Arch. Pharm. Res. DOI 10.1007/s12272-014-0359-8
RESEARCH ARTICLE
Evaluation of multimeric siRNA conjugates for efficient protamine-based delivery into breast cancer cells Hyundong Yoo • Hyejung Mok
Received: 10 December 2013 / Accepted: 16 February 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract Despite the preferable properties of well-defined cationic peptides for small interfering RNA (siRNA) delivery, their application as siRNA carriers remains limited due to their poor binding affinity with short-chain RNAs. In this study, we investigated the feasibility of a novel strategy for circumventing this limitation, by assessing the utility of multimeric conjugates of siRNA for improving the binding affinity of siRNAs with cationic peptides and the extent of intracellular delivery. Protamine, a natural and arginine-rich peptide, was used to produce stably condensed polyelectrolyte complexes (PECs) with multimeric siRNAs (multisiRNA) with a size of 120 nm while conventional siRNA/ protamine particles are over 500 nm. The formulated multisiRNA/protamine PECs showed greatly enhanced stability, intracellular uptake, and biocompatibility compared to conventional, monomeric (mono)-siRNA/protamine particles. With the addition of chloroquine, multi-siRNA/protamine PECs successfully inhibited target gene expression in MDA-MB-435 cells, a breast cancer cell line, even in the presence of serum protein. This study demonstrates that multi-siRNA conjugates greatly facilitate the formulation of nano-sized protamine-based carriers and significantly improve intracellular delivery in vitro compared to common siRNAs, and therefore may provide a platform for the design of peptide-based siRNA delivery systems for in vivo applications. Keywords Functional peptide Protamine Multimeric siRNA Charge density Gene silencing
H. Yoo H. Mok (&) Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea e-mail: [email protected]
Introduction Peptide-based carriers have received attention as promising candidate materials for the delivery of therapeutic genes, drugs, and proteins due to their favorable characteristics, e.g., well-defined structure, ease of synthesis, reproducibility, and biocompatibility (Huang et al. 2013; Yewale et al. 2013). Thus, diverse functional peptides, such as fusogenic peptides and cell-penetrating peptides, have been intensively studied as potential carriers for therapeutic genes such as small interfering RNAs (siRNAs) (Kumar et al. 2008; Meade and Dowdy 2007; Scholz and Wagner 2012). Peptide carriers allow enhanced delivery of nucleic acids by forming nano-sized particles via noncovalent interactions (mainly electrostatic interactions) as well as attaching to nucleotides via direct chemical conjugation (Kim and Kim 2009; Meade and Dowdy 2007). Negatively charged nucleic acids can form nano-sized polyelectrolyte complexes (PECs) with cationic peptides, which is popular formulation method due to its simple processes and the favorable translocation of PECs into the cytoplasm. The biological performances of formulated PECs, e.g. intracellular uptake, transfection efficiency, and cytotoxicity, depend on their physicochemical properties including size, compactness, and stability, which are closely associated with the intensity of the electrostatic interactions between the electrolytes (Fischer et al. 2004, 2003). The strong electrostatic interactions of molecules with high charge density and molecular weight usually generate densely packed PECs, allowing successful intracellular delivery. For example, plasmid DNA can form PECs with insufficiently charged cations such as low molecular weight linear polyethyleneimine, while antisense ODN and siRNAs cannot form nanoparticles under the same conditions. Accordingly, one of the major challenges in the design of
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peptide-based carriers is caused by their lower molecular weight and poorer spatial charge density compared to those of high molecular synthetic/natural polymers and lipids, which hinders firmly compacted complexation with nucleic acids. Loosely fabricated PECs can be easily dissociated by competitive molecules, limiting their intracellular delivery and potential in vivo applications. Thus, the development of novel strategies for the design of peptide-based stable PECs that do not elicit cytotoxicity is highly desirable. Recently, functional peptides have been linked via chemical bonds to generate oligomeric peptides with reducible linkages. This approach appears efficient, as the peptides were found to provide efficient delivery of genes into cells and facilitate appropriate biological activity in vitro and in vivo without noticeable cytotoxicity, compared to common peptides (Kiselev et al. 2013; Mok and Park 2008; Won et al. 2011). It is conceivable that oligomeric peptides might interact and be complexed with genes via strong ionic interactions in the extracellular space, while allowing easy release of incorporated genes for biological processing in the reductive cytoplasm, due to the dissociation of reducible linkages. As an alternative approach, the structure of nucleic acids can be modified to facilitate optimal ionic interactions with peptide carriers. In our previous study, siRNAs were multimerized via chemical conjugation, which has the potential to significantly improve their binding affinity to cationic polymers and the extent of intracellular delivery (Lee et al. 2012; Mok et al. 2010). Here, we present a comparative evaluation of multi-siRNA conjugates and common monomeric siRNAs for use as peptide-based carrier systems, in terms of particle formulation, intracellular delivery, and gene suppression of siRNAs. In this study, a natural and arginine-rich peptide, a protamine, was selected for the condensation of siRNAs. Protamine-based siRNA complexes were characterized by gel electrophoresis and dynamic light scattering (DLS). The extent of intracellular delivery of siRNAs/protamine complexes was visualized using confocal microscopy. In addition, biological activities, including target gene suppression and cell viability of peptide-based particles, were assessed quantitatively using anti-green fluorescence protein (GFP) siRNAs for stably GFP-expressing MDA-MB-435 cells.
Materials and methods Materials Conventional siRNAs and modified siRNAs with a thiolgroup at the distal 30 end were purchased from Bioneer Co. (Daejeon, Republic of Korea). The siRNA sequences were as follows: GFP sense strand siRNA, 50 -GCAAGCUGACC
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CUGAAGUUdTdT-30 ; antisense strand siRNA, 50 AACUUCAGGGUCAGCUUGCdTdT-30 (Mok et al. 2010). Protamine, diethylpyrocarbonate (DEPC), heparin (MW: 12 kDa), and chloroquine were purchased from Sigma (St. Louis, MO). KALA peptide (WEAKLAKALAKALAKHL AKALAKALKACEA) was purchased from Peptron Inc. (Daejeon, South Korea). POPOTM-3 iodide was purchased from Invitrogen (Carlsbad, CA). Dulbecco’s Modified Eagle Medium (DMEM) medium, penicillin/streptomycin (P/S), and fetal bovine serum (FBS) were obtained from Gibco BRL (Grand Island, NY). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). All other chemicals and reagents were of analytical grade.
Preparation of multi-siRNA Multi-siRNAs were prepared as described in our previous study, with slight modifications (Mok et al. 2010). Briefly, free thiol groups were generated at the 30 end of sense and antisense single strand siRNAs (25 nmol) by overnight incubation with a reducing agent, 2 M dithiothreitol (DTT) solution, at pH 8.0. Following the deprotection process, the reactant was purified three times with a desalting column (MWCO 7 k) to remove excess DTT and then dried using a speed-vac. The resulting sense and antisense single-strand siRNAs with free thiol groups were dissolved in 25 lL of PBS solution and reacted with dithiobismaleimidoethane (DTME, 50 nmol) overnight at room temperature with stirring (850 rpm). The resulting dimeric sense and antisense single strands were hybridized together via hydrogen bonding, resulting in multi-siRNAs. The multi-siRNAs and common mono-siRNAs (1 lg) were loaded onto 1 % agarose gels and 15 % polyacrylamide gels for 45 min of gel electrophoresis at 180 V. The siRNAs in the gels were stained with ethidium bromide (EtBr) and visualized using a UV-trans-illuminator. The resulting gel images were used for quantitative analysis of multi-siRNAs using Image J software (National Institute of Health, USA; http://rsb.info. nih.gov/ij/) according to previous study (Mok et al. 2010).
Preparation of siRNA/protamine complexes To prepare PECs, 1 lg of mono- and multi-siRNAs in DEPC-treated deionized water (DEPC-DW) was mixed with predetermined amounts of protamine and KALA peptides by pipetting at weight ratios of 0, 0.1, 0.5, 1, and 2 at room temperature and incubated for 25 min. The PECs prepared were loaded onto 15 % acrylamide gels and gel electrophoresis was performed for 45 min. Migration of each RNA was visualized using a UV trans-illuminator after EtBr staining.
Evaluation of multimeric siRNA conjugates for breast cancer cells
For the competition assay, varying amounts of heparin (MW: 17,000–19,000) were added to each PEC (protamine/siRNA weight ratio = 2) at heparin/RNA weight ratios of 0, 1, 2, 5, and 10 for 30 min. The free siRNAs released from PECs into the solutions were examined by polyacrylamide gel electrophoresis. To prepare PECs, each RNA (10 lg) in DEPC-DW was complexed with protamine (20 lg) for 25 min at room temperature. The hydrodynamic sizes of the PECs containing mono- and multi-siRNAs were determined by DLS (ZEN 3690, Malvern Instruments Ltd., Malvern, UK).
Intracellular uptake of siRNA/protamine complexes Donated human breast cancer MDA-MB-435 cells stably expressing GFP (MDA-MB-GFP) were maintained in DMEM supplemented with 10 % FBS, 100 units/mL penicillin, and 100 lg/mL streptomycin at 37 °C in a humidified atmosphere of 5 % CO2. Cells were plated on 4-well chamber slides at a density of 2 9 105 cells/well for 24 h prior to transfection. To stain RNA, 3 lL of POPOTM3 iodide dyes (1 mM) was incubated with the RNA (10 lg) for 1 h at room temperature. After intercalation, POPO-3labeled siRNAs were purified by ethanol precipitation. After dissolving POPO-3-labeled siRNA in DEPC-DW, the concentration of siRNA in solution was measured using a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE). Labeled mono- and multi- siRNAs (1 lg) were mixed with protamine (12 lg) for 25 min and then added to each well of a chamber slide. After 4 h of incubation, cells were washed three times with fresh PBS solution at room temperature and fixed with 3.7 % formaldehyde solution in PBS at 48C. Cells were visualized using confocal microscopy (FV-1000 spectral, Olympus, Japan).
at an siRNA concentration of 290 nM in 6 % serum-containing medium and the mixtures were incubated for 5 h. Next, the medium was replaced and cells were incubated for a further 24 h. Cell viability was assessed using the CCK-8 assay according to manufacturer’s protocol.
Gene inhibition assay Cells were seeded on 24-well plates at a density of 5 9 104 cells/well for 24 h prior to transfection. The siRNA/protamine complexes were administered to cells at an siRNA concentration of 290 nM in 6 % serum-containing medium with and without chloroquine (50 lM) and incubated for 5 h. The medium was replaced with fresh 10 % serumcontaining medium and cells were incubated for a further 2 days. To obtain cell lysates, cells were treated with PBS solution with 1 % Triton X-100 and centrifuged to remove cell debris. The amount of GFP expression was measured using a spectrofluorophotometer (Molecular Devices, Sunnyvale, CA) at excitation and emission wavelengths of 480 and 520 nm, respectively. To observe GFP gene expression level, cells were seeded on 4-well chamber slide at a density of 5 9 104 cells/well. Next day, The multisiRNA/protamine PECs were transfected to cells (siRNA concentration of 290 nM) in 6 % serum-containing medium with and without chloroquine (50 lM) and incubated for 5 h. The medium was replaced with fresh 10 % serumcontaining medium and cells were incubated for a further 2 days. Cells were washed with PBS two times and treated with 3.7 % formaldehyde solution in PBS for fixation. The cells were visualized using confocal microscopy.
Results Formulation of protamine/multi-siRNA PECs
Cell viability assay MDA-MB-GFP cells were plated on 96-well plates at a density of 5 9 103 cells/well for 24 h prior to transfection. Branched PEI (MW 25 k), linear PEI (MW 25 k), and protamine were administered at predetermined polymer concentrations in 10 % serum-containing medium for 5 h. After replacing the medium with fresh 10 % serum media, cells were incubated for a further 24 h before cell viability was assessed by CCK-8 assay, according to the manufacturers’ instructions. To assess the cytotoxicity of the siRNA/protamine complexes, two types of siRNAs were mixed with protamine at weight ratios of 0, 3, 6, and 12 for 25 min. The formulated siRNA/protamine PECs were added to the cells
siRNAs that had been thiol-functionalized at both 30 -ends were chemically conjugated alone to form multimeric crosslinked siRNAs conjugates via disulfide bonds, using similar methods published in a previous study (Mok et al. 2010). The prepared multi-siRNAs were examined by gel electrophoresis using both agarose and acrylamide gels, as shown in Fig. 1a. As expected, the multi-siRNAs showed obviously retarded mobility in both agarose and acrylamide gels because their molecular weight was higher than that of mono-siRNAs. However, the decreased mobility could be fully recovered in the presence of the reducing agent, DTT, because of cleavage of internal disulfide linkages in multimeric siRNAs, as shown in our previous study (Mok et al. 2010). The difference in gel mobility for common siRNAs and multimeric siRNAs was much greater in polyacrylamide
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a
b
c
Fig. 1 a Gel electrophoresis of common and multimeric siRNAs using (left panel) 1 % agarose gel and (right panel) 15 % acrylamide gel. b–c Gel retardation assays after incubation of two kinds of
siRNAs (common siRNA and multimeric siRNA) with peptide carriers at various peptide/siRNA weight ratios using b KALA and c protamine
gels than in agarose gels. It is well known that the pore size is larger in 1 % agarose gels ([360 nm) than acrylamide gels (*70–130 nm), which may result in a difference in resolution between the two gel types (Heuer et al. 2003; Pernodet et al. 1997). Using gel images, the conjugation yield of multimeric siRNAs was quantitatively analyzed using the Image J program. Approximately 80 % of thiol-modified siRNAs were successfully connected via disulfide bonds. As potential peptide carriers, two types of peptides were assessed in terms of complexation properties with RNAs via ionic interactions. Protamine, a natural cationic peptide with a molecular weight of *4 kDa, is a wellknown DNA-condensing peptide with membrane-translocating ability (Brewer et al. 1999; Reynolds et al. 2005). In addition, protamine is also clinically available peptide as a heparin-neutralizing agent (Makris et al. 2000). KALA peptide (MW: 3130) is a cationic fusogenic peptide that allows pH-dependent membrane destabilization for efficient intracellular gene delivery (Mok and Park 2008; Wyman et al. 1997). Using two kinds of functional peptides, the affinity of peptides to siRNAs was comparatively evaluated via a gel retardation assay after incubation of KALA and protamine with two types of siRNAs, as shown in Fig. 1b, c. Interestingly, despite the similarity in molecular weight, protamine’s binding capacity with both mono- and multi-siRNAs was superior to that of KALA. More than half of the common siRNAs remained free at a KALA/siRNA weight ratio of 2, which indicates that PECs were not produced under those conditions. However, siRNAs successfully interacted and condensed with protamine peptides at a protamine/siRNA weight ratio of 2, and no free siRNA was observed in the gel. It should be noted that more than 60 % of protamine is composed of arginine, while 23.3 % of KALA peptides are cationic lysines (of the total 30 amino acids, 7 are lysines). This suggests that the relatively small portion of cationic amino acids in KALA peptides could provide different affinity with siRNAs to protamine. Thus, in this study, protamine was selected as a carrier peptide due to
its high density of cationic amino acids per single peptide and favorable condensing capability with siRNAs. In addition, two types of siRNAs, mono- and multi-siRNA, were comparatively evaluated for complexation with carrier peptides. Figure 1c shows that multi-siRNAs were completely complexed with protamine and no free multisiRNAs remained at a protamine/siRNA weight ratio of 1, while mono- siRNAs remained free under the same conditions. This result indicates that multi-siRNA structures allow much more improved condensation with cationic peptides than conventional siRNA structures, probably because of their high spatial charge density and flexible internal spacer (Lee et al. 2012).
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Characterization of protamine/multi-siRNA PECs To assess whether multi-siRNAs can form PECs with protamines firmly, particle size and stability were examined, as shown in Fig. 2. Particle sizes of protamine/siRNA PECs were measured using DLS (Fig. 2a). The sizes of the PECs that common mono- and multi-siRNA formed with protamines were 511.2 ± 313.2 and 119.3 ± 66.8 nm, respectively. The multi-siRNA PECs were much smaller in diameter (*4.3-fold) than those formed with mono-siRNAs, probably due to strong ionic interaction and compaction with protamine. Particle stability was investigated by performing gel electrophoresis after incubation of the competitive anionic polymer, heparin, with protamine PECs (Fig. 2b). After completion of interactions between siRNAs and protamine at a weight ratio of 2, different amounts of heparin were administered to determine the minimum amounts of heparin required for dissociation of siRNA/protamine complexes. The release of free siRNAs from siRNA/protamine complexes was observed with an increasing ratio of heparin to siRNA. Figure 2b shows that a fivefold greater concentration of heparin was needed for decomplexation of multi-siRNA/protamine particles than for that of mono-siRNA/protamine particles.
Evaluation of multimeric siRNA conjugates for breast cancer cells Fig. 2 a Diameter of protamine/siRNA PECs at a peptide/siRNA weight ratio of 2 in DW. b Competition assay for protamine/siRNA PECs in the presence of varying amounts of heparin
a
b
Intracellular uptake of protamine/multi-siRNA PECs To compare the extent of intracellular delivery of fabricated PECs, two types of siRNAs with protamine were administered to MDA-MB-GFP cells after fluorescence labeling of siRNAs with POPO-3 and were visualized by confocal microscopy (Fig. 3). Because free POPO-3 was completely removed during the ethanol precipitation process, background fluorescence signal was negligible when cells were treated with dye-labeled siRNAs alone. However, strong red fluorescence was observed in cells treated with multi-siRNA/protamine PECs due to the excellent intracellular uptake, while cells incubated with mono-siRNA PECs had much weaker fluorescence intensity, as shown in Fig. 3. This result indicates that mono-/protamine complexes were taken up less efficiently than multi-/protamine complexes, which may be attributed to their large size and relative instability.
Biocompatibility of protamine/multi-siRNA PECs To determine whether protamines are biocompatible in cells, MDA-MB-GFP cells were treated with various concentrations of protamines and cell viability was evaluated using a CCK-8 assay (Fig. 4a). In this experiment, two well-known cationic polymers for gene delivery, branched PEI (bPEI) and liner PEI (LPEI), were also administered to cells for comparative evaluation of their biocompatibility. The relative cell viabilities after treatment with bPEI and LPEI at a concentration of 80 lg/mL were 8.6 ± 0.2 and 14.7 ± 0.1 %, respectively, indicating severe cytotoxicity. However, treatment with protamine produced a significantly lower cytotoxic effect. In addition, cell viability was examined using protamine PECs with two types of siRNAs using mono- and multi-PECs at various protamine/siRNA weight ratios (Fig. 4b). Interestingly, only mono-siRNA based PECs showed obvious cytotoxicity at a protamine/siRNA
Fig. 3 Confocal microscopic images of intracellular siRNAs after complexation with protamine in MDA-MB-435 cells. siRNAs were stained with the red fluorescence dye POPO-3
weight ratio of 12. The relative cell viabilities of cells treated with common siRNA PECs were 78.2 ± 7.3 %. Considering that protamine/siRNA complexes prepared using the conventional formulation process, with a size of over 500 nm, resulted in *80 % of the cell viability observed in a
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a bPEI LPEI protamine
Cell viability (%)
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Fig. 4 Cell viability after incubation with a varying amounts of cationic polymers and b protamine/siRNA complexes for MDA-MB435 cells (*p \ 0.05)
previous study, this result appears consistent (Kundu et al. 2012). The observed pattern may be attributed to the fact that free protamines that could not participate in complexation with siRNAs elicit cell toxicity because of the poor binding affinity between common siRNAs and protamines.
Gene silencing in GFP expressing MDA-MB-435 cells Using multi-siRNA/protamine PECs with more improved biocompatibility, the extent of gene suppression after treatment was quantitatively evaluated in Fig. 5. Unexpectedly, multi-siRNA/protamine PECs showed negligible GFP gene inhibition under serum conditions, despite their excellent intracellular delivery. In previous studies, endosomal escape and intracellular uptake have been considered crucial determinants for successful biological activity of siRNAs after transfection (Nguyen and Szoka 2012; Tseng et al. 2009). Thus, chloroquine (CQ), a chemical agent that
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Fig. 5 a The extent of GFP gene suppression by multi-siRNA/ protamine PECs with and without CQ (50 lM) in 10 % serumcontaining medium for GFP-expressing MDA-MB-435 cells. b Confocal microscopy images of GFP-expressing MDA-MB-435 cells after treatment of multi-siRNA/protamine PECs with and without CQ (50 lM)
enables endosomal escape, was added to the media with siRNA/protamine complexes, and gene suppression was examined. The extent of GFP gene expression after treatment with multi-siRNA/protamine complexes at a protamine/siRNA weight ratio of 12 was 97.8 ± 9.3 and 67.1 ± 6.3 % in the absence and presence of 50 lM of CQ, respectively, for 5 h. This result shows that addition of the endosomal escape moiety resulted in successful delivery of protamine/multi-siRNA complexes and induction of notable gene inhibition without cytotoxicity to the MDAMB-435 cells. However, incubation of PECs with CQ for 12 h was too toxic to evaluate gene suppression. The GFP gene suppression by multi-siRNA/protamine complexes was also observed by confocal microscopy. Figure 5b shows that multi-siRNA/protamine complexes successfully inhibited target GFP gene expression in the presence of CQ.
Evaluation of multimeric siRNA conjugates for breast cancer cells
Discussion Currently, protamine has received attention as a promising material for formulation of nucleic acid-based drugs in vitro and in vivo because of a relatively low price compared to synthetic functional peptides, clinical availability, efficient translocation through cellular membranes, and high affinity to nucleic acids (Choi et al. 2010; Kundu et al. 2012). In this study, we evaluated multi-siRNA-based ionic complexes with protamine as a new carrier for therapeutic siRNAs. Our results exhibited that multi-siRNA structures are superior to common siRNA structures for the formulation and efficient intracellular uptake of protaminebased PECs. Figure 2a shows that biocompatible protamine formed small compact nanoparticles with multisiRNAs, with a size of 120 nm. Considering that particles smaller than 200 nm possess great advantages in terms of intracellular endocytosis in vitro and passively targeted delivery in vivo via the enhanced permeability and retention (EPR) effect, peptide formulation using multimeric siRNAs appears to be a promising strategy for efficient siRNA delivery (Decuzzi et al. 2009). In addition, Fig. 2b shows that multi-siRNA-based ionic complexes with protamine were stable against exterior competitive polyelectrolytes, while mono-siRNA/protamine complexes were only loosely formed. Multi-siRNAs/protamine PECs were not easily dissociated by anionic molecules probably due to their strong ionic interactions. In addition, it was also demonstrated that protamine-based multi-siRNA PECs with a narrow size distribution and firm compaction provide high intracellular uptake as well as excellent target gene inhibition using anti-GFP siRNAs. Notably, multisiRNA/protamine PECs showed excellent gene suppression without cycotoxicity, which may be favorable for in vivo applications. It should be also noticed that all transfections were performed in the presence of serum proteins to consider nonspecific interference of intracellular delivery by serum proteins in vivo. In our previous study, multi-siRNAs showed superior condensation and delivery efficiency with synthetic polymeric carriers, LPEIs, which had a low charge. This study clearly demonstrated that clinically available natural peptide protamine that pose less safety concerns could replace high molecular weight cationic polymers like LPEIs (25 k). However, for applying the current protamine-based multi-siRNA delivery system in vivo, further studies will be necessary to optimize protamine carriers with endosomal escape moieties.
Conclusion In conclusion, to our knowledge, this is the first study to report that multi-siRNAs are favorable for incorporation in
peptide carriers and subsequent intracellular delivery in vitro. Fabricated multi-siRNA/protamine PECs with a size of 120 nm showed greatly improved intracellular uptake and biocompatibility, compared to conventional siRNA/protamine particles. In addition, the multi-siRNA/ protamine particles effectively suppressed target gene expression in the presence of serum proteins and CQ without cytotoxicity for the MDA-MB-435 breast cancer cells. Thus, a serious issue in peptide-siRNA particle formulation, that is, the poor binding affinity of siRNAs to cationic peptides, could be overcome by using multi-siRNA conjugates, thereby providing a potential platform technology for the design of peptide-based siRNA delivery systems for in vivo applications. Acknowledgments This study was supported by a grant from the National R&D Program for Cancer Control, Ministry for Health and Welfare, Republic of Korea (1220050).
References Brewer, L.R., M. Corzett, and R. Balhorn. 1999. Protamine-induced condensation and decondensation of the same DNA molecule. Science 286: 120–123. Choi, Y.S., et al. 2010. The systemic delivery of siRNAs by a cell penetrating peptide, low molecular weight protamine. Biomaterials 31: 1429–1443. Decuzzi, P., et al. 2009. Intravascular delivery of particulate systems: does geometry really matter? Pharmaceutical Research 26: 235–243. Fischer, D., et al. 2004. Poly(diallyldimethylammonium chlorides) and their N-methyl-N-vinylacetamide copolymer-based DNApolyplexes: role of molecular weight and charge density in complex formation, stability, and in vitro activity. International Journal of Pharmaceutics 280: 253–269. Fischer, D., et al. 2003. In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis. Biomaterials 24: 1121–1131. Heuer, D.M., S. Saha, and L.A. Archer. 2003. Topological effects on the electrophoretic mobility of rigid rodlike DNA in polyacrylamide gels. Biopolymers 70: 471–481. Huang, Y., et al. 2013. Curb challenges of the ‘‘Trojan Horse’’ approach: smart strategies in achieving effective yet safe cellpenetrating peptide-based drug delivery. Advanced drug delivery reviews 65: 1299–1315. Kim, W.J., and S.W. Kim. 2009. Efficient siRNA delivery with nonviral polymeric vehicles. Pharmaceutical Research 26: 657–666. Kiselev, A., et al. 2013. Characterization of reducible peptide oligomers as carriers for gene delivery. International Journal of Pharmaceutics 441: 736–747. Kumar, P., et al. 2008. T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice. Cell 134: 577–586. Kundu, A.K., et al. 2012. Stability of lyophilized siRNA nanosome formulations. International Journal of Pharmaceutics 423: 525–534. Lee, S.H., et al. 2012. Small-interfering RNA (siRNA)-based functional micro- and nanostructures for efficient and selective gene silencing. Accounts of Chemical Research 45: 1014–1025. Makris, M., R.E. Hough, and S. Kitchen. 2000. Poor reversal of low molecular weight heparin by protamine. British Journal of Haematology 108: 884–885.
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H. Yoo, H. Mok Meade, B.R., and S.F. Dowdy. 2007. Exogenous siRNA delivery using peptide transduction domains/cell penetrating peptides. Advanced Drug Delivery Reviews 59: 134–140. Mok, H., et al. 2010. Multimeric small interfering ribonucleic acid for highly efficient sequence-specific gene silencing. Nature Materials 9: 272–278. Mok, H., and T.G. Park. 2008. Self-crosslinked and reducible fusogenic peptides for intracellular delivery of siRNA. Biopolymers 89: 881–888. Nguyen, J., and F.C. Szoka. 2012. Nucleic acid delivery: the missing pieces of the puzzle? Accounts of Chemical Research 45: 1153–1162. Pernodet, N., M. Maaloum, and B. Tinland. 1997. Pore size of agarose gels by atomic force microscopy. Electrophoresis 18: 55–58. Reynolds, F., R. Weissleder, and L. Josephson. 2005. Protamine as an efficient membrane-translocating peptide. Bioconjugate Chemistry 16: 1240–1245.
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Scholz, C., and E. Wagner. 2012. Therapeutic plasmid DNA versus siRNA delivery: common and different tasks for synthetic carriers. Journal of Controlled Release 161: 554–565. Tseng, Y.C., S. Mozumdar, and L. Huang. 2009. Lipid-based systemic delivery of siRNA. Advanced Drug Delivery Reviews 61: 721–731. Won, Y.W., et al. 2011. Poly(oligo-D-arginine) With Internal Disulfide Linkages as a Cytoplasm-sensitive Carrier for siRNA Delivery. Molecular Therapy 19: 372–380. Wyman, T.B., et al. 1997. Design, synthesis, and characterization of a cationic peptide that binds to nucleic acids and permeabilizes bilayers. Biochemistry 36: 3008–3017. Yewale, C., et al. 2013. Proteins: emerging carrier for delivery of cancer therapeutics. Expert Opinion on Drug Delivery 10: 1429–1448.
RESEARCH ARTICLE
Evaluation of multimeric siRNA conjugates for efficient protamine-based delivery into breast cancer cells Hyundong Yoo • Hyejung Mok
Received: 10 December 2013 / Accepted: 16 February 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract Despite the preferable properties of well-defined cationic peptides for small interfering RNA (siRNA) delivery, their application as siRNA carriers remains limited due to their poor binding affinity with short-chain RNAs. In this study, we investigated the feasibility of a novel strategy for circumventing this limitation, by assessing the utility of multimeric conjugates of siRNA for improving the binding affinity of siRNAs with cationic peptides and the extent of intracellular delivery. Protamine, a natural and arginine-rich peptide, was used to produce stably condensed polyelectrolyte complexes (PECs) with multimeric siRNAs (multisiRNA) with a size of 120 nm while conventional siRNA/ protamine particles are over 500 nm. The formulated multisiRNA/protamine PECs showed greatly enhanced stability, intracellular uptake, and biocompatibility compared to conventional, monomeric (mono)-siRNA/protamine particles. With the addition of chloroquine, multi-siRNA/protamine PECs successfully inhibited target gene expression in MDA-MB-435 cells, a breast cancer cell line, even in the presence of serum protein. This study demonstrates that multi-siRNA conjugates greatly facilitate the formulation of nano-sized protamine-based carriers and significantly improve intracellular delivery in vitro compared to common siRNAs, and therefore may provide a platform for the design of peptide-based siRNA delivery systems for in vivo applications. Keywords Functional peptide Protamine Multimeric siRNA Charge density Gene silencing
H. Yoo H. Mok (&) Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea e-mail: [email protected]
Introduction Peptide-based carriers have received attention as promising candidate materials for the delivery of therapeutic genes, drugs, and proteins due to their favorable characteristics, e.g., well-defined structure, ease of synthesis, reproducibility, and biocompatibility (Huang et al. 2013; Yewale et al. 2013). Thus, diverse functional peptides, such as fusogenic peptides and cell-penetrating peptides, have been intensively studied as potential carriers for therapeutic genes such as small interfering RNAs (siRNAs) (Kumar et al. 2008; Meade and Dowdy 2007; Scholz and Wagner 2012). Peptide carriers allow enhanced delivery of nucleic acids by forming nano-sized particles via noncovalent interactions (mainly electrostatic interactions) as well as attaching to nucleotides via direct chemical conjugation (Kim and Kim 2009; Meade and Dowdy 2007). Negatively charged nucleic acids can form nano-sized polyelectrolyte complexes (PECs) with cationic peptides, which is popular formulation method due to its simple processes and the favorable translocation of PECs into the cytoplasm. The biological performances of formulated PECs, e.g. intracellular uptake, transfection efficiency, and cytotoxicity, depend on their physicochemical properties including size, compactness, and stability, which are closely associated with the intensity of the electrostatic interactions between the electrolytes (Fischer et al. 2004, 2003). The strong electrostatic interactions of molecules with high charge density and molecular weight usually generate densely packed PECs, allowing successful intracellular delivery. For example, plasmid DNA can form PECs with insufficiently charged cations such as low molecular weight linear polyethyleneimine, while antisense ODN and siRNAs cannot form nanoparticles under the same conditions. Accordingly, one of the major challenges in the design of
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peptide-based carriers is caused by their lower molecular weight and poorer spatial charge density compared to those of high molecular synthetic/natural polymers and lipids, which hinders firmly compacted complexation with nucleic acids. Loosely fabricated PECs can be easily dissociated by competitive molecules, limiting their intracellular delivery and potential in vivo applications. Thus, the development of novel strategies for the design of peptide-based stable PECs that do not elicit cytotoxicity is highly desirable. Recently, functional peptides have been linked via chemical bonds to generate oligomeric peptides with reducible linkages. This approach appears efficient, as the peptides were found to provide efficient delivery of genes into cells and facilitate appropriate biological activity in vitro and in vivo without noticeable cytotoxicity, compared to common peptides (Kiselev et al. 2013; Mok and Park 2008; Won et al. 2011). It is conceivable that oligomeric peptides might interact and be complexed with genes via strong ionic interactions in the extracellular space, while allowing easy release of incorporated genes for biological processing in the reductive cytoplasm, due to the dissociation of reducible linkages. As an alternative approach, the structure of nucleic acids can be modified to facilitate optimal ionic interactions with peptide carriers. In our previous study, siRNAs were multimerized via chemical conjugation, which has the potential to significantly improve their binding affinity to cationic polymers and the extent of intracellular delivery (Lee et al. 2012; Mok et al. 2010). Here, we present a comparative evaluation of multi-siRNA conjugates and common monomeric siRNAs for use as peptide-based carrier systems, in terms of particle formulation, intracellular delivery, and gene suppression of siRNAs. In this study, a natural and arginine-rich peptide, a protamine, was selected for the condensation of siRNAs. Protamine-based siRNA complexes were characterized by gel electrophoresis and dynamic light scattering (DLS). The extent of intracellular delivery of siRNAs/protamine complexes was visualized using confocal microscopy. In addition, biological activities, including target gene suppression and cell viability of peptide-based particles, were assessed quantitatively using anti-green fluorescence protein (GFP) siRNAs for stably GFP-expressing MDA-MB-435 cells.
Materials and methods Materials Conventional siRNAs and modified siRNAs with a thiolgroup at the distal 30 end were purchased from Bioneer Co. (Daejeon, Republic of Korea). The siRNA sequences were as follows: GFP sense strand siRNA, 50 -GCAAGCUGACC
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CUGAAGUUdTdT-30 ; antisense strand siRNA, 50 AACUUCAGGGUCAGCUUGCdTdT-30 (Mok et al. 2010). Protamine, diethylpyrocarbonate (DEPC), heparin (MW: 12 kDa), and chloroquine were purchased from Sigma (St. Louis, MO). KALA peptide (WEAKLAKALAKALAKHL AKALAKALKACEA) was purchased from Peptron Inc. (Daejeon, South Korea). POPOTM-3 iodide was purchased from Invitrogen (Carlsbad, CA). Dulbecco’s Modified Eagle Medium (DMEM) medium, penicillin/streptomycin (P/S), and fetal bovine serum (FBS) were obtained from Gibco BRL (Grand Island, NY). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). All other chemicals and reagents were of analytical grade.
Preparation of multi-siRNA Multi-siRNAs were prepared as described in our previous study, with slight modifications (Mok et al. 2010). Briefly, free thiol groups were generated at the 30 end of sense and antisense single strand siRNAs (25 nmol) by overnight incubation with a reducing agent, 2 M dithiothreitol (DTT) solution, at pH 8.0. Following the deprotection process, the reactant was purified three times with a desalting column (MWCO 7 k) to remove excess DTT and then dried using a speed-vac. The resulting sense and antisense single-strand siRNAs with free thiol groups were dissolved in 25 lL of PBS solution and reacted with dithiobismaleimidoethane (DTME, 50 nmol) overnight at room temperature with stirring (850 rpm). The resulting dimeric sense and antisense single strands were hybridized together via hydrogen bonding, resulting in multi-siRNAs. The multi-siRNAs and common mono-siRNAs (1 lg) were loaded onto 1 % agarose gels and 15 % polyacrylamide gels for 45 min of gel electrophoresis at 180 V. The siRNAs in the gels were stained with ethidium bromide (EtBr) and visualized using a UV-trans-illuminator. The resulting gel images were used for quantitative analysis of multi-siRNAs using Image J software (National Institute of Health, USA; http://rsb.info. nih.gov/ij/) according to previous study (Mok et al. 2010).
Preparation of siRNA/protamine complexes To prepare PECs, 1 lg of mono- and multi-siRNAs in DEPC-treated deionized water (DEPC-DW) was mixed with predetermined amounts of protamine and KALA peptides by pipetting at weight ratios of 0, 0.1, 0.5, 1, and 2 at room temperature and incubated for 25 min. The PECs prepared were loaded onto 15 % acrylamide gels and gel electrophoresis was performed for 45 min. Migration of each RNA was visualized using a UV trans-illuminator after EtBr staining.
Evaluation of multimeric siRNA conjugates for breast cancer cells
For the competition assay, varying amounts of heparin (MW: 17,000–19,000) were added to each PEC (protamine/siRNA weight ratio = 2) at heparin/RNA weight ratios of 0, 1, 2, 5, and 10 for 30 min. The free siRNAs released from PECs into the solutions were examined by polyacrylamide gel electrophoresis. To prepare PECs, each RNA (10 lg) in DEPC-DW was complexed with protamine (20 lg) for 25 min at room temperature. The hydrodynamic sizes of the PECs containing mono- and multi-siRNAs were determined by DLS (ZEN 3690, Malvern Instruments Ltd., Malvern, UK).
Intracellular uptake of siRNA/protamine complexes Donated human breast cancer MDA-MB-435 cells stably expressing GFP (MDA-MB-GFP) were maintained in DMEM supplemented with 10 % FBS, 100 units/mL penicillin, and 100 lg/mL streptomycin at 37 °C in a humidified atmosphere of 5 % CO2. Cells were plated on 4-well chamber slides at a density of 2 9 105 cells/well for 24 h prior to transfection. To stain RNA, 3 lL of POPOTM3 iodide dyes (1 mM) was incubated with the RNA (10 lg) for 1 h at room temperature. After intercalation, POPO-3labeled siRNAs were purified by ethanol precipitation. After dissolving POPO-3-labeled siRNA in DEPC-DW, the concentration of siRNA in solution was measured using a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE). Labeled mono- and multi- siRNAs (1 lg) were mixed with protamine (12 lg) for 25 min and then added to each well of a chamber slide. After 4 h of incubation, cells were washed three times with fresh PBS solution at room temperature and fixed with 3.7 % formaldehyde solution in PBS at 48C. Cells were visualized using confocal microscopy (FV-1000 spectral, Olympus, Japan).
at an siRNA concentration of 290 nM in 6 % serum-containing medium and the mixtures were incubated for 5 h. Next, the medium was replaced and cells were incubated for a further 24 h. Cell viability was assessed using the CCK-8 assay according to manufacturer’s protocol.
Gene inhibition assay Cells were seeded on 24-well plates at a density of 5 9 104 cells/well for 24 h prior to transfection. The siRNA/protamine complexes were administered to cells at an siRNA concentration of 290 nM in 6 % serum-containing medium with and without chloroquine (50 lM) and incubated for 5 h. The medium was replaced with fresh 10 % serumcontaining medium and cells were incubated for a further 2 days. To obtain cell lysates, cells were treated with PBS solution with 1 % Triton X-100 and centrifuged to remove cell debris. The amount of GFP expression was measured using a spectrofluorophotometer (Molecular Devices, Sunnyvale, CA) at excitation and emission wavelengths of 480 and 520 nm, respectively. To observe GFP gene expression level, cells were seeded on 4-well chamber slide at a density of 5 9 104 cells/well. Next day, The multisiRNA/protamine PECs were transfected to cells (siRNA concentration of 290 nM) in 6 % serum-containing medium with and without chloroquine (50 lM) and incubated for 5 h. The medium was replaced with fresh 10 % serumcontaining medium and cells were incubated for a further 2 days. Cells were washed with PBS two times and treated with 3.7 % formaldehyde solution in PBS for fixation. The cells were visualized using confocal microscopy.
Results Formulation of protamine/multi-siRNA PECs
Cell viability assay MDA-MB-GFP cells were plated on 96-well plates at a density of 5 9 103 cells/well for 24 h prior to transfection. Branched PEI (MW 25 k), linear PEI (MW 25 k), and protamine were administered at predetermined polymer concentrations in 10 % serum-containing medium for 5 h. After replacing the medium with fresh 10 % serum media, cells were incubated for a further 24 h before cell viability was assessed by CCK-8 assay, according to the manufacturers’ instructions. To assess the cytotoxicity of the siRNA/protamine complexes, two types of siRNAs were mixed with protamine at weight ratios of 0, 3, 6, and 12 for 25 min. The formulated siRNA/protamine PECs were added to the cells
siRNAs that had been thiol-functionalized at both 30 -ends were chemically conjugated alone to form multimeric crosslinked siRNAs conjugates via disulfide bonds, using similar methods published in a previous study (Mok et al. 2010). The prepared multi-siRNAs were examined by gel electrophoresis using both agarose and acrylamide gels, as shown in Fig. 1a. As expected, the multi-siRNAs showed obviously retarded mobility in both agarose and acrylamide gels because their molecular weight was higher than that of mono-siRNAs. However, the decreased mobility could be fully recovered in the presence of the reducing agent, DTT, because of cleavage of internal disulfide linkages in multimeric siRNAs, as shown in our previous study (Mok et al. 2010). The difference in gel mobility for common siRNAs and multimeric siRNAs was much greater in polyacrylamide
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a
b
c
Fig. 1 a Gel electrophoresis of common and multimeric siRNAs using (left panel) 1 % agarose gel and (right panel) 15 % acrylamide gel. b–c Gel retardation assays after incubation of two kinds of
siRNAs (common siRNA and multimeric siRNA) with peptide carriers at various peptide/siRNA weight ratios using b KALA and c protamine
gels than in agarose gels. It is well known that the pore size is larger in 1 % agarose gels ([360 nm) than acrylamide gels (*70–130 nm), which may result in a difference in resolution between the two gel types (Heuer et al. 2003; Pernodet et al. 1997). Using gel images, the conjugation yield of multimeric siRNAs was quantitatively analyzed using the Image J program. Approximately 80 % of thiol-modified siRNAs were successfully connected via disulfide bonds. As potential peptide carriers, two types of peptides were assessed in terms of complexation properties with RNAs via ionic interactions. Protamine, a natural cationic peptide with a molecular weight of *4 kDa, is a wellknown DNA-condensing peptide with membrane-translocating ability (Brewer et al. 1999; Reynolds et al. 2005). In addition, protamine is also clinically available peptide as a heparin-neutralizing agent (Makris et al. 2000). KALA peptide (MW: 3130) is a cationic fusogenic peptide that allows pH-dependent membrane destabilization for efficient intracellular gene delivery (Mok and Park 2008; Wyman et al. 1997). Using two kinds of functional peptides, the affinity of peptides to siRNAs was comparatively evaluated via a gel retardation assay after incubation of KALA and protamine with two types of siRNAs, as shown in Fig. 1b, c. Interestingly, despite the similarity in molecular weight, protamine’s binding capacity with both mono- and multi-siRNAs was superior to that of KALA. More than half of the common siRNAs remained free at a KALA/siRNA weight ratio of 2, which indicates that PECs were not produced under those conditions. However, siRNAs successfully interacted and condensed with protamine peptides at a protamine/siRNA weight ratio of 2, and no free siRNA was observed in the gel. It should be noted that more than 60 % of protamine is composed of arginine, while 23.3 % of KALA peptides are cationic lysines (of the total 30 amino acids, 7 are lysines). This suggests that the relatively small portion of cationic amino acids in KALA peptides could provide different affinity with siRNAs to protamine. Thus, in this study, protamine was selected as a carrier peptide due to
its high density of cationic amino acids per single peptide and favorable condensing capability with siRNAs. In addition, two types of siRNAs, mono- and multi-siRNA, were comparatively evaluated for complexation with carrier peptides. Figure 1c shows that multi-siRNAs were completely complexed with protamine and no free multisiRNAs remained at a protamine/siRNA weight ratio of 1, while mono- siRNAs remained free under the same conditions. This result indicates that multi-siRNA structures allow much more improved condensation with cationic peptides than conventional siRNA structures, probably because of their high spatial charge density and flexible internal spacer (Lee et al. 2012).
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Characterization of protamine/multi-siRNA PECs To assess whether multi-siRNAs can form PECs with protamines firmly, particle size and stability were examined, as shown in Fig. 2. Particle sizes of protamine/siRNA PECs were measured using DLS (Fig. 2a). The sizes of the PECs that common mono- and multi-siRNA formed with protamines were 511.2 ± 313.2 and 119.3 ± 66.8 nm, respectively. The multi-siRNA PECs were much smaller in diameter (*4.3-fold) than those formed with mono-siRNAs, probably due to strong ionic interaction and compaction with protamine. Particle stability was investigated by performing gel electrophoresis after incubation of the competitive anionic polymer, heparin, with protamine PECs (Fig. 2b). After completion of interactions between siRNAs and protamine at a weight ratio of 2, different amounts of heparin were administered to determine the minimum amounts of heparin required for dissociation of siRNA/protamine complexes. The release of free siRNAs from siRNA/protamine complexes was observed with an increasing ratio of heparin to siRNA. Figure 2b shows that a fivefold greater concentration of heparin was needed for decomplexation of multi-siRNA/protamine particles than for that of mono-siRNA/protamine particles.
Evaluation of multimeric siRNA conjugates for breast cancer cells Fig. 2 a Diameter of protamine/siRNA PECs at a peptide/siRNA weight ratio of 2 in DW. b Competition assay for protamine/siRNA PECs in the presence of varying amounts of heparin
a
b
Intracellular uptake of protamine/multi-siRNA PECs To compare the extent of intracellular delivery of fabricated PECs, two types of siRNAs with protamine were administered to MDA-MB-GFP cells after fluorescence labeling of siRNAs with POPO-3 and were visualized by confocal microscopy (Fig. 3). Because free POPO-3 was completely removed during the ethanol precipitation process, background fluorescence signal was negligible when cells were treated with dye-labeled siRNAs alone. However, strong red fluorescence was observed in cells treated with multi-siRNA/protamine PECs due to the excellent intracellular uptake, while cells incubated with mono-siRNA PECs had much weaker fluorescence intensity, as shown in Fig. 3. This result indicates that mono-/protamine complexes were taken up less efficiently than multi-/protamine complexes, which may be attributed to their large size and relative instability.
Biocompatibility of protamine/multi-siRNA PECs To determine whether protamines are biocompatible in cells, MDA-MB-GFP cells were treated with various concentrations of protamines and cell viability was evaluated using a CCK-8 assay (Fig. 4a). In this experiment, two well-known cationic polymers for gene delivery, branched PEI (bPEI) and liner PEI (LPEI), were also administered to cells for comparative evaluation of their biocompatibility. The relative cell viabilities after treatment with bPEI and LPEI at a concentration of 80 lg/mL were 8.6 ± 0.2 and 14.7 ± 0.1 %, respectively, indicating severe cytotoxicity. However, treatment with protamine produced a significantly lower cytotoxic effect. In addition, cell viability was examined using protamine PECs with two types of siRNAs using mono- and multi-PECs at various protamine/siRNA weight ratios (Fig. 4b). Interestingly, only mono-siRNA based PECs showed obvious cytotoxicity at a protamine/siRNA
Fig. 3 Confocal microscopic images of intracellular siRNAs after complexation with protamine in MDA-MB-435 cells. siRNAs were stained with the red fluorescence dye POPO-3
weight ratio of 12. The relative cell viabilities of cells treated with common siRNA PECs were 78.2 ± 7.3 %. Considering that protamine/siRNA complexes prepared using the conventional formulation process, with a size of over 500 nm, resulted in *80 % of the cell viability observed in a
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a bPEI LPEI protamine
Cell viability (%)
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Fig. 4 Cell viability after incubation with a varying amounts of cationic polymers and b protamine/siRNA complexes for MDA-MB435 cells (*p \ 0.05)
previous study, this result appears consistent (Kundu et al. 2012). The observed pattern may be attributed to the fact that free protamines that could not participate in complexation with siRNAs elicit cell toxicity because of the poor binding affinity between common siRNAs and protamines.
Gene silencing in GFP expressing MDA-MB-435 cells Using multi-siRNA/protamine PECs with more improved biocompatibility, the extent of gene suppression after treatment was quantitatively evaluated in Fig. 5. Unexpectedly, multi-siRNA/protamine PECs showed negligible GFP gene inhibition under serum conditions, despite their excellent intracellular delivery. In previous studies, endosomal escape and intracellular uptake have been considered crucial determinants for successful biological activity of siRNAs after transfection (Nguyen and Szoka 2012; Tseng et al. 2009). Thus, chloroquine (CQ), a chemical agent that
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Fig. 5 a The extent of GFP gene suppression by multi-siRNA/ protamine PECs with and without CQ (50 lM) in 10 % serumcontaining medium for GFP-expressing MDA-MB-435 cells. b Confocal microscopy images of GFP-expressing MDA-MB-435 cells after treatment of multi-siRNA/protamine PECs with and without CQ (50 lM)
enables endosomal escape, was added to the media with siRNA/protamine complexes, and gene suppression was examined. The extent of GFP gene expression after treatment with multi-siRNA/protamine complexes at a protamine/siRNA weight ratio of 12 was 97.8 ± 9.3 and 67.1 ± 6.3 % in the absence and presence of 50 lM of CQ, respectively, for 5 h. This result shows that addition of the endosomal escape moiety resulted in successful delivery of protamine/multi-siRNA complexes and induction of notable gene inhibition without cytotoxicity to the MDAMB-435 cells. However, incubation of PECs with CQ for 12 h was too toxic to evaluate gene suppression. The GFP gene suppression by multi-siRNA/protamine complexes was also observed by confocal microscopy. Figure 5b shows that multi-siRNA/protamine complexes successfully inhibited target GFP gene expression in the presence of CQ.
Evaluation of multimeric siRNA conjugates for breast cancer cells
Discussion Currently, protamine has received attention as a promising material for formulation of nucleic acid-based drugs in vitro and in vivo because of a relatively low price compared to synthetic functional peptides, clinical availability, efficient translocation through cellular membranes, and high affinity to nucleic acids (Choi et al. 2010; Kundu et al. 2012). In this study, we evaluated multi-siRNA-based ionic complexes with protamine as a new carrier for therapeutic siRNAs. Our results exhibited that multi-siRNA structures are superior to common siRNA structures for the formulation and efficient intracellular uptake of protaminebased PECs. Figure 2a shows that biocompatible protamine formed small compact nanoparticles with multisiRNAs, with a size of 120 nm. Considering that particles smaller than 200 nm possess great advantages in terms of intracellular endocytosis in vitro and passively targeted delivery in vivo via the enhanced permeability and retention (EPR) effect, peptide formulation using multimeric siRNAs appears to be a promising strategy for efficient siRNA delivery (Decuzzi et al. 2009). In addition, Fig. 2b shows that multi-siRNA-based ionic complexes with protamine were stable against exterior competitive polyelectrolytes, while mono-siRNA/protamine complexes were only loosely formed. Multi-siRNAs/protamine PECs were not easily dissociated by anionic molecules probably due to their strong ionic interactions. In addition, it was also demonstrated that protamine-based multi-siRNA PECs with a narrow size distribution and firm compaction provide high intracellular uptake as well as excellent target gene inhibition using anti-GFP siRNAs. Notably, multisiRNA/protamine PECs showed excellent gene suppression without cycotoxicity, which may be favorable for in vivo applications. It should be also noticed that all transfections were performed in the presence of serum proteins to consider nonspecific interference of intracellular delivery by serum proteins in vivo. In our previous study, multi-siRNAs showed superior condensation and delivery efficiency with synthetic polymeric carriers, LPEIs, which had a low charge. This study clearly demonstrated that clinically available natural peptide protamine that pose less safety concerns could replace high molecular weight cationic polymers like LPEIs (25 k). However, for applying the current protamine-based multi-siRNA delivery system in vivo, further studies will be necessary to optimize protamine carriers with endosomal escape moieties.
Conclusion In conclusion, to our knowledge, this is the first study to report that multi-siRNAs are favorable for incorporation in
peptide carriers and subsequent intracellular delivery in vitro. Fabricated multi-siRNA/protamine PECs with a size of 120 nm showed greatly improved intracellular uptake and biocompatibility, compared to conventional siRNA/protamine particles. In addition, the multi-siRNA/ protamine particles effectively suppressed target gene expression in the presence of serum proteins and CQ without cytotoxicity for the MDA-MB-435 breast cancer cells. Thus, a serious issue in peptide-siRNA particle formulation, that is, the poor binding affinity of siRNAs to cationic peptides, could be overcome by using multi-siRNA conjugates, thereby providing a potential platform technology for the design of peptide-based siRNA delivery systems for in vivo applications. Acknowledgments This study was supported by a grant from the National R&D Program for Cancer Control, Ministry for Health and Welfare, Republic of Korea (1220050).
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