Prime STAR®GXL DNA Polymerase was purchased from TaKaRa Company (Japan). Restriction enzymes NdeI and XhoI were purchased from NEB (England). RNeasy Mini Kit was purchased from QIAGEN (Gemany). The SuperScript® III First-Strand Synthesis System was purchased from Invitrogen (USA). SYBR Green kit was purchased from TransGen Biotech (China). Gel extraction kit and plasmid miniprep kit were obtained from the CW Bio Company (China). Ni-NTA agarose were purchased from GE Healthcare (Sweden). Pierce® BCA Protein Assay Kit was purchased from Thermo Company (USA). Cholesterol, hydrogenated soy phosphatidylcholine (HSPC), mPEG2000-DSPE and mal-PEG2000-DSPE were purchased from Avanti polar lipids (USA). Hoechst 33342 and Dio were purchased from Sigma (USA). The human bladder cancer cell line RT112 and T24 were supported by JENNIO Company (China).
Construction of pET-20b-ScFv-Cys expression vector
The PCR primers were designed according to the ScFv sequence (Liu et al. 2015) from GenBank (accession number KP405837), and synthesized by the Sangon Biotech Company (Shanghai). The forward primer (F1) was designed as followed (51nt): 5′-GGAATTCCATATGCATCATCATCATCATC ACGAAGTGCAGCTGCTGGAAAG-3′ according to the 5′ terminal sequence of ScFv. The reverse primer (R1) that contained 3′ terminal sequence of ScFv, linker and Cysteine (Cys) was synthesized as followed (67 nt): 5′-CGCTCGAGTTAGCAACCGCTACCGCTGCTACCACCGCTGCTACCACCG CGTTTAATTTCCACTTTGG-3′. The restriction endonuclease NdeI and XhoI sites were added in the forward and reverse primer, respectively. The target gene was obtained by PCR amplification using F1 and R1 as the forward and reverse primers, using the ScFv gene as the template (Liu et al. 2015). PCR parameters consisted of 5 min of pre-denaturation activation at 98 °C, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 55 °C for 30 s, extension at 72 °C for 60 s, and then a final single extension at 68 °C for 5 min. The PCR product and the pET-20b empty vector were digested with NdeI and XhoI, and ligated using T4 DNA ligase, then the ligated plasmid was transformed into E. coli DH5α competent cells. Finally, the recombinant plasmid was verified by double enzyme digestion and confirmed by DNA sequencing (Sangon, Shanghai).
Inducible expression of recombinant ScFv-Cys
The recombinant plasmid was transformed into E. coli BL21(DE3), and several colonies were selected to induce the recombinant protein expression in a 4 mL fresh Luria–Bertani (LB) medium (1% peptone, 0.5% yeast extract, and 1% sodium chloride, pH 7.0), and cultured in a shaker at 37 °C. When the OD600 attained 0.6 to 0.8, 0.5 mM isopropyl-d-thiogalactoside (IPTG) was added into bacteria medium for 4 h induction at 37 °C along with shaking of 200 rpm. The expression of recombinant ScFv-Cys (rScFv-Cys) was detected by SDS-PAGE, and the bacteria clone with the highest expression level was used to induce the expression in a large scale. The detailed methods have been reported previously (Zhang et al. 2014).
Purification and identification of rScFv-Cys
rScFv-Cys was purified by Ni-NTA chromatography, and the detailed method was consistent with the previous report (Liu et al. 2017). After affinity purification employing Ni-column, rScFv-Cys was desalinated with 3 kDa dialysate bag, and concentrated using an ultrafiltration tube. The protein concentration was determined by BCA protein quantitative assay kit (Thermo, USA), and its purity was analyzed by SDS-PAGE. The target protein was cut off from gel and placed in a 1.5 mL tube containing ddH2O, and was identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS) from Huada Protein Co. Ltd. (Beijing).
Preparation and characterization of liposomes and immunoliposomes
Liposomes were prepared by thin-film dispersion method. In brief, HSPC, cholesterol and mPEG2000-DSPE (20:10:1, molar ratio) were dissolved in chloroform. The chloroform was evaporated slowly at 42 °C by rotary evaporator. After the chloroform was completely dried, 2-hydroxyethyl (HEPES) at the concentration of 10 mM was added into this mixture and performed to ultrasonication for 20 min at 20 °C, then liposome solution was filtered by filter member of 450 nm and 220 nm, and the different particle size of liposomes was produced.
To further synthesize ScFv-PEG2000-DSPE, adding DTT to the previously purified rScFv-Cys protein to final concentration 50 mM at 4 °C for 1 h reaction, the thiol group of cysteine residue of rScFv-Cys was fully recovered. The reduced ScFv-Cys was added to the dialysis bag. The extra DTT was removed by 20 mM Tris–HCl and dialyzed at 4 °C for 10 h. Before dialysis, sufficient nitrogen was added to the dialysate to insulate the oxygen for preventing the single chain antibody from oxidation.
ScFv-Cys and malPEG2000-DSPE were mixed in the ratio of 1:4, and was gently shaken at room temperature for 16 h to ensure the full coupling of two components. Also, before the reaction, full nitrogen protection was applied. The target substance ScFv-PEG2000-DSPE was obtained by this method. The particle size of liposomes and immunoliposomes was measured by Marvin laser particle size analyzer.
RNA isolation, cDNA synthesis and real-time RT-PCR
Total RNA of RT112 and T24 cells was extracted using the RNeasy Mini Kit (QIAGEN), and reversely transcribed into cDNA using reverse transcription (RT) PCR kit (The SuperScript® III First-Strand Synthesis System, Invitrogen). PCR was performed with SYBR Green kit (TransGen Biotech, China) by ABI Prism 7000. The two pair of primers against different regions of FGFR3 were designed and named as FGFR3-1 and FGFR3-2. The sequences of forward and reverse prime of FGFR3-1 are 5′- GGGAGGACGAGGCTGAGGAC-3′ and 5′-GATGGAGGGAGTGGGGTTGC-3′, respectively. The sequences for forward and reverse prime of FGFR3-2 are 5′-CCCACTCCCTCCATCTCCTG-3′ and 5′-GCTGCCAAACTTGTTCTCCA-3′, respectively. Gapdh was used as the internal control, and its forward and reverse prime sequences are 5′-TGCACCACCAACTGCTTAGC-3′ and 5′-GGCATG GACTGTGGTCATGA-3′, respectively. PCR parameters consisted of 5 min of DNA Polymerase activation at 94 °C, followed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s. After the reaction, ABI Prism 7000 software was used to statistically analyze the difference of gene transcription between different groups.
The targeted effect and delivery ability of immunoliposomes
200,000 RT112 cells and T24 cells were cultured in DMEM medium, with 10% FBS at 37 °C in the cell incubator, respectively. The fluorescence-labeled liposomes and immunoliposomes were prepared by mixing 0.1% Dio, and added into cells with 800 μl cell medium for 3 h in the dark. Cell medium was removed, and washed three times with PBS buffer. Hoechst 33342 was added to a final concentration of 5 μg/mL for 10 min in the dark. The staining solution was then removed, and cells were washed three times with PBS, and the medium was replaced for DMEM complete medium. Cells were observed by fluorescent microscopy (Olympus, Japan).