http://informahealthcare.com/drt ISSN: 1061-186X (print), 1029-2330 (electronic) J Drug Target, 2014; 22(2): 156–164 ! 2014 Informa UK Ltd. DOI: 10.3109/1061186X.2013.850502
ORIGINAL ARTICLE
Combined delivery of BCNU and VEGF siRNA using amphiphilic peptides for glioblastoma Na Yi, Binna Oh, Hyun Ah Kim, and Minhyung Lee
Abstract
Keywords
Combined delivery of chemical drug and therapeutic gene has been introduced as an efficient method for the treatment of cancers such as glioblastoma. In this study, bis-chloroethylnitrosourea (BCNU) and vascular endothelial growth factor (VEGF) small interfering RNA (VEGFsiRNA) were co-delivered into C6 glioblastoma cells using a non-toxic peptide-based carrier. The R3V6 peptides, which are composed of 3-arginine and 6-valine, formed self-assembled micelles in aqueous solution. BCNU, a hydrophobic anti-cancer drug, was loaded into the hydrophobic core of the micelles, forming BCNU-loaded R3V6 micelles (R3V6-BCNU). In gel retardation assay, R3V6-BCNU formed a stable complex with siRNA. In vitro transfection assay showed that the VEGF-siRNA/R3V6-BCNU complex had the highest transfection efficiency into C6 cells at a 1:20 weight ratio (VEGF-siRNA:R3V6-BCNU). In addition, the VEGF-siRNA/R3V6BCNU complexes had higher delivery efficiency than lipofectamine or naked siRNA. VEGF expressions were remarkably decreased by transfection of the VEGF-siRNA/R3V6 or VEGFsiRNA/R3V6-BCNU complexes. Furthermore, R3V6-BCNU delivered BCNU more efficiently into the cells than BCNU only. Therefore, R3V6 delivered both VEGF-siRNA and BCNU efficiently into the glioblastoma cells. The results suggest that R3V6-BCNU may be useful for combined delivery of siRNA and chemical drug into cancer cells.
Combined delivery, glioblastoma, peptide micelles, VEGF siRNA
Introduction Glioblastoma multiform (GBM) is considered a devastating malignant primary brain tumor in humans. Although advances in other solid tumor therapies have improved patient survival, GBM prognosis is still poor. With standard treatment, the median survival for patients with GBM is 14 months after diagnosis [1,2]. Conventional treatment usually includes surgery, radiation therapy, and chemotherapy. Generally, the first step in the treatment of GBM is surgery to remove as much tumor tissue as possible [3]. However, complete removal is impossible because GBMs have finger-like tentacles. After surgery, radiotherapy and chemotherapy and/or bio-therapy are then used to treat the remaining tumor. These therapies are useful in the treatment of GBM, but they have undesirable side effects. Therefore, a combination of gene therapy and chemotherapy has been investigated as a new potential treatment for gliomas, suggesting that co-delivery of anticancer drugs and siRNA
Address for correspondence: Minhyung Lee, PhD, Department of Bioengineering, College of Engineering, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133 791, Republic of Korea. Tel: 82 2 2220 4456. Fax: 82 2 2220 1998. E-mail: minhyung@ hanyang.ac.kr
History Received 14 May 2013 Accepted 27 September 2013 Published online 12 November 2013
using non-viral delivery carriers may have tumor-suppressive effects [4]. RNA interference (RNAi) is regarded as an effective technology for gene silencing in gene therapy. The double strand RNA-based molecule, siRNA, has potential as a biopharmaceutical therapeutic [5]. As RNAi interferes with translation, and not with DNA transcription, siRNA does not interact with chromosomal DNA. This lack of DNA interaction greatly reduces concerns about possible adverse gene alterations that might result from DNA-based gene therapy. The interaction of siRNA with mRNA, not proteins, also reduces the production of harmful proteins before synthesis [6]. Vascular endothelial growth factor (VEGF), a critical factor for blood vessel growth, has been implicated as a crucial signal in tumor development by promoting angiogenesis [7–9]. Inhibition of VEGF to interfere with tumor neovascularization is the most effective strategy in angiogenesis [10]. Meanwhile, bis-chloroethylnitrosourea (BCNU) has been widely used for the treatment of several types of brain cancer and is able to form inter-strand crosslinks in DNA, which prevents DNA replication and DNA transcription [11,12]. An amphiphilic peptide with 3-arginine and 6-valine (R3V6) has been evaluated as a gene carrier. R3V6 has been employed to transport both siRNA and DNA into cells without cytotoxicity [13,14]. In addition, R3V6 peptides have a higher transfection efficiency whether a hydrophobic drug was loaded or not [15,16]. In this article, the
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Department of Bioengineering, College of Engineering, Hanyang University, Seoul, Republic of Korea
DOI: 10.3109/1061186X.2013.850502
Combined delivery of siRNA and chemical drug into cancer cells
chemotherapeutic drug BCNU and VEGF small interfering RNA (VEGF-siRNA) were co-delivered to treat glioblastoma using a peptide-based delivery carrier. Despite the anti-cancer effects of BCNU, its use in chemotherapy is limited due to drug resistance and toxic side effects. Therefore, our combined approach may generate significant synergistic effects for the treatment of glioblastoma.
Materials and methods
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Synthesis of R3V6 peptides and siRNA oligonucleotides The R3V6 peptides, composed of 3-arginine and 6-valine, were synthesized chemically and purified by C18 reversephase chromatography (Peptron Co., Daejeon, Korea). The peptides were dissolved in distilled water at 5 mg/ml and were stored at 70 C. VEGF-siRNA and scrambled small interfering RNA (scr-siRNA) were synthesized by Bioneer (Daejeon, Korea). Renilla luciferase small interfering RNA (Rluc-siRNA) was synthesized by Genolution (Seoul, Korea). The siRNA sequences are as follows: VEGF, 50 -AAACCGGGAUUUCUUGCGCUUUCGU-30 ; Scrambled, 50 -UUCUCCGAACGUGUCACGUTT-30 ; Renilla luciferase, 50 -GGCCUUUCACUACUCCUAC UUUU-30 . Cell culture C6 cells, originating from a rat brain glioblastoma, were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (PS). The cells were seeded onto a 75 cm2 cell culture flask and cultured at 37 C in a humidified incubator under 5% CO2 and 95% air. Preparation of the siRNA/R3V6 and siRNA/R3V6-BCNU complexes BCNU was dissolved in distilled water at 5 mg/ml (Sigma, St. Louis, MO). The BCNU solution, R3V6 peptide solution and siRNA were mixed into water one by one and blended gently at each step. Then, the solution was incubated at room temperature for 30 min. To determine the optimal ratios of siRNA to R3V6 and siRNA to R3V6-BCNU, siRNA/R3V6 and siRNA/R3V6-BCNU complexes were prepared at various weight ratios. The weight ratio of R3V6 peptide and BCNU was fixed at 1:0.6 (R3V6:BCNU). For all other in vitro and in vivo experiments, the weight ratio of siRNA to R3V6 was fixed at 1:20 (siRNA:R3V6). The siRNA/PEI25k complex was prepared at a 1:1 weight ratio. The siRNA/Lipofectamine complex was prepared at a 1:5 ratio (w/v) as suggested in the manufacturer’s manual. In vitro transfection of C6 cells with Rluc-siRNA To monitor the silencing effect of siRNA, a Renilla/Firefly luciferase reporter plasmid and a psiCHECK-2 vector were transfected into the C6 cells. The C6 cells were seeded in a 100 20 mm cell culture dish and incubated for 24 h at 37 C in a humidified incubator under 5% CO2 and 95% air. Before transfection, the cell culture medium was replaced
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with FBS-free DMEM, and then the transfection complexes were added. Next, the psiCHECK2/PEI25k complex was added. The cells were incubated for 4 h at 37 C in a humidified incubator under 5% CO2 and 95% air. After 4 h, the cell culture medium was changed to DMEM containing 10% FBS and 1% PS, the cells were incubated for an additional 48 h at 37 C in a humidified incubator under 5% CO2 and 95% air. The C6 cells were washed and seeded in a 12-well plate at a density of 3.5 104 cells/well. For the dual luciferase assay, the cells were seeded at a density of 3.5 104 cells/well in 12-well plates (Falcon Co., Becton Dickenson, Franklin Lakes, NJ). The cell culture medium was replaced with FBS-free DMEM and then the Renilla luciferase siRNA (Rluc-siRNA)/carrier complexes prepared at optimal ratios were added. The cells were incubated for 4 h at 37 C in a humidified incubator under 5% CO2 and 95% air. After 4 h, the cell culture medium was changed to DMEM containing 10% FBS and 1% PS, and the cells were incubated for an additional 48 h at 37 C in a humidified incubator under 5% CO2 and 95% air. Dual luciferase assay After 48 h of incubation, the DMEM was removed from each well of the plate. The C6 cells were washed with PBS, 120 ml of passive lysis buffer (Promega, Madison, WI) was added to each well, and the plate was incubated for 15 min at room temperature. The cells were harvested and centrifuged at 13 000 rpm for 5 min. The supernatant was transferred to fresh tubes. A dual luciferase reporter assay system (Promega) was purchased and used according to the manufacturer’s instructions. Luciferase activities were measured using a 96-well plated luminometer (Berthold Detection System GmbH, Pforzheim, Germany) in relative light units (RLU). The final values of luciferase activity were calculated as (Renilla RLU/Firefly RLU) 100 (%). In vitro transfection of C6 cells with VEGF-siRNA and enzyme-linked immunosorbent assay (ELISA) The transfection efficiency was tested by VEGF ELISA. For the VEGF luciferase assay, the cells were seeded at a density of 3.5 104 cells/well in 12-well plates (Falcon). The cell culture medium was replaced with FBS-free DMEM and the VEGF-siRNA/carrier complexes prepared at optimal ratios were added. The cells were incubated for 4 h at 37 C in a humidified incubator under 5% CO2 and 95% air. After 4 h, the cell culture medium was changed to DMEM containing 10% FBS and 1% PS, and the cells were incubated for an additional 48 h in a hypoxia chamber maintained at 1% oxygen. After 48 h incubation, the culture medium was collected and centrifuged. A human VEGF ELISA kit (PeproTech, Rocky Hill, NJ) was used. After the addition of a 100 ml sample, mediums were then incubated for 2 h at room temperature. Following a brief wash, 100 ml of detection antibody solution was added to each well and incubated for 2 h. Conjugate buffer of 100 ml was added and incubated for 30 min at room temperature. Finally, 100 ml of a substrate solution for color development was added. The absorbance was measured at 450 nm by a Micro plate reader 680 from
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Bio-Rad (Hercules, CA). The protein concentration of the extract was measured by a Pierce BCA Protein Assay Kit (Pierce Biochemical, Rockford, IL).
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MTT assay To evaluate cell viability, C6 cells were seeded in a 24-well plate at a density of 5 104 cells/well. The culture plates were incubated for 24 h at 37 C in a humidified incubator under 5% CO2 and 95% air. After incubation, the siRNA/carrier complexes were transfected at optimal ratios in the same way as previously described. After 24 h, 40 ml of 5 mg/ml MTT solution was added to each well. The plates were then incubated for 4 h at 37 C in a humidified incubator under 5% CO2 and 95% air. After 4 h of incubation, the medium of each well was removed and 700 ml of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals formed by the living cells. Cell viability (%) was calculated according to the following equation: Cell viability ð%Þ ¼ OD570nmðsampleÞ =OD570nmðcontrolÞ 100, where OD570nm(sample) represents the wells treated with the plasmid/peptides complexes and OD570 nm (control) represents the wells treated with a 5% glucose solution. Gel retardation assay The formation of the siRNA/R3V6 and siRNA/R3V6-BCNU complexes was verified by a gel retardation assay. A fixed amount of siRNA (0.5 mg) was mixed with increasing amounts of R3V6 and R3V6-BCNU. After 30 min of incubation at room temperature, the mixtures were analyzed on a 1% agarose gel with ethidium bromide. Heparin competition assay The siRNA/R3V6 and siRNA/R3V6-BCNU complexes were prepared at optimal transfection weight ratios (siRNA:R3V6 ¼ 1:20, siRNA:R3V6:BCNU ¼ 1:20:18). Increasing amounts of heparin were added to these complexes. After 30 min of incubation at room temperature, the mixtures were analyzed on a 1% agarose gel. Size and zeta potential The complexes were prepared at optimal ratios. After 30 min incubation, the particle sizes and zeta potentials were evaluated by the Zeta sizer Nano ZS system (Malvern Instruments, Worcestershire, UK). Serum stability assay Naked siRNA and the complexes were incubated with 50% fetal bovine serum (FBS) under shaking at 37 C, 150 rpm for 30 min. After incubation, heparin was added to complexes to dissociate the siRNA from complexes. siRNA was analyzed by 1% (w/v) agarose gel electrophoresis. Scanning electron microscopy BCNU was dissolved in deionized water at 4 mg/ml (Sigma, St. Louis, MO) and R3V6 peptide were dissolved in deionized water at 5 mg/ml. The R3V6 solution, BCNU solution and
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siRNA were mixed into deionized water one by one and blended gently each step at optimal transfection weight ratios (siRNA:R3V6 ¼ 1:20, siRNA:R3V6-BCNU ¼ 1:20:16). And then, the solution was incubated at room temperature for 30 min. The complexes in deionized water were mounted on carbon tape attached on aluminum holders, dried overnight at room temperature, and then coated with platinum while under a vacuum. The morphology and size of nanoparticles were investigated by SEM (NANO SEM 450, NOVA). Flow cytometry analysis The C6 cells were seeded at 1 105 cells/well in six-well plates. The FITC-siRNA/carriers complexes were prepared at optimal ratios and transfected into cells. After 20 h, cells were washed with PBS. The cells were resuspended in FACS buffer and centrifuged at 1200 rpm for 5 min. After supernatant was removed, the cells were resuspended in fixing buffer. The samples were analyzed by a BD FACS Calibur TM (BD Biosciences Immunocytometry Systems, San Jose, CA). Fluorescence microscopy image The C6 cells were seeded at a density of 1 105 cells/well in six-well plates. The FITC-siRNA/carrier complexes were prepared at optimal ratios. After transfection, cells were observed using fluorescence microscopy. Images were taken using NIS-element software at 20 magnification. Statistical analysis Statistical analysis was conducted using ANOVA followed by the Newman–Keuls test. All data are expressed as the average SEM, and p values less than 0.05 were considered statistically significant.
Results Formation of siRNA/R3V6-BCNU complex and physical characterization The R3V6 peptides, composed of 3-arginine and 6-valine, have a hydrophilic block and a hydrophobic block (Figure 1A). The hydrophobic drug BCNU was loaded into the core of the R3V6 micelles, forming R3V6-BCNU and small interfering RNA (siRNA) were combined with R3V6BCNU (Figure 1B). The sizes of R3V6, R3V6-BCNU, siRNA/R3V6, and siRNA/R3V6-BCNU were measured by dynamic light scattering (Table 1). The size of R3V6 in aqueous condition was around 640 nm. The size of the siRNA/R36 was smaller than that of R3V6. Similar tendency was observed in the sizes of R3V6-BCNU and siRNA/R3V6-BCNU. While the size of R3V6-BCNU was around 650 nm, that of siRNA/R3V6BCNU was around 400 nm. The results suggest that the R3V6 and R3V6-micelles may be condensed with siRNA by charge interaction. To confirm the formation of the siRNA/R3V6 and siRNA/ R3V6-BCNU complexes, a gel retardation assay was performed. siRNA (0.5 mg) was mixed with increasing amounts of the R3V6 and R3V6-BCNU peptides (Figure 2A). In the gel retardation assay, siRNA was completely retarded at a 1:2
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DOI: 10.3109/1061186X.2013.850502
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Figure 1. (A) Structure of the R3V6 peptide, (B) Schematic representation of the siRNA/R3V6-BCNU complex.
Table 1. Particle size and zeta potential.
Only R3V6 R3V6/siRNA R3V6-BCNU R3V6-BCNU/siRNA
Size (nm)
Zeta potential (mV)
644.83 56.56 400.9 70.60 652.1 43.65 401.56 103.23
17.1 0.92 24.34 2.38 20.83 0.95 20.88 1.17
weight ratio (siRNA:carrier). The stability of siRNA/R3V6BCNU was evaluated by a heparin competition assay. In a heparin competition assay, the siRNA/R3V6 and siRNA/ R3V6-BCNU complexes released siRNA in the presence of 7.5 mg of heparin (Figure 2B). The siRNA/PEI25k complex began to de-complex with 5 mg of heparin (Figure 2B). However, these results do not mean that the siRNA/R3V6 or siRNA/R3V6-BCNU complex has higher stability than the siRNA/PEI25k complex, since the siRNA/R3V6 and siRNA/ R3V6-BCNU complexes were prepared with much larger amount of carriers than the siRNA/PEI25k complex. In addition, the stability of siRNA in the presence of serum
nucleases was analyzed by serum stability assay. The siRNA/ R3V6, siRNA/R3V6-BCNU, and siRNA/PEI25k complexes were incubated with serum for 30 min. The results showed that the naked siRNA was completely degraded by nucleases for 30 min (Figure 2C). However, R3V6 and R3V6-BCNU protected siRNA from serum nucleases (Figure 2C). The morphologies of siRNA/R3V6, siRNA/R3V6-BCNU nano-particles were observed by SEM (Figure 3). Figure 3(A) shows the morphology of the siRNA/R3V6 complexes. The complexes seemed to form the tangled moniliform shape. It can be observed that the diameter of siRNA/R3V6 was 100–200 nm each. As shown in Figure 3(B), the morphology and size did not change even when the drug was loaded in particles. In vitro transfection assays The optimal ratio of the siRNA/R3V6-BCNU complex for the highest transfection efficiency was determined by in vitro transfection assays into C6 cells. psi-CHECK2 plasmid expressed Renilla and Firefly luciferases. After the delivery
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Figure 2. The siRNA/R3V6-BCNU complex formation. (A) Gel retardation assay siRNA was mixed with increasing amounts of R3V6 or R3V6-BCNU. After 30 min of incubation at room temperature, the samples were analyzed on a 1% agarose gel. (B) Heparin competition assay. The siRNA/R3V6-BCNU and siRNA/R3V6 complexes were prepared at their optimal transfection ratios (siRNA:R3V6:BCNU ¼ 1:20:18). Increasing amounts of heparin were added to these complexes, and after 30 min incubation at room temperature, the complexes were analyzed on a 1% agarose gel. (C) Serum stability assay. The siRNA/R3V6-BCNU, siRNA/R3V6, and siRNA/PEI25k complexes were incubated with serum for 30 min. After the incubation, the samples were analyzed on a 1% agarose gel.
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of Rluc-siRNA, the Firefly luciferase expression was used as an internal control. As a result, the Rluc-siRNA/R3V6-BCNU complex had the highest transfection efficiency at a 1:20 weight ratio (Rluc-siRNA:R3V6-BCNU; Figure 4). The siRNA delivery efficiency of R3V6-BCNU was compared with those of other carriers such as Lipofectamine, PEI25k, and R3V6. The cellular uptake efficiencies of the siRNA/carrier complexes were measured with FITC labeled siRNA (FITC-siRNA). The siRNA/carrier complexes and naked siRNAs were transfected into C6 cells. The FITC-siRNA cellular uptake events were measured by counting the number of fluorescent C6 cells using fluorescence activated cell sorter (FACS) analysis. The siRNA/R3V6 and siRNA/R3V6-BCNU complexes had higher cellular uptake efficiencies than the siRNA/PEI25k and siRNA/ Lipofectamine complexes (Figure 5A). These results were confirmed by fluorescence microscopy study. The results showed that the siRNA/R3V6 and siRNA/R3V6-BCNU complexes had higher fluorescence than the siRNA/PEI25k and siRNA/Lipofectamine complexes (Figure 5B). In vitro transfection assays were performed to evaluate the siRNA delivery efficiency of R3V6 by a dual luciferase assay. Since BCNU induced cell death and reduced expression of luciferase significantly without Rluc-siRNA, it interfered with proper interpretation of the results from the assays. Therefore, in the initial experiments, the siRNA delivery efficiency of R3V6 without BCNU was determined. The Rluc-siRNA/R3V6 complexes were prepared at a 1:20 weight ratio (siRNA:R3V6). The Rluc-siRNA/ PEI25k and Rluc-siRNA/Lipofectamin complexes were used as controls. As a result, R3V6 delivered Rluc-siRNA and reduced the luciferase expression more efficiently than lipofectamine or naked siRNA (Figure 6A). The delivery efficiency of R3V6 was comparable to that of PEI25k (Figure 6A). This result is not consistent with the results of FACS and fluorescence microscopy study. The siRNA/ PEI25k complex had a strong silencing effect, although the cellular uptake of the siRNA/PEI25k complex was less than the siRNA/R3V6 complex. As proved in Figure 2(B), the siRNA/PEI25k complex releases siRNA in the presence of heparin more easily than the siRNA/R3V6 complex. Therefore, the siRNA/PEI25k complex may release siRNA more easily in cytoplasm than the siRNA/R3V6 complex, inducing higher silencing effect. The delivery efficiency of R3V6 was also confirmed by the delivery of VEGF-siRNA. The VEGF siRNAs were delivered into C6 cells using R3V6, PEI25k, and Lipofectmine. The results suggest that R3V6 and PEI25k had the same delivery efficiency, which was much higher than Lipofectamine (Figure 6B). The siRNA delivery efficiency of R3V6 was also determined in the presence of BCNU. The VEGF-siRNA/ R3V6-BCNU complex was prepared and transfected into the C6 cells. The scr-siRNA/R3V6-BCNU complex and BCNU only were used as controls. The result indicated that R3V6 delivered siRNA into the cells efficiently and reduced the luciferase expression in a sequence-specific manner. The VEGF-siRNA/R3V6-BCNU complex reduced the VEGF expression, compared with the BCNU-only control and scrsiRNA/R3V6-BCNU complex (Figure 7).
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Figure 3. Scanning electron microscopy image. Scanning electron microscopy (SEM): The (A) siRNA/R3V6 and (B) siRNA/R3V6-BCNU complexes were prepared at their optimal transfection ratios.
of the complex (Figure 8A). The complexes were transfected into C6 cells and the results showed that R3V6-BCNU had cytotoxicity on C6 cells in a dose-dependent manner (Figure 8A). The cytotoxicity of siRNA/R3V6-BCNU was compared with R3V6-BCNU, siRNA/BCNU mixture and BCNU only (Figure 8B). The results showed that siRNA/R3V6-BCNU and R3V6-BCNU had similar toxicity, suggesting that the R3V6 peptides delivered BCNU at almost same efficiency, regardless of complex formation with siRNAs. In addition, the siRNA/R3V6-BCNU and R3V6-BCNU complex had higher toxicity than the siRNA/BCNU mixture and BCNU only (Figure 8B). This indicated that the R3V6 peptide delivered BCNU more efficiently than BCNU only.
Discussion Figure 4. In vitro transfection efficiency of Rluc-siRNA/R3V6-BCNU by Rluc-siRNA:R3V6 weight ratio. Rluc-siRNA/R3V6-BCNU complexes were prepared at various weight ratios and subsequently transfected into C6 cells. Transfection efficiency was measured by dual luciferase assay. *p50.05 as compared with the control, 1:10 and 1:20 weight ratios.
In vitro anti-tumor effects of siRNA/R3V6 and siRNA/ R3V6-BCNU complexes To evaluate the cytotoxic effects of the siRNA/R3V6-BCNU, the siRNA/R3V6 and siRNA/R3V6-BCNU complexes were transfected into C6 cells and cell viability was measured by MTT assay. The siRNA/R3V6-BCNU complexes were prepared at various weight ratios (siRNA:R3V6-BCNU) to confirm the dose-dependent cytotoxic effects on C6 cells
The R3V6 peptides are an amphiphilic peptide and form micelles in the aqueous solution. The hydrophobic drug could be loaded into the cores of micelles. In the previous reports, dexamethasone and BCNU were loaded into the R3V6 peptide micelles [14–16]. The reports suggest that dexamethasone and BCNU were delivered more efficiently than dexamethasone and BCNU only, suggesting that the peptide micelles are a good carrier of hydrophobic drug. In addition, plasmid DNAs were delivered using the hydrophobic drug loaded R3V6 peptide micelles more efficiently than the R3V6 peptides without drug [14–16]. However, siRNA delivery using drug-loaded R3V6 micelles has not been evaluated. In the current study, VEGF siRNA was co-delivered with BCNU for glioblastoma therapy. The results confirmed that the R3V6 peptide is an efficient carrier of both siRNA and BCNU.
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Figure 5. Cellular uptake of the siRNA/R3V6-BCNU complex. (A) Fluorescence activated cell sorter (FACS) analysis. The C6 cells transfected with FITC-siRNA/carrier complexes. After 16 h of incubation, cellular uptake efficiency was evaluated by fluorescence activated cell sorter (FACS) analysis. (B) Fluorescence microscopy image. The FITC-siRNA/carrier complexes were transfected into C6 cells. After 24 h of incubation, cellular uptake was observed by fluorescence microscopy.
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Figure 7. Delivery efficiency of R3V6-BCNU in the presence of BCNU The siRNA/R3V6-BCNU complexes were prepared and delivered into C6 cells. The same amount of BCNU as R3V6-BCNU was added to the control and naked VEGF-siRNA. The siRNA delivery efficiency was evaluated with VEGF-siRNA. *p50.05 compared with all other groups.
Figure 6. Delivery efficiencies of naked siRNA, R3V6, PEI25k and Lipofectamine in the absence of BCNU. The siRNA/R3V6, siRNA/ PEI25k, and siRNA/Lipofectamine complexes were prepared and siRNA delivery efficiencies were measured in C6 cells. The siRNA delivery efficiencies were evaluated with (A) Rluc-siRNA and (B) VEGF-siRNA. *p50.05 as compared with all other groups, **p50.0005 as compared with control, VEGF-siRNA only, VEGF-siRNA/Lipofectamine, scrsiRNA/R3V6.
In this study, the ratio between siRNA and R3V6-BCNU was optimized in terms of delivery efficiency. However, the ratio should also be optimized in terms of anti-tumor effect. The in vitro transfection assays showed that R3V6-BCNU reduced the Renilla luciferase expression at a wide range of weight ratios. Although the siRNA/R3V6-BCNU had the highest delivery efficiency at a 1:20 weight ratio, the complexes had significant silencing effect at a 1:10 or 1:30 weight ratio (Figure 4). Therefore, the weight ratio between
siRNA and R3V6-BCNU may be altered in this range, depending on the desired results. For example, the complexes may be prepared at a higher weight ratio for higher cytotoxic effect. Chemotherapy is often a cause of significant side-effects. Efficient drug delivery carriers may increase the delivery efficiency and reduce the required dose for the same effect. This may decrease side effects of chemotherapy. BCNU is commonly used for treatment of brain cancers and can lengthen survival times. However, BCNU has various side -effects, limiting its application. To minimize undesirable side effects, the delivery carrier that enhances the BCNU delivery efficiency should be developed. The MTT assay results showed that R3V6-BCNU enhanced the anti-tumor effect of BCNU (Figure 8B), suggesting that R3V6 may be useful to decrease the required dose of BCNU. In the previous studies, the R3V6 peptides did not have significant cytotoxicity to various cells [13–16]. Therefore, the toxicity of R3V6-BCNU may be originated from BCNU, which was loaded in the cores of the R3V6 peptide micelles. However, the VEGF-siRNA delivery by the siRNA/R3V6-BCNU complex did not have significant effect on the viability of C6 cells, compared with R3V6-BCNU (Figure 8B). This suggests that the binding of siRNA to R3V6-BCNU does not change the BCNU delivery efficiency of the R3V6 peptides. In summary, R3V6-BCNU had higher siRNA delivery efficiency than Lipofectamine or naked siRNA. The level of the delivery efficiency was comparable to that of PEI25k. Furthermore, R3V6-BCNU delivered hydrophobic BCNU efficiently into C6 glioblastoma cells, compared with BCNU only. Therefore, R3V6 may be a useful carrier for the combined delivery of BCNU and VEGF-siRNA for the treatment of glioblastoma.
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Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This work was financially supported by a grant from the National Research Foundation of Korea, funded by the Ministry of Science, ICT and Future Planning (2013K000257).
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References
Figure 8. Anti-tumor effect of R3V6-BCNU. (A) Cytotoxicity of siRNA/ R3V6-BCNU complex, depending on the dose of R3V6-BCNU. The siRNA/R3V6 complexes were prepared by addition of increasing amount of R3V6-BCNU to the fixed amount of Rluc-siRNA. The complexes were added to the cells and then, cell viability was measured by MTT assay. *p50.05 as compared with the control, 1:1 and 1:5 weight ratios. (B) Cytotoxicities of R3V6-BCNU, siRNA/R3V6-BCNU complex, siRNA/BCNU mixture, and BCNU only. The siRNA/R3V6-BCNU complexes were prepared as described in the materials and methods. The complexes were transfected into C6 cells. R3V6-BCNU, siRNA/BCNU mixture, and BCNU only were used as controls. Cell viability was measured by MTT assay. **p50.005 compared with the control, siRNA/ BCNU complex, and BCNU only.
1. Mrugala MM, Chamberlain MC. Mechanisms of disease: temozolomide and glioblastoma – look to the future. Nat Clin Pract Oncol 2008;5:476–86. 2. Van Meir EG, Hadjipanayis CG, Norden AD, et al. Exciting new advances in neuro-oncology: the avenue to a cure for malignant glioma. CA Cancer J Clin 2010;60:166–93. 3. Chamberlain MC, Kormanik PA. Practical guidelines for the treatment of malignant gliomas. West J Med 1998;168: 114–20. 4. Liu S, Guo Y, Huang R, et al. Gene and doxorubicin co-delivery system for targeting therapy of glioma. Biomaterials 2012;33: 4907–16. 5. Meister G, Tuschl T. Mechanisms of gene silencing by doublestranded RNA. Nature 2004;431:343–9. 6. Oh YK, Park TG. siRNA delivery systems for cancer treatment. Adv Drug Deliv Rev 2009;61:850–62. 7. Roskoski Jr R. Vascular endothelial growth factor (VEGF) signaling in tumor progression. Crit Rev Oncol Hematol 2007;62: 179–213. 8. Skobe M, Hawighorst T, Jackson DG, et al. Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat Med 2001;7:192–8. 9. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000;407:249–57. 10. Pralhad T, Madhusudan S, Rajendrakumar K. Concept, mechanisms and therapeutics of angiogenesis in cancer and other diseases. J Pharm Pharmacol 2003;55:1045–53. 11. Westphal M, Hilt DC, Bortey E, et al. A phase 3 trial of local chemotherapy with biodegradable carmustine (BCNU) wafers (Gliadel wafers) in patients with primary malignant glioma. Neuro Oncol 2003;5:79–88. 12. Drablos F, Feyzi E, Aas PA, et al. Alkylation damage in DNA and RNA–repair mechanisms and medical significance. DNA Repair (Amst) 2004;3:1389–407. 13. Ryu DW, Kim HA, Song H, et al. Amphiphilic peptides with arginines and valines for the delivery of plasmid DNA. J Cell Biochem 2011;112:1458–66. 14. Lee J, Hyun H, Kim J, et al. Dexamethasone-loaded peptide micelles for delivery of the heme oxygenase-1 gene to ischemic brain. J Control Rel 2012;158:131–8. 15. Park JH, Kim HA, Cho SH, Lee M. Characterization of hydrophobic anti-cancer drug-loaded amphiphilic peptides as a gene carrier. J Cell Biochem 2012;113:1645–53. 16. Park JH, Kim HA, Park JH, Lee M. Amphiphilic peptide carrier for the combined delivery of curcumin and plasmid DNA into the lungs. Biomaterials 2012;33:6542–50.
ORIGINAL ARTICLE
Combined delivery of BCNU and VEGF siRNA using amphiphilic peptides for glioblastoma Na Yi, Binna Oh, Hyun Ah Kim, and Minhyung Lee
Abstract
Keywords
Combined delivery of chemical drug and therapeutic gene has been introduced as an efficient method for the treatment of cancers such as glioblastoma. In this study, bis-chloroethylnitrosourea (BCNU) and vascular endothelial growth factor (VEGF) small interfering RNA (VEGFsiRNA) were co-delivered into C6 glioblastoma cells using a non-toxic peptide-based carrier. The R3V6 peptides, which are composed of 3-arginine and 6-valine, formed self-assembled micelles in aqueous solution. BCNU, a hydrophobic anti-cancer drug, was loaded into the hydrophobic core of the micelles, forming BCNU-loaded R3V6 micelles (R3V6-BCNU). In gel retardation assay, R3V6-BCNU formed a stable complex with siRNA. In vitro transfection assay showed that the VEGF-siRNA/R3V6-BCNU complex had the highest transfection efficiency into C6 cells at a 1:20 weight ratio (VEGF-siRNA:R3V6-BCNU). In addition, the VEGF-siRNA/R3V6BCNU complexes had higher delivery efficiency than lipofectamine or naked siRNA. VEGF expressions were remarkably decreased by transfection of the VEGF-siRNA/R3V6 or VEGFsiRNA/R3V6-BCNU complexes. Furthermore, R3V6-BCNU delivered BCNU more efficiently into the cells than BCNU only. Therefore, R3V6 delivered both VEGF-siRNA and BCNU efficiently into the glioblastoma cells. The results suggest that R3V6-BCNU may be useful for combined delivery of siRNA and chemical drug into cancer cells.
Combined delivery, glioblastoma, peptide micelles, VEGF siRNA
Introduction Glioblastoma multiform (GBM) is considered a devastating malignant primary brain tumor in humans. Although advances in other solid tumor therapies have improved patient survival, GBM prognosis is still poor. With standard treatment, the median survival for patients with GBM is 14 months after diagnosis [1,2]. Conventional treatment usually includes surgery, radiation therapy, and chemotherapy. Generally, the first step in the treatment of GBM is surgery to remove as much tumor tissue as possible [3]. However, complete removal is impossible because GBMs have finger-like tentacles. After surgery, radiotherapy and chemotherapy and/or bio-therapy are then used to treat the remaining tumor. These therapies are useful in the treatment of GBM, but they have undesirable side effects. Therefore, a combination of gene therapy and chemotherapy has been investigated as a new potential treatment for gliomas, suggesting that co-delivery of anticancer drugs and siRNA
Address for correspondence: Minhyung Lee, PhD, Department of Bioengineering, College of Engineering, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133 791, Republic of Korea. Tel: 82 2 2220 4456. Fax: 82 2 2220 1998. E-mail: minhyung@ hanyang.ac.kr
History Received 14 May 2013 Accepted 27 September 2013 Published online 12 November 2013
using non-viral delivery carriers may have tumor-suppressive effects [4]. RNA interference (RNAi) is regarded as an effective technology for gene silencing in gene therapy. The double strand RNA-based molecule, siRNA, has potential as a biopharmaceutical therapeutic [5]. As RNAi interferes with translation, and not with DNA transcription, siRNA does not interact with chromosomal DNA. This lack of DNA interaction greatly reduces concerns about possible adverse gene alterations that might result from DNA-based gene therapy. The interaction of siRNA with mRNA, not proteins, also reduces the production of harmful proteins before synthesis [6]. Vascular endothelial growth factor (VEGF), a critical factor for blood vessel growth, has been implicated as a crucial signal in tumor development by promoting angiogenesis [7–9]. Inhibition of VEGF to interfere with tumor neovascularization is the most effective strategy in angiogenesis [10]. Meanwhile, bis-chloroethylnitrosourea (BCNU) has been widely used for the treatment of several types of brain cancer and is able to form inter-strand crosslinks in DNA, which prevents DNA replication and DNA transcription [11,12]. An amphiphilic peptide with 3-arginine and 6-valine (R3V6) has been evaluated as a gene carrier. R3V6 has been employed to transport both siRNA and DNA into cells without cytotoxicity [13,14]. In addition, R3V6 peptides have a higher transfection efficiency whether a hydrophobic drug was loaded or not [15,16]. In this article, the
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Department of Bioengineering, College of Engineering, Hanyang University, Seoul, Republic of Korea
DOI: 10.3109/1061186X.2013.850502
Combined delivery of siRNA and chemical drug into cancer cells
chemotherapeutic drug BCNU and VEGF small interfering RNA (VEGF-siRNA) were co-delivered to treat glioblastoma using a peptide-based delivery carrier. Despite the anti-cancer effects of BCNU, its use in chemotherapy is limited due to drug resistance and toxic side effects. Therefore, our combined approach may generate significant synergistic effects for the treatment of glioblastoma.
Materials and methods
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Synthesis of R3V6 peptides and siRNA oligonucleotides The R3V6 peptides, composed of 3-arginine and 6-valine, were synthesized chemically and purified by C18 reversephase chromatography (Peptron Co., Daejeon, Korea). The peptides were dissolved in distilled water at 5 mg/ml and were stored at 70 C. VEGF-siRNA and scrambled small interfering RNA (scr-siRNA) were synthesized by Bioneer (Daejeon, Korea). Renilla luciferase small interfering RNA (Rluc-siRNA) was synthesized by Genolution (Seoul, Korea). The siRNA sequences are as follows: VEGF, 50 -AAACCGGGAUUUCUUGCGCUUUCGU-30 ; Scrambled, 50 -UUCUCCGAACGUGUCACGUTT-30 ; Renilla luciferase, 50 -GGCCUUUCACUACUCCUAC UUUU-30 . Cell culture C6 cells, originating from a rat brain glioblastoma, were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (PS). The cells were seeded onto a 75 cm2 cell culture flask and cultured at 37 C in a humidified incubator under 5% CO2 and 95% air. Preparation of the siRNA/R3V6 and siRNA/R3V6-BCNU complexes BCNU was dissolved in distilled water at 5 mg/ml (Sigma, St. Louis, MO). The BCNU solution, R3V6 peptide solution and siRNA were mixed into water one by one and blended gently at each step. Then, the solution was incubated at room temperature for 30 min. To determine the optimal ratios of siRNA to R3V6 and siRNA to R3V6-BCNU, siRNA/R3V6 and siRNA/R3V6-BCNU complexes were prepared at various weight ratios. The weight ratio of R3V6 peptide and BCNU was fixed at 1:0.6 (R3V6:BCNU). For all other in vitro and in vivo experiments, the weight ratio of siRNA to R3V6 was fixed at 1:20 (siRNA:R3V6). The siRNA/PEI25k complex was prepared at a 1:1 weight ratio. The siRNA/Lipofectamine complex was prepared at a 1:5 ratio (w/v) as suggested in the manufacturer’s manual. In vitro transfection of C6 cells with Rluc-siRNA To monitor the silencing effect of siRNA, a Renilla/Firefly luciferase reporter plasmid and a psiCHECK-2 vector were transfected into the C6 cells. The C6 cells were seeded in a 100 20 mm cell culture dish and incubated for 24 h at 37 C in a humidified incubator under 5% CO2 and 95% air. Before transfection, the cell culture medium was replaced
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with FBS-free DMEM, and then the transfection complexes were added. Next, the psiCHECK2/PEI25k complex was added. The cells were incubated for 4 h at 37 C in a humidified incubator under 5% CO2 and 95% air. After 4 h, the cell culture medium was changed to DMEM containing 10% FBS and 1% PS, the cells were incubated for an additional 48 h at 37 C in a humidified incubator under 5% CO2 and 95% air. The C6 cells were washed and seeded in a 12-well plate at a density of 3.5 104 cells/well. For the dual luciferase assay, the cells were seeded at a density of 3.5 104 cells/well in 12-well plates (Falcon Co., Becton Dickenson, Franklin Lakes, NJ). The cell culture medium was replaced with FBS-free DMEM and then the Renilla luciferase siRNA (Rluc-siRNA)/carrier complexes prepared at optimal ratios were added. The cells were incubated for 4 h at 37 C in a humidified incubator under 5% CO2 and 95% air. After 4 h, the cell culture medium was changed to DMEM containing 10% FBS and 1% PS, and the cells were incubated for an additional 48 h at 37 C in a humidified incubator under 5% CO2 and 95% air. Dual luciferase assay After 48 h of incubation, the DMEM was removed from each well of the plate. The C6 cells were washed with PBS, 120 ml of passive lysis buffer (Promega, Madison, WI) was added to each well, and the plate was incubated for 15 min at room temperature. The cells were harvested and centrifuged at 13 000 rpm for 5 min. The supernatant was transferred to fresh tubes. A dual luciferase reporter assay system (Promega) was purchased and used according to the manufacturer’s instructions. Luciferase activities were measured using a 96-well plated luminometer (Berthold Detection System GmbH, Pforzheim, Germany) in relative light units (RLU). The final values of luciferase activity were calculated as (Renilla RLU/Firefly RLU) 100 (%). In vitro transfection of C6 cells with VEGF-siRNA and enzyme-linked immunosorbent assay (ELISA) The transfection efficiency was tested by VEGF ELISA. For the VEGF luciferase assay, the cells were seeded at a density of 3.5 104 cells/well in 12-well plates (Falcon). The cell culture medium was replaced with FBS-free DMEM and the VEGF-siRNA/carrier complexes prepared at optimal ratios were added. The cells were incubated for 4 h at 37 C in a humidified incubator under 5% CO2 and 95% air. After 4 h, the cell culture medium was changed to DMEM containing 10% FBS and 1% PS, and the cells were incubated for an additional 48 h in a hypoxia chamber maintained at 1% oxygen. After 48 h incubation, the culture medium was collected and centrifuged. A human VEGF ELISA kit (PeproTech, Rocky Hill, NJ) was used. After the addition of a 100 ml sample, mediums were then incubated for 2 h at room temperature. Following a brief wash, 100 ml of detection antibody solution was added to each well and incubated for 2 h. Conjugate buffer of 100 ml was added and incubated for 30 min at room temperature. Finally, 100 ml of a substrate solution for color development was added. The absorbance was measured at 450 nm by a Micro plate reader 680 from
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Bio-Rad (Hercules, CA). The protein concentration of the extract was measured by a Pierce BCA Protein Assay Kit (Pierce Biochemical, Rockford, IL).
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MTT assay To evaluate cell viability, C6 cells were seeded in a 24-well plate at a density of 5 104 cells/well. The culture plates were incubated for 24 h at 37 C in a humidified incubator under 5% CO2 and 95% air. After incubation, the siRNA/carrier complexes were transfected at optimal ratios in the same way as previously described. After 24 h, 40 ml of 5 mg/ml MTT solution was added to each well. The plates were then incubated for 4 h at 37 C in a humidified incubator under 5% CO2 and 95% air. After 4 h of incubation, the medium of each well was removed and 700 ml of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals formed by the living cells. Cell viability (%) was calculated according to the following equation: Cell viability ð%Þ ¼ OD570nmðsampleÞ =OD570nmðcontrolÞ 100, where OD570nm(sample) represents the wells treated with the plasmid/peptides complexes and OD570 nm (control) represents the wells treated with a 5% glucose solution. Gel retardation assay The formation of the siRNA/R3V6 and siRNA/R3V6-BCNU complexes was verified by a gel retardation assay. A fixed amount of siRNA (0.5 mg) was mixed with increasing amounts of R3V6 and R3V6-BCNU. After 30 min of incubation at room temperature, the mixtures were analyzed on a 1% agarose gel with ethidium bromide. Heparin competition assay The siRNA/R3V6 and siRNA/R3V6-BCNU complexes were prepared at optimal transfection weight ratios (siRNA:R3V6 ¼ 1:20, siRNA:R3V6:BCNU ¼ 1:20:18). Increasing amounts of heparin were added to these complexes. After 30 min of incubation at room temperature, the mixtures were analyzed on a 1% agarose gel. Size and zeta potential The complexes were prepared at optimal ratios. After 30 min incubation, the particle sizes and zeta potentials were evaluated by the Zeta sizer Nano ZS system (Malvern Instruments, Worcestershire, UK). Serum stability assay Naked siRNA and the complexes were incubated with 50% fetal bovine serum (FBS) under shaking at 37 C, 150 rpm for 30 min. After incubation, heparin was added to complexes to dissociate the siRNA from complexes. siRNA was analyzed by 1% (w/v) agarose gel electrophoresis. Scanning electron microscopy BCNU was dissolved in deionized water at 4 mg/ml (Sigma, St. Louis, MO) and R3V6 peptide were dissolved in deionized water at 5 mg/ml. The R3V6 solution, BCNU solution and
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siRNA were mixed into deionized water one by one and blended gently each step at optimal transfection weight ratios (siRNA:R3V6 ¼ 1:20, siRNA:R3V6-BCNU ¼ 1:20:16). And then, the solution was incubated at room temperature for 30 min. The complexes in deionized water were mounted on carbon tape attached on aluminum holders, dried overnight at room temperature, and then coated with platinum while under a vacuum. The morphology and size of nanoparticles were investigated by SEM (NANO SEM 450, NOVA). Flow cytometry analysis The C6 cells were seeded at 1 105 cells/well in six-well plates. The FITC-siRNA/carriers complexes were prepared at optimal ratios and transfected into cells. After 20 h, cells were washed with PBS. The cells were resuspended in FACS buffer and centrifuged at 1200 rpm for 5 min. After supernatant was removed, the cells were resuspended in fixing buffer. The samples were analyzed by a BD FACS Calibur TM (BD Biosciences Immunocytometry Systems, San Jose, CA). Fluorescence microscopy image The C6 cells were seeded at a density of 1 105 cells/well in six-well plates. The FITC-siRNA/carrier complexes were prepared at optimal ratios. After transfection, cells were observed using fluorescence microscopy. Images were taken using NIS-element software at 20 magnification. Statistical analysis Statistical analysis was conducted using ANOVA followed by the Newman–Keuls test. All data are expressed as the average SEM, and p values less than 0.05 were considered statistically significant.
Results Formation of siRNA/R3V6-BCNU complex and physical characterization The R3V6 peptides, composed of 3-arginine and 6-valine, have a hydrophilic block and a hydrophobic block (Figure 1A). The hydrophobic drug BCNU was loaded into the core of the R3V6 micelles, forming R3V6-BCNU and small interfering RNA (siRNA) were combined with R3V6BCNU (Figure 1B). The sizes of R3V6, R3V6-BCNU, siRNA/R3V6, and siRNA/R3V6-BCNU were measured by dynamic light scattering (Table 1). The size of R3V6 in aqueous condition was around 640 nm. The size of the siRNA/R36 was smaller than that of R3V6. Similar tendency was observed in the sizes of R3V6-BCNU and siRNA/R3V6-BCNU. While the size of R3V6-BCNU was around 650 nm, that of siRNA/R3V6BCNU was around 400 nm. The results suggest that the R3V6 and R3V6-micelles may be condensed with siRNA by charge interaction. To confirm the formation of the siRNA/R3V6 and siRNA/ R3V6-BCNU complexes, a gel retardation assay was performed. siRNA (0.5 mg) was mixed with increasing amounts of the R3V6 and R3V6-BCNU peptides (Figure 2A). In the gel retardation assay, siRNA was completely retarded at a 1:2
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Figure 1. (A) Structure of the R3V6 peptide, (B) Schematic representation of the siRNA/R3V6-BCNU complex.
Table 1. Particle size and zeta potential.
Only R3V6 R3V6/siRNA R3V6-BCNU R3V6-BCNU/siRNA
Size (nm)
Zeta potential (mV)
644.83 56.56 400.9 70.60 652.1 43.65 401.56 103.23
17.1 0.92 24.34 2.38 20.83 0.95 20.88 1.17
weight ratio (siRNA:carrier). The stability of siRNA/R3V6BCNU was evaluated by a heparin competition assay. In a heparin competition assay, the siRNA/R3V6 and siRNA/ R3V6-BCNU complexes released siRNA in the presence of 7.5 mg of heparin (Figure 2B). The siRNA/PEI25k complex began to de-complex with 5 mg of heparin (Figure 2B). However, these results do not mean that the siRNA/R3V6 or siRNA/R3V6-BCNU complex has higher stability than the siRNA/PEI25k complex, since the siRNA/R3V6 and siRNA/ R3V6-BCNU complexes were prepared with much larger amount of carriers than the siRNA/PEI25k complex. In addition, the stability of siRNA in the presence of serum
nucleases was analyzed by serum stability assay. The siRNA/ R3V6, siRNA/R3V6-BCNU, and siRNA/PEI25k complexes were incubated with serum for 30 min. The results showed that the naked siRNA was completely degraded by nucleases for 30 min (Figure 2C). However, R3V6 and R3V6-BCNU protected siRNA from serum nucleases (Figure 2C). The morphologies of siRNA/R3V6, siRNA/R3V6-BCNU nano-particles were observed by SEM (Figure 3). Figure 3(A) shows the morphology of the siRNA/R3V6 complexes. The complexes seemed to form the tangled moniliform shape. It can be observed that the diameter of siRNA/R3V6 was 100–200 nm each. As shown in Figure 3(B), the morphology and size did not change even when the drug was loaded in particles. In vitro transfection assays The optimal ratio of the siRNA/R3V6-BCNU complex for the highest transfection efficiency was determined by in vitro transfection assays into C6 cells. psi-CHECK2 plasmid expressed Renilla and Firefly luciferases. After the delivery
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Figure 2. The siRNA/R3V6-BCNU complex formation. (A) Gel retardation assay siRNA was mixed with increasing amounts of R3V6 or R3V6-BCNU. After 30 min of incubation at room temperature, the samples were analyzed on a 1% agarose gel. (B) Heparin competition assay. The siRNA/R3V6-BCNU and siRNA/R3V6 complexes were prepared at their optimal transfection ratios (siRNA:R3V6:BCNU ¼ 1:20:18). Increasing amounts of heparin were added to these complexes, and after 30 min incubation at room temperature, the complexes were analyzed on a 1% agarose gel. (C) Serum stability assay. The siRNA/R3V6-BCNU, siRNA/R3V6, and siRNA/PEI25k complexes were incubated with serum for 30 min. After the incubation, the samples were analyzed on a 1% agarose gel.
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of Rluc-siRNA, the Firefly luciferase expression was used as an internal control. As a result, the Rluc-siRNA/R3V6-BCNU complex had the highest transfection efficiency at a 1:20 weight ratio (Rluc-siRNA:R3V6-BCNU; Figure 4). The siRNA delivery efficiency of R3V6-BCNU was compared with those of other carriers such as Lipofectamine, PEI25k, and R3V6. The cellular uptake efficiencies of the siRNA/carrier complexes were measured with FITC labeled siRNA (FITC-siRNA). The siRNA/carrier complexes and naked siRNAs were transfected into C6 cells. The FITC-siRNA cellular uptake events were measured by counting the number of fluorescent C6 cells using fluorescence activated cell sorter (FACS) analysis. The siRNA/R3V6 and siRNA/R3V6-BCNU complexes had higher cellular uptake efficiencies than the siRNA/PEI25k and siRNA/ Lipofectamine complexes (Figure 5A). These results were confirmed by fluorescence microscopy study. The results showed that the siRNA/R3V6 and siRNA/R3V6-BCNU complexes had higher fluorescence than the siRNA/PEI25k and siRNA/Lipofectamine complexes (Figure 5B). In vitro transfection assays were performed to evaluate the siRNA delivery efficiency of R3V6 by a dual luciferase assay. Since BCNU induced cell death and reduced expression of luciferase significantly without Rluc-siRNA, it interfered with proper interpretation of the results from the assays. Therefore, in the initial experiments, the siRNA delivery efficiency of R3V6 without BCNU was determined. The Rluc-siRNA/R3V6 complexes were prepared at a 1:20 weight ratio (siRNA:R3V6). The Rluc-siRNA/ PEI25k and Rluc-siRNA/Lipofectamin complexes were used as controls. As a result, R3V6 delivered Rluc-siRNA and reduced the luciferase expression more efficiently than lipofectamine or naked siRNA (Figure 6A). The delivery efficiency of R3V6 was comparable to that of PEI25k (Figure 6A). This result is not consistent with the results of FACS and fluorescence microscopy study. The siRNA/ PEI25k complex had a strong silencing effect, although the cellular uptake of the siRNA/PEI25k complex was less than the siRNA/R3V6 complex. As proved in Figure 2(B), the siRNA/PEI25k complex releases siRNA in the presence of heparin more easily than the siRNA/R3V6 complex. Therefore, the siRNA/PEI25k complex may release siRNA more easily in cytoplasm than the siRNA/R3V6 complex, inducing higher silencing effect. The delivery efficiency of R3V6 was also confirmed by the delivery of VEGF-siRNA. The VEGF siRNAs were delivered into C6 cells using R3V6, PEI25k, and Lipofectmine. The results suggest that R3V6 and PEI25k had the same delivery efficiency, which was much higher than Lipofectamine (Figure 6B). The siRNA delivery efficiency of R3V6 was also determined in the presence of BCNU. The VEGF-siRNA/ R3V6-BCNU complex was prepared and transfected into the C6 cells. The scr-siRNA/R3V6-BCNU complex and BCNU only were used as controls. The result indicated that R3V6 delivered siRNA into the cells efficiently and reduced the luciferase expression in a sequence-specific manner. The VEGF-siRNA/R3V6-BCNU complex reduced the VEGF expression, compared with the BCNU-only control and scrsiRNA/R3V6-BCNU complex (Figure 7).
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Figure 3. Scanning electron microscopy image. Scanning electron microscopy (SEM): The (A) siRNA/R3V6 and (B) siRNA/R3V6-BCNU complexes were prepared at their optimal transfection ratios.
of the complex (Figure 8A). The complexes were transfected into C6 cells and the results showed that R3V6-BCNU had cytotoxicity on C6 cells in a dose-dependent manner (Figure 8A). The cytotoxicity of siRNA/R3V6-BCNU was compared with R3V6-BCNU, siRNA/BCNU mixture and BCNU only (Figure 8B). The results showed that siRNA/R3V6-BCNU and R3V6-BCNU had similar toxicity, suggesting that the R3V6 peptides delivered BCNU at almost same efficiency, regardless of complex formation with siRNAs. In addition, the siRNA/R3V6-BCNU and R3V6-BCNU complex had higher toxicity than the siRNA/BCNU mixture and BCNU only (Figure 8B). This indicated that the R3V6 peptide delivered BCNU more efficiently than BCNU only.
Discussion Figure 4. In vitro transfection efficiency of Rluc-siRNA/R3V6-BCNU by Rluc-siRNA:R3V6 weight ratio. Rluc-siRNA/R3V6-BCNU complexes were prepared at various weight ratios and subsequently transfected into C6 cells. Transfection efficiency was measured by dual luciferase assay. *p50.05 as compared with the control, 1:10 and 1:20 weight ratios.
In vitro anti-tumor effects of siRNA/R3V6 and siRNA/ R3V6-BCNU complexes To evaluate the cytotoxic effects of the siRNA/R3V6-BCNU, the siRNA/R3V6 and siRNA/R3V6-BCNU complexes were transfected into C6 cells and cell viability was measured by MTT assay. The siRNA/R3V6-BCNU complexes were prepared at various weight ratios (siRNA:R3V6-BCNU) to confirm the dose-dependent cytotoxic effects on C6 cells
The R3V6 peptides are an amphiphilic peptide and form micelles in the aqueous solution. The hydrophobic drug could be loaded into the cores of micelles. In the previous reports, dexamethasone and BCNU were loaded into the R3V6 peptide micelles [14–16]. The reports suggest that dexamethasone and BCNU were delivered more efficiently than dexamethasone and BCNU only, suggesting that the peptide micelles are a good carrier of hydrophobic drug. In addition, plasmid DNAs were delivered using the hydrophobic drug loaded R3V6 peptide micelles more efficiently than the R3V6 peptides without drug [14–16]. However, siRNA delivery using drug-loaded R3V6 micelles has not been evaluated. In the current study, VEGF siRNA was co-delivered with BCNU for glioblastoma therapy. The results confirmed that the R3V6 peptide is an efficient carrier of both siRNA and BCNU.
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Figure 5. Cellular uptake of the siRNA/R3V6-BCNU complex. (A) Fluorescence activated cell sorter (FACS) analysis. The C6 cells transfected with FITC-siRNA/carrier complexes. After 16 h of incubation, cellular uptake efficiency was evaluated by fluorescence activated cell sorter (FACS) analysis. (B) Fluorescence microscopy image. The FITC-siRNA/carrier complexes were transfected into C6 cells. After 24 h of incubation, cellular uptake was observed by fluorescence microscopy.
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Figure 7. Delivery efficiency of R3V6-BCNU in the presence of BCNU The siRNA/R3V6-BCNU complexes were prepared and delivered into C6 cells. The same amount of BCNU as R3V6-BCNU was added to the control and naked VEGF-siRNA. The siRNA delivery efficiency was evaluated with VEGF-siRNA. *p50.05 compared with all other groups.
Figure 6. Delivery efficiencies of naked siRNA, R3V6, PEI25k and Lipofectamine in the absence of BCNU. The siRNA/R3V6, siRNA/ PEI25k, and siRNA/Lipofectamine complexes were prepared and siRNA delivery efficiencies were measured in C6 cells. The siRNA delivery efficiencies were evaluated with (A) Rluc-siRNA and (B) VEGF-siRNA. *p50.05 as compared with all other groups, **p50.0005 as compared with control, VEGF-siRNA only, VEGF-siRNA/Lipofectamine, scrsiRNA/R3V6.
In this study, the ratio between siRNA and R3V6-BCNU was optimized in terms of delivery efficiency. However, the ratio should also be optimized in terms of anti-tumor effect. The in vitro transfection assays showed that R3V6-BCNU reduced the Renilla luciferase expression at a wide range of weight ratios. Although the siRNA/R3V6-BCNU had the highest delivery efficiency at a 1:20 weight ratio, the complexes had significant silencing effect at a 1:10 or 1:30 weight ratio (Figure 4). Therefore, the weight ratio between
siRNA and R3V6-BCNU may be altered in this range, depending on the desired results. For example, the complexes may be prepared at a higher weight ratio for higher cytotoxic effect. Chemotherapy is often a cause of significant side-effects. Efficient drug delivery carriers may increase the delivery efficiency and reduce the required dose for the same effect. This may decrease side effects of chemotherapy. BCNU is commonly used for treatment of brain cancers and can lengthen survival times. However, BCNU has various side -effects, limiting its application. To minimize undesirable side effects, the delivery carrier that enhances the BCNU delivery efficiency should be developed. The MTT assay results showed that R3V6-BCNU enhanced the anti-tumor effect of BCNU (Figure 8B), suggesting that R3V6 may be useful to decrease the required dose of BCNU. In the previous studies, the R3V6 peptides did not have significant cytotoxicity to various cells [13–16]. Therefore, the toxicity of R3V6-BCNU may be originated from BCNU, which was loaded in the cores of the R3V6 peptide micelles. However, the VEGF-siRNA delivery by the siRNA/R3V6-BCNU complex did not have significant effect on the viability of C6 cells, compared with R3V6-BCNU (Figure 8B). This suggests that the binding of siRNA to R3V6-BCNU does not change the BCNU delivery efficiency of the R3V6 peptides. In summary, R3V6-BCNU had higher siRNA delivery efficiency than Lipofectamine or naked siRNA. The level of the delivery efficiency was comparable to that of PEI25k. Furthermore, R3V6-BCNU delivered hydrophobic BCNU efficiently into C6 glioblastoma cells, compared with BCNU only. Therefore, R3V6 may be a useful carrier for the combined delivery of BCNU and VEGF-siRNA for the treatment of glioblastoma.
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J Drug Target, 2014; 22(2): 156–164
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This work was financially supported by a grant from the National Research Foundation of Korea, funded by the Ministry of Science, ICT and Future Planning (2013K000257).
Journal of Drug Targeting Downloaded from informahealthcare.com by Laurentian University on 10/13/14 For personal use only.
References
Figure 8. Anti-tumor effect of R3V6-BCNU. (A) Cytotoxicity of siRNA/ R3V6-BCNU complex, depending on the dose of R3V6-BCNU. The siRNA/R3V6 complexes were prepared by addition of increasing amount of R3V6-BCNU to the fixed amount of Rluc-siRNA. The complexes were added to the cells and then, cell viability was measured by MTT assay. *p50.05 as compared with the control, 1:1 and 1:5 weight ratios. (B) Cytotoxicities of R3V6-BCNU, siRNA/R3V6-BCNU complex, siRNA/BCNU mixture, and BCNU only. The siRNA/R3V6-BCNU complexes were prepared as described in the materials and methods. The complexes were transfected into C6 cells. R3V6-BCNU, siRNA/BCNU mixture, and BCNU only were used as controls. Cell viability was measured by MTT assay. **p50.005 compared with the control, siRNA/ BCNU complex, and BCNU only.
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