Polymer-mediated formation and assembly of silver nanoparticles on silica nanospheres for sensitive surface-enhanced Raman scattering detection.

To impart a desired optical property to metal nanoparticles (NPs) suitable for surface-enhanced Raman scattering (SERS) applications, it is crucial to assemble them in two or three dimensions in addition to controlling their size and shape. Herein, we report a new strategy for the synthesis and direct assembly of Ag NPs on silica nanospheres (AgNPs-SiNS) in the presence of poly(ethylene glycol) (PEG) derivatives such as PEG-OH, bis(amino)-PEGs (DA-PEGs), and O,O'-bis(2-aminopropyl)PEG (DAP-PEG). They exhibited different effects on the formation of Ag NPs with variable sizes (10-40 nm) and density on the silica surface. As the molecular weight (MW) of DA-PEGs increased, the number of Ag NPs on the silica surface increased. In addition, DAP-PEG (MW of 2000), which has a 2-aminopropyl moiety at both ends, promoted the most effective formation and assembly of uniform-sized Ag NPs on a silica surface, as compared to the other PEG derivatives with the same molecular weight. Finally, we demonstrated that AgNPs-SiNS bearing 4-fluorobenzenethiol on its surface induced the strong SERS signal at the single-particle level, indicating that each hybrid particle has internal hot spots. This shows the potential of AgNPs-SiNS for SERS-based sensitive detection of target molecules.

Comparison of endothelialization and neointimal formation with stents coated with antibodies against CD34 and vascular endothelial-cadherin.

Vascular endothelial-cadherin (VE-cadherin) is exclusively expressed on the late endothelial progenitor cells (EPC). Therefore, VE-cadherin could be an ideal target surface molecule to capture circulating late EPC. In the present study, we evaluated whether anti-VE-cadherin antibody-coated stents (VE-cad stents) might accelerate endothelial recovery and reduce neointimal formation more than anti-CD34 antibody-coated stents (CD34 stents) through the superior ability to capture the late EPC. The stainless steel stents were coated with anti-human VE-cadherin antibodies or anti-human CD34 antibodies under the same condition. In vitro, VE-cad stents showed higher number of adhering EPC (823.6 ± 182.2 versus 379.2 ± 137.2 cells per HPF, p < 0.001). VE-cad stents also demonstrated better specific capturing of cells with endothelial lineage markers than CD34 stents did in flow cytometric analysis. VE-cad stents showed more effective re-endothelialization after 1 h, 24 h, and 3 days in vivo. At 42 days, VE-cad stents demonstrated significantly smaller neointima area (0.92 ± 0.38 versus 1.24 ± 0.41 mm(2), p = 0.002) and significantly lower PCNA positive cells in neointima (1684.8 ± 658.8/mm(2) versus 2681.7 ± 375.1/mm(2), p = 0.008), compared with CD34 stents. In conclusion, VE-cad stents captured EPC and endothelial cells more selectively in vitro, accelerated re-endothelialization over stents, and reduced neointimal formation in vivo, compared with CD34 stents.

A universal description for the experimental behavior of salt-(in)dependent oligocation-induced DNA condensation.

We report a systematic study of the condensation of plasmid DNA by oligocations with variation of the charge, Z, from +3 to +31. The oligocations include a series of synthetic linear ε-oligo(L-lysines), (denoted εKn, n = 3­10, 31; n is the number of lysines with the ligand charge Z = n+1) and branched α-substituted homologues of εK10: εYK10, εLK10 (Z = +11); εRK10, εYRK10 and εLYRK10 (Z = +21). Data were obtained by light scattering, UV absorption monitored precipitation assay and isothermal titration calorimetry in a wide range concentrations of DNA and monovalent salt (KCl, CKCl). The dependence of EC50 (ligand concentration at the midpoint of DNA condensation) on C(KCl) shows the existence of a salt-independent regime at low C(KCl) and a salt-dependent regime with a steep rise of EC50 with increase of C(KCl). Increase of the ligand charge shifts the transition from the salt-independent to salt-dependent regime to higher C(KCl). A novel and simple relationship describing the EC50 dependence on DNA concentration, charge of the ligand and the salt-dependent dissociation constant of the ligand­DNA complex is derived. For the ε-oligolysines εK6­ÎµK10, the experimental dependencies of EC50 on C(KCl) and Z are well-described by an equation with a common set of parameters. Implications from our findings for understanding DNA condensation in chromatin are discussed.

The effect of salt on oligocation-induced chromatin condensation.

Condensation of model chromatin in the form of fully saturated 12-mer nucleosome arrays, induced by addition of cationic ligands (ε-oligolysines with charge varied from +4 to +11), was studied in a range of KCl concentrations (10-500mM) using light scattering and precipitation assay titrations. The dependence of EC(50) (ligand concentration at the midpoint of the array condensation) on C(KCl) displays two regimes, a salt-independent at low C(KCl) and a salt-dependent at higher salt concentrations. In the salt-dependent regime EC(50) rises sharply with increase of C(KCl). Increase of ligand charge shifts the transition from the salt-independent to salt-dependent regime to higher salt. In the nucleosome array system, due to the partial neutralization of the DNA charge by histones, a lower oligocation concentration is needed to provoke condensation in the salt-independent regime compared to the related case of DNA condensation by the same cation. In the physiological range of salt concentrations (C(KCl)=50-300mM), K(+) ions assist array condensation by shifting EC(50) of the ε-oligolysines to lower values. At higher C(KCl), K(+) competes with the cationic ligands, which leads to increase of EC(50). Values of salt-dependent dissociation constant for the ε-oligolysine-nucleosome array interaction were obtained, by fitting to a general equation developed earlier for DNA, describing the dependence of EC(50) on dissociation constant, salt and polyelectrolyte concentrations.

Fear conditioning occludes late-phase long-term potentiation at thalamic input synapses onto the lateral amygdala in rat brain slices.

Late-phase long-term potentiation (L-LTP) of excitatory synaptic transmission at thalamic input synapses onto the lateral amygdala (T-LA synapses) has been proposed as a cellular substrate for long-term fear memory. This notion is evidenced primarily by previous reports in which the same pharmacological treatments block both T-LA L-LTP and the consolidation of fear memory. In this study, we report that fear conditioning occludes L-LTP at T-LA synapses in brain slices prepared after fear memory consolidation. L-LTP was restored either when synaptic depotentiation was induced prior to L-LTP induction in brain slices prepared from conditioned rats or when brain slices were prepared from conditioned rats that had been exposed to subsequent fear extinction, which is a behavior paradigm known to induce in vivo synaptic depotentiation at T-LA synapses. These results suggest that fear conditioning recruits L-LTP-like mechanisms that are reversible and saturable at T-LA synapses.

Biophysical properties and supramolecular structure of self-assembled liposome/ε-peptide/DNA nanoparticles: correlation with gene delivery.

Using solid-phase synthesis, lysine can be oligomerized by a reaction of the peptide carboxylate with the ε-amino group to produce nontoxic, biodegradable cationic peptides, ε-oligo(L-lysines). Here α-substituted derivatives of such ε-oligo(L-lysines) containing arginine and histidine in the side chain were tested as vectors for in vitro gene delivery. Combination of ε-oligolysines with the cationic lipid DOTAP and plasmid DNA resulted in transfection efficiency exceeding that of DOTAP alone, without significant increase in cytotoxicity. Synchrotron small-angle X-ray scattering studies revealed self-assembly of the DOTAP, ε-oligolysines, and DNA to ordered lamellar complexes. High transfection efficiency of the nanoparticles correlates with increase in zeta potential above +20 mV and requires particle size to be below 500 nm. The synergistic effect of branched ε-oligolysines and DOTAP in gene delivery can be explained by the increase in surface charge and by the supramolecular structure of the DOTAP/ε-oligolysine/DNA nanoparticles.

Reversible plasticity of fear memory-encoding amygdala synaptic circuits even after fear memory consolidation.

It is generally believed that after memory consolidation, memory-encoding synaptic circuits are persistently modified and become less plastic. This, however, may hinder the remaining capacity of information storage in a given neural circuit. Here we consider the hypothesis that memory-encoding synaptic circuits still retain reversible plasticity even after memory consolidation. To test this, we employed a protocol of auditory fear conditioning which recruited the vast majority of the thalamic input synaptic circuit to the lateral amygdala (T-LA synaptic circuit; a storage site for fear memory) with fear conditioning-induced synaptic plasticity. Subsequently the fear memory-encoding synaptic circuits were challenged with fear extinction and re-conditioning to determine whether these circuits exhibit reversible plasticity. We found that fear memory-encoding T-LA synaptic circuit exhibited dynamic efficacy changes in tight correlation with fear memory strength even after fear memory consolidation. Initial conditioning or re-conditioning brought T-LA synaptic circuit near the ceiling of their modification range (occluding LTP and enhancing depotentiation in brain slices prepared from conditioned or re-conditioned rats), while extinction reversed this change (reinstating LTP and occluding depotentiation in brain slices prepared from extinguished rats). Consistently, fear conditioning-induced synaptic potentiation at T-LA synapses was functionally reversed by extinction and reinstated by subsequent re-conditioning. These results suggest reversible plasticity of fear memory-encoding circuits even after fear memory consolidation. This reversible plasticity of memory-encoding synapses may be involved in updating the contents of original memory even after memory consolidation.

Acid-catalyzed tandem thiol switch for preparing peptide thioesters from mercaptoethyl esters.

An efficient method compatible with Fmoc synthesis for preparing peptide thioesters via an acid-catalyzed tandem "thiol switch" of esters is described first by an intramolecular O-S acyl shift and then by an intermolecular S-S exchange, with concurrent deblocking of side chain protection groups.

A universal description for the experimental behavior of salt-(in)dependent oligocation-induced DNA condensation.

We report a systematic study of the condensation of plasmid DNA by oligocations with variation of the charge, Z, from +3 to +31. The oligocations include a series of synthetic linear epsilon-oligo(l-lysines), (denoted epsilonKn, n = 3-10, 31; n is the number of lysines equal to the ligand charge) and branched alpha-substituted homologues of epsilonK10: epsilonYK10, epsilonLK10 (Z = +10); epsilonRK10, epsilonYRK10 and epsilonLYRK10 (Z = +20). Data were obtained by light scattering, UV absorption monitored precipitation assay and isothermal titration calorimetry in a wide range concentrations of DNA and monovalent salt (KCl, C(KCl)). The dependence of EC(50) (ligand concentration at the midpoint of DNA condensation) on C(KCl) shows the existence of a salt-independent regime at low C(KCl) and a salt-dependent regime with a steep rise of EC(50) with increase of C(KCl). Increase of the ligand charge shifts the transition from the salt-independent to salt-dependent regime to higher C(KCl). A novel and simple relationship describing the EC(50) dependence on DNA concentration, charge of the ligand and the salt-dependent dissociation constant of the ligand-DNA complex is derived. For the epsilon-oligolysines epsilonK3-epsilonK10, the experimental dependencies of EC(50) on C(KCl) and Z are well-described by an equation with a common set of parameters. Implications from our findings for understanding DNA condensation in chromatin are discussed.

Design and biophysical characterization of novel polycationic epsilon-peptides for DNA compaction and delivery.

Design and solid-phase synthesis of novel and chemically defined linear and branched -oligo( l-lysines) (denoted -K n, where n is the number of lysine residues) and their alpha-substituted homologues (epsilon-(R)K10, epsilon-(Y)K10, epsilon-(L)K10, epsilon-(YR)K10, and epsilon-(LYR)K10) for DNA compaction and delivery are reported. The ability to condense viral (T2 and T4) and plasmid DNA as well as the size of -peptide DNA complexes under different conditions was investigated with static and dynamic light scattering, isothermal titration calorimetry, and fluorescence microscopy. Nanoparticle diameters varied from 100 to 150 and 375 to 550 nm for plasmid and T4 DNA peptide complexes, respectively. Smaller sizes were observed for oligo(L-lysines) compared to alpha-poly( L-lysine). The linear -oligo-lysines are less toxic and epsilon-(LYR)K10 showed higher transfection efficiency in HeLa cells than corresponding controls. The results also demonstrate that with a branched design having pendent groups of short alpha-oligopeptides, improved transfection can be achieved. This study supports the hypothesis that available alpha-oligolysine derived systems would potentially have more favorable delivery properties if they are based instead on epsilon-oligo( L-lysines). The flexible design and unambiguous synthesis that enables variation of pendent groups holds promise for optimization of such -peptides to achieve improved DNA compaction and delivery.

Dendritic alpha,epsilon-poly(L-lysine)s as delivery agents for antisense oligonucleotides.

PURPOSE: To evaluate the potential use of dendritic alpha,epsilon-poly(L-lysine)s (DPL) for the efficient cellular delivery of antisense oligonucleotides. METHODS: A series of dendritic alpha,epsilon-polylysines of various generations were prepared. Their physical properties and the ability to form complex with oligonucleotide were investigated by polyacrylamide gel electrophoresis, capillary zone electrophoresis (CZE), agarose gel electrophoresis, fluorescence titration and atomic force microscopy (AFM). The efficiency to deliver oligonucleotide to HeLa cells, stably transfected with plasmid pLuc/705, was evaluated by using antisense splicing correction assay and confocal microscopy. RESULTS: DPLs formed the complexes with antisense oligonucleotide with modest cytotoxicity. The charge ratio of oligonucleotide to DPL and the size (generation) of DPLs were all critical variables for the antisense effect. Compared to low generation DPLs, high generation DPLs were more effective in delivering oligonucleotide into cells. CONCLUSIONS: High generation DPL-oligonucleotide complexes were moderately effective for delivery antisense oligonucleotide. The complex formation provides a promise for in vivo therapeutic application of DPLs or their derivatives in the delivery of gene or oligonucleotide.

A facile synthesis and physical properties of nano-sized dendritic alpha,epsilon-poly(L-lysine)s for the delivery of nucleic acids.

A series of nano-sized dendritic alpha,epsilon-poly(L-lysine)s (DPL) were synthesized by the solid-phase peptide synthesis method, using a core epsilon-peptide structure consisting of eight lysine residues. Surface amines of dendritic alpha,epsilon-poly(L-lysine) were characterized by comparing the retention times of a reverse phase HPLC with the electrophoretic mobilities of capillary zone electrophoresis (CZE) and non-denatured polyacrylamide gel electrophoresis (PAGE). The elution times of alpha,epsilon-poly(Llysine) in HPLC were well correlated with the electrophoretic mobilities of CZE and PAGE. The separation was dependent on size, shape of molecule and the number of surface amine. The alpha,epsilon-poly(L-lysine) formed a complex with nucleic acids at various charge ratios and the degree of complex formation was size- and structure-dependent. Atomic force microscopy of the complex visualized the size and morphology of alpha,epsilon-poly(L-lysine)/DNA complex as a nano-sized spherical shape. The small size in complex formation provides a promise for in vivo therapeutic application of dendritic alpha,epsilon-poly(L-lysine)s or their derivatives in the delivery of gene or oligonucleotide.

Mimicking reverse protein splicing by three-segment tandem peptide ligation.

Here we report a bi-directional and interchangeable three-segment peptide ligation of N, M, and C-segments, mimicking the reverse process of protein splicing to form, in tandem, a tripartite NMC-peptide using a synthetic intein, a role served by the M-segment with an N-terminal Ser or Thr and a C-terminal thioester.

Total synthesis of (+)-asteltoxin.

A convergent synthesis of (+)-asteltoxin (1) has been achieved by the Horner-Emmons olefination of bis(tetrahydrofuran) aldehyde 53 and alpha-pyrone phosphonate 5. A key step features the stereoselective construction of a sterically congested quaternary center embedded in the densely functionalized bis(tetrahydrofuran) subunit by a Lewis acid-catalyzed, pinacol-type rearrangement of an epoxy silyl ether. This pivotal rearrangement methodology parallels the proposed biosynthetic pathway of 1 and is ripe for applications to the stereocontrolled synthesis of structurally complex natural products.

Tandem ligation of multipartite peptides with cell-permeable activity.

To prepare multipartite peptides with several functional cargoes including a cell-permeable sequence or transportant for intracellular delivery, tandem ligation of peptides is a convenient convergent approach with the fewest synthetic steps. It links three or four unprotected segments forming two or more regiospecific bonds consecutively without a deprotection step. This paper describes a tandem ligation strategy to prepare multipartite peptides with normal and branched architectures carrying a novel transportant peptide that is rich in arginine and proline to permit their cargoes to be translocated across membranes to affect their biological functions in cytoplasm. Our strategy consists of three ligation methods specific for amino terminal cysteine (Cys), serine/threonine (Ser/Thr), and N(alpha)-chloroacetylated amine to afford Xaa-Cys, Xaa-OPro (oxaproline) and Xaa-psiGly (pseudoglycine) at the ligation sites, respectively. Assembly of single-chain peptides from three different segments was achieved by the tandem Cys/OPro ligation to form two amide bonds, an Xaa-Cys and then an Xaa-OPro. Assembly of two- and three-chain peptides with branched architectures from four different segments was accomplished by tandem Cys/psiGly/OPro ligation. These NT-specific tandem ligation strategies were successful in generating cell-permeable multipartite peptides with one-, two-, and three-chain architectures, ranging in size from 52 to 75 residues and without the need of a protection or deprotection step. In addition, our results show that there is considerable flexibility in architectural design to obtain cell-permeable multipartite peptides containing a transportant sequence.

Translocating proline-rich peptides from the antimicrobial peptide bactenecin 7.

The intracellular delivery of most peptides, proteins, and nucleotides to the cytoplasm and nucleus is impeded by the cell membrane. To allow simplified, noninvasive delivery of attached cargo, cell-permeant peptides that are either highly cationic or hydrophobic have been utilized. Because cell-permeable peptides share half of the structural features of antimicrobial peptides containing clusters of charge and hydrophobic residues, we have explored antimicrobial peptides as templates for designing cell-permeant peptides. We prepared synthetic fragments of Bac 7, an antimicrobial peptide with four 14-residue repeats from the bactenecin family. The dual functions of cell permeability and antimicrobial activity of Bac 7 were colocalized at the N-terminal 24 residues of Bac 7. In general, long fragments of Bac(1-24) containing both regions were bactericidal and cell-permeable, whereas short fragments with only a cationic or hydrophobic region were cell-permeant without the attendant microbicidal activity when measured in a fluorescence quantitation assay and by confocal microscopy. In addition, the highly cationic fragments were capable of traversing the cell membrane and residing within the nucleus. A common characteristic shared by the cell-permeant Bac(1-24) fragments, irrespective of their number of charged cationic amino acids, is their high proline content. A 10-residue proline-rich peptide with two arginine residues was capable of delivering a noncovalently linked protein into cells. Thus, the proline-rich peptides represent a potentially new class of cell-permeant peptides for intracellular delivery of protein cargo. Furthermore, our results suggest that antimicrobial peptides may represent a rich source of templates for designing cell-permeant peptides.