Peptide-Based Intracellular Shuttle Able To Facilitate Gene Transfer

Bioconjugate Chem. 1999, 10, 745−754

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Peptide-Based Intracellular Shuttle Able To Facilitate Gene Transfer in Mammalian Cells Devender Singh,‡ Stuart K. Bisland, Kim Kawamura, and Jean Garie´py† Division of Molecular and Structural Biology, Department of Medical Biophysics, University of Toronto and the Ontario Cancer Institute, Princess Margaret Hospital, 610 University Avenue, Toronto, Canada M5G 2M9. Received November 3, 1998; Revised Manuscript Received March 11, 1999

Loligomers are peptide-based intracellular vehicles able to penetrate cells and self-localize into distinct cellular compartments. Loligomers can be rapidly assembled by automated solid-phase approaches and were designed to act as nonviral, nonlipophilic intracellular shuttles. One nucleus-directed loligomer, termed loligomer 4, was evaluated for its ability to function as a transfection agent. Loligomer 4 readily associates with plasmids to form noncovalent complexes. The migration of loligomer 4-plasmid complexes into cells was monitored by flow cytometry and fluorescence microscopy. Populations of plasmids labeled with 7-AAD exist either free or in association with loligomer 4 inside cells and are visible throughout the cytosol and nucleus of chinese hamster ovary (CHO) cells. Loligomer 4-plasmid complexes were not cytotoxic to cells and were readily imported by most cells (>70%). CHO cells were transfected with complexes of loligomer 4 and plasmids harboring luciferase, green fluorescent protein or β-galactosidase reporter genes. The transfection efficiency of loligomer 4-plasmid DNA complexes was greater when cells were maintained as suspensions instead of monolayers. Transfections could be performed with cells suspended in serum-containing medium. The observed levels of transfection, however, were modest with 5-10% of CHO cells expressing either a green fluorescent protein or the enzyme β-galactosidase. Loligomers have recently been observed in vesicular compartments [Singh, D., Kiarash, R., Kawamura, K, LaCasse, E. C., and Garie´py, J. (1998) Biochemistry 37, 5798-5809] and differences between levels of cellular import and transfection efficiency may well reflect the need to optimize the release of loligomers and their complexes from these compartments in future designs. In summary, loligomer 4 behaves as a stable, soluble and effective transfection agent. These results demonstrate the feasibility of designing loligomers able to act as intracellular guided agents aimed at gene transfer applications.

INTRODUCTION

Transcription of exogenous genes in mammalian cells has proven invaluable in a wide variety of applications. These uses include the expression of foreign genes in mammalian cells, the production of proteins from cloned cDNAs, the analysis of transcriptional regulatory sequences, and the expression of molecules involved in the intracellular sorting and posttranslational processing of secreted polypeptides. A variety of methods has been developed to transfer genes into eukaryotic cells. These techniques involve the direct physical intake of DNA by the cell, either introduced in the context of a viral particle inside the cell (1, 2), microinjected directly into the cell, or introduced through the use of receptor-mediated gene transfer strategies (3-5) or by utilizing nonviral genetransfer methods in combination with a lysosomotropic agent such as chloroquine (6). The choice of a gene transfer technique is dependent on the cell line or cell type as well as the limitations of the experimental design. Most biochemical methods result in the disruption of cell membranes that allow the import of DNA (7), either through the formation of DNA complexes with inorganic salts and polycations (8) or with lipids (9, 10). Recent examples of peptide- and polycations-DNA complexes * To whom correspondence should be addressed. Phone: (416) 946-2967. Fax: (416) 946-6529. E-mail: [email protected]. ‡ Present address: Gemma Biotechnology, 620 University Avenue, Toronto, Canada M5G 2M9.

able to act as efficient transfection agents have also been reported (11-14). Nonviral DNA transfection techniques have historically resulted in a low efficiency transfer of the DNA into a cell population in comparison to virus-mediated transfer methods involving the use of SV40 vectors, vaccinia virus, adenovirus, and retroviruses. Although retroviral transduction can lead to a higher transfection efficiency in terms of gene transfer and integration (60-90%), the efficacy of retroviral infection itself remains limited by the titer associated with the helper-free packaging cell line, the time of infection, and its facilitated uptake with various chemical substrates (2, 15). Calcium phosphatemediated transfection (16) is one of the oldest approaches and is still the most popular transfection method (17). Other popular methods include DEAE-dextran mediated transfection (18), electroporation (19), liposome technology (20), lipid-mediated transfection (21), and electropermeabilization (22). The need to design defined, guided intracellular vehicles able to act as efficient nonviral gene delivery agents is obvious and can potentially be addressed using solid-phase synthesis strategies. In particular, the concept of loligomers1 (23, 24) was recently developed as a design strategy for creating multitasking agents to target different organelles in cells. Loligomers contain functional domains presented on a tentacular scaffold. More precisely, lysine residues give rise to a branched peptide, which presents multiple domains that code for functions

10.1021/bc980131d CCC: $18.00 © 1999 American Chemical Society Published on Web 08/25/1999

746 Bioconjugate Chem., Vol. 10, No. 5, 1999

Figure 1. Structure of loligomer 4, a peptide-based intracellular vehicle (23, 24). This construct consists of a 5-amino acid long, C-terminal region (analytical arm) and eight identical N-terminal arms linked together through a branched lysine polymer (BP). Each N-terminal arm contains a domain representing the nuclear localization signal of the SV40 large T-antigen (NLS; residues 124-135) and a lysine pentapeptide acting as a cytoplasmic translocation signal (CTS).

such as cellular delivery, cell signaling, and/or cytotoxicity. Prototypic loligomers have been generated that can self-localize in the nucleus of cells (23). The intracellular routing of a nucleus-directed loligomer was recently analyzed by microscopy and flow cytometry, indicating its presence in the cytosol, in endocytic vesicles, and in the nucleus of eukaryotic cells (24). In this report, we demonstrated the ability of one such loligomer, termed loligomer 4, to associate noncovalently with plasmids to form complexes that self-localize inside eukaryotic cells. MATERIALS AND METHODS

Peptide Synthesis. The branched peptide (loligomer 4) was synthesized by classical solid phase peptide synthesis (25), on an Applied Biosystems 430A Synthesizer using tert-butoxycarbonyl (t-Boc) chemistry and phenylacetamidomethyl resin supports. The synthesis, coupling efficiencies, purification, and characterization procedures were described previously (23, 24). Loligomer 4 was labeled with fluorescein via the free thiol group in the C-terminal arm of the molecule (Figure 1) as described previously (23, 24). Fluorescein maleimide was purchased from Molecular Probes (Eugene, Oregon) Gel Mobility Shift Assay. Plasmid DNA (1 µg; pGL2 Luciferase, Promega) was dispensed into microcentrifuge tubes and mixed with increasing amounts of loligomer 4 (5-1000 ηg). Aliquots of each loligomer 4-plasmid mixture (20 µL) were then incubated at room temperature for 5-10 min. A 3 µL volume of 6× loading buffer 1 Abbreviations: CTS, cytoplasmic translocation signal; GFP, green fluorescent protein; loligofection, process of transfecting cells with plasmid DNA-loligomer complexes; loligomer, a squidlike branched peptide construct that incorporates cell penetration and intracellular localization signals; loligomer 4, a loligomer harbouring 8 N-terminal arms that comprise of a pentalysine cytoplasmic translocation domain and the SV40 large T-antigen nuclear localization signal; NLS, nuclear localization signal; RLU, relative light unit.

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(0.25% bromophenol blue, 0.25% Xylene cyanol F. F., and 30% glycerol in water) was subsequently added to each tube. Samples were thereafter loaded into wells of a precast 0.8% agarose gel (1× TBE). Samples were electrophoresed on agarose gels for 1 h at 70 V in TBE buffer (0.09 M Tris-borate and 0.002 M EDTA). Bands corresponding to plasmid DNA were detected by staining the gel with ethidium bromide (10 µg/100 mL TBE buffer). Banding patterns were visualized under a UV light and photographed. Cell Culture. Cells were maintained at 37 °C, 5% (v/ v) CO2, 90% humidity in Nunclon tissue culture plasticware (Gibco-BRL). CHO (Chinese hamster ovary cells subclone AUXB1; ref 26), COS-7 (SV40 transformed African green monkey kidney cells; ref 27), and MCF-7 cells (human breast carcinoma; ref 28) were grown in a MEM supplemented with 10% (v/v) fetal calf serum (FCS; ImmunoCorp, Montreal, Canada), 1 unit/mL penicillin (Sigma), 100 µg/mL streptomycin sulfate (Sigma), and 1 mM L-glutamine (Gibco-BRL). Subconfluent cultures of cells were passaged by washing them with 10 mL of PBS, followed by a trypsinization step for 2 min at 37 °C with 1 mL Trypsin (Gibco-BRL). The reaction was stopped by adding growth medium once cells composing the monolayer were detached from the dish. Cells were then harvested by centrifugation for 5 min at 1500 rpm and grown either as adherent monolayers or as suspensions in plastic tubes (Nunc). DNA Vectors. The following DNA plasmids were used in this study. The pGL2 luciferase (Ctrl) vector (Promega) contains both a SV40 promoter and an enhancer sequence inserted into the pGL2 basic vector, which harbors a luciferase gene. The pCMV β-gal vector consists of both a CMV promoter sequence and a β-galactosidase gene. The pCMV β-gal plasmid contains the major immediate early gene promoter and the enhancer region of the human cytomegalovirus (CMV) and LacZ genes (29). The pGFP vector (Clontech) incorporates a gene coding for a green fluorescent protein. Transfection Methods. Loligofection (Transfection with Loligomer 4-Plasmid Complexes). Loligomer 4 (ranging in concentrations from 1 to 60 µg prepared in PBS) was mixed with 10 µg of either plasmids pGL2 luciferase (Promega), pGFP (Clontech), or pCMV β-gal (Clontech). Sample volumes were then adjusted to 50 µL with PBS, and the mixtures were incubated at room temperature for 10 min. Thereafter, the following methodologies were used for transfections. Treatment of Cells as Monolayers. Two hundred thousand (2 × 105) cells were seeded in individual wells of 6-well plates and incubated overnight at 37 °C. A 50 µL sample of each loligomer 4-plasmid DNA mixture was adjusted to 500 µL with R-medium. The medium was removed from plates seeded with cells and replaced with medium containing a loligomer 4-plasmid mixture. The plates were incubated for 30 min at room temperature with occasional redistribution of the solution from the edges of the dish over the entire surface of the dish every 10 min. A 4.5 mL aliquot of fresh medium was then added and the cells were incubated at 37 °C for 48 h. Treatment of Cells in Suspensions. Cells (2 × 105 cells) suspended in growth medium were mixed with loligomer 4-plasmid preparations giving a final volume of 500 µL. The resulting cell suspensions were incubated for up to 5 h at 37 °C in eppendorf tubes attached to a rotating shaker. The cells were subsequently plated in a 6-well dish for 48 h with an additional 4.5 mL of growth medium.

Loligomers as Transfection Agents

Calcium Phosphate Method. Transfections were performed using the calcium phosphate method (16). Briefly, 15 µL of a 2 M CaCl2 solution was added dropwise to 250 µL of DNA-HBS buffer. The cell suspension method described above was used and the medium was changed after 24 h. The cells were incubated for an additional 24 h period. Other Methods. Superfect (Qiagen), Transfectam (Promega), Tfx-50 (Promega), and Lipofectamine (Gibco-BRL) reagents were used as described by their suppliers using cell suspensions described in the protocol for loligomer transfection (loligofection). Transfection with Lipofectamine was performed in a serum-free medium for the first 24 h. Superfect (Qiagen) is a specifically designed polycation based on dendrimer technology, Tfx-50 (Promega) consists of a synthetic cationic lipid molecule, N,N,N′,N′-tetramethyl-N-N′-bis(2-hydroxyethyl)-2-3,dioleoyloxy-1,4-butane diammonium iodide and L-dioleoyl phosphatidyl ethanolamine (DOPE). Transfectam (Promega) contains dioctacylamidoglycyl spermine and Lipofectamine (Gibco-BRL) reagent contains 3:1 (w/w) liposome formulation of the polycationic lipid 2,3-dioleyloxyN-[2-(spermine carboxamido) ethyl]-N,N-dimethyl-1propaneminium trifluoroacetate (DOSPA) and L-R dioleoyl phosphatidylethanolamine (DOPE). Preparation of Cell Extracts. Preparation of cell extracts was performed as described elsewhere (30). Briefly, cells were washed with PBS, incubated with 200 µL of a cell lysis solution (25 mM Tris, pH 7.8, with H3PO4; 2 mM EDTA; 2 mM DTT; 10% glycerol; 1% Triton X-100; Promega) for 5 min and finally scraped into an eppendorf tube. The cells were disrupted using one to two cycles of freezing in dry ice-ethanol or incubating at -70 °C for 30 min followed by a thawing step at 37 °C for 5 min. The extracts were centrifuged at 12 000 rpm for 5 min at 4 °C, and the resulting supernatants were transferred to new tubes. The protein concentration in cell lysates was determined using the Bradford (31) dye binding protein assay (Bio-Rad, Hercules, CA). The total amount of luciferase activity was reported in terms of light units per milligram of protein in the extracts. Luciferase Assay. Luminescence was detected in cells transfected with a plasmid construct harboring a luciferase reporter gene (pGL2 luciferase control vector; Promega). The luciferase enzyme catalyzes the production of light in fireflies by oxidizing D-(-)-luciferin in the presence of ATP, Mg2+, and O2 (32). The reaction produces oxyluciferin, CO2, and a photon. The level of light emitted during the reaction can be measured with a luminometer. A 20 µL aliquot of a cell extract was mixed with 100 µL of luciferase assay reagent [Promega; 20 mM tricine, 1.07 mM (MgCO3)4 Mg(OH)2‚5H2O, 2.67 mM MgSO4, 0.1 mM EDTA, 33.3 mM DTT, 270 µM Coenzyme A (lithium salt), 470 µM luciferin, and 530 µM ATP; pH 7.8] maintained at room temperature. The test tube containing the reaction mixture was placed in a luminometer (Lumat LB 9507, Berthold, Princeton, NJ), and light produced (luminescence units) for 10 s was recorded. Fluorescence Microscopy. The plasmid pGL2 Luciferase (10 µg) was incubated with the DNA 7-aminoactinomycin (7-AAD; 0.1 µg) for 5 min. The labeled plasmid was subsequently complexed with fluoresceinlabeled loligomer 4 at a 2:1 loligomer 4:plasmid weight ratio. CHO cells (106 cells) grown in suspension were transfected with the dual-labeled complex following the procedure described in the section on transfection methods. Cells were examined by fluorescence microscopy (Leica DM-LB microscope) and photographed 4 h after transfection. Fluorescence excitation filters (fluorescein,

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EX filter 355/425, EM filter LP 470; 7-AAD, EX filter 530/ 595, EM filter LP615) were used to observe the migration of both fluorophores. The GFP fluorescence signal (EX filter 355/425, EM filter LP 470) emitted by CHO cells transfected with a plasmid harboring the gene coding for green fluorescent protein was stable and monitored directly in viable cells. Flow Cytometry. CHO cells (106 cells) were transfected as described in the previous paragraph with the 7-AAD-labeled plasmid pGL2 or the complex of fluoresceinlabeled-loligomer 4 and 7-AAD-labeled plasmid pGL2. Labeled cells were subsequently analyzed by flow cytometry on a FACScan unit (Becton Dickinson) to determine the extent of cell viability (forward scatter, FSC; side scatter, SSC) and the binding/internalization of 7-AAD into CHO cells (argon laser; excitation wavelength, 488 nm; emission signal, LP 650 nm filter). The dye 7-AAD is excluded by viable cells unless complexed to a molecule that is internalized by cells. β-Galactosidase Staining. The detection of β-galactosidase activity in situ in cultured cells was performed using a histochemical stain (33). The substrate, 5-bromo4-chloro-3-indolyl-β-D-galactoside (X-Gal), is hydrolyzed by β-galactosidase to generate an indigo-colored precipitate which facilitates the subcellular localization of the β-galactosidase activity. A monolayer of cells was washed twice in PBS containing 2 mM MgCl2. The cells were fixed in 0.75% (v/v) glutaraldehyde (Sigma) in PBS for 15 min at room temperature, and then washed with PBS/ MgCl2 for 5 min. A 1.5 mL volume of X-Gal (1 mg/mL) diluted in staining solution [5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6‚3H2O, 0.01% (w/v) sodium deoxycholate, 0.02% (v/v) Nonidet P40, and 2 mM MgCl2 in PBS] was added to each one-chamber cover glass slide (LabTek borosilicate; Nunc). Cells were analyzed under a light microscope and photographs were taken. RESULTS

Loligomers are intracellular vehicles able to import large cargos such as plasmids into cells. One of these constructs, loligomer 4, is a nucleus-directed vehicle. It is a structurally defined, synthetic peptide (Figure 1) which possesses properties expected of a transfection agent. Loligomer 4 is water soluble, heat stable and probably associates noncovalently with the phosphate backbone of nucleic acids. Its cellular import is insensitive to the presence of serum or medium. The ability of loligomer 4 to act as a transfection agent was analyzed using reporter plasmids. Design and Synthesis of loligomer 4 Variants. Loligomers (23, 24) are peptide-based multitasking shuttles, which are able to selectively deliver cytotoxic or therapeutic agents to the cytosol or inside cellular compartments. The term loligomer was derived from the latin root loligo referring to members of the squid family, thereby emphasizing the branched or “squid-like” nature of these peptides. In particular, loligomer 4 (Figure 1) contains eight nuclear localization signals (NLS) and eight pentalysine stretches acting as cytoplasmic translocation signals (CTS) which allow it to penetrate cells and relocate to their nucleus. Loligomer 4 (Figure 1) was synthesized by solid-phase peptide synthesis. Peptide branching was introduced following successive rounds of couplings of lysine derivatives harboring identical protecting groups on their R- and -amino groups (23). The concept of peptide branching originated from the work of Tam (34, 35) dealing with the construction of multiple antigenic peptides. The thiol side chain located in the


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Figure 2. A gel-retardation assay illustrating a shift in migration of plasmid DNA (pGL2 luciferase) in agarose gels when complexed with loligomer 4. Lane 1, banding pattern observed for 1 µg of pGL2 luciferase plasmid; lanes 2-11, banding patterns observed for 1 µg of pGL2 luciferase plasmid DNA complexed with increasing amounts of loligomer 4. Lane 2, 5 ng loligomer 4; lane 3, 10 ng; lane 4, 50 ng; lane 5, 100 ng; lane 6, 150 ng; lane 7, 200 ng; lane 8, 250 ng; lane 9, 500 ng; lane 10, 750 ng; lane 11, 1000 ng. Lane 12 was loaded with 0.5 µg of a 1 kb DNA ladder (Gibco BRL).

C-terminal arm of loligomer 4 (Figure 1) was modified with fluorescein maleimide to produce a fluoresceinlabeled loligomer 4 for cell migration studies. Loligomer 4 Forms a Complex with Plasmid DNA. The plasmid pGL2 luciferase was mixed at different ratios with loligomer 4 to generate noncovalently associated complexes. The resulting macromolecular aggregates were easily observed as large molecular weight species on agarose gels. Their noncovalent association is due to the fact that loligomer 4 is a cationic structure that can readily interact with the negatively charged phosphate backbone of plasmid DNA. The migration of plasmid DNA (1µg) in agarose gels was retarded by the addition of loligomer 4, as detected with ethidium bromide (Figure 2). Plasmid DNA containing no loligomer migrated as a pair of bands representing both supercoiled and open circular forms of DNA (lane 1). The mobility of plasmid DNA (1 µg) was not altered by the presence of loligomer 4 ranging in concentrations between 5 and 100 ng (lanes 2-5). The presence of 150 ng of loligomer (lane 6) resulted in the appearance of a band migrating slightly above the supercoiled DNA plasmid band. Upon adding 200 ng of loligomer 4 (lane 7), the uncomplexed supercoiled DNA band disappeared with most of the plasmid DNA remaining in the sample well. The intensity of the plasmid DNA open circular form was further decreased by the addition of 250 ng of loligomer 4 (lane 8) and concentrations of the vehicle in the range 500-1000 ng (lanes 9-11) were sufficient to retard the migration of all the plasmid DNA. In summary, the mixing of loligomer 4 with the plasmid pGL2 luciferase at a minimal weight ratio of 0.5 resulted in an efficient complexation of all the available plasmid DNA as shown by the retardation of the DNA-peptide complex on agarose gels (Figure 2). Complexes of Fluorescein-Labeled Loligomer 4 and 7-AAD-Labeled Plasmids Are Imported into CHO Cells. The migration of plasmids inside CHO cells

occurs only in the presence of loligomer 4. The DNA intercalator 7-AAD does not enter cells that are viable and is typically used in flow cytometry to measure the extent of cell death in a cell population. This fluorescent intercalator was used to label plasmid DNA in an effort to follow the migration of plasmid inside CHO cells. Loligomer 4 was labeled with fluorescein in order to track its intracellular movement in relation to the 7-AADlabeled plasmid. A typical distribution pattern of plasmid (red color) and loligomer 4 (green color) inside CHO cells after 4 h is presented in Figure 3 (Panel A). Zones of yellow color inside cells highlight areas of comigration of the 7-AAD and fluorescein chromophores and suggest that a significant fraction of labeled plasmid exists as complexes with loligomer 4. The overall cellular distribution of 7-AAD fluorescence (red color) also suggests the occurrence of either free plasmid inside these cells or the rapid exchange of 7-AAD originally associated with the plasmid with intracellular pools of DNA. The flow cytometric patterns of CHO cells exposed to a 7-AAD-labeled plasmid for 4 h indicate that the plasmid alone is not imported by these cells (Figure 3, panel B, right side, relative fluorescence signal less than 10 units). In contrast, CHO cells transfected with a complex of loligomer 4 and 7-AAD-labeled plasmid bind and internalize the complex rapidly (in less than a hour) with >70% of cells being labeled with the plasmid harboring the 7-AAD label (Figure 3, panel C, right side, relative fluorescence signal greater than 10). All cells analyzed for the binding and internalization of the 7-AAD-labeled plasmid were viable (gated cell population [R1] based on forward and side scatter properties, Figure 3, panels B and C, left sides), indicating that unbound 7-AAD was not responsible for the fluorescence signal observed. The viability of CHO cells also confirms that loligomer 4 at a concentration of 3 µM is not toxic to these cells when the peptide is associated with a plasmid (30 µg of loligomer 4 and 10 µg of plasmid in 500 µL).



Figure 3. Localization of loligomer 4-plasmid complexes in CHO cells. (A) Photograph of an unfixed preparation of viable CHO cells after a 4 h exposure to loligomer 4-plasmid complexes depicting the intracellular distribution of fluorescein-labeled loligomer 4 (green color) complexed with 7-AAD-labeled plasmid pGL2 (red color) in CHO cells. The comigration of labeled loligomer 4 and 7-AAD-labeled plasmid gives rise to intracellular areas of yellow fluorescence. (B) FACS analysis of CHO cells after a 4 h exposure to 7-AAD labeled plasmid. (Panel B, left side) Flow cytometric pattern (forward scatter and side scatter properties) indicating that most CHO cells remained viable following this treatment. (Panel B, right side) Histogram of viable cells (R1) labeled with the 7-AAD labeled plasmid. Most cells do not bind or internalize 7-AAD (autofluorescence). (C) FACS analysis of CHO cells after a 4-hour exposure to loligomer 4- complexed with the 7-AAD labeled plasmid. (Panel C, left side) Flow cytometric pattern (forward scatter and side scatter properties) indicating that most CHO cells remained viable following their exposure to these complexes. (Panel C, right side) Histogram of viable cells (R1) labeled with the peptide- 7-AAD labeled plasmid complex. Most cells (>70%) bind and internalize 7-AAD (panel A) suggesting that the plasmid requires the presence of loligomer 4 to effectively be imported inside CHO cells.

Micromolar Concentrations of Loligomer 4 Facilitate Efficient Gene Transfer. The transfection efficiency of the pGL2 luciferase vector complexed to loligomer 4 into CHO cells was confirmed by monitoring

the extent of luciferase activity present in the cytosol of CHO transfected cells. The histogram presented in Figure 3A highlights the level of luciferase activity (per milligram of protein) in CHO cell extracts as a function


Figure 4. Levels of luciferase activity detected in extracts of CHO cells transfected with loligomer 4-pGL2 plasmid complexes. The luciferase activity present in cell lysates is reported as relative light units per milligram of protein (RLU/mg of protein). It represents a late stage measurement of the transfection efficiency of loligomer-plasmid complexes in CHO cells. (A) Effect of loligomer 4 concentration on recorded luciferase activity. The pGL2 luciferase plasmid (10 µg) was complexed with increasing amounts of loligomer 4 (4-62 µg). (B) Plasmid concentration on recorded luciferase activity. Loligomer 4 (31 µg) was complexed with increasing amounts of pGL2 luciferase plasmid (0.5-25 µg). Luciferase activity was not observed in CHO cells transfected with naked DNA only.

of the weight ratio of loligomer 4 to plasmid used to initially transfect CHO cells. A 10 µg aliquot of the plasmid pGL2 luciferase was mixed with increasing amounts of loligomer 4. Significant levels of loligofection yielding (0.06-0.3) × 107 relative luciferase unit (RLU)/ mg of protein were observed at loligomer 4 concentrations ranging in values from 4 to 11 µg. However, a 7-fold increase in luciferase units (2.1 × 107 RLU/mg of protein) was obtained when 21 µg of loligomer 4 (1.1 µmol; loligomer 4-to-plasmid weight ratio, 2.1) was used in the transfection procedure. The magnitude of the luciferase signal then reached a plateau between 21 and 62 µg of loligomer 4 (loligomer 4 to plasmid weight ratios between 2.1 and 6.2) with RLU values leveling to between 2.4 × 107 and 2.8 × 107 RLU /mg of protein. Loligomer 4 to Plasmid Weight Ratio of 3 Is Optimal To Achieve Maximal Transfection Efficiency. A fixed concentration of loligomer 4 (31 µg; 1.67 µmol) was mixed with increasing amounts of pGL2 luciferase plasmid DNA ranging 0.5-25 µg (Figure 4B; log scale units). A low level of relative luciferase activity (1.9 × 105 RLU/mg of protein) could be detected in CHO cells transfected with a mixture containing 0.5 µg of plasmid DNA, reaching a maximal value of 2.7 × 107 RLU/mg of protein at a plasmid content of 10.0 µg (loligomer 4 to plasmid weight ratio of 3.1). Further

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Figure 5. Comparison of transfection efficiencies for loligofection in CHO, COS-7, and MCF-7 cells. Transfections were performed on cells grown as monolayers (Panel A) or maintained in suspension (Panel B) with loligomer 4-plasmid complexes. Transfection efficiency is reported in terms of luciferase activity (RLU/mg of protein) present in cell lysates. A 31 µg aliquot of loligomer 4 was complexed with the plasmid pGL2 luciferase (10 µg). Transfection with naked DNA did not result in any luciferase activity.

increase in plasmid DNA content in relation to loligomer 4 resulted in a decreased transfection efficiency down to 1.2 × 107 RLU/mg of protein at 15 µg of pGL2 luciferase plasmid and 0.07 × 107 RLU/mg of protein at 25 µg of plasmid. Transfection Efficiency of Loligomer 4 Is Greater When Cell Suspensions Are Treated with Loligomer:Plasmid Complexes Rather Than Cell Monolayers. Three cell lines (CHO, COS-7, and MCF-7) were used to evaluate the transfection efficiency of our loligomer 4-plasmid complexes. Cell lines maintained as monolayers, then transfected with 10 µg of pGL2 luciferase plasmid DNA and 31 µg of loligomer 4, exhibited very low transfection efficiency as monitored by the resulting levels of luciferase activity (Figure 5 A; log scale units). Recorded light emissions ranged in values between 2.6 and 2.7 × 105 RLU/mg of protein for COS-7 and CHO cells and 2.5 × 103 RLUs with MCF-7 cells. The transfection efficiency was found to be more prominent and efficient (up to 100-fold increases) when cells were transfected as suspensions (Figure 5B), where luciferase activity values of 2.7 × 107 and 0.2 × 107 RLUs were recorded for CHO and COS-7 cells, respectively. Finally, a value of only 2 × 105 RLU/mg of protein was registered when MCF-7 cells were transfected as suspension cells, suggesting that the efficiency of this procedure remains dependent on the cell line. Comparing Loligomer 4 to Other Transfection Agents in the Context of Cells Maintained in Suspension. The efficacy of loligomer 4 as a prototypic transfection agent is significant only in the context of cells presented as suspensions. The relative ability of


Figure 6. Comparison of transfection efficiencies (luciferase activity) in CHO cells for loligofection in relation to other established nonviral transfection methods. The luciferase activity present in cell lysates is reported as relative light units per milligram of protein (RLU/mg of protein). The luciferase activity was monitored using 10 µg of pGL2 luciferase and 31 µg of loligomer 4. Other transfection methods including calcium phosphate and commercially available agents such as Superfect (Qiagen), Transfectam (Promega), Tfx-50 (Promega), and Lipofectamine (Gibco-BRL) were compared to loligofection. No luciferase activity was observed in CHO cells transfected with naked DNA only.

loligomer 4-plasmid complexes to transfect CHO cells in suspension was compared to other available methods (Figure 6) in order to highlight their comparable potential as nonviral transfection strategies. Agents such as Superfect (Qiagen), Transfectam (Promega), and Tfx-50 (Promega) all facilitated the transfection of the pGL2 luciferase plasmid into CHO cells maintained in suspension with RLU/mg of protein values ranging between 0.4 × 107 and 0.6 × 107. Similarly, luciferase activities of the order of 1.0 × 107 RLU/mg of protein were recorded with the calcium phosphate method. Higher values [(2.7-4.8) × 107 RLU/mg of protein) were observed with Lipofectamine (Gibco-BRL) and loligomer 4. All differences in transfection efficiencies must be contrasted with the fact that none of the previous established methods were optimized for transfecting cells in suspension. Nevertheless, there is a clear indication that the loligomer 4 construct represents a useful template for designing alternate classes of transfection agents. β-Galactosidase and Green Fluorescent Protein Are Expressed in Transfected CHO Cells. CHO cells were transfected with 10 µg of pCMV β-gal or pGFP complexed to 31 µg of loligomer 4. The reporter plasmid pCMV β-gal incorporates a β-galactosidase gene. The production of β-galactosidase was monitored by the cleavage of its substrate, 5-bromo-4-chloro-3-indolyl-βD-galactoside (X-gal), which resulted in an indigo color precipitate present in CHO cells successfully transfected with the loligomer 4-plasmid complex (Figure 7B). In contrast, no color was observed for cells exposed to the plasmid alone (Figure 7A). The plasmid pGFP harbors a gene coding for a green fluorescent protein (GFP). The transfection and expression of this plasmid in CHO cells occurs only when complexed to loligomer 4 (Figure 7C). The transfection efficiency of loligomer 4-plasmid complexes in CHO cells in terms of the percentage of cells expressing the reporter proteins ranges in value from 5 to 10% of cells. DISCUSSION

The transfection of eukaryotic cells has become an important technique for the study of cellular genes and for the development of gene transfer studies. Advances in gene therapy depend in large part on the development


of delivery systems capable of introducing DNA into a target cell or tissue. These approaches include the use of carrier molecules, such as liposomes or chemical precipitates, to self-replicating systems, such as viruses (36). In this study, a peptide-based, intracellular vehicle, termed loligomer 4 (23, 24), was tested for its ability to form complexes with plasmid DNA and facilitate their transport into cells. Loligomer 4 contains eight copies of the SV40 large T antigen nuclear localization signal (37) as well as eight pentalysines sequences that act as a cytoplasmic translocation signal (CTS; ref 23) (Figure 1). The branched nature of loligomers is derived from a synthetic approach initially developed for the creation of multiple antigenic peptides (34, 35). This tentacular geometry affords several distinct advantages in terms of incorporating tissue-targeting sequences and cytotoxic moieties. First, branching reduces the impact of proteolytic degradation on loligomers (23, 24) since the cleavage of one or more arms does not destroy the overall localization properties of the construct. Second, multivalency represents an important feature of loligomers and other recent delivery strategies involving dendrimers (14, 38) and peptabodies (39) in terms of enhancing the avidity of such constructs for a cell surface receptor or to promote their localization properties inside cells. For example, it has been demonstrated that, in the case of protein conjugates, the efficiency of their nuclear targeting property increases with the number of NLS sequences coupled to them (40). The attachment of the SV40 Large T antigen nuclear localization signal peptide induced the nuclear accumulation of the conjugated DNA in digitoninpermeabilized cells (41). In the case of loligomers, the display of eight pentalysine sequences dramatically enhances its cellular uptake (23). The cellular uptake and intracellular routing of loligomer 4 has recently been studied by flow cytometry and microscopy (24). Loligomer 4 was shown to be irreversibly retained inside cells when its intracellular routing leads to its accumulation into the nucleus (24). The major objective of this study was to determine if loligomer 4 is able to act as a vehicle in terms of delivering large molecular cargos such as plasmids inside cells. Loligomer 4 is a branched cationic peptide with a theoretical net charge of +71. This construct was thus expected to interact noncovalently with the negatively charged phosphate backbone of plasmid DNA. Loligomer 4 retarded the electrophoretic migration of plasmid DNA in agarose gels (Figure 2) starting at a 1:2 peptide:DNA weight ratio. The minimal theoretical charge ratio (() necessary for loligomer 4 to complex plasmid DNA was calculated to be ∼6.5. In contrast, the optimal level of transfection in CHO cells observed for these peptideDNA complexes occurs at a loligomer 4:plasmid DNA weight ratio of 3: 1 (Figure 4). The efficiency of this transfection approach was monitored using a plasmid incorporating a luciferase reporter gene. Overall, these results suggest that a range of peptide-DNA complexes exist in solution and that efficient levels of transfection are achieved with complexes presenting a charge ratio between 30 and 70 (loligomer 4:plasmid DNA weight ratios between 2:1 and 6:1; Figure 4). Previous studies have shown that the interaction of plasmid DNA with cationic substrates results in a marked condensation of DNA molecules into 80-100 nm diameter toroid structures (8). The ratio of loligomer 4 to DNA may thus regulate the fraction of “packaged” plasmid complexes optimally suited to their penetration into cells and intracellular routing.


Singh et al.

Figure 7. Expression of β-galactosidase and GFP in CHO cells. (A) CHO cells transfected with the naked plasmid pCMV β-gal showing no indigo staining of cells. (B) Indigo-colored cells are observed in this preparation of CHO cells transfected with loligomer 4-pCMV β-gal plasmid. (C) Fluorescent green cells were visualized in a field of CHO cells transfected with loligomer 4-pGFP plasmid.

The import of loligomer 4 into CHO cells occurs through absorptive endocytosis (23, 24). One-arm peptide or loligomer constructs lacking the cytoplasmic translocation domains were either poorly endocytosed or unable to enter cells (23). Loligomer 4 is typically observed in vesicular compartments as well as in the cytosol of CHO cells within the first 30 min of exposing cells to this

construct. After a few hours, a significant fraction of internalized loligomer 4 accumulates in the cell nucleus (23, 24). Free loligomer 4 and loligomer 4-plasmid DNA complexes represent two (or more) distinct presentations of this peptide vehicle. The presentation of loligomer 4 as part of a peptide-DNA complex may alter its ability to enter cells. In addition, plasmid DNA may not be


routed through the same intracellular pathway as loligomer 4. The migration of loligomer 4-plasmid DNA complexes was thus analyzed by fluorescence microscopy and flow cytometry. The migration of the reporter plasmid was monitored by marking the plasmid with the fluorescent DNA intercalator 7-AAD, a dye that does not enter into viable cells. Loligomer 4 was labeled with fluorescein, and dual-labeled complexes of loligomer 4-plasmid DNA were created by mixing both fluorescent entities together. The majority of CHO cells (>70%) rapidly imported the labeled complex (Figure 3, panel A and panel C, right side) while the labeled plasmid alone was not endocytosed by cells (Figure 3, panel B, right side). An image depicting the distribution of the two fluorophores inside CHO cells after 4 h is presented in Figure 3A. No fluorescence signal was observed for cells exposed to the labeled plasmid alone (results not shown). Yellow areas in Figure 3A represent zones where plasmids (7-AAD, red fluorescence) and loligomer 4 (green fluorescence) comigrated inside CHO cells. The intracellular distribution of yellow fluorescence is reminiscent of previous patterns observed for loligomer 4 alone (23, 24), suggesting that its complexation with plasmid DNA has not altered its properties as an intracellular shuttle. Intracellular regions of red fluorescence highlight areas where the labeled plasmid is not complexed to loligomer 4. Alternatively, 7-AAD may rapidly exchange between plasmid DNA and intracellular pools of DNA. Overall, these results suggest the comigration of a significant population of peptide-plasmid DNA complexes inside CHO cells and the possible dissociation of a large fraction of these complexes inside cells yielding free plasmid or the exchange of 7-AAD. The relatively global distribution of fluorescein-labeled loligomer and 7-AAD in these viable CHO cells suggests that both the cytosol and nucleus have been targeted by these agents. The uptake of loligomer 4 into cells is greater for cells either maintained in suspension or gently trypsinized to generate a cell suspension than for cells presented as monolayers (24). This difference in cellular uptake is less significant, however, in the case of nonadherent cell lines such as Daudi and EL4 (24). It was thus predicted that the levels of transfection efficiencies observed for loligomer 4-plasmid DNA complexes would be greater for CHO, COS-7, and MCF-7 cells grown in suspension rather than monolayers. Results presented in Figure 5 confirmed this hypothesis and support the view that the import of plasmid DNA as part of a loligomer 4-plasmid complex follows the internalization mechanism proposed for loligomer 4 itself. The reason the import mechanism is enhanced by cells presented as suspensions remains unclear although effects due to cytoskeletal components have now been ruled out (24). No differences in transfection efficiency were observed for cells grown in spinner flasks as opposed to suspension cells arising from trypsinizing cell monolayers. The value of loligomer 4 as a transfection agent was evaluated using two approaches. The efficiency of transfection of loligomer 4 complexed to a reporter plasmid harboring the luciferase gene was initially compared to other nonviral transfections methods. The major limitation of such an analysis stems from the fact that transfections were performed on cell suspensions (significant levels of transfection for loligomer 4-plasmid complexes are only achieved for cells presented as suspensions; Figure 5). Since other transfection strategies are typically performed on cell monolayers, the resulting transfection conditions for these methods were not optimal. Nevertheless, the transfection efficiency observed in CHO cells


with loligomer 4-plasmid DNA complexes was comparable to results observed for lipofectamine and better than results obtained for other transfection agents used in this study (Figure 6). Comparable results (using luciferase as a reporter protein) have been reported for other recently developed cationic agents (13, 42). A more conclusive method of evaluating the value of loligomer 4-plasmid complexes in transfecting cells is to simply tabulate the percentage of cells that either produce β-galatosidase (reporter plasmid, pCMV β-gal) or GFP (reporter plasmid, pGFP) within a field of cells. Calculated percentages for CHO cells transfected with loligomer 4-plasmid DNA complexes ranged in values from 5 to 10% depending on the vector used. Images of CHO cells expressing these reporter proteins are presented in Figure 7 (panels B and C). Finally, a clear difference exists between the level of import of loligomer 4-plasmid complexes by cells (>70% of viable cells take up the constructs; Figure 3) and the maximal level of transfection observed (∼10% of cells). Reasons for this discrepancy probably lie in the design of loligomer 4 itself. For example, a large fraction of this construct remains trapped in vesicular compartments (24) since we have not yet introduced a signal in this construct to favor its escape from such compartments. Their release from cytoplasmic vesicles is at best inefficient but does occur as demonstrated in this report as well as in previous studies (23, 24). Nevertheless, the fact that a prototypic peptide design such as loligomer 4 can already act as a transfection agent demonstrates the potential of creating biologically effective, multitasking peptide constructs using simple solid-phase strategies. Loligomers may thus represent useful vehicles for gene transfer experiments (43), antisense technologies (38), DNA-based immunization strategies (44), as well as photodynamic therapy (45). ACKNOWLEDGMENT

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BC980131D

Peptide-Based Intracellular Shuttle Able To Facilitate Gene Transfer

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