Selection and identification of transferrin receptor-specific peptides as recognition probes for cancer cells
Since the transferrin receptor (CD71 or TFRC) is known to be highly expressed in numerous cancers, CD71 has become an attractive target in cancer research. Acquiring specific molecular probes for CD71, such as small molecular ligands, aptamers, peptides, or antibodies, is of great importance for cancer cell recognition and capture. In this work, we chose CD71 as the target for phage display, and after four rounds of positive selection and one round of negative selection, the specific phage library was enriched. After verification and sequence analysis, six peptides were identified to be able to bind to CD71 with high specificity. The specific recognition of the CD71-positive cells was confirmed by flow cytometry and confocal microscopy. Competition experiments demonstrated that peptide Y1 and transferrin (TF) were bound to distinct sites on CD71, indicating that peptide Y1 could replace TF as a potential probe for cell imaging and drug delivery, thus avoiding competition by endogenous TF and side effects.
KeywordsPhage display Biopanning CD71 Cancer cell Imaging
Cancer is still one of the most severe diseases, with more than 10 million new patients suffering from cancer every year and cancer-related death rates remaining stubbornly high . Current cancer treatments, including surgical intervention, radiotherapy, thermotherapy, and chemotherapy, have made a certain degree of progress in cancer therapy. However, radiotherapy and chemotherapy also cause negative side effects . In order to reduce the side effects of these therapies, the strategies of targeted drug delivery and targeted therapy have become popular in cancer treatment [3, 4]. These strategies are useful not only in decreasing the required dose and improving drug efficacy but also in helping relieve patients’ pain and improving their life quality. As the key elements for targeted therapy, there is an urgent need for the development of target-specific ligands.
In recent decades, ligand- and receptor-induced delivery systems have attracted great attention. In general, the binding of ligand to its receptor can bring the ligand to the specific location for accurate treatment. In addition, ligand–receptor complexes have good biological compatibility, low toxicity, and low immunogenicity. Some ligand–receptor systems are frequently used in the drug delivery, including apolipoproteins A and E [5, 6], receptor-associated protein , transferrin (TF) , lactotransferrin , melanotransferrin , and leptin . It is worth noting that the receptor of transferrin is CD71, which is an attractive target for cancer diagnosis and therapy. CD71 is expressed abundantly in brain cells, and the capillary endothelial cells of the brain, as well as some proliferative cells, such as cancer cells, activated lymphocytes and serum-induced fibroblasts [12, 13, 14]. CD71 is a type II transmembrane glycoprotein expressed as a homodimer in erythroid blood cell lines and in activated leukocytes. Upon binding of CD71, TF is internalized by clathrin-mediated endocytosis. Considerable research has used TF as the drug carrier to bring the drug to the tumor location [15, 16, 17]. However, high levels of endogenic TF lead to competitive binding that increases the required dose of therapy agents.
To solve this problem, we propose that replacing TF with a specific peptide as the CD71-specific ligand for drug delivery could avoid competition with endogenic TF. Fortunately, antibodies or peptides can be displayed on the surface of phages, and the target-specific antibody or peptide can be acquired via enrichment of a phage library. Thus, phage display is a powerful technique for isolating monoclonal antibodies as well as for discovering specific peptides. Herein, phage display was employed to screen CD71-specific peptides using the Ph.D.™-12 phage display library. Four rounds of positive selection and one round of negative selection were used to enrich the specific phage library. Positive phage clones were isolated for binding characterization by ELISA and then sent for sequencing. After sequence analysis, six peptides were obtained, and the peptide Y1 was chosen for further characterization of its binding capability. The peptide Y1 can bind to CD71 and cancer cells with high CD71 expression with good binding affinity and specificity. All these results indicated that the peptide Y1 has great potential in targeted drug delivery or therapy.
Reagents and materials
The entire selection process was based on the Ph.D.™-12 phage display library kit (New England Biolabs, USA), and the Ph.D.™-12 phage display library kit includes Escherichia coli ER2738 with tetracycline resistance. CD71, APC-labeled CD71 antibody (mAb), and HRP-labeled M13 antibody were purchased from the Sino Biological, Inc. The small interfering RNA (siRNA) of CD71 was purchased from Thermo Fisher Scientific. Polyethylene glycol 8000 (PEG 8000) was purchased from Sigma-Aldrich. Tween 20 and 3,3′,5,5′-tetramethylbenzidine (TMB) were purchased from Xiamen Lulong Co., Ltd. T98G cells (human glioblastoma cell line, ATCC CRL-1690) and HeLa cells (human cervix cancer cell, ATCC CCL-2) were obtained from ATCC, L02 cells (normal human liver cell) were presented by Hunan University, and all the experiments used living cell for further researching. All reagents used in this experiment were analytically pure, and the water was 18.2 MΩ.cm ultrapure water.
Phage display selection procedure for CD71
CD71 was immobilized on 96-well plates (10 μg) overnight at 4 °C. The microplate well was blocked with bovine serum albumin (BSA) (5 mg/mL) for 1 h at room temperature and then washed three times with TBST (Tris-HCl 10 mM, pH 8, 150 mM NaCl, including 5 mg/mL BSA and 0.5% Tween 20) to remove unbound BSA or protein. Afterwards, 10 μL of M13 phage library (1 × 1011) was added to the well for 1-h incubation at room temperature with shaking. After incubation, the well was washed six times with TBST to remove the unbound phages. Then, the bound phages were eluted by adding 200 mM glycine HCl, pH 2.2, with 1 mg/mL BSA for 8 min at room temperature. The collected library was cultured with E. coli for 4.5 h at 37 °C in LB broth (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) with 50 μg/mL tetracycline. Then, the amplified library was purified by centrifuging (4 °C, 10,000 rpm, 10 min). Then, the concentration of phage was determined by phage tittering for calculation of the recovery rate, and the library was stored for the next round. In further rounds of selection, all procedures were performed the same as in the first round, except for the time of incubation and the concentration of Tween 20. All the selection conditions are listed in Table S1 in the Electronic Supplementary Material (ESM). After four rounds of selection, the bound phages were collected and used for a counter selection, using BSA as the negative target, and the unbound phages were collected for further experiments. These phages with 10-fold serial dilutions were incubated with the E. coli, mixed with the top agar, and then poured onto an IPTG/Xgal/LB plate. After overnight culturing, the blue plaques were picked randomly from about 100 plaques on the plate for amplification and further analysis.
Monitoring the progress of library enrichment and identification of the positive phage clones for CD71 with phage ELISA
After each round of selection, the phages before and after amplification were collected and quantified by phage tittering. Phages in TBST were diluted to 10-fold serial gradient concentrations, incubated with E. coli, and poured on the IPTG/Xgal/LB plate. The plate with about 100 plaques was chosen to calculate the number of the phage clones. Finally, the recovery rate (output/input phages) was used to evaluate the enrichment of the selection. In addition, phage ELISA was applied to characterize the binding ability between phage clones and CD71. First, CD71 (0.2 μg/well) was immobilized on 96-well plates overnight at 4 °C. Then, the nonspecific sites were blocked with BSA (5 mg/mL) for 1 h at room temperature. After blocking, the wells were washed three times with TBST, and the phage clones (5 × 105) were incubated with the coated CD71 on 96-well plates for 1 h in a shaker. Then, the wells were washed six times with TBST, and they were incubated with 100 μL M13 bacteriophage antibody labeled with HRP (1:5000) for 1 h at room temperature, followed by washing six times with TBST to remove the unbound antibodies. Finally, the color development reaction was performed with 100 μL TMB, and all the reactions were stopped after 10 min by adding 50 μL of 1 M H2SO4. Then, the OD450 value was recorded with a microplate reader.
Sequencing and peptide synthesis
The positive clones were picked out and purified, and the ssDNA of these phages was extracted and sent to Sangon (Shanghai, China) for sequencing. Resulting sequences were analyzed by DNAMAN 6.0 software. All sequences were aligned to different families according to their homology. Six peptides were chosen for further characterization. Then, six FITC-labeled (Ex = 490 nm, Em = 525 nm) peptides were synthesized (> 95% purity) by SynPeptide Co., Ltd. (Shanghai, China) and used for affinity and specificity analysis. An unrelated control peptide (FITC-TLRDRMSYNMRR) was also synthesized as a negative control.
Binding affinity of peptides with CD71
In order to determine the affinity of peptides, CD71 was coated on Ni beads by His-tag. Different diluted peptides were incubated with these beads (1 × 105) on a roller at room temperature for 1 h and then washed three times with TBST. Finally, the fluorescence intensity of the beads was analyzed by flow cytometry (FACSVerse™; Becton Dickinson). The geo mean fluorescence values of gradient concentration samples were fitted to the equation (Y = B max X/(K d + X)), and the K d was used to evaluate the affinity of these peptides.
Binding specificity of peptide with CD71
Prostate-specific antigen (PSA), BSA, thrombin (THB), C-reactive protein (CRP), human serum albumin (HSA), CD71-coated beads, and protein-free beads were chosen to analyze the specificity of peptides Y1 and Y2. The peptides (50 μM of Y1 or Y2) was incubated with 50 μM PSA, BSA, THB, CRP, or HSA-coated beads and 5 μM CD71-coated beads, and protein-free beads (1 × 105) separately for 30 min, and all samples were washed three times with TBST and analyzed by flow cytometry.
Binding capability of peptide with CD71 high expression HeLa cells
For further application of peptides, we chose HeLa cells, which show high expression of CD71, as a model to test the binding ability of peptides. FITC-labeled peptides Y1 and Y2 were incubated with 1 × 105 living HeLa cells for 30 min at room temperature and washed three times with 1× PBS. The samples were analyzed by flow cytometry and laser scanning confocal microscopy (Leica TCS SP5, Leica Microsystems). As a control, an unrelated sequence was used (FITC-TLRDRMSYNMRR). Meanwhile, APC-labeled CD71 antibody was used to confirm the CD71 expression level of HeLa cells. Furthermore, we tested the influence of different temperatures (4, 25, and 37 °C) and different incubation times (0.5, 1.0, 1.5, and 2.0 h) on the binding of 50 μM Y1 and Y2 to HeLa cells. In addition, in order to verify the specificity of peptides Y1 and Y2, we used CD71 siRNA to knock down the expression level of CD71. After treating with siRNA for 48 h, the HeLa cells were incubated with the 50 μM peptides Y1 and Y2, and laser scanning confocal microscopy and flow cytometry were used to confirm the binding of peptides and HeLa cells.
Competition between TF and peptide Y1
In order to confirm the feasibility of replacing TF with peptide Y1 for drug delivery, the binding competition between FITC-labeled peptide Y1 and TF was analyzed by flow cytometry. Fifty micromolars of peptide Y1 was incubated with HeLa cells, and then different concentrations of TF (0–100 μM) were added. The fluorescence intensity was recorded using flow cytometry.
Results and discussion
Selection of phages against CD71
Sequence analysis and synthesis
Sequences obtained after four rounds of selection
Affinity and specificity of peptides against CD71
To further demonstrate the specificity of Y1 and Y2, other proteins, such as PSA, BSA, THB, CRP, and HSA, were tested with peptides Y1 and Y2. The results in Fig. 2c indicated that the binding of peptides Y1 and Y2 to CD71 is 20 times higher than their binding to other proteins and 8.7 times higher than their binding to protein-free beads, and the nonspecific adsorption of peptides to the naked beads’ surface resulted in high fluorescence signal, demonstrating that Y1 and Y2 have great potential for specific recognition of CD71.
Peptide binding with tumor cells having high expression of CD71
To further characterize the binding specificity of peptides, HeLa cells were treated with siRNA to knock down the expression level of CD71. Then, the CD71-silenced HeLa cells were treated with antibody and peptides. In Fig. S7 (see ESM), the antibody binding result showed that after siRNA treatment, the antibody signal was significantly reduced, demonstrating the successful knockdown of CD71 by siRNA treatment. The signals for peptides Y1 and Y2 also significantly decreased after siRNA treatment, further indication of the specificity of peptides against CD71. In addition, the normal liver cell LO2 cells were chosen as negative control. As shown in Fig. S8 (see ESM), there was no significant fluorescence signal appeared after incubation with peptides, which provided more evidence for the good specificity of peptides.
The binding site of peptide Y1
In summary, specific peptides were successfully isolated using in vitro biopanning against CD71 and HeLa cells with high specificity and affinity, demonstrating that the peptides could be used in clinical applications in a similar way as antibodies. Interestingly, peptides Y1 and Y2, both with α-helix secondary structures, could enter cancer cells with increasing incubation temperature and time, suggesting that Y1 and Y2 can be useful in ligand–receptor-mediated drug delivery and imaging. Furthermore, due to their lower molecular weights and flexibility, Y1 and Y2 promise to be powerful tools in biosensors and in CTC capture and release.
We thank the National Science Foundation of China (81602206, 21325522, 21422506, 21435004, 21521004), National Basic Research Program of China (2013CB933703), Program for Changjiang Scholars and Innovative Research Teams in University (IRT13036), National Found for Fostering Talents of Basic Science (NFFTBS, J1310024), and China Postdoctoral Science Foundation (2016M592089) for their financial support.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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