Name: Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries (Video) | JoVE Journal
Uploaded: 2012-09-06T12:00:00.000+00:00
Duration: 10 min 58 s
Description: Learn combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries for high-throughput protein release screening. This method enables rapid development of drug delivery biomaterials.

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5 minutes

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.

1. Combinatorial Polymer Library Synthesis (Varying in Polymer Chemistry) - see Figure 1 for Robotic Setup
<ol>
 <li>Dissolve each monomer in the appropriate solvent (concentration = 25 mg/ml) and load each into a 10cc gas tight syringe.</li>
 <li>Attach the solvent resistant lure lock capillary tubes to the end of each syringe.</li>
 <li>Place the syringes on the syringe pumps (New Era Programmable Syringe Pumps) and lock into position.</li>
 <li>Set the linear actuators (Zaber) to the starting position.</li>
 <li>Using ring stand clamps, position the end of both capillary tubes into the starting vial/well for monomer deposition.</li>
 <li>Initiate the LabVIEW program, which pumps variable volumes of each monomer into each well depending upon the desired copolymer composition. This is achieved in the program by instructing the Z-axis actuator to lower the capillary tubes into the vial/well and then each pump to dispense the desired volume. Next, the program instructs the Z-actuator to return to its home position and the X- and Y- actuators to move to the position of the next vial/well. This is carried out until each well has the desired volume of monomer deposited into it.</li>
 <li>Following monomer deposition, the multi-well or multi-vial monomer library is transferred to a pre-heated vacuum oven and incubated under vacuum for the duration of the condensation polymerization reaction. For CPH:SA synthesis the reaction is carried out at 180 °C, 0.3 torr, for 1.5 hr but these reaction conditions will vary between different polymer systems.</li>
</ol>
2. Combinatorial Blank and Protein-loaded Polymer Nanoparticle and Film Library Fabrication - see Figure 1 for Robotic Setup
<ol>
 <li>The syringe in the first programmable syringe pump is filled with a solvent (blank library) or a solvent with protein dispersed in it (protein-loaded library) while the syringe in the second syringe pump is left empty. Tubes for nanoparticle fabrication in the adjacent sample holder are filled with the non-solvent (ratio of solvent to non-solvent is 1 to 100). For film fabrication an empty multi-well plate is utilized in place of the tubes in the adjacent sample holder.</li>
 <li>Using the LabVIEW program, the solvent is deposited into all polymer vials/wells of the library (concentration = 20 mg/ml) and incubated for 1-5 min. An optional sonication step (30 s at 40 Hz) can be introduced to ensure complete polymer dissolution.</li>
 <li>Next, by initiating a separate LabVIEW program, the sample is withdrawn into the empty syringe, and deposited into its corresponding tube of non-solvent (nanoparticles) or empty well (films) in the adjacent sample holder.</li>
 <li>This process is carried out for each composition of the discrete polymer library.</li>
 <li>The nanoparticle or film library is then placed in a vacuum chamber for solvent and non-solvent removal (the nanoparticle library can also be recovered using vacuum filtration).</li>
</ol>
3. High-throughput Protein Release Kinetics
<ol>
 <li>For investigating high-throughput protein release kinetics from a polymer library, a 96 deep-welled (2 ml/well), polypropylene plate is modified such that the top 1/3 of the well wall in neighboring columns (ex: A and B, C and D, E and F, G and H) is removed to adjoin the wells. The protein-loaded films must be fabricated in or the nanoparticles transferred into this plate to perform high-throughput release kinetics studies. Protein-loaded nanoparticle or film samples must only be placed in one well of the adjoining columns (ex: A, C, E, and G). See Figure 4 for details.</li>
 <li>Following the transfer of the nanoparticles to the 96 deep-welled release plate, the particles are allowed to settle. PBS buffer (0.1 mM, pH 7.4) is slowly added to each sample well (columns A, C, E and G) and then to each neighboring well (columns B, D, F, and H) until the wells are full and the buffer is free flowing between adjoining wells. For nanoparticles, this process needs to be carried out with extra caution to ensure the particles remain at the bottom of the sample well (columns A, C, E and G) and not transferred into the adjoining release well (columns B, D, F, and H). In some cases, centrifugation is required to localize the nanoparticle samples at the bottom of the sample well (columns A, C, E and G).</li>
 <li>Next, the release plate is sealed with a lid and placed at the desired temperature (i.e. 37 °C) under agitation for the duration of the experiment.</li>
 <li>At incremental time points (i.e. 0.04, 1, 2, 3, 5, 7, 9, 12, 15, 19, 24, and 30 days) the amount of released fluorochrome-conjugated protein is quantified using a 9,400 Typhoon Flatbed Fluorescent Scanner (GE Healthcare). The release plate is placed on the scanner surface, scanned using the appropriate excitation laser and emission filters, and the fluorescence intensity of the released protein in the release wells (columns B, D, F and H) is quantified (Image Quant). It is suggested that protein standards of known concentrations be included in the well plate for calculating protein quantity and accounting for fluorochrome quenching.</li>
</ol>
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.
<img alt="Polymer dispensing setup diagram; capillary tubes, syringe pumps, linear actuators; research use." src="/files/ftp_upload/3882/3882fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Combinatorial polymer film and nanoparticle fabrication apparatus.
<img alt="Cumulative TRBSA release over time; graph analyzing polymer compositions of Poly SA, CPH:SA." src="/files/ftp_upload/3882/3882fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
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. 
 
<img alt="Polymer release study; graph of cumulative mass fraction vs. time for CPTEG:CPH ratios." src="/files/ftp_upload/3882/3882fig3.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
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.
<img alt="Polymer modification diagram; compares surfaces before and after polymer film application." src="/files/ftp_upload/3882/3882fig4.jpg" data-altupdatedat="1787630408000" data-alt="Figure 4"> 
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.

<ol><li>Petersen, L. K., Sackett, C. K., Narasimhan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel%2C+high-throughput+method+to+study+in+vitro+protein+release+from+polymer+nanospheres%5BTitle%5D)+AND+%22J.+Comb.+Chem%22%5BJournal%5D)">A novel, high-throughput method to study in vitro protein release from polymer nanospheres.</a> J. Comb. Chem. 12, 51-56 (2010).</li>
<li>Petersen, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+of+innate+immune+responses+in+a+pathogen-mimicking+manner+by+amphiphilic+polyanhydride+nanoparticle+adjuvants%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Activation of innate immune responses in a pathogen-mimicking manner by amphiphilic polyanhydride nanoparticle adjuvants.</a> Biomaterials. 32, 6815-6822 (2011).</li>
<li>Vogel, B. M., Cabral, J. T., Eidelman, N., Narasimhan, B., Mallapragada, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parallel+synthesis+and+high-throughput+dissolution+testing+of+biodegradable+polyanhydride+copolymers%5BTitle%5D)+AND+%22J.+Comb.+Chem%22%5BJournal%5D)">Parallel synthesis and high-throughput dissolution testing of biodegradable polyanhydride copolymers.</a> J. Comb. Chem. 7, 921-928 (2005).</li>
<li>Petersen, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+evaluation+of+in+vivo+biodistribution+of+polyanhydride+nanoparticles%5BTitle%5D)+AND+%22Adv.+Healthcare+Mater%22%5BJournal%5D)">High-throughput evaluation of in vivo biodistribution of polyanhydride nanoparticles.</a> Adv. Healthcare Mater. , Forthcoming (2012).</li>
<li>Petersen, L. K., Narasimhan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combinatorial+design+of+biomaterials+for+drug+delivery%3A+opportunities+and+challenges%5BTitle%5D)+AND+%22Expert+Opin.+Drug+Deliv%22%5BJournal%5D)">Combinatorial design of biomaterials for drug delivery: opportunities and challenges.</a> Expert Opin. Drug Deliv. 5, 837-846 (2008).</li>
<li>Petersen, L. K., Oh, J., Sakaguchi, D. S., Mallapragada, S. K., Narasimhan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amphiphilic+polyanhydride+films+promote+neural+stem+cell+adhesion+and+differentiation%5BTitle%5D)+AND+%22Tissue+Eng%22%5BJournal%5D)">Amphiphilic polyanhydride films promote neural stem cell adhesion and differentiation.</a> Tissue Eng. 17, 2533-2541 (2011).</li>
<li>Petersen, L. K., Sackett, C. K., Narasimhan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+analysis+of+protein+stability+in+polyanhydride+nanoparticles%5BTitle%5D)+AND+%22Acta+Biomater%22%5BJournal%5D)">High-throughput analysis of protein stability in polyanhydride nanoparticles.</a> Acta Biomater. 6, 3873-3881 (2010).</li>
<li>Petersen, L. K., Xue, L., Wannemuehler, M. J., Rajan, K., Narasimhan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+simultaneous+effect+of+polymer+chemistry+and+device+geometry+on+the+in+vitro+activation+of+murine+dendritic+cells%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">The simultaneous effect of polymer chemistry and device geometry on the in vitro activation of murine dendritic cells.</a> Biomaterials. 30, 5131-5142 (2009).</li>
<li>Thorstenson, J. B., Petersen, L. K., Narasimhan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combinatorial%2Fhigh-throughput+methods+for+the+determination+of+polyanhydride+phase+behavior%5BTitle%5D)+AND+%22J.+Comb.+Chem%22%5BJournal%5D)">Combinatorial/high-throughput methods for the determination of polyanhydride phase behavior.</a> J. Comb. Chem. 11, 820-828 (2009).</li>
<li>Xue, L., Petersen, L., Broderick, S., Narasimhan, B., Rajan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identifying+factors+controlling+protein+release+from+combinatorial+biomaterial+libraries+via+hybrid+data+mining+methods%5BTitle%5D)+AND+%22ACS+Comb.+Sci%22%5BJournal%5D)">Identifying factors controlling protein release from combinatorial biomaterial libraries via hybrid data mining methods.</a> ACS Comb. Sci. 13, 50-58 (2011).</li>
<li>Adler, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+cell-based+screening+of+biodegradable+polyanhydride+libraries%5BTitle%5D)+AND+%22Comb.+Chem.+High+Through.+Screen%22%5BJournal%5D)">High-throughput cell-based screening of biodegradable polyanhydride libraries.</a> Comb. Chem. High Through. Screen. 12, 634-645 (2009).</li>
<li>Determan, A. S., Trewyn, B. G., Lin, V. S., Nilsen-Hamilton, M., Narasimhan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Encapsulation%2C+stabilization%2C+and+release+of+BSA-FITC+from+polyanhydride+microspheres%5BTitle%5D)+AND+%22J.+Control.+Release%22%5BJournal%5D)">Encapsulation, stabilization, and release of BSA-FITC from polyanhydride microspheres.</a> J. Control. Release. 100, 97-109 (2004).</li>
<li>Determan, A. S., Wilson, J. H., Kipper, M. J., Wannemuehler, M. J., Narasimhan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+stability+in+the+presence+of+polymer+degradation+products%3A+consequences+for+controlled+release+formulations%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Protein stability in the presence of polymer degradation products: consequences for controlled release formulations.</a> Biomaterials. 27, 3312-3320 (2006).</li>
<li>Torres, M. P., Determan, A. S., Anderson, G. L., Mallapragada, S. K., Narasimhan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amphiphilic+polyanhydrides+for+protein+stabilization+and+release%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Amphiphilic polyanhydrides for protein stabilization and release.</a> Biomaterials. 28, 108-116 (2007).</li>
<li>Torres, M. P., Vogel, B. M., Narasimhan, B., Mallapragada, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthesis+and+characterization+of+novel+polyanhydrides+with+tailored+erosion+mechanisms%5BTitle%5D)+AND+%22J.+Biomed.+Mater.+Res.+A%22%5BJournal%5D)">Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms.</a> J. Biomed. Mater. Res. A. 76, 102-110 (2006).</li>
<li>Carrillo-Conde, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Encapsulation+into+amphiphilic+polyanhydride+microparticles+stabilizes+Yersinia+pestis+antigens%5BTitle%5D)+AND+%22Acta+Biomater%22%5BJournal%5D)">Encapsulation into amphiphilic polyanhydride microparticles stabilizes Yersinia pestis antigens.</a> Acta Biomater. 6, 3110-3119 (2010).</li>
</ol>

No conflicts of interest declared.

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 c...

<table><tbody><tr><td>Motorized XYZ Stage: 3x T-LSM050A, 50 mm travel per axis</td><td>Zaber Technologies</td><td>T-XYZ-LSM050A-KT04</td><td></td></tr><tr><td>NE-1000 Single Syringe Pump</td><td>New Era Pump Systems</td><td>NE-1000</td><td></td></tr><tr><td>Pyrex* Vista* Rimless Reusable Glass Culture Tubes</td><td>Corning</td><td>07-250-125</td><td></td></tr><tr><td>Glass cuvettes</td><td>Scientific Strategies</td><td>G102</td><td></td></tr><tr><td>LabVIEW</td><td>National Instruments</td><td>776671-35</td><td></td></tr><tr><td>SGE Gas Tight Syringes, Luer Loc</td><td>Sigma Aldrich</td><td>509507</td><td></td></tr><tr><td>U96 DeepWell Plates 1.3 ml &amp;amp; 2.0 ml</td><td>Thermo Scientific: Nunc</td><td>278743</td><td></td></tr><tr><td>Well cap mats</td><td>Thermo Scientific: Nunc</td><td>276000</td><td></td></tr><tr><td>Typhoon 9400</td><td>GE Healthcare</td><td>63-0055-79</td><td></td></tr><tr><td>Whatman Grade 50 Circles 90 mm</td><td>Whatman</td><td>1450-090</td><td></td></tr></tbody></table>

combinatorial synthesis of and high-throughput protein release from polymer film and nanoparticle libraries

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

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.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

This video describes the combinatorial synthesis of biodegradable poly anhydride film and nanoparticle libraries, and the high throughput detection of protein release from these libraries. First, a high throughput automated deposition apparatuses used to deposit a compositionally diverse array of monomers into glass cuvettes. A poly condensation reaction is carried out at 180 degrees Celsius and 0.3 tor for 1.5 hours.These reaction conditions are specific for C-P-H-S-A poly anhydride polymer synthesis. Using the automated deposition apparatus, the solvent and any components for encapsulation are deposited into the cuvettes containing the polymer library. Sonication can be administered to ensure completed dissolution of the polymer and uniform dispersal of the additional components.Then the dissolved polymer is transferred into a tube of nons solvent to fabricate the nanoparticle library by nano precipitation. The nanoparticles are recovered by vacuum drying or filtration, and then transferred to a modified 96 well released plate. Finally, the protein loaded library is incubated under physiological conditions and protein release kinetics are evaluated using a fluorescent space detection method.Ultimately, results can be obtained that show protein release kinetics as a function of polymer properties or release environment conditions. The main advantage of this approach over other conventional one sample at a time techniques is that we're able to synthesize multiple samples simultaneously. These samples can then be screened in high throughput for protein polymer cell polymer, or host polymer interactions.This then expedites the rational design and development of these biomaterials for drug delivery and tissue engineering. Demonstrating the procedure will be La Tricia Peterson, a post-op from my laboratory. In this section of the video, a Hyatt throughput deposition apparatus is used to deposit a compositionally variant monomer library following polymer synthesis.This apparatus is utilized to fabricate a combinatorial library of blank or protein loaded polymer films or nanoparticles. The automated platform consists of two programmable syringe pumps and three linear actuators connected in series to a computer on the computer. LabVIEW software is used to direct commands to the syringe pumps and actuators.10 cc gast tight syringes are loaded into the syringe pumps with metal capillary tubes connected through lure lock fittings, which dispense the polymer solutions into glass cuvettes. In this demonstration, seic acid or SA will be used with one six Biss para oxy heane or CPH to make a library of nanoparticles encapsulating Texas Red Boan serum albumin. The instructions in this video and the accompanying documents can be adapted to fabricate and test a wide array Of polymer film and nanoparticle libraries.To synthesize the libraries begin by dissolving each monomer and chloroform at a final concentration of 25 milligrams per milliliter in a glass vial. Next, load each solution into a 10 cc gast tight syringe by aspirating the solution through the lure lock end of the syringe. Remove any air bubbles.Next, attach the solvent resistant lure lock stainless steel capillary tube to the end of the syringe. Secure the capillary tubes in clamps, affix to a ring stand. Then position the end of both capillary tubes into the starting vete.For monomer deposition. Place the syringes on the programmable syringe pumps and lock them into position in the lab view software. Set the linear actuators to the starting position in the software.Initiate the automated deposition of the SA and CPH monomers into the glass cuvettes the x, y, and Z axis. Actuators will properly position the capillary tubes into each Q, vet and pump programmed volumes of each monomer into them, creating a compositionally variant monomer library. Following monomer deposition.Transfer the monomer library to a preheated vacuum oven and incubate at 180 degrees Celsius and 0.3 tour for 1.5 hours to enable condensation polymerization. Following the incubation, the fabrication of the polymer library is completed. Next protein loaded polymer nanoparticles are generated.See the accompanying protocol for protein loaded polymer film library fabrication for fabricating a protein loaded nanoparticle library. Add the protein in this case, Texas red bovine serum albumin to the appropriate solvent, which should be capable of dissolving the polymer library. Here, methylene chloride is used at a concentration of two milligrams per milliliter.Fill the 10 cc syringe with Texas Red BSA containing solvent and place it in the first syringe pump. Then place a clean, empty two cc syringe in the second syringe pump place tubes containing 15 milliliters of a nons solvent in this case, pentane in the tube holder adjacent to the vial platform. Next in the lab, view software initiate the automated solvent deposition process by selecting the program.Following initiation of the program, the solvent is deposited into the cuvettes containing the polymer library, resulting in a polymer concentration of 20 milligrams per milliliter. When all of the solvent has been deposited, allow it to dissolve the polymer for one to five minutes. Follow this with an optional syndication to ensure a complete polymer dissolution and uniform protein dispersal.Next, initiate the sample transfer program in lab view. Each dissolved polymer sample in the library is then withdrawn into the empty syringe and dispensed into its corresponding tube of nons solvent in the sample holder. Once each sample has been transferred, recover the protein loaded nanoparticles by placing the nanoparticle library in a vacuum chamber at 10 millimeters of mercury vacuum, or by using vacuum filtration until the solvent is removed.To investigate the release of encapsulated BSA, begin with a deep 96 weld polypropylene plate that has been modified by removing the top half inch of each well wall of every other adjoining column in the plate. Removing the top of each well between columns A and B, C and DENF and GNH allows for liquid flow between each pair of adjoined wells when filled to full volume transfer. Texas red BSA standards ranging from 1000 to 10 micrograms per milliliter into the deep 96 well plate.These will be used for calculating protein quantity and will also account for fluorochrome quenching reconstitute the desired mass of nanoparticles in PBS buffer, then sonicate to uniformly disperse the particles. Next, transfer the nanoparticle library into the deep 96 well plate and wait until samples have settled to the bottom of the well plate. Next, using a pipetter slowly fill all of the adjoining wells with PBS buffer up to one fourth of an inch from the top of the well.There should be enough buffer in the adjoining wells so that it is free flowing between the wells. Next, seal the release plate with the lid and place it at 37 degrees Celsius under agitation. For the duration of the experiment at incremental time points, use a 9, 400 Typhoon flatbed fluorescent scanner to scan the plate using the appropriate excitation, laser, and emission filters.Following scanning, use image quant software to quantify the amount of released fluorochrome conjugated protein in each release. Well, Texas Red BSA was encapsulated in a polymer nanoparticle library synthesized from SA and CPH monomers as described in this video to determine protein release kinetics as a function of polymer chemistry. The protein loaded nanoparticles were incubated at 37 degrees Celsius for one month, during which fluorescent scans were obtained at incremental time points for protein release quantification.As shown here, this hydro throughput method was utilized to determine Texas Red BSA release kinetics from A-C-P-H-S-A poly anhydride nanoparticle library. Chemistry dependent trends were observed with increasing release rates from Sarich film chemistries. Similar results were obtained when using standard protein quantification assays.In a separate experiment, the Texas Red BSA release from CPT CPH Poly Anhydride Film Libraries was assessed as shown here near zero order release kinetics were observed with cpeg rich films releasing the protein the most rapidly with potential applications in the medical field as biodegradable drug delivery devices. These results indicate that the desired release profile of a therapeutic drug or vaccine antigen can be controlled by changing the polymer chemistry. Following this procedure, other combinatorial methods have been developed to investigate areas such as protein stability, cellular toxicity, cellular activation, and InVivo biodistribution.These aspects are important for the rational design of biomaterials for drug delivery and tissue engineering. Don't forget to wear proper PPE and practice safe laboratory procedures throughout this entire protocol.

combinatorial-synthesis-high-throughput-protein-release-from-polymer

Research

JoVE Journal

Bioengineering

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Polymer Microarrays for High Throughput Discovery of Biomaterials

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

High-throughput Protein Expression Generator Using a Microfluidic Platform

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

A simple, robust and scalable technique to functionalize and self-assemble macroscopic nanoparticle-ligand monolayer films onto template-free substrates is described in this protocol.

A Technique to Functionalize and Self-assemble Macroscopic Nanoparticle-ligand Monolayer Films onto Template-free Substrates

We report protocols for “polymerizing” various types of polymer-encapsulated metal nanoparticles into long chains of “homo-“ and “co-polymers”.

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

Non-stirred precipitation polymerization provides a rapid, reproducible prototyping approach to the synthesis of stimuli-sensitive poly(N-isopropylacrylamide) microgels of narrow size distribution. In this protocol synthesis, light scattering characterization and single particle fluorescence tracking of these microgels in a wide-field microscopy setup are demonstrated.

Controlled Synthesis and Fluorescence Tracking of Highly Uniform Poly(N-isopropylacrylamide) Microgels

Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation.

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Nanogels are an excellent and versatile nanoparticle platform for the delivery of biologics. Stimuli-responsive poly(ethylene) glycol-based polymeric nano-gels, capable of encapsulating protein-based payloads, were synthesized using a one-step cross-linking co-nanopolymerization strategy in aqueous conditions. The optimal fabrication and characterization of these novel nanoparticles are presented here.

Synthesis of Stimuli-responsive Nanogels using Aqueous One-step Crosslinking and Co-nanopolymerization

Here, we present two protocols for embedding cell-free protein synthesis reactions in macro-scale hydrogel matrices without the need for an external liquid phase.

Methods for Embedding Cell-Free Protein Synthesis Reactions in Macro-Scale Hydrogels

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

Bilayer Microfluidic Device for Combinatorial Plug Production

Learn combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries for high-throughput protein release screening. This method enables rapid development of drug delivery biomaterials.

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

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