Development of Neuropeptide Y and Cell-Penetrating Peptide MAP Adsorbed onto Lipid Nanoparticle Surface

Functionalization of nanoparticles surfaces have been widely used to improve diagnostic and therapeutic biological outcome. Several methods can be applied to modify nanoparticle surface; however, in this article we focus toward a simple and less time-consuming method. We applied an adsorption method on already formulated nanostructured lipid carriers (NLC) to functionalize these nanoparticles with three distinct peptides sequences. We selected a cell-penetrating peptide (CPP), a lysine modified model amphipathic peptide (Lys(N₃)-MAP), CPP/drug complex, and the neuropeptide Y. The aim of this work is to evaluate the effect of several parameters such as peptide concentration, different types of NLC, different types of peptides, and incubation medium on the physicochemical proprieties of NLC and determine if adsorption occurs. The preliminary results from zeta potential analysis indicate some evidence that this method was successful in adsorbing three types of peptides onto NLC. Several non-covalent interactions appear to be involved in peptide adsorption with the possibility of three adsorption peptide hypothesis that may occur with NLC in solution. Moreover, and for the first time, in silico docking analysis demonstrated strong interaction between CPP MAP and NPY Y1 receptor with high score values when compared to standard antagonist and NPY.     

PR_b-targeted PEGylated liposomes for prostate cancer therapy

In recent years, there has been considerable effort in designing improved delivery systems by including site-directed surface ligands to further enhance their selective targeting. The goal of this study is to engineer alpha5beta1-targeted stealth liposomes (nanoparticles covered with poly(ethylene glycol) (PEG)) that will bind to alpha5beta1-expressing LNCaP human prostate cancer cells and efficiently release the encapsulated load intracellularly. For this purpose, liposomes (with and without PEG2000) were functionalized with a fibronectin-mimetic peptide (PR_b) and delivered to LNCaPs. The amount of PEG2000 and other liposomal components were characterized by 1H NMR, and the amount of peptide by the bicinchoninic acid protein assay. Fibronectin is the natural ligand for alpha5beta1, and a promising design for a fibronectinmimetic peptide includes both the primary binding site (RGD) and the synergy site (PHSRN) connected by a linker and extended off a surface by a spacer. We have previously designed a peptide-amphiphile, PRb, that employed a hydrophobic tail, connected to the N-terminus of a peptide headgroup composed of a spacer, the synergy site sequence, a linker mimicking both the distance and hydrophobicity/hydrophilicity present in the native protein fibronectin (thus presenting an overall "neutral" linker), and finally the primary binding sequence. We have examined different liposomal formulations, functionalized only with PR_b or with PR_b and PEG2000. For PR_b-targeted PEGylated liposomes, efficient cell binding was observed for peptide concentrations of 2 mol % and higher. When compared to GRGDSP-targeted stealth liposomes, PR_b functionalization was superior to that of GRGDSP as shown by increased LNCaP binding, internalization efficiency, as well as cytotoxicity after incubation of LNCaPs with tumor necrosis factor-alpha (TNFalpha)-encapsulated liposomes. More importantly, PR_b is alpha5beta1-specific, whereas many integrins bind to small RGD peptides. Thus, the proposed PR_b-targeted delivery system has the potential to deliver a therapeutic payload to prostate cancer cells in an efficient and specific manner.     

Peptide C(alpha)D(alpha) stretch frequencies in a hydrated conformation are perturbed mainly by C(alpha)-D(alpha)...O hydrogen bonding

We have shown (J. Phys. Chem. A 2004, 108, 10923; 2007, 111, 5300) that the C(alpha)D(alpha) stretch frequency, nu(CD), can discriminate between uniform alpha(R), beta, and polyproline II conformations of isolated peptides. Similar results for such peptides to which explicit waters are hydrogen bonded exhibit shifts in nu(CD) from those of the isolated structures. We demonstrate that the main source of these frequency shifts is the formation of C(alpha)-D(alpha)...O hydrogen bonds to water. Taking into account C-H...O(water) hydrogen bonding, together with the traditional bonding of peptide groups to water, can be expected to increase our understanding of the interaction of proteins with their aqueous environment.     

Synthesis of cadmium sulphide in pure and mixed Langmuir-Blodgett films of n -octadecylsuccinic acid

Cadmium sulphide (CdS) nanoparticles were grown by the reaction of sodium sulphide (Na₂S) with Langmuir-Blodgett (LB) films of cadmium salts ofn-octadecylsuccinic acid (ODSU) and with LB films of ODSU in mixtures of octadecylamine and octadecyl alcohol. The results indicate that heterogeneous nucleation and aggregation in the pure ODSU LB films due to processes like Ostwald ripening are destabilized by the presence of the long-chain amine and alcohol in mixed systems. CdS nanoparticles in the LB films were monitored by UV-visible absorption spectra, which allow an estimation of the size of the particles. The morphology, size and nature of the nanocrystallites formed depend on whether the sulphidation was done on the pure film or in the mixed films. It is seen that particles of size around 1.6 nm were formed in ODSU/octadecylalcohol and ODSU/ octadecylamine mixed LB films while in pure ODSU films the size was about 2.7 nm. These films showed typical needle-shaped structures, as observed by the optical microscopic technique. Mean size and morphology were confirmed by transmission and scanning electron microscopy, while selective area electron diffraction patterns showed six-fold symmetry and indicated that the CdS crystals grow epitaxially with respect to the monolayer. Further, the crystallisation enhanced in the mixed LB films showed a characteristic zinc oxide (Wurtzite) structure compared with the pure ODSU matrix.     

净电荷对螺旋型抗癌肽生物活性的影响

以高活性两亲性α-螺旋型阳离子抗癌肽A12L/A20L(多肽P)为模板, 在其亲水面进行氨基酸定点取代, 获得了一系列带有不同净电荷的多肽类似物, 研究了净电荷对螺旋型抗癌肽生物活性的影响. 结果表明, 抗癌肽净电荷的改变对其溶血活性影响较小(最大差异为2倍), 而对抗癌活性和选择性的影响显著(最大差异为10倍). 抗癌肽P的净电荷最适范围为+7到+8, 分子间静电排斥作用的最佳数目为3~5个, 高于或低于此范围, 其抗癌活性和选择性均明显降低. 与人的正常细胞相比, 负电性的癌细胞膜对于抗癌肽的净电荷变化更敏感, 表明两亲性螺旋型抗癌肽针对癌细胞与正常细胞表现出良好的选择特异性.     

液晶聚合物的单层与Langmuir-Blodgett膜

系统研究了手性液晶聚硅氧烷和光致变色液晶聚硅氧烷两个毓的侧链液晶聚合物在空气/水界面的单层行为和Langmuir-Blodgett(LB)膜沉积特性,对LB膜结构与存在的聚集现象进行了系统的表征,并初步探讨了LB膜中液晶聚合物表现的功能性。     

Biological selection of peptides for poly(l-lactide) substrates

Short peptides that recognize the alpha form of poly( l-lactide) (PLLA) crystalline films were identified from a phage-displayed peptide library. An enzyme-linked immunosorbent assay (ELISA) revealed that the apparent binding constants of the phage clones for the alpha form of PLLA were greater than those of the unselected phage library. The specificity index for the alpha form of PLLA referred to a structurally similar atactic poly(methyl methacrylate) (at-PMMA), supporting the alpha form of PLLA specific binding of the selected phage. Amino acid residues with proton-donor lateral groups and hydrophobic alkyl groups were relatively enriched in a sequence of heptapeptides on the specific phage clones, thereby suggesting the presence of hydrogen bonding as well as hydrophobic interactions between the alpha form of PLLA and the peptides. Surface plasmon resonance (SPR) analysis revealed that the binding constant of the freed c22 heptapeptide (Gln-Leu-Met-His-Asp-Tyr-Arg) for the alpha form of PLLA was greater than those for reference at-PMMA, amorphous PLLA, and the beta form of PLLA. It was found that c22 peptide can recognize slight differences in PLLA polymorphs such as a crystalline state and an arrangement of PLLA functional groups.     

Solution NMR studies of the A beta(1-40) and A beta(1-42) peptides establish that the Met35 oxidation state affects the mechanism of amyloid formation

The pathogenesis of Alzheimer's disease is characterized by the aggregation and fibrillation of the 40-residue A beta(1-40) and 42-residue A beta(1-42) peptides into amyloid plaques. The structural changes associated with the conversion of monomeric A beta peptide building blocks into multimeric fibrillar beta-strand aggregates remain unknown. Recently, we established that oxidation of the methionine-35 side chain to the sulfoxide (Met35(red) --> Met35(ox)) significantly impedes the rate of aggregation and fibrillation of the A beta peptide. To explore this effect at greater resolution, we carefully compared the (1)H, (15)N, and (13)C NMR chemical shifts of four A beta peptides that had the Met35 reduced or oxidized (A beta(1-40)Met35(red), A beta(1-40)Met35(ox), A beta(1-42)Met35(red), and A beta(1-42)Met35(ox)). With the use of a special disaggregation protocol, the highly aggregation prone A beta peptides could be studied at higher, millimolar concentrations (as required by NMR) in aqueous solution at neutral pH, remaining largely monomeric at 5 degrees C as determined by sedimentation equilibrium studies. The NOE, amide-NH temperature coefficients, and chemical shift indices of the (1)H alpha, (13)C alpha, and (13)C beta established that the four peptides are largely random, extended chain structures, with the Met35(ox) reducing the propensity for beta-strand structure at two hydrophobic regions (Leu17-Ala21 and Ile31-Val36), and turn- or bendlike structures at Asp7-Glu11 and Phe20-Ser26. Additional NMR studies monitoring changes that occur during aging at 37 degrees C established that, along with a gradual loss of signal/noise, the Met35(ox) significantly hindered upfield chemical shift movements of the 2H NMR signals for the His6, His13, and His14 side chains. Taken together, the present NMR studies demonstrate that the Met35(red) --> Met35(ox) conversion prevents aggregation by reducing both hydrophobic and electrostatic association and that the A beta(1-40)Met35(red), A beta(1-40)Met35(ox), A beta(1-42)Met35(red), and A beta(1-42)Met35(ox) peptides may associate differently, through specific, sharp changes in structure during the initial stages of aggregation.     

Temperature profiling of polypeptides in reversed-phase liquid chromatography. I. Monitoring of dimerization and unfolding of amphipathic alpha-helical peptides

The present study sets out to extend the utility of reversed-phase liquid chromatography (RP-HPLC) by demonstrating its ability to monitor dimerization and unfolding of de novo designed synthetic amphipathic alpha-helical peptides on stationary phases of varying hydrophobicity. Thus, we have compared the effect of temperature (5-80 degrees C) on the RP-HPLC (C8 or cyano columns) elution behaviour of mixtures of peptides encompassing amphipathic alpha-helical structure, amphipathic alpha-helical structure with L- or D-substitutions or non-amphipathic alpha-helical structure. By comparing the retention behaviour of the helical peptides to a peptide of negligible secondary structure (a random coil), we rationalize that "temperature profiling" by RP-HPLC can monitor association of peptide molecules, either through oligomerization or aggregation, or monitor unfolding of alpha-helical peptides with increasing temperature. We believe that the conformation-dependent response of peptides to RP-HPLC under changing temperature has implications both for general analysis and purification of peptides but also for the de novo design of peptides and proteins.     

Efficient molecular mechanics simulations of the folding,orientation, and assembly of peptides in lipid bilayers using an implicit atomic solvation model

Membrane proteins comprise a significant fraction of the proteomes of sequenced organisms and are the targets of approximately half of marketed drugs. However, in spite of their prevalence and biomedical importance, relatively few experimental structures are available due to technical challenges. Computational simulations can potentially address this deficit by providing structural models of membrane proteins. Solvation within the spatially heterogeneous membrane/solvent environment provides a major component of the energetics driving protein folding and association within the membrane. We have developed an implicit solvation model for membranes that is both computationally efficient and accurate enough to enable molecular mechanics predictions for the folding and association of peptides within the membrane. We derived the new atomic solvation model parameters using an unbiased fitting procedure to experimental data and have applied it to diverse problems in order to test its accuracy and to gain insight into membrane protein folding. First, we predicted the positions and orientations of peptides and complexes within the lipid bilayer and compared the simulation results with solid-state NMR structures. Additionally, we performed folding simulations for a series of host–guest peptides with varying propensities to form alpha helices in a hydrophobic environment and compared the structures with experimental measurements. We were also able to successfully predict the structures of amphipathic peptides as well as the structures for dimeric complexes of short hexapeptides that have experimentally characterized propensities to form beta sheets within the membrane. Finally, we compared calculated relative transfer energies with data from experiments measuring the effects of mutations on the free energies of translocon-mediated insertion of proteins into lipid bilayers and of combined folding and membrane insertion of a beta barrel protein.     

Amphiphilic peptides as models for protein-membrane interactions: interfacial behaviour of sequential Lys- and Leu-based peptides and their penetration into lipid monolayers

Synthetic peptides constructed with doublets of hydrophobic residues tandemly repeated with doublets of positively charged residues, (Leu-Lys-Lys-Leu)_n, were used as models for the study of protein-membrane interactions. Their behaviour has been compared with that of their strictly alternating iso peptides, (Leu-Lys)_n. Both peptides present a random coil structure in pure water. In saline solutions, (Leu-Lys-Lys-Leu)_n peptides adopt an α-helical structure whereas (Leu-Lys)_n transit into a β-sheet structure. These peptides form multilayer assemblies on a pure water subphase but they are organized in monomolecular films on a saline aqueous subphase. The stability of these films increases with the peptide length. Structured peptides (α helices and β sheets) penetrate readily into lipid monolayers, whereas the penetration of unordered peptides is very slow. We have not observed any significant difference between the behaviour of a helices and β-sheet structures.     

Design of a novel fibronectin-mimetic peptide-amphiphile for functionalized biomaterials

The interaction of the alpha5beta1 integrin with its ligand, fibronectin, supports numerous adhesive functions and has an important role in health and disease. In recent years, there has been a considerable effort in designing fibronectin-mimetic peptides to target the integrin. However, to date, the therapeutic use of these peptides has been limited, as they cannot accurately mimic fibronectin's binding affinity for alpha5beta1. A peptide-amphiphile (PR_b) was synthesized with a peptide headgroup composed of four building blocks: a spacer; RGDSP, the primary recognition site for alpha5beta1; PHSRN, the synergy binding site; and a linker. The linker was designed to mimic two important criteria: the distance and the hydrophobicity/hydrophilicity between PHSRN and RGD in fibronectin. Human umbilical vein endothelial cells were seeded on different substrates and evaluated in terms of adhesion, spreading, specificity, cytoskeleton organization, focal adhesions, and secretion of extracellular fibronectin. This peptide was shown to perform comparably to fibronectin, indicating that a biomimetic approach can result in the design of novel peptides with therapeutic potential for biomaterial functionalization.     

聚苯胺基LB膜的制备及气敏特性的研究

以聚苯胺 (PAN)为原料 ,利用LB(Langmuir Blodgett)超薄分子膜技术 ,制备了不同层数的纯聚苯胺LB膜及聚苯胺与乙酸 (AA)混合 (PAN AA)LB膜 ,对其进行了紫外 可见光光谱分析研究 ,并对不同层数的聚苯胺基LB膜的二氧化氮 (NO2 )气敏特性进行了研究 ,发现PANLB膜较PAN AALB膜具有更好的敏感性、响应性和可逆性 ,3层和 15层PANLB膜对相对百分比为 2 0× 10 -6NO2 的响应时间分别为 10s和 30s,15层PANLB膜的恢复时间约为 4min     

Thermodynamic analysis of beta-hairpin-forming peptides from the thermal dependence of (1)H NMR chemical shifts

The temperature dependence of the (1)H chemical shifts of six designed peptides previously shown to adopt beta-hairpin structures in aqueous solution has been analyzed in terms of two-state (beta-hairpin left arrow over right arrow coil) equilibrium. The stability of the beta-hairpins formed by these peptides, as derived from their T(m) (midpoint transition temperature) values, parallels in general their ability to adopt those structures as deduced from independent NMR parameters: NOEs, Deltadelta(C)(alpha)(H), Deltadelta(C)(alpha), and Deltadelta(C)(beta) values. The observed T(m) values are dependent on the particular position within the beta-hairpin that is probed, indicating that their folding to a beta-hairpin conformation deviates from a "true" two-state transition. To obtain individual T(m) values for each hairpin region in each peptide, a simplified model of a successive uncoupled two-state equilibrium covering the entire process has been applied. The distribution of T(m) values obtained for the different beta-hairpin regions (turn, strands, backbone, side chains) in the six analyzed peptides reveals a similar pattern. A model for beta-hairpin folding is proposed on the basis of this pattern and the reasonable assumption that regions showing higher T(m) values are the last ones to unfold and, presumably, the first to form. With this assumption, the analysis suggests that turn formation is the first event in beta-hairpin folding. This is consistent with previous results on the essential role of the turn sequence in beta-hairpin folding.     

Periodic sequence distribution of product ion abundances in electron capture dissociation of amphipathic peptides and proteins

The rules for product ion formation in electron capture dissociation (ECD) mass spectrometry of peptides and proteins remain unclear. Random backbone cleavage probability and the nonspecific nature of ECD toward amino acid sequence have been reported, contrary to preferential channels of fragmentation in slow heating-based tandem mass spectrometry. Here we demonstrate that for amphipathic peptides and proteins, modulation of ECD product ion abundance (PIA) along the sequence is pronounced. Moreover, because of the specific primary (and presumably secondary) structure of amphipathic peptides, PIA in ECD demonstrates a clear and reproducible periodic sequence distribution. On the one hand, the period of ECD PIA corresponds to periodic distribution of spatially separated hydrophobic and hydrophilic domains within the peptide primary sequence. On the other hand, the same period correlates with secondary structure units, such as α-helical turns, known for solution-phase structure. Based on a number of examples, we formulate a set of characteristic features for ECD of amphipathic peptides and proteins: (1) periodic distribution of PIA is observed and is reproducible in a wide range of ECD parameters and on different experimental platforms; (2) local maxima of PIA are not necessarily located near the charged site; (3) ion activation before ECD not only extends product ion sequence coverage but also preserves ion yield modulation; (4) the most efficient cleavage (e.g. global maximum of ECD PIA distribution) can be remote from the charged site; (5) the number and location of PIA maxima correlate with amino acid hydrophobicity maxima generally to within a single amino acid displacement; and (6) preferential cleavage sites follow a selected hydrogen spine in an α-helical peptide segment. Presently proposed novel insights into ECD behavior are important for advancing understanding of the ECD mechanism, particularly the role of peptide sequence on PIA. An improved ECD model could facilitate protein sequencing and improve identification of unknown proteins in proteomics technologies. In structural biology, the periodic/preferential product ion yield in ECD of α-helical structures potentially opens the way toward de novo site-specific secondary structure determination of peptides and proteins in the gas phase and its correlation with solution-phase structure.     

Rationalizing the membrane interactions of cationic amphipathic antimicrobial peptides by their molecular shape

Biophysical and structural studies of cationic amphipathic antimicrobial peptides have revealed new mechanistic details concerning their membrane interactions. In interfacial environments the peptides adopt amphipathic conformations and the resulting distribution of polar, charged and hydrophobic residues allows them to partition into the bilayer interface. For several helical peptides it was found that their long axis is oriented parallel to the membrane surface, an arrangement which results in considerable perturbations in the packing of the lipid bilayer. Within the molecular shape concept the peptides act as wedge-like structures which impose positive curvature strain on the membrane. As a consequence a wide variety of morphologies are observed of peptide–lipid mixtures which strongly depend on the detailed peptide sequence, the membrane lipid composition, buffer, temperature and other environmental parameters. Therefore, the peptide–lipid systems are best described by phase diagrams, similar to the ones of detergent–lipid mixtures, encompassing on the one extreme regions where the peptide stabilizes the bilayer and on the other extreme regions where membrane lysis occurs. The effects of peptide sequence, membrane penetration depth, lipid composition and membrane surface charge density on membrane-association, -morphology and the resulting phase boundaries are discussed.     

Structural consequences of metal complexation of cyclo[Pro-Phe-Phe-Ala-Xaa]2 decapeptides

The conformational features of both free and Ca2+-complexed cyclo[Pro-Phe-Phe-Ala-Xaa]2 (with Xaa= Glu(OtBu), Lys(CIZ), Leu, and Ala) in solution have been determined by NMR spectroscopy and extensive distance-geometry calculations. The decapeptides are conformationally homogeneous in solution and show common structural features in their free and complexed forms. The structures of the free form contain only trans peptide bonds and are topologically similar to the structure of gramicidin-S, folded up in two antiparallel extended structures, stabilized by interstrand hydrogen bonds, and closed at both ends by two beta-turns. In contrast, the Ca2+-complexed peptides present two cis peptide bonds and are generally similar to those observed for the metal-complexed forms of antamanide and related analogues, folded into a saddle shape with two beta-turns. The Glu(OtBu)-, Leu-, and Lys(ClZ)-containing peptides examined here maintain the biological activity of the cyclolinopeptide A in their ability to competitively inhibit cholate uptake. The natural antamanide and cyclolinopeptide A are both able to inhibit the uptake of bile salts into hepatocytes. They share the same postulated active sequence Pro-Phe-Phe. Based on our structural results, we conclude that the ability to adopt a global conformation, characterized by a clear amphipathic separation of hydrophobic and hydrophilic surfaces, is an important feature for the functioning of this class of peptides.     

稀土Eu(Ⅲ)双酞菁衍生物LB膜的荧光性

合成了八－４－（四氢糠氧基）酞菁铕（Ⅲ）,通过元素分析、红外光谱、质谱和紫外－可见光谱加以确认。测定了配合物的ＩＩ－Ａ曲线,证明它有很好的成膜性,Ｚ型沉积形成的ＬＢ膜材料有很强的荧光响应,随着ＬＢ膜厚度的增加,荧光性增强。掺杂邻菲咯啉形成的混合ＬＢ膜,其荧光性比纯膜强。但不是邻菲咯啉加入的量越多荧光性越强。配合物：邻菲咯啉＝１：１０时（摩尔比）,有最好的荧光行为。用电子光谱对ＬＢ膜的结构进行了表征     

The Competition between the SRN1 and the SNAr on the β-position of Porphyrin Ring

In our recently reports, a series of amphipathic β-substituted porphyrins are synthesized, And their LB films show some novel properties.^[1] During the preparation of the intermediate of amphipathic porphyrins by the reaction of 2-nitro-5, 10,15,20-tetraphenylporphyrinato copper (Ⅱ) 1 with phenoxide ion 2(eq.l), we found that the product distribution mainly relied on if the electron acceptor was added and which solvent was used. Here we will first report the influence of p-dinitrobenzene (P-DNB) to the above reaction,the influence of the solvents to this reaction and the reaction of 2-naphoxide with 1 will be reported elsewhere.