Nanoparticle-Facilitated Membrane Depolarization-Induced Receptor Activation: Implications on Cellular Uptake and Drug Delivery

Cell-specific uptake of drug delivery systems (DDSs) are crucial to achieve optimal efficacy of many drugs. Widely employed strategies to facilitate targeted intracellular drug delivery involves attachment of targeting ligands (peptides or antibodies) to DDSs. Target receptors mutations can limit the effectiveness of this approach. Herein, we demonstrate, through in vitro inhibitory and drug delivery studies, that graphene nanoribbons (GNRs), water dispersed with the amphiphilic polymer called PEG-DSPE ((1, 2-distearoyl-<i>sn</i>-glycero-3-phosphoethanolamine-N [amino (polyethylene glycol)]) (induce membrane depolarization-mediated epidermal growth factor receptor (EGFR) activation. This phenomenon is ligand-independent and EGFR activation occurs via influx of Ca²⁺ ions from the extracellular space. We further provide evidence, through in vivo studies, that this mechanism could be exploited to facilitate efficacious drug delivery into tumors that overexpress EGFR. The results suggest that transient membrane depolarization-facilitated cell receptor activation can be employed as an alternate strategy for enhanced intracellular drug delivery

Analysis of molecular dynamics trajectories revealed low occupancy of hydrolysis-competent active sites in CP4 either alone or fused to the collagen triple-helix forming sequence (GPP)₁₀.

<p>Black lines indicate instance of one or more clusters of H + S + acid groups in proximity to form a catalytically competent triad over 400 ns of all-atom explicit solvent dynamics. In contrast, CP4-Aβ modeled as three strands in an antiparallel beta-sheet shows significant triad occupancy. The most frequent triad observed in CP4-Aβ shows interactions between S1 and H10 on one chain and the F19 carboxy terminus of the amyloid domain of the adjacent chain.</p

Molecular Self-Assembly Strategy for Generating Catalytic Hybrid Polypeptides

<div><p>Recently, catalytic peptides were introduced that mimicked protease activities and showed promising selectivity of products even in organic solvents where protease cannot perform well. However, their catalytic efficiency was extremely low compared to natural enzyme counterparts presumably due to the lack of stable tertiary fold. We hypothesized that assembling these peptides along with simple hydrophobic pockets, mimicking enzyme active sites, could enhance the catalytic activity. Here we fused the sequence of catalytic peptide CP4, capable of protease and esterase-like activities, into a short amyloidogenic peptide fragment of Aβ. When the fused CP4-Aβ construct assembled into antiparallel β-sheets and amyloid fibrils, a 4.0-fold increase in the hydrolysis rate of <i>p</i>-nitrophenyl acetate (<i>p</i>-NPA) compared to neat CP4 peptide was observed. The enhanced catalytic activity of CP4-Aβ assembly could be explained both by pre-organization of a catalytically competent Ser-His-acid triad and hydrophobic stabilization of a bound substrate between the triad and <i>p</i>-NPA, indicating that a design strategy for self-assembled peptides is important to accomplish the desired functionality.</p></div

Structural analysis of the CP4-Aβ assembly.

<p>(a) TEM observation of nanofibers of the CP4-Aβ assembly. (b) UV/visible absorption of Congo red in the absence and presence of self-assembled CP4-Aβ peptides and Aβ peptides. (c) CD spectrometry of the CP4-Aβ peptide assembly in MeOH.</p

Kinetic studies of <i>p</i>-NPA ester hydrolysis catalyzed by the CP4-Aβ catalytic peptide assembly and the CP4 catalytic peptide monomers.

<p>(a) Monitoring the hydrolysis product of <i>p</i>-NP catalyzed by CP4-Aβ catalytic peptides with and without the self-assembly. (b) Normalized catalytic activities of CP4 peptides for <i>p</i>-NP generation with β-sheet self-assembly (2), without β-sheet self-assembly (3), with triple helical sheet self-assembly (4), and assembled ones where triads were mutated to alanine. (5)–(11) show the mutated position of CP4 peptide (SMESLSKTHHYRFFKLVFF) conjugated with the Aβ motif. (12) shows that the enzyme inhibitor of leupeptin reduces the activity of CP4-Aβ mimicking protease. Catalytic activities were derived from initial reaction rates (raw data were shown in Fig D in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153700#pone.0153700.s001" target="_blank">S1 File</a>) and normalized by the result of control experiment, <i>p</i>-NPA ester hydrolysis in PBS/MeOH without any catalytic peptides.</p

Schematic illustrations of the self-assembled structures of <i>de novo</i> hybrid peptides.

<p>(a) The self-assembly of <i>de novo</i> hybrid peptides that the sequence of CP4 catalytic peptide is fused with amyloid β peptide. The self-assembly of CP4-Aβ (SMESLSKTHHYRFFKLVFF) into 2D hydrophobic antiparallel β-sheet conformation increases the catalytic activity as compared to the non-assembled neat CP4 peptide. (b) Another sheet structure assembly by using <i>de novo</i> triple helix peptide as an assembly motif. The CP4 fused into (G_LP_LP)₁₀ sequence can be assembled into the sheet <i>via</i> isometric association with (G_DP_DP)₁₀.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153700#pone.0153700.ref040" target="_blank">40</a>].</p

Peptide–Metal Organic Framework Swimmers that Direct the Motion toward Chemical Targets

Highly efficient and robust chemical motors are expected for the application in microbots that can selectively swim toward targets and accomplish their tasks in sensing, labeling, and delivering. However, one of major issues for such development is that current artificial swimmers have difficulty controlling their directional motion toward targets like bacterial chemotaxis. To program synthetic motors with sensing capability for the target-directed motion, we need to develop swimmers whose motions are sensitive to chemical gradients in environments. Here we create a new intelligent biochemical swimmer by integrating metal organic frameworks (MOFs) and peptides that can sense toxic heavy metals in solution and swim toward the targets. With the aid of Pb-binding enzymes, the peptide-MOF motor can directionally swim toward PbSe quantum dots (QD) by sensing pH gradient and eventually complete the motion as the swimmer reaches the highest gradient point at the target position in solution. This type of technology could be evolved to miniaturize chemical robotic systems that sense target chemicals and swim toward target locations

Peptide–Metal Organic Framework Swimmers that Direct the Motion toward Chemical Targets

Highly efficient and robust chemical motors are expected for the application in microbots that can selectively swim toward targets and accomplish their tasks in sensing, labeling, and delivering. However, one of major issues for such development is that current artificial swimmers have difficulty controlling their directional motion toward targets like bacterial chemotaxis. To program synthetic motors with sensing capability for the target-directed motion, we need to develop swimmers whose motions are sensitive to chemical gradients in environments. Here we create a new intelligent biochemical swimmer by integrating metal organic frameworks (MOFs) and peptides that can sense toxic heavy metals in solution and swim toward the targets. With the aid of Pb-binding enzymes, the peptide-MOF motor can directionally swim toward PbSe quantum dots (QD) by sensing pH gradient and eventually complete the motion as the swimmer reaches the highest gradient point at the target position in solution. This type of technology could be evolved to miniaturize chemical robotic systems that sense target chemicals and swim toward target locations

Antivesiculation and Complete Unbinding of Tail-Tethered Lipids

We report the effect of tail-tethering on vesiculation and complete unbinding of bilayered membranes. Amphiphilic molecules of a bolalipid, resembling the tail-tethered molecular structure of archaeal lipids, with two identical zwitterionic phosphatidylcholine headgroups self-assemble into a large flat lamellar membrane, in contrast to the multilamellar vesicles (MLVs) observed in its counterpart, monopolar nontethered zwitterionic lipids. The antivesiculation is confirmed by small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cyro-TEM). With the net charge of zero and higher bending rigidity of the membrane (confirmed by neutron spin echo (NSE) spectroscopy), the current membrane theory would predict that membranes should stack with each other (aka “bind”) due to dominant van der Waals attraction, while the outcome of the nonstacking (“unbinding”) membrane suggests that the theory needs to include entropic contribution for the nonvesicular structures. This report pioneers an understanding of how the tail-tethering of amphiphiles affects the structure, enabling better control over the final nanoscale morphology

The Effect of Cage Shape on Nanoparticle-Based Drug Carriers: Anticancer Drug Release and Efficacy via Receptor Blockade Using Dextran-Coated Iron Oxide Nanocages

Although a range of nanoparticles have been developed as drug delivery systems in cancer therapeutics, this approach faces several important challenges concerning nanocarrier circulation, clearance, and penetration. The impact of reducing nanoparticle size on penetration through leaky blood vessels around tumor microenvironments via enhanced permeability and retention (EPR) effect has been extensively examined. Recent research has also investigated the effect of nanoparticle shape on circulation and target binding affinity. However, how nanoparticle shape affects drug release and therapeutic efficacy has not been previously explored. Here, we compared the drug release and efficacy of iron oxide nanoparticles possessing either a cage shape (IO-NCage) or a solid spherical shape (IO-NSP). Riluzole cytotoxicity against metastatic cancer cells was enhanced 3-fold with IO-NCage. The shape of nanoparticles (or nanocages) affected the drug release point and cellular internalization, which in turn influenced drug efficacy. Our study provides evidence that the shape of iron oxide nanoparticles has a significant impact on drug release and efficacy