Kidney-targeted micromolecule polypeptide nano-liposome as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to a kidney-targeted micromolecule polypeptide nano-liposome as well as a preparation method and application thereof. According to the specific technical scheme, the preparation method of the kidney-targeted micromolecule polypeptide nano-liposome comprises the following steps: adding dichloromethane into soybean lecithin and cholesterol, carrying out ultrasonic dissolution, slowly dropwise adding the solution into PBS (Phosphate Buffer Solution) dissolved with micromolecule peptide under continuous stirring, carrying out rotary evaporation to remove an organic solvent dichloromethane, adding a PBS buffer solution into a formed film, carrying out ultrasonic treatment, and carrying out freeze drying to obtain the kidney-targeted micromolecule polypeptide nano-liposome. And eluting the film, adding a certain proportion of high molecular weight chitosan and low molecular weight chitosan, and magnetically stirring to obtain the small molecular polypeptide nano-liposome. The small-molecule polypeptide nano-liposome disclosed by the invention is small in particle size, high in encapsulation efficiency, good in dispersity, free of cytotoxicity, relatively good in biocompatibility and certain in kidney targeting property. According to the invention, the problems of poor stability, early medicine leakage and short residence time of a micromolecule polypeptide oral medicine are solved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly relates to a kidney-targeted small molecule polypeptide nano-liposome, a preparation method thereof and an application thereof. Background Art

[0002] With the rapid progress of biotechnology, more and more proteins and polypeptides have been developed into drugs for treating various diseases. Due to their high selectivity and effectiveness, and relatively low toxicity, etc., protein and polypeptide drugs have become one of the alternatives to small molecule drugs. Usually, the administration route of such drugs is injection. Long-term continuous injection may pose a huge challenge to drug compliance, including pain, aversion to injection, local irritation, etc. Therefore, the oral route is the most attractive alternative route because of its higher safety and compliance. However, the structural organization and physiological functions of the gastrointestinal tract result in low bioavailability and short half-life of protein and polypeptide drugs after oral administration. The absorption and targeted delivery of protein and polypeptide drugs after oral administration have become the bottleneck in the development of the oral administration route of such drugs.

[0003] Liposomes, as a common nano-carrier, have a structure similar to that of biological membranes, good biocompatibility, and also have the advantages of protecting drug activity, reducing drug toxicity, reducing immunogenicity, and being biodegradable in vivo. As an ideal and safe drug delivery system, liposomes can encapsulate various drugs in two hydrophilic inner and outer layers respectively. Although liposome carriers have many advantages, unmodified liposomes still have some deficiencies, such as poor stability, early drug leakage, short residence time, etc. To solve this problem, the surface of liposomes can be modified to improve their stability in different environments.

[0004] Based on the above considerations, the present invention constructs a drug-loaded liposome nanoparticle that not only retains the biological activity of small peptides but also can target the kidney directionally. Liposomes are used as carriers, and small molecule polypeptide drugs are encapsulated therein, and are modified by chitosan to form chitosan-modified small peptide nano-liposomes. The particle size, zeta potential and PDI of the samples are measured by a Malvern laser particle size analyzer to evaluate the quality of the small peptide nano-liposomes. Subsequently, the small peptide nano-liposome drug system is used to encapsulate the small molecule polypeptide SRP (Ser-Arg-Pro), and SRP has been reported in previous studies to potentially alleviate kidney injury. The cytotoxicity, hemolytic safety and kidney targeting of the SRP nano-liposome were observed. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides a kidney-targeted small peptide nano-liposome, a preparation method thereof and an application thereof, which solve the problems of poor stability, early drug leakage and short residence time of the small molecule polypeptide liposome carrier.

[0006] To achieve the above object, the present invention is realized through the following technical solutions:

[0007] The present invention discloses a preparation method of a kidney-targeted small molecule polypeptide nano-liposome. After adding and ultrasonically dissolving dichloromethane into soybean lecithin and cholesterol, it is slowly dropped into PBS containing dissolved small molecule peptide under continuous stirring. After rotary evaporation of the organic solvent dichloromethane, PBS buffer solution is added to the formed film. After ultrasonication, the film is eluted, and chitosan is added, and then the liposome is obtained after magnetic stirring.

[0008] Preferably, the mass ratio of the small molecule peptide to soybean lecithin is 1:5 - 15, and the mass ratio of soybean lecithin to cholesterol is 1 - 10:1.

[0009] Preferably, the pH of the PBS is 5.0 - 9.0, and the ultrasonication time is 10 - 90 min.

[0010] Preferably, the chitosan includes low molecular weight chitosan and high molecular weight chitosan, and the high molecular weight chitosan and low molecular weight chitosan are coated layer by layer according to the mass ratio of 10 - 0:0 - 10.

[0011] Correspondingly, a small molecule peptide nano-liposome prepared by the above preparation method.

[0012] Correspondingly, an application of a small molecule peptide nano-liposome prepared by the above preparation method in the preparation of a drug for treating kidney injury.

[0013] The present invention has the following beneficial effects:

[0014] In order to better maintain the biological activity of the polypeptide drug and target it to the kidney, the present invention uses chitosan nano-liposome to encapsulate and deliver it. The results show that the liposome modified with chitosan (SL) exhibits good biocompatibility and kidney targeting effect in vivo, providing an important theoretical and experimental basis for its clinical application as a new type of kidney protectant. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a graph of the optimization results of the liposome preparation process (A: optimization of the drug-lipid ratio, B: soybean lecithin:cholesterol, C: ultrasonication time, D: pH value of PBS);

[0016] Figure 2 It is a graph of the targeting results of different ratios of chitosan;

[0017] Figure 3 It is a characterization graph of SRP liposome (A: particle size distribution graph, B: TEM graph and fluorescence microscope graph of liposome, C: Fourier transform infrared of liposome);

[0018] Figure 4 For the storage stability of liposomes;

[0019] Figure 5 For the safety results of liposomes (A: Results of the effect of liposomes on HK-2 cell viability, B: Effect of liposomes on red blood cell morphology, C: Hemolytic results of liposomes); Note: ### Indicates a significant difference p < 0.001 compared with the blank group, and *** indicates a significant difference p < 0.05 compared with the positive treatment group;

[0020] Figure 6 For the distribution of liposomes in vivo. Detailed implementation manners

[0021] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0022] If not specifically specified, the technical means used in the implementation examples are conventional means well-known to those skilled in the art.

[0023] The present invention discloses a preparation method of a renal-targeted small molecule polypeptide nanoliposome, and the process is as follows: Add dichloromethane to soybean lecithin and cholesterol and dissolve it by ultrasonic treatment, then slowly drop it into PBS (phosphate buffer solution, pH = 5.0 - 9.0) containing the dissolved small molecule polypeptide drug under continuous stirring. After magnetic stirring for 30 minutes, rotate and evaporate to remove dichloromethane. Add PBS to the formed film, and after ultrasonic treatment (power 100%, ultrasonic treatment for 1 minute, pause for 1 minute) for 10 - 90 minutes, elute the film, add chitosan, and obtain liposomes after magnetic stirring.

[0024] Furthermore, the mass ratio of the small molecule peptide to soybean lecithin is 1:5 - 15, and the mass ratio of soybean lecithin to cholesterol is 1 - 10:1.

[0025] Preferably, the chitosan includes low molecular weight chitosan and high molecular weight chitosan, and the high molecular weight chitosan and low molecular weight chitosan are coated layer by layer according to a mass ratio of 10 - 0:0 - 10.

[0026] Next, the present invention will be further elaborated with specific embodiments.

[0027] The experimental animals used in the following examples were: 60 male SPF-grade C57BL / 6 mice at 6 weeks of age, weighing 18 - 22 g, purchased from Zhuhai Baishitong Biotechnology Co., Ltd., with the license number SCXK(Yue)2020 - 0051. These mice were housed in the Animal Experiment Center of Guangdong Medical University at room temperature (20 - 25 °C), humidity (50 - 60%), with a 12:12-hour light-dark cycle, and provided with standard food and water. The animal protocol was approved by the Ethics Committee of Guangdong Medical University, and all procedures were strictly in accordance with the operating standards approved by the National Animal Ethics Committee and the Animal Ethics Committee of Guangdong Medical University.

[0028] Example 1 Preparation of Small Molecule Polypeptide Nanoliposomes

[0029] Reverse evaporation method: Weigh 0.2820 g of soybean lecithin and 0.094 g of cholesterol, add an appropriate amount of dichloromethane and dissolve it by ultrasound. Slowly drop the organic phase into 20 mL of PBS (pH = 7.2) solution containing small molecule polypeptide (0.01 g) under continuous stirring, stir magnetically for 30 min, rotate and evaporate to remove dichloromethane. Add an appropriate amount of PBS to the formed film, and sonicate for 10 min (power 100%, sonicate for 1 min, pause for 1 min). Wash the film off the bottle wall, add 20 mL of chitosan (0.001 g / mL) and stir magnetically for 2 h. Extrude the liposomes 11 times with a liposome extruder. The results showed that the particle size of the prepared liposomes was 406.8 nm, and the PDI value was 0.366. The lower the PDI value, the more stable the liposome system, and the less likely it is to aggregate and sediment.

[0030] Example 2 Optimization of the Preparation Conditions of Small Molecule Polypeptide Nanoliposomes

[0031] Since liposomes with too large particle size are easily recognized and phagocytosed by macrophages in the body, shortening the half-life in the body, and when the particle size is less than 20 nm, they are easily cleared by the kidneys and excreted from the body and are prone to cause nano-hazards. Therefore, it is necessary to control its particle size within a suitable range to maintain good in vivo stability. Therefore, the present invention takes the particle size as the main standard for evaluating the quality of liposomes.

[0032] 1. Investigation of the drug-lipid ratio

[0033] Under the same fixed experimental conditions, liposomes were prepared by the method of Example 1, and the effects of the mass ratio of small molecule polypeptide to soybean lecithin at 1:5, 1:7.5, 1:10, 1:12.5, and 1:15 on its particle size were studied.

[0034] 2. Study on the mass ratio of soybean lecithin-cholesterol

[0035] Under the same conditions, liposomes were prepared by the method of Example 1, and the effects of the mass ratios of soybean lecithin to cholesterol at 1:1, 3:1, 5:1, 7:1, and 10:1 on the particle size were studied.

[0036] 3. Investigation of the pH value of PBS

[0037] On this basis, liposomes were prepared by the method of Example 1, and small molecule polypeptides were dissolved under the conditions of pH values of 5.0, 6.0, 7.0, 8.0, and 9.0 respectively, and the particle sizes of the prepared liposomes were measured.

[0038] 4. Investigation of the ultrasonic time

[0039] Keeping other conditions unchanged in the fixed experiment, liposomes were prepared by the method of Example 1, and the effects of 10, 30, 50, 70, and 90 minutes on the particle size of the prepared liposomes were studied respectively.

[0040] The results are as Figure 1 (A) shows that in the range of the mass ratio of polypeptide drug to soybean lecithin from 1:5 to 1:12.5, the particle size of the sample shows a gradually decreasing trend, and reaches the lowest value (particle size of 353.3 nm) at 1:12.5. At this time, the liposome system is relatively stable. However, with the increase of the added amount of polypeptide drug, the particle size shows an obvious upward trend, indicating that the liposome system gradually becomes unstable.

[0041] From Figure 1 (B), it can be seen that when the mass ratio of soybean lecithin to cholesterol is in the range of 1:1 to 10:1, the mass ratio of soybean lecithin to cholesterol shows a trend of first increasing and then decreasing for the particle size of liposomes. Generally, the addition of cholesterol can increase the packing of phospholipid molecules, thereby improving the stability of liposomes and enhancing the resistance of vesicles to aggregation. However, when the mass ratio of cholesterol to soybean lecithin is excessive, it will cause the aggregation of liposomes, thereby reducing their stability. On the contrary, if the ratio of cholesterol to soybean lecithin is too small, it may lead to a loose arrangement of the phospholipid molecular layer. At a mass ratio of 10:1, the smallest particle size of 295.6 nm was obtained, and thus the optimal ratio of phospholipid / cholesterol was determined to be 10:1.

[0042] From Figure 1 (C), it can be seen that the ultrasonic time has little effect on the particle size, and an ultrasonic time of 30 min was selected for subsequent experiments. This can effectively save the experimental time and ensure stable and reliable experimental results at the same time.

[0043] From Figure 1As can be seen from (D), the pH of PBS has a great influence on the particle size. Generally, it is lower and the particle size is smaller under weakly acidic or weakly basic pH. When the pH is 6, the particle size is 275.2 nm, and when the pH is 8, the particle size is 256.3 nm. Due to the low pH value, fatty acids in the liposome are protonated, forming hexagonal crystals, which increases the fluidity of the liposome membrane and thus reduces the system stability. Therefore, the pH of PBS is selected to be 8.

[0044] According to the above experimental screening results, when the small molecule polypeptide is 0.0288 g, soy lecithin is 0.36 g, cholesterol is 0.036 g, the ultrasonic time is 30 min, and PBS is 8, the particle size of the prepared liposome (SL) is 256.3 nm.

[0045] 5. Screening of chitosan concentration

[0046] Layer-by-layer coating was carried out using different ratios of low molecular weight chitosan to high molecular weight chitosan (high molecular weight chitosan: low molecular weight chitosan = 10:0; 6:4; 5:5, 0:10). The particle size, PDI and targeting effect were used as evaluation indexes to determine the most suitable chitosan ratio.

[0047] Low molecular weight chitosan (<3000 kDa) has renal targeting effect, but low molecular weight chitosan has less charge, so it is difficult to have electrostatic interaction with liposomes and be loaded on the surface of chitosan. Therefore, a composite solution of high molecular weight and low molecular weight chitosan is selected to modify the targeting of liposomes. As can be seen from Table 1, for liposomes with only low molecular weight chitosan added, the particle size is too large, and visible aggregation will occur, which is not suitable for subsequent experiments, so it is not considered. Using only high molecular weight chitosan to load liposomes can make the liposomes have better particle size and stability, but no targeting was observed. When the mass ratio of high molecular weight chitosan to low molecular weight chitosan is 6:4 and 5:5, they can also have better particle size and PDI, but through in vivo imaging analysis of small animals, Figure 2 As shown, liposomes with a mass ratio of high molecular weight chitosan to low molecular weight chitosan of 5:5 have better targeting. Therefore, in subsequent experiments, a mass ratio of high molecular weight chitosan to low molecular weight chitosan of 5:5 is selected for its loading modification.

[0048] Table 1 Screening results of chitosan concentration

[0049]

[0050] Example 3 Liposome characterization

[0051] Under the optimized preparation conditions of nano-liposomes according to Example 2, reverse evaporation method in Example 1 was used to prepare nano-liposomes of oligopeptide SRP derived from Sipunculus nudus with kidney protection effect (SRP Liposomes, hereinafter referred to as SL).

[0052] 1. Determination of particle size, zeta potential, and PDI

[0053] Particle size, zeta potential, and PDI are the main indicators for evaluating the quality of liposomes. The prepared liposomes were appropriately diluted in ultrapure water, and then the particle size, zeta potential, and PDI of the samples were measured using a Malvern laser particle size analyzer. To ensure the accuracy and reliability of the data, three parallel determinations were performed each time.

[0054] 2. Determination of encapsulation efficiency

[0055] Take 2 mL of liposomes and centrifuge for 30 min using a refrigerated centrifuge. Precisely transfer 0.4 mL of the supernatant to a new centrifuge tube, and add 1.6 mL of methanol for demulsification treatment. After sonication for 3 min, measure and calculate the drug content as W1 under an enzyme-linked immunosorbent assay (ELISA) reader. Precisely transfer 0.4 mL of the liposome suspension to a centrifuge tube, add 1.6 mL of methanol for ultrasonic demulsification treatment. Determine the drug content in the suspension, and calculate the encapsulation efficiency (EE) of the liposomes according to Equation 5-1.

[0056]

[0057] 3. FT-IR analysis

[0058] Functional groups of SRP, oligopeptide liposomes (Control Liposomes, CL), and oligopeptide SRP nano-liposomes (SL) prepared in this invention were determined using a Fourier transform infrared spectrometer. Mix SRP, NL, and SL with KBr powder, and press them into thin slices at a pressure of 12.5 MPa for >1 min using a tablet press. Then, scan in the range of 4000 cm -1 -400 cm -1 .

[0059] 4. Transmission electron microscopy (TEM) analysis

[0060] The morphology of SL was imaged using transmission electron microscopy (TEM) at 100 kV with negative staining method. First, dilute the liposomes with Tris buffer (37 °C; pH = 7.4) at a ratio of 1:10. Take 20 μL of the diluted sample and smear it on a carbon-coated copper grid. Then, after standing for 5 minutes, use forceps to clamp a filter paper to remove the excessive solution, and then negatively stain the sample with 2% (w / v) phosphotungstic acid staining agent. After staining, the sample is left to stand at room temperature for 2 minutes, and can be observed and photographed after drying.

[0061] The results are as follows Figure 3 (A) shows that the SL liposomes were characterized by dynamic light scattering (DLS). The average size of SL was 257.8 ± 0.56 nm, which was less than 300 nm and had a uniform distribution. At the same time, the PDI was 0.204, indicating that the prepared SL liposomes had good stability.

[0062] The Zeta potential of the prepared SRP liposomes was 67.07 mV, indicating good stability. By measuring the encapsulation efficiency of SL, the encapsulation efficiency of the liposomes for SRP was calculated to be 68.2% ± 1.57%, indicating that the liposomes had a good loading of the oligopeptide SRP.

[0063] As shown in Figure 3 (B), the particle size observed by TEM was consistent with the measurement results of DLS. SL presented as closed spherical vesicles and was well dispersed in the suspension. In addition, in order to better observe the morphology of the liposomes, rhodamine B was used instead of SRP and observed under a fluorescence microscope. Except for adding a layer of coating on the surface of SL, the chitosan coating did not change the morphology of the nanoparticles.

[0064] The results of infrared spectroscopy analysis are as shown in Figure 3 (C). There was a primary amine in the SRP structure, and its typical absorption peak was located at 3266 cm -1 . At the same time, characteristic peaks of SRP were also observed at 1666 cm -1 , 1454 cm -1 and 1184 cm -1 . Among them, the peak at 1666 cm -1 was attributed to the stretching vibration of C=O, the peak at 1184 cm -1 was related to the carboxyl functional group of SRP, and 3266 cm -1 represented the O-H stretching vibration of SRP. For the blank liposomes, characteristic peaks were observed at 3423 cm -1 (O-H stretching vibration), 1737 cm -1 (C=O stretching vibration) and 1460 cm -1 (CH2 shear vibration). At the same time, the symmetric and asymmetric stretching vibrations of PO 2- resulted in characteristic peaks at 1056 cm -1 and 1201 cm -1 , which were the characteristic peaks of lecithin. The infrared spectra of SL and CL were almost the same, and no characteristic peaks of SRP were observed, indicating that SRP was well encapsulated in the liposomes and thus did not show signal peaks. However, after chitosan coating, the lecithin characteristic peak at 1735 cm -1 in CL shifted to 1737 cm -1 and 1678 cm-1 (SL). This transformation may be due to the -NH of chitosan 3+ and the PO of phosphatidylcholine 2- resulting from the electrostatic interaction between them, which is consistent with the results of Zeta potential and TEM.

[0065] 5. Study on the particle size stability of liposomes

[0066] Storage stability: The storage stability was studied in an environment of 4°C. Three parallel samples were placed under each storage condition, and the particle size, PDI, and zeta potential were measured by regular sampling.

[0067] In view of the necessity of long-term storage of soybean lecithin under low-temperature conditions, the storage stability of SL liposomes at 4°C was studied. During storage, as shown Figure 4 in the figure, with the extension of storage time, the particle size and PDI of SL also increased correspondingly. It is worth noting that no obvious crystal precipitation occurred in the liposomes during the whole storage period. After 28 days of storage, the particle size of SL increased from 264.1 nm to 444.2 nm, indicating that SL liposomes have good stability and can be used as an effective carrier system for SRP delivery.

[0068] Example 4 Biosafety analysis of oligopeptide SRP nanoliposomes

[0069] 1. Cytotoxicity analysis: For cell viability detection, a 96-well plate was used. The cell suspension was diluted to 10 5 cells / mL, and 100 μL of the sample was added to each well of the 96-well plate, so that the cell concentration in the well plate was 10 4 cells / well. The side wells were filled with PBS, and the plate was placed in a cell culture incubator at 37°C for 24 h until the cells grew to cover the bottom of the wells. Then, different drugs were added according to the experimental grouping and incubated for 24 h. The culture medium was discarded, and the wells were carefully rinsed once with PBS. Then, 10% CCK-8 culture solution prepared with complete medium containing 1% FBS was added, and the plate was placed in a 37°C incubator and continued to be cultured for 2 h. The plate was gently shaken to fully dissolve the product, and the absorbance of each well was measured at a wavelength of 450 nm using a fully automatic enzyme-linked detector. The number of live cells was calculated according to the absorbance values of each group.

[0070] The results of the cell viability experiment are as shown in Figure 5 (A). When the concentration range was 1 - 10 mg / mL, the survival rate of HK-2 cells cultured with SL was higher than 125.2%. According to the ISO10993-5 standard, when the cell survival rate is higher than 75%, it is considered to be non-cytotoxic. Therefore, the cell survival rates of all experimental groups were higher than this threshold, indicating that SL has good safety for HK-2 cells and can be considered a safe biomaterial.

[0071] 2. Hemolytic analysis: The hemolytic activity of the SL nanosystem at different concentrations was evaluated by the release of hemoglobin from mouse blood cells. Blood was collected from the mouse heart into a heparin-containing centrifuge tube and centrifuged at 3000 rpm for 10 min, and then washed three times with physiological saline. The purified red blood cells were resuspended in physiological saline to obtain a 2% (v / v) red blood cell suspension. The samples were successively added to the 2% red blood cell suspension, incubated in an incubator at 37 °C for 1 h, and then the morphology of the red blood cells was observed with an optical microscope. The mixture was centrifuged at 3000 rpm for 15 min. The supernatant was collected and the absorbance was measured at a wavelength of 540 nm. The hemolysis rate of red blood cells was calculated using the following formula 5-2:

[0072]

[0073] where A Sample 、A Saline and A Water represent the absorbances of the substance sample solution, physiological saline solution, and ultrapure aqueous solution at 540 nm, respectively.

[0074] The results are shown in Figure 5 (B, C). By evaluating the hemolytic activity of the SL nanoliposome system at different concentrations, it was found that at lower concentrations, the morphology of red blood cells was good and the release of hemoglobin was low. Only at higher concentrations did some mouse blood cells show inward concavity in morphology, indicating that the nanoliposomes prepared by the present invention have high biocompatibility and are suitable for in vivo applications.

[0075] 3. In vivo imaging analysis

[0076] To evaluate the in vivo biodistribution and kidney targeting efficacy of SL, according to the method for preparing liposomes in Example 1, the fluorescent dye rhodamine B was used to replace SRP to prepare a preparation loaded with rhodamine B, which was freeze-dried and stored. Near-infrared imaging: Healthy male C57BL / 6 mice were injected with rhodamine B-loaded liposomes (0.4 mg / kg) via the tail vein. Four hours after injection, the mice were sacrificed by cervical dislocation, and the heart, liver, spleen, lungs, and kidneys were immediately dissected and exposed. The in vivo time-course distribution of the marker was observed by a small animal in vivo imaging system (excitation wavelength 540 nm, emission wavelength 580 nm).

[0077] In the present invention, the fluorescent agent rhodamine B was used to replace SRP to prepare chitosan liposomes. Different doses of rhodamine B liposomes were injected into C57BL / 6 mice via the vein, and the biodistribution was evaluated using a small animal in vivo imager. Ex vivo fluorescence imaging is shown in Figure 6As shown, the rhodamine B-labeled liposomes selectively accumulate in the kidneys, and no significant fluorescence is observed in other tissues. The targeting of the drug is positively correlated with the fluorescence intensity, and the accumulation amount of the drug in the kidney tissue has a linear relationship with the administration dose. This indicates that the small-sized chitosan liposomes have the targeting ability to the kidneys.

[0078] The embodiments described above are only for describing the preferred mode of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A preparation method of a kidney-targeted small molecule polypeptide nanoliposome, characterized in that: After adding dichloromethane to soy lecithin and cholesterol and dissolving them by ultrasonic treatment, the solution was slowly dropped into PBS containing small molecule peptides under continuous stirring. After rotary evaporation of the organic solvent dichloromethane, PBS buffer was added to the formed film. After ultrasonic treatment, the film was eluted, and chitosan was added. After magnetic stirring, liposomes were obtained.

2. The preparation method according to claim 1, characterized in that: The mass ratio of the small molecule peptide to soy lecithin is 1:5 to 15, and the mass ratio of soy lecithin to cholesterol is 1 to 10:

1.

3. The preparation method according to claim 1, characterized in that: The pH of the PBS is 5.0 to 9.0, and the ultrasonic treatment time is 10 to 90 min.

4. The preparation method according to claim 1, characterized in that: The chitosan includes low molecular weight chitosan and high molecular weight chitosan, and the high molecular weight chitosan and low molecular weight chitosan are coated layer by layer according to a mass ratio of 10 to 0:0 to 10.

5. A small molecule peptide nano-liposome prepared by the preparation method according to any one of claims 1 to 4.

6. Use of a small molecule peptide nano-liposome prepared by the preparation method according to any one of claims 1 to 4 in the preparation of a drug for treating kidney injury.

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