Expanding the Utility of β-Galactosidase Complementation: Piece by Piece

The ability to image and quantify multiple biomarkers in disease necessitates the development of split reporter fragment platforms. We have divided the β-galactosidase enzyme into unique, independent polypeptides that are able to reassemble and complement enzymatic activity in bacteria and in mammalian cells. We created two sets of complementing pairs that individually have no enzymatic activity. However, when brought into close geometric proximity, the complementing pairs associated resulting in detectable enzymatic activity. We then constructed a stable ligand complex composed of reporter fragment, linker, and targeting moiety. The targeting moiety, in this case a ligand, allowed cell surface receptor targeting in vitro. Further, we were able to simultaneously visualize two cell surface receptors implicated in cancer development, epidermal growth factor receptor and transferrin receptor, using complementing pairs of the ligand−reporter fragment complex

Deep Penetration of a PDT Drug into Tumors by Noncovalent Drug-Gold Nanoparticle Conjugates

Efficient drug delivery to tumors is of ever-increasing importance. Single-visit diagnosis and treatment sessions are the goal of future theranostics. In this work, a noncovalent PDT cancer drug-gold nanoparticle (Au NP) conjugate system performed a rapid drug release and deep penetration of the drug into tumors within hours. The drug delivery mechanism of the PDT drug through Au NPs into tumors by passive accumulation was investigated via fluorescence imaging, elemental analysis, and histological staining. The pharmacokinetics of the conjugates over a 7-day test period showed rapid drug excretion, as monitored via the fluorescence of the drug in urine. Moreover, the biodistribution of Au NPs in this study period indicated clearance of the NPs from the mice. This study suggests that noncovalent delivery via Au NPs provides an attractive approach for cancer drugs to penetrate deep into the center of tumors

Supplementary Figure Legend from An Optical Probe for Noninvasive Molecular Imaging of Orthotopic Brain Tumors Overexpressing Epidermal Growth Factor Receptor

PDF file, 83K.</p

Supplementary Table 1 from An Optical Probe for Noninvasive Molecular Imaging of Orthotopic Brain Tumors Overexpressing Epidermal Growth Factor Receptor

PDF file, 46K, Peptides were characterized and verified by MALDI mass spectrometry. Table shows the expected and observed molecular weight of the peptides.</p

Supplementary Figure 1 from An Optical Probe for Noninvasive Molecular Imaging of Orthotopic Brain Tumors Overexpressing Epidermal Growth Factor Receptor

PDF file, 116K, A) Expression levels of the wild type EGFR determined by Western blot in A431, U87-MG, and Gli36Δ5 cell lines. Cell lysates were derived from each cell type and 50 μg of each separated using SDS-PAGE. The gels were then transferred to nitrocellulose membrane and probed for wild type EGFR (DAKO, Carpenteria, CA, clone DAK-H1-WT). Loading control was determined by blotting against β-actin. B) Quantification of the western blot data from S1A. Protein expression levels of wild type EGFR are represented in log10 scale. A431 cells overexpress wild type EGFR more than 60 and 20-fold relative to U87-MG and Gli36Δ5 cells, respectively. C) Saturation binding assay. Compound 2 was incubated with A431 cells at increasing concentrations for 1 hour, washed, and cell associated Cy5.5 was quantified. The Kd determined for the A431 cell line was 8.0�3.0 μM. D) Cell uptake of compound 2 in the presence of hEGF ligand. A431 cells were plated in 96-well plates and were treated with 1 μM of Cy5.5-EGFpep in the presence of increasing amounts of hEGF (or no treatment) for 90 minutes. Uptake increased with increasing EGF stimulation. These data suggest that uptake of labeled ligand is specific to EGF receptors. *, P>0.01; **, P>001.</p

Supplementary Table 2 from An Optical Probe for Noninvasive Molecular Imaging of Orthotopic Brain Tumors Overexpressing Epidermal Growth Factor Receptor

PDF file, 36K, Physical properties of peptides studied in this report. RP-HPLC conditions 10-60% acetonitrile against 0.1% trifluoroacetic acid over 30 minutes through a C18 column (PROTO 300, 10 micron, 250 x 4.6 mm) where the injection peak at 3.6 min. 5-90% acetonitrile against 0.1% trifluoroacetic acid over 30 minutes through a C18 column (PROTO 200, 5 micron, 250x 10 mm). Injection peak at 6.8 min.</p

Deep Penetration of a PDT Drug into Tumors by Noncovalent Drug-Gold Nanoparticle Conjugates

Efficient drug delivery to tumors is of ever-increasing importance. Single-visit diagnosis and treatment sessions are the goal of future theranostics. In this work, a noncovalent PDT cancer drug-gold nanoparticle (Au NP) conjugate system performed a rapid drug release and deep penetration of the drug into tumors within hours. The drug delivery mechanism of the PDT drug through Au NPs into tumors by passive accumulation was investigated via fluorescence imaging, elemental analysis, and histological staining. The pharmacokinetics of the conjugates over a 7-day test period showed rapid drug excretion, as monitored via the fluorescence of the drug in urine. Moreover, the biodistribution of Au NPs in this study period indicated clearance of the NPs from the mice. This study suggests that noncovalent delivery via Au NPs provides an attractive approach for cancer drugs to penetrate deep into the center of tumors

Deep Penetration of a PDT Drug into Tumors by Noncovalent Drug-Gold Nanoparticle Conjugates

Efficient drug delivery to tumors is of ever-increasing importance. Single-visit diagnosis and treatment sessions are the goal of future theranostics. In this work, a noncovalent PDT cancer drug-gold nanoparticle (Au NP) conjugate system performed a rapid drug release and deep penetration of the drug into tumors within hours. The drug delivery mechanism of the PDT drug through Au NPs into tumors by passive accumulation was investigated via fluorescence imaging, elemental analysis, and histological staining. The pharmacokinetics of the conjugates over a 7-day test period showed rapid drug excretion, as monitored via the fluorescence of the drug in urine. Moreover, the biodistribution of Au NPs in this study period indicated clearance of the NPs from the mice. This study suggests that noncovalent delivery via Au NPs provides an attractive approach for cancer drugs to penetrate deep into the center of tumors

Dual Receptor-Targeted Theranostic Nanoparticles for Localized Delivery and Activation of Photodynamic Therapy Drug in Glioblastomas

Targeting gold nanoparticles (AuNPs) with two or more receptor binding peptides has been proposed to address intratumoral heterogeneity of glioblastomas that overexpress multiple cell surface receptors to ultimately improve therapeutic efficacy. AuNPs conjugated with peptides against both the epidermal growth factor and transferrin receptors and loaded with the photosensitizer phthalocyanine 4 (Pc 4) have been designed and compared with monotargeted AuNPs for <i>in vitro</i> and <i>in vivo</i> studies. The (EGF_pep+Tf_pep)-AuNPs-Pc 4 with a particle size of ∼41 nm improved both specificity and worked synergistically to decrease time of maximal accumulation in human glioma cells that overexpressed two cell surface receptors as compared to cells that overexpressed only one. Enhanced cellular association and increased cytotoxicity were achieved. <i>In vivo</i> studies show notable accumulation of these agents in the brain tumor regions

Supplementary Materials and Methods from Isoflavone ME-344 Disrupts Redox Homeostasis and Mitochondrial Function by Targeting Heme Oxygenase 1

Antibodies. Primers. Click Chemistry and Affinity Enrichment Mass Spectrometry. Surface Plasmon Resonance - Biacore 3000 Kinetic Determinations. Co-Immunoprecipitation. Transfections with shRNA and plasmids. Plate colony formation assay. References.</p