Polymeric Amphiphile Branching Leads to Rare Nanodisc Shaped

Aug 9, 2008 - School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N, Department of Pharmaceutical Sciences, University of ...
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Langmuir 2008, 24, 9997-10004

9997

Polymeric Amphiphile Branching Leads to Rare Nanodisc Shaped Planar Self-Assemblies Xiaozhong Qu,‡ Leila Omar,‡ Thi Bich Hang Le,† Laurence Tetley,§ Katherine Bolton,‡ Kar Wai Chooi,† Wei Wang,‡ and Ijeoma F. Uchegbu*,† School of Pharmacy, UniVersity of London, 29-39 Brunswick Square, London WC1N, Department of Pharmaceutical Sciences, UniVersity of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, United Kingdom, and Electron Microscopy Unit, Institute of Biomedical and Life Sciences, UniVersity of Glasgow, Glasgow G12 8QQ, United Kingdom ReceiVed March 12, 2008. ReVised Manuscript ReceiVed June 24, 2008 Self-assembly is fundamental to the biological function of cells and the fabrication of nanomaterials. However, the origin of the shape of various self-assemblies, such as the shape of cells, is not altogether clear. Polymeric, oligomeric, or low molecular weight amphiphiles are a rich source of nanomaterials, and controlling their self-assembly is the route to tailored nanosystems with specific functionalities. Here, we provide direct evidence that a particular molecular architecture, polymeric branching, leads to a rare form of self-assembly, the planar nanodisc. Cholesterol containing self-assemblies formed from amphiphilic linear or branched cetyl poly(ethylenimine) (Mn ∼ 1000 Da) or amphiphilic cetyl poly(propylenimine) dendrimer derivatives (Mn ∼ 2000 Da) show that branching, by reducing the hydrophilic headgroup area, alters the shape of the self-assemblies transforming closed 60 nm spherical bilayer vesicles to rare 50 nm × 10 nm planar bilayer discs. Increasing the hydrophilic headgroup area, by the inclusion of methoxy poly(ethylene glycol) moieties into the amphiphilic headgroup, transforms the planar discs to 100 nm spherical bilayer vesicles. This study provides insight into the key role played by molecular shape on molecular self-organization into rare nanodiscs.

Introduction Self-assembly is fundamental to the biological function of cells and the fabrication of nanomaterials for delivery,1 diagnostic,2 and environmental cleanup3 applications. However, the origin of the shape of various self-assemblies, such as the shape of cells, is not altogether clear; although it is known that some form of membrane amphiphile heterogeneity is responsible for the variability seen in synthetic membrane curvature.4,5 Polymeric, oligomeric, or low molecular weight amphiphiles6,7 are a rich source of nanomaterials, and controlling their self-assembly is the route to tailored nanosystems with specific functionalities.7-16 * Corresponding author. † University of London. ‡ University of Strathclyde. § University of Glasgow. (1) Torchilin, V. P., Nanoparticulates as drug carriers; CRC Press: Boca Raton, FL, 2006. (2) Cuenca, A. G.; Jiang, H. B.; Hochwald, S. N.; Delano, M.; Cance, W. G.; Grobmyer, S. R. Cancer 2006, 107(3), 459–466. (3) Narr, J.; Viraraghavan, T.; Jin, Y. C. Fresenius EnViron. Bull. 2007, 16(4), 320–329. (4) Uchegbu, I. F.; Schatzlein, A.; Vanlerberghe, G.; Morgatini, N.; Florence, A. T. J. Pharm. Pharmacol. 1997, 49(6), 606–610. (5) Baumgart, T.; Hess, S. T.; Webb, W. W. Nature 2003, 425(6960), 821– 824. (6) Israelachvili, J. Intermolecular & Surface Forces, 2nd ed.; Academic Press: London, 1991. (7) Uchegbu, I. F. Expert Opin. Drug DeliVery 2006, 3, 629–640. (8) Wang, W.; Qu, X. Z.; Gray, A. I.; Tetley, L.; Uchegbu, I. F. Macromolecules 2004, 37(24), 9114–9122. (9) Bai, G. Y.; Wang, Y. J.; Yan, H. K.; Thomas, R. K.; Kwak, J. C. T. J. Phys. Chem. B 2002, 106(9), 2153–2159. (10) Wang, W.; Tetley, L.; Uchegbu, I. F. J. Colloid Interface Sci. 2001, 237(2), 200–207. (11) Wang, W.; McConaghy, A. M.; Tetley, L.; Uchegbu, I. F. Langmuir 2001, 17(3), 631–636. (12) Qu, X. Z.; Khutoryanskiy, V. V.; Stewart, A.; Rahman, S.; Papahadjopoulos-Sternberg, B.; Dufes, C.; McCarthy, D.; Wilson, C. G.; Lyons, R.; Carter, K. C.; Schatzlein, A.; Uchegbu, I. F. Biomacromolecules 2006, 7(12), 3452– 3459. (13) Discher, B.; Won, Y. Y.; Ege, J. C. M.; Bates, F. S.; Discher, D.; Hammer, D. A. Science 1999, 284, 1143–1146. (14) Shimizu, T. Polym. J. 2003, 35(1), 1–22.

Here, we provide direct evidence that a particular molecular architecture, branching, leads to a rare form of self-assembly, the planar nanodisc. Cholesterol containing self-assemblies formed from amphiphilic linear or branched cetyl poly(ethylenimine) (Mn ∼ 1000 Da) show that branching, by reducing the hydrophilic headgroup area, alters the shape of the self-assemblies transforming closed 60 nm spherical bilayer vesicles to rare 50 nm × 10 nm planar bilayer discs. Increasing hydrophilic headgroup area by the inclusion of methoxy poly(ethylene glycol) moieties transforms the planar discs to 100 nm spherical bilayer vesicles. This study provides insight into the key role played by molecular shape on molecular self-organization into rare nanodiscs. According to the work of Israelachvili, increasing the relative size of an amphiphile’s hydrophobic moiety (or alternatively diminishing the relative size of its hydrophilic headgroup area) should result in aqueous dispersions of self-assemblies transforming from spherical micelles to rodlike micelles and finally to vesicular bilayers.6 It is known that further increases in amphiphile hydrophobicity result in the transformation of spherical vesicles to dense nanoparticles.8 Direct experimental evidence exists for these spherical7,8,10-13,16,17 (micelles, vesicles, and dense nanoparticles) and rod shaped8,14 aggregates being formed from the self-assembly of polymeric7,8,10-13,16,17 and low molecular weight amphiphiles6,14,15 in aqueous media, with each self-assembly being prepared from specific amphiphile chemistries. Planar disclike self-assemblies, on the other hand, are rare, and the rules governing their formation are poorly understood. We have investigated the origin of such shape determinants, and by using amphiphiles prepared from the polyamine polymer, poly(ethylenimine) (PEI), we are able to (15) Gregoriadis, G. Liposome Technology; CRC Press: Boca Raton, FL, 2006; Vols. I-III. (16) Uchegbu, I. F.; Schatzlein, A. G. Polymers in Drug DeliVery; Taylor and Francis: Boca Raton, FL, 2006. (17) Wang, W.; Tetley, L.; Uchegbu, I. F. Langmuir 2000, 16(20), 7859– 7866.

10.1021/la8007848 CCC: $40.75  2008 American Chemical Society Published on Web 08/09/2008

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Table 1. Synthesis of Branched and Linear PEI Amphiphiles

sample LCPEI 7 LCPEI 14 LCPEI 23 LCPEI 38 LCPEI 42 8 BCPEI 7 BCPEI 14 BCPEI 26 c

methyl CMC initial PEI monomer, no. of cetyl no. of palmitoyl cetyl bromide chains/ chains/ CMC CMC (µM cetyl orange Tm linear or Mn PEI molar ratio cetylation (%)a moleculeb molecule (g L-1) (µM) chains) λmax (nm) (°C)c branched PEI (Da) linear linear linear linear linear branched branched branched

423 423 423 423 423 600 600 600

1:0.12 1:0.13 1:0.23 1:0.45 1:0.49 1:0.09 1:0.15 1:0.32

0.65 14 23 38 42 7 14 26

0.7 1.4 (1.0) 2.3 (1.9) 3.8 (3.6) 4.2 0.9 (1.2) 1.9 (2.3) 3.6 (4.4)

0.019 0.012 0.018 0.018 0.021 0.014 0.017

29 15 14 13 25 13 11

41 35 53 55 23 25 40

428 421 419 n.d. 429 424 420

none none 31d 24 n.d. n.d. n.d.

a Number of cetyl chains per 100 monomer units. b Determined using elemental analysis data;8,20 data in parentheses were determined using 1H NMR. Tm ) phase transition temperature. d ∆H ) 6.8 J g-1.

Table 2. Synthesis of Branched mPEG Containing Amphiphiles

sample PCBPEI 1-1 PCBPEI 1-2 PpBPEI 1-2 PpBPEI 3-1 a

initial mPEG, initial mPEG, no. of PEG no. of cetyl no. of palmitoyl CMC methyl linear or Mn PEI BCPEI molar BPEI, chains/ chains/ chains/ CMC CMC (µM cetyl orange branched PEI (Da) ratio palmitoyl ratio molecule molecule molecule (g L-1) (µM) chains) λmax (nm) branched branched branched branched

600 600 600 600

Synthesized from BCPEI 7.

b

0.4 0.3

0.9a 1.9b

1.4 0.8 1.3 2.9

0.2:1:3.0 2.5:1:1.5

1.7 1.3

0.104 0.030 0.400 0.929

18 5.0 66 59

16 9.5

427 424 427 430

Synthesized from BCPEI 14.

Table 3. Synthesis of Poly(propylenimine) Amphiphiles initial poly(propylenimine), cetyl bromide/palmitoyl N-hydroxysuccinimide molar ratio

sample cetyl poly(propylenimine) acetamide terminated palmitoyl (propylenimine) a

4.9 5

Determined using elemental analysis data.8,20

b

no. of cetyl/palmitoyl chains per molecule

cetylation/palmitoylation levels (mol % per primary amine groups)

CMC (g L-1)

CMC (µM)

6 6

0.27 0.129 ( 0.033

140 50 ( 12

1.09 ( 0.67 1.12 (1.07)b

a

Determined using MALDI-TOF data; data in parentheses calculated using 1H NMR.

show that polymer branching leads to the formation of planar disc morphologies in cholesterol containing self-assemblies, because polymer branching results in a diminished hydrophilic headgroup area at the particle interface.

Materials and Methods Synthesis and Characterization. Cetyl linear poly(ethylenimine) (LCPEI) and cetyl branched poly(ethylenimine) (BCPEI) were synthesized using previously published methods in which poly(ethylenimine) (PEI, Sigma Aldrich Co., U.K.) was reacted with 1-bromo-hexadecane (Sigma Aldrich Co., U.K.)8 in the reactant ratios given in Table 1 and with a starting level of 2 g of PEI. The solvent used for synthesis was chloroform (20 mL, Merck, U.K.), and the product was freeze-dried, presenting as a yellow solid. The yields of LCPEIs were as previously reported,8 while the yields of BCPEIs were as follows: BCPEI 7-45%, BCPEI 14-42%, BCPEI 26-56%. Synthesis of cetyl poly(ethylenimine)-g-methoxy poly(ethylene glycol) (PCBPEI) proceeded by the dissolution of BCPEI (150 mg) in a mixture of sodium tetraborate (0.08 M, 210 mL) and absolute ethanol (90 mL). To this was added methoxy poly(ethylene glycol)p-nitrophenol carbonate (mPEG-p-nitrophenol carbonate, Mw ∼ 5000 Da) in the quantities given in Table 2 and in three divided portions over a 3 h period. The reaction mixture was stirred for 24 h at room temperature, protected from light, and subjected to exhaustive dialysis (Visking tubing, molecular weight cutoff ) 3500 Da) against 40% v/v ethanol (5 L with three changes in 8 h), followed by dialysis against distilled water (5 L with six changes in 24 h). The residue was freeze-dried and presented as a light yellow powder. The yield was 80%. Synthesis of palmitoyl poly(ethylenimine)-g-methoxy poly(ethylene glycol) proceeded by the dissolution of branched PEI (BPEI, 500 mg) in sodium tetraborate (0.08 M, 300 mL). To this was added an amount of mPEG-p-nitrophenol carbonate (MW ∼ 5000 Da) as

Table 4. Molecular Weight and Radius of Gyration of Poly(ethylenimine)

avg molecular mass (Da) Rg (nm) dn/dc

branched PEI (mean ( SD, n ) 3)

linear PEI

900 ( 115 31.7 ( 1.9 0.181 ( 0.020

489 ( 113 76.0 ( 23.4 0.189 ( 0.010

Table 5. Hydrodynamic Diameters of PEI Amphiphile Self-Assemblies Z-average mean diameter of PEI amphiphile (10 g L-1) aggregates (nm)

Z-average mean diameter of PEI amphiphile (10 g L-1), cholesterol (5 g L-1) aggregates (nm)

diameter (nm) polydispersity diameter (nm) LCPEI 14 LCPEI 23 LCPEI 38 BCPEI 7 BCPEI 14 BCPEI 26 PCBPEI 1-1 PCBPEI 1-2 PpBPEI 1-2 PpBPEI 3-1

17 (5.1) Pa 42 (14) Pa 108 (54) Pa 15 (6.2) Pa 47 (10) Pa 47 (36) 139 (60) Pa 165 (80) Pa 96 78

0.33 0.48 0.72 0.74 0.49 0.19 0.61 0.80 0.23 0.51

59 57 82 58 46 43 134 103 176 178

polydispersity 0.24 0.34 0.41 0.17 0.38 0.27 0.12 0.11 0.18 0.25

a P ) two peaks seen in the size distribution plot; numbers in parentheses are the volume mean diameters.

indicated in Table 2. The reaction mixture was stirred for 24 h at room temperature, protected from light, and then subjected to exhaustive dialysis (MW cutoff ) 3500 Da for PpBPEI 1-2, MW cutoff ) 7000 Da for PpBPEI 3-1) against distilled water (5 L with six changes in 24 h). Sodium bicarbonate (1.2 g, 14.3 mmol) was then dissolved in the dialyzed liquid, and an ethanolic solution

Rare Nanodisc Shaped Planar Self-Assemblies (absolute ethanol, 100 mL) of palmitic-N-hydroxysuccinimide (PNS) in the amount given in Table 2 (referenced to the starting amount of BPEI) was added dropwise with stirring for 1 h. The reaction mixture was then left stirring in the dark for 72 h, subsequently dialyzed exhaustively against 40% v/v ethanol (5 L with three changes in 8 h), followed by dialysis against distilled water (5 L with six changes in 24 h). The dialyzate was extracted with diethyl ether and freeze-dried to give a white powder. Cetyl poly(propylenimine) dendrimer was synthesized by reacting cetyl bromide (0.036 mL, 0.12 mmol) with poly(propylenimine) generation 3 dendrimer (1 g, 0.59 mmol) in tetrahydrofuran (50 mL) at 70 °C (reflux) for 48 h with continuous stirring. At the end of which sodium hydroxide pellets (2 g, 50 mmol) dissolved in methanol (25 mL) were added to the cooled reaction mixture, and the reaction was refluxed a second time at 70 °C for a further 24 h. The product was isolated by evaporation of the solvent under reduced pressure, air drying of the product, exhaustive dialysis (Visking dialysis tubing, Medicell International, U.K., MW cutoff ) 12-14 kDa) against distilled water (5 L with six changes over 24 h), and freeze-drying. The yield was 0.332 g (32%) Palmitoyl poly(propylenimine) dendrimer was synthesized by the dropwise addition of palmitic acid N-hydroxysuccinimide (0.524 g, 1.48 mmol) dissolved in ethanol (397 mL) to poly(propylenimine) generation 3 dendrimer (0.5 g, 0.30 mmol) in the presence of sodium bicarbonate (0.375 g, 4.5 mmol). The poly(propylenimine) dendrimer and sodium bicarbonate were dissolved in a 1:1 mixture of ethanol and water (500 mL). The reaction was left stirring for 72 h protected from light, and the product was isolated by evaporation of ethanol under reduced pressure at 60 °C, exhaustive dialysis as detailed above, freeze-drying, and washing of the dry product with diethyl ether (3 × 100 mL). Residual ether was removed by air drying. Acetylation of palmitoyl poly(propylenimine) was performed by the dropwise addition of acetic anhydride (0.557 mL, 5.05 mmol) to ice cooled palmitoyl poly(propylenimine) (0.311 mg, 0.16 mmol) dissolved in phosphate buffered saline (pH ) 7.4, 63 mL) and saturated sodium acetate solution (63 mL) on ice. The reaction mixture was stirred for 24 h, exhaustively dialyzed (Visking tubing, molecular weight cutoff ) 1.35 kDa) against distilled water (5 L with six changes over 24 h), and lyophilized. The yield was 0.949 g (28.4%). Chemical characterization involved analysis of the relative amounts of carbon, hydrogen, and nitrogen using a Perkin Elmer 2400 analyzer8 and 1H NMR analysis (with integration) of solutions of the amphiphiles, in CDCl3 or CD3OD, using a Bruker AMX 400 MHz spectrometer (e.g., Supporting Information S1). Cetyl poly(propylenimine) dendrimer was also analyzed by positive electrospray ionization mass spectrometry: cetyl poly(propylenimine) dendrimer was dissolved in a 1:1 mixture of methanol and formic acid (0.1%) and then infused into a TSQ 7000 quadrupole mass spectrometer (ThermoQuest) with the electrospray ionization needle held at 4.5 kV and the capillary temperature at 250 °C. Complete acetylation of unreacted amines was confirmed via the trinitrobenzene sulfonic acid assay for primary amines.18 Acetamide terminated palmitoyl poly(propylenimine) was analyzed by matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) spectrometry. The experiments were carried out on a Voyager-DE PRO Biospectrometry workstation (PerSeptive Biosystems, Inc.) equipped with a nitrogen laser operating at 337 nm. The matrix used was 2,5-dihydroxybenzoic acid (10 mg) in acetonitrile and trifluoroacetyl (TFA, 1 mL, 30:70 v/v). Prior to measurement, the polymer solution (0.7 µL, 4-8 mg mL-1) in methanol and water (1:1 v/v) was placed onto a stainless steel target. The matrix (0.7 µL) was then added to the sample spot, pipet-mixed, and subsequently allowed to air-dry at room temperature. The instrument was operated in a linear mode at a 20 kV acceleration voltage. Each mass spectrum was acquired from 300 laser shots and 3003 laser intensity. All data were collected and processed using the Voyager Biospectrometry workstation with Delayed Extraction Technology version 5 series software provided with the instrument. (18) Snyder, S.; Sobocinski, P. Anal. Biochem. 1975, 64, 284–288.

Langmuir, Vol. 24, No. 18, 2008 9999 Self-Assembly. Polymer self-assembly was initially probed by measuring the critical micellar concentration (CMC) using the hypsochromic shift experienced by a dilute alkaline solution (borate buffer, 0.02 M, pH ) 9.4) of methyl orange (25 µM) when methyl orange partitions into the polymer aggregates’ nonpolar domain.8,19,20 An amphiphilic polymer dispersion formed by probe sonication (10 min with the instrument set at its maximum output, Soniprep 150, Sanyo MSE) in water (20 g L-1) was subsequently diluted with the methyl orange solution, and the absorbance spectra were recorded. A plot of the wavelength of maximum absorbance against concentration gives a sharp inflection point when aggregation commences (Figure 2). Supramolecular structures were prepared and characterized using established methods,11,17 namely, dispersion by probe sonication of the amphiphiles in aqueous media in the absence and presence of cholesterol and sized and imaged, subsequent to filtration (0.45 µm, Millipore), using photon correlation spectroscopy and transmission electron microscopy, respectively. Transmission electron microscopy with negative staining was performed as follows. Carbon-coated 200 mesh copper grids were glow discharged, and vesicle suspensions were applied, followed by the application of methylamine tungstate negative stain. The grids were dried and imaged using a LEO 902 electron microscope at 80 kV. Furthermore, cholesterol-free samples of the low molecular weight branched amhiphiles were subjected to differential scanning calorimetry: polymer dispersions (40 g L-1) were sealed in 120 µL medium pressure aluminum crucibles and scanned at a heating rate of 1 °C min-1 using a Mettler Toledo DSC 30 instrument (Mettler Toledo, U.K.). A DAWN EOS static laser light scattering instrument (Wyatt Technology) was used to measure the average molecular mass, rootmean-square radius (Rg), and second virial coefficient (A2) of linear PEI (MW ) 423 Da) and branched PEI (MW ) 600 Da) in methanol, NaCl (0.5 M) (1:1). A Rheodyne 7725 sample injector was used to load different concentrations (2.5-30 mg mL-1) of the filtered (0.2 µm) solutions at a pump flow rate of 0.6 mL min-1. The MW, Rg, and A2 values were obtained from Zimm plots processed using Astra for Windows 4.90.08 software (Micro-Batch mode). Refractive index increments (dn/dc) of the polymer solutions in the same solvent were measured with an OPTILAB DSP interferometric refractometer (Wyatt Technology, λ ) 690 nm) at 25 °C. Filtered (0.2 µm) solutions (0.1-0.5 mg mL-1) were loaded onto the instrument at a pump flow rate of 0.3 mL min-1, and the data were processed using DNDC for Windows version 5.90.03 software.

Results and Discussion The hypothesis that aqueous dispersions of disc shaped polymeric nanoassemblies result from a reduced hydrophilic headgroup area and may be accessed via branched polymeric amphiphiles was tested by synthesizing and studying 11 PEI amphiphiles and further confirmed with a branched [poly(propylenimine) dendrimer] amphiphile. PEI is available in branched and linear formats as well as a variety of molecular weights (Figure 1 and Table 1). The synthesis and characterization of cetyl PEI amphiphiles [cetyl linear PEI (LCPEI8) and cetyl branched PEI (BCPEI20)] have been reported previously. Structural characterization was achieved using 1H NMR8,20 (Supporting Information Figure S1a). Elemental analysis data (e.g., Supporting Information Table 2) were used to determine cetylation levels.8,20 Although we have previously used 1H NMR data to estimate levels of hydrophobic group substitution8,10,17 in polymer amphiphiles bearing acyl pendant groups, the overlapping of the cetyl and PEI methylene amino peaks at 2.3-3.1 ppm makes exploitation of the NMR analysis data, for (19) Zhu, D. M.; Wu, X.; Schelly, Z. A. J. Phys. Chem. 1992, 96(17), 7121– 7126. (20) Cheng, W. P.; Gray, A. I.; Tetley, L.; Hang, T. L. B.; Schatzlein, A. G.; Uchegbu, I. F. Biomacromolecules 2006, 7(5), 1509–1520.

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Figure 1. (top) Poly(ethylenimine) amphiphiles and (bottom) poly(propylenimine) amphiphiles.

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Rare Nanodisc Shaped Planar Self-Assemblies

Figure 2. Aggregation of branched PEI amphiphiles: open symbols, mPEG amphiphiles; closed symbols, cetyl PEI amphiphiles. The first inflection point in the λmax versus concentration curve indicates the migration of methyl orange into the apolar domains formed by aggregating polymer molecules, and this first inflection point is the critical micellar concentration (CMC).

estimating the level of hydrophobic substitution, less than straightforward. However, if the cetylation levels are calculated by comparing the integrals of the peaks located at 0.9 and 2.3-3.1 ppm and including a correction for the contribution of the cetyl CH2N protons to the broad multiplet at 2.3-3.1 ppm (subtraction of the equivalent of 2 protons for every 3 methyl protons at 0.9 ppm), there is good agreement between cetylation levels estimated using 1H NMR (Supporting Information Figure S1a) and elemental analysis (comparing the ratio of carbon to nitrogen, e.g., Supporting Information Table 2) data (Table 1). Cetylation levels (Table 1) were efficiently controlled via the reactant ratios,8 and the eventual percentage of PEI monomers substituted was not affected by polymer branching (Table 1). Methoxy(polyethylene glycol) (mPEG) substituted molecules (Figure 1 and Table 2), namely, cetyl poly(ethylenimine)-gmethoxy poly(ethylene glycol) (PCBPEI) and palmitoyl poly(ethylenimine)-g-methoxy poly(ethylene glycol) (PpBPEI), were synthesized in order to examine the effect of increasing the size of the amphiphile’s hydrophilic headgroup area and were prepared using a modification of the method reported previously for poly(L-lysine) polymers;10,17 structural confirmation was obtained using 1H NMR data (e.g., Supporting Information Figure S1b); and proton assignments were as follows: PpBPEI [δ 0.85, -CH3 (palmitoyl); 1.24, -CH2 (palmitoyl); 2.2-2.9 -CH2 (PEI); 3.38, -OCH3 (mPEG); 3.45-3.90, -CH2-O (mPEG); 4.22, -NH (PEI)], PCBPEI [δ 0.95, CH3 (cetyl); 1.33, -CH2 (cetyl); 2.6-3.4, -CH2-N (cetyl and PEI); 3.43, -OCH3 (mPEG); 3.55-3.93, -CH2-O (mPEG); 4.26, -NH (PEI)]. Elemental analysis and 1H NMR data (comparing the O-CH peak at 3.38 pp with the 3 broad multiplet at 2.2-3.4 ppm) were used to estimate the levels of palmitoylation and mPEG substitution, respectively, at the end of each of the reaction steps (Table 2). In the case of the cetylated and mPEGylated polymer (PCBPEI), since the cetyl amphiphile was used as the starting material in the mPEGylation reaction, the level of cetylation was assumed to be unchanged during the mPEGylation reaction. Cetyl poly(propylenimine) was synthesized in order to confirm the hypothesis that branching drives disc shaped self-assemblies and was synthesized using a modification of the method used to prepare cetyl PEIs.8,20 Structural confirmation was obtained using NMR (Supporting Information S1c); proton assignments were as follows: δ 0.89, -CH3 (cetyl); 1.29, -CH2 (cetyl); 1.47,

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-NCH2-CH2-CH2-CH2N [poly(propylenimine)] and CH2CH2-N (cetyl); 1.64, -NCH2-CH2-CH2N [poly(propylenimine)]; 2.48, -CH2-N [poly(propylenimine) and cetyl], 2.75, -CH2-NH2 [poly(propylenimine)]; 3.10 and 3.26, CH2-NH3+ [poly(propylenimine)]. The level of cetylation, estimated from the elemental analysis data,8,20 was 1 cetyl chain per poly(propylenimine) molecule but varied from 0.45 cetyl chains to 1 cetyl chain per molecule. Mass spectrometry data (Supporting Information Figure S1d and Table S1) further confirmed the synthesis of cetyl poly(propylenimine). The synthesis of acetamide terminated palmitoyl poly(propylenimine) was confirmed using NMR (Supporting Information Figure S1f); proton assignments were as follows: δ 0.87, -CH3 (palmitoyl); 1.26, -CH2 (palmitoyl); 1.44, -NCH2-CH2CH2-CH2N [poly(propylenimine)]; 1.59, -CH2-CH2-CO (palmitoyl); 1.70, -NCH2-CH2-CH2N [poly(propylenimine)]; 1.94, -CH3-CO-NH- [acetyl-poly(propylenimine)]; 2.17, -CH2-CO-NH (palmitoyl); 2.44-2.70, -CH2-N [poly(propylenimine)]; 3.19, -CH2-NH-CO [poly(propylenimine)]. The level of palmitoylation was estimated using 1H NMR (comparing the peak intensities at δ 0.87 and 1.70, Supporting Information Figure S1e) and MALDI-TOF (Supporting Information Figure S1f data). LCPEIs (Mn ) 423 Da) with a level of cetylation of 23 mol % per PEI monomer or less (23 out of every 100 PEI monomers substituted with a cetyl group) form micellar aggregates in aqueous media and self-assemble into larger ordered particles (indicated by the presence of a phase transition endotherm) when the level of cetylation is increased to 38-42 mol %8 (Table 1). The branched amphiphiles also aggregate in aqueous media, as evidenced by the hypsochromic shift8,19,20 in the methyl orange absorbance spectra (Table 1 and Figure 2), and the critical micellar concentration (CMC) is determined as the first inflection point in the λmax versus concentration plot (Figure 2). Using this method, the CMC of Triton-X 100 was found to be 0.15 g L-1 (Supporting Information Figure S2), which is in good agreement with published values of 0.19 g L-1.21 The CMC values are not very different for the branched and linear amphiphiles despite the higher Mn for the branched amphiphile (Tables 1 and 4). Poly(propylenimine) dendrimer amphiphiles also aggregate in aqueous media (Table 3). BCPEIs with 14 mol % cetylation or less self-assemble into 5-14 nm micelles, and a minority of larger 20-50 nm particles as two peaks were seen in the size distribution plot (Table 5); however, 26 mol % cetylation in the branched amphiphiles results in the formation of more of the larger nanoparticles (Tables 1 and 5, and Supporting Information Figure S3). Particle sizes in excess of 20 nm cannot be conventional core shell micelles, as this would entail the hydrocarbon chains considerably exceeding their critical chain length (lc),6 which for a C16 chain is estimated to be 2.2 nm.6 Increasing the hydrophilic headgroup area of the branched polymers by mPEG substitution increases the size (Tables 1 and 5) and, in the case of BCPEI 14, the stability of the aggregates (aggregates form at a lower CMC, compare PCBPEI 1-2 with BCPEI 14, Figure 2). It is envisaged that noncovalent bonding involving mPEG groups promotes aggregate stability. The palmitoyl groups have a profound effect on aggregation, decreasing the stability of the aggregates markedly (compare PCBPEI 1-2 and PpBPEI 1-2, Table 5 and Figure 2). It is clear that the carbonyl unit in the palmitoyl group enables hydrogen bonding with water and thus diminishes any entropic gain22 that (21) Rharbi, Y.; Li, M.; Winnik, M. A.; Hahn, K. G. J. Am. Chem. Soc. 2000, 122(26), 6242–6251.

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Figure 3. (a) Negative stained transmission electron micrograph of an aqueous dispersion of LCPEI 7 (20 mg mL-1) and cholesterol (10 mg mL-1) vesicles, bar ) 200 nm. Ellipsoid and spherical vesicles are seen. (b) Freeze fracture electron micrograph of an aqueous dispersion of LCPEI 14 (20 mg mL-1) and cholesterol (10 mg mL-1) vesicles, bar ) 200 nm. Vesicles are clearly seen as well as some unusual fused structures (indicated by arrow). (c) Negative stained electron micrograph of an aqueous dispersion of BCPEI 7 (10 mg mL-1) and cholesterol (5 mg mL-1), bar ) 200 nm. The dispersion is dominated by spherical structures, although some discs are evident (indicated by arrows). (d) Negative stained electron micrograph of an aqueous dispersion of BCPEI 14 (5 mg mL-1) and cholesterol (2.5 mg mL-1), bar ) 200 nm. The dispersion is almost entirely composed of disc shaped structures, pictured from their short and long axes. (e) Negative stained electron micrograph of an aqueous dispersion of PCBPEI 1-2 (10 mg mL-1) and cholesterol (5 mg mL-1), bar ) 200 nm. The amphiphile contains two cetyl chains and one mPEG chain per molecule. Spherical vesicular structures are seen. (f) Negative stained transmission electron micrograph of an aqueous dispersion of PpBPEI 1-2 (10 mg mL-1) and cholesterol (5 mg mL-1), bar ) 100 nm. The amphiphile contains two palmitoyl chains and one mPEG chain per molecule. Spherical vesicles and a few micellar structures are in evidence. The rod shaped micelles seen in the top right-hand corner of the image are unlikely to be discs viewed from their short axis, due to the fact that the dimensions of these rod shaped structures do not correlate with the diameters of the high curvature structures in the center of the image. (g) Negative stained transmission electron micrograph of an aqueous dispersion of cetyl poly(propylenimine) dendrimer (5 mg mL-1) and cholesterol (2.5 mg mL-1) in water, bar ) 200 nm. Nanodiscs are clearly seen in the image.

would be experienced, on aggregation, by the liberation of a cavity of water bounding the hydrocarbon units. Cholesterol promotes membrane formation in both low molecular weight amphiphiles23 and polymeric bilayers,8 and

the inclusion of cholesterol transforms the micellar forming polymers (LCPEI 7 and LCPEI 14) to vesicle forming polymers (Figure 3a and b). The branched polymer/cholesterol selfassemblies (BCPEI 7 and BCPEI 14), however, give rise to rare

Rare Nanodisc Shaped Planar Self-Assemblies

Figure 4. Schematic representation of the molecular arrangement in the nanodiscs (top image) and spherical vesicles (bottom image).

60 nm × 10 nm disc shaped morphologies (Figure 3c and d). As shown in Table 4 (Supporting Information Figure S4) and as has been found by others,24,25 branching reduces the radius of gyration of polymers in dilute solution. This reduced hydrophilic headgroup area of the branched amphiphile is responsible for the disc formation in BCPEI 7 and BCPEI 14 dispersions. It is important to note that the levels of cholesterol in the LCPEI and BCPEI dispersions were similar. Admittedly fewer disc shaped morphologies are contained in the BCPEI 7, when compared to the BCPEI 14 dispersion, and this is due to the fact that an increase in relative hydrocarbon volume, as is found when moving from BCPEI 7 to BCPEI 14, favors disc shaped morphologies. Decreasing the relative hydrocarbon volume or increasing the hydrophilic headgroup area by the incorporation of mPEG moieties in BCPEI 14 to give PCBPEI 1-2 (where the numerals 1 and 2 denote the number of mPEG and cetyl groups per molecule, respectively) gives rise to spherical vesicular type assemblies (Figure 3e). Also, while PpBPEI 1-2 gives predominantly spherical vesicular morphologies, some micellar morphologies are also evident (Figure 3f). Symmetrical branching, as is found in the polypropylenimine dendrimer amphiphiles, also results in the formation of 150 nm × 10 nm disc shaped aggregates (Figure 3g) in the presence of cholesterol, while in the absence of cholesterol spherical aggregates are seen in acetamide terminated palmitoyl poly(propylenimine), as is found with the BCPEIs (Supporting Information Figure S5). Since low levels of cetylation in the branched molecule (BCPEI) result in predominantly spherical assemblies in the presence of cholesterol (Figure 3c), the high curvature edges of the disc must consist of branched polymer chains with low levels of hydrophobic substitution and the planar portion of the disc must consist of the more highly cetylated polymer chains and cholesterol (Figure 4). A schematic representation of the influence of branching on self-assembly is given in Figure 4. Planar nanodiscs are rare self-assemblies:26,27 arising when cationic and anionic surfactants self-assemble in low conductivity (22) Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes; John Wiley and Sons: New York, 1980. (23) New, R. R. C. Liposomes: A Practical Approach; Oxford University Press: Oxford, 1990. (24) Versluis, C.; Hillegers, T. Macromol. Theory Simul. 2002, 11(6), 640– 648. (25) Kharchenko, S. B.; Kannan, R. M.; Cernohous, J. J.; Venkataramani, S. Macromolecules 2003, 36(2), 399–406.

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environments,26 anionic surfactants are partially neutralized by cationic water soluble polyelectrolytes28 and with certain perfluoroalkyl block copolymer amphiphiles,27 and we have previously reported some disc shaped morphologies appearing in the more hydrophobic linear PEI amphiphile dispersions, specifically aqueous dispersions of LCPEI 42 alone.8 However, this is the first time that direct microscopic evidence has shown that polymer branching is a route to nanodisc formation. The main reason that nanodiscs are rare is because of the energetic penalty associated with maintaining the edges of the disc, and hence, by definition, these systems can only stem from a heterogeneous mixture of amphiphiles, as a different population of amphiphiles is required for the edges when compared to the planar contours of the disc. Zemb and colleagues are almost alone in appreciating this subtlety and have opined that low molecular weight surfactants with fully ionized cationic headgroups form the edges of their discs and that ion paired cationic-anionic low molecular weight surfactants form the planar parts.26 What is clear from our data is that a relative reduction in the size of the hydrophilic headgroup is a prerequisite to disc formation; see data from a BCPEI 7/cholesterol dispersion (Figure 3b, some discs) and BCPEI 14/cholesterol dispersion (Figure 3c, numerous discs). Another element which must be present is the heterogeneity of the dispersion. Inevitably, the presence of a critical level of less substituted polymers and cholesterol in the BCPEI 14 (Figure 3d) sample favors disc formation as opposed to other assemblies such as solid nanoparticles, and the presence of cholesterol and some high and low substituted molecules (Supporting Information Figure S1d) in the cetyl poly(propylenimine) dispersions also provides the heterogeneity required for disc formation.

Conclusions The most significant findings of the current report are that a reduced hydrophilic headgroup area in amphiphiles leads to disc shaped self-assemblies and that this reduction in hydrophilic headgroup area may be accessed by employing branched polymers. It is conceivable that discs may also be prepared in nonaqueous solvents, if molecules of the correct architecture are employed. Furthermore, our knowledge of amphiphile selfassembly is enriched, as prior to our work it was widely accepted that as the relative hydrocarbon volume of amphiphilic molecules in a self-assembling system increases, or their relative hydrophilic headgroup area decreases, particles transform from spherical micelles to rods and then to vesicles.6 Our current and previous8 work shows that, as the collective hydrophobic volume in heterogeneous amphiphile dispersions increases, the aqueous dispersions transform from micelles to spherical vesicles to nanodiscs. An array of materials such as composites with superior properties could result from this ability to fabricate nanodiscs and nanospheres on demand. Not only may nanodiscs have specific properties that may be harnessed, for example, the ability to fit through small vascular fenestrae in ViVo and thus extravasate with an active drug payload to diseased cells or explore a larger hydrophilic volume per unit time due to their asymmetry, but also particle shape has a significant influence on the bulk properties of a particle dispersion, for example, polyhedral vesicles form highly viscous dispersions (26) Zemb, T.; Dubois, M.; Deme, B.; Gulik-Krzywicki, T. Science 1999, 283(5403), 816–819. (27) Lodge, T. P.; Bang, J. A.; Li, Z. B.; Hillmyer, M. A.; Talmon, Y. Faraday Discuss. 2005, 128, 1–12. (28) Goldraich, M.; Schwartz, J. R.; Burns, J. L.; Talmon, Y. Colloids Surf., A 1997, 125(2-3), 231–244.

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when compared to their spherical analogues,29 and hence, particle shape may thus be a route to manipulating the properties of a dispersion.

Resonance Laboratory are acknowledged for NMR experiments and the processing of spectra.

Acknowledgment. The Engineering and Physical Sciences Research Council is acknowledged for financial support. Mr. Craig Irving and Ms. Alaine Martin of the University of Strathclyde, Pure and Applied Chemistry Nuclear Magnetic

Supporting Information Available: 1H NMR spectra, electrospray ioniozation mass spectrum, table of prinicpal mass spectrometry peaks, MALDI-TOF spectrum, plot of λmax versus concentration, TEM micrographs, Zimm plots of linear and branched PEI, and table of exemplar elemental analysis data. This material is available free of charge via the Internet at http://pubs.acs.org.

(29) Florence, A. T.; Arunothayanun, P.; Kiri, S.; Bernard, M. S.; Uchegbu, I. F. J. Phys. Chem. B 1999, 103(11), 1995–2000.

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