Abhigyan Som

Cationic Nanoparticles Induce Nanoscale Disruption in Living Cell Plasma Membranes

It has long been recognized that cationic nanoparticles induce cell membrane permeability. Recently, it has been found that cationic nanoparticles induce the formation and/or growth of nanoscale holes in supported lipid bilayers. In this paper, we show that noncytotoxic concentrations of cationic nanoparticles induce 30-2000 pA currents in 293A (human embryonic kidney) and KB (human epidermoid carcinoma) cells, consistent with a nanoscale defect such as a single hole or group of holes in the cell membrane ranging from 1 to 350 nm(2) in total area. Other forms of nanoscale defects, including the nanoparticle porating agents adsorbing onto or intercalating into the lipid bilayer, are also consistent; although the size of the defect must increase to account for any reduction in ion conduction, as compared to a water channel. An individual defect forming event takes 1-100 ms, while membrane resealing may occur over tens of seconds. Patch-clamp data provide direct evidence for the formation of nanoscale defects in living cell membranes. The cationic polymer data are compared and contrasted with patch-clamp data obtained for an amphiphilic phenylene ethynylene antimicrobial oligomer (AMO-3), a small molecule that is proposed to make well-defined 3.4 nm holes in lipid bilayers. Here, we observe data that are consistent with AMO-3 making approximately 3 nm holes in living cell membranes.

Synthetic mimics of antimicrobial peptides

Infectious diseases and antibiotic resistance are now considered the most imperative global healthcare problem. In the search for new treatments, host defense, or antimicrobial, peptides have attracted considerable attention due to their various unique properties; however, attempts to develop in vivo therapies have been severely limited. Efforts to develop synthetic mimics of antimicrobial peptides (SMAMPs) have increased significantly in the last decade, and this review will focus primarily on the structural evolution of SMAMPs and their membrane activity. This review will attempt to make a bridge between the design of SMAMPs and the fundamentals of SMAMP-membrane interactions. In discussions regarding the membrane interaction of SMAMPs, close attention will be paid to the lipid composition of the bilayer. Despite many years of study, the exact conformational aspects responsible for the high selectivity of these AMPs and SMAMPs toward bacterial cells over mammalian cells are still not fully understood. The ability to design SMAMPs that are potently antimicrobial, yet nontoxic to mammalian cells has been demonstrated with a variety of molecular scaffolds. Initial animal studies show very good tissue distribution along with more than a 4-log reduction in bacterial counts. The results on SMAMPs are not only extremely promising for novel antibiotics, but also provide an optimistic picture for the greater challenge of general proteomimetics.

Investigating the Effect of Increasing Charge Density on the Hemolytic Activity of Synthetic Antimicrobial Polymers

The current study is aimed at investigating the effect of fine-tuning the cationic character of synthetic mimics of antimicrobial peptides (SMAMPs) on the hemolytic and antibacterial activities. A series of novel norbornene monomers that carry one, two, or three Boc-protected amine functionalities was prepared. Ring-opening metathesis polymerization (ROMP) of the monomers, followed by deprotection of the amine groups resulted in cationic antimicrobial polynorbornenes that carry one, two, and three charges per monomer repeat unit. Increasing the number of amine groups on the most hydrophobic polymer reduced its hemolytic activity significantly. To understand the membrane activity of these polymers, we conducted dye leakage experiments on lipid vesicles that mimic bacteria and red blood cell membranes, and these results showed a strong correlation with the hemolysis data.

Doubly Selective Antimicrobial Polymers: How Do They Differentiate between Bacteria?

We have investigated how doubly selective synthetic mimics of antimicrobial peptides (SMAMPs), which can differentiate not only between bacteria and mammalian cells, but also between Gram-negative and Gram-positive bacteria, make the latter distinction. By dye-leakage experiments on model vesicles and complementary experiments on bacteria, we were able to relate the Gram selectivity to structural differences of these bacteria types. We showed that the double membrane of E. coli rather than the difference in lipid composition between E. coli and S. aureus was responsible for Gram selectivity. The molecular-weight-dependent antimicrobial activity of the SMAMPs was shown to be a sieving effect: while the 3000 g mol(-1) SMAMP was able to penetrate the peptidoglycan layer of the Gram-positive S. aureus bacteria, the 50000 g mol(-1) SMAMP got stuck and consequently did not have antimicrobial activity.

Mechanism of a Prototypical Synthetic Membrane-Active Antimicrobial: Efficient Hole-Punching Via Interaction With Negative Intrinsic Curvature Lipids

Phenylene ethynylenes comprise a prototypical class of synthetic antimicrobial compounds that mimic antimicrobial peptides produced by eukaryotes and have broad-spectrum antimicrobial activity. We show unambiguously that bacterial membrane permeation by these antimicrobials depends on the presence of negative intrinsic curvature lipids, such as phosphatidylethanolamine (PE) lipids, found in high concentrations within bacterial membranes. Plate-killing assays indicate that a PE-knockout mutant strain of Escherichia coli drastically out-survives the wild type against the membrane-active phenylene ethynylene antimicrobials, whereas the opposite is true when challenged with traditional metabolic antibiotics. That the PE deletion is a lethal mutation in normative environments suggests that resistant bacterial strains do not evolve because a lethal mutation is required to gain immunity. PE lipids allow efficient generation of negative curvature required for the circumferential barrel of an induced membrane pore; an inverted hexagonal HII phase, which consists of arrays of water channels, is induced by a small number of antimicrobial molecules. The estimated antimicrobial occupation in these water channels is nonlinear and jumps from ≈1 to 3 per 4 nm of induced water channel length as the global antimicrobial concentration is increased. By comparing to exactly solvable 1D spin models for magnetic systems, we quantify the cooperativity of these antimicrobials.

Influence of lipid composition on membrane activity of antimicrobial phenylene ethynylene oligomers.

Host defense peptides (HDPs), part of the innate immune system, selectively target the membranes of bacterial cells over that of host cells. As a result, their antimicrobial properties have been under intense study. Their selectivity strongly depends on the chemical and mostly structural properties of the lipids that make up different cell membranes. The ability to synthesize HDP mimics has recently been demonstrated. To better understand how these HDP mimics interact with bilayer membranes, three homologous antimicrobial oligomers (AMOs) 1-3 with an m-phenylene ethynylene backbone and alkyl amine side chains were studied. Among them, AMO 1 is nonactive, AMO 2 is specifically active, and AMO 3 is nonspecifically active against bacteria over human red blood cells, a standard model for mammalian cells. The interactions of these three AMOs with liposomes having different lipid compositions are characterized in detail using a fluorescent dye leakage assay. AMO 2 and AMO 3 caused more leakage than AMO 1 from bacteria membrane mimic liposomes composed of PE/PG lipids. The use of E. coli lipid vesicles gave the same results. Further changes of the lipid compositions revealed that AMO 2 has selectively higher affinity toward PE/PG and E. coli lipids than PC, PC/PG or PC/PS lipids, the major components of mammalian cell membranes. In contrast, AMO 3 is devoid of this lipid selectivity and interacts with all liposomes with equal ease; AMO 1 remains inactive. These observations suggest that lipid type and structure are more important in determining membrane selectivity than lipid headgroup charges for this series of HDP mimics.

Synthetic Mimics of Antimicrobial Peptides with Immunomodulatory Responses

A new series of aryl-based synthetic mimics of antimicrobial peptides (SMAMPs) with antimicrobial activity and selectivity have been developed via systematic tuning of the aromatic groups and charge. The addition of a pendant aromatic group improved the antimicrobial activity against Gram-negative bacteria, while the addition of charge improved the selectivity. SMAMP 4 with six charges and a naphthalene central ring demonstrated a selectivity of 200 against both Staphylococcus aureus and Escherichia coli , compared with a selectivity of 8 for the peptide MSI-78. In addition to the direct antimicrobial activity, SMAMP 4 exhibited specific immunomodulatory activities in macrophages both in the presence and in the absence of lipopolysaccharide, a TLR agonist. SMAMP 4 also induced the production of a neutrophil chemoattractant, murine KC, in mouse primary cells. This is the first nonpeptidic SMAMP demonstrating both good antimicrobial and immunomodulatory activities.

Mechanism of A Prototypical Synthetic Membrane-Active Antimicrobial: Efficient Hole-Punching by Targeting Lipids With Negative Spontaneous Curvature Lipids

a. Department of Materials Science and Engineering, d. Departments of Microbiology and Biochemistry, b. Department of Physics, e.Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801; and c. Department of Polymer Science and Engineering, University of Massachusetts, Amherst, MA 01003

Self‐Activation in De Novo Designed Mimics of Cell‐Penetrating Peptides

The unique ability of cell-penetrating peptides (CPPs), also known as protein transduction domains, to navigate across the nonpolar biological membrane has been under intense investigation. In vitro studies have shown that multiple mechanisms are available, with the precise details being dependent on the peptide and cell line studied. The several clearly demonstrated pathways include various forms of endocytosis, macropinocytosis, lipid-raft-dependent macropinocytosis, and protein-dependent translocation. In addition, an energy-independent pathway, or spontaneous translocation, has also been illustrated. 5] Perhaps the clearest example of an energy-independent pathway is the ability of CPPs, and their synthetic mimics, to cross model phospholipid bilayer vesicle membranes. General consensus in the literature suggests that hydrophobic counterions play an essential role in this transduction by complexation around the guanidinium-rich backbone, thus coating the highly cationic structure with lipophilic moieties. For example, an octamer of arginine in the presence of sodium laurate partitioned into octanol versus water with better than 95% efficiency. Separately, it was shown that the simple peptide nonaarginine ((Arg)9) does not in fact transverse membranes very effectively on its own. However, the presence of hydrophobic counterions “activates” this molecule, thus turning it into a potent transduction peptide. It was shown that n-alkyl chain surfactants were good “activators” and thus efficient at promoting the transport of oligoand polyarginines across biological membranes. 8] After the initial discovery that CPP-like behavior could be emulated in simple norbornene-based polymers, we wondered if the presence of covalently attached hydrophobic residues would increase their translocation activity. To evaluate this hypothesis, we designed and synthesized a series of norbornene-based guanidine-rich polymers, where the hydrophobic groups were introduced through a side chain rather than as counterions (Scheme 1). Remarkably, the guanidine polymers containing certain alkyl side chains exhibited significantly enhanced activity (by three orders of magnitude) without the need for any “counterion activator”. Monomers were prepared by either Mitsunobu coupling or nucleophilic substitution reactions (see the Supporting Information). Random copolymers G1–G12 with 50:50 mol% monomer distribution were targeted at two molecular weights (Mn) using ring-opening metathesis polymerization (ROMP; low Mn 2.9–3.9 kDa and high Mn 11.4– 13.6 kDa of the tert-butyloxycarbonyl (Boc)-protected polymers were obtained). Gel-permeation chromatography gave monomodal signals and narrow molecular-weight indices (1.05–1.15). The Boc-protected polymers were deprotected to obtain G1–G12, and their activities were studied in vesicle assays. Using the standard biophysical assay well-accepted in the CPP literature, the transport activities of G1–G12 were determined. Specifically, 5(6)-carboxyfluorescein (CF) was used as a fluorescent probe in egg yolk phosphatidylcholine large unilamellar vesicles (EYPC-LUVs). The activity of G1– G12 transporters increased with increasing polymer content at a constant vesicle concentration as detected by CF emission intensity, yielding plots of fluorescence intensity versus polymer concentration for the series G1–G12 (Supporting Information, Figure S1). Fitting the Hill equation (Y/ (c/EC50) ) to this data for each individual polymer revealed a nonlinear dependence of the fractional fluorescence intensity Yon the polymer concentration c. This analysis gave Ymax (maximal CF release relative to complete release by Triton X100), EC50 (effective polymer concentration needed to reach Ymax/2), and the Hill coefficient n (Supporting Information, Figure S2, Tables S1 and S2). For direct comparison, it is worth mentioning that the CPPs heptaarginine and polyarginine were inactive under these conditions; it is known that polyarginine needs counterions for activation. Figure 1 collects the EC50 values for this series of copolymers. Polymers with lower EC50 values are said to be Scheme 1. Guanidino copolymers G1–G12.

Protein Transduction Domain Mimics: The Role of Aromatic Functionality

Cell-penetrating peptides (CPPs), or protein transduction domains (PTDs), are a special class of membrane-active proteins that can cross the cell membrane with unusual efficiency. They have attracted considerable attention because of their ability to readily cross biological membranes, in spite of their highly charged nature. While the exact mechanism of this transport remains under intense investigation, energy-independent pathways are known. Perhaps the clearest example is the ability of CPPs, and their synthetic mimics, to cross model phospholipid bilayer vesicle membranes. One suggested mechanism implies that, in fact, CPPs like polyarginine (pR) need assistance to cross the membrane. It suggests that hydrophobic counterions complex around the guanidinium-rich backbone, thus “coating” the highly cationic structure with lipophilic moieties. This process has been termed “activation”, in which the lipophilic anion acts as an activator. In a series of detailed studies it was shown that aromatic activators outperform aliphatic ones. For example, sodium 4-(pyren-1-yl)butane-1-sulfonate gave an EC50 (effective concentration to obtain 50% activity) of 6.7 mm whereas the value for sodium dodecane-1sulfonate was 16 mm. Among other activators studied, the larger aromatic counterion, coronene, was not better than pyrene; however, a fullerene analogue was surprisingly effective. While this work beautifully demonstrated the role of various counterions for pR activation, it was not clear if this better activation was due to general hydrophobicity or to the aromatic nature of these activators. There is good reason to think that aromatic functional groups may play a special role, beyond their general hydrophobicity. It is well recognized that membrane proteins are enriched in aromatic amino acids at the membrane surface. Their central hydrophobic core, composed mostly of aliphatic residues, is flanked on both sides by “aromatic belts”. Although this belt is predominantly composed of tryptophan and tyrosine, as opposed to phenylalanine, it was shown that aromatic residues, including N-methylindole, have favorable free energies of insertion into the bilayer interface. This rules out a dominant effect of hydrogen bonding. It was suggested that the flat-rigid shape, p-electronic structure, and associated quadrupolar moments provide unique and highly favorable interactions with the bilayer interface. Specific interactions that have been proposed include p-cation, electrostatic, dipole–dipole, and entropic factors related to bilayer perturbation. Even HIV-TAT, the original protein that initiated the field of small PTDs, requires tryptophan (Trp11) for translocation. [10] Moreover, an oligoarginine consisting of seven arginine residues with a C-terminal tryptophan (R7W) and a TAT48–60 peptide with residue 59 substituted with a tryptophan (TAT48–60P59W) exhibit cellular internalization through energy-independent pathways. Another classical CPP, penetratin (Pen), contains two tryptophan residues. Substitution of tryptophan by phenylalanine (Pen2W2F) did not significantly impact cell uptake. Among the aromatic amino acids, phenylalanine has the unique ability to partition at the interface and in the membrane core. In fact, aromatic residues, especially phenylalanine, are most effective at anchoring proteins in the membrane due to their “special ability” to form and stabilize essential interactions with the polar elements of the bilayer. As a result, aromatic functionality could be a critical element facilitating the interactions between CPPs and the bilayer during transduction. In the past few years, we and others have reported polymers designed to mimic the transduction activity of PTDs. More recently, we demonstrated that these protein transduction domain mimics (PTDMs) have “self-activation” properties when hydrophobic alkyl side chains were built into the copolymers. Here, a new series of PTDMs was designed to determine if an aromatic functionality provides better transduction efficiency than aliphatic ones, at the same relative hydrophobicity. Given the importance of aromatic amino acids in membrane proteins and their interactions with the bilayer, it was proposed that aromatic side chains would make better activators, given equal relative hydrophobicity. Although aromatic groups have been studied in peptidebased CPPs, demonstration of the importance of aromatic functionality in these synthetic analogues is critical to establishing them as appropriate mimics, or PTDMs. By using reversed-phase HPLC to determine side-chain hydrophobicity and EC50 values in a classic transduction experiment, it is demonstrated here that it was possible to differentiate between side-chain hydrophobicity and aromaticity. As shown in Table 1, a series of new PTDM polymers was prepared by ring-opening metathesis polymerization (see the Supporting Information for detailed synthesis and characterization of monomers and polymers). Reversed-phase HPLC, commonly used to evaluate relative hydrophobicity, was [*] Dr. A. Som, A. Reuter, Prof. G. N. Tew Polymer Science & Engineering Department University of Massachusetts 120 Governors Drive, Amherst, MA 01003 (USA) E-mail: tew@mail.pse.umass.edu Homepage: http://www.pse.umass.edu/gtew/index.html