Targeted Modification of the Cationic Anticancer Peptide HPRP-A1

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Targeted modification of the cationic anticancer peptide HPRP-A1 with iRGD to improve specificity, penetration, and tumor-tissue accumulation Cuihua Hu, Yibing Huang, and Yuxin Chen Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b00854 • Publication Date (Web): 28 Dec 2018 Downloaded from http://pubs.acs.org on January 1, 2019

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Molecular Pharmaceutics

Targeted modification of the cationic anticancer peptide HPRP-A1 with iRGD to improve specificity, penetration, and tumor-tissue accumulation

Cuihua Hu1,2, Yibing Huang1,2, Yuxin Chen1,2* 1. Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, Changchun, China 130021; 2. School of Life Sciences, Jilin University, Changchun, China 130021 ABSTRACT 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 (HUVEC) 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. Keywords: targeted modification, cationic anticancer peptide, tumor penetration, HPRP-A1, iRGD 1

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INTRODUCTION Cancer is one of the highest death rate diseases in the word. Although the treatment of cancer has advanced substantially during the past two decades, limitations, including lower therapeutic index, adverse reactions, and frequently drug resistance are still problems in the use of the presently available treatments 1, 2. In addition, the poor penetration ability of the peptides into tumor tissues is also a weakness in tumor treatment 3, 4. Cationic membrane-active peptides (MAPs) are promising molecules as anticancer agents with the mechanism of action, broad-spectrum anticancer activity, low immunogenicity, and unlikeliness to create resistance 5. However, the relatively low selectivity of MAPs between cancer and normal cells is the primary obstacle for these peptides to be developed as anticancer drugs 6. Hence, increasing the selectivity, penetration, and tumor-tissue targeting of ACPs through effective modification is a promising approach to solve these problems. Many strategies, including targeted modification, polymer modification, and co-administration have been used for ACPs 5, 7, 8. In addition, many different targeting peptides have been used to increase the targeting ability of the ACPs, including RGD 9, 10, iRGD 11, 12, T3 13, 14, Z13 15, TCP-1 16, TMTP-1 17, BRBP-1 18, 19, and Bld-1 20, 21. Most of the ACPs modified by targeting peptides or cell-penetrating peptides (CPPs) are pro-apoptotic peptides or have poor cell-membrane transporting ability, such as 23 and TP5 22, 23. Few studies have carried out on MAPs. D(KLA)2 HPRP-A1 is a cationic α-helical MAP containing 15 amino acids, which was obtained and optimized on the basis of the peptide sequence from the N-terminus of the Helicobacter pylori ribosomal protein L1 24, 25. HPRP-A1 was found to show excellent anticancer activity in vitro and in vivo, and the dual mechanisms of action have been demonstrated to be disruption of the cytoplasmic membrane via micellization and pore formation, and the induction of apoptosis 26, 27. To increase the anticancer activity of HPRP-A1, the cell-penetrating peptide TAT has been used to modify HPRP-A1; the TAT-modified HPRP-A1 peptide increased the therapeutic index significantly 28. However, the TAT modification could only enhance the therapeutic index, which was attributed to enhanced anticancer activity and decreased toxicity toward normal cells, it could not increase the targeting to cancer cells and penetrability to solid tumors. The traditional targeted peptide RGD and homing/penetration peptide iRGD were used to modify HPRP-A1 by chemical conjugation. In addition, two glycines were used as a linker between the targeting peptide and HPRP-A1 to increase the molecular flexibility and optimize the conjugation. iRGD is a homing/penetrating peptide that is widely used to increase vascular and tissue permeability by specifically binding to the neuropilin-1 (NRP-1) receptor, 2


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which is highly expressed in most tumor tissues and angiogenesis 8, 29. iRGD has been widely used for improving tumor targeting 30, 31. In this study, iRGD was chemical conjugated with HPRP-A1, to form HPRP-A1-iRGD, we hypothesized the new pepide could increase the tumor cell specificity, the penetrability, and the tumor-tissue accumulation in cell lines that have high expression of the NRP-1–receptor. The molecular mechanism of action of HPRP-A1-iRGD was systematically investigated.

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RESULTS Peptide properties, stability study and anticancer activity in vitro The traditional targeting peptide, RGD, and cyclic homing peptide, iRGD, were selected to chemically conjugate to the C-terminal of the HPRP-A1 peptide with or without a two-glycine linker. Table 1 shows the peptide sequences in order of decreasing hydrophobicity. Table 1. Amino acid sequences and RP-HPLC retention times of the peptides

a

Peptides

Sequence

tR (min) a

HPRP-A1

FKKLKKLFSKLWNWK

47.2

HPRP-A1-RGD

FKKLKKLFSKLWNWK-RGD

46

HPRP-A1-GG-iRGD

FKKLKKLFSKLWNWK-GG-CRGDKGPD C

45.6

HPRP-A1-GG-RGD

FKKLKKLFSKLWNWK-GG-RGD

45.5

HPRP-A1-iRGD

FKKLKKLFSKLWNWK-CRGDKGPDC

44.9

iRGD

CRGDKGPDC

17.3

RGD

RGD

5.1

RP-HPLC retention time of peptides.

The secondary structures of the series of targeting peptides, parent peptide and free iRGD were examined using CD spectroscopy in PBS (50 mM KH2PO4/K2HPO4 containing 100 mM KCl, pH 7.4) and in the presence of 50% TFE 32. As shown in Figure 1A, in PBS conditions, all of the peptides showed random-coil structures, however, in the presence of 50% TFE (Figure 1B), except iRGD, all of the other targeted modified peptides exhibited different degrees of α-helical structure. The helical content of RGD- and iRGD-modified HPRP-A1 peptides without linker were similar to that of HPRP-A1; however, the helical content of RGD- and iRGD-modified HPRP-A1 peptides with a two-glycine linker were slightly lower than that of the HPRP-A1 peptide alone. The CD results demonstrated that chemical conjugation of RGD/iRGD with HPRP-A1 did not affect the secondary structure of the peptide.

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Figure 1. CD spectra of peptides. (A) In PBS at 25 °C and (B) in the presence of 50% TFE at 25 °C.

Peptide identification and stability study The HPLC chromatograms and MS spectra of the various peptides were in the supporting information Figure S1 to S25. The molecular weight of peptides was same as the theoretical molecular weight. The stability study of peptides in fetal bovine serum (FBS) was shown in the supporting information Figure S26. As the result showed that the peptides were quite stable in FBS for 24 h, the degradation of the peptides was around 10%-20%. The anticancer activity of the modified peptides was screened using different cancer cell lines, as well as normal cell lines, by the MTT method. As shown in Table 2, the IC50 values of HPRP-A1-iRGD in A549 cells and MDA-MB-231 cells were decreased dramatically compared with those of the HPRP-A1 peptide. The IC50 values of the modified peptides in the other three cancer cell lines, H-460, BGC, and MCF-7, were similar compared with those of the HPRP-A1 peptide alone. To investigate the cytotoxicity of the peptides, the IC50 values in the HUVEC cell line and minimum hemolytic concentration (MHC) in human red cells (hRBCs) were tested accordingly. The IC50 values of the modified peptides in normal cells were higher than those in 5



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cancer cells, indicating lower cytotoxicity toward normal cells, and the MHC values of the modified peptides were higher than for the HPRP-A1 peptide, suggesting that modification of HPRP-A1 also decreased the hemolytic activity of HPRP-A1. It has been reported that NRP-1, the targeted receptor of the iRGD peptide, is highly expressed in the A549 cell line 33 and MDA-MB-231 cell line 34; thus, A549 and MDA-MB-231 were selected as the cancer cell lines, and the HUVEC cell line was chosen as the normal cell line for use in the following studies. Table 2. Anticancer activity and hemolytic activity of peptides MHC (µM)b

IC50 a（µM） Peptides A549

MDA-MB-23 H-460 1

BGC

MCF-7

HUVE C

HPRP-A1

14.8

22.9

13.3

14.1

15.7

24.2

32

HPRP-A1-iRGD

8.4

15.6

14.6

10.8

11.8

31.6

125

HPRP-A1-GG-iR GD

21.6

15.2

12.9

14.8

15.8

31.6

125

HPRP-A1-RGD

11.1

27

19.5

15.7

11.8

26.5

125

HPRP-A1-GG-R GD

14.3

22.5

15.2

18.5

14.8

29.1

125

iRGD

>250

-

>250

-

-

-

>500

RGD

>250

-

>250

-

-

-

>500

a

IC50 represents the concentration of peptides at which cell viability was reduced by 50% in

comparison with untreated cells. The MTT assay was repeated for three times and the IC50 value was the average value of b

three separate experiments.

MHC: the minimum hemolytic concentration is the concentration of peptide that result in 10%

hemolysis of human red blood cells (hRBCs)

Expression levels of NRP-1 in the targeted cells As reported, iRGD has high affinity with the NRP-1 receptor 29. The expression of NRP-1 was checked using the western blotting method in HUVEC, A549, and MDA-MB-231 cell lines. As shown in Figure 2A, NRP-1 expression was higher in MDA-MB-231 and A549 cell lines than in the HUVEC cell line. Based on the western blotting results, the NRP-1 expression in A549 and MDA-MB-231 cell lines was investigated by immunofluorescence assay. In Figure 2B, the bright green 6


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fluorescence of these two cell lines can be observed, indicating that NRP-1 was highly expressed on the membrane of these two cancer cell lines. In addition, to further examine if high expression of NRP-1 exists in tumor tissues, the A549 xenograft model was used to make an immunohistochemical assay. As shown in Figure 2C, the brown color in the A549 tumor tissue was significantly deeper than that of the control tumor tissue, suggesting a high NRP-1 expression in A549 xenograft tumor tissue. Thus, the expression of NPR-1 protein in tumor cells was higher than that in normal cells.

Figure 2. Expression levels of NRP-1 in the targeted cells. (A) Expression analysis of NRP-1 in A549, MDA-MB-231, and HUVEC cell lines by western blotting assay and quantitation analysis. (B) Expression analysis of NRP-1 in A549 and MDA-MB-231 cells lines by immunofluorescence. NRP-1 is stained in green. Nuclei are stained in blue. Scale bar is 20 μm. (C) Immunohistochemical assay of the NRP-1 expression in A549 xenograft nude mouse tumor tissue. Negative control is para-carcinoma tissue. Scale bar is 50 μm.

Anticancer activity of peptides in A549 cells in vitro 7



To further characterize the peptides, a scratch test, cell viability assay, and apoptosis induction test were performed using the A549 cell line. As shown in Figure 3A, HPRP-A1-iRGD and HPRP-A1-RGD inhibited cancer cell migration after 24-h incubation, while the other peptides did not inhibit cell migration. In addition, the cell viability of A549 cells incubated with 16 μM iRGD/RGD modified peptide as well as HPRP-A1 peptide for 1, 24, and 48 h was detected using the MTT method. Figure 3B indicates that the cell viability of A549 cells incubated with iRGD modified peptide for both 24 and 48 h was significantly lower than the other two peptides; hence, HPRP-A1-iRGD was selected for comparison with HPRP-A1 in the following studies. An apoptosis induction assay of A549 cells incubated with different concentrations of iRGD modified peptide and none modified peptide for 24 h was performed using flow cytometry (FACSCalibur, Becton-Dickinson, San Jose, CA, USA). As shown in Figure 3C, 8 and 16 μM HPRP-A1-iRGD induced 20% and 80% apoptosis, respectively, while 8 and 16 μM HPRP-A1 only induced 10% and 60% apoptosis, respectively, (P < 0.01), indicating that apoptosis induction may be one of the mechanisms by which HPRP-A1-iRGD kills cancer cells.

Figure 3. In vitro anticancer activities. (A) Scratch test of A549 cells cultured with peptides for 24 h. (B) Cell viability of A549 cells cultured with 16 μM peptides for 1, 24, and 48 h. (C) Apoptosis of A549 cells cultured with HPRP-A1 and HPRP-A1-iRGD peptides. (a) The scatter diagram of the apoptosis of A549 cells cultured with peptides for 24 h. (b) The semi-quantitative analysis of early and late apoptosis percentages of A549 cells incubated with peptides for 24 h. *** P

< 0.001; ** P < 0.01; * P < 0.05. 8


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Cellular uptake and membrane disruption of peptides HPRP-A1, Aas a cationic membrane-active peptide, the cellular uptake of HPRP-A1can internalize cytoplasm was rapidly by disrupting the integrity of the cell membrane 5. In this study, FITC-labeled HPRP-A1 and FITC-labeled HPRP-A1-iRGD were used to determine the cellular uptake rate of peptides by LSCM (Figure 4A and 4B). The results in Figure 4A show that the fluorescence of FITC-labeled HPRP-A1-iRGD in MDA-MB-231 and A549 cell lines was much brighter than that of FITC-labeled HPRP-A1. However, in the HUVEC cell line, the opposite result was observed, indicating that HPRP-A1-iRGD was less cytotoxic than HPRP-A1 toward normal cells. As shown in Figure 4B, the rate of fluorescence intensity changes of HPRP-A1-iRGD increased quickly for both cancer cell lines, compared with HPRP-A1; however, in the HUVEC cell line, the fluorescence intensity changes of HPRP-A1-iRGD were much slower than those of HPRP-A1. Similar results were observed in the membrane disruption assay. As shown in Figure 4Ca, the PI uptake rate of 16 μM HPRP-A1-iRGD in MDA-MB-231 and A549 cell lines was remarkably higher than that of HPRP-A1. Figure 4Cb indicates that the peptides could disrupt the cell membrane rapidly within 15-30 min. The PI uptake rate of 16 μM HPRP-A1-iRGD was significantly higher than that of HPRP-A1 at 15, 30, and 60 min in MDA-MB-231 cells, and at 30 and 60 min in A549 cells. However, the PI uptake rates of the two peptides in the HUVEC cell line were both lower than in the cancer cell lines.

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Figure 4. Cellular uptake and membrane disruption of peptides. (A) Representative LSCM images of A549, MDA-MB-231, and HUVEC cells cultured with 16 μM FITC-labeled HPRP-A1 and FITC-labeled HPRP-A1-iRGD. The LSCM images were obtained with incubation for 0–120 s using the time series model of confocal microscopy. Green color denotes FITC-HPRP-A1 and FITC-HPRP-A1-iRGD. (B) The change rates of green fluorescence intensity change rates were recorded from 0–120 s in three cell lines. The black square indicates HPRP-A1, the red circle indicates HPRP-A1-iRGD. (C) Membrane disruption in A549, MDA-MB-231, and HUVEC cells by peptides. (a) A549, MDA-MB-231, and HUVEC cells were cultured with 4, 8, and 16 μM HPRP-A1 and HPRP-A1-iRGD for 1 h. (b) A549, MDA-MB-231, and HUVEC cells were cultured with 16 μM HPRP-A1 and HPRP-A1-iRGD for 5, 15, 30, and 60 min. The white, grey, and black columns indicate HPRP-A1, HPRP-A1-iRGD, and the control group, respectively. The uptake rate of PI was measured by flow cytometry. *** P < 0.001; ** P < 0.01; * P < 0.05.

Penetrability of FITC-labeled peptides in A549 3D-MCSs 10


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To investigate the penetration ability of HPRP-A1-iRGD and HPRP-A1 peptides, 3D MCSs of A549 cells were established as a model to mimic solid tumors. Figure 5A shows the photographic images of A549 3D MCSs stained by Hoechst 33258. The nucleus were stained by Hoechst 33258, the MCS were cultured with 16 μM FITCHPRP-A1-iRGD and 16 μM FITC- HPRP-A1 for 30 min, and detected by LSCM using the Z scan model (Figure 5B). The distribution of green fluorescence in the center of the MCS could be detecte only in the 15th layer in HPRP-A1 group, while in HPRP-A1-iRGD group, even in the 45th layer, the green fluorescence could be visualized clearly. The result above suggesting the penetration depth of HPRP-A1-iRGD was significantly deeper than HPRP-A1. Figure 5C shows the changes in fluorescence intensity across the MCS in the 45-μm layer, the fluorescence intensity of the MCS incubating with iRGD modified peptide was stronger than that without modified peptide. . Figure 5D shows the 3D simulation of A549 MCSs cultured with FITC-HPRP-A1 and FITC-HPRP-A1-iRGD. These results indicated that conjugation of iRGD with HPRP-A1 enhanced the penetration ability of the HPRP-A1 peptide into A549 3D MCSs.

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Figure 5. Penetration of FITC-labeled peptides in A549 (3D MCS). (A) Representative LSCM image of A549 3D-MCS stained with Hoechst 33258. (B) Representative LSCM images showing the peptides in A549 MCS incubated with 16 μM FITC-HPRP-A1 and 16 μM FITC-HPRP-A1-iRGD for 30 min. The green fluorescence indicates FITC-labelled peptides and blue fluorescence indicates nucleus stained by Hoechst 33258. (C) The corresponding degrees of fluorescence intensity across the spheroids. Blue represents the nucleus stained by Hoechst 33258 and green represents FITC-peptides. (D) The 3D simulation of the A549 MCS cultured with FITC-HPRP-A1 and FITC-HPRP-A1-iRGD.

In vivo tumor targeting assessment The in vivo tumor-targeting ability of RhB-HPRP-A1 and RhB-HPRP-A1-iRGD was evaluated in A549 tumor-bearing nude mice. Figure 6A shows the tumor accumulation at 24 h after injection with saline (control), RhB-HPRP-A1, and 12


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RhB-HPRP-A1-iRGD. The results showed that the red fluorescence was detected in the tumor area in the HPRP-A1-iRGD group. Distribution of the peptides in the major organs is shown in Figure 6B. In the mice tumors, the fluorescence intensity in the RhB-HPRP-A1 group was lower than in the RhB-HPRP-A1-iRGD group, suggesting a dramatic increase in tumor targeting and accumulation of the RhB-HPRP-A1-iRGD peptide in the tumors, because of the tumor-targeting properties of iRGD. These results demonstrated that iRGD chemically conjugated to HPRP-A1 increased the tumor accumulation of the HPRP-A1 peptide in xenograft mice.

Figure 6. In vivo and ex vivo fluorescent imaging of RhB-labeled peptides in A549 tumor bearing nude mice. (A) In vivo images of the A549 tumor-bearing nude mice after intravenous injection of physiological saline (as control), RhB-HPRP-A1, and RhB-HPRP-A1-iRGD. The X-ray images are shown for the control group. The white circle shows the tumor location. (B) Ex vivo images of tumors and other organs. Two mice were tested in each group.

Adhesive, pull-down, and affinity assays of the peptides iRGD can specifically bind with integrin αvβ3 and αvβ5, and after proteolytic cleavage, the truncated peptide (CRGDK) has affinity for NRP-1 29. To demonstrate whether the peptides could bind to integrin-positive cells and promote adhesion, the 13



adhesive assay was performed 35. Figure 7A shows that in the adhesive assay, many more cells were adhered to the Elissa plates after pre-culture with 8 or 16 μM HPRP-A1-iRGD compared with HPRP-A1 at the same concentrations. These data suggest that HPRP-A1-iRGD can attach to A549 cells more effectively than HPRP-A1. To determine the affinity of the iRGD-modified HPRP-A1 peptide for the NRP-1 protein, the pull-down assay was carried out using SAM and the western blot method. As shown in Figure 7B, the quantity of NRP-1 in the HPRP-A1-CRGDK group was significantly higher than in the HPRP-A1 group, which indicated that more NRP-1 protein was coupled with the HPRP-A1-CRGDK peptide. As previously reported, the CRGDK motif is a bingding motif for NRP-1. 4. Thus, in the affinity studies, including the pull-down assay and the biolayer interferometry (BLI) assay, the proteolytic cleavage motif HPRP-A1-CRGDK was used to assess the affinity between the peptide and proteins. The affinity dynamics and calculation of kD values were assessed using the BLI assay by loading the biotin-labeled peptides on the SA sensor. As shown in Figure 7C, the kD value of HPRP-A1-CRGDK was 6 nM compared with 10 nM for HPRP-A1, which indicated that the affinity between HPRP-A1-CRGDK and the NRP-1 protein was almost two times stronger than that of HPRP-A1. These results suggested that the iRGD modified HPRP-A1 peptide increased the binding affinity with the targeted protein compared with the unmodified peptide.

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Figure 7. Adhesive, pull-down, and affinity assays of the peptides. (A) Adhesive assay of peptides with A549 cells. Different concentrations of HPRP-A1 and HPRP-A1-iRGD peptides were coated on 96 well ELISA plates overnight. A549 cells were plated on the ELISA plates for 1 h, stained with Giemsa stain, and images were taken by microscopy. (B) Pull-down assay of biotin-labeled peptides with membrane protein lysis of the MDA-MB-231 cell line. The biotin-labeled HPRP-A1 and HPRP-A1-CRGDK were immobilized on streptavidin-modified magnetic beads, and cultured with membrane protein lysis for 30 min. The peptide-NRP-1 composition was analyzed by western blot assay. (C) Affinity of the peptides with NRP-1 protein analyzed by biolayer interferometry (BLI). BLI curves of (a) HPRP-A1 and (b) HPRP-A1-CRGDK, binding with 12.5 (blue), 6.25 (red), 3.125 (purple) and 1.6 (green) μg/ml NRP-1 proteins. (c) Kinetic parameters of peptides derived from the BLI experiments. Kon, kinetic association rate; koff, kinetic dissociation rate; KD, equilibrium dissociation constant (koff/kon).

DISCUSSION In this study, the cationic membrane-active peptide HPRP-A1 was modified by chemical conjugation with the tumor-homing peptide iRGD. The selectivity of the modified peptide toward cell membranes expressing the NRP-1 receptor, the penetration ability in 2D and 3D cell models, as well as the accumulation in tumor tissues were increased dramatically compared with those of the HPRP-1 peptide. In addition, the affinity of the peptide modified with the targeting protein was increased 15



significantly, which indicated the value of this iRGD-modified peptide approach to enhance tumor-targeting specificity. The modified peptides all had slightly decreased hydrophobicity compared with the HPRP-A1 peptide, which may be attributed to the hydrophilicity of RGD and iRGD. As reported in a previous study, a decrease in the hydrophobicity of ACPs reduced the biological activity 5, which might be the reason why the cytotoxic effect of the modified peptides in normal cells, including HUVEC cells and hRBCs, decreased. However, the IC50 values of iRGD-modified HPRP-A1 against A549 and MDA-MB-231 cell lines decreased, which can possibly be attributed to the high expression of the NRP-1 receptor in these two cell lines (Figure 2), which significantly increased the affinity of the peptides with cancer cells. Based on the IC50 value, the chemically conjugated peptide HPRP-A1-iRGD showed the best activity in the A549 cells scratch test and the cell viability assay of all the peptides. The apoptosis assay demonstrated that chemical conjugation with iRGD did not change the anticancer mechanism of HPRP-A1, but increased the apoptosis induction activity. A possible mechanism for this increase may be that the permeability changes in the cell membrane allow more peptide to internalize into the cytoplasm and induce more apoptosis. To further validate the specificity of HPRP-A1-iRGD to cancer cell membranes over normal cell membranes, cellular uptake and membrane disruption tests were used to assess the selectivity of the peptides between cells with different levels of NRP-1 expression. As shown in Figure 4, the cellular uptake of FITC-labeled HPRP-A1-iRGD and the PI uptake rate were higher in tumor cells that highly expressed NRP-1, and lower in normal cells with a low level of NRP-1 expression. In general, ACPs have a higher affinity with tumor cell membranes compared with normal cell membranes based on the different lipid compositions in the cell membranes of tumor and normal cells, which results in selective disruption of the integrity of cancer cell membranes 5. This selectivity resulted in the lower cellular uptake of HPRP-A1 and lower PI uptake rate in HUVEC cells compared with two tumor cell lines. In this study, this membrane composition-based selectivity toward tumor cells was magnified by the chemically conjugated. The closer ACPs get to the tumor membrane, the more they can disrupt the membrane, and enable more molecules to enter into the cytoplasm. In contrast, after the iRGD modification, HPRP-A1-iRGD exhibited a further decreased cellular uptake rate in HUVEC cells; this phenomenon may possibly be because of the decrease in peptide hydrophobicity with the iRGD modification. As well as in 2D monolayers of tumor cells, the penetration ability of HPRP-A1-iRGD was also investigated in 3D MCSs to mimic penetration into solid tumor tissues. As shown in Figure 5, the iRGD-modified peptide penetrated much 16


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deeper than the HPRP-A1 peptide in A549 3D MCSs. The mechanism of action may be based on a three-step process in which iRGD homes to the tumors: first, the internal RGD specifically binding to αv integrins which are over expressed on tumor cell and tissue; secondly, iRGD was proteolytic cleavage and the C-end motif was exposed to bind with NRP-1; and then, this binding motif helps ACP to penetrate into tumor tissues and cells 4. Tumor accumulation of the peptides was detected in the A549 xenograft model using In Vivo Imaging Systems, the stability study indicated the Rhodamine B labelled peptides were stable within 24 h in FBS. As previously reported, the tumor targeting ability of iRGD to tumors is associated with the overexpression of αv integrins and NRP-14. As αv integrins are higher expressed in most tumor cells , after HPRP-A1-iRGD is intravenously injected into A549 xenograft model mice, the peptides should be located with tumor blood vessels because of the affinity of the RGD sequence, then the C-terminal motif of the peptide will be subsequently exposed by proteolytic cleavage, and binding with NRP-1 will enable permeability and penetration of the tumor vessels and tissues 4, 29. This binding and penetrating ability of an iRGD-modified ACP was further demonstrated by the pull-down and BLI assays. The KD value in the BLI assay for HPRP-A1-CRGDK was 6.46 nM, which was lower than that of HPRP-A1 (10.7 nM), indicating that iRGD-modified HPRP-A1 binds to cancer cells and penetrates into cells using the CRGDK pathway.

MATERIALS AND METHODS Materials FITC-HPRP-A1, FITC-HPRP-A1-iRGD, biotin-HPRP-A1-CRGDK, biotin-HPRP-A1, hydrophilic rhodamine B (RhB)-HPRP-A1, and RhB-HPRP-A1-iRGD were provided by GL Biochem Ltd., (Shanghai, China). The Hoechst 33258 and PI dye detection kits were purchased from Solarbio Life Science (Beijing, China). The membrane protein extraction kit was provided by BestBio (Shanghai, China). The Pierce™ BCA protein assay kit and FITC secondary antibody were provided by Thermo Fisher Scientific (Waltham, USA). Thallium (III) trifluoroacetate was provided by Tokyo Chemical Industry (Tokyo, Japan). Streptavidin-coupled magnetic beads (AIIMAG® SAM-070) were provided by Shanghai Allrun Nano Science & Technology Co., Ltd. (Shanghai, China). NRP-1 antibody was provided by Abcam (Cambridge, USA). Streptavidin (SA) biosensor was provided by ForteBio (Fremont, CA, USA), and NRP-1 protein was purchased from Sino Biological Inc. (Beijing, China). Cancer cell lines, A549, H460, MDA-MB-231, MCF-7, and BGC and normal cell line HUVECs were obtained from the Institute of Biochemistry and Cell Biology, 17



Shanghai Institute for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Female BALB/c nude mice, 6–8 weeks old and weighing 18–22 g, were purchased from Beijing Weitonglihua Co., Ltd., China, and housed in a Good Laboratory Practice (GLP) laboratory according to the guidelines of the animal ethics committee of Jilin University (Approval No. JLUSWLL003, Jilin, China). Peptide synthesis and purification Anticancer peptides HPRP-A1, HPRP-A1-RGD, HPRP-A1-GG-RGD, HPRP-A1-iRGD, HPRP-A1-GG-iRGD and homing peptides RGD and iRGD were synthesized using solid-phase method as described previously 36. Homing peptide iRGD and modified peptides HPRP-A1-iRGD and HPRP-A1-GG-iRGD are disulfide-based cyclic peptides. The linear peptides were synthesized on MBHA rink amide resin by the Fmoc method. Then, the disulfide bridge was formed by oxidation with thallium trifluoroacetate 37. Finally, the peptides were purified by RP-HPLC system as described previously 38 (see Supplementary information for details). Circular dichroism (CD) spectroscopy of peptides The series targeted modified peptides were dissolved in PBS and PBS plus 50% trifluoroethanol (TFE). The helical structure of the peptides was investigated by CD spectroscopy using a Jasco J-810 spectropolarimeter (Jasco, Tokyo, Japan)32. The values of the molar ellipticities of the targeted modified peptides at wavelengths of 208 nm and 222 nm were used to estimate the relative helicity of the peptides. Identification and stability study of the peptides The peptides were diluted to final concentration of 1 mg/mL in pure water and tested on HPLC system using Agilent ZORBAX SB-C8 column (150×4.6 mm, 5μm particle size, 300 Å pone size; Agilent Technologies, CA, USA). The linear AB gradient was 30%-60% for HPRP-A1, HPRP-A1-iRGD, HPRP-A1-RGD, HPRP-A1-GG-iRGD, HPRP-A1-GG-RGD, 5%-20% for iRGD and 1%-20% for RGD, and the flow rate was 1 mL/min, solvent A was 0.1% aqueous TFA, solvent B was 0.1% TFA in acetonitrile, the temperature of column was 25 ℃. The mass spectra of peptides were performed in AB SCIEX TOF/TOF 5800 system (see Supplementary information for details). The peptides were dissolved in pure water to a final concentration of 1 mg/mL, then incubated with 10% fetal bovine serum at 37 ℃. At different time point (0, 2, 6, 24 h), 10 μL peptides were diluted with equal volume of 0.1% aqueous trifluoroacetic acid (TFA) and then analyzed by RP-HPLC for stability study 18


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Cell cultures A549, H460, MDA-MB-231, MCF-7, BGC, and HUVEC cell lines were cultured in DMEM high glucose culture medium (Gibco Life Technologies, Thermo Fisher Scientific, Waltham, USA) with 10% fetal bovine serum (FBS), in a 37 °C, 5% CO2 incubator. The A549 3D MCS was cultured with DMEM/high glucose with 10% FBS, penicillin (100 U/ml), and streptomycin (100 U/ml) in a humid atmosphere at 37 °C with 5% CO2. Fresh culture medium was added in the dish every 3 days. Cell viability assay Briefly, cancer and normal cells (1 × 104) were plated in a 96-well microtiter plate and cultured at 37 °C and 5% CO2 in an incubator overnight. The peptides were dissolved in fresh medium (without FBS) to various concentrations, the medium without peptide was the negative control. After culturing the cells with 100 μL of peptides for 24 h, 20 μl of 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added in 96-well microtiter plate for another 4 h. The solution in 96-well microtiter plate was removed, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well, the OD value at 492 nm was detected using a microplate reader (GF-M3000; Gaomi Caihong Analytical Instruments Co., Ltd., Shandong, China). The IC50 values and cell viability were calculated using the software Origin 6.0. The MTT assays were performed in triplicate. Hemolytic activity Peptides were serially diluted to a final volume of 70 μl. 1-2 mL of human erythrocytes were diluted with 3 mL PBS and centrifugated at 1000 rpm for 5 min, repeat for three times until the supernatant was clear. Then the erythrocytes were diluted in PBS to a final concentration of 2% solution. The erythrocytes that dissolved in purified water were used as positive control. The peptides were dissolved in PBS to several concentrations. 70 μl erythrocytes and 70 μl peptides were added in 96-well plates and incubated at 37 °C incubator with rotation at 90 rpm for 1.5 h. The plates were then centrifuged for 10 min at 3000 rpm at 4 °C, and 90 μl of supernatant was transferred to a flat-bottomed 96-well plate. The absorbance of the supernatant was measured at 540 nm with a microplate reader. Hemolytic activity was determined as the concentration of the peptide to cause 10% hemolysis. Expression levels of the targeting protein NRP-1 expression by western blotting The NRP-1 expression in MDA-MB-231, A549, and HUVEC cells was determined by western blotting. Briefly, MDA-MB-231, A549, and HUVEC cells were washed 19



with ice-cold PBS, scraped off and collected by centrifugation at 1500 rpm for 3 min. The supernatant was removed and the protein precipitate was solubilized in RIPA lysis buffer. After incubation on ice for 2 h, the cell lysis was centrifugated at 12000 rpm for 15 min at 4 °C, the protein in supernatant was collected. Precise concentrations of protein were qualified by bicinchoninic acid (BCA) protein assay kit. 50 μg of protein were loading on 12% SDS-PAGE gels, after separation, the gel was transferred to activated polyvinylidene fluoride membranes by formaldehyde. Membranes were blocked for 2.5 h with 5% skim milk in PBS at room temperature and cultured with anti-NRP-1 antibody (abcam 25998) overnight at 4 °C. After washing for three times, the membranes were incubated with secondary antibodies for 2 h at room temperature, then washed for 3 times. The protein band were visualized on the Tanon-5200 Chemiluminescent Imaging System (Tanon Science & Technology Co., Ltd., Beijing, China) 39. NRP-1 expression by immunofluorescence The NRP-1 expression in MDA-MB-231 and A549 cells was determined by immunofluorescence. Briefly, MDA-MB-231 and A549 cells were fixed with paraformaldehyde for 30 min, and incubated with anti-NRP-1 antibody overnight at 4 °C. After washing with PBS for three times, the cells were cultured with secondary fluorescence antibody for 1 h at room temperature. The nuclei were stained with Hoechst 33258 for 30 min. Finally, the immunofluorescence was investigated using a LSCM (LSCM 710, CarlZeiss,Oberkochen, Germany). NRP-1 expression by immunohistochemistry Tumor sections and para-carcinoma tissue from A549 xenograft mice were dewaxed, dehydrated, and rehydrated. The primary antibody NRP-1 (1:200) was incubated to the sections at 4 °C overnight. Then, the sections were cultured with biotinylated secondary antibodies. Finally, the cell ncleus were stained by hematoxylin, and detected using microscope. Selectivity of the peptides against cancer cell and normal cell membranes Membrane disruption A549, MDA-MB-231, and HUVEC cells in the logarithmic growth phase were plated (2-3 × 105 cells/well) in 6-well plates. After the cells were cultured at CO2 incubator overnight, the culture medium was removed and fresh medium containing different concentrations of HPRP-A1 and HPRP-A1-iRGD were added in 6-well plates. After incubation for different time periods, the cells were collected by trypsin digestion and centrifugation at 1500 rpm for 3 min. The cell pellet was washed three 20


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times with cold PBS and then stained with 5 μL of propidium iodide (PI) for 15 min at 37 ℃. The fluorescence was measured using flow cytometry. Cellular uptake of the peptides A549, MDA-MB-231, and HUVEC cells were cultured in glass-bottomed dishes for 24 h. After washing with PBS for three times, the FITC labeled peptides were added in the dish and the fluorescence images were taken by LSCM using a time series model, at 10 s per cycle, with a total of 12 cycles. The fluorescence intensity of the peptides inside the cells was quantificated using the ZEN lite 2012 image software (CarlZeiss, Oberkochen, Germany). The fluorescence change rate was calculated by the following equation: fluorescence change rate = (fluorescence value at the experimental time point / fluorescence value at time 0) × 100%. Penetrability assay of peptides in a 3-dimension (3D) multiple cell sphere (MCS) The MCSs model was established using A549 cells as reported 40, 41, sterilized agarose was heated to 90 °C and coated on the bottom of 10 cm dish, cooling to room temperature. 1×104 A549 cells were cultured in the agarose coated dish and cultured at incubator for around 7-10 days 42. The MCS was transferred to glass-bottom dish and cultured with Hoechst 33258 for 30 min at room temperature, then cultured with FITC-labeled HPRP-A1 and FITC-labeled HPRP-A1-iRGD peptides for 30 min. The depth of the peptides penetrating in the MCS was evaluated by the fluorescence intensity in each layer. 43 In vivo tumor targeting assessment Approximately 1 × 107 cultured A549 cells suspended in PBS were subcutaneously injected in the right posterior limb of female BALB/c nude mice. When the diameter of the tumors were grow to 0.8–1.0 cm (25–30 days), the tumor-bearing mice were subject to tumor targeting studies. To determine the tumor targeting, RhB-labeled HPRP-A1 and RhB-labeled HPRP-A1-iRGD peptides were intravenously injected to A549 tumor-bearing mice. At 24 h, the tumor accumulation image was investigated by In Vivo Imaging Systems (FX Pro, Kodak, New York, NY, USA). Subsequently, the mice were sacrificed, the major organs including heart, liver, spleen, lung, kidney, and tumor tissue were taken out and the fluorescence image was investigated 44. In vitro binding assays Adhesive assay Evaluation of cell attachment was conducted as described previously 35. Briefly, HPRP-A1 and HPRP-A1-iRGD were diluted to different concentrations by PBS and coated in a 96-well ELISA plate. After the plates were blocked with 2% BSA in 21



DMEM at 37 °C for 1 h, MDA-MB-231 cells were added to the wells (2–3 × 105 cells/well). After incubation for 1 h at 37 °C, the plates were washed three times with PBS to remove non-adherent cells. Adherent cells were then fixed, stained with 0.5% trypan blue, and later photographed. Pull-down assay HPRP-A1 and HPRP-A1-CRGDK were modified by biotin. The membrane protein was extracted using a membrane protein extracting kit. The biotin-peptides were immobilized in streptavidin-coupled magnetic beads (SAM), 100 μl of SAM were washed with PBS three times by attachment to the magnetic separator. Biotin-HPRP-A1 and bitotin-HPRP-A1-CRGDK were mixed with the SAM by vortexing for 1 h. The biotin-labeled peptide loaded SAM were incubated with the membrane protein solution for 1 h at room temperature. The pepetide-protein-SAM compound was selected and resuspeded in the loading buffer. After heated at 100 ℃ for 10 min to separate the peptide-protein compound and SAM, the eluted proteins were used for the western blot assay. Affinity analysis by Biolayer Interferometry (BLI) The affinity between the HPRP-A1/HPRP-A1-CRGDK peptides and the NRP-1 receptor was analyzed on the Octet RED 96 system (ForteBio, Fremont, CA, USA) at 30 ºC using SA biosensors and a kinetic buffer (PBS, containing 0.02% Tween-20 and 0.1% BSA, at pH 7.4). Biotin-labeled HPRP-1 and biotin-labeled HPRP-A1-CRGDK peptides (GL Biochem, Shanghai, China) were used to bind to the SA biosensor. The purified NRP-1 protein was diluted to different concentrations. The kinetic association (kon), dissociation (koff) rates and the equilibrium dissociation constant (KD) between peptides and protein were measured according to a previous study 45. Statistical methods Data are analyzed using software of SPSS and expressed as means ± standard error (SE) of the mean.

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CONCLUSIONS In this study, the cationic ACP HPRP-A1 was modified by chemical conjugation with RGD and iRGD with or without a two-glycine linker. HPRP-A1-iRGD showed the best in vitro anticancer activity. HPRP-A1 modified with iRGD exhibited stronger anticancer activity, and weaker cytotoxicity against normal cells. More importantly, HPRP-A1-iRGD showed stronger activity in inhibiting cancer cell migration and greater binding affinity and selectivity, especially the penetration ability in tumor cells and tissues than HPRP-A1. Bio-distribution studies of the modified peptide in vivo were conducted in the tumor tissue of A549 xenograft mice. The improved penetrability and accumulation of HPRP-A1-iRGD in A549 3D MCSs and xenogeneic tumor tissue indicated the efficacy of using a tumor-homing peptide to modify ACPs. This modification method is a promising approach to improve the tumor-targeting ability of anticancer drugs. Supporting Information: Supplementary Figures: Figure S1 HPLC of HPRP-A1 Figure S2 HPLC of HPRP-A1-iRGD Figure S3 HPLC of HPRP-A1-RGD Figure S4 HPLC of HPRP-A1-GG-RGD Figure S5 HPLC of HPRP-A1-GG-iRGD Figure S6 HPLC of iRGD Figure S7 HPLC of RGD Figure S8 HPLC of FITC-HPRP-A1 Figure S9 HPLC of FITC-HPRP-A1-iRGD Figure S10 HPLC of Rhodamine B -HPRP-A1 Figure S11 HPLC of Rhodamine B -HPRP-A1-iRGD Figure S12 HPLC of Biotin-HPRP-A1 Figure S13 HPLC of Biotin-HPRP-A1-CRGDK Figure S14 MS of HPRP-A1 Figure S15 MS of HPRP-A1-iRGD Figure S16 MS of HPRP-A1-GG-iRGD Figure S17 MS of HPRP-A1-RGD Figure S18 MS of HPRP-A1-GG-RGD Figure S19 MS of iRGD Figure S20 MS of FITC-HPRP-A1 Figure S21 MS of FITC-HPRP-A1-iRGD Figure S22 MS of RhodamineB-HPRP-A1 23



Figure S23 MS of RhodamineB-HPRP-A1-iRGD Figure S24 MS of Biotin-HPRP-A1 Figure S25 MS of Biotin-HPRP-A1-CRGDK Figure S26 Stability study of peptides in serum AUTHOR INFORMATION Corresponding author: Yuxin Chen, Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, 2699 Qianjin St., Changchun, Jilin, P. R. China 130012. Tel.: +86-431-85155220; Fax: +86-431-85155200; E-mail: [email protected]. AUTHOR CONTRIBUTIONS Conceived and designed the experiments: C. H. H. and Y. X. C. Performed the experiments: C. H. H. Analyzed the data: C. H. H. Performed the syntheses of peptides: C. H. H. Drawing the Schematic diagram of the mechanism: C. H. H. Contributed reagents/materials/analysis tools: C. H. H., Y. B. H. and Y. X. C. Wrote the paper: C. H. H. and Y. X. C. NOTES The authors declare that there is no conflict of interest regarding the publication of this manuscript. ACKNOWLEDGMENTS This work was supported by the National Key Science and Technology Project of China (2017ZX09309001 to Y. X. C.) and the Natural Science Foundation of Jilin Province of China (20180101250 JC to Y.B.H) and the “13th Five-year” Science and Technology Project of Jilin Provincal Department of Education (No. JJKH20180178 KJ to Y.B.H)

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Targeted Modification of the Cationic Anticancer Peptide HPRP-A1

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