CN103505419B - Oxygen carrier liposome of a kind of low surface tension and preparation method thereof - Google Patents
Oxygen carrier liposome of a kind of low surface tension and preparation method thereof Download PDFInfo
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- CN103505419B CN103505419B CN201310438079.1A CN201310438079A CN103505419B CN 103505419 B CN103505419 B CN 103505419B CN 201310438079 A CN201310438079 A CN 201310438079A CN 103505419 B CN103505419 B CN 103505419B
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Abstract
The present invention relates to a kind of preparation method adopting the oxygen carrier liposome of low surface tension.The concrete steps of the method are: first by phospholipid, cholesterol, alevaire, hexadecanol, and Palmic acid is dissolved in organic solvent, and ultrasonic mix homogeneously, then adds fluorocarbons in the solution, continues ultrasonic mix homogeneously.Obtained mixed solution is slowly dropped in saline solution, agitating solution, be stirred to organic solvent volatilization completely, namely can obtain the oxygen carrier liposome wrapping up fluorocarbons.Oxygen carrier liposome prepared by the present invention, has relatively low surface tension, is sprawled, enlarge active surface when being convenient to pulmonary administration.Can at Emergency Oxygen Supply medicine, the aspects such as acute blood supply are applied.
Description
Technical field
The invention belongs to the preparation field of nano material, relate to a kind of water-soluble nano level oxygen carrier liposome and technology of preparing thereof.
Technical background
Perfluorocarbon compound is the compound that in a class fluorine atom substituted hydrocarbons, hydrogen atom obtains, be generally colourless transparent liquid under room temperature, density is high, and stable in properties not easily metabolic breakdown occurs, having the function fully carrying oxygen and carbon dioxide, is desirable liquid breathing medium.Adopt approach in lung, can be Acute Respiratory Distress, the situations such as accident provide treatment.
Adopt direct liquid ventilation method, namely gas exchange is carried out directly to the fluorocarbons injecting oxygenate in lung, although lung compliance and the oxygenate situation of respiratory distress syndrome (ivrds) patient can be improved, but easily cause extra injury of lung, clinically application difficult (J.Pediatr., 1990,117:106., Am.J.Pespir.Crit.Care.Med., 2002,165:781).Adopt and suck ventilation, not only can improve lung compliance and oxygenate situation, also can alleviate pneumonia reaction (PLA's medical journal, 2009,34:746.), there is potential researching value.The relevant report of the method is less, and it still needs further research for clinical.
At present, the perfluorocarbon compound of liposome obtains certain research, but still can not provide a kind of and meet clinical required oxygen carrier liposome.Employing injection method can prepare the fluorine carbon liposome compared with small particle diameter, but only adopts phospholipid as one-tenth film base material, and the liposome solutions of acquisition has relatively high surface tension, is difficult to sprawl at alveolar surface, thus limits its application.In the one-tenth film base material of phospholipid, add and can reduce capillary alevaire, hexadecanol, Palmic acid, reduces the surface tension of fluorine carbon liposome, better improves spreadability, increase gas exchange area, thus the efficiency of gas exchange is enhanced.
Summary of the invention
In order to overcome the deficiencies in the prior art, oxygen carrier liposome that the invention provides a kind of low surface tension and preparation method thereof.
A kind of oxygen carrier liposome of low surface tension, it is characterized in that, containing 0.1-50 gram of fluorocarbons in every 100 milliliters of liposome solutions, 1-50 gram of phospholipid, the cholesterol of 1-10 gram, the alevaire of 0-5 gram, the hexadecanol of 0-5 gram, the Palmic acid of 0-5 gram, surplus is water water, and the liposome mean diameter in described solution is in 30-150 nanometer.
A preparation method for the oxygen carrier liposome of low surface tension, is characterized in that, comprise the following steps:
(1) count by weight, by the hexadecanol of the alevaire of the cholesterol of 1-500 part phospholipid, 1-50 part, 0-20 part, 0-20 part, the Palmic acid of 0-20 part, be dissolved in the organic solvent of 1000 parts, ultrasonic mix homogeneously;
(2) in the organic solvent being dissolved with phospholipid, add fluorocarbons 10-500 part, continue ultrasonic mix homogeneously;
(3) solution that step (2) obtains slowly is dropped in the saline solution of 1000-5000 part, agitating solution, mixing speed is 500-2500 rev/min, is stirred to organic solvent volatilization completely, about 2 hours time, namely can obtain the oxygen carrier liposome wrapping up fluorocarbons.
By phospholipid, cholesterol, alevaire, hexadecanol, Palmic acid, is dissolved in organic solvent, adds fluorocarbons and makes its mix homogeneously, the mixed solution of gained joins in saline solution, stirs and organic solvent is volatilized completely, namely can obtain the oxygen carrier liposome wrapping up fluorocarbons.
Described organic solvent is petroleum ether, chloroform, dichloromethane, ether, normal hexane, ethyl acetate, methanol, the one in toluene or its combination.
Described phospholipid is soybean lecithin, Ovum Gallus domesticus Flavus lecithin, hydrolecithin, HSPC, hydrogenation Yolk lecithin, DLPC, two nutmeg phosphatidyl cholines, dipalmitoyl phosphatidyl choline, distearoyl phosphatidylcholine, 1-nutmeg acyl-2-palmitoylphosphatidyl choline, 1-palmityl-2-DSPC, 1-stearoyl-2-palmitoylphosphatidyl choline, POPC, the sub-oleoyl phosphatidylcholine of 1-stearoyl-2-, DOPC, hydrogenation dipalmitoyl phosphatidyl choline, distearoyl phosphatidylcholine, two nutmeg acyl phosphatidic acid, two nutmeg acyl phosphatidic acid, DPPA, DPPA, G 12S3P, two lima bean lotus acyl phosphatidyl ethylene glycol amine, two palmityl phospholipid indulge in ethylene glycol amine, cephalin acyl serine, two nutmeg acyl Phosphatidylserine, two palmityl Phosphatidylserine, E-PG, PE, two nutmeg acyl phosphatidyl glycerols, DPPG, DSPG, DOPG, brain sphingomyelins, one in two palmitoyl sphingomyelin or distearyl sphingomyelins or its combination.
Described saline solution is normal saline, phosphate buffer, acetate buffer, Basionic buffer, barbitol buffer solution, sodium formate buffer, citrate buffer, ammonia-ammonium chloride buffer, Borax-calcium chloride buffer, acetic acid-lithium salts buffer, Acetic acid-sodium acetate buffer, acetic acid-potassium acetate buffer, acetic acid-ammonium acetate buffer, the one in phosphoric acid-triethylamine buffer solution.
Described fluorocarbons is two (F-alkyl) ethylene, is specially two (F-butyl) ethylene, two (F-hexyl) ethylene; F-naphthalane; F-amantadine (FA); F-methyl amantadine (FMA); F-1,3-dimethyl amantadine (FDMA); Perfluoro-2,2,4,4-tetramethylpentane; F-bis-or F-trimethyl bicyclo-[3,3,1] nonane; The fluoridized amine of C7-12, is specially F-tripropyl amine (TPA), F-4-methyl octahydro quinolizine (FMOQ), F-n-methyl-Decahydroisoquinolinpreparation (FMIQ), F-n-methyl decahydroquinoline (FHQ), F-n-cyclohexyl pyrrolidine (FCHP); Bromination perfluorocarbon compound, is specially perfluoro bromide octane (C
8f
17br), 1-bromine 15 fluorine heptane (C
7f
15br), 1-bromine 13 fluorine hexane (C
6f
13br), the one in or its combination.
The object of the present invention is to provide a kind of preparation method of oxygen carrier liposome of low surface tension, prepared this oxygen carrier liposome encapsulation is high, and particle diameter is little, surface tension is low, and toxicity is little, is suitable as pulmonary's oxygen supply and uses, and method is easy, be convenient to large-scale production.
The invention has the advantages that:
(1) the oxygen carrier liposome prepared of the present invention, surface tension is low, better can be sprawled, thus realize pulmonary's oxygen supply on lung surface.
(2) adopt fluorocarbons to be dissolved oxygen material, oxygen content is high, can realize gas exchange rapidly.
Accompanying drawing explanation
The transmission electron microscope picture of oxygen carrier liposome in Fig. 1 embodiment 1.
The Cytotoxic evaluation of oxygen carrier liposome in Fig. 2 embodiment 3.
The Cytotoxic evaluation of oxygen carrier liposome in Fig. 3 embodiment 3.
Left: not have the cell in the phosphate buffered solution of oxygen carrier liposome; Right: the cell in the phosphate buffer containing oxygen carrier liposome.
Detailed description of the invention
Below by way of specific embodiment, technical scheme of the present invention is further described.Following embodiment further illustrates of the present invention, and be not limited to scope of the present invention.
Embodiment 1: the preparation of oxygen carrier liposome.
By weight, formula is as follows:
Normal saline 2000 parts;
Soybean lecithin 200 parts;
Perfluoro bromide octane 200 parts;
Dichloromethane 1000 parts;
Palmic acid 5 parts;
Alevaire 2 parts.
Preparation technology:
(1) count by weight, by 200 parts of soybean lecithins, 5 parts of Palmic acids, 2 parts of alevaires are dissolved in the ether of 2000 parts, ultrasonic mix homogeneously;
(2) in diethyl ether solution, add perfluoro bromide octane 200 parts, continue ultrasonic mix homogeneously;
(3) solution that step (2) obtains slowly is dropped in the normal saline of 2000 parts, agitating solution (mixing speed is 1000 revs/min), be stirred to ether volatilization completely, about 2 hours time.Namely the oxygen carrier liposome wrapping up perfluoro bromide octane can be obtained.This oxygen carrier Liposomal formulation is milky white solution, dilutes the clear solution that 10 times can obtain light white band blue-opalescent.The surface tension of said preparation is that 29mN/m liposome size is about 40
-distribution (its transmission electron microscope picture as shown in Figure 1) in the scope of 100nm, the envelop rate of perfluoro bromide octane is about 95%.
Embodiment 2: the preparation of oxygen carrier liposome.
By weight, formula is as follows:
Phosphate buffer 2000 parts;
Ovum Gallus domesticus Flavus lecithin 200 parts;
The mixture of FC-77(PFO and perfluor ring octyl ether) 200 parts;
Ether 1000 parts;
Hexadecanol 1 part;
Alevaire 1 part.
Preparation technology:
(1) count by weight, by 200 parts of soybean lecithins, 2 parts of hexadecanols, 1 part of alevaire is dissolved in the ether of 1000 parts, ultrasonic mix homogeneously;
(2) in diethyl ether solution, add the mixture of FC-77(PFO and perfluor ring octyl ether) 200 parts, continue ultrasonic mix homogeneously;
(3) solution that step (2) obtains slowly is dropped in the normal saline of 2000 parts, agitating solution (mixing speed is 1000 revs/min), be stirred to ether volatilization completely, about 2 hours time.Namely the oxygen carrier liposome wrapping up perfluoro bromide octane can be obtained.This oxygen carrier Liposomal formulation is milky white solution, dilutes the clear solution that 10 times can obtain light white band blue-opalescent.The surface tension of said preparation is that 33mN/m liposome size distributes in the scope of 50-90nm, and the envelop rate of FC-77 is about 80%.
Embodiment 3 oxygen carrier liposome toxicity assessment.
To take the logarithm the monolayer culture HEK293 epithelial cell of trophophase, with 0.25% membrane proteolytic enzyme and 0.025% disodium EDTA solution digestion single-layer culturing cell, be made into single cell suspension, with every hole 1 × 10 with the DMEM cell culture medium containing 10% hyclone
4individual cell is inoculated in 96 orifice plates, and every pore volume 100 microlitre, moves into culture plate in CO2 gas incubator, and at 37 DEG C, overnight incubation under 5% carbon dioxide and saturated humidity condition, makes cell attachment.Next day, with the fluorine carbon oxygen carrier liposome solutions (adopting the oxygen carrier liposome prepared by embodiment 2) of a series of variable concentrations of phosphate-containing solution preparation.By the culture fluid sucking-off in culture dish, add oxygen carrier liposome solutions, hatch different time for 37 DEG C, inhale and abandon supernatant.Wash twice with phosphate solution, every hole adds the tetrazolium bromide solution that 100 lli are 5 mg/ml, continues cultivation 4 hours.Add the sodium dodecyl sulfate solution of 100 microlitres 10%, cultivate and adopt microplate reader to test after 4 hours, absorbing wavelength adopts 570nm.Result as shown in Figure 2.Can find out, cultivate 4 hours under liposome existence condition, there is not significantly active reduction in cell, shows that this oxygen carrier liposome does not have toxicity substantially to epithelial cell.
Embodiment 4 oxygen carrier liposome toxicity assessment.
To take the logarithm the monolayer culture HEK293 epithelial cell of trophophase, with 0.25% membrane proteolytic enzyme and 0.025% disodium EDTA solution digestion single-layer culturing cell, be made into single cell suspension, with every hole 1 × 10 with the DMEM cell culture medium containing 10% hyclone
5individual cell is inoculated in Tissue Culture Dish, and every pore volume 1 milliliter, moves into culture dish in CO2 gas incubator, and at 37 DEG C, overnight incubation under 5% carbon dioxide and saturated humidity condition, makes cell attachment.Next day, the phosphate solution (adopting the oxygen carrier liposome prepared by embodiment 2) of preparation fluorine carbon oxygen carrier liposome.In content of phospholipid, the concentration of liposome solutions is 20 mg/ml.By the culture fluid sucking-off in culture dish, add oxygen carrier liposome solutions, hatch 4 hours for 37 DEG C, inhale and abandon supernatant.Wash 3 times with phosphate solution, observe under being placed in fluorescence microscope light field, and take pictures.Picture as shown in Figure 3.As can be seen from the figure, adopt the aquicultural cell containing oxygen carrier liposome, the phosphate solution cultured cells with not containing oxygen carrier liposome, quantity and pattern does not have notable difference, shows that this oxygen carrier liposome does not have toxicity substantially to epithelial cell.
Claims (2)
1. a preparation method for oxygen carrier liposome, is characterized in that,
(1) count by weight, by 200 parts of soybean lecithins, 5 parts of Palmic acids, 2 parts of alevaires are dissolved in the ether of 2000 parts, ultrasonic mix homogeneously;
(2) in diethyl ether solution, add perfluoro bromide octane 200 parts, continue ultrasonic mix homogeneously;
(3) solution that step (2) obtains slowly is dropped in the normal saline of 2000 parts, agitating solution, its mixing speed is 1000 revs/min, be stirred to ether volatilization completely, about 2 hours time, namely the oxygen carrier liposome wrapping up perfluoro bromide octane can be obtained, this oxygen carrier Liposomal formulation is milky white solution, dilute the clear solution that 10 times can obtain light white band blue-opalescent, the surface tension of said preparation is 29mN/m, liposome size distributes in the scope of 40-100nm, and the envelop rate of perfluoro bromide octane is about 95%.
2. the oxygen carrier liposome prepared of the preparation method of an oxygen carrier liposome as claimed in claim 1.
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| CN105362220A (en) * | 2014-08-08 | 2016-03-02 | 深圳君圣泰生物技术有限公司 | Preparation, preparation method and uses thereof |
| CN105435227A (en) * | 2014-08-08 | 2016-03-30 | 深圳君圣泰生物技术有限公司 | Liquid preparation composition and preparation method and use and solid preparation thereof |
| WO2016019627A1 (en) * | 2014-08-08 | 2016-02-11 | Shenzhen Hightide Biopharmaceutical, Ltd. | Liquid formulation compositions, medicament delivery devices, and methods of preparation and use thereof |
| CN105434346A (en) * | 2014-08-08 | 2016-03-30 | 深圳君圣泰生物技术有限公司 | Preparation composition and preparation method and use thereof |
| CN105435344A (en) * | 2015-12-15 | 2016-03-30 | 上海纳米技术及应用国家工程研究中心有限公司 | Portable efficient oxygen supply spraying device for lung |
| CN106361701B (en) * | 2016-08-31 | 2019-08-27 | 上海交通大学 | A kind of lipidosome drug carrier and its preparation method and application |
| CN109730965A (en) * | 2019-01-28 | 2019-05-10 | 玉林师范学院 | A kind of preparation method producing oxygen nano liposomes |
| CN113801344B (en) * | 2021-09-15 | 2022-08-12 | 复旦大学 | Oxygen-loaded fluorine-containing temperature-sensitive hydrogel and preparation method and application thereof |
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Patent Citations (4)
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| CN1185103A (en) * | 1995-05-08 | 1998-06-17 | 海马金/全氟碳公司 | Homogeneous water-in-perfluorochemical stable liquid dispersion for administration of a drug to the lung of an animal |
| CN1286081A (en) * | 2000-08-29 | 2001-03-07 | 上海维来现代科技发展有限公司 | High-concentration super-fine perfluorocarbon emulsion for injection and its preparing process |
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