Gene Delivery Using Ternary Lipopolyplexes Incorporating Branched

Dec 4, 2012 - and Alethea B. Tabor*. ,†. †. Department of Chemistry, University College London, Christopher Ingold Laboratories, 20 Gordon Street,...
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Gene Delivery Using Ternary Lipopolyplexes Incorporating Branched Cationic Peptides: The Role of Peptide Sequence and Branching Katharina Welser,† Frederick Campbell,† Laila Kudsiova,‡ Atefeh Mohammadi,† Natalie Dawson,† Stephen L. Hart,§ David J. Barlow,‡ Helen C. Hailes,† M. Jayne Lawrence,‡ and Alethea B. Tabor*,† †

Department of Chemistry, University College London, Christopher Ingold Laboratories, 20 Gordon Street, London WC1H 0AJ, U.K. ‡ Institute of Pharmaceutical Science, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, Waterloo Campus, London SE1 9NH, U.K. § Wolfson Centre for Gene Therapy of Childhood Disease, UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH, U.K. S Supporting Information *

ABSTRACT: Cationic peptide sequences, whether linear, branched, or dendritic, are widely used to condense and protect DNA in both polyplex and lipopolyplex gene delivery vectors. How these peptides behave within these particles and the consequences this has on transfection efficiency remain poorly understood. We have compared, in parallel, a complete series of cationic peptides, both branched and linear, coformulated with plasmid DNA to give polyplexes, or with plasmid DNA and the cationic lipid, DOTMA, mixed with 50% of the neutral helper lipid, DOPE, to give lipopolyplexes, and correlated the transfection efficiencies of these complexes to their biophysical properties. Lipopolyplexes formulated from branched Arg-rich peptides, or linear Lys-rich peptides, show the best transfection efficiencies in an alveolar epithelial cell line, with His-rich peptides being relatively ineffective. The majority of the biophysical studies (circular dichroism, dynamic light scattering, zeta potential, small angle neutron scattering, and gel band shift assay) indicated that all of the formulations were similar in size, surface charge, and lipid bilayer structure, and longer cationic sequences, in general, gave better transfection efficiencies. Whereas lipopolyplexes formulated from branched Argcontaining peptides were more effective than those formulated from linear Arg-containing sequences, the reverse was true for Lys-containing sequences, which may be related to differences in DNA condensation between Arg-rich and Lys-rich peptides observed in the CD studies. KEYWORDS: nonviral gene delivery, cationic peptides, circular dichroism, branched peptides, lipids, small angle neutron scattering, structure−activity correlation

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INTRODUCTION

Within LPD complexes, the peptide component primarily serves to condense DNA within the complex core. However, multifunctional peptides can be designed that additionally serve to target cell surface receptors, promote cellular internalization, aid endosomal escape, and carry the DNA payload to the nucleus.5 In addition, vectors formulated using cationic peptides typically demonstrate lower toxicities than those formulated from other cationic polymers, such as polyethyleneimine (PEI).6 Given their relatively high pKa, cationic poly-L-Lys and linear Lys sequences have commonly been used to condense and protect DNA in lipopolyplex systems.7−9 Cationic sequences, such as the polymeric poly-L-Arg,10 or shorter synthetic Arg11,12

The development of complexes capable of efficient and cellspecific delivery of plasmid DNA in vivo remains an ongoing challenge. A successful DNA gene vector should condense plasmid DNA; yield small particles; protect DNA from extracellular degradation; selectively target a cell type of interest; trigger complex internalization; facilitate endosomal escape; and, ideally, mediate DNA transport to the nucleus and promote nuclear internalization.1 A variety of nonviral gene delivery vectors have been developed, including lipoplexes (LD; complexes of cationic lipid and DNA), polyplexes (PD; complexes of cationic peptide and DNA), and, of particular interest to this study, lipopolyplex (LPD) formulations where cationic peptides and cationic lipids (generally combined with a neutral helper lipid) are coformulated with plasmid DNA to form ternary complexes.2−4 © 2012 American Chemical Society

Received: Revised: Accepted: Published: 127

April 6, 2012 October 24, 2012 November 9, 2012 December 4, 2012 dx.doi.org/10.1021/mp300187t | Mol. Pharmaceutics 2013, 10, 127−141

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allowing the chloroform to slowly evaporate overnight in a vacuum desiccator. The resulting lipid film was hydrated with double-distilled, filtered (cellulose acetate (Millipore) filter 0.20 μm) water at ambient temperature to produce a crude suspension of 1 mg mL−1 based on the DOTMA present. The crude suspension was then sonicated at room temperature for 5 min using a Lucas Dawes probe sonicator (model no.: 7535A) fitted with a tapered microtip operating at 50% of maximum output. Formation of Polyplexes and Lipopolyplexes. gWIZLuc plasmid DNA (pDNA) was purchased from Aldevron (USA), and calf thymus DNA (ctDNA) was obtained from Sigma (U.K.). The absence of any RNA and protein in the ctDNA was confirmed by the ratio of the UV absorbance at 260 and 280 nm. OptiMEM was supplied from Invitrogen (U.K.). DNA (either calf thymus DNA (ctDNA) or gWIZ-Luc plasmid DNA (pDNA)) and peptides were individually diluted to a concentration of 1 mg mL−1 with double-distilled, filtered water (for dynamic light scattering, zeta potential, gel shift assays, and transfection studies) or D2O (circular dichroism and small angle neutron scattering measurements). The DNA and peptide solutions and the lipid suspensions were further diluted, as required, using either double-distilled, filtered water or D2O as appropriate, or in the case of the transfection studies, OptiMEM to give the required DNA and peptide stock solutions, and the lipid stock suspensions for preparation of the PD or LPD complexes. When preparing PDs and LPDs at given charge ratios it was assumed the peptides would be fully ionized under the conditions of use (Supporting Information, Table S1). PD complexes were formulated by adding equal volumes of the required DNA stock solution to a peptide solution containing the amount of peptide required to produce PDs at either a 6:1 or 12:1 charge ratio (see Supporting Information, Table S2, for actual peptide concentrations used). LPD complexes were prepared by mixing the vesicle suspension with peptide and DNA solutions to give LPDs of lipid:peptide:DNA charge ratios of 0.5:6:1 and 0.5:12:1, respectively. For dynamic light scattering and zeta potential measurements, PDs and LPDs were formulated at weight ratios of 2:1, 4:1, and 8:1 and 1:2:1, 1:4:1, and 1:8:1 respectively. These weight ratios correlate to charge ratios of (3−4):1 to (9−16):1 for PDs and 0.5:(3−4):1 to 0.5:(9−16):1 for LPDs. The order of mixing of the components is of critical importance.26,27 An equal volume, for example, 154 μL of peptide solution (see Supporting Information, Table S2, for actual peptide concentrations used), was added to 154 μL (0.024 mg mL−1 of DOTMA and DOPE) of a vesicle suspension. To this lipid:peptide mixture, an equal volume of DNA solution (in this example 308 μL of a 0.01 mg mL−1 DNA solution) was added and the resulting suspension mixed by gentle vortexing. In Vitro Transfection. Lipofectamine was purchased from Invitrogen (U.K.) and was used as a positive control in the following experiments after preparation using the manufacturer's instructions. In brief, Lipofectamine was diluted with OptiMEM before complexing with the required amount of DNA to produce a final Lipofectamine:DNA weight ratio of 5:1. A549 cells were seeded onto 24-well plates at 1.2 × 105 cells per well and incubated at 37 °C in a 5% CO2 and 90% relative humidity environment for 24 h. The culture medium was then removed, and the cells were rinsed with 200 μL of OptiMEM. OptiMEM (200 μL) was then added to each well followed by the addition of 200 μL of a PD suspension at either a 6:1 or 12:1 charge ratio or LPD complexes at either a 0.5:6:1 or 0.5:12:1 charge ratio. Both

sequences, have been less frequently studied despite their prevalence as cell-penetrating peptides (CPP) and/or nuclear localization sequences (NLS).5,13,14 D-Arg sequences have recently generated significant interest for siRNA delivery, however, DNA delivery systems based on D-Arg have not been studied to date.15 Branched and dendritic peptides based on Lys, Arg, histidylated poly-L-Lys, and alternating His and Lys sequences have also been shown to effectively condense, protect, and deliver DNA within PD and LPD systems.16−24 We have recently carried out extensive investigations into the gene delivery properties of LPD complexes, incorporating multifunctional peptides containing both a linear, polycationic (Lys)16 domain and a cyclic target domain.25−27 Peptide and plasmid DNA were complexed and encapsulated in a 1:1 molar ratio mixture of various cationic lipids with the neutral lipid, dioleoylphosphatidylethanolamine (DOPE). The latter is believed to promote fusion with the endosomal membrane and mediate endosomal escape.2 These LPD complexes have been optimized for a number of in vitro applications, including delivery to respiratory cells.28,29 Alternative cationic lipid components with short ethylene glycol oligomer headgroups have also been investigated, 30−32 and used to formulate targeted LPD complexes which are stable in the systemic circulation but which disassemble once internalized in the target cell.33,34 In our previous work, the peptide component has always contained a linear, polycationic (Lys)16 domain to condense DNA. Very few previous studies have compared the effects of branched versus linear peptides on the transfection efficiencies of PD or LPD complexes,18,19 and to our knowledge a complete survey of the cationic amino acid sequences, and degree of branching, that will give the best transfection efficiencies with the lowest toxicities in LPD complexes, has never been undertaken. In the present study we have set out to understand, at a fundamental level, the role and influence of polycationic peptides in an LPD gene vector system. Herein, we have compared, in parallel, a comprehensive set of targeted cationic peptides, varying composition and degree of branching, in both PD and LPD systems, with the 1:1 mixture of the lipids DOTMA and DOPE kept constant throughout the study. For the first time, direct parallels can be drawn between the sequence of cationic amino acids, the degree of peptide branching, the condensation and protection of DNA in LPD complexes, and transfection efficiencies ex vivo.

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METHODS General synthetic methods and the synthesis, purification, and analytical data for the peptides are described in the Supporting Information. Cell Culture. A549 (human alveolar epithelial) cells were supplied by LGC Promochem (U.K.). Cell culture media and reagents were all purchased from Sigma (U.K.). A549 cells were cultured in tissue flasks in RPMI-1640 medium supplemented with 10% v/v fetal bovine serum (FBS), 1% v/v of 100 times strength nonessential amino acids (NEAA), and 1% v/v penicillin/streptomycin solution (10 000 U mL−1/10 mg mL−1) at 37 °C, 5% CO2, and 90% relative humidity. The cells were passaged every four days when ∼70% confluent using 0.25% w/v trypsin-EDTA solution (Sigma, U.K.). Preparation of Cationic Lipid Vesicles. Dioleoylphosphatidylethanolamine (DOPE) was obtained from Avanti Polar Lipids (USA). 1,2-Di-O-octadecenyl-3-trimethylammonium propane (DOTMA) was supplied by TCI Europe (Belgium). The required amounts of DOTMA and DOPE were first dissolved in chloroform, and a thin lipid film was produced by 128

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10 mg mL−1 pAsp solution to 10 μL of the various complexes. Experiments to determine the level of DNA protection afforded by the complexes from enzymatic degradation were prepared by adding first 1 μL of a 0.1 M MgCl2 solution followed by 1 μL of 1000 U mL−1 DNase I to 10 μL of the various complexes. After incubating the mixture for 10 min at 37 °C, 2.5 μL of 0.5 M EDTA solution was added, followed by 1.25 μL of 10 mg mL−1 pAsp solution. Negative and positive controls comprised a solution of pDNA and a solution of pDNA treated with DNase I, respectively. To all samples, 2 μL (or 3 μL in case of the samples measuring pDNA protection) of gel loading buffer (0.25% w/v bromphenol blue and 40% w/v sucrose, pH 7.4) was added before loading the sample onto a 0.8% w/v agarose gel in Tris-acetate-EDTA (TAE) buffer, pH 7.4, containing 3 μL of GelRed DNA gel stain (Invitrogen, U.K.) at 80 V for 60 min (Fisher Brand, model HU12 electrophoresis chamber) and visualized under UV light using an AlphaImage EP MultiImage Light Cabinet (Alpha Innotech, South Africa). In all cases each sample was measured on at least two other occasions to ensure the reproducibility of the data. Circular Dichroism and Ultraviolet Spectroscopy. The circular dichroism (CD) and ultraviolet (UV) spectra of DOTMA:DOPE vesicles, ctDNA, peptide, PDs, and LPDs, all dispersed in D2O (>99% purity, Aldrich, U.K.), were measured at 20 ± 2 °C using a Chirascan Plus spectrometer (Applied Photophysics, U.K.) operated with a scan speed of 30 nm min−1, a bandwidth of 1 nm, and a time per point of 2 s. Spectra were recorded between 320 and 180 nm when a cell of path length of 1 mm was used and 340 and 210 nm when a 1 cm path length cell was used. CD and UV absorbance spectra were acquired simultaneously. All spectra were corrected for D2O background, which was measured periodically throughout the experiment. The CD and UV spectra of ctDNA were measured at a concentration of 0.025 mg mL−1, while the spectra of the peptides K12B0-L1-[Y], K6B1-L1-[Y], K6B2-L1-[Y], R12B0[L1]-Y, R6B1-[L1]-Y, and H6B1-L1-[Y] were measured at a concentration of 6 μM (equivalent to a 0.02−0.04 mg mL−1 of peptide). The CD and UV spectra of the PD and LPD complexes were measured at charge ratios of 6:1 and 0.5:6:1, respectively such that the final concentration of ctDNA in each sample was 0.025 mg mL−1. DOTMA:DOPE vesicles were measured at a DOTMA concentration of 0.028 mg mL−1, i.e., the same concentration of lipid that was present in the LPD complexes. Transmission Electron Microscopy. PD and LPD complexes, prepared at a final pDNA concentration of 0.01 mg mL−1, were examined via transmission electron microscopy (TEM) by negative staining with 4% w/v uranyl acetate (Sigma, U.K.). Briefly, a drop of the sample was placed on a Formvar 200 mesh copper grid for one minute followed by removing excess liquid using a wick of filter paper. A drop of uranyl acetate was then added to the grid for approximately five minutes, after which time the grid was washed with 50% v/v ethanol in water. The dried sample was visualized using an FEI Tecnai transmission electron microscope (USA). Small Angle Neutron Scattering. The small angle neutron scattering (SANS) of the LPDs and the DOTMA:DOPE vesicles from which they were prepared was measured on the LOQ and SANS2D beamlines at the ISIS pulsed neutron source (ISIS Facility, the Rutherford-Appleton Laboratories, Didcot, U.K.). The DOTMA:DOPE vesicles dispersed in D2O were measured at a DOTMA concentration of 1 mg mL−1. LPD samples were freshly prepared in either 8.2 vol % D2O in H2O or D2O at a

PD and LPD complexes were prepared in triplicate. The final amount of DNA in each well of 400 μL final volume of test suspension was 1 μg. The cells were incubated at 37 °C in a 5% CO2 atmosphere and 90% relative humidity for 4 h, after which time the test suspensions were removed and replaced by 1 mL of cell culture medium. The cells were incubated for 48 h at 37 °C in a 5% CO2 and 90% relative humidity environment. Luciferase expression levels were then measured using a luciferase assay kit (Promega, USA) according to the manufacturer’s protocol. Briefly, the cells were rinsed with 200 μL of phosphate buffered saline (PBS) and then lysed by the addition of 200 μL of 1× reporter lysis buffer (provided in the kit) for 1 h at 25 °C. After freezing the cells for 30 min at −80 °C, the lysed cells were thawed, 50 μL of the lysate was transferred to a white 96-well plate, and the luciferase activity was measured over 10 s using a MLX microtiter plate luminometer (Dynex Technologies, USA) fitted with an automated feeding system delivering 100 μL of the reconstituted Luciferase assay reagent into each well. The amount of protein in each cell lysate was determined using a Pierce BCA Protein Assay kit (Thermo Scientific, U.K.) according to the manufacturer’s instructions. Briefly, 20 μL of the cell lysate and 20 μL of bovine serum albumin (BSA) protein standards were transferred into a transparent 96-well plate to which 200 μL of the mix reagent A/B (provided in the kit) was added. The plate was incubated with slight shaking at 37 °C for 30 min, after which time the absorbance was measured at 562 nm using a SpectraMax 190 plate reader (Molecular Device, USA). Luciferase activity was expressed as relative light units (RLU) per milligram of protein (RLU per mg protein). All measurements were carried out in triplicate, and the error bars represent the standard deviation calculated from three different measurements carried out in one experiment. Transfections for the PDs and LPDs have been repeated on at least two, but generally three, different occasions to confirm that the results were reproducible (repeat data sets not shown). Dynamic Light Scattering and Zeta Potential Measurement. The apparent hydrodynamic size (assuming the presence of spherical particles) and the zeta potential of PD and LPD complexes were measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments, U.K.) at 25 ± 0.1 °C. PD and LPD complexes were diluted in filtered (cellulose acetate (Millipore) filter 0.20 μm), double-distilled water, usually at a final DNA concentration range between 0.003 and 0.005 mg mL−1. When the apparent hydrodynamic size of the PD and LPD complexes prepared for the CD spectroscopy and small angle neutron scattering measurements was determined, slightly higher DNA concentrations of 0.025 and 0.1 mg mL−1, respectively, were used. When the complexes were prepared in D2O, the appropriate refractive index and viscosity values of D2O were used. In all cases, three repeat measurements were made for each sample and each sample was measured on at least two other occasions to ensure reproducibility of the data. The error bars represent the standard deviation calculated from three repeat measurements carried out in one experiment. Gel Band Shift Assay. DNase I and poly-L-aspartic acid (pAsp) were purchased from Sigma (U.K.). Suspensions of complexes were prepared as above at a 6:1 charge ratio for the PD complexes and a 0.5:6:1 charge ratio for the LPD complexes. 1 μg of pDNA and 10 μL of complex suspension were used in each experiment. To determine the extent of DNA complexation, PD or LPD complexes were used without further treatment. Experiments to measure the extent of dissociation of DNA from the complexes were prepared by the addition of 1.25 μL of 129

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lipid:peptide:DNA charge ratio of 0.5:6:1 using a ctDNA concentration of 0.1 mg mL−1. LPD complexes or vesicle suspensions were placed in clean, disk-shaped fused silica cells of 2 mm path length and their SANS measured at 25 ± 0.1 °C. The SANS intensity, I(Q), of a sample as a function of the scattering vector, Q = (4π/λ)sin(θ/2), where θ/2 is the scattering angle, was normalized to the appropriate sample transmission after subtraction of the scattering from the corresponding dispersion media. Fitting procedures included flat background corrections to allow for any mismatch in the incoherent and inelastic scattering between the sample and the dispersion media. Fitted background levels were always checked to ensure that they were of a physically reasonable magnitude. The SANS data for the DOTMA:DOPE vesicles and LPD complexes could be well modeled assuming a mixture of (isolated) infinite planar (lamellar) sheets and one-dimensional paracrystals (stacks), to account for the presence, in the suspension, of multilamellar vesicles.35 Samples were generally measured on more than one occasion to ensure the reproducibility of the data. When modeling the vesicles and LPD complexes dispersed in D2O as (single) sheets, the fits to the SANS data were obtained by the least-squares refinement of three parameters, namely L, Rσ, and the absolute scale factor (together with the background, as described above), where Rσ is the Lorentz correction factor which provides information about the extent of rigidity/ curvature of the lamellar sheets. Since the polydispersity on the bilayer thickness (σ(L)/L) can only reliably be fitted if good quality data are available in the high Q regime, and given the limited Q range of the measurements recorded here, this parameter was fixed as 0.1. In the mixed sheet and stack model, the fit to the SANS data is obtained by least-squares refinement of seven parameters, namely, the mean bilayer thickness (L), the Lorentz factor (Rσ), the number of bilayers in the stack (M), their mean separation or d-spacing (D), the width of the Gaussian distribution in the plane, (σ(D)/D), and the absolute scale factors for the unilamellar and multilamellar vesicles. In the present study, σ(L)/L was again fixed as 0.1, σ(D)/D at 0.05, and Rσ at 300. Furthermore, when modeling a mixed population of sheets and stacks, L, σ(L)/L, and Rσ were constrained to be the same for the isolated and stacked lamellae, a not unreasonable assumption. For all models, the least-squares refinements were performed using the model-fitting routines provided in the FISH software.36

Figure 1. Sequences and structures of cationic peptides.

RVRRGA linker.32 All peptides used in this study contained the targeting peptide sequence CYFGLPHKFC, abbreviated here as [Y]. This sequence was previously selected by phage display to bind human airway epithelial cells and shown to mediate gene delivery.28 In addition, a linear K12 peptide (not shown in Figure 2) was also synthesized to assess the contribution of [Y] on transfection efficiency. A 1:1 molar ratio of cationic lipid, DOTMA, and neutral helper lipid, DOPE (Lipofectin), was used in LPD formulations throughout. These are commonly used reagents for the preparation of lipoplexes and lipopolyplexes.3,37 In Vitro Transfection. PD and LPD complexes were formulated at two different charge ratios: 6:1 and 12:1 for PDs and 0.5:6:1 and 0.5:12:1 for LPDs (prepared as described in Methods). Preliminary screens indicated optimal transfection efficiencies occurred within this charge ratio range (data not shown). The ability of the complexes to transfect A549 epithelial lung cells with plasmid DNA (gWIZ-Luc pDNA) was assessed by induced luciferase expression (Figure 3). Whereas all PD complexes show negligible transfection efficiencies compared to the positive control, Lipofectamine (Figure 3a−c), many LPD complexes exhibit signficantly higher transfection efficiencies than Lipofectamine. In addition, LPD complexes formulated at a higher ratio of peptide (0.5:12:1) generally result in higher transfection efficiencies. A clear difference in transfection was observed between LPD complexes formulated with the group 1 peptides (Figure 3d). H6B1-L1-[Y] and H6B1-L2-[Y], formulated in LPD complexes, were relatively ineffective transfection agents. In contrast, LPD complexes containing K6B1-L1-[Y] or R6B1-L1-[Y] were approximately 2 and 4 times more effective than the Lipofectamine positive control. LPD complexes containing R6B1-L1-[Y] were better transfection agents those prepared using dR6B1-L1[Y]. Similar to previous observations,32 LPD complexes formulated with peptides containing the enzymatically cleavable linker, RVRRGA (L1), were in general considerably better transfection agents than those with the noncleavable linker, GAGA (L2). The only exception can be seen for the two polyHis peptides, H6B1L1-[Y] and H6B1-L2-[Y], where similar transfection efficiencies were observed for both RVRRGA and GAGA linkers. All LPD complexes formulated with the group 2 peptides were better transfection agents than the Lipofectamine control (Figure 3e). However, the degree of polycationic character was significant. LPDs formulated using a total of 12 Lys residues, K6B1-L1-[Y] were more effective than those prepared with a total of 8 Lys residues K4B1-L1-[Y]. Likewise, complexes formulated with a linear 12 Lys sequence, K12B0-L1-[Y], were more effective than those prepared with a linear 6 Lys sequence, K6B0-L1-[Y], as reported previously.7 Additional branching of

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RESULTS Peptide Design. To correlate peptide sequence to transfection efficiency, we designed and synthesized a comprehensive set of linear, singly and doubly branched polycationic peptides, for comparison in parallel (Figure 1). Group 1 comprises singly branched (B1) peptides only (Figure 2). These vary in both cationic amino acid character (K vs R vs H) and linker sequence (using either the sequence RVRRGA (L1), which should be cleaved by furin within the endosome32 or a noncleavable control sequence GAGA (L2)). Group 2 comprises linear and singly and doubly branched peptides. These peptides vary in amino acid character (K vs R), degree of branching (B0 vs B1 vs B2) and the number of cationic residues. Group 3 comprises singly branched (B1) peptides containing alternating H and K sequences. These sequences were selected to be similar to those previously studied by Mixson et al. and optimized for the delivery of plasmid DNA or siRNA.18,20,21 Group 2 and 3 peptides exclusively contain the cleavable 130

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LPD complexes formulated with peptides from group 3 were generally less effective transfection agents than the Lipofectamine positive control (Figure 3f). The cationic sequence (HHK)4-B1-L1-[Y], at a 0.5:6:1 charge ratio, provided the best transfection efficiencies when formulated in LPD complexes. This sequence was identified, by Mixson and co-workers,18 as optimal for delivery of plasmid DNA to MDA-MB-435, MDAMB-231, MCF7, and CRL-5800 cancer cell lines. LPD complexes formulated with (H 3 K) 3 H 3 -B1-L1-[Y] and (KH3)4B1-L1-[Y] were ineffective as transfection agents at both a 0.5:6:1 and 0.5:12:1 charge ratio. These latter sequences were identified by Mixson and co-workers22 as being optimal for siRNA delivery to MDA-MB-435, SVR-bag4, and C6 cells, but were found to be approximately 5-fold less effective for DNA delivery against the same cell lines compared to the (HHK)n sequences. The positive influence of the cyclic targeting peptide is evident in comparisons between LPD complexes prepared with K12 and those prepared with K12B0-L1-[Y] (Supporting Information, Figure S1). Similar observations have been made previously using peptides with and without cyclic targeting sequences targeted to MDA-MB 231 breast cancer cells.27 Cell Viability. None of the PD complexes exhibited a significant reduction in the viability of A549 cells compared to the untreated controls (Supporting Information, Figure S2). In contrast, several LPD complexes showed cytotoxicity similar to that of Lipofectamine (Supporting Information, Figure S3). Observed cytotoxicity is more pronounced in LPD complexes formulated with higher amounts of peptide. Cell viability was not compromised by LPDs formulated with His-containing peptides in either group 1 or group 3. Light Scattering and Zeta Potential Measurement. The apparent hydrodynamic size and zeta potential of the LPD complexes studied (prepared as described in Methods) are illustrated in Figure 4. LPD complexes were typically between 60 and 100 nm in diameter, with no correlation between observed size and peptide ratio, sequence composition, or degree of branching (Figure 4a). LPD formulations were stable over a period of at least 1 month at ambient temperature (Supporting Information: Figures S5 and S6), with the exception of formulations containing H6B1-L2-[Y], which aggregated rapidly after 10 days. As expected, all complexes were positively charged, with zeta potentials between 20 and 40 mV, which accounts for their high size stability. Again no correlation was observed between zeta potential and the peptide ratio, sequence composition, or degree of branching (Figure 4b). The apparent hydrodynamic diameters of selected PD complexes (Supporting Information: Figure S4a) were similar to those of the corresponding LPD complexes except for complexes containing H6B1-L1-[Y] which were 150 and 120 nm in diameter when prepared at weight ratios of 2:1 and 4:1 respectively. The zeta potential of PD complexes, although all positive (with the exception of the PD complexes containing H6B1-L1-[Y] at a 2:1 weight ratio), were generally lower than that obtained with the corresponding LPD complexes (15−30 mV) (Supporting Information: Figure S4b). Gel Band Shift Assay. All peptides, within both PD and LPD complexes, appear to completely condense pDNA at the charge ratios employed (lane 1), with the exception of H6B1-L1-[Y] and H6B1-L2-[Y], which show incomplete condensation. PD and LPD complexes were treated with DNase I followed by pAsp (lane 2) to assess stability to enzymatic degradation. While naked DNA was completely degraded by DNase I (Figure 5, lane B), all

Figure 2. Schematic representation of peptides synthesized.

the Lys sequences does not appear to improve transfection efficiency, as is seen for K6B2-L1-[Y]. Interestingly, complexes formulated with the linear 12 Lys residue sequence, K12B0-L1-[Y], showed improved transfection efficiency over the singly branched sequence, K6B1-L1-[Y], despite containing the same number of Lys residues in both peptides. For the analogous Arg-containing peptides, R6B1-L1[Y] and R12B0-L1-[Y], however, the situation is reversed. 131

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Figure 3. Luciferase expression in A549 epithelial lung cells upon transfection with Lipofectamine and PD (a−c) and LPD complexes (d−f) using group 1−3 peptides (Figure 2). Luciferase expression is expressed as relative light units (RLU) per milligram of protein (RLU per mg protein). The Lipofectamine control gave comparable transfection efficiencies in all experiments. All measurements were carried out in triplicate, and the error bars represent the standard deviation calculated from three different measurements carried out in one experiment.

tested formulations withstood DNase I exposure to a significant degree. Some degree of partial DNA degradation by DNase1 (by comparing band brightness between lanes 2 and 3) is, however, evident for all LPD and PD complexes. Treatment of PD and LPD complexes with pAsp resulted in the expected dissociation of DNA from all complexes (lane 3). Circular Dichroism and Ultraviolet Spectroscopy. Circular dichroism (CD) spectra (Figure 6) were measured in D2O to increase the optical transparency of the solution.38 The apparent hydrodynamic sizes of PDs and LPDs prepared for CD spectroscopy were slightly larger than those described previously (Supporting Information: Figure S7), which can be attributed to the higher concentration of DNA (0.025 mg mL−1 and 0.1 mg mL−1, for PDs and LPDs respectively) used for these studies. DNA free in solution usually adopts the B form, with a characteristic positive signal peaking at approximately 275 nm and a negative signal between 230 and 260 nm (Figure 6a,c−f). The CD spectrum of DOTMA/DOPE:ctDNA LD complexes at 0.5:1 charge ratio (at the equivalent lipid content used in LPD formulations) shows a slight red shift in the CD spectrum of ctDNA, becoming progressively more prominent as charge ratio increases to 1:1 and 2:1 (Figure 6a). Although indicative of the ability of DOTMA:DOPE to condense and affect the conformation of ctDNA the contribution of the lipid at LPD charge ratios of 0.5:6:1 and 0.5:12:1, used throughout this study,

can be regarded as insignificant. As anticipated from their structure, no CD signals were recorded for DOTMA:DOPE vesicles dispersed in D2O (Figure 6a). Six peptides, K6B1-L1-[Y], H6B1-L1-[Y], R6B1-L1-[Y], R12B0-L1-[Y], K12B0-L1-[Y], and K6B2-L1-[Y], were selected on the basis of their varying abilities to transfect pDNA. CD spectra taken of each peptide, free in solution (Figure 6b), reveal negative signals peaking at around 240 nm. These are attributed to the disulfide bond and tyrosine residue present in the target sequence and coincide with characteristic CD signals of free ctDNA. Given the small intensity of these signals, however, significant features observed within this region of the CD spectra of PD and LPD complexes can still be analyzed. The observed large negative signal at 200 nm and small positive signal at 215 nm are typical of peptides adopting a polyproline II (PII) conformation.39 Peptides in this conformation are characterized by a left-handed helical turn.39 Significant perturbations of ctDNA confirmation are observed for all PD and LPD complexes formulated at 6:1 and 0.5:6:1 charge ratios respectively (Figure 6c−f). The reduction and red shift of the signal at 275 nm were most prominent in the case of the complex prepared with R6B1-L1-[Y] and least prominent for H6B1-L1-[Y]. For the Lys-branched peptides, the degree of branching, comparing K12B0-L1-[Y], K6B1-L1-[Y], and K6B2L1-[Y], appears to have little additional effect on ctDNA 132

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Figure 4. (a) Apparent hydrodynamic size and (b) zeta potential of LPD complexes in water obtained immediately postformulation (final DNA concentration = 0.003−0.005 mg mL−1). All measurements were carried out in triplicate, and the error bars represent the standard deviation calculated from three different measurements carried out in one experiment.

Figure 5. Gel electrophoresis of PD and LPD complexes containing group 1−3 peptides. PD complexes were prepared with a charge ratio of peptide:pDNA of 6:1, while LPD complexes were prepared with a charge ratio of lipid:peptide:pDNA of 0.5:6:1. The effect of the peptide component on DNA condensation, on protection from degradation by DNase I, and on DNA release was studied. Lane A: DNA. Lane B: DNA treated with DNase I. Lane 1: untreated samples. Lane 2: samples treated with DNase I followed by pAsp. Lane 3: samples treated with pAsp only.

intensity of the negative band as seen at 250 nm in Figure 7c and Figure 7d is thought to be a consequence of cation-induced spectral changes attributed to an increase in the ctDNA widening angle (from 10.4 to 10.2 bp/turn).40,41

conformational change, whereas, in the case of the Arg sequence peptides, R6B1-L1-[Y] shows a significantly greater perturbation of the ctDNA conformation compared to the linear R12B0-L1[Y] sequence. The preservation and even deepening of the 133

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Figure 6. CD spectra of (a) ctDNA in comparison with DOTMA/DOPE vesicles alone and DOTMA/DOPE:DNA (LD) complexes at 0.5:1, 1:1, and 2:1 charge ratios; (b) peptides R6B1-L1-[Y], K6B1-L1-[Y], H6B1-L1-[Y], K6B2-L1-[Y,] K12B0-L1-[Y], and R12B0-L1-[Y] at 6 mM using a 1 cm path length; (c, d) PD and LPD complexes at 6:1 and 0.5:6:1 charge ratios prepared using His, Lys, and Arg peptides H6B1-L1-[Y], K6B1-L1-[Y], R6B1-L1[Y], and R12B0-L1-[Y]; and (e, f) PD and LPD complexes at 6:1 and 0.5:6:1 charge ratios prepared using peptides with different degrees of branching: K12B0-L1-[Y], K6B1-L1-[Y], and K6B2-L1-[Y]. All the DNA-containing samples were prepared at a final DNA concentration of 0.025 mg mL−1 using a 1 mm path length. Figure insets show the CD spectra of the same samples as in panels c−f but measured using a 1 cm path length.

Simultaneous UV absorbance measurements were performed to assess the extent and influence of light scattering to the CD spectra. In a 1 cm cell, light scattering was observed, particularly at wavelengths below 210 nm, when compared to ctDNA and the DOTMA:DOPE vesicles alone. This was reduced to around 1

absorbance unit at 180 nm when a 1 mm path length cell was used (Supporting Information, Figure S8). Reassuringly, the CD spectra in the wavelength region of 210 − 320 nm, obtained using the 1 cm path length cell, were very similar in shape to those obtained using the 1 mm path length cell and scaled with 134

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Figure 7. CD spectra of peptides alone at concentrations equivalent to those in PD and LPD complexes, compared to the spectra of PD and LPD complexes at 6:1 or 0.5:6:1 charge ratios respectively, minus the spectrum of ctDNA at 0.025 mg mL−1, to show the conformation of the peptides within the complexes prepared using (a) K12B0-L1-[Y], (b) K6B1-L1-[Y], (c) K6B2-L1-[Y], (d) H6B1-L1-[Y], (e) R6B1-L1-[Y], and (f) R12B0-L1-[Y]. All measurements were performed in a 1 mm path length cuvette.

Transmission Electron Microscopy. Transmission electron microscopy (TEM) was performed to determine the effect of peptide composition and branching on the shape and morphology of both PD and LPD complexes. The same complexes selected for CD experiments were stained with 4% w/v uranyl acetate and analyzed by TEM. All LPD complexes formed relatively homogeneously sized, spherical particles in the range of 50−200 nm in diameter (Figure 8). Very few, if any, vesicles were left visible in the samples. Interestingly, LPD complexes containing branched peptides, K6B1-L1-[Y] and

path length. This confirms light scattering does not affect the CD spectra obtained at wavelengths above 210 nm. Using a 1 mm path length cell, it was therefore possible to accurately record the CD spectra for samples at wavelengths below 210 nm to probe conformational changes of the peptides upon complexation with ctDNA. For ease of interpretation, the CD spectrum of ctDNA alone was subtracted from those obtained for PD and LPD complexes (Figure 7). However, upon analysis, no significant conformational change was observed for any of the peptides in either PD or LPD complexes. 135

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Figure 8. Transmission electron microscopy of LPD complexes prepared using DOTMA:DOPE with the peptides (a) K12B0-L1-[Y], (b) K6B1-L1-[Y], (c) K6B2-L1-[Y], (d) R6B1-L1-[Y], (e) H6B1-L1-[Y], and (f) R12B0-L1-[Y] at a final DNA concentration of 0.024 mg/mL.

K6B2-L1-[Y], possess more ordered core structures than LPD complexes containing the linear, 12-lysine sequence K12B0-L1[Y]. Less defined internal structures were also observed for LPD complexes prepared using R6B1-L1-[Y], R12B0-L1-[Y], and H6B1-L1-[Y]. No obvious internal structure was observed in PD complexes prepared using the same peptides (Supporting Information, Figure S9). Compared to the LPDs, PDs were more polydisperse with both spherical and rod-shaped particles of varying sizes, as observed in previous studies.42 Rod-shaped particles were substantially absent in the images of LPD complexes. Small Angle Neutron Scattering. Small angle neutron scattering (SANS) profiles were recorded for DOTMA/DOPE vesicles (1:1 molar ratio, at a final DOTMA concentration of 1 mg mL−1 in D2O) and for five of the LPD complexes (charge ratio 0.5:6:1) used for the CD and TEM experiments using the instrument, LoQ (Figure 9). DOTMA/DOPE vesicles studied

by SANS were then used to formulate LPD complexes. SANS experiments were not carried out with PD complexes due to the lack of any discernible internal structure (TEM) and high polydispersity. The variation in the intensity of the SANS as a function of Q for cationic vesicles prepared using a 1:1 molar ratio of DOTMA and DOPE at a DOTMA concentration of 1 mg mL−1 in D2O is shown in Figure 9a. In agreement with our previous studies,27,42 the average size of the cationic vesicles was sufficiently large that they are best approximated as single, flat sheets (i.e., unilamellar vesicles) or stacks (i.e., multilamellar vesicles) rather than hollow spheres (with the wall of the sphere representing the cationic lipid bilayer). The SANS data were fitted using a model comprising a mixture of single, infinite planar (bilayer) sheets and one-dimensional paracrystals (stacks). This model suggested vesicles were predominantly unilamellar in nature, with only a very small 136

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Figure 9. Small angle neutron scattering data (dots) at 298 K and the best fit to the data (solid line) obtained using the mixed sheet and stack model for (a) cationic vesicles prepared from a 1:1 molar ratio of DOTMA:DOPE at a DOTMA concentration of 1 mg mL−1 DOTMA and LPD particles prepared using DOTMA:DOPE vesicles at a concentration of 0.112 mg mL−1 DOTMA and containing ctDNA with (b) R6B1-L1-[Y], (c) H6B1-L1-[Y], (d) K12B0-L1-[Y], (e) K6B2-L1-[Y], and (f) K6B1-L1-[Y] at 0.5:6:1 charge ratio.

∼10:1, suggesting the addition of peptide and ctDNA promotes the formation of multilayers in LPD complexes. The influence of any unreacted lipid vesicles or nonencapsulated PD complexes could not be determined in this study. However, both the large multilayer structures, produced through interaction of cationic vesicles and DNA, and the large, unstructured aggregates common for PD complexes were unobserved in the SANS analysis. The SANS studies of the LPD complexes dispersed in D2O gave no indication of any “internal” structure in any of the LPDs in apparent contradiction to the electron microscopy study. This discrepancy is undoubtedly a consequence of the neutron scattering contrast used in the study (i.e., LPD complexes prepared using hydrogenous lipid and dispersed in D2O) making the experiment very sensitive to the presence of lipid but much less sensitive to the presence of DNA and peptide.27 Consequently SANS studies of the LPD complexes dispersed in 8.2 vol % D2O in H2O were performed on SANS2D. This dispersion medium was selected because the lipid was contrast matched to the medium and so “invisible” to neutrons, and in this way it should be possible to determine the size of the DNA and peptide “core” of the LPD complex and whether there was any ordered structure present in this region. Unfortunately the concentration of the LPD complex used in the present study was too low to enable us to determine either the size or the presence of any internal structure. Further studies are underway to examine the LPD complexes by SANS at higher concentrations.

number of multilamellar vesicles. This correlates well with the small vesicle size as measured by dynamic light scattering, indicative of very few multilamellar vesicles, if any, present in the sample. Table S3 (Supporting Information) gives an assessment of the goodness of fit of the data in the form of the SWSE and the surface area of lipid in the form of (unilamellar) sheets and (multilamellar) stacks. This assumes the thickness of the bilayer (L) is fixed as 41 Å with a polydispersity (σ(L)/L) of 0.1, an Rσ of 300 Å, a D spacing (or repeat distance) of 65 Å, and a polydispersity (σ(D)/D) of 0.05. Due to the absence of a pronounced Bragg peak (Figure 9a) the data were fitted using a stack with a maximum of 2 bilayers. Fits to the data using higher numbers of bilayers comprising the stacks did not improve the quality of the fit. The thickness and repeat distance reported in this study agree well with values, 44.7 Å and 65 Å, for previously reported DOTMA/DOPE vesicles (1:1 molar ratio);27 with ∼39 Å and 65 Å, reported values for related DOTAP/DOPE vesicles (1:1 molar ratio);43 and with values (∼21 ± 1 Å) for the thickness of a compressed DOPE monolayer.44 SANS data for LPD complexes are shown in Figures 9b−f. The lower SANS intensity of the LPD complexes, compared to the DOTMA/DOPE vesicles alone, is a consequence of the dilution made when preparing the complexes, from 1 mg mL−1 (in vesicles) to 0.112 mg mL−1 of DOTMA (in LPD complexes). The scattering patterns recorded for the LPDs are qualitatively very similar to one another and equally to the DOTMA/DOPE vesicles. This suggests LPD particles contain one or more lipid bilayers. A similar observation was made in the preparation of LPD particles from cationic lipids containing unsaturated C14 chains.27 From this, it can be concluded the composition and structure of the peptide do not significantly alter the structure of the lipid bilayer. The only difference noted was that the ratio of unilamellar:multilamellar vesicles decreased from ∼30:1 to

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DISCUSSION In contrast to polyplexes (PD),4−12,16,17,23,24,45,46 the precise role of the peptide component within lipopolyplexes (LPD) has not been subjected to thorough analysis.13,18,19,21 In line with previous work,3,4,26,47 in this study we have consistently observed improved transfection efficiencies for LPD complexes over their PD counterparts. However, between LPD complexes, we observe 137

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differences in the nature and extent of complexation between the peptides and the DNA. His-Rich Peptides Do Not Appear To Contribute Significantly to the Endosomal Release of LPD Complexes. Histidine-rich peptides and polymers, formulated as polyplexes, have been reported to be efficient nonviral gene vectors in vitro and in vivo.17,20 Their proficiency has largely been attributed to the positive influence of the “proton-sponge” effect, defined as osmotic swelling and destabilization of the endosomal membrane following protonation of histidine residues within the acidic environment of the endosome.17 In this study, however, singly branched His-rich peptides, formulated as polyplexes, were generally found to be poorer transfection agents than their Arg or Lys counterparts. (HHK)4B1-L1-[Y] is the only exception (Figure 3c) and shows arguably the best transfection efficiency of any of the polyplex formulations tested. This is in agreement with Mixon et al.,18 who found the identical sequence composition, (HHK)n, to be most efficient for delivery of plasmid DNA to cells. Given the sequence similarity to other group 3 peptides, however, it is difficult to attribute this improved transfection efficiency solely as a result of the “proton-sponge” effect. As suggested by the authors, the improved transfection efficiency of (HHK)4B1-L1[Y], formulated as a polyplex, may be due to favorable structural conformations, that resemble native DNA-binding histone proteins, adopted by an (HHK)n peptide. However, this improved transfection efficiency is not carried over to the LPD complexes formulated with the same peptides, suggesting this structural consequence may have only a marginal bearing on transfection efficiency. Universally, LPD complexes formulated with His-rich peptides were worse transfection agents than those formulated with Argand Lys-rich peptides. The sequence bearing exclusively His residues, H6B1-L2-[Y], showed very poor complexation and protection of DNA (Figure 5a) and also poor long-term stability (Supporting Information: Figures S5 and S6). Adding Arg residues (in the case of H6B1-L1-[Y], which has the RVRRGA linker sequence) or Lys residues (in the case of the group 3 peptides) appears to restore the ability of these peptides to complex and protect DNA, at the resolution afforded by the gel shift assays (Figure 5). However, this has little bearing on the transfection efficiencies of the resulting LPD complexes, which are still very poor. In addition, the CD spectra (Figure 6) of LPD and PD complexes formulated with H6B1-L1-[Y] are similar to those formulated with the branched Lys peptides, implying that the conformational change of the ctDNA induced by both His and Lys peptides is similar. Interestingly, whereas the inclusion of the furin cleavable linker, RVRR, has a significant and positive effect on all branched Lys- and Arg-rich sequences, it appears to have little bearing on the transfection efficiencies of any of the branched His peptides. This may imply that, while the RVRR sequence is a key factor in the separation of the branched Lysand Arg-rich sequences from the cyclic targeting sequence of the peptide, and may also have an additional role as a NLS (vide infra), it is possible that, after internalization, either the endosomal pH or the pH of the cytoplasm is not sufficiently acidic for the His-branched peptides to remain complexed to the DNA, as has been observed previously.19 This explanation would account for the low transfection efficiencies of the His-branched peptides, and would be independent of the cleavage of the peptide by furin. Based on these results, we can reasonably conclude that the proton-sponge effect, previously described for

significant differences in transfection efficiency against an A549 epithelial cell line. Given that, in this study, both lipid and DNA components are kept constant throughout, these differences must be driven by the peptide component. Lipid Structure, Particle Size, and Surface Charge Are Unaffected by the Peptide Component. From the SANS and TEM studies presented here, any significant influence of the peptide component on the structure of the lipid membrane can be ruled out. In our previous work in this area, we have studied in detail the macromolecular structure of LPD complexes. We demonstrated that the cationic sequence of the peptide condenses the DNA, resulting in a tightly bound inner core of peptide:DNA complex, surrounded by the lipid bilayer, from which the integrin-targeting sequence of the peptide is thought to partially protrude, allowing it to interact with its target receptors.25 More recently, we have analyzed the structure of the lipid bilayer in more detail by SANS, and have proposed that it is important to have enough lipid to form a single bilayer on the exterior of the LPD complex but not so much that multiple bilayers are formed, burying the region of the peptide component essential for receptor-mediated uptake.27 In the present study, SANS results were obtained with the lower proportion of peptide (0.5:6:1) and show a slight increase in the number of bilayers present in the LPD complex compared with the vesicles, whereas formulations with the higher proportion of peptide/lower proportion of lipid (0.5:12:1) were shown to be more effective in transfection (Figure 3), probably because they have relatively few lipid bilayers. Other groups have studied the macromolecular structures of different lipopolyplex formulations using similar techniques. Miller and co-workers have demonstrated by cryo-electron microscopy that DC-Chol/DOPE/mu peptide/pDNA lipopolyplexes consist of a peptide/DNA core encapsulated within a cationic bilamellar liposome.3 DOTAP/protamine/ctDNA lipopolyplexes were shown by SAXS to have a similar condensed protamine/DNA core, encapsulated by a lipid envelope composed of an average of 10 DOTAP bilayers, and which protects the DNA from serum.47 Conversely, lipid/protamine/ oligonucleotide lipopolyplexes (mixtures of PC:DC-Chol:PEGDSPE lipids were used) showed cryo-TEM structures consistent with multilamellar vesicles with protamine/oligonucleotide complexes interspersed within the bilayers.48 However, the morphology of lipopolyplexes formulated from the (short) 18mer antisense oligonucleotides used in their study is likely to be significantly different from that observed when complexing pDNA or ctDNA as in the present study. Finally, complexation of pDNA with poly-L-Lys and several different anionic liposome formulations gave lipopolyplexes in which the pDNA/poly-L-Lys complex coats the outside of the anionic liposome:49 again, the very different electrostatic interaction between the polyplex and the anionic liposome is likely to yield LPDs of a significantly different morphology from those obtained in the present study. It is worth noting that, in our analysis, the presence of small amounts of (contaminating) LD, PD, and liposomes cannot be ruled out, although the SANS and TEM data in particular indicate that there is only one major type of complex present and that contaminants, if present, are likely to be at a low level. In general, we have also found that all of the complexes are similar in size (Figure 3a), surface charge (Figure 3b), and stability (Supporting Information: Figures S5 and S6), with the exception of those formulated from the His-containing peptides H6B1-L1-[Y] and H6B1-L2-[Y]. The observed differences in transfection efficiencies must therefore arise from subtle 138

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possible to explain this in terms of size, surface charge, the ability to condense or protect DNA, the conformation of the condensed DNA, or the structure of the lipid bilayer. Significant structural differences are observed in the TEM images of the LPD complexes with Lys-rich peptides. While LPD complexes formulated with K6B1-L1-[Y] or K6B2-L1-[Y] appear to possess an ordered core structure, complexes formulated with K12B0-L1[Y] appear to have a much less well-defined internal structure (Figure 8). However, for the Arg-rich peptides R12B0-L1-[Y] and R6B1-L1-[Y], the resolution of the TEM images was not sufficient to draw conclusions about the structure of the peptide−DNA core. Similar findings have recently been published for polyplexes formulated with linear Arg- and Lys-containing peptides using AFM and fluorimetry-based DNA release assays. The studies reported different morphologies and stabilities for linear homoLys polyplexes and linear homo-Arg polyplexes.5 In this case, differences were attributed to different modes of interactions between lysine/arginine residues and the backbone phosphate groups of DNA.46 This would appear to tie in with the differing CD spectra we obtained for LPD complexes containing Lys-rich and Arg-rich peptides respectively, however, as already stated, the differences in the mode of binding appears to have little, if any, effect on transfection efficiency. Possible Additional Roles of Arg-Rich Sequences in Transfection. While enhancement in transfection efficiency may arise from the observed structural differences observed, it is also important to appreciate ancillary roles played by the peptide that may have an additional positive effect on transfection efficiency. Arg-containing sequences may play a role in the nuclear localization of the DNA cargo,1,5 and this may, in part, account for the enhanced transfection efficiencies observed when peptides containing the RVRR linker are used, as well as the role played by this sequence in allowing the peptide to be enzymatically degraded after internalization.25,33 Related studies with dendrimer/DNA polyplexes,23 and with lipoplexes with cationic peptides attached to the surface,55 have also shown that complexes where Arg residues are close to the surface have superior transfection efficiencies to complexes where His or Lys residues are at the surface. It is also possible that, even when DOPE is included in the lipoplexes, the (Arg)8 sequences play an important role in mediating endosomal escape at both neutral and acidic pH.55

His-rich peptides, is not significant in these lipopolyplexes in A549 cells. Arg-Rich Peptides Condense DNA in a Different Way from Lys-Rich Peptides. The transfection efficiencies for the Arg- and the Lys-rich peptides are broadly comparable. For example, the complex with the best Arg-rich sequence, R6B1-L1[Y], shows similar transfection efficiency to the complex with the best Lys-rich sequence, K12B0-L1-[Y] (Figure 3). However, the CD studies reveal significant differences in the mode of DNA condensation. Greater perturbation of DNA conformation, characterized by a reduction and red shift of the CD signal at 275 nm, are seen with LPD (and PD) complexes containing Argrich peptides compared to their lysine analogues. This is characteristic of a ctDNA conformational change from the Bform to the more tightly packed C-form and is often observed with lipid:DNA complexes.50,51 The ctDNA in these complexes does not adopt a condensed −ψ phase, characterized by a negative signal maximum around 260 nm. This observation is in line with previous studies of PD complexes52 showing that while a linear K16 peptide could interact with DNA to give a complex with a condensed −ψ phase, bifunctional peptides with a cyclic headgroup and polycationic tail did not result in this degree of condensation. Even though it is not possible to determine the exact nature of the conformational change in ctDNA taking place from the CD studies, it is most likely to be a condensed form of ctDNA. This is presumably stabilized through ion pairing between the negatively charged phosphate backbone of the ctDNA and the positively charged side chains of the cationic peptide in the extended PII conformation. Our results are consistent with previous CD studies in which different binding modes of Lys and Arg peptides to DNA were reported.53,54 In these studies it was found that polylysine completely binds to the DNA phosphate backbone with a 1:1 stoichiometry, while not all partial cationic residues on polyarginine bind.53,54 Given the comparable transfection efficiencies between the Arg- and Lysrich peptides, we can conclude perturbation of DNA conformation within the LPD core is neither beneficial nor detrimental to transfection efficiency. Despite the current interest in the use of peptides containing 15 D-Arg containing sequences for siRNA delivery, in our work we have found dR6B1-L1-[Y] to be less effective in LPD complexes than R6B1-L1-[Y]. Further work will be required to determine whether this arises from differences in the mode of binding DNA or in the endosomal escape/nuclear localization properties of DArg peptides. It will also be of interest to examine the in vivo properties of LPD complexes formulated using dR6B1-L1-[Y], which may have greater resistance to enzymatic digestion and hence a longer lifetime in vivo. Peptide Branching Improves Transfection Efficiencies for Arg-Rich Peptides but Is Detrimental to Lys-Rich Sequences in LPD Complexes. Szoka and co-workers16 originally suggested that the geometry of branched peptides might afford more favorable interactions with DNA over their corresponding linear peptides. We have observed this to be true for the Arg-rich peptides used in this study; the LPD complex formulated from R6B1-L1-[Y] is a significantly better transfection agent than the LPD complex formulated from its linear analogue, R12B0-L1-[Y]. However, for the Lys-rich peptides formulated within LPD complexes, the reverse is true. The LPD complex formulated from the linear K12B0-L1-[Y] shows significantly better transfection efficiency than those formulated from either K6B1-L1-[Y] or K6B2-L1-[Y]. As discussed above, on the basis of the biophysical studies presented here, it is not

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CONCLUSIONS In LPD formulations both Arg-rich and Lys-rich peptides condense and protect DNA effectively and can be used to transfect cells efficiently. However, transfection of LPD complexes formulated with His-containing cationic peptide sequences is poor, as these appear to make only a small contribution to endosomal release, and do not provide adequate DNA condensation and/or protection. In the present study, for both Arg-rich and Lys-rich peptides, larger numbers of cationic residues give the most efficient transfection. This reflects the need for efficient condensation of DNA within the LPD core; however, previous studies suggest that much longer sequences than those studied here would be detrimental, as they would prevent efficient release of the plasmid DNA following cellular internalization.16 Importantly, our work suggests that linear sequences give the best transfection results when LPD complexes are formulated from Lys-rich peptides, and recent reports indicate that this may also apply to polyplexes formulated from small branched peptides45 or self-assembling 139

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DNA-binding dendrons.56 Considering the increased toxicities often encountered with dendrimeric gene delivery agents,6 lipopolyplexes formulated from peptides with highly branched cationic sequences based on Lys residues may therefore not prove to be effective for gene therapy. Conversely, branched sequences appear to be best for LPD complexes formulated from Arg-rich peptides. We can, in part, attribute this to the observation that Lys-rich and Arg-rich peptides have different modes of binding to DNA. Finally, while the LPD complexes formulated with the different cationic peptide sequences all have similar lipid bilayer structures, TEM images reveal that, for the Lys-rich peptides, the lipopolyplex with the best transfection efficiencies, formulated from the linear peptide K12B0-L1-[Y], has a much less well-defined core structure than the lipopolyplexes formulated from the branched peptides K16B1-L1[Y] or K6B2-L1-[Y]. The subtle interplay between peptide charge, geometry, and complexation to DNA, will require further study, in particular with a detailed investigation by SANS of the structure of the peptide−DNA core, and for eventual in vivo studies, the effect of the presence of serum on the structure and cellular fate of the lipopolyplexes will also require investigation.

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REFERENCES

(1) Varga, C. M.; Wickham, T. J.; Lauffenburger, D. A. Receptormediated targeting of gene delivery vectors: Insights from molecular mechanisms for improved vehicle design. Biotechnol. Bioeng. 2000, 70, 593−605. (2) Miller, A. D. Cationic liposomes for gene therapy. Angew. Chem., Int. Ed. 1998, 37, 1769−1785. (3) Kostarelos, K.; Miller, A. D. Synthetic, self-assembly ABCD nanoparticles: A structural paradigm for viable synthetic non-viral vectors. Chem. Soc. Rev. 2005, 34, 970−994. (4) Mintzer, M. A.; Simanek, E. E. Nonviral vectors for gene delivery. Chem. Rev. 2009, 109, 259−302. (5) Mann, A.; Thakur, G.; Shukla, V.; Ganguli, M. Peptides in DNA delivery: Current insights and future directions. Drug Discovery Today 2008, 13, 152−160. (6) Jain, K.; Kesharwani, P.; Gupta, U.; Jain, N. K. Dendrimer toxicity: Let’s meet the challenge. Int. J. Pharm. 2010, 394, 122−142. (7) Martin, M. E.; Rice, K. G. Peptide-guided gene delivery. AAPS J. 2007, 9, E18−E29. (8) Wadhwa, M. S.; Collard, W. T.; Adami, R. C.; McKenzie, D. L.; Rice, K. G. Peptide-mediated gene delivery: Influence of peptide structure on gene expression. Bioconjugate Chem. 1997, 8, 81−88. (9) Adami, R. C.; Collard, W. T.; Gupta, S. A.; Kwok, K. Y.; Bonadio, J.; Rice, K. G. Stability of peptide condensed plasmid DNA formulations. J. Pharm. Sci. 1998, 87, 678−683. (10) Koo, H.; Kang, H.; Lee, Y. Analysis of the relationship between the molecular weight and transfection efficiency/cytotoxicity of poly-Larginine on a mammalian cell line. Bull. Korean Chem. Soc. 2009, 30, 927−930. (11) van Rossenberg, S. M. W.; van Keulen, A. C. I.; Drijfhout, J. W.; Vasto, S.; Koerten, H. K.; Spies, F.; van ’t Noordende, J. M.; van Berkel, T. H. C.; Biessen, E. A. L. Stable polyplexes based on arginine-containing oligopeptides for in vivo gene delivery. Gene Ther. 2004, 11, 457−464. (12) Siprashvili, Z.; Scholl, F. A.; Oliver, S. F.; Adams, A.; Contag, C. H.; Wender, P. A.; Khavari, P. A. Gene transfer via reversible plasmid condensation with cysteine-flanked, internally spaced arginine-rich peptides. Hum. Gene Ther. 2003, 14, 1225−1233. (13) Maitani, Y.; Hattori, Y. Oligoarginine-PEG-lipid particles for gene delivery. Expert Opin. Drug Delivery 2009, 6, 1065−1077. (14) Futaki, S. Membrane-permeable arginine-rich peptides and the translocation mechanisms. Adv. Drug Delivery Rev. 2005, 57, 547−558. (15) Kumar, P.; Ban, H. S.; Kim, S. S.; Wu, H. Q.; Pearson, T.; Greiner, D. L.; Laouar, A.; Yao, J. H.; Haridas, V.; Habiro, K.; Yang, Y. G.; Jeong, J. H.; Lee, K. Y.; Kim, Y. H.; Kim, S. W.; Peipp, M.; Fey, G. H.; Manjunath, N.; Shultz, L. D.; Lee, S. K.; Shankar, P. T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice. Cell 2008, 134, 577− 586. (16) Plank, C.; Tang, M. X.; Wolfe, A. R.; Szoka, F. C. Branched cationic peptides for gene delivery: Role of type and number of cationic residues in formation and in vitro activity of DNA polyplexes. Hum. Gene Ther. 1999, 10, 319−332. (17) Midoux, P.; Pichon, C.; Yaouanc, J. J.; Jaffres, P. A. Chemical vectors for gene delivery: A current review on polymers, peptides and lipids containing histidine or imidazole as nucleic acids carriers. Br. J. Pharmacol. 2009, 157, 166−178. (18) Chen, Q. R.; Zhang, L.; Stass, S. A.; Mixson, A. J. Branched copolymers of histidine and lysine are efficient carriers of plasmids. Nucleic Acids Res. 2001, 29, 1334−1340. (19) Chen, Q. R.; Zhang, L.; Luther, P. W.; Mixson, A. J. Optimal transfection with the HK polymer depends on its degree of branching and the pH of endocytic vesicles. Nucleic Acids Res. 2002, 30, 1338− 1345. (20) Leng, Q. X.; Mixson, A. J. Modified branched peptides with a histidine-rich tail enhance in vitro gene transfection. Nucleic Acids Res. 2005, 33, 9. (21) Leng, Q. X.; Scaria, P.; Ioffe, O. B.; Woodle, M.; Mixson, A. J. A branched histidine/lysine peptide, H2K4b, in complex with plasmids encoding antitumor proteins inhibits tumor xenografts. J. Gene Med. 2006, 8, 1407−1415.

ASSOCIATED CONTENT

S Supporting Information *

General methods for synthesis of peptides and HPLC purification. Analytical data for all peptides. Tables S1, S2, and S3 showing peptide charge data, stock solutions, and surface area of lipid and SWSE from SANS data. Additional transfection and protein assay data (Figures S1, S2, and S3). Sizing of complexes over 3−20 days (Figures S4, S5, S6, and S7). UV spectra (Figure S8). TEM of PD complexes (Figure S9). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Department of Chemistry, University College London, Christopher Ingold Laboratories, 20 Gordon Street, London WC1H 0AJ, U.K. Phone: +44 20 7679 4695. Fax: +44 20 7679 7463. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The EPSRC is thanked for the award of a Nanotechnology Grand Challenge grant (EP/G061521/1) to K.W., F.C., and L.K. and for a DTG studentship to A.M. The MRC are thanked for a studentship to N.D. (G0900207). STFC is thanked for the award of neutron beam time at the ISIS Facility, The Rutherford Appleton Laboratories. The expert help of Dr. Alex Drake and Dr. Tam Bui (Biomolecular Spectroscopy Centre, King’s College London) is gratefully acknowledged for the circular dichroism/ UV measurements, and the help of Dr. Alice Warley (Centre for Ultrastructural Imaging, King’s College London) with the TEM measurements is acknowledged. The EPSRC National Mass Spectrometry Service Centre, Swansea University, is thanked for the provision of selected MS data.

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ABBREVIATIONS USED CPP, cell-penetrating peptide; DOPE, dioleoylphosphatidylethanolamine; DOTMA, 1,2-di-O-octadecenyl-3-trimethylammonium propane; NLS, nuclear localization sequence; PEI, polyethyleneimine; SWSE, sum of weighted squared errors 140

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Molecular Pharmaceutics

Article

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