PURPOSE: We developed a Tc-99m and fluorescence-labeled peptide, Tc-99m TAMRA-GHEG-ECG-GNQWFI, to target tumor cells, and evaluated the diagnostic performance as a dual-modality imaging agent for tumor in a murine model.METHODS: TAMRA-GHEG-ECG-GNQWFI was synthesized using Fmoc solid-phase peptide synthesis. Radiolabeling of TAMRA-GHEG-ECG-GNQWFI with Tc-99m was done using ligand exchange via tartrate. Binding affinity and in vitro cellular uptake studies were performed. Gamma camera imaging, biodistribution, and ex vivo imaging studies were performed in murine models with U87MG tumors. Tumor tissue slides were prepared and analyzed with immunohistochemistry using confocal microscopy.RESULTS: After radiolabeling procedures with Tc-99m, Tc-99m TAMRA-GHEG-ECG-GNQWFI complexes were prepared in high yield (> 95%). The K(d) of Tc-99m TAMRA-GHEG-ECG-GNQWFI determined by saturation binding was 29.5 ± 4.5 nM. Confocal microscopy images of U87MG cells incubated with TAMRA-GHEG-ECG-GNQWFI showed strong fluorescence in the cytoplasm. Gamma camera imaging revealed substantial uptake of Tc-99m TAMRA-GHEG-ECG-GNQWFI in tumors. Tumor uptake was effectively blocked by the co-injection of an excess concentration of GNQWFI. Specific uptake of Tc-99m TAMRA-GHEG-ECG-GNQWFI was assessed by biodistribution, ex vivo imaging, and immunohistochemistry stain studies.CONCLUSION: In vivo and in vitro studies revealed substantial and specific uptake of Tc-99m TAMRA-GHEG-ECG-GNQWFI in tumor cells. Tc-99m TAMRA-GHEG-ECG-GNQWFI could be a good candidate dual-modality imaging agent for tumors.
Cytoplasm ; Fluorescence ; Immunohistochemistry ; In Vitro Techniques ; Microscopy, Confocal ; Multimodal Imaging ; Radionuclide Imaging ; Solid-Phase Synthesis Techniques

To find anti-hypertensive lead drug, angiotensin converting enzyme (ACE) inhibitory peptides were synthesized and their effects on inhibiting ACE activity were investigated. ACE inhibitory peptides were synthesized via Fmoc solid-phase synthesis, isolated and purified through reversed phase high-performance liquid chromatography (RP-HPLC), and identified by mass spectrometry. A RP-HPLC analysis method was used to test ACE inhibitory activity in vitro of these ACE inhibitory peptides. Six octapeptides were successfully synthesized, and the analytical results of mass spectrum were consistent with their theoretically calculated data. Among these synthetic octapeptides, the anti-SARS (severe acute respiratory syndromes) octapeptide had the most obvious ACE inhibitory activity with an IC50 value of 3.4 x 10(-5) mol x L(-1). So octapeptide AVLQSGFR-OH (anti-SARS peptide) was found to be the strongest candidate for potential development as an anti-hypertensive drug and had the implication of further study.
Angiotensin-Converting Enzyme Inhibitors ; chemical synthesis ; chemistry ; pharmacology ; Antihypertensive Agents ; chemical synthesis ; chemistry ; pharmacology ; Chromatography, High Pressure Liquid ; methods ; Mass Spectrometry ; Molecular Structure ; Oligopeptides ; chemical synthesis ; chemistry ; pharmacology ; Peptidyl-Dipeptidase A ; drug effects ; Solid-Phase Synthesis Techniques

OBJECTIVETo synthesize a tumor-targeting cell-penetrating peptide (CPP) and evaluate its biological activity and cytotoxicity in vitro.

METHODSWith fluorenylmethyloxycarbonyl (Fmoc) as the protective group of α-amino acid, the tumor-targeting CPP were synthesized with stepwise amino acid extension using solid-phase synthesis method. 5-carboxytetramethylrhodamine was added for fluorescence labeling in the presence of the coupling agents HATU and DMF. The purity of the CPP was measured by high-performance liquid chromatography and its molecular weight measured by mass spectrometry. Fluorescence microscope was used to assess the cell-penetrating activity?of the CPP in hepatocellular carcinoma cell lines SMMC-7721 and normal hepatocellular cell lines LO2. The growth activity of CPP-treated SMMC-7721 cells was measured by MTT assay.

RESULTSWith a purity of 96.05% and a relative molecular mass of 3504.9, the synthesized CPP showed no translocation activity in normal hepatocellular cell lines LO2, but showed strong ability to translocate into SMMC-7721 cells without affecting the biological activity of the cells.

CONCLUSIONUsing Fmoc solid-phase synthesis method, we have successfully synthesized the CPP with tumor-targeting activity.

Cell Line, Tumor ; Cell-Penetrating Peptides ; chemical synthesis ; pharmacology ; Drug Delivery Systems ; Drug Design ; Humans ; Liver Neoplasms ; drug therapy ; metabolism ; pathology ; Matrix Metalloproteinase 2 ; metabolism ; Rhodamines ; chemistry ; Solid-Phase Synthesis Techniques

To investigate the angiotensin I-converting enzyme (ACE) inhibitory activity of beta-chain hemoglobin fragments, 17 fragments were synthesized by microwave-assisted solid-phase synthesis method. Wang resin or Trt(2-Cl) resin, Fmoc and HBTU-HOBt were used as solid carrier, N-terminal amino acid protecting groups and coupling reagents, respectively. The ACE inhibitory, alpha-glucosidase inhibitory, antibacterial and antitumor activities of the synthesized fragments were assayed. In vitro, Val-Val-Tyr-Pro-Trp-Thr showed high ACE inhibitory activity (IC50 = 7.42 micromol x L(-1)). The results indicate that there are two active sites in Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe, one consists of Val-Val-, and the other -Gln-Arg-Phe. Peptides showed high ACE inhibitory activity when the N-terminal was hydrophobic amino acid such as Val and C-terminal tripeptide contained Phe, Trp or Arg. Some of the fragments showed low a-glucosidase inhibitory activity. No antibacterial activity or antitumor activity was detected in vitro. The results indicate that these peptides have a potential antihypertensive effect and possible application in the treatment of hypertension.
Amino Acid Sequence ; Angiotensin-Converting Enzyme Inhibitors ; pharmacology ; Anti-Bacterial Agents ; pharmacology ; Antihypertensive Agents ; pharmacology ; Antineoplastic Agents ; pharmacology ; Cell Line, Tumor ; Glycoside Hydrolase Inhibitors ; Humans ; Peptide Fragments ; chemical synthesis ; chemistry ; pharmacology ; Peptidyl-Dipeptidase A ; drug effects ; Solid-Phase Synthesis Techniques ; methods ; alpha-Glucosidases ; drug effects ; beta-Globins ; chemical synthesis ; chemistry ; pharmacology