CN105617412A - Preparation method of different surface-modified SPECT/CT dual-mode imaging contrast agent based on PEGylated polyethyleneimine - Google Patents

Preparation method of different surface-modified SPECT/CT dual-mode imaging contrast agent based on PEGylated polyethyleneimine Download PDF

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CN105617412A
CN105617412A CN201410717554.3A CN201410717554A CN105617412A CN 105617412 A CN105617412 A CN 105617412A CN 201410717554 A CN201410717554 A CN 201410717554A CN 105617412 A CN105617412 A CN 105617412A
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pei
mpeg
spect
preparation
dtpa
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赵晋华
赵凌舟
史向阳
温诗辉
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FIRST PEOPLE'S HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIVERSITY
Donghua University
National Dong Hwa University
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FIRST PEOPLE'S HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIVERSITY
Donghua University
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Abstract

The invention belongs to the field of medical imaging contrast agents, and in particular relates to a preparation method of a different surface-modified SPECT/CT dual-mode imaging contrast agent based on PEGylated polyethyleneimine (PEI). The preparation method comprises the following steps: firstly, modifying the surface of PEI with a radionuclide <99m>Tc chelating agent DTPA, further modifying with mPEG-COOH activated by EDC, so as to obtain functional PEI; synthesizing functional PEI coated gold nanoparticles by using the in-situ reduction method, and performing acetylization or hydroxylation on amino groups remained on the surfaces; finally, marking <99m>Tc to obtain the SPECT/CT dual-mode imaging contrast agent. The different surface-modified SPECT/CT dual-mode imaging contrast agent based on PEI has an excellent SPECT/CT imaging effect; besides, tests show that SPECT/CT imaging of different tissues in a mouse body can be realized, a solid foundation is laid for development of a novel functional tissue specific contrast agent, and the application prospect is broad.

Description

Preparation method based on the SPECT/CT bimodal image-forming contrast medium that the different surfaces of Pegylation polymine is modified
Technical field
The invention belongs to the preparation field of medical science SPECT/CT bimodal image-forming contrast medium, particularly to the preparation method of the SPECT/CT bimodal image-forming contrast medium that the different surfaces based on Pegylation polymine is modified.
Background technology
Prior art discloses nucleus medical image technology and become so that the new imaging technique of internal physiology, the monitor in real time of biochemical process, nucleus medical image utilizes the developer of radioisotope labeling or radiopharmaceutical to introduce in organism, so that the monitor in real time of internal physiology, biochemical process is possibly realized. Compared with traditional morphology formation method (B ultrasonic, X-ray computer tomography (CT), nuclear magnetic resonance, X ray take the photograph sheet), it has prominent functional imaging advantage, especially in clinical diagnosis and treatment disease technology, it is possible to the blood flow change of monitor in real time reflection metabolism and changes of function, internal organs or tissue, special receptor density and activity change etc. Nucleus medical image utilizes the ray that the reception radionuclide image agent of SPECT or PET picture reproducer or medicine send, contrast agent can be monitored and participate in the function information of Physiology and biochemistry and metabolism aspect in organism, and obtain qualitative, quantitative and location clinical disease results of diagnosis and treatment. But nuclear medicine spatial resolution is low, it is impossible to expliciting the position imaging anatomy position, it is therefore desirable to the auxiliary of morphology formation method simultaneously. Along with the continuous progress of technology, the appearance of PET/CT and SPECT/CT technology and instrument successfully solves this problem. Checked by SPECT/CT, image co-registration and the diagnostic multiplication of the anatomical diagnosis information of internal CT and the functional metabolism information of SPECT can be obtained simultaneously. Therefore, compared with independent CT or SPECT, SPECT/CT more has clinical practice meaning in medical diagnosis on disease and prognostic evaluation etc.
Although SPECT/CT bimodal image instrument is widely used for medical diagnosis on disease, but the development and Application of corresponding bimodal image-forming contrast medium is but without obtaining enough attention so that CT imaging is used only for providing the anatomy location information of SPECT signal. Although the use in conjunction of multiple CT and SPECT image-forming contrast medium can complete respective contrast imaging, but the use of two kinds of contrast agent probes, not only cause great inconvenience to clinical practice, also the contrast agent toxic and side effects to patient can be increased, there is also simultaneously and be difficult to coordinate between contrast agent, different distribution in vivo processes occurs, affects bimodal imaging effect. Therefore, a kind of multi-functional contrast agent system that can complete the diagnosis of SPECT/CT bimodal imaging contrast, sensitivity and the accuracy of medical diagnosis on disease will be greatly enhanced, more abundant body physiological Biochemical Information is provided, and alleviate misery and toxic and side effects that patient is caused, widen its application in clinic.
Polymine (PEI) is simple due to its synthesis technique, it is possible to obtain in a large number in the industrial production, is therefore obtained in every field and is widely applied. Although the amido functional group that surface is numerous so that PEI has bio-toxicity, but its amino can serve as the stabilizer of nano material synthesis, dressing agent and Gene transfer vector, is the excellent carrier material preparing macromolecule image-forming contrast medium. Namely can significantly improve its biocompatibility and blood circulation time additionally by simple Pegylation or acetylation modification, meet various biomedical applications requirement. Because PEI as multifunctional carrier material, at finishing functionalization group, and can wrap up inorganic nanoparticles, it is achieved multi-functional integration, form multifunctional nano probe, meet the requirement of clinical practice. It is that carrier is prepared SPECT/CT bimodal contrast agent and will can be met the demand of clinical practice hence with PEI, the modifiability according to PEI surface simultaneously, carry out different surfaces and modify and realize different in-vivo tissue imaging organs.
Domestic and international document and the patent results about SPECT/CT bimodal contrast agent aspect of retrieval shows: at present, there is not yet the trapping gold nano-particle chelating based on Pegylation PEI99mThe report of the preparation and application of the SPECT/CT contrast agent of Tc.
Summary of the invention
It is an object of the invention to overcome the shortcomings and deficiencies of prior art, it is provided that a kind of with Pegylation PEI carrier material load99mTc chelate trapping gold nano-particle are as SPECT/CT bimodal image-forming contrast medium, to reach the bimodal imaging contrast effect of internal different tissues organ.
The main technical schemes that the present invention adopts is to utilize Pegylation PEI as carrier material, will by covalent bond99mTc chelating agen is connected to its surface, and by the method for fabricated in situ at PEI trapping gold nano-particle, to reach good histoorgan bimodal imaging effect.
The preparation process of the present invention be normal temperature and pressure, easily operated, there is good practical value.
Concrete, the preparation method of the SPECT/CT bimodal image-forming contrast medium that the different surfaces based on Pegylation polymine of the present invention is modified, it includes step:
(1) will99mTc chelating agen diethylenetriamine pentaacetic acid (DTPA) and polyethyleneglycol modified in polymine (PEI) surface, prepares PEI-DPTA-mPEG;
(2) synthesize the gold nano grain of PEI parcel with above-mentioned functionalization PEI for stabilizer, and acetylated for its surface residual or hydroxylating are obtained the nano-particle (Au-AcPENPs and Au-GlyPENPs) that different surfaces is modified;
(3) labelling99mTc, prepares chelating nucleic99mThe functionalization PEI of Tc trapping gold nanoparticle (99mTc-Au-AcPENPs and99mTc-Au-GlyPENPs)��
In step of the present invention (1): PEI (being purchased from SigmaAldrich company) is dissolved in water, and it is added dropwise over the aqueous solution of cDTPAA (being purchased from SigmaAldrich company), at room temperature stirring reaction 6-8h obtains PEI-DTPA, add the mPEG-COOH (being purchased from Yan Yi bio tech ltd, Shanghai) after EDC (being purchased from SigmaAldrich company) activation, stirring reaction 24-52h, obtains functionalization PEI (PEI-DTPA-mPEG) solution;
The preparation method of the aqueous solution of the mPEG-COOH of described EDC activation is: adds the aqueous solution of EDC in the aqueous solution of mPEG-COOH, then stirs 1-3h;
The molecular weight of described mPEG-COOH is 5000;
The mol ratio of described PEI and cDTPAA is 1:4-12;
The mol ratio of described mPEG-COOH and EDC is 1:10-16, and soak time is 1-3h;
The mol ratio of described PEI-DTPA and mPEG-COOH is 1:12-26;
Outside in step of the present invention (1), reaction can carry out in pure water, it is also possible at PBS, dimethyl sulfoxide, dimethylformamide, dimethyl acetylamide polar solvent reacts.
In step of the present invention (2): the PEI-DTPA-mPEG solution obtained in step (1) adds chlorauric acid solution stirring 10-20min, adds sodium borohydride solution, stirring reaction 2-3h; Wherein in acetylation operation, add triethylamine stirring 10-20min, be eventually adding acetic anhydride, stirring reaction 16-24h; Wherein in hydroxylation process, add (+)-2,3-Epoxy-1-propanol, stirring reaction 16-24h, reactant liquor is dialysed, finally the aqueous solution lyophilization of product is obtained Au-AcPENPs and Au-GlyPENPs;
Described PEI-DTPA-mPEG and the mol ratio of gold chloride are 1:160-240;
Described PEI-DTPA-mPEG and the mol ratio of sodium borohydride are 1:800-1300;
Described PEI-DTPA-mPEG and the mass ratio of triethylamine are 1:1-3;
Described PEI-DTPA-mPEG and the mass ratio of acetic anhydride are 1:1-3;
Described PEI-DTPA-mPEG and the mass ratio of (+)-2,3-Epoxy-1-propanol are 1:1.5-4;
Dialysis technique in step of the present invention (2) is: adopting bag filter first to dialyse in the PBS that pH is 7.4, then dialyse in distilled water, bag filter is cellulose dialysis film, and molecular cut off is 8000-14000; Reaction except can carrying out in pure water, it is also possible at PBS, dimethyl sulfoxide, dimethylformamide, reacts in dimethyl acetylamide polar solvent.
In step of the present invention (3): Au-AcPENPs and the Au-GlyPENPs of preparation in step (2) is dissolved in phosphate buffer (PBS, pH=7.2-7.4) respectively, adds SnCl2, then add aseptic radioactivity persalt solution and mix rapidly, column chromatography for separation, obtaining chelating nucleic99mThe functionalization PEI of Tc trapping gold nanoparticle (99mTc-Au-AcPENPs and99mTc-Au-GlyPENPs);
Described Au-AcPENPs and SnCl2Mass ratio be 1:0.2-0.6;
The ratio of described Au-AcPENPs and radioactivity persalt is 1mg:2000-4000MBq;
Described Au-GlyPENPs and SnCl2Mass ratio be 1:0.2-0.6;
The ratio of described Au-GlyPENPs and radioactivity persalt is 1mg:2000-4000MBq;
Outside in step of the present invention (3), reaction can carry out in pure water, it is also possible at PBS, dimethyl sulfoxide, dimethylformamide, dimethyl acetylamide polar solvent reacts;
The technique of described separation is: adopt PD-10 desalination chromatography target product, removes free99mTc and unreacting substance.
In the present invention, will99mTc chelating agen cDTPAA is grafted to PEI surface, and cDTPAA adopts the mode dropwise dripped under fast stirring to join in PEI solution, to ensure the homogeneity of PEI grafting DTPA.
In the present invention, using 5-20 times amount EDC to activate the carboxyl of mPEG-COOH, strengthen and the PEI activity reacted, the mode that the mPEG-COOH of activation is also adopted by dropwise dripping joins in PEI solution, to ensure the homogeneity of PEI grafting PEG; The addition of PEG simultaneously can increase the circulation time in vivo of this material, reaches good blood pool CT contrasting effects.
In the present invention, modify the amino of PEI surface residual respectively with acetylation and hydroxylating, to reduce its surface potential, improve the biocompatibility of material, and give its different surface group, to realize its imaging of tissue performance different in vivo.
The present invention use UV-Vis (uv-vis spectra), TEM (transmission electron microscope), SAED (SEAD), MTT test (cell viability analysis), and the nano-particle with bimodal radiography function that the internal SPECT/CT imaging representation present invention obtains, result shows:
(1) UV-Vis test result
UV-Vis test result shows: surface plasma body resonant vibration (SPR) peak of SPECT/CT bimodal contrast agent particle Au-AcPENPs and the Au-GlyPENPs prepared in the present invention lays respectively at 515 and 500nm (as shown in Figure 2), show that the present invention has prepared gold nano grain, and different finishinges has different SPR peak positions;
(2) TEM test result
TEM test result shows picture and the distribution of sizes (as shown in Figures 3 and 4) of two kinds of gold nano grains, which show two kinds of gold nano grain sizes of Au-AcPENPs and Au-GlyPENPs similar, average diameter is 2.83nm and 2.78nm respectively, and its distribution of sizes is narrower, there is good dispersibility;
(3) SAED test result
SAED test result shows that two kinds of gold nano grains are respectively provided with good crystal property (shown in Fig. 5), (111) in this SAED collection of illustrative plates, (200), (220) and (311) circle shows that two kinds of gold nano grains prepared are respectively provided with the face-centered cubic crystal structure of typical golden nanometer particle;
(4) MTT test result
Cell compatibility with SKOV-3 cell research Au-AcPENPs and Au-GlyPENPs: by difference Au concentration (0,5,10,25,50,75,100 ��Ms) Au-AcPENPs and Au-GlyPENPs and SKOV-3 co-culture of cells 24h after, the vigor (as shown in Figure 6) of cell is detected with mtt assay, figure shows, relative to the comparison SKOV-3 cell processed with PBS, Au-AcPENPs and Au-GlyPENPs complex when Au is at concentrations up to 100 ��Ms still cell growth there is no any impact, show good cell compatibility;
(5) blood compatibility
For the nano-particle being tested the application of its vivo biodistribution by tail vein injection, good blood compatibility it is critical that, therefore this experimental evaluation blood compatibility of Au-AcPENPs and Au-GlyPENPs nano-particle; Fig. 7 shows Au-AcPENPs and Au-GlyPENPs nano-particle hemolytic test result under variable concentrations (50,100,200,400 �� g/mL), by the extinction spectrum of the supernatant measures the hemolytic of quantitative assessment nano material, as figure upper right corner uv-spectrogram shows, when concentration reaches 400 �� g/mL, the hemolysis rate of Au-AcPENPs and Au-GlyPENPs is respectively less than 4%, illustrate that the nano-particle of preparation has good blood compatibility, thus providing guarantee for its SPECT/CT imaging applications in vivo;
(6)99mMicro-SPECT/CT imaging in Tc-Au-AcPENPs Mice Body
By 200 �� L99mTc-Au-AcPENPs([99mTc]=370MBq/mL; [Au]=0.08M) tail vein injections enters in the Mice Body that body weight is 22g; by the SPECT/CT picture (as Suo Shi Fig. 8, Figure 10) that micro-SPECT/CT small animal imaging instrument Scanning Detction obtains, figure shows acetylation surface treatment99mTc-Au-AcPENPs material can realize pulmonary's specificity imaging of mice, its brightness is apparently higher than other major organs such as livers, and simultaneously it can be seen that liver, kidney, the signal of spleen and bladder strengthens, and imaging time can continue at least 2 hours, and metabolic signals weakens (as shown in Figure 8) gradually, it was demonstrated that the method synthesis of the present invention99mTc-Au-AcPENPs has good SPECT/CT bimodal imaging effect;
(7)99mMicro-CT imaging in Tc-Au-GlyPENPs Mice Body
By 200 �� L99mTc-Au-GlyPENPs([99mTc]=370MBq/mL, [Au]=0.08M) tail vein injections enters in the Mice Body that body weight is 22g, obtains mice CT picture (as Suo Shi Fig. 9, Figure 11) by micro-SPECT/CT small animal imaging instrument Scanning Detction; Figure shows what hydroxylated surface processed99mTc-Au-GlyPENPs material can stop the longer time in blood, completes heart, kidney, the blood pool radiographies such as ventral aorta, prolongation over time, material metabolism can enter spleen, bladder and liver (as shown in Figure 9), it was demonstrated that this method synthesis99mTc-Au-GlyPENPs has good SPECT/CT bimodal imaging effect; The in-vivo imaging result of bi-material shows that the contrast agent that synthesized different surfaces is modified has dramatically different internal metabolism distribution behavior, it is possible to complete different tissue and organ specificity radiography functions.
Chelating prepared by the inventive method99mThe functionalization PEI material of Tc trapping gold nanoparticle, its size of nanometer gold grain narrow distribution, there is good dispersibility, external have good cell compatibility, and in vivo test shows its good SPECT/CT bimodal imaging performance.
The present invention utilize a large amount of functional group in PEI surface modifiability and modified after the good biocompatibility that possesses, by PEI, inorganic nanoparticles modified, assemble and biological functional, and the grafting of various little molecular contrast agents, prepare excellent polyfunctional molecule iconography contrast agent for SPECT/CT bimodal imaging, the early diagnosis of disease will be can apply to.
There is advantages that
(1) preparation process is simple, and experiment condition is normal temperature and pressure, it is easy to operation, and the preparation procedure adopted can be used for the preparation of other functionalization PEI, and SPECT and CT contrast agent is for SPECT/CT bimodal imaging, has significantly good use value;
(2) the bimodal contrast agent of the different surfaces group prepared has different internal characteristics, different histoorgan radiographies can be completed, there is significantly good clinical value, and realize, for different surfaces modification, the basis that different tissues organ radiography provides good;
(3) preparation procedure of the present invention can be used for preparing SPECT and the CT contrast agent with targeting for SPECT/CT targeting bimodal imaging, has good use value;
(4) the SPECT/CT bimodal image-forming contrast medium based on Pegylation PEI of the functionalization prepared has good SPECT/CT imaging effect, and the exploitation for Multifunction contrast agent provides good basis.
Accompanying drawing explanation
Fig. 1 is reaction equation sketch of the present invention.
Fig. 2 is the uv absorption spectra of Au-AcPENPs (a) and Au-GlyPENPs (b) prepared by the present invention.
Fig. 3 is TEM picture (a) and particle size distribution rectangular histogram (b) of Au-AcPENPs prepared by the present invention.
Fig. 4 is TEM picture (a) and particle size distribution rectangular histogram (b) of Au-GlyPENPs prepared by the present invention.
Fig. 5 is the SAED spectrogram of Au-AcPENPs (a) and Au-GlyPENPs (b) prepared by the present invention.
Fig. 6 is the MTT analysis of Au-AcPENPs and Au-GlyPENPs prepared by the present invention SKOV-3 cell viability processed in different Au concentration.
Fig. 7 is the hemolytic experiment analysis of Au-AcPENPs and Au-GlyPENPs prepared by the present invention.
Fig. 8 is 200 �� L99mTc-Au-AcPENPs([99mTc]=370MBq/mL) tail vein injections enters in Mice Body, by the mouse lung that micro-SPECT/CT Scanning Detction obtains, liver, kidney, the SPECT/CT picture of spleen and bladder (as shown by arrows), it is followed successively by 0.5h (a), 1h (b), 2h (c) after injection from top to bottom.
Fig. 9 is 200 �� L99mTc-Au-AcPENPs ([Au]=0.08M) tail vein injections enters in Mice Body, by the mouse lung that micro-CT Scanning Detction obtains, heart, liver, the CT picture of kidney and bladder (as shown by arrows), is followed successively by (a) before injection, 0.5h (b) after injection, 1h (c), 2h (d).
Figure 10 is 200 �� L99mTc-Au-GlyPENPs([99mTc]=370MBq/mL) tail vein injections enters in Mice Body, by the mouse heart that micro-SPECT/CT Scanning Detction obtains, postcava, liver, kidney, the SPECT/CT picture of spleen and bladder (as shown by arrows), it is followed successively by 0.5h (a) after injection from top to bottom, 1h (b), 2h (c). Figure 11 is 200 �� L99mTc-Au-GlyPENPs ([Au]=0.08M) tail vein injections enters in Mice Body, by the mouse heart that micro-CT Scanning Detction obtains, liver, the CT picture of kidney and bladder (as shown by arrows), it is followed successively by (a) before injection, 0.5h (b) after injection, 1h (c), 2h (d).
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further. Should be understood that these embodiments are merely to illustrate the present invention rather than restriction the scope of the present invention. In addition, it is to be understood that after having read the content that the present invention lectures, the present invention can be made various changes or modifications by those skilled in the art, and these equivalent form of values fall within the application appended claims limited range equally.
Embodiment 1
As it is shown in figure 1, preparation99mTc-Au-AcPENPs��
(1) by 15.0mg polymine (PEI, it is purchased from SigmaAldrich company) it is dissolved in 15mL water, and it is added dropwise over 1mL diethylenetriamine pentaacetic acid cyclic acid anhydride (cDTPAA, be purchased from SigmaAldrich company) aqueous solution (1.7mg/mL), at room temperature stirring reaction 6h obtains PEI-DTPA; Simultaneously, the aqueous solution (6mg/mL) of 10mLmPEG-COOH adds 5mL1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC, be purchased from SigmaAldrich company) aqueous solution (6mg/mL) and stir 2h, then the mPEG-COOH (being purchased from Yan Yi bio tech ltd, Shanghai) after being activated by EDC adds in PEI-DTPA solution, stirring reaction 48h, obtains functionalization PEI (PEI-DTPA-mPEG) solution;
(2) the PEI-DTPA-mPEG solution obtained in (1) adds 1.65mL tetra-hydration chlorauric acid solution (30mg/mL) and stirs 30min, solution becomes light yellow, add 2.4mL sodium borohydride aqueous solution (10mg/mL), solution moment becomes peony, stirring reaction 40min. It is subsequently adding 75 �� L triethylamine stirring 20min, is eventually adding 50 �� L acetic anhydrides, stirring reaction 15h. Finally product is gradually dialysed 3 days with cellulose dialysis film (MWCO=14000) in PBS solution 2L �� 3 and distilled water 2L �� 3, finally the product lyophilization after purification is obtained Au-AcPENPs;
(3) Au-AcPENPs of preparation in 0.2mg step (2) is dissolved in 0.25mLPBS solution (pH=7.2-7.4), adds 0.05mgSnCl2, then add the aseptic radioactivity persalt (radioactivity of 1.5mL99mTc concentration is 370MBq/mL) solution also hybrid reaction 30min, employing PD-10 desalination chromatography separation rapidly, obtain chelating nucleic99mThe functionalization PEI of Tc trapping gold nanoparticle (99mTc-Au-AcPENPs);
UV-Vis test result shows that surface plasma body resonant vibration (SPR) peak of the SPECT/CT bimodal contrast agent particle Au-AcPENPs prepared in the present invention is positioned at 515nm, as shown in Figure 2 a, show the present invention has prepared gold nano grain, the Au-AcPENPs average diameter respectively 2.83nm of TEM test result display simultaneously, and there is narrower particle size distribution (as shown in Figure 3), SAED test result display gained gold nano grain has good crystal property, wherein (111), (200), (220) enclose show that prepared gold nano grain has the face-centered cubic crystal structure (as shown in Figure 5 a) of typical golden nanometer particle with (311).
Embodiment 2
As it is shown in figure 1, preparation99mTc-Au-GlyPENPs��
(1) by 15.0mg polymine (PEI, it is purchased from SigmaAldrich company) it is dissolved in 15mL water, and it is added dropwise over 1mL diethylenetriamine pentaacetic acid cyclic acid anhydride (cDTPAA, be purchased from SigmaAldrich company) aqueous solution (1.7mg/mL), at room temperature stirring reaction 6h obtains PEI-DTPA; Simultaneously, the aqueous solution (6mg/mL) of 10mLmPEG-COOH adds 5mL1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC, be purchased from SigmaAldrich company) aqueous solution (6mg/mL) and stir 2h, then the mPEG-COOH (being purchased from Yan Yi bio tech ltd, Shanghai) after being activated by EDC adds in PEI-DTPA solution, stirring reaction 1-3d, obtains functionalization PEI (PEI-DTPA-mPEG) solution;
(2) the PEI-DTPA-mPEG solution obtained in (1) adds 1.65mL tetra-hydration chlorauric acid solution (30mg/mL) and stirs 30min, solution becomes light yellow, add 2.4mL sodium borohydride aqueous solution (10mg/mL), solution moment becomes peony, stirring reaction 40min. It is subsequently adding 100 �� L (+)-2,3-Epoxy-1-propanol stirring reaction 24h. Finally product is gradually dialysed 3 days with cellulose dialysis film (MWCO=14000) in phosphate buffered solution 2L �� 3 and distilled water 2L �� 3, finally the product lyophilization after purification is obtained Au-GlyPENPs;
(3) Au-GlyPENPs of preparation in 0.2mg step (2) is dissolved in 0.25mLPBS, adds 0.05mgSnCl2, then add the aseptic radioactivity persalt (radioactivity of 1.5mL99mTc concentration is 370MBq/mL) solution also hybrid reaction 30min, employing PD-10 desalination chromatography separation rapidly, obtain chelating nucleic99mThe functionalization PEI of Tc trapping gold nanoparticle (99mTc-Au-GlyPENPs);
UV-Vis test result shows that surface plasma body resonant vibration (SPR) peak of the SPECT/CT bimodal contrast agent particle Au-GlyPENPs prepared is positioned at 500nm (as shown in Figure 2 b), show that the present invention has prepared gold nano grain, the Au-GlyPENPs average diameter respectively 2.78nm of TEM test result display simultaneously, and there is the particle size distribution (as shown in Figure 4) more narrower than Au-AcPENPs, SAED test result display gained gold nano grain has good crystal property, wherein (111), (200), (220) enclose show that prepared gold nano grain has the face-centered cubic crystal structure (as shown in Figure 5 b) of typical golden nanometer particle with (311).
Embodiment 3
The cytotoxicity of Au-AcPENPs and Au-GlyPENPs prepared by institute is carried out by MTT experiment;
Collecting logarithmic (log) phase human oophoroma cell line (SKOV-3 cell), join in 96 porocyte culture plates, every hole adds the 200 celliferous RPMI1640 culture medium of �� L makes cell density to 8000/ hole; Then at cell culture incubator (5%CO237 DEG C) in hatch 24 hours, outwell culture medium and add 180 �� L fresh cultures, adding 20 �� LPBS buffer (the gold final concentration respectively 0,5 of Au-AcPENPs and Au-GlyPENPs containing variable concentrations, 10,25,50,75,100 ��Ms), to verify the material impact on SKOV-3 Growth of Cells. All of test group is all provided with 5 Kong Weiyi parallel group; After incubator hatches 24h, every hole adds the MTT solution (5mg/mL) of 20 �� L, after cultivating 4h, carefully suck culture fluid in hole, 200 �� LDMSO are added in every hole, put lucifuge vibration 15min on shaking table, then measure the MTT first solution light absorption value in each hole at enzyme-linked immunosorbent assay instrument 570nm place. Analyze result and show Au-AcPENPs and the Au-GlyPENPs toxic action to cell with cell survival rate. The vigor (as shown in Figure 6) of cell after mtt assay detection process, figure shows, relative to untreated SKOV-3 cell, cell is not produced toxicity by Au-AcPENPs and Au-GlyPENPs when gold concentration is up to 100 ��Ms, shows good biocompatibility.
Embodiment 4
Weigh each 1mg of Au-AcPENPs and Au-GlyPENPs of lyophilizing, being scattered in PBS the concentration being configured to 1mg/mL respectively is mother solution, then it is 50 �� g/mL with PBS successively compound concentration, 100 �� g/mL, the nano granule suspension of 200 �� g/mL and 400 �� g/mL evaluates the blood compatibility (as shown in Figure 7) of nano material prepared by the present invention, take appropriate people's fresh blood, first centrifugal (2000rpm/min, 5min) remove supernatant, then erythrocyte PBS is washed 5 times, collect healthy erythrocyte and dilute 10 times with PBS, Au-AcPENPs and Au-GlyPENPs nano material (50-400 �� g/mL) is mixed standing again after 2 hours with erythrocyte, 10000rpm is centrifuged 1min, survey the ultraviolet light absorption spectrum of supernatant. this process is using PBS as negative control, ultra-pure water is as positive control, Fig. 7 shows Au-AcPENPs and Au-GlyPENPs hemolytic test result under concentration 50,100,200,400 �� g/mL, by measuring the hemolytic of the absorbance quantitative assessment nano material of the supernatant, result shows, when concentration reaches 400 �� g/mL, the hemolysis rate of Au-AcPENPs and Au-GlyPENPs is respectively less than 4%, illustrate that the nano-particle of preparation has good blood compatibility, thus providing guarantee for its SPECT/CT imaging applications in vivo.
Embodiment 5
By what 200 �� L embodiments 1 or 2 obtained99mTc-Au-AcPENPs([99mTc]=370MBq/mL; [Au]=0.08M) tail vein injections enters in the Mice Body that body weight is 22g; before medication, it is obtained comparison CT picture (as shown in Figure 10) by CT scan detection by 5min; and after medication 0.5; 1,2h obtains mouse lung respectively through micro-SPECT/CT scanning again, and SPECT and the CT picture of liver and kidney is (such as Fig. 8; shown in Figure 10), figure shows acetylation surface treatment99mTc-Au-AcPENPs material can realize pulmonary and the liver specificity imaging of mice, and liver brightness is apparently higher than other major organs such as lungs, and imaging time can continue at least 2 hours, and metabolic signals weakens (as shown in Figure 8) gradually, it was demonstrated that obtained99mTc-Au-AcPENPs has good SPECT/CT bimodal imaging effect.
Embodiment 6
By 200 �� L embodiments 1 or 299mTc-Au-GlyPENPs([99mTc]=370MBq/mL, [Au]=0.08M) tail vein injections enters in the Mice Body that body weight is 22g, before medication, it is obtained comparison CT picture (as shown in figure 11) by CT scan detection by 5min, and after medication 0.5,1,2h again respectively through micro-SPECT/CT scanning obtain mouse carotid vein, heart, the SPECT picture of liver and postcava and kidney and CT picture (as Suo Shi Fig. 9, Figure 11), show what hydroxylated surface processed in figure99mTc-Au-GlyPENPs material can stop the longer time in blood, completes jugular vein, heart, kidney, the blood pool radiography such as postcava, prolongation over time, material can enter bladder (as shown in Figure 9) by renal metabolism, it was demonstrated that this method synthesis99mTc-Au-GlyPENPs has good SPECT/CT bimodal imaging effect; The in-vivo imaging result of bi-material shows that the contrast agent that synthesized different surfaces is modified has dramatically different internal metabolism distribution behavior, it is possible to complete different tissue and organ specificity radiography functions.

Claims (7)

1. the preparation method of the SPECT/CT bimodal image-forming contrast medium modified based on the different surfaces of Pegylation polymine, it is characterized in that, this contrast agent, will by covalent bond using the polymine (PEI) of pegylation as polymer carrier99mTc chelating agen diethylenetriamine pentaacetic acid is connected to its surface, and by the method trapping gold nano-particle of fabricated in situ: it comprises the following steps:
(1) will99mTc chelating agen diethylenetriamine pentaacetic acid (DTPA) and polyethyleneglycol modified in PEI surface, prepares PEI-DPTA-mPEG;
(2) synthesize the gold nano grain of PEI parcel with prepared functionalization PEI for stabilizer, and acetylated for surface residual or hydroxylating are prepared the nano-particle (Au-AcPENPs and Au-GlyPENPs) that different surfaces is modified;
(3) labelling99mTc, prepares chelating nucleic99mThe trapping gold nanoparticle of Tc functionalization PEI (99mTc-Au-AcPENPs and99mTc-Au-GlyPENPs)��
2. preparation method according to claim 1, it is characterized in that, in described step (1): PEI is dissolved in water, and it is added dropwise over the aqueous solution of diethylenetriamine pentaacetic acid cyclic acid anhydride (cDTPAA), at room temperature stirring reaction 6-8h obtains PEI-DTPA, add the mPEG-COOH after 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) activation, stirring reaction 24-52h, obtains functionalization PEI (PEI-DTPA-mPEG) solution;
The mol ratio of PEI and cDTPAA therein is 1:4-12;
The mol ratio of mPEG-COOH and EDC therein is 1:10-16, and soak time is 1-3h;
The mol ratio of PEI-DTPA and mPEG-COOH therein is 1:12-26.
3. preparation method according to claim 1, it is characterized in that, in described step (2): add chlorauric acid solution stirring 10-20min in the PEI-DTPA-mPEG solution that step (1) prepares, add sodium borohydride solution, stirring reaction 2-3h; In acetylation therein operation, add triethylamine stirring 10-20min, be eventually adding acetic anhydride, stirring reaction 16-24h; In hydroxylation process therein, adding (+)-2,3-Epoxy-1-propanol, stirring reaction 16-24h, hemodialysis reaction liquid, the aqueous solution of desciccate is freezing obtains Au-AcPENPs and Au-GlyPENPs;
The mol ratio of PEI-DTPA-mPEG therein and gold chloride is 1:160-240;
The mol ratio of PEI-DTPA-mPEG therein and sodium borohydride is 1:800-1300;
The mass ratio of PEI-DTPA-mPEG therein and triethylamine is 1:1-3;
The mass ratio of PEI-DTPA-mPEG therein and acetic anhydride is 1:1-3;
The mass ratio of PEI-DTPA-mPEG therein and (+)-2,3-Epoxy-1-propanol is 1:1.5-4.
4. preparation method according to claim 1, it is characterized in that, in described step (3): be dissolved in phosphate buffer by Au-AcPENPs and the Au-GlyPENPs of preparation in step (2) (PBS) pH=7.2-7.4 respectively, add SnCl2, then add aseptic radioactivity persalt solution and mix, column chromatography for separation, obtaining chelating nucleic99mTc functionalization PEI parcel golden nanometer particle (99mTc-Au-AcPENPs and99mTc-Au-GlyPENPs);
Au-AcPENPs and SnCl therein2Mass ratio be 1:0.2-0.6;
The ratio of Au-AcPENPs therein and radioactivity persalt is 1mg:2000-4000MBq;
Au-GlyPENPs and SnCl therein2Mass ratio be 1:0.2-0.6;
The ratio of Au-GlyPENPs therein and radioactivity persalt is 1mg:2000-4000MBq.
5. preparation method according to claim 2, it is characterised in that the molecular weight of described mPEG-COOH is 5000.
6. preparation method according to claim 3, it is characterised in that in described dialysis: adopting bag filter to dialyse in the PBS that pH is 7.4, then dialyse in distilled water, wherein, bag filter is cellulose dialysis film, and molecular cut off is 8000-14000.
7. preparation method according to claim 3, it is characterised in that described separation method is: adopt PD-10 desalination chromatography target product, removes free99mTc and unreacting substance.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107343961A (en) * 2017-03-08 2017-11-14 东华大学 A kind of preparation method of the hyperbranched polyethyleneimine nano-probe based on rgd peptide modification
CN110128666A (en) * 2019-05-27 2019-08-16 南京工业大学 Functionalized polyethy-lene imines wraps up nanogold particle composite material and preparation method

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103143043A (en) * 2013-03-06 2013-06-12 东华大学 Preparation method of Fe3O4/Au composite nanoparticles
CN103239738A (en) * 2013-05-22 2013-08-14 东华大学 Preparation method of pegylation modified hyperbranched poly(ethylene imine) coated nano-gold particles
CN103877597A (en) * 2014-03-19 2014-06-25 中国人民解放军第二军医大学 Pegylated polyethyleneimine macromolecular magnetic resonance imaging contrast agent and preparation method of contrast agent
CN104162175A (en) * 2014-06-18 2014-11-26 东华大学 Functionalized dendrimer-based SPECT-CT bimodal imaging contrast agent and preparation method thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103143043A (en) * 2013-03-06 2013-06-12 东华大学 Preparation method of Fe3O4/Au composite nanoparticles
CN103239738A (en) * 2013-05-22 2013-08-14 东华大学 Preparation method of pegylation modified hyperbranched poly(ethylene imine) coated nano-gold particles
CN103877597A (en) * 2014-03-19 2014-06-25 中国人民解放军第二军医大学 Pegylated polyethyleneimine macromolecular magnetic resonance imaging contrast agent and preparation method of contrast agent
CN104162175A (en) * 2014-06-18 2014-11-26 东华大学 Functionalized dendrimer-based SPECT-CT bimodal imaging contrast agent and preparation method thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107343961A (en) * 2017-03-08 2017-11-14 东华大学 A kind of preparation method of the hyperbranched polyethyleneimine nano-probe based on rgd peptide modification
CN110128666A (en) * 2019-05-27 2019-08-16 南京工业大学 Functionalized polyethy-lene imines wraps up nanogold particle composite material and preparation method
CN110128666B (en) * 2019-05-27 2021-09-28 南京工业大学 Functionalized polyethyleneimine coated nano-gold particle composite material and preparation method thereof

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