Oligonucleotide-Coated Metallic Nanoparticles as a Flexible Platform

Oct 9, 2007 - Chapter 5. Nanomaterial-Antibody Hybrids. Jyothi Menon , Lei Song , Nadia Falzone , Katherine Vallis. 2016,83-110 ...
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Oligonucleotide-Coated Metallic Nanoparticles as a Flexible Platform for Molecular Imaging Agents Nitin Nitin, David J. Javier, and Rebecca Richards-Kortum* Department of Bioengineering, Rice University. Received April 13, 2007; Revised Manuscript Received August 14, 2007

Targeted metallic nanoparticles have shown promise as contrast agents for molecular imaging. To obtain molecular specificity, the nanoparticle surface must be appropriately functionalized with probe molecules that will bind to biomarkers of interest. The aim of this study was to develop and characterize a flexible approach to generate molecular imaging agents based on gold nanoparticles conjugated to a diverse range of probe molecules. We present two complementary oligonucleotide-based approaches to develop gold nanoparticle contrast agents which can be functionalized with a variety of biomolecules ranging from small molecules, to peptides, to antibodies. The size, biocompatibility, and protein concentration per nanoparticle are characterized for the two oligonucleotidebased approaches; the results are compared to contrast agents prepared using adsorption of proteins on gold nanoparticles by electrostatic interaction. Contrast agents prepared from oligonucleotide-functionalized nanoparticles are significantly smaller in size and more stable than contrast agents prepared by adsorption of proteins on gold nanoparticles. We demonstrate the flexibility of the oligonucleotide-based approach by preparing contrast agents conjugated to folate, EGF peptide, and anti-EGFR antibodies. Reflectance images of cancer cell lines labeled with functionalized contrast agents show significantly increased image contrast which is specific for the target biomarker. To demonstrate the modularity of this new bioconjugation approach, we use it to conjugate both fluorophore and anti-EGFR antibodies to metal nanoparticles, yielding a contrast agent which can be probed with multiple imaging modalities. This novel bioconjugation approach can be used to prepare contrast agents targeted with biomolecules that span a diverse range of sizes; at the same time, the bioconjugation method can be adapted to develop multimodal contrast agents for molecular imaging without changing the coating design or material.

1. INTRODUCTION The aim of this study is to develop and characterize a bioconjugation approach which combines the flexibility and specificity of oligonucleotide hybridization with the excellent optical contrast properties of gold nanoparticles to develop biocompatible molecular imaging agents. Gold nanoparticles provide strong contrast when imaged in reflectance mode based on their unique optical absorption and scattering properties (1–5). In current clinical practice, reflectance imaging is used to aid in the diagnosis of various cancers and precancers based on differences in the native optical properties of neoplastic and normal epithelial tissue (6–8). The development of molecularspecific contrast agents which can be imaged in reflectance mode has the potential to improve the sensitivity and specificity of detection of early-stage precancers and cancers by providing the ability to noninvasively image the molecular changes associated with early neoplastic transformation. Studies by Sokolov et al. (4) and El-Sayed et al. (3) have shown the potential of using gold nanoparticles to image receptor overexpression in various tumor model systems. Gold nanoparticle-based contrast agents have distinct advantages due to their biocompatibility, photostability, and ease of coating with proteins and polymers. In these studies, gold nanoparticles were linked to antibodies by direct absorption of protein on the surface of the nanoparticle. It has been established that electrostatically adsorbed gold–protein conjugates are stable only in the presence of excess protein in solution (9). This result suggests that there is a dynamic exchange between coated protein molecules and excess proteins in solution. This exchange is not desired for biological applications, as targeting proteins may be displaced * Corresponding author. E-mail: [email protected]. Ph.: 713-3483823. Fax: 713-348-5877.

by the high concentration of native proteins in biological samples. In addition, electrostatic protein adsorption offers very little control over the orientation of bound molecules; inappropriate orientation and conformational changes in antibodies during adsorption can reduce their binding affinities (10). Further, this approach cannot be easily extended to other potential probe biomolecules of interest including peptides and small molecules; small biomolecules cannot provide enough surface coverage to prevent the aggregation of gold nanoparticles in physiological buffers. As an alternative to direct protein adsorption, other polymeric coatings including dextran, chitosan (1, 11), and self assembled monolayers (SAM) have been developed for surface modifications of nanoparticles. In the case of dextran- and chitosanbased coatings, typically large molecular weight polymers (>50 000 MW) are required to coat nanoparticles. This limitation is due to small numbers of reactive groups present on polymer chains with lower molecular weight. The use of large MW polymers leads to coated nanoparticles with a large effective size. In the case of SAM-based coatings, the solubility of the resulting nanoparticle in an aqueous environment is limited by the hydrophobic nature of the coating polymer, leading to aggregation in high-salt solutions. In addition, various other chemical functionalization techniques have been developed for coating gold nanoparticles in aqueous settings, but in many cases, this has led to aggregation (12). This limitation has been overcome by adding various polymeric stabilizers which nonspecifically interact with the particle surface, but these stabilizers can potentially limit the biological applications as well as interfere with surface conjugations (13). Further disulfide-modified polymer (e.g., OPSS-PEG) coatings may also absorb nonspecifically on the surface of gold nanoparticles, as the affinities for some of these polymer

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Oligonucleotide-Coated Metallic Nanoparticles

backbones are only slightly less than the specific gold–disulfide interactions (12). Thus, the functional groups in these coatings will have very limited accessibility for further surface bioconjugation to probe biomolecules. In some cases, polymer molecules with bifunctional ends have been linked to antibodies or other biomolecules before coating the gold nanoparticles. In this case, it is expected that nonspecific adsorption will play a significant role as compared to the single thiol interaction with the gold nanoparticle. To overcome these limitations, we have developed a bioconjugation approach which combines the stability of thiol-modified oligonucleotide coatings for gold nanoparticles with the ease of bioconjugation using bifunctionally modified oligonucleotides to develop gold-based reflectance contrast agents. This coating approach has been optimized for various analytical assays (14) (15) and is stable at high salt conditions as well as in the presence of other proteins and biomolecules. The approach provides flexibility to conjugate a variety of biomolecules, to control the orientation of conjugated biomolecules, and to control the size of the functionalized particles. Further, the sequence specificity of oligonucleotides can be exploited to develop flexible approaches for assembly of contrast agents and the development of multifunctional contrast agents, with either multiple targeting moieties or multiple reporting agents.

2. EXPERIMENTAL PROCEDURES 2.1. Materials. Gold nanoparticles (∼25 nm) were synthesized using gold chloride (HAUCl4, >99.999% purity, Sigma) and sodium citrate (Sigma). The gold nanoparticles were coated with19-meroligonucleotides(MWG,5′-thiol-AAAAAAAAAAATCCTTTAC-amine) functionalized with a thiol group on the 5′ end and an amino group at the 3′ end. The oligonucleotide was first reduced with TCEP · HCl (Tris(2-carboxyethyl) phosphine hydrochloride, Pierce) before adding it to the gold nanoparticles. Its complementary sequence was also used (MWG, 3′-TAGGAAATGTTATAA-amine) for the conjugation of targeting molecules. Targeting molecules were chosen to represent a wide range of molecular weight molecules which include a 165 kD EGFR antibody (epidermal growth factor receptor antibody, Baylor College of Medicine antibody facility), a short 5.5 kD peptide EGF (epidermal growth factor peptide, US Biological), and a small 0.5 kD molecule (folate, Sigma). The following reagents and cross-linkers were used for bioconjugation of the targeting molecules to the oligonucleotide-coated gold nanoparticles: C6-SANH (C6-SANH (C6-succinimidyl 4-hydrazinonicotinate acetone hydrazone, Pierce), C6-SFB (C6-succinimidyl 4-formylbenzoate, Pierce), DMSO (Sigma), 2-mercaptoethanol (Sigma), EDAC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, Pierce), and sulfo-NHS (Pierce). For characterization, sodium cyanide (Sigma) was used to dissolve the gold nanoparticles. Purification columns that were used include a Zeba desalting column (Pierce), a YM-3 Microcon spin column (Millipore), and an S-400 spin column (GE Healthcare). For cellular targeting, various cancer cell lines obtained from ATCC were cultured using DMEM (Dulbecco’s minimum essential media) or MEM with 5% FBS (fetal bovine serum) with antibiotics as recommended by ATCC. The cell lines include 1483 cells and SiHa cells for EGFR targeting with antibodies and EGF peptides, and Kb cells for targeting folate receptors. A Cy5 dye modified complementary oligonucleotide (MWG) was used to demonstrate the flexibility of the coating and conjugation approach to develop multimodal/multifunctional contrast agents. 2.2. Methods. 2.2.1. Synthesis and Coating of Gold Nanoparticles. Gold nanospheres (∼25 nm) were synthesized using the sodium citrate reduction of gold ions developed by Frens et al. (16). The size of the nanospheres was controlled

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by adjusting the ratio of Au ions (HAuCl4) and sodium citrate. The procedure for coating gold nanoparticles with oligonucleotides was adapted from Mirkin et al. (14). This protocol is based on the Au–S interaction between the gold lattice and thiolfunctionalized oligonucleotides. The colloidal gold nanoparticles were first stabilized and coated using chemisorption of thiolmodified oligonucleotides (14). These oligonucleotides were also modified with NH2 reactive group at the 3′-terminus. Briefly, 100 µL of 30 µM oligonucleotide was reduced with ∼1 mM TCEP for 15 min. The reduced oligonucleotide containing free thiol was then added to 900 µL of gold nanoparticles (∼0.1 nM concentration) and aged for 24–48 h with increasing salt concentration (until 1× PBS concentration is reached). The resulting oligonucleotide-coated nanoparticles were concentrated 10-fold by centrifugation at 6000 rpm for 30 min. To illustrate the flexibility of the approach, oligonucleotide-coated nanoparticles were then functionalized with several types of probe molecules, including anti-EGFR antibodies, EGF peptide, and folate. 2.2.2. Bioconjugation of Oligonucleotides and Targeting Molecules To Gold Nanoparticles. a. Direct Approach. In this method, the targeting molecules were directly incorporated onto the oligonucleotide-coated Au nanoparticles. After first coating the gold nanoparticles with oligonucleotides, complementary oligonucleotides (∼3 uM) were hybridized to increase the number of amino functional sites. Anti-EGFR (1 mg/mL) or EGF (200 ug/mL) proteins were conjugated to oligonucleotidecoated Au (∼1 nM) using heterobifunctional cross-linkers, SANH and SFB. The amino groups present on proteins were functionalized with SFB (20× molar excess), while amino groups present on the oligonucleotide–Au were functionalized with SANH (20× molar excess) for 1–2 h. Excess SANH and SFB were removed by passage on a Zeba desalting column twice. The SFB-modified protein and SANH-modified oligonucleotide Au were then mixed overnight (pH 7.0) at room temperature. The unreacted anti-EGFR or EGF proteins were removed by spinning the gold nanoparticles at 6000 rpm for 30 min. For folate-functionalized nanoparticles, the direct conjugation of folate to oligonucleotide–Au was accomplished using EDAC chemistry. The COOH group present on folate (1 mg/mL, 1 mL) was reacted with EDAC (0.6 mg) and sulfoNHS (1.1 mg) for 15 min. After quenching the reaction with mercaptoethanol, an aliquot of the highly reactive NHS–folate (∼20 uM) was added to oligonucleotide–Au for 2–3 h. Excess NHS–folate was also removed by centrifuging the gold nanoparticles at 6000 rpm for 30 min. b. Indirect Approach. In this method, complementary oligonucleotides and the targeting molecules were conjugated first before hybridizing them to the oligonucleotide-coated gold nanoparticles. Similar to the direct approach, anti-EGFR or EGF proteins were reacted with SFB, while complementary oligonucleotides (∼3 uM) were reacted with SANH. The resulting oligonucleotide–protein was purified using an S-400 spin column and then hybridized to its corresponding oligonucleotide-coated Au nanoparticles overnight at room temperature. For folate conjugation, folate was first added to EDAC/sulfo-NHS, before mixing with complementary oligonucleotides. The oligonucleotide folate conjugate was purified using a YM-3 column and hybridized to its corresponding oligonucleotide–Au. The excess oligonucleotide–protein and oligonucleotide–folate were removed by spinning the gold nanoparticles at 6000 rpm for 30 min. c. Adsorption of Antibodies on Gold Nanoparticle Surface. For the development of antibody-coated gold nanoparticle-based contrast agents using adsorption of biomolecules on the surface, we followed the procedure developed by Slot and Geuze (1985) (17) and adapted by Sokolov et al. (4) and

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El-Sayed et al. (3). Briefly, in this procedure the anti-EGFR antibody was incubated with gold nanoparticles for ∼30 min in a salt-free environment. After incubation, the gold nanoparticles were centrifuged to remove excess antibodies in the presence of 1% PEG (10 000 MW). After centrifugation and decantation of solution, the pellet of gold nanoparticles was dispersed in 1× PBS (phosphate buffered saline) and tested for colloidal stability using the conventional salt test. In this test, the gold nanoparticle solution was tested using 10% NaCl solution, and stability was determined by visual assessment of color change. In the case of antibody-coated particles, we did not observe any color change, indicating colloidal stability based on this salt test procedure. 2.2.3. Characterization. Various qualitative and quantitative approaches were used to characterize the gold nanoparticle conjugates. a. Size Characterization. Dynamic light scattering (DLS, Brookhaven) was used to measure the size of the oligonucleotide-coated nanoparticles, with and without the targeting molecules, and also gold nanoparticles with physically adsorbed antibodies. To evaluate the colloidal stability of gold nanoparticle conjugates, the size measurements were repeated 24 h postsynthesis of contrast agents. b. Biocompatibility. The biocompatibility of oligonucleotidecoated nanoparticles was determined using the MTT assay following the procedure recommended by ATCC. Briefly, cells were cultured in 90-well plates for 24 h prior to incubation with oligonucleotide-conjugated gold nanoparticles. In each experiment, three wells were incubated with oligonucleotide-coated gold nanoparticles along with triplicate control wells. The experiment was carried out using SiHa and 1483 cell lines. Coated gold nanoparticles were incubated at twice the concentration levels used for cell labeling experiments. After 24 h of incubation, the plates were assayed using reagents provided with the MTT kit. The results were analyzed using absorbance measurements at 570 nm. The background level was determined using cells without any treatment and MTT reagents. c. Concentration Estimation for Conjugated Antibodies and Peptides on Oligonucleotide-Coated Gold Nanoparticles. To determine the estimated concentration of antibodies and peptides conjugated to oligonucleotide coated gold nanoparticles, we used a protein quantification assay kit based on Coomassie staining. Prior to staining with Coomassie dye, the gold nanoparticles were dissolved using 100 mM potassium cyanide (KCN). This step was essential to remove the contribution from the gold nanoparticle plasmon band during the absorbance assay. Coomassie staining was carried out using the procedure recommended by the Pierce Protein Quantification kit. In addition, we used known concentrations of antibodies and peptides respectively as standards to establish the linear range for the assay and to calculate the concentration of conjugated proteins/ peptides on gold nanoparticles. 2.2.4. Reflectance Imaging Using Oligonucleotide-Coated Nanoparticles. Oligonucleotide-coated nanoparticles functionalized with the targeting molecules were incubated with different cancer cell lines (1483 and SiHa for EGFR and EGF targeting, and Kb for folate targeting) at 37 °C for 30 min. 1483 and SiHa are oral and cervical cancer cell lines, respectively, which overexpress the EGF receptor (18). Kb cell is an oral cancer cell line which overexpresses the folate receptor (19). The cells were washed three times and centrifuged to remove unbound conjugate. Confocal reflectance images were obtained from the resulting cell pellet using a Zeiss LSM 510 microscope in reflectance mode. All images were acquired under the same imaging conditions. Postacquisition, the image contrast was adjusted in images of the folate-targeted cells to facilitate

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comparison with images acquired from cells labeled with EGFand EGFR-targeted contrast agents. 2.2.5. Bimodal Contrast Agents and Imaging. To demonstrate the flexibility of the coating approach to develop multifunctional contrast agents, we used the indirect method to develop a contrast agent which combines both reflectance and fluorescence imaging capabilities in a single contrast agent. To develop this bimodal contrast agent, gold nanoparticles coated with oligonucleotides were hybridized overnight with complementary oligonucleotides modified with Cy5 dyes and complementary oligonucleotides conjugated with EGFR antibody. To provide efficient hybridization for antibody-conjugated complementary oligonucleotides, an excess of antibody-conjugated oligonucleotide (5:1 ratio) was used as compared with dye-labeled oligonucleotide. The use of a near-IR dye can be potentially useful for in ViVo imaging as well as reduce the quenching effects due to gold nanoparticles. After the hybridization step and removal of excess complementary oligonucleotide, the purified product was used to label 1483 cancer cells. Labeled cells were imaged using both fluorescence and reflectance confocal microscopy.

3. RESULTS AND DISCUSSION Figure 1 shows a schematic representation of the two complementary oligonucleotide-based bioconjugation approaches to develop gold nanoparticle based molecular contrast agents. In the direct approach, a complementary oligonucleotide was hybridized with the oligonucleotide coating on the surface of the gold nanoparticles (Figure 1). This stabilized and extended double-stranded DNA was then further conjugated with target protein or biomolecules of interest using bifunctional crosslinkers. In the indirect approach, a complementary oligonucleotide was first conjugated to targeting molecules (Figure 1). These modified oligonucleotides were then hybridized with the coated oligonucleotides on the surface of gold nanoparticles to generate a molecular contrast agent. These bioconjugation approaches have different advantages for developing molecular contrast agents. In the direct approach, the significant advantage is the ease of separation of small molecules and peptides after a conjugation reaction. In this case, a series of simple centrifugation steps can remove excess unbound targeting molecules from gold-conjugated molecules. In the indirect approach, the significant advantage is the ease with which multifunctional contrast agents can be developed. Using this approach, we can place multiple targeting moieties or multiple reporting moieties on the surface of gold nanoparticles by ratiometric mixing of oligonucleotides conjugated to different moieties. To further enhance the specificity of this process and provide ratiometric control over conjugation processes, different sequences of oligonucleotides can be coated on the surface of gold nanoparticles. These sequences can be hybridized with their respective complementary sequences modified with multiple reporting or targeting agents. In this study, we evaluated contrast agents produced with both the direct and indirect approaches as outlined above. After developing gold nanoparticle conjugates, our next step was to characterize the bioconjugates and compare their size and stability with conjugates made using absorption of antibodies. We used dynamic light scattering (DLS) to analyze the diameter of the conjugated gold nanoparticles with anti-EGFR, EGF peptide, and folate-based targeting. Results for conjugates prepared using the direct approach are shown in Figure 2a; similar results were obtained using indirect conjugates. The size of the contrast agent increased as the size of the conjugated probe molecule was increased from a small molecule (folate) to a peptide (EGF) to an antibody (anti-EGFR). For example, antibody-conjugated gold nanoparticles prepared using oligo-

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Figure 1. Schematic outline of conjugation of biomolecules to the oligonucleotide-functionalized gold nanoparticles. In the direct approach, the complementary oligonucleotides are first hybridized to the surface of the oligonucleotide-coated gold nanoparticles followed by conjugation of targeting molecules using hetero-bifunctional cross-linkers. In the indirect approach, the targeting biomolecules are first conjugated to the complementary oligonucleotides, which are then hybridized to oligonucleotides coated on the surface of gold nanoparticles. The conjugation approaches offer flexibility in processing and developing a molecular-specific contrast agent.

nucleotide coatings show an increase of approximately ∼15–20 nm in diameter as compared with bare gold nanoparticles; this is consistent with an approximate antibody size of 15 nm. In contrast, antibody-targeted contrast agents prepared using adsorption are significantly larger (∼80 nm) and show a wide distribution in size as compared to oligonucleotide-conjugated antibodies (∼40 nm). These results suggest that the adsorption process leads to larger conjugates either as a result of multiple protein layers per nanoparticle or due to aggregation of nanoparticles. To characterize and compare the stability of gold nanoparticle conjugates prepared using the adsorption and oligonucleotide procedure, we measured the change in size of anti-EGFR conjugates at 24 h postsynthesis. Results for conjugates prepared using direct oligonucleotide conjugation are shown in Figure 2b; again, similar results were obtained for those prepared using the indirect method. While the sizes of the oligonucleotidecoated, antibody-targeted contrast agents were stable over this time period, we observed a significant increase in the size of the adsorbed conjugates, suggesting that aggregation occurs over time. These results demonstrate the greater stability and smaller sizeofantibody-targeted,oligonucleotide-coatedgoldnanoparticles. To establish biocompatibility of these agents, we carried out the standard MTT-based cytotoxicity assay. Figure 3 shows results of the assay for oligonucleotide-coated gold nanoparticles prepared using the direct method; similar results were obtained for conjugates prepared using the indirect method. No significant change was observed in cell viability even after 24–48 h incubation of these agents with cells, establishing the biocompatibility of oligonucleotide-coated gold nanoparticles in cell culture models. Future studies are warranted to study similar processes in animal models before clinical translation of these contrast agents. We next estimated the concentration of the proteins or peptides conjugated on the surface of gold nanoparticles using the direct and indirect approaches. The results of this quantification assay are shown in Table 1. Both the direct and indirect

methods yield similar concentrations of biomolecules conjugated to the surface of oligonucleotide-coated gold nanoparticles. This indicates that both approaches have similar efficiency for loading probe biomolecules on the surface of gold nanoparticles. In a next step, we labeled cells in culture with targeted contrast agents; images of labeled cells were obtained using confocal reflectance microscopy. To demonstrate that gold nanoparticles can be conjugated to a wide size range of biomolecules (from antibodies to small molecules), we conjugated gold nanoparticles with anti-EGFR (150 kDa), EGF (6 kDa), and folate (∼0.3 Da) using both direct and indirect approaches. To demonstrate the specificity of the resulting contrast agents, we prepared IgG gold nanoparticle conjugates and oligonucleotide-coated particles without any targeting biomolecules as controls. In this study, we used 1483 and SiHa oral and cervical cancer lines to evaluate EGFR-specific contrast and KB cancer cells for folate-specific imaging. Representative confocal reflectance images obtained from cells labeled with direct and indirect conjugates are shown in Figure 4a,b, respectively. Results show strong reflectance signal from the cell membrane of SiHa cells labeled with both anti-EGFRtargeted gold nanoparticles and EGF peptide-based targeting. Images obtained from cells targeted with both direct and indirect conjugates are similar, indicating that effective contrast agents can be generated with either conjugation approach. With targeting of folate overexpression using folate-conjugated gold nanoparticles, we also obtained similar results with both direct and indirect conjugates. The specificity in this case was evaluated using oligonucleotide-modified gold nanoparticles without any targeting molecules. Based on these results, we have demonstrated that gold nanoparticles can be modified with variety of biomolecules including antibodies, peptides, and small molecules to develop effective reflectance contrast agents for molecular imaging applications. Thus, with this bioconjugation platform, molecular imaging applications with gold nanoparticles are no longer

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Figure 2. (a) Size measurements of oligonucleotide-coated gold nanoparticles and their conjugates using dynamic light scattering (DLS). The conjugates were prepared using the direct approach. Similar sizes were observed for conjugates prepared using the indirect approach (data not shown). Conjugation using the oligonucleotide approach was compared with methods of directly adsorbing large biomolecules on the surface of gold nanoparticles. (2) Size stability of gold nanoparticle conjugates measured using dynamic light scattering (DLS). Over a period of 24 h, results show an increase in size of gold conjugates with adsorbed antibodies as compared to antibodies chemically conjugated to the surface of gold nanoparticles using the direct approach. Conjugates prepared using the indirect approach showed similar size stability (data not shown). Table 1. Estimated Concentration of Conjugated EGFR Antibodies and EGF Peptide on Surface of Gold Nanoparticlesa [protein]

direct µg/mL

indirect µg/mL

anti-EGFR EGF

3.6 3.3

4.6 4.3

a Protein concentration was measured using Coomassie stain after dissolution of protein-conjugated gold nanoparticles in KCN.

Figure 3. Biocompatibility studies of oligonucleotide-coated gold nanoparticles using standard MTT assay. Cells were incubated for 48 h with gold nanoparticles coated with oligonucleotides and analyzed by measuring absorbance at 570 nm using a standard plate reader.

limited to antibody-based approaches for targeting, and these designs can be easily extended to a variety of biomolecules.

To demonstrate the flexibility of this platform to develop bimodality contrast agents, we used the indirect approach to develop a single contrast agent capable of simultaneous reporting in both fluorescence and reflectance modes. The development of multimodality reporting is of significant interest in the area of molecular imaging, as multiple reporters can extend the range of sensitivity, penetration depth, and resolution for reporting molecular events. Figure 5a illustrates the approach to develop a dual-reporting contrast agent directed by the hybridization of complementary oligonucleotides. The cellular assay of this contrast agent was carried under similar conditions as for the direct and indirect conjugates in Figure 4. After

Oligonucleotide-Coated Metallic Nanoparticles

Figure 4. (a) Evaluation of molecular specific targeting of direct conjugates using confocal reflectance imaging. The contrast agents were prepared by conjugating different size-targeting molecules (antibody, peptides, and small molecules) to oligonucleotide-coated gold nanoparticles in various cancer cell lines. IgG-conjugated gold nanoparticles were used as controls in these cell lines (representative control image is shown). The scale bar represents 15 µm. (b) Evaluation of molecularspecific targeting of indirect conjugates using confocal reflectance imaging. The contrast agents were prepared by conjugating different size targeting molecules (antibody, peptides, and small molecules) to oligonucleotide-coated gold nanoparticles in various cancer cell lines. IgG-conjugated gold nanoparticles were used as controls in these cell lines (representative control image is shown). The scale bar represents 15 µm.

incubation with the contrast agent, cells were imaged using both reflectance and fluorescence confocal microscopy. Since the reflectance signal is significantly stronger than the fluorescence signal, the pinhole size and laser power used in reflectance imaging were significantly smaller than that used for fluorescence imaging. Results are shown in Figure 5b and show a similar distribution of reflectance and fluorescence signal on the surface of the cells. The reflectance image collected with a smaller confocal pinhole shows a significantly sharper image than the fluorescence image. This study demonstrates the concept for dual modality imaging, which can be extended to a variety of reporting schemes including MR contrast agents such as Gd3+ or PET contrast agents based on modification of oligonucleotides with chelating groups.

4. SUMMARY AND CONCLUSIONS In this study, we have developed and characterized a bioconjugation approach which combines the flexibility and

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Figure 5. (a) A conceptual approach of using oligonucleotide-coated gold nanoparticles to develop multifunctional contrast agents for targeting, reporting, and delivery. In this example, the ratio of targeting and reporting molecules on the surface of gold nanoparticles can be controlled by controlling the ratio of modified oligonucleotides during the hybridization step. (b) Proof of concept study to illustrate development of bimodal reporting molecular contrast agents. Fluorescence and reflectance confocal images of SiHa cells labeled with bimodal contrast agent targeting the EGF receptor. The scale bar represents 15 µm.

specificity of oligonucleotide hybridization with the excellent optical contrast properties of gold nanoparticles. The approach yields biocompatible molecular imaging agents which can be targeted with a wide variety of probe molecules to yield highcontrast molecular images in cell model systems. The stability and size distribution of the contrast agents produced using this approach are superior to those of contrast agents based on protein absorbed gold nanoparticles, which have been previously been used to produce targeted gold nanoparticles for use as molecular contrast agents. To demonstrate the flexibility of the conjugation approach, we illustrate the development of multimodal contrast agents which can be imaged in both reflectance and fluorescence modes. The results highlight the potential of oligonucleotide coatings for development of multifunctional molecular contrast agents.

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