Method for improving particle size stability of drug-loaded albumin nanoparticles

Abstract

The invention discloses a method for improving the particle size stability of drug-loaded albumin nanoparticles. The method for improving the particle size stability of the drug-loaded albumin nanoparticles comprises the step of sealing sulfydryl-SH on the surfaces of the drug-loaded albumin nanoparticles. The problem that the particle size of the drug-loaded albumin nanoparticles is increased due to the placement process can be effectively solved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological albumin and relates to a method for improving the particle size stability of drug-loaded albumin nanoparticles. Background Art

[0002] The main preparation techniques for albumin nanoparticles include the Nab method, the desolvation method, the gel method, and the self-assembly method. Currently, there are two marketed drugs using albumin nanoparticle technology: paclitaxel albumin nanoparticles and sirolimus albumin nanoparticles. For albumin nanoparticles, particle size and particle stability are key attributes that determine their quality. Excessively large particle size of the prepared albumin nanoparticles directly affects the sample yield during sterile filtration. Poor particle stability of albumin nanoparticles, even if they have a small particle size upon initial preparation, can lead to particle size growth during long-term storage of freeze-dried albumin nanoparticles or during the period after reconstitution and before administration. This increased particle size can not only affect the in vivo disintegration, distribution, and efficacy of the albumin nanoparticles, but can also lead to sample precipitation due to the excessively large particle size, posing a life-threatening risk to patients. Therefore, good particle stability of albumin nanoparticles is essential for maintaining their efficacy and safety. Through research and analysis of the principles of the Nab method, it was found that the initial value and particle size stability of the albumin nanoparticles controlled by the Nab method are mainly related to the number of high-pressure microfluidization cycles. The greater the number of cycles, the higher the temperature of the sample solution, the smaller the nanoparticle size, and the better the stability. However, this method is only applicable to heat-stable drugs. For heat-labile drugs such as sirolimus, its particle size stability cannot be improved by increasing the number of microfluidization cycles. Therefore, commercially available sirolimus albumin nanoparticles can only be stored at 2-8°C and need to be administered immediately after reconstitution.

[0003] In summary, it is necessary to explore the principle of albumin nanoparticle size growth and, based on this principle, improve the particle size stability of nanoparticles so that they can have better drug compliance. Summary of the Invention

[0004] The particle size and particle size stability of albumin nanoparticles may affect their in vivo fate and efficacy. However, for certain drugs or certain preparation technologies, if the particle size stability of albumin nanoparticles cannot be guaranteed, it may affect the clinical application or further development of the product. Therefore, it is necessary to solve the particle size stability problem of albumin nanoparticles based on the principle of albumin nanoparticle size growth.

[0005] The invention blocks the thiol groups on the surface of albumin nanoparticles, thereby converting nanoparticles with poor particle size stability into preparations with good particle size stability.

[0006] The present invention provides a method for improving the particle size stability of drug-loaded albumin nanoparticles, which comprises the steps of blocking the thiol groups -SH on the surface of the drug-loaded albumin nanoparticles;

[0007] The drug-loaded albumin nanoparticles include or consist of the following: albumin in nanoparticle form as a drug carrier and a drug (such as an amorphous drug); wherein the albumin encapsulates the drug to form a core-shell structure, and the shell is formed by cross-linking albumin molecules through disulfide bonds;

[0008] The closure is partial closure or complete closure.

[0009] In some embodiments, in the above method, the particle size of the drug-loaded albumin nanoparticles is 80-220 nm, preferably 90-150 nm, more preferably 120-150 nm, for example 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150 nm, or values and ranges between any two of these values.

[0010] In some embodiments, in any of the above methods, the thiol groups -SH on the surface of the drug-loaded albumin nanoparticles are blocked by chemical reaction.

[0011] In some embodiments, in the above method, the chemical reaction is selected from one or more of a click chemistry reaction, an oxidation reaction, a nucleophilic substitution reaction, a reduction reaction, and a condensation reaction.

[0012] In some embodiments, the method comprises the steps of:

[0013] S1. Centrifuging the solution containing the drug-loaded albumin nanoparticles, aspirating the supernatant for later use, and re-dissolving the precipitate with water to obtain a drug-loaded albumin nanoparticle aqueous solution;

[0014] S2. Adding a compound containing an unsaturated group to the reconstituted solution of the drug-loaded albumin nanoparticles under stirring to perform a click chemistry reaction (the click chemistry reaction is a reaction between a thiol group -SH and an unsaturated group in the compound containing an unsaturated group; the unsaturated group includes but is not limited to a double bond, a triple bond, and a cyclic structure) or adding an oxidant to perform an oxidation reaction (the oxidation reaction is a reaction between a thiol group and an oxidant);

[0015] S3, centrifuging the solution obtained in step S2, discarding the supernatant (and removing unreacted compounds containing unsaturated groups or oxidants, etc.), and re-dissolving with the supernatant in step S1 to obtain a drug-loaded albumin nanoparticle solution with a stable particle size; and optionally

[0016] S4, freeze-drying the solution obtained in step S3;

[0017] Wherein, in step S1, the centrifugal speed is 10000g-25000g;

[0018] In step S1, the solution containing the drug-loaded albumin nanoparticles contains any suitable concentration of drug and albumin, for example, wherein the drug concentration is 1 mg / ml or more and / or the albumin concentration is 5 mg / ml or more, for example, wherein the drug concentration is 1-10 mg / ml (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / ml or a value or range between any two of these values) and / or the albumin concentration is 5-100 mg / ml (for example, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg / ml or a value or range between any two of these values), for example, wherein the drug concentration is 4-8 mg / ml and / or the albumin concentration is 40-60 mg / ml.

[0019] In some embodiments, in the above method, the compound containing an unsaturated group is a compound containing a maleimide fragment (MAL), preferably one or more selected from 3-maleimidopropionic acid, N-methylmaleimide, maleimide, 2-maleimidoacetic acid, 1,6-bis(maleimido)hexane; and / or

[0020] The oxidant is selected from one or more of peroxides and ozone, and is preferably selected from one or more of hydrogen peroxide, sodium peroxide, potassium peroxide, calcium peroxide, and magnesium peroxide.

[0021] In some embodiments, in the above method, the amount of the compound containing an unsaturated group added is related to the amount of total albumin in the reconstituted solution of the drug-loaded albumin nanoparticles. The greater the amount added, the more complete the blocking effect on the sulfhydryl groups contained in the total albumin in the solution. In some embodiments, the molar ratio of the compound containing a maleimide fragment (MAL) to the total albumin in the reconstituted solution of the drug-loaded albumin nanoparticles is not less than 0.5:1, preferably (1-20):1; and / or

[0022] The amount of the oxidant added is related to the amount of total albumin in the reconstituted solution of the drug-loaded albumin nanoparticles; in some embodiments, the mass ratio of hydrogen peroxide to the total albumin in the reconstituted solution of the drug-loaded albumin nanoparticles is not less than 0.5:5, preferably (1-5):5.

[0023] In some embodiments, the method comprises the steps of:

[0024] s1. Adding a compound containing an unsaturated group to the solution containing the drug-loaded albumin nanoparticles under stirring to carry out a click chemistry reaction (the click chemistry reaction is a reaction between a thiol group -SH and an unsaturated group in the compound containing an unsaturated group; the unsaturated group includes but is not limited to a double bond, a triple bond, and a cyclic structure) or adding an oxidant to carry out an oxidation reaction (the oxidation reaction is a reaction between a thiol group and an oxidant);

[0025] s2, concentrating the solution obtained in step s1 (for example, using a tangential flow filtration system), and removing (for example, by diafiltration) the organic solvent (such as ethanol, etc.) and unreacted compounds containing unsaturated groups or oxidants in the solution; and optionally

[0026] s3, freeze-drying the solution obtained in step s2;

[0027] Wherein, in step s1, the solution containing the drug-loaded albumin nanoparticles contains any suitable concentration of drug and albumin, for example, wherein the drug concentration is 5 mg / ml or less and / or the albumin concentration is 50 mg / ml or less, for example, wherein the drug concentration is 0.5-5 mg / ml (for example, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 mg / ml or a value or range between any two of these values) and / or the albumin concentration is 2-50 mg / ml (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 mg / ml or a value or range between any two of these values), for example, wherein the drug concentration is 0.5-2 mg / ml and / or the albumin concentration is 5-15 mg / ml.

[0028] In some embodiments, in the above method, the compound containing an unsaturated group is a compound containing a maleimide fragment (MAL), preferably one or more selected from 3-maleimidopropionic acid, N-methylmaleimide, maleimide, 2-maleimidoacetic acid, 1,6-bis(maleimido)hexane; and / or

[0029] The oxidant is selected from one or more of peroxides and ozone, and is preferably selected from one or more of hydrogen peroxide, sodium peroxide, potassium peroxide, calcium peroxide, and magnesium peroxide.

[0030] In some embodiments, the molar ratio of the compound containing a maleimide fragment (MAL) to the total albumin in the solution containing the drug-loaded albumin nanoparticles is not less than 5:1, preferably (16-320):1; and / or

[0031] The mass ratio of the hydrogen peroxide to the total albumin in the solution containing the drug-loaded albumin nanoparticles is not less than 1:500, preferably 1:(32-325).

[0032] In some embodiments, in any of the above methods, the albumin in the drug-loaded albumin nanoparticles is not particularly limited and can be any albumin known in the art for preparing nanoparticles, for example, can be selected from one or more of human serum albumin, bovine serum albumin, and ovalbumin, preferably human serum albumin;

[0033] The drug in the drug-loaded albumin nanoparticles can be any drug known in the art that can be loaded by albumin, for example, can be selected from one or more of taxanes, macrolides, anthracyclines, irinotecan, tacrolimus, lopinavir, cyclosporine, and semustine;

[0034] Preferably, the taxane drug is selected from one or more of paclitaxel, docetaxel, and cabazitaxel;

[0035] Preferably, the macrolide drug is selected from one or more of sirolimus, epothilone B, tanespiramycin, and everolimus;

[0036] Preferably, the anthracycline is selected from one or more of epirubicin, doxorubicin, aclarubicin, and pirarubicin.

[0037] The present invention provides a method for improving the particle size of drug-loaded albumin nanoparticles. This method blocks the sulfhydryl groups on the surface of the drug-loaded albumin nanoparticles, thereby preventing the drug-loaded albumin nanoparticles from reacting with each other through the sulfhydryl groups on their surfaces to form disulfide bonds. This prevents the nanoparticles from connecting with each other to form dimers or multimers, ultimately achieving the purpose of improving the particle size stability of the drug-loaded albumin nanoparticles. The method of the present invention can ensure the particle size stability of the drug-loaded albumin nanoparticles at 25°C for 48 hours, effectively solving the problem of nanoparticle size growth caused by the storage process during the period between reconstitution and administration of the freeze-dried drug-loaded albumin nanoparticles.

[0038] The present invention can improve the particle size stability of drug-loaded albumin nanoparticles, which originally had poor particle size stability, thereby reducing the process difficulty in preparing drug-loaded albumin nanoparticles, and can also achieve the loading of heat-sensitive drugs and ensure their particle size stability, which can meet the medication needs of a large number of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of drug-loaded albumin nanoparticles forming nanoparticle dimers through the free thiol -SH on the surface of the nanoparticles.

[0040] Figure 2 Schematic diagram of using 3-maleimidopropionic acid containing maleimide (MAL) fragment to block the -SH on the surface of drug-loaded albumin nanoparticles. DETAILED DESCRIPTION

[0041] The technical solutions in the embodiments of the present invention are described in detail below, but the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. In order to better understand the present invention, the present invention will be further explained below through specific embodiments, but it should not be understood as limiting the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content also fall within the scope of the present invention.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0043] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0044] The present invention will be further described below with reference to specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0045] Sirolimus was purchased from Zhejiang Hisun Pharmaceutical Co., Ltd.

[0046] Human albumin solution was purchased from Sichuan Yuanda Shuyang Pharmaceutical Co., Ltd.

[0047] Human albumin reference substance was purchased from SIGMA.

[0048] 3-Maleimidopropionic acid was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., product number BD34019.

[0049] The HPLC method for the determination of albumin in sirolimus albumin nanoparticle solution was based on the Chinese Pharmacopoeia and is briefly described as follows: The chromatographic column was Tosohaas TSK G3000SW XL (300mm×7.8mm,5.0μm), with Tosohaas TSK SW XL(40 mm × 6.0 mm, 7.0 μm) guard column; operating conditions: flow rate 0.5 ml / min, injection volume 10 μL, detection wavelength 228 nm, column temperature 25°C, human albumin haploid retention time 18 ± 10% min. Accurately weigh approximately 35 mg of human albumin reference substance into a 10 ml volumetric flask, add an appropriate amount of diluent to dissolve, dilute to the mark, and shake well to obtain a reference solution with a concentration of 3.5 mg / ml. The mobile phase consists of 0.1 mol / L dipotassium hydrogen phosphate solution, adjusted to pH 7.0 ± 0.1 with dilute hydrochloric acid. The diluent is 0.9% aqueous sodium chloride solution.

[0050] Nanoparticle size determination method: dilute the sample to be tested with 0.9% sodium chloride aqueous solution to a sirolimus concentration of 0.5 mg / mL, use this as the test solution, and measure the particle size of the nanoparticles using a Malvern laser particle size analyzer.

[0051] Example 1

[0052] (1) 1.6 g of sirolimus was weighed and dissolved in 30.40 g of anhydrous ethanol to obtain an organic phase solution with a concentration of 50 mg / g; a human serum albumin solution containing 13.60 g (about 204.5 μmol) of albumin was taken and diluted with water for injection to obtain an aqueous phase solution containing 3 mg / g of albumin and heated to 60°C for use; the organic phase solution and the aqueous phase solution were mixed and loaded with the drug using a linear mixing device, and the resulting solution was then rapidly cooled to room temperature to obtain a dilute solution of sirolimus albumin nanoparticles. The dilute solution was concentrated using a tangential flow filtration system to a sirolimus concentration of 6 mg / mL, thereby obtaining a sirolimus albumin nanoparticle concentrate, at which point the total albumin concentration in the concentrate was 51 mg / mL.

[0053] (2) Take 10 mL of the sirolimus albumin nanoparticle concentrate and centrifuge at 15,000 g for 1 h. The supernatant is aspirated and set aside. The precipitate is reconstituted with 10 mL of purified water to obtain the sirolimus albumin nanoparticle aqueous solution. The sirolimus albumin nanoparticle aqueous solution no longer contains free albumin, and all albumin is involved in the formation of nanoparticles. The albumin concentration in the sirolimus albumin nanoparticle aqueous solution was determined by HPLC and was approximately 1.2 mg / mL.

[0054] (3) Add 0.03 mg of 3-maleimidopropionic acid to 10 mL of the sirolimus albumin nanoparticle reconstituted solution under stirring (the molar ratio of 3-maleimidopropionic acid to total albumin in the solution is about 1:1) and stir for 5 minutes.

[0055] (4) The solution obtained in step (3) was centrifuged at 15000 g for 1 h, the supernatant was discarded, and the supernatant in step (2) was used for re-dissolution to obtain a sirolimus albumin nanoparticle solution with a stable particle size.

[0056] (5) The sirolimus albumin nanoparticle solution with a stable particle size was placed at room temperature. Samples were taken at 0 h, 24 h, and 48 h, and the particle size of the nanoparticles was measured. The particle size measurement results are shown in Table 1.

[0057] Table 1 Sample particle size test results

[0058]

[0059] Table 1 shows that the particle size of the sirolimus albumin nanoparticles prepared in this example is stable.

[0060] The schematic diagram of drug-loaded albumin nanoparticles forming nanoparticle dimers through the free thiol -SH on the surface of the nanoparticles is shown in Figure 1 shown.

[0061] The schematic diagram of using 3-maleimidopropionic acid containing maleimide (MAL) fragment to block the surface of drug-loaded albumin nanoparticles -SH is shown in the figure. Figure 2 shown.

[0062] Example 2

[0063] Repeat the steps of Example 1, except that step (3) is replaced by:

[0064] (3) Add 0.15 mg of 3-maleimidopropionic acid to 10 mL of the sirolimus albumin nanoparticle reconstituted solution under stirring (the molar ratio of 3-maleimidopropionic acid to total albumin in the solution is now approximately 5:1) and stir for 5 minutes.

[0065] The particle size measurement results are shown in Table 2.

[0066] Table 2 Sample particle size test results

[0067]

[0068]

[0069] Table 2 shows that the particle size of the sirolimus albumin nanoparticles prepared in this example is stable.

[0070] Example 3

[0071] Repeat the steps of Example 1, except that step (3) is replaced by:

[0072] (3) Add 0.6 mg of 3-maleimidopropionic acid to 10 mL of the sirolimus albumin nanoparticle reconstituted solution under stirring (the molar ratio of 3-maleimidopropionic acid to total albumin in the solution is about 20:1) and stir for 5 minutes.

[0073] The particle size measurement results are shown in Table 3.

[0074] Table 3 Sample particle size test results

[0075]

[0076] Table 3 shows that the particle size of the sirolimus albumin nanoparticles prepared in this example is stable.

[0077] Example 4

[0078] Repeat the steps of Example 1, except that step (3) is replaced by:

[0079] (3) Add 0.05 mL of hydrogen peroxide solution (concentration of 5 g / 100 mL) to 10 mL of the sirolimus albumin nanoparticle reconstituted solution under stirring (the hydrogen peroxide:total albumin (mass ratio) in the solution is about 1:5) and stir for 5 minutes.

[0080] The results are shown in Table 4.

[0081] Table 4 Sample particle size test results

[0082]

[0083] Table 4 shows that the particle size of the sirolimus albumin nanoparticles prepared in this example is stable.

[0084] Example 5

[0085] Repeat the steps of Example 1, except that step (3) is replaced by:

[0086] (3) Add 0.10 mL of hydrogen peroxide solution (concentration of 5 g / 100 mL) to 10 mL of the sirolimus albumin nanoparticle reconstituted solution under stirring (the hydrogen peroxide:total albumin (mass ratio) in the solution is about 2:5) and stir for 5 minutes.

[0087] The results are shown in Table 5.

[0088] Table 5 Sample particle size test results

[0089]

[0090] Table 5 shows that the particle size of the sirolimus albumin nanoparticles prepared in this example is stable.

[0091] Example 6

[0092] Repeat the steps of Example 1, except that step (3) is replaced by:

[0093] (3) Add 0.25 mL of hydrogen peroxide solution (concentration of 5 g / 100 mL) to 10 mL of the sirolimus albumin nanoparticle reconstituted solution under stirring (the hydrogen peroxide:total albumin (mass ratio) in the solution is about 1:1) and stir for 5 minutes.

[0094] The results are shown in Table 6.

[0095] Table 6 Sample particle size test results

[0096]

[0097] Table 6 shows that the particle size of the sirolimus albumin nanoparticles prepared in this example is stable.

[0098] Example 7

[0099] (1) 1.6 g of sirolimus was weighed and dissolved in 30.40 g of anhydrous ethanol to obtain an organic phase solution with a concentration of 50 mg / g; a human serum albumin solution containing 13.60 g (about 204.5 μmol) of albumin was diluted with water for injection to obtain an aqueous phase solution containing 3 mg / g of albumin and heated to 60°C for use; the organic phase solution and the aqueous phase solution were mixed and loaded with the drug using a linear mixing device, and the resulting solution was then rapidly cooled to room temperature to obtain a dilute solution of sirolimus albumin nanoparticles. The sirolimus concentration in the dilute solution was 1.2 mg / mL, and the total albumin concentration was 10 mg / mL.

[0100] (2) Add 2.67 g of 3-maleimidopropionic acid to 1300 mL of the dilute solution under stirring (the molar ratio of 3-maleimidopropionic acid to total albumin in the solution is about 80:1) and stir for 5 minutes.

[0101] (3) The solution obtained in step (2) was concentrated using a tangential flow filtration system and washed to remove ethanol and unreacted 3-maleimidopropionic acid, thereby obtaining a sirolimus albumin nanoparticle concentrate having a sirolimus concentration of 6 mg / mL.

[0102] (4) The sirolimus albumin nanoparticle concentrate was filled into a 50R controlled vial with a filling volume of 16.7 mL and freeze-dried according to the freeze-drying process in Table 7.

[0103] (5) One bottle of the lyophilized product was reconstituted with 20 mL of 0.9% sodium chloride aqueous solution to obtain a sirolimus albumin nanoparticle reconstitution solution with a sirolimus concentration of 5 mg / mL. The reconstitution solution was placed at 25°C, and samples were taken at 0 h, 24 h, and 48 h to measure the nanoparticle size. The results are shown in Table 8.

[0104] Table 7 Freeze-drying process

[0105]

[0106] Table 8 Sample particle size test results

[0107]

[0108] Table 8 shows that the particle size of the sirolimus albumin nanoparticles prepared in this example is stable.

[0109] Example 8

[0110] Repeat the steps of Example 7, except that step (2) is replaced by:

[0111] (2) Add 0.53 g of 3-maleimidopropionic acid to 1300 mL of the diluted solution under stirring (the molar ratio of 3-maleimidopropionic acid to total albumin in the solution is about 16:1) and stir for 5 minutes.

[0112] The particle size measurement results are shown in Table 9.

[0113] Table 9 Sample particle size test results

[0114]

[0115] Table 9 shows that the particle size of the sirolimus albumin nanoparticles prepared in this example is stable.

[0116] Example 9

[0117] Repeat the steps of Example 7, except that step (2) is replaced by:

[0118] (2) Add 10.68 g of 3-maleimidopropionic acid to 1300 mL of the diluted solution under stirring (the molar ratio of 3-maleimidopropionic acid to total albumin in the solution is now about 320:1) and stir for 5 minutes.

[0119] The particle size measurement results are shown in Table 10.

[0120] Table 10 Sample particle size test results

[0121]

[0122] Table 10 shows that the particle size of the sirolimus albumin nanoparticles prepared in this example is stable.

[0123] Example 10

[0124] Repeat the steps of Example 7, except that steps (2) and (3) are replaced by:

[0125] (2) Add 4 mL of hydrogen peroxide solution (concentration of 5 g / 100 mL) to 1300 mL of the dilute solution under stirring (the mass ratio of hydrogen peroxide to total albumin in the solution is about 1:65) and stir for 5 minutes.

[0126] (3) The solution obtained in step (2) was concentrated using a tangential flow filtration system and washed to remove ethanol and unreacted hydrogen peroxide, thereby obtaining a sirolimus albumin nanoparticle concentrate having a sirolimus concentration of 6 mg / mL.

[0127] The particle size measurement results are shown in Table 11.

[0128] Table 11 Sample particle size test results

[0129]

[0130] Table 11 shows that the particle size of the sirolimus albumin nanoparticles prepared in this example is stable.

[0131] Example 11

[0132] Repeat the steps of Example 7, except that steps (2) and (3) are replaced by:

[0133] (2) Add 0.8 mL of hydrogen peroxide solution (concentration of 5 g / 100 mL) to 1300 mL of the dilute solution under stirring (the mass ratio of hydrogen peroxide to total albumin in the solution is about 1:325) and stir for 5 minutes.

[0134] (3) The solution obtained in step (2) was concentrated using a tangential flow filtration system and washed to remove ethanol and unreacted hydrogen peroxide, thereby obtaining a sirolimus albumin nanoparticle concentrate having a sirolimus concentration of 6 mg / mL.

[0135] The particle size measurement results are shown in Table 12.

[0136] Table 12 Sample particle size test results

[0137]

[0138]

[0139] Table 12 shows that the particle size of the sirolimus albumin nanoparticles prepared in this example is stable.

[0140] Example 12

[0141] Repeat the steps of Example 7, except that steps (2) and (3) are replaced by:

[0142] (2) Add 8 mL of hydrogen peroxide solution (concentration of 5 g / 100 mL) to 1300 mL of the dilute solution under stirring (the mass ratio of hydrogen peroxide to total albumin in the solution is about 1:32) and stir for 5 minutes.

[0143] (3) The solution obtained in step (2) was concentrated using a tangential flow filtration system and washed to remove ethanol and unreacted hydrogen peroxide, thereby obtaining a sirolimus albumin nanoparticle concentrate having a sirolimus concentration of 6 mg / mL.

[0144] The particle size measurement results are shown in Table 13.

[0145] Table 13 Sample particle size test results

[0146]

[0147] Table 13 shows that the particle size of the sirolimus albumin nanoparticles prepared in this example is stable.

[0148] Comparative Example 1

[0149] (1) 1.6 g of sirolimus was weighed and dissolved in 30.40 g of anhydrous ethanol to obtain an organic phase solution with a concentration of 50 mg / g; a human serum albumin solution containing 13.60 g (about 204.5 μmol) of albumin was taken and diluted with water for injection to obtain an aqueous phase solution containing 3 mg / g of albumin and heated to 60°C for use; the organic phase solution and the aqueous phase solution were mixed and loaded with the drug using a linear mixing device, and the resulting solution was then rapidly cooled to room temperature to obtain a dilute solution of sirolimus albumin nanoparticles. The dilute solution was concentrated using a tangential flow filtration system to a sirolimus concentration of 6 mg / mL, thereby obtaining a sirolimus albumin nanoparticle concentrate, at which point the total albumin concentration in the concentrate was 51 mg / mL.

[0150] (2) The sirolimus albumin nanoparticle concentrate prepared in step (1) was placed at room temperature, and samples were taken at 0 h, 24 h, and 48 h, respectively, to measure the particle size of the nanoparticles. The particle size measurement results are shown in Table 14.

[0151] Table 14 Sample particle size test results

[0152]

[0153] Table 14 shows that the particle size of the sirolimus albumin nanoparticles prepared in this comparative example is unstable.

[0154] Comparative Example 2

[0155] (1) 1.6 g of sirolimus was weighed and dissolved in 30.40 g of anhydrous ethanol to obtain an organic phase solution with a concentration of 50 mg / g; a human serum albumin solution containing 13.60 g (about 204.5 μmol) of albumin was diluted with water for injection to obtain an aqueous phase solution containing 3 mg / g of albumin and heated to 60°C for use; the organic phase solution and the aqueous phase solution were mixed and loaded with the drug using a linear mixing device, and the resulting solution was then rapidly cooled to room temperature to obtain a dilute solution of sirolimus albumin nanoparticles. The sirolimus concentration in the dilute solution was 1.2 mg / mL, and the total albumin concentration was 10 mg / mL.

[0156] (2) The solution obtained in step (1) was concentrated using a tangential flow filtration system, and then filtered to remove ethanol, thereby obtaining a sirolimus albumin nanoparticle concentrate having a sirolimus concentration of 6 mg / mL.

[0157] (3) The sirolimus albumin nanoparticle concentrate was filled into a 50R controlled vial with a filling volume of 16.7 mL and freeze-dried according to the freeze-drying process in Table 7.

[0158] (4) One bottle of the lyophilized product was reconstituted with 20 mL of 0.9% sodium chloride aqueous solution to obtain a sirolimus albumin nanoparticle reconstitution solution with a sirolimus concentration of 5 mg / mL. The reconstitution solution was incubated at 25°C. Samples were taken at 0 h, 24 h, and 48 h, and the nanoparticle particle size was measured. The particle size measurement results are shown in Table 15.

[0159] Table 15 Sample particle size test results

[0160]

[0161] Table 15 shows that the particle size of the sirolimus albumin nanoparticles prepared in this comparative example is unstable.

Claims

1. A method for improving the particle size stability of drug-loaded albumin nanoparticles, comprising the step of blocking the thiol groups (-SH) on the surface of the drug-loaded albumin nanoparticles.

2. The method according to claim 1, wherein: The thiol groups -SH on the surface of the drug-loaded albumin nanoparticles are blocked by chemical reaction.

3. The method according to claim 2, wherein: The chemical reaction is selected from one or more of click chemistry reaction, oxidation reaction, nucleophilic substitution reaction, reduction reaction, and condensation reaction.

4. The method according to any one of claims 1 to 3, wherein: The method comprises the following steps: S1. Centrifuging the solution containing the drug-loaded albumin nanoparticles, aspirating the supernatant for later use, and re-dissolving the precipitate with water to obtain a drug-loaded albumin nanoparticle aqueous solution; S2. adding a compound containing an unsaturated group to the reconstituted aqueous solution of the drug-loaded albumin nanoparticles under stirring to perform a click chemistry reaction or adding an oxidant to perform an oxidation reaction; S3. Centrifuge the solution obtained in step S2, discard the supernatant, and re-dissolve with the supernatant in step S1 to obtain a drug-loaded albumin nanoparticle solution with a stable particle size.

5. The method according to claim 4, wherein: The compound containing an unsaturated group is a compound containing a maleimide fragment (MAL), preferably one or more selected from 3-maleimidopropionic acid, N-methylmaleimide, maleimide, 2-maleimidoacetic acid, 1,6-di(maleimido)hexane; and / or The oxidant is selected from one or more of peroxides and ozone, and is preferably selected from one or more of hydrogen peroxide, sodium peroxide, potassium peroxide, calcium peroxide, and magnesium peroxide.

6. The method according to claim 5, wherein: The molar ratio of the compound containing maleimide fragment (MAL) to the total albumin in the reconstituted solution of the drug-loaded albumin nanoparticles is not less than 0.5:1, preferably (1-20):1; and / or The mass ratio of the hydrogen peroxide to the total albumin in the aqueous reconstitution solution of the drug-loaded albumin nanoparticles is not less than 0.5:5, preferably (1-5):

5.

7. The method according to any one of claims 1 to 3, wherein: The method comprises the following steps: s1. adding a compound containing an unsaturated group to the solution containing the drug-loaded albumin nanoparticles under stirring to perform a click chemistry reaction or adding an oxidant to perform an oxidation reaction; s2. Concentrate the solution obtained in step s1 and wash filter.

8. The method according to claim 7, wherein: The compound containing an unsaturated group is a compound containing a maleimide fragment (MAL), preferably one or more selected from 3-maleimidopropionic acid, N-methylmaleimide, maleimide, 2-maleimidoacetic acid, 1,6-di(maleimido)hexane; and / or The oxidant is selected from one or more of peroxides and ozone, and is preferably selected from one or more of hydrogen peroxide, sodium peroxide, potassium peroxide, calcium peroxide, and magnesium peroxide.

9. The method according to claim 8, wherein: The molar ratio of the compound containing maleimide fragment (MAL) to the total albumin in the solution containing the drug-loaded albumin nanoparticles is not less than 5:1, preferably (16-320):1; and / or The mass ratio of the hydrogen peroxide to the total albumin in the solution containing the drug-loaded albumin nanoparticles is not less than 1:500, preferably 1:(32-325).

10. The method according to any one of claims 1 to 9, characterized in that: The albumin in the drug-loaded albumin nanoparticles is selected from one or more of human serum albumin, bovine serum albumin, and ovalbumin, preferably human serum albumin; The drug in the drug-loaded albumin nanoparticles is selected from one or more of taxanes, macrolides, anthracyclines, irinotecan, tacrolimus, lopinavir, cyclosporine, and semustine; Preferably, the taxane drug is selected from one or more of paclitaxel, docetaxel, and cabazitaxel; Preferably, the macrolide drug is selected from one or more of sirolimus, epothilone B, tanespiramycin, and everolimus; Preferably, the anthracycline is selected from one or more of epirubicin, doxorubicin, aclarubicin, and pirarubicin.

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