By T. Phil. Texas A&M University, Texarkana.

Further chemical modifications are needed to make the polyamine derivatives as well as other analogues able to target the amastigote stage of the parasite best pletal 100 mg. In the case of protozoan species that are causative agents of important human diseases such as Leishmaniasis generic pletal 50 mg fast delivery, an exogenous supply of polyamines supports parasite proliferation purchase pletal 100mg without a prescription. A significant reduction of promastigotes and axenic amastigotes growth was observed in the presence of increasing concentrations of the drugs proven 100mg pletal, although the mechanisms leading to the parasite growth arrest seems to be different 100mg pletal free shipping. Indeed, by using a number of biochemical approaches to analyse the alterations that occurred during early stages of parasite-drug interaction (i. The drug inhibitory effect on amastigotes growth and the absence of propidium iodide labelling may suggest that the compounds are acting as cytostatic substances. Although, the mechanisms of action of these compounds have yet to be elucidated, the above data show for the first time that polyamine derivatives may act differentially on the Leishmania parasite stages. Further chemical modifications are needed to make the polyamine derivatives as well as other analogues able to target the amastigote stage of the parasite. Introduction donovani infantum, especially affects children and South American visceral leishmaniasis caused by Leishmania In humans, parasites from the Leishmania donovani donovani chagasi is a disease of both adults and children. Mediterranean Kala-azar, caused by Leishmania antimonials such as sodium stibogluconate (Pentostan), N-methylglucamine (Glucantime), amphotericin B or penta- midine (Murry, 2001). Address: Laboratorio de Bioquımica, Faculdade´ ´ ´ duration of therapy, causing adverse reactions and resistance de Farmacia da Universidade do Porto, Rua Anibal Cunha, 164, 4050-047 Porto, Portugal. However, analyses of the drug-induced alterations agents acting on parasite development is still warranted. In trypanosomatid protozoa, polyamines have an additional role participating in the endogenous redox equilibrium 2. Materials and methods through the compound (N1, N8-bis (glutathionyl) spermi- dine), named trypanothione T(S)2, which is maintained in 2. The parasites (10 /ml) in the logarithmic phase that selective interference with the parasitic polyamine (2 days of culture) were incubated with a serial range of metabolism will lead to an alteration of natural defence concentrations of each drug for 5 days at 28 8C. The percentage of growth inhibition amine analogues, able to mimic the natural polyamines in was calculated as (1-growth rate of the experimental their self-regulatory role, but unable to act as substitutes for culture/growth rate of the control culture)! The growth of parasites was determined by the same After several washes, the parasites were permeabilised with methods described above. Parasites were then To monitor parasite death, short incubation periods were incubated with a rabbit immune serum to a L. Flow cytometric analysis of external in Vectashield (Vector Laboratories) and analysed with a phosphatidylserine exposure fluorescent microscope (Axioskop-Carl Zeiss, Germany) at 1000! Statistical analysis amastigotes treated and untreated with 10 mM of either of the three drugs during 24 h, was determined using The data were analysed using the Student’s t-test. The polyamine derivative drugs control the growth of Leishmania infantum parasites Treatment of L. It can be seen that at R12 mM all the three drugs completely blocked both promastigote and amastigote multiplication. Promastigote (closed square) and amastigote (open square) forms were incubated with a concentration range of 0. Percentages of promastigote and amastigote fragmentation in Leishmania infantum promastigotes growth inhibition were determined after 5 days by counting using a Newbauer chamber under light microscopy. Each point represents the mean In order to determine whether the observed effect was of two replicates G standard deviation. The data shown is representative of due to a leishmanicidal activity and to establish the type at least three independent experiments. The growth rate of the promastigote and amastigote parasite forms without drug treatment was 24 and 100 times related to the initial concentration, respectively. However, examination of a number of slides showed slightly different patterns of labelling. Quantification of phosphatidylserine externalisation in gotes showed no significant increase of Annexin-V positive Leishmania infantum promastigotes and amastigotes cells (Fig. This is likely due the translocation of phosphatidylserine from the inner side to the absence of apoptosis induction since amastigotes to the outer layer of the plasma membrane. Annexin V, 2C treated with 4 mM staurosporine, a drug known to induce aCa dependent phospholipid binding protein with apoptosis-like death in Leishmania showed around 80% affinity for phosphatidylserine, is routinely used in a Annexin-V positive cells (Fig. Flow cytometry analysis of phosphatidylserine exposure to Leismania infantum after polyamine derivative compound treatment.

A variety of engineered nanoparticles effective 50mg pletal, particularly doxorubicin- loaded cyanoacrylate nanoparticles buy cheap pletal 100mg on-line, showed increased renal distribution and thus increased kidney toxicity (88–90) purchase 100mg pletal mastercard. Kidney injury has been demonstrated in many other nanoparticles such nano-zinc particles in which severe histological alterations are observed in murine kidneys (91 discount pletal 50 mg,92) order pletal 100mg with mastercard. These cell lines were used in variety of in vitro assays to evaluate cytotoxicity, mechanistic toxicology and pharmacology, etc. Oxidative Stress The generation of free radicals by nanomaterials of ambient or industrial origin is well documented (94,95). However, engineered nanomaterials such as fullerenes and polystyrene nanoparticles have also been shown to generate oxidative stress (56,96,97). At the end of exposure, dichlorofluorescein fluores- cence was determined at excitation wavelength of 485 nm and emission wavelength of 530 nm, respectively. Control cells cultured in Ag-free media (50 and 100 nm) were run in parallel to the treatment groups. Apoptosis and Mitochondrial Dysfunction Nanoparticle-induced cell death can occur by either necrosis or apoptosis, processes that can be distinguished both morphologically and biochemically. Apoptosis, 212 Murthy and Pathak morphologically, is characterized by perinuclear partitioning of condensed chromatin and budding of the cell membrane (105). The ability of nanoparticles, such as den- drimers and carbon nanotubes, to induce apoptosis has been demonstrated by in vitro studies (106,107). However, this link between oxidative stress, mitochondrial dysfunction, and apoptosis has also been observed in man-made nanoparticles such as quantum dots and metals (76,109), water-soluble fullerenes derivatives (54,114), chitosan nanoparticles (115), and in various in vitro models. The loss of mem- brane potential causes the dye to dissipate from the matrix and can be measured in its monomeric state at emission wavelength 527 nm (green). Thus, the degree of mitochondrial membrane depolarization is measured as the proportion of green to red fluorescence. Pharmacological and Toxicological Characterization of Nanosystems 213 Mitochondrial membrane potential measurement can be an index of toxic- ity, which can be determined by the uptake study of rhodamine 123 according to the method of Wu et al. Cells were exposed to different concentrations of Ag (15 and 100 nm) for 24 hours. The fluorescence was determined at excitation wavelength 485 nm and emission wavelength 530 nm. Control cells cultured in Ag-free media (50 and 100 nm) were run in parallel to the treatment groups. The fluorescence intensity value of control cells (nanoparticle-free medium at 0 hour) was taken as 100% and then calculated as the percentage of reduction of fluorescence in nanoparticle-exposed cells. Computational models may provide a much needed bridge between the two, producing highly realistic in vitro models upon which alternate therapies may be conducted. The power of such models over in vitro monolayer and even spheroid assays lies in their ability to integrate processes over a multitude of scales, approximating the complex in vivo interplay of phenomena such as heterogeneous vascular delivery of drugs and nutrients, diffusion through lesion, heterogeneous lesion growth, apoptosis, necrosis, and cellular uptake, efflux, and target binding. Guidelines for industries – Immunotoxicological evaluation of investiga- tional new drugs. Guidelines for industries – Developing medical imaging drug and biological products; part 1: Conducting safety assessments. Standard Practice F 748-98: Selecting generic biological test methods for materi- als and devices. Understanding the nanoparticle-protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles. Assay cascade protocols, Nanotechnology Characterization Laboratory, National Can- cer Institute. F756-00: Standard practice for assessment of hemolytic properties of materials of materials, 2000. Pharmacological characteristics of solid-phase von Willebrand factor in human platelets. Mechanisms of action of proteinase- activated receptor agonists on human platelets. Membrane type-1 matrix metallo- proteinase stimulates tumour cell-induced platelet aggregation: role of receptor glyco- proteins. Differential regulation of platelet aggregation by matrix metalloproteinases-9 and -2. Plasma protein adsorption patterns on liposomes: Establishment of analytical procedure. Plasma protein adsorption patterns on emulsion for parenteral administration: Establishment of a protocol for two-dimensional polyacrylamide elec- trophoresis. Alternative sample preparation prior to two-dimensional electrophoresis protein analysis on solid lipid nanoparticles.

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For instance order pletal 100mg online, there was only one difference in the top 20 most significant substructures purchase 100mg pletal with amex, and the ordering was virtually the same (compare Table 2 with Table 1 in Supporting Information) discount pletal 100mg with amex. The top most significant substructures are dominated by a few substructural themes that are common to the group as a whole order pletal 50 mg on line. The hypothetical ‘parent’ fragment from which all frequent substructures derive would be an amine connected to an aromatic system through a carbon chain buy pletal 100 mg line. Again, the abundance of this substructure is probably due to the high number of aminergic receptor ligands present in the database (see also below). Even though the top significant substructures provide chemical insights found in the largest number of compounds, they also might reflect an obvious bias. Due to the high attrition rates in drug discovery and 45 development these advanced compounds must have additional features for drug- likeness that made them not fail beforehand, reducing the ‘randomness’ of substructure occurrence. Substructures were sorted according to significance, with the most significant substructure at the top. Ligands having the first substructure are taken out of the two sets (left path, N for ‘No’) or rather kept (right path, Y for ‘Yes’), and the process of substructure mining is repeated. The name of the representation that yielded this substructure is given at the bottom of the box surrounding the substructures. To find a new set of significant features, the experiment was repeated on subsets of the original sets: a subset in which all structures contained the substructure and another set in which they did not. The most significant substructure from the best performing representation was used to split the sets and substructure mining was then repeated for the two subsets. More than half of the molecules containing this aminergic tail also contain a substructure consisting of a heteroatom substituted with a methyl group and a propyl group. This substructure may overlay the butyl amine group or may be located elsewhere in the molecule. Molecules without the butyl amine substructure have a six atom aromatic system, of which one is nitrogen, as most important structural feature. This aromatic chain is not closed to form a six-membered ring, meaning that this substructure is found not only in six- membered rings but also in aromatic systems containing fused five-membered rings etc. Since the ‘normal’ representation does not discriminate between aliphatic and aromatic bonds, it may lead to substructure contributions that are part of an aromatic ring system (Figure 2, e. We therefore tested whether the use of a special aromatic bond type was the more suitable representation. Substructure occurrences for the aromatic bonds representation are listed in Table 3. When comparing the substructures of this representation with those of the ‘normal’ representation, a large overlap of the common motif was observed. For both representations, the most important moiety is the nitrogen substituted with one or more alkane chains. From this data, a second hierarchy (Figure 4) was constructed that did not consider the ‘normal’ representation. This approach resulted in a different substructure set than with the ‘normal’ representation. This carbon tail is probably shorter due to the absence of contributing aromatic bonds that were represented as single bonds in the ‘normal’ representation. The first substructure with an aromatic bond was found at position 24 in the substructure list in Table 3, whereas in the ‘aromatic atoms and bonds’ representation the first aromatic substructure was found much higher in the substructure list, i. Iterating over the substructures with the ‘aromatic atoms and bonds’ representation leaves the impression that an aromatic ring is forming (Supporting Information: substructures 3 to 5 and 10 to 15 in Table 2). The reason for this is that aromatic rings that differ in size contribute the same open substructures to the appearance lists. Since closed five-membered rings and closed six-membered rings cannot be overlaid, the support for these substructures is much lower. Using this representation, a significant enrichment of the source sets can be accomplished, provided that bonds may be part of an aromatic system (Figure 1). Table 4 gives an overview of the most-significant substructures for each representation. The tree is the same for analysis with and without the results of the ‘normal’ representation, since this representation did not produce any most significant substructures.

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Experimental Procedure Place cuvettes into A purchase pletal 100mg amex, B order 100mg pletal mastercard, C cheap pletal 50mg visa, and D test rows on a coagulometer buy pletal 50mg with amex. Add one metal ball into each cuvette and let cuvette with the ball warm for at least 3 minutes before use discount pletal 100mg amex. Start the timer for each of the test rows by pressing A, B, C, or D timer buttons. When time is up, add coagulation activation reagent to each cuvette and record coagulation time. Nanoparticles on Coagulatory Changes Nanomaterials could cause blood coagulation, as modification in surface chem- istry has been shown to improve immunological compatibility at the particle– blood interface. Application of poly(vinyl chloride) resin particles resulted in 19% decrease in platelet count, indicating platelet adhesion/aggregation and increased blood coagulation time. The same particle coated with poly(ethylene glycol) did not affect platelet count and also elements of coagulation cascade. Similarly, folate- coated Gd nanoparticles did not aggregate platelets or activate neutrophils (26). The method has been efficiently used to study plasma protein adsorbed on the sur- face of stealth poly(cyano acrylate) particles (27), liposome (28,29), solid lipid nanoparticles (30), and iron oxide nanoparticles (31). Proteins commonly identi- fied include antithrombine, C3 component of the compliment, 2-macroglobulin, Pharmacological and Toxicological Characterization of Nanosystems 197 heptaglobin, plasminogen, immunoglobulins, albumin, fibrinogen, and apolipopro- tein, of which, albumin, immunoglobulins, and fibrinogen are the most abundant. To investigate the impact of airway exposure to nanoparticles on the coagu- latory system, Inoue et al. Twenty-four hours postadministration, blood was retrieved from each mouse by cardiac puncture, collected into 3. A complement is a system composed of several components (C1, C2, C9) and factors (B, D, H, I, and P). Activation of either one of the three pathways results in the cleavage of C3 component of the complement. These antibodies recog- nize both native C3 component of the complement and its cleaved products. Native C3 and no, or minor, amounts of C3 cleavage products are visualized by Western blot in control human plasma. When a test compound or positive control (cobra venom factor) induces the activation of complement, the majority of C3 component is cleaved and the appearance of C3 cleavage products is documented. This “yes” or “no” protocol is designed for rapid and inexpensive assessment of complement activation. Test nanoparticles found to be positive in this assay will be a subject for more detailed investigation aimed at delineation of specific complement activation pathway. The ability of injection of nanoparticles for complement activation in experi- mental animals is the basis of development of nanoparticles as vaccines. Antigen- bearing nanoparticle vaccines were investigated for two novel features: lymph node–targeting and in situ complement activation. Results showed the accumulation of ultra- small nanoparticles in lymph nodes after subcutaneous injection, while slightly larger ones do not. Polyhydroxylated nanoparticle surfaces activate complement to much higher levels than do polymethoxylated nanoparticles. Pharmacological and Toxicological Characterization of Nanosystems 199 Complement activation by core–shell poly(isobutyl cyanoacrylate)–poly- saccharide nanoparticles coated with different polysaccharides was investigated by Bertholon et al. The results showed the cleavage of C3 increased with the size of dextran bound in a “loops” configuration whereas it decreased when dextran was bound in a “brush” configuration. It was concluded that complement activation was highly sensitive to surface features of the nanoparticles. Type of polysaccharide, configuration on the surface, and accessibility to reactive functions along chains are critical parameters for complement activation. Experimental Procedure The experimental protocol described in the technical manual # 28405 (37) developed by StemCell Technologies Inc. Attach a 16-gauge blunt-ended needle to a 3-mL syringe; place the nee- dle below the surface of the solution and draw up approximately 1 mL. Dispense 100 L of controls and test samples per well on a round-bottomed, 96-well plate.

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Cochleates have been shown to encapsulate plasmid and enhance plasmid stability and transfection efficiency 50mg pletal for sale. A cationic lipid consists of: • a hydrophobic lipid anchor group • a linker group • a positively charged headgroup purchase pletal 50mg on line. Lipid anchors help in forming liposomes (or micellar structures) and determine the physical properties of a lipid bilayer cheap 100 mg pletal with mastercard, such as membrane rigidity and rate of lipid exchange between lipid 341 membranes order pletal 50 mg mastercard. The linker group is an important component pletal 100mg without prescription, which determines the chemical stability and biodegradability of a cationic lipid. The head groups of cationic lipid appear to be critical for transfection and cytotoxicity of corresponding liposome formulations. The cationic amphiphiles differ markedly in structure and may be single or multiple charged as primary, secondary, tertiary and/or quaternary amines. Examples are lipospermine, cationic cholesterol, cationic detergent or lipopolysine. The relative proportions of each component and the structure of the head group influence the physicochemical properties of plasmid/lipid complexes. Many effective cationic lipids contain protonatable polyamines linked to dialkyl or cholesterol anchors. To increase the biodegradability of cationic lipids, a series of cationic lipids have been synthesized in which the ether bonds were replaced with ester bonds. Cationic lipid-based gene delivery systems lack target specificity, which results in low transfection efficiency in certain tissues due to the interference from cationic lipid-binding macromolecules either in the circulation or in the extracellular matrix. To circumvent this problem, neutral plasmid/lipospermine complexes containing a trigalactolipid have been prepared and shown to efficiently transfect hepatoma HepG2 cells bearing asialoglycoprotein receptor. Addition of 25% (mol mol−1) of the triantennary galactolipid increased the transfection efficiency by a thousand fold, compared to the lipid-based system with no targeting ligand. An efficient transfection of β-galactosidase into HeLa cells has been shown with the combination of transferrin and cationic liposome Lipofectin, whereas Lipofectin alone had low transfection efficiency. Asialofetuin is an asialoglycoprotein containing terminal galactosyl residues that have been used to target liposomes to the liver. The resulting complexes retain their ability to interact specifically with target cell receptors, leading to receptor-mediated internalization of the complex into the cells. It is known that the active sites of enzymes, receptor ligands and antibodies usually involve about 5 to 20 amino acids. One example of such a gene delivery system comprises: 343 • a galactosylated peptide that both condenses the plasmid into monodisperse nanoparticles of about 100 nm in diameter and enables specific recognition and binding to asialoglycoprotein receptors; • an amphipathic, pH-selective peptide that enables the plasmid to leave the endosomes prior to their fusion with lysosomes and entry into the cytoplasm. Two general classes of lipopeptide analogs of Tyr-Lys-Ala-Lys -n Trp-Lys peptides have been prepared by including a hydrophobic anchor. The general structures are N, N- dialkyl-Gly-Tyr-Lys-Ala-Lys -Trp-Lys and N ,N -diacyl-Lys-Lys -Trp-Lys. These peptides differ from theα n n parent structures in that they self-associate to form micelles in aqueous solutions. The lytic characteristics are revealed as the carboxyl groups of the aspartyl and glutamyl side chains are protonated, which allows the peptides to assume a a-helical conformation that can be inserted into the membrane bilayer. The hydrophobic face contains only strongly apolar amino acids, while negatively charged glutamic acid residues dominate the hydrophilic face at physiological pH. At a given charge ratio of condensing peptide to plasmid, the transfection efficiency has been shown to be proportional to the concentration of the endosomolytic peptide added to the complex. The increased hydrophobicity of the complex may enhance interaction with cell membranes and facilitate cell uptake. However, these polymers cannot be used for in vivo application due to their poor transfection efficiency and high cytotoxicity. The effect of colloidal and surface characteristics of plasmid/ dendrimer complexes on gene transfer has been examined. These complexes were monodisperse, with a mean hydrodynamic diameter of about 200 nm. The particle size, surface charge and gene transfer efficiency of plasmid/dendrimer complexes prepared with the 5th generation of dendrimers has been shown to be influenced by dendrimer concentration in the complexes. The colloidal and surface properties of plasmid/chitosan complexes have been shown to depend on the molecular weight of chitosan, the ratio of plasmid to chitosan and the preparation medium. Smaller nanoparticles have been observed with low molecular weight chitosan (2 kDa) as compared to high molecular weight chitosan (540 kDa). Interestingly, the transfection efficiency of the complexes was not affected by the presence of serum proteins, even though the presence of serum is known to adversely affect the transfection efficiency.

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