Novel pH-Sensitive Cyclic Peptides

A series of cyclic peptides containing a number of tryptophan (W) and glutamic acid (E) residues were synthesized and evaluated as pH-sensitive agents for targeting of acidic tissue and pH-dependent cytoplasmic delivery of molecules. Biophysical studies revealed the molecular mechanism of peptides action and localization within the lipid bilayer of the membrane at high and low pHs. The symmetric, c[(WE)4WC], and asymmetric, c[E4W5C], cyclic peptides translocated amanitin, a polar cargo molecule of similar size, across the lipid bilayer and induced cell death in a pH- and concentration-dependent manner. Fluorescently-labelled peptides were evaluated for targeting of acidic 4T1 mammary tumors in mice. The highest tumor to muscle ratio (5.6) was established for asymmetric cyclic peptide, c[E4W5C], at 24 hours after intravenous administration. pH-insensitive cyclic peptide c[R4W5C], where glutamic acid residues (E) were replaced by positively charged arginine residues (R), did not exhibit tumor targeting. We have introduced a novel class of cyclic peptides, which can be utilized as a new pH-sensitive tool in investigation or targeting of acidic tissue

Targeted Imaging of Urothelium Carcinoma in Human Bladders by an ICG pHLIP Peptide Ex vivo

Bladder cancer is the fifth most common in incidence and one of the most expensive cancers to treat. Early detection greatly improves the chances of survival and bladder preservation. The pH Low Insertion Peptide (pHLIP® peptide) conjugated with a near infrared fluorescent dye (ICG) targets low extracellular pH allowing visualization of malignant lesions in human bladder carcinoma ex vivo. Cystectomy specimens obtained after radical surgery were immediately irrigated with non-buffered saline and instilled with a solution of the ICG pHLIP® construct, incubated, and rinsed. Bladders were subsequently opened and imaged, the fluorescent spots were marked, and a standard pathological analysis was carried out to establish the correlation between ICG pHLIP® imaging and white light pathological assessment. Accurate targeting of bladder lesions was achieved with a sensitivity of 97%. Specificity is 100%, but reduced to 80%, if targeting of necrotic tissue from previous transurethral resections or chemotherapy are considered as false positives. ICG pHLIP® imaging agent marked high grade urothelial carcinomas, both muscle invasive and non-muscle invasive. Carcinoma in situ (CIS) was accurately diagnosed in 11 cases, whereas only 4 cases were seen using white light, so imaging with the ICG pHLIP® peptide offers improved early diagnosis of bladder cancers, and may also enable new treatment alternatives

Protective Ag :TiO2 thin films for pressure sensors in orthopedic prosthesis: the importance of composition, structural and morphological features on the biological response of the coatings

DC reactive magnetron sputtered Ag:TiO2 nanocomposite thin films were developed to be used as protective coatings in pressure sensor devices. The coatings, with Ag content varying from 0 to about 30 at.%, were prepared and characterized in order to study their biological response. The as-deposited samples were annealed in vacuum at 500 °C in order to evaluate the influence of their morphological and structural differences over the response elicited upon contact with simulated bodily fluids and cultured human cells, as well as selected microorganisms. The results showed that the annealing treatment produced less porous films with an enhanced structure, with a significant reduction in structural defects and improved crystallinity. Additionally, samples with higher Ag contents (≥12.8 at.%) exhibited Ag agglomerates/clusters at the surface, a result anticipated from the XRD data. The crystallization of the TiO2 matrix was also observed by XRD analysis, albeit delayed by the dispersion of Ag into the matrix. Biological characterization showed that the antimicrobial activity and cytotoxicity of the coatings were directly related with their composition, closely followed by the particular structural and morphological features, namely those resulting from annealing process.This research is partially sponsored by FEDER funds through the program COMPETE—Programa Operacional Factores de Competitividade and by national funds through FCT—Fundação para a Ciência e a Tecnologia, under the projects PEst-C/EME/UI0285/2011, PTDC/SAU-ENB/116850/2010, PTDC/CTM-NAN/112574/2009P. T Matamá acknowledges FCT for Grant SFRH/BPD/47555/2008

Comparative Study of Tumor Targeting and Biodistribution of pH (Low) Insertion Peptides (pHLIP® Peptides) Conjugated with Different Fluorescent Dyes

Purpose Acidification of extracellular space promotes tumor development, progression, and invasiveness. pH (low) insertion peptides (pHLIP® peptides) belong to the class of pH-sensitive membrane peptides, which target acidic tumors and deliver imaging and/or therapeutic agents to cancer cells within tumors. Procedures Ex vivo fluorescent imaging of tissue and organs collected at various time points after administration of different pHLIP® variants conjugated with fluorescent dyes of various polarity was performed. Methods of multivariate statistical analyses were employed to establish classification between fluorescently labeled pHLIP® variants in multidimensional space of spectral parameters. Results The fluorescently labeled pHLIP® variants were classified based on their biodistribution profile and ability of targeting of primary tumors. Also, submillimeter-sized metastatic lesions in lungs were identified by ex vivo imaging after intravenous administration of fluorescent pHLIP® peptide. Conclusions Different cargo molecules conjugated with pHLIP® peptides can alter biodistribution and tumor targeting. The obtained knowledge is essential for the design of novel pHLIP®-based diagnostic and therapeutic agents targeting primary tumors and metastatic lesions

Structural and optical properties of Ag: TiO2 nanocomposite films prepared by magnetron sputtering

Three sets of nanocomposite films consisting of different atomic concentrations of Ag dispersed in a TiO2 dielectric matrix were deposited by DC reactive magnetron sputtering, and subjected to several thermal annealing experiments in vacuum, for temperatures ranging from 200 to 600 ºC. The main goal of the present study is to analyse the optical properties of the as-deposited and annealed films in order to clarify the role of Ag inclusions in the TiO2 dielectric matrix. The influence of the thermal annealing in the structural and morphological evolution was then correlated with the changes in the optical behavior of the samples. Significant structural and morphological changes were observed, consisting on the crystallization of Ag and their clustering. Clusters growth as a function of temperature was also observed by the evolution of the diffractograms with the temperature increase. The present study allowed to conclude that at certain concentrations (close to 10 at. %), the films revealed some important changes on the optical properties, commonly known as Surface Plasmon Resonance, SPR. This change in the optical behavior of the films was found to be in accordance with the clusters growth as concluded from the evolution of the diffraction patterns. The optical changes, and the correspondent Surface Plasmon Resonance effect were confirmed by reflectivity and CIELab colour measurements. The samples with lower Ag content (11 at. % and 7 at. %) show typical interferometric behavior on the reflectivity curves, similar to those of “pure” TiO2 samples. After a minimum of reflectance at 300 nm, there was an increase of the reflectivity at higher wavelengths. For the samples annealed between 400 and 600 °C, a red-shift centered at around 500-550 nm is visible in the absorbance spectrum.Fundação para a Ciência e a Tecnologia (FCT) - PTDC/CTM/ 70037/2006

Thin films composed of Ag nanoclusters dispersed in TiO2: Influence of composition and thermal annealing on the microstructure and physical responses

Noble metal powders containing gold and silver have been used for many centuries, providing different colours in the windows of the medieval cathedrals and in ancient Roman glasses. Nowadays, the interest in nanocomposite materials containing noble nanoparticles embedded in dielectric matrices is related with their potential use for a wide range of advanced technological applications. They have been proposed for environmental and biological sensing, tailoring colour of functional coatings, or for surface enhanced Raman spectroscopy. Most of these applications rely on the so-called localised surface plasmon resonance absorption, which is governed by the type of the noble metal nanoparticles, their distribution, size and shape and as well as of the dielectric characteristics of the host matrix. The aim of this work is to study the influence of the composition and thermal annealing on the morphological and structural changes of thin films composed of Ag metal clusters embedded in a dielectric TiO2 matrix. Since changes in size, shape and distribution of the clusters are fundamental parameters for tailoring the properties of plasmonic materials, a set of films with different Ag concentrations was prepared. The optical properties and the thermal behaviour of the films were correlated with the structural and morphological changes promoted by annealing. The films were deposited by DC magnetron sputtering and in order to promote the clustering of the Ag nanoparticles the as-deposited samples were subjected to an in-air annealing protocol. It was demonstrated that the clustering of metallic Ag affects the optical response spectrum and the thermal behaviour of the films.This research was sponsored by FEDER funds through the COMPETE program (Programa Operacional Factores de Competitividade) and by FCT (Fundação para a Ciência e a Tecnologia), under the projects PEST-C/FIS/UI607/2013 and PEst-C/EME/UI0285/2013. The authors also acknowledge the financial support by the project Nano4color – Design and develop a new generation of color PVD coatings for decorative applications (FP7 EC R4SME Project No. 315286). J. Borges also acknowledges the support by the European social fund within the framework of realising the project “Support of inter-sectoral mobility and quality enhancement of research teams at Czech Technical University in Prague”, CZ.1.07/2.3.00/30.0034. C. Lopes acknowledges FCT for the PhD grant SFRH/BD/103373/2014. F.M. Couto acknowledges CAPES – Foundation, Ministry of Education of Brazil, Brasília – DF 70040-20, Brazil, funding by stage sandwich doctorate, through PDSE – Doctoral Program Sandwich

Aquatic Toxicity of Photocatalyst Nanoparticles to Green Microalgae Chlorella vulgaris

In the last years, nanoparticles such as TiO2, ZnO, NiO, CuO and Fe2O3 were mainly used in wastewater applications. In addition to the positive aspects concerning using nanoparticles in the advanced oxidation process of wastewater containing pollutants, the impact of these nanoparticles on the environment must also be investigated. The toxicity of nanoparticles is generally investigated by the nanomaterials’ effect on green algae, especially on Chlorella vulgaris. In this review, several aspects are reviewed: the Chlorella vulgaris culture monitoring and growth parameters, the effect of different nanoparticles on Chlorella vulgaris, the toxicity of photocatalyst nanoparticles, and the mechanism of photocatalyst during oxidative stress on the photosynthetic mechanism of Chlorella vulgaris. The Bold basal medium (BBM) is generally recognized as an excellent standard cultivation medium for Chlorella vulgaris in the known environmental conditions such as temperature in the range 20–30 °C and light intensity of around 150 μE·m2·s−1 under a 16/8 h light/dark cycle. The nanoparticles synthesis methods influence the particle size, morphology, density, surface area to generate growth inhibition and further algal deaths at the nanoparticle-dependent concentration. Moreover, the results revealed that nanoparticles caused a more potent inhibitory effect on microalgal growth and severely disrupted algal cells’ membranes

Toxicity of a Binary Mixture of TiO2 and Imidacloprid Applied to Chlorella vulgaris

Nanoparticles have applications in various fields such as manufacturing and materials synthesis, the environment, electronics, energy harvesting, and medicine. Besides many applications of nanoparticles, further research is required for toxic environmental effect investigation. The toxic effect of titanium dioxide nanoparticles on the physiology of the green alga Chlorella vulgaris was studied with a widely used pesticide, imidacloprid (IMD). Chlorella vulgaris was exposed for 120 h in Bold’s basal medium to different toxic compounds, such as (i) a high concentration of TiO2 nanoparticles, 150–2000 mg/L, usually optimised in the photocatalytic degradation of wastewater, (ii) an extremely toxic pesticide for the aquatic environment, imidacloprid, in concentrations ranging from 5 to 40 mg/L, (iii) TiO2 nanoparticles combined with imidacloprid, usually used in a photocatalytic system. The results show that the TiO2 nanoparticles and IMD inhibited Chlorella vulgaris cell growth and decreased the biovolume by approximately 80% when 2 g/L TiO2 was used, meaning that the cells devised a mechanism to cope with a potentially stressful situation; 120 h of Chlorella vulgaris exposure to 40 mg/L of IMD resulted in a 16% decreased cell diameter and a 41% decrease in cell volume relative to the control sample, associated with the toxic effect of pesticides on the cells. Our study confirms the toxicity of nanoparticles through algal growth inhibition with an effective concentration (EC50) value measured after 72 h of 388.14 mg/L for TiO2 and 13 mg/L for IMD in a single-toxic system. The EC50 of TiO2 slowly decreased from 258.42 to 311.11 mg/L when IMD from 5 to 20 mg/L was added to the binary-toxic system. The concentration of TiO2 in the binary-toxic system did not change the EC50 for IMD, and its value was 0.019 g/L. The photodegradation process of imidacloprid (range of 5–40 mg/L) was also investigated in the algal medium incubated with 150–600 mg/L of titanium dioxide

pH (Low) Insertion Peptide targets 4T1 mammary tumors

Malignant tumors exhibit elevated uptake and use of glucose, as well as metabolite buildup and hypoxia due to inadequate blood supply, leading to tumor acidosis. The extracellular acidity is linked to tumor progression. The most effective pH-sensitive tumor targeting agents should sense pH at the surface of cancer cells, where it is expected to be the lowest. pHLIPs® (pH (Low) Insertion Peptides) belong to the class of pH-sensitive agents capable of delivering imaging and/or therapeutic agents to cancer cells within tumors. Here, we investigated targeting of highly metastatic 4T1 mammary tumors in BALB/c mice with three Alexa546 labeled pHLIP variants including well-characterized WT-pHLIP and, recently introduced, Var3-and Var7-pHLIPs