Preparation method and application of amphotericin B sustained release microspheres

Abstract

The invention discloses a preparation method of amphotericin B sustained release microspheres, which comprises the following steps: S1, suspending AmB in methanol to prepare an AmB suspension; s2, dissolving BSA in water to prepare a BSA aqueous solution; s3, uniformly mixing the AmB suspension with the BSA aqueous solution to obtain a water phase; s4, taking liquid paraffin as an oil phase, adding an emulsifier, then adding the water phase into the oil phase under a mechanical stirring condition, emulsifying to obtain W / O type primary emulsion, and naturally volatilizing to remove methanol; s5, a glutaraldehyde solution with the concentration being 8 wt% is added into the primary emulsion for curing; s6, the cured substance is washed and freeze-dried, and the amphotericin B sustained release microspheres are prepared. The amphotericin B sustained-release microsphere can reduce the administration frequency, alleviate the dose-dependent toxic and side effects and reduce the cost of pharmaceutic adjuvants and preparations.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a preparation method and application of amphotericin B sustained-release microspheres. Background Art

[0002] Fungus is a large class of eukaryotic microorganisms with cell walls, cell membranes, nuclei and complete organelles. Fungal infections can be divided into three types according to the site of the disease: superficial infection, subcutaneous tissue infection and deep infection. Superficial fungal infection mainly refers to infections caused by fungi involving the surface of the skin and appendages (such as nails and hair), mainly caused by fungi such as Trichophyton, Microsporum, Candida and Malassezia. Subcutaneous tissue fungal infection refers to infections caused by fungi involving subcutaneous tissue, mainly caused by Sporothrix Schenckii and pigmented fungi. Deep fungal infection is an infection of internal tissues and organs other than the skin, appendages and subcutaneous tissue caused by fungi, mainly caused by Candida, Aspergillus and Cryptococcus. Because deep fungal infection is highly invasive and harmful, this type of disease is collectively referred to as invasive fungal infection (IFI). Among them, Mucor is the most common invasive fungus, which is a conditional pathogen with a high mortality rate. The organs or tissues most commonly affected by Mucor are the lungs, central nervous system, sinuses, and orbits. Gastrointestinal Mucormycosis is relatively rare. The most common sites of gastrointestinal Mucormycosis are the stomach, colon, and ileum, respectively. Depending on the affected site, symptoms may include abdominal pain, diarrhea, gastrointestinal bleeding, jaundice, fever, nausea, and vomiting.

[0003] Treatment of gastrointestinal mucormycosis usually requires early surgery combined with amphotericin B. Amphotericin B (AmB) is a macrocyclic polyene antibiotic with a broad antifungal spectrum, strong activity, and rare drug-resistant strains. It is of great significance in the clinical treatment of IFI. The mechanism of action of AmB is that the drug combines with sterols (mainly ergosterol) on the cell membrane of sensitive fungi to form a sterol-polyene complex, increase the permeability of the cell membrane, form many micropores on the cell membrane, increase the permeability of the cell membrane to monovalent and divalent cations, and cause the leakage of important substances such as potassium ions, nucleotides and amino acids in the cell, thereby destroying the normal metabolism of fungal cells and inhibiting their growth. Due to its unique chemical properties, AmB has low solubility in water and poor oral bioavailability, and is mainly used as an injection. At the same time, AmB has serious toxic side effects on the body, mainly manifested as dose-dependent hemolytic toxicity and nephrotoxicity. This is because human cell membranes contain cholesterol, which is similar in structure to ergosterol in fungal cell membranes. Therefore, AmB can also bind to ergosterol in mammalian cell membranes, and will inevitably interact with cholesterol in human cell membranes to destroy human cells. In order to improve the solubility of AmB, traditional amphotericin B injections add the surfactant sodium deoxycholate for solubilization. Since the toxicity of the drug itself cannot be effectively avoided, various adverse reactions are easily produced during use, and the scope of use is greatly limited. In order to solve the problem of toxic side effects caused by AMB, AmB lipid preparations have entered the clinic. The lipid preparations currently used in the clinic mainly include: amphotericin B liposomes, amphotericin B lipid complexes and amphotericin B colloidal dispersions. Although these preparations can reduce the toxic side effects of AmB, the excipients are expensive and the preparation cost is high, and more administration times are required, which limits their wide application.

[0004] It can be seen that the existing amphotericin B has problems in the treatment of invasive fungal infections, such as dose-dependent hemolytic toxicity, nephrotoxicity and other side effects, high frequency of administration, expensive pharmaceutical excipients and high preparation costs. Summary of the invention

[0005] In view of the above problems existing in the prior art, the present invention provides a method for preparing amphotericin B sustained-release microspheres and their application. The amphotericin B sustained-release microspheres of the present invention can reduce the number of administrations, reduce dose-dependent toxic side effects, and reduce the cost of pharmaceutical excipients and preparations.

[0006] The technical solution of the present invention is as follows:

[0007] The first object of the present invention is to provide a method for preparing amphotericin B sustained-release microspheres, the preparation method comprising the following steps:

[0008] S1. suspending amphotericin B (AmB) in methanol to prepare an amphotericin B (AmB) suspension;

[0009] S2, dissolving bovine serum albumin (BSA) in water to prepare a bovine serum albumin (BSA) aqueous solution;

[0010] S3, mixing the AmB suspension obtained in step S1 with the BSA aqueous solution obtained in step S2, and mixing them well to form the aqueous phase;

[0011] S4, using liquid paraffin as the oil phase, adding an emulsifier, and then slowly adding the water phase obtained in step S3 dropwise into the oil phase under mechanical stirring conditions, emulsifying to obtain W / O type colostrum, and naturally volatilizing to remove methanol;

[0012] S5, adding 8 wt% glutaraldehyde solution to the colostrum obtained in step S4 for solidification;

[0013] S6. Wash and freeze-dry the solidified material in step S5 to obtain amphotericin B sustained-release microspheres.

[0014] In one embodiment of the present invention, in step S1, the concentration of the AmB suspension is 20-500 mg·mL -1 .

[0015] In one embodiment of the present invention, in step S1, the concentration of the AmB suspension is 125-250 mg·mL -1 .

[0016] In one embodiment of the present invention, in step S1, the concentration of the AmB suspension is 166.67 mg·mL -1 .

[0017] In one embodiment of the present invention, in step S2, the concentration of the BSA aqueous solution is 50-250 mg·mL -1 .

[0018] In one embodiment of the present invention, in step S2, the concentration of the BSA aqueous solution is 250 mg·mL -1 .

[0019] In one embodiment of the present invention, in step S3, the mass ratio of AmB to BSA is 1:(2-10).

[0020] In one embodiment of the present invention, in step S3, the mass ratio of AmB to BSA is 1:(4-8).

[0021] In one embodiment of the present invention, in step S3, the mass ratio of AmB to BSA is 1:8.

[0022] In one embodiment of the present invention, in step S4, the emulsifier is one or more of Tween-80 and Span-80.

[0023] In one embodiment of the present invention, in step S4, the amount of the emulsifier relative to the aqueous phase is 0.05-0.2 g / mL.

[0024] In one embodiment of the present invention, in step S4, the amount of the emulsifier relative to the aqueous phase is 0.1 g / mL.

[0025] In one embodiment of the present invention, in step S4, the speed of mechanical stirring is 500-1000 rpm.

[0026] In one embodiment of the present invention, in step S4, the speed of mechanical stirring is 600-900 rpm.

[0027] In one embodiment of the present invention, in step S4, the speed of mechanical stirring is 600 rpm.

[0028] In one embodiment of the present invention, in step S4, the emulsification time is 10-30 minutes.

[0029] In one embodiment of the present invention, in step S4, the emulsification time is 10 minutes.

[0030] In one embodiment of the present invention, in step S4, the volume ratio of the oil phase to the water phase is (10-50):1.

[0031] In one embodiment of the present invention, in step S4, the volume ratio of the oil phase to the water phase is 50:1.

[0032] In one embodiment of the present invention, in step S5, the volume ratio of the 8 wt % glutaraldehyde solution to the aqueous phase is (0.08-10):1.

[0033] In one embodiment of the present invention, in step S5, the volume ratio of the 8 wt % glutaraldehyde solution to the aqueous phase is (2.8-7.6):1.

[0034] In one embodiment of the present invention, in step S5, the volume ratio of the 8 wt % glutaraldehyde solution to the aqueous phase is 2.8:1.

[0035] In one embodiment of the present invention, in step S5, the curing time is 1-2 hours.

[0036] In one embodiment of the present invention, in step S5, the curing time is 1 hour.

[0037] The second object of the present invention is to provide amphotericin B sustained-release microspheres prepared by the above preparation method.

[0038] The third object of the present invention is to provide an application of the above-mentioned amphotericin B sustained-release microspheres for preparing an antibacterial agent.

[0039] The beneficial technical effects of the present invention are:

[0040] (1) The present invention prepares amphotericin B into a sustained-release microsphere dosage form. The sustained-release preparation prolongs the drug's residence time in the body, reduces the number of dosings, and achieves a long-term antifungal effect; on the other hand, it reduces blood drug concentration and alleviates its dose-dependent toxic side effects.

[0041] (2) The present invention prepares sustained-release microspheres by adding non-toxic, harmless and biodegradable bovine serum albumin, and the preparation process is simple, which greatly reduces the cost of pharmaceutical excipients and preparations while also ensuring good biocompatibility of the microspheres.

[0042] (3) The present invention uses mechanical stirring instead of traditional magnetic stirring, which has stronger mechanical force and is conducive to forming microspheres with more uniform and stable particle size distribution; glutaraldehyde solution as a curing agent can make the amphotericin B wrapped by bovine serum albumin form microspheres more quickly.

[0043] (4) The AmB / BSA obtained by the present invention is a full sphere, with an encapsulation efficiency of (57.43±1.15)%, a particle size of (5.82±0.29) μm, and a potential of (-11.9±0.53) mV. The results of in vitro drug release experiments showed that the microspheres can slowly release for 7 days. The results of in vitro antifungal experiments showed that when the drug concentration was 0.25 μg mL -1 The fungal inhibition rate of free AmB was (48.71±1.35)%, while the inhibition rates of AmB / BSA simulated for 1, 3, 5, and 7 days were (27.05±0.87)%, (38.53±0.36)%, (43.68±0.44)% and (46.63±1.07)%, respectively. Conclusion: AmB / BSA has a long-term sustained release effect for 7 days, and its antibacterial effect is not inferior to that of free AmB. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The X-ray diffraction patterns of the raw materials and the prepared microspheres in Example 1 of the present invention;

[0045] Figure 2 The differential scanning calorimetry analysis diagram of the raw materials and the prepared microspheres in Example 1 of the present invention;

[0046] Figure 3 The Fourier transform infrared spectra of the raw materials and the prepared microspheres in Example 1 of the present invention;

[0047] Figure 4 This is a micrograph of AmB / BSA microspheres prepared in Example 1 of the present invention;

[0048] Figure 5 The particle size distribution diagram of AmB / BSA microspheres prepared in Example 1 of the present invention;

[0049] Figure 6 This is the potential diagram of AmB / BSA microspheres prepared in Example 1 of the present invention;

[0050] Figure 7 This is a SEM image of AmB / BSA microspheres prepared in Example 1 of the present invention;

[0051] Figure 8 The in vitro drug release curve of AmB / BSA obtained in Example 1 of the present invention. DETAILED DESCRIPTION

[0052] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0053] Drugs and reagents: bovine serum albumin (Shanghai Sangon Biotechnology Co., Ltd.); potato dextrose agar medium (Qingdao Haibo Biotechnology Co., Ltd.); yeast extract (OXOID); tryptone (OXOID); other reagents were all commercially available analytical grade.

[0054] Test method:

[0055] X-ray diffraction (XRD): XRD was used to analyze the crystal form of the sample; the scanning rate was 5°·min -1 , the 2θ scanning range was 5°-30°.

[0056] Differential scanning calorimetry (DSC): Differential scanning calorimetry (DSC3+) was used to analyze the samples; the temperature detection range was 30°C-260°C, and the heating rate was 10°C min -1 .

[0057] Fourier transform infrared spectroscopy (FT-IR): Take the API and freeze-dried microsphere samples and use Fourier transform infrared spectroscopy to analyze the -1 The test and analysis were carried out within the wavelength range with a resolution of 4cm -1 .

[0058] Scanning electron microscope (SEM): The freeze-dried microsphere particles were glued to the conductive glue on the sample stage, gold was sprayed under vacuum, and the morphology of the sample was observed at an accelerating voltage of 5 kV.

[0059] Particle size and Zeta potential: The prepared microspheres were centrifuged at 5000 rpm for 10 min, the supernatant was removed, and the precipitate was redispersed in an aqueous solution; the particle size distribution and Zeta potential of the product were measured using a Zeta potential and nanoparticle size analyzer.

[0060] Determination of encapsulation efficiency and drug loading: Accurately weigh 5 mg of microspheres, add 15 mL of dimethyl sulfoxide (DMSO) and ultrasonically disrupt them for 10 min, then continue stirring in a dark environment for 48 h. Using DMSO as a control, measure the absorbance at a wavelength of 416 nm, substitute it into the AmB standard curve equation to calculate the content, and calculate the encapsulation efficiency EE and drug loading rate DL according to the formula.

[0061] EE%=W e / W t *100%

[0062] DL%=W e / W m *100%

[0063] Among them, W t , W e and W m They are the dosage, encapsulated drug amount and weighed drug-loaded microspheres.

[0064] Example 1

[0065] The preparation method of AmB / BSA microspheres comprises the following steps:

[0066] (1) Weigh 66.67 mg of AmB and dissolve it in 0.4 ml of methanol to prepare 166.67 mg mL -1 AmB suspension;

[0067] (2) 400 mg BSA was dissolved in 1.6 ml ultrapure water to prepare 250 mg mL -1 of BSA aqueous solution.

[0068] (3) 0.4 mL of AmB suspension was added to 1.6 mL of BSA aqueous solution as the aqueous phase;

[0069] (4) 100 mL of liquid paraffin was taken as the oil phase, 2.8 g of Span-80 and 0.2 g of Tween-80 were added as emulsifiers, and the aqueous phase was slowly added dropwise to the oil phase under mechanical stirring at 600 rpm. The mixture was emulsified for 10 min to obtain W / O colostrum, and methanol was removed by natural evaporation.

[0070] (5) 5.6 mL of 8 wt% glutaraldehyde solution was added, followed by curing for 1 h, centrifugation at 5000 rpm for 10 min, collecting the lower precipitate, washing the microspheres with anhydrous ethanol and water for multiple times, and freeze-drying to obtain AmB / BSA microspheres.

[0071] The encapsulation efficiency of the obtained AmB / BSA microspheres was (57.43±1.15)%.

[0072] Example 2

[0073] Referring to Example 1, the amount of AmB was adjusted to 50 mg, the volume of the 8 wt % glutaraldehyde solution was adjusted to 7.6 mL, and the other contents remained unchanged to obtain the corresponding AmB / BSA microspheres.

[0074] Example 3

[0075] Referring to Example 1, the volume of the 8 wt % glutaraldehyde solution was adjusted to 5.2 mL, the stirring speed was adjusted to 750 rpm, and the other parameters remained unchanged to obtain the corresponding AmB / BSA microspheres.

[0076] Example 4

[0077] Referring to Example 1, the amount of AmB was adjusted to 100 mg, the volume of the 8 wt % glutaraldehyde solution was adjusted to 2.8 mL, the stirring speed was adjusted to 900 rpm, and the other parameters remained unchanged to obtain the corresponding AmB / BSA microspheres.

[0078] Example 5

[0079] Referring to Example 1, the amount of AmB was adjusted to 100 mg, the volume of the 8 wt % glutaraldehyde solution was adjusted to 7.6 mL, the stirring speed was adjusted to 750 rpm, and the other parameters remained unchanged to obtain the corresponding AmB / BSA microspheres.

[0080] Example 6

[0081] Referring to Example 1, the amount of AmB was adjusted to 50 mg, the volume of the 8 wt % glutaraldehyde solution was adjusted to 5.2 mL, the stirring speed was adjusted to 900 rpm, and the other parameters remained unchanged to obtain the corresponding AmB / BSA microspheres.

[0082] Example 7

[0083] Referring to Example 1, the volume of the 8 wt % glutaraldehyde solution was adjusted to 2.8 mL, and the other parameters remained unchanged to obtain the corresponding AmB / BSA microspheres.

[0084] Example 8

[0085] Referring to Example 1, the volume of the 8 wt % glutaraldehyde solution was adjusted to 7.6 mL, the stirring speed was adjusted to 900 rpm, and the other conditions remained unchanged to obtain the corresponding AmB / BSA microspheres.

[0086] Example 9

[0087] Referring to Example 1, the amount of AmB was adjusted to 100 mg, the volume of the 8 wt % glutaraldehyde solution was adjusted to 5.2 mL, and the other contents remained unchanged to obtain the corresponding AmB / BSA microspheres.

[0088] Example 10

[0089] Referring to Example 1, the amount of AmB was adjusted to 50 mg, the stirring speed was adjusted to 750 rpm, and the other parameters remained unchanged to obtain the corresponding AmB / BSA microspheres.

[0090] The embedding effect of AmB / BSA microspheres obtained in Example 2-10 is shown in Table 1.

[0091] Table 1

[0092] Example Encapsulation rate (%) Example 2 43.07±1.01 Example 3 30.63±0.27 Example 4 39.62±0.52 Example 5 44.94±0.81 Example 6 34.56±0.98 Example 7 53.62±1.25 Example 8 54.86±0.90 Example 9 54.51±0.43 Example 10 54.23±0.55

[0093] Test example:

[0094] (1) Characterization of AmB / BSA microspheres:

[0095] The XRD spectrum is used to evaluate the crystal form of the drug. The XRD spectrum of the raw materials and the prepared AmB / BSA microspheres in Example 1 is as follows: Figure 1 As shown, it can be seen from the figure that BSA exhibits a wide range of typical BSA amorphous peaks; amphotericin B raw material has strong absorption at diffraction angles of 9.52, 14.12, 15.36, 17.36, 18.64 and 21.8°, indicating that amphotericin B raw material exists in a crystalline state; the physical mixture of AmB and blank unloaded microspheres simultaneously shows the amorphous peak of BSA and the characteristic peak of AmB; and in the X-ray diffraction pattern of AmB / BSA, the characteristic peak of amphotericin B raw material disappears, indicating that there is an interaction between amphotericin B raw material and the drug carrier, and amphotericin B loses its crystal structure in the microspheres and exists in an amorphous state.

[0096] The DSC analysis curves of the raw materials and the prepared AmB / BSA microspheres in Example 1 are as follows: Figure 2 As shown, Figure 2 It shows that AmB has two obvious endothermic peaks between 115-135℃ and 200-220℃, indicating that AmB is crystalline. Similar peaks exist simultaneously in the physical mixture of AmB and blank unloaded drug microspheres; while these two peaks are not observed in AmB / BSA, indicating that AmB is uniformly dispersed in the polymer matrix, resulting in the transformation of the drug from crystalline to amorphous.

[0097] The FT-IR spectra of the raw materials and the prepared AmB / BSA microspheres in Example 1 are as follows: Figure 3As shown in the figure, in the spectrum of AmB API, the stretching vibrations of OH, CH, C=O and C=C of AmB appear at 3392, 2935, 1692 and 1559 cm -1 ; At the same time, the characteristic peak of AmB was also observed in the physical mixture of AmB and blank unloaded microspheres; but the characteristic peak of AmB disappeared in AmB / BSA, and only the characteristic peak of BSA was observed. The above results indicate that there is an interaction between the AmB API and the drug carrier, and AmB is successfully encapsulated in the microspheres.

[0098] (2) Particle size, potential and morphology of microspheres:

[0099] The particle size, potential and morphology of the AmB / BSA microspheres obtained in Example 1 are shown in Figure 4-7 As shown. Figure 5 It can be seen that the particle size of AmB / BSA is (5.82±0.29) μm and the PDI is 0.228. Figure 7 It can be seen that the microspheres are uniform in size and have a full spherical shape. Figure 6 It can be seen that the potential of AmB / BSA microspheres is lower than that of BSA from (-9.07±0.29) mV to (-11.9±0.53) mV, indicating that AmB is successfully encapsulated into BSA.

[0100] (3) In vitro drug release assay of AmB / BSA microspheres

[0101] PBS with a pH of 10.0 was selected as the release medium. 20 mL of the release medium was taken from each group into a test tube, and the AmB / BSA microspheres prepared in Example 1 were added, and the tubes were placed in a constant temperature oscillator for oscillation (100 rpm, 37° C.). Equal amounts of the solution were taken out at 1 h, 2 h, 4 h, 6 h, 8 h, 24 h, 48 h, 72 h, 5 d, and 7 d, and the absorbance at 416 nm was measured by ultraviolet. The drug content was calculated by the standard curve regression equation, and the cumulative release rate at different times was calculated.

[0102] like Figure 8 As shown in the figure, the cumulative release of AmB / BSA microspheres within 24 hours reached about 40%, which may be due to the presence of drug adhesion on the surface of the microspheres, resulting in a burst release of the drug. In the next 6 days, the drug can be released slowly. By the 7th day, the cumulative release rate of AmB / BSA reached 84.7%.

[0103] (4) Evaluation of in vitro antibacterial performance

[0104] Minimum inhibitory concentration (MIC) determination:

[0105] The minimum inhibitory concentration of the compound was determined by microdilution method. Take the activated bacterial solution that has been cultured to the logarithmic phase and dilute it with the required liquid culture medium to make the bacterial solution concentration 1.5×10 3 CFU / mL; take a 96-well plate, add 100 μL of liquid culture medium to each well in the 1st to 11th columns, and add 200 μL of liquid culture medium to the 12th column; dilute the microsphere sample prepared in Example 1 to a final drug concentration of 64 μg / mL, simulate the release for 1, 3, 5, and 7 days at a constant temperature of 37°C, draw 100 μL and add it to the first column, pipette and mix in the well, then draw out 100 μL of culture medium and add it to the next well to continue pipetting and mixing, dilute it in multiple proportions to the tenth column, take it out and discard it after mixing so that the final drug concentration of AmB in each column is 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 μg / mL, respectively, do not add the drug to be tested to the 11th column as a bacterial growth control, and only add liquid culture medium to the 12th well as a blank control. Three replicate wells were set up for each group; the diluted bacterial suspension was added to the wells in the 1st to 11th columns, 100 μL was added to each well and mixed; the cells were placed in a 37°C constant temperature incubator for 24 hours, and the OD 600 value was measured using an ELISA instrument to observe the minimum inhibitory concentration. The determination method for the free AmB drug group was the same as above.

[0106] The results showed that the MIC of free AmB 90 The MIC of the microsphere samples was 0.25 μg / mL, simulating release for 1, 3, and 5 days. 90 0.5 μg / mL, while the MIC of the microsphere sample simulated for 7 days 90 This indicates that the microspheres prepared by the present invention have a good long-term sustained release effect, and the effect after 7 days of sustained release is equivalent to that of free AmB drug.

[0107] Dilution spread plate count method:

[0108] The antibacterial properties of the microspheres were evaluated by the dilution plate coating counting method. Free AmB and the AmB / BSA microspheres prepared in Example 1 were diluted to a final drug concentration of 0.25 μg mL -1 Dilute the activated culture solution to 1×10 6 CFU·mL -1 100 μL of bacterial solution and 100 μL of solvent were applied to the PDA medium plate as blank control, and 100 μL of bacterial solution and 100 μL of the final drug concentration were 0.25 μg mL -1 The free AmB or AmB / BSA simulated for 1, 3, 5, and 7 days were coated on PDA plates, and each group was cultured in a 37°C constant temperature biochemical incubator for 24 hours, and the colonies were counted. The inhibition rate of Candida albicans was calculated according to the formula.

[0109] Inhibition rate (%) = (1-CFU exp / CFU con) × 100%

[0110] The experimental results showed that when the drug concentration was 0.25 μg mL -1 The fungal inhibition rate of free AmB was (48.71±1.35)%, while the inhibition rates of AmB / BSA microspheres simulated for 1, 3, 5, and 7 days were (27.05±0.87)%, (38.53±0.36)%, (43.68±0.44)%, and (46.63±1.07)%, respectively. This indicates that AmB / BSA microspheres have a long-term sustained release effect for 7 days, and the antibacterial effect is not inferior to that of free AmB.

[0111] The present invention obtains AmB / BSA microspheres with high encapsulation rate through a large number of experimental explorations, which can release amphotericin B in a week, effectively reduce the dose-dependent toxic and side effects of AmB and reduce the number of administrations. At the same time, the preparation process is simple, the raw materials have good biocompatibility and are easy to obtain, and the requirements for production equipment are not high, which greatly reduces the cost of pharmaceutical excipients and preparations. The results of in vitro antifungal experiments show that the antifungal effect of AmB / BSA microspheres is equivalent to that of free AmB drugs.

[0112] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing amphotericin B sustained-release microspheres, characterized in that: The preparation method comprises the following steps: S1, suspending AmB in methanol to prepare an AmB suspension; S2, dissolving BSA in water to prepare a BSA aqueous solution; S3, mixing the AmB suspension obtained in step S1 with the BSA aqueous solution obtained in step S2, and mixing them well to form the aqueous phase; S4, using liquid paraffin as the oil phase, adding an emulsifier, and then adding the water phase obtained in step S3 to the oil phase under mechanical stirring conditions, emulsifying to obtain W / O type colostrum, and naturally volatilizing to remove methanol; S5, adding 8 wt% glutaraldehyde solution to the colostrum obtained in step S4 for solidification; S6. Wash and freeze-dry the solidified material in step S5 to obtain amphotericin B sustained-release microspheres.

2. The preparation method according to claim 1, characterized in that: In step S1, the concentration of AmB suspension is 20-500 mg·mL -1 .

3. The preparation method according to claim 1, characterized in that: In step S2, the concentration of the BSA aqueous solution is 50-250 mg·mL -1 .

4. The preparation method according to claim 1, characterized in that: In step S3, the mass ratio of AmB to BSA is 1:(2-10).

5. The preparation method according to claim 1, characterized in that: In step S4, the emulsifier is one or more of Tween-80 and Span-80; the amount of the emulsifier relative to the aqueous phase is 0.05-0.2 g / mL.

6. The preparation method according to claim 1, characterized in that: In step S4, the speed of mechanical stirring is 500-1000 rpm; and the emulsification time is 10-30 min.

7. The method according to claim 1, characterized in that In step S4, the volume ratio of the oil phase to the water phase is (10-50):

1.

8. The method according to claim 1, characterized in that In step S5, the volume ratio of the 8 wt % glutaraldehyde solution to the aqueous phase is (0.08-10):1; and the curing time is 1-2 h.

9. Amphotericin B sustained-release microspheres prepared by the preparation method according to any one of claims 1 to 8.

10. A use of the amphotericin B sustained-release microspheres according to claim 9, characterized in that: Used to prepare antibacterial agents.

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