NIR Light-Mediated Mitochondrial RNA Modification for Cancer RNA Interference Therapeutics.

Mitochondrial RNA (mtRNA) plays a critical role in synthesis of mitochondrial proteins. Interfering mtRNA is a highly effective way to induce cell apoptosis. Herein, we report a near-infrared (NIR) light-mediated mitochondrial RNA modification approach for long-term imaging and effective suppression of tumors. A tumor-targetable NIR fluorescent probe f-CRI consisting of a cyclic RGD peptide, a NIR fluorophore IR780, and a singlet oxygen (1 O2 )-labile furan group for RNA modification was rationally designed and synthesized. This probe was demonstrated to dominantly accumulate in cellular mitochondria and could be covalently conjugated onto mtRNA upon 808 nm irradiation resulting in prolonged retention in tumors. More notably, this covalent modification of mtRNA by f-CRI could perturb the function of mitochondria leading to remarkable tumor suppression. We thus envision that our current approach would offer a potential approach for cancer RNA interference therapeutics.

Non-enzymatic detection of miR-21 in cancer cells using a homogeneous mix-and-read smart probe assay.

We report a new assay system for the detection of miR-21 in cancer cells. The new assay works at room temperature and it does not involve enzymatic amplification. It consists a hairpin smart probe, designed to specifically recognize miR-21 target sequence. We tested the performance and sequence recognition capability of the smart probe to confirm desired specifications. We used the smart probe for the sequence-specific recognition of synthetic miR-21 oligonucleotides as well as mismatch sequences and we found that the probe recognizes the target sequence-specifically, while discriminating against mismatched sequences. We determined the limit of detection and limit of quantitation for the miR-21 oligonucleotides to be 1.72 nM and 5.78 nM, respectively, while the sensitivity is 6.90 × 1011 c.p.sM-1. More importantly, we showed that the smart probe-based method is also sensitive and selective for miR-21 when applied to crude extractions from MCF-7 cancer cell line at room temperature, with the results showing high fluorescence signals for the MCF-7 samples while showing much less signals for samples that did not contain miR-21. Thus, this new smart probe system constitutes a homogeneous, mix-and-read detection technique that can provide reliable diagnostics of miR-21 cancer biomarker at room temperature.

Detection of Several Homologous MicroRNAs by a Single Smart Probe System Consisting of Linear Nucleic Acid Blockers.

We report a universal smart probe (SP) that is capable of detecting several homologous let-7 microRNAs (miRNAs). While the SP is complementary to let-7a, and therefore, strongly binds to this target, due to sequence homology, the SP also has equal propensity to non-specifically hybridize with let-7b and let-7c, which are homologous to let-7a. The fluorescence signal of the SP was switched off in the absence of any homologous member target, but the signal was switched on when any of the three homologous members was present. With the assistance of nucleic acid blockers (NABs), this SP system can discriminate between homologous miRNAs. We show that the SP can discriminate between let-7a and the other two sequences by using linear NABs (LNABs) to block non-specific interactions between the SP and these sequences. We also found that LNABs used do not cross-react with the let-7a target due to the low LNABs:SP molar ratio of 6:1 used. Overall, this SP represents a universal probe for the recognition of a homologous miRNA family. The assay is sensitive, providing a detection limit of 6 fmol. The approach is simple, fast, usable at room temperature, and represents a general platform for the in vitro detection of homologous microRNAs by a single fluorescent hairpin probe.

Real-time monitoring of DNA methyltransferase activity using a hemimethylated smart probe.

A real-time assay for DNA methyltransferase (MTase) activity has been developed. A hemimethylated smart probe is used as the substrate for DNA MTase. Cleavage of the methylated product leads to separation of fluorophore from quencher, giving a proportional increase in fluorescence. The method permits real-time monitoring of DNA methylation process and makes it easy to characterize the activity of DNA MTase. It also has the potential to screen suitable inhibitor drugs for DNA MTase.

Site-specific conjugation of the quencher on peptide's N-terminal for the synthesis of a targeted non-spreading activatable optical probe.

Optical imaging offers high sensitivity and portability at low cost. The design of 'smart' or 'activatable' probes can decrease the background noise and increase the specificity of the signal. By conjugating a fluorescent dye and a compatible quencher on each side of an enzyme's substrate, the signal remains in its 'off ' state until it reaches the area where a specific enzyme is expressed. However, the signal can leak from that area unless the dye is attached to a molecule able to bind to a specific target also presented in that area. The aim of this study was to (i) specifically conjugate the quencher on the α-amino group of the peptide's N-terminus, (ii) conjugate the dye on the ε-amino group of a lysine in C-terminus, and (iii) conjugate the carboxyl group of the peptide's C-terminus to an amino group present on an antibody, using carbodiimide chemistry. The use of protecting groups, such as Boc or Fmoc, to allow site-specific conjugation, presents several drawbacks including 'on beads labeling', additional steps required for deprotection and removal from the resin, decreased yield, and dye degradation. A method of preferential labeling of α-amino N-terminal group in slightly acidic solution, proposed by Selo et al. (1996) has partially solved the problem. The present study reports improvements of the method allowing to (i) avoid the homo-bilabeling, (ii) increase the yield of the N-terminal labeling by two folds, and (iii) decrease the cost by 44-fold. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Real-Time Tissue Classification Using a Novel Optical Needle Probe for Biopsy.

Core needle biopsy is a part of the histopathological process, which is required for cancerous tissue examination. The most common method to guide the needle inside of the body is ultrasound screening, which in greater part is also the only guidance method. Ultrasound screening requires user experience. Furthermore, patient involuntary movements such as breathing might introduce artifacts and blur the screen. Optically enhanced core needle biopsy probe could potentially aid interventional radiologists during this procedure, providing real-time information on tissue properties close to the needle tip, while it is advancing inside of the body. In this study, we used diffuse optical spectroscopy in a custom-made core needle probe for real-time tissue classification. Our aim was to provide initial characteristics of the smart needle probe in the differentiation of tissues and validate the basic purpose of the probe of informing about breaking into a desired organ. We collected optical spectra from rat blood, fat, heart, kidney, liver, lungs, and muscle tissues. Gathered data were analyzed for feature extraction and evaluation of two machine learning-based classifiers: support vector machine and k-nearest neighbors. Their performances on training data were compared using subject-independent k-fold cross-validation. The best classifier model was chosen and its feasibility for real-time automated tissue recognition and classification was then evaluated. The final model reached nearly 80% of correct real-time classification of rat organs when using the needle probe during real-time classification.

Application of scanning acoustic microscopy for evaluation of MMP activation in multiple cancer cell lines with a smart probe.

Background/aim: Matrix metalloproteinases (MMPs) play an important role in the evaluation of many cancer types; however, the detection usually presents a challenge. Further assays for a better understanding of the fundamental roles of MMPs in pathophysiology are still needed. We aimed to use an activatable probe in scanning acoustic microscopy (SAM) to evaluate acoustically if the probe can aid the visualization of the effects of in vitro MMP activity. Materials and methods: We applied scanning acoustic impedance microscopy to obtain acoustic impedance maps of the cell line models of HT1080, THP-1, and SK-MEL-28 with and without MMPSense 680 probe incubation. We visually validated our results using confocal laser scanning microscopy imaging. We further analyzed the effects of MMPSense 680 probe on cell viabilities to eliminate any artifacts. Results: This is the first study presenting the applicability of SAM in the acoustical evaluation of MMPSense 680 probe cleavage in a cellular medium through acoustic impedance measurements. We proposed that SAM measurement with the activatable probe can be used as an effective tool for studying the acoustical variations of MMP activities in cell lines. As a result, we detected MMPSense 680 probe cleavage in HT1080 human fibrosarcoma cell line. Conclusion: We showed that SAM with the smart probe can detect proteolytic activity using MMPSense 680 in in vitro HT1080 cell line by acoustic impedance measurements. SAM could be proposed as an alternative tool leading a novel way for a better understanding of the roles of MMPs in cancer progression before clinical settings.

Novel Glutathione Activated Smart Probe for Photoacoustic Imaging, Photothermal Therapy, and Safe Postsurgery Treatment.

Preventing tumor recurrence is the most important target for cancer treatment. However, the current effective and advanced technology relies on the use of near-infrared region (NIR), and the equipment of NIR-I and NIR-II fluorescence imaging technique-based fluorescent-guided surgery is expensive and complicated to operate. Here, we report a safe and effective strategy of an organic-inorganic hybrid gold nanoparticle-based novel smart probe (Au@PDA-ss-PEGm NPs) which is appropriate for photoacoustic imaging (PAI) and plasmonic photothermal therapy (PPTT) of tumors in vivo. After intravenous injection, the probe would be transported to the tumor to penetrate the cellular membrane. Then the disulfide bond on the probe surface would be broken with the help of a high concentration of glutathione in the tumor cell. The remaining Au@PDA NPs would aggregate to form plasmonic nanoclusters and exhibit a notable plasmon coupling enhanced photothermal (PCEPT) effect. Besides, the results further proved its good biosafety and pharmacokinetic characteristics in vivo and, more important, a short time exposure under 808 nm laser after surgical removal of the tumor, which would be effective to prevent tumor recurrence and bring dawn to the high-efficiency treatment of tumors.

A NIR fluorescent smart probe for imaging tumor hypoxia.

BACKGROUND: Tumor hypoxia is a characteristic of paramount importance due to low oxygenation levels in tissue negatively correlating with resistance to traditional therapies. The ability to noninvasively identify such could provide for personalized treatment(s) and enhance survival rates. Accordingly, we recently developed an NIR fluorescent hypoxia-sensitive smart probe (NO2 -Rosol) for identifying hypoxia via selectively imaging nitroreductase (NTR) activity, which could correlate to oxygen deprivation levels in cells, thereby serving as a proxy. We demonstrated proof of concept by subjecting a glioblastoma (GBM) cell line to extreme stress by evaluating such under radiobiological hypoxic (pO2 ≤ ~0.5%) conditions, which is a far cry from representative levels for hypoxia for brain glioma (pO2  = ~1.7%) which fluctuate little from physiological hypoxic (pO2  = 1.0-3.0%) conditions. AIM: We aimed to evaluate the robustness, suitability, and feasibility of NO2 -Rosol for imaging hypoxia in vitro and in vivo via assessing NTR activity in diverse GBM models under relevant oxygenation levels (pO2  = 2.0%) within physiological hypoxic conditions that mimic oxygenation levels in GBM tumor tissue in the brain. METHODS: We evaluated multiple GBM cell lines to determine their relative sensitivity to oxygenation levels via measuring carbonic anhydrase IX (CAIX) levels, which is a surrogate marker for indirectly identifying hypoxia by reporting on oxygen deprivation levels and upregulated NTR activity. We evaluated for hypoxia via measuring NTR activity when employing NO2 -Rosol in in vitro and tumor hypoxia imaging studies in vivo. RESULTS: The GBM39 cell line demonstrated the highest CAIX expression under hypoxic conditions representing that of GBM in the brain. NO2 -Rosol displayed an 8-fold fluorescence enhancement when evaluated in GBM39 cells (pO2  = 2.0%), thereby establishing its robustness and suitability for imaging hypoxia under relevant physiological conditions. We demonstrated the feasibility of NO2 -Rosol to afford tumor hypoxia imaging in vivo via it demonstrating a tumor-to-background of 5 upon (i) diffusion throughout, (ii) bioreductive activation by NTR activity in, and (iii) retention within, GBM39 tumor tissue. CONCLUSION: We established the robustness, suitability, and feasibility of NO2 -Rosol for imaging hypoxia under relevant oxygenation levels in vitro and in vivo via assessing NTR activity in GBM39 models.

Assembly Transformation Jointly Driven by the LAP Enzyme and GSH Boosting Theranostic Capability for Effective Tumor Therapy.

Developing intelligent and morphology-transformable nanomaterials that can spatiotemporally undergo stimulus-responsive size transformation holds great promise for improving the tumor delivery efficiency of drugs in vivo. Here, we report a smart size-transformable theranostic probe Ce6-Leu consisting of a leucine amino peptidase (LAP) and glutathione (GSH) dual-responsive moiety, an 1,2-aminothiol group, and a clinically used photosensitizer Ce6. This probe tends to self-assemble into uniform nanoparticles with an initial size of ∼80 nm in aqueous solution owing to the amphiphilic feature. Surprisingly, taking advantage of the biocompatible CBT-Cys condensation reaction, the large nanoprobes can be transformed into tiny nanoparticles (∼23 nm) under the joint action of LAP and GSH in a tumor microenvironment, endowing them with great tumor accumulation and deep tissue penetration. Concomitantly, this LAP/GSH-driven disassembly and size shrinkage of Ce6-Leu can also activate the fluorescence/magnetic resonance signals and the photodynamic effect for enhanced multimodal imaging-guided photodynamic therapy of human liver HepG2 tumors in vivo. More excitingly, the Mn2+-chelating probe (Ce6-Leu@Mn2+) was demonstrated to have the capability to catalyze endogenous H2O2 to persistently release O2 at the hypoxic tumor site, as a consequence improving the oxygen supply to boost the radiotherapy effect. We thus believe that this LAP/GSH-driven size-transformable nanosystem would offer a novel advanced technology to improve the drug delivery efficiency for achieving precise tumor diagnosis and treatment.

Tumor Microenvironment Responsive "Head-to-Foot" Self-Assembly Nanoplatform for Positron Emission Tomography Imaging in Living Subjects.

Sensitivity and specificity of molecular probes are two important factors in determining the accuracy of cancer diagnosis or the efficacy of cancer treatment. However, the development of probes with high sensitivity and strong specificity still poses many challenges. Herein, we report an 18F-labeled smart tracer ([18F]1) targeting cancer-associated biotin receptor (BR) and self-assembling into nanoparticles in response to intracellular glutathione. The tracer [18F]1 selectively targeted BR-positive cancer cells A549 and Hela and formed nanoparticles through self-assembly with an average diameter of 138.2 ± 16.3 nm. The character of self-assembly into nanoparticles enhanced the uptake and extended the retention of probe [18F]1 in the target tissue and hence improved the quality of positron emission tomography (PET) images. Thus, [18F]1 is a promising PET tracer for accurately detecting BR-positive cancers. Moreover, the tumor microenvironment responsive "head-to-foot" self-assembly nanoplatform is particularly attractive for development of other smart molecular probes.

Smart fluorescent probes for in situ imaging of enzyme activity: design strategies and applications.

Enzymes play critical roles in the physiological and pathological processes of living systems. To provide detailed pictures of enzyme activity at the molecular and cellular levels, interdisciplinary studies of chemistry and biology have led to the emergence of many smart fluorescent probes, which emit fluorescence or show a shifted signal only upon interaction with their targets. With distinct advantage of a higher signal-to-noise ratio than traditional 'always on' probes, smart fluorescent probes enable sensitive detection of enzymes with clinical significance. In this review, we summarize the design strategies and selected applications of smart fluorescent probes for in situ imaging of enzyme activity. Current challenges and future developments in this field are also discussed.

Design and Characterization of a Singly Labeled Fluorescent Smart Probe for In Vitro Detection of miR-21.

A sensitive hairpin smart probe (SP) has been developed and tested for its sequence-specificity and sensitivity for detecting microRNAs (miRNAs). The loop sequence of this SP is perfectly complementary to microRNA-21 (miR-21) sequence. This miRNA regulates certain biological processes and has been implicated in certain forms of cancer. The stem of the new SP consists of a fluorophore on one end and multiple guanine bases on the opposing end are used as quenchers. The fluorescence of the SP is significantly quenched by the guanine bases at room temperature and in the absence of the miR-21 target. The presence of miR-21 switches on the fluorescence due to spontaneous hybridization of the SP with this target, which also forces the stem hybrid of the SP apart. This new SP successfully discriminated between the perfect miR-21 target and two closely similar single-base mismatch sequences. When the SP was incubated with the miR-21 at 37 ℃, the hybridization kinetics increased seven times, compared to room temperature hybridization. Overall, this new SP shows good detection sensitivity and gives a limit of detection and limit of quantitation of 14.0 nM and 46.7 nM, respectively. This detection platform represents a simple, fast, mix-and-read homogeneous assay for sequence-specific detection of miR-21, and it can be adapted for other related diagnostic applications.

Activatable fluorescence: From small molecule to nanoparticle.

Molecular imaging has emerged as an indispensable technology in the development and application of drug delivery systems. Targeted imaging agents report the presence of biomolecules, including therapeutic targets and disease biomarkers, while the biological behaviour of labelled delivery systems can be non-invasively assessed in real time. As an imaging modality, fluorescence offers additional signal specificity and dynamic information due to the inherent responsivity of fluorescence agents to interactions with other optical species and with their environment. Harnessing this responsivity is the basis of activatable fluorescence imaging, where interactions between an engineered fluorescence agent and its biological target induce a fluorogenic response. Small molecule activatable agents are frequently derivatives of common fluorophores designed to chemically react with their target. Macromolecular scale agents are useful for imaging proteins and nucleic acids, although their biological delivery can be difficult. Nanoscale activatable agents combine the responsivity of fluorophores with the unique optical and physical properties of nanomaterials. The molecular imaging application and overall complexity of biological target dictate the most advantageous fluorescence agent size scale and activation strategy.

FRET Imaging of Enzymatic Activities Using Smart Probes.

Tumor-related enzymes are extensively involved in the occurrence, development, invasion, and metastasis of tumors, indicating they hold the potential to serve as biomarkers for tumor diagnosis and treatment. Smart probes based on characteristic activities of these enzymes and fluorescence resonance energy transfer (FRET) have been widely developed for fluorescent imaging of enzymatic activities. Here, we describe the detailed chemical strategies for construction of smart probe and its application for FRET imaging of fibroblast activation protein alpha in vitro and in vivo.

A Smart Near-Infrared Fluorescence Probe for Selective Detection of Tau Fibrils in Alzheimer's Disease.

Development of a novel, tau-selective smart near-infrared fluorescence (NIRF) probe was attempted by combining the previously identified core scaffold 3,5-dimethoxy-N,N-dimethylaniline-4-yl moiety, with the characteristic donor-π-acceptor architecture of the smart NIRF Aß probes DANIR-2c and MCAAD-3. A series of compounds (2 and 3) were prepared, which were identified as "turn-on" NIRF probes for the visual detection of tau aggregates and Aß fibrils (λem = 650 nm, Stokes shifts = 70-110 nm). In particular, combination of the 3,5-dimethoxy-N,N-dimethylanilin-4-yl moiety and the donor part of MCAAD-3 endowed the resulting probes, 3g and 3h, with significant selectivity toward tau aggregates (selectivity for tau over Aß = 5.7 and 3.8); they showed much higher fluorescence intensities upon binding to tau aggregates (FItau = 49 and 108) than when bound to Aß fibrils (FIAß = 9 and 28). Quantitative analysis of binding affinities and fluorescence properties of 3g and 3h revealed that microenvironment-sensitive molecular rotor-like behavior, rather than binding affinity to the target, is responsible for their selective turn-on fluorescence detection of tau fibrils. Selective fluorescent labeling of tau fibrils by 3g and 3h was further demonstrated by immunofluorescence staining of human Alzheimer's disease brain sections, which showed colocalization of the probes (3g and 3h) and phosphorylated tau antibody.

Protease-activated ratiometric fluorescent probe for pH mapping of malignant tumors.

A protease-activated ratiometric fluorescent probe based on fluorescence resonance energy transfer between a pH-sensitive fluorescent dye and biocompatible Fe3O4 nanocrystals was constructed. A peptide substrate of MMP-9 served as a linker between the particle quencher and the chromophore that was covalently attached to the antitumor antibody. The optical response of the probe to activated MMP-9 and gastric cell line SGC7901 tumor cells was investigated, followed by in vivo tumor imaging. Based on the ratiometric pH response to the tumor microenvironment, the resulting probe was successfully used to image the pH of subcutaneous tumor xenografts.