Materials
Fluorescein isothiocyanate (FITC), bobo-3 iodide (570/602), and Lipofectamine™ Plus were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Exfection™ LE Mini was purchased from GeneAll Biotechnology (Seoul, Korea). Minimum Essential Medium (MEM), RPMI 1640 and Dulbecco’s Modified Eagle’s Medium (DMEM), Dulbecco’s Phosphate Buffered Saline (PBS), and trypsin were purchased from Sigma Aldrich (Taufkirchen, Germany), and fetal bovine serum (FBS) was purchased from EMD Millipore (US Origin). Caco-2 and HT-29 (human epithelial colorectal adenocarcinoma cell lines), HEK-293 (human embryonic kidney cell line), and HeLa (human cervical carcinoma cell line) were purchased from the Korean Cell Line Bank (KCLB, Korea). The GLP-1 ELA kit was purchased from Sigma-Aldrich (St. Louis, MO, USA). Ultrasensitive mouse insulin ELISA kit was purchased from Morinaga (Yokohama, Japan). A pAcGFP-N1 expression plasmid was kindly provided by Dr. Young Jun Kim (Konkuk University, Chungju, Korea). A pβ-sp-GLP-1 expression vector was obtained from Dr. Minhyung Lee (Hanyang University, Seoul, Korea).
Animals
Balb/c mice (5–7 weeks old) and Lepdb/db mice (male, 7–9 weeks old) were purchased from Daehan Bio Link, Inc. (Chungbuk, Korea). All mice were maintained in sterile, autoclaved cages, 3 per cage, on a standard chow diet. All animal experiments followed the guidelines established by Chonnam University Institutional Animal Care Use Committee and other proper approvals were obtained before the study.
Preparation of hIgG1-Fc-9Arg
hIgG1-Fc-9Arg protein was prepared by a transient cell-based protein expression system. Briefly, the C-terminus of a hIgG1-Fc expression plasmid (Korea Research Institute of Bioscience & Biotechnology, South Korea) was extended with nine-arginine (9 Arg) sequences by PCR. The extension of 9 Arg from hIgG1-Fc was confirmed by both PCR amplification and DNA sequencing [28,29,30,31]. The hIgG1-Fc-9Arg expression plasmid was further introduced into Expi293F cells using ExpiFectamine™ 293 transfection reagent according to manufacturer’s instructions. At 6–7 days of cell culture after transfection, the cleared supernatants were harvested by centrifugation at 8000 rpm for 15 min and microfiltration with 0.22 μm microfilter. A-HiTrap Mabselect SuRe column (GE Lifesciences, Buckinghamshire, England) was used to separate the hIgG1-Fc-9Arg protein from the supernatants. The hIgG1-Fc-9Arg protein eluted from the column was neutralized with 1 M Tris (pH 8.5). After buffer exchange of the neutralized eluent with PBS containing 5% trehalose (pH 7.4), the hIgG1-Fc-9Arg protein solution was concentrated with ultracentrifugal filter (3 kDa Amicon Ultra 2 mL centrifugal filter, Millipore-Sigma, Burlington, MA, USA). The size of hIgG1-Fc-9Arg was confirmed by both SDS-PAGE and PAGE.
Complexation of hIgG1-Fc-9Arg with pAcGFP-N1
Various weight ratios of hIgG1-Fc-9Arg /pAcGFP-N1 complexes were prepared in microcentrifuge tubes by addition of pAcGFP-N1 (1 μg/μl) into hIgG1-Fc-9Arg (9 μg/μl) following adjustment of final volume by PBS (pH 7.4) (Additional file 2). After stabilization of the complex with 30-min incubation at room temperature, the complex was applied to a DNA retardation assay to ensure DNA-complexation by electrophoresis on 1% agarose gels under tris-acetate-EDTA (TAE) buffer at 100 V for 30 min.
Characterization of hIgG1-Fc-9Arg/ pAcGFP-N1 complex
The surface properties of hIgG1-Fc-9Arg/pAcGFP-N1 complexes (20/1, 50/1, 100/1, w/w) were analyzed by an atomic force microscope (AFM, Multimode-N3-AM, Bruker crop., Germany) in Tapping Mode™. The shape and size of an hIgG1-Fc-9Arg/ pAcGFP-N1 complex (50/1, w/w) were observed by a Field Emission Transmission Electron Microscope (FE-TEM, JEM-2100F, JEOL Ltd., Japan) at 20 kV in high vacuum mode [30]. The samples were prepared by spreading 1000-fold diluted hIgG1-Fc-9Arg/pAcGFP-N1 complexes on a copper grid and drying the samples.
pH and serum stability of hIgG1-Fc-9Arg/pAcGFP-N1 complex
Orally administered biological compounds need to be stable without loss of their activity from pH-fluctuations during gastrointestinal transit and from serum instability during systemic circulation. To estimate the stability of hIgG1-FC-9Arg during gastrointestinal transit, hIgG1-FC-9Arg was incubated for 0, 30, and 60 min at various pH conditions. SDS-PAGE was performed to check the stability of hIgG1-FC-9Arg after incubation. The stability of hIgG1-Fc-9Arg/pAcGFP-N1 (50/1, w/w) complexes were also assessed by 1% agarose gel electrophoresis with samples that were prepared after incubation at various times at pH 2, the average pH of the stomach.
Various ratios of the complex were incubated for up to 24 h with 10% fetal bovine serum (FBS) at 37 °C to assess serum stability of the complexes. After incubation, samples were treated with EDTA to stop the further enzymatic reaction and were subjected to 1% agarose gel electrophoresis.
Samples were treated with 1% SDS to release plasmid DNA to estimate either pH or serum effect of the pAcGFP-N1 plasmid in the complex. Noncomplexed pAcGFP-N1 plasmid, a naked pDNA, was used as a comparison with the samples.
FcRn-mediated cellular uptake of hIgG1-Fc-9Arg/pDNA complex
The expression of FcRn was measured by immunoblotting the cell extracts obtained from human epithelial colorectal adenocarcinoma cells (Caco-2, HT-29), human embryonic kidney cells (HEK 293), and human cervical carcinoma cells (HeLa). HEK293-FcRn cell extracts played the role of positive FcRn expression controls to ensure the expression of FcRN in the experimental samples.
Ten μg FITC-hIG1-Fc-9Arg conjugate was added to the HeLa, HEK293, and Caco2 cells for 1 h to check whether the FcRn cell surface receptor played a role in cellular uptake of the hIgG1-Fc-9Arg complex. The confocal microscopic analysis confirmed the green fluorescence emitted from FITC in the conjugate. In addition to FITC conjugates, 20:1, 50:1 and 100:1 ratio of the hIgG1-FC-9Arg/pAcGFP-N1 complex was added to the Caco-2 cells for 48 h, and GFP expression was monitored. CaCo-2 cells transfected with pAcGFP-N1 using lipofectamine were transfection controls to compare GFP expression from the experimental samples treated with the complex.
Cellular toxicity of hIgG1-Fc-9Arg/pDNA complex
To determine the cellular toxicity of the complex, an MTT-based cell viability assay was performed in Caco-2 cells according to the manufacturer’s instructions. Caco-2 cells that were plated in the 96-well plates at 0.1 million cells per well at 24 h before the treatment were treated with the complex for 24 h. After treatment, culture media was replaced with MTT solution consisting of 20 μl MTT (5 mg/ml, w/v) solution and 180 μl media and were further incubated for 30 min to an hour at 37 °C to observe insoluble formazan crystals in the cells. After finishing MTT treatment, DMSO treatment completely solubilized the formazan crystals in the cells. The absorbance at 570 nm of the solution proportionally increased with the numbers of viable cells. The percentage cell viability was described by the following equation:
$$ \% viable\ cells=\frac{\left({abs}_{sample}-{abs}_{blank}\right)}{\left({abs}_{control}-{abs}_{blank}\right)}\times 100 $$
abssample: absorbance of the sample with a treatment.
abscontrol: absorbance of the sample without treatment.
absblank: absorbance of DMSO.
Cellular transport of hIgG1-Fc-9Arg/pDNA complex across the Caco-2 cell monolayer
A transwell permeability assay with monolayered Caco-2 cells simulated cellular transport of materials from the apical region to basolateral region of the gastrointestinal tract [32,33,34]. Briefly, the complex prepared from various ratios of hIgG1-Fc-9Arg and bobo-3 pDNA was applied to the apical site of the Caco-2 cells monolayered on a transwell system (Corning® Transwell® polyester membrane pore size 0.4 μm, diameter 12 mm cell culture inserts, Millipore). The determination of apical-to-basolateral translocation of the complex was the measurement of fluorescence intensity emitted from the bobo-3 pDNA complex in the basolateral medium (HBSS, pH 7.4).
Endosomal trafficking of hIgG1-Fc-9Arg complex
The experimental cells were the Caco-2 cell line for treatment of 10 μg FITC-hIgG1-Fc-9Arg complex for either 30 min or 4 h. The additional 10-min treatment of Caco-2 cells with lysotracker® led to staining late endosomes and lysosomes in the cells. The fluorescent images obtained from FITC and lysotracker in the cells indicated the endosomal locations of the complex.
Intracellular fluorescence emitted from the various ratios of hIgG1-Fc-9Arg/bobo-3 pDNA complex demonstrated the endosomal fate of the complexes. A positive control to monitor endosomal trafficking of the complexes was the cells treated with a commercial transfection reagent, Lipofectamine.
Biodistribution of a FITC- hIGg1-Fc-9Arg complex
Balb/c mice (n = 3, male, 5–7 weeks) were the experimental animal given the oral administration of 10 μg FITC-hIgG1-Fc-9Arg complex. At either 1 or 3 h after oral feeding, various parts of organs from the mice were harvested and imaged to observe biodistribution of the complex by using a Kodak Digital Science™ Image Station 440CF (IS440CF) system. After imaging, the organs were further processed into a solution obtained from grinding the liquid nitrogen-frozen organ with mortar and pestle. Quantification of the organ distribution of the complex was performed by measurement of the fluorescence intensity emitted from FITC in the complex by microplate spectrometry.
Antidiabetic effect of a hIgG1-Fc-9Arg/pGLP-1 complex
Lepdb/db (n = 4, male, 7 weeks) and Balb/c (n = 3, male, 5–7 weeks) mice received oral administration of hIgG1-Fc-9Arg/ pβ-sp-GLP-1 complexes (50/1). The complex contained 20 μg of pβ-sp-GLP-1 and was given to the mice on 0, 7, 14, 21, 35, 44 days. Control mice received PBS by oral route following the same dosing schedule. At every oral dosing time, mice underwent measurement of glucose levels and body weight with a glucose meter and weight balance. Experimental mice supplied their blood from their abdominal-inferior vena cava after 8 h fasting for further processing of the serum by centrifugation. The performance of EIA (Sigma-Aldrich) and ELISA (Morinaga) with the serum provided information regarding the level of GLP-1 and insulin. HE histology analysis estimated intestinal tissue toxicity after oral administration of the complex to the mice.
Statistical analysis
All applicable data are expressed as a mean ± standard deviation unless otherwise noted. Statistical analysis was performed with Prism software (version 7.04; GraphPad Software, La Jolla, CA, USA). Significance was set at p < 0.05.