CN114209881B - Functional biological material and preparation method and application thereof - Google Patents

Functional biological material and preparation method and application thereof Download PDF

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CN114209881B
CN114209881B CN202111536838.9A CN202111536838A CN114209881B CN 114209881 B CN114209881 B CN 114209881B CN 202111536838 A CN202111536838 A CN 202111536838A CN 114209881 B CN114209881 B CN 114209881B
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functional biomaterial
functional
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dibenzocyclooctyne
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CN114209881A (en
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董群
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Jiangsu Dubu Biotechnology Co ltd
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Abstract

The invention provides a functional biological material and a preparation method and application thereof; the functional biological material comprises ordered collagen modified by azide derivatives and a liposome modified by dibenzocyclooctyne derivatives loaded on the ordered collagen modified by the azide derivatives; the liposome carries cholesterol ester synthetase inhibitor. The functional biological material provided by the invention regulates and controls a nerve regeneration microenvironment by regulating and controlling local cholesterol ester anabolism of a spinal cord injury part, and promotes spinal cord injury repair; the functional biomaterial provided by the invention has good tensile resistance.

Description

Functional biological material and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a functional biomaterial, and a preparation method and application thereof.
Background
Spinal cord injury repair is a worldwide medical problem, and spinal cord injury causes loss of sensory and motor functions below the plane of injury, which places a heavy burden on patients and families. According to the world health organization and epidemiological studies of Europe and America, 10-40 persons in each million persons in the world are suffered from spinal cord injury. Data indicate that 50-60 spinal cord injuries occur in each million people every year in China.
Research finds that the spinal cord injury part forms an inhibitory microenvironment, so that the regeneration of neuron axons is inhibited, and the differentiation of neural stem cells to neurons is inhibited. Many proteins, including myelin proteins and the like, are currently considered to be a major component of the spinal inhibitory microenvironment.
CN110833543A discloses a medicine for promoting recovery from chronic spinal cord injury, a preparation method and application thereof. The invention discloses a medicine for promoting spinal cord injury recovery, which mainly comprises the active ingredient of a specific histone deacetylase 3 inhibitor RGFP966. The invention discloses novel functions of RGFP966 in treating diseases such as inhibiting inflammatory response, promoting anti-inflammatory response, promoting axonal regeneration and promoting functional recovery after chronic spinal cord injury by inhibiting HDAC 3.
CN105311620a discloses a spinal cord injury therapeutic agent, a spinal cord injury therapeutic agent and a demyelinating disease therapeutic agent containing HGF protein as an active ingredient.
CN113171369A discloses an application of polypyrimidine sequence binding protein in preparation of a spinal cord injury repair drug. The polypyrimidine sequence binding Protein (PTB) is silenced in vitro through viruses, and meanwhile, micromolecule Retinoic Acid (RA) and Purmorphamine (PMA) related to motor neuron differentiation are added in a combined mode, so that mouse spinal cord reactive astrocytes are successfully reprogrammed into motor neurons, help is provided for further in vivo research on the action of a PTB combined micromolecule reprogramming strategy in spinal cord injury repair, and further better spinal cord injury repair and function reconstruction effects are achieved.
In addition to proteins, lipids are an important component of spinal cord tissue, but the role of lipids in nerve regeneration following spinal cord injury remains unclear. Lipids are generally classified into cholesterol esters, cholesterol, phospholipids and glycolipids. The synthesis, transfer, transformation and storage of cholesterol play an important role in the physiological regulation of organisms, free cholesterol and cholesterol ester exist in the central nervous system, the change of the cholesterol ester has certain correlation with Alzheimer disease, amyotrophic lateral sclerosis and the like, but the promotion of spinal cord injury repair by means of regulating lipid metabolism is not reported yet.
Therefore, how to provide a method for promoting spinal cord injury repair by regulating lipid metabolism is an urgent problem to be solved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a functional biological material and a preparation method and application thereof. The functional biological material provided by the invention regulates and controls a nerve regeneration microenvironment by regulating and controlling local cholesterol ester anabolism of a spinal cord injury part, and promotes spinal cord injury repair.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a functional biomaterial, comprising an azide derivative-modified ordered collagen and a dibenzocyclooctyne derivative-modified liposome loaded on the azide derivative-modified ordered collagen; the liposome carries cholesterol ester synthetase inhibitor.
The functional biological material provided by the invention regulates and controls a nerve regeneration microenvironment by regulating and controlling local cholesterol ester anabolism of a spinal cord injury part, and promotes spinal cord injury repair; the functional biomaterial provided by the invention has the effects of promoting the migration of endogenous stem cells to the damaged part after spinal cord injury, promoting nerve bridging after spinal cord injury and promoting the recovery of motor function after spinal cord injury; the functional biomaterial provided by the invention has good tensile resistance.
In the invention, the azide derivative modified ordered collagen and the dibenzocyclooctyne derivative modified liposome are connected through a triazole bond formed by the azide derivative and the dibenzocyclooctyne derivative.
Preferably, the cholesterol ester synthase inhibitor is a cholesterol acyltransferase inhibitor, preferably Avasimibe.
The functional biological material containing Avasimibe in the invention regulates and controls the nerve regeneration microenvironment by regulating and controlling the local cholesterol ester anabolism of the spinal cord injury part, and promotes the spinal cord injury repair.
Preferably, the functional biomaterial is filaments arranged in bundles.
Preferably, each bundle of functional biomaterial has a diameter of 2-4mm, and may be, for example, 2mm, 2.2mm, 2.4mm, 2.6mm, 2.8mm, 3mm, 3.2mm, 3.4mm, 3.6mm, 3.8mm, 4mm, etc.; the length is 8-15cm, and may be, for example, 8cm, 9cm, 10cm, 11cm, 12cm, 13cm, 14cm, 15cm, or the like.
Preferably, each bundle of functional biomaterial comprises 20-400 filaments, which may be, for example, 20, 50, 70, 100, 120, 150, 170, 200, 220, 250, 270, 300, 320, 350, 370, 400, etc.
Preferably, the filaments have a diameter of 10-100 μm, and may be, for example, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, or the like; the length is 8-15cm, and may be, for example, 8cm, 9cm, 10cm, 11cm, 12cm, 13cm, 14cm, 15cm, or the like.
In the present invention, the azide derivative-modified ordered collagen accounts for 90 to 99% of the total mass of the functional biomaterial, and may be, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or the like.
Preferably, the azide derivative accounts for 10-30% of the total mass of the azide derivative modified ordered collagen, and may be, for example, 10%, 15%, 20%, 25%, 30%, etc.
Preferably, the azide derivative comprises azido- (N-hydroxysuccinimide) (N) 3 -NHS) and/or azido-polyethylene glycol- (N-hydroxysuccinimide) (N 3 -PEG-NHS), preferably azido-polyethylene glycol- (N-hydroxysuccinimide); the English language of polyethylene glycol is abbreviated as PEG.
Preferably, the number average molecular weight of the polyethylene glycol in the azide-polyethylene glycol- (N-hydroxysuccinimide) is 2000 to 5000, and may be, for example, 2000, 2500, 3000, 3500, 4000, 4500, 5000, and the like.
In the present invention, the dibenzocyclooctyne derivative-modified liposome accounts for 1 to 10% of the total mass of the functional biomaterial, and may be, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or the like.
Preferably, the dibenzocyclooctyne derivative accounts for 10-20% of the total mass of the dibenzocyclooctyne derivative-modified liposome, and may be, for example, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, etc.
Preferably, the dibenzocyclooctyne derivative comprises dibenzocyclooctyne-hydrophobic polymer-polyethylene glycol (DBCO-hydrophobic polymer-PEG).
Preferably, the hydrophobic polymer in the dibenzocyclooctyne-hydrophobic polymer-polyethylene glycol comprises any one of Distearoylphosphatidylethanolamine (DSPE), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylethanolamine (DPPE) or Dipalmitoylphosphatidylcholine (DPPC) or a combination of at least two thereof.
Preferably, the number average molecular weight of the polyethylene glycol in the dibenzocyclooctyne-hydrophobic polymer-polyethylene glycol is 2000-5000, such as 2000, 2500, 3000, 3500, 4000, 4500, 5000 and the like.
In the present invention, the N-hydroxysuccinimide in the azide derivative (azide- (N-hydroxysuccinimide) and/or azide-polyethylene glycol- (N-hydroxysuccinimide)) can form a stable amide bond with an amino acid residue in collagen; in addition, the polyethylene glycol in the azide-polyethylene glycol- (N-hydroxysuccinimide) can enhance the solubility and stability, reduce the nonspecific binding of the surface of a charged molecule and reduce the immunogenicity of the polypeptide; the azide in the azide derivative can react with dibenzocyclooctyne in a dibenzocyclooctyne derivative (dibenzocyclooctyne-hydrophobic polymer-polyethylene glycol) to generate a stable triazole bond through click chemistry; the hydrophobic polymer and the polyethylene glycol in the dibenzocyclooctyne derivative can be connected with the liposome through hydrophilic and hydrophobic effects.
In the invention, the dibenzocyclooctyne derivative modified liposome comprises soybean phospholipid and cholesterol.
Preferably, the mass ratio of the soybean phospholipids to the cholesterol is (2-5): 1, and the soybean phospholipids can be 2:1, 3:1, 4:1, 5:1 and the like.
Preferably, the particle size of the dibenzocyclooctyne derivative-modified liposome is 30-70nm, and may be, for example, 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, or the like.
Preferably, the drug loading of the dibenzocyclooctyne derivative-modified liposome is 60-80%, for example, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, etc.
Preferably, the encapsulation efficiency of the dibenzocyclooctyne derivative-modified liposome is 50-70%, for example, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70% or the like.
In a second aspect, the present invention provides a method for preparing a functional biomaterial according to the first aspect, the method comprising the steps of: mixing the ordered collagen modified by the azide derivative and the liposome modified by the dibenzocyclooctyne derivative carrying the cholesterol ester synthetase inhibitor, and reacting to obtain the functional biological material.
Preferably, the reaction temperature is 20-35 ℃, for example can be 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃,29 ℃,30 ℃,31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃ etc.; the time is 10 to 15 hours, and may be, for example, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, or the like.
Preferably, the obtained functional biological materials are bundled into bundles, each bundle comprises 20 to 400 functional biological materials, for example, 20, 50, 70, 100, 120, 150, 170, 200, 220, 250, 270, 300, 320, 350, 370, 400 functional biological materials can be used.
In the invention, the preparation method of the azide derivative modified ordered collagen comprises the following steps: dissolving the azide derivative and the ordered collagen in a buffer solution, and reacting to obtain the ordered collagen modified by the azide derivative.
Preferably, the buffer solution comprises a phosphate buffer, the pH of the buffer solution being 6-8, and may be, for example, 6, 6.2, 6.4, 6.6, 6.8, 7, 7.2, 7.4, 7.6, 7.8, 8, and the like.
Preferably, the azide derivative in the mixed solution obtained after dissolution is at a concentration of 1 to 10mg/mL, for example, 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL, 10mg/mL, etc., and the ordered collagen is at a concentration of 1 to 5g/mL, for example, 1g/mL, 2g/mL, 3g/mL, 4g/mL, 5g/mL, etc.
Preferably, the reaction temperature is 35-39 deg.C, such as 35 deg.C, 36 deg.C, 37 deg.C, 38 deg.C, 39 deg.C, etc., and the reaction time is 3.5-4.5h, such as 3.5h, 3.6h, 3.7h, 3.8h, 3.9h, 4.0h, 4.1h, 4.2h, 4.3h,. 4.4h, 4.5h, etc.
Preferably, the ordered collagen modified by the azide derivative is obtained by washing.
Preferably, the solvent used for cleaning is water, the number of times of cleaning is 2-5, such as 2, 3, 4, 5, etc., and the time of each cleaning is 8-15min, such as 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, etc.
In the invention, the ordered collagen is prepared by the following preparation method: and sequentially carrying out coarse purification treatment, enzymolysis treatment, nucleic acid removal treatment, organic solvent treatment, detergent treatment, surfactant treatment, freezing and freeze-drying on the membrane tendon tissue to obtain the ordered collagen.
Preferably, said crude purification comprises the removal of muscle and solid adipose tissue.
Preferably, the pretreatment is followed by water treatment, the number of times of water treatment is 2-5, such as 2, 3, 4, 5, etc., and the time of each time of water treatment is 12-17min, such as 12min, 13min, 14min, 15min, 16min, 17min, etc.
Preferably, the protease adopted by the enzymolysis treatment comprises papain, and the enzyme activity of the protease is 200-400U/g, such as 200U/g, 250U/g, 300U/g, 350U/g, 400U/g and the like; the buffer solution adopted by the enzymolysis treatment comprises a phosphate buffer solution, wherein the pH of the buffer solution is 5.5-6.5, such as 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 and the like; the treatment time of the enzymolysis treatment is 0.1-1h, and can be, for example, 0.1h, 0.2h, 0.3h, 0.4h, 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1h and the like.
Preferably, the enzymes used in the nucleic acid removing treatment comprise RNase A and DNase I, and the enzyme activity of the enzymes is 80-120U/g, such as 80U/g, 90U/g, 100U/g, 110U/g, 120U/g and the like; the time for the nucleic acid removal treatment is 2.5 to 3.5 hours, and may be, for example, 2.5 hours, 2.6 hours, 2.7 hours, 2.8 hours, 2.9 hours, 3.0 hours, 3.1 hours, 3.2 hours, 3.3 hours, 3.4 hours, 3.5 hours, or the like.
Preferably, the organic solvent used for the organic solvent treatment comprises n-heptane and ethanol, wherein the volume ratio of n-heptane to ethanol is (0.5-1.5): 2, and can be, for example, 0.5; the organic solvent treatment time is 20-30h, for example, 20h, 22h, 24h, 26h, 28h, 30h and the like.
Preferably, the organic solvent treatment is further followed by water treatment, the number of water treatments is 2-5, such as 2, 3, 4, 5, etc., and the time of each water treatment is 15-25min, such as 15min, 16min, 17min, 18min, 19min, 20min, 21min, 22min, 23min, 24min, 25min, etc.
Preferably, the detergent used for the detergent treatment comprises an aqueous sodium deoxycholate solution, and the mass content of the aqueous sodium deoxycholate solution is 2-6%, such as 2%, 3%, 4%, 5%, 6% and the like; the time for the descaler treatment is 1 to 3 hours, and may be, for example, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, etc.
The detergent is further treated with water, the number of water treatments is 4-7, such as 4, 5, 6, 7, etc., and the time of each water treatment is 8-15min, such as 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, etc.
Preferably, the surfactant used in the surfactant treatment comprises an aqueous solution of sodium dodecyl sulfate, the mass content of the aqueous solution of sodium dodecyl sulfate is 0.1-0.3%, for example, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, etc.; the time for the surfactant treatment is 0.1 to 1 hour, and may be, for example, 0.1 hour, 0.2 hour, 0.3 hour, 0.4 hour, 0.5 hour, 0.6 hour, 0.7 hour, 0.8 hour, 0.9 hour, 1 hour, or the like.
Preferably, the surfactant treatment is followed by water treatment, the number of water treatments is 4-7, such as 4, 5, 6, 7, etc., and the time of each water treatment is 8-15min, such as 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, etc.
In the present invention, the fascia is first subjected to a crude treatment to remove muscle and solid adipose tissue; after the rough treatment, water treatment is carried out to clean adhered substances; enzymolysis treatment, in order to remove collagen telopeptide, less immunogenicity; a nucleic acid removal treatment for removing DNA and RNA; organic solvent treatment to remove lipids; after the organic solvent treatment, water treatment is carried out to remove the organic solvent; the detergent treatment, the water treatment after the detergent treatment, the surfactant treatment and the water treatment after the surfactant treatment are all for removing impurity proteins.
Preferably, the freezing temperature is-90 to-70 ℃, for example, -90 ℃, -85 ℃, -80 ℃, -75 ℃, -70 ℃ and the like, and the time is 0.8 to 1.5h, for example, 0.8h, 0.9h, 1.0h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h and the like.
Preferably, the freeze-drying pressure is 0.05-0.15mbar, such as 0.05mbar, 0.07mbar, 0.09mbar, 0.11mbar, 0.13mbar, 0.15mbar, etc., for 20-30h, such as 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, etc.
In the invention, the dibenzocyclooctyne derivative modified liposome is prepared by adopting a film dispersion method.
Preferably, the preparation method of the dibenzocyclooctyne derivative modified liposome comprises the following steps: dissolving soybean phospholipid, cholesterol and dibenzocyclooctyne derivatives in an organic solvent, and concentrating to obtain an intermediate; and mixing the obtained intermediate, a cholesterol ester synthetase inhibitor and a buffer solution to obtain the dibenzocyclooctyne derivative modified liposome.
Preferably, the organic solvent comprises chloroform and methanol.
Preferably, the volume ratio of chloroform to methanol is (1.5-2.5): 1, and can be, for example, 1.5.
Preferably, the mixing further comprises sequentially performing the ultrasonic treatment and the filtering.
Preferably, the power of the ultrasound is 250-350W, such as 250W, 270W, 290W, 310W, 330W, 350W and the like, and the time is 15-25min, such as 15min, 17min, 19min, 21min, 23min, 25min and the like.
Preferably, the filtration is performed with a filter membrane of 0.18-0.30. Mu.m, such as 0.18. Mu.m, 0.20. Mu.m, 0.22. Mu.m, 0.24. Mu.m, 0.26. Mu.m, 0.28. Mu.m, 0.30. Mu.m, etc.
In a third aspect, the present invention provides a use of the functional biomaterial of the first aspect in the preparation of a material for promoting nerve regeneration after spinal cord injury.
Compared with the prior art, the invention has the following beneficial effects:
(1) The functional biological material provided by the invention regulates and controls a nerve regeneration microenvironment by regulating and controlling local cholesterol ester anabolism of a spinal cord injury part, and promotes spinal cord injury repair;
(2) The functional biomaterial provided by the invention has more Nestin proteins at the spinal cord injury part, which indicates that neural stem cells appear at the injury part, and the appearance of positive neural stem cells indicates that the functional biomaterial provided by the invention has the effect of promoting the migration of endogenous stem cells to the injury part after the spinal cord injury;
(3) The functional biomaterial provided by the invention has more NF positive neurons at the damaged part, which lays a foundation for nerve bridging after spinal cord injury;
(4) By adopting the functional biological material provided by the invention, the BBB score of a rat is higher, which shows that the functional biological material provided by the invention can promote the recovery of motor function after spinal cord injury;
(5) The functional biomaterial provided by the invention has good tensile resistance.
Drawings
FIG. 1 is a scanning electron microscope image of the functional biomaterial provided in example 1.
Fig. 2 is a tensile curve of the functional biomaterial provided in example 1.
FIG. 3A is a photograph showing immunofluorescence staining of Nestin-positive cells at a lesion site 7 days after the control group rats without transplanted material; wherein the white bright areas are staining of neural stem cell marker Nestin;
FIG. 3B is an immunofluorescence staining pattern of Nestin positive cells at the injury site 7 days after the functional biomaterial provided in comparative example 1 was transplanted into a spinal cord injured rat; wherein the white bright areas are staining of neural stem cell marker Nestin;
FIG. 3C is a photograph of immunofluorescence staining of Nestin-positive cells at the injury site 7 days after the functional biomaterial provided in example 1 was transplanted into spinal cord injured rats; wherein the white bright areas are staining of neural stem cell marker Nestin.
FIG. 4A is a photograph of immunofluorescence staining of NF positive cells at the injury site 30 days after control rats without transplanted material; wherein the brightened area is the staining of the neuronal marker NF;
FIG. 4B is an immunofluorescence staining pattern of NF positive cells at the injury site 30 days after the functional biomaterial provided in comparative example 1 was transplanted into a spinal cord injured rat; wherein the brightened areas are staining of neuronal markers NF;
FIG. 4C is an immunofluorescence staining pattern of NF positive cells at the injury site 30 days after the functional biomaterial provided in example 1 was transplanted into spinal cord injured rats; wherein the brightened areas are staining for the neuronal marker NF.
FIG. 5 is a BBB score of the motor function of rats after transplantation of the functional biomaterials provided in the blank control group, example 1 and comparative example 1 into spinal cord injured rats.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitation of the present invention.
The sources of the components in the following examples are as follows:
Figure BDA0003412851890000111
example 1
The embodiment provides a functional biomaterial, and a preparation method of the functional biomaterial comprises the following steps:
(one) preparation of azide derivative modified ordered collagen:
(1) Taking 200g of fresh bovine membranous tendon tissue, and removing muscle and solid adipose tissue; then soaking the mixture in 1000mL of deionized water for 3 times, and changing the solution every 15 min;
(2) Adding 300U/g of 200mL papain in phosphate buffer PBS (pH = 6) into the product obtained in the step (1), and treating for 30min;
(3) Adding 100U/mL RNase A and 100U/mL DNase I into the product obtained in the step (2) for 200mL for treatment for 3h;
(4) Adding 500mL of a mixed solution of n-heptane and ethanol (V) to the product obtained in the step (3) N-heptane :V Ethanol = 1:2) for 24h; then washing with 1000mL deionized water for 3 times, each time for 20min;
(5) Treating the product obtained in the step (4) with 500mL of 4% sodium deoxycholate solution for 2h, and then washing with deionized water for 5 times, wherein each time lasts for 10min;
(6) Treating the product obtained in the step (5) with 500mL of 0.2% sodium dodecyl sulfate for 30min; then washing with deionized water for 5 times, each time for 10min;
(7) Freezing the product obtained in step (6) in a refrigerator at-80 deg.C for 1h, then freeze-drying in a freeze dryer, and freeze-drying at 0.1mbar for 24h to obtain 2g of ordered collagen;
(8) Mixing 2g of the ordered collagen of step (7) with 500mg of N 3 PEG5k-NHS was dissolved in 100mL PBS (pH = 7.2) solution and reacted at 37 ℃ for 4h; then washing with deionized water for 3 times, each time for 10 minutes, to obtain 2.4g of azide derivative modified ordered collagen;
preparation of (di) dibenzocyclooctyne derivative modified liposome:
dissolving 50mg soybean phospholipid, 15mg cholesterol, 12mg DBCO-DSPE-PEG2k in 8mL chloroform methanol solution (V) Chloroform :V Methanol = 2:1), evaporating to dryness in a rotary evaporator, adding 4mL of PBS solution, adding 1mg of cholesterol ester synthetase inhibitor Avasimibe, and performing ultrasonic treatment at 300W for 20min; then filtering by using a 0.22um filter membrane to obtain 60mg of the liposome modified by the dibenzocyclooctyne derivative;
(III) preparation of functional biological Material
The obtained 2.4g of ordered collagen modified by azide derivatives and 60mg of liposomes modified by dibenzocyclooctyne derivatives were mixed and reacted at 25 ℃ for 12 hours to obtain 2.46g of functional biomaterials, which were bundled into bundles each containing 30 functional biomaterials.
Example 2
The embodiment provides a functional biomaterial, and the preparation method of the functional biomaterial comprises the following steps:
(one) preparation of azide derivative modified ordered collagen:
(1) Taking 200g of fresh bovine membranous tendon tissue, and removing muscle and solid adipose tissue; then soaking the mixture in 950mL of deionized water for 4 times, and changing the solution every 16 min;
(2) Adding 300U/g papain 205mL into the product obtained in the step (1), wherein the buffer is phosphate buffer PBS (pH = 6), and treating for 35min;
(3) Adding 100U/mL of RNase A and 100U/mL of DNase I205 mL into the product obtained in the step (2) for treatment for 2.9h;
(4) 490mL of a mixed solution of n-heptane and ethanol (V) was added to the product obtained in step (3) N-heptane :V Ethanol = 1:2) for 25h; then washing with 1100mL deionized water for 4 times, each for 18min;
(5) Treating the product obtained in the step (4) with 505mL of 4% sodium deoxycholate solution for 2.1h, and then washing with deionized water for 6 times, wherein each time lasts for 8min;
(6) Treating the product obtained in the step (5) with 505mL of 0.2% sodium dodecyl sulfate for 27min; then washing with deionized water for 6 times, 8min each time;
(7) Freezing the product obtained in step (6) in a refrigerator at-80 deg.C for 1h, and freeze-drying in a freeze dryer at 0.11mbar for 25h to obtain 2.1g order collagen;
(8) Mixing 2.1g of the ordered collagen of step (7) with 505mg of N 3 PEG5k-NHS was dissolved in 100mL PBS (pH = 7.2) solution and reacted at 37 ℃ for 4.2h; then washing with deionized water for 3 times, and each time for 9 minutes to obtain 2.5g of azide derivative modified ordered collagen;
preparation of (di) dibenzocyclooctyne derivative modified liposome:
dissolving soybean phospholipid 55mg, cholesterol 17mg, and DBCO-DSPE-PEG2k 15mg in chloroform-methanol solution 9mL (V) Chloroform :V Methanol = 2:1), after evaporation to dryness on a rotary evaporator, add 4mL PBS solution, add 1.2mg cholesteryl ester synthase inhibitor Avasimibe, sonicate 1W under 310W8min; then filtering by using a 0.22um filter membrane to obtain 60mg of the liposome modified by the dibenzocyclooctyne derivative;
(III) preparation of functional biological Material
The obtained 2.5g of ordered collagen modified by azide derivative and 60mg of liposome modified by dibenzocyclooctyne derivative were mixed and reacted at 27 ℃ for 11.8 hours to obtain 2.56g of functional biomaterial, which was bundled into bundles each containing 30 functional biomaterials.
Example 3
The embodiment provides a functional biomaterial, and the preparation method of the functional biomaterial comprises the following steps:
(one) preparation of azide derivative modified ordered collagen:
(1) Taking 200g of fresh bovine membranous tendon tissue, and removing muscle and solid adipose tissue; then soaking the fabric in 1050mL of deionized water for 3 times, and changing the solution every 17 min;
(2) Adding 195mL of 300U/g papain into the product obtained in the step (1), wherein the buffer is Phosphate Buffered Saline (PBS) (pH = 6), and treating for 35min;
(3) Adding 100U/mL of RNase A and 100U/mL of DNase I into the product obtained in the step (2) for treatment for 3.2h;
(4) Adding 510mL of a mixed solution of n-heptane and ethanol (V) to the product obtained in the step (3) N-heptane :V Ethanol = 1:2) for 23h; then washing with 950mL deionized water for 3 times, each time for 25min;
(5) Treating the product obtained in the step (4) with 495mL of 4% sodium deoxycholate solution for 1.8h, and then washing with deionized water for 5 times, each time for 9min;
(6) Treating the product obtained in the step (5) with 495mL of 0.2% sodium dodecyl sulfate for 33min; then washing with deionized water for 5 times, each time for 12min;
(7) Freezing the product obtained in step (6) in a refrigerator at-80 deg.C for 1h, then freeze-drying in a freeze dryer, freeze-drying at 0.12mbar for 23.5h to obtain 1.9g of ordered collagen;
(8) Mixing 1.9g of the ordered collagen obtained in step (7) with 495mg of N 3 -PEG5k-NHS in 105mL PBS (pH = 7.2) solution, 37 deg.CReacting for 3.8h; then washing with deionized water for 3 times, each time for 12 minutes, to obtain 2.3g of azide derivative modified ordered collagen;
preparation of (di) dibenzocyclooctyne derivative modified liposome:
dissolving soybean phospholipid 48mg, cholesterol 13mg, and DBCO-DSPE-PEG2k 10mg in chloroform-methanol solution (7 mL) (V) Chloroform :V Methanol = 2:1), after evaporating to dryness in a rotary evaporator, adding 4mL of PBS solution, adding 0.9mg of cholesteryl ester synthetase inhibitor Avasimibe, and performing ultrasonic treatment at 295W for 25min; then filtering by using a 0.22um filter membrane to obtain 53mg of the liposome modified by the dibenzocyclooctyne derivative;
(III) preparation of functional biological Material
The obtained 2.3g of ordered collagen modified with azide derivatives and 53mg of liposomes modified with dibenzocyclooctyne derivatives were mixed and reacted at 24 ℃ for 13 hours to obtain 2.35g of functional biomaterials, which were bundled into bundles each containing 30 functional biomaterials.
Example 4
This example provides a functional biomaterial, differing from example 1 only in that, in step (8), N is added 3 Replacement of PEG5k-NHS with an equal weight of azido-
Figure BDA0003412851890000151
-NHS, the other steps are as in example 1.
Example 5
This example provides a functional biomaterial, which is different from example 1 only in that DBCO-DSPE-PEG2k is replaced with DBCO-
Figure BDA0003412851890000161
MAL, other steps as in example 1.
Comparative example 1
This comparative example provides a functional biomaterial, differing from example 1 only in that it comprises only steps (1) to (7), resulting in ordered collagen.
Comparative example 2
This comparative example provides a functional biomaterial, differing from example 1 only in the absence of step (8), and the other synthetic steps being the same as example 1.
Comparative example 3
This comparative example provides a functional biomaterial, which is different from example 1 only in that DBCO-DSPE-PEG2k is not included in the preparation of the dibenzocyclooctyne derivative-modified liposome, and the other synthetic steps are the same as those of example 1.
Comparative example 4
This example provides a functional biomaterial, which is different from example 1 only in that the cholesterol ester synthase inhibitor Avasimibe is replaced with paclitaxel of the same weight in the preparation of dibenzocyclooctyne derivative-modified liposomes, and the other steps are the same as example 1.
Test example 1
Dibenzo cyclooctyne derivative modified liposome performance test
Testing a sample: dibenzocyclooctyne derivative-modified liposomes provided in examples 1 to 5, comparative examples 2 and 4; comparative example 3 liposomes
The test method comprises the following steps: the freeze dryer and the microplate reader are used for testing and calculating the drug loading rate and the encapsulation efficiency, the dynamic light scattering laser particle size analyzer is used for testing the particle size distribution, and the specific test results are shown in table 1:
TABLE 1
Sample(s) Encapsulation efficiency/% Loading capacity/%) Average particle diameter/nm
Example 1 75 68 50
Example 2 76 70 48
Example 3 80 60 30
Example 4 60 72 70
Example 5 72 50 60
Comparative example 2 68 70 40
Comparative example 3 62 54 36
Comparative example 4 65 65 56
As is clear from the data in Table 1, the dibenzocyclooctyne derivative-modified liposomes provided by the present invention (examples 1 to 5) had an entrapment rate of 60 to 80%, a drug loading of 50 to 72%, and an average particle diameter of 30 to 70nm.
Test example 2
Surface topography testing of functional biomaterials
Testing a sample: example 1 provides a functional biomaterial
The test method comprises the following steps: the surface morphology of the functional biomaterial provided in example 1 was observed by scanning electron microscopy, and the test results are shown in fig. 1.
As shown in FIG. 1, in the scanning electron microscope image of the functional biomaterial provided in example 1, it can be seen that the fibers in the functional biomaterial are longitudinally and orderly arranged.
Test example 3
Tensile Strength test
Testing a sample: examples 1 to 5 and comparative examples 1 to 4 provide functional biomaterials
The test method comprises the following steps: the functional biomaterial was subjected to a tensile test in a universal material testing machine, the tensile strength of the sample was tested, and the test results are shown in table 2:
TABLE 2
Test specimen Tensile Strength (MPa)
Example 1 277
Example 2 230
Example 3 280
Example 4 198
Example 5 292
Comparative example 1 232
Comparative example 2 210
Comparative example 3 262
Comparative example 4 294
As is clear from the data in Table 2, the functional biomaterials provided by the present invention (examples 1-5) had a tensile strength of 198-292MPa.
As shown in fig. 2, the tensile curve of the functional biomaterial provided in example 1 revealed that the tensile strength of the functional biomaterial provided in example 1 was 277MPa.
Test example 4
Animal experiments
Testing a sample: examples 1-5 and comparative examples 1-4 provide functional biomaterials
Establishing a rat thoracic section 2mm full-transverse spinal cord injury model: selecting 50 female adult SD rats with the body weight of 200 + -25 g, and dividing into 10 groups of 5 rats; after the pentobarbital sodium is injected into the abdominal cavity for anesthesia, the prone position is taken, the lumbar and the back of a rat are unhaired, the rat is disinfected, T7-T8 skin and subcutaneous tissues are cut, muscles beside spinous processes on two sides are separated, the muscles beside the spinous processes are pulled open by a draw hook, and the T7-T8 spinous processes and vertebral plates are exposed. The small needle holder bites off the spinous process, and then the left vertebral plate is cut off, the inner side of the left vertebral plate passes through the midline, and the outer side of the left vertebral plate reaches the inner side of the articular process until the half spinal cord and the posterior median vein of the spinal cord are exposed. The dura mater is longitudinally opened along the posterior midline by a microsciscope, a microscisco-retractor and a microscisco. The spinal cord was transected and approximately 2mm long. After hemostasis, each group was filled with the functional biomaterials provided in examples 1-5 and comparative examples 1-4, respectively, and injured rats without implanted materials were used as control groups.
Evaluation of Effect of regenerating rat posterior nerve
The test method comprises the following steps: 7 days after transplantation, nestin was recognized using GeneTex antibody GTX 630201; after 1 month of transplantation, NF-positive neurons were recognized by using antibody MAB1651 from Merck.
As shown in fig. 3A, 3B and 3C, by performing a staining analysis on the rats 7 days after the transplantation, it was found that Nestin was more present at the spinal cord injury site using the functional biomaterial provided in example 1 (fig. 3C) than the functional biomaterial provided in the control group (fig. 3A) and comparative example 1 (fig. 3B); nestin is a protein of the intermediate filament type, can be specifically expressed on a molecular marker of a neuroepithelial stem cell, and can play a role in the differentiation of neurons; the appearance of positive neural stem cells indicates that the functional biomaterial provided in example 1 has the effect of promoting the migration of endogenous stem cells to the injury site after spinal cord injury.
As shown in fig. 4A, 4B and 4C, by performing a staining analysis on the rats 1 month after transplantation, it was found that the injured site of the rats using the functional biomaterial provided in example 1 (fig. 4C) had more NF-positive neurons than the functional biomaterial provided in the control group (fig. 4A) and the comparative example 1 (fig. 4B), which laid the foundation for nerve bridging after spinal cord injury.
(II) behavioral anthropological examination of rats
The test method comprises the following steps: all rats were subjected to behavioral tests 1-12 weeks after transplantation and scored using the BBB scoring scale method, on a scale of 0-21, for a total of 22. The 21 score was normal in function and the 0 score was complete loss of function. The BBB scoring adopts a single blind detection method, the behavior of the injured lower limb, namely the left lower limb, of the animal on the open ground is observed, the scoring is carried out according to a scoring standard, the observation time is 4 minutes, and the test result is shown in a table 3:
TABLE 3
Figure BDA0003412851890000201
As can be seen from the data in Table 3, the BBB scores of the rats transplanted with the functional biomaterials provided by the present invention (examples 1-5) were 3-4,8 and 6-6.67 after 4 weeks, and 6-8 after 12 weeks.
As can be seen from a comparison of example 1 and example 5, DBCO-DSPE-PEG2k was replaced with DBCO-
Figure BDA0003412851890000202
After MAL, the effect of functional biomaterials is affected.
It can be seen from a comparison of example 1 and comparative example 1 that when the functional biomaterial is only an ordered collagen, recovery of motor function in rats is poor.
As shown in FIG. 5, the BBB score of the rats transplanted with the functional biomaterial provided in example 1 was significantly better than that of the rats transplanted with the functional biomaterial provided in comparative example 1 and the rats of the control group.
It is understood from the comparison between example 1 and comparative examples 2-3 that no covalent bond is formed between the azide-derivative-modified ordered collagen and the dibenzocyclooctyne-derivative-modified liposome, which affects the performance of the functional biomaterial.
It can be seen from the comparison between example 1 and comparative example 4 that the recovery of motor function in rats is affected when the cholesterol ester synthase inhibitor Avasimibe is replaced with paclitaxel of the same weight.
The applicant states that the present invention is illustrated by the above examples to a functional biomaterial and its preparation method and application, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must rely on the above examples to be carried out. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims (43)

1. A functional biomaterial capable of regulating and controlling a nerve regeneration microenvironment to promote spinal cord injury repair, which is characterized in that the functional biomaterial comprises ordered collagen modified by azide derivatives and a liposome modified by dibenzocyclooctyne derivatives loaded on the ordered collagen modified by the azide derivatives; a cholesterol ester synthase inhibitor is entrapped in the liposome;
the azide derivative modified ordered collagen and the dibenzocyclooctyne derivative modified liposome are connected through a triazole bond formed by the azide derivative and the dibenzocyclooctyne derivative;
the liposome modified by the dibenzocyclooctyne derivative comprises soybean phospholipid and cholesterol;
the functional biological material is filaments arranged in bundles;
the azide derivative comprises azide-, (N-hydroxysuccinimide) and/or azido-polyethylene glycol-, (N-hydroxysuccinimide);
the dibenzocyclooctyne derivatives comprise dibenzocyclooctyne-hydrophobic polymer-polyethylene glycol;
the hydrophobic polymer in the dibenzocyclooctyne-hydrophobic polymer-polyethylene glycol comprises any one or the combination of at least two of distearoylphosphatidylethanolamine, distearoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine or dipalmitoylphosphatidylcholine.
2. The functional biomaterial of claim 1, wherein the cholesterol ester synthase inhibitor is a cholesterol acyltransferase inhibitor.
3. The functional biomaterial of claim 2, wherein the cholesterol ester synthase inhibitor is Avasimibe.
4. The functional biomaterial of claim 1, wherein each bundle of functional biomaterial has a diameter of 2-4mm and a length of 8-15 cm.
5. The functional biomaterial of claim 4, wherein each bundle of functional biomaterial comprises 20-400 filaments.
6. The functional biomaterial of claim 1, wherein the filaments have a diameter of 10-100 μm and a length of 8-15 cm.
7. The functional biomaterial of claim 1, wherein the azide-modified ordered collagen accounts for 90-99% of the total mass of the functional biomaterial.
8. The functional biomaterial of claim 1, wherein the azide derivative accounts for 10-30% of the total mass of the azide derivative-modified ordered collagen.
9. The functional biomaterial of claim 1, wherein the azide-polyethylene glycol-,(s) (i) is/areN-hydroxysuccinimide) the polyethylene glycol has a number average molecular weight of 2000-5000.
10. The functional biomaterial of claim 1, wherein the dibenzocyclooctyne derivative-modified liposome comprises 1 to 10% of the total mass of the functional biomaterial.
11. The functional biomaterial of claim 1, wherein the dibenzocyclooctyne derivative comprises 10-20% of the total mass of the dibenzocyclooctyne derivative-modified liposome.
12. The functional biomaterial of claim 1, wherein the number average molecular weight of the polyethylene glycol in the dibenzocyclooctyne-hydrophobic polymer-polyethylene glycol is 2000-5000.
13. The functional biomaterial according to claim 1, wherein the mass ratio of the soybean phospholipids to the cholesterol is (2-5): 1.
14. The functional biomaterial of claim 1, wherein the particle size of the dibenzocyclooctyne derivative-modified liposome is 30 to 70nm.
15. The functional biomaterial of claim 1, wherein the drug loading of the dibenzocyclooctyne derivative-modified liposome is 60 to 80%.
16. The functional biomaterial of claim 1, wherein the encapsulation efficiency of the dibenzocyclooctyne derivative-modified liposome is 50 to 70%.
17. A method for preparing a functional biomaterial according to any one of claims 1-16, comprising the steps of: mixing the ordered collagen modified by the azide derivative and the liposome modified by the dibenzocyclooctyne derivative which is coated with the cholesterol ester synthetase inhibitor, and reacting to obtain the functional biological material;
the obtained functional biological material is bound into bundles, each bundle comprises 20-400 of the functional biological material.
18. The method for preparing functional biomaterial according to claim 17, wherein the reaction temperature is 20-35 ℃ and the reaction time is 10-15 h.
19. The method for preparing functional biomaterial according to claim 17, wherein the method for preparing the azide-derivative-modified ordered collagen comprises the following steps: dissolving the azide derivative and the ordered collagen in a buffer solution, and reacting to obtain the ordered collagen modified by the azide derivative.
20. The method for preparing functional biomaterial according to claim 19, wherein the buffer solution comprises phosphate buffer, and the pH of the buffer solution is 6-8.
21. The method for preparing functional biomaterial according to claim 19, wherein the concentration of the azide derivative in the mixed solution obtained after dissolution is 1-10mg/mL, and the concentration of the ordered collagen is 1-5 g/mL.
22. The method for preparing functional biomaterial according to claim 19, wherein the reaction temperature is 35-39 ℃ and the reaction time is 3.5-4.5 h.
23. The method for preparing functional biomaterial according to claim 19, wherein the ordered collagen modified by azide derivative is obtained by washing.
24. The method for preparing functional biomaterial according to claim 23, wherein the solvent used for washing is water, the number of washing is 2-5, and the time for each washing is 8-15 min.
25. The method for preparing functional biomaterial according to claim 19, wherein the ordered collagen is prepared by the following preparation method: and sequentially carrying out coarse purification treatment, enzymolysis treatment, nucleic acid removal treatment, organic solvent treatment, detergent treatment, surfactant treatment, freezing and freeze-drying on the membrane tendon tissue to obtain the ordered collagen.
26. The method of claim 25, wherein the coarse purification comprises removal of muscle and solid adipose tissue.
27. The method of claim 25, wherein the water treatment is performed after the coarse purification treatment, the number of water treatments is 2-5, and the time for each water treatment is 12-17 min.
28. The method for preparing functional biomaterial according to claim 25, wherein the protease used in the enzymatic hydrolysis treatment comprises papain, and the enzyme activity of the protease is 200-400U/g; the buffer solution adopted by the enzymolysis treatment comprises a phosphate buffer solution, and the pH value of the buffer solution is 5.5-6.5; the treatment time of the enzymolysis treatment is 0.1-1 h.
29. The method for preparing functional biomaterial according to claim 25, wherein the enzymes used in the nucleic acid removal treatment comprise RNase a and DNase I, and the enzyme activity of the enzymes is 80 to 120U/g; the time of the nucleic acid removing treatment is 2.5-3.5 h.
30. The method for preparing functional biomaterial according to claim 25, wherein the organic solvent used in the organic solvent treatment comprises n-heptane and ethanol, the volume ratio of n-heptane to ethanol is (0.5-1.5): 2, and the time of the organic solvent treatment is 20-30 h.
31. The method for preparing functional biomaterial according to claim 25, wherein the organic solvent treatment is followed by water treatment, the number of the water treatments is 2-5, and the time for each water treatment is 15-25 min.
32. The method for preparing functional biomaterial according to claim 25, wherein the detergent used in the detergent treatment comprises an aqueous solution of sodium deoxycholate, the mass content of the aqueous solution of sodium deoxycholate is 2-6%; the time of the detergent treatment is 1-3 h;
and after the detergent treatment, water treatment is carried out, the water treatment is carried out for 4-7 times, and the time of each water treatment is 8-15 min.
33. The method for preparing the functional biomaterial according to claim 25, wherein the surfactant used for the surfactant treatment comprises an aqueous solution of sodium dodecyl sulfate, and the mass content of the aqueous solution of sodium dodecyl sulfate is 0.1-0.3%; the time of the surfactant treatment is 0.1-1 h.
34. The method for preparing functional biomaterial according to claim 25, wherein the surfactant treatment is followed by water treatment, the number of the water treatment is 4-7, and the time for each water treatment is 8-15 min.
35. The method for preparing functional biomaterial according to claim 25, wherein the freezing temperature is-90 to-70 ℃ and the freezing time is 0.8 to 1.5 h.
36. The method for preparing functional biomaterial according to claim 25, wherein the pressure for lyophilization is 0.05-0.15mbar and the time is 20-30 h.
37. The method for preparing functional biomaterial according to claim 17, wherein the dibenzocyclooctyne derivative-modified liposome is prepared by a thin film dispersion method.
38. The method for preparing functional biomaterial according to claim 37, wherein the method for preparing the dibenzocyclooctyne derivative-modified liposome comprises the following steps: dissolving soybean phospholipid, cholesterol and dibenzocyclooctyne derivatives in an organic solvent, and concentrating to obtain an intermediate; mixing the obtained intermediate, cholesterol ester synthetase inhibitor and buffer solution to obtain the liposome modified by the dibenzocyclooctyne derivative;
and the ultrasonic treatment and the filtration are sequentially carried out after the mixing.
39. The method for preparing functional biomaterial according to claim 38, wherein the organic solvent comprises chloroform and methanol.
40. The method for preparing a functional biomaterial according to claim 39, wherein the volume ratio of chloroform to methanol is (1.5-2.5): 1.
41. The method for preparing functional biomaterial according to claim 38, wherein the power of the ultrasound is 250-350W and the time is 15-25 min.
42. The method for preparing functional biomaterial according to claim 38, wherein the filtration membrane used for the filtration is 0.18-0.30 μm.
43. Use of a functional biomaterial according to any one of claims 1-16 for the preparation of a material for promoting nerve regeneration after spinal cord injury.
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