http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–8 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2014.881439

ORIGINAL ARTICLE

Six-cell penetrating peptide-based fusion proteins for siRNA delivery Hua Li1,2 and TungYu Tsui1 Department of General, Visceral and Thoracic Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany and 2Department of Basic Medical Sciences, Huzhou University Schools of Medicine and Nursing Sciences, Huzhou, Zhejiang, China

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Abstract

Keywords

The siRNA deliveries, for the siRNA’s high negative charge, short serum half life, poor cellular internalization, etc, are still the key barriers for its application in clinic. In this study, several cell penetrating peptide (CPP) and dsRNA binding domain (dsRBD)-based fusion proteins have been developed and screened as the siRNA vector. The siRNA binding ability was measured by the agarose gel retardation, the cell uptaking was characterized under fluorescence microscopy, and further more RNAi effect was evaluated on the endogenous (GAPDH, western blot) and exogenous (GFP, flow cytometry analysis) genes in HeLa cell. Finally, the cytotoxicity was assessed on HeLa cells using cell counting kit-8. The efficiency of siRNA delivery by the CPP-dsRBD fusion protein was the CPP and the dsRBD dependent. Three fusion proteins showed similar efficiency of siRNA delivery when comparing to Lipofectamine RNAi Max as the siRNA carrier. These results indicated that these CPP-dsRBD-based fusion proteins were promising candidates as siRNA carriers.

Calcium, cell penetrating peptide, intracellular delivery, RNA interference, siRNA

Introduction Although the viral vector can deliver siRNA with high efficiency, a long time to produce the viral vector, high cost cytotoxicity, and others also limit its application in clinic. Except less efficiency in vivo (Li & Huang, 2007), the nonviral vector (compared to the viral vector) is a convenient delivery strategy with low toxicity and ease of large-scale production. Cell penetrating peptide (CPP) can transfer various cargos including protein (Choi et al., 2006; ElAndaloussi et al., 2007; Nakase et al., 2008), nuclear acid (Eguchi et al., 2001; Endoh & Ohtsuki, 2009), and the other drug vector (Koppelhus et al., 2008) into a range of cell types as well as the blood–brain barrier (Lindgren et al., 2000). However, the negative charge of siRNA may neutralize the positive charges of CPPs either by covalent attachment (Meade & Dowdy, 2007) or noncovalent conjugated (Veldhoen et al., 2006; Meade & Dowdy, 2008), and result to reduce efficiency of CPP. Until now, the siRNA delivery is still the key barrier for its application in clinic. DsRNA binding domain (dsRBD) was an important regulator of gene expression in many eukaryotes. It was approximately 70 amino acids in length with two a helices and three antiparallel b sheet (Bycroft et al., 1995; Kharrat et al., 1995; Nanduri et al., 1998; Ramos et al., 1999; Gan et al., 2005), and divided into two groups: group A dsRBD demonstrate a strong homology at their N terminals Address for correspondence: TungYu Tsui, Department of General, Visceral and Thoracic Surgery, University Medical Center HamburgEppendorf, Martinistr. 52, 20246 Hamburg, Germany. Tel: +4940-741050822. Fax: +49-40-7410 46756. Email: [email protected]

History Received 5 December 2013 Revised 6 January 2014 Accepted 6 January 2014

(Jantsch, 1996), whereas group B dsRBD are conserved only at the C terminals. The type A dsRBD could bind dsRNA with high affinity, but the type B is opposite (Doyle & Jantsch, 2003). Here, we have designed several fusion proteins, CPP linked to a dsRBD, as the siRNA carrier. In order to develop vectors with high efficiency and low cytotoxicity, different CPPs, including the extensively utilized CPP-Tat, efficiency transduction for primary T cell CPP-Hph1 (Choi et al., 2006, 2008), and the CPP-PTD4 were designed in the fusion protein and evaluated. The dsRBD was also designed from different proteins, such as Protein kinase R (PKR), Dicer and Escherichia coli Ribonuclease III. The dsRBD was replaced with the two repeat segments of CPP-MPG: MPG-MPG (Endoh & Ohtsuki, 2009) to investigate if the magnitude of dsRBD parts combining with siRNA molecules in the complex would influence siRNA delivery. Six fusion proteins, as shown in Table 1, were designed as CPP-dsRBD-based siRNA vector. In addition, calcium condensed step was also added to optimize siRNA delivery efficiency.

Materials and methods Materials The compete cell of Rosetta (DE3)pLysS and Top 10 were purchased from Novagen (Wisconsin, USA) and Invitrogen (Karlsruhe, Germany), respectively, the LB, IPTG, and imidazole, complete protease inhibitor, Ni-NTA column, and PD-10 column were from Fluka (Steinheim, Germany), Sigma (Steinheim, Germany), Roch (Mannheim, Germany), Qiagen (Hilden, Germany), Invitrogen (Karlsruhe, Germany)

H. Li & T.Y. Tsui

Tat, PTD4, and Hph-1 are CPP. dsRBD is the dsRNA binding domain from PKR. Dicer and E. coli RNase are the dsRBD from human Dicer and E. coli Ribonuclease III. MPG is also CPP.

M ar ke Pr r ot ei n 5 Pr ot ei n 7 Pr ot ei n 11

Tat-Tat-dsRBD Tat-Tat-Tat-dsRBD PTD4-PTD4-E. coli RNAse PTD4-PTD4-MPG-MPG Hph1-Hph1-Dicer Hph1-Hph1-E. coli RNAse

ot ei n

2 3 5 7 10 11

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Protein Protein Protein Protein Protein Protein

M ar

ke r

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Table 1. List of proposed recombinant proteins for siRNA delivery.

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individually. Fetal bovine serum (FBS), Dulbecco’s modified eagle’s medium (DMEM), dimethyl sulfoxide (DMSO), mercaptoethanol, penicillin/streptomycin, pyruvat, and glutamine were ordered from Invitrogen (Karlsruhe, Germany). The primers of GAPDH and R18S for real-time PCR, GAPDH siRNA (siGAPDH) were purchased from Qiagen (Hilden, Germany). The GFP siRNA (siGFP) and carboxyfluorescein-labeled siRNA (FAM-siRNA) was ordered from Ambion (Darmstadt, Germany). HeLa cells were from American type culture collection (ATCC), the GFP HeLa cells were prepared according to the instruction of pcDNA3.1+ from Invitrogen (Steinheim, Germany). CPP-dsRBD fusion proteins designing and production To obtain a series of our desired genes, the gene of TatdsRBD (from PKR), PTD4-PTD4-dsRBD (from Dicer), PTD4-PTD4-dsRBD (from E. coli RNAse), PTD4PTD4-MPG-MPG, and Hph1-Hph1-TAT-TAT-dsRBD (from PKR), CM9-TAT-dsRBD (from PKR)-PTD were initially synthesized from Eurofin (Ebensburg, Germany) and subcloned into the prokaryotic expression vector (PET28b+) with the restriction sites Nhe I and EcoRI. In order to obtain various diverse CPP-dsRBD fusion proteins, the restriction site Pst I was inserted between the domains of CPP and dsRBD (supplement Figure 1A). Based on theories of restriction digestion and ligation, the above target genes could be subcloned again between the restriction sites of Pst I and EcoRI as shown in supplement Figure 1B). Finally, the plasmid containing protein gene (2, 3, 5, 7, 8, 10, or11) was constructed. The constructed plasmids of the proteins 2, 3, and 10 were cloned into the Rosetta, while the proteins 5, 7, and 11 were transformed into BL21 (DE3) plysS strain. The protein are expressed and isolated as follows: cells were cultured at 37  C overnight, and 20 ml of overnight cells were transferred to 500 ml fresh LB medium and cultured 3 h at 25  C, then cells were induced with 500 mM IPTG for 6 h. Cells were centrifuged down (10 min, 8000g) and stored at 80  C for at least 12 h and the cells were then resuspended in the lysis buffer (50 mM NaH2PO4, 2 M NaCl, 1  protease inhibitor, and 20 mM imidazole, pH 7.9–8.3) plus 1 mg/ml lysozyme and sonicated at 60% energy for 5 min. As to the proteins 5, 7, 10, and 11, 8 M urea was added into the lysis buffer and sonicated at 60% energy for 5 min. The lysate was centrifuged at 50 000 g and 4  C for 20 min, the supernatant was loaded on the Ni-NTA column, the protein was further eluted with lysis buffer including 500 mM Imidazole. Purified protein was finally desalted (using PD10) into 100 mM NaCl including 10% glycerol and stored at 80  C.

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Figure 1. SDS Page analysis of the purity of the protein. Recombinant proteins (2 and 3) produced by Escherichia coli were purified by HisTrap from GE in the native condition. The other proteins (5, 7, 10, and 11) were purified via the denature method.

Preparation of protein/siRNA Protein/siRNA was prepared as described previously (Eguchi et al., 2009) with minor modification: various portions of protein/siRNA were mixed at different molar rates and incubated on ice for 30 min to allow formation of protein/ siRNA complex. Preparation of protein/siRNA condensed with CaCl2 Protein/siRNA was prepared as previous (Baoum et al., 2012) with slight modifications: various ratio of protein/siRNA were mixed at different molar rates, the 300 mM CaCl2 was then added to the final concentration of 23.1 mM, and finally were incubated on ice for 30 min. Agarose gel retardation Protein/siRNA (100 pmol siRNA was used) complexes (1/1, 5/1, 10/1, 20/1 mol/mol) were analyzed with gel retardation assay. Electrophoresis was performed on the 1% agarose gel containing 0.01% ethidium bromide and kept running at 65 V for 1–2 h in the RNase free TBE buffer. Any retardation of the complexes could be visualized under UV. Cell culture Culturing of HeLa cell, steadily expressing GFP, was performed in DMEM supplemented with 10% heat inactivated FBS, 1% penicillin/streptomycin, and 1.5 mg/ml gentamicin (G418) at 37  C and 5% CO2. The HeLa cell was cultured as the same protocol as the HeLa steadily expressing GFP, but the medium without the G418. Cell uptaking fluorescence microscopy About 100 ml  104 HeLa cells were preplated into the 96-well cell culture plates at one day before experiments, and then the media was aspirated and rinsed three times with fresh DMEM. After that, 80 ml fresh DMEM (combined with FAM-siRNA/protein complexes; final concentration of siRNA was 100 nM) was added to the cell for cell transfection. In order to check transfection efficiency, all samples

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were observed and photos were taken under Leica DMI4000 B Microscope (Leica, Wetzlar, Germany) at 1 h after transfection.

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Cell viability assay

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Statistical analysis All experiments were assayed in triplicate on at least three independent occasions to get accurate results. Experimental data were analyzed using Student’s t test.

The cytotoxicity of fusion protein was determined by Cell Counting kit-8. To begin with, cells were seeded into 96-well plates at the density of 1  104 HeLa cells/well in the complete growth medium and incubated for 24 h. Then, fusion proteins with different concentrations (30/1: 3 mM; 20/1: 2 mM; 1/1: 0.1 mM) were all diluted to a total volume of 20 ml with growth medium (without serum) and these solutions were added to the cell, which was incubated for another 48 h, followed by the addition of 10 ml Cell Counting Kit-8 solution to each well. After 1 h of incubation, cells were placed in the cell incubator at 37  C, cell viability were detected at 450 nm using InfiniteÕ M200 from Tecan (Ma¨nnedorf, Schweiz). Each concentration was triplicated for the sake of better accuracy in all experiments.

Results

Gene knocked down in vitro

Determining the siRNA-binding ability

For siRNA delivery into cells, the method was modified by Dowdy SF (Eguchi et al., 2009), explained as follows:100 ml (1  104) HeLa cells or GFP-expressing HeLa cells were preplated in 96 wells plate 24 h before experiments, the cells were rinsed using serum free-DMEM two times, then the protein/siRNA complex was added to the cells surrounding by the serum-free DMEM and coincubated for 4 h with final siRNA concentration at 50 nM (siGFP) or 100 nM (siGAPDH). Medium replacement was followed in replacing serum-free DMEM with fresh complete cell culture medium (containing of FBS), the efficiency of RNAi on the GFP was measured by flow cytometry at 48 h after treatment. And to the RNAi on the gene of GAPDH, cells were harvested for western blot analysis at 48 h after treatment.

The siRNA binding ability was screened by gel retardation assays, siRNAs were incubated with different protein at different molar ratio. Gel retardation results, as shown in Figure 2, exhibited that all six fusion proteins could efficiently associate with siRNA and the entirely siRNA binding was protein dependent: proteins 3, 10 and 11 exhibited the lowest molar ratio (5/1) of protein/siRNA, while protein 2 needed at rate of 10/1 and proteins 5 and 7 needed 20/1. This phenomenon might be decided based on different affinity abilities of protein to siRNA molecular, strength of positive charges, and the location of the binding site (Lee & Kim, 2010), the ability of siRNA binding with different CPPs (Mo et al., 2012).

Protein preparing Six fusion proteins with numbers 2, 3, 5, 7, 10, and 11 (the protein sequences were shown in the supplement) were expressed in Escherichia coli with one (proteins 5, 7, 10, and 11) or two his tags (proteins 2 and 3). The proteins 2 and 3 could bind and be isolated from the Ni-NTA column in the form of its native structure. The remaining proteins 5, 7, 10, and 11 could bind with high efficiency to the Ni-NTA in denature condition, instead of the native condition. All the proteins were eluted from His-TrapTM from GE, and immediately desalted using PD-10. The purities of these proteins were summarized in the Figure 1, as they were isolated with high purity.

Cell uptaking Western Blot The protein was first transferred from SDS-page gel to PVDF membrane using an electric current of 100 mA for 1 h. The western blotting was carried out according to the instruction of two-component kit -ECLTM Western Blot Detection Reagents from Invitrogen. The first antibody (anti-HSC70 and anti GAPDH) was used at a dilution ratio of 1/2000.

Figure 2. ?Gel retardation assays of protein 2, 3, 5, 7, 10, and 11 binding siRNA (100 pmol of siRNA) at different molar rates of protein/ siRNAs.

The cell uptaking of the complex of protein/siRNA was next detected under the florescence microscopy using fluorescently labeled siRNA molecules (FAM-siRNA). The fusion protein mediated cell uptaking of siRNA at ratio of 30/1 was analyzed and compared at 1 h after incubating with HeLa cell. As shown in Figure 3, proteins 3, 5, 10, and 11, except protein 2, could rapidly transfer siRNA, due to the low concentration of protein 7, it was not applied at a ratio of 30/1 with

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Protein 2/siRNA(30:1)

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Calcium condenced Protein 3/siRNA(1:1)

Protein 3/siRNA(30:1)

Calcium condenced Protein 5/siRNA(1:1)

Protein5/siRNA(30:1)

Calcium condenced Protein 7/siRNA(1:1)

Calcium condenced Protein 10/siRNA(1:1)

Protein 10/siRNA(30:1)

Figure 3. Fluorescent microscope analysis concerning protein delivery of siRNA into HeLa cell. Fluorescent images regarded proteins 2, 3, 5, 10, and 11 combined with FAM-siRNA (green) at a ratio of 30:1 without existence of CaCl2, respectively; or proteins 3, 5, 7, 10, and 11 with FAM-siRNA at a ratio of 1:1 condensed with 23.1 mM CaCl2, respectively. From left to right: figures were taken under bright field and green filter, and an overlay of the above was then presented. The scale bar was defined as 20 mm.

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Calcium condenced Protein 11/siRNA(1:1)

PBS with FAM-siRNA

Figure 3. Continued. (A)

(B) 100 90 80

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Protein11/siRNA(30:1)

70 60 50 40 30 20

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Figure 4. In vitro screening of proteins for siRNA delivery in GFP-expression HeLa cells. (A) The cells were treated with protein/siRNA complexes at mol rate of 20:1. (B) 48 h after treatment, the average reduction percentages of GFP expression in cells. These complexes of protein/siGFP were prepared at mol rate of 1:1 and condensed in the 23.1 mM CaCl2 buffer, as calculated from at least triplicate parallel samples and showed above (A, n ¼ 3; B, n ¼ 4). Asterisk (**) indicates p50.01 based on analysis of one sample t test and one-way ANOVA post-hoc multiple comparisons (LSD as the variances assumed).

FAM-siRNA. On the other hand, the use of calcium to condense protein/siRNA complex improved cell uptaking. Exogenous GFP knocked down in GFP-expression HeLa cells To investigate the RNA interference (RNAi) effect of protein/ siRNA, stably GFP expressing HeLa cell was transferred by protein (2, 3, 5, 7, 10, or 11)/siGFP, a ratio of 20/1. The suppression level of GFP expression was 5.63%, 42.7%, 9.3%, 36.37%, 70.4%, and 64.83% separately (Figure 4A). These results demonstrated that proteins 3, 5, 7, 10, and 11, except protein 2, were the efficient siRNA vectors. Furthermore,

compared to the commercially available reagents Lipofectamine RNAi Max (74.5 %), siGFP transferred by proteins 3, 7, 10, and 11 induced extremely similar or much higher RNAi’s efficiency on GFP gene. Notably, calcium condensed complex of protein (2, 3, 5, 7, 10, or 11)/siGFP exhibited significantly higher gene inhibition effect (40.2%, 63.8%, 52.88%, 86.45%, 86.2%, and 84.15%, respectively) even at molar ratio of 1/1 (Figure 4B). Endogenous gene knocked down in HeLa cell The RNAi effect was further studied on the endogenous gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

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Figure 5. Screening the efficiency of the recombinant protein siRNA delivery GAPDH siRNA on HeLa cell GAPDH. The Western blot was used to analyze the RNAi efficiency at protein/siRNA ratio of 1:1 condensed in the 23.1 mM CaCl2 or at a ratio of 30:1. Lipofectamine RNAi Max used as the positive control. After 48 h treatment, the expressions of GAPDH after siRNA delivery were shown in contrast to the expression of HSC70 in the control sample.

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Protein/siRNA 1/1 condensed with 23.1mM CaCl2 2

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GAPDH

50nM GAPDH siRNA with different amount Lipofectamine RNAi Max Negeve 0.6 1 1.5 0 µl siRNA

GAPDH

Figure 6. The viability of cell treatment of these recombinant proteins with siRNA. The OD 450 of cells was measured 48 h after the treatment with different amounts of proteins (0.1 mM, 2 mM, or 3 mM) as well as the protein amount used in the knockdown efficiency assay with protein/siRNA at a ratio of 1:1 combined with 23.1 mM CaCl2, of 20:1 or 30:1, respectively. The data are presented as mean ± standard deviation calculated from parallel triplicate samples (n ¼ 3).

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HSC 70

SiGAPDH, transferred by protein 3 or 11 at a ratio of 30/1 as well as the Lipofectamine RNAi Max, could efficiently inhibit GAPDH expression (Figure 5). In accordance with the cell uptaking result, the high cell uptake of protein 10/siRNA at a ratio of 30/1 led to cell death and low concentration of protein 7, these two proteins were not performed this section. Similar to exogenous GFP gene, endogenous gene GAPDH was significantly knocked down by calcium condensed complex of protein/siGAPDH even at a molar ratio of 1/1.

protein was used (30/1: 3 mM; 20/1: 2 mM; 1/1: 0.1 mM), notably without siRNA, was incubated with HeLa cell, as shown in Figure 6: compared to PBS group, proteins 2, 10, and 11 did not significantly affect cell proliferation at all these protein conditions. However, proteins 3, 5, and especially 7, inhibited proliferation ability at protein amount of 30/1 (3 mM). In consideration of the result, it revealed almost no evidence of cytotoxicity and cells maintained high viability, when treated with these fusion proteins or using calciumcondensed complex in HeLa cell.

Cell viability To investigate the cytotoxicity of these recombinant proteins, the viability of HeLa cells, using the Cell Counting Kit-8, were monitored 48 h after treatment. The same amount of

Discussions Naked siRNA is difficult to apply in pharmacokinetics research due to its easy degradation by RNase

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DOI: 10.3109/10717544.2014.881439

(Haupenthal et al., 2006), short serum half-life (Dykxhoorn et al., 2006; Raemdonck et al., 2008), negligible cellular internalization (Aliabadi et al., 2012), etc. The CPP, an efficient carrier for diverse cargos, could deliver siRNA via covalent conjugated or noncovalent charge interaction; however, the negatively charged siRNA can abolish the cell penetrating ability of CPP (Lee et al., 2013) via charge neutralizing. The potential toxicity of high positively charged CPP was an additional obstacle to siRNA delivery in vitro and vivo. As reported, a dsRBD from PKR can bind siRNA with high avidity, and the fusion protein with three CPP of Tat could efficiently deliver siRNA (Eguchi et al., 2009; Palm-apergi et al., 2011) and overcome the charge neutralizing by siRNA. In order to further improve the siRNA delivery, here, we design six fusion proteins of CPP-dsRBDs, using different CPP and dsRBD to study the CPP and dsRBD effect on siRNA delivery. Our protein 3 (Tat-Tat-Tat-dsRBD), similar construct of the fusion protein PTD-dsRBD from S. Dowdy (Eguchi et al., 2009; Palmapergi et al., 2011), could efficiently deliver siRNA, which was highly agreed with their results. siGFP transferred with proteins 10 and 11, respectively, showed similar or higher efficiency of GFP gene-silencing effect than the commercial siRNA carrier Lipofectamine RNAi Max. Protein 2 (Tat-Tat-dsRBD) and protein 3 (Tat-Tat-Tat-dsRBD), which have two or three CPPs at the N terminal of protein, respectively, were mixed with siGFP at a molar ratio of 20/ 1. Flow cytometry analysis demonstrated that the siGFP delivered by protein 3 has greatly enhanced the GFP silencing effect compared to protein 2. This result indicated that the addition of three CPPs to N terminal of protein might be more efficient than two CPPs. In parallel, protein 10 (Hph1-Hph1-dsRBD from Dicer) as well as protein 11 (Hph1-Hph1-dsRBD from Ribonuclease III), which have different dsRBD from different types of protein, can transfer siGFP into HeLa cells, and showed higher efficiency than the protein 3 (Tat-Tat-Tat-dsRBD). Notably, the use of calcium to condense protein/siRNA complex resulted in high RNAi efficiency, the similar result was reported on the CPP of ‘‘double’’ Tat (Baoum et al., 2012). The cell viability assays with these fusion proteins indicated low cytotoxicity; however, the complex of protein10/siRNA at ratio 30/1 induced HeLa cell death in Figure 3. The possible reasons for abnormal cell death was high amounts of siRNA entering into the cell, because both the GFP knock down efficiency experiment at a ratio of 20/1 and cytotoxicity assay further confirmed that protein 10 showed low cytotoxicity. Here, we reported six CPP-dsRBD-based fusion proteins as siRNA carrier, and four of them show high efficiency and have great potent to apply in vivo or clinic. The efficiency of siRNA delivery by the CPP-dsRBD fusion protein depends on the number of the CPP and the type of dsRBD. The complex of protein/siRNA could further improve the efficiency of RNAi by addition of calcium condensed step. Although these vectors, CPP-dsRBD fusion protein, show high efficiency in vitro, in the future, the in vivo experiment and target delivery strategy should be further studied and constructed.

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Conclusion Four of the six CPP-dsRBD-based fusion proteins, as siRNA delivery vector, have been developed and could efficiently deliver siRNA inhibit gene expression by the mechanism of RNAi. They also showed similar efficiency of RNAi comparing to Lipofectamine RNAi Max as the siRNA carrier with low cytotoxicity. These results indicated that the CPPdsRBD-based fusion proteins were promising candidates as siRNA carriers. The calcium condensed step will also improve the efficiency of RNAi.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

References Aliabadi HM, Landry B, Sun C, et al. (2012). Supramolecular assemblies in functional siRNA delivery: where do we stand? Biomaterials 33: 2546–69. Baoum A, Ovcharenko D, Berkland C. (2012). Calcium condensed cell penetrating peptide complexes offer highly efficient, low toxicity gene silencing. Int J Pharmaceut 427:134–42. Bycroft M, Gru¨nert S, Murzin AG, et al. (1995). NMR solution structure of a dsRNA binding domain from Drosophila staufen protein reveals homology to the N-terminal domain of ribosomal protein S5. EMBO J 14:3563–71. Choi J-M, Ahn M-H, Chae W-J, et al. (2006). Intranasal delivery of the cytoplasmic domain of CTLA-4 using a novel protein transduction domain prevents allergic inflammation. Nat Med 12:574–9. Choi J-M, Kim S-H, Shin J-H, et al. (2008). Transduction of the cytoplasmic domain of CTLA-4 inhibits TcR-specific activation signals and prevents collagen-induced arthritis. Proc Nat Acad Sci USA 105:19875–80. Doyle M, Jantsch MF. (2003). New and old roles of the double-stranded RNA-binding domain. J Struct Biol 140:147–53. Dykxhoorn DM, Palliser D, Lieberman J. (2006). The silent treatment: siRNAs as small molecule drugs. Gene Therapy 13:541–52. Eguchi A, Akuta T, Okuyama H, et al. (2001). Protein Transduction Domain of HIV-1 Tat Protein Promotes Efficient Delivery of DNA into Mammalian Cells. J Biol Chem 276:26204–10. Eguchi A, Meade BR, Chang Y-C, et al. (2009). Efficient siRNA delivery into primary cells by a peptide transduction domain-dsRNA binding domain fusion protein. Nat Biotechnol 27:567–71. El-Andaloussi S, Johansson HJ, Holm T, et al. (2007). A novel cellpenetrating peptide, M918, for efficient delivery of proteins and peptide nucleic acids. Mol Ther 15:1820–6. Endoh T, Ohtsuki T. (2009). Cellular siRNA delivery using cellpenetrating peptides modified for endosomal escape. Advanced Drug Deliv Rev 61:704–9. Gan J, Tropea JE, Austin BP, et al. (2005). Intermediate states of ribonuclease III in complex with double-stranded RNA. Structure 13: 1435–42. Haupenthal J, Baehr C, Kiermayer S, et al. (2006). Inhibition of RNAse A family enzymes prevents degradation and loss of silencing activity of siRNAs in serum. Biochem Pharmacol 71:702–10. Jantsch MF. (1996). Comparative mutational analysis of the doublestranded RNA binding domains of Xenopus laevis RNA-binding protein A. J Biol Chem 271:28112–19. Kharrat A, Macias MJ, Gibson TJ, et al. (1995). Structure of the dsRNA binding domain of E. coli RNase III. EMBO J 14:3572–84. Koppelhus U, Shiraishi T, Zachar V, et al. (2008). Improved cellular activity of antisense peptide nucleic acids by conjugation to a cationic peptide-lipid (CatLip) domain. Bioconjugate Chem 19:1526–34. Lee SH, Castagner B, Leroux J-C. (2013). Is there a future for cellpenetrating peptides in oligonucleotide delivery? Eur J Pharmaceut Biopharm Offic J Arbeitsgemeinschaft fu¨r Pharmazeutische Verfahrenstechnik e.V 85:5–11.

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Lee YW, Kim WT. (2010). Tobacco GTBP1, a homolog of human heterogeneous nuclear ribonucleoprotein, protects telomeres from aberrant homologous recombination. Plant Cell 22:2781–95. Li S-D, Huang L. (2007). Non-viral is superior to viral gene delivery. J Control Release 123:181–3. Lindgren M, Gallet X, Soomets U, et al. (2000). Translocation properties of novel cell penetrating transportan and penetratin analogues. Bioconjugate Chem 11:619–26. Meade BR, Dowdy SF. (2007). Exogenous siRNA delivery using peptide transduction domains/cell penetrating peptides. Adv Drug Deliv Rev 59:134–40. Meade BR, Dowdy SF. (2008). Enhancing the cellular uptake of siRNA duplexes following noncovalent packaging with protein transduction domain peptides. Advanced Drug Deliv Rev 60:530–6. Mo RH, Zaro JL, Shen W-C. (2012). Comparison of cationic and amphipathic cell penetrating peptides for siRNA delivery and efficacy. Mol pharmaceut 9:299–309. Nakase I, Takeuchi T, Tanaka G, et al. (2008). Methodological and cellular aspects that govern the internalization mechanisms of

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arginine-rich cell-penetrating peptides. Adv Drug Delivery Rev 60: 598–607. Nanduri S, Carpick BW, Yang Y, et al. (1998). Structure of the doublestranded RNA-binding domain of the protein kinase PKR reveals the molecular basis of its dsRNA-mediated activation. EMBO J 17: 5458–65. Palm-apergi C, Eguchi A, Dowdy SF. (2011). PTD–DRBD siRNA delivery. Methods Mol Biol 683:339–47. Raemdonck K, Vandenbroucke RE, Demeester J, et al. (2008). Maintaining the silence: reflections on long-term RNAi. Drug Discov Today 13:917–31. Ramos A, Bayer P, Varani G. (1999). Determination of the structure of the RNA complex of a double-stranded RNAbinding domain from Drosophila Staufen protein. Biopolymers 52: 181–96. Veldhoen S, Laufer SD, Trampe A, et al. (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.

Six-cell penetrating peptide-based fusion proteins for siRNA delivery.

The siRNA deliveries, for the siRNA's high negative charge, short serum half life, poor cellular internalization, etc, are still the key barriers for ...
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