The signal-to-background ratio (SBR) is the key determinant of sensitivity, detectability, and linearity in optical imaging. As signal strength is often constrained by fundamental limits, background reduction becomes an important approach for improving SBR. We recently reported that a zwitterionic near-infrared (NIR) fluorophore, ZW800-1, exhibits low background. Here we show that this fluorophore provides much-improved SBR when targeted to cancer cells or proteins by conjugation with a cyclic RGD peptide, fibrinogen, or antibodies. ZW800-1 outperforms the commercially available NIR fluorophores IRDye800-CW and Cy5.5 in vitro for immunocytometry, histopathology and immunoblotting, and in vivo for image-guided surgery. In tumor model systems, tumor-to-background ratios of 17.2 are achieved after only 4 h post-injection, compared with 5.1 for IRDye800-CW and 2.7 for Cy5.5. Our results suggest that introducing zwitterionic properties into targeted fluorophores may be a general strategy for improving the SBR in diagnostic and therapeutic applications.

SUMMARYNanoparticles (NPs) have the potential to revolutionize drug delivery, however, administering them to the human body without the need for intravenous injection remains a major challenge. In this study, a series of near-infrared (NIR) fluorescent NPs were systematically varied in chemical composition, shape, size, and surface charge, and their biodistribution and elimination were quantified in rat models after lung instillation. We demonstrate that NPs with hydrodynamic diameter (HD) less than ≈ 34 nm and a non-cationic surface charge translocate rapidly from lung to mediastinal lymph nodes. NPs of HD < 6 nm can traffic rapidly from the lungs to lymph nodes and the bloodstream, and then be subsequently cleared by the kidneys. We discuss the importance of these findings to drug delivery, air pollution, and carcinogenesis. KeywordsNanoparticles; nanomedicine; drug delivery; air pollution; lymph node uptake; biodistribution; renal clearance * Co-Senior Authors: Beth Israel Deaconess Medical Center 330 Brookline Avenue, Room SL-B05 Boston, MA 02215 Phone: 617-667-0692 Fax: 617-667-0981 jfrangio@bidmc.harvard.edu Harvard School of Public Health 665 Huntington Avenue Boston, MA 02115 Phone: 617-432-0127 Fax: 617-432-4710 atsuda@hsph.harvard.edu . AUTHOR CONTRIBUTIONS H.S.C., Y.A., J.H.L., S.H.K., A.M., N.I., and A.T. performed the experiments. H.S.C., M.G.B., M.S.B., A.T., and J.V.F. reviewed, analyzed, and interpreted the data. H.S.C., A.T., and J.V.F. wrote the paper. All authors discussed the results and commented on the manuscript. Nanoparticles (NPs) have been proposed as diagnostic, therapeutic, and theragnostic agents for a wide variety of human diseases. 1-3 Lung-based drug delivery of NPs is receiving increased attention due to the large surface area available and the minimal anatomical barriers limiting access to the body. 4 In this study, we explore whether it would be possible to administer NPs via the lung, and in so doing, attempt to define the key parameters that mediate lung to body NP translocation and subsequent elimination (i.e., clearance). COMPETING INTERESTS STATEMENTLung-administered NPs also have significant implications for air pollution. Recent toxicological studies have confirmed that nano-sized or ultrafine particles reach deep into the alveolar region of the lungs 5,6 and cause severe inflammation reactions due to their large surface areas per mass. 6 Inhalation of NPs is increasingly recognized as a major cause of adverse health effects, and has especially strong influence on the cardiovascular system and hemostasis, leading to increased cardiovascular morbidity and mortality. [6][7][8] The standard approach for studying the translocation of inhaled NPs and ultrafine air pollutants from the lungs to extrapulmonary compartments in animals is to perform postmortem analysis of tissues after inhalation of carbon-based particles, 9 radiotracers, 10 or neutron-activated metal particles. 11-13 Recently, Moller et al. reported that ultrafine NPs could pass from the lungs into bloodstream an...

To address two fundamental and unsolved problems in optical imaging (nonspecific uptake of near‐infrared fluorophores by normal tissues and organs and incomplete elimination of unbound targeted fluorophores from the body), novel zwitterionic near‐infrared fluorophores (e.g., ZW800‐1) were synthesized and their performance compared in vivo to conventional molecules (e.g., ICG) as a function of charge, charge distribution, and hydrophobicity (see picture).

CpG-binding protein (CXXC finger protein 1 (CFP1)) binds to DNA containing unmethylated CpG motifs and is required for mammalian embryogenesis, normal cytosine methylation, and cellular differentiation. Studies were performed to identify proteins that interact with CFP1 to gain insight into the molecular function of this protein. Immunoprecipitation and mass spectrometry reveal that human CFP1 associates with a ϳ450-kDa complex that contains the mammalian homologues of six of the seven components of the Set1/COMPASS complex, the sole histone H3-Lys 4 methyltransferase in yeast. In vitro assays demonstrate that the human Set1/CFP1 complex is a histone methyltransferase that produces mono-, di-, and trimethylated histone H3 at Lys 4 . Confocal microscopy reveals that CFP1 and Set1 co-localize to nuclear speckles associated with euchromatin. A Set1 complex of reduced mass persists in murine embryonic stem cells lacking CFP1. These cells carry elevated levels of methylated histone H3-Lys 4 and reduced levels of methylated histone H3-Lys 9 . Together with the previous finding of reduced levels of cytosine methylation, these data indicate that cells lacking CFP1 contain reduced levels of heterochromatin. Furthermore, ES cells lacking CFP1 exhibit a 4-fold excess of histone H3-Lys 4 methylation following induction of differentiation, indicating that CFP1 restricts the activity of the Set1 histone methyltransferase complex. These results reveal a mammalian counterpart to the yeast Set1/COMPASS complex. The presence of CFP1 in this complex implicates this protein as a critical epigenetic regulator of histone modification in addition to cytosine methylation and reveals one mechanism by which this protein intersects with the epigenetic machinery.CpG-binding protein exhibits a unique DNA binding specificity for unmethylated CpG motifs and acts as a transcriptional activator (1). This factor, encoded by the CXXC1 gene, has recently been designated CXXC finger protein 1, and will hereafter be referred to as CFP1.2 Originally identified in mammals, homologues of CFP1 have been detected in Drosophila, Caenorhabditis elegans, and both Saccharomyces cerevisiae and Schizosaccharomyces pombe (1, 2). CFP1 contains a cysteinerich CXXC DNA-binding domain (1, 3), which is present in several other proteins, including DNA methyltransferase 1 (Dnmt1) (4), the major maintenance DNA methyltransferase; human trithorax (HRX) (also known as ALL-1 or MLL), a histone H3-Lys 4 methyltransferase encoded by a gene frequently involved in chromosomal translocations in leukemia (5-10); methyl-binding domain protein 1, which binds to methylated CpG dinucleotides (11, 12); leukemia-associated protein LCX (13); and MLL-2, a histone H3-Lys 4 methyltransferase, which is often amplified in solid tumors (14, 15). CFP1 also contains two plant homeodomain (PHD) motifs (1, 3), which are characteristic of chromatin-associated proteins and/or regulators of gene expression (1, 16) and mediate protein/protein interactions (17-19). CFP1 is a component of the nuclear...

Trimethylation of histone H3 Lys 4 (H3K4me3) is a mark of active and poised promoters. The Set1 complex is responsible for most somatic H3K4me3 and contains the conserved subunit CxxC finger protein 1 (Cfp1), which binds to unmethylated CpGs and links H3K4me3 with CpG islands (CGIs). Here we report that Cfp1 plays unanticipated roles in organizing genome-wide H3K4me3 in embryonic stem cells. Cfp1 deficiency caused two contrasting phenotypes: drastic loss of H3K4me3 at expressed CGI-associated genes, with minimal consequences for transcription, and creation of “ectopic” H3K4me3 peaks at numerous regulatory regions. DNA binding by Cfp1 was dispensable for targeting H3K4me3 to active genes but was required to prevent ectopic H3K4me3 peaks. The presence of ectopic peaks at enhancers often coincided with increased expression of nearby genes. This suggests that CpG targeting prevents “leakage” of H3K4me3 to inappropriate chromatin compartments. Our results demonstrate that Cfp1 is a specificity factor that integrates multiple signals, including promoter CpG content and gene activity, to regulate genome-wide patterns of H3K4me3.