CN111617045B - Taxol nano preparation and preparation method thereof - Google Patents

Taxol nano preparation and preparation method thereof Download PDF

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CN111617045B
CN111617045B CN202010310859.8A CN202010310859A CN111617045B CN 111617045 B CN111617045 B CN 111617045B CN 202010310859 A CN202010310859 A CN 202010310859A CN 111617045 B CN111617045 B CN 111617045B
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CN111617045A (en
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顾全荣
袁守军
李琳娜
王东海
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Beijing Sunlight Pharmaceutical Co ltd
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Abstract

The invention discloses a taxol nanometer preparation and a preparation method thereof, wherein the preparation method comprises the following steps: s1, dissolving paclitaxel in a first organic solvent to obtain a first solution; s2, dissolving the mPEG-b-PCL amphiphilic block copolymer in a second organic solvent to obtain a second solution; s3, dissolving the fatty acid in a third organic solvent to obtain a third solution; s4, mixing the first solution, the second solution and the third solution, and removing the organic solvent to obtain a mixture; s5, hydrating the mixture, and filtering the mixture by using a nano filter to obtain filtrate; s6, freezing the filtrate to obtain paclitaxel nano ice blocks; s7, freeze-drying the paclitaxel nanometer ice blocks to obtain the paclitaxel nanometer preparation. The taxol nanometer preparation has the characteristics of low toxic and side effects, good anti-tumor effect and reduction of tumor metastasis rate, and the preparation method has the advantages of simple process flow, low production cost and easy realization of large-scale production.

Description

Taxol nano preparation and preparation method thereof
Technical Field
The invention belongs to the technical field of biological medicines, and relates to a taxol nano preparation and a preparation method thereof.
Background
Paclitaxel (PTX) is a natural drug extracted from yew trees and has important antitumor activity. The action mechanism is as follows: the polymerization of tubulin is promoted to inhibit depolymerization, so that the tubulin is kept stable, and the mitosis of the cell is inhibited, so that the cell is prevented from replicating and dying. Paclitaxel has poor water solubility and is difficult to directly administer, so researchers and researchers have developed various paclitaxel water-soluble injections. Taxol (Taxol) is a first generation paclitaxel formulation, and the main components of the antitumor drug Taxol developed by Bristol-Myers Squibb, Inc. include paclitaxel, polyoxyethylated castor oil, and absolute alcohol, each comprising 6mg paclitaxel, 527mg purified polyoxyethylated castor oil, and 49.7% (v/v) absolute alcohol per ml sterile, non-pyrogenic solution. Taxol is usually used for treating lung cancer, breast cancer, ovarian cancer and the like, but has adverse reactions such as myelosuppression toxicity, fever complicated with infection, anemia, severe anaphylactic reaction, peripheral neuropathy, arthralgia, myalgia, abnormal liver function and the like. These toxic side effects are mainly caused by the drug itself and the adjuvant of the drug, so that the second generation paclitaxel formulation reduces the toxic side effects by changing the adjuvant.
Abraxane (albumin paclitaxel) and Genex-PM (micellar paclitaxel) belong to the second generation paclitaxel formulations. In Abraxane, paclitaxel and albumin are complexed to form stable particles with an average particle size of 130 nm. Abraxane is typically stored in a 50ml vial as a sterile lyophilized cake containing 100mg paclitaxel and about 900mg human albumin. When the preparation method is used, each vial is dissolved by 20mL of 0.9% sodium chloride injection to prepare a suspension containing 5mg/mL albumin-bound particles, the particle size formed by paclitaxel and albumin is 130nm, the nanoparticles have negative potential, and the system is stable. The Genex-PM is a micelle system formed by paclitaxel and PEG-b-PLA, the paclitaxel and the PEG-b-PLA are compounded to form stable particles of 20-50 nm, and when the composition is used, the composition is dissolved by 0.9% sodium chloride injection, so that the system is stable. Compared with the first generation paclitaxel preparation, the second generation paclitaxel preparation changes the pharmaceutical auxiliary agent and reduces the toxic and side effects, but still has the problems to be improved, such as the albumin auxiliary agent of Abraxane is expensive and has a complex preparation process, Genex-PM still has toxic and side effects and poor tumor targeting property. Therefore, in order to better apply paclitaxel to tumor therapy, development of novel paclitaxel formulations is required.
Disclosure of Invention
The invention aims to overcome the defects of the existing paclitaxel preparation and provides a novel paclitaxel preparation with low toxic and side effects, good anti-tumor effect and simple preparation process and a preparation method thereof. In order to achieve the above purpose, the invention provides the following technical scheme: a paclitaxel nano preparation comprises paclitaxel and an amphiphilic block copolymer, wherein the amphiphilic block copolymer is mPEG-b-PCL amphiphilic block copolymer.
Preferably, the mass ratio of the paclitaxel to the mPEG-b-PCL amphiphilic block copolymer is as follows: 1: 4-100.
A method for preparing the taxol nanometer preparation comprises the following steps:
s1, dissolving paclitaxel in a first organic solvent to obtain a first solution;
s2, dissolving the mPEG-b-PCL amphiphilic block copolymer in a second organic solvent to obtain a second solution;
s3, dissolving the fatty acid in a third organic solvent to obtain a third solution;
s4, mixing the first solution, the second solution and the third solution, and removing the organic solvent to obtain a mixture;
s5, hydrating the mixture, and filtering the mixture by using a nano filter to obtain filtrate;
s6, freezing the filtrate to obtain paclitaxel nano ice blocks;
s7, freeze-drying the paclitaxel nanometer ice blocks to obtain a paclitaxel nanometer preparation;
wherein the mass ratio of the paclitaxel to the mPEG-b-PCL amphiphilic block copolymer is as follows: 1: 4-100.
Preferably, the first organic solvent and the third organic solvent are absolute ethyl alcohol, and the second organic solvent is acetonitrile.
Preferably, the fatty acid in step S3 is selected from any one of lauric acid, palmitic acid, oleic acid and linoleic acid.
Preferably, step S4 is specifically: and stirring and mixing the first solution, the second solution and the third solution in a water bath at 60 ℃ for 30 minutes, and removing the organic solvent at the temperature of 60 ℃ under negative pressure to obtain a mixture of paclitaxel and the amphiphilic block copolymer of mPEG-b-PCL.
Preferably, the step S5 is specifically: hydrating the mixture with distilled water, shaking and mixing for 15-30 minutes to obtain a mixed solution, and filtering the mixed solution with a nano filter to obtain a filtrate.
Preferably, the nano filter in step S5 is a 200 nano filter.
Preferably, the temperature at which the filtrate is frozen in step S6 is-40 ℃.
Preferably, step S7 is specifically: and drying the paclitaxel nanometer ice blocks for 48-72 hours by using a freeze dryer to obtain the paclitaxel nanometer preparation.
The taxol nano preparation comprises taxol and mPEG-b-PCL amphiphilic block copolymer with low tin content, and all components act synergistically, so that the taxol nano preparation has the effects of lower toxic and side effects and better anti-tumor effect. Meanwhile, the preparation method disclosed by the invention is simple in process flow, low in production cost and easy to realize large-scale production.
Drawings
FIG. 1 shows the bioluminescence intensity distribution of Day1 in each group of tumor-bearing nude mice in the experimental examples of the present invention.
FIG. 2, Experimental example of the invention Each group of tumor-bearing nude mice Day1 bioluminescence 2D imaging.
FIG. 3 shows the bioluminescence intensity distribution of Day7 in each group of tumor-bearing nude mice in the experimental examples of the present invention.
FIG. 4, Experimental examples of the invention groups of tumor-bearing nude mice Day7 bioluminescence 2D imaging.
FIG. 5 shows the bioluminescence intensity distribution of Day15 in each group of tumor-bearing nude mice in the experimental examples of the present invention.
FIG. 6, Experimental example of the invention Each group of tumor-bearing nude mice Day15 bioluminescence 2D imaging.
FIG. 7 is a comparison of survival time of tumor-bearing nude mice of each group in the experimental example of the present invention.
FIG. 8 is a graph showing the comparison of the increase times of bioluminescence intensity of each group of tumor-bearing nude mice in the experimental examples of the present invention.
FIG. 9 is a graph showing the comparison of the fold increase of body weight of each group of tumor-bearing nude mice in the experimental examples of the present invention.
Detailed Description
In order to make the skilled person better understand the present invention, the following examples of the present invention are clearly and specifically described, but not intended to limit the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The taxol nano preparation comprises taxol and mPEG-b-PCL amphiphilic block copolymer, wherein the mass ratio of the taxol to the mPEG-b-PCL amphiphilic block copolymer is as follows: 1: 4-100.
In specific embodiments, the paclitaxel and mPEG-b-PCL amphiphilic block copolymer of the invention may be present in a mass ratio of, for example, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100, etc.
The method for preparing the taxol nano preparation comprises the following steps:
s1, dissolving paclitaxel in the first organic solvent to obtain a first solution. In a specific embodiment, the paclitaxel is paclitaxel powder, the first organic solvent is absolute ethyl alcohol, and the concentration of the paclitaxel in the obtained first solution is 0.1-0.5 g/L, preferably 0.2 g/L.
S2, dissolving the mPEG-b-PCL amphiphilic block copolymer in a second organic solvent to obtain a second solution. In a specific embodiment, the mPEG-b-PCL amphiphilic block copolymer has the characteristics of narrow molecular weight distribution and low tin content, wherein the tin content is 3-45 ppm; the second organic solvent is acetonitrile, and the concentration of the mPEG-b-PCL amphiphilic block copolymer in the obtained second organic solvent is 0.4-5 g/L, preferably 2 g/L.
And S3, dissolving the fatty acid in a third organic solvent to obtain a third solution. In a specific embodiment, the fatty acid is any one of lauric acid, palmitic acid, oleic acid and linoleic acid, the third organic solvent is absolute ethyl alcohol, and the concentration of the fatty acid in the obtained third solution is 0.1-2 g/L, preferably 1 g/L.
And S4, mixing the first solution, the second solution and the third solution, and removing the organic solvent to obtain a mixture. In a specific embodiment, the first solution, the second solution and the third solution are mixed according to the following volume ratio: 1-100: 1, preferably 25:25: 1. In a specific embodiment, the first solution, the second solution and the third solution are stirred and mixed for 30 minutes under the condition of water bath at the temperature of 60 ℃, and then the organic solvent is removed under negative pressure at the temperature of 60 ℃ to obtain a mixture of paclitaxel and mPEG-b-PCL amphiphilic block copolymer.
S5, hydrating the mixture, and filtering the mixture by using a nano filter to obtain filtrate. In a specific embodiment, the mixture is hydrated by distilled water, shaking and mixing are carried out for 15-30 minutes to obtain a mixed solution, and the mixed solution is filtered by a 200-nanometer filter to obtain a filtrate.
And S6, freezing the filtrate to obtain paclitaxel nano ice blocks. In a specific embodiment, the filtrate is frozen at-40 ℃ to form paclitaxel nano ice blocks.
S7, freeze-drying the paclitaxel nanometer ice blocks to obtain the paclitaxel nanometer preparation. In a specific embodiment, the paclitaxel nanometer ice blocks are dried for 48-72 hours by a freeze dryer to obtain the paclitaxel nanometer preparation.
Example 1
Preparation of mPEG-b-PCL amphiphilic block copolymer
The paclitaxel nano preparation provided by the invention is prepared from paclitaxel and mPEG-b-PCL amphiphilic block copolymer, wherein the mPEG-b-PCL amphiphilic block copolymer has the characteristic of low tin content, and in the embodiment, the preparation method of the mPEG-b-PCL amphiphilic block copolymer comprises the following steps:
(1) 10g mPEG (molecular weight 2000) was dried under vacuum at 35 ℃ for 8 hours.
(2) mPEG was mixed with 0.1ml of a methylene chloride solution of stannous isooctanoate, sealed, and then high-purity nitrogen gas was blown at 70 ℃ for 2 hours to form N2And (3) adding 5-10 g of epsilon-CL into the mixture in the atmosphere, uniformly mixing, and reacting for 3 hours at the temperature of 170 ℃ to obtain a reaction product.
(3) And cooling to room temperature under the nitrogen atmosphere, adding the reaction product into dichloromethane with the volume of 2 times, adding anhydrous ether with the volume of 10 times after complete dissolution, dissolving unreacted caprolactone and caprolactone homopolymer generated by reaction, and filtering to obtain a crude product of the mPEG (2K) -b-PCL (xK) amphiphilic block copolymer.
(4) And (3) drying the crude product of the mPEG (2K) -b-PCL (xK) amphiphilic block copolymer in vacuum at the temperature of 35 ℃ for 35 hours.
(5) Dissolving the dried mPEG (2K) -b-PCL (xK) amphiphilic block copolymer crude product in water, adding a disodium EDTA solution with the concentration of 0.15g/L, wherein the addition amount of the disodium EDTA solution is that the mass of the disodium EDTA accounts for 2% of the mass of the mPEG (2K) -b-PCL (xK) amphiphilic block copolymer crude product, complexing stannous ions to form a complex, then filling the complex into a dialysis bag, putting the dialysis bag into ultrapure water for dialysis for 20 hours, replacing the ultrapure water every 5 hours, and freeze-drying the dialyzed product for 24 hours to obtain the mPEG-b-PCL amphiphilic block copolymer of the embodiment.
The amphiphilic block copolymer of mPEG-b-PCL prepared by the preparation method has the tin ion content of 3-45 ppm, and has the advantages of narrow molecular weight distribution, low tin element content and high safety.
Example 2
Preparation of taxol nano preparation
In this embodiment, the preparation method of the paclitaxel nano-preparation includes the following steps:
a) 50mg of paclitaxel powder was dissolved in 250ml of anhydrous ethanol solvent.
b) 0.4g of mPEG (2K) -b-PCL (yK) amphipathic copolymer and 0.1g of mPEG (2K) -b-PCL (xK) amphipathic copolymer powder were dissolved in 250ml of acetonitrile solvent.
c)10mg of lauric acid was dissolved in 10ml of absolute ethanol.
d) The three solutions were mixed: in a glass bottle of a rotary evaporator, rotating and mixing for 30 minutes in a water bath at 60 ℃; and negative pressure at 60 deg.c for about 2 hours to remove the ethanol and acetonitrile solvent, leaving the paclitaxel and amphiphilic polymer mixture.
e) Hydrating the mixture of paclitaxel and amphiphilic mPEG-b-PCL polymer with 25ml of distilled water to form a mixed solution, and shaking and mixing uniformly during hydration for 15-30 minutes.
f) The mixture was filtered through a 200 nm filter, and the filtrate was placed in a glass bottle.
g) And (4) freezing the filtrate in the glass bottle at-40 ℃ to form paclitaxel nano ice blocks.
h) The frozen paclitaxel nano ice blocks are dried for 48 hours by a freeze dryer to obtain the paclitaxel nano preparation of the embodiment.
In the paclitaxel nano-preparation prepared in this example, the mass ratio of paclitaxel to the amphiphilic polymer of mPEG-b-PCL is 1: 10. The embodiment also provides a paclitaxel nano injection, which is prepared by adopting normal saline to prepare the paclitaxel nano preparation into the injection, and the preparation method comprises the following steps: 25ml of 0.9% physiological saline dissolves the paclitaxel nano preparation to obtain a transparent liquid, which has a stability of 5 hours and can be used for intravenous injection.
Example 3
Preparation of taxol nano preparation
In this embodiment, the preparation method of the paclitaxel nano-preparation includes the following steps:
(a) 50mg of paclitaxel powder was dissolved in 250ml of anhydrous ethanol solvent.
(b) 0.4g of mPEG (2K) -b-PCL (yK) amphiphilic block copolymer and 0.1g of powders of mPEG (2K) -b-PCL (xK) amphiphilic block copolymer were dissolved in 250ml of acetonitrile solvent.
(c) 10mg of palmitic acid was dissolved in 10ml of absolute ethanol.
(d) Mixing the three solutions, and carrying out rotary mixing in a glass bottle of a rotary evaporator for 30 minutes in a water bath at 60 ℃; subsequently, the ethanol and acetonitrile solvents were removed at 60 ℃ under negative pressure for about 2 hours, leaving a mixture of paclitaxel and mPEG-b-PCL amphiphilic polymer.
(e) And hydrating the mixture of paclitaxel and the amphiphilic mPEG-b-PCL polymer with 25ml of distilled water, and shaking for 15-30 minutes to obtain a mixed solution.
(f) Filtering the mixed solution by using a 200-nanometer filter to obtain filtrate, and placing the filtrate in a glass bottle.
(g) And (4) freezing the filtrate in the glass bottle at-40 ℃ to form paclitaxel nano ice blocks.
(h) The paclitaxel nano ice blocks were dried for 48 hours by a freeze dryer to obtain the paclitaxel nano preparation of this example.
In the paclitaxel nano-preparation prepared in this example, the mass ratio of paclitaxel to the amphiphilic polymer of mPEG-b-PCL is 1: 10. The preparation method of the paclitaxel nano injection of the embodiment is as follows: 25ml of 0.9% physiological saline dissolves the paclitaxel nano preparation to obtain a transparent liquid, which has a stability of 5 hours and can be used for intravenous injection.
Example 4
Preparation of taxol nano preparation
In this embodiment, the preparation method of the paclitaxel nano-preparation includes the following steps:
(1) 50mg of paclitaxel powder was dissolved in 250ml of anhydrous ethanol solvent.
(2) 0.41g of mPEG (2K) -b-PCL (yK) amphiphilic block copolymer powder and 0.09g of mPEG (2K) -b-PCL (xK) amphiphilic block copolymer powder were dissolved in 250ml of acetonitrile solvent.
(3) 10mg of oleic acid are dissolved in 10ml of absolute ethanol.
(4) Mixing the three solutions in a glass bottle of a rotary evaporator, carrying out water bath at 60 ℃, and carrying out rotary mixing for 30 minutes; the ethanol and acetonitrile solvents were removed under reduced pressure at 60 deg.C for about 2 hours, leaving a mixture of paclitaxel and amphiphilic polymer.
(5) Hydrating the mixture of the paclitaxel and the amphiphilic mPEG-b-PCL polymer by using 25ml of distilled water to form a mixed solution, and shaking for 15-30 minutes in the hydrating process.
(6) Filtering the mixed solution by using a 200-nanometer filter to obtain filtrate, and placing the filtrate in a glass bottle.
(7) And (4) freezing the filtrate in the glass bottle at-40 ℃ to form paclitaxel nano ice blocks.
(8) Drying the paclitaxel nanometer ice blocks for 48 hours by using a freeze dryer to obtain the paclitaxel nanometer preparation.
In the paclitaxel nano-preparation prepared in this example, the mass ratio of paclitaxel to the amphiphilic polymer of mPEG-b-PCL is 1: 10. The preparation method of the paclitaxel nano injection of the embodiment is as follows: 25ml of 0.9% physiological saline dissolves the paclitaxel nano preparation to obtain a transparent liquid, which has a stability of 5 hours and can be used for intravenous injection.
Example 5
Preparation of taxol nano preparation
In this embodiment, the preparation method of the paclitaxel nano-preparation includes the following steps:
a) 50mg of paclitaxel powder was dissolved in 250ml of anhydrous ethanol solvent.
b) 0.38g of mPEG (2K) -b-PCL (yK) amphiphilic block copolymer and 0.12g of mPEG (2K) -b-PCL (xK) amphiphilic block copolymer powder were dissolved in 250ml of acetonitrile solvent.
c)10mg of linoleic acid was dissolved in 10ml of absolute ethanol.
d) Mixing the three solutions in a glass bottle of a rotary evaporator, carrying out water bath at 60 ℃, and carrying out rotary mixing for 30 minutes; the ethanol and acetonitrile solvents were then removed under negative pressure at 60 ℃ for about 2 hours, leaving a mixture of paclitaxel and amphiphilic polymer.
e) The mixture of paclitaxel and mPEG-b-PCL was hydrated with 25ml of distilled water and shaken for 15-30 minutes to obtain a mixed solution.
f) Filtering the mixed solution by using a 200-nanometer filter to obtain filtrate, and placing the filtrate in a glass bottle.
g) The filtrate in the glass bottle is frozen at-40 ℃ to form paclitaxel nano ice blocks.
h) The paclitaxel nanometer ice blocks were dried by a freeze dryer for 48 hours to obtain the paclitaxel nanometer preparation of this example.
In the paclitaxel nano-preparation prepared in this example, the mass ratio of paclitaxel to the amphiphilic polymer of mPEG-b-PCL is 1: 10. The preparation method of the paclitaxel nano injection comprises the following steps: 25ml of 0.9% physiological saline dissolves the paclitaxel nano preparation to obtain a transparent liquid, which has a stability of 5 hours and can be used for intravenous injection.
Example 6
Preparation of taxol nano preparation
In this embodiment, the preparation method of the paclitaxel nano-preparation includes the following steps:
a) 12.5mg of paclitaxel powder was dissolved in 250ml of anhydrous ethanol solvent.
b) 0.38g of mPEG (2K) -b-PCL (yK) amphiphilic block copolymer and 0.12g of mPEG (2K) -b-PCL (xK) amphiphilic block copolymer powder were dissolved in 250ml of acetonitrile solvent.
c)10mg of linoleic acid was dissolved in 10ml of absolute ethanol.
d) Mixing the three solutions in a glass bottle of a rotary evaporator, carrying out water bath at 60 ℃, and carrying out rotary mixing for 30 minutes; the ethanol and acetonitrile solvents were then removed under negative pressure at 60 ℃ for about 2 hours, leaving a mixture of paclitaxel and amphiphilic polymer.
e) The mixture of paclitaxel and mPEG-b-PCL was hydrated with 25ml of distilled water and shaken for 15-30 minutes to obtain a mixed solution.
f) Filtering the mixed solution by using a 200-nanometer filter to obtain filtrate, and placing the filtrate in a glass bottle.
g) The filtrate in the glass bottle is frozen at-40 ℃ to form paclitaxel nano ice blocks.
h) The paclitaxel nanometer ice blocks were dried by a freeze dryer for 48 hours to obtain the paclitaxel nanometer preparation of this example.
In the paclitaxel nano-preparation prepared in this example, the mass ratio of paclitaxel to the amphiphilic polymer of mPEG-b-PCL is 1: 4. The preparation method of the paclitaxel nano injection comprises the following steps: 25ml of 0.9% physiological saline dissolves the paclitaxel nano preparation to obtain a transparent liquid, which has a stability of 3 hours and can be used for intravenous injection.
Example 7
Preparation of taxol nano preparation
In this embodiment, the preparation method of the paclitaxel nano-preparation includes the following steps:
a) 50mg of paclitaxel powder was dissolved in 250ml of anhydrous ethanol solvent.
b) 3.8g of mPEG (2K) -b-PCL (yK) amphiphilic block copolymer and 1.2g of powders of mPEG (2K) -b-PCL (xK) amphiphilic block copolymer were dissolved in 250ml of acetonitrile solvent.
c)10mg of linoleic acid was dissolved in 10ml of absolute ethanol.
d) Mixing the three solutions in a glass bottle of a rotary evaporator, carrying out water bath at 60 ℃, and carrying out rotary mixing for 30 minutes; the ethanol and acetonitrile solvents were then removed under negative pressure at 60 ℃ for about 2 hours, leaving a mixture of paclitaxel and amphiphilic polymer.
e) The mixture of paclitaxel and mPEG-b-PCL was hydrated with 25ml of distilled water and shaken for 15-30 minutes to obtain a mixed solution.
f) Filtering the mixed solution by using a 200-nanometer filter to obtain filtrate, and placing the filtrate in a glass bottle.
g) The filtrate in the glass bottle is frozen at-40 ℃ to form paclitaxel nano ice blocks.
h) The paclitaxel nanometer ice blocks were dried by a freeze dryer for 48 hours to obtain the paclitaxel nanometer preparation of this example.
In the paclitaxel nano-preparation prepared in this example, the mass ratio of paclitaxel to the amphiphilic polymer of mPEG-b-PCL is 1: 100. The preparation method of the paclitaxel nano injection comprises the following steps: 25ml of 0.9% physiological saline dissolves the paclitaxel nano preparation to obtain a transparent liquid, which has 24-hour stability and can be used for intravenous injection.
Example 8
Preparation of taxol nano preparation
In this embodiment, the preparation method of the paclitaxel nano-preparation includes the following steps:
a) 10mg of paclitaxel powder was dissolved in 250ml of anhydrous ethanol solvent.
b) 0.38g of mPEG (2K) -b-PCL (yK) amphiphilic block copolymer and 0.12g of mPEG (2K) -b-PCL (xK) amphiphilic block copolymer powder were dissolved in 250ml of acetonitrile solvent.
c)10mg of linoleic acid was dissolved in 10ml of absolute ethanol.
d) Mixing the three solutions in a glass bottle of a rotary evaporator, carrying out water bath at 60 ℃, and carrying out rotary mixing for 30 minutes; the ethanol and acetonitrile solvents were then removed under negative pressure at 60 ℃ for about 2 hours, leaving a mixture of paclitaxel and amphiphilic polymer.
e) The mixture of paclitaxel and mPEG-b-PCL was hydrated with 25ml of distilled water and shaken for 15-30 minutes to obtain a mixed solution.
f) Filtering the mixed solution by using a 200-nanometer filter to obtain filtrate, and placing the filtrate in a glass bottle.
g) The filtrate in the glass bottle is frozen at-40 ℃ to form paclitaxel nano ice blocks.
h) The paclitaxel nanometer ice blocks were dried by a freeze dryer for 48 hours to obtain the paclitaxel nanometer preparation of this example.
In the paclitaxel nano-preparation prepared in this example, the mass ratio of paclitaxel to the amphiphilic polymer of mPEG-b-PCL is 1: 50. The preparation method of the paclitaxel nano injection comprises the following steps: 25ml of 0.9% physiological saline dissolves the paclitaxel nano preparation to obtain a transparent liquid, which has 24-hour stability and can be used for intravenous injection.
Examples of the experiments
Pharmacodynamic test
The taxol nano preparation is applied to animal experiments to detect the treatment effect on lung orthotopic transplantation tumor and systemic metastasis tumor of nude mice bearing human lung cancer cells H460. The main research method of the pharmacodynamic test comprises the steps of constructing a bioluminescent human lung cancer cell H460 and a lung in-situ tumor mouse model; measuring bioluminescence intensity by using an animal living body imaging system (IVIS SPECTRUM CT) to monitor tumorigenesis and development in real time; and performing drug effect evaluation by using methods such as bioluminescence 2D imaging, bioluminescence 3D imaging, lung weight comparison, life cycle analysis, weight change trend comparison and the like.
First, tumor models were constructed and administered in groups. H460 cells are injected into the lungs of nu/nu nude mice by puncture in situ, bioluminescent signals of tumor-bearing nude mice are collected 7 days later, and are grouped according to the bioluminescent signals, and intravenous injection administration is started and is marked as Day 1. Tumor-bearing nude mice were divided into control group, Paclitaxel-treated group, PTX-facial Paclitaxel-treated group 1, PTX-facial Paclitaxel-treated group 2, and 10 mice per group. Wherein the control mice did not receive any treatment; the mice in the Paclitaxel group received Taxol (Taxol) intravenous injection therapy in an amount of 20mg per kg of mouse body weight; the injection prepared by the paclitaxel nano preparation is intravenously injected into the mice in the PTX-paclitaxel treatment group 1, and the dosage is 10 mg/kg; PTX-Fatacid paclitaxel treatment group 2 mice also received the paclitaxel nanoformulation of the present invention at a dose of 20 mg/kg. FIG. 1 shows the bioluminescence intensity distribution of each group, and FIG. 2 shows the bioluminescence 2D imaging of each group, which shows that the number and distribution of H460 cancer cells of each group of tumor-bearing nude mice are similar in Day 1.
On Day7 of dosing, designated Day7, bioluminescence signals were collected from each group of tumor-bearing nude mice. FIG. 3 shows the bioluminescence intensity distribution of each group of tumor-bearing nude mice by Day7, FIG. 4 shows the bioluminescence 2D imaging of each group of tumor-bearing nude mice by Day7, and the luminescent signals in the two PTX-facial paclitaxel treatment groups are weak, thus showing that the paclitaxel nano-preparation of the invention can effectively inhibit the rapid progression of diseases. In FIG. 4, boxes indicate that the nude mice died, 1 nude mouse died in the control group at Day3 and Day7, respectively, and 2 nude mice died in the Paclixel-treated group at Day2 and Day7, respectively, and Taxol showed acute toxicity.
On Day15 of dosing, designated Day15, bioluminescence signals were collected from each group of tumor-bearing nude mice. FIG. 5 shows bioluminescence intensity of DAY15 groups of tumor-bearing nude mice, FIG. 6 is 2D image of bioluminescence of DAY15 groups of tumor-bearing nude mice, wherein the box in FIG. 6 indicates that the nude mice have died, and it can be seen that only 4 tumor-bearing nude mice survived in the DAY15 in the control group, 6 tumor-bearing nude mice survived in the Paclixel-treated group, and all nude mice survived in the two PTX-Fatacid Paclitaxel-treated groups. In addition, the Paclitaxel-treated nude mice showed signs of injection site necrosis, indicating that Paclitaxel can induce marked acute toxicity and irreversible local irritation, and is less safe than PTX-facid Paclitaxel. From the bioluminescence signal, the bioluminescence intensity of each tumor-bearing nude mouse of the control group is high, the diffusion and transfer conditions of tumor cells are obvious, the bioluminescence intensity of the two PTX-facial paclitaxel treatment groups is obviously weaker, and the paclitaxel nano preparation can effectively inhibit the development process of the ex-situ tumor.
The survival time, the bioluminescence intensity increase multiple and the body weight increase multiple of each group of tumor-bearing nude mice are statistically analyzed, and the results are respectively shown in fig. 7, fig. 8 and fig. 9. FIG. 7 shows the survival of each group of tumor-bearing nude mice As described earlier, the experimental models of both PTX-Fatacid Paclitaxel-treated groups remained alive, only 4 tumor-bearing nude mice of the control group survived, and the tumor-bearing nude mice of the Paclitaxel-treated group died earlier; FIG. 9 shows the body weight changes of tumor-bearing nude mice in each group, reflecting their health status, the mice of both PTX-Fatacid Paclitaxel treated groups maintained stable body weight, while the mice of the control group and Paclitaxel treated group showed significant weight loss; the result shows that Taxol is easy to cause acute toxic reaction, and the Taxol has low toxic and side effects and high safety, and is favorable for keeping a good health state of tumor-bearing nude mice. FIG. 8 shows the changes in bioluminescence intensity of tumor-bearing nude mice in each group from Day1 to Day15, which reflects the changes in tumor size of mice in the Control, Paclitaxel and PTX-Fatacid groups at different times. The contrast group tumor-bearing nude mice have rapid increase of bioluminescence intensity, the Paclitaxel treatment group shows an inhibition effect on growth, but the two PTX-facial Paclitaxel treatment groups have more obvious tumor inhibition effect, and the treatment effect of the Paclitaxel nano preparation on lung orthotopic transplantation tumor and systemic metastasis tumor is reflected.
Table 1 demonstrates the therapeutic effect of paclitaxel formulations on lung orthotopic transplantations and systemic metastases by multiple sets of data. The inventor analyzes the theoretical tumor inhibition rate by calculating the lung tumor weight (the calculation is disclosed as: calculating the lung tumor weight as the total lung weight of tumor-bearing nude mice- (normal nude mice lung weight index x actual body weight of tumor-bearing nude mice)), and the result shows that both Taxol and the Taxol nano-preparation have the tumor inhibition effect, and the theoretical lung tumor inhibition rates of each group are 84.8%, 80.9% and 86.2% respectively; the PTX-Fatacid Paclitaxel treated group showed better tumor suppression than the Paclitaxel treated group with the same therapeutic dose. Meanwhile, the comparison of bioluminescence intensity analyzes the cancer inhibition effect of each group of Paclitaxel preparations, and the 2D imaging and 3D imaging results show that the cancer inhibition rate of the Paclitaxel nano preparation treatment group is higher than that of the Paclitaxel nano preparation treatment group. In addition, 3D imaging can show the tumor systemic metastasis, and the result can be seen that the systemic metastasis incidence of the tumor-bearing nude mice of the two PTX-Fatacid Paclitaxel treatment groups is far lower than that of the control group and the Paclitaxel treatment group.
TABLE 1 therapeutic Effect of paclitaxel on lung orthotopic transplantation tumors and systemic metastasis
Figure BDA0002457543080000131
According to pharmacodynamic test results, the taxol nano preparation can remarkably delay the malignant progress of lung orthotopic transplantation tumor and improve the survival period of tumor-bearing mice. Compared with Taxol, the Taxol nanometer preparation has better treatment effect on controlling the systemic metastasis of tumors, has no obvious toxic or side effect, and is favorable for increasing the physique of tumor-bearing nude mice.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims (8)

1. The preparation method of the taxol nano preparation is characterized by comprising the following steps:
s1, dissolving paclitaxel in a first organic solvent to obtain a first solution;
s2, dissolving the low-tin-content mPEG-b-PCL amphiphilic block copolymer in a second organic solvent to obtain a second solution;
s3, dissolving the fatty acid in a third organic solvent to obtain a third solution;
s4, mixing the first solution, the second solution and the third solution, and removing the organic solvent to obtain a mixture;
s5, hydrating the mixture, and filtering the mixture by using a nano filter to obtain filtrate;
s6, freezing the filtrate to obtain paclitaxel nano ice blocks;
s7, freeze-drying the paclitaxel nanometer ice blocks to obtain a paclitaxel nanometer preparation;
wherein the mass ratio of the paclitaxel to the mPEG-b-PCL amphiphilic block copolymer is as follows: 1: 4-100.
2. The method for preparing the paclitaxel nanoformulation according to claim 1, wherein the first organic solvent and the third organic solvent are absolute ethanol, and the second organic solvent is acetonitrile.
3. The method for preparing paclitaxel nanoformulation according to claim 1, wherein the fatty acid in step S3 is selected from any one of lauric acid, palmitic acid, oleic acid and linoleic acid.
4. The method for preparing paclitaxel nano-formulation according to claim 1, wherein the step S4 specifically comprises: and stirring and mixing the first solution, the second solution and the third solution in a water bath at 60 ℃ for 30 minutes, and removing the organic solvent at the temperature of 60 ℃ under negative pressure to obtain a mixture of paclitaxel and the amphiphilic block copolymer of mPEG-b-PCL.
5. The method for preparing paclitaxel nano-formulation according to claim 1, wherein the step S5 specifically comprises: hydrating the mixture with distilled water, shaking and mixing for 15-30 minutes to obtain a mixed solution, and filtering the mixed solution with a nano filter to obtain a filtrate.
6. The method for preparing paclitaxel according to claim 5, wherein the nanofilter of step S5 is a 200 nanofilter.
7. The method for preparing paclitaxel according to claim 1, wherein the temperature for freezing the filtrate in step S6 is-40 ℃.
8. The method for preparing paclitaxel nano-formulation according to claim 1, wherein the step S7 specifically comprises: and drying the paclitaxel nanometer ice blocks for 48-72 hours by using a freeze dryer to obtain the paclitaxel nanometer preparation.
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Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
Copolymer Micelles and Nanospheres with Different In Vitro Stability Demonstrate Similar Paclitaxel Pharmacokinetics;Kevin Letchford;《Molecular Pharmaceutics》;20111228;第248-260页 *
Effect of PEG conformation and particle size on the cellular uptake efficiency of nanoparticles with the HepG2 cells;Y.Hu,et al;《Journal of Controlled Release》;20071231;第118卷;第7-17页 *
Taxol-loaded block copolymer nanospheres composed of methoxy poly(ethylene glycol) and poly(ε-caprolactone) as novel anticancer drug carriers;S.Y.Kim,et al;《Biomaterials》;20011231;第22卷;第1697-1704页 *
The combined use of paclitaxel-loaded nanoparticles with a low-molecular-weight copolymer inhibitor of P-glycoprotein to overcome drug resistance;Wan,et al;《International Journal of Nanomedicine》;20131231;第379-391页 *
紫杉醇MPEG-PCL 纳米粒的制备、表征及其体外释药行为研究;郑毅等;《中国现代应用药学》;20140930;第31卷(第9期);第1093-1097页 *
郑毅等.紫杉醇MPEG-PCL 纳米粒的制备、表征及其体外释药行为研究.《中国现代应用药学》.2014,第31卷(第9期),第1093-1097页. *

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