Photoenhancement of Transfection Efficiency Using Novel Cationic

Bioconjugate Chem. 2003, 14, 513−516

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Photoenhancement of Transfection Efficiency Using Novel Cationic Lipids Having a Photocleavable Spacer Takeshi Nagasaki,*,† Akinobu Taniguchi, and Seizo Tamagaki Department of Applied and Bioapplied Chemistry, Graduate School of Engineering, Osaka City University, SORST (JST), Osaka, 558-8585, Japan . Received December 26, 2002

New cationic lipids having an o-nitrobenzyl moiety as a photocleavable spacer between its hydrophilic and hydrophobic region were synthesized. To improve the efficiency of transfection with lipoplexes, after transfecting the cationic lipid aggregate/DNA complex, photoirradiation was performed. Photochemical decomposition of lipids would not only make the vector’s membrane unstable to facilitate the fusion with endocytic vesicles, but also promote dissociation of cationic lipid-DNA complex, thus aiding the escape of DNA from the endocytic vesicles. Using a luciferase gene as a model, we show that UV irradiation of photoresponsive lipoplex-treated COS-1 cells induces a substantial increase in the efficiency of transfection. Herein, we show a novel photoresponsive gene delivery system.

In such techniques as gene therapy, when transducing and expressing exogenous genes in mammalian cells, viral vectors, which are natural DNA complex nanoparticles, are used in the overwhelming majority of the cases. The reason for this is their high efficiency, but their disadvantages include high cost, inability to transduce large genes, and safety issues. As a result, there is an urgent need to develop highly efficient nonviral vectors using artificial nanoparticles, such as liposome (lipid aggregate)/DNA complexes (lipoplexes) or polymer/DNA complexes (polyplexes) (1). To improve the transfection efficiency of nonviral vectors, it is necessary to improve several problems (2-5). With lipoplexes, one serious obstacle is the escape of DNA from endocytic vesicles after passing through the cell membrane by endocytosis (6-10). Factors affecting the endocytic pathway could either facilitate escape of plasmid DNA into the cytosol or protect these molecules from degradation by lysosomal nucleases, thereby influencing the efficiency of transfection (11-15). Prasmickaite et al. used a phthalocyanine derivative as a photosensitizer, and by destabilizing the membrane of endocytic vesicles with light irradiation, they successfully facilitated the escape of an exogenous gene and improved transfection efficiency (16). However, this method is highly toxic, and although the transfection efficiency of polyplexes is improved, the transfection efficiency of lipoplexes is reduced (17). With the exception of this type of photochemical internalization using photosensitizers, no studies have investigated the delivery of exogenous genes into the cytoplasm using the photoresponsive system. In the hope of facilitating the escape of a gene from endocytic vesicles and improving transfection efficiency, we have investigated photoresponsive gene carriers and using UV irradiation during gene delivery to destabilize endocytic vesicle membranes and to facilitate membrane fusion. In the present study, to improve the efficiency of * To whom correspondence should be addressed. E-mail: [email protected]. Phone: 81 6 6605 2696. Fax: 81 6 6605 2785. † SORST.

Scheme 1. Synthesis of Boc-derivative of 3-Nitro-4-aminomethylbenzoic Acid

transfection with lipoplexes, after transfecting cationic lipid aggregate/DNA complexes including a photocleavable section in the lipid component, photoirradiation was performed. Photocleavage of cationic lipids would make vector’s membranes unstable to facilitate the fusion with endocytic vesicles, thus resulting in the induction of the destabilization of vesicular membrane and aiding the escape of DNA from the endocytic vesicles. Key intermediate, Boc-derivative of 3-nitro-4-aminomethylbenzoic acid (18), was prepared in 43% total yield according with a modified synthetic route (Scheme 1). New photocleavable lipids, having an o-nitrobenzyl structure as a spacer, a lysine or arginine residue in its hydrophilic region, and a didodecyl tertiary amide structure in its hydrophobic region, were synthesized in the dark. New photocleavable lipids (KNBN12 and RNBN12) were obtained in 36% and 41% total yield from Boc-derivative of 3-nitro-4aminomethylbenzoic acid, respectively. Nonphotocleavable compound (KBN12) was also synthesized as control in 45% total yield (Scheme 2).1 These lipids were dissolved in a chloroform/MeOH (50/50) solution, and through the use of an evaporator, a thin membrane was formed along the wall of a glass tube. Then, through the use of a voltex, the thin membrane was subjected to ultrasound at 50 °C and dispersed in a Tris buffer (pH 7.5) to attempt lipid aggregate formation. Then, complexes were formed using 1 mM lipid dispersion (7.7 µL) and pGL3-control plasmid encoding luciferase (1 µg). After that, transfection was carried out for 3 h using COS-1 cells in a 24well plate, and the activity of luciferase in lysed cell solutions was measured 48 h later to assess transfection efficiency. The measurement of transfection efficiencies

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Scheme 2. Syntheses of Cationic Lipids

was performed in triplicate (Figure 1).2 To investigate the effects of photoirradiation on transfection, after allowing cells to come in contact with complexes for 3 h, UV irradiation (365 nm, 3.5 mW/cm2) for 0 or 10 min was performed, and the results were compared. Transfection was performed using a prepared lipid aggregate solution consisting of photocleavable lipids: when compared to 1 Selected data for KNBN12: mp 127-130 °C; ν max (KBr)/ cm-1 2922, 2850 (C-H), 1701 (CONH), 1618 (ArCON), 1535 and 1340 (NO2); δ (DMSO-d6, 25 °C, TMS) 0.85 (t, J ) 6.8 Hz, 6H, CH3), 1.16-1.46 (m, 44H, CCH2C, γ- and δ-CH2 of lysyl), 1.55 and 1.71 (m × 2, 2H, β-CH2 of lysyl), 3.13 and 3.40 (t, 4H, CONCH2C), 3.17 (m, 2H, -CH2 of lysyl), 3.81 (t, J ) 6.2 Hz, 1H, CH), 4.66 (m, 2H, ArCH2NH), 7.64 (d, J ) 8.0 Hz, 1H, o-ArH to CH2NH), 7.70 (d, 1H, J ) 8.0 Hz, m-ArH to CH2NH), 7.85 (br s, 3H, NH), 7.97 (s, 1H, o-ArH to NO2); positive FAB-MS m/z 660.6 ([M + H]+; calcd for C38H70N5O4, 660.5). Anal. Calcd for C38H69N5O4‚2CF3CO2H: C, 56.81; H, 8.06; N, 7.89%. Found: C, 56.96; H, 8.23; N, 7.89. Data for RNBN12: mp 7778 °C; νmax (KBr)/cm-1 2926, 2855 (C-H), 1675 (CONH), 1620 (ArCON), 1553 and 1367 (NO2); δ (DMSO-d6, 20 °C, TMS) 0.85 (t, J ) 6.4 Hz, 6H, CH2CH3), 1.16-1.30 (m, 40H, CCH2C), 1.51.8 (m, 4H, β-and γ-CH2 of arginyl), 3.13 and 3.44 (m, 4H, CONCH2C), 3.17 (m, 2H, δ-CH2 of arginyl), 3.90 (s, 1H, CH), 4.67 (m, 2H, ArCH2NH), 7.33 (m, 4H, NH of guanidium), 7.67 (m, 2H, o-ArH and m-ArH to CH2NH), 7.98 (s, 1H, o-ArH to NO2), 9.17 (s, 1H, ArCH2NH); positive FAB-MS m/z 688.6 ([M + H]+; calcd for C38H70N7O4, 688.6). Anal. Calcd for C38H69N7O4‚ 2CF3CO2H: C, 55.07; H, 7.81; N, 10.70%. Found: C, 54.76; H, 7.90; N, 10.73. Data for KBN12: viscous oil; νmax (neat)/cm-1 2922, 2855 (C-H), 1680 (CONH), 1630 (ArCON); δ (DMSO-d6, 25 °C, TMS) 0.86 (t, J ) 6.8 Hz, 6H, CH3), 1.10-1.46 (m, 44H, CCH2C, γ-and δ-CH2 of lysyl), 1.59 and 1.77 (m × 2, 2H, β-CH2 of lysyl), 2.75 and 3.41 (t × 2, 4H, CONCH2C), 3.12 (m, 2H, -CH2 of lysyl), 3.83 (t, J ) 6.2 Hz, 1H, CH), 4.38 (m, 2H, ArCH2NH), 7.27 (d, J ) 8.4 Hz, 2H, o-ArH to CH2NH), 7.35 (d, J ) 8.4 Hz, 2H, m-ArH to CH2-3NH), 8.16 (br s, 6H, NH3), 9.18 (s, 1H, CONH); positive FAB-MS m/z 615.2 ([M + H]+; calcd for C38H71N4O2, 615.6). Anal. Calcd for C38H69N4O2‚2HCl‚3.5H2O: C, 60.73; H, 10.52; N, 7.41%. Found: C, 60.64; H, 10.47; N, 7.41. 2 COS-1 cells ((3-4) × 104 cells for an each well) were grown to just before confluence in 24-well culture plates in DMEM with 10% FBS and 100 U/mL penicillin and 100 mg/mL streptomycin in an atmosphere of 5% CO2 at 37 °C and washed twice with 0.5 mL of PBS. Transfection was performed with 1 µg of plasmid DNA (pGL3-control) for each well. Luciferase assays were performed as described in the protocol of Steady-Glo Luciferase Assay System (Promega, Madison, WI). Luciferase relative light units (RLU) were analyzed by luminometer (Fluoroskan AscentFL, Thermo Labsystems, Finland). The protein concentrations of the cell lysates were measured as described in the protocol of NanoOrange Protein Quantitation Kit (Molecular Probes, Eugene, OR) using bovine serum albumin as a standard. The expressed luciferase shown in Figure 1 represent the amount (mole quantity) which is standardized for total protein content of the cell lysate.

Figure 1. Comparison of transfection efficiency of DNA complexes with cationic lipids. Open bars indicate the transfection efficiency in the dark conditions. Closed bars indicate that under UV irradiation.

Figure 2. Complexes that protect pGL3-control against DNase I digestion. Lane 1, DNA (UV+); lane 2, DNA (UV-); lane 3, DNA recovered from KNBN12 complex (UV+); lane 4, DNA recovered from KNBN12 complex (UV-); lane 5, DNA recovered from RNBN12 complex (UV+); lane 6, DNA recovered from RNBN12 complex (UV-); lane 7, undigested DNA. Band marked by arrow mean intact supercoiled plasmid.

lipofectin (a commercially available tertiary ammonium lipid/DOPE, 1,2-dioleyl-sn-glycero-3-phosphoethanolamine, mixture), the transfection efficiency of KNBN12 and RNBN12 was 13 and 3 times greater without the UV irradiation and more than 19 and 10 times greater with the UV irradiation, respectively. Also, since the photoirradiation did not increase the transfection efficiency of KBN12 (an analogue without a photocleavable structure), the decomposition of lipid structures must be involved in improved transfection efficiency. The higher basicity of RNBN12 than KNBN12 would result in lower efficiency due to less DNA release. DNA protection is also an important factor involved in the high transfection efficiency for nonviral gene delivery (19). Therefore, to ascertain whether cationic lipids protect DNA from nuclease, the protection ability of KNBN12 and RNBN12 against the hydrolysis of DNase I was investigated (Figure 2).3 Without UV irradiation to cationic lipid aggregate/DNA complexes the intact supercoiled plasmid DNA band was still observed 3 Lipid/DNA (3 µg of pGL3-control) complexes were prepared in 17 µL of PBS (phosphate buffered saline) at a charge ratio of 5 with or without UV irradiation (365 nm, 5min, 3.5 mW/cm2). The samples were incubated at 37 °C in the presence of DNase I (30 unit in 3 µL, Wako Chemicals, Tokyo, Japan) for 15 min. The samples were mixed with 5 µL of stop buffer (250 mM EDTA in PBS) under mild vortexing, followed by adding 5 µL of 10% sodium dodecyl sulfate and incubated at room temperature for 1 h. Sample solutions (10 µL) were analyzed by gel electrophoresis (0.8% agarose).

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Figure 3. Photostimulation of membrane fusion. Fluorescent intensity of rhodamine in DPPC liposomes is quenched by cationic lipids when membrane fusion occurs.

after DNase I treatment (lane 4 and 6), although some nicking and decomposition of plasmid DNA occurred under recovering conditions with SDS (lane 7). On the other hand, intact band completely disappeared with UV irradiation (lane 3 and 5). Both KNBN12 and RNBN12 exhibited high protection ability, but 5-min UV irradiation (365 nm, 3.5 mW/cm2) lowered this ability. These findings suggest that photoirradiation lowers the affinity of lipids toward DNA and makes it easier for the nuclease to access to DNA. The hypothesis proposed by Szoka et al. (20) suggests that dissociation of transfecting DNA from lipidic vectors is an important step for the successful nuclear transport and final expression of a transgene. In the case of such photocleavable lipids, the photoenhanced DNA release also contributes to the increase of transfection efficiency. Photoirradiation may improve transfection efficiency by facilitating the fusion between endocytic vesicles and unstable photocleaved lipids, thus making the endocytic vesicles unstable. Therefore, the ability of photoirradiation to facilitate membrane fusion was investigated: Liposomes, consisting solely of 1,2-dipalmitoyl-sn-glycero3-phosphocholine (DPPC), were labeled using 1 mol % fluorescent rhodamine-modified phosphotidylethanolamine. Furthermore, a lipid dispersion consisting solely of photocleavable lipids was prepared and mixed with the above-mentioned liposomes. Then, the fluorescent intensity of rhodamine was measured after UV irradiation (365 nm, 3.5 mW/cm2) (Figure 3). The results showed that only when KNBN12 or RNBN12 was used and UV was irradiated was a large degree of decrease in fluorescent intensity seen because the fluorescence of rhodamine was quenched by the electron transfer from the amino group of uncleaved amino acid in the vicinity of fusion. Furthermore, the intracellular trafficking of added lipids were examined by confocal fluorescence microscopy. First of all, COS-1 cells were transfected with the pEGFP-Endo gene (Clonethech, Palo Alto, CA) which expresses a RhoB-fused green fluorescent protein. RhoB has been known to be an endosomal localization signaling protein (21). At transfected COS-1 cells, therefore, the lipid membrane of endocytic vesicles was visualized green. Next, lipid aggregate/DNA complexes, where photocleavable lipids were mixed with 1 mol % rhodaminemodified phosphotidylethanolamine, were prepared and added to cells. As was the case with the transfection, after

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3 h of incubation, the cells were fixed and examined by confocal fluorescent microscopy. The result (data not shown) represented that the localization of the red fluorescence for administered lipids mostly matched the green fluorescence for the lipid membranes of cytoplasmic vesicles, such as endosomes. This finding confirms not only the internalization of the DNA complex by endocytosis, but also the existence of DNA complexes in endocytic vesicles when photoirradiation improves transfection efficiency. In conclusion, we succeeded in developing a new transfection technique using cationic lipids having photocleavable spacer by irradiating UV after adding DNA complexes to cells. The transfection efficiency of this technique was substantially enhanced compared to nonirradiating transfection. The reason for this increased transfection efficiency would be that when lipoplexes enter cells, the photocleavage of lipids facilitates the escape of DNA from endocytic vesicles by not only the stimulated fusion with endocytic vesicles, but also enhanced dissociation of the cationic lipid-DNA complex. ACKNOWLEDGMENT

This study was supported by a Grant from Osaka City University (Special Research Promotion Program) and a Health Sciences Research Grant for Human Genome, Tissue Engineering Food Biotechnology from the Ministry of Health, Labor and Welfare of Japan (13I-5). LITERATURE CITED (1) Felgner, P. L., Barenholz, Y., Behr, J. P., Cheng, S. H., Cullis, P., Huang, L., Jessee, J. A., Seymour, L., Szoka, F., Thierry, A. R., Wagner, E., and Wu, G. (1997) Nomenclature for synthetic gene delivery systems. Hum. Gene Ther. 8, 511512. (2) MacLachlan, I., Cullis, P., and Graham, R. W. (1999) Progress towards a synthetic virus for systemic gene therapy. Curr. Opin. Mol. Ther. 1, 252-259. (3) Brown, M. D., Scha¨tzlein, A. G., and Uchegbu, I. F. (2001) Gene delivery with synthetic (non viral) carriers. Int. J. Pharm. 229, 1-21. (4) Nishikawa, M., and Huang, L. (2001) Nonviral vectors in the new millennium: delivery barriers in gene transfer. Hum. Gene Ther. 12, 861-870. (5) Harashima, H., Shinohara, Y., and Kiwada, H. (2001) Intracellular control of gene trafficking using liposomes as drug carriers. Eur. J. Pharm. Sci. 13, 85-89. (6) Zhou, X., and Huang, L. (1994) DNA transfection mediated by cationic liposomes containing lipopolylysine: characterization and mechanism of action. Biochim. Biophys. Acta 1189, 195-203. (7) Ouahabi, A. El, Thiry, M., Pector, V., Fuks, R., Ruysschaert, J. M., and Vandenbranden, M. (1997) The role of endosome destabilizing activity in the gene transfer process mediated by cationic lipids. FEBS Lett. 414, 187-192. (8) Noguchi, A., Furuno, T., Kawaura, C., and Nakanishi, M. (1998) Membrane fusion plays an important role in gene transfection mediated by cationic liposomes. FEBS Lett. 433, 169-173. (9) Mui, B., Ahkong, Q. F., Chow, L., and Hope, M. J. (2000) Membrane perturbation and the mechanism of lipid-mediated transfer of DNA into cells. Biochim. Biophys. Acta 1467, 281292. (10) Wattiaux, R., Laurent, N., Coninck, S. W., and Jadot, M. (2000) Endosomes, lysosomes: their implication in gene transfer. Adv. Drug Deliv. Rev. 41, 201-208. (11) Farhood, H., Serbina, N., and Huang, L. (1995) The role of dioleoyl phosphatidylethanolamine in cationic liposome mediated gene transfer. Biochim. Biophys. Acta 1235, 289295.

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