CN104861047B - Single function magnetic nanoparticle based on ferritin - Google Patents

Abstract

The invention discloses a ferritin-based monofunctional magnetic nanoparticle, which comprises a monofunctional ferritin shell and a magnetic nanoparticle core, the monofunctional ferritin shell is an asymmetric globulin with a tetrahedral structure, The asymmetric globulin is mainly composed of 1 mutant Dps subunit and 11 wild-type Dps subunits, and the Dps is a starvation-induced DNA-binding protein. The present invention uses biomacromolecule-protein as a template material to construct ferritin-based monofunctional magnetic nanoparticles, which are easy to transform, easy to manipulate, and convenient to obtain in large quantities, and they only have one functional group, which is useful for subsequent controllable assembly great significance.

Description

Ferritin-based monofunctional magnetic nanoparticles

technical field

The invention specifically relates to a ferritin-based monofunctional magnetic nanoparticle, which belongs to the field of nanobiotechnology.

Background technique

Magnetic nanoparticles are an important part of nanomaterials. Because of their special size distribution, magnetic nanoparticles have surface and interface effects, small size effects, quantum size effects, macroscopic quantum tunneling, etc., so they are widely used, involving machinery, electronics, etc. , optics, magnetism, chemistry and biology and other fields. A necessary condition for the application of magnetic nanoparticles in nuclear magnetic resonance imaging, magnetic separation, drug delivery carriers, and tumor hyperthermia is good superparamagnetism. Although the unmodified one has good superparamagnetism, due to the small particle size, large specific surface area and the influence of van der Waals force, the particles themselves are easy to aggregate and have poor stability. It is swallowed by organs and has a short half-life, which limits its application. Therefore, one of the keys to develop the application of magnetic nanoparticles in medicine is to modify and coat the nanoparticles.

In recent years, a series of novel and effective strategies have been developed for the modification and coating of magnetic nanoparticles. These methods can externally modify the magnetic nanoparticles with amphiphilic polymer molecules, or wrap biopolymers, thereby enhancing biocompatibility, non-toxic to cells, and greatly extending the circulation time in blood vessels. Biomacromolecules such as protein and DNA are natural nanomaterials. They have various structures, can replicate themselves, are highly uniform, and are easy to manipulate and mass-produce. DNA has shown unique advantages in the development of nanotechnology; compared with DNA, proteins carry richer structural information, so it is expected to provide a more flexible operating platform for nanotechnology. Among them, protein cage structures, such as viral capsids, ferritin, and heat shock proteins, have been widely used in the coating of nanoparticles or as reaction vessels to synthesize magnetic nanoparticles.

However, because the morphology and surface properties of magnetic nanoparticles have spherical symmetry, the general method can only form a uniform modified layer, and cannot form anisotropic magnetic nanostructures. In recent years, a series of effective asymmetric modification and coating methods for magnetic nanoparticles have been reported successively, which can modify magnetic nanoparticles that are isotropic to anisotropic, making this bottleneck a breakthrough. However, obtaining asymmetric magnetic nanoparticles, controlling their self-assembly at the molecular level, and accurately analyzing their assembly products are still challenging work.

Contents of the invention

In view of the deficiencies in the prior art, the purpose of the present invention is to provide a monofunctional magnetic nanoparticle based on ferritin, which can realize the monofunctionalization of magnetic nanoparticles at the molecular level, so that the magnetic nanoparticles can be used as building blocks , providing a better strategy and development space for subsequent controllable assembly, which in turn enables more complex, controllable, and directional assembly.

In order to realize the above-mentioned purpose of the invention, the present invention has adopted following technical scheme:

A ferritin-based monofunctional magnetic nanoparticle, comprising a monofunctional ferritin shell and a magnetic nanoparticle core, the monofunctional ferritin shell is an asymmetric globulin with a tetrahedral structure, and the asymmetric sphere The protein is mainly composed of 1 mutant Dps subunit and 11 wild-type Dps subunits (wtDps for short), and the Dps is a starvation-induced DNA-binding protein.

Further, the mutant Dps is obtained by inserting a His-tag and a specific biotinylated 15 peptide having the sequence shown in SEQ ID No. 1 at the N-terminus of the wild-type Dps, and can be referred to as HBDps for short.

Further, the functional group of the starvation-induced DNA binding protein is the 15-peptide contained in the mutant Dps subunit that can be specifically biotinylated, and the separation group is His-tag.

Further, the outer diameter of the monofunctional ferritin shell is 8-10 nm, and the inner diameter is 4.5-5 nm.

Further, the average thickness of the monofunctional ferritin shell is 2nm.

Further, the core of the magnetic nanoparticles includes iron oxide nanoparticles with a diameter of 2-3 nm.

Compared with the prior art, the beneficial effects of the present invention include: by using biomacromolecule-protein as a template material, ferritin-based monofunctional magnetic nanoparticles are constructed, which are easy to transform, easy to manipulate, and convenient to obtain in large quantities, and The ferritin-based monofunctional magnetic nanoparticles have only one functional group, which is of great significance for the subsequent controllable assembly.

Description of drawings

Fig. 1 is the SDS-PAGE gel figure in a typical embodiment of the present invention;

Fig. 2 is a transmission electron microscope image of an asymmetric protein nanoparticle prepared by starvation-induced DNA-binding protein assembly in a typical embodiment of the present invention;

Figure 3 is a transmission electron microscope image of the starvation-induced DNA-binding protein adsorbed AuNPs shown in Figure 2;

Figure 4 is a transmission electron microscope image of the positive control (mutant starvation-induced DNA binding protein) adsorbed AuNPs in a typical example of the present invention;

Fig. 5 is a transmission electron microscope image of the negative control (wild-type starvation-induced DNA-binding protein) adsorbed AuNPs in a typical example of the present invention.

Figure 6 is a transmission electron microscope image of mineralized iron oxide magnetic nanoparticles in asymmetric protein nanoparticles in a typical embodiment of the present invention (negatively stained with phosphotungstic acid);

Figure 7 is a transmission electron microscope image of mineralized iron oxide magnetic nanoparticles in asymmetric protein nanoparticles in a typical embodiment of the present invention (not negatively stained with phosphotungstic acid);

Fig. 8 is an agar gel electrophoresis figure in a typical embodiment of the present invention;

Fig. 9 is a nuclear magnetic resonance weighted imaging image in a typical embodiment of the present invention.

Detailed ways

The gist of the present invention is to provide a ferritin-based monofunctional magnetic nanoparticle. Transformation, the functional groups/separation groups are skillfully coupled in structure, and then the modified ferritin and wild-type ferritin are fully mixed in a specific ratio, depolymerized and reassembled, and realized by controlling the ratio of the two proteins Controlled assembly of starvation-induced DNA-binding proteins to an asymmetrically functionalized nanoparticle. According to the separation groups on the asymmetric particles, the separation and purification of the target product can be further realized. Finally, the mineralized magnetic nanoparticles in the asymmetrically functionalized nanoparticles were separated, and the functional groups of AuNPs (gold nanoparticles) were preferably used to characterize the obtained products to confirm that the obtained products were monofunctional magnetic nanoparticles.

Specifically, the preparation method includes the following steps:

1) Genetic modification of the starvation-induced DNA-binding protein to construct a protein mutant vector with functional groups and separation groups;

2) Transform the constructed vector into the organism for expression, purify and identify starvation-induced DNA-binding protein mutants;

3) Fully mix the mutant with wild-type ferritin, and then perform protein depolymerization and mixed assembly under pH-adjusted conditions;

4) Purify the assembly product according to the characteristics of the separation group;

5) Mineralize the obtained asymmetric functionalized nanoparticles, and synthesize iron oxide magnetic nanoparticles in the particle center. The single-bonded magnetic nanoparticles of the target product are obtained.

In order to make the ferritin-based monofunctional magnetic nanoparticles of the present invention easier to understand its substantive characteristics and practicability, the structure and specific implementation methods of the present invention will be described below in conjunction with a typical embodiment and Figures 1-9 For further detailed description, but the following descriptions and illustrations about the embodiments do not constitute any limitation to the protection scope of the present invention.

Step 1, constructing, expressing, and purifying a mutant starvation-induced DNA-binding protein. Specific steps are as follows:

Insert His-tag and specific biotinylated 15-peptide GLNDIFEAQKIEWHE (shown in SEQ ID No.1) at the N-terminus of the Loop of the starvation-induced DNA-binding protein, and construct the vector PET32a-His-Dps (His-Dps) ; Confirm the correctness of the target gene sequence by sequencing. Transfer PET32a-His-Dps into E.coli Rosetta (DE3) competent cells by the calcium chloride method, spread on the plate (with ampicillin and chloramphenicol added to the plate) at least 12 hours, pick out single clones from the plate and inoculate Add ampicillin (final concentration 100 μg/mL) and chloramphenicol (final concentration 68 μg/mL) into 5 mL LB test tube medium, and incubate overnight at 37°C and 180 r/min. According to the 1% (v/v) inoculum amount, transfer to 5 mL LB test tube culture medium (parallel 3 tubes), add corresponding antibiotics, and culture at 37°C constant temperature, 180 r/min shaking for about 2.5 h (OD600 at 0.4~ 0.6), one of the tubes was used as a blank control (without induction agent IPTG), and the other two tubes were added with IPTG with a final concentration of 1 mM inducer, and continued induction culture at 25°C and 37°C for 10 h and 2 h, respectively. The bacteria were collected by centrifugation, and the expression of His-Dps was detected by SDS-PAGE. It was found that it could be expressed at both temperatures, and inclusion bodies were also produced, and there were more inclusion bodies at 37°C. 25°C was chosen as the induction temperature for mass production of proteins.

Inoculate E.coli Rosetta (DE3)/PET32a-His-Dps into 5 mL LB test tube culture medium, add corresponding antibiotics and incubate overnight at 37°C and 180 r/min. The next day, transfer 5 mL of the bacterial solution into a 500 mL LB Erlenmeyer flask, add ampicillin (final concentration 100 μg/mL) and chloramphenicol (final concentration 68 μg/mL), and keep the temperature at 37°C and 180r/min After about 2.5 h of shaking culture (OD600 between 0.4 and 0.6), add inducer IPTG with a final concentration of 1 mM, continue shaking induction culture at 25 °C for 10 h, and collect the bacteria by centrifugation. The bacteria pellet was washed once with binding buffer and resuspended in a certain volume of binding buffer for ultrasonic disruption (conditions: ultrasonic power 400W, working 4s, intermittent 4s, total ultrasonic disruption time 60 min), and the broken bacterial liquid was dissolved in 12000 Centrifuge at r/min for 30 min, put the supernatant on the Ni ²⁺ --NTA affinity chromatography column equilibrated with binding buffer, then wash with imidazole at low concentration (20mM, 40mM, 60mM, 120mM) successively Impurities are removed by the column, and the target protein is finally eluted with elution buffer (500mM imidazole).

The prepared HBDps and wtDps exist in the form of spherical nanoparticles. The concentrations of HBDps and wtDps were determined by the kit Bradford Protein Assay (Coomassie Brilliant Blue staining method), and the steps were performed according to the instructions of the kit. At the same time combined with SDS-PAGE verification.

Step 2, assembly and purification of monofunctional Dps. Specific steps are as follows:

1. Mix 2 mL (16.24 mg) of wtDps (18KD) with a concentration of 8.12 mg/mL and 0.224 mL (0.57 mg) of His-Dps (21KD) with a concentration of 1.776 mg/mL, shake slowly at 4°C for 2 hours;

2. Dilute the total protein solution to a final concentration of 1mg/mL, adjust the pH value to disaggregate and remix to assemble the asymmetric protein structure. Specific steps are as follows:

ⅰ. Put the protein solution into a dialysis bag and put it into 900mL water, add 0.1M HCl to the water at a speed of 1.5mL/min with a syringe pump until the pH value is adjusted to 2. Add water to 1L, and dialyze at 4°C for 12 h. The principle of this process is that Dps will be depolymerized into protein monomers from assembled globulins under the condition of pH=2.

ⅱ. Prepare 1 L of phosphate buffer (0.001M/L), adjust pH=2 with concentrated hydrochloric acid, put it into the above-mentioned dialysis bag, add 2M/L NaOH to the water at a speed of 0.5mL/min with a syringe pump until the The pH value was adjusted to 7, and then dialyzed at 4°C for 12 h. The principle of this process is that Dps will reassemble into globulin when pH=2 is adjusted to pH=7.

3. The obtained product contains mixed assembled Dps, His-Dps and wtDps globulins as well as HBDps and wtDps monomers. To isolate the asymmetric mixed-assembled Dps nanoparticles, the following specific steps are required:

ⅰ. Add 4.2 mL of 5×binding buffer to the assembly solution. At this time, the total volume is 21. mL. The assembly solution is loaded on the Ni ²⁺ -NTA affinity chromatography column equilibrated with the binding buffer, only with HBDps It can be adsorbed to the chromatography column, use binding buffer, 15mM, 30mM imidazole to remove the starvation-induced DNA binding protein non-specifically adsorbed on the column, then use 100mM imidazole to elute the monofunctional Dps, and then use 500-1000mM imidazole to elute Globulin with multiple His-Dps monomers. The principle of this process is: an NTA (nitrilotriacetic acid, nitrogen triacetic acid) is connected to the Ni ²⁺ -NTA chromatography gel matrix, which can be combined with Ni ions, and the Ni ions are generated between the 6-his amino acids of the fusion protein Strong chelation, thus distinguishing proteins with his-tag histidine tags from other proteins. Therefore, when eluted with a high concentration of imidazole solution (such as 300 mM or higher), imidazole will compete with the imidazole ring of the protein his-tag for binding, and finally the fusion protein will be eluted from the gel.

4. Dialyze the monofunctional Dps eluted with 100mM imidazole with 0.05M Tris, 0.5M NaCl, 5% glycerol, pH=8 buffer solution, and dilute to more than 10000 times.

Single function Dps characterization:

There are two ways to verify whether the obtained Dps are monofunctional. The specific method is as follows:

1. Run the obtained target protein on SDS-PAGE gel. Since the molecular weight of the wild-type Dps is 18KD and the molecular weight of the mutant Dps is 21KD, the two are in different positions in the SDS-PAGE gel. According to the gray of the two in the SDS-PAGE gel The ratio of the amount of the two substances can be determined to determine whether the target protein is monofunctional. As shown in Figure 1, the two substances are calculated according to the gray value of wtDps and HBDps in the monofunctional Dps The calculated ratio of the two is about 30:1, which proves that the obtained target protein is indeed a monofunctional Dps.

2. NTA (nitrilotriacetic acid, nitrilotriacetic acid)-Ni modified AuNPs can efficiently bind to the his-tag of HBDps, but there is no specific binding to wild-type Dps. The samples were characterized by NTA-Ni-AuNPs adsorption experiments (Figure 3), while positive controls (Figure 4) and negative controls (Figure 5) were prepared. When the amount of AuNPs and the obtained target protein is 2:1, the target protein basically only adsorbs one gold particle, the wild-type Dps cannot adsorb gold particles, and the full mutant Dps can adsorb 3-6 gold particles. of gold particles. The results indicated that the target protein only contained one His-Dps monomer with his-tag, and the remaining 11 were wild-type Dps monomers.

Step 3, synthesizing iron oxide magnetic particles in the asymmetric protein nanoparticles. Specific steps:

1. Add 4.6 ml (330 μg/mL) of the above target protein and 15 mL 0.1M NaCl into a jacketed reactor filled with N2 protection, which is connected with an automatic titrator (TITRINO, Metrohm AG) and a thermometer. The pH of the reaction mixture was adjusted to 8.5 with 30 mM/L NaOH. The reactor was fixed on the SHT-type stirring and display thermostat electric heating mantle, and stirred and heated to 65°C.

2. Add freshly prepared 0.1mL 5mM/L ferrous ammonium sulfate and 0.075mL 2.5mM/L hydrogen peroxide, and adjust the pH of the reaction mixture to 8.5 with 30mM/L NaOH, react for 5min, repeat 8 times, and finally react for 10min .

3. Cool the product to room temperature and centrifuge at 12000rpm for 30min. The supernatant was filtered with a 0.2 μm filter, and the filtered liquid was the target product: monofunctional magnetic nanoparticles.

Characterization of magnetic nanoparticles, that is, to verify whether magnetic particles are contained in monofunctional Dps, has the following methods:

First of all, as shown in Figure 6, the samples negatively stained with phosphotungstic acid can clearly see the protein and the particles in the center of the protein under the electron microscope, while the samples that have not been negatively stained with phosphotungstic acid can only be seen as shown in Figure 7. Small particles of -3nm. This proves that there are particles in the inner cavity of the protein.

When ferric ions encounter ferrocyanide, blue ferric ferrocyanide will be produced, so Prussian blue staining can be used to verify whether the protein contains iron oxide. As shown in Figure 8: a is an agarose gel stained with Prussian blue, and b is an agarose gel stained with Coomassie brilliant blue with the same control sample. Among them, a3 and b3 are unmineralized monofunctional Dps; a2 and b2 are mineralized wt-Dps, and a1 and b1 are mineralized monofunctional Dps. It can be seen that ferritin without mineralization can only be considered Maas brilliant blue staining can not be stained with Prussian blue, but mineralized wild-type ferritin and monofunctional ferritin can be stained blue with Mas brilliant blue and Prussian blue. It shows that iron oxide nanoparticles are indeed formed in the protein after mineralization.

Finally, the NMR signal detection of the sample is carried out. As shown in Figure 9: Wells 2, 3, and 4 of T1 and T2 weighted imaging grayscale images are blank buffer, 2×mineralized monofunctional Dps (1.5μm/L), and 1×mineralized monofunctional Dps (0.75um/l). Well 1 is an irrelevant sample. The results show that the mineralized monofunctional Dps can significantly shorten the T1 relaxation time, and can also significantly shorten the T2 relaxation time. In summary, it can be judged that the mineralized and synthesized ultrafine paramagnetic iron oxide in the asymmetric monofunctional Dps (Ustrasmall superparamagnetic iron oxide, USPIO).

The application prospect of USPIO contrast agent in receptor-mediated magnetically labeled probe imaging has attracted widespread attention. The so-called magnetic labeling probe refers to the combination of paramagnetic particles with specific receptors, antibodies or genes into cells in a certain way. , the distribution of MR signals caused by paramagnetic substances represents the distribution of receptors, antibodies or genes, and the ligands labeled with paramagnetism, mediated by receptors, produce specific concentrations to achieve the purpose of selective enhancement and further imaging . The ferritin-based monofunctional magnetic nanoparticles obtained in the present invention can be used as magnetic labeling probes, and a suitable ligand can be added to the functionalized subunit of monofunctional Dps to achieve selective signal enhancement and further imaging the goal of.

The above are only examples of numerous specific application examples of the present invention, and do not constitute any limitation to the protection scope of the present invention. All technical solutions formed by equivalent transformation or equivalent replacement fall within the protection scope of the present invention.

<110> Suzhou Institute of Nanotechnology and Nanobionics, Chinese Academy of Sciences

<120> Ferritin-based monofunctional magnetic nanoparticles

<160> 1

<210> 1

<211> 15

<212> PRT

<213> Artificial sequence

<400> 1

Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu

1 5 10 15

Claims (4)

1. A kind of 1. single function magnetic nanoparticle based on ferritin, it is characterised in that including single function ferritin shell and Magnetic nanoparticle kernel, the single function ferritin shell are the asymmetric globulin with tetrahedral structure, it is described not Symmetrical globulin is mainly made up of 1 saltant type Dps subunit and 11 wild type Dps subunits, and the Dps is hungry induction DBP, wherein saltant type Dps are N-terminal His-tag and one section of sequence of insertion such as SEQ ID in wild type Dps Can be obtained by specific biotinylated 15 peptide shown in No.1.
2. 2. the single function magnetic nanoparticle according to claim 1 based on ferritin, it is characterised in that single work( The external diameter that ferritin shell can be changed is 8-10nm, internal diameter 4.5-5.5nm.
3. 3. single function ferritin shell according to claim 1, it is characterised in that the single function ferritin shell Average thickness be 2nm.
4. 4. the single function magnetic nanoparticle according to claim 1 based on ferritin, it is characterised in that the magnetic Nano particle kernel includes a diameter of 2-3nm ferric oxide nanometer particle.