70 research outputs found
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
IFNL3 polymorphisms and HCV infection in patients with beta thalassemia
Background and relationale for the study. Genome-wide association studies have identified host genetic variation to be critical for spontaneous clearance and treatment response in patients infected with hepatitis C virus. Recently, the role of the IFNL3 polymorphisms in influencing the spontaneous clearance of HCV, the response to interferon and the progression of liver fibrosis, was also demonstrated in patients with thalassemia major infected by genotype 1b. In the present study we retrospectively analyzed 368 anti-HCV positive patients with beta-thalassemia at two Italian major centers in Cagliari and Torino. Results. C/C variant of polymorphism rs12979860 was related to response to interferon treatment and, above all, to spontaneous clearance of the virus. However, the positive predictive power was stronger for viral persistence than spontaneous clearance and in such respect the TT allele was more predictive than CC. The methylation associated polymorphism rs4803221 had independent effects with respect to rs12979860 and the haplotype tagged by SNP rs12979860 and rs4803221 significantly could improve the viral clearance prediction in infected patients. Neither necroinflammation or bilirubin values in the chronic phase of the hepatitis C were related to IFNL3 polymorphisms. No relation among IFNL3 polymorphisms and fibrosis stage directly shown by the liver biopsy was found. Conclusions. Also in thalassemia the SNPs on chromosome 19q13 closely associates with spontaneous and treatment-induced HCV clearance. The viral clearance prediction is significantly improved by the haplotype tagged by SNP rs12979860 and rs4803221. Neither necroinflammation, bilirubin values or fibrosis stage seem to be related to IFNL3 polymorphisms
Inhomogeneous Diastereomeric Composition of Mongersen Antisense Phosphorothioate Oligonucleotide Preparations and Related Pharmacological Activity Impairment
Mongersen is a 21-mer antisense oligonucleotide designed to downregulate Mothers against decapentaplegic homolog 7 (SMAD7) expression to treat Crohn's disease. Mongersen was manufactured in numerous batches at different scales during several years of clinical development, which all appeared identical, using common physicochemical analytical techniques, while only phosphorous-31 nuclear magnetic resonance (P-31-NMR) in solution showed marked differences. Close-up analysis of 27 mongersen batches revealed marked differences in SMAD7 downregulation in a cell-based assay. Principal component analysis of P-31-NMR profiles showed strong correlation with SMAD7 downregulation and, therefore, with pharmacological efficacy in vitro. Mongersen contains 20 phosphorothioate (PS) linkages, whose chirality (Rp/Sp) was not controlled during manufacturing. A different diastereomeric composition throughout batches would lead to superimposable analytical data, but to distinct P-31-NMR profiles, as indeed we found. We tentatively suggest that this may be the origin of different biological activity. As similar manifolds are expected for other PS-based oligonucleotides, the protocol described here provides a general method to identify PS chirality issues and a chemometric tool to score each preparation for this elusive feature
The westward lithospheric drift, its role on the subduction and transform zones surrounding Americas. Andean to cordilleran orogenic types cyclicity
We investigate the effect of the westerly rotation of the lithosphere on the active margins that surround the Americas and find good correlations between the inferred easterly-directed mantle counterflow and the main structural grain and kinematics of the Andes and Sandwich arc slabs. In the Andes, the subduction zone is shallow and with low dip, because the mantle flow sustains the slab; the subduction hinge converges relative to the upper plate and generates an uplifting doubly verging orogen. The Sandwich Arc is generated by a westerly-directed SAM (South American) plate subduction where the eastward mantle flow is steepening and retreating the subduction zone. In this context, the slab hinge is retreating relative to the upper plate, generating the backarc basin and a low bathymetry single-verging accretionary prism. In Central America, the Caribbean plate presents a more complex scenario: a) To the East, the Antilles Arc is generated by westerly directed subduction of the SAM plate, where the eastward mantle flow is steepening and retreating the subduction zone. b) To the West, the Middle America Trench and Arc are generated by the easterly-directed subduction of the Cocos plate, where the shallow subduction caused by eastward mantle flow in its northern segment gradually steepens to the southern segment as it is infered by the preexisting westerly-directed subduction of the Caribbean Plateau. In the frame of the westerly lithospheric flow, the subduction of a divergent active ridge plays the role of introducing a change in the oceanic/continental plate's convergence angle, such as in NAM (North American) plate with the collision with the Pacific/Farallon active ridge in the Neogene (Cordilleran orogenic type scenario). The easterly mantle drift sustains strong plate coupling along NAM, showing at Juan de Fuca easterly subducting microplate that the subduction hinge advances relative to the upper plate. This lower/upper plate convergence coupling also applies along strike to the neighbor continental strike slip fault systems where subduction was terminated (San Andreas and Queen Charlotte). The lower/upper plate convergence coupling enables the capture of the continental plate ribbons of Baja California and Yakutat terrane by the Pacific oceanic plate, transporting them along the strike slip fault systems as para-autochthonous terranes. This Cordilleran orogenic type scenario, is also recorded in SAM following the collision with the Aluk/Farallon active ridge in the Paleogene, segmenting SAM margin into the eastwardly subducting Tupac Amaru microplate intercalated between the proto-Liquiñe-Ofqui and Atacama strike slip fault systems, where subduction was terminated and para-autochthonous terranes transported. In the Neogene, the convergence of Nazca plate with respect to SAM reinstalls subduction and the present Andean orogenic type scenario
Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication
Directed evolution is a powerful tool to improve the characteristics of biomolecules. Here we present a protocol for the intracellular evolution of proteins with distinct differences and advantages in comparison with established techniques. These include the ability to select for a particular function from a library of protein variants inside cells, minimizing undesired coevolution and propagation of nonfunctional library members, as well as allowing positive and negative selection logics using basally active promoters. A typical evolution experiment comprises the following stages: (i) preparation of a combinatorial M13 phagemid (PM) library expressing variants of the gene of interest (GOI) and preparation of the Escherichia coli host cells; (ii) multiple rounds of an intracellular selection process toward a desired activity; and (iii) the characterization of the evolved target proteins. The system has been developed for the selection of new orthogonal transcription factors (TFs) but is capable of evolving any gene—or gene circuit function—that can be linked to conditional M13 phage replication. Here we demonstrate our approach using as an example the directed evolution of the bacteriophage λ cI TF against two synthetic bidirectional promoters. The evolved TF variants enable simultaneous activation and repression against their engineered promoters and do not cross-react with the wild-type promoter, thus ensuring orthogonality. This protocol requires no special equipment, allowing synthetic biologists and general users to evolve improved biomolecules within ~7 weeks
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 141Â keV, T1/2 6.02Â h), 123I (Emax 529Â keV, T1/2 13.0Â h) and 111In (Emax 245Â keV, T1/2 67.2Â 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 (<0.5Â mm) is higher than that of microPET cameras (>1.5Â mm). In this report, studies on new radiolabelled tracers for SPECT imaging of angiogenesis in tumours are reviewed
Inhibition of IL-6+IL-6 soluble receptor-stimulated aromatase activity by the IL-6 antagonist, Sant 7, in breast tissue-derived fibroblasts
Post-translational deregulation of YAP1 is genetically controlled in rat liver cancer and determines the fate and stem-like behavior of the human disease
Previous studies showed that YAP1 is over-expressed in hepatocellular carcinoma
(HCC). Here we observed higher expression of Yap1/Ctgf axis in dysplastic nodules and
HCC chemically-induced in F344 rats, genetically susceptible to hepatocarcinogenesis,
than in lesions induced in resistant BN rats. In BN rats, highest increase in Yap1-
tyr357, p73 phosphorylation and Caspase 3 cleavage occurred. In human HCCs with
poorer prognosis (< 3 years survival after partial liver resection, HCCP), levels of
YAP1, CTGF, 14–3–3, and TEAD proteins, and YAP1-14-3-3 and YAP1-TEAD complexes
were higher than in HCCs with better outcome (> 3 years survival; HCCB). In the
latter, higher levels of phosphorylated YAP1-ser127, YAP1-tyr357 and p73, YAP1
ubiquitination, and Caspase 3 cleavage occurred. Expression of stemness markers
NANOG, OCT-3/4, and CD133 were highest in HCCP and correlated with YAP1 and
YAP1-TEAD levels. In HepG2, Huh7, and Hep3B cells, forced YAP1 over-expression
led to stem cell markers expression and increased cell viability, whereas inhibition
of YAP1 expression by specific siRNA, or transfection of mutant YAP1 which does
not bind to TEAD, induced opposite alterations. These changes were associated, in
Huh7 cells transfected with YAP1 or YAP1 siRNA, with stimulation or inhibition of cell
migration and invasivity, respectively. Furthermore, transcriptome analysis showed
that YAP1 transfection in Huh7 cells induces over-expression of genes involved
in tumor stemness. In conclusion, Yap1 post-translational modifications favoring
its ubiquitination and apoptosis characterize HCC with better prognosis, whereas
conditions favoring the formation of YAP1-TEAD complexes are associated with
aggressiveness and acquisition of stemness features by HCC cells
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
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