Synthesis and Characterization of Novel Cationic Lipid and

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Synthesis and Characterization of Novel Cationic Lipid and Cholesterol-Coated Gold Nanoparticles and Their Interactions with Dipalmitoylphosphatidylcholine Membranes Santanu Bhattacharya* and Aasheesh Srivastava Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, India Received December 4, 2002. In Final Form: February 17, 2003 Novel gold nanoparticles bearing cationic single-chain, double-chain, and cholesterol based amphiphilic units have been synthesized. These nanoparticles represent size-stable entities in which various cationic lipids have been immobilized through their thiol group onto the gold nanoparticle core. The resulting colloids have been characterized by UV-vis, 1H NMR, FT-IR spectroscopy, and transmission electron microscopy. The average size of the resultant nanoparticles could be controlled by the relative bulkiness of the capping agent. Thus, the average diameters of the nanoparticles formed from the cationic singlechain, double-chain, and cholesterol based thiolate-coated materials were 5.9, 2.9, and 2.04 nm, respectively. We also examined the interaction of these cationic gold nanoparticles with vesicular membranes generated from dipalmitoylphosphatidylcholine (DPPC) lipid suspensions. Nanoparticle doped DPPC vesicular suspensions displayed a characteristic surface plasmon band in their UV-vis spectra. Inclusion of nanoparticles in vesicular suspensions led to increases in the aggregate diameters, as evidenced from dynamic light scattering. Differential scanning calorimetric examination indicated that incorporation of single-chain, double-chain, and cholesteryl-linked cationic nanoparticles exert variable effects on the DPPC melting transitions. While increased doping of single-chain nanoparticles in DPPC resulted in the phases that melt at higher temperatures, inclusion of an incremental amount of double-chain nanoparticles caused the lowering of the melting temperature of DPPC. On the other hand, the cationic cholesteryl nanoparticle interacted with DPPC in membranes in a manner somewhat analogous to that of cholesterol itself and caused broadening of the DPPC melting transition.

1. Introduction Colloidal gold has been known from the middle ages, but a basic understanding about it was achieved by the work of Faraday1 in the nineteenth century. Though techniques to make nanoparticles in a size-selective manner were available, their instability significantly limited the exploitation of these interesting systems for real applications until recently.2 The stabilization of gold nanoparticles with alkanethiols was first reported by Mulvaney and Giersig.3 This showed the possibility of using thiols of different chain lengths for the preparation of gold nanoparticles as well as allowed the analysis of thiols on nanoparticle surfaces. Brust and co-workers prepared thiol-coated gold nanoparticles using reverse micelles.4 The resulting nanoparticles were very stable and almost monodisperse. The size control on the preparation of gold nanoparticles was also achieved by judicious variation of the gold/thiol ratio.5,6 Metal nanoparticles are of interest because of their interesting electronic properties.7 These systems display intense color attributed to the oscillations of surface * Address correspondence to this author. Also at Chemical Biology Unit, JNCASR, Bangalore 560012, India. E-mail: [email protected]. ernet.in. Fax: +91-80-360-0529. (1) Faraday, M. Philos. Trans. R. Soc. London 1857, 147, 145-181. (2) Enu¨stu¨n, B. V.; Turkevich, J. J. J. Am. Chem. Soc. 1963, 85, 3317-3319. (b) Slot, J. W.; Gueze, H. J. Eur. J. Cell Biol. 1985, 38, 87-95. (3) Giersig, M.; Mulvaney, P. Langmuir 1993, 9, 3408-3413. (4) Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. J. Chem. Soc., Chem. Commun. 1994, 801-802. (b) Brust, M.; Fink, J.; Bethell, D.; Schiffrin, D. J.; Keily, C. J. J. Chem. Soc., Chem. Commun. 1995, 1665-1666. (5) Leff, D. V.; Ohara, P. C.; Heath, J. R.; Gelbart, W. M. J. Phys. Chem. 1995, 99, 7036-7041.

electrons (the surface plasmon resonance).8,9 They also show “Coulomb blockade” behavior in electron conduction and have capacitance in atto-farad range.10-12 This phenomenon is observed due to their small size as well as the presence of insulating layers of thiols used as stabilizers. To render them water soluble, nanoparticles coated with cationic and anionic moieties have been synthesized.13 Some of these nanoparticle systems have also been utilized in sensing metal ions.14 Even their utility in a number of biological applications has been demonstrated.15 For instance, suitably tailored nanoparticles (6) Hostetler, M. J.; Wingate, J. E.; Zhong, C. J.; Harris, J. E.; Vachel, R. W.; Clark, M. R.; London, J. D.; Green, S. J.; Stokes, J. J.; Wignal, G. D.; Glish, G. L.; Porter, M. D.; Ebans, N. D.; Murray, R. W. Langmuir 1998, 14, 17-30. (b) Sarathy, K. V.; Raina, G.; Yadav, R. T.; Kulkarni, G. U.; Rao, C. N. R. J. Phys. Chem. B 1997, 101, 9876-9879. (7) Templeton, A. C.; Wuelfing, P. W.; Murray, R. W. Acc. Chem. Res. 2000, 33, 27-36 and references therein. (8) Link, S.; El-Syed, M. A. J. Phys. Chem. B 1999, 103, 4212-4217. (9) Alvarez, M. M.; Khoury, J. T.; Schaaff, T. G.; Shafigullin, M. N.; Vezmar, I.; Whetten, R. L. J. Phys. Chem. B 1997, 101, 3706-3712. (10) Hicks, J. F.; Templeton, A. C.; Chen, S.; Sheran, K. M.; Jasti, R.; Murray, R. W. Anal. Chem. 1999, 71, 3703-3711. (11) Ingram, R. S.; Hostetler, M. J.; Murray, R. W.; Schaaff, T. G.; Khoury, J. T.; Whetten, R. L.; Bigioni, T. P.; Guthrie, D. K.; First, P. N. J. Am. Chem. Soc. 1997, 119, 9279-9280. (12) Chen, S. W.; Ingram, R. S.; Hostetler, M. J.; Pietron, J. J.; Murray, R. W.; Schaaff, T. G.; Khoury, J. T.; Alvarez, M. M.; Whetten, R. L. Science 1998, 280, 2098. (13) Kumar, A.; Pattarkine, M.; Bhadbhade, M.; Mandale, A. B.; Ganesh, K. N.; Datar, S. S.; Dharmadhikari, C. V.; Sastry, M. Adv. Mater. 2001, 13, 341-344. (b) Shon, Y.; Weulfing, W. P.; Murray, R. W. Langmuir 2001, 17, 1255-1261. (c) Cliffel, D. E.; Zamborini, F. P.; Gross, S. M.; Murray, R. W. Langmuir 2000, 16, 9699-9702. (d) Templeton, A. C.; Cliffel, D. E.; Murray, R. W. J. Am. Chem. Soc. 1999, 121, 7081-7089. (e) Sastry, M.; Mayya, K. S.; Patil, V.; Paranjpe, D. V.; Hegde, S. G. J. Phys. Chem. 1997, 101, 4954-4958. (14) Templeton, A. C.; Zamborini, F. P.; Wuelfing, W. P.; Murray, R. W. Langmuir 2000, 16, 6682-6686.

10.1021/la0269513 CCC: $25.00 © 2003 American Chemical Society Published on Web 04/11/2003

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displayed efficient DNA recognition behavior.16,17 Gold nanoparticles modified with oligonucleotides were shown to be an efficient system for colorimetric detection of singlenucleotide mismatches in DNA.18 At the same time, DNA and oligonucleotides were used as templates for forming organized assemblies of nanoparticles.19 They have also been utilized in the inhibition of DNA transcription as well as in transfection of mammalian cells.20 These systems therefore hold tremendous potential in developing a variety of smart materials. We have earlier reported the aggregation properties of a range of cationic amphiphiles.21 Several of these amphiphilic aggregates induced efficient transfection in the eukaryotic cells.22 Herein we consider the design of cationic amphiphile-coated gold nanoparticles and report the synthesis and characterization of nanoparticles stabilized by reduced forms of lipid-analogous cationic disulfides 1-3. We also present the results of an investigation on the interaction of all the three cationic nanoparticles with membranes of a natural phospholipid, dipalmitoylphosphatidylcholine (DPPC). In particular, we examined the physical changes in membranes comprising different mixtures of these nanoparticles upon solubilization in DPPC membrane by using UV-vis spectroscopy (plasmon band), transmission electron microscopy (TEM), dynamic light scattering (DLS), and differential scanning calorimetry (DSC). Depending upon the nature of the nanoparticle, differential effects were observed on the mainchain melting phase transition of DPPC. 2. Experimental Section 2.1. Materials. Hydrogen tetrachloroaurate(III) trihydrate, H[AuCl4]‚3H2O, and sodium borohydride (NaBH4) were obtained from Aldrich Chemical Co. and were used as received. DPPC was purchased from Sigma Chemical Company. Disulfides 1-3 were synthesized as shown in Scheme 1. All solvents were distilled prior to use. Water was distilled twice over KMnO4 to remove the organic and inorganic impurities. 2.2. General. All melting points reported are uncorrected.1H NMR studies of nanoparticles and their precursors were recorded on a JEOL LAMBDA 300 FT spectrophotometer. FT-IR was performed on a JASCO FT/IR 410 spectrometer by drop-coating a concentrated sample solution on the cell and air-drying it (neat and as also as KBr pellet). For preparing the KBr pellet for a nanoparticle, a concentrated chloroform solution of the nanoparticle was added to solid KBr. This mixture was then ground in a mortar and pestle. A pellet was made out of this pink mixture by using a KBr press. Dynamic light scattering was done on a Malvern Zetasizer 3000 instrument. Energy minimized struc(15) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128-4158. (16) Mahtab, R.; Harden, H. H.; Murphy, C. J. J. Am. Chem. Soc. 2000, 122, 14-17. (17) Lackowicz, J. R.; Gryczynski, I.; Gryczynski, Z.; Nowaczyk, K.; Murphy, C. J. Anal. Biochem. 2000, 280, 128-136. (18) Storhoff, J. J.; Mirkin, C. A. Chem. Rev. 1999, 99, 1849-1862. (b) Elghaninan, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 227, 1078-1081. (c) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607-609. (19) Alivisatos, A. P.; Johnson, K. P.; Peng, X.; Wilson, T. E.; Loweth, C. J.; Bruchez, M. P., Jr.; Schultz, P. G. Nature 1996, 382, 609-612. (b) Taton, T. A.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 2000, 122, 6305-6306. (c) Niemeyer, C. M.; Burger, W.; Peplies, J. Angew. Chem., Int. Ed. Engl. 1998, 37, 2265-2267. (20) McIntosh, C. M.; Esposito, E. A., III; Boal, A. K.; Simard, J. M.; Martin, C. T.; Rotello, V. M. J. Am. Chem. Soc. 2001, 123, 7626-7629. (b) Sandhu, K. K.; McIntosh, C. M.; Simard, J. M.; Smith, S. W.; Rotello, V. M. Bioconjugate Chem. 2002, 13, 3-6. (21) Haldar, J.; Aswal, V. K.; Goyal, P. S.; Bhattacharya, S. Angew. Chem., Int. Ed. 2001, 40, 1228-1232. (b) Bhattacharya, S.; Haldar, S. Langmuir 1995, 11, 4748-4757. (22) Ghosh, Y. K.; Visweswariah, S.; Bhattacharya, S. Bioconjugate Chem. 2002, 13, 378-384. (b) Dileep, P. V.; Antony, A.; Bhattacharya, S. FEBS Lett. 2001, 509, 327-331. (c) Ghosh, Y. K.; Visweswariah, S.; Bhattacharya, S. FEBS Lett. 2000, 473, 341-344.

Bhattacharya and Srivastava tures were generated using INSIGHT II (Version 97.5) on a Silicon Graphics’ Octane workstation. 2.3. Synthesis. N,N′-(Dithio-2,1-ethanediyl)-bis(11-bromoundecanamide) (1a). Cystamine dihydrochloride (225 mg, 1 mmol) was added to dry acetonitrile (10 mL). This was kept in an icebath, and triethylamine (1 g, 10 mmol) was added to it. This mixture was stirred for 3 h, after which freshly prepared 11bromoundecanoyl chloride (800 mg, 2.8 mmol) was added to it. The resulting mixture was stirred at 0 °C for 1 h and then at room temperature for 14 h. CH3CN was removed by rotary evaporation from the reaction mixture to leave a residue that was extracted in chloroform (2 × 20 mL). The CHCl3 layer was washed with 2 N HCl (2 × 5 mL) and then with water (2 × 5 mL) and finally dried via passing through anhydrous Na2SO4. Column chromatography using 1% MeOH in CHCl3 over silica gel afforded 1a in 75% isolated yield. 1H NMR (CDCl3, 300 MHz): δ 3.56 (q, 2 × CH2NH, 4H), 3.41 (t, 2 × CH2Br, 4H), 2.83 (t, 2 × CH2S, 4H), 2.21 (t, 2 × CH2CONH, 4H), 1.8-1.2 (m, 2 × (CH2)8, 32H). FT-IR (thin film) [cm-1] 3296, 2922, 2851, 1637, 1544, 1464, 1376, 1115. MALDI-TOF MS for C26H50N2O2S2Br2 (M+): calcd 646.3; obsd 646.4 (M+). Bis(N,N,N-trimethyl-11-undecanoylaminoethyl) Disulfide (1). Dry NMe3 was introduced into dry THF (5 mL) in a screw-top pressure tube kept in an ice-bath. This resulted in an ∼2-fold increase in the volume. N,N′-(Dithio-2,1-ethanediyl)bis(11-bromoundecanamide) (150 mg, 0.24 mmol) was added into it, and the mixture was heated at 80 °C for 24 h. After this, the mixture was cooled, THF was decanted, and the brown gum sticking to the edges of the tube was dissolved in MeOH. This was added to the THF soluble fraction, and the mixture was concentrated. Finally, the compound was isolated by precipitation using 1:5 MeOH/acetone to furnish bis(N,N,N-trimethyl-11-undecanoylaminoethyl) disulfide22 as a brown gum in 70% yield. 1H NMR (D2O, 300 MHz): δ 3.4 (t, 2 × CH2-NH, 4H), 3.2 (t, 2 × (CH2)N+, 4H), 3.0 (s, 2 × (CH3)3N+, 18H), 2.8 (t, 2 × CH2-S, 4H), 2.2 (t, CH2-CO, 2H), 1.2-1.8 (br m, CH2, 16H). FT-IR (neat) [cm-1]: 3049, 2918, 2852, 1648, 1549, 1464, 1209, 1037. ESI-MS for C32H68N4O2S2Br2 M+: calcd 764.87; obsd 684.06 (M+-Br). Anal. Calcd for C32H68N4O2Br2S2‚H2O: C, 49.09; H, 9.01; N, 7.16. Found: C, 49.23; H, 9.16; N, 6.93. Bis(N,N-dimethyl-N-tetradecylammonio-11-undecanoylaminoethyl) Disulfide (2). N,N′-(Dithio-2,1-ethanediyl)bis(11-bromoundecanamide) (100 mg, 0.16 mmol) and N,N-dimethyl-Ntetradecylamine (100 mg, 0.42 mmol) were dissolved in dry MeOH (4 mL), and the resulting mixture was heated at 70 °C in a screwtop pressure tube for 60 h. The brownish reaction mixture was cooled, concentrated, and then precipitated using a MeOH/ acetone mixture (1:10) to isolate bis(N,N-dimethyl-N-tetradecylammonio-11-undecanoylaminoethyl) disulfide as a yellowish gummy oil in 60% yield. 1H NMR (CDCl3, 300 MHz): δ 3.5 (br m, 2 × CH2-N+-CH2, 8H), 3.4 (m, 2 × CH2-NH, 4H), 3.3 (s, 2 × (CH3)2N+, 12H), 2.8 (t, CH2-S, 4H), 2.3 (t, 2 × CH2-CO, 4H), 1.2-1.8 (br m, 2 × (CH2)40, 80H), 0.9 (t, 2 × CH3, 6H). FT-IR (neat) [cm-1]: 3049, 2918, 2852, 1648, 1549, 1464, 1209,1037. ESI-MS for C58H120N4O2Br2S2 M+: calcd 1129.6; obsd 485.2 ((M2+/ 2) - 2Br). Anal. Calcd for C58H120N4O2Br2S2‚0.5H2O: C, 61.18; H, 10.71; N, 4.92. Found: C, 60.91; H, 10.86; N, 4.56. Bis(N,N-dimethyl-N-(cholest-5-en-3-ol-(3β)-(3-ammoniopropyl)carbamate(11-undecanoylaminoethyl)) Disulfide (3). A methanolic solution of N,N′-(dithio-2,1-ethanediyl)bis(11-bromoundecanamide) (100 mg, 0.15 mmol) and cholest-5-en-3-ol-3β-[3(dimethylamino)propyl]carbamate (200 mg, 0.38 mmol) (prepared in 55% yield from reaction of cholest-5-en-3β-chloroformate with N,N-dimethyl-1,3-propylenediamine in dry chloroform) was heated at 75 °C in a screw-top pressure tube. After 72 h, TLC indicated no further change. The reaction mixture was then cooled and concentrated, and product was precipitated using acetone. A solid was isolated in 28% yield. Mp: 170 °C. 1H NMR (CDCl3, 300 MHz): δ 2.8 (t, 2 × CH2-S, 4H), 3.4 (m, 2 × CH2-NH, 4H), 2.3 (t, 2 × CH2-CO, 4H), 3.3 (s, 2 × (CH3)2 N+, 12H), 3.5 (br t, 2 × CH2-N+-CH2, 8H), 0.67-1.7 (2 × (CH3 + CH2, 10H)). FT-IR (neat) [cm-1]: 3395, 2928, 2851, 1698, 1645, 1540, 1467, 1253, 1032. Anal. Calcd for C92H174N6O8S2Br2‚0.5H2O: C, 65.08; H, 10.42; N, 4.95. Found: C, 65.40; H, 10.35; N, 4.52. Gold Nanoparticles. To a well-stirred methanolic solution of HAuCl4‚3H2O (7.5 mmol/L, 2 mL) was added a solution (2 mL)

Cationic Lipid and Cholesterol-Coated Au Nanoparticles

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Scheme 1. Synthesis Scheme for Disulfides 1, 2, and 3a

a Reagents, conditions and yields: (i) Br(CH2)10COCl, Et3N, CH3CN, 0 °C, 1 h f RT, 14 h, 75%. (ii) NMe3, THF, pressure tube, 80 °C, 24 h, 70%. (iii) C14H29NMe2, MeOH, pressure tube, 70 °C, 60 h, 60%. (iv) Chol-OC(O)NH(CH2)3NMe2, MeOH, pressure tube, 75 °C, 72 h, 28%.

of either disulfide 1, 2, or 3 (3.75 mmol/L) in methanol. This mixture was stirred for 15 min to ensure complete mixing of disulfide/Au(III). Freshly prepared aqueous NaBH4 (0.25 mol/L, 1 mL) was added to this mixture. To ensure quantitative in situ reduction of disulfides to thiols and also to achieve complete reduction of Au(III) to Au(0), a 15-fold excess of the reducing agent was employed. This also facilitated the formation of thiolprotected colloidal gold in a size-controlled manner, which was evident from the color change of the solution from transparent yellow to dark red. The stirring was continued for another 2 h to ensure maximal attachment of the thiols to nascent nanoparticles. Inducing precipitation using 1:2 acetone/hexane isolated the particles from the solution. This process was repeated thrice to ensure the removal of any unadsorbed organic disulfide. Complete removal of unadsorbed disulfide from the nanoparticles was ascertained by TLC. Finally, all the nanoparticles were characterized by 1H NMR, FT-IR, and UV-vis spectroscopy as well as by TEM. Au-1. 1H NMR (D2O, 300 MHz): 3.16 (br m, CH2N+), 3.06 (br m, CH3N+), 2.2 (br t, CH2-CO), 1.2-1.8 (br m, CH2). λmax ∼ 515 nm (MeOH). Au-2. 1H NMR (CDCl3, 300 MHz): 3.34 (br m, CH2-NH), 3.23 (br s, (CH3)2N+), 3.5 (br m, CH2-N+-CH2), 1.2-1.8 (br m, CH2), 0.9 (t, CH3). FT-IR (neat) [cm-1]: 3049, 2918, 2852, 1648, 1549, 1464, 1209,1037. FT-IR (KBr) [cm-1]: 2924, 2854, 1644, 1543, 1465, 1378, 1219, 1046. λmax ∼ 525 nm (CHCl3). Au-3. 1H NMR (CDCl3, 300 MHz): 3.5 (br m, CH2-N+-CH2), 3.3 (br m, (CH3)2 N+), 2.3 (br m, CH2-CO), 0.67-1.7 (br m, CH3s + CH2s). No distinct maximum in the plasmon band absorbance was seen. FT-IR (neat) [cm-1]: 3290, 2930, 2853, 1700, 1646,

1538, 1465, 1253, 1034. FT-IR (KBr) [cm-1]: 2926, 2852, 1700, 1647, 1541, 1466, 1377, 1034. 2.4. Transmission Electron Microscopy (TEM). The shape and size of nanoparticles was determined by TEM on a JEOL 200 CX electron microscope. The colloid as a methanolic solution was drop-coated on a formvar-coated copper grid and was airdried. The micrographs were then analyzed using image analysis software (Mocha, Jandel Scientific Corp.). Approximately 200 particles were used in the statistics to determine the size distribution, which indicated the mean diameter and its standard deviation. 2.5. UV-Visible Spectroscopy. UV-vis spectra were taken using a Shimadzu UV2100 spectrophotometer in the visible region to find the location and intensity of the surface plasmon resonance peak for the colloids. The samples for spectra were prepared by dissolving 1 mg of colloid in 10 mL of H2O in the case of Au-1 and by dissolving 1 mg of colloid in 10 mL of CHCl3 in the case of Au-2 and Au-3. 2.6. Light Scattering. For measuring hydrodynamic radius, 3 mL of a 0.33 mM aqueous suspension of either DPPC vesicles or nanoparticle-doped-DPPC vesicles was taken and analyzed in a Malvern Zetasizer 3000. For ζ-potential measurements, this suspension was further diluted with water to 10 mL volume. 2.7. Differential Scanning Calorimetry. Individual mixtures of Au-1 (0.1-0.5 mg) and DPPC (1.1 mg) were dissolved together in MeOH (100 µL). MeOH was then removed from the mixture under a steady stream of N2 to generate a film of the lipid containing Au-1. For Au-2 and Au-3, the same procedure was followed using chloroform as solvent instead of MeOH. The last traces of solvent were eliminated by evaporation under high vacuum for 6 h. Then, the requisite amount of water was added

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to make the resulting mixture 1 mM with respect to DPPC. After these films were hydrated for 8 h, the mixture was vortexed, frozen to ∼0 °C for 5 min, and then thawed to 55 °C. After three freeze-thaw cycles, the suspensions were sonicated at 55 °C for 2 min and then loaded inside the cells of a multicell differential calorimeter, Calorimetry Science Corp. (CSC, Provo, UT). The heating rate was maintained at 0.3 K/min.

3. Results and Discussion There have been recent attempts to use gold nanoparticles for biological applications.15-18 For instance, gold nanoparticles have been shown to inhibit DNA transcription successfully.20a But the nanoparticles in this case were prepared first using a long-chain thiol which was then place-exchanged with R,ω-ammonium thiols. We attempted to circumvent this two-step procedure by preparing nanoparticles using disulfides 1-3. These disulfides themselves contain hydrophobic chains as well as quaternary ammonium ion. For instance, 2 contains a long hydrocarbon chain as in lipids and 3 contains a cholesterol moiety, and hence these should be biocompatible. 3.1. Synthesis of Cationic Disulfide Amphiphiles. Cationic disulfide amphiphiles 1-3 were synthesized as shown in Scheme 1. Briefly, cystamine dihydrochloride was coupled with 11-bromoundecanoyl chloride (2.8 equiv) in the presence of Et3N in dry acetonitrile to afford N,N′(dithio-2,1-ethanediyl)bis-(11-bromoundecanamide), 1a, in 75% yield. Quaternization of 1a using excess Me3N in dry THF in a screw-top pressure tube afforded 1 in 70% yield. Similarly, quaternization of 1a using N,N-dimethylN-tetradecylamine in dry MeOH in a pressure tube afforded 2 as a gum in 60% yield. For the synthesis of 3, first cholest-5-en-3-ol-(3β)[3-(dimethylamino)propyl]carbamate (3a) was synthesized upon coupling of N,Ndimethyl-1,3-propylenediamine with cholesteryl chloroformate in chloroform. 3a was then reacted with 1a to furnish the desired cationic cholesteryl disulfide derivative (3) in 28% isolated yield. All the new compounds were fully characterized by satisfactory TLC, FT-IR, 1H NMR, mass spectrometry, and elemental analysis. We computed the energy minimized preferred conformations of the thiol forms of the three disulfide amphiphiles using the INSIGHT II (version 97.5) package. These are shown in Figure 1a. It can be seen that 1 has a linear shape with a molecular length of ∼19.32 Å. 2 has an angular shape with a 129° angle between the two hydrocarbon chains attached to the quaternary ammonium group in its most energy minimized conformation in the gas phase. The total length of the molecule (from the S of the thiol group to the end methyl carbon of the tetradecyl chain) was calculated to be ∼32 Å. The most stable conformer of 3 has a “loop” shape with the cholesterol moiety bent toward the thiol group. This structure has a length of ∼18.71 Å from the S of the thiol group to the methyl of the ammonium group. 3.2. Colloid Preparation. Upon modification of a reported procedure,23 a methanolic solution of cationic gold nanoparticles, Au-1, was prepared by reduction of HAuCl4 (7.5 mM) by NaBH4 (0.25 mM) in the presence of disulfide 1 at an Au/1 ratio of 1.0. Similarly, nanoparticles Au-2 and Au-3 were prepared in MeOH as described in the Experimental Section. The respective gold nanoparticles were collected as dark, gummy material by addition of a mixture of acetone/hexane (1:2) followed by filtration. Solubilization of the gum in MeOH/CHCl3 regenerated the colloids. Only Au-1 could be resuspended in water (23) Yonezawa, T.; Onoue, S.; Kunitake, T. Chem. Lett. 1999, 10611062.

Bhattacharya and Srivastava

while aqueous suspensions of Au-2 and Au-3 resulted in precipitation. The fact that Au-2 and Au-3 are insoluble in water and could be dissolved in CHCl3 indicates that the location of the charge on the ammonium cation of the amphiphilic thiols on the gold nanoparticle surfaces would be different from that in Au-1, which is water soluble. It would therefore be reasonable to expect that all three ligands share a similar, fully extended conformation on the Au surfaces. In such a situation in Au-1, the terminal cationic ammonium group residues would be accessible to significant hydration, rendering them water soluble. On the other hand, in Au-2 and Au3, the free unanchored long hydrocarbon chain or the cholesteryl unit, respectively, extends far beyond the cationic charge in their extended conformation. This increases the hydrophobic character of the particle peripheral surface and mitigates their solubility in water. Taking these into consideration, a schematic illustration of various thiol-coated colloids is given in Figure 1b. The Brust method4 uses a phase transfer reagent containing quaternary ammonium ion (such as tetraoctylammonium bromide) to bring the Au(III) salt to the organic layer. A long-chain thiol is then used to cap the gold colloids, rendering them stable to further manipulations. The disulfides employed by us possess both of these functionalities in the same molecule, viz. a quaternary ammonium ion at one end and a disulfide at the other linked through a hydrophobic polymethylene chain. The nanoparticles prepared herein are therefore expected to show amphiphilic behavior. 3.3. Colloid Characterization. 3.3.1. UV-vis Spectroscopy. Figure 2 shows the UV-vis spectra of the darkred solution of different cationic gold nanoparticles (0.1 mg/mL) in the wavelength region 400-800 nm. Broad bands with λmax at ∼525 nm were observed for both the colloids Au-1 and Au-2. These are the characteristic “surface plasmon bands” ascribed to the collective oscillations of the conduction electrons in response to optical excitation.8,9 Freshly prepared Au-3 did not show any peak in the surface plasmon band, owing to its small size (not shown). This is in line with previous reports that the surface plasmon band is dependent on the particle size.6,9,24 Upon prolonged aging, some morphological change was observed only in the case of Au-3, and the particle size increased when Au-3 was kept at ambient temperature for nearly a year. The sample visible spectrum shown in Figure 2c is that obtained after keeping the nanoparticle in solution for 8 months after its preparation. It shows a broad band in the 500-600 nm region. All the particles could be kept as waxlike material or powder and gave visually homogeneous solutions when resuspended in MeOH or in CHCl3 (for Au-2 and Au-3). 3.3.2. IR Spectroscopy. The FT-IR spectra in the transmission mode of neat samples of various cationic amphiphile thiolate-stabilized gold nanoparticles were collected over the range 600-4000 cm-1 (not shown). The C-H stretching region (2800-3000 cm-1) has been used in earlier IR studies on self-assembled monolayers to determine the orientation of the polymethylene chains.6,25 The valleys observed at ∼2920 and 2850 cm-1 in Au-2 could be assigned to symmetric and antisymmetric CH2 stretching vibration modes. With a drop-coated neat (24) Mulvaney, P. Langmuir 1996, 12, 788-800. (25) Nuzzo, R. G.; Fusco, F. A.; Allara, D. L. J. Am. Chem. Soc. 1987, 109, 2358-2370. (b) Hoffman, A. J.; Mills, G.; Yee, H.; Hoffman, M. R. J. Phys. Chem. 1991, 95, 525-530. (c) Sellers, H.; Ulman, A.; Schnidman, Y.; Eilers, J. E. J. Am. Chem. Soc. 1993, 115, 9389-9401. (d) Ulman, A. Chem. Rev. 1996, 96, 1533-1554. (e) Hostetler, M. J.; Stokes, J. J.; Murray, R. W. Langmuir 1996, 12, 3604-3612.


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Figure 1. (a) Energy-minimized conformation of 1, 2, and 3. (b) Schematic illustration of Au-1, Au-2, and Au-3.

sample of Au-2, the values in the observed IR spectrum were 2918 and 2852 cm-1 while, in KBr pellet, these were 2924 and 2854 cm-1, respectively. This indicated that, in the KBr pellet, the thiols were more fluidlike as compared to that in neat conditions. This might be due to the distortions induced during KBr pellet sample preparation. However, in both neat and KBr samples, the capping thiolate appears to exist in a semicrystalline state. We also observed a similar semicrystalline state in the scissoring motion of CH2s. The value of 1464 cm-1 was between that of an all-trans polymethylene chain and that of a methylene unit next to a gauche type conformation. It may be noted that a band at 1419 cm-1 was observed in both the samples of Au-2. This has been ascribed to the scissoring of the methylene group adjacent to the Au-S bond.25e This further confirms the attachment of 2 to Au. A CH3 symmetric bending (umbrella) vibration at 1378 cm-1 was observed in both the samples. Also, the chain end-gauche defect at 1339 cm-1 was observed in the case of a KBr pellet. These results show that the chains were

between melted and crystalline states, and the use of pressure in the KBr pellet preparation might have caused the thiol to become more disordered with the manifestation of the end-gauche defect. Similarly, in the case of Au-3, vibrations at 2930 and 2853 cm-1 were observed in the neat state while, in KBr pellet form, the values were 2926 and 2852, respectively. This result indicates that in Au-3 the polymethylene chain is already in a melted state, and pressure did not significantly alter that. The carbamate carbonyl of 3 showed a peak at 1700 cm-1, and this also remained unaffected upon coating on the nanoparticle surface. The amide-I and amide-II peaks were observed at 1647 and 1541 cm-1, respectively. A peak at 1467 cm-1 indicated that the polymethylene chain was present in an all-trans conformation. This specimen also had a new band at 1418 cm-1. There were peaks at 1377 cm-1, indicating the umbrella transition of the CH3 group, as well as a transition at 1364 cm-1, indicating an internal kink. Therefore, the IR experiments showed that the thiol in

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Figure 2. Visible spectra of solutions of gold colloid (0.1 mg/ mL): (a) Au-1 (in MeOH); (b) Au-2 (in CHCl3); (c) Au-3 (in CHCl3).

Au-3 is already in a disordered, melted state and that the exerted pressure did not significantly change its packing order around the core of the nanoparticle. 3.3.3. NMR Spectroscopy. The 1H NMR spectrum of Au-1 in D2O showed broadened resonances, except for the terminal N(CH3)3 resonance. The 1H NMR spectra of the colloidal particles Au-2 and Au-3 were recorded in CDCl3 as solvent. It also showed line broadening that is characteristic of the thiols adsorbed to colloids (Figure 3b and d). The peaks near the gold core, viz. those for CH2-S and CH2-NH, were found to be indistinguishable from the background noise. This may be explained due to the combined effects of spin-lattice-relaxational broadening (T1) and the chemical shift distributions owing to discontinuous changes in the magnetic susceptibility at the gold-hydrocarbon interface.26-28 NMR spectra also confirmed the absence of any unadsorbed disulfides, as was earlier observed using TLC. 3.3.4. Transmission Electron Microscopy. Direct evidence concerning the size and morphology of nanoparticles came from TEM. Figure 4 shows the TEM images and the size distribution histograms (inset) of all three nanoparticles. For Au-1, the particles have a spherical form and the average particle diameter and the standard deviation in the particle size are 5.9 and 1.8 nm, respectively. Au-2 has particles that are spherical with an average diameter of 2.9 nm and a standard deviation of 0.7 nm. The cationic cholesterol-coated sample has a smaller size (∼2.04 nm) and a deviation of 0.4 nm. Measuring the diameters of a population of 200 particles and doing statistical analysis on the same made us arrive at the figures on the average particle size and standard deviation. Depending upon the molecular structures of the stabilizing disulfide amphiphiles, the nanoparticles Au-1, Au-2, and Au-3 displayed average diameters of 5.9, 2.9, and 2.04 nm, respectively. It is interesting to note that there is an inverse relationship between the bulkiness of the thiol and the size of the nanoparticle formed by it. Thus, the average particle size decreased from Au-1 to Au-3. This is in accord with a previous report where the (26) Terrill, R. H.; Postlethwaile, T. A.; Chen, C. H.; Poon, C. D.; Terzis, A.; Chen, A.; Hutchingston, J. E.; Clark, M. R.; Wignall, G.; Londono, J. D.; Superfine, R.; Falvo, M.; Johnson, C. S., Jr.; Samulski, E. J.; Murray, R. W. J. Am. Chem. Soc. 1995, 117, 12537-12548. (27) Badia, A.; Lennox, R. B.; Reven, L. Acc. Chem. Res. 2000, 33, 475-481. (28) Badia, A.; Singh, S.; Demers, L.; Cuccia, L.; Reven, L. Langmuir 1996, 12, 1262-1269.

Figure 3. 1H NMR spectra (CDCl3, 300 MHz) of (a) 2, (b) Au2, (c) 3, and (d) Au-3.

use of a double-chain disulfide molecule resulted in the formation of a smaller nanoparticle.29 3.3.5. Cationic Nanoparticle-DPPC Coaggregates. To understand the way such nanoparticles coated with cationic lipid entities interact with phospholipids in cell membranes, we prepared various binary mixtures of nanoparticles by doping them in dipalmitoylphosphatidylcholine (DPPC) vesicles. The nanoparticles Au-1 were quite soluble in water and could be solubilized readily into water at a wide range of its concentrations. These solutions were also stable at any pH of the aqueous media. Cosonication of hydrated films of Au-1/DPPC mixtures afforded nanoparticleloaded vesicles, and the resulting suspensions were found to be quite stable. Nanoparticles Au-2 and Au-3 were insoluble in water on their own. However, they could be doped in DPPC vesicles, and the resulting mixtures formed stable aqueous suspensions depending on the relative amount of nanoparticle incorporated. Figure 5 shows the UV-vis spectra of Au-1, Au-2, and Au-3 doped in DPPC vesicles. The presence of a surface plasmon band for these nanoparticle-doped vesicular suspensions clearly demonstrates the presence of a gold nanoparticle in the aggregates (Figure 5). It may be noted that Au-3 had (29) Yonezawa, T.; Yasui, K.; Kimizuka, N. Langmuir 2001, 17, 271273.


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Figure 4. TEM images of nanoparticles: (a) Au-1; (b) Au-2; (c) Au-3 (freshly prepared); (d) Au-3 (after 8 months of preparation).

Figure 5. Visible spectrum of DPPC vesicles doped with nanoparticles (0.2 mg nanoparticles in 1.1 mg of DPPC): (a) Au-1; (b) Au-2; (c) Au-3.

changed morphologically upon aging. But it still gave optically clear chloroform solutions and was used for UVvis to show the presence of a surface plasmon band over the scattering by the vesicular suspension. To further characterize the incorporation of such lipidcoated nanoparticles in DPPC membranes, we examined

the resulting mixtures by TEM. Representative TEM images are shown in Figure 6. In the absence of stain, only nanoparticles and no lipid lamellae were observed (Figure 6a). A negatively stained micrograph of Au-2 inclusion in DPPC is shown in Figure 6b. It shows the presence of Au-2 on DPPC aggregates. The DPPC aggregate lamella are seen, and the presence of Au-2 is evidenced from the existence of dense particulates on the vesicular layers. The use of phosphotungstic acid stain was found to be essential to observe the presence of vesicular structures. Light scattering experiments (figure not shown) revealed that DPPC vesicles were formed with small size distribution and had a mean hydrodynamic diameter of 77 nm, while a similar solution containing 0.3 mg of Au-2 showed a broad size distribution with a mean diameter of 185 nm. This showed that nanoparticle incorporation caused some change in the assembly of the amphiphile. Also, there was a significant change in the ζ-potential of the aggregates. While the DPPC vesicles alone had a ζ-potential close to zero, the nanoparticle-doped assemblies displayed a positive ζ-potential. For example, doping 0.3 mg of Au-2 in 1.1 mg of DPPC gave a ζ-potential of +19.8 mV. This indicates that the assemblies attain a net positive charge on doping with cationic nanoparticles. Differential Scanning Calorimetry (DSC). DSC studies of the sonicated suspensions of neat disulfide amphiphiles in water showed flat traces without any peak in the region 25-55 °C that was examined (not shown). Dispersal of the DPPC or its mixtures with the nanoparticles in water by brief sonication afforded membranous aggregates having the solid-to-fluid thermal phase transition properties summarized in Table 1. The temperature

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Figure 6. TEM images of Au-2 doped in DPPC vesicles. (a) Without staining agent; (b) in the presence of 1% phosphotungstic acid. Table 1. Thermal Phase Transition Parameters of DPPC and Its Coaggregates with Various Nanoparticles As Determined by Differential Scanning Calorimetrya wt % in nanoparticle DPPC Tmb (°C) ∆Hc (kcal/mol) ∆S (cal/(K mol)) DPPC Au-1 Au-2

Au-3

100 9 27 9 18 36 45 9 27

41.6 41.9 41.8 41.5 41.1 40.9 40.5 41.0 40.1

8.5 6.7 8.3 5.6 6.8 4.9 2.3 4.4 3.8

27.0 21.0 26.5 18.0 22.0 15.5 7.5 14.0 12.0

a See text for experimental details on DSC; [DPPC] ) 1 mM; scan rate used was 0.3 K/min. b Accuracy of Tm was (0.1 °C between successive runs of the same sample; two different sample preparations gave a difference of (1 °C. c Calorimetric data are the average of two independent experiments; the error in ∆H is (0.2 kcal/mol.

at which each of these coaggregates is half-converted to the fluid phase is shown under Tm. Transition enthalpy (∆H) and entropy (∆S) values of DPPC coaggregates with nanoparticles were generally lower than those of pure DPPC suspensions. Importantly, in no instance was any evidence of phase separation observed among the blends examined, indicating a practically homogeneous distribution of such nanoparticles on the DPPC membranes. The DSC traces containing the heating and cooling thermograms of the DPPC suspensions doped with various amounts of nanoparticles Au-1, Au-2, and Au-3 are shown in Figure 7. Inclusion of an increasing percentage of Au-1 led to progressive broadening of the gel-to-liquid crystalline phase transition of DPPC, leading to small increases in the Tm values. A gradual decrease in the transition enthalpy was also seen generally, although the resulting Tm values were slightly higher than that of DPPC alone (Table 1). Due to the complex nature of the transition, it is, however, less certain why the transition enthalpy and entropy values of DPPC coaggregates with 0.3 mg of Au-1 were higher. It should also be mentioned that pure suspensions of Au-1 in water did not display a melting transition on their own in the investigated temperature range (25-55 °C) under DSC. Inclusion of Au-2 in DPPC had interesting consequences. In this case, inclusion of incremental Au-2 led to progressive lowering of the gel-to-liquid crystalline temperature (Tm) of DPPC with concomitant broadening (Figure 7b). However, even after inclusion of 0.5 mg of

Au-2 in DPPC, distinct melting transitions were observed. It is important to recognize that in Au-2 only one hydrocarbon chain is anchored on the gold interface through the thiol function, while the other n-tetradecyl chain is free to undergo heat-induced s-trans to s-cis conformational isomerization. The presence of such a dynamic hydrocarbon chain in Au-2 enables it to be solubilized in DPPC membranes more effectively without significantly disturbing the palmitoyl chain motions of DPPC. Incorporation of cholesterol-containing cationic nanoparticle Au-3 in DPPC effected >1 °C lowering of Tm and eventual abolition of the melting phase transition of DPPC (Figure 7c). Clearly, while a melting transition was visible upon incorporation of 0.5 mg of Au-2 in 1.1 mg of DPPC, addition of 0.3 mg of Au-3 in 1.1 mg of DPPC caused total abolition of DPPC melting by completely suppressing the chain melting of the palmitoyl chains of DPPC. This suggests that despite attachment of a hydrocarbon chain and a charged segment to the cholesterol and its immobilization on the Au interface, the resulting systems exert an effect that is comparable to that of natural cholesterol in DPPC membranes.30,31 It must be, therefore, the presence of rigid, fused rings in the steroid in the extended conformational orientation in Au-3 which induces quenching of fatty acid chain melting in phosphatidylcholine membranes. Taken together, these results indicate that more lipophilic cationic nanoparticles interact with DPPC membranes more effectively irrespective of whether the hydrophobic segments are based on a flexible hydrocarbon chain or a rigid cholesteryl skeleton. Thus, significantly higher amounts of cationic lipid units immobilized on gold nanoparticles of Au-2 and Au-3 could be successfully incorporated into naturally occurring membranes of DPPC. It is also noteworthy that the incorporation of hydrophilic nanoparticles Au-1 in DPPC had the opposite effect in that it raised the Tm values by ∼1 °C while Au-2 and Au-3 lowered the DPPC Tm values. Considerably less amount of Au-1 could be incorporated in the DPPC. These findings provide key insights for the design of cationic lipid-coated nanoparticles that should be able to (30) Ghosh, Y. K.; Indi, S. S.; Bhattacharya, S. J. Phys. Chem. B 2001, 105, 10257-10265. (31) Bhattacharya, S.; Ghosh, Y. K Langmuir 2001, 17, 2067-2072. (b) Bhattacharya, S.; Haldar, S. Biochim. Biophys. Acta 2000, 1467, 39-48.


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Figure 7. Effect of nanoparticle inclusion on the DPPC vesicle melting transition: (a) Au-1; (b) Au-2; (c) Au-3.

interact with natural membrane components effectively for specific biological applications. 4. Conclusions In summary, we have reported the controlled synthesis of cationic gold nanoparticles coated with different types of amphiphilic disulfide based stabilizers. These particles display a small size distribution. This is the first report of the preparation and characterization of cholesterolderivatized gold nanoparticles. These nanoparticles, owing to the presence of cationic charge and a cholesterol moiety, will be of interest in biological studies, for example, transfection, interactions with natural and artificial membranes, and so forth. Differential scanning calorimetric studies indicated that the hydrophobic nanoparticles interact strongly with DPPC vesicles and cause their melting points to decrease gradually while the hydrophilic nanoparticles exert almost the reverse effect and cause formation of phases that melt at higher temperatures. The cholesterol moiety in Au-3 also caused the broadening of the melting transition, and this result is similar to the effect of free cholesterol.31

Cationic liposomes made of cholesterol derivatives have been shown to be efficient DNA transfection agents.22 This prompted us to synthesize disulfides 2 and 3, since the nanoparticles synthesized using these disulfide as stabilizers would provide access to “size-stable analogues” of cationic liposomes. They would provide a cationic surface for strong interaction with negatively charged DNA but would be stable to changes in size or shape, since they would be anchored to the nanoparticle surface by the sulfur atom. Moreover, due to the chemical inertness of gold, such nanoparticles should be biologically safe. Also, these nanoparticles are electron-dense and can be easily observed by TEM. So the trafficking of these systems across biological membranes should be monitored by electron microscopy. Work is underway to accomplish such applications. Acknowledgment. This work is supported by the Swarnajayanti Fellowship grant of the DST. A.S. thanks CSIR for a Senior Research Fellowship. LA0269513

Synthesis and Characterization of Novel Cationic Lipid and

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