carbocyanines and Hemolysis

An amphipathic α-helical peptide, Hp1404, was isolated from the venomous gland of the scorpion

Topics: 1-Naphthylamine; Acinetobacter baumannii; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Benzothiazoles; Biofilms; Carbocyanines; Cell Survival; Circular Dichroism; Drug Resistance, Multiple, Bacterial; Escherichia coli; Gram-Negative Bacteria; Gram-Positive Bacteria; Hemolysis; Listeria monocytogenes; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Protein Conformation, alpha-Helical; Pseudomonas aeruginosa; Salmonella; Scorpions

Timely diagnosis of acute myocardial infarction (AMI) strongly impacts the survival rate of patients. The authors report the development of a two-shell hollow silica contrast agent useful for ultrasound (US) imaging, which is able to provide ultra-early diagnosis of AMI. To target the characterization of fast blood flow and high blood pressure in the heart, two shells of hollow silica are adopted with opposite polarities, which assemble based on amino and perfluorodecyl silanes. The external amino silane facilitates the attachment of disease-targeted groups, while the internal perfluorodecyl silane provides great US imaging contrast. The material also possesses superior water dispersity, controllable morphology, low toxicity, and biodegradability both in vitro and in vivo, thus promoting its applications in the ultra-early diagnosis of AMI in rats, and is particularly useful for delineation of myocardial necrosis sites.

Topics: Animals; Carbocyanines; Cell Line; Cell Survival; Contrast Media; Early Diagnosis; Female; Fluorescein-5-isothiocyanate; Hemolysis; Male; Mice; Microscopy, Electron, Transmission; Myocardial Infarction; Nanospheres; Rats, Sprague-Dawley; Silicon Dioxide; Spectroscopy, Fourier Transform Infrared; Ultrasonography

Clinical application of cisplatin (CDDP) against various solid tumors is often limited due to its poor selectivity and severe side effect. Considering this, in our study, CDDP was incorporated in fluorescent PEG amine grafted aldehyde hyaluronic acid by imine bond and metal ion coordination bond linking and formed a complex, the complex was then self-assembled into nanoparticles in water simply. FT-IR, XRD, DLS and SEM analysis demonstrated that the nanoparticles were prepared successfully and exhibited a spherical structure with size ranged from 216.4 to 372.3 nm in diameter. CDDP releasing from the nanoparticles was in a controlled manner, and had faster release rate at lower pH, indicating the nanoparticles were responsive to tumor micro-acid environment. Since fluorescent Cy5.5 and targeting hyaluronic acid existed on the surface of the nanoparticles, CLSM images clearly showed that the nanoparticles could target and internalize into HeLa cells, and then inhibited the growth of HeLa cells. In addition, MTT, AO-EB staining, and hemolysis assay showed that the nanoparticles had good cyto-/hemo-compatibility. Hence, the nanoparticles had the potential to be used for cancer therapy and diagnosis. The further in vivo experiment will be shown in the next work. pH responsible and fluorescent Cy5.5-PEG-g-A-HA/CDDP complex nanoparticles were facilely fabricated for controlled and targeted delivery of CDDP.

Topics: Aldehydes; Antineoplastic Agents; Carbocyanines; Cell Line, Tumor; Cisplatin; Drug Carriers; Drug Delivery Systems; Fluorescent Dyes; HeLa Cells; Hemolysis; Humans; Hyaluronic Acid; Hydrogen-Ion Concentration; Imines; Nanoparticles; Neoplasms; Polyethylene Glycols; Spectroscopy, Fourier Transform Infrared; Thermogravimetry; X-Ray Diffraction

N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers have been presented as nanoscale drug/gene delivery systems and imaging probes, and the well-defined HPMA copolymers prepared via reversible addition-fragmentation chain transfer (RAFT) polymerization promote their to clinical trials, as the significant enhanced anticancer efficacy. The biosafety is another issue associated with the carriers. In this study, we prepared the linear and branched HPMA copolymers labeled with Cy5.5 via RAFT polymerization and click chemistry, and their potential biosafety was studied. The linear copolymer was prepared via RAFT polymerization mediated by the ends-functionalized peptide chain transfer agent (peptide2CTA), resulting in well-defined and block linear HPMA copolymer with molecular weight (MW) of 98 kDa. Additionally, the branched HPMA copolymer was also prepared via RAFT polymerization. Followed by Cy5.5 labeling, the two copolymers showed negative zeta potential and their accumulation into tumor was studied by in vivo optical fluorescence imaging in the nude mice with breast tumors. The biosafety studies on in vitro cytotoxicity and hemocompatibility studies, including hemolysis tests, plasma coagulation and thromboelastography assay were carried out well, demonstrating that the linear HPMA copolymer-Cy5.5 with MW around 100 kDa and biodegradable moiety in the main chain might be utilized as safe nanoscale carrier.

Topics: 3T3 Cells; Animals; Blood Coagulation; Carbocyanines; Drug Carriers; Drug Design; Female; Hemolysis; Humans; Mice; Molecular Imaging; Nanostructures; Polymerization; Polymethacrylic Acids; Safety; Thrombelastography

The antimicrobial activity of analogs obtained by substituting arginine and lysine in CL(14-25), a cationic α-helical dodecapeptide, with alanine against Porphyromonas gingivalis, a periodontal pathogen, varied significantly depending on the number and position of cationic amino acids. The alanine-substituted analogs had no hemolytic activity, even at a concentration of 1 mM. The antimicrobial activities of CL(K20A) and CL(K20A, K25A) were 3.8-fold and 9.1-fold higher, respectively, than that of CL(14-25). The antimicrobial activity of CL(R15A) was slightly lower than that of CL(14-25), suggesting that arginine at position 15 is not essential but is important for the antimicrobial activity. The experiments in which the alanine-substituted analogs bearing the replacement of arginine at position 24 and/or lysine at position 25 were used showed that arginine at position 24 was crucial for the antimicrobial activity whenever lysine at position 25 was substituted with alanine. Helical wheel projections of the alanine-substituted analogs indicate that the hydrophobicity in the vicinity of leucine at position 16 and alanines at positions 18 and/or 21 increased by substituting lysine at positions 20 and 25 with alanine, respectively. The degrees of diSC3 -5 release from P. gingivalis cells and disruption of GUVs induced by the alanine-substituted analogs with different positive charges were not closely related to their antimicrobial activities. The enhanced antimicrobial activities of the alanine-substituted analogs appear to be mainly attributable to the changes in properties such as hydrophobicity and amphipathic propensity due to alanine substitution and not to their extents of positive charge (cationicity).

Topics: Alanine; Amino Acid Sequence; Amino Acid Substitution; Animals; Anti-Infective Agents; Arginine; Benzothiazoles; Carbocyanines; Carbon-Nitrogen Lyases; Cations; Hemolysis; Inhibitory Concentration 50; Lysine; Microbial Sensitivity Tests; Molecular Sequence Data; Oligopeptides; Oryza; Peptide Fragments; Porphyromonas gingivalis; Sheep; Time Factors; Unilamellar Liposomes

For effective ovarian cancer gene therapy, systemic administrated tumor-targeting siRNA/folic acid-poly(ethylene glycol)-chitosan oligosaccharide lactate (FA-PEG-COL) nanoparticles is vital for delivery to cancer site(s). siRNA/FA-PEG-COL nanoparticles were prepared by ionic gelation for effective FA receptor-expressing ovarian cancer cells transfection and in vivo accumulation. The chemical structure of FA-PEG-COL conjugate was characterized by MALDI-TOF-MS, FT-IR and (1)H NMR. The average size of siRNA/FA-PEG-COL nanoparticles was approximately 200 nm, and the surface charge was +8.4 mV compared to +30.5 mV with siRNA/COL nanoparticles. FA-PEG-COL nanoparticles demonstrated superior compatibility with erythrocytes in terms of degree of aggregation and haemolytic activity and also effects on cell viability was lower when compared with COL nanoparticles. FA grafting significantly facilitated the uptake of nanoparticles via receptor mediated endocytosis as demonstrated by flow cytometry. The in vitro transfection and gene knockdown efficiency of HIF-1α were superior to COL nanoparticles (76-62%, respectively) and was comparable to Lipofectamine 2000 (79%) as demonstrated by RT-qPCR and Western blot. Gene knockdown at the molecular level translated into effective inhibition of proliferation in vitro. Accumulation efficiency of FA-PEG-COL nanoparticles was investigated in BALB/c mice bearing OVK18 #2 tumor xenograft using in vivo imaging. The active targeting FA-PEG-COL nanoparticles showed significantly greater accumulation than the passive targeting COL nanoparticles. Based on the results obtained, siRNA/FA-PEG-COL nanoparticles show much potential for effective ovarian cancer treatment via gene therapy.

Topics: Animals; Carbocyanines; Cell Line; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chitosan; Female; Fluorescent Dyes; Folic Acid; Gene Knockdown Techniques; Genetic Therapy; Hemolysis; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mice, Nude; Nanoparticles; Ovarian Neoplasms; Polyethylene Glycols; RNA, Small Interfering

Receptor targeted therapy is advantageous in overcoming the toxicity burden of conventional cancer chemotherapeutics. Over expression of epidermal growth factor receptor (EGFR) on cancer cells and its role in metastasis, malignancy and drug resistance in many human cancers lead to its selection as a promising target for cancer treatment. The present work investigated the preparation and characterization of docetaxel (DTXL) loaded gamma-poly (glutamic acid) (gamma-PGA) nanoparticles (Nps) conjugated with EGFR antibody (Cetuximab, CET) targeted to colon cancer cells (HT-29), highly over expressing EGFR. The flow cytometric analysis revealed two fold increased cellular uptake of CET-DTXL-gamma-PGA Nps by HT-29 (EGFR +ve) cells compared to that of IEC-6 (EGFR-ve) cells confirming the active targeting. Cytotoxicity assays (MTT and LDH) showed superior anti-proliferative activity of CET-DTXL-gamma-PGA NPs over DTXL-gamma-PGA Nps against HT-29 cells. The cell cycle analysis indicated that CET-DTXL-gamma-PGA NPs induced cell death in enhanced percentage of HT-29 cells by undergoing cell cycle arrest in G2/M phase compared to that of DTXL-gamma-PGA Nps. The mechanism of cancer cell death was analyzed via apoptotic and mitochondrial membrane potential assays and showed that targeted Nps treatment reduced the mitochondrial membrane potential thereby inducing enhanced HT-29 cell death (apoptosis and necrosis). The biodistribution of targeted and non-targeted Nps were analyzed in vivo in Swiss albino mice using NIR imaging. ICG-CET-DTXL-gamma-PGA Nps (targeted) and ICG-DTXL-gamma-PGA Nps (non-targeted) followed the similar biodistribution pattern in vivo, but with different elimination time. In short, CET-DTXL-gamma-PGA nanoparticles enhance the tumor selective therapeutic efficacy for colon cancer.

Topics: Animals; Annexins; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Benzimidazoles; Carbocyanines; Cell Cycle; Cell Death; Cetuximab; Colonic Neoplasms; Docetaxel; Drug Carriers; ErbB Receptors; Hemolysis; HT29 Cells; Humans; Mice; Nanomedicine; Nanoparticles; Polyglutamic Acid; Taxoids; Tissue Distribution

In this study, the antifungal activity and mode of action(s) of hibicuslide C derived from Abutilon theophrasti were investigated. Antifungal susceptibility testing showed that hibicuslide C possessed potent activities toward various fungal strains and less hemolytic activity than amphotericin B. To understand the antifungal mechanism(s) of hibicuslide C in Candida albicans, flow cytometric analysis with propidium iodide was done. The results showed that hibicuslide C perturbed the plasma membrane of the C. albicans. The analysis of the transmembrane electrical potential with 3,3'-dipropylthiacarbocyanine iodide [DiSC3(5)] indicated that hibicuslide C induced membrane depolarization. Furthermore, model membrane studies were performed with calcein encapsulating large unilamellar vesicles (LUVs) and FITC-dextran (FD) loaded LUVs. These results demonstrated that the antifungal effects of hibicuslide C on the fungal plasma membrane were through the formation of pores with radii between 2.3 nm and 3.3 nm. Finally, in three dimensional flow cytometric contour plots, a reduced cell sizes by the pore-forming action of hibicuslide C were observed. Therefore, the present study suggests that hibicuslide C exerts its antifungal effect by membrane-active mechanism.

Topics: Amphotericin B; Antifungal Agents; Benzothiazoles; Candida albicans; Carbocyanines; Cell Membrane; Cell Membrane Permeability; Dextrans; Erythrocytes; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Dyes; Hemolysis; Humans; Malvaceae; Membrane Potentials; Phenylpropionates; Plant Extracts; Unilamellar Liposomes

Targeted nano-particulate systems hold extraordinary potential for delivery of therapeutics across blood brain barrier (BBB). In this work, we investigated the potential of novel bi-ligand (transferrin-poly-l-arginine) liposomal vector for delivery of desired gene to brain, in vivo. The in vivo evaluation of the delivery vectors is essential for clinical translation. We followed an innovative approach of combining transferrin receptor targeting with enhanced cell penetration to design liposomal vectors for improving the transport of molecules into brain. The biodistribution profile of 1, 1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine iodide(DiR)-labeled liposomes was evaluated in adult rats after single intravenous injection at dose of 15.2μmoles of phospholipids/kg body weight. We demonstrated that bi-ligand liposomes accumulated in rat brain at significantly (p<0.05) higher concentrations as compared to the single-ligand (transferrin) or plain liposomes. In addition, the bi-ligand liposomes resulted in increased expression of β-galactosidase(β-gal) plasmid in rat brain tissue in comparison to the single-ligand liposomes. Histological examination of the transfected tissues did not show any signs of tissue necrosis or inflammation. Hemolysis assay further authenticated the biocompatibility of bi-ligand liposomes in blood up to 600 nmoles of phospholipids/1.4×10(7) erythrocytes. The findings of this study provide important and detailed information regarding the distribution of bi-ligand liposomes in vivo and accentuate their ability to demonstrate improved brain penetration and transfection potential over single-ligand liposomes.

Topics: Animals; beta-Galactosidase; Brain; Carbocyanines; Cell-Penetrating Peptides; Erythrocytes; Fluorescent Dyes; Hemolysis; Ligands; Liposomes; Peptides; Rats; Rats, Sprague-Dawley; Tissue Distribution; Transfection; Transferrin

Lariciresinol is an enterolignan precursor isolated from the herb Sambucus williamsii, a folk medicinal plant used for its therapeutic properties. In this study, the antifungal properties and mode of action of lariciresinol were investigated. Lariciresinol displays potent antifungal properties against several human pathogenic fungal strains without hemolytic effects on human erythrocytes. To understand the antifungal mechanism of action of lariciresinol, the membrane interactions of lariciresinol were examined. Fluorescence analysis using the membrane probe 3,3'-diethylthio-dicarbocyanine iodide (DiSC(3)-5) and 1,6-diphenyl-1,3,5-hexatriene (DPH), as well as a flow cytometric analysis with propidium iodide (PI), a membrane-impermeable dye, indicated that lariciresinol was associated with lipid bilayers and induced membrane permeabilization. Therefore, the present study suggests that lariciresinol possesses fungicidal activities by disrupting the fungal plasma membrane and therapeutic potential as a novel antifungal agent for the treatment of fungal infectious diseases in humans.

Topics: Antifungal Agents; Benzothiazoles; Candida albicans; Carbocyanines; Cell Membrane; Cells, Cultured; Diphenylhexatriene; Erythrocytes; Flow Cytometry; Fluorescent Dyes; Furans; Hemolysis; Hemolytic Agents; Humans; Lignans; Propidium; Sambucus

We report on encapsulation of cyanine IR-768 in oil-in-water (o/w) microemulsion, i.e. fabrication of templated polymeric nanocapsules as effective nanocarriers for a new generation of photodynamic agents suitable for photodynamic therapy (PDT). Discussed here are nanocapsule imaging, their in vitro biological evaluation, cyanine encapsulation potential, and the cellular localization of cyanine IR-768 delivered in the nanocapsules to MCF-7 cancer cells. Oil-cored poly(n-butyl cyanoacrylate) (PBCA) nanocapsules were prepared by interfacial polymerization in o/w microemulsions formed by the nonionics Tween 80 (polysorbate 80, polyoxyethylene 20 sorbitan monooleate), and Brij 96 (polyoxyethylene 10 oleyl ether). Iso-propyl myristate (IPM), ethyl oleate (EOl), iso-octane (IO), and oleic acid (OA) were used as the oil phases and iso-propanol (IP) and propylene glycol (PG) as the cosurfactants. Such o/w droplets, also containing hydrophobic IR-768 in the oil phase, were applied in the interfacial polymerization of n-butyl cyanoacrylate at 10 degrees C at pH 5.0. The isolated cyanine-loaded nanoparticles were visualized by atomic force microscopy (AFM) and scanning electron microscopy (SEM), which proved their unimodal size distribution and spherical shape, with diameters dependent upon the monomer content and the template type. The entrapment efficiency of cyanine increased with increasing n-butyl cyanoacrylate concentration and varied from 65.7% to 91.7%. The results of in vitro erythrocyte hemolysis and the cell viability of breast cancer MCF-7 cells showed that the PBCA nanocapsules are quite safe carriers of IR-768 in the circulation, having a very low hemolytic potential and being non-toxic to the studied cells. Fluorescence microscopy visualized the cyanine intracellular distribution and, furthermore, demonstrated that PBCA-nanocarriers effectively delivered the IR-768 molecules to the MCF-7 doxorubicin-sensitive and -resistant cell lines. Photoirradiation of the cancer cells with entrapped photosensitizer decreased cell viability, demonstrating that this effect may be utilized in PDT.

Topics: Carbocyanines; Cell Line, Tumor; Drug Carriers; Hemolysis; Humans; Microscopy, Atomic Force; Microscopy, Electron, Scanning; Nanoparticles; Photochemotherapy; Photosensitizing Agents

Gramicidin S (GS) is a 10-residue cyclic beta-sheet peptide with lytic activity against the membranes of both microbial and human cells, i.e. it possesses little to no biologic specificity for either cell type. Structure-activity studies of de novo-designed 14-residue cyclic peptides based on GS have previously shown that higher specificity against microbial membranes, i.e. a high therapeutic index (TI), can be achieved by the replacement of a single L-amino acid with its corresponding D-enantiomer [Kondejewski, L.H. et al. (1999) J. Biol. Chem. 274, 13181]. The diastereomer with a D-Lys substituted at position 4 caused the greatest improvement in specificity vs. other L to D substitutions within the cyclic 14-residue peptide GS14, through a combination of decreased peptide amphipathicity and disrupted beta-sheet structure in aqueous conditions [McInnes, C. et al. (2000) J. Biol. Chem. 275, 14287]. Based on this information, we have created a series of peptide diastereomers substituted only at position 4 by a D- or L-amino acid (Leu, Phe, Tyr, Asn, Lys, and achiral Gly). The amino acids chosen in this study represent a range of hydrophobicities/hydrophilicities as a subset of the 20 naturally occurring amino acids. While the D- and L-substitutions of Leu, Phe, and Tyr all resulted in strong hemolytic activity, the substitutions of hydrophilic D-amino acids D-Lys and D-Asn in GS14 at position 4 resulted in weaker hemolytic activity than in the L-diastereomers, which demonstrated strong hemolysis. All of the L-substitutions also resulted in poor antimicrobial activity and an extremely low TI, while the antimicrobial activity of the D-substituted peptides tended to improve based on the hydrophilicity of the residue. D-Lys was the most polar and most efficacious substitution, resulting in the highest TI. Interestingly, the hydrophobic D-amino acid substitutions had superior antimicrobial activity vs. the L-enantiomers although substitution of a hydrophobic D-amino acid increases the nonpolar face hydrophobicity. These results further support the role of hydrophobicity of the nonpolar face as a major influence on microbial specificity, but also highlights the importance of a disrupted beta-sheet structure on antimicrobial activity.

Topics: 1-Naphthylamine; Amino Acid Substitution; Anti-Infective Agents; Benzothiazoles; Candida albicans; Carbocyanines; Cell Membrane Permeability; Circular Dichroism; Glycine; Gram-Negative Bacteria; Gram-Positive Bacteria; Gramicidin; Hemolysis; Humans; Hydrophobic and Hydrophilic Interactions; Membrane Potentials; Microbial Sensitivity Tests; Peptides, Cyclic; Protein Structure, Secondary; Structure-Activity Relationship; Yeasts

A synthetic peptide with the sequence of the first 20 residues of melittin and terminating with an additional cysteine amide was found to have cytolytic activity similar to that of melittin. It was apparent from MS data that the cysteine-terminating peptides had formed disulphide dimers. A peptide in which the thiol was blocked by iodoacetate showed no activity, whereas the same peptide blocked by acetamidomethyl showed activity marginally less haemolytic than that of melittin. Cytolytic activity of melittin analogues comprising the full 26 residues could be obtained with wide sequence permutations providing that a general amphipathic helical structure was preserved. In contrast, the activity of the dimers was dependent not only on retention of an amphipathic helix but also on certain individual residues and a free positive charge. A free N-terminus was essential for haemolytic activity. In addition, a lysine or arginine residue at position 7 and a proline at position 14 were found to be necessary for activity, although it was apparent that additional residues are important for retention of the full lytic potential.

Topics: Amino Acid Sequence; Benzothiazoles; Carbocyanines; Cell Survival; Cysteine; Disulfides; Flow Cytometry; Fluorescent Dyes; Hemolysis; Humans; Melitten; Membrane Potentials; Molecular Sequence Data; Peptides; Protein Conformation; Tumor Cells, Cultured

Human red blood cells (RBCs) adhere to and are lysed by schistosomula of Schistosoma mansoni. We have investigated the mechanism of RBC lysis by comparing the dynamic properties of transmembrane protein and lipid probes in adherent ghost membranes with those in control RBCs and in RBCs treated with various membrane perturbants. Fluorescence photobleaching recovery was used to measure the lateral mobility of two integral membrane proteins, glycophorin and band 3, and two lipid analogues, fluorescein phosphatidylethanolamine (Fl-PE) and carbocyanine dyes, in RBCs and ghosts adherent to schistosomula. Adherent ghosts manifested 95-100% immobilization of both membrane proteins and 45-55% immobilization of both lipid probes. In separate experiments, diamide-induced cross-linking of RBC cytoskeletal proteins slowed transmembrane protein diffusion by 30-40%, without affecting either transmembrane protein fractional mobility or lipid probe lateral mobility. Wheat germ agglutinin- and polylysine-induced cross-linking of glycophorin at the extracellular surface caused 80-95% immobilization of the transmembrane proteins, without affecting the fractional mobility of the lipid probe. Egg lysophosphatidylcholine (lysoPC) induced both lysis of RBCs and a concentration-dependent decrease in the lateral mobility of glycophorin, band 3, and Fl-PE in ghost membranes. At a concentration of 8.4 micrograms/ml, lysoPC caused a pattern of protein and lipid immobilization in RBC ghosts identical to that in ghosts adherent to schistosomula. Schistosomula incubated with labeled palmitate released lysoPC into the culture medium at a rate of 1.5 fmol/h per 10(3) organisms. These data suggest that lysoPC is transferred from schistosomula to adherent RBCs, causing their lysis.

Topics: Animals; Carbocyanines; Cell Adhesion; Cross-Linking Reagents; Erythrocyte Membrane; Hemolysis; Humans; Lysophosphatidylcholines; Membrane Fluidity; Membrane Lipids; Membrane Proteins; Schistosoma mansoni