CN115029310B - Cell membrane bionic nanoparticle of osteoclast precursor, and preparation method and application thereof - Google Patents
Cell membrane bionic nanoparticle of osteoclast precursor, and preparation method and application thereof Download PDFInfo
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- CN115029310B CN115029310B CN202210471438.2A CN202210471438A CN115029310B CN 115029310 B CN115029310 B CN 115029310B CN 202210471438 A CN202210471438 A CN 202210471438A CN 115029310 B CN115029310 B CN 115029310B
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Abstract
The invention discloses an osteoclast precursor cell membrane bionic nanoparticle, a preparation method and application thereof, wherein the bionic nanoparticle of a targeted delivery gene transfection material is B-PDEAEA-siRNA/shRNA nanoparticle wrapped by the osteoclast precursor cell membrane, and the preparation method and application thereof are provided. The cell membrane bionic nano-particles contain proteins on the cell membrane surface of the osteoclast precursor, have the targeting recognition and fusion capabilities, and endow the targeted osteoclast precursor with the function. It solves the technical difficulty that the existing materials can not selectively inhibit specific cell stages in the osteoclast lineage. According to the invention, the osteoclast precursor cell membrane bionic nano particles are adopted for delivery, so that the osteoclast precursor cells can be specifically identified, a gene transfection material enters the cells in a membrane fusion mode, and after response to intracellular ROS, charge inversion is realized, so that nucleic acid medicines are released, and the gene transfection effect is achieved. Provides a safe and efficient gene delivery material, and has wide application prospect.
Description
Technical Field
The invention belongs to biological materials and application thereof, and in particular relates to an osteoclast precursor cell membrane bionic nanoparticle for targeted delivery of a gene transfection material and a preparation method thereof.
Background
Excessive activation of cells by polynucleation is an important factor in the pathological process of various diseases, including osteolytic diseases, granulomatous tissue destruction in chronic infections, excessive inflammatory response of polynuclear giant cells, etc. Osteoporosis is a clinically common chronic skeletal disease characterized by low bone mass and severe destruction of bone microstructure, often increasing bone fragility and susceptibility to fracture. As a unique bone resorption cell, mature polynuclear osteoclasts secrete large amounts of enzymes and acids to perform the function of bone resorption due to their high transcriptional activity. However, excessive multinucleation of osteoclasts can lead to an imbalance in bone homeostasis and thus is a major factor in causing osteoporosis. Currently, first line treatments for osteolytic diseases, such as bisphosphonates, indiscriminately inhibit the osteoclast lineage, leading to apoptosis of all bone resorption cells, thereby disrupting the necessary bone turnover. Thus, there is a need to develop a material to selectively target osteoclast precursor cells at specific stages of disease-related in the osteoclast lineage.
Cell membrane engineering techniques have been widely used in a variety of fields ranging from drug delivery, imaging, to photoactivation therapy. Bionic nanoparticles camouflaged with cell membranes confer cell-like functions, thereby prolonging the circulation of the nanoparticles in the blood, enabling them to escape from the clearance of the immune system. Furthermore, in cancer therapy, cancer cell membrane coated nanoparticles can specifically target homologous cells because they inherit the necessary fusion proteins and adhesion molecules from the cells. Thus, homotypic targeted delivery of these cell membrane materials is a useful strategy for future disease treatment. However, although cell membrane engineering techniques have been used in various fields, selective targeted delivery of cells with specific stages during cell multi-nuclear process has not been achieved. Osteoclast precursor cells are derived from RANKL-induced 3-day macrophages, with specific proteins involved in osteoclast circulation, recruitment, intercellular recognition and fusion, proteins involved in migration and targeting, such as CXCR4, CDC42 and RAC2, fusion related proteins such as CD44 and OSCAR. Thus, the cell membrane with the specific stage marker protein contributes to the selective targeted delivery of osteoclast precursor cells.
Cell membrane engineering techniques have shown an important role in homotypic targeting, but currently few use this property to deliver gene transfection materials. Because therapeutic nucleic acids typically carry a negative charge, encapsulation with negatively charged cell membranes is difficult to achieve a desired entrapment rate, and therefore suitable materials are required to bind therapeutic nucleic acids. The boric acid (ester) benzyl quaternary cationic polymer (B-PDEAEA) can neutralize the negative charge of DNA or RNA and compress it into nanoparticles to protect the nucleic acid from degradation. Previous studies have shown that nanoparticles of B-PDEAEA-entrapped nucleic acids are capable of responding to intracellular reactive oxygen species
(ROS), thereby rapidly releasing nucleic acid drugs, promoting gene expression and silencing (Liu X, et al adv Mater,2016.28 (9): 1743-52;Li Y,et al.Nanoscale,2017.10 (1): 203-214). The osteoclast lineage contains a large number of ROS physiological properties that trigger the release of nucleic acids from nanoparticles. These chemical properties make B-PDEAEA an ideal candidate for gene delivery.
Therefore, the invention relates to an osteoclast precursor cell membrane bionic nanoparticle for targeted delivery of gene transfection materials, which has great significance and value in osteoporosis treatment, but has no public report so far.
Disclosure of Invention
The primary purpose of the invention is to solve the technical problem that the existing material does not specifically target cells at a specific stage in an osteoclast lineage and efficiently deliver gene transfection materials.
The invention also aims to provide a preparation method of the osteoclast precursor cell membrane bionic nano-particles for targeted delivery of the gene transfection material. Synthesizing a gene transfection material capable of reversing charges through electrostatic self-assembly; then coating the gene transfection material in the cell membrane of the osteoclast precursor by an extrusion method to prepare the bionic nanoparticle of the cell membrane of the osteoclast precursor.
It is a further object of the present invention to provide such osteoclast precursor cell membrane biomimetic nanoparticle delivery applications.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
an osteoclast precursor cell membrane bionic nanoparticle comprises an osteoclast precursor cell membrane capable of targeting an osteoclast precursor cell and a gene transfection material capable of reversing charges and coated in the osteoclast precursor cell membrane; the charge-reversible gene transfection material is a nano-composite formed by electrostatic self-assembly of a charge-reversible cationic polymer and a nucleic acid drug; the osteoclast precursor cell membrane bionic nano-particles realize charge reversal under a cell microenvironment so as to release nucleic acid medicines.
Preferably, the particle size of the charge-reversible gene transfection material is 30-200 nm.
Preferably, the nucleic acid drug is at least one of siRNA, microRNA or DNA.
The preparation method of the osteoclast precursor cell membrane bionic nano-particle specifically comprises the following steps:
(1) Preparation of Gene transfection Material
Dissolving B-PDEAEA in 10mM HEPES buffer solution, wherein the pH of the HEPES buffer solution is 7.4, incubating for 10 minutes at 37 ℃, preparing a nucleic acid medicament into 40 mug/mL solution, diluting the B-PDEAEA into 250-1500 mug/mL by using the 10mM HEPES buffer solution, adding the pH of the HEPES buffer solution into the nucleic acid medicament solution of 40 mug/mL according to the volume ratio of 1:1, vortex oscillating for 10-30 seconds, standing for 20-30 minutes at room temperature, and obtaining a nano-composite solution with the nitrogen-phosphorus molar ratio of 5-30;
(2) Extraction of osteoclast precursor cell membranes
Extracting mammal mononuclear cells, inducing for 3-5 days by 15-30 ng/mL M-CSF to obtain macrophages, and inducing for 3-5 days by 30-80 ng/mL RANKL to obtain osteoclast precursor cells; after washing, pancreatin digestion, the cells were resuspended in TM buffer at 4℃which was 1mM MgCl 2 And 10mM Tris plus water and the pH was adjusted to 7.4. Extruding for 30-40 times by using a micro extruder after re-suspending to destroy cells;
1M sucrose was added and mixed with the cell homogenate to a final sucrose concentration of 0.25M. Centrifuging the obtained mixture for 10 minutes at the temperature of 4 ℃ and 2000xg, collecting supernatant, centrifuging for 30-35 minutes at the temperature of 4 ℃ and 3000xg, re-suspending and washing the precipitate by using a 0.25M sucrose solution, centrifuging for 30-35 minutes at the temperature of 3000xg again at the temperature of 4 ℃ to obtain the precipitate which is a cell membrane, and re-suspending the cell membrane to 1-2 mg/mL by using a 10mM HEPES buffer solution, wherein the pH of the HEPES buffer solution is 7.4.
(3) Preparation of osteoclast precursor cell membrane bionic nano-particles
Mixing the osteoclast precursor cell membrane obtained in the step (2) with the charge-reversible gene transfection material obtained in the step (1) in a volume ratio of 0.5-2, preferably 0.5-1.5. The mixture is ultrasonically mixed for 1 to 3 minutes, and finally the mixture is repeatedly extruded through the polycarbonate porous membrane.
Preferably, the molar ratio of nitrogen to phosphorus is 10.
Preferably, the mammalian mononuclear cells are 6-8 weeks C57BL/6J mouse tibia and femur mononuclear cells.
Preferably, the pancreatin digestion is performed after 3 to 5 minutes of pancreatin digestion, and the digestion is stopped and centrifuged at 1000rpm for 5 minutes.
Preferably, the micro extruder is a 1ml insulin syringe.
Preferably, the number of repeated pressing is 5 to 20.
Preferably, the polycarbonate has a pore size of 100 to 400nm.
Application of osteoclast precursor cell membrane bionic nano particles as transfection material carriers or for preparing targeted drugs.
The invention combines the charge-reversible gene transfection material with cell bionics to successfully construct the osteoclast precursor cell membrane biomimetic nanoparticle. The charge-reversible gene transfection material forms a nano-composite with the nucleic acid drug through electrostatic self-assembly, and then realizes charge reversal through responding to intracellular ROS, thereby releasing the nucleic acid drug. Meanwhile, the stability is enhanced by utilizing the cell membrane of the osteoclast precursor to wrap the gene transfection material, the in vivo long circulation of the gene medicine is realized, and the nucleic acid medicine release can be carried out by targeting the cell of the osteoclast precursor.
The invention has the beneficial effects that:
compared with the prior art, the invention has the remarkable progress that the target delivery gene transfection material of the invention is characterized in that:
(1) The cell biomembrane is used for coating the gene transfection material for delivering the nucleic acid medicine, so that the technical problem of poor long-circulating stability of the existing gene medicine is successfully solved.
(2) The particle size of the gene transfection material is about 50nm, nucleic acid medicines are released through charge reversal, and the products before and after the material reaction have no toxicity to cells.
(3) The osteoclast precursor cell membrane bionic nano particles can retain relevant proteins such as targeted recognition and fusion in osteoclast precursor cells, have obvious in-vivo and in-vitro osteoclast precursor cell targeting effect, solve the problem that the traditional medicine does not selectively target osteoclast cell lineages, and have the prospect of becoming an efficient targeted nano delivery material in osteoporosis gene therapy.
(4) The preparation method of the cell membrane bionic nano delivery material is simple to operate, does not pollute the environment, has good safety, is used as a high-efficiency low-toxicity nano delivery material with a targeting effect, and is expected to be applied to the research and treatment fields on a large scale.
(5) The cell membrane bionic nano particles at a specific differentiation stage of polynuclear cells are used for targeting cells at the specific differentiation stage in the cell lineage, and provide a new research thought for drug delivery of various polynuclear cells.
Description of the drawings:
FIG. 1 is a graph of H for B-PDEAEA and siRNA/shRNA nanocomposites with different nitrogen to phosphorus ratios 2 O 2 Agarose gel electrophoresis patterns before and after stimulation.
FIG. 2 is a Western Blot identification of osteoclast precursor cell membranes.
FIG. 3 is a comparison of osteoclast precursor cell membrane and macrophage membrane proteomic profile by GO enrichment analysis.
FIG. 4 is a particle size potentiometric analysis and electron microscopy image of B-PDEAEA and siRNA Nanocomposite (NPs), osteoclast precursor cell membrane (POCM), and B-PDEAEA and siRNA/shRNA nanocomposite (POCM-NPs) encapsulated by osteoclast precursor cell membrane.
Fig. 5 is a stability analysis of osteoclast precursor cell membrane biomimetic nanoparticles.
FIG. 6 is a graph of laser confocal co-localization results of fusion of osteoclast precursor cell membrane biomimetic nanoparticles with osteoclast precursor cells.
FIG. 7 is a graph of laser confocal results of nucleic acid drug release process after fusion of osteoclast precursor cell membrane biomimetic nanoparticles with osteoclast precursor cells.
FIG. 8 is a graph showing the fluorescence distribution and statistics of various tissues of an in vivo mouse imaging experiment of an osteoclast precursor cell membrane biomimetic material.
Fig. 9 is a graph of confocal fluorescence co-localization of osteoclast precursor cell membrane biomimetic nanoparticles with osteoclast lineage laser within femoral tissue of mice.
Detailed Description
The following examples are provided to further describe the osteoclast precursor cell membrane biomimetic nanoparticle for targeted delivery of gene transfection material and the preparation method thereof, but the following examples should not be construed as limiting the scope of the present invention.
Example 1 preparation of Gene transfection Material NPs@siRNA (NPs)
B-PDEAEA white solid was dissolved in HEPES buffer (pH 7.4, 10 mM) to prepare 1500. Mu.g/mL, incubated at 37℃for 10 min, and siRNA was selected NC As nucleic acid drugs, siRNA was dissolved in DEPC water to prepare a solution of 40. Mu.g/mL, and B-PDEAEA was diluted with HEPES buffer (pH 7.4, 10 mM) to 250, 500, 750, 1000,1, respectively250 1500 mug/mL is rapidly added into 40 mug/mL siRNA solution according to the volume ratio of 1:1, vortex oscillation is carried out for 30s, and standing is carried out for 30 minutes at room temperature, thus obtaining NPs@siRNA nano-composite solution with nitrogen-phosphorus ratio (N/P molar ratio) of 5, 10, 15, 20, 25 and 30.
Example 2 preparation of Gene transfection Material NPs@shRNA (NPs)
B-PDEAEA white solid was dissolved in HEPES buffer (pH 7.4, 10 mM) to prepare 1500. Mu.g/mL, incubated at 37℃for 10 min, and shRNA was selected NC As a nucleic acid drug, shRNA was dissolved in HEPES buffer (pH 7.4, 10 mM) to prepare a solution of 40. Mu.g/mL, and B-PDEAEA was diluted with HEPES buffer (pH 7.4, 10 mM) to 250, 500, 750, 1000, 1250, 1500. Mu.g/mL, respectively, and rapidly added to the shRNA solution of 40. Mu.g/mL at a volume ratio of 1:1, vortexing was performed for 10 seconds, and the mixture was allowed to stand at room temperature for 30 minutes to obtain NPs@shRNA nanocomposite solutions having nitrogen-phosphorus ratios (N/P molar ratios) of 5, 10, 15, 20, 25, 30.
EXAMPLE 3 Gene transfection Material NPs FITC @siRNA cy5 Is prepared from
B-PDEAEA FITC Yellow solid was dissolved in HEPES buffer (pH 7.4, 10 mM) and set at 1500. Mu.g/mL, incubated at 37℃for 10 min, and cy 5-labeled siRNA was selected NC (siRNA cy5 ) siRNA as a nucleic acid drug cy5 Dissolving in DEPC water to 40 μg/mL, and adding B-PDEAEA FITC Diluting with HEPES buffer solution (pH 7.4, 10 mM) to 500 μg/mL, rapidly adding into siRNA solution of 40 μg/mL according to volume ratio of 1:1, vortex oscillating for 30 seconds, standing at room temperature for 30 minutes to obtain NPs with nitrogen-phosphorus ratio (N/P mole ratio) of 10 FITC @siRNA cy5 Nanocomposite solution.
EXAMPLE 4 preparation of osteoclast precursor cell membrane (POCM)
Collection of osteoclast precursor cells: the femur and tibia of C57BL/6J mice were washed with alpha-MEM+10% FBS+1% penicillin G and streptomycin (complete alpha-MEM medium) for 6-8 weeks, and the washed cells were cultured in complete alpha-MEM medium containing 15ng/mL M-CSF for 3 days, and the cells were changed once during the culture to obtain macrophages. The resulting macrophages were cultured in complete alpha-MEM medium containing 15ng/mL M-CSF and 30ng/mL RANKL for 5 days to give osteoclast precursor cells.
Extraction of osteoclast precursor cell membranes: after washing the osteoclast precursor cells and centrifuging by pancreatin digestion, re-suspending in ice-cold TM buffer, 1mM MgCl 2 And 10mM Tris plus water and the pH was adjusted to 7.4. After resuspension, the cells were destroyed by extrusion through a mini-extruder 30 times. The cell homogenate was then mixed with 1M sucrose to a sucrose concentration of 0.25M and the mixture was centrifuged at 2000xg for 10 minutes at 4 ℃. Collecting the supernatant, further centrifuging at 4deg.C and 3000xg for 30 min, re-suspending the precipitate with 0.25M sucrose solution, and centrifuging at 4deg.C and 3000xg for 30 min to obtain precipitate as cell membrane.
EXAMPLE 5 preparation of osteoclast precursor cell membrane (POCM)
Collection of osteoclast precursor cells: the femur and tibia of C57BL/6J mice were washed with alpha-MEM+10% FBS+1% penicillin G and streptomycin (complete alpha-MEM medium) for 6-8 weeks, and the washed cells were cultured in complete alpha-MEM medium containing 30ng/mL M-CSF for 4 days, and the cells were changed once during the culture to obtain macrophages. The resulting macrophages were cultured in complete alpha-MEM medium containing 30ng/mL M-CSF and 80ng/mL RANKL for 4 days to give osteoclast precursor cells.
Extraction of osteoclast precursor cell membranes: after washing the osteoclast precursor cells and centrifuging by pancreatin digestion, re-suspending in ice-cold TM buffer, 1mM MgCl 2 And 10mM Tris plus water and the pH was adjusted to 7.4. After resuspension, the cells were destroyed by extrusion through a mini-extruder 40 times. The cell homogenate was then mixed with 1M sucrose to a sucrose concentration of 0.25M and the mixture was centrifuged at 2000xg for 10 minutes at 4 ℃. Collecting the supernatant, further centrifuging at 4deg.C and 3000xg for 35 min, and washing with 0.25M sucrose solution to obtain precipitate as cell membrane.
Example 6 preparation of cell membrane biomimetic nanoparticles of osteoclast precursor (POCM-NPs)
The osteoclast precursor cell membrane extracted according to example 4 was dissolved in HEPES buffer solution (pH 7.4, 10 mM) to 1mg/mL. Meanwhile, a nanocomposite having a nitrogen-to-phosphorus ratio of 10 was prepared according to the nps@sirna nanocomposite preparation method of example 1. Mixing 1mg/mL of the osteoclast precursor cell membrane with 250 mug/mL of the nano-composite with the nitrogen-phosphorus ratio of 10 in a volume ratio of 1, carrying out ultrasonic treatment on the mixture for 1 minute, and repeatedly extruding the mixture through a 200nm polycarbonate porous membrane for 5 times to obtain the osteoclast precursor cell membrane bionic nano-particles.
Example 7 preparation of cell membrane biomimetic nanoparticles of osteoclast precursor (POCM-NPs)
The osteoclast precursor cell membrane extracted according to example 4 was dissolved in HEPES buffer solution (pH 7.4, 10 mM) to 1mg/mL. Meanwhile, a nanocomposite having a nitrogen-to-phosphorus ratio of 10 was prepared according to the nps@sirna nanocomposite preparation method of example 1. Mixing 1mg/mL of the osteoclast precursor cell membrane with 250 mug/mL of the nano-composite with the nitrogen-phosphorus ratio of 10 in a volume ratio of 1, carrying out ultrasonic treatment on the mixture for 1 minute, and repeatedly extruding the mixture through a 100nm polycarbonate porous membrane for 5 times to obtain the osteoclast precursor cell membrane bionic nano-particles.
Example 8 preparation of cell membrane biomimetic nanoparticles of osteoclast precursor (POCM-NPs)
The osteoclast precursor cell membrane extracted according to example 4 was dissolved in HEPES buffer solution (pH 7.4, 10 mM) to 1mg/mL. Meanwhile, a nanocomposite having a nitrogen-to-phosphorus ratio of 10 was prepared according to the nps@sirna nanocomposite preparation method of example 1. Mixing 1mg/mL of the osteoclast precursor cell membrane with 250 mug/mL of the nano-composite with the nitrogen-phosphorus ratio of 10 in a volume ratio of 1, carrying out ultrasonic treatment on the mixture for 3 minutes, and repeatedly extruding the mixture through a 200nm polycarbonate porous membrane for 5 times to obtain the osteoclast precursor cell membrane bionic nano-particles.
Example 9 preparation of cell membrane biomimetic nanoparticles of osteoclast precursor (POCM-NPs)
The osteoclast precursor cell membrane extracted according to example 4 was dissolved in HEPES buffer solution (pH 7.4, 10 mM) to 1mg/mL. Meanwhile, a nanocomposite having a nitrogen-to-phosphorus ratio of 10 was prepared according to the nps@sirna nanocomposite preparation method of example 1. Mixing 1mg/mL of the osteoclast precursor cell membrane with 250 mug/mL of the nano-composite with the nitrogen-phosphorus ratio of 10 in a volume ratio of 1, carrying out ultrasonic treatment on the mixture for 3 minutes, and repeatedly extruding the mixture through a 100nm polycarbonate porous membrane for 5 times to obtain the osteoclast precursor cell membrane bionic nano-particles.
Example 10 preparation of fluorescent-labeled osteoclast precursor cell membrane biomimetic nanoparticles (POCM-NPs) FITC @siRNA cy5 )
The osteoclast precursor cell membrane extracted according to example 4 was dissolved in HEPES buffer solution (pH 7.4, 10 mM) to 1mg/mL. 1mg/mL of osteoclast precursor cell membrane was combined with 250. Mu.g/mL NPs of example 3 FITC @siRNA cy5 Mixing the nano-composite with the volume ratio of 1, carrying out ultrasonic treatment on the mixture for 1 minute, and repeatedly extruding the mixture through a 200nm polycarbonate porous membrane for 5 times to obtain the osteoclast precursor cell membrane bionic nano-particles.
The application of the osteoclast precursor cell membrane bionic nano-particles prepared by the method in preparation of the target delivery gene transfection material-based osteoclast lineage treatment is that the osteoclast precursor cell membrane is extracted, the gene transfection material is prepared, and the osteoclast precursor cell membrane bionic nano-particle delivery system is prepared. The preparation method is stable and feasible, and the raw materials are simple to prepare. In-vivo and in-vitro experiments show that the prepared osteoclast precursor cell membrane bionic nano-particles can specifically target osteoclast precursor cells, successfully release nucleic acid medicines, have the characteristics of good stability and low immunogenicity, provide a new strategy for clinical osteoporosis treatment, and have the following related experimental data:
test example 1 Gene transfection nanocomposites at different H 2 O 2 Agarose gel electrophoresis at concentration
NPs@siRNA nanocomposite was obtained as in example 1, 20. Mu.L of NPs@siRNA nanocomposite was taken, 10. Mu.L of siRNA was taken as a control, 7 samples were respectively loaded into gel wells prepared with 1% agarose containing 0.5mg/mL ethidium bromide, 1XTAE buffer was added, 100mV was used, and electrophoresis was performed for 30 minutes. And after the end, the image is shot by a gel imaging system. As shown in fig. 1A, the polymer well compressed the siRNA, preventing migration of the siRNA. 7 samples were added to H prior to electrophoresis 2 O 2 Incubation was carried out at 37℃for 1 hour, followed by electrophoresis and photography under the same conditions. As shown in FIG. 1B at H 2 O 2 The visible siRNA release of the nano-composite with the nitrogen-phosphorus ratio of 5, 10 and 15 in the presence shows that the gene transfection nano-composite NPThe s@siRNA can respond to ROS to realize the release of nucleic acid drug siRNA.
The NPs@shRNA nanocomposite was obtained as in example 2, 20. Mu.L of the NPs@shRNA nanocomposite was taken, 10. Mu.L of shRNA was taken as a control, 7 samples were respectively loaded into gel wells prepared with 1% agarose containing 0.5mg/mL ethidium bromide, 1XTAE buffer was added, 100mV was added, and electrophoresis was performed for 30 minutes. And after the end, the image is shot by a gel imaging system. As shown in fig. 1C, the polymer also compressed shRNA well, preventing migration of shRNA. Adding different concentrations of H into the nano-composite with the nitrogen-phosphorus ratio of 10 before electrophoresis 2 O 2 Incubation was carried out at 37℃for 1 hour, and each sample was electrophoresed and photographed under the same conditions. As shown in FIG. 1D with H 2 O 2 The concentration is increased to more than 1mM, and the nano-composite shRNA is released, which shows that the gene transfection nano-composite NPs@shRNA can also respond to ROS to realize the release of nucleic acid medicament shRNA.
Test example 2 cell membrane extraction validation analysis of osteoclast precursor
The osteoclast precursor cell membrane was obtained as in example 4, and was compared with the cytoplasmic and whole cell lysis solution by Western Blot detection, as shown in FIG. 2, and the cell membrane fraction of the osteoclast precursor cell contained the membrane protein Na-K-Atpase, but not the cytoplasmic protein GAPDH, demonstrating successful isolation of the cell membrane from which the osteoclast precursor cells were extracted.
Experimental example 3 proteomic analysis of cell membrane of osteoclast precursor
The osteoclast precursor cell membrane obtained in example 4 was compared with the macrophage membrane prepared by the same method in proteomics as shown in fig. 3, and graphs a and B show that the osteoclast precursor cell membrane has a higher expression of a protein involved in the recognition of intercellular adhesion and a higher osteoclast differentiation index, indicating the homotypic fusion potential with the osteoclast precursor cell, by using GO enrichment analysis to show the difference of the functions of the osteoclast precursor cell membrane and the macrophage membrane on cell adhesion, recognition and osteoclast differentiation proteins. Panels C and D show that with respect to functional aspects, proteins involved in migration and targeting, such as CXCR4, CDC42 and RAC2, are significantly highly expressed in the osteoclast precursor cell membrane, as are fusion related proteins such as CD44 and OSCAR. Indicating that the osteoclast precursor cell membrane has the potential for osteoclast lineage targeting.
Test example 4 cell membrane bionic nanoparticle particle size potential of osteoclast precursor and transmission electron microscope measurement test
Gene transfection Nanocomposite (NPs) with a nitrogen-phosphorus ratio of 10 was obtained as in example 1, and was dropped one drop on a 400 mesh copper mesh, the excess liquid was sucked off with filter paper, then the negative staining was performed by dropping one drop of phosphotungstic acid, and after the excess liquid was sucked off, naturally dried at room temperature, and the sample was observed with a projection electron microscope. Preparation of the osteoclast precursor cell membrane biomimetic nanoparticles (POCM-NPs) obtained in example 6A copper mesh was treated for 120s by glow discharge to increase its hydrophilicity, and then 2.5. Mu.L of POCM-NPs were respectively dropped onto the mesh to form a thin liquid layer, which was rapidly frozen by immersion in liquid ethane for 200kV freeze-projection electron microscopy. And meanwhile, detecting particle sizes of NPs, POCM and POCM-NPs laser particle diameter instruments, and detecting surface potentials by using a Zeta potentiometer. As shown in FIG. 4A, the gene transfection nano-complexes (NPs) formed by self-assembly are spherical-like nano-particles with regular morphology. As shown in fig. 4B, the osteoclast precursor cell membrane biomimetic nanoparticle has a spherical structure wrapped by a lipid bilayer. As shown in FIG. 4C, NPs particle size around 50nm, the osteoclast precursor cell membrane encapsulation increased its size, and POCM-NPs average particle size was about 123.6nm, but slightly smaller than 141.9nm of POCM. As shown in FIG. 4D, the Zeta potential of NPs was about 14.1mV, and the Zeta potential of POCM-NPs-26.2 mV was close to-27.1 mV of POCM. Indicating successful synthesis of the osteoclast precursor cell membrane bionic nano-particles.
Test example 5: analysis of stability of osteoclast precursor cell membrane bionic nano-particles
Gene transfection Nanocomposites (NPs) with a nitrogen to phosphorus ratio of 10 were obtained as in example 1, and stored at room temperature in PBS and alpha-MEM medium+10% fetal bovine serum (serum), respectively, to obtain Bare (PBS) and Bare (serum); preparation of the osteoclast precursor cell membrane bionic nanoparticle (POCM-NPs) obtained in example 5, respectively, stored in PBS and alpha-MEM medium+10% fetal bovine serum (serum) at room temperature to obtain Coated (PBS) and Coated (serum), and after 0,1,3,5,7 days of storage, the particle size was detected by a particle size detector, respectively. As shown in FIG. 5, the particle size of the gene transfected nano-composite without the osteoclast membrane wrapping becomes obviously larger after 1 day, which indicates that the nano-composite starts to dissociate, and the particle size of the nano-particles is stabilized at about 140nm within 7 days after the osteoclast precursor membrane wrapping, which indicates that the stability of the bionic nano-particles after the osteoclast precursor membrane wrapping is enhanced.
Test example 6: evaluation of in vitro targeting fusion ability of osteoclast precursor cell membrane
The osteoclast precursor cell membrane obtained in example 4 was labeled with DiI at 37℃for 15 minutes. Centrifugation was performed at 10,000Xg for 5 minutes to isolate DiI-labeled osteoclast precursor cell membranes, washed twice with PBS and resuspended in PBS at 1mg/mL for use. The osteoclast precursor cells were cultured in glass bottom dishes at a density of 50,000 cells per dish according to the cell culture method described above. Osteoclast precursor cells were labeled with DiO at 37 ℃ for 15 min and washed twice with PBS. Then, the DiI-labeled osteoclast precursor cell membrane solution was added to the medium and the mixture was incubated at 37℃for 1 hour. After incubation, the nuclei were stained by washing 2 times with PBS and incubating for 15 minutes at 37℃with hoechst33342, and finally the co-localization results were imaged by laser confocal imaging. As shown in fig. 6, the osteoclast precursor cell membrane was successfully bound to the osteoclast precursor cell in a membrane fusion manner.
Test example 7: evaluation of release capacity of osteoclast precursor cell membrane bionic nanoparticle nucleic acid drug
The fluorescent-labeled osteoclast precursor cell membrane biomimetic nanoparticles (POCM-NPs) obtained in example 10 FITC @siRNA cy5 ) The mixture was marked with DiI at 37℃for 15 minutes. Centrifugation at 10,000Xg for 5min to isolate DiI-labeled osteoclast precursor cell membranes, washing twice with PBS and re-suspending in PBS at 1mg/mL to give POCM DiI -NPs FITC @siRNA cy5 And (5) standby. The osteoclast precursor cells were cultured in glass bottom dishes at a density of 50,000 cells per dish according to the cell culture method described above. Then, the labeled osteoclast precursor cell membrane biomimetic nanoparticles (POCM DiI -NPs FITC @siRNA cy5 ) The solution was added to the medium and the mixture was incubated at 37℃for 1,6 hours. After incubation, the nuclei were stained by washing with PBS 2 times, incubating for 15 minutes at hoechst33342 37 ℃and finally confocal with laserAnd shooting a result graph. As shown in fig. 7, the osteoclast precursor cell membrane biomimetic nanoparticles were bound to the osteoclast precursor cells in a membrane fusion manner after 1h of culture, and FITC-labeled transfection material was not separated from cy 5-labeled nucleic acid drug. After 6 hours, the visible gene transfected nanocomplex enters the cells and the FITC-labeled transfection material was separated from the cy 5-labeled nucleic acid drug, indicating successful release of the nucleic acid drug.
Test example 8: in vivo targeting ability assessment of bone precursor cell membrane biomimetic nanoparticles
Establishing a mouse osteoporosis animal model: c57BL/6J mice (females, 11 weeks) were anesthetized with 4% chloral hydrate and subjected to bilateral ovariectomy after skin disinfection to induce osteoporosis. The reduction of the microCT detection bone mass index after 8 weeks indicates successful modeling.
Detection of in vivo targeting ability of bone precursor cell membrane biomimetic nanoparticles: the osteoclast precursor cell membrane obtained in example 4 was mixed with indocyanine green (ICG) in an equal volume of 1mg/mL, sonicated for 3 minutes, and then sequentially pushed through a 200nm polycarbonate filter 5 times. The ICG alone and the macrophage membrane group served as control. After the osteoporosis mice are successfully molded, ICG wrapped by the cell membrane of an osteoclast precursor is injected into tail veins, and after 6, 24 and 72 hours, the fluorescence intensity of heart, liver, spleen and kidney, two lower limbs and spine is detected by a small animal living body imager, and the excitation wavelength is 710nm and the emission wavelength is 785nm. As a result, as shown in fig. 8A, the fluorescence intensity of both lower limbs and the spinal column of the mice was significantly higher than that of the control group, and the fluorescence intensity of both lower limbs and the spinal column of the macrophage membrane group was rapidly attenuated, while the fluorescence intensity of the cell membrane group of the osteoclast precursor was still present after 72 hours. As shown in fig. 8B, fluorescence statistics at each period show that the fluorescence intensity of the osteoclast precursor cell membrane group is higher than that of the control group and the macrophage membrane group in both the lower limbs and the spinal column, indicating that the osteoclast precursor cell membrane bionic nano-particles have in vivo targeting effect.
Further verifying in vivo targeting ability, marking DiO on the osteoclast precursor cell membrane by the method, and injecting DiO-marked osteoclast precursor cell membrane bionic nanoparticles (POCM-NPs) into a tail vein injection osteoporosis mouse model, taking femur tissue after 1 day, fixing 4% paraformaldehyde, 14% EDTA,37 ℃, decalcifiing for 12 hours, and embedding paraffin. After slicing, the cells were incubated overnight at 4℃with DC-STAMP (labeled osteoclast precursor cells), washed 4 times with PBST for 5 minutes each, then stained with Alexa Fluor 594 secondary antibody, washed 4 times with PBST for 5 minutes each, capped with DAPI-containing anti-fluorescence quencher, and the results were imaged with laser confocal. As shown in fig. 9, green spots (DiO) overlapped with red spots (DC-STAMP) in bone surface and bone marrow suggesting in vivo osteoclast precursor cell targeting of osteoclast precursor cell membrane biomimetic nanoparticles.
The embodiment shows that the preparation method of the osteoclast precursor cell membrane bionic nano-particles for targeted delivery of the gene transfection material has the characteristics of simple and convenient operation, low cost and the like. The material can actively and specifically target the osteoclast precursor cells in the osteoclast lineage, and the gene transfection nano-composite enters the cells in a membrane fusion mode, and after responding to the microenvironment of the cells, the charge of the transfection material is reversed, so that the nucleic acid medicine is released. The invention aims at the characteristics of cells at different stages in an osteoclast lineage to accurately target gene administration, and is a potential cell membrane bionic targeting material.
The foregoing is merely a preferred embodiment of the invention, and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (11)
1. A cell membrane biomimetic nanoparticle of an osteoclast precursor, which is characterized in that: comprises an osteoclast precursor cell membrane capable of targeting an osteoclast precursor cell and a gene transfection material capable of reversing charges and coated in the osteoclast precursor cell membrane;
the cell membrane of the osteoclast precursor is from an osteoclast precursor cell obtained by inducing macrophages for 3-5 days by using 30-80 ng/mL RANKL; the charge-reversible gene transfection material is a nano-composite formed by electrostatic self-assembly of a charge-reversible cationic polymer B-PDEAEA and a nucleic acid drug; the osteoclast precursor cell membrane bionic nano-particles realize charge reversal under a cell microenvironment so as to release nucleic acid medicines.
2. The osteoclast precursor cell membrane biomimetic nanoparticle of claim 1, wherein: the particle size of the gene transfection material capable of reversing charges is 30-200 nm.
3. The osteoclast precursor cell membrane biomimetic nanoparticle of claim 1, wherein: the nucleic acid drug is at least one of siRNA, microRNA or DNA.
4. A method for preparing an osteoclast precursor cell membrane biomimetic nanoparticle, which is characterized in that the method comprises the following steps of:
(1) Preparation of Gene transfection Material
Dissolving B-PDEAEA in 10mM HEPES buffer solution, wherein the pH of the HEPES buffer solution is 7.4, incubating for 10 minutes at 37 ℃, preparing a nucleic acid medicament into 40 mug/mL solution, diluting the B-PDEAEA into 250-1500 mug/mL by using the 10mM HEPES buffer solution, adding the pH of the HEPES buffer solution into the nucleic acid medicament solution of 40 mug/mL according to the volume ratio of 1:1, vortex oscillating for 10-30 seconds, standing for 20-30 minutes at room temperature, and obtaining a nano-composite solution with the nitrogen-phosphorus molar ratio of 5-30;
(2) Extraction of osteoclast precursor cell membranes
Extracting mammal mononuclear cells, inducing for 3-5 days by 15-30 ng/mL M-CSF to obtain macrophages, and inducing for 3-5 days by 30-80 ng/mL RANKL to obtain osteoclast precursor cells; after washing, pancreatin digestion, the cells were resuspended in TM buffer at 4℃which was 1mM MgCl 2 Mixing with 10mM Tris plus water and adjusting pH to 7.4; extruding for 30-40 times by using a micro-injector after re-suspending to destroy cells;
adding 1M sucrose and mixing with the cell homogenate to reach the final concentration of 0.25M sucrose; centrifuging the obtained mixture for 10 minutes at the temperature of 4 ℃ and the temperature of 2000xg, collecting supernatant, centrifuging for 30-35 minutes at the temperature of 4 ℃ and the temperature of 3000xg, re-suspending and washing the precipitate by using a 0.25M sucrose solution, centrifuging for 30-35 minutes at the temperature of 3000xg again to obtain the precipitate which is a cell membrane, and re-suspending the precipitate to 1-2 mg/mL by using a 10mM HEPES buffer solution, wherein the pH value of the HEPES buffer solution is 7.4;
(3) Preparation of osteoclast precursor cell membrane bionic nano-particles
Mixing the osteoclast precursor cell membrane obtained in the step (2) with the charge-reversible gene transfection material obtained in the step (1) in a volume ratio of 0.5-2; the mixture is ultrasonically mixed for 1 to 3 minutes, and finally the mixture is repeatedly extruded through the polycarbonate porous membrane.
5. The method for preparing the osteoclast precursor cell membrane bionic nanoparticle according to claim 4, wherein the method comprises the following steps: the molar ratio of nitrogen to phosphorus is 10.
6. The method for preparing the osteoclast precursor cell membrane bionic nanoparticle according to claim 4, wherein the method comprises the following steps: the mammal mononuclear cells are tibia mononuclear cells and femur mononuclear cells of a 6-8 week C57BL/6J mouse.
7. The method for preparing the osteoclast precursor cell membrane bionic nanoparticle according to claim 4, wherein the method comprises the following steps: the pancreatin digestion is carried out for 3-5 min, then the digestion is stopped and centrifuged at 1000rpm for 5 min.
8. The method for preparing the osteoclast precursor cell membrane bionic nanoparticle according to claim 4, wherein the method comprises the following steps: the micro-injector is a 1ml insulin injector.
9. The method for preparing the osteoclast precursor cell membrane bionic nanoparticle according to claim 4, wherein the method comprises the following steps: the repeated extrusion times are 5-20 times.
10. The method for preparing the osteoclast precursor cell membrane bionic nanoparticle according to claim 4, wherein the method comprises the following steps: the aperture of the polycarbonate is 100-400 nm.
11. Use of the osteoclast precursor cell membrane biomimetic nanoparticle according to any one of claims 1-3 for the preparation of a medicament, characterized in that: the application of the osteoclast precursor cell membrane bionic nano-particles as an osteoclast precursor cell transfection material carrier or in preparation of an osteoclast precursor cell targeting drug.
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