Automated High-Throughput Synthesis of Protein-Loaded Polyanhydride Nanoparticle Libraries

Abstract

The development of high-throughput techniques and combinatorial libraries can facilitate rapid synthesis and screening of biomaterial-based nanocarriers for drug and vaccine delivery. This study describes a high-throughput method using an automated robot for synthesizing polyanhydride nanoparticles encapsulating proteins. Polyanhydrides are a class of safe and biodegradable polymers that have been widely used as drug and vaccine delivery vehicles. The robot contains a multiplexed homogenizer and has the capacity to handle parallel streams of monomer or polymer solutions to synthesize polymers and/or nanoparticles. Copolymer libraries were synthesized using the monomers sebacic acid, 1,6-bis( p-carboxyphenoxy)hexane, and 1,8-bis( p-carboxyphenoxy)-3,6-dioxactane and compared to conventionally synthesized copolymers. Nanoparticle libraries of varying copolymer compositions encapsulating the model antigen ovalbumin were synthesized using flash nanoprecipitation. The amount of the surfactant Span 80 was varied to test its effect on protein encapsulation efficiency as well as antigen release kinetics. It was observed that, although the amount of surfactant did not significantly affect protein release rate, its presence enhanced protein encapsulation efficiency. Protein burst and release kinetics from conventionally and combinatorially synthesized nanoparticles were similar even though particles synthesized using the high-throughput technique were smaller. Finally, it was demonstrated that the high-throughput method could be adapted to functionalize the surface of particle libraries to aid in the design and screening of targeted drug and vaccine delivery systems. These results suggest that the new high-throughput method is a viable alternative to conventional methods for synthesizing and screening protein and vaccine delivery vehicles.

High-Throughput Synthesis and Screening of Rapidly Degrading Polyanhydride Nanoparticles

“…High-throughput synthesis of CPTEG:SA nanoparticle libraries was carried out using a modified protocol from that reported by Goodman et al 9 Methylene chloride solutions containing appropriate concentrations of the model drugs, RhoB or CBB, were dispensed by the robot into polymer library test tubes to dissolve polymer at 20 mg/mL. Fifty mL centrifuge tubes were 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 filled with 45 mL of hexanes chilled to -10°C and placed in the robot's stainless-steel rack, which had been chilled in a -80°C freezer and filled with dry ice.…”

“…Previous work from our laboratories described the development of a high-throughput method to synthesize nanoparticle libraries with comparable properties to conventionally synthesized particles. 9 We adapted this method for CPTEG:SA copolymers, increasing the mass scale to improve yields and to enable a wider battery of tests to be performed on individual nanoparticle lots. Methylene chloride and chloroform were tested as polymer solvents, and pentane and hexanes at 4°C, -10°C, and -20°C were tested as anti-solvents ( Figure S6).…”

“…An automated, high-throughput methodology was recently developed in our laboratory for synthesizing copolymer libraries, protein-encapsulating nanoparticle libraries, and surface-functionalized nanoparticle libraries, as well as validating through comparisons with conventionally synthesized polymers and nanoparticles. 9 Small molecule drug release from CPH:SA and CPTEG:CPH copolymer films and microparticles have been investigated conventionally. [10][11][12] Polyanhydride nanoparticles have also shown promising capabilities for delivery of small molecule drugs, [13][14][15] but the pace of understanding and rational design of these nanomedicines is limited by the low throughput of conventional methods.…”

“…Additionally, newly synthesized polyanhydride copolymers, such as CPTEG:SA, 16 have limited supporting literature to guide understanding of their nano-scale drug 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 delivery properties. These CPTEG:SA copolymers are hypothesized to combine the favorable thermal properties of SA-rich polymers, such as high melting point (T m ), that enables room temperature nanoparticle synthesis and discrete nanoparticle morphology, 9 with the amphiphilicity, payload-stabilizing, 6 and high cellular internalization 15 properties of CPTEG-rich polymers. Furthermore, the relatively low hydrophobicity of these monomers is predicted to provide rapid polymer erosion, corresponding with faster release kinetics of encapsulated payloads from nanoparticles.…”

Data Analytics Approach for Rational Design of Nanomedicines with Programmable Drug Release

“…A total of 68 nanoformulations were tested, spanning drug loadings between 1% and 20% loading (% w/w). To validate the informatics analysis, nanoparticles encapsulating meropenem or ceftazidime were synthesized using a high-throughput method adapted from Goodman et al 23 Briefly, polymer and drugs were dissolved/dispersed in methylene chloride and dispensed via highthroughput robot into 10 mL borosilicate tubes at a final polymer concentration of 20 mg/mL. In this case, x = 5 because 5 PCs captured >90% of the variance in the data.…”