Journal: Biophysical Journal

Article Title: Determination of the Membrane Translocation p K of the pH-Low Insertion Peptide

doi: 10.1016/j.bpj.2017.06.065

Figure Lengend Snippet: The pHLIP does not have a unique pK value. Bars correspond to the pK determined by W fluorescence SM, spectral CM, or FI, by CD, or by Nt-NBD or Ct-NBD of pHLIP. Mean values are shown, for experiments repeated three to six times, and error bars are the SD. Color shading represents groups with no statistical difference. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.005; NS; no significance.

Article Snippet: For the conjugation of NBD to the N-terminus of pHLIP, we used instead succinimidyl 6- n -7-nitrobenz-2-oxa-1,3-diazol-4-yl amino hexanoate (NBD-X SE; Anaspec, Fremont, CA).

Techniques: Fluorescence

Journal: Biophysical Journal

Article Title: Determination of the Membrane Translocation p K of the pH-Low Insertion Peptide

doi: 10.1016/j.bpj.2017.06.065

Figure Lengend Snippet: NBD conjugates of pHLIP display different pK values based on location. (A and B) Representative emission spectra of pHLIP-NBD (A) and NBD-pHLIP (B) show an increase in fluorescence intensity as acidity is increased (shaded arrows). (C and D) Normalized fluorescence intensity at 540 nm is plotted versus pH to determine pKCt-NBD (C) and pKNt-NBD (D). Lines in (C) and (D) show the fits of Eq. 2 to the data. Solid arrows mark the approximate pH values for the start and end of the titrations.

Article Snippet: For the conjugation of NBD to the N-terminus of pHLIP, we used instead succinimidyl 6- n -7-nitrobenz-2-oxa-1,3-diazol-4-yl amino hexanoate (NBD-X SE; Anaspec, Fremont, CA).

Techniques: Fluorescence

Journal: Biophysical Journal

Article Title: Determination of the Membrane Translocation p K of the pH-Low Insertion Peptide

doi: 10.1016/j.bpj.2017.06.065

Figure Lengend Snippet: Intrinsic W, Nt-NBD, and Ct-NBD fluorescence display similar kinetics during pHLIP membrane insertion. Given here are average spectra of W (dark shaded), Nt-NBD (dashed light shaded), and Ct-NBD (solid) in POPC.

Article Snippet: For the conjugation of NBD to the N-terminus of pHLIP, we used instead succinimidyl 6- n -7-nitrobenz-2-oxa-1,3-diazol-4-yl amino hexanoate (NBD-X SE; Anaspec, Fremont, CA).

Techniques: Fluorescence, Membrane

Journal: Biophysical Journal

Article Title: Determination of the Membrane Translocation p K of the pH-Low Insertion Peptide

doi: 10.1016/j.bpj.2017.06.065

Figure Lengend Snippet: Qualitative comparison of fluorescence pK titrations with ssNMR pKa titrations for the individual aspartate residues. The individual titrations for D14 (magenta), D25 (blue), D31 (green), and D34 (red) obtained by Hanz et al. (10) are overlaid with the fluorescence data obtained following tryptophan SM (A) and FI (B), and pHLIP-NBD (C). The ssNMR data were represented by the fitting to the Boltzmann equation (10). Normalized chemical shifts are shown for a better comparison, and values were inverted in (B) and (C) to align with the fluorescence data changes.

Article Snippet: For the conjugation of NBD to the N-terminus of pHLIP, we used instead succinimidyl 6- n -7-nitrobenz-2-oxa-1,3-diazol-4-yl amino hexanoate (NBD-X SE; Anaspec, Fremont, CA).

Techniques: Comparison, Fluorescence

Journal: Biophysical Journal

Article Title: Determination of the Membrane Translocation p K of the pH-Low Insertion Peptide

doi: 10.1016/j.bpj.2017.06.065

Figure Lengend Snippet: Tentative model showing the transition from State II to State III during the membrane insertion of pHLIP, highlighting the insertion step/s that each pK value reports on. The solid arrows represent the transitions between each step in the membrane insertion process, with the corresponding pH midpoints. The change of the D residues from shaded to solid represents the protonation. CD, and potentially SM and CM, reports on the insertion process encompassing D33, D31, and D25, and are then shown labeling the first three transitions. FI and Nt-NBD follow the protonation of D14, and to a lesser extent D25 and D33 (not shown in figure). Finally, Ct-NBD reports on the last insertion step, where the C-terminal end translocates across the bilayer.

Article Snippet: For the conjugation of NBD to the N-terminus of pHLIP, we used instead succinimidyl 6- n -7-nitrobenz-2-oxa-1,3-diazol-4-yl amino hexanoate (NBD-X SE; Anaspec, Fremont, CA).

Techniques: Membrane, Labeling

Journal: Scientific Reports

Article Title: pHLIP ICG for delineation of tumors and blood flow during fluorescence-guided surgery

doi: 10.1038/s41598-020-75443-5

Figure Lengend Snippet: Characterization of pHLIP ICG and blood clearance in mice and dogs. ( a ) The Var3 pHLIP sequence is shown with the chemical structure of the ICG dye coupled to a single Cys residue at the N-terminal part of pHLIP peptide. ( b ) Normalized absorbance (black line) and fluorescence (red line) spectra (the excitation is at 805 nm) of pHLIP ICG (5 µM) measured in DMF. ( c ) Circular dichroism (CD) spectra of pHLIP ICG measured in phosphate buffer (pH 8) in absence (pHLIP ICG, pH 8—black line) and presence (pHLIP ICG + PC, pH 8—blue line) of POPC liposomes, and in the presence of POPC liposomes at pH 5.2 (pHLIP ICG + PC, pH 5.2—red line) indicate on formation of helical structure in membrane at low pH. ( d ) pH-dependent insertion of pHLIP ICG into the bilayers of POPC liposomes monitored by changes of the CD spectral signal at 222 nm. The data were fitted with the Henderson-Hasselbach equation (red line with 95% confidence interval) to establish the midpoint of the transition (pK). ( e ) The kinetics signal (multiplied by − 1) of pHLIP ICG insertion into lipid bilayers of POPC liposomes as a result of a pH drop from pH 8 (blue line in insert) to pH 5 (red line in insert) was monitored in real-time by the tryptophan fluorescence changes (black line). The gray line is a baseline obtained by mixing pHLIP ICG with POPC liposomes at pH 8 with pH 8 buffer. The red line is a bi-exponential fit of the experimental data (85% of the signal changes occur with a rate of 5 s −1 and about 15% of final adjustment is completed at a rate of 0.077 s −1 ). ( f , g ) Blood clearance profile and single exponential fitting curves (red lines) obtained from mice ( f ) and dogs ( g ) after a single i.v. injection of 12.3 mg/kg of pHLIP ICG to mice and 0.064 mg/kg of pHLIP ICG to dogs are shown.

Article Snippet: All manufacturing procedures were completed in accordance with good laboratory practice (GLP) quality control specifications. pHLIP ICG was synthesized and the agent was produced by Iris Biotech, GmbH (Germany) in partnership with Chemical and Biopharmaceutical Laboratories (CBL) (Greece) and then further optimized by CordenPharma.

Techniques: Sequencing, Residue, Fluorescence, Circular Dichroism, Liposomes, Membrane, Injection

Journal: Scientific Reports

Article Title: pHLIP ICG for delineation of tumors and blood flow during fluorescence-guided surgery

doi: 10.1038/s41598-020-75443-5

Figure Lengend Snippet: Imaging of blood vessels in mice. Representative NIR pHLIP ICG fluorescent images of blood vessels at different time points after a single i.v. injection of ICG-Cys ( a ) or pHLIP ICG ( b ).

Article Snippet: All manufacturing procedures were completed in accordance with good laboratory practice (GLP) quality control specifications. pHLIP ICG was synthesized and the agent was produced by Iris Biotech, GmbH (Germany) in partnership with Chemical and Biopharmaceutical Laboratories (CBL) (Greece) and then further optimized by CordenPharma.

Techniques: Imaging, Injection

Journal: Scientific Reports

Article Title: pHLIP ICG for delineation of tumors and blood flow during fluorescence-guided surgery

doi: 10.1038/s41598-020-75443-5

Figure Lengend Snippet: Imaging of blood vessels in pigs. Representative NIR pHLIP ICG (verification batch #1912127) fluorescent images and overlay of fluorescent and color images of blood vessels at different time points after injection of pHLIP ICG in PBS (dose 1—0.052 mg/kg; dose 2—additional 0.12 mg/kg; dose 3—additional 0.24 mg/kg) are shown. ( a ) Imaging inside the pig body cavity was performed using a Stryker 1688 AIM system (808 nm excitation) for endoscopic/laparoscopic imaging (gain 8/10 for dose 2, gain 7/10 for dose 3). ( b ) Imaging of a pedicle flap was performed using a Stryker SPY-PHI handheld device (805 nm excitation) for open-field imaging.

Article Snippet: All manufacturing procedures were completed in accordance with good laboratory practice (GLP) quality control specifications. pHLIP ICG was synthesized and the agent was produced by Iris Biotech, GmbH (Germany) in partnership with Chemical and Biopharmaceutical Laboratories (CBL) (Greece) and then further optimized by CordenPharma.

Techniques: Imaging, Injection

Journal: Scientific Reports

Article Title: pHLIP ICG for delineation of tumors and blood flow during fluorescence-guided surgery

doi: 10.1038/s41598-020-75443-5

Figure Lengend Snippet: Tumor targeting. Representative photos and overlay of photos and in vivo NIR pHLIP ICG fluorescent images of athymic female nude mice bearing murine and human tumors are shown with skin removed from the tumor site. Targeting of human breast MDA-MB-231 tumor ( a ), murine breast 4T1 tumor ( b ), human A549 lung tumor ( c ), murine LLC Lewis lung carcinoma ( d ), human M4A4 epithelial tumor ( e ), human HeLa cervical tumor ( f ), human UMUC3 urinary bladder tumor ( g ) and human prostate LNCaP tumor ( h ) are shown. The imaging was performed at 24 h after a single i.v. administration of pHLIP ICG. Arrows indicate tumor locations. In the case of the MDA-MB231 breast tumor model, both a small tumor (arrow at the right) and an inguinal lymph node (indicated as LN) are targeted.

Article Snippet: All manufacturing procedures were completed in accordance with good laboratory practice (GLP) quality control specifications. pHLIP ICG was synthesized and the agent was produced by Iris Biotech, GmbH (Germany) in partnership with Chemical and Biopharmaceutical Laboratories (CBL) (Greece) and then further optimized by CordenPharma.

Techniques: In Vivo, Imaging

Journal: Scientific Reports

Article Title: pHLIP ICG for delineation of tumors and blood flow during fluorescence-guided surgery

doi: 10.1038/s41598-020-75443-5

Figure Lengend Snippet: Ex vivo imaging of tumors with surrounding muscle. Representative photos, and overlays of photos and ex vivo NIR pHLIP ICG fluorescent images of tumors resected with surrounding tissue immediately after in vivo imaging. Targeting of human breast MDA-MB-231 tumor ( a ), murine breast 4T1 tumor ( b ), human A549 lung tumor ( c ), murine LLC Lewis lung carcinoma ( d ), human M4A4 epithelial tumor ( e ), human HeLa cervical tumor ( f ), human UMUC3 urinary bladder tumor ( g ) and human prostate LNCaP tumor ( h ) are shown. In the case of the MDA-MB231 breast tumor model, both a small tumor and an inguinal lymph node (indicated as LN) are targeted.

Article Snippet: All manufacturing procedures were completed in accordance with good laboratory practice (GLP) quality control specifications. pHLIP ICG was synthesized and the agent was produced by Iris Biotech, GmbH (Germany) in partnership with Chemical and Biopharmaceutical Laboratories (CBL) (Greece) and then further optimized by CordenPharma.

Techniques: Ex Vivo, Imaging, In Vivo Imaging

Journal: Scientific Reports

Article Title: pHLIP ICG for delineation of tumors and blood flow during fluorescence-guided surgery

doi: 10.1038/s41598-020-75443-5

Figure Lengend Snippet: Ex vivo imaging and imaging of sections. ( a ) Representative images of tumors with surrounding tissue: photos, NIR pHLIP ICG fluorescent images, overlay of photos with NIR pHLIP ICG fluorescent images, and images of HE-stained sections obtained from the same tumor-muscle pieces are shown. ( b ) Bright field image of HE-stained section and NIR pHLIP ICG fluorescent images of the adjacent non-processed section obtained on IR scanner shown in monochrome (green) and 16-color (white and red are the highest intensity, and black and dark blue are the lowest intensity) presentations. T indicates tumor; M indicates muscle, and S indicates stroma. ( c ) Magnified bright field images (using 20 × objective) of tumor (T), muscle (M1 and M2), tumor stroma (TS1, TS2 and TS3) of HE-stained section are presented in panel ( b ).

Article Snippet: All manufacturing procedures were completed in accordance with good laboratory practice (GLP) quality control specifications. pHLIP ICG was synthesized and the agent was produced by Iris Biotech, GmbH (Germany) in partnership with Chemical and Biopharmaceutical Laboratories (CBL) (Greece) and then further optimized by CordenPharma.

Techniques: Ex Vivo, Imaging, Staining

Journal: ACS Omega

Article Title: Tools of the Trade: Image Analysis Programs for Confocal Laser-Scanning Microscopy Studies of Biofilms and Considerations for Their Use by Experimental Researchers

doi: 10.1021/acsomega.2c07255

Figure Lengend Snippet: Overview of Image Analysis Programs for Biofilm Confocal Micrographs with a Focus on Tool Scope and Selection of Relevance to Experimental Researchers a

Article Snippet: PHLIP , free, open-access, available at https://sourceforge.net/projects/phlip/ , Windows, Linux, macOS , MATLAB toolbox-based script , GUI , segmentation; biofilm volume, morphology, and surface characterization , allows batch processing; offers both manual and automated (Otsu’s Method) thresholding; multichannel analysis; biofilm spatial and temporal calculations: biovolume, substratum coverage, thickness, roughness and colocalization; offers a data conversion option (PHLIP-ML) to convert different image formats to input format; has direct connectivity to PHLIP-ML (file convertor) for Leica Systems , MATLAB-based and requires MATLAB licensing; requires preprocessing steps such as image inversion and x-section resolution.

Techniques: Selection, Fluorescence, Bacteria, Staining, Cytometry, Labeling, Cell Counting, Introduce, Filtration, Activity Assay, Software, Microscopy