We thank Phil Griffin and Tera Lavoie of the Soft Matter Characterization Facility and Advanced Electron Microscopy Facility, respectively, at The University of Chicago. We also thank Xiaobing Zuo and Soenke Seifert of the Advanced Photon Source at Argonne National Laboratory and NIST Center for Hierarchical Materials Design (CHiMaD) for support. We thank Jeff Ting and Michael Lueckheide for their contributions to this work.

The authors have nothing to disclose.

As mentioned above, the protocols presented here are written with a focus on oligonucleotides as the polyanion component and pLys-PEG as the cationic-neutral block copolymer, but we have tested them with a variety of polymers, such as poly(acrylic acid), polyglutamate, and PEG-poly(vinylbenzyl trimethylammonium), and believe they will be generally applicable for most polyelectrolyte pairs. One parameter that may need to be optimized is the salt concentration used for annealing, because it should be high enough that PCMs ...

When oppositely charged polyelectrolytes are mixed in aqueous solution, entropy gain from release of their counterions causes demixing of the solution into a polymer-rich condensed phase and a polymer-depleted supernatant1,2,3,4,5, a phenomenon known as polyelectrolyte complexation. If a neutral hydrophilic block is conjugated to one or both of the polyelectrolytes, nanoscale phase separation occurs instead (Figure 1A). The resulting self-assembled core-shell nanoparticles are variously referred to as polyelectrolyte complex micelles (PCMs), polyion complex micelles, block ionomer complexes, or coacervate-core micelles by analogy to surfactant micellization, even though all components of the system are hydrophilic6,7. A PCM&#39;s ability to encapsulate hydrophilic molecules such as proteins and nucleic acids, as well as the extensive tunability offered by the block copolymer carrier architecture makes them attractive candidates for delivering therapeutic molecules in vivo8,9,10,11,12,13.
Delivering therapeutic nucleic acids to cellular targets is a particularly important challenge, and one for which PCMs offer several advantages. Therapeutic nucleic acids (genetic DNA, mRNA, and oligonucleotides such as siRNA) have immense potential for improving human health, but must overcome numerous biological and physical barriers to realize that potential14,15,16. Bare nucleic acids are degraded by serum and cellular nucleases, are quickly cleared from circulation, and their strong negative charge makes it difficult for them to penetrate cell membranes without assistance. Current approaches for overcoming these barriers include costly chemical modifications to prevent damage from nucleases and/or encapsulation into various lipid nanoparticles assembled via hydrophobic interactions15,17,18. While these methods have proven effective for local injections and liver targeting, systemic use presents significant limitations of toxicity, immunogenicity, and limited biodistribution16. By contrast, PCMs use the negative charge of nucleic acids to condense them within the phase-separated core, while the neutral corona provides a steric barrier against degradation as well as a platform for incorporating ligands to enhance targeting or internalization11,19. In vitro and animal studies have shown that PCMs can effectively deliver various nucleic acid payloads20,21,22,23,24, but weaknesses in our ability to predict PCM properties such as size, shape, and stability from the properties of the constituent polymers have hindered their wider adoption.
Recent work by our group and others in the field has begun to address this problem by developing structure-property, and in some cases structure-property-function relationships for PCMs formed from nucleic acids and various cationic-neutral polymers7,25,26,27. Two consistent themes that have emerged from these studies are the importance of developing well-controlled, repeatable protocols for PCM assembly and the benefit of using multiple techniques to characterize the resulting nanoparticles. Polyelectrolytes, particularly those with high charge density like nucleic acids, interact with each other very strongly, and appear to readily become kinetically trapped upon mixing, resulting in PCM preparations that are highly sensitive to small variations in procedure and display high polydispersity and poor repeatability from batch to batch. PCMs have also been shown to adopt a wide range of shapes and sizes depending on the atomic-level configurations of their components, and capturing this diversity with any individual characterization technique is very difficult, particularly since some common techniques such as dynamic light scattering (DLS) require assumptions about particle shape for their interpretation.
In this article, we discuss material design and selection for PCMs, with a focus on oligonucleotides and cationic-neutral diblock copolymers. We then describe a salt annealing protocol that uses high salt concentrations followed by slow dialysis to avoid kinetic trapping during PCM assembly. The polyelectrolytes are mixed in high salt conditions where electrostatic attractions are screened, then the salt concentration is slowly lowered to allow the polyelectrolytes to settle into their most energetically favorable configurations, analogous to the slow cooling process of thermal annealing. Using this protocol, we are regularly able to achieve exceptionally low polydispersity and high repeatability for oligonucleotide PCMs7,26. Finally, we describe how four separate measurement techniques can be used to characterize PCMs over a very wide range of length scales, from external morphology to internal structure: DLS, multi-angle light scattering (MALS), small angle X-ray scattering (SAXS), and transmission electron microscopy (TEM). We hope that these protocols will enable more researchers to effectively explore the capabilities of these interesting nanoparticles.
Polymer Selection and Preparation 
PCM properties are strongly influenced by the physical and chemical characteristics of the constituent polymers, making polymer selection a critical step in the design process. The most well-characterized block copolymers for nucleic acid PCMs are linear diblocks such as poly(lysine)-poly(ethylene glycol) (pLys-PEG), but PCMs can be formed between polyelectrolytes and a variety of hydrophilic neutral-charged polymers, which can be generated in a high throughput manner28. The choice of charged group strongly affects the stability of ion pairing and shape of micelles26, and PCM size has been shown to increase with the length of the charged block5,7,26 (Figure 2), thus allowing PCM properties to be tuned for the requirements of a desired application. For linear diblocks, we have found that the charged block should have at least 10 charges and be strongly charged at the desired pH. Longer charged blocks may promote PCM formation with oligonucleotides such as siRNA, which are difficult to complex with shorter blocks21. We have successfully observed PCM formation with block lengths up to 200, and the literature describes longer polymers. More flexibility is available in the choice of neutral blocks24, but experience has shown that very short neutral blocks lead to aggregation rather than nanoparticle formation, and that the minimum neutral length increases with charged block length. For pLys-PEG, a PEG MW of at least 3,000&#8211;5,000 is required for pLys lengths below ~50, and longer lengths are required as the charged block is increased further. Increased neutral block length results in increased PCM size, particularly shell thickness, due to steric crowding of the neutral polymers.
This manuscript presents a protocol for preparing PCMs from lyophilized high-purity pLys-PEG and oligonucleotides of known quantity, but should be readily adaptable to other systems as well. We have tested it successfully with several charged polypeptides, including polyarginine and polyglutamic acid, as well as several synthetic polyelectrolytes, such as polyacrylic acid and poly(vinylbenzyl trimethylammonium). We also describe preparing PCMs with a stoichiometric ratio of polyelectrolyte charges, but this is easily modified. We find it easiest to work in charge concentration units (c.c.), which also naturally accommodates polymers that are not fully charged. If either polymer is not well-characterized, care should be taken to accurately determine the polymer lengths/masses and ensure that excess salt is not present beyond that needed for charge neutralization by dialysis, for example. The presence of any retained water should also be accounted for when concentrations are calculated. Nucleic acid concentration can be conveniently quantified by absorbance at 260 nm, and the presence or absence of terminal phosphates should be considered when calculating the c.c.
When using oligonucleotides as polyanions, the hybridization state and chemical structure help determine the propensity for self-assembly and the characteristics of the resulting PCM5,7,26. Optimizing these, within the requirements for biological efficacy if using PCMs for delivery, will increase the likelihood of forming the desired structures. Helpful tools for analyzing hybridization include MATLAB functions for nucleic acids, NUPACK29, and IDT OligoAnalyzer. We recommend analyzing a candidate sequence to understand the strength of binding to 1) itself in a hairpin formation; 2) another copy of the same sequence (self-dimer); and 3) to other oligonucleotides present in the system. DNA and RNA melting temperatures for a specific sequence can also be calculated using the nearest-neighbor method30,31. Thermal annealing of nucleic acids (step 2.3) denatures any residual secondary structure in the individual nucleotides and promotes equilibrium folding.
PCM Characterization and Analysis 
A wide range of techniques are available for characterizing nanoparticles, including static and dynamic light scattering, small angle scattering of electrons or neutrons, and electron microscopy. In this article, we provide protocols for two light scattering techniques, small angle X-ray scattering, and two electron microscopy techniques.
DLS measures the autocorrelation of temporal fluctuations in scattering intensity at one angle from Brownian motion of the sample. Fitting this data can provide hydrodynamic radius and polydispersity for spherical micelles (Figure 3). Multiple angle light scattering (MALS) measures the static scattering intensity at many angles. This angular dependence describes the shape of the nanoparticle but is limited to length scales longer than ~50 nm for visible light, which limits its effectiveness for smaller nanoparticles. Both techniques are based on refractive index mismatch and primarily describe the outside dimensions of the nanoparticle.
Small angle X-ray scattering (SAXS) uses X-rays as the scattering probe, and their shorter wavelength allows measurements over a range of ~0.1&#8211;100 nm. Fitting the observed scattering intensity vs. angle (conventionally expressed as momentum transfer q) provides information on PCM morphology (i.e., size and shape) and also internal structure. If an absolute intensity calibration is available, and if the scattering intensity can be extrapolated to zero angle, PCM mass and aggregation number can also be estimated32, making SAXS an extremely versatile and valuable method. Small angle neutron scattering (SANS) is sensitive over a similar range of length scales but is only available at specialized facilities and will not be explicitly discussed in this article33,34,35.
Recent years have seen the advent of benchtop SAXS instruments, but we find that synchrotron sources are better suited for PCM characterization, as their higher intensity allows data to be collected much faster for these low-contrast samples. We provide a brief protocol for acquiring PCM SAXS data at Beamline 12-ID-B at the Advanced Photon Source (Argonne National Laboratory, USA) from a user perspective. This protocol should be applicable to most synchrotron sources, but consulting with local staff before proposing an experiment is highly recommended. We also provide a data reduction and analysis protocol using Irena36, a free set of macros written for Igor Pro. Irena includes a versatile set of form factors for modeling SAXS data and allows for construction of multicomponent models that are capable of describing the complex scattering profile of PCMs (see Representative Results, Figure 4). Irena also has comprehensive documentation and tutorials available online. Before attempting the procedures below, we recommend familiarization with these, particularly the tutorial &#34;Modeling of SAXS data with two main scatterer populations&#34;.
Radiation damage is a concern for X-ray scattering, but several measures can be employed to minimize it. In particular, we recommend using a flow cell setup with a syringe pump and PCM sample flowing during data acquisition, rather than a sealed capillary. This also greatly simplifies background subtraction. We also suggest taking multiple exposures of the flowing sample rather than one longer one in order to limit the flux that any single volume of sample sees and to allow for comparison of the exposure data to identify any damage.
In contrast to the scattering techniques, which generally require fitting to interpret, transmission electron microscopy (TEM) provides a real space visual image of the nanoparticles by passing an electron beam through the sample and projecting an image on a scintillation screen (Figure 5). We present protocols for two TEM techniques in this article. Cryo TEM freezes micelle samples into a thin layer of vitreous ice, preserving structural conformation with minimal foreign substances, optimal for micelles &#8804;~10-100 nm in radius. Negative stain TEM uses a heavy metal salt (e.g., uranium) to surround the sample after it has been dried on the surface of a grid. The dense stain will scatter more electrons than the sample, adding contrast and producing a negative image of the sample. Cryo TEM is recommended for high-quality images. However, it is more costly, time consuming, and may not provide sufficient contrast. When this is a concern, negative stained samples should be used. Examples of each are shown in Figure 5.
Each of these techniques reports on slightly different aspects of the nanoparticles, with different strengths and limitations. Light scattering is readily available, and is often the fastest approach, but has substantial limitations in size and shape resolution. SAXS can provide information over a large range of length scales at reasonably high throughput, but requires specialized equipment to acquire the data, as well as modeling to interpret it. TEM images are straightforward to interpret but can be limited in contrast and are inherently low throughput. Our experience has shown that using multiple techniques for characterization greatly increases the information that can be obtained about PCM properties and simplifies interpretation of data sets obtained from each one alone. For example, SAXS and TEM primarily examine a PCM&#39;s dense core, while light scattering reports on the overall dimensions of the nanoparticle. Thus, combining them allows measurement of both core and corona size. TEM&#39;s ability to acquire real space images can provide ground truth data to enable selection of appropriate form factors for modeling SAXS data that might otherwise be ambiguous. This article describes protocols for all four techniques, and an example process for using them to characterize an unknown sample is given in the Discussion section.

<table><tbody><tr><td>70 mm circle filter paper</td><td>Whatman</td><td>1001-070</td><td>Filter paper for wicking during grid prep</td></tr><tr><td>Carbon Film TEM grid</td><td>Electron Microscopy Sciences</td><td>CF200-Cu</td><td>TEM grid</td></tr><tr><td>DAWN</td><td>Wyatt Technology</td><td>DAWN</td><td>MALS instrument</td></tr><tr><td>DNA oligonucleotide</td><td>Integrated DNA Nanotechnologies Inc</td><td></td><td>Custom oligonucleotide</td></tr><tr><td>Lacey Carbon TEM grid</td><td>Electron Microscopy Sciences</td><td>LC200-Cu</td><td>TEM grid</td></tr><tr><td>Methoxy-poly(ethylene glycol)-block-poly(l-lysine hydrochloride) PEG5k - PLKC50</td><td>Alamanda Polymers Inc</td><td>mPEG5K-b-PLKC50</td><td>Example block copolymer</td></tr><tr><td>Milli-Q</td><td>Millipore Sigma</td><td></td><td>Ultrapure water</td></tr><tr><td>NanoDrop</td><td>Thermo Scientific</td><td></td><td>For measuring nucleic acid concentration</td></tr><tr><td>negative-action tweezers</td><td>Dumont</td><td>N7</td><td>Tweezers for grid preparation</td></tr><tr><td>Parafilm &quot;M&quot;</td><td>Bemis Company Inc</td><td>PM996</td><td>Laboratory film</td></tr><tr><td>Quantifoil Holey Carbon TEM grid</td><td>Electron Microscopy Sciences</td><td>Q210CR1.3</td><td>TEM grid</td></tr><tr><td>Research Goniometer and Laser Light Scattering System</td><td>Brookhaven Instruments</td><td>BI-200SM</td><td>DLS/MALS instrument</td></tr><tr><td>Slide-A-Lyzer G2 2K 0.5 mL</td><td>Thermo Scientific Pierce Protein Biology</td><td>87723</td><td>Dialysis cartridge</td></tr><tr><td>small volume cuvette</td><td>Brookhaven Instruments</td><td>BI-SVC</td><td>Cuvette for DLS/MALS</td></tr><tr><td>Solarus 950 Advanced Plasma System</td><td>Gatan</td><td>Solarus 950</td><td>Plasma system for TEM grids</td></tr><tr><td>Talos TEM</td><td>FEI</td><td>Talos</td><td>TEM used for cryo samples</td></tr><tr><td>Tecnai Spirit TEM</td><td>FEI</td><td>Spirit</td><td>TEM used for dry samples</td></tr><tr><td>Uranyl Formate</td><td>SPI-Chem</td><td>16984-59-1</td><td>For negative staining samples for TEM</td></tr><tr><td>Vitrobot</td><td>FEI</td><td>Vitrobot</td><td>Vitrification robot for cryo grid preparation</td></tr></tbody></table>

assembly and characterization of polyelectrolyte complex micelles

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful platform for exploring the physics of polyelectrolyte interactions and also offer a promising solution to the pressing problem of delivering therapeutic oligonucleotides in vivo. Developing predictive structure-property relationships for PCMs has proven difficult, in part due to the presence of strong kinetic traps during nanoparticle self-assembly. This article discusses criteria for choosing polymers for PCM construction and provides protocols based on salt annealing that enable assembly of repeatable, low-polydispersity nanoparticles. We also discuss PCM characterization using light scattering, small-angle X-ray scattering, and electron microscopy.

In order to illustrate the characterization methods described above, we show typical results for PCMs assembled from oligonucleotides and block copolymers of various lengths and chemistries (Figure 1). Figure 2 provides an example of how PCM core size (as determined from SAXS and TEM, Figure 4 and Figure 5) varied with charged block length. Figure 3 shows DLS data and fitti...

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Assembly and Characterization of Polyelectrolyte Complex Micelles

We provide protocols and representative data for designing, assembling, and characterizing polyelectrolyte complex micelles, core-shell nanoparticles formed by polyelectrolytes and hydrophilic charged-uncharged block copolymers.

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful ...

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10.7K Views.  The University of Chicago. We provide protocols and representative data for designing, assembling, and characterizing polyelectrolyte complex micelles, core-shell nanoparticles formed by polyelectrolytes and hydrophilic charged-uncharged block copolymers.

Video: Assembly and Characterization of Polyelectrolyte Complex Micelles

Controlling the Size, Shape and Stability of Supramolecular Polymers in Water

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do so is highly important in view of a number of applications in functional soft matter including electronics, biomedical engineering, and sensors. In the past, successful strategies to control the size and shape of supramolecular polymers typically focused on the use of templates2,3, end cappers4 or selective solvent techniques5. 
Here we disclose a strategy based on self-assembling discotic amphiphiles that leads to the control over stack length and shape of ordered, chiral columnar aggregates. By balancing electrostatic repulsive interactions on the hydrophilic rim and attractive non-covalent forces within the hydrophobic core of the polymerizing building block, we manage to create small and discrete spherical objects6,7. Increasing the salt concentration to screen the charges induces a sphere-to-rod transition. Intriguingly, this transition is expressed in an increase of cooperativity in the temperature-dependent self-assembly mechanism, and more stable aggregates are obtained. 
For our study we select a benzene-1,3,5-tricarboxamide (BTA) core connected to a hydrophilic metal chelate via a hydrophobic, fluorinated L-phenylalanine based spacer (Scheme 1). The metal chelate selected is a Gd(III)-DTPA complex that contains two overall remaining charges per complex and necessarily two counter ions. The one-dimensional growth of the aggregate is directed by &pi;-&pi; stacking and intermolecular hydrogen bonding. However, the electrostatic, repulsive forces that arise from the charges on the Gd(III)-DTPA complex start limiting the one-dimensional growth of the BTA-based discotic once a certain size is reached. At millimolar concentrations the formed aggregate has a spherical shape and a diameter of around 5 nm as inferred from 1H-NMR spectroscopy, small angle X-ray scattering, and cryogenic transmission electron microscopy (cryo-TEM). The strength of the electrostatic repulsive interactions between molecules can be reduced by increasing the salt concentration of the buffered solutions. This screening of the charges induces a transition from spherical aggregates into elongated rods with a length &gt; 25 nm. Cryo-TEM allows to visualise the changes in shape and size. In addition, CD spectroscopy permits to derive the mechanistic details of the self-assembly processes before and after the addition of salt. Importantly, the cooperativity -a key feature that dictates the physical properties of the produced supramolecular polymers- increases dramatically upon screening the electrostatic interactions. This increase in cooperativity results in a significant increase in the molecular weight of the formed supramolecular polymers in water.

The goal of this experiment is to determine and control the size, shape and stability of self-assembled discotic amphiphiles in water. For aqueous based supramolecular polymers such level of control is very difficult. We apply a strategy using both repulsive and attractive interactions. The experimental techniques applied to characterize this system are broadly applicable.

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do ...

Engineering

Using Polystyrene-block-poly(acrylic acid)-coated Metal Nanoparticles as Monomers for Their Homo- and Co-polymerization

We present a template-free method for &#8220;polymerizing&#8221; nanoparticles into long chains without side branches. A variety of nanoparticles are encapsulated in polystyrene-block-poly(acrylic acid) (PSPAA) shells and then used as monomers for their self-assembly. Spherical PSPAA micelles upon acid treatment are known to assemble into cylindrical micelles. Exploiting this tendency, the core-shell nanoparticles are induced to aggregate, coalesce, and then transform into long chains. When more than one type of nanoparticles are used, random and block &#8220;copolymers&#8221; of nanoparticles can be obtained. Detailed procedures are reported for the PSPAA encapsulation of nanoparticles, homo- and co-polymerization of the core-shell nanoparticles, separation and purification of the resulting nanoparticle chains. Transformations of single-line chains into double- and triple-line chains are also presented. The synergy between the polymer shell and the embedded nanoparticles leads to an unusual chain-growth polymerization mode, giving long nanoparticle chains that are distinct from the products of the traditional step-growth aggregation process.

Using Polystyrene-block-polyacrylic acid-coated Metal Nanoparticles as Monomers for Their Homo- and Co-polymerization

We report protocols for &#8220;polymerizing&#8221; various types of polymer-encapsulated metal nanoparticles into long chains of &#8220;homo-&#8220; and &#8220;co-polymers&#8221;.

We present a template-free method for &#8220;polymerizing&#8221; nanoparticles into long chains without side branches. A variety of nanoparticles are ...

Preparation and Characterization of Individual and Multi-drug Loaded Physically Entrapped Polymeric Micelles

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar concentration forming hydrophobic cores surrounded by hydrophilic shells in aqueous environments. The core of these micelles can be utilized to load hydrophobic, poorly water soluble drugs like docetaxel (DTX) and everolimus (EVR). Systematic characterization of the micelle structure and drug loading capabilities are important before in vitro and in vivo studies can be conducted. The goal of the protocol described herein is to provide the necessary characterization steps to achieve standardized micellar products. DTX and EVR have intrinsic solubilities of 1.9 and 9.6 &#181;g/ml respectively Preparation of these micelles can be achieved through solvent casting which increases the aqueous solubility of DTX and EVR to 1.86 and 1.85 mg/ml, respectively. Drug stability in micelles evaluated at room temperature over 48 hr indicates that 97% or more of the drugs are retained in solution. Micelle size was assessed using dynamic light scattering and indicated that the size of these micelles was below 50 nm and depended on the molecular weight of the polymer. Drug release from the micelles was assessed using dialysis under sink conditions at pH 7.4 at 37 oC over 48 hr. Curve fitting results indicate that drug release is driven by a first order process indicating that it is diffusion driven.

The goal of this protocol is to describe the preparation and characterization of physically entrapped, poorly water soluble drugs in micellar drug delivery systems composed of amphiphilic block copolymers.

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar ...

Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins with the synthesis of a hydrophilic poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) homopolymer using reversible addition-fragmentation chain-transfer (RAFT) polymerization. Under mild visible light irradiation (&#955; = 460 nm, 0.7 mW/cm2), this macro-chain transfer agent (macro-CTA) in the presence of a ruthenium based photoredox catalyst, Ru(bpy)3Cl2 can be chain extended with a second monomer to form a well-defined block copolymer in a process known as Photoinduced Electron Transfer RAFT (PET-RAFT). When PET-RAFT is used to chain extend POEGMA with benzyl methacrylate (BzMA) in ethanol (EtOH), polymeric nanoparticles with different morphologies are formed in situ according to a polymerization-induced self-assembly (PISA) mechanism. Self-assembly into nanoparticles presenting POEGMA chains at the corona and poly(benzyl methacrylate) (PBzMA) chains in the core occurs in situ due to the growing insolubility of the PBzMA block in ethanol. Interestingly, the formation of highly pure worm-like micelles can be readily monitored by observing the onset of a highly viscous gel in situ due to nanoparticle entanglements occurring during the polymerization. This process thereby allows for a more reproducible synthesis of worm-like micelles simply by monitoring the solution viscosity during the course of the polymerization. In addition, the light stimulus can be intermittently applied in an ON/OFF manner demonstrating temporal control over the nanoparticle morphology.

This article describes a process for producing polymeric self-assembled nanoparticles using visible light mediated dispersion polymerization. Using low energy visible light to control the polymerization allows for the reproducible formation of self-assembled worm-like micelles at high solids content.

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins ...

Anionic Polymerization of an Amphiphilic Copolymer for Preparation of Block Copolymer Micelles Stabilized by &#960;-&#960; Stacking Interactions

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE). Characterization of the copolymer revealed a narrow molecular distribution (PDI &#60; 1.03) and confirmed the degree of polymerization of mPEG122-b-(PheGE)15. The critical micelle concentration of the copolymer was evaluated using an established fluorescence method with the aggregation behavior evaluated by dynamic light scattering and transmission electronic microscopy. The potential of the copolymer for use in drug delivery applications was evaluated in a preliminary manner including in vitro biocompatibility, loading and release of the hydrophobic anti-cancer drug doxorubicin (DOX). A stable micelle formulation of DOX was prepared with drug loading levels up to 14% (wt%), drug loading efficiencies &#62; 60% (w/w) and sustained release of drug over 4 days under physiologically relevant conditions (acidic and neutral pH, presence of albumin). The high drug loading level and sustained release is attributed to stabilizing &#960;-&#960; interactions between DOX and the core-forming block of the micelles.

The key steps of living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE) are described. The resulting block copolymer micelles (BCMs) were loaded with doxorubicin 14% (wt%) and sustained release of drug over 4 days under physiologically relevant conditions was obtained.

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl ...

Self-assembling Morphologies Obtained from Helical Polycarbodiimide Copolymers and Their Triazole Derivatives

A facile method for the preparation of polycarbodiimide-based secondary structures (e.g., nano-rings, &#34;craters,&#34; fibers, looped fibers, fibrous networks, ribbons, worm-like aggregates, toroidal structures, and spherical particles) is described. These aggregates are morphologically influenced by extensive hydrophobic side chain-side chain interactions of the singular polycarbodiimide strands, as inferred by atomic force microscopy (AFM) and scanning electron microscopy (SEM) techniques. Polycarbodiimide-g-polystyrene copolymers (PS-PCDs) were prepared by a combination of synthetic methods, including coordination-insertion polymerization, copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) &#34;click&#34; chemistry, and atom transfer radical polymerization (ATRP). PS-PCDs were found to form specific toroidal architectures at low concentrations in CHCl3. To determine the influence of a more polar solvent medium (i.e., THF and THF/EtOH) on polymer aggregation behavior, a number of representative PS-PCD composites have been tested to show discrete concentration-dependent spherical particles. These fundamental studies are of practical interest to the development of experimental procedures for desirable architectures by directed self-assembly in thin film. These architectures may be exploited as drug carriers, whereas other morphological findings represent certain interest in the area of novel functional materials.

Here, we present a protocol to prepare and visualize secondary structures (e.g., fibers, toroidal architectures, and nano-spheres) derived from helical polycarbodiimides. The morphology characterized by both atomic force microscopy (AFM) and scanning electron microscopy (SEM) was shown to depend on molecular structure, concentration, and the solvent of choice.

A facile method for the preparation of polycarbodiimide-based secondary structures (e.g., nano-rings, &#34;craters,&#34; fibers, looped fibers, fibrous ...

Fabrication Procedures and Birefringence Measurements for Designing Magnetically Responsive Lanthanide Ion Chelating Phospholipid Assemblies

Bicelles are tunable disk-like polymolecular assemblies formed from a large variety of lipid mixtures. Applications range from membrane protein structural studies by nuclear magnetic resonance (NMR) to nanotechnological developments including the formation of optically active and magnetically switchable gels. Such technologies require high control of the assembly size, magnetic response and thermal resistance. Mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and its lanthanide ion (Ln3+) chelating phospholipid conjugate, 1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine-diethylene triaminepentaacetate (DMPE-DTPA), assemble into highly magnetically responsive assemblies such as DMPC/DMPE-DTPA/Ln3+ (molar ratio 4:1:1) bicelles. Introduction of cholesterol (Chol-OH) and steroid derivatives in the bilayer results in another set of assemblies offering unique physico-chemical properties. For a given lipid composition, the magnetic alignability is proportional to the bicelle size. The complexation of Ln3+ results in unprecedented magnetic responses in terms of both magnitude and alignment direction. The thermo-reversible collapse of the disk-like structures into vesicles upon heating allows tailoring of the assemblies&#39; dimensions by extrusion through membrane filters with defined pore sizes. The magnetically alignable bicelles are regenerated by cooling to 5 &#176;C, resulting in assembly dimensions defined by the vesicle precursors. Herein, this fabrication procedure is explained and the magnetic alignability of the assemblies is quantified by birefringence measurements under a 5.5 T magnetic field. The birefringence signal, originating from the phospholipid bilayer, further enables monitoring of polymolecular changes occurring in the bilayer. This simple technique is complementary to NMR experiments that are commonly employed to characterize bicelles.

Fabrication procedures for highly magnetically responsive lanthanide ion chelating polymolecular assemblies are presented. The magnetic response is dictated by the assembly size, which is tailored by extrusion through nanopore membranes. The assemblies&#39; magnetic alignability and temperature-induced structural changes are monitored by birefringence measurements, a complimentary technique to nuclear magnetic resonance and small angle neutron scattering.

Bicelles are tunable disk-like polymolecular assemblies formed from a large variety of lipid mixtures. Applications range from membrane protein structural ...

Synthesis of Monodisperse Cylindrical Nanoparticles via Crystallization-driven Self-assembly of Biodegradable Block Copolymers

The production of monodisperse cylindrical micelles is a significant challenge in polymer chemistry. Most cylindrical constructs formed from diblock copolymers are produced by one of three techniques: thin film rehydration, solvent switching or polymerization-induced self-assembly, and produce only flexible, polydisperse cylinders. Crystallization-driven self-assembly (CDSA) is a method which can produce cylinders with these properties, by stabilizing structures of a lower curvature due to the formation of a crystalline core. However, the living polymerization techniques by which most core-forming blocks are formed are not trivial processes and the CDSA process may yield unsatisfactory results if carried out incorrectly. Here, the synthesis of cylindrical nanoparticles from simple reagents is shown. The drying and purification of reagents prior to a ring-opening polymerization of &#949;-caprolactone catalyzed by diphenyl phosphate is described. This polymer is then chain extended by methyl methacrylate (MMA) followed by N,N-dimethyl acrylamide (DMA) using reversible addition&#8722;fragmentation chain-transfer (RAFT) polymerization, affording a triblock copolymer that can undergo CDSA in ethanol. The living CDSA process is outlined, the results of which yield cylindrical nanoparticles up to 500 nm in length and a length dispersity as low as 1.05. It is anticipated that these protocols will allow others to produce cylindrical nanostructures and elevate the field of CDSA in the future.

Crystallization-driven self-assembly (CDSA) displays the unique ability to fabricate cylindrical nanostructures of narrow length distributions. The organocatalyzed ring-opening polymerization of &#949;-caprolactone and subsequent chain extensions of methyl methacrylate and N,N-dimethyl acrylamide are demonstrated. A living CDSA protocol that produces monodisperse cylinders up to 500 nm in length is outlined.

The production of monodisperse cylindrical micelles is a significant challenge in polymer chemistry. Most cylindrical constructs formed from diblock ...

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery vehicles and enzyme scaffolds. Due to their biocompatibility and tunability on the genetic level, Elastin-like proteins (ELP) are ideal building blocks for biotechnological and biomedical applications. Nevertheless, the assembly of protein based supramolecular structures with distinct physiochemical properties and good encapsulation potential remains challenging.
Here we provide two efficient protocols for guided self-assembly of amphiphilic ELPs into supramolecular protein architectures such as spherical coacervates, fibers and stable vesicles. The presented assembly protocols generate Protein Membrane-Based Compartments (PMBCs) based on ELPs with adaptable physicochemical properties. PMBCs demonstrate phase separation behavior and reveal method dependent membrane fusion and are able to encapsulate chemically diverse fluorescent cargo molecules. The resulting PMBCs have a high application potential as a drug formulation and delivery platform, artificial cell, and compartmentalized reaction space.

At the interface of organic and aqueous solvents, tailored amphiphilic elastin-like proteins assemble into complex supramolecular structures such as vesicles, fibers and coacervates triggered by environmental parameters. The described assembly protocols yield Protein Membrane-Based Compartments (PMBCs) with tunable properties, enabling the encapsulation of various cargo.

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery ...

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...