Amphotericin B nano-composite based on sphingosine coupled polyethylene glycol carrier as well as preparation method and application of amphotericin B nano-composite

Abstract

The invention relates to an amphotericin B nano-composite based on sphingosine coupled polyethylene glycol carrier as well as a preparation method and application thereof, and belongs to the technical field of pharmaceutical preparations. The water-soluble sphingosine-polyethylene glycol conjugate is obtained by carrying out coupling reaction on amino on sphingosine and polyethylene glycol activated ester. The amphotericin B / sphingosine nano-composite is used as a carrier material, and forms a nano-composite in a highly aggregate form with drug molecules under the intermolecular acting force of amphotericin B and sphingosine. Compared with the prior art, the amphotericin B compound can effectively reduce hemolytic toxicity, kidney cell toxicity and acute toxicity of amphotericin B, and has good in-vivo and in-vitro antifungal activity. In addition, the invention also provides a preparation method of the carrier and the nano-composite, and the carrier and the nano-composite have a wide market prospect in the aspect of treating systemic deep fungal infection.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparation preparation, and specifically relates to an amphotericin B nanocomposite and a preparation method and application thereof. Background Art

[0002] Amphotericin B (AmB) is a broad-spectrum, potent polyene antifungal drug that has long been the "gold standard" for the treatment of invasive fungal infections. However, it can cause nephrotoxicity, hemolytic toxicity, electrolyte disturbances, and infusion-related adverse reactions in clinical applications, which limits its application. Currently, the main commercial preparations include amphotericin B deoxycholate (AmB-D), amphotericin B liposomes (L-AmB, Ambisome), amphotericin B colloidal dispersion (ABCD), and amphotericin B lipid complex (ABLC). These preparations have significant differences in microstructure, physicochemical properties, in vivo metabolism, and adverse reactions. Among them, L-AmB preparations have strong binding force between drugs and carrier materials, high stability, and low acute toxicity. At present, only ABCD generic drugs are on the market in China, while L-AmB generic drugs are still in the application stage.

[0003] Due to the difference in carrier materials, the UV-visible absorption graphs of various amphotericin B preparation solutions are obviously different, but they all contain 4 main absorption peaks, of which 320-330nm and 330-340nm are soluble aggregates, 340-350nm are non-covalently bound dimers or polymers, and 410-420nm are characteristic absorptions in the form of monomers. Therefore, the ratio of the absorption peak intensities at 320-330nm and 410-420nm is used as an indicator to measure the aggregation degree of amphotericin B molecules. DSPG and amphotericin B molecules in Ambisome form a highly aggregated state, with a maximum absorption peak at around 320nm. The maximum absorption peak of the aggregate formed by cholesterol sulfate and amphotericin B molecules in ABCD shifts to the right to around 330nm. Polyethylene glycol-phosphatidylethanolamine and amphotericin B molecules can form different forms such as monomers, dimers or polymers. Studies have found that the acute toxicity of each preparation is related to the aggregate form of drug molecules. Acute toxicity tests on mice found that when Ambisome was administered at a dose of 60 mg / kg or above (up to 100 mg / kg), no mice died within 2 weeks after a single administration, while other commercially available preparations could cause death in mice at a dose of 20 mg / kg, and some preparations could even cause death in mice 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 polyethylene glycol active ester, sphingosine and organic base and dissolve them in an organic solvent, stir at 20-40°C for 1-12h under N2 protection, evaporate and remove the solvent to obtain a reactant, add a mixed solution (for dissolution) with a volume ratio of dichloromethane:methanol = 7:1 to dissolve the reactant for column chromatography. 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 and remove the solvent, and vacuum dry to obtain a sphingosine-coupled polyethylene glycol carrier.

[0013] Further preferably, the polyethylene glycol active ester in the step is methoxy polyethylene glycol succinimide ester (MPEG 2000 -NHS);

[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 with a volume ratio of ethanol:chloroform = 1:1;

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

[0018] The mass volume ratio of the polyethylene glycol active ester to the organic solvent is 1:10-30, mg / ml;

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

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

[0021] 4. The method for preparing the above-mentioned amphotericin B nanocomplex based on sphingosine coupled to a polyethylene glycol carrier comprises the following steps:

[0022] Amphotericin B and sphingosine-coupled polyethylene glycol carrier were weighed and dispersed in a mixed solvent with a volume ratio of methanol: dichloromethane = 1:1, 0.1 mol / l dilute hydrochloric acid was added until the drug was dissolved, and then a hydrogenated soybean phosphatidylcholine and cholesterol solution dissolved in a mixed solvent with a volume ratio of methanol: dichloromethane = 1:1 were added, mixed evenly, the solvent was evaporated under reduced pressure at 60-65° C., and then an organic base aqueous solution at 60-65° C. was added to adjust the pH of the solution to 5-6, and a suspension was obtained after stirring and dispersing at 60-65° C. for 1.5-2.5 hours, and an amphotericin B nanocomplex solution was obtained after high-pressure homogenization.

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

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

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

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

[0027] 5. Use of the above-mentioned amphotericin B nanocomplex 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, comprising the above-mentioned amphotericin B nanocomplex 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 lyophilized powder injection.

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

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

[0033] 2. The present invention also discloses a sphingosine-coupled polyethylene glycol carrier and a method for preparing an amphotericin B nanocomplex based on the carrier. The preparation method is simple and can be prepared into a liposome preparation, which is highly feasible for product development.

[0034] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

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

[0037] Figure 2 The UV-visible absorption spectra of amphotericin B nanocomplex, amphotericin B and control preparation;

[0038] Figure 3 The in vitro drug release curves of amphotericin B nanocomplex and control preparation;

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

[0040] Figure 5 The results of Cryptococcal fungal load experiments in the lungs and brains of infected mice;

[0041] Figure 6 The results of the Candida albicans fungal load experiment in the kidneys of infected mice;

[0042] Figure 7 The chemical structure diagram of sphingosine coupled to polyethylene glycol carrier. DETAILED DESCRIPTION

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

[0044] Example 1

[0045] The method for preparing a sphingosine-coupled polyethylene glycol carrier comprises the following steps:

[0046] Weigh 1g MPEG 2000 -NHS, 317mg phytosphingosine and 101mg triethylamine were dissolved in 20ml of a mixed solvent with a volume ratio of methanol: dichloromethane = 1:1, stirred at 30°C for 6h under N2 protection, and the solvent was evaporated to obtain the reactant, and 5ml of a mixed solution with a volume ratio of dichloromethane: methanol = 7:1 was added to dissolve the reactant for column chromatography. Silica gel powder was used as the stationary phase and a mixed solution with a volume ratio of dichloromethane: methanol = 7:1 was used as the mobile phase. The reactant was separated and purified by column chromatography, the eluent was collected, the solvent was evaporated, and vacuum dried to obtain a sphingosine-coupled polyethylene glycol carrier, which 1 H NMR (400MHz) spectrum is attached Figure 1 shown.

[0047] Example 2

[0048] Preparation of an amphotericin B nanocomplex based on sphingosine coupled to a polyethylene glycol carrier specifically comprises the following steps:

[0049] 100 mg of amphotericin B and 250 mg of sphingosine-coupled polyethylene glycol carrier were weighed and dispersed in 130 ml of a mixed solvent with a volume ratio of methanol: dichloromethane = 1:1, 2.8 ml of 0.1 mol / l dilute hydrochloric acid was added until the drug was dissolved, and then 45 ml of a solution of hydrogenated soybean phosphatidylcholine (426 mg) and cholesterol (106 mg) dissolved in a mixed solvent with a volume ratio of methanol: dichloromethane = 1:1 was added, mixed evenly, and the solvent was removed by reduced pressure evaporation at 60°C, and then 20 ml of a 10% triethanolamine aqueous solution preheated to 60°C was added to adjust the pH of the solution to 5-6, and a suspension was obtained after stirring and dispersing at 60°C for 2 hours, and an amphotericin B nanocomposite solution was obtained after high-pressure homogenization. Then 1.8 g of trehalose was added to dissolve and clarify, and after mixing evenly, it was filtered through a 0.45 μm filter membrane, and the filtrate was collected and freeze-dried to obtain amphotericin B nanocomposite freeze-dried powder.

[0050] Example 3

[0051] The performance of the amphotericin B nanocomplex prepared in Example 2 was tested:

[0052] 1. Particle size and zeta potential determination of amphotericin B nanocomplexes

[0053] Weigh 10 mg of amphotericin B nanocomplex lyophilized powder, add pure water to reconstitute and dilute to a certain concentration, draw an appropriate amount of sample into the sample pool, and use 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 encapsulation efficiency and drug loading of amphotericin B nanocomplexes

[0055] Weigh 5 mg of amphotericin B nanocomplex lyophilized powder into a 25 ml volumetric flask, add 1 ml of DMSO for ultrasonic dispersion, then add methanol to dilute to the mark, and use HPLC to determine the content of amphotericin B in the solution. The drug loading (LC) and encapsulation efficiency (EE) calculation formulas are: drug loading (LC, %) = encapsulated drug mass / mass of drug-loaded liposomes × 100; encapsulation efficiency (EE, %) = encapsulated drug mass / feed drug mass × 100. The measurement results show that the encapsulation efficiency of amphotericin B nanocomplex is 93.01 ± 0.73%, and the drug loading is 3.48 ± 0.34%.

[0056] 3. UV-visible absorption spectrum measurement

[0057] Weigh appropriate amounts of amphotericin B nanocomplex lyophilized powder, domestic ABCD preparation, and imported Ambisome preparation, add pure water to reconstitute and dilute to a certain concentration, place in a UV-visible spectrophotometer for detection, and set the scanning wavelength range to 300-450nm. The measurement results are shown in the attached Figure 2 As shown, compared with Ambisome, the maximum absorption peak of ABCD shifts to the right, while the maximum absorption peak position of the amphotericin B nanocomplex prepared by us is consistent with that of Ambisome, reflecting a similar highly aggregated state.

[0058] 4. In vitro drug release study

[0059] Amphotericin B nanocomplex, ABCD domestic preparation and Ambisome imported preparation solution with a concentration of 1 μg / ml were prepared respectively, placed in a shaker at 37°C, and the UV-visible absorption curve was scanned at 0, 1, 3, 6, 12 and 24 hours, with a wavelength range of 300-450 nm. The ratio (a / b) of the maximum absorption peak intensities at 320-340 nm (a) and 400-420 nm (b) was calculated. The change in the absorption peak ratio reflects the process of amphotericin B from aggregates to monomers, that is, the release process. As can be seen from the results (see Appendix Figure 3 ), the release process of the above preparations is biphasic, with a faster release in the first hour and then gradually slowing down. The cumulative amount of amphotericin B nanocomplex in 24 hours is close to that of Ambisome, while the release rate of ABCD is faster, with a cumulative release rate of 85.7% in 24 hours.

[0060] 5. In vitro hemolysis test

[0061] Weigh the amphotericin B nanocomplex lyophilized powder and ABCD preparation respectively, reconstitute with pure water and dilute to different drug concentrations with 5% glucose solution, then mix evenly with 2% red blood cell suspension at a volume ratio of 1:1, pure water plus 2% red blood cell suspension as positive control, 5% glucose solution plus 2% red blood cell suspension as negative control, the final drug concentration of the sample solution is 800, 400, 200, 100, 50, 25 and 12.5 μg / ml, the mixed sample is placed in a 37°C constant temperature incubator for 3 hours, then take out the sample and centrifuge at 2000rpm for 10 minutes, observe the hemolysis of red blood cells, and take 200 μl of supernatant to measure the absorbance value, and set the wavelength to 574nm. Hemolysis rate (%) = (A sample-A negative control) / (A positive control-A negative control).

[0062] The experimental results showed that the amphotericin B nanocomplex had no hemolytic toxicity at a drug concentration of 800 μg / ml or less, while obvious hemolysis was observed in the ABCD group. At a drug concentration of 400 μg / ml, the in vitro hemolysis rate of ABCD reached 22.6%, and when the concentration increased to 800 μg / ml, its hemolysis rate was as high as 100%. This result shows that the nanocomplex can significantly reduce the hemolytic toxicity of amphotericin B.

[0063] 6. In vitro pharmacodynamic evaluation

[0064] Amphotericin B nanocomplex lyophilized powder and ABCD preparation were weighed separately, reconstituted with pure water and diluted to different drug concentrations with 5% glucose solution. 40 μl of preparation solution was added to a 96-well plate, and then 160 μl of bacterial solution (1.25×10 3CFU / ml), 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 no drug, and a positive control well was set up with drug but no bacteria. The 96-well plate was placed in a 30°C constant temperature incubator for culture, and the fungal growth was observed after 48 hours. It can be seen from the experimental results (Table 1) that the amphotericin B nanocomplex has a good in vitro antifungal effect.

[0065] Table 1 Evaluation of in vitro antifungal activity

[0066]

[0067] 7. Evaluation of renal cell toxicity

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

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

[0070] 8. Acute toxicity test

[0071] Healthy KM mice were randomly divided into groups of 6 mice each. The drug was injected into the tail vein of the mice at a dose of 40 mg / kg. The symptoms of the mice in each group and the deaths within 14 days were observed and recorded. 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 (see attached). Figure 4 ). When the dosage was within the range of 5-30 mg / kg, no mice died.

[0072] 9. In vivo pharmacodynamic evaluation 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 solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in 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 below:

2. The method for preparing the sphingosine-coupled polyethylene glycol carrier according to claim 1, characterized in that: The reaction formula of the method is:

3. The preparation method according to claim 2, characterized in that: The method comprises the steps of: Weigh polyethylene glycol active ester, sphingosine and organic base and dissolve them in an organic solvent, stir at 20-40°C for 1-12h under N2 protection, evaporate and remove the solvent to obtain a reactant, add a mixed solution (for dissolution) with a volume ratio of dichloromethane:methanol = 7:1 to dissolve the reactant for column chromatography. 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 and 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: The polyethylene glycol active ester in the step is methoxy polyethylene glycol succinimide ester (MPEG) with a molecular weight of 2000. 2000 -NHS), 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 polyethylene glycol active ester, sphingosine and the organic base is 1:1.5~3:1.5~3, the mass volume ratio of the polyethylene glycol active ester and the organic solvent is 1:10~30, mg / ml, and the mass volume ratio of the polyethylene glycol active ester and a mixed solution with a volume ratio of dichloromethane:methanol=7:1 (for dissolution) is 1:5~10, mg / ml.

5. An amphotericin B nanocomplex based on sphingosine coupled to a polyethylene glycol carrier, characterized in that: The molar ratio of amphotericin B to the sphingosine-coupled polyethylene glycol carrier in the nanocomposite is 1:0.8-3.

6. The method for preparing amphotericin B nanocomplex based on sphingosine coupled to polyethylene glycol carrier according to claim 5, characterized in that: The method comprises the steps of: Amphotericin B and sphingosine-coupled polyethylene glycol carrier were weighed and dispersed in a mixed solvent with a volume ratio of methanol: dichloromethane = 1:1, 0.1 mol / l dilute hydrochloric acid was added until the drug was dissolved, and then a hydrogenated soybean phosphatidylcholine and cholesterol solution dissolved in a mixed solvent with a volume ratio of methanol: dichloromethane = 1:1 were added, mixed evenly, and the solvent was removed by reduced pressure evaporation at 60-65°C, and then an organic base aqueous solution preheated to 60-65°C was added to adjust the pH of the solution to 5-6, and a suspension was obtained after stirring and dispersing at 60-65°C for 1.5-2.5 hours, and an amphotericin B nanocomplex solution was obtained after high-pressure homogenization.

7. The preparation method according to claim 6, characterized in that: The mass volume ratio of the 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, the mass volume ratio of the cholesterol to the methanol:dichloromethane mixed solvent is 5:1-3, mg / ml, the molar ratio of the amphotericin B to hydrogenated soybean phosphatidylcholine and cholesterol is 1:5-6:2-3, the organic base is any one of triethanolamine, tromethamine, and ethylenediamine, and the mass volume of the amphotericin B to the organic base aqueous solution is 5:1-5, mg / ml.

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

9. A pharmaceutical composition, characterized in that A nanocomplex of amphotericin B based on a sphingosine-coupled polyethylene glycol carrier as claimed in claim 5.

10. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition comprises a lyophilization protectant, and the dosage form of the pharmaceutical composition is a lyophilized powder injection.

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