Systemic Administration of siRNA via cRGD-containing Peptide

Although small interfering RNAs (siRNAs) have been demonstrated to specifically silence their target genes in disease models and clinical trials, in vivo siRNA delivery is still the technical bottleneck that limits their use in therapeutic applications. In this study, a bifunctional peptide named RGD10-10R was designed and tested for its ability to deliver siRNA in vitro and in vivo. Because of their electrostatic interactions with polyarginine (10R), negatively charged siRNAs were readily complexed with RGD10-10R peptides, forming spherical RGD10-10R/siRNA nanoparticles. In addition to enhancing their serum stability by preventing RNase from attacking siRNA through steric hindrance, peptide binding facilitated siRNA transfection into MDA-MB-231 cells, as demonstrated by FACS and confocal microscopy assays and by the repressed expression of target genes. When RGD10 peptide, a receptor competitor of RGD10-10R, was added to the transfection system, the cellular internalization of RGD10-10R/siRNA was significantly compromised, suggesting a mechanism of ligand/receptor interaction. Tissue distribution assays indicated that the peptide/siRNA complex preferentially accumulated in the liver and in several exocrine/endocrine glands. Furthermore, tumor-targeted delivery of siRNA was also demonstrated by in vivo imaging and cryosection assays. In summary, RGD10-10R might constitute a novel siRNA delivery tool that could potentially be applied in tumor treatment.

Scientific RepoRts | 5:12458 | DOi: 10.1038/srep12458 have been found to mediate adhesion between cells and the extracellular matrix (ECM) and to stimulate intracellular signaling and gene expression involved in cell growth, migration and survival 20 . Integrin α v β 3 is the predominant receptor of the RGD motif and is highly expressed in tumor cells and neovascular endothelial cells [21][22][23] . The diverse applications of RGD peptides include inhibiting apoptosis, angiogenesis and tumor formation 24 ; imaging for diagnostic purposes 25,26 ; and coating surfaces for use as biomaterials to enhance drug delivery systems 27 . Although RGD-based peptides have been used to facilitate siRNA transportation in vitro and in vivo, they were only incorporated into certain carriers and simply played a role of specific recognition [28][29][30][31][32] . Such types of delivery systems are typically multicomponent, complex and difficult to develop as potential drug delivery formulations.
In this study, RGD10, a representative RGD-motif-containing peptide with an amino acid sequence of DGARYCRGDCFDG, identified by Kontermann and colleagues using phage display technology 33 , was fused with decu-L-arginine residues (10R) to form a bifunctional CPP termed RGD10-10R. RGD10 is a cyclic peptide that contains a disulfide linkage between the two cysteines. It was previously reported that the cyclic RGD peptide exhibited considerably higher affinity toward α v β 3 compared with the linear RGD peptide 34 . In this delivery system, RGD10 was designed to recognize integrin(s) expressed on tumor cells and tumor-associated endothelial cells, and 10R was employed to bind siRNA through electrostatic interactions (Fig. 1a). The physicochemical properties of the RGD10-10R/siRNA complex, including siRNA retarding ability, morphology, hydrodynamic diameter, zeta potential, and RNase resistance, were characterized. Cytotoxicity, intracellular uptake and localization, and specific gene silencing were assessed in vitro. Furthermore, we evaluated the in vivo distribution and tumor-targeting profile through fluorescent imaging and cryosection observations.

Results
Gel retardation assay. To evaluate the loading capability of RGD10-10R, a gel retardation assay was performed using si-NC-67M, a serum-resistant siRNA. The use of this siRNA could exclude the possibility that degradation of the siRNA affected its gel shift. Two and a half microliters of siRNA (20 μ M, ~50 pmol) was mixed with a given amount of the peptide and incubated at room temperature for 15-20 min to form RGD10-10R/siRNA complexes with different molar ratios. The complexes were adjusted to identical volumes with DEPC water; then, they were loaded into a 4% agarose gel and separated for 40 min at a constant voltage of 140 V. UV imaging revealed that when the molar ratio of peptide and siRNA increased from 25:1 to 200:1, increasingly less siRNAs moved in the gel from the negative pole to positive gel to form electrophoresis bands, suggesting that more siRNAs were entrapped in the complexes with increasing amounts of the peptide. Moreover, when the molar ratio reached 25:1, the majority of the siRNAs were already entrapped in the complexes (Fig. 2a, 1b-left panel). As expected, when the molar ratio increased to more than 100:1, the complexes ran in the opposite direction (from the positive pole to the negative pole) (Fig. 2b-left panel). Compared to RGD10-10R, compromised siRNA binding capacity was observed for polyarginine (10R) peptide without the RGD10 motif at the N terminal ( Fig. 2b-right panel). The influence of Opti-MEM, an optimized medium used for siRNA transfection, on the stability of the complex was further assessed. The results indicated that RGD10-10R could bind siRNA and form stable complexes in this medium, as well as in DEPC water (Fig. 2c).

RNase resistance assay.
To test the hypothesis that the formation of complexes could protect siR-NAs from RNase attack and therefore enhance their stability, an RNase resistance assay was performed using an unstable double-stranded small RNA termed mmu-miR-672. The results indicated that when incubated in 10% FBS at 37 °C, these small RNAs were rapidly degraded ( Fig. 2d-left). In contrast, when the small RNAs were encapsulated by the peptides, full-length bands were clearly observed, even with a prolonged incubation of 12 hours. In addition, if the peptide/siRNA complexes were not treated with proteinase K, the peptides were not digested and the complexes were not destroyed, and they could not move in the gel from the negative pole to the positive pole, which further confirmed that the siRNAs were thoroughly entrapped by the peptides (Fig. 2d-right panel, the second lane). Here, RGD10-10R/ siRNA with a molar ratio of 100:1 was not observed to run from the positive pole to the negative pole because no gel existed in the opposite direction, which was different from the agarose gel used in the gel retardation assay.
Physicochemical properties of RGD10-10R/siRNA complexes. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to investigate the physicochemical properties of the complexes, including their morphology, size, polydispersity index (PDI), and zeta potential. The TEM images revealed that all three formulations, with molar ratios of 20:1, 50:1 and 100:1, exhibited regularly spherical nanostructures, with a diameter from a few tens of nanometers to three-hundred nanometers (Figs 3a and 1a). Moreover, the DLS measurements indicated that the Z-average particle sizes of the three complexes were between 110-150 nm with a PDI of less than 0.20 (Fig. 3b, Table 1). As a reference for the measurements, the DLS size of Lipofectamine 2000/siRNA complex was approximately 140 nm. As expected, the peptides alone or naked siRNAs showed no statistical size ( Table 1). The zeta potentials were 5.20, 21.40, and 20.08 mV for the complexes with molar ratios of 20:1, 50:1, and 100:1, respectively. The zeta potential of the Lipofectamine 2000/siRNA complex was 24.40 mV. As expected, naked siRNA presented a negative charge of − 8.38 mV ( Table 1). Note that peptide/siRNA complexes  , the receptor of the RGD10 motif. Taking advantage of this ligand/receptor interaction, drug delivery approaches targeting α v β 3 have been extensively investigated [37][38][39] . As the molar ratio of peptide/siRNA increased, the percentage of the cells that internalized FAM-labeled siRNA was greatly increased (Fig. 4a,b). Accordingly, the mean fluorescence intensity (MFI) was also gradually enhanced ( Fig. 4a,b). When the molar ratio reached 50:1, the peptide/siRNA complex exhibited transfection efficiency that was equal to that of Lipofectamine 2000, a gold standard for in vitro siRNA transfection (Fig. 4a,b). To confirm that this internalization was triggered through ligand/receptor recognition and interaction (Fig. 1b), RGD10 peptide without polyarginine at the C terminal, and thus no siRNA binding capacity, was used as a competitor of RGD10-10R for receptor binding. The results indicated that adding RGD10 reduced both the transfection efficiency and the MFI of the complexes with various molar ratios (Fig. 4b).
Subcellular localization. Confocal microscopy was used to investigate the subcellular localization of the peptide/siRNA complexes. According to the confocal microscopy observations, FAM-labeled siR-NAs were internalized by the cells after being transfected for 8-10 hours, with a distribution pattern and fluorescence intensity that were similar to those of commercial Lipofectamine 2000 (Fig. 5). Magnified images of RGD10-10R/siRNA-treated cells exhibited both granular-shaped and dispersive signals. The granular signals in yellow represented the co-localization of the siRNAs (green signals) and the endosome/lysosome complexes (red signals), indicating that those siRNAs were still located in the endosomes/lysosomes. In contrast, the dispersive siRNA signals within the cytoplasm were hypothesized to be the active molecules that escaped from the endosomes/lysosome. Note that although it has been reported The complex formed in Opti-MEM was examined to assess the siRNA binding capability of the peptide and the stability of the complexes (c). An RNase resistance assay was performed to evaluate the protection effect resulting from encapsulation of the peptides using an unstable small RNA termed mmu-miR-672. Both naked RNAs (d-left panel) and complexes (d-right panel) were separated in a 20% native PAGE. These data demonstrated that RGD10-10R binds siRNAs and effectively protects them from RNase degradation. that polyarginine is a type of cell-penetrating peptide 40,41 , cells treated with only 10R/siRNA complexes showed faint siRNA signals around the cell surface (Fig. 5). This result might be attributed to the relatively weaker siRNA binding capability for 10R compared with RGD10-10R (Fig. 1b), or the peptide lost its penetrating capacity after complexing with siRNA. Similar observations were obtained for the cells treated with naked-siRNA (Fig. 5).
The FACS and confocal data (Figs 4 and 5) indicated that RGD10-10R mediated efficient siRNA transfection in vitro and showed that this activity was greatly compromised by the receptor competitor RGD10. Furthermore, polyarginine (10R) was unable to deliver siRNA into MDA-MB-231 cells. Collectively, siRNA transfection mediated by RGD10-10R into α v β 3 -highly expressed cells was a robust and specific process.

Knockdown efficiency in vitro.
Real-time quantitative PCR (RT-qPCR) and Western blot were performed to assess the in vitro activity of the RGD10-10R/siRNA complexes. Anti-Lamin A/C siRNA was used in this study. The RT-qPCR and Western blot data, collected from MDA-MB-231, both indicated that approximately 25% knockdown efficiency could be achieved for the complexes with high molar ratios of 100:1 and 200:1 (Fig. 6a,b). However, the complex with a lower molar ratio of 20:1 suppressed Lamin A/C gene expression with a 45% knockdown efficiency (Fig. 6a). Lipofectamine 2000/siLamin A/C, the positive control, showed a significant knockdown of targeted gene expression (~80% for mRNA, ~60% for protein) (Fig. 6a,b). In addition, human umbilical vein endothelial cell (HUVEC), another α v β 3 -highly expressing cell 42,43 , was further employed in the Western blot assay. Another commercial transfection  reagent, X-TremeGENE (Roche), was included as an additional control because HUVEC is well known as being difficult to transfect. Western blot indicated that RGD10-10R/siRNA with a molar ratio of 20:1 inhibited the expression of Lamin A/C with a 41% knockdown efficiency. By contract, Lipofectamine 2000 and X-TremeGENE only exhibited 21% and 25% knockdown efficiencies, respectively (Fig. 6c). These data highlighted the pivotal role of the ligand/receptor interaction that was employed by RGD10-10R during siRNA transfection.
Cytotoxicity. An MTT assay was conducted to investigate the biocompatibilities of the peptide/siRNA complexes in MDA-MB-231 cells. The data (Fig. S1) showed that all the formulations with molar ratios of less than 100:1 and Lipofectamine 2000/siRNA achieved nearly 100% cell viability. Complexes with a molar ratio of 200:1 and X-TremeGENE/siRNA exhibited weak and equal cytotoxicities (88% cell viability).

In vivo biodistribution.
In vivo biodistribution of RGD10-10R/siRNA complexes was performed in mice after tail vein injection. Mice were anesthetized with a gas mixture of oxygen and isoflurane to perform fluorescent imaging at indicated time points post-administration (Fig. 7). By merging the fluorescence image with an X-ray image, we found that the peptide/siRNA complexes, in contrast to naked-siRNA, remarkably accumulated in the liver and were maintained for a longer period of time.
After normalizing to the MFI of saline-treated mice, the MFIs of the naked-siRNA, 'RGD10-10R/ siRNA 50:1' and 'RGD10-10R/siRNA 100:1' were 1062, 4809, and 7396, respectively, in the liver 8 hours post-administration. The normalized MFIs in the spleen for the three groups were 1332, 3145, and 4795, respectively. Both naked siRNA and complex-treated mice showed strong fluorescence signals in the kidney, via which siRNAs would be eliminated from the body. Furthermore, mice treated with naked siRNA showed notable accumulation in several glands, including submandibular glands and the pancreas. In contrast, the mice treated with the peptide/siRNA exhibited relatively weaker signal intensity in these glands compared to the naked-siRNA. These results suggested that RGD10-10R influenced the in vivo biodistribution pattern of siRNAs after forming complexes.
Tumor-targeting properties of complexes. It was previously reported that integrin α v β 3 is constitutively highly expressed in tumor cells and neovascular endothelial cells 21 , suggesting its pivotal role in tumor-associated angiogenesis and potential value as a target for anti-tumor therapy. In this study, we have shown that RGD10-10R could mediate effective and specific siRNA delivery in MDA-MB-231 and HUVEC (Figs 4-6) in vitro. Therefore, in vivo tumor-targeting properties were investigated using the MDA-MB-231 xenograft murine model. Tumor cells were injected subcutaneously into the right axillary fossa of female BALB/C nude mice (Fig. 8a, as indicated by kermesinus circles). The data demonstrated that peptide/siRNA complexes remarkably accumulated in the tumor after i.v. injection (Fig. 8a). Isolated-organ-imaging at 24 hours post-administration indicated that RGD10-10R/siRNA with a molar ratio of 50:1 exhibited the highest fluorescence intensity compared with those with molar ratios of 20:1 and 100:1. For clarification, the absolute MFIs in Figs 7 and 8 are not comparable because these two assays were performed under different experimental conditions. Furthermore, cryosections were prepared and stained with DAPI and FITC-labeled phalloidin. Confocal scanning microscopy revealed that the red signals, representing Cy5-labeled siRNAs, were localized between the blue and green areas, representing nuclei and cell outlines, respectively (Fig. 8d). Therefore, it is highly likely that siRNAs were delivered into cytoplasm of the tumor cells, where RNA interference (RNAi) occurs.

Discussion
The development of siRNA therapeutics is severely hindered by inadequate clinically suitable, safe and effective drug delivery vehicles. In this work, we designed a bifunctional peptide, RGD10-10R. The RGD10 motif is the specific ligand of integrin α v β 3 , an integrin that is highly expressed in tumor cells and neovascular endothelial cells, and decu-L-arginine (10R) can bind siRNA through electrostatic interactions.
It was demonstrated that RGD10-10R can effectively form regular nanostructures with siRNA, and it protected siRNA from RNase attack. We observed that RGD10-10R/siRNA complexes might run in the opposite direction (from the positive pole to the negative pole) when the molar ratio increased to more than 100:1 (Fig. 2b-left panel), suggesting that the net charge of the complexes with this molar ratio was converted from negative to positive. Polyarginine (10R) without the RGD10 polypeptide at the N terminal showed a weaker siRNA binding capacity than RGD10-10R, which might be because the longer peptide chain was beneficial for siRNA complexation and complex stabilization. In addition, the RNase resistance assay performed with unstable RNA, mmu-miR-672, should be attributed to the steric hindrance present within the peptide/siRNA complexes.
RGD10, as a receptor competitor of RGD10-10R, was introduced into the cellular uptake assay to validate the transfection mechanism of RGD10-10R. The addition of RGD10 remarkably reduced the cellular uptake of RGD10-10R/siRNA, suggesting that the transfection of siRNAs into the cell was indeed dependent on the ligand/receptor interaction. Furthermore, RT-PCR and Western blot assays revealed that anti-Lamin A/C siRNA repressed target gene expression in vitro after being transfected with RGD10-10R. Lamin A/C, also known as LMNA, is a component of the nuclear membrane structure that, in humans, is encoded by the LMNA gene 44 . Lamins are important for maintaining normal cell functions, such as cell cycle control, DNA replication and chromatin organization. During apoptosis, Lamin A/C is specifically cleaved to a large (40-45kD) and a small (28kD) fragment. The cleavage of lamins results in nuclear dysregulation and cell death 45 . Dysfunction of Lamin A/C is associated with a series of diseases 46 . However, the in vitro activity of RGD10-10R/siRNA was compromised, which might result from insufficient particles escaping from the endosome/lysosome after being internalized by the cells. This was supported by the subcellular localization data (Fig. 5, yellow granule). It is also possible that the binding force between the peptides and the siRNAs is too strong to release siRNA, even though the complexes had successfully escaped from the endosomes/lysosomes. The in vivo distribution profile revealed that the complexes not only remarkably accumulated in the liver in normal C57BL/6 mice but could also effectively deliver siRNA into tumor tissue in the xenograft murine model. The size distribution of the peptide/siRNA complexes ranged from 110-150 nm (Fig. 1b,  Table 1). It was previously reported that the particle size should be less than 200 nm if it is intended to deliver drugs to hepatocytes because larger particles are more likely processed and eliminated by Kupffer cells and filtered into the spleen, for which the cut-off point extends up to 250 nm 47 . However, it was also reported that fenestrations in the liver sinusoidal endothelium permitted particles of 100-200 nm in diameter to exit the bloodstream and gain access to hepatocytes and other liver cells 48,49 , which might facilitate the accumulation of siRNAs or particles in the liver. In addition, this size distribution pattern also met the requirements of the EPR (enhanced permeability and retention) effect, which facilitates the accumulation of nanoparticles in tumor tissue in a passive-targeting manner 50 . Together with the scenario that integrin was highly expressed in both MDA-MB-231 and neovascular endothelial cells, RGD10-10R/ . The data were normalized to corresponding tissues from saline-treated animals. Each bar represents the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. corresponding tissue from naked siRNA-treated mice. siRNA could be entrapped in the tumor tissue and cross the cell membrane via receptor-mediated endocytosis. Furthermore, the in vivo imaging data revealed that RGD10-10R/siRNA with a molar ratio of 50:1 showed more tumor accumulation than the complexes with molar ratios of 20:1 and 100:1. The DLS size of 'RGD10-10R/siRNA 50:1′ was 117 nm, the smallest particle size among the three formulations (Table 1), indicating that particle size indeed played an important role in facilitating tumor-targeted transport of siRNA 50 .
In conclusion, RGD10-10R provided a promising siRNA delivery tool for further development of RNAi-based therapeutics, particularly for cancer treatment.

Materials and methods
Materials. RGD10 RGD10-10R/siRNA complex preparation. According to the requirements of the present experiment, RGD10-10R powder was dissolved in 200-1000 μ M (approximately 0.60-3.0 μ g/μ l) (for in vitro testing) or 10-30 μ g/μ l (for in vivo study) with DEPC (diethylpyrocarbonate)-treated water. Moreover, siRNA was dissolved in 20 μ M (approximately 0.266 μ g/μ l) and 1 μ g/μ l with DEPC water for in vitro tests and in vivo studies, respectively. The peptide and siRNA solutions were mixed directly according to different desired molar ratios. After electrostatic interaction for 15-20 min at room temperature, peptide/ siRNA complexes were formed (Fig. 1a). Additional DEPC water or 1 × PBS or normal saline (NS) could be added to the complex solution to obtain the final formulation used for transfection or injection.
Gel retardation assay. To assess the siRNA loading ability of RGD10-10R, a gel retardation assay was performed. Here, a serum-stable siRNA, si-NC67-M 51 , was used to prepare the peptide/siRNA complexes according to the aforementioned protocol. Twenty-two and a half microliters of complexes containing 50 pmol siRNA (20 μ M, 2.5 μ l) was mixed with 4 μ l of 6 × loading buffer and loaded into a 4 wt% agarose gel containing 5 μ g/ml ethidium bromide. Electrophoresis was set up in 1 × TAE buffer at 120 V and run for 40 min. siRNA retardation was analyzed on a UV illuminator to show the location of the siRNA.
RNase resistance. Theoretically, peptides can protect siRNAs form RNase attack because of the steric hindrance effect. Therefore, siRNAs entrapped in the complexes would be more stable than naked siRNAs. Hence, an RNase resistance assay was performed. mmu-miR-672, a microRNA that was previously proven to be unstable in fetal bovine serum (FBS) 52 , was employed in this assay. Chemically synthesized microRNA is similar to siRNA in terms of their nucleotide base constituents (cytosine, guanine, adenine and uracil) and biological structure properties (double strand, 19-25nt). A RGD10-10R/ RNA complex with a molar ratio of 100:1 and containing 22.5 pmol of mmu-miR-672 was prepared. Additionally, a 22.5 pmol naked mmu-miR-672 solution without peptide binding was used as a control. Peptide/RNA complexes and naked mmu-miR-672 were incubated in 10% FBS (v/v, in 1 × PBS) at 37 °C. Complexes and naked RNA were collected and immediately frozen at − 20 °C at 0 h, 3 h, 6 h and 12 h post-incubation. Then, all samples were centrifuged for 5 min at 10000 rpm and 4 °C to precipitate complexes or naked RNA. The supernatants were discarded, and 15 μ l of DEPC water was added to resuspend the precipitate. To destroy complexes and release RNA, proteinase K (0.2 μ l, 1 mg/ml), Tris-HCl (1 μ l, 50 mM, pH = 7.0) and CaCl 2 (1 μ l, 5 mM) were added to the resuspended complex solution, followed by incubating for 4-5 hours at 37 °C. After adding loading buffer, the complexes and naked RNA solution were both separated in native 20% PAGE (polyacrylamide gel electrophoresis) for 150 min at a constant voltage of 150 V. Finally, the gels were stained with SYBR Gold for 20 min, exposed by Vilber Lourmat imaging system (France) and analyzed with Biocap software to show the location of the small RNA.
Characterization of particle sizes and zeta potential. The particle sizes and zeta potentials of the peptide/siRNA complexes were measured using a Zetasizer 3000HS (Malvern Instruments, Inc., Worcestershire, UK) at a wavelength of 677 nm with a constant angle of 90° at room temperature. Complexes were prepared by mixing 2 μ g of si-NC67-M (7.69 μ l, 20 μ M) and the required amount of peptide (1000 μ M) at room temperature. The solutions were then diluted with 0.8 ml of double-distilled water prior to characterization. Moreover, the morphologies and sizes of the complexes were further analyzed by transmission electron microscopy (TEM) (Tecnai G2 20 STWIN transmission electron microscope, Philips, Netherlands) with an acceleration voltage of 200 kV. Briefly, samples were prepared by adding 7 μ l of complex solutions onto a carbon-coated copper grid. Then, they were negatively stained with 1% (wt/v) phosphotungstic acid (pH adjusted to 7.3 with 1N NaOH) and air-dried prior to collecting images.
Cell lines and culture. The breast carcinoma cell line MDA-MB-231 was obtained from the Cell Resource Center of Peking Union Medical College (Beijing, China). Cells were cultured with L15 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 μ g/ml streptomycin at 37 °C in a 100% air humidified atmosphere (without CO 2 ). CO 2 was isolated from the cells by covering the culture plates or bottles with Parafilm. Human umbilical vein endothelial cells (HUVECs) were grown in medium 199 (Invitrogen) containing fibroblast growth factor, heparin and 20% fetal bovine serum (HyClone, Ogden, UT) at 37 °C in a humidified atmosphere of 5% CO 2 .

Fluorescence-activated cell sorting (FACS). MDA-MB-231 cells were plated in 12-well plates
(1 × 10 5 per well) one day before transfection and incubated in L15 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin to 60-70% confluence. Then, the medium was removed and replaced with Opti-MEM, a reduced serum medium designed for cationic lipid transfections. Subsequently, complexes containing 100 pmol of FAM-labeled siRNA were added into each well. After incubation at 37 °C for 4 hours, 2 ml of fresh L15 containing 10% FBS was added, followed by further incubation for 4 hours. Then, the cells were washed three times with 1 ml of precooled 1 × PBS to remove residual free complexes and siRNAs, subsequently suspended in 400 μ l of precooled 1 × PBS and introduced into a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). Subcellular localization. MDA-MB-231 cells were plated in 6-well plates (2 × 10 5 per well) with glass coverslips (one coverslip per well) at the bottom one day prior to transfection. RGD10-10R/siRNA complexes containing 150 pmol (7.5 μ l, 20 μ M) of FAM-labeled siRNA were transfected into cells according to the above protocol. Approximately 10 hours later, the subcellular distribution of siRNA was observed in living cells using a Zeiss confocal microscope (LSM510, Carl Zeiss, Germany). Moreover, LysoTracker Red DND-99 (Invitrogen, Carlsbad, CA) was used to indicate endosomes and lysosomes.
MTT assay. An MTT assay was employed to evaluate the cytotoxicity of the peptide/siRNA complexes. MDA-MB-231 cells were seeded at 10000 cells per 100 μ l of L15 per well on a 96-well plate and allowed to adhere overnight at 37 °C. Cells were transfected with complexes containing 6.25 pmol of siRNA in 25 μ l of Opti-MEM. Four hours later, 200 μ l of fresh complete L15 medium was added. After an additional 20 hours of culture in the presence of 10% FBS, 200 μ l of medium was removed and 2 μ l of MTT was added to each well, followed by an additional 4 hours of incubation under the same incubation conditions. Then, all medium was removed and 50 μ l of DMSO was added, followed by incubation for an additional 30 min at 37 °C to dissolve formazan. Finally, the absorbance was read at 540 nm with a reference wavelength of 650 nm, and the absolute absorbance (OD net540 ) was OD 540 minus OD 650 . For comparison of relative viability, all data were presented as the mean percentage ± S.D. in two replicate samples compared to the absorbance value of mock-treated cells. The cell viability was calculated as follows: where OD net540(sample) was the absorbance at 540 nm of the transfected cells and OD net540(control) was the absorbance at 540 nm of the mock control (nontransfected cells).
Real-time PCR. Real-time PCR was performed to determine whether siLamin A/C (against the LMNA gene) transfected by RGD10-10R could suppress the expression of the targeted gene. MDA-MB-1231 cells were plated in 12-well plates (1 × 10 5 per well) one day prior to transfection and incubated in L15 to approximately 60% confluence. Then, the medium was removed and replaced with 1 ml of Opti-MEM. Subsequently, 200 μ l of complexes containing 100 pmol siLamin A/C was added to each well (the final transfection concentration of siRNA was ~80 nM). Here, Lipofectamine 2000 was the positive control, and each sample was tested in two replicate wells. After 4 hours of incubation, 2 ml of