Ayman El-Sayed

Delivery of macromolecules using arginine-rich cell-penetrating peptides: ways to overcome endosomal entrapment.

Arginine-rich cell-penetrating peptides (AR-CPPs) are very promising tools for the delivery of therapeutic macromolecules such as peptides, proteins, and nucleic acids. These peptides allow efficient internalization of the linked cargos intracellularly through the endocytic pathway. However, when linked to bulky cargos, entrapment in the endocytic vesicles is a major limitation to the application of these peptides in cytosolic delivery. Attachment of a compatible endosomal escape device is, therefore, necessary to allow cytosolic delivery of the peptide-attached cargo. This review presents different endosomal escape devices currently in application in combination with AR-CPPs. Applications of fusogenic lipids, membrane-disruptive peptides, membrane-disruptive polymers, lysosomotropic agents, and photochemical internalization to enhance the cytosolic delivery of AR-CPPs-attached cargos are presented. The properties of each system and its mechanism of action for the enhancement of endosomal escape are discussed, together with its applications for the delivery of different macromolecules in vitro and, if applicable, in vivo.

Octaarginine- and octalysine-modified nanoparticles have different modes of endosomal escape.

The present study examines the role of surface modification with an octaarginine peptide (R8) in liposomal escape from endocytic vesicles, using octalysine (K8) as a control cationic peptide; the mechanism of endosomal escape of liposomes was also investigated. Gene expression of condensed plasmid DNA encapsulated in R8-modified nanoparticles was more than 1 order of magnitude higher than that of K8-modified nanoparticles, and 2 orders of magnitude higher than gene expression using unmodified nanoparticles. The difference in gene expression could not be attributed to differences in uptake, as R8- and K8-modified liposomes were taken up primarily via macropinocytosis with comparable efficiency. The extent of R8-nanoparticle escape to the cytosol was double that of K8-nanoparticles. Suppression of endosome acidification inhibited R8-nanoparticle endosomal escape, but enhanced that of K8-nanoparticles. Using spectral imaging in live cells, we showed that R8- and K8-liposomes escaped from endocytic vesicles via fusion between the liposomes and the endosomal membrane. R8-liposomes fused efficiently at both acidic and neutral pH, whereas K8-liposomes fused only at neutral pH. Similar behavior was observed during in vitro lipid mixing and calcein-release experiments. Co-incubation of cells with distinctly labeled K8- and R8-modified nanoparticles confirmed a common uptake pathway and different rates of endosomal escape particularly at longer time intervals. Therefore, it was concluded that R8 on the liposome surface stimulates efficient escape from endocytic vesicles via a fusion mechanism that works at both neutral and acidic pH; in contrast, K8 mediates escape mainly at neutral pH.

Endocytosis of Gene Delivery Vectors: From Clathrin-dependent to Lipid Raft-mediated Endocytosis

The ideal nonviral vector delivers its nucleic acid cargo to a specific intracellular target. Vectors enter cells mainly through endocytosis and are distributed to various intracellular organelles. Recent advances in microscopy, lipidomics, and proteomics confirm that the cell membrane is composed of clusters of lipids, organized in the form of lipid raft domains, together with non-raft domains that comprise a generally disordered lipid milieu. The binding of a nonviral vector to either region can determine the pathway for its endocytic uptake and subsequent intracellular itinerary. Given this model of the cell membrane structure, endocytic pathways should be reclassified in relation to lipid rafts. In this review, we attempt to assess the currently recognized endocytic pathways in mammalian cells. The endocytic pathways are classified in relation to the membrane regions that make up the primary endocytic vesicles. This review covers the well-recognized clathrin-mediated endocytosis (CME), phagocytosis, and macropinocytosis in addition to the less addressed pathways that take place in lipid rafts. These include caveolae-mediated, flotillin-dependent, GTPase regulator associated with focal adhesion kinase-1 (GRAF1)-dependent, adenosine diphosphate-ribosylation factor 6 (Arf6)-dependent, and RhoA-dependent endocytic pathways. We summarize the regulators associated with each uptake pathway and methods for interfering with these regulators are discussed. The fate of endocytic vesicles resulting from each endocytic uptake pathway is highlighted.

Enhanced gene expression by a novel stearylated INF7 peptide derivative through fusion independent endosomal escape

An octaarginine-modified multifunctional envelope-type nano device (R8-MEND) was previously reported to be an efficient nonviral vector for the delivery of plasmid DNA, in vitro and after topical administration. We report herein on a novel stearylated derivative of the INF7 peptide, a derivative of the N-terminal domain of the HA2 protein of the influenza virus envelope, which enhances the endosomal escape of R8-MEND through a mechanism independent of fusion between the MEND coat and the endosomal membrane. The use of the novel peptide derivative would permit the gene expression of the R8-MEND to be improved, both in vitro and in vivo. R8-MEND modified with stearylated INF7 resulted in gene expression levels that were 77-fold higher than unmodified and 20-fold higher than the free INF7 peptide-modified R8-MEND with no cellular toxicity. Spectral imaging in live cells confirmed that the stearylated INF7 modification did not mediate fusion between liposomes and the endosomal membrane. The inclusion of DOPE to the R8-MEND coat was synergistic with the peptide in improving gene transfection. The intravenous injection of an R8-MEND modified with stearylated INF7 to ICR mice resulted in luciferase expression levels 240-fold higher in liver and 115-fold higher in spleen than that of the R8-MEND.

Lipid envelope-type nanoparticle incorporating a multifunctional peptide for systemic siRNA delivery to the pulmonary endothelium

A system that permits the delivery of cargoes to the lung endothelium would be extraordinarily useful in terms of curing a wide variety of lung-related diseases. This study describes the development of a multifunctional envelope-type nanodevice (MEND) that targets the lung endothelium, delivers its encapsulated siRNA to the cytoplasm, and eradicates lung metastasis. The key to the success can be attributed to the presence of a surface-modified GALA peptide that has dual functions: targeting the sialic acid-terminated sugar chains on the pulmonary endothelium and subsequently delivering the encapsulated cargoes to the cytosol via endosomal membrane fusion, analogous to the influenza virus. The active targeting of MENDs without the formation of large aggregates was verified by intravital real-time confocal laser scanning microscopy in living lung tissue. The GALA-modified MEND is a promising carrier that opens a new generation of therapeutic approaches for satisfying unmet medical needs in curing lung diseases.

Stearylated INF7 Peptide Enhances Endosomal Escape and Gene Expression of PEGylated Nanoparticles both In Vitro and In Vivo

We previously reported on a stearylated INF7 peptide (str-INF7), which enhances the endosomal escape of an octaarginine (R8)-modified liposomal particle encapsulating plasmid DNA (pDNA) in a fusion-independent manner. This study examined whether this peptide derivative enhanced the endosomal escape and gene expression of PEGylated liposomes encapsulating pDNA. We used a PEGylated, R8-modified multifunctional envelope-type nanodevice (R8-MEND) as a model for PEGylated liposomes. Polyethylene glycol 2000 (PEG2000) attached to two different anchors, distearoylphosphatidylethanolamine (DSPE-PEG) or dimyristoylphosphatidylethanolamine (DMPE-PEG), was used to modify the R8-MEND in the presence or absence of two different concentrations of str-INF7. Modification of the PEGylated R8-MEND with str-INF7 resulted in luciferase gene expression levels in HeLa cells that were 73-fold and 24-fold higher than the corresponding value for an unmodified MEND in the case of DSPE-PEG and DMPE-PEG, respectively. The endosomal escape of the PEGylated R8-MEND was improved by str-INF7, as confirmed by confocal laser scanning microscopy. Furthermore, modification with str-INF7 enhanced the hepatic gene expression of the R8-MEND modified with DSPE-PEG and DMPE-PEG by 95-fold and 1885-fold, respectively, after intravenous injection in mice. Collectively, these data demonstrate that str-INF7 can be a useful device for enhancing the endosomal escape even for PEGylated liposomes encapsulating pDNA.

Transcytosis‐Targeting Peptide: A Conductor of Liposomal Nanoparticles through the Endothelial Cell Barrier

The ultimate goal in the area of drug-delivery systems is the development of a nanoparticle that can penetrate the endothelial cell monolayer for the targeting of tissue parenchyma. In the present study, we identify a transcytosis-targeting peptide (TTP) that permits polyethyleneglycol (PEG)-modified liposomes (PEG-LPs) to penetrate through monolayers of brain-derived endothelial cells. These endothelial cells were layered on a gelatin nanofiber sheet, a nanofiber meshwork that allows the evaluation of transcellular transport of nanosized particles (ca. 100 nm). Systematic modification of the sequences results in the identification of the consensus sequence of TTP as L(R/K)QZZZL, where Z denotes hydrophilic amino acids (R/K/S and partially D). The TTP-modified liposomes are bound on the heparin sulfate proteoglycan, and are then taken up via lipid raft-mediated endocytosis. Subsequent intracellular imaging of the particles reveals a unique intracellular sorting of TTP-modified PEG liposomes (TTP-PEG-LPs); namely the TTP-LPs are not localized with the lysosomes, whereas this co-localization is dominant in the unmodified PEG liposomes (PEG-LPs). The in vivo endothelial penetration of liposomes in adipose tissue is conferred by the dual modification of the particles with TTP and tissue-targeting ligands. This technology promises innovations in intravenously available delivery system to tissue parenchyma.

A new genus and a new species of Schizomyiina (Diptera: Cecidomyiidae: Asphondyliini) inducing petiole galls on Macaranga bancana (Miq.) in Borneo, Malaysia

We describe a gall midge Macarangamyia itiokai Elsayed Tokuda gen. n., sp. n. belonging to the subtribe Schizomyiina (Diptera: Cecidomyiidae: Asphondyliini) inducing petiole galls on Macaranga bancana (Miq.) in Lambir Hills National Park, Borneo, Malaysia. The new genus is distinguishable from all known genera of Schizomyiina by the unique dorsally-placed aedeagus slit, the short, membranous, protrusible ovipositor, with scattered strong setae ventrally and dorsally, and the presence of spiracles on all larval thoracic segments. It is compared and separated from its closely related Oriental genera of Schizomyiina.

A new Asphondylia species (Diptera: Cecidomyiidae) and a eulophid wasp (Hymenoptera) inducing similar galls on leaf buds of Schoepfia jasminodora (Schoepfiaceae), with reference to their ecological traits and a description of the new gall midge

Different gall inducers belonging to distinct insect orders are rarely known to induce similarly shaped galls on the same host plant organs. We report that Asphondylia tojoi Elsayed & Tokuda sp. nov. (Diptera: Cecidomyiidae) and Ceratoneura sp. (Hymenoptera: Eulophidae) induce galls on leaf buds of Schoepfia jasminodora Sieb. et Zucc. (Schoepfiaceae). We describe the gall midge species as new to science and report a phylogenetic analysis for known Japanese Asphondylia species. We also describe life histories of the two species, based on monthly surveys during 2015–2017: although both species are multivoltine, A. tojoi overwinters as first instars in galls, whereas Ceratoneura sp. possibly does so as adults outside the galls. In addition, the internal structure of galls differed between the two species. Galls containing A. tojoi consist of a single chamber with inner walls clearly covered with whitish fungal mycelia after the gall midges develop into second instars. Those containing the Ceratoneura sp. have multiple chambers with hard black inner walls. Although some eulophids are known to be inquilines of galls induced by Asphondylia species, we consider that the Ceratoneura sp. is probably a true gall inducer because of the different gall structure and absence of fungal mycelia in their galls. This is the first report detailing the annual life history of a Ceratoneura species. Asphondylia tojoi represents the first example of monophagous Asphondylia species with a multivoltine life history on a deciduous tree.

A new Gephyraulus species (Diptera: Cecidomyiidae) inducing flower bud galls on the European sea rocket Cakile maritima Scop. (Brassicaceae)

The European sea rocket Cakile maritima Scop. (Brassicaceae) is a common herb growing on sandy coastlines worldwide and is considered a useful plant because of its medicinal importance, its edibility, and potential as an oilseed crop. However, C. maritima is an invasive plant over a wide range, e.g., eastern South America, North America, northern Iran, Australia and New Zealand, and has a limited number of associated herbivorous insects. During investigations on gall midges (Diptera: Cecidomyiidae) in Egypt, we found a gall midge inducing flower bud galls on C. maritima and preventing fruit production, which suggested that this gall midge is a potential pest of this plant. In this paper, we describe this gall midge species, Gephyraulus zewaili Elsayed and Tokuda sp. nov., as new to science by comparing its morphology with that of close congeners. Partial sequence data of the mitochondrial DNA cytochrome oxidase subunit I gene are also provided.