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Osteoporosis is a debilitating skeletal disorder that is characterized by loss of bone densityover time. It affects one in two women and one in four men, age 50 and older. New treatmentsthat specifically drive bone formation are desperately needed. We developed a peptide, CK2.3, thatacts downstream of the bone morphogenetic protein receptor type Ia and it induces osteogenesisin-vitro and in-vivo. However, its mechanism of action, especially its mode of uptake by cellsremains unknown. To demonstrate CK2.3 internalization within a cell, we conjugated CK2.3to Quantum Dot&amp;reg;s (Qdot&amp;reg;s), semiconductor nanoparticles. We purified CK2.3-Qdot&amp;reg;s by sizeexclusion chromatography and verified the conjugation and stability using UV/VIS and Fouriertransform infrared spectroscopy. Our results show that CK2.3 was conjugated to the Qdot&amp;reg;s andthe conjugate was stable for at least 4 days at 37 &amp;deg;C. Moreover, CK2.3-Qdot&amp;reg;s exerted biologicalresponse similar to CK2.3. Addition of CK2.3-Qdot&amp;reg;s to cells followed by confocal imaging revealedthat CK2.3-Qdot&amp;reg;s were internalized at 6 h post stimulation. Furthermore, using pharmacologicalinhibitors against endocytic pathways, we demonstrated that CK2.3-Qdot&amp;reg;s were internalized bycaveolae. These results show for the first time that the novel peptide CK2.3 is taken up by the cellthrough caveolae mediated endocytosis.

Mechanisms of Cellular Internalization of Quantum Dot® Conjugated Bone Formation Mimetic Peptide CK2.3