CN105861418A - Application of simian virus 40 capsid protein VP1 assembled virus-like particles in mediating microparticles into cells - Google Patents

Abstract

The invention discloses the application of virus-like particles assembled by simian virus 40 capsid protein VP1 in mediating the entry of micron particles into cells. The virus-like particles assembled by simian virus 40 capsid protein VP1 are combined with inorganic microparticles through chemical modification, so that the microparticles are covered, and the microparticles can be brought into mammalian cells to realize the cell delivery of inorganic microparticles. By packaging nanoparticles with different functions and combining microparticles with different properties, a method for introducing microparticles into cells was established, and a multi-level, multifunctional nanostructure was realized. It has potential application value in targeted diagnosis of diseases, targeted delivery, high-capacity drug delivery, hyperthermia, etc.

Description

Application of virus-like particles assembled by simian virus 40 capsid protein VP1 in mediating the entry of microparticles into cells

technical field

The invention relates to the technical field of cell transduction, and more specifically relates to the application of virus-like particles assembled by simian virus 40 capsid protein VP1 in mediating micron particles into cells.

Background technique

Bionanoparticles have both nanometer size and structural characteristics, and unique bioactive functions, so they have great potential in nanotechnology applications. At present, most of the nano-research is still focused on artificial chemical nanostructures, less use of natural biological nanostructures, and biological nanoparticles have the advantages of convenient production, uniform structure, strong biocompatibility, etc., which are very suitable for life and health medicine aspect. For example, viruses, as natural nano-scale organisms, have regular and uniform structures and unique biological effects. The capsid proteins of some viruses can self-assemble into hollow particles, which do not have viral nucleic acid and cannot replicate, but are similar in shape to natural viruses. This cage particle can be used as a carrier to package various exogenous particles, including organic particles, inorganic particles and small molecules, so as to transport "cargo" to cells or living bodies as a carrier system.

The viral capsid is a regular hierarchy formed by many identical proteins. Virus-based nanomaterials are an exciting research field in nanoscience, and have become a research hotspot at the intersection of materials science, biochemistry, and virus physical chemistry. The key element of this class of nanomaterials is to obtain functionalized viral nanoparticles (VNPs) based on the self-assembly or controllable assembly of viral protein capsids (shells) and organic or inorganic cargo. VNPs can carry polymers, enzymes, and other organic cargo, as well as encapsulate metal ions. There are two feasible strategies for the design of nanomaterials loaded with inorganic particles on the viral capsid: 1, the inorganic particles are packaged inside the capsid; 2, the inorganic material is combined on the outside of the viral capsid. In both cases it is the interior or exterior of the capsid, or some part of the viral protein that directly forms, packages or binds inorganic cargo. Continuing with the study of phages, the formation of plant and animal viral VNPs packaging inorganic cargo was also explored. Plant viruses include BMV, CPMV, TMV, CCMV, and RCNMV. Except for the rod-shaped TMV, other viruses have an icosahedral structure. Animal viruses that have packaged inorganic cargo include SV40, Alphavirus and HIV-1.

From a materials science point of view, viral capsids can play different roles in VNPs. If the inorganic particles are formed on the outside of the viral capsid, and the viral capsid is used as a template or scaffold to decorate the inorganic nanoparticles, then a hollow capsule structure, tubular structure, etc. with inorganic materials on the surface can be obtained. If an inorganic particle is nucleated in the viral capsid, the viral capsid can act as a nanoreactor whose inner wall can promote, catalyze, and control the growth of nanoparticles. A third possibility is that when viral capsids self-assemble around inorganic nanoparticles, viral capsids self-assemble due to nanoparticle nucleation. There are many kinds of VNPs, and we focus on VNPs as cell transport carriers of inorganic nanoparticles and their broad application prospects in biomedicine such as imaging, drug delivery, hyperthermia and nanomaterial design. For example, bioimaging is one of the most promising applications of inorganic particle-based VNPs. VNP packaging or binding to gold nanoparticles can be visually tracked in vitro. VNP packaging or binding quantum dots can be used for in vitro or in vivo experiments by fluorescence observation. In addition, the composite with quantum dots outside the CPMV has a high local concentration of quantum dots, and the fluorescence quantum yield is increased by 15% compared with the free quantum dots in the solution. A large number of iron oxide nanoparticles combined on the surface of CPMV have enhanced local magnetic field strength, which is promising for magnetic resonance imaging. But most of the above examples have not been fully proved. Virus particles can be used for drug delivery, and many VLPs and VNPs loaded with organic cargoes have been successfully used for in vitro and in vivo drug delivery applications, while inorganic cargoes have only just been considered. In packaging inorganic particles, we can adsorb drug molecules on the surface of functionally modified (especially amphiphilic) nanoparticles and release them when VNPs are depolymerized. Inorganic particles can be imaged and magnetically loaded simultaneously. When inorganic particles are bound to the outer surface of the viral capsid, the interior of the VNP can be loaded with drug molecules, but such experiments have not been reported so far. In terms of tumor hyperthermia, various shell-packed magnetic nanoparticles have been studied more and more, which can not only deliver magnetic nanoparticles, but also control their therapeutic response according to the properties of the organic shell.

The perfect outer surface of the virus capsid can promote the binding of specific molecules, which can make it specifically target certain cancer cells, so VNP packaged with magnetic particles can be used for hyperthermia. Of course, virus particles can also be used to construct new nano-device materials. For example, VNPs such as monodisperse packaging nano-gold and cobalt oxide are self-assembled into dielectric metamaterials to manufacture gold-cobalt oxide hybrid nanowires that can improve battery capacity, and prepare nano-electrodes or batteries with extremely excellent performance.

The cell membrane allows the passage of small molecules, the uptake of nutrients by the cell, and all communication with the microenvironment occurs during the passage of the cell membrane using a variety of mechanisms. Oxygen, carbon dioxide, water and small hydrophobic or nonpolar molecules are able to diffuse freely across cell membranes through concentration gradient differences. Small molecules such as ions and amino acids pass through the cell membrane through active transport systems such as membrane protein pumps or ion channels. Nanoscale hydrophilic biomacromolecules usually rely on endocytosis to enter cells, which uses transport vesicles derived from cell membranes to internalize cargo into cells. There have been many studies on the cellular uptake and mechanism of nanoparticles. For example, engineered nanoparticles, such as metal clusters, carbon nanotubes, fullerenes, and quantum dots, can penetrate cell membranes and enter endothelial cells, lung epithelial cells, intestinal epithelial cells, alveolar macrophages, other macrophages and neurons and other cells. However, the mechanisms proposed by different laboratories for cellular uptake of nanoparticles are sometimes inconsistent or even outright conflicting. Moreover, each nanoparticle exhibited a preference for different cellular internalization pathways. A systematic understanding of the uptake and transport of nanoparticles in cells thus becomes of paramount importance.

The size and shape of nanoparticles are also key parameters governing their properties and thus also determine the process of cellular uptake. For example, the uptake of gold nanoparticles by HeLa cells was changed with the size of gold nanoparticles. Internalization of Herceptin-modified gold nanoparticles (from 2nm to 100nm) is largely determined by its size: the most efficient size range for endocytosis is 25-50nm, which is during receptor-mediated endocytosis, Resulting from a directional balance between membrane receptors and multivalent cross-links of the membrane packaging process. Aggregation of endosomes in cells was found to enhance their magnetic capabilities and lead to better MRI contrast. The shape of the nanoparticles is another important factor, which directly affects the route of uptake. Still using gold nanoparticles as an example, some experiments have studied the effect of the geometry of gold nanorods (NRs) on cell uptake, and found that the geometry of NRs has a great impact on cell uptake: short Nanorods enter cells more easily than long nanorods. Spherical nanoparticles of the same size entered cells more easily than rod-shaped nanoparticles, possibly because rod-shaped nanoparticles required longer membrane bending time.

Iron oxide particles can be used in magnetic resonance imaging, tumor hyperthermia, etc., and are a promising functional material. According to particle size, iron oxide particles can be roughly divided into superparamagnetic iron oxide particles (superparamagnetic iron oxide, SPIO), ultra-small superparamagnetic iron oxide particles (ultra-small superparamagnetic iron oxide, USPIO) and single crystal iron oxide nanoparticles (monocrystalline iron oxide nanoparticles). ironoxide nanoparticles. MION). In 2004, Shapiro et al. discovered that micron-sized superparamagnetic iron oxide particles (micron-sized superparamagnetic iron oxide, MPIO) can label cells, generate stronger signal contrast, and achieve single-cell tracking imaging, which has attracted many scientists. research interests. In recent years, MPIO-based research and applications have achieved remarkable results and breakthroughs. However, because the micron-scale superparamagnetic iron oxide particles (MPIO) are large and the coating is inert, it is more difficult to introduce into cells. MPIO is combined with some transfection media to enter cells, but the efficiency is very low, and the transfection media used may inhibit cell proliferation and differentiation. Viral capsid, as an introduction carrier, is expected to become a new efficient and safe transduction method.

Contents of the invention

The purpose of the present invention is to provide the application of virus-like particles assembled by simian virus 40 capsid protein VP1 in mediating the entry of inorganic micro-particles into cells. The virus-like particles assembled by simian virus 40 capsid protein VP1 are combined with inorganic microparticles through chemical modification, so that the microparticles are covered, and the microparticles can be brought into mammalian cells to realize the cell delivery of inorganic microparticles.

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

The application of virus-like particles assembled by simian virus 40 capsid protein VP1 in mediating the entry of micron particles into cells, the application process is as follows:

1) Combining the virus-like particles assembled by the simian virus 40 capsid protein VP1 with the inorganic microparticles through chemical modification, so as to coat the microparticles to obtain composite microparticles.

2) Incubate the composite microparticles obtained in step 1) with mammalian cells, and the microparticles can be mediated by virus-like particles into the cells.

The virus-like particles assembled by the simian virus 40 capsid protein VP1 can be obtained by using methods known in the art or references (Small, 2009, 5(6):718-726) to obtain the simian virus 40 capsid protein VP1 Assembled virus-like particles, the virus-like particles can be packaged with conventional tracer particles in the field such as quantum dots or gold nanoparticles as required.

According to the above application, the application of the virus-like particles assembled by using the simian virus 40 capsid protein VP1 in mediating the entry of micron-scale magnetic iron oxide particles MPIO into cells is as follows:

(1) Construct the expression plasmid (PET-VP1) of VP1 protein, express and purify VP1 in Escherichia coli (E. coli Rosetta), and characterize its pentameric form by electron microscope.

(2) Quantum dots were packaged in the VP1 protein, and then purified through a sucrose density gradient to obtain virus-like particles (SVQDs) packaged with quantum dots, which were characterized by electron microscopy.

(3) Mixing SVQDs with a biotinylation reagent to remove excess unbound biotin to obtain Biotin-SVQDs. Streptavidin-modified MPIO was mixed with Biotin-SVQDs, and magnetic beads wrapped by SVQDs (MPIOs-VLP-QDs) were purified by magnetic stand.

(4) Observing and taking pictures with a fluorescence microscope system to obtain a microscopic image of a single particle.

(5) Mix MPIOs-VLP-QDs with the cultured Vero cells and incubate on ice, wash off excess MPIOs-VLP-QDs, and then put them into an incubator to continue culturing.

(6) Observe the granule localization of MPIOs-VLP-QDs cells incubated at 4 degrees at different times by fluorescence microscope.

(7) Place a magnet outside the culture dish to continue the culture, use a fluorescence microscope to observe the localization of MPIOs-VLP-QDs in the cells, and at the same time "after the infected cells are digested and suspended with trypsin, observe the control of the magnetic field on the direction of cell movement.

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

The present invention utilizes the main capsid protein VP1 self-assembly system of monkey virus 40 to package quantum dots, and then through the interaction between biotin and streptavidin, the virus capsid self-assembly particles are wrapped and combined on the surface of magnetic microparticles, and the virus The ability of capsids to bring micron-sized particles into mammalian cells has led to the development of a method capable of carrying micron particles into mammalian cells. The viral capsid protein itself has good biocompatibility and is easy to degrade. Inorganic nanoparticles can be wrapped in the capsid, and biological functional molecules can be modified chemically or genetically on the capsid, and then the functional microparticles can be loaded to construct multi-level, multifunctional micron functional particles, which can be used in the targeted diagnosis of diseases , Targeted transport, high-capacity drug delivery, hyperthermia and other aspects have potential application value.

The system can not only load nanoparticles itself, but also can be combined with microparticles and delivered into mammalian cells. After entering the cells, the magnetic microparticles can also be controlled by an external magnetic field, including controlling the arrangement of the magnetic particles in the cells and the direction of movement of the suspended cells. The method is a good biocompatibility, multi-level and multifunctional particle introduction system.

Description of drawings

Fig. 1 Schematic diagram of VP1 protein and its pentamer.

Among them, a is the SDS-PAGE electrophoresis image of VP1 protein, b is the western blot identification image of VP1 protein, and c is the electron microscope schematic diagram of VP1 pentamer.

Figure 2 is a schematic diagram of electron microscopy of SVQDs.

Figure 3 is a schematic diagram of MPIOs-VLP-QDs.

Among them, a is the schematic diagram of MPIOs-VLP-QDs simulation, and b is the schematic diagram of MPIOs-VLP-QDs fluorescence microscope.

Figure 4 is a schematic diagram of the fluorescence localization of MPIOs-VLP-QDs incubated with cells for different times.

Where a is the fluorescence localization map of MPIOs-VLP-QDs incubated with cells for 0 hours, and b is the fluorescence localization map of cells incubated with cells for 16 hours.

Fig. 5 is a fluorescence image of the arrangement direction of MPIOs-VLP-QDs particles in cells controlled by an external magnetic field.

Fig. 6 is a fluorescence image of the movement direction of the suspended cells containing MPIOs-VLP-QDs particles controlled by an external magnetic field.

detailed description

In the embodiment of the present invention, the virus-like particle assembled by the simian virus 40 capsid protein VP1 mediates the entry of MPIO into the cell as an example to further illustrate the operation process, but the scope of the present invention is not limited to the following examples. The technical solutions described in the present invention are conventional solutions in the art unless otherwise specified.

Example 1:

Preparation of packaged quantum dot virus-like particles

(1) Construct the expression plasmid (PET-VP1) of VP1 protein, express and purify VP1 in Escherichia coli (E. coli Rosetta), and characterize its pentameric form by electron microscope.

Using pSV21SphI-N1 (J Virol Methods, 1997, 64 (1): 1-9) as a template to amplify VP1, after BglII-XhoI double enzyme digestion, it was connected into the pQE-30 vector (Qiagen Corporation) of BamHI-SalI double enzyme digestion purchased), constructed into pQEVP1; using pQEVP1 as a template, amplify the VP1 encoding gene fused with Histag at the N-terminal, and insert it into the pET32a(+) vector through the NdeI-XhoI site (the vector contains two NdeI sites, and NdeI is fully digested) , constructed into pET32a-hisVP1. All enzyme digestion and PCR operation steps were carried out according to relevant product instructions. Both pQEVP1 and pET32a-hisVP1 were sequenced to confirm the correctness of the target gene sequence.

Transform pET32a-hisVP1 into E.coli Rosetta (DE3) competent cells using the CaCl ₂ method, pick a single clone from the plate and insert it into a 5 mL LB test tube culture medium, add ampicillin and chloramphenicol, and keep the temperature at 37°C, Cultivate overnight at 200r/min. On the next day, transfer 1% of the inoculum into 5 mL LB test tube culture medium (4 tubes in parallel), add corresponding antibiotics, and culture at a constant temperature of 37°C and 200 r/min shaking until the OD600 is between 0.4 and 0.6, of which 1 tube As a blank control, IPTG was added to the other three tubes to a final concentration of 1mM, and after induction culture was continued at 20°C, 25°C, and 37°C for 16h, 8h, and 3h, the bacteria were collected, and the expression of VP1 was detected by SDS-PAGE. VP1 can be expressed at three temperatures, and inclusion bodies are produced, and 25°C is selected as the induction temperature for mass production of proteins.

E. coli Rosetta (DE3) (pET32a-hisVP1) was inoculated into 5 mL of LB test tube culture medium, ampicillin and chloramphenicol were added, and cultured overnight at 37°C and 200 r/min. On the next day, transfer to a 500mL LB Erlenmeyer flask, add the corresponding antibiotics, and culture at a constant temperature of 37°C and 200r/min shaking until the OD600 is between 0.4 and 0.6, add IPTG to a final concentration of 1mM, and continue to induce culture at 25°C for 8h Collect the bacteria, wash the bacteria pellet once, resuspend and ultrasonically disrupt, centrifuge at 10,000r/min for 30min, apply the supernatant to a Ni ²⁺ -NTA affinity chromatography column to purify the target protein (Figure 1a), Western The blot was validated (Fig. 1b) and the VP1 pentamer was characterized by electron microscopy (Fig. 1c).

(2) Add quantum dots (emission light at 605nm) to VP1 protein, dialyze into packaging buffer (10mM Tris-HCl pH7.2, 250mMNaCl, 1mM CaCl ₂ , 5% glycerol), and then purify through sucrose density gradient to obtain Virus-like particles (SVQDs) packed with quantum dots were characterized by electron microscopy (Fig. 2).

Example 2:

Preparation of MPIOs-VLP-QDs particles

(1) Mix SVQDs with biotinylation reagent (EZ-Link Sulfo-NHS-LC-LC-Biotin, Thermo Company, product number: Pierce Part no.21343) (molar ratio 1:3), and let stand at room temperature for 30 minute. Dilute the mixture 104 times using 100KD ultrafiltration tubes and PBS to remove excess unbound biotin. Magnetic particles (MPIO, MyOne ^TM Streptavidin C1, Thermo Fisher Company, Cat. No.: 65001) Adsorbed on the wall of the EP tube with a magnetic stand, sucked off the supernatant, and then added PBS, after washing for 3 times, massage the magnetic particles (MPIO) and Biotin-SVQDs The molar ratio was 1:1000, or the Biotin-SVQDs were mixed in absolute excess, shaken slowly on a vortex shaker, and reacted for 30 minutes at room temperature. After the reaction, the sample was placed on a magnetic stand for adsorption, magnetic beads for 3 minutes, magnetic nanoparticles for 30 minutes, the supernatant was removed, and washed twice with PBS to obtain SVQDs-coated particles bound to magnetic beads (MPIOs-VLP-QDs). Suspend the final magnetic beads in 500 μL PBS and store at 4°C for later use.

(2) Observing and taking pictures through the fluorescence microscope system, using a 60 times oil lens, and setting the filter as the red channel and the bright field channel, to obtain the single particle microscopic image of MPIOs-VLP-QDs (Fig. 3).

Example 3:

Cell introduction of MPIOs-VLP-QDs particles and controllable manipulation

(1) Vero cells were cultured, and 200 μL of 5 mg/mL MPIOs-VLP-QDs were taken out and added to glass-bottom dishes with 80-90% confluence of Vero cells. Place the small dish in a refrigerator at 4°C for 1.5 h, then wash the small dish three times with DMEM medium, add 5 μL Hoechst 33258 and 500 μL Opti-MEM medium and incubate at room temperature for 5 minutes, wash the small dish three times with DMEM medium, then add 1.5 mL of fresh DMEM medium, and placed in a 37°C carbon dioxide incubator to continue culturing for 24 hours.

(2) After incubating the cells in MPIOs-VLP-QDs at 4°C for 1.5 h, use a fluorescence microscope and a 60X objective lens to observe the samples in the small dish, use the DAPI filter set to image Hochest-stained nuclei, and the TRITC filter set to image magnetic composite particles , the cell morphology was photographed in bright field, and the results showed that MPIOs-VLP-QDs could be adsorbed on the cell surface (Fig. 4a). After the cells were further cultured in a 37°C carbon dioxide incubator for 16 hours, microscopic imaging revealed that MPIOs-VLP-QDs entered the cells and were located near the nucleus (Fig. 4b).

If a magnetic field is applied to the side of the small dish during cell culture, the magnetic particles and their fluorescence in the cells are oriented in the direction of the magnetic field after MPIOs-VLP-QDs "infect" for 24 hours (Figure 5). After MPIOs-VLP-QDs "infected" cells were digested and rounded with 200 microliters of trypsin, 2 mL of DMEM medium was added, and the cells were blown up and suspended with a pipette gun. The dynamic process was photographed in the bright field, and the cells containing the magnetic particles MPIOs-VLP-QDs could be seen to move according to the direction of the magnetic field under the control of the external magnetic field (Figure 6).

Claims (4)

1. Application of virus-like particles assembled by simian virus 40 capsid protein VP1 in mediating microparticles into cells. 2. The application according to claim 1, wherein the micron particles are inorganic micron particles. 3. The application according to claim 1, characterized in that: the application of virus-like particles assembled by simian virus 40 capsid protein VP1 in mediating micron-scale magnetic iron oxide particles MPIO into cells. 4. The application according to claim 3, the steps comprising: (1) Construct the expression plasmid of VP1 protein, express and purify VP1 in Escherichia coli, and characterize its pentamer morphology by electron microscope; (2) Quantum dots were packaged in VP1 protein, and then purified through a sucrose density gradient to obtain virus-like particles SVQDs packaged with quantum dots, which were characterized by electron microscopy; (3) Mix SVQDs with a biotinylation reagent to remove excess unbound biotin to obtain Biotin-SVQDs; Streptavidin-modified MPIO was mixed with Biotin-SVQDs, and the magnetic beads MPIOs-VLP-QDs wrapped by SVQDs were purified through a magnetic stand; (4) Observing and taking pictures with a fluorescence microscope system to obtain a single particle microscopic image; (5) Mix and incubate MPIOs-VLP-QDs with the cultured Vero cells on ice, wash off excess MPIOs-VLP-QDs, and then put them into the incubator to continue culturing; (6) Observe the particle localization of MPIOs-VLP-QDs incubated cells under different incubation times by fluorescence microscope; (7) Place a magnet outside the culture dish to continue the culture, use a fluorescence microscope to observe the localization of MPIOs-VLP-QDs in the cells, and at the same time, trypsinize and suspend the infected cells, and observe the control of the magnetic field on the direction of cell movement.