CN103230596A - Gold nanometer medicament carrier with synergistic antineoplastic effect - Google Patents
Gold nanometer medicament carrier with synergistic antineoplastic effect Download PDFInfo
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Abstract
The invention discloses a gold nanometer medicament carrier with synergistic antineoplastic effect, which is composed of double-function group targeting ligands and function groups for attaching a hydrophobic medicament, wherein, the double-function group targeting ligands respectively contains a thioctic acid modified folic acid molecule and a substituent of thioctic acid modified tyrosine, and the function group for attaching the hydrophobic medicament is thioctic acid modified beta-cyclodextrin. The experiment confirms that the gold nanometer carrier provided by the invention can absorb doxorubicin and combine with X-ray which selectively enhances the tumor cells toxicity and induces more DNA damages in the targeting cell, and the gold nanometer carrier is safer for the non-targeting cells, therefore the invention provides a latent new method for treating cancer.
Description
Technical field
The present invention relates to a kind of gold nano-material pharmaceutical carrier, relate in particular to the gold nano-material pharmaceutical carrier that a kind of couple of chemical functional group modifies, and the Synergistic anti-cancer effect of this system under the X-ray irradiation.
Background technology
Except excision, chemotherapy and radiation is main treatment of cancer means.But chemotherapy still is radiotherapy is not to treat at tumor cell specifically, can not avoid the damage to normal cell and tissue, all can be owing to serial toxic and side effects is subjected to application limitations.Therefore, people are badly in need of a kind of can the breakthrough yet micromolecular compound is difficult to realization in above-mentioned field specially at cytotoxic drug and a kind of method that can be limited in harmful radiation tumor cell of cancerous cell.In contrast be, nanotechnology provides the possibility of finishing above-mentioned target for people, form multi-functional nanometer material as multiple functional molecular being attached to same nano-material surface, these functional moleculars comprise but are not confined to the targeting molecule makes nano material be gathered in tumor locus more, fluorescence molecule comes the spike nano material and therapeutic effect is followed the tracks of, and high molecular polymer increases the water solublity of nano material and increases biological similarity.Simultaneously the attached targeted nanometer material surface of antitumor drug can be reached better anticancer effect by the targeting of combining nano material and the anti-tumor activity of medicine.Wherein gold nano-material has had years of researches history.With respect to the nano material of other kind, the advantage of gold nano-material is to be easy to synthesize; Its particle diameter and water solublity all can be controlled by control reactant ratio and reaction condition; The finishing of gold nano-material can be regulated and control by utilizing sulfydryl and gold to form golden sulfide linkage.The characteristics that the what is more important gold nano-material is low owing to its cytotoxicity, biocompatibility is high are widely used in the living things system.
The another one outstanding feature of gold nano-material is to strengthen ionizing radiation, as the injury of X-ray to cell, destroys the hereditary material of cell, thereby reaches the effect that stops the cell growth.If use targeting to experimentize in the gold nano-material of tumor cell, thereby the X-ray reaches anticancer effect with specificity kill tumor cell.On the other hand, a lot of antitumor drug as cisplatin and doxorubicin, itself namely are radiosensitizers.
Doxorubicin and amycin are the combinations of a kind of topoisomerase II inhibitor, can cause that the dna double chain interruption reaches the kill tumor cell.But retrieval shows the two targeting gold nano-materials of utilization as the carrier of doxorubicin, and the document and the patent that strengthen the antineoplastic effect in conjunction with the X-ray yet there are no report.
Summary of the invention
At the deficiencies in the prior art, the problem to be solved in the present invention provides a kind of novel gold nano pharmaceutical carrier with synergistic antitumor effect; Be the gold nano-material pharmaceutical carrier that a kind of couple of chemical functional group modifies, and the Synergistic anti-cancer effect of this system under the X-ray irradiation.
Gold nano pharmaceutical carrier with synergistic antitumor effect of the present invention is used for the functional group of appendix hydrophobic drug by difunctional targeting part and one and constitutes; It is characterized in that: described difunctional targeting part is respectively the folate molecule of thioctic acid modification and the substituent of the tyrosine that thioctic acid is modified, and the described functional group that is used for the appendix hydrophobic drug is the beta-schardinger dextrin-that thioctic acid is modified; The pattern of gold nano pharmaceutical carrier is spherical, particle size distribution is between 3-6nm, dynamically particle diameter determines that with dynamic light scattering (DLS) method it is 16-17nm in the PBS buffer, the surface potential Zeta potential value of gold nano pharmaceutical carrier in aqueous solution is distributed as from-35 to-46mV, when it is suspended in when containing in the 1% serum aqueous solution, the Zeta potential value moves to-15mV, shows that this carrier is to the non-specific adsorption of albumen.
Wherein: the substituent preferable methyl 2-(3-(4-(5-(1 of the tyrosine that above-mentioned thioctic acid is modified, 2-dithiolane-3-yl) valeryl amido) 4-chlorphenyl)-2-Benzamido-propiono)-the 2-(4-hydroxy phenyl) methyl acetate.
Above-mentioned gold nano pharmaceutical carrier with synergistic antitumor effect is made by following method:
1) be dissolved in ligand-1 thioctic acid-beta-schardinger dextrin-, ligand-2 thioctic acid-folic acid (TA-FA) in the high purity water respectively, ligand-3 thioctic acid-tyrosine substituent is dissolved in the methanol, and makes ligand-1: ligand-2: the mol ratio of ligand-3 is 1:1:1; Gold chloride (III) trihydrate (being the tetra chlorauric acid trihydrate) is dissolved in the high purity water, and wherein the consumption of gold chloride (III) trihydrate is ligand-1, ligand-2 and ligand-3 mole sum; Sodium borohydride is dissolved in the water, and wherein the sodium borohydride consumption is 2 times of gold chloride (III) trihydrate consumption;
Above-mentioned ligand-1, ligand-2 and ligand-3 structural formula are as follows:
Ligand-1 ligand-2 ligand-3
2) under the stirring condition ligand-1, ligand-2 and ligand-3 solution for preparing is mixed, slowly join then in the chlorauric acid solution, under room temperature condition, stirred 30 ± 5 minutes;
3) sodium borohydride solution is dropwise joined in gold chloride and the ligand mixture, and at room temperature stirred 4-9 hour;
4) in the reactant mixture of step 3), dropwise add 1mol/L hydrochloric acid, the excessive sodium borohydride that neutralizes, and make pH value reach 7.0, cessation reaction:
5) reactant mixture with step 4) places centrifugal filter, with 100K molecular retention under the 4000r.p.m. rotating speed centrifugal 30 ± 5 minutes, separates and removes excessive ligand; The solid that separates is dissolved in the deionized water, after the conventional supersound process, again with 100K molecular retention under the 4000r.p.m. rotating speed centrifugal 30 ± 5 minutes, and so repeated centrifugation washing 5 ± 1 times; Behind last washing step, obtain solids and be the gold nano pharmaceutical carrier.
6) further, the gold nano pharmaceutical carrier is dissolved in the 5mL high purity water, and as stock solution, clears up the back and can measure gold element content with inductively coupled plasma mass spectrometry (ICP-MS) method.
Wherein: the described mixing time of step 3) preferred 4-6 hour.
The application that has the gold nano pharmaceutical carrier absorption doxorubicin of synergistic antitumor effect and strengthen cytotoxicity in conjunction with X-ray selectivity of the present invention.
(I) gold nano pharmaceutical carrier provided by the invention is to absorption and the release of doxorubicin
Novel gold nano pharmaceutical carrier involved in the present invention can be used for the absorption doxorubicin, and the while doxorubicin can rely on different pH and carry out drug release.Doxorubicin carries out combination by the β-cyclodextrin on spontaneous and the gold nano carrier surface ligand 1, and this neutrality that is combined in arrives under the alkaline pH situation highly stable.Each gold nano carrier granular can adsorb about 50 doxorubicins.Carrier is distinguished to some extent with the release of doxorubicin complex under different pH situations, and neutrallty condition reaction is after 32 hours, and 20% the doxorubicin of only having an appointment obtains discharging, and (pH5.5) is released more than 60% doxorubicin under acid condition.
In the absorption and method for releasing of above-mentioned many gold nano carrier to doxorubicin, described doxorubicin and carrier be 8.6 in conjunction with the condition optimization pH value, preferred 50 doxorubicins of its doxorubicin adsorption rate/gold nano carrier.
(II) gold nano pharmaceutical carrier provided by the invention has two targeting groups, and its selectivity target tumor cell is better than single targeting gold nano carrier.
The present invention has related to the method that multiple qualitative characterize cells is taken in the gold nano carrier simultaneously.Select cervical cancer cell and the HeLa cell of high expressed folacin receptor (FR) as target cell to be in the experiment, select the A549 cell of the low FR of expression as non-target cell to be simultaneously.In order to promote the HeLa cell to the absorption of gold nano carrier, modify ligand-2 and ligand-3 simultaneously on gold nano carrier GNP-1 surface as the targeting molecule.Can observe individual cells to the absorption of gold nano carrier by transmission electron microscope.In the HeLa cell, find the aggregation of a large amount of gold nano carriers, but in the A549 cell, do not find above-mentioned gathering (Fig. 5), simultaneously, can observe most of gold nano carriers under high-resolution-ration transmission electric-lens concentrates in cell endocytic body, lysosome sample vesicle and the Cytoplasm.
As negative control, we design the GNP-2 surface and only contain folic acid ligand-2 as the targeting molecule.After cell and gold nano carrier hatched certain hour altogether, with chloroazotic acid with cell dissociation after with the accurate quantitative concentration of cell Endothelium corneum nano-carrier of ICP-MS, and then calculate the absolute magnitude that contains the gold nano carrier in each cell or the number of taking in the gold nano carrier.The HeLa cell is 4 times of A549 cell intake to the absorption of GNP-1, and simultaneously, the HeLa cell is 3.5 times of GNP-2 to the absorption of GNP-1.
(III) the invention provides novel gold nano carrier absorption doxorubicin and reach the Cytotoxic method of enhancing in conjunction with the X-ray simultaneously
The present invention strengthens cytotoxicity with X-ray and gold nano carrier and the combined effect of doxorubicin complex.Doxorubicin acts on the supercoiled topoisomerase II of DNA.Topoisomerase II was untied the DNA chain in order to copy after, doxorubicin meeting stable topology isomerase II prevented that dna double thigh spiral from combining again, thereby stops reproduction process.The gold nano carrier can strengthen ionizing radiation simultaneously, as the X-ray, to the injury of cell, destroys the hereditary material of cell, thereby reaches the effect that stops the cell growth.Therefore use targeting in the gold nano of the tumor cell carrier as doxorubicin, the doxorubicin selectivity is transported in the tumor cell, auxiliary with x-ray irradiation, under the multiple action of doxorubicin, gold nano-material and X-ray, produce and to play the enhancing apoptosis of tumor cells to the powerful double attack of cell DNA.Reduce non-targeting cell toxicant simultaneously and pay the effect of effect.After GNP-1 and doxorubicin complex or doxorubicin itself hatched a period of time altogether with HeLa and A549 cell, wash unnecessary gold nano carrier off with PBS, with doses x-ray irradiation cell.Cell is used XTT measuring cytoactive after hatching in 48 hours.In control experiment, cell only passes through x-ray irradiation without any processing.Experimental result shows that gold nano carrier and doxorubicin compound system not only can improve the safety to non-targeted cells (as normal structure), more can improve the cytotoxicity of complex greatly in conjunction with the X-ray, and most of potentiation comes from two targeting gold nano carriers.
The preferred 3-5GY of above-mentioned x-ray dose.
Simultaneous verification of the present invention strengthen because the DNA damage aggravation causes with caused cytotoxicity behind x-ray irradiation gold nano carrier and the doxorubicin complex.The quantitative analysis that detects by γ-H2AX shows that as carrier, doxorubicin in conjunction with x-ray irradiation, can cause more DNA damage as the system of medicine in targeted cells, and safer to non-target cell with two target spot gold nano-materials.
For better essence of the present invention and the using value set forth, further specify two target spot gold nano carriers with experiment and interpretation of result in detail below and be combined X-ray selectivity enhancing cytotoxicity and application with the doxorubicin complex.
One. the assemble method of gold nano carrier of the present invention and analysis and characterization, its structure is seen Fig. 1, its number of assembling steps comprises:
1) synthetic thioctic acid-beta-schardinger dextrin-(ligand-1), thioctic acid-folic acid (TA-FA, ligand-2), and thioctic acid-tyrosine substituent (ligand-3) (synthesis step is seen embodiment in detail);
2) ligand-1 and ligand-2 are dissolved in the high purity water, part-3 is dissolved in the methanol, ligand-1 wherein, and ligand-2, the mol ratio of ligand-3 are 1:1:1;
3) gold chloride (III) trihydrate is dissolved in the certain volume high purity water, wherein the consumption of gold chloride (III) trihydrate is ligand-1, ligand-2, ligand-3 mole sum;
4) sodium borohydride is dissolved in the certain volume water, wherein the sodium borohydride consumption is 2 times of gold chloride (III) trihydrate consumption;
5) under the stirring condition with ligand-1, ligand-2, and ligand-3(comprises fluorescent ligand sometimes) mix, said mixture is slowly joined in the chlorauric acid solution, under room temperature condition, stirred 30 minutes;
6) sodium borohydride solution is dropwise joined in gold chloride and the ligand mixture, this solution has become redness immediately, and at room temperature stirs 4 hours;
7) come cessation reaction with following operation: dropwise add 1mol/L hydrochloric acid in the reactant mixture and excessive sodium borohydride, reach 7.0 up to pH value;
8) come the gold nano pharmaceutical carrier is carried out purification with following method: a) reactant mixture is placed Millipore centrifugal filter (100K molecular retention), the centrifugal ligand of coming excessive separation in 30 minutes under the 4000r.p.m. rotating speed; B) with colourless incline supernatant, solid is dissolved in the deionized water of 3.0mL, pass through supersound process; C) again with washing in centrifugal 30 minutes in the Millipore centrifugal filter; D) this washs centrifugal circulation repetition five times; E) behind last washing step, the gold nano pharmaceutical carrier is dissolved in the 5mL high purity water, and as stock solution, clears up the back and measure gold element content with inductively coupled plasma mass spectrometry (ICP-MS) method.
Novel gold nano pharmaceutical carrier involved in the present invention all can be suspended in the phosphate buffer (PBS) equably, can stablize and deposit at least three months.
The resulting novel gold nano pharmaceutical carrier of the present invention (TEM) under high resolution transmission electron microscopy characterizes, and the result shows that this carrier is spherical in shape, the about 5nm(Fig. 2 of diameter).Because the existence of finishing group, it is 16-17nm(Fig. 3 through the dynamic diameter that dynamic light scattering (DLS) is measured in PBS).The Zeta potential value of this carrier in aqueous solution is-46mV(Fig. 4).When being suspended in when containing in the 1% serum aqueous solution, its Zeta potential value moves to-15mV, shows that this carrier to albumen non-specific adsorption has taken place.
Two. gold nano carrier of the present invention is to absorption and the release of doxorubicin
In order to measure the gold nano carrier to the envelop rate of doxorubicin, doxorubicin (mother solution is that 10mg/mL is dissolved among the DMSO) is diluted to different final concentrations (4～500 μ g/mL) with the pH8.6 buffer, it is 1mg/mL that carrier is diluted to final concentration with the pH8.6 buffer, the equal-volume doxorubicin is mixed with the gold nano carrier, in room temperature vibration 4 hours.By centrifugal gold nano carrier and the doxorubicin complex removed under the 13000r.p.m. rotating speed, collect supernatant and also measure the concentration of excessive doxorubicin in the supernatant by the HPLC-MS method, measure its envelop rate (Fig. 5) by its content in the doxorubicin consumption removal supernatant of participating in reaction.Each gold nano carrier granular can adsorb about 50 doxorubicin molecules.
Gold nano carrier and the release conditions of doxorubicin complex under different pH have further been studied.By situation in pH7.4 buffer simulation human normal tissue and the blood, (nano material is taken in by cell through the endocytosis mode usually with situation in the pH5.5 buffer indication tumor cell, concentrate in the organelles such as the endocytosis body of cell and lysosome, its environment is acid).By centrifugal gold nano carrier and the doxorubicin complex removed under the 13000r.p.m. rotating speed, collect supernatant and pass through the concentration that the HPLC-MS method is measured excessive doxorubicin in the supernatant, account for gold nano carrier absorption doxorubicin content by doxorubicin concentration in the supernatant and measure release rate.Reaction is after 32 hours under the neutrallty condition, and 20% the doxorubicin of only having an appointment obtains discharging, and (pH5.5) is released (Fig. 5) more than 60% doxorubicin under acid condition.
Three. tumor cell is taken in the selectivity of gold nano carrier of the present invention
After the gold nano carrier of HeLa cell or A549 cell and 50 μ g/mL hatched 8 hours altogether, do not take in carrier with the PBS flush away, will collect after the cell fixation, by transmission electron microscope, can observe individual cells to the absorption of nano-carrier.In the HeLa cell, find the aggregation of a large amount of gold nano carriers, but in the A549 cell, do not find above-mentioned gathering (Fig. 6), simultaneously, observing most of gold nano carriers under high-resolution-ration transmission electric-lens concentrates in cell endocytic body, lysosome sample vesicle and the Cytoplasm.
The present invention has related to the method that multiple quantitatively characterizing cell is taken in the gold nano carrier simultaneously.Cell and gold nano carrier are hatched certain hour altogether, clean behind the cell three times the numeration of cell eluting and collect with cold PBS, accurately judge the concentration of cell Endothelium corneum nano-carrier with ICP-MS with chloroazotic acid after with cell dissociation, and then can calculate the absolute magnitude that contains the gold nano carrier in each cell or the number of taking in the gold nano carrier.The combination of two targeting part GNP-1 and HeLa cell has time dependence, hatches after 6 hours cell combination and absorption reach capacity (Fig. 7) altogether.The HeLa cell is 4 times of A549 cell to the absorption of GNP-1, and simultaneously, the HeLa cell is 3.5 times of single targeting GNP-2 to the absorption of GNP-1.
Four. gold nano carrier absorption doxorubicin of the present invention reaches the enhancing cytotoxicity in conjunction with the X-ray simultaneously
A desirable pharmaceutical carrier should have lower toxicity and better biocompatibility.Use is carried out cytotoxicity experiment far above the gold nano carrier concn of follow-up cell experiment.The gold nano carrier of 100 μ g/mL and tumor cell hatched 48 altogether in 96 orifice plates after, test to detect cytoactive by XTT, all gold nano carriers there is no overt toxicity (Fig. 8) under this concentration.
The present invention strengthens cytotoxicity with X-ray and gold nano carrier and the combined effect of doxorubicin complex.Doxorubicin acts on the supercoiled topoisomerase II of DNA.Topoisomerase II was untied the DNA chain in order to copy after, doxorubicin meeting stable topology isomerase II prevented that dna double thigh spiral from combining again, thereby stops reproduction process.The gold nano carrier can strengthen ionizing radiation simultaneously, as the injury of X-ray to cell, destroys the hereditary material of cell, thereby reaches the effect that stops the cell growth.Therefore if use targeting in the gold nano of the tumor cell carrier as doxorubicin, the doxorubicin selectivity is transported in the tumor cell, auxiliary with x-ray irradiation, multiple action by doxorubicin, gold nano and X-ray, generation can be played the enhancing apoptosis of tumor cells to the powerful double attack of cell DNA, reduces the effect that non-targeting cell toxicant is paid effect simultaneously.Measure doxorubicin to the IC of HeLa cell
50Be 1.2 μ M.Selection is lower than IC
50Doxorubicin dosage carry out x-ray dose optimization experiment (the gold nano carrier of 10 μ g/mL and doxorubicin complex, the doxorubicin concentration of equivalence is 100 nM), after carrier-doxorubicin complex and HeLa cell hatched 8 hours altogether, the carrier that flush away is unnecessary is with the x-ray irradiation cell of various dose.Cell is used XTT measuring cytoactive after hatching in 48 hours.In control experiment, cell only passes through x-ray irradiation without any processing, and the X-ray of 5Gy does not cause obvious cytotoxicity.Under this dosage, GNP-1 and doxorubicin complex cytoactive obviously reduce (Fig. 9).For effective kill cancer cell, simultaneously Normocellular infringement is down to minimumly, selecting 3Gy in subsequent experimental is x-ray dose.
By research doxorubicin and gold nano carrier and doxorubicin complex to the dose-dependence of HeLa cell and A549 cell obtain drawing a conclusion (all experiments are divided into normal group and x-ray irradiation group (Figure 10)):
1. in the A549 cell, the complex of GNP-1 and doxorubicin has demonstrated the toxicity lower than doxorubicin itself, even x-ray irradiation does not have obvious enhancing yet, show that gold nano carrier and doxorubicin compound system are to the raising of non-targeted cells (as normal structure) safety;
2.X-handling cell to doxorubicin, ray (for example shown slight potentiation, in the A549 cell that doxorubicin is handled, the IC50 that passes through and do not pass through x-ray irradiation is respectively 3.0 μ M and 5.7 μ M), show that doxorubicin itself also is the weak reinforcing agent of a kind of X-ray;
3. cross in the HeLa cell of expression at folacin receptor, GNP-1 and doxorubicin complex all show reinforced effects through x-ray irradiation and (for example, in the HeLa cell of GNP-1 and the processing of doxorubicin complex, pass through and do not pass through the IC of x-ray irradiation
50Be respectively 0.3 μ M and 1.5 μ M).Doxorubicin only causes that slight cytotoxicity strengthens, and most of potentiation comes from targeting gold nano carrier.
Simultaneous verification of the present invention strengthen because the DNA damage aggravation causes with caused cytotoxicity behind x-ray irradiation gold nano carrier and the doxorubicin complex.The DNA that doxorubicin is induced embeds and the damage to cell DNA under x-ray irradiation of gold nano carrier may be that this system strengthens Cytotoxic mechanism.Analyze quantitative assay dna double chain interruption (can prove dna double aggressive mechanism Figure 11) by γ-H2AX.The quantitative analysis that detects by γ-H2AX shows, through the HeLa cell of GNP-1 and doxorubicin Combined Processing, under normal circumstances with under the x-ray irradiation produced 10.1% and the 25.2%DNA double-strand break respectively.Under the equal conditions x-ray irradiation, GNP-1 compares with only passing through doxorubicin processing HeLa cell with the HeLa cell of doxorubicin Combined Processing, and the NNA double-strand break has strengthened 2.5 times.These data show, two target spot gold nano carriers-doxorubicin drug system in conjunction with x-ray irradiation, can cause more DNA damage in targeted cells, and safer to non-target cell, are a kind of new methods of potential treatment cancer.
Description of drawings
Fig. 1: the structural representation of novel gold nano pharmaceutical carrier.
Fig. 2: the high resolution transmission electron microscopy photo of novel gold nano pharmaceutical carrier.
Fig. 3: the particle size distribution that novel gold nano pharmaceutical carrier is recorded by dynamic light scattering.
Fig. 4: the Zeta potential of novel gold nano pharmaceutical carrier.
Fig. 5: novel gold nano pharmaceutical carrier is to appendix and the release of doxorubicin under condition of different pH of doxorubicin.
Wherein:
A: the gold nano pharmaceutical carrier is to the appendix of doxorubicin;
B: the gold nano pharmaceutical carrier is to the release under condition of different pH of doxorubicin.
Fig. 6: cell is to the absorption of gold nano carrier under the transmission electron microscope.
Wherein:
A: the A549 cell is to the absorption of gold nano carrier under the transmission electron microscope;
B: the HeLa cell is to the absorption of gold nano carrier under the transmission electron microscope;
C: under the high-resolution multiple, the HeLa cell is to the absorption zone 1 of gold nano carrier under the transmission electron microscope;
D: under the high-resolution multiple, the A549 cell is to the absorption zone 2 of gold nano carrier under the transmission electron microscope.
Fig. 7: the ICP-MS quantization cell is to the absorption of gold nano carrier.
Wherein:
A:ICP-MS quantitative assay HeLa cell to the absorption of gold nano carrier over time;
B:ICP-MS quantitative assay A549 and the absorption of HeLa cell to different gold nano carriers.
Fig. 8: the gold nano pharmaceutical carrier is to the influence of A549 and HeLa cytoactive.
Fig. 9: gold nano pharmaceutical carrier and doxorubicin complex and various dose x-ray irradiation influence the HeLa cytoactive.
Figure 10: various dose gold nano carrier and doxorubicin are to A549 and the influence of HeLa cytoactive under the x-ray irradiation.
Figure 11: the damage of HeLa cell DNA under doxorubicin or gold nano carrier and doxorubicin complex and x-ray irradiation.
Wherein:
A: the dna double chain interruption that is shown by γ-H2AX under the fluorescence microscope;
The quantification of DNA damage in the b:HeLa cell.
The specific embodiment
Ligand 1(thioctic acid-beta-schardinger dextrin-) preparation:
(CD-OTs, 1.0g 0.77mmol) are dissolved in the ethyl diamidogen (5.0mL) β-cyclodextrin of tosylation, at room temperature stir it is dissolved fully, and mixture is in 60 ℃ of stirred overnight.Mixture is cooled to room temperature, and dropwise joins acetone in (100mL), occur a large amount of yellow mercury oxides at once.With mixture at 4000rpm centrifugal 5 minutes, collecting precipitation, and with washing with acetone precipitation 2 times, obtain intermediate 2(860mg, productive rate 82.8%).This intermediate can be directly used in next step reaction, need not to be further purified.
With thioctic acid (206mg, 1.0mmol) be dissolved in the oxolane (2mL), N, the N'-dicyclohexyl carbodiimide (DCC, 247mg, 1.2mmol), N-hydroxy-succinamide (NHS, 137mg, 1.2mmol) and triethylamine (279 μ L 2.0mmol) join in the above-mentioned solution successively.Mixture removes by filter precipitate after at room temperature stirring 4 hours.Intermediate 2(750mg 0.63mmol) joins in the solution, under the room temperature mixture stirring is spent the night.After reaction is finished, mixture is poured in the acetone (100mL), a large amount of yellow mercury oxides occurred.At 4000rpm centrifugal 5 minutes, by centrifugal collecting precipitation, and with twice of washing with acetone.Crude product by purification by flash chromatography, is obtained ligand 1 thioctic acid-beta-schardinger dextrin-.
1HNMR(400MHz,DMSO-d6)δ6.22–5.53(m,7H),4.84(s,4H),4.47(d,J=5.4Hz,3H),3.64(s,14H),3.23–2.96(m,3H),2.90–2.55(m,2H),2.41(s,2H),2.31–2.16(m,1H),2.06(s,2H),1.89(d,J=6.2Hz,1H),1.73–1.15(m,6H)。
Ligand 2(thioctic acid-folic acid, preparation TA-FA):
Reagent and reaction condition: (I) Boc
2O, NEI
3, MeOH; (II) Thiotic acid, DCC, NHS; (III) TFA/DCM, 1:4; (IV) Folic acid, DCC, Py, DMSO
Synthetic NH
2-EG-NHBoc
4,7,10-, three oxygen-1,13-tridecane diamidogen (2,874 μ L, 4.00mmol) and triethylamine (1.39mL 8.00mmol) is dissolved in 10.0mL methanol.(474 μ L, 10mL methanol solution 2.00mmol) dropwise adds in the said mixture with Bis(tert-butoxycarbonyl)oxide under the stirring condition.With reactant mixture further stirring at room 8 hours.Crude product is carried out purification by silica gel chromatography, use as eluant 1:10 methanol/dichloromethane solution.Obtaining NH2-EG-NHBoc is colorless oil.Yield: 72.4%.
1H?NMR(400MHz,CDCl
3)δ5.22(s,1H),3.80-3.38(m,10H),3.21(d,J=6.1,2H),2.79(t,J=6.7,2H),1.88-1.63(m,4H),1.48(s,2H),1.42(s,9H).ESI-MS?m/z321(MH
+).。
Synthetic TA-EG-NHBoc
Thioctic acid (309mg, 1.50mmol), N, the N'-dicyclohexyl carbodiimide (474mg, 2.30mmol) and N-hydroxy-succinamide (264mg 2.30mmol) is dissolved in the 10.0mL oxolane, and at room temperature stirs 1 hour.With NH2-EG-NHBoc(463mg, 1.45mmol) join in the mixture.At room temperature mixture is stirred and spend the night.Under reduced pressure after the desolventizing, crude product extracts between ethyl acetate and water, and uses the salt water washing.Organic layer is concentrated, use the 1:16 ethanol/methylene as the silica gel chromatography purification of eluant.Obtain 602mgTA-EG-NHBoc, be yellow oil, yield: 81.8%.
1H?NMR(400MHz,CDCl
3)δ6.91-6.56(b,1H),5.25-4.91(b,1H),3.57(m,12H),3.35(m,2H),3.19(m,3H),2.46(m,2H),2.19(m,1H),1.84(m,1H),1.84-1.71(m,4H),?1.71-1.47(m,4H),1.42(s,9H),1.25(d,J=6.4,4H),1.12(d,J=6.8,2H).ESI-MS?m/z509(MH
+).
TA-FA's is synthetic
TA-EG-NHBoc(602mg 1.18mmol) is dissolved in the 1:4(volume of 10mL) the dichloromethane solution of trifluoroacetic acid in.This mixture at room temperature stirred 3 hours.The vapourisation under reduced pressure solvent.With the crude product dilute with water and use dichloromethane extraction.Organic layer is concentrated, and be used for next step and need not purification.
Folic acid (800mg 2.06mmol) is dissolved in the 10mL dimethyl sulfoxine, adds N, the N'-dicyclohexyl carbodiimide (800mg, 3.88mmol) and pyridine (500 μ L, 6.18mmol), stirring at room activated in 30 minutes.With TA-EG-NH
2Solution in join in the mixture.Reaction is 24 hours under 60 ° of C, and reactant is at room temperature stirred.After being cooled to room temperature, pour mixture into the 150mL ether.Leach precipitate, and be dissolved in the 15mL10%NaOH aqueous solution.At 4000rpm centrifugal 30 minutes, by this solid of centrifugalize.Solid is dissolved in the methanol.Adding trifluoroacetic acid regulator solution pH value is about 4.0.A large amount of precipitations occur immediately, precipitate by 4000rpm centrifugalize in centrifugal 5 minutes.This product by acetonitrile washing 2 times and methanol wash once.Pure product obtain 500mgTA-FA in the vacuum dried overnight.More than the productive rates of 2 step reactions be 50.9%.
1HNMR(400MHz,DMSO)δ12.12(b,1H),11.41(s,1H),8.64(s,1H),8.06(d,J=7.7,1H),7.81(d,J=5.0,1H),7.74(s,1H),7.65(d,J=8.6,2H),6.92(s,3H),6.63(d,J=8.7,2H),4.48(d,J=5.6,2H),4.32(s,1H),4.10(d,J=5.2,1H),3.73-3.55(m,1H),3.45(dd,J=11.3,3.7,10H),3.22-2.96(m,6H),2.73(d,J=47.8,2H),2.31(m,3H),2.04(t,J=7.0,2H),1.99-1.74(m,3H),1.70-1.41(m,7H),1.40-1.15(m,2H).ESI-MS?m/z833(MH
+).
Ligand 3(thioctic acid-tyrosine substituent: methyl 2-(3-(4-(5-(1,2-dithiolane-3-yl) 4-chlorphenyl valeryl amido))-2-Benzamido-propiono)-the 2-(4-hydroxy phenyl) methyl acetate) preparation:
Reagent and reaction condition: (I) R
2-COCl, TEA, DCM; (II) Zn, AcOH, EtOH; (III) Oxatyl chloride, DMF, DCM, Thioctic acid, TEA; (IV) LiOH, H
2O, THF; (V) R-NH
2, DCC, NHS, THF
Methyl 2-acyl group acylamino--3-(4-nitro-phenyl) ethyl propionate (NO
2-Tyr-Ac) synthesis step.
Methyl 2-amino-3-(4-nitrobenzophenone) ethyl propionate (1,1.50g, 5.7mmol) and triethylamine (1.5mL 11.4mmol) is dissolved in the anhydrous N of 12mL, in the dinethylformamide.In ice bath, needed acyl chlorides (7.5mmol) is dropwise joined in this mixture.After interpolation finishes, remove ice bath, and mixture was further stirred 2-3 hour in room temperature.Adopt the thin layer chromatography monitoring reaction.White depositions is by removing by filter, and washs by ethyl acetate.By concentrating under reduced pressure filtrate, this intermediate can be directly used in next step and need not purification.
Methyl 2-acyl group acylamino--3-(4-amino-phenyl) ethyl propionate (NH
2-Tyr-Ac) synthetic general procedure.
Methyl 2-acyl group acylamino--3-(4-nitro-phenyl) ethyl propionate (obtaining from the reaction of front) is dissolved in the mixture of the ethanol of 60mL and acetic acid (volume ratio 1:1).(2.20g 34.2mmol) is divided into and joins several times in the above-mentioned solution with zinc powder.At room temperature mixture is stirred and spend the night.Remove by filter undissolved solid, filtrate is concentrated, and be dissolved in again in the ethyl acetate, the water extraction.Organic layer low pressure is dry and concentrated.Crude product is carried out purification with chromatography, use hexane and ethyl acetate as eluant during purification.
Methyl 3-(4-(5-(1,2-dithiolane-3-yl) valeryl) phenyl)-the 2-amide groups ethyl propionate (synthesis step of TA-Tyr-Ac (1-5)-OMe).
(1.41g 6.8mmol) is dissolved in the 6mL anhydrous methylene chloride thioctic acid, adds the anhydrous N of 300 μ L, and dinethylformamide is as catalyst.(2.0M, 6.8mmol) the oxalyl chloride anhydrous methylene chloride solution that in ice bath, dropwise add 3.4mL in the mixture.Remove ice bath after the interpolation, and with mixture further stirring at room 2 hours.
Methyl 2-acyl group acylamino--3-(4-amino-phenyl) (1.7mL 12.0mmol) is dissolved in the anhydrous methylene chloride of 10mL for ethyl propionate (6.0mmol) and triethylamine.Under the condition of ice bath, the solution of above-mentioned thioctic acid is dropwise joined in this mixture.After the interpolation, remove ice bath, with the further stirring at room of reactant mixture 2 hours.Adopt the thin layer chromatography monitoring reaction.Precipitate by removing by filter, and is washed by ethyl acetate.Organic layer is under reduced pressure concentrated.Crude product carries out purification with flash chromatography, uses hexane and ethyl acetate as eluant.
Synthetic N-(4-(2-amide groups-3-(alkylamino)-and the 3-oxopropyl) phenyl)-5-(1,2-dithiolane-3-yl) general step of pentanamide (TA-Tyr-Am (1-6)-Ac (1-5)).
Methyl 3-(4-(5-(1,2-dithiolane-3-yl) valeryl) phenyl)-2-amide groups ethyl propionate (4,10.0mmol) be dissolved in 48mL oxolane and the 8mL water.(1.16g 28.5mmol) slowly joins in the mixture, stirs under the room temperature and spends the night with Lithium hydrate.After this mixture under reduced pressure concentrates, by chromatography purification, use methanol and dichloromethane as eluent crude product.
(0.42mmol) is dissolved in the 2mL oxolane with above-mentioned product.With N, the N'-dicyclohexyl carbodiimide (DCC, 65mg, 0.315mmol), N-hydroxy-succinamide (NHS, 36mg, 0.315mmol) and triethylamine (44 μ L 0.315mmol) join in the mixture, stir simultaneously.At room temperature mixture is stirred and spend the night.Reaction produces precipitate by after removing by filter, and crude product is extracted between ethyl acetate and water, and organic layer is concentrated.Solid is dissolved in the 1mL two sulfur sulfoxides, and the Biotage HPLC system that is loaded into 96 orifice plates carries out automatic purification.This pure products is checked by HPLC/MS and NMR's.Confirm to make ligand 3 thioctic acid-tyrosine substituent: methyl 2-(3-(4-(5-(1,2-dithiolane-3-yl) the valeryl amido) the 4-chlorphenyl)-2-Benzamido-propiono)-the 2-(4-hydroxy phenyl) methyl acetate.
Methyl 2-(3-(4-(5-(1,2-dithiolane-3-yl) the valeryl amido) the 4-chlorphenyl)-2-Benzamido-propiono)-the 2-(4-hydroxy phenyl) methyl acetate.1H?NMR(400MHz,MeOD)δ7.72-7.62(m,2H),7.39(ddd,J=23.1,18.0,10.8Hz,5H),6.99(d,J=8.5Hz,3H),6.76-6.61(m,2H),5.27(s,1H),4.82(t,J=7.1Hz,1H),3.66-3.53(m,3H),3.55-3.45(m,1H),3.38(ddd,J=10.6,6.7,4.1Hz,2H),?3.18-2.87(m,4H),2.39(dd,J=12.4,5.9Hz,1H),2.29(t,J=7.5Hz,2H),1.89-1.73(m,4H),1.54-1.34(m,3H)。
Novel gold nano preparation of drug carriers:
(ligand-2,5.0mg 0.006mmol) are dissolved in the water of 1.0mL to thioctic acid-folic acid.(ligand-1,8.0mg 0.006mmol) are dissolved in the water of 1.0mL to thioctic acid-beta-schardinger dextrin-.Ligand-3(4.0mg 0.006mmol) is dissolved in the methanol of 1.0mL.Under agitation above-mentioned solution is mixed.(7.5mg 0.018mmol) is dissolved in the 1.0mL water gold chloride (III) trihydrate.Under the room temperature said mixture is slowly joined in the chlorauric acid solution.After at room temperature stirring 30 minutes, (1.44mg, 0.038mmol) aqueous solution of dissolving 2.0mL dropwise joins in this mixture sodium borohydride.This solution has become redness immediately, and at room temperature stirs 4 hours.Dropwise add 1mol/L hydrochloric acid in the reactant mixture and excessive sodium borohydride, reach 7.0 up to pH value.Reactant mixture is placed Millipore centrifugal filter (100K molecular retention), the centrifugal part that came excessive separation in 30 minutes under the 4000rpm rotating speed, supernatant inclines, solid is dissolved in the deionized water of 3.0mL, pass through supersound process, and washed in centrifugal 30 minutes in the centrifugal filter of recentrifuge Millipore company, this washs centrifugal circulation and repeats 5 times, obtains solids and be the gold nano pharmaceutical carrier behind last washing step.
Behind last washing step, the gold nano carrier is dissolved in the 5mL high purity water, and as stock solution, clears up the back and measure gold element content by the ICP-MS method.
The gold nano pharmaceutical carrier is to appendix and the release of doxorubicin:
The PBS solution of the doxorubicin (4.0 μ g/mL are to 500 μ g/mL) of 100 μ L variable concentrations is mixed with the multi-functional gold nano carrier aqueous solution of 100 μ L (500 μ g/mL).With this mixture jolting at room temperature 6 hours.At 13000rpm centrifugal 10 minutes, by the complex of centrifugalize gold nano carrier and doxorubicin.Collect supernatant, the content of doxorubicin in the usefulness HPLC/MS method detection supernatant (the doxorubicin standard solution: 500,250,125,62.5, the dimethyl sulfoxide solution of 31.3,15.6,8.3,4.2 μ mol/L).The content that the doxorubicin content of reaction is removed in the supernatant is the doxorubicin medicament contg that the gold nano carrier adsorbs, and can calculate the gold nano carrier like this to the envelop rate of doxorubicin.
Gold nano carrier and doxorubicin complex are suspended among the PBS again, are that 5.5 and 7.4 buffer dilutes with pH.The burst size of different time points doxorubicin is measured after centrifugal 10 minutes under 13000rpm.
The mensuration of cellular uptake gold nano carrier:
The Hela cell with the MEM culture fluid cultivate (Invitrogen company, IL).A549 cell RPMI1640 culture medium (Invitrogen company, IL).Each cell culture medium is supplemented with 10% hyclone (FBS), 10U/mL penicillin and 10mg/mL streptomycin.
So be used for cell culture 2 orifice plates that the gold nano carrier is taken in.All cells is cultivated in 12 orifice plates with the density of 20,000 cells/well, and culture dish remains under 37 ℃, contains 5%CO
2Wet condition under.Inoculate after 24 hours, with cell with cold PBS washing once, add the fresh culture (using the gold nano-material that contains fluorescence in the flow cytometer experiment) that contains gold nano carrier (50 μ g/mL), cell and gold nano carrier are hatched certain hour (being generally 6 hours, except the cellular uptake of time dependence is measured) altogether.With cold PBS washed cell 3 times to remove extra gold nano carrier.For flow cytometer and ICP-MS experiment, cell is carried out eluting by trypsin-EDTA solution (0.25% trypsin, 1mM EDTA).The cell that comes off is counted, and prepares flow cytometer and ICP-MS experiment then, and each tests triplicate.When carrying out the experiment of transmission electron microscope and dark field microscope, cell is at room temperature fixed 30 minutes with 2.5% glutaraldehyde at the Na-of 0.1M dimethyl arsenic acid buffer liquid (Tousimis research company).Be fixed on the cell direct observation under darkfield microscope on the microscope slide.Intracellular gold nano carrier is shown as bright yellow spotting.
Embodiment 7
The cytotoxic assay of gold nano carrier and doxorubicin:
Cell is cultivated in 96 orifice plates with the density of 5000 cells/well.Inoculate after 24 hours, with cell with cold PBS washing once.The gold nano carrier of variable concentrations or gold nano carrier and doxorubicin complex are joined in the cell, add culture medium in contrast, hatched altogether 8 hours with cell, then with cold PBS washing 3 times, to remove extra gold nano carrier, add fresh culture medium.After cell is hatched 48 hours, with the XTT method cell survival rate is measured, each experiment is carried out 3 times.XTT operates by the experimental procedure that company provides.
The cytotoxic assay of gold nano carrier and doxorubicin under the x-ray irradiation:
Cell is cultivated in 96 orifice plates with the density of 5000 cells/well.Inoculate after 24 hours, with cell with cold PBS washing once.The gold nano carrier of variable concentrations or gold nano carrier and doxorubicin complex are joined in the cell, add culture medium in contrast, hatched altogether 8 hours with cell, then with cold PBS washing 3 times, to remove extra gold nano carrier, add fresh culture medium.96 orifice plates are placed The AXR Minishot160X-ray generator, place obstacles 60KV voltage and mA electric current, and with the 1mm aluminium flake X-ray is disturbed, finally obtain 27.0R/ minute X-ray of stable mean dose rate.In the optimization experiment, fixedly doxorubicin and gold nano carrier doxorubicin complex dosage are 1mmol/L, change irradiation time and obtain different X-transmitted intensities.In toxicity test subsequently, when fixedly X-ray accumulated dose was 3Gy, corresponding irradiation time was 11.1 minutes, changed the dosage of the doxorubicin of doxorubicin or gold nano carrier absorption.Shine and measure with the cytoactive of XTT after 48 hours.In all experiments, use same material to handle cell, but the cytoactive of not passing through x-ray irradiation is as contrast, all test triplicate.
Embodiment 9
DNA damage is measured under the different condition:
The damage of DNA is measured by (phosphorylation of H2AX) method of γ-H2AX.Cell is inoculated in the 35mm culture dish by appropriate density, after the incubated overnight, handled cell 8 hours with doxorubicin or gold nano carrier doxorubicin complex (0.1 μ mol/L doxorubicin dosage), and with PBS washing 3 times, to remove extra gold nano carrier.Cell and was cultivated 48 hours after the X-ray shines again.There is not the cell of x-ray irradiation under the equal conditions in contrast.Use 4% paraformaldehyde of prepared fresh under 4 ° of C, to carry out fixed cell 15 minutes, destroyed permeability of cell membrane in 10 minutes with 0.25% Triton X-100PBS solution-treated then.Cell cleans 3 times with PBS, and at room temperature handles with the PBS solution of 1% bovine serum albumin (BSA) and to interrupt in 30 minutes., (Millipore blocks in the Bill, and MA) incubated cell is 1 hour with the antibody of the mouse anti γ-H2AX of 100 times of the PBS dilutions that contains 1%BSA.Wash three times to remove unconjugated first antibody with PBS.Handled cell 1 hour with 500 times of diluent lucifuges of the anti-mouse IgG antibody of FITC labelled goat (Jackson Immunoresearch Labs), cell cleans 3 times with PBS.Use DAPI(Vector Laboratories at last, Burlingame CA) comes the imaging nucleus.(Carl Zeiss MicroImaging, Jena Germany) takes pictures to the result with Zeiss LSM510 fluorescence microscope.For the quantitative analysis of γ-H2AX, each sample counting is at least the percentage ratio sum that 500 nuclears and dna double chain interruption are expressed as the number that the positive cell of γ-H2AX examines.
Claims (5)
1. gold nano pharmaceutical carrier with synergistic antitumor effect, described gold nano pharmaceutical carrier are used for the functional group of appendix hydrophobic drug by difunctional targeting part and one and constitute; It is characterized in that: described difunctional targeting part is respectively the folate molecule of thioctic acid modification and the substituent of the tyrosine that thioctic acid is modified, and the described functional group that is used for the appendix hydrophobic drug is the beta-schardinger dextrin-that thioctic acid is modified; The pattern of gold nano pharmaceutical carrier is spherical, particle size distribution is between 3-6nm, dynamically particle diameter determines that with dynamic light scattering (DLS) method it is 16-17nm in the PBS buffer, the surface potential Zeta potential value of gold nano pharmaceutical carrier in aqueous solution is distributed as from-35 to-46mV, when it is suspended in when containing in the 1% serum aqueous solution, the Zeta potential value moves to-15mV.
2. the gold nano pharmaceutical carrier that has the synergistic antitumor effect according to claim 1; it is characterized in that: the substituent of the tyrosine that described thioctic acid is modified is methyl 2-(3-(4-(5-(1,2-dithiolane-3-yl) the valeryl amido) the 4-chlorphenyl)-2-Benzamido-propiono)-the 2-(4-hydroxy phenyl) methyl acetate.
3. have the gold nano pharmaceutical carrier of synergistic antitumor effect according to claim 1, it is characterized in that: described gold nano pharmaceutical carrier is made by following method:
1) ligand-1 thioctic acid-beta-schardinger dextrin-, ligand-2 thioctic acid-folic acid are dissolved in respectively in the high purity water, ligand-3 thioctic acid-tyrosine substituent is dissolved in the methanol, and make ligand-1: ligand-2: the mol ratio of ligand-3 is 1:1:1; Gold chloride (III) trihydrate (being the tetra chlorauric acid trihydrate) is dissolved in the high purity water, and wherein the consumption of gold chloride (III) trihydrate is ligand-1, ligand-2 and ligand-3 mole sum; Sodium borohydride is dissolved in the water, and wherein the sodium borohydride consumption is 2 times of gold chloride (III) trihydrate consumption;
Above-mentioned ligand-1, ligand-2 and ligand-3 structural formula are as follows:
Ligand-1 ligand-2 ligand-3
2) under the stirring condition ligand-1, ligand-2 and ligand-3 solution for preparing is mixed, slowly join then in the chlorauric acid solution, under room temperature condition, stirred 30 ± 5 minutes;
3) sodium borohydride solution is dropwise joined in gold chloride and the ligand mixture, and at room temperature stirred 4-9 hour;
4) in the reactant mixture of step 3), dropwise add 1mol/L hydrochloric acid, the excessive sodium borohydride that neutralizes, and make pH value reach 7.0, cessation reaction:
5) reactant mixture with step 4) places centrifugal filter, with 100K molecular retention under the 4000r.p.m. rotating speed centrifugal 30 ± 5 minutes, separates and removes excessive ligand; The solid that separates is dissolved in the deionized water, after the conventional supersound process, again with 100K molecular retention under the 4000r.p.m. rotating speed centrifugal 30 ± 5 minutes, and so repeated centrifugation washing 5 ± 1 times; Behind last washing step, obtain solids and be the gold nano pharmaceutical carrier.
4. as having the gold nano pharmaceutical carrier of synergistic antitumor effect as described in the claim 3, it is characterized in that: the described mixing time of step 3) is 4-6 hour.
5. the described application that has the gold nano pharmaceutical carrier absorption doxorubicin of synergistic antitumor effect and strengthen cytotoxicity in conjunction with X-ray selectivity of claim 1.
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Application publication date: 20130807 |