Materials
Zoledronic acid (PHR1893), melatonin (PHR1767), Dulbecco’s Modified Eagle’s Medium (DMEM) (D5796), Dulbecco′s Phosphate Buffered Saline (PBS) (D8537), and ultrapure Nuclease-free water (W4502) were purchased from Sigma-Aldrich. Ultrapure 1 M Tris-HCl (J22638), Magnesium chloride hexahydrate (213832), 50 bp DNA Ladder (10416014), and TrackIt™ Cyan/Orange Loading Buffer (10482028) were purchased from Thermo Fisher Scientific. Powder-formed ssDNA was purchased from Thermo Fisher Scientific oligonucleotide synthesis service with the sequence shown in Table 1.
Agarose gel electrophoresis
Agarose gel electrophoresis was run with 2% agarose gel (1613100, Bio-Rad) at 120 V in TAE buffer for 60 min. After electrophoresis, the bands of DNA were stained with 1× SYBR Safe and the image was visualized using ChemiDoc XRS+ Molecular Imager and processed using Image Lab Software (Bio-Rad).
Transmission electron microscopy (TEM)
Two micrometer TDNs were synthesized and subsequently fixed using a 2% glutaraldehyde solution before TEM. TDNs were stained with phosphotungstic acid (PTA) and imaged with CM10 TEM (PHILIPS) with a maximum magnification of 13,000×.
Molecular simulation
ICM-Pro was utilized for the simulation of molecular interactions between TDN and small-molecule drugs, including ZA and vancomycin. The 3D molecular model of ZA and vancomycin was obtained from PubChem. The molecular docking simulation was performed by treating linear dsDNA or TDN as a receptor and ZA or vancomycin as a ligand. The docking score was calculated by the ICM Pro software. For each simulation, 100 top hits with lower binding energy scores were generated, and the result with the highest affinity was selected. Each simulation was repeated 5 times.
Preparation of drug-loaded TDNs and the measurement of drug-loading capacity
Drug-loading was conducted using an established protocol32. 100 μL of 2 μM DNA TDN was prepared 24 h before the experiment and then mixed with 100 μL of 1 mM ZA or vancomycin. The final concentration was 1 μM TDN and 500 μM free drug. The mixture was incubated at 37 °C, 60 rpm for 3 h to make sure each drug was sufficiently intercalated into TDNs before the miscible liquids were passed through Amicon ultracentrifuge filters by centrifuging at 14,000g for 10 min. Next, the unloaded drug concentration was measured by UV-Vis absorbance at 209 nm for ZA and 280 nm for vancomycin using a SPARK microplate multimode reader (Tecan). The drug-loading capacity was calculated by the following formula:
\(\text{Drug}-\text{loading}\,\text{capacity}=\,\frac{\left[\text{Drug}\,\text{added}\right]-\left[\text{Unload}\,\text{drug}\right]}{[\text{TDN}]}\)
Cell viability and proliferation studies
Cell viability and cell proliferation studies were performed by Calcein AM staining (Thermo Fisher Scientific, cat. No. C1430) and MTS assay (Promega, Cat. No. G3580). THP-1 monocytic cells were seeded into 96-well plates with a seeding density of 1 × 105 per well and cultured for 12 h. Fresh media were added, and the cells were treated with 100, 250, 500, 750, and 1000 nM TDN. All the experiments were performed following the manufacturer’s protocol, and the live cell images were taken using the fluorescent Nikon Eclipse Ti2-E microscope (Nikon).
Hemolysis studies
Mouse red blood cells (mRBCs) were isolated from whole blood samples purchased from Innovative Research. The mRBCs were separated from the serum and washed three times with sterile DPBS by centrifugation at 3460 rpm for 10 min at 4 °C. Next, mRBCs were diluted with DPBS to a final concentration of 0.16% (v/v) mRBCs suspension. Then, 0.16% mRBCs suspension was incubated with DPBS, 100 nM TDNs, 500 nM TDNs, 1000 nM TDNs, and with 0.2% Triton-X as a positive control. The plate was incubated at 37 °C with agitation for 1 h, after which the supernatant was transferred to Eppendorf tubes and centrifuged at 14,000 rpm at 4 °C for 10 min to remove any intact cells. Hemolytic activity was assessed by measuring the amount of hemoglobin liberated into the surrounding solution due to membrane rupture. The amount of hemoglobin released was determined by measuring the absorbance at 415 nm. Controls defining 0 and 100% hemolysis were plated in DPBS in the absence or presence of 0.2% Triton-X, respectively. Representative images of mRBCs in the supernatant were taken using a Nikon Eclipse Ti2-E microscope.
Cell apoptosis study in the presence of TDNs
Cellular apoptosis was determined using Annexin V and a propidium iodide staining kit (Biotium, Cat. No. 30061). In brief, THP-1 cells were seeded into a six-well plate in suspension at a density of 1 × 105 per well in a 96-well plate and incubated for 24 h. The next day, the cells were treated for 24 h with TDNs (500 nM) and CPG-TDNs (500 nM). The positive control was made by exposing the cells to 55°C for 20 min, followed by overnight incubation at 37 °C in a CO2 incubator14. All the samples were stained according to the manufacturer’s specified protocol and analyzed using the Cytoflex flow cytometer (Beckman Coulter).
Cell genotoxicity assessment using alkaline comet assay in the presence of TDNs
The alkaline comet assay was carried out according to the manufacturer’s protocol (R&D Systems, Cat. No. 4250-050-ESK). Briefly, human mesenchymal stem cells (hMSCs) purchased from Lonza were seeded in a 48-well plate with a seeding density of 30,000 cells/well and cultured overnight. The next morning, cell media was replaced with fresh media and was either left untreated (negative control) or treated with 30 µg/mL NiO (positive control) or 0.5 µM TDNs and exposed for 24 h. Cells were then harvested by trypsinization and resuspended in cold PBS. 50 µL of the cell suspension was added to 500 µL of LMAgarose. 50 µL of the resulting cell/gel solution was pipetted onto CometSlides. Cells were lysed for 1 h in the dark and placed in an alkaline buffer for unwinding. This was followed by electrophoresis at 25 V for 30 min in an alkaline buffer. Samples were then washed, dried overnight, and fixed in methanol. DNA was stained using 1:10,000 SYBR-Green in TAE buffer for 15 min. DNA was imaged using a fluorescence microscope (Nikon) and scored using OpenComet ImageJ software. A minimum of 30 comets were scored per sample, and results were expressed as a mean of percent of DNA in tail ± SD. Three individual experiments were performed for each group.
Inflammation studies with hPBMCs and TDNs
Human peripheral blood mononuclear cells (hPBMCs) were purchased from Lonza. Cells were cultured in a T-75 flask for 6 days in RPMI medium with 10% FBS, 100 U/ml penicillin, 2 mM l-glutamine, and 25 ng/mL M-CSF for macrophage differentiation. hPBMCs were seeded in a 96-well plate at a density of 5 × 104 cells and incubated overnight. PBS, 100, 500, or 1000 nM TDNs were added to the cells, and 1 µg/ml lipopolysaccharides (LPS) (Sigma Aldrich, Cat. No. L6529-1MG) were added separately as a positive control. Supernatants collected at 1 h after hPBMC seeding without differentiation media were used as negative controls. After 6 h, the supernatant was collected, and TNF-α and IL-6 secretion were quantified using the Quantikine ELISA Kit (R&D Biosystems) according to the manufacturer’s protocol.
In vitro osteogenic differentiation and osteoclast inhibition assay
Human MSCs were cultured on 100 mm petri dishes in stem cell culture media until reaching 100% confluency. hMSCs were seeded in a 48-well plate at a density of 3 × 104 cells/well in hMSC media supplemented with 5% FBS and 1% PS. Upon reaching 100% confluency, 0.5 μM ZA or 0.5 μM Z-TDN was added or replaced by osteogenic media supplemented with 10 mM BGP and 0.5 mM l-ascorbic acid (Sigma). After 14 days, ARS staining was performed according to the manufacturer’s protocol (ScienCell). hPBMCs were differentiated into macrophages for 6 days using 20 ng/mL M-CSF and then seeded onto a 96-well plate at a density of 2 × 104 per well. Seeded macrophages were differentiated into osteoclasts using 20 ng/mL M-CSF and RANK-L for 6 days. Next, the media was replaced with LGM media (Lonza) and treated with ZA, Z-TDN, or left untreated for 3 days. Osteoclast activity was visualized by TRAP staining according to the manufacturer’s protocol33 (Sigma). TRAP-positive osteoclasts were counted using OpenCFU version 3.9.
Antibacterial activity of V-TDN
Antibacterial assay of V-TDN was performed following the previous publication34. Overnight cultures of MRSA were grown in TSB medium at 37°C with shaking. Cultures were pelleted at 13,000g for 3 min, washed twice with DPBS, and resuspended in DPBS. The optical density at 600 nm (OD600) of the bacterial suspension in each group was determined, and the cells were normalized to an OD600 equal to 0.1 in DPBS. A 0.2 mL suspension of TSB and the experimental group was inoculated with MRSA at 37 °C in a 96-well plate with shaking for 24 h. The OD600 values of bacterial cultures were measured before diluting and plating onto TS-agar to enumerate CFUs at 24 h post-inoculation.
Fabrication of 3D bioprinted, injectable, and sprayable TDN-integrated GelMA hydrogel
3D bioprinted scaffold
To prepare 3D bioprintable TDN-integrated GelMA hydrogels, GelMA prepolymer (15% w/v), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (1% w/v), Tartrazine (0.1% w/v), and 0.5 μM TDNs were dissolved in PBS at 70 °C. TDN-integrated GelMA pre-gel solution was 3D bioprinted using a Lumen X DLP 3D printer (CELLINK) following an established protocol35.
Injectable scaffold
To prepare TDN-integrated GelMA pre-gel solution for in situ gelation, GelMA prepolymer (5% w/v), lithium phenyl-2,4,6-trimethylbenzoylphophinate (1% w/v), Tartrazine (0.1%, w/v) and 1 μM TDNs were dissolved in DPBS at 70 °C. A 6 mm biopsy punch was made on the surface of a porcine heart. 300 µL of TDN-integrated GelMA pre-gel solution was injected with a 1 ml syringe, and the solution was crosslinked with a 405 nm blue light for 10 min.
Sprayable scaffold
To prepare a TDN-integrated GelMA sprayable scaffold, the same hydrogel pre-gel solution preparation procedure was performed as in situ gelation. The pre-gel solution was sprayed using a 10 mL spray bottle onto the surface of a porcine heart and exposed to a 405 nm blue light for 10 min.
Characterization of sprayable and injectable TDN-integrated GelMA hydrogel
Rheological analysis
Rheological analysis was performed using a HAAKE Modular Advanced Rheometer System (MARS) (Fisher Scientific). Sprayed and injected TDN-integrated GelMA hydrogels or GelMA hydrogels with the same reparation method were tested using a P20/Ti titanium plate. Stress sweep tests were performed at 37 °C from 0.1 to 104 Pa34.
TDN release study
TDN-integrated GelMA (15% w/v) hydrogels were prepared and printed into cylinders with a volume of 0.2 mL. TDN-integrated GelMA hydrogels were then incubated at physiological conditions (1.5 mL of PBS at 37 °C on an orbital shaker) for 10 days until a plateau was reached. TDN release was measured by the absorbance of the supernatant at 260 nm and percentage release was calculated by blanking with the measurements of GelMA hydrogels in the same conditions.
Statistical analysis
The variations among the experimental groups were quantified using ordinary one-way ANOVA with Tukey post hoc comparisons. GraphPad Prism 10 was used for all statistical analyses. All the experimental data were calculated as mean ± standard deviation. Values of p < 0.05 were considered to be statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
