Esterase-Activated, pH-Responsive, and Genetically Targetable Nano-Prodrug for Cancer Cell Photo-Ablation.

Activatable prodrugs have drawn considerable attention for cancer cell ablation owing to their high specificity in drug delivery systems. However, phototheranostic prodrugs with dual organelle-targeting and synergistic effects are still rare due to low intelligence of their structures. Besides, the cell membrane, exocytosis, and diffusional hindrance by the extracellular matrix reduce drug uptake.

Monotherapy and Combination Therapy Using Anti-Angiogenic Nanoagents to Fight Cancer.

Anti-angiogenic therapy, targeting vascular endothelial cells (ECs) to prevent tumor growth, has been attracting increasing attention in recent years, beginning with bevacizumab (Avastin) through its Phase II/III clinical trials on solid tumors. However, these trials showed only modest clinical efficiency; moreover, anti-angiogenic therapy may induce acquired resistance to the drugs employed. Combining advanced drug delivery techniques (e.

Subcellular Singlet Oxygen and Cell Death: Location Matters.

We developed a tool for targeted generation of singlet oxygen using light activation of a genetically encoded fluorogen-activating protein complexed with a unique dye molecule that becomes a potent photosensitizer upon interaction with the protein. By targeting the protein receptor to activate this dye in distinct subcellular locations at consistent per-cell concentrations, we investigated the impact of localized production of singlet oxygen on induction of cell death. We analyzed light dose-dependent cytotoxic response and characterized the apoptotic vs.

Nanocrystal Formulation Improves Vaginal Delivery of CSIC for HIV Prevention.

5-Chloro-3-phenylsulfonylindole-2-carboxamide (CSIC) is a highly potent non-nucleoside reverse transcriptase inhibitor (NNRTI) with potential for use in topical prophylaxis against HIV transmission. However, the hydrophobic nature of CSIC limits its administration through vaginal route. In this study, we developed nanocrystals of CSIC to potentially improve the aqueous solubility and intracellular uptake of CSIC in vitro and in vivo.

Permeation of macromolecules into the renal glomerular basement membrane and capture by the tubules.

How the kidney prevents urinary excretion of plasma proteins continues to be debated. Here, using unfixed whole-mount mouse kidneys, we show that fluorescent-tagged proteins and neutral dextrans permeate into the glomerular basement membrane (GBM), in general agreement with Ogston's 1958 equation describing how permeation into gels is related to molecular size. Electron-microscopic analyses of kidneys fixed seconds to hours after injecting gold-tagged albumin, negatively charged gold nanoparticles, and stable oligoclusters of gold nanoparticles show that permeation into the lamina densa of the GBM is size-sensitive.

Affibody-targeted fluorogen activating protein for in vivo tumor imaging.

Molecular imaging using near-infrared (NIR) fluorescence is useful for intraoperative imaging and real-time margin identification. Directly conjugated IR dyes possess useful properties for in vivo imaging, but conjugation often substantially alters the circulation dynamics of targeting moieties. We developed and characterized a new tumor-targeting probe, affiFAP, which consists of a protein that specifically binds EGFR (affibody) and a fluorogen activating protein (FAP).

Simultaneous multi-species tracking in live cells with quantum dot conjugates.

Quantum dots are available in a range of spectrally separated emission colors and with a range of water-stabilizing surface coatings that offers great flexibility for enabling bio-specificity. In this study, we have taken advantage of this flexibility to demonstrate that it is possible to perform a simultaneous investigation of the lateral dynamics in the plasma membrane of i) the transmembrane epidermal growth factor receptor, ii) the glucosylphospatidylinositol-anchored protein CD59, and iii) ganglioside GM1-cholera toxin subunit B clusters in a single cell. We show that a large number of the trajectories are longer than 50 steps, which we by simulations show to be sufficient for robust single trajectory analysis.

Nanoparticle transport from mouse vagina to adjacent lymph nodes.

To test the feasibility of localized intravaginal therapy directed to neighboring lymph nodes, the transport of quantum dots across the vaginal wall was investigated. Quantum dots instilled into the mouse vagina were transported across the vaginal mucosa into draining lymph nodes, but not into distant nodes. Most of the particles were transported to the lumbar nodes; far fewer were transported to the inguinal nodes.

Imaging vasculature and lymphatic flow in mice using quantum dots.

Quantum dots are ideal probes for fluorescent imaging of vascular and lymphatic tissues. On injection into appropriate sites, red- and near-infrared-emitting quantum dots provide excellent definition of vasculature, lymphoid organs, and lymph nodes draining both normal tissues and tumors. We detail methods for use with commercially available quantum dots and discuss common difficulties.

Long-term persistence and spectral blue shifting of quantum dots in vivo.

Quantum dots are a powerful fluorophore family with desirable attributes for fluorescence imaging. They have been used in several animal models with direct clinical relevance, including sentinel lymph node mapping, tracing vasculature and lymphatics, and targeting specific lesions for diagnosis and removal. (1-12) Despite significant interest for use in translational applications, little is known about the persistence and long-term fate of quantum dots in vivo.

Cholera toxin B conjugated quantum dots for live cell labeling.

Cholera toxin subunit B (CTB)--quantum dot conjugates were developed for labeling mammalian cells. The conjugates were internalized by all tested cell lines into small vesicles dispersed throughout the cytoplasm, while commercially available polyarginine conjugates rapidly accumulated in large perinuclear endosomes. Although a large proportion of CTB conjugates eventually also accumulated in perinuclear endosomes, this accumulation required several days, and even then many CTB conjugated quantum dots remained in small vesicles dispersed throughout the cytoplasm.

Sentinel lymph node imaging using quantum dots in mouse tumor models.

We demonstrate that quantum dots injected into two model tumors rapidly migrate to sentinel lymph nodes. PEG-coated quantum dots having terminal carboxyl, amino, or methoxyl groups all migrated from the tumor to surrounding lymph nodes similarly. Passage from the tumor through lymphatics to adjacent nodes could be visualized dynamically through the skin; at least two nodes could usually be defined.

Fluorescent DNA nanotags: supramolecular fluorescent labels based on intercalating dye arrays assembled on nanostructured DNA templates.

Fluorescence detection and imaging are vital technologies in the life sciences and clinical diagnostics. The key to obtaining high-resolution images and sensitive detection is to use fluorescent molecules or particles that absorb and emit visible light with high efficiency. We have synthesized supramolecular complexes consisting of a branched DNA template and fluorogenic intercalating dyes.

Fluorescence imaging of tumors in vivo.

We review recent progress in tumor imaging in vivo using fluorescent tags, highlight the problems of fluorescence imaging in small animals, discuss recent advances in near-infrared fluorochromes and quantum dots, and point to some future possibilities. GFP-based fluorescence imaging is briefly discussed. The authors believe that improvements in near-infrared fluorochromes are required to enable practical imaging in tissues at centimeter depths.

Noninvasive imaging of quantum dots in mice.

Quantum dots having four different surface coatings were tested for use in in vivo imaging. Localization was successfully monitored by fluorescence imaging of living animals, by necropsy, by frozen tissue sections for optical microscopy, and by electron microscopy, on scales ranging from centimeters to nanometers, using only quantum dots for detection. Circulating half-lives were found to be less than 12 min for amphiphilic poly(acrylic acid), short-chain (750 Da) methoxy-PEG or long-chain (3400 Da) carboxy-PEG quantum dots, but approximately 70 min for long-chain (5000 Da) methoxy-PEG quantum dots.

Epitope tagging genomic DNA using a CD-tagging Tn10 minitransposon.

Here, we describe an efficient system for epitope tagging cloned genes by CD tagging using a mini-Tn10 transposon delivery vector. The system was tested against a lambdaFIX genomic clone of the human nucleolin gene. Transfection of HeLa cells with the tagged gene led to the expression of both the appropriately spliced tagged transcript and the appropriately localized tagged protein.

Cellular adhesion mediated by factor J, a complement inhibitor. Evidence for nucleolin involvement.

Factor J (FJ) is a complement inhibitor that acts on the classical and the alternative pathways. We demonstrated FJ-cell interactions in fluid phase by flow cytometry experiments using the cell lines Jurkat, K562, JY, and peripheral blood lymphocytes. FJ bound to plastic plates was able to induce in vitro adhesion of these cells with potency equivalent to fibronectin.

Cyanine fluorochrome-labeled antibodies in vivo: assessment of tumor imaging using Cy3, Cy5, Cy5.5, and Cy7.

Monoclonal antibodies to two different targetable antigens were conjugated to each of four commercially available cyanine fluorochromes. Equal amounts of all four antibodies were coinjected into tumor-bearing animals and imaged. Small, superficial tumors were adequately labeled using all four fluorochromes.

Tumor detection and visualization using cyanine fluorochrome-labeled antibodies.

Tumor localization using fluorescence has been made practical by current improvements in tumor targeting molecules, especially monoclonal antibodies and their derivatives, by the development of convenient near-infrared emitting fluorochromes and by the availability of digital cameras having high sensitivity in this spectral region. Recent studies in animals have demonstrated that fluorochrome labeling of monoclonal antibodies confers adequate sensitivity and improved resolution. Distribution and catabolism of fluorochrome-labeled and radiolabeled antibodies are similar.

Expression of SSEA-1 (Lewis(x)) on transitional cell carcinoma of the bladder.

The expression of stage-specific embryonic antigen 1 (SSEA-1) in transitional cell carcinomas of the bladder (TCCB) has been reported to correlate with tumor grade and the likelihood of lymphatic metastases. We examined the expression of this antigen in TCCBs to evaluate if staining correlated with grade, stage, recurrence, progression and response to intravesical chemotherapy. We studied the expression of SSEA-1 in TCCBs from 74 patients by staining with two different monoclonal antibodies (Mabs), P-12 and anti-SSEA-1, to evaluate if staining correlated with grade, stage and tumor recurrence.

Internalization of anti-nucleolin antibody into viable HEp-2 cells.

Anti-nucleolin antibodies have been detected in patients with systemic connective tissue diseases (SCTD) including systemic sclerosis (SSc) and systemic lupus erythematosus (SLE). In vivo bound autoantibodies to nucleoli of epidermal keratinocytes have been demonstrated in skin from patients with SCTD. In this study, monoclonal antibody to nucleolin (D-3) was used to determine the distribution of nucleolin in different culture cells including HEp-2, HepG2, HRCC, Molt-4 and Wil2 cells.

Tumor labeling in vivo using cyanine-conjugated monoclonal antibodies.

Far-red-emitting cyanine fluorochromes have many properties desirable for in vivo imaging: absorption and emission at wavelengths where blood and tissue are relatively transparent, high quantum yields, and good solubility even at high molar ratios of fluorochrome to antibody. Potentially, conjugation by multiple linkages should minimize hydrolysis in vivo. We conjugated two tumor-targeting monoclonal antibodies: anti-SSEA-1 (IgM, kappa) at ratios of 1.

Improved lymphocyte cytotoxicity against murine renal cell carcinoma.

In this paper we describe the generation of antibody dependent cellular cytotoxicity against a murine renal cell carcinoma. Using human recombinant interleukin-2 and in vitro adherence to plastic, we generated lymphokine activated killer and adherent lymphokine activated killer cells. Adherent lymphokine activated killer cells had significant (p less than 0.

Tissue localization of methotrexate-monoclonal-IgM immunoconjugates: anti-SSEA-1 and MOPC 104E in mouse teratocarcinomas and normal tissues.

Methotrexate (MTX) was coupled to the tumor-targeting monoclonal IgM, anti-SSEA-1 and the non-targeting myeloma IgM, MOPC 104E. At 24-h intervals following injection, drug deposition in MH-15 teratocarcinomas and in several normal tissues was followed by immunoperoxidase microscopy using the M16 monoclonal antibody to MTX. MTX-anti-SSEA-1 was deposited on the surface and in the interior of living tumor cells 24 h after injection; at 48 h and after, only low-level binding to necrotic tissue was found.