Live-cell Fluorescence Imaging Research Articles

Synthesis, Photophysical Properties, and Living Cell Imaging of Theranostic Half-Sandwich Iridium–4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) Dyads

We report the synthesis, characterization, and photophysical properties of two new cyclometalated half-sandwich iridium(III) complexes having the general formula [(η5-Cp*)Ir(ppy)Z]PF6 where η5-Cp* = pentamethylcyclopentadienyl and ppy = 2-phenyl-pyridine as C∧N-chelating ligand and Z = 3- or 4-pyridyl-BODIPY (BODIPY = 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dye containing a 3- or 4-pyridyl group at the meso position). The molecular structure of both complexes has been determined by X-ray crystallography. The photophysical properties of the dyads were investigated and compared to the pyridyl-BODIPY precursors. Antiproliferative studies demonstrated that one of the compounds was highly active with submicromolar IC50 on a panel of cancer cell lines. The replacement of the chlorido ligand by the pyridyl-BODIPY increased the lipophilicity of the complexes and slowed down the hydrolysis rate, which in turn increased the cytotoxicity of the metallodrug candidate. For the first time, cell uptake of one of the dyads was monitored by living cell fluorescence imaging. Interestingly, extremely fast internalization was observed the rate of which was temperature-dependent.

\u2018Gardos Channelopathy\u2019: a variant of hereditary Stomatocytosis with complex molecular regulation

The Gardos channel is a Ca2+ sensitive, K+ selective channel present in several tissues including RBCs, where it is involved in cell volume regulation. Recently, mutations at two different aminoacid residues in KCNN4 have been reported in patients with hereditary xerocytosis. We identified by whole exome sequencing a new family with two members affected by chronic hemolytic anemia carrying mutation R352H in the KCNN4 gene. No additional mutations in genes encoding for RBCs cytoskeletal, membrane or channel proteins were detected. We performed functional studies on patients’ RBCs to evaluate the effects of R352H mutation on the cellular properties and eventually on the clinical phenotype. Gardos channel hyperactivation was demonstrated in circulating erythrocytes and erythroblasts differentiated ex-vivo from peripheral CD34+ cells. Pathological alterations in the function of multiple ion transport systems were observed, suggesting the presence of compensatory effects ultimately preventing cellular dehydration in patient’s RBCs; moreover, flow cytometry and confocal fluorescence live-cell imaging showed Ca2+ overload in the RBCs of both patients and hypersensitivity of Ca2+ uptake by RBCs to swelling. Altogether these findings suggest that the ‘Gardos channelopathy’ is a complex pathology, to some extent different from the common hereditary xerocytosis.

Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons

Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP. Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies.

Deconvolution of Voltage Sensor Time Series and Electro-Diffusion Modeling of Synaptic Input in Dendritic Spines

Inward current flowing inside the neuronal post-synaptic terminal modulates the membrane voltage potential. Most of synaptic excitatory connections are made on dendritic spines, characterized by a large diversity in their geometry. How the voltage in a spine is modulated by its geometry remains unclear due in part to the absence of direct measurements. To investigate the voltage-current relation and determine the electrical properties, we combine live cell fluorescent imaging data with electro-diffusion model. We first deconvolve the genetically encoded voltage sensor expressed in hippocampal neurons, then use the electrodiffusion theory to compute the electric field and the current-voltage conversion. We further extract the effective spine neck resistance and investigate numerically using the electro-diffusion model in non-cylindrical geometry the effect of changing the geometry. We conclude that the postsynaptic current cannot only be modulated by changing the number of receptors, but also by changing the spine geometry, that could be a key step in synaptic transduction and plasticity.

PEGylated Anionic Magnetofluorescent Nanoassemblies: Impact of Their Interface Structure on Magnetic Resonance Imaging Contrast and Cellular Uptake.

Controlling the interactions of functional nanostructures with water and biological media represents high challenges in the field of bioimaging applications. Large contrast at low doses, high colloidal stability in physiological conditions, the absence of cell cytotoxicity, and efficient cell internalization represent strong additional needs. To achieve such requirements, we report on high-payload magnetofluorescent architectures made of a shell of superparamagnetic iron oxide nanoparticles tightly anchored around fluorescent organic nanoparticles. Their external coating is simply modulated using anionic polyelectrolytes in a final step to provide efficient magnetic resonance imaging (MRI) and fluorescence imaging of live cells. Various structures of PEGylated polyelectrolytes have been synthesized and investigated, differing from their iron oxide complexing units (carboxylic vs phosphonic acid), their structure (block- or comblike), their hydrophobicity, and their fabrication process [conventional or reversible addition-fragmentation chain transfer (RAFT)-controlled radical polymerization] while keeping the central magnetofluorescent platforms the same. Combined photophysical, magnetic, NMRD, and structural investigations proved the superiority of RAFT polymer coatings containing carboxylate units and a hydrophobic tail to impart the magnetic nanoassemblies (NAs) with enhanced-MRI negative contrast, characterized by a high r2/r1 ratio and a transverse relaxation r2 equal to 21 and 125 s-1 mmol-1 L, respectively, at 60 MHz clinical frequency (∼1.5 T). Thanks to their dual modality, cell internalization of the NAs in mesothelioma cancer cells could be evidenced by both confocal fluorescence microscopy and magnetophoresis. A 72 h follow-up showed efficient uptake after 24 h with no notable cell mortality. These studies again pointed out the distinct behavior of RAFT polyelectrolyte-coated bimodal NAs that internalize at a slower rate with no adverse cytotoxicity. Extension to multicellular tumor cell spheroids that mimic solid tumors revealed the successful internalization of the NAs in the periphery cells, which provides efficient deep-imaging labels thanks to their induced T2* contrast, large emission Stokes shift, and bright dotlike signal, popping out of the strong spheroid autofluorescence.

Oxytocin is involved in cellular signaling within the retina

BackgroundOxytocin (OXT) is a neuropeptide that activates the oxytocin receptor (OXTR), a rhodopsin family G‐protein coupled receptor. We have recently localized OXTR to the retinal pigment epithelium (RPE) and OXT to the adjacent cone photoreceptors.ObjectiveWe sought to characterize the signaling mechanism of OXTR in the RPE and explore the downstream effects of OXT, focusing on the regulation of inwardly rectifying K+ channel Kir7.1.MethodsCa2+ response to OXT was measured in cultured human fetal RPE cells (hfRPE) using Fura‐2 AM in the presence of 2‐APB and nifedipine, pharmacological inhibitors of Ca2+ signaling pathways. HEK‐293 cells where used for the stable expression of human OXTR. Cellular signaling was visualized using live cell fluorescence imaging following transient expression of PH‐GFP and PKC‐GFP, monitors of GPCR metabolites PIP2 and DAG. Whole cell patch clamp electrophysiology was performed on HEK‐OXTR cells transfected with GFP‐fused Kir7.1 as well as freshly isolated mouse RPE cells to study Kir7.1 channel function.ResultsOXT treatment of RPE cells in culture resulted in a transient increase in cytoplasmic Ca2+ that was reduced by 95% in the presence of the IP3R antagonist, 2‐APB (P < 0.0001). Upon bathing the cells in Ca2+‐free extracellular solution or nifedipine, the Ca2+ response to OXT was not inhibited, although amplitude was reduced. Time to the rise in [Ca2+]i was not altered but recovery was slowed down with time constants (τ) of 0.35 ± 0.02 (r2 =.99), 0.34 ± 0.03 (r2 =.98), and 0.20 ± 0.005 (r2=.99) min for Ca2+‐free, nifedipine, and Ringer's solution, respectively. We also demonstrate that OXTR activation blunted Kir7.1 channel current. In isolated mouse RPE, we observed an average 61.81 ± 4.77 % decrease in K+ inward current amplitude at ‐160mV and an average 11.4 ± 3.2 mV depolarization in resting membrane potential by OXT.ConclusionWe propose that OXTR stimulates a mobilization of intracellular Ca2+, independent of extracellular Ca2+, through 2nd messengers coupled to OXTR/G‐protein in the RPE. This OXT‐OXTR signaling in the RPE cell also integrated the parallel modulation of the Kir7.1 channel. Kir7.1 channel is responsible for controlling RPE resting membrane potential and can influence retinal health and vision. We suggest that novel OXT‐OXTR signaling pathways in the outer retina will be of fundamental importance for eye development, health and visual function.Support or Funding InformationNIH EY024995, UnityPoint Health Meriter Foundation, Pediatrics, Graduate School, School of Medicine and Public Health

Effect of probe diffusion on the SOFI imaging accuracy

Live-cell super-resolution fluorescence imaging is becoming commonplace for exploring biological systems, though sample dynamics can affect the imaging quality. In this work we evaluate the effect of probe diffusion on super-resolution optical fluctuation imaging (SOFI), using a theoretical model and numerical simulations based on the imaging of live cells labelled with photochromic fluorescent proteins. We find that, over a range of physiological conditions, fluorophore diffusion results in a change in the amplitude of the SOFI signal. The magnitude of this change is approximately proportional to the on-time ratio of the fluorophores. However, for photochromic fluorescent proteins this effect is unlikely to present a significant distortion in practical experiments in biological systems. Due to this lack of distortions, probe diffusion strongly enhances the SOFI imaging by avoiding spatial undersampling caused by the limited labeling density.

Engineering of mCherry variants with long Stokes shift, red-shifted fluorescence, and low cytotoxicity.

MCherry, the Discosoma sp. mushroom coral-derived monomeric red fluorescent protein (RFP), is a commonly used genetically encoded fluorophore for live cell fluorescence imaging. We have used a combination of protein design and directed evolution to develop mCherry variants with low cytotoxicity to Escherichia coli and altered excitation and emission profiles. These efforts ultimately led to a long Stokes shift (LSS)-mCherry variant (λex = 460 nm and λem = 610 nm) and a red-shifted (RDS)-mCherry variant (λex = 600 nm and λem = 630 nm). These new RFPs provide insight into the influence of the chromophore environment on mCherry’s fluorescence properties, and may serve as templates for the future development of fluorescent probes for live cell imaging.

The Mechanisms and Biomedical Applications of an NIR BODIPY-Based Switchable Fluorescent Probe

Highly environment-sensitive fluorophores have been desired for many biomedical applications. Because of the noninvasive operation, high sensitivity, and high specificity to the microenvironment change, they can be used as excellent probes for fluorescence sensing/imaging, cell tracking/imaging, molecular imaging for cancer, and so on (i.e., polarity, viscosity, temperature, or pH measurement). In this work, investigations of the switching mechanism of a recently reported near-infrared environment-sensitive fluorophore, ADP(CA)2, were conducted. Besides, multiple potential biomedical applications of this switchable fluorescent probe have been demonstrated, including wash-free live-cell fluorescence imaging, in vivo tissue fluorescence imaging, temperature sensing, and ultrasound-switchable fluorescence (USF) imaging. The fluorescence of the ADP(CA)2 is extremely sensitive to the microenvironment, especially polarity and viscosity. Our investigations showed that the fluorescence of ADP(CA)2 can be switched on by low polarity, high viscosity, or the presence of protein and surfactants. In wash-free live-cell imaging, the fluorescence of ADP(CA)2 inside cells was found much brighter than the dye-containing medium and was retained for at least two days. In all of the fluorescence imaging applications conducted in this study, high target-to-noise (>5-fold) was achieved. In addition, a high temperature sensitivity (73-fold per Celsius degree) of ADP(CA)2-based temperature probes was found in temperature sensing.

Exploring the Mechanism of Microtubule Depolymerization by the Kinesin 13 KLP10A and it's Phosphoregulation

Kinesins-13s are non-motile molecular motors that regulate microtubule (MT) dynamics by promoting MT depolymerization activity at the MT tips. We have used cryo-electron microscopy and functional assays to investigate the mechanism of action and regulation of the Drosophila melanogaster kinesin-13 KLP10A. Previous work has shown that phosphorylation of serine 573 in the KLP10A motor domain down regulates depolymerization activity. We have obtained cryo-EM sub-nanometer resolution maps of constructs that include the KLP10A motor and N-terminal neck domains bound to MTs and to tubulin depolymerization intermediates. Comparison of these structures revealed key features of the depolymerization process. To study the down-regulation mechanism by phosphorylation we have combined a variety of cell biology and biophysical methods and compared the behavior of phosphomutant (S573E) and wild-type (WT) KLP10A. Surprisingly, we found that the binding affinity and ATPase activity stimulated by curved tubulin polymers (mimicking MT ends) of WT-KLP10A and S573E-KLP10A were relatively similar (kcat of 1.8 s−1 and 2.5 s−1 respectively). In contrast, we found significant differences in their MT-lattice stimulated ATPases (kcat of 0.56 s−1 and 0.14 s−1 respectively). We also found that the mutant S573E had shorter residence time relative to WT (τ of 5 s and 28 s respectively) while undergoing one-dimensional diffusion over the MT lattice. These results suggest a model in which phosphorylation of S573 reduce KLP10 MT depolymerization activity by decreasing the probability of reaching the MT ends by one dimensional diffusion. Consistent with this model we found by fluorescent live cell imaging that KLP10A-S573E MRFP labeled constructs accumulate significantly less at the tip of growing MTs than WT-KLP10A MRFP labeled constructs.

ADNP/NAP dramatically increase microtubule end-binding protein-Tau interaction: a novel avenue for protection against tauopathy.

Activity-dependent neuroprotective protein (ADNP), vital for brain formation and cognitive function, is mutated in autism and linked to neurodegenerative/psychiatric diseases. An eight-amino-acid peptide snippet of ADNP, NAP (NAPVSIPQ), identified as a smallest active fragment, includes the SxIP microtubule (MT) end-binding protein (EB) association motif, and enhances ADNP-EB3 interaction. Depletion of EB1 or EB3 abolishes NAP protection against zinc intoxication. Furthermore, NAP enhances Tau-MT interaction, and Tau regulates the localization and function of EB1 and EB3 in developing neuronal cells. Here, we asked how NAP (ADNP) enhances Tau-MT interactions and whether this is mediated by EBs. We showed, for we believe the first time, that NAP augmented endogenous EB1 comet density in the N1E-115 neuroblastoma neuronal model. This finding was substantiated by cell transfection with fluorescent EB1 and live cell imaging. NAP increased comet amounts, length and speed. At the molecular level, NAP enhanced EB3 homodimer formation, while decreasing EB1-EB3 heterodimer content and driving EB1- and EB3-Tau interactions (dramatic 20-fold increases), leading to recruitment of EB1/EB3 and Tau to MTs under zinc intoxication. Our previous results showed that while NAP protected neuronal-like cells against oxidative stress, it did not protect NIH3T3 fibroblasts. Here, NAP did not protect NIH3T3 cells against zinc intoxication, unless these cells were transfected with Tau. Interestingly, other MT associated proteins (MAPs) may replace Tau, thus, EB-Tau (MAPs) interaction is identified as a novel target for endogenous ADNP neuroprotection, and a future target for drug development, with NAP as a prototype.

Quantification of growth factor signaling and pathway cross talk by live-cell imaging.

Peptide growth factors stimulate cellular responses through activation of their transmembrane receptors. Multiple intracellular signaling cascades are engaged following growth factor-receptor binding, leading to short- and long-term biological effects. Each receptor-activated signaling pathway does not act in isolation but rather interacts at different levels with other pathways to shape signaling networks that are distinctive for each growth factor. To gain insights into the specifics of growth factor-regulated interactions among different signaling cascades, we developed a HeLa cell line stably expressing fluorescent live-cell imaging reporters that are readouts for two major growth factor-stimulated pathways, Ras-Raf-Mek-ERK and phosphatidylinositol (PI) 3-kinase-Akt. Incubation of cells with epidermal growth factor (EGF) resulted in rapid, robust, and sustained ERK signaling but shorter-term activation of Akt. In contrast, hepatocyte growth factor induced sustained Akt signaling but weak and short-lived ERK activity, and insulin-like growth factor-I stimulated strong long-term Akt responses but negligible ERK signaling. To address potential interactions between signaling pathways, we employed specific small-molecule inhibitors. In cells incubated with EGF or platelet-derived growth factor-AA, Raf activation and the subsequent stimulation of ERK reduced Akt signaling, whereas Mek inhibition, which blocked ERK activation, enhanced Akt and turned transient effects into sustained responses. Our results reveal that individual growth factors initiate signaling cascades that vary markedly in strength and duration and demonstrate in living cells the dramatic effects of cross talk from Raf and Mek to PI 3-kinase and Akt. Our data further indicate how specific growth factors can encode distinct cellular behaviors by promoting complex interactions among signaling pathways.

A novel acidic pH fluorescent probe based on a benzothiazole derivative

A novel acidic pH fluorescent probe 1 based on a benzothiazole derivative has been designed, synthesized and developed. The linear response range covers the acidic pH range from 3.44 to 6.46, which is valuable for pH researches in acidic environment. The evaluated pKa value of the probe 1 is 4.23. The fluorescence enhancement of the studied probe 1 with an increase in hydrogen ions concentration is based on the hindering of enhanced photo-induced electron transfer (PET) process. Moreover, the pH sensor possesses a highly selective response to H+ in the presence of metal ions, anions and other bioactive small molecules which would be interfere with its fluorescent pH response. Furthermore, the probe 1 responds to acidic pH with short response time that was less than 1min. The probe 1 has been successfully applied to confocal fluorescence imaging in live HeLa cells and can selectively stain lysosomes. All of such good properties prove it can be used to monitoring pH fluctuations in acidic environment with high sensitivity, pH dependence and short response time.

Functional Viability: Measurement of Synaptic Vesicle Pool Sizes.

Neurons and their function of conveying information across a chemical synapse are highly regulated systems. Impacts on their functional viability can occur independently from changes in morphology. Here we describe a method to assess the size of synaptic vesicle pools using live cell fluorescence imaging and a genetically encoded probe (pHluorin). Assessing functional parameters such as the size of synaptic vesicle pools can be a valuable addition to common assays of neuronal cell viability as they demonstrate that key cellular functions are intact.

Reversible Cryo-arrests of Living Cells to Pause Molecular Movements for High-resolution Imaging.

Fluorescence live-cell imaging by single molecule localization microscopy (SMLM) or fluorescence lifetime imaging microscopy (FLIM) in principle allows for the spatio-temporal observation of molecular patterns in individual, living cells. However, the dynamics of molecules within cells hamper their precise observation. We present here a detailed protocol for consecutive cycles of reversible cryo-arrest of living cells on a microscope that allows for a precise determination of the evolution of molecular patterns within individual living cells. The usefulness of this approach has been demonstrated by observing ligand-induced clustering of receptor tyrosine kinases as well as their activity patterns by SMLM and FLIM (Masip et al., 2016).

A Schiff's base receptor for red fluorescence live cell imaging of Zn2+ ions in zebrafish embryos and naked eye detection of Ni2+ ions for bio-analytical applications.

An interesting dual chemoreceptor (QAMP) was synthesized to quantify the presence of two environmentally as well as biologically important Zn2+/Ni2+ ions in a highly selective manner using different analytical techniques. The fluorescence profile was enhanced to 663 nm (red region) due to the formation of its 1 : 1 complex with Zn2+ at room temperature even in the presence of other interfering ions such as Cd2+ and Hg2+. Moreover, it exhibits excellent chromo-isomerism with Ni2+ ions. Meanwhile, this assay could be successfully oriented with molecular logic functions of AND, OR, NOR and NOT logic gates. The obtained large Stokes shift (∼274 nm) value and red emission promoted this receptor as a potential tool for biological studies (e.g. live cell imaging in cancer cell line and zebrafish models).

Multi-Parametric Imaging of Hypoxia and Cell Cycle in Intestinal Organoid Culture.

Dynamics of oxygenation of tissue and stem cell niches are important for understanding physiological function of the intestine in normal and diseased states. Only a few techniques allow live visualization of tissue hypoxia at cellular level and in three dimensions. We describe an optimized protocol, which uses cell-penetrating O2-sensitive probe, Pt-Glc and phosphorescence lifetime imaging microscopy (PLIM), to analyze O2 distribution in mouse intestinal organoids. Unlike the other indirect and end-point hypoxia stains, or point measurements with microelectrodes, this method provides high-resolution real-time visualization of O2 in organoids. Multiplexing with conventional fluorescent live cell imaging probes such as the Hoechst 33342-based FLIM assay of cell proliferation, and immunofluorescence staining of endogenous proteins, allows analysis of key physiologic parameters under O2 control in organoids. The protocol is useful for gastroenterology and physiology of intestinal tissue, hypoxia research, regenerative medicine, studying host-microbiota interactions and bioenergetics.

Real-time and quantitative fluorescent live-cell imaging with quadruplex-specific red-edge probe (G4-REP)

The development of quadruplex-directed molecular diagnostic and therapy rely on mechanistic insights gained at both cellular and tissue levels by fluorescence imaging. This technique is based on fluorescent reporters that label cellular DNA and RNA quadruplexes to spatiotemporally address their complex cell biology. The photophysical characteristics of quadruplex probes usually dictate the modality of cell imaging by governing the selection of the light source (lamp, LED, laser), the optical light filters and the detection modality. Here, we report the characterizations of prototype from a new generation of quadruplex dye termed G4-REP (for quadruplex-specific red-edge probe) that provides fluorescence responses regardless of the excitation wavelength and modality (owing to the versatility gained through the red-edge effect), thus allowing for diverse applications and most imaging facilities. This is demonstrated by cell images (and associated quantifications) collected through confocal and multiphoton microscopy as well as through real-time live-cell imaging system over extended period, monitoring both non-cancerous and cancerous human cell lines. Our results promote a new way of designing versatile, efficient and convenient quadruplex-reporting dyes for tracking these higher-order nucleic acid structures in living human cells. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Local pulsatile contractions are an intrinsic property of the myosin 2A motor in the cortical cytoskeleton of adherent cells.

The role of nonmuscle myosin 2 (NM2) pulsatile dynamics in generating contractile forces required for developmental morphogenesis has been characterized, but whether these pulsatile contractions are an intrinsic property of all actomyosin networks is not known. Here we used live-cell fluorescence imaging to show that transient, local assembly of NM2A "pulses" occurs in the cortical cytoskeleton of single adherent cells of mesenchymal, epithelial, and sarcoma origin, independent of developmental signaling cues and cell-cell or cell-ECM interactions. We show that pulses in the cortical cytoskeleton require Rho-associated kinase- or myosin light chain kinase (MLCK) activity, increases in cytosolic calcium, and NM2 ATPase activity. Surprisingly, we find that cortical cytoskeleton pulses specifically require the head domain of NM2A, as they do not occur with either NM2B or a 2B-head-2A-tail chimera. Our results thus suggest that pulsatile contractions in the cortical cytoskeleton are an intrinsic property of the NM2A motor that may mediate its role in homeostatic maintenance of tension in the cortical cytoskeleton of adherent cells.

Highly efficient gene silencing and bioimaging based on fluorescent carbon dots in vitro and in vivo

Small interfering RNA (siRNA) is an attractive therapeutic candidate for sequence-specific gene silencing to treat incurable diseases using small molecule drugs. However, its efficient intracellular delivery has remained a challenge. Here, we have developed a highly biocompatible fluorescent carbon dot (CD), and demonstrate a functional siRNA delivery system that induces efficient gene knockdown in vitro and in vivo. We found that CD nanoparticles (NPs) enhance the cellular uptake of siRNA, via endocytosis in tumor cells, with low cytotoxicity and unexpected immune responses. Real-time study of fluorescence imaging in live cells shows that CD NPs favorably localize in cytoplasm and successfully release siRNA within 12 h. Moreover, we demonstrate that CD NP-mediated siRNA delivery significantly silences green fluorescence protein (GFP) expression and inhibits tumor growth in a breast cancer cell xenograft mouse model of tumor-specific therapy. We have developed a multifunctional siRNA delivery vehicle enabling simultaneous bioimaging and efficient downregulation of gene expression, that shows excellent potential for gene therapy.