Protein crystal based materials for nanoscale applications in medicine and biotechnology.

The porosity, order, biocompatibility, and chirality of protein crystals has motivated interest from diverse research domains including materials science, biotechnology, and medicine. Porous protein crystals have the unusual potential to organize guest molecules within highly ordered scaffolds, enabling applications ranging from biotemplating and catalysis to biosensing and drug delivery. Significant research has therefore been directed toward characterizing protein crystal materials in hopes of optimizing crystallization, scaffold stability, and application efficacy. In this overview article, we describe recent progress in the field of protein crystal materials with special attention given to applications in nanomedicine and nanobiotechnology. This article is categorized under: Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Therapeutic Approaches and Drug Discovery > Emerging Technologies Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials.

Characterizing the Cytocompatibility of Various Cross-Linking Chemistries for the Production of Biostable Large-Pore Protein Crystal Materials.

With rapidly growing interest in therapeutic macromolecules, targeted drug delivery, and in vivo biosensing comes the need for new nanostructured biomaterials capable of macromolecule storage and metered release that exhibit robust stability and cytocompatibility. One novel possibility for such a material are engineered large-pore protein crystals (LPCs). Here, various chemically stabilized LPC derived biomaterials were generated using three cross-linking agents: glutaraldehyde, oxaldehyde, and 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide. LPC biostability and in vitro mammalian cytocompatibility was subsequently evaluated and compared to similarly cross-linked tetragonal hen egg white lysozyme crystals. This study demonstrates the ability of various cross-linking chemistries to physically stabilize the molecular structure of LPC materials-increasing their tolerance to challenging conditions while exhibiting minimal cytotoxicity. This approach produces LPC-derived biomaterials with promising utility for diverse applications in biotechnology and nanomedicine.

Adsorption-Coupled Diffusion of Gold Nanoclusters within a Large-Pore Protein Crystal Scaffold.

Large-pore protein crystals (LPCs) are ordered biologically derived nanoporous materials exhibiting pore diameters greater than 8 nm. These substantial pores distinguish LPCs from typical nanoporous scaffolds, enabling engineered LPC materials to readily uptake, immobilize, and release macromolecular guests. In this study, macromolecular transport within an LPC environment was experimentally and computationally investigated by studying adsorption-coupled diffusion of Au25(glutathione)18 nanoclusters within a cross-linked LPC scaffold via time-lapse confocal microscopy, bulk equilibrium adsorption, and hindered diffusion simulation. Equilibrium adsorption data is congruent with a Langmuir adsorption model, exhibiting strong binding behavior between nanoclusters and the scaffold. The standard Gibbs free energy of binding is equivalent to -37.2 kJ/mol, and the maximum binding capacity of 1.25 × 103 mg/g corresponds to approximately 29 nanoclusters per LPC unit cell. The hindered diffusion model showed good agreement with experimental data, revealing a pore diffusion coefficient of 3.7 × 10-7 cm2/s under low nanocluster concentration. Furthermore, the model was sufficient to determine adsorption and desorption kinetic values for ka and kd equal to 13 cm3/mol·s and 1.7 × 10-7 s-1, respectively. At higher nanocluster concentrations, the simulated pore diffusion coefficient could be reduced by 3 orders of magnitude to 3.4 × 10-10 cm2/s due to the effects of pore occlusion. This study demonstrates a strategy to analyze adsorption-coupled diffusion data to better understand complex transport of fluorescent macromolecules into LPCs. This approach fits the observable fluorescence data to the key molecular details and will benefit downstream efforts to engineer LPC-based nanoporous materials.

Programmed Assembly of Host-Guest Protein Crystals.

The binding and release of guest fluorescent proteins inside a protein crystal with 13 nm axial pores is controlled. Spatially segregated guest protein loading is achieved via sequential binding and release stages. Additionally, selective stabilization of the crystal exterior results in hollow crystalline shells.

Gold nanoparticle capture within protein crystal scaffolds.

DNA assemblies have been used to organize inorganic nanoparticles into 3D arrays, with emergent properties arising as a result of nanoparticle spacing and geometry. We report here the use of engineered protein crystals as an alternative approach to biologically mediated assembly of inorganic nanoparticles. The protein crystal's 13 nm diameter pores result in an 80% solvent content and display hexahistidine sequences on their interior. The hexahistidine sequence captures Au25(glutathione)∼17 (nitrilotriacetic acid)∼1 nanoclusters throughout a chemically crosslinked crystal via the coordination of Ni(ii) to both the cluster and the protein. Nanoparticle loading was validated by confocal microscopy and elemental analysis. The nanoparticles may be released from the crystal by exposure to EDTA, which chelates the Ni(ii) and breaks the specific protein/nanoparticle interaction. The integrity of the protein crystals after crosslinking and nanoparticle capture was confirmed by single crystal X-ray crystallography.