The six amino acid antimicrobial peptide bLFcin6 penetrates cells and delivers siRNA

Authors

  • Bing Fang,

    1. Key Laboratory of Functional Dairy, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China
    2. Beijing Higher Institution Engineering Research Center of Animal Product, China
    3. Beijing Key Laboratory of Nutrition, Health & Food Safety, China
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  • Hui Y Guo,

    1. Key Laboratory of Functional Dairy, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China
    2. Beijing Key Laboratory of Nutrition, Health & Food Safety, China
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  • Ming Zhang,

    1. Key Laboratory of Functional Dairy, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China
    2. Beijing Higher Institution Engineering Research Center of Animal Product, China
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  • Lu Jiang,

    1. Key Laboratory of Functional Dairy, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China
    2. Beijing Higher Institution Engineering Research Center of Animal Product, China
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  • Fa Z Ren

    Corresponding author
    1. Beijing Higher Institution Engineering Research Center of Animal Product, China
    2. Beijing Key Laboratory of Nutrition, Health & Food Safety, China
    • Key Laboratory of Functional Dairy, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China
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Correspondence

F. Z. Ren, College of Food Science & Nutritional Engineering; China Agricultural University, PO Box 287, No. 17 Qinghua East Road, Haidian, Beijing 100083, China

Fax: +86 10 62736344

Tel.: +86 10 62736344

E-mail: renfazheng@263.net

Abstract

Cell-penetrating peptides (CPPs) are a new class of vectors with high pharmaceutical potential to deliver bioactive cargos into cells. Here, we characterized bLFcin6, a six amino acid peptide derived from bovine lactoferricin, as a CPP. Uptake of bLFcin6 was measured by flow cytometry. The ability to delivery siRNA was analyzed in HeLa cells. bLFcin6 exhibited concentration-dependent uptake and intracellular distribution. Below 7.5 μm, uptake of bLFcin6 was significantly lower than uptake of TAT (P < 0.05) because bLFcin6 has fewer cationic amino acids. Compared to CPP5 (RLRWR) and CPP6 (PFVYLI), bLFcin6 had a significantly higher internalization ratio above 2.5 μm because it has two tryptophan residues. Uptake of bLFcin6 starts with an ionic cell-surface interaction. It is then rapidly internalized by lipid raft-dependent macropinocytosis, followed by release from macropinosomes into the cytosol and nucleus. Moreover, bLFcin6 formed stable electrostatic complexes with siRNA and delivered siRNA into cells, resulting in significant knockout activity at both the mRNA and protein levels. The knockout activity of siRNA delivered by bLFcin6 was similar to that mediated by TAT, although knockout by bLFcin6 required a higher molar ratio. In this study, bLFcin6 was tested for its ability to act as an siRNA-delivering CPP.

Abbreviations
AMP

antimicrobial peptides

bLFcin

bovine lactoferricin

bLFcin6

residues 4-9 of bLFcin

CPP

cell-penetrating peptides

5-FAM

5-carboxyfluorescein

GAPDH

glyceraldehyde phosphate dehydrogenase

Introduction

Cell-penetrating peptides (CPPs) typically have 5–30 amino acids, and have the ability to traverse cell membranes at low concentrations [1, 2]. CPPs are widely used in the delivery of peptides [3], proteins [4], oligonucleotides [5], plasmid DNA [6], liposomes [7, 8] and iron beads [9], both in vitro and in vivo. Several CPPs are the subject of clinical trials [10].

CPPs have been identified and designed [3, 10-20] with varying amino acid compositions and secondary structures. Most CPPs, such as TAT [14], penetratin [15] and polyarginines [16], have a net positive charge. Although the minimum positive net charge of polyargineines for efficient cell penetration is +8 [12, 16], the shortest CPP is CPP5(RKRWR) [21]. Amphipathic CPPs such as pVEC [17], transportan [13], MPG [18] and Pep-1 [19] have both cationic and hydrophobic amino acids. VPTLK and VPALR, which contain only a single cationic amino acid, have been reported to have cell-penetrating properties [22, 23]. This indicates that hydrophobic amino acids such as tryptophan (Trp) are also important in cell penetration. A few hydrophobic CPPs have been discovered, such as Pep-7 [20] and PFVYLI [24], which contain only apolar residues and have a low net charge.

CPPs are derived from natural proteins, signal peptides or antimicrobial peptides (AMP) [10] that interact strongly with cell membranes and influence membrane permeabilization [25]. AMPs and CPPs have consistent functional characteristics. CPPs show antimicrobial activity because of their positive charge [26], and AMPs have been reported to be taken up into mammalian cells [25]. Several AMP-derived CPPs have been identified, including Bac7 [27], pyrrhocoricin [28], crotamine [29], melittin [30] and human lactoferrin(19-40) [31]. The penetrating activity of these AMPs is reported to be due to their positive and aromatic amino acids [27-31].

Bovine lactoferricin (bLFcin) is an AMP derived from pepsin digestion of bovine lactoferrin, and corresponds to amino acids 17-41 [11]. bLFcin shows activity against a wide range of microorganisms [32, 33], and has been reported to be the antimicrobial center of lactoferrin [34]. One of the microbicidal mechanisms of bLFcin is membrane perturbation [33, 35]. bLfcin has five arginine (Arg) and three lysine (Lys) residues that confer a net charge of +8. The strong cationic nature of Arg may enable it to form multiple hydrogen bonds with anionic membranes [36]. In addition, an aromatic Trp and a phenylalanine (Phe) residue provide a hydrophobic motif for membrane interaction [37]. The presence of Trp allows bLFcin to interact with membrane lipid bilayers [38]. The amphipathic nature of bLFcin, with its net positive charge and several hydrophobic residues, is common to other non-AMP-derived CPPs [39-41].

Residues 4-9 of bLFcin (hereafter bLFcin6), with the amino sequence Arg-Arg-Trp-Gln-Trp-Arg, are responsible for the majority of its antimicrobial activity [34, 42-44]. Replacement of Arg with Lys and of Trp with Phe or Tyr considerably reduces the antimicrobial activity of bLfcin [44, 45]. In this study, we evaluated bLFcin6 as a new CPP. The cell-penetrating and siRNA delivery capacity of bLFcin6 was tested and compared to that of other CPPs. The intracellular distribution and penetrating mechanism of bLFcin6 was evaluated. The six amino acid bLFcin6 is easy to synthesize, and its ability to deliver siRNA gives it potential for peptide therapy.

Results

Cellular uptake

The internalization efficiency of bLFcin6, CPP5, CPP6 and TAT were measured by flow cytometry using HeLa cells (Fig. 1). All four peptides entered the cells in a concentration-dependent manner (Fig. 1B). The internalization ratio of bLFcin6 below 7.5 μm was significantly lower than that of TAT (< 0.05). However, when compared to CPP5 and CPP6, bLFcin6 exhibited a higher internalization ratio, which became significant above 2.5 μm (< 0.05).

Figure 1.

Internalization ratio of bLFcin6, CPP5, CPP6 and TAT in HeLa cells. (A) Flow cytometry histograms of untreated cells (top) and cells incubated with 10 μm bLFcin6 (bottom). The arrows indicate the proportion of cells showing efficient peptide uptake. (B) Internalization ratio of cells incubated with 1–20 μm bLFcin6 (diamond), CPP5 (triangle), CPP6 (square) and TAT (circle). Error bars correspond to the standard error of three independent experiments. Significant differences were determined by Student's t test. Asterisks indicate a statistically significant difference for bLFcin6 versus CPP5 and CPP6 at = 0.05. The crossed-out open diamond symbols indicate a statistically significant difference for bLFcin6 versus TAT at = 0.05.

Cytotoxicity

The cell toxicity of the peptides was assessed on HeLa cells using methylene blue (Fig. 2A) and methyl thiazolyl tetrazolium assays (Fig. 2B). No significant decrease in cell viability or cell proliferation was seen after 48 h of incubation with bLFcin6, CPP5, CPP6 or TAT (> 0.05).

Figure 2.

Cytotoxicity of bLFcin6 (diamond), CPP5 (triangle), CPP6 (square) and TAT (circle) in HeLa cells. HeLa cells were incubated with various concentrations of CPPs for 48 h. (A) Cell viability measured by the methylene blue assay. (B) Cell proliferation determined using the MTT assay.

Delivery of siRNA

The ability of bLFcin6, CPP5, CPP6 and TAT to form an electrostatic complex with siRNA was monitored by agarose gel-shift assays (Fig. 3A). For bLFcin6, CPP5 and TAT, formation of stable CPP/siRNA complexes was initiated at a 1 : 1 molar ratio (CPP:siRNA), and increased with increasing concentrations of CPP. At a molar ratio of 40 : 1, siRNA molecules were entirely associated with bLFcin6, and no free siRNA was detected on agarose gels. TAT and CPP5 gave similar results at molar ratios of 40 : 1 (TAT) and 80 : 1 (CPP5). CPP6 did not form electrostatic complexes at any tested molar ratios.

Figure 3.

Delivery of siRNA into HeLa cells by bLFcin6, CPP5, CPP6 and TAT. (A) CPP/siRNA electrostatic complexes formed at various molar ratios were tested by agarose gel-shift assay. Lane 1, siRNA alone; lanes 2–12, CPP/siRNA complexes at molar ratios of 1, 2, 4, 8, 16, 20, 30, 40 and 60. (B) Internalization of 5-FAM-labeled siRNA delivered into HeLa cells by bLFcin6 (diamond), CPP5 (triangle), CPP6 (square) and TAT (circle). Significant differences were determined by Student's t test. Asterisks indicate a statistically significant difference for bLFcin6 versus CPP5 and CPP6 at = 0.05. The crossed-out open diamond symbols indicate a statistically significant difference for bLFcin6 versus TAT at = 0.05. (C) Distribution of bLFcin6/siRNA complexes at various molar ratios in HeLa cells. Molar ratio = 20 (left), 40 (middle) and 60 (right). Each image is composed of two single channels and an overlay channel: red, propidium iodide stain; green, 5-FAM labled-siRNA; yellow, co-localization of red and green. Scale bar corresponds to 20 μm.

The ability to deliver siRNA into HeLa cells was evaluated using 5-carboxyfluorescein (5-FAM)-labeled siRNA. A fixed concentration of siRNA was incubated with bLFcin6, CPP5, CPP6 or TAT at molar ratios ranging from 1 : 1 to 80 : 1 (CPP:siRNA). Internalization ratios were monitored by flow cytometry. As shown in Fig. 3B, cellular uptake efficiency was directly related to the CPP:siRNA molar ratio. The internalization ratio increased with the bLFcin6:siRNA ratio up to a ratio of 80 : 1. CPP5 and TAT also delivered siRNA into cells, with maximum internalization ratios at molar ratios of 100 : 1 for CPP5 and 60 : 1 for TAT. CPP6 did not delivery siRNA into HeLa cells at any of the tested CPP concentrations.

The cellular distribution of bLFcin6/siRNA complexes was monitored by confocal fluorescence microscopy (Fig. 3C). As the molar ratio increased, siRNA was transferred from the cytoplasm to the nucleus and the total intensity increased. At a molar ratio of 20 : 1 (CPP : siRNA), the siRNA was localized predominantly in the cytoplasm, while, at 40 : 1, siRNA started to be transferred into the nucleus. At 60 : 1, siRNA was mainly localized in the nucleus, which is where siRNA targets mRNA and gene silencing activity occurs.

Knockout activity of the delivered siRNA

The knockout activity of anti-glyceraldehyde phosphate dehydrogenase (GAPDH) siRNA delivered into HeLa cells by bLFcin6, CPP5 or TAT was investigated at the mRNA level (Fig. 4A) and the protein level (Fig. 4B). Lipofectamine 2000 served as a positive control. The three CPPs all induced significant knockout activity in a time-dependent way. As shown in Fig. 4A, GAPDH-targeting siRNA delivered by bLFcin6 resulted in a 33% decrease in mRNA after 24 h and a 48% decrease after 48 h. This was a significantly greater effect than seen for siRNA delivered by CPP5 (< 0.05). CPP5-mediated delivery resulted in a 22% decrease in mRNA after 24 h and a 37% decrease after 48 h. The positive control, Lipofectamine 2000, gave the best knockout activity, with a 49% decrease in mRNA after 24 h and a 40% decrease after 48 h. TAT showed similar knockout activity as bLFcin6, resulting in a 35% decrease after 24 h and a 46% decrease after 48 h. The knockout activity at the protein level (Fig. 4B) was similar to the results for mRNA.

Figure 4.

Knockout activity of anti-GAPDH siRNA delivered by bLFcin6, CPP5, TAT or Lipofectamine 2000. (A) Suppression of GAPDH mRNA in HeLa cells at 24 h (black) and 48 h (white) after treatment with 50 nm siRNA mediated by bLfcin6 (molar ratio of 60), CPP5 (molar ratio of 80), TAT (molar ratio of 40) or 5 μL Lipofectamine 2000. Suppression was normalized to β-actin mRNA levels; 0% suppression indicates the mRNA level in cells treated with negative control siRNA. Significant differences were determined by Student's t test. Asterisks indicate a statistically significant difference for bLFcin6 versus CPP5 and CPP6 at = 0.05. The crossed-out open diamond symbols indicate a statistically significant difference for bLFcin6 versus Lipofectamine 2000 at = 0.05. (B) Suppression of GAPDH protein in HeLa cells 24 and 48 h after treatment with 50 nm siRNA mediated by bLfcin6 (molar ratio of 60), CPP5 (molar ratio of 80), TAT (molar ratio of 40) or 5 μL Lipofectamine 2000.

Cell-penetrating mechanism of bLFcin6

The intracellular distribution of bLFcin6 in HeLa cells was investigated by confocal microscopy (Fig. 5A). At 1 μm, the peptide was localized predominantly in the cytoplasm. With increasing concentration, fluorescence was observed in the nucleus. When the concentration was above 10 μm, almost all cells showed a homogeneous cytoplasmic and nuclear distribution.

Figure 5.

Mechanism of bLFcin6 penetration. (A) Concentration-dependent distribution of bLFcin6. HeLa cells were incubated with various concentrations of FITC-labeled bLFcin6 for 30 min, and analyzed by confocal laser scanning microscopy. Scale bars correspond to 20 μm. (B–E) Internalization ratio of bLfcin in the presence of various endosomal pathway inhibitors, analyzed by flow cytometry. Cells were pre-treated for 30 min with 0–25 μg·mL−1 heparin (B), 0–5 mm methyl-β-cyclodextrin (C), 0–50 μg·mL−1 nystatin (D) or 0–200 μm chlorchinolin (E), and incubated with 10 μm FITC-labeled bLFcin6 for another 2 h. Values are means of three separate experiments. Significant differences were determined by Student's t test. Asterisks indicate (*= 0.05; **= 0.01).

The internalization ratio of bLFcin6 in the presence of internalization inhibitors was tested by flow cytometry (Fig. 5B–E). As shown in Fig. 5B, heparin strongly inhibited internalization in a dose-dependent way. Disruption of lipid rafts by either methyl-β-cyclodextrin or nystatin resulted in dose-dependent inhibition of internalization (Fig. 5C,D). Treatment with chloroquine resulted in a marked and dose-dependent increase in bLFcin6 internalization efficiency (Fig. 5E).

Discussion

The results of this paper characterize bLFcin6 as a new CPP. bLFcin6 comprises amino acids 4-9 of bLfcin (FKCRRWQWRMKKLGAPSITCVRRAF) [46]. The anti-parallel β-sheet structure of bLfcin enables it to interact with biological membranes [47]. Nuclear magnetic resonance studies showed that hydrophilic and positively charged residues surround a hydrophobic surface [47]. Although disulfide bonds have been reported to be essential for stabilizing the β-sheet structure of bLfcin, the shorter ‘active center’ bLFcin6 appears to govern the antimicrobial activity [48]. In addition, spectroscopic studies have shown that bLFcin6 causes membrane disturbances in model membranes similar to bLfcin [34]. Considering the structure similarity between AMPs and CPPs, bLFcin6 may be a potent CPP.

The cell-penetrating property of bLFcin6 was found to be concentration-dependent, similar to other CPPs such as TAT [17]. The internalization efficiency of bLFcin6 was lower than that of TAT (Fig. 1B), possibly because bLFcin6 has fewer Arg residues. Arg improves electrostatic cell-surface interactions and enhances CPP cellular entry [12, 16]. Studies on poly-Arg peptides (Arg3 to Arg12) have shown that increasing the number of Arg residues increases uptake [16]. The minimal number of positive charges for efficient uptake is reported to be eight [12], and the minimal sequence for cellular uptake is eight arginines [16]. Although positive charges are crucial for CPP uptake, other residues may also be important. Uptake of penetratin (RQIKIWFQNRRMKWKK) is abolished by mutation of the codon for Trp [49]. Therefore, we hypothesized that the higher internalization efficiency of bLFcin6 compared to CPP5 may be because bLFcin6 has more Trp residues. Trp is reported to have the ability to interact with lipid or cholesterol molecules within the membrane [50], making it essential for cellular uptake. Although Trp is hydrophobic because of its uncharged side chain, and may insert into membranes [51, 52], it is located towards the more hydrophilic side of lipid bilayers [10, 53]. This activity may favored the peptide entering membranes.

Although conjugation strategies using either transportan [54] or penetratin [55] improved their delivery into cultured cells, non-covalent electrostatic strategies using TAT [56] and MPG [57] appear to be more appropriate for siRNA delivery in vivo. Because of the positive charges on Arg, bLFcin6 forms stable complexes with siRNA. For this reason, hydrophobic CPP6 does not complex with siRNA. Trp is reported to be involved in stabilizing complexes in non-covalent contexts [51, 58]. This may be the reason why uptake of siRNA was higher when mediated by bLFcin6 than by CPP5 at the same molar ratio. Although CPP5 has one more positive net charge than bLFcin6, the additional Trp in bLFcin6 gave it better siRNA delivery ability. The formation of bLFcin6/siRNA complexes and consequent delivery into cells were directly associated with molar ratio (Fig. 3A,B). Although agarose gel-shift assays demonstrated that nearly all siRNA molecules formed complexes at a bLFcin6:siRNA molar ratio of 60 : 1, flow cytometry experiments showed that optimal cellular uptake was achieved at a molar ratio of 80 : 1. CPP5 and TAT showed the same results. These findings indicated that the delivery of siRNA requires positive charges, not only to neutralize the negative charge of siRNA but also to interact with the cell membrane. Moreover, the confocal laser scanning microscopy results demonstrated that cellular uptake increased with the molar ratio of CPP to siRNA, but, at ratios above 60 : 1 (Fig. 3C), the internalized siRNA aggregated in the nucleus.

We showed that bLFcin6-mediated siRNA delivery was associated with significant knockdown of the target gene at both the mRNA and protein levels (Fig. 4A,B,E). bLFcin6-mediated siRNA delivery showed knockdown activity similar to that of TAT, which has more Arg residues. Moreover, the knockdown activity at the protein level for siRNA delivered by bLFcin6 was similar to that for Lipofectamine 2000, although bLFcin6-mediated mRNA knockdown activity was lower than that for Lipofectamine 2000 (< 0.05). The siRNA binding and penetrating ability of bLFcin6 may be related to its ability to interfere with RNA and DNA synthesis [59-61]. Similar results were obtained for adherent cell lines (HeLa, HepG2, HT29, Tca, MCF, L02), suspension cell lines (HL60) and primary cell lines (HUVEC) (data not shown). Together, these results indicate that bLFcin6 is capable of delivering siRNA and inducing gene silencing in a variety of mammalian cells.

To better define bLFcin6 as a CPP, the cellular entry mechanism of bLFcin6 was investigated in the presence of various inhibitors of the uptake pathway. The generally accepted model is that, when CPPs interact with cell membranes, an initial electrostatic interaction is followed by association of hydrophobic amino acids with the hydrophobic core [62, 63]. In this study, cells were first treated with heparin, a known extracellular glycosaminoglyan [64] that neutralizes positive charges, and uptake of bLFcin6 was measured. Pre-treatment with heparin resulted in a decrease of bLFcin6 internalization (Fig. 5B), similar to other CPPs [65-69]. These results indicate that cell-surface binding, probably by electrostatic interaction between Arg residues of bLFcin6 and negatively charged cell-surface constituents, is necessary before internalization.

Endocytosis, an essential cellular process for internalization of a wide variety of extracellular factors, occurs through functionally distinct mechanisms [70]. Removal of cholesterol from the plasma membrane disrupts several lipid raft-mediated endocytic pathways [65, 71, 72]. Treatment with β-cyclodextrin or nystatin depletes or sequesters cholesterol [65]. A decrease in the internalization ratio (Fig. 5C,D) showed that internalization of bLFcin6 requires interaction with lipid rafts in the cell membrane in a receptor-independent manner. After entering the membrane, cargos are enveloped into vesicles termed macropinosomes [65, 70-72]. Chloroquine is an ion-transporting ATPase inhibitor that prevents macropinosome acidification [73]. Uptake of bLFcin6 was significantly enhanced after treatment with chloroquine (Fig. 5E), indicating that uptake of bLFcin6 requires release from macropinosomes. Taken together, these results indicate that, after rapid binding to the negatively charged membrane and lipid raft-mediated endocytic internalization, followed by destabilization of the integrity of the macropinosome vesicle lipid bilayer, the bLFcin6 cargo was released into the cytoplasm for nuclear transport.

In summary, the results presented here show that the bLFcin6 peptide is a new CPP of short length with siRNA-delivering ability. Investigations of CPP have attracted attention during the last decade, achieving delivery of biologically active cargoes even to live animals [3-9]. Application of a novel CPP to deliver macromolecules and drugs against a range of diseases such as cancer may allow the development of more effective therapeutic compounds. The bLFcin6 peptide described in this study may be a promising CPP for these purposes, as it has siRNA transfer ability without immunogenicity or toxicity. Although additional studies are required to characterize the potential of bLfcin6 for in vivo nucleic acid delivery, the features of bLFcin6 are consistent with several key requirements for such applications.

Experimental procedures

Materials

1-hydroxybenzotriazole, thioanisole, N,N-diisopropylcarbodiimide, ethanedithiol, trifluoroacetic acid, triisopropylsilane, diisopropylethylamine and all Fmoc-protected amino acids were obtained from GL Biochem (Shanghai, China). Wang resin was obtained from Tianjin Nankai Hecheng Science & Technology Co. Ltd (Tianjin, China). HPLC-grade methanol and acetonitrile were obtained from Fisher Scientific (Hanover Park, IL, USA). All other reagents were of analytical grade.

Peptide synthesis and purification

bLFcin6 (RRWQWR), CPP5 (RLRWR), CPP6 (PFVYLI) and TAT (RKKRRQRRR) [with or without fluorescein isothiocyanate (FITC)] were purified by semi-preparative HPLC after solid-phase synthesis. FITC was coupled to the N-terminus of each peptide before cleavage by the same procedure. The peptide purity was confirmed to be above 95% by reversed-phase HPLC using a C18 column (Kramsil (AkzoNobel, Separation Products, Shanghai, China); internal diameter 4.6 mm, length 250 mm). Peptides were verified by electrospray mass spectrometry (Agilent 1100 series LC/MSD).

Cell cultures

HeLa cells were from the American Type Culture Collection (Manassas, VA, USA), and were maintained in high-glucose Dulbecco's modified Eagle's medium supplemented with 20 U·mL−1 penicillin, 20 μg·mL−1 streptomycin, 2 mm l-glutamine and 10% fetal bovine serum (Gibco/Invitrogen, Beijing, China). Cells were grown at 37 °C in an atmosphere of 5% CO2.

Flow cytometry

Cells were seeded in 12-well culture plates in 1 mL medium at 1 × 105 cells per well and incubated at 37 °C. After 24 h, medium was removed and the cells were washed with NaCl/Pi (10 mm, pH 7.4). To investigate the internalization of bLFcin6, CPP5, CPP6 and TAT, FITC-labeled peptides were dissolved in ultrapure water (Merck Millipore, Billerica, MA, USA), added to wells at final concentrations of 1, 2.5, 5, 7.5, 10 and 20 μm, and incubated for 2 h. To study the delivery ability of bLFcin6, CPP5, CPP6 and TAT, cells were treated with the indicated CPP:siRNA concentrations for 5 h at 37 °C. For experiments using uptake inhibitors, cells were pre-incubated with inhibitors in serum-free medium for 30 min at 37 °C, followed by addition of 10 μm bLFcin6 and 2 h incubation at 37 °C. After washing by NaCl/Pi for three times at room temperture, each time 3 min, cells were centrifuged (4° C, 1000 g, 5 min) and re-suspended in 300 μl NaCl/Pi. Cell pellets were kept on ice until measurements were performed. Immediately before analysis, 8 μL 0.5% trypan blue per 0.5 mL volume was added to reduce extracellular fluorescence. Cells were analyzed using a FACSCalibur flow cytometer (Beckton Dickinson, CA, USA) equipped with an FLT 1 FITC signal detector (excitation = 488 nm). Viable cells for analysis were gated by dot plots based on sideward and forward scatter. A minimum of 15 000 events per sample was analyzed. Each experiment was performed in duplicate or triplicate.

Confocal laser scanning microscopy

HeLa cells were seeded in glass-bottomed culture dishes and grown to 60% confluence. After treatment with bLFcin6 or bLFcin6/siRNA(5-FAM labled) complexes, cells were washed with NaCl/Pi (3 min for 3 times at room temperature) to remove surface-associated FITC, and fixed with 4% formaldehyde at 4 °C for 15 min. Cells were stained with propidium iodide and imaged using a LSM710 laser scanning inverted microscope (Zeiss, Göttingen, Germany) after washing. FITC was excited using 488 nm light from an argon ion laser (Melles-Griot), and propidium iodide was excited using a 633 nm helium/neon laser (Melles-Griot). Untreated cells were used as a negative control.

Cell viability assay

Cells were seeded in 96-well plates at 1 × 104 cells per well and grown for 24 h. Medium was removed, and 5, 10, 15, 20, 40, 60 or 80 μm bLFcin6, CPP5, CPP6 or TAT were added to new medium at the indicated final concentrations. Untreated cells were used as a negative control. After 48 h, cell viability was tested by a methylene blue assay [74].

Cell proliferation assay

The cell proliferation of HeLa cells after treatment with bLFcin6, CPP5, CPP6 or TAT was investigated. Cells were plated in 96-well plates at 1 × 104 cells per well in medium in the presence or absence of 5, 10, 15, 20, 40, 60 or 80 μm bLFcin6, CPP5, CPP6 or TAT. Following a 48 h incubation, a final concentration of 0.5 mg·mL−1 MTT (Sigma, St Louis, MO, USA) dissolved in NaCl/Pi was added to each well for a 4 h incubation. The attenuance at 490 nm (D490) was measured, and cell viability was expressed as the D490 for cells treated with complex divided by the D490 for control samples, as a percentage. Values shown are the means of three separate experiments.

Agarose gel-shift assay

Anti-GAPDH siRNA was incubated for 30 min at 37 °C in NaCl/Pi with bLFcin6, CPP5, CPP6 or TAT at molar ratios between 1 and 80 : 1 (1, 2 : 1, 4 : 1, 8 : 1, 16 : 1, 20 : 1, 30 : 1, 40 : 1, 60 : 1 and 80 : 1). Pre-formed complexes were analyzed by electrophoresis on agarose gels (1.5% w/v) and stained with ethidium bromide.

Delivery of siRNA into cells

The siRNA that targets the GAPDH gene was incubated for 30 min at 37 °C with bLFcin6, CPP5 and TAT at a molar ratio of 60 : 1 (CPP:siRNA). Lipofectamine 2000 (Invitrogen, Beijing, China) was used as a positive control, with 5 μL Lipofectamine 2000 being mixed with 20 μm siRNA and incubated at 37 °C for 20 min. Solutions were added to the cells for a final concentration of 50 nm siRNA per well. Cells were seeded in six-well plates and grown in complete culture medium for 24 h at 37 °C. Medium was removed, cells were washed once by NaCl/Pi for 3 min at room temperature, and fresh medium was added. Anti-GAPDH siRNA transfer complexes were added to cells and incubated for 5 h. Cells were washed and fresh medium was added for 24 or 48 h at 37 °C. GAPDH mRNA and protein expression were analyzed by RT-PCR and western blot. Complexes of non-specific siRNA were prepared as described above.

RNA isolation and quantitative PCR analysis

Cells were lysed using TRIzol (Qiagen, Shanghai, China) after washing, and RNA was isolated according to the manufacturer's instructions for cultured cells. Isolated RNA was reverse-transcribed using a PrimeScript 1st Strand cDNA synthesis kit (Takara, Dalian, China). Products were stored at −80 °C until use. For real-time PCR, cDNA was added to SYBR Green MasterMix (Applied Biosystems, Foster City, CA, USA). Quantitative PCR reactions were run in triplicate on a Techne Quantica PCR system using the program 5 min at 95 °C, 45 cycles of 15 s at 95 °C, 30 s at 60 °C and 1 min at 72 °C, then 5 s at 80 °C. Amplification was quantified during the 72 °C step. Dissociation curves were obtained by subjecting samples to 1 min at 95 °C, 30 s at 55 °C and 30 s at 95 °C, and monitoring fluorescence during heating from 65 to 95 °C. Expression ratios were calculated relative to β-actin according to an efficiency-corrected relative quantification model [75]. The primer sequences used were 5′-GGATCCGACTTCGAGCAAGAGATGGCCAC-3′ (forward) and 5′-CAATGCCAGGGTACATGGTGGTG-3′ (reverse) for β-actin, and 5′-GAAGGTGAAGGTCGGAGTC-3′ (forward) and 5′-GAAGATGGTGATGGGATTTC-3′ (reverse) for GAPDH.

Western blotting

At 24 and 48 h after treatment with anti-GAPDH siRNA/bLFcin6 complexes, cells were lysed using RIPA buffer (Cell Signaling Technology, Shanghai, China) containing the protease inhibitor phenylmethanesulfonyl fluoride (Beyotime, Beijing, China) for 10 min on ice. Cell lysates were collected and total protein was tested using a Micro BCA assay (Pierce, Rockford, IL, USA). After SDS/PAGE, protein samples were transferred by electroblotting onto a poly(vinylidene difluoride) membrane (Millipore, Billerica, MA, USA). Membranes were blocked using 5% skim milk in PBST buffer (NaCl/Pi with 0.1% Tween-20) at room temperature for 1 h with constant shaking. Membranes were incubated with mouse monoclonal GAPDH antibody (Novus Littleton, CO, USA) and horseradish peroxidase-labeled goat anti-mouse secondary antibody (Novus Littleton, CO, USA). After washing with PBST, membranes were detected using an ECL Plus kit (Millipore, Billerica, MA, USA), and imaged using X-ray film (Kodak, Beyotime, Beijing, China).

Cellular entry mechanism of bLfcin6

To measure the internalization mechanism of bLFcin6, HeLa cells were seeded at 1 × 105 cells per well in 12-well plates. Cells were treated in serum-free medium for 30 min with 0–25 μg·mL−1 heparin (Sigma, St. Louis, MO, USA), 0–5 mm methyl-β-cyclodextrin (Sigma, St. Louis, MO, USA), 0–50 μg·mL−1 nystatin (Fluka, St. Gallen, Switzerland) or 0–200 μm chlorchinolin (Sigma, St. Louis, MO, USA) at 37 °C. Cells were incubated for 30 min with 5 μm FITC-labeled bLFcin6 in the presence of endocytosis inhibitors. After 1 h incubation, cells were washed, trypsinized, centrifuged, re-suspended in NaCl/Pi and analyzed by flow cytometry.

Statistics

Statistical differences between bLFcin6, CPP5, CPP6 and TAT internalization were determined independently for the various culture conditions using Student's t-test. Comparisons were performed using one-way ANOVA followed by Duncan's post-hoc tests.

Acknowledgments

We gratefully acknowledge the financial support of the Ministry of Science and Technology of China (2011AA100903 and 2012BAD28B08), and the Beijing Science and Technology Project (D101105046010001).

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