Highly Efficient Delivery of Functional Cargoes by a Novel Cell

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Highly efficient delivery of functional cargoes by a novel cell-penetrating peptide derived from SP140-like protein Hu Wang, Jielan Ma, Yinggui Yang, Fanhui Zeng, and Changbai Liu Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00161 • Publication Date (Web): 12 Apr 2016 Downloaded from http://pubs.acs.org on April 17, 2016

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Bioconjugate Chemistry

Highly efficient delivery of functional cargoes by a novel cell-penetrating peptide derived from SP140-like protein Hu Wang1,2,3*, Jielan Ma2,3, Yinggui Yang2,3, Fanhui Zeng4, Changbai Liu1,2,3* 1. The Institute of Cell Therapy, China Three Gorges University, Yichang 443002, China. 2. Department of Pathology, Biology and Immunology, Medical School, China Three Gorges University, Yichang 443002, China. 3. Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang 443002, China. 4. The Central Hospital of Enshi Tujia and Miao Autonomous Prefecture, Enshi 445000, China. *Corresponding author: Chang-Bai Liu, MD., PhD., Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, 8 Daxue Road, Yichang, China. 443002. Tel.:+86 717 639 7179. E-mail: [email protected]. Hu Wang, PhD., Department of Pathology and Immunology, Medical School, China Three Gorges University, Yichang, China. 443002. Tel.:+86 717 639 7438. E-mail: [email protected].

ABSTRACT Cell-penetrating peptides (CPPs) have been successfully applied to deliver various functional macromolecules into cells in recent. Here, we describe a novel CPP designated as hPP3 (KPKRKRRKKKGHGWSR), which derived from human nuclear body protein SP140-like protein. The location of hPP3-FITC in cells was investigated using the fluorescence microscopy, and the internalization of hPP3 was quantitatively measured using a fluorescence spectrophotometer. The results showed that hPP3-FITC could enter into culturing cells, following a concentration-, incubation time-, serum- and temperature-dependent manner. Uptake of hPP3-FITC into cells was significantly enhanced by DMSO pretreatment, and inhibited by heparin and the endocytosis inhibitors (chlorpromazine and sodium azide), while the potent lysosomotropic agent, chloroquine showed a little positive effects on hPP3-FITC penetrating. Moreover, hPP3 could mediate functional GFP, KLA or NBD penetration. The findings of this study showed that human origin peptide hPP3 have potential to act as a macromolecular carrier penetrating cellular membranes and promising delivery peptide as drug delivery vectors. KEYWORDS: cell-penetrating peptide, drug delivery, endocytosis

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Introduction The majority of biological molecules need to be delivered into cells to efficiently exert their activity.1 However, the barriers of cell membranes restrict the access of hydrophilic macromolecules and drugs. Thus, improving cellular uptake is important for therapeutics application.2 Since the last two decades, the development of methods for therapeutics like drugs, nucleic acids and proteins delivery has gained much a attention in biomedical research.3 Cell-penetrating peptides (CPPs) that can act as appropriate vectors for the nonviral-based intracellular delivery of therapeutics have opened a new horizon in the drug delivery.4, 5 CPPs, alternatively named protein transduction domains (PTDs) are short, cationic and/or amphipathic peptides (about 30 amino acids, arginine and/or lysine-rich), able to penetrate cell membranes, and can transfer different kinds of cargos (ranging from small drug molecules to large proteins and nucleic acids) as effective vehicles both in vitro and in vivo.6-9 The exploitation of cargos delivery against a range of diseases, including cancer despite problems and controversies in characterizing and the entry of CPP,10 remain a key focus in biomedical research as well as the pharmaceutical industry. Most of CPPs used to date are of viral origins and result in undesired effects,11 when CPPs conjugated with therapeutics, these CPPs may give rise to an adaptive immune response in vivo. Therefore, novel CPPs derived from human are considered highly attractive import vehicles, such as human derived penetrating peptide-hCT,12 hCLOCK,13 Hph-1,14 BagP,1 p14ARF,9 human lactoferrin (hLF),15 Cytc86-101,16 and TCTP,11 etc. They have show promising potential for protein transduction as well as gene delivery agents. In general, the most important feature of CPPs is the existence of high cationic residues (arginine and lysine), which are essential for binding to cell surfaces and contacting with cell membrane or negatively charged cargo molecule.3, 17-20 The apoptosis inducing KLA peptide, (KLAKLAK)2, possesses an ability to initiate apoptosis in mammalian cells by disrupting mitochondrial membranes.21 However, this peptide has a poor cell penetrating potential, thus, it requires facilitated intracellular delivery tools such as cell penetrating peptides for effective translocation. NF-κB (nuclear factor-κB) activation is a crucial step for activating hepatic stellate cells (HSCs) in the pathogenesis of hepatic fibrosis,22 while, NBD (NF-κB essential modulator-binding domain) peptide can block the activation of the IκB kinase complex. When we were engaged in the research of cell-penetrating peptides, it was found that a 16-amino acid sequence from human nuclear body protein SP140-like protein was rich in Arg and Lys residues of the primary structure through the protein database search, and it was similar with the known CPPs. Then we analyze its secondary structure and found this peptide could form a classical α-helical conformation. In present study, we found that hPP3 as novel CPP derived from human origin protein, can act as a carrier translocating cellular membranes efficiently. Our results demonstrated that human derived peptides hPP3 could be used as a novel and promising delivery peptide for drug delivery.

Results hPP3 peptide prediction


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Prediction of CPPs using bioinformatic method can significantly accelerate the CPPs identification process more speedily and conveniently as well as help us to find novel CPP. Firstly, hPP3 (amino acids residue 380-395, as is shown in Figure 1A) from SP140-like protein was identified by bioinformatic software, and then characterized the penetrating efficiency. The Schiffer-Edmundson helical wheel modeling of hPP3 by DNAstar program showed the possible amphipathic α-helical conformation (Figure 1B). CellPPD was used to predict highly efficient CPPs. Prediction of hPP3 as a CPP along with all possible residue motifs scanned within this peptide (Figure 1C). Physicochemical properties hPP3 including molecular weight, hydrophobicity, hydrophilicity, hydropathicity, amphipathicity, stearic hindrance, net hydrogen, side bulk, charge and pI were determined (Supplementary Table S1). Solvent accessibility analyzed by NetSurfP web server (Supplementary Figure S1A) and Secondary conformations (Supplementary Figure S1B), 3D predicted models from I-TASSER server (Supplementary Figure S1C-1D) are developed. Furthermore, the 3D structure of hPP3 had a finger-shape like structure, the hydrogen exchange space, electrostatic surface distribution and hydrophobicity were displayed (Figure 1D). Moreover, the peptide was synthesized, purified and evaluated by HPLC and MS analysis (Supplementary Figure S2).

Figure 1 hPP3.

Prediction and three-dimensional (3-D) characterization of



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(A) Amino acid sequence and location of hPP3 peptide in SP140-like protein. (B) The helical wheel representation of hPP3. (C) SVM score from CPPred server based on predicted motifs, full length of hPP3, 15 to 10 amino acid fragments and control peptide TAT and NCO were represent by different color respectively. (D) hPP3 structure, energy map (green color, steric favorable region; blue color, hydrogen acceptor favorable; yellow color: hydrogen donor favorable region associated with hPP3), surface electrostatics and surface hydrophobicity.

The translocating ability of hPP3-FTIC hPP3-FTIC and TAT-FITC at different concentrations from 2.5 µM to 10 µM were incubated with ECV304 cells, the result from fluorescence microscope revealed that at the concentration of 2.5 µM hPP3-FTIC, the fluorescence was little, when the concentration was 5 µM , the fluorescence was sporadic, until 10 µM, the fluorescence was apparent (Figure 2A). The majority of internalized hPP3-FITC was located in the cytosol (nucleus and cytoplasm). To investigate the precise ability of hPP3-FTIC penetration, different concentration of hPP3-FITC uptake in cells were subjected to quantitative analysis. The hPP3-FITC (10 µM) could penetrate into cells significantly weaker compared with TAT-FITC penetration (Figure 2B). It is clearly indicated that hPP3-FTIC could entry into different cells (Figure 2C), while, at different time points, there are no significant difference of fluorescence intensity between different cell lines.


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Figure 2

Uptaken of hPP3-FITC by ECV304 cells.

(A) ECV304 cells were incubated with different concentrations (from 2.5 µM to 10 µM) of hPP3-FITC for 1 h. The cells were washed with PBS for 5 times and observed under fluorescence microscope. (B) Fluorescence quantization analysis was used to detect the penetration of hPP3-FITC. TAT treatment was used as a positive control. (C) Effects of incubation time on the internalization of hPP3 (10 µM) in different cell lines, fluorescence quantization was determined after peptide incubation for 0.5, 1, 2, 4, 5, 10, 20 and 30 h.



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Effects of pretreatment with DMSO on the internalization of hPP3 In our previous work, we found that DMSO (dimethyl sulfoxide) could efficiently enhance the penetrating of TAT with low cytotoxicity.23 And here, we attempted to explore whether DMSO could enhance the penetrating efficiency of hPP3. It was found that 5 µM hPP3-FITC uptake into different types of cells with 5% DMSO was significantly increased, and well-distribute in the cytosol and nucleus (Figure 3A), while hPP3-FITC uptake was weaker than TAT-FITC without DMSO treatment (Figure 3B). And the result of fluorescence quantization analysis revealed DMSO could significantly enhance the levels of peptides (hPP3 and TAT) internalized (Figure 3C). In order to evaluate that the pretreated with DMSO enhancing the peptides uptake was dose-dependent, B16 and ECV304 cells were incubated with different concentrations of DMSO (1.25 % to 10 %). Result indicated that the fluorescence of hPP3 pre-treating at the concentration of 1.25 % DMSO was apparent, the fluorescence were markedly increased with the advance of DMSO uptake into cells (Supplementary Figure S3).

Figure 3 Effects of hPP3-FITC uptake pretreated with DMSO. (A) hPP3-FITC (5 µM) distribution in ECV304, HeLa and MG63 cells with or without


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DMSO treatment. (B) Fluorescence quantization analysis of hPP3-FITC (5 µM) penetration without DMSO treatment, 5 µM TAT-FITC internalization was used as positive control. (C) Fluorescence quantization analysis of hPP3-FITC (5 µM) penetration with 5% DMSO treatment, 5 µM TAT-FITC internalization was used as positive control.

Effects of different conditions on the internalization of hPP3 Cultured cells were treated with 10 µM hPP3-FITC in culture medium with- or without serum. Quantization results have shown that hPP3-FITC internalization was significantly decreased in serum containing medium compared to the serum-free, which indicated that the cellular uptake of hPP3-FITC could be affected by the serum (Figure 4A). It is widely believed that the lipid membrane minimizes active processes of cellular transporting and should inhibit uptake of CPPs at low temperature.24 In present study, ECV304, Caski and HeLa were selected to assess penetration of hPP3-FITC at different temperatures. Our result revealed that the fluorescence (Supplementary Figure S4) was much weaker at the low temperature (4 °C) from the fluorescence quantization analysis (Figure 4B). Sodium azide can be used to inhibit ATP production by blocking oxidative phosphorylation within the cell membrane.25, 26 In order to determine whether hPP3 penetrate into cells by an ATP-dependent process, as shown in Figure 4C, the result suggest that cellular uptake of the hPP3-FITC was ATP-independent. Generally, clathrin-mediated endocytosis represents the main endocytotic pathway for cell uptake. To investigate whether clathrin-mediated endocytosis was involved in hPP3-FITC uptake, the internalization of hPP3-FITC was measured with or without chlorpromazine (known inhibitors) treatment. After incubation for 1 h, hPP3-FITC uptake had no significant changes in the presence or without chlorpromazine treatment shown in Figure 4D, which indicated that hPP3-FITC internalization involves clathrin-independent endocytic pathway. Membrane-associated HSPGs (heparan sulfate proteoglycans) plays an important role in endocytic uptake of CPPs,27, 28 heparin, as a competitor for HSPG, was used to investigate the cellular uptake of hPP3-FITC. After incubation for 1 h, hPP3-FITC uptake prevented remarkably in the presence of heparin (Figure 4E). These results suggest that hPP3-FITC uptake was HSPG-dependent. Furthermore, chloroquine, as a lysosomotropic agent, can inhibit endocytosis by preventing the acidification of endosome.29 The fluorescence intensity of uptake hPP3-FITC was higher in the presence of chloroquine in Caski cells, but not in PC3 and ECV304 (Figure 4F), which suggested that hPP3-FITC was partly delivered into acidic cellular compartments through intracellular endosomes and lysosomes.



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Figure 4 Effects of hPP3-FITC uptaken under different conditions. (A) (B) (C) (D)

Effects of serum on the internalization of hPP3 in HepG2, ECV304 and B16 cells. hPP3-FITC penetration in ECV304, Caski and HeLa cells at 4 °C or 37 °C. With or without sodium azide treatment, hPP3-FITC uptake was investigated. The cellular uptake of hPP3-FITC with or without chlorpromazine incubation. CPM, chlorpromazine. (E) Involvement of heparan sulfate receptors in the internalization of hPP3-FITC. hPP3-FITC internalization was detected in the absence or presence of heparin. (F) The cellular uptake of hPP3-FITC with or without chloroquine treatment. CQ, chloroquine.

Cytotoxicity and LDH release analysis of hPP3 To investigate the effect of hPP3 on cultured cell growth, the cell viability of ECV304 and HepG2 was evaluated through the MTT assay. After various concentrations (10 µM to 50 µM) of hPP3 were used to treat cells for 2 h, MTT assay was performed. Data have shown that there was little repressive effect on cell growth (Figure 5A), which suggested that hPP3 penetrated into cells without cytotoxicity. Furthermore, LDH release assay was performed to detect the membrane disturbance, our data suggested there was not membrane disturbance to different cell lines with different concentration of hPP3 treatment for 1 h (Figure 5B) and 2 h (Figure 5C).


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Figure 5 Cell membrane perforation or cytotoxicity evaluation on hPP3-FITC. (A) ECV304 and HepG2 cells were plated into 96-well plates and cultured for 15 h, different test concentrations (10 µM to 50 µM) of hPP3 were incubated for 24 h. Viability of different cells was examined by MTT assay. (B) LDH release quantity was determined after hPP3 (2.5 µM to 20 µM) incubation for 1 h, positive control using 0.2% NP-40. (C) LDH release quantity was determined after different concentrations of hPP3 incubation for 2 h, positive control using 0.2% NP-40.



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hPP3-GFP internalization In order to analyze the delivering capability of hPP3 fused with macromolecules, recombinant hPP3-GFP protein was expressed using prokaryotic expression systems, purified and then analyzed by SDS-PAGE (Figure 6A), hPP3-GFP fusion protein not only can be uptake by primary cultured mouse spleen cells (Figure 6B), but also can penetrate into cultured B16 melanoma cells in the present DMSO, although TAT-GFP can penetrate into cells inefficient in the present of 2.5% DMSO (Supplementary Figure S5), the fluorescent of hPP10-GFP was well-distributed in the cytosol of cells (Figure 6C) with 5% DMSO treatment, hPP10-GFP fusion protein uptake efficiency was similar with TAT-GFP treatment in ECV304 and Cos7 cells (Supplementary Figure S6A), however, the penetration efficiency were different in different cells (Supplementary Figure S6B).

Figure 6 hPP3-GFP fusion protein expression and its penetration in primary or cancer cells. (A) Recombinant hPP3-GFP and GFP protein expression were detected by SDS-PAGE. High-level expression of hPP3-GFP was prepared using prokaryotic expression plasmid pET-15b. Lane 1 and 6: without IPTG, lane 2 and 5: IPTG induction, lane 3 and 4 purified fusion protein. (B) Cell-permeability of hPP3-GFP (5 µM) in primary cultured mouse spleen lymphocyte


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with 5% DMSO treatment. The fluorescence microscopy was detected at three times. (C) hPP3-GFP fusion proteins' (from 0.5 to 5 µM) penetration with (0, 2.5% and 4%) or without DMSO observed under fluorescence microscopy.

hPP3-KLA and hPP3-NBD mediated cell apoptosis In order to analyze whether the biological activity of cargos can be delivered by hPP3 conjugation, caski cells or HSC-T6 cells apoptosis induced by hPP3-KLA or hPP3-NBD was analyzed by TUNEL assay. Result from TUNEL assay showed that hPP3-KLA or hPP3-NBD not only can enter into the cells, but also retain their bioactivity of apoptotic induction (hPP3-KLA in Caski cells shown in Figure 7A, hPP3-NBD in HSC-T6 cells shown in Figure 7B), while, nearly no apoptotic induction effect on Caski cells and HSC-T6 cell treated only using hPP3 (Supplementary Figure S7). Moreover, their apoptotic induction ability was nearly the same (hPP3-KLA in Caski cells shown in Figure 7C and hPP3-NBD in HSC-T6 cells shown in Figure 7D). These findings suggested that hPP3 could mediate functional KLA or NBD penetration into cells.

Figure 7 hPP3-KLA or hPP3-NBD induced Caski or HSC-T6 cell apoptosis. (A) TUNEL assay of hPP3-KLA (5 µM or 10 µM) induced Caski cells apoptosis.



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(B) TUNEL assay of hPP3-NBD (5 µM or 10 µM) induced HSC-T6 cells apoptosis. (C) The quantification of cell apoptosis rates of hPP3-KLA induction in Caski cells are represented as mean ± SEM for three separate experiments. (D) The quantification of cell apoptosis rates of hPP3-NBD induction in HSC-T6 cells are represented as mean ± SEM for three separate experiments.

Discussion Eukaryotic cells have a membrane barrier to deliver biologically active cargos to their cytoplasm; however, previous approaches for cargos internalization still have limitations, such as increased toxicity and poor cellular uptake. During the last 30 years, CPPs attracted extensive scientific attention in the field drug delivery. Although definition of CPPs is constantly evolving, CPPs are able to penetrate cell membranes and deliver various types of therapeutics both in vitro and in vivo.5 Because viral CPPs may result in undesired effects, human-derived CPPs are need to be developed. In our study, we found that a 16-amino acid in length and sequence rich in Lys and Arg residues from human nuclear body protein SP140-like protein (hPP3) had good transduction ability. The internalization of hPP3 was found to be in a concentration-, incubation time- and serum-dependent fashion, and the penetration efficiency could be enhanced by DMSO pretreatment, just like enhancing the TAT penetrating.23 The details of CPP internalization remain not well understood,30 in a number of reports; low temperature (4 °C) may minimize active processes of cellular transport. Our result showed that low temperature affect the internalization of hPP3. Cell surface HSPG has been reported to play an important role in the endocytic pathway.31-33 Heparin can be used as a competitor for cell surface HSPG leading to weak uptake. This result indicates that cell surface HSPGs is involved in hPP3 uptake. Although different CPPs may internalize with different pathway, chloroquine can be used to enhance the transduction efficiency through lysosomal escape. Chloroquine treatment aid hPP3 releasing from lysosomes by preventing its degradation. Furthermore, macromolecules could be delivered by hPP3 in different cell lines even in primary cultured cells. Despite CPPs are broadly accepted as useful tools, the mechanism of CPPs conjugated cargo internalization remains unclear. In the present study, the hPP3 may represent a promising CPP, as it combines cell internalization capability without toxicity concerns and immunogenicity. Further, we will further analyze the ability of hPP3 delivering DNA, siRNA and protein to play the corresponding biological activity and the mechanism of this peptide.

Materials and methods Drugs and reagents Heparin and NaN3 were supplied by Shandong Wanbang Biochem Co. Ltd (Shandong, China). Chloroquine and chlorpromazine were purchased from HongJing Chem Co. Ltd (Hubei, China). Trypsin was obtained from Sigma-Aldrich (USA) and dimethyl sulfoxide (DMSO) was obtained from Sigma.


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Cells and cell culture HeLa and Caski (Human Cervical Cancer Cells), MG63 (human osteosarcoma cell line), ECV304 (human umbilical vein endothelial cell), HepG2 (human hepatocellular carcinoma cell line), COS7 (African Green Monkey SV40-transfered kidney fibroblast cell line), PC3 (human prostate cancer cell line), HSC-T6 (a rat hepatic stellate cell line) and B16 cells were maintained in our lab. All cells were maintained in RPMI 1640 medium, and cultured at 37 °C in a 5% CO2 incubator. Bioinformatic analysis of hPP3 Cell-penetrating peptide or non-cell-penetrating could be predicted by bioinformatic approach, such as the support vector machine (SVM)-based CellPPD web server.34 The Motif-based and SVM-based method (SVM threshold of -0.1 and a motif e-value of 10) were used. The 3D structure of hPP3 was predicted by peptide tertiary structure prediction server (Pepstr, 7 to 25 residues length small peptides modeling).35 Helical properties of hPP3 were delineated with Schiffer Edmundson wheel modeling using DNASTAR Lasergene 7 programme. The 3D structure of hPP3 peptide also used I-TASSER program (http://zhanglab.ccmb.med.umich.edu/I-TASSER) to re-predict. The modeled structure is refined with energy minimization and molecular dynamic simulations based on Pepstr server, and then validation the structure by VADAR (Volume Area Dihedral Angle Reporter) web server,36 the energy map and surface electrostatics of hPP3 were analyzed with Molegro molecular viewer. Synthesis of fluorescein-labeled peptides AND fusion protein expression N-terminal labeled FITC of hPP3 (KPKRKRRKKKGHGWSR), N-terminal labeled FITC of NCO (KALGISYGRKK), N-terminal labeled FITC of TAT (YGRKKRRQRRRK), and without FITC labeled hPP3-NBD (KPKRKRRKKKGHGWSRGGTALDWSWLQTE) as well as hPP3-KLA (KPKRKRRKKKGHGWSRKLAKLAKKLAKLAK) were synthesized from SBS Genetech (Beijing, China) following the protocol shown in the supplementary method. The peptides were purified using reversed phase analytical HPLC to more than 99 % purity then diluted to 500 mM in PBS for stock solution. hPP3-GFP, TAT-GFP as well as GFP DNA fragment was cloned into pET-15b vector, and fusion protein of them were prepared following the protocol described.23, 37 Peptide internalization HeLa, MG63, ECV304 or B16 cells (5×105 cells per well) were seeded into 24-well-plates (Greiner, Germany) and cultivated to semi-confluence in an RPMI-1640 medium in a humidified air atmosphere for 24 h at 37 °C. Cells in the well were washed with phosphate-buffered saline (0.1 M, pH 7.4) for 3 times and then added 10 µM FITC-labeled peptides for 1 h incubation at 37 °C. The cells were further washed 5 times with PBS, the fluorescence was observed under fluorescence microscope (Nikon, Japan) using a band-pass filter (detects FITC). To quantify the intracellular fluorescence intensity of FITC-labeled peptides uptake in cells, Multimode Spectrophotometry (Tecan, Mannedorf, Switzerland) were used for quantitative analysis. Briefly, after the incubation of FITC-labeled peptide finish, cells were



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washed twice with PBS, lysed by adding 300 µl of 0.1 M NaOH and incubated for 10 min at room temperature, the lysed cells were centrifuged (14000 g for 5 min), the fluorescence intensity of the supernatant was determined at 494/518 nm in a Tecan 2000 Multimode Microplate Reader. The total cellular protein concentration was measured using the Bradford protein assay with BSA as standard (Bio-Rad). The fluorescence intensity of cellular uptake is expressed as fluorescence intensity per mg of total cellular protein. All experiments were performed in triplicate and repeated at least three times. The hPP3-FITC were incubated with seeded cells for 0.5 h, 1 h, 2 h, 4 h, 5 h, 10 h, 20 h and 30 h, the cellular uptake were determined quantitative analysis by Multimode Spectrophotometry. To assess the effect of temperatures (4 °C and 37 °C), serum (v/v 10%) and endocytic inhibitors such as heparin (10 µM), chlorpromazine (30 µM), chloroquine (10 µM) or sdium azide (40 µM) on the internalization of hPP3-FITC, the same procedure above was used. MTT assay ECV304 and HepG2 cells (4×104 cells per well) were seeded in 96-well plates, and then cultured in a humidified incubator for 24 h at 37 °C. Cells in plate were washed three times with PBS, adding different concentrations of hPP3 in serum-free medium, incubated for 2 h at 37 °C, washed with PBS three times, and then adding fresh medium containing 10% serum for 24 h incubation. MTT assay was used to assess cell viability. Briefly, 20 µl of 5.5 mg/ml MTT in serum-free media was added and maintained for 4 h at 37 °C. After removing the supernatant and then adding 100 µl of DMSO, the samples were incubated for 30 min at 37 °C before quantifying at the wavelength of 550 nm. LDH (lactate dehydrogenase) leakage assays The integrity of membrane was assessed by lactate dehydrogenase (LDH) leakage. After cells treated in 96-well plate for apropriate time under various concentrations of hPP3, cell-free supernatant was obtained, and then substrate mixture was added to each well for another 10 min enzymatic reaction. Absorbance was measured at 560/590 nm using a Multiskan Spectrum (Thermo, USA) plate-reader. TUNEL Analysis of Cell Apoptosis The procedure of hPP3-KLA or hPP3-NBD treatment was same shown above. Briefly, after the incubation of these two peptides, the Caski or HSC-T6 cells were maintained with 5 µM hPP3-KLA or hPP3-NBD for 4 h, washed 3 times using PBS, fixed with 4% paraformaldehyde (PFA), and then subjected to TUNEL (terminal dUTP-digoxigenin nick-end labeling) staining according to the instructions of the TUNEL kit, and followed by observation under a light microscope. Statistical analysis All results from quantitative analysis are expressed as means ± standard deviation (SD). Statistical significance between different groups was calculated using SPSS software. A student’s t-test was performed for data analysis and p value < 0.05 was taken as the level of statistically significant.


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Supporting Information Additional figures and tables showing the effect of hPP3 structure on bioinformatic prediction, different conditions on observation, as well as the key physicochemical properties hPP3 and its internal fragments. Acknowledgements We thank John R Hood, BSc (Hons), PhD (GlaxoSmithKline, Stevenage) for the helpful suggestions. We are thankful for the financial support of National Nature Science Foundation of China (NO. 81501330), the Science Foundation of CTGU (KJ2014B066) and Nature Science Foundation of Hubei Province (No. 2010CDB10705). References (1) Niarchos, D. K., Perez, S. A., and Papamichail, M. (2006) Characterization of a novel cell penetrating peptide derived from Bag-1 protein. Peptides. 27, 2661-9. (2) Machova, Z., Muhle, C., Krauss, U., Trehin, R., Koch, A., Merkle, H. P., and Beck-Sickinger, A. G. (2002) Cellular internalization of enhanced green fluorescent protein ligated to a human calcitonin-based carrier peptide. Chembiochem. 3, 672-7. (3) Li, H., and Qian, Z. M. (2002) Transferrin/transferrin receptor-mediated drug delivery. Med Res Rev. 22, 225-50. (4) Foerg, C., Ziegler, U., Fernandez-Carneado, J., Giralt, E., Rennert, R., Beck-Sickinger, A. G., and Merkle, H. P. (2005) Decoding the entry of two novel cell-penetrating peptides in HeLa cells: lipid raft-mediated endocytosis and endosomal escape. Biochemistry. 44, 72-81. (5) Liu, H., Zeng, F., Zhang, M., Huang, F., Wang, J., Guo, J., Liu, C., and Wang, H. (2016) Emerging landscape of cell penetrating peptide in reprogramming and gene editing. J Control Release. 226, 124-137. (6) Schwarze, S. R., Ho, A., Vocero-Akbani, A., and Dowdy, S. F. (1999) In vivo protein transduction: delivery of a biologically active protein into the mouse. Science. 285, 1569-72. (7) Joliot, A., and Prochiantz, A. (2004) Transduction peptides: from technology to physiology. Nat Cell Biol. 6, 189-96. (8) Veldhoen, S., Laufer, S. D., Trampe, A., and Restle, T. (2006) Cellular delivery of small interfering RNA by a non-covalently attached cell-penetrating peptide: quantitative analysis of uptake and biological effect. Nucleic Acids Res. 34, 6561-73. (9) Johansson, H. J., El-Andaloussi, S., Holm, T., Mae, M., Janes, J., Maimets, T., and Langel, U. (2008) Characterization of a novel cytotoxic cell-penetrating peptide derived from p14ARF protein. Mol Ther. 16, 115-23. (10) Johansson, H. J., Andaloussi, S. E., and Langel, U. (2011) Mimicry of protein function with cell-penetrating peptides. Methods Mol Biol. 683, 233-47. (11) Kim, M., Kim, M., Kim, H. Y., Kim, S., Jung, J., Maeng, J., Chang, J., and Lee, K. (2011) A protein transduction domain located at the NH2-terminus of human translationally



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

controlled tumor protein for delivery of active molecules to cells. Biomaterials. 32, 222-30. (12) Krauss, U., Muller, M., Stahl, M., and Beck-Sickinger, A. G. (2004) In vitro gene delivery by a novel human calcitonin (hCT)-derived carrier peptide. Bioorg Med Chem Lett. 14, 51-4. (13) Peng, T., Liu, Y. H., Yang, C. L., Wan, C. M., Wang, Y. Q., and Wang, Z. R. (2004) A new peptide with membrane-permeable function derived from human circadian proteins. Acta Biochim Biophys Sin (Shanghai). 36, 629-36. (14) Choi, J. M., Ahn, M. H., Chae, W. J., Jung, Y. G., Park, J. C., Song, H. M., Kim, Y. E., Shin, J. A., Park, C. S., Park, J. W., Park, T. K., Lee, J. H., Seo, B. F., Kim, K. D., Kim, E. S., Lee, D. H., Lee, S. K., and Lee, S. K. (2006) Intranasal delivery of the cytoplasmic domain of CTLA-4 using a novel protein transduction domain prevents allergic inflammation. Nat Med. 12, 574-9. (15) Duchardt, F., Ruttekolk, I. R., Verdurmen, W. P., Lortat-Jacob, H., Burck, J., Hufnagel, H., Fischer, R., van den Heuvel, M., Lowik, D. W., Vuister, G. W., Ulrich, A., de Waard, M., and Brock, R. (2009) A cell-penetrating peptide derived from human lactoferrin with conformation-dependent uptake efficiency. J Biol Chem. 284, 36099-108. (16) Jones, S., Holm, T., Mager, I., Langel, U., and Howl, J. (2010) Characterization of bioactive cell penetrating peptides from human cytochrome c: protein mimicry and the development of a novel apoptogenic agent. Chem Biol. 17, 735-44. (17) Rusnati, M., Urbinati, C., Caputo, A., Possati, L., Lortat-Jacob, H., Giacca, M., Ribatti, D., and Presta, M. (2001) Pentosan polysulfate as an inhibitor of extracellular HIV-1 Tat. J Biol Chem. 276, 22420-5. (18) Suzuki, T., Futaki, S., Niwa, M., Tanaka, S., Ueda, K., and Sugiura, Y. (2002) Possible existence of common internalization mechanisms among arginine-rich peptides. J Biol Chem. 277, 2437-43. (19) Wadia, J. S., Stan, R. V., and Dowdy, S. F. (2004) Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat Med. 10, 310-5. (20) Tyagi, M., Rusnati, M., Presta, M., and Giacca, M. (2001) Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans. J Biol Chem. 276, 3254-61. (21) Ellerby, H. M., Arap, W., Ellerby, L. M., Kain, R., Andrusiak, R., Rio, G. D., Krajewski, S., Lombardo, C. R., Rao, R., Ruoslahti, E., Bredesen, D. E., and Pasqualini, R. (1999) Anti-cancer activity of targeted pro-apoptotic peptides. Nat Med. 5, 1032-8. (22) Nieto, N., Friedman, S. L., Greenwel, P., and Cederbaum, A. I. (1999) CYP2E1-mediated oxidative stress induces collagen type I expression in rat hepatic stellate cells. Hepatology. 30, 987-96. (23) Wang, H., Zhong, C. Y., Wu, J. F., Huang, Y. B., and Liu, C. B. (2010) Enhancement of TAT cell membrane penetration efficiency by dimethyl sulphoxide. J Control Release. 143, 64-70. (24) Sheng, J., Oyler, G., Zhou, B., Janda, K., and Shoemaker, C. B. (2009) Identification and characterization of a novel cell-penetrating peptide. Biochem Biophys Res Commun. 382, 236-40. (25) Drin, G., Cottin, S., Blanc, E., Rees, A. R., and Temsamani, J. (2003) Studies on the internalization mechanism of cationic cell-penetrating peptides. J Biol Chem. 278,


Page 16 of 18

Page 17 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


31192-201. (26) Lewis, H. D., Husain, A., Donnelly, R. J., Barlos, D., Riaz, S., Ginjupalli, K., Shodeinde, A., and Barton, B. E. (2010) Creation of a novel peptide with enhanced nuclear localization in prostate and pancreatic cancer cell lines. BMC Biotechnol. 10, 79. (27) Montrose, K., Yang, Y., Sun, X., Wiles, S., and Krissansen, G. W. (2013) Xentry, a new class of cell-penetrating peptide uniquely equipped for delivery of drugs. Sci Rep. 3, 1661. (28) Montrose, K., Yang, Y., and Krissansen, G. W. (2014) The tetrapeptide core of the carrier peptide Xentry is cell-penetrating: novel activatable forms of Xentry. Sci Rep. 4, 4900. (29) Zaro, J. L., Vekich, J. E., Tran, T., and Shen, W. C. (2009) Nuclear localization of cell-penetrating peptides is dependent on endocytosis rather than cytosolic delivery in CHO cells. Mol Pharm. 6, 337-44. (30) Ma, D. X., Shi, N. Q., and Qi, X. R. (2011) Distinct transduction modes of arginine-rich cell-penetrating peptides for cargo delivery into tumor cells. Int J Pharm. 419, 200-8. (31) Lundin, P., Johansson, H., Guterstam, P., Holm, T., Hansen, M., Langel, U., and S, E. L. A. (2008) Distinct uptake routes of cell-penetrating peptide conjugates. Bioconjug Chem. 19, 2535-42. (32) Jones, A. T. (2008) Gateways and tools for drug delivery: endocytic pathways and the cellular dynamics of cell penetrating peptides. Int J Pharm. 354, 34-8. (33) Kong,

H.

J.,

and

Mooney,

D.

J.

(2007)

Microenvironmental

regulation

of

biomacromolecular therapies. Nat Rev Drug Discov. 6, 455-63. (34) Gautam, A., Chaudhary, K., Kumar, R., Sharma, A., Kapoor, P., Tyagi, A., Open source drug discovery, c., and Raghava, G. P. (2013) In silico approaches for designing highly effective cell penetrating peptides. J Transl Med. 11, 74. (35) Kaur, H., Garg, A., and Raghava, G. P. (2007) PEPstr: a de novo method for tertiary structure prediction of small bioactive peptides. Protein Pept Lett. 14, 626-31. (36) Willard, L., Ranjan, A., Zhang, H., Monzavi, H., Boyko, R. F., Sykes, B. D., and Wishart, D. S. (2003) VADAR: a web server for quantitative evaluation of protein structure quality. Nucleic Acids Res. 31, 3316-9. (37) Ma, J. L., Wang, H., Wang, Y. L., Luo, Y. H., and Liu, C. B. (2015) Enhanced Peptide Delivery into Cells by Using the Synergistic Effects of a Cell-Penetrating Peptide and a Chemical Drug to Alter Cell Permeability. Mol Pharm. 12, 2040-8.



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Highly Efficient Delivery of Functional Cargoes by a Novel Cell

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