CN107867677B - One-dimensional calcium phosphate nano/micron material and preparation method and application thereof - Google Patents
One-dimensional calcium phosphate nano/micron material and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a one-dimensional calcium phosphate nano material and a preparation method and application thereof, the one-dimensional calcium phosphate nano/micron material is prepared by microwave-assisted hydrothermal reaction of water-soluble calcium salt and water-soluble phosphate, the one-dimensional calcium phosphate nano/micron material is of a rod-shaped structure, the length of the one-dimensional calcium phosphate nano/micron material is 50nm-500 mu m, and the length-diameter ratio is more than 5:1, it can be mixed with nucleic acid and then deliver the nucleic acid into cells, and has good biocompatibility, high gene transfection efficiency and long duration.
Description
Technical Field
The invention relates to the technical field of gene vectors, in particular to a one-dimensional calcium phosphate nano/micron material and a preparation method and application thereof.
Background
Gene therapy achieves the purpose of treating diseases by introducing foreign genes into target nuclei to repair defective genes causing diseases or to suppress harmful genes causing diseases. Successful gene therapy relies on effective gene vectors, and common vectors are classified into viral vectors and non-viral vectors. Viral vectors have great safety hazards, which limits the development of viral vectors. In recent years, the appearance of Minicircle DNA (mcDNA) has become a large highlight in gene vectors, and Minicircle DNA is a circular expression cassette, which is a product obtained by removing bacterial framework DNA of a standard plasmid by a DNA recombination technique, and contains only one gene expression cassette without an external bacterial framework sequence. However, since naked DNA is rapidly cleared in a physiological environment and cannot efficiently enter target cells, a suitable delivery system is still required to deliver the minicircle DNA to the target tissue or organ.
Commonly used gene delivery systems include cationic liposomes, cationic polymers (polyethyleneimine PEI), and the like. Such delivery systems have low immunogenicity, large gene loading capacity, but low transfection efficiency, greatly limiting their clinical application. Therefore, the key to the clinical application of gene medicine is to develop a gene delivery system with high efficiency, good biocompatibility and targeting.
The calcium phosphate is used as a gene vector, has higher biological safety, and has no obvious cytotoxicity while well protecting and wrapping DNA. But generally has the problems of uncontrollable particle size, easy agglomeration, low transfection efficiency and the like. However, the transfection efficiency of the calcium phosphate-gene coprecipitation method is very low (about 10-20%), and the prepared calcium phosphate particles are large in particle size, difficult to control, poor in stability, easy to agglomerate and difficult to use for in vivo transfection research. Leaf Huang et al propose a phospholipid bilayer-coated DNA/calcium phosphate nanoparticle system prepared by a reverse microemulsion method, but the purification steps of calcium phosphate are complicated, the cationic liposome used in the preparation process has high cytotoxicity, and the phospholipid material is expensive, thus being not beneficial to further clinical application.
Disclosure of Invention
In view of the above, the invention prepares the one-dimensional calcium phosphate nano/micron material by a microwave hydrothermal synthesis method, the one-dimensional calcium phosphate nano/micron material has a larger length-diameter ratio (the one-dimensional calcium phosphate nano/micron material is a nanorod or a needle-shaped object with a tip), and the one-dimensional calcium phosphate nano/micron material can be efficiently transfected in the abdominal cavity of a mouse by intraperitoneal injection after being mixed with micro-ring DNA. Can be used as a gene vector for high-efficiency transfection.
In a first aspect, the invention provides a preparation method of a one-dimensional calcium phosphate nano/micron material, which comprises the following steps:
(1) respectively dissolving water-soluble calcium salt and water-soluble phosphate in water to prepare water-soluble calcium salt and water-soluble phosphate solution, adding the water-soluble calcium salt and the water-soluble phosphate solution into a first solvent, and mixing to obtain a first mixed solution, wherein the first solvent is water or a mixed solvent of water and alcohol, and the alcohol comprises one or more of ethanol, ethylene glycol and glycerol;
(2) adjusting the pH value of the first mixed solution to 5-11, and placing the mixed solution after the pH adjustment in a microwave hydrothermal kettle for microwave-assisted hydrothermal reaction, wherein the reaction temperature is 110-;
(3) and (3) centrifugally separating a product obtained after the reaction, and washing and drying the separated solid matter to obtain the one-dimensional calcium phosphate nano/micron material.
Preferably, the water-soluble calcium salt is calcium chloride and/or its hydrate, calcium nitrate and/or its hydrate (Ca (NO)3)2·4H2O), and/or calcium acetate and/or its hydrate (Ca (CH)3COO)2·H2O), but not limited to.
Preferably, the water-soluble phosphate is ammonium phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate or the like; but are not limited to the soluble inorganic phosphates listed above.
I.e., including but not limited to (NH)4)3PO4，(NH4)2HPO4，(NH4)H2PO4，Na3PO4，Na2HPO4，NaH2PO4，K3PO4，K2HPO4，KH2PO4Or K2HPO4。
Preferably, the water-soluble calcium salt and the water-soluble phosphate solution have a Ca: the P molar ratio is (0.5-5): 1.
further preferably, the water-soluble calcium salt and the water-soluble phosphate solution have a Ca: the P molar ratio is (0.5-2): 1. further preferred is (0.5-1.5): 1.
preferably, the first solvent is a mixed solvent of water and alcohol.
Further preferably, in the first solvent, the volume ratio of water to ethanol is (0.1-10): 1.
further preferably, in the first solvent, the volume ratio of water to ethanol is (0.5-3): 1.
preferably, the pH value of the first mixed solution is 7 to 11.
Preferably, the pH is adjusted by using an acid or a base.
Further preferably, the acid used for adjusting the pH is hydrochloric acid, nitric acid or acetic acid; the alkali used for adjusting the pH is ammonia water, NaOH or KOH.
Preferably, in step (2), the reaction temperature is 120-160 ℃, for example 130, 140, 150 ℃.
In the step (2), the reaction time may be 20, 30, 45, 50 or 60 min.
Preferably, the one-dimensional calcium phosphate nano/micron material is in a rod-like structure.
Preferably, the diameter of the one-dimensional calcium phosphate nano/micron material is 10-1000 nm.
Preferably, the length of the one-dimensional calcium phosphate nano/micron material is 50nm-500 μm. More preferably 100nm to 100 μm; or 350-30 μm. More preferably 150-350 nm.
Preferably, the aspect ratio of the one-dimensional calcium phosphate nano/micron material is more than 5: 1. more preferably greater than 10: 1.
the one-dimensional calcium phosphate nano/micron material can be a nano/micron rod with two flat ends, a nano/micron rod structure with two pointed ends (also called a needle structure in the case), or a structure with one flat end and the other pointed end.
Further preferably, the one-dimensional calcium phosphate nano/micron material is in a nano/micron rod structure with two pointed ends. In this case the aspect ratio is greater than 20: 1. more preferably (20-50): 1.
furthermore, the one-dimensional calcium phosphate nano/micron material is also modified with a ligand for cell specific recognition.
The cell-specific recognition ligand includes, but is not limited to, modifications such as lactose or galactose ligands, hyaluronic acid ligands, folate ligands, transferrin ligands, lipopolysaccharide ligands, polypeptide ligands (e.g., arginine-glycine-aspartic acid series polypeptides), and the like. It is understood that the cell types targeted by the specific ligands include, but are not limited to, various tumor cells, immune cells (e.g., macrophages, DC cells, T cells, B cells, NK cells, etc.), normal tissue cells (e.g., stem cells, liver cells, muscle cells, epithelial cells, etc.). The ligand may be selected by the destination of delivery of the one-dimensional calcium phosphate nano/micro material when used as a gene carrier. The ligand specifically identified by the cells can be modified after the one-dimensional calcium phosphate nano/micron material is prepared by a microwave-assisted hydrothermal method, and can also be added into the first mixed solution. However, if the ligand is added during the preparation process, it is necessary to ensure that the ligand-modified one-dimensional calcium phosphate material structure (without changing its one-dimensional structure) can still be obtained after the ligand is added to the first mixed solution. Preferably, after the one-dimensional calcium phosphate nano/micron material is prepared, the ligand is modified.
In one embodiment of the present invention, the target protein and/or polypeptide may be prepared into a water solution with a certain concentration, the prepared one-dimensional calcium phosphate nano/micron material is dispersed in the protein/polypeptide solution, and after fully stirring overnight, the one-dimensional calcium phosphate material with the surface modified with the protein/polypeptide is obtained by centrifugation.
In another embodiment of the invention, the one-dimensional calcium phosphate nano/micron material is firstly reacted in a polyamino compound (such as polyethyleneimine) solution overnight, a great amount of amino groups are distributed on the surface of the calcium phosphate through a positive and negative charge attraction process, and then the purpose of grafting the ligand is achieved through covalent coupling of the connected amino groups and the ligand (RGD cyclopeptide and/or folic acid molecules) with carboxyl groups under the action of a coupling agent 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and the like.
The preparation method of the one-dimensional calcium phosphate nano/micron material provided by the first aspect of the invention has the advantages of simple preparation process and high yield, does not need to use expensive raw materials, and can control the morphology, the size and the like of the obtained calcium phosphate nano/micron material by adjusting conditions such as reaction temperature, time, system pH, the ratio of a calcium source to a phosphorus source and the like.
The obtained one-dimensional calcium phosphate nano/micron material has higher length-diameter ratio and good biocompatibility, can be used as a gene carrier and directly mixed with DNA (such as minicircle DNA) to obtain a DNA-calcium phosphate solution, when the DNA-calcium phosphate solution is injected into the abdominal cavity of a mouse through the abdominal cavity, the one-dimensional calcium phosphate material can easily stimulate mesothelial cells due to the one-dimensional structure with higher length-diameter ratio, the permeability of cell membranes of the cells is increased in the process of division or proliferation, so that the DNA can rapidly enter the cells, and the cells can proliferate and divide when being stimulated, at the moment, the nuclear membranes of the cell nuclei are opened, and the cell nuclei are divided into two parts, so that the DNA can enter the cell nuclei to realize efficient transfection.
In a second aspect, the present invention provides a one-dimensional calcium phosphate nano/micro material prepared by the preparation method of the first aspect of the present invention.
In a third aspect, the present invention provides a pharmaceutical composition (or called gene vector complex, gene delivery system) comprising the above one-dimensional calcium phosphate nano/micro material and a nucleic acid. The one-dimensional calcium phosphate nano/micron material loads the nucleic acid, and the loading can be blending or combining.
Preferably, the mass ratio of the one-dimensional calcium phosphate nano/micron material to the nucleic acid is (1-200): 1.
further preferably, the mass ratio of the one-dimensional calcium phosphate nano/micron material to the nucleic acid is (10-150): 1.
further preferably, the mass ratio of the one-dimensional calcium phosphate nano/micron material to the nucleic acid is (15-100): 1.
more preferably, the mass ratio of the one-dimensional calcium phosphate nano/micro material to the nucleic acid is 100: 1.
preferably, the nucleic acid includes one or more of a DNA fragment and an RNA fragment, but is not limited thereto.
As used herein, "DNA fragment" includes one or more of DNA fragments artificially synthesized and purified in a biological sample; "RNA fragment" includes one or more of RNA fragments that are artificially synthesized and purified in a biological sample. Specifically, the artificially synthesized or purified DNA fragment in the biological sample may be plasmid DNA, minicircle DNA, DNA fragment, or the like; specifically, the RNA fragment artificially synthesized or purified in the biological sample may be microRNA (microRNA), siRNA (small interfering RNA), shRNA, or the like.
Further preferably, the nucleic acid is a micro-loop DNA, but is not limited thereto.
Preferably, the pharmaceutical composition further comprises at least one of a biological drug and a chemical drug, wherein the biological drug includes, but is not limited to, one or more of a polypeptide, a protein, and a vaccine; including but not limited to one or more of anti-cancer drugs, imaging agents, and tracers. The tracer may be a fluorescent dye, a lymphatic tracer, or the like.
In a fourth aspect, the present invention provides a preparation method of the above pharmaceutical composition, comprising the following steps:
mixing the one-dimensional calcium phosphate nano/micron material solution and the nucleic acid solution, and incubating to obtain the pharmaceutical composition. Wherein, the one-dimensional calcium phosphate nano/micron material loads the nucleic acid, and the loading can be blending or combining.
Preferably, the mass ratio of the one-dimensional calcium phosphate nano/micron material to the nucleic acid is (1-200): 1.
further preferably, the mass ratio of the one-dimensional calcium phosphate nano/micron material to the nucleic acid is (10-150): 1.
further preferably, the mass ratio of the one-dimensional calcium phosphate nano/micron material to the nucleic acid is (15-100): 1.
more preferably, the mass ratio of the one-dimensional calcium phosphate nano/micro material to the nucleic acid is 100: 1.
preferably, at least one of a biological drug and a chemical drug can be added when the one-dimensional calcium phosphate nano/micron material solution and the nucleic acid solution are mixed, wherein the biological drug includes but is not limited to one or more of polypeptide, protein and vaccine; including but not limited to one or more of anti-cancer drugs, imaging agents, and tracers.
Further preferably, the one-dimensional calcium phosphate nano/micro material solution is an aqueous solution of one-dimensional calcium phosphate nano/micro material, but is not limited thereto. Specifically, the calcium phosphate nano/micron material comprises but is not limited to an aqueous solution of a one-dimensional calcium phosphate nano/micron material, a physiological saline solution of a one-dimensional calcium phosphate nano/micron material, a glucose solution of a one-dimensional calcium phosphate nano/micron material, a phosphate buffer solution of a one-dimensional calcium phosphate nano/micron material and the like.
Further preferably, the nucleic acid solution is an aqueous solution of nucleic acid, but is not limited thereto. Specifically, the aqueous solution of nucleic acid includes, but is not limited to, an aqueous solution of nucleic acid, a physiological saline solution of nucleic acid, a glucose solution of nucleic acid, a phosphate buffer solution of nucleic acid, and the like.
According to the invention, the incubation is directly carried out at room temperature without heating or cooling, and the incubation temperature is 20-37 ℃.
Preferably, the incubation time is 10-100 min.
Preferably, the pharmaceutical composition can be diluted and concentrated to prepare an aqueous injection for gene therapy.
Preferably, the pharmaceutical composition can also be prepared into freeze-dried powder injection by freeze-drying and other processes for gene therapy.
In a fifth aspect, the present invention provides the use of a one-dimensional calcium phosphate nano/micro material according to the first aspect of the invention or a pharmaceutical composition according to the third aspect of the invention for the preparation of a nucleic acid delivering medicament.
When the one-dimensional calcium phosphate nano/micron material or the pharmaceutical composition is used as a nucleic acid delivery drug, the one-dimensional calcium phosphate nano/micron material has low toxicity, high biocompatibility and high delivery efficiency and expression efficiency of nucleic acid.
In a sixth aspect, the invention also provides a method of delivering a nucleic acid molecule to a cell comprising contacting the cell with a pharmaceutical composition according to the third aspect of the invention.
Preferably, the cell is in vivo, but is not so limited.
Further preferably, the contacting of the cell with the pharmaceutical composition of the third aspect of the present invention is by: contacting the cells with the pharmaceutical composition according to the third aspect of the invention by in vitro transfection or in vivo subcutaneous injection (e.g., intraperitoneal injection) or intramuscular injection.
Wherein, when the pharmaceutical composition is injected into a mouse body by intraperitoneal injection, high-concentration DNA expression products can be detected in ascites and blood.
Preferably, the pharmaceutical composition is administered in an amount of not less than 0.1 to 10. mu.g of nucleic acid per kg of body weight of the animal in the case of contact by subcutaneous or intramuscular injection in vivo, but is not particularly limited.
The beneficial effects of the invention comprise the following aspects:
1) the one-dimensional calcium phosphate nano/micron material has a unique structure, good biocompatibility, high transfection efficiency and low toxicity when used as a gene vector;
2) the pharmaceutical composition formed by the one-dimensional calcium phosphate nano/micron material and nucleic acid (such as micro-ring DNA) has good stability, and can efficiently express the micro-ring DNA for a long time;
3) the preparation method of the one-dimensional calcium phosphate nano/micron material and the pharmaceutical composition is simple, convenient and feasible, and has high controllable degree.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. The specific embodiments described herein are merely illustrative of the invention and do not delimit the invention.
FIG. 1 shows the XRD results of one-dimensional calcium phosphate nano/micro materials provided by different experimental groups of the present invention;
FIG. 2 is an electron microscope photograph of one-dimensional calcium phosphate nano/micro material provided by different experimental groups and comparative examples of the present invention, and the transfection result of the mediated micro-ring DNA with fluorescein after intraperitoneal injection;
FIG. 3 shows the result of agarose gel electrophoresis experiment of one-dimensional calcium phosphate nano/micron material and DNA prepared by the embodiment of the present invention;
FIG. 4 is a graph showing the effect of different mass ratios of one-dimensional calcium phosphate nano/micro material/DNA on luciferase transfection in mouse abdominal cavity;
FIG. 5 shows the transfection result of one-dimensional calcium phosphate nano/micro material mediated micro-loop DNA in the peritoneal cavity with CD3/Cmet dual-targeting antibody, the left graph shows the antibody concentration in serum; the right panel shows the antibody concentration in the abdominal cavity.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Preparation examples: preparation of one-dimensional calcium phosphate nano/micron material
The invention provides a preparation method of a one-dimensional calcium phosphate nano/micron material, which comprises the following steps:
(1) 3mLNaH2PO4Solution (0.05mol/L) and 3mL of CaCl2Adding the solution (0.1mol/L) into a mixed solvent consisting of 15mL of deionized water and 15mL of absolute ethyl alcohol, and uniformly mixing at room temperature; then, the pH value of the mixed solution is adjusted to 7.0 +/-0.5, and a reaction solution A is obtained. (2) Pouring the reaction solution A into a microwave hydrothermal reaction kettle, heating for 10min at 110 ℃ by microwave, and taking out the reaction kettle when the system is naturally cooled to below 60 ℃; (3) centrifuging the product in the reaction kettle at 12000g for 10min, washing the separated solid with anhydrous ethanol for 3 times, and freeze drying to obtain calcium phosphateNano/micro material, this sample was named sample 1.
It should be noted that, according to different needs, the pH of the solution a, the reaction temperature and time, the Ca of the water-soluble calcium salt and the water-soluble phosphate solution: p molar ratio, water system volume and the like to obtain one-dimensional calcium phosphate samples with different grain diameters. In this embodiment, 1 to 10 calcium phosphate samples prepared under different experimental conditions are provided, and the specific formulations are shown in table 1 below:
TABLE 1
| Experimental group | pH of solution A | T/℃ | t/ |
| 1 | 7.0 | 110 | 10 |
| 2 | 11.0 | 110 | 10 |
| 3 | 5.0 | 110 | 10 |
| 4 | 6.0 | 110 | 10 |
| 5 | 8.0 | 110 | 10 |
| 6 | 9.0 | 110 | 10 |
| 7 | 10.0 | 110 | 10 |
| 8 | 7.0 | 110 | 30 |
| 9 | 10.0 | 150 | 30 |
| 10 | 10.0 | 180 | 10 |
Calcium phosphate sample characterization experiment:
the calcium phosphate samples prepared in each experimental group of example 1 were placed on an XRD tester and characterized by XRD powder diffraction, and the results are shown in fig. 1. From XRD in FIG. 1, it can be seen that the crystal phase of the reaction product obtained was hydroxyapatite (P63/m, card No. 25-0166) at pH 7, 11, 6, while the crystal phase of the reaction product was brushite (la (9), card No. 11-0293) at pH 5 of the reaction solution, both of which are the common crystal structures of calcium phosphate.
Calcium phosphate samples prepared in each experimental group were dispersed in an aqueous solution, sonicated for more than 10min until the samples were completely dispersed, and then dropped on a copper mesh, and an electron microscope photograph of each sample was obtained using a FEI Tecnai G2F20S-Twin model electron microscope at a voltage of 110V, with the results shown in fig. 2.
Cellular activity assay of calcium phosphate samples: after incubating one-dimensional calcium phosphate samples prepared in experimental group 1 of the present invention in table 1 with 293T cells for 24 hours, the cytotoxicity was tested using CCK8 kit. The experimental results prove that the concentration of the calcium phosphate sample is 1 multiplied by 10-5The survival rate of 283T cells reaches 90-100% when the cell density is about 1mg/mL, which indicates that the calcium phosphate sample has no obvious toxicity to 283T cells and normal cell morphology and proliferation.
Agarose gel electrophoresis experiment: each test group was controlled to contain 0.5. mu.g of DNA, and the DNA solution was mixed with the calcium phosphate sample (sample 1) to a total volume of 20. mu.L at different mass ratios of calcium phosphate sample to DNA (0.25:1, 0.5:1, 1:1, 2:1, 4:1) while agarose gel containing DNA dye (1%, w/v) was added (while DNA solution alone was used as a control). Then, 2. mu.L of loading buffer was added and mixed well, and the mixture was added to the well. The electrophoresis was carried out in Tris-acetate buffer (TAE), electrophoresis was carried out at 80V for 40 minutes, and then visualized by imaging with a gel imager under UV irradiation, the results are shown in FIG. 3.
DNA is a negatively charged macromolecule and will move to the positive pole under the action of an electric field. In the agarose gel experiment of FIG. 3, it was found that the DNA could move under the action of the electric field after the DNA was incubated with calcium phosphate, which indicates that the calcium phosphate did not block the movement of the DNA, and this experiment demonstrated that there was no chemical force between the DNA and the calcium phosphate.
Calcium phosphate samples mediated micro-loop DNA intraperitoneal transfection (luciferase):
dispersing 2mg of each group of calcium phosphate samples in 200 μ L of ultrapure water, and performing ultrasonic treatment for 10 minutes to form a calcium phosphate solution; dispersing 20 μ g of micro-loop DNA carrying a luciferase (luciferase) expression gene in 200 μ L of water to form a micro-loop DNA solution B;
adding the micro-ring DNA solution B into the calcium phosphate solution to form a pharmaceutical composition solution C; the pharmaceutical composition solution C was intraperitoneally injected into mice with age of 6-8 weeks and weight of 20g, and 24 hours later, luciferase expression in the mice was observed using an IVIS small animal imager, and the result is shown in FIG. 2, in which red circles represent ROI (region of interest).
Comparative example 1: (1) dissolving 200mg of egg yolk lecithin in 15ml of absolute ethyl alcohol to obtain an egg yolk lecithin solution; 3mL of NaH2PO4Solution (0.05mol/L) and 3ml CaCl2Adding the solution (0.1mol/L) into a mixed solvent consisting of 15mL of deionized water and 15mL of absolute ethyl alcohol, and uniformly mixing at room temperature to obtain a first mixed solution; (2) adding the egg yolk lecithin solution into the first mixed solution B to obtain a second mixed solution; placing the obtained second mixed solution in a microwave hydrothermal reaction kettle, heating for 10min at 110 ℃, and taking out the reaction kettle when the system is naturally cooled to below 60 ℃; (3) centrifuging the product in the reaction kettle for 10min at 12000g, washing the separated solid with anhydrous ethanol for 3 times, and freeze-drying to obtain lecithin-modified calcium phosphate nanoparticles, which are named as pair 1.
Comparative example 2: (1) 0.111g adenosine disodium triphosphate (ATP) and 0.111g CaCl2Dissolving in 50ml distilled water, magnetically stirring to dissolve completely, and adjusting pH value of the solution to 7.0; (2) placing the pH solution in a microwave hydrothermal reaction kettle, heating at 110 deg.C for 10min, and taking out the reaction kettle when the system is naturally cooled to below 60 deg.C; (3) centrifuging the product in the reaction kettle for 10min at 12000g, washing the separated solid by adopting absolute ethyl alcohol for 3 times, and finally freeze-drying to obtain the calcium phosphate nano-particles taking adenosine triphosphate as a phosphorus source, wherein the sample is named as a pair 2 sample.
FIG. 2 is the electron micrographs of the calcium phosphate samples prepared in the examples of the present invention and comparative examples and the results of the calcium phosphate samples in each case mediating the transfection of luciferase into the micro-loop DNA abdominal cavity. As shown in fig. 2, the calcium phosphate samples prepared by the method provided by the present invention are all one-dimensional structures, wherein, the samples 1 and 2 are both one-dimensional nano/micro rod structures (in this case, they may also be referred to as needle structures) with tips at both ends, and the samples 1 and 2 have diameters of 10nm and 20nm, and lengths of about 150nm and 300nm, respectively. Samples 3, 10 are nano/micro rods with flat ends. Sample 3 had a diameter of 600nm and a length of about 20 μm; sample 10 had a diameter of 30nm and a length of about 400 nm. In addition, each one-dimensional calcium phosphate nano/micron material prepared by the embodiment of the invention can well mediate the abdominal cavity transfection of the micro-ring DNA. The samples prepared in comparative examples 1 and 2 were round particles with diameters of about 100nm and 150nm, respectively; this is probably due to the fact that organic substances capable of chelating calcium ions are added during the calcium phosphate crystallization process, the calcium ions may be preferentially chelated with the organic substances, and the long-range ordered crystallization process of the calcium phosphate crystals is interrupted during the growth process to form amorphous spherical calcium phosphate particles. When they were used to transfect the minicircle DNA carrying the luciferase (luciferase) expression gene in vivo, no expressed luciferase was observed in the abdominal cavity of mice, which suggests that the calcium phosphate material of the round particles did not function well to stimulate cells and thus effective transfection of DNA could not be achieved.
Effect of one-dimensional calcium phosphate nano/micron material/DNA with different mass ratios on luciferase transfection effect in abdominal cavity of mouse
Respectively dispersing 0.3, 0.6 and 1mg of each group of calcium phosphate samples in 200 mu L of ultrapure water, and carrying out ultrasonic treatment for 10 minutes to form a calcium phosphate solution; dispersing 20 μ g of micro-loop DNA carrying a luciferase (luciferase) expression gene in 200 μ L of water to form a micro-loop DNA solution; adding the micro-ring DNA solution into each calcium phosphate solution to form a pharmaceutical composition solution; the solution of the pharmaceutical composition was intraperitoneally injected into Balb/C mice aged 6-8 weeks and weighing about 20g, and after 24 hours, luciferase expression in the mice was observed using an IVIS small animal imager, and the results are shown in FIG. 4.
As can be seen from FIG. 4, the calcium phosphate dose had a greater effect on the transfection effect in vivo, and the same mice were injected with 20. mu.g of DNA, the greater the calcium phosphate dose, the greater the transfection effect. This is probably because large amounts of calcium phosphate more readily stimulate the stress response of mesothelial cells, favoring DNA phagocytosis.
Calcium phosphate samples mediated micro-loop DNA intraperitoneal transfection (anti-CD 3/anti-Cmet dual targeting antibody): dispersing 2mg of each group of calcium phosphate samples in 200 μ L of ultrapure water, and performing ultrasonic treatment for 10 minutes to form a calcium phosphate solution; dispersing 20 mu g of micro-ring DNA with the anti-CD 3/anti-Cmet double-targeting antibody expression gene in 200 mu L of ultrapure water to form a micro-ring DNA solution C;
adding the micro-ring DNA solution C into the calcium phosphate solution to form a pharmaceutical composition solution D; the pharmaceutical composition solution D was intraperitoneally injected into a mouse with a weight of 20g, the mouse was sacrificed 24 hours later, and the concentration of the dual targeting antibody expressed in ascites and serum of the mouse was monitored, and the results are shown in fig. 5. Wherein, the left graph in FIG. 5 is the antibody concentration in serum; the right panel shows the antibody concentration in the abdominal cavity.
The results of fig. 5 show that: by calcium phosphate mediation, the target protein can be expressed in the abdominal cavity by injecting the micro-ring DNA into the abdominal cavity, such as anti-CD 3/anti-Cmet double-targeting antibody with high concentration can be detected in serum and ascites, and the concentration of the antibody in the serum is higher than that in the ascites.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (7)
1. A preparation method of one-dimensional calcium phosphate nano/micron material is characterized by comprising the following steps:
(1) respectively dissolving water-soluble calcium salt and water-soluble phosphate in water to prepare water-soluble calcium salt and water-soluble phosphate solution, adding the water-soluble calcium salt and the water-soluble phosphate solution into a first solvent, and mixing to obtain a first mixed solution, wherein the first solvent is a mixed solvent of water and alcohol, and the alcohol comprises one or more of ethanol, ethylene glycol and glycerol; the volume ratio of the water to the ethanol is (0.1-10): 1; the water-soluble phosphate is inorganic phosphate;
(2) adjusting the pH value of the first mixed solution to 5-11, and placing the mixed solution after the pH adjustment in a microwave hydrothermal kettle for microwave-assisted hydrothermal reaction, wherein the reaction temperature is 110-;
(3) centrifugally separating a product obtained after the reaction, and washing and drying a separated solid to obtain a one-dimensional calcium phosphate nano/micron material; the one-dimensional calcium phosphate nano/micron material is of a rod-shaped structure, the length of the one-dimensional calcium phosphate nano/micron material is 50nm-500 mu m, and the length-diameter ratio is more than 5: 1; the one-dimensional calcium phosphate nano/micron material is in a nano/micron rod structure with two pointed ends.
2. A pharmaceutical composition comprising a nucleic acid and the one-dimensional calcium phosphate nano/micro material obtained by the method for preparing a one-dimensional calcium phosphate nano/micro material according to claim 1.
3. The pharmaceutical composition of claim 2, wherein the mass ratio of the one-dimensional calcium phosphate nano/micro material to the nucleic acid is (1-200): 1.
4. the pharmaceutical composition of claim 2, wherein the one-dimensional calcium phosphate nano/micro material is further modified with a cell-specific recognition ligand comprising one or more of a lactose ligand, a galactose ligand, a hyaluronic acid ligand, a folic acid ligand, a transferrin ligand, and a lipopolysaccharide ligand.
5. The pharmaceutical composition of claim 2, further comprising at least one of a biologic drug and a chemical drug, wherein the biologic drug comprises one or more of a polypeptide, a protein, and a vaccine; the chemical drug includes one or more of an anti-cancer drug, an imaging agent, and a tracer.
6. A method for preparing a pharmaceutical composition, wherein the one-dimensional calcium phosphate nano/micro material obtained by the preparation method of claim 1 is mixed with a nucleic acid solution, and then incubated to prepare the pharmaceutical composition.
7. Use of a pharmaceutical composition according to any one of claims 2-5 in the manufacture of a nucleic acid delivering medicament.
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| CN110974979B (en) * | 2019-11-05 | 2020-12-15 | 中国人民解放军第四军医大学 | Preparation method and application of functionalized calcium phosphate gene delivery system |
| CN113456886B (en) * | 2020-03-31 | 2023-07-18 | 中国人民解放军第四军医大学 | Nucleic acid-calcium phosphate nanoparticle complexes and their use in biomineralization |
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