In vivo cancer imaging by poly(ethylene glycol)-b-poly(ɛ-caprolactone) micelles containing a near-infrared probe.

Noninvasive near-infrared (NIR) fluorescence imaging is a promising technique for the intraoperative assessment of solid tumor removal. We incorporated a lipophilic NIR probe, 1,1'-dioctadecyltetramethyl indotricarbocyanine iodide (DiR), in poly(ethylene glycol)-b-poly(ɛ-caprolactone) (PEG-b-PCL) micelles, resulting in DiR solubilization in water, occupying nanoscopic PEG-b-PCL micelles. DiR in a self-quenched or nonquenched state showed different kinetics of release from PEG-b-PCL micelles in vitro; however, both obtained high tumor delineation (tumor-to-muscle ratio of 30-43 from collected organs). These results suggest that PEG-b-PCL micelles with DiR are a promising nanosized imaging agent that will provide a basis for enhanced surgical guidance via NIR visualization of tumors. FROM THE CLINICAL EDITOR: In this paper, noninvasive near-infrared fluorescence imaging coupled with specific lipophilic probes is discussed as a promising technique for intraoperative assessment of solid tumor removal, leading to optimized outcomes for in toto removal of tumors.

Photophysical, photochemical, and tumor-selectivity properties of bromine derivatives of rhodamine-123.

The conceptual basis for the development of mitochondrial targeting as a novel therapeutic strategy for both chemotherapy and photochemotherapy of neoplastic diseases rests on the observation that enhanced mitochondrial membrane potential is a common tumor cell phenotype. The potential of this strategy is highlighted by the fact that the toxic effects associated with a number of cationic dyes known to localize in energized cell mitochondria are much more pronounced in tumor cells than in normal cells. Here we evaluate the phototoxic properties of four bromine derivatives of rhodamine-123 toward human uterine sarcoma (MES-SA) and green monkey kidney (CV-1) cells and compare the degrees of tumor cell selectivity associated with these dyes with those associated with two model mitochondrial triarylmethanes (crystal violet and ethyl violet). Selective phototoxicity toward tumor cells was found to be highly dependent upon the lipophilic/hydrophilic character of the cationic photosensitizer. Our experimental data have indicated that the probability of success of mitochondrial targeting in (photo)chemotherapy of neoplastic diseases is higher when the octan-1-ol/water partition coefficient of the drug candidate falls within approximately two orders of magnitude from that of the prototypical mitochondria-specific dye rhodamine-123.

Effect of dye aggregation on triarylmethane-mediated photoinduced damage of hexokinase and DNA.

The observation that enhanced mitochondrial membrane potential is a prevalent cancer cell phenotype has provided the conceptual basis for the development of mitochondrial targeting as a novel therapeutic strategy for both chemo- and photochemotherapy of neoplastic diseases. Cationic triarylmethane (TAM(+)) dyes represent a series of photosensitizers whose phototoxic effects develop at least in part at the mitochondrial level. In this report we describe how the molecular structure of four representative TAM(+) dyes (Crystal Violet, Ethyl Violet, Victoria blue R, and Victoria pure blue BO) affects their efficiency as mediators of the photoinduced inactivation of two model mitochondrial targets, hexokinase (HK) and DNA. Our results have indicated that TAM(+) dyes efficiently bind to HK and DNA in aqueous media both as dye monomers and aggregates, with the degree of aggregation increasing with increasing the lipophilic character of the photosensitizer. The efficiency with which HK and DNA are damaged upon 532 nm photolysis of biopolymer-TAM(+) complexes was found to decrease upon increasing the degree of dye aggregation over these macromolecular templates. Comparative experiments carried out both in water and in D(2)O, and in air-equilibrated and nitrogen-purged samples have also indicated that, at least when Crystal Violet is used as the photosensitizer, the mechanism of macromolecular damage does not require the involvement of molecular oxygen to operate. This finding makes Crystal Violet a potential candidate for use in photochemotherapy of hypoxic or poorly perfused tumor areas.

Solvent and concentration effects on the visible spectra of tri-para-dialkylamino-substituted triarylmethane dyes in liquid solutions.

We have characterized the spectroscopy properties of crystal violet (CV+) and ethyl violet (EV+) in liquid solutions as a function of the solvent type and dye concentration. The analysis of how solvent properties and dye concentration affects the electronic spectra of these tri-para-dialkylamino substituted tryarylmethane (TAM+) dyes was performed on the basis of two spectroscopic parameters, namely the difference in wavenumber (deltanu) between the maximum and the shoulder that appears in the short-wavelength side of the respective maximum visible band (deltanu = 1/lambda(shoulder)-1/lambda(max) cm(-1)), and the wavelength of the maximum absorption (lambda(max)). The solvent and the concentration effects on lambda(max) and deltanu have indicated that both solute/solute (ion-pairing and dye aggregation) and solute/solvent (H-bonding type) interactions modulate the shape of the visible electronic spectra of these dyes in solution. In solvent with small dieletric constant (epsilon < approximately 10), the formation of ion-pairs represents a major contribution to the shaping of these spectra. Upon increasing dye concentration the formation of ion-pairs was characterized by an increase in deltanu observed concomitantly with a red shift in lambda(max) In chloroform and chlorobenzene the ion-pair association constant of CV+ and EV+ with Cl- ions were found to be in the order of 10(6) and 10(5) M(-1), respectively. In trichloroethylene the association constant for the CV+Cl- pair was 10(8) M(-1). In water, dye aggregation instead of ion-pairing represents a major contribution to the shaping of the visible spectra of CV+ and EV+. Dye aggregation was indicated by an increase in deltanu observed concomitantly with a blue shift in lambda(max) upon increasing dye concentration. The distinct behavior of deltanu for dye aggregation and ion-pairing as a function of dye concentration can therefore assist in the characterization of these two distinct phenomena. The solute/solvent interactions were studied in a series of polar solvents in which solute/solute interactions do not occur in any detectable extent. The dependence found for deltanu as a function of the Kamlet-Tafts solvatochromic parameters (alpha, beta and pi*) is in keeping with previous inferences indicating that the splitting in the overlapped absorption band of CV+ and EV+ in hydroxilated solvents arises from a perturbation in the molecular symmetry induced by hydrogen bonding (donor-acceptor) type interactions with solvent molecules. A distinction between the effects of solute/solute and solute/solvent interactions on the visible spectra of these dyes is provided.

Polymeric micelles for apoptosis-targeted optical imaging of cancer and intraoperative surgical guidance.

In a two-step strategy, an intraperitoneal (IP) injection of poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-b-PCL) micelles containing paclitaxel (PTX), cyclopamine (CYP), and gossypol (GSP) at 30, 30, and 30 mg/kg, respectively, debulked tumor tissues by 1.3-fold, based on loss of bioluminescence with <10% body weight change, and induced apoptosis in peritoneal tumors when used as neoadjuvant chemotherapy (NACT) in an ES-2-luc-bearing xenograft model for ovarian cancer. In a second step, a single intravenous (i.v.) injection of apoptosis-targeting GFNFRLKAGAKIRFGS-PEG-b-PCL micelles containing a near-infrared (NIR) fluorescence probe, DiR (1,1'-dioctadecyltetramethyl indotricarbocyanine iodide), resulted in increased peritoneal DiR accumulation in apoptosis-induced ES-2-luc tumor tissues (ex vivo) by 1.5-fold compared with DiR molecules delivered by methoxy PEG-b-PCL micelles (non-targeted) at 48 h after i.v. injection in a second step. As a result, a tandem of PEG-b-PCL micelles enabled high-resolution detection of ca. 1 mm diameter tumors, resulting in resection of approximately 90% of tumors, and a low peritoneal cancer index (PCI) of ca. 7. Thus, a tandem of PEG-b-PCL micelles used for NCAT and NIR fluorescence imaging of therapy-induced apoptosis for intraoperative surgical guidance may be a promising treatment strategy for metastatic ovarian cancer.

Major determinants of photoinduced cell death: Subcellular localization versus photosensitization efficiency.

We present a study on whether and to what extent subcellular localization may compete favorably with photosensitization efficiency with respect to the overall efficiency of photoinduced cell death. We have compared the efficiency with which two cationic photosensitizers, namely methylene blue (MB) and crystal violet (CV), induce the photoinduced death of human cervical adenocarcinoma (HeLa) cells. Whereas MB is well known to generate singlet oxygen and related triplet excited species with high quantum yields in a variety of biological and chemical environments (i.e., acting as a typical type II photosensitizer), the highly mitochondria-specific CV produces triplet species and singlet oxygen with low yields, acting mostly via the classical type I mechanism (e.g., via free radicals). The findings described here indicate that the presumably more phototoxic type II photosensitizer (MB) does not lead to higher degrees of cell death compared to the type I (CV) photosensitizer. In fact, CV kills cells with the same efficiency as MB, generating at least 10 times fewer photoinduced reactive species. Therefore, subcellular localization is indeed more important than photochemical reactivity in terms of overall cell killing, with mitochondrial localization representing a highly desirable property for the development of more specific/efficient photosensitizers for photodynamic therapy applications.

Search for cytotoxic agents in multiple Laurencia complex seaweed species (Ceramiales, Rhodophyta) harvested from the Atlantic Ocean with emphasis on the Brazilian State of Espírito Santo

The development of new anti-cancer drugs of algal origin represents one of the least explored frontiers in medicinal chemistry. In this regard, the diversity of micro- and macroalgae found in Brazilian coastal waters can be viewed as a largely untapped natural resource. In this report, we describe a comparative study on the cytotoxic properties of extracts obtained from the Laurencia complex: Laurencia aldingensis, L. catarinensis, L. dendroidea, L. intricata, L. translucida, L. sp, and Palisada flagellifera. All of these species were collected in the coastal waters of the State of Espírito Santo, Brazil. Four out of the twelve samples initially investigated were found to show significant levels of toxicity towards a model tumor cell line (human uterine sarcoma, MES-SA). The highest levels of cytotoxicity were typically associated with non-polar (hexane) algal extracts, while the lowest levels of cytotoxicity were found with the corresponding polar (methanol) extracts. In this report, we also describe a biological model currently in development that will not only facilitate the search for new anti-cancer drug candidates of algal origin, but also permit the identification of compounds capable of inducing the destruction of multi-drug resistant tumors with greater efficiency than the pharmaceuticals currently in clinical use.

Effect of molecular structure on the selective phototoxicity of triarylmethane dyes towards tumor cells.

In response to transmembrane potentials which are negative on the inner side of both the plasma and mitochondrial membranes, cationic dyes displaying appropriate structural features naturally accumulate in the cytosol and inside the mitochondria. Because enhanced mitochondrial membrane potential is a prevalent tumor cell phenotype, a number of cationic dyes preferentially accrue and are retained for longer periods in the mitochondria of tumor cells as compared to normal cells. The opportunities brought about by this phenomenon in chemo- and photochemotherapy of neoplastic diseases is highlighted by the observation that the phototoxic effects associated with some of the cationic photosensitizers known to accumulate in cell mitochondria are much more pronounced in tumor cells than in normal cells. However, the structural determinants of selective phototoxicity towards tumor cells are not well understood, and the lack of a robust model to describe the relationship between molecular structure and tumor selectivity has prevented mitochondrial targeting from becoming a more dependable therapeutic strategy. In this report we describe how the lipophilic/hydrophilic character of a series of cationic triarylmethane dyes affects the selectivity with which these photosensitizers mediate the destruction of tumor cells. Our results indicated that only the more hydrophilic triarylmethanes show tumor selectivity, presumably because these are the only dyes capable of staining energized mitochondria with a high degree of specificity. The partition of the more lipophilic dyes into a variety of extra-mitochondrial subcellular compartments occurs with comparable efficiencies in tumor and in normal cells, and this less specific subcellular localization precludes tumor selectivity from taking place.