CN107281504A - A kind of preparation method of the SPECT/CT bimodal image-forming contrast mediums based on second generation polyamide-amine dendrimer - Google Patents

A kind of preparation method of the SPECT/CT bimodal image-forming contrast mediums based on second generation polyamide-amine dendrimer Download PDF

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CN107281504A
CN107281504A CN201710434129.7A CN201710434129A CN107281504A CN 107281504 A CN107281504 A CN 107281504A CN 201710434129 A CN201710434129 A CN 201710434129A CN 107281504 A CN107281504 A CN 107281504A
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rgd
nota
peg
spect
preparation
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沈明武
许晓迎
李鑫
王鹏
史向阳
赵凌舟
赵晋华
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Shanghai First Peoples Hospital
Donghua University
National Dong Hwa University
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Donghua University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/06—Macromolecular compounds, carriers being organic macromolecular compounds, i.e. organic oligomeric, polymeric, dendrimeric molecules
    • A61K51/065—Macromolecular compounds, carriers being organic macromolecular compounds, i.e. organic oligomeric, polymeric, dendrimeric molecules conjugates with carriers being macromolecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/04—X-ray contrast preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
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    • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
    • A61K51/0482—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group chelates from cyclic ligands, e.g. DOTA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/082—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins the peptide being a RGD-containing peptide

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Abstract

The present invention relates to a kind of preparation method of the SPECT/CT bimodal image-forming contrast mediums based on second generation polyamide-amine dendrimer, including：Using second generation polyamide-amine dendrimer as mould material, radionuclide is modified successively on its surface99mTc chelating agents NOTA and Pegylation RGD molecules, then by in-situ reducing synthetic method parcel gold nano grain, finally mark radionuclide99mTc, is produced.SPECT/CT contrast agent prepared by the present invention possesses good colloidal stability, excellent biocompatibility, longer blood circulation time and good inside and outside SPECT/CT imaging effects, has α to cell membrane surfacevβ3The C6 cells of integrin receptor have selectively targeted effect, and good thinking is provided to develop a kind of new bimodal nano-contrast agent.

Description

A kind of SPECT/CT bimodals based on second generation Polyamidoamine Dendrimers into As the preparation method of contrast agent
Technical field
It is more particularly to a kind of based on second generation Polyamidoamine Dendrimers the invention belongs to contrast preparation field The preparation method of SPECT/CT bimodal image-forming contrast mediums.
Background technology
SPECT or PET picture reproducers can receive the ray that radionuclide is sent in nucleus medical image, can clearly observe The function signal in organism in terms of various Physiology and biochemistries and metabolism is participated in contrast agent, qualitative, quantitative and positioning face is obtained Bed diagnosis of disease result.This imaging modality can reflect dirty compared with traditional morphology imaging method (CT, B ultrasound, MRI) The blood flow change of device or tissue, Rd and activity change, metabolism and changes of function, as the most advanced of clinical diagnosis and treatment disease One of technology.Meanwhile, there is also spatial resolution is low and be unable to the shortcoming of expliciting the position imaging anatomy position for nuclear medicine. PET/CT and SPECT/CT bimodals imaging technique can obtain internal SPECT functional metabolism information and CT anatomical diagnosis simultaneously The image co-registration of information and diagnostic multiplication (Delbeke et al., J.Nucl.Med.2006,47 (7), 1227-1234). Therefore, compared with single SPECT or CT, SPECT/CT more has clinic in terms of diagnosing the illness and evaluating prognosis Application value.
Although SPECT/CT bimodals image documentation equipment clinically has great popularization, corresponding SPECT/ The development of CT bimodal image-forming contrast mediums does not obtain enough attention also.Although two kinds of image-forming contrast mediums of CT and SPECT Use can complete corresponding contrast imaging, but can be brought inconvenience to clinical manipulation, can also increase poison of the contrast agent to patient Side effect, while being difficult to coordinate between also there is contrast agent, is respectively present the phenomenon of difference in vivo.Therefore, a kind of many work(are developed The contrast preparation system of energy is diagnosed suitable for SPECT/CT bimodals imaging contrast, it will be greatly enhanced sensitivity and the standard of diagnosis Exactness, and less pain and toxic side effect are caused to patient, there will be very big application value in clinic.
The dendrimer of low algebraically because its algebraically is low and the features such as there is open structure and less surface group. Liu etc. (Liu et al., Chem.-Eur.J.2013,19,6409-6416) is made with second generation Polyamidoamine Dendrimers Gold nano grain (＞ 5nm) is prepared for stabilizer and folate-targeted agent is modified, and realizes and identification is imaged to the CT of tumor tissues.Cao Second generation Polyamidoamine Dendrimers bag is prepared for Deng (Cao etal., J.Mater.Chem.B, 2015,3,286-295) Wrapping up in gold nano grain has the CT nano-contrast agents of lactobionic acid liver cancer targeting function, with good selectively targeted function and good Good CT imaging effects.History face south etc. (history faces south, Wen Shihui, Zhao Jinhua, Zhao Ling boat functionalization based on dendrimer SPECT-CT bimodal image-forming contrast mediums and preparation method thereof：China, 201410271238.8 [P] .2014.06.18) with the 5th Gold nano grain is wrapped up for dendrimer and is marked99mTc, realize the SPECT/CT at different tissues position in Mice Body into Picture, and imaging effect is good.
Retrieval shows about the document and patent results in terms of SPECT/CT bimodal contrast agent both at home and abroad：At present, do not have also It is found and has modified chelating agent NOTA and polypeptide RGD second generation Polyamidoamine Dendrimers and wrapped up gold nano grain simultaneously Radionuclide is chelated99mReport in terms of the preparation and application of Tc SPECT/CT bimodal image-forming contrast mediums.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of based on second generation Polyamidoamine Dendrimers The preparation method of SPECT/CT bimodal image-forming contrast mediums, contrast agent preparation technology is simple, and reaction condition is gentle, it is easy to operate Separation, used is environment-friendly material, with the commercialized prospect of implementation.
A kind of SPECT/CT bimodal image-forming contrast mediums based on second generation Polyamidoamine Dendrimers of the present invention Preparation method, including：
(1) by second generation Polyamidoamine Dendrimers G2.NH2And radionuclide99mTc chelating agents NOTA difference is molten In dimethyl sulfoxide (DMSO) DMSO, after being well mixed, lasting stirring reaction 24~28 hours, dialysis, after freeze-drying at room temperature Obtain the second generation Polyamidoamine Dendrimers of chelating agent NOTA modifications, i.e. G2-NOTA；
(2) it is the peg molecule COOH-PEG-MAL and polypeptide that the carboxyl other end is maleimide base group by one end RGD is dissolved in dimethyl sulfoxide (DMSO) DMSO respectively, after being well mixed, stirring reaction 24~28 hours, and dialysis is obtained after freeze-drying The peg molecule of polypeptide RGD modifications, i.e. RGD-PEG-COOH；
(3) RGD-PEG-COOH is dissolved in DMSO, is firstly added 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides Hydrochloride EDC is activated, and is then added n-hydroxysuccinimide NHS and is continued to activate, then by the polyglycol solution after activation It is added dropwise in G2-NOTA DMSO solution, in stirring reaction under room temperature condition 72~76 hours, finally dialyses, freezing is dry The second generation Polyamidoamine Dendrimers of RGD modifications, i.e. G2-NOTA-PEG-RGD are obtained after dry；
(4) above-mentioned G2-NOTA-PEG-RGD is dissolved in water, adds aqueous solution of chloraurate, stir at ambient temperature 30~60 minutes, the sodium borohydride aqueous solution of precooling is then added, stirring reaction is dialysed after 3~5 hours in frozen water, freezing RGD modifications are obtained after drying and the second generation Polyamidoamine Dendrimers of gold nano grain, i.e. G2-NOTA-PEG- have been wrapped up RGD@Au；
(5) above-mentioned G2-NOTA-PEG-RGD@Au are dissolved in radiopertechnetate solution, obtain RGD modifications and wrap Wrap up in gold nano grain and chelate radionuclide99mTc SPECT/CT bimodal image-forming contrast mediums, i.e. G2-NOTA (99mTc)- PEG-RGD@Au。
G2.NH in the step (1)2Mol ratio with NOTA is 1:4.
The mol ratio of COOH-PEG-MAL and RGD in the step (2) are 1:1.2.
RGD-PEG-COOH in the step (3) is dissolved in the concentration after DMSO for 6mg/mL；G2-NOTA DMSO solution Concentration be 4.5mg/mL.
The mol ratio of RGD-PEG-COOH, EDC, NHS and G2-NOTA in the step (3) are 10:50:50:1.
The mol ratio of sodium borohydride, gold chloride and G2-NOTA-PEG-RGD in the step (4) is 30:6:1.
The quality of G2-NOTA-PEG-RGD@Au in the step (5) and the exit dose of radiopertechnetate solution Ratio is 28mg:35-78MBq.
Dialysis in step (1)~(4) is specially：With the bag filter dialysis 3 that molecular cut off is 1000~3000 My god, dialysis distilled water 2L used, changes water 9 times altogether every time.
Present invention use proton nmr spectra (1H NMR), ultraviolet-visible absorption spectroscopy (UV-Vis), high-resolution transmission electricity The methods such as sub- microscope (TEM), inductively coupled plasma emission spectrography (ICP-OES), Zeta electric potential, hydration particle diameter are characterized The physicochemical properties of material, are then evaluated by Cell Counting Kit-8 (CCK-8) methods and fluorescence inverted microscope The cell compatibility of material, is finally imaged to characterize the low with bimodal radiography function of preparation using inside and outside SPECT/CT Algebraically dendrimer is to tumour cell and the diagnosis effect of tumor tissues.
The present invention utilizes the specific structure and property of second generation Polyamidoamine Dendrimers, by radionuclide99mTc Chelating agent NOTA is grafted on dendrimer surface to chelate99mTc realizes that SPECT is imaged, and is then grafted RGD-PEG-COOH Its biocompatibility can be both improved on dendrimer surface, extends blood circulation time, it is possessed to α againvβ3Integrin The function of receptor-specific targeting, is finally imaged in dendrimer internal package nanogold particle for CT, so as to prepare The SPECT/CT bimodal contrast agent of premium properties, realizes the contrast agent demand of different imaging techniques.
Beneficial effect
(1) present invention using gentle reaction condition prepare G2-NOTA (99mTc)-PEG-RGD@Au nano particles are used for SPECT/CT bimodal image-forming contrast mediums, preparation technology is simple, and reaction condition is gentle, it is easy to which operation separation, used is environment Friendly materials, with the commercialized prospect of implementation；
(2) G2-NOTA of the invention (99mTc)-PEG-RGD@Au nano particles can be stably dispersed in water for a long time, Be not in reunion or deposited phenomenon, show good SPECT/CT imaging effects in testing in vitro, be multi-functional make The exploitation of shadow agent is laid a good foundation；
(3) G2-NOTA of the invention (99mTc)-PEG-RGD@Ac granules in rats neuroglial cytoma has preferable Selectively targeted effect, is that a kind of potential bimodal is imaged new radiography while having SPECT and CT imaging advantage Agent.
Brief description of the drawings
The intermediate product that Fig. 1 is prepared for the present invention1H NMR, wherein a are G2-NOTA, and b is RGD-PEG-COOH, and c is G2-NOTA-mPEG, d are G2-NOTA-PEG-RGD；
The G2-NOTA-mPEG and G2-NOTA-PEG-RGD that Fig. 2 is prepared for the present invention are before and after parcel gold nano grain UV-Vis spectrograms；
G2-NOTA-PEG-RGD@Au (a and c) and G2-NOTA-mPEG@Au (b and d) nanometer that Fig. 3 is prepared for the present invention UV-Vis spectrogram of the particle in the range of different temperatures (4-60 DEG C) and pH (5-8)；
Fig. 4 for the present invention prepares G2-NOTA-mPEG@Au and G2-NOTA-PEG-RGD@Au nano particles TEM scheme (a, C) with grain size distribution (b, d)；
Fig. 5 is that G2-NOTA-PEG-RGD Au nano particles (2) prepared by medical Iohexol (1) and the present invention are dense in material Spend to scan obtained CT images (a) and corresponding signal value changes (b) by CT imagers in the range of 0.005 to 0.04M； Fig. 6 is that CCK-8 methods measure C6 cells by PBS, G2-NOTA-PEG-RGD@Au and control material G2-NOTA-mPEG@ Cell viabilities of the Au after being handled 24 hours under gold concentration is 200 to 4000nM；
Fig. 7 is that C6 cells pass through PBS, G2-NOTA-PEG-RGD@Au (targeting) and control material G2-NOTA- MPEG@Au (non-targeted) are in the Fluorescence microscopy Cells shape appearance figure that gold concentration is respectively under 200 to 4000nM after processing 24 hours；
Fig. 8 for the G2-NOTA-PEG-RGD@Au particles that prepare of the present invention respectively with αvβ3The C6 of the high and low expression of integrin is thin Born of the same parents and G2-NOTA-mPEG@Au particles and αvβ3After the C6 cells of integrin height expression are co-cultured 4 hours, the gold of cell phagocytosis Content；
The G2-NOTA-PEG-RGD@Au (targeting) and control material G2-NOTA-mPEG@Au that Fig. 9 is prepared for the present invention are (non- Targeting) cell suspending liquid of post processing in 3 hours is co-cultured with C6 cells, by CT imagers scan the CT images (a) that measure and Corresponding signal value changes (b)；Wherein, 1-3 refers to G2-NOTA-mPEG@Au, RGD blockings+G2-NOTA-PEG- respectively in Fig. 9 a RGD@Au and G2-NOTA-PEG-RGD@Au；
Figure 10 for the present invention prepare G2-NOTA (99mTc)-PEG-RGD@Au (a-d) and control material G2-NOTA(99mTc) the radiochemical purity analysis of-mPEG@Au (e-h) different time points (0,1,6,12 hour) after mark；
Figure 11 for the present invention prepare G2-NOTA (99mTc)-PEG-RGD@Au (targeting) and control material G2-NOTA (99mTc)-mPEG@Au (non-targeted) co-culture the cell suspending liquid of post processing in 3 hours with C6 cells, are swept by SPECT imagers Retouch the radioactive dosage that 1-6 in obtained SPECT images (a) and corresponding signal value changes (b), wherein Figure 11 a refers to material The μ Ci of respectively 37.25,72.5,125,150,300 and 600；
Figure 12 is preparation principle figure of the invention.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limitation the scope of the present invention.In addition, it is to be understood that after the content of the invention lectured has been read, people in the art Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited Scope.
Embodiment 1
(1) by 20.0mg second generation polyamide-amine dendrimers (G2.NH2) be dissolved in 3.0mL dimethyl sulfoxide (DMSO)s, and 3.0mL radionuclides are added dropwise99mTc chelating agents NOTA dimethyl sulphoxide solution (5.5mg/mL), is stirred at room temperature Reaction 24 hours, 3 days (dialysis distilled water 2L used, changes water 9 times altogether every time) is dialysed with molecular cut off for 1000 bag filter, G2-NOTA is obtained after freeze-drying.1H NMR test results show：Appear in 2.3-3.2ppm's and 3.3-3.4ppm in spectrogram Chemical shift peak corresponds to G2.NH respectively2With NOTA characteristic peak.It can be calculated by integral area, each G2.NH2Surface modification About 3.1 NOTA (Fig. 1 a).
(2) 26.9mg polypeptides RGD and 65mg COOH-PEG-MAL are mixed in DMSO solution, stirring reaction 24 hours, COOH-PEG-MAL is obtained, being dialysed 3 days with the bag filter that molecular cut off is 1000, (dialysis distilled water 2L used, is changed altogether every time Water 9 times), the polyethylene glycol of rgd peptide modification is obtained after freeze-drying；Wherein, SH-RGD：COOH-PEG-MAL mol ratio is 1：1.2；1H NMR test results show：The chemical shift peak that 3.6ppm and 7.1-7.4ppm is appeared in spectrogram is corresponded to respectively The characteristic peak of polyethylene glycol and RGD.It can be calculated by integral area, about 0.6 RGD molecule of each polyethylene glycol surface modification (Fig. 1 b).
(3) (2) products obtained therefrom 50mg is dissolved in 5mL DMSO, 5mL EDC DMSO solution is added while stirring (7.7mg/mL) is activated 30 minutes, 5mL NHS DMSO solution (4.6mg/mL) activation 3 hours is and then added dropwise, then In the DMSO solution (5.0mg/mL) for being added dropwise to 2mL (1) products obtained therefrom, stirring reaction 72 hours under room temperature condition.Reaction terminates Afterwards, it is 3000 bag filter dialysis 3 days (dialysis distilled water 2L used, changes water 9 times altogether every time), freeze-drying with molecular cut off The dendrimer G2-NOTA-PEG-RGD of NOTA and RGD- polyethylene glycol has been modified afterwards.1H NMR test results show： The chemical shift peak that 3.6ppm and 2.3-3.2ppm is appeared in spectrogram corresponds to the spy of polyethylene glycol and dendrimer respectively Peak is levied, 7.1-7.4ppm chemical shift peak corresponds to RGD characteristic peak.It can be calculated by integral area, it is each tree-shaped big point About 7.0 RGD-PEG (Fig. 1 d) of sub- surface modification.
(4) (3) products obtained therefrom 26mg is dissolved in 26mL ultra-pure waters, 312.14 μ L aqueous solution of chloraurate is added while stirring (10mg/mL), magnetic agitation adds the sodium borohydride aqueous solution (19mg/mL) of 75.78 μ L ice baths pretreatment, room after 30 minutes Temperature reaction 3 hours.After reaction terminates, with 3 days (distilled water used of dialysing every time of the bag filter dialysis that molecular cut off is 1000 2L, changes water 9 times altogether), obtain wrapping up the dendrimer for having modified NOTA and RGD-PEG of gold nano grain after freeze-drying G2-NOTA-PEG-RGD@Au.There is ultraviolet feature at 500-550nm in the G2-NOTA-PEG-RGD@Au particles prepared Absworption peak, but due to gold filled amount less and size of nanometer gold grain it is small (<2nm) cause its surface plasma body resonant vibration (SPR) peak not Substantially (Fig. 2).This shows that G2-NOTA-PEG-RGD prepared by the present invention has successfully wrapped up gold nano grain.TEM test result tables It is bright：The Size Distribution of the gold nano grain prepared is narrower, with good water dispersible, and average grain diameter is about 1.87nm (Fig. 4 c, 4d).
(5) (4) products obtained therefrom 28mg is dissolved in the sterile radioactivity Gao Technetium hydrochlorate (radioactivity of 0.10mL99mTc concentration is 740MBq/mL) solution, vibration mixing obtains parcel golden nanometer particle and chelates nucleic99mTc SPECT/CT bimodals imaging Contrast agent G2-NOTA (99mTc)-PEG-RGD@Au。
Embodiment 2
Stability of material test result
The aqueous solution (0.4mg/mL) of (4) products obtained therefrom in Example 1, after being placed in different temperatures lower 0.5 hour, is determined Its uv-spectrogram, determines the stability of product at different temperatures.In addition, in Example 1 (4) products obtained therefrom the aqueous solution (0.4mg/mL) is respectively the solution that pH is 5-8 with NaAc_HAc buffer solution and PBS buffer preparations, determines it purple Outer collection of illustrative plates, determines stability of the product under different pH.As a result show：In fig. 3 it can be seen that prepared in the present invention G2-NOTA-PEG-RGD@Au (Fig. 3 a and 3c) and control material G2-NOTA-mPEG@Au (Fig. 3 b and 3d) nano particle are in 500- Existing characteristics absworption peak at 550nm, in the range of certain temperature (4 DEG C -60 DEG C) and pH (5-8), absworption peak does not occur substantially Skew, this shows that the G2-NOTA-PEG-RGD@Au of the invention prepared and control material G2-NOTA-mPEG@Au nano particles exist There is good colloidal stability in the range of this temperature and pH.
Embodiment 3
(4) products obtained therefrom prepares the mother liquor that gold concentration is 0.04M using distilled water as solvent in Example 1.Gradient afterwards Dilute 0.02,0.01 and 0.005M sample.Meanwhile, with Iohexol it is that control material plus distilled water diluting go out accordingly using clinic The sample of iodine concentration, and CT imaging tests are carried out respectively to two groups of materials.Test result is shown：Prepared in the present invention G2-NOTA-PEG-RGD@Au particles show to decline better than the CT imaging effects (Fig. 5 a) and X-ray of traditional contrast agent Iohexol Subtract coefficient (Fig. 5 b), show that it possesses good potential CT contrast abilities.
Embodiment 4
(4) products obtained therefrom is configured to 40000nM mother liquor with sterile PBS buffer in Example 1.It is diluted to afterwards 10000nM sample solution.It is another that G2-NOTA- obtained by (2) in the comparative example 1 without targeted molecular RGD is prepared with same scheme MPEG@Au sample solution.Cultured C6 cells kind is taken in 96 orifice plates, is inoculated with according to the density of 10,000 cells/wells, per hole The μ L of volume 200.After overnight incubation, the sample of above-mentioned each dilution gradient is added, is co-cultured 24 hours with cell.Each gradient training Nutrient solution dilutes 10 times, i.e., per hole gold final concentration be respectively 200,500,1000,2000,4000nM.Each gradient do 5 it is parallel Hole, blank control is used as using PBS.Then with CCK-8 methods detection cell viability, CCK-8 solution, 37 DEG C of hatchings are added per hole 4 hours.Afterwards absorbance at 450nm is detected with ELIASA.CCK-8 test results show, G2-NOTA-PEG-RGD@Au and G2- NOTA-mPEG@Au nano particles do not show cytotoxicity within this range, and cell survival rate is held in more than 94%, Show good cell compatibility (Fig. 6).Equally, G2-NOTA-PEG-RGD the@Au and G2-NOTA- of various concentrations are taken MPEG@Au nano materials (gold concentration is respectively 200,500,1000,2000,4000nM) co-cultured 24 hours with C6 cells, with PBS is control.Then, 20 μ LCalcein-AM fluorescent dyeing reagents are dispersed in the culture medium of 8mL serum-frees, 200 μ LCalcein-AM solution are added into each cultivation plate hole, continues to dye 15 minutes at 37 DEG C, uses PBS Calcein-AM solution is washed away, and adds in each hole the culture medium of 200 μ L serum-frees, in fluorescence microscopy Microscopic observation, with Verify whether the material prepared can produce influence to cellular morphology.As shown in fig. 7, the G2-NOTA-PEG- of various concentrations Cellular morphology after RGD@Au are handled 24 hours with G2-NOTA-mPEG@Au is compared with the cell after PBS processing, is not had Obvious change, further illustrates the good cell compatibility of the material of synthesis.
Embodiment 5
(4) products obtained therefrom is configured to the mother liquor that concentration is 40000nM with sterile PBS buffer in Example 1, dilute afterwards It is interpreted as 10000nM sample solution.Cultured C6 cells kind is taken in 12 orifice plates, is inoculated with according to the density of 200,000 cells/wells, It is 1mL per pore volume.After overnight incubation, one group of C6 cell adds 2 μM of RGD co-cultivations, and (C6 for being defined as RGD blockings is thin within 3 hours Born of the same parents).Then, the material of above-mentioned each dilution gradient is added, is co-cultured 4 hours with cell, another group of C6 cell does not add RGD to train altogether Support and be defined as αvβ3The C6 cells of integrin receptor height expression.Then, add after above-mentioned 10 times of dilution sample (4000nM and 1000nM) respectively with αvβ3The C6 cells of the high and low expression of integrin receptor are co-cultured 3 hours at 37 DEG C.(2) institute in comparative example 1 G2-NOTA-mPEG@Au be also configured to sterile PBS buffer concentration for 4000 and 1000nM respectively with αvβ3Integrin receptor The C6 cells of height expression are co-cultured 3 hours at 37 DEG C.Blank control is used as using PBS.Culture uses PBS after terminating 3 times, then pancreatin digestion collected after centrifugation cell, add 2mL chloroazotic acid and digest 24 hours, then detected by ICP-OES in cell The phagocytosis amount of Au elements.As shown in figure 8, in research concentration range, the α of G2-NOTA-PEG-RGD@Au culturesvβ3Integrin by The C6 cells of body height expression (non-RGD block) are to the phagocytosis amount of gold apparently higher than low αvβ3Integrin receptor expresses (being blocked by RGD) C6 cells and G2-NOTA-mPEG@Au to αvβ3The phagocytosis amount of the C6 cells of integrin receptor height expression.As a result show, target Molecule RGD modification causes G2-NOTA-PEG-RGD@Au to αvβ3The C6 cells of integrin receptor height expression have special target energy Power.
Embodiment 6
(4) products obtained therefrom is configured to the mother liquor that concentration is 40000nM with sterile PBS buffer in Example 1, dilute afterwards It is interpreted as 10000nM sample solution.Cultured C6 cells kind is taken in 6 orifice plates, is inoculated with according to the density of 2,000,000 cells/wells, It is 2mL per pore volume.After overnight incubation, the sample (4000nM and 1000nM) added after above-mentioned dilution 10 respectively with αvβ3Integrate The C6 cells of the plain high and low expression of acceptor are co-cultured 3 hours at 37 DEG C.G2-NOTA-mPEG@Au obtained by (2) in comparative example 1 It is 4000nM and 1000nM solution that concentration is configured to sterile PBS buffer, then respectively with αvβ3Integrin receptor height expression C6 cells co-cultured 3 hours at 37 DEG C.Blank control is used as using PBS.Pancreatin digests collected after centrifugation cell, and Cell is uniformly dispersed in 200 μ L PBSs.Afterwards, CT imaging tests are carried out to this three groups of samples.Such as Fig. 9 institutes Show, in research concentration range, the α of G2-NOTA-PEG-RGD@Au culturesvβ3The CT values of the C6 cells of integrin receptor height expression Apparently higher than low αvβ3The C6 cells of integrin receptor expression and the α of G2-DTPA-mPEG@Au culturesvβ3The high table of integrin receptor The C6 cells reached.Targeted molecular RGD modification is demonstrated so that G2-NOTA-PEG-RGD@Au are to αvβ3Integrin receptor height expression C6 cells have a special target ability, and can be used as and be used for the potential contrast agent that internal CT is imaged.
Embodiment 7
In Example 1 obtained by (5) G2-NOTA (99mTc) G2-NOTA obtained by (3) in-PEG-RGD@Au and comparative example 1 (99mTc)-mPEG Au gel filtration column separating purifications, its mark rate is 70%, is then existed respectively99m0,1,6 after Tc marks, Carry out radiochemical purity analysis (Figure 10) within 12 hours.12 hours after mark, G2-NOTA (99mTc)-PEG-RGD@Au are put Penetrate chemical purity and be still maintained at more than 98.909%, show that it has good radiostability.
Embodiment 8
In Example 1 obtained by (5) G2-NOTA (99mTc)-PEG-RGD@Au are configured to difference with sterile PBS buffer The sample solution (37.25,72.5,125,150,300 and 600 μ Ci) of radiological dose.It is another to prepare to be free of RGD targets with same scheme Into the comparative example 1 of molecule G2-NOTA obtained by (3) (99mTc)-mPEG@Au sample solution.Cultured C6 cells kind is taken in 6 In orifice plate, it is inoculated with according to the density of 2,000,000 cells/wells, is 2mL per pore volume.After overnight incubation, different radiological doses are added Above two material respectively with αvβ3The C6 cells of integrin receptor height expression are co-cultured 3 hours at 37 DEG C.Pancreatin digestion centrifugation After collect cell, and cell is uniformly dispersed in 200 μ L PBSs.Afterwards, this two groups of samples are carried out SPECT into As test (Figure 11 a) and corresponding signal value analysis (Figure 11 b).In research concentration range, G2-NOTA (99mTc)-PEG- The α of RGD@Au culturesvβ3Integrin receptor height expression C6 cells SPECT imaging effects apparently higher than G2-NOTA (99mTc)- The α of mPEG@Au culturesvβ3The C6 cells of integrin receptor height expression.Targeted molecular RGD modification is demonstrated so that G2-NOTA (99mTc)-PEG-RGD@Au have more preferable SPECT imaging effects to the C6 cells of RGD acceptors height expression.
Comparative example 1
(1) 60mg polyethylene glycol (Mw=2000, mPEG-COOH) is dissolved in 10mL DMSO, added dropwise while stirring The DMSO solution (11.5mg/mL) for entering 5mL EDC is activated 30 minutes, and 5mL NHS DMSO solution is and then added dropwise (6.7mg/mL) is activated 3 hours, and then (1) products obtained therefrom 20mg in embodiment 1 is dissolved in 5mL ultra-pure waters, and by after activation Polyglycol solution be added dropwise to wherein, in stirring reaction under room temperature condition 72 hours, be then with molecular cut off 2000 bag filter is dialysed 3 days (dialysis distilled water 2L used, changes water 9 times altogether every time), and poly- second has been modified after freeze-drying The dendrimer G2-NOTA-mPEG of glycol.1H NMR test results show：3.6ppm and 2.3- are appeared in spectrogram 3.2ppm chemical shift peak corresponds to the characteristic peak of polyethylene glycol and dendrimer respectively.It can be calculated by integral area, About 7.1 peg molecules (Fig. 1 c) of each dendrimer surface modification.
(2) (1) products obtained therefrom 22mg is dissolved in 22mL ultra-pure waters, the μ L of aqueous solution of chloraurate 296.62 is added while stirring (10mg/mL), magnetic agitation adds the μ L (19mg/mL) of sodium borohydride solution 72.02 of ice bath pretreatment, room temperature after 30 minutes Reaction 3 hours.After reaction terminates, with molecular cut off for 2000 bag filter dialyse 3 days (dialysis distilled water 2L used every time, Water is changed altogether 9 times), obtain wrapping up the dendrimer G2-NOTA- for having modified polyethylene glycol of gold nano grain after freeze-drying mPEG@Au.TEM test results show：The Size Distribution of the gold nano grain prepared is narrower, is dissipated with good moisture Property, average grain diameter about 1.91nm (Fig. 4 a and 4b).
(3) (2) products obtained therefrom 25mg is dissolved in the sterile radioactivity Gao Technetium hydrochlorate (radioactivity of 100 μ L99mTc concentration is 740MBq/mL) solution, obtains parcel golden nanometer particle chelating nucleic99mTc SPECT/CT bimodal image-forming contrast mediums G2- NOTA(99mTc)-mPEG@Au。

Claims (8)

1. a kind of preparation method of the SPECT/CT bimodal image-forming contrast mediums based on second generation Polyamidoamine Dendrimers, Including：
(1) by second generation Polyamidoamine Dendrimers G2.NH2And radionuclide99mTc chelating agents NOTA is dissolved in diformazan respectively In base sulfoxide DMSO, after being well mixed, persistently stirring reaction 24~28 hours, dialysis, chela is obtained after freeze-drying at room temperature The second generation Polyamidoamine Dendrimers of mixture NOTA modifications, i.e. G2-NOTA；
(2) it is the peg molecule COOH-PEG-MAL and polypeptide RGD that the carboxyl other end is maleimide base group by one end It is dissolved in respectively in dimethyl sulfoxide (DMSO) DMSO, after being well mixed, stirring reaction 24~28 hours, dialysis obtains many after freeze-drying The peg molecule of peptide RGD modifications, i.e. RGD-PEG-COOH；
(3) RGD-PEG-COOH is dissolved in DMSO, is firstly added 1- (3- dimethylamino-propyls) -3- ethyl carbodiimide hydrochlorides Salt EDC is activated, and is then added n-hydroxysuccinimide NHS and is continued to activate, then by the polyglycol solution after activation dropwise It is added in G2-NOTA DMSO solution, in stirring reaction under room temperature condition 72~76 hours, finally dialyses, after freeze-drying Obtain the second generation Polyamidoamine Dendrimers of RGD modifications, i.e. G2-NOTA-PEG-RGD；
(4) above-mentioned G2-NOTA-PEG-RGD is dissolved in water, add aqueous solution of chloraurate, at ambient temperature stir 30~ 60 minutes, the sodium borohydride aqueous solution of precooling is then added, stirring reaction is dialysed after 3~5 hours in frozen water, freeze-drying RGD modifications are obtained afterwards and have wrapped up the second generation Polyamidoamine Dendrimers of gold nano grain, i.e. G2-NOTA-PEG-RGD@ Au；
(5) above-mentioned G2-NOTA-PEG-RGD@Au are dissolved in radiopertechnetate solution, obtain RGD modifications and parcel gold Nano particle and radionuclide is chelated99mTc SPECT/CT bimodal image-forming contrast mediums, i.e. G2-NOTA (99mTc)-PEG- RGD@Au。
2. a kind of SPECT/CT bimodals based on second generation Polyamidoamine Dendrimers according to claim 1 into As the preparation method of contrast agent, it is characterised in that：G2.NH in the step (1)2Mol ratio with NOTA is 1:4.
3. a kind of SPECT/CT bimodals based on second generation Polyamidoamine Dendrimers according to claim 1 into As the preparation method of contrast agent, it is characterised in that：The mol ratio of COOH-PEG-MAL and RGD in the step (2) are 1: 1.2。
4. a kind of SPECT/CT bimodals based on second generation Polyamidoamine Dendrimers according to claim 1 into As the preparation method of contrast agent, it is characterised in that：RGD-PEG-COOH in the step (3) is dissolved in the concentration after DMSO and is 6mg/mL；The concentration of G2-NOTA DMSO solution is 4.5mg/mL.
5. a kind of SPECT/CT bimodals based on second generation Polyamidoamine Dendrimers according to claim 1 into As the preparation method of contrast agent, it is characterised in that：RGD-PEG-COOH, EDC, NHS and G2-NOTA's in the step (3) Mol ratio is 10:50:50:1.
6. a kind of SPECT/CT bimodals based on second generation Polyamidoamine Dendrimers according to claim 1 into As the preparation method of contrast agent, it is characterised in that：Sodium borohydride, gold chloride and G2-NOTA-PEG-RGD in the step (4) Mol ratio be 30:6:1.
7. a kind of SPECT/CT bimodals based on second generation Polyamidoamine Dendrimers according to claim 1 into As the preparation method of contrast agent, it is characterised in that：The quality and radioactivity of G2-NOTA-PEG-RGD@Au in the step (5) The ratio of the exit dose of pertechnetate solution is 28mg:35-78MBq.
8. a kind of SPECT/CT bimodals based on second generation Polyamidoamine Dendrimers according to claim 1 into As the preparation method of contrast agent, it is characterised in that：Dialysis in step (1)~(4) is specially：It is with molecular cut off 1000~3000 bag filter is dialysed 3 days, and dialysis distilled water 2L used, changes water 9 times altogether every time.
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CN108148129A (en) * 2018-02-09 2018-06-12 浙江工商大学 A kind of application of the preparation method and NOTA of heavy metal zinc artificial antigen in heavy metal zinc artificial antigen reagent is prepared
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CN108553653A (en) * 2018-04-13 2018-09-21 东华大学 There is one kind RGD target functions to be based on G2.NH2Manganese base MR/CT bimodal image-forming contrast mediums preparation method
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CN105665736A (en) * 2016-01-12 2016-06-15 东华大学 Preparation method for functionalized gold nanostar/siRNA compound modified through RGD and stabilized through dendritic macromolecules

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CN105251028A (en) * 2015-11-05 2016-01-20 东华大学 Method for preparing low-generation dendrimer-based SPECT/CT bimodal imaging contrast agent with FA target function
CN105665736A (en) * 2016-01-12 2016-06-15 东华大学 Preparation method for functionalized gold nanostar/siRNA compound modified through RGD and stabilized through dendritic macromolecules

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CN108148129A (en) * 2018-02-09 2018-06-12 浙江工商大学 A kind of application of the preparation method and NOTA of heavy metal zinc artificial antigen in heavy metal zinc artificial antigen reagent is prepared
CN108148130A (en) * 2018-02-09 2018-06-12 浙江工商大学 A kind of application of the preparation method and NOTA of heavy metal Hg artificial antigen in heavy metal Hg artificial antigen reagent is prepared
CN108148130B (en) * 2018-02-09 2020-10-27 浙江工商大学 Preparation method of heavy metal mercury artificial antigen and application of NOTA in preparation of heavy metal mercury artificial antigen reagent
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CN109045310A (en) * 2018-08-17 2018-12-21 东华大学 A kind of dendrimer composite material of amphoteric ion modification and its preparation and application
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