CN104940959B - Hyaluronic acid decorated single-walled carbon nanotube prepares method and the application of the reduction-sensitive medicament nano agent of diagnoses and treatment - Google Patents
Hyaluronic acid decorated single-walled carbon nanotube prepares method and the application of the reduction-sensitive medicament nano agent of diagnoses and treatment Download PDFInfo
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 title claims abstract description 93
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Abstract
Method and the application of the reduction-sensitive medicament nano agent of diagnoses and treatment are prepared the present invention relates to hyaluronic acid decorated single-walled carbon nanotube,The tumor-targeting that can effectively solve anticancer drugs, doxorubicin is poor,Toxic side effect is big and targeting is poor,Relaxivity is low,The problem of half-life short,The transport system passes through 3 by the single-walled carbon nanotube after hyaluronic acid decorated,3 ' dithiodipropionic acids are connected as linking arm with adriamycin,Then gadolinium trichloride is adsorbed onto on single-walled carbon nanotube,Formed again with surfactant-dispersed with hyaluronic acid decorated single-walled carbon nanotube,Nanometer formulation for the reduction-sensitive medicine of diagnoses and treatment,The present invention possesses good biocompatibility,Tumor-targeting is good,Thermotherapy is combined with chemotherapy,The advantages of Clinics and Practices are integrated,It is the innovation in diagnosis and tumor,Economic and social benefit is huge.
Description
Technical field
The present invention relates to medicine, particularly a kind of hyaluronic acid (HA) modification single-walled carbon nanotube (SWCNTs) prepares diagnosis
The method of the reduction-sensitive medicament nano agent for the treatment of and application.
Background technology
Malignant tumour serious threat the health and lives safety of the mankind, and its treatment turns into focus of concern, according to
China's epidemiology survey shows that either city or rural area, mortality of malignant tumors are occupied first of all dead diseases.
Doxorubicin hydrochloride (Doxorubicin, DOX) is used as broad-spectrum anti-tumor chemotherapeutics, to the tumour cells of various growth cycles all
There is killing action, clinic is mainly used in treating the superficial solid tumors such as breast cancer, prostate cancer, sarcoma.At present in doxorubicin hydrochloride
City's formulation is injection, larger to the toxicity caused by hemopoietic system and heart due to lacking targeting in Clinical practice.
CNT is the cylinder for being crimped by graphite and being formed, and internal diameter is from 1 nanometer to more than ten nanometers, and external diameter is also several
Nanometer is between more than ten nanometers, and length is typically all in nanoscale or micron order.It is reported that CNT toxicity depends on its table
The functionalization in face, the CNT with high functionalization is generally with smaller toxicity.And without the modification of any hydrophilic radical
Single-walled carbon nanotube (SWCNTs), due to the high hydrophobicity on its surface, cause phase occurs with cell in cell cultivation process
Interaction, including with the polymerization such as intracellular protein, nucleic acid, so as to produce certain cytotoxicity.Hyaluronic acid
(hyaluronic acid, HA) is linear polysaccharide present in a kind of nature, has high-biocompatibility, biodegradable
Property, is eliminated by lymphatic system in vivo, and the advantages that hypotoxicity, HA is widely used in biomedicine field.In addition
Research finds that HA acceptors are related to tumour, and kinds of tumors is overexpressed hyaluronic acid receptor CD44, although normal cell also has CD44
Acceptor, but the CD44 of normal structure is in silence state, is not activated.
Up to the present, the magnetic resonance contrast agent clinically commonly used lacks targeting more, half-life period is shorter, relaxivity is low
And the shortcomings of can causing toxic side effect when largely using, therefore invent a kind of targeting, long half time, relaxivity height and toxicity
Low magnetic resonance contrast agent is technical problem urgently to be resolved hurrily.
The content of the invention
For the above situation, to overcome the defect of prior art, the purpose of the present invention is just to provide a kind of hyaluronic acid and repaiied
Method and the application of the reduction-sensitive medicament nano agent of single-walled carbon nanotube preparation diagnoses and treatment are adornd, can effectively solve anticarcinogen
The problem of tumor-targeting of thing adriamycin is poor, toxic side effect is big poor with targeting, relaxivity is low, half-life short
The technical scheme that the present invention solves is that the transport system is by the single-walled carbon nanotube after hyaluronic acid (HA) modification
(SWCNTs) it is connected by 3,3 '-dithiodipropionic acid as linking arm with adriamycin, gadolinium trichloride is then adsorbed onto single wall
On CNT, then formed with hyaluronic acid decorated single-walled carbon nanotube with surfactant-dispersed, for diagnoses and treatment
The nanometer formulation of reduction-sensitive medicine;The hyaluronic acid is that molecular weight is more than or equal to 600 dalton and is less than or equal to 400kd
Low-molecular-weight hyaluronic acid;The length of the single-walled carbon nanotube is 1~3 μm, a diameter of 1~2nm;The linking arm is carbon
The 3,3 '-dithiodipropionic acid or suberic acid of atomicity 2~12;Disulfide bond in the 3,3 '-dithiodipropionic acid has also
Former sensitiveness, the rapid fracture release medicine under the glutathione effect of tumour cell high concentration.Its preparation method is by following step
It is rapid to realize:
(1) carboxylation carbon pipe (i.e. carboxylic carbon nano-tube) is connected (HA-SWCNTs) with ammonification hyaluronic acid:
25~75mg carboxylation carbon pipes are weighed in the beaker of 15~45ml reaction dissolvents, ice bath, which is visited, surpasses 15~45min, into carboxylic
Change carbon pipe solution;Weigh ammonification hyaluronic acid (HA-NH2) 60~120mg is dissolved in 5-15ml solvent, into ammonification hyaluronic acid
Solution;Weigh EDC (1- ethyls-(3- dimethylaminopropyls) carbodiimide) 120~240mg, NHS (HOSu NHS)
80~160mg, it is vortexed and is dissolved with 1~7ml solvents, is added in carboxylation carbon pipe solution, stirring 5~25min of room temperature reaction, into carboxylic
Change carbon pipe mixed solution;The μ l of triethylamine 100~260 are added in ammonification hyaluronic acid solution, then by ammonification hyaluronic acid and
The mixed solution fast drop of triethylamine reacts at room temperature 8~40h into carboxylation carbon pipe mixed solution, and it is pre- to add 3~4 times of volumes
Cold acetone, cooling crystallization, filtering, acetone washing precipitation, sediment is obtained, sediment is redissolved with water, and dialyse 2d, freezes;
Described solvent is water, formamide or N,N-dimethylformamide and water or formamide and water or N, N- diformazan
The mixed solvent of base formamide and formamide, or the mixture of other similar solvents and other similar solvents;
Described ammonification hyaluronic acid is, by hyaluronic acid 95-105mg, 1- ethyl-(3- dimethylaminopropyls) carbon two
Inferior amine salt hydrochlorate 254-264mg and n-hydroxysuccinimide 150-160mg, is dissolved in 8-12ml organic solvent, room temperature
30min is stirred, reaction solution is obtained, reaction solution is slowly dropped into the formamide solution of 0.4-0.6ml ethylenediamines, 1h is added dropwise in ice bath,
Reaction 6-48h is warmed to room temperature, adds 50ml acetone precipitations, filters, obtains sediment, sediment adds water to redissolve, and dialyse 2d, and freezing is dry
It is dry, ammonification hyaluronic acid is produced, organic solvent is formamide, DMF, one kind of dimethyl sulfoxide (DMSO);
(2) SWCNTs-HA-ss-COOH (i.e. the SWCNTs load 3,3'- dithiodipropionic acids of HA modifications) is prepared:
The 3 of 500~1500mg carbon numbers 2~12 are taken, 3 '-dithiodipropionic acid or suberic acid are linking arm, are dissolved in
In reaction dissolvent, 500~1500mg of EDC, 200~1000mg of NHS are vortexed are dissolved in reaction dissolvent respectively, by above-mentioned solution
5~25min of activation is mixed, into activated solution;By HA-SWCNTs 50~350mg ultrasonic dissolutions, mixed with activated solution,
1~8h is stirred under room temperature condition, products therefrom filtering, collects precipitation, precipitation is redissolved with water, is dialysed by acetone ice bath crystallization
2d, unnecessary 3,3'- dithiodipropionic acids, EDC, NHS salt are removed, freezed, (HA is modified into SWCNTs-HA-ss-COOH
SWCNTs loads 3,3'- dithiodipropionic acids), 4 DEG C of preservations;
Described reaction dissolvent is water or formamide or N,N-dimethylformamide and water or formamide and water or N,
The mixed solvent of dinethylformamide and formamide, or the mixture of other similar solvents and other similar solvents;
(3) SWCNTs-HA-ss-COOH and DOX (adriamycin) connect into SWCNTs-HA-ss-DOX:
Take 1~8mg DOXHCL be dissolved in 500~1500 μ l pH be 7~10 PBS solution in, into the first solution;
2~15mg SWCNTs-HA-ss-COOH ultrasonic dissolutions are weighed in 200~800 μ l reaction dissolvents, into the second solution, by two kinds
Solution mixes 1~4h of reaction, into mixed liquor;Take 10~60mg hydroxylamine hydrochlorides be dissolved in 200~2000 μ l pH for 7~
In 10 PBS solution, adding and 0.5~5h is reacted in mixed liquor, obtain product, product is dialysed with ultra-pure water after 2d, is freezed, into
SWCNTs-HA-ss-DOX;
Described reaction dissolvent is thionyl chloride or N,N-dimethylformamide or thionyl chloride and N, N- dimethyl methyl
The mixed solvent of acid amides, or the mixture of other similar solvents and other similar solvents;
(4) Gd (gadolinium) load:
5~10mg SWCNTs-HA-ss-DOX is added to the water, and 5~60min of ultrasound disperses in ice bath, and centrifugation discards
Undispersed precipitation, into single-walled carbon nanotube suspension;By 50~80mg GdCl under the conditions of room temperature magnetic agitation3Powder is dissolved in
In water, be added drop-wise in finely dispersed single-walled carbon nanotube suspension, produce flocculent deposit, Gd/SWCNTs-HA-ss-
DOX;
(5) Gd/SWCNTs-HA-ss-DOX medicament nano agent is prepared:
Mixture of the step (4) containing flocculent deposit is centrifuged into 2~30min, abandoning supernatant must precipitate, and add in precipitation
Enter surfactant solution, 5~55min of ice-bath ultrasonic disperses, and is then centrifuged for 2~25min, discards precipitation, supernatant dialysis 10-
16h (overnight), then 2~30min is centrifuged, precipitation is discarded, hyaluronic acid decorated single-walled carbon nanotube is produced and prepares diagnoses and treatment
Reduction-sensitive medicament nano agent (Gd/SWCNTs-HA-ss-DOX);
Described surfactant is soybean lecithin, or the mixture of soybean lecithin and poloxamer, surface-active
The water that agent solution is added 1ml by every 10mg surfactant is made.
The reduction-sensitive medicament nano agent that the hyaluronic acid decorated single-walled carbon nanotube of the present invention prepares diagnoses and treatment is being made
Application in standby tumor chemotherapeutic drug and magnetic resonance imaging.
The present invention by HA modifications SWCNTs by the sensitive compound 3,3 ' of the reproducibility environment that provides glutathione-
Dithiodipropionic acid is connected as linking arm with the active group of adriamycin, then receives gadolinium trichloride physical absorption to single wall carbon
Mitron, then formed with surfactant-dispersed with nanometers of the SWCNTs of HA modifications for the reduction-sensitive medicine of diagnoses and treatment
Preparation.This nanometer formulation possesses good good biocompatibility, tumor-targeting, thermotherapy and change as newtype drug transport system
The advantages for the treatment of is combined, Clinics and Practices are integrated, is the innovation in diagnosis and tumor, and economic and social benefit is huge
Greatly.
Embodiment
The embodiment of the present invention is elaborated with reference to embodiments.
The present invention in specific implementation, is realized by following examples.
Embodiment 1
The present invention is realized in specific implementation by following steps:
(1) carboxylation carbon pipe (i.e. carboxylic carbon nano-tube) is connected (HA-SWCNTs) with ammonification hyaluronic acid:
Weigh 50mg carboxylation carbon pipes to be dissolved in 20ml formamides, ice bath visits super 25min, into carboxylation carbon pipe solution;Weigh ammonia
Change hyaluronic acid (HA-NH2) 80mg is dissolved in 10ml formamide, into ammonification hyaluronic acid solution;Weigh EDC (1- ethyls-
(3- dimethylaminopropyls) carbodiimide) 160mg, NHS (HOSu NHS) 90mg, it is vortexed with 2.5ml formamides molten
Solution, is added in carboxylation carbon pipe solution, stirring room temperature reaction 15min, into carboxylation carbon pipe mixed solution;It is molten in ammonification hyaluronic acid
The μ l of triethylamine 150 are added in liquid, then mix the mixed solution fast drop of ammonification hyaluronic acid and triethylamine to carboxylation carbon pipe
Closing in solution, react at room temperature 24h, add the acetone of 3 times of volume precoolings, cooling crystallization, organic membrane filter, acetone, which washs, to be precipitated,
Sediment is obtained, sediment is redissolved with water, and dialyse 2d, freezes;
(2) SWCNTs-HA-ss-COOH (i.e. the SWCNTs load 3,3'- dithiodipropionic acids of HA modifications) is prepared:
The 3 of 1000mg carbon numbers 2~12 are taken, 3 '-dithiodipropionic acid or suberic acid are linking arm, are dissolved in formamide
In, EDC 1000mg, NHS 600mg are vortexed are dissolved in formamide respectively, and above-mentioned solution is mixed into activation 15min, into
Activated solution;HA-SWCNTs 200mg Probe Ultrasonic Searchings are dissolved, mixed with activated solution, 1~8h, institute are stirred under room temperature condition
Product is obtained by acetone ice bath crystallization, filtering, collects precipitation, precipitation is redissolved with water, and dialyse 2d, removes the unnecessary sulphur of 3,3'- bis-
For dipropionic acid, EDC, NHS salt, freeze, into SWCNTs-HA-ss-COOH, (SWCNTs of HA modifications loads 3,3'- dithio dipropyls
Acid), 4 DEG C of preservations;
(3) SWCNTs-HA-ss-COOH and DOX (adriamycin) connect into SWCNTs-HA-ss-DOX:
Take 6.3mg DOXHCL be dissolved in 1400 μ l pH be 7.4 PBS solution in, into the first solution;Weigh
12.6mg SWCNTs-HA-ss-COOH ultrasonic dissolutions, into the second solution, two kinds of solution are blended in 500 μ l thionyl chlorides
One reacts 2h, into mixed liquor;Take 49mg hydroxylamine hydrochlorides be dissolved in 2000 μ l pH be 7.4 PBS solution in, add mixed liquor
Middle reaction 2.5h, product is obtained, product is dialysed with ultra-pure water after 2d, is freezed, into SWCNTs-HA-ss-DOX;
(4) Gd (gadolinium) load:
8mg SWCNTs-HA-ss-DOX is added to the water, and Probe Ultrasonic Searching 35min disperses in ice bath, and centrifugation discards not
Scattered precipitation, into single-walled carbon nanotube suspension;By 64mg GdCl under the conditions of room temperature magnetic agitation3Powder is soluble in water,
Be added drop-wise in finely dispersed single-walled carbon nanotube suspension, produce flocculent deposit, Gd/SWCNTs-HA-ss-DOX;
(5) Gd/SWCNTs-HA-ss-DOX medicament nano agent is prepared:
Mixture of the step (4) containing flocculent deposit is centrifuged into 10min, abandoning supernatant must be precipitated, added in precipitation
Surfactant solution, ice bath Probe Ultrasonic Searching 30min disperse, and are then centrifuged for 20min, discard precipitation, supernatant dialysis 10-16h
(overnight), then 15min is centrifuged, precipitation is discarded, the reduction for producing hyaluronic acid decorated single-walled carbon nanotube preparation diagnoses and treatment is quick
Sensitive drug nanometer agent (Gd/SWCNTs-HA-ss-DOX);
Described surfactant solution is that soybean lecithin and poloxamer 1 ︰ 1 by weight mixture 10mg are dissolved in
It is made in 1ml water.
Embodiment 2
The present invention can also be realized in specific implementation by following steps:
(1) carboxylation carbon pipe (i.e. carboxylic carbon nano-tube) is connected (HA-SWCNTs) with ammonification hyaluronic acid:
Weigh 60mg carboxylation carbon pipes to be dissolved in 20ml formamides, ice bath visits super 30min, into carboxylation carbon pipe solution;Weigh ammonia
Change hyaluronic acid (HA-NH2) 96mg is dissolved in 10ml formamide, into ammonification hyaluronic acid solution;Weigh EDC (1- ethyls-
(3- dimethylaminopropyls) carbodiimide) 160mg, NHS (HOSu NHS) 108mg, it is vortexed with 3ml formamides molten
Solution, is added in carboxylation carbon pipe solution, stirring room temperature reaction 15min, into carboxylation carbon pipe mixed solution;It is molten in ammonification hyaluronic acid
The μ l of triethylamine 180 are added in liquid, then mix the mixed solution fast drop of ammonification hyaluronic acid and triethylamine to carboxylation carbon pipe
Closing in solution, react at room temperature 24h, add the acetone of 3 times of volume precoolings, cooling crystallization, organic membrane filter, acetone, which washs, to be precipitated,
Sediment is obtained, sediment is redissolved with water, and dialyse 2d, freezes;
(2) SWCNTs-HA-ss-COOH (i.e. the SWCNTs load 3,3'- dithiodipropionic acids of HA modifications) is prepared:
The 3 of 1200mg carbon numbers 2~12 are taken, 3 '-dithiodipropionic acid or suberic acid are linking arm, are dissolved in formamide
In, EDC 1200mg, NHS 720mg are vortexed are dissolved in formamide respectively, and above-mentioned solution is mixed into activation 18min, into
Activated solution;HA-SWCNTs 240mg Probe Ultrasonic Searchings are dissolved, mixed with activated solution, 5h, gained production are stirred under room temperature condition
Thing organic membrane filter, collects precipitation, precipitation is redissolved with water, and dialyse 2d, removes unnecessary 3,3'- bis- by acetone ice bath crystallization
Thio-2 acid, EDC, NHS salt, freeze, into SWCNTs-HA-ss-COOH, (SWCNTs of HA modifications loads 3,3'- bis- thio two
Propionic acid), 4 DEG C of preservations;
(3) SWCNTs-HA-ss-COOH and DOX (adriamycin) connect into SWCNTs-HA-ss-DOX:
Take 5.4mg DOXHCL be dissolved in 1200 μ l pH be 8 PBS solution in, into the first solution;Weigh 9mg's
Two kinds of solution, into the second solution, are mixed reaction by SWCNTs-HA-ss-COOH ultrasonic dissolutions in 600 μ l thionyl chlorides
2h, into mixed liquor;Take 42mg hydroxylamine hydrochlorides be dissolved in 1920 μ l pH be 8 PBS solution in, add mixed liquor in react 2h,
Product is obtained, product is dialysed with ultra-pure water after 2d, is freezed, into SWCNTs-HA-ss-DOX;
(4) Gd (gadolinium) load:
6mg SWCNTs-HA-ss-DOX is added to the water, and Probe Ultrasonic Searching 30min disperses in ice bath, and centrifugation discards not
Scattered precipitation, into single-walled carbon nanotube suspension;By 48mg GdCl under the conditions of room temperature magnetic agitation3Powder is soluble in water,
Be added drop-wise in finely dispersed single-walled carbon nanotube suspension, produce flocculent deposit, Gd/SWCNTs-HA-ss-DOX;
(5) Gd/SWCNTs-HA-ss-DOX medicament nano agent is prepared:
Mixture of the step (4) containing flocculent deposit is centrifuged into 15min, abandoning supernatant must be precipitated, added in precipitation
Surfactant solution, ice bath Probe Ultrasonic Searching 30min disperse, and are then centrifuged for 20min, discard precipitation, supernatant dialysis 10-16h
(overnight), then 15min is centrifuged, precipitation is discarded, the reduction for producing hyaluronic acid decorated single-walled carbon nanotube preparation diagnoses and treatment is quick
Sensitive drug nanometer agent (Gd/SWCNTs-HA-ss-DOX);
Described surfactant solution is that soybean lecithin and poloxamer 2 ︰ 1 by weight mixture 10mg are dissolved in
It is made in 1ml water.
Embodiment 3
The present invention is realized in specific implementation by following steps:
(1) carboxylation carbon pipe (i.e. carboxylic carbon nano-tube) is connected (HA-SWCNTs) with ammonification hyaluronic acid:
Weigh 70mg carboxylation carbon pipes to be dissolved in 28ml DMFs, ice bath visits super 35min, into carboxylation carbon pipe
Solution;Weigh ammonification hyaluronic acid (HA-NH2) 112mg is dissolved in 14ml DMF, into ammonification hyaluronic acid
Solution;Weigh EDC (1- ethyls-(3- dimethylaminopropyls) carbodiimide) 224mg, NHS (HOSu NHS)
126mg, it is vortexed and is dissolved with 3.5ml DMFs, is added in carboxylation carbon pipe solution, stirring room temperature reaction
18min, into carboxylation carbon pipe mixed solution;The μ l of triethylamine 210 are added in ammonification hyaluronic acid solution, then by ammonification hyalomitome
The mixed solution fast drop of acid and triethylamine reacts at room temperature 36h, adds 3 times of volume precoolings into carboxylation carbon pipe mixed solution
Acetone, cooling crystallization, organic membrane filter, acetone washing precipitation, obtain sediment, sediment is redissolved with water, dialyse 2d, freeze;
(2) SWCNTs-HA-ss-COOH (i.e. the SWCNTs load 3,3'- dithiodipropionic acids of HA modifications) is prepared:
The 3 of 1400mg carbon numbers 2~12 are taken, 3 '-dithiodipropionic acid or suberic acid are linking arm, are dissolved in N, N- bis-
In NMF, EDC 1400mg, NHS 840mg are vortexed are dissolved in DMF respectively, by above-mentioned solution
Activation 18min is mixed, into activated solution;HA-SWCNTs 280mg Probe Ultrasonic Searchings are dissolved, mixed with activated solution, room
4h is stirred under the conditions of temperature, products therefrom organic membrane filter, collects precipitation, precipitation is redissolved with water, is dialysed by acetone ice bath crystallization
2d, unnecessary 3,3'- dithiodipropionic acids, EDC, NHS salt are removed, freezed, (HA is modified into SWCNTs-HA-ss-COOH
SWCNTs loads 3,3'- dithiodipropionic acids), 4 DEG C of preservations;
(3) SWCNTs-HA-ss-COOH and DOX (adriamycin) connect into SWCNTs-HA-ss-DOX:
Take 6.3mg DOXHCL be dissolved in 1400 μ l pH be 9.2 PBS solution in, into the first solution;Weigh
12.6mg SWCNTs-HA-ss-COOH ultrasonic dissolutions, into the second solution, two kinds of solution are blended in 500 μ l thionyl chlorides
One reacts 2h, into mixed liquor;Take 49mg hydroxylamine hydrochlorides be dissolved in 2000 μ l pH be 9.2 PBS solution in, add mixed liquor
Middle reaction 2h, product is obtained, product is dialysed with ultra-pure water after 2d, is freezed, into SWCNTs-HA-ss-DOX;
(4) Gd (gadolinium) load:
8mg SWCNTs-HA-ss-DOX is added to the water, and Probe Ultrasonic Searching 35min disperses in ice bath, and centrifugation discards not
Scattered precipitation, into single-walled carbon nanotube suspension;By 64mg GdCl under the conditions of room temperature magnetic agitation3Powder is soluble in water,
Be added drop-wise in finely dispersed single-walled carbon nanotube suspension, produce flocculent deposit, Gd/SWCNTs-HA-ss-DOX;
(5) Gd/SWCNTs-HA-ss-DOX medicament nano agent is prepared:
Mixture of the step (4) containing flocculent deposit is centrifuged into 10min, abandoning supernatant must be precipitated, added in precipitation
Surfactant solution, ice bath Probe Ultrasonic Searching 30min disperse, and are then centrifuged for 20min, discard precipitation, supernatant dialysis 10-16h
(overnight), then 15min is centrifuged, precipitation is discarded, the reduction for producing hyaluronic acid decorated single-walled carbon nanotube preparation diagnoses and treatment is quick
Sensitive drug nanometer agent (Gd/SWCNTs-HA-ss-DOX);
Described surfactant solution is that soybean lecithin and poloxamer 3 ︰ 1 by weight mixture 10mg are dissolved in
It is made in 1ml water.
The reduction-sensitive medicament nano agent that the hyaluronic acid decorated single-walled carbon nanotube of the present invention prepares diagnoses and treatment is being made
Application in standby tumor chemotherapeutic drug and magnetic resonance imaging.
The inventive method is easy to operate, and prepared product has good biocompatibility as tumour medicine transport system, swollen
Knurl targeting is good, thermotherapy is with chemotherapy is combined, Clinics and Practices are integrated the advantages of.
From the above, it is seen that the present invention uses the SWCNTs after HA is modified, and it is carbon nano-tube modified using HA, not only carry
The high water solubility and biocompatibility of CNT, also improves its targeting ability to tumour cell, based on this HA's
By 3 containing disulfide bond, 3 '-dithiodipropionic acid it is linking arm on amino, introduces the DOX containing amino:Disulfide bond is also
Former response type chemical bond, by the effect of the glutathione of high expression in tumour cell, rapid fracture so that antineoplastic is high
Effect release.Further, since SWCNTs is aromatic skeleton, Gd3+ is selectively deposited on ultrashort single-walled carbon nanotube, overcome
The defects of current clinical conventional radiography agent carrier targeting difference, its imaging performance is greatly improved, and has cancer target concurrently, nuclear-magnetism into
Picture, thermotherapy are combined with chemotherapy, the newtype drug system Gd/SWCNTs-HA-ss-DOX (hyalomitomes that Clinics and Practices are integrated
Acid modification single-walled carbon nanotube prepare diagnoses and treatment the agent of reduction-sensitive medicament nano), and through experiment achieve it is very satisfied
Advantageous effects, relevant information is as follows:
The measure of Gd contents in Gd/SWCNTs-HA-ss-DOX nanometer formulations
Using the content of ICP-OES assay gadolinium elements, with the drugloading rate of formula (1) calculating sample, drugloading rate reaches
To 12.0%,
The particle diameter and current potential of Gd/SWCNTs-HA-ss-DOX nanometer formulations characterize
After taking appropriate Gd/SWCNTs-HA-ss-DOX nanometer formulations to be diluted with water to suitable concentration, swashed with Nano-ZS90 types
It is respectively 199.3nm and -27.2 ± 0.26mv that light nano-particle size analysis instrument, which measures its particle diameter and current potential,.
Gd/SWCNTs-HA-ss-DOX nanometers agent of the present invention is tested the Proliferation Ability of MCF-7 cells
Single cell suspension is arrived into the processing of MCF-7 cells according to the step of passage, is inoculated with 5 × 103 after counting per hole
For cell on 96 orifice plates, incubator culture (37 DEG C, 5%CO2) 24h discards dosing after former culture medium after cell attachment is complete,
Gd/SWCNTs-HA-ss-DOX groups, SWCNTs-HA-ss-DOX groups, drug concentration added by DOX groups are in a series of ladders with DOX concentration
Degree is formulated with the culture medium without serum, DOX concentration gradient 0, and 0.039,0.078,0.156,0.313,0.625,
1.25,2.5,5,10 μ g/ml, the group for the single-walled carbon nanotube being connected with after modification are provided with 808nm laser control groups, and after dosing
A period of time irradiation 808nm laser 3min (power 2.0), continue to take out culture plate after cultivating 48h, 50 μ l precoolings are added per hole
50% trichloroacetic acid, final concentration of 10%, stand 5min;Move in 4 DEG C of refrigerators and stand 1h, take out and washed 5 times with ultrapure, room temperature
After air drying is complete, the SRB50 μ l of 1% peracetic acid formulation are added in every hole, 20min is dyed at room temperature, outwells dye liquor, are used
1% acetic acid is washed 5 times, removes the dyestuff being not associated with hole, and air at room temperature uses pH10.5 10mmol/LTris alkali lye 150 after drying
μ l dissolve, and 10min are shaken in air heats shaking table, in determining light absorbs of each hole at 515nm on enzyme-linked immunosorbent assay instrument
Angle value.Calculate inhibiting rate (%)=(1- experimental group A/ control group As) × 100%, it follows that the half of above-mentioned sample suppresses dense
Degree (IC50) is followed successively by:The non-thermotherapy groups of Gd/SWCNTs-HA-ss-DOX, the non-thermotherapy groups of SWCNTs-HA-ss-DOX, DOX groups are
1.008,0.987,1.626 μ g/ml;Gd/SWCNTs-HA-ss-DOX thermotherapy groups, SWCNTs-HA-ss-DOX thermotherapy groups:
0.5934,0.6186 μ g/ml.
Gd/SWCNTs-HA-ss-DOX nanometer formulations of the present invention are absorbed to MCF-7 cells streaming and tested
Experiment is divided into SWCNT/FITC groups, HA-SWCNTs/FITC groups, SWCNTs-HA-ss-DOX groups, Gd/SWCNTs-
HA-ss-DOX groups, it is additionally provided with blanc cell group and pure FITC groups.Wherein contained by FITC groups in FITC concentration and remaining group containing FITC
Concentration containing FITC is consistent.The MCF-7 cells in growth period of taking the logarithm are processed into single cell suspension according to the step of passage, count
After spread six orifice plates, be 3 × 10 per hole cell number5Individual/hole, and incubator (37 DEG C, 5%CO2) in culture 24h it is adherent completely after, discard
Culture medium, the medicine prepared with the culture medium without serum is added, 0.5h, 1h, 2h are set respectively, after 4h, 6h period progress
Phase is handled, and in the corresponding EP pipes of decoction suction, PBS is washed 2 times, then with the 0.5ml pancreatin digestion process 1min without EDTA, is added
Enter culture medium 1ml, EP corresponding to suction is managed, and after 1000rpm/10min centrifugations, is abandoned supernatant and is stayed precipitation, it is equal to add 2mlPBS piping and druming
It is even, after 1000rpm/10min centrifugations, abandon supernatant and stay precipitation, add after 0.5mlPBS is blown and beaten uniformly to be transferred in 1.5mlEP pipes and put
In ice chest, machine testing in streaming is waited, measures and MCF-7 streaming intake is followed successively by:35.2%, 89.6%, 95.6%,
96.2%.
The pharmacodynamics test of Gd/SWCNTs-HA-ss-DOX nanometer formulations of the present invention
10 female mices are subjected to S-180 sarcoma ascites cultures, extract ascites after two weeks, the kind for mouse entity knurl
Plant, when the volume of tumour reaches 100mm3During the above, tumor model is inoculated with successfully, and tumor-bearing mice is randomly divided into nine groups, every group
Eight, packet situation is as follows:(1) blank group;(2) blank-laser group;(3) DOX groups;(4) DOX- laser groups;(5)SWCNT-
HA-ss-DOX groups;(6) SWCNTs-HA-ss-DOX- laser groups;(7) Gd/SWCNTs-HA-ss-DOX groups;(8)Gd/SWCNTs-
HA-ss-DOX laser groups.Then the next day of carrying out administration, dosage is 4mg/kg by people mouse dose lonvestion, and tail vein injection is given
Medicine.Observe the survival state of mouse daily and the amount of weighing knurl volume (passes through gross tumor volume (V)=A × B2/ 2), Ran Houtong
Cross relative knurl volume and carry out R=V/V0Evaluate the change (V of tumour0For the size of the previous day knurl volume is administered) evaluation tumour is anti-swollen
Tumor activity.The as shown by data of record, compared at the end of administration with before administration, each group knurl volume inhibiting rate is followed successively by 0.01%,
2.09%th, 48.09%, 52.50%, 71.27%, 82.97%, 70.14%, 83.17%.
The magnetic resonance imaging experiment of the gadolinium class contrast agent of Gd/SWCNTs-HA-ss-DOX nanometer formulations of the present invention
12 tumor-bearing mices are randomly divided into three groups and marked, takes isoflurane suction-type to anaesthetize, then fixes mouse
It is unenhanced in being carried out in fixed plate with the special NMR of 7.0T toys;Three groups of tumor-bearing mices inject Gd/SWCNTs- respectively
HA-ss-DOX, Gd/SWCNTs-COOH, Gd-DTPA, Gd3+Injection volume is 0.15mmol/Kg, inject successfully after 0h,
Mouse is fixed on the up each time spot scan of fixed plate by 0.5h, 1h, 3h, 6h, 8h again, and three groups of mouse are injecting different magnetic
Enhancing scanning is carried out after resonance contrast agent, three groups of mouse signal after 30min is injected strengthens, but Gd-DTPA group signals
Strengthen maximum in 0.5h, 1h signals substantially reduce, and subsequent time point signal intensity recovers normal value;Gd/SWCNTs-HA-ss-
The signal intensity of DOX groups and Gd/SWCNTs-COOH groups strengthens with time lengthening, and Gd/SWCNTs-HA-ss-DOX group signals
It is maximum for intensity enhancing, hence it is evident that more than Gd/SWCNTs-COOH groups.Gd/SWCNTs-HA-ss-DOX shows obvious high relaxation
Efficiency and sustained release performance.
Experiment shows that the present invention compared with prior art, has advantageous effects following prominent:
1st, the bioreductive glutathione of the invention based on high concentration provides tumor tissues reproducibility microenvironment, selects
Linking arm connection single-walled carbon nanotube and antineoplastic containing disulfide bond, antitumor system can be realized using this trigger mechanism
Agent quick, Targeting delivery medicine and reduces the toxic side effect of antineoplastic in vivo;
2nd, the single-walled carbon nanotube after the present invention is modified from hyaluronic acid as target head carries out load medicine, the carrier formed
Have the advantages that good biocompatibility, Drug-loading Pattern are various, tumor-targeting is good, thermotherapy effect is good;
3rd, the anti-tumor medicinal preparation prepared by the present invention also has the prospect for being applied to clinical tumor diagnosis, contrast agent gadolinium
It is carried on carbon material and tumor-targeting enhancing, relaxivity enhancing, biological half-life is extended, with reference to antineoplastic
The novel medicine feeding system that is combined of Clinics and Practices can be achieved in treatment, is the innovation in diagnosis and tumor, it is economical and
Social benefit is huge.
Claims (4)
1. a kind of method that hyaluronic acid decorated single-walled carbon nanotube prepares the reduction-sensitive medicament nano agent of diagnoses and treatment, its
It is characterised by, by the single-walled carbon nanotube after hyaluronic acid decorated by 3,3 '-dithiodipropionic acid as linking arm and Ah mould
Element is connected, and then gadolinium trichloride is adsorbed onto on single-walled carbon nanotube, then is repaiied with surfactant-dispersed formation with hyaluronic acid
The single-walled carbon nanotube of decorations, the nanometer formulation for the reduction-sensitive medicine of diagnoses and treatment;The hyaluronic acid is molecular weight
Low-molecular-weight hyaluronic acid more than or equal to 600 dalton and less than or equal to 400 kd;The length of the single-walled carbon nanotube is 1
~3 μm, a diameter of 1~2nm;The linking arm is the 3,3 '-dithiodipropionic acid or suberic acid of carbon number 2~12;It is described
3, the disulfide bond in 3 '-dithiodipropionic acid has reduction-sensitive, fast under the glutathione effect of tumour cell high concentration
Release medicine is split in quick-break, is realized by following steps:
(1)Carboxylation carbon pipe is connected with ammonification hyaluronic acid:
25~75mg carboxylation carbon pipes are weighed in the beaker of 15~45ml reaction dissolvents, ice bath, which is visited, surpasses 15~45min, into carboxylation carbon
Pipe solution;Weigh 60~120mg of ammonification hyaluronic acid to be dissolved in 5-15ml solvent, into ammonification hyaluronic acid solution;Weigh EDC
120~240mg, 80~160mg of NHS, it is vortexed and is dissolved with 1~7ml solvents, is added in carboxylation carbon pipe solution, stirring room temperature is anti-
5~25min is answered, into carboxylation carbon pipe mixed solution;The μ l of triethylamine 100~260 are added in ammonification hyaluronic acid solution, then will
The mixed solution fast drop of ammonification hyaluronic acid and triethylamine reacts at room temperature 8~40h, added into carboxylation carbon pipe mixed solution
Enter the acetone of 3~4 times of volume precoolings, cooling crystallization, filtering, acetone washing precipitation, obtain sediment, sediment is redissolved with water, thoroughly
2d is analysed, is freezed;
Described solvent is water, formamide or N,N-dimethylformamide and water or formamide and water or N, N- dimethyl methyl
The mixed solvent of acid amides and formamide;
Described ammonification hyaluronic acid is, by hyaluronic acid 95-105mg, 1- ethyl-(3- dimethylaminopropyls) carbon two
Inferior amine salt hydrochlorate 254-264mg and N- HOSu NHS 150-160mg, is dissolved in 8-12ml organic solvent, room
Temperature 30 min of stirring, obtain reaction solution, reaction solution are slowly dropped into the formamide solution of 0.4-0.6ml ethylenediamines, ice bath is added dropwise
1h, reaction 6-48h is warmed to room temperature, adds 50ml acetone precipitations, filters, obtains sediment, sediment adds water to redissolve, and dialyse 2d, cold
It is lyophilized dry, ammonification hyaluronic acid is produced, organic solvent is formamide, N, N- dimethylformamides, one kind of dimethyl sulfoxide (DMSO);
(2)Prepare SWCNTs-HA-ss-COOH:
The 3 of 500~1500mg carbon numbers 2~12 are taken, 3 '-dithiodipropionic acid or suberic acid are linking arm, are dissolved in reaction
In solvent, 500~1500mg of EDC, 200~1000mg of NHS are vortexed are dissolved in reaction dissolvent respectively, and above-mentioned solution is mixed
5~25min of stir-activating, into activated solution;By HA-SWCNTs 50~350mg ultrasonic dissolutions, mixed with activated solution, room temperature
Under the conditions of stir 1~8h, products therefrom filtering, collects precipitation by acetone ice bath crystallization, and precipitation is redissolved with water, and dialyse 2d, removes
Unnecessary 3,3'- dithiodipropionic acids, EDC, NHS salt are removed, is freezed, into SWCNTs-HA-ss-COOH, 4 DEG C of preservations;
Described reaction dissolvent is water or formamide or N,N-dimethylformamide and water or formamide and water or N, N- bis-
The mixed solvent of NMF and formamide;
(3)SWCNTs-HA-ss-COOH and DOX connects into SWCNTs-HA-ss-DOX:
Take 1~8mg DOX HCL be dissolved in 500~1500 μ l pH be 7~10 PBS solution in, into the first solution;Weigh 2
~15mg SWCNTs-HA-ss-COOH ultrasonic dissolutions, into the second solution, two kinds of solution are mixed in 200~800 μ l reaction dissolvents
1~4h of reaction is combined, into mixed liquor;It is 7~10 to take 10~60mg hydroxylamine hydrochlorides to be dissolved in 200~2000 μ l pH
In PBS solution, add and 0.5~5h is reacted in mixed liquor, obtain product, product is dialysed with ultra-pure water after 2d, is freezed, into SWCNTs-
HA-ss-DOX;
Described reaction dissolvent is thionyl chloride or N,N-dimethylformamide or thionyl chloride and N,N-dimethylformamide
Mixed solvent;
(4)Gd load:
5~10mg SWCNTs-HA-ss-DOX is added to the water, and 5~60min of ultrasound disperses in ice bath, and centrifugation, which discards, not to divide
Scattered precipitation, into single-walled carbon nanotube suspension;By 50~80mg GdCl under the conditions of room temperature magnetic agitation3Powder is dissolved in water
In, be added drop-wise in finely dispersed single-walled carbon nanotube suspension, produce flocculent deposit, Gd/SWCNTs-HA-ss-DOX;
(5)Prepare Gd/SWCNTs-HA-ss-DOX medicament nano agent:
By step(4)Mixture containing flocculent deposit centrifuges 2~30min, and abandoning supernatant must be precipitated, and table is added in precipitation
Face activator solution, 5~55min of ice-bath ultrasonic disperse, and are then centrifuged for 2~25min, discard precipitation, supernatant dialysis 10-16h,
2~30min is centrifuged again, discards precipitation, produces the reduction-sensitive that hyaluronic acid decorated single-walled carbon nanotube prepares diagnoses and treatment
Medicament nano agent;
Described surfactant is soybean lecithin, or the mixture of soybean lecithin and poloxamer, surfactant are molten
The water that liquid is added 1ml by every 10mg surfactant is made.
2. hyaluronic acid decorated single-walled carbon nanotube according to claim 1 prepares the reduction-sensitive medicine of diagnoses and treatment
The method of nanometer agent, it is characterised in that realized by following steps:
(1)Carboxylation carbon pipe is connected with ammonification hyaluronic acid:
Weigh 50mg carboxylation carbon pipes to be dissolved in 20ml formamides, ice bath visits super 25min, into carboxylation carbon pipe solution;It is saturating to weigh ammonification
Bright matter acid 80mg is dissolved in 10ml formamide, into ammonification hyaluronic acid solution;EDC 160mg, NHS 90mg are weighed, is used
2.5ml formamides, which are vortexed, to be dissolved, and is added in carboxylation carbon pipe solution, stirring room temperature reaction 15min, is mixed into carboxylation carbon pipe molten
Liquid;Addition triethylamine 150 μ l, then fast by the mixed solution of ammonification hyaluronic acid and triethylamine in ammonification hyaluronic acid solution
Speed is added dropwise in carboxylation carbon pipe mixed solution, reacts at room temperature 24h, adds the acetone of 3 times of volume precoolings, cooling crystallization, organic film
Filtering, acetone washing precipitation, obtains sediment, and sediment is redissolved with water, and dialyse 2d, freezes;
(2)Prepare SWCNTs-HA-ss-COOH:
The 3 of 1000mg carbon numbers 2~12 are taken, 3 '-dithiodipropionic acid or suberic acid are linking arm, are dissolved in formamide,
EDC 1000mg, NHS 600mg are vortexed are dissolved in formamide respectively, above-mentioned solution are mixed into activation 15min, into activation
Solution;HA-SWCNTs 200mg Probe Ultrasonic Searchings are dissolved, mixed with activated solution, 1~8h, gained production are stirred under room temperature condition
Thing filtering, collects precipitation, precipitation is redissolved with water, and dialyse 2d, removes unnecessary 3,3'- bis- thio two by acetone ice bath crystallization
Propionic acid, EDC, NHS salt, freeze, into SWCNTs-HA-ss-COOH, 4 DEG C of preservations;
(3)SWCNTs-HA-ss-COOH and DOX connects into SWCNTs-HA-ss-DOX:
Take 6.3mg DOX HCL be dissolved in 1400 μ l pH be 7.4 PBS solution in, into the first solution;Weigh 12.6mg's
Two kinds of solution, into the second solution, are mixed reaction by SWCNTs-HA-ss-COOH ultrasonic dissolutions in 500 μ l thionyl chlorides
2h, into mixed liquor;Take 49mg hydroxylamine hydrochlorides be dissolved in 2000 μ l pH be 7.4 PBS solution in, add mixed liquor in react
2.5h, product is obtained, product is dialysed with ultra-pure water after 2d, is freezed, into SWCNTs-HA-ss-DOX;
(4)Gd load:
8mg SWCNTs-HA-ss-DOX is added to the water, and Probe Ultrasonic Searching 35min disperses in ice bath, and centrifugation, which discards, not to be disperseed
Precipitation, into single-walled carbon nanotube suspension;By 64mg GdCl under the conditions of room temperature magnetic agitation3Powder is soluble in water, is added dropwise
Into finely dispersed single-walled carbon nanotube suspension, produce flocculent deposit, Gd/SWCNTs-HA-ss-DOX;
(5)Prepare Gd/SWCNTs-HA-ss-DOX medicament nano agent:
By step(4)Mixture centrifugation 10min containing flocculent deposit, abandoning supernatant must be precipitated, and surface is added in precipitation
Activator solution, ice bath Probe Ultrasonic Searching 30min disperse, and are then centrifuged for 20min, discard precipitation, supernatant dialysis 10-16h, then from
Heart 15min, precipitation is discarded, produce the reduction-sensitive medicament nano that hyaluronic acid decorated single-walled carbon nanotube prepares diagnoses and treatment
Agent;
Described surfactant solution is that soybean lecithin and poloxamer 1 ︰ 1 by weight mixture 10mg are dissolved in 1ml
Water in be made.
3. hyaluronic acid decorated single-walled carbon nanotube according to claim 1 prepares the reduction-sensitive medicine of diagnoses and treatment
The method of nanometer agent, it is characterised in that realized by following steps:
(1)Carboxylation carbon pipe is connected with ammonification hyaluronic acid:
Weigh 60mg carboxylation carbon pipes to be dissolved in 20ml formamides, ice bath visits super 30min, into carboxylation carbon pipe solution;It is saturating to weigh ammonification
Bright matter acid 96mg is dissolved in 10ml formamide, into ammonification hyaluronic acid solution;EDC 160mg, NHS 108mg are weighed, uses 3ml
Formamide, which is vortexed, to be dissolved, and is added in carboxylation carbon pipe solution, stirring room temperature reaction 15min, into carboxylation carbon pipe mixed solution;In ammonia
Change and the μ l of triethylamine 180 are added in hyaluronic acid solution, then by ammonification hyaluronic acid and the mixed solution fast drop of triethylamine
Into carboxylation carbon pipe mixed solution, room temperature reaction 24h, the acetone of 3 times of volume precoolings of addition, cooling crystallization, organic membrane filter, third
Ketone washing precipitation, obtains sediment, and sediment is redissolved with water, and dialyse 2d, freezes;
(2)Prepare SWCNTs-HA-ss-COOH:
The 3 of 1200mg carbon numbers 2~12 are taken, 3 '-dithiodipropionic acid or suberic acid are linking arm, are dissolved in formamide,
EDC 1200mg, NHS 720mg are vortexed are dissolved in formamide respectively, above-mentioned solution are mixed into activation 18min, into activation
Solution;HA-SWCNTs 240mg Probe Ultrasonic Searchings are dissolved, is mixed with activated solution, 5h is stirred under room temperature condition, products therefrom leads to
Acetone ice bath crystallization is crossed, organic membrane filter, collects precipitation, precipitation is redissolved with water, and dialyse 2d, and it is thio to remove unnecessary 3,3'- bis-
Dipropionic acid, EDC, NHS salt, freeze, into SWCNTs-HA-ss-COOH, 4 DEG C of preservations;
(3)SWCNTs-HA-ss-COOH and DOX connects into SWCNTs-HA-ss-DOX:
Take 5.4mg DOX HCL be dissolved in 1200 μ l pH be 8 PBS solution in, into the first solution;Weigh 9mg SWCNTs-
Two kinds of solution into the second solution, are mixed reaction 2h in 600 μ l thionyl chlorides by HA-ss-COOH ultrasonic dissolutions, into mixed
Close liquid;Take 42mg hydroxylamine hydrochlorides be dissolved in 1920 μ l pH be 8 PBS solution in, add mixed liquor in react 2h, obtain product,
Product is dialysed with ultra-pure water after 2d, is freezed, into SWCNTs-HA-ss-DOX;
(4)Gd load:
6mg SWCNTs-HA-ss-DOX is added to the water, and Probe Ultrasonic Searching 30min disperses in ice bath, and centrifugation, which discards, not to be disperseed
Precipitation, into single-walled carbon nanotube suspension;By 48mg GdCl under the conditions of room temperature magnetic agitation3Powder is soluble in water, is added dropwise
Into finely dispersed single-walled carbon nanotube suspension, produce flocculent deposit, Gd/SWCNTs-HA-ss-DOX;
(5)Prepare Gd/SWCNTs-HA-ss-DOX medicament nano agent:
By step(4)Mixture centrifugation 15min containing flocculent deposit, abandoning supernatant must be precipitated, and surface is added in precipitation
Activator solution, ice bath Probe Ultrasonic Searching 30min disperse, and are then centrifuged for 20min, discard precipitation, supernatant dialysis 10-16h, then from
Heart 15min, precipitation is discarded, produce the reduction-sensitive medicament nano that hyaluronic acid decorated single-walled carbon nanotube prepares diagnoses and treatment
Agent;
Described surfactant solution is that soybean lecithin and poloxamer 2 ︰ 1 by weight mixture 10mg are dissolved in 1ml
Water in be made.
4. hyaluronic acid decorated single-walled carbon nanotube according to claim 1 prepares the reduction-sensitive medicine of diagnoses and treatment
The method of nanometer agent, it is characterised in that realized by following steps:
(1)Carboxylation carbon pipe is connected with ammonification hyaluronic acid:
Weigh 70mg carboxylation carbon pipes to be dissolved in 28ml DMFs, ice bath visits super 35min, into carboxylation carbon pipe solution;
Weigh ammonification hyaluronic acid 112mg to be dissolved in 14ml DMF, into ammonification hyaluronic acid solution;Weigh EDC
224mg, NHS 126mg, it is vortexed and is dissolved with 3.5ml DMFs, is added in carboxylation carbon pipe solution, teeter chamber
Temperature reaction 18min, into carboxylation carbon pipe mixed solution;The μ l of triethylamine 210 are added in ammonification hyaluronic acid solution, then by ammonification
The mixed solution fast drop of hyaluronic acid and triethylamine reacts at room temperature 36h into carboxylation carbon pipe mixed solution, adds 3 times of bodies
The acetone of product precooling, cooling crystallization, organic membrane filter, acetone washing precipitation, sediment being obtained, sediment is redissolved with water, and dialyse 2d,
It is lyophilized;
(2)Prepare SWCNTs-HA-ss-COOH:
The 3 of 1400mg carbon numbers 2~12 are taken, 3 '-dithiodipropionic acid or suberic acid are linking arm, are dissolved in N, N- dimethyl
In formamide, EDC 1400mg, NHS 840mg are vortexed are dissolved in DMF respectively, and above-mentioned solution is mixed
Stir-activating 18min, into activated solution;HA-SWCNTs 280mg Probe Ultrasonic Searchings are dissolved, mixed with activated solution, room temperature bar
4h is stirred under part, products therefrom is redissolved by acetone ice bath crystallization, organic membrane filter, collection precipitation, precipitation with water, and dialyse 2d,
Unnecessary 3,3'- dithiodipropionic acids, EDC, NHS salt are removed, is freezed, into SWCNTs-HA-ss-COOH, 4 DEG C of preservations;
(3)SWCNTs-HA-ss-COOH and DOX connects into SWCNTs-HA-ss-DOX:
Take 6.3mg DOX HCL be dissolved in 1400 μ l pH be 9.2 PBS solution in, into the first solution;Weigh 12.6mg's
Two kinds of solution, into the second solution, are mixed reaction by SWCNTs-HA-ss-COOH ultrasonic dissolutions in 500 μ l thionyl chlorides
2h, into mixed liquor;Take 49mg hydroxylamine hydrochlorides be dissolved in 2000 μ l pH be 9.2 PBS solution in, add mixed liquor in react
2h, product is obtained, product is dialysed with ultra-pure water after 2d, is freezed, into SWCNTs-HA-ss-DOX;
(4)Gd load:
8mg SWCNTs-HA-ss-DOX is added to the water, and Probe Ultrasonic Searching 35min disperses in ice bath, and centrifugation, which discards, not to be disperseed
Precipitation, into single-walled carbon nanotube suspension;By 64mg GdCl under the conditions of room temperature magnetic agitation3Powder is soluble in water, is added dropwise
Into finely dispersed single-walled carbon nanotube suspension, produce flocculent deposit, Gd/SWCNTs-HA-ss-DOX;
(5)Prepare Gd/SWCNTs-HA-ss-DOX medicament nano agent:
By step(4)Mixture centrifugation 10min containing flocculent deposit, abandoning supernatant must be precipitated, and surface is added in precipitation
Activator solution, ice bath Probe Ultrasonic Searching 30min disperse, and are then centrifuged for 20min, discard precipitation, supernatant dialysis 10-16h, then from
Heart 15min, precipitation is discarded, produce the reduction-sensitive medicament nano that hyaluronic acid decorated single-walled carbon nanotube prepares diagnoses and treatment
Agent;
Described surfactant solution is that soybean lecithin and poloxamer 3 ︰ 1 by weight mixture 10mg are dissolved in 1ml
Water in be made.
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