Combinatorial design and development of biomaterials for use as drug delivery devices and immune adjuvants

There are several challenges associated with current strategies for drug and vaccine delivery. These include the need for multiple-dose administrations, which can hinder patience compliance, the requirements for specific storage conditions due to the fragile structure of protein-based molecules, and the need for additional excipients to enhance protein stability or adjuvant the immune response. This work has focused on the development of a high throughput, combinatorial approach to optimize degradable polymeric biomaterials, specifically polyanhydrides, to overcome these challenges associated with drug and vaccine delivery. We have developed high throughput techniques to rapidly fabricate polymer film and nanoparticle libraries to carry out detailed investigations of protein/biomaterial, cell/biomaterial, and host/biomaterial interactions. By developing and employing a highly sensitive fluorescence-based assay we rapidly identified that protein release kinetics are dictated by polymer chemistry, pH, and hydrophobicity, and thus can be tailored for the specific application to potentially eliminate the need for multiple-dose treatments. Further investigation of protein/biomaterial interactions identified polymer chemistry, pH, hydrophobicity, and temperature to be integral factors controlling protein stability during fabrication of the delivery device, storage, and delivery. Amphiphilic polymer chemistries were specifically identified to preserve the structure of both robust and fragile proteins from device fabrication to release. Our investigations of cell/biomaterial interactions revealed that all nanoparticle and polymer film chemistries studied were non-toxic at concentrations expected for human use. Furthermore, cellular activation studies were carried out with antigen presenting cells co-incubated with the polymer libraries which indicated that polymer films do not possess immune stimulating properties; however, the nanoparticles do, in a chemistry dependent manner. Combining these insights with informatics analysis, we discovered the molecular basis of the pathogen-mimicking behavior of amphiphilic polyanhydride nanoparticles. Specific molecular descriptors that were identified for this pathogen-mimicking behavior include alkyl ethers, % hydroxyl end groups, backbone oxygen content, and hydrophobicity. These findings demonstrated the stealth properties of polyanhydride films for tissue engineering and the pathogen-mimicking adjuvant properties of the nanoparticles for vaccine delivery. Finally, host/biomaterial interactions were studied, which indicated that polymer chemistry and administration route affect nanoparticle biodistribution and mucoadhesion. Amphiphilic nanoparticles were identified to reside longest at parenteral administration routes and adhere best to mucosal surfaces. These results point to their ability to provide a long-term antigen depot in vivo. In summary, the studies described in this thesis have created a rational design paradigm for materials selection and optimization for use as drug delivery vehicles and vaccine adjuvants, which will overcome the challenges associated with administration frequency, protein instability, and insufficient immune stimulation

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 polyanhydrides(1). 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 previously(1). This combinatorial platform has been validated with conventional methods(2) and the polyanhydride film and nanoparticle libraries have been characterized with (1)H 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 mucoadhesion(1-11). 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

Identifying factors controlling protein release from combinatorial biomaterial libraries via hybrid data mining methods

Polyanhydrides are a class of degradable biomaterials that have shown much promise for applications in drug and vaccine delivery. Their properties can be tailored for controlled drug release, drug/protein stability, and immune regulation (adjuvant effect). Identifying the relationship between the molecular structures of the polymers and the drug release kinetics profiles would help understand the release mechanism and aid in the accurate prediction of drug release and the rational design of polymer-based drug carrier systems. The molecular structure descriptors that had the most impact on the release kinetics were identified using a prediction/optimization data mining approach. Using this new approach for modeling nonlinear release kinetics behavior, we determined that the descriptors which had the greatest effect on the release kinetics were the number of backbone -COO- nonconjugated bonds, the number of aromatic rings, and the number of -CH 2- bonds

Amphiphilic polyanhydride films promote neural stem cell adhesion and differentiation

Several challenges currently exist for rational design of functional tissue engineering constructs within the host, which include appropriate cellular integration, avoidance of bacterial infections, and low inflammatory stimulation. This work describes a novel class of biodegradable, amphiphilic polyanhydrides with many desirable protein-material and cell-material attributes capable of confronting these challenges. The biocompatible amphiphilic polymer films were shown to release laminin in a stable and controlled manner, promote neural cell adhesion and differentiation, and evade inflammatory responses of the immune system. Using high-throughput approaches, it was shown that polymer chemistry plays an integral role in controlling cell-film interactions, which suggests that these polyanhydrides can be tailored to achieve the desired cell adhesion and differentiation while minimizing immune recognition. These findings have important implications for development of engineered constructs to regulate differentiation and target the growth of transplanted cells in stem cell-based therapies to treat nervous system disorders

Retention of structure, antigenicity, and biological function of pneumococcal surface protein A (PspA) released from polyanhydride nanoparticles

Pneumococcal surface protein A (PspA) is a choline-binding protein which is a virulence factor found on the surface of all Streptococcus pneumoniae strains. Vaccination with PspA has been shown to be protective against a lethal challenge with S. pneumoniae, making it a promising immunogen for use in vaccines. Herein, the design of a PspA-based subunit vaccine using polyanhydride nanoparticles as a delivery platform is described. Nanoparticles based on sebacic acid (SA), 1,6-bis-(p-carboxyphenoxy)hexane (CPH) and 1,8-bis-(p-carboxyphenoxy)-3,6- dioxaoctane (CPTEG), specifically 50:50 CPTEG:CPH and 20:80 CPH:SA, were used to encapsulate and release PspA. The protein released from the nanoparticle formulations retained its primary and secondary structure as well as its antigenicity. The released PspA was also biologically functional based on its ability to bind to apolactoferrin and prevent its bactericidal activity towards Escherichia coli. When the PspA nanoparticle formulations were administered subcutaneously to mice, the animals elicited a high titer and high avidity anti-PspA antibody response. Together, these studies provide a framework for the rational design of a vaccine against S. pneumoniae based on polyanhydride nanoparticles

Sichuan 512 catastrophe: rebuilding libraries after the earthquake

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Rational Design of Pathogen-Mimicking Amphiphilic Materials as Nanoadjuvants

An opportunity exists today for cross-cutting research utilizing advances in materials science, immunology, microbial pathogenesis, and computational analysis to effectively design the next generation of adjuvants and vaccines. This study integrates these advances into a bottom-up approach for the molecular design of nanoadjuvants capable of mimicking the immune response induced by a natural infection but without the toxic side effects. Biodegradable amphiphilic polyanhydrides possess the unique ability to mimic pathogens and pathogen associated molecular patterns with respect to persisting within and activating immune cells, respectively. The molecular properties responsible for the pathogen-mimicking abilities of these materials have been identified. The value of using polyanhydride nanovaccines was demonstrated by the induction of long-lived protection against a lethal challenge of Yersinia pestis following a single administration ten months earlier. This approach has the tantalizing potential to catalyze the development of next generation vaccines against diseases caused by emerging and re-emerging pathogens

Combinatorial design and development of biomaterials for use as drug delivery devices and immune adjuvants

There are several challenges associated with current strategies for drug and vaccine delivery. These include the need for multiple-dose administrations, which can hinder patience compliance, the requirements for specific storage conditions due to the fragile structure of protein-based molecules, and the need for additional excipients to enhance protein stability or adjuvant the immune response. This work has focused on the development of a high throughput, combinatorial approach to optimize degradable polymeric biomaterials, specifically polyanhydrides, to overcome these challenges associated with drug and vaccine delivery. We have developed high throughput techniques to rapidly fabricate polymer film and nanoparticle libraries to carry out detailed investigations of protein/biomaterial, cell/biomaterial, and host/biomaterial interactions. By developing and employing a highly sensitive fluorescence-based assay we rapidly identified that protein release kinetics are dictated by polymer chemistry, pH, and hydrophobicity, and thus can be tailored for the specific application to potentially eliminate the need for multiple-dose treatments. Further investigation of protein/biomaterial interactions identified polymer chemistry, pH, hydrophobicity, and temperature to be integral factors controlling protein stability during fabrication of the delivery device, storage, and delivery. Amphiphilic polymer chemistries were specifically identified to preserve the structure of both robust and fragile proteins from device fabrication to release. Our investigations of cell/biomaterial interactions revealed that all nanoparticle and polymer film chemistries studied were non-toxic at concentrations expected for human use. Furthermore, cellular activation studies were carried out with antigen presenting cells co-incubated with the polymer libraries which indicated that polymer films do not possess immune stimulating properties; however, the nanoparticles do, in a chemistry dependent manner. Combining these insights with informatics analysis, we discovered the molecular basis of the "pathogen-mimicking" behavior of amphiphilic polyanhydride nanoparticles. Specific molecular descriptors that were identified for this pathogen-mimicking behavior include alkyl ethers, % hydroxyl end groups, backbone oxygen content, and hydrophobicity. These findings demonstrated the stealth properties of polyanhydride films for tissue engineering and the pathogen-mimicking adjuvant properties of the nanoparticles for vaccine delivery. Finally, host/biomaterial interactions were studied, which indicated that polymer chemistry and administration route affect nanoparticle biodistribution and mucoadhesion. Amphiphilic nanoparticles were identified to reside longest at parenteral administration routes and adhere best to mucosal surfaces. These results point to their ability to provide a long-term antigen depot in vivo. In summary, the studies described in this thesis have created a rational design paradigm for materials selection and optimization for use as drug delivery vehicles and vaccine adjuvants, which will overcome the challenges associated with administration frequency, protein instability, and insufficient immune stimulation.</p