Sonochemically-Produced Metal-Containing Polydopamine Nanoparticles and Their Antibacterial and Antibiofilm Activity

A facile one-pot sonochemical synthesis of Cu-, Ag-, and hybrid Cu/Ag-based polydopamine nanoparticles (Cu-, Ag-, and Cu/Ag-PDA-NPs) and the mechanisms by which they exert antibacterial and antibiofilm activities are reported. We showed that the nanoparticles are spherical with a core–shell structure. Whereas Cu is chelated to the shell of Cu-PDA-NPs in oxidation states of +1/+2, the core of Ag-PDA-NPs is filled with elemental Ag°. Sonochemical irradiation of dopamine in the presence of both Cu²⁺ and Ag⁺ generates hybrid Cu/Ag-PDA-NPs, whose shells are composed of Cu-chelated PDA with Ag° in the core. The redox potential of the metals was found to be the main determinant of the location and oxidation state of the metals. Leaching studies under physiological conditions reveal a relatively fast release of Cu ions from the shell, whereas Ag leaches very slowly from the core. The metal-containing PDA-NPs are highly microbicidal and exhibit potent antibiofilm activity. The combination of both metals in Cu/Ag-PDA-NPs is especially effective against bacteria and robust biofilms, owing to the dual bactericidal mechanisms of the metals. Most importantly, both Ag- and Cu/Ag-PDA-NPs proved to be significantly more antibacterial than commercial Ag-NPs while exhibiting lower toxicity toward NIH 3T3 mouse embryonic fibroblasts. Mechanistically, the metal-containing PDA-NPs generate stable PDA-semiquinone and reactive oxygen species under physiological conditions, which contribute at least partly to the antimicrobial activity. We also demonstrated that simple treatment of surfaces with Ag-PDA-NPs converts them to antibacterial, the activity of which was preserved even after prolonged storage under ambient conditions

Effective Targeting of Aβ to Macrophages by Sonochemically Prepared Surface-Modified Protein Microspheres

Imbalanced homeostasis and oligomerization of the amyloid-β (Aβ) peptide in the brain are hallmarks of Alzheimer’s disease (AD). Microglia and macrophages play a critical role in the etiology of AD either by clearing Aβ from the brain or inducing inflammation. Recent evidence suggests that clearance of Aβ by microglia/macrophages via the phagocytic pathway is defective in AD, which can contribute to the accumulation of Aβ in the brain. We have recently demonstrated that protein microspheres modified at their surface with multiple copies of an Aβ-recognition motif can strongly bind Aβ, inhibit its aggregation, and directly reduce its toxicity by sequestering it from the medium. Here, we describe how microsphere-bound Aβ can stimulate microglial cells and be phagocytosed through a mechanism that is distinct from that of Aβ removal and, thus, contribute to the clearance of Aβ, even by defective microglial cells. The phagocytosis was most effective, with microspheres having a diameter of <1 μm. The introduction of polyethylene glycol to the surface of the microspheres changed the kinetics of the phagocytosis. Moreover, while aggregated Aβ induced a significant inflammatory response that was manifested by the release of TNF-α, the microsphere-bound Aβ dramatically reduced the amount of cytokine released from microglial cells

Synthetic double labeled oligo-peptides.

<p>Two oligopeptides were prepared in order to demonstrate the resolution of two sub-populations characterized by two different intramolecular diffusion coefficients in a mixed ensemble by trFRET measurements. (A) oligo-proline based rigid peptide (DA₁). (B) oligo(Ser-Gly) based flexible peptide (DA₂). Both peptides were labeled with naphthyl-alanine and dansyl-alanine.</p

End-to-end distance distributions obtained for the mixtures of the model peptides by the joint global analysis.

<p>Results of joint analysis of trFRET data given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143732#pone.0143732.t003" target="_blank">Table 3</a>. Top panel: sum of 2 sub-populations with different rigid:flexible mixture ratios: brown 4:1, light blue 3:2, green 2:3, magenta 1:4. Bottom panel: Separate measurement of single population distance distributions of the flexible peptide (red) and the rigid peptide (black). The recovered single population parameters from the top panel were equal to those obtained by the separate measurement of each one of the peptides as shown at the bottom panel.</p

Limitations of an analysis based on single composition of the mixture of peptides.

<p>Uncertainty ranges of the two diffusion coefficients obtained by rigorous analysis of two simulated trFRET datasets. (A) The range of the value of the diffusion coefficient of the rigid peptide obtained at analysis of experiments simulated for different mole fractions of that peptide (the compositions are shown in the inset). The input parameters used for the simulations are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143732#pone.0143732.t001" target="_blank">Table 1</a>. Each trace represents the extreme values of the diffusion coefficient obtained for the indicated combinations of the molar ratios of the two sub-populations ((A) and (B) The same procedure was applied in search for the uncertainty range of the values of the diffusion coefficient for the second sub-population (D = 20Ų/ns). Greater reduction of the uncertainty of the two determined diffusion coefficients was obtained when experiments with low and high molar fractions of the rigid peptide 0.1 & 0.9 (red) were used. The horizontal dashed line represents 1 SD confidence level.</p

Fluorescence decay curves obtained for the two model peptides included in the global analysis.

<p>Green trace <b>(DO):</b> The donor emission decay without acceptor; the same traces were obtained for the flexible and the rigid peptide. Blue trace (<b>AO)</b> the acceptor emission decay in the absence of the donor; the same traces were obtained for the two <b>AO</b> model peptides. Purple trace (<b>DA</b>_<b>1</b><b>)</b> the time resolved donor emission in the flexible peptide in the presence of acceptor. Red trace <b>(DA</b>_<b>2</b><b>)</b> the time resolved donor emission in the rigid peptide in the presence of acceptor. Light brown trace <b>(DAA</b>_<b>1</b><b>)</b> the acceptor emission in the flexible peptide in the presence of a donor under excitation at the wavelength of the donor absorption. Orange. Trace <b>(DAA</b>_<b>2</b><b>)</b> the acceptor emission in the rigid peptide in the presence of a donor excited at the donor absorption wavelength. The black traces are the calculated theoretical curves of the best fit. Upper right inset: The right hand box: The autocorrelation of the residuals between each one of the above experimental emission decay curve and the corresponding best fit theoretical emission decay curves (black traces) obtained by the global analysis.</p

Simulated combined distance distribution of two peptides and expected trFRET data.

<p>A mixture of two peptides at a molar fraction of 0.5 each was simulated using the parameters given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143732#pone.0143732.t002" target="_blank">Table 2</a> were used as input. (A) Plot of the combined end to end distance distribution expected for the ensemble formed by the mixture of the two peptides. The gray window marks the range of distances around the Förster critical distance where a significant FRET effect is expected ((0.5–1.5)R_o). (B) Simulated fluorescence decay curves to be used in the global analysis procedure: blue (DO), fluorescence decay of the donor in the absence of acceptor; green (AO), fluorescence decay of the acceptor in the absence of a donor; red (DA), fluorescence decay of the donor in the presence of a acceptor; and black (DAA), fluorescence decay of the acceptor in the presence of the donor and under excitation at the wavelength of the donor absorption.</p

In Vitro and Mechanistic Studies of an Antiamyloidogenic Self-Assembled Cyclic d,l‑α-Peptide Architecture

Misfolding of the Aβ protein and its subsequent aggregation into toxic oligomers are related to Alzheimer’s disease. Although peptides of various sequences can self-assemble into amyloid structures, these structures share common three-dimensional features that may promote their cross-reaction. Given the significant similarities between amyloids and the architecture of self-assembled cyclic d,l-α-peptide, we hypothesized that the latter may bind and stabilize a nontoxic form of Aβ, thereby preventing its aggregation into toxic forms. By screening a focused library of six-residue cyclic d,l-α-peptides and optimizing the activity of a lead peptide, we found one cyclic d,l-α-peptide (<b>CP-2</b>) that interacts strongly with Aβ and inhibits its aggregation. In transmission electron microscopy, optimized thioflavin T and cell survival assays, <b>CP-2</b> inhibits the formation of Aβ aggregates, entirely disassembles preformed aggregated and fibrillar Aβ, and protects rat pheochromocytoma PC12 cells from Aβ toxicity, without inducing any toxicity by itself. Using various immunoassays, circular dichroism spectroscopy, photoinduced cross-linking of unmodified proteins (PICUP) combined with SDS/PAGE, and NMR, we probed the mechanisms underlying <b>CP-2</b>’s antiamyloidogenic activity. NMR spectroscopy indicates that <b>CP-2</b> interacts with Aβ through its self-assembled conformation and induces weak secondary structure in Aβ. Upon coincubation, <b>CP-2</b> changes the aggregation pathway of Aβ and alters its oligomer distribution by stabilizing small oligomers (1–3 mers). Our results support studies suggesting that toxic early oligomeric states of Aβ may be composed of antiparallel β-peptide structures and that the interaction of Aβ with <b>CP-2</b> promotes formation of more benign parallel β-structures. Further studies will show whether these kinds of abiotic cyclic d,l-α-peptides are also beneficial as an intervention in related in vivo models

Multifunctional Cyclic d,l‑α-Peptide Architectures Stimulate Non-Insulin Dependent Glucose Uptake in Skeletal Muscle Cells and Protect Them Against Oxidative Stress

Oxidative stress directly correlates with the early onset of vascular complications and the progression of peripheral insulin resistance in diabetes. Accordingly, exogenous antioxidants augment insulin sensitivity in type 2 diabetic patients and ameliorate its clinical signs. Herein, we explored the unique structural and functional properties of the abiotic cyclic d,l-α-peptide architecture as a new scaffold for developing multifunctional agents to catalytically decompose ROS and stimulate glucose uptake. We showed that His-rich cyclic d,l-α-peptide <b>1</b> is very stable under high H₂O₂ concentrations, effectively self-assembles to peptide nanotubes, and increases the uptake of glucose by increasing the translocation of GLUT1 and GLUT4. It also penetrates cells and protects them against oxidative stress induced under hyperglycemic conditions at a much lower concentration than α-lipoic acid (ALA). In vivo studies are now required to probe the mode of action and efficacy of these abiotic cyclic d,l-α-peptides as a novel class of antihyperglycemic compounds