CN104940959A - Method for preparing reduction-sensitive medicine nano-agent for diagnoses and treatment through single-walled carbon nanotubes modified through hyaluronic acid and application of method - Google Patents

Abstract

The invention relates to a method for preparing a reduction-sensitive medicine nano-agent for diagnoses and treatment through single-walled carbon nanotubes modified through hyaluronic acid and application of the method. The method can effectively solve the problems that tumor targeting of the anti-cancer drug adriamycin is poor, toxic and side effects are large, targeting is poor, relaxivity is low and the half-life period is short. According to a transport system, the single-walled carbon nanotubes modified through the hyaluronic acid are connected with the adriamycin through 3,3'-dithio-thiodipropionic acid serving as a joint arm, then gadolinium chloride is adsorbed to the single-walled carbon nanotubes, and the single-walled carbon nanotubes modified through the hyaluronic acid are formed through dispersion of a surface active agent. The reduction-sensitive medicine nano-agent used for diagnoses and treatment has the advantages that biocompatibility and tumor targeting are good, thermal therapy and chemotherapy are combined, and diagnosis and treatment are combined, is an innovation of tumor diagnosis and treatment medicine, and is large in economic and social benefit.

Description

Hyaluronic acid decorated SWCN prepares method and the application of the reduction-sensitive medicament nano agent of diagnoses and treatment

Technical field

The present invention relates to medicine, particularly a kind of hyaluronic acid (HA) modifies method and the application that SWCN (SWCNTs) prepares the reduction-sensitive medicament nano agent of diagnoses and treatment.

Background technology

The health and lives safety of the mankind in malignant tumor serious threat, and its treatment becomes the focus of people's concern, and show according to China's Epidemiological study, no matter be city or rural area, mortality of malignant tumors all occupies first of whole dead disease.Doxorubicin hydrochloride (Doxorubicin, DOX), as broad-spectrum anti-tumor chemotherapeutics, has killing action to the tumor cell of various growth cycle, and clinical being mainly used in treats the shallow table solid tumors such as breast carcinoma, carcinoma of prostate, sarcoma.Current doxorubicin hydrochloride listing dosage form is injection, larger owing to lacking the toxicity that targeting causes hemopoietic system and heart when Clinical practice.

CNT is curling by graphite and cylinder that is that formed, and internal diameter is from 1 nanometer to tens nanometers, and external diameter is also between several nanometer to tens nanometers, and length is generally all at nanoscale or micron order.It is reported that CNT toxicity depends on the functionalization on its surface, the CNT with high functionalization has less toxicity usually.And without the SWCN (SWCNTs) that any hydrophilic group is modified, due to the high hydrophobicity on its surface, cause interacting with cell in cell cultivation process, comprise and the polymerization such as intracellular protein, nucleic acid, thus produce certain cytotoxicity.Hyaluronic acid (hyaluronic acid, HA) is the linear polysaccharide that a kind of occurring in nature exists, and has high-biocompatibility, biodegradability, be eliminated by lymphsystem in vivo, the advantages such as hypotoxicity, HA is widely used in biomedicine field.Research finds that HA receptor is relevant to tumor in addition, and kinds of tumors process LAN hyaluronic acid receptor CD44, although normal cell also has CD44 receptor, the CD44 of normal structure is in silence state, is not activated.

Up to the present, magnetic resonance contrast agent many shortages targeting conventional clinically, the half-life is shorter, relaxivity is low and can cause the shortcomings such as toxic and side effects when using in a large number, therefore invents a kind of targeting, long half time, relaxivity is high and toxicity is low magnetic resonance contrast agent is technical problem urgently to be resolved hurrily.

Summary of the invention

For above-mentioned situation, for overcoming the defect of prior art, the object of the present invention is just to provide method and the application that a kind of hyaluronic acid decorated SWCN prepares the reduction-sensitive medicament nano agent of diagnoses and treatment, effectively can solve the problem that tumor-targeting is poor, toxic and side effects is large and targeting is poor, relaxivity is low, the half-life is short of anticancer drugs, doxorubicin

The technical scheme that the present invention solves is, this transport system by hyaluronic acid (HA) modify after SWCN (SWCNTs) by 3,3 '-dithiodipropionic acid is connected with amycin as linking arm, then gadolinium trichloride is adsorbed onto on SWCN, formed with hyaluronic acid decorated SWCN, for the nanometer formulation of the reduction-sensitive medicine of diagnoses and treatment by surfactant-dispersed again; Described hyaluronic acid is that molecular weight is more than or equal to 600 dalton and is less than or equal to the low-molecular-weight hyaluronic acid of 400kd; The length of described SWCN is 1 ~ 3 μm, diameter is 1 ~ 2nm; Described linking arm is 3 of carbon number 2 ~ 12,3 '-dithiodipropionic acid or suberic acid; Described 3, the disulfide bond in 3 '-dithiodipropionic acid has reduction-sensitive, and under the glutathion effect of tumor cell high concentration, fracture discharges medicine rapidly.Its preparation method is realized by following steps:

(1) carboxylation carbon pipe (i.e. carboxylic carbon nano-tube) is connected (HA-SWCNTs) with ammonification hyaluronic acid:

Take 25 ~ 75mg carboxylation carbon pipe in the beaker of 15 ~ 45ml reaction dissolvent, ice bath is visited and is surpassed 15 ~ 45min, becomes carboxylation carbon pipe solution; Take ammonification hyaluronic acid (HA-NH ₂) 60 ~ 120mg is dissolved in the solvent of 5-15ml, becomes ammonification hyaluronic acid solution; Take EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) 120 ~ 240mg, NHS (N-Hydroxysuccinimide) 80 ~ 160mg, dissolve with 1 ~ 7ml solvent vortex, join in carboxylation carbon pipe solution, stir room temperature reaction 5 ~ 25min, become carboxylation carbon pipe mixed solution; Triethylamine 100 ~ 260 μ l is added in ammonification hyaluronic acid solution, then by the mixed solution fast drop of ammonification hyaluronic acid and triethylamine in carboxylation carbon pipe mixed solution, room temperature reaction 8 ~ 40h, adds the acetone of 3 ~ 4 times of volume pre-coolings, cooling crystallization, filter, washing with acetone precipitates, and obtain precipitate, precipitate water redissolves, dialysis 2d, lyophilizing;

Described solvent is the mixed solvent of water, Methanamide or DMF and water or Methanamide and water or DMF and Methanamide, or the mixture of other similar solvent and other similar solvent;

Described ammonification hyaluronic acid is, by hyaluronic acid 95-105mg, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride 254-264mg and N-hydroxy-succinamide 150-160mg, be dissolved in the organic solvent of 8-12ml, stirring at room temperature 30min, obtain reactant liquor, reactant liquor is slowly instilled in the formamide solution of 0.4-0.6ml ethylenediamine, ice bath drips 1h, rise to room temperature reaction 6-48h, add 50ml acetone precipitation, sucking filtration, obtain precipitate, precipitate adds water redissolution, dialysis 2d, lyophilization, obtain ammonification hyaluronic acid, organic solvent is Methanamide, N, dinethylformamide, the one of dimethyl sulfoxide,

(2) SWCNTs-HA-ss-COOH (i.e. SWCNTs load 3, the 3'-dithiodipropionic acid of HA modification) is prepared:

Get 3 of 500 ~ 1500mg carbon number 2 ~ 12,3 '-dithiodipropionic acid or suberic acid are linking arm, be dissolved in reaction dissolvent, EDC 500 ~ 1500mg, NHS 200 ~ 1000mg respectively vortex are dissolved in reaction dissolvent, by above-mentioned solution mix and blend activation 5 ~ 25min, become activated solution; By HA-SWCNTs 50 ~ 350mg ultrasonic dissolution, mix with activated solution, under room temperature condition, stir 1 ~ 8h, products therefrom, by acetone ice bath crystallize, filters, collecting precipitation, precipitation water redissolves, dialysis 2d, removes 3,3'-unnecessary dithiodipropionic acids, EDC, NHS salt, lyophilizing, become SWCNTs-HA-ss-COOH (SWCNTs load 3, the 3'-dithiodipropionic acid that HA modifies), 4 DEG C of preservations;

Described reaction dissolvent is the mixed solvent of water or Methanamide or DMF and water or Methanamide and water or DMF and Methanamide, or the mixture of other similar solvent and other similar solvent;

(3) SWCNTs-HA-ss-COOH and DOX (amycin) connect into SWCNTs-HA-ss-DOX:

Getting the pH that 1 ~ 8mg DOXHCL is dissolved in 500 ~ 1500 μ l is in the PBS solution of 7 ~ 10, becomes the first solution; The SWCNTs-HA-ss-COOH ultrasonic dissolution taking 2 ~ 15mg, in 200 ~ 800 μ l reaction dissolvents, becomes the second solution, two kinds of solution is mixed reaction 1 ~ 4h, becomes mixed liquor; Getting the pH that 10 ~ 60mg oxammonium hydrochloride. is dissolved in 200 ~ 2000 μ l is in the PBS solution of 7 ~ 10, and add in mixed liquor and react 0.5 ~ 5h, obtain product, product is with after ultra-pure water dialysis 2d, and lyophilizing, becomes SWCNTs-HA-ss-DOX;

Described reaction dissolvent is the mixed solvent of thionyl chloride or DMF or thionyl chloride and DMF, or the mixture of other similar solvent and other similar solvent;

(4) load of Gd (gadolinium):

The SWCNTs-HA-ss-DOX of 5 ~ 10mg is added to the water, ultrasonic 5 ~ 60min dispersion in ice bath, centrifugally discards the precipitation of not disperseing, and becomes SWCN suspension; By the GdCl of 50 ~ 80mg under room temperature magnetic agitation condition ₃powder is soluble in water, is added drop-wise in finely dispersed SWCN suspension, produce flocculent deposit, Gd/SWCNTs-HA-ss-DOX;

(5) agent of Gd/SWCNTs-HA-ss-DOX medicament nano is prepared:

By the centrifugal 2 ~ 30min of step (4) mixture containing flocculent deposit, abandoning supernatant must precipitate, surfactant solution is added in precipitation, ice-bath ultrasonic 5 ~ 55min disperses, and then centrifugal 2 ~ 25min, discards precipitation, supernatant dialysis 10-16h (spending the night), centrifugal 2 ~ 30min, discards precipitation again, obtains the reduction-sensitive medicament nano agent (Gd/SWCNTs-HA-ss-DOX) that hyaluronic acid decorated SWCN prepares diagnoses and treatment;

Described surfactant is soybean lecithin, or the mixture of soybean lecithin and poloxamer, and surfactant solution is that the water adding 1ml by the surfactant of every 10mg is made.

The application in tumor chemotherapeutic drug and nuclear magnetic resonance is being prepared in the reduction-sensitive medicament nano agent that the hyaluronic acid decorated SWCN of the present invention prepares diagnoses and treatment.

The compound 3 of reproducibility environment sensitive of the SWCNTs that the present invention is modified by HA by providing glutathion, 3 '-dithiodipropionic acid is connected with the active group of amycin as linking arm, then by gadolinium trichloride physical absorption to SWCN, then form by surfactant-dispersed the nanometer formulation of reduction-sensitive medicine that the SWCNTs modified with HA is used for diagnoses and treatment.This nanometer formulation is as newtype drug transport system, and possess good biocompatibility, tumor-targeting is good, thermotherapy combines with chemotherapy, Clinics and Practices is integrated advantage, be the innovation on Diagnosis and Treat tumour medicine, economic and social benefit is huge.

Detailed description of the invention

Below in conjunction with embodiment, the specific embodiment of the present invention is elaborated.

The present invention, in concrete enforcement, is realized by following examples.

Embodiment 1

The present invention, in concrete enforcement, is realized by following steps:

(1) carboxylation carbon pipe (i.e. carboxylic carbon nano-tube) is connected (HA-SWCNTs) with ammonification hyaluronic acid:

Taking 50mg carboxylation carbon pipe is dissolved in 20ml Methanamide, and ice bath visits super 25min, becomes carboxylation carbon pipe solution; Take ammonification hyaluronic acid (HA-NH ₂) 80mg is dissolved in the Methanamide of 10ml, becomes ammonification hyaluronic acid solution; Take EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) 160mg, NHS (N-Hydroxysuccinimide) 90mg, dissolve with 2.5ml Methanamide vortex, join in carboxylation carbon pipe solution, stir room temperature reaction 15min, become carboxylation carbon pipe mixed solution; Triethylamine 150 μ l is added in ammonification hyaluronic acid solution, then by the mixed solution fast drop of ammonification hyaluronic acid and triethylamine in carboxylation carbon pipe mixed solution, room temperature reaction 24h, adds the acetone of 3 times of volume pre-coolings, cooling crystallization, organic membrane filter, washing with acetone precipitates, and obtain precipitate, precipitate water redissolves, dialysis 2d, lyophilizing;

(2) SWCNTs-HA-ss-COOH (i.e. SWCNTs load 3, the 3'-dithiodipropionic acid of HA modification) is prepared:

Get 3 of 1000mg carbon number 2 ~ 12,3 '-dithiodipropionic acid or suberic acid are linking arm, are dissolved in Methanamide, and EDC 1000mg, NHS 600mg respectively vortex are dissolved in Methanamide, by above-mentioned solution mix and blend activation 15min, become activated solution; HA-SWCNTs 200mg Probe Ultrasonic Searching is dissolved, mixes with activated solution, under room temperature condition, stir 1 ~ 8h, products therefrom, by acetone ice bath crystallize, filters, collecting precipitation, precipitation water redissolves, dialysis 2d, removes 3,3'-unnecessary dithiodipropionic acids, EDC, NHS salt, lyophilizing, become SWCNTs-HA-ss-COOH (SWCNTs load 3, the 3'-dithiodipropionic acid that HA modifies), 4 DEG C of preservations;

(3) SWCNTs-HA-ss-COOH and DOX (amycin) connect into SWCNTs-HA-ss-DOX:

Getting the pH that 6.3mg DOXHCL is dissolved in 1400 μ l is in the PBS solution of 7.4, becomes the first solution; The SWCNTs-HA-ss-COOH ultrasonic dissolution taking 12.6mg, in 500 μ l thionyl chlorides, becomes the second solution, two kinds of solution is mixed reaction 2h, becomes mixed liquor; Getting the pH that 49mg oxammonium hydrochloride. is dissolved in 2000 μ l is in the PBS solution of 7.4, and add in mixed liquor and react 2.5h, obtain product, product is with after ultra-pure water dialysis 2d, and lyophilizing, becomes SWCNTs-HA-ss-DOX;

(4) load of Gd (gadolinium):

The SWCNTs-HA-ss-DOX of 8mg is added to the water, and in ice bath, Probe Ultrasonic Searching 35min disperses, and centrifugally discards the precipitation of not disperseing, and becomes SWCN suspension; By the GdCl of 64mg under room temperature magnetic agitation condition ₃powder is soluble in water, is added drop-wise in finely dispersed SWCN suspension, produce flocculent deposit, Gd/SWCNTs-HA-ss-DOX;

(5) agent of Gd/SWCNTs-HA-ss-DOX medicament nano is prepared:

By the centrifugal 10min of step (4) mixture containing flocculent deposit, abandoning supernatant must precipitate, surfactant solution is added in precipitation, ice bath Probe Ultrasonic Searching 30min disperses, and then centrifugal 20min, discards precipitation, supernatant dialysis 10-16h (spending the night), centrifugal 15min, discards precipitation again, obtains the reduction-sensitive medicament nano agent (Gd/SWCNTs-HA-ss-DOX) that hyaluronic acid decorated SWCN prepares diagnoses and treatment;

Described surfactant solution be soybean lecithin and poloxamer by weight the mixture 10mg of 1 ︰ 1 be dissolved in the water of 1ml and make.

Embodiment 2

The present invention, in concrete enforcement, also can be realized by following steps:

(1) carboxylation carbon pipe (i.e. carboxylic carbon nano-tube) is connected (HA-SWCNTs) with ammonification hyaluronic acid:

Taking 60mg carboxylation carbon pipe is dissolved in 20ml Methanamide, and ice bath visits super 30min, becomes carboxylation carbon pipe solution; Take ammonification hyaluronic acid (HA-NH ₂) 96mg is dissolved in the Methanamide of 10ml, becomes ammonification hyaluronic acid solution; Take EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) 160mg, NHS (N-Hydroxysuccinimide) 108mg, dissolve with 3ml Methanamide vortex, join in carboxylation carbon pipe solution, stir room temperature reaction 15min, become carboxylation carbon pipe mixed solution; Triethylamine 180 μ l is added in ammonification hyaluronic acid solution, then by the mixed solution fast drop of ammonification hyaluronic acid and triethylamine in carboxylation carbon pipe mixed solution, room temperature reaction 24h, adds the acetone of 3 times of volume pre-coolings, cooling crystallization, organic membrane filter, washing with acetone precipitates, and obtain precipitate, precipitate water redissolves, dialysis 2d, lyophilizing;

(2) SWCNTs-HA-ss-COOH (i.e. SWCNTs load 3, the 3'-dithiodipropionic acid of HA modification) is prepared:

Get 3 of 1200mg carbon number 2 ~ 12,3 '-dithiodipropionic acid or suberic acid are linking arm, are dissolved in Methanamide, and EDC 1200mg, NHS 720mg respectively vortex are dissolved in Methanamide, by above-mentioned solution mix and blend activation 18min, become activated solution; HA-SWCNTs 240mg Probe Ultrasonic Searching is dissolved, mixes with activated solution, under room temperature condition, stir 5h, products therefrom passes through acetone ice bath crystallize, organic membrane filter, collecting precipitation, precipitation water redissolves, dialysis 2d, removes 3,3'-unnecessary dithiodipropionic acids, EDC, NHS salt, lyophilizing, become SWCNTs-HA-ss-COOH (SWCNTs load 3, the 3'-dithiodipropionic acid that HA modifies), 4 DEG C of preservations;

(3) SWCNTs-HA-ss-COOH and DOX (amycin) connect into SWCNTs-HA-ss-DOX:

Getting the pH that 5.4mg DOXHCL is dissolved in 1200 μ l is in the PBS solution of 8, becomes the first solution; The SWCNTs-HA-ss-COOH ultrasonic dissolution taking 9mg, in 600 μ l thionyl chlorides, becomes the second solution, two kinds of solution is mixed reaction 2h, becomes mixed liquor; Getting the pH that 42mg oxammonium hydrochloride. is dissolved in 1920 μ l is in the PBS solution of 8, and add in mixed liquor and react 2h, obtain product, product is with after ultra-pure water dialysis 2d, and lyophilizing, becomes SWCNTs-HA-ss-DOX;

(4) load of Gd (gadolinium):

The SWCNTs-HA-ss-DOX of 6mg is added to the water, and in ice bath, Probe Ultrasonic Searching 30min disperses, and centrifugally discards the precipitation of not disperseing, and becomes SWCN suspension; By the GdCl of 48mg under room temperature magnetic agitation condition ₃powder is soluble in water, is added drop-wise in finely dispersed SWCN suspension, produce flocculent deposit, Gd/SWCNTs-HA-ss-DOX;

(5) agent of Gd/SWCNTs-HA-ss-DOX medicament nano is prepared:

By the centrifugal 15min of step (4) mixture containing flocculent deposit, abandoning supernatant must precipitate, surfactant solution is added in precipitation, ice bath Probe Ultrasonic Searching 30min disperses, and then centrifugal 20min, discards precipitation, supernatant dialysis 10-16h (spending the night), centrifugal 15min, discards precipitation again, obtains the reduction-sensitive medicament nano agent (Gd/SWCNTs-HA-ss-DOX) that hyaluronic acid decorated SWCN prepares diagnoses and treatment;

Described surfactant solution be soybean lecithin and poloxamer by weight the mixture 10mg of 2 ︰ 1 be dissolved in the water of 1ml and make.

Embodiment 3

The present invention, in concrete enforcement, is realized by following steps:

(1) carboxylation carbon pipe (i.e. carboxylic carbon nano-tube) is connected (HA-SWCNTs) with ammonification hyaluronic acid:

Taking 70mg carboxylation carbon pipe is dissolved in 28ml DMF, and ice bath visits super 35min, becomes carboxylation carbon pipe solution; Take ammonification hyaluronic acid (HA-NH ₂) 112mg is dissolved in the DMF of 14ml, becomes ammonification hyaluronic acid solution; Take EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) 224mg, NHS (N-Hydroxysuccinimide) 126mg, use 3.5ml N, dinethylformamide vortex dissolves, join in carboxylation carbon pipe solution, stir room temperature reaction 18min, become carboxylation carbon pipe mixed solution; Triethylamine 210 μ l is added in ammonification hyaluronic acid solution, then by the mixed solution fast drop of ammonification hyaluronic acid and triethylamine in carboxylation carbon pipe mixed solution, room temperature reaction 36h, adds the acetone of 3 times of volume pre-coolings, cooling crystallization, organic membrane filter, washing with acetone precipitates, and obtain precipitate, precipitate water redissolves, dialysis 2d, lyophilizing;

(2) SWCNTs-HA-ss-COOH (i.e. SWCNTs load 3, the 3'-dithiodipropionic acid of HA modification) is prepared:

Get 3 of 1400mg carbon number 2 ~ 12,3 '-dithiodipropionic acid or suberic acid are linking arm, are dissolved in N, in dinethylformamide, EDC 1400mg, NHS 840mg respectively vortex are dissolved in DMF, by above-mentioned solution mix and blend activation 18min, become activated solution; HA-SWCNTs 280mg Probe Ultrasonic Searching is dissolved, mixes with activated solution, under room temperature condition, stir 4h, products therefrom passes through acetone ice bath crystallize, organic membrane filter, collecting precipitation, precipitation water redissolves, dialysis 2d, removes 3,3'-unnecessary dithiodipropionic acids, EDC, NHS salt, lyophilizing, become SWCNTs-HA-ss-COOH (SWCNTs load 3, the 3'-dithiodipropionic acid that HA modifies), 4 DEG C of preservations;

(3) SWCNTs-HA-ss-COOH and DOX (amycin) connect into SWCNTs-HA-ss-DOX:

Getting the pH that 6.3mg DOXHCL is dissolved in 1400 μ l is in the PBS solution of 9.2, becomes the first solution; The SWCNTs-HA-ss-COOH ultrasonic dissolution taking 12.6mg, in 500 μ l thionyl chlorides, becomes the second solution, two kinds of solution is mixed reaction 2h, becomes mixed liquor; Getting the pH that 49mg oxammonium hydrochloride. is dissolved in 2000 μ l is in the PBS solution of 9.2, and add in mixed liquor and react 2h, obtain product, product is with after ultra-pure water dialysis 2d, and lyophilizing, becomes SWCNTs-HA-ss-DOX;

(4) load of Gd (gadolinium):

The SWCNTs-HA-ss-DOX of 8mg is added to the water, and in ice bath, Probe Ultrasonic Searching 35min disperses, and centrifugally discards the precipitation of not disperseing, and becomes SWCN suspension; By the GdCl of 64mg under room temperature magnetic agitation condition ₃powder is soluble in water, is added drop-wise in finely dispersed SWCN suspension, produce flocculent deposit, Gd/SWCNTs-HA-ss-DOX;

(5) agent of Gd/SWCNTs-HA-ss-DOX medicament nano is prepared:

By the centrifugal 10min of step (4) mixture containing flocculent deposit, abandoning supernatant must precipitate, surfactant solution is added in precipitation, ice bath Probe Ultrasonic Searching 30min disperses, and then centrifugal 20min, discards precipitation, supernatant dialysis 10-16h (spending the night), centrifugal 15min, discards precipitation again, obtains the reduction-sensitive medicament nano agent (Gd/SWCNTs-HA-ss-DOX) that hyaluronic acid decorated SWCN prepares diagnoses and treatment;

Described surfactant solution be soybean lecithin and poloxamer by weight the mixture 10mg of 3 ︰ 1 be dissolved in the water of 1ml and make.

The application in tumor chemotherapeutic drug and nuclear magnetic resonance is being prepared in the reduction-sensitive medicament nano agent that the hyaluronic acid decorated SWCN of the present invention prepares diagnoses and treatment.

The inventive method is easy to operate, and prepared product has good biocompatibility, tumor-targeting is good, thermotherapy combines with chemotherapy, Clinics and Practices is integrated advantage as tumour medicine transport system.

As can be seen from above-mentioned, the present invention uses the SWCNTs after HA modifies, use HA carbon nano-tube modified, not only increase water solublity and the biocompatibility of CNT, also improve its targeting ability to tumor cell, pass through to contain 3 of disulfide bond on the amino of HA based on this, 3 '-dithiodipropionic acid is linking arm, introduce containing amino DOX: disulfide bond is reduction response type chemical bond, by the effect of the glutathion of high expressed in tumor cell, rapid fracture, makes antitumor drug efficiently discharge.In addition, because SWCNTs is virtue skeleton, Gd3+ is optionally deposited on ultrashort SWCN, overcome the defects such as current clinical conventional contrast agent carrier targeting difference, its imaging performance greatly improves, have cancer target concurrently, NMR (Nuclear Magnetic Resonance) imaging, thermotherapy combines with chemotherapy, the newtype drug system Gd/SWCNTs-HA-ss-DOX (hyaluronic acid decorated SWCN prepares the reduction-sensitive medicament nano agent of diagnoses and treatment) that Clinics and Practices is integrated, and achieve very satisfied Advantageous Effects through test, interrelated data is as follows:

The mensuration of Gd content in Gd/SWCNTs-HA-ss-DOX nanometer formulation

Adopt the content of ICP-OES assay gadolinium element, the drug loading of (1) calculation sample with the formula, drug loading reaches 12.0%,

The particle diameter of Gd/SWCNTs-HA-ss-DOX nanometer formulation and current potential characterize

Get after appropriate Gd/SWCNTs-HA-ss-DOX nanometer formulation is diluted with water to suitable concentration, record its particle diameter with Nano-ZS90 type laser nano Particle Size Analyzer and current potential is respectively 199.3nm and-27.2 ± 0.26mv.

Gd/SWCNTs-HA-ss-DOX nanometer of the present invention agent is tested the Proliferation Ability of MCF-7 cell

According to the step of passage by the process of MCF-7 cell to single cell suspension, after counting, every hole inoculation 5 × 103 cells are on 96 orifice plates, incubator cultivates (37 DEG C, 5%CO2) 24h until cell attachment completely after discard dosing after former culture medium, Gd/SWCNTs-HA-ss-DOX group, SWCNTs-HA-ss-DOX group, the added drug level of DOX group is that a series of gradient is formulated by the culture medium not containing serum with DOX concentration, the Concentraton gradient 0 of DOX, 0.039, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 μ g/ml, the group being connected with the SWCN after modification is provided with 808nm laser matched group, and after dosing a period of time irradiate 808nm laser 3min (power 2.0), culture plate is taken out after continuing to cultivate 48h, every hole adds 50% trichloroacetic acid of 50 μ l pre-coolings, final concentration is 10%, leave standstill 5min, move in 4 DEG C of refrigerators and leave standstill 1h, take out with ultrapure washing 5 times, in air at room temperature after drying completely, add the SRB50 μ l of 1% peracetic acid formulation in every hole, dye under room temperature 20min, outwell dye liquor, wash 5 times with 1% acetic acid, unconjugated dyestuff in removing hole, air at room temperature drying is dissolved with the 10mmol/LTris alkali liquor 150 μ l of pH10.5 afterwards, in air heat shaking table, shake 10min, on enzyme-linked immunosorbent assay instrument, measure the light absorption angle value of each hole at 515nm place.Calculate suppression ratio (%)=(1-experimental group A/ control group A) × 100%, show that the half-inhibition concentration (IC50) of above-mentioned sample is followed successively by thus: the non-thermotherapy group of Gd/SWCNTs-HA-ss-DOX, the non-thermotherapy group of SWCNTs-HA-ss-DOX, DOX group is 1.008,0.987,1.626 μ g/ml; Gd/SWCNTs-HA-ss-DOX thermotherapy group, SWCNTs-HA-ss-DOX thermotherapy group: 0.5934,0.6186 μ g/ml.

Gd/SWCNTs-HA-ss-DOX nanometer formulation of the present invention is to MCF-7 cell streaming picked-up test

Experiment is divided into SWCNT/FITC group, HA-SWCNTs/FITC group, SWCNTs-HA-ss-DOX group, Gd/SWCNTs-HA-ss-DOX group, is separately provided with blanc cell group and pure FITC group.Wherein the contained FITC concentration of FITC group is consistent containing containing FITC concentration in FITC group with all the other.The MCF-7 cell of trophophase of taking the logarithm becomes single cell suspension according to the step process of passage, and paving six orifice plates after counting, every porocyte number is 3 × 10 ⁵individual/hole, and incubator (37 DEG C, 5%CO ₂) in cultivate 24h adherent completely after, discard culture medium, add and use not containing the medicine that the culture medium of serum is prepared, 0.5h is set respectively, 1h, 2h, 4h, the time period of 6h carries out post-processed, medicinal liquid sucks in corresponding EP pipe, PBS washes 2 times, then with the trypsinization process 1min not containing EDTA of 0.5ml, add culture medium 1ml, suck corresponding EP pipe, after 1000rpm/10min is centrifugal, abandon supernatant and stay precipitation, add 2mlPBS piping and druming evenly, after 1000rpm/10min is centrifugal, abandon supernatant and stay precipitation, transfer in 1.5mlEP pipe after adding 0.5mlPBS piping and druming evenly and be placed in ice chest, wait for machine testing in streaming, record and the streaming intake of MCF-7 is followed successively by: 35.2%, 89.6%, 95.6%, 96.2%.

The pharmacodynamics test of Gd/SWCNTs-HA-ss-DOX nanometer formulation of the present invention

10 female mices are carried out S-180 sarcoma ascites to cultivate, extract ascites after two weeks, for the plantation of mouse entity tumor, when the volume of tumor reaches 100mm ³time above, tumor model is inoculated successfully, and tumor-bearing mice is divided into nine groups at random, often organizes eight, and grouping situation is as follows: (1) blank group; (2) blank-laser group; (3) DOX group; (4) DOX-laser group; (5) SWCNT-HA-ss-DOX group; (6) SWCNTs-HA-ss-DOX-laser group; (7) Gd/SWCNTs-HA-ss-DOX group; (8) Gd/SWCNTs-HA-ss-DOX laser group.Then administration next day of carrying out, dosage is 4mg/kg by people Mus dose lonvestion, tail vein injection administration.Observe the survival state of mice every day and the amount of weighing tumor volume (by gross tumor volume (V)=A × B ²/ 2), then R=V/V is carried out by relative tumor volume ₀evaluate the change (V of tumor ₀size for administration tumor the previous day volume) evaluate tumor anti-tumor activity.The data of record show, compare at the end of administration with before administration, and each group tumor volume suppression ratio is followed successively by 0.01%, 2.09%, 48.09%, 52.50%, 71.27%, 82.97%, 70.14%, 83.17%.

The nuclear magnetic resonance test of the gadolinium class contrast agent of Gd/SWCNTs-HA-ss-DOX nanometer formulation of the present invention

12 tumor-bearing mices are divided into three groups and labelling at random, take isoflurane suction-type to anaesthetize, then mice is fixed on fixing head and carries out unenhanced with 7.0T toy specific core magnetic resonance device; Three groups of tumor-bearing mices inject Gd/SWCNTs-HA-ss-DOX, Gd/SWCNTs-COOH, Gd-DTPA, Gd respectively ³⁺injection volume is 0.15mmol/Kg, inject successfully and again mice is fixed on the up each time point scanning of fixing head in 0h, 0.5h, 1h, 3h, 6h, 8h, three groups of mices carry out enhanced ct scans after the magnetic resonance contrast agent that injection is different, three groups of mices signal after injection 30min all strengthens, but Gd-DTPA group signal strengthens maximum at 0.5h, 1h signal obviously reduces, and time point signal intensity subsequently recovers normal value; The signal intensity of Gd/SWCNTs-HA-ss-DOX group and Gd/SWCNTs-COOH group extends enhancing in time, and Gd/SWCNTs-HA-ss-DOX group signal is that intensity enhancing is maximum, is obviously greater than Gd/SWCNTs-COOH group.Gd/SWCNTs-HA-ss-DOX shows obvious high relaxation usefulness and sustained release performance.

Test shows, the present invention compared with prior art, has following outstanding Advantageous Effects:

1, the bioreductive glutathion that the present invention is based on high concentration provides tumor tissues reproducibility microenvironment, select the linking arm containing disulfide bond to connect SWCN and antitumor drug, utilize this trigger mechanism can realize anti-tumor agent in vivo fast, Targeting delivery medicine and reduce the toxic and side effects of antitumor drug;

2, the SWCN after the present invention selects hyaluronic acid to modify as target head carries out medicine carrying, the advantages such as the carrier formed has good biocompatibility, Drug-loading Pattern is various, tumor-targeting good, thermotherapy effect is good;

3, the anti-tumor medicinal preparation prepared by the present invention also has the prospect being applied to clinical tumor diagnosis, contrast agent gadolinium is carried on material with carbon element and makes tumor-targeting enhancing, relaxivity strengthens, biological half-life extends, treatment in conjunction with antitumor drug can realize the Novel medicine feeding system that Clinics and Practices combines, be the innovation on Diagnosis and Treat tumour medicine, economic and social benefit is huge.

Claims (6)

1. a hyaluronic acid decorated SWCN prepares the reduction-sensitive medicament nano agent of diagnoses and treatment, it is characterized in that, this transport system by the SWCN after hyaluronic acid decorated by 3,3 '-dithiodipropionic acid is connected with amycin as linking arm, then gadolinium trichloride is adsorbed onto on SWCN, formed with hyaluronic acid decorated SWCN, for the nanometer formulation of the reduction-sensitive medicine of diagnoses and treatment by surfactant-dispersed again; Described hyaluronic acid is that molecular weight is more than or equal to 600 dalton and is less than or equal to the low-molecular-weight hyaluronic acid of 400 kd; The length of described SWCN is 1 ~ 3 μm, diameter is 1 ~ 2nm; Described linking arm is 3 of carbon number 2 ~ 12,3 '-dithiodipropionic acid or suberic acid; Described 3, the disulfide bond in 3 '-dithiodipropionic acid has reduction-sensitive, and under the glutathion effect of tumor cell high concentration, fracture discharges medicine rapidly.

2. hyaluronic acid decorated SWCN according to claim 1 prepares the method for the reduction-sensitive medicament nano agent of diagnoses and treatment, it is characterized in that, is realized by following steps:

(1) carboxylation carbon pipe is connected with ammonification hyaluronic acid:

Take 25 ~ 75mg carboxylation carbon pipe in the beaker of 15 ~ 45ml reaction dissolvent, ice bath is visited and is surpassed 15 ~ 45min, becomes carboxylation carbon pipe solution; Taking ammonification hyaluronic acid 60 ~ 120mg is dissolved in the solvent of 5-15ml, becomes ammonification hyaluronic acid solution; Take EDC 120 ~ 240mg, NHS 80 ~ 160mg, dissolve with 1 ~ 7ml solvent vortex, join in carboxylation carbon pipe solution, stir room temperature reaction 5 ~ 25min, become carboxylation carbon pipe mixed solution; Triethylamine 100 ~ 260 μ l is added in ammonification hyaluronic acid solution, then by the mixed solution fast drop of ammonification hyaluronic acid and triethylamine in carboxylation carbon pipe mixed solution, room temperature reaction 8 ~ 40h, adds the acetone of 3 ~ 4 times of volume pre-coolings, cooling crystallization, filter, washing with acetone precipitates, and obtain precipitate, precipitate water redissolves, dialysis 2d, lyophilizing;

Described solvent is the mixed solvent of water, Methanamide or DMF and water or Methanamide and water or DMF and Methanamide;

Described ammonification hyaluronic acid is, by hyaluronic acid 95-105mg, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride 254-264mg and N-N-Hydroxysuccinimide 150-160mg, be dissolved in the organic solvent of 8-12ml, stirring at room temperature 30 min, obtain reactant liquor, reactant liquor is slowly instilled in the formamide solution of 0.4-0.6ml ethylenediamine, ice bath drips 1h, rise to room temperature reaction 6-48h, add 50ml acetone precipitation, sucking filtration, obtain precipitate, precipitate adds water redissolution, dialysis 2d, lyophilization, obtain ammonification hyaluronic acid, organic solvent is Methanamide, N, N-dimethyl formamide, the one of dimethyl sulfoxide,

(2) SWCNTs-HA-ss-COOH is prepared:

Get 3 of 500 ~ 1500mg carbon number 2 ~ 12,3 '-dithiodipropionic acid or suberic acid are linking arm, be dissolved in reaction dissolvent, EDC 500 ~ 1500mg, NHS 200 ~ 1000mg respectively vortex are dissolved in reaction dissolvent, by above-mentioned solution mix and blend activation 5 ~ 25min, become activated solution; By HA-SWCNTs 50 ~ 350mg ultrasonic dissolution, mix with activated solution, under room temperature condition, stir 1 ~ 8h, products therefrom is by acetone ice bath crystallize, filter, collecting precipitation, precipitation water redissolves, dialysis 2d, remove 3,3'-unnecessary dithiodipropionic acids, EDC, NHS salt, lyophilizing, become SWCNTs-HA-ss-COOH, 4 DEG C of preservations;

Described reaction dissolvent is the mixed solvent of water or Methanamide or DMF and water or Methanamide and water or DMF and Methanamide;

(3) SWCNTs-HA-ss-COOH and DOX connects into SWCNTs-HA-ss-DOX:

Getting the pH that 1 ~ 8mg DOXHCL is dissolved in 500 ~ 1500 μ l is in the PBS solution of 7 ~ 10, becomes the first solution; The SWCNTs-HA-ss-COOH ultrasonic dissolution taking 2 ~ 15mg, in 200 ~ 800 μ l reaction dissolvents, becomes the second solution, two kinds of solution is mixed reaction 1 ~ 4h, becomes mixed liquor; Getting the pH that 10 ~ 60mg oxammonium hydrochloride. is dissolved in 200 ~ 2000 μ l is in the PBS solution of 7 ~ 10, and add in mixed liquor and react 0.5 ~ 5h, obtain product, product is with after ultra-pure water dialysis 2d, and lyophilizing, becomes SWCNTs-HA-ss-DOX;

Described reaction dissolvent is the mixed solvent of thionyl chloride or DMF or thionyl chloride and DMF;

(4) load of Gd:

The SWCNTs-HA-ss-DOX of 5 ~ 10mg is added to the water, ultrasonic 5 ~ 60min dispersion in ice bath, centrifugally discards the precipitation of not disperseing, and becomes SWCN suspension; By the GdCl of 50 ~ 80mg under room temperature magnetic agitation condition ₃powder is soluble in water, is added drop-wise in finely dispersed SWCN suspension, produce flocculent deposit, Gd/SWCNTs-HA-ss-DOX;

(5) agent of Gd/SWCNTs-HA-ss-DOX medicament nano is prepared:

By the centrifugal 2 ~ 30min of step (4) mixture containing flocculent deposit, abandoning supernatant must precipitate, surfactant solution is added in precipitation, ice-bath ultrasonic 5 ~ 55min disperses, and then centrifugal 2 ~ 25min, discards precipitation, supernatant dialysis 10-16h, centrifugal 2 ~ 30min, discards precipitation again, obtains the reduction-sensitive medicament nano agent that hyaluronic acid decorated SWCN prepares diagnoses and treatment;

Described surfactant is soybean lecithin, or the mixture of soybean lecithin and poloxamer, and surfactant solution is that the water adding 1ml by the surfactant of every 10mg is made.

3. hyaluronic acid decorated SWCN according to claim 2 prepares the method for the reduction-sensitive medicament nano agent of diagnoses and treatment, it is characterized in that, is realized by following steps:

(1) carboxylation carbon pipe is connected with ammonification hyaluronic acid:

Taking 50mg carboxylation carbon pipe is dissolved in 20ml Methanamide, and ice bath visits super 25min, becomes carboxylation carbon pipe solution; Taking ammonification hyaluronic acid 80mg is dissolved in the Methanamide of 10ml, becomes ammonification hyaluronic acid solution; Take EDC 160mg, NHS 90mg, dissolve with 2.5ml Methanamide vortex, join in carboxylation carbon pipe solution, stir room temperature reaction 15min, become carboxylation carbon pipe mixed solution; Triethylamine 150 μ l is added in ammonification hyaluronic acid solution, then by the mixed solution fast drop of ammonification hyaluronic acid and triethylamine in carboxylation carbon pipe mixed solution, room temperature reaction 24h, adds the acetone of 3 times of volume pre-coolings, cooling crystallization, organic membrane filter, washing with acetone precipitates, and obtain precipitate, precipitate water redissolves, dialysis 2d, lyophilizing;

(2) SWCNTs-HA-ss-COOH is prepared:

Get 3 of 1000mg carbon number 2 ~ 12,3 '-dithiodipropionic acid or suberic acid are linking arm, are dissolved in Methanamide, and EDC 1000mg, NHS 600mg respectively vortex are dissolved in Methanamide, by above-mentioned solution mix and blend activation 15min, become activated solution; HA-SWCNTs 200mg Probe Ultrasonic Searching is dissolved, mixes with activated solution, under room temperature condition, stir 1 ~ 8h, products therefrom is by acetone ice bath crystallize, filter, collecting precipitation, precipitation water redissolves, dialysis 2d, remove 3,3'-unnecessary dithiodipropionic acids, EDC, NHS salt, lyophilizing, become SWCNTs-HA-ss-COOH, 4 DEG C of preservations;

(3) SWCNTs-HA-ss-COOH and DOX connects into SWCNTs-HA-ss-DOX:

Getting the pH that 6.3mg DOXHCL is dissolved in 1400 μ l is in the PBS solution of 7.4, becomes the first solution; The SWCNTs-HA-ss-COOH ultrasonic dissolution taking 12.6mg, in 500 μ l thionyl chlorides, becomes the second solution, two kinds of solution is mixed reaction 2h, becomes mixed liquor; Getting the pH that 49mg oxammonium hydrochloride. is dissolved in 2000 μ l is in the PBS solution of 7.4, and add in mixed liquor and react 2.5h, obtain product, product is with after ultra-pure water dialysis 2d, and lyophilizing, becomes SWCNTs-HA-ss-DOX;

(4) load of Gd:

The SWCNTs-HA-ss-DOX of 8mg is added to the water, and in ice bath, Probe Ultrasonic Searching 35min disperses, and centrifugally discards the precipitation of not disperseing, and becomes SWCN suspension; By the GdCl of 64mg under room temperature magnetic agitation condition ₃powder is soluble in water, is added drop-wise in finely dispersed SWCN suspension, produce flocculent deposit, Gd/SWCNTs-HA-ss-DOX;

(5) agent of Gd/SWCNTs-HA-ss-DOX medicament nano is prepared:

By the centrifugal 10min of step (4) mixture containing flocculent deposit, abandoning supernatant must precipitate, surfactant solution is added in precipitation, ice bath Probe Ultrasonic Searching 30min disperses, and then centrifugal 20min, discards precipitation, supernatant dialysis 10-16h, centrifugal 15min, discards precipitation again, obtains the reduction-sensitive medicament nano agent that hyaluronic acid decorated SWCN prepares diagnoses and treatment;

Described surfactant solution be soybean lecithin and poloxamer by weight the mixture 10mg of 1 ︰ 1 be dissolved in the water of 1ml and make.

4. hyaluronic acid decorated SWCN according to claim 2 prepares the method for the reduction-sensitive medicament nano agent of diagnoses and treatment, it is characterized in that, is realized by following steps:

(1) carboxylation carbon pipe is connected with ammonification hyaluronic acid:

Taking 60mg carboxylation carbon pipe is dissolved in 20ml Methanamide, and ice bath visits super 30min, becomes carboxylation carbon pipe solution; Taking ammonification hyaluronic acid 96mg is dissolved in the Methanamide of 10ml, becomes ammonification hyaluronic acid solution; Take EDC 160mg, NHS 108mg, dissolve with 3ml Methanamide vortex, join in carboxylation carbon pipe solution, stir room temperature reaction 15min, become carboxylation carbon pipe mixed solution; Triethylamine 180 μ l is added in ammonification hyaluronic acid solution, then by the mixed solution fast drop of ammonification hyaluronic acid and triethylamine in carboxylation carbon pipe mixed solution, room temperature reaction 24h, adds the acetone of 3 times of volume pre-coolings, cooling crystallization, organic membrane filter, washing with acetone precipitates, and obtain precipitate, precipitate water redissolves, dialysis 2d, lyophilizing;

(2) SWCNTs-HA-ss-COOH is prepared:

Get 3 of 1200mg carbon number 2 ~ 12,3 '-dithiodipropionic acid or suberic acid are linking arm, are dissolved in Methanamide, and EDC 1200mg, NHS 720mg respectively vortex are dissolved in Methanamide, by above-mentioned solution mix and blend activation 18min, become activated solution; HA-SWCNTs 240mg Probe Ultrasonic Searching is dissolved, mixes with activated solution, under room temperature condition, stir 5h, products therefrom is by acetone ice bath crystallize, organic membrane filter, collecting precipitation, precipitation water redissolves, dialysis 2d, remove 3,3'-unnecessary dithiodipropionic acids, EDC, NHS salt, lyophilizing, become SWCNTs-HA-ss-COOH, 4 DEG C of preservations;

(3) SWCNTs-HA-ss-COOH and DOX connects into SWCNTs-HA-ss-DOX:

Getting the pH that 5.4mg DOXHCL is dissolved in 1200 μ l is in the PBS solution of 8, becomes the first solution; The SWCNTs-HA-ss-COOH ultrasonic dissolution taking 9mg, in 600 μ l thionyl chlorides, becomes the second solution, two kinds of solution is mixed reaction 2h, becomes mixed liquor; Getting the pH that 42mg oxammonium hydrochloride. is dissolved in 1920 μ l is in the PBS solution of 8, and add in mixed liquor and react 2h, obtain product, product is with after ultra-pure water dialysis 2d, and lyophilizing, becomes SWCNTs-HA-ss-DOX;

(4) load of Gd:

The SWCNTs-HA-ss-DOX of 6mg is added to the water, and in ice bath, Probe Ultrasonic Searching 30min disperses, and centrifugally discards the precipitation of not disperseing, and becomes SWCN suspension; By the GdCl of 48mg under room temperature magnetic agitation condition ₃powder is soluble in water, is added drop-wise in finely dispersed SWCN suspension, produce flocculent deposit, Gd/SWCNTs-HA-ss-DOX;

(5) agent of Gd/SWCNTs-HA-ss-DOX medicament nano is prepared:

By the centrifugal 15min of step (4) mixture containing flocculent deposit, abandoning supernatant must precipitate, surfactant solution is added in precipitation, ice bath Probe Ultrasonic Searching 30min disperses, and then centrifugal 20min, discards precipitation, supernatant dialysis 10-16h, centrifugal 15min, discards precipitation again, obtains the reduction-sensitive medicament nano agent that hyaluronic acid decorated SWCN prepares diagnoses and treatment;

Described surfactant solution be soybean lecithin and poloxamer by weight the mixture 10mg of 2 ︰ 1 be dissolved in the water of 1ml and make.

5. hyaluronic acid decorated SWCN according to claim 2 prepares the method for the reduction-sensitive medicament nano agent of diagnoses and treatment, it is characterized in that, is realized by following steps:

(1) carboxylation carbon pipe is connected with ammonification hyaluronic acid:

Taking 70mg carboxylation carbon pipe is dissolved in 28ml DMF, and ice bath visits super 35min, becomes carboxylation carbon pipe solution; Taking ammonification hyaluronic acid 112mg is dissolved in the DMF of 14ml, becomes ammonification hyaluronic acid solution; Take EDC 224mg, NHS 126mg, dissolve with 3.5ml DMF vortex, join in carboxylation carbon pipe solution, stir room temperature reaction 18min, become carboxylation carbon pipe mixed solution; Triethylamine 210 μ l is added in ammonification hyaluronic acid solution, then by the mixed solution fast drop of ammonification hyaluronic acid and triethylamine in carboxylation carbon pipe mixed solution, room temperature reaction 36h, adds the acetone of 3 times of volume pre-coolings, cooling crystallization, organic membrane filter, washing with acetone precipitates, and obtain precipitate, precipitate water redissolves, dialysis 2d, lyophilizing;

(2) SWCNTs-HA-ss-COOH is prepared:

Get 3 of 1400mg carbon number 2 ~ 12,3 '-dithiodipropionic acid or suberic acid are linking arm, are dissolved in N, in dinethylformamide, EDC 1400mg, NHS 840mg respectively vortex are dissolved in DMF, by above-mentioned solution mix and blend activation 18min, become activated solution; HA-SWCNTs 280mg Probe Ultrasonic Searching is dissolved, mixes with activated solution, under room temperature condition, stir 4h, products therefrom is by acetone ice bath crystallize, organic membrane filter, collecting precipitation, precipitation water redissolves, dialysis 2d, remove 3,3'-unnecessary dithiodipropionic acids, EDC, NHS salt, lyophilizing, become SWCNTs-HA-ss-COOH, 4 DEG C of preservations;

(3) SWCNTs-HA-ss-COOH and DOX connects into SWCNTs-HA-ss-DOX:

Getting the pH that 6.3mg DOXHCL is dissolved in 1400 μ l is in the PBS solution of 9.2, becomes the first solution; The SWCNTs-HA-ss-COOH ultrasonic dissolution taking 12.6mg, in 500 μ l thionyl chlorides, becomes the second solution, two kinds of solution is mixed reaction 2h, becomes mixed liquor; Getting the pH that 49mg oxammonium hydrochloride. is dissolved in 2000 μ l is in the PBS solution of 9.2, and add in mixed liquor and react 2h, obtain product, product is with after ultra-pure water dialysis 2d, and lyophilizing, becomes SWCNTs-HA-ss-DOX;

(4) load of Gd:

The SWCNTs-HA-ss-DOX of 8mg is added to the water, and in ice bath, Probe Ultrasonic Searching 35min disperses, and centrifugally discards the precipitation of not disperseing, and becomes SWCN suspension; By the GdCl of 64mg under room temperature magnetic agitation condition ₃powder is soluble in water, is added drop-wise in finely dispersed SWCN suspension, produce flocculent deposit, Gd/SWCNTs-HA-ss-DOX;

(5) agent of Gd/SWCNTs-HA-ss-DOX medicament nano is prepared:

By the centrifugal 10min of step (4) mixture containing flocculent deposit, abandoning supernatant must precipitate, surfactant solution is added in precipitation, ice bath Probe Ultrasonic Searching 30min disperses, and then centrifugal 20min, discards precipitation, supernatant dialysis 10-16h, centrifugal 15min, discards precipitation again, obtains the reduction-sensitive medicament nano agent that hyaluronic acid decorated SWCN prepares diagnoses and treatment;

Described surfactant solution be soybean lecithin and poloxamer by weight the mixture 10mg of 3 ︰ 1 be dissolved in the water of 1ml and make.

6. the application in tumor chemotherapeutic drug and nuclear magnetic resonance is being prepared in the reduction-sensitive medicament nano agent that claim 1 or the hyaluronic acid decorated SWCN described in any one of 2-5 prepare diagnoses and treatment.