中央研究院 原子與分子科學研究所

Cell therapy is a promising strategy for the treatment of human diseases.  While the first use of cells for therapeutic purposes can be traced to the 19th century, there has been a lack of general and reliable methods to study the biodistribution and associated pharmacokinetics of transplanted cells in various animal models for preclinical evaluation.  Here, we present a new platform using albumin-conjugated fluorescent nanodiamonds (FNDs) as biocompatible and photostable labels for quantitative tracking of human placenta choriodecidual membrane-derived mesenchymal stem cells (pcMSCs) in miniature pigs by magnetic modulation.  With this background-free detection technique and time-gated fluorescence imaging, we have been able to precisely determine the numbers as well as positions of the transplanted FND-labeled pcMSCs in organs and tissues of the miniature pigs after intravenous administration.  The method is applicable to single-cell imaging and quantitative tracking of human stem/progenitor cells in rodents and other animal models as well. 

Much of the current understanding of thermal effects in biological systems is based on macroscopic measurements.  There is little knowledge about the local thermostability or heat tolerance of subcellular components at the nanoscale.  Herein, we show that gold nanorod-fluorescent nanodiamond (GNR-FND) hybrids are useful as a combined nanoheater-nanothermometer in living cells.  With the use of a 594 nm laser for both heating and probing, we measured the temperature changes by recording the spectral shifts of the zero-phonon lines of negatively charged nitrogen-vacancy centers in FNDs.  The technique allows us to determine the rupture temperatures of individual membrane nanotubes in human embryonic kidney cells, as well as to generate high temperature gradients on the cell membrane for photoporation and optically controlled hyperthermia.  Our results demonstrate a new paradigm for hyperthermia research and application.

Fluorescent nanodiamond (FND) has recently played a central role in fueling new discoveries in interdisciplinary fields spanning biology, chemistry, physics, and materials sciences.  The nanoparticle is unique in that it contains a high density ensemble of negatively charged nitrogen-vacancy (NV^–) centers with outstanding optical and magnetic properties.  Firstly, NV^– has an absorption maximum at ~550 nm and when exposed to green-orange light, it emits bright fluorescence at ~700 nm with a lifetime of longer than 10 ns.  These spectroscopic properties are little affected by surface modification and allow background-free imaging of FNDs in tissue sections.  Next, as an artificial atom in the solid state, the NV^– center is perfectly photostable, without photobleaching and blinking.  Therefore, the NV-containing FND is suitable as a contrast agent for super-resolution imaging by stimulated emission depletion (STED).  Last, the NV^– center in diamond is an atom-like quantum system with a total electron spin of 1 and the ground states of the spins show a crystal field splitting of 2.87 GHz, separating the m_s = 0 and ±1 sublevels.  Nanothermometry with both high spatial and temporal resolution can be achieved with a technique known as optically detected magnetic resonance (ODMR).  This account provides a summary of the recent advances in FND-enabled technologies with a special focus on long-term cell tracking, super-resolution imaging, and nanoscale temperature sensing.  These emerging and multifaceted technologies are in synchronicity with modern imaging modalities.  

Recent advances in quantum technology have demonstrated the potential use of negatively charged nitrogen-vacancy (NV^–) centers in diamond for temperature and magnetic sensing at sub-cellular levels.  Fluorescent nanodiamonds (FNDs) containing high-density ensembles of NV^– centers are appealing for such applications because they are inherently biocompatible and non-toxic.  Here, we show that FNDs conjugated with gold nanorods (GNRs) are useful as a combined nanoheater and nanothermometer for highly localized hyperthermia treatment using near-infrared (NIR) lasers as the heating source.  A temperature rise of ~10 K can be readily achieved at a NIR laser power of 0.4 mW in cells.  The technique is compatible with the presence of static magnetic fields and allows for simultaneous temperature and magnetic sensing with nanometric spatial resolution.  To elucidate the nanoscale heating process, numerical simulations are conducted with finite element analysis, providing an important guideline for the use of this new tool for active and high-precision control of temperature under diverse environmental conditions.   

News/Highlights:
http://epjqt.epj.org/210-epj-qt/977-epjqt-highlight-gold-diamond-nanodevice-for-hyperlocalised-cancer-therapy

Measuring temperature in nanoscale spatial resolution either at or far from equilibrium is of importance in many scientific and technological applications.  Although negatively charged nitrogen-vacancy (NV^–) centers in diamond have recently emerged as a promising nanometric temperature sensor, the technique has been applied only under steady state conditions so far.  Here, we present a three-point sampling method that allows real-time monitoring of the temperature changes over ±100 K and a pump-probe-type experiment that enables the study of nanoscale heat transfer with a temporal resolution of better than 10 μs.  The utility of the time-resolved luminescence nanothermometry was demonstrated with 100-nm FNDs spin-coated on a glass substrate and submerged in gold nanorod solution heated by a near-infrared laser, and the validity of the measurements was verified with finite-element numerical simulations.  The combined theoretical and experimental approaches will be useful to implement time-resolved temperature sensing in laser processing of materials and even for devices in operation at the nanometer scale.

Lung stem/progenitor cells are potentially useful for regenerative therapy, for example in repairing damaged or lost lung tissue in patients.  Several optical imaging methods and probes have been used to track how stem cells incorporate and regenerate themselves in vivo over time.  However, these approaches are limited by photobleaching, toxicity and interference from background tissue autofluorescence.  Here we show that fluorescent nanodiamonds, in combination with fluorescence-activated cell sorting, fluorescence lifetime imaging microscopy and immunostaining, can identify transplanted CD45^–CD54⁺CD157⁺ lung stem/progenitor cells in vivo, and track their engraftment and regenerative capabilities with single-cell resolution.  Fluorescent nanodiamond labelling did not eliminate the cells’ properties of self-renewal and differentiation into type I and type II pneumocytes.  Time-gated fluorescence imaging of tissue sections of naphthalene-injured mice indicates that the fluorescent nanodiamond-labelled lung stem/progenitor cells preferentially reside at terminal bronchioles of the lungs for 7 days after intravenous transplantation.

News/Highlights:
http://www.nature.com/nnano/journal/v8/n9/nnano.2013.147/metrics/news