CN102234679B - Preparation method of tumor detection nanoprobe - Google Patents

Abstract

The invention discloses a method for preparing a nano-probe for detecting tumors. The steps include: (1) constructing an expression plasmid expressing ferritin heavy chain, expressing and purifying ferritin in Escherichia coli, and verifying and characterizing its cage structure with an electron microscope. (2) A green fluorescent protein was fused to the amino terminal of ferritin, and the ferritin cage structure of the fluorescent protein was displayed on the surface prepared by expression and purification. (3) A short tumor-targeting peptide RGD was fused to the amino-terminus of the green fluorescent protein ferritin H-chain to prepare a ferritin cage structure displaying both fluorescent protein and tumor-targeting peptide on the surface. (4) Based on the ferritin cage structure displaying both fluorescent protein and tumor-targeting peptide on the surface, Fe ₃ O ₄ nanoparticles were synthesized in its inner cavity to prepare multifunctional ferritin with tumor targeting, fluorescence and magnetism probe. The nanoprobe integrates the functions of tumor targeting, fluorescence imaging and magnetic resonance imaging, and can realize multi-mode simultaneous detection.

Description

A preparation method for detecting tumor nanoprobes

technical field

The present invention relates to the technical field of tumor detection, more specifically to a method for preparing a nanoprobe for detecting tumors, by displaying fluorescent proteins and RGD tumor-targeting short peptides on the surface of a ferritin cage structure, and synthesizing Fe ₃ O in its inner cavity ₄ Nanoparticles, preparing nanoparticle probes with three functions of tumor targeting, magnetism and fluorescence. This trifunctional nanoprobe can be used for the detection of tumor cells or living tumors in magnetic resonance imaging (MRI) and fluorescence imaging.

Background technique

Cancer is one of the leading causes of human death. Despite great advances in tumor biology and oncology medicine, such as the discovery of tumor biomarkers, facile surgical procedures, and the development of radiotherapy and chemotherapy, overall cancer survival rates have not significantly improved over the past two decades. improve. In order to improve the survival rate of tumor patients, we urgently need to develop new methods for early diagnosis and treatment of tumors.

The development of nanoscience and technology has led to the development of nanomaterials for molecular cell imaging and cancer treatment, as well as the development of nanodevices for cancer detection and screening. Nanoparticles can not only provide highly sensitive and specific imaging information of cancer patients, but also transport anticancer drugs to tumor sites. Currently, our knowledge is limited in three areas: first, the biomarkers suitable for imaging; second, the choice of imaging targets and contrast-enhancing materials; and third, the chemical methods used to biolize the imaging probes . We also encountered many difficulties in the development of cancer-specific imaging reagents, such as: 1) delivery of probes targeting tissues or tumors; 2) biocompatibility and biotoxicity; 3) probe stability and in vivo signal Enhanced effectiveness; 4) Enough imaging methods and strategies.

Today, the development of tumor-targeted nanoprobes also presents great challenges. Magnetic resonance imaging (MRI) is an excellent imaging modality that provides fine soft-tissue contrast, can reveal tissue morphological and anatomical details, and can even be used for whole-body imaging in animals and humans. Magnetic contrast agents are often used to enhance contrast and amplify signal. Although gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA) has a strong T1-shortening effect and is widely accepted clinically, its contrast effect is relatively low and its retention time in the body is too short. The toxicity and biocompatibility of gadolinium during and after endocytosis are unknown. Recently, magnetic iron oxide nanoparticles have emerged as a new generation of target-specific MRI T2 contrast agents. Magnetic iron oxide nanoparticles are more effective than Gd-DTPA in promoting relaxation. The magnetic properties of magnetic iron oxide nanoparticles can be manipulated by controlling the size of the core and surface modifications. More importantly, the magnetic iron oxide nanoparticles have the characteristics of long blood retention time, biodegradability and low biotoxicity.

Despite our efforts in developing MRI contrast agents based on magnetic iron oxide nanoparticles, many challenges still remain. The main challenge is to develop a surface modification material that not only stabilizes the nanoparticles but also provides active functional groups for the controllable biocrosslinking of probe molecules. Traditional molecules used to stabilize magnetic iron oxide nanoparticles (such as dextran) are easily detached from the surface of nanoparticles due to weak interaction with nanoparticles, resulting in the loss of nanoparticles under physiological conditions and storage. The reunion and precipitation during the period.

Also, while MRI is a major imaging modality in oncology, it has limited resolution when used for molecular and cellular imaging. Moreover, MRI contrast agents alone, such as magnetic iron oxide nanoparticles, are difficult to target to tumor cells or tissues.

Ferritin is an octahedral symmetric globular protein cage structure formed by self-assembly of 24 subunits with the same structure. The outer diameter of ferritin is 12nm, and the inside is a cavity of 6-8nm. Like SV40, ferritin is also a typical protein cage structure. Endogenous human ferritin consists of two different subunits, H chain and L chain, and the molecular weights of H chain and L chain are different, 21kDa and 19kDa, respectively. The H chain contains a conserved ferrous oxidase enzyme active site, which can catalyze the oxidation of two Fe ²⁺ ; the L chain has a large number of negative charges inside and participates in the formation of the iron oxide core. In the H chain of mammalian ferritin, there is a ferrous oxidase that can catalyze the oxidation of Fe ²⁺ to Fe ³⁺ . The H chain of mammalian ferritin alone can also self-assemble into a 24-mer globular protein cage structure. The amino terminal of ferritin extends to the outer surface, which is very easy for genetic manipulation and fusion of various proteins and short peptides.

Therefore, try to develop a nanoparticle with three functions of tumor targeting, magnetism and fluorescence based on ferritin cage structure, which can be used for tumor targeting magnetic resonance imaging and fluorescence imaging.

Contents of the invention

The purpose of the present invention is to provide a method for preparing a nano-probe for detecting tumors. A green fluorescent protein (GFP) is fused to the amino terminal of the ferritin H chain, and then a segment of RGD tumor-targeting short peptide is fused. Purified ferritin with tumor-targeting peptide and GFP, and then synthesized Fe ₃ O ₄ nanoparticles in its inner cavity, so as to develop a nanoparticle probe with three functions such as tumor targeting, magnetism, and fluorescence. Trifunctional nanoparticles. This trifunctional nanoprobe can be used for the convenient detection of tumors such as tumor-targeted magnetic resonance imaging (MRI) and fluorescence imaging in living cells of tumors or in vivo.

In order to achieve the above object, the present invention adopts the following technical measures:

A preparation method for detecting tumor nanoprobes, the steps of which are:

(1) Construct the expression plasmid (pET-rHF) expressing ferritin heavy chain, express and purify ferritin in Escherichia coli (E.coli BL21) (purchased from promega), and verify and characterize its cage structure by electron microscopy.

(2) A green fluorescent protein was fused to the amino terminal of ferritin, expressed and purified to prepare a ferritin cage structure of fluorescent protein on the surface, and the structure and fluorescent properties were characterized.

(3) A short tumor-targeting peptide RGD (purchased from Shanghai Shenggong) was fused to the amino-terminus of the green fluorescent protein ferritin H chain, and a fluorescent protein and tumor-targeting peptide were prepared on the surface at the same time. ferritin cage structure.

(4) Based on the ferritin cage structure displaying both fluorescent protein and tumor targeting peptide on the surface, Fe ₃ O ₄ nanoparticles were synthesized in its inner cavity to prepare multifunctional ferritin with tumor targeting, fluorescence and magnetism probe.

A kind of nanoprobe detects tumor, and its steps are:

(1) Using tumor cell verification probes such as A549 (purchased from the China Center for Type Culture Collection of Wuhan University) and U87MG (purchased from the China Center for Type Culture Collection of Wuhan University) for tumor targeting recognition functions.

(2) Microscopic imaging detection The trifunctional probe is used for the fluorescence imaging function of the tumor.

(3) Nuclear magnetic resonance imaging verifies the magnetic resonance imaging function of the three-function probe used in tumors.

(4) The detection of three functional probes can specifically identify tumor cells, and realize fluorescence imaging and magnetic resonance imaging of tumor cells

Compared with the prior art, the present invention has the following advantages and effects:

In this study, using gene manipulation technology, a tumor-targeting peptide and green fluorescent protein were fused to the amino terminal of the ferritin H chain, and based on the function of ferritin's original ferrous oxidase, ferritin can be biomineralized in vitro, _Fe3O4 nanoparticles were synthesized in its lumen to prepare an organic/inorganic hybrid trifunctional nanoprobe for _tumor detection. Ferritin itself has excellent biocompatibility, and as the surface protection group of Fe ₃ O ₄ nanoparticles, it can also stabilize the inner cavity of Fe ₃ O ₄ nanoparticles. The nanoprobe integrates tumor targeting, fluorescence imaging, and magnetic resonance imaging. It is easy to use and can realize multi-mode simultaneous detection. It is expected to be applied to specific tumor cell research and multi-functional timely imaging for clinical diagnosis of tumors. and drug-loaded targeted therapy for tumors.

Description of drawings

Fig. 1 is a schematic diagram of a method for preparing a nanoprobe for detecting tumors.

Tumor-targeting peptide and green fluorescent protein were displayed on the surface of ferritin cage particles, and Fe ₃ O ₄ nanoparticles were synthesized in its lumen.

Figure 2A-a is a purified ferritin electrophoresis characterization diagram

Figure 2A-b 2B is an electrophoresis characterization diagram of a fusion protein of purified ferritin and green fluorescent protein

Figure 2A-c is an electrophoretic characterization diagram of a fusion protein of purified ferritin, green fluorescent protein and tumor targeting peptide RGD

Figure 2B-a is an electron microscope representation of a ferritin cage particle structure

Figure 2B-b is an electron microscope representation of ferritin cage particles displaying green fluorescent protein on the surface

Figure 2B-c is an electron microscope representation of ferritin cage particles displaying green fluorescent protein and tumor targeting peptide RGD on the surface

Figure 3A is an electron microscope representation of Fe ₃ O ₄ nanoparticles synthesized in the cavity of ferritin cage particles

Figure 3B is an electron microscope representation of ferritin cage particles with green fluorescent protein on the surface and Fe ₃ O ₄ nanoparticles synthesized in the inner cavity

Fig. 3C is a ferritin cage structure with fluorescent protein and tumor targeting peptide displayed on the surface, which is a complete three-functional nanoprobe surface electron microscope characterization diagram.

Figure 4A is a trifunctional nanoprobe used for specific fluorescence imaging of tumor cell A549.

Figure 4B is a bright field image of A549 tumor cells imaged with fluorescence in Figure 4A.

Figure 4C is a trifunctional nanoprobe used for specific fluorescence imaging of tumor cell U87MG.

Figure 4D is a bright field image of U87MG tumor cells imaged with fluorescence in Figure 4A.

Fig. 5A is the results of magnetic resonance imaging of three functional probes T2 at different concentrations (0, 2, 6, 20, 60, 200nM)

Figure 5B is the T2 relaxation time corresponding to the magnetic resonance imaging of the three functional probes at different concentrations (0, 2, 6, 20, 60, 200nM)

Figure 5C-a is the T2 magnetic resonance imaging result map of untreated A549 cells

Figure 5C-b is the result of T2 magnetic resonance imaging of A549 cells treated with ferritin particles without targeting function

Figure 5C-c is the T2 magnetic resonance imaging results of A549 cells treated with targeting, fluorescent and magnetic trifunctional ferritin particle probes

Figure 5C-d is the result of T2 magnetic resonance imaging of untreated U87MG cells

Figure 5C-e are T2 magnetic resonance imaging results of U87MG cells treated with ferritin particles without targeting function

Figure 5C-f are T2 magnetic resonance imaging results of U87MG cells treated with targeting, fluorescent and magnetic trifunctional ferritin particle probes.

Detailed ways

Ferritin is a hollow protein sphere with a diameter of 12 nanometers, which has three different interfaces: the outer surface, the subunit interface and the inner cavity, and these three interfaces can be endowed with new functions through artificial design. RGD tumor-targeting short peptide and green fluorescent protein were fused at the amino terminal of ferritin to prepare cage-type ferritin particles to display tumor-targeting short peptide and green fluorescent protein on the surface, and then based on the original ferrous oxidation of ferritin function of the enzyme, and synthesize Fe ₃ O ₄ nanoparticles in its inner cavity, thereby manufacturing a multifunctional nanoparticle for tumor-targeted magnetic resonance imaging and fluorescence imaging, which is expected to be applied to specific tumor cell research and clinical diagnosis of tumors. Multifunctional timely imaging and drug-loaded targeted therapy of tumors, etc.

Example 1:

A preparation method for detecting tumor nanoprobes, the steps of which are:

(1) Amplify the synthetic human ferritin heavy chain gene rHF by polymerase chain reaction (PCR), insert it into the expression vector pET-28a (purchased from Promega Company), and construct the expression plasmid pET- rHF, transformed into Escherichia coli E.coli BL21 (λDE3) (purchased from Promega Company), 37 ℃ constant temperature, 200r/min shaking culture to OD600 between 0.4 ~ 0.6, add IPTG to the culture to a final concentration of 1mmol/L After the cultures were induced for 8 hours at 25°C, the cells were collected by centrifugation at 6000g at 4°C, and the cell pellet was washed once with Tris-HCl buffer (20mM Tris-HCl, 50mM NaCl, pH 8.0), and then resuspended in In 30mL Tris-HCl buffer solution, after ultrasonic crushing, centrifuge at 12000r/min for 30min, collect the supernatant and place it in a 60°C water bath for 10min, then centrifuge at 12000r/min for 20min, collect the supernatant with an Amicon Ultra15 ultrafiltration tube (100KDa MW cut-off) (purchased from millipore company) concentrated. The concentrated protein sample was purified with a molecular sieve column Superdex 200 10/300GL or Superose 6 10/300GL (purchased from GE), and the elution peaks were collected to obtain purified cage-shaped iron eggs.

(2) Insert the green fluorescent protein gene (PCR amplification and synthesis) into the plasmid pET-rHF, construct the expression plasmid pET-GFP-rHF expressing the fusion protein of ferritin and green fluorescent protein, and express it in E. coli BL21 (λDE3) 1. Purify the fusion protein (the specific test method is the same as above), and prepare a ferritin cage structure displaying the fluorescent protein on the surface.

(3) Insert the RGD short peptide expression gene (purchased from Shanghai Sangong) into the plasmid pET-GFP-rHF, and construct the expression plasmid pET28a-RGF expressing the fusion protein of ferritin-green fluorescent protein-tumor targeting peptide RGD, large intestine Bacillus E.coli BL21(λDE3) expresses and purifies the fusion protein (the specific test method is the same as above), and prepares a ferritin cage structure displaying both fluorescent protein and tumor targeting peptide on the surface.

(4) Synthesize Fe ₃ O ₄ nanoparticles in its inner cavity based on the ferritin cage structure with fluorescent protein and tumor targeting peptide displayed on the surface at the same time. The specific method is: 30mL 0.4μM rHF protein (or GFP-rHF protein or RGF protein) NaCl (0.1M) solution was degassed and added to the reaction cup of the titrator, fed with high-purity nitrogen, and placed in a 65°C constant temperature water bath. Using the constant titration mode of the titrator, the pH value of the reaction solution was controlled at 8.5 with 50mM NaOH titration during the entire reaction process. 12.5mM ferrous ammonium sulfate ((NH ₄ ) ₂ Fe(SO ₄ ) ₂ ·6H ₂ O) and 4.17 The freshly prepared mM H ₂ O ₂ solution was added to the above reaction solution at a rate of 0.16 mL/min at the same time, and the dropwise addition was stopped after 30 minutes, so that the theoretical iron atomic loading of each protein cage structure in the reaction solution reached 5000. The reaction solution continued to react for 5 minutes after the dropwise addition was stopped, and then 0.6mL of 0.3M sodium citrate solution was added to chelate excess iron ions, and then the temperature was lowered to room temperature, that is, the iron for synthesizing Fe ₃ O ₄ nanoparticles in the inner cavity was successfully obtained. Protein probe, ferritin cage structure surface displays fluorescent protein and tumor-targeting peptide, because the surface already has fluorescent protein and tumor-targeting peptide, plus Fe ₃ O ₄ nanoparticles in the inner cavity, it is complete A tumor-targeting, fluorescent, and magnetic multifunctional probe for ferritin.

Example 2:

The three-function tumor detection probe is used for specific fluorescence imaging of tumor cells, and the steps are:

(1) Select human malignant glioblastoma cell line U87MG (purchased from China Center for Type Culture Collection of Wuhan University) and human non-small cell lung cancer cell line A549 cells (purchased from China Center for Type Culture Collection of Wuhan University), the cells All of them have up-regulated expression of tumor marker αvβ3 integrin on the surface. The cells were subcultured in DMEM medium containing 10% fetal bovine serum at 37°C in an atmosphere of 5% carbon dioxide. The passage ratio was 1:2.

(2) U87MG and A549 cells were placed in the culture dish with a cover glass glued on the center, at 37°C, 5% carbon dioxide environment for 24-36 hours, after reaching 50% cell confluence, washed 3 times with PBS buffer, Change to binding buffer (20mM Tris-HCl, 150mM NaCl, 1mM Ca ²⁺ , 1mM Mg ²⁺ , 1% BSA, pH 7.4), add the prepared trifunctional ferritin nanoprobe to the cells to a final concentration of 50nM , incubated at 37°C for 1 hour, and detected its fluorescence under a fluorescence microscope, and ferritin particles without tumor targeting peptide fusion were used as a control.

(3) Fluorescent detection found that there were obvious green fluorescent signals on the surface of U87MG and A549 cells (Figure 4), while ferritin particles without tumor targeting peptides could not generate fluorescent signals on U87MG and A549 cells, which indicated the above The prepared multifunctional probes can be used for specific target recognition and fluorescence imaging of tumor cells.

Example 3:

Trifunctional tumor detection probe as contrast agent for magnetic resonance imaging of tumor cells. The steps are:

(1) Select human malignant glioblastoma cell line U87MG (purchased from China Center for Type Culture Collection of Wuhan University) and human non-small cell lung cancer cell line A549 cells (purchased from China Center for Type Culture Collection of Wuhan University), the cells All of them have up-regulated expression of tumor marker αvβ3 integrin on the surface. The cells were subcultured in DMEM medium containing 10% fetal bovine serum at 37°C in an atmosphere of 5% carbon dioxide. The passage ratio was 1:2.

(2) 10 ⁶ U87MG and A549 cells (purchased from China Center for Type Culture Collection, Wuhan University) were washed 3 times with PBS buffer, and replaced with binding buffer (20mM Tris-HCl, 150mM NaCl, 1mM Ca ²⁺ , 1mM Mg ^{2 +} , 1% BSA, pH 7.4), the prepared trifunctional ferritin nanoprobe or ferritin particles without tumor targeting peptide but fused with fluorescent protein and containing Fe ₃ O ₄ nanoparticles were added to the cells for The final probe concentration was 50nM, and incubated at 37°C for 30 minutes.

(3) The cells treated with the trifunctional nanoprobes were imaged with a 4.7T imager for T2, and the T2 relaxation times of U87MG and A549 cells were 283.8 ms and 314.0 ms, respectively. Whereas, the T2 relaxation times of U87MG and A549 cells treated with ferritin particles without tumor-targeting peptide fusion were 457.3 ms and 451.3 ms, respectively. The T2 relaxation times of U87MG and A549 cells not treated with any contrast agent were 454.4 ms and 447.6 ms, respectively. It shows that the three-functional nanoprobe prepared above can cause a significant decrease in T2 relaxation time when used in magnetic resonance imaging of tumor cells. Therefore, the trifunctional nanoprobe can be used for specific target recognition and magnetic resonance imaging of tumor cells.

Claims (1)

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.

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