CN102920660A - Novel liver cancer treatment preparation with physical and biological double-targeting effect - Google Patents
Novel liver cancer treatment preparation with physical and biological double-targeting effect Download PDFInfo
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- CN102920660A CN102920660A CN2012103992688A CN201210399268A CN102920660A CN 102920660 A CN102920660 A CN 102920660A CN 2012103992688 A CN2012103992688 A CN 2012103992688A CN 201210399268 A CN201210399268 A CN 201210399268A CN 102920660 A CN102920660 A CN 102920660A
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Abstract
The invention discloses a novel liver cancer treatment preparation with a physical and biological double-targeting effect. The preparation is docetaxel targeted nano particles prepared by an emulsifying-solvent volatilization method, and the preparation method comprises the following steps of: dissolving mono-methoxy polyoxyethylene-poly(lactic-co-glycolic acid), docetaxel and galactose cholesterol in dichloromethane to form an organic phase; absorbing the organic phase, and slowly injecting the organic phase into a polyvinyl alcohol aqueous solution; performing ultrasonic emulsification; pouring the obtained emulsion into another polyvinyl alcohol aqueous solution; stirring to remove dichloromethane; filtering with a polycarbonate membrane; and performing vacuum freeze drying and irradiation sterilization at 60 DEG C, and storing for later use. The average particle size of the prepared docetaxel targeted nano particles is 209.3nm, and the multi-element dispersion coefficient is 0.40; and the drug loading ratio of the docetaxel targeted nano particles is 3.79%. Through the invention, the particle size of the docetaxel targeted nano particles is about 200nm; and the in vivo anti-tumor experiments indicate that the treatment effect is better by combining the docetaxel targeted nano particles with ultrasonic irradiation, and the anti-tumor rate reaches 74.2%.
Description
Technical field
The present invention relates to a kind of learn a skill hepatoma-targeting treatment novel formulation of preparation of modern medicinal agents that adopts.The biological effect that ultrasonic irradiation produces increases the tumor blood flow amount, vascular permeability strengthens, and then make docetaxel nano-particle be gathered in a large number tumor by local, and enter in the tumor cell by receptor-mediated endocytic pathway, from physics, double route performance biology hepatoma-targeting therapeutical effect.
Background technology
Hepatocarcinoma whole world sickness rate has surpassed for 62.6 ten thousand/year, and wherein 55% case occurs in China.At present the treatment means of hepatocarcinoma mainly contains surgical resection and partial interventional therapy, and various technology especially tumor local ablation technical development are swift and violent and be widely used in clinical.But result of study shows that 5 years survival rates of liver cancer patient are not significantly improved.Therefore, explore more efficiently liver cancer treatment method to prolonging patient's life, improving the quality of living has very important meaning.
Docetaxel is a kind of semi-synthetic antitumor drug, has been widely used in the chemotherapy such as Several Kinds of Malignancies such as breast carcinoma, nonsmall-cell lung cancer, carcinoma of prostate, gastric cancer and tumor of head and neck.In vivo and in vitro shows that Docetaxel has stronger anti-hepatocarcinoma effect.But because Docetaxel is insoluble in water, use clinically at present preparation to adopt the tween solubilising more, cause allergic reaction easily.In addition, the untoward reaction such as bone marrow depression, nerve, heart and Liver and kidney toxicity that cause of system's chemotherapy are more obvious.Therefore, change the Docetaxel dosage form, improve its water solublity and tumor-targeting, to improve its antitumor curative effect, to reduce toxic and side effects significant.
Neoplasm targeted therapy is the emphasis and one of focus of modern oncotherapy research, and it utilizes idiosyncratic carrier, and the active component of medicine or other killing tumor cells optionally is transported to the tumor target site, has efficiently, the antitumor advantage of low toxicity.There is asialoglycoprotein receptor in the hepatoma carcinoma cell surface, but the specific recognition end again by endocytosis, initiatively changes performance antitumor action in the cell with glycoprotein and the with it combination of galactose residue over to.Asialoglycoprotein receptor has become one of important target spot of hepatoma-targeting treatment research.
Mono methoxy polyethylene glycol-polylactic-co-glycolic acid is a kind of bi-block copolymer, is usually used in preparing various drug-carrying nanometer particles.Hydrophobic polylactic-co-glycolic acid is positioned at the core of nanoparticle, wraps up various insoluble anti-tumor medicaments.Hydrophilic Polyethylene Glycol is distributed in the nanoparticle surface, has obviously improved hydrophilic and the surface stability of nanoparticle, and can significantly reduce reticuloendothelial system to the picked-up scavenging action of nanoparticle, makes nanoparticle have macrocyclic effect.
Summary of the invention
The purpose of this invention is to provide a kind of
Have physics, biological dual-target effect the liver cancer treatment novel formulation and the preparationApplication in the medicine of alkene paclitaxel targeted nano granule combining ultrasonic radiation treatment tumor.
The object of the present invention is achieved like this, described a kind of
Liver cancer treatment novel formulation with physics, biological dual-target effect, be the Docetaxel targeted nano granule of emulsifying-solvent evaporation method preparation, adopt mono methoxy polyethylene glycol-polylactic-co-glycolic acid, Docetaxel and glycosyl galactose cholesterol to be dissolved in dichloromethane and form organic facies; Draw organic facies, slowly be injected in the polyvinyl alcohol water solution, ultrasonic emulsification, the emulsion of acquisition is poured in another polyvinyl alcohol water solution, and stir and remove dichloromethane, through the Merlon membrane filtration, vacuum lyophilization,
60Obtain the Docetaxel targeted nano granule behind the Co irradiation sterilization.
The mean diameter of prepared Docetaxel targeted nano granule is 209.3nm, and the polydisperse number is 0.40; The carrying drug ratio of Docetaxel targeted nano granule is 3.79%.
Of the present invention a kind of
Liver cancer treatment novel formulation with physics, biological dual-target effectPreparation method, step is: precision takes by weighing 100mg mono methoxy polyethylene glycol-polylactic-co-glycolic acid, 5mg Docetaxel, 15mg glycosyl galactose cholesterol Gal-C4-Chol, is dissolved in 1 ml dichloromethane, 60 ℃ of vortex concussions, it is fully dissolved, be organic facies; Draw organic facies with syringe, slowly be injected in 3ml 5 % (volume fraction) polyvinyl alcohol water solution ultrasonic emulsification, 50 W, 30 sec work, 5 sec intermittently, emulsifying 5 times, emulsion is poured into 8ml 1 %(volume fraction) poly-vinyl alcohol solution, magnetic agitation is removed dichloromethane, the Merlon membrane filtration, vacuum lyophilization
604 ℃ of preservations behind the Co irradiation sterilization.
Of the present invention a kind of
Has the liver cancer treatment novel formulation of physics, biological dual-target effect in preparationApplication in the medicine of alkene paclitaxel targeted nano granule combining ultrasonic radiation treatment tumor.
Advantage of the present invention is: when ultrasound wave is propagated in vivo, can produce heat effect, cavitation effect, mechanical effect etc., and then the Tissue Blood flow is increased, vascular permeability strengthens.Normal structure blood vessel endothelium gap is many less than 100nm, and particle diameter is difficult to arrive interstice by blood vessel endothelium greater than the granule of 100nm.The present invention adopts emulsion-solvent evaporation method to prepare the carrying docetaxel high molecular nanometer grain of liver cancer targeting, the about 200nm of particle diameter, be difficult to enter the normal structure gap after the intravenous injection, improve the tumor by local blood flow by ultrasonic irradiation again, expansion tumor vessel permeability, make nanoparticle be gathered in a large number tumor by local, and enter in the hepatoma carcinoma cell by asialoglycoprotein receptor, performance is efficient, the hepatoma-targeting therapeutic effect of low toxicity.Anti-tumor in vivo experiment draws, and Docetaxel targeted nano granule combining ultrasonic irradiation carries out therapeutic effect the best, inhibitory rate 74.2%, and apparently higher than other each group, the two is united and has the synergistic antitumor effect.
Description of drawings
Fig. 1 isThe scanning electron microscope (SEM) photograph of Docetaxel targeted nano granule.
Fig. 2 is Docetaxel targeted nano granule release graphics in phosphate buffer, and 6h has discharged 11.3%, 3 day cumulative release 78.1%.
The specific embodiment
1, the preparation of Docetaxel targeted nano granule
Adopt emulsifying-solvent evaporation method to prepare the Docetaxel targeted nano granule.Precision takes by weighing 100mg mono methoxy polyethylene glycol-polylactic-co-glycolic acid, 5mg Docetaxel, 15mg glycosyl galactose cholesterol Gal-C4-Chol, is dissolved in 1 ml dichloromethane, and the vortex concussion is fully dissolved it, is organic facies; Draw organic facies with syringe, slowly inject 3ml 5 %(volume fractions) polyvinyl alcohol water solution, ultrasonic emulsification, 50 W, 30 sec work, 5 sec intermittently, emulsifying 5 times, emulsion is poured 8ml 1 % (volume fraction) poly-vinyl alcohol solution into, magnetic agitation is removed dichloromethane, the Merlon membrane filtration, vacuum lyophilization
604 ℃ save backup behind the Co irradiation sterilization.
2, the sign of Docetaxel targeted nano granule
Preparation Docetaxel targeted nano granule aqueous solution fully behind the ultra-sonic dispersion, drips on the copper mesh that covers carbon film, spatters gold with the ion film plating instrument after the drying, observes visible nanoparticle good dispersion under scanning electron microscope, is the spherical (see figure 1) of class.The mean diameter that laser particle instrument records the Docetaxel targeted nano granule is 209.3nm, and the polydisperse number is 0.40.
Adopt high performance liquid chromatography to detect the carrying drug ratio of Docetaxel targeted nano granule.Mobile phase: methanol/water=75/25(volume ratio), flow velocity: 1.0ml/min, 30 ℃ of column temperatures detect wavelength 227nm.Precision takes by weighing Docetaxel targeted nano granule 10mg in the 5ml centrifuge tube, adds the 0.5ml dichloromethane, fully adds 2.5ml methanol after the dissolving, vortex 10min, and the centrifugal 10min of 5000rpm gets supernatant 20 μ l sample introductions, and recording carrying drug ratio is 3.79%.
3, the release in vitro of Docetaxel targeted nano granule
Phosphate buffer (pH 7.2) preparation Docetaxel targeted nano granule solution with 0.02 mol/L (contains 0.02 % (volume fraction) NaN
3, 0.1 %(volume fraction) Tween 80), in 37 ℃, 80rpm constant-temperature table concussion is carried out release in vitro, timing sampling, centrifugal 10 minutes of 5000rpm ultrafiltration, filtrate row high-performance liquid chromatogram determination Docetaxel content.The Docetaxel targeted nano granule steadily discharges in phosphate buffer as a result, 6h has discharged 11.3%, 3 day cumulative release 78.1%(Fig. 2).
4, the acute toxicity test of Docetaxel targeted nano granule
Get 80 of Kunming mouses, weight is 18～22 g, male and female half and half, be divided at random 10 groups, every group 8, respectively through caudal vein injection Docetaxel targeted nano granule solution (Docetaxel dosage is respectively 80,120,180,270,405 mg/kg) and Docetaxel injection (Docetaxel dosage is respectively 15,23,34,51,76 mg/kg).The conventional raising for 2 weeks after the administration, outward appearance, activity and the death condition etc. of record mice.It is 47.3mg/kg that the improvement karber's method records Docetaxel injection mice median lethal dose(LD 50), and the mice median lethal dose(LD 50) of Docetaxel targeted nano granule is elevated to 219.5mg/kg.
5, ultrasonic irradiation is on the impact of tumor blood flow perfusion
Human Hepatocarcinoma Cell line in vitro HepG2 adjusts cell density about 1 * 10 with normal saline after a large amount of amplifications
7/ ml, 0.2 ml cell suspension is injected in every right oxter of nude mice.Choose 12 of tumor maximum diameter 1.7～2.0cm tumor bearing nude mices after 2 weeks, be divided into matched group and ultrasonic irradiation group, 6 every group.Acoustic contrast agent SonoVue(5ml/kg annotates in nude mice of control group tail vein group), SEQUOIA 512 diasonographs carry out ultrasonic contrast.The ultrasonic irradiation group nude mice tumor ultrasonic irradiation of going ahead of the rest, frequency 840kHz, sound intensity 0.75w/cm
2, carry out immediately ultrasonic contrast behind the irradiation 2min.Start Angiographic software Syngo US Workplace, relatively two groups of tumor imagings beginnings increase the parameters such as time (AT), peak time (TTP), peak value-underlying strength poor (PBD).As a result, behind the ultrasonic irradiation, tumor peak value-underlying strength is poor apparently higher than matched group (P＜0.05), shows tumor blood flow perfusion showed increased.
The impact that table 1 ultrasonic irradiation pours into tumor blood flow (
± s)
6, ultrasonic irradiation is on the impact of drug distribution
Get 12 of tumor maximum diameter 1.7～2.0cm tumor bearing nude mices, be divided into Docetaxel targeted nano granule and Docetaxel targeted nano granule combining ultrasonic irradiation group, 6 every group.Behind the nude mice tail vein injection nanoparticle (Docetaxel 10mg/kg), combining ultrasonic irradiation group tumor row ultrasonic irradiation, frequency 840kHz, sound intensity 0.75w/cm
2Ultrasonic irradiation 2min, interval 2h repetitive irradiation 1 time, totally 3 times.Get tumor tissues homogenate extracting Docetaxel, high performance liquid chromatography detects.As a result, combining ultrasonic irradiation group tumor tissues Docetaxel medication amount is 2.9 ± 0.5 μ g/g albumen, is 1.8 times of simple Docetaxel targeted nano granule group.
7, anti-tumor in vivo effect
Choose 25 of the tumor bearing nude mices of about 1.0 cm of tumor maximum diameter, be divided at random 5 groups, 5 every group, give corresponding treatment.Nanoparticle adopts the tail vein injection administration, and Docetaxel dosage is 10mg/kg.Combining ultrasonic irradiation group after administration, frequency 840kHz, sound intensity 0.75w/cm
2Ultrasonic irradiation 2min, interval 2h repetitive irradiation 1 time, totally 3 times.
A group: model group
B group: docetaxel nano-particle group
C group: Docetaxel targeted nano granule group
D group: docetaxel nano-particle+ultrasonic irradiation group
E group: Docetaxel targeted nano granule+ultrasonic irradiation group
More than the treatment weekly treatment is 1 time, continuous 4 times.Nude mice is put to death in rear the 28th day for the treatment of, peels off tumor and claims quality, calculates tumour inhibiting rate, Docetaxel targeted nano granule combining ultrasonic radiation treatment best results, and inhibitory rate 74.2% is apparently higher than other each group (table 2).
Table 2 respectively organize the oncotherapy effect relatively (
± s)
*
P＜0.05, compare with the A group.
Claims (4)
1. one kind
Liver cancer treatment novel formulation with physics, biological dual-target effect, be the Docetaxel targeted nano granule of emulsifying-solvent evaporation method preparation, adopt mono methoxy polyethylene glycol-polylactic-co-glycolic acid, Docetaxel and glycosyl galactose cholesterol to be dissolved in dichloromethane and form organic facies; Draw organic facies, slowly be injected in the polyvinyl alcohol water solution, ultrasonic emulsification, the emulsion of acquisition is poured in another polyvinyl alcohol water solution, and stir and remove dichloromethane, through the Merlon membrane filtration, vacuum lyophilization,
60Obtain the Docetaxel targeted nano granule behind the Co irradiation sterilization.
2.
According to claim 1A kind of
Liver cancer treatment novel formulation with physics, biological dual-target effect, the mean diameter that it is characterized in that prepared Docetaxel targeted nano granule is 209.3nm, the polydisperse number is 0.40; The carrying drug ratio of Docetaxel targeted nano granule is 3.79%.
3. claimed in claim 1 a kind of
Liver cancer treatment novel formulation with physics, biological dual-target effectPreparation method, step is: precision takes by weighing 100mg mono methoxy polyethylene glycol-polylactic-co-glycolic acid, 5mg Docetaxel, 15mg glycosyl galactose cholesterol Gal-C4-Chol, is dissolved in 1 ml dichloromethane, the vortex concussion, it is fully dissolved, be organic facies; Draw organic facies with syringe, slowly be injected in the 3ml 5 % polyvinyl alcohol water solutions ultrasonic emulsification, 50 W, 30 sec that work, 5 sec intermittently, emulsifying 5 times, emulsion is poured into 8ml 1 % poly-vinyl alcohol solution, magnetic agitation is removed dichloromethane, Merlon membrane filtration, vacuum lyophilization
604 ℃ of preservations behind the Co irradiation sterilization.
4. claimed in claim 1 a kind of
Has the liver cancer treatment novel formulation of physics, biological dual-target effect in preparationApplication in the medicine of alkene paclitaxel targeted nano granule combining ultrasonic radiation treatment tumor.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101057831A (en) * | 2006-05-15 | 2007-10-24 | 沈阳药科大学 | Docetaxel liposome novel preparations and its preparation method |
| CN101301479A (en) * | 2007-05-08 | 2008-11-12 | 上海华明高技术（集团）有限公司 | Tumor-targeted developing composition for magnetic resonance image-forming diagnose and preparation thereof |
| CN101735410A (en) * | 2009-12-11 | 2010-06-16 | 武汉大学 | Reduction-sensitive amphiphilic block copolymer and micelle thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101057831A (en) * | 2006-05-15 | 2007-10-24 | 沈阳药科大学 | Docetaxel liposome novel preparations and its preparation method |
| CN101301479A (en) * | 2007-05-08 | 2008-11-12 | 上海华明高技术（集团）有限公司 | Tumor-targeted developing composition for magnetic resonance image-forming diagnose and preparation thereof |
| CN101735410A (en) * | 2009-12-11 | 2010-06-16 | 武汉大学 | Reduction-sensitive amphiphilic block copolymer and micelle thereof |
Non-Patent Citations (1)
| Title |
|---|
| 于雷，牛俊奇: "药物载体系统中肝靶向药物的研究与进展", 《中国组织工程研究与临床康复》, 4 November 2007 (2007-11-04), pages 8932 - 8935 * |
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