Protein particle with a poorly water-soluble drug encapsulated therein and preparation method thereof

Abstract

The present invention relates to a protein particle with a poorly water-soluble drug encapsulated therein and a preparation method therefor. The preparation method comprises the following steps: i) dissolving a poorly water-soluble drug and a liquid solubilizer into a good solvent; ii) partially or fully removing said good solvent from the product of step i); iii) mixing the product of step ii) with a protein; and iv) dispersing the product of step iii) in a poor solvent. The preparation method is simple and is suitable for industrial production.

Claims

1. A method for preparing a protein particle with a poorly water-soluble drug encapsulated therein, comprising the following steps: i) dissolving a poorly water-soluble drug and a liquid solubilizer into a first solvent, wherein said first solvent comprises one of more selected from the group consisting of ethanol, methanol, diethyl ether, dichloromethane, and chloroform; ii) partially or fully removing said first solvent from the product of step i) to form a liquid complex comprising the poorly water-soluble drug and the liquid solubilizer; iii) mixing the product of step ii) with a protein, wherein said protein is albumin; iv) dispersing the product of step iii) in a second solvent, wherein said second solvent is selected from the group consisting of water, phosphate buffer saline buffer solution and citric acid buffer solution; wherein said poorly water-soluble drug has solubility as follows: 1 gram of said poorly water-soluble drug is incapable of being completely dissolved in 30 ml or more of water.

2. The method according to claim 1, wherein said liquid solubilizer comprises one of more selected from the group consisting of propanol, liquid polyethylene glycol, polysorbates, sorbitan esters and glycerol.

3. The method according to claim 1, wherein step ii) comprises: partially or fully removing said first good solvent from the product of step i) by one or more operations selected from the group consisting of rotating evaporation, heating and vacuum pumping.

4. The method according to claim 1, wherein in step iii), said protein is in powder form.

5. The method according to claim 1, wherein the albumin comprises one or more selected from the group consisting of recombinant albumin and natural albumin.

6. The method according to claim 1, wherein step iv) comprises: dispersing the product of step iii) in the second solvent by one or more operations selected from the group consisting of stirring, shearing, ultrasonic treatment, homogenizing, and grinding.

7. The method according to claim 1, further comprising removing the liquid solubilizer from the product of step iv).

8. The method according to claim 1, further comprising separating an albumin particle from the product of step iv).

9. The method according to claim 1, wherein said poorly water-soluble drug comprises one or more selected from the group consisting of paclitaxel, docetaxel, camptothecin, hydroxycamptothecin, rapamycin, doxorubicin, itraconazole and nimodipine.

10. The method according to claim 1, wherein said liquid solubilizer is selected from the group consisting of liquid polyethylene glycol, and polysorbates.

11. The method according to claim 1, wherein said protein particle with a poorly water-soluble drug encapsulated therein has an average particle size of 100 nm-1000 nm.

12. The method according to claim 1, wherein said albumin comprises one or more selected from the group consisting of natural albumin and modified albumin.

13. A protein particle with a poorly water-soluble drug encapsulated therein, prepared by the method according to claim 1.

Description

DESCRIPTION OF FIGURES

(1) The drawings described herein are provided for a further understanding of the present disclosure and are intended to be a part of the present disclosure, however, the illustrative examples of the disclosure and the description thereof are used for explaining the present disclosure and are not intended to limit the disclosure. In the figures:

(2) FIG. 1 depicts curves reflecting intensity variation of transmitted light of paclitaxel protein particle diluted by 50 times with PBS;

(3) FIG. 2 depicts curves reflecting intensity variation of backscattered light of paclitaxel protein particles diluted by 50 times with PBS;

(4) FIG. 3 depicts curves reflecting intensity variation of transmitted light of paclitaxel protein particles diluted by 100 times with PBS;

(5) FIG. 4 depicts curves reflecting intensity variation of backscattered light of paclitaxel protein particles diluted by 100 times with PBS;

(6) FIG. 5 depicts curves reflecting intensity variation of transmitted light of paclitaxel protein particles diluted by 50 times with complete medium;

(7) FIG. 6 depicts curves reflecting intensity variation of backscattered light of paclitaxel protein particles diluted by 50 times with complete medium;

(8) FIG. 7 depicts curves reflecting intensity variation of transmitted light of paclitaxel protein particles diluted by 100 times with complete medium;

(9) FIG. 8 depicts curves reflecting intensity variation of backscattered light of paclitaxel protein particles diluted by 100 times with complete medium.

EXAMPLES

(10) The embodiments of the disclosure are specifically described by the following figures and examples.

(11) Instruments used in the following examples are shown in the following table:

(12) TABLE-US-00001 Name of Instruments Manufacture and Brands Rotary Evaporator RE-2000, Shanghai Yarong Biochemical Instrument Factory Circulating Water SHD-D(III), Gongyi City Yingyu Yuhua Vacuum Pump Instrument Factory High Performance degasser G1379B; Bin Pump SL G1312B, Liquid Hip-ALSSL G1367C, TCCSL G1316B, UV Chromatography G1314C, U.S. Agilent Company; chroma- tographic column (Agilent Eclipse plus-C.sub.8 4.6 × 250 mm 5 μm, U.S. Agilent Company) Ultrasonic Cell SCIENTZ (IID), Ningbo Scientz Biotech- Disruptor nology Co,. LTD High-pressure AVESTIN, Canada, EmulsiFlex-C3 Homogenizer Stability Meter Formulation TURBISCAN TOWER Particle Size Meter FLUOSTAR, Germany Symptec Centrifugal Micromax, U.S. Equipment Human Serum Batch Number: VNA1Q087, Baxter AG (U.S. Albumin Baxter Company)

Example 1

(13) 50 mg of paclitaxel was weighed, added with 400 μL of PEG600, added with 10 mL of anhydrous ethanol, and the mixture was subjected to ultrasonic treatment (with a water temperature of 30˜45° C.) until it became transparent, ethanol was removed by rotary evaporation under reduced pressure, and a paclitaxel-polyethylene glycol complex was formed with a residual amount of ethanol of less than or equal to 0.5 wt %;

(14) 100 mg of human serum albumin powder was weighed and added to the paclitaxel-polyethylene glycol complex, mixed well, to obtain a mixture.

(15) The abovementioned mixture was added to 2 mL of PBS solution with a pH of 7.4, and the mixture was subjected to ultrasonic treatment in an ultrasonic disruptor with 45% of total power for 200 s (ultrasonic treatment for 2 s—pausing for 2 s, in a loop), to obtain a suspension.

(16) The suspension was put into a dialysis bag containing a PBS solution having a pH of 7.4, and repeatedly dialyzed to remove PEG600 and free albumin. Subsequently, the suspension was subjected to configuration with a speed of 3000 rpm for 10 min to remove drug participants that were not encapsulated, then the product was subjected to freeze drying, to obtain protein particles with paclitaxel encapsulated therein.

Example 2

(17) 100 mg of 10-hydroxycamptothecin was weighed, added with 1 mL of PEG400, added with a appropriate amount of anhydrous ethanol, and the mixture was subjected to ultrasonic treatment until it became transparent, ethanol was removed by rotary evaporation under reduced pressure, and a 10-hydroxycamptothecin-polyethylene glycol complex was formed with a residual amount of ethanol of less than or equal to 0.5 wt %;

(18) 300 mg of human serum albumin powder was weighed and added to the complex, mixed well, to obtain a mixture.

(19) The abovementioned mixture is added into 10 mL of citric acid buffer solution having a pH of 5.0, the mixture was homogenized by passing through a membrane using a high-pressure homogenizer, to obtain a suspension.

(20) The suspension was put into a dialysis bag and repeatedly dialyzed to remove PEG400 and free albumin.

(21) The suspension was subjected to centrifugation with a speed of 3000 rpm for 10 min to remove drug participants that were not encapsulated, then the product was subjected to freeze drying, to obtain protein particles with 10-hydroxycamptothecin encapsulated therein.

Example 3

(22) Approximately 100 mg of docetaxel was weighed, added with 500 μL of PEG400, added with an appropriate amount of anhydrous methanol, and the mixture was subjected to ultrasonic treatment until it became transparent, methanol was removed by rotary evaporation under reduced pressure, and a docetaxel-polyethylene glycol complex was formed with a residual amount of ethanol of less than or equal to 0.5 wt %;

(23) 300 mg of human serum albumin powder was weighed and added to the complex, mixed well, to obtain a mixture.

(24) The abovementioned mixture is added into 10 mL of citric acid buffer solution having a pH of 5.0, and the mixture was homogenized by passing through a membrane using a high-pressure homogenizer, to obtain a suspension.

(25) The suspension was put into a dialysis bag and repeatedly dialyzed to remove PEG400 and free albumin. The suspension was subjected to centrifugation with a speed of 3000 rpm for 10 min to remove drug participants that were not encapsulated, then the product was subjected to freeze drying, to obtain protein particles with docetaxel encapsulated therein.

Example 3b

(26) Approximately 100 mg of docetaxel was weighed, added with 500 μL of PEG400, added with an appropriate amount of anhydrous methanol, the mixture was subjected to ultrasonic treatment until it became transparent, methanol was removed by rotary evaporation under reduced pressure, and a docetaxel-polyethylene glycol complex was formed with a residual amount of ethanol of less than or equal to 0.5 wt %;

(27) 300 mg of human serum albumin powder was weighed and added to the complex, mixed well, to obtain a mixture;

(28) The abovementioned mixture was added into 10 mL of PBS solution having a pH of 7.4, and the mixture was homogenized by passing through a membrane using a high-pressure homogenizer, to obtain a suspension.

(29) The suspension was put into a dialysis bag and repeatedly dialyzed to remove PEG400 and free albumin. The suspension was subjected to centrifugation with a speed of 3000 rpm for 10 min to remove drug participants that were not encapsulated, then the product was subjected to freeze drying, to obtain protein particles with docetaxel encapsulated therein.

Example 4

(30) Approximately 100 mg of rapamycin was weighed, added with 1 mL of PEG400, added with an appropriate amount of anhydrous ethanol, and the mixture was subjected to ultrasonic treatment until it became transparent, ethanol was removed by rotary evaporation under reduced pressure, and a rapamycin-polyethylene glycol complex was formed with a residual amount of ethanol of less than or equal to 0.5 wt %.

(31) 300 mg of human serum albumin powder was weighed and added to the complex, to obtain a mixture by mixing.

(32) The abovementioned mixture was added into 10 mL of citric acid buffer solution having a pH of 5.0, and the mixture was homogenized by passing through a membrane using a high-pressure homogenizer, to obtain a suspension. The suspension was put into a dialysis bag, and repeatedly dialyzed to remove PEG400 and free albumin.

(33) The suspension was subjected to centrifugation with a speed of 3000 rpm for 10 min to remove drug participants that were not encapsulated, then the product was subjected to freeze drying, to obtain protein particles with rapamycin encapsulated herein.

Example 5

(34) Approximately 100 mg of itraconazole was weighed, added with 1 mL of PEG400, added with an appropriate amount of anhydrous ethanol, and the mixture was subjected to ultrasonic treatment until it became transparent, ethanol was removed by rotary evaporation under reduced pressure, and an itraconazole-polyethylene glycol complex was formed with a residual amount of ethanol of less than or equal to 0.5 wt %;

(35) 300 mg of human serum albumin powder was weighed and added to the complex, mixed well, to obtain a mixture.

(36) The abovementioned mixture was added into 10 mL of citric acid buffer solution having a pH of 5.0, and the mixture was homogenized by passing through a membrane using a high-pressure homogenizer, obtaining a suspension.

(37) The suspension was put into a dialysis bag and repeatedly dialyzed to remove PEG400 and free albumin. The suspension was subjected to centrifugation with a speed of 3000 rpm for 10 min to remove drug participants that were not encapsulated, then the product was subjected to freeze drying, to obtain protein particles with itraconazole encapsulated therein.

Example 5b

(38) Approximately 100 mg of itraconazole was weighed, added with 1 mL of PEG400, added with an appropriate amount of anhydrous ethanol, the mixture was subjected to ultrasonic treatment until it became transparent, ethanol was removed by rotary evaporation under reduced pressure, and an itraconazole-polyethylene glycol complex was formed with a residual amount of ethanol of less than or equal to 0.5 wt %;

(39) 300 mg of human serum albumin powder was weighed and added to the complex, mixed well, to obtain a mixture.

(40) The abovementioned mixture is added into 10 mL of PBS solution having a pH of 7.4 solution, and the mixture was homogenized by passing through a membrane using a high-pressure homogenizer, obtaining a suspension.

(41) The suspension was put into a dialysis bag and repeatedly dialyzed to remove PEG400 and free albumin. The suspension was subjected to centrifugation with a speed of 3000 rpm for 10 min to remove drug participants that were not encapsulated, then the product was subjected to freeze drying, to obtain protein particles with itraconazole encapsulated therein.

Example 6

(42) Approximately 100 mg of hydroxycamptothecin was weighed, added with 1 mL of Tween80, added with an appropriate amount of anhydrous ethanol, and the mixture was subjected to ultrasonic treatment until it became transparent, ethanol was removed by rotary evaporation under reduced pressure, and a hydroxycamptothecin-Tween80 complex was formed with a residual amount of ethanol of less than or equal to 0.5 wt %;

(43) 300 mg of human serum albumin powder was weighed and added to the complex, mixed well, to obtain a mixture.

(44) The abovementioned mixture is added into 10 mL of citric acid buffer solution having a pH of 5.0, and the mixture was homogenized by passing through a membrane using a high-pressure homogenizer, obtaining a suspension.

(45) The suspension was put into a dialysis bag and repeatedly dialyzed to remove Tween80 and free albumin. The suspension was subjected to centrifugation with a speed of 3000 rpm for 10 min to remove drug participants that were not encapsulated, then the product was subjected to freeze drying, to obtain protein particles with hydroxycamptothecin encapsulated therein.

(46) Yields of nearly 100% were achieved by the methods of Example 1˜6.

(47) Assay 1—The Measurement of Encapsulation Efficiency and Drug Loading

(48) (1) Approximately 10 mg of albumin particles were provided, an appropriate amount of ethanol was added to completely dissolve the albumin particles, and they were diluted with the mobile phase to a certain volume, the concentration of the drug was measured by HPLC method, and the total amount of drug in the particles (A) was calculated.

(49) (2) Another approximately 10 mg of albumin particles were provided, an appropriate amount of water was added, mixed well, and the mixture was centrifuged for 3000 rpm for 10 min. After the centrifugation, if there were any, the precipitates were collected, dissolved with an appropriate amount of ethanol, diluted with mobile phase to a certain volume, and the concentration of the drug was measured by HPLC method, and the total amount of drug in the precipitates (B) was calculated.

(50) After the centrifugation, the upper layer of the suspension was collected and centrifuged with a super-high speed of 16,000 rpm for 20 min, the supernatant was collected, diluted with mobile phase to a certain volume, the concentration of the drug in the upper layer of the suspension was measured by HPLC method, and the total amount of drug in the upper layer of the suspension (C) was calculated.

(51) The drug in the precipitates after low-speed centrifugation and the drug in the supernatant after the super-high centrifugation are the drugs that were not encapsulated.

(52) The encapsulation efficiency and the drug loading were calculated.
Encapsulation Efficiency=(A−B−C)/A×100%
Drug Loading=EE×W.sub.drug/(W.sub.drug+W.sub.albumin)

(53) EE is Encapsulation Efficiency. W.sub.drug is the total amount of drug that is fed during the preparation process of the particles. W.sub.albumin is the total amount of albumin that is fed during the preparation process of the particles albumin.

(54) TABLE-US-00002 TABLE 1 The drug loading and encapsulation efficiency of the albumin particles example example example example example example example example 1 2 3 4 5 6 3b 5b encapsulation 99.2% 98.6% 94.5% 96.8% 94.4% 92.3% 98.7 99.2% efficiency (%) drug loading 33.1% 24.6% 23.6% 24.2% 23.6% 21.4% 24.7% 24.8% (%)

(55) Assay 2—Measurement of Particle Size of Albumin Particles

(56) Albumin particles of examples 1˜6 were provided, added with water to form suspensions, the suspensions were put in a laser particle size analyzer to measure the particle sizes of the particles. The results are as follows:

(57) TABLE-US-00003 TABLE 2 particle sizes of albumin particles example example example example example example example example 1 2 3 4 5 6 3b 5b Particle 186 173 192 204 193 153 195 202 size (nm)

(58) According to table 2, protein particles with poorly water-soluble drug encapsulated therein can be obtained by methods of example 1˜6, for example protein nanoparticle with poorly water-soluble drug encapsulated therein having particle sizes from 153 nm to 204 nm were obtained.

(59) Assay 3—Measurement of Stability of Paclitaxel Albumin Particles

(60) The paclitaxel albumin particles of Example 1 were provided and diluted with phosphate buffer solution (PBS) and minimum essential medium (MEM) containing fetal calf serum respectively by 50 times and by 100 times. The stability of the stock solution and the diluted solutions were measured by a stability meter. The results are shown in FIGS. 1˜8. FIG. 1 depicts curves reflecting intensity variation of transmitted light of paclitaxel protein particle diluted by 50 times with PBS. FIG. 2 depicts curves reflecting intensity variation of backscattered light of paclitaxel protein particles diluted by 50 times with PBS. FIG. 3 depicts curves reflecting intensity variation of transmitted light of paclitaxel protein particles diluted by 100 times with PBS. FIG. 4 depicts curves reflecting intensity variation of backscattered light of paclitaxel protein particles diluted by 100 times with PBS. FIG. 5 depicts curves reflecting intensity variation of transmitted light of paclitaxel protein particles diluted by 50 times with complete medium. FIG. 6 depicts curves reflecting intensity variation of backscattered light of paclitaxel protein particles diluted by 50 times with complete medium. FIG. 7 depicts curves reflecting intensity variation of transmitted light of paclitaxel protein particles diluted by 100 times with complete medium. FIG. 8 depicts curves reflecting intensity variation of backscattered light of paclitaxel protein particles diluted by 100 times with complete medium. The results indicate that the intensity variation of both transmitted light and backscattered light of paclitaxel albumin particles diluted by various medium at 37° C. are no more than 3%, which indicates the paclitaxel protein particles have good stability.

(61) Finally, it should be noted that the above examples are only for illustrating the embodiments of the present disclosure rather than limiting them; although the present disclosure is described in detail with reference to the preferred examples, those of ordinary skill in the art should understand: the detailed examples can be modified and some of the technical features can be equivalently substituted as long as they do not depart from the spirit of the embodiments of the present disclosure. These modifications and substitutions should all be covered within the scope of the claims claimed by the disclosure.