Automated High Throughput Synthesis of Protein-Loaded

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Automated High Throughput Synthesis of ProteinLoaded Polyanhydride Nanoparticle Libraries Jonathan T Goodman, Adam Mullis, Lucas Dunshee, Akash Mitra, and Balaji Narasimhan ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.8b00008 • Publication Date (Web): 04 Apr 2018 Downloaded from http://pubs.acs.org on April 4, 2018

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ACS Combinatorial Science

Automated High Throughput Synthesis of Protein-Loaded Polyanhydride Nanoparticle Libraries Jonathan T. Goodman, Adam S. Mullis, Lucas Dunshee, Akash Mitra, and Balaji Narasimhan Department of Chemical and Biological Engineering and Nanovaccine Institute, Iowa State University, Ames, IA 50011, U.S.A. Email: [email protected], [email protected], [email protected], [email protected], [email protected] Corresponding Author: Dr. Balaji Narasimhan

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Abstract The development of high throughput techniques and combinatorial libraries can facilitate rapid synthesis and screening of biomaterial-based nano-carriers for drug and vaccine delivery. This study describes an 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 1,8bis(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 while 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.


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Keywords Polyanhydride; high throughput synthesis; protein delivery; nanoparticles; surface functionalization

Introduction The use of combinatorial libraries and high throughput screening methods gained widespread support in the 1980s when the pharmaceutical industry successfully pioneered these techniques to synthesize and screen small molecules that would fill their drug pipelines.1 High throughput technologies are needed because of limitations in our ability to make accurate predictions about the activity and efficacy of drugs or drug delivery vehicles. They allow for a greater number of compounds to be screened enabling the selection of a drug or drug delivery vehicle with the most favorable properties. Combinatorial libraries are advantageous relative to conventional “onesample-at-a-time” synthesis because they have the potential to integrate multiple components including the synthesis and the screening process together, thereby reducing the overall workload.2,3 Key challenges in this regard are related to experimental design and the design of high throughput methods and automation equipment that most efficiently screen and select drugs or drug delivery vehicles for their optimal properties. Combinatorial methods have been used to optimize biomaterials design and drug delivery vehicles for many complex biomedical end-use applications, including targeting cancer, 3 ACS Paragon Plus Environment


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optimizing gene delivery, penetrating the blood brain barrier (BBB), and modulating the immune response.2–5 In particular, techniques that automate and accelerate the synthesis of libraries of polymeric nanoparticles and microparticles for use as drug delivery vehicles are of great interest. This is because there are many complex interactions between the drug, the delivery vehicle, and the cells or tissue interacting with the delivery vehicle.6,7 High throughput technologies have the ability to rapidly screen these interactions to identify and select those that are favorable. Previous work from our laboratory and other research groups has utilized these technologies to study the effect of polymer composition and device geometry on protein release.7–14 Studies with libraries of polyanhydrides based on sebacic acid (SA), 1,6-bis(p-carboxyphenoxy)hexane (CPH), and 1,8-bis(p-carboxyphenoxy)-3,6-dioxactane (CPTEG) demonstrated their high biocompatibility and their ability to activate macrophages.15–17 These studies demonstrated that polyanhydride formulations have excellent properties as drug delivery vehicles owing to their ability to stabilize proteins, to provide sustained release of a protein or drug (driven by the surface erosion mechanism of polyanhydrides), and to target specific cells.18–21 Additionally, polyanhydride chemistry is highly tunable (i.e., by varying copolymer composition), which allows the rate of drug release to be modulated.22 This work builds upon the previous studies via the design of a new and automated high throughput system for synthesizing and characterizing both polymer libraries as well as proteinloaded nanoparticle libraries. The high throughput method described herein efficiently synthesized polymer libraries, synthesized and functionalized nanoparticle libraries, and enabled rapid analysis of protein release kinetics from these libraries. Several processing parameters were investigated for their effect on nanoparticle size and antigen release, including nozzle velocity


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during anti-solvent nanoprecipitation and use of surfactants. The high throughout data was validated using data gathered from conventionally synthesized polymers and nanoparticles.

Results & Discussion Polymer and Nanoparticle Library Synthesis and Characterization This work describes the design and validation of a novel high throughput method, using an automated robot, for efficient synthesis of libraries of polymers and protein-loaded nanoparticles for rapid screening for properties that optimize their use as drug/vaccine delivery vehicles. Representative copolymers were synthesized with the high throughput method and characterized using proton nuclear magnetic resonance (1H NMR) spectroscopy and differential scanning calorimetry (DSC) (Table 1, Figure 1). The high throughput method enabled tight control over the molar composition of CPTEG:CPH (Figure 1a) and CPH:SA (Figure 1b) copolymers. It was observed that the glass transition temperature (Tg) gradually increased as the CPH content within the CPTEG:CPH copolymer increased. No defined melting point (Tm) was observed for these CPTEG:CPH copolymers (Figure 1c), in agreement with previous studies that showed that these copolymers were amorphous.23 Additionally, increasing the CPH composition generally increased the molecular weight of the CPTEG:CPH copolymers, suggesting that the CPH monomer is more reactive than the CPTEG monomer (Table 1). The semicrystalline CPH:SA copolymers showed well-defined Tg’s and Tm’s (Figure 1d). Both the Tg and Tm increased as the SA content increased within the copolymer (Table 1, Figure 1d). The molar composition, molecular weight, Tg, and Tm of copolymers synthesized at high throughput were in good agreement with the same properties of conventionally synthesized copolymers (Table 1).



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The high throughput method enabled facile synthesis of polymer libraries with properties that were consistent with that of conventionally synthesized polymers, but at significantly faster times. For example, in conventional synthesis, the reacted polymer is dissolved in methylene chloride and precipitated in hexanes.21,22 The polymer is filtered, dried, and stored until it is used for nanoparticle synthesis. In the high throughput method, the number of steps required to convert monomer or prepolymer to nanoparticles is drastically reduced. Herein, the monomer or prepolymer and its solvent are pumped directly into its corresponding test tube. This test tube is then heated under vacuum to evaporate the solvent and react the prepolymer, while the solvent is collected in a chilled dewar flask. This test tube is then stored until it is used for nanoparticle synthesis. As nanoparticle synthesis involves precipitation in pentane, unreacted monomers partition into the anti-solvent. This eliminated a separate polymer purification step. Moreover, the high throughput method does not utilize a rotary evaporator, removing a time-consuming bottleneck to conventional polymer synthesis. It is estimated that conventional synthesis of a single CPTEG:CPH copolymer requires approximately 2-3 h for laboratory preparation, six hours for monomer polymerization, 6-8 hours for copolymer to dissolve in methylene chloride, and six hours for copolymer precipitation in hexanes, filtration, and drying. This conventional method of polymer synthesis also requires a silicone bath and vacuum pump for each polymer synthesized each time, adding another timeconsuming bottleneck. In contrast, the high throughput method can simultaneously synthesize a library of 24 copolymers ranging in composition within 1-2 h of laboratory time and an additional 5 h for the monomers to polymerize with no time spent on precipitating the polymer. An equivalent library of 24 conventionally-synthesized copolymers would require approximately 144 hours of reaction time alone, assuming a one-sample-at-a-time method. Therefore, the high 6 ACS Paragon Plus Environment

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throughput synthesis method is more time-efficient relative to the conventional method because more copolymer compositions can be synthesized simultaneously in a much shorter period of time. The high throughput method also reduced material costs associated with polymer library synthesis. By eliminating the polymer precipitation step, the cost of hexanes is eliminated. Additionally, the ability to synthesize up to 24 copolymers at a time facilitates screening more polymer compositions, at small scale (90% of the encapsulated antigen was released in the first several days from the particles synthesized by both methods (Figure 4). This formulation also had the largest antigen burst, with 70-80% of the antigen being released in the first few hours. With its backbone consisting of aromatic rings and hexane, CPH is more hydrophobic relative to SA or CPTEG. As a result polymer chemistries rich in CPH are more hydrophobic and have the slowest protein release 9 ACS Paragon Plus Environment


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rates.18 Polymer chemistry also significantly affected antigen encapsulation efficiency (Table 3). Regardless of surfactant incorporation, the 20:80 CPTEG:CPH nanoparticles consistently had higher protein encapsulation efficiencies compared to that of the 20:80 CPH:SA nanoparticles. This is likely due to the presence of the less hydrophobic CPTEG monomer, which contains hydrophilic ethylene glycol moieties, and amphiphilic polymers have been shown to stabilize proteins in previous studies.19,37–42 Incorporating the surfactant into the nanoparticles had a weak effect on antigen release. The antigen burst was slightly increased when 0.2% Span 80 was used to synthesize 20:80 CPTEG:CPH nanoparticles, but the slope of the release rate was nearly identical compared to 20:80 CPTEG:CPH nanoparticles synthesized in the absence of any surfactant (Figure 4). Incorporating 0.2% or 1% Span 80 into 20:80 CPH:SA nanoparticles had minimal effect on antigen burst and antigen release rate. Incorporating surfactant into the nanoparticles did affect the antigen encapsulation efficiency. As expected, adding surfactant increased the amount of antigen encapsulated within the nanoparticles for both polymer chemistries (Table 3). This is because the surfactant enhances the stability of the insoluble protein as it is dispersed in the methylene chloride oil phase just prior to nanoprecipitation. Span-based surfactants have been extensively used for stabilization of emulsions.43 Surfactant function is quantified by its hydrophilic-lipophilic balance (HLB) value. Generally, surfactants with an HLB

Automated High Throughput Synthesis of Protein-Loaded

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