Nanoparticle-based Cell Trackers for Biomedical Applications

The continuous or real-time tracking of biological processes using biocompatible contrast agents over a certain period of time is vital for precise diagnosis and treatment, such as monitoring tissue regeneration after stem cell transplantation, understanding the genesis, development, invasion and metastasis of cancer and so on. The rationally designed nanoparticles, including aggregation-induced emission (AIE) dots, inorganic quantum dots (QDs), nanodiamonds, superparamagnetic iron oxide nanoparticles (SPIONs), and semiconducting polymer nanoparticles (SPNs), have been explored to meet this urgent need. In this review, the development and application of these nanoparticle-based cell trackers for a variety of imaging technologies, including fluorescence imaging, photoacoustic imaging, magnetic resonance imaging, magnetic particle imaging, positron emission tomography and single photon emission computing tomography are discussed in detail. Moreover, the further therapeutic treatments using multi-functional trackers endowed with photodynamic and photothermal modalities are also introduced to provide a comprehensive perspective in this promising research field.

Currently, two major categories of cell labeling strategies, direct labeling and indirect labeling, have been implemented in practice. Each strategy has its own advantages and disadvantages. In general, direct labeling approach enjoys the advantages of easy preparation, high labeling efficiency, and abundant availability of exogenous contrast agents, while indirect labeling strategy involving genetic modification can afford permanent cell tagging. Among them, bioluminescence, a natural light source based on luciferase catalysis oxidation of its luciferin substrate, is a typical and most well-adapted indirect labeling technology. Luciferase catalyzes the oxidization of luciferin by intramolecular oxygen, leading to oxyluciferin molecule in the excited state. After emitting in the excited state, the molecule reduces back to luciferin substrate. This technique has shown promising potentials in a wide range of in vitro and in vivo applications, including immunoassays, gene expression analyses, drug screening, bioimaging of living systems, as well as diagnosis and microenvironmental monitoring of tumors [26]. Bioluminescence does not need external light irradiation, which helps avoid interference from background fluorescence and biological auto-fluorescence signals during imaging. Thus, bioluminescence-based methods are extremely sensitive to provide good spatial resolution in a wide dynamic range. Inspired by the unique property of bioluminescence, Miyawaki et al. designed a bioluminescence imaging system (named AkaBLI) that produces in vivo emission signals 100 to 1000-fold brighter as compared with conventional technology (Figure 1) [27]. They recorded video-rate bioluminescent signals from neurons in the striatum, a deep brain area, for more than a year. This study indicates that the red-emissive and highly deliverable luciferin analog (AkaBLI) can serve as a bioengineered light source to motivate unidentified scientific, medical, and engineering applications. Advances in bioluminescence imaging methods allowed researchers to measure tumor growth, visualize growing processes, and track cell-cell interactions [28,29].
Nevertheless, many challenges and limitations still exist in bioluminescence imaging technology. For example, the imaging requires highly sensitive CCD lens and unstable bioluminescence suffers from signal decay. In addition, long detection time due to their weak signals, high cost owing to the repeated luciferin injection from time to time, and the risk of transgenic markers transfecting on cells, genes, or antibodies are all of major concerns that impede their progress in translational research. On the other hand, green fluorescent protein (GFP) and its variants, another major category of genetic cell tagging in indirect labeling strategies, are restricted by their poor photostability, inherent susceptibility to enzymes and interference from bio-substrate autofluorescence [30,31]. Alternatively, exploration of exogenous contrast agents, such as nanoparticle (NP)-based cell trackers, for biomedical and/or preclinical investigation has attracted a broad research interest. For now, several nanoparticles, including superparamagnetic iron oxide NPs (SPIONs), carbon NPs, silver NPs and CdS/ZnS quantum dots, have been tested in the clinical trials (Table 1). Therein, SPIONs for MRI-guiding pancreatic cancer and silver NPs-based central venous catheters for catheter related infections have been completed in phase 4 clinical trials. This indicates that the preclinical development and exploration of NP-based trackers or agents are of great importance. In this review, we thus mainly focus on the recent research and application of NP-based direct labeling strategy for long-term cell-tracking of biomedical progress. Their potential theranostic applications are also briefly discussed to inspire the future exploration of multi-functional cell trackers.
Recently, Ashraf et al. labeled mouse bone marrow-derived mesenchymal stem cells (MSCs) with SPIONs encapsulated by polyelectrolyte multilayers. At a low feeding concentration (100 μg Fe mL −1 ), the encapsulated SPIONs served as an efficient labeling agent for stem cells, which had a 2-fold higher uptake efficiency of Fe in comparison with that labeled with bare SPIONs (Figure 2A) [39]. The bio-distribution after intra-cardiac injection of labeled cells was monitored longitudinally by MRI. Through 2 weeks of in vivo tracking, the particles released from dead cells mainly accumulated in liver and spleen without complete clearance. To further enhance the cellular uptake and biocompatibility, Rivas et al. used glucosamine to modify polyacrylic acid (PAA)-coated ultrasmall iron oxide nanoparticles (USPIO-PAA-GlcN) [43] and label MSCs for tracking by MRI, while PAA-coated SPIONs (SPIONs-PAA) and PAA-coated USPIONs (USPIONs-PAA) were used for comparison ( Figure 2B) [2]. After incubation with MSCs, the SPIONs-PAA were only found in a portion of cells with nonuniform distribution due to their severe aggregation behavior in cell culture medium. On the other hand, USPIONs-PAA showed good in vitro colloidal stability but low intracellular internalization efficiency. By contrast, USPIO-PAA-GlcN displayed good biocompatibility, high cellular uptake (56.5 pg Fe/cell) and excellent sensitivity in both in vitro and in vivo MRI experiments, showing promises as a labeling agent for cell tracking applications in animal models of cerebral ischemia [44,45]. Lately, Daldrup-Link et al. utilized ferumoxytol-labeled matrix-associated stem cell implants (MASIs) to predict cartilage repair outcomes with MRI in a minipig model. The ferumoxytol-labeled MASIs showed significant T2 shortening compared with unlabeled MASIs (22.2 msec ± 3.2 vs 27.9 msec ± 1.8; P < 0.001), facilitating MR imaging of the MASI upon transplantation in cartilage defects. In addition, ferumoxytol-labeled apoptotic MASIs showed a higher T2 relaxation time compared with the labeled viable MASIs 2 weeks post implantation (26.6 msec ± 4.9 vs 20.8 msec ± 5.3; P = 0.001) due to the rapid iron loss post cell death. Correspondingly, standard MRI showed incomplete cartilage defect repair in the apoptotic MASI-treated group at 24 weeks. These results suggested that the commercial ferumoxytol as MR trackers can facilitate early diagnosis of failed MASIs within 2 weeks in a large-animal model [46]. In  addition  to  single-modality  imaging  applications,  MRI  synergized with PET or fluorescence imaging  modalities  have  great  potentials for dual-/multi-modality tracking to provide more comprehensive biological information, especially in translational research and clinical practice [6,36].

Cell tracking via magnetic particle imaging (MPI)
Over the last decade, magnetic particle imaging (MPI) has been an emerging imaging approach with near-ideal image contrast, penetration depth and robustness to image IONs-labeled cells in vivo. Especially, it has shown ultra-sensitive and linearly quantitative imaging in practice. As compared to MRI, MPI only requires non-ionizing and low frequency magnetic fields that are safer for clinical translation. In 2016, Zheng et al. used MPI technique to dynamically monitor and quantify the biodistribution of human mesenchymal stem cells (hMSCs) labeled by SPIONs (Figure 3) [47]. They first demonstrated that the MPI signal was not significantly dependent on surrounding tissue depth and hence can be clinically translatable for applications in molecular imaging ( Figure 3B). After tail vein injection, the labeled hMSCs were immediately entrapped in lung tissues with the Fe concentration of ~52 ng Fe mm -3 , and then migrated to the liver within one day. In stark contrast, the standard SPIONs upon tail vein injection were directly taken up by liver and spleen ( Figure 3C) [48,49]. Through monitoring in vivo distribution and clearance of labeled hMSCs over a period of 12 days, they obtained a gradual decay of MPI signal in liver with a half-life of 4.6 days and 95% confidence intervals between 3.7 and 6.0 days ( Figure 3D).
To further improve MPI performance, Rao et al. utilized a semiconducting polymer (SP; PFODBT) and IONs to produce Janus NPs with an average size of 27 nm in diameter, which showed both magnetic and optical properties in MPI and fluorescence imaging ( Figure 4A) [12]. At the same Fe concentration, MPI signal of the Janus NPs showed a 3-fold and 7-fold enhancement as compared to that of the commercial MPI tracker (Vivotrax) and MRI contrast agent (Feraheme), respectively. During in vivo tumor-growth tracking, fluorescence and MPI signals only decreased by 20% from day 10 to day 20 post-implantation ( Figure 4B and C). These results indicate that MPI can allow imaging of the magnetic tracers in deep tissues with an outstanding linearity between tracer amount and signal intensity for quantitative detection. Recently, they developed a NIR-emissive Janus NPs (MMPF NPs) serving as magneto-optical multimodal nanoplatform to image xenografted tumors in living mice [50]. MMPF NPs with an average diameter of 42 nm by TEM possessed a long-term blood circulation time (49 h of half-life) and high tumor uptake efficiency (18% ID/g), leading to high tumor to normal tissue contrast in subcutaneous and orthotopic tumor models. Interestingly, using MPI technique, they observed the disappeared NPs signal in the liver upon day 14 post injection and the visible signal in the spleen after day 85. Among the existing molecular imaging modalities, MPI shows a unique combination of high sensitivity, quantitative accuracy, and longitudinal monitoring in long-term cell tracking study. As such, the multi-modal technology combining MRI, PA and fluorescence imaging can simultaneously enhance the detecting and tracking accuracy, which will further promote the development of preclinical and/or clinical cell therapies [11,13].

Inorganic quantum dots: QDs
Quantum dots (QDs) are a class of inorganic semiconductor nanocrystals with unique photophysical properties, including long fluorescence lifetime, high fluorescence quantum yield, and excellent photostability [66][67][68]. However, traditional inorganic QDs have shown inherent limitations such as high toxicity and short circulation time [69]. For instance, a study has reported that QDs could induce abnormalities during embryo development [70] or depolarize mitochondria membranes to cause potential cytotoxicity [71]. In addition, QDs encapsulated by exotic polymer materials have been reported to cause inevitable immune response [72], while QDs modified by biomacromolecules such as proteins and genes led to drug tolerance and systematic toxicity [73]. To address this critical issue, series of novel QDs, such as silver sulfide (Ag2S) [54,[74][75][76][77], silver selenide (Ag 2 Se) [78], and lead sulfide (PbS) [79,80] QDs, have been explored for a variety of biomedical applications including cell tracking [53,[81][82][83]. Among these QDs, Ag 2 S QDs have been most successfully used for cell tracking due to their superior photostability and high quantum yields in the second near-infrared (NIR-II) window [53,66,84]. Wang et al. encapsulated an Ag 2 S QD with fluorescence in the NIR-II window (1000-1700 nm) with protein nanocages (PNCs), using simian virus 40 (SV40) PNC (PNC SV40 ) as a model, to monitor the in vivo behaviors of PNCs [54,74]. Benefiting from the high spatiotemporal resolution and deep tissue penetration of NIR-II fluorescence imaging [85], the dynamic in vivo distribution of PNC SV40 in living mice was tracked in a real time manner [54]. Besides, they successfully used Ag 2 S QD-labeled hMSCs to continuously monitor the fluorescence signal in animals up to 30 days [76]. The in vivo NIR-II imaging clearly revealed the heparin-facilitated translocation of hMSCs from lung to liver and the long-term retention of hMSCs in the liver for treatment of liver failure [76]. Furthermore, they used Ag2S QDs to monitor lymphatic drainage and vascular networks with high spatial resolution, and track angiogenesis mediated by a tiny tumor in vivo [75]. Soon afterwards, Achilefu et al. conjugated a tumor-avid cyclic pentapeptide (Arg-Gly-Asp-DPhe-Lys, cRGDfk) to ultra-small Ag2S QDs (named cRGDfK-Ag 2 S QDs; Figure 5A) with tunable light emission from 500 to 1200 nm and a hydrodynamic diameter below 10 nm [86]. Compared with the non-conjugated ones, the water-soluble cRGDfK-Ag 2 S QDs showed selective integrin-mediated internalization in cancer cells ( Figure 5B). From in vivo and ex vivo fluorescence images ( Figure 5C and D), an exceptionally high tumor-to-liver uptake ratio can be observed 24 h post injection, indicating that cRGDfK-Ag2S QDs had high targeting ability to the α v β 3 integrin receptors to promote their accumulation in tumor tissues via receptor-mediated endocytosis [87]. As a result, such cRGDfK-Ag 2 S QDs provide an efficient approach to construct nanoplatforms that are able to selectively target disease biomarkers in living organisms [86,88].
In regenerative medicine, promoted differentiation of the transplanted stem cells can facilitate the overall healing progresses. To address this issue, Bain et al. developed a QD-based multifunctional NP (RGD-β-CD-QDs) with an average diameter of 4~5 nm [89]. Its compact size facilitates the cellular uptake of QDs and further delivers the hydrophobic osteogenic dexamethasone and siRNA into the stem cells to enhance their osteogenesis differentiation. Moreover, the in vivo fluorescent signal of the RGD-β-CD-QDs labeled hMSCs could be observed on day 21 post implantation. These results clearly evidence that such functional QD-based trackers can provide a powerful tool to simultaneously enhance differentiation and long-term tracking of hMSCs in vitro and in vivo. Besides QDs, the same group also reported a multifunctional nanocarrier based on inorganic upconversion nanoparticles for controlled differentiation and long-term tracking of hMSCs. Upon exposure of NIR light, the emission in UV region leads to photocleavage of the photocaged linker and intracellular release of differentiation-inducing kartogenin, which further triggered chondrogenic differentiation of hMSCs for promoted neocartilage formation in vivo [90,91].

Organic material-based nanoparticles
As compared to QDs, organic material-based NPs with nontoxic luminogens are potential alternatives for in vivo cell tracking tasks due to their excellent biocompatibility and comparable fluorescent stability [92][93][94]. However, most organic luminogens face the challenge of aggregation caused quenching (ACQ) due to π-π stacking of hydrophobic organic dyes in aqueous media [95], which is a main obstacle in construction of high-performance fluorescent nanoparticles. Opposing to the ACQ effect, luminogens with aggregation-induced emission (AIE) characteristics exhibit bright fluorescence in aggregate state and weak fluorescence in molecular state [25,[96][97][98][99]. With the discovery of AIE effect, organic luminogens have been held up to a new level in biomedical application, especially in cell imaging and tracking [21,[100][101][102][103][104]. Recently, Tang et al. synthesized an AIEgen (BPN-BBTD) and prepared its NPs through encapsulation in amphiphilic Pluronic F-127 [105]. The BPN-BBTD NPs showed a broad emission spectrum covering 800 to 1300 nm with a QY (~1.8%) in the second near-infrared (NIR-II) window (>1000 nm), and a photothermal conversion efficiency of 39.8% under a 785 nm laser irradiation (Figure 6). Upon tail vein injection of NPs, the capillary with a small diameter of ~0.37 mm can be detected through NIR-II fluorescence imaging with a high spatial resolution. The NPs can preferably accumulate in tumor sites 24 h post intravenous injection in the subcutaneous and orthotopic bladder tumor-bearing mice, allowing effective photothermal ablation of tumors upon 785 nm laser irradiation (0.6 W cm -2 ). Noteworthy is that the intense and stable NIR-II fluorescence of BPN-BBTD NPs can ensure long-term tracking of bladder tumors for 32 days without any abnormalities or lesions in the main organs.
In addition to AIEgens, semiconducting polymers (SPs) represent another category of materials for synthesis of advanced fluorescent NPs. Their good photostability and large absorption cross-section will greatly benefit long-term biological studies. In this contribution, Li et al. used SP-based NPs (Tat-PFBD) as noninvasive fluorescent trackers with high brightness and low cytotoxicity for in vivo cell tracking to reveal the mechanism of transplanted MSCs in promoting skin regeneration [64,106,107]. The obtained Tat-PFBD NPs had an average size of 37 nm in water, showing an emission maximum at 583 nm with a quantum yield of 42%. The arithmetic average number of emitted photons from each individual Tat-PFBD NP was determined to be 9.72 × 10 5 counts per 100 s, which can promote the real-time single dot tracking and long-term bioimaging applications. After continuous incubation at 37 °C in Dulbecco's modified Eagle's medium, the fluorescence intensity of Tat-PFBD NPs remained above 90% after 35 days, whereas that of the most widely used commercial QD-based tracker (Qtracker ® 585) dropped to only 10% after 9 days. Moreover, Tat-PFBD showed significantly higher labeling efficiency and better long-term tracking ability without compromising the features of MSCs in terms of proliferation, migration, differentiation, and secretum. As a result, Tat-PFBD NPs showed an excellent in vivo tracking for 21 days (Figure 7), revealing that the transplanted MSCs can promote skin regeneration mainly through paracrine signaling effect. Besides, they also realized far-red-absorbing and NIR-emissive PIDT-DBT-Tat NPs to monitor orthotopic liver tumor growth for more than 27 days in a real-time manner [108]. Through both in vitro and in vivo results, PIDT-DBT-Tat NPs as fluorescent probes showed better performance as compared to commercial QD tracker (Qtracker ® 705), whose fluorescent signal disappeared at day 6 in the orthotopic liver tumor growth study.

Fluorescent nanodiamonds (FNDs)
FNDs are unique because they contain a high-density ensemble of negatively charged nitrogen-vacancy (NV -) centers as built-in fluorophores, in which the maximum wavelengths of absorption and emission are at 550 and 700 nm, respectively [55]. Based on the special NVcenters, FNDs possess various inherent properties: (i) higher spatial resolution than normal fluorescent dyes, which is particularly suitable to monitor cellular and subcellular components [55]; (ii) optically detected magnetic resonance (ODMR) character, which is contributed by a sp 3 nano-carbon allotrope [55]; (iii) three-dimensional (3D) lattice structure, which can benefit the coating of functional groups on FND surface [109]; (iv) the size between 35 and 100 nm, which is suitable for cell imaging [55]. Thanks to the perfect photostability without photobleaching in NVcenter and low background in their emission region, FNDs are ideal materials in long-term cell tracking with good biocompatibility to maintain the self-renewal and differentiation of stem cells [110].
Recently, Wang et al. targeted FNDs with transforming growth factor (TGF) to yield imaging probes for endogenous TGF-beta (TGF-β) receptor labeling and 3D single molecule imaging [111]. To minimize aggregation, they used bovine serum albumin (BSA) to coat the FND surface via physical adsorption to reduce nonspecific interactions, yielding FND-TGF-BSA. The FND-TGF-BSA had an average hydrodynamic diameter of 46 nm in water, exhibiting strongly far-red fluorescence as well as high signal-to-noise ratio (SNR; ~48) passing through a single-band bandpass filter (675/67 nm). Interestingly, the probe can efficiently bind to HCC827 cells as a result of specific interaction with TGF-β receptor, demonstrating that FND-TGF-BSA is stable in live cells even under small molecule kinase inhibitor (SMI) treatment [111]. As a result, the FNDs can be used as specific endogenous protein tags to study transmembrane signaling function and dynamics in 3D, which is useful in investigating the influence of drug on dynamic behaviors of the target proteins in living cells. Soon after, Chang et al. conjugated FNDs with a recombinant envelope protein of vaccinia virus (VacV) which has been used to track and image the glycosaminoglycans (GAGs) in targeted living cells (Figure 8) [112]. Specifically, the recombinant A27 (rA27) proteins from VacV were used to functionalize FND surface because wild-type rA27 proteins (containing aa 21-84; named rA27-FND) are stable and can be easily produced in a large quantity [113]. A mutant rA27 protein (containing aa 33-84) was employed to afford rDA27-FND as the negative control, due to its defective binding ability to heparan sulfates [114]. Flow cytometric analysis revealed that about 75% of rA27-FND fed cells showed far-red emission owing to the specific targeting of GAG receptors by rA27-FND, whereas only 5% of rDA27-FND fed cells showed positive signals ( Figure 8A). The rA27-FNDs thus showed more than 30-fold higher fluorescence intensity at far-red channel in living Hela cells as compared to that of rDA27-FNDs ( Figure 8B). Similar results were observed when comparing the cellular internalization by GAG-overexpressed L cells (86% positive) and GAG-lacking Sog9 cells (1.5% positive) ( Figure 8C). Furthermore, Schirhagl et al. took the advantage of unique optical property of FNDs to reveal the causes of cell aging at a molecular level and the exact function of free radicals in the aging process [115]. Transmission electron microscopy (TEM) images revealed that FNDs were accumulated in cytoplasm around nucleus. The chronological life span assay suggested that FND internalization was effective to monitor free radicals in stationary phase yeast cell aging process, according to free radical theory of cellular aging [116].
The excellent biocompatibility of FNDs is ideal for long-term cell tracking applications, particularly in stem cell research. Yu and Chang et al. used FND-labeled lung stem cells (LSCs) to identify their in vivo behaviors post transplantation and track the cellular engraftment and regenerative capabilities by fluorescence lifetime imaging microscopy (FLIM) (Figure 9) [117]. Confocal fluorescence microscopy images revealed that LSCs labeled with FNDs did not show obvious difference from unlabeled LSCs in terms of self-renewal and differentiation ability. The result in time-gated fluorescence imaging of tissue section indicated that FDN-labeled LSCs trended to engraft at the terminal bronchioles of injury lungs after intravenous transplantation for 7 days. In addition, Chao et al. introduced FND-labeled embryonal carcinoma stem cells (ECSCs) to track their neuronal differentiation [118]. After internalized by ECSCs, FNDs showed negligible effect on their proliferation and expression. Particularly, FNDs did not alter neuronal differentiation and neuron cells derived from ECSCs. In this contribution, the highly biocompatible FNDs could track neuronal differentiation processes for ~7 days using confocal microscope or flow cytometer.
FNDs have also been used to track cancer stem cells (CSCs), which is considered as a source of tumor initiation [119]. Geno-toxicity tests with comet and micronucleus assays for human fibroblasts and breast cancer cells indicated that FNDs had no influence on DNA or cell growth. After tracking human CSCs for 20 days, FNDs showed excellent intracellular retention capability, comparing with the commonly used cell trackers, carboxyfluorescein diacetate succinimidyl ester (CFSE) and 5-ethynyl-2′-deoxyuridine (EdU) (Figure 10). On day 4, flow cytometric analysis of dissociated mammospheres of AS-B145-1R cells showed a proportion of 9.5%, 12.4%, and 82.5% for EdU+, CFSE+, and FND+ cells, respectively. Specifically, 5.0% CFSE+ cells and 10.6 % FND+ cells were still detectable on day 20, whereas no EdU+ cells could be found by day 12 (Figure 10B). Interestingly, FND+ cells showed a mammosphere-forming efficiency nearly twice as high as that of the FND− cells ( Figure  10C), resulting from slow-proliferating and/or quiescent CSCs for the former. These results indicated that FND-based cell tracking platform is an effective tool in distinguishing fast-proliferating and slow-proliferating/quiescent cells as well as tracking the clonal expansion of CSCs or other stem cells.

Cell tracking via nuclear imaging
A combination technology of positive emission tomography (PET) and single-photon emission computed tomography (SPECT) for cell tracking is used to record functional processes in cells by detecting biologically active radiotracers, which can provide an image of targeted distribution [120]. PET/SPECT lacks spatial resolution and cannot provide anatomic information, and they thus are usually combined with computed tomography (CT) technique [121] to precisely monitor the location of tracked cells [120]. The cell tracking through PET/SPECT can be realized through either imaging intracellular reporter genes with radioactive reporter probes or labeling of cells with radioactive isotope-incorporated molecules or NPs [122].   Although T-cell therapy has made a wide impact on clinic, monitoring the fate of T cells remains a major challenge in cancer immunotherapy research. Thus, tracking transplantation of T-cells in vivo is necessary to evaluate the ability of T-cells to penetrate and traffic to tumors. Recently, Wu et al. labeled 89 Zr-desferrioxamine with anti-CD8 cys-diabody (named 89 Zr-malDFO-169 cDb) to track endogenous CD8 + T cells via noninvasive immune-PET [123]. The 169 cDb can bind to CD8α, which is expressed on cytotoxic lymphocytes of all mouse strains, to realize lymph node and spleen targeting. To overcome the limitation of small region detection using PET, anti-CD8 immuno-PET based on 89 Zr-malDFO-169 cDb not only detected lymph nodes in mice via targeting tumor-infiltrating CD8 + T cells, but also provided a preclinical evaluation for antitumor immune responses of immunotherapy through tracking dynamic distribution of T-cells. Disis et al. used 111 In labeled HER-2/neu specific T-cells to monitor egress and traffic of the specific T-cells as assessed by concurrent SPECT/PET-CT imaging [124]. The results showed a fluorodeoxyglucose flare at metastatic site and an increase of tumor uptake to 32% at 48 h post T-cell infusion (Figure 11A-C). This result provided evidence of T-cell homing to disease sites and a tumor metabolism flare response. Moreover, Weber et al. utilized 86 Y/ 177 Lu-AABD to continuously track DAbR1-positive chimeric antigen receptor T (CAR T) cells via PET/SPECT [125]. They observed that 86 Y-AABD could be maintained in T cells and cleared by normal tissue, which ensured a high contrast of in vivo cell tracking via PET and SPECT.
In  4 remained associated with the cells in liver, spleen and bone marrow after 7 days in vivo ( Figure 11D) 4 was highly specific and sensitive to track cancer cells with better pharmacokinetics (e.g., faster tumor uptake, quicker and more complete clearance from circulation). Accordingly, [ 18 F]BF 4 via PET/CT imaging has a preclinical potential in in vivo cell tracking for NIS expressing disease models.

Cell tracking via photoacoustic imaging
Photoacoustic (PA) imaging as a noninvasive imaging modality has great potential in biomedical and clinical applications, owing to its unique advantages of superb contrast, high spatial resolution, and high sensitivity to tissue functional characteristics [129][130][131]. PA signals are similar to ultrasound (US) waves in imaging, where the light scattering and dissipation by tissue can be minimized. PA imaging thus can afford deeper tissue penetration in comparison with other optical imaging technologies. However, to increase the light intensity and PA SNR in deep tissues, PA-imaging contrast agents (PA-trackers) have only been developed in recent years [132]. An excellent PA-trackers should own special photophysical properties, such as low fluorescence quantum yield, high molar-extinction coefficient in the near-infrared (NIR) window absorption, and good photostability. To date, a variety of PA-trackers have been developed, including carbon nanotubes, graphene-based agents, gold nanorods, organic small molecules, and SPNs.
For the recent cell-tracking using PA imaging, Wu and Li et al. developed a NIR-absorbing SP-based PA tracker to monitor the transplantation of human embryonic stem cell-derived cardiomy-ocytes (hESC-CMs) in living mouse hearts [18]. The PA tracker exhibited an excellent sensitivity in detecting 2000 labeled CMs to distinguish them from background tissues without significant impact on the structure and function of hESC-CMs. Moreover, the high labeling efficiency and PA signal allowed assess the delivery and engraftment of hESC-CMs in beating hearts, providing a facile approach to evaluate the efficacy of cell transplantation in cardiac regenerative therapy. Later, Bian et al. used a second near-infrared (NIR-II) absorptive organic SP-based nanoprobe (OSPN) as PA tracker to label hMSCs for in vivo tracking ( Figure 12A) [14]. PA signal from the OSPNs in cytoplasm was measured by optical-resolution photoacoustic microscopy (OR-PAM), revealing that hMSCs had more efficient uptake of positively charged OSPNs + than that of the negatively charged OSPNs -. Quantitative analysis revealed that the PA signal intensity of OSPNs + -labeled cells was 3-fold higher than that of OSPNs --labeled cells. In addition, PA imaging excited by NIR-II light irradiation (1064 nm) showed higher SNR (1.4-fold) in comparison with that excited by the first near-infrared (NIR-I) light irradiation (860 nm) at the same power intensity (5.0 mJ cm −2 ), implying that OSPN + was superior in deep brain imaging with NIR-II absorption. Noteworthy is that the PA contrast was still visualized clearly upon 2 weeks post-injection, even the intensity decreased by ~61.4% as a result of cell proliferation. Thus, the NIR-II SPN-based PA-trackers were demonstrated to show significant advantages for labeling and tracking of hMSCs in biological tissue, providing a powerful method to understand the mechanisms in stem cell therapy.
In addition, Suggs et al. used inert gold nanorods (AuNRs) coated with a reactive oxygen species (ROS) sensitive NIR dye (IR775c) as the PA-tracker to label and track the viability of MSCs (Figure 12B) [17]. After cell death, MSCs produced ROS to degrade the cells. Accordingly, the viability of MSCs can be measured by PA signal ratio of IR775c (795 nm) to AuNRs (920 nm) because the latter are insensitive to ROS. The ratiometric heatmap of PA signal revealed the location of MSCs, where the high ratio value indicated living MSC populations while the low ratio value implied dying or dead cell populations ( Figure   12C). After transplanted into the gastrocnemius muscle, the PA intensity of MSCs for IR775c decreased by ~22% at day 1 and ~38% at day 10 based on the normalized intensity for AuNRs. As a result, the relative viability of MSCs was estimated to be 0.92 and 0.05 for day 0 and day 10, respectively. In addition, PA imaging results revealed that the viability of cell population within the first day post transplantation decreased rapidly. Such ROS-sensitive PA-tracker can provide a faster process to optimize their therapeutic condition in a preclinical research, including cell transplantation timing, number of transplanted stem cells, and stem cell viability after administration.
Blood vessels play a crucial role in living bodies as a physical barrier and functional network, which facilitate the transportation of solutes, nutrients, and cells among tissues. A number of diseases involve the disfunction of blood vessel. As such, tracking vascular structures by imaging is of high important in theranostic applications. Recently, Liu et al. developed a NIR-II absorptive conjugated polymer (PTD) and fabricated PTD NPs by a modified nanoprecipitation method using a coaxial microfluidic glass capillary mixer ( Figure 13A) [133]. Under optimized microfluidic conditions (320 of Reynolds number and 40% EtOH), the formulated NPs exhibited the smallest average diameter of 40 nm by TEM image. PTD NPs showed a strong NIR-II absorption peak at 1160 nm with an extinction coefficient of 48.1 L g −1 cm −1 in aqueous media, which could contribute to their strong PA amplitude under a pulsed laser irradiation of 1064 nm. To evaluate in vivo angiography performance of PTD NPs, 3D OR-PAM imaging, a decipherable technology for wide-field 3D biological structures with deep penetration and large signal-to-background ratio (SBR), was constructed on a mice ear and cerebral vasculatures with an excitation wavelength of 1064 nm. The regular ear vasculatures with a resolution of 19.2 µm and an SBR of 29.3 dB at the maximal imaging depth of 539 µm were visualized clearly ( Figure 13B). Moreover, 3D whole-cortex cerebral vasculatures with a large imaging area (48 mm 2 ), good resolution (25.4 µm), and high SBR (22.3 dB) at a depth up to 1001 µm are also clearly resolved through the intact skull ( Figure 13C). These results suggested that ORPAMI using NIR-II NPs as PA-trackers will be a competitive imaging technology in enhancing the spatiotemporal resolution of cell-tracking in future. In addition, Liu and Zheng et al. developed AIE dots to achieve precise brain cancer diagnostics. The AIE dots showed an NIR-I absorption maximum at 740 nm with a large absorptivity (10.2 L g −1 cm −1 ) and NIR-II emission maximum at 975 nm with a high quantum yield (6.2%), promoting the synergetic NIR-I photoacoustic and NIR-II fluorescence imaging [134]. Among them, NIR-II fluorescence imaging showed a high SBR (4.4) and resolution (38 µm), whereas PA signal of tumor region could reach 2.0 mm of tissue depth. Collectively, the synergetic PA and fluorescence imaging based on AIE dots in NIR window will own great potential for monitoring and visualizing the abnormalities in blood vessel and tissue of brain toward precise brain-tumor diagnosis [135].

Theranostic applications of multifunctional trackers
In addition to cell tracking, NPs can also serve as theranostic agents for disease diagnosis and treatments. For example, some luminogens can generate ROS or heat upon light irradiation, which have the capability to ablate cancer cells, simultaneously realizing both diagnostic and therapeutic effects [136,137]. Besides, many types of leukocytes, including macrophages, neutrophils, and dendritic cells, can sense chemokine and cytokine cues and thus can play the role as nanocarriers to target tumors [138,139]. Inspired by this concept, Xie and Li et al. developed silica nanocapsules, consisting of doxorubicin (Dox) as a representative drug, IONs as MRI tracker, 63 Cu-PTSM as PET tracker and DiD dye as fluorescent tracker (named DSN). The DSNs can be efficiently loaded into macrophages. After intravenous injection, the DSN-laden macrophages showed high accumulation in tumor and further efficiently suppressed tumor growth upon drug release in situ with low systematic toxicity [140]. As such, the multifunctional cell trackers can be used to carry therapeutic agents and track the loaded cells simultaneously, opening a new door in disease theranostics. In this section, the new treatment methods based on NPs will be discussed, mainly covering photodynamic therapy (PDT) [141] and photothermal therapy (PTT) [62,142]. Inspired by the pioneering studies, further combination of cell trackers with novel therapeutic modalities will motivate new research activities in biomedical studies.

Photodynamic therapy (PDT)
Photosensitizers (PSs) generating ROS have attracted a broad research interest in different application fields, including PDT [143], organic synthesis [144], and sewage wastewater treatment  [145,146]. PDT denotes a well-consolidated but gradually expanding approach in the cancer treatment, owing to its high spatiotemporal precision, controllability, and noninvasive nature [147]. To enhance the accuracy of therapy, Liu et al. developed an activatable AIE PS for the simultaneous imaging and photodynamic ablation of cancer cells [148]. Upon conjugation to cRGD [22,149] with an enzyme-activatable spacer [150,151], PSs simultaneously showed light-up fluorescence imaging and activated PDT for specific cancer cells (Figure 14) [152]. In this work, the PSs could specifically target lysosomes of MDA-MB-231 cells overexpressing αvβ3 integrin rather than MCF-7 and 293T cells. After PSs were activated by cathepsin B, a lysosomal protease, under light irradiation, the aggregation of released AIEgens showed a fluorescence turn-on signature. This process further facilitated the ROS production to damage the lysosomal membranes and cause cell apoptosis, showing considerable potentials for targeted and image-guided PDT [151,[153][154][155]. Later, the same group synthesized a self-reporting AIE nanoprobe for real-time monitoring ROS and self-tracking photodynamic ablation of cancer cells [156]. The AIE molecule (named TPETP-AA-Rho-cRGD) composed of TPETP as an AIE luminogen (AIEgen), aminoacrylate (AA) as a singlet oxygen cleavable linker, a fluorogenic rhodol dye (Rho), and cRGD as targeting group (Figure 15). They self-assembled into NPs in a mixture of DMSO and phosphate buffered saline (1/199, v/v) with an average size of ~140 nm, exhibiting a high singlet oxygen QY of 0.68 [157]. Under light irradiation, the AA linker was cleaved as a result of ROS generation and green fluorescence from rhodol dye was then observed. Upon imaging-guided photodynamic therapy (PDT), the nanoprobe can efficiently monitor singlet oxygen generation via fluorescent signal changes.
In clinical study, especially in fluorescenceguided surgery [158,159], the implementation of highly sensitive and specific imaging modalities for timely cancer diagnosis and progression monitoring has been of great significance. Unlike traditional methods of endogenous targeting molecules (antibodies, peptides, or aptamers) labeled with imaging probes [160,161], bioorthogonal chemistry has shown powerful applications in biological fields in combination with metabolic glycoengineering. Through the intrinsic metabolism of metabolic glycoengineering, the cancer cell surface labeling with unnatural sialic acids can be specifically targeted through chemically binding of in vivo biorthogonal click chemistry [162][163][164][165][166][167][168][169][170]. Inspired by the unique properties of AIEgens, Liu et al. developed a water-soluble AIEgen tying triple-bond, a reactive site with azido-functionalized glycans on cancer cell surface, to achieve bioorthogonal turn-on imaging ( Figure 16) [164]. In aqueous media, the bioorthogonal probes showed very weak fluorescence. However, a "turn-on" red fluorescence was clearly observed upon click reaction with azido-functionalized cancer surface owing to the restriction of intramolecular motion. Moreover, photodynamic ablation for targeting cancer cells can be further accomplished at the same time. Compared to previously reported bioorthogonal probes [167][168][169], AIEgen-based probes can realize a unique alternative for real-time live cell labeling, imaging, and therapy. To improve in vivo bioorthogonal efficiency, the copper-free click reactive probe [166,170] and cancer-cell-specific metabolic precursor [165] would be subsequently developed for in vivo labeling. These studies suggest that the bioorthogonal probes targeting to tumor cell surface with covalent bond can serve as long-term cell trackers as well as fluorescent guidance for clinical surgery.

Photothermal therapy (PTT)
Photothermal therapy (PTT), another light-activatable therapy, collocating PA or NIR-II imaging has exhibited advantages over conventional theranostics such as fluorescence-guided chemotherapy [15,16,[171][172][173][174][175][176][177][178][179][180][181][182]. PA-trackers naturally have photothermal functions, which thus can be regarded as PTT agents. They can synergistically offer deep penetration, high spatial resolution, and effective therapy with minimal side-effects. So far, various NIR-II PTT agents have been prepared from inorganic (copper sulfide, plasma metal clusters, and carbon nanotubes) [183][184][185] and organic materials (small molecules and semiconducting polymers) [186][187][188]. The organic agents usually have relatively excellent biodegradation and biocompatibility, which are desired in in vivo biomedical applications [60]. Compared to small molecules, donor-acceptor (D-A) structured SPs have advantages in good photostability and large extinction coefficient, which can benefit PTT efficacy [62,[180][181][182]. Hence, Liu and Zheng et al. developed NIR-II absorptive SPNs (named P1 NPs, 50-64 nm) with an active targeting cRGD ligand (named P1RGD NPs) for selective targeting of integrin on tumor cell surface ( Figure  17A) [181]. Under laser irradiation (1064 nm, 1 W cm −2 ), the temperature of P1 NPs (0.1 mg mL −1 ) rapidly increased to 64.8 ℃ within 5 min and the photothermal conversion efficiency (PTCE) was calculated to be 30.1%. In addition, PA signals of P1RGD NPs exhibited 3.5-fold higher than that of P1 NPs at tumor sites ( Figure 17B), suggesting that cRGD did significantly enhance tumor uptake. For orthotopic tumor model (Figure 17C), upon 24 h post-injection, both P1 NPs and P1RGD NPs showed high PA signals in tumors with a detectable depth up to 3.2 and 2.8 mm, respectively. Moreover, P1RGD NP-based PA imaging performed a higher SBR of 90 in comparison with P1 NP-based one (SBR of 61), implying that active brain-tumor targeting could be realized by cRGD-modified NPs for improved therapeutic efficacy. This work demonstrated that photostable and biocompatible NIR-II SPNs will be promising for precise tracking as well as further treatment of brain tumors.    In addition, to enhance accumulation of NPs in tumor, Pu et al. synthesized a NIR photothermal active SP nanoenzyme (named PCB1-Bro) (Figure 18A), which contained a SP amphiphile (PCB-1) and bromelain (Bro), serving as a photothermal nano-transducer and a temperature-sensitive enzyme, respectively [175]. Under NIR laser irradiation, PCB1-Bro underwent efficient photothermal conversion to increase the temperature around tumor and activated Bro enzyme to digest the most abundant tumor extracellular-matrix proteins, leading to enhanced NPs accumulation in tumor with boosted PTT effect (Figure 18B). At 6 h post-injection, the fluorescence intensity from PCB1-Bro enriched in tumor reached a maximum value, which was 1.4-fold higher than other groups ( Figure 18C). This can be attributed to the improved EPR effect after digesting collagen by enzymatic activation of PCB1-Bro. Consequently, PCB1-Bro completely inhibited the tumor growth for 16 d due to higher PTT efficacy ( Figure 18D). Thus, the strategy of using nanoenzyme will provide an innovative approach to enhance the drug delivery efficacy for PTT and other therapies such as PDT, chemotherapy, and gene therapy [155].

Conclusions and perspectives
In this review, we summarized the recent advances in exploration of nanoparticles as long-term cell trackers for cell therapies and cancer theranostics. The advantages and limitations of currently available cell trackers for various diagnostic technologies, including MRI, MPI, PA, PET, SPECT and fluorescence imaging, have been discussed and summarized in Table 2. Successive in vitro and in vivo cell tracking over a long period of time can provide valuable evidence on cellular processes and biomedicinal therapies, especially on stem cell therapy [189,190] and immunotherapy. Although the traditional clinical techniques such as MRI and PET/SPECT have excellent advantages in the near-ideal and/or limitless in vivo depth detection and quantitative analysis, they face the challenge of high cost and relatively slow imaging speed except for radioisotope safety. By contrast, PA and fluorescence imaging techniques are emerging research fields, enjoying much lower cost for imaging reagents, faster imaging processes within seconds-to-minutes and more maneuverable instruments. These trackers based on organic small-molecules and/or semiconducting polymers have advantages in in vivo real-time and long-term cell tracking, owing to their good biocompatibility. However, further efforts should be made to enhance the in vivo tissue penetration depth without compromising the imaging resolution, which is of high importance for quantitative analysis. To date, some promising results have been shown with the revolutionary development of imaging techniques from software to hardware. For instance, thanks to the minimized tissue absorbance and interference in NIR-II window, the problem of detection depth can be alleviated. Previous studies in NIR-II window have shown a penetration depth of 11.6 cm with 18 dB of SNR in PA imaging [191] and 1.3 μm with 12 of T/NT ratio for fluorescence imaging [192], which demonstrated significant superiority than NIR-I trackers. On the other hand, multi-modal NPs hold great potentials in deep tissue imaging and precise disease diagnosis, particularly in the cerebrovascular and brain tissues. Through combination of different imaging modalities, such multi-modal NPs can round off the strong points of each imaging technique to acquire more comprehensive information than single-modal NPs. In addition, utilizing stimuli-responsive in situ self-assembly or release in tumor microenvironment to design smart trackers and/or probes can also increase contrast signal, tracing veracity and guide-monitoring real-time performance of the targets. Meanwhile, some trackers holding capacity of ROS generation, drug release or heat production under light irradiation can realize precise and efficient treatment. Such trackers have attracted broad attention owing to the integration of diagnosis and therapy in a single formulation. Thus, for modern and even future theranostics in particular of stem-cell therapy and immunotherapy, the in vivo cell tracking combining multimodal technologies with integrated smart probes will be of great potentials.  Trackers: inorganic QDs-, FNDs-and organic material-based NPs. Advantages: (1) Easy operation, low cost, non-invasive and fast imaging from seconds to minutes; (2) Relatively good biocompatibility for FNDs and organic material-based NPs; (3) High photostability for SP-based NPs and AIE dots; (4) Several weeks tracking for in vivo studies; (5) Theranostic capability based on some luminogens owning ROS generation. Disadvantages: (1) Limited tissue penetration depth in several micrometers for traditional optical probes, except for NIR-II probes (several millimeters); (2) Interference from biological auto-fluorescence; (3) Difficult to quantitative analysis. PET/SPECT imaging Trackers: radioisotopes-based materials such as 18 F, 64 Cu, 89 Zr, 111 In and 86 Y/ 177 Lu-based reagents. Advantages: (1) Non-invasive inspection for early stage cancers and further precise medication with high quality; (2) Unlimited detection depth, spatial resolution (1~2 mm) and quantitative analysis; (3) Short tracking period depending on the half-life of radioactive reagents; (4) Whole-body imaging with high sensitivity. Disadvantages: (1) High cost; (2) Use of radioisotopes leading to hard-to-assess in vivo security; (3) Low sensitive to tumors in lung, liver and gastrointestinal tract in comparison with MRI or CT. PA imaging Trackers: Au nanorods, graphene, carbon nanotubes, organic smell-molecules and SPNs etc. Advantages: (1) Non-invasive and non-ionizing detection and fast imaging from seconds to minutes; (2) Deep tissue penetration depth in centimeters with good SNR and spatial resolution in tens to hundred micrometers, rich contrast and high sensitivity; (3) Days to weeks tracking for in vivo fate and aging of stem cells; (4) Low cost and portable system. Disadvantages: (1) Limited imaging resolution in comparison with optical imaging; (2) Lack of stability in detecting methods, and shallow detection depth in comparison with mature clinic detection technology such as CT and MRI.