Abstract
Abstract. An effective cancer control strategy requires improved early detection methods, patient-specific drug selection, and the ability to assess response to targeted therapeutics. Recently, plasmon resonance coupling between closely spaced metal nanoparticles has been used to develop ultrasensitive bioanalytical assays in vitro. We demonstrate the first in vivo application of plasmon coupling for molecular imaging of carcinogenesis. We describe molecular-specific gold bioconjugates to image epidermal growth factor receptor ͑EGFR͒; these conjugates can be delivered topically and imaged noninvasively in real time. We show that labeling with gold bioconjugates gives information on the overexpression and nanoscale spatial relationship of EGF receptors in cell membranes, both of which are altered in neoplasia. EGFR-mediated aggregation of gold nanoparticles in neoplastic cells results in more than a 100-nm color shift and a contrast ratio of more than tenfold in images of normal and precancerous epithelium in vivo, dramatically increasing contrast beyond values reported previously for antibody-targeted fluorescent dyes.
Introduction
A number of different types of nanoparticles have recently been proposed for use as optical imaging agents to aid in cancer detection, including quantum dots ͑qdots͒, [1] [2] [3] nanoshells, 4 and metal nanoparticles. 5 The use of nanoparticles for molecular imaging of cancer has several important advantages. Their surface chemistry is well understood, so they can be easily targeted using antibodies or aptamers to bind to cancer biomarkers. By adjusting the particle geometry, the optical properties can be tuned over a large spectral region to provide multicolor labeling. The bright reflectance or luminescence of nanoparticles provides a strong source of signal for in vivo imaging. Last, multiplexing different types of particles and targeting molecules provides a common platform for multiple applications with a high degree of flexibility. 2 While qdots have shown promise in preclinical cellular and animal studies, they possess a number of disadvantages for clinical use. Qdots are generally composed of very toxic materials, raising concerns about their potential use in humans. Although qdots offer greatly improved photostability, they still are not completely resistent to photobleaching; 6 this, in combination with "blinking" effects, can complicate the development of qdot-based ultrasensitive imaging techniques. Last, tissue autofluorescence in vivo occurs in the same spectral region as the luminescence of some of the most widely used qdots and can be difficult to separate from the contrast agent signal. 1 The use of metal nanoparticles as contrast agents for in vivo molecular imaging offers a number of advantages over both qdots and organic fluorescent dyes. Similar to qdots, the optical properties of metal particles can be systematically changed by varying particle size and geometry. 5, 7 However, the very high scattering cross section of metal nanoparticles as compared with the fluorescence cross sections of organic dyes and even qdots provides a much brighter source of signal with complete immunity to photobleaching. 5, 8, 9 Metal nanoparticles, particularly gold, offer an inert, biocompatibile alternative to qdots; gold is already used in routine clinical treatment of arthritis. 10 Last, the strength and the wavelength characteristics of the scattered signal from metal nanoparticles is sensitive to the nanoscale spacing of individual particles. 9, 11, 12 This property provides a tool not only to monitor overexpression of a biomarker, but also to monitor biomarker interactions and activation and to increase contrast between images of normal and cancerous tissue beyond the limited contrast provided by a fewfold overexpression level.
Introduction
Nanotechnology offers an opportunity to probe the molecular and cellular events of carcinogenesis in real time and the ability to monitor the impact of therapeutic interventions. However, there are a number of barriers to achieve this potential, including development of:
Introduction
• bright molecular specific contrast agents that can be safely used in humans; • delivery formulations for these contrast agents in vivo; • imaging tools. We present a study to address these barriers in detection of epithelial precancers, which account for up to 90% of all initial malignant changes. Recently, the potential of gold nanoparticles for molecular specific imaging was demonstrated in cell culture models of oral cancer using a combination of dark field optical microscopy and white light illumination. 13 In this paper, we bring together molecular-specific contrast agents that are based on gold nanoparticles, topical formulations for their delivery in vivo, and noninvasive confocal reflectance microscopy to demonstrate detection of molecular changes in models of epithelial precancers ranging from cell tissue cultures to preclinical in vivo animal models, as shown conceptually in Fig. 1 . Integrating these tools, we focus on imaging one of the hallmarks of cancer-the uncoupling of a cell's growth program from environmental signals. Specifically, we describe imaging of the epidermal growth factor receptor ͑EGFR͒, which is associated with carcinogenesis in many epithelial cancers including lung, oral cavity, and cervix.
Introduction
14 There is good consensus that EGFR levels demonstrate a statistically significant increase as lesion severity progresses from earlier dysplasia to invasion. 15, 16 It has been also demonstrated that EGFR function and activation is associated with dimerization and concurrent higher order aggregation of receptor molecules. 17 
Materials and Methods

Anti-EGFR Contrast Agents
We synthesized 25-nm gold nanoparticles via sodium citrate reduction of gold tetrachloride ͑Sigma͒. The conjugates of gold nanoparticles with anti-EGFR monoclonal antibodies were prepared using the method that was originally developed for preparation of gold immunostains for electron microscopy. 18, 19 Briefly, 0.1 mg/ mL of anti-EGFR antibodies ͑clone 225, Neomarkers, or clone 29.1, Sigma͒, in a 40-mM HEPES buffer, pH 7.4, were mixed for 20 to 30 min with an equal volume of gold nanoparticles at approximately 10 12 particles per mL. Conjugates were centrifuged at 3000 rcf for 1 to 1.5 h in the presence of 1% PEG and resuspended at ϳ2 ϫ 10 12 particle concentration in a PBS buffer, pH 7.4 containing 1% BSA and 0.2% PEG. Bifunctional contrast agents were synthesized via a monoclonal antibody labeling kit ͑Mo-lecular Probes͒ to attach fluorescein or Alexa Fluor 647 dye to purified antibodies via a succinimidyl ester reaction that forms linkages between the dye and primary amines on the antibody. Fluorescently labeled antibodies were then conjugated with gold nanoparticles as described earlier.
Cell Cultures
Cells were cultured in DMEM ͑A-431, MDA-MB-435͒ or MEM ͑MDA-MB-468͒ plus 10% FBS and grown at 37°C in 5% CO 2 . Cells suspensions were labeled in a 50:50 ͑v/v͒ mixture of phenol/serum-free DMEM and contrast agents for 30 min at room temperature.
Cell Specificity Assays
For the competition assay, SiHa cervical cancer cells were incubated with a mixture of anti-EGFR gold conjugates and unlabeled anti-EGFR mAbs at ϳ1:25 molar ratio in phenolfree medium for a period of 30 min at room temperature under mild mixing. The cells were then washed in the media and imaged using confocal microscopy in reflectance mode. For the displacement assay, SiHa cells were first labeled with anti-EGFR gold nanoparticles for 30 min at room temperature. The cells were then washed and exposed to an excess of unlabeled anti-EGFR mAbs ͑concentration 2.6 M͒ for 45 min.
Cell Specificity Assays
Following incubation, the cells were imaged using reflectance confocal microscopy. Cells labeled with anti-EGFR gold nanoparticles alone were used as a positive control, and cells incubated with gold conjugates with nonspecific IgG were used as a negative control for labeling efficiency. The assays showed a tenfold or more decrease in the scattering signal of cells either labeled in the presence of unlabeled anti-EGFR mAbs or incubated with the unlabeled Abs after labeling with anti-EGFR gold conjugates.
Tissue Phantoms
In vivo molecular imaging requires development of efficient delivery systems of contrast agents to their targets. We used dimethyl sulfoxide ͑DMSO͒ to promote topical delivery of anti-EGFR gold conjugates. DMSO was chosen because of its permeation efficiency and high aqueous solubility and because it is already FDA-approved for human use 20 in certain applications.
Tissue Phantoms
We fabricated heterogeneous three-dimensional ͑3-D͒ tissue constructs that consisted of a 1:1 high-density mixture of EGFR͑+͒ MDA-MB-468 and EGFR͑−͒ MDA-MB-435 breast cancer cells. EGFR͑+͒ and EGFR͑−͒ cell lines were labeled with membrane-permeable derivatives of fluorescein and seminaphthorhodafluors ͑SNARF͒, respectively, which fluoresce only when they are inside the cells. Cells were incubated with membrane permeable derivatives of fluorescein and seminaphthorhodafluors ͑SNARF; Molecular Probes͒, respectively, in DMEM for 1 h and washed. The dyes are rendered membrane permeable via a chloromethyl group that is enzymatically cleaved upon entering a viable cell. Thus, once in the cytosol, the dye becomes membrane impermeable and does not exit the cell. A 1:1 ͑v/v͒ mixture of the two cell lines was resuspended in a 1-mg/mL buffered ͑pH 7.4͒ collagen solution ͑Roche͒ at a concentration of approximately 80 to 100ϫ 10 6 cells per mL. A 200 to 500-m-thick disk of the cell/collagen mixture was coated onto a permeable membrane ͑Corning͒ and left to proliferate overnight in the incubator. Phantoms were topically exposed to 100 L of contrast agents ͑at ϳ10 12 particles per mL͒ in a 1ϫ PBS solution containing 10% DMSO for 30 min, briefly washed in media, and transversely sectioned using a Krumdieck slicer.
Tissue Phantom Specificity Assays
For homogeneous phantoms used in the specificity assays, 3-D tissue cultures with SiHa cells were prepared using the method described earlier for the heterogeneous phantoms. First, phantoms were treated with unconjugated anti-EGFR mAbs. The mAbs were topically delivered in PBS with 10% DMSO at the concentration of 0.4 M for 45 min at 37°C.
Tissue Phantom Specificity Assays
Immediately after this initial application, anti-EGFR gold nanoparticles were topically applied in the same solution for 45 min. The cultures were then washed in PBS, transversely sliced, and imaged using reflectance confocal microscopy. Tissue cultures that were not preexposed to the unlabeled mAbs were used as a positive control for labeling efficiency. Each of these assays was repeated at least three times. The average grayscale values of reflectance signal intensity were computed by using the NIH ImageJ software. The ratio of unlabeled anti-EGFR mAbs to anti-EGFR gold nanoparticles was calculated based on the amount of mAbs conjugated to the nanoparticles. The amount of the attached Abs was determined using the number of nanoparticles ͑ϳ10 15 particles/l͒, their diameter ͑ϳ25 nm͒, and a previously published value of IgG antibody density on a gold surface ͑ϳ2.5 pM/ cm 2 ͒ ͑Ref. 21͒.
Tissue Phantom Specificity Assays
We observed a more than tenfold decrease in the scattering signal of the cultures pretreated with unlabeled mAbs as compared to the positive control. These results illustrate the specificity of the anti-EGFR gold bioconjugates for EGFRs in 3-D tissue cultures.
Human Cervical Biopsies
Clinically normal and abnormal cervical biopsies were obtained from consenting women seen at the Department of Gynecologic Oncology, M. D. Anderson Cancer Center. Biopsies were placed in chilled DMEM and later embedded in a 3% agarose gel such that only the epithelial surface was exposed.
Human Cervical Biopsies
We applied 200 L of contrast agents ͑at ϳ10 12 particles per mL͒ in 1ϫ PBS containing 10% DMSO ͑v/v͒ to the surface for 45 min at room temperature. Biopsies were then transversely sectioned and imaged. After imaging, samples were placed in 10% formalin and submitted for histological evaluation.
Human Cervical Biopsies
To evaluate whether the presence of dysplasia can compromise the barrier function of the epithelium, we prepared dual fluorescent/reflectance contrast agents using anti-EGFR mAbs labeled with Alexa Fluor 647 dye and then conjugated to gold particles. The bifunctional contrast agents were topically applied to a pair of normal and precancerous ͑CIN 2͒ cervical biopsies. The excess contrast agent was removed, and two types of optical measurements were performed. First, a ball lens fiber optic probe 22 was used to measure the fluorescence of the Alexa Fluor 647 dye that is proportional to the total uptake of the dual contrast agent. Second, the biopsies were transversely sliced and imaged using fluorescence/reflectance confocal microscopy to study the spatial distribution of the agent.
Hamster Cheek Pouch Model
Ten Golden Syrian hamsters were treated 2 to 3 times weekly with a 0.5% solution of dimethyl benz͓␣͔anthracene ͑DMBA͒ in mineral oil on the right mucosal side of the cheek pouch under the supervision of a small animal veterinarian. Two nontreated, separately housed animals functioned as normal controls. After anesthetization with ketamine, the cheek pouch was inverted, and 200 to 400 L of contrast agent ͑at ϳ10 12 particles per mL͒ was applied topically in 1ϫ PBS containing 10% DMSO ͑v/v͒ for 15 to 20 min. Contrast agent exposure times were shorter than those used for biopsies due to the relatively short anesthetization times that are safely attained using ketamine. After washing, the hamsters were imaged using a portable reflectance confocal microscope. 23 Immediately following, biopsies were taken from the imaged area of the cheek pouch and submitted for histological analysis. All animals were treated under guidance from the institutionally approved IACUC protocol study standards.
Topical Delivery of Polystyrene Spheres Ex Vivo
Pig tissue from the oral cavity was obtained from a freshly harvested pig head. Cheek oral epithelial samples were cut in approximately 6-mm cubes. The specimens were embedded in an agarose gel with the epithelium exposed. Polystyrene fluorescent nanoparticles conjugated with neutravidin ͑Molecular Probes͒ were then topically applied for 45 min in solutions containing 10% DMSO or 5% Transcutol as permeation enhancers. Nanoparticles with 40-and 100-nm diam were used. After incubation, the samples were washed, transversely sliced using the Krumdieck Tissue Slicer, and imaged under a fluorescence confocal microscope. The fluorophors were excited with a 488-nm Ar laser and detected using a 560-nm-long bandpass filter.
Optical Imaging
Darkfield and fluorescence imaging of cells was performed using a Leica DM6000 upright microscope configured with a 20ϫ, 0.5-NA darkfield objective, fluorescence filter cubes ͑Chroma͒, and Xe light source. RGB images were captured using a Q-Imaging Retiga EXi CCD camera with color LCD attachment. Hyperspectral images were captured with a SpectraCube ® spectral imaging camera ͑Applied Spectral Imaging͒. Tissue phantoms were imaged using a Leica TCS-4D confocal microscope using 488-nm excitation and 515/10-nm bandpass filter ͑Fluorescein͒ and 514-nm excitation and 660-nm long pass filters ͑SNARF͒. Reflectance was acquired with 647-nm excitation. Cervical biopsies were imaged using a Leica SP2 AOBS confocal microscope. Reflectance images were obtained with 594-nm and 633-nm illumination. Bifunctional contrast agents were imaged with 633-nm excitation and 650 to 700-nm emission bandwidth. Imaging of hamsters in vivo was carried out using a portable reflectance confocal microscope, which is described in Ref. 23 .
Scanning Transmission Electron Microscopy
Labeled A-431 cells were absorbed onto a gold/carbon film grid and exposed to 5% buffered, pH 7.4 formaldehyde for 10 min at room temperature ͑RT͒. The cells were then dehydrated in a series of water/ethanol mixtures with increasing ethanol percentage from 10% to 25%, 50%, 75%, and finally 100% for 10 min in each solution. Subsequently, cells were fixed in 2% glutaraldehyde for 10 min, and the samples were then subjected to a series of ethanol/acetone exchanges in the same manner as described earlier for water/ethanol. After this treatment, the cells were placed in an automated CO 2 /acetone critical point drying apparatus ͑Samdri-790, Tousimis Corp.͒, which avoids surface tension-induced disturbances of the cell surface during drying. 24 Cells were imaged with a JEOL 2010F STEM equipped to image in Z-contrast mode. A HAADF detector ͑50 to 110 mrad͒ was used for image acquisition with a probe size of 0.2 nm. In addition, the JEOL STEM system contains atom-level probes connected to an Oxford INCA Energy Dispersive Spectrometer ͑EDS͒ to positively identify the presence of gold on the cellular surface.
Modeling of Light Scattering from Metal Nanoparticles
For incident collimated light of intensity I 0 , the total power P scattered into a given differential solid angle d⍀͑ , ͒ from an assembly of N particles may be expressed as:
Modeling of Light Scattering from Metal Nanoparticles
where S͑ , ͒ is a factor analogous to the structure factor used to express scattering from large molecules or from refractive-index fluctuations in solids, 25 and d 0 /d⍀ is the differential scattering cross section for a single particle in the forward direction ͑i.e., =0͒. The structure factor includes the scattering effects of the geometry of the assembly of particles, including effects due to the phase shift of light scattered from distinct particles in the assembly.
Modeling of Light Scattering from Metal Nanoparticles
For aggregates of size sufficiently less than /2, the optical phase shift across the aggregate is minimal. 26 In this case, taking the wavelength and angular integration of Eq. ͑1͒ results in:
Modeling of Light Scattering from Metal Nanoparticles
where s is the wavelength-integrated total scattering cross section. In the present analysis, the effects of plasmon coupling contribute at the second order and, therefore, can be neglected. This approximation was used to model the expected behavior of the scattering signal intensity from living cells labeled with anti-EGFR gold bioconjugates and indicates a quadratic dependence on the scattering cross section with respect to the number of particles in an aggregate. Simulation of the spectral shift for nanoparticle aggregate cross-sections was implemented through the use of a simple two-parameter effective-field model. The two model parameters correspond to the mean interparticle spacing in an aggregate and the largest aggregate size that contributes to the observed red shift. This model is a first-order approximation of a complex system of nanoparticle arrangement on the cytoplasmic membrane; however, it can be used to illustrate the major tendencies in scattering by nanoparticle aggregates. It is important to note that the contribution to the spectrum from large aggregates is significantly enhanced over that from smaller aggregates due to the coherent scattering enhancement discussed earlier. The aggregates were assumed to have planar geometry, which is appropriate for an arrangement of particles on the plasma membrane. A particle center-to-center spacing of 2.25 times the particle radius and a 91-particle aggregate size were found to give a spectrum consistent with the largest experimentally observed spectral shift.
Modeling of Light Scattering from Metal Nanoparticles
All simulations use the most widely accepted experimental permittivity data for gold, with appropriate corrections for the small particle size. The gold particle dimer simulation includes coupling through the dipole-dipole term, and this simulation is known to be quite accurate for the small particles that were used in this study. For the calculation of the total cross section, the average value is taken over polarization and dimer orientation.
Results

Optical Properties of Gold Nanoparticles for Molecular Imaging of Cancer In Vivo
We imaged SiHa cells with a darkfield microscope equipped with a hyperspectral imaging system and color CCD cameras to study the color and spectral changes associated with labeling using anti-EGFR gold conjugates ͑Fig. 2͒. The conjugates were prepared using monoclonal antibodies, which do not interfere with biological activity of cells. 28 The unlabeled cells appear blue due to the intrinsic cellular scattering ͓Fig. 2͑a͔͒. The labeled cells have dim green regions and bright regions that are different shades of orange ͓Fig. 2͑b͔͒. The green tinge is the color of the isolated nanoparticles, and it corresponds to regions with low density of bound contrast agent. The orange color corresponds to the closely spaced assemblies of anti-EGFR gold conjugates that interact with EGF receptors on the plasma membrane of SiHa cells. EGF receptors are known to form closely spaced assemblies in cancer cell membranes. 29 Hyperspectral imaging reveals that there is more than a 100-nm red shift in light scattering associated with the orange-colored assemblies of the particles in SiHa cancer cells ͓Fig. 2͑c͔͒.
Optical Properties of Gold Nanoparticles for Molecular Imaging of Cancer In Vivo
The wavelength shift of the maximum scattering cross section is attributed to changes in the local electric field in the vicinity of an individual particle due to the combined effects of the scattered electric fields from all other particles in the imaging voxel. The dominant contribution to interparticle coupling is from the dipole-dipole interaction, which depends on the cube of the interparticle distance. 27 Therefore, the spatial arrangement of gold bioconjugates is key to understanding the observed color changes. To determine if experimental results correlate with theory, high-angle annular darkfield ͑HAADF͒ scanning transmission electron microscopy ͑STEM͒ pictures were obtained of whole labeled cells ͓Figs. 3͑a͒ and 3͑b͔͒. The observed distribution of the nanoparticles is in good agreement with previously published reports on the organization of EGF receptors. 29 The ϳ100-nm wavelength shift of the maximum scattering cross section from a nanoparticle aggregate is consistent with published theoretical results 27 when estimates of mean interparticle distance are taken from the electron microscopy images ͓Fig. 3͑c͔͒. A smaller red shift ͑5 to 8 nm͒ is predicted in the case of two isolated interacting nanoparticles ͓Fig. 3͑c͔͒. Hyperspectral images reveal spectral information ͑c͒ at individual pixels, as indicated by the ͓colored͔ arrows. The scattering peak from individual gold bioconjugates ͑maximum ϳ530 nm͒ cannot be resolved in the hyperspectral data on the background of the scattering signal from cells ͑C, ͓green͔ curve͒. EGFR mediated aggregation of gold bioconjugates results in a significant increase in scattering intensity and shift of scattering maximum to the ͓red͔ region at ϳ650 nm ͑C, ͓red͔ curve͒. Scale bar is ϳ20 m.
Optical Properties of Gold Nanoparticles for Molecular Imaging of Cancer In Vivo
To study the intensity behavior of optical scattering from nanoparticle labeled cells, we labeled cancer cells using bifunctional anti-EGFR contrast agents ͑Fig. 4͒. These agents consisted of fluorescently labeled mAbs conjugated with gold nanoparticles. We carried out fluorescence and darkfield reflectance measurements from the same labeled cells and plotted the values of reflectance signal versus fluorescence signal on a pixel-by-pixel basis. While the fluorescence signal intensity is proportional to the number of gold particles in each pixel, the intensity of darkfield reflectance is proportional to the scattering cross section of the gold nanoparticle assemblies. The experimental data of Fig. 4 agree well with the theoretically predicted quadratic dependence of the scattering cross section on aggregate size ͑see Sec. 2.11͒. Because labeling with gold bioconjugates can give information on both the overexpression and nanoscale spatial relationship of EGF receptors in the cell membrane, both of which are altered in neoplasia, it can significantly increase the image contrast between normal and EGFR overexpressing cancer cells. In contrast, fluorescent labeling results in signal intensities that are linearly related to receptor concentration. Figure 5 shows transverse sections of topically labeled heterogeneous tissue phantoms and indicates that the reflectance signal ͑white͒ is localized to the periphery of the green EGFR͑+͒ cells. Little to no reflectance signal is associated with red EGFR͑−͒ cells. Results demonstrate that DMSOmediated delivery does not adversely alter molecular specificity of the contrast agents and enables penetration through a thickness that corresponds to a typical epithelial layer.
3-D Tissue Cultures

Human Cervical Biopsies
While 3-D tissue cultures remain a useful, reproducible preclinical tool, the use of human ex vivo tissue represents a more realistic and complex model. We analyzed a total of four colposcopically normal/abnormal biopsy pairs. Figure 6͑c͒ summarizes the histopathologic diagnoses and the abnormal to normal reflectance intensity ratios after labeling. The reflectance intensity was integrated over the entire epithelial thickness. The histopathologic diagnoses range from changes associated with human papillomavirus infection to CIN 3 and represents several important steps in the progression of precancerous changes in the cervix. The reflectance intensity ratio increases with progression of carcinogenesis ͓Fig. 6͑c͔͒; this correlates well with the increase of EGFR expression seen in precancer progression in the cervix. 30 Figure 6͑a͒ shows representative confocal reflectance images of a normal cervical biopsy and a biopsy with severe dysplasia ͑CIN 3͒ labeled with anti-EGFR gold contrast agents. The abnormal specimen clearly shows a distinct "honeycomb" structure characteristic of EGFR labeling on cellular membranes. No distinct structures are visible in the normal biopsy. We observed signal intensity differences of up to 21-fold between normal and abnormal cases. The confocal reflectance and H&E stained images of the abnormal biopsy reveal very similar tissue architecture ͓Fig. 6͑b͔͒.
Human Cervical Biopsies
It is known that epithelial dysplasia can compromise the barrier function of the epithelium. To evaluate whether the "leaky roof" of dysplasia partially accounts for the increased signal intensity of precancerous biopsies, we used dual fluorescent/reflectance contrast agents topically applied to a pair of normal and precancerous ͑CIN 2͒ cervical biopsies. The abnormal to normal fluorescence intensity ratio, as measured by a ball lens fiber optic probe, 22 was 1.4± 0.1, indicating delivery of ϳ40% more contrast agent to the abnormal biopsy. The transversely sliced abnormal biopsy showed both fluorescence associated with the Alexa Fluor 647 dye ͓Fig. 7͑b͔͒ and the strong reflectance signal associated with gold ͓Fig. 7͑a͔͒ throughout the entire epithelial thickness of ϳ250 m. The fluorescence and reflectance were perfectly colocalized, as seen in Fig. 7͑c͒ ; this demonstrates that both signals are associated with the dual contrast agents. In contrast, fluorescence was observed to a depth of 160 m in the normal sample ͓Fig. 7͑e͔͒, and only very weak reflectance was detected, as shown in Fig. 7͑d͒ . The reflectance intensity was ϳ11.6 times higher in the abnormal sample than in the normal, even in regions with similar fluorescence intensity of the Alexa Fluor dye ͑and therefore similar amounts of the contrast agent͒. The difference in the reflectance signal between the two samples can be attributed to EGFR-mediated clustering of gold bioconjugates in the abnormal sample. In the normal biopsy, nonbasal epithelial cells do not express substantial levels of EGFR molecules; 30 therefore, the effect of contrast agent clustering is not significant.
In Vivo Hamster Cheek Pouch Model
Last, we demonstrated the efficacy of molecular-specific imaging with gold bioconjugates in vivo in the hamster cheek pouch carcinogenesis model. Histologically, the 16-week treatment with dimethyl benz͓␣͔anthracene ͑DMBA͒ pushes the epithelial lining of the cheek pouch through progressive dysplastic changes and ultimately carcinoma in situ. 31 Anesthetized hamsters were imaged with a portable reflectance confocal microscope described in Ref. 23 immediately before and after topical application of anti-EGFR gold nanoparticles. Figure 8 shows results of in vivo imaging of the cheek pouch of the same hamster at different points of the study. The images in Fig. 8͑a͒ were obtained before the beginning of treatment with the carcinogen, and the images in Fig. 8͑b͒ show the results after 3 weeks of treatment. Although the contrast agents were applied in both imaging sessions, cell-membrane morphology characteristic for labeling of EGF receptors and a significant increase in scattering intensity after the application of gold bioconjugates were observed only after the animal was treated with DMBA ͓Fig. 8͑b͒ right͔. The contrast in the images between the normal animal ͓Fig. 8͑a͒ right͔ and the animal with early neoplastic changes ͓Fig. 8͑b͒ right͔ after the application of the contrast agents exceeds 10 times. Figure  8͑a͒ , left, and ͑b͒, left, indicate the appearance before application of contrast agents. The imaging results were confirmed using EGFR immuno-histochemical staining of cheek pouch 6 Confocal reflectance images of transverse sections of abnormal ͓͑a͒, left͔ and normal ͓͑a͒, right͔ cervical biopsies labeled with anti-EGFR gold conjugates. The image of the abnormal biopsy was obtained just above the basement membrane. Images of tissue intrinsic fluorescence were also collected ͑not shown͒ to independently confirm the presence of epithelial cells in the same field of view. Corresponding H&E staining of the abnormal ͓͑b͒, left͔ and normal ͓͑b͒, right͔ biopsies. An excitation wavelength of 647 nm was used in ͑a͒. Scale bars are ϳ25 m. In ͑c͒, the average signal intensity ratios between the abnormal and normal biopsy pairs were compared to the abnormal diagnosis in the four sample pairs collected. The abnormal/ normal signal ratio increased monotonically with pathological severity.
In Vivo Hamster Cheek Pouch Model
biopsies taken from the areas that were imaged in vivo ͓Fig. 8͑c͔͒. These results clearly demonstrate the possibility of molecular-specific imaging with topically delivered gold bioconjugates. However, they were not always repeatable. We attribute the difficulty in topical delivery of the nanoparticles to extremely high levels of superficial keratin expressed in the hamster buccal mucosa. The thick layer of keratin represents a significant barrier against topical delivery of not only nanoparticles and macromolecules but even small molecules like acetic acid. Fortunately, such elevated levels of surface keratin are not characteristic for human epithelial tissues. Therefore, future preclinical evaluation of topical delivery of contrast agents should be focused on animal models with levels of keratin expression that are more representative of human epithelium.
Conclusion
It is known that the optical properties of metal nanoparticles undergo dramatic changes when two or more particles are in close vicinity 12 to each other. These include an increase in the scattering cross section per particle and a red shift in the plasmon resonance frequency ͑color change͒. Recently, these properties were exploited to develop ultrasensitive assays in vitro, 5, 8, 9, 11, 32, 33 as well as for photothermolysis applications. 34 Some of the most promising directions in this area focus on exploiting plasmon resonance coupling between adjacent nanoparticles for DNA probe assays. 11, 32, 33 Here, we used cancer cells labeled with anti-EGFR gold conjugates to demonstrate how these nonlinear optical properties can translate into high-contrast molecular imaging of cancer biomarkers in living cells.
Conclusion
EGFR-mediated nanoparticle aggregation results in plasmon coupling between gold bioconjugates, producing a spectral shift of more than 100 nm in light scattering of the nanoparticles. In addition, coherent scattering effects result in a quadratic increase in the total scattering cross section of closely spaced nanoparticles for an assembly with total aggregate size sufficiently less than /2, where is the wavelength of light in the media; 26 this occurs because an assembly of nanoparticles manifests an electric polarization in response Fig. 7 Topical delivery of bifunctional fluorescent/reflectance anti-EGFR gold nanoparticles in abnormal ͑CIN 2͒ ͓͑a͒, ͑b͒, ͑c͔͒ and normal ͓͑d͒, ͑e͔͒ cervical biopsies. The dual contrast agents were prepared using anti-EGFR mAbs labeled with Alexa Fluor 647 dye and then conjugated to gold particles. Reflectance confocal images ͓͑a͒ and ͑d͔͒ were obtained using 633-nm excitation, and fluorescence confocal images ͓͑b͒ and ͑e͔͒ were excited with 633 nm and collected using a 650 to 700-nm filter. Image ͑c͒ is the overlap of images ͑a͒ and ͑b͒. The absolute intensity of the reflectance signal ͑a͒ is ϳ50-fold higher than the intensity of the fluorescence signal ͑b͒. The detector gain was adjusted to obtain both images at the same intensity level. The scale bar is ϳ25 m.
Conclusion
to the incident electric field. The magnitude of this polarization is proportional to the magnitude of the incident field and also to the total number of nanoparticles. The scattered electric field is proportional to this polarization, and therefore the scattered power and the scattering cross section are proportional to the square of the number of the particles in the aggregate.
Conclusion
The combination of the plasmon redshift and the nonlinear increase in the total scattering cross section of aggregates leads to a large increase in the scattering signal from labeled cells in the red optical region; this allows simple optimization of the excitation wavelength for detection of highly proliferating cancerous cells, even in the presence of isolated gold bioconjugates. Additionally, the large red optical shift in the plasmon resonances provides the possibility to decrease the contribution from the endogenous scattering of cells and epithelial tissue that is predominant in the blue spectral region. Indeed, high contrast was observed in the images of normal and precancerous human cervical biopsies ͑Figs. 6 and 7͒ and between normal and abnormal hamster cheek pouch in vivo ͑Fig. 8͒ without implementation of any washing steps, which would strongly limit the potential of in vivo applications. We observed contrast ratio in excess of tenfold in vivo, providing substantial improvement over recently reported values of less than 2 times obtained in the same animal model of carcinogenesis using antibody-targeted near-infrared ͑NIR͒ fluorescent dyes. 35 In fluorescence labeling, signal intensity is linear with the number of fluorophors, and unbound dye can create strong background signal. To overcome this limitation, "smart" fluorescent probes which emit light only in the presence of particular biomolecules such as proteolytic enzymes have been developed. 36 Although very promising, this approach can be used for only a limited number of different biomolecules. It is important to note that the nonlinear properties of gold nanoparticles cannot be fully exploited for detection of biological molecules that do not form closely spaced assemblies in the natural environment of living cells or the human body. In these situations, enzyme-activated molecular beacons and fluorophor-quencher pairs may provide better contrast.
Conclusion
We believe that the combination of the contrast agents based on gold nanoparticles, topical delivery formulation, and noninvasive optical systems is feasible for use in human clinical trials in the near future because each component of this combination has already been approved for different applications in humans. The bioreactivity and toxicity of metallic gold has long been established as low, as evidenced by the use of metallic gold implants, the use of monovalent gold salts in treatment of arthritis, 10 the exploration of gold nanoparticles as gene therapy carriers for HIV patients with up to 500 mg of gold applied during treatment, 37 and preclinical studies of a new minimally invasive thermal treatment of cancer using gold nanoshells. 21 However, it is important to note that metallic gold is not metabolized. In some cases, a premalignant lesion can require a long patient follow-up; therefore, potential interference between the initial labeling with gold nanoparticles and subsequent imaging should be considered. It is well known that EGFR is regularly internalized and degraded within the cell. 38 The degraded receptors are replaced with newly synthesized EGF receptors that can be targeted by the contrast agents.
Conclusion
Delivery of nanoparticulates through mucosal boundaries has been demonstrated in a number of studies for a variety of size ranges. For example, 150 to 300-nm diameter chitosan derivative-based nanoparticles have been successfully used as therapeutic carriers 39 for peanut allergy immunization in the intestinal epithelium. DMSO has been shown to facilitate transmucosal transport of several materials such as antibodies, 40 anticancer drugs, 41 and photosensitizers. 42 The FDA has approved DMSO as a treatment for interstitial cystitis via bladder instillation, 20 and the toxicity of DMSO has been shown to be quite low, with an LD 50 of 17 400 mg/ kg. Further, there are several other compounds that have been shown to promote transmucosal permeability, such as polyvinyl pyrrolidone ͑PVP͒ ͑Ref. 43͒, chitosan, 39 and lipid surfactants 44 that may be utilized for promoting topical delivery of gold nanoparticle contrast agents. We found that two permeation enhancers, DMSO and Transcutol, can deliver neutravidin-conjugated 40-nm-diameter polymeric fluorescent spheres through the whole thickness of fresh porcine oral epithelium ͑Fig. 9͒. However, delivery of 100-nm particles was significantly inhibited.
Conclusion
We demonstrated that impairment of the epithelial barrier function by precancerous lesions facilitates topical delivery of gold bioconjugates in two ways: overall, approximately 40% more contrast agents are delivered to the abnormal tissue as compared to normal epithelium, and the contrast agents pen- 8 In vivo confocal reflectance imaging of EGFR overexpression in a hamster model. Images shown are obtained from the same hamster before the beginning of the treatment with carcinogen DMBA ͑a͒ and after 3 weeks of DMBA treatment ͑b͒. The images were taken immediately before ͓͑a͒, left, and ͑b͒, left͔ and after ͓͑a͒, right, and ͑b͒, right͔ topical application of the anti-EGFR gold conjugates. EGFR immunohistochemical staining ͑c͒ reveals elevated EGFR levels in the DMBAtreated animal ͓͑c͒, right͔ and very low levels of EGFR expression before DMBA treatment ͓͑c͒, left͔. The scale bars are ϳ50 m.
Conclusion
etrated through the whole epithelial thickness ͑more than 250 m͒ in the abnormal tissue and about 160 m deep in the normal epithelium. However, the 40% increase in the amount of the topically delivered contrast agent between the abnormal and normal samples cannot account for ϳ11-fold increase in the intensity of the reflectance signal. Even in the areas where the amount of the contrast agent is the same in the normal and precancerous samples, we still observed the 11-fold increase in the reflectance signal in the abnormal tissue. Therefore, the specific binding of the contrast agents to EGF receptors in the abnormal sample is the determining factor in producing high reflectance intensity and increasing image contrast.
Conclusion
Thus, gold bioconjugates in combination with topical delivery formulations and noninvasive real-time optical imaging systems are uniquely positioned to fulfill the need for sensitive detection and monitoring of epithelial cancers and their precursors, which account for approximately 85% of all cancer-related deaths. This can impact clinical practice, providing new tools to assess the risk of cancer progression based on molecular profiling, rational choice of therapeutic agents, and rapid molecular assessment of cancer response to therapy. Fig. 9 Topical delivery of fluorescently labeled polymeric nanospheres coated with neutravidin to the oral cavity epithelium of a pig ex vivo using 10% DMSO as a permeation agent ͑a͒ and 5% Transcutol ͑b͒. Fluorescence confocal images are shown in the left column, while the fluorescence signal overlaid with the transmittance signal is shown in the right column. The scale bar is ϳ100 m.

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