Surface Molecular Composition and Electrical Property of Cationic

Article pubs.acs.org/JPCC

Surface Molecular Composition and Electrical Property of Cationic Solid Lipid Nanoparticles with Assembled Lipid Layer Mediated by Noncovalent Interactions Yung-Chih Kuo* and Cheng-Chin Wang Department of Chemical Engineering, National Chung Cheng University, Chia-Yi, Taiwan 62102, Republic of China ABSTRACT: Understanding of charged groups on nanostructured lipid particles is an important issue in nanomedicine development. This study presents the relation between noncovalent interactions and the composition of ionogenic groups in the assembled lipid layer of cationic solid lipid nanoparticles (CSLNs). Innovated CSLNs containing cacao butter, cholesterol, stearylamine, and esterquat 1 (EQ 1) were fabricated by modified solvent diffusion method. The results revealed that the average diameter of CSLNs decreased when the weight percentage of cholesterol and EQ 1 increased. X-ray photoelectron spectroscopy and Fourier-transform infrared spectroscopy evidenced strong interactions between cholesterol and EQ 1. These noncovalent interactions exhibited hydrophobic and cation−aromatic characteristics and mediated the distribution of lipids in the external layer of CSLNs. In addition, an increase in the weight percentage of cholesterol and EQ 1 enhanced the zeta potential, electrophoretic mobility, and fixed charge density on CSLNs. The noncovalent interactions in the assembled lipid layer affect the chemical composition on the lipid surface and yield intriguing electrokinetic behavior of CSLNs.

1. INTRODUCTION The advancement of nanotechnology attracts intensive investigations on lipid nanocarrier, which often exhibits a complex structure with unique surface characteristics.1 Cationic solid lipid nanoparticles (CSLNs) containing a hydrophobic core encompassed by a positively charged surface layer, for instance, are a newly developed carrier system for entrapping and delivering pharmaceuticals.2 This is mainly because the lipid core of CSLNs shows high affinity to hydrophobic drugs. In addition to lipid matrix, the organic cations condensed on the external surface of CSLNs can bind to cell membranes via electrostatic interaction and favor particle infusion across physiological barriers such as the blood−brain barrier.3 In fact, CSLNs can grasp antibodies and DNA for specific functionalization, rendering an extension to the blood circulation and an efficacious cell targeting effect after administration.4 The interior architecture and exterior charge are also crucial to the physicochemical property of CSLNs.5 During fabrication, the composition of lipids and surfactants may affect the particle size, zeta potential, charge distribution, and packing pattern of the assembled lipid layer on CSLNs.6,7 Surface charge of biomimetic nanoparticles is one of the fundamental traits for estimating the efficiency of tissue uptake and body distribution.8 The particulate charge is also influential to the nanoparticle−cell interaction and intracellular transport. For example, stem cells could internalize mesoporous silica nanoparticles with low surface charge via the normal pathway of clathrin- and actin-dependent mechanisms; however, above a charge threshold, a shift of the uptake process to a new chargedependent mechanism was observed.9 In a study on gene © 2012 American Chemical Society

transfection using cationic lipids, it has been concluded that positively charged vectors were more attractive to mammalian COS-1 cells than neutral vectors.10 Cholesterol is an indispensable component in mammalian cells and plays an important role in several membrane functions. In a study on molecular transport kinetics, it has been drawn that the highly hydrophobic and rigid sterol skeleton could enhance the mechanical stability of cholesterolincorporating lipid bilayers.11 In addition to mechanical property, cholesterol in natural and artificial lipids could induce a thermal effect and alter the vesicle permeability.12 However, the influence of cholesterol-mediated structure on the electrical property of CSLNs has not been examined. This study aims to unveil the distribution of cationic molecules in assembled lipid layer with cholesterol medication and the effect of surface molecular composition on the electrokinetic behavior of CSLNs. Because ionogenic groups are the key ingredients of surface charge on nanoparticles, a disclosure of the distribution of the organic cations on SCLNs is ineluctable. We examine the influence of the weight percentage of esterquat 1 (EQ 1) in cationic surfactant mixture and the weight percentage of cholesterol in internal lipid matrix on the formation of noncovalent interactions, particle size, zeta potential, and electrophoretic mobility of SCLNs. Finally, the fixed charge density on CSLNs was evaluated using Ohshima’s soft particle theory. Received: April 20, 2012 Revised: July 17, 2012 Published: July 25, 2012 16999

dx.doi.org/10.1021/jp303803m | J. Phys. Chem. C 2012, 116, 16999−17007

The Journal of Physical Chemistry C

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(Fluka) to desorb the bound fluorescein dye. After removal of CSLNs by centrifugation, the supernatant was mixed with 0.1 M aqueous phosphate buffer (Sigma) at pH 8.0 in a ratio of 9:1. The absorbance of the resultant solution at 501 nm was detected by an ELISA reader. The quantity of fluorescein bound to quaternary amine on CSLNs was calculated using a value of 77 mM−1 cm−1 as the extinction coefficient.16 The loading efficiency of EQ 1 containing quaternary amine on the surface of CSLNs, LEEQ 1, could be evaluated by LEEQ 1 (%) = [(weight of EQ 1 on the surface)/(total weight of EQ 1)] × 100%. Particle Size and Zeta Potential of CSLNs. The cumulant Z-average diameter (D) and zeta potential (ζ) of CSLNs were analyzed by a zetasizer 3000 HSA (Malvern, Worcestershire, U.K.) with a photon correlation spectroscope and a laser Doppler velocimeter at 25 °C. The concentration of CSLNs in 0.1 M tris-hydroxyl methyl-amino-methane buffer (Tris buffer; Riedel-de Haën) was 2 mg/mL for this analysis. Morphology of CSLNs. CSLNs (0.2% w/v) in Tris buffer were loaded on a carbon-coated 200-mesh copper grid for 2 min. The samples were pretreated with 2% (w/v) phosphortungstic acid (Sigma) to stain highly electron-transmissible atoms for 24 h. The images were obtained by a transmission electron microscope (TEM, JEM-1400, Jeol, Tokyo, Japan). Electrophoretic Mobility of CSLNs. Electrophoresis of CSLNs were analyzed by a high-performance capillary electrophoresis with Gold data acquisition software (P/ACE2100, Beckman Coulter, Palo Alto, CA), followed by a UV detector (Beckman Coulter) at 214 nm. Electrophoretic mobility, μ, of CSLNs was evaluated by μ = (l/t)/(V/L), where l, t, V, and L are the effective capillary length, the migration time, the applied electrical potential, and the total capillary length, respectively. In this study, l = 39.7 cm, L = 46.6 cm, and V = 10 kV. The inner and outer diameters of the capillary were 75 and 375 μm, respectively. Fixed Charge Density on CSLNs. On the basis of Ohshima’s soft particle theory, the electrophoretic mobility of CSLNs can be expressed by17

2. EXPERIMENTAL SECTION Preparation of CSLNs. CSLNs were fabricated by modified solvent diffusion method in an aqueous system.13 We dissolved 40 mg lipid matrix (w/w), including cacao butter (OCG Cacao, Whitinsville, MA) and cholesterol (Sigma, St. Louis, MO), and 10 mg (w/w) cationic surfactants, including stearylamine (SA; Fluka, Buchs, Switzerland) and EQ 1 (Gerbu Biotechnik, Gaiberg, Germany), in organic medium, including 3 mL of acetone (Mallinckrodt Baker, Hazelwood, MO) and 2 mL of ethanol (Tedia, Fairfield, OH), over a water bath at 70 °C. The weight percentage of cholesterol in lipids was at 0, 25, or 75%. The weight percentage of EQ 1 in cationic surfactants was at 0, 33, 67, or 100%. For preparing SLNs, cationic surfactants were not used. The resultant organic solution was mixed in 50 mL of ultrapure water (Barnstead, Dubuque, IA) containing 0.02% (w/v) Tween 80 (FisherScientific, Fair Lawn, NJ) at 400 rpm and 75 °C for 20 min. CSLNs were obtained after the emulsified fluid was cooled to room temperature. The suspension containing CSLNs was centrifuged at 7500g for 20 min using a 100 kDa Amicon Ultra centrifugal filter (Millipore, Madrid, Spain). After removal of supernatant, the bottom pellet was washed three times with ultrapure water. Resuspended CSLNs in ultrapure water were mixed with 2% (w/v) D-mannitol (Sigma), frozen at 4 °C for 30 min, at −20 °C for 30 min, and at −80 °C for 1 h, and lyophilized by a freeze-dryer (Eyela, Tokyo, Japan) at 2−4 Torr and −80 °C over 24 h to obtain powder products. Surface Elements on CSLNs. An X-ray photoelectron spectroscope (XPS; Kratos, Kanagawa, Japan) with a vacuum grade of 2 × 10−7 Pa and 300 W was used to resolve the atoms on CSLNs. The sample on a cover slide of 5 × 5 mm was vacuum-dried for 15 min before test. Functional Groups of CSLNs. The Fourier-transform infrared (FTIR) spectra of CSLNs were obtained by an IRabsorption spectrophotometer (Shimadzu, Columbia, MD). CSLNs were compressed with KBr powders in a ratio of 1:5. Quantification of Primary and Quaternary Amines on CSLNs. The quantity of primary amine on the surface of CSLNs was determined by the chemical assay of 2,4,6trinitrobenzene sulfonic acid (TNBS).14 In brief, 100 μL of the suspension containing 0.5% (w/v) CSLNs was mixed with 200 μL of 0.8 M sodium hydrogen carbonate solution (pH 8.5; Riedel-de Haën, Seelze, Germany) and 20 μL of aqueous solution containing 1% (w/v) TNBS (Sigma) and incubated at 37 °C for 2 h. After reaction, 100 μL of the mixture was analyzed by an ultraviolet−visible detector (Biotek, Winooski, VT) connected to an enzyme-linked immunosorbent assay (ELISA, Biotek) reader at 420 nm and calibrated with glycine solution (J. T. Baker, Phillipsburg, NJ). The loading efficiency of SA containing primary amine on the surface of CSLNs, LESA, could be evaluated by LESA (%) = [(weight of SA on the surface)/(total weight of SA)] × 100%. The quantity of quaternary amine on the surface of CSLNs was evaluated by the fluorescein-binding method.15 We mixed 100 μL of the suspension containing 0.5% (w/v) CSLNs with 5 mL of ultrapure water containing 1% (w/v) fluorescein (sodium salt, Sigma) for 10 min and incubated at 37 °C for 2 h. Fluorescein-binding CSLNs were recovered by centrifugation at 7500g for 20 min using a 100 kDa Amicon Ultra centrifugal filter, washed three times with ultrapure water, and ultrasonically vibrated for 15 min in 3 mL of aqueous solution containing 0.25% (w/v) cetyltrimethylammonium chloride

μ=

⎤ ρ 2εoεr φo /κ m + φDON /λ ⎡ 1 + fix2 ⎢1 + 3⎥ 3η 1/κ m + 1/λ ⎣ 2(1 + d /a) ⎦ ηλ (1)

where ε0, εr, η, φ0, φDON, κm, ρfix, λ, d, and a are, respectively, the permittivity of a vacuum, the relative permittivity of the suspension of CSLNs, the fluid viscosity, the surface potential on CSLNs, the Donnan potential, the Debye screening length for the surface layer, the fixed charge density on CSLNs, the surface softness, the thickness of the surface layer, and the radius of the lipid core. The surface potential, Donna potential, the Debye screening length for the surface layer, and the surface softness in eq 1 are defined as φo =

17000

⎛ ⎡ kT ⎜ ⎢ ρfix ln z e ⎜ ⎢⎣ 2z en ⎝ ⎡ 2z en ⎢ + 1− ρfix ⎢⎣

⎫1/2 ⎤⎞ ⎧⎛ ρ ⎞2 + ⎨⎜ fix ⎟ + 1⎬ ⎥⎟ ⎭ ⎥⎦⎟⎠ ⎩⎝ 2z en ⎠ ⎫1/2 ⎤ ⎧⎛ ρ ⎞2 fix ⎟ ⎜ ⎨ + 1⎬ ⎥ ⎭ ⎥⎦ ⎩⎝ 2z en ⎠

(2)


The Journal of Physical Chemistry C φDON

⎛ ⎡ ⎫1/2 ⎤⎞ ⎧⎛ ρ ⎞2 kT ⎜ ⎢ ρfix fix ⎥⎟ ⎟ + 1⎬ ln = + ⎨⎜ ⎥⎟ ⎝ 2z en ⎠ z e ⎜ ⎢⎣ 2z en ⎭ ⎩ ⎦⎠ ⎝

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mobility, surface softness, and viscosity into eq 7, the fixed charge density on CSLNs can be evaluated. (3)

1/4 ⎡ ⎛ ρ ⎞2 ⎤ κ m = κ ⎢1 + ⎜ fix ⎟ ⎥ ⎝ 2z en ⎠ ⎦ ⎣

(4)

⎛ γ ⎞1/2 λ=⎜ ⎟ ⎝η⎠

(5)

⎛ 2I e 2ν 2N ⎞0.5 A ⎟ κ=⎜ kT ε ε ⎠ ⎝ r O

(6)

3. RESULTS AND DISCUSSION Distribution of Atoms on the Surface of CSLNs. Figure 1 shows the influence of cholesterol or EQ 1 on the XPS

where k, T, z, e, n, κ, γ, I, v, and NA are, respectively, the Boltzmann constant, the absolute temperature, the valence of the cationic surface groups, the elementary charge, the number concentration of ions in the bulk, the reciprocal of Debye screening length (double-layer thickness), the friction coefficient of the surface layer, the ionic strength of the electrolyte solution, the valence of symmetric electrolyte ions, and the Avogadro number, respectively.18 In this study, four ionic species, including Tris+, Cl−, H+, and OH−, contributed to κ in Tris buffer and κ = 6.86 × 108 m−1.19 The surface layer thickness of CSLNs was ∼4 nm.20 The core radii of CSLNs ranged from 83.5 to 271 nm, which were obtained by subtracting 4 nm from the D value (data shown in Figure 4). Hence, the cationic surface on CSLNs satisfied the assumption of a thin polyelectrolyte layer, that is, a ≫ d. Equation 1 reduces to21 μ=

ρfix ⎡ ⎛ λ ⎞⎛ 1 + λ /2κ ⎞⎤ ⎟⎥ 1 + ⎜ ⎟⎜ 2⎢ ⎝ κ ⎠⎝ 1 + λ / κ ⎠⎦ ηλ ⎣

(7)

In addition, the charged segments on the surface of CSLNs can assume as spheres with radius r and distribute in the external layer with a volume concentration n.22 Because each segment generates the Stokes resistance, that is, γ = 6πηrm, eq 5 can be expressed by

λ = (6πrm)1/2

(8)

Moreover, r = (3M/4πρsNA) and m = ϕρsNA/M, where M, ρs, NA, and ϕ are the average molecular weight of the segments, the density of the segments, the Avogadro number, and the volume fraction of the segments in the surface layer.23 Thus, eq 8 can be recast as 1/3

⎛ ρp NA ⎞1/3 ⎟ ϕ1/2 λ ≈ 3.4⎜ ⎝ M ⎠

Figure 1. XPS spectra (in C 1s) of SLNs and CSLNs. (a) SLNs with PCH/(CB+CH) = 0%; (b) CSLNs with PCH/(CB+CH) = 0%, PEQ 1/(SA+EQ 1) = 100%; (c) SLNs with PCH/(CB+CH) = 25%; and (d) CSLNs with PCH/(CB+CH) = 25%, PEQ 1/(SA+EQ 1) = 100%.

(9)

The polyelectrolyte layer is composed of SA and EQ 1, where the quantity of primary and quaternary amines on the surface can determine the average molecular weight of the positively charged segments. Therefore, the average molecular weights of the segments for PEQ 1/(SA+EQ 1) = 0, 33, 67, and 100% are 269, 368, 535, and 634, respectively. In addition, ϕ can assume 0.1.23 Adopting ρp = 1 g/cm3, λ can be calculated by eq 9. This leads to λ = (1.40, 1.27, 1.12, and 1.05) × 107 cm−1 for PEQ 1/(SA+EQ 1) = 0, 33, 67, and 100%, respectively. Furthermore, an MCR 500 rheometer (Anton 168 Paar, Graz, Austria) with US 200 software at a shear rate of 500 s−1 resolved the fluid viscosity. Substituting the electrophoretic

spectra (in C 1s) of SLNs and CSLNs. As revealed in Figure 1a, when cholesterol was not incorporated in the lipid matrix, the carbon signals of SLNs could be decomposed into four peaks at 282.60, 283.29, 284.31, and 286.81 eV, which were attributed to C−C, C−O−C, C−O−CO, and O−CO, respectively.24 When Figure 1a was compared with Figure 1b, without the interference from cholesterol, the presence of EQ 1 would not appreciably alter the binding energy of the four decomposed peaks. As indicated in Figure 1b, the peak area at 284.31 eV 17001



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resulting from C−O−CO of EQ 1 increased and the peak area at 286.81 eV resulting from O−CO (carboxyl group) of cacao butter reduced when the lipids interacted with EQ 1. This demonstrated that EQ 1 was incorporated in the surface layer of CSLNs during the particulate preparation. However, the quantity of surface nitrogen was relatively low (data not shown) when compared with surface carbon. Therefore, the nitrogen peak in these spectra could not split analytically. In fact, the peak at 283.31 eV included the contributions from C− O−C and C−N.25 In the cases of PCH/(CB+CH) = 25%, the carbon signals can be also decomposed into the four peaks, as displayed in Figure 1c,d. The peak at 283.31 eV (C−O−C) included the contribution from C−OH of cholesterol; that is, the difference in the binding energy between the two functional groups is indistinguishable.25 Moreover, as compared with Figure 1a, the binding energy of C−O−C/C−OH in Figure 1c slightly shifted from 283.29 to 283.31 eV, attributed to the interaction between OH of cholesterol and COOH of cacao butter. In the presence of 25% (w/w) cholesterol, the peak position of O−CO in Figure 1c also shifted to a higher level from 286.81 to 287.02 eV. However, no difference in the peak position of O−CO (at 286.81 eV) was observed in Figure 1a,b. Moreover, when cholesterol was incorporated, the area of the peak at 283.31 eV (C−O−C/C−OH) increased and the area of the peak at 287.02 eV (O−CO) reduced, as exhibited in Figure 1a,c. This evidenced that cholesterol could interact with cacao butter and affect the internal structure of lipid core. When Figure 1c was compared with Figure 1d, the presence of EQ 1 shifted the binding energy of C−O−CO from 284.31 to 283.95 eV, C−O−C/C−OH slightly from 283.31 to 283.34 eV, and O−CO from 287.02 to 286.9 eV Figure 2 shows the XPS spectra (in C 1s) of SLNs and CSLNs at PCH/(CB+CH) = 25% when the weight percentage of EQ 1 varies. For a clear presentation, the structure of particle surface with relevant functional groups is displayed. When SLNs (without cationic surfactants) were compared with CSLNs at PEQ 1/(SA+EQ 1) = 0, the presence of SA would not appreciably alter the binding energies of C−C, C−O−C, C− O−CO, and O−CO on the lipid nanoparticles, as revealed in Figure 2a,b. However, the peak area of O−CO (peak 4) reduced when SA was incorporated. In a study on the structure of mixed surfactants including double-tailed didodecyldimethylammonium bromide (DDAB) and single-tailed dodecyltrimethylammonium bromide (DTAB) in aqueous solution, surfactant aggregates contained more DDAB than DTAB; that is, DTAB tended to disperse in the bulk liquid phase.26 In addition to free surfactants, DDAB was localized in the inner membrane of lipid bilayer with the exclusion of DTAB, and the outer membrane comprised the mixture of DDAB and DTAB. Hence, single-tailed SA was more likely to accumulate on the surface of CSLNs and expelled cacao butter from the surface. Therefore, the content of carboxyl groups (from cacao butter) on the surface of CSLNs reduced, rendering a weak signal of O−CO in Figure 2b. This could also yield a thick and irregular surfactant layer on CSLNs (image shown in Figure 5b). As exhibited in Figure 2b−d, the binding energy of C−O− CO (peak 3) and C−O−C/C−OH (peak 2) shifted, respectively, to a lower and higher level, when the weight percentage of EQ 1 increased. This suggested that the hydrophobic interaction could be crucial in the surface layer of CSLNs. In fact, the hydrophobic trait of cholesterol is stronger than dodecyl, cyclododecyl, 1-adamantyl, and 1naphthyl.27 Thus, cholesterol could play an important role in

Figure 2. Effect of the weight percentage of EQ 1 on the variation in XPS spectra (in C 1s) of SLNs and CSLNs. PCH/(CB+CH) = 25%. 1, 2, 3, and 4 denote C−C, C−O−C, C−O−CO, and O−CO, respectively. Schematic representation of functional groups on the surface of CSLNs is illustrated beside the corresponding spectrum. (a) no SA and EQ 1; (b) PEQ 1/(SA+EQ 1) = 0%; (c) PEQ 1/(SA+EQ 1) = 33%; (d) PEQ 1/(SA+EQ 1) = 67%; and (e) PEQ 1/(SA+EQ 1) = 100%.

the hydrophobic interaction with EQ 1 in CSLNs. Moreover, it has been found that the cation−aromatic interaction between aromatic rings and positively charged groups was probable to impel a specific arrangement and improve the stability of the lipid bilayer.28 In addition, it was noteworthy that the van der Waals attraction and hydrophobic force were regarded as the key contributions to the interactions between cholesterol and lipids.29,30 As a result, the main noncovalent interactions between EQ 1 and cholesterol included the van der Waals attraction, hydrophobic interaction, and cation−aromatic interaction. Hence, an increase in the weight percentage of EQ 1 could enhance commensurately the quantity of EQ 1 on the surface. In a study on the adsorption of lipids carrying ester group, it has been concluded that carboxyl-containing surface could significantly reduce the adsorbed quantity of dodecyl esterquat.31 In addition to the decrease in carboxyl attraction, ester group enhanced hydroxyl affinity.31 These literatures explained that EQ 1 could be more attractive to cholesterol than cacao butter. Furthermore, the binding energy of O−C O (peak 4) shifted to a lower level with an increasing weight percentage of EQ 1. This could be attributed to the fact that the mole percentage of cacao butter abundant with carboxyl groups was high in CSLNs. Therefore, a certain degree of interaction between cacao butter and EQ 1 could be possible. Distribution of Cationic Surfactants on CSLNs. Table 1 lists the content of primary and quaternary amines on the surface of CSLNs. As shown in this Table, the quantity of quaternary amine increased with an increase in the weight percentage of EQ 1. This is because EQ 1 is the only species possessing quaternary amine in these CSLNs. In addition, an 17002



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Table 1. Distribution of Primary and Quaternary Amines on the Surface of CSLNs CPA (mmol/mL)a

PCH/(CB+CH) (%) PEQ 1/(SA+EQ 1) (%) 0 25 75

CQA (mmol/mL)b

100

67

33

0

100

67

33

0

0 0 0

0.49 ± 0.11 0.36 ± 0.09 0.59 ± 0.13

1.14 ± 0.17 0.93 ± 0.11 1.46 ± 0.18

2.57 ± 0.15 2.69 ± 0.11 2.61 ± 0.14

1.02 ± 0.04 1.32 ± 0.03 1.16 ± 0.06

0.59 ± 0.05 0.82 ± 0.04 0.73 ± 0.03

0.24 ± 0.07 0.33 ± 0.05 0.28 ± 0.03

0 0 0

a

CPA is the concentration of primary amine on the surface of CSLN and was determined by the TNBS assay. bCQA is the concentration of quaternary amine on the surface of CSLNs and was determined by the fluorescein assay.

Table 2. Percentage of Loaded SA and EQ 1 on the Surface of CSLNs LESA (%)a

PCH/(CB+CH) (%) PEQ 1/(SA+EQ 1) (%) 0 25 75

LEEQ 1 (%)b

100

67

33

0

100

67

33

0

0 0 0

41 ± 8 27 ± 7 48 ± 11

46 ± 6 38 ± 4 59 ± 7

70 ± 3 73 ± 3 70 ± 4

65 ± 3 84 ± 2 74 ± 3

57 ± 5 77 ± 4 70 ± 7

47 ± 9 62 ± 8 55 ± 6

0 0 0

a LESA is the percentage of loaded SA on the surface of CSLNs. LESA (%) = [(weight of SA on the surface)/(total weight of SA)] × 100%. bLEEQ 1 is the percentage of loaded EQ 1 on the surface of CSLNs. LEEQ 1 (%) = [(weight of EQ 1 on the surface)/(total weight of EQ 1)] × 100%.

Figure 3. FTIR spectra of SLNs and CSLNs. (a) SLNs with PCH/(CB+CH) = 0%; (b) CSLNs with PCH/(CB+CH) = 0%, PEQ 1/(SA+EQ 1) = 100%; (c) SLNs with PCH/(CB+CH) = 25%; (d) and CSLNs with PCH/(CB+CH) = 25%, PEQ 1/(SA+EQ 1) = 100%.

addition of 25% (w/w) cholesterol into lipid emulsion yielded an increase in the quantity of quaternary amine on the surface of CSLNs when compared with the case without cholesterol. The rationale behind this behavior was the noncovalent interactions between EQ 1 and cholesterol. In fact, the emergence of strong hydrophobic and cation−aromatic interactions in lipid droplet improved the lipid−surfactant association and enhanced the affinity of cationic EQ 1 to CSLNs. As a result, EQ 1 could be preferentially adsorbed and locate at the medium−CSLN interface. However, the quantity of quaternary amine on CSLNs decreased when the weight percentage of cholesterol increased from 25% to 75% (w/w). Three probable explanations of this result were described as follows. First, when cholesterol was not included in the formulation, the inner lipid core of CSLNs was occupied mostly by double-tailed EQ 1 and cacao butter (fatty acid), which has

the smallest surface/oil value. When the cholesterol content increased to 25% (w/w), the EQ 1-cholesterol associates, which have higher surface/oil values, appeared in the outer layer and the percentage of SA−fatty acid pair increased in the inner layer. As the cholesterol content further increased, the fatty acid content was not enough to grasp SA in the inner layer. Therefore, the percentage of SA in the outer layer increased. Second, the aggregation rate of EQ 1 was faster than that of SA.32 From this point of view, EQ 1 could rapidly shape the contour of CSLNs, in general. In fact, the self-assembly capability of cholesterol moieties is very strong.33 Therefore, cholesterol was liable to accumulate and was difficult to form strong interactions to grasp EQ 1 at a high level of cholesterol. However, a complete expulsion of EQ 1 by SA in the external layer could be implausible in practice.34 Third, the electrical repulsion between two adjacent EQ 1 head groups could repel 17003



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EQ 1 from the surface layer and reduce the quantity of quaternary amine on CSLNs when cholesterol was 75% (w/w). In addition to hydrophobic force, assembly capability, and charge interaction, cholesterol could also affect the competition between SA and EQ 1 for the attachment sites on CSLNs when the quantity of quaternary amine was compared with that of primary amine on the particle surface. As indicated in Table 1, the quantity of primary amine decreased when 25% (w/w) cholesterol was used. This was because 25% (w/w) cholesterol could prevent SA from incorporation into external lipid layer. Table 2 lists the loading efficiency of SA and EQ 1 on CSLNs. As shown in this Table, an increase in the weight percentage of EQ 1 reduced the loading efficiency of SA and enhanced the loading efficiency of EQ 1. An addition of 25% (w/w) cholesterol yielded the highest loading efficiency of EQ 1 when the concentration of cholesterol varied, that is, a further increase in cholesterol content to 75% (w/w) reduced the loading efficiency of EQ 1. The reverse behavior was true for the loading efficiency of SA, in general. Therefore, it can be drawn that the hydrophobic and cation−aromatic interactions regulated the distribution of cationic surfactants on the surface of CSLNs. Functional Groups of CSLNs. Figure 3 shows the FTIR spectra of SLNs and CSLNs, where the samples are the same as those used in Figure 1. As revealed in Figure 3b,d, a weak band at 1488 cm−1 was the typical absorption of EQ 1, representing the characteristics of antisymmetric bend of trimethyl ammonium compounds.35 As revealed in the magnified graph on the right-hand side of the main spectra, the absorption at 1743 cm−1 indicated the vibration of cacao butter CO. In addition, a new CO vibration peak at 1734 cm−1 appeared. This resulted from the and formation of intermolecular hydrogen-bonding functionalities between hydroxyl of cholesterol and ester group of EQ 1.36,37 Hydrogen bonds between hydroxyl of cholesterol and headgroup or functional chainlinkage group of lipids were found to be substantially important.38 In a study on the interaction between cholesterol and lipids, the formation of hydrogen bonds was observed between hydroxyl of cholesterol and ester carbonyl group of diacyl phosphatidylcholine.39 However, cholesterol could not form ample hydrogen bonds with lipids without ester group in their chain linkage such as dimethyldioctadecylammonium bromide.39 An association of EQ 1, a kind of cationic diacyl lipids, could also induce a competition between cholesterol and cacao butter for grasping EQ 1 because the three ingredients were melted and mixed simultaneously during the preparation. However, cholesterol could preferentially interact with EQ 1. It has been concluded that the interaction between cholesterol and N-(1-(2,3-dioleoyloxy) propyl)-N,N,N-trimethylammonium chloride (a kind of diacyl lipids) was strong.36 Particle Size of CSLNs. Figure 4 shows the Z-average of the diameter of CSLNs. As exhibited in this Figure, the average diameter decreased with an increasing weight percentage of EQ 1. Two reasons could be attributed to this behavior. First, the hydrophobic effect of double-tailed EQ 1 was stronger than that of single-tailed SA.40 Thus, the former yielded a lower surface tension and a better fluid flow mobility to produce small particles. Second, noncovalent interactions between cholesterol and EQ 1 induced stabilization in cationic surface layer, reduced the van der Waal attraction of lipids/surfactants between different CSLNs, increased the net particulate repulsion force, and reduced colloidal agglomeration. A similar variation in the particle size was also concluded in a liposome

Figure 4. Average diameter of CSLNs. ○: PCH/(CB+CH) = 0%, PCH/(CB+CH) = 25%, □: PCH/(CB+CH) = 75%.

△:

system.41 However, the average diameter could be larger than 200 nm when the weight percentage of SA in CSLNs was high. This was a drawback for delivering drug because a large particle with diameter larger than 200 nm would provoke a body defense mechanism.42,43 In addition, an increase in the weight percentage of cholesterol decreased the average diameter of CSLNs. This was mainly because the hydrophobic interaction between cacao butter and cholesterol was high, yielding dense hydrophobic lipid cores and small particles.44 Moreover, the interaction between cholesterol and SA/EQ 1 could increase the surface curvature of lipid particles and reduce the diameter of CSLNs.45 This cholesterol effect on particle size was consistent with the literature results.46 Morphology of CSLNs. Figure 5 shows the structure of CSLNs composed of various ingredients. As revealed in Figure 5a, SLNs without cationic surfactants exhibited a spherical geometry with uniform size. CSLNs contained dark solid cores covered with gray exterior layers comprising cationic surfactants, as revealed in Figure 5b−d. As compared with Figure 5a, CSLNs in Figure 5b−d demonstrated slightly rugged external layers. In addition, CSLNs without EQ 1 in Figure 5b displayed a roughest surface layer among other CSLNs. As displayed in Figure 5b−d, a higher weight percentage of EQ 1 yielded a thinner and smoother exterior on CSLNs by more ordered packing of cationic surfactants in the surface layer. This was due to high affinity of EQ 1 to cholesterol and hydrophobic interactions between EQ 1 and cacao butter. These images were consistent with the XPS analysis exhibited in Figure 2. Moreover, the particle diameters in Figure 5b−d were slightly smaller than the data shown in Figure 4. This was because the pretreatment steps for TEM visualization eliminated moisture adsorbed on the surface of CSLNs, whereas zetasizer 3000 HSA detected the diameter of CSLNs in hydrated state.47 Electrical Properties of CSLNs. Figure 6 shows the variation in zeta potential of CSLNs as a function of the weight percentage of EQ 1. As indicated in this Figure, CSLNs exhibited positive zeta potentials, demonstrating that charged 17004



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Figure 5. TEM images of SLNs and CSLNs. PCH/(CB+CH) = 25%. (a) SLNs; (b) CSLNs with PEQ 1/(SA+EQ 1) = 0%; (c) CSLNs with PEQ 1/(SA+EQ 1) = 33%; (d) CSLNs with PEQ 1/(SA+EQ 1) = 67%; and (e) CSLNs with PEQ 1/(SA+EQ 1) = 100%.

of quaternary amine of EQ 1 is greater than 12, which is larger than 10.6, the pKa value of primary amine of SA. Second, a higher weight percentage of SA in CSLNs produced a thicker and more irregular surfactant layer (images shown in Figure 5b−e). A thick adsorption layer on particle surface could shift the slip plane toward the bulk phase and decrease the zeta potential.49 Third, noncovalent interactions between cholesterol and EQ 1 promoted the content of EQ 1 on CSLNs (data shown in Tables 1 and 2) and enhanced the electrostatic characteristics. Fourth, from the point view of particle size, a higher PEQ 1/(SA+EQ 1) produced smaller CSLNs (data shown in Figure 4). Therefore, protonated amines were concentrated on a small area, raising the surface charge density. As indicated in Figure 6, an increase in the weight percentage of cholesterol in CSLNs enhanced the zeta potential. Two reasons could be attributed to this behavior. First, hydroxyl of cholesterol was more efficient in clutching cationic surfactants on CSLNs than carboxyl groups of cacao butter. This fixation of amines on the surface could refer to the evidence of hydrophobic and cation− aromatic interactions shown in Figures 1 and 2. Second, a higher weight percentage of cholesterol in CSLNs yielded a smaller particle size (data shown in Figure 4) and concentrated surface charge. Figure 7 shows the electrophoretic mobility of CSLNs. The fixed charge density evaluated from the corresponding electrophoretic mobility and eq 6 is shown in Figure 8. As revealed in Figures 7 and 8, a higher content of cholesterol and EQ 1 yielded larger electrophoretic mobility of CSLNs and a higher fixed charge density in the surface layer. This behavior was similar to that of zeta potential shown in Figure 6. It was worth noting that COOH of cacao butter, composed of linolenic acid, oleic acid, palmitic acid, and stearic acid, could be partially deprotonated at pH 7.4, and the resultant carboxylate groups were liable to attract cationic surfactants.50 However, these minor carboxylate groups produced from cacao butter were not the dominant factor for grasping SA and EQ 1 on CSLNs. The detailed discussion of the interaction between

Figure 6. Zeta potential of CSLNs as a function of the weight percentage of EQ 1. ○: PCH/(CB+CH) = 0%, △: PCH/(CB+CH) = 25%, and □: PCH/(CB+CH) = 75%.

amines of SA and EQ 1 were incorporated in the external zone of CSLNs. This positively charged surface layer encompassing lipid core improved the stability of CSLNs in aqueous medium via electrostatic repulsion. In addition, an increase in the weight percentage of EQ 1 raised the zeta potential of CSLNs. The rationale behind this tendency was discussed as follows. First, the ionization degree of EQ 1 was higher than that of SA. This was consistent with the literature, where the ionization degree of amines was in the order of primary amine < secondary amine ≪ tertiary amine ≈ quaternary amine.48 In fact, the pKa value 17005



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The hydrophobic affinity and cation−aromatic interaction strongly influenced the distribution of cationic surfactants in the surface layer. At 25% (w/w) cholesterol, the noncovalent interactions yielded the highest quantity of doubled-tailed EQ 1 on the surface of CSLNs. In addition, a higher weight percentage of cholesterol and EQ 1 produced smaller CSLNs. An increase in the weight percentage of cholesterol and EQ 1 in CSLNs enhanced zeta potential, electrophoretic mobility, and fixed charge density. These cholesterol-mediated CSLNs demonstrate controllable charged behavior and can be a promising nonviral drug delivery system in future nanomedicine.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 886-5-272-0411, ext. 33459. Fax: 886-5-272-1206. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science Council of the Republic of China.

Figure 7. Mobility of CSLNs as a function of the weight percentage of EQ 1. ○: PCH/(CB+CH) = 0%, △: PCH/(CB+CH) = 25%, and □: PCH/(CB+CH) = 75%.

Nomenclature

PCH/(CB+CH) weight percentage of cholesterol in lipids (%) PEQ 1/(SA+EQ 1) weight percentage of EQ 1 in cationic surfactants (%) Abbreviation

CSLN EQ 1 SA SLN

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cationic solid lipid nanoparticle esterquat 1 stearylamine solid lipid nanoparticle

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Figure 8. Fixed charge density in the assembled lipid layer as a function of the weight percentage of EQ 1. ○: PCH/(CB+CH) = 0%, △: PCH/(CB+CH) = 25%, and ○: PCH/(CB+CH) = 75%.

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4. CONCLUSIONS The composition of cationic surfactants in the external shell of CSLNs was unveiled by considering the cholesterol-mediated noncovalent interactions. Effects of the content of cationic surfactants and internal lipids on the electrical properties of CSLNs were also investigated. XPS and FTIR spectra revealed that strong interactions existed between cholesterol and EQ 1. 17006



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Surface Molecular Composition and Electrical Property of Cationic

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