Improving Dermal Delivery of Rose Bengal by Deformable Lipid Nanovesicles for Topical Treatment of Melanoma

Cutaneous melanoma is one of the most aggressive and metastatic forms of skin cancer. However, current therapeutic options present several limitations, and the annual death rate due to melanoma increases every year. Dermal delivery of nanomedicines can effectively eradicate primary melanoma lesions, avoid the metastatic process, and improve survival. Rose Bengal (RB) is a sono-photosensitizer drug with intrinsic cytotoxicity toward melanoma without external stimuli but the biopharmaceutical profile limits its clinical use. Here, we propose deformable lipid nanovesicles, also known as transfersomes (TF), for the targeted dermal delivery of RB to melanoma lesions to eradicate them in the absence of external stimuli. Considering RB's poor ability to cross the stratum corneum and its photosensitizer nature, transfersomal carriers were selected simultaneously to enhance RB penetration to the deepest skin layers and protect RB from undesired photodegradation. RB-loaded TF dispersion (RB-TF), prepared by a modified reverse-phase evaporation method, were nanosized with a ζ-potential value below -30 mV. The spectrophotometric and fluorimetric analysis revealed that RB efficiently interacted with the lipid phase. The morphological investigations (transmission electron microscopy and small-angle X-ray scattering) proved that RB intercalated within the phospholipid bilayer of TF originating unilamellar and deformable vesicles, in contrast to the rigid multilamellar unloaded ones. Such outcomes agree with the results of the in vitro permeation study, where the lack of a burst RB permeation peak for RB-TF, observed instead for the free drug, suggests that a significant amount of RB interacted with lipid nanovesicles. Also, RB-TF proved to protect RB from undesired photodegradation over 24 h of direct light exposure. The ex vivo epidermis permeation study proved that RB-TF significantly increased RB's amount permeating the epidermis compared to the free drug (78.31 vs 38.31%). Finally, the antiproliferative assays on melanoma cells suggested that RB-TF effectively reduced cell growth compared to free RB at the concentrations tested (25 and 50 μM). RB-TF could potentially increase selectivity toward cancer cells. Considering the outcomes of the characterization and cytotoxicity studies performed on RB-TF, we conclude that RB-TF represents a valid potential alternative tool to fight against primary melanoma lesions via dermal delivery in the absence of light

Granitos anatécticos de las Sierras Pampeanas de Córdoba (Argentina): edades U-Pb SHRIMP y estudio LAICP- MS de elementos traza en circón de metamorfismo y cristalización diacrónicos

In this contribution we present new U-Pb SHRIMP ages and in situ LA-ICP-MS trace element geochemistry of zircon crystals from the Río de los Sauces anatectic granite, Córdoba, Argentina. Notable difference in texture and composition allowed us to identify two zircon populations in a single granite sample that are interpreted as reflecting metamorphic and igneous origins. Zircons regarded as restitic crystals entrained during the melt segregation yielded a slightly older concordia age of 537.1 ± 4.8 Ma (2σ) than those interpreted as igneous, dated at 529 ± 6 (2σ) Ma. Inherited metamorphic zircons are interpreted to represent solid-state growth during high temperature metamorphism of the Pampean orogeny at the onset of the anatexis or metamorphic peak. By contrast, igneous zircons would record the crystallization age of Zr within the Río de los Sauces granite. The textural, compositional and geochronological data of both zircon populations suggest that the inception of the anatexis, the melt segregation and crystallization occurred during a short period of time of 8 myEn este trabajo se aportan nuevos datos de edades U-Pb SHRIMP y análisis in situ LA-ICP-MS de elementos traza de circones provenientes del granito Río de los Sauces, Córdoba, Argentina. A partir de marcadas diferencias texturales y composicionales se pudieron identificar dos poblaciones de circones en una misma muestra del granito, las cuales sugieren orígenes metamórficos e ígneos. Las edades concordia obtenidas en los circones metamórficos e ígneos fueron de 537,1 ± 4,8 Ma (2σ) y 529 ± 6 (2σ) Ma, respectivamente. Se interpreta que los circones metamórficos representan el crecimiento en estado sólido durante el metamorfismo de alta temperatura de la orogenia Pampeana, durante el inicio o el clímax de la anatexia. Por su parte, las edades de los circones ígneos representan la edad de cristalización del granito Río de los Sauces. Los datos texturales, composicionales y geocronológicos de ambas poblaciones de circones sugieren que el inicio de la anatexia, la segregación del fundido y la cristalización ocurrieron durante un periodo breve de tiempo de 8 ma

Geochemical and metallogenetical study of the pegmatites and associated granites from southern Comechingones pegmatitic field, Córdoba

El distrito pegmatítico Comechingones, ubicado en el faldeo oriental de la sierra homónima, en la provincia de Córdoba, involucra pegmatitas graníticas correspondientes a la clase de Elementos Raros, tipo berilo, subtipo berilo-columbita-fosfatos, algunas en transición a la clase muscovítica, con mineralizaciones de Be-Nb- Ta-U y minerales industriales. Dos tipos de pegmatitas graníticas han sido descriptas en el sector sur del distrito: pegmatitas tipo I, con tamaños que en total pueden alcanzan los 1000 metros de longitud y superar los 50 de ancho, internamente zonadas y portadoras de Be, Nb-Ta y U; y pegmatitas tipo II, de menores dimensiones, no zonadas, ricas en cuarzo de alta pureza, carentes de mineralizaciones metalíferas, y asociadas espacial y genéticamente con leucogranitos aplíticos. En este trabajo se presentan y discuten los datos geoquímicos preliminares de ambos tipos de pegmatitas y granitos asociados. Los datos geoquímicos obtenidos, apoyados con descripciones de campo y petrográficas, permiten establecer que las dos tipologías de pegmatitas corresponden a dos eventos magmáticos diferentes (muy probablemente diacrónicos). El primero generó las pegmatitas tipo I, las cuales de sur a norte presentan un aumento en el grado de fraccionamiento desde pegmatitas poco evolucionadas y sin mineralizaciones de elementos raros, hasta pegmatitas evolucionadas con depósitos metalíferos de interés económico. El segundo evento dio origen a las pegmatitas tipo II y a los granitos aplíticos, y carece de especialización metalogenética, evidenciado por los indicadores de diferenciación magmática sistemáticamente inferiores a los de las pegmatitas tipo I y a la carencia de mineralizaciones metalíferas.The Comechingones pegmatitic field (CPF) is located in the eastern flank of the Sierra de Comechingones, Córdoba province. It is composed of granite pegmatites belonging to the Rare-Element class, beryl type, beryl-columbite-phosphate subtype; some of them are transitional into the Muscovite class. Beryllium, Nb, Ta and U deposits, as well as high-quality industrial mineral deposits, are frequently associated with these pegmatites. In the southern part of the CPF two different pegmatite types have been described. Type I pegmatites constitute large zoned bodies with up to 1000 m long and 50 m thick, and may constitute rare element deposits, whereas type II pegmatites occur as small, unzoned quartz-rich dykes, without metalliferous mineralizations, spatial and genetically associated with aplitic leucogranites. Preliminary geochemical data from both pegmatites types and granites are presented and discussed in this contribution. Geochemical evidences, supported by field and petrographic observations, suggest that the two types of pegmatites identified in the study area represent two different, probably diachronic, magmatic stages. Type I pegmatites display a geochemical gradation in a S-N direction, from barren pegmatites in the south to fractionated pegmatites in the northern part of the study area, and are the lithological product of the first magmatic stage. The second stage lead to the crystallization of aplitic granites and barren type II pegmatites, geochemically less fractionated than type II pegmatites.Centro de Investigaciones Geológica

A Single-Molecule Bioelectronic Portable Array for Early Diagnosis of Pancreatic Cancer Precursors

A cohort of 47 patients is screened for pancreatic cancer precursors with a portable 96-well bioelectronic sensing-array for single-molecule assay in cysts fluid and blood plasma, deployable at point-of-care (POC). Pancreatic cancer precursors are mucinous cysts diagnosed with a sensitivity of at most 80% by state-of-the-art cytopathological molecular analyses (e.g., KRASmut DNA). Adding the simultaneous assay of proteins related to malignant transformation (e.g., MUC1 and CD55) is deemed essential to enhance diagnostic accuracy. The bioelectronic array proposed here, based on single-molecule-with-a-large-transistor (SiMoT) technology, can assay both nucleic acids and proteins at the single-molecule limit-of-identification (LOI) (1% of false-positives and false-negatives). It comprises an enzyme-linked immunosorbent assay (ELISA)-like 8 × 12-array organic-electronics disposable cartridge with an electrolyte-gated organic transistor sensor array, and a reusable reader, integrating a custom Si-IC chip, operating via software installed on a USB-connected smart device. The cartridge is complemented by a 3D-printed sensing gate cover plate. KRASmut, MUC1, and CD55 biomarkers either in plasma or cysts-fluid from 5 to 6 patients at a time, are multiplexed at single-molecule LOI in 1.5 h. The pancreatic cancer precursors are classified via a machine-learning analysis resulting in at least 96% diagnostic-sensitivity and 100% diagnostic-specificity. This preliminary study opens the way to POC liquid-biopsy-based early diagnosis of pancreatic-cancer precursors in plasma

Characterisation and radioimmunotherapy of L19-SIP, an anti-angiogenic antibody against the extra domain B of fibronectin, in colorectal tumour models

Angiogenesis is a characteristic feature of tumours and other disorders. The human monoclonal antibody L19- SIP targets the extra domain B of fibronectin, a marker of angiogenesis expressed in a range of tumours. The aim of this study was to investigate whole body distribution, tumour localisation and the potential of radioimmunotherapy with the L19-small immunoprotein (SIP) in colorectal tumours. Two colorectal tumour models with highly different morphologies, the SW1222 and LS174T xenografts, were used in this study. Localisation and retention of the L19-SIP antibody at tumour vessels was demonstrated using immunohistochemistry and Cy3-labelled L19-SIP. Whole body biodistribution studies in both tumour models were carried out with 125I-labelled L19-SIP. Finally, 131I-labelled antibody was used to investigate the potential of radioimmunotherapy in SW1222 tumours. Using immunohistochemistry, we confirmed extra domain B expression in the tumour vasculature. Immunofluorescence demonstrated localisation and retention of injected Cy3-labelled L19-SIP at the abluminal side of tumour vessels. Biodistribution studies using a 125I-labelled antibody showed selective tumour uptake in both models. Higher recorded values for localisation were found in the SW1222 tumours than in the LS174T (7.9 vs 6.6 %ID g−1), with comparable blood clearance for both models. Based on these results, a radioimmunotherapy study was performed in the SW1222 xenograft using 131I-Labelled L19-SIP (55.5 MBq), which showed selective tumour uptake, tumour growth inhibition and improved survival. Radio- and fluorescence-labelled L19-SIP showed selective localisation and retention at vessels of two colorectal xenografts. Furthermore, 131I-L19-SIP shows potential as a novel treatment of colorectal tumours, and provides the foundation to investigate combined therapies in the same tumour models

Molecular imaging of angiogenesis with SPECT

Single-photon emission computed tomography (SPECT) and position emission tomography (PET) are the two main imaging modalities in nuclear medicine. SPECT imaging is more widely available than PET imaging and the radionuclides used for SPECT are easier to prepare and usually have a longer half-life than those used for PET. In addition, SPECT is a less expensive technique than PET. Commonly used gamma emitters are: 99mTc (Emax 141 keV, T1/2 6.02 h), 123I (Emax 529 keV, T1/2 13.0 h) and 111In (Emax 245 keV, T1/2 67.2 h). Compared to clinical SPECT, PET has a higher spatial resolution and the possibility to more accurately estimate the in vivo concentration of a tracer. In preclinical imaging, the situation is quite different. The resolution of microSPECT cameras (<0.5 mm) is higher than that of microPET cameras (>1.5 mm). In this report, studies on new radiolabelled tracers for SPECT imaging of angiogenesis in tumours are reviewed

Human monoclonal antibodies targeting carbonic anhydrase IX for the molecular imaging of hypoxic regions in solid tumours

BACKGROUND: Hypoxia, which is commonly observed in areas of primary tumours and of metastases, influences response to treatment. However, its characterisation has so far mainly been restricted to the ex vivo analysis of tumour sections using monoclonal antibodies specific to carbonic anhydrase IX (CA IX) or by pimonidazole staining, after the intravenous administration of this 2-nitroimidazole compound in experimental animal models.METHODS: In this study, we describe the generation of high-affinity human monoclonal antibodies (A3 and CC7) specific to human CA IX, using phage technology.RESULTS: These antibodies were able to stain CA IX ex vivo and to target the cognate antigen in vivo. In one of the two animal models of colorectal cancer studied (LS174T), CA IX imaging closely matched pimonidazole staining, with a preferential staining of tumour areas characterised by little vascularity and low perfusion. In contrast, in a second animal model (SW1222), distinct staining patterns were observed for pimonidazole and CA IX targeting. We observed a complementary pattern of tumour regions targeted in vivo by the clinical-stage vascular-targeting antibody L19 and the anti-CA IX antibody A3, indicating that a homogenous pattern of in vivo tumour targeting could be achieved by a combination of the two antibodies.CONCLUSION: The new human anti-CA IX antibodies are expected to be non-immunogenic in patients with cancer and may serve as broadly applicable reagents for the non-invasive imaging of hypoxia and for pharmacodelivery applications. British Journal of Cancer (2009) 101, 645-657. doi: 10.1038/sj.bjc.6605200 www.bjcancer.com Published online 21 July 2009 (C) 2009 Cancer Research U

ENDODONTIC TREATMENT AND HYPOTHESES ON AN UNUSUAL CASE OF DENS INVAGINATUS

This work describes a case of ‘‘dens invaginatus’’ and analyzes the possible aspects of this malformation. An unusual type of dens invaginatus was detected in a young patient corresponding to the maxillary lateral incisor and showing extensive periradicular radiolucency and a vestibular fistula. The radiographic and tomographic examination revealed two apices: one wide open in the distal part of the root and the other normally formed in the mesial. Nonsurgical endodontic treatment was performed by using the ‘‘one-step apexification technique,’’ filling both apexes with mineral trioxide aggregate followed by composite resin. The follow-up examination 6 months later showed the healing of the radiolucent area and the healing of the sinus tract. Hypotheses on which was the type of dens invaginatus we had to deal with are formulated. (J Endod 2009;35: 417–42

Synergistic trans-activation of the human C-reactive protein promoter by transcription factor HNF-1 binding at two distinct sites.

The promoter region of the human C-reactive protein (CRP) gene comprises two distinct regions (APREs, for Acute Phase Responsive Elements) each one containing information necessary and sufficient for liver specific and IL-6 inducible expression in human hepatoma Hep3B cells. In this paper we show that both APREs contain a low affinity binding site for the liver specific transcription factor HNF-1/LF-B1. The two sites are separated by approximately 80 bp. Mutations in either of the two sites abolish inducible expression. The same effect is specifically obtained in cotransfection competition experiments when the human albumin HNF-1 site is used as competitor. However, HNF-1 is not the intranuclear mediator of IL-6 because synthetic promoters formed by multimerized copies of different HNF-1 binding sites are not transcriptionally activated by this cytokine. An expression vector encoding full length HNF-1 is capable of trans-activating transcription from the wild-type CRP promoter but not from mutants which have lost the ability to bind HNF-1. Moreover, the level of trans-activation observed with the natural promoter containing both HNF-1 binding sites is far greater than the level of mutated variants containing only one of the two sites. This result strongly suggests that two HNF-1 molecules bound simultaneously to sites distant from each other can act synergistically to activate gene expression