NUCLEIC ACID-CALCIUM PHOSPHATE NANOPARTICLE COMPLEXES AND APPLICATION THEREOF IN BIOMINERALIZATION

Abstract

Disclosed are nucleic acid-calcium phosphate nanoparticle complexes and application thereof in biomineralization. Specifically, disclosed are a biological mineralizer and a preparation method thereof. The mineralizer contains a complex formed by nucleic acid and amorphous calcium phosphate nanoparticles. Further, disclosed is a collagen fiber product containing the biological mineralizer or treated with the biological mineralizer, such as a medical device for being implanted into a patient. Further, disclosed is use of the biological mineralizer or the collagen fiber product in treatment of bone-associated diseases or disorders or improvement of bone conditions of patients. Further, disclosed is a method of using the biological mineralizer to induce biomimetic mineralization of collagen fibers or a preparation method of a mineralized collagen fiber product.

Claims

1. A biological mineralizer, containing a complex formed by nucleic acid and amorphous calcium phosphate nanoparticles, preferably, formed by means of electrostatic adsorption.

2. The biological mineralizer according to claim 1, wherein the complex is formed by the nucleic acid and the amorphous calcium phosphate nanoparticles by the following method: electrostatically adsorbing, by phosphate groups of the nucleic acid, calcium ions of the nanoparticles, and further electrostatically adsorbing, by the calcium ions adsorbed onto the nucleic acid, free phosphate ions.

3. The biological mineralizer according to claim 1, formed by mixing a solution of nucleic acid with a solution of calcium chloride and a solution of dipotassium hydrogen phosphate.

4. The biological mineralizer according to claim 1, wherein the calcium ion concentration is 1 to 10 mM, preferably 2 to 5 mM, and more preferably 1.67 to 3.5 mM; and the phosphate ion concentration is 0.5 to 10 mM, preferably 1 to 5 mM, and more preferably 1.0 to 2.1 mM.

5. The biological mineralizer according to claim 1, wherein a ratio of the calcium ions to the phosphate ions is 10:1 to 1:5, preferably 5:1 to 1:3, preferably 3.5:1 to 1:1.25, and more preferably is 2:1 to 1:1, such as 1.67:1.

6. The biological mineralizer according to claim 1, wherein the complex has a particle size of 1 to 100 nm, preferably 10 to 100 nm, and more preferably 20 to 100 nm, such as 40 to 60 nm and 60 to 100 nm.

7. The biological mineralizer according to claim 1, wherein the nucleic acid is DNA or RNA, such as total DNA or total RNA isolated from mammalian cells or plasmid DNA; or the nucleic acid includes nucleic acid promoting osteogenic differentiation and/or bone regeneration, such as miR-17-92, miR-26a, miR-148b, and BMP2-plasmid DNA.

8. The biological mineralizer according to claim 1, wherein the mammalian cells are osteoprogenitor cells, pre-osteoblasts, osteocytes, osteogenitor cells, osteoblasts, osteoclasts, or bone marrow stromal cells.

9. The biological mineralizer according to claim 1, wherein the working concentration of the nucleic acid is 10 to 500 μg/mL, preferably 50 to 500 μg/mL, and more preferably 100 to 300 μg/mL, such as 150 to 250 μg/mL.

10. The biological mineralizer according to claim 1, having a pH value of 5.5 to 7, and preferably 6.0 to 6.5.

11. The biological mineralizer according to claim 1, being in a liquid state such as a solution and a colloidal solution; or in a semisolid state such as a gel; or in a solid state such as powder and lyophilized powder.

12. A mineralized collagen fiber product, containing the biological mineralizer according to claim 1, or treated with the biological mineralizer according to claim 1.

13. The collagen fiber product according to claim 12, selected from collagen scaffolds, collagen films, collagen fiber sheets, demineralized bone tissues, demineralized dentin slices, mouse tail, tooth or bone repair materials, tooth or bone scaffold materials, tooth or bone regeneration materials, and tooth or bone implant materials.

14. A method of treating for a bone-associated disease or disorder or improving bone conditions of a patient comprising administering a therapeutically effective amount of the mineralized collagen fiber product of claim 12, or a drug or medical device comprising a therapeutically effective amount of the mineralized collagen fiber product of claim 12 to the patient.

15. The method according to claim 14, wherein the disease or disorder is a bone defect or bone loss, or the method of improving bone conditions is promoting bone repair, osteogenic differentiation or bone regeneration in the patient.

16. A method of mineralizing osteocollagenous fibers in a patient comprising administering a therapeutically effective amount of the biological mineralizer of claim 1 to the patient.

17. A preparation method of the biological mineralizer according to claim 1, comprising: at step (1), obtaining nucleic acid, and preferably extracting total DNA (comprising various types of DNA) or total RNA (comprising various types of RNA) from mammalian cells or extracting plasmid DNA, at step (2), mixing the nucleic acid obtained at step (1) with a solution of calcium chloride, and at step (3), adding a solution of dipotassium hydrogen phosphate into the mixture obtained at step (2) to obtain a complex formed by nucleic acid and amorphous calcium phosphate nanoparticles.

18. The method according to claim 17, wherein the initial concentration of the nucleic acid obtained at step (1) is 100 to 5,000 ng/L, preferably 500 to 5,000 ng/μL, and more preferably 1,000 to 3,000 ng/μL, such as 1,500 to 2,500 ng/μL; and the nucleic acid has a wide molecular weight range, and preferably has a molecular weight of greater than 40 kDa.

19. A method for inducing biomimetic mineralization of collagen fibers or a preparation method of a mineralized collagen fiber product, comprising a step of allowing the biological mineralizer according to claim 1 to be in contact with collagen fibers or a product containing collagen fibers.

20. A nucleic acid delivery or transfection system, wherein the nucleic acid delivery system contains the biological mineralizer according to claim 1.

21. The nucleic acid delivery or transfection system according to claim 20, which is used to transfect cells, control and maintain the expression of osteogenic proteins at bone defects, or promote osteogenic differentiation and bone regeneration.

22. The nucleic acid delivery or transfection system according to claim 20, wherein the biological mineralizer contains nucleic acid promoting osteogenic differentiation and/or bone regeneration.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0079] FIG. 1 is a transmission electron microscopy image of RNA-ACP nanoparticles in low concentration, wherein the upper right corner of (a) is a selected area electron diffraction image of the RNA-ACP nanoparticles; (c) to (f) are images of distribution of elements of oxygen, calcium, phosphorus, and nitrogen, respectively; and (b) is a composite image of distribution of the above four elements.

[0080] FIG. 2 is a diagram of potential and particle size distribution of calcium phosphate in low concentration, wherein (a) and (b) show potential and particle size distribution of a pure mixed solution of calcium and phosphate without stabilizers; (c) and (d) show potential and particle size distribution of an RNA-ACP mineralizing solution; and (e) and (f) show potential and particle size distribution of a DNA-ACP mineralizing solution.

[0081] FIG. 3 is a Fourier infrared spectrogram, wherein from top to bottom, infrared spectrograms of hydroxyapatite lyophilized powder in low concentration, DNA-ACP lyophilized powder in low concentration, and RNA-ACP lyophilized powder in low concentration are shown in sequence.

[0082] FIG. 4 is a transmission electron microscopy image of collagen fibers, wherein (a) shows that after the collagen fibers are mineralized with an RNA-ACP mineralizing solution in low concentration for 5 days, intra- and extrafibrillar mineralization of collagen can be observed; (b) shows that after the collagen fibers are mineralized with a DNA-ACP mineralizing solution in low concentration for 5 days, intra- and extrafibrillar mineralization of collagen is completed.

[0083] FIG. 5 is a scanning electron microscopy image of a collagen fiber scaffold, wherein (a) shows surface topography of mineralized collagen fibers of a femur of a 4-month-old mouse; and (b) shows surface topography of a pure collagen fiber scaffold mineralized with an RNA-ACP mineralizing solution in low concentration for 5 days, and intra- and extrafibrillar mineralization of collagen can be observed.

[0084] FIG. 6 is a scanning electron microscopy image of collagen fibers mineralized with an RNA-ACP mineralizing solution in low concentration for 5 hours, wherein (a) shows that calcium phosphate is deposited in order in the collagen fibers to complete intrafibrillar mineralization of collagen, which is observed by using a dark-field microscope; the lower left corner of (b) is a selected area electron diffraction image showing that calcium phosphate has been transformed into hydroxyapatite crystals arranged along a C axis of collagen; and (c) and (d) are images of distribution of elements of calcium and phosphorus, respectively.

[0085] FIG. 7 is a transmission electron microscopy image of collagen fibers stained with ruthenium red, wherein (a) shows pure collagen fibers that are co-incubated with pure RNA (200 μg/mL) for 48 hours, washed, and stained with ruthenium red; and (b) shows pure collagen fibers that are co-incubated with nuclease-free water for 48 hours, washed, and stained with ruthenium red.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0086] The present disclosure extracts biomacromolecules having anionic properties, such as RNA and DNA, from cells, and use the same in vitro to stabilize minerals such as calcium phosphate by means of biomimetic mineralization so as to prepare a nucleic acid-ACP nanoparticle complex. Meanwhile, as an agent for inducing mineralization of collage fibers, nucleic acid makes ACP enter the collage fibers and be deposited between collagen fibers to complete biomimetic intra- and extrafibrillar mineralization of collagen. Thus, an organic/inorganic hydroxyapatite-collagen composite material having high biocompatibility, low immunogenicity, fast mineralizing speed, and high mechanical strength is prepared, which provides an unique and efficient solution for repair of bone tissue defects.

[0087] The following specific examples are provided by the inventor to further explain and describe the technical solutions of the present disclosure.

Example 1

[0088] (1) Mouse BMSC were inoculated into T75 according to an initial coating density of 5×10.sup.4 cells/cm.sup.2, and cultured with αMEM containing 10% FBS for 2 days. When the cells were in good condition and at a confluence of about 70 to 80%, an osteogenic induction medium was added and changed every 2 to 3 days. [0089] (2) After osteogenic differentiation was induced for 7 days, total RNA of the cells was extracted by a Trizol method when the cell grew well. Because RNA is easily degraded, the extraction is performed at low temperature on ices, and the used tools need to be subjected to enzyme-free treatment. Finally, the obtained RNA precipitates were dissolved in nuclease-free water. [0090] (3) the concentration of solution of RNA in each EP tube was determined by using an ELISA reader, and the solutions of RNA having an initial concentration of greater than 1,500 ng/μL were selected and mixed together to obtain 1 mL of solution of RNA in a high concentration of 1,600 to 2,500 ng/μL. [0091] (4) Equal amounts of nuclease-free water was respectively added into 2 centrifuge tubes with a volume of 15 mL, and calcium chloride dihydrate powder (having a molecular weight of 147) was prepared into solutions containing calcium ions in concentrations of 7 mM and 3.5 mM, respectively. [0092] (5) Equal amounts of nuclease-free water was respectively added into 2 centrifuge tubes with a volume of 15 mL, and dipotassium hydrogen phosphate powder (having a molecular weight of 174) was prepared into solutions containing phosphate ions in concentrations of 4.2 mM and 2.1 mM, respectively. The solutions used in the present example for preparing reagents are nuclease-free water. [0093] (6) 100 μL of RNA was mixed with the solution (450 μL) of calcium chloride dihydrate in the concentration of 7 mM, and then equal volume of solution (450 μL) of dipotassium hydrogen phosphate in the concentration of 4.2 mM was slowly dropwise added into the mixture. An RNA-ACP mineralizing solution in high concentration having a concentration ratio of calcium to phosphorus of 3.5:2.1 was prepared. Similarly, 100 μL of RNA was mixed with the solution (450 μL) of calcium chloride dihydrate in the concentration of 3.5 mM, and then equal volume of solution (450 μL) of dipotassium hydrogen phosphate in the concentration of 2.1 mM was slowly dropwise added into the mixture. An RNA-ACP mineralizing solution in low concentration having a concentration ratio of calcium to phosphorus of 1.67:1 was prepared. [0094] (7) A solution (8 mg/mL) of rat tail tendon collagen/acetic acid was placed into a dialysis bag, and the dialysis bag was fastened. [0095] (8) The dialysis bag was placed into a phosphate buffer solution (PBS, pH 7.4). The dialysis solution was changed at 37° C. every 12 hours. [0096] (9) After 72 hours, the self-assembly of collagen fibers was completed, in order to reduce the influence of phosphate ions on the self-assembled collagen fibers, the PBS dialysis solution was replaced with deionized water (pH 7.4), and reverse dialysis was performed. [0097] (10) After 48 hours, an appropriate amount of solution (8 mg/mL) of collagen in the dialysis bag was taken by using a pipette and dropwise added onto a 400-mesh nickel/gold net covered with carbon support film, and the net faced up and was dried at room temperature. [0098] (11) The collagen fibers were cross-linked with a solution of 0.3 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.06 M N-hydroxysuccinimide (NHS) for 4 hours, washed with deionized water 3 times, and dried at room temperature for later use. [0099] (12) About 450 μL of nucleic acid-ACP nanoparticle mineralizing solution was dropwise added into an EP tube cap to form a uniform spherical shape. [0100] (13) A front surface of the nickel/gold net carrying collagen fibers was in contact with the mineralizing solution for mineralization for 5 days.

[0101] In an optimal solution, the selected matrix to be mineralized may be a solution of rat tail tendon type I collagen/acetic acid, a self-assembled rat tail tendon, a self-assembled 3D collagen scaffold, a collagen film, a demineralized bone tissue, a demineralized dentin slice, etc.

Example 2

[0102] Differences between this example and Example 1 are: [0103] (1) Total DNA of cells is extracted using a kit by a Trizol method, and the initial concentration of the obtained DNA was determined by using the ELISA reader, which may be greater than 700 ng/μL. [0104] (2) 150 μL of DNA was uniformly mixed with 425 μL of solutions of calcium chloride dihydrate in high and low concentrations respectively, 425 μL of solutions of dipotassium hydrogen phosphate in high and low concentrations were respectively slowly dropwise added into the mixed solutions of DNA-calcium chloride dihydrate. Two groups of stable and clear DNA-ACP nanoparticle mineralizing solutions in high and low concentrations were respectively synthesized, which were applied to later mineralization.

[0105] According to Examples 1 and 2, the biomimetic mineralization of collagen fibers induced by the nucleic acid-ACP nanoparticles constructed in the present disclosure has the following characteristics.

[0106] As shown in FIG. 1, a result of transmission electron microscopy shows that particles of the calcium phosphate complex constructed in Example 1 are spherical and have a relatively uniform size of about 50 nm (FIG. 1, a). A result of selected area electron diffraction shows that calcium phosphate is non-crystalline and amorphous (FIG. 1, the upper right corner of a). Results of energy-dispersive spectroscopy of elements show that main components of the electron-dense particles are oxygen, calcium, phosphorus, and nitrogen (FIG. 1, c to f). A composite image of elements (FIG. 1, b) shows that the distribution of nitrogen is consistent with those of other elements. Because nitrogen only exists in organic nucleic acids, this indicates that under this method, RNA can participate in stabilizing calcium and phosphorus particles, and finally form RNA-ACP nanoparticles having a relatively uniform particle size.

[0107] As shown in FIG. 2, results of Zeta potential show that in a pure solution of calcium and phosphorus without any stabilizer (FIG. 2, a), the potential is basically 0; in a solution of calcium and phosphate added with RNA (150 to 250 μg/mL) as a stabilizer (FIG. 2, c), the potential is negative and about −10 mV; and in a solution of calcium and phosphorus added with DNA (150 μg/mL) as a stabilizer (FIG. 2, e), the potential is about −20 mV. This indicates that, because nucleic acids are negatively charged, the ACP nanoparticle complexes under the stabilization of nucleic acids are also negatively charged. Results of particle sizes show that in the pure solution of calcium and phosphorus without any stabilizer (FIG. 2, b), the calcium and phosphorus particles have a particle size of greater than 1 μm, which indicates that the calcium and phosphorus particles are directly separated from the solution in the form of precipitates. While the DNA-ACP nanoparticles have a particle size of about 60 to 100 nm (FIG. 2, f), and the RNA-ACP nanoparticles have a smaller and more uniform particle size, which is about 40 to 60 nm (FIG. 2, d). This indicates that both RNA and DNA can participate in stabilizing calcium phosphate, and meanwhile, because DNA is of a double-stranded structure and has a larger molecular weight, the diameter of the DNA-ACP nanoparticles presented in space is slightly larger than that of the RNA-ACP nanoparticles.

[0108] As shown in FIG. 3, results of Fourier infrared spectroscopy show that RNA-ACP and DNA-ACP have vibration peaks of O—P—O bonds and phosphate groups at 500 cm-1, 600 cm-1, and 1,000 cm-1. This indicates that the RNA-ACP and DNA-ACP complexes contain calcium phosphate. Meanwhile, vibration peaks of characteristic bases of RNA and DNA, such as guanine, adenine, and cytosine, do not change. Therefore, it indicates that structures of RNA and DNA in the two types of complexes have not been damaged and still remain stability.

[0109] As shown in FIG. 4, results of transmission electron microscopy (FIG. 4, a to b) show that when nucleic acid-ACP is used as a mineralizing solution, ACP nanoparticles can enter collagen fibers, further grow in order along a C axis of the collagen fibers, and finally are transformed into thermodynamically stable hydroxyapatite to complete intrafibrillar mineralization of collagen fibers. At the same time, calcium and phosphorus can also be deposited between or on surfaces of the collagen fibers, and grow disorderly along various directions to finally complete extrafibrillar mineralization of collagen fibers. This indicates that the nucleic acid-ACP has the effect of inducing intra- and extrafibrillar mineralization of collagen fibers, and can simulate the fine characteristics of microstructures of mineralized collagen of natural bone tissues, which lays a firm foundation for the preparation of biomimetic bone repair materials.

[0110] As shown in FIG. 5, a result of scanning electron microscopy shows that after a pure collagen fiber scaffold is mineralized with RNA-ACP for 5 days (FIG. 5, b), the collagen fiber scaffold has expanded in volume in many places, and meanwhile, surfaces of the expanded places are not smooth, and an original characteristic transverse striation structure is covered. It indicates that the space inside and outside the fibers is occupied by inorganic minerals, and intra- and extrafibrillar mineralization of collagen occurs. It is worthwhile to note that a result of scanning electron microscopy (FIG. 5, a) shows that the surface morphology of mineralized collagen of a femur of a natural mouse is also very rough, and the surface is covered with hydroxyapatite, which is extremely consistent with the morphology of the mineralized collagen fibers induced by the RNA-ACP mineralizing solution. This indicates that the stabilizers, the preparation methods, and the mineralization processes used in Examples 1 and 2 are similar to the real in vivo mineralization of collagen fibers. Therefore, the preparation of the material is relatively bionic in terms of mineralization mode.

[0111] As shown in FIG. 6, a result of scanning transmission electron microscopy shows that a large area of collagen can be intrafibrillarly mineralized within only 5 hours of contact with an RNA-ACP mineralizing solution (FIG. 6, a). A result of distribution of elements shows that calcium and phosphorus are deposited in order in the collagen fibers, and meanwhile, a result of selected area electron diffraction also shows that the minerals deposited in the collagen fibers are ordered hydroxyapatite crystals. This indicates that RNA, as an agent for stabilizing calcium and phosphorus and inducing collagen fibers, can rapidly induce intrafibrillar mineralization of a large area of collagen, with extremely short mineralization time and extremely high efficiency. This provides strong support for the efficient preparation and clinical transformation of bone repair materials.

[0112] As shown in FIG. 7, after being co-incubated with pure RNA for 48 hours, pure collagen fibers are washed and stained with ruthenium red. Results of transmission electron microscopy show that the electron-dense RNA-ruthenium red stain solution is uniformly adsorbed onto a larger area of collagen fibers (FIG. 7a), and the collagen fibers that are not co-incubated with RNA have no electron-dense particles (FIG. 7b). This indicates that there is an adsorption force between RNA and collagen, which further verifies the bionics of the experimental model involved in the present patent, and provides an important idea for verifying the real mechanism of in vivo intrafibrillar mineralization of collagen.

[0113] The present disclosure has been further described above in detail with reference to specific preferred embodiments, but the specific embodiments of the present disclosure are not limited thereto. For those of ordinary skill in the art to which the present disclosure belongs, several simple deductions or substitutions can be made without departing from the concept of the present disclosure, and all of these deductions or substitutions shall be regarded as belonging to the present disclosure, and fall within the scope of patent protection determined by the submitted claims.