CN101231233B - Method for controlling release velocity of fluorescent probe molecule in emulsions - Google Patents
Method for controlling release velocity of fluorescent probe molecule in emulsions Download PDFInfo
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- CN101231233B CN101231233B CN2008100076611A CN200810007661A CN101231233B CN 101231233 B CN101231233 B CN 101231233B CN 2008100076611 A CN2008100076611 A CN 2008100076611A CN 200810007661 A CN200810007661 A CN 200810007661A CN 101231233 B CN101231233 B CN 101231233B
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Abstract
A method for controlling the speed of releasing the fluorescent probe molecules in the latex emulsion belongs to the material controlled release technical field. The art is as follows: a fluorescent probe molecule water solution or a polymer-fluorescent probe molecule water solution taken as the water phase is added into a mineral oil phase containing a polylol non-ionic surface promoting agent, the volume ratio of the polylol non-ionic surface promoting agent to the mineral oil ranges from 1:19 to 1:10, the solution is evenly stirred at a rotating speed of 8000 to 12000 turns per minute under 273 to 298 K for 1 to 5 minutes to get a water-in-oil latex emulsion, a 0.8 milliliters of water solution containing two to four percent of polyoxyethylene sorbitan laurate is added into to the prepared water-in-oil latex emulsion to be emulsionized for the second time for 2 to 5 minutes to obtain the water-in-oil or oil-in-water latex double emulsion. The sample is analyzed by a fluorescent energetic cell sorter and the release speed of the fluorescent probe molecules is calculated by a formula. The invention has the advantages of high speed, high efficiency, directness and accuracy.
Description
Technical field
The invention belongs to control of material release tech field, particularly a kind of method of controlling release velocity of fluorescent probe molecule in the emulsion, suitable polymers material.
Background technology
The sustained release of material is extremely important in medicine sustained release and slowly-releasing, protein and cosmetic field.But analyzing detecting method is difficult to accurately measure the release rate of material from emulsion usually.Fluorescence activity cell sorting method (FACS: the common instrument in a kind of biochemical research) be a kind of fluorescent probe molecule (comprising solute such as medicine, protein, gene etc.) new method of release rate from water-in-oil-in-water compositions (W/O/W emulsion) or O/w emulsion (O/W) of measuring fast, directly, accurately, polymkeric substance can be controlled the release rate of fluorescent probe molecule from the W/O/W emulsion, thereby reach the purpose of control of material slowly-releasing, provide theoretical and the actual foundation that instructs for studying medicament slow release and drug mechanism.
The character of fluorescence activity cell sorting method (FACS) research water-in-oil-in-water compositions water-in-oil-in-water compositions (W/O/W emulsion) is adopted in hamming pools etc., and has obtained release rate and the releasing mechanism of fluorescent probe molecule from the W/O/W emulsion.Bernath, K and hamming pool etc. utilize the fluorescence activity cell sorting method in the W/O/W emulsion, to separate and enrichment gene.The fluorescence activity cell sorting method can be studied the character of W/O/W (W/O/W) or oil-in-water (O/W) emulsion, the drop of aqueous phase carries out data acquisition and analysis for cell unit in addition, and it not only can study water-soluble solute in the character of W/O/W emulsion but also can study the character of hydrophobicity solute in the O/W emulsion.Adopt Water-In-Oil (W/O) emulsion can solubilising and protect water-soluble solute (fluorescent probe molecule, medicine, protein, gene etc.) in water in oil water nuclear, aqueous phase contains the two emulsions (W/O/W emulsion) of W/O/W of fluorescent probe molecule and solutes such as medicine, protein or gene in obtaining by the second emulsifying technology.The applied water-in-oil emulsion system of the present invention is Pan of polyol-based non-ionic surfactant department type (ester that Span-sorbitol anhydride and various fatty acid form) or tween type (Tween-polyoxyethylene groups sorbitan carboxylic esters); the Water-In-Oil system has solubilising and protective effect to water-soluble solute (fluorescent probe molecule, medicine, protein, gene etc.), and non-water-soluble medicine (taxol PTX etc.) solubilising and protection are in the oil phase of O/W emulsion.The method can study water-soluble solutes such as medicine, protein and gene in the W/O/W emulsion or sustained release and the releasing mechanism of water-insoluble cancer therapy drug in oil-in-water (O/W) emulsion.
Summary of the invention
The object of the present invention is to provide a kind of method of controlling release velocity of fluorescent probe molecule in the emulsion, polymkeric substance is the sustained release fluorescent probe molecule in water-in-oil-in-water compositions (W/O/W), be implemented in the W/O/W emulsion control solute release rate and improve the stability of emulsion, for the sustained release and the releasing mechanism research of medicine, protein (bovine serum albumin(BSA) BSA etc.), gene and other water-soluble solutes provides new method.Water-insoluble cancer therapy drug (taxol PTX; many Xi Tabin TXT; oxaliplatin L-OHP; Vinorelbine NVB etc.) and solute solubilising such as hydrophobicity fluorescent probe molecule and the oil phase of protection in oil-in-water (O/W) emulsion, by release rate and the releasing mechanism of fluorescence activity cell sorting method research solute in the O/W emulsion.
Concrete technical scheme of the present invention is as follows: earlier with fluorescent probe molecule (the bovine serum albumin(BSA) FITC-BSA of fluorescein isothiocyanate (fitc) mark, uranin etc.) aqueous solution or polymkeric substance (polyoxyethylene, sanlose, polyacrylamide etc.)-the fluorescent probe molecule aqueous solution joins as water and contains polyol-based non-ionic surfactant [first-selected this Pan's series, (volume ratio is 1: 19 to 1: 10 in the mineral oil phase of the sorbitol monooleate Span80 of 4%-5% weight, cumulative volume is 1 milliliter], at 273-298K, at the uniform velocity stir under 8000-12000 rev/min the rotating speed and obtained water-in-oil emulsion (W/O emulsion) in 1-5 minute, again 0.8 milliliter of aqueous solution that contains 2%-4%Tween20 (polyoxyethylene sorbitol acid anhydride laurate) is joined in the prepared W/O emulsion and carry out second emulsifying, 273-298K obtained the two emulsions (W/O/W emulsion) of W/O/W in emulsification 2-5 minute under 8000-12000 rev/min the rotating speed; After the water-in-oil-in-water compositions of getting 10 microlitres dilutes in 1 milliliter aqueous solution, by fluorescence activity cell sorter analytic sample, obtain the fluorescence intensity of sample, the time dependent tables of data of emulsion droplet Size Distribution is by the release rate of formula calculating fluorescent probe molecule.Described formula is: R=(Y
T=0-Y
T=t) * 100/Y
T=0(Y
T=0: time t is the fluorescence intensity of fluorescent probe molecule in the 0 o'clock pair of emulsions; Y
T=t: the fluorescence intensity of fluorescent probe molecule in two emulsions when time t is t; R is the release rate percentage of fluorescent probe molecule).
Described polyol-based non-ionic surfactant is this Pan's series of polyol-based non-ionic surfactant or polyol-based non-ionic surfactant tween series, this Pan of polyol-based non-ionic surfactant is the sorbitol monooleate (Span80) that column selection contains 4%-5% weight, polyol-based non-ionic surfactant tween series, selection contains the polyoxyethylene sorbitol acid anhydride laurate (Tween20) of 2%-4% weight.
Raw material among the present invention comprises: non-ionic surfactant (hydrophilic lipophilic balance can form water-in-oil emulsion between the 4-7 and hydrophilic lipophilic balance can form O/w emulsion between 12-17), first-selected this Pan's series non-ionic surfactants Span80[sorbitol monooleate] and the first-selected Tween20[polyoxyethylene sorbitol of tween series non-ionic surfactants acid anhydride laurate], mineral oil (or vegetable oil), fluorescent probe molecule (the bovine serum albumin(BSA) FITC-BSA of fluorescein isothiocyanate (fitc) mark, uranin etc.), polymkeric substance (polyoxyethylene, sodium carboxymethyl cellulose, polyacrylamide etc.), deionized water.
Analytical approach described in the present invention is: the thin separating method of fluorescence activity.
Solvent described in the present invention comprises deionized water or phosphate PBS buffer solution (pH value is 7.4).
This analytical approach is compared with existing method, has directly, advantage fast and accurately, can directly obtain the release rate of analyzed sample and the data of emulsion droplet Size Distribution.Adopt emulsion method, utilize aqueous phase to contain in the water-soluble polymers of fluorescent probe molecule and variety classes and concentration or the oil phase and contain hydrophobicity fluorescent probe molecule and the anticancer class medicine of hydrophobicity, prepared two emulsions of W/O/W W/O/W or O/w emulsion O/W respectively.The drop of W/O/W emulsion or O/W emulsion can be used as a cell unit becomes the object that the thin sorter of fluorescence activity is analyzed, obtain the fluorescence intensity distributed data in time of fluorescent probe molecule, and then can calculate release rate and the releasing mechanism of fluorescent probe molecule from emulsion by formula, the sustained release and the releasing mechanism that contain water soluble drug for research in the water-in-oil-in-water compositions provide new method, and for containing the sustained release of hydrophobic anticancer drug in the research O/w emulsion (O/W) and the research of releasing mechanism provides new method.
Compare with prior art, the present invention has following useful result:
1, the present invention adopt fluorescence activity cell sorting method research fluorescent probe molecule (water-soluble or hydrophobicity) from two emulsion (W/O/W) systems of W/O/W or from O/w emulsion sustained release and releasing mechanism, compare with the method for traditional indirect research emulsion property, have rapidly and efficiently, advantage directly and accurately.
2, the present invention successfully controls fluorescent probe molecule sustained release from two emulsion (W/O/W) systems of W/O/W by adding polymkeric substance, for sustained release research of solute (medicine, protein, the gene) material of etc.ing that needs protection provides new method and theoretical foundation.
3, the present invention adopts sustained release and the releasing mechanism of fluorescence activity cell sorting method research hydrophobic anticancer drug (taxol PTX etc.) in oil-in-water (O/W) emulsion, for the toxic and side effect of studying the oral of anticancer class medicine and reducing anticancer class medicine provides scientific basis.
Specific implementation method
Embodiment 1
Earlier fluorescent probe molecule (the bovine serum albumin(BSA) FITC-BSA of fluorescein isothiocyanate (fitc) mark or uranin etc.) aqueous solution is joined as water that (volume ratio is 1: 19 to 1: 10 in the mineral oil phase of the sorbitol monooleate Span80 that contains non-ionic surfactant 5%, cumulative volume is 1 milliliter), at 273K, at the uniform velocity stir under 9500 rev/mins the rotating speed and obtained water-in-oil emulsion (W/O emulsion) in 5 minutes, 0.8 milliliter of aqueous solution that contains 2.7% polyoxyethylene sorbitol acid anhydride laurate Tween20 is joined in the prepared W/O emulsion again and carry out second emulsifying, emulsification obtained the two emulsions (W/O/W emulsion) of W/O/W in 2 minutes under 8000 rev/mins the rotating speed under 273K.After the water-in-oil-in-water compositions of getting 10 microlitres dilutes in 1 milliliter aqueous solution, by fluorescence activity cell sorter analytic sample, obtain the fluorescence intensity of fluorescent probe molecule in the sample, the stable time dependent tables of data of W/O/W emulsion droplet Size Distribution and emulsion is calculated the release rate of fluorescent probe molecule from emulsion by formula.
Embodiment 2
Earlier with water-soluble polymers (sodium carboxymethyl cellulose, polyoxyethylene etc.)-fluorescence probe (the bovine serum albumin(BSA) FITC-BSA of fluorescein isothiocyanate (fitc) mark or uranin etc.) molecular water solution joins as water in the mineral oil phase that contains surfactant (5% sorbitol monooleate Span80) that (volume ratio is 1: 19 to 1: 10, cumulative volume is 1 milliliter), at 273K, at the uniform velocity stir under 9500 rev/mins the rotating speed and obtained water-in-oil emulsion (W/O emulsion) in 1-5 minute, 0.8 milliliter of aqueous solution that contains 2.7% polyoxyethylene sorbitol acid anhydride laurate Tween20 is joined in the prepared W/O emulsion again and carry out second emulsifying, emulsification obtained the two emulsions (W/O/W emulsion) of W/O/W in 2 minutes under 273K8000 rev/min the rotating speed.After the water-in-oil-in-water compositions of getting 10 microlitres dilutes in 1 milliliter aqueous solution, by fluorescence activity cell sorter analytic sample, obtain the fluorescence intensity of fluorescent probe molecule in the sample, time dependent tables of data such as W/O/W emulsion droplet Size Distribution and stability of emulsion are by the release rate of formula calculating fluorescent probe molecule.
Embodiment 3
Adopt O/w emulsion (O/W), anticancer class medicine (hydrophobic material such as taxol PTX) is dissolved in the oil phase (vegetable oil or salad oil), join in the aqueous solution that contains 3%-6% non-ionic surfactant (first-selected food-grade tween series polyoxyethylene sorbitol acid anhydride laurate Tween20), under 9500 rev/mins rotating speed, be prepared into the O/W emulsion at 273K.Change the volume ratio of water and oil phase, prepared the O/W stable emulsion that contains the variable concentrations cancer therapy drug in the oil phase respectively, by the fluorescence intensity of fluorescence activity cell sorting method analysis of fluorescence probe molecule and cancer therapy drug and the size and the stability of O/W drop, realize the sustained release of cancer therapy drug and obtain the dual purpose of cancer therapy drug releasing mechanism.
Embodiment 4
Adopt O/w emulsion (O/W), with anticancer class medicine (hydrophobic material such as taxol PTX) and fluorescence probe (thiazole orange etc.) molecular melting in oil phase (vegetable oil or salad oil), join and contain the 3%-6% non-ionic surfactant (food-grade tween series: aqueous solution first-selected food-grade tween series polyoxyethylene sorbitol acid anhydride laurate Tween20) or contain in 3-6% surfactant and polymer (poly(ether-urethane) etc. the have biocompatible polymer) aqueous solution is prepared into the O/W emulsion under 273K9500 rev/min rotating speed.Change the volume ratio of water and oil phase, prepared the O/W stable emulsion that contains the variable concentrations cancer therapy drug in the oil phase respectively, by the fluorescence intensity of fluorescence activity cell sorting method analysis of fluorescence probe molecule and cancer therapy drug and the size and the stability of O/W drop, realize the sustained release of cancer therapy drug and obtain the dual purpose of cancer therapy drug releasing mechanism.
Claims (1)
1. method of controlling release velocity of fluorescent probe molecule in the emulsion, it is characterized in that, earlier join polymkeric substance-fluorescent probe molecule aqueous solution in the mineral oil phase that contains polyol-based non-ionic surfactant as water, the volume ratio of polyol-based non-ionic surfactant and mineral oil is 1: 19～1: 10, cumulative volume is 1 milliliter, at 273-298K, at the uniform velocity stir under 8000-12000 rev/min the rotating speed and obtained water-in-oil emulsion in 1-5 minute, again 0.8 milliliter of aqueous solution that contains 2%-4% polyoxyethylene sorbitol acid anhydride laurate is joined and carry out second emulsifying in the prepared water-in-oil emulsion, 273-298K obtained the two emulsions of W/O/W in emulsification 2-5 minute under 8000-12000 rev/min the rotating speed; After the water-in-oil-in-water compositions of getting 10 microlitres dilutes in 1 milliliter aqueous solution, by fluorescence activity cell sorter analytic sample, obtain the fluorescence intensity of sample, the time dependent tables of data of emulsion droplet Size Distribution is by the release rate of formula calculating fluorescent probe molecule;
Described fluorescent probe molecule is uranin or fluorescein isothiocyanate (fitc) mark bovine serum albumin(BSA);
Described polymkeric substance is polyoxyethylene, sanlose or polyacrylamide;
Described polyol-based non-ionic surfactant is this Pan's series of polyol-based non-ionic surfactant or polyol-based non-ionic surfactant tween series, this Pan of polyol-based non-ionic surfactant is the sorbitol monooleate that column selection contains 4%-5% weight, polyol-based non-ionic surfactant tween series, selection contains the polyoxyethylene sorbitol acid anhydride lauric acid of 2%-4% weight;
The formula that calculates fluorescent probe molecule release rate from emulsion is: R=(Y
T=0-Y
T=t) * 100%/Y
T=0Wherein, Y
T=0: time t is the fluorescence intensity of 0 o'clock fluorescent probe molecule; Y
T=t: the fluorescence intensity of fluorescent probe molecule when time t is t; R is the release rate percentage of fluorescent probe molecule.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2008100076611A CN101231233B (en) | 2007-12-18 | 2008-03-04 | Method for controlling release velocity of fluorescent probe molecule in emulsions |
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| CN200710179752 | 2007-12-18 | ||
| CN2008100076611A CN101231233B (en) | 2007-12-18 | 2008-03-04 | Method for controlling release velocity of fluorescent probe molecule in emulsions |
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| CN101231233B true CN101231233B (en) | 2010-12-08 |
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| CN102419340B (en) * | 2011-08-25 | 2014-06-25 | 王群威 | Standard substance for detecting impurity elements in crude oil and petroleum products and detection method thereof |
| EP3736572A1 (en) * | 2016-04-05 | 2020-11-11 | Université de Strasbourg | Intra-droplet surface engineering to capture a molecular target |
| CN112341998A (en) * | 2020-11-16 | 2021-02-09 | 成都理工大学 | Plugging material and preparation method thereof |
| CN113846141A (en) * | 2021-07-26 | 2021-12-28 | 浙江工商大学 | Rapid physiological toxicity detection method based on emulsion interface energy induced release and application |
| CN114306642A (en) * | 2021-11-23 | 2022-04-12 | 安徽中医药大学 | Three-phase double-probe fluorescent nano-emulsion tracing system and preparation and imaging methods |
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