Abstract
Hydroxyapatite nanoparticles (HA-NPs) are proposed for a miRNA delivery system with additional value in bone engineering because of its osteogenic activity. HA-NPs were synthesized by hydrothermal method, and surface-modified with two cationic functional groups 3-aminopropyltriethoxysilane (HA-NPs-APTES) and poly2-(dimethylamino)ethyl methacrylate (HA-NPs-PDMAEMA). The particles were visualized under a transmission electron microscope and then characterized for their physical properties, cell compatibility, and efficiency of miRNA binding. The FitC-tagged particles internalized in human mandibular osteoblasts (HMOBs) demonstrated insufficient miRNA binding and internalization of the unmodified particles. HA-NPs-APTES particles were in 150–200 nm elongated shape and demonstrated positive surface charge with the highest miRNA binding efficiency. HA-NPs-APTES was highly internalized in HMOBs without significantly changing cell metabolism. In contrast, PDMAEMA-modified particles demonstrated an increase in size and surface charge, and are thus potentially toxic to cells. The level of delivered miRNA in total RNA was determined by qPCR, and the HA-NP delivery system was analyzed. The HA-NP delivery system efficiently delivered miRNA, and the miRNA-targeted GAPDH expression was diminished by three- to tenfold. The GAPDH level was suppressed after 50 μg/mL or 100 μg/mL HA-NPs-APTES, significantly lower than the effect by Dharmafect. In conclusion, HA-NPs-APTES demonstrates optimal efficiency in miRNA delivery and gene regulation in the osteoblast in vitro model.
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Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Acknowledgments
We would like to thank Assoc. Prof. Anak Khantachawana and Assoc. Prof. Kwanchanok Pasuwat for providing access to live cell time-lapse microscopy at the Automated Tissue Culture Lab, King Mongkut’s University of Technology Thonburi (Bangkhuntian). This research is funded by Royal Golden Jubilee Ph.D. scholarship from the Thailand Research Fund and Faculty of Dentistry Research Fund, Chulalongkorn University (DRT 61020). N. Thepphanao received financial support from Graduate School Thesis Grant, Chulalongkorn University, and Center of Excellence on Petrochemical and Materials Technology.
Funding
This research is funded by Royal Golden Jubilee Ph.D. scholarship from the Thailand Research Fund and the research fund by the Faculty of Dentistry, Chulalongkorn University (DRT 61020). N. Thepphanao received financial support from Graduate School Thesis Grant, Chulalongkorn University, and Center of Excellence on Petrochemical and Materials Technology.
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1. Study conception and design:
Pirawish Limlawan, Anjalee Vacharaksa
2. Acquisition of data:
Pirawish Limlawan, Nathaya Thepphanao
3. Analysis and interpretation of data:
Pirawish Limlawan, Nathaya Thepphanao, Numpon Insin, Anjalee Vacharaksa
4. Drafting of manuscript:
Pirawish Limlawan, Nathaya Thepphanao, Anjalee Vacharaksa
5. Critical revision:
Pirawish Limlawan, Numpon Insin, Anjalee Vacharaksa
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Supplemental Fig. 1
. Flow Cytometry plotting of FitC-positive HmOBs. Cells were incubated (24 h) with various concentration (20, 50, 100 μg/mL) of (a) FitC-tag HA-NPs, (b) APTES-modified (HA-NPs-APTES), (c) PDMAEMA-modified (HA-NPs-PDMAEMA) particles. The cells incubated with particles without FitC are included in the control. The total number of gated cells is approximately 10,000. The control cells are shown to demonstrate FigC-negative cells. The number of FitC-positive cells is determined from the cell population above the cut point line that is distinct from the control group. (PNG 2957 kb)
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Limlawan, P., Thepphanao, N., Insin, N. et al. Surface-modified hydroxyapatite nanoparticle for microRNA delivery to regulate gene expression in human mandibular osteoblast cells. J Nanopart Res 23, 12 (2021). https://doi.org/10.1007/s11051-020-05121-0
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DOI: https://doi.org/10.1007/s11051-020-05121-0