An amphotericin B nanocomplex based on a sphingosine-coupled polyethylene glycol carrier, its preparation method and applications

Abstract

The present invention relates to an amphotericin B nanocomplex based on a sphingosine-coupled polyethylene glycol carrier, and a preparation method and application thereof, belonging to the technical field of pharmaceutical preparations. In the present invention, an amino group on sphingosine is coupled with a polyethylene glycol active ester through a coupling reaction to obtain a water-soluble sphingosine-polyethylene glycol conjugate. Using this as a carrier material, under the intermolecular force between amphotericin B and sphingosine, a nanocomplex in the form of a highly aggregated body is formed with the drug molecule. Compared with the prior art, the present invention can effectively reduce the hemolytic toxicity, renal cell toxicity and acute toxicity of amphotericin B, and at the same time has good in vitro and in vivo antifungal activities. In addition, the present invention also provides a preparation method for the carrier and the nanocomplex, which has broad market prospects in the treatment of systemic deep fungal infections.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparation, and particularly relates to an amphotericin B nano - complex, a preparation method thereof and an application thereof. Background Art

[0002] Amphotericin B (AmB) is a broad - spectrum and potent polyene antifungal drug and has always been the "gold standard" for the treatment of invasive fungal infections. However, in clinical applications, it can cause nephrotoxicity, hemolytic toxicity, electrolyte disorders, infusion - related adverse reactions, etc., which limits its application to a certain extent. Currently, the commercially available preparations mainly include amphotericin B deoxycholate (AmB - D), amphotericin B liposome (L - AmB, Ambisome), amphotericin B colloidal dispersion (ABCD), and amphotericin B lipid complex (ABLC). These several preparations have significant differences in microstructure, physicochemical properties, in - vivo metabolism and adverse reactions. Among them, the L - AmB preparation has a strong binding force between the drug and the carrier material, high stability and low acute toxicity. Currently, only the ABCD generic drug is on the market in China, while the L - AmB generic drug is still in the declaration stage.

[0003] Due to different carrier materials, the ultraviolet - visible absorption spectra of each amphotericin B preparation solution are significantly different, but all contain 4 main absorption peaks. Among them, the 320 - 330 nm and 330 - 340 nm peaks are for soluble aggregates, the 340 - 350 nm peak is for non - covalently bound dimers or polymers, and the 410 - 420 nm peak is the characteristic absorption of the monomer form. Therefore, the ratio of the absorption peak intensities at 320 - 330 nm and 410 - 420 nm is used as an index to measure the aggregation degree of amphotericin B molecules. In Ambisome, DSPG forms a highly aggregated state with amphotericin B molecules, and the maximum absorption peak is around 320 nm. In ABCD, the maximum absorption peak of the aggregate formed by cholesterol sulfate and amphotericin B molecules shifts to around 330 nm. Polyethylene glycol - phosphatidylethanolamine can form different forms such as monomers, dimers or polymers with amphotericin B molecules. It is found that the acute toxicity of each preparation is somewhat related to the aggregate form of the drug molecule. Through the acute toxicity test of mice, it is found that when the administration dose of Ambisome is 60 mg / kg or more (up to 100 mg / kg), no mice die within 2 weeks after a single administration, while other commercially available preparations can cause mouse death at a dose of 20 mg / kg, and some preparations can even cause mouse death at a low dose of 1 mg / kg.

[0004] As a classic L-AmB preparation, Ambisome has obvious advantages over other preparations in improving the hemolytic toxicity, cytotoxicity and acute toxicity of drugs. Among them, the highly aggregated form formed by DSPG phospholipids and amphotericin B molecules is crucial. Limited by the difficulty of substituting DSPG and the high price, as well as the complex preparation process, it is of great significance to develop a liposomal preparation of amphotericin B with high cost performance and beneficial to patients. After a large number of experiments, we screened a sphingosine-coupled polyethylene glycol carrier material, which can not only promote the solubility of amphotericin B in organic solvents, but also form a highly aggregated form similar to Ambisome with amphotericin B molecules, thereby achieving efficient drug encapsulation, delaying the release rate of drugs, effectively reducing the hemolytic toxicity, renal cytotoxicity and acute toxicity of amphotericin B, prolonging the circulation time of drugs in the blood, reducing the distribution of drugs in tissues and organs, and having good antifungal activity in vivo and in vitro. In addition, the preparation method of the amphotericin B nanocomplex is simple, and it can be prepared into a liposomal preparation, which is very feasible for product development. Summary of the invention

[0005] In view of this, one of the objects of the present invention is to provide a sphingosine-coupled polyethylene glycol carrier; the second object of the present invention is to provide a method for preparing a sphingosine-coupled polyethylene glycol carrier; the third object of the present invention is to provide an amphotericin B nanocomplex based on a sphingosine-coupled polyethylene glycol carrier; the fourth object of the present invention is to provide a method for preparing an amphotericin B nanocomplex based on a sphingosine-coupled polyethylene glycol carrier; the fifth object of the present invention is to provide an application of an amphotericin B nanocomplex based on a sphingosine-coupled polyethylene glycol carrier in the treatment of fungal infectious diseases; the sixth object of the present invention is to provide a pharmaceutical composition of an amphotericin B nanocomplex based on a sphingosine-coupled polyethylene glycol carrier.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] 1. A sphingosine-coupled polyethylene glycol carrier, the structural formula of the carrier is shown below:

[0008]

[0009] 2. The preparation method of the above-mentioned sphingosine-coupled polyethylene glycol carrier, the reaction formula of the method is:

[0010]

[0011] Preferably, the method comprises the following steps:

[0012] Weigh poly(ethylene glycol) active ester, sphingosine and organic base, dissolve them in an organic solvent, stir at 20 - 40 °C for 1 - 12 h under N₂ protection, evaporate the solvent to obtain the reactant, add a mixed solution of dichloromethane:methanol = 7:1 (for dissolution) to dissolve the reactant for column chromatography. Use silica gel powder as the stationary phase and a mixed solution of dichloromethane:methanol = 7:1 as the mobile phase, separate and purify the reactant by column chromatography, collect the eluate, evaporate the solvent, and dry it under vacuum to obtain the sphingosine-coupled poly(ethylene glycol) carrier.

[0013] Further preferably, the poly(ethylene glycol) active ester in the step is methoxypoly(ethylene glycol) succinimidyl ester (MPEG 2000 -NHS) with a molecular weight of 2000;

[0014] The sphingosine is phytosphingosine;

[0015] The organic base is triethylamine or N,N-diisopropylethylamine;

[0016] The organic solvent is ethanol or a mixed solution of ethanol:chloroform = 1:1 by volume;

[0017] The molar ratio of the poly(ethylene glycol) active ester, sphingosine and organic base is 1:1.5 - 3:1.5 - 3;

[0018] The mass-volume ratio of the poly(ethylene glycol) active ester and the organic solvent is 1:10 - 30, mg / ml;

[0019] The mass-volume ratio of the poly(ethylene glycol) active ester and the mixed solution of dichloromethane:methanol = 7:1 (for dissolution) is 1:5 - 10, mg / ml.

[0020] 3. An amphotericin B nanocomplex based on a sphingosine-coupled poly(ethylene glycol) carrier, wherein the molar ratio of amphotericin B to the sphingosine-coupled poly(ethylene glycol) carrier in the nanocomplex is 1:0.8 - 3.

[0021] 4. A preparation method of the above amphotericin B nanocomplex based on a sphingosine-coupled poly(ethylene glycol) carrier, the method comprising the following steps:

[0022] Weigh amphotericin B and sphingosine-coupled polyethylene glycol carrier and disperse them in a mixed solvent with a volume ratio of methanol:dichloromethane = 1:1. Add 0.1 mol / l dilute hydrochloric acid until the drug dissolves, then add a solution of hydrogenated soy phosphatidylcholine and cholesterol dissolved in a mixed solvent with a volume ratio of methanol:dichloromethane = 1:1. Mix evenly, evaporate the solvent under reduced pressure at 60 - 65 °C, then add an aqueous solution of organic base at 60 - 65 °C to adjust the pH of the solution to 5 - 6, and stir and disperse for 1.5 - 2.5 h at 60 - 65 °C to obtain a suspension. After high-pressure homogenization, an amphotericin B nanocomposite solution is obtained.

[0023] Further preferably, in the step, the mass-volume ratio of amphotericin B to the methanol:dichloromethane mixed solvent and 0.1 mol / l dilute hydrochloric acid is 5:6 - 10:0.13 - 0.18, mg / ml;

[0024] The mass-volume ratio of cholesterol to the methanol:dichloromethane mixed solvent is 5:1 - 3, mg / ml;

[0025] The molar ratio of amphotericin B to hydrogenated soy phosphatidylcholine and cholesterol is 1:5 - 6:2 - 3;

[0026] The organic base is any one of triethanolamine, tromethamine, and ethylenediamine. The mass-volume of amphotericin B to the aqueous solution of organic base is 5:1 - 5, mg / ml.

[0027] 5. Use of the above amphotericin B nanocomposite based on sphingosine-coupled polyethylene glycol carrier in the preparation of a pharmaceutical preparation for inhibiting fungal growth or treating fungal infectious diseases.

[0028] 6. A pharmaceutical composition, which comprises the above amphotericin B nanocomposite based on sphingosine-coupled polyethylene glycol carrier.

[0029] Preferably, the pharmaceutical composition comprises a lyoprotectant;

[0030] Preferably, the dosage form of the pharmaceutical composition is a freeze-dried powder injection.

[0031] The beneficial effects of the present invention are as follows:

[0032] 1. The present invention discloses a sphingosine-conjugated polyethylene glycol carrier and an amphotericin B nanocomplex based on this carrier. By coupling the amino group on sphingosine with polyethylene glycol activated ester, a water-soluble sphingosine-polyethylene glycol conjugate is obtained. Using it as a carrier material to form a nanocomplex in the form of highly aggregated particles with amphotericin B can effectively reduce the hemolytic toxicity, renal cell toxicity and acute toxicity of amphotericin B, prolong the circulation time of the drug in the blood, reduce the distribution of the drug in tissues and organs, and at the same time has good antifungal activity in vitro and in vivo, showing good application prospects in the treatment of fungal infectious diseases.

[0033] 2. The present invention also discloses a preparation method of a sphingosine-conjugated polyethylene glycol carrier and an amphotericin B nanocomplex based on this carrier. This preparation method is simple and can be prepared into liposomal preparations, showing great feasibility for product development.

[0034] Other advantages, objectives and features of the present invention will be elaborated to some extent in the subsequent specification, and to some extent, will be obvious to those skilled in the art based on the study of the following text, or can be learned from the practice of the present invention. The objectives and other advantages of the present invention can be achieved and obtained through the following specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in detail preferably with reference to the accompanying drawings, where:

[0036] Figure 1 is the 1H NMR spectrum of the sphingosine-conjugated polyethylene glycol carrier;

[0037] Figure 2 is the UV-Vis absorption spectra of the amphotericin B nanocomplex, amphotericin B and the control preparation;

[0038] Figure 3 is the in vitro drug release curve of the amphotericin B nanocomplex and the control preparation;

[0039] Figure 4 is the result of the acute toxicity evaluation in mice;

[0040] Figure 5 is the result of the cryptococcal fungal burden experiment in the lungs and brains of infected mice;

[0041] Figure 6 is the result of the candida albicans fungal burden experiment in the kidneys of infected mice;

[0042] Figure 7 is the chemical structural formula diagram of the sphingosine-conjugated polyethylene glycol carrier. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0043] The following specific examples illustrate the embodiments of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the drawings provided in the following examples only illustrate the basic concept of the present invention in a schematic manner. Without conflict, the following examples and the features in the examples can be combined with each other.

[0044] Example 1

[0045] Prepare a sphingosine-coupled polyethylene glycol carrier, which specifically includes the following steps:

[0046] Weigh 1 g of MPEG 2000 -NHS, 317 mg of phytosphingosine and 101 mg of triethylamine are dissolved in 20 ml of a mixed solvent with a volume ratio of methanol:dichloromethane = 1:1. Under N2 protection, stir at 30 °C for 6 h, evaporate to remove the solvent to obtain a reactant, add 5 ml of a mixed solution with a volume ratio of dichloromethane:methanol = 7:1 to dissolve the reactant for use in column chromatography. Using silica gel powder as the stationary phase and a mixed solution with a volume ratio of dichloromethane:methanol = 7:1 as the mobile phase, separate and purify the reactant by column chromatography, collect the eluate, evaporate to remove the solvent, and vacuum dry to obtain the sphingosine-coupled polyethylene glycol carrier, the 1 1H NMR (400 MHz) spectrum is as shown in the appendix Figure 1 as follows.

[0047] Example 2

[0048] Prepare an amphotericin B nanocomplex based on the sphingosine-coupled polyethylene glycol carrier, which specifically includes the following steps:

[0049] Weigh 100 mg of amphotericin B and 250 mg of the sphingosine-coupled polyethylene glycol carrier and disperse them in 130 ml of a mixed solvent with a volume ratio of methanol:dichloromethane = 1:1. Add 2.8 ml of 0.1 mol / l dilute hydrochloric acid until the drug dissolves, then add 45 ml of a solution of hydrogenated soy phosphatidylcholine (426 mg) and cholesterol (106 mg) dissolved in a mixed solvent with a volume ratio of methanol:dichloromethane = 1:1, mix evenly, evaporate to remove the solvent under reduced pressure at 60 °C, then add 20 ml of a preheated 10% triethanolamine aqueous solution at 60 °C to adjust the pH of the solution to 5-6, stir and disperse at 60 °C for 2 h to obtain a suspension, and obtain an amphotericin B nanocomplex solution after high-pressure homogenization. Then add 1.8 g of trehalose to dissolve and clarify, mix evenly, filter through a 0.45 μm filter membrane, collect the filtrate and freeze-dry to obtain the amphotericin B nanocomplex lyophilized powder.

[0050] Example 3

[0051] Perform performance tests on the amphotericin B nanocomplex prepared in Example 2:

[0052] 1. Determination of the particle size and zeta potential of the amphotericin B nanocomplex

[0053] Weigh 10 mg of the freeze-dried powder of the amphotericin B nanocomplex, add pure water to re-dissolve and dilute to a certain concentration. Pipette an appropriate amount of the sample into the sample cell, and use a Malvern laser particle size analyzer to measure its particle size and potential. The average particle size of the nanocomplex is 133.53 nm, and the Zeta potential is 0.95 mV.

[0054] 2. Determination of the encapsulation efficiency and drug loading of the amphotericin B nanocomplex

[0055] Weigh 5 mg of the freeze-dried powder of the amphotericin B nanocomplex and place it in a 25-ml volumetric flask. Add 1 ml of DMSO and disperse it by ultrasound, then add methanol to dilute to the scale. Use HPLC to determine the content of amphotericin B in the solution. The calculation formulas for drug loading (LC) and encapsulation efficiency (EE) are: drug loading (LC, %) = mass of the encapsulated drug / mass of the drug-loaded liposome × 100; encapsulation efficiency (EE, %) = mass of the encapsulated drug / mass of the fed drug × 100. The measurement results show that the encapsulation efficiency of the amphotericin B nanocomplex is 93.01 ± 0.73%, and the drug loading is 3.48 ± 0.34%.

[0056] 3. Ultraviolet-visible absorption spectrum determination

[0057] Weigh appropriate amounts of the freeze-dried powder of the amphotericin B nanocomplex, the domestic ABCD preparation, and the imported Ambisome preparation. Add pure water to re-dissolve and dilute to a certain concentration, and place them in a UV-visible spectrophotometer for detection. Set the scanning wavelength range to 300 - 450 nm. The measurement results are as follows Figure 2 shown. Compared with Ambisome, the maximum absorption peak of ABCD shows a right shift, while the position of the maximum absorption peak of the amphotericin B nanocomplex we prepared is the same as that of Ambisome, indicating a similar highly aggregated state.

[0058] 4. In vitro drug release study

[0059] Prepare amphotericin B nanocomplexes, domestic ABCD preparations, and Ambisome imported preparation solutions with a concentration of 1 μg / ml respectively, place them in a shaker at 37°C and shake. Sampling and scanning the ultraviolet-visible absorption curves are carried out at 0, 1, 3, 6, 12, and 24 h respectively. The wavelength range is 300 - 450 nm. Calculate the ratio (a / b) of the maximum absorption peak intensities at 320 - 340 nm (a) and 400 - 420 nm (b). The change in the absorption peak ratio is used to reflect the process of amphotericin B changing from aggregates to monomers, that is, the release process. It can be seen from the results (attached Figure 3 ) that the external release processes of the above preparations are all biphasic releases, with a faster release within the first 1 h and then gradually leveling off. The cumulative amount of amphotericin B nanocomplexes at 24 h is close to that of Ambisome, while the drug release rate of ABCD is faster, and the cumulative release rate at 24 h is 85.7%.

[0060] 5. In vitro hemolysis test

[0061] Weigh the freeze-dried powder of amphotericin B nanocomplexes and ABCD preparations respectively, reconstitute them with pure water and then dilute them to different drug concentrations with 5% glucose solution. Then mix them evenly with 2% red blood cell suspension at a volume ratio of 1:1. Use pure water plus 2% red blood cell suspension as the positive control, and 5% glucose solution plus 2% red blood cell suspension as the negative control. The final drug concentrations of the sample solutions are 800, 400, 200, 100, 50, 25, and 12.5 μg / ml. Incubate the mixed samples in a constant temperature incubator at 37°C for 3 h, then take out the samples and centrifuge them at 2000 rpm for 10 min. Observe the hemolysis of red blood cells, and pipette 200 μl of the supernatant to measure the absorbance value, with the set wavelength being 574 nm. Hemolysis rate (%) = (A_sample - A_negative control) / (A_positive control - A_negative control).

[0062] The experimental results show that within a drug concentration of 800 μg / ml and below, the amphotericin B nanocomplexes have no hemolytic toxicity, while obvious hemolysis phenomena can be observed in the ABCD group. When the drug concentration is 400 μg / ml, the in vitro hemolysis rate of ABCD reaches 22.6%, and when the concentration increases to 800 μg / ml, its hemolysis rate is as high as 100%. This result indicates that the nanocomplex can significantly reduce the hemolytic toxicity of amphotericin B.

[0063] 6. In vitro pharmacodynamic evaluation

[0064] Weigh the freeze-dried powder of amphotericin B nanocomplexes and ABCD preparations respectively, reconstitute them with pure water and then dilute them to different drug concentrations with 5% glucose solution. Pipette 40 μl of the preparation solution into a 96-well plate, and then add 160 μl of bacterial solution (1.25×10 3(CFU / ml), and the final concentrations of the drugs in the wells were 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / ml, respectively. A negative control well was set up with bacteria but without drugs, and a positive control well was set up with drugs but without bacteria. The 96-well plate was placed in a constant temperature incubator at 30 °C and cultured. After 48 h, it was taken out to observe the fungal growth. It can be seen from the experimental results (Table 1) that the amphotericin B nanocomplex has good in vitro antifungal effects.

[0065] Table 1 Evaluation of in vitro antifungal activity

[0066]

[0067] 7. Evaluation of renal cytotoxicity

[0068] Weigh the freeze-dried powder of the amphotericin B nanocomplex and the ABCD preparation respectively. After reconstituting with pure water, dilute them to different drug concentrations with 5% glucose solution. Weigh amphotericin B and dissolve it in DMSO, and dilute it to different drug concentrations with 5% glucose solution. Weigh HK-2 cells in good growth state, aspirate 160 μl of cell suspension and add it to a 96-well plate, with 3000 cells in each well. The plated cells were placed in an incubator at 37 °C and cultured for 24 h, then 40 μl of the preparation solution was added, and the final concentration of the drug in the well was 200 - 3.125 μg / ml. After culturing the 96-well plate in an incubator at 37 °C for 24 h, add MTT (20 μl) with a concentration of 5 mg / ml, incubate in the incubator for 4 h, then remove the solution in the well, add 150 μl of DMSO solution, and measure the OD value with an enzyme-linked immunosorbent assay (ELISA) reader, with the wavelength set at 490 nm.

[0069] The experimental results showed that amphotericin B has strong renal cytotoxicity, with the IC50 in the range of 6.25 - 12.25 μg / ml, while the amphotericin B nanocomplex and ABCD did not show renal cytotoxicity even at a concentration as high as 200 μg / ml, indicating that the nanocomplex can significantly reduce the renal cytotoxicity of amphotericin B.

[0070] 8. Acute toxicity experiment

[0071] Healthy KM mice were randomly divided into groups of 6 each. The drugs were administered by tail vein injection to the mice, with the dosage of 40 mg / kg. Observe and record the symptoms of each group of mice and the death situation within 14 days. Using the commercially available ABCD preparation as a control, 2 mice in the amphotericin B nanocomplex group died within one week, and 3 mice in the ABCD group died (attached Figure 4 ). When the dosage range was 5 - 30 mg / kg, no mice died.

[0072] 9. Evaluation of pharmacodynamics in mice

[0073] Cryptococcal fungal load experiment in mice: Cryptococcus neoformans H99 was injected into the tail vein to establish a mouse lung and brain fungal infection model. The specific method is as follows:

[0074] (1) Absorb the H99 Cryptococcus culture (5×10 6 CFU / ml), and the model was established by injecting bacterial solution into the tail vein of KM mice (dose 100μl / 20g). 24h after infection, amphotericin B nanocomplex solution and control commercial ABCD preparation were injected into the tail vein, once a day, for 3 consecutive times, with a dose of 5mg / kg. 24h after the third administration, the mice were dissected on a sterile operating table, the lungs and brain tissues were removed, washed with sterilized PBS, and dried with filter paper. PBS was added according to the weight of lung or brain tissue and PBS mass volume ratio of 1:3 (g / ml), and then homogenized and diluted. 50μl was drawn and spread on YPD solid culture medium, placed in a 30℃ incubator for 48h, and the fungal growth in each culture plate was observed, and the colony count was performed.

[0075] From the attached Figure 5 It can be seen that compared with the untreated model group, the amphotericin B nanocomplex has obvious antifungal activity and significantly reduces the number of fungi in the lungs and brain of mice, and there is no significant difference in the therapeutic effect with the commercially available ABCD preparation.

[0076] In vivo Candida albicans fungal load experiment in mice: The mouse kidney fungal infection model was established by tail vein injection of Candida albicans ATCC 90028. The entire experimental method was the same as the Cryptococcal fungal load experiment. The results are shown in the attached Figure 6 Compared with the untreated model group, the amphotericin B nanocomplex had significant antifungal activity and significantly reduced the number of fungi in the kidneys of mice, and there was no significant difference in the therapeutic effect with the commercially available ABCD preparation.

[0077] The above results show that the amphotericin B nanocomplex prepared by the present invention has good in vivo antifungal activity.

[0078] In summary, the present invention obtains a sphingosine-coupled polyethylene glycol carrier, and uses the carrier to encapsulate amphotericin B to form a nanocomposite, and the highly aggregated form thereof can delay the release rate of the drug, effectively reduce the hemolytic toxicity, renal cell toxicity and acute toxicity of amphotericin B, and has good in vitro and in vivo antifungal activity. In addition, the present invention also provides a sphingosine-coupled polyethylene glycol carrier and a method for preparing an amphotericin B nanocomposite based on the carrier, and the prepared amphotericin B nanocomposite has broad market prospects in the treatment of systemic deep fungal infections.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the present technical solution, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A sphingosine-coupled polyethylene glycol carrier, characterized in that, The structural formula of the carrier is shown in the following figure: 。 2. The preparation method of the sphingosine-coupled polyethylene glycol carrier according to claim 1, wherein The reaction formula of the method is: 。 3. The preparation method according to claim 2, wherein The method comprises the following steps: Weigh methoxypolyethylene glycol succinimide ester, sphingosine and organic base, dissolve them in an organic solvent, stir at 20-40 °C for 1-12 h under N2 protection, evaporate to remove the solvent to obtain a reactant, add a mixed solution with a volume ratio of dichloromethane:methanol = 7:1 to dissolve the reactant, wait for column chromatography use. Use silica gel powder as the stationary phase, and a mixed solution with a volume ratio of dichloromethane:methanol = 7:1 as the mobile phase, separate and purify the reactant by column chromatography, collect the eluent, evaporate to remove the solvent, and vacuum dry to obtain a sphingosine-coupled polyethylene glycol carrier.

4. The preparation method according to claim 3, characterized in that, In the step, the methoxypolyethylene glycol succinimide ester is methoxypolyethylene glycol succinimide ester with a molecular weight of 2000, the sphingosine is phytosphingosine, the organic base is triethylamine or N,N-diisopropylethylamine, the organic solvent is ethanol or a mixed solution with a volume ratio of ethanol:chloroform = 1:1, the molar ratio of the methoxypolyethylene glycol succinimide ester, sphingosine and organic base is 1:1.5-3:1.5-3, the mass-volume ratio (mg / ml) of the methoxypolyethylene glycol succinimide ester and the organic solvent is 1:10-30, and the mass-volume ratio (mg / ml) of the methoxypolyethylene glycol succinimide ester and the mixed solution with a volume ratio of dichloromethane:methanol = 7:1 is 1:5-10.

5. An amphotericin B nanocomplex based on a sphingosine-coupled polyethylene glycol carrier, characterized in that, The structural formula of the sphingosine-coupled polyethylene glycol carrier is shown in the following figure: ， The molar ratio of amphotericin B to the sphingosine-coupled polyethylene glycol carrier in the nanocomplex is 1:0.8-3, and the preparation method of the nanocomplex comprises the following steps: Weigh amphotericin B and the sphingosine-coupled polyethylene glycol carrier, disperse them in a mixed solvent with a volume ratio of methanol:dichloromethane = 1:1, add 0.1 mol / l dilute hydrochloric acid until the drug dissolves, then add a solution of hydrogenated soy phosphatidylcholine and cholesterol dissolved in a mixed solvent with a volume ratio of methanol:dichloromethane = 1:1, mix evenly, evaporate to remove the solvent under reduced pressure at 60-65 °C, then add an aqueous solution of organic base preheated to 60-65 °C to adjust the pH of the solution to 5-6, stir and disperse at 60-65 °C for 1.5-2.5 h to obtain a suspension, and obtain an amphotericin B nanocomplex solution after high-pressure homogenization.

6. The amphotericin B nanocomplex according to claim 5, wherein, The mass-volume ratio (mg / ml / ml) of amphotericin B to the methanol:dichloromethane mixed solvent and 0.1 mol / l dilute hydrochloric acid is 5:6-10:0.13-0.18, the mass-volume ratio (mg / ml) of cholesterol to the methanol:dichloromethane mixed solvent is 5:1-3, the molar ratio of amphotericin B to hydrogenated soy phosphatidylcholine and cholesterol is 1:5-6:2-3, the organic base is any one of triethanolamine, tromethamine, ethylenediamine, and the mass-volume ratio (mg / ml) of amphotericin B to the aqueous solution of organic base is 5:1-5.

7. Use of the amphotericin B nanocomplex based on the sphingosine-coupled polyethylene glycol carrier according to claim 5 in the preparation of a pharmaceutical preparation for inhibiting fungal growth or treating fungal infectious diseases.

8. A pharmaceutical composition, characterized in that, Containing the amphotericin B nanocomplex with a sphingosine-coupled polyethylene glycol carrier as described in claim 5.

9. The pharmaceutical composition according to claim 8, wherein, The pharmaceutical composition includes a lyoprotectant, and the dosage form of the pharmaceutical composition is a freeze-dried powder injection.

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