(556d) Bioactive DNA-Peptide Nanotubes As Artificial Extracellular Matrices for Bone Tissue Engineering

Introduction: The extracellular matrix (ECM) is a complex network composed of collagen, fibronectin and other macromolecules that not only provides structural support but also relays crucial biochemical and biomechanical signals¹. It dictates how cells attach, behave and interact with one another, thus, it plays a key role in tissue morphogenesis, differentiation and repair. In this context, the construction of artificial matrices that mimic the structure of the naturally occurring ECM and display biological signals might prove to be highly promising in controlling tissue regeneration². Although electrospun fibers, polymeric hydrogels and self-assembled peptide amphiphiles have been widely investigated for cell adhesion and proliferation, thereâ??s a need for scaffolds that are structurally programmable and easy to modify. DNA has emerged as one of the most promising building blocks for such nanoconstruction due to the high predictability of Watson-Crick base pairing, enabling the generation of one, two, three-dimensional assemblies for templated growth of nanowires, the organization of nanoparticles and as drug delivery carriers³. In the present study, DNA nanotubes were constructed by thermal annealing based on double-crossover motifs⁴ and were further covalently functionalized with a BMP-7 derived peptide. The structure of the nanotubes was inspired by collagen with similar size and stiffness, and the BMP-7 derived peptide was selected because of its role in regulating the proliferation, differentiation and apoptosis of bone cells. The objective of the present study was to develop an extracellular microenvironment that can mimic the structural and regulatory characteristics of natural ECMs. Results showed that the DNA nanotube coated glass coverslips revealed a tangled network of fiber-like materials that resemble the morphology of natural ECM, suggesting DNA-peptide nanotubes could be a promising candidate as artificial extracellular matrices. Further experiments are still need to fully investigate their effects on osteoblast cell adhesion and proliferation.

Materials and Methods: Preparation of oligonucleotides stocks: All DNA strands were purchased with polyacrylamide gel electrophoresis (PAGE) purification from Integrated DNA Technology (IDT), dissolved at 500 µM in IDTE buffer (IDT) and stored at -20 ºC prior to use. For generating fluorescent tubes, the S3 sequence was ordered from IDT with a 3â??-fluorescein (FAM) modification. Preparation of DNA-peptide hybrid conjugates: The BMP-7 derived peptide Ser-Asn-Val-Ile-Leu-Lys-Lys-Tyr-Arg-Asn (SNVILKKYRN) was conjugated to a DNA strand by Biosynthesis using a previously reported² Huisgen 1,3-dipolar cycloaddition reaction between a strained cyclooctyne and an azide. The resulting conjugate was purified by multiple high-performance liquid chromatography (HPLC) and characterized by mass spectrometry. DNA nanotube annealing: To form the DNA nanotubes, oligonucleotide stocks were further diluted to 50 µM in a TAE/Mg buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA and 12.5 mM Mg(OAc)₂) and equal amounts of all constituent strands were mixed. The mixture was annealed from 95 ºC to 25 ºC over ~6 hours in a thermal cycler. After annealing, the solution was stored at 4 ºC. Preparation of APTES-treated glass coverslips: In order to bind negatively charged DNA nanotubes, glass coverslips were treated with (3-Aminopropyl) triethoxysilane (APTES). The resulting coverslips had a positive charge at physiological pH. Briefly, to coverslips (12 mm in diameter) in a 12-well tissue culture plate, 100 µl of APTES were added into the spaces between wells (600 µl total). The plate was sealed with two layers of tape, wrapped in double layer of aluminum foil and then placed in oven at 90 ºC for 18 hours. The coverslips were washed by ethanol and then DI water. Excess water was blown off by nitrogen gas and the coverslips were dried in a vacuum oven at 115 ºC. Surface coating: To coat the surface of glass coverslips, 400 µl of the nanotube stock solutions were added on APTES-treated glass coverslips in a 24 well-plate and were incubated at 37 ºC overnight to allow adhesion on the surface. Following incubation, the solution was removed and the coverslips were rinsed twice with sterile PBS before seeding cells. Transmission electron microscopy: To characterize size and morphology of DNA nanotubes, they were visualized using transmission electron microscopy operated at 80 kV. Briefly, 10 µl of the stock solution was applied on 300-mesh copper-coated carbon grids for 5 min. Following wicking and rinsing with deionized water, the grids were then stained with 10 µl of a 1.5% uranyl acetate solution twice for 10 s and dried for 15 min prior to imaging. Fluorescent imaging of the surface coating: Following annealing, the DNA nanotube solution was coated on APTES-treated coverslips according to the method described in the surface coating section. Images of the coatings were taken using a fluorescence microscope under 40X objective. A Z-stack was acquired to estimate the approximate thickness of the coating and a 3D reconstruction of the z stack was obtained using a 3D viewer plugin in ImageJ. Cell viability assay: To determine cell viability, human osteoblasts (HOB, Promocell) were seeded at a density of 20,000 cells/cm² in 96-well plate and subsequently co-cultured with the DNA nanotubes at various concentrations (0/1.25/2.5/3.75/5 µM) for 24 hours under standard cell culture conditions (37 ºC, humidified, 5% CO₂/95% air) . MTS assays were used to determine cell density after incubation. Briefly, 20 µl of MTS dye solution was added per 100 µl of solution, and the absorbance readings were taken at 490 nm after 4 hours of incubation. Cell density was then determined with correlation to a standard curve. Statistical analysis: All experiments were conducted in triplicate and repeated at least three different times. Statistical differences were determined using analysis of variance (ANOVA) where p< 0.01 was considered statistically significant.

Results and Discussion: TEM images confirmed that the rationally designed DNA tiles can self-assemble and form long tubelike structures about 10-12 nm in diameter and often many micrometers in length with both walls and central pore clearly visible. Incorporating hairpin into one of the strands yields an almost indistinguishable structure, indicating that modifications at selected positions are not likely to affect tube formation. This offers the largest advantage of this system over other alternatives in that there is potential to vary the biological epitope without affecting the core structure. Stability of the nanotubes and nanotube-peptide hybrids against temperature, cation depletion and nuclease degradation were shown to be better than DNA double helices using TEM.The fibrillar morphology of the coating resembles that of extracellular matrices, indicating the potential of these DNA nanotubes to act as mimics of a native extracellular matrix. Finally, these nanotubes were shown to have no toxic effect on osteoblast cells with a concentration of up to 5 µM. Conversely, at low concentrations, specifically 1.25 and 2.5 µM, increases in cell density were observed.

Conclusions: Through above experiments, DNA nanotubes self-assembled from DNA tiles were successfully designed, constructed and characterized. The ECM-like fibrillar morphology and the minimal toxicity to osteoblast cells indicated their potential to be used as improved artificial extracellular matrices. 

References:

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2. Stephanopoulos N, Freeman R, North HA, et al. Bioactive DNA-peptide nanotubes enhance the differentiation of neural stem cells into neurons. Nano Lett. 2015;15(1):603-609.

3. Aldaye FA, Senapedis WT, Silver PA, Way JC. A Structurally Tunable DNA-Based Extracellular Matrix. J Am Chem Soc. 2010;132(42):14727-14729.

4. Rothemund PWK, Ekani-Nkodo A, Papadakis N, Kumar A, Fygenson DK, Winfree E. Design and Characterization of Programmable DNA Nanotubes. J Am Chem Soc. 2004;126(50):16344-16352.