrollable and repair-free, the chymosin cannot be used for clinical treatment, excessive matrix degradation can be caused by singly using chymosin in the prior art to accelerate intervertebral disc collapse, compact crosslinking matrixes and inflammatory barriers exist in the degeneration microenvironment of growth factors such as TGF-beta 3 and the like singly using TGF-beta 3, the target cells are difficult to effectively contact, and the bioavailability is extremely low. Disclosure of Invention The present invention has been made in view of the above-described problems occurring in the prior art. Therefore, the invention provides a chymosin composite preparation for disc degeneration by ultrasonic injection, which solves the problems that in the existing model research, chymosin (papain) is only used for non-selectively degrading proteoglycan by high-dose injection, irreversible disc injury is induced, the action mechanism is destructive, uncontrollable and repair-free, the chymosin cannot be used for clinical treatment, excessive matrix degradation can be caused by single use of chymosin in the prior art, disc collapse is accelerated, compact crosslinked matrixes and inflammation barriers exist in the degeneration microenvironment due to single use of growth factors such as TGF-beta 3, and the like, target cells are difficult to effectively contact, and the bioavailability is extremely low. In order to solve the technical problems, the invention provides the following technical scheme: In a first aspect, the present invention provides a chymosin complex formulation for use in disc degeneration by ultrasound injection, made of: Freeze-dried recombinant chymosin powder, biodegradable nano-microspheres loaded with chymosin and matrix-promoting synthetic factors, and microenvironment responsive hydrogel precursor solution. As a preferred scheme of the ultrasonic injection of the chymosin composite preparation for disc degeneration, the freeze-dried recombinant chymosin powder comprises chymosin, trehalose, mannitol and a nonionic surfactant, wherein the mass ratio of chymosin to trehalose to mannitol to the nonionic surfactant is 1:10:5:1. As a preferred scheme of the chymosin complex preparation for disc degeneration under ultrasonic injection, the matrix-promoting synthesis factor is selected from one or more of TGF-beta 3, BMP-7 or SOX9 activation peptide, the nano microsphere is composed of polylactic acid-glycolic acid copolymer (PLGA) with the particle size of 200-400 nm, and cavitation microbubble precursors or sound-sensitive liposomes are doped in the PLGA matrix to enhance ultrasonic responsiveness. As a preferred scheme of the ultrasonic injection chymosin composite preparation for disc degeneration, the micro-environment and ultrasonic double-response type hydrogel precursor solution comprises methacrylic acylated hyaluronic acid, thioketal diacrylate, acetal modified gelatin and an acoustic-sensitive cross-linking agent which can be triggered to break by ultrasonic shearing force or cavitation effect, the freeze-dried recombinant chymosin powder and the nano-microspheres are jointly dispersed in the hydrogel precursor solution after being reconstituted before use to form an injectable composite precursor solution, and the precursor solution can be subjected to in-situ cross-linking to form resident gel in the disc, and can accelerate initial burst release of chymosin under the irradiation of external low-intensity focused ultrasound (LIFU), and then the matrix-promoting factor is slow