(K-440) Characterization of a Composite Magnetic Nanoparticle System for In Vivo Joint Tracking

Introduction::

Nanoparticles (NPs) are a promising vehicle for drug delivery because of their tunable physicochemical properties which enable better localization and retention of the NPs at the target site. However, the long-term fate (i.e., biodistribution, retention, and clearance) of nanoparticles in vivo is still not well understood. Current methods of evaluating long-term NP fate involve fluorescently tracking NPs via optical methods. Unfortunately, these optical methods are limited by several factors, such as signal attenuation, photobleaching, and autofluorescence, which impede our ability to adequately understand the long-term fate of the NPs in vivo. Magnetic particle imaging (MPI) is an emerging imaging modality that leverages the behavior of magnetic nanoparticles (MNPs) in a magnetic field to generate a 3D visualization and quantification of the MNPs in vivo. Furthermore, it can overcome the limitations of optical NP tracking methods mentioned earlier. To better understand the fate profiles suggested by these two imaging modalities, a composite MNP system was synthesized that was capable of both MPI and optical imaging. However, a significant decrease in MPI signal intensity of the composite MNPs was observed compared to base MNPs capable of MPI only. Since MPI signal intensity is linearly proportional to the iron content of the MNPs the decrease suggests a loss of iron from the composite MNPs following formulation. Thus, the objective of this study was to investigate how iron loss was occurring in our composite MNP synthesis process.

Materials and Methods::

Composite NP synthesis involves first formulating base MNPs by incorporating superparamagnetic iron oxide nanoparticles (SPIONs) into poly(lactic-co-glycolic acid) NPs via single emulsification solvent evaporation and then conjugating the base MNPs with an Alexa Fluor 750 (A750) succinimidyl ester dye via carbodiimide chemistry (Fig 1). The composite MNPs were characterized for their hydrodynamic size and charge via dynamic light scattering (DLS) and iron mass via o-phenanthroline UV assay. The ultracentrifugation of the composite MNPs was investigated first because after ultracentrifugation, material is removed from the synthesis process in the form of supernatant waste. Thus, it is a key step in determining where iron loss occurs and is potentially the cause of the iron loss. To investigate the contribution of ultracentrifugation to iron loss, MNPs were categorized into two different groups. A control MNP group that received the normal amount of ultracentrifugation washes based on previously published work [1] and an experimental MNP group that received a reduced amount of ultracentrifugation washes in each stage of synthesis (Fig. 2). The control group MNPs received a total of five washes across the entire synthesis process, while the experimental group MNPs received a reduced total of three washes. For each ultracentrifugation wash the resulting supernatant was collected and analyzed for iron mass. Composite MNPs were analyzed for iron mass and iron content after formulation and after conjugation, where iron content is the iron mass normalized based on NP mass.

Results, Conclusions, and Discussions::

Base MNPs had an iron content of 3.63%w/w while composite MNPs had an iron content of 2.60%w/w confirming the statistically significant loss of iron after fluorescent conjugation (Fig 3A). However, there was not a significant difference between the iron content of the control group and experimental group composite MNPs indicating that ultracentrifugation is not a significant contributing factor to the iron loss of the composite MNPs (Fig 3B).

Additionally, iron quantification of the collected supernatant samples only observed iron mass in the initial ultracentrifugation washes of formulation and conjugation. To elaborate, the first ultracentrifugation of formulation and conjugation had an iron mass of 0.053mg and 0.062mg, respectively, while the other ultracentrifugation’s had an iron mass near 0 (Fig 4). This indicates that the observed iron loss after conjugation occurs between the last ultracentrifugation of formulation and first ultracentrifugation of conjugation. Furthermore, the data supports the conclusion that ultracentrifugation is not a significant contributing factor to the iron loss because there is not an equal distribution of iron mass found throughout the supernatant samples. If ultracentrifugation was the major contributor to iron loss, it is expected that following every ultracentrifugation there would be iron mass found in the supernatant.

In conclusion, this study confirms the relationship between decreased MPI signal intensity and iron loss. Furthermore, investigation into the composite MNP synthesis process showed that ultracentrifugation is not a major contributor towards the iron loss. Thus, the other major steps in composite MNP synthesis should be further investigated, such as freezing of the NPs or addition of the Alexa Fluor dye. As MPI technology becomes more widely adopted, optimization of the MNP tracers used for MPI is increasingly important. This study advances our understanding of the cause of iron loss during composite MNP synthesis through identification of the approximate location where iron loss occurs and elimination of a major step of the synthesis process as a possible contributor to the iron loss. Ultimately, improving our understanding of the MNP biodistribution, retention, and clearance in vivo.

Acknowledgements (Optional): : I would like to thank my amazing mentors Dr. Blanka Sharma and Tolu Ajayi for their support and guidance throughout this project.

References (Optional): : [1] Brown, et al. “Effects of Cartilage-Targeting Moieties on Nanoparticle Biodistribution in Healthy and Osteoarthritic Joints.” Acta Biomaterialia, Elsevier, 4 Oct. 2019, https://www.sciencedirect.com/science/article/pii/S1742706119306701?via%3Dihub.