CN104667290A - Targeting peptide-modified gold nanoparticle and preparation method thereof as well as application of targeting peptide-modified gold nanoparticle as platinic pro-drug carrier - Google Patents
Targeting peptide-modified gold nanoparticle and preparation method thereof as well as application of targeting peptide-modified gold nanoparticle as platinic pro-drug carrier Download PDFInfo
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Abstract
The invention discloses a hepatoma carcinoma cell targeting peptide-modified gold nanoparticle and a preparation method thereof and an application of the hepatoma carcinoma cell targeting peptide-modified gold nanoparticle as a platinic prodrug carrier and an antitumor medicine carrier. The hepatoma carcinoma cell targeting peptide-modified gold nanoparticle has selectivity. A hepatoma carcinoma cell targeting peptide is connected to the surface of the gold nanoparticle by forming a gold-sulfur covalent bond. The hepatoma carcinoma cell targeting peptide-modified gold nanoparticle is used for carrying a platinic prodrug, so that the selectivity on the hepatoma carcinoma cell is effectively improved; the bioavailability of the gold nanoparticle is improved; the targeting peptide is very helpful to application of the nanoparticle on treatment of the liver cancer. According to the preparation method of the hepatoma carcinoma cell targeting peptide, the targeting action of the gold nanoparticle on tumor cells is improved; the non-specific combination on normal cells is reduced; and the method is simple, feasible and low in cost, and can be prepared on a large scale.
Description
Technical field
The invention belongs to cancer target and safe treatment technical field.Particularly, relate to the nanogold particle that a kind of hepatoma carcinoma cell targeting peptides is modified, with and preparation method thereof with its application in preparation tetravalence platinum prodrug carrier.
Background technology
Hepatocarcinoma (abbreviation hepatocarcinoma) is one of modal malignant tumor in the world, simultaneously one of modal malignant tumor of Ye Shi China, and Chinese onset of liver cancer rate accounts for 45% of the whole world, and mortality rate is in the 3rd of malignant tumor.Hepatocarcinoma onset is hidden, and lacks classical symptom in early days, and progress is fast, and grade malignancy is high, poor prognosis, belongs to middle and advanced stage when the overwhelming majority finds.The treatment of hepatocarcinoma is mainly based on operation, chemotherapy and radiation, but chemotherapeutics effect is mostly non-selective, distribution in vivo is wide, and under therapeutic dose, normal tissue organ toxic and side effects is large, comprises gastrointestinal reaction, bone marrow inhibition, nephrotoxicity and Drug tolerance reduction etc.; According to statistics, the liver cancer patient Endodontic failure of more than 95%.
Cisplatin (Cisplatin, CDDP) be the antitumor drug of a platiniferous, namely CDDP (II), belongs to cell cycle nonspecific agent (CCNSA), has therapeutic efficiency to sarcoma, carcinoma, lymphoma and germ cell tumor.It is one that is synthesized the earliest in a large class platinum medicine, and structure is the simplest and the mechanism of action is clear and definite.But the side effect of cisplatin is a lot, comprising: Toxicity of Kidney, Nausea and vomiting, neurotoxicity, ototoxicity, alopecia and electrolyte disturbance; The time of cisplatin use simultaneously, time longer, most patients all can produce resistance to cisplatin, thus palindromia; It is necessary for finding new method for improvement of the treatment of cisplatin.
Tetravalence platinum compounds, as the prodrug of cisplatin, has that good water solubility, toxicity are low, oral availability advantages of higher, shows good anti-tumor activity, and still keep good activity to cisplatin-resistant cell in clinical trial.Meanwhile, tetravalence platinum compounds has great advantage in drug modification and medicament transport System Design.
It is high and be easy to modify the advantages such as chemical group that nanogold particle has low toxicity, biocompatibility, can safety be applied to human tumor treatment.Research shows, because the size of nanometer gold is less, tumor tissues improves the permeability to it, can assemble after making nanogold particle intravenous injection at tumor locus.
According to document 1 (naturally summarize cancer---Nature Reviews Cancer, 2005,5:161-171) report, blood vessel endothelium near tumor tissues has higher permeability compared with normal blood vessel endothelium, thus gold nano grain can penetrate in tumor tissues, then is entered in tumor cell by endocytosis.Medicine is loaded into nano grain surface, the object of transporting drugs can be reached.Bar-shaped gold nano grain has higher photoabsorption cross-section and excellent photo-thermal conversion efficiency near infrared band, research shows, nanometer gold bar and relevant nanostructured can pass through photo-thermal therapy, cancerous cell is killed under less illumination dose, and the cell of surrounding can not be destroyed, thus, nanogold particle has the function of pharmaceutical carrier.
SP94 sequence is the polypeptide of a liver cancer targeting cell.It is the polypeptide found by Phage display in vivo, and shows selective binding to multiple hepatoma carcinoma cell, and only has a small amount of combination for normal cell (normal liver cell, endotheliocyte, immunocyte etc.).In the mouse experiment of the combined immunodeficiency of transplanting human hepatocarcinoma cells, SP94 can be combined but not internal organs with liver cancer tissue by selectivity; Good selectivity makes SP94 can become a kind of hepatocarcinoma targeting part of practicality.
The nanogold particle that targeting peptides is modified can significantly improve its bio-compatibility, increases the selectivity to cancerous cell, reduces toxic and side effects.In prior art, the peptide modified method to nanogold particle surface there are two kinds, one is by peptide modified surperficial to nanogold particle by link agent, such as document 2 (Langmuir, 2001,17 (20): 6368-74) can be combined with polypeptide after using Polyethylene Glycol (PEG) decorated nanometer gold grain.The second is such as, by forming golden sulfide linkage directly by peptide modified surperficial at nanogold particle, (GCGC) sequence that document 3 (Bioconjugate Chemistry, 2012,23 (7): 1494-501) is used.
Therefore, develop a kind of nanogold particle with hepatoma carcinoma cell targeting, as pharmaceutical carrier, platinum medicine is transported to inside tumor cells, helpful to the chemotherapy of tumor, current this kind of technology is more, but with the gold nano grain that hepatoma carcinoma cell targeting peptides SP94 modifies, it is not yet seen report.
Summary of the invention
The object of this invention is to provide pharmaceutical carrier of the gold nano grain that a kind of hepatoma carcinoma cell targeting peptides being used as delivery platinum medicine is modified and preparation method thereof, to improve the selectivity to hepatoma carcinoma cell of gold nano grain, reduce it to Normocellular non-specific binding and controllable release platinum medicine.The nanogold particle simultaneously providing this hepatoma carcinoma cell targeting peptides to modify is preparing the application in pharmaceutical carrier.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
The gold nano grain that hepatoma carcinoma cell targeting peptides is modified, the gold nano grain that described targeting peptides is modified has selectivity to hepatoma carcinoma cell.
According to the gold nano grain that described a kind of hepatoma carcinoma cell targeting peptides is modified, wherein said targeting peptides contains 14-20 aminoacid, wherein comprise SFSIIHTPILPL and the SP94 sequence with hepatoma carcinoma cell targeting, containing 1-3 cysteine residues in described peptide sequence.
According to the gold nano grain that described a kind of hepatoma carcinoma cell targeting peptides is modified, the gold nano grain that described hepatoma carcinoma cell targeting peptides is modified forms by the sulfydryl in peptide molecule and gold the surface that peptide to be connected to gold nano grain by golden sulfur covalent bond.
According to the gold nano grain that described a kind of hepatoma carcinoma cell targeting peptides is modified, the gold nano grain that this targeting peptides is modified is obtained as follows:
(1) prepare the gold nano grain that cetyl trimethyl ammonia bromide (CTAB) is stable: with cetyl trimethyl ammonia bromide as soft stamp agent, adopt seed mediated growth method to prepare gold nano grain or prepare gold nano grain with sodium citrate as reducing agent;
(2) prepare the gold nano grain that targeting peptides combines: to step (1) gold nano particle colloidal sols, in add hepatoma carcinoma cell targeting peptides 1 ~ 5mg/ml and react; React after 4 ~ 8 hours, removed by responseless peptide by centrifugal method, by reactant liquor under 5000 ~ 14000 revs/min centrifugal 3 ~ 30 minutes, the gold nano grain that targeting peptides combines was deposited in bottom centrifuge tube; And then by nano-particle ultrasonic disperse in water, so repeatedly, clean the gold nano grain that targeting peptides combines.
The preparation method of the gold nano grain that described a kind of hepatoma carcinoma cell targeting peptides is modified, comprises the steps:
(1) prepare the gold nano grain that cetyl trimethyl ammonia bromide CTAB is stable: with cetyl trimethyl ammonia bromide as soft stamp agent, adopt seed mediated growth method to prepare gold nano grain or prepare gold nano grain with sodium citrate as reducing agent;
(2) prepare the gold nano grain that targeting peptides combines: to step (1) gold nano particle colloidal sols, in add hepatoma carcinoma cell targeting peptides 1 ~ 5mg/ml and react; React after 4 ~ 8 hours, removed by responseless peptide by centrifugal method, by reactant liquor under 5000 ~ 14000 revs/min centrifugal 3 ~ 30 minutes, the gold nano grain that targeting peptides combines was deposited in bottom centrifuge tube; And then by nano-particle ultrasonic disperse in water, so repeatedly, clean the gold nano grain that targeting peptides combines.
According to described preparation method, the method comprises the steps:
(1) preparation of gold nano grain:
The preparation of gold nano seed solution: take cetyl trimethyl ammonium bromide CTAB and be dissolved in 7.5ml water, make it in the hot water to dissolve completely, be then cooled to room temperature, by sodium borohydride (NaBH
4) be dissolved in frozen water, be configured to the solution of 10 mM/ls of concentration, in CTAB solution, add the chlorauric acid solution of 250 μ l, slightly shake, add rapidly the NaBH that 600 μ l brand-news are standby, freezing
4, within ultrasonic 2 minutes, get rid of bubble, constant temperature more than 2 hours in 37 DEG C of water;
The preparation of the gold nano grain that CTAB is stable: first weigh CTAB soluble in water, hot bath makes it to dissolve completely, then room temperature is cooled to, hydrochloric acid, silver nitrate, gold chloride, ascorbic acid is added successively in solvent CTAB solution, shake up as after colourless, add seed solution, within ultrasonic 2 minutes, get rid of bubble, put into 37 DEG C of water-baths and spend the night;
(2) preparation of Jenner's rod of targeting peptides modification
Get hepatoma carcinoma cell targeting peptides soluble in water, add nanometer gold bar colloidal sol, be slowly warming up to 55 DEG C, water bath with thermostatic control is after 3 hours, gentle agitation 2 hours under room temperature, and with 6000 revs/min, within 10 minutes, centrifugal secondary removes precipitation;
(3) nanometer gold bar that the targeting peptides of fluorophor is modified is with
Get rhodamine B, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide (N-Hydroxysuccinimide, NHS) soluble in water, lucifuge places 20 minutes, add the nanometer gold bar solution that targeting peptides is modified, lucifuge stirring reaction 24 hours;
With centrifuge speed 6000 revs/min, centrifugation time 10 minutes repeatedly centrifugal segregation supernatant concentrated volume;
(4) cell-targeting experiment
I. recover
The cell of recovery has hepatoma carcinoma cell (HepG2 cell), normal liver cell (7702 cell), Human Lung adenocarcinoma epithelial cell (A549 cell).
Open from-80 DEG C of refrigerators and take out frozen cell, tepidarium, until melt, draws cell suspension with liquid-transfering gun, and the culture fluid instilling more than 10 times mixes centrifugal afterwards and removes supernatant; Then with cell is resuspended and count containing the culture fluid of 10% hyclone, adjustment density, then inoculates, cultivates for 37 DEG C in culture bottle, and culture fluid is changed once every day;
II. targeting experiment
Exponential phase cell 2 × 10 is selected in cell-targeting experiment
4individual/ml is seeded in 24 orifice plates, every hole 100 μ l, cultivate and change fresh culture medium after 24 hours, experimental group, matched group and blank group are set, experimental group adds the medicine of variable concentrations gradient, matched group does not add medicine, and blank group does not have inoculating cell and do not add medicine, continues cultivation and removes culture medium after 4 hours;
Collect logarithmic (log) phase cell, adjustment concentration of cell suspension, every hole adds 100 μ l, and bed board makes cell to be measured adjust density to 10 ten thousand/hole, and edge hole sterile phosphate buffer is filled; 5%CO
2, hatch for 37 DEG C, cultivate 12 hours; Within second day, carry out application of sample, 5%CO
2, hatch 4 hours for 37 DEG C; Discard old culture medium and add fresh culture 100 μ l, wash once with phosphate buffer; Every hole adds 100 μ l Digestive systems, makes lysis, adds a large amount of phosphate buffer and is placed in 1.5ml centrifuge tube, carry out centrifugal with centrifuge speed 1500 revs/min, 5 minutes time after digestion terminates, removing supernatant; Add 1ml phosphate buffer centrifuge washing 1 time, abandoning supernatant, add 0.5ml phosphate buffer after cleaning, after being configured to suspension, use flow cytometer analysis.
The preparation method of the gold nano grain that a kind of hepatoma carcinoma cell targeting peptides of the present invention is modified can more specifically be expressed as follows: the surface by the covalent that forms golden sulfur effect, hepatoma carcinoma cell targeting peptides being connected to gold nano grain.
Comprise following concrete steps:
The first step, (this step is prior art to prepare the stable gold nano grain of cetyl trimethyl ammonia bromide (CTAB), can list of references 4 (chemical material---Chem.Mater.2003,15,1957-1962)): with cetyl trimethyl ammonia bromide as soft stamp agent, seed mediated growth method is adopted to prepare gold nano grain or prepare gold nano grain with sodium citrate as reducing agent;
Second step, prepares the gold nano grain that targeting peptides combines: in gold nano particle colloidal sols, add hepatoma carcinoma cell targeting peptides 1 ~ 5mg/ml react; React after 4 ~ 8 hours, by centrifugal method, responseless peptide is removed, by reactant liquor under 5000 ~ 14000 revs/min centrifugal 3 ~ 30 minutes, the gold nano grain that targeting peptides combines was deposited in bottom centrifuge tube, removes unreacted targeting peptides in supernatant; Then by nano-particle ultrasonic disperse in water, so repeatedly, clean targeting peptides modify nanogold particle;
3rd step, cell-targeting is tested: cell-targeting experiment selects exponential phase cell to be seeded in 96 orifice plates, cultivate and change fresh culture medium after 24 hours, experimental group, matched group and blank group are set, experimental group adds the medicine of variable concentrations gradient, matched group does not add medicine, and blank group does not have inoculating cell and do not add medicine; Continue cultivation and use flow cytometer analysis after 4 hours.
The gold nano grain that hepatoma carcinoma cell targeting peptides is modified is used as tetravalence platinum prodrug carrier.
The gold nano grain that hepatoma carcinoma cell targeting peptides is modified is used as antineoplastic drug carrier.
The application of gold nano grain in preparation tetravalence platinum prodrug carrier that hepatoma carcinoma cell targeting peptides is modified.
The gold nano grain that hepatoma carcinoma cell targeting peptides is modified is preparing the application in antineoplastic drug carrier.
Tetravalence platinum prodrug carrier or antineoplastic drug carrier is used as according to the gold nano grain that described hepatoma carcinoma cell targeting peptides is modified, the gold nano grain that described hepatoma carcinoma cell targeting peptides is modified is that the prodrug tetravalence platinum of cisplatin is connected on hepatoma carcinoma cell targeting peptides by the mode by forming amido link, and platinum medicine is transported to inside tumor cells by the mode of cell endocytic by gold nano grain.
Be used as tetravalence platinum prodrug carrier or antineoplastic drug carrier according to the gold nano grain that described hepatoma carcinoma cell targeting peptides is modified, the gold nano grain that described hepatoma carcinoma cell targeting peptides is modified is the surface by forming golden sulfur covalent bond hepatoma carcinoma cell targeting peptides being connected to gold nano grain.
According to the application of gold nano grain in preparation tetravalence platinum prodrug carrier or antineoplastic drug carrier that described hepatoma carcinoma cell targeting peptides is modified, described application is that the prodrug tetravalence platinum of cisplatin is connected on hepatoma carcinoma cell targeting peptides by the mode by forming amido link, and platinum medicine is transported to inside tumor cells by the mode of cell endocytic by gold nano grain.
According to the application of gold nano grain in preparation tetravalence platinum prodrug carrier or antineoplastic drug carrier that described hepatoma carcinoma cell targeting peptides is modified, described application is the gold nano grain delivery platinum medicine modified with hepatoma carcinoma cell targeting peptides.
Preparing the application in antineoplastic drug carrier according to the gold nano grain that described hepatoma carcinoma cell targeting peptides is modified, wherein said tumor is hepatocarcinoma.
14-20 aminoacid is contained according in described hepatoma carcinoma cell targeting peptides, wherein comprise SFSIIHTPILPL (SP94) sequence with hepatoma carcinoma cell targeting, in addition, be used in conjunction with gold nano grain containing 1-3 cysteine residues in peptide sequence.
The method of the gold nano grain that hepatoma carcinoma cell targeting peptides described in preparation is modified, being the surface by forming golden sulfur covalent bond hepatoma carcinoma cell targeting peptides being connected to gold nano grain, comprising the steps:
(1) prepare the gold nano grain that cetyl trimethyl ammonia bromide CTAB is stable: with cetyl trimethyl ammonia bromide as soft stamp agent, adopt seed mediated growth method to prepare gold nano grain or prepare gold nano grain with sodium citrate as reducing agent;
(2) gold nano grain that targeting peptides combines is prepared: in gold nano particle colloidal sols, add hepatoma carcinoma cell targeting peptides 1 ~ 5mg/ml react, react after 4 ~ 8 hours, by centrifugal method, responseless peptide is removed, by reactant liquor under 5000 ~ 14000 revs/min centrifugal 3 ~ 30 minutes, the gold nano grain that targeting peptides combines is deposited in bottom centrifuge tube, remove unreacted targeting peptides in supernatant, then by nano-particle ultrasonic disperse in water, so repeatedly, the gold nano grain that targeting peptides combines is cleaned;
(3) cell-targeting experiment: cell-targeting experiment selects exponential phase cell to be seeded in 96 orifice plates, train and change fresh culture medium after 24 hours, experimental group, matched group and blank group are set, experimental group adds the medicine of variable concentrations gradient, matched group does not add medicine, blank group does not have inoculating cell and does not add medicine, continues cultivation and uses flow cytometer analysis after 4 hours;
(4) the prodrug compound 1 [Pt (NH of cisplatin is prepared
3)
2cl
3(OOCCH
2cH
2cOOH)]: the aqueous hydrogen peroxide solution being 30% by 0.6mmol ~ 6mmol cisplatin and 5 ~ 50ml volumetric concentration joins in 6 ~ 60ml water, is heated to 40 ~ 80 DEG C, stirring reaction 1 ~ 10 hour, yellow solid slowly dissolves, and solution is faint yellow; Concentration of reaction solution to 0.5 ~ 5ml is also cooled to 0 ~ 8 DEG C, has the solid crystal solid of ecru to separate out, then recrystallization in boiling water, obtains compound 2 [Pt (NH
3)
2cl
3oH]; 1 ~ 5mmol succinic anhydride and 1 ~ 5mmol compound 2 are dissolved room temperature reaction 1 ~ 15 hour in 1 ~ 5ml dimethyl sulfoxide, solid material slowly dissolves, to reactant liquor in yellow, dimethyl sulfoxide is drained with vacuum pump, after remaining grease is dissolved in 0.1 ~ 0.5ml acetone, in this solution, add 30 ~ 50ml ether has faint yellow product to be precipitated out, dry in vacuum drying oven, obtains flaxen compound 1;
(5) gold nano grain that the targeting peptides preparing load platinum medicine combines: to the gold nano grain solution that combines of targeting peptides in add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl) and the N-hydroxysuccinimide (NHS) of 0.1 ~ 1mg compound 1 and 1 ~ 2 times of mole thereof, react 12 ~ 24 hours at 30 ~ 70 DEG C, by centrifugal method, responseless platinum medicine is removed, reactant liquor under 5000 ~ 14000 revs/min centrifugal 3 ~ 30 minutes, the gold nano grain in conjunction with targeting peptides of load platinum medicine is deposited in bottom centrifuge tube, remove unreacted platinum medicine in supernatant, then by the gold nano grain ultrasonic disperse in conjunction with targeting peptides of load platinum medicine in water, so repeatedly, clean load platinum medicine hepatoma carcinoma cell targeting peptides combine gold nano grain,
(6) targeting peptides of load platinum medicine is to platinic controllable release: add 0.1 ~ 10 mM/l of guanyl combining in platinic hepatoma carcinoma cell targeting peptides solution, 0.1 ~ 10 mM/l of vitamin C reacts, and in the sampling of differential responses stage, carry out high performance liquid chromatography detection.
Preparation method of the present invention can more specifically be expressed as follows: be connected on hepatoma carcinoma cell targeting peptides by the prodrug tetravalence platinum of cisplatin by the mode forming amido link, and platinum medicine is transported to inside tumor cells by the mode of cell endocytic by gold nano grain.By forming golden sulfur covalent bond hepatoma carcinoma cell targeting peptides is connected to the surface of gold nano grain.Comprise following concrete steps:
The first step, (this step is prior art to prepare the stable gold nano grain of cetyl trimethyl ammonia bromide (CTAB), can list of references 2 (chemical material---Chem.Mater.2003,15,1957-1962)): with cetyl trimethyl ammonia bromide as soft stamp agent, seed mediated growth method is adopted to prepare gold nano grain or prepare gold nano grain with sodium citrate as reducing agent;
Second step, prepares the gold nano grain that targeting peptides combines: in gold nano particle colloidal sols, add hepatoma carcinoma cell targeting peptides SP941 ~ 5mg/ml react; React after 4 ~ 8 hours, by centrifugal method, responseless peptide is removed, by reactant liquor under 5000 ~ 14000 revs/min centrifugal 3 ~ 30 minutes, the gold nano grain that targeting peptides combines was deposited in bottom centrifuge tube, removes unreacted targeting peptides in supernatant; Then by nano-particle ultrasonic disperse in water, so repeatedly, clean clean targeting peptides combine gold nano grain;
3rd step, cell-targeting is tested: cell-targeting experiment selects exponential phase cell to be seeded in 96 orifice plates, cultivate and change fresh culture medium after 24 hours, experimental group, matched group and blank group are set, experimental group adds the medicine of variable concentrations gradient, matched group does not add medicine, and blank group does not have inoculating cell and do not add medicine; Continue cultivation and use flow cytometer analysis after 4 hours.
4th step, prepares the prodrug compound 1 [Pt (NH of cisplatin
3)
2cl
3(OOCCH
2cH
2cOOH)] (this step is prior art, can list of references 3 (pharmaceutical chemistry---J.Med.Chem.2007,50,6692 – 6699)).Compound 1 [Pt (NH
3)
2cl
3(OOCCH
2cH
2cOOH) preparation]: the aqueous hydrogen peroxide solution being 30% by 0.6mmol ~ 6mmol cisplatin and 5 ~ 50ml volumetric concentration joins in 6 ~ 60ml water, be heated to 40 ~ 80 DEG C, stirring reaction 1 ~ 10 hour, yellow solid slowly dissolves, and solution is faint yellow; Concentration of reaction solution to 0.5 ~ 5ml is also cooled to 0 ~ 8 DEG C, has the solid crystal solid of ecru to separate out, then in boiling water recrystallization, obtain compound 2 [Pt (NH
3)
2cl
3oH]; By 1 ~ 5mmol succinic anhydride and 1 ~ 5mmol compound 2 [Pt (NH
3)
2cl
3oH] in 1 ~ 5ml dimethyl sulfoxide, dissolve room temperature reaction 1 ~ 15 hour, solid material slowly dissolves, to reactant liquor in yellow, dimethyl sulfoxide is drained with vacuum pump, after remaining grease is dissolved in 0.1 ~ 0.5ml acetone, in this solution, add 30 ~ 50ml ether has faint yellow product to be precipitated out, dry in vacuum drying oven, obtains flaxen compound 1;
5th step, the gold nano grain of the hepatoma carcinoma cell targeting peptides combination of preparation load platinum medicine: 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl) and the N-hydroxysuccinimide (NHS) that add 0.1 ~ 1mg compound 1 and 1 ~ 2 times of mole thereof in the gold nano grain solution that hepatoma carcinoma cell targeting peptides combines, react 12 ~ 24 hours at 30 ~ 70 DEG C, removed by the responseless platinum medicine of centrifugal method, by reactant liquor under 5000 ~ 14000 revs/min centrifugal 3 ~ 30 minutes, the gold nano grain in conjunction with targeting peptides of load platinum medicine is deposited in bottom centrifuge tube, remove unreacted platinum medicine in supernatant, then by the gold nano grain ultrasonic disperse in conjunction with targeting peptides of load platinum medicine in water, so repeatedly, clean load platinum medicine hepatoma carcinoma cell targeting peptides combine gold nano grain,
6th step, the targeting peptides of load platinum medicine is to platinic controllable release.In in conjunction with platinic targeting peptides solution, add 0.1 ~ 10 mM/l of guanyl, 0.1 ~ 10 mM/l of vitamin C reacts, and in the sampling of differential responses stage, carries out high performance liquid chromatography detection.
Compared with prior art, the present invention possesses following advantage and good effect:
The gold nano grain that hepatoma carcinoma cell targeting peptides of the present invention is modified, effectively improve its selectivity to hepatoma carcinoma cell, improve the bioavailability of gold nano grain, preparation method of the present invention is simple simultaneously, with low cost, can prepare on a large scale.
Namely peptide itself has targeting, has again the Stabilization of nanometer gold, and material bio-compatibility is high; Avoid other chemical modification, reduce toxicity.
(GCGC) sequence can guarantee hepatoma carcinoma cell targeting peptides to be directly connected on nanogold particle, and need not engage in addition link agent react, this greatly reducing the time of reaction, improves reaction efficiency.
The gold nano grain delivery tetravalence platinum prodrug that hepatoma carcinoma cell targeting peptides of the present invention is modified, effectively improve its selectivity to hepatoma carcinoma cell, improve the bioavailability of gold nano grain, helpful at the treatment use of hepatocarcinoma to gold nano grain, preparation method of the present invention is simple simultaneously, with low cost, can prepare on a large scale.
Accompanying drawing explanation
Fig. 1 is the gold nano grain Electronic Speculum figure that hepatoma carcinoma cell targeting peptides of the present invention is modified;
Fig. 2 is the gold nano grain uv-visible absorption spectra figure that hepatoma carcinoma cell targeting peptides of the present invention is modified;
Fig. 3 is the gold nano grain cell-targeting experimental result that hepatoma carcinoma cell targeting peptides of the present invention is modified, and wherein 7702 cells are normal liver cell system, and HepG2 cell is hepatoma cell line, and A549 is human body adenocarcinoma of lung epithelial cell line;
The tetravalence platinum controllable release that Fig. 4 (a), (b) are the hepatoma carcinoma cell targeting peptides of load platinum medicine of the present invention;
Fig. 5 is the tetravalence platinum controllable release reaction rate of the hepatoma carcinoma cell targeting peptides of load platinum medicine of the present invention.
Detailed description of the invention
Below in conjunction with accompanying drawing, further illustrate essentiality content of the present invention with embodiments of the invention, but do not limit content of the present invention with this.
Embodiment 1:
One, the preparation of gold nano grain
The preparation of gold nano seed solution: take 273.3mg cetyl trimethyl ammonium bromide (CTAB) and be dissolved in 7.5ml water, make it in the hot water to dissolve completely, be then cooled to room temperature.Weigh 2.2 ~ 2.3mg sodium borohydride (NaBH
4) be dissolved in 6ml frozen water, be configured to the solution of 10 mM/ls of concentration.Gold chloride (the HAuCl of 250 μ l is added in CTAB solution
44H
2o) solution (10 mM/ls), slightly shakes, and adds rapidly the NaBH that 600 μ l brand-news are standby, freezing
4, within ultrasonic 2 minutes, get rid of bubble.Constant temperature more than 2 hours in 37 DEG C of water;
The preparation of the gold nano grain that CTAB is stable: first weigh 1.3692g CTAB and be dissolved in the water of 38ml, hot bath makes it to dissolve completely, is then cooled to room temperature.591 μ l hydrochloric acid (1 mol/L), 0.4ml silver nitrate (10 mM/ls), 2ml gold chloride (10 mM/ls), 0.2ml ascorbic acid (0.1 mol/L) is added successively in solvent CTAB solution, shake up as after colourless, add 96 μ l seed solutions, within ultrasonic 2 minutes, get rid of bubble, put into 37 DEG C of water-baths and spend the night.
Two, the preparation of the gold nano grain of targeting peptides modification
Get 2mg Seq1 sequence (SFSIIHTPILPLGC) to be dissolved in 2ml water.Add 10ml nanogold particle colloidal sol, be slowly warming up to 55 DEG C, water bath with thermostatic control is after 2 hours, gentle agitation 2 hours under room temperature.With 5000 revs/min, within 3 minutes, centrifugal secondary removes precipitation.
Embodiment 2:
One, the preparation of gold nano grain
The preparation of gold nano seed solution: take 273.3mg cetyl trimethyl ammonium bromide (CTAB) and be dissolved in 7.5ml water, make it in the hot water to dissolve completely, be then cooled to room temperature.Weigh 2.2 ~ 2.3mg sodium borohydride (NaBH
4) be dissolved in 6ml frozen water, be configured to the solution of 10 mM/ls of concentration.Gold chloride (the HAuCl of 250 μ l is added in CTAB solution
44H
2o) solution (10 mM/ls), slightly shakes, and adds rapidly the NaBH that 600 μ l brand-news are standby, freezing
4, within ultrasonic 2 minutes, get rid of bubble.Constant temperature more than 2 hours in 37 DEG C of water;
The preparation of the gold nano grain that CTAB is stable: first weigh 1.3692g CTAB and be dissolved in the water of 38ml, hot bath makes it to dissolve completely, is then cooled to room temperature.591 μ l hydrochloric acid (1 mol/L), 0.4ml silver nitrate (10 mM/ls), 2ml gold chloride (10 mM/ls), 0.2ml ascorbic acid (0.1 mol/L) is added successively in solvent CTAB solution, shake up as after colourless, add 96 μ l seed solutions, within ultrasonic 2 minutes, get rid of bubble, put into 37 DEG C of water-baths and spend the night.Hydrochloric acid herein add the productive rate that can significantly improve nanogold particle, and than the more seed solution of addition in document ensure that gold plant smooth generation.
Two, the preparation of the gold nano grain of targeting peptides modification
Get 10mg Seq2 sequence (SFSIIHTPILPLGCGC) to be dissolved in 2ml water.Add 10ml nanogold particle colloidal sol, be slowly warming up to 55 DEG C, water bath with thermostatic control is after 3 hours, gentle agitation 2 hours under room temperature.With 6000 revs/min, within 10 minutes, centrifugal secondary removes precipitation.The gold nano grain Electronic Speculum figure that Fig. 1 targeting peptides is modified.The gold nano grain uv-visible absorption spectra figure that Fig. 2 targeting peptides is modified, shows that gold nano grain still keeps original extinction effect after targeting peptides is modified.
Embodiment 3:
One, the preparation of gold nano grain
The preparation of gold nano seed solution: take 273.3mg cetyl trimethyl ammonium bromide (CTAB) and be dissolved in 7.5ml water, make it in the hot water to dissolve completely, be then cooled to room temperature.Weigh 2.2 ~ 2.3mg sodium borohydride (NaBH
4) be dissolved in 6ml frozen water, be configured to the solution of 10 mM/ls of concentration.In CTAB solution, add the chlorauric acid solution (10 mM/ls) of 250 μ l, slightly shake, add rapidly the NaBH that 600 μ l brand-news are standby, freezing
4, within ultrasonic 2 minutes, get rid of bubble.Constant temperature more than 2 hours in 37 DEG C of water;
The preparation of the gold nano grain that CTAB is stable: first weigh 1.3692g CTAB and be dissolved in the water of 38ml, hot bath makes it to dissolve completely, is then cooled to room temperature.591 μ l hydrochloric acid (1 mol/L), 0.4ml silver nitrate (10 mM/ls), 2ml gold chloride (10 mM/ls), 0.2ml ascorbic acid (0.1 mol/L) is added successively in solvent CTAB solution, shake up as after colourless, add 96 μ l seed solutions, within ultrasonic 2 minutes, get rid of bubble, put into 37 DEG C of water-baths and spend the night.
Two, the preparation of the gold nano grain of targeting peptides modification
Get 4mg Seq3 sequence (SFSIIHTPILPLGCGCGC) to be dissolved in 2ml water.Add 10ml nanogold particle colloidal sol, be slowly warming up to 55 DEG C, water bath with thermostatic control is after 4 hours, gentle agitation 2 hours under room temperature.With 8000 revs/min, within 15 minutes, centrifugal secondary removes precipitation.
Embodiment 4:
One, the preparation of gold nano grain
The preparation of gold nano seed solution: take 273.3mg cetyl trimethyl ammonium bromide (CTAB) and be dissolved in 7.5ml water, make it in the hot water to dissolve completely, be then cooled to room temperature.Weigh 2.2 ~ 2.3mg sodium borohydride (NaBH
4) be dissolved in 6ml frozen water, be configured to the solution of 10 mM/ls of concentration.In CTAB solution, add the chlorauric acid solution (10 mM/ls) of 250 μ l, slightly shake, add rapidly the NaBH that 600 μ l brand-news are standby, freezing
4, within ultrasonic 2 minutes, get rid of bubble.Constant temperature more than 2 hours in 37 DEG C of water;
The preparation of the gold nano grain that CTAB is stable: first weigh 1.3692g CTAB and be dissolved in the water of 38ml, hot bath makes it to dissolve completely, is then cooled to room temperature.591 μ l hydrochloric acid (1 mol/L), 0.4ml silver nitrate (10 mM/ls), 2ml gold chloride (10 mM/ls), 0.2ml ascorbic acid (0.1 mol/L) is added successively in solvent CTAB solution, shake up as after colourless, add 96 μ l seed solutions, within ultrasonic 2 minutes, get rid of bubble, put into 37 DEG C of water-baths and spend the night.
Two, the preparation of the gold nano grain of targeting peptides modification
Get 6mg Seq4 sequence (SFSIIHTPILPLMGGGCCC) to be dissolved in 2ml water.Add 10ml nanogold particle colloidal sol, be slowly warming up to 55 DEG C, water bath with thermostatic control is after 5 hours, gentle agitation 2 hours under room temperature.With 10000 revs/min, within 20 minutes, centrifugal secondary removes precipitation.
Embodiment 5:
One, the preparation of gold nano grain
The preparation of gold nano seed solution: take 273.3mg cetyl trimethyl ammonium bromide (CTAB) and be dissolved in 7.5ml water, make it in the hot water to dissolve completely, be then cooled to room temperature.Weigh 2.2 ~ 2.3mg sodium borohydride (NaBH
4) be dissolved in 6ml frozen water, be configured to the solution of 10 mM/ls of concentration.In CTAB solution, add the chlorauric acid solution (10 mM/ls) of 250 μ l, slightly shake, add rapidly the NaBH that 600 μ l brand-news are standby, freezing
4, within ultrasonic 2 minutes, get rid of bubble.Constant temperature more than 2 hours in 37 DEG C of water;
The preparation of the gold nano grain that CTAB is stable: first weigh 1.3692g CTAB and be dissolved in the water of 38ml, hot bath makes it to dissolve completely, is then cooled to room temperature.591 μ l hydrochloric acid (1 mol/L), 0.4ml silver nitrate (10 mM/ls), 2ml gold chloride (10 mM/ls), 0.2ml ascorbic acid (0.1 mol/L) is added successively in solvent CTAB solution, shake up as after colourless, add 96 μ l seed solutions, within ultrasonic 2 minutes, get rid of bubble, put into 37 DEG C of water-baths and spend the night.
Two, the preparation of the gold nano grain of targeting peptides modification
Get 8mg Seq5 sequence (SFSIIHTPILPLTGCRGCCG) to be dissolved in 2ml water.Add 10ml nanogold particle colloidal sol, be slowly warming up to 55 DEG C, water bath with thermostatic control is after 6 hours, gentle agitation 2 hours under room temperature.With 14000 revs/min, within 30 minutes, centrifugal secondary removes precipitation.
Embodiment 6:
One, the preparation of gold nano grain
Be add the trisodium citrate that 1ml concentration is 1% in the chlorauric acid solution of 0.01% in 100ml concentration, heated and boiled 20 minutes, solution becomes redness, then obtain nanogold particle.
Two, the preparation of the gold nano grain of targeting peptides modification
Get 10mgSeq2 sequence (SFSIIHTPILPLGCGC) to be dissolved in 2ml water.Add 10ml nanogold particle colloidal sol, gentle agitation 2 hours under room temperature.With 6000 revs/min, within 10 minutes, centrifugal secondary removes precipitation.
Embodiment 7:
The gold nano grain of the hepatoma carcinoma cell targeting modification of preparation band fluorophor
Get 1 ~ 2mg rhodamine B, 22mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl), 5.3mg N-hydroxysuccinimide (N-Hydroxysuccinimide, NHS) be dissolved in 2ml water, lucifuge places 20 minutes, add the gold nano grain solution of preparation in embodiment 2, lucifuge stirring reaction 24 hours.
With centrifuge (rotating speed 6000 revs/min, centrifugation time 10 minutes) repeatedly centrifugal segregation supernatant concentrated volume to 1ml.
The serial overview table of hepatoma carcinoma cell targeting peptides of the present invention
| Title | Aminoacid number | Sequence |
| Seq1 | 14 | SFSIIHTPILPLGC |
| Seq2 | 16 | SFSIIHTPILPLGCGC |
| Seq3 | 18 | SFSIIHTPILPLGCGCGC |
| Seq4 | 19 | SFSIIHTPILPLMGGGCCC |
| Seq5 | 20 | SFSIIHTPILPLTGCRGCCG |
Embodiment 8:
Cell-targeting is tested
I. recover
The cell of recovery has hepatoma carcinoma cell (HepG2 cell), normal liver cell (7702 cell), Human Lung adenocarcinoma epithelial cell (A549 cell).
Open from-80 DEG C of refrigerators and take out frozen cell, tepidarium is until melt.Draw cell suspension with liquid-transfering gun, the culture fluid instilling more than 10 times mixes centrifugal afterwards and removes supernatant.Then with cell is resuspended and count containing the culture fluid of 10% hyclone, adjustment density, then inoculates, cultivates for 37 DEG C in culture bottle, and culture fluid needs change once every day.
II. targeting experiment
Cell-targeting experiment selects exponential phase cell 20,000/ml to be seeded in 24 orifice plates, every hole 100 μ l, cultivate and change fresh culture medium after 24 hours, experimental group, matched group and blank group are set, experimental group adds the medicine of variable concentrations gradient, matched group does not add medicine, and blank group does not have inoculating cell and do not add medicine; Continue cultivation and remove culture medium after 4 hours.
1, collect logarithmic (log) phase cell, adjustment concentration of cell suspension, every hole adds 100 μ l, and bed board makes cell to be measured adjust density to 10 ten thousand/hole, (edge hole sterile phosphate buffer is filled).
2,5%CO
2, 37 DEG C of overnight incubation.
3, sample prepared in example 7 within second day, is carried out adding, 5%CO
2, hatch 4 hours for 37 DEG C.
4, discard old culture medium and add fresh culture 100 μ l, wash once with phosphate buffer (PBS).
5, every hole adds 100 μ l Digestive systems, makes lysis, adds a large amount of phosphate buffer and is placed in 1.5ml centrifuge tube, carry out centrifugal with centrifuge (rotating speed 1500 revs/min, 5 minutes time) after digestion terminates, removing supernatant.
6,1ml phosphate buffer centrifuge washing is added 1 time, abandoning supernatant.Add 0.5ml phosphate buffer after cleaning, after being configured to suspension, use flow cytometer analysis.
In Fig. 3,7702 cells are normal liver cell system, and HepG2 cell is hepatoma cell line, and A549 is human body adenocarcinoma of lung epithelial cell line, and the nanogold particle that targeting peptides is modified as can be seen from the results has certain selectivity to hepatoma carcinoma cell.The gold nano grain that hepatoma carcinoma cell targeting peptides of the present invention is modified, effectively improve it to hepatoma carcinoma cell selectivity, improve the bioavailability of gold nano grain, helpful at the treatment use of hepatocarcinoma to gold nano grain, preparation method of the present invention is simple simultaneously, with low cost, can suitability for industrialized production.
Embodiment 9:
One, the prodrug compound 1 [Pt (NH of cisplatin is prepared
3)
2cl
3(OOCCH
2cH
2cOOH)]
The aqueous hydrogen peroxide solution being 30% by 0.6mmol cisplatin and 50ml volumetric concentration joins in 60ml water, is heated to 40 DEG C, stirring reaction 10 hours, and yellow solid slowly dissolves, and solution is faint yellow; Be cooled to 8 DEG C with Rotary Evaporators concentration of reaction solution to 5ml, have yellow solid crystal solid to separate out, then in boiling water recrystallization, obtain bright yellow solid product; 5mmol succinic anhydride and 5mmol bright yellow solid product are dissolved in 5ml dimethyl sulfoxide, room temperature reaction more than 15 hours, solid material slowly dissolves, to reactant liquor in yellow, drain dimethyl sulfoxide with vacuum pump, after remaining grease is dissolved in 0.5ml acetone, in this solution, add 50ml ether has faint yellow product to be precipitated out, dry in vacuum drying oven, obtain flaxen object product.
Two, the preparation of the gold nanorods of the targeting peptides modification of load platinum medicine
0.1mg compound 1,0.1mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl), 0.1mg N-hydroxysuccinimide (NHS) is added in the nanogold particle solution of preparation in embodiment 2, room temperature reaction 24 hours, reactant liquor is centrifugal 30 minutes at 14000 revs/min, repeatedly centrifugal segregation supernatant, by the gold nanorods ultrasonic disperse in conjunction with targeting peptides that is deposited in bottom centrifuge tube in water, the aqueous solution of the gold nanorods that the targeting peptides namely obtaining Supported Pt Nanoparticles class prodrug combines.The platinum concentration loading to gold nanorods surface is come quantitatively by inductive coupling plasma mass spectrometry or atomic absorption spectrum.
Embodiment 10:
One, the prodrug compound 1 [Pt (NH of cisplatin is prepared
3)
2cl
3(OOCCH
2cH
2cOOH)]
The aqueous hydrogen peroxide solution being 30% by 1mmol cisplatin and 5ml volumetric concentration joins in 6ml water, is heated to 60 DEG C, stirring reaction 3 hours, and yellow solid slowly dissolves, and solution is faint yellow; Be cooled to 0 DEG C with Rotary Evaporators concentration of reaction solution to 0.5ml, have yellow solid crystal solid to separate out, then in boiling water recrystallization, obtain bright yellow solid product; 1mmol succinic anhydride and 1mmol bright yellow solid product are dissolved in 5ml dimethyl sulfoxide, room temperature reaction more than 12 hours, solid material slowly dissolves, to reactant liquor in yellow, drain dimethyl sulfoxide with vacuum pump, after remaining grease is dissolved in 0.1ml acetone, in this solution, add 30ml ether has faint yellow product to be precipitated out, dry in vacuum drying oven, obtain flaxen object product.
Two, the preparation of the gold nanorods of the targeting peptides modification of load platinum medicine
1mg compound 1,1mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl), 1mg N-hydroxysuccinimide (NHS) is added in the nanogold particle solution of preparation in embodiment 2, room temperature reaction 12 hours, reactant liquor is centrifugal 10 minutes at 6000 revs/min, repeatedly centrifugal segregation supernatant, by the gold nanorods ultrasonic disperse in conjunction with targeting peptides that is deposited in bottom centrifuge tube in water, the aqueous solution of the gold nanorods that the targeting peptides namely obtaining Supported Pt Nanoparticles class prodrug combines.The platinum concentration loading to gold nanorods surface is come quantitatively by inductive coupling plasma mass spectrometry or atomic absorption spectrum.
Embodiment 11:
One, the prodrug compound 1 [Pt (NH of cisplatin is prepared
3)
2cl
3(OOCCH
2cH
2cOOH)]
The aqueous hydrogen peroxide solution being 30% by 6mmol cisplatin and 25ml volumetric concentration joins in 30ml water, is heated to 80 DEG C, stirring reaction 1 hour, and yellow solid slowly dissolves, and solution is faint yellow; Be cooled to 4 DEG C with Rotary Evaporators concentration of reaction solution to 2.5ml, have yellow solid crystal solid to separate out, then in boiling water recrystallization, obtain bright yellow solid product; 2.5mmol succinic anhydride and 2.5mmol bright yellow solid product are dissolved in 2.5ml dimethyl sulfoxide, room temperature reaction more than 18 hours, solid material slowly dissolves, to reactant liquor in yellow, drain dimethyl sulfoxide with vacuum pump, after remaining grease is dissolved in 0.3ml acetone, in this solution, add 40ml ether has faint yellow product to be precipitated out, dry in vacuum drying oven, obtain flaxen object product.
Two, the preparation of the gold nanorods of the targeting peptides modification of load platinum medicine
0.5mg compound 1,0.5mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl), 0.5mg N-hydroxysuccinimide (NHS) is added in the nanogold particle solution of preparation in embodiment 2, room temperature reaction 15 hours, reactant liquor is centrifugal 3 minutes at 10000 revs/min, repeatedly centrifugal segregation supernatant, by the gold nanorods ultrasonic disperse in conjunction with targeting peptides that is deposited in bottom centrifuge tube in water, the aqueous solution of the gold nanorods that the targeting peptides namely obtaining Supported Pt Nanoparticles class prodrug combines.The platinum concentration loading to gold nanorods surface is come quantitatively by inductive coupling plasma mass spectrometry or atomic absorption spectrum.
Embodiment 12:
In conjunction with the preparation of platinic targeting peptides solution.
Get compound 1,22mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl) of preparation in 1mg embodiment 10,5.3mg N-hydroxysuccinimide (N-Hydroxysuccinimide, NHS) be dissolved in 2ml water, lucifuge places 20 minutes, add Seq2 sequence (SFSIIHTPILPLGCGC) 10mg, lucifuge stirring reaction 24 hours.
With centrifuge (rotating speed 6000 revs/min, centrifugation time 10 minutes) repeatedly centrifugal segregation supernatant.
Embodiment 13:
In embodiment 12, preparation adds 0.1 mM/l of guanyl in conjunction with in platinic targeting peptides solution, 10 mM/ls of vitamin Cs react, carry out littlely sampling 100 μ l constantly in 0 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 30 in reaction, carried out high performance liquid chromatography detection.Using the aqueous solution of 1 ‰ trifluoroacetic acids as A phase, the methanol (CH of 100%
3oH) as B phase; The performance liquid chromatographic column used is Jupiter C18 post, and gradient is the methanol-eluted fractions of 30% ~ 90%, and arranging determined wavelength is 260nm.
Embodiment 14:
In embodiment 12, preparation adds 1.33 mM/ls of guanyls in conjunction with in platinic targeting peptides solution, 0.57 mM/l of vitamin C reacts, mol ratio between tetravalence platinum, guanyl, vitamin C three is about 1:7:3, and carried out littlely sampling 100 μ l constantly in 0 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 30 in reaction, carry out high performance liquid chromatography detection.Using the aqueous solution of 1 ‰ trifluoroacetic acids as A phase, the methanol (CH of 100%
3oH) as B phase; The performance liquid chromatographic column used is JupiterC18 post, and gradient is the methanol-eluted fractions of 30% ~ 90%, and arranging determined wavelength is 260nm.
Embodiment 15:
In embodiment 12, preparation adds 10 mM/ls of guanyls in conjunction with in platinic targeting peptides solution, 0.1 mM/l of vitamin C reacts, carry out littlely sampling 100 μ l constantly in 0 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 30 in reaction, carried out high performance liquid chromatography detection.Using the aqueous solution of 1 ‰ trifluoroacetic acids as A phase, the methanol (CH of 100%
3oH) as B phase; The performance liquid chromatographic column used is Jupiter C18 post, and gradient is the methanol-eluted fractions of 30% ~ 90%, and arranging determined wavelength is 260nm.
The release of the platinum of the nanogold particle load tetravalence platinum prodrug system of being modified by the targeting peptides of load platinum medicine and the reaction simulation of guanyl targeting peptides, result shows to obtain platinic controllable release under reducing agent effect, and is combined with DNA further and reaches antineoplastic effect.
The tetravalence platinum controllable release of the hepatoma carcinoma cell targeting peptides that Fig. 4 (a), (b) are load platinum medicine; Fig. 5 is the tetravalence platinum controllable release speed of the targeting peptides of load platinum medicine, draw according to Fig. 4 (a), (b), as can be seen from curve, before the reaction in 15 hours, the Pt-guanyl complex of generation is little, and reaction rate is slow, after 15 hours, reaction rate improves rapidly, and almost exponentially trend increases, and at this moment a large amount of products generates.The reason that this situation occurs is because whole course of reaction is made up of two steps: tetravalence platinum is sloughed from peptide, and being reduced agent reduction becomes cisplatin; Cisplatin and guanyl react.Reaction rate indicates first step reaction, and namely platinic reduction reaction is relatively slower, and second step reaction is then very rapid.
Claims (18)
1. a gold nano grain for hepatoma carcinoma cell targeting peptides modification, is characterized in that, the gold nano grain that described targeting peptides is modified has selectivity to hepatoma carcinoma cell.
2. the gold nano grain of a kind of hepatoma carcinoma cell targeting peptides modification according to claim 1, it is characterized in that, described targeting peptides contains 14-20 aminoacid, wherein comprise SFSIIHTPILPL and the SP94 sequence with hepatoma carcinoma cell targeting, containing 1-3 cysteine residues in described peptide sequence.
3. the gold nano grain of a kind of hepatoma carcinoma cell targeting peptides modification according to claim 1, it is characterized in that, the gold nano grain that described hepatoma carcinoma cell targeting peptides is modified forms by the sulfydryl in peptide molecule and gold the surface that peptide to be connected to gold nano grain by golden sulfur covalent bond.
4. the gold nano grain of a kind of hepatoma carcinoma cell targeting peptides modification according to claim 1, is characterized in that, the gold nano grain that this targeting peptides is modified is obtained as follows:
(1) prepare the gold nano grain that cetyl trimethyl ammonia bromide CTAB is stable: with cetyl trimethyl ammonia bromide as soft stamp agent, adopt seed mediated growth method to prepare gold nano grain or prepare gold nano grain with sodium citrate as reducing agent;
(2) gold nano grain that targeting peptides combines is prepared: to step (1) gold nano particle colloidal sols, it locates absorption value at about 800nm is add hepatoma carcinoma cell targeting peptides 1 ~ 5mg/ml in 1 to react; React after 4 ~ 8 hours, removed by responseless peptide by centrifugal method, by reactant liquor under 5000 ~ 14000 revs/min centrifugal 3 ~ 30 minutes, the gold nano grain that targeting peptides combines was deposited in bottom centrifuge tube; And then by nano-particle ultrasonic disperse in water, so repeatedly, clean the gold nano grain that targeting peptides combines.
5. the preparation method of the gold nano grain that a kind of hepatoma carcinoma cell targeting peptides described in claim 1 or 2 or 3 is modified, comprises the steps:
(1) prepare the gold nano grain that cetyl trimethyl ammonia bromide is stable: with cetyl trimethyl ammonia bromide as soft stamp agent, adopt seed mediated growth method to prepare gold nano grain or prepare gold nano grain with sodium citrate as reducing agent;
(2) gold nano grain that targeting peptides combines is prepared: to step (1) gold nano particle colloidal sols, it locates absorption value at about 800nm is add hepatoma carcinoma cell targeting peptides 1 ~ 5mg/ml in 1 to react; React after 4 ~ 8 hours, removed by responseless peptide by centrifugal method, by reactant liquor under 5000 ~ 14000 revs/min centrifugal 3 ~ 30 minutes, the gold nano grain that targeting peptides combines was deposited in bottom centrifuge tube; And then by nano-particle ultrasonic disperse in water, so repeatedly, clean the gold nano grain that targeting peptides combines.
6. preparation method according to claim 5, is characterized in that, the method comprises the steps:
(1) preparation of gold nano grain:
The preparation of gold nano seed solution: take cetyl trimethyl ammonium bromide) be dissolved in 7.5ml water, make it in the hot water to dissolve completely, be then cooled to room temperature, by sodium borohydride (NaBH
4) be dissolved in frozen water, be configured to the solution of 10 mM/ls of concentration, in CTAB solution, add the gold chloride (HAuCl of 250 μ l
44H
2o) solution, slightly shakes, and adds rapidly the NaBH that 600 μ l brand-news are standby, freezing
4, within ultrasonic 2 minutes, get rid of bubble, constant temperature more than 2 hours in 37 DEG C of water;
The preparation of the gold nano grain that CTAB is stable: first weigh CTAB soluble in water, hot bath makes it to dissolve completely, is then cooled to room temperature, adds hydrochloric acid (HCl), silver nitrate (AgNO in solvent CTAB solution successively
3), gold chloride (HAuCl
44H
2o), ascorbic acid, shake up as after colourless, add seed solution, within ultrasonic 2 minutes, get rid of bubble, put into 37 DEG C of water-baths and spend the night;
(2) preparation of Jenner's rod of targeting peptides modification
Get hepatoma carcinoma cell targeting peptides soluble in water, add nanometer gold bar colloidal sol, be slowly warming up to 55 DEG C, water bath with thermostatic control is after 3 hours, gentle agitation 2 hours under room temperature, and with 6000 revs/min, within 10 minutes, centrifugal secondary removes precipitation;
(3) nanometer gold bar that the targeting peptides of fluorophor is modified is with
Get rhodamine B, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide (N-Hydroxysuccinimide, NHS) soluble in water, lucifuge places 20 minutes, add the nanometer gold bar solution that targeting peptides is modified, lucifuge stirring reaction 24 hours;
With centrifuge speed 6000 revs/min, centrifugation time 10 minutes repeatedly centrifugal segregation supernatant concentrated volume;
(4) cell-targeting experiment
I. recover
The cell of recovery has hepatoma carcinoma cell (HepG2 cell), normal liver cell (7702 cell), Human Lung adenocarcinoma epithelial cell (A549 cell); Open from-80 DEG C of refrigerators and take out frozen cell, tepidarium, until melt, draws cell suspension with liquid-transfering gun, and the culture fluid instilling more than 10 times mixes centrifugal afterwards and removes supernatant; Then with cell is resuspended and count containing the culture fluid of 10% hyclone, adjustment density, then inoculates, cultivates for 37 DEG C in culture bottle, and culture fluid is changed once every day;
II. targeting experiment
Exponential phase cell 2 × 10 is selected in cell-targeting experiment
4individual/ml is seeded in 24 orifice plates, every hole 100 μ l, cultivate and change fresh culture medium after 24 hours, experimental group, matched group and blank group are set, experimental group adds the medicine of variable concentrations gradient, matched group does not add medicine, and blank group does not have inoculating cell and do not add medicine, continues cultivation and removes culture medium after 4 hours;
Collect logarithmic (log) phase cell, adjustment concentration of cell suspension, every hole adds 100 μ l, and bed board makes cell to be measured adjust density to 10 ten thousand/hole, and edge hole sterile phosphate buffer is filled; 5%CO
2, hatch for 37 DEG C, cultivate 12 hours; Within second day, carry out application of sample, 5%CO
2, hatch 4 hours for 37 DEG C; Discard old culture medium and add fresh culture 100 μ l, wash once with phosphate buffer (PBS); Every hole adds 100 μ l Digestive systems, makes lysis, adds a large amount of phosphate buffer and is placed in 1.5ml centrifuge tube, carry out centrifugal with centrifuge speed 1500 revs/min, 5 minutes time after digestion terminates, removing supernatant; Add 1ml phosphate buffer centrifuge washing 1 time, abandoning supernatant, add 0.5ml phosphate buffer after cleaning, after being configured to suspension, use flow cytometer analysis.
7. gold nano grain described in claim 1 is used as tetravalence platinum prodrug carrier.
8. the application of gold nano grain described in claim 1 in preparation tetravalence platinum prodrug carrier.
9. described in claim 1, gold nano grain is used as antineoplastic drug carrier.
10. gold nano grain described in claim 1 is preparing the application in antineoplastic drug carrier.
The gold nano grain that 11. hepatoma carcinoma cell targeting peptides according to claim 7 or 9 are modified is used as tetravalence platinum prodrug carrier or antineoplastic drug carrier, it is characterized in that, the gold nano grain that described hepatoma carcinoma cell targeting peptides is modified is the surface by forming golden sulfur covalent bond hepatoma carcinoma cell targeting peptides being connected to gold nano grain.
The gold nano grain that 12. hepatoma carcinoma cell targeting peptides according to claim 7 or 9 are modified is used as tetravalence platinum prodrug carrier or antineoplastic drug carrier, it is characterized in that, the gold nano grain that described hepatoma carcinoma cell targeting peptides is modified is that the prodrug tetravalence platinum of cisplatin is connected on hepatoma carcinoma cell targeting peptides by the mode by forming amido link.
The application of gold nano grain in preparation tetravalence platinum prodrug carrier or antineoplastic drug carrier that hepatoma carcinoma cell targeting peptides described in 13. according to Claim 8 or 10 is modified, it is characterized in that, described application is the gold nano grain delivery platinum medicine modified with hepatoma carcinoma cell targeting peptides.
The application of gold nano grain in preparation tetravalence platinum prodrug carrier or antineoplastic drug carrier that hepatoma carcinoma cell targeting peptides described in 14. according to Claim 8 or 10 is modified, it is characterized in that, described application is that the prodrug tetravalence platinum of cisplatin is connected on hepatoma carcinoma cell targeting peptides by the mode by forming amido link.
The gold nano grain that 15. hepatoma carcinoma cell targeting peptides according to claim 10 are modified is preparing the application in antineoplastic drug carrier, and it is characterized in that, described tumor is hepatocarcinoma.
16. application according to claim 7 or 8 or 9 or 10 or 15, it is characterized in that, described targeting peptides contains 14-20 aminoacid, wherein comprises SFSIIHTPILPL and the SP94 sequence with hepatoma carcinoma cell targeting, containing 1-3 cysteine residues in described peptide sequence.
The method of the gold nano grain that 17. preparation claim 7 or 8 or 9 or the hepatoma carcinoma cell targeting peptides described in 10 or 15 are modified, it is characterized in that, by the covalent that forms golden sulfur effect, hepatoma carcinoma cell targeting peptides is connected to the surface of gold nano grain, comprises the steps:
18. (1) prepare the stable gold nano grain of cetyl trimethyl ammonia bromide CTAB: with cetyl trimethyl ammonia bromide as soft stamp agent, adopt seed mediated growth method to prepare gold nano grain or prepare gold nano grain with sodium citrate as reducing agent;
(2) gold nano grain that targeting peptides combines is prepared: in purple gold nano particle colloidal sols, add hepatoma carcinoma cell targeting peptides 1 ~ 5mg/ml react, react after 4 ~ 8 hours, by centrifugal method, responseless peptide is removed, by reactant liquor under 5000 ~ 14000 revs/min centrifugal 3 ~ 30 minutes, the gold nano grain that targeting peptides combines is deposited in bottom centrifuge tube, remove unreacted targeting peptides in supernatant, then by nano-particle ultrasonic disperse in water, so repeatedly, the gold nano grain that targeting peptides combines is cleaned;
(3) gold nano grain that the hepatoma carcinoma cell targeting peptides preparing load platinum medicine combines: add 0.1 ~ 1mg Pt (NH in the gold nano grain solution that hepatoma carcinoma cell targeting peptides combines
3)
2cl
3(OOCCH
2cH
2and the 1-ethyl of 1 ~ 2 times of mole-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl) and N-hydroxysuccinimide (NHS) COOH), react 12 ~ 24 hours at 30 ~ 70 DEG C, by centrifugal method, responseless platinum medicine is removed, reactant liquor under 5000 ~ 14000 revs/min centrifugal 3 ~ 30 minutes, the gold nano grain in conjunction with targeting peptides of load platinum medicine is deposited in bottom centrifuge tube, removes unreacted platinum medicine in supernatant; Then by the gold nano grain ultrasonic disperse in conjunction with targeting peptides of load platinum medicine in water, so repeatedly, clean load platinum medicine hepatoma carcinoma cell targeting peptides combine gold nano grain;
(4) targeting peptides of load platinum medicine is to platinic controllable release: add 0.1 ~ 10 mM/l of guanyl combining in platinic hepatoma carcinoma cell targeting peptides solution, 0.1 ~ 10 mM/l of vitamin C reacts, and in the sampling of differential responses stage, carry out high performance liquid chromatography detection.
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