WO2022095151A1 - 一种治疗中枢神经损伤的可注射水凝胶及其制备方法 - Google Patents

一种治疗中枢神经损伤的可注射水凝胶及其制备方法 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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王绪化
陈作兵
叶婧佳
靳爽
蔡万雄
张天芳
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Zhejiang University
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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    • 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
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    • 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
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    • A—HUMAN NECESSITIES
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    • 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
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    • 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

一种治疗中枢神经损伤的可原位注射水凝胶及其制备方法，所述水凝胶以精氨酸-甘氨酸-天冬氨酸修饰过的多臂聚乙二醇-X与多臂聚乙二醇-Y通过官能团对X-Y点击化学偶联而形成，所述水凝胶内包覆有通过化学偶联反应负载在其上的缓释免疫调节和/或抗氧化药物的纳米/微米粒子与生长因子。注射水凝胶在损伤部位局部精确缓释免疫调节和/或抗氧化药物与生长因子，阻止了脊髓损伤后囊性空腔的形成，减轻了神经炎症导致的二次损伤，保护了残留的脊髓神经组织和轴突，减少了胶质瘢痕组织的形成，为本体神经轴突的再生提供了一种可穿透性的细胞外基质环境，进而促进电生理和运动功能的恢复，可用于脊髓等软组织损伤的修复。

Description

一种治疗中枢神经损伤的可注射水凝胶及其制备方法 技术领域
本发明涉及软组织修复工程技术领域，具体涉及一种治疗脊髓损伤的可注射水凝胶的制备及应用。
背景技术
脊髓损伤(SCI)是一种严重致残性疾病，往往导致损伤节段以下肢体严重的生理功能障碍。据统计分析每年世界范围内SCI的发生概率是40-50人每百万人，目前全世界有超过2700万患者遭受瘫痪等后遗症的折磨，其中中国约有400万患者，并以每年12万左右的速度增加。
SCI导致的严重神经炎症反应会诱导损伤部位神经元的凋亡产生有毒的细胞残体，进而破坏那些病灶附近未受物理损伤的脊髓组织，这些病理改变最终破坏了连接大脑和脊髓的神经环路，导致患者永久性的感觉和运动功能障碍，甚至出现致命的并发症，从而极大地影响患者的生命及其生活质量(Young et al.,1993,J Emerg Med)。最近的治疗SCI的临床试验发现，全身性的甲基强的松龙激素冲击疗法没有显示任何显著的好处，甚至有一些是有害的(Liu et al.,2019,Neurology；Fehlings et al.,2014,Neurosurgery；Hurlbert et al.,2000,J Neurosurg)。最近的研究发现，急性期的神经炎症信号也可能是有益的，因为它能促进坏死细胞碎片的清除，维持组织稳态，并限制进一步的组织/轴突损伤(Bellver-Lndete et al.,2019,Nat Comm；Anderson et al.,2016,Nature；Aguzzi et al.2013,Science；Donnelly et al.,2008,Exp Neurol)。实验证明中枢神经创伤导致的神经炎症信号随着疾病的进展是一个动态的过程。急性SCI期(1-3天)神经炎症信号介导坏死细胞碎片的清除，帮助脊髓组织的修复，在3-7天时神经炎症反应在达到高峰，引起神经元和胶质细胞凋亡，进而导致后急性期(1-2周)抑制神经再生的囊腔空腔及星形胶质瘢痕形成(David et al,2011,Nat Rev Neurol；Simon et al.,2017,Nat Rev Neurol)。因此，如何通过组织工程技术在精确的时间节点调节过度的神经炎性反应，既不影响坏死细胞碎片的清除，组织的修复，又保护残存的神经组织免受过度炎症反应的侵害及减少抑制神经再生的囊性空腔和胶质瘢痕的形成是一个世界性的难题(Donnelly et al.,2008,Exp Neurol；Simon et al.,2017,Nat Rev Neurol)。
最近，人们致力于开发人工脊髓样移植物，通过这种人工脊髓移植，损伤的脊髓神经可通过这种移植再生和介导功能恢复。例如，开发了以神经祖细胞为种子的三维打印仿生水凝胶脊髓支架，以支持SCI后的神经再生(Koffler et al.,2019/02,Nat.Med.)。目前，以干细胞为种子的方法在细胞资源可得性和安全性方面还存在很大争议(Sing et al.,2013/07,US 8,475,788 B2；Abbot et al.,2012/06,US 2012/0156230 A1；Wirth III et al.,2019/08,US 2019/0262405 A1)，硬性植入移植物的外科手术容易损伤残存的组织/轴突，加重功能缺陷(Langer et al,2015/04,US9,440,008 B2；Dai et al,2020/05,CN 1111109424 A；Dai et al,2019/11,CN 110404078 A；Cheng et al,2019/08,CN 110101918 A)。另一方面，为了在手术过程中尽量减少组织损伤，注射合成聚合物原位形成水凝胶被建议为SCI临床治疗的首选。例如，基于持续递送神经营养因子和重建必须的细胞外基质(ECM)的方法，提出了可注射材料促进SCI动物模型轴突的再生的策略(Song et al.,2020/10,US10,799,529 B2；Wang et al,2012/12,US8,333,962 B2；Criscione et al.,2016/02,US 2016/0045439 A1；Pritchard et al,2013/12,US 2013/0324500 A1)。尽管这些生物材料具有巨大的潜力，但在这些生物材料的临床治疗方面取得的成功十分有限，主要是没有综合考虑SCI组织愈合过程中神经炎症微环境的时空特性，精准调节SCI导致的免疫微环境失衡。
技术问题
针对现有技术存在的问题，本发明提供了一种治疗中枢神经损伤的可注射水凝胶及其制备方法。根据疾病的进展，该水凝胶能在正确的时间节点，在病灶部位精确缓释免疫调控药物抑制过度的炎症反应，保护残留神经组织或轴突，抑制囊性空腔和瘢痕组织的形成，并长时间地缓释细胞生长因子，促进神经的再生，从而达到一种无瘢痕的组织愈合。水凝胶注射可以让脊髓受损后的动物运动功能恢复达到一个较高的水平，而且皮层电刺激和肌电图记录实验证实脑到肌肉的神经回路的重新建立。本发明开发了一种精确缓释药物及生长因子的可注射水凝胶体系，有望用于中枢神经损伤的临床治疗。
技术解决方案
为解决上述技术问题，本发明采用了以下技术方案：一种治疗中枢神经损伤的可注射水凝胶，以精氨酸-甘氨酸-天冬氨酸修饰过的多臂聚乙二醇-X与多臂聚乙二醇-Y通过点击化学反应得到用于病灶原位形成水凝胶支架的聚合物，所述聚合物还负载有纳米/微米粒子和细胞生长因子，所述纳米/微米颗粒负载有免疫调控药物和/或抗氧化药物；
所述多臂聚乙二醇-X和多臂聚乙二醇-Y中发生点击化学反应的官能团对X与Y选自：巯基与马来酰胺基、巯基与烯、叠氮与炔环、共轭双烯与取代烯烃、醛与酰肼、四嗪与降冰片烯、四嗪与异腈；
所述多臂聚乙二醇-X为4-臂聚乙二醇-X或3-臂聚乙二醇-X；所述多臂聚乙二醇-Y为4-臂聚乙二醇-Y或3-臂聚乙二醇-Y；
所诉多臂聚乙二醇-X与多臂聚乙二醇-Y中聚乙二醇的聚合度n为1-1000。
作为优选方案，所述纳米/微米粒子选自反应基团修饰过的高分子微米/纳米粒子和/或脂质体中一种或多种的混合。
作为优选方案，所述纳米/微米粒子采用聚乙烯、明胶、胶原聚乳酸或脂质体粒子中的一种或多种的混合。
作为优选方案，所述免疫调控类/抗氧化类药物选自甲基强的松龙琥珀酸钠(MPSS)、异丙嗪、地塞米松、氢化可的松、布洛芬、羟基保泰松、环孢菌素A、藤霉素、硫唑嘌呤、6-巯基嘌呤、环磷酰胺、他克莫司(KF506)、雷帕霉素、霉酚酸脂、茶多酚(TP)、生育酚、丁基羟基茴香醚(BHA)、二丁基羟基甲苯(BHT)和/或叔丁基对苯二酚(TBHQ)中一种或多种的混合。
作为优选方案，所述细胞生长因子选自：碱性成纤维生长因子(bFGF)、脑源性神经营养因子(BDNF)、血管内皮生长因子(VEGF)、酸性成纤维生长因子(aFGF)、肝细胞生长因子(HGF)、睫状神经营养因子(CNTF)、胶质细胞衍生神经营养因子(GDNF)、神经营养因子-3(NT-3)、表皮生长因子(EGF)、白介素3(IL-3)、转化生长因子-α(TGF-α)、血小板衍生的生长因子(PDGF)、胰岛素样生长因子-1(IGF-1)、骨形态发生蛋白(BMP)、结缔组织生长因子(CTGF)、骨桥蛋白(OPN)和/或生长激素释放因子(GRF)中一种或多种的混合。
本发明还提供了一种优选的上述治疗中枢神经损伤的可注射水凝胶的制备方法，包括以下步骤：
步骤1：高分子纳米粒子(NP)的合成：在反应容器中，将DMAEMA、ST、ACLT-PEG-NHS、NaSS、MBA加入水中，然后加入氧化还原引发剂KPS和SPS，在温度为50±10℃、氮气保护和搅拌下聚合反应3h以上，通过透析膜去除未反应完的单体得到纳米粒子NP；
步骤2：在纳米粒子NP的表面修饰马来酰亚胺：继续加入摩尔比为1:1～1.1的MAL-NH2和ACLT-PEG-NHS，在搅拌下进行马来酰亚胺改性反应至少1h，继续通过透析膜去除未反应完单体并冻干，得到冻干的马来酰亚胺改性纳米粒子NP-MAL；
步骤3：NP-MAL粒子负载MPSS：在MPSS的PBS溶液中，加入上述得到的纳米粒子NP-MAL并静置过夜，然后经过离心收集处理得到负载MPSS的纳米粒子，并经过冻干处理得到负载MPSS的纳米粒子MM-NPs；
步骤4：生长因子GFs的马来酰亚胺改性：将生长因子GFs按照1:30～70的摩尔比与sulfo-SMCC混合反应，得到马来酰亚胺改性的生长因子GF-MAL；
步骤5：将4a-PEG-MAL和RGD-PEG-SH按1:1摩尔比混合，得到RGD修饰过的4a-PEG-MAL，然后加入步骤3得到MM-NPs纳米粒子与步骤4得到的马来酰亚胺改性的生长因子GF-MAL并混合均匀形成溶液A；
步骤6：溶液B的制备：将4a-PEG-SH溶于缓冲溶液，混合均匀形成溶液B；步骤7：将步骤5与步骤6中的溶液A和B按1:1比例混合反应，即得到偶联负载药物的纳米粒子与生长因子的可注射水凝胶MPG-HD。
作为优选方案，步骤1聚合反应中各反应物浓度为5％(w/v)DMAEMA、10％(w/v)ST、3％(w/v)ACLT-PEG-NHS、0.4％(w/v)NaSS、0.4％(w/v)MBA、0.2％(w/v)KPS和0.1％(w/v)SPS；
作为优选方案，步骤2，4，5，6，7提供一对点击化学偶联的官能团巯基与马来酰胺基，选自：巯基与马来酰胺基、巯基与烯、叠氮与炔环、共轭双烯与取代烯烃、醛与酰肼；
作为优选方案，步骤3中作为免疫调节或抗氧化药物的MPSS可由异丙嗪、地塞米松、氢化可的松、布洛芬、羟基保泰松、环孢菌素A、藤霉素、硫唑嘌呤、6-巯基嘌呤、环磷酰胺、他克莫司(KF506)、雷帕霉素、霉酚酸脂、茶多酚(TP)、生育酚、丁基羟基茴香醚(BHA)、二丁基羟基甲苯(BHT)和/或叔丁基对苯 二酚(TBHQ)中一种或多种的混合代替。
有益效果
有益效果：本发明开发了一种可注射的空腔填充和ECM形成支架的用于治疗中枢神经损伤的可注射水凝胶MPG-HD，并具有以下效果：1)MPSS在急性期的快速释放以抑制炎症；2)长时间持续释放GFs以促进轴突再生；3)有效减少了囊性空腔与瘢痕组织的形成，促进了神经再生；4)皮层刺激和肌电图记录显示神经元从大脑到肌肉的神经回路连接。
附图说明
图1a为本发明所述马来酰亚胺改性MPSS负载纳米粒子MM-NPs的合成路线图。
图1b为本发明实施例制备的马来酰亚胺改性前纳米粒子NP的微观形貌图。
图1c为本发明实施例制备的马来酰亚胺改性后纳米粒子NP-MAL的微观形貌图。
图1d为本发明实施例制备的马来酰亚胺改性MPSS负载纳米粒子MM-NPs的电位变化图。
图1e为本发明实施例制备的MM-NPs的粒径分布图。
图1f为本发明实施例制备的MM-NPs表面马来酰亚胺定量图。
图1g为本发明实施例制备的MM-NPs与4a-PEG-SH反应后的巯基减少定量图。
图2a为本发明所述可注射水凝胶MPG-HD的合成制备机理示意图。
图2b为本发明所述不同浓度可注射水凝胶的弹性模量图。
图2c为本发明所述可注射水凝胶的降解速率图。
图2d为本发明所述可注射水凝胶的溶胀速率图。
图2e为本发明所述可注射水凝胶中MPSS缓释速度图。
图2f为本发明所述可注射水凝胶的生长因子缓释速度图。
图2g为本发明所述可注射水凝胶浸出物用于细胞培养的活细胞指标图。
图3a为本发明实施例大鼠损伤部位注射PBS、G-HD和MPG-HD的试验流程示意图。
图3b为本发明实施例大鼠损伤部位注射PBS、G-HD和MPG-HD的试验示意图；
图3c为本发明实施例大鼠损伤部位注射PBS、G-HD和MPG-HD后损伤部位的HE染色图。
图3d为本发明实施例大鼠损伤部位注射PBS、G-HD和MPG-HD后脊髓形状变化图。
图3e为本发明实施例大鼠损伤部位注射PBS、G-HD和MPG-HD后脊髓三维重建图。
图3f为本发明实施例大鼠损伤部位注射PBS、G-HD和MPG-HD后各组织和空洞体积图。
图3g为本发明实施例大鼠损伤部位注射PBS、G-HD和MPG-HD后行为学评分图。
图4a为本发明所述损伤部位及前后的5-HT轴突再生状况免疫染色图。
图4b为本发明所述损伤部位及前后的本体神经轴突再生状况免疫染色图。
图4c为本发明所述损伤部位及前后的神经纤维再生状况免疫染色图。
图4d为本发明所述损伤前后的5-HT轴突的数量统计图。
图4e为本发明所述损伤前后的本体神经轴突的数量统计图。
图4f为本发明所述损伤前后的神经纤维的数量统计图。
图5a为本发明实施大鼠损伤部位注射PBS、G-HD和MPG-HD后大脑皮层诱发电位图。
图5b为本发明实施大鼠损伤部位注射PBS、G-HD和MPG-HD后后肢运动和肌电发放情况图。
本发明的最佳实施方式
下面结合附图并以具体实施例，进一步阐明本发明。应理解这些实施例仅用于说明本发明而不用于限制本发明的范围，在阅读了本发明之后，本领域技术人员对本发明的各种等价形式的修改均落于本申请所附权利要求所限定的范围。以下是物质英文缩写和中文名称注释：
Figure PCTCN2020131330-appb-000001
一、马来酰亚胺改性MPSS纳米粒子的合成
马来酰亚胺改性MPSS负载纳米粒子MM-NPs的制备：采用无皂乳液一步聚合 法。
纳米粒子NP的合成(采用DMAEMA、ST、ACLT-PEG-NHS、NaSS和MBA合成纳米颗粒的核)：将DMAEMA(w/v5％)、ST(w/v10％)、ACLT-PEG-NHS(w/v3％)、NaSS(w/v0.4％)、MBA(w/v0.4％)和水的混合物加入四颈烧瓶内，烧瓶设有氮气出口、进口和进料口；然后，将氧化还原引发剂KPS(w/v0.2％)和SPS(w/v0.1％)溶于水中，在反应混合物溶液和引发剂溶液中分别加入缓慢的氮气流1小时，接着将引发剂溶液加入反应混合物溶液中，并在160rpm和50±5℃下进行聚合反应，反应5h后，使用透析膜(MWCO 14kDa)在去离子水中透析反应混合物48h，以去除未反应的单体。
马来酰亚胺改性：在反应混合物中加入1mg/ml MAL-NH2，摩尔比为1:1的ACLT-PEG-NHS，室温搅拌4h。然后，使用透析膜(14KDa)对去离子水透析反应混合物，并冻干，得到冻干的纳米粒子NP-MAL。
马来酰亚胺改性后的MPSS负载：将0.1g冻干纳米粒NP-MAL浸入溶于PBS中的4ml浓度为5mg/ml的MPSS中过夜，并以30000rpm离心收集60min(美国Beckman Beckman的Optimal MAX-XP)，然后冻干(图1a)。
如图1b和1c分别是马来酰亚胺改性前后纳米粒子的微观形貌，从图可知：马来酰亚胺改性前观察到边界清晰的粒子(图1b)，改性后观察到具有亲水层的粒子(图1c)，纳米颗粒的粒径为110nm，与动态光散射的研究结果接近(图1e)。
用Elleman试验也证实了本发明马来酰亚胺在纳米粒子表面的改性(图1f)以及粒子与4a-PEG-SH中硫醇基的反应(图1g)。计算了纳米颗粒表面马来酰亚胺基团浓度为0.85mmol/g，加入纳米颗粒后，4a-PEG-SH中巯基含量降低了0.12mmol。
二、马来酰亚胺改性的生长因子及效率评价
生长因子GFs的马来酰亚胺改性：所述生长因子采用浓度为50ng/ml的bFGF、BDNF和VEGF，生长因子GFs与sulfo-SMCC以1:50的摩尔比反应5min，然后用3T3、C6和HUVEC细胞检测马来酰亚胺修饰bFGF、BDNF和VEGF的活性。将细胞分别接种于96孔无生长因子培养基上，孵育24h后，用PBS洗涤细胞。然后，将0.1ml改性或未改性的含有生长因子的培养基按所示形式加入 每个孔中，对照样品加入无生长因子培养基，将培养板在37℃、含5％CO2的增湿空气中培养24小时，并使用细胞增殖试剂盒I(MTT)测量细胞活性，在550nm波长下用微板阅读器测定吸光度。
细胞系 NIH 3T3 C6 HUVEC
中等 DMEM DMEM EGM-2
生长因子 bFGF(30ng/ml) BDNF(100ng/ml) VEGF(30 ng/ml)
细胞数(细胞/孔) 40000个 60000个 40000个
结果显示：经修饰的GFs与正常GFs培养的细胞活力无明显差异。在所有三种GFs中，非GFs条件下的存活率低于添加GFs的条件。
三、可注射水凝胶溶液MPG-HD的制备
MPG-HD按以下方法制备：将RGD-PEG-SH以1∶1的摩尔比添加到4a-PEG-MAL(5％，wt％)中，然后与MM-NPs(20％，wt％)混合，将马来酰亚胺修饰的VEGF(10ng/μl)、BDNF(50ng/μl)、bFGF(10ng/μl)加入形成溶液A。将4a-PEG-SH溶于缓冲溶液中形成溶液B(5％，wt％)。将溶液A和B按1∶1的比例混合，进行聚合反应制成MPG-HD(图2a)。巯基马来酰亚胺Michael加成反应可在高浓度(高于2.5％)下以小于3秒的凝胶速率形成水凝胶，在稀释条件下(1％)未观察到凝胶化。在弹性模量测试中，测量了浓度2.5％到10％的凝胶，5％的凝胶显示出与脊髓组织相似的模量(图2b)。
本发明的实施方式
本实施例可注射水凝胶MPG-HD的制备是通过具有反应基团对巯基(-SH)与马来酰胺基(-MAL)的4-臂聚乙二醇进行点击化学反应耦合而成，因此需要指出的是：能发生点击化学反应耦合的反应基团对均可以代替，也属于本发明的保护范围。例如：巯基与烯、叠氮与炔环、共轭双烯与取代烯烃、醛与酰肼、四嗪与降冰片烯、四嗪与异腈。
另外，本发明可注射水凝胶MPG-HD制备过程通过多臂聚乙二醇-X和多臂聚乙二醇-Y中发生点击化学反应的官能团对X-Y进行耦联，而精氨酸-甘氨酸-天冬氨酸(RGD)对多臂聚乙二醇-X和/或多臂聚乙二醇-Y进行修饰，目的是用于提高MPG-HD的粘性和生物亲近性。
本实施例制备的可注射水凝胶MPG-HD负载的免疫调节或抗氧化药物采用的是 MPSS，而例举的异丙嗪、地塞米松、氢化可的松、布洛芬、羟基保泰松、环孢菌素A、藤霉素、硫唑嘌呤、6-巯基嘌呤、环磷酰胺、他克莫司(KF506)、雷帕霉素、霉酚酸脂、茶多酚(TP)、生育酚、丁基羟基茴香醚(BHA)、二丁基羟基甲苯(BHT)和/或叔丁基对苯二酚(TBHQ)中一种或多种的混合作为免疫调节或抗氧化药物同样可以替换，属于本发明的保护范围。
同样的，本实施例制备的可注射水凝胶MPG-HD负载的细胞生长因子GFs可选用：碱性成纤维生长因子(bFGF)、脑源性神经营养因子(BDNF)、血管内皮生长因子(VEGF)、酸性成纤维生长因子(aFGF)、肝细胞生长因子(HGF)、睫状神经营养因子(CNTF)、胶质细胞衍生神经营养因子(GDNF)、神经营养因子-3(NT-3)、表皮生长因子(EGF)、白介素3(IL-3)、转化生长因子-α(TGF-α)、血小板衍生的生长因子(PDGF)、胰岛素样生长因子-1(IGF-1)、骨形态发生蛋白(BMP)、结缔组织生长因子(CTGF)、骨桥蛋白(OPN)和/或生长激素释放因子(GRF)中一种或多种的混合。
工业实用性
四、可注射水凝胶溶液MPG-HD的在SCI治疗的治疗效果表征
a、可注射水凝胶MPG-HD的物化性质表征
采用万能材料测试机，根据应力-应变曲线的线性部分计算了水凝胶的弹性模量。简单地说，用A溶液和B溶液按1:1的比例混合，制备了厚度为1cm的水凝胶(w/v2.5％，5％，10％)。采用万能材料试验机(Roell Z020，Wick，Germany)在50N静态称重传感器下，以0.5mm/min的应变率测量应力-应变曲线。另外，水凝胶的降解和溶胀通过以下方式测量，将2ml水凝胶(5％)在5ml PBS中37℃孵育60天，以评估水解降解。在0、0.5、1、3、5、7、15、30、60天，分别测量湿凝胶和冻干凝胶的质量。冻干前，水凝胶在蒸馏水中清洗，以除去表面可能积聚的残留盐。通过与0天样品的干重进行比较，确定每个样品的总干聚合物质量损失。溶胀率计算为(Ms-Md)/Md。
在降解实验中，水凝胶在7天内呈线性降解，2周后显著降低，2个月时降解达到80％(图2c)，这说明水凝胶可以支持细胞在2个月的慢性期迁移。
溶胀研究表明，在PBS中浸泡1天后，平衡低膨胀率达到31％，然后在7天后保持不变(图2d)。低溶胀率可避免大体积溶胀造成的组织损伤。因此，本发 明制备的水凝胶具有适合脊髓损伤注射的特性。
b、可注射水凝胶MPG-HD在SCI治疗过程中的MPSS和生长因子的持续释放通过以下方法研究并验证MPSS和生长因子在水凝胶中的释放动力学。按上述方法制备200μl水凝胶，其中含有上述MPSS和生长因子。在每根试管中加入2ml PBS，并在37℃下孵育。0、0.5、1、3、5、7、15、30、60天后，提取上清液，并在-80℃下保存在Eppendorf管中。根据现有程序使用高效液相色谱法分析0、1、3、5、7天的MPSS浓度。生长因子浓度的检测则使用bFGF-ELISA试剂盒按说明书测定bFGF浓度。结果显示：通过马来酰亚胺与硫醇的相互作用，使bFGF缓释1个月。在未改性条件下，GFs以高速释放，在第7天达到最终浓度(图2f)。与没有纳米颗粒的情况(1天)相比，包埋在纳米颗粒中的MPSS显示出较慢的释放速率(3天)(图2e)。MPSS在NPs条件下可以缓释3天，这足够覆盖脊髓损伤的急性期。
c、细胞毒性
细胞毒性评价：采用水凝胶和纳米颗粒复合浸泡培养基。用MTT比色法测定细胞活力，结果显示：水凝胶MPG-HD浸泡悬浮液培养的细胞显示出与非凝胶培养基相似的细胞活力(图2g)，这说明我们使用的材料没有或非常低的细胞毒性，并且在体内不会引起细胞死亡。
d、MPG-HD减少挫伤性脊髓损伤后的空洞形成
雌性sprague-dawley大鼠(200-250g，二级，证书号：SCXK2008-0033，浙江省医学科学院实验动物中心，杭州市)撞伤后1周，在损伤部位注射PBS、G-HD或MPG-HD(图3a,b)。伤后8周，观察注射后脊髓组织空腔大小。
结果显示：
(1)脊髓注射PBS后，其形状严重变形，损伤部位完整组织大量丢失(图3c-e)。
(2)G-HD注射显示出比PBS对照组更小的空腔大小和更完整的脊髓组织(图3c-e)。但是空腔仍然很大，大约20％的脊髓组织消失了。
(3)与其他两种情况相比，注射MPG-HD的脊髓更完整，变形更小(图3c-e)，在横切面图像中，注射MPG-HD使动物囊腔几乎完全消失(图c,f)并充满了ECM组织。
在脊髓三维重建的定量结果中，G-HD和MPG-HD注射后，空腔体积减小， MPG-HD注射使空腔体积减少到总体积的0.8％，约为PBS腔的1/40和G-HD注射的1/20。G-HD和MPG-HD注射后，脊髓中病理组织体积(定义为无正常组织结构的体积)显著增加，残存的完整组织(包括白质和灰质)在这些脊髓中也显著增加(图3f)。
在功能和行为方面，我们发现在治疗后的前2-3周内，这些治疗都没有使运动能力有显著改善。然而，与PBS对照组动物(主要表现为后肢瘫痪)相比，接受G-HD治疗的大鼠在5周时间点首先表现出大范围的踝关节运动，并在注射后8周达到平台，BBB评分为4-5分，这和形态学上比PBS组表现出的更小的空腔大小和更少的脊柱形状变形是吻合的。而在MPG-HD治疗组中，动物表现出一致的后肢足底位置和负重步态，在6周的时间点后，10只动物中有6只的BBB得分为8-10(图3g)，这表明MPG-HD在脊髓损伤疾病进展后释放GFs和MPSS可显著改善后肢运动功能恢复，在严重损伤模型中，负重步行能力被认为是功能恢复的限制步骤。
e、可注射水凝胶MPG-HD抑制囊性空腔与瘢痕组织形成并促进轴突再生
为了进一步评估水凝胶注射诱导的再生，我们收集了损伤部位的脊髓和头、尾侧组织(图4a-c)。取3种神经纤维，用AAV2/9-mCherry注入上胸椎脊髓追踪降支轴突(脊髓上或长本体脊髓)，免疫染色观察5-HT轴突和神经丝阳性轴突(NF轴突)。
结果显示：在注射PBS的对照脊髓中，损伤部位存在向头侧延伸的大空腔，没有任何组织基质，宿主和移植体之间形成的腔边缘致密的GFAP +瘢痕(图4a，b,c,PBS，箭头标示)，所有动物(n＝10)均无轴突伸入腔内(图4a,b,c,PBS)。相反，在MPG-HD治疗中，囊性空腔被ECM所替代，宿主和移植体之间形成的腔边缘呈现疏松状，没有致密GFAP+瘢痕(图4a,b,c,MPG-HD,箭头标示),在运动恢复中起重要作用的5-羟色胺能(5-HT)轴突生长并延伸到纤维化基质中(图4a，MPG-HD线，a’，a”)。此外，纤维化基质中央区域也观察到mcherry标记的红色固有脊髓轴突(图4b，MPG-HD，b’，b”)。在MPG-HD治疗中，大量NF+的轴突纤维明显地重新长入纤维化基质，穿过损伤部位并延伸到损伤下方的脊髓节段，这在PBS注射的动物中没有观察到(图4c，c’，c”)。另一方面，与预期的PBS注射动物相比，G-HD处理的脊髓显示出更小的空腔形成 和一些轴突再生。脊髓冠状面图像和损伤部位下方轴突密度的定量分析表明，MPG-HD治疗的动物在损伤处的5-HT、本体脊髓和NF +轴突明显多于G-HD或PBS注射的动物，这可能不仅是由于改善了轴突再生造成的，也与有更多的轴突残留有关。
MPG-HD治疗促进脊髓神经回路重建
电生理记录，以评估从中枢到外周神经元的神经回路的连接情况。结果显示：在皮层电刺激同时记录TA肌肉的电信号，证实经MGP-HD治疗的脊髓能更有效地将下行神经信号传递到腰椎段脊髓。本实验仅在注射MGP-HD的动物身上记录到与正常动物相比延迟更大，幅度更微弱的肌电信号，表明通过MGP-HD治疗重建的神经环路存在多个突触连接，而且能将部分皮层电刺激产生的电信号，传递给受伤动物腰段脊髓的运动神经元(图5a)。在自由行走过程中，注射PBS的大鼠不能支撑体重，后肢无关节活动。在注射G-HD的大鼠中，捕捉到膝关节的轻微运动和体重的支撑。与之相对应，注射MGP-HD的大鼠能支撑体重，并能做与正常大鼠相似的步态。肌电图显示，注射MPG-HD后，大鼠TA、GS活动明显，后肢总步幅明显增加。与正常大鼠一样，记录了步态时TA和GS的交替激活。但GS信号明显弱于正常大鼠，提示MGP-HD注射大鼠出现了次优的后肢运动功能恢复(图5b)。结果表明，注射MGP-HD可使严重脊髓挫伤模型动物的建立新的伸进环路连接，恢复部分负重步行能力。
以上结果表明本发明成功制备了具有药物释放能力的可注射水凝胶，能用于SCI的修复，具体特征如下：
1、通过本发明制备的可注射水凝胶MPG-HD能使脊髓损伤的原位凝胶化，确保了其能适配空腔的形状，从而使脊髓组织和凝胶之间的间隙最小化。在损伤部位注入的水凝胶为成纤维细胞的迁移和侵袭提供了支架，形成富含成纤维细胞的ECM，从而减少孔洞产生。
2、本发明的可注射水凝胶MPG-HD具有合适的溶胀比，降低了损伤部位残留组织二次损伤风险。
3、本发明通过反应基团修饰的负载药物的纳米粒子/GFs与水凝胶的反应基团通过点击化学耦合，使药物或生长因子能在损伤部位分时段释放，适应脊髓损伤后病理变化。
4、本发明制备的可注射水凝胶MPG-HD有助于减少瘢痕组织生成，促进脊髓神经再生。
5、本发明制备的可注射水凝胶MPG-HD有助于脊髓神经回路的重建，促进功能恢复。

Claims (9)

  1. 一种治疗中枢神经损伤的可注射水凝胶，其特征在于：以精氨酸-甘氨酸-天冬氨酸修饰过的多臂聚乙二醇-X与多臂聚乙二醇-Y通过点击化学反应得到用于病灶原位形成水凝胶支架的聚合物，所述聚合物还负载有纳米/微米粒子和细胞生长因子，所述纳米/微米颗粒负载有免疫调控药物和/或抗氧化药物；
    所述多臂聚乙二醇-X和多臂聚乙二醇-Y中发生点击化学反应的官能团对X与Y选自：巯基与马来酰胺基、巯基与烯、叠氮与炔环、共轭双烯与取代烯烃、醛与酰肼、四嗪与降冰片烯、四嗪与异腈；
    所述多臂聚乙二醇-X为4-臂聚乙二醇-X或3-臂聚乙二醇-X；所述多臂聚乙二醇-Y为4-臂聚乙二醇-Y或3-臂聚乙二醇-Y；
    所诉多臂聚乙二醇-X与多臂聚乙二醇-Y中聚乙二醇的聚合度n为1-1000。
  2. 根据权利要求1所述治疗中枢神经损伤的可注射水凝胶，其特征在于，所述纳米/微米粒子选自反应基团修饰过的高分子微米/纳米粒子和/或脂质体中一种或多种的混合。
  3. 根据权利要求2所述治疗中枢神经损伤的可注射水凝胶，其特征在于，所述纳米/微米粒子采用聚乙烯、明胶、胶原聚乳酸或脂质体粒子中的一种或多种的混合。
  4. 根据权利要求1所述治疗中枢神经损伤的可注射水凝胶，其特征在于，所述免疫调控类/抗氧化类药物选自甲基强的松龙琥珀酸钠(MPSS)、异丙嗪、地塞米松、氢化可的松、布洛芬、羟基保泰松、环孢菌素A、藤霉素、硫唑嘌呤、6-巯基嘌呤、环磷酰胺、他克莫司(KF506)、雷帕霉素、霉酚酸脂、茶多酚(TP)、生育酚、丁基羟基茴香醚(BHA)、二丁基羟基甲苯(BHT)和/或叔丁基对苯二酚(TBHQ)中一种或多种的混合。
  5. 根据权利要求1所述治疗中枢神经损伤的可注射水凝胶，其特征在于，所述细胞生长因子选自：碱性成纤维生长因子(bFGF)、脑源性神经营养因子(BDNF)、血管内皮生长因子(VEGF)、酸性成纤维生长因子(aFGF)、肝细胞生长因子(HGF)、睫状神经营养因子(CNTF)、胶质细胞衍生神经营养因子(GDNF)、神经营养因子-3(NT-3)、表皮生长因子(EGF)、白介素3(IL-3)、转化生长因子-α(TGF-α)、血小板衍生的生长因子(PDGF)、胰岛素样生长因子-1(IGF-1)、骨形态发生蛋白(BMP)、结缔组织生长因子(CTGF)、骨桥蛋 白(OPN)和/或生长激素释放因子(GRF)中一种或多种的混合。
  6. 一种权利要求1～5任一所述治疗中枢神经损伤的可注射水凝胶的制备方法，包括以下步骤：
    步骤1：高分子纳米粒子(NP)的合成：在反应容器中，将DMAEMA、ST、ACLT-PEG-NHS、NaSS、MBA加入水中，然后加入氧化还原引发剂KPS和SPS，在温度为50±10℃、氮气保护和搅拌下聚合反应3h以上，通过透析膜去除未反应完的单体得到纳米粒子NP；
    步骤2：在纳米粒子NP的表面修饰马来酰亚胺：继续加入摩尔比为1:1～1.1的MAL-NH2和ACLT-PEG-NHS，在搅拌下进行马来酰亚胺改性反应至少1h，继续通过透析膜去除未反应完单体并冻干，得到冻干的马来酰亚胺改性纳米粒子NP-MAL；
    步骤3：NP-MAL粒子负载MPSS：在MPSS的PBS溶液中，加入上述得到的纳米粒子NP-MAL并静置过夜，然后经过离心收集处理得到负载MPSS的纳米粒子，并经过冻干处理得到负载MPSS的纳米粒子MM-NPs；
    步骤4：生长因子GFs的马来酰亚胺改性：将生长因子GFs按照1:30～70的摩尔比与sulfo-SMCC混合反应，得到马来酰亚胺改性的生长因子GF-MAL；
    步骤5：将4a-PEG-MAL和RGD-PEG-SH按1:1摩尔比混合，得到RGD修饰过的4a-PEG-MAL，然后加入步骤3得到MM-NPs纳米粒子与步骤4得到的马来酰亚胺改性的生长因子GF-MAL并混合均匀形成溶液A；
    步骤6：溶液B的制备：将4a-PEG-SH溶于缓冲溶液，混合均匀形成溶液B；
    步骤7：将步骤5与步骤6中的溶液A和B按1:1比例混合反应，即得到偶联负载药物的纳米粒子与细胞生长因子的可注射水凝胶MPG-HD。
  7. 根据权利要求5所述治疗中枢神经损伤的可注射水凝胶的制备方法，其特征在于：步骤1聚合反应中各反应物浓度为5％(w/v)DMAEMA、10％(w/v)ST、3％(w/v)ACLT-PEG-NHS、0.4％(w/v)NaSS、0.4％(w/v)MBA、0.2％(w/v)KPS和0.1％(w/v)SPS。
  8. 根据权利要求5所述治疗中枢神经损伤的可注射水凝胶的制备方法，其特征在于：步骤3、7中的载药纳米/微米粒子通过偶联反应负载在水凝胶上。
  9. 根据权利要求6所述治疗中枢神经损伤的可注射水凝胶的制备方法，其特征在于：步骤4、7中生长因子通过偶联反应负载在水凝胶上。
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