Microscopic investigation of" topically applied nanoparticles for molecular imaging of fresh tissue surfaces

Figure S1. Wide-area nanoparticle-based molecular imaging (NMI) of fresh tissue topically stained with SERS NPs. A 10-mW 785-nm diode laser illuminates tissue with a spot size of 0.5 mm. A custom spectrometer disperses the collected signal onto a cooled deep-depletion spectroscopic CCD. Raster-scanned spectral imaging of the sample is performed by scanning the tissue sample, while keeping the fiberbundle imaging probe stationary. A direct classical least-squares (DCLS) algorithm is used to compute the relative SERS NP weights and the weights of all broadband background components. The ratio of the weights of targeted vs untargeted SERS NPs is used to obtain a ratiometric image of the tissue specimen. Additional details about the imaging system have been described previously [1–6]

Figure S2. TEM images of (A) unconjugated NPs and (B) antibody-conjugated NPs with uniform size distribution around 120-nm in diameter

Figure S3. We hypothesize that the design of conjugated NPs with larger sizes could reduce the diffusion of the NPs and therefore reduce the nonspecific accumulation of the NPs in tissue. This could in turn allow for molecular imaging of fresh tissue surfaces with higher NP ratios (targeted vs untargeted) compared with our original 120-nm NPs

Figure S4. In order to ensure that the relative brightness of the fluorescence from each of the NP sizes was well matched, the NP concentrations used in the conjugation reactions were tuned such that total surface area of the NPs was identical to that of the 120-nm NPs used in previous experiments. Here, we used a spectrofluorometer to show that the different NP sizes exhibit comparable levels of fluorescence for aliquots with identical total NP surface areas. Background subtraction was applied using the fluorescence intensity of stock NPs that have not been reacted with fluorophores

Figure S5. Flow cytometry validation of conjugated NPs of various sizes with cell lines of varying biomarker (EGFR) expression levels. Flow cytometry experiments were performed to verify that the binding of the EGFR-targeted NPs to EGFR-positive cell lines was comparable in magnitude for various NP sizes when staining cells with NP concentrations that were matched in terms of total NP surface areas. EGFR-NPs and isotype-NPs of various sizes were individually used to stain 3T3 (top row, EGFR−), U251 (middle row, EGFR+), or A431 (bottom row, EGFR++). The columns are separated by NP size (120, 200, and 300-nm diameter). The fourth column shows the mean fluorescence intensity between the EGFRtargeted NP vs untargeted NP to compare binding levels of the various NP sizes

Figure S6. A mathematical model of the diffusion of NPs topically applied in fresh tissue. (A) An illustration of our simplified model of NP diffusion and binding. The NP solution is an infinite reservoir that establishes a constant NP concentration at the tissue surface. NPs are allowed to diffuse into the tissue, where they bind to cell-surface biomarkers or remain unbound. (B) A representation of our mathematical model showing targeted and untargeted NPs diffusing into the tissue with diffusion coefficient D, where the targeted NPs bind to and unbind from cell-surface biomarkers with rate constants ka and kd, respectively. The compartments in the reaction network are defined as unbound targeted NPs, Ctu; bound targeted NPs, Ctb; and untargeted NPs, Cuu. The total concentration of targeted NPs is Ctar = Ctu + Ctb, while the concentration of untargeted NPs is Cuntar = Cuu. (C) Our kinetic model was run using the implicit and unconditionally stable Crank-Nicolson method and fit to the concentration curves obtained experimentally with the micro-NMI method (Figure 4) using a nonlinear least-squares algorithm (lsqnonlin in MATLAB). We note that this simple model of diffusion and binding of topically stained NPs on fresh tissue, while not a perfect fit to our experimental data, does predict a binding-site barrier for tumor tissue. Improved fitting of the model with our experimental data may potentially be achieved by taking into account the nonspecific binding of NPs to cell-surface receptors with a nonspecific binding compartment for both targeted and untargeted NPs, as previously described in the literature [7]

APPENDIX S1. REFERENCES