Dimitris Missirlis

Laser-Activated Gene Silencing via Gold Nanoshell−siRNA Conjugates

The temporal and spatial control over the delivery of materials such as siRNA into cells remains a significant technical challenge. We demonstrate the pulsed near-infrared (NIR) laser-dependent release of siRNA from coated 40 nm gold nanoshells. Tat-lipid coating mediates the cellular uptake of the nanomaterial at picomolar concentration, while spatiotemporal silencing of a reporter gene (green fluorescence protein) was studied using photomasking. The NIR laser-induced release of siRNA from the nanoshells is found to be power- and time-dependent, through surface-linker bond cleavage, while the escape of the siRNA from endosomes occurs above a critical pulse energy attributed to local heating and cavitation. NIR laser-controlled drug release from functional nanomaterials should facilitate more sophisticated developmental biology and therapeutic studies.

Targeting of albumin-embedded paclitaxel nanoparticles to tumors.

We have used tumor-homing peptides to target abraxane, a clinically approved paclitaxel-albumin nanoparticle, to tumors in mice. The targeting was accomplished with two peptides, CREKA and LyP-1 (CGNKRTRGC). Fluorescein (FAM)-labeled CREKA-abraxane, when injected intravenously into mice bearing MDA-MB-435 human cancer xenografts, accumulated in tumor blood vessels, forming aggregates that contained red blood cells and fibrin. FAM-LyP-1-abraxane co-localized with extravascular islands expressing its receptor, p32. Self-assembled mixed micelles carrying the homing peptide and the label on different subunits accumulated in the same areas of tumors as LyP-1-abraxane, showing that Lyp-1 can deliver intact nanoparticles into extravascular sites. Untargeted, FAM-abraxane was detected in the form of a faint meshwork in tumor interstitium. LyP-1-abraxane produced a statistically highly significant inhibition of tumor growth compared with untargeted abraxane. These results show that nanoparticles can be effectively targeted into extravascular tumor tissue and that targeting can enhance the activity of a therapeutic nanoparticle.

Mechanisms of peptide amphiphile internalization by SJSA-1 cells in vitro.

Self-assembly of peptide amphiphiles into nanostructures makes them attractive for a variety of applications in drug and peptide delivery. We here report on the interactions of micelles composed of a palmitoylated, pro-apoptotic peptide derived from p53 tumor suppressor protein with a human cancer cell line. Characterization of self-assembly in aqueous buffered solutions revealed formation of elongated rod-like micelles above a critical micelle concentration. Our results however demonstrate that monomers instead of micelles are internalized, a finding that correlates with the dynamic nature of the assemblies and the noncovalent interactions that hold them together. Internalization is shown to occur via adsorption-mediated, energy-dependent pathways, resulting in accumulation of the material in endocytic vesicles. We conclude that palmitoylation of peptides is an efficient way to increase peptide permeability inside SJSA-1 cells and that increased micelle stability would be required for intact micelle internalization.

Effect of the Peptide Secondary Structure on the Peptide Amphiphile Supramolecular Structure and Interactions

Bottom-up fabrication of self-assembled nanomaterials requires control over forces and interactions between building blocks. We report here on the formation and architecture of supramolecular structures constructed from two different peptide amphiphiles. Inclusion of four alanines between a 16-mer peptide and a 16 carbon long aliphatic tail resulted in a secondary structure shift of the peptide headgroups from α helices to β sheets. A concomitant shift in self-assembled morphology from nanoribbons to core-shell worm-like micelles was observed by cryogenic transmission electron microscopy (cryo-TEM) and atomic force microscopy (AFM). In the presence of divalent magnesium ions, these a priori formed supramolecular structures interacted in distinct manners, highlighting the importance of peptide amphiphile design in self-assembly.

Thermodynamic and Kinetic Stability of DSPE-PEG(2000) Micelles in the Presence of Bovine Serum Albumin

This work investigated the stability of DSPE-PEG(2000) micelles in the presence of bovine serum albumin (BSA). DSPE-PEG(2000) was found to exist in equilibrium among monomeric, micellar, and BSA-bound states, and this equilibrium shifted toward the BSA-bound state when the temperature increased from 20 to 37 °C. The micellar state is thermodynamically unstable at both temperatures when the concentration of BSA approaches that of DSPE-PEG(2000), and micelle breakup occurs with a first-order time constant of 130 ± 9 min at 20 °C and 7.8 ± 1.6 min at 37 °C. Thus, previous targeting experiments that demonstrate synergistic effects in multiply functionalized DSPE-PEG(2000) micelles are likely due to targeting that occurs on a timescale faster than that of micelle breakup. Micelle breakup was limited by diffusion at 20 °C whereas at 37 °C monomer desorption from the micelle was the rate-limiting step. These findings give clear guidance concerning the lifetimes of micelles that may be used as diagnostic and therapeutic nanoparticles.

Combined Effects of PEG Hydrogel Elasticity and Cell-Adhesive Coating on Fibroblast Adhesion and Persistent Migration

The development and use of synthetic, cross-linked, macromolecular substrates with tunable elasticity has been instrumental in revealing the mechanisms by which cells sense and respond to their mechanical microenvironment. We here describe a hydrogel based on radical-free, cross-linked poly(ethylene glycol) to study the effects of both substrate elasticity and type of adhesive coating on fibroblast adhesion and migration. Hydrogel elasticity was controlled through the structure and concentration of branched precursors, which efficiently react via Michael-type addition to produce the polymer network. We found that cell spreading and focal adhesion characteristics are dependent on elasticity for all types of coatings (RGD peptide, fibronectin, vitronectin), albeit with significant differences in magnitude. Importantly, fibroblasts migrated slower but more persistently on stiffer hydrogels, with the effects being more pronounced on fibronectin-coated substrates. Therefore, our results validate the hydrogels presented in this study as suitable for future mechanosensing studies and indicate that cell adhesion, polarity, and associated migration persistence are tuned by substrate elasticity and biochemical properties.

Linker Chemistry Determines Secondary Structure of p5314−29 in Peptide Amphiphile Micelles

Biofunctional micelles formed via self-assembly of synthetic peptide-lipid conjugates are a class of promising biomaterials with applications in drug delivery and tissue engineering. The micelle building block, termed peptide amphiphile, consists of a lipid-like chain covalently linked through a spacer to a peptide headgroup. Self-assembly results in formation of a hydrophobic core surrounded by a dense shell with multiple, functional peptides. We report here on the effect that different linkers between a palmitic tail and a bioactive peptide (p5314-29) have on headgroup secondary structure. Peptide p5314-29 may act as an inhibitor of the interaction between tumor suppressor p53 and human double minute-2 (hDM2) proteins by binding hDM2 in a partially helical form, leading to the release of p53 and the induction of apoptosis in certain tumors. Circular dichroism and fluorescence spectroscopy data revealed that the extent and type of secondary structure of p5314-29 are controlled through size and hydrogen bond potential of the linker. In addition, the structure of the self-assembled micelles was influenced through linker-dependent altered headgroup interactions. This study provides insight into the mechanisms through which headgroup structuring occurs on peptide amphiphile micelles, with implications on the bioactivity, stability, and morphology of the self-assembled entities.

Substrate engagement of integrins α5β1 and αvβ3 is necessary, but not sufficient, for high directional persistence in migration on fibronectin.

The interplay between specific integrin-mediated matrix adhesion and directional persistence in cell migration is not well understood. Here, we characterized fibroblast adhesion and migration on the extracellular matrix glycoproteins fibronectin and vitronectin, focusing on the role of α5β1 and αvβ3 integrins. Fibroblasts manifested high directional persistence in migration on fibronectin-, but not vitronectin-coated substrates, in a ligand density-dependent manner. Fibronectin stimulated α5β1-dependent organization of the actin cytoskeleton into oriented, ventral stress fibers, and assembly of dynamic, polarized protrusions, characterized as regions free of stress fibers and rich in nascent adhesions at their edge. Such protrusions correlated with persistent, local leading edge advancement, but were not sufficient, nor necessary for directional migration over longer times. Selective blocking of αvβ3 or α5β1 integrins using small molecule integrin antagonists reduced directional persistence on fibronectin, indicating integrin cooperativity in maintaining directionality. On the other hand, patterned substrates, designed to selectively engage either integrin, or their combination, were not sufficient to establish directional migration. Overall, our study demonstrates adhesive coating-dependent regulation of directional persistence in fibroblast migration and challenges the generality of the previously suggested role of β1 and β3 integrins in directional migration.

Structural Effects and Lipid Membrane Interactions of the pH-Responsive GALA Peptide with Fatty Acid Acylation

GALA is a pH-responsive, membrane-perturbing peptide designed to fold from a random coil at physiological pH to an amphipathic α-helix under mildly acidic conditions. Because of its pH-activated function, GALA has been sought-after as a component of intracellular drug delivery systems that could actively propel endosomal escape. In this study, we conjugated GALA with lauryl and palmitoyl fatty acid tails as model hydrophobic moieties and examined the physicochemical characteristics and activities of the resulting peptide amphiphiles (PAs). The fatty acid variants of GALA exhibited distinctly different membrane perturbing mechanisms at pH 7.5 and 5.5. At physiological pH, the PAs ruptured liposomes through a surfactant-like mechanism. At pH 5.5, lauryl-GALA was shown to form transmembrane pores with a higher potency as compared to its unmodified peptide counterpart; however, after prolonged exposure it also caused liposome lysis. The lytic activity of fatty acid-conjugated GALA did not impair cell viability. Lauryl-GALA was tolerated well by SJSA-1 osteocarcinoma cells and enhanced cell internalization of the PA was observed. Our findings are discussed with the overarching goal of developing efficient therapeutic delivery systems.

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.