Enhanced EPR directed and Imaging guided Photothermal Therapy using Vitamin E Modified Toco-Photoxil

Herein we report synthesis, characterization and preclinical applications of a novel hybrid nanomaterial Toco-Photoxil developed using vitamin E modified gold coated poly (lactic-co-glycolic acid) nanoshells incorporating Pgp inhibitor d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) as a highly inert and disintegrable photothermal therapy (PTT) agent. Toco-Photoxil is highly biocompatible, physiologically stable PTT material with an average diameter of 130 nm that shows good passive accumulation (2.3% ID) in solid tumors when delivered systemically. In comparison to its surface modified counterparts such as IR780-Toco-Photoxil, FA-Toco-Photoxil or FA-IR780-Toco-Photoxil accumulation are merely ~0.3% ID, ~0.025% ID and ~0.005% ID in folate receptor (FR) negative and positive tumor model. Further, Toco-Photoxil variants are prepared by tuning the material absorbance either at 750 nm (narrow) or 915 nm (broad) to study optimal therapeutic efficacy in terms of peak broadness and nanomaterial’s concentration. Our findings suggest that Toco-Photoxil tuned at 750 nm absorbance is more efficient (P = 0.0097) in preclinical setting. Toco-Photoxil shows complete passiveness in critical biocompatibility test and reasonable body clearance. High tumor specific accumulation from systemic circulation, strong photothermal conversion and a very safe material property in body physiology makes Toco-Photoxil a superior and powerful PTT agent, which may pave its way for fast track clinical trial in future

IHC Profiler: An Open Source Plugin for the Quantitative Evaluation and Automated Scoring of Immunohistochemistry Images of Human Tissue Samples

<div><p>In anatomic pathology, immunohistochemistry (IHC) serves as a diagnostic and prognostic method for identification of disease markers in tissue samples that directly influences classification and grading the disease, influencing patient management. However, till today over most of the world, pathological analysis of tissue samples remained a time-consuming and subjective procedure, wherein the intensity of antibody staining is manually judged and thus scoring decision is directly influenced by visual bias. This instigated us to design a simple method of automated digital IHC image analysis algorithm for an unbiased, quantitative assessment of antibody staining intensity in tissue sections. As a first step, we adopted the spectral deconvolution method of DAB/hematoxylin color spectra by using optimized optical density vectors of the color deconvolution plugin for proper separation of the DAB color spectra. Then the DAB stained image is displayed in a new window wherein it undergoes pixel-by-pixel analysis, and displays the full profile along with its scoring decision. Based on the mathematical formula conceptualized, the algorithm is thoroughly tested by analyzing scores assigned to thousands (n = 1703) of DAB stained IHC images including sample images taken from human protein atlas web resource. The IHC Profiler plugin developed is compatible with the open resource digital image analysis software, ImageJ, which creates a pixel-by-pixel analysis profile of a digital IHC image and further assigns a score in a four tier system. A comparison study between manual pathological analysis and IHC Profiler resolved in a match of 88.6% (<i>P</i><0.0001, CI = 95%). This new tool developed for clinical histopathological sample analysis can be adopted globally for scoring most protein targets where the marker protein expression is of cytoplasmic and/or nuclear type. We foresee that this method will minimize the problem of inter-observer variations across labs and further help in worldwide patient stratification potentially benefitting various multinational clinical trial initiatives.</p></div

Dietary curcumin post-treatment enhances the disappearance of B(a)P-derived DNA adducts in mouse liver and lungs

To study the post-treatment effects of dietary curcumin on the levels of benzo(a)pyrene [B(a)P]-induced DNA adducts, mice were administered oil or B(a)P and randomized into 7 subgroups after 24 h. One of the subgroups from both the oil and B(a)P groups was killed at 24 h while the remaining 6 subgroups were shifted to powdered control or 0.05% curcumin diet and killed after 24, 72 and 120 h (experiment 1), and 7, 14, and 28 days (experiment 2). Quantitative comparisons of BPDE-DNA nuclear adducts (area and intensity) in immunohistochemically stained lungs and liver sections was carried out by IHC profiler. A time-dependent decrease in the levels of adducts in B(a)P-treated animals was further enhanced by curcumin exposure compared to the levels in time-matched controls. To assess the contribution of apoptosis and cell proliferation in observed curcumin-mediated enhanced decrease of BPDE-DNA adducts, comparative evaluation of apoptosis and cell proliferation markers was undertaken. Results suggested enhancement of B(a)P-induced apoptosis in liver and lungs by curcumin during 24–120 h while no such enhancement was observed at 7–28 days. Results suggest curcumin-mediated enhancement in apoptosis (experiment 1) and adduct dilution (experiment 2) to be the reason for the observed higher decrease of BPDE-DNA adducts

Comparison of IHC profiler with available IHC image analysis tools.

<p>Comparison of IHC profiler with available IHC image analysis tools.</p

Comparison chart showing automated vs. manual scoring.

<p>Table shows the distribution of samples and a comparison study between the automated and the manual scoring. Total number of cases determines the sample size taking into account for the study. The difference of significance was obtained by two-tailed chi-square test resulting into values of <i>P</i><0.0001 (CI  = 95%).</p

Representative histogram profile and score of a cytoplasmic and nuclear stained image using IHC Profiler.

<p><b>A:</b> Profiling of the DAB stained cytoplasmic image sample. The histogram profile corresponds to the pixel intensity value vs. corresponding number counts of a pixel intensity. The log given below the histogram profile shows the accurate percentage of the pixels present in each zone of pixel intensity and the respective computed score. <b>B:</b> Profiling of the DAB stained nuclear stained image sample. The red spots on the DAB image indicate the threshold selection of the nucleus areas using the threshold function of ImageJ. The representative histogram profile corresponds to the number of pixels vs. the corresponding value at which the pixel of the respective intensity is present.</p

Representation of color deconvolution using the old and the new optical density (OD) vectors.

<p><b>A:</b> Color deconvolution using the old OD vectors. <b>B:</b> Color deconvolution using the new OD vectors. <b>C:</b> Scatter plot comparing the intensities on the complimentary image with the old OD vectors (blue) and the new OD vectors (red). <b>D:</b> Plot comparing the number of pixels with the intensity value of 255. An improvement between 2 to 10 fold is shown using 7 different samples. Each data plot represents an individual sample with its respective pixel count of the intensity value of 255.</p

Agreement of scores between manual vs. IHC Profiler assessment.

<p>Table summarize the agreement of scores between the manual scoring process vs. IHC profiler assessment. This table excludes the samples wherein the inter-observer score did not match with each other. Kappa statistics was performed and the value of Kappa  = 0.843 (95% CI: From 0.819 to 0.867).</p

Variability of scores in between the pathological opinions.

<p>Table summarizes the inter-observer variability of two pathologists whose opinions were taken into consideration during this study (383 cases as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096801#pone-0096801-t002" target="_blank">Table 2</a>). Sample number was rounded off to 380 for statistical comparison between the two groups. Kappa statistics was performed and the value of Kappa  = −0.669 (95% CI: From −0.702 to −0.637) indicates the strength of agreement is worse than what one would expect to see by chance alone.</p

Impact of magnification on image scoring.

<p><b>A:</b> Analysis of a 10X image area where a significant amount of stroma and fatty tissue is present. After color deconvolution, the score assigned by IHC profiler on the DAB image was determined as low positive. <b>B:</b> Scoring analysis of the same tissue area where image captured was by using a 20X lens in the marked area, focusing more on the actual tumor mass resolute a score of positive. <b>C:</b> Scoring analysis of the same tissue area wherein the image was captured using a 40X lens, focusing more on eliminating the stromal and fatty tissue region increases the percentage of the positive pixels in the positive and high positive zones.</p