[Show abstract][Hide abstract] ABSTRACT: S-thanatin (Ts) was a short antimicrobial peptide with selective antibacterial activity. In this study, we aimed to design a drug carrier with specific bacterial targeting potential. The positively charged Ts was modified onto the liposome surface by linking Ts to the constituent lipids via a PEG linker. The benefits of this design were evaluated by preparing a series of liposomes and comparing their biological effects in vitro and in vivo. The particle size and Zeta potential of the constructed liposomes were measured with a Zetasizer Nano ZS system and a confocal laser scanning microscope. The in vitro drug delivery potential was evaluated by measuring the cellular uptake of encapsulated levofloxacin using HPLC. Ts-linked liposome or its conjugates with quantum dots favored bacterial cells, and increased the bacterial uptake of levofloxacin. In antimicrobial assays, the Ts and levofloxacin combination showed a synergistic effect, and Ts-LPs-LEV exhibited excellent activity against the quality control stain Klebsiella pneumoniae ATCC 700603 and restored the susceptibility of multidrug-resistant K. pneumoniae clinical isolates to levofloxacin in vitro. Furthermore, Ts-LPs-LEV markedly reduced the lethality rate of the septic shock and resulted in rapid bacterial clearance in mouse models receiving clinical multidrug resistant (MDR) isolates. These results suggest that the Ts-functionalized liposome may be a promising antibiotic delivery system for clinical infectious disorders caused by MDR bacteria, in particular the sepsis related diseases.
Preview · Article · Oct 2015 · Frontiers in Pharmacology
[Show abstract][Hide abstract] ABSTRACT: This study was aimed to investigate the effect of a single-chain fragment variable antibody of connective tissue growth factor (anti-CTGF scFv) against the differentiation of fibroblast into myofibroblast. The scFv antibody was firstly expressed in Escherichia coli cells and was then purified by affinity chromatography. The yield scFv protein reached a purity over 95% after purification. Immunoreactivity assay demonstrated that scFv possessed a special affinity toward CTGF. RT-PCR, western blot, and immunofluorescence experiments showed that increased expression of α-smooth muscle actin induced by TGF-β1 could be suppressed by this scFv antibody through inhibiting the phosphorylation of Akt.
No preview · Article · Dec 2011 · Applied Microbiology and Biotechnology
[Show abstract][Hide abstract] ABSTRACT: To investigate the targeting activity of the peptide (named P1c) derived from connective tissue growth factor (CTGF) to αvβ3-rich tumor cells.
P1c was synthesized and conjugated with ultrasuperparamagnetic iron oxide particles (USPIOs) coated with meso-2,3-dimercaptosuccinic acid (DMSA). The specific binding activity of P1c-USPIOs to αvβ3 was verified by solid phase binding assay. The combination of P1c-USPIOs with a human primary liver cancer cell (Bel 7402) with αvβ3-positive expression and uptake of P1c-USPIOs by cells was investigated by Prussian blue staining, transmission electron microscopy (TEM), and magnetic resonance imaging (MRI). The targeting activity of the probe in vivo was also evaluated using a small-animal tumor model by MRI.
The cell uptake of P1c-USPIOs was observed in a dose-dependent manner, whereas no significant particle uptake was found in the plain USPIOs group. The differences on T2*-weighted imaging were also found by MRI and the signal intensity (SI) was statistically reduced after coculture of Bel 7402 cells with P1c-USPIOs at a concentration of 20-80 μg/mL compared with plain USPIOs (P < 0.05). The in vivo study showed that the signal reduction was distributed mainly in the periphery and some central areas of the tumor. The tumor-to-muscle CNR (contrast-to-noise ratio) at 12 hours after the administration of the P1c-USPIOs was statistically significantly different compared to those at 0 hour, 1 hour, or the plain USPIO group (P < 0.05).
The peptide P1c might be a good candidate as a targeting carrier for drugs or tracers.
No preview · Article · Aug 2011 · Journal of Magnetic Resonance Imaging
[Show abstract][Hide abstract] ABSTRACT: S-thanatin, a small antimicrobial peptide with 21 amino acid residues, was expressed as a fusion protein containing thrombin cleavage site in Escherichia coli BL21 (DE3). To reduce the production cost, immobilization of thrombin in polyacrylamide gel for cleavage was studied in this work. The immobilized thrombin exhibited excellent activity within wider ranges of pH value and temperature for reaction than free enzyme, and the residual activity could remain above 75% after ten times of usage. Tricine-SDS-PAGE result showed that the immobilized thrombin could cleave the S-thanatin fusion protein effectively. After cleavage, recombinant S-thanatin was purified by preparative reversed-phase high-performance liquid chromatography and mass spectrum showed that the molecular weight (2,448.86) was close to the theoretical value (2,448.98). After purification, about 7 mg of S-thanatin was obtained from 1 l of culture and the recombinant exhibited excellent bioactivity to E. coli ATCC 25922, with the minimum inhibitory concentration of 12 μg/ml. The purification method could be applied to prepare other peptides with similar properties at low cost.
No preview · Article · Jun 2011 · Applied Microbiology and Biotechnology
[Show abstract][Hide abstract] ABSTRACT: In this study, antimicrobial peptide S-thanatin (Ts) was chemically synthesized and linked to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) by carbodiimide reagent. Rabbits were immunized with Ts-KLH and polyclonal antibody against Ts was purified by fractional precipitation of ammonium sulfate, coupled with anion-exchange chromatography. The purified antibody specifically binding to Ts residues but not BSA molecules was observed by Western-Blot analysis. Ts-BSA was selected as immobilized antigen and reacted with the residual antibody after the excess of anti-Ts antibody was combined with Ts in the sample. The binding antibody was recognized by HRP-conjugated secondary antibody. Finally, the horseradish peroxidase in the complex could catalyze the TMB substrate, resulting in color development. The method was evaluated by analysis of linearity, precision and accuracy and successfully applied in determination of Ts in rat plasma. The data of the pharmacokinetic parameters were also obtained. The proposed ELISA has a great value in routine analysis of Ts for its therapeutic monitoring and pharmacokinetic studies.
[Show abstract][Hide abstract] ABSTRACT: Connective tissue growth factor (CTGF) has been identified as playing critical roles in fibrosis and is a promising therapeutic target. In a previous study, we used a phage display library to develop a humanized single-chain variable fragment antibody (scFv) against CTGF. In the present study, the protective effect of anti-CTGF scFv against bleomycin (BL)-induced pulmonary fibrosis was investigated in mice.
The expression of α-smooth muscle actin in human embryonic lung fibroblast (HELF) cells was analysed by western blotting. A mouse model of pulmonary fibrosis was established by tracheal injection of BL (5 mg/kg). Mice received anti-CTGF scFv (4 mg/kg, three times a week) by i.v. injection. The effects of anti-CTGF scFv were evaluated by leukocyte counts in BAL fluid, hydroxyproline measurements in lung tissue and pathological examination.
α-Smooth muscle actin expression was decreased in HELF cells treated with anti-CTGF scFv. Anti-CTGF scFv significantly reduced the numbers of inflammatory leukocytes (total and differential count) in BAL fluid, as well as the hydroxyproline content of lung tissue. The severity of alveolitis and fibrosis in the mouse model was markedly attenuated by treatment with anti-CTGF scFv.
Anti-CTGF scFv may potentially be developed as a useful inhibitor of pulmonary fibrosis.