CN102397564B - 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
- CN102397564B CN102397564B CN 201010286508 CN201010286508A CN102397564B CN 102397564 B CN102397564 B CN 102397564B CN 201010286508 CN201010286508 CN 201010286508 CN 201010286508 A CN201010286508 A CN 201010286508A CN 102397564 B CN102397564 B CN 102397564B
- Authority
- CN
- China
- Prior art keywords
- contrast agent
- tumor
- polypeptide
- magnetic resonance
- nuclear magnetic
- 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.)
- Expired - Fee Related
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 tumor-targeted diagnosis nuclear magnetic resonance contrast agent and preparation method thereof, especially a kind of peptide modified tumor-targeted diagnosis nuclear magnetic resonance contrast agent and preparation method thereof.
Background technology
Tumor is adopted the magnetic resonance imaging diagnosis and studies efficient contrast agent is noticeable focus in recent years.The contrast agent that uses clinically at present mostly is hydrophilic micromolecule, can not enter cell, often rests on interstice and blood vessel internal cavity by the blood vessel endothelium space, remove very fast, for a long time video picture; And owing to lack specific conveying, only depend on vascular space to penetrate into tissue, cause contrast agent to be accumulated in inflammation part, cause easily the mistaken diagnosis to inflammation.The contrast agent Magnevist Solution (Gd-DTPA) of selling in the market 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, providing efficient tumor-targeted diagnosis nuclear magnetic resonance contrast agent, effectively contrast agent is transported to tumor locus single-mindedly and enters cell, efficiently to the tumor tissues video picture, reduce simultaneously the whole body toxic and side effects 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 the class Performances of Novel Nano-Porous meter level that occurs in recent years, the height branch, be monodispersity, its terminal amino group is abundant, and is easy to through the suitable bioactive substances such as modification connection targeting head base.There is research to find that the novel nano level dendrimer of this class can be connected with diethylenetriamine pentaacetic acid (DTPA) and then chelating gadolinium ion (Gd
3+), significantly improve 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 specific basic modification system, and the receptor of specific binding specific cells surface overexpression is to reach the effect of targeted.Studies show that, tumor cell surface overexpression series of receptors, wherein transferrins (transferrin is referred to as Tf) receptor is used as the existing long research history 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 affect cancer target efficient.In the recent period medical research uses display technique of bacteriophage to filter out a kind of novel polypeptide---and T7 peptide, its sequence are HAIYPRH, and be suitable to affinity and the Tf of Tf receptor; The T7 peptide is different from binding site and the Tf of Tf receptor, can not suppress and not affect with Tf competition the physiological function of Tf itself, opposite Tf and Tf receptor enter born of the same parents' efficient in conjunction with meeting promotion T7 peptide.So far, there is not yet the report of relevant peptide modified tumor-targeted diagnosis nuclear magnetic resonance contrast agent.
Summary of the invention
The objective of the invention is to overcome defective of the prior art and deficiency, a kind of tumor-targeted diagnosis nuclear magnetic resonance contrast agent and preparation method thereof is provided, a kind of peptide modified tumor-targeted diagnosis nuclear magnetic resonance contrast agent specifically is provided.
The present invention is by peptide modified macromolecular material, and take polypeptide as targeting head base, the dendrimer material is basic macromolecule carrier, and the surface connects the micromolecule contrast agent, makes tumor-targeted diagnosis nuclear magnetic resonance contrast agent.This contrast agent enters cell in the endocytosis mode, improves tumor cell to the picked-up of contrast agent, and safety.
Particularly, a kind of tumor-targeted diagnosis nuclear magnetic resonance contrast agent of the present invention is characterized in that it is made by macromolecular material, Polyethylene Glycol, polypeptide, double function ligand and gadolinium trichloride.
Among the present invention, the molecular proportion of described 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 dendroid, and there is cavity its inside; The macromolecular material of selecting 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 by 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 preparation method of tumor-targeted diagnosis nuclear magnetic resonance contrast agent, it is characterized in that, it comprises step:
Step 1:Macromolecular material is dissolved in an amount of suitable solvent is mixed with storing solution, get in right amount and in cillin bottle, dry up, take by weighing the solution that is mixed with suitable concentration in the phosphate buffer that an amount of Polyethylene Glycol is dissolved into pH value 7.8～8.2, join in the said vesse, with the macromolecular material ratio be 1:2～1:10, stirring reaction a few hours get 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:The polypeptide solution that above-mentioned steps 2 is made mixes in the ratio of 1:236 ~ 1:256 with double function ligand, 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:The polypeptide solution that above-mentioned steps 3 is made mixes in the ratio of 1:236 ~ 1:1416 with chelating ion, 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 the peptide T 7 peptide modified macromolecule materials of utilization specific binding tumor cell surface Tf receptor, connect contrast agent and make the cancer target mri contrast agent, make the distribution of contrast agent have cancer target feature and the cellular uptake feature of T7 peptide, improve the ingestion efficiency of tumor cell and the diagnosis efficiency of tumor; This cancer target mri contrast agent has Tf as the characteristics of 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 represents relaxation rate, and slope is larger, and relaxation rate is larger.
Fig. 5 be human tumor cells Bel-7402 to the picked-up situation of contrast agent carrier, cell culture is in 24 orifice plates, when treating that density reaches 80 ~ 90%, hatch the carrier 30 as one kind min of green fluorescence probe BODIPY labelling, suck medicinal liquid, buffer solution for cleaning, 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 be rat Glioma cells C6 to the picked-up situation of contrast agent carrier, cell culture is in 24 orifice plates, when treating that density reaches 80 ~ 90%, hatch the carrier 30 as one kind min of green fluorescence probe BODIPY labelling, suck medicinal liquid, buffer solution for cleaning, 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 the red fluorescence probe BODIPY 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 cells C6 intracranial Situ model of nude mice tail vein gives the red fluorescence probe BODIPY 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 cells 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 made 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, stirring at room is reacted 24 h, specific reaction occurs in sulfydryl on MAL group and the T7 peptide cysteine residues, make 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 made 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 occurs 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.
Embodiment 3
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 made 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 occurs 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, 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 mri contrast agent is made 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 made 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 occurs 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, 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 mri contrast agent is made in the dissolving of pH 7.4 physiology phosphate buffers.
Embodiment 5
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 made 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, stirring at room is reacted 24 h, specific reaction occurs in sulfydryl on MAL group and the T7 peptide cysteine residues, make 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, 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 occurs 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, 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 made in the dissolving of pH 7.4 physiology phosphate buffers.
Make targeted magnetic resonance contrast agent and non-targeted mri contrast agent, adopt commercially available Magnevist Solution in 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 made 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, stirring at room is reacted 24 h, specific reaction occurs in sulfydryl on MAL group and the T7 peptide cysteine residues, make 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, 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 occurs 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, 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 made in the dissolving of pH 7.4 physiology phosphate buffers.Targeted magnetic resonance contrast agent and commercially available Magnevist Solution dilution are diluted to respectively a series of concentration, 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 dry up in cillin bottle, 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-targeted carrier of green fluorescence labelling.
Get 3 mg polyamide-amides (PAMAM, 77.35 mg/ml methanol solutions) and dry up in cillin bottle, 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, react 2 h in stirring at room, polyamide-amide-Polyethylene Glycol (PAMAM-PEG is made 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-targeted carrier of green fluorescence labelling.
Get 3 mg polyamide-amides (PAMAM, 77.35 mg/ml methanol solutions) and dry up in cillin bottle, 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, react 2 h in stirring at room, polyamide-amide-Polyethylene Glycol (PAMAM-PEG is made 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, stirring at room is reacted 24 h, specific reaction occurs in sulfydryl on MAL group and the T7 peptide cysteine residues, fluorescently-labeled polyamide-amide-the 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
Non-targeted carrier and the targeting vector of green fluorescence labelling are diluted to debita spissitudo with buffer respectively.People's tumor Bel-7402 cell culture when treating that density reaches 80 ~ 90%, is hatched the carrier 30 as one kind min of green fluorescence probe BODIPY labelling in 24 orifice plates, suck medicinal liquid, 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
Non-targeted carrier and the targeting vector of green fluorescence labelling are diluted to debita spissitudo with buffer respectively.Rat glioma C 6 cells is incubated at 24 orifice plates, when treating that density reaches 80 ~ 90%, hatches the carrier 30 as one kind min of green fluorescence probe BODIPY labelling, suck medicinal liquid, 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, add 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-targeted carrier of red fluorescent labeling.
Embodiment 14
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, add red fluorescence probe (BODIPY, 0.67 mg 67 μ l DMSO solution), molar ratio is 1:10, room temperature reaction 1 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 made 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-targeted carrier of red fluorescent labeling.
Embodiment 15
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, add red fluorescence probe (BODIPY, 0.67 mg 67 μ l DMSO solution), molar ratio is 1:10, room temperature reaction 1 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 made 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, stirring at room is reacted 24 h, specific reaction occurs in sulfydryl on MAL group and the T7 peptide cysteine residues, fluorescently-labeled polyamide-amide-the 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 the red fluorescence probe BODIPY carrier of labelling, and different time CRI living imaging is observed the interior distribution situation of body of each carrier.
Embodiment 17
Rat Glioma cells C6 intracranial Situ model of nude mice tail vein gives the red fluorescence probe BODIPY carrier of labelling, and different time CRI living imaging is observed the interior distribution situation of body of each carrier.
Embodiment 18
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 mri contrast agent and commercially available mri contrast agent Magnevist Solution are in contrast.
Embodiment 19
Different time is to the diagnosis situation of rat Glioma cells 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 mri contrast agent and commercially available mri contrast agent Magnevist Solution are in contrast.
Experimental result of the present invention shows: but the peptide T 7 peptide modified macromolecule materials of utilization specific binding tumor cell surface Tf receptor, connect contrast agent and make the cancer target mri contrast agent, make the distribution of contrast agent have cancer target feature and cellular uptake feature, can improve the ingestion efficiency of tumor cell and the diagnosis efficiency of tumor; Have Tf as the characteristics of 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 (5)
1. a tumor-targeted diagnosis nuclear magnetic resonance contrast agent is characterized in that, is made by macromolecular material, Polyethylene Glycol, polypeptide, double function ligand and gadolinium trichloride;
Described macromolecular material is polyamide-amide type arborization thing, and described Polyethylene Glycol is diethylenetriamine pentaacetic acid from maleimide-Polyethylene Glycol 3500-butanimide, described double function ligand, and its aminoacid sequence of described polypeptide is HAIYPRH;
The preparation process of described contrast agent is as follows:
Step 1: macromolecular material is dissolved in an amount of suitable solvent is mixed with storing solution, get in right amount and in cillin bottle, dry up, take by weighing the solution that is mixed with suitable concentration in the phosphate buffer that an amount of Polyethylene Glycol is dissolved into pH value 7.8～8.2, join in the said vesse, with the macromolecular material ratio be 1:2～1:10, stirring reaction a few hours get 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, react 24h under the uniform temperature, make the cancer target nano-carrier, transfer in the MWCO5000 ultra-filtration centrifuge tube and remove unreacted PEG and T7 peptide with 12000rpm ultrafiltration 30min, the phosphate buffer of pH value 9.0 redissolves;
Step 3: the polypeptide solution that above-mentioned steps 2 is made mixes in the ratio of 1:236～1:256 with double function ligand, stirring reaction 48h at ambient temperature, transfer in the MWCO3000 ultra-filtration centrifuge tube and remove the double function ligand that does not connect with 12000rpm ultrafiltration 30min, the acetate buffer of pH value 6.0 redissolves;
Step 4: the polypeptide solution that above-mentioned steps 3 is made mixes in the ratio of 1:236～1:1416 with chelating ion, 4
oReact 24h under the C condition, transfer in the MWCO3000 ultra-filtration centrifuge tube and remove the not gadolinium ion of chelating with 12000rpm ultrafiltration 30min, make the cancer target mri contrast agent.
2. by tumor-targeted diagnosis nuclear magnetic resonance contrast agent claimed in claim 1, it is characterized in that described peptide modified described dendrimer material forms nanoparticle.
3. by tumor-targeted diagnosis nuclear magnetic resonance contrast agent claimed in claim 1, it is characterized in that there is cavity its inside of described macromolecular material.
4. by tumor-targeted diagnosis nuclear magnetic resonance contrast agent claimed in claim 1, it is characterized in that described polypeptid specificity is in conjunction with TfR.
5. the tumor-targeted diagnosis nuclear magnetic resonance contrast agent of claim 1 is in preparation targeting diagnosis human body source or the tumor cell of liver of animal origin or the purposes in other cancerous cell medicine.
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 CN102397564A (en) | 2012-04-04 |
| CN102397564B true 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 (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Families Citing this family (8)
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101080240A (en) * | 2004-12-17 | 2007-11-28 | 皇家飞利浦电子股份有限公司 | Targeting contrast agents or targeting therapeutic agents for molecular imaging and therapy |
-
2010
- 2010-09-19 CN CN 201010286508 patent/CN102397564B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102397564A (en) | 2012-04-04 |
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 | |
| Tan et al. | Peptide-targeted nanoglobular Gd-DOTA monoamide conjugates for magnetic resonance cancer molecular imaging | |
| US8697031B2 (en) | Dual function polymer micelles | |
| 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 | |
| Kim et al. | Gadolinium-chelate nanoparticle entrapped human mesenchymal stem cell via photochemical internalization for cancer diagnosis | |
| Zhou et al. | Mn-Loaded apolactoferrin dots for in vivo MRI and NIR-II cancer imaging | |
| Yang et al. | GE11-PDA-Pt@ USPIOs nano-formulation for relief of tumor hypoxia and MRI/PAI-guided tumor radio-chemotherapy | |
| US20100209353A1 (en) | Tumor targeting protein conjugate and a method for preparing the same | |
| Zhou et al. | Dendritic nanoglobules with polyhedral oligomeric silsesquioxane core and their biomedical applications | |
| CN111205411B (en) | Blood vessel and tumor enhancement macromolecule magnetic resonance contrast agent and preparation method and application thereof | |
| Huang et al. | Sericin-based gadolinium nanoparticles as synergistically enhancing contrast agents for pH-responsive and tumor targeting magnetic resonance imaging | |
| 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 | |
| Wei et al. | Biocompatible low-retention superparamagnetic iron oxide nanoclusters as contrast agents for magnetic resonance imaging of liver tumor | |
| Sun et al. | Facile synthesis of melanin-dye nanoagent for NIR-II fluorescence/photoacoustic imaging-guided photothermal therapy | |
| 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 | |
| CN109620974B (en) | Osteoarthritis cartilage targeted gadolinium-based magnetic resonance imaging contrast agent and preparation method thereof | |
| Zhang et al. | Arsenite-loaded albumin nanoparticles for targeted synergistic chemo-photothermal therapy of HCC | |
| Li et al. | Safe and efficient magnetic resonance imaging of acute myocardial infarction with gadolinium-doped carbon dots | |
| Sun et al. | A dual-modality MR/PA imaging contrast agent based on ultrasmall biopolymer nanoparticles for orthotopic hepatocellular carcinoma imaging | |
| Li et al. | VEGF mimetic peptide-conjugated nanoparticles for magnetic resonance imaging and therapy of myocardial infarction | |
| CN103877597A (en) | Pegylated polyethyleneimine macromolecular magnetic resonance imaging contrast agent and preparation method of contrast agent |
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 |