Article pubs.acs.org/Langmuir
Target-Specific Copper Hybrid T7 Phage Particles Siva Sai Krishna Dasa,#,† Qiaoling Jin,†,‡ Chin-Tu Chen,†,§ and Liaohai Chen*,† †
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah Division of X-ray Science, Argonne National Laboratory, Argonne, Illinois 60439, United States § Department of Radiology, University of Chicago, Chicago, Illinois 60637, United States ‡
S Supporting Information *
ABSTRACT: Target-specific nanoparticles have attracted significant attention recently, and have greatly impacted life and physical sciences as new agents for imaging, diagnosis, and therapy, as well as building blocks for the assembly of novel complex materials. While most of these particles are synthesized by chemical conjugation of an affinity reagent to polymer or inorganic nanoparticles, we are promoting the use of phage particles as a carrier to host organic or inorganic functional components, as well as to display the affinity reagent on the phage surface, taking advantage of the fact that some phages host well-established vectors for protein expression. An affinity reagent can be structured in a desired geometry on the surface of phage particles, and more importantly, the number of the affinity reagent molecules per phage particle can be precisely controlled. We previously have reported the use of the T7 phage capsid as a template for synthesizing target-specific metal nanoparticles. In this study herein, we reported the synthesis of nanoparticles using an intact T7 phage as a scaffold from which to extend 415 copies of a peptide that contains a hexahistidine (6His) motif for capture of copper ions and staging the conversion of copper ions to copper metal, and a cyclic Arginine-Glycine-Aspartic Acid (RGD4C) motif for targeting integrin and cancer cells. We demonstrated that the recombinant phage could load copper ions under low bulk copper concentrations without interfering with its target specificity. Further reduction of copper ions to copper metal rendered a very stable copper hybrid T7 phage, which prevents the detachment of copper from phage particles and maintains the phage structural integrity even under harsh conditions. Cancer cells (MCF-7) can selectively uptake copper hybrid T7 phage particles through ligand-mediated transmembrane transportation, whereas normal control cells (MCF-12F) uptake 1000-fold less. We further demonstrated that copper hybrid T7 phage could be endocytosed by cancer cells in culture.
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INTRODUCTION Nanoparticles are the cornerstones for nanotechnology, and have transformed and revolutionized both physical and life sciences.1−6 Target-specific nanoparticles play critical roles in enabling the application of nanoparticles in disease diagnosis and treatment, as well as the synthesis of nanoparticle-based complex materials.7−10 In biomedical research, although a generic nanoparticle could be disease-specific in some cases (for example, tumors may take up more nanoparticles compared to normal tissues, due to their leaky vascularity and the size of nanoparticles), in most cases, target-specific nanoparticles are essential to achieve the use of nanoparticles in applications ranging from therapeutics (such as hypothermia),11−13 therapeutic enhancement (such as radiation therapeutic efficacy enhancement),14 target-specific drug delivery,15−17 and imaging contrast agents or probes.18−21 Because they are target-specific, the local concentration of the nanoparticles at disease-affected tissues can be much higher, while bulk concentration of the nanoparticles can be low, thus significantly reducing their toxicity to normal tissue.22−25 In order to generate target-specific nanoparticles, an affinity reagent must be incorporated on the surface of nanoparticles. With advances in organic and inorganic synthesis, this can be © 2012 American Chemical Society
routinely achieved by conjugating the affinity reagent to the particle surface chemically. The challenge is how to generate uniformly functional particles, i.e., to precisely control the number of affinity reagents per particle, and to make sure that the recognition function of the affinity reagent is not altered due to the chemical conjugation. While the combination of affinity reagent mutagenesis and conjugation chemistry could partially address the problem,26−29 one of the definitive ways is to use molecular biology approaches to incorporate the affinity reagent to biological nanoparticles. One promising candidate of biological nanoparticles is the viral particle, especially bacteriophage particles. Bacteriophages (“phage(s)” thereafter) have been used as templates to synthesize target-specific imaging reagents, magnetic nanoparticles, and to assemble novel catalytic systems.30−32 Phage particles are not only robust and uniform in structure, but also easy to amplify and economic to produce. Another intrinsic advantage of using phage as a template for target-specific nanoparticle synthesis is that phage particles Received: June 19, 2012 Revised: November 14, 2012 Published: November 19, 2012 17372
dx.doi.org/10.1021/la3024919 | Langmuir 2012, 28, 17372−17380
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Article
TEM grids, the grids were glow discharged so that the samples would bind easily to the grids. Aliquots of approximately 10 μL of the samples were placed on TEM grids and allowed to sit for 30 s. After this period, the TEM grids were washed with sterile water followed by staining the samples with 2% uranyl acetate solution for 10 s. The stain was dried using filter paper and imaging was carried out using FEI Tecnai F30 scanning transmission electron microscope (STEM) at the Electron Microscopy Center, University of Chicago. For samples without stain, the grids were dried after washing and then used for imaging. Dynamic Light Scattering (DLS). T7 samples were diluted to less than 106 phage/mL, and 10 μL aliquots of the samples were placed in a 384-well glass-bottom plate. The measurement was taken at 22 °C, and for every sample, 25 measurements were taken using a Wyatt microplate DLS. αVβ3 Integrin Binding Assay. An aliquot of 1 μg/mL of integrins (human integrin αVβ3, Millipore) was prepared in saline, and 100 μL was added to each well in a 96-well ELISA black plate. The plate was incubated at 4 °C overnight, and the next day the excess integrins were removed by tapping the plate on paper towels, and the wells were washed twice with PBS. An aliquot of 100 μL of phage dilution was added to wells coated with integrins and incubated for 2 h at room temperature. The excess phage was removed, and the wells were rinsed twice with PBS, followed by incubation with primary antibody (antiT7 antibody, gift from Dr. Toshiyuki Mori, Biomedical Research Laboratories, Takeda Pharmaceutical Company Ltd., Osaka, Japan) directed against T7 phage for 2 h at room temperature. The excess antibody was removed, and washed twice with PBS followed by incubation with secondary antibody labeled with Alexa fluor 488, for 2 h at room temperature. The excess antibody was removed and rinsed using PBST (0.005% Tween 20), and finally, 200 μL of PBS was added to the wells. The fluorescence intensity at 535 nm was measured using a fluorescence ELISA plate reader. A curve was plotted with fluorescence intensity vs phage concentration to analyze phage binding to integrins. Copper Ion Reduction to Copper Metal Using Sodium Borohydride. Copper chloride at 15 μM concentration was used as a copper precursor, and was reduced using sodium borohydride. The reaction was carried out at room temperature for 1 h with continuous stirring.35−38 After the reaction, the samples were centrifuged to separate the phage particles, and were quantified for copper association using bathocuproine solution as described below. Before using bathocuproine, the copper metal was oxidized to Cu2+ with nitric acid. Copper Quantification Using Bathocuproine Disulfonate Solution. Cuprous ion forms a water-soluble orange-colored chelate with bathocuproine disulfonate (2,9-dimethyl-4,7-diphenyl-1,10-phenanthrolinedisulfonic acid, disodium salt), while the color forms over the pH range between 4 and 5. The sample was buffered at a pH of approximately 4.3, and was then reduced with hydroxylamine hydrochloride. The absorbance was measured at 484 nm. The absorbance at 484 nm correlates with the copper concentration, with higher absorbance values correlating to higher copper concentration.39−43 The method can be applied to measure copper concentrations of up to at least 5 mg/L with sensitivity up to 20 μg/L. All of the chemicals used for this protocol were obtained from Sigma (St. Louis, MO), unless described otherwise. To 1 mL of sample (phage pellet, obtained by spinning at 60 000 rpm for 1 h, 153 000 × g), 20 μL HCl, 100 μL hydroxylamine hydrochloride, 100 μL sodium acetate solution, and 100 μL bathocuproine solution were added and incubated for 5 min. The absorbance was measured at 484 nm, and the copper concentration from the T7 sample was obtained from a standard copper concentration curve. Nitric acid was used to oxidize copper metal or copper +1 to copper +2 and pH (between 4 and 5) was adjusted using hydroxylamine hydrochloride and sodium acetate followed by bathocuproine addition. Phen-green fluorescent indicator (Molecular Probes/Invitrogen) for copper ions was also used to quantify copper ions.. The assay was conducted based on the manufactory’s protocol. The fluorescence of Phen-green is quenched by copper ions, and thus, the concentration of
specific to virtually any molecules (ranging from proteins, oligos, organic polymers to inorganics) can be readily generated via phage display technology.30−33 In phage display, ligands (such as recombinant antibody fragments, cDNAencoded segments, or combinatorial peptides) are expressed as fusions to a capsid protein present on the surface of viral particles. Libraries of millions to billions of phage particles, each displaying a different fusion protein, are screened (usually by affinity selection) for members displaying the desired properties or binding affinities, which will lead to the generation of targetspecific phage particles. In this paper, we report the use of an intact T7 phage as a template to synthesize target-specific metal nanoparticles. T7 is a virulent phage with an icosahedral head (capsid) with a diameter of ∼50 nm, a short noncontractile tail (∼20 nm), and a dsDNA genome of 39 937 bp. The head, which is made of protein gp10A and gp10B, presents an icosahedral symmetry with triangulation number (T) = 7. T7 phage was chosen as it is easy to isolate and purify and, most importantly, hosts one of the most commonly used vectors for protein expression. A multicomponent peptide that contains both a copper ion binding polyhistidine (6His) motif and a cancer-specific cyclic Arginine-Glycine-Aspartic Acid (RGD4C)34 motif was displayed on the surface of T7 phage. The RGD4C motif is placed at the end of c-terminal (exterior of the capsid) of major capsid protein, and the 6His motif is present right before RGD4C separated by a spacer arm GGGS. Copper ions were chosen with the aim of developing a target-specific positron emission tomography (PET) imaging probe, as copper-64 has become a popular isotope for PET imaging. We demonstrated that the copper-loaded T7 phage due to the presence of the metal binding motif could specifically interact with cancer cells. By converting copper ions to copper metal on the phage surface, we were able to generate stable, cancer-specific, copper hybrid T7 phage particles. The behavior of these particles against both normal and tumor cells was investigated.
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EXPERIMENTAL PROCEDURES
Display of 6His-RGD4C Peptide on T7 Phage Capsid. For displaying peptides on T7 phage, we used the T7select415 cloning kit (Emd Chemicals). We displayed 6His peptide (HHHHHH) and 6His along with RGD4C (HHHHHHGGGS-CDCRGDCFC) sequence as a single peptide, as part of the T7 capsid protein. The DNA sequence that codes for 6His and RGD4C peptide was synthesized, modified, and ligated into precut vectors arms as described by the manufacturer (Emd Chemicals). The DNA inserts that were cloned were as follows: Copper binding/6His DNA insert sequence Forward-[Phos] AAT TCG CAT CAT CAC CAT CAC CAC TAA and reverse-[Phos] AGC TTT AGT GGT GAT GGT GAT GAT GCG; and for 6His and RGD4C, forward-[Phos] AAT TCG CAT CAT CAC CAT CAC CAC GGA GGT GGA TCT TGT GAT TGT CGT GGT GAT TGC TTC TGT TAA and reverse-[Phos] A GCT TTA ACA GAA GCA ATC ACC ACG ACA ATC ACA AGA TCC ACC TCC GTG GTG ATG GTG ATG ATG CG. These oligonucleotides were mixed to form dsDNA fragments, which were then ligated into T7 select vector that was precut with EcoR1 and Hind III restriction enzymes. The DNA fragment ligated into the vector becomes part of the c-terminus major capsid protein. Amplification, purification, and plaque assay were carried out as described by the manufacturer (T7 Select, Emd Chemicals). Recombinant phage samples were characterized by TEM and DLS to determine the purity of the phage preparation. Transmission Electron Microscopy (TEM) of T7 Phage. To image T7 phage particles using TEM, 200-mesh copper TEM grids coated with carbon were used. Before the samples were placed on 17373
dx.doi.org/10.1021/la3024919 | Langmuir 2012, 28, 17372−17380
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Figure 1. (a) TEM image of recombinant 6His-RGD4C T7 and (b) ELISA results of 6HisRGD4C T7 and wild-type T7 against αVβ3 integrins. PBS. The cells were fixed using 4% paraformaldehyde and incubating for 20 min at room temperature, followed by two washes with PBS. The cells were then stained for nuclei using Hoechst 33342 for 1 min, followed by 2−3 washes with PBS. Images were taken using the 100× oil immersion objective, and the glass coverslips from the cell culture dishes were imaged using an Olympus DSU spinning disk confocal (IX81) at the Microscope Facility at the University of Chicago. MTT Assay. The MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay was conducted by following the manufacturer's protocol. In brief, 12 mM MTT (Invitrogen) stock solution was prepared by adding 1 mL of sterile PBS to 5 mg of MTT. The mixture was vortexed or sonicated until dissolved. The cells were grown on a 96-well plate, and recombinant phage, copper, and copper hybrid phage samples at different concentrations were added in triplicate for 3, 8, and 24 h. The toxicity of copper analyzed in this assay ranged from 1 μM to 1 mM copper chloride solution, and for recombinant phage or copper-hybrid phage, it ranged from 108 to 1012 pfu/mL concentration. The copper hybrid phage that was used for imaging cancer cells was prepared using 1010 pfus and 1 μM copper chloride solution. Following the incubation periods, the media was replaced with media without phenol red. Ten microliters of 12 mM MTT stock solution was then added to each well along with negative and positive controls. The plate was then incubated at 37 °C for 4 h followed by addition of 100 μL of SDS-HCl solution (1 g of SDS in 10 mL of 0.01 M HCl). The contents of each well were mixed thoroughly with the help of a pipet and incubated at 37 °C for 16 h in a humidified chamber. The absorbance was read at 570 nm and toxicity was assessed. The higher the absorbance value at 570 nm, the higher the cell proliferation capacity of the culture is. Statistical Analysis. Quantitative data were expressed as mean ± SD. Means were compared using one-way ANOVA and student’s t test. P values of
