CN104383555B - Folic acid-cyclodextrin conjugate, drug delivery vehicle, Preparation method and use - Google Patents
Folic acid-cyclodextrin conjugate, drug delivery vehicle, Preparation method and use Download PDFInfo
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Abstract
The invention belongs to field of medicine and chemical technology, are related to a kind of folic acid cyclodextrin conjugate, folic acid cyclodextrin fullerene drug delivery vehicle, Preparation method and use.Specifically, the folic acid cyclodextrin conjugate, is connected with the hydroxyl of cyclodextrin by the carboxyl of folic acid and is formed, and specifically can connect to be formed with 6 hydroxyls of γ cyclodextrin by the γ carboxyls of folic acid.The invention further relates to conjugate embedding fullerene C60The drug delivery vehicle formed.Moreover, it relates to folic acid cyclodextrin fullerene platinum medicine composition, Preparation method and use.The folic acid cyclodextrin fullerene platinum medicine composition of the present invention has height targeting to tumour cell, can be used in inhibiting tumor cell proliferation.
Description
Technical field
The invention belongs to field of medicine and chemical technology, are related to a kind of folic acid-cyclodextrin conjugate, conjugate embedding fullerene is formed
Drug delivery vehicle, pharmaceutical composition, Preparation method and use.
Background technology
Active constituents of medicine often due to can not effectively reach lesions position or it is excessive act on normal cell and tissue, and
It can not realize therapeutic purposes or generate toxic side effect.Such as have platinum medicine than more typical, it is widely used in oophoroma, small thin
Born of the same parents' lung cancer, carcinoma of testis, G. cephalantha, cervix cancer, non-small cell lung cancer, carcinoma of urinary bladder, mesothelioma of pleura, melanoma and son
The chemotherapeutic treatment of endometrial carcinoma.But the targeting of platinum medicine is poor, general drug dose is unable to reach therapeutic effect, crosses multi-agent
Amount can cause drug largely to act on normal cell and tissue again, cause human body bone marrow suppression work, gastrointestinal reaction, kidney after chemotherapy
The adverse reactions such as toxicity, neurotoxicity, thus limit the use of platinum-based chemotherapy drug.
To mitigate side effect caused by active constituents of medicine targeting difference, drug delivery vehicle comes into being, and acts on
Active ingredient is delivered in blood or histocyte to treat disease by mainly medicament-carried active ingredient.For example, in cancer
Chemotherapeutic treatment in, chemotherapeutics is delivered in cancer cell by delivery vector, makes active constituents of medicine and DNA in cancer cell mutual
Effect generates inhibitory action to tumour.Currently used platinum-based chemotherapy drug delivery vehicle include liposome, micella, nanocapsule,
Polymer-platinum conjugate and carbon nanotubes etc..
Strong and with slow controlled-release effect the drug delivery vehicle it is still necessary to a kind of new targeting at present.
The content of the invention
The present inventor has createed a kind of new folic acid-cyclodextrin conjugate, and has utilized after many experiments
It has prepared the strong drug delivery vehicle and drug of targeting.
One aspect of the present invention provides a kind of folic acid-cyclodextrin conjugate, is connected by the carboxyl of folic acid and the hydroxyl of cyclodextrin
It connects to be formed.
In an embodiment of the invention, the cyclodextrin is γ cyclodextrin.
In an embodiment of the invention, the molar ratio of folic acid and γ cyclodextrin is 8 in folic acid-cyclodextrin conjugate:
1。
In an embodiment of the invention, the folic acid-γ cyclodextrin conjugate is pasted by the γ carboxyls and γ rings of folic acid
6 hydroxyls of essence connect to be formed, structure such as following formula, wherein, 8 units of composition γ cyclodextrin are identical, 6 hydroxyls of each unit
Base is connected with the γ carboxyls of a folate molecule,
Further aspect of the present invention provides a kind of drug delivery vehicle, it includes the folic acid-cyclodextrin conjugate and
Fullerene；Specifically, the drug delivery vehicle is formed by the folic acid-cyclodextrin conjugate embedding fullerene.
In an embodiment of the invention, the fullerene is C60。
In an embodiment of the invention, the molar ratio of the folic acid-cyclodextrin conjugate and fullerene is 1:1.
Another aspect of the present invention provides a kind of pharmaceutical composition (drug delivery system), is carried including the drug delivery
Body and can be by the active constituents of medicine of fullerene adsorption.
In an embodiment of the invention, the weight ratio of the drug delivery vehicle and active constituents of medicine is (1:1)-
(10:1) it is preferably, 5:1.
In an embodiment of the invention, the active constituents of medicine is platinum medicine active ingredient；Further, institute
Platinum medicine active ingredient is stated as platinum medicine and/or platinum class prodrug；It is preferred that the platinum medicine includes Pt (NH3)2Cl2It is (suitable
Platinum, relative molecular mass 300.05), C6H12N2O4Pt (carboplatin, relative molecular mass 371.3), C8H14N2O4Pt (oxaliplatin,
Relative molecular mass 397.29), C2H8N2O3Pt (Nedaplatin, relative molecular mass 303.18), C9H18N2O3Pt (Lobaplatin, relatively
Molecular mass 397.33) and C10H22Cl2N2O4One or more in Pt (satraplatin, relative molecular mass 500.28)；It is preferred that
The platinum class prodrug includes glucuronic acid-platinum and/or thio-platinum, structural formula are as follows：
The present invention also provides the preparation methods of the folic acid-cyclodextrin conjugate, comprise the following steps：(1) by folic acid,
Cyclodextrin is protected from light with solvent and mixes, with 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and N- maloyls
Reaction under the conditions of imines is protected from light for activator is until terminate；Preferably, step is further included：(2) product in step (1) is carried out
Dialysis, drying.
In an embodiment of the invention, chosen from the followings 1) -5) any one or multinomial in item：
1) cyclodextrin is γ cyclodextrin；
2) the γ cyclodextrin, folic acid, 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and N- hydroxyl fourths
The molar ratio of imidodicarbonic diamide is 1:8:(2-10):(2-10) is preferably 1:8:4:4；
3) solvent is dimethyl sulfoxide (DMSO), sodium hydrate aqueous solution or aqueous sodium carbonate；Preferably dimethyl sulfoxide (DMSO)；
4) dialysis is carried out successively with phosphate buffer and second distillation water as medium；
5) drying is freeze-drying.
The present invention gives the preparation method of drug delivery vehicle：
Method one, by folic acid-cyclodextrin conjugate and fullerene C60Stirring is protected from light in methyl alcohol for 24 hours, with 16000r/min
Speed centrifuges 5min, collects sediment, is cleaned 3-5 times with methanol, and sediment 20mL distilled waters are dissolved in nitrogen drying, then
2min is centrifuged with 5000r/min, removes remaining C60Product is finally freeze-dried by fragment.
Method two, by folic acid-cyclodextrin conjugate and fullerene C60Grinding 3h is protected from light in methyl alcohol, collects grinding product,
Nitrogen dries up, and sediment 20mL distilled waters are dissolved, then centrifuges 2min with 5000r/min, removes remaining C60Fragment, 4 DEG C
It is kept in dark place.
Specifically, the conjugate is folic acid-γ cyclodextrin conjugates；Specifically, the conjugate and fullerene C60's
Molar ratio is (1:1)-(5:1) it is preferably, 1.5:1.
Another aspect of the invention provides the preparation method of described pharmaceutical composition, comprises the following steps：By the leaf
Acid-cyclodextrin conjugate with pre- adsorbent is protected from light and mixes in methyl alcohol, through separating, cleaning, drying, obtains pharmaceutical composition；Its
In, the pre- adsorbent is made as follows：Fullerene, active constituents of medicine are protected from light with solvent and mixed, through dialysing, doing
It is dry to form pre- adsorbent；Specifically, the solvent is 5% (w/w) glucose injection or 0.9% (w/w) sodium chloride injection.
In an embodiment of the invention, any one or multinomial in the item of (1) chosen from the followings-(7)：
(1) charged material weight of active constituents of medicine and fullerene ratio is (1:10)–(1:1) it is preferably, 1:4；
(2) the charged material weight ratio of folic acid-cyclodextrin conjugate and pre- adsorbent is (1:1)-(5:1) it is preferably, 1.5:1；
(3) fullerene is C60；
(4) mixing independently is stirring or grinding；
(5) it is described to be separated into centrifugation；
(6) cleaning is using methanol or redistilled water；
(7) drying is freeze-drying or nitrogen drying.
The present invention gives the folic acid-cyclodextrin conjugate, the drug delivery vehicle or described pharmaceutical composition
Purposes in antitumor or anticancer medicine is prepared；Specifically, the tumour is the tumour of the high expression of folacin receptor, the leaf
The high expression of acid acceptor refers to that folacin receptor is significantly higher than normal tissue cell in the expression quantity of tumor cell surface；Specifically, institute
It is the medicine for inhibiting tumor cell proliferation and/or inhibiting the increase of tumour sphere volume and/or tumour ball being promoted to disintegrate to state antitumor drug
Object；Specifically, the tumour for lymph cancer, cervical carcinoma, breast cancer, oral cavity epidermal carcinoma, stomach cancer, lung cancer, nasopharyngeal carcinoma, cancer of the esophagus,
In carcinoma of urinary bladder, carcinoma of testis, oophoroma, prostate cancer, osteosarcoma, chronic myelocytic leukemia cancer, melanoma and head and neck cancer
It is at least one；Specifically, the tumour cell be lymphocytic cancer cell, cervical cancer cell, breast cancer cell, oral cavity epidermal carcinoma it is thin
Born of the same parents, stomach cancer cell, lung carcinoma cell, nasopharyngeal carcinoma cell, esophageal cancer cell, transitional cell bladder carcinoma cell line, testicular cancer cell, ovarian cancer cell,
At least one of prostate gland cancer cell, osteosarcoma cell, chronic marrow original leukaemia, melanoma and head ＆ neck cancer cell；
It is preferred that the cervical cancer cell behaviour source cervical cancer cell HeLa or people source cervical cancer cell HeLa -229；It is preferred that the mammary gland
Cancer cell behaviour source MCF-7 Breast Cancer Cell；It is preferred that the oral cavity epidermis cancer cell behaviour source oral cavity epidermoid carcinoma cell KB is thin
Born of the same parents.
The embedding of the present invention, which refers to γ cyclodextrin in conjugate, has void structure, and hole can utilize intermolecular force
Some molecules (such as fullerene) are packed in the substance wherein, formed and are also referred to as super molecular compound.
Advantageous effect of the invention
(1) in folic acid of the present invention-cyclodextrin conjugate, the carboxyl of folic acid is coupled with the hydroxyl of γ cyclodextrin, is beneficial to
The folacin receptor of folic acid and tumor cell surface interacts, and improves targeting of the conjugate for tumour cell, and is coupled
Object has high-hydrophilic.
(2) in drug delivery vehicle of the present invention, fullerene can adsorb active constituents of medicine by delocalized electron, carefully
Intracellular sour environment makes the active constituents of medicine of fullerene adsorption easily release in the cell.Cyclodextrin inner hydrophobic pocket is easy
It is adapted in fullerene, while cyclodextrin makes delivery vector have good external hydrophilic.
Description of the drawings
Fig. 1：Pharmaceutical composition (drug delivery system) synthetic route schematic diagram of the present invention.
Fig. 2：The infrared absorption spectrum of γ cyclodextrin in drug materialization detection of the present invention.
Fig. 3：The infrared absorption spectrum of folic acid in drug materialization detection of the present invention.
Fig. 4：The infrared absorption spectrum of folic acid-γ cyclodextrin conjugates in drug materialization detection of the present invention.
Fig. 5：The drug release patterns of drug delivery system 1 in drug materialization detection of the present invention.
Fig. 6：The atomic force microscopy diagram of drug delivery system 1 in drug materialization detection of the present invention.
Fig. 7：Intensity-grain size curve figure of 1 dynamic light scattering of drug delivery system in drug materialization detection of the present invention.
Fig. 8：Drug delivery system 1 and carboplatin are to the inhibiting rate graph of HeLa cells in cell in vitro of the present invention evaluation and test.
Fig. 9：HeLa cells are to folic acid-γ cyclodextrin-C in cell in vitro evaluation and test of the present invention60- FITC, γ cyclodextrin-C60-
The intake graph of FITC, FITC and control group.
Figure 10：The HeLa cell tow -dimensions atom power without the processing of drug delivery system 1 is shown in cell in vitro evaluation and test of the present invention
Micro mirror figure.
Figure 11：The HeLa cell three-dimensional atomic forces without the processing of drug delivery system 1 are shown in cell in vitro evaluation and test of the present invention
Micro mirror figure.
Figure 12：Through drug delivery system 1, treated that HeLa cell tow -dimensions atom power is shown in cell in vitro of the present invention evaluation and test
Micro mirror figure.
Figure 13：Through drug delivery system 1, treated that HeLa cell three-dimensional atomic forces are shown in cell in vitro of the present invention evaluation and test
Micro mirror figure.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment specific
Condition person, the condition suggested according to normal condition or manufacturer carry out.Reagents or instruments used without specified manufacturer is
It can be with conventional products that are commercially available.
(1) medicine preparation
Preparation example 1：
(1) raw material：
Fullerene C60：Purchased from fullerene Science and Technology Ltd. of Puyang Yongxin, purity >=98%.
Folic acid：Purchased from Sigma companies, purity >=97%, Lot：081M1546V.
γ cyclodextrin：Purchased from Adamas Reagent companies, purity >=98%, Lot：P1064982.
Carboplatin：Science and Technology Ltd., purity >=98%, lot number are coupled purchased from Beijing：120330.
1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides：Purchased from Adamas Reagent companies, purity >=
97%, Lot：P1064982.
N- hydroxysuccinimides：Purchased from Sinopharm Chemical Reagent Co., Ltd., purity >=97%, lot number：
F20101019。
5% (w/w) glucose injection：Purchased from four medicine group of Shijiazhuang, lot number：110505702.
(2) preparation process：Pharmaceutical composition (drug delivery system) synthetic route is as shown in Figure 1.
A) synthesis of folic acid-γ cyclodextrin conjugate：Synthetic route is as follows.
8 molfraction folic acid and 1 molfraction γ cyclodextrin in dimethyl sulfoxide (DMSO) are protected from light stirring for 24 hours, add in 4 moles
Part 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and 4 molar part N- hydroxysuccinimides make activator into
Row reaction.It is dialysed with the bag filter that molecular cut off is 500, is first situated between with phosphate buffer (pH=7.4) work after reaction
Matter dialysis 48h, then medium dialysis is made for 24 hours with redistilled water (distilled water), it is last cold to remove unreacting material and by-product
It is lyophilized dry.
B) fullerene C60Adsorb carboplatin：
The fullerene of 4 parts by weight and the carboplatin of 1 parts by weight are protected from light stirring for 24 hours in 5% glucose injection, with retention
The bag filter that molecular weight is 500 is dialysed, and is dialysed for 24 hours by medium of 5% glucose injection, to remove carboplatin not to be adsorbed,
It is finally freeze-dried, forms pre- adsorbent.
C) folic acid-γ cyclodextrin conjugate embeds pre- adsorbent：
By the folic acid-γ cyclodextrin conjugate of 1.5 parts by weight and the pre- adsorbent of 1 parts by weight methanol (analysis is pure, >=
99.5%) be protected from light in stirring for 24 hours, with 16000r/min speed centrifuge 5min, collect sediment, with methanol (analysis it is pure, >=
99.5%) clean 3-5 times, nitrogen drying adds the stirring of 20mL distilled waters, centrifuges 2min with 5000r/min speed, removal is residual
Remaining C60Sediment is finally freeze-dried by fragment, obtains drug delivery system 1.
Preparation example 2：
By the folic acid-γ cyclodextrin conjugate of 1.5 parts by weight and the pre- adsorbent of 1 parts by weight methanol (analysis is pure, >=
99.5%) grinding 3h is protected from light in, collects grinding product, nitrogen drying adds in the stirring of 20mL distilled waters, then with 5000r/min from
Heart 2min, to remove remaining C60Fragment is kept in dark place at 4 DEG C.It is other same as Example 1, obtain drug delivery system 2.
The present invention includes but not limited to use with the carboplatin (C in above-described embodiment6H12N2O4Pt) as pharmaceutical activity into
Point, cis-platinum (Pt (NH3)2Cl2), oxaliplatin (C8H14N2O4Pt), Nedaplatin (C2H8N2O3Pt), Lobaplatin (C9H18N2O3Pt), match
Special platinum (C10H22Cl2N2O4Pt) and platinum class prodrug and other kinds of active constituents of medicine can be employed as preparing drug of the present invention.Its
In, platinum class prodrug includes but not limited to glucuronic acid-platinum and/or thio-platinum.The claimed drug of the present invention includes but unlimited
In antitumor or anticancer class drug.
(2) drug materialization detects
(1) infrared detection：
Folic acid-γ cyclodextrin the conjugate weighed respectively in the folic acid, γ cyclodextrin and preparation example 1 of 2mg carries out potassium bromide
Tabletting, using the infrared absorption spectrum of the above-mentioned each substance of Brooker determination of infrared spectroscopy, as in Figure 2-4.
Spectrogram shows：Folic acid-γ cyclodextrin conjugate is in functional group region (4000~1300cm-1) and fingerprint region (1300~
400cm-1) occur characteristic absorption peak have 3424~3330.4cm-1Hydroxyl absorption peak, 2929~2851cm-1CH2, CH stretches
Contracting vibration absorption peak, 1626.5~1575.5cm-1The vibration absorption peak of phenyl ring skeleton, 1157.6~1069cm-1C-O- in ester group
The stretching vibration peak of C.Compared with γ cyclodextrin in 3700~3000cm-1Wide absworption peak, the hydroxyl group absorption of folic acid-γ cyclodextrin
Peak narrows, and illustrates hydroxyl on γ cyclodextrin because with being consumed during carboxyl reaction in folic acid, quantity is reduced so absworption peak narrows.
There is phenyl ring, the CH of folic acid in folic acid-γ cyclodextrin conjugates2, the stretching vibration peak of CH and stretching for reaction site ester group
Contracting vibration peak illustrates that folic acid is coupled with γ cyclodextrin.
Theoretical limitation is not limited to, the active low and α carboxyls of α carboxyl reactions of folic acid are easy to thin by the high expression of folacin receptor
Born of the same parents are absorbed, and folic acid γ carboxyl reactions activity is high compared with α carboxyls, is easily coupled as graft site with other compounds.
The reactivity order of hydroxyl is 6 in γ cyclodextrin>>2>3, except the hydroxyl of 6 outer other positions is inertia.
(2) drugloading rate and drug release patterns detection：
A) compound concentration is respectively the carboplatin solution of 0.64,1.6,3.2,6.4,12.8,25.6,32,64,112 μ g/ml,
The absorbance of carboplatin in each solution is measured at wavelength 229nm using 2800 ultraviolet specrophotometers of You Nike, with mass concentration
It is that ordinate establishes linear relationship y=0.0105x+0.0082, R for abscissa, absorbance2=0.9984.
B) drug delivery system 1 in 1mg preparation examples 1 is dissolved in 5ml, 5% glucose injection, with 40kHz frequencies
Rate, which is ultrasonically treated 2h, makes carboplatin desorption in drug, is measured with 2800 ultraviolet specrophotometers of You Nike at wavelength 229nm molten
The absorbance of carboplatin in liquid calculates the mass concentration of carboplatin in solution according to mass concentration-absorbance linear relationship in a),
It is 20% (w/w) to calculate drugloading rate according to the following formula.
Carboplatin quality/drug delivery system quality × 100% in drugloading rate (%)=solution
Wherein, carboplatin quality=solution carboplatin mass concentration × liquor capacity in solution.
C) 1mg drug delivery systems 1 are dissolved in 5ml, 5% glucose injection, be packed into molecular cut off be 500 it is saturating
Analyse bag in, using the bag filter equipped with pharmaceutical liquid be put into wide-mouth bottle add in simultaneously 35mL phosphate buffers (pH=5.3) as
Extracellular fluid dialysis, wide-mouth bottle 37 DEG C with 100r/min constant temperature oscillations for a period of time, respectively 20min, 40min, 2h, 4h, 8h,
12h and 3mL extracellular fluid dialysis are taken at time point for 24 hours, while 3mL phosphate buffers (pH=5.3) are supplemented into bottle, use You Nike
2800 ultraviolet specrophotometers measure the absorbance of carboplatin in each sub-sampling solution at the wavelength 229nm, according to mass concentration-
Absorbance linear relationship calculates carboplatin mass concentration in each sampling solution.Bag filter is finally opened to discharge pharmaceutical liquid completely
Into extracellular fluid dialysis, carboplatin mass concentration in solution when calculating release completely using same procedure calculates drug and releases according to the following formula
Rate is put, and using drug release rate as ordinate, the time draws drug release patterns for abscissa, as shown in Figure 5.
Wherein,
Carboplatin mass concentration × sample volume in certain time point burst size=certain point in time sampling solution；
Carboplatin mass concentration × sample volume in solution during complete burst size=complete release.
The result shows that in phosphate-buffered medium, drug release rate is interior when initial 2 is small to be less than 45%, illustrates without prominent
Release effect.The period when 2-10 is small, drug release rate slowly raise up, and change in the range of 30%~60%, illustrate that drug is in
Slow release conditions.At 24 hours, release amount of medicine reaches more than 70%.It can be seen that the drug delivery vehicle of the present invention has
Good slow controlled-release effect.
(3) morphological feature is observed：
A) a small amount of drug delivery system 1 is dissolved with distilled water, 40kHz ultrasonic disperses 30 minutes are dripped in fresh cloud
Matrix face, is dried at room temperature for, and morphological observation is carried out using German JPK Nanowizard atomic force microscope, using tapping
Mode scans, as shown in Figure 6.
B) a small amount of drug delivery system 1 is dissolved with distilled water, 40KHz ultrasonic disperses 30 minutes, using Malvern dynamic
Light scattering apparatus measures grain size, and the results are shown in Figure 7.
The result shows that：For the drug of the present invention in monodisperse state, atomic force microscope and dynamic light scattering survey particle diameter distribution
Value is in the range of 190~200nm.Also, it is found after detecting drug 2 using same method, 1 and 2 nothing of drug delivery system
Significant difference.
(3) cell in vitro is evaluated and tested
(1) material and drug are used：
HeLa Cells, purchased from institute of materia medica of consonance medical university of the Chinese Academy of Medical Sciences.
The culture medium of HeLa Cells is folic acid deficiency type RPMI-1640 cell culture mediums, hyclone, mould
Element and streptomysin, match as folic acid deficiency type RPMI-1640 cell culture mediums：Hyclone：Penicillin：Streptomysin=1ml：
0.1ml：100U：100μg.
Negative control：5% glucose injection.
Drug delivery system 1 made from preparation example 1.
(2) to the inhibitory action of human cervical carcinoma cell HeLa：
Using the HeLa Cells of the high expression of folacin receptor as research object, using tetramethyl azo azoles salt (3- (4,
5-dimcthylthioazol-2-yl) -2,5-diphenyl-terazolium bromide, MTT) method measure drug delivery system
The inhibiting rate of 1 pair of tumour cell of system.
Mtt assay：HeLa cell dissociations in exponential phase are resuspended, with 1 × 104Cells/well is inoculated in 96 orifice plates
In (contain 100 μ L cell suspensions per hole), by 96 orifice plates be put into constant incubator culture 24 it is small when (37 DEG C, 5% (v/v) CO2)。
Again by negative control sample and 1 μ g/mL, 10 μ g/mL, 25 μ g/mL, 50 μ g/mL, 80 μ g/mL drug delivery system 1 and card
Platinum liquor sample is separately added into orifice bore, and each sample repeats 6 holes, and 80 μ L are added in per hole, continue afterwards culture 48 it is small when.
Then add in the MTT that 20 μ L concentration are 5mg/mL into each hole, 37 DEG C incubate 4 it is small when after, suction abandons in hole solution and into each hole
Add in 150 μ L dimethyl sulfoxide (DMSO)s, shake 15 minutes at 37 DEG C after bluish violet crystallization is completely dissolved in hole, is existed using microplate reader
The absorbance in each hole is measured at 490nm.Experiment is averaged in triplicate.Inhibiting rate calculation formula is as follows, as a result such as Fig. 8 institutes
Show.
Inhibiting rate (%)=(negative control absorbance-drug delivery system or carboplatin absorbance)/negative control is inhaled
Shading value × 100%
The result shows that：For HeLa cells, 1 solution pair of carboplatin and drug delivery system of 1,10,25,50,80 μ g/mL
The inhibiting rate of HeLa cell Proliferations is respectively 18% ± 1%, 32% ± 7%, 53% ± 2%, 65% ± 2%, 66% ± 4% He
19% ± 3%, 40% ± 7%, 71% ± 3%, 80% ± 2%, 82% ± 1%.When concentration is more than 25 μ g/mL, carboplatin and
Drug delivery system 1 all has HeLa cells apparent depression effect, but the two has significant difference.A represents drug
(folic acid-γ cyclodextrin-the C of delivery system 160- carboplatin) compared with carboplatin, p<0.05；M ± SD, n=3.
(3) to the statistics of human cervical carcinoma cell HeLa apoptotic effects：
Using the HeLa Cells of the high expression of folacin receptor as research object, drug is measured using flow cytometer and is passed
Send the influence of system 1 and carboplatin to apoptosis of tumor cells.
Specific method is：HeLa cell dissociations resuspension in exponential phase is inoculated in 6 orifice plates and (contains 500 μ per hole
L cell suspensions), be put into constant incubator culture 24 it is small when (37 DEG C, 5% (v/v) CO2).Again drug is added in into orifice plate to pass
System 1 and carboplatin is sent to make the final concentration of 50 μ g/mL of drug in hole, continue culture 48 it is small when.It will be used per hole inner cell before test
Phosphate buffer is rinsed and digested and is resuspended in 500 μ L buffer solutions, and gained sample inner cell number reaches 2 × 106Cell/mL,
Add 5 μ LAnnexin V-FITC and 5 μ LPI (Annexin-V-Fluos Staining kit, Sigma Aldrich,
USA examination with computer after) being protected from light 15 minutes, experiment are averaged in triplicate, and the results are shown in Table 1.
Table 1
Note：* drug delivery system 1 is represented compared with negative control (5% glucose), p<0.05；+ represent drug delivery
System 1 is compared with carboplatin, p<0.05；M ± SD, n=3.
The result shows that：Drug concentration is that the drug delivery system 1 of 50 μ g/mL and carboplatin can cause apparent cell to wither
It dies.Always apoptosis rate is respectively：Negative control (11.0%), carboplatin (70.4%), drug delivery system 1 (91.5%), drug are passed
Send system 1 that there is conspicuousness inducing cell early apoptosis compared with carboplatin.
(4) intake of human cervical carcinoma cell HeLa
A) standard curve of fluorescein isothiocynate (Fluorescein isothiocyanate, FITC) is measured：Accurately
The storing solution that 3mgFITC is configured to 300 μ g/mL with 10mL dimethyl sulfoxide (DMSO)s (DMSO) is weighed, takes 500 μ L, 100 μ L, 50 μ respectively
L, the storing solution of 30 μ L and 10 μ L are settled to 10mL, and it is respectively 15 μ g/mL, 3 μ g/mL, 1.5 μ g/mL, 0.9 μ to obtain FITC concentration
The standard sample of g/mL and 0.3 μ g/mL measure the ultraviolet light absorption angle value of each concentration standard sample, with FITC concentration at 495nm
It is that abscissa is drawn to obtain standard curve for ordinate, absorbance.
B) FITC labeled drugs：Two parts of label solutions, folic acid-γ cyclodextrin-C are obtained with DMSO dissolvings FITC60(preparation side
Method is shown in Summary method one, wherein, conjugate and fullerene C60Molar ratio be 1.5:And γ cyclodextrin 1)-
C60(except without using folic acid, preparation method and folic acid-γ cyclodextrin-C60It is identical) it is blended in keeps away with two parts of label solutions respectively
It when stirring 4 is small under the conditions of light, is rinsed after precipitation is collected by centrifugation with phosphate buffer solution, two kinds of drugs for obtaining FITC marks are passed
Send carrier.
C) measure of carrier adsorption FITC amounts：The 1mgFITC two kinds of drug delivery vehicles marked are dissolved in 5ml's respectively
In DMSO, FITC desorptions are made with 40kHz frequency ultrasound 30min processing, the ultraviolet absorptivity of solution is measured at 495nm wavelength
Value, solution FITC concentration values are obtained according to standard curve, calculate folic acid-γ cyclodextrin-C according to the following formula60With γ cyclodextrin-C60
FITC adsorbances.
FITC adsorbances=solution FITC concentration values × liquor capacity
D) by HeLa cell inoculations in 6 orifice plates, 2 × 105A cells/well, when cell density reaches 70%, respectively to
Different holes add in the folic acid-γ cyclodextrin-C of FITC marks60, γ cyclodextrin-C60, FITC and HeLa Cells culture
Base (as control), 37 DEG C incubate 4 it is small when after cell rinsed 2~3 times using phosphate buffer, with 300 μ L phosphate-buffereds
Liquid suspension examination with computer.
For flow cytometer measurement result as shown in figure 9, wherein a is control, b is γ cyclodextrin-C60- FITC, c FITC, d
For folic acid-γ cyclodextrin-C60-FITC.HeLa cells are to folic acid-γ cyclodextrin-C60- FITC, γ cyclodextrin-C60-FITC、
The intake of FITC is：Folic acid-γ cyclodextrin-C60-FITC>FITC>γ cyclodextrin-C60-FITC.This illustrates HeLa cells
A large amount of folacin receptors can increase HeLa cells to folic acid-γ cyclodextrin-C60Intake, so as to using folic acid-γ rings paste
Essence-C60When-platinum medicine is the drug delivery system of the present invention, the targeting of drug is further improved.
(5) to the morphology influence of human cervical carcinoma cell HeLa
HeLa cell inoculations are cultivated in 6 orifice plates containing coverslip.After cell attachment, added in into a part of hole
Drug concentration be the drug delivery system 1 of 25 μ g/mL incubates altogether 24 it is small when, fixed afterwards with 4% paraformaldehyde in all holes carefully
Born of the same parents 30 minutes after rinsing cell using phosphate buffer are dried, are observed using atomic force microscope at room temperature.Test mould
Formula：Tapping, sweep speed：0.5-1Hz, image processing and analysis software：SPM Version 3.3.25.
As a result as shown in figures 10-13, the HeLa cells without drug-treated have normal tumour cell form, including complete
Whole coating, nucleus and cytoplasm, visible cytoskeleton fiber and lamellipodium.And through folic acid-γ cyclodextrin-C60- card
After platinum processing, significant change occurs for cellular morphology, and surface is more coarse, cell shrinkage, and a large amount of holes, pseudopodium occurs in cell surface
It disappears, illustrates folic acid-γ cyclodextrin-C60- carboplatin changes the original structure of HeLa cells, destroys tumour cell structure, promotes
Its apoptosis embodies its lethal effect to tumour cell
Drug delivery vehicle provided by the invention can be reduced by the chemotherapeutics targeted delivery such as platinum class into tumour cell
The accumulation of chemotherapeutic in the normal tissue solves the problems, such as that chemotherapeutics lacks active targeting, and reduces chemotherapeutic
Into in normal structure, have the function that reduce toxicity, a kind of effective way is provided for clinical treatment.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.Root
According to all introductions having disclosed, those details can be carry out various modifications and replaced, these change in the guarantor of the present invention
Within the scope of shield.The four corner of the present invention is provided by appended claims and its any equivalent.
Claims (30)
1. a kind of pharmaceutical composition it includes drug delivery vehicle and by the active constituents of medicine of fullerene adsorption and can appoint
The pharmaceutically acceptable auxiliary material of choosing；The drug delivery vehicle includes folic acid-cyclodextrin conjugate and fullerene, the medicine
Object delivery vector is formed by the folic acid-cyclodextrin conjugate embedding fullerene；Wherein, the folic acid-cyclodextrin conjugate by
The carboxyl of folic acid connects to be formed with the hydroxyl of cyclodextrin；The active constituents of medicine is platinum medicine active ingredient；
Wherein, the preparation method of described pharmaceutical composition, comprises the following steps：
Folic acid-cyclodextrin conjugate with pre- adsorbent is protected from light and mixed in methyl alcohol, through separating, cleaning, drying, obtains medicine group
Close object；Wherein, the pre- adsorbent is made as follows：Fullerene, active constituents of medicine are protected from light with solvent and mixed, is passed through
Dialysis, drying form pre- adsorbent；
Also, the charged material weight of active constituents of medicine and fullerene ratio is (1:10)-(1:1)；Folic acid-cyclodextrin conjugate with it is pre-
The charged material weight ratio of adsorbent is (1:1)-(5:1).
2. pharmaceutical composition according to claim 1, wherein, the cyclodextrin is γ cyclodextrin.
3. pharmaceutical composition according to claim 2, wherein, the molar ratio of folic acid and γ cyclodextrin is in the conjugate
8:1；Folic acid-γ cyclodextrin the conjugate is connected with 6 hydroxyls of γ cyclodextrin by the γ carboxyls of folic acid and formed, and structure is such as
Under,
4. pharmaceutical composition according to claim 1, wherein, the fullerene is C60。
5. pharmaceutical composition according to claim 1, wherein, mole of the folic acid-cyclodextrin conjugate and fullerene
Than for 1:1.
6. pharmaceutical composition according to claim 1, wherein, the weight of the drug delivery vehicle and active constituents of medicine
Than for (1:1)-(10:1).
7. pharmaceutical composition according to claim 1, wherein, the weight of the drug delivery vehicle and active constituents of medicine
Than for 5:1.
8. pharmaceutical composition according to claim 1, wherein, the platinum medicine active ingredient for platinum medicine and/or
Platinum class prodrug.
9. pharmaceutical composition according to claim 8, wherein, the platinum medicine is selected from Pt (NH3)2Cl2、
C6H12N2O4Pt、C8H14N2O4Pt、C2H8N2O3Pt、C9H18N2O3Pt and C10H22Cl2N2O4One or more in Pt.
10. pharmaceutical composition according to claim 8, wherein, the platinum class prodrug for glucuronic acid-platinum and/or thio-
Platinum.
11. pharmaceutical composition according to claim 1, wherein, the solvent in the preparation method of the pre- adsorbent
For 5% (w/w) glucose injection or 0.9% (w/w) sodium chloride injection.
12. the pharmaceutical composition according to claim 1 or 11, it is characterised in that any one in following (1)-(4) item
Item is multinomial：
(1) mixing independently is stirring or grinding；
(2) it is described to be separated into centrifugation；
(3) cleaning is using methanol or redistilled water；
(4) drying is freeze-drying or nitrogen drying.
13. pharmaceutical composition according to claim 1, wherein, the charged material weight ratio of active constituents of medicine and fullerene is
1:4。
14. pharmaceutical composition according to claim 1, wherein, the weight that feeds intake of folic acid-cyclodextrin conjugate and pre- adsorbent
Amount is than being 1.5:1.
15. pharmaceutical composition according to claim 1, wherein, the preparation method of the folic acid-cyclodextrin conjugate includes
Following step：
(1) folic acid, cyclodextrin are protected from light with solvent and mixed, with 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides
It is reacted under the conditions of being protected from light for activator until terminating with N- hydroxysuccinimides.
16. pharmaceutical composition according to claim 15, wherein, the conjugate preparation method further includes step (2)：
(2) dialysed, dried to the product in step (1).
17. the pharmaceutical composition according to claim 15 or 16, wherein, the conjugate preparation method further includes as follows
1) item and/or 2) item：
1) cyclodextrin is γ cyclodextrin；
2) solvent is dimethyl sulfoxide (DMSO), sodium hydrate aqueous solution or aqueous sodium carbonate.
18. pharmaceutical composition according to claim 16, wherein, the conjugate preparation method further includes following 4)
Item and/or 5) item：
4) dialysis is carried out successively with phosphate buffer and second distillation water as medium；
5) drying is freeze-drying.
19. pharmaceutical composition according to claim 17, wherein, 1) in item, the γ cyclodextrin, folic acid, 1- (3- bis-
Methylaminopropyl) molar ratio of -3- ethyl-carbodiimide hydrochlorides and N- hydroxysuccinimides is 1:8:(2-10):
(2-10)。
20. pharmaceutical composition according to claim 19, wherein, the γ cyclodextrin, folic acid, 1- (3- dimethylaminos third
Base) molar ratio of -3- ethyl-carbodiimide hydrochlorides and N- hydroxysuccinimides is 1:8:4:4.
21. pharmaceutical composition according to claim 17, wherein, the 2) in item, and the solvent is dimethyl sulfoxide (DMSO).
22. purposes of the pharmaceutical composition in antitumor or anticancer medicine is prepared any one of claim 1 to 21.
23. purposes according to claim 22, wherein, the tumour is the tumour of the high expression of folacin receptor, the folic acid
The high expression of receptor refers to that folacin receptor is significantly higher than normal tissue cell in the expression quantity of tumor cell surface.
24. purposes according to claim 22, wherein, the antitumor drug is to inhibit tumor cell proliferation and/or suppression
Tumour sphere volume increase processed and/or the drug that tumour ball is promoted to disintegrate.
25. purposes according to claim 22, wherein, the tumour is lymph cancer, cervical carcinoma, breast cancer, oral cavity epidermis
Cancer, stomach cancer, lung cancer, nasopharyngeal carcinoma, cancer of the esophagus, carcinoma of urinary bladder, carcinoma of testis, oophoroma, prostate cancer, osteosarcoma, chronic granulocyte are white
At least one of blood disease cancer, melanoma and head and neck cancer.
26. the purposes according to claim 23 or 24, wherein, the tumour cell is lymphocytic cancer cell, cervical cancer cell,
Breast cancer cell, oral cavity epidermis cancer cell, stomach cancer cell, lung carcinoma cell, nasopharyngeal carcinoma cell, esophageal cancer cell, transitional cell bladder carcinoma cell line,
Testicular cancer cell, ovarian cancer cell, prostate gland cancer cell, osteosarcoma cell, chronic marrow original leukaemia, melanoma and
At least one of head ＆ neck cancer cell.
27. purposes according to claim 26, wherein, the cervical cancer cell behaviour source cervical cancer cell HeLa.
28. purposes according to claim 26, wherein, the cervical cancer cell behaviour source cervical cancer cell HeLa -229.
29. purposes according to claim 26, wherein, the breast cancer cell behaviour source MCF-7 Breast Cancer Cell.
30. purposes according to claim 26, wherein, the oral cavity epidermis cancer cell behaviour source oral cavity epidermoid carcinoma cell
KB cells.
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| CN101809039A (en) * | 2007-09-28 | 2010-08-18 | 那诺蒂克株式会社 | Cyclodextrin compound modified with folic acid, process for production thereof, drug delivery agent for targeting drug delivery system, pharmaceutical composition, and imaging agent |
| CN102675655A (en) * | 2012-06-04 | 2012-09-19 | 郑州大学 | Water-soluble fullerene and preparation and application methods thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101809039A (en) * | 2007-09-28 | 2010-08-18 | 那诺蒂克株式会社 | Cyclodextrin compound modified with folic acid, process for production thereof, drug delivery agent for targeting drug delivery system, pharmaceutical composition, and imaging agent |
| CN102675655A (en) * | 2012-06-04 | 2012-09-19 | 郑州大学 | Water-soluble fullerene and preparation and application methods thereof |
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