We have synthesized a series of copolymers containing both positively charged (amine, guanidine) and hydrophobic side chains (amphiphilic antimicrobial peptide mimics). To investigate the structure-activity relationships of these polymers, low polydispersity polymethacrylates of varying but uniform molecular weight and composition were synthesized, using a reversible addition-fragmentation chain transfer (RAFT) approach. In a facile second reaction, pendant amine groups were converted to guanidines, allowing for direct comparison of cation structure on activity and toxicity. The guanidine copolymers were much more active against Staphylococcus epidermidis and Candida albicans compared to the amine analogues. Activity against Staphylococcus epidermidis in the presence of fetal bovine serum was only maintained for guanidine copolymers. Selectivity for bacterial over mammalian cells was assessed using hemolytic and hemagglutination toxicity assays. Guanidine copolymers of low to moderate molecular weight and hydrophobicity had high antimicrobial activity with low toxicity. Optimum properties appear to be a balance between charge density, hydrophobic character, and polymer chain length. In conclusion, a suite of guanidine copolymers has been identified that represent a new class of antimicrobial polymers with high potency and low toxicity.
The influence of mechanical stiffness of biomaterials on bacterial adhesion is only sparsely studied and the mechanism behind this influence remains unclear. Here, bacterial adhesion on polydimethylsiloxane (PDMS) samples, having four different degrees of stiffness with Young's modulus ranging from 0.06 to 4.52 MPa, is investigated. Escherichia coli and Pseudomonas aeruginosa are found to adhere in greater numbers on soft PDMS (7‐ and 27‐fold increase, respectively) than on stiff PDMS, whereas Staphylococcus aureus adheres in similar numbers on the four tested surfaces. To determine whether the observed adhesion behavior is caused by bacteria‐specific mechanisms, abiotic polystyrene (PS) beads are employed as bacteria substitutes. Carboxylate‐modified PS (PS‐COOH) beads exhibit the same adhesion pattern as E. coli and P. aeruginosa with four times more adhered beads on soft PDMS than on stiff PDMS. In contrast, amine‐modified PS (PS‐NH2) beads adhere in similar numbers on all tested samples, reminiscent of S. aureus adhesion. This work demonstrates for the first time that the intrinsic physicochemical properties associated with PDMS substrates of different stiffness strongly influence bacterial adhesion and challenge the previously reported theory on active bacterial mechanosensing, which provides new insights into the design of antifouling surfaces.
Cardiac tissue engineering approaches can deliver large numbers of cells to the damaged myocardium and have thus increasingly been considered as a possible curative treatment to counteract the high prevalence of progressive heart failure after myocardial infarction (MI). Optimal scaffold architecture and mechanical and chemical properties, as well as immune-and bio-compatibility, need to be addressed. We demonstrated that radio-frequency plasma surface functionalized electrospun poly (e-caprolactone) (PCL) fibres provide a suitable matrix for bone-marrow-derived mesenchymal stem cell (MSC) cardiac implantation. Using a rat model of chronic MI, we showed that MSC-seeded plasma-coated PCL grafts stabilized cardiac function and attenuated dilatation. Significant relative decreases of 13% of the ejection fraction (EF) and 15% of the fractional shortening (FS) were observed in sham treated animals; respective decreases of 20% and 25% were measured 4 weeks after acellular patch implantation, whereas a steadied function was observed 4 weeks after MSC-patch implantation (relative decreases of 6% for both EF and FS).
Background— Everolimus-eluting bioresorbable vascular scaffolds have been developed to improve late outcomes after coronary interventions. However, recent registries raised concerns regarding an increased incidence of scaffold thrombosis (ScT). The mechanism of ScT remains unknown. Methods and Results— The present study investigated angiographic and optical coherence tomography findings in patients experiencing ScT. Fifteen ScT (14 patients, 79% male, age 59±10 years) occurred at a median of 16 days (25%–75% interquartile range: 1–263 days) after implantation. Early ScT (<30 days) occurred in 8 cases (53%). Possible causal factors in these patients included insufficient platelet inhibition in 2 cases and procedural factors (scaffold underexpansion, undersizing, or geographical miss) in 4 cases. No obvious cause could be found in 2 early ScT. In late (>1 month) and very late (>1 year) ScT (respectively, 5 and 2 cases), 5 scaffolds showed intimal neovessels or marked peristrut low-intensity areas. Scaffold fractures were additionally found in 2 patients, and scaffold collapse was found in 1 patient with very late ScT. Extensive strut malapposition was the presumed cause for ScT in 1 case. One scaffold did not show any morphological abnormality. Thrombectomy specimens were analyzed in 3 patients and did not demonstrate increased numbers of inflammatory cells. Conclusions— The mechanisms of early ScT seem to be similar to metallic stents (mechanical and inadequate antiplatelet therapy). The predominant finding in late and very late ScT is peristrut low-intensity area.
Biofouling on silicone implants causes serious complications such as fibrotic encapsulation, bacterial infection, and implant failure. Here we report the development of antifouling, antibacterial silicones through covalent grafting with a cell-membrane-inspired zwitterionic gel layer composed of 2-methacryolyl phosphorylcholine (MPC). To investigate how substrate properties influence cell adhesion, we cultured human-blood-derived macrophages and Escherichia coli on poly(dimethylsiloxane) (PDMS) and MPC gel surfaces with a range of 0.5-50 kPa in stiffness. Cells attach to glass, tissue culture polystyrene, and PDMS surfaces, but they fail to form stable adhesions on MPC gel surfaces due to their superhydrophilicity and resistance to biofouling. Cytokine secretion assays confirm that MPC gels have a much lower potential to trigger proinflammatory macrophage activation than PDMS. Finally, modification of the PDMS surface with a long-term stable hydrogel layer was achieved by the surface-initiated atom-transfer radical polymerization (SI-ATRP) of MPC and confirmed by the decrease in contact angle from 110 to 20° and the >70% decrease in the attachment of macrophages and bacteria. This study provides new insights into the design of antifouling and antibacterial interfaces to improve the long-term biocompatibility of medical implants.
Pseudomonas aeruginosa MPAO1 is the parental strain of the widely utilized transposon mutant collection for this important clinical pathogen. Here, we validate a model system to identify genes involved in biofilm growth and biofilm-associated antibiotic resistance. Our model employs a genomics-driven workflow to assemble the complete MPAO1 genome, identify unique and conserved genes by comparative genomics with the PAO1 reference strain and genes missed within existing assemblies by proteogenomics. Among over 200 unique MPAO1 genes, we identified six general essential genes that were overlooked when mapping public Tn-seq data sets against PAO1, including an antitoxin. Genomic data were integrated with phenotypic data from an experimental workflow using a user-friendly, soft lithography-based microfluidic flow chamber for biofilm growth and a screen with the Tn-mutant library in microtiter plates. The screen identified hitherto unknown genes involved in biofilm growth and antibiotic resistance. Experiments conducted with the flow chamber across three laboratories delivered reproducible data on P. aeruginosa biofilms and validated the function of both known genes and genes identified in the Tn-mutant screens. Differential protein abundance data from planktonic cells versus biofilm confirmed the upregulation of candidates known to affect biofilm formation, of structural and secreted proteins of type VI secretion systems, and provided proteogenomic evidence for some missed MPAO1 genes. This integrated, broadly applicable model promises to improve the mechanistic understanding of biofilm formation, antimicrobial tolerance, and resistance evolution in biofilms.
Photodynamic therapy (PDT), which is based on the activation of photosensitizers with light, can be used to reduce plaque burden. We hypothesized that intra-arterial photosensitizer administration and photo-activation will lead to high and rapid accumulation within the plaque with reduced systemic adverse effects. Thus, this “intra-arterial” PDT would be expected to have less side effects and due to the short time involved would be compatible with percutaneous coronary interventions.Aim: We characterized the dose-dependent uptake and efficacy of intra-arterial PDT using Liposomal Verteporfin (Visudyne®), efficient for cancer-PDT but not tested before for PDT of atherosclerosis.Methods and Results: Visudyne® (100, 200, and 500 ng/ml) was perfused for 5–30 min in atherosclerotic aorta isolated from ApoE−/− mice. The fluorescence Intensity (FI) after 15 min of Visudyne® perfusion increased with doses of 100 (FI-5.5 ± 1.8), 200 (FI-31.9 ± 1.9) or 500 ng/ml (FI-42.9 ± 1.2). Visudyne® (500 ng/ml) uptake also increased with the administration time from 5 min (FI-9.8 ± 2.5) to 10 min (FI-23.3 ± 3.0) and 15 min (FI-42.9 ± 3.4) before reaching saturation at 30 min (FI-39.3 ± 2.4) contact. Intra-arterial PDT (Fluence: 100 and 200 J/cm2, irradiance-334 mW/cm2) was applied immediately after Visudyne® perfusion (500 ng/ml for 15 min) using a cylindrical light diffuser coupled to a diode laser (690 nm). PDT led to an increase of ROS (Dihydroethidium; FI-6.9 ± 1.8, 25.3 ± 5.5, 43.4 ± 13.9) and apoptotic cells (TUNEL; 2.5 ± 1.6, 41.3 ± 15.3, 58.9 ± 6%), mainly plaque macrophages (immunostaining; 0.3 ± 0.2, 37.6 ± 6.4, 45.3 ± 5.4%) respectively without laser irradiation, or at 100 and 200 J/cm2. Limited apoptosis was observed in the medial wall (0.5 ± 0.2, 8.5 ± 4.7, 15.3 ± 12.7%). Finally, Visudyne®-PDT was found to be associated with reduced vessel functionality (Myogram).Conclusion: We demonstrated that sufficient accumulation of Visudyne® within plaque could be achieved in short-time and therefore validated the feasibility of local intravascular administration of photosensitizer. Intra-arterial Visudyne®-PDT preferentially affected plaque macrophages and may therefore alter the dynamic progression of plaque development.
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