Constructing a better binding peptide for drug delivery targeting the interleukin-4 receptor.

Targeted delivery of antitumor drugs is especially important for tumour therapy. Tumour targeting peptides have been shown to be very effective drug carriers for tumour therapy. Interleukin-4 receptor (IL-4R) is overexpressed on the surface of various human solid tumours. To obtain a better targeting peptide, we first designed a novel targeting peptide derived from interleukin-4 (IL-4), ILBP-b. ILBP-b contains the key high-affinity binding residue E9 of IL-4 to IL-4R. Compared with a reported targeting peptide ILBP-a (containing another key high affinity residue R88), ILBP-b was proved to be a better targeting peptide by the fluorescence experiments. Then, we further fused ILBP-b and ILBP-a to increase the multisite-binding ability of ILBP-b and got a better targeting peptide ILBP-ba. ILBP-ba showed a stronger preferential binding ability to IL-4R high-expressing cells than ILBP-a and ILBP-b. Competitive binding experiments demonstrated ILBP-ba specifically targets IL-4R. By fusing ILBP-ba with drug protein trichosanthin (TCS), in vitro drug carrying experiments showed that ILBP-ba could specifically enhance the killing effect of TCS on IL-4R high-expressing tumour cells (more than 10 folds). These results indicated that ILBP-ba has great potential for drug delivery applications targeting IL-4R and will be beneficial for the development of tumour therapeutic agents.

Screening and characterization of a novel high-efficiency tumor-homing cell-penetrating peptide from the buffalo cathelicidin family.

Targeted delivery of antitumor drugs is especially important for tumor therapy. Cell-penetrating peptides (CPPs) have been shown to be very effective drug carriers for tumor therapy. However, most CPPs lack tumor cell specificity. Here, we identified a highly efficient CPP, CAT, from the newly identified buffalo-derived cathelicidin family, which exhibits a preferential binding capacity for multiple tumor cell lines and delivers carried drug molecules into cells. CAT showed an approximately threefold to sixfold higher translocation efficiency than some reported cell-penetrating antimicrobial peptides, including the well-known classical CPP TAT. Moreover, the delivery efficiency of CAT was greater in a variety of tested tumor cells than in normal cells, especially for the human hepatoma cell line SMMC-7721, for which delivery was 7 times more efficient than the normal human embryonic lung cell line MRC-5, according to fluorescent labeling experiment results. CAT was conjugated to the Momordica charantia-derived type-I ribosome-inactivating protein MAP 30, and the cytotoxicity of the MAP 30-CAT fusion protein in the tumor cell line SMMC-7721 was significantly enhanced compared with that of the unconjugated MAP 30. The IC50 value of MAP 30-CAT was approximately 83 times lower than the IC50 value of the original MAP 30. Interestingly, the IC50 value of MAP 30 alone for MRC-5 was approximately twofold higher than the value for SMMC-7721, showing a small difference. However, when MAP 30 was conjugated to CAT, the difference in IC50 values between the two cell lines was significantly increased by 38-fold. The results of the flow cytometric detection of apoptosis revealed that the increase in cytotoxicity after CAT conjugation was mainly caused by the increased induction of apoptosis by the fusion protein. These results suggest that CAT, as a novel tumor-homing CPP, has great potential in drug delivery applications in vivo and will be beneficial to the development of tumor therapeutics.

Structure optimisation to improve the delivery efficiency and cell selectivity of a tumour-targeting cell-penetrating peptide.

Cell-penetrating peptide (CPP) is used for the delivery of biomacromolecules across the cell membrane and is limited in cancer therapy due to the lack of cell selectivity. Epidermal growth factor receptor (EGFR) has been widely used in clinical targeted therapy for tumours. Here, we reported a novel tumour targeting cell-penetrating peptide (TCPP), EHB (ELBD-C6H) with 20-fold and 3000-fold greater transmembrane ability and tumour cell selectivity than our previously reported S3-HBD and classic CPP TAT, respectively. In this new TCPP, a specific alpha helix structure was inserted into a repeated amino acid (AA) sequence formed by tandem multiple selected key AA residues of vaccinia growth factor (VGF), and this sequence was then fused to a tailored heparin binding domain sequence (C6H) derived from heparin-binding epidermal growth factor-like growth factor to intensify its targeting delivery ability. EHB could carry anticancer proteins such as MAP30 (Momordica Antiviral Protein 30 kDa) into EGFR-overexpressing cancer cell and inhibit cell growth, but it had a greatly reduced interaction with normal cells. These results indicated that EHB, as a novel efficient TCPP for the selective delivery of drug molecules into cancer cells, would help to improve the efficacy and safety of anti-tumour drugs.

Recombinant expression and purification of a MAP30-cell penetrating peptide fusion protein with higher anti-tumor bioactivity.

MAP30 (Momordica Antiviral Protein 30 Kd), a single-stranded type-I ribosome inactivating protein, possesses versatile biological activities including anti-tumor abilities. However, the low efficiency penetrating into tumor cells hampers the tumoricidal effect of MAP30. This paper describes MAP30 fused with a human-derived cell penetrating peptide HBD which overcome the low uptake efficiency by tumor cells and exhibits higher anti-tumor bioactivity. MAP30 gene was cloned from the genomic DNA of Momordica charantia and the recombinant plasmid pET28b-MAP30-HBD was established and transferred into Escherichia coli BL21 (DE3). The recombinant MAP30-HBD protein (rMAP30-HBD) was expressed in a soluble form after being induced by 0.5mM IPTG for 14h at 15°C. The recombinant protein was purified to greater than 95% purity with Ni-NTA affinity chromatography. The rMAP30-HBD protein not only has topological inactivation and protein translation inhibition activity but also showed significant improvements in cytotoxic activity compared to that of the rMAP30 protein without HBD in the tested tumor cell lines, and induced higher apoptosis rates in HeLa cells analyzed by Annexin V-FITC with FACS. This paper demonstrated a new method for improving MAP30 protein anti-tumor activity and might have potential applications in cancer therapy area.

Evaluating the translocation properties of a new nuclear targeted penetrating peptide using two fluorescent markers.

Human-derived cell penetrating peptides (CPPs) have attracted much more attentions than other CPPs which are limited by their potential toxicity and immunogenicity. Previously, we identified a novel human-originated CPP (named heparin-binding domain (HBD) in this article), which derived from the C-terminus of human extracellular superoxide dismutase, and demonstrated HBD is an efficient vector for delivering exogenous drug molecules such as apoptin into HeLa cells. In this study, we found this novel CPP showed differentiated efficiency in several tested cell lines. Heparin competitive inhibition experiment and heparanase pre-incubation experiment showed cell surface polysaccharides play an important role for the transmembrane transport. The results of endocytosis inhibitors suggested that HBD penetrates the cell membrane via a direct translocation, which is different from that of TAT, a classical clathrin-mediated endocytosis. HBD could deliver up to 90 kD protein cargoes into cells. Different conjugated modes with cargo molecules greatly affect their translocation efficiency. HBD also showed significant nuclear transport capacity when it was incubated with HeLa cells. Furthermore, the core region for HBD possessing membrane-penetrating ability was identified by deletion analyses. These results would be helpful for developing HBD as a new nuclear delivery tool for therapeutic biomolecules.