Scientific Reports 5, Article number: 14768 (2015)
A Nanoparticle-based Sensor Platform for Cell Tracking and Status/Function Assessment
David Yeo1, Christian Wiraja1, Yon Jin Chuah1, Yu Gao1 & Chenjie Xu1,2
1School of Chemical & Biomedical Engineering, Nanyang Technological University, Singapore
2NTU-Northwestern Institute of Nanomedicine, Nanyang Technological University, Singapore
Nanoparticles are increasingly popular choices for labeling and tracking cells in biomedical applications such as cell therapy. However, all current types of nanoparticles fail to provide real-time, noninvasive monitoring of cell status and functions while often generating false positive signals. Herein, a nanosensor platform to track the real-time expression of specific biomarkers that correlate with cell status and functions is reported. Nanosensors are synthesized by encapsulating various sensor molecules within biodegradable polymeric nanoparticles. Upon intracellular entry, nanosensors reside within the cell cytoplasm, serving as a depot to continuously release sensor molecules for up to 30 days. In the absence of the target biomarkers, the released sensor molecules remain ‘Off’. When the biomarker(s) is expressed, a detectable signal is generated (On). As a proof-of-concept, three nanosensor formulations are synthesized to monitor cell viability, secretion of nitric oxide, and β-actin mRNA expression.
Keywords: Mesenchymal stem cells, Biomaterials – cells, Biosensors, Nanoparticles
Micro/nano-particle based contrast agents are popular choice for examining cell biodistribution in cell therapy and regenerative medicine . However, all current particle-based nanosensors only emit passive signals and cannot provide any information regarding the cell’s real-time status (e.g. viability, differentiation). Thus, there is a need to ascertain the therapeutic function and/or efficacy of implanted cells . Herein, we introduce a novel ‘nanosensor’ concept that circumvents the above drawbacks of current nanoparticles used in cell tracking.
This platform is based on a generalized concept of encapsulating sensor molecules (fluorophore derivatives, oligonucleotides etc.) within biodegradable polymeric micro/nano-particles. These nanosensors are readily uptaken by cells, and reside in the cell cytoplasm as a release depot for sensor molecules. Upon the degradation of the polymeric matrix, released molecules interact with the intracellular elements (e.g. esterase, secreted metabolites and mRNA) that are closely related with the cell status and/or function. This interaction would cause the change of fluorescence intensity or the color, which allows the identification of cell status and/or function change in real-time.
As a proof-of-concept, we prepared a viability nanosensor by encapsulating calcein AM (CAM) within the biodegradable polymeric nanoparticles. Released CAM molecules are originally weakly fluorescent. Upon interaction with esterases within living cells, AM groups are cleaved off to generate fluorescent calcein. Through monitoring the changes of fluorescent signal, the expression level of intracellular esterases is revealed and indicates the survival of cells. This viability nanosensor was utilized to assess the cell viability under the exposure to various concentrations of DMSO. A high correlation of fluorescence signal and cell viability was observed (R2=0.977).
It is reasonable to question the necessity of encapsulating CAM within the particles, given that CAM itself can be used to detect cell viability. We compared the performance of nanosensors versus free CAM in 2 respects – signal retention and labeled cell health/function. During the cell labeling, CAM provided stronger fluorescence signal, approximately 3-fold higher than nanosensors (Figure 1). However, 6 days post-labeling, the fluorescence signal in cells with nanosensors maintained the original level while the fluorescence signal in cells with CAM decreased almost 10-fold from its original intensity (Figure 1B). This reveals that free CAM is poorly retained within cells. Besides the poor retention in the cells, CAM labeling significantly reduced normal cell proliferation. As shown in Figure 1C, both nanosensor labeled cells and unlabeled cells exhibited a >6-fold increase in cell density after a period of 10 days. On the other hand, CAM labeling did not allow the cells to proliferate over the same period (ascertained through cell counting).
Figure 1: Signal retention and proliferation activity in free calcein AM (CAM) and nanosensor labeled cells. (A) Representative images of free CAM and nanosensor labeled cells (i.e. mesenchymal stem cells) at day 1 and 6; (B) Quantified fluorescence intensities of cells in A; (C) Proliferation of free CAM and nanosensor labeled cells compared to unlabeled cells. Scale bar: 100 μm
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