Session V55: Soft Materials Containing Synthetic Polymers, Peptides, Proteins, Biomachinery and Beyond II: Nucleic Acids and Solutions

Lessons from In-Silico Experiments

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Quantifying Cargo Partitioning and Exchange in Block Copolymer Micelles

Block-copolymer micelles (BCMs) can carry molecular cargo in a nanoscopic package that is tunable using polymer structure in combination with cargo properties. For example, BCMs are used in targeted drug delivery due to their biocompatibility, in vivo degradability and prolonged circulation time. We are using NMR spectroscopy and diffusometry combined with SANS to investigate diblock poly (ethylene oxide)-b-(caprolactone) (PEO-PCL) and commercial triblock (PEO-PPO-PPO, aka Pluronic) micelle systems. We quantify populations and diffusion coefficients of coexisting micelles and free unimers over a range of temp. and solvent composition by NMR diffusometry, including mapping of micelle-unimer phase diagrams. Furthermore, we will present NMR measurements that quantify cargo (e.g., drug) partitioning and exchange rates in BCMs as a function of cargo hydrophobicity and size, and we observe that more hydrophobic cargos partition more strongly into micelles. We also observe with SANS that cargo loading and type differentially affects micelle structure. In summary, our NMR and SANS studies shed light on how intermolecular interactions fundamentally affect cargo partitioning and release, and micelle structure and dynamics.   

Chitosan as a Yield Stress Fluid: Concentration Dependent Rheology and Microdynamics

Chitosan and its derivatives are of interest due to their versatility and potential both as a biomaterial and additive for rheological control. For chitosan concentrations ranging from the dilute to concentrated regime, we report two regions of shear thinning at high and low shear rates. The shear thinning at low shear rates, although reported for chitosan derivatives, has not been investigated in aqueous chitosan and is consistent with the existence of an apparent yield-stress. We probe the physicochemical and microdynamical origins of this behavior by the addition of urea – a hydrogen bond and hydrophobic interaction disrupter – and by dynamic light scattering (DLS). Our findings suggest that urea is not able to fully disrupt chitosan associations and only marginally lowers the yield stress parameter and viscosity. The microstructure of highly concentrated chitosan solutions, as revealed by DLS, is dominated by extremely slow microdynamics, suggesting a network of large, associating aggregates. At the highest concentration analyzed by DLS, the dynamics are consistent with weak gel behavior.   

Synthesis Of Lysine Mimicking Antibacterial Polymers

Antibiotic resistance in bacteria is a major health care problem. Antimicrobial Peptides (amps) are efficient in killing most microbes and yet development of resistance to amps is rare due to their unique mode of action. Although amps show promising antimicrobial activities, their transition from bench to bedside has been hindered as in-vitro extraction, purification and synthesis in large scale is complicated and expensive. To overcome these issues, polymeric AMP analogs can be synthesized retaining the components necessary for antimicrobial efficacy. Herein we have developed methacrylamide based polymers with amps mimicking features possessing high antimicrobial activities with low cytotoxicity. Antibacterial assay, confocal and scanning electron microscopy studies showed synthesized polymers are effective against both gram positive and gram negative bacteria. These polymers are found to be lethal to bacteria being non cytotoxic to mammalian cells thus enhancing their potential as antibiotics.   

Transition Between Collective Mechanical Instabilities in 3D Printed Microtissues

Collective cell migration and multicellular forces in monolayers have been studied extensively, but collective behaviors in 3D systems are relatively unexplored due to the challenges of creating well defined 3D structures. Simple 3D systems for studying collective cell behavior include spheroids and cells dispersed in extracellular matrix. Spherical aggregates act like fluid drops, exhibiting an effective surface tension created by cell-cell cohesion. Cells embedded in ECM contract their surroundings forming a stressed structure, which likely comes to a balance between cell tension and ECM compression. To systematically study collective mechanical behavior, we 3D print microbeams of collagen and glioblastoma cells at different collagen concentrations. These cell/ECM microbeams are printed directly into a jammed microgels of varying material properties. In microgels with large elastic moduli, the microtissues are immobile. As we lower the modulus of the microgel, the microtissues become unstable and break into drop-like shapes. When the modulus is lowered further, the microbeams undulate with a wavelength predicted by Euler-Bernoulli beam theory. Measurements of these phenomena allow us to predict stress of cells as a function of material properties of their microenvironment.   

Stabilization of helical polymer phases with different bending and torsional constraints

We performed extensive parallel-tempering Monte Carlo simulations for elastic polymers, employing a generic coarse-grained bead-spring model. We systematically investigate the structural transitions into all possible helical phases by suitable order parameters, temperature, bending and torsional barriers. The dominant conformations contain disordered helical arrangements, single helices and ordered tertiary helix bundles. We also studied how different bending constraints and torsional strengths affect the stability of the structural phases. The results can be accumulated in suitable hyperphase diagrams. This aids in understanding why the restricted flexibility of biological polymers such as double-stranded DNA tends to form helical structures and proteins often exhibit helical structures.
  

Helix Formation by Poly(amino acid)s in Neat Ionic Liquids and Their Mixtures with Buffer

Many polypeptides and proteins dissolve in neat ionic liquids (ILs) or aqueous buffers containing a significant level of IL as added electrolyte. Conformation and activity are difficult to assess when the IL content is high, but numerous experiments suggest that native secondary structures are stabilized while native tertiary structures are not. In the mixed IL/buffer solvents examined, insolubility was always noted across a window of intermediate IL content. To understand structure and solubility in the presence of ILs more systematically, two helix-forming poly(amino acids)s, poly(l-lysine) and poly(l-glutamic acid), were adopted as model systems to probe structure and solubility as IL content and temperature varied; the IL was tributylehtylphosphonium diethylphosphate, which at high concentration, dissolves both poly(amino acid)s as α-helix. The corresponding mixed solvent solubility/conformation maps, determined by circular dichroism and FTIR, show regions of solubility and order (α-helix, β-sheet) positioned differently than for an aqueous buffer containing added simple electrolyte (i.e., KCl).
  

The Role of Repulsion in Colloidal Crystal Engineering with DNA

Hybridization interactions between DNA-functionalized nanoparticles (DNA-NPs) can be used to program the crystallization behavior of superlattices, yielding access to complex three-dimensional structures. Yet the role of repulsive interactions in guiding structure formation is still largely unexplored. Here, a comprehensive approach is taken to study the role of the DNA shell, enabling the calculation of interparticle interaction potentials based on experimental results. In this work, we used two different means to assemble DNA-NPs—Watson-Crick base-pairing interactions and depletion interactions—and systematically varied the salt concentration to study the effective interactions in DNA-NP superlattices. A comparison between the two systems allows us to decouple the repulsive forces from the attractive hybridization interactions that are sensitive to the ionic environment. To better understand such behavior, we propose a model that provides a mathematical description for repulsive interaction caused by the polyelectrolyte shell.   

DNA Droplets: Design, Characterization, and Manipulation

We describe a single-component coacervate droplet comprised entirely of DNA. These DNA droplets are based on a multi-arm geometry termed a DNA nanostar. In our system, every nanostar exhibits four arms that each terminates in a single-stranded DNA overhang (sticky end) with a palindromic sequence. The sticky ends enable transient base-pairing interactions between nanostars, leading to a liquid-liquid phase separation in low-temperature, high-salt conditions. We observe that the viscosity and surface tension of DNA droplets can be controlled by altering the ionic strength of the solution as well as the sequence of the overhangs. We further show that mixtures of two types of nanostars, with different, non-interacting sequences, form two immiscible liquid phases. However, when there is partial complementarity between the two sticky ends, the two types of droplets can adhere, forming low curvature interfaces. We present correlations between the interfacial angles and hybridization energy for various pairs of overhang sequences.   

Salt Dependent Characteristics of DNA droplets

We investigate the salt-dependent behavior of self-assembled, liquid droplets of DNA. The droplets are composed of four-armed DNA nanostars. Each arm terminates in a palindromic sticky end that allows the nanostars to bind to each other, creating self-assembled networks. Inserting a single stranded gap before each sticky end increases the flexibility of the nanostar-nanostar connections. This flexibility makes network rearrangement more accessible and causes the DNA network to condense into liquid droplets. We examine the sedimentation of liquid droplets and compare it to droplet size to determine DNA volume fraction within the droplets. The volume fraction of DNA within the droplets increases with salt concentration, consistent with a screening effect. We discuss models of this behavior, and compare our results to other biomolecular coacervate systems.