CN100570333C - A kind of method of measuring drug-loading rate of bacteria nano magnetosomes - Google Patents
A kind of method of measuring drug-loading rate of bacteria nano magnetosomes Download PDFInfo
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- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 44
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Abstract
The present invention relates to a kind of method of measuring drug-loading rate of bacteria nano magnetosomes, adopt ultraviolet spectrum or fluorescence spectrum to determine the uv-absorption maximum wavelength or the maximum emission wavelength λ of medicine to be measured
MaxThe drug solution of measuring variable concentrations is at this λ
MaxThe ultraviolet absorptivity value or the fluorescent emission intensity value at place are made its standard working curve; With the medicine and the bacillus nanometer magnetosome hybrid reaction of concentration known, isolate reacted magnetic corpusculum, get supernatant; In addition preparation and said medicine with concentration with bacillus nanometer magnetosome reaction equivalent drug solution, as blank reaction comparative solution; Measure supernatant and blank reaction comparative solution respectively at λ
MaxThe ultraviolet absorptivity value or the fluorescent emission intensity value at place are calculated the drug loading of bacillus nanometer magnetosome by standard working curve according to the two difference.
Description
Technical field
The present invention relates to a kind of assay method of drug-loading rate of bacteria nano magnetosomes, particularly relate to a kind of method that adopts ultraviolet spectrum or fluorescence spectrum indirect determination drug-loading rate of bacteria nano magnetosomes.
Background technology
Magnetictaxis bacteria (magnetotactic bacterium) is the peculiar microorganism of a class of U.S. biologist Bu Lake More (Blakemore) in 1975 nature existence serendipitous, it can move along the magnetic line of force, its cell contains the bacillus nanometer magnetosome (magnetosomes) that magnetic field is had susceptibility, it has played guiding in cell, and can move by means of the flagellum of self.The bacillus nanometer magnetosome size is about 35-120nm, and within the brilliant range of size of permanent single magnetic domain, principal ingredient is Fe
3O
4Or Fe
3S
4, there is the phosphatide lipid bilayer bag quilt by chimeric protein the outside, after the separation and purification in solution good dispersion, stability is high.Japan scholar Matsunaga will be a kind of new living resources during new and high technology is used in just estimating magnetotactic bacteria magnetic corpusculum 10 years in future in 1991.At fixed enzyme vector, immune detection, transgenosis, the result of study of aspects such as the separation of DNA, RNA and mark shows, the performance of bacillus nanometer magnetosome is better than the synthetic magnetic nanoparticle, the former immobilised enzymes amount big (but intelligent worker's synthesizing magnetic nano particle 100 times), antibody connection amount is big, the sensitivity of immune detection height, the DNA adsorbance is big, and application prospect is boundless.Compare with the synthetic magnetic nanoparticle, bacillus nanometer magnetosome is owing to from live bacteria, have superior Bc, and toxic and side effect is little; Its size is all at nanoscale, uniform particles, and stable crystal form has bigger specific surface area; The big and scalable of electronegativity is difficult for assembling; Because adipose membrane bag quilt is arranged, reactive group is abundant simultaneously, and various chemical modifications can be carried out in the surface, may become a kind of better nano-medicament carrier, are showing tempting application prospect aspect the target locating therapy tumour.Existing Primary Study result shows, the bacillus nanometer magnetosome epigranular, and change of size is little before and after its carrying medicament adriamycin, than the little 1-20 of medicine carrying magnetic particle of traditional synthetic doubly.Adopt biological nano magnetic corpusculum as pharmaceutical carrier, the exceeding more than 10 times of the comparable traditional synthetic magnetic particle of drug loading.
But because it is just at the early-stage to adopt bacillus nanometer magnetosome to carry out the research work of medicine carrying aspect, the method for mensuration drug-loading rate of bacteria nano magnetosomes does not have ready-made experience yet and can follow.Bibliographical information is arranged, and the drug loading for measuring synthetic nanometer magnetic particle adopts strong acid and intensive polar solvent that it is dissolved usually, measures after the extraction again.But for the bacillus nanometer magnetosome system, these strong acid and intensive polar solvent also very easily make the character of contained medicine change and decompose in dissolved destruction bacillus nanometer magnetosome film and magnetic nuclear, thereby influence the accurate mensuration of its drug loading.Therefore, how not destroying under character of medicine own and the active condition, accurately measuring the drug loading of bacillus nanometer magnetosome to medicine, is technical barrier anxious to be solved in the research work of present bacillus nanometer magnetosome medicine carrying.
Summary of the invention
The object of the present invention is to provide a kind of assay method of drug-loading rate of bacteria nano magnetosomes.Specifically, provide a kind of method that adopts ultraviolet spectrum or fluorescence spectrum indirect determination drug-loading rate of bacteria nano magnetosomes.The said bacillus nanometer magnetosome of the present invention is the nano magnetic particle that forms in the magnetotactic bacteria cell, particle diameter 35-120nm, and principal ingredient is Fe
3O
4Or Fe
3S
4, single magnetic particle has adipose membrane bag quilt.The medicine that this method relates to be can be coupled with bacillus nanometer magnetosome medicine.
The assay method of drug-loading rate of bacteria nano magnetosomes of the present invention comprises the steps:
(1) water or buffer solution are added in the medicine to be measured, make the pharmaceutical standards solution of a series of variable concentrations;
(2) be reference with pure water or corresponding buffer solution, measure said medicine standard solution ultraviolet scanning spectrum or fluorescence emission spectrum (wavelength coverage 190-900nm), determine uv-absorption maximum wavelength or maximum emission wavelength λ
MaxThe drug solution of measuring above-mentioned a series of known accurate concentration is at this λ
MaxThe ultraviolet absorptivity value or the fluorescent emission intensity value at place are made its standard working curve;
(3) drug solution with known accurate concentration mixes with bacillus nanometer magnetosome, makes them quantitatively decide volumetric reaction, and reaction is drawn to container bottom with the magnetic corpusculum in the reaction vessel with magnet after finishing outside reaction vessel, collect supernatant, and is to be measured; Again preparation a identical with above-mentioned mixed solution concentration not with the drug solution of bacillus nanometer magnetosome reaction as reacting blank comparative solution, stand-by;
(4) treat that measured reaction supernatant and the blank comparative solution of reaction are at λ in the determination step 3 respectively
MaxThe ultraviolet absorptivity value or the fluorescent emission intensity value at place;
(5) according to treating that measured reaction supernatant and the blank comparative solution of reaction are at λ
MaxThe ultraviolet absorptivity value at place or the difference of fluorescent emission intensity value by the respective standard working curve of contained medicine, calculate the drug loading of bacillus nanometer magnetosome.
Adopt the assay method of the drug-loading rate of bacteria nano magnetosomes of the invention described above, traditional ultraviolet spectrum or fluorescence spectrum are combined with the actual conditions of bacillus nanometer magnetosome medicine carrying reaction, variation indirect determination drug-loading rate of bacteria nano magnetosomes by reactant liquor Chinese traditional medicine concentration before and after the reaction, can under the condition of not destroying character of medicine own and activity, measure the drug loading of bacillus nanometer magnetosome to medicine.This method is simple to operate, and reliable results, and can guarantee the continuation of pharmaceutically active and reaction is for the mensuration of drug-loading rate of bacteria nano magnetosomes provides novel, reliable detection technological means.
Description of drawings
Fig. 1 is an adriamycin standard solution ultraviolet spectrogram.
Fig. 2 is the standard working curve (λ of adriamycin aqueous solution
Max=480nm).
Fig. 3 is anti-CEA standard solution fluorescence emission spectrogram.
Fig. 4 is the standard working curve of anti-CEA phosphate buffer solution.
Embodiment
Below the reagent and the instrument that adopt among each embodiment be described as follows:
Bacillus nanometer magnetosome (BMP): got by MSR-1 magnetotactic bacteria strain culturing, particle diameter is 40-50nm, is provided by Biology College, Chinese Agriculture Univ.'s department of microbiology.
Monoclonal anti CEA: available from SIGMA company.
Anticancer chemotherapeutic agent adriamycin (ADM): purchase in Pharmaceutical Technology Co., Ltd of Beijing Yun Di Tontru.
Water: secondary deionized water.It is pure that solvent for use and chemical reagent are analysis.
Instrument: day island proper Tianjin UV-3100 type ultraviolet-visible spectrometer, the thick 1cm of quartz cell; KQ-100 type ultrasonic oscillator; Perkin Elmer LS50B type fluorescence spectrophotometer, the thick 1cm of quartz cell.
Embodiment 1: adopt ultraviolet spectroscopy to measure the drug loading of the contained anticancer drugs, doxorubicin of bacillus nanometer magnetosome (BMP) (ADM).
(1) standard solution of preparation adriamycin: accurately take by weighing the 0.02g adriamycin, be dissolved in pure water, its standard solution of preparation in the 25ml volumetric flask.The above-mentioned adriamycin standard solution that pipettes different volumes is in the 25ml volumetric flask, and thin up accurately makes the adriamycin standard solution of a series of concentration known to scale.
(2) set up the standard working curve of contained medicine: do reference with pure water, measure above-mentioned adriamycin standard solution ultraviolet scanning spectrum, obtain its ultraviolet spectrum as shown in Figure 1.Can determine adriamycin maximum absorption wavelength λ in pure water solution according to Fig. 1
MaxBe 480nm.Measure the absorbance of the adriamycin solution of above-mentioned a series of known accurate concentration, make its standard working curve as shown in Figure 2 at the 480nm place.Determine that its equation of linear regression is A=23.73*C.A is an absorbance, and C is the adriamycin concentration of standard solution, and the ie in solution absorbance is directly proportional with the content of adriamycin in the solution.
(3) with medicine and bacillus nanometer magnetosome quantitative reaction: the bacillus nanometer magnetosome that pentanedial decoration is crossed mixes with the adriamycin solution of known accurate concentration, make that the bacillus nanometer magnetosome reaction density is 0.02mg/ml in the mixed liquor, the concentration of adriamycin is 0.2mg/ml, open ultrasonic oscillator, the two reaction, reaction is drawn to reaction container bottom with magnet with the magnetic corpusculum after finishing, collect supernatant, to be measured.
(4) the reaction sky of preparation adriamycin is from comparative solution: a mixed solution with step (3) of preparation is the adriamycin medicine solution of 0.2mg/ml with the concentration of volume, as not with the blank comparative solution of the adriamycin of bacillus nanometer magnetosome reaction.
(5) ultraviolet spectroscopy: measure the ultraviolet absorptivity value of the blank comparative solution of reaction of supernatant and step (4) after the reaction of above-mentioned steps (3) gained respectively at the 480nm place.Recording the two difference in the ultraviolet absorptivity value at 480nm place is 0.254.
(6) calculating of drug loading: according to treating measured reaction supernatant (3) and the blank comparative solution of reaction (4) difference in the ultraviolet absorptivity value at 480nm place, by the determined equation of linear regression of respective standard working curve (Fig. 2) of contained medicine, the drug loading that calculates bacillus nanometer magnetosome is 536 μ gADM/mgBMP.
It should be noted that, no matter be to adopt ultraviolet spectroscopy or fluorescent spectrometry, all should prepare the blank comparative solution of reaction of adriamycin when carrying out medicine and bacillus nanometer magnetosome quantitative reaction simultaneously at every turn, fully consistent with the preparation condition that guarantees the blank comparative solution of reaction with reactant liquor, reduce experimental error.
Embodiment 2: adopt fluorescent spectrometry bacterial detection nanometer magnetic corpusculum to carry the anti-CEA content of monoclonal antibody.
(1) the anti-CEA standard solution of preparation: add phosphoric acid buffer solution, prepare the anti-CEA standard solution of a series of known accurate concentration.
(2) set up the standard working curve of anti-CEA: measure above-mentioned anti-CEA standard solution fluorescence emission spectrum, obtain its fluorescence emission spectrum as shown in Figure 3.Determine its maximum emission wavelength λ
MaxBe 336nm.Measure the fluorescent emission intensity value of the anti-CEA solution of above-mentioned a series of known accurate concentration, make its standard working curve as shown in Figure 4 at the 336nm place.Determine that its regression equation is I=8.047*10
3* C.I is an absorbance, and C is anti-CEA concentration of standard solution, and the ie in solution absorbance is directly proportional with the content of anti-CEA in the solution.
(3) will resist CEA and bacillus nanometer magnetosome quantitative reaction: the bacillus nanometer magnetosome that pentanedial decoration is crossed mixes with the anti-solution of the CEA of known accurate concentration, make that the bacillus nanometer magnetosome reaction density is 0.02mg/ml in the mixed liquor, the concentration of anti-CEA is 0.14mg/ml, open ultrasonic oscillator, the two reaction, reaction is drawn to reaction container bottom with magnet with the magnetic corpusculum after finishing, collect supernatant, to be measured.
(4) the blank comparative solution of reaction of the anti-CEA of preparation: a mixed solution with step (3) of preparation is the anti-CEA drug solution of 0.14mg/ml with the concentration of volume, as not with the blank comparative solution of the anti-CEA of bacillus nanometer magnetosome reaction.
(5) spectroscopic assay: the blank comparative solution of reaction that difference determination step (3) gained reaction supernatant and step (4) are prepared is in the fluorescent emission intensity value at 336nm place, and recording the two difference in the fluorescent emission intensity at 336nm place is 724.
(6) calculating of drug loading: according to treating measured reaction supernatant (3) and the blank comparative solution of reaction (4) difference in the fluorescent emission intensity value at 336nm place, respective standard working curve by contained medicine) determined equation of linear regression, the amount that calculates the anti-CEA of connection of bacillus nanometer magnetosome are the anti-CEA/mgBMP of 4.5mg.
Embodiment 3: the precision experiment
With the fluorescent spectrometry is example, as stated above same batch anti-CEA solution and pentanedial decoration bacillus nanometer magnetosome carried out parallel laboratory test simultaneously with same operating conditions, fluorescent emission intensity value according to each experiment, calculate the amount of the anti-CEA of connection of bacillus nanometer magnetosome, the result is as shown in table 1 below.This result shows that detection method of the present invention can reach desirable precision requirement.
Table 1 precision experimental result
Table 1 shows, through six parallel testings, records the drug loading error with a collection of raw material in allowed band.
Claims (1)
1. method of measuring drug-loading rate of bacteria nano magnetosomes is characterized in that comprising following step:
(1), take by weighing a certain amount of medicine to be measured, be dissolved in pure water or damping fluid, in the 25ml volumetric flask, be mixed with pharmaceutical standards solution; This pharmaceutical standards solution of getting different volumes moves to respectively in each 25ml volumetric flask, and each volumetric flask thin up respectively makes this pharmaceutical standards solution of a series of variable concentrations to scale;
(2) be reference with pure water or corresponding buffer solution, measure ultraviolet scanning spectrum or the fluorescence emission spectrum of said medicine standard solution in the 190-900nm wavelength coverage, determine uv-absorption maximum wavelength or maximum emission wavelength λ
MaxThe pharmaceutical standards solution of measuring above-mentioned a series of variable concentrations is at this λ
MaxThe ultraviolet absorptivity value or the fluorescent emission intensity value at place are made its standard working curve;
(3) drug solution with known accurate concentration mixes with bacillus nanometer magnetosome, make drug solution and bacillus nanometer magnetosome quantitative reaction in the mixed liquor, reaction is drawn to container bottom with the magnetic corpusculum in the reaction vessel with magnet after finishing outside reaction vessel, collect supernatant, to be measured; Again preparation a with above-mentioned mixed liquid concentration with not with the drug solution of bacillus nanometer magnetosome reaction as the blank comparative solution of reaction, stand-by;
(4) treat that measured reaction supernatant and the blank comparative solution of reaction are at λ in the determination step 3 respectively
MaxThe ultraviolet absorptivity value or the fluorescent emission intensity value at place;
(5) according to treating that measured reaction supernatant and the blank comparative solution of reaction are at λ
MaxThe ultraviolet absorptivity value at place or the difference of fluorescent emission intensity value by the respective standard working curve of contained medicine, calculate the drug loading of bacillus nanometer magnetosome.
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| CN101666032B (en) * | 2009-08-24 | 2010-12-01 | 中华人民共和国苏州出入境检验检疫局 | Quantitative detection method of nanometer metal oxide substrate (MOX) on textile |
| CN104048739B (en) * | 2013-03-14 | 2017-04-19 | 飞旸生医有限公司 | Medicament dosage monitoring method and medicament dosage monitoring device |
| CN103776808A (en) * | 2014-01-24 | 2014-05-07 | 鼎泰（湖北）生化科技设备制造有限公司 | Method for detecting hydroxyl radical concentration |
| CN113777297A (en) * | 2021-08-14 | 2021-12-10 | 浙江大学 | Fluorescence differential rapid detection method based on magnetic nanoparticles |
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| 细菌磁小体形成机制和应用研究进展. 刘朋明，高梅影.华中农业大学学报，第26卷第1期. 2007 |
| 细菌磁小体形成机制和应用研究进展. 刘朋明，高梅影.华中农业大学学报，第26卷第1期. 2007 * |
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Effective date of registration: 20160420 Address after: 210000 No. 213, Guangzhou Road, Gulou District, Jiangsu, Nanjing Patentee after: Nanjing Rui Rui Jie Biochemical Technology Co., Ltd. Address before: 210093 Hankou Road, Jiangsu, China, No. 22, No. Patentee before: Nanjing University |