Targeted Modification of the Cationic Anticancer Peptide HPRP-A1 with iRGD To Improve Specificity, Penetration, and Tumor-Tissue Accumulation

The chimeric peptide HPRP-A1-iRGD, composed of a chemically conjugated tumor-homing/penetration domain (iRGD) and a cationic anticancer peptide domain (HPRP-A1), was used to study the effect of targeted modification to enhance the peptide’s specificity, penetration, and tumor accumulation ability. The iRGD domain exhibits tumor-targeting and tumor-penetrating activities by specifically binding to the neuropilin-1 receptor. Acting as a homing/penetration domain, iRGD contributed to enhancing the tumor selectivity, permeability, and targeting of HPRP-A1 by targeted receptor dependence. As the anticancer active domain, HPRP-A1 kills cancer cells by disrupting the cell membrane and inducing apoptosis. The in vitro membrane selectivity toward cancer cells, such as A549 and MDA-MB-23, and human umbilical vein endothelial cells (HUVECs), normal cells, the penetrability assessment in the A549 3D multiple cell sphere model, and the in vivo tumor-tissue accumulation test in the A549 xenograft model indicated that HPRP-A1-iRGD exhibited significant increases in the selectivity toward membranes that highly express NRP-1, the penetration distance in 3D multiple cell spheres, and the accumulation in tumor tissues after intravenous injection, compared with HPRP-A1 alone. The mechanism of the enhanced targeting ability of HPRP-A1-iRGD was demonstrated by the pull-down assay and biolayer interferometry test, which indicated that the chimeric peptide could specifically bind to the neuropilin-1 protein with high affinity. We believe that chemical conjugation with iRGD to increase the specificity, penetration, and tumor-tissue accumulation of HPRP-A1 is an effective and promising approach for the targeted modification of peptides as anticancer therapeutics

H&E, TUNEL and Ki 67 staining in tumour tissues and histological examination of major organs.

(A) H&E, Ki67, and TUNEL staining of tumour tissues. (B) Histological examination by H&E staining of major organs (heart, liver, spleen, lungs, and kidneys) in the breast cancer mouse model.</p

Coadministration of kla peptide with HPRP-A1 to enhance anticancer activity

The apoptosis-inducing peptide kla (KLAKLAK)2 possesses the ability to disrupt mitochondrial membranes and induce cancer cell apoptosis, but this peptide has a poor eukaryotic cell-penetrating potential. Thus, it requires the assistance of other peptides for effective translocation at micromolar concentrations. In this study, breast and lung cancer cells were treated by kla peptide co-administrated with membrane-active anticancer peptide HPRP-A1. HPRP-A1 assisted kla to enter cancer cells and localized on mitochondrial membranes to result in cytochrome C releasing and mitochondrial depolarization which ultimately induced apoptosis.The apoptosis rate was up to 65%and 45% on MCF-7 and A549 cell lines, respectively, induced by HPRP-A1 coadministration with kla group. The breast cancer model was constructed in mice, and the anticancer peptides were injected to observe the changes in cancer volume, andimmunohistochemical analysis was performed on the tissues and organs after the drug was administered. Both the weight and volume of tumor tissue were remarkable lower in HPRP-A1 with kla group compared with thosepeptidealonggroups. The results showed that the combined drug group effectively inhibited the growth of cancer and did not cause toxic damage to normal tissues, as well as exhibited significantly improvement on peptide anticancer activity in vitro and in vivo.</div

Phase estimation of Mach-Zehnder interferometer via Laguerre excitation squeezed state

Quantum metrology has an important role in the fields of quantum optics and quantum information processing. Here we introduce a kind of non-Gaussian state, Laguerre excitation squeezed state as input of traditional Mach-Zehnder interferometer to examine phase estimation in realistic case. We consider the effects of both internal and external losses on phase estimation by using quantum Fisher information and parity detection. It is shown that the external loss presents a bigger effect than the internal one. The phase sensitivity and the quantum Fisher information can be improved by increasing the photon number and even surpass the ideal phase sensitivity by two-mode squeezed vacuum in a certain region of phase shift for realistic case

Co-localization of peptides in MCF-7 cells.

Various concentrations of FITC-kla with or without 4 μM HPRP-A1 were applied to MCF-7 cells for 1 h. The blue colour was nuclei stained by Hoechst 33258, green colour was FITC-labelled kla and red colour was mitochondria stained by Mito-Tracker® Red, respectively. Images were obtained by confocal microscopy.</p

Mitochondrial depolarization and protein level of cytochrome C.

(A) MCF-7 cells were treated with various concentrations kla with or without 4 μM HPRP-A1.MitoCapture has aggregated in the mitochondria of healthy cells shown as red. In apoptotic cells, MitoCapture cannot accumulate in mitochondria, which remains as monomers in the cytoplasm and fluoresces green. (B) Protein level of cytochrome C detected by western blotting in MCF-7 cells after treatment with kla with or without HPRP-A1 for 6 h. COX IV served as a mitochondrial reference control, and tubulin served as a cytoplasmic reference control.</p

Cytotoxic effects of kla with or without HPRP-A1 against NIH3T3, A549, and MCF-7 cell lines.

(A) Normal mouse cell line NIH 3T3, (B) human lung cancer line A549, and (C) human breast cancer cell line MCF-7 were treated with various concentrations of kla with or without 4 μM HPRP-A1 for 12 h, and then cell viability was detected by MTT assays.</p

Antitumor activity of kla and HPRP-A1 <i>in vivo</i>.

(A) Photographs of tumours. (B) Tumour weights. (C) Changes in tumour sizes.</p

Apoptosis induced by kla with or without HPRP-A1.

(A) MCF-7 and A549 cells were treated with various concentrations of kla with or without 4 μM HPRP-A1 for 12 h, and then the rates of early and late apoptosis of (B) MCF-7 and (C) A549 cells were measured using a FITC-Annexin V/PI kit and flow cytometry (***PP<0.01).</p

Cellular uptake of co-administration peptides by MCF-7 cells.

The blue colour was nuclei stained by Hoechst33258, green colour was FITC-labelled kla. MCF-7 cells were treated with various concentrations of FITC-kla with or without 4 μM HPRP-A1 for 1 h. Fluorescence was measured by confocal microscopy.</p