In Vivo Targeting of Hydrogen Peroxide by Activatable Cell-Penetrating Peptides

A hydrogen peroxide (H₂O₂)-activated cell-penetrating peptide was developed through incorporation of a boronic acid-containing cleavable linker between polycationic cell-penetrating peptide and polyanionic fragments. Fluorescence labeling of the two ends of the molecule enabled monitoring its reaction with H₂O₂ through release of the highly adhesive cell-penetrating peptide and disruption of fluorescence resonance energy transfer. The H₂O₂ sensor selectively reacts with endogenous H₂O₂ in cell culture to monitor the oxidative burst of promyelocytes and in vivo to image lung inflammation. Targeting H₂O₂ has potential applications in imaging and therapy of diseases related to oxidative stress

An Optimized Triple Modality Reporter for Quantitative <i>In Vivo</i> Tumor Imaging and Therapy Evaluation

<div><p>We present an optimized triple modality reporter construct combining a far-red fluorescent protein (E2-Crimson), enhanced firefly luciferase enzyme (Luc2), and truncated wild type herpes simplex virus I thymidine kinase (wttk) that allows for sensitive, long-term tracking of tumor growth <i>in vivo</i> by fluorescence, bioluminescence, and positron emission tomography. Two human cancer cell lines (MDA-MB-231 breast cancer and HT-1080 fibrosarcoma cancer) were successfully transduced to express this triple modality reporter. Fluorescence and bioluminescence imaging of the triple modality reporter were used to accurately quantify the therapeutic responses of MDA-MB-231 tumors to the chemotherapeutic agent monomethyl auristatin E <i>in vivo</i> in athymic nude mice. Positive correlation was observed between the fluorescence and bioluminescence signals, and these signals were also positively correlated with the <i>ex vivo</i> tumor weights. This is the first reported use of both fluorescence and bioluminescence signals from a multi-modality reporter construct to measure drug efficacy <i>in vivo</i>.</p></div

Final triple reporter tumor optical signals <i>in vivo</i> and weights <i>ex vivo</i> following therapy.

<p>Tumors from the therapy experiment were imaged then resected and weighed after 28 days of growth <i>in vivo</i>. Optical signals of the triple reporter tumors were representative of the tumor mass. Tumors in the MMAE-treated group were too small to be measured by a caliper after day 19. Significant decreases (p<0.005) in the tumor weight and optical signals in the MMAE-treated group compared to the untreated and MMAF-treated groups are indicated by*.</p

<i>In vivo</i> validation of the triple reporter components.

<p>Representative live animal images from the coronal plane of six MDA-MB-231 triple reporter tumors and two HT-1080 triple reporter tumors confirm the activity of all three imaging modalities in both cell lines <i>in vivo.</i> The fluorescence signal is shown as the radiant efficiency (p/s/cm²/str)/(mW/cm²). The bioluminescence signal is shown as the radiance photons (p/s/cm²/sr). The PET signal is shown as the% injected dose of ¹⁸F-FHBG per gram. High gut (GI) retention is characteristic of ¹⁸F-FHBG in microPET imaging, which required that tumors be placed away from the abdomen of each mouse.</p

Correlation between triple reporter tumor optical signals <i>in vivo</i> and weights <i>ex vivo</i> following therapy.

<p>(A) Fluorescence signal [(p/s)/(cm²/sr)] vs. bioluminescence signal (p/s) of the MDA-MB-231 triple reporter tumors on day 28 of tumor growth. (B) Fluorescence signal [(p/s)/(cm²/sr)] vs. weight (mg) of the MDA-MB-231 triple reporter tumors on day 28 of tumor growth. (C) Bioluminescence signal (p/s) vs. weight (mg) of the MDA-MB-231 triple reporter tumors on day 28 of tumor growth.</p

Sizes and modality signals of the triple reporter tumors after two weeks of growth.

<p>The tumor size, fluorescence signal, bioluminescence signal, and PET signal were quantified for the MDA-MB-231 and HT-1080 triple reporter tumors expressing all three modalities <i>in vivo</i>. Only tumors that were healthy and large enough to be detectable by all three modalities of the triple reporter construct were averaged and included in this dataset.</p

Comparison of four far-red and infrared fluorescent proteins.

<p>(A) The mean quartile (mean 25%) fluorescence intensities (FLI) of E2-Crimson (C), infrared fluorescent protein (I), mNeptune (N), and mPlum (P) in HT-1080 cells measured by fluorescent-activated cell sorting (FACS) were compared (100 mW laser with ex 568 nm and em 650–670 nm for C, N, and P; ex 690 nm and em 710–900 nm for I). (B) The top 5% brightest HT-1080 cells from each fluorescent protein cell type were injected into athymic nude mice (1×10⁶ cells/injection; exposure time: 500 msec; ex 590/23 nm and em 645LP for C, N, and P; ex 640/48 nm and em 700LP for I).</p

Quantification of the triple reporter optical signals to monitor therapy responses <i>in vivo</i>.

<p>(A) Average fluorescence signal [(p/s)/(cm²/sr)] of each MDA-MB-231 triple reporter tumor treatment group over time. (B) Average bioluminescence signal (p/s) of each MDA-MB-231 triple reporter tumor treatment group over time. (C) Average size (mm³) of each MDA-MB-231 triple reporter tumor treatment group over time based on caliper measurements. MMAE or MMAF (0.5 nmol/g) was administered on days 7, 10, 13, 16, 19, and 22. Significant decreases (p<0.005) in the tumor optical signals in the MMAE-treated group compared to the untreated and MMAF-treated groups are indicated by*.</p