CN102397564A - Tumor-targeted diagnosis nuclear magnetic resonance contrast agent and preparation method thereof - Google Patents
Tumor-targeted diagnosis nuclear magnetic resonance contrast agent and preparation method thereof Download PDFInfo
- Publication number
- CN102397564A CN102397564A CN2010102865084A CN201010286508A CN102397564A CN 102397564 A CN102397564 A CN 102397564A CN 2010102865084 A CN2010102865084 A CN 2010102865084A CN 201010286508 A CN201010286508 A CN 201010286508A CN 102397564 A CN102397564 A CN 102397564A
- Authority
- CN
- China
- Prior art keywords
- contrast agent
- cancer target
- polypeptide
- mri contrast
- macromolecular material
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention belongs to the technical field of biotechnology, and relates to a tumor-targeted diagnosis nuclear magnetic resonance contrast agent and a preparation method thereof. The tumor-target diagnosis nuclear magnetic resonance contrast agent is prepared from a high molecular material, polyethylene glycol, a polypeptide, a dual-function ligand and gadolinium chloride by taking the polypeptide as a target head group, taking the tree-like high polymer material as a basic high molecular vector and connecting a small molecular contrast agent to the surface. In the invention, a polypeptide-modified high molecular material screened by using a phage display technology enters cells in an endocytic way, so that the introjection of the contrast agent by tumor cells is increased, and the characteristic of high safety is achieved. The target head group polypeptide used in the invention has the advantages of transferrin, can be used for effectively avoiding the interference of endogenous transferrin, has high targeting and diagnosing efficiencies, is simple to prepare, and can be further applied to target diagnosis of other tumor tissues.
Description
Technical field
The invention belongs to biological technical field, relate to mri contrast agent, be specifically related to a kind of cancer target diagnosis mri contrast agent and preparation method thereof, especially a kind of peptide modified cancer target diagnosis mri contrast agent and preparation method thereof.
Background technology
Tumor adopted the magnetic resonance imaging diagnosis and studies efficiently contrast agent is noticeable in recent years focus.Mostly the contrast agent that uses clinically at present is hydrophilic micromolecule, can not get into cell, often rests on interstice and blood vessel internal cavity through the blood vessel endothelium space, remove very fast, video picture for a long time; And because the conveying of lack of specific only depends on vascular space to penetrate into tissue, cause contrast agent to be accumulated in inflammation part, cause mistaken diagnosis easily inflammation.The contrast agent Magnevist Solution of selling in the market (Gd-DTPA) remains at stability problem, often body normal organ such as kidney etc. is caused very large infringement; In addition, the relaxation rate of Magnevist Solution is lower, needs to improve magnetic field intensity, the danger when this has also increased patient diagnosis.Therefore, the mri contrast agent of cancer target diagnosis efficiently being provided, effectively contrast agent being transported to tumor locus and getting into cell single-mindedly, efficiently to the tumor tissues video picture, reduce the whole body toxic and side effects simultaneously for a long time, is the key of present diagnosing tumor.
Dendrimer material such as polyamide-amide arborization thing (polyamidoamine; Be called for short PAMAM) and polylysine (dendri-graft polylysines; Be called for short DGL) be the synthetic high polymer of one type of Performances of Novel Nano-Porous meter level occurring in recent years, the height branch is monodispersity; Its terminal amino group is abundant, and is easy to through suitable bioactive substances such as modification connection targeting head base.Have and discover that the novel nano level dendrimer of this type can be connected with diethylenetriamine pentaacetic acid (DTPA) and then chelating gadolinium ion (Gd
3+), significantly improve the relaxation rate and the imaging results of contrast agent.
Initiatively targeting is to make up the main policies that targeting is passed release system, utilizes a specific basic modification system, and specificity combines the receptor of specific cells surface overexpression to reach the effect of targeted.There is research to show, a series of receptors of tumor cell surface overexpression, it is historical that wherein transferrins (transferrin abbreviates Tf as) receptor is used as the existing long research of target spot.But the endogenous transferrins is dense, is about 25 mM, can with the Tf receptor of Tf as the drug-supplying system of targeting head base competition tumor cell surface, thereby influence cancer target efficient.Medical research in the recent period uses display technique of bacteriophage to filter out a kind of novel polypeptide---and T7 peptide, its sequence are HAIYPRH, and be suitable to the affinity and the Tf of Tf receptor; The T7 peptide is different with the binding site and the Tf of Tf receptor, can not suppress and not influence the physiological function of Tf itself with Tf competition, on the contrary Tf and Tf receptor combine to promote the T7 peptide go into born of the same parents' efficient.So far, the relevant peptide modified cancer target of Shang Weijian is diagnosed the report of mri contrast agent.
Summary of the invention
The objective of the invention is to overcome defective of the prior art and deficiency, a kind of cancer target diagnosis mri contrast agent and preparation method thereof is provided, specifically provide a kind of peptide modified cancer target diagnosis mri contrast agent.
The present invention is targeting head base through peptide modified macromolecular material with the polypeptide, and the dendrimer material is basic macromolecule carrier, and the surface connects the micromolecule contrast agent, processes cancer target diagnosis mri contrast agent.This contrast agent gets into cell with the endocytosis mode, improves the picked-up of tumor cell to contrast agent, and safety.
Particularly, a kind of cancer target diagnosis mri contrast agent of the present invention is characterized in that it is processed by macromolecular material, Polyethylene Glycol, polypeptide, double function ligand and gadolinium trichloride.
Among the present invention, the molecular proportion of said macromolecular material and Polyethylene Glycol is 1:2～1:10; The molecular proportion of macromolecular material and polypeptide is 1:1～1:5; The molecular proportion of macromolecular material and double function ligand is 1:236～1:256; The molecular proportion of macromolecular material and gadolinium trichloride is 1:236～1:1416.
Among the present invention, macromolecular material is a dendroid, and there is cavity its inside; The macromolecular material of selecting for use in one embodiment of the present of invention is polyamide-amide type arborization thing and polylysine.
Among the present invention, described carrier system adopts peptide modified macromolecular material to form nanoparticle; Its aminoacid sequence of described polypeptide (T7) is HAIYPRH, and this polypeptide obtains through the display technique of bacteriophage screening.
Among the present invention, described Polyethylene Glycol is selected from maleimide-Polyethylene Glycol 3500-butanimide (MAL-PEG3500-NHS);
Among the present invention, described double function ligand comprises difunctional diethylenetriamine pentaacetic acid (p-SCN-Bn-DTPA), p-SCN-Bn-DOTA, p-SCN-Bn-NOTA, p-SCN-Bn-oxo-DO3A or p-SCN-Bn-PCTA;
Among the present invention, described chelating ion is gadolinium trichloride (GdCl
3).
The invention provides the method for preparing of cancer target diagnosis mri contrast agent, it is characterized in that it comprises step:
Step 1:Macromolecular material is dissolved in an amount of appropriate solvent is mixed with storing solution; Get in right amount and in cillin bottle, dry up; Take by weighing an amount of Polyethylene Glycol and be dissolved into the solution that is mixed with suitable concentration in the phosphate buffer of pH value 7.8～8.2; Join in the said vesse, with the macromolecular material ratio be 1:2～1:10, the stirring reaction number hour gets final product under the uniform temperature;
Step 2:Polypeptide is dissolved in an amount of phosphate buffer; Be mixed with the polypeptide solution of debita spissitudo, join in macromolecule-polyglycol solution, with macromolecular material ratio 1:1～1:5; Reaction 24 h under the uniform temperature; Make the cancer target nano-carrier, transfer in MWCO 5000 ultra-filtration centrifuge tubes and remove unreacted PEG and T7 peptide with 12000 rpm ultrafiltration, 30 min, the phosphate buffer of pH value 9.0 redissolves;
Step 3:Polypeptide solution that above-mentioned steps 2 is made and double function ligand are by the mixed of 1:236 ~ 1:256; Stirring reaction 48 h at ambient temperature; Transfer in MWCO 3000 ultra-filtration centrifuge tubes and remove the double function ligand that does not connect with 12000 rpm ultrafiltration, 30 min, the acetate buffer of pH value 6.0 redissolves;
Step 4:Polypeptide solution that above-mentioned steps 3 is made and chelating ion are by the mixed of 1:236 ~ 1:1416,4
oReaction 24 h transfer in MWCO 3000 ultra-filtration centrifuge tubes and remove the not gadolinium ion of chelating with 12000 rpm ultrafiltration, 30 min under the C condition, make the cancer target mri contrast agent.
Advantage of the present invention is: but utilize specificity to combine the peptide T 7 peptide modified macromolecule materials of tumor cell surface Tf receptor; Connect contrast agent and process the cancer target mri contrast agent; Make the distribution of contrast agent have the cancer target characteristic and the cellular uptake characteristic of T7 peptide, improve the ingestion efficiency of tumor cell and the diagnosis efficiency of tumor; This cancer target mri contrast agent has the characteristics of Tf as targeting head base, can effectively avoid the interference of endogenous Tf, and targeting and diagnosis efficiency are high, is applicable to tumor cell and other tumor cell of targeting human body source and animal origin.
Description of drawings
Fig. 1 is that hydrogen nuclear magnetic resonance characterizes cancer target nano-carrier PAMAM-PEG-T7.
Fig. 2 is that hydrogen nuclear magnetic resonance characterizes tumor target direction contrast agent intermediate DTPA-PAMAM-PEG-T7.
Fig. 3 is that the external haemolysis situation of mri contrast agent compares.
Fig. 4 is that the external relaxation rate of mri contrast agent compares, and slope is represented relaxation rate, and slope is big more, and relaxation rate is big more.
Fig. 5 is the picked-up situation of human tumor cells Bel-7402 to the contrast agent carrier, and cell culture treats that in 24 orifice plates density reaches at 80 ~ 90% o'clock; Hatch the carrier 30 as one kind min of green fluorescence probe BODIPY labelling; Medicinal liquid is removed in suction, buffer solution for cleaning, and OLYMPUS IX 71 fluorescence microscopes are also taken pictures:
Wherein, A and D are PAMAM, and B and E are PAMAM-PEG, and C and F are PAMAM-PEG-T7, and G and J are Tf+PAMAM, and H and K are Tf+PAMAM-PEG, and I and L are Tf+PAMAM-PEG-T7.
Fig. 6 is the picked-up situation of rat glioma cell C6 to the contrast agent carrier, and cell culture treats that in 24 orifice plates density reaches at 80 ~ 90% o'clock; Hatch the carrier 30 as one kind min of green fluorescence probe BODIPY labelling; Medicinal liquid is removed in suction, buffer solution for cleaning, and OLYMPUS IX 71 fluorescence microscopes are also taken pictures:
Wherein, A and D are PAMAM, and B and E are PAMAM-PEG, and C and F are PAMAM-PEG-T7, and G and J are Tf+PAMAM, and H and K are Tf+PAMAM-PEG, and I and L are Tf+PAMAM-PEG-T7.
Fig. 7 is that human tumor cells Bel-7402 subcutaneous transplantation nude mice model tail vein gives red fluorescence probe BODIPY the carrier of labelling, and different time CRI living imaging is observed the interior distribution situation of body of each carrier.
Fig. 8 is that rat glioma cell C6 intracranial original position nude mice model tail vein gives red fluorescence probe BODIPY the carrier of labelling, and different time CRI living imaging is observed the interior distribution situation of body of each carrier.
Fig. 9 adopts Bruker Biospec 4.7 T/30 cm Scanner to observe behind the mri contrast agent tail vein injection different time to the diagnosis situation of human tumor cells Bel-7402 subcutaneous transplantation tumor (A) and rat glioma cell C6 intracranial primary tumor (B).
The specific embodiment
Get 3 mg polyamide-amide (PAMAM; 77.35 the mg/ml methanol solution) in cillin bottle, dry up, add 3.64 mg Polyethylene Glycol (MAL-PEG3500-NHS, 1 mg/ml, 0.035 M pH, 8.0 phosphate solutions); The two molar ratio is 1:10; React 2 h in stirring at room, polyamide-amide-Polyethylene Glycol (PAMAM-PEG is processed in the amino specific reaction on NHS group and PAMAM surface
10) complex solution; Remove unreacted PEG with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes, pH 7.0 phosphate buffers redissolve, and add 0.52 mg T7 peptide (2 mg/ml pH, 7.0 phosphate solutions); Molar ratio is 1:5; Stirring at room is reacted 24 h, and specific reaction takes place sulfydryl on MAL group and the T7 peptide cysteine residues, processes polyamide-amide-Polyethylene Glycol-polypeptide (PAMAM-PEG-T7) complex; Remove unreacted T7 peptide with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes, proton nmr spectra characterizes the synthetic of targeting vector.
Get 3 mg polyamide-amide (PAMAM; 77.35 the mg/ml methanol solution) in cillin bottle, dry up, add 3.64 mg Polyethylene Glycol (MAL-PEG3500-NHS, 1 mg/ml, 0.035 M pH, 8.0 phosphate solutions); The two molar ratio is 1:10; React 2 h in stirring at room, polyamide-amide-Polyethylene Glycol (PAMAM-PEG is processed in the amino specific reaction on NHS group and PAMAM surface
10) complex solution, remove unreacted PEG with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes, pH 9.0 phosphate buffers redissolve; With the difunctional targeting part of 13.4 mg diethylenetriamine pentaacetic acid (p-SCN-Bn-DTPA; 4 mg/ml pH, 9.0 phosphate solutions), molar ratio is 1:236, and stirring at room is reacted 48 h; Specific reaction takes place in the amino on SCN group and PAMAM surface; Obtain the mri contrast agent intermediate, remove unreacted double function ligand with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes, proton nmr spectra characterizes the joint efficiency of cheland on the carrier.
Get 3 mg polyamide-amide (PAMAM; 77.35 the mg/ml methanol solution) in cillin bottle, dry up, add 3.64 mg Polyethylene Glycol (MAL-PEG3500-NHS, 1 mg/ml, 0.035 M pH, 8.0 phosphate solutions); The two molar ratio is 1:10; React 2 h in stirring at room, polyamide-amide-Polyethylene Glycol (PAMAM-PEG is processed in the amino specific reaction on NHS group and PAMAM surface
10) complex solution, remove unreacted PEG with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes, pH 9.0 phosphate buffers redissolve; With the difunctional targeting part of 13.4 mg diethylenetriamine pentaacetic acid (p-SCN-Bn-DTPA, 4 mg/ml pH, 9.0 phosphate solutions), molar ratio is 1:236; Stirring at room is reacted 48 h; Specific reaction takes place in the amino on SCN group and PAMAM surface, obtains the mri contrast agent intermediate, removes unreacted double function ligand with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes; PH 6.0 acetate buffers redissolve, with gadolinium trichloride (GdCl
3, 100 mg/ml pH, 6.0 acetate solutions), molar ratio is 1:236,4
oC reacts 24 h; Remove unreacted gadolinium trichloride with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes; Non-targeted magnetic resonance contrast agent is processed in the dissolving of pH 7.4 physiology phosphate buffers, adopts inductively coupled plasma atomic emission spectrum to characterize the chelating efficient of gadolinium.
Get 3 mg polyamide-amide (PAMAM; 77.35 the mg/ml methanol solution) in cillin bottle, dry up, add 3.64 mg Polyethylene Glycol (MAL-PEG3500-NHS, 1 mg/ml, 0.035 M pH, 8.0 phosphate solutions); The two molar ratio is 1:10; React 2 h in stirring at room, polyamide-amide-Polyethylene Glycol (PAMAM-PEG is processed in the amino specific reaction on NHS group and PAMAM surface
10) complex solution, remove unreacted PEG with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes, pH 9.0 phosphate buffers redissolve; With the difunctional targeting part of 13.4 mg diethylenetriamine pentaacetic acid (p-SCN-Bn-DTPA, 4 mg/ml pH, 9.0 phosphate solutions), molar ratio is 1:236; Stirring at room is reacted 48 h; Specific reaction takes place in the amino on SCN group and PAMAM surface, obtains the mri contrast agent intermediate, removes unreacted double function ligand with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes; PH 6.0 acetate buffers redissolve, with gadolinium trichloride (GdCl
3, 100 mg/ml pH, 6.0 acetate solutions), molar ratio is 1:236,4
oC reacts 24 h, removes unreacted gadolinium trichloride with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes, and non-targeted magnetic resonance contrast agent is processed in the dissolving of pH 7.4 physiology phosphate buffers.
Get 3 mg polyamide-amide (PAMAM; 77.35 the mg/ml methanol solution) in cillin bottle, dry up, add 3.64 mg Polyethylene Glycol (MAL-PEG3500-NHS, 1 mg/ml, 0.035 M pH, 8.0 phosphate solutions); The two molar ratio is 1:10; React 2 h in stirring at room, polyamide-amide-Polyethylene Glycol (PAMAM-PEG is processed in the amino specific reaction on NHS group and PAMAM surface
10) complex solution, remove unreacted PEG with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes, pH 7.0 phosphate buffers redissolve; Add 0.52 mg T7 peptide (2 mg/ml pH, 7.0 phosphate solutions), molar ratio is 1:5, and stirring at room is reacted 24 h; Specific reaction takes place in sulfydryl on MAL group and the T7 peptide cysteine residues, processes polyamide-amide-Polyethylene Glycol-polypeptide (PAMAM-PEG-T7) complex, removes unreacted T7 peptide with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes; PH 9.0 phosphate buffers redissolve; With the difunctional targeting part of 13.4 mg diethylenetriamine pentaacetic acid (p-SCN-Bn-DTPA, 4 mg/ml pH, 9.0 phosphate solutions), molar ratio is 1:236; Stirring at room is reacted 48 h; Specific reaction takes place in the amino on SCN group and PAMAM surface, obtains the mri contrast agent intermediate, removes unreacted double function ligand with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes; PH 6.0 acetate buffers redissolve, with gadolinium trichloride (GdCl
3, 100 mg/ml pH, 6.0 acetate solutions), molar ratio is 1:236,4
oC reacts 24 h, removes unreacted gadolinium trichloride with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes, and the targeted magnetic resonance contrast agent is processed in the dissolving of pH 7.4 physiology phosphate buffers.
Process targeted magnetic resonance contrast agent and non-targeted magnetic resonance contrast agent; Adopt commercially available Magnevist Solution as contrast; Each group contrast agent is diluted to 0,2.52,5.03,10.1,25.2,50.3,100.7,503 μ g Gd/ml, with 2% red blood cell suspension 37
oC is hatched 1 h, measures the haemolysis situation.
Get 3 mg polyamide-amide (PAMAM; 77.35 the mg/ml methanol solution) in cillin bottle, dry up, add 3.64 mg Polyethylene Glycol (MAL-PEG3500-NHS, 1 mg/ml, 0.035 M pH, 8.0 phosphate solutions); The two molar ratio is 1:10; React 2 h in stirring at room, polyamide-amide-Polyethylene Glycol (PAMAM-PEG is processed in the amino specific reaction on NHS group and PAMAM surface
10) complex solution, remove unreacted PEG with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes, pH 7.0 phosphate buffers redissolve; Add 0.52 mg T7 peptide (2 mg/ml pH, 7.0 phosphate solutions), molar ratio is 1:5, and stirring at room is reacted 24 h; Specific reaction takes place in sulfydryl on MAL group and the T7 peptide cysteine residues, processes polyamide-amide-Polyethylene Glycol-polypeptide (PAMAM-PEG-T7) complex, removes unreacted T7 peptide with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes; PH 9.0 phosphate buffers redissolve; With the difunctional targeting part of 13.4 mg diethylenetriamine pentaacetic acid (p-SCN-Bn-DTPA, 4 mg/ml pH, 9.0 phosphate solutions), molar ratio is 1:236; Stirring at room is reacted 48 h; Specific reaction takes place in the amino on SCN group and PAMAM surface, obtains the mri contrast agent intermediate, removes unreacted double function ligand with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes; PH 6.0 acetate buffers redissolve, with gadolinium trichloride (GdCl
3, 100 mg/ml pH, 6.0 acetate solutions), molar ratio is 1:236,4
oC reacts 24 h, removes unreacted gadolinium trichloride with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes, and the targeted magnetic resonance contrast agent is processed in the dissolving of pH 7.4 physiology phosphate buffers.Targeted magnetic resonance contrast agent and commercially available Magnevist Solution dilution are diluted to a series of concentration respectively, adopt Bruker Biospec 4.7 T/30 cm Scanner to measure the relaxation rate of contrast agent.
Get 3 mg polyamide-amides (PAMAM, 77.35 mg/ml methanol solutions) and in cillin bottle, dry up, the dissolving of 600 μ l, 100 mM sodium bicarbonate solutions adds green fluorescence probe (BODIPY, 0.56 mg, 600 μ l DMSO solution), and molar ratio is 1:10, and 4
oC reacts 12 h, obtains the non-targeting vector of green fluorescence labelling.
Get 3 mg polyamide-amides (PAMAM, 77.35 mg/ml methanol solutions) and in cillin bottle, dry up, the dissolving of 600 μ l, 100 mM sodium bicarbonate solutions adds green fluorescence probe (BODIPY, 0.56 mg, 600 μ l DMSO solution), and molar ratio is 1:10, and 4
oC reacts 12 h; Add 3.64 mg Polyethylene Glycol (MAL-PEG3500-NHS; 1 mg/ml, 0.035 M pH, 8.0 phosphate solutions), the two molar ratio is 1:10, reacts 2 h in stirring at room; Polyamide-amide-Polyethylene Glycol (PAMAM-PEG is processed in the amino specific reaction on NHS group and PAMAM surface
10) complex solution, remove unreacted PEG with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes, obtain the non-targeting vector of green fluorescence labelling.
Get 3 mg polyamide-amides (PAMAM, 77.35 mg/ml methanol solutions) and in cillin bottle, dry up, the dissolving of 600 μ l, 100 mM sodium bicarbonate solutions adds green fluorescence probe (BODIPY, 0.56 mg, 600 μ l DMSO solution), and molar ratio is 1:10, and 4
oC reacts 12 h; Add 3.64 mg Polyethylene Glycol (MAL-PEG3500-NHS; 1 mg/ml, 0.035 M pH, 8.0 phosphate solutions), the two molar ratio is 1:10, reacts 2 h in stirring at room; Polyamide-amide-Polyethylene Glycol (PAMAM-PEG is processed in the amino specific reaction on NHS group and PAMAM surface
10) complex solution; Remove unreacted PEG with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes, pH 7.0 phosphate buffers redissolve, and add 0.52 mg T7 peptide (2 mg/ml pH, 7.0 phosphate solutions); Molar ratio is 1:5; Stirring at room is reacted 24 h, and specific reaction takes place sulfydryl on MAL group and the T7 peptide cysteine residues, the fluorescently-labeled polyamide-amide-Polyethylene Glycol of row yielding-polypeptide (PAMAM-PEG-T7) complex; Remove unreacted T7 peptide with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes, obtain green fluorescence labelling targeting vector.
Embodiment 11
The non-targeting vector and the targeting vector of green fluorescence labelling are diluted to debita spissitudo with buffer respectively.People's tumor Bel-7402 cell culture treats that in 24 orifice plates density reaches at 80 ~ 90% o'clock, hatches the carrier 30 as one kind min of green fluorescence probe BODIPY labelling; Medicinal liquid is removed in suction; Buffer solution for cleaning, OLYMPUS IX 71 fluorescence microscopes are also taken pictures, and observe the picked-up situation of contrast agent carrier.
Embodiment 12
The non-targeting vector and the targeting vector of green fluorescence labelling are diluted to debita spissitudo with buffer respectively.Rat glioma C6 cell culture treats that in 24 orifice plates density reaches at 80 ~ 90% o'clock, hatches the carrier 30 as one kind min of green fluorescence probe BODIPY labelling; Medicinal liquid is removed in suction; Buffer solution for cleaning, OLYMPUS IX 71 fluorescence microscopes are also taken pictures, and observe the picked-up situation of contrast agent carrier.
Embodiment 13
Get 3 mg polyamide-amide (PAMAM; 77.35 the mg/ml methanol solution) in cillin bottle, dry up, the dissolving of 300 μ l, 100 mM sodium bicarbonate solutions adds red fluorescence probe (BODIPY; 0.67 mg 67 μ l DMSO solution); Molar ratio is 1:10, and room temperature reaction 1 h obtains the non-targeting vector of red fluorescent labeling.
Embodiment 14
Get 3 mg polyamide-amides (PAMAM, 77.35 mg/ml methanol solutions) and in cillin bottle, dry up, the dissolving of 300 μ l, 100 mM sodium bicarbonate solutions; Add red fluorescence probe (BODIPY, 0.67 mg, 67 μ l DMSO solution), molar ratio is 1:10; Room temperature reaction 1 h adds 3.64 mg Polyethylene Glycol (MAL-PEG3500-NHS, 1 mg/ml, 0.035 M pH, 8.0 phosphate solutions); The two molar ratio is 1:10; React 2 h in stirring at room, polyamide-amide-Polyethylene Glycol (PAMAM-PEG is processed in the amino specific reaction on NHS group and PAMAM surface
10) complex solution, remove unreacted PEG with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes, obtain the non-targeting vector of red fluorescent labeling.
Get 3 mg polyamide-amides (PAMAM, 77.35 mg/ml methanol solutions) and in cillin bottle, dry up, the dissolving of 300 μ l, 100 mM sodium bicarbonate solutions; Add red fluorescence probe (BODIPY, 0.67 mg, 67 μ l DMSO solution), molar ratio is 1:10; Room temperature reaction 1 h adds 3.64 mg Polyethylene Glycol (MAL-PEG3500-NHS, 1 mg/ml, 0.035 M pH, 8.0 phosphate solutions); The two molar ratio is 1:10; React 2 h in stirring at room, polyamide-amide-Polyethylene Glycol (PAMAM-PEG is processed in the amino specific reaction on NHS group and PAMAM surface
10) complex solution; Remove unreacted PEG with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes, pH 7.0 phosphate buffers redissolve, and add 0.52 mg T7 peptide (2 mg/ml pH, 7.0 phosphate solutions); Molar ratio is 1:5; Stirring at room is reacted 24 h, and specific reaction takes place sulfydryl on MAL group and the T7 peptide cysteine residues, the fluorescently-labeled polyamide-amide-Polyethylene Glycol of row yielding-polypeptide (PAMAM-PEG-T7) complex; Remove unreacted T7 peptide with 12000 rpm ultrafiltration, 30 min in MWCO 5000 ultra-filtration centrifuge tubes, obtain red fluorescent labeling targeting vector.
Embodiment 16
Human tumor cells Bel-7402 subcutaneous transplantation nude mice model tail vein gives red fluorescence probe BODIPY the carrier of labelling, and different time CRI living imaging is observed the interior distribution situation of body of each carrier.
Rat glioma cell C6 intracranial original position nude mice model tail vein gives red fluorescence probe BODIPY the carrier of labelling, and different time CRI living imaging is observed the interior distribution situation of body of each carrier.
Different time is to the diagnosis situation of human tumor cells Bel-7402 subcutaneous transplantation tumor behind employing Bruker Biospec 4.7 T/30 cm scanner observation cancer target mri contrast agent tail vein injection.Non-targeted magnetic resonance contrast agent and commercially available mri contrast agent Magnevist Solution are as contrast.
Embodiment 19
Different time is to the diagnosis situation of rat glioma cell C6 intracranial orthotopic transplantation tumor behind employing Bruker Biospec 4.7 T/30 cm scanner observation cancer target mri contrast agent tail vein injection.Non-targeted magnetic resonance contrast agent and commercially available mri contrast agent Magnevist Solution are as contrast.
Experimental result of the present invention shows: but utilize specificity to combine the peptide T 7 peptide modified macromolecule materials of tumor cell surface Tf receptor; Connect contrast agent and process the cancer target mri contrast agent; Make the distribution of contrast agent have cancer target characteristic and cellular uptake characteristic, can improve the ingestion efficiency of tumor cell and the diagnosis efficiency of tumor; Have the characteristics of Tf as targeting head base, can effectively avoid the interference of endogenous Tf, targeting and diagnosis efficiency are high, are applicable to tumor cell and other tumor cell of targeting human body source and animal origin.
Claims (10)
1. a cancer target diagnosis mri contrast agent is characterized in that, is processed by macromolecular material, Polyethylene Glycol, polypeptide, double function ligand and gadolinium trichloride;
The molecular proportion of said macromolecular material and Polyethylene Glycol is 1:2～1:10; The molecular proportion of macromolecular material and polypeptide is 1:1～1:5; The molecular proportion of macromolecular material and double function ligand is 1:236～1:256; The molecular proportion of macromolecular material and gadolinium trichloride is 1:236～1:1416.
2. by the described cancer target diagnosis of claim 1 mri contrast agent, it is characterized in that described carrier system adopts peptide modified dendrimer material to form nanoparticle.
3. by the described cancer target diagnosis of claim 1 mri contrast agent, it is characterized in that described macromolecular material is polyamide-amide type arborization thing and polylysine.
4. by the described cancer target diagnosis of claim 1 mri contrast agent, it is characterized in that described macromolecular material is a dendroid, there is cavity its inside.
5. by the described cancer target diagnosis of claim 1 mri contrast agent, it is characterized in that described Polyethylene Glycol is selected from maleimide-Polyethylene Glycol 3500-butanimide.
6. by the described cancer target diagnosis of claim 1 mri contrast agent, it is characterized in that its aminoacid sequence of described polypeptide is HAIYPRH.
7. by the described cancer target diagnosis of claim 1 mri contrast agent; It is characterized in that described double function ligand is selected from difunctional diethylenetriamine pentaacetic acid, p-SCN-Bn-DOTA, p-SCN-Bn-NOTA, p-SCN-Bn-oxo-DO3A or p-SCN-Bn-PCTA.
8. by the described cancer target diagnosis of claim 1 mri contrast agent, it is characterized in that described polypeptid specificity combines TfR.
9. the described cancer target diagnosis of claim 1 mri contrast agent is being used for absorbing human body source and the tumor cell of animal origin and the purposes of other cancerous cell.
10. the cancer target of claim 1 is diagnosed the method for preparing of mri contrast agent, it is characterized in that it comprises step:
Step 1:Macromolecular material is dissolved in an amount of appropriate solvent is mixed with storing solution; Get in right amount and in cillin bottle, dry up; Take by weighing an amount of Polyethylene Glycol and be dissolved into the solution that is mixed with suitable concentration in the phosphate buffer of pH value 7.8～8.2; Join in the said vesse, with the macromolecular material ratio be 1:2～1:10, the stirring reaction number hour gets final product under the uniform temperature;
Step 2:Polypeptide is dissolved in an amount of phosphate buffer; Be mixed with the polypeptide solution of debita spissitudo, join in macromolecule-polyglycol solution, with macromolecular material ratio 1:1～1:5; Reaction 24 h under the uniform temperature; Make the cancer target nano-carrier, transfer in MWCO 5000 ultra-filtration centrifuge tubes and remove unreacted PEG and T7 peptide with 12000 rpm ultrafiltration, 30 min, the phosphate buffer of pH value 9.0 redissolves;
Step 3:Polypeptide solution that above-mentioned steps 2 is made and double function ligand are by the mixed of 1:236 ~ 1:256; Stirring reaction 48 h at ambient temperature; Transfer in MWCO 3000 ultra-filtration centrifuge tubes and remove the double function ligand that does not connect with 12000 rpm ultrafiltration, 30 min, the acetate buffer of pH value 6.0 redissolves;
Step 4:Polypeptide solution that above-mentioned steps 3 is made and chelating ion are by the mixed of 1:236 ~ 1:1416,4
oReaction 24 h transfer in MWCO 3000 ultra-filtration centrifuge tubes and remove the not gadolinium ion of chelating with 12000 rpm ultrafiltration, 30 min under the C condition, make the cancer target mri contrast agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010286508 CN102397564B (en) | 2010-09-19 | 2010-09-19 | Tumor-targeted diagnosis nuclear magnetic resonance contrast agent and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010286508 CN102397564B (en) | 2010-09-19 | 2010-09-19 | Tumor-targeted diagnosis nuclear magnetic resonance contrast agent and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102397564A true CN102397564A (en) | 2012-04-04 |
| CN102397564B CN102397564B (en) | 2013-05-29 |
Family
ID=45880456
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010286508 Expired - Fee Related CN102397564B (en) | 2010-09-19 | 2010-09-19 | Tumor-targeted diagnosis nuclear magnetic resonance contrast agent and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102397564B (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103705464A (en) * | 2012-10-09 | 2014-04-09 | 复旦大学 | Tumor active targeting nano drug delivery system controlled by slightly acidic environment, and preparation method thereof |
| CN103705465A (en) * | 2012-10-09 | 2014-04-09 | 复旦大学 | Slightly acidic environment targeted polypeptide modified tumor targeted nano drug delivery system, and preparation method thereof |
| CN104758954A (en) * | 2015-03-16 | 2015-07-08 | 北京化工大学 | Double-functional nano-composite spheres based on metal ion-inducing polypeptide self-assembly and preparation method thereof |
| CN105126127A (en) * | 2015-10-14 | 2015-12-09 | 常州大学 | Preparation method of colon cancer nuclear magnetic resonance multifunctional contrast agent |
| CN105343900A (en) * | 2015-09-16 | 2016-02-24 | 中国海洋大学 | Lymph targeted nuclear magnetic resonance imaging contrast agent with brown algal polysaccharide serving as carrier and preparation method and application of lymph targeted nuclear magnetic resonance imaging contrast agent with brown algal polysaccharide serving as carrier |
| CN105727318A (en) * | 2014-12-09 | 2016-07-06 | 中国科学院苏州纳米技术与纳米仿生研究所 | Polypeptide molecule image probe targeting mesenchymal stem cell, preparation method of polypeptide molecule image probe, and mesenchymal stem cell marked by polypeptide molecule image probe |
| CN106890345A (en) * | 2015-12-17 | 2017-06-27 | 中国科学院苏州纳米技术与纳米仿生研究所 | A kind of contrast agent molecule of targetted mitochondria is used as T2The purposes of contrast agent |
| CN110836879A (en) * | 2019-11-06 | 2020-02-25 | 东南大学 | Cell membrane long-time multicolor fluorescence imaging reagent and preparation method and application thereof |
| CN112043839A (en) * | 2020-08-21 | 2020-12-08 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Radioisotope-labeled polypeptide imaging agent targeting transferrin receptor and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090238767A1 (en) * | 2004-12-17 | 2009-09-24 | Koninklijke Philips Electronics, N.V. | Targeting contrast agents or targeting therapeutic agents for molecular imaging and therapy |
| CN101549161A (en) * | 2009-05-13 | 2009-10-07 | 中南大学 | Liver, spleen specific positive magnetic nuclear resonance contrast agent and method of preparing the same |
| CN101612407A (en) * | 2009-07-14 | 2009-12-30 | 山东大学 | Polymer nanoparticle magnetic resonance contrast medium and preparation method thereof |
-
2010
- 2010-09-19 CN CN 201010286508 patent/CN102397564B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090238767A1 (en) * | 2004-12-17 | 2009-09-24 | Koninklijke Philips Electronics, N.V. | Targeting contrast agents or targeting therapeutic agents for molecular imaging and therapy |
| CN101549161A (en) * | 2009-05-13 | 2009-10-07 | 中南大学 | Liver, spleen specific positive magnetic nuclear resonance contrast agent and method of preparing the same |
| CN101612407A (en) * | 2009-07-14 | 2009-12-30 | 山东大学 | Polymer nanoparticle magnetic resonance contrast medium and preparation method thereof |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103705465B (en) * | 2012-10-09 | 2016-01-13 | 复旦大学 | Cancer target administration nano-drug administration system that a kind of micro-acid environment target polypeptide is modified and preparation method thereof |
| CN103705465A (en) * | 2012-10-09 | 2014-04-09 | 复旦大学 | Slightly acidic environment targeted polypeptide modified tumor targeted nano drug delivery system, and preparation method thereof |
| CN103705464A (en) * | 2012-10-09 | 2014-04-09 | 复旦大学 | Tumor active targeting nano drug delivery system controlled by slightly acidic environment, and preparation method thereof |
| CN103705464B (en) * | 2012-10-09 | 2015-10-07 | 复旦大学 | A kind of micro-acid environment controls tumor-targeting administration nano-drug administration system opened and preparation method thereof |
| CN105727318A (en) * | 2014-12-09 | 2016-07-06 | 中国科学院苏州纳米技术与纳米仿生研究所 | Polypeptide molecule image probe targeting mesenchymal stem cell, preparation method of polypeptide molecule image probe, and mesenchymal stem cell marked by polypeptide molecule image probe |
| CN104758954B (en) * | 2015-03-16 | 2017-08-25 | 北京化工大学 | A kind of dual-functional nanometer composite balls based on metal ion inducing polypeptide self assembly and preparation method thereof |
| CN104758954A (en) * | 2015-03-16 | 2015-07-08 | 北京化工大学 | Double-functional nano-composite spheres based on metal ion-inducing polypeptide self-assembly and preparation method thereof |
| CN105343900A (en) * | 2015-09-16 | 2016-02-24 | 中国海洋大学 | Lymph targeted nuclear magnetic resonance imaging contrast agent with brown algal polysaccharide serving as carrier and preparation method and application of lymph targeted nuclear magnetic resonance imaging contrast agent with brown algal polysaccharide serving as carrier |
| CN105126127A (en) * | 2015-10-14 | 2015-12-09 | 常州大学 | Preparation method of colon cancer nuclear magnetic resonance multifunctional contrast agent |
| CN105126127B (en) * | 2015-10-14 | 2017-11-10 | 常州大学 | A kind of preparation method of the multi-functional contrast agent of colon cancer nuclear magnetic resonance |
| CN106890345A (en) * | 2015-12-17 | 2017-06-27 | 中国科学院苏州纳米技术与纳米仿生研究所 | A kind of contrast agent molecule of targetted mitochondria is used as T2The purposes of contrast agent |
| CN106890345B (en) * | 2015-12-17 | 2020-09-01 | 中国科学院苏州纳米技术与纳米仿生研究所 | Contrast agent molecule targeting mitochondria as T2Use of contrast agents |
| CN110836879A (en) * | 2019-11-06 | 2020-02-25 | 东南大学 | Cell membrane long-time multicolor fluorescence imaging reagent and preparation method and application thereof |
| CN110836879B (en) * | 2019-11-06 | 2021-12-07 | 东南大学 | Cell membrane long-time multicolor fluorescence imaging reagent and preparation method and application thereof |
| CN112043839A (en) * | 2020-08-21 | 2020-12-08 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Radioisotope-labeled polypeptide imaging agent targeting transferrin receptor and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102397564B (en) | 2013-05-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102397564B (en) | Tumor-targeted diagnosis nuclear magnetic resonance contrast agent and preparation method thereof | |
| CN102406949B (en) | Target tracing multi-mode diagnostic nano imaging medicine | |
| US8697031B2 (en) | Dual function polymer micelles | |
| Peng et al. | Targeted tumor CT imaging using folic acid-modified PEGylated dendrimer-entrapped gold nanoparticles | |
| Tan et al. | Peptide-targeted nanoglobular Gd-DOTA monoamide conjugates for magnetic resonance cancer molecular imaging | |
| Jin et al. | Upconversion Nanoparticles Conjugated with Gd3+‐DOTA and RGD for Targeted Dual‐Modality Imaging of Brain Tumor Xenografts | |
| Yan et al. | Ultrasmall hybrid protein–copper sulfide nanoparticles for targeted photoacoustic imaging of orthotopic hepatocellular carcinoma with a high signal-to-noise ratio | |
| US8741615B2 (en) | Magnetic nanoparticle with biocompatibility | |
| CN111205411B (en) | Blood vessel and tumor enhancement macromolecule magnetic resonance contrast agent and preparation method and application thereof | |
| Zhou et al. | Dendritic nanoglobules with polyhedral oligomeric silsesquioxane core and their biomedical applications | |
| CN103041407B (en) | Core-shell type nano-contrast agent, preparation method and application thereof | |
| CN101569751A (en) | Tumor-targeted nonionic dendritic macromolecule magnetic resonance imaging contrast agent | |
| CN102048694B (en) | Polypeptide-modified liver tumor-targeted nano medicine delivery system and preparation method thereof | |
| US20230087639A1 (en) | Biogenic hemin-based mri contrast agents, and compositions and methods thereof | |
| Huang et al. | Sericin-based gadolinium nanoparticles as synergistically enhancing contrast agents for pH-responsive and tumor targeting magnetic resonance imaging | |
| Wei et al. | Biocompatible low-retention superparamagnetic iron oxide nanoclusters as contrast agents for magnetic resonance imaging of liver tumor | |
| CN111004307A (en) | Indocyanine green compound for treating early brain glioma and preparation method and application thereof | |
| CN104436220A (en) | Preparation method of magnetic chitosan nanoparticles and application of magnetic chitosan microspheres | |
| CN102058891A (en) | Fluorescent magnetic silicon nano carrier modified with polyethylene glycol and preparation method and application | |
| Li et al. | Safe and efficient magnetic resonance imaging of acute myocardial infarction with gadolinium-doped carbon dots | |
| CN109620974B (en) | Osteoarthritis cartilage targeted gadolinium-based magnetic resonance imaging contrast agent and preparation method thereof | |
| Sun et al. | A dual-modality MR/PA imaging contrast agent based on ultrasmall biopolymer nanoparticles for orthotopic hepatocellular carcinoma imaging | |
| CN103877597A (en) | Pegylated polyethyleneimine macromolecular magnetic resonance imaging contrast agent and preparation method of contrast agent | |
| Li et al. | VEGF mimetic peptide-conjugated nanoparticles for magnetic resonance imaging and therapy of myocardial infarction | |
| Wang et al. | Multifunctional nanoprobe for multi-mode imaging and diagnosis of metastatic prostate cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130529 Termination date: 20150919 |
|
| EXPY | Termination of patent right or utility model |