Efficient delivery of antigens is of paramount concern in immunotherapies. We aimed to target antigen presenting cells (APCs) by conjugating CpG oligonucleotides to an E2 protein nanoparticle surface (CpG-PEG-E2). Compared to E2 alone, we observed ∼4-fold increase of in vitro APC uptake of both CpG-PEG-E2 and E2 conjugated to non-CpG DNA. Furthermore, compared to E2-alone or E2 functionalized solely with polyethylene glycol (PEG), the CpG-PEG-E2 showed enhanced lymph node retention up to at least 48 h and 2-fold increase in APC uptake in vivo, parameters which are advantageous for vaccine success. This suggests that enhanced APC uptake of nanoparticles mediated by oligonucleotide display may help overcome delivery barriers in vaccine development.Keywords: biodistribution; CpG; dendritic cell targeting; E2; nanoparticle vaccine; oligonucleotide; polyethylene glycol;

AbstractLeukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is an inhibitory receptor broadly expressed on immune cells, with its ligands residing within the extracellular matrix protein collagen. In this study, surfaces are modified with a LAIR-1 ligand peptide (LP), and it is observed that macrophages cultured on LAIR-1 LP-conjugated surfaces exhibit significantly reduced secretion of inflammatory cytokines in response to proinflammatory stimuli that reflect an injured environment. These downregulated mediators include TNF-α, MIP-1α, MIP-1β, MIP-2, RANTES, and MIG. Knockdown of LAIR-1 using siRNA abrogates this inhibition of cytokine secretion, supporting the specificity of the inhibitory effect to this receptor. These results are the first to demonstrate that integration of LAIR-1 ligands with biomaterials could suppress inflammatory responses.

Collagen is the most abundant protein in extracellular matrices and is commonly used as a tissue engineering scaffold. However, collagen and other biopolymers from native sources can exhibit limitations when tuning mechanical and biological properties. Cysteines do not naturally occur within the triple-helical region of any native collagen. We utilized a novel modular synthesis strategy to fabricate variants of recombinant human collagen that contained 2, 4, or 8 non-native cysteines at precisely defined locations within each biopolymer. This bottom-up approach introduced capabilities using sulfhydryl chemistry to form hydrogels and immobilize bioactive factors. Collagen variants retained their triple-helical structure and supported cellular adhesion. Hydrogels were characterized using rheology, and the storage moduli were comparable to fibrillar collagen gels at similar concentrations. Furthermore, the introduced cysteines functioned as anchoring sites, with TGF-β1-conjugated collagens promoting myofibroblast differentiation. This approach demonstrates the feasibility to produce custom-designed collagens with chemical functionality not available from native sources.

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 nonviral 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 codelivering peptide epitopes and CpG activator in a particle of optimal DC-uptake size, we demonstrate the ability of a noninfectious protein nanoparticle to mimic viral properties and facilitate enhanced DC activation and cross-presentation.Keywords: biomimetic; CpG; cross-presentation; dendritic cell; nanoparticle vaccine; protein cage; virus-like particle

Self-assembling protein nanocapsules can be engineered for various bionanotechnology applications. Using the dodecahedral scaffold of the E2 subunit from pyruvate dehydrogenase, we introduced non-native surface cysteines for site-directed functionalization. The modified nanoparticle’s structural, assembly, and thermostability properties were comparable to the wild-type scaffold (E2-WT), and after conjugation of poly(ethylene glycol) (PEG) to these cysteines, the nanoparticle remained intact and stable up to 79.7 ± 1.8 °C. PEGylation of particles reduced uptake by human monocyte-derived macrophages and MDA-MB-231 breast cancer cells, with decreased uptake as PEG chain length is increased. In vitro C4-depletion and C5a-production assays yielded 97.6 ± 10.8% serum C4 remaining and 40.1 ± 6.0 ng/mL C5a for E2-WT, demonstrating that complement activation is weak for non-PEGylated E2 nanoparticles. Conjugation of PEG to these particles moderately increased complement response to give 79.7 ± 6.0% C4 remaining and 87.6 ± 10.1 ng/mL C5a. Our results demonstrate that PEGylation of the E2 protein nanocapsules can modulate cellular uptake and induce low levels of complement activation, likely via the classical/lectin pathways.

Two-dimensional non-close-packed crystals of the protein streptavidin, grown on phospholipid membranes, can serve as nanoscale templates capable of directing the formation of ordered nanoparticle arrays through site-specific electrostatic adsorption. Here we examine the effects of both interparticle and nanoparticle/lipid membrane electrostatic interactions on the degree of structural order exhibited by the templated nanoparticle array. Interparticle electrostatic repulsion is shown to have only marginal influence on nanoparticle ordering. In contrast, the degree of order exhibited by the templated array can be tuned by controlling the charge on the lipid membrane. Analysis of the local and global structure of arrays generated with negatively charged gold nanoparticles (∼6 nm) indicate improved long-range order when the lipid membrane supporting the protein crystal is derived from cationic lipid molecules as opposed to zwitterionic phospholipids. Furthermore, as nanoparticle size is reduced (∼3 nm), the presence of a charged lipid membrane is found to be essential, as smaller particles do not adhere to streptavidin crystals grown on zwitterionic membranes. These findings demonstrate that the composition of the lipid support can influence the efficacy of directed-assembly processes which utilize protein templates and are important results toward enhancing control over bottom-up nanofabrication applications.

A collagen-mimetic polymer that can be easily engineered with specific cell-responsive and mechanical properties would be of significant interest for fundamental cell-matrix studies and applications in regenerative medicine. However, oligonucleotide-based synthesis of full-length collagen has been encumbered by the characteristic glycine-X-Y sequence repetition, which promotes mismatched oligonucleotide hybridizations during de novo gene assembly. In this work, we report a novel, modular synthesis strategy that yields full-length human collagen III and specifically defined variants. We used a computational algorithm that applies codon degeneracy to design oligonucleotides that favor correct hybridizations while disrupting incorrect ones for gene synthesis. The resulting recombinant polymers were expressed in Saccharomyces cerevisiae engineered with prolyl-4-hydroxylase. Our modular approach enabled mixing-and-matching domains to fabricate different combinations of collagen variants that contained different secretion signals at the N-terminus and cysteine residues imbedded within the triple-helical domain at precisely defined locations. This work shows the flexibility of our strategy for designing and assembling specifically tailored biomimetic collagen polymers with re-engineered properties.

The protein streptavidin exhibits unique properties advantageous for “bottom-up” nanofabrication applications. It self-assembles into various 2-D crystalline lattices onto which nanoparticles can be attached through both electrostatic and ligand−receptor mechanisms. We examine the electrostatic adsorption of gold nanoparticles onto non-close-packed streptavidin crystals and show that site-specific attachment preferentially occurs in between protein molecules. The resulting nanoparticle arrangement consequently displays a long-range structural coherence with the underlying protein lattice, although with a reduced degree of order relative to that of the biological template. Monte Carlo simulations reveal that this remittent ordering is due to (1) the random offset between the nanoparticles and their respective adsorption sites and (2) nonspecific binding to the surface of the protein molecules. Overall, our results indicate that streptavidin crystals are capable of templating ordered nanoparticle arrays.

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.

Self-assembling protein cage structures have many potential applications in nanotechnology, one of which is therapeutic delivery. For intracellular targeting, pH-controlled disassembly of virus-like particles and release of their molecular cargo is particularly strategic. We investigated the potential of using histidines for introducing pH-dependent disassembly in the E2 subunit of pyruvate dehydrogenase. Two subunit interfaces likely to disrupt stability, an intratrimer interface (the N-terminus) and an intertrimer interface (methionine-425), were redesigned. Our results show that changing the identity of the putative anchor site 425 to histidine does not decrease stability. In contrast, engineering non-native pH-dependent behavior and modulating the transition pH at which disassembly occurs can be accomplished by mutagenesis of the N-terminus and by ionic strength changes. The observed pH-triggered disassembly is due to electrostatic repulsions generated by histidine protonation. These results suggest that altering the degree of electrostatic repulsion at subunit interfaces could be a generally applicable strategy for designing pH-triggered assembly in protein macromolecular structures.

The geometric and physicochemical properties of the protein streptavidin make it a useful building block in the construction and manipulation of nanoscale structures and devices. However, one requirement in exploiting streptavidin for “bottom-up” assembly is the capability to modulate protein−nanoparticle interactions. This work examines the effects of pH and the biotin−streptavidin interaction on the adsorption of colloidal gold onto a two-dimensional streptavidin crystal. Particle deposition was carried out below (pH 6), at (pH 7), and above (pH 8) the protein’s isoelectric point with both biotinylated and nonbiotinylated nanoparticles. Particle surface coverage depends on deposition time and pH, and increases by 1.4−10 times when biotin is incorporated onto the particle surface. This coverage is highest for both particle types at pH 6 and decreases monotonically with increasing pH. Calculations of interparticle potentials based on Derjaguin−Landau−Verwey−Overbeek (DLVO) theory demonstrate that this trend in surface coverage is most likely due to alterations in particle−surface electrostatic interactions and not a result of changes in interparticle electrostatic repulsion. Furthermore, post-adsorption alterations in pH demonstrate that electrostatically adsorbed particles can be selectively desorbed from the surface. Evaluation of the nonspecifically adsorbed fraction of biotinylated particles indicates that the receptor−ligand adsorption mechanism gives a higher rate of attachment to the substrate than nonspecific, electrostatic adsorption. This results in faster adsorption kinetics and higher coverages for biotinylated particles relative to the nonbiotinylated case.

Highly ordered protein arrays have been proposed as a means for templating the organization of nanomaterials. Toward this end, we investigate the ability of the protein streptavidin to self-assemble into various configurations on solid-supported phospholipids. We identify two genetic variants of streptavidin (comprising amino acids 14−136 and 13−139) and examine their molecular organization at the liquid−solid interface. Our results demonstrate that the structural differences between these two protein variants affect both crystalline lattice and domain morphology. In general, these results for the liquid−solid interface are similar and consistent with those at the air−water interface with a few notable differences. Analogous to crystallization at the air−water interface, both forms of streptavidin yield H-like domains with lattice parameters that have C222 symmetry at pH 7. At pH 4, the native, truncated form of streptavidin yields needle-like domains consisting of molecules arranged in P1 symmetry. Unlike crystalline domains grown at the air−water interface, however, the lattice parameters of this P1 crystal are unique and have not yet been reported. The presence of a solid substrate does not appear to dramatically alter streptavidin’s two-dimensional crystallization behavior, suggesting that local intermolecular interactions between proteins are more significant than interactions between the interface and protein. Our results also demonstrate that screening the electrostatic repulsion between protein molecules by modulating ionic strength will increase growth rate while decreasing crystalline domain size and macroscopic defects. Finally, we show that these domains are indeed functional by attaching biotinylated gold nanoparticles to the crystals. The ability to modulate molecular configuration, crystalline defects, and domain size on a functional array supports the potential application of this system toward materials assembly.

•Caged proteins can overcome limitations of other nanoparticles for drug delivery.•Unique physical, chemical, and recombinant methods allow drug loading/release.•Simultaneously display of drug and targeting molecules can improve drug efficacy.•Caged proteins are well-suited for immunotherapy because of similarities with viruses.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.Download high-res image (392KB)Download full-size image

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.