An amorphous solid dispersion of doxorubicin and a method of making and using the same

Abstract

The application discloses an adriamycin amorphous solid dispersion and a preparation method and application thereof, and belongs to the field of pharmaceutical preparations. The adriamycin amorphous solid dispersion is formed by intermolecular p-p interaction between one arginine guanidinium-rich protamine and an aromatic ring-containing hydrophobic molecule adriamycin, wherein the amino acid sequence of the arginine-rich protamine is: MPRRRRASRR VRRRRRPRVS RRRRRGGRRR R, and the aromatic ring-containing hydrophobic molecule adriamycin has the structure shown in the specification. The obtained amorphous solid dispersion does not need to introduce an exogenous carrier, the protamine and the adriamycin are both marketed drugs, the safety problem caused by the exogenous carrier is avoided, meanwhile, the water solubility of the hydrophobic adriamycin is improved, the cell uptake is enhanced, the antitumor therapeutic effect is enhanced through the amorphous solid dispersion, and the amorphous solid dispersion has potential clinical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to an amorphous solid dispersion of doxorubicin, a preparation method thereof, and an application thereof. Background Art

[0002] In the pharmaceutical field, approximately 40% of marketed drugs and 75% of drugs under development are poorly soluble hydrophobic drugs. Their minimal solubility in water makes them difficult to absorb, significantly limiting their application. Solubilizing and improving the bioavailability of hydrophobic drugs, as well as diversifying dosage forms and routes of administration, have long been key research areas and challenges.

[0003] Doxorubicin is an anti-tumor drug with a broad anti-tumor spectrum, but its hydrophobicity greatly limits its application. Generally, doxorubicin is prepared in the form of hydrochloride to increase the solubility of doxorubicin, but due to the drug resistance problem of biological cells, the utilization of doxorubicin hydrochloride is reduced. How to overcome the problem of cell resistance while increasing the solubility of hydrophobic doxorubicin is the focus and difficulty of research. Chinese patent CN117065036A increases the lethality of doxorubicin to tumor cells and reduces its toxicity to normal cells by encapsulating doxorubicin in a two-dimensional nanomaterial vanadium carbide. Chinese patent CN114558144A obtains black fungus polysaccharide-encapsulated carbon nanotubes loaded with doxorubicin by mixing a black fungus polysaccharide solution, a carbon nanotube solution modified by acid oxidation, and a doxorubicin solution, and then stirring the mixture to react. This significantly improves the dispersibility and stability of the carbon tubes and hydrophobic doxorubicin, has photothermal and pH-responsive sustained-release effects, greatly reduces the toxic side effects of normal cells, and improves the anti-tumor efficacy.

[0004] With the emerging rise of nanomedicine and the development of new protein carriers, some scholars have developed protein-doxorubicin nanomedicine. For example, Chinese patent CN102357077A discloses a protein nanoparticle for wrapping insoluble drugs, and the formula of the particle contains the following substances in percentage by weight: 0.1-10% insoluble drug, 0.1-40% water-soluble carrier material, and 50-90% proteinaceous substance; a proteinaceous substance capable of being cross-linked by a thiol group and / or a disulfide bond, such as albumin, is selected to prepare a carrier shell of the protein nanoparticle, which is wrapped outside the insoluble drug. Chinese patent CN111821280A discloses a preparation method of a pH-responsive silk sericin-doxorubicin nanomedicine carrier construction method, and the main steps of the preparation method are: (1) preparation of ZIF-8, loading of doxorubicin drug and modification of silk sericin, and (2) water washing and purification of ZIF-8@DOX@SS. The obtained drug is named ZIF-8@DOX@SS, and the obtained inorganic nanoparticles can form a uniformly distributed nanoparticle aqueous solution in water, have high stability, low toxic side effects, good drug control, and other advantages, and effectively solve the problem of low solubility of hydrophobic drug molecules. Chinese patent CN111939151A discloses a composite doxorubicin albumin nanoparticle (VES-DOX-BSANPs), which uses bovine serum albumin (BSA) as a carrier material, and then wraps the drug doxorubicin (DOX) and vitamin E succinate (VES) to prepare VES-DOX-BSANPs by a high-pressure homogenization method. The composite doxorubicin albumin nanoparticle has the advantages of uniform size, suitable particle size, good dispersibility, and stable physicochemical properties; in in vivo and in vitro drug efficacy experiments, VES-DOX-BSANPs have the effects of synergistic attenuation and reversal of tumor multidrug resistance. However, these methods have the problems of complex preparation method, uncontrollable drug quality, and drug safety.

[0005] A solid dispersion system refers to a dispersion system formed by uniformly dispersing a drug in a certain solid carrier material in a molecular, colloidal, amorphous, microcrystalline or other state. Preparing a solid dispersion of a poorly soluble drug is an effective method for increasing the solubility and dissolution rate of the drug, and improving the absorption and bioavailability of the drug. The present application aims to provide a doxorubicin solid dispersion system using protein as a carrier. SUMMARY

[0006] In order to overcome the deficiencies in the prior art, the purpose of the present application is to provide a doxorubicin amorphous solid dispersion, a preparation method and application thereof, and to select protamine rich in guanidino groups as a carrier, and connect with hydrophobic doxorubicin through π-π interaction, thereby serving as a carrier for hydrophobic drugs and forming an amorphous solid dispersion with doxorubicin, solubilizing hydrophobic doxorubicin, overcoming cell drug resistance, enhancing cell uptake, and improving antitumor efficacy.

[0007] One of the objectives of the present invention is to provide a doxorubicin solid dispersion comprising free doxorubicin and a guanidine-rich protein capable of being linked to the free doxorubicin via π-π conjugation.

[0008] As one of the preferred embodiments of the present invention, the protein containing a guanidine group that can be linked to free doxorubicin by π-π conjugation is protamine; further, the amino acid sequence of protamine is MPRRRRASRR VRRRRRPRVSRRRRRGGRRR R (SEQ ID NO.1); further, it is protamine sulfate.

[0009] The mass ratio of protamine sulfate to doxorubicin is 40-200:1.

[0010] A second object of the present invention is to provide a method for preparing the above-mentioned doxorubicin amorphous solid dispersion PRTA-DOX, which specifically comprises the following steps:

[0011] (1) Take the required amount of protamine solid powder, dissolve it in deionized water to obtain a protamine solution, and dilute it to the required concentration;

[0012] (2) Free doxorubicin was added to the protamine solution in step (1), and the solution was placed in a constant temperature shaker at 20-35°C for 18-24 hours to obtain a dispersed protamine-doxorubicin (PRTA-DOX) solution, which was stored at 4°C in the dark.

[0013] (3) The protamine-doxorubicin (PRTA-DOX) solution obtained in step (2) was pre-frozen at -80°C for 30 minutes, taken out and freeze-dried for 24 hours to obtain a PRTA-DOX amorphous solid dispersion.

[0014] Among them, the preparation of free doxorubicin: accurately weigh doxorubicin hydrochloride and dissolve it in deionized water, and shake it until it is completely dissolved. Slowly add NaOH to adjust the pH and observe whether a precipitate is formed. Centrifuge the mixture and collect the precipitate. Use deionized water to slowly wash the precipitate until it is colorless. Add an appropriate amount of deionized water to cover the precipitate and pre-freeze for 30 minutes. Take it out and freeze-dry it for 24 hours to obtain hydrophobic doxorubicin solid, and store it in the dark at 4°C. All steps must be completed under light-proof conditions. The concentration of doxorubicin hydrochloride is 2 mg / mL; the pH value is adjusted to 9.5-10 to form a precipitate. The mixture is centrifuged at 10,000 rpm for 10 minutes.

[0015] In the PRTA-DOX solution, the concentration of protamine is 10-50 mg / mL, and the concentration of doxorubicin is 0.01-1 mg / mL.

[0016] A third object of the present invention is to provide the use of the above-mentioned doxorubicin amorphous solid dispersion in the preparation of anti-tumor drugs.

[0017] A pharmaceutical composition comprises the above-mentioned doxorubicin amorphous solid dispersion as one of the main active ingredients and a pharmaceutically acceptable carrier.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention targets hydrophobic doxorubicin (DOX) containing an aromatic benzene ring structure and selects guanidine-rich proteins, especially protamine, which is mainly composed of arginine. The hydrophobic drug can be solubilized through the π-π interaction between the hydrophobic surface of the guanidine part and the hydrophobic molecules rich in aromatic rings, and can be easily stacked into a carrier modified with similar aromatic groups through π-π conjugation.

[0020] This invention uses commercially available protamine sulfate and doxorubicin hydrochloride as raw materials to develop a safe and simple amorphous solid dispersion of doxorubicin. Guanidine-rich protamine (PRTA) is linked to the hydrophobic drug doxorubicin (DOX) via π-π conjugation to form the amorphous solid dispersion. This improves the water solubility of the hydrophobic doxorubicin while enhancing cellular uptake, overcoming cellular drug resistance and improving anti-tumor efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 These are the full-scan fluorescence spectra of the doxorubicin group (free-DOX), protamine-doxorubicin group (PRTA-DOX), bovine serum albumin-doxorubicin group (BSA-DOX), and small molecule collagen-doxorubicin group (COL-DOX) in the 480nm-700nm band.

[0022] Figure 2 The fluorescence values ​​of doxorubicin group (free-DOX), protamine-doxorubicin group (PRTA-DOX), bovine serum albumin-doxorubicin group (BSA-DOX), and small molecule collagen-doxorubicin group (COL-DOX) at 590 nm.

[0023] Figure 3 : These are the appearance pictures of PRTA-DOX solution and amorphous solid dispersion.

[0024] Figure 4 (A) UV images and (B) fluorescence images of PRTA-DOX solutions with different concentrations.

[0025] Figure 5 This is the fluorescence quenching diagram between doxorubicin and protamine.

[0026] Figure 6 (A) XRD pattern and (B) DSC pattern of amorphous solid dispersion.

[0027] Figure 7The results of cytotoxicity test of PRTA-DOX solution on MCF-7 cells.

[0028] Figure 8 The results of the PRTA-DOX solution uptake test on MCF-7 cells.

[0029] Figure 9 This is a microscopic image of the PRTA-DOX solution's inhibitory effect on MCF-7 cell tumor spheres.

[0030] Figure 10 This is the volume change curve of MCF-7 cell tumor spheres induced by PRTA-DOX solution.

[0031] Figure 11 The results of the PRTA-DOX solution uptake test on MCF-7 / adr cells.

[0032] Figure 12 The solid dispersion of doxorubicin is in the freeze-dried state and the reconstituted liquid state at 7 days, 14 days, and 30 days.

[0033] Figure 13 The results of the uptake test of the reconstituted doxorubicin solid dispersion into MCF-7 cells are shown in FIG. DETAILED DESCRIPTION

[0034] The technical solutions of the present invention are further described below with reference to specific embodiments and accompanying drawings to facilitate understanding by those skilled in the art, but the present invention is not limited in any way.

[0035] Protamine, D20061, was purchased from Beijing Biosun Biotechnology Co., Ltd.

[0036] Bovine serum albumin, BS114, was purchased from Biosharp, a subsidiary of Lanjieke Technology Co., Ltd.

[0037] Small molecule collagen, TTB02-02SC, was purchased from Jiangsu Chuangjian Medical Technology Co., Ltd. Example 1

[0038] Fluorescence spectrometer was used to screen and explore the connection relationship between doxorubicin and various protein carriers, and doxorubicin group (free-DOX), protamine-doxorubicin group (PRTA-DOX), bovine serum albumin-doxorubicin group (BSA-DOX), small molecule collagen-doxorubicin group (COL-DOX) were set up. Figure 1 The full scan fluorescence spectra of each group in the 480nm-700nm band are shown. Figure 2The fluorescence values ​​of each group at 590 nm (the characteristic absorption wavelength of doxorubicin) are shown. According to the principle of aggregation-induced fluorescence quenching, when doxorubicin forms π-π stacking with the hydrophobic surface of the carrier, the high-energy molecules in the excited state transfer energy to the low-energy molecules in the ground state, thereby reducing the overall energy. As shown in the figure, doxorubicin has a very high autofluorescence value. However, when protamine is mixed with doxorubicin, the fluorescence value is greatly reduced, indicating that protamine can bind to doxorubicin through π-π stacking. The fluorescence values ​​of other proteins mixed with doxorubicin show a slight downward trend or no decrease, indicating that these proteins cannot bind to doxorubicin through π-π stacking, thus failing to solubilize the protein. Example 2

[0039] (1) Preparation of free doxorubicin: Accurately weigh doxorubicin hydrochloride (10 mg) and dissolve it in deionized water (5 mL). Shake until completely dissolved. Slowly add NaOH to adjust the pH to 9.6 and observe whether a precipitate is formed. Centrifuge the mixture (10,000 rpm, 10 min) and collect the precipitate. Slowly wash the precipitate with deionized water until it is colorless. Add an appropriate amount of deionized water (1-2 mL) to cover the precipitate and pre-freeze it in a -80°C ultra-low temperature refrigerator for 30 min. Take it out and freeze-dry it in a freeze dryer for 24 hours to obtain hydrophobic doxorubicin solid, which is stored at 4°C in the dark. All steps must be completed under light-proof conditions.

[0040] (2) Accurately weigh 1 mg of hydrophobic doxorubicin and mix with 4 mL of protamine solution (10 mg / mL). Place on a shaker in the dark for 24 h. Remove and centrifuge (4000 rpm, 10 min). Collect the supernatant to obtain a dispersed protamine-doxorubicin (PRTA-DOX) solution.

[0041] (3) The obtained PRTA-DOX solution was pre-frozen in a -80°C refrigerator for 30 minutes and freeze-dried for 24 hours to obtain an amorphous solid dispersion of PRTA-DOX, which was then stored at 4°C in the dark. Example 3

[0042] The difference between this embodiment and embodiment 2 is that the concentration of the protamine solution is 20 mg / mL. Example 4

[0043] The difference between this embodiment and embodiment 2 is that the concentration of the protamine solution is 40 mg / mL. Example 5

[0044] The difference between this embodiment and embodiment 2 is that the concentration of the protamine solution is 50 mg / mL. Comparative Example 1

[0045] The difference between this comparative example and Example 2 is that 4 mL of deionized water is used instead of the protamine solution, and the obtained solid is marked as free-DOX. Example 6

[0046] The solubility of DOX in the PRTA-DOX solution of step (3) in each embodiment and comparative example was calculated using an ultraviolet spectrophotometer and scanned at a wavelength of 480 nm. The absorbance curve is shown in FIG. Figure 4 (A), doxorubicin concentrations are shown in Table 1. As shown in Table 1, as the protamine solution concentration increases, the absorbance of the group also increases, indicating that the amount of hydrophobic doxorubicin dissolved therein also increases. Based on the standard curve of doxorubicin, the mass of doxorubicin dissolved in each group can be calculated from the absorbance values ​​of each group. The solubility of doxorubicin in water is 0.006 mg / mL. When the protamine solution concentration is 50 mg / mL, the amount of doxorubicin dissolved therein is 0.0473 mg / mL, which is 8 times that dissolved in water.

[0047] Table 1 Doxorubicin concentration in the system

[0048]

[0049] The fluorescence content of PRTA-DOX solution was determined by fluorescence spectrometer. Figure 4 As shown in (B), the fluorescence value of this group increases with the increase in the concentration of the protamine solution, indicating that the amount of hydrophobic doxorubicin dissolved therein also increases. The overall trend is upward. Example 7

[0050] Fluorescence spectrometry was used to investigate the connection between doxorubicin and protamine. Figure 5 As shown in the figure. According to the principle of aggregation fluorescence quenching, when doxorubicin and protamine form π-π stacking on their hydrophobic surfaces, the high-energy molecules in the excited state transfer energy to the low-energy molecules in the ground state, thereby reducing the overall energy. As can be seen from the figure, protamine itself is non-fluorescent, while doxorubicin has a very high fluorescence value. However, when protamine and doxorubicin are mixed, the fluorescence value decreases, indicating that the connection between doxorubicin and protamine is π-π conjugation. Example 8

[0051] X-ray diffraction (XRD) was used to analyze the state of the protamine-doxorubicin solid system. Figure 6 As shown in (A), the hydrophobic doxorubicin exhibits strong diffraction peaks at 6.79°, 10.14°, 22.20°, 25.45°, and 43.94°, indicating that the hydrophobic doxorubicin is in a crystalline state. After being prepared into a solid dispersion PRTA-DOX with protamine, the characteristic diffraction peaks essentially disappear except for a diffraction peak at 10.14°, and are almost identical to those of protamine, indicating that after the solid dispersion PRTA-DOX is prepared, the hydrophobic doxorubicin is dispersed in the carrier in an amorphous state.

[0052] Differential scanning calorimetry (DSC) was used to analyze the state of the protamine-doxorubicin system. Figure 6 As shown in (B), the hydrophobic doxorubicin exhibits a decomposition melting peak at 208.5°C. After being prepared into PRTA-DOX with protamine, the decomposition melting peak almost disappears and is almost identical to the peak pattern of protamine, indicating that after the solid dispersion PRTA-DOX is prepared, the hydrophobic doxorubicin is dispersed in the carrier in an amorphous state. Example 9

[0053] The cytotoxicity was detected by MTT assay. A blank control group (blank well), negative control group (DMEM), 1% Triton X-ray diffraction (TX) positive control group, free-DOX group, 10 mg / mL PRTA-DOX group, 20 mg / mL PRTA-DOX group, 40 mg / mL PRTA-DOX group, 50 mg / mL PRTA-DOX group, 20 mg / mL PRTA group, and 50 mg / mL PRTA group were set up, with 5 replicate wells in each group. MCF-7 (human breast cancer) cells in the logarithmic growth phase were seeded in 96-well plates, with 8,000 cells and 200 μL of cell solution per well. After approximately 16 hours of culture, the culture medium was aspirated and 100 μL of each sample was added. After incubation with the cells for 24 hours, 20 μL of MTT reagent was added directly to each well. After 4 hours, the liquid in the plate was aspirated and 150 μL of DMSO was added to each well. The plate was shaken for 15 minutes. The OD value at 490 nm was measured using a microplate reader to calculate cell viability. The calculation formula is: Cell viability (%) = [A(drug added) - A(blank)] / [A(0 drug added) - A(blank)] × 100, [A(drug added): OD value of wells with cells, MTT solution, and drug solution; A(0 drug added): OD value of wells with cells, MTT solution, but no drug solution; A(blank): OD value of wells without cells]. The effects of each drug group on MCF-7 cell viability are shown in the following table. Figure 7 As shown in the figure, the protamine solution itself has a weak ability to inhibit tumor cell proliferation. In the PRTA-DOX solution group, cell survival decreased with increasing protamine concentration, indicating that protamine enhanced the cell growth inhibitory effect of doxorubicin. This may be due to the protamine solution's solubilizing effect on hydrophobic doxorubicin, which increases the amount of doxorubicin it contains. Example 10

[0054] Flow cytometry was used to detect the cellular uptake effect. A control group (no drug addition group), a DOX·HCL group, a free-DOX group, a 10mg / mL PRTA-DOX group, a 20mg / mL PRTA-DOX group, a 40mg / mL PRTA-DOX group, a 50mg / mL PRTA group, and two replicate wells were set up for each group. MCF-7 cells in the logarithmic growth phase were seeded in a 24-well plate, with 80,000 cells and 1mL of cell solution per well. After culturing for about 16 hours to allow cells to adhere, the medium was removed and 500μL of each sample was added to the cells for incubation for 4 hours. Cell uptake was quantitatively analyzed by flow cytometry. Figure 8 As shown in the figure, the protamine solution itself is nonfluorescent. However, as the protamine concentration in the PRTA-DOX solution group increases, the fluorescence value of the cells increases, indicating that the group contains more doxorubicin. This demonstrates that the protamine solution solubilizes the hydrophobic doxorubicin. However, due to the high solubility of doxorubicin hydrochloride, the PRTA-DOX sample group failed to demonstrate superior uptake of doxorubicin hydrochloride in MCF-7 cells. Example 11

[0055] An MCF-7 cell tumor sphere inhibition experiment was conducted. A control group (no drug group), a free-DOX group, a 50mg / mL PRTA-DOX group, and a 50mg / mL PRTA group were set up, with 2 replicate wells in each group. Weigh 75mg of low-electrosmotic agarose and dissolve it in 5mL of water. Boil until the whole is clear and transparent, quickly add it to a 96-well plate, 60μL per well, and wait for the agarose gel to solidify. MCF-7 cells in the logarithmic growth phase were inoculated into this 96-well plate with agarose gel, 1000 cells and 200μL of cell fluid per well, and cultured in an incubator for 2-3 days. After uniform spherical tumor spheres were formed in the observation wells, samples from each group were added and incubated with the tumor spheres. The size of the tumor spheres was recorded daily using a fluorescent inverted microscope for 7 days, and the volume of the tumor spheres was calculated using ImageJ software. Figure 9 、 10 As shown in the figure, over 7 days, the control and PRTA groups had no inhibitory effect on tumor spheroid growth, the free-DOX group showed slight signs of inhibition, and the PRTA-DOX group had the most pronounced inhibitory effect. Furthermore, the tumor spheroids showed signs of disintegration and cell fragmentation. This suggests that protamine significantly solubilizes hydrophobic doxorubicin, which is minimally soluble in water, thereby effectively inhibiting tumor spheroid growth. Example 12

[0056] Flow cytometry was used to detect the cellular uptake effect. A control group (no drug addition), a DOX·HCL group, a free-DOX group, a 50mg / mL PRTA-DOX group, and a 50mg / mL PRTA group were set up, with two replicate wells set up in each group. MCF-7 / adr (a drug-resistant human breast cancer cell line) cells in the logarithmic growth phase were seeded in a 24-well plate, with 80,000 cells and 1mL of cell solution per well. After culturing for approximately 16 hours, the cells adhered to the wall. The medium was aspirated, and 500μL of each sample was added to the cells and incubated for 4 hours. Cell uptake was quantitatively analyzed by flow cytometry. Figure 11 As shown in the figure, the protamine solution itself has no fluorescence. In the PRTA-DOX solution group, as the concentration of the protamine solution increases, the fluorescence value of the cells increases, indicating that the amount of doxorubicin contained in the group is greater. This shows that the protamine solution has a solubilizing effect on hydrophobic doxorubicin. In addition, the PRTA-DOX sample group shows a better effect than the uptake of doxorubicin hydrochloride in MCF-7 / adr cells. This is because the resistant cells are doxorubicin-resistant cells and will pump out doxorubicin. The excellent uptake effect of PRTA-DOX also shows that the system enhances the effect of cell uptake. Example 13

[0057] 1 mg of the lyophilized solid dispersion was taken out at 7 days, 14 days, and 30 days and dissolved in 4 mL of water. The lyophilized solid dispersion dissolved rapidly, and the solution was clear, transparent, and uniformly dispersed. Figure 12 The solid dispersion of doxorubicin is in the freeze-dried state and the reconstituted liquid state at 7 days, 14 days, and 30 days. Example 14

[0058] Flow cytometry was used to detect the cellular uptake effect of the solid dispersion after reconstitution. A control group (no drug addition group), a free-DOX group, a PRTA-DOX group, and a re-PRTA-DOX group (freeze-dried and stored for 30 days and reconstituted group) were set up, with two replicate wells in each group. MCF-7 cells in the logarithmic growth phase were seeded in a 24-well plate, with 80,000 cells and 1 mL of cell fluid per well. After culturing for about 16 hours, the cells adhered to the wall, the culture medium was removed, and 500 μL of each sample was added to the cells and incubated for 4 hours. Flow cytometry was used to quantitatively analyze the cellular uptake, as shown in Figure 2. Figure 13 As shown in the figure, the reconstituted PRTA-DOX solution still has a good uptake effect, indicating that the solid dispersion is very stable.

[0059] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An amorphous solid dispersion of doxorubicin, characterized in that: The amorphous doxorubicin solid dispersion includes free doxorubicin and a guanidine-rich protein capable of connecting to the free doxorubicin via a π-π conjugation. The amorphous doxorubicin solid dispersion is formed by connecting the guanidine hydrophobic plane of the protein to the free doxorubicin via intermolecular π-π interactions. The protein is protamine, and the amino acid sequence of the protamine is: MPRRRRASRRVRRRRRPRVSRRRRRGGRRRR. The mass ratio of the sulfate of the protamine to the free doxorubicin is 40-200:

1.

2. A method for preparing the amorphous solid dispersion of doxorubicin according to claim 1, characterized in that: The steps include: (1) Add free doxorubicin to the protamine solution and shake on a constant temperature shaker for 18-24 hours to obtain a protamine-doxorubicin solution; (2) The protamine-doxorubicin solution is pre-frozen and then freeze-dried to obtain an amorphous solid dispersion of doxorubicin.

3. The method for preparing the amorphous solid dispersion of doxorubicin according to claim 2, characterized in that: The preparation method of the free doxorubicin comprises the following steps: accurately weighing doxorubicin hydrochloride and dissolving it in deionized water, shaking until completely dissolved, slowly adding sodium hydroxide solution to adjust the pH to 9.5-10, centrifuging the mixture, collecting the precipitate, washing the precipitate with deionized water until it is colorless, adding deionized water to cover the precipitate, and freeze-drying to obtain the free doxorubicin.

4. The method for preparing the amorphous solid dispersion of doxorubicin according to claim 2, wherein: The protamine solution is obtained by dissolving protamine in deionized water, and its concentration is 10-50 mg / mL.

5. The method for preparing the amorphous solid dispersion of doxorubicin according to claim 2, characterized in that: The suitable temperature of the constant temperature shaker is 20-35℃.

6. The method for preparing the amorphous solid dispersion of doxorubicin according to claim 2, characterized in that: The pre-freezing temperature in step (2) is -80°C, and the pre-freezing time is more than 30 minutes.

7. Use of the amorphous solid dispersion of doxorubicin according to claim 1 in the preparation of anti-tumor drugs.

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