Abstract
Osteoporosis is a debilitating skeletal disorder that is characterized by loss of bone density over time. It affects one in two women and one in four men, age 50 and older. New treatments that specifically drive bone formation are desperately needed. We developed a peptide, CK2.3, that acts downstream of the bone morphogenetic protein receptor type Ia and it induces osteogenesis in-vitro and in-vivo. However, its mechanism of action, especially its mode of uptake by cells remains unknown. To demonstrate CK2.3 internalization within a cell, we conjugated CK2.3 to Quantum Dot®s (Qdot®s), semiconductor nanoparticles. We purified CK2.3-Qdot®s by size exclusion chromatography and verified the conjugation and stability using UV/VIS and Fourier transform infrared spectroscopy. Our results show that CK2.3 was conjugated to the Qdot®s and the conjugate was stable for at least 4 days at 37 °C. Moreover, CK2.3-Qdot®s exerted biological response similar to CK2.3. Addition of CK2.3-Qdot®s to cells followed by confocal imaging revealed that CK2.3-Qdot®s were internalized at 6 h post stimulation. Furthermore, using pharmacological inhibitors against endocytic pathways, we demonstrated that CK2.3-Qdot®s were internalized by caveolae. These results show for the first time that the novel peptide CK2.3 is taken up by the cell through caveolae mediated endocytosis.
Highlights
Osteoporosis is a silent disease due to its gradual progression of loss in bone mass and deterioration of the microarchitecture of the bone tissue
We employed size exclusive chromatography to separate the conjugated CK2.3-Quantum Dot®s (Qdot®s) from other undesired conjugates
The conjugation between CK2.3 and Qdot®s was determined by Fourier transform infrared (FTIR) Spectroscopy
Summary
Osteoporosis is a silent disease due to its gradual progression of loss in bone mass and deterioration of the microarchitecture of the bone tissue. 10 million Americans are diagnosed with osteoporosis and another 44 million are at an increased risk of a fracture due to their low bone density. C2C12 cells is an ideal cell line to study the role of BMPRIa in inducing osteogenesis, since it is reported that BMPRIa is expressed in C2C12 cells [29] and BMPRIa is the key mediator in inducing C2C12 differentiation into osteoblasts [30]. Significance of the study: Currently prescribed drugs against osteoporosis (e.g., PTH, bisphosphonates, SERMS) have failed to find a lasting treatment for this disease, and in many cases, have led to adverse side effects [8]. Using this probe, coupled with confocal microscopy we were able to determine the time frame of CK2.3 uptake. This further demonstrates that CK2.3 is directing the uptake and localization within the cell
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