CN102225051A - pH sensitive doxorubicin nanoliposome modified by folic acid-carboxymethyl chitosan - Google Patents
pH sensitive doxorubicin nanoliposome modified by folic acid-carboxymethyl chitosan Download PDFInfo
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- CN102225051A CN102225051A CN2011101542935A CN201110154293A CN102225051A CN 102225051 A CN102225051 A CN 102225051A CN 2011101542935 A CN2011101542935 A CN 2011101542935A CN 201110154293 A CN201110154293 A CN 201110154293A CN 102225051 A CN102225051 A CN 102225051A
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- carboxymethyl chitosan
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Abstract
The invention relates to pH sensitive doxorubicin nanoliposome modified by folic acid-carboxymethyl chitosan and its preparation method. The liposome provided in the invention comprises doxorubicin, phosphatide, cholesterol, vitamin E, folic acid-carboxymethyl chitosan. The prepared liposome has pH sensitivity, tumor targeting performance and long cyclic active targeting drug delivery functions.
Description
Technical field
The present invention relates to responsive doxorubicin nanometer liposome of the sugar-modified pH of a kind of folic acid-carboxymethyl chitosan and preparation method thereof.
Background technology
Doxorubicin (Doxorubicin), be commonly called as amycin again, doxorubicin, be antibiotics antineoplastic agent commonly used clinically, anticancer spectrum is wide, and curative effect is wide, can treat kinds of tumors, but this medicine has serious toxic and side effects to heart, especially serious heart damage, and side reactions such as bone marrow depression have limited its application clinically.For many years, the various countries scientist is seeking the effective ways that reduce its toxicity always, as adjusting dosage regimen, develops the low amycin analog of cardiac toxicity, and the mode of drug combination and change pharmaceutical dosage form alleviates its serious cardiac toxicity.The toxicity of liposome amycin specific ionization medicine reduces 50-70% (Budai M, et al.Liposomes as drug carrier systems:preparation, classification and therapeutic advantages of liposomes.Acta Pharm Hung., 2001,71 (1): but 114-8.) liposome is because it is easily by macrophage phagocytic, content in normal structure is still very high, in order further to reduce the drug toxicity of Evacet, utilize targeting substance or particle that surface of liposome or inside are modified, make liposome overcome the various physiologic barriers that medicine is run in the course of conveying in vivo, medicine is delivered to specific target position, improve the targeting of medicine.PH sensitivity liposome is exactly wherein a kind of, utilize tumor tissues when ischemia, unusual acidify phenomenon can appear in mesenchyma stroma of tumors liquid, pH value is lower than normal structure, utilize this characteristic scientist to adopt distinct methods to carry out finishing, the phospholipid material that has pH sensitivity as the bioabsorbable polymer material that has the pH sensitivity in the surface of liposome grafting or use prepares liposome.
Carboxymethyl chitosan (CMCT) is a kind of soluble derivative that chitosan generates behind carboxymethylation reaction, its stable in properties, and biocompatibility and degradability are good, and toxicity is less.It had both contained weak base cation (NH
3 +) group, contain weak acid anion (COO again
-) group, be a kind of amphipathic electrolyte.Because it has the pH sensitivity, under different pH value conditions, its segmental conformation difference, utilize this characteristic, carboxymethyl chitosan is sugar-modified to surface of liposome, make under different pH value because the drug release that different its active forces of conformation wrap up in liposome reaches the pH sensitivity.Tong Xin bravely wait (the sugar-modified influence that Taxdol self assembled precusor liposome rat in-vivo tissue is distributed of carboxymethyl chitosan. Chinese natural drug, 2007,5 (1): 45-49) utilize this rule to carry out the modification of Paclitaxel liposome and its medicine is studied for distribution, result of study shows, after the interpolation carboxymethyl chitosan carries out finishing, obvious variation has taken place in configuration in the body of rat, and the ingestion of medicines amount in liver, spleen, lung, ovary, uterus etc. obviously increases.
The targeting form of nano-grade medicine carrier is based on passive target, promptly utilize the chemical factors such as size, quality, pH value of hydrophobicity (or hydrophilic), electrostatic interaction, the carrier of carrier to be positioned at the monokaryon of liver, spleen, lung and bone marrow, the picked-up of huge macrophage system easily, strengthen the chemotherapy effect of antitumor drug the tumor that is positioned at mononuclear phagocyte system.In order to strengthen targeting, can design nanoparticle with active targeting, combine with the cell surface specific receptor by targeted molecular, medicine can accurately be delivered in the tumor cell, realize the targeted therapy of malignant tumor.Because folacin receptor is in the overexpression of tumor cell adventitia, having known has special substantial connection between tumor cell and the folic acid.When folic acid is connected in the carboxyl (folic acid-carboxymethyl chitosan) of carboxymethyl chitosan by side group, it still keeps normal receptor affinity, therefore can be by the receptor endocytosis by internalization.And folacin receptor also provides a kind of effective means (Sumanta K., et al. In vitro evaluation of folic acid modified carboxymethyl chitosan nanoparticles loaded with doxorubi for specific medicament to the targeting of tumor in multiple human tumor cell's overexpression
Summary of the invention
The objective of the invention is to disclose responsive nanometer Mycocet of the sugar-modified pH of a kind of folic acid-carboxymethyl chitosan and preparation method thereof.The responsive nanometer Mycocet of the sugar-modified pH of folic acid-carboxymethyl chitosan is prepared from as component by doxorubicin hydrochloride, lecithin, cholesterol, vitamin E, folic acid-carboxymethyl chitosan etc.
The responsive nanometer Mycocet of the sugar-modified pH of folic acid-carboxymethyl chitosan of the present invention, form by the component of following mass fraction ratio:
Wherein, preferably be grouped into by the one-tenth of following ratio of quality and the number of copies:
The preparation of the responsive nanometer Mycocet of the sugar-modified pH of folic acid-carboxymethyl chitosan of the present invention may further comprise the steps:
(1) takes by weighing corresponding phospholipids, cholesterol and vitamin E by prescription and put into appropriate containers, add the ultrasonic concussion of dehydrated alcohol then and be dissolved into solution, this solution is changed in the rotary evaporator; Remove organic solvent, film forming at 40 ℃ of water-bath rotary evaporations; Add buffer solution (pH=4.0) and wash film, aquation, 0.5h is once ultrasonic at interval, and aquation 2h makes blank liposome;
(2) ultrasonic or high pressure homogenize makes nano level blank liposome with ultrasonic cell pulverization machine gap with the blank liposome of preparation, and mistake 0.22 μ m filter membrane obtains the blank nanometer liposome of uniform particle diameter;
(3) regulate the outer aqueous pH values of blank nanometer liposome to 6.8-7.4 with buffer solution (pH=8.0), add the isotonic sodium chlorrde solution that is dissolved with doxorubicin hydrochloride again, by the pH gradient method doxorubicin hydrochloride is written in the blank nanometer liposome, hydration 3h under 37 ℃ of water-baths makes Doxil;
(4) folic acid-carboxymethyl chitosan sugar juice is joined in the Doxil, hydration 0.5h under 37 ℃ of water-baths makes the responsive nanometer Mycocet of the sugar-modified pH of folic acid-carboxymethyl chitosan.
Described lecithin is selected from Ovum Gallus domesticus Flavus lecithin, soybean lecithin, hydrogenated soy phosphatidyl choline or hydrogenated yolk lecithin.
The degree of substitution by carboxymethyl of described carboxymethyl chitosan is 0.6-1.2, and molecular weight is 3000-100000; Described folic acid-carboxymethyl chitosan be by folic acid behind room temperature activation 2h under the condition that N-hydroxy-succinamide (NHS) and carbodiimide hydrochloride (EDCHCl) exist, generate with the carboxymethyl chitosan reaction again.
Described buffer is selected from the normal saline of phosphate buffered solution or 0.9% sodium chloride.
The described cell pulverization machine intermittently optimization power of ultrasonic blank liposome is 200-300w, off time 5s, ultrasonic time 3s, the working time is 30min; Or to adopt the optimization pressure parameter of high pressure homogenize be 40-90MPa, and cycle-index is 6-15 time.
Adopt method of the present invention, the responsive nanometer Mycocet of the sugar-modified pH of the folic acid-carboxymethyl chitosan preparation that can prepare small particle diameter, stable performance, the modification of folic acid-carboxymethyl chitosan can make it possess pH sensitivity, tumor-targeting, and prolongs long cycle performance in its body.The present invention adopts dehydrated alcohol as organic solvent, has got rid of the residual of bigger organic solvent chloroform of the toxicity used in the document or methanol, has reduced environmental pollution, has reduced production cost, is more suitable for suitability for industrialized production.
Description of drawings
Fig. 1 is Fourier's infrared spectrum of folic acid-carboxymethyl chitosan.
Fig. 2 is the drug release rate figure of the sugar-modified doxorubicin nanometer liposome of folic acid-carboxymethyl chitosan in different pH buffer.
The specific embodiment
By following embodiment the specific embodiment of the present invention is described, but protection scope of the present invention is not limited to this.
Under aseptic condition, get soybean lecithin for injection 2g, cholesterol 0.5g, vitamin E 0.025g.Above-mentioned material is placed appropriate containers, add the ultrasonic concussion of dehydrated alcohol 100ml to being dissolved into solution fully, above-mentioned solution is transferred in the pyriform bottle, the decompression rotary evaporation is to forming transparent faint yellow thin film in water bath with thermostatic control.Get PBS (pH=4.0) buffer solution 100ml eluting, slowly wash film.Washing behind the film with ultrasonic cell pulverization machine is 200-300w at power, off time 5s, ultrasonic time 3s, working time is the 30min Ultrasonic Pulverization, the control particle diameter is between 50nm-200nm, with 0.22 micron membrane filtration degerming, regulate its outer water to pH=6.8 with PBS (pH=8.0) buffer solution, add with the dissolved doxorubicin hydrochloride 0.2g hydration of 0.9% sodium chloride solution 3h, add a certain amount of folic acid-carboxymethyl chitosan sugar juice, hydration 0.5h obtains the responsive doxorubicin nano-lipid of pH liquid solution.
Under aseptic condition, get injection Ovum Gallus domesticus Flavus lecithin 1.6g, cholesterol 0.4g, vitamin E 0.020g.Above-mentioned material is placed appropriate containers, add the ultrasonic concussion of dehydrated alcohol 80ml to being dissolved into solution fully, above-mentioned solution is transferred in the pyriform bottle, the decompression rotary evaporation is to forming transparent faint yellow thin film in water bath with thermostatic control.Get PBS (pH=4.0) buffer solution 80ml eluting, slowly wash film.Wash and adopt behind the film high pressure homogenize in pressure parameter circulation 10 times during for 80MPa, regulate its outer water to pH=7.0 with PBS (pH=8.0) buffer solution, add with the dissolved doxorubicin hydrochloride 0.16g hydration of 0.9% sodium chloride solution 3h, add a certain amount of folic acid-carboxymethyl chitosan sugar juice, hydration 0.5h obtains the responsive doxorubicin nano-lipid of the sugar-modified pH of folic acid-carboxymethyl chitosan liquid solution.
Embodiment 3
Under aseptic condition, get injection Ovum Gallus domesticus Flavus lecithin 0.4g, cholesterol 0.1g, vitamin E 0.005g.Above-mentioned material is placed appropriate containers, add the ultrasonic concussion of dehydrated alcohol 20ml to being dissolved into solution fully, above-mentioned solution is transferred in the pyriform bottle, the decompression rotary evaporation is to forming transparent faint yellow thin film in water bath with thermostatic control.Get PBS (pH=4.0) buffer solution 20ml eluting, slowly wash film.Washing behind the film with ultrasonic cell pulverization machine is 200-300w at power, off time 5s, ultrasonic time 3s, working time is the 30min Ultrasonic Pulverization, the control particle diameter is between 50nm-200nm, with 0.22 micron membrane filtration degerming, regulate its outer water to pH=7.4 with PBS (pH=8.0) buffer solution, add with the dissolved doxorubicin hydrochloride 0.04g hydration of 0.9% sodium chloride solution 3h, add a certain amount of folic acid-carboxymethyl chitosan sugar juice, hydration 0.5h obtains the responsive doxorubicin nano-lipid of the sugar-modified pH of folic acid-carboxymethyl chitosan liquid solution.
Claims (6)
1. the responsive doxorubicin nanometer liposome of the sugar-modified pH of a folic acid-carboxymethyl chitosan, it is characterized in that, form by following components in weight percentage: doxorubicin hydrochloride 3.2-16 part, lecithin 16-80 part, cholesterol 4-20 part, vitamin E 0.16-0.8 part, the carboxymethyl chitosan of folic acid modification (folic acid-carboxymethyl chitosan) 1-5 part, buffer 800-1000 part; With lecithin, cholesterol and vitamin E, take by weighing corresponding weight respectively in proportion and put into appropriate containers, add the ultrasonic concussion of quantitative dehydrated alcohol then and be dissolved into solution, this solution is changed in the rotary evaporator, film forming after organic solvent is removed in 40 ℃ of water-bath rotary evaporation decompressions, the buffer solution of additional proportion amount (pH=4.0) is washed film, aquation, 0.5h is once ultrasonic at interval, aquation 2h, make blank liposome, with blank liposome with ultrasonic cell pulverization machine intermittently ultrasonic or high pressure homogenize make nanometer liposome, after crossing 0.22 μ m filter membrane, regulate outer aqueous pH values with buffer solution (pH=8.0) and to 6.8-7.4, add the isotonic sodium chlorrde solution that is dissolved with doxorubicin hydrochloride, by the pH gradient method doxorubicin hydrochloride is written in the blank nanometer liposome, hydration 3h under 37 ℃ of water-baths joins folic acid-carboxymethyl chitosan sugar juice in the doxorubicin hydrochloride nanometer liposome, hydration 0.5h under 37 ℃ of water-baths makes the responsive doxorubicin nanometer liposome of pH.
2. the responsive doxorubicin nanometer liposome of the sugar-modified pH of a kind of folic acid-carboxymethyl chitosan according to claim 1, it is characterized in that, form by following components in weight percentage: doxorubicin hydrochloride 4-12 part, lecithin 20-60 part, cholesterol 4-15 part, vitamin E 0.2-0.6 part, folic acid-carboxymethyl chitosan 1.25-3.75 part, buffer 800-1000 part.
3. according to the responsive doxorubicin nanometer liposome of the sugar-modified pH of the described a kind of folic acid-carboxymethyl chitosan of claim 1-2, it is characterized in that described lecithin is selected from Ovum Gallus domesticus Flavus lecithin, soybean lecithin, hydrogenated soy phosphatidyl choline or hydrogenated yolk lecithin.
4. according to the responsive doxorubicin nanometer liposome of the sugar-modified pH of the described a kind of folic acid-carboxymethyl chitosan of claim 1-2, it is characterized in that the degree of substitution by carboxymethyl in the described carboxymethyl chitosan is 0.6-1.2, molecular weight is 3000-100000; Described folic acid-carboxymethyl chitosan be by folic acid behind room temperature activation 2h under the condition that N-hydroxy-succinamide (NHS) and carbodiimide hydrochloride (EDCHCl) exist, generate with the carboxymethyl chitosan reaction again.
5. according to the method for preparing the responsive nanometer Mycocet of the sugar-modified pH of folic acid-carboxymethyl chitosan described in the claim 1, it is characterized in that described buffer is selected from the sodium chloride normal saline of phosphate buffered solution or 0.9%.
6. according to the method for preparing the responsive nanometer Mycocet of the sugar-modified pH of folic acid-carboxymethyl chitosan described in the claim 1, when it is characterized in that using the cell pulverization machine to pulverize the gained blank liposome, its power is 200-300w, off time 5s, ultrasonic time 3s, the working time is 30min; Or the gained blank liposome carried out high pressure homogenize, and pressure is 40-90MPa, cycle-index is 6-15 time.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102429868A (en) * | 2011-12-09 | 2012-05-02 | 南开大学 | Liposome medicinal composition with tumor targeting, in-vivo tracing and treating functions and preparation method thereof |
| CN102488658A (en) * | 2011-12-23 | 2012-06-13 | 华东理工大学 | Folacin-carboxymethyl chitosan modified pH sensitive taxol nanoliposome |
| CN105708848A (en) * | 2016-02-04 | 2016-06-29 | 武汉艾美博特医疗用品有限公司 | Environmentally responsive tumor targeted combined administration transfer system |
| CN107049953A (en) * | 2017-06-05 | 2017-08-18 | 福州大学 | A kind of pH/ near infrared lights response bubble liposome and preparation method thereof |
| CN107260676A (en) * | 2017-06-05 | 2017-10-20 | 福州大学 | A kind of application of pH/ near infrared lights response bubble liposome |
-
2011
- 2011-06-09 CN CN2011101542935A patent/CN102225051A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102429868A (en) * | 2011-12-09 | 2012-05-02 | 南开大学 | Liposome medicinal composition with tumor targeting, in-vivo tracing and treating functions and preparation method thereof |
| CN102488658A (en) * | 2011-12-23 | 2012-06-13 | 华东理工大学 | Folacin-carboxymethyl chitosan modified pH sensitive taxol nanoliposome |
| CN105708848A (en) * | 2016-02-04 | 2016-06-29 | 武汉艾美博特医疗用品有限公司 | Environmentally responsive tumor targeted combined administration transfer system |
| CN105708848B (en) * | 2016-02-04 | 2018-07-20 | 武汉艾美博特医疗用品有限公司 | A kind of environment-responsive cancer target administering drug combinations transmission system |
| CN107049953A (en) * | 2017-06-05 | 2017-08-18 | 福州大学 | A kind of pH/ near infrared lights response bubble liposome and preparation method thereof |
| CN107260676A (en) * | 2017-06-05 | 2017-10-20 | 福州大学 | A kind of application of pH/ near infrared lights response bubble liposome |
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Application publication date: 20111026 |