iRGD peptide conjugation potentiates intraperitoneal tumor delivery of paclitaxel with polymersomes

Polymersomes are versatile nanoscale vesicles that can be used for cytoplasmic delivery of payloads. Recently, we demonstrated that pH-sensitive polymersomes exhibit an intrinsic selectivity towards intraperitoneal tumor lesions. A tumor homing peptide, iRGD, harbors a cryptic C-end Rule (CendR) motif that is responsible for neuropilin-1 (NRP-1) binding and for triggering extravasation and tumor penetration of the peptide. iRGD functionalization increases tumor selectivity and therapeutic efficacy of systemic drug-loaded nanoparticles in many tumor models. Here we studied whether intraperitoneally administered paclitaxel-loaded iRGD-polymersomes show improved efficacy in the treatment of peritoneal carcinomatosis. First, we demonstrated that the pH-sensitive polymersomes functionalized with RPARPAR (a prototypic CendR peptide) or iRGD internalize in the cells that express NRP-1, and that internalized polymersomes release their cargo inside the cytosol. CendR-targeted polymersomes loaded with paclitaxel were more cytotoxic on NRP-1-positive cells than on NRP-1-negative cells. In mice bearing peritoneal tumors of gastric (MKN-45P) or colon (CT26) origin, intraperitoneally administered RPARPAR and iRGD-polymersomes showed higher tumor-selective accumulation and penetration than untargeted polymersomes. Finally, iRGD-polymersomes loaded with paclitaxel showed improved efficacy in peritoneal tumor growth inhibition and in suppression of local dissemination compared to the pristine paclitaxel-polymersomes or Abraxane. Our study demonstrates that iRGD-functionalization improves efficacy of paclitaxel-polymersomes for intraperitoneal treatment of peritoneal carcinomatosis

Combinatorial targeting and discovery of ligand-receptors in organelles of mammalian cells

Phage display screening allows the study of functional protein–protein interactions at the cell surface, but investigating intracellular organelles remains a challenge. Here we introduce internalizing-phage libraries to identify clones that enter mammalian cells through a receptor-independent mechanism and target-specific organelles as a tool to select ligand peptides and identify their intracellular receptors. We demonstrate that penetratin, an antennapedia-derived peptide, can be displayed on the phage envelope and mediate receptor-independent uptake of internalizing phage into cells. We also show that an internalizing-phage construct displaying an established mitochondria-specific localization signal targets mitochondria, and that an internalizing-phage random peptide library selects for peptide motifs that localize to different intracellular compartments. As a proof-of-concept, we demonstrate that one such peptide, if chemically fused to penetratin, is internalized receptor-independently, localizes to mitochondria, and promotes cell death. This combinatorial platform technology has potential applications in cell biology and drug development

VEGF165-induced vascular permeability requires NRP1 for ABL-mediated SRC family kinase activation.

The vascular endothelial growth factor (VEGF) isoform VEGF165 stimulates vascular growth and hyperpermeability. Whereas blood vessel growth is essential to sustain organ health, chronic hyperpermeability causes damaging tissue edema. By combining in vivo and tissue culture models, we show here that VEGF165-induced vascular leakage requires both VEGFR2 and NRP1, including the VEGF164-binding site of NRP1 and the NRP1 cytoplasmic domain (NCD), but not the known NCD interactor GIPC1. In the VEGF165-bound receptor complex, the NCD promotes ABL kinase activation, which in turn is required to activate VEGFR2-recruited SRC family kinases (SFKs). These results elucidate the receptor complex and signaling hierarchy of downstream kinases that transduce the permeability response to VEGF165. In a mouse model with choroidal neovascularisation akin to age-related macular degeneration, NCD loss attenuated vessel leakage without affecting neovascularisation. These findings raise the possibility that targeting NRP1 or its NCD interactors may be a useful therapeutic strategy in neovascular disease to reduce VEGF165-induced edema without compromising vessel growth

Interplay between transglutaminases and heparan sulphate in progressive renal scarring

Transglutaminase-2 (TG2) is a new anti-fibrotic target for chronic kidney disease, for its role in altering the extracellular homeostatic balance leading to excessive build-up of matrix in kidney. However, there is no confirmation that TG2 is the only transglutaminase involved, neither there are strategies to control its action specifically over that of the conserved family-members. In this study, we have profiled transglutaminase isozymes in the rat subtotal nephrectomy (SNx) model of progressive renal scarring. All transglutaminases increased post-SNx peaking at loss of renal function but TG2 was the predominant enzyme. Upon SNx, extracellular TG2 deposited in the tubulointerstitium and peri-glomerulus via binding to heparan sulphate (HS) chains of proteoglycans and co-associated with syndecan-4. Extracellular TG2 was sufficient to activate transforming growth factor-β1 in tubular epithelial cells, and this process occurred in a HS-dependent way, in keeping with TG2-affinity for HS. Analysis of heparin binding of the main transglutaminases revealed that although the interaction between TG1 and HS is strong, the conformational heparin binding site of TG2 is not conserved, suggesting that TG2 has a unique interaction with HS within the family. Our data provides a rationale for a novel anti-fibrotic strategy specifically targeting the conformation-dependent TG2-epitope interacting with HS

Expression pattern of the epithelial V\u2010like antigen (Eva) transcript suggests a possible role in placental morphogenesis

Adhesive mechanisms are considered to be of crucial importance for blastocyst adherence to the uterine wall, as well as for the interactions between embryonal and decidual tissues during hemochorial placenta formation. Epithelial V-like Antigen (Eva) is a novel homophilic adhesion molecule of the immunoglobulin superfamily, which during mouse embryonic development is expressed by various differentiating epithelia. In the present paper we describe Eva expression during mouse trophoblast invasion and placental morphogenesis, analysing day 5.5 to 18.5 postcoitum (p.c.) placentas and deciduomas by in situ hybridization. Eva transcripts were detected in spongiotrophoblast cells from 7.5 to 18.5 days p.c. Expression was uniform at early stages, but after day 11.5, p.c. became limited to the invasive subpopulation of spongiotrophoblasts (known as glycogen cells). Trophoblast giant cells did not express Eva in any of the stages analysed. Besides trophoblasts, also early postimplantation decidua was positive for Eva transcripts. In decidual tissue, Eva expression was present at day 5.5 p.c., peaked at day 7.5 p.c., and declined on successive days. The expression pattern of Eva transcripts suggests that during mouse placenta formation, its protein product may play a role in the processes of trophoblast invasion, decidual response, and trophoblast-decidual interaction

The non-peptidic part determines the internalization mechanism and intracellular trafficking of peptide amphiphiles

BACKGROUND: Peptide amphiphiles (PAs) are a class of amphiphilic molecules able to self-assemble into nanomaterials that have shown efficient in vivo targeted delivery. Understanding the interactions of PAs with cells and the mechanisms of their internalization and intracellular trafficking is critical in their further development for therapeutic delivery applications. METHODOLOGY/PRINCIPAL FINDINGS: PAs of a novel, cell- and tissue-penetrating peptide were synthesized possessing two different lipophilic tail architectures and their interactions with prostate cancer cells were studied in vitro. Cell uptake of peptides was greatly enhanced post-modification. Internalization occurred via lipid-raft mediated endocytosis and was common for the two analogs studied. On the contrary, we identified the non-peptidic part as the determining factor of differences between intracellular trafficking and retention of PAs. PAs composed of di-stearyl lipid tails linked through poly(ethylene glycol) to the peptide exhibited higher exocytosis rates and employed different recycling pathways compared to ones consisting of di-palmitic-coupled peptides. As a result, cell association of the former PAs decreased with time. CONCLUSIONS/SIGNIFICANCE: Control over peptide intracellular localization and retention is possible by appropriate modification with synthetic hydrophobic tails. We propose this as a strategy to design improved peptide-based delivery systems