Preparation and in Vitro Evaluation of Novel Lipopeptide Transfection

In an attempt to construct more efficient nonviral gene delivery vectors, we have designed a series of novel lipopeptide transfection agents, consisti...
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Bioconjugate Chem. 2008, 19, 940–950

Preparation and in Vitro Evaluation of Novel Lipopeptide Transfection Agents for Efficient Gene Delivery Tarwadi,† Jalal A. Jazayeri,† Richard J. Prankerd,‡ and Colin W. Pouton*,† Department of Pharmaceutical Biology and Department of Pharmaceutics, Victorian College of Pharmacy, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia. Received December 14, 2007; Revised Manuscript Received February 1, 2008

Gene therapy by delivery of nonviral expression vectors is highly desirable, due to their safety, stability, and suitability for production as bulk pharmaceuticals. However, low transfection efficiency remains a limiting factor in application on nonviral gene delivery. Despite recent advances in the field, there are still major obstacles to overcome. In an attempt to construct more efficient nonviral gene delivery vectors, we have designed a series of novel lipopeptide transfection agents, consisting of an alkyl chain, one cysteine, 1 to 4 histidine and 1 to 3 lysine residues. The lipopeptides were designed to facilitate dimerization (by way of the cysteine residues), DNA binding at neutral pH (making use of charged lysine residues), and endosomal escape (by way of weakly basic histidine residues). DNA/lipopeptide complexes were evaluated for their biophysical properties and transfection efficiencies. The number and identity of amino acids incorporated in the lipopeptide construct affected their DNA/lipopeptide complex forming capacity. As the number of lysine residues in the lipopeptide increased, the DNA complexes formed became more stable, had higher zeta potential (particle surface charge), and produced smaller mean particle sizes (typically 110 nm at a charge ratio of 5.0 and 240 nm at a charge ratio of 1.0). The effect of inclusion of histidines in the lipopeptide moiety had the opposite effect on complex formation to lysine, but was necessary for high transfection efficiency. In vitro transfection studies in COS-7 cells revealed that the efficiency of gene delivery of the luciferase encoding plasmid, pCMV-Luc, mediated by all the lipopeptides, was much higher than poly(Llysine) (PLL), which has no endosomal escape system, and in two cases was slightly higher than that of branched polyethylenimine (PEI). Lipopeptides with at least two lysine residues and at least one histidine residue produced spontaneous transfection complexes with plasmid DNA, indicating that endosomal escape was achieved by incorporation of histidine residues. These low molecular weight peptides can be readily synthesized and purified and offer new insights into the mechanism of action of transfection complexes.

INTRODUCTION There are several strategies for mediating gene delivery to the nucleus of eukaryotic cells. One approach has been direct injection of naked DNA into a tissue. This has been attractive due to simplicity and lack of toxicity. However, the size and hydrophilic nature of DNA prevents it from being taken up by cells efficiently. Approximately 106 plasmid DNA are needed for a single cell transfection in vitro with less than 100 reaching the nucleus (1). This is due to several physical, chemical, and metabolic barriers in the cell interior, with the final obstacle being the nuclear membrane which, in the absence of cell division, is a major impediment to gene delivery. The DNA delivery system needs to prevent degradation and facilitate intracellular transport on its journey to the nucleus. To achieve this target, several strategies are being exploited by researchers in the field of gene therapy. These include (i) physical methods, using mechanical or electrical forces, such as particle bombardment, gene guns, electroporation, microinjection, ultrasound, and hydrodynamic; (ii) chemical means (condensing DNA using cationic lipids or cationic polymers); and (iii) viral vectors (e.g., using adenoviruses). Viral vectors, although comparatively efficient, are considered to have safety * Author for correspondence: Professor Colin W. Pouton, Department of Pharmaceutical Biology, Victorian College of Pharmacy, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia. E-mail: [email protected], Tel: +613-99039562, Fax: +613-99039638. † Department of Pharmaceutical Biology. ‡ Department of Pharmaceutics.

problems associated with recombination events. Nonviral delivery vectors, on the other hand, are relatively inefficient in their current form and have not been successful for in vivo gene therapy. Nevertheless, they are attractive alternatives to viral and physical methods, because of their relative safety (for recent reviews, see refs 2–6). Systematic efforts will be required to improve the efficiency of nonviral delivery before they can be used clinically. Among the nonviral vehicles, cationic lipids have been explored most extensively, and many have been commercialized as in vitro transfection agents (7–10). Felgner and co-workers were the first to report a highly efficient lipid-mediated DNAtransfection agent using a synthetic cationic lipid, N-(1-(2,3dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (11). They found that small unilamellar liposomes containing DOTMA interacted spontaneously with DNA to form lipid-DNA complexes. They also reported that, depending on the cell line used, DOTMA was 5- to 100-fold more efficient, compared to either the calcium phosphate or DEAE dextran transfection methods. In designing a cationic lipid as a transfection agent, they showed that the alkyl chain length and the number of double bonds could either increase or decrease the transfection efficiency (C14:0 > C18:1 > C16:0 > C18:0) (7). Using a different set of cationic lipids, Wheeler et al. formulated a liposome composing of GAP-DLRIE; (()-N-(3aminopropyl)-N,N-dimethyl-2,3-bis-(dodecyloxy)-1-propanaminium bromide, and DOPE (dioleoylphosphatidyl-ethanolamine). This formulation enhanced chloramphenicol acetyl-transferase (CAT) expression in vivo by more than 100-fold (observed in mouse lung) (12). Recently, Slimani and co-workers reported

10.1021/bc700463q CCC: $40.75  2008 American Chemical Society Published on Web 03/12/2008

Novel Lipopeptide Transfection Agents

a ligand-bearing lipopeptide specifically targeting neurophilia fen-1 (NRPAL1) receptor, which is overexpressed by human breast cell lines (13). The lipopeptide was based on a palmitoyl fatty chain (C16) linked to a sequence of seven amino acids (ATWLPPR). Upon transfection, they showed that the lipopeptide delivery of either a β-galactosidase reporter gene or a green fluorescence protein was selectively enhanced in NRPAL1 positive cells as compared to NRPAL1 negative cells. The synthetic polycation polyethylenimine (PEI) has also been used widely in transfection studies, either alone (14) or by bonding to adenovirus particles (15) or to other viral protein and targeting devices (16, 17). However, although PEI and its derivatives have a prominent position in gene delivery research, due to their ability to facilitate endosomal escape which results in high transfection efficiency in vitro, the toxicity of PEI remains the drawback, especially for in vivo gene delivery applications (14, 18). Low-molecular-weight cationic peptide-based nonviral vectors have considerable potential but are probably the least explored, whereas cationic polymers such as polyethylenimine (PEI) or poly(L-lysine) (PLL) have been investigated regularly (19–25). Recently, there have been several publications on peptide-based structures for delivery of DNA to mammalian cells (8, 26–29). Chen et al. reported that, in combination with cationic liposomes such as DOTAP, Lipofectin, Lipofectamine, and DOSPER branched copolymers of histidine and lysine were able to condense and enhance transfection efficiency in MDA-MB-435 cells (30). In addition, histidine-rich amphiphatic peptides were reported to transfect several cell lines with efficiencies comparable to those of DOTAP and PEI (28). Meanwhile, Lentz and co-workers reported the use of highly branched histidine-lysine peptides as carriers for small interfering RNA molecules (29). Arginine-rich peptides have also been exploited in nonviral gene transfer (31, 32). Arginine-rich peptides can facilitate the cellular uptake of macromolecules such as proteins, liposomes, and iron nanoparticles. They have also been shown to enhance DNA delivery by making use of protein transduction sequences, including hepta-arginine and TAT47–57 (31). A group led by Rice has been exploring the application of copolymers incorporating peptide sequences of cysteine, lysine, histidine, and tryptophan residues to mediate nonviral gene delivery (33–35). McKenzie et al. reported that a minimal repeating lysine chain of 18 residues, followed by tryptophan and an alkylated cysteine residue (AlKCMK18, MW 2672 Da), formed small particles with DNA (∼80 nm) and mediated efficient in vitro gene transfer to a level higher than that of PLL (MW of 1–4 kDa) (34). This group developed peptide-mediated gene delivery agents by inserting multiple cysteine residues into a short (dp 20) synthetic peptide (33). They found that the stability of cross-linked peptide DNA condensates increased in proportion to the number of cysteines incorporated into the peptide. They suggested that disulfide bond formation led to a decrease in particle size, relative to control peptide DNA condensates, and prevented dissociation of peptide DNA condensates (33). In addition, they demonstrated that substitution of His for Lys resulted in an optimal peptide of Cys-His-(Lys)6His-Cys, which provided a buffering condition capable of enhancing in vitro gene expression in the absence of chloroquine (33). Despite recent advances in the design of nonviral gene delivery systems, transfection agents based on nonviral reagents are still very inefficient, particularly for in vivo gene delivery applications, where typically a very small proportion of cells are transfected. In this study, a series of lipopeptide transfection agents were designed and constructed. In addition to an investigation of transfection efficiency, these complexes were also evaluated in terms of their physical properties, since

Bioconjugate Chem., Vol. 19, No. 4, 2008 941 Table 1. Lipopeptide Structures and Their State of Ionization at pH 7.4 lipopeptidea

molecular weight (Da)b

chargec

purity %d

LauCKH2-NH2 LauCK2H-NH2 PalCKH2-NH2 PalCK2H2-NH2 PalCK2H3-NH2 PalCK2H4-NH2 PalCK2H5-NH2 PalCK3H2-NH2 PalCK3H3-NH2

761 696 761 889 1026 1163 1300 1017 1154

1 2 1 2 2 2 2 3 3

>96 >97 >95 >95 >95 >95 >95 >97 >95

a Lau: Lauryl; Pal: Palmitoyl. b Determined by mass spectrometry. Determined as per number of protons. d The purity of the compounds was measured by HPLC using 218 nm UV absorption; SH was detected by Ellman’s test.

c

complex size and charge are known to influence processes involved in gene delivery. A better understanding of the physical properties of DNA complexes, in particular, the factors which promote DNA condensation, cellular uptake, and endosomal escape, will help design more efficient gene delivery vectors.

EXPERIMENTAL PROCEDURES Materials. The lipopeptides (Table 1) were synthesized by Auspep Pty. Ltd. (Parkville, Victoria, Australia). The purities of the compounds were examined by HPLC (Waters, USA) with nonspecific short-wavelength UV detection and found to be >95%. Their molecular weights were confirmed by mass spectrometry. The plasmid pCMV-Luc was a gift from Dr. David Melroy (University of Bath, UK) and the plasmid pCMVβ-gal stock solution (1 mg/mL in water) was obtained from Clontech (NSW, Australia). Lipofectamine was from Invitrogen (Melbourne, VIC, Australia); PLL (average Mw 15–30 kDa), branched PEI (average Mw 750 kDa), decamethonium (dication C10), dodecyltrimethylammonium bromide (DDTAB), and hexadecyltrimethylammonium bromide (HDTAB) were from SigmaAldrich (Sydney, Australia). Dulbecco’s Modified Eagle’s Medium (DMEM) was from Invitrogen (Melbourne, VIC, Australia). All tissue culture flasks, including black polystyrene 96-well plates, were obtained from Corning Costar (Melbourne, VIC, Australia). All other chemicals were commercially obtained and were of analytical grade. Design and Construction of Lipopeptides. The basic structure comprises an N-terminal alkylamide, one cysteine, 1 to 4 histidine and 1 to 3 lysine residues. The addition of each lysine provided a unit positive charge on the lipopeptide at neutral pH, which was able to interact with the negatively charged sugar–phosphate backbone of the DNA molecule. Histidine was included because its side hain is weakly basic and predominantly un-ionized at neutral pH, but is more highly protonated in the weakly acidic environment of the endosome. It was hypothesized that this phenomenon would facilitate release of the DNA-lipopeptide complex from the endosome. The number of lysine and histidine residues included in the lipopeptide was varied to explore the effect on transfection efficiency. The inclusion of an alkyl amide chain, either lauryl (C12) or palmitoyl (C16), in the lipopeptide was intended to promote hydrophobic interactions to stabilize the lipopeptide-plasmid complex, subsequent to an initial ion interaction between primary amine and phosphate groups. To inform the choice of alkyl chain length, a range of dicationic molecules were investigated using the ethidium bromide exclusion assay (Table 2). The thiol-bearing cysteine residue was included with the intention of facilitating dimerization by disulfide formation on the DNA molecular template. Plasmid DNA Isolation. The plasmid pCMV-Luc, a luciferase mammalian expression vector, was cultivated in E. coli,

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Table 2. Structure of Molecules Used in DNA Condensation assays

strain DH5-R, and isolated using a Qiagen Maxiprep kit (Qiagen Pty. Ltd., Clifton Hill, Victoria, Australia). The purity of each plasmid was checked electrophoretically on a 1% agarose gel. Their concentrations were determined from UV absorbance at 260 and 280 nm. Purified plasmids were resuspended in Milli-Q water (MQW) and frozen (-20 °C) until use. Determination of Charge and N/P Ratios. The charge ratio (+/-) of DNA complexes refers to the number of positively charged (protonated) nitrogens provided by the transfection agent per negatively charged nucleotide unit. An average mass of 330 Da per nucleotide was used for the calculations. For example, to obtain a theoretical charge ratio of 1:1 between PalCK2H2 (889 Da, 2NH+/molecule) and DNA, 1 µg of DNA (3 nmol of nucleotide) was mixed with 1.3 µg of PalCK2H2 (1.5 nmol), since every molecule of PalCK2H2 generates 2 NH+ groups. For the cationic polymer PEI, rather than using charge ratio, complexes were described in terms of the total nitrogen/ phosphate (N/P) ratio. DNA Condensation Assay. The ability of the lipopeptide constructs to condense DNA was evaluated using the ethidium bromide (EtBr) exclusion assay, as described by Murphy et al. (36). This assay was carried out in 96-well black plates; the fluorescence intensities were measured using a FluoStar OPTIMA plate reader (FluoStar OPTIMA, BMG Laboratory Technology, Sydney, NSW, Australia). A sample containing 5 µg plasmid DNA and an excess of EtBr (20 µL; 100 µg/mL) was used to calibrate the spectrofluorometer to 100% fluorescence intensity (λex ) 520 nm; λem ) 610 nm). For the assays; 50 µL of 60 mM Tris HCl buffer (pH 7.4) was added to each well containing plasmid DNA. A series of charge ratios from 0 to 10 (or N/P ratios for PEI) of the DNA complexes were prepared. Milli-Q water was added to a total volume of 230 µL

per well. The samples were left to stabilize at ambient temperature for 3 min after which 20 µL of EtBr solution was added. The 96-well plate was shaken orbitally for 30 s and the fluorescence intensity measured. Gel Mobility Shift Assay. The formation of the DNAtransfection agent complex was studied using a gel mobility shift assay. A series of charge ratios from 0 to 5 (or N/P ratios for PEI) of the complexes were prepared in HEPES glucose buffer pH 7.4 (15 mM HEPES and 5.13% w/v glucose). Complexes of DNA (20 µg/mL) and transfection agent were incubated for 30 min at 37 °C. Subsequently, the samples (15 µL) were mixed with 3 µL of loading buffer and analyzed by 1% agarose gel electrophoresis. DNA bands were visualized by UV transillumination. DNA-Lipopeptide Stability Assay. The susceptibility of the condensed DNA to enzymatic degradation was assessed by exposing the complex to nucleases (Turbo DNase, Ambion, Scoresby, VIC, Australia). The plasmid DNA sample (20 µg/ mL) and the appropriate lipopeptide were diluted separately in HGB1pH 7.4 in a series of charge ratios from 0 to 5. The samples were then mixed and incubated in the presence of DNaseI (2U/µg DNA) on ice (4 °C) for 15 min prior to 1% agarose gel electrophoresis. DNA bands were visualized by UV transillumination. 1 Abbreviations: HGB, HEPES glucose buffer; PBS, phosphate buffered saline, PEI, polyethylenimine; PLL, poly(L-lysine); EtBr, ethidium bromide; DOPE, (dioleoyl phosphatidyl-ethanolamine); DOTMA, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride.

Novel Lipopeptide Transfection Agents

Particle Sizing and Zeta Potential. The mean particle size and zeta potential (ζ) of each DNA-lipopeptide complex was determined with a Zetasizer Nano Series instrument (Malvern Instruments, Worcestershire, UK). Calibration of particle sizes was carried out with 60 nm ( 2.7 nm NIST/Nanosphere (Duke Scientific Corp. Palo Alto, CA, USA) standard polystyrene spheres. For calibration of zeta potential, the -50 mV ( 5 mV zeta potential standard was used. DNA-lipopeptide complexes were prepared in HEPES glucose buffer pH 7.4. The mean particle sizes were measured at 25 °C using 1.5 mL disposable cuvettes. Zeta potentials were determined at 25 °C using the folded capillary (Smoluchowski) cell (Malvern Instruments, Worcestershire, UK). To study the physical stability of each DNA-lipopeptide complex, 40 µg of DNA (pCMV-Luc) was combined in a cuvette with each of PalCK2H2, PalCK2H3, PalCK3H2, or PalCK3H3 (at a charge ratio of 1.5), in a total of 500 µL of HEPES glucose buffer, pH 7.4. The mean particle size distributions were measured at 0.05, 0.5, 1, 2, 8, 30, 60, and 120 h after complex formation. The effect of DNA concentration on particle aggregation was studied at a fixed charge ratio of 1.5; DNA (2.5, 5, 10, 15, 20, or 40 µg) was diluted with 250 µL HGB pH 7.4 and then added dropwise into 250 µL HGB pH 7.4 containing the appropriate mass of PalCK3H2, PalCK3H3, PEI, or Lipofectamine to obtain DNA at a final concentration of 7.6, 15.2, 30.4, 45.6, 60.8, or 121.6 nM, respectively. The effect of increasing charge ratio on particle size and zeta potential was also studied. This was achieved by diluting 5 µg DNA in 250 µL of HGB pH 7.4 dropwise into 250 µL HGB pH 7.4 in a microtube containing the appropriate mass of PalCK2H, PalCK2H2, PalCK3H2, or PalCK3H3 to obtain charge ratios of 0.5, 0.75, 1.0, 2.0, 3.0, 4.0, or 5.0, respectively. Preparation of Lipopeptide/Plasmid DNA Complexes for Transfection. The plasmid pCMV-Luc (2 µg) was diluted in 12.5 µL HGB pH 7.4 and added to the lipopeptide solution (diluted in the 12.5 µL of the same buffer) in dropwise manner. Lipofectamine, PLL (MW 70–150 kDa) and branched PEI (MW 750 kDa) were used as positive controls. In Vitro Transfection. COS-7 cells were cultured in DMEM media supplemented with 10% FCS, 100 units/mL penicillin, and 100 µg/mL streptomycin in a humidified incubator with 5% CO2 at 37 °C. The day before transfection, cells were seeded at 5 × 104 cells/well in the 24-well plates. After reaching a confluency of ∼60–70%, the cells were washed twice with PBS, and the medium replaced with Opti-MEM prior to addition of the DNA/lipopeptide complexes. After 24 h of incubation in Opti-MEM, cells were washed twice with PBS, then harvested by adding 115 µL/well of Reporter Lysis Buffer (Promega, NSW, Australia) and incubated for 15 min at room temperature. The cells were then scraped, collected, and centrifuged, and the supernatants were used for protein and luciferase assays. Luciferase and Protein assays. After harvest, cells were centrifuged at 13 000 g for 2 min at 4 °C. The supernatant (50 µL) was used to measure luciferase activity using a luciferase detection kit (Promega, Annandale, NSW, Australia). Quantilum Recombinant Luciferase (QRL) (Promega, Annandale, NSW, Australia) was used as a standard for luciferase assays. Total protein concentrations were measured using the Bradford protein assay (Sigma Aldrich, Sydney, NSW, Australia). Bovine serum albumin (BSA; Sigma-Aldrich Sydney, NSW, Australia) was used as a standard for protein assays.

RESULTS DNA Condensation Studies. The results of DNA condensation experiments showed that the alkyltrimethylammonium bromide with a C16 alkyl chain (HDTAB) condensed DNA as efficiently as PLL (Figures 1, 2A), whereas the C12 compound

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Figure 1. Representative structures of the lipopeptide-based transfection agents and their protonation at physiological pH 7.4: (A) Lauryl (C12)-CK2H2 and (B) Palmitoyl (C-16)-CK3H3.

(DDTAB) and the decamethonium dication did not. Similarly, a lipopeptide bearing a palmitoyl (C16) alkyl chain condensed DNA more efficiently than the corresponding lauryl (C12) equivalent (Figure 2B). Lipopeptides with a single lysine residue, such as PalCKH2, were weak condensers of DNA when compared to lipopeptides with two or three lysine residues (Figure 2C). The extent of DNA condensation by palmitoylated compounds with two lysine residues was a function of the number of histidine residues. At each charge ratio, the extent of ethidium exclusion decreased with increasing histidinyl chain length over the range 2–5 (Figure 2D). Gel Mobility Shift Assay. Condensation of DNA with PalCK2H2 and PalCK2H5 caused retardation of DNA migration at charge ratios above 1 (Figure 3A,B). At a charge ratio of 0.5, migration was substantially retarded, though some DNA appeared to migrate into the gel from the PalCK2H5 complex, indicating less efficient condensation. Figure 3C shows that an N/P ratio of at least 1.5 was required to effect gel retardation by PEI. At charge ratios of 5.0 or above, the DNA-PalCK2H2 and DNA-PalCK2H5 complexes could not be detected on the agarose gel (Figure 3A,B). Similarly, DNA-PEI complexes, at an N/P ratio of 5 or above, were not identified on the gel (Figure 3C). This can be explained by the previous observation that more complete condensation at higher charge ratio prevents the binding of ethidium to the complex, as exemplified by the results of the ethidium exclusion experiments (Figure 2). The results of the gel-shift experiments suggest that immobilization of the complex occurred before the DNA was fully condensed. Complex Stability in the Presence of DNase. The stability of lipopeptide-DNA complexes was evaluated in the presence of DNase to model the protection to enzymatic degradation resulting from condensation. Figure 4 shows a representative experiment using PalCK2H2 complexes. The results show that the majority of the mass of DNA in free solution was degraded after 30 min of incubation in the presence of DNase. At charge ratios greater than 1.0, the lipopeptide PalCK2H2 fully protected DNA from DNase degradation. The protection from nuclease degradation at this low charge ratio was superior to the protection given by cationic polymers and liposomes. This level of DNA protection by PalCK2H2 was also more pronounced than that offered by the cationic amphiphile guanidinocysteine N-decylamide (C10-CG+). The latter compound has been reported to protect DNA from serum degradation at a charge ratio of 3.0 (37), but as indicated by our ethidium exclusion studies (Figure 2), DNA condensation was less efficient than that achieved with PalCK2H2.

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Figure 2. Condensation studies of DNA-transfection agents using ethidium bromide exclusion assay (measured at λex ) 520 nm and λem ) 610 nm; FluoSTAR plate reader). Data are presented as mean ( SD (n ) 3). (A) Comparison of alkyl chains, as the hydrophobic anchors, on ethidium exclusion during DNA condensation. (B) Effect of lauryl (C-12) and palmitoyl (C-16) chains on ethidium exclusion. (C) Effect of the number of lysine residues on ethidium exclusion by lipopeptides containing two histidine residues. (D) Effect of the number of histidine residues on the ability of lipopeptides with two lysine residues to condense DNA molecules.

Figure 3. Gel mobility shift assays of DNA complexes formed by (A) PalCK2H2, (B) PalCK2H5, and (C) PEI. At a charge ratio of 1.0 or 1.5, both PalCK2H2 and PalCK2H5 effectively condensed the plasmid DNA to an extent analogous with condensation by PEI.

Physical stability of DNA-Lipopeptide Complexes. Particle size was determined over a 120 h period after formation of the complexes in HGB pH 7.4. This was of practical significance, particularly with regard to transfection studies, to ensure that the complexes did not aggregate during the period between formation and use, a common problem with polyelectrolyte complexes. Complexes formed at a charge ratio of 1.5, using 40 µg of DNA (243 nM) and the lipopeptides PalCK2H2, PalCK2H3, PalCK3H2, and PalCK3H3 in 500 µL HGB pH 7.4. These were completely stable for 1 h and had particle diameters in the range 250–300 nm. After longer time periods, some aggregation was evident with PalCK3H2 complexes (forming aggregates approximately twice the original diameter), but the other complexes remained unchanged for 60 h (Figure 5A). More aggregation was observed after 120 h, but PalCK3H3

complexes remained unchanged. A major advantage of the lipopeptides introduced in this study is their ability to form 200–300 nm complexes at relatively high DNA concentrations. Unlike complexes formed by Lipofectamine or PEI, the diameter of complexes of DNA with PalCK3H2 or PalCK3H3 was independent of DNA concentration over the range 10–120 µm (Figure 5B). Mean Particle Size, Polydispersity Index, and Zeta Potential of Complexes. The mean particle size and zeta potential of complexes formed by DNA (15.2 nM) and lipopeptides (in 500 µL HGB pH 7.4) were examined as a function of increasing charge ratio (Figure 6A,B). As the charge ratios increased from 0.5 to 5.0, the mean particle sizes of the complexes formed with PalCK2H2, PalCK3H2, and PalCK3H3 decreased slightly (Figure 6A), from 230-260 nm down to

Novel Lipopeptide Transfection Agents

Figure 4. Stability of DNA-PalCK2H2 complexes against enzymatic degradation by DNase (Turbo DNase). In the absence of PalCK2H2 (a charge ratio ) 0), the plasmid DNA was almost completely degraded. At a charge ratio of >1.0, the majority of the plasmid DNA remained intact after 30 min incubation with DNase at 4 °C.

110–150 nm at a charge ratio of 5.0 (Table 3). Complexes formed by PalCKH2 were larger (Figure 2C), which may reflect a lower degree of DNA condensation by this lipopeptide, a hypothesis which is supported by the ethidium exclusion data. When PalCKH2 was used to condense DNA the extent of ethidium exclusion was considerably lower than exclusion caused by K2 or K3 lipopeptides at the same charge ratio. The zeta potential of complexes was a function of charge ratio and was similar for complexes formed by all lipopeptides. The zeta potential was negative at charge ratios below 1.0 and approached zero at a charge ratio of unity. When the charge

Bioconjugate Chem., Vol. 19, No. 4, 2008 945

ratio was increased above 1.0, the zeta potential became positive, reaching a maximum value of approximately +20 mV for charge ratios between 2.0 and 5.0 (Figure 6B). Figure 7 shows typical values of the PDI, mean particle size, and zeta potential for 40 µg DNA complexed with (A) lipopeptide PalCK2H2, (B) PEI, and (C) Lipofectamine in HGB pH 7.4, at a charge ratio of 1.5 (N/P of 9.0 for PEI). For complexes of DNA-transfection agents shown, the PDI for the DNA-Lipofectamine complex was the highest (PDI ) 1.0), indicating a wide size distribution. The Nanosizer data suggested that PEI and Lipofectamine complexes may have been multiphasic as well as polydisperse. DNA complexes formed by PalCK2H2 and other lipopeptides typically had more consistent particle size, narrower size distributions, and more precise zeta potentials than complexes formed with PEI or Lipofectamine. For lipopeptides with the same number of histidine residues (2 or 3 histidines), we observed that, as the number of lysine residues in the lipopeptide increased, their mean particle sizes and polydispersities decreased (Tables 4 and 5). However, zeta potential was increased as the number of lysines was increased. In Vitro Transfection Studies. The transfection efficiency of PalCK2H2 in Cos7 cells was found to be 80-fold greater than LauCK2H2 (p < 0.001) (Figure 8A), which correlated with its superior properties with respect to DNA condensation, complex stability, and resistance to nuclease digestion. PalCK3H2 was twice as effective as PalCK3H3 (Figure 8B). When the lipopeptides containing two histidine residues were compared, there were clear differences. The compound with one lysine residue was significantly less active than those with two or three lysine residues, which had similar activity (Figure 8C). For lipopeptides with three histidine residues, the K3 lipopeptide was over twice as active as the K2 lipopeptide (Figure 8D). When the best lipopeptides were compared with PEI and Lipofectamine,

Figure 5. Physical stability of DNA complexes at a charge ratio of 1.5 formed in HEPES glucose buffer (HGB pH 7.4) at ambient temerapture: (A) Effect of storage time on mean particle size of complexes formed by four lipopeptides; (B) Effect of DNA concentration on mean particle size of complexes formed by lipopeptides, PEI, and Lipofectamine. Data are presented as mean ( SD (n ) 3).

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Figure 6. Effect of charge ratio on (A) mean particle size and (B) zeta potential of DNA-lipopeptide complexes formed in HGB at pH 7.4. Data are presented as mean ( SD (n ) 3). Table 3. Effect of Charge Ratio on Mean Particle Size of DNA-Lipopeptide Complexes charge ratio particle size lipopeptide

0.5

5

PalCK2H2 PalCK3H2 PalCK3H3

229 ( 23 nm 256 ( 22 nm 240 ( 27 nm

112 ( 5 nm 146 ( 50 nm 117 ( 3 nm

activity was comparable, although generally Lipofectamine was a marginally better transfection agent in vitro (Figure 8E).

DISCUSSION This project investigated the effect of lipopeptide composition (alkyl chain length, number of lysines, and histidines) on DNA complex formation and transfection efficiency, and compared these novel lipopeptides with PEI and Lipofectamine. DNA condensation studies indicated that the addition of an alkyl chain to the lipopeptide molecule promoted complexation, and that the palmitoyl peptides were more effective than the corresponding lauryl lipopeptides. We hypothesize that the presence of the alkyl chain promotes complex formation resulting from the increased entropy derived by loss of water structure around the alkyl chain, commonly called the hydrophobic effect. The longer hydrophobic palmitoyl (C16) chain would be expected to promote a stronger hydrophobic interaction, and this results in more compact and stable particles that have improved transfection efficiency. The lysine residues on the lipopeptides were included to provide a protonated amine group at neutral pH that effected condensation of plasmid DNA. The amino acid side chain of lysine has a pKa ) 10.80, and is therefore 99.96% ionized at pH 7.4, enabling ionic interaction with the negatively charged sugar–phosphate backbone of DNA. DNA condensation with

lipopeptides such as PalCK2H2 took place efficiently, and zeta potential data suggested that the complex formed included all lysine residues in a one-to-one stoichiometric relationship with the nucleotides. At least two lysine residues were required for efficient condensation and good transfection efficiency. The advantage of including a third lysine residue was limited. Histidine residues were included to provide a source of weakly basic groups. It was hypothesized that these residues would become protonated in the endosome, thereby promoting endosomal escape in a manner analogous to the mechanism by which PEI is thought to achieve transfection. This approach led to efficient DNA condensation and transfection by the compound PalCK2H2. Additional histidine residues might be expected to improve the efficiency of endosomal escape, but this strategy resulted in less efficient condensation, when compared to PalCK2H2 (Figure 2D). There was a clear rank order, H5 < H4 < H3 < H2, in condensation efficiency as determined by ethidium exclusion, for palmitoylated lipopeptides containing two lysine residues. The lipopeptide PalCK2H4, for example, with four histidine residues per lipopeptide, resulted in only 10% ethidium exclusion at a charge ratio of 2.0. In contrast the DNA-ethidium fluorescence intensities for PalCK2H3 and PalCK2H2 complexes, at the same charge ratio, were reduced to 40% and 55% of the initial value, respectively. We suggest that the reduction in efficiency of DNA condensation with increase in histidine chain length may be explained by steric hindrance or a decrease in the entropic driving force for complexation. Cysteine was included in the design if the lipopeptides to promote complexation by formation of disulfide bonds in a manner analogous to the compounds described previously by Behr and colleagues (36). We hypothesized that, in the presence of the DNA template, the Cys-lipopeptide is likely to dimerize, leading to in situ formation of a transfection agent with a double alkyl chain. This is the main advantage of including cysteine in the lipopeptide structure, since most single alkyl chain

Novel Lipopeptide Transfection Agents

Bioconjugate Chem., Vol. 19, No. 4, 2008 947

Figure 7. Typical size distributions and zeta potentials of DNA complexes formed at a charge ratio of 1.5 in HGB pH 7.4: with PalCK2H2 (A-1 and A-2), PEI (B-1 and B-2), and Lipofectamine (C-1 and C-2). The particle size data are expressed as a histogram showing the size distribution and as the cumulative % undersize, shown as the solid line plotted against particle size. Table 4. Effect of Lipopeptide Structure on Mean Particle Sizes, Zeta Potential, and Polydispersity of DNA-Lipopeptide Complexes at a Charge Ratio of 1.5 in HGB pH 7.4a

lipopeptide

particle size (nm) (mean ( SD)

PDI (mean ( SD)

zeta potential (mV) (mean ( SD)

PalCKH2 PalCK2H2 PalCK2H3 PalCK3H3

688 ( 28 240 ( 4.1 254 ( 6.2 247 ( 3.3

0.77 ( 0.05 0.31 ( 0.03 0.35 ( 0.04 0.27 ( 0.001

3.6 ( 1.7 13.2 ( 0.7 5.4 ( 0.3 6.3 ( 0.5

a Complexes were formed by diluting 40 µg DNA (∼243 nM); data are represented as mean ( SD of triplicate measurements (n ) 3).

Table 5. Effect of Coupling Histidine to PalCK2 Lipopeptides on Mean Particle Size, Zeta Potential, and Polydispersity of DNA-Lipopeptide Complexes at a Charge Ratio of 1.5 in HGB pH 7.4a

lipopeptide

particle size (nm) mean ( SD

PDI mean ( SD

zeta potential (mV) mean ( SD

PalCK2H2 PalCK2H3 PalCK2H4 PalCK2H5

240 ( 4.1 254 ( 6.2 724 ( 317.0 Sedimentationb

0.31 ( 0.03 0.35 ( 0.04 0.67 ( 0.18 -

13.17 ( 0.74 5.44 ( 0.28 1.53 ( 0. 25 -

a Complexes were formed by diluting 40 µg DNA (∼243 nM); data are presented as mean ( SD of triplicate measurements (n ) 3). b The complex formed large aggregates that sedimented rapidly, therefore their mean particle sizes and other particle properties could not be determined with the Zetasizer Nano ZS.

transfection agents are (a) less efficient at condensing DNA and (b) generally regarded as toxic to cells (38). We have not established yet whether dimerization is indeed occurring after complexation, and this is the subject of further investigation in our laboratories.

In our studies, the mean diameters of the DNA-lipopeptide complexes were between 180 and 275 nm and 250–400 nm for DNA-PalCK3H3 and DNA-PalCK3H2, respectively. Particle mean size was approximately independent of DNA concentration, up to 120 nM. Typically, DNA complexes aggregate at high concentration. For example, Duguid et al. have reported that the diameter of DNA complexes formed at a charge ratio of 3.0 with synthetic cationic peptide analogues (YKAKnWK) and the amphipathic peptide (GLFEALLELLESLWELLLEA) increased as DNA concentration increased (39). The lipopeptides described here are unlikely to bind more than one primary condensed DNA particle at once. This reduces the opportunity for particle cross-linking, which we suggest may explain the particle growth at high concentrations observed in this study with DNA-PEI or DNA-Lipofectamine complexes. The mean particle sizes of the DNA-PEI complexes were