CN102614522A - Preparation of dendrimer-modified multi-walled carbon nanotube type composite material-loaded adriamycin - Google Patents
Preparation of dendrimer-modified multi-walled carbon nanotube type composite material-loaded adriamycin Download PDFInfo
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Abstract
The invention relates to a preparation method of dendrimer-modified multi-walled carbon nanotube type composite material-loaded adriamycin, which comprises the steps of: (1) preparing G5-FI; (2) preparing G5-FI-FA; (3) preparing MWCNT-G5-FI-FA; (4) preparing MWCNT-G5-FI-FA-Ac; and (5) preparing a drug-loaded complex DOX/MWCNT-G5-FI-FA-Ac. The preparation process is simple, experiments are implemented at room temperature and under atmospheric pressure, operation is facilitated, and the applied preparation procedures can be applied in preparation of other functional dendrimers and have good practical values.
Description
Technical field
The invention belongs to the adriablastina target transport field, particularly a kind of method for preparing of dendrimer modifying multiwall carbon nano-tube composite load amycin.
Background technology
CNT (CNTs) is a kind of novel nano material with carbon element that is curled and formed by single or multiple lift carbon graphite lamella, is divided into SWCN (SWCNTs) and multi-walled carbon nano-tubes (MWCNTs).The tubular structure that CNTs is special makes it have unique mechanical, optics, electromagnetic property, an emissivity and thermal characteristic, and at numerous areas such as high strength composite, field emission apparatus, pick off, detectors potential application prospect is arranged all.Research shows, CNTs can get into cell and toxigenicity (Yang W., Thordarson P. not through passive diffusion and endocytosis initiatively; Gooding J.J.; Ringer S.P., Braet F., Carbon nanotubes for biological and biomedical applications.Nanotechnology; 2007.18:412001.), and among the CNTs carbon atom with SP
2Hybridism is main, and its surface is made up of a large amount of aromatic structures, can combine with form covalently or non-covalently with multiple inorganic and organic molecule.The hollow structure that CNTs is unique makes it than ball shaped nano carrier bigger specific surface area arranged, and higher medicine useful load (Li Shanshan, He Hua, Jiao Qingcai, CNT is in the application in medicine and gene delivery field. chemical progress, 2008.20 (11): 1798-1803).CNTs good hydrophilic property after function modified, good biocompatibility, and have long blood circulation time; Can be through metabolism Safety Sweep (Bianco A.; Kostarelos K., Partidos C.D., Prato M.; Biomedical applications of functionalised carbon nanotubes.Chemical Communications, 2005 (5): 571-577).Therefore CNTs as carrier can drug loading, immobilized enzyme, conveying DNA etc., obtain application more and more widely at functional material, biotechnology and biomedical sector.
Amycin (DOX) is clinical antitumor drug commonly used, is widely used in the chemotherapy of leukemia, pulmonary carcinoma, malignant lymphoma, soft tissue neoplasms, osteosarcoma, bladder cancer, breast carcinoma, thyroid carcinoma etc.Owing to DOX can the intercalation of DNA suppress the synthetic of nucleic acid, so it can produce biochemical effect widely to body, has intensive cytotoxicity.But in traditional chemotherapy process, owing to lack selectivity, in kill cancer cell, it also produces very big murder by poisoning to normal structure, and its clinical modal toxic and side effects is exactly a cardiac toxicity.Nearest research shows that CNTs can pass through pi-pi accumulation used load amycin, and this load is that pH is dependent, can under the low environment of pH value, discharge drug molecule.And tumor tissues compares with normal structure and has faint acidity, and therefore, this load effect of CNTs can be released in tumor locus with drug molecule effectively; Thereby reduce toxic and side effects (the Liu Z. of medicine to normal structure and organ; Sun X., Nakayama-Ratchford N., Dai H.; Supramolecular chemistry on water-soluble carbon nanotubes for drug loading and delivery.ACS Nano, 2007.1 (1): 50-56).Combine CNTs under the irradiation of near-infrared laser, can cause the characteristic of controlled delivery of pharmaceutical agents release and himself heat production in addition, can realize targeted chemotherapy and thermotherapy (Chakravarty P., Marches R. tumor cell; Zimmerman N.S., Swafford A.D.E., Bajaj P.; Musselman I.H.; Pantano P., Draper R.K., Vitetta E.S.; Thermal ablation of tumor cells with antibody-functionalized single-walled carbon nanotubes.Proceedings of the National Academy of Sciences, 2008.105 (25): 8697-8702).
The monodispersity that the PAMAM dendrimer is possessed, internal cavity structures and the characteristics such as good biocompatibility that after modifying, have; Make that it can be modified effectively, assembling and biological functional inorganic nano material; Be widely used in the polyfunctional molecule imaging, and the early diagnosis and therapy of tumor.(Shi X., Wang S.H., Shen M. such as Shi; Antwerp M.E., Chen X., Li C.; Petersen E.J., Huang Q., Weber Jr W.J.; Baker Jr J.R., Multifunctional dendrimer-modified multiwalled carbon nanotubes:Synthesis, characterization; And in vitro cancer cell targeting and imaging.Biomacromolecules, 2009.10 (7): 1744-1750) utilize multifunction the 5th generation PAMAM dendrimer to modify MWCNTs, not only improved dispersibility and the biocompatibility of MWCNTs; Simultaneously can also connect fluorophor and targeting group, realizing fluorescent tracing and to increase tissue specificity, and be successfully used to the targeted imaging of relevant cancerous cell.
Yet; Retrieval shows about the document and the patent of DOX targeting transportation aspect both at home and abroad: the MWCNTs that modifies with the multifunction dendrimer is a carrier material; Through pi-pi accumulation used load amycin, the research that reaches the multi-efficiency aspect of targeting, pH controlled release and fluorescence imaging is not also reported.It not only can realize the imaging of cancerous cell, kill cancer cell optionally, and normal structure and cell are not produced murder by poisoning.Therefore this method will be expanded the range of application of DOX on clinical medicine.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method for preparing of dendrimer modifying multiwall carbon nano-tube composite load amycin; It is simple that this method prepares process; Experiment condition is a normal temperature and pressure; Easy operating, the preparation procedure that is adopted can be used for preparing the macromolecular preparation of other functional dendritic, have good practical value.
The method for preparing of a kind of dendrimer modifying multiwall carbon nano-tube composite load amycin of the present invention comprises:
(1) end with 11.92-17.88mg is the 5th amino generation polyamide-amide dendrimer G5-NH
2Be dissolved in the DMSO solution of 4-6mL, and dropwise add the DMSO solution that 2-3mL contains the Fluorescein isothiocyanate FI of 0.893-1.340mg, at room temperature stirring reaction 12-24h obtains G5-FI;
(2) the folic acid FA with 1.012-1.653mg is dissolved in the DMSO solution of 5-7.5mL; Add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride of 4.393-6.590mg then, at room temperature behind the stirring reaction 2-4h, dropwise join in the middle of the DMSO solution of above-mentioned G5-FI; Stirring reaction 1-3 days; The dialysis reactant liquor is removed reaction dissolvent DMSO, excessive reactant and byproduct of reaction, and the aqueous solution lyophilization with product at last obtains G5-FI-FA;
(3) the carboxylated multi-walled carbon nano-tubes MWCNTs with 40.00-60.00mg is dissolved in the 20-30mL water; 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride that adds 32.39-48.59mg then; At room temperature behind the stirring reaction 2-4h; Add 5-7.5mL then and contain the aqueous solution of the G5-FI-FA of above-mentioned 9.17-13.76mg, obtained MWCNT-G5-FI-FA in stirring reaction 1-3 days;
(4) in the reactant liquor of step (3), add 20-30 μ L triethylamine; After mixing 20-40min; The acetic anhydride that adds 13.44-20.16 μ L again, stirring reaction 12-24h under the room temperature, dialysis reactant liquor; Excessive reactant and byproduct of reaction are removed, and the aqueous solution lyophilization with product at last obtains MWCNT-G5-FI-FA-Ac;
(5) take by weighing the above-mentioned MWCNT-G5-FI-FA-Ac of 16-24mg and being dispersed in the ultra-pure water, be made into the dispersion liquid of 2mg/mL, it is even to be placed in the ultrasonic washing instrument ultra-sonic dispersion; The aqueous solution that adds the doxorubicin hydrochloride DOX of isopyknic 2mg/mL then; Regulate mixed solution pH to 9.5 then, under the condition of lucifuge, continue to stir 12-24h, DOX is fully combined with MWCNT-G5-FI-FA-Ac; Centrifugal, remove supernatant, reuse pH is 9.5 water washing and precipitating thing, is colourless until supernatant, promptly gets medicine carrying complex DOX/MWCNT-G5-FI-FA-Ac.
The concrete technology of dialysis in said step (2) and (4) is for adopting bag filter, in pH is 7.4 PBS buffer, dialyses earlier, in distilled water, dialyses again.
The mass ratio of MWCNT-G5-FI-FA-Ac and DOX is 1: 1 in the said step (5), and the concentration of MWCNT-G5-FI-FA-Ac and DOX is 1mg/mL.
Adopt the sodium hydroxide solution of 0.1M to regulate pH to 9.5 in the said step (5).
Among the present invention with FI be grafted to terminal for amino the 5th generation polyamide-amide dendrimer surface, FI is employed in the mode that dropwise drips under the quick stirring and joins in the dendrimer solution, to guarantee the homogeneity of dendrimer grafting FI.
Use 5-10 doubly to measure EDC activated carboxyl and amino reaction among the present invention, intensified response is active.To reduce its surface potential, improve the biocompatibility of material with the remaining amino in acetylation dendrimer surface.Keep the alkaline environment of solution to guarantee carrying out smoothly of acetylization reaction with triethylamine.
Preparation, medicament slow release and the neoplasm targeted therapy research of the MWCNTs load DOX that multi-functional dendrimer is modified, carried out UV-Vis (uv-vis spectra), TEM (transmission electron microscope), TGA (thermogravimetic analysis (TGA)), MTT (cell viability analysis) and laser confocal microscope analysis:
(1)
1H NMR test result
With
1H NMR test characterizes the modification of multifunction dendrimer, and the result shows and successfully synthesizes multi-functional dendrimer G5-FI-FA and G5-FI.Average each strong FI that closes in dendrimer surface and the quantity of FA are respectively 5.0 and 4.6.Referring to description of drawings 1.
(2) UV-Vis test result
FA has stronger absworption peak at the 280nm place, and FI has stronger absworption peak at the 501nm place, and the UV-Vis collection of illustrative plates has verified that these base group modifications are on the dendrimer, referring to accompanying drawing 2 and 3.As can be seen from Figure 2; G5-FI has absworption peak at the 501nm place; Simultaneously a less absorption is arranged, and G5-FI-FA there is stronger absworption peak at 280nm and 501nm place, compares with G5-FI at 280nm; The absorption at 280nm place is more increased, and shows FI and FA molecule being connected on the dendrimer of success.As can beappreciated from fig. 3; MWCNT-G5-FI-Ac has absworption peak at the 501nm place; MWCNT-G5-FI-FA-Ac has stronger absworption peak at 280nm and 501nm place; Compare the absorption at 280nm place with MWCNT-G5-FI-Ac and more increase, show G5-FI-FA molecule being connected on the MWCNTs of success.
(3) TGA test result
With the TGA analysis grafting amount of functional dendritic macromole on MWCNTs analyzed, referring to accompanying drawing 4.With 566 ℃ of temperature as the loss of calculating thermogravimetric, under this temperature, MWCNTs only has 5% the loss in weight, and the loss of the thermogravimetric of MWCNT-G5-FI-Ac and MWCNT-G5-FI-FA-Ac is respectively 22% and 22.2%.So nearly 17% the loss in weight comes from G5-FI-Ac and the G5-FI-FA-Ac that is modified at the MWCNTs surface.
(4) TEM characterizes
Use the not influence of MWCNTs structure in order to verify that tree-shaped macromolecular modification turns into, the pattern of MWCNT-G5-FI-FA-Ac has been done the TEM sign, referring to accompanying drawing 5.As can be seen from the figure, MWCNTs remains hollow tubular structure after modifying through the multifunction dendrimer, i.e. modification turns the pattern of using MWCNTs into and do not exert an influence.
(5) drug release result
The release behavior of DOX in research DOX/MWCNT-G5-FI-FA-Ac and the DOX/MWCNT-G5-FI-Ac complex in the phosphate buffer of the acetate buffer of pH 5.4 and pH 7.4 respectively, its release profiles is referring to accompanying drawing 6.The result shows: DOX/MWCNT-G5-FI-FA-Ac and DOX/MWCNT-G5-FI-Ac medicine carrying composite all show tangible pH controlled-release effect.For DOX/MWCNT-G5-FI-FA-Ac, the burst size of medicine is respectively 66.7% and 12.1% behind the 12h in the sustained-release liquid of pH=5.4 and pH=7.4.Behind the slow release 24h, the former has 77.4% DOX to discharge, and the latter has only 13.5% release.Therefore there is bigger influence in pH value to the release of DOX, when pH value is relatively lower, and the DOX good water solubility, the MWCNTs that modifies from the multifunction dendrimer more easily separates, and can reach the purpose of pH sustained release.
(6) MTT cytotoxicity analysis
Study the drug effect of DOX/MWCNT-G5-FI-FA-Ac and DOX/MWCNT-G5-FI-Ac with the KB cell.Behind free drug DOX, DOX/MWCNT-G5-FI-FA-Ac complex and the DOX/MWCNT-G5-FI-Ac complex and KB co-culture of cells 48h with different DOX concentration (0.5,1,2,4 μ M), with the vigor of mtt assay detection cell, referring to accompanying drawing 7.As can be seen from the figure; With respect to untreated KB cell; Free drug DOX, DOX/MWCNT-G5-FI-Ac complex and DOX/MWCNT-G5-FI-FA-Ac complex all have very big toxicity to the KB cell, and the drug effect of DOX does not have too big difference between them.The cytotoxicity MTT of resulting not medicine carrying MWCNT-G5-FI-FA-Ac and MWCNT-G5-FI-Ac carrier material analyzes and finds (referring to accompanying drawing 8 simultaneously; What wherein the concentration of multifunction MWCNTs was got is to reach 1,2, the concentration of MWCNTs during 4 μ M when DOX concentration); These two materials itself are nontoxic (comparing with contrast; P value＞0.05), show that DOX/MWCNT-G5-FI-FA-Ac is only relevant with the DOX medicine to the drug effect of KB cell with DOX/MWCNT-G5-FI-Ac, and irrelevant with carrier material.
(7) the MTT drug targeting is analyzed
Behind DOX/MWCNT-G5-FI-Ac and DOX/MWCNT-G5-FI-FA-Ac (DOX concentration is 1 μ M) and KB-HFAR (homofolic acid expression of receptor) and KB-LFAR (low folacin receptor is expressed) co-culture of cells 1h; Outwell the culture medium that contains material; Add the corresponding culture medium that does not contain material again after giving a baby a bath on the third day after its birth time with the PBS buffer and then cultivate 48h; Detect the vigor of cell with the MTT method, referring to accompanying drawing 9.The cell viability of the KB-HFAR cell of handling with DOX/MWCNT-G5-FI-FA-Ac significantly reduces (approximately be control cells vigor 67%) (p value＜0.05).On the contrary, do not contain the KB-HFAR cell that the DOX/MWCNT-G5-FI-Ac complex of FA handled and approximately still have 90% survival.Simultaneously, about 96% KB-LFAR cell still survival after DOX/MWCNT-G5-FI-FA-Ac handles.This shows that DOX/MWCNT-G5-FI-FA-Ac can targeting ground suppresses the growth of the cancerous cell of surperficial high expressed folacin receptor.
(8) laser confocal microscope analysis
Fluorescein isothiocyanate be connected to the 5th generation dendrimer surface, be for when DOX/MWCNT-G5-FI-FA-Ac is taken in by cell, can follow the tracks of detection through fluorescence imaging, the endocytosis of observation medicine and carrier molecule.Because drug molecule DOX itself can rubescent color fluorescence; Can green-emitting fluorescence and contain fluorescein (FI) on our carrier material, so we further observe the endocytosis situation of medicine carrying material in different cells (KB-HFAR and KB-LFAR) with laser confocal microscope.Behind 1h after DOX/MWCNT-G5-FI-FA-Ac (DOX concentration is 1 μ M) and KB-HFAR and the KB-LFAR co-culture of cells; Outwell the culture medium that contains material; Add the corresponding culture medium that does not contain material again after giving a baby a bath on the third day after its birth time with PBS and then cultivate 4h; Come the endocytosis effect of observation of cell to medicine with laser confocal microscope, have only those KB-HFAR that stronger fluorescence signal is arranged, this explanation DOX/MWCNT-G5-FI-FA-Ac complex targeting enters into cell interior.On the contrary, under the same conditions, KB-LFAR does not but have tangible fluorescence signal.This explanation, DOX/MWCNT-G5-FI-FA-Ac has good targeting effect.
The present invention makes full use of the numerous functional group in PAMAM dendrimer surface; Through covalent bond it is modified and functionalization; Connect fluorescence imaging molecule and targeted molecular; Modify the MWCNTs surface again, not only improve the aqueous solution dispersibility and the biocompatibility of CNT, also give CNT fluorescence imaging and targeting property.Can follow the tracks of detection through the fluorescence molecule imaging, observe the endocytosis of medicine and carrier molecule.Through targeted molecular, can the medicine orientation be transported to cancerous cell.Utilize the pi-pi accumulation effect of MWCNTs and DOX to realize the high-efficient carrier of DOX and the advantage that the tumor slightly acidic environment discharges simultaneously, further realize the cancerous cell targeted and the treatment of medicine.
Beneficial effect
(1) preparation process of the present invention is simple, and experiment condition is a normal temperature and pressure, and easy operating, the preparation procedure that is adopted can be used for preparing the macromolecular preparation of other functional dendritic, have good practical value;
(2) the present invention has realized the cancerous cell targeted of DOX medicine, can effectively reduce its toxic and side effects; Utilize the pi-pi accumulation effect of MWCNTs and DOX to realize that the high-efficient carrier of DOX and tumor slightly acidic environment discharge, and have good practical value simultaneously;
(3) the present invention utilizes fluorescein to be fluorescent tag molecule, and then observes the endocytosis of medicine and carrier material.
Description of drawings
Fig. 1 is for the multi-functional dendrimer G5-FI (a) of the present invention preparation and G5-FI-FA (b)
1H NMR collection of illustrative plates;
(curve a) and the UV-Vis collection of illustrative plates of G5-FI-FA (curve b) for the multi-functional dendrimer G5-FI of the present invention preparation for Fig. 2;
Fig. 3 be MWCNTs (curve a), the UV-Vis collection of illustrative plates of MWCNT-G5-FI-Ac (curve b) and MWCNT-G5-FI-FA-Ac (curve c);
Fig. 4 is that (curve a), the TGA of MWCNT-G5-FI-FA-Ac (curve b) and MWCNT-G5-FI-Ac (curve c) analyzes for MWCNTs;
Fig. 5 is the TEM picture of MWCNT-G5-FI-FA-Ac;
Fig. 6 is under different pH condition, the elution profiles of DOX on dendrimer modificationization MWCNTs;
Fig. 7 is that the MTT of the KB cell viability of variable concentrations DOX and the processing of DOX multifunction carbon mano-tube composite analyzes;
Fig. 8 is that the KB cell viability MTT of the multifunction CNT of not medicine carrying analyzes;
Fig. 9 is contrast KB-HFAR cell (a), the vigor figure of the KB-HFAR cell (d) that KB-LFAR cell (c) that KB-HFAR cell of handling with DOX/MWCNT-G5-FI-FA-Ac (b) and DOX/MWCNT-G5-FI-FA-Ac handled and DOX/MWCNT-G5-FI-Ac handled.
The specific embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.Should be understood that in addition those skilled in the art can do various changes or modification to the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
With G5-NH
2(11.92mg) be dissolved in the DMSO solution of 4mL, under condition of stirring, dropwise add the DMSO solution that 2mL contains FI (0.893mg).After this reaction is at room temperature stirred 12-24h, obtain the DMSO solution of unpurified G5-FI.FA (1.012mg) is dissolved in the DMSO solution of 5mL, adds EDC (4.393mg) then, at ambient temperature behind the stirring reaction 2h; Mixed solution is dropwise joined in the middle of the DMSO solution of unpurified G5-FI, stirring reaction 1 day is put into bag filter (MWCO=14 with reaction solution at last; 000) in, reaction dissolvent DMSO, excessive reactant and byproduct of reaction are removed, earlier with PBS buffer dialysis 3 times through dialysis; Each 2L; Reuse distill water dialysis 5 times, each 2L, the aqueous solution lyophilization with product at last obtains G5-FI-FA;
Carboxylated MWCNTs (40.00mg) is dissolved in the 20mL water, adds EDC (32.39mg) then, behind the stirring reaction 2h, add the aqueous solution that 5mL contains G5-FI-FA (9.17mg), stirring reaction 2 days at ambient temperature.Add 20 μ L triethylamines again, mix 30min after, add 13.44 μ L acetic anhydrides again, stirring reaction 15h under the room temperature.At last reaction solution is put into bag filter (MWCO=50; 000) in, excessive reactant and byproduct of reaction are removed, earlier with PBS buffer dialysis 3 times through dialysis; Each 2L; Reuse distill water dialysis 5 times, each 2L, the aqueous solution lyophilization with product at last obtains MWCNT-G5-FI-FA-Ac;
Product G5-FI-FA's
1H NMR collection of illustrative plates such as accompanying drawing 1b, result show average 5.0 FI molecules of each dendrimer surface grafting and 4.6 FA molecules.The characteristic absorption peak that while UV-Vis collection of illustrative plates (seeing accompanying drawing 2, curve b) also can be seen FI is at 501nm, and the characteristic absorption peak of FA explains that at 280nm FI and FA successfully have been grafted on the dendrimer surface.The characteristic absorption peak that also can see FI from the UV-Vis collection of illustrative plates (seeing accompanying drawing 3, curve c) of product MWCNT-G5-FI-FA-Ac is at 501nm, and the characteristic absorption peak of FA explains that at 280nm G5-FI-FA successfully is grafted to the multi-wall carbon nano-tube tube-surface.
With TGA the multifunction dendrimer is analyzed in the grafting amount on MWCNTs surface, referring to accompanying drawing 4b.With 566 ℃ of temperature as the loss of calculating thermogravimetric, under this temperature, MWCNTs only has 5% the loss in weight, and the loss of the thermogravimetric of MWCNT-G5-FI-FA-Ac is 22.2%.So nearly 17% the loss in weight comes from the G5-FI-FA-Ac that is modified at the MWCNTs surface.
Further MWCNT-G5-FI-FA-Ac has been done the TEM sign, referring to accompanying drawing 5.As can be seen from the figure, MWCNTs remains hollow tubular structure after modifying through the multifunction dendrimer, promptly modifies the pattern that turns into not changing MWCNTs.
With G5-NH
2(17.88mg) be dissolved in the DMSO solution of 6mL, under condition of stirring, dropwise add the DMSO solution that 3mL contains FI (1.340mg).After this reaction is at room temperature stirred 24h, obtain the DMSO solution of unpurified G5-FI.FA (1.653mg) is dissolved in the DMSO solution of 7.5mL, adds EDC (6.590mg) then, at ambient temperature behind the stirring reaction 4h; Mixed solution is dropwise joined in the middle of the DMSO solution of unpurified G5-FI, stirring reaction 3 days is put into bag filter (MWCO=14 with reaction solution at last; 000) in, reaction dissolvent DMSO, excessive reactant and byproduct of reaction are removed, earlier with PBS buffer dialysis 3 times through dialysis; Each 2L; Reuse distill water dialysis 5 times, each 2L, the aqueous solution lyophilization with product at last obtains G5-FI-FA;
Carboxylated MWCNTs (60.00mg) is dissolved in the 30mL water, adds EDC (48.59mg) then, behind the stirring reaction 3h, add the aqueous solution that 7.5mL contains G5-FI-FA (9.17-13.76mg), stirring reaction 3 days at ambient temperature.Add 30 μ L triethylamines again, mix 40min after, add 20.16 μ L acetic anhydrides again, stirring reaction 24h under the room temperature.At last reaction solution is put into bag filter (MWCO=50; 000) in, excessive reactant and byproduct of reaction are removed, earlier with PBS buffer dialysis 3 times through dialysis; Each 2L; Reuse distill water dialysis 5 times, each 2L, the aqueous solution lyophilization with product at last obtains MWCNT-G5-FI-FA-Ac;
Product G5-FI-FA's
1H NMR collection of illustrative plates (accompanying drawing 1b) result shows average 5.0 FI molecules of each dendrimer surface grafting and 4.6 FA molecules.The characteristic absorption peak that while UV-Vis collection of illustrative plates also can be seen FI is at 501nm, and the characteristic absorption peak of FA is at 280nm (seeing accompanying drawing 2, curve b).Simultaneously, the characteristic absorption peak that the UV-Vis collection of illustrative plates of product MWCNT-G5-FI-FA-Ac (seeing accompanying drawing 3, curve c) also can be seen FI is at 501nm, and the characteristic absorption peak of FA explains that at 280nm G5-FI-FA successfully is grafted to the multi-wall carbon nano-tube tube-surface.
Embodiment 3
Take by weighing MWCNT-G5-FI-Ac and MWCNT-G5-FI-FA-Ac (20.0mg) respectively and be dispersed in the ultra-pure water, be made into the dispersion liquid of 2mg/mL, it is even to be placed in the ultrasonic washing instrument ultra-sonic dispersion.The aqueous solution that adds the DOX of isopyknic 2mg/mL then, making the mass ratio of DOX and MWCNTs is 1: 1, concentration is 1mg/mL.Using the 0.1M sodium hydrate aqueous solution to regulate pH then is 9.5.Under the condition of lucifuge, continue to mix solution 24h, DOX is fully combined with MWCNT-G5-FI-Ac or MWCNT-G5-FI-FA-Ac.Afterwards; Mixed solution is centrifugal, remove supernatant, the water washing and precipitating thing of reuse pH=9.5; Until supernatant is colourless; Collect the supernatant of all eluting, the amount of the DOX that washes off through calculating is calculated the percentage ratio that uploads to the DOX on the multifunction CNT, and its envelop rate and drug loading are 98%.
The phosphate buffer of acetate buffer and pH=7.4 of selecting pH=5.4 is respectively analyzed the release performance of DOX under two kinds of pH environment as the slow release media.Take by weighing MWCNT-G5-FI-Ac and MWCNT-G5-FI-FA-Ac (2.0mg) at first respectively, add acetate buffer (pH=5.4) or the PBS buffer (pH=7.4) of 2mL respectively.Scattered solution is inserted (the molecular retention amount is 14,000) in the bag filter, insert the bag filter that sample is housed in the ampoule that the corresponding buffer of 13mL is housed then, overall solution volume is 15mL in the bottle, and three in each sample is parallel.At constant temperature (37 ℃) shaking table, test its release performance down for 90 rpms.For all slow release samples, according to the interval that designs (0.5,1,2,4,6,9,12,24,48 and 72h), from sample bottle, take out the 5mL sustained-release liquid at each time point, add the new buffer of 5mL again, keep the constancy of volume of solution.According to light absorption value and DOX the concentration-light absorption value standard curve under two kind buffer of the DOX that discharges at the 481nm place, obtain DOX in the total amount that different time point accumulative total discharges, analyze the release dynamics characteristic of medicine.
Its release profiles is referring to accompanying drawing 6.As can be seen from the figure, be the DOX/MWCNT-G5-FI-FA-Ac of band folic acid or all do not show tangible pH controlled-release effect with the medicine carrying composite of the DOX/MWCNT-G5-FI-Ac of folic acid.For DOX/MWCNT-G5-FI-Ac, in the sustained-release liquid of pH=5.4 and pH=7.4, the burst size of medicine is respectively 68.5% and 13.1% behind the 12h.Behind the slow release 24h, the former has 80.4% DOX to discharge, and the latter has only 14.3% release.For DOX/MWCNT-G5-FI-FA-Ac, the burst size of medicine is respectively 66.7% and 12.1% behind the 12h in the sustained-release liquid of pH=5.4 and pH=7.4.Behind the slow release 24h, the former has 77.4% DOX to discharge, and the latter has only 13.5% release.Therefore there is bigger influence in pH value to the release of DOX; When pH value is relatively lower, the DOX good water solubility, the MWCNTs that modifies from the multifunction dendrimer more easily separates; Can reach the purpose of pH sustained release, realize directed release of slightly acidic environment of tumor tissues.
Embodiment 5
Collect the logarithmic (log) phase cell earlier, join 96 porocyte culture plates, every hole adds the celliferous culture medium of 200 μ L makes cell density to 8000/ hole, and edge hole is filled with aseptic PBS buffer; Then at cell culture incubator (5% CO
2, 37 ℃) in hatch 24h, be paved with the hole to cell monolayer at the bottom of (96 hole flat underside); Outwelled culture medium in second day, add 180 μ L culture medium then, (each material is that benchmark is made as 4 concentration with DOX to add the 20 μ LPBS buffer of DOX, DOX/MWCNT-G5-FI-Ac or the DOX/MWCNT-G5-FI-FA-Ac contain variable concentrations again; Promptly 0.5; 1,2,4 μ M).Design not medicine carrying MWCNT-G5-FI-FA-Ac and MWCNT-G5-FI-Ac materials control group simultaneously, every hole adds the PBS buffer 20 μ L of the material that contains respective concentration, adds culture medium 180 μ L again, with the influence of the cell growth of checking material own.All test group and matched group are all established 3 Kong Weiyi parallel-group; After in incubator, hatching 48h, each group cellular morphology is observed with inverted phase contrast microscope.Every then hole adds the MTT solution of 20 μ L, continues to cultivate 4h, and cell and MTT are fully reacted.Stop at last cultivating, the careful suction removed culture fluid in the hole, adds 200 μ L DMSO in every hole, puts lucifuge low-speed oscillation 15min on the shaking table, and crystal is fully dissolved, and measures the MTT Jia Za solution light absorption value in each hole at enzyme-linked immunosorbent assay instrument 492nm place.Statistical analysis is implemented by means of the ANOVA method.In all assessments, think P＜0.05 o'clock, the difference between the sample has significance,statistical.Analysis result shows DOX, DOX/MWCNT-G5-FI-Ac and the DOX/MWCNT-G5-FI-FA-Ac toxic action to the KB cell with cell survival rate.
Mtt assay detects the vigor of handling the back cell, referring to accompanying drawing 7.As can be seen from the figure, with respect to untreated KB cell, pure medicine DOX, DOX/MWCNT-G5-FI-Ac and DOX/MWCNT-G5-FI-FA-Ac have very big toxicity to the KB cell, and the drug effect of DOX does not have too big difference between them.The MTT analysis of our resulting not medicine carrying MWCNT-G5-FI-FA-Ac and MWCNT-G5-FI-Ac material simultaneously can be known (referring to accompanying drawing 8; What wherein the concentration of multifunction MWCNTs was got is to reach 1 when DOX concentration; The concentration of MWCNTs during 2,4 μ M), these two materials itself are nontoxic (comparing with contrast; P value＞0.05), shows that DOX/MWCNT-G5-FI-FA-Ac is only relevant with the DOX of its load to the drug effect of KB cell with DOX/MWCNT-G5-FI-Ac.
Embodiment 6
Collect the logarithmic (log) phase cell; Join 96 porocyte culture plates; Every hole adds the celliferous culture medium of 200 μ L (cell 8000/ hole); Wherein the KB-HFAR group is used the culture medium that does not normally contain folic acid, and the KB-LFAR group is used the culture medium (the folic acid final concentration is 2.5 μ M in Tissue Culture Plate) that contains folic acid; Then at cell culture incubator (5% CO
237 ℃) in hatch 24h; After outwelling original culture medium, add the corresponding culture medium of 180 μ L, add again and contain the DOX/MWCNT-G5-FI-Ac of 10 μ M DOX or the 20 μ L PBS buffer of DOX/MWCNT-G5-FI-FA-Ac (final concentration that with DOX is benchmark is 1 μ M).PBS buffer matched group is cultivated in design simultaneously, and its culture medium is not for containing the culture medium of folic acid.All test group and matched group are all established 3 Kong Weiyi parallel-group; After in incubator, hatching 1h; Outwell the culture medium that contains material, add the corresponding culture medium that does not contain material after giving a baby a bath on the third day after its birth time with the PBS buffer again and continue to cultivate 48h, every then hole adds the MTT solution of 20 μ L; Continue to cultivate 4h; Do the MTT test, the cell of handling with the PBS buffer is contrast, the survival rate of test material group cell.
Accompanying drawing 9 is that the cell viability MTT of the KB-LFAR cell handled of untreated KB-HFAR cell and the KB-HFAR cell of handling with DOX/MWCNT-G5-FI-Ac and DOX/MWCNT-G5-FI-FA-Ac and DOX/MWCNT-G5-FI-FA-Ac analyzes.Can be clear that from figure the cell viability of the KB-HFAR cell of handling with DOX/MWCNT-G5-FI-Ac significantly reduces (approximately be control cells vigor 67%) (p value＜0.05).On the contrary, the KB-HFAR cell handled of about 90% DOX/MWCNT-G5-FI-Ac is still survived.Simultaneously, about 96% KB-LFAR cell still survival after DOX/MWCNT-G5-FI-FA-Ac handles.This result shows, DOX/MWCNT-G5-FI-FA-Ac can targeting ground suppresses the growth of the cancerous cell of surface expression folacin receptor, and this targeting is folacin receptor mediated by the cancerous cell surface.
Observe the endocytosis situation of medicine in different cells (KB-HFAR and KB-LFAR) with laser confocal microscope: experiment uses diameter to be 6cm Laser Scanning Confocal Microscope culture dish cultured cell.The phase cell of promptly taking the logarithm; Each culture dish adds the celliferous culture medium of 2000 μ L (cell 20000/ culture dish); Wherein the KB-HFAR group is used the culture medium that does not contain folic acid, and the KB-LFAR group is used the culture medium (the folic acid final concentration is 2.5 μ M in Tissue Culture Dish) that contains folic acid; Then at 5% CO
2, hatch 24h under 37 ℃, outwell original culture medium after, add the corresponding culture medium of 1800 μ L, add the 200 μ L PBS solution (final concentration that with DOX is benchmark is 1 μ M) of the DOX/MWCNT-G5-FI-FA-Ac that contains 10 μ M DOX again.After in incubator, hatching 1h; Outwell the culture medium that contains material; Add corresponding culture medium again after giving a baby a bath on the third day after its birth time with PBS and then cultivate 4 h; Observe the endocytosis situation of medicine in each group cell with laser confocal microscope then, have only those KB-HFAR that stronger fluorescence signal is arranged, this explanation DOX/MWCNT-G5-FI-FA-Ac complex targeting enters into cell interior.On the contrary, under the same conditions, KB-LFAR does not but have tangible fluorescence signal.This explanation, DOX/MWCNT-G5-FI-FA-Ac has good targeting effect.
Comparative Examples 1
With G5-NH
2(11.82mg) be dissolved in the DMSO solution of 4mL, under condition of stirring, dropwise add the DMSO solution that 2mL contains FI (0.885mg).24h is at room temperature stirred in this reaction.At last reaction solution is put into bag filter (MWCO=14; 000) in, reaction dissolvent DMSO, excessive reactant and byproduct of reaction are removed, earlier with PBS buffer dialysis 4 times through dialysis; Each 2L; Reuse distill water dialysis 4 times, each 2L, the aqueous solution lyophilization with product at last obtains G5-FI.
Carboxylated MWCNTs (40.00mg) is dissolved in the 20mL water, adds EDC (32.39mg) then, behind the stirring reaction 3h, add the aqueous solution that 5mL contains G5-FI (8.5mg), stirring reaction 3 days at ambient temperature.Afterwards, after reaction mixes adding 20 μ L triethylamines in the liquid, mixes 30min, add 13.44 μ L acetic anhydrides again, stirring reaction 24h under the room temperature.At last reaction solution is put in the bag filter (MWCO=50,000), through dialysis excessive reactant and byproduct of reaction are removed, the aqueous solution lyophilization with product at last obtains MWCNT-G5-FI-Ac.
Product G5-FI's
1H NMR collection of illustrative plates such as accompanying drawing 1a, result show average 5.0 FI molecules of each dendrimer surface grafting.The characteristic absorption peak that while UV-Vis collection of illustrative plates (seeing accompanying drawing 2a) also can be seen FI explains that at 501nm FI successfully has been grafted on the dendrimer surface.The characteristic absorption peak that the UV-Vis collection of illustrative plates of product MWCNT-G5-FI-Ac also can be seen FI explains that at 501nm G5-FI successfully is grafted to the MWCNTs surface.
With TGA the multifunction dendrimer is analyzed in the grafting amount on MWCNTs surface, referring to accompanying drawing 4c.With 566 ℃ of temperature as the loss of calculating thermogravimetric, under this temperature, MWCNTs only has 5% the loss in weight, and the loss of the thermogravimetric of MWCNT-G5-FI-Ac is 22%.So nearly 17% the loss in weight comes from the G5-FI-Ac that is modified at the MWCNTs surface.
Claims (4)
1. the method for preparing of a dendrimer modifying multiwall carbon nano-tube composite load amycin comprises:
(1) end with 11.92-17.88mg is the 5th amino generation polyamide-amide dendrimer G5-NH
2Be dissolved in the DMSO solution of 4-6mL, and dropwise add the DMSO solution that 2-3mL contains the Fluorescein isothiocyanate FI of 0.893-1.340mg, at room temperature stirring reaction 12-24h obtains G5-FI;
(2) the folic acid FA with 1.012-1.653mg is dissolved in the DMSO solution of 5-7.5mL; 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride that adds 4.393-6.590mg then; At room temperature behind the stirring reaction 2-4h, dropwise join in the middle of the DMSO solution of above-mentioned G5-FI stirring reaction 1-3 days; The dialysis reactant liquor, the aqueous solution lyophilization with product at last obtains G5-FI-FA;
(3) the carboxylated MWCNTs with 40.00-60.00mg is dissolved in the 20-30mL water; 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride that adds 32.39-48.59mg then; At room temperature behind the stirring reaction 2-4h; Add 5-7.5mL then and contain the aqueous solution of the G5-FI-FA of above-mentioned 9.17-13.76mg, obtained MWCNT-G5-FI-FA in stirring reaction 1-3 days;
(4) in the reactant liquor of step (3), add 20-30 μ L triethylamine; After mixing 20-40min, add the acetic anhydride of 13.44-20.16 μ L again, stirring reaction 12-24h under the room temperature; The dialysis reactant liquor, the aqueous solution lyophilization with product at last obtains MWCNT-G5-FI-FA-Ac;
(5) take by weighing the above-mentioned MWCNT-G5-FI-FA-Ac of 16-24mg and being dispersed in the ultra-pure water, be made into the dispersion liquid of 2mg/mL, add the aqueous solution of the doxorubicin hydrochloride DOX of isopyknic 2mg/mL then; Regulate mixed solution pH to 9.5 then, under the condition of lucifuge, continue to stir 12-24h; Centrifugal, remove supernatant, reuse pH is 9.5 water washing and precipitating thing, is colourless until supernatant, promptly gets medicine carrying complex DOX/MWCNT-G5-FI-FA-Ac.
2. the method for preparing of a kind of dendrimer modifying multiwall carbon nano-tube composite load amycin according to claim 1; It is characterized in that: the concrete technology of dialysis in said step (2) and (4) is for adopting bag filter; In pH is 7.4 PBS buffer, dialyse earlier, in distilled water, dialyse again.
3. the method for preparing of a kind of dendrimer modifying multiwall carbon nano-tube composite load amycin according to claim 1; It is characterized in that: the mass ratio of MWCNT-G5-FI-FA-Ac and DOX is 1: 1 in the said step (5), and the concentration of MWCNT-G5-FI-FA-Ac and DOX is 1mg/mL.
4. the method for preparing of a kind of dendrimer modifying multiwall carbon nano-tube composite load amycin according to claim 1 is characterized in that: adopt the sodium hydroxide solution of 0.1M to regulate pH to 9.5 in the said step (5).
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