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.
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Method Article
Assembly and Characterization of Polyelectrolyte Complex Micelles
In This Article
Summary
Abstract
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.
Introduction
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 n....
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Protocol
1. Preparation of Materials
- Weigh out lyophilized diblock polymer and add water up to nearly the volume required for a stock solution of 10 mg/mL final concentration. Vortex at maximum speed for 2 min.
- Sonicate for 5 min. Very long diblocks may require additional sonication. The stock solution should appear completely transparent and homogeneous.
- Adjust pH to 7.4 using NaOH or HCl as needed. Add water to the final volume. pLys-PEG solutions are fairly stable but should be refrigerated for longer-term storage and the pH must be checked before use. Lyophilization is preferable to freezing.
- Resuspend lyophilized oligonucleotide(s....
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Results
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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Discussion
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 .......
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Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 70 mm circle filter paper | Whatman | 1001-070 | Filter paper for wicking during grid prep |
| Carbon Film TEM grid | Electron Microscopy Sciences | CF200-Cu | TEM grid |
| DAWN | Wyatt Technology | DAWN | MALS instrument |
| DNA oligonucleotide | Integrated DNA Nanotechnologies Inc | Custom oligonucleotide | |
| Lacey Carbon TEM grid | Electron Microscopy Sciences | LC200-Cu | TEM grid |
| Methoxy-poly(ethylene glycol)-block-poly(l-lysine hydrochloride) PEG5k - PLKC50 | Alamanda Polymers Inc | mPEG5K-b-PLKC50 | Example block copolymer |
| Milli-Q | Millipore Sigma | Ultrapure water | |
| NanoDrop | Thermo Scientific | For measuring nucleic acid concentration | |
| negative-action tweezers | Dumont | N7 | Tweezers for grid preparation |
| Parafilm "M" | Bemis Company Inc | PM996 | Laboratory film |
| Quantifoil Holey Carbon TEM grid | Electron Microscopy Sciences | Q210CR1.3 | TEM grid |
| Research Goniometer and Laser Light Scattering System | Brookhaven Instruments | BI-200SM | DLS/MALS instrument |
| Slide-A-Lyzer G2 2K 0.5 mL | Thermo Scientific Pierce Protein Biology | 87723 | Dialysis cartridge |
| small volume cuvette | Brookhaven Instruments | BI-SVC | Cuvette for DLS/MALS |
| Solarus 950 Advanced Plasma System | Gatan | Solarus 950 | Plasma system for TEM grids |
| Talos TEM | FEI | Talos | TEM used for cryo samples |
| Tecnai Spirit TEM | FEI | Spirit | TEM used for dry samples |
| Uranyl Formate | SPI-Chem | 16984-59-1 | For negative staining samples for TEM |
| Vitrobot | FEI | Vitrobot | Vitrification robot for cryo grid preparation |
References
- Spruijt, E., Westphal, A. H., Borst, J. W., Cohen Stuart, M. A., van der Gucht, J. Binodal compositions of polyelectrolyte complexes. Macromolecules. 43 (15), 6476-6484 (2010).
- van der Gucht, J., Spruijt, E., Lemmers, M., Cohen Stuart, M. A. Polyelectrolyte complexes: bulk ph....
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