Gerard C. L. Wong

Arginine-rich cell-penetrating peptides.

Arginine‐rich cell‐penetrating peptides are short cationic peptides capable of traversing the plasma membranes of eukaryotic cells. While successful intracellular delivery of many biologically active macromolecules has been accomplished using these peptides, their mechanisms of cell entry are still under investigation. Recent dialogue has centered on a debate over the roles that direct translocation and endocytotic pathways play in internalization of cell‐penetrating peptides. In this paper, we review the evidence for the broad range of proposed mechanisms, and show that each distinct process requires negative Gaussian membrane curvature as a necessary condition. Generation of negative Gaussian curvature by cell‐penetrating peptides is directly related to their arginine content. We illustrate these concepts using HIV TAT as an example.

Reversible Cell‐Specific Drug Delivery with Aptamer‐Functionalized Liposomes

cis-Diamminedichloroplatinum(II) (cisplatin) is a potent chemotherapeutic agent for the treatment of a broad range of cancerous tumors. 2] Despite its excellent antitumor efficacy, the major drawbacks of cisplatin include its lack of tumor specificity and severe side effects. In addition, certain tumor-cell types develop resistance to cisplatin from exposure to the drug. Strategies that allow the delivery of cisplatin specifically to tumor cells are highly desirable. Several strategies have been reported for the delivery of cisplatin specifically to tumor sites, the most common of which is to use antibody (Ab) recognition against different cell-surface targets. The binding of Abs to the cell-membrane receptors triggers receptor-mediated endocytosis, with the result of improved therapeutic efficacy. Despite this success, the use of Abs as cell-specific homing agents poses significant challenges. Ab conjugations are difficult to control and typically show poor site specificity for the conjugation and inconsistent binding affinity. The antibody-based drugdelivery system also tends to be immunogenic, so it requires extra humanization steps, which make it more difficult for clinical application. Nucleic acid based aptamers provide excellent alternatives to antibodies as cell-specific agents. They are singlestranded oligonucleotides identified through an in vitro selection process, termed system evolution of ligands by exponential enrichment (SELEX), to bind the target molecules selectively. 12] Many aptamers identified by SELEX have nearly identical binding affinity and specificity to those of Abs. Aptamers are much easier to prepare and to scale up. They are generally considered nonimmunogenic and can be gradually degraded by nucleases and cleared from the blood to cause minimal system toxicity. Functionalizations of aptamers to facilitate site-specific conjugation are also straightforward. Thus, aptamers are promising targeting ligands and have been used in targeted drug-delivery systems, most of which are block-copolymer nanoparticles. Although these new nanotechnology-based platforms look promising, the clinical benefit of nanoparticles for targeted cancer therapy is yet to be demonstrated. Liposomes are by far the most successful drug-delivery systems; a number of liposome-based systems have been approved by the US Food and Drug Administration for disease treatment in the clinic. Liposomes have been shown to increase the plasma residence time of aptamers. Previous efforts on liposomal drug delivery have focused on developing long-circulating liposomes that target cancerous tumor tissues through the enhanced permeation and retention (EPR) effect, 34] a passive targeting mechanism. However, cancer targeting entirely based on EPR still has undesirable systemic side effects and suboptimal antitumor efficacy: clinical studies of a cisplatin-containing liposome showed only poor to moderate therapeutic efficacy. 38] Delivery vehicles with active tumor-targeting capability could, in principle, improve this significantly. Personalized chemotherapy is an unmet challenge in cancer treatment. Despite the existence of rough empirical dosage guidelines, the individual patient response can deviate strongly from average behavior. This problem is especially acute for chemotherapy agents, for which drug overdosage can have severe consequences. At present, once an initial dosage is administered, the side effects and drug effectiveness can no longer be modulated if there are no “antidotes” to the treatment. However, the discovery of good antidotes or neutralizers for each individual drug molecule is not an easy task, if even practical. Moreover, there are no known ways to “multiplex” different antidotes to control complex treatment profiles with multiple drugs. We report here the controlled formulation of aptamerconjugated, cisplatin-encapsulating multifunctional liposomes. Cancer-cell-specific targeting and drug delivery are demonstrated by using this delivery platform. Furthermore, we also show for the first time that a complementary DNA (cDNA) of the aptamer can function as an antidote to disrupt [*] R. Tong, A. Mishra, Prof. G. C. L. Wong, Prof. J. Cheng, Prof. Y. Lu Department of Materials Science and Engineering University of Illinois at Urbana-Champaign 1304 W. Green Street, Urbana, IL 61801 (USA) Fax: (+ 1)217-333-2736 E-mail: gclwong@illinois.edu jianjunc@illinois.edu Homepage: http://www.matse.illinois.edu/faculty/wong/profile.html http://www.matse.illinois.edu/faculty/cheng/profile.html Dr. Z. Cao, W. Xu, Prof. Y. Lu Department of Chemistry University of Illinois at Urbana-Champaign 600 S. Mathews Avenue, Urbana, IL 61801 (USA) Fax: (+ 1)217-244-3186 E-mail: yi-lu@illinois.eduyi-lu@illinois.edu Homepage: http://www.chemistry.illinois.edu/faculty/Yi_Lu.html [] Z.C. and R.T. contributed equally to this work.

The Pel Polysaccharide Can Serve a Structural and Protective Role in the Biofilm Matrix of Pseudomonas aeruginosa

Bacterial extracellular polysaccharides are a key constituent of the extracellular matrix material of biofilms. Pseudomonas aeruginosa is a model organism for biofilm studies and produces three extracellular polysaccharides that have been implicated in biofilm development, alginate, Psl and Pel. Significant work has been conducted on the roles of alginate and Psl in biofilm development, however we know little regarding Pel. In this study, we demonstrate that Pel can serve two functions in biofilms. Using a novel assay involving optical tweezers, we demonstrate that Pel is crucial for maintaining cell-to-cell interactions in a PA14 biofilm, serving as a primary structural scaffold for the community. Deletion of pelB resulted in a severe biofilm deficiency. Interestingly, this effect is strain-specific. Loss of Pel production in the laboratory strain PAO1 resulted in no difference in attachment or biofilm development; instead Psl proved to be the primary structural polysaccharide for biofilm maturity. Furthermore, we demonstrate that Pel plays a second role by enhancing resistance to aminoglycoside antibiotics. This protection occurs only in biofilm populations. We show that expression of the pel gene cluster and PelF protein levels are enhanced during biofilm growth compared to liquid cultures. Thus, we propose that Pel is capable of playing both a structural and a protective role in P. aeruginosa biofilms.

Translocation of HIV TAT peptide and analogues induced by multiplexed membrane and cytoskeletal interactions

Cell-penetrating peptides (CPPs), such as the HIV TAT peptide, are able to translocate across cellular membranes efficiently. A number of mechanisms, from direct entry to various endocytotic mechanisms (both receptor independent and receptor dependent), have been observed but how these specific amino acid sequences accomplish these effects is unknown. We show how CPP sequences can multiplex interactions with the membrane, the actin cytoskeleton, and cell-surface receptors to facilitate different translocation pathways under different conditions. Using “nunchuck” CPPs, we demonstrate that CPPs permeabilize membranes by generating topologically active saddle-splay (“negative Gaussian”) membrane curvature through multidentate hydrogen bonding of lipid head groups. This requirement for negative Gaussian curvature constrains but underdetermines the amino acid content of CPPs. We observe that in most CPP sequences decreasing arginine content is offset by a simultaneous increase in lysine and hydrophobic content. Moreover, by densely organizing cationic residues while satisfying the above constraint, TAT peptide is able to combine cytoskeletal remodeling activity with membrane translocation activity. We show that the TAT peptide can induce structural changes reminiscent of macropinocytosis in actin-encapsulated giant vesicles without receptors.

Like-charge attraction between polyelectrolytes induced by counterion charge density waves

Electrostatics in aqueous media is commonly understood in terms of screened Coulomb interactions, where like-charged objects, such as polyelectrolytes, always repel. These intuitive expectations are based on mean field theories, such as the Poisson–Boltzmann formalism, which are routinely used in colloid science and computational biology [Israelachvili, J. (1992) Intermolecular and Surface Forces (Academic, London), 2nd ed.]. Like-charge attractions, however, have been observed in a variety of systems [Gelbart, W. M., Bruinsma, R. F., Pincus, P. A. & Parsegian, V. A. (2000) Phys. Today 53, 38–44]. Intense theoretical scrutiny over the last 30 years suggests that counterions play a central role, but no consensus exists for the precise mechanism. We have directly observed the organization of multivalent ions on cytoskeletal filamentous actin (a well defined biological polyelectrolyte) by using synchrotron x-ray diffraction and discovered an unanticipated symmetry-breaking collective counterion mechanism for generating attractions. Surprisingly, the counterions do not form a lattice that simply follows actins helical symmetry; rather, the counterions organize into “frozen” ripples parallel to the actin filaments and form 1D charge density waves. Moreover, this 1D counterion charge density wave couples to twist distortions of the oppositely charged actin filaments. This general cooperative molecular mechanism is analogous to the formation of polarons in ionic solids and mediates attractions by facilitating a “zipper-like” charge alignment between the counterions and the polyelectrolyte charge distribution. We believe these results can fundamentally impinge on our general understanding of electrostatics in aqueous media and are relevant to a wide range of colloidal and biomedical processes.

Electrostatics of Strongly Charged Biological Polymers: Ion-Mediated Interactions and Self-Organization in Nucleic Acids and Proteins

Charges on biological polymers in physiologically relevant solution conditions are strongly screened by water and salt solutions containing counter-ions. However, the entropy of these counterions can result in surprisingly strong interactions between charged objects in water despite short screening lengths, via coupling between osmotic and electrostatic interactions. Widespread work in theory, experiment, and computation has been carried out to gain a fundamental understanding of the rich, yet sometimes counterintuitive, behavior of these polyelectrolyte systems. Examples of polyelectrolyte association in biology include DNA packaging and RNA folding, as well as aggregation and self-organization phenomena in different disease states.

Psl trails guide exploration and microcolony formation in Pseudomonas aeruginosa biofilms

Bacterial biofilms are surface-associated, multicellular, morphologically complex microbial communities. Biofilm-forming bacteria such as the opportunistic pathogen Pseudomonas aeruginosa are phenotypically distinct from their free-swimming, planktonic counterparts. Much work has focused on factors affecting surface adhesion, and it is known that P. aeruginosa secretes the Psl exopolysaccharide, which promotes surface attachment by acting as ‘molecular glue’. However, how individual surface-attached bacteria self-organize into microcolonies, the first step in communal biofilm organization, is not well understood. Here we identify a new role for Psl in early biofilm development using a massively parallel cell-tracking algorithm to extract the motility history of every cell on a newly colonized surface. By combining this technique with fluorescent Psl staining and computer simulations, we show that P. aeruginosa deposits a trail of Psl as it moves on a surface, which influences the surface motility of subsequent cells that encounter these trails and thus generates positive feedback. Both experiments and simulations indicate that the web of secreted Psl controls the distribution of surface visit frequencies, which can be approximated by a power law. This Pareto-type behaviour indicates that the bacterial community self-organizes in a manner analogous to a capitalist economic system, a ‘rich-get-richer’ mechanism of Psl accumulation that results in a small number of ‘elite’ cells becoming extremely enriched in communally produced Psl. Using engineered strains with inducible Psl production, we show that local Psl concentrations determine post-division cell fates and that high local Psl concentrations ultimately allow elite cells to serve as the founding population for initial microcolony development.

HIV TAT Forms Pores in Membranes by Inducing Saddle‐Splay Curvature: Potential Role of Bidentate Hydrogen Bonding

The TAT protein transduction domain (PTD) of the human immunodeficiency virus (HIV-1) can cross cell membranes with unusual efficiency and has many potential biotechnological applications. Extant work has provided important clues to the molecular mechanism underlying the activity of this peptide, which consists of 11 amino acids, 8 of which are cationic and 6 of these are arginines. TAT PTD synthesized with d-amino acids enters cells as efficiently as the native form, thereby indicating that the mechanism of transduction is receptor independent; this conclusion is consistent with recent results that suggest that the TAT PTD may enter cells through receptor-independent macropinocytosis. Substitution of any of the PTD1s cationic residues with neutral alanine decreases activity, while substitution of neutral residues has no effect. This indicates the importance of electrostatic interactions between cationic TAT PTD and anionic phospholipid membranes. Recent work has shown that the physics of electrostatic interactions can drive a rich polymorphism of self-assembled structures that depend on parameters such as charge density and intrinsic membrane curvature. However, although arginine-rich polycations can enter cells, cationic polylysine cannot. This shows that electrostatic interactions alone are insufficient for PTD activity and that the arginine residues play a specific, essential role. We use confocal microscopy and synchrotron X-ray scattering (SAXS) to study the interaction of the TAT PTD with model membranes at room temperature. We find that the transduction activity correlates with induction of negative Gaussian (“saddle-splay”) membrane curvature, which is topologically required for pore formation. Moreover, we show that the TAT PTD can drastically remodel vesicles into a porous bicontinuous phase with analogues in block-copolymer systems, and we propose a geometric mechanism facilitated by both electrostatics and bidentate hydrogen bonding. The latter is possible for the TAT PTD but not for similarly cationic, nonarginated polypeptides. Cell membranes are composed of lipids that have fundamentally different interactions with cationic macroions such as TAT PTD. We examine representative model membranes composed of lipids with different charges and intrinsic curvatures: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) have zwitterionic headgroups, while 1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] (sodium salt) (DOPS) and 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG) have anionic headgroups; all have zero intrinsic curvature (C0 = 0, “cylinder-shaped”) except for DOPE, which has negative intrinsic curvature (C0< 0, “cone-shaped”). When rhodamine-tagged TAT PTD (Rh-PTD) is applied to the exterior of giant unilamellar vesicles (GUVs, diameters of 5–30 mm) with low DOPE content (0 and 20%), rhodamine fluorescence is seen only outside the GUVs (Figure 1a), thereby indicating that the Rh-PTD has not crossed these membranes. However, when Rh-PTD is applied to GUVs with 40% DOPE content, the rhodamine intensity equilibrates across the membrane over tens of seconds (Figure 1b and c; see also the movie in the Supporting Information). This shows that Rh-PTD has crossed the GUV membranes, which remain intact (Figure 1c). Thus, we see that the membrane transduction activity of Rh-PTD requires the presence of a threshold amount of DOPE in the membrane.

Living in the matrix: assembly and control of Vibrio cholerae biofilms

Nearly all bacteria form biofilms as a strategy for survival and persistence. Biofilms are associated with biotic and abiotic surfaces and are composed of aggregates of cells that are encased by a self-produced or acquired extracellular matrix. Vibrio cholerae has been studied as a model organism for understanding biofilm formation in environmental pathogens, as it spends much of its life cycle outside of the human host in the aquatic environment. Given the important role of biofilm formation in the V. cholerae life cycle, the molecular mechanisms underlying this process and the signals that trigger biofilm assembly or dispersal have been areas of intense investigation over the past 20 years. In this Review, we discuss V. cholerae surface attachment, various matrix components and the regulatory networks controlling biofilm formation.

Bacteria Use Type IV Pili to Walk Upright and Detach from Surfaces

A searchable database of images allows detailed analysis of bacterial motility. Bacterial biofilms are structured multicellular communities involved in a broad range of infections. Knowing how free-swimming bacteria adapt their motility mechanisms near surfaces is crucial for understanding the transition between planktonic and biofilm phenotypes. By translating microscopy movies into searchable databases of bacterial behavior, we identified fundamental type IV pili–driven mechanisms for Pseudomonas aeruginosa surface motility involved in distinct foraging strategies. Bacteria stood upright and “walked” with trajectories optimized for two-dimensional surface exploration. Vertical orientation facilitated surface detachment and could influence biofilm morphology.