pH-Responsive Micelles Based on Caprylic Acid - American Chemical

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pH-Responsive Micelles Based on Caprylic Acid Stefan Salentinig,*,† Stephanie Phan,† Tamim A. Darwish,‡,§ Nigel Kirby,∥ Ben J. Boyd,*,† and Elliot P. Gilbert*,‡

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†

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, VIC 3052, Australia ‡ Bragg Institute, Australian Nuclear Science and Technology Organisation, Locked Bag 2001, Kirrawee DC, NSW 2232, Australia § National Deuteration Facility, Australian Nuclear Science and Technology Organisation, Locked Bag 2001, Kirrawee DC, NSW 2232, Australia ∥ SAXS/WAXS Beamline, Australian Synchrotron, 800 Blackburn Rd, Clayton, VIC 3168, Australia S Supporting Information *

ABSTRACT: Free fatty acids play a vital role as fuel for cells and in lipid metabolism. During lipid digestion in the gastrointestinal tract, triglycerides are hydrolyzed, resulting in free fatty acid and monoglyceride amphiphilic products. These components, together with bile salts, are responsible for the transport of lipids and poorly water-soluble nutrients and xenobiotics from the intestine into the circulatory system of the body. In this study, we show that the self-assembly of digestion products from medium-chain triglycerides (tricaprylin) in combination with bile salt and phospholipid is highly pH-responsive. Individual building blocks of caprylic acid within the mixed colloidal structures are mapped using a combination of small-angle X-ray and neutron scattering combined with both solvent contrast variation and selective deuteration. Modeling of the scattering data shows transitions in the size and shape of the micelles in combination with a transfer of the caprylic acid from the core of the micelles to the shell or into the bulk water upon increasing pH. The results help to understand the process of lipid digestion with a focus on colloidal structure formation and transformation for the delivery of triglyceride lipids and other hydrophobic functional molecules.

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INTRODUCTION

When food arrives in the small intestine, the body secretes bile including bile salts and phospholipids. These components can form mixed micelles or vesicles depending on their ratio and dilution. Bile salt-phospholipid micelles have been reported to form globular or wormlike structures, with the phospholipid molecules oriented radially along the axis surrounded by the bile salt at the aggregate−water interface.6 Elongation of these mixed micelles and a transition to vesicles were found to occur upon dilution or addition of phospholipids.6−12 At the end of the lipid digestion process in the small intestine, these mixed micelles are solubilizing fatty acids and monoglycerides.13−16 These assemblies then diffuse through the unstirred water layer, which separates the brush-border membrane of the enterocytes (the absorptive cells in the small intestine) from the bulk fluid phase of the intestinal lumen.17,18 The unstirred water layer is proposed to have a reduced pH environment compared to the bulk lumen; thus a pH gradient exists, across which the degree of ionization of fatty acids may vary. This has been proposed as a mechanism for driving lipid absorption through increases in thermodynamic activity.19,20

Fatty acids are a major end product of triglyceride digestion. They are composed of a hydrophilic carboxylic acid group and an aliphatic hydrocarbon chain with carbon numbers between 4 and 28.1 Most of the naturally occurring fatty acids have an even number of carbon atoms due to their biosynthesis with coenzyme acetyl-CoA. In the case of unsaturated fatty acids, the dominant configuration in naturally occurring species is cis; the trans configuration is mostly the result of industrial hydrogenation processes or bacterial metabolism.2 It has been reported that diets rich in saturated fatty acids or trans-fatty acids raise cholesterol levels more than the polyunsaturated cisfatty acids.3 Free fatty acids have important biological properties: Part of the energy for the heart muscle is fueled by free fatty acids delivered via the plasma compartment from the adipocytes in distal sites. High concentrations have been shown to induce diabetes through the lipotoxic effect of free fatty acids on the islets of Langerhans.4 A knowledge and understanding of how major food components are treated, transported, and utilized during the digestion process opens opportunities for functional food products that help to avoid, or even cure, such health issues.5 © 2014 American Chemical Society

Received: March 4, 2014 Revised: May 27, 2014 Published: June 6, 2014 7296

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Columbia, MD, USA) was used for benchtop column chromatography. Methods. Deuteration of Caprylic Acid. A mixture of caprylic acid (13 g, 90.5 mmol), Pt/activated carbon (0.48 g, 10 wt % of the substrate), and 40% w/w NaOD (9.5 g, 92.6 mmol) in D2O (120 mL) was loaded into a Parr pressure reactor (600 mL size). The contents of the reactor were degassed by purging with N2 gas and then sealed and heated to 220 °C (23 bar), with constant stirring for 3 days. The reactor was cooled to room temperature, and the contents were filtered through a short plug of Celite to remove the catalyst, which was further washed with H2O (100 mL). The aqueous filtrate was acidified to pH 2 using 1 M HCl. The aqueous phase was extracted with diethyl ether (100 mL × 3), and the combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo to give an oily substance of caprylic acid (12 g, 92% D by MS). The aforementioned quantity of acid (12 g) was reloaded into the reactor, and the above method was repeated using fresh reagents to give 10.5 g of caprylic acid (73% yield on the basis of the original amount of the protonated caprylic acid, 98% D). Thin-layer chromatography was used (referenced with the protonated compound) to estimate the purity and to develop separation protocols. 1H NMR (400 MHz) and 2 H NMR (61.4 MHz) spectra were recorded on a Bruker 400 MHz spectrometer at 298 K. More details on the preparation and characterization are presented in the Supporting Information. The 1 H NMR and 2H NMR spectra are shown in Figures SI-1 and SI-2, respectively. Sample Preparation. Bile salt micelles were prepared using bile salt (BS, sodium taurodeoxycholate) and phospholipid (DOPC) concentrations in a ratio of 20 mM/5 mM in digestion buffer (50 mM Tris maleate, 150 mM NaCl). Caprylic acid (20 mM) and monocaprylin (10 mM) were added to the solution. Sonication with a tip sonicator for 30 s at 100 W was used to solubilize the components. The pH of the micellar systems was adjusted by the addition of 1 M HCl or NaOH. pH values in this study are the direct pH meter readings in H2O and D2O using a glass electrode.26 Samples were equilibrated for a minimum of 24 h before the experiments. Small-Angle Neutron Scattering (SANS). SANS experiments were performed on the Quokka instrument at OPAL27 at a wavelength of 5.0 Å and 10% wavelength resolution. Two instrument configurations were used with a source-to-sample distance of 8 m and sample-todetector distances of 8 and 2 m, the latter with a 300 mm detector offset to increase the maximum q. These configurations provide a continuous q range of 0.01 to 0.5 Å −1 where q is the magnitude of the scattering vector, defined by q = 4π/λ sin(θ/2), λ is the wavelength, and θ is the scattering angle. All samples were enclosed in Hellma cuvettes with a 1 mm path length. Samples were studied at several temperatures with control provided with a Julabo thermostated bath. All data were corrected for blocked beam measurements, normalized, radially averaged, placed on an absolute scale using a package of macros in Igor software (Wavemetrics, Lake Oswego, OR, U.S.A.), and modified to accept HDF5 data files from Quokka.28 Small-Angle X-ray Scattering (SAXS). SAXS measurements were performed at the SAXS/WAXS beamline at the Australian Synchrotron.29 An X-ray beam with a wavelength of 1.1271 Å (11 keV) was used. A sample-to-detector distance of 1015 mm gave the q range of 0.007 < q < 0.7 Å−1. The 2D SAXS patterns were acquired within 1 s using a Pilatus 1 M detector with an active area of 169 × 179 mm2 and a pixel size of 172 μm. All experiments were performed at T = 27 °C (hutch temperature at the beamline). All samples were degassed under vacuum and centrifuged at 3500 rpm for 5 min before the measurement in the quartz capillary mounted in the beam. To avoid beam damage, the solution was pumped continuously during the measurement. For each curve, 10 frames were recorded and averaged manually after inspection for beam damage; no beam damage was observed in the samples. Two-dimensional scattering patterns were integrated into one-dimensional scattering function I(q) using inhouse-developed software package scatterBrain. Scattering curves are plotted as a function of relative (SAXS) or absolute (SANS) intensity, I, versus q.

More-polar, low-molecular-weight fatty acids such as caprylic acid have higher rates of penetration across the intestine and gallbladder than larger, less-polar compounds. This implies that in addition to the passive absorption of molecules across the lipid membrane of the mucosal cell, there is also a movement of small molecules through a more-polar diffusion pathway through the epithelial surface.21 This study shows that the pH response of the mixed colloidal digestion products can influence the fatty acid delivery through modification of the micelle−water interface. The aim is to systematically study the influence of pH modification on the self-assembly of caprylic acid in biologically relevant systems containing the corresponding monoglyceride monocaprylin, bile salt, and phospholipid. Somewhat tangential to the low-pH microclimate at the enterocytes, during the digestion process in mammals, the pH of the food bolus gradually increases as it passes along the gastrointestinal tract; the pH of the gastric contents varies from 1.0−3.0 and that of duodenal contents, from 4.8−8.2.22 Thus, a complete understanding of the impact of pH on such colloidal structures is imperative. With increasing pH, the polarity of the amphiphilic molecule caprylic acid increases upon deprotonation of its carboxylic group. The pH modifies the protonation state of the fatty acid, with a shift in the pKa of the carboxylic group to higher values having been observed for fatty acids in self-assembled systems.23 The thermodynamic model of the transfer properties of charged and uncharged fatty acids from bulk water to the surface of the micelle, based on the Poisson−Boltzmann equation, has highlighted the major role of the dielectric discontinuity in the protonation behavior of fatty acids.24 Calorimetric measurements during the titration of fatty acids solubilized in micelles showed that this process is endothermic at 298 K, contrary to fatty acids solubilized in water (ΔH ≈ 0).24 This has been attributed to a dielectric interface caused by lower static permittivity in the core of the micelles compared to that of the surrounding water solution; the presence of a dielectric interface results in large energies for charged molecules located near the micelle−water interface.25 This study aims to identify the location of the fatty acids within the micellar structures using contrast variation from SANS in combination with SAXS and DLS to explain the faster absorption rates of short-chain fatty acids such as caprylic acid. The pH response is of relevance to this process as the fatty acid molecules change their location within the mixed micelles with changing polarity due to the modified protonation state.

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MATERIALS AND METHODS

Materials. Deuterated caprylic acid (C8D16O2) was prepared as described below. Tris maleate (reagent grade), bile salt (sodium taurodeoxycholate >95%), monocaprylin (purity ≥98%) (C11H22O4), NaOH (p.a. grade), and HCl (p.a. grade) were purchased from SigmaAldrich (St. Louis, MO, USA). Phospholipid (dioleoylphosphatidylcholine, DOPC, >94%) was from Trapeze Associates Pty Ltd. (Clayton, Victoria, Australia). Calcium chloride (>99%) was obtained from Ajax Finechem (Seven Hills, NSW, Australia), and sodium chloride (>99%) was obtained from Chem Supply (Gillman, SA, Australia). Ultrapure water (resistivity >18 MΩ cm) was used for the preparation of all samples. Caprylic acid (purity ≥98%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). D2O (99.8%) was supplied by AECL (Ontario, Canada). Thin-layer chromatography (TLC) was performed on Fluka Analytical silica gel aluminum sheets (25 F254) (product of Sigma-Aldrich, St. Louis, MO, USA). Davisil silica gel (LC60 Å 40-63 micrometer) (product of W. R. Grace & Co., 7297

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Table 1. Summary of Chemical Formulae and X-ray and Neutron Scattering Length Densities (ρ) of the Individual Componentsa

a

component

physical density [g/cm−3]

D2O H2O C8D16O2 C11H22O4 C26H44NO6SNa C42H82NO8P

1.1 1.0 0.9 0.9 0.9 0.9

X-ray scattering length density [Å−2] 9.37 9.47 7.64 8.43 8.29 8.47

× × × × × ×

neutron scattering length density [Å−2]

10−6 10−6 10−6 10−6 10−6 10−6

6.38 −5.61 5.80 3.49 6.12 2.38

× × × × × ×

neutron contrast in H2O Δρ (H2O)

10−6 10−7 10−6 10−7 10−7 10−7

6.36 9.10 1.17 7.99

× × × ×

neutron contrast in D2O Δρ (D2O)

10−6 10−7 10−6 10−7

−5.80 −6.03 −5.77 −6.14

× × × ×

10−7 10−6 10−6 10−6

Also shown is the neutron contrast Δρ = (ρ − ρsolvent); the highest contrast (strongest scatterers) in D2O and H2O are underlined.

Dynamic Light Scattering (DLS). DLS was used to determine the mean hydrodynamic radius (RH) and the size distribution width of the dispersed particles. Measurements were carried out on a Malvern Zetasizer NanoZS with a He−Ne laser (λ = 633 nm) and a laser power of 4 mW at a backscattering angle of 173° at 25 °C. From the correlation functions, the average diffusion coefficient D was obtained by cumulant analysis.30 The hydrodynamic radius RH was deduced from the diffusion coefficient using the Stokes−Einstein equation: RH =

kBT 6πηD

the cross-section pair distance distribution function pc(r). The radial electron density profile Δρc(r) is related to pc(r) via pc(r) = rΔρ̃2(r).36 pc(r) can be calculated from I(q) with the following equation:

I(q) =

(1)

μ2 Γ̅ 2

(2)

where μ2 is the second cumulant and Γ̅ is the mean of the inverse decay time. Before the DLS measurements, all samples were centrifuged at 3500 rpm for 5 min. The samples were measured without further dilution to avoid the possible affect of dilution on the mixed micelles.7−11 Consequently, the hydrodynamic radii presented in this study are “apparent” values. The total concentration of BS + DOPC + caprylic acid + monocaprylin micelles is approximately 2 wt %. Evaluation of SANS and SAXS Data. Constant background after buffer subtraction (e.g., from the incoherent neutron scattering by 1H) was determined with a Porod extrapolation, I(q) ∝ q−4, and subtracted from the corresponding data set. Where applicable, Guinier analysis, from the Guinier approximation and further analysis of the inverse Fourier transform of the scattering data (see below), was performed to calculate the radius of gyration (Rg). Note that Rg represents the rootmean-square distance of the scattering density from the center of an object, and it is thus weighted by the associated distribution of electron density (SAXS) or nuclear density (SANS) with the particle of interest. This will become apparent below when Rg values extracted from the two scattering methods are compared. The generalized indirect Fourier transformation method (GIFT) was used to analyze the scattering data.31−33 For particles of arbitrary shape with an electron density or scattering length density difference of Δρ(r) relative to the mean value, the pair-distance distribution function p(r) is given by p(r) = r2Δρ̃2(r), where Δρ̃2(r) is the convolution square of Δρ̃(r) averaged for all directions in space. This averaging causes no loss of information in the case of particles with spherical symmetry. p(r) is calculated from the scattered intensity I(q) using following equation34 I(q) = 4π

∫0

∞

p(r )

sin(qr ) dr qr

∫0

∞

pc (r ) J0 (qr ) dr

(4)

Here, J0(qr) is the zeroth-order Bessel function and L is the length of the cylinder. The deconvolution of p(r) for spherical particles and pc(r) for cylindrical micelles gives the radial contrast profile Δρ(r) or Δρc(r) in electron density or scattering length density, which gives information about the internal structure of the scattering particles. Most spherical micelles have a core−shell-type structure, with a hydrophobic core and a hydrophilic shell also containing counterions and bound solvent molecules. If there is a difference in scattering length, the radius of the core and the thickness of the shell can be discerned directly from the radial contrast profile. Interparticle effects described by the structure factor S(q) can influence the scattering function at higher concentration (volume fraction). In the case of monodisperse, homogeneous, and spherical particles, the scattering intensity can then be expressed by

kB is the Boltzmann constant, T is the absolute temperature (298.15 K), and η is the viscosity of the solvent (η(D2O) = 1.095 cP). The polydispersity index (PDI) of the size distribution was determined from the second cumulant

PDI =

2π 2L q

I(q) = NS(q) P(q)

(5)

with N being the number of particles and P(q) being the form factor describing the intraparticle interactions. The generalized indirect Fourier transformation (GIFT) method allows the separation of form and structure factors.31,37 This technique uses the model-free approach for the p(r) described above, which corresponds only to the form factor. Simultaneously, a model for the structure factor is fitted to eq 5. As the colloids in this study are charged, the Ornstein−Zernike equation38 with the hypernetted-chain (HNC) approximation for the closure relation was employed.37 The radius of gyration was also calculated from p(r) using

Rg =

∫ p(r)r 2 dr 2 ∫ p(r ) dr

(6)

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RESULTS AND DISCUSSION SANS is a powerful method for studying colloidal structures in food-relevant systems39 and benefits from the possibility of enhancing the scattering contrast through the replacement of hydrogen with its heavier isotope deuterium. Since neutrons Table 2. Radius of Gyration Calculated from the SANS Data of the Mixed Micelles in D2O at pH 4.0 sample

(3)

bile bile bile bile

and enables the determination of the overall shape and size of the scattering objects.35 In the case of cylindrical micelles with a sufficient length/diameter ratio (>3), the cross section can be investigated using 7298

salt salt salt salt

micelles micelles monoglyceride micelles + fatty acid micelles + monoglyceride and fatty acid

Rg [Å] 16.5 20.4 24.2 59.3

± ± ± ±

0.5 0.8 0.5 2.7

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Figure 1. SANS curves for the bile salt micelles containing monocaprylin and deuterated caprylic acid at different pH values in D2O (a) and H2O (b). The scattering of bile salt micelles in both solvents is also presented.

solvent resulted in the generation of scattering arising primarily from the bile salt, phospholipid, and monoglyceride, whereas in H2O the scattering signal is dominated by the scattering of the deuterated fatty acid. The X-ray and neutron scattering length densities of the components are summarized in Table 1. The radius of gyration (Rg) from Guinier analysis for the bile salt−phospholipid micelles assembled in D2O at pH 4 is presented in Table 2. Before the addition of the fatty acid and monoglyceride, the Rg was approximately 16 Å. This Rg is comparable to literature values for similar systems (i.e., 19 Å for the lecithin/sodium taurocholate in a ratio of 0.4 and 21 mM total concentration).7 On addition of fatty acid or monoglyceride, the Rg of the micelles increased to approximately 20 Å. Interestingly, the addition of a mixture of both fatty acid and monoglyceride resulted in a dramatic increase in Rg to 60 Å. The change in the equilibrium packing geometry in this multicomponent system is thought to be responsible for this behavior. At this pH, the fatty acid is mostly protonated. Increasing the concentration of rather hydrophobic components in the micelles results in an increase in particle size and elongation. This effect is comparable to the dilution of phospholipid/bile salt systems; indeed, prior studies on the dilution of the lecithin/bile-salt system found particle elongation due to the leaching of bile salt from the micelles and, as a result, increasing the hydrophobic material (lecithin)/bile salt ratio within the mixed micelles.7,10 The influence of varying the pH on the scattering from the caprylic acid + monocaprylin micellar systems with simulated intestinal fluid in D2O and H2O is presented in Figure 1a,b, respectively. With increasing pH, a decrease in the forward scattering was observed. Increasing S(q) due to the partial deprotonation of fatty acids (increase in charge) as well as an decrease in the size of the micelles and a shape transition from elongated to spherical can explain this change in the I(q) in the D2O-based system. This transition from elongated to approximate spherical shape with increasing pH values is clearly shown in the crosssection Guinier plots of the SANS data in D2O presented in the Supporting Information in Figure SI-3. The scattering intensity for the H2O-based system decreased dramatically between pH 3.8 and 6.5, showing no further changes at higher pH values. As the I(q) in the H2O-based system mainly results from the scattering of the deuterated caprylic acid, this suggests that most of the fatty acid is

Figure 2. pH dependence of the radius of gyration calculated from SANS curves presented in Figure 1 using the Gunier approximation (full symbols) and from p(r) obtained from model-independent fitting with generalized indirect Fourier transformation using eq 6 (open symbols).

Table 3. Measured Hydrodynamic Radii and Polydispersity in D2O for the Deuterated Caprylic Acid-Based Mixed Micelle System at Various pH Valuesa pH

RH [Å]

PDI

Rg/RH

Rg*/RH

2.2 5.3 6.4 7.1 bile salt micelles

50 37 32 25 28

0.18 0.24 0.29 0.15 0.15

1.39 0.70 0.41 0.51 0.57

1.48 0.83 0.60 0.70 0.67

a The included Rg/RH and Rg*/RH were calculated with Rg from the Guinier approximation from I(q) directly (Rg) and from P(q) (Rg*).

scatter from the atomic nucleus, the associated isotopic dependence enables both solvent contrast variation (e.g., mixtures of H2O and D2O) and, typically more costly, selective deuteration of nonsolvent molecules. Contrast variation with Xrays is typically limited to anomalous methods, which are unsuitable for low-atomic-number elements. In this study, mixed micelles formed by custom-synthesized fully deuterated fatty acids in combination with nondeuterated monoglyceride, bile salt, and phospholipid were employed. Using D2O as a 7299

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Figure 3. (a) Measured SANS profiles in D2O and H2O and SAXS profile for the deuterated caprylic acid-based mixed micelle system at pH 2.2 (symbols) and fit (red line). (b) Corresponding p(r) profiles calculated from (a) using eq 3 and the structure factor model.

measured with DLS are summarized in Table 3. The corresponding correlation functions showing rather monomodal and uniformly distributed particles are presented in Figure SI-6. The RH follows the same trend as Rg, where it decreases with increasing pH. The Rg/RH ratios, which provide further details on particle shape, are presented in the same table. The theoretical Rg/RH value for homogenuous spheres is 0.775, with elongated particles deviating to values of >1.41 With Rg calculated directly from the experimental I(q), lower Rg/RH ratios than those for homogeneous spheres were observed in some systems (e.g., as low as 0.4 for the system at pH 6.4 and 0.5 for the bile salt−DOPC mixed micelles). As mentioned above, a structure factor can lead to an underestimation of the Rg values due to the excluded volume effect decreasing the forward scattering intensity. Consequently, S(q) and P(q) were determined from the I(q) for all systems (eq 5). Details on the interaction parameters resulting from this calculation are presented in the Supporting Information. The resultant values, which we denote as Rg*, were then calculated with the Guinier extrapolation from P(q). All Rg*/RH ratios are higher than Rg/RH, indicating that the structure factor was a major cause of the deviation. However, Rg*/RH for the system at high pH >6.4 and the bile salt−DOPC mixed micelles still deviates to lower values of 6 toward the micelle−water interface and dispersed into the aqueous bulk phase. This result has important implications for fatty acid release during the digestion of lipids: the fast release of short-chain fatty acids to the aqueous bulk phase may provide a mechanism for the fast digestion kinetics observed for corresponding short-chain triglyceride systems. The method of localizing individual components in complex colloidal systems presented in this work might also be of interest for studying and optimizing colloidal drug-delivery systems. ASSOCIATED CONTENT

S Supporting Information *

Detailed description of the preparation of deuterated caprylic acid. Control experiments employing additional SAXS, SANS, and DLS data. Cross-sectional Guinier plots. Details on the interaction parameters for the structure factor model. This material is available free of charge via the Internet at http:// pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank AINSE Ltd. for providing financial assistance to enable work on the Quokka SANS instrument at ANSTO. The studies were funded by the Australian Research Council through the Discovery Projects scheme (DP120104032). The SAXS measurements were conducted on the SAXS/WAXS beamline at the Australian Synchrotron. 7302

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dx.doi.org/10.1021/la500835e | Langmuir 2014, 30, 7296−7303