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1.  Biomimetic Design of Protein Nanomaterials for Hydrophobic Molecular Transport 
Advanced functional materials  2012;22(15):3170-3180.
Biomaterials such as self-assembling biological complexes have demonstrated a variety of applications in materials science and nanotechnology. The functionality of protein-based materials, however, is often limited by the absence or locations of specific chemical conjugation sites. In this investigation, we developed a new strategy for loading organic molecules into the hollow cavity of a protein nanoparticle that relies only on non-covalent interactions, and we demonstrated its applicability in drug delivery. Based on a biomimetic model that incorporates multiple phenylalanines to create a generalized binding site, we retained and delivered the antitumor compound doxorubicin by redesigning a caged protein scaffold. Through an iterative combination of structural modeling and protein engineering, we obtained new variants of the E2 subunit of pyruvate dehydrogenase with varying levels of drug-carrying capabilities. We found that an increasing number of introduced phenylalanines within the scaffold cavity generally resulted in greater drug loading capacities. Drug loading levels could be achieved that were greater than conventional nanoparticle delivery systems. These protein nanoparticles containing doxorubicin were taken up by breast cancer cells and induced significant cell death. Our novel approach demonstrates a universal strategy to design de novo hydrophobic binding domains within protein-based scaffolds for molecular encapsulation and transport, and it broadens the ability to attach guest molecules to this class of materials.
doi:10.1002/adfm.201200052
PMCID: PMC3603581  PMID: 23526705
biomimetic material; protein cage; bionanotechnology; drug delivery; hydrophobicity
2.  Complement Activation and Cell Uptake Responses toward Polymer-Functionalized Protein Nanocapsules 
Biomacromolecules  2012;13(4):974-981.
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 polyethylene 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.
doi:10.1021/bm300083e
PMCID: PMC3322319  PMID: 22416762
pyruvate dehydrogenase; E2; virus-like particle; polyethylene glycol; cell uptake; complement activation
3.  Assaying proline hydroxylation in recombinant collagen variants by liquid chromatography-mass spectrometry 
BMC Biotechnology  2012;12:51.
Background
The fabrication of recombinant collagen and its prescribed variants has enormous potential in tissue regeneration, cell-matrix interaction investigations, and fundamental biochemical and biophysical studies of the extracellular matrix. Recombinant expression requires proline hydroxylation, a post-translational modification which is critical for imparting stability and structure. However, these modifications are not native to typical bacterial or yeast expression systems. Furthermore, detection of low levels of 4-hydroxyproline is challenging with respect to selectivity and sensitivity.
Results
We have developed a new liquid chromatography-mass spectrometry (LC-MS) method to evaluate proline hydroxylation in recombinant collagen. This assay was tested in different Saccharomyces cerevisiae expression systems to evaluate the effect of gene ratio between prolyl-4-hydroxylase and collagen on the extent of hydroxylation. These systems used a human collagen III gene that was synthesized de novo from oligonucleotides. The LC-MS assay does not require derivatization, uses only picomoles of sample, and can measure proline hydroxylation levels in recombinant and native collagen ranging from approximately 0% to 40%. The hydroxylation values obtained by LC-MS are as accurate and as precise as those obtained with the conventional method of amino acid analysis.
Conclusions
A facile, derivatization-free LC-MS method was developed that accurately determines the percentage of proline hydroxylation in different yeast expression systems. Using this assay, we determined that systems with a higher collagen-to-hydroxylase gene copy ratio yielded a lower percentage of hydroxylation, suggesting that a specifically balanced gene ratio is required to obtain higher hydroxylation levels.
doi:10.1186/1472-6750-12-51
PMCID: PMC3443662  PMID: 22901055
Hydroxyproline; Liquid chromatography-mass spectrometry; LC-MS assay; Recombinant collagen
4.  Protein Nanocapsules Containing Doxorubicin as a pH-Responsive Delivery System 
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.
doi:10.1002/smll.201002242
PMCID: PMC3118673  PMID: 21456086
Drug delivery; 6-maleimidocaproyl hydrazone derivative of doxorubicin; virus-like particle; pyruvate dehydrogenase; pH-dependent hydrolysis

Results 1-4 (4)