CN110393809A - A kind of nanoscale magnetic resonance imaging contrast and preparation method thereof - Google Patents
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Abstract
The invention discloses a kind of nanoscale magnetic resonance imaging contrast and preparation method thereof, the synthesis material of the contrast agent is DNA (DNA) and metal radiography element.Specific preparation method is using cyclic DNA as template, drive metalloenzyme to amplify with being repeated cyclically the long single stranded DNA of sequence by metal radiography element, with duration single stranded DNA with react in byproduct metal difficulty soluble salt hydridization formation partial size 200~400nm or so nanoscale magnetic resonance imaging contrast.The contrast agent that the present invention is prepared has good biocompatibility and biodegradability, has active/passive targeting and the easy resolution characteristic of acid, can be used in the fields such as magnetic resonance imaging, gene diagnosis and treatment, chemomotive force therapy.
Description
Technical field
The invention belongs to the preparations of magnetic resonance imaging nano material, are applied to bio-imaging field, are related to a kind of nanoscale
Magnetic resonance imaging contrast and preparation method thereof.
Background technique
Magnetic resonance imaging (MRI) is wide on current clinical medicine due to its high spatial resolution and deep tissue penetration
General use.Clinically used magnetic resonance imager disturbs doctor usually because its muting sensitivity and low precision etc. limit at present
The raw accurate judgement for sufferer disease sites.In order to solve this problem, doctor usually requires to make sufferer injection magnetic resonance
Shadow agent is to improve the imaging effect of sufferer.Currently, having been achieved with commercialized magnetic resonance contrast agent is Gd-DOPA (gadolinium chelate compound)
With SPIO (Superparamagnetic Iron Oxide), but these two types of contrast agent usually there will be the disadvantages such as shortage specificity and potential toxic side effect
End.Mn2+As a kind of microelement of human body, because of its high paramagnetism and low potential source biomolecule toxicity, to make it in magnetic resonance
Imaging field obtains extensive concern.
Rolling circle amplification (rolling circle amplification, RCA) is the one of invention application in 1998
Kind nucleic acid amplification technologies.2013 Nian Luodan seminars are by carrying out microstructure table to the DNA hydrogel obtained by rolling circle amplification
In present hydrogel network of levying dense arrangement similar flower-shaped nanoparticle [1], subsequent researcher pass through to this
Microstructure carries out analysis and finds, this flower-like nanometer particle be by rolling circle amplification long chain DNA with react in by-product
Mg2Produced by PPi is interacted by non-state-set prices [2].According to its component and pattern, there is researcher to call it as DNA nanometers
Flower [3].The unique hybrid inorganic-organic structure of DNA nano flower imparts this nanoparticle multifrequency nature, inorganic skeleton
Mg2PPi has pH responsiveness, while also giving the certain physiological stability of DNA chain can be in complicated fluid environment
Under be stabilized；DNA itself has editability and designability, to impart material diversity and designability [4].
Currently, DNA nano flower is applied to gene therapy by existing research personnel, and cell imaging, RNA analysis in living cells,
The fields such as protein delivery.It is worth noting that, research sight is usually concentrated on the programming of DNA and is set in existing research
On meter, [5] of simultaneously probe into application are rarely modified to inorganic skeleton.How rationally and effectively to change inorganic skeleton is to widen
The effective means in DNA nano flower biologic applications field.
Bibliography:
1.Lee,J.B.,et al.,A mechanical metamaterial made from a DNA
hydrogel.Nat Nanotechnol,2012.7(12):p.816-20.
2.Kim,E.,et al.,One-Pot Synthesis of Multiple Protein-Encapsulated
DNA Flowers and Their Application in Intracellular Protein Delivery.Adv
Mater,2017.29(26).
3.Zhu,G.,et al.,Noncanonical self-assembly of multifunctional DNA
nanoflowers for biomedical applications.J Am Chem Soc,2013.135(44):p.16438-
45.
4.Jin,Y.,et al.,Biodegradable,multifunctional DNAzyme nanoflowers for
enhanced cancer therapy.NPG Asia Materials,2017.9(3):p.e365.
5.Lee,J.S.,et al.,Enzyme-Driven Hasselback-Like DNA-Based Inorganic
Superstructures.Advanced Functional Materials,2017.27(45):p.1704213.
Summary of the invention
The purpose of the present invention is widening the biologic applications field of DNA nano flower, is applied in field of magnetic resonance imaging, mentioned
For a kind of nanoscale magnetic resonance imaging contrast and preparation method thereof.
Technical solution of the present invention is summarized as follows:
A kind of preparation method of nanoscale magnetic resonance imaging contrast, comprising the following steps:
(1) in the reaction vessel, the circular DNA template with DNA primer of final concentration of 300nM~1 μM is added, it is dense eventually
Degree is the MnCl of 6~8mM2Solution, (dATP, dTTP, dGTP, dCTP are 1:1:1:1 according to molar ratio to atriphos mixed liquor
Mixing obtains atriphos mixed liquor), phi 29DNA polymerase buffer (does not include MgCl2), BSA, KCl aqueous solution
And 29 polymerase of phi, residual volume are supplied with deionized water, 400~600rpm, are reacted 3-6h under the conditions of 30 DEG C, are obtained white
Color turbid solution；
(2) ultrasonic cleaner is to obtained 5~10min of white opacity solution ultrasound；
(3) 10000rpm is centrifuged 3~5min, after removing supernatant, (10 μ L matter is added in 10mL deionized water using weak aqua ammonia
Measure the concentrated ammonia liquor that score is 33%) washing lower layer's white product 1~3 time, centrifugation obtains magnetic resonance contrast agent after removing weak aqua ammonia.
Circular DNA template in step (1) with DNA primer is made with the following method:
A. according to the molar ratio of 1:2,5 ' ends are mixed by the linear DNA template and DNA primer of phosphorylation modification, institute
It states 5 ' end regions of DNA primer and passes through 5 ' end regions complementary pairings of the linear DNA template of phosphorylation modification, the 3 ' of DNA primer
3 ' end regions complementary pairings of end regions and the linear DNA template by phosphorylation modification；The NaCl of final concentration of 120mM is added
Reaction volume is complemented to 20 μ L with deionized water by aqueous solution；
B. above-mentioned mixed reaction system is placed in PCR instrument, high annealing reaction is carried out, to obtained DNA product
Middle addition T4DNA ligase reacts 3~8h at 22 DEG C, obtains the circular DNA template with DNA primer.
The magnetic resonance contrast agent prepared by the above method.
Advantages of the present invention:
Magnetic resonance contrast agent prepared by the present invention, can active/passive accumulate in tumor locus improve tumor region at
As effect.Meanwhile preparation process is simple, prepared contrast agent has high biocompatibility and low bio-toxicity.
Detailed description of the invention
Fig. 1 is the electrophoretogram of circular DNA template of the verifying synthesis with DNA primer；Wherein 1- gradient is referring to band；2- is mono-
Chain DNA；3- cyclic DNA；4- reaction product；
Fig. 2 is the scanning electron microscopic picture of the contrast agent of synthesis；
Fig. 3 is the result of the cytotoxicity experiment of the contrast agent of synthesis；
Fig. 4 is the result being imaged in vitro.
Specific embodiment
Below by specific embodiment, the present invention is further illustrated.The following examples are to make this field
Technical staff better understood when the present invention, but not impose any restrictions to the present invention.
Embodiment 1:
(1) preparation method of the circular DNA template with DNA primer, includes the following steps:
A.20 in the reaction system of μ L, it is 100 μM that 2 μ L concentration, which are added, and the linear DNA mould of phosphorylation modification is passed through at 5 ' ends
Plate, the DNA primer that 4 μ L concentration are 100 μM, 4 μ L concentration are the NaCl aqueous solution and 10 μ L deionized waters of 800mM, carry out high temperature
Annealing reaction, wherein 5 ' end regions of the linear DNA template of phosphorylation modification are passed through at 5 ' end regions of DNA primer and the 5 ' end
Complementary pairing, 3 ' end regions of DNA primer and 3 ' end complementary pairings of linear DNA template, the effect of NaCl are to improve DNA double
The stability of chain structure.
b.The linear DNA template of 5 ' end phosphorylation and the base sequence of DNA primer are as shown in table 1
The base sequence of table 1.DNA sample
C. sample is mixed by table 2
The cyclisation system component of 2. linear die of table
Mixed liquid is placed in PCR instrument and carries out following thermal annealing processes:
In annealing process, the both ends of linear DNA template can with DNA primer by base pair complementarity in conjunction with, realize pair
The cyclisation of linear DNA template, at this point, there are notches between the both ends of template；The circular DNA template concentration of acquisition is 10 μM.To
The T4DNA ligase of 1U is added in every 200ng DNA product obtained above, mixes sample by table 3.
The synthetic system of 3. circular DNA template of table
Mixed liquor is placed at 22 DEG C and stands 4h, T4DNA ligase is connected by the notch of template, obtains drawing with DNA
The circular DNA template of object, concentration are 1 μM.
(2) synthesis of circular DNA template is verified:
The synthesis of circular DNA template is verified by 12% polyacrylamide gel electrophoresis experiment.Deposition condition: electricity
Pressure, 110V；Time, 110min.
Applied sample amount is as shown in table 4:
4. polyacrylamide gel electrophoresis sample of table
Run glue the result is shown in Figure 1.
Linear DNA template is preferentially selected as 60-120bp without specific length requirement；Meanwhile preparing nanoscale magnetic resonance
The template sequence of image-forming contrast medium can be designed without specified base demands according to design requirement.
A kind of preparation method of magnetic resonance imaging contrast, includes the following steps:
(1) in the reaction vessel, the circular DNA template with DNA primer of final concentration of 300nM is added, it is final concentration of
The MnCl of 6mM2Solution, the atriphos mixed liquor (dNTPs) of final concentration of 2mM, final concentration of 1 × phi 29DNA polymerization
Enzyme buffer liquid (does not include MgCl2), the KCl aqueous solution and final concentration of the BSA of final concentration of 0.2mg/mL, final concentration of 40mM
For 29 polymerase of phi of 1U/ μ L, residual volume is supplied with deionized water, 400~600rpm, is reacted 3h under the conditions of 30 DEG C, is obtained
To white opacity solution；
(2) ultrasonic cleaner is to obtained 5~10min of white opacity solution ultrasound；
(3) 10000rpm is centrifuged 3~5min, after removing supernatant, (10 μ L matter is added in 10mL deionized water using weak aqua ammonia
Measure the concentrated ammonia liquor that score is 33%) washing lower layer's white product 1~3 time, centrifugation obtains magnetic resonance contrast agent after removing weak aqua ammonia.
Embodiment 2
The preparation method of circular DNA template with DNA primer is referring to embodiment 1.
A kind of preparation method of magnetic resonance imaging contrast, includes the following steps:
(1) in the reaction vessel, the circular DNA template with DNA primer of final concentration of 600nM is added, it is final concentration of
The MnCl of 7mM2Solution, the atriphos mixed liquor (dNTPs) of final concentration of 2mM, final concentration of 1 × phi 29DNA polymerization
Enzyme buffer liquid (does not include MgCl2), the KCl aqueous solution and final concentration of the BSA of final concentration of 0.2mg/mL, final concentration of 40mM
For 29 polymerase of phi of 1U/ μ L, residual volume is supplied with deionized water, 400~600rpm, is reacted 3h under the conditions of 30 DEG C, is obtained
To white opacity solution；
(2) ultrasonic cleaner is to obtained 5~10min of white opacity solution ultrasound；
(3) 10000rpm is centrifuged 3~5min, after removing supernatant, (10 μ L matter is added in 10mL deionized water using weak aqua ammonia
Measure the concentrated ammonia liquor that score is 33%) washing lower layer's white product 1~3 time, centrifugation obtains magnetic resonance contrast agent after removing weak aqua ammonia.
Embodiment 3:
The preparation method of circular DNA template with DNA primer is referring to specific embodiment 1.
A kind of preparation method of magnetic resonance imaging contrast, includes the following steps:
(1) in the reaction vessel, final concentration of 1 μM of the circular DNA template with DNA primer, final concentration of 8mM is added
MnCl2Solution, the atriphos mixed liquor (dNTPs) of final concentration of 2mM, final concentration of 1 × phi 29DNA polymerase are slow
Fliud flushing (does not include MgCl2), the KCl aqueous solution of the BSA of final concentration of 0.2mg/mL, final concentration of 40mM and final concentration of
29 polymerase of phi of 1U/ μ L, residual volume are supplied with deionized water, 400~600rpm, are reacted 3h under the conditions of 30 DEG C, are obtained
White opacity solution；
(2) ultrasonic cleaner is to obtained 5~10min of white opacity solution ultrasound；
(3) 10000rpm is centrifuged 3~5min, after removing supernatant, (10 μ L matter is added in 10mL deionized water using weak aqua ammonia
Measure the concentrated ammonia liquor that score is 33%) washing lower layer's white product 1~3 time, centrifugation obtains magnetic resonance contrast agent after removing weak aqua ammonia.
Embodiment 4:
The preparation method of circular DNA template with DNA primer is referring to specific embodiment 1.
A kind of preparation method of magnetic resonance imaging contrast, includes the following steps:
(1) in the reaction vessel, the circular DNA template with DNA primer of final concentration of 1mM, final concentration of 8mM is added
MnCl2 solution, the atriphos mixed liquor (dNTPs) of final concentration of 2mM, final concentration of 1 × phi29DNA polymerase is slow
Fliud flushing (do not include MgCl2), the KCl aqueous solution of the BSA of final concentration of 0.1mg/mL, final concentration of 20mM and final concentration of
29 polymerase of phi of 1U/mL, residual volume are supplied with deionized water, 400~600rpm, are reacted 3h under the conditions of 30 DEG C, are obtained
White opacity solution；
(2) ultrasonic cleaner is to obtained 5~10min of white opacity solution ultrasound；
(3) 10000rpm is centrifuged 3~5min, after removing supernatant, (10mL matter is added in 10mL deionized water using weak aqua ammonia
Measure the concentrated ammonia liquor that score is 33%) washing lower layer's white product 1~3 time, centrifugation obtains magnetic resonance contrast agent after removing weak aqua ammonia.
Embodiment 5:
The preparation method of circular DNA template with DNA primer is referring to specific embodiment 1.
A kind of preparation method of magnetic resonance imaging contrast, includes the following steps:
(1) in the reaction vessel, the circular DNA template with DNA primer of final concentration of 600nM is added, it is final concentration of
The MnCl2 solution of 8mM, the atriphos mixed liquor (dNTPs) of final concentration of 1mM, final concentration of 1 × phi29DNA polymerase
Buffer (do not include MgCl2), the KCl aqueous solution of the BSA of final concentration of 0.1mg/mL, final concentration of 60mM and final concentration of
29 polymerase of phi of 1U/mL, residual volume are supplied with deionized water, 400~600rpm, are reacted 5h under the conditions of 30 DEG C, are obtained
White opacity solution；
(2) ultrasonic cleaner is to obtained 5~10min of white opacity solution ultrasound；
(3) 10000rpm is centrifuged 3~5min, after removing supernatant, (10mL matter is added in 10mL deionized water using weak aqua ammonia
Measure the concentrated ammonia liquor that score is 33%) washing lower layer's white product 1~3 time, centrifugation obtains magnetic resonance contrast agent after removing weak aqua ammonia.
Embodiment 6:
The preparation method of circular DNA template with DNA primer is referring to specific embodiment 1.
A kind of preparation method of magnetic resonance imaging contrast, includes the following steps:
(1) in the reaction vessel, the circular DNA template with DNA primer of final concentration of 300nM is added, it is final concentration of
The MnCl2 solution of 8mM, the atriphos mixed liquor (dNTPs) of final concentration of 2mM, final concentration of 1 × phi29DNA polymerase
Buffer (does not include MgCl2), the KCl aqueous solution and final concentration of the BSA of final concentration of 0.05mg/mL, final concentration of 40mM
For 29 polymerase of phi of 2U/mL, residual volume is supplied with deionized water, 400~600rpm, is reacted 6h under the conditions of 30 DEG C, is obtained
To white opacity solution；
(2) ultrasonic cleaner is to obtained 5~10min of white opacity solution ultrasound；
(3) 10000rpm is centrifuged 3~5min, after removing supernatant, (10mL matter is added in 10mL deionized water using weak aqua ammonia
Measure the concentrated ammonia liquor that score is 33%) washing lower layer's white product 1~3 time, centrifugation obtains magnetic resonance contrast agent after removing weak aqua ammonia.
Embodiment 7:
The preparation method of circular DNA template with DNA primer is referring to specific embodiment 1.
A kind of preparation method of magnetic resonance imaging contrast, includes the following steps:
(1) in the reaction vessel, the circular DNA template with DNA primer of final concentration of 1mM, final concentration of 8mM is added
MnCl2 solution, the atriphos mixed liquor (dNTPs) of final concentration of 2mM, final concentration of 1 × phi29DNA polymerase is slow
Fliud flushing (do not include MgCl2), the KCl aqueous solution of the BSA of final concentration of 0.3mg/mL, final concentration of 40mM and final concentration of
29 polymerase of phi of 3U/mL, residual volume are supplied with deionized water, 400~600rpm, are reacted 4h under the conditions of 30 DEG C, are obtained
White opacity solution；
(2) ultrasonic cleaner is to obtained 5~10min of white opacity solution ultrasound；
(3) 10000rpm is centrifuged 3~5min, after removing supernatant, (10mL matter is added in 10mL deionized water using weak aqua ammonia
Measure the concentrated ammonia liquor that score is 33%) washing lower layer's white product 1~3 time, centrifugation obtains magnetic resonance contrast agent after removing weak aqua ammonia.
The nanoscale magnetic resonance imaging contrast obtained with embodiment 1 does following experiment:
1. the microscopic appearance of contrast agent characterizes
By sample solution drop on clean silicon wafer, its apparent pattern is observed using scanning electron microscope after freeze-drying, is such as schemed
Shown in piece 2, a plurality of line petal-shaped is presented in particle, and particle diameter distribution is in 200-400nm or so.
2. cytotoxicity experiment
10000 cells are added in every hole in (1) 96 orifice plate, altogether 1 control group of setting and 5 experimental groups, and every group parallel 5
It is a；It is PBS that sample is added in control group, and the contrast agent of various concentration, final concentration of 5,10,15,20,25 μ g/ are added in experimental group
ML, the contrast agent of addition is according to prepared by embodiment 1.96 orifice plates after the completion of sample-adding are in 5%CO2, in 37 DEG C of incubator
Culture is for 24 hours；After removing supernatant waste liquid, the fresh medium of 90 μ L is added, adds 10 μ L MTT reagents, continues to cultivate 4h；It inhales
After taking supernatant, 110 μ L Formazan lysates are added in every hole, and 10min is vibrated under 400rpm, measure suction of the sample at 490nm
Shading value calculates cell viability with experimental group/control group ratio.
(2) two kinds of cells, respectively MCF-7 and MDA-MB-231 cell, experimental result such as Fig. 3 institute are tested in this experiment altogether
Show, prepared magnetic resonance contrast agent is without obvious cytotoxicity.
3. external imaging experiment
Contrast agent prepared by embodiment 1 is subjected to imaging test according to different manganese contents, as shown in figure 4, manganese content
Higher, material imaging effect is better, meanwhile, it is above the imaging effect of water, illustrates that contrast agent has good imaging effect.
Sequence table
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acgggcgtca gtgcttccga aatgcttgcc accaccacca acaccaccac caccgtagtt 60
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cccgttgcag ctacctcgcc tt 22
Claims (4)
1. a kind of nanoscale magnetic resonance imaging contrast, which is characterized in that prepare by the following method:
(1) in the reaction vessel, the circular DNA template of final concentration of 300nM~1 μM, the MnCl of final concentration of 6~8mM is added2
Solution, atriphos mixed liquor, 29 DNA polymerase buffer liquid of phi, BSA, KCl aqueous solution and phi29 polymerase remain
Remaining volume is supplied with deionized water, 400~600rpm, reacts 3-6h under the conditions of 30 DEG C, obtains white opacity solution；
(2) ultrasonic cleaner is to obtained 5~10min of white opacity solution ultrasound；
(3) 10000rpm is centrifuged 3~5min, after removing supernatant, is washed lower layer's white product 1~3 time using weak aqua ammonia, centrifugation is gone
Except obtaining magnetic resonance contrast agent after weak aqua ammonia.
2. nanoscale magnetic resonance imaging contrast according to claim 1, which is characterized in that step (1) cyclic DNA
Template is the circular DNA template with DNA primer, is made with the following method:
A. according to the molar ratio of 1:2,5 ' ends are mixed by the linear DNA template and DNA primer of phosphorylation modification, it is described
5 ' end regions complementary pairings of 5 ' end regions of DNA primer and the linear DNA template by phosphorylation modification, 3 ' ends of DNA primer
3 ' the end regions complementary pairings in region and the linear DNA template by phosphorylation modification, are added the NaCl water of final concentration of 120mM
Reaction volume is complemented to 20 μ L with deionized water by solution；
B. above-mentioned mixed reaction system is placed in PCR instrument, carries out high annealing reaction, added into obtained DNA product
Enter T4 DNA ligase, reacts 3~8h at 22 DEG C, obtain the circular DNA template with DNA primer.
3. a kind of preparation method of nanoscale magnetic resonance imaging contrast, which comprises the following steps:
(1) in the reaction vessel, the circular DNA template of final concentration of 300nM~1 μM, the MnCl of final concentration of 6~8mM is added2
Solution, atriphos mixed liquor, phi29 DNA polymerase buffer liquid, BSA, KCl aqueous solution and phi29 polymerase, it is remaining
Volume is supplied with deionized water, 400~600rpm, reacts 3-6h under the conditions of 30 DEG C, obtains white opacity solution；
(2) ultrasonic cleaner is to obtained 5~10min of white opacity solution ultrasound；
(3) 10000rpm is centrifuged 3~5min, after removing supernatant, is washed lower layer's white product 1~3 time using weak aqua ammonia, centrifugation is gone
Except obtaining magnetic resonance contrast agent after weak aqua ammonia.
4. the preparation method of nanoscale magnetic resonance imaging contrast according to claim 3, which is characterized in that the step
(1) circular DNA template is the circular DNA template with DNA primer, is made with the following method:
A. according to the molar ratio of 1:2,5 ' ends are mixed by the linear DNA template and DNA primer of phosphorylation modification, it is described
5 ' end regions complementary pairings of 5 ' end regions of DNA primer and the linear DNA template by phosphorylation modification, 3 ' ends of DNA primer
3 ' the end regions complementary pairings in region and the linear DNA template by phosphorylation modification；The NaCl water of final concentration of 120mM is added
Solution is supplied reaction volume with deionized water；
B. above-mentioned mixed reaction system is placed in PCR instrument, carries out high annealing reaction, added into obtained DNA product
Enter T4 DNA ligase, reacts 3~8h at 22 DEG C, obtain the circular DNA template with DNA primer.
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| CN116059356A (en) * | 2022-08-18 | 2023-05-05 | 湖南省人民医院(湖南师范大学附属第一医院) | DNA nano-enzyme with self-oxygen production capability and preparation method and application thereof |
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| CN116059356A (en) * | 2022-08-18 | 2023-05-05 | 湖南省人民医院(湖南师范大学附属第一医院) | DNA nano-enzyme with self-oxygen production capability and preparation method and application thereof |
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