Versatile peptide dendrimers for nucleic acid delivery

Dendrimers are nonviral vectors that have attracted interest on account of a number of features. They are structurally versatile because their size, shape, and surface charge can be selectively altered. Here we examine the functions of a new family of composite dendrimers that were synthesized with lipidic amino acid cores. These dendrimers are bifunctional because they are characterized by positively charged (lysine) modules for interaction with nucleic acids and neutral lipidic moieties for membrane lipid-bilayer transit. We assessed their structure-function correlations by a combination of molecular and biophysical techniques. Our assessment revealed an unexpected pleitropy of functions subserved by these vectors that included plasmid and oligonucleotide delivery. We also generated a firefly luciferase cell line in which we could modulate luciferase activity by RNA interference. We found that these vectors could also mediate RNA suppression of luciferase expression by delivering double-stranded luciferase transcripts generated in vitro. The structural uniqueness of these lipidic peptide dendrimers coupled with their ease and specificity of assembly and the versatility in their choice of cargo, puts them in a new category of macromolecule carriers. These vectors, therefore, have potential applications as epigenetic modifiers of gene function. (C) 2004 Wiley-Liss, Inc. and the American Pharmacists Association.

The stability of lipidic analogues of GnRH in plasma and kidney preparations: the stereoselective release of the parent peptide

The conjugation of a lipoamino acid to the N-terminus of Gonadotropin releasing hormone (GnRH) produces a lipophilic peptide from which the parent GnRH peptide is released into solution on treatment with plasma and kidney enzyme preparation. Our findings show that one stereoisomer of the Laa is cleaved very rapidly, providing a bolus dose of the peptide while the opposite stereoisomer is cleaved much more slowly, providing prolonged elevation of peptide concentration. The Laa-Glu linkage appears to act as a two phase prodrug system. © 2005 Elsevier Ltd. All rights reserved.

Lipoamino acid-based adjuvant carrier system: Enhanced immunogenicity of group a streptococcal peptide epitopes

Lipoamino acid-based synthetic peptides (lipid core peptides, LCP) derived from the type-specific and conserved region determinants of group A streptococci (GAS) were evaluated as potential candidate sequences in a vaccine to prevent GAS-associated diseases, including rheumatic heart, disease and poststreptococcal acute glomerulonephritis. The LCP peptides had significantly enhanced immunogenicity as compared with the monomeric peptide epitopes. Furthermore, the peptides incorporated into the LCP system generated epitope-specific antibodies without the use of any conventional adjuvant.

Interaction of a synthetic antimicrobial peptide with model membrane by fluorescence spectroscopy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Inhibition of in vitro VEGF expression and choroidal neovascularization by synthetic dendrimer peptide mediated delivery of a sense oligonucleotide

Ocular neovascularisation is the leading cause of blindness in developed countries and the most potent angiogenic factor associated with neovascularisation is vascular endothelial growth factor (VEGF). We have previously described a sense oligonucleotide (ODN-1) that possesses anti-human and rat VEGF activity. This paper describes the synthesis of lipid-lysine dendrimers and their subsequent ability to delivery ODN-1 to its target and mediate a reduction in VEGF concentration both in vitro and in vivo. Positively charged dendrimers were used to deliver ODN-1 into the nucleus of cultured D407 cells. The effects on VEGF mRNA transcription and protein expression were analysed using RT-PCR and ELISA, respectively. The most effective dendrimers in vitro were further investigated in vivo using an animal model of choroidal neovascularisation (CNV). All dendrimer/ODN-1 complexes mediated in a significant reduction in VEGF expression during an initial 24 hr period (40-60%). Several complexes maintained this level of VEGF reduction during a subsequent, second 24 hr period, which indicated protection of ODN-1 from the effects of endogenous nucleases. In addition, the transfection efficiency of dendrimers that possessed 8 positive charges (chi = 81(.)51%) was significantly better (P = 0(.)0036) than those that possessed 4 positive charges (chi = 56(.)8%). RT-PCR revealed a correlation between levels of VEGF protein mRNA. These results indicated that the most effective structural combination was three branched chains of intermediate length with 8 positive charges such as that found for dendrimer 4. Dendrimer 4 and 7/ODN-1 complexes were subsequently chosen for in vivo analysis. Fluorescein angiography demonstrated that both dendrimers significantly (P < 0(.)0001) reduced the severity of laser mediated CNV for up to two months post-injection. This study demonstrated that lipophilic, charged dendrimer mediated delivery of ODN-1 resulted in the down-regulation of in vitro VEGF expression. In addition, in vivo delivery of ODN-1 by two of the dendrimers resulted in significant inhibition of CNV in an inducible rat model. Time course studies showed that the dendrimer/ODN-1 complexes remained active for up to two months indicating the dendrimer compounds provided protection against the effects of nucleases. (C) 2004 Elsevier Ltd. All rights reserved.

Synthesis of a highly pure lipid core peptide based self-adjuvanting triepitopic group A Streptococcal vaccine, and subsequent immunological evaluation

We have developed a highly pure, self-adjuvanting, triepitopic Group A Streptococcal vaccine based on the lipid core peptide system, a vaccine delivery system incorporating lipidic adjuvant, carrier, and peptide epitopes into a single molecular entity. Vaccine synthesis was performed using native chemical ligation. Due to the attachment of a highly lipophilic adjuvant, addition of 1% (w/v) sodium dodecyl sulfate was necessary to enhance peptide solubility in order to enable ligation. The vaccine was synthesized in three steps to yield a highly pure product (97.7% purity) with an excellent overall yield. Subcutaneous immunization of B10. BR (H-2(k)) mice with the synthesized vaccine, with or without the addition of complete Freund's adjuvant, elicited high serum IgG antibody titers against each of the incorporated peptide epitopes.

Method for the synthesis of highly pure vaccines using the lipid core peptide system

Traditional vaccines consisting of whole attenuated microorganisms, killed microorganisms, or microbial components, administered with an adjuvant (e.g. alum), have been proved to be extremely successful. However, to develop new vaccines, or to improve upon current vaccines, new vaccine development techniques are required. Peptide vaccines offer the capacity to administer only the minimal microbial components necessary to elicit appropriate immune responses, minimizing the risk of vaccination associated adverse effects, and focusing the immune response toward important antigens. Peptide vaccines, however, are generally poorly immunogenic, necessitating administration with powerful, and potentially toxic adjuvants. The attachment of lipids to peptide antigens has been demonstrated as a potentially safe method for adjuvanting peptide epitopes. The lipid core peptide (LCP) system, which incorporates a lipidic adjuvant, carrier, and peptide epitopes into a single molecular entity, has been demonstrated to boost immunogenicity of attached peptide epitopes without the need for additional adjuvants. The synthesis of LCP systems normally yields a product that cannot be purified to homogeneity. The current study describes the development of methods for the synthesis of highly pure LCP analogs using native chemical ligation. Because of the highly lipophilic nature of the LCP lipid adjuvant, difficulties (e.g. poor solubility) were experienced with the ligation reactions. The addition of organic solvents to the ligation buffer solubilized lipidic species, but did not result in successful ligation reactions. In comparison, the addition of approximately 1% (w/v) sodium dodecyl sulfate (SDS) proved successful, enabling the synthesis of two highly pure, tri-epitopic Streptococcus pyogenes LCP analogs. Subcutaneous immunization of B10.BR (H-2(k)) mice with one of these vaccines, without the addition of any adjuvant, elicited high levels of systemic IgG antibodies against each of the incorporated peptides. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.

The Crystal-t Algorithm: A New Approach To Calculate The Sle Of Lipidic Mixtures Presenting Solid Solutions.

Lipidic mixtures present a particular phase change profile highly affected by their unique crystalline structure. However, classical solid-liquid equilibrium (SLE) thermodynamic modeling approaches, which assume the solid phase to be a pure component, sometimes fail in the correct description of the phase behavior. In addition, their inability increases with the complexity of the system. To overcome some of these problems, this study describes a new procedure to depict the SLE of fatty binary mixtures presenting solid solutions, namely the Crystal-T algorithm. Considering the non-ideality of both liquid and solid phases, this algorithm is aimed at the determination of the temperature in which the first and last crystal of the mixture melts. The evaluation is focused on experimental data measured and reported in this work for systems composed of triacylglycerols and fatty alcohols. The liquidus and solidus lines of the SLE phase diagrams were described by using excess Gibbs energy based equations, and the group contribution UNIFAC model for the calculation of the activity coefficients of both liquid and solid phases. Very low deviations of theoretical and experimental data evidenced the strength of the algorithm, contributing to the enlargement of the scope of the SLE modeling.

Sim-xl: A Powerful And User-friendly Tool For Peptide Cross-linking Analysis.

Chemical cross-linking has emerged as a powerful approach for the structural characterization of proteins and protein complexes. However, the correct identification of covalently linked (cross-linked or XL) peptides analyzed by tandem mass spectrometry is still an open challenge. Here we present SIM-XL, a software tool that can analyze data generated through commonly used cross-linkers (e.g., BS3/DSS). Our software introduces a new paradigm for search-space reduction, which ultimately accounts for its increase in speed and sensitivity. Moreover, our search engine is the first to capitalize on reporter ions for selecting tandem mass spectra derived from cross-linked peptides. It also makes available a 2D interaction map and a spectrum-annotation tool unmatched by any of its kind. We show SIM-XL to be more sensitive and faster than a competing tool when analyzing a data set obtained from the human HSP90. The software is freely available for academic use at http://patternlabforproteomics.org/sim-xl. A video demonstrating the tool is available at http://patternlabforproteomics.org/sim-xl/video. SIM-XL is the first tool to support XL data in the mzIdentML format; all data are thus available from the ProteomeXchange consortium (identifier PXD001677).

Study of the antimicrobial peptide indolicidin and a mutant in micelle medium by molecular dynamics simulation

The antimicrobial peptide indolicidin (IND) and the mutant CP10A in hydrated micelles were studied using molecular dynamics simulations in order to observe whether the molecular dynamics and experimental data could be sufficiently correlated and a detailed description of the interaction of the antimicrobial peptides with a model of the membrane provided by a hydrated micelle system could be obtained. In agreement with the experiments, the simulations showed that the peptides are located near the surface of the micelles. Peptide insertions agree with available experimental data, showing deeper insertion of the mutant compared with the peptide IND. Major insertion into the hydrophobic core of the micelle by all tryptophan and mutated residues of CP10A in relation to IND was observed. The charged residues of the terminus regions of both peptides present similar behavior, indicating that the major differences in the interactions with the micelles of the peptides IND and CP10A occur in the case of the hydrophobic residues.

Sensitization Prevalence, Antibody Cross-Reactivity and Immunogenic Peptide Profile of Api g 2, the Non-Specific Lipid Transfer Protein 1 of Celery

Background: Celery (Apium graveolens) represents a relevant allergen source that can elicit severe reactions in the adult population. To investigate the sensitization prevalence and cross-reactivity of Api g 2 from celery stalks in a Mediterranean population and in a mouse model. Methodology: 786 non-randomized subjects from Italy were screened for IgE reactivity to rApi g 2, rArt v 3 (mugwort pollen LTP) and nPru p 3 (peach LTP) using an allergen microarray. Clinical data of 32 selected patients with reactivity to LTP under investigation were evaluated. Specific IgE titers and cross-inhibitions were performed in ELISA and allergen microarray. Balb/c mice were immunized with purified LTPs; IgG titers were determined in ELISA and mediator release was examined using RBL-2H3 cells. Simulated endolysosomal digestion was performed using microsomes obtained from human DCs. Results: IgE testing showed a sensitization prevalence of 25.6% to Api g 2, 18.6% to Art v 3, and 28.6% to Pru p 3 and frequent co-sensitization and correlating IgE-reactivity was observed. 10/32 patients suffering from LTP-related allergy reported symptoms upon consumption of celery stalks which mainly presented as OAS. Considerable IgE cross-reactivity was observed between Api g 2, Art v 3, and Pru p 3 with varying inhibition degrees of individual patients' sera. Simulating LTP mono-sensitization in a mouse model showed development of more congruent antibody specificities between Api g 2 and Art v 3. Notably, biologically relevant murine IgE cross-reactivity was restricted to the latter and diverse from Pru p 3 epitopes. Endolysosomal processing of LTP showed generation of similar clusters, which presumably represent T-cell peptides. Conclusions: Api g 2 represents a relevant celery stalk allergen in the LTP-sensitized population. The molecule displays common B cell epitopes and endolysosomal peptides that encompass T cell epitopes with pollen and plant-food derived LTP.

Bothrops jararaca Peptide with Anti-Hypertensive Action Normalizes Endothelium Dysfunction Involved in Physiopathology of Preeclampsia

Preeclampsia, a pregnancy-specific syndrome characterized by hypertension, proteinuria and edema, is a major cause of fetal and maternal morbidity and mortality especially in developing countries. Bj-PRO-10c, a proline-rich peptide isolated from Bothrops jararaca venom, has been attributed with potent anti-hypertensive effects. Recently, we have shown that Bj-PRO-10c-induced anti-hypertensive actions involved NO production in spontaneous hypertensive rats. Using in vitro studies we now show that Bj-PRO-10c was able to increase NO production in human umbilical vein endothelial cells from hypertensive pregnant women (HUVEC-PE) to levels observed in HUVEC of normotensive women. Moreover, in the presence of the peptide, eNOS expression as well as argininosuccinate synthase activity, the key rate-limiting enzyme of the citrulline-NO cycle, were enhanced. In addition, excessive superoxide production due to NO deficiency, one of the major deleterious effects of the disease, was inhibited by Bj-PRO-10c. Bj-PRO-10c induced intracellular calcium fluxes in both, HUVEC-PE and HUVEC, which, however, led to activation of eNOS expression only in HUVEC-PE. Since Bj-PRO-10c promoted biological effects in HUVEC from patients suffering from the disorder and not in normotensive pregnant women, we hypothesize that Bj-PRO-10c induces its anti-hypertensive effect in mothers with preeclampsia. Such properties may initiate the development of novel therapeutics for treating preeclampsia.

Role of the gp85/Trans-Sialidases in Trypanosoma cruzi Tissue Tropism: Preferential Binding of a Conserved Peptide Motif to the Vasculature In Vivo

Background: Transmitted by blood-sucking insects, the unicellular parasite Trypanosoma cruzi is the causative agent of Chagas' disease, a malady manifested in a variety of symptoms from heart disease to digestive and urinary tract dysfunctions. The reasons for such organ preference have been a matter of great interest in the field, particularly because the parasite can invade nearly every cell line and it can be found in most tissues following an infection. Among the molecular factors that contribute to virulence is a large multigene family of proteins known as gp85/trans-sialidase, which participates in cell attachment and invasion. But whether these proteins also contribute to tissue homing had not yet been investigated. Here, a combination of endothelial cell immortalization and phage display techniques has been used to investigate the role of gp85/trans-sialidase in binding to the vasculature. Methods: Bacteriophage expressing an important peptide motif (denominated FLY) common to all gp85/trans-sialidase proteins was used as a surrogate to investigate the interaction of this motif with the endothelium compartment. For that purpose phage particles were incubated with endothelial cells obtained from different organs or injected into mice intravenously and the number of phage particles bound to cells or tissues was determined. Binding of phages to intermediate filament proteins has also been studied. Findings and Conclusions: Our data indicate that FLY interacts with the endothelium in an organ-dependent manner with significantly higher avidity for the heart vasculature. Phage display results also show that FLY interaction with intermediate filament proteins is not limited to cytokeratin 18 (CK18), which may explain the wide variety of cells infected by the parasite. This is the first time that members of the intermediate filaments in general, constituted by a large group of ubiquitously expressed proteins, have been implicated in T. cruzi cell invasion and tissue homing.

Purification and characterization of an antimicrobial peptide produced by Pseudomonas sp strain 4B

An antimicrobial peptide produced by a bacterium isolated from the effluent pond of a bovine abattoir was purified and characterized. The strain was characterized by biochemical profiling and 16S rDNA sequencing as Pseudomonas sp. The antimicrobial peptide was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. Direct activity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was observed. A major band on SDS-PAGE suggested that the antimicrobial peptide has a molecular mass of about 30 kDa. The substance was inhibitory to a broad range of indicator strains, including pathogenic and food spoilage bacteria such as Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, among other. The partially purified antimicrobial substance remained active over a wide temperature range and was resistant to all proteases tested. This substance showed different properties than other antimicrobials from Pseudomonas species, suggesting a novel antimicrobial peptide was characterized.