Self-assembling biological complexes such as viral capsids have been manipulated to function in innovative nanotechnology applications. The E2 component of pyruvate dehydrogenase from Bacillus stearothermophilus forms a dodecahedral complex and potentially provides another platform for these purposes. In this investigation, we show that this protein assembly exhibits unusual stability and can be modified to encapsulate model drug molecules. To distill the E2 protein down to its structural scaffold core, we synthesized a truncated gene optimized for expression in Escherichia coli. The correct assembly and dodecahedral structure of the resulting scaffold was confirmed with dynamic light scattering and transmission electron microscopy. Using circular dichroism and differential scanning calorimetry, we found the thermostability of the complex to be unusually high, with an onset temperature of unfolding at 81.1 +/- 0.9 degrees C and an apparent midpoint unfolding temperature of 91.4 +/- 1.4 degrees C. To evaluate the potential of this scaffold for encapsulation of guest molecules, we made variants at residues 381 and 239 which altered the physicochemical properties of the hollow internal cavity. These mutants, yielding 60 and 120 mutations within this cavity, assembled into the correct architecture and exhibited high thermostability that was comparable to the wild-type scaffold. To show the applicability of this scaffold, two different fluorescent dye molecules were covalently coupled to the cysteine mutant at site 381. We demonstrate that these mutations can introduce non-native functionality and enable molecular encapsulation within the cavity while still retaining the dodecahedral structure. The unusually robust nature of this scaffold and its amenability to internal changes reveal its potential for nanoscale applications.

Many current cancer vaccine strategies suffer from the inability to mount a CD8 T cell response that is strong enough to overcome the low immunogenicity of tumors. Viruses naturally possess the sizes, geometries, and physical properties for which the immune system has evolved to recognize, and mimicking those properties with nanoparticles can produce robust platforms for vaccine design. Using the non-viral E2 core of pyruvate dehydrogenase, we have engineered a viral-mimicking vaccine platform capable of encapsulating dendritic cell (DC)-activating CpG molecules in an acid-releasable manner and displaying MHC I-restricted SIINFEKL peptide epitopes. Encapsulated CpG activated bone marrow-derived DCs at a 25- fold lower concentration in vitro when delivered with the E2 nanoparticle than with unbound CpG alone. Combining CpG and SIINFEKL within a single multifunctional particle induced ~ 3-fold greater SIINFEKL display on MHC I by DCs over unbound peptide. Importantly, combining CpG and SIINFEKL to the E2 nanoparticle for simultaneous temporal and spatial delivery to DCs showed increased and prolonged CD8 T cell activation, relative to free peptide or peptide-bound E2. By co-delivering peptide epitopes and CpG activator in a particle of optimal DC-uptake size, we demonstrate the ability of a non-infectious protein nanoparticle to mimic viral properties and facilitate enhanced DC activation and cross-presentation.

The E2 component of pyruvate dehydrogenase has been engineered to form a caged, hollow dodecahedral protein assembly, and we have examined the feasibility of this scaffold to be used as a drug delivery system by introducing cysteines to the internal cavity (D381C). Fluorescent dye Alexa Fluor 532 (AF532M) and the antitumor drug doxorubicin were coupled to this internal cavity through maleimides on the guest molecules. The virus-like particle’s structure and stability remained intact after binding of the molecules within the interior of the nanocapsule. The pH-dependent hydrolysis of a hydrazone linkage to doxorubicin allowed 90% drug release from the D381C scaffold within 72 hrs at pH 5.0. Fluorescence microscopy of MDA-MB-231 breast cancer cells indicated significant uptake of the D381C scaffold incorporating AF532M and doxorubicin and suggested internalization of the nanoparticles through endocytosis. We observed that the protein scaffold does not induce cell death, but doxorubicin encapsulated in D381C is indeed cytotoxic, yielding an IC50 of 1.3 ± 0.3 μM. While the majority of particulate-based drug delivery strategies encapsulates drugs within polymeric nanoparticles, our results show the potential of using macromolecular protein assemblies. This approach yields a promising new opportunity for designing highly-defined nanomaterials for therapeutic delivery.

Caged protein nanoparticles possess many desirable features for drug delivery, such as ideal sizes for endocytosis, non-toxic biodegradability, and the ability to functionalize at three distinct interfaces (external, internal, and inter-subunit) using the tools of protein engineering. Researchers have harnessed these attributes by covalently and non-covalently loading therapeutic molecules through mechanisms that facilitate release within specific microenvironments. Effective delivery depends on several factors, including specific targeting, cell uptake, release kinetics, and systemic clearance. The innate ability of the immune system to recognize and respond to proteins has recently been exploited to deliver therapeutic compounds with these platforms for immunomodulation. The diversity of drugs, loading/release mechanisms, therapeutic targets, and therapeutic efficacy are discussed in this review.

The extracellular matrix (ECM) is a complex and dynamic structural scaffold for cells within tissues and plays an important role in regulating cell function. Recently it has become appreciated that the ECM contains bioactive motifs that can directly modulate immune responses. This review describes strategies for engineering immunomodulatory biomaterials that utilize natural ECM‐derived molecules and have the potential to harness the immune system for applications ranging from tissue regeneration to drug delivery. A top‐down approach utilizes full‐length ECM proteins, including collagen, fibrin, or hyaluronic acid‐based materials, as well as matrices derived from decellularized tissue. These materials have the benefit of maintaining natural conformation and structure but are often heterogeneous and encumber precise control. By contrast, a bottom‐up approach leverages immunomodulatory domains, such as Arg–Gly–Asp (RGD), matrix metalloproteinase (MMP)‐sensitive peptides, or leukocyte‐associated immunoglobulin‐like receptor‐1(LAIR‐1) ligands, by incorporating them into synthetic materials. These materials have tunable control over immune cell functions and allow for combinatorial approaches. However, the synthetic approach lacks the full natural context of the original ECM protein. These two approaches provide a broad range of engineering techniques for immunomodulation through material interactions and hold the potential for the development of future therapeutic applications.

We studied the molecular arrangement of two-dimensional streptavidin crystals at the air-water interface over a range of pH values. We quantified the varying amounts of coexisting P1, P2, and C222 crystals in the different morphologies observed at pH 4.5-6.5. Chiral, needlelike crystals at pH 4.5 consist of P1 crystals with frequent line defects. Larger chiral domains near pH 5 are essentially all P1 coexisting with a small amount of P2, whereas at slightly higher pH values (near pH 5.5), H-shaped domains contain 4 times as much P1 coexisting with a P2/C222 mixture. Morphologies intermediate to these shapes exhibit intermediate compositions. Between pH ∼6-7, crystals all display a characteristic dendritic-X morphology, but arrangement at the molecular level is quite different compared with lower pH values. Crystals are mostly P2 in symmetry near pH 6, but at pH 7 and above, crystals have C222 symmetry. Coexistence of P2 and C222 crystals occurs at intermediate pH values. We determined the orientation and arrangement of streptavidin molecules in P1, P2, and C222 crystals relative to the directions exhibiting faster growth. The direction of faster growth in P1 crystals includes both interactions between biotin-free subunits and interactions between biotin-bound subunits. In the P2 arrangement, growth in the direction of intermolecular biotin-free subunits is preferred, whereas growth is faster along the biotin-bound direction of C222 crystals. We developed a model of the molecular arrangement for the observed solid-phase coexistence in these crystals.

Self-assembling protein cages provide a wide range of possible applications in nanotechnology. We report the first example of an engineered pH-dependent molecular switch in a virus-like particle. By genetically manipulating the subunit-subunit interface of the E2 subunit of pyruvate dehydrogenase, we introduce pH-responsive assembly into a scaffold that is natively stable at both pH 5.0 and 7.4. The redesigned protein module yields an intact, stable particle at pH 7.4 that dissociates at pH 5.0. This triggered behavior is especially relevant for applications in therapeutic delivery.

The immune system is a powerful resource for the eradication of cancer, but to overcome the low immunogenicity of tumor cells, a sufficiently strong CD8+ T cell-mediated adaptive immune response is required. Nanoparticulate biomaterials represent a potentially effective delivery system for cancer vaccines, as they can be designed to mimic viruses, which are potent inducers of cellular immunity. We have been exploring the non-viral pyruvate dehydrogenase E2 protein nanoparticle as a biomimetic platform for cancer vaccine delivery. Simultaneous conjugation of a melanoma-associated gp100 epitope and CpG to the E2 nanoparticle (CpG-gp-E2) yielded an antigen-specific increase in the CD8+ T cell proliferation index and IFN-γ secretion by 1.5-fold and 5-fold, respectively, compared to an unbound peptide and CpG formulation. Remarkably, a single nanoparticle immunization resulted in a 120-fold increase in the frequency of melanoma epitope-specific CD8+ T cells in draining lymph nodes and a 30-fold increase in the spleen, relative to free peptide with free CpG. Furthermore, in the very aggressive B16 melanoma murine tumor model, prophylactic immunization with CpG-gp-E2 delayed the onset of tumor growth by approximately 5.5 days and increased animal survival time by approximately 40%, compared to PBS-treated animals. These results show that by combining optimal particle size and simultaneous co-delivery of molecular vaccine components, antigen-specific anti-tumor immune responses can be significantly increased.