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CN-121971705-A - Biomineralization-based aptamer functionalized DNA nano-structure complex and preparation method and application thereof

CN121971705ACN 121971705 ACN121971705 ACN 121971705ACN-121971705-A

Abstract

The invention belongs to the technical field of biomedicine and nanointersection, and particularly relates to a biomineralization-based nucleic acid aptamer functionalized DNA nanostructure composite, and a preparation method and application thereof. The compound has a tetrahedron structure, comprises a functionalized DNA tetrahedron frame and a calcium phosphate coating coated on the surface of the functionalized DNA tetrahedron frame, wherein the functionalized DNA tetrahedron frame is formed by self-assembling four DNA single chains, and the 5' end of one DNA single chain is modified with a nucleic acid aptamer. The bionic calcium coating is used as a protective shell, so that the stability of Apt02 in the body and the angiogenesis promoting activity of the Apt02 are remarkably improved. Meanwhile, by virtue of the special spatial configuration of the tetrahedral DNA nano structure, the complex can release calcium ions after being ingested by cells, so that osteogenic differentiation is further promoted, and a novel strategy for synergetic promotion is provided for vascularized bone regeneration.

Inventors

  • GAO FEI
  • MA BAOJIN
  • ZHANG TING

Assignees

  • 山东大学

Dates

Publication Date
20260505
Application Date
20260204

Claims (10)

  1. 1. A biomineralization-based aptamer functionalized DNA nanostructure complex, characterized in that the biomineralization-based aptamer functionalized DNA nanostructure complex has a tetrahedral structure, comprising a functionalized DNA tetrahedral framework and a calcium phosphate coating coated on the surface thereof; The functional DNA tetrahedral framework is formed by self-assembling four DNA single chains, wherein the 5' end of one DNA single chain is modified with a nucleic acid aptamer.
  2. 2. The biomineralization-based aptamer functionalized DNA nanostructure complex of claim 1, wherein the functionalized DNA tetrahedral framework has a particle size of 10-20 nm; the biomineralization nucleic acid aptamer functionalized DNA nano-structure complex has a tetrahedral structure, and the particle size of the complex is 50-60 nm.
  3. 3. A method of preparing a biomineralization-based aptamer functionalized DNA nanostructure complex according to claim 1 or 2, comprising the steps of: (1) Adding four DNA single strands into a buffer solution, mixing, and performing self-assembly reaction to obtain a functional DNA tetrahedral framework; (2) And (3) dropwise adding the calcium chloride solution into a phosphate-containing functionalized DNA tetrahedral framework buffer solution to prepare a mixed solution, performing calcium mineralization reaction, and purifying to prepare the biomineralization aptamer functionalized DNA nano-structure compound.
  4. 4. The method according to claim 3, wherein in the step (1), the buffer solution is Tris-CaCl 2 buffer solution, and the concentration of each of the four DNA single strands is 90-110. Mu.M.
  5. 5. A method according to claim 3, wherein in step (1), the self-assembly reaction is carried out by heating to 90-95 ℃ for 3-6 min and then cooling to 3-4 ℃ for 20-40 min; Preferably, the self-assembly reaction is to heat 95 ℃ to maintain 5 min, cool to 4 ℃ and maintain more than 30 min.
  6. 6. The method according to claim 3, wherein in the step (2), the buffer solution is Tris buffer having a pH of 8.5 to 9.5, the final concentration of the DNA tetrahedral framework in the mixed solution is 3 to 5. Mu.M, the final concentration of Ca 2+ is 1.5 to 2.5 mM, and the final concentration of PO 4 3- is 1.0 to 1.5 mM; Preferably, the Tris buffer has a pH of 9.0, the final concentration of DNA tetrahedral framework in the mixed solution is 4. Mu.M, the final concentration of Ca 2+ is 2.0 mM, and the final concentration of PO 4 3- is 1.2 mM.
  7. 7. A method according to claim 3, wherein in step (2), the calcium mineralization is carried out at a temperature of 35-40 ℃ for a time of 6-48 h; preferably, the temperature of the calcium mineralization reaction is 37 ℃ and the time is 6 h.
  8. 8. The method according to claim 3, wherein in the step (2), the purification is an ultrafiltration centrifugation method using an ultrafiltration centrifuge tube having a molecular weight cut-off of 25 to 35 kDa, centrifuging 8 to 12min at 25 to 30 ℃ under 2500 to 3500×g, and repeating the operation 2 to 4 times.
  9. 9. Use of a biomineralization-based aptamer functionalized DNA nanostructure complex according to claim 1 or 2 in bone defect repair.
  10. 10. A bone defect repair product comprising the biomineralization-based aptamer functionalized DNA nanostructure complex of claim 1 or 2.

Description

Biomineralization-based aptamer functionalized DNA nano-structure complex and preparation method and application thereof Technical Field The invention belongs to the technical field of biomedicine and nanointersection, and particularly relates to a biomineralization-based nucleic acid aptamer functionalized DNA nanostructure composite, and a preparation method and application thereof. Background In the field of tissue regeneration medicine, efficient repair of bone defects not only relies on osteoblast differentiation and bone matrix deposition (osteogenesis), but also entails the simultaneous formation of a functional vascular network (vascularization) to supply the new bone tissue with sufficient nutrients and oxygen. This spatiotemporal synergy of "vascularization-osteogenesis" is a central condition for successful regeneration of bone tissue. However, existing biomaterials and therapeutic strategies often have difficulty achieving efficient coupling of both processes in time and space, resulting in limited bone regeneration effects. Currently, the delivery of bioactive components such as growth factors, polypeptides or small nucleic acid molecules to promote vascularization or osteogenesis is a common intervention. Among them, nucleic acid aptamers are attracting attention because of their high affinity, ease of modification, and low immunogenicity. For example, nucleic acid aptamers with pro-angiogenic functions (e.g., apt 02) have shown potential for use in therapeutic angiogenesis. However, as an oligonucleotide, the aptamer is easily and rapidly degraded by nuclease in a complex in vivo environment, so that the aptamer has poor stability, short half-life and low bioavailability, and the clinical transformation of the aptamer is severely restricted. To enhance stability and achieve targeted delivery, nucleic acid aptamers are often loaded into nanocarriers. DNA nanotechnology, particularly framed nucleic acids with precise three-dimensional structures (e.g., DNA tetrahedra), has become a promising delivery platform by virtue of its good biocompatibility, programmability, ease of functionalization, and efficient cellular uptake capacity. However, unmodified DNA nanostructures themselves remain susceptible to nuclease degradation in serum, and they act primarily as carriers of loading and localization, and there is a lack of effective strategies to actively enhance the biological function of loading factors, synergistically regulate multiple biological processes (e.g., like protecting aptamers and providing osteogenic signals). Meanwhile, the biomimetic strategy of biomimetic mineralization provides a new way for improving the bioactivity and stability of the material. For example, the biomimetic deposition of a calcium phosphate coating on the surface of a material, which mimics the inorganic components of natural bone matrix, has been shown to be beneficial for cell adhesion and osteogenic differentiation. If the strategy can be combined with the DNA nanotechnology, a composite system with structural stability, bioactivity and multiple functions is expected to be constructed on the nanometer scale. However, how to realize accurate and controllable biomimetic mineralization on the surface of a DNA nano-carrier with a fine structure, form a uniform and ultrathin protective functional coating, and systematically evaluate the active protective effect of the DNA nano-carrier on a nucleic acid aptamer and the cooperative regulation and control effect of the DNA nano-carrier on a vascularization-bone formation process, and the technical problem to be broken through is still needed in the current field. Disclosure of Invention The invention aims to provide a biomineralization-based nucleic acid aptamer functionalized DNA nano-structure compound and a preparation method and application thereof, so that the defects of the prior art are overcome, the nucleic acid aptamer can be effectively protected, the function of the nucleic acid aptamer can be enhanced, and a novel multifunctional nano-compound capable of simultaneously providing osteogenesis induction signals can be provided, and the novel multifunctional nano-structure compound has important scientific significance and application value for promoting the development of bone tissue regeneration treatment. In order to achieve the above purpose, the technical scheme of the invention is as follows: In a first aspect, the present invention provides a biomineralization-based aptamer functionalized DNA nanostructure complex, the biomineralization-based aptamer functionalized DNA nanostructure complex having a tetrahedral structure comprising a functionalized DNA tetrahedral framework and a surface-coated calcium phosphate coating thereof; The functional DNA tetrahedral framework is formed by self-assembling four DNA single chains, wherein the 5' end of one DNA single chain is modified with a nucleic acid aptamer. As an example, the four DNA single strands S1-S4