CN114652898A - Hydrophilic negative electricity porous nano-film for chronic nephropathy restoration and preparation method and application thereof - Google Patents
Hydrophilic negative electricity porous nano-film for chronic nephropathy restoration and preparation method and application thereof Download PDFInfo
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- CN114652898A CN114652898A CN202210302002.0A CN202210302002A CN114652898A CN 114652898 A CN114652898 A CN 114652898A CN 202210302002 A CN202210302002 A CN 202210302002A CN 114652898 A CN114652898 A CN 114652898A
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- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
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Abstract
The invention discloses a hydrophilic negative electricity porous nano-film for repairing chronic nephropathy and a preparation method and application thereof; the method takes sucrose and L-lactide as reactants to synthesize an 8-arm star-shaped polylactic acid element with carboxylated tail ends, and prepares a negative electricity porous nano-film with the aperture of 23.5 +/-4.5 nm by self-assembly with the element; by utilizing amide reaction between carboxyl and amino, polyethylene glycol (PEG) and glomerular specific antibody (Ab) are successfully modified on the surface of the film in sequence, so that the film has good hydrophilicity and kidney targeting property; the pore diameter of the hydrophilic negative electricity porous nano-film for repairing chronic nephropathy is 19-28 nm. The hydrophilic negative electricity porous nano-film prepared by the invention can simulate the filtration barrier of glomeruli, directly repair the damaged glomerular filtration membrane of a rat and achieve the effect of treating chronic nephropathy.
Description
Technical Field
The invention belongs to the technical field of preparation of biological functional materials, and particularly relates to a hydrophilic negative electricity porous nano-film for repairing chronic nephropathy and a preparation method and application thereof.
Background
With the increase of work and life pressure and the increase of bad living habits of people, the global incidence rate of chronic kidney diseases is increasing year by year, and the chronic kidney diseases become one of the global public health problems. When chronic kidney disease progresses to the end of renal failure, patients can only rely on hemodialysis or even kidney transplantation to sustain life, which also means that the patients need to pay expensive treatment costs. Studies have shown that the underlying cause of chronic kidney disease is a decrease in glomerular filtration rate caused by glomerular injury. Among them, the structural change of the glomerular filtration barrier is mainly manifested by decrease of endothelial cell glycoprotein, thickening of basement membrane, decrease of podocyte number and disappearance of charge barrier, thereby leading to the protein in blood being filtered out. The current clinical method for treating chronic nephropathy is mainly to inhibit the renin-angiotensin-aldosterone system and kidney fibrosis through drugs, and control hypertension, acid-base balance, vitamin D deficiency and the like. However, the above treatment methods often only have the effect of delaying nephropathy, have poor treatment effects and obvious side effects, and cannot fundamentally repair the damaged glomerular filtration membrane.
In recent years, the nano material has great potential in development and application of nephropathy treatment due to the characteristics of controllable morphology and property, diversified surface modification, good biocompatibility and the like. CN103933615A discloses a self-assembled porous polymer membrane for repairing a glomerular filtration membrane, but the preparation method is complex, the surface of the membrane is hydrophobic, and thrombus is easily caused.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a hydrophilic negative electricity porous nano-film for repairing chronic nephropathy and a preparation method and application thereof.
According to the method, the octahydroxyl sucrose with low cost is used as a reactant, so that the synthesis steps are simplified, and on the basis, the surface hydrophilic and hydrophobic modification is carried out on the material, so that the hydrophilic negative electricity porous nano-film with smaller pore diameter (23.5 +/-4.5 nm) is synthesized, the hydrophilicity and the biocompatibility are improved, the mechanical barrier and the charge barrier of the glomerular filtration membrane can be simulated, the damaged glomerular filtration membrane can be directly repaired, and the renal functions of albumin mice and adriamycin nephrosis mice can be effectively improved.
The purpose of the invention is realized by the following technical scheme:
the hydrophilic negative electricity porous nano-film for repairing the chronic kidney disease has the pore diameter of 19-28 nm; the hydrophilic negative electricity porous nano film for repairing the chronic kidney disease is formed by connecting polyethylene glycol (PEG) and a glomerular specific antibody Ab on a negative electricity porous nano film. The hydrophilic negative electricity porous nano film for repairing chronic kidney diseases has good biocompatibility, can simulate the mechanical barrier and the charge barrier of a glomerular filtration membrane, reduces the occurrence of proteinuria, and fundamentally treats the chronic kidney diseases.
Preferably, the negative electricity porous nano film is prepared by self-assembly of a terminal carboxylated 8-arm star-shaped polylactic acid motif.
Preferably, the preparation method of the end-carboxylated 8-arm star-shaped polylactic acid motif comprises the following steps:
(1) adding stannous isooctanoate as catalyst into sucrose and L-lactide, introducing inert gas for 20-40 min to remove air in the reaction system, sealing, and reacting at 80-120 deg.C for 20-30 hr while stirring to obtain 8-arm star-shaped polylactic acid G with hydroxyl at the end1-(OH)8；
(2) G obtained in the step (1)1-(OH)8Dissolving with dichloromethane and precipitating with methanol, respectively, collecting the product by suction filtration, repeating for 2-5 times, vacuum drying at 30-50 deg.C for 5-10 hr to obtain G1-(OH)8White powder;
(3) g obtained in the step (2)1-(OH)8Dissolving white powder in anhydrous 1, 4-dioxane, adding reactant methacrylic anhydride and catalyst 4-dimethylaminopyridine and 3-ethylamine, stirring at normal temperature for reaction for 12-24 hours to obtain the product with the tail end8-arm star-shaped polylactic acid G with methacrylic acid double bond2-(methacrylate)8；
(4) G obtained in the step (3)2-(methacrylate)8Dissolving with dichloromethane and precipitating with diethyl ether, respectively, collecting the product by suction filtration, repeating for 2-5 times, vacuum drying at 30-50 deg.C for 5-10 hr to obtain G2-(methacrylate)8Light yellow powder;
(5) g obtained in the step (4)2-(methacrylate)8Dissolving the light yellow powder in N, N-dimethylformamide, adding reactants of thiomalic acid and photosensitizer benzoin dimethyl ether, stirring and reacting for 12-24 hours at 40-50 ℃ under the irradiation of ultraviolet light to obtain 8-arm star-shaped polylactic acid G with the terminal of carboxyl3-(COOH)16；
(6) G obtained in the step (5)3-(COOH)16Dissolving with dichloromethane and precipitating with methanol, respectively, collecting the product by suction filtration, repeating for 2-5 times, vacuum drying at 30-50 deg.C for 5-10 hr to obtain terminal carboxylated 8-arm star-shaped polylactic acid elementary G3-(COOH)16White powder.
Further preferably, the molar ratio of the sucrose to the stannous isooctanoate in the step (1) is 1: 0.002-0.01; the molar ratio of the sucrose to the L-lactide is 1: 50-100 parts of; the inert gas is nitrogen;
further preferably, G in step (3)1-(OH)8The molar ratio to methacrylic anhydride was 1: 3-8; the G is1-(OH)8The molar ratio of 4-dimethylaminopyridine to 4-dimethylaminopyridine is 1: 1-3; the G is1-(OH)8The mol ratio of the compound to 3-ethylamine is 1: 1-3;
further preferably, G in the step (5)2-(methacrylate)8The molar ratio of the compound to the thiomalic acid is 1: 2-5; the G is2-(methacrylate)8The mol ratio of the benzoin dimethyl ether to the benzoin dimethyl ether is 1: 0.5 to 2; the wavelength of the ultraviolet light is 350-400 nm.
Preferably, the self-assembly preparation process of the negative electricity porous nano film comprises the following steps:
dissolving the end-carboxylated 8-arm star-shaped polylactic acid element in tetrahydrofuran, starting magnetic stirring, dropwise adding glycerol at the temperature of 30-80 ℃ at the speed of 2-5 drops/second, quenching with liquid nitrogen, exchanging solvent in distilled water, and finally performing suction filtration and freeze drying to obtain the negative electricity porous nano film.
Further preferably, the mass-to-volume ratio of the terminally carboxylated 8-arm star-shaped polylactic acid motif to tetrahydrofuran is 10-20: 5-15 mg/ml; the volume mass ratio of the glycerol to the terminal carboxylated 8-arm star-shaped polylactic acid motif is 10-20: 10-20 ml/mg; the volume-mass ratio of the distilled water to the end-carboxylated 8-arm star-shaped polylactic acid unit is 500-800: 10-20 ml/mg.
The preparation method of the hydrophilic negative electricity porous nano-film for repairing chronic kidney disease is characterized by comprising the following steps:
(a) dispersing the negative electricity porous nano film into a PB buffer solution with the pH value of 5-6, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide EDC and N-hydroxysuccinimide NHS, reacting for 1-2 hours at room temperature, centrifuging, collecting precipitates, re-dispersing by using the PB buffer solution with the pH value of 7-8, adding PEG, reacting for 8-15 hours at room temperature, centrifuging, washing for 3-5 times by using deionized water, and freeze-drying to obtain a hydrophilic negative electricity porous nano film;
(b) dispersing the hydrophilic negative electricity porous nano film obtained in the step (a) in a PB buffer solution with the pH value of 5-6, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide EDC and N-hydroxysuccinimide NHS, reacting for 1-2 hours at room temperature, centrifuging to collect precipitates, re-dispersing by using a PB buffer solution with the pH value of 7-8, adding an Ab solution, reacting for 8-15 hours at room temperature, centrifuging, washing for 3-5 times by using deionized water, and freeze-drying to obtain the hydrophilic negative electricity porous nano film for repairing chronic nephrosis.
Preferably, in the step (a), the mass-to-volume ratio of the electronegative porous nano-film to the PB buffer solution is 10-20: 5-10 mg/ml; the mass ratio of the 1-ethyl- (3-dimethylamino-C) methylamine to the electronegative porous nano film is 20-30: 10-20 parts of; the mass ratio of the N-hydroxysuccinimide to the negative electricity porous nano film is 2-3: 10-20 parts of; the mass ratio of the PEG to the negative electricity porous nano film is 12-18: 10-20.
Preferably, in the step (b), the mass-to-volume ratio of the hydrophilic negative electricity porous nano film to the PB buffer solution is 10-20: 5-10 mg/ml; the mass ratio of the 1-ethyl- (3-dimethylamino-C) carbodiimide to the hydrophilic negative electricity porous nano film is 20-30: 10-20 parts of; the mass ratio of the N-hydroxysuccinimide to the hydrophilic negative electricity porous nano film is 2-3: 10-20 parts of; the volume mass ratio of the Ab solution to the negative electricity porous nano film is 200: 10-20 μ L/mg; the mass concentration of the Ab solution is 1-2%.
The hydrophilic negative electricity porous nano-film for repairing chronic kidney diseases is applied to the preparation of a chronic kidney disease repairing reagent.
Dispersing the hydrophilic negative electricity porous nano film with the surface modified with PEG and Ab in physiological saline, and injecting the hydrophilic negative electricity porous nano film into an adriamycin model mouse body through intravenous injection; the contents of urine protein and blood creatinine of the mice are tracked and monitored, and the treatment effect of the material on the functions of the glomerular filtration membrane and the kidney is evaluated.
The invention has the beneficial effects that:
(1) the negative electricity porous nano film prepared by the invention is prepared by taking sucrose containing octahydroxyl as a reactant, preparing star-shaped polylactic acid with the terminal carboxyl number of 8 through ring-opening polymerization reaction and sulfydryl-methacrylic double bond click reaction, and finally performing self-assembly, wherein the surface of the film contains a large number of carboxyl groups, so that the surface modification is easy to perform, and the method has the advantages of lower raw material price, simple and clear route, convenience for synthesis and better repeatability;
(2) the surface of the hydrophilic negative electricity porous nano film prepared by the invention is modified with polyethylene glycol (PEG), so that the hydrophilicity of the material can be effectively improved, nonspecific protein adsorption or deposition on a biomaterial-blood interface is reduced, the biocompatibility, the blood circulation time and the utilization rate of the material are enhanced, and thrombosis after intravenous injection of a mouse is avoided;
(3) the prepared podocyte-targeted hydrophilic negative electricity porous nano film has the pore diameter (23.5 +/-4.5 nm) smaller than that of a glomerular fissure pore diaphragm (40nm), has the surface with electronegativity, can simulate the mechanical barrier and the charge barrier of the glomerular filtration membrane, effectively reduces the generation of albuminuria of an albumin model rat, reduces the ratio of urine protein to creatinine of an adriamycin nephropathy model, and is expected to become an injectable potential medicament for treating nephropathy.
Drawings
Fig. 1 is a scanning electron microscope image of the negatively charged porous nanomembranes before and after the modification of PEG and Ab and the hydrophilic negatively charged porous nanomembranes targeted by podocytes prepared in example 1 of the present invention, and a pore size distribution and a water contact angle image thereof.
FIG. 2 is Zeta potential diagram of the hydrophilic porous nano-film prepared in example 1 of the present invention.
Fig. 3 is a cytotoxicity plot of different concentrations of podocyte-targeted hydrophilic porous nanofilms of example 2 of the present invention.
Fig. 4 is an organ toxicity diagram of the negatively charged porous nanomembrane and the podocyte-targeted porous nanomembrane before and after PEG and Ab modification in example 3 of the present invention.
FIG. 5 is a graph showing the results of urine protein changes in a rat model of albumin nephropathy after intervention of the podocyte-targeted hydrophilic negative-charge porous nano-film of the present invention.
FIG. 6 is a graph showing the results of the change in the ratio of urine protein to creatinine in a mouse model of podocyte-targeted hydrophilic negative-charge porous nano-film dry prognosis doxorubicin nephropathy according to example 5 of the present invention.
Detailed Description
The following description of the embodiments of the present invention is provided in connection with the accompanying drawings and examples, but the invention is not limited thereto. It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.
Example 1
(1) Adding 0.125mmol of sucrose and 0.1mol of L-lactide into a round-bottom flask, adding 1mmol of catalyst stannous isooctanoate, introducing nitrogen for 40 minutes to remove air in the reaction flask, sealing, and stirring at 100 ℃ for 24 hours to obtain 8-arm star-shaped polylactic acid (G) with a hydroxyl end1-(OH)8)；
(2) Subjecting the above to obtain G1-(OH)8Dissolving with dichloromethane and precipitating with methanol respectively, collecting the product by suction filtration, repeating for 3 times, vacuum drying at 45 deg.C for 8 hr to obtain G1-(OH)8A white powder;
(3) 1mmol of G1-(OH)8Dissolving white powder in anhydrous 1, 4-dioxane, adding 2mmol of methacrylic anhydride, 2mmol of 4-dimethylaminopyridine and 2mmol of 3-ethylamine, and reacting at room temperature under vigorous stirring for 24 hr to obtain 8-arm star-shaped polylactic acid (G) with terminal methacrylic double bond2-(methacrylate)8)；
(4) G is to be2-(methacrylate)8Dissolving with dichloromethane and precipitating with diethyl ether, respectively, collecting the product by suction filtration, repeating for 3 times, and vacuum drying at 45 deg.C for 8 hr to obtain G2-(methacrylate)8Light yellow powder;
(5) 1mmol of G2-(methacrylate)8Dissolving light yellow powder in N, N-dimethylformamide, adding 4mmol of thiomalic acid and 1mmol of benzoin dimethyl ether, stirring at 45 deg.C under 365nm ultraviolet lamp for 12 hr to obtain 8-arm star-shaped polylactic acid (G) with carboxyl at end3-(COOH)16)；
(6) G is to be3-(COOH)16Dissolving with dichloromethane and precipitating with methanol, respectively, collecting the product by suction filtration, repeating for 3 times, vacuum drying at 45 deg.C for 8 hr to obtain terminal carboxylated 8-arm star-shaped polylactic acid elementary G3-(COOH)16A white powder;
(7) 15mg of G3-(COOH)16Dissolving the white powder in 10ml tetrahydrofuran, stirring vigorously, adding 15ml glycerol dropwise at 50 deg.C at a speed of 4 s/drop, quenching with liquid nitrogen, exchanging solvent in 500ml distilled water, filtering, and lyophilizing to obtain negatively charged porous nano-film;
(8) dispersing 10mg of negative electricity porous nano film in 5ml of phosphate buffer solution (PB, pH5.0), adding 20mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) and 2mg of N-hydroxysuccinimide (NHS), reacting at room temperature for 2 hours, centrifuging, collecting precipitate, re-dispersing by using PB buffer solution with pH7.4, adding 15mg of PEG, reacting at room temperature for 12 hours, centrifuging, washing for 3 times by using deionized water, and freeze-drying to obtain the hydrophilic negative electricity porous nano film;
(9) dispersing 10mg of hydrophilic negative electricity porous nano-film into 5ml of PB buffer solution with the pH value of 5.0, adding 20mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) and 2mg of N-hydroxysuccinimide (NHS), reacting at room temperature for 2 hours, centrifuging, collecting precipitates, re-dispersing by using PB buffer solution with the pH value of 7.4, adding 200 microliter of 1% Ab solution, reacting at room temperature for 12 hours, centrifuging, washing by using deionized water for 3 times, and freeze-drying to obtain the podocyte-targeted hydrophilic negative electricity porous nano-film.
The results of the negative electricity porous nano-film (a and C in fig. 1) prepared in step (7) and the hydrophilic negative electricity porous nano-film (B and D in fig. 1) prepared in step (9) in this example are shown in fig. 1, and can be obtained from a scanning electron micrograph and a pore diameter statistical chart, and with the modification of PEG and Ab, the pore diameter of the hydrophilic negative electricity porous nano-film is reduced from 27.6 ± 6.3nm to 23.5 ± 4.5nm, which are both smaller than that of a glomerular fissure pore diaphragm (40 nm); furthermore, the hydrophilicity of the material surface was significantly improved (from 93 ° down to 37 °) as can be seen from the water contact angle. The results of Zeta potential are shown in FIG. 2, the surface of the nano-film is negative, and the Zeta potential is-47 mV, because the surface is modified by Ab protein with a large amount of anions.
Example 2
Podocytes (purchased from ATCC in USA) were grown to a confluence of at least 80% and trypsinized to 5 x 104And (2) planting 100ul of the cell suspension in a 96-well plate, continuously culturing for 24h, changing to a serum-free basic culture medium, culturing for 6 h to synchronize the cell state, configuring the podocyte targeted hydrophilic negative electricity porous nano-film prepared in the example 1 into different concentrations (0.1mg/ml, 1mg/ml, 2mg/ml, 5mg/ml and 10mg/ml) by using the basic culture medium, and dropwise adding 10ul of the cell targeted hydrophilic negative electricity porous nano-film into the 96-well plate, wherein each concentration is provided with 3 multiple wells. And a normal control group without material intervention and a basal medium blank group without cells are added for comparison. After culturing the cells in each well for 24 hours,10ul cck-8 reagent is dripped in the mixture, the absorbance of the 450nm wavelength is measured by an enzyme-linked immunosorbent assay (ELIAS) reader after 1h, and the experiment is repeated for 3 times. The results are shown in FIG. 3, and as the concentration of the material increased, the activity of the cells did not change significantly. The cell activity of the 10mg/ml material group was 91.4% compared to the control group without material. The result indicates that the obtained podocyte-targeted hydrophilic negative electricity porous nano film has excellent biocompatibility.
Example 3
BALB/c mice were accurately weighed and randomly divided into 3 groups of 5 mice each, a control group, a group of negative charged porous nanoplates of unmodified PEG (prepared according to step (7) of example 1) and a group of podocyte-targeted hydrophilic negative charged porous nanoplates of modified PEG of example 1, and the mice were injected with physiological saline, negative charged porous nanoplates of unmodified PEG (30mg/kg) and hydrophilic negative charged porous nanoplates of modified PEG (30mg/kg) through the tail vein, respectively. All mice in the unmodified PEG group have different degrees of suffocating asthma, asphyxia and other manifestations within one minute after injection, and lung thrombosis is prompted; the vital signs of the control group and the PEG-modified group mice were normal, all mice were sacrificed immediately and their lungs and liver were collected, and H & E sections of the organs were prepared, and finally pathological changes of the organs were observed under a microscope. The results are shown in figure 4, compared with the control group, the unmodified PEG group mice have alternate red and white lung lobe colors due to pulmonary embolism and hepatic congestion, which indicates that the blood perfusion at the embolism part is incomplete, and the liver color is deep, which indicates that the perfusion effect is poor; h & E staining showed thrombus formation in liver and lung capillaries (arrows) in unmodified PEG group mice. The blood perfusion effect of the mice in the modified PEG material group is good, the color of the lung lobes is uniform and white, the color of the liver is uniform, and the color of the liver is not obviously different from that of the control group, so that the smooth perfusion and the non-extravasated blood embolism are prompted; h & E staining shows that the liver and lung are normal, and the difference with a control group is not obvious, which indicates that no obvious lesion exists.
Example 4
In order to simulate the nephropathy caused by a large amount of clinical proteinuria, an albumin nephropathy model is prepared to be used as an experimental animal model. The process is as follows: 18 SPF SD rats are randomly divided into 3 groups and intervened in different modes, wherein the first group is a sham operation group, the abdomen is opened and closed after a renal membrane is stripped, and the rats are fed with normal diet; the second group is albumin model group, after one kidney is removed by operation, 15mg/kg albumin is injected into the abdomen every day, and the animal is fed with normal diet; the third group was an experimental group, in which after one kidney was surgically removed, 15mg/kg of albumin was injected into the abdomen every day, food and water were administered, and from the third day, 10mg/kg of body weight of the podocyte-targeted hydrophilic negative-charge porous nanomembrane prepared in example 1 was injected into the tail vein every four days. All rats were kept in urine for 24 hours a week starting from the day of model creation for 6 weeks, and the total protein content in the urine was measured by coomassie blue color development as follows:
(1)10mg of bovine serum albumin was diluted with 10ml of pure water to a 1mg/ml standard protein solution;
(2) respectively sucking 2ul, 4ul, 6ul, 8ul, 10ul and 12ul of standard protein solution, adding into Coomassie brilliant blue solution, and mixing to obtain a final volume of 800 ul;
(3) adding a proper volume of urine sample into Coomassie brilliant blue solution, and mixing uniformly to obtain a final volume of 800 ul;
(4) sucking 200ul of each tube and adding into a 96-well plate;
(5) measuring OD values of the standard protein solution and the sample solution at 595nm by using an ultraviolet spectrophotometer method;
(6) and (4) fitting a standard Curve by using standard Curve software Curve Expert 1.3, calculating the protein concentration of the sample according to the OD value of the sample solution, and multiplying the OD value by the urine amount of 24h to obtain the urine protein quantification of 24 h.
The experimental result is shown in fig. 5, and as can be seen from the line graph, the kidney of the rat in the pseudo-operation group is not obviously damaged, and no urine protein leaks out, which indicates that the pseudo-operation of the rat is successful; the urine protein of the albumin model group mice begins to rise at one week and reaches the peak (400-600mg/d) at 24 days and lasts for 41 days, which indicates that the model building of the experimental model is successful; the proteinuria of the rats in the experimental group fluctuated at a low level (100mg/d) all the time in the experimental period of 7 to 14 days after the administration of the hydrophilic negative electricity porous nano-film, suggesting a decrease in the urine protein, compared to the model group. The result indicates that the podocyte-targeted hydrophilic negative electricity porous nano film can effectively prevent the albumin of the rat with the disease from leaking out on the premise of ensuring the biocompatibility, and the improvement and the repair of the kidney function of the rat with the albumin are realized.
Example 5
In order to simulate the clinical glomerular filtration membrane injury, an adriamycin nephropathy model is prepared as an experimental animal model, and the process is as follows: preparing 18 SPF (specific pathogen free) grade C57BL/6 mice, randomly dividing the mice into 3 groups, and performing intervention in different modes, wherein the first group is an NS group, and the normal diet observation life index is performed after single tail vein injection of 12mg/kg body weight normal saline; the second group is an adriamycin model group, and the life index of normal diet observation is carried out after single injection of 12mg/kg body weight adriamycin; the third group is an experimental group, which is a normal diet after a single injection of 12mg/kg body weight of doxorubicin, and a tail vein injection of 10mg/kg body weight of the podocyte-targeted hydrophilic negative-charge porous nano-film prepared in example 1 is started every 1 day after the doxorubicin kidney disease model is stabilized on day 21. All mice were left with 24 hour urine weekly for 5 weeks starting on the molding day. In order to reflect the change of the kidney function of the mice more truly, the experimental data takes the urinary creatinine of the mice as correction, and the ratio change of the urinary creatinine and the albumin of each group of mice is calculated and counted.
The experimental result is shown in fig. 6, and it can be seen from the line graph a that the urine protein creatinine ratio of the mice in the experimental group is obviously improved compared with the adriamycin model group after the podocyte-targeted hydrophilic negative-electricity porous nano-film is subjected to dry treatment. As shown in B in fig. 6 and C in fig. 6, the serum creatinine value of the mouse significantly decreased and the glomerular filtration rate significantly increased after the intervention of the hydrophilic negative-charge porous nano-film, while the serum creatinine value of the pathological group tended to increase and the filtration rate thereof remained at a low level. The result indicates that the podocyte-targeted hydrophilic negative electricity porous nano film can obviously improve the glomerular filtration function of a disease mouse on the premise of ensuring biocompatibility, and realizes the glomerular filtration membrane repair of an adriamycin nephropathy mouse.
The above examples of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. The hydrophilic negative electricity porous nano-film for chronic kidney disease repair is characterized in that the pore diameter of the hydrophilic negative electricity porous nano-film for chronic kidney disease repair is 19-28 nm; the hydrophilic negative electricity porous nano film for repairing the chronic kidney disease is formed by connecting polyethylene glycol (PEG) and a glomerular specific antibody Ab on a negative electricity porous nano film.
2. The hydrophilic negative-charge porous nanomembrane for chronic kidney disease repair of claim 1, wherein the negative-charge porous nanomembrane is prepared by self-assembly of a terminally carboxylated 8-arm star-shaped polylactic acid motif.
3. The hydrophilic negative-charged porous nanomembrane for chronic kidney disease repair of claim 2, wherein the method for preparing the end-carboxylated 8-arm star-shaped polylactic acid moiety comprises the following steps:
(1) adding catalyst stannous isooctanoate into reactant sucrose and L-lactide, introducing inert gas for 20-40 min to remove air in the reaction system, sealing, and stirring at 80-120 ℃ for reaction for 20-30 h to obtain 8-arm star-shaped polylactic acid G with the terminal being hydroxyl1-(OH)8；
(2) G obtained in the step (1)1-(OH)8Dissolving with dichloromethane and precipitating with methanol, respectively, collecting the product by suction filtration, repeating for 2-5 times, vacuum drying at 30-50 deg.C for 5-10 hr to obtain G1-(OH)8A white powder;
(3) g obtained in the step (2)1-(OH)8Dissolving white powder in anhydrous 1, 4-dioxane, adding reactant methacrylic anhydride, catalyst 4-dimethylaminopyridine and 3-ethylamine, stirring at normal temperature for reaction for 12-24 hours to obtain 8-arm star-shaped polylactic acid G with the end of methacrylic double bond2-(methacrylate)8；
(4) G obtained in the step (3)2-(methacrylate)8Dissolving with dichloromethane and precipitating with diethyl ether, respectively, collecting the product by suction filtration, repeating for 2-5 times, vacuum drying at 30-50 deg.C for 5-10 hr to obtain G2-(methacrylate)8Light yellow powder;
(5) g obtained in the step (4)2-(methacrylate)8Dissolving light yellow powder in N, N-dimethylformamide, adding reactants of thiomalic acid and photosensitizer benzoin dimethyl ether, stirring and reacting for 12-24 hours at 40-50 ℃ under the irradiation of ultraviolet light to obtain 8-arm star-shaped polylactic acid G with the tail end being carboxyl3-(COOH)16；
(6) G obtained in the step (5)3-(COOH)16Dissolving with dichloromethane and precipitating with methanol, respectively, collecting the product by suction filtration, repeating for 2-5 times, vacuum drying at 30-50 deg.C for 5-10 hr to obtain terminal carboxylated 8-arm star-shaped polylactic acid elementary G3-(COOH)16White powder.
4. The hydrophilic negative-electricity porous nano-film for repairing chronic kidney disease as claimed in claim 3, wherein the molar ratio of sucrose to stannous isooctanoate in step (1) is 1: 0.002-0.01; the molar ratio of the sucrose to the L-lactide is 1: 50-100 parts of; the inert gas is nitrogen;
step (3) said G1-(OH)8The molar ratio to methacrylic anhydride was 1: 3-8; the G is1-(OH)8The molar ratio of 4-dimethylaminopyridine to 4-dimethylaminopyridine is 1: 1-3; the G is1-(OH)8The mol ratio of the compound to 3-ethylamine is 1: 1-3;
g in step (5)2-(methacrylate)8The molar ratio of the compound to the thiomalic acid is 1: 2-5; the G is2-(methacrylate)8The mol ratio of the benzoin dimethyl ether to the benzoin dimethyl ether is 1: 0.5-2; the wavelength of the ultraviolet light is 350-400 nm.
5. The hydrophilic negative-charge porous nano-film for chronic kidney disease repair of claim 2, wherein the self-assembly preparation process of the negative-charge porous nano-film comprises the following steps:
dissolving the end-carboxylated 8-arm star-shaped polylactic acid element in tetrahydrofuran, starting magnetic stirring, dropwise adding glycerol at the temperature of 30-80 ℃ at the speed of 2-5 drops/second, quenching with liquid nitrogen, exchanging solvent in distilled water, and finally performing suction filtration and freeze drying to obtain the negative electricity porous nano film.
6. The hydrophilic negative-charged porous nanomembrane for chronic kidney disease repair of claim 5, wherein the mass to volume ratio of the terminally carboxylated 8-arm star-shaped polylactic acid motif to tetrahydrofuran is 10 to 20: 5-15 mg/ml; the volume-mass ratio of the glycerol to the end-carboxylated 8-arm star-shaped polylactic acid element is 10-20: 10-20 ml/mg; the volume-mass ratio of the distilled water to the end-carboxylated 8-arm star-shaped polylactic acid unit is 500-800: 10-20 ml/mg.
7. The method for preparing a hydrophilic negative-charged porous nano-film for repairing chronic kidney disease as claimed in claim 1, comprising the steps of:
(a) dispersing the negative electricity porous nano film into a PB buffer solution with the pH value of 5-6, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide EDC and N-hydroxysuccinimide NHS, reacting for 1-2 hours at room temperature, centrifuging, collecting precipitates, re-dispersing by using the PB buffer solution with the pH value of 7-8, adding PEG, reacting for 8-15 hours at room temperature, centrifuging, washing for 3-5 times by using deionized water, and freeze-drying to obtain a hydrophilic negative electricity porous nano film;
(b) dispersing the hydrophilic negative electricity porous nano film obtained in the step (a) in a PB buffer solution with the pH value of 5-6, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide EDC and N-hydroxysuccinimide NHS, reacting for 1-2 hours at room temperature, centrifuging to collect precipitates, re-dispersing by using a PB buffer solution with the pH value of 7-8, adding an Ab solution, reacting for 8-15 hours at room temperature, centrifuging, washing for 3-5 times by using deionized water, and freeze-drying to obtain the hydrophilic negative electricity porous nano film for repairing chronic nephrosis.
8. The preparation method according to claim 7, wherein in the step (a), the mass-to-volume ratio of the electronegative porous nano-film to the PB buffer solution is 10-20: 5-10 mg/ml; the mass ratio of the 1-ethyl- (3-dimethylamino-C) methylamine to the electronegative porous nano film is 20-30: 10-20 parts of; the mass ratio of the N-hydroxysuccinimide to the negative electricity porous nano film is 2-3: 10-20; the mass ratio of the PEG to the negative electricity porous nano film is 12-18: 10-20.
9. The preparation method according to claim 7, wherein in the step (b), the mass-to-volume ratio of the hydrophilic negative-electricity porous nano-film to the PB buffer solution is 10-20: 5-10 mg/ml; the mass ratio of the 1-ethyl- (3-dimethylamino-propyl) methylamine to the hydrophilic negative electricity porous nano film is 20-30: 10-20 parts of; the mass ratio of the N-hydroxysuccinimide to the hydrophilic negative electricity porous nano film is 2-3: 10-20 parts of; the volume mass ratio of the Ab solution to the negative electricity porous nano film is 200: 10-20 μ L/mg; the mass concentration of the Ab solution is 1-2%.
10. Use of the hydrophilic negative-charged porous nano-film for chronic kidney disease repair according to any one of claims 1 to 6 in the preparation of a chronic kidney disease repair agent.
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