atom-probe-tomography-studies-on-the-cuingase2-grain-boundaries

Atom Probe Tomography Studies on the Cu(In,Ga)Se<sub>2</sub> Grain Boundaries

Compared with the existent techniques, atom probe tomography is a unique technique able to chemically characterize the internal interfaces at the ...

Max-Planck-Institut für Eisenforschung GmbH

Cell-penetrating peptides (CPPs) are able to translocate problematic therapeutic cargoes across cellular membranes. The exact mechanisms of translocation are still under investigation. However, evidence for endocytic uptake is increasing. We investigated the interactions of CPPs with phospholipid bilayers as first step of translocation. To this purpose, we employed four independent techniques, comprising (i) liposome buffer equilibrium dialysis, (ii) Trp fluorescence quenching, (iii) fluorescence polarization, and (iv) determination of zeta-potentials. Using unilamellar vesicles (LUVs) of different phospholipid composition, we compared weakly cationic human calcitonin (hCT)-derived peptides with the oligocationic CPPs pVEC and penetratin (pAntp). Apparent partition coefficients of hCT-derived peptides in neutral POPC LUVs were dependent on amino acid composition and secondary structure; partitioning in negatively charged POPC/POPG (80:20) LUVs was increased and mainly governed by electrostatic interactions. For hCT(9-32) and its derivatives, D values raised from about 100-200 in POPC to about 1000 to 1500 when negatively charged lipids were present. Localization profiles of CPPs obtained by Trp fluorescence quenching were dependent on the charge density of LUVs. In POPC/POPG, hCT-derived CPPs were located on the bilayer surface, whereas pVEC and pAntp resided deeper in the membrane. In POPG LUVs, an increase of fluorescence polarization was observed for pVEC and pAntp but not for hCT-derived peptides. Generally, we found strong peptide-phospholipid interactions, especially when negatively charged lipids were present.

Bilayer interaction and localization of cell penetrating peptides with model membranes: a comparative study of a human calcitonin (hCT)-derived peptide with pVEC and pAntp(43-58).

Peptides de p&eacute;n&eacute;tration cellulaire (CPPs) sont en mesure d&#39;effectuer des transferts des cargaisons th&eacute;rapeutiques probl&eacute;matiques &agrave; travers les membranes cellulaires. Les m&eacute;canismes exacts de la translocation sont toujours sous enqu&ecirc;te. Toutefois, les &eacute;l&eacute;ments de preuve pour l&#39;absorption de l&#39;endocytose augmente. Nous avons &eacute;tudi&eacute; les interactions de PPC avec bicouches phospholipidiques comme premi&egrave;re &eacute;tape de translocation. &Agrave; cette fin, nous avons utilis&eacute; quatre techniques ind&eacute;pendants, comprenant la dialyse &agrave; l&#39;&eacute;quilibre tampon liposome (i), (ii) Trp extinction de la fluorescence, polarisation de fluorescence (iii) et (iv) la d&eacute;termination du potentiel z&ecirc;ta. &Agrave; l&#39;aide de v&eacute;sicules unilamellaires (LUVs) de composition phospholipidique diff&eacute;rents, nous avons compar&eacute; la calcitonine humaine cationique faiblement (hCT)-d&eacute;riv&eacute; de peptides avec le pVEC CPPs oligocationic et penetratin (pAntp). Coefficients de partage apparent des peptides d&eacute;riv&eacute;s hCT en neutre POPC LUVs d&eacute;pendaient de la composition en acides amin&eacute;s et la structure secondaire ; partitionnement dans n&eacute;gativement charg&eacute; POPC/le POPG (80/20) LUVs a &eacute;t&eacute; augment&eacute; et r&eacute;gi principalement par les interactions &eacute;lectrostatiques. Pour hCT(9-32) et ses d&eacute;riv&eacute;s, valeurs D &eacute;lev&eacute;s d&#39;environ 100-200 dans POPC &agrave; environ 1000 &agrave; 1500 lorsque n&eacute;gativement charg&eacute;s de lipides &eacute;taient pr&eacute;sents. Profils de localisation de CPPs obtenues par extinction de la fluorescence Trp d&eacute;pendaient de la densit&eacute; de charge de LUVs. Dans POPC/le POPG, d&eacute;riv&eacute; de hCT CPPs &eacute;taient situ&eacute;es sur la surface de la bicouche, consid&eacute;rant que pVEC et pAntp r&eacute;sident plus profond&eacute;ment dans la membrane. Dans le POPG LUVs, on a observ&eacute; une augmentation de la polarisation de fluorescence pour pVEC et pAntp mais pas pour les peptides d&eacute;riv&eacute;s hCT. G&eacute;n&eacute;ralement, nous avons trouv&eacute; des interactions fortes peptide-phospholipides, surtout quand les lipides charg&eacute;s n&eacute;gativement &eacute;taient pr&eacute;sents.

Interaction de la bicouche et localisation de cellule p&eacute;n&eacute;trant des peptides avec membranes mod&egrave;les : une &eacute;tude comparative d&#39;une humaine de la calcitonine (hCT)-d&eacute;riv&eacute; de peptide avec pVEC et pAntp(43-58).

Cellula-penetrante peptidi (CPPs) sono in grado di traslocano nel problematici carichi terapeutici attraverso le membrane cellulari. L&#39;esatto meccanismo di traslocazione &egrave; ancora sotto inchiesta. Tuttavia, prove di assorbimento endocitico sono in aumento. Abbiamo studiato le interazioni di CPPs con bilayer del fosfolipide come primo passo della traslocazione. A questo scopo, abbiamo impiegato quattro tecniche indipendenti, composto da dialisi di equilibrio buffer liposoma (i), (ii) Trp fluorescenza tempra, polarizzazione di fluorescenza (iii) e (iv) la determinazione del potenziale zeta. Utilizzando le vescicole unilamellari (LUVs) di composizione differente fosfolipide, abbiamo confrontato la calcitonina umana debolmente cationica (hCT)-derivato di peptidi con la oligocationic CPPs pVEC e penetratin (pAntp). Coefficienti di partizione apparente di peptidi derivati hCT in neutro POPC LUVs erano dipendenti dalla composizione in amminoacidi e struttura secondaria; partizionamento negativamente caricato POPC/POPG (80: 20) LUVs era aumentato e soprattutto governato da interazioni elettrostatiche. Per hCT(9-32) e suoi derivati, i valori D generati da circa 100-200 in POPC a circa 1000-1500 quando negativamente caricato lipidi erano presenti. Profili di localizzazione di CPPs ottenuti da Trp fluorescenza tempra erano dipendenti dalla densit&agrave; di carica di LUVs. Nel POPC/POPG, CPPs hCT-derivati sono stati situati sulla superficie del doppio strato, mentre pVEC e pAntp risiedette pi&ugrave; profondo nella membrana. In POPG LUVs, &egrave; stato osservato un aumento della polarizzazione di fluorescenza per pVEC e pAntp, ma non per peptidi derivati hCT. In generale, abbiamo trovato le interazioni forti del peptide-fosfolipide, soprattutto quando caricate negativamente lipidi erano presenti.

Interazione a doppio strato e la localizzazione della cella penetrante peptidi con membrane modello: uno studio comparativo di una calcitonina umana (hCT)-derivato del peptide con pVEC e pAntp(43-58).

P&eacute;ptidos de penetraci&oacute;n celular (CPPs) son capaces de translocan problem&aacute;ticas cargas terap&eacute;uticas a trav&eacute;s de las membranas celulares. Los mecanismos exactos de desplazamiento est&aacute;n todav&iacute;a bajo investigaci&oacute;n. Sin embargo, la evidencia de absorci&oacute;n endoc&iacute;ticas est&aacute; aumentando. Investigamos las interacciones de la CPPs con bicapas de fosfol&iacute;pidos como primer paso de desplazamiento. Para ello, empleamos cuatro t&eacute;cnicas independientes, que comprende i liposoma tamp&oacute;n equilibrio di&aacute;lisis, Trp (ii) Temple de fluorescencia, polarizaci&oacute;n de la fluorescencia (iii) y (iv) la determinaci&oacute;n de potenciales zeta. Con ves&iacute;culas unilaminar (pa&ntilde;ales) de la composici&oacute;n de los diferentes fosfol&iacute;pidos, comparamos la calcitonina humana cati&oacute;nica d&eacute;bil (hCT)-derivado de p&eacute;ptidos oligocationic CPPs pVEC y penetratin (pAntp). Coeficientes de partici&oacute;n aparente de p&eacute;ptidos derivados del hCT en neutral POPC pa&ntilde;ales depend&iacute;an de composici&oacute;n de amino&aacute;cidos y la estructura secundaria; repartir en negativamente cargado POPC/POPG (80: 20) pa&ntilde;ales fue aumentado y principalmente gobernada por interacciones electrost&aacute;ticas. Para hCT(9-32) y sus derivados, valores D aument&oacute; de unos 100-200 en POPC a cerca de 1000 a 1500 cuando negativamente cargado l&iacute;pidos estaban presentes. Perfiles de localizaci&oacute;n de CPPs obtenidos por Trp fluorescencia amortiguamiento depend&iacute;an de la densidad de la carga de pa&ntilde;ales. En POPC/POPG, hCT-derivado de la CPPs fueron situada en la superficie de la bicapa, mientras que pVEC y pAntp residieron m&aacute;s profundo en la membrana. En POPG de pa&ntilde;ales, se observ&oacute; un aumento de la polarizaci&oacute;n de la fluorescencia para pVEC y pAntp pero no para p&eacute;ptidos derivados del hCT. En general, encontramos las interacciones fuertes p&eacute;ptido-fosfol&iacute;pido, especialmente cuando los l&iacute;pidos cargados negativamente estaban presentes.

Interacci&oacute;n de la bicapa y localizaci&oacute;n de c&eacute;lula penetrando p&eacute;ptidos con membranas modelo: un estudio comparativo de un humano calcitonina (hCT)-derivado p&eacute;ptido con pVEC y pAntp(43-58).

hCT(9-32) is a human calcitonin (hCT)-derived cell-penetrating peptide that has been shown to translocate the plasma membrane of mammalian cells. It has been suggested as a cellular carrier for drugs, green fluorescent protein, and plasmid DNA. Because of its temperature-dependent cellular translocation resulting in punctuated cytoplasmatic distribution, its uptake is likely to follow an endocytic pathway. To gain insight into the molecular orientation of hCT(9-32) when interacting with lipid models, and to learn more about its mode of action, various biophysical techniques from liposome partitioning to high-resolution NMR spectroscopy were utilized. Moreover, to establish the role of individual residues for the topology of its association with the lipid membrane, two mutants of hCT(9-32), i.e., W30-hCT(9-32) and A23-hCT(9-32), were also investigated. Although unstructured in aqueous solution, hCT(9-32) adopted two short helical stretches when bound to dodecylphosphocholine micelles, extending from Thr10 to Asn17 and from Gln24 to Val29. A23-hCT(9-32), in which the helix-breaking Pro23 was replaced by Ala, displayed a continuous alpha-helix extending from residue 12 to 26. Probing with the spin label 5-doxylstearate revealed that association with dodecylphosphocholine micelles was such that the helix engaged in parallel orientation to the micelle surface. Moreover, the Gly to Trp exchange in W30-hCT(9-32) resulted in a more stable anchoring of the C-terminal segment close to the interface, as reflected by a twofold increase in the partition coefficient in liposomes. Interestingly, tighter binding to model membranes was associated with an increase in the in vitro uptake in human cervix epithelial adenocarcinoma cell line cells. Liposome leakage studies excluded pore formation, and the punctuated fluorescence pattern of internalized peptide indicated vesicular localization and, in conclusion, strongly suggested an endocytic pathway of translocation.

Membrane surface-associated helices promote lipid interactions and cellular uptake of human calcitonin-derived cell penetrating peptides.

hCT(9-32) est une humaine de la calcitonine (hCT)-d&eacute;riv&eacute; de peptide de p&eacute;n&eacute;tration cellulaire qui a &eacute;t&eacute; d&eacute;montr&eacute; que la translocation de la membrane plasmique des cellules de mammif&egrave;res. Il a &eacute;t&eacute; sugg&eacute;r&eacute; comme un transporteur cellulaire des drogues, prot&eacute;ine fluorescente verte et l&#39;ADN de plasmide. En raison de sa d&eacute;pend de la temp&eacute;rature translocation cellulaire ayant pour r&eacute;sultat une distribution cytoplasmique ponctu&eacute;e, son absorption est susceptible de suivre une voie d&#39;endocytose. Pour avoir un aper&ccedil;u de l&#39;orientation mol&eacute;culaire des hCT(9-32) lors de l&#39;interaction avec les mod&egrave;les de lipides et en savoir plus sur son mode d&#39;action, diverses techniques biophysiques de liposome partitionnement &agrave; la spectroscopie RMN haute r&eacute;solution ont &eacute;t&eacute; utilis&eacute;es. En outre, afin d&#39;&eacute;tablir le r&ocirc;le des r&eacute;sidus individuels pour la topologie de son association avec la membrane lipidique, deux mutants de hCT(9-32), c&#39;est-&agrave;-dire, W30-hCT(9-32) et A23-hCT(9-32), ont aussi &eacute;t&eacute; examin&eacute;es. Bien que non structur&eacute;es en solution aqueuse, hCT(9-32) adopte deux tron&ccedil;ons h&eacute;lico&iuml;dales courtes lorsqu&#39;elle est li&eacute;es &agrave; des micelles de dod&eacute;cylphosphocholine, s&#39;&eacute;tendant de niveau Thr10 &agrave; Asn17 et de Gln24 &agrave; Val29. A23-hCT(9-32), dans lequel le Pro23 h&eacute;lice r&eacute;volutionnaire a &eacute;t&eacute; remplac&eacute; par Ala, affiche une h&eacute;lice alpha continue s&#39;&eacute;tendant de 12 &agrave; 26 de r&eacute;sidus. Sondage avec l&#39;&eacute;tiquette de spin 5-doxylst&eacute;arate a r&eacute;v&eacute;l&eacute; que liaison avec dod&eacute;cylphosphocholine micelles &eacute;tait tel que l&#39;h&eacute;lice engag&eacute;e dans une orientation parall&egrave;le &agrave; la surface de la micelle. En outre, la Gly Trp &eacute;change en W30-hCT(9-32) a entra&icirc;n&eacute; un ancrage plus stable du segment C-terminal &agrave; proximit&eacute; de l&#39;interface, comme en t&eacute;moigne une augmentation du double dans le coefficient de partage dans les liposomes. Fait int&eacute;ressant, la liaison plus serr&eacute;e &agrave; membranes mod&egrave;les &eacute;tait associ&eacute;e &agrave; une augmentation de l&#39;absorption in vitro dans le col de l&#39;ut&eacute;rus humain ad&eacute;nocarcinome &eacute;pith&eacute;lial ligne cellules. Liposome fuite &eacute;tudes exclues de la formation de pores, et le mod&egrave;le de fluorescence ponctu&eacute; de peptide int&eacute;rioris&eacute;e indiqu&eacute; localisation v&eacute;siculaire et, en conclusion, fortement sugg&eacute;r&eacute; une voie endocytose de translocation.

H&eacute;lices de surface-associated membrane promouvoir des interactions lipides et assimilation de peptides p&eacute;n&eacute;trante des cellules humaines d&eacute;riv&eacute; de la calcitonine.

hCT(9-32) &egrave; una calcitonina umana (hCT)-derivato del peptide cellula-penetrante che &egrave; stato dimostrato che traslocano nella membrana plasmatica delle cellule di mammiferi. &Egrave; stato suggerito come un cellulare carrier per farmaci, proteina fluorescente verde e DNA del plasmide. A causa della sua temperatura-dipendente traslocazione cellulare con conseguente distribuzione citoplasmatiche punteggiato, l&#39;assorbimento &egrave; probabile che a seguire un percorso endocitico. Per guadagnare comprensione l&#39;orientamento molecolare di hCT(9-32) durante l&#39;interazione con i modelli di lipidi e per saperne di pi&ugrave; sulle sue modalit&agrave; d&#39;azione, sono state utilizzate varie tecniche biofisiche da liposomi di partizionamento per spettroscopia NMR ad alta risoluzione. Inoltre, per stabilire il ruolo dei singoli residui per la topologia della sua associazione con la membrana lipidica, due mutanti di hCT(9-32), cio&egrave;, W30-hCT(9-32) e A23-hCT(9-32), sono stati anche studiati. Anche se non strutturati in soluzione acquosa, hCT(9-32) ha adottato due brevi tratti elicoidale quando associati a micelle dodecylphosphocholine, che si estende da Thr10 a Asn17 e da Gln24 a Val29. A23-hCT(9-32), in cui l&#39;elica-rottura Pro23 fu sostituito da Ala, visualizzato un continuo alfa-elica che si estende da 12 a 26 di residui. Sondaggio con l&#39;etichetta di spin 5-doxylstearate, ha rivelato che associazione con micelle dodecylphosphocholine era tale che l&#39;elica impegnati nell&#39;orientamento parallelo alla superficie della micella. Inoltre, la glicina a Trp exchange in W30-hCT(9-32) ha provocato un ancoraggio pi&ugrave; stabile del segmento C-terminale vicino all&#39;interfaccia, come riflesso di un duplice aumento il coefficiente in liposomi. &Egrave; interessante notare che, pi&ugrave; stretta associazione delle membrane modello era associata con un aumento dell&#39;assorbimento in vitro in cellule di cervice umana epiteliali adenocarcinoma cell line. Studi di perdita di liposomi escluso formazione dei pori, e il pattern di fluorescenza punteggiata di peptide interiorizzato indicato localizzazione vescicolare dei suini e, in conclusione, fortemente suggerito un percorso endocitico di traslocazione.

Eliche di superficie-associata di membrana promuovono interazioni lipidi e assorbimento cellulare dei peptidi penetranti cellula derivata della calcitonina umana.

hCT(9-32) es una humana calcitonina (hCT)-derivado de p&eacute;ptido de penetrar en la c&eacute;lula que se ha demostrado que se translocan a la membrana plasm&aacute;tica de las c&eacute;lulas mam&iacute;feras. Se ha sugerido como un portador celular para drogas, prote&iacute;na verde fluorescente y ADN pl&aacute;smido. Debido a su desplazamiento celular dependiente de la temperatura dando por resultado la distribuci&oacute;n citoplasm&aacute;tica puntuado, su absorci&oacute;n es probable que siga un camino endoc&iacute;ticos. Para profundizar en la orientaci&oacute;n molecular de hCT(9-32) al interactuar con modelos de l&iacute;pidos y para aprender m&aacute;s sobre su modo de acci&oacute;n, se utilizaron diversas t&eacute;cnicas biof&iacute;sicas de liposoma repartir a espectroscop&iacute;a de RMN de alta resoluci&oacute;n. Por otra parte, para establecer el papel de los residuos para la topolog&iacute;a de su asociaci&oacute;n con la membrana lip&iacute;dica, dos mutantes de hCT(9-32), es decir, W30-hCT(9-32) y A23-hCT(9-32), tambi&eacute;n investigaron. Aunque no estructurados en soluci&oacute;n acuosa, hCT(9-32) adopt&oacute; dos tramos helicoidales cortos cuando obligados a micelas de dodecylphosphocholine, que se extiende desde Thr10 a Asn17 y de Gln24 a Val29. A23-HCT(9-32), en el que el Pro23 de h&eacute;lice-&uacute;ltima hora fue reemplazado por Ala, muestran un continuo h&eacute;lice alfa que se extiende desde el residuo 12 a 26. Sondeo con la etiqueta de vuelta 5-doxylstearate revel&oacute; que la asociaci&oacute;n con micelas dodecylphosphocholine era tal que la h&eacute;lice se dedica a la orientaci&oacute;n en paralelo a la superficie de la micela. Por otra parte, Gly Trp intercambio en W30-hCT(9-32) dio lugar a un anclaje m&aacute;s estable del segmento C-terminal cerca de la interfaz, como se refleja por un aumento del doble en el coeficiente de partici&oacute;n en liposomas. Curiosamente, m&aacute;s vinculante a las membranas modelo se asoci&oacute; con un aumento en la captaci&oacute;n in vitro en c&eacute;lulas del cuello uterino humano adenocarcinoma epitelial celular l&iacute;nea. Liposoma fuga estudios excluidos formaci&oacute;n de poros, y el patr&oacute;n de fluorescencia puntuado de p&eacute;ptido internalizado indica localizaci&oacute;n vesicular y, en conclusi&oacute;n, sugiere fuertemente un camino endoc&iacute;ticos de desplazamiento.

H&eacute;lices de superficie asociados de membrana promoci&oacute;n interacciones de l&iacute;pidos y la absorci&oacute;n celular de p&eacute;ptidos penetrante c&eacute;lulas calcitonina humana.

The cell penetrating peptide (CPP) pVEC has been shown to translocate efficiently the plasma membrane of different mammalian cell lines by a receptor-independent mechanism without exhibiting cellular toxicity. This ability renders CPPs of broad interest in cell biology, biotechnology, and drug delivery. To gain insight into the interaction of CPPs with biomembranes, we studied the interaction of pVEC and W2-pVEC, an Ile --> Trp modification of the former, with phase-separated supported phospholipid bilayers (SPB) by atomic force microscopy (AFM). W2-pVEC induced a transformation of dipalmitoyl phosphatidylcholine (DPPC) domains from a gel phase state via an intermediate state with branched structures into essentially flat bilayers. With pVEC the transformation followed a similar pathway but was slower. Employing fluorescence polarization, we revealed the capability of the investigated peptides to increase the fluidity of DPPC domains as the underlying mechanism of transformation. Due to their tighter packing, sphingomyelin (SM) domains were not transformed. By combination, AFM observations, dynamic light scattering studies, and liposome leakage experiments indicated that bilayer integrity was not compromised by the peptides. Transformation of gel phase domains in SPB by CPPs represents a novel aspect in the discussion on uptake mechanisms of CPPs.

The cell penetrating peptides pVEC and W2-pVEC induce transformation of gel phase domains in phospholipid bilayers without affecting their integrity.

La cellule p&eacute;n&eacute;trante peptide (RPC) pVEC a d&eacute;montr&eacute; d&#39;effectuer des transferts efficacement la membrane plasmique des lign&eacute;es cellulaires de mammif&egrave;res diff&eacute;rents par un m&eacute;canisme ind&eacute;pendant du r&eacute;cepteur sans exposer la toxicit&eacute; cellulaire. Cette possibilit&eacute; rend CPPs de large int&eacute;r&ecirc;t en biologie cellulaire, la biotechnologie et la d&eacute;livrance de m&eacute;dicaments. Pour mieux comprendre l&#39;interaction des CPPs avec biomembranes, nous avons &eacute;tudi&eacute; l&#39;interaction de pVEC et W2-pVEC, une Ile--&gt; Trp modification de l&#39;ancienne, avec des bicouches phospholipidiques pris en charge &agrave; phases s&eacute;par&eacute;es (SPB) par microscopie &agrave; force atomique (AFM). W2-pVEC induit une transformation de dipalmitoyl phosphatidylcholine (DPPC) domaines d&#39;un &eacute;tat de phase de gel par un &eacute;tat interm&eacute;diaire avec structures ramifi&eacute;es en essentiellement plats bicouches. Avec pVEC la transformation a suivi un parcours similaire mais a &eacute;t&eacute; plus lente. Employant la polarisation de fluorescence, nous a r&eacute;v&eacute;l&eacute; la capacit&eacute; des peptides &eacute;tudi&eacute;s afin d&#39;accro&icirc;tre la fluidit&eacute; des domaines de la DPPC, comme le m&eacute;canisme sous-jacent de transformation. En raison de leur emballage plus serr&eacute;, sphingomy&eacute;line (SM) domaines ne &eacute;taient pas transform&eacute;s. Par combinaison, AFM observations, &eacute;tudes de diffusion dynamique de la lumi&egrave;re et des exp&eacute;riences de fuite de liposome a indiqu&eacute; que l&#39;int&eacute;grit&eacute; bicouche n&#39;&eacute;tait pas compromise par les peptides. Transformation des domaines phase gel &agrave; SPB de CPPs repr&eacute;sente une nouveaut&eacute; dans le d&eacute;bat sur les m&eacute;canismes d&#39;absorption des CPPs.

La cellule p&eacute;n&eacute;trante peptides pVEC et W2-pVEC induisent la transformation des domaines de phase de gel dans les bicouches phospholipidiques sans affecter leur int&eacute;grit&eacute;.

La cella penetrante pVEC peptide (CPP) ha dimostrato di traslocano nel efficacemente la membrana plasmatica delle linee di cellule di mammifero diverso da un meccanismo recettore-indipendente senza esporre tossicit&agrave; cellulare. Questa abilit&agrave; rende CPPs di ampio interesse in biologia cellulare e biotecnologie drug delivery. Per comprendere l&#39;interazione di CPPs con biomembrane, abbiamo studiato l&#39;interazione di pVEC e W2-pVEC, un Ile--&gt; Trp modifica del precedente, con bilayer fosfolipidico supportati fase separata (SPB) di microscopia a forza atomica (AFM). W2-pVEC indotta da una trasformazione di dipalmitoilfosfatidiletanolamina domini di fosfatidilcolina (DPPC) da uno stato di fase gel attraverso uno stato intermedio con strutture ramificate in essenzialmente piatte fosfolipidici. Con pVEC la trasformazione seguito un percorso simile ma era pi&ugrave; lenta. Impiegando la polarizzazione di fluorescenza, abbiamo evidenziato la capacit&agrave; dei peptidi studiati per aumentare la fluidit&agrave; dei domini DPPC come il meccanismo sottostante di trasformazione. A causa della loro imballaggio pi&ugrave; stretto, domini di sfingomielina (SM) non sono stati trasformati. Di combinazione, AFM osservazioni, studi di diffusione dinamica della luce ed esperimenti di dispersione liposoma indicato che l&#39;integrit&agrave; doppio strato non &egrave; stata compromessa dai peptidi. Trasformazione dei domini fase gel in SPB da CPPs rappresenta un aspetto romanzo nella discussione sui meccanismi di assorbimento di CPPs.

La cella penetrante peptidi pVEC e W2-pVEC indurre trasformazione dei domini fase gel nel bilayer fosfolipidico senza compromettere la loro integrit&agrave;.

La c&eacute;lula penetrante p&eacute;ptido (CPP) pVEC se ha demostrado a translocan eficientemente la membrana plasm&aacute;tica de las c&eacute;lulas mam&iacute;feros diferentes por un mecanismo independiente del receptor sin que exhibe toxicidad celular. Esta capacidad hace CPPs de amplio inter&eacute;s en la biolog&iacute;a celular, biotecnolog&iacute;a y administraci&oacute;n de f&aacute;rmacos. Para profundizar en la interacci&oacute;n de la CPPs con biomembranas, hemos estudiado la interacci&oacute;n de pVEC y W2-pVEC, un Ile--&gt; Trp modificaci&oacute;n del anterior, con bicapas de fosfol&iacute;pidos admitidos fase separada (SPB) por microscop&iacute;a de fuerza at&oacute;mica (AFM). W2-pVEC inducida por una transformaci&oacute;n de dipalmitoyl fosfatidilcolina (DPPC) dominios de un estado de fase gel mediante un estado intermedio con estructuras ramificadas en esencialmente planas bicapas. Con pVEC la transformaci&oacute;n sigui&oacute; un camino similar pero m&aacute;s lenta. El empleo de polarizaci&oacute;n de la fluorescencia, revelamos la capacidad de los p&eacute;ptidos investigados para aumentar la fluidez de los dominios DPPC como el mecanismo subyacente de la transformaci&oacute;n. Debido a su embalaje m&aacute;s apretado, dominios de esfingomielina (SM) no se transformaron. Por combinaci&oacute;n, AFM observaciones, estudios de dispersi&oacute;n de la luz din&aacute;mica y liposoma fuga experimentos indicaron que la integridad de la bicapa no fue comprometida por los p&eacute;ptidos. Transformaci&oacute;n de dominios de fase gel en SPB por CPPs representa un aspecto novedoso en la discusi&oacute;n sobre los mecanismos de captaci&oacute;n de la CPPs.

La c&eacute;lula penetrante p&eacute;ptidos pVEC y W2-pVEC inducen transformaci&oacute;n de dominios de fase gel en bicapas fosfolip&iacute;dicas sin afectar su integridad.

The discovery of cell-penetrating peptides (CPPs), which have the ability to translocate across the plasma membranes of mammalian cells, has led to widespread optimism for delivery of problematic therapeutic cargoes to cells. These cargoes include peptide, protein, and nucleic acid biopharmaceuticals and even nano-sized vectors such as liposomes and nanoparticles. Research on CPPs includes biophysical studies of membrane models to investigate fundamental principles of CPP-lipid membrane interactions as well as cell studies focusing on the efficiency of uptake, mechanisms of translocation, and toxicity. However, both lines of research have suffered from misinterpretation as well as premature extrapolations. In this review, we provide a critical evaluation of the potential and limitations of selected biophysical methodologies such as fluorescence spectroscopy, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, atomic-force microscopy (AFM), and non-spectroscopic methods. We include a discussion of the most important bilayer membrane models in CPP research. We then evaluate important cell biological methodologies, in particular confocal laser scanning microscopy (CLSM) and fluorescence-associated cell sorting (FACS) in combination with various techniques to distinguish between translocated and non-translocated CPPs. Moreover, we discuss the diverse methodologies for tracing the pathways of CPP translocation and their routes of intracellular trafficking.

Reviewing biophysical and cell biological methodologies in cell-penetrating peptide (CPP) research.

La d&eacute;couverte des peptides de p&eacute;n&eacute;tration cellulaire (CPPs), qui ont la capacit&eacute; d&#39;effectuer des transferts &agrave; travers les membranes plasmiques des cellules de mammif&egrave;res, a conduit &agrave; un optimisme r&eacute;pandu pour la livraison des cargaisons th&eacute;rapeutiques probl&eacute;matiques aux cellules. Ces cargaisons incluent des peptides, prot&eacute;ines et acides nucl&eacute;iques produits biopharmaceutiques et vecteurs de m&ecirc;me taille nanom&eacute;trique comme les liposomes et nanoparticules. Recherche sur PPC comprend les &eacute;tudes biophysiques de membrane mod&egrave;les pour &eacute;tudier les principes fondamentaux des interactions de RPC-lipides membranaires ainsi que des &eacute;tudes de cellule en se concentrant sur l&#39;efficacit&eacute; d&#39;absorption, de m&eacute;canismes de translocation et la toxicit&eacute;. Toutefois, les deux axes de recherche ont souffert de la mauvaise interpr&eacute;tation, mais aussi les extrapolations pr&eacute;matur&eacute;es. Dans cette revue, nous fournissons une &eacute;valuation critique du potentiel et les limites de certaines m&eacute;thodologies biophysiques telles que la spectroscopie de fluorescence, dichro&iuml;sme circulaire (CD) et la spectroscopie de r&eacute;sonance magn&eacute;tique nucl&eacute;aire (RMN), microscopie &agrave; force atomique (AFM) et des m&eacute;thodes spectroscopiques-non. Nous incluons une discussion des mod&egrave;les de membrane bicouche plus importants dans la recherche de la RPC. Nous &eacute;valuons ensuite m&eacute;thodologies biologiques cellulaires importantes, microscopie &agrave; balayage laser confocal en particulier (CLSM) et cellule associ&eacute;e &agrave; fluorescence (FACS) de tri en combinaison avec des techniques diff&eacute;rentes pour distinguer la CPPs transloqu&eacute;s et non-transloqu&eacute;e. De plus, nous discutons les diverses m&eacute;thodes pour tracer les voies de la translocation de la RPC et leurs itin&eacute;raires de trafic intracellulaire.

Examen biophysiques et m&eacute;thodologies biologiques cellulaires dans la recherche de peptide (RPC) p&eacute;n&eacute;tration cellulaire.

La scoperta della cellula-penetrante peptidi (CPPs), che hanno la capacit&agrave; di traslocano nel attraverso la membrana plasmatica delle cellule di mammiferi, ha portato alla diffusa ottimismo per la consegna delle merci terapeutiche problematici per le cellule. Questi carichi comprendono anche nano-sized vettori quali liposomi e nanoparticelle e biofarmaceutici peptidi, proteine e acidi nucleici. Ricerca su PPC include studi biofisici di modelli di membrana per indagare i principi fondamentali delle interazioni membrana lipidica CPP nonch&eacute; studi sulle cellule concentrandosi sull&#39;efficienza di assorbimento, meccanismi di traslocazione e tossicit&agrave;. Tuttavia, entrambe le linee di ricerca hanno sofferto di errata interpretazione, nonch&eacute; estrapolazioni premature. In questa recensione, forniamo una valutazione critica delle potenzialit&agrave; e dei limiti delle metodologie biofisiche selezionati come spettroscopia di fluorescenza, Dicroismo circolare (CD) e spettroscopia di risonanza magnetica nucleare (NMR), microscopia a forza atomica (AFM) e metodi spettroscopici-non. Includiamo una discussione dei modelli di membrana doppio strato pi&ugrave; importante nella ricerca CPP. Valutiamo quindi metodologie biologiche cellulari importanti, microscopia a scansione laser confocale in particolare (CLSM) e cella fluorescenza associata cernita (FACS) in combinazione con varie tecniche per distinguere tra traslocato e non traslocata CPPs. Inoltre, discutiamo le diverse metodologie per tracciare i percorsi della traslocazione CPP e le rotte del traffico intracellulare.

Esame biofisico e metodologie biologiche delle cellule nella cellula-penetrante ricerca peptide (CPP).

El descubrimiento de penetraci&oacute;n celular p&eacute;ptidos (CPPs), que tienen la capacidad de translocan a trav&eacute;s de las membranas del plasma de las c&eacute;lulas mam&iacute;feras, gener&oacute; optimismo generalizado para la entrega de cargas terap&eacute;uticas problem&aacute;ticas a las c&eacute;lulas. Estas cargas son p&eacute;ptidos, prote&iacute;nas y &aacute;cidos nucleicos de biof&aacute;rmacos e incluso de tama&ntilde;o nanom&eacute;trico vectores como liposomas y las nanopart&iacute;culas. Investigaci&oacute;n sobre CPPs incluye estudios biof&iacute;sicos de modelos de membrana para investigar los principios fundamentales de las interacciones de membrana de l&iacute;pidos CPP, as&iacute; como estudios de celular, centr&aacute;ndose en la eficiencia de la absorci&oacute;n, mecanismos de desplazamiento y toxicidad. Sin embargo, ambas l&iacute;neas de investigaci&oacute;n han sufrido de interpretaci&oacute;n as&iacute; como extrapolaciones prematuras. En esta revisi&oacute;n, le ofrecemos una evaluaci&oacute;n cr&iacute;tica de las posibilidades y limitaciones de las metodolog&iacute;as biof&iacute;sicas como espectroscopia de fluorescencia, dicro&iacute;smo circular (CD) y espectroscopia de resonancia magn&eacute;tica nuclear (RMN), microscop&iacute;a de fuerza at&oacute;mica (AFM) y m&eacute;todos no espectrosc&oacute;picos. Se incluye una discusi&oacute;n de los modelos m&aacute;s importantes de la membrana de bicapa en investigaci&oacute;n CPP. Luego evaluamos metodolog&iacute;as biol&oacute;gica celular importante, microscop&iacute;a l&aacute;ser confocal en particular (CLSM) y c&eacute;lula asociado de fluorescencia clasificaci&oacute;n (FACS) en combinaci&oacute;n con varias t&eacute;cnicas para distinguir entre la CPPs desplazados y no desplazado. Adem&aacute;s, se discuten las diversas metodolog&iacute;as para trazar las rutas de desplazamiento de CPP y sus rutas de tr&aacute;fico intracelular.

Revisar biof&iacute;sicos y metodolog&iacute;as biol&oacute;gica celular en investigaci&oacute;n de p&eacute;ptido (CPP) de penetrar en la c&eacute;lula.

Michael Herbig

Department of Microstructure Physics and Alloy Design

Max-Planck-Institut für Eisenforschung GmbH

Atom Probe Tomography Studies on the Cu(In,Ga)Se₂ Grain Boundaries

Michael Herbig

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Microstructure Physics and Alloy Design

Max-Planck-Institut für Eisenforschung GmbH

Atom Probe Tomography Studies on the Cu(In,Ga)Se2 Grain Boundaries

Department of Microstructure Physics and Alloy Design

Atom Probe Tomography Studies on the Cu(In,Ga)Se₂ Grain Boundaries