Advertisement

Analytical and Bioanalytical Chemistry

, Volume 410, Issue 3, pp 1071–1077 | Cite as

Selection and identification of transferrin receptor-specific peptides as recognition probes for cancer cells

  • Yuyu Tan
  • Wenli Liu
  • Zhi Zhu
  • Lijun Lang
  • Junxia Wang
  • Mengjiao Huang
  • Mingxia Zhang
  • Chaoyong Yang
Research Paper
Part of the following topical collections:
  1. ABCs 16th Anniversary

Abstract

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.

Graphical abstract

Six peptides were successfully isolated using in vitro biopanning against CD71 with high specificity and affinity. Peptides Y1 and Y2 would be powerful tools in biosensors and biomedicine due to their unique properties.

Keywords

Phage display Biopanning CD71 Cancer cell Imaging 

Introduction

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 [1]. 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 [2]. 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 [7], transferrin (TF) [8], lactotransferrin [9], melanotransferrin [10], and leptin [11]. 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.

Experimental

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

Phage display is a powerful tool that enables the acquisition of a specific ligand that binds to the target of interest. Here, we used the M13 phage library to acquire the peptides with high affinity for CD71. The entire selection procedure is illustrated in Fig. 1. The target was immobilized on the 96-well plate. After incubating with phage library and washing away unbound phages, the bound phages were eluted with low pH buffer and amplified by infecting E. coli ER2738. Once purified, the products were used as the library for the next round. After four rounds of selection, the number of phages was determined by phage tittering (ESM Table S2). The recovery rate was used for evaluating the enrichment of the library. As shown in Fig. S1A (see ESM), the recovery rate of the fourth library was 100 times higher than that of the first library, suggesting that the phage library was enriched after four rounds of selection. In addition, the products of each selection round were verified with ELISA, which indicated that the binding ability of the fourth library was stronger than that of the first library, further indication of successful enrichment of the library (ESM Fig. S1B). The fourth selection products were incubated with a BSA-coated 96-well plate for one round of counter selection, and the unbound phages were collected and cultured on an IPTG/Xgal/LB plate. Twenty phage clones were selected randomly from this plate for further analysis. These monoclonal phages were amplified and purified as the candidate phage clones, and their binding ability was evaluated by ELISA (ESM Fig. S2). Phage clones with S/N ≥ 3 were considered to be positive clones, which were chosen for DNA sequencing and further investigation.
Fig. 1

Schematic illustration of biopanning for transferrin receptor. (1) Immobilization of the target on a 96-well plate and incubation with phage library. (2) Removal of unbound phages. (3) Elution of bound phages with low pH buffer. (4) Amplification of eluted phages by infecting E. coli and purification as the pool for the next round. (5) Repetition of the processes of incubation, washing, elution, and amplification for further enrichment of the library. (6) After four rounds of selection, use of one counter selection round to improve the specificity of the pool. (7) Sequencing of the enriched phage library

Sequence analysis and synthesis

Twenty clones were sent for sequencing, and the obtained six sequences are listed in Table 1, in which sequence Y1 is highly enriched with 15 copies and there is one copy each of the remaining five sequences. All these six peptides were synthesized for further study. We used the software Scratch Protein Predictor (http://scratch.proteomics.ics.uci.edu/) to analyze the structures of these peptides. Interestingly, peptides Y1 (VHWDFRQWWQPS) and Y2 (AWYSNLLPLARF) both contain α-helices. Furthermore, we used CD spectrometry to confirm the structures. Consistent with the structure predicted by the software Scratch Protein Predictor, the CD result (ESM Fig. S3) indicated that the α-helix structure occurs in peptides Y1 and Y2.
Table 1

Sequences obtained after four rounds of selection

No.

Peptides

Copies

Structures

Y1

VHWDFRQWWQPS

15

CCCCHHHHCCCC

Y2

AWYSNLLPLARF

1

CCHHCHHHHCCC

Y3

LPAQVGQGPLGT

1

CCCCCCCCCCCC

Y4

WFPTSRWTSGWI

1

CCCCCCCCCCCC

Y5

VDARYWTRGTYM

1

CCCEEEECCCCC

Y6

AKHPDHPLTVGG

1

CCCCCCCCECCC

H α-helix, E extended strand, C random coil

Affinity and specificity of peptides against CD71

To investigate the peptide affinities, FITC-labeled peptides were incubated with CD71-coated beads, and the fluorescence intensity was recorded by flow cytometry. The K ds of peptides Y1 (Fig. 2a) and Y2 (Fig. 2b) were determined to be 7.5 ± 1.0 and 5.3 ± 1.2 μM, respectively. Lee et al. [18], Kim and Choi [19], and Dai et al. [20] made great contribution to the CD71 affinity peptide screening, and some peptides were acquired through screening, such as peptides HAIYPRH, THRPPMWSPVWP, 3T14 (CFGQSSLPRDGPNC), and BP9 (AHLHNRS). Compared with these peptides, Y1 and Y2 have the comparable affinity at the micromole range, except peptide THRPPMWSPVWP, the K d of which is as low as 1.5 × 10−8, so Y1 and Y2 could provide more choices for CD71-related researches.
Fig. 2

Binding affinities of Y1 (a), Y2 (b), and CD71-coated beads determined by flow cytometry. (c) Responses of Y1 and Y2 to 5 μM of CD71 or 50 μM of other proteins (including PSA, BSA, THB, CRP, and HSA) or protein-free beads. Results are averages of three independent measurements

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

In order to verify peptide binding with CD71 on the cell surface, we chose HeLa cells, known to exhibit high expression of CD71, as the model. After confirming the high CD71 expression level on HeLa cells with CD71 antibody, six peptides were tested to verify their binding ability to HeLa cells. Flow cytometry (Fig. 3a) results verified the binding of peptides to HeLa cells. In addition, we also tested another high CD71 expression cell line, T98g, again with positive results (Fig. 3b). The K d of CD71 antibody was obtained by flow cytometry to be 0.738 ± 0.076 μM (ESM Fig. S4). The K ds of Y1 and Y2 binding to HeLa cells were also determined (ESM Fig. S5) and found to be 21.1 ± 3.4 and 16.4 ± 3.9 μM, respectively. The K ds of peptides Y1 and Y2 were 29-fold and 22-fold higher than those of the antibody, respectively. Considering the dramatic difference in molecular weight and size between peptide and antibody, the slightly lower affinity of the peptides is reasonable.
Fig. 3

Flow cytometry results showed the peptides bind to the HeLa cells (a) and T98G cells (b). (c) Confocal images showed the FITC-labeled peptide and APC-labeled CD71 antibody bind to HeLa cells at 4 °C. An unrelated peptide was used as control. The scale bar represents 10 μm

Due to their higher affinity to CD71, Y1 and Y2 were also chosen for further confocal characterization as representative, and both peptides showed binding to HeLa cells (Fig. 3c). Interestingly, we found that peptides Y1 and Y2 could gradually enter HeLa cells when the temperature was increased (Fig. 4). In addition, when the incubation time was prolonged to more than 1.5 h (ESM Fig. S6), peptides Y1 and Y2 significantly internalized into HeLa cells even at 4 °C, indicating that both peptides have potential for targeted and internalized drug delivery.
Fig. 4

Internalization of Y1 and Y2 into HeLa cells. FITC-labeled peptides were incubated with HeLa cells at different temperatures for 1 h. The number of peptides entering the cells increased with increasing temperature. The scale bar represents 15 μm

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

Competition assays were also performed using TF and the antibody (mAb) of CD71. As shown in Fig. 5, after blocking with peptide Y1, different concentrations of TF or mAb were incubated with the HeLa cells. There was no obvious shift in the flow cytometry histogram, indicating no competition between Y1 and the TF or mAb and demonstrating that the binding site of Y1 is different from that of TF or mAb. These results suggested that peptide Y1 has great potential to replace TF for drug delivery, while avoiding endogenous competition between the TF drug and the endogenous TF.
Fig. 5

Competition experiment between peptides Y1 and TF (a) or mAb (b) against CD71. After blocking with FITC-labeled peptide Y1, different concentrations of TF or mAb were added to the solution

Conclusions

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.

Notes

Acknowledgments

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.

Supplementary material

216_2017_664_MOESM1_ESM.pdf (670 kb)
ESM 1 (PDF 669 kb)

References

  1. 1.
    Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61(2):69–90.CrossRefGoogle Scholar
  2. 2.
    Oken MM, Creech RH, Tormey DC, Horton J, Davis TE, McFadden ET, et al. Toxicity and response criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol Cancer. 1982;5(6):649–55.CrossRefGoogle Scholar
  3. 3.
    Ferrari M. Cancer nanotechnology: opportunities and challenges. Nat Rev Cancer. 2005;5(3):161–71.CrossRefGoogle Scholar
  4. 4.
    Allen TM, Cullis PR. Drug delivery systems: entering the mainstream. Science. 2004;303(5665):1818–22.CrossRefGoogle Scholar
  5. 5.
    Mo Z-C, Ren K, Liu X, Tang Z-L, Yi G-H. A high-density lipoprotein-mediated drug delivery system. Adv Drug Deliv Rev. 2016;106:132–47.CrossRefGoogle Scholar
  6. 6.
    Neves AR, Queiroz JF, Reis S. Brain-targeted delivery of resveratrol using solid lipid nanoparticles functionalized with apolipoprotein E. J Nanobiotechnol. 2016;14:27–43.CrossRefGoogle Scholar
  7. 7.
    Pan WH, Kastin AJ, Zankel TC, van Kerkhof P, Terasaki T, Bu GJ. Efficient transfer of receptor-associated protein (RAP) across the blood-brain barrier. J Cell Sci. 2004;117(21):5071–8.CrossRefGoogle Scholar
  8. 8.
    Salvati A, Pitek AS, Monopoli MP, Prapainop K, Bombelli FB, Hristov DR, et al. Transferrin-functionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface. Nat Nanotechnol. 2013;8(2):137–43.CrossRefGoogle Scholar
  9. 9.
    Tomita M, Wakabayashi H, Shin K, Yamauchi K, Yaeshima T, Iwatsuki K. Twenty-five years of research on bovine lactoferrin applications. Biochimie. 2009;91(1):52–7.CrossRefGoogle Scholar
  10. 10.
    Kuo Y-C, Chao I-W. Conjugation of melanotransferrin antibody on solid lipid nanoparticles for mediating brain cancer malignancy. Biotechnol Prog. 2016;32(2):480–90.CrossRefGoogle Scholar
  11. 11.
    Farooqi IS, Jebb SA, Langmack G, Lawrence E, Cheetham CH, Prentice AM, et al. Effects of recombinant leptin therapy in a child with congenital leptin deficiency. N Engl J Med. 1999;341(12):879–84.CrossRefGoogle Scholar
  12. 12.
    Jefferies WA, Brandon MR, Hunt SV, Williams AF, Gatter KC, Mason DY. Transferrin receptor on endothelium of brain capillaries. Nature. 1984;312(5990):162–3.CrossRefGoogle Scholar
  13. 13.
    Shindelman JE, Ortmeyer AE, Sussman HH. Demonstration of the transferrin receptor in human breast cancer tissue. Potential marker for identifying dividing cells. Int J Cancer. 1981;27(3):329–34.CrossRefGoogle Scholar
  14. 14.
    Walker RA, Day SJ. Transferrin receptor expression in non-malignant and malignant human breast tissue. J Pathol. 1986;148(3):217–24.CrossRefGoogle Scholar
  15. 15.
    Dixit S, Novak T, Miller K, Zhu Y, Kenney ME, Broome A-M. Transferrin receptor-targeted theranostic gold nanoparticles for photosensitizer delivery in brain tumors. Nano. 2015;7(5):1782–90.Google Scholar
  16. 16.
    Singh R, Norret M, House MJ, Galabura Y, Bradshaw M, Ho D, et al. Dose-dependent therapeutic distinction between active and passive targeting revealed using transferrin-coated PGMA nanoparticles. Small. 2016;12(3):351–9.CrossRefGoogle Scholar
  17. 17.
    Liu K, Dai L, Li C, Liu J, Wang L, Lei J. Self-assembled targeted nanoparticles based on transferrin-modified eight-arm-polyethylene glycol–dihydroartemisinin conjugate. Sci Rep. 2016;6:29461–72.CrossRefGoogle Scholar
  18. 18.
    Lee JH, Engler JA, Collawn JF, Moore BA. Receptor mediated uptake of peptides that bind the human transferrin receptor. Eur J Biochem. 2001;268(7):2004–12.CrossRefGoogle Scholar
  19. 19.
    Kim S, Choi SJ. Identification of a transferrin receptor binding peptide from a phage-displayed peptide library. J Life Sci. 2008;18(3):298–303.CrossRefGoogle Scholar
  20. 20.
    Dai XY, Xiong YL, Xu DD, Li LY, Su ZJ, Zhang QH, et al. TfR binding peptide screened by phage display technology—characterization to target cancer cells. Trop J Pharm Res. 2014;13(3):331–8.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Yuyu Tan
    • 1
  • Wenli Liu
    • 1
  • Zhi Zhu
    • 1
  • Lijun Lang
    • 1
  • Junxia Wang
    • 1
  • Mengjiao Huang
    • 1
  • Mingxia Zhang
    • 1
  • Chaoyong Yang
    • 1
  1. 1.MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, Collaborative Innovation Center of Chemistry for Energy Materials, Key Laboratory for Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical EngineeringXiamen UniversityXiamenChina

Personalised recommendations