Poster D5 - Concentration-Dose Response of PEGylated C-Rings and C-Cages in Living Bone

Thursday, October 24, 2024

2:45 PM – 3:45 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Nuzhat Mukul

Undergraduate Student
Cornell University, College of Engineering
Ithaca, New York, United States

Co-Author(s)

Melia D. Matthews (she/her/hers)

BBS PhD Candidate
Cornell University, United States

Primary Investigator(s)

Karl Lewis, PhD

Assisant Professor
Cornell University, United States

Introduction: Osteocytes are cells which reside in the bone matrix and they make up 90-95% of all bone cells and are crucial for bone homeostasis. Osteocytes are the principal sensors for mechanical loading of bone. They contribute to the regulation of bone remodeling and mineral homeostasis all of which are crucial in maintaining strong and healthy bones. Their mechanisms have not been well studied in vivo. This is because the bone is made of a mineralized matrix that is very difficult to see into. There is a need for novel ways to study osteocytes and their subcellular dynamics. Previous research by the Lewis Lab and Wiesner Lab demonstrated that C’Dots are non-toxic at biologically relevant concentrations and can be used for real-time imaging. This study investigates the concentration-dose response of PEGylated C-Rings and C-Cages conjugated with DEAC fluorescent dye. PEGylation enhances the biocompatibility and solubility of these fluorescent probes, making them suitable for various biomedical applications. We focus on understanding how different concentrations of these PEGylated structures influence their fluorescent properties and biological interactions with osteocytes. Through in vivo experiments, we analyze the fluorescence intensity and cellular uptake of PEGylated C-Rings and C-Cages in cells in living bone. It was previously found that C’Dot intensity and uptake in osteocytes depend on concentration and incubation time. To characterize the use of C-Rings and C-Cages for in vivo imaging and potential drug delivery, we will establish how fluorescent intensity and cell uptake vary in response to initial concentration.

Materials and

Methods: We inject a local, subcutaneous bolus of 15 µL of C-rings/C-cages above the 3rd metatarsal of a mouse paw, performed on both paws to reduce the number of mice needed. Post-incubation, local surgery exposes the 3rd metatarsal for imaging. Z-stack images of the bone are taken with two-photon microscopy at a wavelength of 820 nm to observe C’Dot fluorescence. A C’Dot Filtering and Cleaning plugin was used for quantification. Intensity quantification is performed in ImageJ using 3D object segmentation and Mean Grey value/cell count.

Results, Conclusions, and Discussions: The uptake of C-Rings showed a dose-dependent increase, with both male and female groups displaying similar trends. At lower concentrations (0.1 to 1 µM), the cell count remained low. However, a significant increase in cell count was observed at higher concentrations, with the highest uptake noted at 100 µM, reaching around 75-100 cells. Similar to C-Rings, C-Cages also exhibited a dose-dependent increase in cellular uptake. The male group showed slightly higher uptake at concentrations above 10 µM compared to the female group, with a peak at 50 µM.
The average cell intensity of C-Rings remained relatively constant across the concentration range, with slight increases observed at higher concentrations (50 and 100 µM). The intensity response for C-Cages was similar to that of C-Rings, with a steady fluorescence intensity observed across all concentrations.
The results indicate that both PEGylated C-Rings and C-Cages demonstrate a dose-dependent increase in cellular uptake in osteocytes, with higher concentrations leading to greater cell counts. The consistent fluorescence intensity across varying concentrations suggests that the PEGylation process maintains the fluorescent properties of the DEAC dye, making these probes effective for imaging purposes at different doses. These findings support the potential use of PEGylated C-Rings and C-Cages for real-time imaging and possibly for targeted drug delivery applications in bone tissue. The consistent intensity per cell across concentrations implies that the probes are uniformly taken up by osteocytes, ensuring reliable imaging at various doses.
Furthermore, the slightly higher uptake observed in male groups for C-Cages may indicate a gender-related difference in cellular interactions with these PEGylated structures. However, further studies are needed to explore the underlying mechanisms. Overall, the study contributes valuable insights into the application of PEGylated C-Rings and C-Cages in osteocyte imaging. The ability to track these structures at varying concentrations paves the way for advanced studies in osteocyte function, bone remodeling, and potential therapeutic applications.

Acknowledgements (Optional): Thank you to my mentor Melia Matthews and my PI Dr.Karl J Lewis. I would like to thank the Lewis Lab and the McNair Scholars Program.