Novel Polyamine−Dialkyl Phosphate Conjugates for Gene Carriers

Nakai, T., Kanamori, T., Sando, S., and Aoyama, Y. (2003) Remarkably size-regulated Cell invasion by artificial viruses. Saccharide-dependent self-agg...
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Bioconjugate Chem. 2004, 15, 824−830

Novel Polyamine-Dialkyl Phosphate Conjugates for Gene Carriers. Facile Synthetic Route via an Unprecedented Dialkyl Phosphate Takehisa Dewa,*,†,‡ Yukari Ieda,† Kazuyuki Morita,† Li Wang,§ Robert C. MacDonald,§ Kouji Iida,⊥ Keiji Yamashita,† Naoto Oku,| and Mamoru Nango*,† Materials Science and Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555 Japan, PRESTO (JST), Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208, Nagoya Municipal Industrial Research Institute, 3-4-41 Rokuban-cho, Atsuta-ku, Nagoya 456-0058 Japan, and Department of Medical Biochemistry and COE Program in the 21st Century, University of Shizuoka School of Pharmaceutical Sciences, 52-1 Yoda, Shizuoka, Japan. Received March 25, 2004; Revised Manuscript Received April 22, 2004

To develop a novel nonviral gene carrier, three types of polyamine-dialky phosphates conjugates were synthesized via an unprecedented synthetic intermediate, dimerized dicetyl phosphate (DCP) anhydride, and the transfection efficiency and the complexation properties of the conjugate-DNA were evaluated. Condensation of DCP by 1,3,5-triisopropylbenzenesulfonyl chloride, TPSCl, gives the dimerized anhydride, which is stable enough to isolate by column chromatography in ∼90% yield. The anhydride is reactive with various amines, i.e., spermidine, spermine, and polyethylenimine (PEI(1800)), providing corresponding polyamine-DCP conjugates via phosphoramidate linkage. The polyamine-DCP conjugates exhibited moderate transfection efficacy evaluated by β-galactosidase assay. The conjugate-DNA complex was observed by using an atomic force microscope (AFM), revealing that the PEI(1800)-DCP conjugate, which showed the most efficient transfection, enables the formation of the more compact complex with DNA.

INTRODUCTION

Development of more efficient and safer gene-transfer systems is one of the most challenging aspects of gene therapy (1). The two major approaches at present for gene transfer are viral and nonviral systems. The latter is safer and easier for mass production despite the higher efficiency of the former. Therefore, nonviral systems, such as cationic liposomes and polycations (2-8) as well as nonionic carriers (9), continue to be developed to improve their efficiency. The mechanism of gene delivery by such cationic carriers probably involves an endosomal pathway (10); however, the details remain to be clarified. In this regard, the polymorphism including lamellar, multilamellar, and hexagonal structures of polycation-DNA complexes have been studied (11-16). A nucleosome-like structure in which DNA wraps around an aggregate of dioctadeylamidoglycylspermine (DOGS)1 molecules has been reported by Yoshikawa et al. by using fluorescence and electron microscopy (11). The nucleosome-like assemblies further associate with each other to form a network, which may play a role in effective transfection. However, * To whom correspondence should be addressed. Tel/Fax: +81-52-735-5226. E-mail: [email protected]. † Nagoya Institute of Technology. ‡ PRESTO (JST). § Northwestern University. ⊥ Nagoya Municipal Industrial Research Institute. | University of Shizuoka School of Pharmaceutical Sciences. 1 Abbreviations: DOGS, dioctadeylamidoglycylspermine; PCL, polycation liposome; PEI, polyethylenimine; cetyl-PEI, cetylated polyethylenimine; DCP, dicetyl phosphate; AFM, atomic force microscopy; EtBr, ethidium bromide; DLS, dynamic light scatterig; TPSCl, 1,3,5-triisopropylbenzenesulfonyl chloride; FBS, fetal bovine serum.

the relationship between polycation-DNA assembly and the efficiency of gene transfection is far from wellunderstood. We have recently reported that polycation liposomes (PCL) composed of cetylated polyethylenimine (cetyl-PEI) possess high gene transfer activity (17-20). In the most recent report, we proposed a possible mechanism of PCLmediated gene transfer wherein PCL-DNA complexes are taken up by endosomal pathway; this was based on tracking of fluorescence-labeled components, PCL lipid, cetyl-PEI, and DNA, as cetyl PEI-DNA complex is released and transferred into the nucleus via the cytosol (20). The key step of gene transfer mediated by cationic liposomes is thought to be entry into the nucleus (21). Thus, compaction of DNA should be crucial; both electrostatic and hydrophobic interactions in the cetyl PEIDNA complex are likely to be responsible for its effective compaction. PEI is used as a gene transfer vector by itself; however, it has inherent disadvantages, i.e., cytotoxicity and polydispersity. In this report, we synthesize a series of polyamine-dicetyl phosphate (DCP) conjugates via reaction of a novel synthetic intermediate, dimerized DCP anhydride, with various polyamines, spermidine, spermine, and PEI (Scheme 1). Since spermidine and spermine are naturally occurring polyamines, we expected low cytotoxicity. These conjugates exhibited moderate gene transfer activity. We also observed the morphology of the conjugate-DNA complex by using atomic force microscopy (AFM). We briefly discuss the relationship between the assembling structure of the conjugate-DNA and their transfection efficiency. EXPERIMENTAL PROCEDURES

General Methods. Unless stated otherwise, all chemicals and reagents were obtained commercially and used

10.1021/bc049925k CCC: $27.50 © 2004 American Chemical Society Published on Web 06/05/2004

Novel Polyamine Gene Carriers Scheme 1.

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Synthetic Strategy for Polyamine-DCP Conjugates via Synthetic Intermediate, 1, DCP Anhydride

without further purification. DCP was purchased from Sigma. PEI(1800) with an average molecular weight of 1800 was kindly provided by Nippon Shokubai Co., Ltd (Osaka, Japan). The PEI(1800) was purified by ultrafiltration with an ultrafilter membrane (YM-1, Amicon Corp., Danvers, MA). NMR spectra were recorded on either a Varian Gemini-2000 (300 MHz) or a Bruker AVANCE600 (600 MHz) instrument. Secondary ion mass spectra were obtained by using HITACHI M-2000S. Infrared spectroscopy was carried out on a PerkinElmer Spectrum 2000 FT-IR spectrometer. Suspensions of polyamine conjugates were prepared in Tris-HCl buffer (15 mM, pH 8.4) by ultrasonication for 3 min. To the suspension was added 5 µg of ColE1 plasmid DNA (6646 bp, Nippon Gene), followed by equilibration for 15 min at room temperature. Displacement of ethidium bromide, EtBr, intercalated into DNA was measured with a fluorescence spectrophotometer FP-777 (JASCO) at EtBr/ nucleotide ) 50/1 (mol/mol) with varying polyamine contents. EtBr fluorescence at 590 nm (excitation wavelength: 260 nm) was recorded to obtain the relative fluorescence intensity, I/I0, where I and I0 are fluorescence intensities in the presence and the absence of polyamine derivatives, respectively. Measurement of dynamic light scattering was carried out on a Nicomp Model 370 particle size analyzer. AFM images of the conjugate-DNA complexes were acquired with a JEOL JSPM-4210 equipped with a cantilever (MikroMasch, type A: 110 µm, 7.5 N/m), using the discrete contact mode (AC mode). A sample was deposited on a dry mica surface by spin coating, followed by drying at ambient pressure. For observation of a negatively charged specimen, e.g., ColE1 plasmid DNA, a mica surface was premodified with polylysine. For DLS and AFM measurements, suspensions of the polyamine conjugates were prepared in distilled and deionized water. Synthesis of Dimerized Dicetyl Phosphate Anhydride (1). Dicetyl phosphate, DCP (50 mg, 90 µmol), was dissolved in 1 mL of anhydrous pyridine. To this solution was added 1,3,5-triisopropylbenzenesulfonyl chloride (TP-

SCl, 277 mg, 0.91 mmol) as a powder. After the reaction mixture was stirred for a few hours at room temperature under a nitrogen atmosphere, DCP was converted into DCP anhydride, whose molybdenum blue-active spot appeared at Rf 0.97 (SiO2, CHCl3/MeOH/H2O, 13/6/1). The solvent was evaporated, and the crude product was purified by column chromatography (Aldrich silica gel, eluted by CHCl3). After being dried in vacuo, the isolated pure product, 1, was obtained as a white powder (44.3 mg, 41 µmol, 90% yield). 1H NMR (600 MHz, CDCl3) δ: 0.88 (t, 12H), 1.25 (br s, 96H), 1.37 (m, 8H), 1.70 (m, 8H), 4.0-4.2 (m, 8H); 31P NMR (243 MHz, CDCl3, 10% H3PO4 in D2O as an external standard) δ: -12.27, -0.13; 13 C NMR (150 MHz, CDCl3) 14.01, 22.68, 25.36, 25.45, 29.16, 29.36, 29.54, 29.60, 29.66, 29.71, 30.31, 31.92, 67.67, 69.12; FT-IR (KBr) ν (cm-1): 2849, 1471, 1294, 1108, 1021, 952; SIMS for (C64H133O7P2)+ calcd 1075.9, found 1075.4. Synthesis of Dicetyl Phosphate-Spermidine Conjugate (2). To a solution of spermidine (6.74 mg, 46 µmol) in 0.5 mL of dry pyridine was added 1 (10 mg, 9.3 µmol) in 0.1 mL of anhydrous pyridine. The reaction mixture was stirred under nitrogen atmosphere for 5 h at room temperature. Removal of solvent under reduced pressure, followed by column chromatography [1 g of silica gel, eluting with CHCl3/MeOH/Et3N (10/1/0.1, v/v/v) and then CHCl3/MeOH/Et3N (1/1/0.02, v/v/v), gave 4.9 mg (78%) of 2 as a white powder. 1H NMR (300 MHz, CDCl3) δ: 0.88 (t, 6H), 1.26 (s, 52H), 1.56 (m, 4H), 1.70 (m, 6H), 2.61-3.12 (bm, 12H), 3.98 (bm, 4H); FT-IR (KBr) ν (cm-1): 3235, 2917, 2850, 1636, 1467, 1210, 959; SIMS for (C39H85N3O3P)+ calcd 674.6, found 674.8. Synthesis of Spermine-Dicetyl Phosphate Conjugate (3). To a solution of spermine (10 mg, 49 µmol) in 0.5 mL of dry pyridine was added 1 (10 mg, 9.3 µmol) in 0.1 mL of anhydrous pyridine. The reaction mixture was stirred under a nitrogen atmosphere for 5 h at room temperature. Removal of solvent under reduced pressure was followed by column chromatography [1 g of silica gel, eluting with CHCl3/MeOH/Et3N (10/1/0.1, v/v/v)] to re-

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move free DCP component. The product absorbed on the silica gel was eluted by washing with CHCl3/MeOH/Et3N (1/1/0.02, v/v/v), giving 4.5 mg (66%) of 3 as a white powder. 1H NMR (300 MHz, CDCl3) δ: 0.88 (t, 6H), 1.26 (s, 52H), 1.42 (bm, 8H), 1.55 (bm, 4H), 2.15 (br, 5H), 3.17 (bm, 8H), 3.63 (bm, 4H), 3.97 (m, 4H). 31P NMR (243 MHz, CDCl3, 10% H3PO4 in D2O as external standard) δ: 9.76. FT-IR (KBr) ν (cm-1): 3426, 3235, 2917, 2850, 1637, 1466, 1212, 1012; SIMS for (C42H92N4O3P)+ calcd 731.7, found 731.5. Synthesis of PEI(1800)-Dicetyl Phosphate Conjugate (4). To a solution of PEI(1800) (16.7 mg, 9.3 µmol) in 0.5 mL of dry pyridine was added 1 (10 mg, 9.3 µmol) in 0.1 mL of anhydrous pyridine. The reaction mixture was stirred under a nitrogen atmosphere for 5 h at room temperature. Removal of solvent under reduced pressure was followed by column chromatography [1 g of silica gel, eluting with CHCl3/MeOH/Et3N (10/1/0.1, v/v/v)] to remove free DCP component. The product absorbed on the silica gel was extracted by washing out with CHCl3/ MeOH/Et3N (1/1/0.02, v/v/v), giving 3.5 mg (20.7%) of 4. 1 H NMR (300 MHz, CDCl3) δ: 0.88 (t, 6H), 1.1-1.6 (br, 83H), 1.65 (bm, 4H), 2.5-3.1 (bm, 125H), 4.1 (m, 4H); FT-IR (KBr) ν (cm-1): 3253, 2915, 2846, 1637, 1466, 1214, 1033. Cell Culture. Primary vascular smooth muscle cells were obtained by removal of the thoracoabdominal aortas of Wistar rats, which were then stripped of endothelium and adventitia (22). Medial VSMCs were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM), 20% fetal bovine serum (FBS), 200 units/mL penicillin, 200 µg/mL streptomycin (all Gibco, Gaithersburg, MD) at 37 °C with 5% CO2. At confluence, the cells were passaged using 0.25 mg/mL trypsin/EDTA (Clonetics, Walkersville, MD). Transfection Procedure. The cells were seeded in 96-well plates at 24 h before transfection at densities to give about 80% confluence at the time of transfection. β-Galactosidase plasmid DNA (purchased from Clontech Labtories Inc. (Palo Alto, CA) and propagated and purified by Bayou Biolabs (Harahan, LA)) was diluted in serum-free cell culture medium to 20 µg/mL. The transfection reagents were suspended in D-PBS at 1 mg/ mL, diluted in the same volume of serum-free cell culture medium as that of DNA (the dilution factor was varied as appropriate to give the desired ratio of the transfection reagent to DNA), and then pipetted into plasmid DNA. The resultant complexes were incubated at room temperature for 15 min and then added to the cells that were either in medium lacking serum or medium containing 20% fetal bovine serum (FBS). At 3 h after addition of complexes, the cells were washed with PBS and fresh medium containing 20% FBS was added. Cells were assayed for β-galactosidase activity 24 h after transfection with a microplate fluorometric assay (23), modified by inclusion of a heating step (50 °C, 45 min) to inactivate endogenous enzyme activity. Following aspiration of the medium from each well, the cells were washed once with PBS and then lysed by addition of 100 µL of lysis buffer (0.03% Triton X-100 in 100 mM HEPES, pH 7.8, containing 1 mM MgSO4, 10 mM KCl). The plates were placed at 50 °C for 45 min and then allowed to cool to room temperature. Into each well was added 10 µL of fluorescein di-β-D-galactopyranoside, FDG (100 µM). Fluorescence was measured with a microplate fluorimeter (Model 7620, Cambridge Technology Inc.) after incubation at 37 °C overnight. Under these assay conditions, 0.1 milliunit of β-galactosidase corresponded to 16 000 fluorescence units.

Dewa et al. RESULTS AND DISCUSSION

Synthetic Strategy for Polyamine-Dialkyl Phosphate Conjugates via a Synthetic Intermediate. Our preliminary idea for the syntheses of polyamine-DCP conjugates was to use the bromoethylated compound, 5, as a synthetic intermediate via condensation of DCP and 2-bromoethanol (Scheme 1) by using TPSCl (24, 25). Instead of the expected adduct, 5, however, we obtained another product having a high Rf value, 0.97 (Rf ) 0.56 for DCP by eluting CHCl3/MeOH/H2O ) 13/6/1, v/v/v). The resulting product is stable enough to be isolated by column chromatography. SIMS indicates almost double molecular mass, 1075.4 (546.9 for DCP). The observed IR absorption band at 952 cm-1 is assignable to the P-O-P stretching mode. The 31P NMR spectrum of the product, whose signals appear at δ ) -12.27 and -0.13 ppm, is clearly distinguishable from that of DCP having a signal at δ ) 2.15 ppm. This evidence, taken together, leaves no doubt that the product is dimerized DCP in anhydrous form, 1 [(C64H133O7P2)+ calcd 1075.9], connected via P-O-P bonding. Hydrolytic decomposition of 1 is negligible for a few days in chloroform or anhydrous pyridine at room temperature. In wet chloroform, however, the decomposition is accelerated, and under acidic and basic conditions, e.g., chloroform containing small amount of diluted HCl, sodium carbonate, ammonia, or triethylamine, the decomposition is further accelerated. There are some prior examples in the synthesis of pyrophosphate derivatives bearing small alkyl moieties, methyl, ethyl, propyl, and butyl (26); however, the dimerized anhydride, 1, bearing phospholipid-like long alkyl chains is an unprecedented compound. This compound is a convenient synthetic intermediate for formimg phosphoramidate bonds, as described below. The anhydride 1 readily react with amines, e.g., spermidine, spermine, and even polymer, PEI(1800), to form the phosphoramidate, P-N bond, providing the corresponding adducts, 2, 3, and 4, respectively. The reactivity toward these nucleophiles indicates that the anhydrous compound 1 is potentially a good synthetic intermediate for making polyamine-dialkyl phosphate conjugates via the P-N bond. When reacted with spermine, for example, the adduct 3 readily forms, concomitantly with the loss of DCP. The 31P NMR signal of compound 3 is shifted downfield, to 9.76 ppm compared with that of DCP at 2.15 ppm, indicating the formation of the P-N bond in the compound. The downfield-shifted signal is attributed to the lower electronegativity of the nitrogen atom in spermine relative to the oxygen atom in DCP. Although structural ambiguity of the resulting adducts, 2, 3, and 4,2 cannot be completely eliminated, anhydride 1 is a useful synthetic intermediate because it is (1) stable yet reactive with amino groups, (2) very simple and easy to prepare, and (3) produced in high yield (∼90%). In addition, it should be noted that a potential advantage of the conjugation via P-N bond is that it may cleave in endosomes, because the linkage is very unstable under acidic conditions. Such a cleavable bond would have advantages in terms of disruption of endosomes: the cleaved DCP portion could facilitate release of the polyamine-DNA complex from the endosome. In addition, it should confer biodegradability on the compound. 2 The asymmetric molecule, spermidine, has two distinguishable primary amines. Product 2, bearing a spermidine moiety, has two isomers, which, to date, have not been identified spectroscopically. Along the similar lines, content of primary amine of PEI(1800) in the product 4 is 25% in total amino moieties; thus, the structural uncertainty cannot be avoided.

Novel Polyamine Gene Carriers

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Figure 2. Changes in fluorescence intensity of ethidium bromide intercalated into Col E1 plasmid DNA-polyamine compounds: (A), spermidine (b), spermine (2), and PEI(1800) (9); (B), spermidine-DCP (O), spermine-DCP (4), and PEI(1800)-DCP (0). Figure 1. Transfection efficacy of polyamine-DCP conjugates 2-4 on VSMC, in the absence (A) and the presence of 20% of FBS (B). The ratios of compound-β-galactosidase plasmid DNA (w/w) were 1.5/1, 3/1, and 6/1, respectively.

These compounds could hence provide new category of polycationic lipids. Transfection Efficacy. The polyamine-DCP conjugates 2-4 were subjected to transfection assay using the β-galactosidase expression system. Figure 1A shows the transfection efficiency of 2, 3, and 4 evaluated according to β-galactosidase activity (milliunit/well), in the absence of FBS. Compared with the commercially available transfection reagent, O-ethyl DOPC (E-DOPC) (5), which produced 0.1 milliunit/well transgene expression, compounds 2-4 exhibit moderate efficiency, ∼30 to 50% of the efficiency of E-DOPC. The transfection efficiencies of the components themselves, i.e., DCP, spermidine, spermine, and PEI(1800), were almost negligible (data not shown). Thus, the transfection activity results from the conjugation of two moieties: a hydrophilic polyamine and a hydrophobic DCP. Compound 4 shows the highest efficiency at 3/1 (w/w) of 4-DNA, whereas the efficiency of the other compounds are comparable and insensitive to the compound-DNA ratio within the range of error. In Figure 1B is shown the transfection efficiency in the presence of 20% FBS. The efficiency of these compounds was not influenced by the presence of 20% FBS, retaining 80-100% of the activity (except compound 4 at 3/1 (w/ w)). Such serum-resistant activity was also observed for the PCL gene transfection system previously reported (19). It is well-known that serum often inhibits transfection; such inhibition is due to binding of negatively charged serum proteins to the cationic transfection reagents resulting in forming aggregates ineffective to the transfection. Although it is not clear why the polyamine-DCP conjugates are not influenced by the presence of the serum, the polyamine part may be assumed to efficiently interact with DNA via electrostatic interactions.

Compound 4 exhibits maximum efficacy at a 3/1 of 4-DNA ratio in this series (Figure 1A). We examine complexation of the polyamine-DCP conjugates with DNA by intercalation of EtBr, DLS, and AFM as described below. Complexation with ColE1 Plasmid DNA. It was found that DNA (ColE1 plasmid DNA) complexes with various polyamines (A) and polyamine-DCP conjugates (B) by monitoring the decrease of fluorescence from EtBr initially intercalated into DNA (Figure 2A and 2B). With an increase in the cation (N)/anion (P) ratio, defined as N/P, the relative fluorescence intensity decreased as a result of complexation of DNA and polyamine. As shown in Figure 2A, the polymeric molecule, PEI(1800) (N ∼ 42 per molecule) most efficiently forms a complex with DNA; the complexation was almost complete at N/P ) 3. Spermidine (N ) 3 per molecule) and spermine (N ) 4 per molecule) are less effective than PEI(1800), however, with complexation being complete at around N/P ∼ 7. For polyamine-DCP conjugates (Figure 2B), the tendency for the fluorescence intensity to decrease is due to the complexation with DNA in the same way as that of the free amines, although influence of the hydrophobic alkyl chain in the complexation is not at all pronounced. The mean particle diameters of various polyamineDCP conjugates in an aqueous suspension, as given by dynamic light scattering, is summarized in Table 1: 155 ( 54 nm for 2, 173 ( 46 nm for 3, and 128 ( 38 nm for 4. AFM images for the suspension of these compounds exhibited spherical or ellipsoidal particles, whose sizes, defined according to diameter for the spheres and major × minor axes for the ellipsoid, were 132 nm for 2, 156 nm for 3, and 209 × 145 nm for 4 (Figure 3A, B, and C, respectively). The particle sizes for these compounds as observed by AFM show good agreement with those obtained by DLS. The molecular shapes of these compounds are regarded to be the “cone” type, due to the attachment of the large polyamine moiety. We have endeavored to make liposome from these compounds; however, entrapment of a fluorescence probe, calcein, was

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Dewa et al.

Table 1. Estimated Size and Shape of 2-4 and Complexes with ColE1 DNA Suspended in Aqueous Solutiona AFM/shape (size (nm)) and height (nm)b

DLS (nm) compound

conjugate alone

complex with DNA

2 3 4

155 ( 54 173 ( 46 128 ( 38

409 ( 115 237 ( 127 115 ( 40

conjugate alone sphere (132) sphere (156) ellipsoid (209 × 145)

complex with DNA 21 24 12

aggregate (∼1000 × 437) aggregate (569 × 317) sphere (120)

16 20 20

a The ratio of polyamine conjugate-DNA was 3/1 (w/w). Complexation of polyamine conjugate with DNA was carried out in water. Sample solution was spreaded on a mica surface and dried. AFM images were obtained under dry condition. Width and height of the complexes were estimated from the AFM images in Figures 3 and 4.

b

Figure 3. AFM images of arrays of compounds 2 (A), 3 (B), and 4 (C). The compound was suspended in distilled water and then dropped onto a mica surface by spin coating. All scale bars represent 200 nm. The object indicated by the arrow is discussed in the text.

Figure 4. AFM images of polyamine-ColE1 plasmid DNA complexes: (A), ColE1 plasmid DNA alone on polylysine-treated mica; (B), spermine-DNA; (C), PEI(1800)-DNA; (D), spermidine-DCP-DNA; (E), spermine-DCP-DNA; (F), PEI(1800)-DCP-DNA. The polyamine-DNA ratio was 3/1 (w/w). Scale bars inserted in these images represent (A) 200, (B) 1000, (C) 200, (D) 500, (E) 500, and (F) 200 nm, respectively. The objects indicated by arrow(s) are discussed in the text.

impossible. Therefore, we assume that the particle consisting of polyamine-DCP conjugates is a micellelike aggregate. Particle sizes of the conjugate-DNA (3/1: w/w) complex evaluated by DLS is 409 ( 115 nm for 2, 237 ( 127 nm for 3, and 115 ( 40 nm for 4, respectively (Table 1). With increase in the size of polyamine portion, the particle size significantly decreases. AFM images support

this conclusion. Figure 4 shows an AFM image of DNA (A) and of the complexes it forms with various polyamines (B-F). Figure 4A reveals a clear image of partially coiled ColE1 plasmid DNA (6646 bp), whose size is estimated to be 300-400 nm. The height is ∼2 nm, corresponding to the diameter of B-form DNA, ca. 2.4 nm. When DNA was complexed with compound 2, a “spider nest”-like structure was observed (Figure 4D). The size of the

Novel Polyamine Gene Carriers

Figure 5. Hypothetical complexation of polyamine-DCP conjugates 2-4, with ColE1 plasmid DNA based on the AFM images as shown in Figures 3 and 4. The bright-gray sphere represents micelle of polyamine-DCP conjugate 2, 3, or 4. When the plasmid DNA complexes with the conjugate micelles via electrostatic interaction, different types of conjugate-DNA, 2-DNA, 3-DNA, and 4-DNA, form depending on the conjugates. The solid lines and the dark-gray spheres depicted in the complexes represent the plasmid DNA and aggregates consisting of the conjugate and DNA, respectively.

“quasi-ellipsoidal core region” is ∼1000 × 437 nm and the height is ∼16 nm. The height of the radiating peripheral “nest” region is ∼2 nm, suggesting the nest region consists of free DNA. The height of the core region (∼16 nm) is clearly larger than that of the DNA part; therefore, the core region of the structure must consist of the 2-DNA complex. Thus, complexation of compound 2 and DNA gives rise to a segregated array, having complex and free DNA portions. Compound 3 forms a similar but distinguishably different complex structure with DNA (Figure 4E), which resembles a “pearl necklacelike” aggregate (11), 569 × 317 nm in the plane of the substrate and 4-20 nm in height. The size of the pearl parts is 120-170 nm in diameter and 11-20 nm in height. These parts are connected by a region that is 4-5 nm in height. It appears from the image that the assembly consists of a tightly packed 3-DNA complex and regions of partially compacted DNA parts. Pronounced compaction of the complex was observed for the 4-DNA, which formed a spherical cluster (120 nm in width and ∼20 nm in height) (Figure 4F). The order of increasing compaction of DNA, 2 < 3 < 4, is consistent with the extent of intercalation of EtBr (Figure 2B); that is, the more tightly packed was the DNA complex, the lower was the extent of intercalation of EtBr. On the basis of the AFM images of the polyamine conjugateDNA complexes, the hypothetical shapes of the complexes are as depicted in Figure 5. Upon comparison of the observed DNA complex with free polyamines, spermine (Figure 4B) and PEI (1800) (Figure 4C), the complex size and shape are obviously distinguishable from the corresponding conjugate forms; much larger complexes form with these free polyamines. The dimension of the spermine-DNA complex (Figure 4B) is ∼3 µm; the compaction of DNA is obviously

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incomplete, judging from the presence of the “nest” portion of DNA that is similar to peripheral part of the 2-DNA complex (Figure 4D). The PEI(1800)-DNA complex is smaller (416 × 218 nm by 22 nm high) than the former complex, suggesting that the greater cationic charge makes the complex smaller. When one considers the effect of the hydrophobic portion in the polyamine compound on the size of the complex, it is clear that hydrophobic alkyl parts in the conjugates play an important role in the compaction of DNA, which is most prominently observed in conjugate 4 (Figure 4C vs 4F). From these results, the prominent transfection efficiency of 4 likely results from the more efficient compaction of DNA in the complex 4-DNA, whereas the lower activity of 2 and 3 is likely due to their much weaker compaction. Because high compaction is needed for transfection, 2 and 3 exhibited only moderate transfection activity. The efficiency of 2 and 3 does not depend on the dose in the range of 1.5/1 to 6/1 (w/w), whereas the efficacy of conjugate 4 decreases at 6/1 (w/w). This may be a consequence of the cytotoxicity of PEI portion. One may wonder why the morphologies of the complexes of 2 and 3 with DNA are so different despite the small structural difference in the cationic portion. It might come from the difference in delicate balance of hydrophilic/hydrophobic factor and/or molecular shapes which reflect a packing parameter. A wide variety of similar polyamine-dialkyl phosphate derivatives bearing different type of hydrophobic alkyl chain, e.g., shorter or unsaturated, is synthetically available. It should be intriguing to see the effect of hydrophilic/hydrophobic balance on not only the morphology of the complex with DNA but also transfection efficacy. Although the precise mechanism remains to be clarified, the compaction by these polyamine compounds probably plays an important role because entry into the nucleus is thought to be a key step and a smaller complex is likely to be associated with more efficient transfection. The conjugates in this study possibly decompose into separate polyamine and DCP portions in the endosome. We assume that two critically important factors are involved in the present transfection system: compaction of DNA by the polyamine part and with the hydrophobic dialkyl portion and decomposition of the conjugate in the endosome so as to liberate the DNA-polyamine complex to be transported into nucleous. As previously reported, DNA accompanied by cetyl-PEI enters into the nucleus. In conclusion, we described herein novel types of polyamine-dialkyl phosphate conjugates that have moderate gene transfection activity for the β-galactosidase assay. These conjugates are easy to prepare via a novel synthetic intermediate, dimerized DCP anhydride, 1. The synthetic approaches described herein are flexible and possess potential for the rationale design of highly efficient gene carriers with single or narrow ranges of molecular weight. It is one of critical aspects for practical usage. ACKNOWLEDGMENT

The authors thank professor Akihiro Yoshino, Nagoya Institute of Technology, for helpful discussion and comments. This study was supported by PRESTO (JST), the NITECH 21st Century COE Program “World Ceramics Center for Environmental Harmony”, NEDO, and Takemoto Oil and Fat Co. LITERATURE CITED (1) Kay, M. A., Liu, D., and Hoogerbrugge, P. M. (1997) Gene therapy. Proc. Natl. Acad. Sci. U.S.A. 94, 12744-12746.

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