ZEBRA cell‐penetrating peptide as an efficient delivery system in Candida albicans

There is increasing interest in drug delivery systems, such as nanoparticles, liposomes, and cell‐penetrating peptides, for the development of new antimicrobial treatments. In this study, we investigated the transduction capacity of a carrier peptide derived from the Epstein–Barr virus ZEBRA protein in the pathogenic fungus Candida albicans. ZEBRA‐minimal domain (MD) was able to cross the cell wall and cell membrane, delivering eGFP to the cytoplasm. Uptake into up to 70% of the cells was observed within two hours, without toxicity. This new delivery system could be used in C. albicans as a carrier for different biological molecules including peptides, proteins, and nucleic acids. Thereby, in antifungal therapy, MD may carry promising bioactive fungal inhibitors that otherwise penetrate poorly into the cells. Furthermore, MD will be of interest for deciphering molecular pathways involving cell‐cycle control in yeast or signaling pathways. Short interfering peptides could be internalized using MD, providing new tools for the inhibition of metabolic or signaling cascades essential for the growth and virulence of C. albicans, such as yeast‐to‐hyphae transition, cell wall remodeling, stress signaling and antifungal resistance. These findings create new possibilities for the internalization of cargo molecules, with applications for both treatment and functional analyses.     

Interactions of the Anticancer Drug Tamoxifen with Lipid Membranes

Interactions of the hydrophobic anticancer drug tamoxifen (TAM) with lipid model membranes were studied using calcein-encapsulated vesicle leakage, attenuated total reflection Fourier transform infrared (FTIR) spectroscopy, small-angle neutron scattering (SANS), atomic force microscopy (AFM) based force spectroscopy, and all-atom molecular dynamics (MD) simulations. The addition of TAM enhances membrane permeability, inducing calcein to translocate from the interior to the exterior of lipid vesicles. A large decrease in the FTIR absorption band’s magnitude was observed in the hydrocarbon chain region, suggesting suppressed bond vibrational dynamics. Bilayer thickening was determined from SANS data. Force spectroscopy measurements indicate that the lipid bilayer area compressibility modulus K_A is increased by a large amount after the incorporation of TAM. MD simulations show that TAM decreases the lipid area and increases chain order parameters. Moreover, orientational and positional analyses show that TAM exhibits a highly dynamic conformation within the lipid bilayer. Our detailed experimental and computational studies of TAM interacting with model lipid membranes shed new light on membrane modulation by TAM.     

Coarse-grained modeling study of nonpeptide RGD ligand density and PEG molecular weight on the conformation of poly(γ-glutamyl-glutamate) paclitaxel conjugates

Molecular shape, flexibility, and surface hydrophilicity are thought to influence the ability of nanoparticles to cross biological barriers during drug delivery. In this study, coarse-grained (CG) molecular dynamics (MD) simulations were used to study these properties of a polymer-drug construct in potential clinical development: poly(γ-glutamyl-glutamate)-paclitaxel-poly(ethylene glycol) nonpeptide RGD (PGG-PTX-PEG-npRGD), a linear glutamyl-glutamate polymer with paclitaxel and poly(ethylene glycol)-nonpeptide RGD side groups. It was hypothesized that the PEG molecular weight (MW) (500 Da; 1,000 Da; and 2,000 Da) and nonpeptide RGD ligand density (4, 8, 12, and 16 per molecule), respectively, may have advantageous effects on the shape, flexibility, and surface hydrophilicity of PGG-PTX-PEG-npRGD. Circular dichroism spectroscopy was used to suggest initial structures for the all-atom (AA) models of PGG-PTX-PEG-npRGD, which were further converted to CG models using a commercially available mapping algorithm. Due to its semi-flexibility, PGG-PTX-PEG-npRGD is not limited to one specific conformation. Thus, CG MD simulations were run until statistical equilibrium, at which PGG-PTX-PEG-npRGD is represented as an ensemble of statistically similar conformations. The size of a PGG-PTX-PEG-npRGD molecule is not affected by the PEG MW or the nonpeptide RGD density, but higher PEG MW results in increased surface density of a PGG-PTX-PEG-npRGD molecule. Most PGG-PTX-PEG-npRGD shapes are globular, although filamentous shapes were also observed in the PEG500 and PEG1000 molecules. PEG500 and PEG1000 molecules are more flexible than PEG2000 systems. A higher presence of npRGD ligands results in decrease surface hydrophilicity of PGG-PTX-PEG-npRGD. These results indicate that the PGG-PTX-PEG1000-npRGD₄ and PGG-PTX-PEG1000-npRGD₈ molecules are the most efficacious candidates and are further recommended for experimental preclinical studies.     

Surface charge of nanoparticles determines their endocytic and transcytotic pathway in polarized MDCK cells

A major challenge in drug delivery is the internalization through the apical plasma membrane of the polarized epithelial cells lining organs facing the external environment, e.g., lungs and the gastrointestinal tract. The reduced permeation of drugs entering through this pathway is in part due to the mucosal barrier and low rate of endocytosis at these membranes. We investigated the possible role of nanoparticle surface charge on its entry through the apical plasma membrane and its intracellular pathway. We found that both cationic and anionic nanoparticles are targeted mainly to the clathrin endocytic machinery. A fraction of both nanoparticle formulations is suspected to internalize through a macropinocytosis-dependent pathway. A significant amount of nanoparticles transcytose and accumulate at the basolateral membrane. Some anionic but not cationic nanoparticles transited through the degradative lysosomal pathway. Taken together, these observations indicate that cationic nanoparticles, in addition to their potential for drug delivery to epithelia, may be promising carriers for transcytosing drugs to the blood stream.     

Coating of magnetic nanoparticles affects their interactions with model cell membranes

BackgroundThe use of functionalized iron oxide nanoparticles of various chemical properties and architectures offers a new promising direction in theranostic applications. The increasing applications of nanoparticles in medicine require that these engineered nanomaterials will contact human cells without damaging essential tissues. Thus, efficient delivery must be achieved, while minimizing cytotoxicity during passage through cell membranes to reach intracellular target compartments.MethodsDifferential Scanning Calorimetry (DSC), molecular modeling, and atomistic Molecular Dynamics (MD) simulations were performed for two magnetite nanoparticles coated with polyvinyl alcohol (PVA) and polyarabic acid (ARA) in order to assess their interactions with model DPPC membranes.ResultsDSC experiments showed that both nanoparticles interact strongly with DPPC lipid head groups, albeit to a different degree, which was further confirmed and quantified by MD simulations. The two systems were simulated, and dynamical and structural properties were monitored. A bimodal diffusion was observed for both nanoparticles, representing the diffusion in the water phase and in the proximity of the lipid bilayer. Nanoparticles did not enter the bilayer, but caused ordering of the head groups and reduced the area per lipid compared to the pure bilayer, with MAG-PVA interacting more strongly and being closer to the lipid bilayer.ConclusionsResults of DSC experiments and MD simulations were in excellent agreement. Our findings demonstrate that the external coating is a key factor that affects nanoparticle-membrane interactions. Magnetite nanoparticles coated with PVA and ARA did not destabilize the model membrane and can be considered promising platforms for biomedical applications.General significanceUnderstanding the physico-chemical interactions of different nanoparticle coatings in contact with model cell membranes is the first step for assessing toxic response and could lead to predictive models for estimating toxicity. DSC in combination with MD simulations is an effective strategy to assess physico-chemical interactions of coated nanoparticles with lipid bilayers.     

The Selective Interaction between Silica Nanoparticles and Enzymes from Molecular Dynamics Simulations

Nanoscale particles have become promising materials in many fields, such as cancer therapeutics, diagnosis, imaging, drug delivery, catalysis, as well as biosensors. In order to stimulate and facilitate these applications, there is an urgent need for the understanding of the interaction mode between the nano-particles and proteins. In this study, we investigate the orientation and adsorption between several enzymes (cytochrome c, RNase A, lysozyme) and 4 nm/11 nm silica nanoparticles (SNPs) by using molecular dynamics (MD) simulation. Our results show that three enzymes are adsorbed onto the surfaces of both 4 nm and 11 nm SNPs during our MD simulations and the small SNPs induce greater structural stabilization. The active site of cytochrome c is far away from the surface of 4 nm SNPs, while it is adsorbed onto the surface of 11 nm SNPs. We also explore the influences of different groups (-OH, -COOH, -NH₂ and CH₃) coated onto silica nanoparticles, which show significantly different impacts. Our molecular dynamics results indicate the selective interaction between silicon nanoparticles and enzymes, which is consistent with experimental results. Our study provides useful guides for designing/modifying nanomaterials to interact with proteins for their bio-applications.     

Molecular dynamics of nanoparticle translocation at lipid interfaces

We report molecular dynamics simulations of bare and hydrophilic C60 nanoparticles at a dipalmitoylphosphatidylcholine–water interface representing a model lung surfactant layer. Bare C60 particles penetrate into the lipid layer from the vapour to sit just before the lipid head groups while hydrophilic nanoparticles penetrate into the head-group–water interface. The potential of mean force shows how the preferred position varies with the density of the lipid layer (in the physiological range) and with hydrophilicity. We conclude that C60 nanoparticles will not spontaneously diffuse across a surfactant monolayer but that functionalised nanoparticles may, depending on the membrane density, translocate the membrane to reach the water phase in 10–20 ns.     

Translocation or membrane disintegration? Implication of peptide-membrane interactions in pep-1 activity.

The Cell membrane is impermeable for most peptides, proteins, and oligonucleotides. Moreover, some cationic peptides, the so-called cell-penetrating peptides (CPPs), are able to translocate across the membrane. This observation has attracted much attention because these peptides can be covalently coupled to different macromolecules, which are efficiently delivered inside the cell. The mechanism used by these peptides to pass across the membrane is a controversial matter of debate. It has been suggested that endocytosis is the main mechanism of internalization and this was confirmed by several studies for different peptides. Pep-1 is an exception worthy of attention for its ability to translocate cargo macromolecules without the need to be covalently attached to them. A preferential internalization by an endocytosis-independent mechanism was demonstrated both in vitro and in vivo. Pep-1 has a high affinity to lipidic membranes, it is able to insert and induce local destabilization in the lipidic bilayer, although without pore formation. No cytotoxic effects were found for pep-1 concentrations where translocation is fully operative. At much higher concentrations, membrane disintegration takes place by a detergent-like mechanism that resembles anti-microbial peptide activity. In this review, the ability of pep-1 to transverse the membrane by an endocytosis-independent mechanism, not mediated by pores as well as an ability to induce membrane disintegration at high peptide concentration, is demonstrated.     

Enhancement of Thermal Properties of Polyvinylpyrrolidone (PVP)-Coated Silver Nanoparticles by Using Plasmid DNA and their Localized Surface Plasmon Resonance (LSPR) Characteristics

In this paper, the enhancement of thermal properties of polymer-coated silver nanoparticles by the addition of plasmid DNA is described. Nanoparticles of noble metals such as gold and silver possess specific characteristics by virtue of their quantum size effects. Therefore, noble metal nanoparticles are used for chemical sensing and biosensing applications based on their localized surface plasmon resonance absorption that can be measured in the visible region. The polyvinylpyrrolidone (PVP)-coated noble metal nanoparticles, in particular, with high dispersion ability in water, offer several advantages for sensing applications. However, some difficulties are encountered in the use of these PVP-coated noble metal nanoparticles for sensing applications due to their poor thermal properties. To improve the thermal properties of PVP-coated noble metal nanoparticles, we found that the addition of plasmid DNA to PVP-coated silver nanoparticles enhances their thermal properties due to good thermal stability of DNA. The introduction of plasmid DNA into PVP-coated silver nanoparticle dispersion enhanced the thermal properties through the formation of a complex between the nanoparticles and plasmid DNA. Furthermore, other polymers such as proteins and polyethylene glycol did not enhance the thermal properties of PVP-coated silver nanoparticles. Thus, the PVP-coated silver nanoparticle–plasmid DNA complex with enhanced thermal properties has a great potential for use in medical and drug delivery applications.     

Insights into the molecular basis of action of the AT1 antagonist losartan using a combined NMR spectroscopy and computational approach

The drug:membrane interactions for the antihypertensive AT1 antagonist losartan, the prototype of the sartans class, are studied herein using an integrated approach. The pharmacophore arrangement of the drug was revealed by rotating frame nuclear Overhauser effect spectroscopy (2D ROESY) NMR spectroscopy in three different environments, namely water, dimethyl sulfoxide (DMSO), and sodium dodecyl sulfate (SDS) micellar solutions mimicking conditions of biological transport fluids and membrane lipid bilayers. Drug association with micelles was monitored by diffusion ordered spectroscopy (2D DOSY) and drug:micelle intermolecular interactions were characterized by ROESY spectroscopy. The localisation of the drug in the micellar environment was investigated by introducing 5-doxyl and 16-doxyl stearic acids. The use of spin labels confirmed that losartan resides close to the micelle:water interface with the hydroxymethyl group and the tetrazole heterocyclic aromatic ring facing the polar surface with the potential to interact with SDS charged polar head groups in order to increase amphiphilic interactions. The spontaneous insertion, the diffusion pathway and the conformational features of losartan were monitored by Molecular Dynamics (MD) simulations in a modeled SDS micellar aggregate environment and a long exploratory MD run (580 ns) in a phospholipid dipalmitoylphosphatidylcholine (DPPC) bilayer with the AT₁ receptor embedded. MD simulations were in excellent agreement with experimental results and further revealed the molecular basis of losartan:membrane interactions in atomic-level detail. This applied integrated approach aims to explore the role of membranes in losartan's pathway towards the AT₁ receptor.     

Nonenzymatic rapid control of GIRK channel function by a G protein-coupled receptor kinase

G protein-coupled receptors (GPCRs) respond to agonists to activate downstream enzymatic pathways or to gate ion channel function. Turning off GPCR signaling is known to involve phosphorylation of the GPCR by GPCR kinases (GRKs) to initiate their internalization. The process, however, is relatively slow and cannot account for the faster desensitization responses required to regulate channel gating. Here, we show that GRKs enable rapid desensitization of the G protein-coupled potassium channel (GIRK/Kir3.x) through a mechanism independent of their kinase activity. On GPCR activation, GRKs translocate to the membrane and quench channel activation by competitively binding and titrating G protein βγ subunits away from the channel. Of interest, the ability of GRKs to effect this rapid desensitization depends on the receptor type. The findings thus reveal a stimulus-specific, phosphorylation-independent mechanism for rapidly downregulating GPCR activity at the effector level.     

pVEC hydrophobic N‐terminus is critical for antibacterial activity

Cell‐penetrating peptides (CPPs) are commonly defined by their shared ability to be internalized into eukaryotic cells, without inducing permanent membrane damage, and to improve cargo delivery. Many CPPs also possess antimicrobial action strong enough to selectively lyse microbes in infected mammalian cultures. pVEC, a CPP derived from cadherin, is able to translocate into mammalian cells, and it is also antimicrobial. Structure‐activity relationship and sequence alignment studies have suggested that the hydrophobic N‐terminus (LLIIL) of pVEC is essential for this peptide's uptake into eukaryotic cells. In this study, our aim was to examine the contribution of these residues to the antimicrobial action and the translocation mechanism of pVEC. We performed antimicrobial activity and microscopy experiments with pVEC and with del5 pVEC (N‐terminal truncated variant of pVEC) and showed that pVEC loses its antimicrobial effect upon deletion of the LLIIL residues, even though both peptides induce membrane permeability. We also calculated the free energy of the transport process using steered molecular dynamic simulations and replica exchange umbrella sampling simulations to compare the difference in uptake mechanism of the 2 peptides in atomistic detail. Despite the difference in experimentally observed antimicrobial activity, the simulations on the 2 peptides showed similar characteristics and the energetic cost of translocation of pVEC was higher than that of del5 pVEC, suggesting that pVEC uptake mechanism cannot be explained by simple passive transport. Our results suggest that LLIIL residues are key contributors to pVEC antibacterial activity because of irreversible membrane disruption.     

Effect of the surface charge density of nanoparticles on their translocation across pulmonary surfactant monolayer: a molecular dynamics simulation

Interaction between nanoparticles (NPs) and pulmonary surfactant monolayer plays a very significant role in nanoparticle-based pulmonary drug delivery system. Previous researches have indicated that different properties of nanoparticles can affect their translocation across pulmonary surfactant monolayer. Here we performed coarse-grained molecular dynamics simulation aimed at nanoparticles’ surface charge density effect on their penetration behaviours. Several hydrophilic nanoparticles with different surface charge densities were modelled in the simulations. The results show that NPs’ surface charge density affects their translocation capability: the higher the surface charge densities of NPs are, the worse their translocation capability is. It will cause the structural changes of pulmonary surfactant monolayer, and inhibit the normal phase transition of the monolayer during the compression process. Besides, charged NPs can be adsorbed on the surface of the monolayer after translocation as a stable state, and the adsorption capability of NPs increases generally with the increase of surface charge densities. Our simulation results suggest that the study of nanoparticle-based pulmonary drug delivery system should consider the nanoparticles’ surface charge density effect in order to avoid biological toxicity and improve efficacy.     

Cryo-electron tomography of nanoparticle transmigration into liposome

Nanoparticle transport across cell membrane plays a crucial role in the development of drug delivery systems as well as in the toxicity response induced by nanoparticles. As hydrophilic nanoparticles interact with lipid membranes and are able to induce membrane perturbations, hypothetic mechanisms based on membrane curvature or hole formation have been proposed for activating their transmigration. We report on the transport of hydrophilic silica nanoparticles into large unilamellar neutral DOPC liposomes via an internalization process. The strong adhesive interactions of lipid membrane onto the silica nanoparticle triggered liposome deformation until the formation of a curved neck. Then the rupture of this membrane neck led to the complete engulfment of the nanoparticle. Using cryo-electron tomography we determined 3D architectures of intermediate steps of this process unveiling internalized silica nanoparticles surrounded by a supported lipid bilayer. This engulfing process was achieved for a large range of particle size (from 30 to 200 nm in diameter). These original data provide interesting highlights for nanoparticle transmigration and could be applied to biotechnology development.     

Lipid domain separation,bilayer thickening and pearling induced by the cell penetrating peptide penetratin

Protein membrane transduction domains are able to translocate through cell membranes. This capacity resulted in new concepts on cell communication and in the design of vectors for internalization of active molecules into cells. Penetratin crosses the plasma membrane by a receptor and metabolic energy-independent mechanism which is at present unknown. A better knowledge of its interaction with phospholipids will help to understand the molecular mechanisms of cell penetration. Here, we investigated the role of lipid composition on penetratin induced membrane perturbations by X-ray diffraction, microscopy and ³¹P-NMR. Penetratin showed the ability to induce phospholipid domain separation, membrane bilayer thickening, formation of vesicles, membrane undulations and tubular pearling. These data demonstrate its capacity to increase membrane curvature and suggest that dynamic phospholipid–penetratin complexes can be organized in different structural arrangements. These properties and their implications in peptide membrane translocation capacity are discussed.     

Molecular dynamics simulation of the size-dependent morphological stability of cubic shape silver nanoparticles

The morphological stability of sharp-edged silver nanoparticles is examined by the classical molecular dynamics (MD) simulations. The crystalline structure and the perfect fcc atom packing of a series of silver nanocubes (AgNC) of different sizes varying from 63 up to 1099 atoms are compared against quasi-spherical nanoparticles of the same sizes at temperature 303 K. Our MD simulations demonstrate that starting from the preformed perfect crystalline structures the cubic shape is preserved for AgNCs composed of 365–1099 atoms. Surprisingly, the rapid loss of the cubic shape morphology and transformation into the non-fcc-structure are found for smaller AgNCs composed of less than ~256 atoms. No such loss of the preformed crystalline structure is seen for quasi-spherical nanoparticles composed of 38–1007 atoms. The analysis of the temperature dependence and the binding energy of outermost Ag surface atoms suggests that the loss of the perfect cubic shape, rounding and smoothing of sharp edges and corners are driven by the tendency towards the increase in their coordination number. In addition, we revealed that AgNC₁₀₉₉ partially loses its sharp edges and corners in the aqueous environment; however, the polymer coating with poly(vinyl alcohol) (PVA) was able to preserve the well-defined cubic morphology. Finally, these results help improve the understanding of the role of surface capping agents in solution phase synthesis of Ag nanocubes.     

Regulation of adenovirus membrane penetration by the cytoplasmic tail of integrin beta5

Adenovirus (Ad) cell entry involves sequential interactions with host cell receptors that mediate attachment (CAR), internalization (alphavbeta3 and alphavbeta5), and penetration (alphavbeta5) of the endosomal membrane. These events allow the virus to deliver its genome to the nucleus. While integrins alphavbeta3 and alphavbeta5 both promote Ad internalization into cells, integrin alphavbeta5 selectively facilitates Ad-mediated membrane permeabilization and endosome rupture. In the experiments reported herein, we demonstrate that the intracellular domain of the integrin beta5 subunit specifically regulates Ad-mediated membrane permeabilization and gene delivery. CS-1 melanoma cells expressing a truncated integrin beta5 or a chimeric (beta5-beta3) cytoplasmic tail (CT) supported normal levels of Ad endocytosis but had reduced Ad-mediated gene delivery and membrane permeabilization relative to cells expressing a wild-type integrin beta5. Thin-section electron microscopy revealed that virion particles were capable of being endocytosed into cells expressing a truncated beta5CT, but they failed to escape cytoplasmic vesicles and translocate to the nucleus. Site-specific mutagenesis studies suggest that a C-terminal TVD motif in the beta5CT plays a major role in Ad membrane penetration.     

Homology modeling of dopamine d(2) and d(3) receptors: molecular dynamics refinement and docking evaluation

Dopamine (DA) receptors, a class of G-protein coupled receptors (GPCRs), have been targeted for drug development for the treatment of neurological, psychiatric and ocular disorders. The lack of structural information about GPCRs and their ligand complexes has prompted the development of homology models of these proteins aimed at structure-based drug design. Crystal structure of human dopamine D(3) (hD(3)) receptor has been recently solved. Based on the hD(3) receptor crystal structure we generated dopamine D(2) and D(3) receptor models and refined them with molecular dynamics (MD) protocol. Refined structures, obtained from the MD simulations in membrane environment, were subsequently used in molecular docking studies in order to investigate potential sites of interaction. The structure of hD(3) and hD(2L) receptors was differentiated by means of MD simulations and D(3) selective ligands were discriminated, in terms of binding energy, by docking calculation. Robust correlation of computed and experimental K(i) was obtained for hD(3) and hD(2L) receptor ligands. In conclusion, the present computational approach seems suitable to build and refine structure models of homologous dopamine receptors that may be of value for structure-based drug discovery of selective dopaminergic ligands.     

Sulfonates-PMMA nanoparticles conjugates: A versatile system for multimodal application

We report herein the viability of a novel nanoparticles (NPs) conjugated system, namely the attachment, based on ionic and hydrophobic interactions, of different sulfonated organic salts to positively charged poly(methylmethacrylate) (PMMA)-based core-shell nanoparticles (EA0) having an high density of ammonium groups on their shells. In this context three different applications of the sulfonates@EA0 systems have been described. In detail, their ability as cytotoxic drugs and pro-drugs carriers was evaluated in vitro on NCI-H460 cell line and in vivo against human ovarian carcinoma IGROV-1 cells. Besides, 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS) was chosen for NPs loading, and its internalization as bioimaging probe was evaluated on Hep G2 cells. Overall, the available data support the interest for these PMMA NPs@sulfonates systems as a promising formulation for theranostic applications. In vivo biological data strongly support the potential value of these core-shell NPs as delivery system for negatively charged drugs or biologically active molecules. Additionally, we have demonstrated the ability of these PMMA core-shell nanoparticles to act as efficient carriers of fluorophores. In principle, thanks to the high PMMA NPs external charge density, sequential and very easy post-loading of different sulfonates is achievable, thus allowing the preparation of nanocarriers either with bi-modal drug delivery behaviour or as theranostic systems.     

Dynamics of Receptor-Mediated Nanoparticle Internalization into Endothelial Cells

Nanoparticles offer a promising medical tool for targeted drug delivery, for example to treat inflamed endothelial cells during the development of atherosclerosis. To inform the design of such therapeutic strategies, we develop a computational model of nanoparticle internalization into endothelial cells, where internalization is driven by receptor-ligand binding and limited by the deformation of the cell membrane and cytoplasm. We specifically consider the case of nanoparticles targeted against ICAM-1 receptors, of relevance for treating atherosclerosis. The model computes the kinetics of the internalization process, the dynamics of binding, and the distribution of stresses exerted between the nanoparticle and the cell membrane. The model predicts the existence of an optimal nanoparticle size for fastest internalization, consistent with experimental observations, as well as the role of bond characteristics, local cell mechanical properties, and external forces in the nanoparticle internalization process.