CN103480009A - Bi-modal tumor targeted nano particle photographic developer and method for preparing same - Google Patents
Bi-modal tumor targeted nano particle photographic developer and method for preparing same Download PDFInfo
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Abstract
The invention provides a bi-modal tumor targeted nano particle photographic developer and a method for preparing the bi-modal tumor targeted nano particle photographic developer. Near-Infrared fluorescence core-cross-linked polymeric micelles (NIRF-CCPM) with good biological performance serve as a carrier, coupling of polypeptide RGD (arginine-glycine-aspartic acid) with tumor targeting and nano particles is carried out to obtain a novel tumor targeting nano molecular imaging probe NIRF-CCPM-RGD, labeling is conducted on the NIRF-CCPM-RGD through a radionuclide 111In to obtain 111In-NIRF-CCPM-RGD, specific binding can be conducted on the 111In-NIRF-CCPM-RGD and an antigen alpha v beta 3 of a tumor neoangiogenesis endothelial cell, a tissue with high expression through the alpha v beta 3 is accurately positioned respectively through an infrared mode and a nuclear medicine mode to achieve tumor targeting molecule image diagnoses, and researches of a plurality of molecule image probes are conducted on the same molecule to achieve tumor early detection, early diagnosing and early treatment targets.
Description
Technical field
The present invention relates to radiopharmaceutical chemistry and clinical nuclear medicine technical field, specifically, relate to a kind of bimodal cancer target nanoparticle developer and preparation method thereof.
Background technology
In radiopharmaceutical chemistry and clinical nuclear medicine technical field, lasting growth, Invasion and Metastasis and the tumor-blood-vessel growth of malignant tumor are closely related, integrin alpha in this process
vβ
3play an important role.α
vβ
3main by and extracellular matrix (ECM) in some containing RGD(Arg-Gly-Asp) ligand binding plays a role, studies confirm that its high expressed in the new vessels endotheliocyte, and express seldom or shortage at normal cell.Radioactive label RGD peptide carries out nuclear medicine image as the label of high specific to malignant tumor, can reach the early diagnosis and therapy purpose.But, because RGD is annular tripeptides, molecular weight is little, in body, to remove comparatively fast, tumor uptake is relatively low, is difficult to long-term video picture continuously.The combination of tumor-targeting drug and nanotechnology will be the study hotspot of the novel diagnosis of Future Development and medicine for treating tumor thing.
Summary of the invention
The purpose of this invention is to provide a kind of infrared/nuclear medicine bimodal cancer target nanoparticle developer and preparation method thereof.
In order to realize the object of the invention, at first the present invention provides a kind of cancer target nanoparticle, and it has the NIRF-CCPM nanoparticle of RGD peptide, i.e. NIRF-CCPM-RGD for coupling.Described RGD peptide is arginine-glycine-aspartic acid (Arg-Gly-Asp).
Wherein, the NIRF-CCPM nanoparticle, it is near-infrared core cross-linked polymeric micellar nanoparticles, its construction method list of references ([1] Z Yang, SY Zheng, WJ Harrison, J Harder, XX Wen, JG Gelovani, A Qiao, C Li*.Long-Circulating Near-Infrared Fluorescence Core-Cross-Linked Polymeric Micelles:Synthesis, Characterization, and Dual Nuclear/Optical Imaging.Biomacromolecules.2010,8,3422-3428).Be summarized as follows:
Adopting the tert-butyl group-aminoethyl ethanol is raw material, synthetic 2-bromo acid t-Boc-amino ethyl ester.After taking off the t-Boc-protection by trifluoroacetic acid, obtained highly purified reacting precursor 2-bromo acid 2-amino ethyl ester trifluoroacetate.Poly--(PEG-acrylic acid)-b-is poly--and (triethoxysilicane alkyl acrylate) (PPEGMA-b-PESPMA) synthetic be by atom transfer polymerization (ATRP), react with Catalysts Cu Br and prepare by the different acrylate of lead compound 2-amino-ethyl 2-bromo.Surface is with both sexes block polymer PPEGMA-b-PESPMA for amino CCPM nanoparticle and NIRF dyestuff 3-(triethoxysilyl) propyl-Cy7 is synthetic obtains.Water is added in the THF containing block polymer (10mg/mL) and hydrophobic NIRF fuel 3-(triethoxysilyl) propyl-Cy7 to the self-assembling formation polymeric micelle.After in solution, adding acetic acid, polymeric micelle has experienced a sol gel reaction process.At first Ethoxysilane precursor fast hydrolyzing, PESPMA core and Cy7-derivant are cross-linked to form the Si-O key.Crosslinked core provides a stable micelle nano structure, thus the unstability of having avoided the critical micell polymerization to produce.The CCPM of preparation adopts the dialysis purification, and carry out sequential filtration with the filter membrane of 0.7 μ m, 0.45 μ m, 0.2 μ m and 0.1 μ m, then adopt the daltonian film of molecular cut off 30000 to carry out ultrafiltration, centrifugal concentrating obtains the near-infrared fluorescent of desired concn-core cross-linked polymeric micellar nanoparticles (NIRF-CCPM).Building-up process as shown in Figure 5.
Hydrophobic fluorescent dye is embedded in nanometer core, be conducive to improve the nanoparticle surface hydrophilic, reduce engulfing of macrophage.NIRF-CCPM nanoparticle provided by the invention has tumor uptake and longer blood retention time preferably, and the netted epithelial tissue picked-up such as liver spleen is low, and tumor imaging is very clear.Provide good platform and nano-carrier system for further carrying out the molecular targeted research of tumor.
The present invention also provides the method for the described cancer target nanoparticle of preparation, with chelating agen DTPA, NIRF-CCPM nanoparticle surface amino is modified, then with cross-linking agent GMBS, NIRF-CCPM nanoparticle surface amino is further modified, the gained nanoparticle, by sulfydryl and the coupling of RGD peptide, obtains.
The present invention also provides the application of described cancer target nanoparticle in preparation tumor bimodal developer.
The present invention also provides described cancer target nanoparticle preparing integrin alpha
vβ
3application in receptor tumor bimodal developer.
The present invention also provides a kind of bimodal cancer target nanoparticle developer, and it is with the described cancer target nanoparticle precursor that serves as a mark, and during use, carries out
111the In isotope labeling.Its preparation method comprises the following steps:
1) with NCS-Bz-DTPA, NIRF-CCPM nanoparticle surface amino is modified, obtain DTPA-NIRF-CCPM, then with cross-linking agent GMBS, nanoparticle surface amino is further modified, obtained DTPA-NIRF-CCPM-GMBS, and separate with the PD-10 post;
2) targeting modification of nanoparticle: the RGD peptide is carried out to sulfydryl modification, obtain RGD-SH, then RGD-SH is coupled to the nanoparticle surface that above-mentioned separation obtains, form cancer target nanoparticle DTPA-NIRF-CCPM-RGD(or NIRF-CCPM-RGD), precursor serves as a mark after separating with the PD-10 post;
3) labelling: DTPA-NIRF-CCPM-RGD is carried out
111the In isotope labeling, obtain bimodal cancer target nanoparticle developer,
111in-NIRF-CCPM-RGD.
Wherein, step 1) is specially: according to number of amino groups on the NIRF-CCPM surface, with the mol ratio 1:1-3 of NCS-Bz-DTPA, mix, lucifuge reaction 24h, then mix with the mol ratio 1:3-8 of GMBS according to the NIRF-CCPM nanoparticle, lucifuge reaction 2h; Having reacted rear separates with the PD-10 post.
Step 2) be specially: mix with the mol ratio 1:10-50 of RGD-SH according to the DTPA-NIRF-CCPM-GMBS nanoparticle, separate with the PD-10 post after having reacted.Nanoparticle is carried out to packing, with N
2sealing after protection, packing 0.5mL/ bag, standing under 4 ℃.
Step 3) is specially: in labelled precursor, add successively 0.1M NaAc(5-10 μ L), 185MBq
111inCl
3, room temperature reaction 20-30min, obtain
111in-NIRF-CCPM-RGD.Measure mark rate and radiochemical purity, when mark rate is less than 90%, need to separate with the PD-10 post gained
111the radiochemical purity of In-NIRF-CCPM-RGD is greater than 95%.
Before the PD-10 post is used, use through the past metal ion, 0.01M PBS buffer (pH7.4) drip washing.
Provided by the invention
111the SPECT video picture result of In-NIRF-CCPM-RGD shows,
111in-NIRF-CCPM-RGD can accurately locate α
vβ
3the receptor positive tumor, and high-visible in the nude mice that human glioma cell U87 kind is planted in 6-96h.
111the infrared video picture demonstration of In-NIRF-CCPM-RGD,
111in-NIRF-CCPM-RGD can accurately locate α
vβ
3the receptor positive tumor, and high-visible in the nude mice of human glioma cell (U87) plantation in 6-96h.
The present invention using there is good biology performance near-infrared core cross-linked polymeric micellar nanoparticles (NIRF-CCPM) as carrier, the polypeptide RGD(arginine-glycine-aspartic acid that will there is tumor-targeting) with the nanoparticle coupling, obtain the nano molecular image probe NIRF-CCPM-RGD of novel cancer target, and with radionuclide
111in carries out labelling, obtains
111in-NIRF-CCPM-RGD can with the α of endothelial cells in tumor neogenetic blood vessels
vβ
3the antigenic specificity combination, accurately locate α by infrared/nuclear medicine means respectively
vβ
3the tissue of high expressed, realize the targeted molecular diagnostic imaging purpose of tumor, carries out the research of different kinds of molecules image probe on the same molecule, to reach tumor, early finds, the target of diagnosis, early treatment early.
The accompanying drawing explanation
Fig. 1 is novel nano particle of the present invention
111the synthetic route of In-NIRF-CCPM-RGD.
Fig. 2 shows
111distribution (n=3) in the BALB/C nude mouse that In-NIRF-CCPM-RGD plants in the U87 kind.
Fig. 3 is
111sPECT video picture figure (arrow is designated as tumor) in the BALB/C nude mouse that In-NIRF-CCPM-RGD plants in the U87 kind.
Fig. 4 is
111infrared video picture figure in the BALB/C nude mice that In-NIRF-CCPM-RGD plants in the U87 kind (left figure means the blocking-up group of common injection 100 μ g RGD).
Fig. 5 is the synthetic schematic diagram of NIRF-CCPM nanoparticle.
The specific embodiment
Following examples are used for the present invention is described, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art, the raw materials used commercial goods that is.
Construction method list of references ([1] Z Yang of the near-infrared core cross-linked polymeric micellar nanoparticles (NIRF-CCPM) related in following examples, SY Zheng, WJ Harrison, J Harder, XX Wen, JG Gelovani, A Qiao, C Li*.Long-Circulating Near-Infrared Fluorescence Core-Cross-Linked Polymeric Micelles:Synthesis, Characterization, and Dual Nuclear/Optical Imaging.Biomacromolecules.2010,8,3422-3428).
The preparation method list of references of RGD-SH (Cai W, Chen X.Preparation of peptide-conjugated quantum dots for tumor vasculature-targeted imaging.Nat Protoc.2008; 3 (1): 89-96).
Embodiment 1 bimodal cancer target nanoparticle developer and preparation method thereof
Bimodal cancer target nanoparticle developer (
111in-NIRF-CCPM-RGD) preparation comprises the following steps:
(1) according to amino on the NIRF-CCPM surface, with the NCS-Bz-DTPA mol ratio, be that 1:1 mixes, lucifuge reaction 24h, obtain DTPA-NIRF-CCPM; Then according to NIRF-CCPM nanoparticle and GMBS mol ratio, be that 1:3 mixes, lucifuge reaction 2h, obtain DTPA-NIRF-CCPM-GMBS; Having reacted rear separates with the PD-10 post.Before the PD-10 post is used, use through the past metal ion, 0.01M PBS buffer (pH7.4) drip washing.
(2) be that 1:10 mixes according to DTPA-NIRF-CCPM-RGD nanoparticle and RGD-SH mol ratio, separate with the PD-10 post after having reacted, obtain the tumor imaging labelled precursor.Nanoparticle is carried out to packing, with N
2sealing after protection, packing 0.5mL/ bag, standing under 4 ℃.
(3) add successively 0.1M NaAc5-10 μ L, 185MBq to labelled precursor
111inCl
3, room temperature reaction 20-30min.Measure mark rate and radiochemical purity, when mark rate is less than 90%, need carry out the PD-10 column separating purification, obtain
111in-NIRF-CCPM-RGD.
111the synthetic route of In-NIRF-CCPM-RGD as shown in Figure 1.
The mensuration of mark rate adopts the instant thin-layer chromatography method.Quick silica gel thin-layer chromatography-the silica gel paper of system: ITLC-SG(); Developing solvent: 4mM EDTA, 0.01M PBS solution, pH7.4; Label is at initial point, free
111inCl
3ahead of the curve.The result demonstration, mark rate and radiochemical purity are greater than 95%.
Embodiment 2 bimodal cancer target nanoparticle developers and preparation method thereof
Preparation with embodiment 1, only by amino on NIRF-CCPM surface in step (1), with the NCS-Bz-DTPA mol ratio, change 1:3 into, NIRF-CCPM nanoparticle and GMBS mol ratio change 1:8 into, in step (2), DTPA-NIRF-CCPM-RGD nanoparticle and RGD-SH mol ratio change 1:50 into, the remaining reaction condition is identical
Measure mark rate and radiochemical purity, the result demonstration, mark rate and radiochemical purity are greater than 95%.
Preparation with embodiment 1, only by amino on NIRF-CCPM surface in step (1), with the NCS-Bz-DTPA mol ratio, change 1:2 into, NIRF-CCPM nanoparticle and GMBS mol ratio change 1:4 into, in step (2), DTPA-NIRF-CCPM-RGD nanoparticle and RGD-SH mol ratio change 1:25 into, the remaining reaction condition is identical
Measure mark rate and radiochemical purity, the result demonstration, mark rate and radiochemical purity are greater than 95%.
Embodiment 4 bimodal cancer target nanoparticle developer distributions in animal body
111in-NIRF-CCPM-RGD distribution is in animal body tested, and gets the BALC/C nude mice of the U87 tumor cell plantation of 25g left and right front oxter plantation, treats that tumor tissues grows to 0.5cm
3after, tail vein injection 1.11MBq/200 μ L's
111in-NIRF-CCPM-RGD(embodiment 1 preparation), respectively at 24h, 72h, 120h, put to death.Get respectively blood, the heart, liver,spleen,kidney, lung, stomach, small intestinal, large intestine, muscle, bone, brain, tumor and measure its radiocounting and corresponding weight, calculate %ID/g, concrete outcome is shown in Fig. 2.
Embodiment 5 bimodal cancer target nanoparticle developer SPECT video pictures in animal body
Get the BALC/C nude mice of the U87 tumor cell plantation of 25g left and right front oxter plantation, treat that tumor tissues grows to 0.8cm
3after, tail vein injection 18.5MBq/200 μ L's
111in-NIRF-CCPM-RGD(embodiment 1 preparation), respectively at 48h, 96h, 144h after injection, adopting the E.cam SPECT of Siemens instrument to carry out image acquisition, can peak be 246keV, can window be 20%, matrix 128 * 128, image is enlarged into 3, carries out the normotopia video picture.Observe
111the picked-up variation of In-NIRF-CCPM-RGD and whole body distribution situation, its video picture the results are shown in Figure 3.As seen from Figure 3, injection
111after In-NIRF-CCPM-RGD, all can see the video picture of tumor locus after 24h.In whole videograph process, except liver, other non-specific picked-ups are less.For this medicine is used for integrin alpha
vβ
3the video picture of receptor positive tumor provides support.
Embodiment 6 bimodal cancer target nanoparticle developer infrared video pictures in animal body
Get the BALC/C nude mice of the U87 tumor cell plantation of 25g left and right front oxter plantation, treat that tumor tissues grows to 0.8cm
3after, tail vein injection 18.5MBq/200 μ L's
111in-NIRF-CCPM-RGD(embodiment 1 preparation), after the SPECT video picture, adopt smart Nuo Zhen-infrared display of IVIS Lumina II to carry out infrared video picture in 96h, the visual field is 12.5cm, utilize ICG order (exciting light 710nm), CCD camera lens 1.3 * 1.3cm, 1024 * 1024 pixels, time of exposure 1s.Concrete outcome is shown in Fig. 4.As seen from Figure 4, when 96h, tumor locus has showed very significantly obviously, and other position is without any video picture.This result shows, infrared video picture is at integrin alpha
vβ
3effective application in the video picture of receptor positive tumor.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. the cancer target nanoparticle, is characterized in that, it has the NIRF-CCPM nanoparticle of RGD peptide for coupling; Described RGD peptide is arginine-glycine-aspartic acid.
2. the method for preparing the described nanoparticle of claim 1, it is characterized in that, with chelating agen DTPA, NIRF-CCPM nanoparticle surface amino is modified, then with cross-linking agent GMBS, NIRF-CCPM nanoparticle surface amino is further modified, the gained nanoparticle, by sulfydryl and the coupling of RGD peptide, obtains.
3. the application of the described nanoparticle of claim 1 in preparation tumor bimodal developer.
4. the described nanoparticle of claim 1 is preparing integrin alpha
vβ
3application in receptor tumor bimodal developer.
5. a bimodal cancer target nanoparticle developer, is characterized in that, with the nanoparticle claimed in claim 1 precursor that serves as a mark, during use, carries out
111the In isotope labeling.
6. prepare the method for the described developer of claim 5, it is characterized in that, comprise the following steps:
1) with NCS-Bz-DTPA, NIRF-CCPM nanoparticle surface amino is modified, obtain DTPA-NIRF-CCPM, then with cross-linking agent GMBS, nanoparticle surface amino is further modified, obtained DTPA-NIRF-CCPM-GMBS, and separate with the PD-10 post;
2) targeting modification of nanoparticle: the RGD peptide is carried out to sulfydryl modification, obtain RGD-SH, then RGD-SH is coupled to the nanoparticle surface that above-mentioned separation obtains, forms cancer target nanoparticle DTPA-NIRF-CCPM-RGD, precursor serves as a mark after separating with the PD-10 post;
3) labelling: DTPA-NIRF-CCPM-RGD is carried out
111the In isotope labeling, obtain bimodal cancer target nanoparticle developer,
111in-NIRF-CCPM-RGD.
7. method according to claim 6, it is characterized in that, step 1) is specially: according to number of amino groups on the NIRF-CCPM surface, with the mol ratio 1:1-3 of NCS-Bz-DTPA, mix, lucifuge reaction 24h, then mix lucifuge reaction 2h with the mol ratio 1:3-8 of GMBS according to the NIRF-CCPM nanoparticle; Having reacted rear separates with the PD-10 post.
8. method according to claim 6, is characterized in that step 2) be specially: mix with the mol ratio 1:10-50 of RGD-SH according to the DTPA-NIRF-CCPM-GMBS nanoparticle, separate with the PD-10 post after having reacted.
9. method according to claim 6, is characterized in that, step 3) is specially: in labelled precursor, add successively 0.1M NaAc, 185MBq
111inCl
3, room temperature reaction 20-30min, obtain
111in-NIRF-CCPM-RGD.
10. method according to claim 6, is characterized in that,
111the radiochemical purity of In-NIRF-CCPM-RGD is greater than 95%.
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| CN105249929A (en) * | 2015-11-06 | 2016-01-20 | 广西大学 | Breast tumor blood oxygen nano enhancing functional imaging and examination system |
| CN107899027A (en) * | 2017-11-07 | 2018-04-13 | 北京肿瘤医院 | Neuroendocrine tumor targeted nano-particle developer and preparation method and application |
| CN112933249A (en) * | 2021-03-26 | 2021-06-11 | 福建医科大学附属第一医院 | PD-L1 targeted bimodal molecular probe and preparation method and application thereof |
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| HUA ZHU等: "Design, Synthesis and Evaluation of Dual-Modality Glyco-Nanoparticles for Tumor Imaging", 《MOLECULES》, 30 May 2013 (2013-05-30), pages 6425 - 6438 * |
| 杨志: "肿瘤靶向纳米探针NIRF-CCPM-RGD的多功能分子显像研究（2010年获批国家自然基金项目）", 《网页，HTTP://RESEARCH.BJMU.EDU.CN/ART/2011/3/15/ART_1921_55617.HTML》, 15 March 2011 (2011-03-15), pages 1 - 2 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105249929A (en) * | 2015-11-06 | 2016-01-20 | 广西大学 | Breast tumor blood oxygen nano enhancing functional imaging and examination system |
| CN107899027A (en) * | 2017-11-07 | 2018-04-13 | 北京肿瘤医院 | Neuroendocrine tumor targeted nano-particle developer and preparation method and application |
| CN112933249A (en) * | 2021-03-26 | 2021-06-11 | 福建医科大学附属第一医院 | PD-L1 targeted bimodal molecular probe and preparation method and application thereof |
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