CN117624581A - Polymer material with renal active targeting and preparation method and application thereof - Google Patents
Polymer material with renal active targeting and preparation method and application thereof Download PDFInfo
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- CN117624581A CN117624581A CN202311647099.XA CN202311647099A CN117624581A CN 117624581 A CN117624581 A CN 117624581A CN 202311647099 A CN202311647099 A CN 202311647099A CN 117624581 A CN117624581 A CN 117624581A
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Abstract
The invention belongs to the technical field of organic chemical synthesis and biomedical materials. Discloses a high molecular material (A) with renal active targeting, a preparation method and application thereof. The preparation method has the advantages of cheap and easily obtained starting materials, mild reaction conditions, high product yield and high purity by using a green solvent, and is suitable for industrial mass production. The high polymer material (A) has very remarkable renal active targeting, and can be used for preparing the renal fibrosis medicine for treating renal diseases.
Description
Technical Field
The invention belongs to the technical field of organic chemical synthesis and biomedical materials, and particularly relates to a high polymer material with renal active targeting as well as a preparation method and application thereof.
Technical Field
Kidneys are one of the major organs susceptible to fibrosis, the incidence is very high, next to the liver (on average, the annual incidence of renal fibrosis exceeds 10%), and this proportion increases with age. Chronic kidney disease (Chronic kidney disease, CKD) has been recognized as a global leading public health problem, and according to the relevant literature, the prevalence of CKD is about 11%. Liu et al, according to 18 literature summaries and 78671 investigation studies, showed that Chinese adult CKD prevalence is as high as 13.4%. Xu et al showed by clinical case analysis studies: CKD patients in China still show fold increase, and the onset age tends to be younger. CKD directly affects the incidence and mortality of global kidney disease by affecting cardiovascular risk and end-stage kidney disease, and is expected to be one of five causes of death in 2040 years. CKD patients show persistent renal fibrosis with progressive loss of renal function, eventually progressing to end stage renal disease (End stage renal disease, ESRD). Therefore, it is important to find effective and conservative treatments to delay the progression of CKD.
Fibrosis is an evolutionary and dynamic process with complex mechanisms triggered by a number of different stimuli and causative factors, which are generally thought to undergo four indivisible stages: initiation, activation, execution and progression, which occasionally occur concomitantly and overlap each other, these stimuli and pathogenic factors trigger a cascade of repair, accumulating in the molecular signal responsible for initiating and driving fibrosis. The fibrotic process is a response to different types of tissue damage, critical to normal healing, closely related to inflammation and tissue regeneration that may occur during and after the inflammatory response, and may play a defensive role under some pathogen-encapsulated infectious conditions. Fibrosis may also play a protective role in the event of damage to vital tissues, such as renal infarction, pyelonephritis, and myocardial infarction. Although fibrosis may play a defensive role, persistent chronic inflammation can lead to the pooling of inflammatory cells into the kidney, including but not limited to macrophages, mast cells, T cells and additional fibroblasts. At some stage, it may gradually become an uncontrolled irreversible and self-sustaining process, known as pathologic fibrosis. Here we will simply refer to pathological fibrosis as-fibrosis.
Renal fibrosis is a common pathway for almost all kidney disease to progress to end-stage renal failure, as well as a common endpoint of kidney injury and many CKDs. Renal fibrosis involves multiple links such as inflammatory responses, oxidative stress, immune cell apoptosis, and the like, which are more likely to lead to progressive deterioration of renal function than the primary disease. Renal fibrosis is a hallmark of CKD, the severity of which is generally related to the extent to which renal fibrosis progresses. It is characterized by persistent nephron damage, massive proliferation of fibroblasts in the stroma and formation of myofibroblasts, excessive and sedimentary extracellular matrix (Extracellular matrix, ECM) and Glomerulosclerosis (GS), renal mesenchyme fibrosis (Renal Interstitial Fibrosis, RIF), leading to irreversible scarring of the kidneys and eventual loss of renal function.
Renal fibrosis requires comprehensive medical control, however, no clinically effective anti-fibrosis therapies exist at present. The origin, functional heterogeneity and regulation of scar forming cells that occur during human kidney fibrosis remain poorly understood. The therapeutic devices available to nephrologists to arrest the progression of chronic kidney disease are very limited. In chronic kidney disease, patients have only two expensive options: dialysis and renal replacement therapy (Renal replacementtherapy, RRT). RRT has limitations of organ donors and ethical requirements. Therefore, it is urgent to find effective early prevention and treatment measures for kidney diseases.
At present, a therapeutic scheme of combining glucocorticoid with tripterygium glycosides, tacrolimus, cyclophosphamide and other medicaments for maintaining kidney diseases is often adopted in clinical conservation treatment. The toxic side effects of large doses of glucocorticoids and maintenance drugs severely limit the continued treatment of kidney disease. While a decrease in renal glomerular filtration rate can significantly alter the pharmacokinetics and pharmacodynamics of drugs excreted by renal and non-renal mechanisms.
The traditional Chinese medicine is based on the treatment concept of diagnosis and treatment, and the pathogenesis of kidney fibrosis is considered to be principal deficiency, principal deficiency and principal deficiency of spleen and kidney, principal excess and principal turbid toxin, damp-heat and blood stasis, namely-deficiency, stasis, toxin and damp-turbidity. In recent years, research on prevention and treatment of renal fibrosis by traditional Chinese medicine components has been greatly progressed, including experimental and clinical researches on single traditional Chinese medicines (astragalus membranaceus, ligusticum wallichii, radix salviae miltiorrhizae, cordyceps sinensis and the like), traditional Chinese medicine extracts (emodin, schisandrin B, astragaloside IV and the like) and compound preparations (Zhenwu decoction, anti-fiber prescription, kidney qi pill, flavored astragalus mongholicus and red wind decoction and the like). The Chinese medicinal active ingredients can effectively prevent and treat renal fibrosis and protect residual renal function. For thousands of years, herbal medicines rich in medicinal ingredients have been used for preventing and treating diseases, and they have become a major source of medicines for treating kidney-related diseases. However, because no accurate pathogenesis and pathogenic factors are found, the treatment effect of treating renal fibrosis by using the active ingredients of the traditional Chinese medicine is affected by lack of targeting.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a high polymer material (A) with renal active targeting, and a preparation method and application thereof. The polymer material (A) has the characteristics of kidney active targeting, has the advantages of mild preparation process conditions, rich raw material sources, environment-friendly solvent, high product purity and the like, and the nano preparation prepared by the polymer material (A) comprises microcapsules, microspheres, submicron emulsion, liposome, nanoemulsion, nanoparticles, polymer micelles and the like, can actively release traditional Chinese medicine active ingredients to focus positions of kidney diseases, improves the treatment effect of the traditional Chinese medicine active ingredients, reduces the toxic and side effects of the traditional Chinese medicine active ingredients, and is suitable for treating various kidney diseases.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a high polymer material (A) with renal active targeting has a structural formula:
n is greater than 2.
The preparation method of the high polymer material (A) with the renal active targeting comprises the following steps:
s1, dissolving 4-methoxybenzoyl chloride in an organic solvent to obtain a 4-methoxybenzoyl chloride solution; dissolving 2-bromoethylamine hydrobromide in an organic solvent to obtain a 2-bromoethylamine hydrobromide solution; adding the 4-methoxybenzoyl chloride solution and the 2-bromoethylamine hydrobromide solution into a round bottom flask, uniformly mixing, and stirring for 2-4h at room temperature.
S2, sucking the solution after the reaction in the step S1, and spotting the solution on a GF254 model silica gel plate, wherein the reaction mixture is used as a control. Taking methylene dichloride-methanol (according to the volume ratio of 5:1-1:1) mixed solution as a developing agent for developing upwards, taking out when the front trace of the solvent is 0.5-1.5 cm away from the top end of the thin plate, and airing; observing under 254nm ultraviolet lamp, scraping corresponding spots of the product, soaking in methanol for several times, and spin-drying by a rotary evaporator to obtain the product.
S3, respectively dissolving the product obtained in the step S2 and distearoyl phosphatidyl ethanolamine-polyethylene glycol-amino cross-linked substance (DSPE-PEG-NH 2) in an organic solvent to respectively obtain a product solution obtained in the step S2 and a DSPE-PEG-NH2 solution; and (3) adding the product solution obtained in the step (S2) and the DSPE-PEG-NH2 solution into a round bottom flask, uniformly mixing, and stirring for 2-4h at 35-50 ℃.
S4, transferring the sample obtained in the step S3 into a centrifuge tube, adding an organic solvent, uniformly mixing, and storing for 3-4 hours at a low temperature; centrifuging the centrifuge tube, discarding supernatant and drying in vacuum overnight; the obtained solid is redissolved in water, centrifuged, and the supernatant is collected.
S5, placing the supernatant obtained in the step S4 on a 1.5-6.0 kDa ultrafiltration membrane, and purifying by pressurizing through an ultrafilter, wherein a part to be reserved on the lyophilized membrane is obtained to obtain the high polymer material (A) with renal active targeting.
Based on the above technical scheme, further, in the step S1, the molar ratio of the 4-methoxybenzoyl chloride to the 2-bromoethylamine hydrobromide is 1:0.5-3, preferably 1:1-1.5.
Based on the technical scheme, in the step S2, methanol is soaked for 2-6 times, and each time is 2-5 min.
Based on the above technical scheme, in step S3, the molar ratio of the product obtained in step S2 to DSPE-PEG-NH2 is 10-100:1, preferably 30-60:1.
Based on the above technical scheme, in step S3, the molecular weight of DSPE-PEG-NH2 used is 1000-6000, preferably 1500-3000.
Based on the above technical scheme, further, in the step S4, the low temperature means-80 ℃ to-5 ℃, preferably-20 ℃ to-5 ℃.
Based on the technical scheme, the organic solvent is one or more of acetonitrile, ethanol, acetic acid, acetone, dimethyl sulfoxide and dichloromethane.
Based on the above technical scheme, in step S5, the ultrafiltration time is 5min to 30min.
The application of the polymer material (A) with renal active targeting in preparing medicines for treating renal diseases.
Based on the technical scheme, further, the application forms comprise microcapsules, microspheres, submicron emulsion, liposome, nanoemulsion, nanoparticles and polymer micelles.
Based on the above technical scheme, preferably, the kidney disease comprises kidney fibrosis caused by chronic kidney disease. The high polymer material (A) has very remarkable renal active targeting, and can be used for preparing the renal fibrosis medicine for treating renal diseases.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides a high polymer material (A) with renal active targeting.
2. The invention provides a preparation method of a high polymer material (A) with renal active targeting, which has the advantages of mild preparation process conditions, rich raw material sources, low cost, easy obtainment, environment-friendly solvent, high product yield, high purity and the like, and is suitable for industrial mass production.
3. The polymer material (A) provided by the invention can be used for preparing nano preparations such as microcapsules, microspheres, submicron emulsion, liposome, nanoemulsion, nanoparticles, polymer micelles and the like, so that the nano preparations have the characteristic of targeting the kidney. The polymer material (A) can prevent plasma protein from being adsorbed on the surface of the liposome, protect the liposome from being recognized and ingested by a mononuclear phagocyte system (Mononuclearphagocyte system, MPS), and improve the stability and the circulation half-life of the liposome in vivo.
4. The nano preparation prepared by the polymer material (A) can actively release the active ingredients of the traditional Chinese medicine to the focus of kidney diseases, improves the treatment effect of the active ingredients of the traditional Chinese medicine, reduces the toxic and side effects of the active ingredients of the traditional Chinese medicine, and is suitable for treating various kidney diseases.
Drawings
FIG. 1 is an IR spectrum of product 1;
FIG. 2 is product 1 1 H-NMR spectrum;
FIG. 3 is an IR spectrum of the polymer material (A);
FIG. 4 shows the polymer material (A) 1 H-NMR spectrum;
FIG. 5 is an external view of the samples of test examples 1-3;
FIG. 6 is a scanning electron micrograph of test example 1-3;
FIG. 7 is a transmission electron micrograph of test example 1-3;
FIG. 8 is a sample differential scanning calorimeter curve;
FIG. 9 is an in vitro release behavior study of test examples;
FIG. 10 is a graph showing the change in body weight of test mice;
FIG. 11 is a photograph of kidneys of test mice;
FIG. 12 is a photograph of glycogen PAS from a test mouse;
FIG. 13 is a photograph of each major organ of a test mouse.
Detailed Description
The present invention is further described below by way of examples, but the present invention is not limited thereto. The present invention is described in detail below by way of specific examples, but the scope of the present invention is not limited thereto. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and all experimental equipment, materials, reagents, etc. used can be obtained from commercial sources.
Example 1
Preparation of Polymer Material (A)
1g of 4-methoxybenzoyl chloride was dissolved in 5ml of acetone, and 1.5g of 2-bromoethylamine hydrobromide was dissolved in 5ml of acetonitrile, and the above two solutions were added to a round-bottomed flask and mixed uniformly, and stirred at 300rpm for 2 hours at room temperature. Mu.l of the solution after each pipetting reaction was spotted on a GF254 model silica gel plate and the reaction mixture was used as a control. Dichloromethane-methanol (5:1) is used as developing agent for developing upwards, and when the front edge trace of the solvent is 1.2cm away from the top end of the thin plate, the thin plate is taken out and dried. Observing under 254nm ultraviolet lamp, scraping corresponding spots of the product, soaking in methanol for 3 times and 2min each time, and spin-drying by a rotary evaporator to obtain product 1. 1.3g of the obtained product 1 and 0.5g of DSPE-PEG2000-NH2 are weighed and dissolved in 2ml of acetone respectively, added into a round bottom flask and uniformly mixed, and stirred for 2 hours at 35 ℃. The sample was transferred to a centrifuge tube and 20ml acetonitrile was added. After mixing well, store at-8deg.C for 3h. Centrifuge at 4500g for 10min. The supernatant was discarded and dried under vacuum overnight. The product was dissolved in 15mLH 2 O. After thorough mixing, the mixture was centrifuged at 4500g for 15min. The supernatant was collected and the particles discarded. The resulting supernatant was the final product. And (3) placing the obtained supernatant on a 3.0kDa ultrafiltration membrane, pressurizing by using an ultrafilter, and purifying for 10min, wherein the part of the lyophilized membrane needs to be reserved, so as to obtain the high polymer material (A). The total yield of the polymer material (A) was 82.1%.
An appropriate amount of product 1 was added to the sample well and infrared absorption was measured by IR. Its infrared spectrum is 3300cm -1 A broad peak (amino, -NH-) characteristic peak at 3030cm -1 Shoulder peaks (aromatic hydrocarbon, v=ch) appear, with 1640cm at the same time -1 And 1570cm -1 (benzene ring, C=C), 750cm -1 (benzene ring, -Ar-) can prove to contain benzene ring and be para-substituted. 1600cm -1 、1580cm -1 And 1694cm -1 (aldehyde group, C=O), 1265cm -1 (aryl ketone, ar-c=o), in combination with 3450cm -1 ，1290cm -1 ，400cm -1 700cm -1 Proved to be secondary amide (Ar-CO-NH) and 650cm -1 (halocarbons, C-Br).
IR and product 1 1 H-NMR analysis
Weighing a proper amount of product 1 sample in a nuclear magnetic tube, adding a proper amount of deuterated DMSO, and utilizing after the sample is completely dissolved 1 The chemical shift of various hydrogen atoms in the material was determined by H-NMR. 1 H NMR(500MHz,DMSO-d6)：δ(ppm)8.02(s,1H)，7.78(d,J＝8.9Hz,1H)，6.99(d,J＝8.9Hz,1H)，4.34(t,J＝9.5Hz,1H)，3.89(t,J＝9.5Hz,1H)，3.79(s,1H)。
Integrated IR 1 H-NMR results demonstrate successful synthesis of product 1, see FIGS. 1 and 2.
IR and Polymer Material (A) 1 H-NMR analysis
And adding a proper amount of polymer material (A) into the sample hole, and measuring infrared absorption by adopting IR. Its infrared spectrum is 3300cm -1 A broad peak (amino, -NH-) characteristic peak at 3030cm -1 Shoulder peaks (aromatic hydrocarbon, v=ch) appear, with 1640cm at the same time -1 And 1570cm -1 (benzene ring, C=C), 750cm -1 (benzene ring, -Ar-) can prove to contain benzene ring and be para-substituted. 1735cm -1 (ester group, C=O), 1680cm -1 (amide, c=o) and 1580cm -1 (amide, -NH-), 1180cm -1 (C-O-C)。2960cm -1 、2800cm -1 (alkane, -CH) 3 ) And 1120-940 cm -1 With strong and broad Peaks (PO) 4 3- ) And an isocharacteristic peak.
Weighing a proper amount of polymer material (A) sample into a nuclear magnetic tube, adding a proper amount of deuterated DMSO, and utilizing after the sample is completely dissolved 1 The chemical shift of various hydrogen atoms in the material was determined by H-NMR. 1 H NMR(500MHz,DMSO-d6)：δ(ppm)7.86(d,J＝8.9Hz，2H)，6.98(d，J＝8.9Hz，2H)，4.36(t，J＝9.5Hz，1H)，3.90(s，1H)，3.80(s，1H)，3.60-3.54(m，7H)，3.50(s，90H)，2.08(s，64H)，1.22(s，6H)。
Integrated IR 1 The H-NMR results showed that the polymer material (A) was successfully synthesized, see FIGS. 3 and 4.
Example 2
Preparation of Polymer Material (A)
1g of 4-methoxybenzoyl chloride was dissolved in 3ml of acetonitrile, 3.5g of 2-bromoethylamine hydrobromide was dissolved in 6ml of acetonitrile, and the above two solutions were added to a round-bottomed flask and mixed uniformly, and stirred at 200rpm for 3 hours at room temperature. Mu.l of the solution after each pipetting reaction was spotted on a GF254 model silica gel plate and the reaction mixture was used as a control. Dichloromethane-methanol (4:1) is used as developing agent for developing upwards, and when the front edge trace of the solvent is 1cm away from the top end of the thin plate, the thin plate is taken out and dried. Observing under 254nm ultraviolet lamp, scraping corresponding spots of the product, soaking in methanol for 2 times and 3min each time, and spin-drying by a rotary evaporator to obtain product 1. 2.1g of the obtained product 1 and 0.5g of DSPE-PEG1500-NH2 are weighed and dissolved in 5ml of acetone respectively, added into a round bottom flask and uniformly mixed, and stirred for 2 hours at 50 ℃. The sample was transferred to a centrifuge tube and 15ml of acetone was added. After mixing well, store at-15℃for 2h. Centrifuge at 4500g for 8min. The supernatant was discarded and dried under vacuum overnight. The product was dissolved in 20mLH 2 O. After thorough mixing, the mixture was centrifuged at 4500g for 15min. The supernatant was collected and the particles discarded. The resulting supernatant was the final product. And (3) placing the obtained supernatant on a 2.5kDa ultrafiltration membrane, pressurizing by using an ultrafilter, purifying for 6min, and obtaining the polymer material (A) on the part needing to be reserved on the freeze-dried membrane. The total yield of the polymer material (A) was 91.1%.
Example 3
Preparation of Polymer Material (A)
1g of 4-methoxybenzoyl chloride was dissolved in 2.5ml of methylene chloride, 0.8g of 2-bromoethylamine hydrobromide was dissolved in 3ml of acetone, and the two solutions were added to a round bottom flask and mixed well, and stirred at 250rpm for 2.5h at room temperature. Mu.l of the solution after each pipetting reaction was spotted on a GF254 model silica gel plate and the reaction mixture was used as a control. Dichloromethane-methanol (4.5:1) is used as developing agent for developing upwards, and when the front trace of the solvent is 0.8cm away from the top end of the thin plate, the thin plate is taken out and dried. Observing under 254nm ultraviolet lamp, scraping corresponding spots of the product, soaking in methanol for 4 times, each time for 2min, and spin-drying by a rotary evaporator to obtain product 1. Weighing 2.1g of the obtained product 1, and weighing DSPE-PEG2500-NH21g of each of the above-mentioned components was dissolved in 5ml of acetone, and the mixture was added to a round-bottomed flask and stirred at 45℃for 2 hours. The sample was transferred to a centrifuge tube and 26ml of dichloromethane was added. After mixing well, storing for 2h at-20 ℃. Centrifuge at 4000g for 13min. The supernatant was discarded and dried under vacuum overnight. The product was dissolved in 30mL H 2 O. After thorough mixing, the mixture was centrifuged at 4000g for 13min. The supernatant was collected and the particles discarded. The resulting supernatant was the final product. And (3) placing the obtained supernatant on a 4.0kDa ultrafiltration membrane, pressurizing by an ultrafilter for purification, wherein the ultrafiltration time is 10min, and obtaining the polymer material (A) from the part needing to be reserved on the freeze-dried membrane. The total yield of the polymer material (A) was 86.1%.
Example 4
Preparation of Polymer Material (A)
1.5g of 4-methoxybenzoyl chloride was dissolved in 5ml of ethanol, 2.1g of 2-bromoethylamine hydrobromide was dissolved in 8ml of methylene chloride, and the two solutions were added to a round bottom flask and mixed well, and stirred at 150rpm for 3 hours at room temperature. Mu.l of the solution after each pipetting reaction was spotted on a GF254 model silica gel plate and the reaction mixture was used as a control. Dichloromethane-methanol (3.5:1) is used as developing agent for developing upwards, and when the front trace of the solvent is 1.2cm away from the top end of the thin plate, the thin plate is taken out and dried. Observing under 254nm ultraviolet lamp, scraping corresponding spots of the product, soaking in methanol for 3 times and 3min each time, and spin-drying by a rotary evaporator to obtain product 1. 5.4g of the obtained product 1 and 1g of DSPE-PEG2500-NH2 are weighed and respectively dissolved in 10ml of ethanol, added into a round bottom flask, uniformly mixed and stirred for 3 hours at 40 ℃. The sample was transferred to a centrifuge tube and 40ml of ethanol was added. After mixing well, store at-30℃for 2.5h. Centrifuge at 5000g for 7min. The supernatant was discarded and dried under vacuum overnight. The product was dissolved in 50mL H 2 O. After thorough mixing, centrifuge at 5000g for 7min. The supernatant was collected and the particles discarded. The resulting supernatant was the final product. And (3) placing the obtained supernatant on a 2.5kDa ultrafiltration membrane, pressurizing by an ultrafilter for purification, wherein the ultrafiltration time is 8min, and obtaining the polymer material (A) from the part needing to be reserved on the freeze-dried membrane. The total yield of the polymer material (A) was 92.6%.
Example 5
Preparation of Polymer Material (A)
2.8g of 4-methoxybenzoyl chloride was dissolved in 14ml of dimethyl sulfoxide, 3.8g of 2-bromoethylamine hydrobromide was dissolved in 9ml of methylene chloride, and the above two solutions were added to a round bottom flask and mixed uniformly, and stirred at 300rpm for 2 hours at room temperature. Mu.l of the solution after each pipetting reaction was spotted on a GF254 model silica gel plate and the reaction mixture was used as a control. Dichloromethane-methanol (2:1) is used as developing agent for developing upwards, and when the front edge trace of the solvent is 1.2cm away from the top end of the thin plate, the thin plate is taken out and dried. Observing under 254nm ultraviolet lamp, scraping corresponding spots of the product, soaking in methanol for 4 times, each time for 3min, and spin-drying by a rotary evaporator to obtain product 1. 3.36g of the obtained product 1 and 0.8g of DSPE-PEG3000-NH2 are weighed and dissolved in 15ml of acetone respectively, added into a round bottom flask and uniformly mixed, and stirred for 2 hours at 45 ℃. The sample was transferred to a centrifuge tube and 35ml acetonitrile was added. After mixing well, storing for 2h at-50 ℃. Centrifuge at 4000g for 20min. The supernatant was discarded and dried under vacuum overnight. The product was dissolved in 45mLH 2 O. After thorough mixing, the mixture was centrifuged at 4000g for 20min. The supernatant was collected and the particles discarded. The resulting supernatant was the final product. And (3) placing the obtained supernatant on a 4kDa ultrafiltration membrane, pressurizing by using an ultrafilter, purifying for 8min, and obtaining the polymer material (A) on the part needing to be reserved on the freeze-dried membrane. The total yield of the polymer material (A) was 92.7%.
Test example 1
Preparation of tanshinone IIA liposome with different functions
Test example 1-1 preparation of general tanshinone IIA liposome
Weighing 10g of soybean lecithin and 1g of cholesterol, putting into a eggplant-shaped bottle, adding 0.55g of tanshinone IIA, fully dissolving with a proper amount of mixed solution of methanol and chloroform (1:2, v:v), and removing the organic solvent by vacuum reduced pressure rotary evaporation at 40 ℃ to ensure that lipid forms a uniform film on the inner wall of the eggplant-shaped bottle. Adding PBS buffer solution (0.01M, pH 7.4), slowly rotating on a rotary evaporator for hydration for 2h, carefully transferring the obtained liposome suspension into an ultrasonic tube, placing under a probe of a cell breaker, positioning the probe at the center of a test tube, treating with an ultrasonic cell pulverizer for 5min (the ultrasonic process is 400w×5 min), and sequentially extruding through 0.45 and 0.22 μm microporous filter membranes to obtain the common tanshinone IIA liposome. Transferring to penicillin bottle, and placing in refrigerator at 4deg.C for use.
Test examples 1-2 preparation of blank liposomes
The rest processes are unchanged, and tanshinone IIA is not added, thus obtaining the blank liposome.
Test examples 1-3 preparation of tanshinone IIA liposomes with renal active targeting
Adding 0.55g of polymer material (A) into the common tanshinone IIA liposome prepared in test example 1-1, stirring at room temperature for 10min, incubating in water bath at 60 ℃ for 2h, and sequentially extruding to pass through 0.45 and 0.22 mu m microporous filter membranes. Test examples 1-3 were observed to be translucent with orange opalescence, see FIG. 5.
Test example 2
Test examples 1 to 3 determination of encapsulation efficiency
The encapsulation efficiency is measured by adopting an n-hexane extraction method, and the tanshinone IIA content is measured by adopting an HPLC method.
100 mu L of the kidney-active targeting tanshinone IIA liposome prepared in test example 1-3 is sucked, placed in a 10mL centrifuge tube, 2mL of normal hexane is added, vortex mixing is carried out for 1min, 5000g is centrifuged for 10min, the upper normal hexane liquid is taken, repeated extraction is carried out for 3 times, the normal hexane liquid is combined in a 10mL volumetric flask, and normal hexane is used for volume fixing. Sample injection analysis, and determination of tanshinone IIA content as C in sample 0 . Adding methanol 10mL into the extracted liposome, mixing for 10s by vortex, adding 2mL of n-hexane, centrifuging for 10min at 5000g, collecting upper n-hexane solution, extracting repeatedly for 3 times until the liposome is milky, mixing n-hexane solutions, continuously metering volume with n-hexane to 10mL, and determining tanshinone IIA content in sample as C 1 . EE (encapsulation efficiency) was calculated by the following formula:
TABLE 1 encapsulation efficiency measurement results
According to the guidelines of the 9014 microparticle preparation of the Chinese pharmacopoeia-four parts of 2020 edition, the encapsulation efficiency is generally not lower than 80 percent. Test results show that the encapsulation efficiency of test examples 1-3 meets the standard.
Test example 3
Test examples 1 to 3 measurement of particle diameter and zeta potential
Test examples 1 to 3 were measured for particle size and zeta potential using a Markov laser particle size analyzer (Nano-zs 90 laser particle size analyzer, malvern, UK) and a zeta potential analyzer (Coulter zeta potential analyzer, malvern, UK). And taking a proper amount of sample, diluting with deionized water by a certain multiple, and then placing the sample into a sample cell for measurement, wherein the sample is added with the attention to avoid generating bubbles and influencing the measurement result.
TABLE 2 measurement results of particle size and zeta potential
| Particle size (nm) | Zeta potential (mV) | PDI |
| 107.63±1.09 | -35.36±0.47 | 0.23±0.01 |
The test results showed that the average particle diameter of test examples 1-3 was about 107nm, the particle diameter dispersibility coefficient (Particle dispersion index, PDI) was about 0.23, which indicated uniform particle diameter distribution, and the zeta potential was about-35.36 mV, which indicated that the sample was relatively stable.
Test example 4
Scanning electron microscope (Scanning electron microscope, SEM) observation of the surface morphology of test examples 1 to 3
A small amount of samples of test examples 1-3 were adhered to one side of the double faced adhesive tape, the surface floating or redundant lyophilized samples were blown off, the other side of the double faced adhesive tape was adhered to the sample holder with care along the edge with forceps, and the samples were subjected to a metal spraying treatment by an ion beam sputtering apparatus and were observed by a scanning electron microscope (KYKY-1000B scanning electron microscope, scientific instrument factory of China academy of sciences).
The surface morphology of test examples 1-3 under scanning electron microscope is shown in FIG. 6.
The test results show that the test examples 1-3 are good in form, spherical, uniformly dispersed and basically distributed at about 110nm in particle size, and basically accord with the measurement results of a particle sizer.
Test example 5
The morphology of test examples 1 to 3 was observed by a transmission electron microscope (Transmission electron microscope, TEM)
An appropriate amount of samples of test examples 1-3 were diluted to an appropriate multiple with PBS buffer (0.01M, pH 7.4), the diluted solution was pipetted in suspension and dropped onto a copper mesh, and deposited for 10min, and the excess liquid was blotted off with a strip of filter paper. And then the 2% phosphotungstic acid prepared at present is dropped on a copper mesh in a hanging way, and is negatively dyed for 3min, the excessive liquid is sucked by a filter paper strip and then dried, and the morphology of the preparation is observed under a TEM (JEM-2000 EX transmission electron microscope, JEOL company, japan) and recorded by photographing.
The morphology of TEM observation test examples 1 to 3 is shown in FIG. 7.
The test results show that the liposome in test examples 1-3 is spherical or spheroid, has obvious phospholipid bilayer structure, has the particle size of about 100nm, and is basically consistent with the particle size measurement result.
Test example 6
Differential scanning calorimetry (Differential scanning calorimetry, DSC) was performed to observe the coupling state of the polymer (A) with test example 1-1
The polymer material (A), test examples 1 to 3 and 5mg of lyophilized sample powder of the physical mixture of the polymer material (A) and test example 1 to 1 were weighed into a crucible, respectively, and atmosphere: nitrogen gas;rate of temperature rise: 5 ℃ min -1 The method comprises the steps of carrying out a first treatment on the surface of the Temperature measurement range: 20-350 ℃. Thermogravimetric analysis curves of the samples were recorded separately.
DSC analysis As shown in FIG. 8, the DSC chart of the physical mixture of the polymer (A) and test example 1-1 shows that the mixture exhibits melting endotherm peaks at 60.5℃and 127℃during the temperature programming, indicating the presence of crystalline free particles in the components thereof. In contrast, in both test examples 1-1 and 1-3, no melting peak was observed at this temperature, and only a dehydration peak was observed at 100℃to indicate that test examples 1-1 and 1-3 were in an amorphous state, and that the polymer material (A) and test example 1-1 were well coupled.
Test example 7
Test examples 1-3 in vitro Release behavior Studies
Two portions of each of test examples 1-1 and test examples 1-32mL were removed and one portion was placed in a dialysis bag and incubated in 200mL of PBS buffer at pH 7.4. 1mL of the dialysate was removed at different time points, and simultaneously an equal amount of release medium was immediately replenished, and the dialysate was diluted with methanol to an appropriate multiple and vortexed. The other part is directly fixed with methanol, the peak area of the sample is measured by high performance liquid chromatography, the concentration of tanshinone IIA is calculated, and the cumulative release rate (Cumulative release rate, CRR) is calculated according to a formula.
Wherein Cn: drug concentration (μg.mL) of nth sample -1 ) V0: total volume of dialysis medium (mL), V: volume per sample (mL), Q: total release amount (μg).
In vitro release behavior As shown in FIG. 9, under the condition of pH 7.4, the cumulative drug release rates of test example 1-1 and test example 1-336h are 80.58% and 54.93%, respectively, and the drug release rate of test example 1-3 is significantly lower than that of test example 1-1 (P < 0.05), which indicates that the polymer material (A) can significantly increase the stability of test example 1-1.
Test example 8
Test examples 1 to 3 inhibition of renal fibrosis in mice
1 establishment of kidney fibrosis model
BALB/c mice (6-8 week old, male, 20-25g, offered by Liaoning long Biotechnology Co., ltd.) were kept at room temperature and were free to drink water. Intravenous doxorubicin hydrochloride (Doxorubicin Hydrochloride, DOX) solution (13 mg/kg) -1 ) Two injections were performed together, 14 days apart. Mice in the blank group were injected with an equal volume of physiological saline each time.
2 dosing regimen
After the DOX solution injection on day 0, the modeled mice were randomly divided into a blank group, a control group, test 1-1 groups, and test 1-3 groups of 7 animals each. Intravenous injection of 10mg/kg on day 2, day 4, and day 6, respectively -1 Test examples 1-1 and 1-3, and test examples 1-2 (control group) of the same volume, the blank mice were injected with an equal volume of physiological saline each time. Mice were sacrificed on day 24 of the trial.
3 observation of weight change and State
Each group of mice was weighed from day 0 at intervals, the weight change of each group of mice during the whole modeling and treatment process was recorded, and the hair gloss, tail status, mental status and death of the mice were observed.
The body weight change of the mice is shown in fig. 10. Test results show that the groups 1-3 can effectively control the weight loss of mice, so that the mice maintain a relatively healthy weight range.
From day 0, DOX was injected, and the state of hair and activity of each group of mice was observed, as shown in Table 3.
TABLE 3 Condition summary of hair and activity for mice of each group
The DOX can cause serious tissue injury, and the tail vein injection mode is adopted for administration during molding and treatment, so that the tail of each group of mice is injured to different degrees. Healthy mice have smooth and bright hair, control mice have dull hair and uneven hair color, and tail and hind limb injury is gradually aggravated with time, and serious patients have mobility inconvenience and death. Test example 1-1 mice in group were kept low in body weight and listless after DOX injection on day 14. Test examples 1-3 mice were free to move, uniform in hair color and good in mental state.
The test results show that the mice in the test examples 1-1 have the same weight reduction trend as the mice in the control group after DOX injection, and the weight change of the mice in the test examples 1-3 is stable and slightly increased, which shows that the targeting and sustained release effects of the test examples 1-3 in the mice are obvious, and the high polymer material (A) can inhibit the occurrence and development of renal fibrosis for a long time.
Mice in the control group and mice in the test examples 1-1 die to different degrees, the hind limbs of individual mice in the control group are inconvenient to move, and the mice in the test examples 1-3 do not die, limb is inconvenient and the like, so that the hair color is uniform and bright, and the high polymer material (A) has targeting long circulation and has low toxic and side effects.
4 blood collection and detection
After the molding is finished and the free feeding is finished for one week, the feeding is fasted for 12 hours, water is forbidden for 1 hour before the experiment, the blood is taken from the eyeground vein, the blood is centrifuged for 10 minutes at 2000g, the serum is separated, the mark is made, and the blood is put into a refrigerator at the temperature of minus 20 ℃ for storage. To detect each serum biochemical index.
After the thawed samples are centrifuged again, the serum creatinine (SCr) and urea nitrogen (Blood urea nitrogen, BUN) levels are detected by an endpoint method at a primary and secondary wavelength of 540nm/670nm and a two-point method at a primary and secondary wavelength of 340nm/405nm, respectively.
The results of the biochemical blood test BUN and SCr are shown in Table 4.
TABLE 4 detection results of BUN and SCr
| Group of | BUN average value (mg. Dl) -1 ) | SCr mean value (mu mol L) -1 ) |
| Blank group | 20.5 | 19.7 |
| Control group | 29.4** | 24.8* |
| Test example 1-1 group | 26.8** | 23.4* |
| Test examples 1 to 3 groups | 21.3 | 20.5 |
Note that: * P <0.05 is shown in comparison with the test 1-3 groups, and P <0.01 is shown in comparison with the test 1-3 groups.
As can be seen from the data in the table, the BUN and SCr of the mice in the experimental group were observed to be higher than those in the normal blank group, the BUN content of the mice in the control group was obviously increased to about 1.43 times that of the healthy mice in the blank group, the BUN content of the mice in the test examples 1-1 group was lower than that in the control group but still 1.3 times that in the blank group, and the BUN content of the mice in the test examples 1-3 group was almost the same as that in the blank group. The SCr content was not much different for each group, but the SCr content was still lower for the mice of test 1-3 groups than for the control group and test 1-1 groups (P < 0.05).
Blood biochemical detection is the main means of kidney function detection, and common indexes are BUN and SCr. From the statistical analysis of renal function, test examples 1-3 were found to reduce the levels of SCr and BUN, thus demonstrating that the polymeric material (a) can well reduce the damage of renal fibrosis to rat renal function through renal active targeting.
Separation of viscera and staining of glycogen PAS
After the animals are killed, the double kidneys are separated, the double kidneys are washed by normal saline, the surface moisture is absorbed by absorbent paper, the surface fat and fascia of the viscera are removed, the mass is accurately weighed by an electronic balance, and the photograph is taken, and is shown in figure 11. Left kidneys were fixed in 4% paraformaldehyde solution and stained for glycogen PAS. The right kidney is packaged in a self-sealing bag and stored in a refrigerator at the temperature of minus 20 ℃.
The organ coefficient is the ratio of the weight of a certain organ of the experimental animal to the weight of the experimental animal, and the calculation formula of the organ coefficient of the double kidneys of the mice is as follows:
the fixed left kidney is dehydrated by ethanol and is soaked in wax for embedding after being transparent by dimethylbenzene, and the embedded paraffin block is cut into slices with the size of 5 mu m and is stuck on an anti-drop glass slide. The prepared paraffin slice is placed in a baking oven at 37 ℃ for overnight, and dewaxed and hydrated by xylene I, xylene II, absolute ethyl alcohol I, absolute ethyl alcohol II, 95% ethyl alcohol, 90% ethyl alcohol, 85% ethyl alcohol, 75% ethyl alcohol and distilled water in sequence. The specific dyeing steps are as follows:
(1) Washing with tap water for 2-3min, and soaking with distilled water for 2 times for 10s each time.
(2) The slices are placed in the oxidizing agent Schiff snow reagent and left at room temperature for 5-8min, generally not more than 10min.
(3) Washing with tap water for 1 time, and soaking with distilled water for 2 times for 10s each time.
(4) The sections were placed in a Schiffreagent and stained in shade at room temperature for 15min.
(5) Washing with tap water for 10min.
(6) The sections were placed in hematoxylin staining solution and stained for 90s.
(7) Hematoxylin differentiation fluid was differentiated for 5s.
(8) After washing for 10-15min with tap water, the double distilled water is replaced for cleaning, so that the blue color of the double distilled water is returned.
(9) And (5) step-by-step conventional ethanol dehydration. The dimethylbenzene is transparent, and the neutral resin is sealed.
(10) The images were observed under a microscope and photographed, and the photographs are shown in fig. 12.
As can be seen from fig. 11, the kidneys of normal mice were purplish red, the envelopes were flat and glossy, the kidneys of the kidney fibrosis model group (i.e., the control group) were reddish brown, the envelopes were uneven, and the kidneys were large. Test example 1-1 the kidneys were somewhat brighter than the model group, with slightly dilated left kidneys and normal right kidneys. The kidneys of test examples 1-3 groups were all relatively similar to the kidneys of normal mice in terms of color, size and envelope smoothness.
The results of the organ coefficients of the double kidneys of each group of mice are shown in Table 5.
Table 5 comparison of organ coefficients of double kidney of mice (n=3)
| Group of | Mean value of double kidney coefficients of mice |
| Blank group | 1.24 |
| Control group | 1.72 |
| Test example 1-1 group | 1.51 |
| Test examples 1 to 3 groups | 1.27 |
As can be seen from table 5, the organ coefficients of the control group are significantly increased according to the comparison of the organ coefficients of the blank group, and the renal fibrosis condition of the mice is stable; the kidney fibrosis conditions of mice are improved in different degrees in the test example 1-1 group and the test example 1-3 group, the viscera coefficients of the test example 1-3 group are closer to those of the blank group mice, and the mice inhibit fibrosis to a greater degree, so that the treatment effect is obvious.
The results of staining glycogen PAS in kidney tissue sections are shown in FIG. 12. Control kidney tissue sections showed increased infiltration of mesenchymal cells, dilation of distal tubular, significant glomerular sclerosis, and also interstitial fibrosis (the presence of both fibroblasts and fibroblasts). Test example 1-1 group tubular dilation, glomerular hypertrophy, and the presence of transparent deposits. And in the test examples 1-3, the glomerulus is slightly mesangial hyperplasia lesion, the tubular has no obvious lesion, and the structure is complete and clear.
Organ coefficients are commonly used indexes in toxicological experiments, and the method is simple and easy to implement and is sensitive to test. The organ factor is also called the viscera-body ratio, which is the ratio of the weight of an organ of an experimental animal to the weight of the organ. The ratio of each organ to body weight is constant in normal state. After animal contamination, the weight of the damaged viscera can be changed, so the viscera coefficients are also changed. Compared with a blank group, the experimental results of each group are obviously increased in the organ coefficients of the kidney of the control group, which indicates that the kidney has congestion, edema or hyperplasia and hypertrophy; the toxicity and side effects of fibrosis on the kidneys of mice are inhibited in test examples 1-3, the kidney organ coefficients of the mice with renal fibrosis treated in test examples 1-3 are similar to those of normal mice, and the polymer material (A) is proved to inhibit the development of fibrosis to a greater extent through renal active targeting, so that the toxicity and side effects on the kidneys are reduced.
The gold index for diagnosing the renal fibrosis is a renal histopathological examination, the renal tissue section is dyed by glycogen PAS, the changes of renal tubules, glomeruli and interstitium in the renal tissue are observed, the renal fibrosis degree of each group of mice is clear, and the control group shows obvious glomerulosclerosis and interstitium fibrosis; while test 1-1 group had less glomerulosclerosis but significantly dilated tubular ducts; the test examples 1-3 only have mild mesangial hyperplasia, so that the high polymer material (A) has obvious effect of inhibiting renal fibrosis through renal active targeting.
6. Observation of the states of the organs of the Experimental mice
After the animals were sacrificed, the main organs were dissected and arranged in a blank group, a control group, test example 1-1 group, and test example 1-3 group, photographs were taken, and the test results are shown in fig. 13. As can be seen from fig. 13, there was no significant difference between the groups of livers; the heart is basically the same in other groups except that the kidneys of the control group are smaller; the spleen control group is obviously bigger and possibly related to inflammatory reaction, the test example 1-1 group is slightly improved, and the test example 1-3 group is basically close to the spleen of a normal mouse; the lungs of the control group had different degrees of atrophy and darkening than those of the test 1-1 groups, and the test 1-3 groups were normal.
The test results show that the main organs of the mice in the test examples 1-3 are not obviously changed, which indicates that the polymer material (A) can obviously reduce the damage of DOX to the organs of the mice in the test, and has the effect of attenuation.
MDA content and SOD activity detection
Taking out right kidney, cutting appropriate amount of kidney tissue, adding physiological saline according to the mass to volume ratio of 1:9, homogenizing in ice bath, preparing 10% tissue homogenate, centrifuging 2500g for 10min, taking supernatant, and respectively measuring MDA content and SOD activity in each tissue homogenate according to the description of the kit. The detection result is calculated by the following company formula.
Wherein C is Label (C) And C Measuring Respectively representing the protein concentration of the standard substance and the protein concentration of the sample to be tested.
Calculation of SOD activity in kidney tissue homogenate:
wherein V is Total (S) 、V 0 、V n V and C represent the total volume of the reaction solution, the total amount of the extract, the measured extract, the blood sampling amount and the hemoglobin content, respectively.
The MDA and SOD detection data are shown in Table 6.
Table 6 MDA content and SOD activity in kidney tissue (n=3)
| Group of | MDA(nmol·mgprot -1 ) | SOD(U·mgprot -1 ) |
| Blank group | 4.92 | 604.78 |
| Control group | 7.85** | 384.57** |
| Test example 1-1 group | 6.46** | 439.21** |
| Test examples 1 to 3 groups | 5.13 | 587.06 |
Note that: * Comparison with test examples 1-3, P <0.01
As can be seen from Table 6, the MDA content in the kidney tissue of the animals in the experimental group was increased to different degrees, wherein the MDA content in the control group was significantly increased, and the MDA content in the test examples 1-3 was closer to that in the blank group, and was smaller than that in the control group and the test examples 1-1 (P < 0.01). The SOD activity in the kidney tissue of the experimental group is lower than that of the blank group, and the SOD activity of the experimental group 1-3 is obviously increased (P is less than 0.01) compared with that of the control group and the experimental group 1-1, and is more approximate to that of the kidney tissue of the healthy mouse.
MDA content and SOD activity in kidney tissue are important indicators of oxidative stress. MDA is a secondary product of lipid peroxidation of oxygen free radicals and erythrocyte membrane polyunsaturated fatty acids, is a final product of injury of the biological cell membrane by the free radicals, has strong biotoxicity, and has the production amount parallel to that of the oxygen free radicals, and the MDA is increased to cause membrane injury and influence erythrocyte deformability, so that the MDA can reflect the oxidation resistance degree of organisms. The amount of free radical generation can be indirectly judged through MDA and SOD, and the increase of MDA level and the decrease of SOD level reflect the decrease of free radical scavenging ability and the enhancement of antioxidant reaction of tissue cells in vivo.
After DOX molding was injected in this study, MDA value in the kidney tissue of mice was increased, while SOD activity was decreased. The increased MDA content can indirectly reflect the increased degree of lipid peroxidation in the body, and the decreased SOD activity can indicate the decreased antioxidant capacity of the body. In the case of test examples 1 to 3, MDA value of kidney tissue of mice was decreased, SOD activity was increased, and renal function and renal pathology were improved. From this, it was inferred that the protective mechanisms of test examples 1-3 on DOX kidney fibrosis model mice might trigger the swing through antioxidant stress. Injection test examples 1-3 can alleviate the hemodynamic and functional changes caused by fibrosis, thus proving that the polymer material (A) obviously inhibits the development of fibrosis through renal active targeting.
The above-described embodiments are only preferred embodiments of the invention, and not all embodiments of the invention are possible. Any obvious modifications thereof, which would be apparent to those skilled in the art without departing from the principles and spirit of the present invention, should be considered to be included within the scope of the appended claims.
Claims (10)
1. A high polymer material (A) with renal active targeting has a structural formula:
n is greater than 2.
2. The method for preparing the polymer material (A) with renal active targeting as claimed in claim 1, which is characterized by comprising the following steps:
s1, dissolving 4-methoxybenzoyl chloride in an organic solvent to obtain a 4-methoxybenzoyl chloride solution; dissolving 2-bromoethylamine hydrobromide in an organic solvent to obtain a 2-bromoethylamine hydrobromide solution; uniformly mixing 4-methoxybenzoyl chloride solution and 2-bromoethylamine hydrobromide solution, and stirring for 2-4 hours at room temperature;
s2, sucking the solution after the reaction in the step S1, spotting the solution on a silica gel plate, taking the reaction mixture as a contrast, taking methylene dichloride-methanol mixed solution as a developing agent according to the volume ratio of 5:1-1:1, developing the solution upwards, taking out the solution when the front edge trace of the solvent is 0.5-1.5 cm away from the top end of the thin plate, and airing the solution; observing under 254nm ultraviolet lamp, scraping corresponding spots of the product, soaking in methanol, and spin-drying by a rotary evaporator to obtain the product;
s3, respectively dissolving the product obtained in the step S2 and DSPE-PEG-NH2 in an organic solvent to respectively obtain a product solution and a DSPE-PEG-NH2 solution obtained in the step S2; and (3) uniformly mixing the product solution obtained in the step (S2) with the DSPE-PEG-NH2 solution, and stirring for 2-4h at the temperature of 35-50 ℃.
S4, adding an organic solvent into the sample obtained in the step S3, uniformly mixing, and storing for 3-4 hours at a low temperature; centrifuging, removing supernatant, and drying; redissolving the obtained solid in water, centrifuging, and collecting supernatant;
s5, placing the supernatant obtained in the step S4 on a 1.5-6.0 kDa ultrafiltration membrane, and purifying by pressurizing through an ultrafilter, wherein a part to be reserved on the lyophilized membrane is obtained to obtain the high polymer material A with renal active targeting.
3. The preparation method according to claim 2, characterized in that: in the step S1, the molar ratio of the 4-methoxybenzoyl chloride to the 2-bromoethylamine hydrobromide is 1:0.5-3, preferably 1:1-1.5.
4. The preparation method according to claim 2, characterized in that: in the step S3, the molar ratio of the product obtained in the step S2 to DSPE-PEG-NH2 is 10-100:1, preferably 30-60:1.
5. The preparation method according to claim 2, characterized in that: in the step S3, the molecular weight of PEG in the DSPE-PEG-NH2 is 1000-6000, preferably 1500-3000; low temperature means-80 ℃ to-5 ℃, preferably-20 ℃ to-5 ℃.
6. The preparation method according to claim 2, characterized in that: the organic solvent is one or more of acetonitrile, ethanol, acetic acid, acetone, dimethyl sulfoxide and dichloromethane.
7. The preparation method according to claim 2, characterized in that: in the step S2, methanol is soaked for 2 to 6 times, and each time is 2 to 5 minutes; in the step S5, the ultrafiltration time is 5-30 min.
8. The use of a polymer material (a) with renal active targeting as claimed in claim 1 for the preparation of a medicament for the treatment of renal diseases.
9. The application of the polymer material (A) with renal active targeting according to claim 8 in preparing medicines for treating renal diseases, wherein the application forms comprise microcapsules, microspheres, submicron emulsion, liposome, nanoemulsion, nanoparticles and polymer micelles.
10. The use of a polymer material (a) with renal active targeting according to claim 8, in the preparation of a medicament for the treatment of renal diseases, including renal fibrosis due to chronic kidney disease.
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