Remarkable Enhancement of Chemi­lumin­escent Signal by Dioxetane–Fluorophore Conjugates: Turn-ON Chemi­lumin­escence Probes with Color Modulation for Sensing and Imaging

Chemi­lumin­escence is among the most sensitive methods for achieving a high signal-to-noise ratio in various chemical and biological applications. We have developed a modular practical synthetic route for preparation of turn-ON fluorophore-tethered dioxetane chemi­lumin­escent probes. The chemi­lumin­escent emission of the probes was significantly amplified through an energy-transfer mechanism under physiological conditions. Two probes were composed with green and near-infrared (NIR) fluorescent dyes tethered to Schaap’s dioxetane. While both probes were able to provide chemi­lumin­escence <i>in vivo</i> images following sub­cutaneous injection, only the NIR probe could provide a chemi­lumin­escence image following intra­peritoneal injection. These are the first <i>in vivo</i> images produced by Schaap’s dioxetane chemi­lumin­escence probes with no need of an enhancer. Previously, chemi­lumin­escence cell images could only be obtained with a luciferin-based probe. Our NIR probe was able to image cells transfected with β-galactosidase gene by chemi­lumin­escence microscopy. We also report, for the first time, the instability of dioxetane–fluorophore conjugates to ambient light. Our synthetic route effectively overcomes this limitation through a late-stage functionalization of the dioxetane intermediate. We anticipate that our practical synthetic methodology will be useful for preparation of various chemi­lumin­escent probes for numerous applications

Inhibition of Gene Expression and Cancer Cell Migration by CD44v3/6-Targeted Polyion Complexes

In recent years, siRNA technology has emerged as a promising strategy for gene silencing in cancer therapy. We have designed novel CD44-targeted polyion complexes (PICs) composed of poly­(ethylene glycol)-<i>block</i>-polyethylenimine (PEG-<i>b</i>-PEI) and laminin-derived peptides (mA5G27D or mA5G27F) for in vivo siRNA delivery and gene silencing in tumors. The full-length A5G27 peptide (RLVSYNGIIFFLK), from which mA5G27D and mA5G27F are derived, binds to CD44v3 and CD44v6 and inhibits tumor cell migration, invasion, and angiogenesis. Thus, when attached to the surface of PICs, A5G27-based peptides can serve both as targeting ligands to navigate siRNA molecules directly to CD44-overexpressing tumors, and as anti-migratory agents to inhibit tumor progression. The mA5G27D- or mA5G27F-harboring PEG-<i>b</i>-PEI copolymers strongly condensed siRNA molecules into nanosized PICs presenting positive surface charges, low in vitro cytotoxicity, and high serum stability. mA5G27D- or mA5G27F-bearing PICs demonstrated high efficacy and selectivity in delivering siRAC1 into CD44-overexpressing cells, thereby silencing RAC1 mRNA and protein levels in such cells. These PICs presented substantial anti-migratory features in vitro and accumulated significantly in SK-OV-3 tumor-bearing mice, following 3 sequential intraperitoneal (i.p.) injections. Treatment of mice with 8 or 9 sequential parenteral (intravenous, (i.v.) or i.p.) injections of mA5G27F-PEG-<i>b</i>-PEI/siRNA efficiently inhibited tumor growth in two different CD44-overexpressing tumor mouse models (A549 and SK-OV-3), regardless of the type of siRNA (siPLK1 or siLUC) used. The results thus reveal the potential utility of this system for targeted delivery of siRNA molecules into solid tumors to prolong the survival time of mice, while at the same time reducing potential toxicity

Novel Pullulan Bioconjugate for Selective Breast Cancer Bone Metastases Treatment

A novel polysaccharide bioconjugate was designed to selectively target breast cancer bone metastases using a bisphosphonate moiety (alendronate, ALN). Paclitaxel (PTX) was first covalently conjugated to pullulan (Pull) through a Cathepsin K-sensitive tetrapeptide spacer followed by a self-immolative aminobenzyl alcohol spacer to obtain Pull-(GGPNle-φ-PTX). ALN was then conjugated to the polymeric backbone of Pull-(GGPNle-φ-PTX) via a PEG spacer. The final bioconjugate Pull-(GGPNle-φ-PTX)-(PEG-ALN) was found to assemble into colloidal spherical structures, which were physically and chemically stable under physiological conditions. In vitro studies showed that Pull-(GGPNle-φ-PTX)-(PEG-ALN) had strong affinity for hydroxyapatite, which simulates the bone tissue. Paclitaxel was rapidly released from the bioconjugate by Cathepsin K cleavage under pathological conditions. All studies performed using human MDA-MB-231-BM (bone metastases-originated clone), murine 4T1 breast cancer cells, murine K7M2, and human SAOS-2 osteosarcoma cells showed that the bioconjugate exerted an enhanced antiproliferative activity compared to the conjugate without the ALN. Furthermore, the nanoconjugate inhibited the migration of cancer cells and further displayed potent anti-angiogenic activity. In conclusion, the results showed that this conjugate has an excellent potential for selective treatment of bone neoplasms such as breast cancer bone metastases and osteosarcoma

Near-Infrared Dioxetane Luminophores with Direct Chemiluminescence Emission Mode

Chemiluminescent luminophores are considered as one of the most sensitive families of probes for detection and imaging applications. Due to their high signal-to-noise ratios, luminophores with near-infrared (NIR) emission are particularly important for <i>in vivo</i> use. In addition, light with such long wavelength has significantly greater capability for penetration through organic tissue. So far, only a few reports have described the use of chemiluminescence systems for <i>in vivo</i> imaging. Such systems are always based on an energy-transfer process from a chemiluminescent precursor to a nearby emissive fluorescent dye. Here, we describe the development of the first chemiluminescent luminophores with a direct mode of NIR light emission that are suitable for use under physiological conditions. Our strategy is based on incorporation of a substituent with an extended π-electron system on the excited species obtained during the chemi­excitation pathway of Schaap’s adamant­ylidene-dioxetane probe. In this manner, we designed and synthesized two new luminophores with direct light emission wavelength in the NIR region. Masking of the luminophores with analyte-responsive groups has resulted in turn-ON probes for detection and imaging of β-galactosidase and hydrogen peroxide. The probes’ ability to image their corresponding analyte/enzyme was effectively demonstrated <i>in vitro</i> for β-galactosidase activity and <i>in vivo</i> in a mouse model of inflammation. We anticipate that our strategy for obtaining NIR luminophores will open new doors for further exploration of complex biomolecular systems using non-invasive intra­vital chemiluminescence imaging techniques

Tagging the Untaggable: A Difluoroalkyl-Sulfinate Ketone-Based Reagent for Direct C–H Functionalization of Bioactive Heteroarenes

We have developed a new difluoroalkyl ketal sulfinate salt reagent suitable for direct derivatization of heteroarene C–H bonds. The reagent is capable of introducing a ketone functional group on heteroarene bioactive compounds via a one-pot reaction. Remarkably, in three examples the ketone analog and its parent drug had almost identical cytotoxicity. In a representative example, the ketone analog was bioconjugated with a delivery vehicle via an acid-labile semicarbazone linkage and with a photolabile protecting group to produce the corresponding prodrug. Controlled release of the drug–ketone analog was demonstrated in vitro for both systems. This study provides a general approach to obtain taggable ketone analogs directly from bioactive heteroarene compounds with limited options for conjugation. We anticipate that this sodium ketal-sulfinate reagent will be useful for derivatization of other heteroarene-based drugs to obtain ketone-taggable analogs with retained efficacy

Structure–Function Correlation of Aminated Poly(α)glutamate as siRNA Nanocarriers

It has been two decades since cationic polymers were introduced to the world of oligonucleotides delivery. However, the optimal physicochemical properties to make them a successful delivery vehicle are yet unknown. An ideal system became particularly interesting and necessary with the introduction of RNA interference as a promising therapeutic approach. Such nanocarrier should overcome challenges such as low plasma stability, poor cellular internalization and endosomal escape to induce gene silencing. To that end, we synthesized a library of biodegradable aminated poly­(α)­glutamate varied by amine moieties. In an attempt to elucidate the structure–function relationship, our polyplexes were physicochemically characterized and their silencing activity and cytotoxicity were evaluated. We found several structures that demonstrated improved cellular internalization. These candidates silenced gene expression to less than 50% of their initial levels, while being safe to cells and mice. Based on our research, an improved and promising tailor-designed siRNA delivery platform can be developed

Orthotopic tumor growth patterns.

<p><b>A.</b> Dormancy periods of mCherry-labeled U-87 MG (red, n = 6) and Clone #1 (black, n = 7) orthotopic tumors (total signal (counts/s) as measured by CRI Maestro™ imaging system). <b>B.</b> Twenty-four days post intracranial inoculation, U-87 MG tumors are significantly larger as detected by increased fluorescent signal (∼750 counts/s) compared with Clone #1 tumors (∼300 counts/s) (<i>p</i> = 0.006).</p

Prospective Identification of Glioblastoma Cells Generating Dormant Tumors

<div><p>Although dormant tumors are highly prevalent within the human population, the underlying mechanisms are still mostly unknown. We have previously identified the consensus gene expression pattern of dormant tumors. Here, we show that this gene expression signature could be used for the isolation and identification of clones which generate dormant tumors. We established single cell-derived clones from the aggressive tumor-generating U-87 MG human glioblastoma cell line. Based only on the expression pattern of genes which were previously shown to be associated with tumor dormancy, we identified clones which generate dormant tumors. We show that very high expression levels of thrombospondin and high expression levels of angiomotin and insulin-like growth factor binding protein 5 (IGFBP5), together with low levels of endothelial specific marker (ESM) 1 and epithelial growth factor receptor (EGFR) characterize the clone which generates dormant U-87 MG derived glioblastomas. These tumors remained indolent both in subcutaneous and orthotopic intracranial sites, in spite of a high prevalence of proliferating cells. We further show that tumor cells which form U-87 MG derived dormant tumors have an impaired angiogenesis potential both <em>in vitro</em> and <em>in vivo</em> and have a slower invasion capacity. This work demonstrates that fast-growing tumors contain tumor cells that when isolated will form dormant tumors and serves as a proof-of-concept for the use of transcriptome profiles in the identification of such cells. Isolating the tumor cells that form dormant tumors will facilitate understanding of the underlying mechanisms of dormant micro-metastases, late recurrence, and changes in rate of tumor progression.</p> </div

Comparison of size-matched tumors generated from U-87 MG and Clone #1 cells.

<p><b>A.</b> Sixteen days post subcutaneous inoculation of mCherry labeled U-87 MG and Clone #1 cells, both tumor types were approximately 2 mm³ in diameter and detectable by non-invasive CRI Maestro™ imaging system (<b>upper panel</b>). Flipped skin of tumor-bearing mice revealed highly vascularized U-87 MG-generated tumors, while blood vessels were only detectable in the surrounding skin of Clone #1-generated tumors (<b>lower panel</b>). <b>B.</b> H&E, CD34 (merged image. The separate images are provided as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044395#pone.0044395.s002" target="_blank">Fig. S2</a>) and TSP-1 staining of U-87 MG and Clone #1 tumor sections. TSP-1 staining was done on size-matched tumors from day 16 (2 mm³) and on large tumors (U-87 MG tumors at end point of experiment and Clone #1 tumors after escape from dormancy) (1800 mm³). <b>C.</b> Contrast-enhanced US imaging of U-87 MG and Clone #1 subcutaneous tumors show high vascularization of the U-87 MG fast-growing tumor (red bar, n = 5) compared with Clone #1 dormant tumors (black bar, n = 3) (<i>p</i> = 0.008). Data represent mean ± s.d. <i>*** p<0.01</i>.</p

Gene expression analysis of single cell-derived clones from U-87 MG glioblastoma cell line.

<p>All RT-PCR measurements were normalized according to expression in the parental U-87 MG cell line. <b>A.</b> Thrombospondin-1 (TSP-1) relative level in U-87 MG derived clones. <b>B.</b> Expression level of genes previously shown to be upregulated in dormant tumors is shown on the left panel. Expression level of genes previously shown to be upregulated in fast-growing tumors is shown on the right panel.</p