- Email: [email protected]
Dynamic control of nanofluidic channels in protein drug delivery vehicles
J. DRUG DEL. SCI. TECH., 18 (1) 41-45 2008
Dynamic control of nanofluidic channels in protein drug delivery vehicles A. Angelova1*, B. Angelov2, 3, S. Lesieur1, R. Mutafchieva3, M.Ollivon1, C. Bourgaux1, R. Willumeit2, P. Couvreur1 CNRS UMR8612 Physicochimie, Pharmacotechnie, Biopharmacie, Université Paris-Sud, 92290 Châtenay-Malabry, France 2 Institute of Materials Research, GKSS Research Center, 21502 Geesthacht, Germany 3 Institute of Biophysics, Bulgarian Academy of Sciences, BG-1113 Sofia, Bulgaria *Correspondence: [email protected]
1
Proteocubosomic nanocarriers are three-dimensional (3D) self-assembly periodic nanofluidic structures that have a surface topology of open nanochannels, which is apparent also in natural and self-assembled viral capsids. Here, proteins are nanoconfined in a hydrated self-assembly mixture of amphiphilic monoolein (MO) and octylglucoside (OG) forming cubosomic nanostructures. The generated periodic 3D nanochannel network architectures are investigated by means of synchrotron X-ray diffraction. The structural behavior of the ternary MO/OG/transferrin and MO/OG/immunoglobulin systems as well as the binary MO/OG mixture is studied in excess aqueous environment in the temperature range from 1 to 99°C. The results indicate that 5% OG molar fraction in protein-containing monoolein systems favors the formation of a swollen bicontinuous cubic liquid crystalline structure with large water channels (DLarge). Thus, it is feasible to dynamically adjust the diameters of the nanofluidic aqueous channels in the proteocubosomic carrier via the incorporation of a suitable amphiphilic additive. The obtained results may inspire the development of novel stimuli-responsive protein drug delivery nanovehicles and biocompatible self-regulating nanofluidic devices. Key words: Self-assembly nanofluidic device – Stimuli-responsive protein nanocarrier – Bicontinuous cubic phase – Aqueous nanochannel network.
Nanofluidic devices for drug delivery are at the forefront of scientific research [1]. In mechanical nanofluidics, the water channels are static and with a fixed size [2]. They do not manifest a capacity to swellshrink water in a reversible manner. In nature, the nanofluidic channel systems are not statically fixed and they may be tunably regulated in cellular assemblies [3]. An ultimate nanofluidic device requires unprecedented control over transport and mixing behaviors [4, 5]. In order to advance current fluidics design into the single-molecule regime, nanocarrier systems and devices have to be developed that have physical dimensions at the nanometer scale. To design and create such devices, we must explore propensity in self-assembly as well as knowledge obtained from biological systems [6]. For example, the Golgi endoplasmic reticulum in eukaryotic cells has many attractive features for sorting and routing single molecules such as ultrasmall-scale dimension, transport control, and the ability to recognize different molecular species. It is capable of performing chemical transformations in nanometer-sized compartments with minimal dilution. An area of great opportunity is that the nano- and microfluidic flows are not Newtonian but viscoelastic. Examples include the on-chip manipulation of cells, of surfactant mesostructures, and of relatively concentrated protein systems [7]. Self-assembly from building blocks at nanoscale to functional systems and beyond is closely related to nanofluidics since the process of self-assembly occurs in water or in another liquid [7-19]. Selfassembly is an increasingly significant part of the technology that mass-produces devices, harvests energy, manipulates information or delivers molecular nanostructures. Native nanoporous assemblies such as viral shells [20-22] are selfassembled spontaneously from their molecular components and they inspire biomimetic nanofluidic structures. In virus self-assembly, the cowpea chlorotic mottle virus (CCMC) virion could be considered as a model of a native stimuli-responsive nanoporous system. The surface of the virion particle exposes open channels to the aqueous environment (Figure 1b). Its supramolecular structure is pH sensitive.
In this work, a similarity is suggested between the nanofluidic organizations of viral capsids and cubosome nanoparticles, both of them being self-assembly objects that could adjust their surface exposed nanopores. Figure 1 presents the surface analogy of nanochannels in the two kinds of biomolecular-carrier systems. It should be emphasized that the size of the virion particle is about 28 nm, while the size of the cubosome nanovehicle could be varied upon its growth in a “bottom-up” approach [26] and via optimization of the technology for preparation/dispersion of nanoparticulate devices [11, 23]. The unique advantages of soft-matter, self-assembly cubosomic nanocarriers as nanocompartment delivery systems have received considerable recent interest [23-30]. Cubosomic nanostructures of a diamond type (Q224 space group) (Figure 1a) display greater structural flexibility with respect to the CCMV virions. The self-assembly viral capsid shown in Figure 1b has an icosahedral symmetry, which does not produce a periodic tilling. The virion particle lacking a periodic internal organization could swell only by 10% upon pH increase from pH 5 to pH above 7 (which represents a rather large pH stress). The cubic unit cell dimension in a self-assembly monoolein/octylglucoside system swells up to 50% as we have recently demonstrated by structural synchrotron-radiation small-angle X-ray scattering (SAXS) analysis [27]. This structural effect may serve as an efficient tool for enhancing the solubilization capacity of the 3D nanochannel network of the cubosomic nanodevices due to the increase in the aqueous nanochannel diameters. Here, the cubosomic nanochannels structures in MO/OG/protein systems are examined from the point of view of dynamic water channel organization controlled by temperature. Our purpose is to demonstrate that by incorporating a monolayer curvature-modulating agent (OG), the diameter of the water channels (Dw) and the temperature of the structural phase transition from a water-swollen (DLarge) to a normal diamond cubic (DNormal) phase can be controlled via external temperature stimulus close to body temperature. For the purpose of comparison with the pure MO/water system [31], the thermal phase behavior 41
J. DRUG DEL. SCI. TECH., 18 (1) 41-45 2008
Dynamic control of nanofluidic channels in protein drug delivery vehicles A. Angelova, B. Angelov, S. Lesieur, R. Mutafchieva, M. Ollivon, C. Bourgaux, R. Willumeit, P. Couvreur
Figure 1 - Three-dimensional presentations of a small cubosomic nanoparticle with a diameter 55 nm and a diamond-type structure (Pn3m space group) (a), and a reconstructed cryo-electron microscopy image of a cowpea chlorotic mottle virus (CCMV) particle with a diameter 28 nm and icosahedral symmetry (Copyright 1998, Elsevier) (b).
Figure 2 - Synchrotron radiation small-angle X-ray diffraction (SAXD) patterns of a MO/OG (95/5, mol/mol) liquid crystalline system selfassembled in 4 mg/mL human transferrin solution in phosphate buffer. Top panel: sequence of dynamic heating (from 1 to 99°C) and cooling (from 99 to 10°C) temperature scans showing a hysteresis in the reversible structural transition between a water-swollen (DLarge) diamond cubic phase and a normal (DNormal) bicontinuous cubic Q224 phase. Bottom panel: selected SAXD patterns from the heating (T = 20°C (a) and T = 60°C (b)) and the cooling scan (T = 20°C (c)). The Bragg peaks belong to the set of (110), (111), (200), (211), (220), (221), (310), (222) reflections (Pn3m space group).
of the MO/OG/protein mixtures is studied in a broad temperature range despite the fact that the high temperatures are not of practical interest for protein encapsulation. The nanochannel structure in the water-swollen DLarge cubic system appears to be stable and reversible in thermal scans. The thermal stability of solubilized proteins is out of the scope of the present work.
principal to that used in the measurements reported in refs. 17, 18, and 27. Two detectors (one-dimensional) covering the small-angle (SAXS) and the wide-angle (WAXS) regions were used. The temperature scans from 1 to 99°C, and the scan rate of 2°C/min, were programmed and controlled automatically via a Microcalix system. The cooling scans were performed immediately after the heating scans. The recorded one-dimensional X-ray diffraction data were determined as intensities versus wave vector (q). The latter was defined as q = 2 π s = (4π/l) Sin(q) = 2 π/d, where 2q is the scattering angle, l = 1.5 Å is the X-ray wavelength, and d is the repeat spacing (d = 1/s). For determination of the s-values, tristearin (d001 = 4.497 nm) was employed as a SAXS calibration sample.
I. Materials and Methods
The samples preparation employed a similar methodology as described in refs. 17, 18, and 27. A powder of 1-monooleoyl-rac-glycerol [C18:1, cis-9] (MO) (MW 356.5) (purity > 99.5%, Sigma-Aldrich Co.) was hydrated and dispersed in excess aqueous phase containing n-octyl beta-D-glucopyranoside (OG) (MW 292.4) (purity > 99.5%, Sigma-Aldrich Co.) dissolved in phosphate buffer (NaH2PO4/Na2HPO4 (1:1 mol/mol) pH 7, p.a. grade, Merck), yielding lipid-to-detergent molar ratios of 95/5 and 80/20. Full hydration of the lipid MO was achieved under excess aqueous phase conditions (20 wt % dry lipid and 80 wt % buffer solution). Hydration of the lipid powder, to yield dispersion of lipid in the OG solution, was performed at ambient temperature (21°C). Incorporation of transferrin (Sigma) and immunoglobulin (Sigma) in MO/OG systems was performed at 4 mg/mL protein concentration. For every sample, eight cycles of vortexing (for 1 min) and incubation (for 5 min) at room temperature were applied. After homogenization, the hydrated samples were stored at 4°C before SAXS measurements.
II. Results and Discussion
The temperature dependence of the small-angle X-ray diffraction (SAXD) patterns characterizing the structural phase behavior of a fully hydrated MO/OG/transferrin system is presented in Figure 2. Throughout the entire temperature range from 1 to 99°C, characteristic diffraction peaks of bicontinuous cubic phases of the Pn3m (Q224) space group are observed. The estimation of the cubic unit cell dimension, a, reveals essential swelling to a = 14 nm at low temperatures (T < 35°C). This value is considerably larger (by about 35%) with regard to that of pure MO cubic phase (a = 10.4 nm) at same temperature. The observed transition in the heating scan from a DLarge (T = 20°C, Figure 2a) to a DNormal (T = 60°C, Figure 2b) bicontinuous cubic phase proves that
Small-angle X-ray diffraction (SAXD)
The supramolecular structure of the amphiphilic MO/OG/water and MO/OG/protein/water self-assembly systems was investigated by means of Synchrotron X-ray diffraction performed at beam line D24 at Lure, Orsay, France. The experimental setup had an analogous 42
Dynamic control of nanofluidic channels in protein drug delivery vehicles A. Angelova, B. Angelov, S. Lesieur, R. Mutafchieva, M. Ollivon, C. Bourgaux, R. Willumeit, P. Couvreur
the incorporation of amphiphilic OG molecules into the MO bilayer at T < 30°C plays a role in lowering the monolayer curvature and thus in enhancing the water swelling of the diamond cubic structure. Upon cooling from 99°C to below 20°C, a hysteresis of the cubic lattice size is observed (Figure 2c) due to the difference in the kinetics of the hydration and dehydration processes. Giving enough time between the heating and the cooling scans should reduce the hysteresis, as shown by de Campo et al. [30] for binary monoglyceride/water systems. Figure 3 indicates an analogous structural behavior with the fully hydrated MO/OG/IgG system in heating and cooling scans. At 37°C, a swollen DLarge cubic nanostructure is also observed. Upon heating, the transition from a DLarge to a DNormal bicontinuous cubic phase begins near 45°C and it terminates at around 50°C, Ttransition being ~ 45°C. The temperature dependence of the cubic lattice parameter, a, for both systems is shown in Figure 4. The presented structural results do not display specificity in the lattice dimension with regard to the protein size (MW 75 kDa for transferrin and MW 150 kDa for immunoglobulin). This confirms the dominating role of the amphiphilic OG in curvature modulation and hydration enhancement of the nanochannels in the investigated systems. It should be emphasized that in MO/OG systems lacking protein, OG induces a DLarge swollen cubic phase at 10% OG molar fraction (a = 15.05 nm at T = 25°C). At 5% OG molar fraction in the self-assembly MO/OG system, a DNormal cubic phase forms in the absence of protein (a = 12.5 nm at T = 25°C). When the system is at the critical OG concentration (ca. 10% OG), an increase is established in the curvature of the MO monolayers upon increase in temperature. This causes a partial release of OG molecules from the MO monolayers on heating in order to maintain the stability of the bicontinuous nanochannels system. After squeezing out a certain amount of OG, the systems transforms into a DNormal cubic phase at T > 45°C. However, at least 5 mol.% OG remains in the bilayer membrane as it is stable at such OG concentration. Thus, by adding a protein at the critical OG concentration for the induction of DLarge-to-DNormal cubic-phase structural transition, the system could become even more sensitive to small amounts of additives. Therefore, 5% OG molar fraction and protein concentration 4 mg/mL appears to be an optimum condition for the entrapment of proteins in the fluidic nanochannels network of the self-assembly cubosomic nanocarrier. The DLarge cubic structure is stable in the temperature interval from 0 to 45°C. This permits storage of the proteocubosome nanocarriers at low temperature and delivery of proteins at body temperature. At 20% molar content of the OG amphiphile, the self-assembly MO/OG system transforms from a cubic to a lamellar phase at low and body temperatures (Figure 5). Heating beyond 52°C, which is well above body temperature, recovers the cubic structure but these conditions are not optimal either for generation of proteocubosomes nor for drug delivery. The estimated repeat spacings of the periodic supramolecular organizations are shown as a function of temperature in Figure 6. For the lamellar MO/OG 80/20 (mol/mol) system, the repeat spacing is d = 5.4 nm and it appears without noticeable temperature dependence. It is known from our previous calculations that the thickness of the mixed MO/OG bilayer is around 3.5 nm. Then, the thickness of the water region that separates the lipid bilayers in the lamellae will be around 1.9 nm. For the cubic phase formed by the MO/OG 80/20 (mol/ mol) assembly upon heating, the lattice parameter is found to exhibit a maximum value a = 13.5 nm at T = 52°C, and it decreases to a = 9.5 nm at T = 99°C (Figure 6). A hexagonal phase was not observed with this sample. This proves that a small amount of OG remains in the amphiphilic bilayer thus modifying its phase behavior with regard to the pure monoolein system, which forms an inverted hexagonal phase at T > 95°C [31]. The frames at 50 and 52°C (Figure 5, bottom panel) show intermediate states in the lamellar-to-cubic structural phase transition for
J. DRUG DEL. SCI. TECH., 18 (1) 41-45 2008
Figure 3 - Synchrotron radiation SAXD patterns of a MO/OG (95/5, mol/mol) liquid crystalline system self-assembled in 4 mg/mL human immunoglobulin (IgG) solution in phosphate buffer. Top panel: sequence of dynamic heating (from 1 to 99°C) and cooling (from 99 to 10°C) temperature scans showing a hysteresis in the reversible structural transition between a water-swollen (DLarge) diamond cubic phase and a normal (DNormal) bicontinuous cubic Q224 phase. Bottom panel: SAXS pattern selected from the heating scan at body temperature (T = 37°C).
Figure 4 - Cubic lattice parameter, a, versus temperature, T, for the investigated MO/OG/transferrin (bright circles) and MO/OG/IgG (filled black circles) self-assembly systems.
the MO/OG 80/20 system. In previous literature reports [32], such intermediates were accounted for as a scattering background. Certain authors [33] consider them to be a disordered bicontinuous emulsion. A special further investigation appears to be necessary in order to characterize the structure of such intermediates. Even though they resemble certain emulsion features, they are very likely to be bicontinuous cubic structures with some disorder of the nanochannel networks. In the higher temperature region (T > 52°C) the X-ray patterns correspond to a cubic phase of the Pn3m symmetry (Figure 5, top panel). * The present work demonstrates the dynamic control of the aqueous nanochannels in protein-containing monoolein bicontinuous cubic 43
J. DRUG DEL. SCI. TECH., 18 (1) 41-45 2008
Dynamic control of nanofluidic channels in protein drug delivery vehicles A. Angelova, B. Angelov, S. Lesieur, R. Mutafchieva, M. Ollivon, C. Bourgaux, R. Willumeit, P. Couvreur
stimulus, the virion shell breaks and a release of active substance occurs due to the fact that the swelling of the virus is not infinite. In MO/OG systems, the increase in the OG concentration causes swelling of the aqueous nanochannel diameters. Above a critical OG concentration, this swelling reaches a maximum and the supramolecular nanochannel structure will transform into a lamellar phase involving two-dimensional aqueous sheets instead of curved water nanochannels for nanoconfinement of guest biomolecules. These features suggest the feasibility of creation of soft-matter, self-regulating nanodevices.
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5. 6. Figure 5 - Synchrotron radiation SAXD patterns of a MO/OG (80/20, mol/mol) liquid crystalline system self-assembled in a phosphate buffer (no protein added). Top panel: dynamic heating scan from 1 to 99°C. Bottom panel: selected SAXD patterns from the heating scan showing an intermediate phase at T = 50°C (b) and at T = 52°C (c) during the transition between lamellar (T = 20°C (a)) and cubic phase (T > 50°C).
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Figure 6 - Temperature dependence of the repeat distance, d, of the lamellar (L) phase and of the lattice parameter, a, of the Pn3m cubic phase of MO/OG 80/20 (mol/mol) mixture self-assembled in phosphate buffer solution.
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Dynamic control of nanofluidic channels in protein drug delivery vehicles A. Angelova, B. Angelov, S. Lesieur, R. Mutafchieva, M. Ollivon, C. Bourgaux, R. Willumeit, P. Couvreur
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Acknowledgements B.A. and R.M. acknowledge support from the Bulgarian Academy of Sciences internal project 2004/5. A.A. and B.A. acknowledge support by project LURE BD 001-03. The authors thank Dr. Geneviève Lebas for continuous cooperation and Gerard Keller for instrumental design and facilities.
Manuscript Received 25 June 2007, accepted for publication 13 August 2007.
45
Dynamic control of nanofluidic channels in protein drug delivery vehicles A. Angelova1*, B. Angelov2, 3, S. Lesieur1, R. Mutafchieva3, M.Ollivon1, C. Bourgaux1, R. Willumeit2, P. Couvreur1 CNRS UMR8612 Physicochimie, Pharmacotechnie, Biopharmacie, Université Paris-Sud, 92290 Châtenay-Malabry, France 2 Institute of Materials Research, GKSS Research Center, 21502 Geesthacht, Germany 3 Institute of Biophysics, Bulgarian Academy of Sciences, BG-1113 Sofia, Bulgaria *Correspondence: [email protected]
1
Proteocubosomic nanocarriers are three-dimensional (3D) self-assembly periodic nanofluidic structures that have a surface topology of open nanochannels, which is apparent also in natural and self-assembled viral capsids. Here, proteins are nanoconfined in a hydrated self-assembly mixture of amphiphilic monoolein (MO) and octylglucoside (OG) forming cubosomic nanostructures. The generated periodic 3D nanochannel network architectures are investigated by means of synchrotron X-ray diffraction. The structural behavior of the ternary MO/OG/transferrin and MO/OG/immunoglobulin systems as well as the binary MO/OG mixture is studied in excess aqueous environment in the temperature range from 1 to 99°C. The results indicate that 5% OG molar fraction in protein-containing monoolein systems favors the formation of a swollen bicontinuous cubic liquid crystalline structure with large water channels (DLarge). Thus, it is feasible to dynamically adjust the diameters of the nanofluidic aqueous channels in the proteocubosomic carrier via the incorporation of a suitable amphiphilic additive. The obtained results may inspire the development of novel stimuli-responsive protein drug delivery nanovehicles and biocompatible self-regulating nanofluidic devices. Key words: Self-assembly nanofluidic device – Stimuli-responsive protein nanocarrier – Bicontinuous cubic phase – Aqueous nanochannel network.
Nanofluidic devices for drug delivery are at the forefront of scientific research [1]. In mechanical nanofluidics, the water channels are static and with a fixed size [2]. They do not manifest a capacity to swellshrink water in a reversible manner. In nature, the nanofluidic channel systems are not statically fixed and they may be tunably regulated in cellular assemblies [3]. An ultimate nanofluidic device requires unprecedented control over transport and mixing behaviors [4, 5]. In order to advance current fluidics design into the single-molecule regime, nanocarrier systems and devices have to be developed that have physical dimensions at the nanometer scale. To design and create such devices, we must explore propensity in self-assembly as well as knowledge obtained from biological systems [6]. For example, the Golgi endoplasmic reticulum in eukaryotic cells has many attractive features for sorting and routing single molecules such as ultrasmall-scale dimension, transport control, and the ability to recognize different molecular species. It is capable of performing chemical transformations in nanometer-sized compartments with minimal dilution. An area of great opportunity is that the nano- and microfluidic flows are not Newtonian but viscoelastic. Examples include the on-chip manipulation of cells, of surfactant mesostructures, and of relatively concentrated protein systems [7]. Self-assembly from building blocks at nanoscale to functional systems and beyond is closely related to nanofluidics since the process of self-assembly occurs in water or in another liquid [7-19]. Selfassembly is an increasingly significant part of the technology that mass-produces devices, harvests energy, manipulates information or delivers molecular nanostructures. Native nanoporous assemblies such as viral shells [20-22] are selfassembled spontaneously from their molecular components and they inspire biomimetic nanofluidic structures. In virus self-assembly, the cowpea chlorotic mottle virus (CCMC) virion could be considered as a model of a native stimuli-responsive nanoporous system. The surface of the virion particle exposes open channels to the aqueous environment (Figure 1b). Its supramolecular structure is pH sensitive.
In this work, a similarity is suggested between the nanofluidic organizations of viral capsids and cubosome nanoparticles, both of them being self-assembly objects that could adjust their surface exposed nanopores. Figure 1 presents the surface analogy of nanochannels in the two kinds of biomolecular-carrier systems. It should be emphasized that the size of the virion particle is about 28 nm, while the size of the cubosome nanovehicle could be varied upon its growth in a “bottom-up” approach [26] and via optimization of the technology for preparation/dispersion of nanoparticulate devices [11, 23]. The unique advantages of soft-matter, self-assembly cubosomic nanocarriers as nanocompartment delivery systems have received considerable recent interest [23-30]. Cubosomic nanostructures of a diamond type (Q224 space group) (Figure 1a) display greater structural flexibility with respect to the CCMV virions. The self-assembly viral capsid shown in Figure 1b has an icosahedral symmetry, which does not produce a periodic tilling. The virion particle lacking a periodic internal organization could swell only by 10% upon pH increase from pH 5 to pH above 7 (which represents a rather large pH stress). The cubic unit cell dimension in a self-assembly monoolein/octylglucoside system swells up to 50% as we have recently demonstrated by structural synchrotron-radiation small-angle X-ray scattering (SAXS) analysis [27]. This structural effect may serve as an efficient tool for enhancing the solubilization capacity of the 3D nanochannel network of the cubosomic nanodevices due to the increase in the aqueous nanochannel diameters. Here, the cubosomic nanochannels structures in MO/OG/protein systems are examined from the point of view of dynamic water channel organization controlled by temperature. Our purpose is to demonstrate that by incorporating a monolayer curvature-modulating agent (OG), the diameter of the water channels (Dw) and the temperature of the structural phase transition from a water-swollen (DLarge) to a normal diamond cubic (DNormal) phase can be controlled via external temperature stimulus close to body temperature. For the purpose of comparison with the pure MO/water system [31], the thermal phase behavior 41
J. DRUG DEL. SCI. TECH., 18 (1) 41-45 2008
Dynamic control of nanofluidic channels in protein drug delivery vehicles A. Angelova, B. Angelov, S. Lesieur, R. Mutafchieva, M. Ollivon, C. Bourgaux, R. Willumeit, P. Couvreur
Figure 1 - Three-dimensional presentations of a small cubosomic nanoparticle with a diameter 55 nm and a diamond-type structure (Pn3m space group) (a), and a reconstructed cryo-electron microscopy image of a cowpea chlorotic mottle virus (CCMV) particle with a diameter 28 nm and icosahedral symmetry (Copyright 1998, Elsevier) (b).
Figure 2 - Synchrotron radiation small-angle X-ray diffraction (SAXD) patterns of a MO/OG (95/5, mol/mol) liquid crystalline system selfassembled in 4 mg/mL human transferrin solution in phosphate buffer. Top panel: sequence of dynamic heating (from 1 to 99°C) and cooling (from 99 to 10°C) temperature scans showing a hysteresis in the reversible structural transition between a water-swollen (DLarge) diamond cubic phase and a normal (DNormal) bicontinuous cubic Q224 phase. Bottom panel: selected SAXD patterns from the heating (T = 20°C (a) and T = 60°C (b)) and the cooling scan (T = 20°C (c)). The Bragg peaks belong to the set of (110), (111), (200), (211), (220), (221), (310), (222) reflections (Pn3m space group).
of the MO/OG/protein mixtures is studied in a broad temperature range despite the fact that the high temperatures are not of practical interest for protein encapsulation. The nanochannel structure in the water-swollen DLarge cubic system appears to be stable and reversible in thermal scans. The thermal stability of solubilized proteins is out of the scope of the present work.
principal to that used in the measurements reported in refs. 17, 18, and 27. Two detectors (one-dimensional) covering the small-angle (SAXS) and the wide-angle (WAXS) regions were used. The temperature scans from 1 to 99°C, and the scan rate of 2°C/min, were programmed and controlled automatically via a Microcalix system. The cooling scans were performed immediately after the heating scans. The recorded one-dimensional X-ray diffraction data were determined as intensities versus wave vector (q). The latter was defined as q = 2 π s = (4π/l) Sin(q) = 2 π/d, where 2q is the scattering angle, l = 1.5 Å is the X-ray wavelength, and d is the repeat spacing (d = 1/s). For determination of the s-values, tristearin (d001 = 4.497 nm) was employed as a SAXS calibration sample.
I. Materials and Methods
The samples preparation employed a similar methodology as described in refs. 17, 18, and 27. A powder of 1-monooleoyl-rac-glycerol [C18:1, cis-9] (MO) (MW 356.5) (purity > 99.5%, Sigma-Aldrich Co.) was hydrated and dispersed in excess aqueous phase containing n-octyl beta-D-glucopyranoside (OG) (MW 292.4) (purity > 99.5%, Sigma-Aldrich Co.) dissolved in phosphate buffer (NaH2PO4/Na2HPO4 (1:1 mol/mol) pH 7, p.a. grade, Merck), yielding lipid-to-detergent molar ratios of 95/5 and 80/20. Full hydration of the lipid MO was achieved under excess aqueous phase conditions (20 wt % dry lipid and 80 wt % buffer solution). Hydration of the lipid powder, to yield dispersion of lipid in the OG solution, was performed at ambient temperature (21°C). Incorporation of transferrin (Sigma) and immunoglobulin (Sigma) in MO/OG systems was performed at 4 mg/mL protein concentration. For every sample, eight cycles of vortexing (for 1 min) and incubation (for 5 min) at room temperature were applied. After homogenization, the hydrated samples were stored at 4°C before SAXS measurements.
II. Results and Discussion
The temperature dependence of the small-angle X-ray diffraction (SAXD) patterns characterizing the structural phase behavior of a fully hydrated MO/OG/transferrin system is presented in Figure 2. Throughout the entire temperature range from 1 to 99°C, characteristic diffraction peaks of bicontinuous cubic phases of the Pn3m (Q224) space group are observed. The estimation of the cubic unit cell dimension, a, reveals essential swelling to a = 14 nm at low temperatures (T < 35°C). This value is considerably larger (by about 35%) with regard to that of pure MO cubic phase (a = 10.4 nm) at same temperature. The observed transition in the heating scan from a DLarge (T = 20°C, Figure 2a) to a DNormal (T = 60°C, Figure 2b) bicontinuous cubic phase proves that
Small-angle X-ray diffraction (SAXD)
The supramolecular structure of the amphiphilic MO/OG/water and MO/OG/protein/water self-assembly systems was investigated by means of Synchrotron X-ray diffraction performed at beam line D24 at Lure, Orsay, France. The experimental setup had an analogous 42
Dynamic control of nanofluidic channels in protein drug delivery vehicles A. Angelova, B. Angelov, S. Lesieur, R. Mutafchieva, M. Ollivon, C. Bourgaux, R. Willumeit, P. Couvreur
the incorporation of amphiphilic OG molecules into the MO bilayer at T < 30°C plays a role in lowering the monolayer curvature and thus in enhancing the water swelling of the diamond cubic structure. Upon cooling from 99°C to below 20°C, a hysteresis of the cubic lattice size is observed (Figure 2c) due to the difference in the kinetics of the hydration and dehydration processes. Giving enough time between the heating and the cooling scans should reduce the hysteresis, as shown by de Campo et al. [30] for binary monoglyceride/water systems. Figure 3 indicates an analogous structural behavior with the fully hydrated MO/OG/IgG system in heating and cooling scans. At 37°C, a swollen DLarge cubic nanostructure is also observed. Upon heating, the transition from a DLarge to a DNormal bicontinuous cubic phase begins near 45°C and it terminates at around 50°C, Ttransition being ~ 45°C. The temperature dependence of the cubic lattice parameter, a, for both systems is shown in Figure 4. The presented structural results do not display specificity in the lattice dimension with regard to the protein size (MW 75 kDa for transferrin and MW 150 kDa for immunoglobulin). This confirms the dominating role of the amphiphilic OG in curvature modulation and hydration enhancement of the nanochannels in the investigated systems. It should be emphasized that in MO/OG systems lacking protein, OG induces a DLarge swollen cubic phase at 10% OG molar fraction (a = 15.05 nm at T = 25°C). At 5% OG molar fraction in the self-assembly MO/OG system, a DNormal cubic phase forms in the absence of protein (a = 12.5 nm at T = 25°C). When the system is at the critical OG concentration (ca. 10% OG), an increase is established in the curvature of the MO monolayers upon increase in temperature. This causes a partial release of OG molecules from the MO monolayers on heating in order to maintain the stability of the bicontinuous nanochannels system. After squeezing out a certain amount of OG, the systems transforms into a DNormal cubic phase at T > 45°C. However, at least 5 mol.% OG remains in the bilayer membrane as it is stable at such OG concentration. Thus, by adding a protein at the critical OG concentration for the induction of DLarge-to-DNormal cubic-phase structural transition, the system could become even more sensitive to small amounts of additives. Therefore, 5% OG molar fraction and protein concentration 4 mg/mL appears to be an optimum condition for the entrapment of proteins in the fluidic nanochannels network of the self-assembly cubosomic nanocarrier. The DLarge cubic structure is stable in the temperature interval from 0 to 45°C. This permits storage of the proteocubosome nanocarriers at low temperature and delivery of proteins at body temperature. At 20% molar content of the OG amphiphile, the self-assembly MO/OG system transforms from a cubic to a lamellar phase at low and body temperatures (Figure 5). Heating beyond 52°C, which is well above body temperature, recovers the cubic structure but these conditions are not optimal either for generation of proteocubosomes nor for drug delivery. The estimated repeat spacings of the periodic supramolecular organizations are shown as a function of temperature in Figure 6. For the lamellar MO/OG 80/20 (mol/mol) system, the repeat spacing is d = 5.4 nm and it appears without noticeable temperature dependence. It is known from our previous calculations that the thickness of the mixed MO/OG bilayer is around 3.5 nm. Then, the thickness of the water region that separates the lipid bilayers in the lamellae will be around 1.9 nm. For the cubic phase formed by the MO/OG 80/20 (mol/ mol) assembly upon heating, the lattice parameter is found to exhibit a maximum value a = 13.5 nm at T = 52°C, and it decreases to a = 9.5 nm at T = 99°C (Figure 6). A hexagonal phase was not observed with this sample. This proves that a small amount of OG remains in the amphiphilic bilayer thus modifying its phase behavior with regard to the pure monoolein system, which forms an inverted hexagonal phase at T > 95°C [31]. The frames at 50 and 52°C (Figure 5, bottom panel) show intermediate states in the lamellar-to-cubic structural phase transition for
J. DRUG DEL. SCI. TECH., 18 (1) 41-45 2008
Figure 3 - Synchrotron radiation SAXD patterns of a MO/OG (95/5, mol/mol) liquid crystalline system self-assembled in 4 mg/mL human immunoglobulin (IgG) solution in phosphate buffer. Top panel: sequence of dynamic heating (from 1 to 99°C) and cooling (from 99 to 10°C) temperature scans showing a hysteresis in the reversible structural transition between a water-swollen (DLarge) diamond cubic phase and a normal (DNormal) bicontinuous cubic Q224 phase. Bottom panel: SAXS pattern selected from the heating scan at body temperature (T = 37°C).
Figure 4 - Cubic lattice parameter, a, versus temperature, T, for the investigated MO/OG/transferrin (bright circles) and MO/OG/IgG (filled black circles) self-assembly systems.
the MO/OG 80/20 system. In previous literature reports [32], such intermediates were accounted for as a scattering background. Certain authors [33] consider them to be a disordered bicontinuous emulsion. A special further investigation appears to be necessary in order to characterize the structure of such intermediates. Even though they resemble certain emulsion features, they are very likely to be bicontinuous cubic structures with some disorder of the nanochannel networks. In the higher temperature region (T > 52°C) the X-ray patterns correspond to a cubic phase of the Pn3m symmetry (Figure 5, top panel). * The present work demonstrates the dynamic control of the aqueous nanochannels in protein-containing monoolein bicontinuous cubic 43
J. DRUG DEL. SCI. TECH., 18 (1) 41-45 2008
Dynamic control of nanofluidic channels in protein drug delivery vehicles A. Angelova, B. Angelov, S. Lesieur, R. Mutafchieva, M. Ollivon, C. Bourgaux, R. Willumeit, P. Couvreur
stimulus, the virion shell breaks and a release of active substance occurs due to the fact that the swelling of the virus is not infinite. In MO/OG systems, the increase in the OG concentration causes swelling of the aqueous nanochannel diameters. Above a critical OG concentration, this swelling reaches a maximum and the supramolecular nanochannel structure will transform into a lamellar phase involving two-dimensional aqueous sheets instead of curved water nanochannels for nanoconfinement of guest biomolecules. These features suggest the feasibility of creation of soft-matter, self-regulating nanodevices.
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5. 6. Figure 5 - Synchrotron radiation SAXD patterns of a MO/OG (80/20, mol/mol) liquid crystalline system self-assembled in a phosphate buffer (no protein added). Top panel: dynamic heating scan from 1 to 99°C. Bottom panel: selected SAXD patterns from the heating scan showing an intermediate phase at T = 50°C (b) and at T = 52°C (c) during the transition between lamellar (T = 20°C (a)) and cubic phase (T > 50°C).
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Figure 6 - Temperature dependence of the repeat distance, d, of the lamellar (L) phase and of the lattice parameter, a, of the Pn3m cubic phase of MO/OG 80/20 (mol/mol) mixture self-assembled in phosphate buffer solution.
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Acknowledgements B.A. and R.M. acknowledge support from the Bulgarian Academy of Sciences internal project 2004/5. A.A. and B.A. acknowledge support by project LURE BD 001-03. The authors thank Dr. Geneviève Lebas for continuous cooperation and Gerard Keller for instrumental design and facilities.
Manuscript Received 25 June 2007, accepted for publication 13 August 2007.
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