An organic molecular compound for in situ identification of mitochondrial G-quadruplexes in live cells.

Emerging studies have shown that mitochondrial G-quadruplex plays a critical role in regulating mitochondrial gene replication and transcription, which makes it a promising target for the diagnosis and treatment of cancer or other major diseases. Molecular compounds that can highly target the mitochondrial G-quadruplexes in live cells are essential for further revealing the function and mechanism of these G-quadruplexes. Here, we have developed an organic molecular compound that can highly target the mitochondria of living cells by virtue of the membrane potential mechanism. Then it shows high selectivity to the G-quadruplex structure in the mitochondria, and its fluorescence overlaps well with that of the BG4 antibody. Moreover, the compound has extremely low cytotoxicity and does not interfere with the natural state of G-quadruplex structure. With these good properties, this compound will have great potential in mitochondrial G-quadruplex tracking research or targeted drug screening.

Cell Coding Arrays Based on Fluorescent Glycan Nanoparticles for Cell Line Identification and Cell Contamination Evaluation.

Cell lines are applied on a large scale in the field of biomedicine, but they are susceptible to issues such as misidentification and cross-contamination. This situation is becoming worse over time due to the rapid growth of the biomedical field, and thus there is an urgent need for a more effective strategy to address the problem. As described herein, a cell coding method is established based on two types of uniform and stable glycan nanoparticles that are synthesized using the graft-copolymerization-induced self-assembly (GISA) method, which further exhibit distinct fluorescent properties due to elaborate modification with fluorescent labeling molecules. The different affinity between each nanoparticle and various cell lines results in clearly distinguishable differences in their endocytosis degrees, thus resulting in distinct characteristic fluorescence intensities. Through flow cytometry measurements, the specific signals of each cell sample can be recorded and turned into a map divided into different regions by statistical processing. Using this sensing array strategy, we have successfully identified six human cell lines, including one normal type and five tumor types. Moreover, cell contamination evaluation of different cell lines with HeLa cells as the contaminant in a semiquantitative analysis has also been successfully achieved. Notably, the whole process of nanoparticle fabrication and fluorescent testing is facile and the results are highly reliable.

Dextran-polylactide micelles loaded with doxorubicin and DiR for image-guided chemo-photothermal tumor therapy.

Image-guided chemo-photothermal therapy based on near-infrared (NIR) theranostic agents has found promising applications in treating tumors. In this multimodal treatment, it is of critical importance to image real-time distribution of photothermal agents in vivo and to monitor therapeutic outcomes for implementing personalized treatment. In this study, an optimally synthesized dextran-polylactide (DEX-PLA) copolymer was assembled with doxorubicin (DOX) and DiR, a kind of NIR dye, to construct desirable micelles ((DiR + DOX)/DEX-PLA) for performing image-guided chemo-photothermal therapy. These (DiR + DOX)/DEX-PLA micelles had good physical and photothermal stability in aqueous media and showed high photothermal efficiency in vivo. Based on the H22-tumor-bearing mouse model, (DiR + DOX)/DEX-PLA micelles were found to accumulate inside tumors sustainably and to emit strong fluorescence signals for more than three days. The (DiR + DOX)@DEX-PLA micelles together with NIR laser irradiation were able to highly inhibit tumor growth or even eradicate tumors with one injection and two dose-designated 5-minute laser irradiations at the tumor site during 14 days of treatment. Furthermore, they showed almost no impairment to the body of the treated mice. These (DiR + DOX)@DEX-PLA micelles have confirmative translational potential in clinical tumor therapy on account of their persistent image-guided capacity, high antitumor efficacy and good in vivo safety.

Cascade signal amplification sensing strategy for highly specific and sensitive detection of homologous microRNAs in different molecular subtypes of breast cancer.

Discriminative identification of homologous miRNAs in miRNA family with high specificity and sensitivity is crucial for accurate classification, diagnosis and prognosis of breast cancer. Herein, we report a reliable, sensitive, and selective assay by coupling fluorescence resonance energy transfer (FRET) with cascade signal amplification. The strategy is developed by designing two programmable DNA probes that can be triggered to shift from "off" to "on" state in a cascade hybridization reaction in the presence of target miRNA let-7a, leading to the generation of an amplified signal. The assay can detect concentrations as low as ∼3.0 pM let-7a and discriminate let-7a from other highly homologous members in the let-7 miRNA family. Moreover, it can also be used to determine let-7a levels at single-cell resolution and evaluate the drug efficacy of let-7a expression among various molecular types of breast cancer cell lines. The advantage of this assay is a combined result of signal generation and amplification triggered by target miRNA, which can satisfy an assay of analogous miRNA in a downregulated manner with high specificity. It has promising potential as a selective assay for homologous miRNAs in precision medicine.

Zwitterionic Cross-Linked Biodegradable Nanocapsules for Cancer Imaging.

Zwitterionic cross-linked biodegradable nanocapsules (NCs) were synthesized for cancer imaging. A polylactide (PLA)-based diblock copolymer with two blocks carrying acetylenyl and allyl groups respectively was synthesized by ring-opening polymerization (ROP). Azide-alkyne "click" reaction was conducted to conjugate sulfobetaine (SB) zwitterions and fluorescent dye Cy5.5 onto the acetylenyl-functionalized first block of the diblock copolymer. The resulting copolymer with a hydrophilic SB/Cy5.5-functionalized PLA block and a hydrophobic allyl-functionalized PLA block could stabilize miniemulsions because of its amphiphilic diblock structure. UV-induced thiol-ene "click" reaction between a dithiol cross-linker and the hydrophobic allyl-functionalized block of the copolymer at the peripheral region of nanoscopic oil nanodroplets in the miniemulsion generated cross-linked polymer NCs with zwitterionic outer shells. These NCs showed an average hydrodynamic diameter ( Dh) of 136 nm. They exhibited biodegradability, biocompatibility and high colloidal stability. In vitro study indicated that these NCs could be taken up by MIA PaCa-2 cancer cells. In vivo imaging study showed that, comparing to a small molecule dye, NCs had a longer circulation time, facilitating their accumulation at tumors for cancer imaging. Overall, this work demonstrates the applicability of zwitterionic biodegradable polymer-based materials in cancer diagnosis.

Assembly of DNA Probes into Superstructures for Dramatically Enhancing Enzymatic Stability and Signal-to-Background Ratio.

DNA fluorescent probes are versatile tools that are widely used for biological detection in tubes. Using DNA probes in living systems, however, represents a significant challenge because of the endogenous nuclease-induced DNA degradation and strong background fluorescence in complex biological environments. Here, we show that assembling DNA probes into core-satellite gold nanoparticle (AuNP) superstructures could unprecedentedly enhance enzymatic stability and reduce background interference. The embedded DNA probes are protected from interaction with nuclease, eliminating the enzymatic degradation. In the meantime, the AuNP superstructures show extremely high quenching efficiency (>98%) toward the embedded DNA probes, whose fluorescence can be instantly turned on by the target binding, resulting in high signal-to-background ratio. To demonstrate these distinct properties, we made use of the assembled nanoprobes to monitor the ATP levels under different stimuli in living cells. The assembly strategy leads to a new opportunity for accurately sensing targets in living systems.

A core-shell nanoparticle-peptide@metal-organic framework as pH and enzyme dual-recognition switch for stepwise-responsive imaging in living cells.

A core-shell nanostructure is fabricated with a pH-sensitive metal-organic framework shell and a peptide functionalized gold nanoparticle core via a mild synthetic route. The nanostructure can be applied as a dual-recognition switch in response to an acidic environment and enzyme activity, sequentially, leading to a stepwise-responsive strategy for imaging lysosomal cathepsin B.

Fluorescence and photoacoustic dual-mode imaging of tumor-related mRNA with a covalent linkage-based DNA nanoprobe.

We developed a dual-mode DNA nanoprobe based on covalent linkage for fluorescence imaging and photoacoustic imaging of tumor-related mRNA.

Synthesis, characterization, in vitro SAR and in vivo evaluation of N,N'bisnaphthylmethyl 2-alkyl substituted imidazolium salts against NSCLC.

Alkyl- and N,N'-bisnaphthyl-substituted imidazolium salts were tested in vitro for their anti-cancer activity against four non-small cell lung cancer cell lines (NCI-H460, NCI-H1975, HCC827, A549). All compounds had potent anticancer activity with 2 having IC50 values in the nanomolar range for three of the four cell lines, a 17-fold increase in activity against NCI-H1975 cells when compared to cisplatin. Compounds 1-4 also showed high anti-cancer activity against nine NSCLC cell lines in the NCI-60 human tumor cell line screen. In vitro studies performed using the Annexin V and JC-1 assays suggested that NCI-H460 cells treated with 2 undergo an apoptotic cell death pathway and that mitochondria could be the cellular target of 2 with the mechanism of action possibly related to a disruption of the mitochondrial membrane potential. The water solubilities of 1-4 was over 4.4mg/mL using 2-hydroxypropyl-ß-cyclodextrin as a chemical excipient, thereby providing sufficient solubility for systemic administration.

Near-infrared fluorescent peptide probes for imaging of tumor in vivo and their biotoxicity evaluation.

Optical imaging techniques are becoming increasingly urgent for the early detection and monitoring the progression of tumor development. However, tumor vasculature imaging has so far been largely unexplored because of the lack of suitable optical probes. In this study, we demonstrated the preparation of near-infrared (NIR) fluorescent RGD peptide probes for noninvasive imaging of tumor vasculature during tumor angiogenesis. The peptide optical probes combined the advantages of NIR emission and RGD peptide, which possesses minimal biological absorption and specially targets the integrin, which highly expressed on activated tumor endothelial cells. In vivo optical imaging of nude mice bearing pancreatic tumor showed that systemically delivered NIR probes enabled us to visualize the tumors at 24 hours post-injection. In addition, we have performed in vivo toxicity study on the prepared fluorescent RGD peptide probes formulation. The blood test results and histological analysis demonstrated that no obvious toxicity was found for the mice treated with RGD peptide probes for two weeks. These studies suggest that the NIR fluorescent peptide probes can be further designed and employed for ultrasensitive fluorescence imaging of angiogenic tumor vasculature, as well as imaging of other pathophysiological processes accompanied by activation of endothelial cells.

In vivo biocompatibility of a new cyanine dye for ILM peeling.

PURPOSE: To investigate the biocompatibility of the new cyanine dye: 3,3'-Di-(4-sulfobutyl)-1,1,1',1'-tetramethyl-di-1H-benz[e]indocarbocyanine (DSS) as a vital dye for intraocular application in an in vivo rat model and to evaluate the effects of this dye on retinal structure and function. METHODS: DSS at a concentration of 0.5% was applied via intravitreal injections to adult Brown Norway rats with BSS serving as a control. Retinal toxicity was assessed 7 days later by means of retinal ganglion cell (RGC) counts, light microscopy, optical coherence tomography (OCT), and electroretinography (ERG). RESULTS: No significant decrease in RGC numbers was observed. No structural changes of the central retina were observed either in vivo (OCT) or under light microscopy. ERGs detected a temporary reduction of retinal function 7 days after injection; this was no longer evident 14 days after injection. CONCLUSIONS: DSS showed good biocompatibility in a well-established experimental in vivo setting and may be usable for intraocular surgery as an alternative to other cyanine dyes. In contrast to indocyanine green, it additionally offers fluorescence in the visual spectrum. Further studies with other animal models are needed before translation into clinical application.

Endogenous subventricular zone neural progenitors contribute to the formation and hyperexcitability of experimental model of focal microgyria.

Microgyria is associated with epilepsy and due to developmental disruption of neuronal migration. However, the role of endogenous subventricular zone-derived neural progenitors (SDNPs) in formation and hyperexcitability has not been fully elucidated. Here, we establish a neonatal cortex freeze-lesion (FL) model, which was considered as a model for focal microgyria, and simultaneously label SDNPs by CM-DiI. Morphological investigation showed that SDNPs migrated into FL and differentiated into neuronal and glia cell types, suggesting the involvement of endogenous SDNPs in the formation of FL-induced microgyria. Patch-clamp recordings in CM-DiI positive (CM-DiI(+)) pyramidal neurons within FL indicated an increase in frequency of spontaneous action potentials, while the resting membrane potential did not differ from the controls. We also found that spontaneous excitatory postsynaptic currents (EPSCs) increased in frequency but not in amplitude compared with controls. The evoked EPSCs showed a significant increase of 10-90% in rise time and decay time in the CM-DiI(+) neurons. Moreover, paired-pulse facilitation was dramatically larger in CM-DiI(+) pyramidal neurons. Western blotting data showed that AMPA and NMDA receptors were increased to some extent in the FL cortex compared with controls, and the NMDA/AMPA ratio of eEPSCs at CM-DiI(+) pyramidal neurons was significantly increased. Taken together, our findings provide novel evidence for the contribution of endogenous SDNPs in the formation and epileptogenicity of FL-induced focal microgyria.

Synthesis of a novel L-methyl-methionine-ICG-Der-02 fluorescent probe for in vivo near infrared imaging of tumors.

PURPOSE: A novel near infrared fluorescent probe, L-methyl-methionine (Met)-ICG-Der-02, was synthesized and characterized for in vivo imaging of tumors and early diagnosis of cancers. METHOD: Met was conjugated with ICG-Der-02 dye through the amide bond function by ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide catalysis chemistry. Met-ICG-Der-02 probe uptake was evaluated on PC3, MDA-MB-231, and human embryonic lung fibroblast cell lines. The dynamics of Met-ICG-Der-02 was investigated in athymic nude mice prior to evaluation of the probe targeting capability in prostate and breast cancer models. RESULTS: Met-ICG-Der-02 was successfully synthesized. Cell experiments demonstrated excellent cellular uptake of Met-ICG-Der-02 on cancer cell lines without cytotoxicity. Optical imaging showed a distinguishable fluorescence signal in the tumor area at 2 h while maximal tumor-to-normal tissue contrast ratio was at 12 h Met-ICG-Der-02 post-injection. Additionally, dynamic study of the probe indicated intestinal and liver-kidney clearance pathways. CONCLUSION: Met-ICG-Der-02 probe is a promising optical imaging agent for tumor diagnosis, especially in their early stage.

Synthesis, staining properties, and biocompatibility of a new cyanine dye for ILM peeling.

BACKGROUND: The aim of this work is to investigate the biocompatibility and staining properties of DSS: 3,3'-Di-(4-sulfobutyl)-1,1,1',1'-tetramethyl-di-1H-benz[e]indocarbocyanine (DSS). METHODS: Dye concentrations of 0.5, 0.25, and 0.1% were evaluated (290 and 295 mOsm). Toxicity was assessed using a colorimetric test measuring the inhibition of ARPE 19 cell, human primary RPE cell, and human Müller cell proliferation. Exposure time was 30, 60, 120, and 300 s. Indocyanine green (ICG) (0.5, 0.25, and 0.1%) served as a control. Cells were also illuminated with plain white light (750 mW/cm(2)) for 10 min to assess phototoxic effects. Besides staining of porcine and human lens capsule, internal limiting membrane (ILM)-staining was assessed by applying 0.25 and 0.5% DSS over the macula in two human post-mortem eyes. RESULTS: DSS of 0.25 and 0.1% showed no toxic effect on primary RPE cells and MIO-M1cells, and 0.5, 0.25, and 0.1% for ARPE-19 cells. In MIO-M1cells, 0.5% dye showed a significant reduction of mitochondrial dehydrogenase activity only following an exposure time of 300 s. Following illumination, ICG showed a significantly more pronounced effect on cell viability in primary RPE cells and MIO-M1cells compared to DSS. The absorption maximum is found at 591 nm; the even more bathochromic fluorescence proceeds with a common Stokes' shift where maxima at 620 and 660 nm with a quantum yield of 32% were found. The fluorescence is sufficiently hypsochromic and the fluorescence quantum yield high enough for an easy visual detection. The contrast and staining properties at the ILM were excellent and allowed for a controlled removal of the ILM during surgery. No penetration into deeper retinal layers was noted. CONCLUSIONS: Our results indicate that this new cyanine dye DSS may represent an alternative for ILM staining due to its matched absorption concerning visibility and fluorescence qualities as well as its good biocompatibility.

Cyanine dyes as contrast agents for near-infrared imaging in vivo: acute tolerance, pharmacokinetics, and fluorescence imaging.

We compare pharmacokinetic, tolerance, and imaging properties of two near-IR contrast agents, indocyanine green (ICG) and 1,1(')-bis-(4-sulfobutyl) indotricarbocyanine-5,5(')-dicarboxylic acid diglucamide monosodium salt (SIDAG). ICG is a clinically approved imaging agent, and its derivative SIDAG is a more hydrophilic counterpart that has recently shown promising imaging properties in preclinical studies. The rather lipophilic ICG has a very short plasma half-life, thus limiting the time available to image body regions during its vascular circulation (e.g., the breast in optical mammography where scanning over several minutes is required). In order to change the physicochemical properties of the indotricarbocyanine dye backbone, several derivatives were synthesized with increasing hydrophilicity. The most hydrophilic dye SIDAG is selected for further biological characterization. The acute tolerance of SIDAG in mice is increased up to 60-fold compared to ICG. Contrary to ICG, the pharmacokinetic properties of SIDAG are shifted toward renal elimination, caused by the high hydrophilicity of the molecule. N-Nitrosomethylurea (NMU)-induced rat breast carcinomas are clearly demarcated, both immediately and 24 h after intravenous administration of SIDAG, whereas ICG shows a weak tumor contrast under the same conditions. Our findings demonstrate that SIDAG is a high potential contrast agent for optical imaging, which could increase the sensitivity for detection of inflamed regions and tumors.

Cyanine dyes as optical contrast agents for ophthalmological surgery.

Cyanine dyes were prepared as optical contrast media for supporting the surgery of the lamina limitans interna (LLI) of the retina and other structures of the human eye. Their absorption spectra were adapted both to the spectral sensitivity of the human eye and to standard illumination. The contrast could be further amplified by the application of the strong fluorescence of the dyes used. The binding of the dyes to various surfaces was studied. No toxic effects could be detected for the applied dyes.

ABCG2-dependent dye exclusion activity and clonal potential in epithelial cells continuously growing for 1 month from limbal explants.

PURPOSE: To determine changes in ABCG2-transport-dependent dye exclusion in outgrowths from limbal explants. METHODS: Human or rabbit limbal strips were deposited onto inserts. Over a month, the segments were twice transferred to new inserts. Fresh tissue (FT) cells, obtained by sequential dispase-trypsin digestion and the cells growing from the explant cultures, were characterized for ABCG2-dependent efflux by flow cytometry using a newly identified substratum, JC1. Rabbit cells were sorted into JC1-excluding (JC1(low)) and main (JC1(main)) cohorts and seeded with feeder 3T3 cells to determine colony formation efficiency (CFE). RESULTS: The JC1(low) cells were all Hoechst 33342-excluding cells and vice versa, establishing the physical equivalence between JC1(low) and the side population (SP). JC1(low) cell content was reduced by three ABCG2-specific inhibitors: FTC, Ko143, and glafenine. JC1(low) percentiles for the fresh human and rabbit cells were 1.4% and 4.1% and CFEs for rabbit JC1(low) and JC1(main) were 1.2% and 5.3%. In contrast, the respective JC1(low) percentiles in the first and second outgrowths were 19.5% and 27.4% and 25.8% and 32.5%, and the rabbit JC1(low) and JC1(main) CFEs were 12.3% and 0.9%. Thus, although in FT the contribution of the JC1(low) cohort to the CFE is minimal, in the explant culture the phenotype incorporates >80% of the CFE. CONCLUSIONS: ABCG2-dependent dye exclusion undergoes a large expansion in explant culture and becomes associated with a high CFE. The transport increase is more pronounced at late outgrowth times, suggesting permanence of stem cells within the explant.

In vivo detection of embryonic stem cell-derived cardiovascular progenitor cells using Cy3-labeled Gadofluorine M in murine myocardium.

OBJECTIVES: The aim of the current study is to test the ability to label and detect murine embryonic stem cell-derived cardiovascular progenitor cells (ES-CPC) with cardiac magnetic resonance (CMR) using the novel contrast agent Gadofluorine M-Cy3 (GdFM-Cy3). BACKGROUND: Cell therapy shows great promise for the treatment of cardiovascular disease. An important limitation to previous clinical studies is the inability to accurately identify transplanted cells. GdFM-Cy3 is a lipophilic paramagnetic contrast agent that contains a perfluorinated side chain and an amphiphilic character that allows for micelle formation in an aqueous solution. Previous studies reported that it is easily taken up and stored within the cytosol of mesenchymal stem cells, thereby allowing for paramagnetic cell labeling. Investigators in our laboratory have recently developed techniques for the robust generation of ES-CPC. We reasoned that GdFM-Cy3 would be a promising agent for the in vivo detection of these cells after cardiac cell transplantation. METHODS: ES-CPC were labeled with GdFM-Cy3 by incubation. In vitro studies were performed to assess the impact of GdFM-Cy3 on cell function and survival. A total of 500,000 GdFM-Cy3-labeled ES-CPC or control ES-CPC were injected into the myocardium of mice with and without myocardial infarction. Mice were imaged (9.4-T) before and over a 2-week time interval after stem cell transplantation. Mice were then euthanized, and their hearts were sectioned for fluorescence microscopy. RESULTS: In vitro studies demonstrated that GdFM-Cy3 was easily transfectable, nontoxic, stayed within cells after labeling, and could be visualized using CMR and fluorescence microscopy. In vivo studies confirmed the efficacy of the agent for the detection of cells transplanted into the hearts of mice after myocardial infarction. A correspondence between CMR and histology was observed. CONCLUSIONS: The results of the current study suggest that it is possible to identify and potentially track GdFM-Cy3-labeled ES-CPC in murine infarct models via CMR.

Pulmonary toxicity and kinetic study of Cy5.5-conjugated superparamagnetic iron oxide nanoparticles by optical imaging.

Recent advances in the development of nanotechnology and devices now make it possible to accurately deliver drugs or genes to the lung. Magnetic nanoparticles can be used as contrast agents, thermal therapy for cancer, and be made to concentrate to target sites through an external magnetic field. However, these advantages may also become problematic when taking into account safety and toxicological factors. This study demonstrated the pulmonary toxicity and kinetic profile of anti-biofouling polymer coated, Cy5.5-conjugated thermally cross-linked superparamagnetic iron oxide nanoparticles (TCL-SPION) by optical imaging. Negatively charged, 36 nm-sized, Cy5.5-conjugated TCL-SPION was prepared for optical imaging probe. Cy5.5-conjugated TCL-SPION was intratracheally instilled into the lung by a non-surgical method. Cy5.5-conjugated TCL-SPION slightly induced pulmonary inflammation. The instilled nanoparticles were distributed mainly in the lung and excreted in the urine via glomerular filtration. Urinary excretion was peaked at 3 h after instillation. No toxicity was found under the concentration of 1.8 mg/kg and the half-lives of nanoparticles in the lung and urine were estimated to be about 14.4+/-0.54 h and 24.7+/-1.02 h, respectively. Although further studies are required, our results showed that Cy5.5-conjugated TCL-SPION can be a good candidate for use in pulmonary delivery vehicles and diagnostic probes.

Uptake and phototoxicity of tricarbocyanine indolenine dye covalently bound with glucose (TICS) under acidification of tumor cells environment.

AIM: To evaluate the influence of pH on accumulation and photocytotoxicity of the novel tricarbocyanine indolenine dye covalently bound with glucose (TICS). METHODS: For in vitro experiments, cultured HeLa cells were incubated with TICS at pH 7.1, 6.5 or 6.2 and then scored for dye uptake, viability and sensitivity to laser (740 nm) irradiation. In vivo TICS was injected to rats with implanted subcutaneously SM-1 tumor and then dye accumulation and tumor necrosis depth after laser irradiation were defined. RESULTS: Reduction of medium pH in vitro was shown to increase in cellular TICS contents and to enhance as its "dark" cytotoxicity, and photocytotoxicity. The ratio of "dark" cytotoxicity to photocytotoxicity parameters increased 2-fold with decreasing pH value. In vivo infusion of glucose (10 g/kg) to rats resulted in improved selectivity of TICS accumulation and increase in tumor necrosis depth after laser irradiation. CONCLUSION: pH reduction of tumor cells environment improves efficacy of photodynamic treatment with TICS.