Department of Chemical and Biological Engineering, Iowa State University
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Petersen, L. K., Chavez-Santoscoy, A. V., Narasimhan, B. Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries. J. Vis. Exp. (67), e3882, doi:10.3791/3882 (2012).
Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.
1. Combinatorial Polymer Library Synthesis (Varying in Polymer Chemistry) - see Figure 1 for Robotic Setup
2. Combinatorial Blank and Protein-loaded Polymer Nanoparticle and Film Library Fabrication - see Figure 1 for Robotic Setup
3. High-throughput Protein Release Kinetics
4. Representative Results
Upon fabrication of the polymer library, characterization has been carried out with 1H NMR, GPC, and FTIR to validate this combinatorial method1,7,8,11. Molecular weights range from 10,000-20,000 g/mol, polydispersity index ranges from 1.5-3.0, and chemical composition has been shown to be accurate and in agreement with conventional methods of polyanhydride synthesis12-15. Similarly, SEM images of the nanoparticles libraries showed similar surface morphology, size, and size distribution as that of conventionally fabricated nanoparticles2. Protein release kinetics from polyanhydride nanoparticles or films is carried out in a modified well plate as described previously1. The results revealed an approximate zero order release with or without a burst dependent upon protein loading and polymer chemistry (Figures 2 and 3) 1,12,14,16.
Figure 1. Combinatorial polymer film and nanoparticle fabrication apparatus.
Figure 2. High-throughput release of Texas Red bovine serum albumin (TRBSA) from a CPH:SA polymer nanoparticle library. SA-rich polymer chemistries release encapsulated TRBSA the most rapidly, whereas CPH-rich polymer chemistries release the slowest. Error bars represent standard deviation and n=4. Reprinted with permission from Petersen et. al.1. Copyright 2010 American Chemical Society.
Figure 3. High-throughput release of Texas Red bovine serum albumin (TRBSA) from a CPTEG:CPH polymer film library. CPTEG-rich polymer chemistries release encapsulated TRBSA the most rapidly, whereas CPH-rich polymer chemistries release the slowest. Error bars represent standard deviation and n=3.
Figure 4. Image showing two neighboring wells "before" and "after" modification in the 96 deep-welled polypropylene plate. The "after" modification image on the right also depicts the addition of a polymer film (bottom of left well) with an encapsulated fluorescent molecule being released between the two wells into a buffer solution. The released fluorescent molecule is then detected in the right well.
Knowledge of the necessary synthesis conditions and the glass transition temperatures (Tgs) of the polymers being synthesized are essential for library fabrication. If the Tgs are below room temperature, the nanoparticle fabrication step may need to be carried out in a controlled temperature environment below the Tg of the polymers. Additionally, caution should be taken to ensure that all equipment that comes in contact with high temperatures and the solvents must be fit to handle those conditions. Several of the parameters of this protocol can be adjusted (i.e., temperature, vacuum, incubation times, solvents, non-solvents, polymer concentration, solvent to non-solvent ratios, etc.) to accommodate different polymer systems for synthesis or particle/film fabrication. In some cases, nanoparticles are not stable in solvents for long time periods (sufficient for solvent removal by vacuum drying) so two alternate solvent removal methods can be used. 1) The particles can be slowly centrifuged, the supernatant solvent decanted, and the remaining particles dried or 2) the particles can be separated by vacuum filtration and then dried. Following fabrication of blank or protein-loaded nanoparticles/films, high-throughput characterization or testing can be conducted to screen the biomaterials for protein, cellular, or host interactions. This high-throughput methodology enables the rapid optimization of biomaterial performance for the desired application.
No conflicts of interest declared.
The authors acknowledge the ONR-MURI Award (NN00014-06-1-1176) and the U.S. Army Medical Research and Materiel Command (Grant No. W81XWH-10-1-0806) for financial support. This material is based upon work supported by the National Science Foundation under Grant No. EEC 0552584 and 0851519.
|Motorized XYZ Stage: 3x T-LSM050A, 50 mm travel per axis||Zaber Technologies||T-XYZ-LSM050A-KT04|
|NE-1000 Single Syringe Pump||New Era Pump Systems||NE-1000|
|Pyrex* Vista* Rimless Reusable Glass Culture Tubes||Corning||07-250-125|
|Glass cuvettes||Scientific Strategies||G102|
|SGE Gas Tight Syringes, Luer Loc||Sigma Aldrich||509507|
|U96 DeepWell Plates 1.3 ml & 2.0 ml||Thermo Scientific: Nunc||278743|
|Well cap mats||Thermo Scientific: Nunc||276000|
|Typhoon 9400||GE Healthcare||63-0055-79|
|Whatman Grade 50 Circles 90 mm||Whatman||1450-090|