DNA Complexing Lipopolythiourea - American Chemical Society

Isabelle Tranchant, Nathalie Mignet, Estelle Crozat, Jeanne Leblond, Christian Girard ... Biologiques 4, avenue de l'Observatoire 75270 Paris Cedex 06...
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Bioconjugate Chem. 2004, 15, 1342−1348

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DNA Complexing Lipopolythiourea Isabelle Tranchant, Nathalie Mignet, Estelle Crozat, Jeanne Leblond, Christian Girard, Daniel Scherman, and Jean Herscovici* Laboratoire de Pharmacologie Chimique et Ge´ne´tique CNRS-UMR 8115, INSERM-U640, ENSCP 11 rue Pierre et Marie Curie 75231 Paris Cedex 05, France, and Faculte´ des Sciences Pharmaceutiques Biologiques 4, avenue de l’Observatoire 75270 Paris Cedex 06, France. Received March 31, 2004; Revised Manuscript Received October 1, 2004

We present a neutral lipopolythiourea (DTTU) as a potential DNA-binding agent. Light scattering experiments showed that mixing a lipopolythiourea with dipalmitoylphosphatidylcholine (DPPC/DTTU) led to small particles with sizes ranging from 100 to 150 nm at optimum conditions. Setting a fixed DNA amount, an increasing amount of DTTU/DPPC or DPPC lipids was added. Particle size increased only with DTTU/DPPC, indicating that interaction occurred between the DTTU/DPPC particles and DNA. In the same way, only DTTU/DPPC limited the ethidium bromide accessibility to plasmid DNA. These data suggest that DTTU/DPPC liposomes associate to DNA, which was confirmed by agarose gel experiments. To prove the active part of the DTTU lipid itself in DNA compaction, pegoylatedlipid was used. Cholesterol-PEG2000 alone was not able to condense DNA. In contrast, DTTU/PEGcholesterol was able to retain plasmid DNA on an agarose gel. In vivo injection of DTTU/DPPC/ complexes was studied. Circulation time increase for noncationic particles as compared to cationic. More obvious was the lack of nonspecific accumulation in the lung, where a gain of 3 to 40 fold was measured.

INTRODUCTION

Among the many constructs that have been used to facilitate oligonucleotides, RNA, and plasmids intracellular delivery, cationic lipids and cationic polymers have received the greatest attention (1). Over the course of the past several years, it has become clear that the use of cationic lipids for nucleic acid in vivo delivery is compromised by substantial problems such as interaction with negatively charged extracellular components, complement activation, platelet aggregation, and related toxicity (2, 3) To overcome these hurdles, PEG-lipid was added to cationic lipoplexes aiming at reducing their blood clearance (4, 5) as was previously shown for conventional liposomes (6). Unexpectedly, the coating providing a steric barrier against opsonising proteins did not necessarily extend blood circulation time (7, 8). The necessity of new nonviral delivery methods prompted us and others to investigate nucleic acid complexation by noncationic systems. Efficient encapsulation of DNA plasmids inside small, neutral liposomes was achieved by the addition of optimized amounts of ethanol and calcium chloride to an aqueous mixture of small unilamellar vesicles (SUVs) and plasmid (9). High plasmid confinement into neutral lamellar phase was shown to be possible, without electrostatic interactions, at high lipid/DNA weight ratios (10). In the present approach, we present a neutral lipopolythiourea as a potential nucleic acid binding agent. The relatively acidic thiourea NH protons (11), with a strong hydrogen-bond donor capability, can establish multipoint hydrogen-bonded patterns with complementary acceptor groups in a specific and predictable manner. Moreover, * To whom correspondence should be addressed. E-mail: [email protected].

bis-thiourea moieties selectively bind to dihydrogenphosphate via multitopic hydrogen bonding, giving stronger complexes with H2PO4- than any synthetic neutral receptor known so far (12). Finally, thioureas show a lower tendency to self-associate than the corresponding urea analogues (11). All these data prompted us to prepare and study the physicochemical properties of lipopolythiourea/DNA complexes. To compare these complexes to cationic lipoplexes, we based the design on lipopolyamine the previously reported RPR 122766 (13). We chose the same hydrophobic anchor connected to a trithiourea by a succinic linker to obtain a comparable size for the two lipids (Figure 1). MATERIALS AND METHODS

All solvents were purchased from SDS (Peypin France). Dichloromethane was distilled from P2O5. DMF was dried over 3 Å molecular sieves and pyridine over KOH. All chemicals were purchased from Sigma-Aldrich-Fluka or Lancaster. Other solvents and products were used without further purification. Reactions were monitored by thin-layer chromatography using Merck precoated 60 F254 silica gel plates. Column chromatography was performed over SDS (Peypin France) 35-70 µm silicagel according to the method of Still, Khan, and Mitra (14). 1H and 13C NMR spectra were recorded on a BRUKER Avance DRX300 spectrometer at 300.13 MHz for the proton and 75.47 MHz for the carbon. NMR spectra were processed using xwinnmr (Bruker) or SwaN-MR (15). MS and elemental analysis were carried out at the Analysis Department of Aventis, Vitry sur Seine France. Dipalmytoylphosphatidylcholine (DPPC), egg phosphatidylcholine (EPC), and phosphatidylethanolamine-lissamine rhodamine were purchased from Aventi Polar Lipids (Alabaster Alabama). All in-vivo experiments were performed with distilled water for injection. Size measurements were performed

10.1021/bc049920n CCC: $27.50 © 2004 American Chemical Society Published on Web 10/22/2004

DNA Complexing Lipopolythiourea

Bioconjugate Chem., Vol. 15, No. 6, 2004 1343

Figure 1. RPR 122766 and DTTU.

by dynamic light scattering (HPPS, Malvern Intruments). Ethidium Bromide assays were performed in a fluorospectrophotometer (Fluoromax2, Jobin Yvon). Synthetic Procedures. [2-(3-Ditetradecylcarbamoylpropionylamino)-ethyl]-carbamic Acid tert-Butyl Ester (4). PyBop (4.6 g, 8.84 mmol), (2-amino-ethyl)-carbamic acid tert-butyl ester (3) (1.56 g, 9.72 mmol), and diisopropylethylamine (4.24 mL, 24.31 mmol) were added successively to a stirred solution of N,N-ditetradecyl-succinamic acid (2) in dichloromethane (88 mL) at room temperature. After 4 h the solution was filtrated and evaporated to dryness. The resulting oil was purified by chromatography (heptane/ethyl acetate 1:1 then 2:8) to yield 3.79 g of 4 (66%). 1 H NMR (CDCl3): δ (ppm) 0.87 (t, 6H, J ) 6.6 Hz, H-14′), 1.25 (m, 44H, H-4′-H-11′), 1.43 (s, 9H, (CH3)3), 1.48 (m, 4H, H-2′), 2.56 (t, 2H, J ) 6.7 Hz, H-2), 2.69 (t, 2H, J ) 6.2 Hz, H-3), 3.28 (m, 4H, H-1′), 3.3 (m, 4H, H-5 and H-6). 13 C NMR (CDCl3): δ (ppm) 14.07 (C-14′), 22.56 (C-13′), 26.99 ((CH3)3), 27.73 (C-3′), 28.29 (C-2), 28.59 (C-2′), 29.27 (C-4′-C-11′), 29.55 (C-3), 31.41 (C-12′), 39.85 (C-5), 40.39 (C-6), 46.21 et 47.98 (C-1′), 78.77 (C-8), 156.33 (C-7), 171.46 (C-1), 173.14 (C-4).MS (IS) calcd. for C39H77N3O4 652,05, found 653.05 (MH+), Anal. Calcd. C ) 71.84; H ) 11.90; N ) 6.44, found C ) 70.42; H ) 12.45; N ) 6.49. 2-(3-Ditetradecylcarbamoyl-propionylamino)-ethylammonium Trifluoroacetate (5). The carbamate 4 (1.59 g, 2.44 mmol) was dissolved in trifluoroacetic acid then the solution was stirred for 4 h. The acid was removed by evaporation then the residue was coevaporated twice with cyclohexane at room temperature. The resulting oil was dried overnight over NaOH pellets under vacuum to afford 5 (100%) 1 H NMR (CDCl3): δ (ppm) 0.91 (t, 6H, J ) 6.6 Hz, H-14′), 1.29 (m, 44 H, H-4′-H-11′), 1.51 (m, 4H, H-2′), 2.59 (t, 2H, J ) 6.7 Hz, H-2), 2.71 (t, 2H, J ) 6.2 Hz, H-3), 3.29 (m, 4H, H-1′), 3.31 (m, 4H, H-5 and H-6). 13 C NMR (CDCl3): δ (ppm) 14.00 (C-14′), 22.67 (C′-13′), 27.35 (C-3′), 27.95 (C-2), 28.53 (C-2′), 29.65 (C-4′-C-11′), 30.70 (C-3), 31.94 (C-12′), 37.83 (C-5), 40.08 (C-6), 47.85 et 49.42 (C-1′), 171.72 (C-1), 173.26 (C-4). (2-{3-[2-(3-Ditetradecylcarbamoyl-propionylamino)-ethyl]-thioureido}-ethyl)-carbamic Acid tert-Butyl Ester (7). The amine 5 (1.62 g, 2.44 mmol) was treated by triethylamine (1.36 mL, 9.76 mmol) for 15 min then the mixture was dissolved in dichloromethane (24.4 mL) and 6 was added (0.59 g, 2.92 mmol). The reaction mixture was stirred at room temperature for 12 h then the solvent was removed. The resulting oil was purified by chromatography (ethyl acetate/heptane 6:4 then pure ethyl acetate) to give 1.34 g of the thiourea 7 (yield 73%). 1 H NMR (CDCl3): δ (ppm) 0.67 (t, 6H, J ) 6.4 Hz, H-14′), 1.05 (m, 44 H, H-4′-H-11′), 1.26 (s, 9H, CH3)3), 1.35 (m, 4H, H-2′), 2.31 (m, 2H, H-2), 2.49 (m, 2H, H-3),

3.06 (m, 4H, H-1′), 3.11 (m, 4H, H-5 and H-9), 3.47 (m, 4H, H-6 and H-8), 7.14 (2H, H thiourea). 13 C NMR (CDCl3): δ (ppm) 13.95 (C-14′), 22.57 (C-13′), 26.92 ((CH3)3), 27.07 (C-3′), 27.78 (C-2), 28.39 (C-2′), 28.82 (C-4′-C-11′), 29.55 (C-3), 31.83 (C-12′), 39.45 (C-5, C-9), 43.63 (C-6 et C-8), 46.39 and 48.16 (C-1′), 79.24 (C-11), 156.53 (C-10), 171.72 (C-1), 173.71 (C-4), 182.97 (C-7). 2-{3-[2-(3-Ditetradecylcarbamoyl-propionylamino)-ethyl]-thioureido}-ethylammonium Trifluoroacetate (8). The carbamate 7 (1.5 g, 9.86 mmol) was reacted with trifluoroacetic acid (0.76 mL, 9.86 mmol) for 3 h, and then the mixture worked-up as for 5 afforded the amine 8 (100%). 1 H NMR (CDCl3): δ (ppm) 0.67 (t, 6H, J ) 6.4 Hz, H-14′), 1.05 (m, 44 H, H-4′-H-11′), 1.31 (m, 4H, H-2′), 2.31 (m, 2H, H-2), 2.49 (m, 2H, H-3), 3.06 (m, 4H, H-1′), 3.11 (m, 4H, H-5 and H-9), 3.44 (m, 4H, H-6 and H-8), 7.10, (2H, H thiourea). 13 C NMR (CDCl3): δ (ppm) 13.85 (C-14′), 22.49 (C-13′), 27.01 (C-3′), 27.72 (C-2), 28.29 (C-2′), 28.79 (C-4′-C-11′), 29.49 (C-3), 31.77 (C-12′), 39.42 (C-5, C-9), 43.75 (C-6 and C-8), 46.29 and 48.09 (C-1′), 171.72 (C-1), 173.71 (C-4′), 182.97 (C-7). (2-{3-[2-(2-{3-[2-(3-Ditetradecylcarbamoyl-propionylamino)-ethyl]-thioureido}-ethylamino)-ethyl]-thioureido}ethyl)-carbamic Acid tert-Butyl Ester (9). The amine 8 (1.47 g, 1.98 mmol) was treated by triethylamine (1.1 mL, 7.92 mmol) for 15 min then the mixture was dissolved in dichloromethane (20 mL) and 6 was added (0.46 g, 2.38 mmol). The reaction mixture was stirred at RT for 12 h, and then the solvent was removed. The resulting oil was purified by chromatography (ethyl acetate/heptane 6:4 then pure ethyl acetate/methanol 98:2) to give 1.14 g of the di-thiourea 9 (yield 67%). 1 H NMR (CDCl3): δ (ppm) 0.74 (t, 6H, J ) 6.6 Hz, H-14′), 1.12 (m, 44 H, H-4′-H-11′), 1.30 (s, 9H, CH3)3), 1.45 (m, 4H, H-2′), 2.41 (m, 2H, H-2), 2.58 (m, 2H, H-3), 3.12 (m, 4H, H-1′), 3.25 (m, 4H, H-5 and H-12), 3.56 (m, 8H, H-6, H-8, H-9, H-11), 7.14 (4H, H thiourea). 13 C NMR (CDCl3): δ (ppm) 14.06 (C-14′), 22.57 (C-13′), 27.11 (C-3′), 26.93 ((CH3)3), 27.79 (C-2), 28.38 (C-2′), 28.81 (C-4′-C-11′), 29.56 (C-3), 31.83 (C-12′), 39.55 (C-5, C-12), 43.66 (C-6, C-8, C-9, C-11), 46.49 and 48.23 (C-1′), 79.28 (C-14), 156.61 (C-13), 171.96 (C-1), 173.72 (C-4), 182.93 (C-7, C-10). ms (IS), Anal. Calcd. for C45H89N7O4S2 856.38, found 857.38 (MH+). 2-{3-[2-(2-{3-[2-(3-Ditetradecylcarbamoyl-propionylamino)-ethyl]-thioureido}-ethylamino)-ethyl]-thioureido}-ethylammonium Trifluoroacetate (10). The carbamate 9 (1 g, 1.15 mmol) was reacted with trifluoroacetic acid (0.45 mL, 5.84 mmol) for 3 h, and then the mixture worked-up as for 5 afforded the amine 10 (100%). 1 H NMR (CDCl3): δ (ppm) 0.85 (t, 6H, J ) 6.6 Hz, H-14′), 1.25 (m, 44 H, H-4′-H-11′), 1.48 (m, 4H, H-2′), 2.65 (m, 2H, H-2), 2.77 (m, 2H, H-3), 3.26 (m, 4H, H-1′), 3.43 (m, 4H, H-5, H-12), 3.85 (m, 8H, H-6, H-8, H-9, H-11), 7.44 (4H, H thiourea).

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C NMR (CDCl3): δ (ppm) 14.05 (C-14′), 22.68 (C-13′), 27.05 (C-3′), 27.58 (C-2), 28.71 (C-2′), 29.36 (C-4′-C-11′), 29.67 (C-3), 31.94 (C-12′), 40.49 (C-5), 43.49 (C-6, C-8, C-9, C-11), 47.40 and 49.07 (C-1′), 172.16 (C-1), 174.00 (C-4), 182.73 (C-7, C-10). N-(2-{3-[2-(2-{3-[2-(3-Methyl-thioureido)-ethyl]-thioureido}-ethylamino)-ethyl]-thioureido}-ethyl)-N′,N′-ditetradecyl-succinamide (11). The amine 10 (0.24 g, 0.28 mmol) was treated by triethylamine (1.1 mL, 7.92 mmol) for 15 min, and then the mixture was dissolved in dichloromethane (3 mL) and methyl isothiocyanate was added (0.024 g, 0.34 mmol). The reaction mixture was stirred at RT for 12 h, and then the solvent was removed. The resulting oil was purified by chromatography (ethyl acetate/heptane 6:4 then pure ethyl acetate/methanol 98:5) to yield the tri-thiourea 11 (0.12 g, 51%). 1 H NMR (CDCl3): δ (ppm) 0.86 (t, 6H, J ) 6.7 Hz, H-14′), 1.24 (m, 44 H, H-4′-H-11′), 1.44 (m, 4H, H-2′), 2.52 (m, 2H, H-2), 2.67 (m, 2H, H-3), 3.05 (m, 3H, H-14), 3.21 (m, 4H, H-1′), 3.32 (m, 2H, H-5), 3.75 (m, 10H, H-6, H-8, H-9, H-11, H-12), 7.14 (6H, H thiourea). 13 C NMR (CDCl3): δ (ppm) 14.09 (C-14′), 22.69 (C-13′), 27.24 (C-3′), 27.89 (C-2′), 28.87 (C-2), 29.38 (C-4′ C-11′), 29.67 (C-3), 31.26 (C-14), 31.94 (C-12′), 39.71 (C-5), 43.63 (C-6, C-8, C-9, C-11, C-12), 46.67 and 48.40 (C-1′), 172.16 (C-1), 174.00 (C-4), 182.73 (C-7, C10, C-13). MS (IS) calcd. for C42H84N8O2S3 829.38, found 830.38 (MH+). Anal. Calcd. for C42H84N8O2S3 C ) 60.82; H ) 10.21; N ) 13.51; S ) 11.60, found C ) 60.73; H ) 11.16; N ) 12.47; S ) 9.38. Preparation of the Liposomes DTTU/DPPC by the Film/Hydration Method. Compound 11 (2 mg, 2.4 µmol) and DPPC (3.5 mg, 4.8 µmol) were dissolved in 500 µL of CHCl3. After solvent removal under vacuum, the film obtained was hydrated with 240 µL of H2O at 4 °C to give a 30 mM multilamellar liposome. Liposomes were successively heated at 45 °C and sonicated (Branson) during 10 min to form a polydisperse population of liposomes with a mean diameter of 142 ( 41 nm. Preparation of the Liposomes DTTU/DPPC by the Ethanolic Injection Method. Compound 11 (2 mg, 2.4 µmol) and DPPC (3.5 mg, 4.8 µmol) were dissolved in 500 µL of a 1/1 mixture of acetone/ethanol. This mixture was dropped on stirred water (5 mL). Most of the solvents were removed under vacuum to obtain a fairly concentrated suspension (580 µL of H2O, 12.4 mM) of liposomes exhibiting a mean diameter of 101 ( 34 nm. Preparation of the Liposomes DTTU/PEG-Cholesterol. Compound 11 (2 mg, 2.4 µmol) and cholesterolPEG2000 (0.12 µmol) were dissolved in 500 µL of CHCl3. After solvent removal under vacuum, the film obtained was hydrated with 250 µL of H2O at 4 °C to give a 10 mM multilamellar liposome. Liposomes were successively warmed and sonicated (Branson) during 10 min to form a polydisperse population of liposomes with a mean diameter of 200 ( 45 nm. Preparation of the Complexes DPPC/DTTU/DNA. Plasmid DNA (pXL3031, from Gencell S.A) (400 µL, 0.02 g/L in Hepes 40 mM, pH 7.4; NaCl 300 mM) was added to various amounts of DPPC/DTTU liposomes (400 µL, 5 mM total lipids, H2O) at RT, and the mixture was vortexed during 10 s and left at RT for 2 h before size measurements, which were performed on a HPPS (Malvern Intruments), using water viscosity and refractive index. Ethidium Bromide Displacement Assays. A JobinYvon Spex Fluoromax-2 spectrofluorometer (Longjumeau, France) was used to measure ethidium bromide fluorescence (Excitation 260 nm, emission 590 nm) at 25 °C.

Tranchant et al.

EtBr (3 µL, 1 mg/mL) was added to the complex suspension (800 µL, 10 µg/mL DNA). Values are expressed as a fluorescence percentage of ethidium bromide with free plasmid DNA, taken as a 100%. Agarose Gel Electrophoresis. Samples were prepared as described above (20 µL) and 5 µL of 30% glucose was added. The mixtures were loaded into 0.8% (w/v) agarose gel at 70 V/cm in TBE buffer (1 M Tris, 0.9 M boric acid, 0.01 M EDTA). DNA was revealed with EtBr and visualized under UV light. Zeta Potential Analysis. The zeta potential was determined using a ZetaSizer 3000HS (Malvern Instruments, Southborough, MA). The system was calibrated using a -50 ( 5 mV standard (DTS 50/50 Standard; Malvern Instruments). Complex samples prepared as described above in 20 mM NaCl subjected to 3 × 30 s (1000 Hz, no field correction) measurement. DNA Stability in Mouse Serum. A solution of complexes was prepared in 5% glucose, [DNA] ) 0.25 g/L, with a total lipid/DNA charge ratio ) 40 or 60 nmol/µg. For each complex tested, 50 µL of lipoplexes was incubated for 30 min at 37 °C/5% CO2 in 1 mL serum diluted or not in 150 mM NaCl. Phenol/chloroform (1/1, v/v, 200 µL) and a solution of TRIS-EDTA (100 µL; 100 mM10 mM, pH 8) were added to the lipoplex mixture in serum (200 µL). The mixture was vortexed and then centrifuged (Jouan MR 18 22, 12 000 rpm, 5 min, 4 °C) in microtubes. The aqueous phase was isolated and washed with a same volume of CHCl3, and then centrifuged (Jouan MR 18 22, 12 000 rpm, 5 min, 4 °C). A solution of AcONa (10 µL, 3M) and EtOH (300 µL) was added in aqueous phase and stored at -20 °C. After 30 min, the mixture was centrifuged (Jouan MR 18 22, 12 000 rpm, 20 min, 4 °C), and the solvent was delicately removed. DNA was resuspended in H2O and 1 µg was loaded on 1% agarose gel in 1% TBE buffer and electrophorized for 2 h at 70 V/cm in TBE buffer. DNA was revealed with ethidium bromide and visualized under UV light. In Vivo Distribution in Tumor-Bearing Mice. Lewis Lung carcinoma tumors were implanted subcutaneously in the right flank of 6-week-old female C57Bl/6 mice (Charles River), and 10-14 days after biodistribution studies were undertaken as previously described. Six-week-old female C57Bl/6 mice (Charles River) were anesthetized by intraperitoneal injection of a mix of ketamine (85.8 mg/kg; Merial) and xylazine (3.1 mg/kg; Bayer) diluted in 150 mM NaCl. Two hundred microliters of rhodamine-labeled complexes (10 µg of DNA, 400 nmol lipids in 5% glucose) were injected into the mouse tail vein. Blood was collected by cardiac puncture at appropriate intervals (30 min, 1, 2, 4, and 6 h after injection) and mice were euthanized at the end of the experiment. In the distribution study, mice were sacrificed at 30 min and 8 h after injection and the liver, spleen, lungs, and tumors were removed, weighed, and homogenized in pH 7.4 PBS (5 mL/g tissue). Experiments were conducted following the NIH recommendation for animal experimentation and Aventis local ethic committee on animal care and experimentation. Rhodamine-labeled lipids were extracted from 100 µL of blood or tissue homogenates with 3 mL of CHCl3/ MeOH (1/1, v/v) by vigorously mixing during 30 min for blood and 40 min for tissue homogenates, followed by centrifugation (3000 rpm, 10 min). The fluorescence intensity was assayed on the supernatant with a fluorospectrophotometer (Fluoromax 2, Jobin Yvon). The amount of lipoplexes in the blood or tissue homogenates

DNA Complexing Lipopolythiourea

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Scheme 1. Preparation of the Lipopolythioureaa

a Reagents and conditions: (i) succinic anhydride, DMAP, pyridine, CH Cl , 98%; (ii) PyBop, diisopropylethylamine, RT, 66%; (iii) 2 2 trifluoroacetic acid, RT, 100%; (iv) triethylamine, 67-72%; (v) triethylamine, methyl isothiocyanate, 51%.

was evaluated with a calibration curve and expressed as the remaining percentage of injected dose. RESULTS

The lipopolythiourea was prepared as shown in Scheme 1. First, ditetradecylamine was reacted with succinic anhydride in the presence of DMAP and pyridine to give the succinate 2 in 98% yield. Reaction of 2 with the monoprotected diethylamine 3 in the presence of PyBop afforded 4 in 66% yield. After removal of the tert-butoxy carbamate, using trifluoroacetic acid, the resulting amine 5 was treated with the isothiocyanate 6 (16) to yield the thiourea 7 (73%). Deprotection of 7, then condensation with 6, led to the amino thiourea 9. Repetition of the same sequence on 9 afforded the di-thiourea 10. Finally, removal of the protecting group then treatment with methyl isothiocyanate gave the ditetradecyl amino trithiourea 11 (DTTU) in 51% yield. Formulation Study. The ability of lipopolythiourea (DTTU) 11 to form micellar or liposomal particles was then evaluated. The DTTU hydrophobicity prevents its solubilization in H2O and particle formation. We added phosphatidylcholine (PC) as an amphiphilic co-lipid

because of its high hydration potency and its zwitterionic nature. We evaluated the association to DTTU of EPC and of DPPC. Both EPC and DPPC formed mixed vesicles with DTTU. A ratio DTTU/DPPC of 1/2 is optimal to obtain a stable lipidic suspension. Energy gained by sonication to reduce particle size resulted in particles exhibiting a 142 ( 41 nm size distribution, as found by dynamic light scattering experiments. The potential primary amine containing lipid due to DTTU degradation was checked before and after sonication using the fluorescamine test (17) and zeta potential measurements (ζ ) +7.1 ( 2.8 mV before sonication, ζ ) +8.8 ( 3.4 mV after sonication). The formation of liposomes by ethanol injection, which does not require any energy gain by sonication, was also performed with the same ratio DTTU/DPPC and spontaneously gave smaller particles (diameter 101 ( 34 nm; ζ ) +5.4 ( 4.3 mV). Interaction of DTTU/DPPC with DNA. The capacity of the DTTU/DPPC particles to associate to plasmid DNA was then studied and compared to DPPC/DNA association. Setting a fixed DNA amount, we added an increasing amount of DTTU/DPPC or DPPC lipids and measured the size of the particles obtained by dynamic

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Figure 3. DNA was mixed with various amounts of DTTU/ DPPC (1/2) liposomes. Ethidium bromide was then added and fluorescence was monitored at 590 nm.

Figure 2. Diameter of the (a) DPPC/DNA particles and (b) DTTU/DPPC/DNA particles measured by dynamic light scattering. Plasmid DNA (8 µg) was added to various amounts of total lipid.

light scattering. Increasing the amount of DPPC from 5 to 20 nmol/µg DNA did not lead to any particle size modification (Figure 2). In contrast, above a ratio of 5 nmol of DTTU/DPPC lipids/µg of DNA, particle size increase was observed, leading to micrometer-range particles (Figure 2). This diameter change clearly indicated that in the conditions used an interaction occurred between the DTTU/DPPC particles and DNA and not between DPPC and DNA. Interestingly, by increasing the amount of total lipid (over 20 nmol of lipid/µg of DNA), we observed stabilized particles with a size distribution of 170 ( 45 nm. We had previously observed such an interaction profile with a cationic lipid, by increasing the ratio lipid over DNA ratio (18), aggregation then stabilization. We observed that addition of DPPC to DNA did not alter significantly ethidium bromide intercalation into DNA. In contrast, DTTU/DPPC presence limited the ethidium bromide accessibility to plasmid DNA, reducing the ethidium bromide fluorescence by a factor of 4 when the ratio lipid/DNA was increased from 0 to 20 nmol/µg. The observed decrease of ethidium bromide fluorescence reveals a lower DNA intercalation. It suggests that DTTU/DPPC liposomes associate to DNA, leading to a DNA compacted state preventing ethidium bromide access to DNA. Agarose gel electrophoresis of the liposome/DNA complexes confirmed that plasmid DNA associates to DTTU/ DPPC liposomes, with a decreasing amount of DNA migrating in the gel. In contrast, mixing the same lipid amount of DPPC alone with DNA did not retain DNA in the wells (Figure 4). The signal UV active in the wells might indicate that part of the nonmigrating DNA, which is associated to DTTU/DPPC liposomes, is accessible to ethidium bromide intercalation. Addition of cations such

Figure 4. Migration of the DPPC/DNA and DTTU/DPPC/DNA complexes on a 0.8% agarose gel in TBE (1×). Complexes were revealed with ethidium bromide staining. From left to right, the lanes correspond to 0, 5, 10, 20 nmol of lipid/µg of DNA.

Figure 5. Complexes DTTU/cholesterol-PEG2000/DNA and cholesterol-PEG2000/DNA were loaded on a 0.8% agarose gel in TBE (1×) and revealed with EtBr staining. Total amount of lipids used is indicated (nmol of lipid/µg of DNA).

as Ca2+ instead of Na+ did not modify DNA retention on an agarose gel under the same conditions (not shown). To prove the active part of the DTTU lipid itself in DNA compaction, we used a pegoylated-lipid (7) (cholesterol-PEG2000) neutral as a co-lipid for DTTU lipid. We then checked by agarose gel electrophoresis the ability of this lipid mixture to associate with plasmid DNA. As observed in Figure 5, within the amount of lipid used, cholesterol-PEG2000 alone was not able to condense DNA and allowed DNA electrophoresis in the gel. In contrast, the mixture DTTU/PEG-cholesterol was able to retain plasmid DNA in deposit wells at 10, 20, and particularly 40 nmol of lipid/µg of DNA. This experiment confirms the active role played by the DTTU lipid in DNA association. Zeta potential of the mixture DTTU/DPPC ( DNA was compared to the one obtained for lipopolyamine/DPPC ( DNA. Addition of DNA to DTTU/DPPC induced a

Bioconjugate Chem., Vol. 15, No. 6, 2004 1347

DNA Complexing Lipopolythiourea Table 1. Zeta Potential (mV) of DTTU/DPPC ( DNA, DTTU/Cholesterol-PEG2000 ( DNA Complexes and RPR209120/DPPC/DNA Complexes Measured in 20 mM NaCl on a Malvern Instruments 3000HSa

lipopolyamine (L/D 5) DTTU/DPPC 1/2 (L/D 20) DTTU/DPPC 1/2 (L/D 20) DTTU/CholPEG (L/D 20) a

method

20 mM NaCl

+DNA/ 20 mM NaCl

ethanolic injection film + hydration ethanolic injection film + hydration

+60.1 ( 2.4

+30 ( 3.5

+8.8 ( 3.4

-20.3 ( 3.5

+5.4 ( 4.3

-16.6 ( 2.5

+1.8 ( 4.2

-0.2 ( 3.2

L/D ) ratio lipid/DNA in nmol of lipid/µg of DNA.

negatively charged zeta potential of -20.3 ( 3.5 mV for the DTTU/DPPC particles prepared by the film hydration method and -16.6 ( 2.5 mV for the particles prepared by the ethanolic injection method (Table 1). Zeta potentials obtained by the two methods are quite comparable (with or without DNA) and far below the one obtained for the lipopolyamine control and other previously described cationic lipids (19). As expected, addition of 10% cholesterol-PEG2000 to the DTTU lipid gave a zeta potential close to neutrality in the presence and absence of DNA. To explain the slightly positive value of potential zeta obtained for the DTTU/DPPC formulation in 20 mM NaCl, we searched for a potential amine contamination in each formulation using the fluorescamine test. Reaction of fluorescamine with alanine was taken as the calibration curve for amine quantification. We found 0.2% mol of amine in the DTTU/DPPC liposome prepared by the film method after sonication. This amount of amine could be responsible for the slightly positive zeta potential obtained, although this amount of amine could undeniably not be responsible for the DNA retention on an agarose gel or lipoplex size increase. Indeed, the amount of DTTU/DPPC used for DNA association: 20 nmol/µg lipid would represent 0.013 nmol of amine content/nmol of DNA phosphate, which is clearly not enough to account for the association effect observed. DNA Protection toward Serum Enzymatic Degradation. DNA protection toward serum degradation was investigated in 20 and 100% serum in the absence and presence of DTTU/DPPC. After 30 min incubation at 37 °C, DNA was extracted by a phenol/chloroform process and loaded onto an agarose gel (Figure 6). In these two conditions, free DNA is fully degraded (lanes 4 and 7), while DNA integrity is conserved in 20% serum and partially maintained in 100% serum when associated to DTTU/DPPC (40 and 60 nmol/µg). In Vivo Injection of DTTU/DPPC/DNA Complexes. Our interest in DTTU as complexing agent lies in the passive accumulation into tumor that could arise from longer circulation time, as previously reported with PEG-coated liposome (20). Indeed, reducing the charge of the lipid/DNA complexes should result in less protein interaction. Complexes including 5% PEG-cholesterol for stability and 1% DOPE-rhodamine for detection were injected into mice subcutaneously implanted with a Lewis lung carcinoma model. The lipids were extracted from the organs and treated for rhodamine recovery as described (Figure 7) (12). Circulation time increase for noncationic particles as compared to cationic particles was noticeable after 30 min; 34 versus 17% was recovered in the blood. More obvious was the lack of nonspecific accumulation in the lung, where a gain of 3-40 was measured for noncationic

Figure 6. Integrity of plasmid DNA after complex exposure to mouse serum. DNA (A), DTTU/DPPC/DNA, 40 nmol/µg (B), and 60 nmol/µg (C) were incubated at 37 °C for 30 min in 150 mM NaCl, 20% serum or 100% serum. Plasmid was then extracted with phenol/CHCl3 and its integrity was analyzed on a 0.8% agarose gel.

Figure 7. Biodistribution of fluorescently labeled particles. Cationic particles RPR209120/DPPC/cholesterol-PEG2000/PErhodamine (triangles) used for the control were prepared via the ethanolic injection method as DTTU/DPPC/cholesterolPEG2000/PE-rhodamine particles (squares). Each point is an average of 5 mice + SD.

particles with regard to the cationic controls. Noncationic particles were also recovered in the reticuloendothelial system (liver + spleen) even at the early times. Passive accumulation seems to have occurred since a factor of 6.5 increase was found in the tumor at 30 min postinjection and was maintained after 1 h for noncationic particles versus cationic controls. No increase in transgene expression was found associated to the lipid accumulation, despite the co-injection of lipids/DNA where the plasmid-DNA expressed chloramphenicol acetyl transferase. It has to be noted that a low amount of DNA was injected (10 µg) per mouse. DISCUSSION

The above data show that the lipopolythiourea DTTU can associate with plasmid DNA and protect it from degradation in serum. To date, this is the first report of a noncationic lipid able to associate to polyanionic DNA at low lipid concentration (10) and without addition of divalent cationic ions (21). DNA was shown to bind to DPPC zwitterionic lipid bilayers strongly enough to perform AFM studies (9, 22). However, in our conditions, the DPPC binding is not sufficient to retain DNA on agarose wells, compete with ethidium bromide intercalation between DNA base pairs, nor induce a DPPC particle

1348 Bioconjugate Chem., Vol. 15, No. 6, 2004

size increase. The active part played by the lipopolythiourea in the interaction between DNA and DTTU/DPPC was evidenced by formulating DTTU with a cholesterolPEG instead of DPPC. Several processes could explain how DNA interaction occurs with DTTU/DPPC particles. The slightly positive zeta potential observed with DTTU/DPPC particles might be explained by the strong polarization of the NH linkage in the thiourea functions, which might be involved in the hydrogen bonds with DNA phosphates. As hypothesized, hydrogen bonds between the DNA phosphate groups and the thiourea headgroups might occur (23). An alternative hypothesis to explain DNA association to neutral lipopolythiourea is to consider the polarization of the lipopolythiourea moiety. One can assume that the negatively polarized sulfur interacts with cationic monovalent or divalent cations from the solution. Such an interaction might account for the positive zeta potential of the DTTU/DPPC liposomes. It might also account for the DNA complexation effect. The involvement of cations has been demonstrated in the case of cochleates which are delivery particles composed of anionic lipids (20). In conclusion, we describe here a new “noncationic” DNA interacting self-assembling system. Because of their very low zeta potential precluding nonspecific binding to plasmatic membrane and adsorption mediated endocytosis, we did not expect DTTU/DPPC/DNA particles to display high transfection efficiency. Indeed, preliminary experiments led to very weak transfection. Targeting DTTU/DPPC/DNA particles to membrane receptor and receptor-mediated transfection might represent the most suitable way to use these new DNA complexing particles for gene transfer. This lipid family might prove to be better tolerated in vivo than cationic DNA delivery vectors. Its ultimate capacity to efficiently deliver plasmid DNA in vitro and in vivo through the use of targeting moieties as well as the study of new polythiourea structures (24) is under investigation. ACKNOWLEDGMENT

This work was supported by CNRS, ENSCP, and Gencell S. A. I.T. thanks Aventis Pharma for a graduate fellowship. We thank Dr. Michel Bessodes for providing us the pegoylated cholesterol. LITERATURE CITED (1) Miller, A. D. (1998) Cationic liposomes for gene therapy. Angew. Chem., Int. Ed. 37, 1768-1785. (2) Zelphati, O., Uyechi, L. S., Barron, L. G., and Szoka R. C., Jr. (1998) Effect of serum components on the physico-chemical properties of cationic lipid/oligonucleotide complexes and on their interactions with cells. Biochim. Biophys. Acta 1390, 119-133. (3) Tousignant, J. D., Gates, A. L., Ingram, L. A., Johnson, C. L., Nietupski, J. B., Cheng, S. H., Eastman, S. J., and Scheule, R. K. (2000) Comprehensive analysis of the acute toxicities induced by systemic administration of cationic lipid: plasmid DNA complexes in mice. Hum. Gene Ther. 11, 2493-2513. (4) Wheeler, J., Palmer, L., Ossanlou, M., MacLachlan, M., Graham, R., Zhang, Y., Hope, M., Scherrer, P., and Cullis, P. (1999) Stabilized plasmid-lipid particles: construction and characterization. Gene Ther. 6, 271-281. (5) Hofland, H. E., Masson, C., Iginla, S., Osetinsky, I., Reddy, J. A., Leamon, C. P., Scherman, D., Bessodes, M., and Wils, P. (2002) Folate-targeted gene transfer in vivo. Mol. Ther. 6, 739-744. (6) Papahadjopoulos, D., Allen, T, Gabizon, A., Mayhew, E., Matthay, K., Huang, S., Lee, K., Woodle, M., Lasic, D., Redemann, C., and Martin, F. (1991) Sterically stabilized

Tranchant et al. liposomes: improvements in pharmacokinetics and antitumor therapeutic efficacy. Proc. Natl. Acad. Sci. U.S.A. 88, 1146011464. (7) Nicolazzi, C., Mignet, N., De la Figuera, N., Cadet, M., Torero-Ibad, R., Seguin, J., Scherman, D., and Bessodes, M. (2003) Anionic polyethylene glycol lipids added to cationic lipoplexes increase their plasmatic circulation time. J. Controlled Release 88, 429-443. (8) Oupicky, D., Ogris, M., Howard, K. A., Dash, P. R., Ulbrich, K., and Seymour, L. W. (2002) Importance of lateral and steric stabilization of polyelectrolyte gene delivery vectors for extended systemic circulation Mol. Ther. 5, 463-472. (9) Bailey, A. L., and Sullivan, S. M. (2000) Efficient encapsulation of DNA plasmids in small neutral liposomes induced by ethanol and calcium. Biochim. Biophys. Acta 1468, 239-252. (10) Pott, T., and Roux, D. (2002) DNA intercalation in neutral multilamellar membrane. FEBS Lett. 5, 150-154. (11) Bordwell, F. G., Algrim, D. J., and Harrelson, J. A., Jr. (1988) The relative ease of removing a proton, a hydrogen atom, or an electron from carboxamides versus thiocarboxamides. J. Am. Chem. Soc. 110, 5903-5904. (12) Buhlmann, P., Nishizawa, S., Xiao, K. P., and Umezawa, Y. (1997) Strong hydrogen bond-mediated complexation of H2PO4- by neutral bis-thiourea hosts. Tetrahedron 53, 16471654. (13) Byk, G., Dubertret, C., Escriou, V., Frederic, M., Jaslin, G., Rangara, R., Pitard, B., Crouzet, J., Wils, P., Schwartz, B., and Scherman, D. (1998) Synthesis, Activity, and Structure-Activity Relationship Studies of Novel Cationic Lipids for DNA Transfer. J. Med. Chem. 41, 224-235. (14) Still, W. C., Kahn, M., and Mitra, A. J. Org. Chem. 1978, 43, 2923-2926. (15) Balacco, G. J. Chem. Inf. Comput. Sci. 1994, 34, 12351241. (16) Kneeland, D. M., Ariga, K., Lynch, V. M., Huang, C. Y., Anslyn, E. V. (1993) Bis(alkylguanidinium) receptors for phosphodiesters: effect of counterions, solvent mixtures, and cavity flexibility on complexation. J. Am. Chem. Soc. 115, 10042-10055. (17) Stocks, S. J., Jones, A. J., Ramey, C. W., and Brooks, D. E. (1986) A fluorometric assay of the degree of modification of protein primary amines with polyethylene glycol. Anal. Biochem. 154, 232-234. (18) Pitard, B., Aguerre, O., Airiau, M., Lachage`s, A. M., Boukhnikachvili, T., Byk, G., Dubertret, C., Herviou, C., Scherman, D., Mayaux, J. F., and Crouzet, J. (1997) Virussized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer. Proc. Natl. Acad. Sci. U.S.A. 94, 14412-14417. (19) Son, K., Patel, D., Tkach, D., and Park, A. (2000) Cationic liposome and plasmid DNA complexes formed in serum-free medium under optimum transfection condition are negatively charged. Biochim. Biophys. Acta 1466, 11-15. (20) Tam, P., Monck, M., Lee, D., Ludkovski, O., Leng, E., Clow, K., Stark, H., Scherrer, P., Graham, R., and Cullis, P. (2000) Stabilized plasmid-lipid particles for systemic gene therapy. Gene Ther. 7, 1867-1874. (21) Tilcock, C., Bally, M., Farren, S., Cullis, P., and Gruner, S. (1984) Cation-dependent segregation phenomena and phase behavior in model membrane systems containing phosphatidylserine: influence of cholesterol and acyl chain composition. Biochemistry 23, 2696-2703. (22) Malghani, M., and Yang, J. (1998) Stable binding of DNA to zwitterionic lipid bilayers in aqueous solutions. J. Phys. Chem. B 102, 8930-8933. (23) Erkang, F., Scott, A., Van, Arman, S., Kincaid, S., and Hamilton, A. (1993) Molecular recognition: hydrogen-bonding receptors that function in highly competitive solvents. J. Am. Chem. Soc. 115, 369-370. (24) Girard, C., Tranchant, I., Gorteau, V., Potey, L., and Herscovici, J. (2004) Development of a DNA interaction test with small molecules still grafted on solid phase. J. Comb. Chem. 6, 275-278.

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