carbocyanines and Carcinoma--Lewis-Lung

Clinical applications of siRNA are being hindered by poor intracellular uptake and enzymatic degradation. To address these problems, we devised an oral delivery system for telomerase reverse transcriptase siRNA using N-((2-hydroxy-3-trimethylammonium) propyl) chitosan chloride (HTCC) nanoparticles (HNP). Both the porous structure and the positive charge of HNP facilitated siRNA encapsulation. The outer coating of HTCC not only protected siRNA from enzymatic degradation, but also improved siRNA permeability in intestine tract. In vivo and in vitro experiments proved that HNP could effectively deliver siRNA to lesion site and further into tumor cells. On the basis of confirming the antitumor activity of HNP:siRNA, we continued to encapsulate a hydrophobic chemotherapeutic drug-paclitaxel (PTX) into HNP to form a "two-in-one" nano-complex (HNP:siRNA/PTX). We demonstrated that HNP:siRNA/PTX could simultaneously ferry siRNA and PTX into tumor cells and increase drug concentration, which, in particular, was much more effective in tumor suppression than that of traditional cocktail therapy. These results suggested that the HNP, as a powerful delivery system for both siRNA and chemotherapeutic drug, would have a far-reaching application in human cancer therapy.

Topics: Absorption; Animals; Antineoplastic Agents; Caco-2 Cells; Carbocyanines; Carcinoma, Lewis Lung; Cell Death; Cell Proliferation; Chitosan; Drug Delivery Systems; Endocytosis; Flow Cytometry; Gene Silencing; Gene Transfer Techniques; Humans; Intracellular Space; Male; Mice; Nanoparticles; Neoplasms; Paclitaxel; Permeability; Quaternary Ammonium Compounds; RNA, Small Interfering; Telomerase; Tissue Distribution; Xenograft Model Antitumor Assays

A revolutionary probe was constructed by conjugating Cy5 on a graphene oxide sheet. The fluorescence was quenched owing to its proximity to graphene oxide in normal circumstances, but lit on arriving at the tumor site. Both in vitro and in vivo studies demonstrated that the probe exhibits great potential for tumor diagnosis.

Topics: Animals; Carbocyanines; Carcinoma, Lewis Lung; Cell Line, Tumor; Enzyme Inhibitors; Fluorescent Dyes; Graphite; Humans; Injections, Intralesional; Maleimides; Metalloproteases; Mice; Molecular Imaging; Molecular Probes; Nanoparticles; Neoplasm Proteins; Oligopeptides; Oxides; Protein Interaction Domains and Motifs; Spectrometry, Fluorescence

Cell death plays a central role in normal physiology and in disease. Common to apoptotic and necrotic cell death is the eventual loss of plasma membrane integrity. We have produced a small organoarsenical compound, 4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid, that rapidly accumulates in the cytosol of dying cells coincident with loss of plasma membrane integrity. The compound is retained in the cytosol predominantly by covalent reaction with the 90 kDa heat shock protein (Hsp90), the most abundant molecular chaperone of the eukaryotic cytoplasm. The organoarsenical was tagged with either optical or radioisotope reporting groups to image cell death in cultured cells and in murine tumors ex vivo and in situ. Tumor cell death in mice was noninvasively imaged by SPECT/CT using an (111)In-tagged compound. This versatile compound should enable the imaging of cell death in most experimental settings.

Topics: Animals; Arsenicals; Carbocyanines; Carcinoma, Lewis Lung; Cell Death; Colorectal Neoplasms; HSP90 Heat-Shock Proteins; Humans; Jurkat Cells; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasms; Pentetic Acid; Peptides; Protein Binding; Radioisotopes

To microscopically analyze the chemotherapeutic response of tumors using in vivo staining based on an annexinV-Cy5.5 probe and independently asses their apoptotic count using quantitative histological analysis.. Lewis Lung Carcinomas cells, that are sensitive (CS-LLC) and resistant (CR-LLC) to chemotherapy were implanted in nude mice and grown to tumours. Mice were treated with cyclophosphamide and injected with a Cy5.5-annexinV fluorescent probe. In vivo imaging was performed using Fluorescence Molecular Tomography. Subsequently tumours were excised and prepared for histology. The histological tumour sections were stained for apoptosis using a terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. A minimum of ten tissue sections were analyzed per tumour for apoptosis quantification by TUNEL staining and corresponding Cy5.5 distribution.. We detected higher levels of apoptosis and corresponding higher levels of Cy5.5 fluorescence in the CS-LLC vs. the CR-LLC tumours. The cell count rate on CS-LLC sections over CR-LLC was found to be approximately 2 :1 where the corresponding area observed on Cy5.5 distribution measurements revealed a approximately 1.7 :1 ratio of CS-LLC over CR-LLC. These observations are consistent with the higher apoptotic index expected from the CS-LLC cell line.. Quantitative analysis of histological slices revealed higher fluorescence and higher apoptotic count in the CS-LLC tumour images compared to the CR-LLC tumour images. These observations demonstrate that the annexinV-Cy5.5 probe sensed the chemotherapeutic effect of cyclophospamide and further confirmed in vivo FMT measurements.

Topics: Animals; Annexin A5; Annexins; Apoptosis; Carbocyanines; Carcinoma, Lewis Lung; Cell Count; Cell Line, Tumor; Cyclophosphamide; Drug Resistance, Neoplasm; Feasibility Studies; Female; Fluorescent Dyes; Immunohistochemistry; In Situ Nick-End Labeling; Mice; Mice, Nude; Neoplasm Transplantation; Tomography, Optical

We designed a fluorescent peptide-magnetic nanoparticle conjugate that images E-selectin expression in mouse xenograft models of Lewis lung carcinoma (LLC) by fluorescence reflectance imaging. It was synthesized by attaching the E-selectin-binding peptide (ESBP; CDSDSDITWDQLWDLMK) to a CLIO(Cy5.5) nanoparticle to yield ESBP-CLIO(Cy5.5). Internalization by activated human umbilical vein endothelial cells (HUVECs) was rapid and mediated by E-selectin, indicated by the lack of uptake of nanoparticles bearing similar numbers of a scrambled peptide (Scram). To demonstrate the specificity of E-selectin targeting to ESBP-CLIO(Cy5.5) in vivo, we coinjected ESBP-CLIO(Cy5.5) and Scram-CLIO(Cy3.5) and demonstrated a high Cy5.5/Cy3.5 fluorescence ratio using the LLC. Histology showed that ESBP-CLIO was associated with tumor cells as well as endothelial cells, but fluorescence-activated cell sorter analysis showed a far less expression of E-selectin on LLC than on HUVECs. Using immunohistochemistry, we demonstrated E-selectin expression in both endothelial cells and cancer cells in human prostate cancer specimens. We conclude that ESBP-CLIO(Cy5.5) is a useful probe for imaging E-selectin associated with the LLC tumor, and that E-selectin is expressed not only on endothelial cells but also on LLC cells and human prostate cancer specimens.

Topics: Animals; Carbocyanines; Carcinoma, Lewis Lung; Cell Line; Cell Line, Tumor; Cell Nucleus; Cell Separation; E-Selectin; Edetic Acid; Endothelial Cells; Endothelium, Vascular; Flow Cytometry; Humans; Immunohistochemistry; Interleukin-1; Male; Mice; Microscopy, Confocal; Microscopy, Fluorescence; Nanostructures; Nanotechnology; Neoplasm Transplantation; Peptides; Platelet Endothelial Cell Adhesion Molecule-1; Prostatic Neoplasms; Sensitivity and Specificity; Substrate Specificity; Time Factors; Umbilical Veins

In vivo imaging of treatment responses at the molecular level could have a significant impact on the speed of drug discovery and ultimately lead to personalized medicine. Strong interest has been shown in developing quantitative fluorescence-based technologies with good molecular specificity and sensitivity for noninvasive 3D imaging through tissues and whole animals. We show herein that tumor response to chemotherapy can be accurately resolved by fluorescence molecular tomography (FMT) with a phosphatidylserine-sensing fluorescent probe based on modified annexins. We observed at least a 10-fold increase of fluorochrome concentration in cyclophosphamide-sensitive tumors and a 7-fold increase of resistant tumors compared with control studies. FMT is an optical imaging technique developed to overcome limitations of commonly used planar illumination methods and demonstrates higher quantification accuracy validated by histology. It is further shown that a 3-fold variation in background absorption heterogeneity may yield 100% errors in planar imaging but only 20% error in FMT, thus confirming tomographic imaging as a preferred tool for quantitative investigations of fluorescent probes in tissues. Tomographic approaches are found essential for small-animal optical imaging and are potentially well suited for clinical drug development and monitoring.

Topics: Animals; Annexin A5; Carbocyanines; Carcinoma, Lewis Lung; Cyclophosphamide; Drug Resistance, Neoplasm; Female; Fluorescence; Fluorescent Dyes; Mice; Mice, Nude; Phantoms, Imaging; Tomography, Optical

Noninvasive imaging using radioactive annexin V is an emerging strategy for the assessment of cell death in vivo (F. G. Blankenberg, and H. W. Strauss. Apoptosis, 6: 117-123, 2001.). Therefore, we investigated whether annexin V labeled with the fluorophore Cy5.5 (Cy) could serve as a probe for imaging of tumor apoptosis using near infrared fluorescence (NIRF). We prepared active Cy-annexin (an equimolar dye:protein ratio) that bound to apoptotic Jurkat T cells and an inactive Cy-annexin probe (>2 dyes/mol protein) that did not. Active Cy annexin was used to image a 9L gliosarcoma, constitutively expressing green fluorescent protein marker, and the CR8 variant of Lewis lung carcinoma, stably transfected to express DsRed2. The expression of transfected fluorescent protein provided an indication of tumor margins and a means of defining tumor-associated NIRF signal intensity with both tumor models. Tumors were imaged with and without cyclophosphamide treatment. In both tumor models active Cy-annexin V tumor NIRF signal increased two to three times after the treatment. Tumor NIRF signal developed by 75 min after active Cy-annexin injection and remained for a 20-h observation period. Inactive annexin V was used as a control in the CR8 carcinoma experiments and resulted in a low nonspecific signal. With the 9L gliomosacrcoma model, active Cy-annexin V bound to both tumor cells (Cy-annexin V staining only) and endothelial cells (costained with Cy-annexin V and antibody to the endothelial marker CD31). Our results demonstrate that active Cy-annexin can be used as a NIRF probe to image apoptosis from outside an intact living animal and may provide nonradioactive method of measuring the antiproliferative effects of cancer chemotherapeutic regimens.

Topics: Animals; Annexin A5; Apoptosis; Carbocyanines; Carcinoma, Lewis Lung; Cyclophosphamide; Female; Fluorescent Dyes; Gliosarcoma; Green Fluorescent Proteins; Humans; Jurkat Cells; Luminescent Proteins; Mice; Mice, Nude; Rats; Spectroscopy, Near-Infrared; Transfection