CN105237658B - A kind of fluorescent decoration cyclodextrin and its preparation method and application - Google Patents
A kind of fluorescent decoration cyclodextrin and its preparation method and application Download PDFInfo
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- CN105237658B CN105237658B CN201510617902.4A CN201510617902A CN105237658B CN 105237658 B CN105237658 B CN 105237658B CN 201510617902 A CN201510617902 A CN 201510617902A CN 105237658 B CN105237658 B CN 105237658B
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Abstract
It is low for cyclodextrin content assay method sensitivity in the prior art and the technical issues of cellular uptake can not be evaluated, present invention fluorescence probe fluorescein isothiocynate (FITC) modification cyclodextrin, FITC：Cyclodextrin=0.1~20:1 (molecule molar ratio), preferably 0.5~10:1, most preferably 1:1；Cyclodextrin is preferably hydroxypropyl beta cyclodextrin.Compared with prior art, FITC modification cyclodextrins provided by the invention do not influence the effect of cyclodextrin encapsulated drug, and the content of FITC fluorescence spectrophotometries detection cyclodextrin can be used, its cyclodextrin concentration lower limit detected is 27.5~110ng/mL, compared with the method that prior art effect liquid phase chromatogram differential refraction detector measures cyclodextrin (concentration limit 0.1mg/mL), sensitivity significantly improves.Therefore the present invention solves the technical barrier of cyclodextrin difficult quantitation, is investigated in pharmaceutical properties such as assay, the releases of the novel form containing cyclodextrin and the cellular uptake behavior of cyclodextrin, targeting drug delivery effect etc. have a wide range of applications.
Description
Technical field
The invention belongs to pharmaceutical fields, and in particular to the fluorescent decoration method and its application of cyclodextrin.
Background technology
Cyclodextrin (cyclodextrin) is with the cyclic oligomer of 1,4- glucosides key connections by 6~12 D-Glucose molecules
Sugar compounds, structure are hollow circle tube, are the most common carrier materials for preparing inclusion compound.Drug molecule and cyclodextrin molecular
After forming inclusion compound by Van der Waals force, the solubility and stability of drug can be improved, realizes liquid medicine such as volatile oil
Solid dosage forms covers the bad smell or taste of drug, adjusts rate of release, improves the bioavilability of drug, reduces medicine
The irritation and toxic side effect of object, therefore, cyclodextrin material is with a wide range of applications in pharmaceutical field.Common cyclodextrin
Material has beta-cyclodextrin, hydroxypropyl-β-cyclodextrin, methyl-B-cyclodextrin etc..Cyclodextrin, can be in addition to directly applying
It is used to prepare the novel forms such as inclusion liposome, inclusion compound nanoparticle.
In order to which the load drug effect fruit of Accurate Determining cyclodextrin and cyclodextrin are from the releasing effect in novel form, except measuring medicine
Beyond object content, often also need to measure the content of cyclodextrin.But cyclodextrin does not have UV absorption, this gives assay band
Very big problem is carried out.At present, the analysis method of cyclodextrin is mainly surveyed using high performance liquid chromatography-differential refraction detector
Fixed, the lower limit that this method can measure hydroxypropyl-β-cyclodextrin is 2mmol/L (being approximately equivalent to 3.1mg/mL) (Zhao Qing etc., Tianjin
University of Science and Technology's journal, 2012,27 (5)：15-17)；The lower limit that beta-cyclodextrin can be measured is 0.1mg/mL (, drug prosperous etc. in praising
Analyze magazine, 2011,31 (4)：749-751).As it can be seen that the sensitivity of this method is not high, the dense of the mg/mL orders of magnitude can only be measured
Degree, can not apply for the sample of low concentration.
Another problem that the assay problem of cyclodextrin is brought is exactly the difficulty of cellular uptake evaluation.At present to being based on
The research of cyclodextrin structure targeted nano carrier it is more (the brilliant magnitude of department, macromolecule circular, 2013, (11)：1-10), in evaluation target
Often to carry out the evaluation that cyclodextrin enters born of the same parents' efficiency during tropism, but due to the quantitative problem of cyclodextrin, conventional cell experiment
Instrument such as flow cytometer or laser co-focusing are all difficult to measure the content of cyclodextrin in the cell.
The content of the invention
It is low for existing cyclodextrin content assay method sensitivity and the technical issues of cellular uptake can not be evaluated, the present invention
It provides using fluorescence probe --- fluorescein isothiocynate (fluorescein isothiocyanate, FITC) modification cyclodextrin
Method, by after fluorescent decoration apply fluorescence spectrophotometry or high performance liquid chromatography-fluorescence device analysis can be notable
The sensitivity that cyclodextrin content measures is improved, so as to be conducive to study the load drug effect fruit containing cyclodextrin dosage form and release behavior,
After FITC modification cyclodextrins, ring can also be easily studied by conventional instruments such as flow cytometer and laser confocal microscopes
The cellular uptake behavior of dextrin, so as to provide evaluation method for the dosage form optimization containing cyclodextrin.
The present invention provides a kind of preparation method of fluorescent decoration cyclodextrin as a result, includes the following steps, is required to be protected from light behaviour
Make：
1) FITC is dissolved in dimethyl sulfoxide (DMSO) and solution is made；
2) according to the molecule molar ratio 0.1~20 of FITC and cyclodextrin:1 weighs cyclodextrin or derivatives thereof, and is dissolved in water
In concentration is made as 10~500mg/mL cyclodextrin solutions, the volume of solution is 9~11 times of FITC solution；
3) FITC solution is added in cyclodextrin solution, 12~36h of magnetic agitation；
4) absolute ethyl alcohol of 5~10 times of volumes is added in, rotary evaporation removes solvent, and product is dissolved with pure water；
5) reaction mixture is transferred in the bag filter that molecular weight is 1500~2000D, it is saturating with 100 times of volume pure water room temperatures
Analysis 6~10 times, every time 8~12h, to remove free FITC；
6) cyclodextrin powder that liquid freezing drying is modified to get FITC in bag filter.
Described cyclodextrin or derivatives thereof is selected from alpha-cyclodextrin, Hydroxyproply-α-cyclodextrin, beta-cyclodextrin, hydroxy propyl-Beta-ring
One in dextrin, gamma-cyclodextrin, hydropropyl-y-cyclodextrin, 2,6- dimethyl-β-cyclodextrins or malt sugar group-beta-cyclodextrin
Kind.
As a preferred embodiment of the present invention, the molecule molar ratio of above-mentioned FITC and cyclodextrin is 0.5~10:1.
As a kind of more preferable scheme of the present invention, the molecule molar ratio of above-mentioned FITC and cyclodextrin is 1:1.As this hair
Bright is further improved, and above-mentioned cyclodextrin or derivatives thereof is preferably hydroxypropyl-β-cyclodextrin.Its concentration is preferably 100mg/
ML, while FITC concentration is 30mg/mL.
As the further improvement of the present invention, magnetic agitation temperature described in step (3) is 25 DEG C；Step (3) always passes through
Between lasting, i.e. the reaction time is for 24 hours.
The present invention provides the fluorescent decoration cyclodextrin of the preparation method preparation gained of above-mentioned fluorescent decoration cyclodextrin simultaneously.With
And the pharmaceutical composition containing the fluorescent decoration cyclodextrin and pharmaceutically acceptable excipient.
Advantageous effect
Compared with prior art, the FITC modification cyclodextrins prepared by the present invention have no effect on the inclusion effect of cyclodextrin,
And after modifying, due to the high sensitivity of FITC fluoroscopic examinations so that compared with unmodified cyclodextrin, by measuring fluorescence intensity
The sensitivity of detection cyclodextrin content greatly improves.The lower limit that FITC fluorescence spectrophotometries can measure is 0.11ng/mL, and
The content containing FITC is about 0.4%~1% in cyclodextrin obtained, you can the lower limit with the concentration of the cyclodextrin of measure is about
27.5~110ng/mL, compared with measuring the method for cyclodextrin using effect liquid phase chromatogram-differential refraction detector at present (under concentration
Limit can reach 0.1mg/mL), sensitivity improves 909~2636 times, so as to easily measure containing the new agent of cyclodextrin
The pharmaceutical properties such as content, the release of type.
In addition, after cyclodextrin fluorescent decoration, the cellular uptake behavior of cyclodextrin also can be easily detected, it can be with
Easily evaluate the targeting drug delivery effect of cyclodextrin.
Description of the drawings
Fig. 1 infared spectrums are verified：A in figure:HP- β-CD (hydroxypropyl-β-cyclodextrin) Dark grey, B:FITC (isothiocyanic acids
Fluorescein) in grey, C:FITC-HP- β-CD (hydroxypropyl-β-cyclodextrin of fluorescein isothiocynate modification) are light grey.
Fig. 2 cyclodextrin is from the release research in liposome：FITC-HP- β-CD groups are free cyclodextrin from bag filter
Release, to verify that it can be quickly through bag filter.
The cellular uptake research (flow cytometer measurement result) of Fig. 3 cyclodextrin：P-C-L is cyclodextrin liposome, P generations
Table surface is modified using PEG, and C represents cyclodextrin, and L represents liposome.
The cellular uptake research (laser co-focusing photo) of Fig. 4 cyclodextrin：P-C-L-s is cyclodextrin liposome, and P is represented
Surface is modified using PEG, and C represents cyclodextrin, and L represents liposome, and behalf phosphatide composition is SPC, that is, soybean lecithin.
Specific embodiment
It further illustrates the present invention with reference to the accompanying drawings and examples, which part preparation condition is only as typical feelings
The explanation of condition, is not limited to the present invention.
The preparation of 1 FITC of embodiment modification hydroxypropyl-β-cyclodextrins (FITC-HP- β-CD) and assay
Precision weighs FITC30mg and is dissolved in DMSO1mL, is added into 10mL1%HP- β-CD solution, 25 DEG C of magnetic agitations
After for 24 hours, absolute ethyl alcohol 100mL is added in, revolving removal solvent is settled to 10mL with pure water, solution is transferred to 1500~
In the bag filter of 2000Da, 25 DEG C of dialysis 72h (dialyzate is the pure water of 1000ml, and a dialyzate is replaced per 8h) are swum with removing
From FITC, be freeze-dried and FITC-HP- β-CD be made, all operationss need to be protected from light.It is measured, surveyed with sepectrophotofluorometer
Fixed condition is excitation wavelength 495nm, launch wavelength 525nm, and the standard curve of fluorescence spectrometry FITC is：Y=208.1X-6.7
(R2=0.999) range of linearity：0.11~2.60ng/mL, it is 0.96% to measure FITC contents in product.
The preparation of 2 FITC of embodiment modification beta-cyclodextrins (FITC- β-CD) and assay
Precision weighs FITC100mg and is dissolved in DMSO1mL, is added into 10mL1.5% β-CD solution, 25 DEG C of magnetic agitations
After 36h, absolute ethyl alcohol 100mL is added in, revolving removal solvent is settled to 10mL with pure water, solution is transferred to 1500~
In the bag filter of 2000Da, 25 DEG C of dialysis 60h (dialyzate is the pure water of 1000ml, and a dialyzate is replaced per 10h) are with removing
Free FITC, is freeze-dried and FITC- β-CD is made, and all operationss need to be protected from light.It is measured with sepectrophotofluorometer, method
With embodiment 1, it is 0.42% to measure FITC contents in product.
The preparation of 3 FITC of embodiment modification hydropropyl-y-cyclodextrins (FITC-HP- γ-CD) and assay
Precision weighs FITC10mg and is dissolved in DMSO1mL, is added into 10mL0.5%HP- γ-CD solution, 25 DEG C of magnetic force
After stirring 12h, absolute ethyl alcohol 100mL is added in, revolving removal solvent is settled to 10mL with pure water, solution is transferred to 1500~
In the bag filter of 2000Da, 25 DEG C of dialysis 80h (dialyzate is the pure water of 1000ml, and a dialyzate is replaced per 8h) are swum with removing
From FITC, be freeze-dried and FITC-HP- γ-CD be made, all operationss need to be protected from light.It is measured with sepectrophotofluorometer, side
For method with embodiment 1, it is 0.63% to measure FITC contents in product.
The inclusion effect of 4 FITC of embodiment modification hydroxypropyl-β-cyclodextrins (FITC-HP- β-CD)
The preparation of FITC-HP- β-CD such as embodiment 1.
Saturated water solution method prepares 9-nitrocamptothecin (9-NC) cyclodextrin inclusion compound：Precision weighs 9-NC, and to be dissolved in acetone molten
For use in liquid, FITC-HP- β-CD are dissolved in (9-NC in pure water 6m:FITC-HP- β-CD=1：120, molar ratio), 60 DEG C of water-baths,
9-NC acetone solns are instilled in FITC-HP- β-CD solution and continue stirring to waving most acetone by 500r/min magnetic agitations.It will system
The inclusion complex in solution got ready while hot cross 0.45 μm miillpore filter remove free drug, be freeze-dried to get.High performance liquid chromatography
Method measures contents of the 9-NC in inclusion compound obtained as 2.66mg/g.
9-NC is included using hydroxypropyl-β-cyclodextrin (HP- β-CD) with method, the 9-NC contents measured are 2.64mg/g.It says
Cyclodextrin encapsulated effect is not influenced after bright FITC modifications.
5 FITC of embodiment modifies the verification of hydroxypropyl-β-cyclodextrin
The preparation of FITC-HP- β-CD such as embodiment 1.
(Fig. 1) is compared in the infared spectrum of FITC, HP- β-CD and FITC-HP- β-CD, FITC is in 1593cm-1Occur
The peak of phenyl ring does not have phenyl ring in HP- β-CD structures therefore in 1593cm-1There is no peak, the FITC-HP- β-CD of the two synthesis exist
1593cm-1There is the peak of phenyl ring, therefore can prove that FITC successfully modifies HP- β-CD.
The application of 6 present invention of embodiment：FITC modifies hydroxypropyl-β-cyclodextrin from the release research in liposome
It is prepared using alcohol injection.Precision weighs soybean lecithin SPC180mg, DSPE-PEG200034mg is dissolved in anhydrous
In ethyl alcohol 5mL, precision weighs FITC-HP- β-CD 250mg and is dissolved in pure water 5mL, 60 DEG C of water-bath magnetic agitations, by ethanol solution
It being injected into aqueous solution, is stirred continuously to most ethyl alcohol is waved, taking-up is settled to 5mL, 400W Probe Ultrasonic Searching 5min with pure water,
1000rpm centrifuges 10min to get cyclodextrin liposome.
Dissolution medium for pH7.4 isotonic phosphate buffers liquid (in prescription containing 137mmol/LNaCl, 3mmol/LKCl,
8mmol/LNaHPO4、1mmol/LKH2PO4).Precision draws 1.5mLFITC-HP- β-CD and cyclodextrin liposome in bag filter
In (FITC total concentrations be 12000ng), be placed in 100mL dissolution mediums, 37 DEG C, 500r/min dialysis, in different time points essence
It is close to draw appropriate medium respectively by the high performance liquid chromatography-differential refraction detector (Zhao Qing of 1 time method of embodiment and document report
Deng, University Of Science and Technology Of Tianjin's journal, 2012,27 (5)：FITC concentration 15-17) is measured, and supplements same amount of isothermal blank release
Medium.
As a result because sensitivity problem, high performance liquid chromatography-differential refraction detector fails to measure the concentration of cyclodextrin, and
The concentration of cyclodextrin has been measured using fluorescence spectrophotometry and has calculated release, as shown in Figure 2.FITC-HP- β in Fig. 2-
CD groups are free cyclodextrin from the release in bag filter, it is known that it can be quickly through bag filter.It can thus be appreciated that pass through fluorescence mark
Note cyclodextrin can investigate the release behavior of cyclodextrin.
The application of 7 present invention of embodiment：The cellular uptake research of FITC modification hydroxypropyl-β-cyclodextrins
The preparation method such as embodiment 6 of cyclodextrin liposome (P-C-L) containing FITC-HP- β-CD.
The Proliferation of Human Ovarian Cell A2780 cells normally cultivated are taken, through digesting, counting, adjustment cell density to 1 × 106/
ML is planted into 6 orifice plates, and fluorescent decoration cyclodextrin or cyclodextrin liposome P-C-L (FITC final concentrations are separately added into after cell attachment
For 50ng/mL), 1h is incubated at 37 DEG C, sucks culture medium, is cleaned 3 times with PBS (PH7.4), with trypsin digestion cell, will be received
The cell suspension collected is blown and beaten uniformly in 500 μ LPBS, and flow cytometry analysis cell fluorescence intensity is investigated into born of the same parents' effect.Knot
Fruit is as shown in Figure 3, Figure 4, it is seen that cyclodextrin is loaded into can significantly improve cyclodextrin after liposome enter born of the same parents' effect, pass through fluorescence
Mark cyclodextrin can investigate the cellular uptake of cyclodextrin.
Advantageous effect
Compared with prior art, the FITC modification cyclodextrins prepared by the present invention have no effect on the inclusion effect of cyclodextrin,
And after modifying, due to the high sensitivity of FITC fluoroscopic examinations so that compared with unmodified cyclodextrin, by measuring fluorescence intensity
The sensitivity of detection cyclodextrin content greatly improves.The lower limit that FITC fluorescence spectrophotometries can measure is 0.11ng/mL, and
The content containing FITC is about 0.4%~1% in cyclodextrin obtained, you can the lower limit with the concentration of the cyclodextrin of measure is about
27.5~110ng/mL, compared with measuring the method for cyclodextrin using effect liquid phase chromatogram-differential refraction detector at present (under concentration
Limit can reach 0.1mg/mL), sensitivity improves 909~2636 times, so as to easily measure containing the new agent of cyclodextrin
The pharmaceutical properties such as content, the release of type.
In addition, after cyclodextrin fluorescent decoration, the cellular uptake behavior of cyclodextrin also can be easily detected, it can be with
Easily evaluate the targeting drug delivery effect of cyclodextrin.
Description of the drawings
Fig. 1 infared spectrums are verified：A in figure:HP- β-CD (hydroxypropyl-β-cyclodextrin) Dark grey, B:FITC (isothiocyanic acids
Fluorescein) in grey, C:FITC-HP- β-CD (hydroxypropyl-β-cyclodextrin of fluorescein isothiocynate modification) are light grey.Fig. 2 rings
Dextrin is from the release research in liposome：FITC-HP- β-CD groups are to dissociate cyclodextrin from the release in bag filter, with verification
It can be quickly through bag filter.
The cellular uptake research (flow cytometer measurement result) of Fig. 3 cyclodextrin：P-C-L is cyclodextrin liposome, P generations
Table surface is modified using PEG, and C represents cyclodextrin, and L represents liposome.
The cellular uptake research (laser co-focusing photo) of Fig. 4 cyclodextrin：P-C-L-s is cyclodextrin liposome, and P is represented
Surface is modified using PEG, and C represents cyclodextrin, and L represents liposome, and behalf phosphatide composition is SPC, that is, soybean lecithin.
Specific embodiment
It further illustrates the present invention with reference to the accompanying drawings and examples, which part preparation condition is only as typical feelings
The explanation of condition, is not limited to the present invention.
The preparation of embodiment 1FITC modification hydroxypropyl-β-cyclodextrins (FITC-HP- β-CD) and assay
Precision weighs FITC30mg and is dissolved in DMSO1mL, is added into 10mL1%HP- β-CD solution, 25 DEG C of magnetic agitations
After for 24 hours, absolute ethyl alcohol 100mL is added in, revolving removal solvent is settled to 10mL with pure water, solution is transferred to 1500~
In the bag filter of 2000Da, 25 DEG C of dialysis 72h (dialyzate is the pure water of 1000ml, and a dialyzate is replaced per 8h) are swum with removing
From FITC, be freeze-dried and FITC-HP- β-CD be made, all operationss need to be protected from light.It is measured, surveyed with sepectrophotofluorometer
Fixed condition is excitation wavelength 495nm, launch wavelength 525nm, and the standard curve of fluorescence spectrometry FITC is：Y=208.1X-6.7
(R2=0.999) range of linearity：0.11~2.60ng/mL, it is 0.96% to measure FITC contents in product.
The preparation of 2 FITC of embodiment modification beta-cyclodextrins (FITC- β-CD) and assay
Precision weighs FITC100mg and is dissolved in DMSO1mL, is added into 10mL1.5% β-CD solution, 25 DEG C of magnetic agitations
After 36h, absolute ethyl alcohol 100mL is added in, revolving removal solvent is settled to 10mL with pure water, solution is transferred to 1500~
In the bag filter of 2000Da, 25 DEG C of dialysis 60h (dialyzate is the pure water of 1000ml, and a dialyzate is replaced per 10h) are with removing
Free FITC, is freeze-dried and FITC- β-CD is made, and all operationss need to be protected from light.It is measured with sepectrophotofluorometer, method
With embodiment 1, it is 0.42% to measure FITC contents in product.
The preparation of 3 FITC of embodiment modification hydropropyl-y-cyclodextrins (FITC-HP- γ-CD) and assay
Precision weighs FITC10mg and is dissolved in DMSO1mL, is added into 10mL0.5%HP- γ-CD solution, 25 DEG C of magnetic force
After stirring 12h, absolute ethyl alcohol 100mL is added in, revolving removal solvent is settled to 10mL with pure water, solution is transferred to 1500~
In the bag filter of 2000Da, 25 DEG C of dialysis 80h (dialyzate is the pure water of 1000ml, and a dialyzate is replaced per 8h) are swum with removing
From FITC, be freeze-dried and FITC-HP- γ-CD be made, all operationss need to be protected from light.It is measured with sepectrophotofluorometer, side
For method with embodiment 1, it is 0.63% to measure FITC contents in product.
The inclusion effect of 4 FITC of embodiment modification hydroxypropyl-β-cyclodextrins (FITC-HP- β-CD)
The preparation of FITC-HP- β-CD such as embodiment 1.
Saturated water solution method prepares 9-nitrocamptothecin (9-NC) cyclodextrin inclusion compound：Precision weighs 9-NC, and to be dissolved in acetone molten
For use in liquid, FITC-HP- β-CD are dissolved in (9-NC in pure water 6m:FITC-HP- β-CD=1：120, molar ratio), 60 DEG C of water-baths,
9-NC acetone solns are instilled in FITC-HP- β-CD solution and continue stirring to waving most acetone by 500r/min magnetic agitations.It will system
The inclusion complex in solution got ready while hot cross 0.45 μm miillpore filter remove free drug, be freeze-dried to get.High performance liquid chromatography
Method measures contents of the 9-NC in inclusion compound obtained as 2.66mg/g.
9-NC is included using hydroxypropyl-β-cyclodextrin (HP- β-CD) with method, the 9-NC contents measured are 2.64mg/g.It says
Cyclodextrin encapsulated effect is not influenced after bright FITC modifications.
5 FITC of embodiment modifies the verification of hydroxypropyl-β-cyclodextrin
The preparation of FITC-HP- β-CD such as embodiment 1.
(Fig. 1) is compared in the infared spectrum of FITC, HP- β-CD and FITC-HP- β-CD, FITC is in 1593cm-1Occur
The peak of phenyl ring does not have phenyl ring in HP- β-CD structures therefore in 1593cm-1There is no peak, the FITC-HP- β-CD of the two synthesis exist
1593cm-1There is the peak of phenyl ring, therefore can prove that FITC successfully modifies HP- β-CD.
The application of 6 present invention of embodiment：FITC modifies hydroxypropyl-β-cyclodextrin from the release research in liposome
It is prepared using alcohol injection.Precision weighs soybean lecithin SPC180mg, DSPE-PEG200034mg is dissolved in anhydrous
In ethyl alcohol 5mL, precision weighs FITC-HP- β-CD 250mg and is dissolved in pure water 5mL, 60 DEG C of water-bath magnetic agitations, by ethanol solution
It being injected into aqueous solution, is stirred continuously to most ethyl alcohol is waved, taking-up is settled to 5mL, 400W Probe Ultrasonic Searching 5min with pure water,
1000rpm centrifuges 10min to get cyclodextrin liposome.
Dissolution medium for pH7.4 isotonic phosphate buffers liquid (in prescription containing 137mmol/LNaCl, 3mmol/LKCl,
8mmol/LNaHPO4、1mmol/LKH2PO4).Precision draws 1.5mLFITC-HP- β-CD and cyclodextrin liposome in bag filter
In (FITC total concentrations be 12000ng), be placed in 100mL dissolution mediums, 37 DEG C, 500r/min dialysis, in different time points essence
It is close to draw appropriate medium respectively by the high performance liquid chromatography-differential refraction detector (Zhao Qing of 1 time method of embodiment and document report
Deng, University Of Science and Technology Of Tianjin's journal, 2012,27 (5)：FITC concentration 15-17) is measured, and supplements same amount of isothermal blank release
Medium.
As a result because sensitivity problem, high performance liquid chromatography-differential refraction detector fails to measure the concentration of cyclodextrin, and
The concentration of cyclodextrin has been measured using fluorescence spectrophotometry and has calculated release, as shown in Figure 2.FITC-HP- β in Fig. 2-
CD groups are free cyclodextrin from the release in bag filter, it is known that it can be quickly through bag filter.It can thus be appreciated that pass through fluorescence mark
Note cyclodextrin can investigate the release behavior of cyclodextrin.
The application of 7 present invention of embodiment：The cellular uptake research of FITC modification hydroxypropyl-β-cyclodextrins
The preparation method such as embodiment 6 of cyclodextrin liposome (P-C-L) containing FITC-HP- β-CD.
The Proliferation of Human Ovarian Cell A2780 cells normally cultivated are taken, through digesting, counting, adjustment cell density to 1 × 106/
ML is planted into 6 orifice plates, and fluorescent decoration cyclodextrin or cyclodextrin liposome P-C-L (FITC final concentrations are separately added into after cell attachment
For 50ng/mL), 1h is incubated at 37 DEG C, sucks culture medium, is cleaned 3 times with PBS (PH7.4), with trypsin digestion cell, will be received
The cell suspension collected is blown and beaten uniformly in 500 μ LPBS, and flow cytometry analysis cell fluorescence intensity is investigated into born of the same parents' effect.Knot
Fruit is as shown in Figure 3, Figure 4, it is seen that cyclodextrin is loaded into can significantly improve cyclodextrin after liposome enter born of the same parents' effect, pass through fluorescence
Mark cyclodextrin can investigate the cellular uptake of cyclodextrin.
Claims (7)
1. a kind of preparation method of fluorescent decoration cyclodextrin, includes the following steps, it is required to be protected from light operation：
1) FITC is dissolved in dimethyl sulfoxide (DMSO) and solution is made；
2) according to the molecule molar ratio 0.1~20 of FITC and cyclodextrin:1 weighs hydroxypropyl-β-cyclodextrin, and soluble in water is made
Concentration is 10~500mg/mL hydroxypropyl-β-cyclodextrin solution, and the volume of solution is 9~11 times of FITC solution；
3) FITC solution is added in hydroxypropyl-β-cyclodextrin solution, 12~36h of magnetic agitation；
4) absolute ethyl alcohol of 5~10 times of volumes is added in, rotary evaporation removes solvent, and product is dissolved with pure water；
5) reaction mixture is transferred in the bag filter that molecular weight is 1500~2000D, with 100 times of volume pure water room temperatures dialysis 6
~10 times, every time 8~12h, to remove free FITC；
6) cyclodextrin powder that liquid freezing drying is modified to get FITC in bag filter.
2. the preparation method of fluorescent decoration cyclodextrin as described in claim 1, it is characterised in that：The FITC and hydroxy propyl-Beta-
The molecule molar ratio of cyclodextrin is 0.5~10:1.
3. the preparation method of fluorescent decoration cyclodextrin as claimed in claim 2, it is characterised in that：The FITC and hydroxy propyl-Beta-
The molecule molar ratio of cyclodextrin is 1:1.
4. the preparation method of fluorescent decoration cyclodextrin as claimed in claim 3, it is characterised in that：The hydroxypropyl-β-cyclodextrin
Concentration for 100mg/mL, FITC concentration is 30mg/mL.
5. the preparation method of fluorescent decoration cyclodextrin as claimed in claim 4, it is characterised in that：Magnetic force stirs described in step (3)
Temperature is mixed as 25 DEG C；Step (3) total elapsed-time standards, i.e. reaction time is for 24 hours.
6. a kind of fluorescent decoration cyclodextrin, it is characterised in that：With any one of Claims 1 to 5 fluorescent decoration cyclodextrin
Preparation method prepares gained.
7. a kind of pharmaceutical composition, it is characterised in that：Containing the fluorescent decoration cyclodextrin described in claim 6 and pharmaceutically may be used
The excipient of receiving.
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| WO1995002700A1 (en) * | 1993-07-13 | 1995-01-26 | Abbott Laboratories | Fluorescent polymer labeled conjugates and intermediates |
| DK1828772T3 (en) * | 2004-12-06 | 2010-08-23 | Biogen Idec Inc | Detection and quantification of cyclodextrins |
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