CN102234679B

CN102234679B - Preparation method of tumor detection nanoprobe

Info

Publication number: CN102234679B
Application number: CN 201110203537
Authority: CN
Inventors: 张先恩; 崔宗强; 李可; 张治平; 危宏平
Original assignee: Wuhan Institute of Virology of CAS
Current assignee: Wuhan Institute of Virology of CAS
Priority date: 2011-07-20
Filing date: 2011-07-20
Publication date: 2013-03-20
Anticipated expiration: 2031-07-20
Also published as: CN102234679A

Abstract

The invention discloses a preparation method of a tumor detection nanoprobe. The method comprises the following steps of: (1) constructing expression plasmids for expressing a ferritin heavy chain, expressing with colon bacillus, purifying ferritins and verifying and representing a cage structure with an electron microscope; (2) fusing a green fluorescent protein at an amino terminal of each ferritin and performing expression and purification to obtain a ferritin cage structure of which the surface expresses the fluorescent protein; (3) fusing a segment of tumor targeting small peptides RGD (Arginine-Glycine-Aspartate) at the amino terminal of a ferritin H chain of which the amino terminal is fused with the green fluorescent protein to prepare a ferritin cage structure of which the surface expresses the fluorescent protein and the tumor targeting peptides; and (4) synthesizing Fe3O5 nanoparticles in the cavity of the ferritin cage structure of which the surface expresses the fluorescent protein and the tumor targeting peptides simultaneously to obtain a multifunctional ferritin probe with tumor targeting, fluorescence and magnetism. In the nanoprobe, a tumor targeting function, a fluorescent imaging function and a magnetic resonance imaging function are fused integrally, so that synchronous detection in multiple modes can be realized.

Description

A kind of preparation method who detects the tumour nano-probe

Technical field

The present invention relates to the lesion detection technical field, more specifically relate to a kind of preparation method who detects the tumour nano-probe, by at ferritin cagelike structure surface display fluorescin and RGD cancer target small peptide, and the synthetic Fe in chamber within it ₃O ₄Nano particle, prepare have cancer target, the nanoparticle probes of magnetic, three kinds of functions of fluorescence.This three-function nano probe can be used in the detections such as the nuclear magnetic resonance (MRI) of the cancer target of tumour cell or live body and fluorescence imaging.

Background technology

Cancer is one of human main cause of death.Although oncobiology and tumour medical science have a great development, such as: the discovery of knubble biological marker, easy surgical procedure, the development of radiation and chemotherapy, total cancer survival rate does not all have significant the raising in the past twenty years.In order to improve the survival rate of tumour patient, we are badly in need of developing new early diagnosis of tumor method and methods for the treatment of.

The development of Nanometer scale science and technology has driven the development that is used for the nano material of molecular cell imaging and cancer therapy, has also driven the exploitation that is used for the nano-device of cancer detection and examination.Nano particle can not only provide the highly sensitive of cancer patient and specificity image-forming information, and can also transport cancer therapy drug to the position of tumour.Current, our understanding aspect following these three or limited: the one, be fit to the biomarker of imaging; The 2nd, the selection of imaging target and contrast strongthener; The 3rd, be used for making the chemical process of image probe bioid.We run into a lot of difficulties equally on developing cancer specificity imaging agents, such as: the 1) transportation of the probe of target tissue or tumour; 2) biocompatibility and bio-toxicity; 3) validity that signal strengthens in the stability of probe and the body; 4) abundant formation method and strategy.

Now, also there is very large challenge in the development of the nano-probe of tumor-targeting.Nuclear magnetic resonance (Magneticresonance imaging, MRI) is a kind of good imaging mode, and fine and smooth soft tissue contrast is provided, and can disclose the details on techtology and the anatomy, even can be used for animal and human's whole body imaging.Magnetic contrast medium often is used to enhanced contrast and amplifying signal.Although gadolinium-diethylene triamine pentacetic acid (DTPA) (Gd-DTPA) has the effect of very strong shortening T1 value and is widely accepted clinically, its contrast effect is relatively low and the interior retention time of body is too short.During the cell endocytic gadolinium and the toxicity behind the endocytosis gadolinium and biocompatibility or unknown.Recently, superparamag-netic iron oxide becomes the MRI T2 contrast medium of the targeting specific of a new generation.Superparamag-netic iron oxide than Gd-DTPA promote aspect the relaxation more effective.The magnetic property of superparamag-netic iron oxide can be controlled by size and the finishing thing of control core.The more important thing is that superparamag-netic iron oxide has very long blood retention time, the characteristics such as biological degradability and low bio-toxicity.

Although we have made a lot of effort in development aspect the MRI contrast medium of superparamag-netic iron oxide, a lot of difficult problems still exist.A main difficult problem is a kind of face finish material of development, and this material not only can also be provided for the crosslinked activity functional groups of probe molecule controllable biological by stable nanoparticles.Traditional molecule (as: dextran) that is used for the stabilized magnetic ferric oxide nanometer particle because and a little less than the interaction force between nano particle, cause nano particle under the physiological condition and the reunion between the shelf lives, precipitation so be easy to split away off from the surface of nano particle.

Simultaneously, although MRI is a kind of main formation method of oncology, its resolving power is very limited when being used for molecule and cell imaging.And independent MRI contrast medium such as superparamag-netic iron oxide are difficult to the target tumor cell or tissue.

Ferritin is an octahedra symmetrical globular preteins cage structure that is formed by 24 identical subunit self-assemblies of structure.The outer dia of ferritin is 12nm, and inside is the cavity of a 6-8nm.Ferritin is the same with SV40 also to be a kind of typical albumen cage structure.The endogenous human ferritin is comprised of two kinds of different subunits, H chain and L chain, and the H chain is different with the molecular size range of L chain, is respectively 21kDa and 19kDa.The H chain contains the enzyme active sites of a conservative ferrous oxidase, can two Fe of catalysis ²⁺Oxidation; L chain inside participates in the formation of ferriferous oxide core with a large amount of negative charges.An energy catalysis Fe is arranged in the H of Mammals ferritin chain ²⁺Be oxidized to Fe ³⁺Ferrous oxidase.The H chain of Mammals ferritin also can be self-assembled into separately 24 aggressiveness globular preteins cage structures.The aminoterminal of ferritin stretches to outside surface, is highly susceptible to genetic manipulation, merges various albumen and small peptide.

So, attempt being used for nuclear magnetic resonance and the fluorescence imaging of cancer target based on a kind of nano particle with three kinds of functions such as cancer target, magnetic, fluorescence of ferritin cagelike structure development.

Summary of the invention

The objective of the invention is to be to provide a kind of preparation method who detects the tumour nano-probe, at an aminoterminal fusion green fluorescent protein (GFP) of ferritin H chain, and then merge one section RGD cancer target small peptide.The ferritin with cancer target peptide and GFP that purifying is good, Fe is synthesized in the chamber within it again ₃O ₄Nano particle, thereby a kind of three-function nano particle of nanoparticle probes that development has three kinds of functions such as cancer target, magnetic, fluorescence.This three-function nano probe can be used in the convenient of tumour such as the nuclear magnetic resonance (MRI) of cancer target of the viable cell of tumour or live body and fluorescence imaging and detects.

In order to realize above-mentioned purpose, the present invention adopts following technical measures:

A kind of preparation method who detects the tumour nano-probe the steps include:

(1), the expression plasmid (pET-rHF) of construction expression ferritin heavy chain, intestinal bacteria (E.coli BL21) (available from promega) are expressed, the purifying ferritin, the electron microscope checking characterizes its cagelike structure.

(2), merge a green fluorescent protein at the ferritin aminoterminal, the ferritin cagelike structure of fluorescin that expression and purification has prepared surface display is carried out structure and fluorescence property and is characterized.

(3), merge the aminoterminal that green fluorescent protein ferritin H chain is arranged at aminoterminal and merge again one section cancer target small peptide RGD (giving birth to the worker available from Shanghai), prepare the ferritin cagelike structure that the surface has been showed fluorescin and cancer target peptide simultaneously.

(4), the ferritin cagelike structure of fluorescin and cancer target peptide, the synthetic Fe in chamber have within it been showed simultaneously based on the surface ₃O ₄Nano particle, be prepared into have cancer target, the ferritin multiprobe of fluorescence, magnetic.

A kind of nano-probe detects tumour, the steps include:

(1), utilize the tumour cell checking probes such as A549 (available from Wuhan University Chinese Typical Representative culture collection center), U87MG (available from Wuhan University Chinese Typical Representative culture collection center) to be used for the target recognition function of tumour.

(2), micro-imaging detects the fluorescence imaging function that this three functional probe is used for tumour.

(3), Magnetic resonance imaging verifies that three functional probes are used for the nuclear magnetic resonance function of tumour.

(4), three functional probes detect and can identify specifically tumour cell, and realize fluorescence imaging and the nuclear magnetic resonance of tumour cell

The present invention compared with prior art, concrete following advantage and effect:

This research and utilization genetically manipulated technology has merged cancer target peptide and green fluorescent protein at the aminoterminal of ferritin H chain, and based on the function of the original ferrous oxidase of ferritin, can carry out external biological mineralising, the synthetic Fe in chamber within it to ferritin ₃O ₄Nano particle, thus prepare a kind of three function lesion detection nano-probes of organic/inorganic heterozygosis.Ferritin itself has good biocompatibility, as Fe ₃O ₄The surface protection group of nano particle can also be stablized the Fe of inner chamber ₃O ₄Nano particle.This nano-probe cancer target, fluorescence imaging, nuclear magnetic resonance is multi-functional combines together, easy to use and can realize multimodal synchronous detection, be expected to be applied to special tumour cell research, the multi-functional in good time imaging of clinical tumor diagnosis and the medicine carrying targeted therapy of tumour etc.

Description of drawings

Fig. 1 is a kind of preparation method's synoptic diagram that detects the tumour nano-probe.

Cancer target peptide and green fluorescent protein are showed in ferritin cage modle particle surface, within it the synthetic Fe in chamber ₃O ₄Nano particle.

Fig. 2 A-a is a kind of ferritin electrophoresis phenogram of purifying

Fig. 2 A-b 2B is the electrophoresis phenogram of the fusion rotein of a kind of ferritin of purifying and green fluorescent protein

Fig. 2 A-c is the electrophoresis phenogram of fusion rotein of a kind of ferritin, green fluorescent protein and cancer target peptide RGD of purifying

Fig. 2 B-a is a kind of Electronic Speculum phenogram of ferritin cage modle grain pattern

Fig. 2 B-b the has been a kind of surface display ferritin cage modle particle Electronic Speculum phenogram of green fluorescent protein

The Electronic Speculum phenogram of the ferritin cage modle particle of Fig. 2 B-c has been a kind of surface display green fluorescent protein and cancer target peptide RGD

Fig. 3 A is the synthetic Fe of a kind of ferritin cage modle particle inner chamber ₃O ₄The Electronic Speculum phenogram of nano particle

Fig. 3 B be a kind of surface display green fluorescent protein, inner chamber synthesized Fe ₃O ₄The ferritin cage modle particle Electronic Speculum phenogram of nano particle

Fig. 3 C is the ferritin cagelike structure that a kind of surface display has fluorescin and cancer target peptide, both complete three-function nano probe table Electronic Speculum phenogram.

Fig. 4 A is that a kind of three-function nano probe is used for the special fluorescence imaging of tumour cell A549.

Fig. 4 B is the light field image of the A549 tumour cell of fluorescence imaging among a kind of and Fig. 4 A.

Fig. 4 C is that a kind of three-function nano probe is used for the special fluorescence imaging of tumour cell U87MG.

Fig. 4 D be with Fig. 4 A in the light field image of U87MG tumour cell of fluorescence imaging.

Fig. 5 A is that (0,2,6,20,60, three functional probe T2 nuclear magnetic resonances 200nM) are figure as a result for different concns

Fig. 5 B be different concns (0,2,6,20,60, T2 relaxation time corresponding to three functional probe nuclear magnetic resonances 200nM)

Fig. 5 C-a is the T2 nuclear magnetic resonance figure as a result of untreated A549 cell

Fig. 5 C-b is without the T2 nuclear magnetic resonance of the A549 cell of target function ferritin particle disposal figure as a result

Fig. 5 C-c is the T2 nuclear magnetic resonance figure as a result with A549 cell that three function ferritin particle probe of target, fluorescence, magnetic process

Fig. 5 C-d is the T2 nuclear magnetic resonance figure as a result of untreated U87MG cell

Fig. 5 C-e is without the T2 nuclear magnetic resonance of the U87MG cell of target function ferritin particle disposal figure as a result

Fig. 5 C-f is the T2 nuclear magnetic resonance figure as a result with U87MG cell that three function ferritin particle probe of target, fluorescence, magnetic process.

Embodiment

Ferritin is that diameter is the hollow protein ball of 12 nanometers, has three different interfaces: outside surface and subunit interface and inner chamber, these three interfaces can be endowed new function by artificial design.Aminoterminal at ferritin has merged RGD cancer target small peptide and green fluorescent protein, preparation cage modle ferritin particle, make its surface display cancer target small peptide and green fluorescent protein, then based on the function of the original ferrous oxidase of ferritin, Fe is synthesized in the chamber within it ₃O ₄Nano particle, thus manufacture a kind of nuclear magnetic resonance of cancer target and the multifunctional nanoparticles of fluorescence imaging, be expected to be applied to special tumour cell research, the multi-functional in good time imaging of clinical tumor diagnosis and the medicine carrying targeted therapy of tumour etc.

Embodiment 1:

(1) by the synthetic people's ferritin heavy chain gene rHF of polymerase chain reaction (PCR) amplification, be inserted on the expression vector pET-28a (available from promega company), construct the expression plasmid pET-rHF that expresses ferritin heavy chain, be transformed into intestinal bacteria E.coli BL21 (λ DE3) (available from promega company), 37 ℃ of constant temperature, the 200r/min shaking culture is cultured to OD600 between 0.4～0.6, add IPTG to final concentration 1mmol/L to culture, culture is all behind 25 ℃ of continuation inducing culture 8h, 4 ℃, the 6000g centrifugal collecting cell, cell precipitation is with Tris-HCl damping fluid (20mM Tris-HCl, 50mM NaCl, pH 8.0) clean thalline once, then be resuspended in the 30mLTris-HCl damping fluid, after the ultrasonication, with the centrifugal 30min of 12000r/min, collecting supernatant liquor places 60 ℃ of water-baths to react 10min, then the centrifugal 20min of 12000r/min collects supernatant liquor and concentrates with Amicon Ultra15 super filter tube (100KDa MW cut-off) (available from mil1ipore company).Concentrated protein sample is collected elution peak with molecular sieve column Superdex 200 10/300GL or Superose 6 10/300GL (available from GE company) purifying, obtains the iron egg of the cagelike structure of purifying.

(2) insert green fluorescence protein gene (pcr amplification is synthetic) among the plasmid pET-rHF, the expression plasmid pET-GFP-rHF of the fusion rotein of construction expression ferritin and green fluorescent protein, intestinal bacteria E.coli BL21 (λ DE3) expresses, purified fusion protein (concrete test method is the same), the ferritin cagelike structure of fluorescin of having prepared surface display.

(3) in plasmid pET-GFP-rHF, insert RGD small peptide expressing gene (giving birth to the worker available from Shanghai), the expression plasmid pET28a-RGF of the fusion rotein of construction expression ferritin-green fluorescent protein-cancer target peptide RGD, intestinal bacteria E.coli BL21 (λ DE3) expresses, purified fusion protein (concrete test method is the same), prepares the ferritin cagelike structure that the surface has been showed fluorescin and cancer target peptide simultaneously.

(4) showed simultaneously the ferritin cagelike structure of fluorescin and cancer target peptide based on the surface, within it the synthetic Fe in chamber ₃O ₄Nano particle, concrete grammar is: be added in the reaction cup of titration apparatus after NaCl (0.1M) solution of 30mL 0.4 μ M rHF albumen (or GFP-rHF albumen or RGF albumen) is degassed, pass into high pure nitrogen, place 65 ℃ of waters bath with thermostatic control.Adopt the permanent titration mode of titration apparatus, the pH value of whole reaction process reaction solution is controlled at 8.5 with the NaOH titration of 50mM.12.5mM ferrous ammonium sulphate ((NH ₄) ₂Fe (SO ₄) ₂6H ₂O) and the H of 4.17mM ₂O ₂Fresh obtain solution joins in the above-mentioned reaction solution with the speed of 0.16mL/min simultaneously, stops after 30 minutes dripping, and makes that the theoretical iron atom carrying capacity of each albumen cage structure reaches 5000 in the reaction solution.Reaction solution continues reaction 5 minutes after stopping to drip, the sodium citrate solution chelating that then adds 0.6mL 0.3M falls unnecessary iron ion, then cools to room temperature, namely successfully obtains the synthetic Fe of inner chamber ₃O ₄The ferritin probe of nano particle, ferritin cagelike structure surface display fluorescin and cancer target peptide because its surface had fluorescin and cancer target peptide, add the Fe of inner chamber ₃O ₄Nano particle is the complete ferritin multiprobe with cancer target, fluorescence, magnetic.

Embodiment 2:

Three function lesion detection probes are used for the specificity fluorescent imaging of tumour cell, the steps include:

(1) select people pernicious glioblastoma cells strain U87MG (available from Wuhan University Chinese Typical Representative culture collection center) and Non-small cell lung carcinoma cell line A549 cell available from Wuhan University Chinese Typical Representative culture collection center), its cell surface all has the special up-regulated expression of tumor markers α v β 3 integrins.The cell DMEM nutrient solution that contains 10% foetal calf serum, at 37 ℃, the cultivation of going down to posterity in 5% the carbon dioxide environment.The ratio that goes down to posterity 1: 2.

(2) U87MG and A549 cell are layered in the culture dish that central authorities glue cover glass, at 37 ℃, and in 5% the carbon dioxide environment 24-36 hour, after reaching 50% cell degree of converging, the PBS damping fluid is washed 3 times, is changed to binding buffer liquid (20mM Tris-HCl, 150mM NaCl, 1mM Ca ²⁺, 1mM Mg ²⁺, 1%BSA, pH 7.4), it is 50nM that the three function ferritin nano-probes that prepare are added cell to final concentration, hatches 1 hour for 37 ℃, detects its fluorescence under fluorescent microscope, and with the ferritin particle that does not merge the cancer target peptide in contrast.

(3) surface at U87MG and A549 cell is found in fluoroscopic examination, has green fluorescence signal (Fig. 4) clearly, and the ferritin particle that does not carry the cancer target peptide can not produce fluorescent signal at U87MG and A549 cell, illustrates that the multiprobe of above-mentioned preparation can be used for selectively targeted identification and the fluorescence imaging of tumour cell.

Embodiment 3:

Three function lesion detection probes are used for the nuclear magnetic resonance of tumour cell as contrast medium.The steps include:

(2) 10 ⁶Individual U87MG and A549 cell (available from Wuhan University Chinese Typical Representative culture collection center) PBS damping fluid is washed 3 times, is changed to binding buffer liquid (20mM Tris-HCl, 150mM NaCl, 1mMCa ²⁺, 1mM Mg ²⁺, 1%BSA, pH 7.4), the three function ferritin nano-probes that prepare or do not merge the cancer target peptide but merge and fluorescin is arranged and contain Fe ₃O ₄Making its probe final concentration in the nano particle ferritin particle adding cell is 50nM, hatches 30 minutes for 37 ℃.

(3) cell of three-function nano probe processing utilizes the 4.7T imager to carry out the T2 imaging, and the T2 relaxation time of U87MG and A549 cell is respectively 283.8ms and 314.0ms.And be respectively 457.3ms and 451.3ms with the U87MG of the ferritin particle disposal that does not merge the cancer target peptide and the T2 relaxation time of A549 cell.Be not respectively 454.4ms and 447.6ms with the U87MG of any contrast medium processing and the T2 relaxation time of A549 cell.The three-function nano probe that above-mentioned preparation is described can cause the remarkable reduction in T2 relaxation time when being used for the tumour cell nuclear magnetic resonance.So this three-function nano probe can be used for selectively targeted identification and the nuclear magnetic resonance of tumour cell.

Claims

1. a preparation method who detects the tumour nano-probe the steps include:

A, by the synthetic people's ferritin heavy chain gene of polymerase chain reaction (PCR) amplification RHF,Be inserted on the expression vector pET-28a, construct the expression plasmid pET-that expresses ferritin heavy chain RHF, be transformed into intestinal bacteria E. coliBL21 (λ DE3), 37 ℃ of constant temperature, 200 r/min shaking culture are cultured to OD600 between 0.4 ~ 0.6, add IPTG to final concentration 1 mmol/L to culture, culture is all behind 25 ℃ of continuation inducing culture 8 h, 4 ℃, 6000 g centrifugal collecting cells, cell precipitation is with the Tris-HCl damping fluid: 20 mM Tris-HCl, 50 mM NaCl, pH 8.0, clean thalline once, then be resuspended in the 30 mLTris-HCl damping fluids, after the ultrasonication, with centrifugal 30 min of 12000 r/min, collect supernatant liquor and place 60 ℃ of water-bath reactions 10 min, then centrifugal 20 min of 12000 r/min, it is concentrated with Amicon Ultra15 super filter tube to collect supernatant liquor, concentrated protein sample is collected elution peak with molecular sieve column Superdex 200 10/300 GL or Superose 6 10/300 GL purifying, obtains the ferritin of the cagelike structure of purifying;

B, plasmid pET- RHFMiddle insertion green fluorescence protein gene, the expression plasmid pET-of the fusion rotein of construction expression ferritin and green fluorescent protein GFP-rHF, intestinal bacteria E. coliBL21 (λ DE3) expresses, purified fusion protein, the ferritin cagelike structure of fluorescin of having prepared surface display;

C, at plasmid pET- GFP-rHFMiddle insertion RGD small peptide expressing gene, the expression plasmid pET28a-of the fusion rotein of construction expression ferritin-green fluorescent protein-cancer target peptide RGD RGF, intestinal bacteria E. coliBL21 (λ DE3) expresses, purified fusion protein, prepares the ferritin cagelike structure that the surface has been showed fluorescin and cancer target peptide simultaneously;

D, the ferritin cagelike structure of having showed simultaneously fluorescin and cancer target peptide based on the surface, the within it synthetic Fe in chamber ₃O ₄Nano particle, method is: be added in the reaction cup of titration apparatus after the NaCl solution of 30 mL, 0.4 μ M rHF albumen or GFP-rHF albumen or RGF albumen is degassed, pass into high pure nitrogen, place 65 ℃ of waters bath with thermostatic control, adopt the permanent titration mode of titration apparatus, the pH value of whole reaction process reaction solution is controlled at the ferrous ammonium sulphate of 8.5,12.5 mM and the H of 4.17 mM with the NaOH titration of 50 mM ₂O ₂Fresh obtain solution joins in the above-mentioned reaction solution with the speed of 0.16 mL/min simultaneously, stop after 30 minutes dripping, make that the theoretical iron atom carrying capacity of each albumen cage structure reaches 5000 in the reaction solution, reaction solution continues reaction 5 minutes after stopping to drip, then the sodium citrate solution chelating that adds 0.6 mL, 0.3 M falls unnecessary iron ion, then cool to room temperature, successfully obtain the synthetic Fe of inner chamber ₃O ₄The ferritin probe of nano particle, ferritin cagelike structure surface display fluorescin and cancer target peptide, add the Fe of inner chamber ₃O ₄Nano particle is the ferritin multiprobe of complete cancer target, fluorescence, magnetic.