Article pubs.acs.org/Biomac
On the Design of Composite Protein−Quantum Dot Biomaterials via Self-Assembly Ravish Majithia,†,‡ Jan Patterson,§ Sarah E. Bondos,*,§ and Kenith E. Meissner*,†,‡ †
Material Science and Engineering Interdisciplinary Program, Texas A&M University, College Station, Texas 77843, United States Department of Biomedical Engineering, Texas A&M University, College Station, Texas 77843, United States § Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas 77840, United States ‡
S Supporting Information *
ABSTRACT: Incorporation of nanoparticles during the hierarchical selfassembly of protein-based materials can impart function to the resulting composite materials. Herein we demonstrate that the structure and nanoparticle distribution of composite fibers are sensitive to the method of nanoparticle addition and the physicochemical properties of both the nanoparticle and the protein. Our model system consists of a recombinant enhanced green fluorescent protein−Ultrabithorax (EGFPUbx) fusion protein and luminescent CdSe−ZnS core−shell quantum dots (QDs), allowing us to optically assess the distribution of both the protein and nanoparticle components within the composite material. Although QDs favorably interact with EGFP-Ubx monomers, the relatively rough surface morphology of composite fibers suggests EGFP-Ubx-QD conjugates impact self-assembly. Indeed, QDs templated onto EGFP-Ubx film post-self-assembly can be subsequently drawn into smooth composite fibers. Additionally, the QD surface charge impacts QD distribution within the composite material, indicating that surface charge plays an important role in self-assembly. QDs with either positively or negatively charged coatings significantly enhance fiber extensibility. Conversely, QDs coated with hydrophobic moieties and suspended in toluene produce composite fibers with a heterogeneous distribution of QDs and severely altered fiber morphology, indicating that toluene severely disrupts Ubx self-assembly. Understanding factors that impact the protein−nanoparticle interaction enables manipulation of the structure and mechanical properties of composite materials. Since proteins interact with nanoparticle surface coatings, these results should be applicable to other types of nanoparticles with similar chemical groups on the surface.
■
the forces that influence protein−nanoparticle interactions potentially affect macroscale protein−nanoparticle composite materials synthesized by bottom-up self-assembly. Efforts to understand the interaction of protein monomers with the surface of nanoparticles suggest that factors including nanoparticle size, hydrophobicity, surface charge, and surface functional groups affect protein−nanoparticle interactions. 12−16 However, it is unclear whether these nanoscale results can be extrapolated to understand the synthesis of macroscale protein−nanoparticle composite materials. Differences in the structure and oligomeric state of the protein may alter the protein’s interaction with the nanoparticles. Conversely, the presence of the nanoparticles at early stages of protein assembly could impact the protein’s self-assembly process. In this work, we explore protein−nanoparticle interactions on the microscale by varying nanoparticle surface chemistry, protein sequence, and assembly technique while monitoring the impact on materials structure, protein and nanoparticle distribution, and the mechanical properties of the resulting
INTRODUCTION Composite protein−nanoparticle materials are an important class of hybrid biomaterials with potential applications in the fields of medicine, device design, and sensors.1,2 Polymeric protein materials, such as collagen, silk, and elastin, have intrinsic chemical or mechanical properties 3 which can complement the electrical, mechanical, and optical properties commonly found in inorganic nanoparticles. Recently, hierarchical protein self-assembly, from nanoscale monomers to ordered macroscale polymers, has been found particularly useful for bottom-up engineering of polymeric protein materials.4−11 During protein folding, unfolded polypeptides self-assemble to form unique nanoscale structures determined by the amino acid sequence of the polypeptide. In a subset of proteins, these folded structures further interact to generate homo- or hetero-oligomers of a defined size, and in special cases even extended oligomers are produced to form ordered materials. Such design motifs, which rely on bottom-up selfassembly, can also be adapted for the generation of polymeric protein−nanoparticle composites. Nanoparticles can be incorporated before, during, or after protein self-assembly, potentially engineering a wide array of composite materials from the same components. In addition, © 2011 American Chemical Society
Received: June 28, 2011 Revised: September 2, 2011 Published: September 5, 2011 3629
dx.doi.org/10.1021/bm200889k | Biomacromolecules 2011, 12, 3629−3637
Biomacromolecules
Article
materials. We introduce and explore one such polymeric protein−nanoparticle composite, an Ultrabithorax (Ubx) protein fiber containing luminescent semiconductor quantum dots (QDs), generated using a bottom-up hierarchical selfassembly technique. Ubx is a 380 amino acid Drosophila melanogaster Hox transcription factor containing a structured DNA-binding homeodomain and a significant amount of intrinsic disorder.6,17,18 During animal development, Hox proteins instigate position-specific developmental programs to differentiate repeated segments into unique body structures.17 Macroscale materials in the form of fibers, ropes, and sheets can be generated from recombinant Ubx protein which selfassembles under gentle conditions. 6 Ubx materials are mechanically robust. By altering fiber diameter, the breaking strength, breaking strain, and Young’s modulus can be tuned to values spanning an order of magnitude, ultimately changing the mechanism of extension.10 Recombinant Ubx also enables incorporation of full-length functional proteins into materials via gene fusion, in which the gene encoding the functional protein and the Ubx gene are placed in tandem without intervening stop codons. Expression of this fusion gene in E. coli creates a single polypeptide that maintains both Ubxmediated self-assembly and the functionality of the appended protein.19 In this study, we use an enhanced green fluorescent protein (EGFP)−Ubx fusion protein to render the protein component of the composite materials optically active, 19 allowing analysis of the distribution of protein within the composite materials. Ubx self-assembly offers an opportunity to incorporate and template nanoparticles to form macroscale materials. In this work, inorganic semiconductor nanoparticles, CdSe−ZnS core−shell QDs emitting at 620 nm, are incorporated with EGFP-Ubx (emission maximum at 509 nm) to form composite materials. QDs are chosen as representative nanoparticles because they have a narrow emission, broad absorption, and resistance to photobleaching. Since the QD emission does not overlap that of the EGFP protein, we can evaluate the distribution of the protein and nanoparticle components within the composite materials. In this work, factors perceived to be important in governing protein−nanoparticle interactions, such as nanoparticle surface functional groups, surface charge, and hydrophobicity, are evaluated in terms of their effects on the composite material on a micro- to macroscale, in contrast to nanoscale studies presented elsewhere. Typically, nanoparticle surfaces are chemically modified for compatibility, solubility, or reactivity in their intended application. Because these surface modifications cover the entire nanoparticle, it is the chemical properties of these surface groups, and not the underlying nanoparticle, that will determine the ability of nanoparticles to bind protein and be incorporated into composite materials. In addition to their photoluminescence, QDs may be functionalized with a wide variety of surface chemistries commonly used with other nanoparticles. Here, positively charged polyethylenimine (PEI)coated QDs (PEI-QDs)20 exhibiting amine surface groups (−NH2), negatively charged dihydrolipoic acid (DHLA)-coated QDs (DHLA-QDs)21 presenting carboxylic surface groups (−COOH), and trioctylphosphine oxide (TOPO)-coated QDs (TOPO-QDs)22,23 exhibiting hydrophobic alkane surface groups have been used to generate composite Ubx·QD materials. Changing surface chemistries (and consequently surface charges) on the QDs allows for simple optical evaluation of surface charge and hydrophobicity on the
structural and mechanical properties of the resulting composite materials. While these studies are specific to Ubx·QD composite materials, it is believed that similar design motifs can be applied to any self-assembling protein and nanoparticles with similar surface chemistries, to generate polymeric protein− nanoparticle composites.
■
MATERIALS AND METHODS
EGFP-Ubx Protein Expression and Purification. A fused gene encoding enhanced green fluorescent protein-Ubx1a (EGFP-Ubx) was cloned into the pET19b vector (Novagen), which appends a His-tag to the N-terminus of the corresponding fusion protein.19 The plasmid construct was transformed into BL21(DE3)pLysS E. coli cells. E. coli cultures were grown in Luria broth containing 50 μg/mL carbenicillin and 30 μg/mL chloramphenicol (LB) at 37 °C. 8 mL of an overnight culture, inoculated from a single colony, was used to inoculate a 1 L LB culture. Cultures were grown to an optical density at 600 nm of 0.6−0.8 and then cooled to 26 °C. EGFP-Ubx1a protein expression was induced with 1 mM IPTG and grown for an additional 105 min. Cells were harvested by centrifugation at 3500g for 30 min at 4 °C. Cell pellets corresponding to 1 L of culture were aliquoted and stored at −20 °C. Each aliquot was thawed at room temperature and lysed in 10 mL of lysis buffer (50 mM sodium phosphate buffer, pH 8.0, 5% glucose w/v, 500 mM NaCl, 1 Roche Complete Mini Protease Inhibitor Cocktail tablet, 0.8 mg/L DNase I). Cell lysates were centrifuged at 17000g for 30 min at 4 °C. The supernatant was loaded on 3 mL bed volume nickel−nitrilotriacetic acid (Ni-NTA) agarose resin column (Qiagen), which was pre-equilibrated with 30 mL of incubation buffer (5% glucose w/v, 500 mM NaCl, 50 mM sodium phosphate buffer, pH 8.0). The column was then washed with 50 mL volumes each of W1 buffer, W2 buffer, and W3 buffer, and 25 mL of W4 buffer (incubation buffer containing 0, 20, 40, and 80 mM imidazole, respectively). Protein was eluted with 14 mL of elution buffer (200 mM imidazole dissolved in equilibration buffer). Concentrations of the purified EGFP-Ubx1a protein samples were determined using the BioRad protein assay (BioRad). Approximately 2 mg of dithiothreitol (DTT) was added to each 2 mL elution volume to maintain the protein in the reduced state. On average Ubx purifications yielded 3 mg of protein per liter of culture at 70% purity. SDS-PAGE of disassembled fibers reveals fibers contain only full-length Ubx. Synthesis of QDs. Core−shell CdSe−ZnS QDs were synthesized in a coordinating solvent tri-n-octylphosphine oxide (TOPO, 99%, Aldrich) in accordance with previously published procedures. 22,23 The synthesis was performed in a single mode CEM Discover microwave reactor operating at 300 W, 2.45 GHz. Cadmium oxide (CdO, 99.99%, Alfa Aesar, 0.0514 g, 0.4 mmol) along with tetradecylphosphonic acid (TDPA,98%, Alfa Aesar, 0.2232 g, 0.8 mmol) and TOPO (3.7768, 9 mmol) were heated with continuous stirring in a 125 mL glass flask. The mixture was heated to ∼300 °C under argon (Ar) flow for 15 min. A selenium stock solution (0.0411 g, 0.5 mmol, Aldrich, 99%) dissolved in 2.4 mL (2 g) of tri-n-octylphosphine (TOP, 99%, Aldrich) TOP) was injected at 270 °C, and QDs were allowed to grow for 4 min. A ZnS shell was grown on the CdSe cores by injecting a mixture of Zn and S precursors: 1.6 mL (12 mmol) of dimethylzinc (DMZ, 1 M in heptane, Aldrich), 0.42 mL (2 mmol) of hexamethyldisilathiane (HMDS, Aldrich), and 6.3 mL (14 mmol) of TOP. The reaction mixture was heated for 30 min at 200 °C. The quantum yield of QDs increases on annealing the core−shell particles at temperatures ∼100 °C for a period of 2 h. The resulting TOPO coated CdSe−ZnS QDs (TOPO-QDs) were either used directly for experiments or further modified to generate DHLA-QDs or PEI-QDs. DHLA Coating of QDs. CdSe-ZnS QDs were coated with dihydrolipoic acid (DHLA) to suspend them in aqueous buffers. The ZnS shell on QDs was not annealed to increase coating efficiencies and stability of DHLA-QDs. DHLA was freshly prepared in accordance with previously reported procedures.21 Briefly, 4 g of (±)-α-lipoic acid (98%, Sigma) was reduced with a fresh stock of 2.96 g of sodium borohydride (NaBH4, 98%). The reaction mixture was 3630
dx.doi.org/10.1021/bm200889k | Biomacromolecules 2011, 12, 3629−3637
Biomacromolecules
Article
allowed to stir for 2 h, acidified with ∼15 mL of 12 M hydrochloric acid to pH 12. DHLA was extracted with ∼100 mL of toluene, with excess water removed by addition of anhydrous MgSO4. Toluene was removed by evaporation leaving behind a ∼4−5 mL of clear DHLA solution. About 0.5 mL of pure DHLA was added to few hundred milligrams of QDs and heated at ∼80 C for 10−12 h on a hot plate with continuous stirring. The mixture was suspended in methanol. Approximately 1 g of potassium tert-butoxide (K-tBuO) was added to the mixture. K-tBuO deprotonates the carboxylic groups of DHLA on the surface of the QDs and imparts ionic stability to the nanocrystals in a basic aqueous buffer. QDs were then suspended in a phosphate buffer at pH 8. To remove excess K-tBuO, DHLA-QDs were filtered using first a 0.2 μm syringe filter (Nalgene, PES 0.2 μm) and then a 100 kDa centrifuge filter. PEI Coating of QDs. TOPO-QDs were coated with high molecular weight branched polyethylenimine (PEI). (Aldrich, MW 25 000) using similarly reported procedures.20 Briefly, 1 mL of 10 mg/ mL solution of PEI in chloroform was mixed with 1 mL of 4−5 μM QDs solution in chloroform. The mixture was tumbled overnight at room temperature. QDs were precipitated from the mixture by addition of excess cyclohexane (Sigma-Aldrich, >99%) followed by centrifugation and resuspended in DI water. Excess PEI was extracted from the aqueous QD solution by addition of fresh chloroform. Chloroform and water, being immiscible, can be phase separated, a process which can be accelerated by centrifugation. QDs, suspended in the lighter aqueous phase, were removed by pipetting.
■
RESULTS AND DISCUSSION Surface Self-Assembly of EGFP-Ubx. Previously, Bondos et al. have reported recombinant Ubx protein (non-EGFP) self-assembles under gentle conditions at the air−water interface to form nanometer-scale fibrils and films. Films can be drawn into robust and highly extensible fibers by means of a needle or a pipet tip.6,10,19 Unlike most other techniques, the method reported by Bondos and co-workers allows for facile synthesis of protein in E. coli cells, purification to near homogeneity, and subsequent self-assembly under mild conditions in aqueous buffer. Production of recombinant Ubx in E. coli cells, an organism with well-established molecular biology protocols, enables manipulation of the Ubx amino acid sequence to create fusion proteins such as EGFP-Ubx, which is used here. Synthesis details of EGFP-Ubx functional materials are reported elsewhere.19 EGFP-Ubx purification generates protein concentrations ≥0.75 mg/mL. EGFP-Ubx protein self-assembles at this concentration at the air−water interface when drops of proteins are exposed to ambient conditions for a period of at least 2 h. Although later stages of Ubx-mediated self-assembly have been previously observed by scanning electron microscopy, the addition of nanoparticles at different stages of EGFP-Ubx assembly requires understanding the oligomeric state of EGFPUbx at early assembly stages. To observe the hierarchical selfassembly under ambient conditions, the “sessile drop” technique was used to pull EGFP-Ubx fibers.10 In this approach, drops of EGFP-Ubx protein (100 μL) were placed on a strip of Parafilm. After 15 min, 1 h, and 2 h, the surface film was sampled by floating a 100 nm thick carbon films (on previously unsampled drops), which were subsequently lifted using TEM grids, stained with phospotungstic acid (PTA, 2% w/v), and imaged by a JEOL 1200 transmission electron microscope (TEM). The protein monomers interact with each other and initiate self-assembly. After 15 min of incubation, the surfaces of the protein drops show the formation of small globular protein aggregates typically sub-25 nm in size (Figure 1). These aggregates interact to form small protofibrils ∼50 nm
Figure 1. TEM images and micrographs showing hierarchical bottomup self-assembly of EGFP-Ubx protein at the air−water interface. EGFP-Ubx aggregates are formed initially after 15 min incubation and self-assemble to form protofibrils within 1 h. After 2 h, lateral association among protofibrils generates fibrils
