A Bioresponsive Near‐Infrared Fluorescent Probe for Facile and Persistent Live‐Cell Tracking

Molecular imaging significantly transforms the field of biomedical science and facilitates the visualization, characterization, and quantification of biologic processes. However, it is still challenging to monitor cell localization in vivo, which is essential to the study of tumor metastasis and in the development of cell‐based therapies. While most conventional small‐molecule fluorescent probes cannot afford durable cell labeling, transfection of cells with fluorescent proteins is limited by their fixed fluorescence, poor tissue penetration, and interference of autofluorescence background. Here, a bioresponsive near‐infrared fluorescent probe is reported as facile and reliable tool for real‐time cell tracking in vivo. The design of this probe relies on a new phenomenon observed upon fluorobenzene‐conjugated fluorescent dyes, which can form complexes with cytosolic glutathione and actively translocates to lysosomes, exhibiting enhanced and stable cell labeling. Fluorobenzene‐coupled hemicyanine, a near‐infrared fluorophore manifests to efficiently staining tumor cells without affecting their invasive property and enables persistent monitoring of cell migration in metastatic tumor murine models at high resolution for one week. The method of fluorobenzene functionalization also provides a simple and universal “add‐on” strategy to render ordinary fluorescent probes suitable for long‐term live‐cell tracking, for which currently there is a deficit of suitable molecular tools.     

Prolonged Dye Release from Mesoporous Silica‐Based Imaging Probes Facilitates Long‐Term Optical Tracking of Cell Populations In Vivo

Nanomedicine is gaining ground worldwide in therapy and diagnostics. Novel nanoscopic imaging probes serve as imaging tools for studying dynamic biological processes in vitro and in vivo. To allow detectability in the physiological environment, the nanostructure‐based probes need to be either inherently detectable by biomedical imaging techniques, or serve as carriers for existing imaging agents. In this study, the potential of mesoporous silica nanoparticles carrying commercially available fluorochromes as self‐regenerating cell labels for long‐term cellular tracking is investigated. The particle surface is organically modified for enhanced cellular uptake, the fluorescence intensity of labeled cells is followed over time both in vitro and in vivo. The particles are not exocytosed and particles which escaped cells due to cell injury or death are degraded and no labeling of nontargeted cell populations are observed. The labeling efficiency is significantly improved as compared to that of quantum dots of similar emission wavelength. Labeled human breast cancer cells are xenotransplanted in nude mice, and the fluorescent cells can be detected in vivo for a period of 1 month. Moreover, ex vivo analysis reveals fluorescently labeled metastatic colonies in lymph node and rib, highlighting the capability of the developed probes for tracking of metastasis.     

Intracellular Imaging with a Graphene‐Based Fluorescent Probe

Graphene is an increasingly important nanomaterial that has shown great promise in the area of nanotechnology. In this study, fluorescein‐functionalized graphene oxide (GO) is synthesized via a polyethylene glycol (PEG) bridge and its application in intracellular imaging is explored. GO is an oxide form of graphene that provides an ideal platform to prepare graphene‐based functional nanomaterials via chemical modification. The PEG bridge was introduced to prevent GO‐induced quenching of conjugated fluorescein. The fluorescein–PEG–GO conjugate thus prepared exhibits excellent pH‐tunable fluorescent properties and, more significantly, can be efficiently taken up by cells and serve as a fluorescent nanoprobe for intracellular imaging.     

A Highly Efficient Tumor‐Targeting Nanoprobe with a Novel Cell Membrane Permeability Mechanism

Efficient tumor targeting has been a great challenge in the clinic for a very long time. The traditional targeting methods based on enhanced permeability and retention (EPR) effects show only an ≈5% targeting rate. To solve this problem, a new graphene‐based tumor cell nuclear targeting fluorescent nanoprobe (GTTN), with a new tumor‐targeting mechanism, is developed. GTTN is a graphene‐like single‐crystalline structure amphiphilic fluorescent probe with a periphery that is functionalized by sulfonic and hydroxyl groups. This probe has the characteristic of specific tumor cell targeting, as it can directly cross the cell membrane and specifically target to the tumor cell nucleus by the changed permeability of the tumor cell membranes in the tumor tissue. This new targeting mechanism is named the cell membrane permeability targeting (CMPT) mechanism, which is very different from the EPR effect. These probes can recognize tumor tissue at a very early stage and track the invasion and metastasis of tumor cells at the single cell level. The tumor‐targeting rate is improved from less than 5% to more than 50%. This achievement in efficient and accurate tumor cell targeting will speed up the arrival of a new era of tumor diagnosis and treatment.     

An Activatable Nano‐Prodrug for Treating Tyrosine‐Kinase‐Inhibitor‐Resistant Non‐Small Cell Lung Cancer and for Optoacoustic and Fluorescent Imaging

Non‐small cell lung cancer (NSCLC) is the most common type of lung cancer and the cause of high rate of mortality. The epidermal growth factor receptor (EGFR)‐targeted tyrosine kinase inhibitors are used to treat NSCLC, yet their curative effects are usually compromised by drug resistance. This study demonstrates a nanodrug for treating tyrosine‐kinase‐inhibitor‐resistant NSCLC through inhibiting upstream and downstream EGFR signaling pathways. The main molecule of the nanodrug is synthesized by linking a tyrosine kinase inhibitor gefitinib and a near‐infrared dye (NIR) on each side of a disulfide via carbonate bonds, and the nanodrug is then obtained through nanoparticle formation of the main molecule in aqueous medium and concomitant encapsulation of a serine threonine protein kinase (Akt) inhibitor celastrol. Upon administration, the nanodrug accumulates at the tumor region of NSCLC‐bearing mice and releases the drugs for tumor inhibition, and the dye for fluorescence and optoacoustic imaging. Through suppressing the phosphorylation of upstream EGFR and downstream Akt in the EGFR pathway by gefitinib and celastrol, respectively, the nanodrug exhibits high inhibition efficacy against orthotopic NSCLC in mouse models.     

Layer‐By‐Layer Films as a Biomimetic Reservoir for rhBMP‐2 Delivery: Controlled Differentiation of Myoblasts to Osteoblasts

Efficient delivery of growth or survival factors to cells is one of the most important long‐term challenges of current cell‐based tissue engineering strategies. The extracellular matrix acts as a reservoir for a number of growth factors through interactions with its components. In the matrix, growth factors are protected against circulating proteases and locally concentrated. Thus, the localized and long‐lasting delivery of a matrix‐bound recombinant human bone morphogenetic protein 2 (rhBMP‐2) from a biomaterial surface would mimic in vivo conditions and increase BMP‐2 efficiency by limiting its degradation. Herein, it is shown that crosslinked poly(L ‐lysine)/hyaluronan (HA) layer‐by‐layer films can serve as a reservoir for rhBMP‐2 delivery to myoblasts and induce their differentiation into osteoblasts in a dose‐dependent manner. The amount of rhBMP‐2 loaded in the films is controlled by varying the deposition conditions and the film thickness. Its local concentration in the film is increased up to ≈500‐fold when compared to its initial solution concentration. Its adsorption on the films, as well as its diffusion within the films, is evidenced by microfluorimetry and confocal microscopy observations. A direct interaction of rhBMP‐2 with HA is demonstrated by size‐exclusion chromatography, which could be at the origin of the rhBMP‐2 “trapping” in the film and of its low release from the films. The bioactivity of rhBMP‐2‐loaded films is due neither to film degradation nor to rhBMP‐2 release. The rhBMP‐2‐containing films are extremely resistant and could sustain three successive culture sequences while remaining bioactive, thus confirming the important and protective effect of rhBMP‐2 immobilization. These films may find applications in the local delivery of immobilized growth factors for tissue‐engineered constructs and for metallic biomaterial surfaces, as they can be deposited on a wide range of substrates with different shapes, sizes, and composition.     

Aptamer‐Based Multicolor Fluorescent Gold Nanoprobes for Multiplex Detection in Homogeneous Solution

The design of a novel multicolor fluorescent gold nanoprobe for homogeneous detection of small‐molecule targets is reported, which combines the specific binding abilities of aptamers with the ultrahigh quenching ability of gold nanoparticles (AuNPs). Dye‐tagged aptamers and their complementary sequence with thiol labels are co‐assembled at the surface of AuNPs. As a proof of concept, it is demonstrated that such a multicolor fluorescent gold nanoprobe can simultaneously detect adenosine, potassium ion, and cocaine with high selectivity. This potentially generic strategy is shown to be promising for rapid screening of small molecular targets.     

Chromophore‐Modified Highly Selective Ratiometric Upconversion Nanoprobes for Detection of ONOO−‐Related Hepatotoxicity In Vivo

Acute hepatitis is a major problem affecting public health and has attracted more and more attention. Generally, as the standard means, blood tests are taken for evaluating hepatitis. However, such tests fail to accurately reflect the level of hepatitis in vivo. Herein, two highly selective ratiometric fluorescent probes are designed to track peroxynitrite (ONOO^?) as the hepatitis indicator, and further evaluate acute liver injury in vivo through dye‐grafted upconversion nanoparticles (UCNPs). Specifically, upconversion luminescence of nanoprobes at 540 or 660 nm can be quenched by the designed and synthesized chromophore E‐CC or H‐CC, that can be destroyed by ONOO^? via energy transfer (ET) process, while the upconversion luminescence intensity at 810 nm remains the same. Thus, the developed nanoprobes can be used for ratiometric detection (I₅₄₀/I₆₆₀ or I₆₆₀/I₈₁₀) of ONOO^?. Moreover, the developed near infrared ratiometric nanoprobes can highly selectively detect ONOO^?, which can eliminate the interference of HOCl and SO₃^2?. Finally, it is demonstrated that this highly selective ratiometric nanosystem can achieve effective detection of ONOO^? in living cells and CCl₄‐induced acute liver injury models. It provides some reference value for clinical detection of hepatotoxicity.     

Polysaccharide‐Blend Multilayers Containing Hyaluronan and Heparin as a Delivery System for rhBMP‐2

It is shown that blend multilayers of hyaluronan (HA) and heparin (HEP) as polyanions and poly(L‐lysine) (PLL) as a polycation can be used to prepare films with different thicknesses and chemical compositions. The amounts of recombinant human BMP‐2 (rhBMP‐2) loaded and the fraction initially released from the films depend on the film's chemical composition. The amounts of rhBMP‐2 loaded in the films are much higher for HA mass fractions of more than 0.4. The bioactivity of the rhBMP‐2‐loaded films is investigated on C2C12 myoblasts, which differentiates into osteoblasts in contact with the films. The alkaline phosphatase expression for cells grown on nanoblend films of various compositions falls over a unique curve. This suggests that the cells “sensing” the rhBMP‐2 are not influenced by the film's chemistry. The rhBMP‐2 can sustain at least three successive culture sequences while remaining bioactive, thus confirming the important and protective effect of rhBMP‐2. Altogether, these results indicate that crosslinked PLL/HA films have superior properties for the incorporation of rhBMP‐2 and on its long‐lasting bioactivity.     

Bright Single‐Chain Conjugated Polymer Dots Embedded Nanoparticles for Long‐Term Cell Tracing and Imaging

Single‐chain conjugated polymer (CP) dots embedded nanoparticles (NPs) bearing cell penetration peptide (TAT) as surface ligands are synthesized for long term cancer cell tracing applications. The CPNPs are fabricated by matrix‐encapsulation method and the embedded CPs can be modulated into spherical dots with different size upon alteration of feed concentrations. Single‐chain CP dots are formed upon decreasing feed concentration to 0.2 mg/mL, where CPNPs exhibit highest fluorescence quantum yield of 32%. Maleimide is introduced as the new NP surface functional group, which favors easy conjugation with cell penetration peptide via click chemistry to preserve its biofunctions. The obtained CPNPs show high brightness and good biocompatibility, which allow cell tracing for over 9 generations, superior to commercial cell tracker Qtracker 585.     

A Targeted and FRET‐Based Ratiometric Fluorescent Nanoprobe for Imaging Mitochondrial Hydrogen Peroxide in Living Cells

Hydrogen peroxide (H₂O₂) is a prominent member of the reactive oxygen species family and plays crucial roles in living organisms, thus detecting H₂O₂ and elucidating its biological functions has become an important area of biological and biomedical research. Herein, a multifunctional fluorescent nanoprobe is demonstrated for detecting mitochondrial H₂O₂. The nanoprobe is prepared by covalently linking a mitochondria‐targeting ligand (triphenylphosphonium, TPP) and a H₂O₂ recognition element (PFl) onto carbon dots (CDs). For this nanoprobe, the CD serves as the carrier and the FRET donor. In the presence of H₂O₂, the PFl moieties on a CD undergo structural and spectral conversion, affording the nanoplatform a FRET‐based ratiometric probe for H₂O₂. The nanoprobe displays excellent water dispersibility, high sensitivity and selectivity, satisfactory cell permeability, and very low cytotoxicity. Following the living cell uptake, this nanoprobe can specifically target and stain the mitochondria; and it can detect the exogenous H₂O₂ in L929 cells, as well as the endogenously produced mitochondrial H₂O₂ in Raw 264.7 cells upon stimulation by PMA. This study shows that CDs can serve as promising nano‐carriers for fabricating practical multifunctional fluorescent nanosensors.     

Development of Blood‐Cell‐Selective Fluorescent Biodots for Lysis‐Free Leukocyte Imaging and Differential Counting in Whole Blood

Complete blood count with leukocyte (white blood cell, WBC) differential is one of the most frequently ordered clinical test for disease diagnosis. Herein, multifunctional fluorescent carbon dots derived from biomolecules (biodots) for rapid lysis‐free whole blood analysis are developed. Specifically, two types of biodots are molecularly engineered through hydrothermal synthesis for differential blood cells labeling. Type I biodots synthesized from amino acid (serine/threonine) precursors and passivated with polyethylenimine can label both red blood cells (RBCs) and WBCs with excellent contrast in fluorescence intensity, enabling direct counting of leukocytes in whole blood samples without a tedious RBC lysis step. It also allows three‐part leukocyte differential counting by flow cytometry without using expensive fluorophore‐conjugated antibodies. On the other hand, Type II biodots synthesized from the same amino acid precursors but passivated with a biopolymer (chitosan) are able to selectively lyse RBCs with greater than 98% efficiency to allow simultaneous fluorescent labeling of leukocytes for WBC counting in whole blood. It is envisioned that these novel nanoreagents, which eliminate the cumbersome lysis and antibody conjugation steps for selective labeling of different blood cells, would revolutionize disease diagnostics toward achieving faster, cheaper, and more accurate whole blood analyses in one test.