Linear Short Histidine and Cysteine Modified Arginine Peptides

Jan 29, 2014 - CSIR-Institute of Genomics and Integrative Biology, Mall Road (near Jubilee Hall), Delhi 110 007, India. §. Indian Institute of Techno...
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Linear Short Histidine and Cysteine Modified Arginine Peptides Constitute a Potential Class of DNA Delivery Agents Anita Mann,†,‡ Vasundhara Shukla,‡,§ Richa Khanduri, Spoorti Dabral, Harpal Singh,§ and Munia Ganguli*,† †

CSIR-Institute of Genomics and Integrative Biology, Mall Road (near Jubilee Hall), Delhi 110 007, India Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110 016, India

§

S Supporting Information *

ABSTRACT: The success of gene therapy relies on the development of safe and efficient multifunctional carriers of nucleic acids that can overcome extraand intracellular barriers, protect the nucleic acid and mediate its release at the desired site allowing gene expression. Peptides bear unique properties that are indispensable for any carrier, e.g., they can mediate DNA condensation, cellular targeting, membrane translocation, endosomal escape and nuclear localization. In an effort to design a multifunctional peptide, we have modified an arginine homopeptide R16 by replacement of seven arginines with histidines and addition of one cysteine at each end respectively to impart endosomal escape property while maintaining the DNA condensation and release balance. Addition of histidines imparts endosomal escape property to arginine homopeptide, but their arrangement with respect to arginines is more critical in controlling DNA condensation, release and transfection efficiency. Intriguingly, R5H7R4 peptide where charge/arginine is distributed in blocks is preferred for strong condensation while more efficient transfection is seen in the variants R9H7 and H4R9H3, which exhibit weak condensation and strong release. Addition of cysteine to each of these peptides further fine-tuned the condensation−release balance without application of any oxidative procedure unlike other similar systems reported in the literature. This resulted in a large increase in the transfection efficiency in all of the histidine modified peptides irrespective of the arginine and histidine positions. This series of multifunctional peptides shows comparable transfection efficiency to commercially available transfection reagent Lipofectamine 2000 at low charge ratios, with simple preparative procedure and exhibits much less toxicity. KEYWORDS: gene delivery, multifunctional peptides, histidine and cysteine, arginine peptides, DNA condensation and release balance, endosomal escape, atomic force microscopy

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INTRODUCTION Development of nonviral carriers for delivery of nucleic acid therapeutics in vivo is an active area of research in recent times and holds enormous potential for possible application in the treatment of various genetic as well as complex diseases.1−3 Such carriers mediate safer delivery as compared to the more commonly used viral carriers. However although different classes of molecules like lipids, polymers and peptides have been used as nonviral carriers, their nucleic acid delivery efficiencies are usually quite low primarily because of their inability to efficiently overcome all the extracellular and intracellular barriers encountered during delivery.4,5 Intracellular DNA delivery involves several critical steps like efficient condensation of DNA, delivery across plasma membrane via endocytotic or nonendocytotic route, escape from the endosomes to the cytosol, unpackaging of the complex and nuclear import followed by accessibility to the transcription machinery. In addition, when used in vivo, different systemic barriers like instability in serum and lack of target organ specificity need to be overcome.5 Thus, an integration of multiple features to © 2014 American Chemical Society

address all the barriers is desired for generating novel and efficient vectors. In this regard, peptides have become a popular choice for DNA delivery.6 Peptides offer ease in synthesis and flexibility in design, can be easily degraded into biocompatible components and show minimal cellular toxicity. Different classes of peptides possess the ability to overcome some of the barriers discussed above and can also be conjugated with other functional moieties like fusogenic lipids or peptides and targeting ligands to create multifunctional integrated systems.6−10 In order to design such systems with improved efficiency, it is important to understand and optimize each of the components involved in the carrier design and how they can help in first overcoming different barriers in vitro. For example, the first step in intracellular DNA delivery is efficient condensation of DNA Received: Revised: Accepted: Published: 683

June 18, 2013 January 6, 2014 January 8, 2014 January 29, 2014 dx.doi.org/10.1021/mp400353n | Mol. Pharmaceutics 2014, 11, 683−696

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multifunctional peptide carrier utilizing the unique properties of arginine, histidine and cysteine as discussed above. In order to achieve this, we have modified the 9-mer arginine homopeptide (minimal length required for efficient condensation of DNA) by adding 7 histidine residues to incorporate the endosomal escape property in this peptide. Several variants were created by keeping the composition the same and changing the position of histidines with respect to arginine residues; and the effect of these variations on complex formation, cellular uptake, endosomal escape and overall transfection efficiency of the peptides was analyzed. Additionally, we modified these peptides with cysteine residues at both ends with an aim to improve their intracellular nucleic acid delivery efficiency further. We have shown that addition of histidines improves the transfection efficiency of arginine peptide by imparting the endosomal escape property, but subtle changes in DNA condensation and release during such modifications can have a strong effect on the overall transfection efficiency. Our work also highlights that the short and linear arginine-histidine (HR) combinations have higher DNA delivery capabilities than the corresponding HK peptides. Our results reveal certain combinations of arginine peptides with histidine and cysteine residues which show significantly high transfection efficiency comparable to that of the commercially available transfection reagent Lipofectamine 2000 in different cancerous cell lines like MCF-7, A-549, HeLa and MDA-MB 231 in vitro with substantially lower toxicity. The salient feature of this system is that such high transfection efficiency is achieved even without any oxidative procedure during the preparation of the peptide− DNA complexes to introduce strong S−S bonds in the system as has been shown in the literature. This makes the system easy to synthesize as well. Such a system offers sufficient scope for further chemical modification and applicability as an effective gene delivery agent in vivo.

into nanoparticles. Lysine and arginine based cationic peptides in linear or branched polymeric form are well-documented to mediate DNA condensation for effective DNA delivery.11,12 However such peptides fail to overcome the subsequent steps like endosomal escape and are usually effective only in the presence of choloroquine (a weak base that can buffer the endosomes), limiting their further applicability. In order to overcome the endosomal barrier, these peptides can be conjugated with fusogenic moieties, e.g., peptides like HA-2 or KALA, or with fusogenic lipids as lipopolyplexes.13−15 Another way of ensuring endosomal escape is to incorporate histidine residues in the peptide. The imidazole ring of histidine is a weak base and can scavenge protons inside the endosomes generating the proton sponge effect to mediate endosomal escape. Incorporation of imidazole moiety or histidine has been used primarily for enhancement of transfection efficiency in peptides, polymers/reducible polycations and lipids.16−20 In addition to strong DNA condensation and endosomal escape, it is also important for the carrier to efficiently release the DNA in the intracellular compartments. Cysteines have been used to impart controlled intracellular release property to the carrier. Cysteines can be oxidized to create cross-linking disulfide bonds in the polyplexes which makes them stable in the extracellular environment but allow easy disassembly under the reducing intracellular conditions mediating controlled release of the payload.21,22 The histidine-lysine (HK) combinations for nucleic acid delivery have been discussed exhaustively in the literature where the potential of various HK linear and branched polymeric forms in delivering plasmid DNA and ODN/ siRNA has been described.16−20 The linear HK peptides with fewer than 20 amino acids seem to have poor DNA delivery efficiency. The efficacy of the linear HK systems has been improved by converting them to reducible polycations by using disulfide cross-linking to overcome their poor DNA binding efficiency and provide a controlled DNA release ability.23,24 A series of histidine-rich amphipathic peptides has also been shown to possess significant DNA delivery capabilities.25 Although arginine-histidine combinations have not been studied much in the literature, there are few very recent examples where arginines have been used instead of lysines as condensing moieties in multifunctional carriers. For example, the modification of TAT peptide with histidines and cysteines shows remarkable improvement in its DNA delivery efficiency and exhibits in vivo applicability.26 Presence of cysteines with reducible poly(arginines) has also been shown to enhance efficiency of delivery of DNA and siRNA in vitro and in vivo.27−30 In a recent study from our laboratory, we have compared a series of lysine and arginine homopeptides and shown that arginine homopeptides not only form stable complexes with DNA but also exhibit higher transfection efficiency. Our results indicate that arginines should be preferred over lysines in a DNA condensation moiety as they form smaller and monodisperse complexes with DNA and also show higher cellular uptake and stronger intracellular release.31 In addition there is a multitude of reports where arginine based peptides have been utilized as cell penetrating peptides with the aim to improve delivery across plasma membrane.32 There are other studies which discuss their DNA condensation properties as well as the use of arginine/guanidinium modification to improve nuclear uptake of peptide−DNA complexes.33,34 Our aim in the current manuscript was to create an efficient

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MATERIALS AND METHODS Materials. Peptides (>95% purity) were custom synthesized from G L Biochem Ltd. The plasmids pEGFP C1 (clonetech) and pMIR REPORT (ambion) (referred to as pDNA in the text) were purified using GenElute HP endotoxin free plasmid maxiprep kit (Sigma), and Luciferase assay kit was purchased from Promega. All the chemicals used were purchased from Sigma-Aldrich unless stated otherwise. Preparation of the Peptide−DNA Complex (Polyplex). Peptide−pDNA complexes (referred to as polyplexes in the text) were prepared at different charge ratios expressed as ratio of amino nitrogen (NH3+) of peptide per phosphate (PO4−) group of DNA and designated as (N/P) or Z (±). The pDNA stock was diluted to a concentration of (20−40) ng/μL and added dropwise to an equal volume of the appropriate peptide dilutions while vortexing, as already reported.31 To prepare polyplexes at different pH, pDNA and peptides were equilibrated in MQ or buffer of different pH for 5 min before making the polyplexes. DNA Condensation Experiments. The electrophoretic mobility of the polyplexes prepared at different charge ratios Z (±) ranging from 0 to 5 was studied using agarose gel electrophoresis. 20 μL of the polyplex having 200 ng total pDNA was loaded onto 1% agarose gel containing ethidium bromide in each case. Electrophoresis was carried out at 100 V in 1× TAE buffer (pH 7.4) for 30 min. DNA condensation was also measured via fluorimetry. The assay was carried out in black 96-well format plates (Nunc) 684

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225 μm long, resonance frequency of around 75 kHz and force constant of 2.8 N/m were used for imaging. The polyplexes were imaged by simply depositing 2 μL of the polyplex solution on freshly cleaved mica and drying it in air. Minimum image processing (first order flattening and contrast settings) was employed, and image analysis was done using PicoImage software. Zeta Potential. Polyplexes of arginine homopeptides and histidine and cysteine modified arginine peptides were prepared at charge ratio 10.0 in MQ and buffers with pH 5.0 and 7.4. Zeta potential for the polyplexes was measured by Zetasizer Nano ZS (Malvern Instruments, U.K.) at a fixed angle of 173° at 25 °C. A minimum 3 readings were recorded for each sample with replicates. Cell Culture and Transfection. Chinese hamster ovary cells (CHO-K1) were maintained in Ham’s F-12K medium, human cervical cancer cells, Henrietta Lacks (HeLa), in minimum essential medium (MEM), and human breast cancer cells, Michigan Cancer Foundation-7 (MCF-7) and MDA MB231, were cultured in Dulbecco’s modified Eagle medium (DMEM) and human alveolar basal epithelial cells (A549) also in Dulbecco’s modified Eagle medium (DMEM), all supplemented with 10% (v/v) fetal bovine serum (Life Technologies, USA) at 37 °C and 5% CO2 in a humidified incubator. Cells were seeded in 24-well plate format, and the experiment was carried out after 24 h when a confluency of 70% was reached. Polyplexes were prepared at different charge ratios Z (±) with final pDNA concentration of 20 ng/μL (pMIR-Report Luciferase) and incubated for one hour. 100 μL of polyplex (2 μg of pDNA/well) was added to cells with and without chloroquine (100 μM/well final concentration) in serum free medium (OptiMEM). For some peptides transfection was also performed in the presence of 10% serum in CHO-K1 and MCF-7 cell lines at charge ratio 10.0. After 5 h of incubation, the medium containing polyplexes was aspirated and cells were rinsed with 1× PBS (pH 7.4) and finally supplemented with complete growth medium. After 24 h of transfection, cells were lysed with 100 μL of cell culture lysis buffer (1× CCLR, Promega) and luciferase activity was measured by mixing luciferase assay substrate (Promega) and cell lysate in 1:1 ratio using a luminometer (Berthold Detection System, Germany). Transfection efficiency was normalized with total protein content of the cells, estimated using bicinchoninic acid assay (BCA). For transfection experiments carried out in the presence of bafilomycin, the polyplexes were prepared at charge ratio 10.0 and incubated for one hour. CHO-K1 cells were pretreated with 100 nM bafilomycin A1 (Sigma) for 15 min in 300 μL of optiMEM, and then the polyplexes were added (2 μg of pDNA/well). After 5 h of incubation at 37 °C, 5% CO2, polyplexes were removed and complete Ham’s F12 K medium was added to each well. After 24 h luciferase activity was measured as described above. To study the effect of free thiols of the polyplex, either ACM modified peptides were used or cysteine polyplexes prepared at charge ratio 10.0 were incubated with 1 mM DTNB [5,5′dithiobis(2-nitrobenzoic acid]. Similarly, cells were pretreated with DTNB (1 mM) for 1 h before adding polyplexes. In both cases transfection efficiency was measured after 24 h by luciferase assay as described above. Transfection efficiency for all experiments is presented as mean value ± standard deviation (SD) of three independent

where 10 μL of ethidium bromide (EtBr) (4.22 ng/μL) and 20 μL of pDNA (20 ng/μL) i.e., 1 EtBr molecule per 6 base pairs of pDNA, was dispensed in each well and incubated in the dark for 5 min at room temperature. 20 μL of peptide solution corresponding to different charge ratios was subsequently added. Two controls were also prepared where pDNA with EtBr and only EtBr were used. Further the plate was incubated for 15 min in the dark and fluorescence was recorded using 535 SL EXP 1 excitation and 595 SL EMP 1 emission filters (Beckman Coulter plate reader, DTX 880, USA). The fluorescence values of pDNA with EtBr were taken as maximum, i.e., 100%, and the relative percentage decrease in fluorescence signal was calculated at increasing charge ratios and plotted as percentage of maximum (% of Max). To study the effect of pH on DNA condensation the peptides were first equilibrated for 5 min in milli Q water or buffers of different pH [10 mM PBS (pH 7.4), 10 mM HEPES (pH 6.5), 10 mM HBS (pH 6.5), 10 mM Na·acetate (pH 5.0) and 10 mM Na·citrate (pH 5.0)]; then peptide−pDNA complexes were prepared at different charge ratio Z (±) ranging from 0 to 10.0 and DNA condensation was measured by fluorimetry as described above. DNA Release/Polyplex Stability. Polyplex stability was studied by monitoring DNA release under different conditions like treating the polyplexes with anionic agent heparin and varying the pH of polyplex solution, and also DNA protection ability of peptides was assessed by treating the polyplexes with DNase I. Polyplexes were prepared at charge ratio Z (±) 10 and treated for 30 min with increasing amount of heparin (H3149100KU) ranging from wt/wt ratios (anionic agent/peptide) of 0.25:1 to 2:1 and analyzed on 1% agarose gel. The amount of the pDNA released from the polyplexes was compared with that of the native uncomplexed pDNA. DNA release or stability of polyplexes was also assessed by fluorimetry.31 Briefly, heparin at increasing amount was added to different wells of black 96-well plate followed by addition of 20 μL of polyplexes prepared at charge ratio 10 and EtBr (at the same concentration used for the condensation assay), and the plate was incubated in the dark for 15 min. Three controls, namely, pDNA with EtBr, heparin and pDNA with EtBr and EtBr alone were also prepared. The fluorescence was recorded as described in the earlier section. The fluorescence value of pDNA control was taken as 100%, and the relative percentage increase in fluorescence signal calculated at increasing concentration of heparin was plotted as percentage of maximum (% of Max). Similarly the stability of polyplexes prepared at different pH was also assessed. DNA protection ability of different peptides from nucleases was estimated by incubating the polyplexes prepared at charge ratio 10 with different concentrations of DNase I for 30 min at 37 °C in reaction buffer (10 mM Tris-HCl, pH 7.5, 0.1 mM CaCl2, and 2.5 mM MgCl2). DNase I was inactivated by heating at 75 °C for 10 min, and the protected DNA was released from the polyplexes by adding heparin (4 μg) and checked by agarose gel electrophoresis as mentioned above in DNA release experiments. Atomic Force Microscopy (AFM). Morphology of the polyplexes at different charge ratios was imaged by atomic force microscopy (AFM) using a 5500 scanning probe microscope (Agilent Technologies, Inc., AZ). Imaging was performed in air using AAC mode to ensure minimum sample damage with scan speed of 1 line/s. Cantilevers (Molecular Imaging), TypeII, Si, 685

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replaced with complete medium after 4 h) of transfection. Briefly, 100 μL of MTT reagent (0.5 mg/mL) was added to each well and incubated at 37 °C for 2 h to allow formation of formazan crystals. The medium containing the MTT reagent was decanted carefully from the wells such that the crystals do not get detached. Following incubation 100 μL of MTT detergent buffer (0.5 mL of 10% SDS and 0.06 mL of 12 N HCl in 10 mL of isopropanol) was added to each well, and crystals were dissolved by gentle shaking for 30 min. Finally the absorbance was taken at 550 and 670 nm (to negate debris) along with the blank. The percentage viability was calculated by

experiments, each performed in duplicate, and significance was calculated using a nonparametric, Mann−Whitney U test (independent samples, two tailed). Flow Cytometry (FACS). Plasmid DNA (pEGFPC1) was labeled with tetramethyl Rhodamine (TM-Rh) using Label IT nucleic acid labeling kit (Cat No. MIR 7013, Mirus Bio, Madison, USA) at 1:1 (v:w) ratio, i.e., 1 μL of labeling reagent for 1 μg of pDNA according to the manufacturer’s protocol. The pDNA concentration was estimated using UV spectroscopy at 260 nm. The entire amount of pDNA used was labeled in one reaction to avoid the fluctuations in labeling densities among different labeling reactions. For measuring polyplex uptake, CHO-K1 cells were grown for 24 h in a 24-well plate as mentioned above and fluorescently labeled polyplexes (having TM-Rh labeled pDNA) at Z (±) of 10 were added to cells. After four hours of incubation, cells were washed twice with ice-cold 1× PBS (pH 7.4) having 1 mg/mL heparin to remove the extracellular bound polyplex. Cells were collected by trypsinization (100 μL of 0.25% trypsin) and washed with ice-cold 1× PBS supplemented with 1% BSA and resuspended in 500 μL of 1× PBS. Measurements were carried out on FACS- Calibur (Becton Dickinson, USA) using CellQuest Pro software. The TM-Rh labeled polyplexes were excited using 488 nm laser and detected with 585/42 nm (FL2) band-pass filter. Total 10,000 events were acquired in each case, and the data shows the average of three independent experiments. Transfected cells with the unlabeled polyplexes were used to correct for the autofluorescence of cells. To study the mode of cellular entry of the different polyplexes, CHO-K1 cells were pretreated for 45 min at 37 °C with various endocytosis inhibitors, 10 μg/mL chlorpromazine, 200 μM genistein, 2 mM methyl beta cyclodextrin (MβCD) and 200 μM dimethyl amiloride (DMA) in 300 μL of optiMEM. TM-Rh labeled polyplexes (75 μL, i.e., 1.5 μg of pDNA/well) were added to the cells and further incubated for 3 h along with the drugs. After incubation the cells were processed as above and analyzed for uptake by flow cytometry. Gating was done on FSC−SSC plot to negate the debris from the analysis. Also the specificity of various drugs toward inhibition of their specific endocytotic pathway was evaluated by studying the uptake of positive markers in each case. The positive markers used in the study were transferrin−Alexa Fluor 633 conjugate (for clathrin mediated endocytosis), BODIPY FL C5-lactosylceramide (for caveolae mediated endocytosis) and neutral dextran−tetramethylrhodamine (for macropinocytosis). The positive markers were incubated with the cells for one hour and analyzed via FACS. To remove the transferrin ligand from the cell surface after incubation, cells were washed twice with ice cold HMEM-G+I medium (DMEM without glucose with 5 mM/L NaN3, buffered with 10 mM/L HEPES to a pH of 7.4) to inhibit metabolic activity followed by 1 min incubation in ice-cold acid wash buffer (0.2 mol/L acetic acid + 0.2 mol/L NaCl) and finally washed twice with ice cold HMEM-G+I medium. Cells were treated with defatted BSA and 1× PBS to remove surface associated LacCer and dextran fluorescence respectively.34 Cell Viability Assay. The toxic effects of various polyplexes were measured using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] assay. CHO-K1 cells were seeded at 7000 cells per well in 96-well plate format. After 24 h cells were treated with different polyplexes at charge ratio 10.0 (in quadruplicate) for 4 h. The viability was assayed after 4 and 24 h (in the latter case medium containing polyplex was

{treated well (OD550 − OD670 )/untreated well (OD550 − OD670 )} × 100

The average of 2 independent experiments is plotted where each treatment is performed in quadruplicate.

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RESULTS Our earlier study has shown that in a series of 9−16-mer arginine peptides, R16 shows the best transfection efficiency owing to its better DNA condensation and intracellular DNA release ability while R9 shows the strongest DNA condensation.31 In the current study we have taken 9 arginine residues to ensure strong condensation and added 7 histidine residues to create a histidine modified equivalent of R16, i.e., R9H7. In order to check the effect of the position of histidines, two more positional variants of this 16-mer peptide were created by changing the arrangement of histidine residues with respect to arginines: H4R9H3 where the arginines were arranged together in a stretch flanked by the histidines and R5H7R4 where the histidines were arranged together in a stretch flanked by blocks of arginines. Two corresponding lysine variants K9H7 and H4K9H3 were also generated for comparison. All the sequences are listed in Table 1. We presumed that the presence of histidines would impart endosomal escape property to the polyplexes formed with DNA but would also possibly alter the DNA condensation and release properties of the peptide. In particular, it is likely to make the polyplexes loosely packed and susceptible to nuclease degradation. In order to fine-tune the condensation−release balance, we also modified all these arginine-histidine variants with cysteine residues at both ends (peptides used are listed in Table 1a). Addition of Histidines Improves Transfection Efficiency of Arginine Peptides. To study the effect of histidine modification on the transfection efficiency of the arginine peptides, transfection was carried out with all the histidine modified peptides as a function of charge ratio in the CHO-K1 cell line (data not shown). All the peptides show maximum efficiency around charge ratio 10.0, and this charge ratio was used to compare the transfection efficiency of this set of peptides. Our previous results have demonstrated that 16-mer arginine homopeptides show higher transfection efficiency than lysine homopeptides.31 In order to check whether this holds good for histidine modified arginine and lysine peptides also, we have compared the transfection efficiency of two of the histidine modified arginine peptides, R9H7 and H4R9H3, with the respective lysine counterparts K9H7 and H4K9H3. The histidine modified arginine peptides exhibited more than 2- to 3-fold higher transfection efficiency as compared to their respective histidine-lysine variants (Figure 1a). We therefore carried out all further experiments with the arginine-histidine combinations. Next we have evaluated the relative transfection 686

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transfection experiments in the presence of bafilomycin A1 (Figure 2a). Bafilomycin A1 is a specific inhibitor of vacuolar ATPase proton pump, which inhibits the protonation of the histidine vector inside endosomes and thus prevents the endosomal escape of the polyplex. Bafilomycin A1 does not have any effect on the transfection efficiency of R16, but it strongly reduces the transfection efficiency of R9H7 (10 times drop in transfection with respect to the control). Even in the case of the two other histidine modified peptides around 8 times drop in transfection efficiency was observed. This further confirms that the histidine modified peptides show better endosomal buffering than the arginine counterparts. We then evaluated the relative condensation ability of the histidine modified peptides and stability against anionic challenge of the complexes formed by them with DNA, using agarose gel electrophoresis and EtBr exclusion assay. In agarose gel electrophoresis assay the retardation in the electrophoretic mobility of DNA as a function of charge ratio is a measure of DNA condensation ability of the peptide. On the other hand, in EtBr exclusion assay, decrease in the EtBr fluorescence upon exclusion from DNA due to binding of the cationic peptide to the anionic DNA molecules is used as a measure of DNA condensation. The aim of these experiments was to check whether differences in DNA condensation and particle stability also contribute to the transfection efficiency of the histidine modified peptides. Gel retardation assay (Figure 2b, left panel) shows that R9H7 retards DNA completely at a very high charge ratio of 5.0; maximum drop in fluorescence is also observed in the EtBr exclusion experiment around charge ratio 5.0 (Figure 2c, upper panel). This is higher than what has been observed in our earlier study with R16.31 The other histidine modified peptides H4R9H3 and R5H7R4 retard DNA completely at a much lower charge ratio of 3.0 and 1.75 respectively (Figure 2b). A similar pattern is evident from the EtBr exclusion assay also where the slope of drop in fluorescence becomes steeper as the positions of the histidines are altered R9H7 < H4R9H3 < R5H7R4 indicating stronger compaction at lower charge ratios as we move across the peptide series (Figure 2c, upper panel). The relative stability of the polyplexes formed with histidine modified peptides was next evaluated (Figure 2b, right panel, and Figure 2c, lower panel) by adding increasing amounts of anionic agent heparin to the polyplex and observing the destabilization of the polyplex and release of DNA on an agarose gel. The DNA release from the polyplexes is found to be consistent with the DNA condensation results where a weaker condensing peptide like R9H7 shows complete DNA release at the lowest concentration of heparin, i.e., wt/wt ratio (anionic agent/peptide) of 0.25 as compared to R16 which shows complete release at a ratio of 1.25 as seen in our earlier study.31 Similarly the stronger condensing peptide R5H7R4 shows complete DNA release at an anionic agent:peptide ratio of 1.0. We further explored whether there are differences in the morphology of the polyplexes formed with the histidine modified peptides (Figure 2d). The AFM image of polyplexes formed with R9H7 shows loose flowerlike structures (with DNA strands hanging out of the core) whose sizes range between 50 and 200 nm whereas we have shown in an earlier study that R16 forms 64−70 nm sized spherical and condensed polyplexes. More compact structures (although still flowerlike in nature) are present in the case of H4R9H3 and R5H7R4 but are smaller and lie in the size range of 50−100 nm. The trend in the particle size and overall particle morphology are in concordance

Table 1. (a) Different Histidines, Cysteine and Other Modified Peptides Studied in This Work and (b) Zeta Potential of Homo Arginines and Modified Arginine Based Polyplexes (a) Different Histidines, Cysteine and Other Modified Peptides Studied in This Work peptide

sequence

charge density

K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K +16.0 K16 K9H7 K-K-K-K-K-K-K-K-K-H-H-H-H-H-H-H +9.6 H4K9H3 H-H-H-H-K-K-K-K-K-K-K-K-K-H-H-H +9.6 R-R-R-R-R-R-R-R-R +9.0 R9 R16 R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R +16.0 R9H7 R-R-R-R-R-R-R-R-R-H-H-H-H-H-H-H +9.6 H-H-H-H-R-R-R-R-R-R-R-R-R-H-H-H +9.6 H4R9H3 R5H7R4 R-R-R-R-R-H-H-H-H-H-H-H-R-R-R-R +9.6 C-R9H7-C C-R-R-R-R-R-R-R-R-R-H-H-H-H-H-H-H-C +9.5 C-H4R9H3-C C-H-H-H-H-R-R-R-R-R-R-R-R-R-H-H-H-C +9.5 C-R-R-R-R-R-H-H-H-H-H-H-H-R-R-R-R-C +9.5 C-R5H7R4-C ACM-(CACM-(C-R-R-R-R-R-H-H-H-H-H-H-H-R+9.5 R5H7R4-C)a R-R-R-C)-ACM (b) Zeta Potential of Homo Arginines and Modified Arginine Based Polyplexes Prepared at Charge Ratio 10.0 under Different pH Conditions zeta potential peptide R9 R16 R9H7 H4R9H3 R5H7R4 C-R9H7-C C-H4R9H3-C C-R5H7R4-C ACM-(C-R5H7R4-C) a

MQ +17.6 ± 3.0 +23.3 ± 5.0 +9.07 ± 3.0 +15.4 ± 2.0 +11.31 ± 5.0 +34.7 ± 2.1 +29.3 ± 2 +32.8 ± 3.0 +20.4 ± 2.0

pH 5.0 +22.7 ± 2.0 +27.7 ± 3.3 +25.2 ± 3.7 +24.6 ± 4.2 +31 ± 5.0 +34.5 ± 6.2 +37.4 ± 5.3 +34.0 ± 5.1 +29.4 ± 4.4

pH 7.4 +0.45 ± 0.2 +9.1 ± 2 −0.6 ± 0.3 +2.5 ± 0.5 +2.2 ± 0.3 +13.3 ± 0.5 +12.6 ± 2 +14.2 ± 0.6 +4.1 ± 1.2

ACM: acetomidomethyl group.

efficiencies of arginine homopeptides and the three histidine modified arginine peptides in the absence or presence of endoosmolytic agent chloroquine in CHO-K1 and MCF-7 cells (Figures 1b and 1c). In the absence of chloroquine, the histidine modified variants show substantially higher transfection efficiency (1−3 orders of magnitude) as compared to R9. When compared to R16, R9H7 shows enhancement in transfection efficiency in both the cell lines by about 1 order of magnitude in the absence of chloroquine. However, in the case of H4R9H3 and R5H7R4, the transfection efficiencies are higher than that of R9 in both the cell lines but not higher than R16 in all the cases. The presence of chloroquine significantly increases the transfection efficiency in the case of homopeptides R9 and R16 (Figures 1b and 1c). However, in the case of all the histidine modified peptides, there is only a marginal increase in transfection efficiency in the presence of chloroquine,0 suggesting that addition of histidines possibly enhances the inherent capacity of endosomal buffering. Histidine Modified Peptides Show Endosomal Escape, Differences in DNA Condensation and Release and Differences in Particle Size and Morphology as Compared to the Arginine Homopeptide. It is clear from the results described above that the presence of histidines results in improved endosomal release, which may or may not translate into increased transfection efficiency. In order to further explore this observation, we have carried out the 687

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Figure 1. (a) In vitro transfection efficiency of the luciferase gene in CHO-K1 cell line. Comparison of 16-mer homopeptide of lysine and arginine with histidine modified counterparts (** p value < 0.005, *** p value < 0.0005). The polyplexes were prepared using 2 μg of DNA at charge ratio 10.0 and added to cells; after 24 h of transfection luciferase activity was measured and expressed as RLU/mg total protein. (b, c) Transfection efficiency of 16-mer arginine homopeptide and histidine modified counterparts in CHO-K1 (** p value < 0.005) and MCF-7 (* p value ≤ 0.05) cell lines. Transfection was performed at charge ratio 10.0 in the absence (white bars) and presence (gray bars) of chloroquine (100 μM), and luciferase activity was measured after 24 h. The data represents the mean ± SD of luciferase activity (n = 3).

with those observed from the DNA condensation and release studies. All the data presented above indicate that R9H7 in which all the arginines and histidines are arranged together in one stretch shows relatively weaker condensation and easy release in vitro and forms relatively loose structures, with higher particle size, as compared to the other histidine modified peptides and also the arginine homopeptide R16. On the other hand, alteration in the distribution of arginines and histidines in blocks results in stronger condensation of DNA and weaker release against anionic challenge. We have also checked the nature of the polyplexes formed at lower charge ratios (charge ratio 3 and 5) with all these peptides and observed the predominance of multimolecular structures (i.e., multiple DNA molecules and multiple peptide molecules in each polyplex, data not shown) as seen with R16 in our earlier study.31 This indicates that although the mechanism of condensation does not possibly vary by adding the histidine residues, there is a strong effect of the relative positions of the arginine and histidine residues on their efficiency of DNA condensation and ease of DNA release. This has been deliberated upon in the Discussion. Addition of Cysteine Residues Enhances Transfection Efficiency of Histidine Modified Arginine Peptides. The

arginine-histidine polyplexes show substantial differences in their DNA condensation and release abilities depending upon the position of the arginines and histidines in them. However, they are similar in the endosomal escape properties as seen from Figure 2a. We also checked the overall cellular uptake efficiency and the pathway of uptake (by labeling the DNA with a fluorophore) in which all the peptides show high cellular uptake (more than 90% cells show positive fluorescence) and similar nature of internalization (data not shown). Thus it is likely that the altered condensation and release balance in these peptides on altering the position of the arginines and histidines could be the primary reason for limiting their transfection efficiency. Cysteines have been used in the literature to impart polyplex stability in the extracellular milieu through increased compaction by cross-linking peptides via S−S bonds when oxidized and at the same time exhibit controlled DNA release in the intracellular reducing environment by the reduction of the S−S bonds.21−30 Such fine-tuning of the condensation and release balance is likely to improve the transfection efficiency. Keeping this in account, two cysteine residues were added at both the terminal ends of all the peptides studied. The effect of cysteine modification on the transfection efficiency was next evaluated in cell lines: CHO-K1, MCF-7 and MDA-MB-231 688

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Figure 2. (a) Comparison of endosome buffering efficiency of 16-mer arginine homopeptide with its histidine modified counterparts. Transfection was performed at charge ratio 10.0 in CHO-K1 cells in the absence (white bars) and presence (gray bars) of bafilomycin A1 (100nM) and luciferase activity was measured after 24 h. The data represents the mean ± SD of luciferase activity (n = 3) (** p value = 0.009). (b, c) Comparison of DNA condensation and release ability of histidine polyplexes using (b) agarose and (c) fluorimetry based assays. (b) Left panel represents the electrophoretic mobility shift of DNA as a function of charge ratio for the different histidine modified peptides. Right panel represents the destabilization of histidine polyplexes prepared at charge ratio 10.0 with increasing amount of heparin (mentioned as anionic agent/peptide: wt/wt ratio). Lane 1 (ctrl) is the uncomplexed pDNA in both cases. (c) Relative DNA condensation ability (left panel) and stability against anionic challenge (right panel) of histidine modified arginine peptides measured using fluorimetry based EtBr assay. The fluorescence of free uncomplexed DNA is set as maximum, i.e., 100%, and relative decrease or increase in fluorescence is measured in DNA condensation and stability assays respectively. (d) Representative atomic force microscopy (AFM) images of histidine polyplexes prepared at charge ratio 10.0 (image size is 4 μm × 4 μm). Zoomed images depict the morphology of particles/polyplexes.

compared to the parent histidine modified peptides (Figure 4a). In gel retardation assay also all these three cysteine variants show early retardation as compared to their parent peptides (Supplementary Figure 1a in the Supporting Information), which corroborates the finding of the EtBr exclusion assay. The changes in DNA condensation ability however do not follow an exactly similar trend in their stability against anionic challenge (Figure 4b and Supplementary Figure 1b in the Supporting Information). Polyplexes prepared with C-R9H7-C and CH4R9H3-C which are strongly condensed resist destabilization at lower heparin concentration like 0.5 whereas their parent histidine peptides show 40% DNA release at this concentration. On the contrary C-R5H7R4-C shows similar destabilization in comparison to its histidine counterpart although it possesses much stronger DNA condensation ability in comparison to its histidine counterpart. We next examined the effect of DNase I to determine the stability of cysteine polyplexes (Supplementary Figure 1c in the Supporting Information). We observed that while native pDNA is completely digested at 1 unit of DNase I enzyme (data not shown), all the cysteine polyplexes provide significant

(Figure 3). Transfection efficiency increases in all the peptides by several orders of magnitude from the parent argininehistidine peptides when compared at charge ratio 10 even in the absence of chloroquine irrespective of the arginine and histidine positions. Thus it is likely that this increase in the transfection efficiency and almost similar transfection efficiencies in all the cysteine modified peptides are likely to have arisen from the differences in DNA condensation and packaging of the polyplexes. However, other factors like differences in cellular uptake and uptake pathways as well as differences in endosomal release efficiency could also play a role. We next set out to delineate which are the factors that play a major role in this increase in transfection efficiency. We first explored the DNA condensation ability and stability against anionic challenge of cysteine modified peptides and polyplexes as compared to the respective parent histidine modified peptides. The DNA condensation experiments show that modification with cysteine improves the DNA condensation ability of all the peptides R9H7, H4R9H3 and R5H7R4 since CR9H7-C, C-H4R9H3-C and C-R5H7R4-C show steeper slope in the drop of fluorescence intensity in EtBr exclusion assay as 689

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Figure 3. Effect of cysteine modification on transfection efficiency of histidine modified peptides in (a) CHO-K1 (* p value < 0.05, ** p value = 0.009), (b) MCF-7 (*p value ≤ 0.05) and (c) MDA MB-231 cells (*p value ≤ 0.05). All polyplexes were prepared at charge ratio 10.0, and transfection was carried out in the absence of chloroquine.

protection even at 2 U of DNase I while their histidine modified counterpart does not. Atomic force microscopy was also carried out to study the morphology of all the polyplexes formed with the cysteine modified peptides (Figure 5). The polyplexes were prepared at lower charge ratio of 3.0 and 5.0 as they show stronger condensation as compared to their parent peptides in several cases. Surprisingly even at a low charge ratio of 3.0 no loose flowerlike structures are seen in the polyplexes of any of the cysteine modified peptides. Only condensed flowers are observed at charge ratio 3.0 (Figure 5, left panel) which show complete condensation upon further increase in charge ratio, i.e., 5.0 (Figure 5, right panel). The particle sizes are in the range of 50−100 nm in all the cases. Thus in contrast to their parent histidine peptides, the cysteine modified variants show complete condensation at much lower charge ratios as seen by differences in the morphology as well as the sizes of the complexes formed. Also cysteine modified polyplexes prepared in MQ and PBS (pH 7.4) at charge ratio 10.0 showed 3 to 4 times higher zeta potential than their histidine variants. The possible explanation could be that S−S bonds formed under autoxidation conditions help in tethering of peptides together on the polyplex surface and increase the overall surface charge. Moreover blocking of cysteine residues by acetomidomethyl (ACM) group in C-R5H7R4-C peptide decreases its zeta potential significantly. Since all the peptides used in this study are highly charged at pH 5.0, the variation in zeta potential is

not expected among peptides, which is evident from the high zeta potential values (Table 1b). We also checked the endosomal buffering efficiencies of all cysteine modified peptides. In the presence of bafilomycin A1 we found substantial decrease in the transfection efficiency of all the peptides to similar levels as seen with histidine modified peptides which does not vary much across the peptide series (Figure 6). We have also estimated the cellular uptake of cysteine modified peptides and found no significant difference in cellular uptake and uptake pathways among them (data not shown). Moreover, these polyplexes also seem to be adopting both caveolae and clathrin mediated entry similar to that in the case of histidine modified arginine peptides (data for representative peptides R9H7 and C-R9H7-C are shown in Supplementary Figure 2 in the Supporting Information). However, we observed larger inhibition (both in the percentage of cells and in the relative intensity) in uptake of C-R9H7-C polyplexes in the presence of chlorpromazine, genistein and MβCD as compared to the histidine counterparts. This perhaps indicates that while different endocytotic pathways are the exclusive modes of entry of the cysteine containing polyplexes, additional pathways might be a minor component in the polyplexes with only histidines. Thus, it is clear that while the cysteine modified peptides show stronger condensation as compared to the histidine modified ones and the complexes are similar in size, morphology, cellular uptake efficiency and pathways across 690

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Figure 4. Comparison of (a) DNA condensation efficiency and (b) stability against anionic challenge of histidine peptides with their respective cysteine modified variants measured using fluorimetry based EtBr assay. The fluorescence of free uncomplexed DNA is set as maximum, i.e., 100% and relative decrease or increase in fluorescence is measured in DNA condensation and stability assays respectively.

ate to dark purple colored formazan product). Since the reduction of MTT is possible only in metabolically active cells this activity is a measure of the viability of cells. All the cysteine modified peptides show 80% or more viability at both 4 and 24 h whereas Lipofectamine 2000 has only 40% viable cell population (Supplementary Figure 3 in the Supporting Information). However, in the presence of serum, transfection efficiency of the cysteine modified arginine peptides is compromised (Supplementary Figure 6 in the Supporting Information). All these data seem to indicate that addition of histidine improves the endosomal buffering efficiency of arginine peptides but the differences in the DNA condensation and release of the complexes possibly affect some of the postuptake processes and are likely to be the major contributors to the differences in transfection efficiency in the case of histidine modified peptides. In case of the cysteine modified peptides, there is substantial improvement in the transfection efficiency over the histidine counterpart. We would like to mention here

the whole series, their DNA release efficiencies seem to be mildly altered. This is further analyzed in the Discussion. Arginine-Histidine-Cysteine Peptides Are a Novel Set of Transfection Agents. Cysteine modified peptides not only show efficient DNA condensation and resistance against anionic challenge at low charge ratios in several cases but also exhibit high transfection efficiency at low charge ratio (charge ratio of 5.0) (Figure 7). All of them show high transfection efficiency in relatively hard to transfect cell lines like HeLa and MDA-MB231, and several of them are equivalent to a commercial agent like Lipofectamine 2000. Thus, this system offers the potential for nucleic acid delivery at low charge ratios, thereby significantly lowering problems associated with toxicity due to use of higher amount of cationic agent. We have next evaluated the toxicity of these complexes at a charge ratio of 10.0 after 4 and 24 h of transfection using MTT assay (a calorimetric assay that measures the reduction of yellow color 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT, by mitochondrial succinate dehydrogen691

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effect of free thiols can be observed by blocking the cell membrane bound thiols or the free thiols of the peptides. If the internalization is due to free thiol interaction of peptides with cell membrane bound thiols, then the transfection efficiency will be affected by the blocking of free thiols. So we blocked the terminal cysteine thiols of the peptides by acetomidomethyl (ACM) group and studied the trasfection efficiency in the CHO-K1 cell line. There was a large drop (almost 2.5 orders) in transfection efficiency (Supplementary Figure 4a in the Supporting Information), suggesting that free thiols of the polyplexes can also be one of the reasons contributing the higher transfection efficiency with the peptides containing cysteines. We have further substantiated this observation by using DTNB, which is cell impermeable nonspecific thiol blocker. We determined the transfection efficiency of polyplexes at charge ratio 10.0 after incubating them with DTNB for one hour and added to cells. We observed that DTNB does not have any effect on transfection efficiency of only histidine modified peptide R5H7R4 and ACM-blocked CR5H7R4-C peptide whereas it has significant effect on native CR5H7R4-C polyplexes. However, blocking of cell surface thiols with DTNB does not alter the transfection efficiency of CR5H7R4-C peptide (Supplementary Figure 4b in the Supporting Information).

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Figure 5. Representative atomic force microscopy (AFM) images of cysteine polyplexes prepared at charge ratio 3.0 and 5.0 (image size is 4 μm × 4 μm). Zoomed images depict the morphology of particles/ polyplexes.

DISCUSSION In this work, we have attempted to make multifunctional peptides which show strong condensation, efficient endosomal escape and intracellular release and thereby high transfection efficiency. For the DNA condensing moiety, we have taken arginine instead of conventionally used lysine since our earlier study has shown that arginine homopeptides show better condensation and release balance and form small and monodisperse polyplexes.31 Lysine-histidine based systems (as polymers or dendrimers) have been largely explored in the literature and shown to be efficient in delivery of DNA as well as siRNA.16−20 However, we also noted in this study that the arginine-histidine peptides show higher transfection efficiency than the lysine-histidine peptides and improved transfection in comparison to their parent homopeptides as well. Thus, our study clearly shows that small and linear peptides with argininehistidine combination are superior to lysine-histidine peptides, and all further experiments and modifications have been carried out on these peptides. Histidines have been used to help in endosomal escape of the polyplexes. The imidazole ring of histidine is a weak base and has pKa around 6.0, which makes it charged at low pH encountered in the endosomes and allows it to scavenge protons. This unique property of histidine allows the histidinebearing polyplexes to be released from the endosomes. Histidines have thus been incorporated with DNA condensing cationic polymers, peptides and lipids for improving the endosomal release and increasing the transfection efficiency.16−20 Similarly, in the arginine-histidine peptides that we have studied, the presence of histidines is critical for endosomal release of the polyplexes. All the polyplexes show endosomal buffering efficiency as they exhibit minimal jump in transfection efficiency in the presence of chloroquine and a large drop in transfection efficiency in the presence of bafilomycin A1. However we observed that the position of histidines with respect to arginine, or in other words, the distribution of charge along the peptide length controls the DNA condensation and in vitro DNA release of the respective

Figure 6. Comparison of endosome buffering efficiency of histidine modified arginine peptides and their cysteine modified counterparts. Transfection was performed at charge ratio 10.0 in CHO-K1 cells in the absence (white bars) and presence (gray bars) of bafilomycin A1 (100 nM) and luciferase activity was measured after 24 h. The data represents the mean ± SD of luciferase activity (n = 3) (* p value ≤ 0.05).

that, in the literature, cysteine modified peptides have only been used under oxidative conditions (either through formation of reducible polycations by oxidizing the peptide itself or introducing S−S linkages by oxidizing the polyplex). However, we have not employed any such condition in our cysteine modified peptides and all polyplexes were prepared under autoxidation conditions. Another point that has been noted in the literature is that cysteines contain free thiols which can interact with the cell membrane bound thiols helping the polyplexes to internalize in the cellular compartment.39 The 692

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Figure 7. Transfection efficiency of different cysteine modified peptides at charge ratio 5.0 (white bars) and 10.0 (gray bars) with Lipofectamine 2000 (black bars) in the absence of chloroquine in different cell lines (a) MCF-7, (b) MDA-MB-231 and (c) HelLa.

have also noted that all the histidine variants show comparable cellular uptake and follow caveolae-mediated endocytosis as the most predominant route of entry. We have seen similar entry pathway for R16 and R9 polyplexes as well (unpublished data). Differences in pathways of entry have often been found to be dictated by particle size.36 Such an effect does not seem to be occurring here. Having identified that the arrangement of the charged arginines and uncharged histidines is critical for the condensation−release balance and in turn influences the transfection efficiency, we also explored the effect of change in pH on the transfection efficiency. The histidine residues are not charged around physiological pH, i.e., 7.4 (the condition at which the polyplexes were prepared). Alteration in pH affects the peptide charge density distribution and hence the DNA condensation and, in turn, might have an effect on the transfection efficiency. On the other hand, the protonation status of the histidines will also have an effect on the endosomal escape efficiency of the polyplexes. There are examples in the literature where preparation of the complexes at low pH has shown higher transfection efficiency.37 In order to check the effect of pH, we have prepared the polyplexes in different buffers ranging from pH 5.0 to 7.4 and measured their transfection efficiencies. A representative example is shown in Supplementary Figure 5a in the Supporting Information. The transfection efficiency does not show much variation when the complexes are prepared at pH 6.5 and 7.4. However, there is a drop in transfection efficiency when the polyplexes are prepared in sodium citrate and sodium acetate at pH 5.0. We checked the DNA condensation and release ability of R9H7 peptide at different pH (Supplementary Figure 5b in the Supporting Information). We did not observe any major difference in DNA

polyplexes and has a great influence on the overall transfection efficiency. In our system R9H7 and H4R9H3, which have contiguous arginine residues in one block/stretch, weak DNA condensation and strong DNA release ability are observed. Similar effect was also observed in histidine-lysine variants (data not shown). It seems that when all positively charged residues are present together in a bunch, they are not able to interact with anionic DNA due to the steric hindrance caused by long stretches of uncharged and bulky histidine residues, which results in weaker condensation and formation of loosely assembled complexes. On the other hand, in the case of peptide R5H7R4 the spacing of charged residues allows better interaction with anionic DNA and less hindrance from uncharged histidines. These peptides thus form strongly packed particles with smaller sizes. An earlier report has also discussed a possible effect of the histidine position on the transfection efficiency due to altered pKa in lysine-histidine peptides.24 The other interesting observation is that, among the argininehistidine peptides, the maximum transfection efficiency is seen in the case of R9H7 polyplexes which are loosely packed and release DNA easily. Our earlier study and results from several other groups have shown in recent times that, while efficient DNA condensation is important, intracellular release, albeit in the correct intracellular compartment, is also a critical determinant of transfection efficiency.31,35 In cases like the arignine-histidine peptides, all of which can overcome the endosomal barrier, the less condensed particles possibly release DNA after endosomal escape with better efficiency and thereby show better transfection efficiency. On the other hand the complexes formed with peptide R5H7R4 are strongly packed, which might result in comparably less intracellular release and could be responsible for their lower transfection efficiency. We 693

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indicate that the addition of cysteines may not be affecting the DNA condensation ability of the peptides only through formation of S−S bonds but instead probably acts by altering the spatial availability of the arginines responsible for condensation in the weaker condensing peptides like R9H7 and H4R9H3. In the case of the other peptides where the arginines are already present such that optimum condensation is achieved, further compaction possibly does not occur. However, it needs to be noted that in peptides CR5H7R4C the difference in release ability with the histidine counterparts is not substantially large to completely account for the 4 orders of magnitude increase in transfection. It is also observed that blocking the free thiols in the polyplex causes a large drop in the transfection efficiency while blocking cell surface thiols does not have any effect. Thus it is unlikely that the free thiols of the polyplex interact with cell surface thiols. Instead only the polyplex thiols are likely to be important in ways described above. We are currently exploring other possible effects of cysteines in the postuptake events of the polyplexes which might constitute additional factors responsible for enhanced transfection. In a very recent report, TAT peptide modified with 10 histidine residues showed very good transfection efficiency which is comparable to that of poly(ethyleneimine). But among its cysteine variants the one having all histidines at one end C-TAT-10H-C showed poor efficiency whereas the other having five histidines flanking TAT, C-5H-TAT-5H-C, showed comparable or better efficiency as compared to its parent peptide TAT-10H depending upon the cell line used.25 Our observation is also similar, i.e., the best transfection is seen in the case of R9H7 peptide among all histidine variants while in cysteine modified peptides the one having spaced out histidines shows the highest efficiency. The advantage in our case seems to be that the cysteine modified peptides do not require any extra trigger like oxidation of complexes in order to show high transfection efficiency. In summary, our work highlights the arginine-histidinecysteine peptide system as an efficient in vitro DNA carrier. While arginines were chosen because of their DNA condensation, cell penetration and even endosomal escape property,31,38 histidines and cysteines were chosen for further enhancement in endosomal escape and balanced DNA condensation and release. Interestingly, we observed very high transfection efficiency in this system where the number and arrangement of arginines and histidines along with the presence of cysteines seem to alter the nature of the complex formed with DNA and control the transfection efficiency. This system is also attractive because of the minimal toxicity as well as the high efficiency of transfection achieved even at low charge ratios in a wide range of cell lines. Such a system holds enormous potential to be developed further as a cell-type specific in vivo DNA delivery system with attachment of appropriate ligands.

condensation ability of R9H7 peptide at different pH including pH 5.0 although a very minute difference was seen in DNA release ability at low pH. Decrease in transfection efficiency at low pH could be perhaps controlled more by poor buffering by histidines and altered condensation and release balance might be a less important factor. However, this can vary from peptide to peptide since peptides with spaced out arginine and histidine residues showed relatively better DNA condensation at low pH (data not shown) suggesting that pH can alter the overall charge of the peptides and can affect the DNA condensation and release ability in selected cases. We did not observe any improvement in transfection ability of any of the histidine peptides by changing pH. Although our data indicates that strong DNA release is important for enhancing the intracellular DNA availability in the arginine-histidine peptides, such systems may be difficult to use in vivo since they are likely to possess poor extracellular stability. As mentioned earlier, addition of cysteines or other cleavable linkages can impart a combination of strong DNA condensation along with controlled intracellular release property to DNA delivery systems. Use of cysteines in peptide gene delivery carriers has been primarily through formation of polymeric reducible polycations (pre-RPCs) from small 8−9 lysine and histidine containing peptides where the S−S linkages have been introduced chemically in the peptide. In other cases, S−S linkages have been introduced after the formation of the complexes.23−30 Intriguingly, in our case, the enhancement in transfection efficiency on addition of the cysteines is observed even under normal conditions where no pre-RPCs or postRPCs have been formed. To look for possible reasons for this observation, we have analyzed the DNA condensation and release profiles of these peptides in detail since they are likely to be altered in the presence of cysteines. It is interesting to note that addition of cysteines does not have a similar effect on all the arginine-histidine peptides. It significantly increases the ability of DNA condensation of arginine-histidine peptides which show weak condensation R9H7 and H4R9H3 and even R5H7R4. Cysteine modification not only enhances the condensation but also reduces the DNA release ability of the peptides CR9H7C and CH4R9H3C in comparison to their histidine counterparts. On the other hand, although the condensation ability of the CR5H7R4C is slightly higher, the DNA release ability is also higher comparable to its histidine counterparts. Thus, the presence of cysteines does not enhance the DNA condensation and reduce the DNA release in all the cases as would have been expected. We analyzed whether formation of disulfide bonds (under autoxidation conditions) was responsible for the increase in condensation ability of some of the cysteine modified peptides. Treatment of the polyplexes with a reducing agent like glutathione (GSH) is likely to increase DNA release in vitro if S−S bonds are present in the complexes.23 However, we did not observe any enhanced DNA release in vitro in the presence of GSH (data not shown). As shown in Figure 6 and Supplementary Figure 1b in the Supporting Information, all the polyplexes made using cysteine modified peptides can be destabilized in the presence of anionic agents like heparin. This could be because, under autoxidation conditions, only few S−S linkages are formed which might not require extra trigger to release DNA. Consistent with this effect, we have not observed any improvement in transfection efficiency in the presence of extra GSH or reduction in their transfection ability in presence of an oxidizing agent like buthionine sulfoximine, BSO (data not shown). This seems to

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ASSOCIATED CONTENT

* Supporting Information S

Supplementary Figure 1, showing the comparison of histidine peptides and their respective cysteine modified peptides in DNA condensation and stability of the polyplexes. Supplementary Figure 2, showing the effect of different endocytic inhibitors on the uptake of polyplexes. Supplementary Figure 3, a cell viability assay by MTT. Supplementary Figure 4, representing the effect of cysteine in the peptide sequence on transfection efficiency. Supplementary Figure 5, effect of pH 694

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(16) Midoux, P.; Pichon, C.; Yaouanc, J. J.; Jaffrs, 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. (17) Pichon, C.; Goncalves, C.; Midoux, P. Histidine-rich peptides and polymers for nucleic acids delivery. Adv. Drug Delivery Rev. 2001, 53, 75−94. (18) Chen, Q. R.; Zhang, L.; Stass, S. A.; Mixson, A. J. Branched copolymers of histidine and lysine and efficient carriers of plasmids. Nucleic Acids Res. 2001, 29, 1334−1340. (19) Kumar, V. V.; Pichon, C.; Refregiers, M.; Guerin, B.; Midoux, P.; Chaudhuri, A. Single histidine residue in head-group region is sufficient to impart remarkable gene transfection properties to cationic lipids: evidence for histidine mediated membrane fusion at acidic pH. Gene Ther. 2003, 10, 1206−1215. (20) Leng, Q.; Goldgeier, L.; Zhu, J.; Cambell, P.; Ambulos, N.; Mixson, A. J. Histidine-Lysine peptides as carriers of nucleic acids. Drug News Perspect. 2007, 20, 77−86. (21) Bauhuber, S.; Hozsa, C.; Breunig, M.; Gopferich, A. Delivery of nucleic acids via disulfide-based carrier systems. Adv. Mater. 2009, 21, 3286−3306. (22) West, K. R.; Otto, S. Reversible covalent chemistry in drug delivery. Curr. Drug Discovery Technol. 2005, 2, 123−160. (23) Read, M. L.; Singh, S.; Ahmed, Z.; Stevenson, M.; Briggs, S. S.; Oupicky, D.; Barrett, L. B.; Spice, R.; Kendall, M.; Berry, M.; Preece, J. A.; Logan, A.; Seymour, L. W. A versatile reducible polycation-based system for efficient delivery of a broad range of nucleic acids. Nucleic Acids Res. 2005, 33, e86. (24) Nasanit, R.; Iqbal, P.; Soliman, M.; Spencer, N.; Allen, S.; Davies, M. C.; Briggs, S. S.; Seymour, L. W.; Preece, J. A.; Alexander, C. Combination dual responsive polypeptide vectors for enhanced gene delivery. Mol. BioSyst. 2008, 4, 741−745. (25) Mason, A. J.; Leborgne, C.; Mouley, G.; Martinez, A.; Danos, O.; Bechinger, B.; Kichler, A. Optimising histidine rich peptides for efficient DNA delivery in the presence of serum. J. Controlled Release 2007, 118, 95−104. (26) Lo, S. L.; Wang, S. An endosomolytic Tat peptide produced by incorporation of histidine and cysteine residues as a nonviral vector for DNA transfection. Biomaterials 2008, 29, 2408−2414. (27) Won, Y. W.; Kim, H. A.; Lee, M.; Kim, Y. H. Reducible Poly (oligo-D-arginine) for enhanced gene expression in mouse lung by intratracheal injection. Mol. Ther. 2010, 18, 734−742. (28) Tanaka, K.; Kanazawa, T.; Ogawa, T.; Suda, Y.; Takashima, Y.; Fukuda, T.; Okada, H. A novel, bio-reducible gene vector containing arginine and histidine enhances gene transfection and expression of plasmid DNA. Chem. Pharm. Bull. 2011, 59, 202−207. (29) Tanaka, K.; Kanazawa, T.; Ogawa, T.; Suda, Y.; Takashima, Y.; Fukuda, T.; Okada, H. Disulfide crosslinked stearoyl carrier peptides containing arginine and histidine enhance siRNA uptake and gene silencing. Int. J. Pharm. 2010, 398, 219−224. (30) Won, Y. W.; Yoon, S. M.; Lee, K. M.; Kim, Y. H. Poly (oligo-Darginine) with internal disulfide linkages as a cytoplasm sensitive carrier for siRNA delivery. Mol. Ther. 2011, 19, 372−380. (31) Mann, A.; Thakur, G.; Shukla, V.; Singh, A. K.; Khanduri, R.; Naik, R.; Jiand, Y.; Kalara, N.; Dwarakanath, B. S.; Langel, U.; Ganguli, M. Differences in DNA condensation and release by lysine and arginine homopeptides govern their DNA delivery efficiencies. Mol. Pharmaceutics 2011, 8, 1729−1741. (32) Futaki, S. Arginine-rich peptides: potential for intracellular delivery of macromolecules and the mystery of the translocation mechanisms. Int. J. Pharm. 2002, 245, 1−7. (33) van Rossenberg, S. M.; van Keulen, A. C.; Drijfhout, J. W.; Vasto, S.; Koerten, H. K.; Spies, F.; van’t Noordende, J. M.; van Berkel, T. J.; Biessen, E. A. Stable polyplexes based on arginine-containing oligopeptides for in vivo gene delivery. Gene Ther. 2004, 11, 357−464. (34) Kim, T. I.; Lee, M.; Kim, S. W. A guanidinylated bioreducible polymer with high nuclear localization ability for gene delivery systems. Biomaterials 2010, 31, 1798−1804.

variation on transfection efficiency and condensation−release behavior of R9H7 polyplexes. Supplementary Figure 6, showing the effect of serum on transfection efficiency. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 91-011-27666156/157, 91-011-27667602, 91-01127667439. Fax: 91-011-27667471. E-mail: [email protected]. in; [email protected]. Author Contributions ‡

Equal contribution.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the project entitled “Nanomaterials and nanodevices for application in health and disease” (NWP35); Council of Scientific and Industrial Research (CSIR), India. Fellowships from CSIR, India, to A.M., V.S. and R.K. are acknowledged.

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