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WO2022095151A1 - Hydrogel injectable pour le traitement d'une lésion du système nerveux central et son procédé de préparation - Google Patents

Hydrogel injectable pour le traitement d'une lésion du système nerveux central et son procédé de préparation Download PDF

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WO2022095151A1
WO2022095151A1 PCT/CN2020/131330 CN2020131330W WO2022095151A1 WO 2022095151 A1 WO2022095151 A1 WO 2022095151A1 CN 2020131330 W CN2020131330 W CN 2020131330W WO 2022095151 A1 WO2022095151 A1 WO 2022095151A1
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growth factor
polyethylene glycol
hydrogel
arm polyethylene
central nervous
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Chinese (zh)
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王绪化
陈作兵
叶婧佳
靳爽
蔡万雄
张天芳
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Zhejiang University ZJU
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/16—Macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/222—Gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A61L2300/414—Growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/426—Immunomodulating agents, i.e. cytokines, interleukins, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/602—Type of release, e.g. controlled, sustained, slow
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction

Definitions

  • the invention relates to the technical field of soft tissue repair engineering, in particular to the preparation and application of an injectable hydrogel for treating spinal cord injury.
  • SCI Spinal cord injury
  • Severe neuroinflammation induced by SCI induces the apoptosis of neurons at the site of injury to produce toxic cellular remnants, which in turn destroys the non-physically damaged spinal cord tissue in the vicinity of the lesion.
  • These pathological changes ultimately disrupt the neural circuits connecting the brain and spinal cord. , resulting in permanent sensory and motor dysfunction, and even fatal complications, which greatly affects the patient's life and quality of life (Young et al., 1993, J Emerg Med).
  • the neuroinflammatory signal caused by CNS trauma is a dynamic process with the progression of the disease.
  • CNS trauma In the acute SCI stage (1-3 days), neuroinflammatory signals mediate the removal of necrotic cell debris and help the repair of spinal cord tissue.
  • the neuroinflammatory response peaks at 3-7 days, causing neuronal and glial cell apoptosis, which in turn leads to The post-acute phase (1-2 weeks) inhibits the regenerated cystic cavity and astroglial scarring (David et al, 2011, Nat Rev Neurol; Simon et al., 2017, Nat Rev Neurol).
  • the present invention provides an injectable hydrogel for treating central nervous system injury and a preparation method thereof.
  • the hydrogel can precisely release immunomodulatory drugs at the correct time node to suppress excessive inflammatory response, protect residual nerve tissue or axons, and inhibit the formation of cystic cavity and scar tissue. And slow release of cell growth factor for a long time, promote nerve regeneration, so as to achieve a scar-free tissue healing.
  • Hydrogel injections can restore motor function to a higher level in animals after spinal cord injury, and cortical electrical stimulation and EMG recording experiments demonstrate the re-establishment of brain-to-muscle neural circuits.
  • the present invention develops an injectable hydrogel system with precise sustained release of drugs and growth factors, which is expected to be used for clinical treatment of central nervous system injury.
  • an injectable hydrogel for the treatment of central nervous system injury a multi-arm polyethylene glycol-X modified with arginine-glycine-aspartic acid and Multi-arm polyethylene glycol-Y obtains polymers for in situ formation of hydrogel scaffolds in lesions through click chemistry, the polymer is also loaded with nano/micro particles and cell growth factors, and the nano/micro particles are loaded There are immunomodulatory drugs and/or antioxidant drugs;
  • the functional group pair X and Y in the multi-arm polyethylene glycol-X and the multi-arm polyethylene glycol-Y that have a click chemical reaction are selected from: mercapto group and maleamide group, mercapto group and alkene, azide and alkyne ring, Conjugated diene and substituted alkene, aldehyde and hydrazide, tetrazine and norbornene, tetrazine and isonitrile;
  • Described multi-arm polyethylene glycol-X is 4-arm polyethylene glycol-X or 3-arm polyethylene glycol-X;
  • Described multi-arm polyethylene glycol-Y is 4-arm polyethylene glycol- Y or 3-arm polyethylene glycol-Y;
  • the polymerization degree n of polyethylene glycol in the multi-arm polyethylene glycol-X and multi-arm polyethylene glycol-Y is 1-1000.
  • the nano/micro particles are selected from a mixture of one or more of polymer micro/nano particles and/or liposomes modified by reactive groups.
  • the nano/micro particles are mixed with one or more of polyethylene, gelatin, collagen polylactic acid or liposome particles.
  • the immunomodulatory/antioxidant drugs are selected from methylprednisolone sodium succinate (MPSS), promethazine, dexamethasone, hydrocortisone, ibuprofen, hydroxybutazone , cyclosporine A, tacrolimus, azathioprine, 6-mercaptopurine, cyclophosphamide, tacrolimus (KF506), rapamycin, mycophenolate mofetil, tea polyphenols (TP), fertility A mixture of one or more of phenol, butylated hydroxyanisole (BHA), dibutylhydroxytoluene (BHT) and/or tert-butylhydroquinone (TBHQ).
  • MPSS methylprednisolone sodium succinate
  • promethazine dexamethasone
  • hydrocortisone hydrocortisone
  • ibuprofen hydroxybutazone
  • cyclosporine A tacrolimus
  • azathioprine 6-mer
  • the cell growth factor is selected from: basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), acidic fibroblast growth factor (aFGF), liver Cell Growth Factor (HGF), Ciliary Neurotrophic Factor (CNTF), Glial Cell-Derived Neurotrophic Factor (GDNF), Neurotrophic Factor-3 (NT-3), Epidermal Growth Factor (EGF), Interleukin-3 (IL- 3), transforming growth factor- ⁇ (TGF- ⁇ ), platelet-derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1), bone morphogenetic protein (BMP), connective tissue growth factor (CTGF) A mixture of one or more of , osteopontin (OPN) and/or growth hormone releasing factor (GRF).
  • bFGF basic fibroblast growth factor
  • BDNF brain-derived neurotrophic factor
  • VEGF vascular endothelial growth factor
  • aFGF acidic fibro
  • the present invention also provides a preferred preparation method of the above-mentioned injectable hydrogel for the treatment of central nervous system injury, comprising the following steps:
  • Step 1 Synthesis of Polymer Nanoparticles (NP): In a reaction vessel, add DMAEMA, ST, ACLT-PEG-NHS, NaSS, MBA to water, then add redox initiators KPS and SPS, at a temperature of 50 ⁇ The polymerization reaction is carried out at 10°C under nitrogen protection and stirring for more than 3 hours, and the unreacted monomers are removed through a dialysis membrane to obtain nanoparticle NPs;
  • Step 2 Modify maleimide on the surface of nanoparticle NP: Continue to add MAL-NH2 and ACLT-PEG-NHS with a molar ratio of 1:1 to 1.1, and carry out the maleimide modification reaction under stirring for at least For 1 h, continue to remove unreacted monomers through a dialysis membrane and freeze-dry to obtain freeze-dried maleimide-modified nanoparticles NP-MAL;
  • Step 3 NP-MAL particles loaded with MPSS: In the PBS solution of MPSS, the nanoparticles NP-MAL obtained above were added and allowed to stand overnight, and then collected by centrifugation to obtain MPSS-loaded nanoparticles, and lyophilized to obtain the loaded nanoparticles Nanoparticle MM-NPs of MPSS;
  • Step 4 maleimide modification of growth factor GFs: the growth factor GFs is mixed and reacted with sulfo-SMCC in a molar ratio of 1:30-70 to obtain a maleimide-modified growth factor GF-MAL;
  • Step 5 Mix 4a-PEG-MAL and RGD-PEG-SH at a molar ratio of 1:1 to obtain RGD-modified 4a-PEG-MAL, and then add the MM-NPs nanoparticles obtained in step 3 and the horses obtained in step 4.
  • the imide-modified growth factor GF-MAL was mixed uniformly to form solution A;
  • Step 6 Preparation of solution B: Dissolve 4a-PEG-SH in the buffer solution, and mix evenly to form solution B;
  • Step 7 Mix and react the solutions A and B in step 5 and step 6 at a ratio of 1:1 to obtain Injectable hydrogel MPG-HD conjugated with drug-loaded nanoparticles and growth factors.
  • the concentration of each reactant in the polymerization reaction in step 1 is 5% (w/v) DMAEMA, 10% (w/v) ST, 3% (w/v) ACLT-PEG-NHS, 0.4% (w/v) v) NaSS, 0.4% (w/v) MBA, 0.2% (w/v) KPS and 0.1% (w/v) SPS;
  • steps 2, 4, 5, 6, and 7 provide a pair of click-chemically coupled functional groups thiol and maleamide, selected from: thiol and maleamide, thiol and alkene, azide and alkyne ring, Conjugated dienes and substituted alkenes, aldehydes and hydrazides;
  • the MPSS as an immunomodulatory or antioxidant drug in step 3 can be selected from promethazine, dexamethasone, hydrocortisone, ibuprofen, hydroxybutazone, cyclosporine A, taupomycin, sulfur Azathioprine, 6-mercaptopurine, cyclophosphamide, tacrolimus (KF506), rapamycin, mycophenolate mofetil, tea polyphenols (TP), tocopherol, butylated hydroxyanisole (BHA), bismuth A mixture of one or more of butylated hydroxytoluene (BHT) and/or tert-butylhydroquinone (TBHQ) instead.
  • promethazine promethazine
  • dexamethasone hydrocortisone
  • ibuprofen hydroxybutazone
  • cyclosporine A taupomycin
  • sulfur Azathioprine 6-mercaptopurine
  • cyclophosphamide tacroli
  • the present invention develops an injectable hydrogel MPG-HD for treating central nervous system injury with an injectable cavity filling and ECM forming scaffold, and has the following effects: 1) Rapid release of MPSS in acute phase 2) Long-term sustained release of GFs to promote axonal regeneration; 3) Effectively reduced the formation of cystic cavities and scar tissue, and promoted nerve regeneration; 4) Cortical stimulation and EMG recordings showed that neurons from Brain-to-muscle neural circuit connections.
  • Figure 1a is a synthetic route diagram of the maleimide-modified MPSS-loaded nanoparticles MM-NPs according to the present invention.
  • Fig. 1b is a microscopic topography diagram of the maleimide-modified nanoparticle NP prepared in the embodiment of the present invention.
  • Figure 1c is a microscopic topography diagram of the maleimide-modified nanoparticle NP-MAL prepared in the embodiment of the present invention.
  • Figure 1d is a graph of the potential change of the maleimide-modified MPSS-loaded nanoparticles MM-NPs prepared in the embodiment of the present invention.
  • Figure 1e is a particle size distribution diagram of the MM-NPs prepared in the embodiment of the present invention.
  • Figure 1f is a quantitative diagram of maleimide on the surface of MM-NPs prepared in the embodiment of the present invention.
  • Figure 1g is a quantitative diagram of the reduction of sulfhydryl groups after the MM-NPs prepared in the embodiment of the present invention reacted with 4a-PEG-SH.
  • Figure 2a is a schematic diagram of the synthesis and preparation mechanism of the injectable hydrogel MPG-HD of the present invention.
  • Figure 2b is a graph of the elastic modulus of the injectable hydrogels with different concentrations according to the present invention.
  • Figure 2c is a graph of the degradation rate of the injectable hydrogel of the present invention.
  • Figure 2d is a graph of the swelling rate of the injectable hydrogel of the present invention.
  • Figure 2e is a graph of the slow release rate of MPSS in the injectable hydrogel of the present invention.
  • Figure 2f is a graph showing the slow release rate of growth factors of the injectable hydrogel of the present invention.
  • Figure 2g is a graph showing the viable cell index of the injectable hydrogel extract of the present invention used for cell culture.
  • Fig. 3a is a schematic diagram of the test flow of injecting PBS, G-HD and MPG-HD into the injured site of the rat according to the embodiment of the present invention.
  • Figure 3b is a schematic diagram of the experiment of injecting PBS, G-HD and MPG-HD into the injured part of the rat according to the embodiment of the present invention
  • Figure 3c is the HE staining diagram of the injured site after injection of PBS, G-HD and MPG-HD into the injured site of the rat according to the embodiment of the present invention.
  • Figure 3d is a graph of the shape change of the spinal cord after injection of PBS, G-HD and MPG-HD into the injured site of the rat according to the embodiment of the present invention.
  • Figure 3e is a three-dimensional reconstruction image of the spinal cord after injection of PBS, G-HD and MPG-HD into the injured site of the rat according to the embodiment of the present invention.
  • Figure 3f is the volume diagram of each tissue and cavity after injection of PBS, G-HD and MPG-HD at the injury site of the rat according to the embodiment of the present invention.
  • Figure 3g is a graph of behavioral scores after injection of PBS, G-HD and MPG-HD into the injured site of the rat according to the embodiment of the present invention.
  • Figure 4a is an immunostaining diagram of the regeneration of 5-HT axons at the injury site and before and after the present invention.
  • Fig. 4b is an immunostaining diagram of the regeneration of the body's axon at the injury site and before and after the injury according to the present invention.
  • Fig. 4c is an immunostaining diagram of the regeneration of nerve fibers before and after the injury site according to the present invention.
  • Figure 4d is a statistical diagram of the number of 5-HT axons before and after injury according to the present invention.
  • Fig. 4e is a statistical graph of the number of proprioceptive nerve axons before and after the injury according to the present invention.
  • Figure 4f is a statistical graph of the number of nerve fibers before and after injury according to the present invention.
  • Figure 5a is a graph of evoked potentials in the cerebral cortex after injection of PBS, G-HD and MPG-HD into the injured site of the rat in accordance with the present invention.
  • Fig. 5b is a graph showing the hindlimb movement and myoelectric discharge after the rats were injected with PBS, G-HD and MPG-HD at the injury site according to the present invention.
  • Synthesis of nanoparticle NPs cores of nanoparticles were synthesized using DMAEMA, ST, ACLT-PEG-NHS, NaSS, and MBA): DMAEMA (w/v 5%), ST (w/v 10%), ACLT-PEG-NHS ( w/v 3%), NaSS (w/v 0.4%), MBA (w/v 0.4%) and water were added into a four-necked flask equipped with a nitrogen outlet, an inlet and a feeding port; then, the Redox initiators KPS (w/v 0.2%) and SPS (w/v 0.1%) were dissolved in water, and a slow nitrogen stream was added to the reaction mixture solution and the initiator solution for 1 hour, respectively, followed by the initiator solution.
  • reaction mixture was added to the solution and polymerized at 160 rpm and 50 ⁇ 5°C. After 5 h of reaction, the reaction mixture was dialyzed against deionized water for 48 h using a dialysis membrane (MWCO 14 kDa) to remove unreacted monomers.
  • MWCO 14 kDa dialysis membrane
  • Maleimide modification add 1 mg/ml MAL-NH2 to the reaction mixture, ACLT-PEG-NHS with a molar ratio of 1:1, and stir at room temperature for 4 h. Then, the reaction mixture was dialyzed against deionized water using a dialysis membrane (14KDa) and lyophilized to obtain lyophilized nanoparticles NP-MAL.
  • Figures 1b and 1c are the microscopic morphologies of the nanoparticles before and after maleimide modification. It can be seen from the figure that particles with clear boundaries were observed before maleimide modification ( Figure 1b). Particles with a hydrophilic layer (Fig. 1c), the nanoparticles have a particle size of 110 nm, which is close to the results of dynamic light scattering studies (Fig. 1e).
  • the modification of the maleimide of the present invention on the nanoparticle surface (Fig. 1f) and the reaction of the particle with the thiol group in 4a-PEG-SH (Fig. 1g) were also confirmed by Elleman test.
  • the concentration of maleimide groups on the surface of nanoparticles was calculated to be 0.85 mmol/g. After adding nanoparticles, the content of sulfhydryl groups in 4a-PEG-SH decreased by 0.12 mmol.
  • Maleimide modification of growth factor GFs the growth factors used bFGF, BDNF and VEGF at a concentration of 50ng/ml, and the growth factor GFs and sulfo-SMCC were reacted at a molar ratio of 1:50 for 5min, and then 3T3, The activities of maleimide-modified bFGF, BDNF and VEGF were detected in C6 and HUVEC cells. The cells were seeded on 96-well growth factor-free medium, and after 24 h incubation, the cells were washed with PBS.
  • modified or unmodified growth factor-containing medium was added to each well as indicated, control samples were added to growth factor-free medium, and the plates were incubated at 37°C in a 5% CO2-enhanced medium. The cells were incubated in humid air for 24 hours, and cell viability was measured using Cell Proliferation Kit I (MTT), and absorbance was measured with a microplate reader at a wavelength of 550 nm.
  • MTT Cell Proliferation Kit I
  • MPG-HD was prepared as follows: RGD-PEG-SH was added to 4a-PEG-MAL (5%, wt%) at a molar ratio of 1:1 and then mixed with MM-NPs (20%, wt%) , maleimide-modified VEGF (10 ng/ ⁇ l), BDNF (50 ng/ ⁇ l), bFGF (10 ng/ ⁇ l) were added to form solution A. 4a-PEG-SH was dissolved in buffer solution to form solution B (5%, wt%). The solutions A and B were mixed at a ratio of 1:1 and polymerized to prepare MPG-HD (Fig. 2a).
  • the mercaptomaleimide Michael addition reaction can form hydrogels at high concentrations (greater than 2.5%) with a gelation rate of less than 3 seconds, and no gelation is observed at dilute conditions (1%).
  • gels at concentrations ranging from 2.5% to 10% were measured, and the 5% gel showed a similar modulus to spinal cord tissue (Fig. 2b).
  • the preparation of the injectable hydrogel MPG-HD in this example is obtained by coupling the 4-arm polyethylene glycol with reactive groups to thiol (-SH) and maleamido (-MAL) through a click chemical reaction, Therefore, it should be pointed out that all pairs of reactive groups capable of coupling with a click chemical reaction can be replaced, which also belongs to the protection scope of the present invention.
  • thiol thiol
  • -MAL maleamido
  • the preparation process of the injectable hydrogel MPG-HD of the present invention couples X-Y through the functional groups in multi-arm polyethylene glycol-X and multi-arm polyethylene glycol-Y that undergo a click chemical reaction, while arginine- Multi-arm polyethylene glycol-X and/or multi-arm polyethylene glycol-Y were modified with glycine-aspartic acid (RGD) for the purpose of improving the viscosity and bio-affinity of MPG-HD.
  • RGD glycine-aspartic acid
  • the immunomodulatory or antioxidant drug loaded by the injectable hydrogel MPG-HD prepared in this example adopts MPSS, while promethazine, dexamethasone, hydrocortisone, ibuprofen, hydroxybutyrate are exemplified.
  • pine pine, cyclosporine A, taupomycin, azathioprine, 6-mercaptopurine, cyclophosphamide, tacrolimus (KF506), rapamycin, mycophenolate, tea polyphenols (TP), Mixtures of one or more of tocopherol, butylated hydroxyanisole (BHA), dibutylated hydroxytoluene (BHT) and/or tert-butylhydroquinone (TBHQ) are also acceptable as immunomodulatory or antioxidant drugs Replacement belongs to the protection scope of the present invention.
  • BHA butylated hydroxyanisole
  • BHT dibutylated hydroxytoluene
  • TBHQ tert-butylhydroquinone
  • the cell growth factor GFs loaded by the injectable hydrogel MPG-HD prepared in this example can be selected from: basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), acidic fibroblast growth factor (aFGF), hepatocyte growth factor (HGF), ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), neurotrophic factor-3 (NT-3) , epidermal growth factor (EGF), interleukin-3 (IL-3), transforming growth factor- ⁇ (TGF- ⁇ ), platelet-derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1), bone morphology A mixture of one or more of gene gene (BMP), connective tissue growth factor (CTGF), osteopontin (OPN) and/or growth hormone releasing factor (GRF).
  • BMP gene gene gene
  • CTGF connective tissue growth factor
  • OPN osteopontin
  • GRF growth hormone releasing factor
  • the elastic modulus of the hydrogel was calculated from the linear part of the stress-strain curve. Briefly, a hydrogel with a thickness of 1 cm (w/v 2.5%, 5%, 10%) was prepared by mixing the A solution and the B solution in a ratio of 1:1. Stress-strain curves were measured using a universal material testing machine (Roell Z020, Wick, Germany) under a 50 N static load cell with a strain rate of 0.5 mm/min. Additionally, the degradation and swelling of the hydrogel was measured by incubating 2 ml of hydrogel (5%) in 5 ml of PBS at 37°C for 60 days to assess hydrolytic degradation.
  • the masses of wet and lyophilized gels were measured, respectively. Before lyophilization, the hydrogels were washed in distilled water to remove residual salts that may have accumulated on the surface. The total dry polymer mass loss for each sample was determined by comparison to the dry weight of the 0-day samples. The swelling ratio was calculated as (Ms-Md)/Md.
  • the hydrogels degraded linearly within 7 days, decreased significantly after 2 weeks, and reached 80% degradation at 2 months (Fig. 2c), indicating that the hydrogels can support cell migration in the chronic phase at 2 months .
  • the hydrogel prepared by the present invention has the properties suitable for spinal cord injury injection.
  • Cytotoxicity evaluation The hydrogel and nanoparticle composite soaking medium was used. Cell viability was measured by MTT colorimetry, and the results showed that the cells cultured in the hydrogel MPG-HD soaked suspension showed similar cell viability to the non-gel medium ( Figure 2g), indicating that the material we used did not or very much. Low cytotoxicity and does not cause cell death in vivo.
  • MPG-HD reduces cavitation after contusive spinal cord injury
  • mice Female sprague-dawley rats (200-250g, class II, certificate number: SCXK2008-0033, Experimental Animal Center, Zhejiang Academy of Medical Sciences, Hangzhou) were injected with PBS, G-HD or MPG- HD (Fig. 3a,b). Eight weeks after injury, the size of the spinal cord tissue cavity after injection was observed.
  • the cavity volume was reduced after G-HD and MPG-HD injection, and MPG-HD injection reduced the cavity volume to 0.8% of the total volume, about 1/40 of the PBS cavity and 1/20 of the G-HD injection.
  • the volume of pathological tissue defined as the volume without normal tissue structures
  • was significantly increased in the spinal cord as was the residual intact tissue (including white and gray matter) in these spinal cords (Fig. 3f).
  • Injectable hydrogel MPG-HD inhibits cystic cavity and scar tissue formation and promotes axonal regeneration
  • Fig. 4a–c spinal cord and cranial and caudal tissues from the injury site.
  • Three kinds of nerve fibers were taken and injected into the upper thoracic spinal cord with AAV2/9-mCherry to trace the descending axons (supraspinal or long body spinal cord). Immunostaining was used to observe 5-HT axons and neurofilament positive axons (NF axons).
  • serotonergic (5-HT) axons that play an important role in motor recovery grow and extend into the fibrotic matrix (Fig. 4a, MPG-HD lines, a', a").
  • the fibrotic matrix MCherry-labeled red intrinsic spinal axons were also observed in the central region (Fig. 4b, MPG-HD, b', b").
  • MPG-HD treatment axonal fibers of abundant NF+ re-grown markedly into the fibrotic matrix, traversing the injury site and extending into the spinal cord segment below the injury, which was not observed in PBS-injected animals (Fig. 4c, c', c").
  • G-HD-treated spinal cords showed smaller cavity formation and some axonal regeneration compared to the expected PBS-injected animals.
  • Coronal image of the spinal cord and axons below the injury site Quantitative analysis of densities showed that MPG-HD-treated animals had significantly more 5-HT, bulk spinal cord, and NF + axons at the lesion than G-HD or PBS-injected animals, possibly not only due to improved axonal regeneration , also associated with having more axon residues.
  • MPG-HD treatment promotes spinal cord neural circuit reconstruction
  • Electrophysiological recordings to assess the connectivity of neural circuits from central to peripheral neurons The results showed that the electrical signals of the TA muscles were recorded simultaneously with cortical electrical stimulation, confirming that the spinal cord treated with MGP-HD could more effectively transmit descending nerve signals to the lumbar spinal cord.
  • EMG signals with greater delay and weaker amplitude were recorded only in MGP-HD-injected animals, indicating that the neural circuits reconstructed by MGP-HD treatment have multiple synaptic connections and can
  • the electrical signals generated by partial cortical electrical stimulation were transmitted to motor neurons in the lumbar spinal cord of injured animals (Fig. 5a).
  • the PBS-injected rats were unable to support their body weight and had no joint movement in the hind limbs.
  • the injectable hydrogel MPG-HD prepared by the present invention enables in situ gelation of spinal cord injury, ensuring that it can adapt to the shape of the cavity, thereby minimizing the gap between the spinal cord tissue and the gel .
  • Injected hydrogel at the injury site provides a scaffold for fibroblast migration and invasion, forming a fibroblast-rich ECM that reduces pore generation.
  • the injectable hydrogel MPG-HD of the present invention has a suitable swelling ratio, which reduces the risk of secondary damage to the residual tissue at the damaged site.
  • the drug-loaded nanoparticles/GFs modified by the reactive group of the present invention are coupled with the reactive group of the hydrogel through click chemistry, so that the drug or growth factor can be released at the injured site in different periods of time to adapt to the pathological changes after spinal cord injury.
  • the injectable hydrogel MPG-HD prepared by the present invention helps to reduce the formation of scar tissue and promote the regeneration of spinal cord nerves.
  • the injectable hydrogel MPG-HD prepared by the present invention is helpful for the reconstruction of spinal nerve circuits and promotes functional recovery.

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Abstract

La présente invention concerne un hydrogel injectable in situ pour le traitement d'une lésion du système nerveux central et son procédé de préparation. L'hydrogel est formé par exécution d'un couplage de chimie click sur du polyéthylène glycol-X multi-bras et du polyéthylène glycol-Y multi-bras modifiés à l'arginine-glycine-acide aspartique à l'aide de groupes fonctionnels, et l'hydrogel est revêtu de nano/micro particules et de facteurs de croissance de médicaments immunomodulateurs et/ou antioxydants à libération prolongée chargés dessus au moyen d'une réaction de couplage chimique. L'hydrogel injectable fournit une libération locale précise et prolongée de médicaments immunomodulateurs et/ou antioxydants et de facteurs de croissance à l'emplacement d'une lésion, de sorte qu'il est possible d'empêcher la formation d'une cavité cystique après une lésion de la moelle épinière, une lésion secondaire causée par la neuro-inflammation est atténuée, les tissus des nerfs de la moelle épinière et les axones résiduels sont protégés, et la formation des tissus cicatriciels gliaux est réduite. L'hydrogel fournit un environnement de matrice extracellulaire perméable pour la régénérescence des axones de nerf corporel, favorisant ainsi la récupération de fonctions électrophysiologiques et motrices, et peut être utilisé pour la réparation de lésions aux tissus mous tels que la moelle épinière.
PCT/CN2020/131330 2020-11-06 2020-11-25 Hydrogel injectable pour le traitement d'une lésion du système nerveux central et son procédé de préparation Ceased WO2022095151A1 (fr)

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