CN101849909B - Preparation method of magnetic disodium clodronate liposome - Google Patents
Preparation method of magnetic disodium clodronate liposome Download PDFInfo
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- CN101849909B CN101849909B CN2010101240907A CN201010124090A CN101849909B CN 101849909 B CN101849909 B CN 101849909B CN 2010101240907 A CN2010101240907 A CN 2010101240907A CN 201010124090 A CN201010124090 A CN 201010124090A CN 101849909 B CN101849909 B CN 101849909B
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Abstract
The invention discloses a preparation method of a magnetic disodium clodronate liposome, belonging to the technical field of preparation of targeted medicine carriers. The preparation method comprises the following steps: (1) preparing ferroferric oxide nanoparticles; and (2) preparing the liposome for cladding the medicine and ferroferric oxide nanoparticles. Ferroferric oxide nanoparticles prepared by thermal decomposition has uniform dimension, regular shapes and good magnetism, the partical size of disodium clodronate liposome prepared by reverse phase evaporation is smaller than 200nm and meets the requirements on the dimension of medical preparation, the encapsulation on magnetic particals and medicines is good, the cladded matter is not easy to leak, the preparation method is simple, the requirements on the process equipment are fewer, and prospect for medical purposes is good.
Description
Technical field
The present invention relates to the preparation of the liposome of a kind of coated magnetic nanoparticle and cancer therapy drug, belong to the target medicine carrier preparing technical field.
Technical background
To be phospholipid molecule act on a kind of molecular assembly assembly of spontaneous formation in water or the solution through hydrophobic association to liposome, and diameter 25～1000nm does not wait, because its particle size is in nano level Jie and sees scope; The physics that many uniquenesses are arranged, chemical property, and also the preparation technology of liposome is simple; Characteristics such as nontoxic in human body, non-immunogenicity, degradable, slow release, institute's loaded drugs kind is extensive, can reduce required dose; Strengthen its body internal stability and pharmacological action; Reduce the toxic and side effects of medicine, make medicine have the passive target characteristic, can also become immune form to realize initiatively targeting property liposome preparation.
The construction features of liposome is the phospholipid bilayer film with biocompatibility, and medicine through number of ways administrations such as intravenous injection, oral, skin, eye, nasal cavity, pulmonary and mucosas, with the prolong drug half-life, improves bioavailability after load.Depend on route of administration owing to distribute in the body of liposome, so liposome is main with infusion application still at present, but in recent years along with the expansion with application approach of improving of technology of preparing, liposome non-injection administration mode becomes the focus of research gradually.
The ferriferrous oxide nano microgranule is a kind of magnetic material with inverse spinel structure, in its crystal, by O
2-The space that surrounds has two types, i.e. octahedral build and positive tetrahedron, wherein 1/2 Fe
3+Occupy in octahedron, and other 1/2 Fe
3+With whole Fe
2+Occupy in positive tetrahedron.Because its special crystal structure, so ferroso-ferric oxide can be used as a kind of typical semi-conducting material, removes in addition, and the unique magnetic adjustable function of ferroso-ferric oxide is also by people's broad research.In recent years, ferroso-ferric oxide is widely used in the systems such as nuclear magnetic resonance, drug-supplying system, biological medicine, biosensor.But; Because independent magnetic particle biocompatibility is poor; Be prone to reasons such as reunion; The surface is carried out magnetic particle that the other biological compatibility is good, side effect is little material coats and is replaced independent magnetic particle just gradually and be applied in the organism, as the magnetic material of magnetic targeting drug delivery system, has good application prospects in therefore that its embedding and biocompatibility is the good liposome.
The magnetic target drug-supplying system comprises medicine and suitable magnetic material and necessary adjuvant, and under the external magnetic field guiding, with blood flow, the arrival of selection also is positioned lesions position.After medicine discharges, on the cell of lesion tissue or subcellsular level, bring into play drug effect from carrier, therefore reduce to the influence of normal structure minimum.The magnetic target liposome is a kind of newtype drug oriented carrier in the magnetic steering drug delivery system, can wrap up 10 in liposome interior
3～10
4Individual drug molecule and nano ferriferrous oxide molecule, thereby can the magnetic target administration.The magnetic target liposomes damages human body hardly, and the magnetisable material of internal package can safe excreting.Therefore the magnetic target liposomes gets more and more people's extensive concerning in the pharmaceutical carrier field.
The method for preparing of liposome mainly contains film dispersion method, reverse phase evaporation, freeze-drying etc., and novel targeted liposome has long circulating liposomes, temperature-sensitive/photosensitive liposome, immunoliposome, magnetic liposome etc.Shi Liping etc. (659-661) send out through anti-phase evaporation and prepared the acid-sensitive liposome, and studied its research of the distribution in the important organ in the mice body by Chinese critical illness emergency medicine 2001,13,11.In the CN100998563 patent, utilize lecithin, cholesterol and magnetic particle are sent out through the anti-phase evaporation and are prepared magnetic liposome.(Langmuir 2002,18,3134-3141) prepare the thermo-responsive type liposome of big single chamber of knowing clearly through membrane process for Vincent Chan etc.(Langmuir 2004,20,7917-7925) prepare through squeezing and pressing method and coat the actin liposome, and considered that actin content and liposome size are to the influence of its Atomic Mechanics microscope to liposome structure for Shuliang Li etc.
Summary of the invention
The object of the invention is to provide a kind of method for preparing of magnetic disodium clodronate liposome, and liposome can improve the targeting property of its medicament transport through the coated magnetic particle, thereby improves the absorption of lesions position to medicine.
The key step that the present invention realizes comprises:
(1) preparation of nano ferriferrous oxide:
The adding mol ratio is 100: 1: 1 ferric acetyl acetonade, aniline, an oleic acid mixed liquor in the three-neck flask; Feed protector under the magnetic agitation to remove oxygen; System is warming up to 80-120 ℃ and keep 20-30min and stir making the reactant mix homogeneously; After removing nitrogen, continue to be heated to temperature and reach 260-270 ℃, keep reflux state 2-4h.The mixture that obtains after the reaction is cooled to room temperature, and absolute ethanol washing for several times, and is centrifugal, and the black product that finally obtains is scattered in dehydrated alcohol, obtains ferroferric oxide magnetic fluid;
(2) preparation of the liposome of coating medicine and nano ferriferrous oxide:
Take by weighing lecithin and cholesterol at 5: 1 by mass ratio, be dissolved in the chloroform, vibrations make the solid dissolving, and the solution rotating evaporation to remove solvent chloroform, will be placed 60 ℃ of oven dry of thermostatic drying chamber with the flask of immobilized artificial membrane.
Clodronate disodium is dissolved in the PBS (PBS) of pH=7.4, according to the clodronate disodium mass ratio be 1: 1 ratio, add the ferroferric oxide magnetic fluid that has prepared in the step (1); This mixture is transferred in the flask that has immobilized artificial membrane of drying the step preparation; With the vibrations of this system, ultrasonic after, use 0.7% sodium chloride solution (normal saline) to wash respectively with PBS; Centrifugal, place 4 ℃ of refrigerators of constant temperature to store end product.
The performance evaluation of product: utilize transmission electron microscope (TEM); X-ray diffractometer (XRD) is analyzed the appearance and size of nano ferriferrous oxide and liposome; Ultraviolet-visible spectrophotometer is measured the envelop rate of medicine, to assess the load performance of prepared liposome as pharmaceutical carrier.
Technological merit of the present invention:
The present invention adopts the method for anti-phase evaporation successfully to prepare magnetic disodium clodronate liposome, and the whole technique process approach is simple, the low pollution, and prepared liposome is good to the containment of medicine, can reach industrial production requirement.
Description of drawings
Fig. 1 is the XRD diffraction spectrogram of prepared nano ferriferrous oxide, and diffraction maximum is the characteristic diffraction peak of nano ferriferrous oxide among the figure.
Fig. 2 is the transmission electron microscope photo of prepared nano ferriferrous oxide.
Fig. 3 is the transmission electron microscope photo of prepared magnetic disodium clodronate liposome.
Embodiment 1
Add the 0.035g ferric acetyl acetonade in the three-neck flask, 0.1mL aniline, 0.32mL oleic acid feeds nitrogen under the magnetic agitation, and system is warming up to 100 ℃ and keep 20min, remove nitrogen after, continue to be heated to temperature and reach 260 ℃, keep reflux state 2h.
The mixture that obtains after the reaction is cooled to room temperature, and absolute ethanol washing for several times, and is centrifugal, and the black product that finally obtains is scattered in dehydrated alcohol, makes it subsequent use with the form of magnetic fluid.
Dissolving 0.34g lecithin in the 20mL chloroform, the 0.068g cholesterol, to remove solvent chloroform, can with one deck monomolecular phosphorus adipose membrane in 60 ℃ thermostatic drying chamber dry this flask on the flask walls this moment with this mixed liquor rotary evaporation.
Add 0.2g clodronate disodium and the above-mentioned nano ferriferrous oxide magnetic fluid that has prepared of 0.2g among the 20mLPBS; This mixture is transferred in the flask that has immobilized artificial membrane of having dried; With continuous ultrasound 1h behind this system strenuous vibration so that the spontaneous formation phospholipid bilayer of monomolecular phosphorus adipose membrane; The liposome that obtains is respectively with normal saline and PBS washing, centrifugal, to remove the not phospholipid molecule of encystation.
Add the 0.11g ferric acetyl acetonade in the three-neck flask, 0.3mL aniline, 0.96mL oleic acid feeds argon under the magnetic agitation, and system is warming up to 120 ℃ and keep 20min, remove nitrogen after, continue to be heated to temperature and reach 260 ℃, keep reflux state 2h.
The mixture that obtains after the reaction is cooled to room temperature, and absolute ethanol washing for several times, and is centrifugal, and the black product that finally obtains is scattered in dehydrated alcohol, makes it subsequent use with the form of magnetic fluid.
Dissolving 0.14g lecithin in the 15mL chloroform, the 0.028g cholesterol, to remove solvent chloroform, can with one deck monomolecular phosphorus adipose membrane in 60 ℃ thermostatic drying chamber dry this flask on the flask walls this moment with this mixed liquor rotary evaporation.
Add the nano ferriferrous oxide magnetic fluid that has prepared among 0.1g clodronate disodium and the 0.1g embodiment 1 among the 15mLPBS; This mixture is transferred in the flask that has immobilized artificial membrane of having dried; With continuous ultrasound 1h behind this system strenuous vibration so that the spontaneous formation phospholipid bilayer of monomolecular phosphorus adipose membrane; The liposome that obtains is respectively with normal saline and PBS washing, centrifugal, to remove the not phospholipid molecule of encystation.
Embodiment 4
Add the 0.035g ferric acetyl acetonade in the three-neck flask, 0.1mL aniline, 0.32mL oleic acid feeds nitrogen under the magnetic agitation, and system is warming up to 100 ℃ and keep 20min, remove nitrogen after, continue to be heated to temperature and reach 260 ℃, keep reflux state 2h.
The mixture that obtains after the reaction is cooled to room temperature, and absolute ethanol washing for several times, and is centrifugal, and the black product that finally obtains is scattered in dehydrated alcohol, makes it subsequent use with the form of magnetic fluid.
Dissolving 0.14g lecithin in the 15mL chloroform, the 0.028g cholesterol, to remove solvent chloroform, can with one deck monomolecular phosphorus adipose membrane in 60 ℃ thermostatic drying chamber dry this flask on the flask walls this moment with this mixed liquor rotary evaporation.
Add the nano ferriferrous oxide magnetic fluid that has prepared among 0.1g clodronate disodium and 0.1 embodiment 1 among the 15mLPBS; This mixture is transferred in the flask that has immobilized artificial membrane of having dried; With continuous ultrasound 1h behind this system strenuous vibration so that the spontaneous formation phospholipid bilayer of monomolecular phosphorus adipose membrane; The liposome that obtains is respectively with normal saline and PBS washing, centrifugal, to remove the not phospholipid molecule of encystation.
Embodiment 5
Add the 0.11g ferric acetyl acetonade in the three-neck flask, 0.3mL aniline, 0.96mL oleic acid feeds argon under the magnetic agitation, and system is warming up to 120 ℃ and keep 20min, remove nitrogen after, continue to be heated to temperature and reach 260 ℃, keep reflux state 2h.
The mixture that obtains after the reaction is cooled to room temperature, and absolute ethanol washing for several times, and is centrifugal, and the black product that finally obtains is scattered in dehydrated alcohol, makes it subsequent use with the form of magnetic fluid.
Dissolving 0.34g lecithin in the 20mL chloroform, the 0.068g cholesterol, to remove solvent chloroform, can with one deck monomolecular phosphorus adipose membrane in 60 ℃ thermostatic drying chamber dry this flask on the flask walls this moment with this mixed liquor rotary evaporation.
Add 0.2g clodronate disodium and the above-mentioned nano ferriferrous oxide magnetic fluid that has prepared of 0.2g among the 20mLPBS; This mixture is transferred in the flask that has immobilized artificial membrane of having dried; With continuous ultrasound 1h behind this system strenuous vibration so that the spontaneous formation phospholipid bilayer of monomolecular phosphorus adipose membrane; The liposome that obtains is respectively with normal saline and PBS washing, centrifugal, to remove the not phospholipid molecule of encystation.
The benchmark test of product:
Prepared magnetic disodium clodronate liposome is dissolved in 18mL chloroform and the 2mL methanol mixed solution; Remove the clodronate disodium that liposome membrane is coated to discharge, through the envelop rate of ultraviolet-visible spectrophotometer detection of drugs concentration with the calculating medicine.
Envelop rate computational methods: envelop rate=(the drug/lipid body Chinese medicine total amount of sealing in the liposome) * 100%
Envelop rate through calculating embodiment 1-5 is respectively 24.8%, 25.3%, and 22.9%, 26.8%, 26.4%.
Claims (1)
1. the method for preparing of a magnetic disodium clodronate liposome is characterized in that its key step comprises:
(1) preparation of nano ferriferrous oxide:
The adding mol ratio is 100: 1: 1 ferric acetyl acetonade, aniline, an oleic acid mixed liquor in the three-neck flask; Feed protection gas under the magnetic agitation to remove oxygen; System is warming up to 80-120 ℃ and keep 20-30min, and stirs and make the reactant mix homogeneously, remove nitrogen after; Continue to be heated to temperature and reach 260-270 ℃, keep reflux state 2-4h; The mixture that obtains after the reaction is cooled to room temperature, and absolute ethanol washing for several times, and is centrifugal, and the black product that finally obtains is scattered in dehydrated alcohol, obtains ferroferric oxide magnetic fluid; Wherein said protection gas is nitrogen;
(2) preparation of the liposome of coating medicine and nano ferriferrous oxide:
Take by weighing lecithin and cholesterol at 5: 1 by mass ratio, be dissolved in the chloroform, vibrations make the solid dissolving, and the solution rotating evaporation to remove solvent chloroform, will be placed 60 ℃ of oven dry of thermostatic drying chamber with the flask of immobilized artificial membrane;
Clodronate disodium is dissolved in the PBS of pH=7.4; Add with the clodronate disodium mass ratio be the ferroferric oxide magnetic fluid that has prepared in 1: 1 the step (1); This mixture is transferred in the flask that has immobilized artificial membrane of the last step preparation of having dried, with these system vibrations, ultrasonic after; Respectively with the PBS washing of 0.7% sodium chloride solution and pH=7.4; Centrifugal, place 4 ℃ of refrigerators of constant temperature to store end product, wherein said lecithin and clodronate disodium mass ratio are 1.7: 1 or 1.4: 1.
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| CN1771972A (en) * | 2004-11-09 | 2006-05-17 | 胡才忠 | Clodronate liposome and its prepn |
| WO2008016172A1 (en) * | 2006-08-04 | 2008-02-07 | Kurume University | Metastasis inhibitor |
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| CN1771972A (en) * | 2004-11-09 | 2006-05-17 | 胡才忠 | Clodronate liposome and its prepn |
| WO2008016172A1 (en) * | 2006-08-04 | 2008-02-07 | Kurume University | Metastasis inhibitor |
Non-Patent Citations (1)
| Title |
|---|
| Shouheng Sun,等.monodisperse MFe2O4(M=Fe,Co,Mn) Nanoparticles.《journal of the american chemical society》.2004,第126卷(第1期),273-279. * |
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