Contrast Agents and Cell Labeling Strategies for in Vivo Imaging

Regenerative medicine has become a new therapeutic approach in which stem cells or genetically reprogrammed cells are delivered to diseased areas in the body with the intention that such multipotent cells will differentiate into healthy tissue and exchange damaged tissue. The success of such cell-based therapeutic approaches depends on precise dosing and delivery of the cells to the desired site in the human body. To determine the accuracy and efficacy of the therapy, tracking of the engrafted cells in an intact living organism is crucial. There is a great need for sensitive, noninvasive imaging methods, which would allow clinicians to monitor viability, migration dynamics, differentiation towards specific cell type, regeneration potential and integration of transplanted cells with host tissues for an optimal time period. Various in vivo tracking methods are currently used including: MRI (Magnetic Resonance Imaging), PET (Positron Emission Tomography), SPECT (Single Photon Emission Computer Tomography), optical imaging (OI), photoacoustic imaging (PAI) and ultrasound (US). In order to carry out the detection with each of the aforementioned techniques, the cells must be labeled either exogenously (ex vivo) or endogenously (in vivo). For tracking the administrated cells, scientists usually manipulate cells outside the living organism by incorporating imaging contrast agents (CAs) or reporter genes. Strategies for stem cell labeling using CAs will be reviewed in the light of various imaging techniques.

Water-soluble Cu30 nanoclusters as a click chemistry catalyst for living cell labeling via azide-alkyne cycloaddition

Cu(I)-catalyzed azide-alkyne cycloadditions(CuAAC)have gained increasing interest in the selective labeling of living cells and organisms with biomolecules.However,their application is constrained either by the high cytotoxicity of Cu(I)ions or the low activity of CuAAC in the internal space of living cells.This paper reports the design of a novel Cu-based nanocatalyst,watersoluble thiolated Cu30 nanoclusters(NCs),for living cell labeling via CuAAC.The Cu30 NCs offer good biocompatibility,excellent stability,and scalable synthesis(e.g.,gram scale),which would facilitate potential commercial applications.By combining the highly localized Cu(I)active species on the NC surface and good structural stability,the Cu30 NCs exhibit superior catalytic activities for a series of Huisgen cycloaddition reactions with good recyclability.More importantly,the biocompatibility of the Cu30 NCs enables them to be a good catalyst for CuAAC,whereby the challenging labeling of living cells can be achieved via CuAAC on the cell membrane.This study sheds light on the facile synthesis of atomically precise Cu NCs,as well as the design of novel Cu NCs-based nanocatalysts for CuAAC in intracellular bioorthogonal applications.

Superparamagnetic Iron Oxide Labeling of Neural Stem Cells and 4.7T MRI Tracking in vivo and in vitro

Neural stem cells were labeled with superparamagnetic iron oxide （SPIO） and tracked by MRI in vitro and in vivo after implantation, Rat neural stem cells were labeled with SPIO combined with PLL by the means of receptor-mediated endocytosis. Prussian blue staining and electron microscopy were conducted to identify the iron particles in these neural stem cells. SPIO-labeled cells were tracked by 4.7T MRI in vivo and in vitro after implantation, The subjects were divided into 5 groups, including 5× 10^5 labeled cells cultured for one day after labeling, 5 × 10^5 same phase unlabeled cells, cell culture medium with 25μg Fe/mL SPIO, cell culture medium without SPIO and distilled water. MR/scanning sequences included TIWI, T2WI and T2＊WI. R2 and R2＊ of labeled cells were calculated. The results showed： （1） Neural stem cells could be labeled with SPIO and labeling efficiency was 100%. Prussian blue staining showed numerous blue-stained iron particles in the cytoplasm; （2） The average percentage change of signal intensity of labeled cells on TIWI in 4.7T MRI was 24.06%, T2WI 50.66% and T2＊WI 53.70% respectively; （3） T2 of labeled cells and unlabeled cells in 4.7T MRI was 516 ms and 77 ms respectively, R2 was 1.94 s^-1 and 12.98 s^-1 respectively, and T2＊ was 109 ms and 22.9 ms, R2＊ was 9.17 s^-1 and 43.67 s^-1 respectively; （4） Remarkable low signal area on T2WI and T2＊WI could exist for nearly 7 weeks and then disappeared gradually in the left brain transplanted with labeled cells, however no signal change in the right brain implanted with unlabeled cells. It was concluded that neural stem cells could be labeled effectively with SPIO. R2 and R2＊ of labeled cells were increased obviously. MRI can be used to track labeled cells in vitro and in vivo.

Artificial light-harvesting systems and their applications in photocatalysis and cell labeling

Photosynthesis is the basis for the survival of organisms in nature;consequently,the fabrication of artificial light-harvesting systems(LHSs)that simulate natural photosynthesis is of significant interest.Recently,a variety of artificial LHSs have been successfully constructed using fluorescence resonance energy transfer(FRET).However,it is crucial to fabricate artificial LHSs with a sequential energy transfer process when considering that the natural photosynthetic process involves a multistep sequential energy transfer process rather than a simple one-step energy transfer.Moreover,many previously reported LHSs have been used as imaging agents for cell labeling and bioimaging or as catalysts in photocatalytic reactions,showing promise for applications simulating natural photosynthesis.In this review,we have summarized recently published representative work on artificial LHSs.In addition,the application of LHSs in photocatalysis and cell labeling has been described in detail.

Bone marrow mesenchymal stem cell transplantation for treatment of spinal cord injury An in vivo magnetic resonance imaging tracking study

Non-invasive tracing in vivo can be used to observe the migration and distnbution of grafted stem cells, and can provide experimental evidence for treatment. This study utilized adenovirus-carrying enhanced green fluorescent protein （AD5/F35-eGFP） and superparamagnetic iron oxide （SPIO）-Iabeled bone marrow mesenchymal stem cells （BMSCs）. BMSCs, double-labeled by AD5/F35-eGFP and SPIO, were transplanted into rats with spinal cord injury via the subarachnoid space. MRI tracing results demonstrated that BMSCs migrated to the injured spinal cord over time （T2 hypointensity signals）. This result was verified by immunofluorescence. These results indicate that MRI can be utilized to trace in vivo the SPIO-labeled BMSCs after grafting.

Magnetic resonance evaluation of human mesenchymal stem cells in corpus cavernosa of rats and rabbits

Aim： To investigate whether the biological process of superparamagnetic iron oxide （SPIO）-labeled human mesenchymal stem cells （hMSCs） may be monitored non-invasively by using in vivo magnetic resonance （MR） imaging with conventional 1.5-T system examinations in corpus cavernosa of rats and rabbits. Methods： The labeling efficiency and viability of SP10-labeled hMSCs were examined with Prussian blue and Tripan blue, respectively. After SPIO-labeled hMSCs were transplanted to the corpus cavernosa of rats and rabbits, serial T2-weighted MR images were taken and histological examinations were carried out over a 4-week period. Results： hMSCs loaded with SPIO compared to unlabeled cells had a similar viability. For SPIO-labeled hMSCs more than lx 105 concentration in vitro, MR images showed a decrease in signal intensity. MR signal intensity at the areas of SPIO-labeled hMSCs in the rat and rabbit corpus cavernosa decreased and was confined locally. After injection of SPIO-labeled hMSCs into the corpus cavernosum, MR imaging demonstrated that hMSCs could be seen for at least 12 weeks after injection. The presence of iron was confirmed with Prussian blue staining in histological sections. Conclusion： SP10-labeled hMSCs in corpus cavernosa of rats and rabbits can be evaluated non-invasively by molecular MR imaging. Our findings suggest that MR imaging has the ability to test the long-term therapeutic potential of hMSCs in animals in the setting of erectile dysfunction.

Magnet-targeted delivery of bone marrow-derived mesenchymal stem cells improves therapeutic efficacy following hypoxic-ischemic brain injury

hypoxicischemic brain injury;however,the therapeutic efficacy of bone marrow-derived mesenchymal stem cells largely depends on the number of cells that are successfully transferred to the target.Magnet-targeted drug delivery systems can use a specific magnetic field to attract the drug to the target site,increasing the drug concentration.In this study,we found that the double-labeling using superparamagnetic iron oxide nanoparticle and poly-L-lysine(SPIO-PLL)of bone marrow-derived mesenchymal stem cells had no effect on cell survival but decreased cell proliferation 48 hours after labeling.Rat models of hypoxic-ischemic brain injury were established by ligating the left common carotid artery.One day after modeling,intraventricular and caudal vein injections of 1×105 SPIO-PLL-labeled bone marrow-derived mesenchymal stem cells were performed.Twenty-four hours after the intraventricular injection,magnets were fixed to the left side of the rats’heads for 2 hours.Intravoxel incoherent motion magnetic resonance imaging revealed that the perfusion fraction and the diffusion coefficient of rat brain tissue were significantly increased in rats treated with SPIO-PLL-labeled cells through intraventricular injection combined with magnetic guidance,compared with those treated with SPIO-PLL-labeled cells through intraventricular or tail vein injections without magnetic guidance.Hematoxylin-eosin and terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)staining revealed that in rats treated with SPIO-PLL-labeled cells through intraventricular injection under magnetic guidance,cerebral edema was alleviated,and apoptosis was decreased.These findings suggest that targeted magnetic guidance can be used to improve the therapeutic efficacy of bone marrow-derived mesenchymal stem cell transplantation for hypoxic-ischemic brain injury.This study was approved by the Animal Care and Use Committee of The Second Hospital of Dalian Medical University,China(approval No.2016-060)on March 2,2016.

Magnetic resonance imaging and cell-based neurorestorative therapy after brain injury

Restorative cell-based therapies for experimental brain injury, such as stroke and traumatic brain injury,substantially improve functional outcome. We discuss and review state of the art magnetic resonance imaging methodologies and their applications related to cell-based treatment after brain injury. We focus on the potential of magnetic resonance imaging technique and its associated challenges to obtain useful new information related to cell migration, distribution, and quantitation, as well as vascular and neuronal remodeling in response to cell-based therapy after brain injury. The noninvasive nature of imaging might more readily help with translation of cell-based therapy from the laboratory to the clinic.

Tracking of Labelled Stem Cells Using Molecular MR Imaging in a Mouse Burn Model <i>in Vivo</i>as an Approach to Regenerative Medicine

Therapies based on stem cell transplants offer significant potential in the field of regenerative medicine. Monitoring the fate of the transplanted stem cells in a timely manner is considered one of the main limitations for long-standing success of stem cell transplants. Imaging methods that visualize and track stem cells in vivo non-invasively in real time are helpful towards the development of successful cell transplantation techniques. Novel molecular imaging methods which are non-invasive particularly such as MRI have been of great recent interest. Hence, mouse models which are of clinical relevance have been studied by injecting contrast agents used for labelling cells such as super-paramagnetic iron-oxide (SPIO) nanoparticles for cellular imaging. The MR techniques which can be used to generate positive contrast images have been of much relevance recently for tracking of the labelled cells. Particularly when the off-resonance region in the vicinity of the labeled cells is selectively excited while suppressing the signals from the non-labeled regions by the method of spectral dephasing. Thus, tracking of magnetically labelled cells employing positive contrast in vivo MR imaging methods in a burn mouse model in a non-invasive way has been the scope of this study. The consequences have direct implications for monitoring labeled stem cells at some stage in wound healing. We suggest that our approach can be used in clinical trials in molecular and regenerative medicine.

THE STUDY OF DIFFERENCE OF PCNA LABELLED CELL INDEXES BETWEEN ENDOMETRIA OF HUMAN AND MOUSE

40 human endometrial tissues of benign diseases were diviided into 8 groups (phases),and 50 BDF1 mousy endometrial tissues were divided into 5 groups (phases).Immunohistochenical staining was performed to show the PCNA (proliferating cell nuclear antigen)positive cell .In human ,the results suggested that the PCNA layer(48%)of mid-proliferative hpase.The PCNA labelled index was low in superficial epithelium.But in mouse, the results suggested that the Pcna labelled index was the highest(22%)in endometrium of estrus and the labelled cells were distributed mainly in superficial epithelium (60%).The results suggested that times that times and positions of cellular proliferation in endometrial tissues of human and mouse differed greatly.

Cobalt protoporphyrin-induced nano-self-assembly for CT imaging,magnetic-guidance,and antioxidative protection of stem cells in pulmonary fibrosis treatment

Mesenchymal stem cells(MSCs)transplantation is a promising approach for pulmonary fibrosis(PF),however it is impeded by several persistent challenges,including the lack of long-term tracking,low retention,and poor survival of MSCs,as well as the low labeling efficiency of nanoprobes.Herein,a cobalt protoporphyrin IX(CoPP)aggregation-induced strategy is applied to develop a multifunctional nano-self-assembly(ASCP)by combining gold nanoparticle(AuNPs),superparamagnetic iron oxide nanoparticles(SPIONs),and CoPP through a facile solvent evaporation-driven approach.Since no additional carrier materials are employed during the synthesis,high loading efficiency of active ingredients and excellent biocompatibility are achieved.Additionally,facile modification of the ASCPs with bicyclo[6.1.0]nonyne(BCN)groups(named as ASCP-BCN)enables them to effectively label MSCs through bioorthogonal chemistry.The obtained ASCP-BCN could not only help to track MSCs with AuNP-based computed tomography(CT)imaging,but also achieve an SPIONs-assisted magnetic field based improvement in the MSCs retention in lungs as well as promoted the survival of MSCs via the sustained release of CoPP.The in vivo results demonstrated that the labeled MSCs improved the lung functions and alle-viated the fibrosis symptoms in a bleomycin–induced PF mouse model.Collectively,a novel ASCP-BCN multi-functional nanoagent was developed to bioorthogonally-label MSCs with a high efficiency,presenting a promising potential in the high-efficient MSC therapy for PF.

Covalent stabilization of DNA nanostructures on cell membranes for efficient surface receptor-mediated labeling and function regulations

One major challenge of using DNA nanostructures for cellular and in vivo applications is their insufficiently structural integrity that stems from the non-covalent base pairing and stacking in complex cellular and physiological environment. The establishment of covalent bonds in DNA nanostructures can link individual strands more stably and therefore should improve the performance of DNA nanostructures in different scenarios where structural integrity is required. Here, we developed a convenient and effective method for constructing covalently stabilized DNA nanostructures by chemically inserting photo-crosslinker(^(CNV)K) in DNA sequences. These covalently linked DNA nanostructures were found to be more resistant to external interference, such as low cation concentrations and unspecific displacement on cell membranes. We also demonstrated that our strategy could improve the efficiency of cell surface receptor-mediated labeling and function regulations in living cells, which sheds light on broadening the biomedical applications of DNA nanostructures.

Inhibition of cerebral ischemia/reperfusion injuryinduced apoptosis:nicotiflorin and JAK2/STAT3 pathway

Nicotiflorin is a flavonoid extracted from Carthamus tinctorius.Previous studies have shown its cerebral protective effect,but the mechanism is undefined.In this study,we aimed to determine whether nicotiflorin protects against cerebral ischemia/reperfusion injury-induced apoptosis through the JAK2/STAT3 pathway.The cerebral ischemia/reperfusion injury model was established by middle cerebral artery occlusion/reperfusion.Nicotiflorin（10 mg/kg） was administered by tail vein injection.Cell apoptosis in the ischemic cerebral cortex was examined by hematoxylin-eosin staining and terminal deoxynucleotidyl transferase d UTP nick end labeling assay.Bcl-2 and Bax expression levels in ischemic cerebral cortex were examined by immunohistochemial staining.Additionally,p-JAK2,p-STAT3,Bcl-2,Bax,and caspase-3 levels in ischemic cerebral cortex were examined by western blot assay.Nicotiflorin altered the shape and structure of injured neurons,decreased the number of apoptotic cells,down-regulates expression of p-JAK2,p-STAT3,caspase-3,and Bax,decreased Bax immunoredactivity,and increased Bcl-2 protein expression and immunoreactivity.These results suggest that nicotiflorin protects against cerebral ischemia/reperfusion injury-induced apoptosis via the JAK2/STAT3 pathway.

Peptide-Conjugated AggregationInduced Emission Fluorogen:Precise and Firm Cell Membrane Labeling by Multiple Weak Interactions

The cell membrane is a vital barrier that protects the cell from external damage and is involved in many biochemical processes.Thus,it is of great significance to label the cell membrane to explore its function.However,due to its complex and dynamic nature,precise and firm cell membrane labeling simultaneously is still a challenge.Herein,we report the fabrication of a peptide-conjugated aggregationinduced emission fluorogen(AIEgen),RTP,consisting of three main components:(1)An integrin-targeting peptide(RGD,R),which could bind specifically to integrinαvβ3 on cell membranes through ligand–receptor interaction.(2)An AIE-active tetraphenylethene derivative(T-MY,T)for fluorescent imaging.(3)Palmitic acid-modified peptide(Pal-RRRR,P),in which Pal isinserted into the lipid on the cellmembrane by hydrophobic interaction,and RRRR interacted with the negatively charged cell membrane components(proteins and lipids)through electrostatic forces.RTP could precisely label tumor cells with high integrinαvβ3 expression andfirmly trace the cellmembrane for up to 4 h;it also has a strong resistance to photobleaching.Moreover,RTP achieved in vivo tumor-specific imaging via cell membrane labeling.Thereby,utilizing multiple weak interactions between the fluorescent probe and the cell membrane provided a new strategy for precise and firm imaging of the cell membrane simultaneously.

Long-term live-cell microscopy with labeled nanobodies delivered by laser-induced photoporation

Fluorescence microscopy is the method of choice for studying intracellular dynamics.However,its success depends on the.availability of specific and stable markers.A prominent example of markers that are rapidly gaining interest are nanobodies(Nbs.-15 kDa),which can be functionalized with bright and photostable organic fluorophores.Due to their relatively small size and high specificity,Nbs offer great potential for high-quality long-term subcellular imaging,but suffer from the fact that they cannot spontaneously cross the plasma membrane of live cells.We have recently discovered that laser-induced photoporation is well suited to deliver extrinsic labels to living cells without compromising their viability.Being a laser-based technology,it is readily compatible with light microscopy and the typical cell recipients used for that.Spurred by these promising initial results,we demonstrate here for the first time successful long-term imaging of specific subcellular structures with labeled nanobodies in living cells.We illustrate this using Nbs that target GFP/YFP-protein constructs accessible in the cytoplasm,actin-bundling protein Fascin,and the histone H2A/H2B heterodimers.With an efficiency of more than 80%labeled cells and minimal toxicity(-2%),photoporation proved to be an excellent intracellular delivery method for Nbs.Time-lapse microscopy revealed that cell division rate and migration remained unaffected,confirming excellent cell viability and functionality.We conclude that laser-induced photoporation labeled Nbs can be easily delivered into living cells,laying the foundation for further development of a broad range of Nbs with intracellular targets as a toolbox for long-term live-cell microscopy.

Spy chemistry enables stable protein immobilization on iron oxide nanoparticles with enhanced magnetic properties

Iron oxide nanoparticles(IONPs)modified with functional proteins hold great promise in the biomedical field.However,conventional protein modification strategies,such as adsorption and covalent coupling,are either unstable or nonspecific,or may result in the changes of protein structure and ultimately the loss of protein activity.Modification of active proteins on small-sized IONPs with a particle size of less than 30 nm is especially difficult due to their high surface energy.Herein,we developed a universal modifica-tion method based on Spy chemistry for rapid and stable protein immobilization on small-sized IONPs,which only requires the presence of active groups on the surface of nanoparticles that can couple with SpyCatcher.In short,the SpyCatcher peptides were first coated on the surface of IONPs by cross-linking with activated groups,and then the SpyTag peptide fused with a model protein(enhanced green fluo-rescent protein,EGFP)was engineered(SpyTag-EGFP)and directly coupled to SpyCatcher-modified IONPs by self-assembly,which is spontaneous and robust while avoiding the effect of chemical reactions on functional protein activity.The obtained EGFP-functionalized IONPs exhibited enhanced and stable green fluorescence and improved magnetic properties.In addition,the cell internalization efficiency of EGFP-functionalized IONPs was significantly increased as compared to unmodified IONPs,providing an ideal solution for efficient cell labeling and tracking.In conclusion,here we report a rapid and easy strategy for EGFP immobilization on IONPs based on Spy chemistry,which could be further adapted to other functional proteins in the future.SpyCatcher-modified IONPs and SpyTag-X(arbitrary functional fusion proteins)hold great potential to be applied as a versatile platform for protein immobilization on IONPs and enable its multifunctional application in the future.