Comparison of Nanocomplexes with Branched and Linear Peptides

Jan 22, 2013 - UCL School of Pharmacy, 29-39 Brunswick Square, London, WC1N 1AX, U.K. ... (MW: 3486) and BR (MW: 3823) were synthesized by UCL...
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Comparison of Nanocomplexes with Branched and Linear Peptides for SiRNA Delivery Aristides D. Tagalakis,† Luisa Saraiva,† David McCarthy,‡ Kenth T. Gustafsson,† and Stephen L. Hart*,† †

Wolfson Centre for Gene Therapy of Childhood Disease, UCL Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, U.K. ‡ UCL School of Pharmacy, 29-39 Brunswick Square, London, WC1N 1AX, U.K. S Supporting Information *

ABSTRACT: Efficient delivery of small interfering RNA (siRNA) remains the greatest technological barrier to the clinical implementation of RNA interference strategies. We are investigating the relationship between the biophysical properties of siRNA nanocomplexes and their transfection efficiency as an approach to the generation of improved formulations. Peptide-based formulations are of great interest, and so in this study we have compared nanocomplex formulations for siRNA delivery containing linear and branched oligolysine or oligoarginine peptides. Peptides were combined with cationic liposomes in siRNA formulations and compared for transfection efficiency, siRNA packaging efficiency, biophysical properties, and particle stability. Nanocomplexes containing linear peptides were more condensed and stable than branched peptide formulations; however, their silencing activity was lower, suggesting that their greater stability might limit siRNA release within the cell. Thus, differences in transfection appeared to be associated with differences in packaging and stability, indicating the importance of optimizing this feature in siRNA nanocomplexes.

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the intracellular pathogen Legionella pneumophila,13 suggesting that this sequence may have specific qualities for inducing cell binding and internalization, although the identity of the receptor is at present unknown. The modular structures of cationic lipids and peptides in the LPRs lend themselves to optimization for siRNA packaging and delivery. In addition, the combination of the lipid and peptide components in the LPR nanocomplexes at optimal ratios act synergistically and are both essential for the formation of stable siRNA complexes and for efficient receptor-targeted knockdown of different genes.11 Apart from linear oligolysine peptides, other cationic peptides such as synthetic arginine peptides14,15 have been used to package and protect plasmid DNA. Furthermore, reports suggested that branched peptides16,17 or branched PEI18 were able to package siRNA more effectively than their linear counterparts. The aim of this study was to compare LPRs with branched peptides with those containing linear peptides in terms of their biophysical properties and stability, as well as their transfection efficiencies.

INTRODUCTION RNA interference is an endogenous cellular process for the regulation of gene expression.1 Small interfering RNA (siRNA) molecules have been used for basic biological research into gene function2 and for therapeutic purposes.3 SiRNA delivery to target cells requires a vector system, as cells do not normally take up siRNA on their own4 and the molecule itself can be degraded by RNases in serum.5 In particular, in vivo siRNA delivery remains challenging,6 and there is therefore a need to better understand, characterize, and improve siRNA vector formulations. We have previously described the use of receptor-targeted nanocomplexes (RTNs) for plasmid DNA delivery.7−10 The RTN formulations in those studies consisted of one of various cationic liposomes and a targeting peptide optimized for the target cell or tissue type. More recently, we took advantage of the modular structures of cationic lipids (L) and peptides (P) to prepare complexes with siRNA (R) (LPR) to make LPR selfassembling nanocomplexes, and optimized them for siRNA packaging and delivery.11 These formulations used a linear targeting peptide, K16GACYGLPHKFCG, which binds siRNA through the K16 motif and targets receptors on multiple cell types though the net cationic charge of the nanocomplex and the targeting motif YGLPHKF.12 This peptide ligand was originally identified by biopanning a phage peptide library for binding to cell surface receptors on airway epithelial cells13 and is similar to a seven amino acid motif in a protein expressed by © 2013 American Chemical Society

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EXPERIMENTAL SECTION

Transfection Reagents and Cells. Neuro-2A cells9 and Neuro2A-Luc cells18 stably expressing luciferase were maintained in Received: November 29, 2012 Revised: January 21, 2013 Published: January 22, 2013 761

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Table 1. Structures of Branched and Linear Peptides Used in This Study

Dulbecco’s minimal essential medium (DMEM; Invitrogen, Paisley, UK) supplemented with 10% fetal calf serum, 1% nonessential amino acids, and 1% sodium pyruvate. All cells were maintained at 37 °C in a humidified atmosphere in 5% carbon dioxide. The RNAs used were Silencer GAPDH siRNA and a Cy-3 labeled version (Applied Biosystems, Warrington, UK), Silencer Firefly Luciferase (GL2+GL3; Applied Biosystems, Warrington, UK), and Silencer Negative Control #1 siRNA (Applied Biosystems, Warrington, UK). Peptide structures are presented in Table 1. Peptide KY (MW: 3303) and Peptide RY (MW: 3752) were both synthesized by Alta Biosciences (Birmingham, UK). The branched peptides BK (MW: 3486) and BR (MW: 3823) were synthesized by UCL Department of Chemistry. The cationic liposome of 1,2-di-Ooctadecenyl-3-trimethylammonium propane (DOTMA) and dioleoylphosphatidylethanolamine (DOPE) at a 1:1 weight ratio was made by Avanti Polar Lipids (Alabaster, Alabama, USA). Lipofectamine 2000 (L2K) was purchased from Invitrogen (Paisley, UK). DNA and siRNA Transfections. LPR formulations were prepared at a weight ratio of 1 (liposome):4 (peptide):1 (siRNA) by first mixing, the liposome (1 mg/mL in water) with the peptide diluted at 0.1 mg/mL in OptiMEM (Invitrogen, Paisley, UK), followed by the addition and thorough mixing of siRNA (5 μM in OptiMEM). The mixture was incubated at room temperature for 30 min to allow complex formation, and then additional OptiMEM was added to give a final siRNA concentration of between 30 nM and 100 nM in a final volume of 200 μL per well of a 96-well plate sufficient for replicates of 6 per formulation group. The LPR consisting of DOTMA/DOPE, linear peptide KY, and siRNA was termed LKY. The other LPRs differed in the peptide constituent and were termed LBK (branched peptide BK), LBR (branched peptide BR), and LRY (linear peptide RY). Neuro-2A-Luc cells were seeded in 96-well plates at 2 × 104 per well 24 h prior to transfection. Following removal of growth medium on the Neuro-2A-Luc cells, the complexes were added to the cells in replicates of six and incubated for 4 h at 37 °C, then medium was replaced by the full growth medium and incubated for an additional 24 h. L2K was used as a positive control for siRNA transfections, and the transfection procedures were performed in accordance with the manufacturer’s instructions, formulated at a weight ratio of 4 (L2K):1 (siRNA). Luciferase activity was analyzed 24 h after transfection, the minimum time for effective gene knockdown.18 Luciferase Assay. Luciferase expression was measured in cell extracts with a luciferase assay (Promega, Southampton, UK) in a FLUOstar Optima luminometer (BMG Labtech, Aylesbury, UK). The amount of protein present in each cell lysate was determined with the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hemel Hempstead, UK). Luciferase activity was expressed as relative light units per milligram of protein (RLU/mg). Each measurement was performed in groups of six and the mean determined. Particle Size and Charge Measurements. LPRs were prepared as above and diluted to a final volume of 1 mL with 5 μg/mL siRNA in distilled water. They were then analyzed for size and charge (ζ

potential) using a Malvern Nano ZS (Malvern, UK). The data were then processed by software provided by the manufacturer, DTS version 5.03, and the data shown had a polydispersity index (PDI) of less than 0.3. Cell Proliferation Assay. Cell viability was assessed in 96-well plates using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Southampton, UK), which is a colorimetric method for determining the number of viable cells in proliferation or cytotoxicity assays. Neuro-2A-Luc cells were seeded and transfected with L2K/siRNA or LPRs as above. After 24 h, the media were changed with growth media containing 20 μL of CellTiter 96 Aqueous One Solution reagent. Finally, after incubation for 2 h, absorbance was measured on a FLUOstar Optima spectrophotometer (BMG Labtech, Aylesbury, UK). Cell viability for each complex was expressed as a percentage of the viability of control cells. PicoGreen Fluorescence Quenching Experiments. Briefly, 0.2 μg siRNA was mixed with PicoGreen reagent (1:150) (Invitrogen, Paisley, UK) at room temperature in TE buffer, and the siRNA/ PicoGreen mixture was then formulated into nanocomplexes with cationic peptides (PR) or with cationic liposome and cationic peptides into LPRs as described above. Fluorescence was analyzed using a fluorescence plate reader, FLUOstar Optima (BMG Labtech, Aylesbury, UK). In complex dissociation assays, heparin sulfate (Sigma, Poole, UK) was added to the complexes formulated in the PicoGreen fluorescence quenching experiment in a range of concentrations (0.05−5 U/mL) to assess the stability to dissociation of the siRNA from LPRs. In each experiment, naked siRNA stained with PicoGreen was used to normalize the PicoGreen signal detected from the complexes. Transmission Electron Microscopy (TEM). For the electron microscopy investigations, the liposomes or nanoparticles prepared as described above were applied onto a 300-mesh copper grid coated with a Formvar/carbon support film (Agar Scientific). Prior to preparation, the grids were “glow discharged” in an Emitech K350G system (Emitech Ltd.) for 15 s at 30 mA (negative polarity). After a few seconds, the grid was dried by blotting with filter paper. The sample was then negatively stained with 1% uranyl acetate for 2−3 s, before blotting with filter paper and air-dried. Imaging was performed with a Philips CM120 BioTwin transmission electron microscope and operated at an accelerating voltage of 120 kV. The images were captured using an AMT 5MP digital TEM camera (Deben UK Limited, Bury St. Edmunds, Suffolk). Confocal Microscopy. 1.5 × 105 Neuro-2A cells were seeded onto poly-L-lysine-coated slides (SLS, Dublin, Ireland). The following day, they were transfected with Cy3-labeled GAPDH siRNA (100 pmol; final concentration of siRNA = 200nM; Applied Biosystems, Warrington, UK) complexed with L2K at a 4:1 weight ratio or LBK and LBR at a 1:4:1 weight ratio. After 4 h of incubation, the slides were washed with phosphate-buffered saline (PBS) and fixed in 4% formaldehyde, permeabilized with 0.5% Triton, and stained for 45 min with AlexaFluor 488 phalloidin (Invitrogen, Paisley, UK). The 762

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slides were washed and sealed in mounting media containing 4',6diamidino-2-phenylindole (DAPI; Invitrogen, Paisley, UK) before visualizing on a Carl Zeiss LSM710 laser scanning microscope system (Jena, Germany) at a magnification of ×400. For the GAPDH silencing experiments, the Neuro-2A cells were transfected as above with GAPDH siRNA or irrelevant control siRNA complexed with L2K at a 4:1 weight ratio or LKY, LRY, LBK, and LBR at a 1:4:1 weight ratio. After 48 h incubation, the slides were washed with PBS and fixed in 4% formaldehyde, permeabilized with 0.5% Triton, and stained for 45 min with anti-GAPDH (Invitrogen, Paisley, UK). The slides were washed and sealed in mounting media containing DAPI before visualizing on a Carl Zeiss LSM710 laser scanning microscope system at a magnification of ×400. Statistical Analysis. All data in this article are expressed as means ± SD. Data were analyzed using a two-tailed, unpaired Student t test where applicable. Probability values of p < 0.05 were marked with *, p < 0.01 values were marked with **, and p < 0.001 values were marked with ***.

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RESULTS AND DISCUSSION Biophysical Characterization of LPR Nanocomplexes. Physical properties such as size and charge were evaluated for Table 2. Particle Size Measurements, ζ Potential and PDI of Complexes Made in Water complex LKY LRY LBK LBR

particle size (nm) 71.1 69.4 68.1 73.4

± ± ± ±

0.3 1.1 0.6 0.8

ζ potential (mV)

PDI

± ± ± ±

0.249 0.244 0.259 0.297

60.9 50.9 49.4 52.8

0.2 0.6 1.0 0.8

Figure 1. Binding properties and synergistic effect of the different components of PRs and LPRs. Relative fluorescence unit (RFU) of the siRNA complexes as a percentage of signal from free siRNA in fluorescence quenching experiments. (a) Weight ratios of different PR formulations with the peptide weight ratio varying. (b) Weight ratios of LPR formulations with the peptide weight ratio varying and liposome and siRNA weight ratios constant (at 1:1).

LPRs made at a 1:4:1 weight ratio, which corresponds to the following molar ratios: 19.7:10.3:1 (LKY), 19.7:9.5:1 (LRY), 19.7:10:1 (LBK), and 19.7:9.4:1 (LBR). All LPR formulations proved to be strongly cationic (ca. +49−61 mV) with sizes of less than 75 nm with no significant differences between the formulations and all PDIs less than 0.3 (Table 2 and Figure S1), indicating a monodisperse population of particles.19,20 The next objective was to determine the effect that the cationic sequence, and branching, had on the condensation of siRNA within the nanoparticles. The three-dimensional folding options of the polycationic components in siRNA formulations may be an important issue in packaging since siRNA itself is short and rigid.21,22 Polycationic formulations where alternative structures have illustrated the importance of folding include branched polyethyleneimine (PEI) compared to linear PEI,18,23 and high molecular weight chitosan compared to a low molecular weight homologue.24 PicoGreen fluorescence quenching studies were performed to evaluate the effectiveness of packaging (>95%) of siRNA within the PR and LPR nanocomplex. The KY/siRNA complexes achieved 50% quenching of fluorescence at a weight ratio of 0.4:1 (0.36:1 charge ratio) and achieved greater than 95% fluorescence quenching at 2:1 weight ratio (Figure 1a). The other PR complexes as seen in Figure 1a achieved 50% quenching of fluorescence at higher weight ratios: BK/siRNA at 1.32:1 (1.22:1 charge ratio), BR/siRNA at 1.16:1 (1.15:1 charge ratio), and RY/siRNA at 1.03:1 (1:1 charge ratio). Therefore, the linear peptides KY and RY were better at siRNA packaging in polyplexes than branched peptides, and lysine was much better for siRNA fluorescence quenching than arginine in the linear format, although there was no significant difference between lysine and arginine branched peptides. Greatest fluorescence quenching of siRNA was achieved to similar

levels by the linear peptides RY (95.9%) and KY (96.0%). Branched arginine BR (94.5%) quenched more efficiently than its lysine counterpart BK (84.9%). This suggests that the branching of the lysine and arginine peptides suppresses compaction of the siRNA within the nanocomplex compared to linear peptides. Although the 50% quenching data suggested arginine was an inferior compaction agent compared to lysine, at sufficient amounts, it can achieve the same level of maximal quenching. The presence of liposome in all LPR formulations enhanced packaging of the siRNA compared to PR formulations (Figure 1b). For example, when comparing the 0.25:1 weight ratio of PRs (Figure 1a) with the 1:0.25:1 weight ratio for LPRs (Figure 1b), 5.6, 3.6, 3.8, and 3.4-fold more quenching was achieved by addition of the liposome in KY, BK, RY, and BR formulations, respectively. As in PR formulations, LPRs containing linear lysine were quenched more efficiently, particularly at lower peptide:siRNA ratios up to 1:1 weight ratio. For example, a 1:0.5:1 weight ratio for LKY gave >92.5% quenching, whereas the other three peptides at that weight ratio were quenched 80% or less. Maximal quenching for all peptides was achieved at a 1:4:1 ratio (>95% for LKY and LRY, > 94% for LBR and >91% for LBK). Therefore, while the peptide alone in PR complexes achieves the same maximal level of fluorescence quenching as in LPR complexes, at low peptide:siRNA ratios better packaging was achieved by the addition of liposome in LPR formulations. The combination of the lipid and peptide 763

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Figure 2. The dissociation properties of LKY, LBK, LBR, and LRY nanocomplexes. PicoGreen fluorescence of complexes, after incubation with heparin (0−5 U/mL), was expressed as a percentage of RFU relative to free siRNA. (a) LBK at four different weight ratios, (b) LBR at four different weight ratios, (c) LKY at four different weight ratios, (d) LRY at four different weight ratios, and (e) LPR complexes at a 1:4:1 weight ratio.

The linear, lysine-rich peptide KY was the best peptide for quenching at low peptide:siRNA weight ratios in both PR and LPR formulations, while at higher ratios, as optimal for use in

components in the LPR formulations at optimal ratios act synergistically to package siRNA and are both essential for the formation of stable siRNA complexes. 764

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assessed their stability and potential for dissociation and release of siRNA in the cytoplasm. While extracellular stability is an essential requirement for an effective siRNA delivery formulation, more effective cellular delivery is achieved if the vectors are able to release the cargo nucleic acid easily following internalization in the cell.30 Cationic nanocomplexes may be dissociated by high concentrations of polyanions, which occur widely in biological environments and include proteins, mRNA, and glycosaminoglycans, which are linear polysaccharides such as chondroitin sulfate, heparan sulfate, and hyaluronic acid.31 The higher concentration of these polyanions within cellular compartments (e.g., glycosaminoglycans, mRNA, tRNA) and at the cell surface (e.g., anionic polysaccharides, sialic acid residues) compared to the extracellular environment provides an appropriate selective biological trigger for intracellular nanocomplex dissociation. Heparin is used widely in studies of dissociation of nucleic acids from polycationic complexes.11,32−34 The stability and dissociation properties of the complexes were investigated by exposure to a range of concentrations of heparin sulfate (0.05−5 U/mL). PicoGreen-labeled complexes were prepared as above at a weight ratio of 1:4:1 (lipid:peptide:siRNA) based on previous packaging (Figure 1b) and transfection data.11 Fluorescence RFU values were determined for each complex after heparin treatment (Figure 2a-e). Different LPR weight ratios were then formulated in order to investigate heparin dissociation when the peptide to siRNA weight ratio was kept constant and the liposome to siRNA ratio changed. We had hypothesized that, based on previous observations, increasing lipid content would result in better packaging,11 and, therefore, more heparin would be needed for dissociation. Indeed, for all LPRs, an increase in the liposome ratio (from 1:1 to 2:1, 3:1, and 4:1) with the peptide to siRNA ratio kept constant at 4:1, showed that more heparin was needed to dissociate the siRNA from LBK (Figure 2a), LBR (Figure 2b), LKY (Figure 2c), and LRY (Figure 2d). For

Table 3. Summary of the Heparin Dissociation Experiments formulation

weight ratio

50% dissociation heparin (U/mL)

LKY LKY LKY LKY LRY LRY LRY LRY LBK LBK LBK LBK LBR LBR LBR LBR

1:4:1 2:4:1 3:4:1 4:4:1 1:4:1 2:4:1 3:4:1 4:4:1 1:4:1 2:4:1 3:4:1 4:4:1 1:4:1 2:4:1 3:4:1 4:4:1

0.41 0.65 0.68 0.71 0.21 0.56 0.70 0.77 0.18 0.43 1.00 1.70 0.20 0.31 0.40 0.46

transfections, there was no significant difference between quenching levels of linear lysine and linear arginine in both PR and LPR formulations. No information is published on the interaction of oligolysine and oligoarginine peptides with siRNA, but in DNA studies it was shown that arginine binds more tightly to the phosphate backbone of DNA, resulting in stronger complexation,25,26 which appears to conflict with the data presented here on siRNA. Previous studies showed different binding modes of lysine and arginine peptides to the backbone phosphate groups of DNA.27−29 Whether these findings would hold for siRNA is unknown bearing in mind the differences between the two nucleic acids, but further studies are needed into the differences of binding between these two nucleic acids and cationic peptides. Heparin Dissociation Studies. Having demonstrated nanocomplex structure and packaging of siRNA, to further characterize the behavior of LPR nanocomplexes, we next

Figure 3. Electron microscopy of nanocomplexes. Negative staining TEM was used to visualize LKY, LBK, LRY, and LBR nanoparticles. Scale bar = 100 nm. 765

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Figure 4. SiRNA knockdown and cell viability. (a) LKY, LBK, LR, and LBR complexes at 1:4:1 weight ratio using siRNA targeting luciferase in Neuro-2A-Luc cells at two concentrations, 50 and 100 nM. (b) LKY, LBK, LRY, and LBR complexes at 1:4:1 and 3:4:1weight ratio using siRNA targeting luciferase in Neuro-2A-Luc cells at a concentration of 50 nM. LPR formulations with irrelevant control (siRNA IRR) were formulated for all silencing transfections shown in (a-b). Untreated cells received no siRNA complexes, while L2K/siRNA was used as a positive control. All formulations achieved significant silencing when compared to their respective irrelevant control formulations (p < 0.05 and in some cases p < 0.01 or p < 0.001). (c) Viability of Neuro-2A-Luc cells following transfection for 24 h with L2K/siRNA, LKY, LBK, LBR, and LRY (all siRNAs were targeting luciferase). Viability values were normalized to the untransfected control cells. All transfections were performed in groups of six, and mean values were calculated. Asterisks indicate comparisons of specific formulations with statistical significance (*, p < 0.05 and ***, p < 0.001).

example, at a 2:4:1 ratio, 0.65 U/mL of heparin was required for 50% dissociation of LKY versus 0.41 U/mL at a 1:4:1 weight ratio (Table 3), and the same was true for LRY (0.56 vs

0.21 U/mL heparin), LBK (0.43 vs 0.18 U/mL heparin), and LBR (0.31 vs 0.20 U/mL heparin). 766

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Figure 5. Confocal microscopy for the localization of siRNA. Neuro-2A cells were transfected with LBK, LBR (at a 1:4:1 weight ratio), or L2K/ siRNA (at a 4:1 weight ratio) complexes, where the siRNA was Cy3-labeled GAPDH (red). Pictures were taken 4 h after transfection. The cells were stained with phalloidin for F-actin on the cytoskeleton in the cytoplasm (green) and DAPI for the nucleus (blue). UNTR = untreated cells. Scale bar = 20 μm.

SiRNA Transfection Studies. Linear and branched oligolysine and oligoarginine LPR formulations at a 1:4:1 (L:P:R) weight ratio were compared for silencing of luciferase in transfections of Neuro-2A-Luc cells. This weight ratio was selected from the biophysical data in this study and from previous studies on linear lysine LPR formulations where 1:4:1 (L:P:R) was optimal.11 The branched arginine formulation, LBR, was significantly better by approximately 2-fold than the linear arginine formulation, LRY, at 100 nM siRNA (p < 0.001) (Figure 4a), whereas the branched lysine formulation, LBK, showed a significant difference in silencing efficiency from the linear lysine formulation (Figure 4a), LKY, at 50 nM siRNA, albeit with a smaller difference of approximately 1.27-fold (p < 0.05). The LPR formulations at 1:4:1 weight ratios were then compared in transfections with formulations at 3:4:1 weight ratios using 50 nM siRNA (Figure 4b). All formulations at both ratios gave similar luciferase silencing, except for LBK, where the 3:4:1 ratio was significantly less efficient than the 1:4:1 ratio (p < 0.05). This result is consistent with Figure 2a, where LBK at a 1:4:1 weight ratio was less stable to heparin challenge than the same nanocomplex at 3:4:1. This suggests the possibility of improved intracellular release of siRNA from LBK at the lower weight ratio in the cytoplasm, leading to improved silencing. For the other nanocomplexes, the enhancement of stability at the higher ratio of lipid was less pronounced (Table 3), corresponding with the similar transfection efficiencies. In MTS toxicity assays of transfected cells, LKY was the least toxic formulation (81−90% cell viability), followed by LRY (70−79%), LBK (68−82%), and LBR (61−76%), while L2K was most toxic at 50 nM (48.8% cell viability, p < 0.001) and

In comparing the stability of each of the formulations at the 1:4:1 ratio (Table 3 and Figure 2e), LKY formulations showed 50% dissociation at a heparin concentration of 0.41 U/mL, which was more than double the heparin concentration needed for 50% dissociation of LRY. The dissociation characteristics of the branched peptides, LBK and LBR, were very similar to each other at the 1:4:1 ratio, while at all higher weight ratios, LBK nanocomplexes were more stable than the other formulations, requiring more heparin for dissociation (Table 3). Reduced stability was observed here for linear arginine peptides compared to linear lysine peptides (Figure 2e). However, for branched arginine peptides compared to branched lysine peptides, stability was reduced only at higher weight ratios of liposome to siRNA (Figure 2a, b). The reduced nanocomplex stability, could be due to the fact that arginine binds heparin with higher affinity than lysine under a variety of conditions.35 Electron Microscopy. All four LPR nanoparticle formulations were analyzed by negative staining TEM to determine differences in shape and morphology due to the peptides (branched or linear). All samples contained material that appeared in various pleomorphic conformations and sizes (Figure 3). Numerous spherical particles were observed for each formulation, with the majority of the particles being less than 100 nm in diameter. Some particles appeared to be solid (white arrow in LKY picture), while others displayed a multilayered structure (Figure 3, inset in LKY picture, and pictures for LRY, LBK, and LBR). There were no obvious differences between the nanocomplexes containing linear and branched peptides, or between the nanocomplexes with arginine or lysine peptides. 767

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Figure 6. SiRNA silencing of GAPDH gene expression. GAPDH siRNA and irrelevant control siRNA transfected into Neuro-2A cells, and analyzed by fluorescence microscopy with an anti-GAPDH antibody. Blue: DAPI for the nucleus; green: fluorescein-labeled antibody to GAPDH. SiRNA silencing of GAPDH expression in Neuro-2A cells is demonstrated following transfection with LKY, LBK, LBR and LRY complexes with siRNA targeting GAPDH. Irrelevant control siRNA has no effect on GAPDH protein levels (LKY IRR). UNTR = untreated cells. Scale bar = 20 μm.

Internalization of the siRNA Complexes and Silencing Effect. Cellular uptake and distribution of siRNA delivered by LPR nanocomplexes was studied by fluorescence microscopy in Neuro-2A cells transfected for 4 h with a formulation of LBK or LBR with Cy3-labeled GAPDH siRNA. The cells were stained with phalloidin for the cytoplasm and DAPI for the nucleus, then assessed by confocal microscopy (Figure 5). Fluorescent LPR nanoparticles appeared inside the cytoplasm at 4 h. No fluorescent siRNA was observed in the nucleus. L2K/Cy3labeled GAPDH siRNA complexes showed similar cytoplasmic distribution at 4 h. These data are in agreement with our previous report where we showed intracellular detection of L2K and LKY nanocomplexes as early as 30 min.11 In silencing experiments, LPR formulations targeted GADPH gene expression (Figure 6). Neuro-2A cells were transfected with GAPDH siRNA and irrelevant control siRNA and

100 nM (41.5% cell viability, p < 0.001) (Figure 4c). The linear peptides LKY and LRY were both significantly less toxic than their branched counterparts, LBK at 50 and 100 nM (p < 0.05 in both cases) and LBR at 100 nM (p < 0.05). The branched peptides contain extra free amines stemming from the first lysine and arginine on each branch, and this might explain the higher cytotoxicity observed when compared to linear peptides. Increased toxicities are often encountered with branched dendrimers used for gene delivery, but when the peripheral primary amines are masked with chemical groups, their cytotoxicity is significantly reduced.36 Oligoarginine has been reported to be cytotoxic previously,37 but in this study, toxicity of branched and linear lysine and arginine formulations was not significantly different. L2K formulations were more toxic than LPRs at 50 and 100 nM, which is consistent with previously reported studies on L2K cytotoxicity.11,38,39 768

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analyzed by fluorescence microscopy with an anti-GAPDH antibody. SiRNA silencing of GAPDH expression in Neuro-2A cells was evident following transfection with LKY, LBK, LBR, and LRY complexes, therefore providing further evidence for the silencing potency of the LPR formulations. GAPDH is known for its key enzyme role in glycolysis, but it also participates in telomere protection, proliferation, apoptosis, and transcription.40−43 GAPDH is localized mainly in the cytoplasm of nondividing cells, although it is also found in the nucleus of dividing cells.44 Similarly, in this study, GAPDH was observed (Figure 6) in both the cytoplasm and nucleus of untransfected cells and of cells transfected with irrelevant siRNA in LKY formulations.

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CONCLUSIONS We have developed LPR nanocomplexes containing a liposome mixed with a targeting peptide containing either linear or branched sequences of oligoarginine or oligolysine of the same net charge for delivery of siRNA. They effectively packaged the siRNA into nanoparticles of less than 75 nm in size, which were dissociated by anionic heparin, suggesting that they could potentially disassemble within the cell in response to high concentrations of anions in the cytoplasm. The nanocomplexes containing linear peptides appeared to be more compact and stable than those containing branched peptides, but the transfection efficiency of LPR complexes containing branched peptides, particularly those containing oligoarginine, was significantly better and achieved better silencing than the linear peptide formulations. Thus packaging capacity and stability parameters are important factors for nanoparticle formation, which can affect silencing efficiency, but the relationship may be complex, particularly with branched or dendrimeric structures due to the wider structural potential of branched peptides. The silencing effect of LPRs was comparable with that of the commercially available L2K, but without any significant cytotoxicity, which is associated with the latter. The LPR nanoparticle delivery system may be used to advance the field of siRNA therapeutics.

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ASSOCIATED CONTENT

* Supporting Information S

Figure S1 on particle distribution. This material is available free of charge via the Internet at http://pubs.acs.org

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AUTHOR INFORMATION

Corresponding Author

*Tel: +44 (0) 207-9052228; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the Engineering and Physical Sciences Research Council (EPSRC; EP/G061521/1). L.S. was funded by the Fundacao para a Ciencia e a Tecnologia, Portugal. We would like to thank Miss Atefeh Mohammadi, Dr. Frederick Campbell, and Dr. Alethea Tabor (Department of Chemistry, UCL, UK) for providing us with peptides BR and BK.

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