Intracellular Fluorescence Imaging Research Articles

Endogenous Stimuli-Responsive Nucleus-Targeted Nanocarrier for Intracellular mRNA Imaging and Drug Delivery.

Drug resistance arising from overexpressed efflux transporters increases the efflux of drugs and accordingly restricts the efficacy of chemotherapy. Advances in nanocarriers have provided potential strategies to cope with drug resistance. Herein, endogenous stimuli-responsive nucleus-targeted nanocarrier is developed for intracellular multidrug resistance protein 1 (MRP1) mRNA imaging and drug delivery. This nanocarrier (AuNP-mRS-DSs) is composed of three parts: (i) gold nanoparticle (AuNP), for loading DNA and quenching fluorescence; (ii) mRNA recognition sequence (mRS) modified on the surface of gold nanoparticle by gold-thiol bond, for the specific recognition of MRP1 mRNA; (iii) detachable subunit (DS), hybridized with Cy5-labeled DNA linker and nucleolin recognition motif and grafted onto mRS via the DNA linker for loading doxorubicin (Dox), binding to nucleolin, and reporting signal. First, nucleolin recognition motif of this nanocarrier targets nucleolin, which is overexpressed on cancer cells surface; subsequently, the whole nanocarrier enters the cell via nucleolin-mediated internalization. Subsequently, mRS will specifically recognize overexpressed MRP1 mRNA, leading to the release of trapped DS and followed by AuNP-quenched Cy5 fluorescence recovery. Finally, by translocation of nucleolin from cytoplasm to nucleus, the DS targets nucleus to delivery Dox. By intracellular fluorescence imaging, the differentiation of drug-resistant and nondrug-resistant cells could be achieved. Compared with free Dox (IC50 > 8.00 μM), Dox-loaded AuNP-mRS-DSs (IC50 = 2.20 μM) performed superior suppression efficacy toward drug-resistant cancer cells. Such a nanocarrier provides an effective strategy to synergistically sense and circumvent drug resistance, which may be exploited as a candidate for personalized medicine.

Self-Nucleation and Self-Assembly of Highly Fluorescent Au5 Nanoclusters for Bioimaging

Fluorescent Au nanoclusters (NCs) are new excellent nanomaterials for biomedical applications but plagued by the problems of limited emission efficiency, unclear emission mechanism, and poor biological application ability. Herein, a novel strategy was developed to facilely synthesize poly(amidoamine) (PAMAM) dendrimer-hosted Au5 NCs (poly-Au5) with a high fluorescence quantum yield of 25%. Most importantly, a two-stage growth process of poly-Au5 was demonstrated through in situ time-course experiments. Stage I was a simultaneous self-nucleation and self-assembly with a rapid rate of fluorescence increase; stage II was a sole self-assembly after the end of reduction with a relatively slower rate of fluorescence increase but contributed 30% to the overall emission intensity of end products. In both stages, enhanced aurophilic interactions promoted the excited state relaxation dynamics; enhanced rigid structures reduced the level of nonradiative relaxition of excited states, and these two factors ensured high emission efficiency of poly-Au5. To further evidence the inference above, we successfully used PAMAM to realize the self-assembly of presynthesized, separated, and red-emitting Au-GSH NCs through electrostatic interaction between negative charges of carboxylic groups in Au-GSH NCs and positive charges of amine groups in PAMAM. As expected, the emission efficiency of Au-GSH NCs was obviously enhanced by PAMAM-mediated self-assembly. Moreover, the as-synthesized poly-Au5 assemblies exhibited excellent cell permeability and great biostability against various metal ions, high REDOX stress, and complex intracellular environments. By virtue of MnO4– as an intermediary agent, poly-Au5 was successfully used for sensitive and stable intracellular fluorescent imaging of endogenous GSH. This study lights up the emission origin of dendrimer-hosted Au NCs with strong emission and implies their huge applications in biomedical sensing and imaging.

Fabrication of Positively Charged Fluorescent Polymer Nanoparticles for Cell Imaging and Gene Delivery.

Development of efficient non-viral gene delivery vector has aroused great attention in the past few decades. In this study, we reported a new gene delivery vector, positively charged fluorescent conjugated polymer nanoparticles (CPNPs), for efficient gene transfection and in-situ intracellular fluorescence imaging. The microscopic and spectroscopic characterizations demonstrated that these CPNPs possess decent fluorescence performance (e.g. with fluorescence quantum yield of 70.7±0.3%) and small size dimension of ~3.6±0.3 nm (DLS result). Fast and efficient cellular translocation capability was observed according to the time-dependent living cell imaging experiments. Nearly all of the cells were loaded with CPNPs after co-incubation for 2 h regardless of the cell type. In comparison with the commonly used gene delivery vector, lipofectamine 2000 (with gene transfection efficiency of 55±5% for pEGFP), the gene expression efficiency with the positively charged CPNPs (70±3% for pEGFP) was improved significantly. Intracellular fluorescence imaging results demonstrated that the CPNPs could actively assemble close to the periphery of nuclei. Disassembly was not observed even 36 h later, which greatly facilitates releasing of pDNA close to the periphery of nuclei and thus promotes the gene transfection efficiency.

Two-photon fluorescent probe for lysosome-targetable hypochlorous acid detection within living cells

The design of robust tools for hypochlorous acid (HClO) detection at subcellular level is highly desired for the better understanding of its biological function in cellular signaling pathways. Herein, we report the development of a two-photon fluorescent probe (TPFP) for lysosome-targetable HClO detection through the integration of a lysosome-targetable group (aminoethyl)morpholine, two-photon fluorophore 1,8-naphthalimide and HClO capturing phenyl-thiourea together. In the presence of HClO, the thiourea moiety is oxidized to urea and thereby restores the fluorescence of 1,8-naphthalimide(at 538nm) due to the inhibition of photo-induced electron transfer (PET) effect. As is demonstrated in the spectroscopic characterizations, the probe TPFP exhibits excellent probing performance toward HClO, including short response time (<30s), outstanding selectivity, high photostability and low detection limit (∼5.7nM). In addition, it is confirmed that TPFP displays promising cell-membrane permeability and high specificity toward HClO in lysosomes through the one-photon and two-photon intracellular fluorescent microscopic imaging experiments. As a consequence, this lysosome-targetable two-photon fluorescent probe provides a promising platform for HClO detection at subcellular level and would inspire the design of plummy tools for other analytes detection.

A ratiometric two-photon fluorescent probe for detecting carboxylesterase in living cells

Abnormal regulation or deficiency of carboxylesterase (CaE) is directly linked to human diseases such as hepatic steatosis, hyperlipidemia, and cancer. To understand the physiological and pharmacological roles of CaE, it is crucial to develop a simple and rapid method for CaE detection in biological samples. In this work, we report a red-light two-photon fluorescent probe for highly selective and ratiometric detection of CaE based on intramolecular charge transfer. CaE-mediated hydrolysis can produce the deacetylated derivative of the probe, and the fluorescence spectra of the sensing system were significantly changed due to enzymatic reaction, thus realizing the ratiometric detection of CaE. This ratiometric probe system showed several advantages including better selectivity, lower detection limit and a wide pH range. Moreover, intracellular two-photon fluorescence imaging experiments demonstrated that the probe DCM-CE was able to facilitate visualization of endogenous CaE in living cells.

Versatile synthesis of end-reactive polyrotaxanes applicable to fabrication of supramolecular biomaterials.

Cyclodextrin (CD)-threaded polyrotaxanes (PRXs) with reactive functional groups at the terminals of the axle polymers are attractive candidates for the design of supramolecular materials. Herein, we describe a novel and simple synthetic method for end-reactive PRXs using bis(2-amino-3-phenylpropyl) poly(ethylene glycol) (PEG-Ph-NH2) as an axle polymer and commercially available 4-substituted benzoic acids as capping reagents. The terminal 2-amino-3-phenylpropyl groups of PEG-Ph-NH2 block the dethreading of the α-CDs after capping with 4-substituted benzoic acids. By this method, two series of azide group-terminated polyrotaxanes (benzylazide: PRX-Bn-N3, phenylazide: PRX-Ph-N3,) were synthesized for functionalization via click reactions. The PRX-Bn-N3 and PRX-Ph-N3 reacted quickly and efficiently with p-(tert-butyl)phenylacetylene via copper-catalyzed click reactions. Additionally, the terminal azide groups of the PRX-Bn-N3 could be modified with dibenzylcyclooctyne (DBCO)-conjugated fluorescent molecules via a copper-free click reaction; this fluorescently labeled PRX was utilized for intracellular fluorescence imaging. The method of synthesizing end-reactive PRXs described herein is simple and versatile for the design of diverse functional PRXs and can be applied to the fabrication of PRX-based supramolecular biomaterials.

A novel ratiometric fluorescent probe based on 1, 8-naphthalimide for the detection of Ho3 + and its bioimaging

A ratiometric fluorescent probe for the detection of Ho3+ in DMSO-aqueous medium was designed and synthesized based on 1, 8-naphthalimide. The probe displayed response to Ho3+ with a fluorescence decrease at 512nm and enhancement at 480nm, accompanying with a distinct fluorescence change from bright yellow-green to cyan. Besides, the probe exhibited a lower detection limit (6×10−8M) and could be used in intracellular fluorescence imaging. To the best of the knowledge, it was the first ratiometric fluorescent probe for Ho3+ detection. This probe was expected to be a useful tool for further elucidating the roles of Ho3+ in materials, biology and environment.

A theranostic prodrug based on FRET for real-time drug release monitoring in response to biothiols

The clinical applications of tradition prodrug are restricted to a certain extent due to its some defects such as side effects, low selectivity and single function. Herein, we designed a theranostic strategy based on the fluorescence resonance energy transfer (FRET) mechanism. The theranostic prodrug was constructed with an anticancer drug camptothecin (CPT), a cleavable linker based on disulfide bonds (SS) and a fluorophore naphthalimide derivative (NAP). For this drug delivery and reporting system (NAP-SS-CPT), FRET occurs between CPT (energy donor) and NAP (energy receptor); and upon cellular uptake by GSH-overexpressing cancer cells, disulfide bonds could be successfully cleaved by the high concentration of GSH, and FRET process was interrupted to achieve dual fluorescence response and specifically release of CPT. The drug delivery system features some favorable properties, like excellent pH-stability, high selectivity and cytotoxicity towards GSH-overexpressing cells and relatively lower cytotoxicity towards normal cells. Fluorescence analysis reveals that NAP-SS-CPT shows a ratiometric fluorescence signal under excitation at 400nm for quantitative detection of GSH. Intracellular fluorescence imaging studies indicate that the prodrug can be efficiently internalized in HeLa cells and used for real-time monitoring of drug release. Moreover, MTT and flow cytometry assay indicate that NAP-SS-CPT exhibits high pro-apoptotic effect for cancer cells. This strategy may provide a new approach for cancer diagnosis and therapy.

Polymer nanoparticle-based ratiometric fluorescent probe for imaging Hg2+ ions in living cells

Mercury pollutant brings huge threat to human health and the environment because of its bioaccumulation and permanent damage. Thus, highly selective and ratiometric detection and imaging of Hg2+ in organisms is quite important. Herein, we report on a polymer nanoparticle-based ratiometric fluorescent probe (NP3) for highly selective recognition of Hg2+ in totally aqueous solution and imaging in living cells. This probe comprises the reference fluorescent dye (4-ethoxy-9-allyl-1,8-naphthalimide: EANI), which resides in the core of nanoparticle, and the Hg2+-recognition group (fluorescein derivative: AEMH-FITC) on the surface of nanoparticle. It displays distinct dual emissions at 432nm and 528nm under a single-wavelength excitation. The fluorescence of the FITC moieties can be effectively quenched by Hg2+ due to photoinduced electron transfer (PET) mechanism, while that of the EANI remains constant, resulting in a ratiometric fluorescent detection of Hg2+. The as-prepared NP3 not only shows favorable water dispersibility, excellent long-term photostability, but also exhibits highly selective ratiometric detection of Hg2+ over other interfering ions (such as Cu2+, Na+, Mg2+, Zn2+, Co2+, Mn2+, K+, Ca2+, Fe3+, Cd2+, Ag+, Fe2+), and quite low detection limit (∼75nM). Furthermore, intracellular fluorescence imaging experiments demonstrate that NP3 has good permeability of cell-membrane, and is able to facilitate visualization of Hg2+ changes in living cells.

Fluorescent amphiphilic copolymer-based tumor theranostics for facile DOX-loading and tumor microenvironment-triggered release

Novel reduction/pH dual-stimuli responsive fluorescent amphiphilic copolymer PEG-Cys-P(DEAEMA-co-Rh6GEAm) with detachable poly(ethylene glycol) (PEG) corona through bio-reducible disulfide linkage and pendent cationic groups was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization for tumor theranostic application. The doxorubicin (DOX)-loaded micelles could be easily fabricated via facile micellization in the presence of DOX in water, with a high drug-loading capacity (DLC) of 39.2%. They exhibited fast release ratio with the synergetic effect of reductive agent and pH in the simulated tumor micro-environment, while the leakage can be efficiently avoided in the physiological media. Furthermore, the fluorescence intensity of the copolymer possessed a reversibly transformation in different pH media, indicating the potential application for intracellular fluorescence imaging of tumors.

Intracellular uptake and fluorescence imaging potential in tumor cell of zinc phthalocyanine

A near IR absorbing phthalocyanine bearing four binaphtyl group has been synthesized in order to investigate its cytotoxicity and intracellular uptake of sensitizer on MCF-7 (human breast cancer), MDAH (ovarian cancer), HeLa (human epitheloid cervix carcinoma), EMT-6 (mouse breast cancer) and WI-38 (human fibroblast lung) cell lines. ZnPc showed four time higher intracellular uptake in carcinoma cells (MCF-7) than normal (WI-38) cell lines. With the aim of studying in detail the biodistribution feature and tumor nuclear imaging capacity, ZnPc was also labeled with I-131. The efficiency of radiolabeled compound was 95±4.6%. In addition, ZnPc reveals to be very efficient singlet oxygen generators (ΦΔ=0.612 in DMSO) and promising PS for PDT application. In vitro fluorescence imaging study with MCF-7 cells showed that ZnPc localized in cytoplasm of the cells. This results showed that synthesized ZnPc is promising candidate for dual fluorescence/nuclear imaging breast cancer and shows potential PS for PDT application.

Water-Soluble 8-Hydroxyquinoline Conjugate of Amino-Glucose As Receptor for La(3+) in HEPES Buffer, on Whatman Cellulose Paper and in Living Cells.

A water-soluble glucopyranosyl conjugate, L, has been synthesized and characterized by different analytical and spectral techniques. The L has been demonstrated to have switch-on fluorescence enhancement of ∼75 fold in the presence of La(3+) among the nine lanthanide ions studied in the HEPES buffer at pH 7.4. A minimum detection limit of 140 nM (16 ± 2 ppb) was shown by L for La(3+) in the buffer at physiological pH. The utility of L has been demonstrated by showing its sensitivity toward La(3+) on Whatman filter paper strips. The reversible and reusable action of L has been demonstrated by monitoring the fluorescence changes as a function of the addition of La(3+) followed by F(-) and HPO4(2-) ions. The complexation of L by La(3+) was shown by absorption spectra wherein isosbestic behavior was observed. The Job's plot suggests a 2:1 complex between L and La(3+), and the same was supported by ESI-MS. The control molecular study revealed the necessity of hydroxy quinoline and the amine group for La(3+) ion binding and the glyco-moiety to bring water solubility and biocompatibility. The structural features of the [2L+La(3+)] complex were established by DFT computational calculations. The chemo-ensemble, [2L+La(3+)], is shown responsible for providing intracellular fluorescence imaging in HepG2 cells.

Development of Functional Fluorescent Molecular Probes for the Detection of Biological Substances.

This review is confined to sensors that use fluorescence to transmit biochemical information. Fluorescence is, by far, the most frequently exploited phenomenon for chemical sensors and biosensors. Parameters that define the application of such sensors include intensity, decay time, anisotropy, quenching efficiency, and luminescence energy transfer. To achieve selective (bio)molecular recognition based on these fluorescence phenomena, various fluorescent elements such as small organic molecules, enzymes, antibodies, and oligonucleotides have been designed and synthesized over the past decades. This review describes the immense variety of fluorescent probes that have been designed for the recognitions of ions, small and large molecules, and their biological applications in terms of intracellular fluorescent imaging techniques.

A novel graphene oxide-based fluorescent nanosensor for selective detection of Fe(3+) with a wide linear concentration and its application in logic gate.

A graphene oxide-based fluorescent nanosensor AGO has been designed and synthesized by covalent grafting allylamine onto GO surface. In aqueous media, AGO displays a highly selective and sensitive discrimination of Fe(3+) from Fe(2+) and other metal ions through electron transfer-induced fluorescence quenching. The quenching of AGO fluorescence is linearly proportional to Fe(3+) concentration in a wide range of 0-120 μM (correlation coefficient R(2)=0.9994). Moreover, AGO can be used to construct a combinational three-input logic gate to discriminate Fe(3+) and Fe(2+). The logic gate works well in intracellular fluorescence imaging, which shows a potential as a promising platform for biosensing analysis.

A sensitive and selective fluorescent probe for detection of glutathione in the presence of Cu2+ and its application to biological imaging

A selective and sensitive method to detect glutathione (GSH) based on the complex of a coumarin-based probe and Cu2+ is reported. In response to GSH, the system provides remarkable fluorescence intensity change with the color change from colorless to yellow, and the detect limit is determined to be 3.6×10−7M. The background amino acids show small or no interference on the detection of GSH. Moreover, this probe could be applied to detect GSH via intracellular fluorescent imaging.

Fe3O4@carbon@zeolitic imidazolate framework-8 nanoparticles as multifunctional pH-responsive drug delivery vehicles for tumor therapy in vivo.

Controlled drug release is a promising approach for cancer therapy due to its merits of reduced systemic toxicity and enhanced antitumor efficacy. Here, multifunctional Fe3O4@carbon@zeolitic imidazolate framework-8 (FCZ) hybrid nanoparticles (NPs) were successfully constructed. Owing to the porosity and acid-sensitivity of zeolitic imidazolate framework-8 (ZIF-8), FCZ NPs not only displayed an improved drug loading capacity compared to most of the polymeric nanocarriers, but also exhibited excellent pH-triggered release of doxorubicin (DOX) in vitro. Moreover, carbon dots (CDs) embedded in the porous carbon shell and superparamagnetic iron oxide nanocrystals could simultaneously function as intracellular fluorescence imaging and T2*-weighted magnetic resonance imaging (MRI) contrast agents, respectively. The results obtained from the MTT assay demonstrated good biocompatibility of FCZ NPs. DOX release experiments showed pH regulation-dominated drug release kinetics: a weak acidic pH in tumor areas could trigger sustained drug release, suggesting that FCZ NPs are ideal drug delivery systems. Moreover, the remarkable inhibition of tumor growth without side effects was confirmed in vivo. These results provide convincing evidence establishing the multifunctional FCZ NPs as promising candidates for tumor therapy.

A Cu-free clickable fluorescent probe for intracellular targeting of small biomolecules.

We synthesized a novel cyclooctyne-based clickable fluorescent probe with versatile properties such as high cell-membrane permeability and free diffusibility in the cell. Our probe "FC-DBCO" was conjugated to an azide-modified mannose via a Cu-free click reaction in living HeLa cells and displayed intracellular specific fluorescence imaging with low background signals.

Design and synthesis of functionalized rhodamine based probes for specific intracellular fluorescence imaging of Fe3+

Two functionalized rhodamine based probes have been rational designed and synthesized. The probes have sufficiently high selectivity and sensitivity for the detection of Fe3+ ion both by fluorescence and ultraviolet spectroscopy with strong ability of anti-interference performance. The detection process has been shown to be reversible and involves the fluoran ring-opening upon complexation with Fe3+. The obvious fluorescence image obtained from confocal laser scanning microscopy of the detection of Fe3+ in living L929 cells demonstrate that the probes could contribute to significant breakthroughs in understanding the critically important functions of Fe3+ in related cells and biological organs.

Live-cell imaging of biothiols via thiol/disulfide exchange to trigger the photoinduced electron transfer of gold-nanodot sensor

Biothiols have been reported to involve in intracellular redox-homeostasis against oxidative stress. In this study, a highly selective and sensitive fluorescent probe for sensing biothiols is explored by using an ultrasmall gold nanodot (AuND), the dendrimer-entrapped Au8-cluster. This strategy relies upon a thiol/disulfide exchange to trigger the fluorescence change through a photoinduced electron transfer (PET) process between the Au8-cluster (as an electron donor) and 2-pyridinethiol (2-PyT) (as an electron acceptor) for sensing biothiols. When 2-PyT is released via the cleavage of disulfide bonds by biothiols, the PET process from the Au8-cluster to 2-PyT is initiated, resulting in fluorescence quenching. The fluorescence intensity was found to decrease linearly with glutathione (GSH) concentration (0–1500μM) at physiological relevant levels and the limit of detection for GSH was 15.4μM. Compared to most nanoparticle-based fluorescent probes that are limited to detect low molecular weight thiols (LMWTs; i.e., GSH and cysteine), the ultrasmall Au8-cluster-based probe exhibited less steric hindrance and can be directly applied in selectively and sensitively detecting both LMWTs and high molecular weight thiols (HMWTs; i.e., protein thiols). Based on such sensing platform, the surface-functionalized Au8-cluster has significant promise for use as an efficient nanoprobe for intracellular fluorescence imaging of biothiols including protein thiols in living cells whereas other nanoparticle-based fluorescent probes cannot.

Drug Delivery: Enzyme‐Responsive Cell‐Penetrating Peptide Conjugated Mesoporous Silica Quantum Dot Nanocarriers for Controlled Release of Nucleus‐Targeted Drug Molecules and Real‐Time Intracellular Fluorescence Imaging of Tumor Cells (Adv. Healthcare Mater. 8/2014)

Drug Delivery: Enzyme‐Responsive Cell‐Penetrating Peptide Conjugated Mesoporous Silica Quantum Dot Nanocarriers for Controlled Release of Nucleus‐Targeted Drug Molecules and Real‐Time Intracellular Fluorescence Imaging of Tumor Cells (Adv. Healthcare Mater. 8/2014)