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.
