Polymer Chain Length Effects on Fibroblast Attachment on Nylon-3-Modified Surfaces

Nylon-3 polymers have a polyamide backbone reminiscent of that found in proteins (β- vs α-amino acid residues, respectively), which makes these materials interesting for biological applications. Because of the versatility of the ring-opening polymerization process and the variety of β-lactam starting materials available, the structure of nylon-3 copolymers is highly amenable to alteration. A previous study showed that relatively subtle changes in the structure or ratio of hydrophobic and cationic subunits that comprise these polymers can result in significant changes in the ability of nylon-3-bearing surfaces to support cell adhesion and spreading. In the present study, we have exploited the highly tailorable nature of these polymers to synthesize new versions possessing a wide range of chain lengths, with the intent of optimizing these materials for use as cell-supportive substrates. We find that longer nylon-3 chains lead to better fibroblast attachment on modified surfaces and that at the optimal chain lengths less hydrophobic subunits are superior. The best polymers we identified are comparable to an RGD-containing peptide in supporting fibroblast attachment. The results described here will help to focus future efforts aimed at refining nylon-3 copolymer substrates for specific tissue engineering applications

Nylon‑3 Polymers That Enable Selective Culture of Endothelial Cells

Substrates that selectively encourage the growth of specific cell types are valuable for the engineering of complex tissues. Some cell-selective peptides have been identified from extracellular matrix proteins; these peptides have proven useful for biomaterials-based approaches to tissue repair or regeneration. However, there are very few examples of synthetic materials that display selectivity in supporting cell growth. We describe nylon-3 polymers that support in vitro culture of endothelial cells but do not support the culture of smooth muscle cells or fibroblasts. These materials may be promising for vascular biomaterials applications

Table1_Ginsenoside Rc ameliorated atherosclerosis via regulating gut microbiota and fecal metabolites.DOCX

Atherosclerosis (AS) and the accompanied cardiovascular diseases (CVDs) were the leading cause of death worldwide. Recently, the association between CVDs, gut microbiota, and metabolites had aroused increasing attention. In the study, we headed our investigation into the underlying mechanism of ginsenoside Rc (GRc), an active ingredient of ginsenosides used for the treatment of CVDs, in apolipoprotein E-deficient (ApoE−/−) mice with high-fat diet (HFD). Seven-week-old male ApoE−/− mice were randomly divided into four groups: the normal control (NC) group, the HFD group, the GRc group (40 mg/kg/d), and the atorvastatin (Ato) group (10 mg/kg/d). Atherosclerotic injury was evaluated by aortic lesions, serum lipid levels, and inflammatory factors. The composition of gut microbiota and fecal metabolite profile were analyzed using 16S rRNA sequence and untargeted metabolomics, respectively. The results showed that GRc significantly alleviated HFD-induced aortic lesions, reduced serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), tumor necrosis factor-α (TNF-α), and interleukin (IL)-6 and IL-1β, and increased high-density lipoprotein cholesterol (HFD-C) level, as well as the alteration of gut microbiota composition, function, and metabolite profile. GRc also reversed HFD change of Bacteroidetes and Firmicutes at the phylum level, Muribaculaceae, Lactobacillus, Ileibacterium, Bifidobacterium, Faecalibaculum, Oscillibacter, Blautia, and Eubacterium_coprostanoligenes_group at the genus level, and 23 key metabolites involved in taurine and hypotaurine metabolism, arginine biosynthesis, ATP-binding cassette (ABC) transporters, primary bile acid biosynthesis, purine metabolism, tricarboxylic acid (TCA) cycle, and glucagon signaling pathways. Additionally, eight differential intestinal floras at the genus level were associated with 23 key differential metabolites involving atherosclerotic injury. In conclusion, our results demonstrated that GRc ameliorated atherosclerotic injury, regulated microbial and metabolomic changes in HFD-induced ApoE−/− mice, and suggested a potential correlation among gut microbiota, metabolites, and atherosclerotic injury regarding the mechanisms of GRc against AS.</p

Single-Cell, Time-Resolved Antimicrobial Effects of a Highly Cationic, Random Nylon‑3 Copolymer on Live <i>Escherichia coli</i>

Synthetic random copolymers based on the nylon-3 (β-peptide) backbone show promise as inexpensive antimicrobial agents resistant to proteolysis. We present a time-resolved observational study of the attack of a particular copolymer <b>MM</b>_<b>63</b><b>:CHx</b>_<b>37</b> on single, live <i>Escherichia coli</i> cells. The composition and chain length of <b>MM</b>_<b>63</b><b>:CHx</b>_<b>37</b> (63% cationic subunits, 37% hydrophobic subunits, 35-subunit average length) were optimized to enhance antibacterial activity while minimizing lysis of human red blood cells. For <i>E. coli</i> cells that export GFP to the periplasm, we obtain alternating phase-contrast and green fluorescence images with a time resolution of 12 s over 60 min following initiation of copolymer flow. Within seconds, cells shrink and exhibit the same plasmolysis spaces that occur following abrupt external osmotic upshift. The osmoprotection machinery attempts to replenish cytoplasmic water, but recovery is interrupted by permeabilization of the cytoplasmic membrane (CM) to GFP. Evidently, the highly cationic copolymer and its counterions rapidly translocate across the outer membrane without permeabilizing it to GFP. The CM permeabilization event is spatially localized. Cells whose CM has been permeabilized never recover growth. The minimum inhibitory concentration (MIC) for cells lacking the osmolyte importer ProP is 4-fold smaller than for normal cells, suggesting that osmoprotection is an important survival strategy. In addition, at the time of CM permeabilization, we observe evidence of oxidative stress. The MIC under anaerobic conditions is at least 8-fold larger than under aerobic conditions, further implicating oxidative damage as an important bacteriostatic effect. Once the copolymer reaches the periplasm, multiple growth-halting mechanisms proceed in parallel

Minimum inhibitory concentrations (MICs) of three nylon-3 polymers against <i>E. coli</i> for different media.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104500#pone-0104500-g001" target="_blank">Fig. 1</a> for structures of <b>A</b>, <b>B</b>, and <b>C</b>. Vertical arrows mark bars that are lower limits only. EZRDM, LB, and BHI as described in main text. The designation “EZ + trypt” refers to EZRDM supplemented with 10 g/L of dialyzed tryptone powder (1X tryptone). “EZ + trypt – PA” refers to EZRDM supplemented with anion-exchanged tryptone at the equivalent of 10 g/L. “EZ + FQS…” refers to EZRDM supplemented with 400 µM of the single anionic peptide FQSEEQQTEDELQDK (net –5 charge).</p

Medium Effects on Minimum Inhibitory Concentrations of Nylon-3 Polymers against <i>E. coli</i>

<div><p>Minimum inhibitory concentrations (MICs) against <i>E. coli</i> were measured for three nylon-3 polymers using Luria-Bertani broth (LB), brain-heart infusion broth (BHI), and a chemically defined complete medium (EZRDM). The polymers differ in the ratio of hydrophobic to cationic subunits. The cationic homopolymer is inert against <i>E. coli</i> in BHI and LB, but becomes highly potent in EZRDM. A mixed hydrophobic/cationic polymer with a hydrophobic <i>t</i>-butylbenzoyl group at its N-terminus is effective in BHI, but becomes more effective in EZRDM. Supplementation of EZRDM with the tryptic digest of casein (often found in LB) recapitulates the LB and BHI behavior. Additional evidence suggests that polyanionic peptides present in LB and BHI may form electrostatic complexes with cationic polymers, decreasing activity by diminishing binding to the anionic lipopolysaccharide layer of <i>E. coli</i>. In contrast, two natural antimicrobial peptides show no medium effects. Thus, the use of a chemically defined medium helps to reveal factors that influence antimicrobial potency of cationic polymers and functional differences between these polymers and evolved antimicrobial peptides.</p></div

Structures of the random, heterochiral nylon-3 polymers used in this study.

<p><b>A</b> bears a hydrophobic p-<i>t</i>-butylbenzoyl group at the N-terminus. <b>B</b> shares the 37∶63 CH:MM ratio of <b>A</b>, but lacks a hydrophobic group at the N-terminus. <b>C</b> is a homopolymer of the cationic MM subunit.</p

Synthetic Polymers Active against <i>Clostridium difficile</i> Vegetative Cell Growth and Spore Outgrowth

Nylon-3 polymers (poly-β-peptides) have been investigated as synthetic mimics of host-defense peptides in recent years. These polymers are attractive because they are much easier to synthesize than are the peptides themselves, and the polymers resist proteolysis. Here we describe <i>in vitro</i> analysis of selected nylon-3 copolymers against <i>Clostridium difficile</i>, an important nosocomial pathogen that causes highly infectious diarrheal disease. The best polymers match the human host-defense peptide LL-37 in blocking vegetative cell growth and inhibiting spore outgrowth. The polymers and LL-37 were effective against both the epidemic 027 ribotype and the 012 ribotype. In contrast, neither vanco­mycin nor nisin inhibited outgrowth for the 012 ribotype. The best polymer was less hemolytic than LL-37. Overall, these findings suggest that nylon-3 copolymers may be useful for combatting <i>C. difficle</i>

Interplay among Subunit Identity, Subunit Proportion, Chain Length, and Stereochemistry in the Activity Profile of Sequence-Random Peptide Mixtures

Fmoc-based solid-phase synthesis methodology was used to prepare peptide mixtures containing one type of hydrophobic residue and one type of cationic residue. Each mixture was random in terms of sequence but highly controlled in terms of length. Analysis of the antibacterial and hemolytic properties of these mixtures revealed that selective antibacterial activity can be achieved with heterochiral binary mixtures but not homochiral binary mixture, if the proper amino acid residues are used

Nylon‑3 Polymers Active against Drug-Resistant Candida albicans Biofilms

Candida albicans is the most common fungal pathogen in humans, and most diseases produced by C. albicans are associated with biofilms. We previously developed nylon-3 polymers with potent activity against planktonic C. albicans and excellent C. albicans versus mammalian cell selectivity. Here we show that these nylon-3 polymers have strong and selective activity against drug-resistant C. albicans in biofilms, as manifested by inhibition of biofilm formation and by killing of C. albicans in mature biofilms. The best nylon-3 polymer (poly-<b>βNM</b>) is superior to the antifungal drug fluconazole for all three strains examined. This polymer is slightly less effective than amphotericin B (AmpB) for two strains, but the polymer is superior against an AmpB-resistant strain