CN101884618A - A long circulation paclitaxel nanoparticle and its preparation method - Google Patents

Abstract

The invention discloses a long-circulation paclitaxel nanoparticle with strong and long-lasting effect, high curative effect, low toxicity and side effects, and can be used for intravenous injection or intravenous infusion. It is made of the following raw materials in parts by weight: 1 part of paclitaxel, carrier material 5-20 parts, 34-132 parts of emulsifier; the particle size range of nanoparticles is 50-450nm; the carrier material is PLA-PEG or PLGA-PEG. The preparation method is as follows: the carrier material and paclitaxel are dissolved together in an organic solvent as the oil phase, and the emulsifier is dissolved in double distilled water as the water phase; then the oil phase and the water phase are mixed according to a certain ratio, and the milky white initial phase is obtained by high-speed shearing. Milk: homogenize the colostrum with a high-pressure homogenizer to obtain a homogenized liquid; stir at a low speed for 3 to 5 hours to completely volatilize the organic solvent to obtain long-cycle paclitaxel nanoparticles.

Description

A long circulation paclitaxel nanoparticle and its preparation method

technical field

The invention relates to a long-cycle paclitaxel nanoparticle and a preparation method thereof.

Background technique

Paclitaxel is a complex secondary metabolite in the Taxus genus, and it is one of the first-line drugs in clinical application. It is mainly suitable for ovarian cancer and breast cancer, and also has a certain effect on lung cancer, colorectal cancer, melanoma, head and neck cancer, lymphoma, and brain tumor. However, traditional taxane drugs are difficult to be administered clinically due to their low solubility, and auxiliary solvents are required, but most of the auxiliary solvents have toxic side effects, such as polyoxyethylene castor oil, which has been reported to produce hypersensitivity reactions, nephrotoxicity, neurotoxicity, and cardiotoxicity, etc. side effect. This obviously increases the toxicity of the drug, which seriously limits the improvement of the curative effect and safe application of this type of drug.

In recent years, the use of polymer materials to prepare nanoparticles as anticancer drug carriers has received widespread attention, mainly including polylactic acid (polylactic acid, PLA), poly(lactic-co-glycolic acid), poly(lactic-co-glycolic acid), PLGA) etc. The polymer carrier can slowly release drugs, reduce drug toxicity, promote drug absorption at tumor sites, and have a high ability to cross biofilm barriers to achieve targeted and sustained release of drugs. Among them, PLA is non-toxic, non-irritating, has good biocompatibility, and can be biodegraded and absorbed. Due to the excellent biocompatibility of PLA, its degradation products can participate in human metabolism, so it has been approved by the US Food and Drug Administration (FDA) as medical surgical sutures, injection capsules, microspheres, nanoparticles and implants. PLGA is a degradable functional polymeric organic compound with good biocompatibility, non-toxicity, good encapsulation and film-forming properties, and is widely used in pharmaceuticals, medical engineering materials and modern industrial fields. In the United States, PLGA has passed FDA certification and has been officially included in the United States Pharmacopoeia as a pharmaceutical excipient.

However, because PLA and PLGA nanoparticles are easy to be recognized and phagocytized by macrophages, the circulation time in the body is short, and they cannot exert sufficient drug effects. The use of polyethylene glycol (polyethylene glycol, PEG) to modify nanoparticles to escape the opsonization in vivo to increase the circulation time in nanoparticles has become a hot spot in the preparation of long-circulating nanoparticles in recent years, but the current technology in this area is not Mature, there is not yet a mature preparation process that can prepare ideal (long residence time in the body, good effect) nanoparticles, and can be used for large-scale production.

Contents of the invention

Aiming at the above-mentioned prior art, the present invention uses PLA-PEG and PLGA-PEG as materials, and provides a long-circulation paclitaxel nanoparticle with strong and long-lasting effect, high curative effect, low toxicity and side effects, which can be used for intravenous injection or intravenous infusion. , and the preparation method thereof, the paclitaxel nanoparticles prepared by the present invention can achieve the effect of long circulation, thereby increasing the residence time of the nanoparticles in the body and increasing the therapeutic effect of the nanoparticles.

A long-circulation paclitaxel nanoparticle is made of the following raw materials in parts by weight: 1 part of paclitaxel, 5-20 parts of carrier material, 34-132 parts of emulsifier; the particle size range of the nano-particle is 50-450nm;

The carrier material is PLA-PEG (polylactic acid-polyethylene glycol) or PLGA-PEG (polylactic acid/glycolic acid-polyethylene glycol), wherein the PLA or PLGA block in PLA-PEG or PLGA-PEG The molecular weight of the PEG block is 25000-80000, and the molecular weight of the PEG block is 2000-5000;

The emulsifier is Pluronic F-68.

A preparation method of long-cycle paclitaxel nanoparticles, the process is as follows: the carrier material and paclitaxel are dissolved together in an organic solvent as an oil phase, and the emulsifier is dissolved in double distilled water as a water phase; then the oil phase and the water phase are mixed according to a certain ratio Mix and shear at high speed to obtain milky white colostrum; homogenize the colostrum with a high-pressure homogenizer to obtain a homogenized liquid; stir at a low speed for 3 to 5 hours to completely volatilize the organic solvent, and obtain long-cycle paclitaxel nanoparticles;

Described organic solvent is one or any two or two mixtures in arbitrary proportions in methylene dichloride, chloroform or ethyl acetate;

The concentration of the carrier material in the oil phase is 10-100 mg/mL, and the concentration of paclitaxel in the oil phase is 0.2-20 mg/mL;

The mass concentration of the emulsifier Pluronic F-68 in the water phase is 0.1% to 5%;

When the oil phase and the water phase are mixed, the volume ratio of the oil phase and the water phase is 1: (5-40).

The speed of the high-speed shearing is 14000-33000 r/min, and the shearing time is 1-5 min.

The number of homogenization by the high-pressure homogenizer is 5-20 times, the homogenization pressure range is 300-1500 bar, and the final homogenization pressure is 800-1500 bar.

The speed of the low-speed stirring is 300-700 r/min.

The long-circulation paclitaxel nanoparticle of the present invention can be used for intravenous injection and intravenous infusion because the particle diameter is 50-450nm. The carrier material used is a biodegradable polymer material, which is non-toxic in itself, and its metabolites are _CO2 and water, and its intermediate products are also normal metabolites of the human body. Non-irritating and has good biocompatibility. Because the PLA or PLGA segment in the carrier material can be slowly degraded, the drug can be released slowly, thereby prolonging the administration time; the PEG block can reduce the conditioning effect of the blood on the nanoparticles, thereby prolonging the circulation time. In addition, no auxiliary solvent polyoxyethylene castor oil or the like is used to avoid side effects such as hypersensitivity, nephrotoxicity, neurotoxicity and cardiotoxicity, and the administration is convenient and the adaptability of patients is increased. At present, the methods for preparing nanoparticles in the prior art include in-liquid drying method, automatic emulsification method, emulsion polymerization method, spray drying method and high-pressure homogenization method, etc. Among them, the high-pressure homogenization method has a simple and stable preparation process, can be applied to large-scale production, and is a preparation method with broad application prospects. The method adopts the high-pressure homogenization method to prepare long-cycle paclitaxel nanoparticles, and the preparation process is simple and stable; the invention also optimizes the specific process flow for preparation.

Although there are many materials and combinations for preparing nanoparticles in the prior art, for paclitaxel, not every combination is applicable, and not every combination can be used to prepare nanoparticles. Not all can play the desired effect; the present invention is the optimal proportion and process condition obtained by the inventor after a large number of experimental screenings, and the nanoparticles prepared by adopting the proportion of the present invention and the process condition can significantly increase the paclitaxel The residence time in the body enhances the therapeutic effect, has low toxicity and side effects, and has good biocompatibility. It can be seen that the present invention has selected a specific combination of paclitaxel, carrier material and emulsifier and a specific preparation process, which has played an ideal effect and is inventive.

Description of drawings

Fig. 1 is the transmission electron micrograph (× 24K) of the long circulation paclitaxel nanoparticle prepared in embodiment 1;

Fig. 2 is the particle size distribution diagram of the long circulation paclitaxel nanoparticles prepared in embodiment 1;

Figure 3 is a high performance liquid chromatogram of paclitaxel.

Detailed ways

The present invention will be further described below in conjunction with embodiment and test example, but the present invention is not limited in any form.

Example 1: Preparation of Long Circulation Paclitaxel Nanoparticles

540mg PLA-PEG and 27mg paclitaxel were dissolved in 10mL dichloromethane as the oil phase, 2g F68 was dissolved in 100mL double-distilled water as the water phase; the oil phase was mixed with the water phase (the ratio of oil phase to water was 1:10), 20,000rpm high-speed shearing for 10 minutes to obtain milky white colostrum; use a high-pressure homogenizer to homogenize the colostrum 5 times at 300bar, 10 times at 600bar, and 20 times at 1000bar to obtain the homogenized liquid; use magnetic stirring at 300rpm to stir for 3 hours, The organic solvent was completely volatilized to obtain long-cycle paclitaxel nanoparticles, the transmission electron microscope photo of which is shown in FIG. 1 , and the particle size distribution diagram thereof is shown in FIG. 2 .

Example 2: Preparation of Long Circulation Paclitaxel Nanoparticles

5.4g PLA-PEG and 270mg paclitaxel were dissolved together in 100mL dichloromethane as the oil phase, 20g F68 was dissolved in 1000mL double-distilled water as the water phase; the oil phase was mixed with the water phase (the ratio of oil phase to water was 1:10), 33000rpm high-speed shearing for 10 minutes to obtain milky white colostrum; use a high-pressure homogenizer to homogenize the colostrum 5 times at 300bar, 10 times at 600bar, 5 times at 1200bar, and 20 times at 1500bar to obtain the homogenized liquid; use magnetic force Stir at 500 rpm for 5 hours to completely volatilize the organic solvent to obtain long-circulation paclitaxel nanoparticles.

Example 3: Preparation of Long Circulation Paclitaxel Nanoparticles

540mg PLA-PEG and 54mg paclitaxel were dissolved in 5mL dichloromethane together as the oil phase, 5g F68 was dissolved in 100mL double distilled water as the water phase; the oil phase was mixed with the water phase (the ratio of oil phase to water was 1:20), 14000rpm High-speed shearing for 5 minutes to obtain milky white colostrum; use a high-pressure milk homogenizer to homogenize the colostrum 5 times at 300 bar, 10 times at 600 bar, 15 times at 1000 bar, and 20 times at 1500 bar to obtain the homogenized liquid; use magnetic stirring Stir at 700rpm for 3 hours to completely volatilize the organic solvent to obtain long-circulation paclitaxel nanoparticles.

Example 4: Preparation of Long Circulation Paclitaxel Nanoparticles

135mg PLA-PEG and 27mg paclitaxel were dissolved in 20mL dichloromethane as the oil phase, 2g F68 was dissolved in 100mL double distilled water as the water phase; the oil phase was mixed with the water phase (the ratio of oil phase to water was 1:5), 20000rpm High-speed shearing for 2 minutes to obtain milky white colostrum; use a high-pressure homogenizer to homogenize the colostrum 5 times at 300 bar, 10 times at 600 bar, and 20 times at 1200 bar to obtain the homogenized liquid; use magnetic stirring at 400 rpm for 3 hours to make The organic solvent is completely volatilized to obtain long-circulation paclitaxel nanoparticles.

Example 5: Preparation of Long Circulation Paclitaxel Nanoparticles

270mg PLGA-PEG and 27mg paclitaxel were dissolved in 10mL dichloromethane as the oil phase, 2g F68 was dissolved in 100mL double distilled water as the water phase; the oil phase was mixed with the water phase (the ratio of oil phase to water was 1:10), 20000rpm High-speed shearing for 10 minutes to obtain milky white colostrum; use a high-pressure homogenizer to homogenize the colostrum 5 times at 300 bar, 10 times at 600 bar, and 20 times at 800 bar to obtain the homogenized liquid; use magnetic stirring at 500 rpm for 3 hours to make The organic solvent is completely volatilized to obtain long-circulation paclitaxel nanoparticles.

Test Example 1: Determination of Drug Loading Capacity and Encapsulation Efficiency of Long Circulation Paclitaxel Nanoparticles

Adopt high-performance liquid chromatography to measure the content of paclitaxel: chromatographic column: Hypersil ODS post (Dalian Yilite Scientific Instrument Co., Ltd.; 250mm * 4.6mm, 10 μ m); Mobile phase: acetonitrile-distilled water (60: 40, V/V); Detection Wavelength: 227nm; Flow rate: 1.0mL/min; Injection volume: 20μL; Column temperature: room temperature; The number of theoretical plates is not less than 6000.

Paclitaxel standard solution with a concentration of 0.25-25 μg/ml was taken respectively, tested according to the chromatographic conditions, the peak area was used to fit the paclitaxel concentration, and a regression equation was established.

The obtained nanoparticle suspension (prepared in Example 1) was filtered using a 0.45 μm microporous membrane, and 200 μL of the filtrate was taken, 800 μL of methanol was added, vortexed for 3 min, ultrasonicated for 5 min, and the content of paclitaxel was determined according to high performance liquid chromatography ( The high-performance liquid chromatogram is shown in Figure 3), obtains the amount of nanoparticle-encapsulated medicine.

At the same time, take 200 μL of the unfiltered nanoparticle suspension, add 800 μL of methanol, vortex for 3 minutes, and sonicate for 5 minutes, and determine the content of paclitaxel according to high performance liquid chromatography to obtain the amount of the total drug.

Drug loading %=(amount of nanoparticle-encapsulated drug/input carrier material mass+amount of nanoparticle-encapsulated drug)×100%

Encapsulation efficiency%=(amount of nanoparticle-encapsulated drug/total drug amount)×100%

The average encapsulation efficiency of the obtained microspheres is 60-99%, and the drug loading is 0.5-20%.

Test Example 2: Determination of appearance, zate point, particle size and distribution of long-cycle paclitaxel nanoparticles

Use a transmission electron microscope to observe the appearance of the nanoparticles (prepared in Example 1), as shown in Figure 1, and use a laser particle size analyzer to measure the particle size distribution and zeta potential of the nanoparticles, as shown in Figure 2.

The obtained nanoparticles have a rounded appearance, a particle diameter of 50-450 nm, and uniform distribution.

Claims (5)

1. a long-circulating paclitaxel nanoparticles is characterized in that, is to be made by the raw material of following weight portion: 1 part of paclitaxel, 5～20 parts of carrier materials, 34～132 parts of emulsifying agents; The particle size range of nanoparticle is 50～450nm;

Described carrier material is PLA-PEG or PLGA-PEG, wherein, the PLA among PLA-PEG or the PLGA-PEG or

The molecular weight of PLGA block is 25000～80000, and the molecular weight of PEG block is 2000～5000;

Described emulsifying agent is that general youth Buddhist nun restrains F-68.

2. the preparation method of a kind of long-circulating paclitaxel nanoparticles according to claim 1 is characterized in that, technology is as follows: carrier material and paclitaxel are dissolved in the organic solvent jointly as oil phase, emulsifiers dissolve in distilled water as water; Then oil phase is mixed according to a certain percentage with water, high speed shear obtains milky colostrum; Use the high pressure dispersing emulsification machine to carry out breast colostrum and spare, obtain the even back of breast liquid; Stirring at low speed volatilized organic solvent in 3～5 hours fully, promptly obtained long-circulating paclitaxel nanoparticles;

Described organic solvent is a kind of or any two kinds or the two kinds of mixture with arbitrary proportion in dichloromethane, chloroform or the ethyl acetate;

The concentration of carrier material is 10～100mg/mL in the described oil phase, and the concentration of paclitaxel is 0.2～25mg/mL in the oil phase;

The mass concentration that the general youth Buddhist nun of described aqueous phase emulsifying agent restrains F-68 is 0.1～5%;

The volume ratio of oil phase and water was 1 when described oil phase and water mixed: (5～40).

3. the preparation method of long-circulating paclitaxel nanoparticles according to claim 2, it is characterized in that: the speed of described high speed shear is 14000～33000r/min, shear time is 1～5min.

4. the preparation method of long-circulating paclitaxel nanoparticles according to claim 2 is characterized in that: the even number of times of described employing high pressure dispersing emulsification machine breast is 5～20 times, and the even pressure limit of breast is 300～1500bar, and the even pressure of final breast is 800～1500bar.

5. the preparation method of long-circulating paclitaxel nanoparticles according to claim 2, it is characterized in that: the speed of described stirring at low speed is 300～700r/min.

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