Molecular photoacoustic imaging with ultrasmall gold nanoparticles

: Gold nanoparticles (AuNPs) below 10 nm in size can undergo renal clearance, which could facilitate their clinical translation. However, due to non-linear, direct relationship between their absorption and size, use of such “ultra-small” AuNPs as contrast agents for photoacoustic imaging (PAI) is challenging. This problem is complicated by the tendency of absorption for ultra-small AuNPs to be below the NIR range, which is optimal for in vivo imaging. Herein, we present 5-nm molecularly activated plasmonic nanosensors (MAPS) that produce a strong photoacoustic signal in labeled cancer cells in the NIR, demonstrating the feasibility of sensitive PAI with ultra-small AuNPs.


Introduction
Gold nanoparticles (AuNPs) are widely used as molecularly targeted contrast agents for photoacoustic imaging (PAI) because of their high absorption cross-sections [1][2][3][4][5][6][7]. For example, an absorption cross-section of 40nm spherical AuNPs is up to 5 orders of magnitude higher than the cross-section of commonly used absorbing organic dyes, such as rhodamine-6G or indocyanine green [8]. Therefore, labeling a molecular target with a single such nanoparticle would be theoretically equivalent to labeling it with thousands of organic dye molecules. Molecularly specific labeling of a single target with thousands of organic chromophores is challenging. However, recent studies have reported development of organic dyes and dye aggregates encapsulated either in micelles or liposomes that can facilitate delivery of a large quantity of chromophores for biomolecular labeling [9][10][11][12]; this is a very promising direction in molecular PAI that is still in early stages of development.
A number of studies in the literature report molecularly specific PAI using AuNPs with a core diameters greater than 20 nm [13][14][15][16][17], which is well above the renal clearance threshold of 5 -10 nm [18][19][20][21]. Retention of non-biodegradable AuNPs can result in side effects such as chronic inflammation and associated complications [22,23]. The lack of efficient body clearance of AuNPs has been a long-standing problem in clinical translation of many promising technologies that are based on in vivo administration of gold nanomaterials [24][25][26][27]. Note that AuNPs for in vivo applications consist of a non-biodegradable gold core and an organic coating that can eventually degrade in the body. During in vivo administration, the overall hydrodynamic diameter of AuNPs could be larger than the renal clearance threshold, preventing fast renal clearance. As the organic coating degrades, the AuNPs with small core sizes could undergo accelerated excretion from the body. However, the excretion process is still very poorly understood and requires significant further investigation.
Development of ultra-small, targeted AuNPs with core diameters below 10 nm can address the body-clearance problem. In addition to overcoming body clearance concerns, the use of ultra-small particles can also greatly improve organ biodistribution and depth of tissue penetration. For example, when particle size increased from 15 nm to 150 nm, a higher level (EMD Millipore) for 20 mins at 3,100 g at 4 °C, and the antibodies were labeled with Alexa Fluor 647 (AF647) fluorophores (A20173, Invitrogen) using the manufacturer's protocol. Then, 100 µL of fluorescently labeled antibodies at 1 mg/mL in PBS buffer were mixed with 4 µL of 23.25mM (~150-fold molar excess) heterofunctional hydrazide-PEG-dithiol linker (dithiolalkanearomatic-PEG 6 -NHNH 2 , SPT-0014B, SensoPath Technologies), and the mixture was incubated in the dark for 1 hr at RT on a shaker at 350 rpm. Unreacted linker molecules were removed by a 10kDa MWCO centrifuge filter at 3,100 g for 20 min at 4°C, and the linker-antibody conjugates were reconstituted at 100 µg/mL in PBS.
Antibody-linker solution at 100 µg/mL in PBS was added to 5nm and 40nm AuNPs, both at optical density (OD) = 1, to achieve final antibody concentrations of 47 and 9.1 µg/mL, respectively, corresponding to 5-and 680-fold molar excesses of antibodies, respectively. The suspensions were incubated in the dark for 1 hr at RT on a shaker at 350 rpm. Then, 5 kDa mPEG-SH (MPEG-SH-5000, Laysan Bio) at 0.05 mg/mL in PBS was added to the suspensions of 5nm and 40nm nanoparticles to achieve final concentrations of 3.8 and 4.2 µg/mL, respectively, followed by an additional 30min incubation at RT on a shaker at 350 rpm. Then, antibody-conjugated 5nm AuNPs were spun down at 100,000 g for 1 hr at 4 °C. Forty nanometer AuNPs were centrifuged in the presence of 2% w/v PEG copolymer (Polyethyleneglycol Bisphenol A Epichlorohydrin Copolymer, P2263, Sigma) at 3,100 g for 30 min at 4 °C. The final antibody conjugated nanoparticles were resuspended in PBS and stored at 4 °C for future experiment. UV-vis spectrophotometry (Synergy HT, BioTek Instruments), DLS (Zetasizer Nano, Malvern) and z-potential analysis (Delsa TM Nano C, Beckman Coulter) were used to characterize spectral properties, size, and surface charge of the nanoparticles, respectively.
For transmittance electron microscopy (TEM), samples were placed on 100-mesh, carboncoated, formvar-coated copper grids treated with poly-l-lysine for approximately 1 hour. Samples were then negatively stained with Millipore-filtered aqueous 1% uranyl acetate for 1 min. Stain was blotted dry from the grids with filter paper, and samples were allowed to dry. Samples were then examined in a JEM 1010 transmission electron microscope (JEOL, USA, Inc., Peabody, MA) at an accelerating voltage of 80 Kv. Digital images were obtained using the AMT Imaging System (Advanced Microscopy Techniques Corp., Danvers, MA). Size distribution of nanoparticles in TEM images was carried out using IMARIS software (Bitplane).

Characterization of antibody binding to 5nm gold nanoparticles
Antibodies pre-labeled with AF647 were used to quantify antibody binding based on the dye's prominent absorbance peak at 650 nm. The number of bound antibodies was determined by measuring the difference in the 650nm absorbance between the antibody solution at the concentration used in the conjugation reaction and the supernatant, which was obtained after centrifuging antibody-conjugated 5nm AuNPs at the completion of the antibody conjugation. Then, a linear calibration curve correlating 650nm absorbance with antibody concentration was used to determine the total amount of antibodies conjugated to the nanoparticles. Finally, the number of antibodies per nanoparticle was calculated by dividing the number of antibodies by the number of 5nm AuNPs, with the latter being estimated from its optical density (OD).
Fluorescence quenching was used to measure the dissociation constant (K d ) between 5nm AuNPs and fluorescently labeled antibodies. Serial 2-fold dilutions of citrate-coated 5nm AuNPs with concentrations ranging from 105 nM to 1.6 pM in deionized water (18 MΩ) were mixed at a 1:1 v/v ratio with a 5nM solution of AF647-labeled antibodies conjugated with hydrazide-PEG-dithiol linker. Each reaction mixture was allowed to interact for 20 min at RT, and fluorescence of the AF647 dye was measured as a function of nanoparticle concentration using a Monolith NT.115 (NanoTemper Technologies); this system requires a very small sample volume (~20 µL), which greatly minimizes the amount of reagent needed for the titration curve. Using the Hill equation, the resulting data were fitted with the Monolith NT.115's analysis software to yield a K d . One microliter of dithiothreitol (DTT) at 1 mM in PBS was added to the 100 µL of 100 µg/mL antibody solution before mixing with AuNPs to confirm that the observed fluorescence quenching was due to thiol-mediated antibody binding to the nanoparticles.

Dark-field and fluorescence imaging of labeled cells
EGFR-positive A431 cells (human epidermoid carcinoma) and EGFR-negative MDA-MB-435 cells (human melanoma) were cultured in HyClone DMEM/High glucose (GE Healthcare Life Sciences) media supplemented with 10% FBS and 1% PS penicillin-streptomycin (Life Technologies) in a humidified atmosphere and 5% CO 2 at 37 °C. Cells were seeded in a Lab-Tek II Chambered Coverglass System (Nalge Nunc International) at 10,000 cells/well and incubated overnight. Then, 5nm or 40nm MAPS were added to the cells at a final concentration of 2.36 µg/mL and incubated at 37 °C. After incubation for various time periods ranging from 1 to 24 hours, the cells were washed with warm PBS containing Ca 2+ and Mg 2+ to remove free nanoparticles. To quantify changes in scattering of gold nanoparticles in dark-field images, cell outlines were determined using ImageJ and signal intensities of R, G and B channels were obtained from the cell regions. A background signal was acquired outside the cells and was subtracted from the cellular signal. Then, the scattering changes associated with plasmon resonance coupling between AuNPs were quantified as a relative increase in the intensity of the red channel [38] by applying the following formula: where R and B are scattering intensities of the red and blue channels of the color camera, respectively. Fluorescence imaging was used to characterize molecular specificity of the antibody-targeted nanoparticles. Dark-field images were acquired using a Leica DM 6000 upright microscope with a 20X 0.5 NA objective, a SPOT Pursuit XS mosaic color CCD camera (Diagnostic Instruments), and a 100W halogen lamp. Fluorescence images were obtained using a CY5 filter cube with 620/60 nm excitation and 700/75 nm emission filters and a 660nm dichromatic mirror. Three independent 453 µm x 341 µm fields of view were analyzed for each sample. All optical imaging data are presented as means and standard deviations of (R/B -1) values or fluorescence intensities from all analyzed cells for each sample.

Tissue-mimicking phantom preparation and PAI setup
Photoacoustic imaging of cells was carried out in tissue-mimicking phantoms [40] that each consisted of a gelatin background with 36 gelatin/cell inclusions. Using a polydimethylsiloxane (PDMS, Dow Corning) mold that was fabricated using a 3D-printed template, phantom backgrounds were cast to have 36 hemispherical 57µL injection wells. The phantom background was comprised of a mixture of 8% w/v gelatin (Sigma-Aldrich), 1% v/v propanol (Sigma-Aldrich) and 0.1% v/v glutaraldehyde (Sigma-Aldrich); this mixture was prepared at 45°C and cooled at 4°C before removing from the PDMS mold. Gelatin/cell suspensions were then aliquoted to phantom wells in triplicate followed by deposition of a top (~3mm thick) gelatin layer. In a typical experiment, one million cells were labeled with antibody-conjugated AuNPs at ~65 µg Au for time periods ranging from 1 to 24 hours. After washing of unbound particles, the cell pellet was reconstituted in PBS and counted. For each sample, ~90 µL of a cell suspension in PBS at a desired concentration was mixed with 16% w/v gelatin in a 1:1 v/v ratio at 40°C. A Vevo 2100 LAZR (FUJIFILM VisualSonics) highfrequency ultrasound and photoacoustic imaging system equipped with a liner array transducer (LZ250; 20-MHz center frequency) was used for PAI. Volumetric (0.47mm step size) B-mode 830, and 870 imaging slice intensity with statistics were optical and PA

Characte
Monoclonal glycosylated F allows the an Antibodies w and to charact in the absorba 650nm absorb clearly seen a peak, it was nanoparticle (  (Fig. 1(B) ye on the conj es ( Fig. 1(C) Size-exclusion chromatography of antibody-conjugated 5nm AuNPs before and after removal of unreacted antibodies by ultracentrifugation confirmed successful washing ( Fig. 1  (D-F)). DLS measurements of the citrate-stabilized 5nm AuNPs prior to functionalization and 5nm AuNPs conjugated with antibodies and mPEG molecules, referred to as "5nm MAPS," show sizes of 7 ± 2 nm and 22 ± 6 nm, respectively ( Fig. 1(G)). TEM images of 5nm MAPS reveal particles with predominantly 5 nm core size and with no sign of particle aggregation ( Fig. 1(H)). Zeta potential measurements of 5nm MAPS revealed a negative 47.10 ± 0.99 mV surface charge, indicating a significant amount of residual citrate anions on the gold surface.
We noticed a partial quenching of fluorescence upon binding of antibodies labeled with AF647 dyes to the gold nanoparticles that is most likely associated with a non-radiative energy transfer to the metal surface [41,42]. We used this effect to quantify the dissociation constant of antibody binding to the nanoparticles (Fig. 1(H)). To this end, changes in intensity of fluorescence were recorded as a function of concentration of 5nm AuNPs added to a solution of fluorescently labeled anti-EGFR antibodies at a fixed concentration. These measurements resulted in a classical sigmoidal binding curve that was used to determine a K d of 4.6 ± 2.7 nM for antibody-nanoparticle interactions. To confirm that antibody binding to AuNPs was responsible for the fluorescence quenching, DTT was added to the antibody solution before nanoparticle titration. DTT is known to effectively disturb binding of any thiolated molecules to a gold surface [43,44]. Fluorescence quenching was not observed in the presence of DTT, indicating that the observed binding curve is associated with antibody attachment to AuNPs. Previous studies reported a K d ranging from 10 to 1,000 nM for interactions between 5nm AuNPs and various blood proteins [45]; these studies were focused on non-specific interactions that were not mediated by any conjugation chemistry. The significantly lower dissociation constant in our study indicates a stronger antibody binding through a thiolated linker [46].

Specificity of 5nm MAPS
Molecular specificity of 5nm MAPS was assessed in EGFR-overexpressing A431 skin cancer cell line and EGFR-negative MDA-MB-435 melanoma cells. Both cells were labeled with either 5 nm MAPS or control 5 nm AuNPs conjugated with non-specific RG16 antibodies (RG16-AuNPs); the latter is an IgG 1 monoclonal anti-rabbit antibody developed in mouse and, therefore, does not have any cross-reactivity with human or mouse antigens. Both antibodies were pre-labeled with AF647 dyes to enable fluorescence imaging. Cells were imaged using dark-field and fluorescence microscopies to concurrently detect the plasmon resonance scattering of AuNPs and the fluorescence signal from the antibodies (Fig. 2). The plasmon resonance scattering increased over time reaching its saturation after approximately 16 hours of incubation with 5 nm MAPS (Fig. 3, A and B). Previously, we showed that this increase is associated with intracellular uptake and accumulation of nanoparticles in endosomal compartments that results in a progressive red shift in plasmon resonance scattering over time [38]. To quantify cell labeling with 5 nm MAPS in dark-field cell images, we used relative changes in intensity of red and blue channels of a RGB color camera by applying the following metric: [(R/B)-1], where R and B are integrated intensities of the red and the blue channels, respectively (Fig. 2(B)). Both the dark-field and the fluorescence cell images showed a strong signal after labeling of EGFR + A431 cells with 5 nm MAPS whereas only background signal was detected in all controls including EGFR -MDA-MB-435 cells incubated with 5 nm MAPS and EGFR + A431 cells labelled with AuNPs conjugated with RG16 antibodies (Fig. 2). The experiments were run in a triplicate, with all data sets showing a high specificity in labeling with 5 nm MAPS.

Time dependence of photoacoustic imaging (PAI) with 5 nm MAPS
The PA signal of EGFR + A431 cells was measured as a function of labeling time with 5 nm MAPS (Fig. 3) (Fig. 2). PA to spacing betw ve [38,47]. Note rnible in the p 3(A)). PA sign s in scattering uNPs inside l ral region. Inde . 4(A)).  Fig. 4(B)). As A linear regr approximately e 57 µL well. 3